Design, fabrication and test of a pneumatically controlled, renewable, microfluidic bead trapping device for sequential injection analysis applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shao, Guocheng; Lu, Donglai; Fu, Zhifeng

This paper describes the design, fabrication, and testing of a pneumatically controlled,renewable, microfluidic device for conducting bead-based assays in an automated sequential injection analysis system. The device used a “brick wall”-like pillar array (pillar size: 20 μm length X 50 μm width X 45 μm height) with 5 μm gaps between the pillars serving as the micro filter. The flow channel where bead trapping occurred is 500 μm wide X 75 μm deep. An elastomeric membrane and an air chamber were located underneath the flow channel. By applying pressure to the air chamber, the membrane is deformed and pushed upwardmore » against the filter structure. This effectively traps beads larger than 5 μm and creates a “bed” or micro column of beads that can be perfused and washed with liquid samples and reagents. Upon completion of the assay process, the pressure is released and the beads are flushed out from underneath the filter structure to renew the device. Mouse IgG was used as a model analyte to test the feasibility of using the proposed device for immunoassay applications. Resulting microbeads from an on-chip fluorescent immunoassay were individually examined using flow cytometry. The results show that the fluorescence signal intensity distribution is fairly narrow indicating high chemical reaction uniformity among the beads population. Electrochemical onchip assay was also conducted. A detection limit of 0.1 ng/mL1 ppb was achieved and good device reliability and repeatability were demonstrated. The novel microfluidic-based beadstrapping device thus opens up a new pathway to design micro-bead based biosensor immunoassays for clinical and othervarious applications.« less

Steady-state force-velocity relation in the ATP-dependent sliding movement of myosin-coated beads on actin cables in vitro studied with a centrifuge microscope.

PubMed Central

Oiwa, K; Chaen, S; Kamitsubo, E; Shimmen, T; Sugi, H

1990-01-01

To eliminate the gap between the biochemistry of actomyosin in solution and the physiology of contracting muscle, we developed an in vitro force-movement assay system in which the steady-state force-velocity relation in the actin-myosin interaction can be studied. The assay system consists of the internodal cells of an alga, Nitellopsis obtusa, containing well-organized actin filament arrays (actin cables); tosyl-activated polystyrene beads (diameter, 2.8 microns; specific gravity, 1.3) coated with skeletal muscle myosin; and a centrifuge microscope equipped with a stroboscopic light source and a video system. The internodal cell preparation was mounted on the rotor of the centrifuge microscope, so that centrifugal forces were applied to the myosin-coated beads moving along the actin cables in the presence of ATP. Under constant centrifugal forces directed opposite to the bead movement ("positive" loads), the beads continued to move with constant velocities, which decreased with increasing centrifugal forces. The steady-state force-velocity curve thus obtained was analogous to the double-hyperbolic force-velocity curve of single muscle fibers. The unloaded velocity of bead movement was 1.6-3.6 microns/s (20-23 degrees C), while the maximum "isometric" force generated by the myosin molecules on the bead was 1.9-39 pN. If, on the other hand, the beads were subjected to constant centrifugal forces in the direction of bead movement ("negative" loads), the bead also moved with constant velocities. Unexpectedly, the velocity of bead movement did not increase with increasing negative loads but first decreased by 20-60% and then increased towards the initial unloaded velocity until the beads were eventually detached from the actin cables. Images PMID:2236007

Steady-state force-velocity relation in the ATP-dependent sliding movement of myosin-coated beads on actin cables in vitro studied with a centrifuge microscope.

PubMed

Oiwa, K; Chaen, S; Kamitsubo, E; Shimmen, T; Sugi, H

1990-10-01

To eliminate the gap between the biochemistry of actomyosin in solution and the physiology of contracting muscle, we developed an in vitro force-movement assay system in which the steady-state force-velocity relation in the actin-myosin interaction can be studied. The assay system consists of the internodal cells of an alga, Nitellopsis obtusa, containing well-organized actin filament arrays (actin cables); tosyl-activated polystyrene beads (diameter, 2.8 microns; specific gravity, 1.3) coated with skeletal muscle myosin; and a centrifuge microscope equipped with a stroboscopic light source and a video system. The internodal cell preparation was mounted on the rotor of the centrifuge microscope, so that centrifugal forces were applied to the myosin-coated beads moving along the actin cables in the presence of ATP. Under constant centrifugal forces directed opposite to the bead movement ("positive" loads), the beads continued to move with constant velocities, which decreased with increasing centrifugal forces. The steady-state force-velocity curve thus obtained was analogous to the double-hyperbolic force-velocity curve of single muscle fibers. The unloaded velocity of bead movement was 1.6-3.6 microns/s (20-23 degrees C), while the maximum "isometric" force generated by the myosin molecules on the bead was 1.9-39 pN. If, on the other hand, the beads were subjected to constant centrifugal forces in the direction of bead movement ("negative" loads), the bead also moved with constant velocities. Unexpectedly, the velocity of bead movement did not increase with increasing negative loads but first decreased by 20-60% and then increased towards the initial unloaded velocity until the beads were eventually detached from the actin cables.

Effects of topography on the functional development of human neural progenitor cells.

PubMed

Wu, Ze-Zhi; Kisaalita, William S; Wang, Lina; Zachman, Angela L; Zhao, Yiping; Hasneen, Kowser; Machacek, Dave; Stice, Steven L

2010-07-01

We have fabricated a topographical substrate with a packed polystyrene bead array for the development of cell-based assay systems targeting voltage-gated calcium channels (VGCCs). Human neural progenitor cells (H945RB.3) cultured on both flat and topographical substrates were analyzed in terms of morphological spreading, neuronal commitment, resting membrane potential (V(m)) establishment and VGCC function development. We found, by SEM imaging, that arrayed substrates, formed with both sub-micrometer (of 0.51 microm in mean diameter) and micrometer (of 1.98 microm in mean diameter) beads, were capable of promoting the spreading of the progenitor cells as compared with the flat polystyrene surfaces. With the micrometer beads, it was found that arrayed substrates facilitated the neural progenitor cells' maintenance of less negative V(m) values upon differentiation with bFGF starvation, which favored predominant neuronal commitment. Almost all the progenitor cells were responsive to 50 mM K(+) depolarization with an increase in [Ca(2+)](i) either before or upon differentiation, suggesting the expression of functional VGCCs. Compared to the flat polystyrene surfaces, microbead arrayed substrates facilitated the development of higher VGCC responsiveness by the progenitor cells upon differentiation. The enhancement of both VGCC responsiveness and cell spreading by arrays of micrometer beads was most significant on day 14 into differentiation, which was the latest time point of measurement in this study. This study thus rationalized the possibility for future substrate topography engineering to manipulate ion channel function and to meet the challenge of low VGCC responsiveness found in early drug discovery.

An optimized library for reference-based deconvolution of whole-blood biospecimens assayed using the Illumina HumanMethylationEPIC BeadArray.

PubMed

Salas, Lucas A; Koestler, Devin C; Butler, Rondi A; Hansen, Helen M; Wiencke, John K; Kelsey, Karl T; Christensen, Brock C

2018-05-29

Genome-wide methylation arrays are powerful tools for assessing cell composition of complex mixtures. We compare three approaches to select reference libraries for deconvoluting neutrophil, monocyte, B-lymphocyte, natural killer, and CD4+ and CD8+ T-cell fractions based on blood-derived DNA methylation signatures assayed using the Illumina HumanMethylationEPIC array. The IDOL algorithm identifies a library of 450 CpGs, resulting in an average R 2  = 99.2 across cell types when applied to EPIC methylation data collected on artificial mixtures constructed from the above cell types. Of the 450 CpGs, 69% are unique to EPIC. This library has the potential to reduce unintended technical differences across array platforms.

Detection of mitochondrial DNA with the compact bead array sensor system (cBASS)

NASA Astrophysics Data System (ADS)

Mulvaney, Shawn P.; Ibe, Carol N.; Caldwell, Jane M.; Levine, Jay F.; Whitman, Lloyd J.; Tamanaha, Cy R.

2009-02-01

Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.

Multicomponent mesofluidic system for the detection of veterinary drug residues based on competitive immunoassay.

PubMed

Hu, Lei; Zuo, Peng; Ye, Bang-Ce

2010-10-01

An automated multicomponent mesofluidic system (MCMS) based on biorecognitions carried out on meso-scale glass beads in polydimethylsiloxane (PDMS) channels was developed. The constructed MCMS consisted of five modules: a bead introduction module, a bioreaction module, a solution handling module, a liquid driving module, and a signal collection module. The integration of these modules enables the assay to be automated and reduces it to a one-step protocol. The MCMS has successfully been applied toward the detection of veterinary drug residues in animal-derived foods. The drug antigen-coated beads (varphi250 microm) were arrayed in the PDMS channels (varphi300 microm). The competitive immunoassay was then carried out on the surface of the glass beads. After washing, the Cy3-labeled secondary antibody was introduced to probe the antigen-antibody complex anchored to the beads. The fluorescence intensity of each bead was measured and used to determine the residual drug concentration. The MCMS is highly sensitive, with its detection limits ranging from 0.02 (salbutamol) to 3.5 microg/L (sulfamethazine), and has a short assay time of 45 min or less. The experimental results demonstrate that the MCMS proves to be an economic, efficient, and sensitive platform for multicomponent detection of compound residues for contamination in foods or the environment. Copyright 2010 Elsevier Inc. All rights reserved.

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling*

PubMed Central

Poetz, Oliver; Henzler, Tanja; Hartmann, Michael; Kazmaier, Cornelia; Templin, Markus F.; Herget, Thomas; Joos, Thomas O.

2010-01-01

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity. PMID:20682761

The optics inside an automated single molecule array analyzer

NASA Astrophysics Data System (ADS)

McGuigan, William; Fournier, David R.; Watson, Gary W.; Walling, Les; Gigante, Bill; Duffy, David C.; Rissin, David M.; Kan, Cheuk W.; Meyer, Raymond E.; Piech, Tomasz; Fishburn, Matthew W.

2014-02-01

Quanterix and Stratec Biomedical have developed an instrument that enables the automated measurement of multiple proteins at concentration ~1000 times lower than existing immunoassays. The instrument is based on Quanterix's proprietary Single Molecule Array technology (Simoa™ ) that facilitates the detection and quantification of biomarkers previously difficult to measure, thus opening up new applications in life science research and in-vitro diagnostics. Simoa is based on trapping individual beads in arrays of femtoliter-sized wells that, when imaged with sufficient resolution, allows for counting of single molecules associated with each bead. When used to capture and detect proteins, this approach is known as digital ELISA (Enzyme-linked immunosorbent assay). The platform developed is a merger of many science and engineering disciplines. This paper concentrates on the optical technologies that have enabled the development of a fully-automated single molecule analyzer. At the core of the system is a custom, wide field-of-view, fluorescence microscope that images arrays of microwells containing single molecules bound to magnetic beads. A consumable disc containing 24 microstructure arrays was developed previously in collaboration with Sony DADC. The system cadence requirements, array dimensions, and requirement to detect single molecules presented significant optical challenges. Specifically, the wide field-of-view needed to image the entire array resulted in the need for a custom objective lens. Additionally, cost considerations for the system required a custom solution that leveraged the image processing capabilities. This paper will discuss the design considerations and resultant optical architecture that has enabled the development of an automated digital ELISA platform.

Optical Manipulation of Single Magnetic Beads in a Microwell Array on a Digital Microfluidic Chip.

PubMed

Decrop, Deborah; Brans, Toon; Gijsenbergh, Pieter; Lu, Jiadi; Spasic, Dragana; Kokalj, Tadej; Beunis, Filip; Goos, Peter; Puers, Robert; Lammertyn, Jeroen

2016-09-06

The detection of single molecules in magnetic microbead microwell array formats revolutionized the development of digital bioassays. However, retrieval of individual magnetic beads from these arrays has not been realized until now despite having great potential for studying captured targets at the individual level. In this paper, optical tweezers were implemented on a digital microfluidic platform for accurate manipulation of single magnetic beads seeded in a microwell array. Successful optical trapping of magnetic beads was found to be dependent on Brownian motion of the beads, suggesting a 99% chance of trapping a vibrating bead. A tailor-made experimental design was used to screen the effect of bead type, ionic buffer strength, surfactant type, and concentration on the Brownian activity of beads in microwells. With the optimal conditions, the manipulation of magnetic beads was demonstrated by their trapping, retrieving, transporting, and repositioning to a desired microwell on the array. The presented platform combines the strengths of digital microfluidics, digital bioassays, and optical tweezers, resulting in a powerful dynamic microwell array system for single molecule and single cell studies.

Wetting behavior on hexagonally close-packed polystyrene bead arrays with different topographies.

PubMed

Park, Yi-Seul; Yoon, Seo Young; Lee, Jin Seok

2016-01-21

Herein, we investigated the wetting behavior of hexagonally close-packed polystyrene bead arrays with different bead diameters and surface flatness. The contact angle was found to be influenced by the surface roughness as well as the contact area of the polystyrene bead array with a water droplet.

Integrating a high-force optical trap with gold nanoposts and a robust gold-DNA bond.

PubMed

Paik, D Hern; Seol, Yeonee; Halsey, Wayne A; Perkins, Thomas T

2009-08-01

Gold-thiol chemistry is widely used in nanotechnology but has not been exploited in optical-trapping experiments due to laser-induced ablation of gold. We circumvented this problem by using an array of gold nanoposts (r = 50-250 nm, h approximately 20 nm) that allowed for quantitative optical-trapping assays without direct irradiation of the gold. DNA was covalently attached to the gold via dithiol phosphoramidite (DTPA). By using three DTPAs, the gold-DNA bond was not cleaved in the presence of excess thiolated compounds. This chemical robustness allowed us to reduce nonspecific sticking by passivating the unreacted gold with methoxy-(polyethylene glycol)-thiol. We routinely achieved single beads anchored to the nanoposts by single DNA molecules. We measured DNA's elasticity and its overstretching transition, demonstrating moderate- and high-force optical-trapping assays using gold-thiol chemistry. Force spectroscopy measurements were consistent with the rupture of the strepavidin-biotin bond between the bead and the DNA. This implied that the DNA remained anchored to the surface due to the strong gold-thiol bond. Consistent with this conclusion, we repeatedly reattached the trapped bead to the same individual DNA molecule. Thus, surface conjugation of biomolecules onto an array of gold nanostructures by chemically and mechanically robust bonds provides a unique way to carry out spatially controlled, repeatable measurements of single molecules.

BeadArray Expression Analysis Using Bioconductor

PubMed Central

Ritchie, Matthew E.; Dunning, Mark J.; Smith, Mike L.; Shi, Wei; Lynch, Andy G.

2011-01-01

Illumina whole-genome expression BeadArrays are a popular choice in gene profiling studies. Aside from the vendor-provided software tools for analyzing BeadArray expression data (GenomeStudio/BeadStudio), there exists a comprehensive set of open-source analysis tools in the Bioconductor project, many of which have been tailored to exploit the unique properties of this platform. In this article, we explore a number of these software packages and demonstrate how to perform a complete analysis of BeadArray data in various formats. The key steps of importing data, performing quality assessments, preprocessing, and annotation in the common setting of assessing differential expression in designed experiments will be covered. PMID:22144879

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen

PubMed Central

van der Wal, Fimme J.; Achterberg, René P.; van Solt-Smits, Conny; Bergervoet, Jan H. W.; de Weerdt, Marjanne; Wisselink, Henk J.

2017-01-01

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population. PMID:28980519

Rapid Identification of Shiga Toxin-Producing Escherichia coli O Serogroups from Fresh Produce and Raw Milk Enrichment Cultures by Luminex Bead-Based Suspension Array.

PubMed

Kase, Julie A; Maounounen-Laasri, Anna; Lin, Andrew

2016-09-01

The U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 4a describes a Luminex microbead-based suspension array used to screen colonies for 11 clinically relevant Shiga toxin-producing Escherichia coli (STEC) serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. We evaluated the usefulness of this method to identify STEC-positive enrichment samples before agar plating. Twelve E. coli strains were added to three types of fresh produce (bagged baby spinach, alfalfa sprouts, and cilantro) at levels near the detection limit of the test. A subset of these strains (six O serogroups) was similarly evaluated in raw milk. For comparison, portions of each of the 168 enrichment cultures were analyzed for serogroup by a real-time PCR assay and a Bio-Plex 200 assay with the bead-based suspensions. No false-positive results were obtained. Of the 112 samples with a reported cycle threshold (C T ) value, 101 undiluted, diluted, or extracted enrichment cultures also produced ratios above 5.0 in the Bio-Plex assay. When PCR C T values approached or were greater than 35, Bio-Plex detection became less reliable. Using undiluted or extracted enrichment cultures resulted in a significantly larger number of positive results. With the same enrichment material prepared for real-time PCR analysis as described in the BAM Chapter 4a, the STEC microbead-based suspension array can accurately screen food enrichment cultures.

Identification of Brucella genus and eight Brucella species by Luminex bead-based suspension array.

PubMed

Lusk Pfefer, Tina S; Timme, Ruth; Kase, Julie A

2018-04-01

Globally, unpasteurized milk products are vehicles for the transmission of brucellosis, a zoonosis responsible for cases of foodborne illness in the United States and elsewhere. Existing PCR assays to detect Brucella species are restricted by the resolution of band sizes on a gel or the number of fluorescent channels in a single real-time system. The Luminex bead-based suspension array is performed in a 96-well plate allowing for high throughput screening of up to 100 targets in one sample with easily discernible results. We have developed an array using the Bio-Plex 200 to differentiate the most common Brucella species: B. abortus, B. melitensis, B. suis, B. suis bv5, B. canis, B. ovis, B. pinnipedia, and B. neotomae, as well as Brucella genus. All probes showed high specificity, with no cross-reaction with non-Brucella strains. We could detect pure DNA from B. abortus, B. melitensis, and genus-level Brucella at concentrations of ≤5 fg/μL. Pure DNA from all other species tested positive at concentrations well below 500 fg/μL and we positively identified B. neotomae in six artificially contaminated cheese and milk products. An intra-laboratory verification further demonstrated the assay's accuracy and robustness in the rapid screening (3-4 h including PCR) of DNA. Published by Elsevier Ltd.

Towards High Throughput Cell Growth Screening: A New CMOS 8 × 8 Biosensor Array for Life Science Applications.

PubMed

Nabovati, Ghazal; Ghafar-Zadeh, Ebrahim; Letourneau, Antoine; Sawan, Mohamad

2017-04-01

In this paper we present a CMOS capacitive sensor array as a compact and low-cost platform for high-throughput cell growth monitoring. The proposed biosensor, consists of an array of 8 × 8 CMOS fully differential charge-based capacitive measurement sensors. A DC-input Σ∆ modulator is used to convert the sensors' signals to digital values for reading out the biological/chemical data and further signal processing. To compensate the mismatch variations between the current mirror transistors, a calibration circuitry is proposed which removes the output voltage offset with less than 8.2% error. We validate the chip functionality using various organic solvents with different dielectric constants. Moreover, we show the response of the chip to different concentrations of Polystyrene beads that have the same electrical properties as the living cells. The experimental results show that the chip allows the detection of a wide range of Polystyrene beads concentrations from as low as 10 beads/ml to 100 k beads/ml. In addition, we present the experimental results from H1299 (human lung carcinoma) cell line where we show that the chip successfully allows the detection of cell attachment and growth over capacitive electrodes in a 30 h measurement time and the results are in consistency with the standard cell-based assays. The capability of proposed device for label-free and real-time detection of cell growth with very high sensitivity opens up the important opportunity for utilizing the device in rapid screening of living cells.

SERS based immuno-microwell arrays for multiplexed detection of foodborne pathogenic bacteria

NASA Astrophysics Data System (ADS)

Sun, Jian; Hankus, Mikella E.; Cullum, Brian M.

2009-05-01

A novel surface enhanced Raman scattering (SERS)-based immuno-microwell array has been developed for multiplexed detection of foodborne pathogenic bacteria. The immuno-microwell array was prepared by immobilizing the optical addressable immunomagnetic beads (IMB) into the microwell array on one end of a fiber optic bundle. The IMBs, magnetic beads coated with specific antibody to specific bacteria, were used for immunomagnetic separation (IMS) of corresponding bacteria. The magnetic separation by the homemade magnetic separation system was evaluated in terms of the influences of several important parameters including the beads concentration, the sample volume and the separation time. IMS separation efficiency of the model bacteria E.coli O157:H7 was 63% in 3 minutes. The microwell array was fabricated on hydrofluoric acid etched end of a fiber optic bundle containing 30,000 fiber elements. After being coated with silver, the microwell array was used as a uniform SERS substrate with the relative standard deviation of the SERS enhancement across the microwell array < 2% and the enhancement factor as high as 2.18 x 107. The antibody modified microwell array was prepared for bacteria immobilization into the microwell array, which was characterized by a sandwich immunoassay. To demonstrate the potential of multiplexed SERS detection with the immuno-microwell array, the SERS spectra of different Raman dye labeled magnetic beads as well as mixtures were measured on the mircrowell array. In bead mixture, different beads were identified by the characteristic SERS bands of the corresponding Raman label.

Jeffamine derivatized TentaGel beads and poly(dimethylsiloxane) microbead cassettes for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound combinatorial small molecule libraries.

PubMed

Townsend, Jared B; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S

2010-09-13

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting trifunctional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry resulting in beads with increased loading capacity, hydrophilicity, and porosity at the outer layer. We have found that such bead configuration can facilitate ultrahigh-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 min) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel were then layered over the microbead cassette to immobilize the compound-beads. After 24 h of incubation at 37 °C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

Large-scale femtoliter droplet array for digital counting of single biomolecules.

PubMed

Kim, Soo Hyeon; Iwai, Shino; Araki, Suguru; Sakakihara, Shouichi; Iino, Ryota; Noji, Hiroyuki

2012-12-07

We present a novel device employing one million femtoliter droplets immobilized on a substrate for the quantitative detection of extremely low concentrations of biomolecules in a sample. Surface-modified polystyrene beads carrying either zero or a single biomolecule-reporter enzyme complex are efficiently isolated into femtoliter droplets formed on hydrophilic-in-hydrophobic surfaces. Using a conventional micropipette, this is achieved by sequential injection first with an aqueous solution containing beads, and then with fluorinated oil. The concentration of target biomolecules is estimated from the ratio of the number of signal-emitting droplets to the total number of trapped beads (digital counting). The performance of our digital counting device was demonstrated by detecting a streptavidin-β-galactosidase conjugate with a limit of detection (LOD) of 10 zM. The sensitivity of our device was >20-fold higher than that noted in previous studies where a smaller number of reactors (fifty thousand reactors) were used. Such a low LOD was achieved because of the large number of droplets in an array, allowing simultaneous examination of a large number of beads. When combined with bead-based enzyme-linked immunosorbent assay (digital ELISA), the LOD for the detection of prostate specific antigen reached 2 aM. This value, again, was improved over that noted in a previous study, because of the decreased coefficient of variance of the background measurement determined by the Poisson noise. Our digital counting device using one million droplets has great potential as a highly sensitive, portable immunoassay device that could be used to diagnose diseases.

Jeffamine Derivatized TentaGel Beads and PDMS Microbead Cassettes for Ultra-high Throughput in situ Releasable Solution-Phase Cell-based Screening of OBOC Combinatorial Small Molecule Libraries

PubMed Central

Townsend, Jared B.; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S.

2011-01-01

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated polydimethylsiloxane (PDMS) cassette for high-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting tri-functional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry, resulting in beads with increased loading capacity, hydrophilicity and porosity at the outer layer. We have found that such bead configuration can facilitate ultra high-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 minutes) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel® were then layered over the microbead cassette to immobilize the compound-beads. After 24 hours of incubation at 37°C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were re-synthesized and found to be cytotoxic (IC50 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultra high-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays. PMID:20593859

Magnetic bead droplet immunoassay of oligomer amyloid β for the diagnosis of Alzheimer's disease using micro-pillars to enhance the stability of the oil-water interface.

PubMed

Kim, Jeong Ah; Kim, Moojong; Kang, Sung Min; Lim, Kun Taek; Kim, Tae Song; Kang, Ji Yoon

2015-05-15

Despite scientific progress in the study of Alzheimer's disease (AD), it is still challenging to develop a robust and sensitive methodology for the early diagnosis of AD due to the lack of a decisive biomarker in blood. Recent reports on the oligomer amyloid β (Aβ) as a biomarker demonstrated its possibility for identifying early onset of AD in patients, but its low concentration in blood requires highly reliable detection techniques. To overcome the low reliability and labor-intensive procedures of conventional enzyme-linked immunosorbent assay (ELISA), we present a magnetic bead-droplet immunoassay platform for simple and highly sensitive detection of oligomer Aβ for the diagnosis of AD. This microchip consists of chambers that contain water-based reagents or oil for consecutive assay procedures, and there are arrays of micro-pillars fabricated between the two adjacent chambers to form robust water-oil interfaces. With the aid of these micro-pillars, magnetic beads can stably pass through each chamber by linearly actuating a magnet along the microchip. The robust water-oil interface and simple procedures of the assay make it possible to obtain reliable results from this microchip. The intensity of the fluorescence at the read-out chamber increased quantitatively and linearly, depending on the amount of serially-diluted standard Aβ solution. The results of the assay indicated that the limit of detection was about 10 pg/mL even though it was done with manual manipulation of the magnet. This platform simplified the complicated ELISA procedure and achieved high sensitivity that was no lower than that of the conventional magnetic bead immunoassay. The magnetic bead-droplet platform reduced the assay time to 45 min, and it also reduced the amount of antibody usage in a single diagnosis significantly (10-30 ng of antibody per single assay). Consequently, this microfluidic chip has strong potential as a feasible system for use in the diagnosis of AD with a fast and easy immunoassay process, since the suggested platform can be automated with ease for point-of-care testing as well as high-throughput diagnostic equipment. Copyright © 2014 Elsevier B.V. All rights reserved.

Digital detection of multiple minority mutants and expression levels of multiple colorectal cancer-related genes using digital-PCR coupled with bead-array.

PubMed

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed "multiplex ligation-dependent probe amplification-digital amplification coupled with hydrogel bead-array" (MLPA-DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA-DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA-DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.

Automated methods for multiplexed pathogen detection.

PubMed

Straub, Timothy M; Dockendorff, Brian P; Quiñonez-Díaz, Maria D; Valdez, Catherine O; Shutthanandan, Janani I; Tarasevich, Barbara J; Grate, Jay W; Bruckner-Lea, Cynthia J

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.

Automated Methods for Multiplexed Pathogen Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Straub, Tim M.; Dockendorff, Brian P.; Quinonez-Diaz, Maria D.

2005-09-01

Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cyclermore » where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides ''live vs. dead'' capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.« less

Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (μCHAMPs) for the detection of amyloid beta oligomers.

PubMed

Park, Min Cheol; Kim, Moojong; Lim, Gun Taek; Kang, Sung Min; An, Seong Soo A; Kim, Tae Song; Kang, Ji Yoon

2016-06-21

Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 μL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (μCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this μCHAMP-based immunoassay platform, small volumes (30-50 μL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 μm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this μCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aβ) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 μL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aβ oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the μCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity.

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

PubMed Central

2017-01-01

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. PMID:28199790

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

PubMed

MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

2017-03-13

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

Breath-Taking Patterns: Discontinuous Hydrophilic Regions for Photonic Crystal Beads Assembly and Patterns Revisualization.

PubMed

Du, Xuemin; Wang, Juan; Cui, Huanqing; Zhao, Qilong; Chen, Hongxu; He, Le; Wang, Yunlong

2017-11-01

Surfaces patterned with hydrophilic and hydrophobic regions provide robust and versatile means for investigating the wetting behaviors of liquids, surface properties analysis, and producing patterned arrays. However, the fabrication of integral and uniform arrays onto these open systems remains a challenge, thus restricting them from being used in practical applications. Here, we present a simple yet powerful approach for the fabrication of water droplet arrays and the assembly of photonic crystal bead arrays based on hydrophilic-hydrophobic patterned substrates. Various integral arrays are simply prepared in a high-quality output with a low cost, large scale, and uniform size control. By simply taking a breath, which brings moisture to the substrate surface, complex hydrophilic-hydrophobic outlined images can be revisualized in the discontinuous hydrophilic regions. Integration of hydrogel photonic crystal bead arrays into the "breath-taking" process results in breath-responsive photonic crystal beads, which can change their colors upon a mild exhalation. This state-of-the-art technology not only provides an effective methodology for the preparation of patterned arrays but also demonstrates intriguing applications in information storage and biochemical sensors.

Corneal Protection for Burn Patients

DTIC Science & Technology

2014-11-01

multiplex immunoassays utilizing the Luminex bead array were procured. Tested chemokines/cytokines include EGF, FGF-2, Eotaxin, IFN, GRO, MDC, PDGF-BB, IL...17A, IL-1RA, IL- 3, IL-6, IL-8, MP-1a and VEGF. A separate assay was used to test for matrix metalloproteinase 9 (MMP 9) given its known up...regulation in dry eye models and clinical scenarios. The tested cytokines were chosen based on their reported prevalence in dry eye states and the

Label-free voltammetric detection of MicroRNAs at multi-channel screen printed array of electrodes comparison to graphite sensors.

PubMed

Erdem, Arzum; Congur, Gulsah

2014-01-01

The multi-channel screen-printed array of electrodes (MUX-SPE16) was used in our study for the first time for electrochemical monitoring of nucleic acid hybridization related to different miRNA sequences (miRNA-16, miRNA-15a and miRNA-660, i.e, the biomarkers for Alzheimer disease). The MUX-SPE16 was also used for the first time herein for the label-free electrochemical detection of nucleic acid hybridization combined magnetic beads (MB) assay in comparison to the disposable pencil graphite electrode (PGE). Under the principle of the magnetic beads assay, the biotinylated inosine substituted DNA probe was firstly immobilized onto streptavidin coated MB, and then, the hybridization process between probe and its complementary miRNA sequence was performed at MB surface. The voltammetric transduction was performed using differential pulse voltammetry (DPV) technique in combination with the single-use graphite sensor technologies; PGE and MUX-SPE16 for miRNA detection by measuring the guanine oxidation signal without using any external indicator. The features of single-use sensor technologies, PGE and MUX-SPE16, were discussed concerning to their reproducibility, detection limit, and selectivity compared to the results in the earlier studies presenting the electrochemical miRNA detection related to different miRNA sequences. © 2013 Elsevier B.V. All rights reserved.

A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma.

PubMed

Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John; Theander, Thor G; Jensen, Anja T R; Turner, Louise

2008-06-12

The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the problem of storage of antigen can influence ELISA results. The bead-based assay described here uses the BioPlex100 (BioRad, Hercules, CA, USA) system which can quantify multiple antibodies simultaneously in a small plasma volume. A total of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1 proteins were covalently coupled onto beads each having its own unique detection signal and the human hyper-immune plasma reactivity was detected for each individual protein using a BioPlex100 system. Protein-coupled beads were analysed at two time points seven months apart, before and after lyophilization and the results compared to determine the effect of storage and lyophilization respectively on the beads. Multiplexed protein-coupled beads from twenty eight unique bead populations were evaluated on the BioPlex100 system against pooled human hyper-immune plasma before and after lyophilization. The bead-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both analyses were highly correlated. The Spearman's rank correlation coefficients (Rho) were > or = 0.86, (P < 0.0001) for all comparisons. Bead-based assays gave similar results regardless of whether they were performed on individual beads or on multiplexed beads; lyophilization had no impact on the assay performance. Spearman's rank correlation coefficients (Rho) were > or = 0.97, (P < 0.0001) for all comparisons. Importantly, the reactivity of protein-coupled non-lyophilized beads decreased with long term storage at 4 degrees C in the dark. Using this lyophilized multiplex assay, antibody reactivity levels to twenty eight different recombinant PfEMP1 proteins were simultaneously measured using a single microliter of plasma. Thus, the assay reported here provides a useful tool for rapid and efficient quantification of antibody reactivity against PfEMP1 variants in human plasma.

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

PubMed Central

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC. PMID:25880764

Detection of Neisseria meningitidis in cerebrospinal fluid using a multiplex PCR and the Luminex detection technology.

PubMed

Møller, Jens Kjølseth

2012-01-01

Rapid clinical and laboratory diagnoses are the foundation for a successful management of serious infections with Neisseria meningitidis. A species-specific multiplex polymerase chain reaction (PCR) coupled with fluidic microarrays using microbeads (the Luminex xMAP™ Technology) can detect pathogens most frequently found in the cerebrospinal fluid of patients. The Luminex suspension array system uniquely combines flow cytometry, microspheres, laser technology, digital signal processing, and traditional chemistry. In this method, the reaction is carried out in one vessel, in which distinctly color-coded bead sets, each conjugated with a different specific nucleic acid reactant, are hybridized with the PCR products, and a reporter molecule is used to quantify the interaction. The flow-based Luminex array reader identifies each reaction (bead set) after excitation by a red classification laser. Reporter signals from each reaction are simultaneously quantified by fluorescence generated by a green reporter laser. This nonculture, multiplex assay may prove to be an important tool for optimal laboratory diagnosis, not only of meningococcal meningitis, but also of meningitis caused by other bacterial or viral pathogens.

Optical Encoding Technology for Viral Screening Panels Final Report CRADA No TC02132.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lenhoff, R.; Haushalter, R.

This was a collaborative effort between Lawrence Livermore National Security, LLC, Lawrence Livermore National Laboratory (LLNL) and Parallel Synthesis Technologies, Inc. (PSTI), to develop Optical Encoding Technology for Viral Screening Panels. The goal for this effort was to prepare a portable bead reader system that would enable the development of viral and bacterial screening panels which could be used for the detection of any desired set of bacteria or viruses in any location. The main objective was to determine if the combination of a bead-based, PCR suspension array technology, formulated from Parallume encoded beads and PSTI’s multiplex assay reader systemmore » (MARS), could provide advantages in terms of the number of simultaneously measured samples, portability, ruggedness, ease of use, accuracy, precision or cost as compared to the Luminexbased system developed at LLNL. The project underwent several no cost extensions however the overall goal of demonstrating the utility of this new system was achieved. As a result of the project a significant change to the type of bead PSTI used for the suspension system was implemented allowing better performance than the commercial Luminex system.« less

Particle-Based Microarrays of Oligonucleotides and Oligopeptides.

PubMed

Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F Ralf; Breitling, Frank; Loeffler, Felix F

2014-10-28

In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

Particle-Based Microarrays of Oligonucleotides and Oligopeptides

PubMed Central

Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K.; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F. Ralf; Breitling, Frank; Loeffler, Felix F.

2014-01-01

In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches. PMID:27600347

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

PubMed

Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

2002-01-01

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

PubMed

Kamburova, E G; Wisse, B W; Joosten, I; Allebes, W A; van der Meer, A; Hilbrands, L B; Baas, M C; Spierings, E; Hack, C E; van Reekum, F E; van Zuilen, A D; Verhaar, M; Bots, M L; Drop, A C A D; Plaisier, L; Seelen, M A J; Sanders, J S F; Hepkema, B G; Lambeck, A J; Bungener, L B; Roozendaal, C; Tilanus, M G J; Vanderlocht, J; Voorter, C E; Wieten, L; van Duijnhoven, E M; Gelens, M; Christiaans, M H L; van Ittersum, F J; Nurmohamed, A; Lardy, N M; Swelsen, W; van der Pant, K A; van der Weerd, N C; Ten Berge, I J M; Bemelman, F J; Hoitsma, A; van der Boog, P J M; de Fijter, J W; Betjes, M G H; Heidt, S; Roelen, D L; Claas, F H; Otten, H G

2016-09-01

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

On-chip immune cell activation and subsequent time-resolved magnetic bead-based cytokine detection.

PubMed

Kongsuphol, Patthara; Liu, Yunxiao; Ramadan, Qasem

2016-10-01

Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of triple-layered microfluidic chambers was fabricated to enable simultaneous cell culture under perfusion flow and detection of the induced cytokines at multiple time-points. Each culture chamber comprises three fluidic compartments which are dedicated to, cell culture, perfusion and immunoassay. The three compartments are separated by porous membranes, which allow the diffusion of fresh nutrient from the perfusion compartment into the cell culture compartment and cytokines secretion from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture and without minimizing benefit gain from perfusion. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The cytokines released from differential stimuli were quantified in situ in non-differentiated U937 monocytes and differentiated macrophages.

The recomBead Borrelia antibody index, CXCL13 and total IgM index for laboratory diagnosis of Lyme neuroborreliosis in children.

PubMed

Skogman, B H; Lager, M; Henningsson, A J; Tjernberg, I

2017-11-01

For laboratory diagnostics of Lyme neuroborreliosis (LNB), the recomBead Borrelia antibody index (AI) assay has shown promising results in a mixed age population, but has not previously been evaluated with specific focus on paediatric patients. The aim of the study was to evaluate the recomBead Borrelia AI assay in cerebrospinal fluid (CSF) for the laboratory diagnosis of LNB in children. We also wanted to explore whether early markers, such as CXCL13 in CSF and/or total IgM index could be useful as complementary diagnostic tools. Children being evaluated for LNB in a Swedish Lyme endemic area were included in the study (n = 146). Serum and CSF were collected on admission. Patients with other specific diagnoses were controls (n = 15). The recomBead Borrelia AI assay and the recomBead CXCL13 assay (Mikrogen) were applied together with total IgM index. The overall sensitivity for recomBead Borrelia AI (IgM and IgG together) was 74% and the specificity was 97%. However, the highest sensitivity (91%) at an acceptable level of specificity (90%) was obtained by recomBead Borrelia AI together with CXCL13 and total IgM index, showing a positive predictive value of 84% and a negative predictive value of 95%. Thus, the recomBead Borrelia AI assay performs with moderate sensitivity and high specificity in paediatric LNB patients. The major advantage seems to be increased sensitivity in the possible LNB group compared to the IDEIA assay. The diagnostic sensitivity may be further increased by using a combination of early markers, such as CXCL13 in CSF and total IgM index.

[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

PubMed

Wen, Hai-yan; Wang, Jing; Liu, Heng-chuan; Sun, Xiao-hong; Yang, Yu; Hu, Kong-xin; Shan, Lin-jun

2009-10-01

To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 ‘shared epitope’ alleles

PubMed Central

Wagner, Catriona A; Sokolove, Jeremy; Lahey, Lauren J; Bengtsson, Camilla; Saevarsdottir, Saedis; Alfredsson, Lars; Delanoy, Michelle; Lindstrom, Tamsin M; Walker, Roger P; Bromberg, Reuven; Chandra, Piyanka E; Binder, Steven R; Klareskog, Lars; Robinson, William H

2015-01-01

Introduction A hallmark of rheumatoid arthritis (RA) is the development of autoantibodies targeting proteins that contain citrulline. Anticitrullinated protein antibodies (ACPAs) are currently detected by the commercial cyclic citrullinated peptide (CCP) assay, which uses a mix of cyclised citrullinated peptides as an artificial mimic of the true antigen(s). To increase the sensitivity of ACPA detection and dissect ACPA specificities, we developed a multiplex assay that profiles ACPAs by measuring their reactivity to the citrullinated peptides and proteins derived from RA joint tissue. Methods We created a bead-based, citrullinated antigen array to profile ACPAs. This custom array contains 16 citrullinated peptides and proteins detected in RA synovial tissues. We used the array to profile ACPAs in sera from a cohort of patients with RA and other non-inflammatory arthritides, as well as sera from an independent cohort of RA patients for whom data were available on carriage of HLA-DRB1 ‘shared epitope’ (SE) alleles and history of cigarette smoking. Results Our multiplex assay showed that at least 10% of RA patients who tested negative in the commercial CCP assay possessed ACPAs. Carriage of HLA-DRB1 SE alleles and a history of cigarette smoking were associated with an increase in ACPA reactivity—in anti-CCP+ RA and in a subset of anti-CCP− RA. Conclusions Our multiplex assay can identify ACPA-positive RA patients missed by the commercial CCP assay, thus enabling greater diagnostic sensitivity. Further, our findings suggest that cigarette smoking and possession of HLA-DRB1 SE alleles contribute to the development of ACPAs in anti-CCP− RA. PMID:24297382

Rapid, highly sensitive detection of herpes simplex virus-1 using multiple antigenic peptide-coated superparamagnetic beads.

PubMed

Ran, Ying-Fen; Fields, Conor; Muzard, Julien; Liauchuk, Viktoryia; Carr, Michael; Hall, William; Lee, Gil U

2014-12-07

A sensitive, rapid, and label free magnetic bead aggregation (MBA) assay has been developed that employs superparamagnetic (SPM) beads to capture, purify, and detect model proteins and the herpes simplex virus (HSV). The MBA assay is based on monitoring the aggregation state of a population of SPM beads using light scattering of individual aggregates. A biotin-streptavidin MBA assay had a femtomolar (fM) level sensitivity for analysis times less than 10 minutes, but the response of the assay becomes nonlinear at high analyte concentrations. A MBA assay for the detection of HSV-1 based on a novel peptide probe resulted in the selective detection of the virus at concentrations as low as 200 viral particles (vp) per mL in less than 30 min. We define the parameters that determine the sensitivity and response of the MBA assay, and the mechanism of enhanced sensitivity of the assay for HSV. The speed, relatively low cost, and ease of application of the MBA assay promise to make it useful for the identification of viral load in resource-limited and point-of-care settings where molecular diagnostics cannot be easily implemented.

A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

PubMed

Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

2015-02-01

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

Single-bead arrays for fluorescence-based immunoassays on capillary-driven microfluidic chips

NASA Astrophysics Data System (ADS)

Temiz, Yuksel; Lim, Michel; Delamarche, Emmanuel

2016-03-01

We report a concept for the simple fabrication of easy-to-use chips for immunoassays in the context of point-of-care diagnostics. The chip concept comprises mainly three features: (1) the efficient integration of reagents using beads functionalized with receptors, (2) the generation of capillary-driven liquid flows without using external pumps, and (3) a high-sensitivity detection of analytes using fluorescence microscopy. We fabricated prototype chips using dry etching of Si wafers. 4.5-μm-diameter beads were integrated into hexagonal arrays by sedimentation and removing the excess using a stream of water. We studied the effect of different parameters and showed that array occupancies from 30% to 50% can be achieved by pipetting a 250 nL droplet of 1% bead solution and allowing the beads sediment for 3 min. Chips with integrated beads were sealed using a 50-μm-thick dry-film resist laminated at 45 °C. Liquids pipetted to loading pads were autonomously pulled by capillary pumps at a rate of 0.35 nL s-1 for about 30 min. We studied ligand-receptor interactions and binding kinetics using time-lapse fluorescence microscopy and demonstrated a 5 pM limit of detection (LOD) for an anti-biotin immunoassay. As a clinically-relevant example, we implemented an immunoassay to detect prostate specific antigen (PSA) and showed an LOD of 108 fM (i.e. 3.6 pg mL-1). While a specific implementation is provided here for the detection of PSA, we believe that combining capillary-driven microfluidics with arrays of single beads and fluorescence readout to be very flexible and sufficiently sensitive for the detection of other clinically-relevant analytes.

Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

PubMed Central

Tait, Brian D.

2016-01-01

This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques. PMID:28018342

Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead-based applications

NASA Astrophysics Data System (ADS)

Kim, Hojin; Choi, In Ho; Lee, Sanghyun; Won, Dong-Joon; Oh, Yong Suk; Kwon, Donghoon; Sung, Hyung Jin; Jeon, Sangmin; Kim, Joonwon

2017-04-01

This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead-encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin-biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules.

Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead-based applications.

PubMed

Kim, Hojin; Choi, In Ho; Lee, Sanghyun; Won, Dong-Joon; Oh, Yong Suk; Kwon, Donghoon; Sung, Hyung Jin; Jeon, Sangmin; Kim, Joonwon

2017-04-10

This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead-encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin-biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules.

A Liquid Array Platform For the Multiplexed Analysis of Synthetic Molecule-Protein Interactions

PubMed Central

Doran, Todd M.; Kodadek, Thomas

2014-01-01

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative comprised of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships and for certain types of serological analyses. PMID:24245981

Design criteria for developing low-resource magnetic bead assays using surface tension valves

PubMed Central

Adams, Nicholas M.; Creecy, Amy E.; Majors, Catherine E.; Wariso, Bathsheba A.; Short, Philip A.; Wright, David W.; Haselton, Frederick R.

2013-01-01

Many assays for biological sample processing and diagnostics are not suitable for use in settings that lack laboratory resources. We have recently described a simple, self-contained format based on magnetic beads for extracting infectious disease biomarkers from complex biological samples, which significantly reduces the time, expertise, and infrastructure required. This self-contained format has the potential to facilitate the application of other laboratory-based sample processing assays in low-resource settings. The technology is enabled by immiscible fluid barriers, or surface tension valves, which stably separate adjacent processing solutions within millimeter-diameter tubing and simultaneously permit the transit of magnetic beads across the interfaces. In this report, we identify the physical parameters of the materials that maximize fluid stability and bead transport and minimize solution carryover. We found that fluid stability is maximized with ≤0.8 mm i.d. tubing, valve fluids of similar density to the adjacent solutions, and tubing with ≤20 dyn/cm surface energy. Maximizing bead transport was achieved using ≥2.4 mm i.d. tubing, mineral oil valve fluid, and a mass of 1-3 mg beads. The amount of solution carryover across a surface tension valve was minimized using ≤0.2 mg of beads, tubing with ≤20 dyn/cm surface energy, and air separators. The most favorable parameter space for valve stability and bead transport was identified by combining our experimental results into a single plot using two dimensionless numbers. A strategy is presented for developing additional self-contained assays based on magnetic beads and surface tension valves for low-resource diagnostic applications. PMID:24403996

Growth of arrays of oriented epitaxial platinum nanoparticles with controlled size and shape by natural colloidal lithography

DOE PAGES

Komanicky, Vladimir; Barbour, Andi; Lackova, Miroslava; ...

2014-07-05

Here, we developed a method for production of arrays of platinum nanocrystals of controlled size and shape using templates from ordered silica bead monolayers. Silica beads with nominal sizes of 150 and 450 nm were self-assembl into monolayers over strontium titanate single crystal substrates. The monolayers were used as shadow masks for platinum metal deposition on the substrate using the three-step evaporation technique. Produced arrays of epitaxial platinum islands were transformed into nanocrystals by annealing in a quartz tube in nitrogen flow. The shape of particles is determined by the substrate crystallography, while the size of the particles and theirmore » spacing are controlled by the size of the silica beads in the mono- layer mask. As a proof of concept, arrays of platinum nanocrystals of cubooctahedral shape were prepared on (100) strontium titanate substrates. We also characterized the nanocrystal arrays by atomic force microscopy, scanning electron microscopy, and synchrotron X-ray diffraction techniques.« less

A user-friendly workflow for analysis of Illumina gene expression bead array data available at the arrayanalysis.org portal.

PubMed

Eijssen, Lars M T; Goelela, Varshna S; Kelder, Thomas; Adriaens, Michiel E; Evelo, Chris T; Radonjic, Marijana

2015-06-30

Illumina whole-genome expression bead arrays are a widely used platform for transcriptomics. Most of the tools available for the analysis of the resulting data are not easily applicable by less experienced users. ArrayAnalysis.org provides researchers with an easy-to-use and comprehensive interface to the functionality of R and Bioconductor packages for microarray data analysis. As a modular open source project, it allows developers to contribute modules that provide support for additional types of data or extend workflows. To enable data analysis of Illumina bead arrays for a broad user community, we have developed a module for ArrayAnalysis.org that provides a free and user-friendly web interface for quality control and pre-processing for these arrays. This module can be used together with existing modules for statistical and pathway analysis to provide a full workflow for Illumina gene expression data analysis. The module accepts data exported from Illumina's GenomeStudio, and provides the user with quality control plots and normalized data. The outputs are directly linked to the existing statistics module of ArrayAnalysis.org, but can also be downloaded for further downstream analysis in third-party tools. The Illumina bead arrays analysis module is available at http://www.arrayanalysis.org . A user guide, a tutorial demonstrating the analysis of an example dataset, and R scripts are available. The module can be used as a starting point for statistical evaluation and pathway analysis provided on the website or to generate processed input data for a broad range of applications in life sciences research.

Viscosity of dilute suspensions of rigid bead arrays at low shear: accounting for the variation in hydrodynamic stress over the bead surfaces.

PubMed

Allison, Stuart A; Pei, Hongxia

2009-06-11

In this work, we examine the viscosity of a dilute suspension of irregularly shaped particles at low shear. A particle is modeled as a rigid array of nonoverlapping beads of variable size and geometry. Starting from a boundary element formalism, approximate account is taken of the variation in hydrodynamic stress over the surface of the individual beads. For a touching dimer of two identical beads, the predicted viscosity is lower than the exact value by 5.2%. The methodology is then applied to several other model systems including tetramers of variable conformation and linear strings of touching beads. An analysis is also carried out of the viscosity and translational diffusion of several dilute amino acids and diglycine in water. It is concluded that continuum hydrodynamic modeling with stick boundary conditions is unable to account for the experimental viscosity and diffusion data simultaneously. A model intermediate between "stick" and "slip" could possibly reconcile theory and experiment.

Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead–based applications

PubMed Central

Kim, Hojin; Choi, In Ho; Lee, Sanghyun; Won, Dong-Joon; Oh, Yong Suk; Kwon, Donghoon; Sung, Hyung Jin; Jeon, Sangmin; Kim, Joonwon

2017-01-01

This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead–encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin–biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules. PMID:28393911

Ultraflexible nanostructures and implications for future nanorobots

NASA Astrophysics Data System (ADS)

Cohn, Robert W.; Panchapakesan, Balaji

2016-05-01

Several high aspect ratio nanostructures have been made by capillary force directed self-assembly including polymeric nanofiber air-bridges, trampoline-like membranes, microsphere-beaded nanofibers, and intermetallic nanoneedles. Arrays of polymer air-bridges form in seconds by simply hand brushing a bead of polymeric liquid over an array of micropillars. The domination of capillary force that is thinning unstable capillary bridges leads to uniform arrays of nanofiber air-bridges. Similarly, arrays of vertically oriented Ag2Ga nanoneedles have been formed by dipping silvercoated arrays of pyramidal silicon into melted gallium. Force-displacement measurements of these structures are presented. These nanostructures, especially when compressively or torsionally buckled, have extremely low stiffnesses, motion due to thermal fluctuations that is relatively easily detected, and the ability to move great distances for very small changes in applied force. Nanofibers with bead-on-a-string structure, where the beads are micron diameter and loaded with magnetic iron oxide (maghemite), are shown to be simply viewable under optical microscopes, have micronewton/ m stiffness, and have ultralow torsional stiffnesses enabling the bead to be rotated numerous revolutions without breaking. Combination of these high aspect ratio structures with stretched elastomers offer interesting possibilities for robotic actuation and locomotion. Polydimethylsiloxane loaded with nanomaterials, e.g. nanotubes, graphene or MoS2, can be efficiently heated with directed light. Heating produces considerable force through the thermoelastic effect, and this force can be used for continuous translation or to trigger reversible elastic buckling of the nanostructures. The remote stimulation of motion with light provides a possible mechanism for producing cooperative behavior between swarms of semiautonomous nanorobots.

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

PubMed Central

Taniuchi, Mami; Verweij, Jaco J.; Noor, Zannatun; Sobuz, Shihab U.; van Lieshout, Lisette; Petri, William A.; Haque, Rashidul; Houpt, Eric R.

2011-01-01

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

PubMed

Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

2015-01-01

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray.

PubMed

Yang, Liu; Wang, Zhihua; Deng, Yuliang; Li, Yan; Wei, Wei; Shi, Qihui

2016-11-15

Circulating tumor cells (CTCs) shed from tumor sites and represent the molecular characteristics of the tumor. Besides genetic and transcriptional characterization, it is important to profile a panel of proteins with single-cell precision for resolving CTCs' phenotype, organ-of-origin, and drug targets. We describe a new technology that enables profiling multiple protein markers of extraordinarily rare tumor cells at the single-cell level. This technology integrates a microchip consisting of 15000 60 pL-sized microwells and a novel beads-on-barcode antibody microarray (BOBarray). The BOBarray allows for multiplexed protein detection by assigning two independent identifiers (bead size and fluorescent color) of the beads to each protein. Four bead sizes (1.75, 3, 4.5, and 6 μm) and three colors (blue, green, and yellow) are utilized to encode up to 12 different proteins. The miniaturized BOBarray can fit an array of 60 pL-sized microwells that isolate single cells for cell lysis and the subsequent detection of protein markers. An enclosed 60 pL-sized microchamber defines a high concentration of proteins released from lysed single cells, leading to single-cell resolution of protein detection. The protein markers assayed in this study include organ-specific markers and drug targets that help to characterize the organ-of-origin and drug targets of isolated rare tumor cells from blood samples. This new approach enables handling a very small number of cells and achieves single-cell, multiplexed protein detection without loss of rare but clinically important tumor cells.

Noncompetitive Homogeneous Detection of Small Molecules Using Synthetic Nanopeptamer-Based Luminescent Oxygen Channeling.

PubMed

Lassabe, Gabriel; Kramer, Karl; Hammock, Bruce D; González-Sapienza, Gualberto; González-Techera, Andrés

2018-05-15

Our group has previously developed immunoassays for noncompetitive detection of small molecules based on the use of phage borne anti-immunocomplex peptides. Recently, we substituted the phage particles by biotinylated synthetic anti-immunocomplex peptides complexed with streptavidin and named these constructs nanopeptamers. In this work, we report the results of combining AlphaLisa, a commercial luminescent oxygen channeling bead system, with nanopeptamers for the development of a noncompetitive homogeneous assay for the detection of small molecules. The signal generation of AlphaLisa assays relies on acceptor-donor bead proximity induced by the presence of the analyte (a macromolecule) simultaneously bound by antibodies immobilized on the surface of these beads. In the developed assay, termed as nanoAlphaLisa, bead proximity is sustained by the presence of a small model molecule (atrazine, MW = 215) using an antiatrazine antibody captured on the acceptor bead and an atrazine nanopeptamer on the donor bead. Atrazine is one of the most used pesticides worldwide, and its monitoring in water has relevant human health implications. NanoAlphaLisa allowed the homogeneous detection of atrazine down to 0.3 ng/mL in undiluted water samples in 1 h, which is 10-fold below the accepted limit in drinking water. NanoAlphaLisa has the intrinsic advantages for automation and high-throughput, simple, and fast homogeneous detection of target analytes that AlphaLisa assay provides.

Novel Multiplex Oligonucleotide-Conjugated Bead Suspension Array for Rapid Identification of Enterovirus 71 Subgenogroups▿ §

PubMed Central

Wu, Y.; Tan, E. L.; Yeo, A.; Chan, K. P.; Nishimura, H.; Cardosa, M. J.; Poh, C. L.; Quak, S. H.; Chow, Vincent T.

2011-01-01

A high-throughput multiplex bead suspension array was developed for the rapid subgenogrouping of EV71 strains, based on single nucleotide polymorphisms observed within the VP1 region with a high sensitivity as low as 1 PFU. Of 33 viral isolates and 55 clinical samples, all EV71 strains were successfully detected and correctly subgenogrouped. PMID:21084510

Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool

PubMed Central

Asati, Atul; Kachurina, Olga; Kachurin, Anatoly

2012-01-01

Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders. PMID:22952605

Single-analyte to multianalyte fluorescence sensors

NASA Astrophysics Data System (ADS)

Lavigne, John J.; Metzger, Axel; Niikura, Kenichi; Cabell, Larry A.; Savoy, Steven M.; Yoo, J. S.; McDevitt, John T.; Neikirk, Dean P.; Shear, Jason B.; Anslyn, Eric V.

1999-05-01

The rational design of small molecules for the selective complexation of analytes has reached a level of sophistication such that there exists a high degree of prediction. An effective strategy for transforming these hosts into sensors involves covalently attaching a fluorophore to the receptor which displays some fluorescence modulation when analyte is bound. Competition methods, such as those used with antibodies, are also amenable to these synthetic receptors, yet there are few examples. In our laboratories, the use of common dyes in competition assays with small molecules has proven very effective. For example, an assay for citrate in beverages and an assay for the secondary messenger IP3 in cells have been developed. Another approach we have explored focuses on multi-analyte sensor arrays with attempt to mimic the mammalian sense of taste. Our system utilizes polymer resin beads with the desired sensors covalently attached. These functionalized microspheres are then immobilized into micromachined wells on a silicon chip thereby creating our taste buds. Exposure of the resin to analyte causes a change in the transmittance of the bead. This change can be fluorescent or colorimetric. Optical interrogation of the microspheres, by illuminating from one side of the wafer and collecting the signal on the other, results in an image. These data streams are collected using a CCD camera which creates red, green and blue (RGB) patterns that are distinct and reproducible for their environments. Analysis of this data can identify and quantify the analytes present.

Digital microfluidics-enabled single-molecule detection by printing and sealing single magnetic beads in femtoliter droplets.

PubMed

Witters, Daan; Knez, Karel; Ceyssens, Frederik; Puers, Robert; Lammertyn, Jeroen

2013-06-07

Digital microfluidics is introduced as a novel platform with unique advantages for performing single-molecule detection. We demonstrate how superparamagnetic beads, used for capturing single protein molecules, can be printed with unprecedentedly high loading efficiency and single bead resolution on an electrowetting-on-dielectric-based digital microfluidic chip by micropatterning the Teflon-AF surface of the device. By transporting droplets containing suspended superparamagnetic beads over a hydrophilic-in-hydrophobic micropatterned Teflon-AF surface, single beads are trapped inside the hydrophilic microwells due to their selective wettability and tailored dimensions. Digital microfluidics presents the following advantages for printing and sealing magnetic beads for single-molecule detection: (i) droplets containing suspended beads can be transported back and forth over the array of hydrophilic microwells to obtain high loading efficiencies of microwells with single beads, (ii) the use of hydrophilic-in-hydrophobic patterns permits the use of a magnet to speed up the bead transfer process to the wells, while the receding droplet meniscus removes excess beads off the chip surface and thereby shortens the bead patterning time, and (iii) reagents can be transported over the printed beads multiple times, while capillary forces and a magnet hold the printed beads in place. High loading efficiencies (98% with a CV of 0.9%) of single beads in microwells were obtained by transporting droplets of suspended beads over the array 10 times in less than 1 min, which is much higher than previously reported methods (40-60%), while the total surface area needed for performing single-molecule detection can be decreased. The performance of the device was demonstrated by fluorescent detection of the presence of the biotinylated enzyme β-galactosidase on streptavidin-coated beads with a linear dynamic range of 4 orders of magnitude ranging from 10 aM to 90 fM.

Micromagnetic Architectures for On-chip Microparticle Transport

NASA Astrophysics Data System (ADS)

Ouk, Minae; Beach, Geoffrey S. D.

2015-03-01

Superparamagnetic microbeads (SBs) are widely used to capture and manipulate biological entities in a fluid environment. Chip-based magnetic actuation provides a means to transport SBs in lab-on-a-chip devices. This is usually accomplished using the stray field from patterned magnetic microstructures, or domain walls in magnetic nanowires. Magnetic anti-dot arrays are particularly attractive due to the high-gradient stray fields from their partial domain wall structures. Here we use a self-assembly method to create magnetic anti-dot arrays in Co films, and describe the motion of SBs across the surface by a rotating field. We find a critical field-rotation frequency beyond which bead motion ceases and a critical threshold for both the in-plane and out-of-plane field components that must be exceeded for bead motion to occur. We show that these field thresholds are bead size dependent, and can thus be used to digitally separate magnetic beads in multi-bead populations. Hence these large-area structures can be used to combine long distance transport with novel functionalities.

Use of a Novel Fluidics Microbead Trap/Flow-cell Enhances Speed and Sensitivity of Bead-Based Bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozanich, Rich M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.

2007-09-15

Automated devices and methods for biological sample preparation often utilize surface functionalized microbeads (superparamagnetic or non-magnetic) to allow capture, purification and pre-concentration of trace amounts of proteins, cells, or nucleic acids (DNA/RNA) from complex samples. We have developed unique methods and hardware for trapping either magnetic or non-magnetic functionalized beads that allow samples and reagents to be efficiently perfused over a micro-column of beads. This approach yields enhanced mass transport and up to 5-fold improvements in assay sensitivity or speed, dramatically improving assay capability relative to assays conducted in more traditional “batch modes” (i.e., in tubes or microplate wells). Summarymore » results are given that highlight the analytical performance improvements obtained for automated microbead processing systems utilizing novel microbead trap/flow-cells for various applications, including: 1) simultaneous capture of multiple cytokines using an antibody-coupled polystyrene bead assay with subsequent flow cytometry detection; 2) capture of nucleic acids using oligonucleotide coupled polystyrene beads with flow cytometry detection; and 3) capture of Escherichia coli 0157:H7 (E. coli) from 50 mL sample volumes using antibody-coupled superparamagnetic microbeads with subsequent culturing to assess capture efficiency.« less

Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.

PubMed

Hartwell, Supaporn Kradtap; Somprayoon, Duangporn; Kongtawelert, Prachya; Ongchai, Siriwan; Arppornchayanon, Olarn; Ganranoo, Lucksagoon; Lapanantnoppakhun, Somchai; Grudpan, Kate

2007-09-26

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.

Highly Stable Lyophilized Homogeneous Bead-Based Immunoassays for On-Site Detection of Bio Warfare Agents from Complex Matrices.

PubMed

Mechaly, Adva; Marx, Sharon; Levy, Orly; Yitzhaki, Shmuel; Fisher, Morly

2016-06-21

This study shows the development of dry, highly stable immunoassays for the detection of bio warfare agents in complex matrices. Thermal stability was achieved by the lyophilization of the complete, homogeneous, bead-based immunoassay in a special stabilizing buffer, resulting in a ready-to-use, simple assay, which exhibited long shelf and high-temperature endurance (up to 1 week at 100 °C). The developed methodology was successfully implemented for the preservation of time-resolved fluorescence, Alexa-fluorophores, and horse radish peroxidase-based bead assays, enabling multiplexed detection. The multiplexed assay was successfully implemented for the detection of Bacillus anthracis, botulinum B, and tularemia in complex matrices.

The novel Group A Streptococcus antigen SpnA combined with bead-based immunoassay technology improves streptococcal serology for the diagnosis of acute rheumatic fever.

PubMed

Hanson-Manful, Paulina; Whitcombe, Alana L; Young, Paul G; Atatoa Carr, Polly E; Bell, Anita; Didsbury, Alicia; Mitchell, Edwin A; Dunbar, P Rod; Proft, Thomas; Moreland, Nicole J

2018-04-01

Streptococcal serology provides evidence of prior Group A Streptococcus (GAS) exposure, crucial to the diagnosis of acute rheumatic fever (ARF) and post-streptococcal glomerulonephritis. However, current tests, which measure anti-streptolysin-O and anti-DNaseB antibodies, are limited by false positives in GAS endemic settings, and incompatible methodology requiring the two tests to be run in parallel. The objective was to improve streptococcal serology by combining the novel GAS antigen, SpnA, with streptolysin-O and DNaseB in a contemporary, bead-based immunoassay. Recombinant streptolysin-O, DNAseB and SpnA were conjugated to polystyrene beads with unique fluorescence positions so antibody binding to all three antigens could be detected simultaneously by cytometric bead array. Multiplex assays were run on sera collected in three groups: ARF; ethnically matched healthy children; and healthy adults. The ability of the antigens to detect a previous GAS exposure in ARF was assessed using the 80th centile of the healthy children group as cut-off (upper limit of normal). SpnA had the highest sensitivity at 88%, compared with 75% for streptolysin-O and 56% for DNaseB. SpnA has favorable immunokinetics for streptococcal serology, and can be combined with anti-streptolysin-O and anti-DNaseB in a multiplex format to improve efficiency and accuracy. Copyright © 2017 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Molecular diagnostics using magnetic nanobeads

NASA Astrophysics Data System (ADS)

Zardán Gómez de la Torre, Teresa; Strömberg, Mattias; Göransson, Jenny; Gunnarsson, Klas; Nilsson, Mats; Svedlindh, Peter; Strømme, Maria

2010-01-01

In this paper, we investigate the volume-amplified magnetic nanobead detection assay with respect to bead size, bead concentration and bead oligonucleotide surface coverage in order to improve the understanding of the underlying microscopic mechanisms. It has been shown that: (i) the immobilization efficiency of the beads depends on the surface coverage of oligonucleotides, (ii) by using lower amounts of probe-tagged beads, detection sensitivity can be improved and (iii) using small enough beads enables both turn-off and turn-on detection. Finally, biplex detection was demonstrated.

Periodic assembly of nanoparticle arrays in disclinations of cholesteric liquid crystals.

PubMed

Li, Yunfeng; Prince, Elisabeth; Cho, Sangho; Salari, Alinaghi; Mosaddeghian Golestani, Youssef; Lavrentovich, Oleg D; Kumacheva, Eugenia

2017-02-28

An important goal of the modern soft matter science is to discover new self-assembly modalities to precisely control the placement of small particles in space. Spatial inhomogeneity of liquid crystals offers the capability to organize colloids in certain regions such as the cores of the topological defects. Here we report two self-assembly modes of nanoparticles in linear defects-disclinations in a lyotropic colloidal cholesteric liquid crystal: a continuous helicoidal thread and a periodic array of discrete beads. The beads form one-dimensional arrays with a periodicity that matches half a pitch of the cholesteric phase. The periodic assembly is governed by the anisotropic surface tension and elasticity at the interface of beads with the liquid crystal. This mode of self-assembly of nanoparticles in disclinations expands our ability to use topological defects in liquid crystals as templates for the organization of nanocolloids.

Dual-mode lensless imaging device for digital enzyme linked immunosorbent assay

NASA Astrophysics Data System (ADS)

Sasagawa, Kiyotaka; Kim, Soo Heyon; Miyazawa, Kazuya; Takehara, Hironari; Noda, Toshihiko; Tokuda, Takashi; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

2014-03-01

Digital enzyme linked immunosorbent assay (ELISA) is an ultra-sensitive technology for detecting biomarkers and viruses etc. As a conventional ELISA technique, a target molecule is bonded to an antibody with an enzyme by antigen-antibody reaction. In this technology, a femto-liter droplet chamber array is used as reaction chambers. Due to its small volume, the concentration of fluorescent product by single enzyme can be sufficient for detection by a fluorescent microscopy. In this work, we demonstrate a miniaturized lensless imaging device for digital ELISA by using a custom image sensor. The pixel array of the sensor is coated with a 20 μm-thick yellow filter to eliminate excitation light at 470 nm and covered by a fiber optic plate (FOP) to protect the sensor without resolution degradation. The droplet chamber array formed on a 50μm-thick glass plate is directly placed on the FOP. In the digital ELISA, microbeads coated with antibody are loaded into the droplet chamber array, and the ratio of the fluorescent to the non-fluorescent chambers with the microbeads are observed. In the fluorescence imaging, the spatial resolution is degraded by the spreading through the glass plate because the fluorescence is irradiated omnidirectionally. This degradation is compensated by image processing and the resolution of ~35 μm was achieved. In the bright field imaging, the projected images of the beads with collimated illumination are observed. By varying the incident angle and image composition, microbeads were successfully imaged.

Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and single cells.

PubMed

Rampini, S; Kilinc, D; Li, P; Monteil, C; Gandhi, D; Lee, G U

2015-08-21

Nonlinear magnetophoresis (NLM) is a novel approach for on-chip transport and separation of superparamagnetic (SPM) beads, based on a travelling magnetic field wave generated by the combination of a micromagnet array (MMA) and an applied rotating magnetic field. Here, we present two novel MMA designs that allow SPM beads to be focused, sorted, and separated on-chip. Converging MMAs were used to rapidly collect the SPM beads from a large region of the chip and focus them into synchronised lines. We characterise the collection efficiency of the devices and demonstrate that they can facilitate on-chip analysis of populations of SPM beads using a single-point optical detector. The diverging MMAs were used to control the transport of the beads and to separate them based on their size. The separation efficiency of these devices was determined by the orientation of the magnetisation of the micromagnets relative to the external magnetic field and the size of the beads and relative to that of micromagnets. By controlling these parameters and the rotation of the external magnetic field we demonstrated the controlled transport of SPM bead-labelled single MDA-MB-231 cells. The use of these novel MMAs promises to allow magnetically-labelled cells to be efficiently isolated and then manipulated on-chip for analysis with high-resolution chemical and physical techniques.

Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array.

PubMed

Qi, Zongtai; Ma, Yinjiao; Deng, Lili; Wu, Haiping; Zhou, Guohua; Kajiyama, Tomoharu; Kambara, Hideki

2011-06-07

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the β-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC.

Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes.

PubMed

Merbah, Mélanie; Onkar, Sayali; Grivel, Jean-Charles; Vanpouille, Christophe; Biancotto, Angélique; Bonar, Lydia; Sanders-Buell, Eric; Kijak, Gustavo; Michael, Nelson; Robb, Merlin; Kim, Jerome H; Tovanabutra, Sodsai; Chenine, Agnès-Laurence

2016-04-01

The prevailing method to assess HIV-1 replication and infectivity is to measure the production of p24 Gag protein by enzyme-linked immunosorbent assay (ELISA). Since fluorescent bead-based technologies offer a broader dynamic range and higher sensitivity, this study describes a p24 capture Luminex assay capable of detecting HIV-1 subtypes A-D, circulating recombinant forms (CRF) CRF01_AE and CRF02_AG, which together are responsible for over 90% of HIV-1 infections worldwide. The success of the assay lies in the identification and selection of a cross-reactive capture antibody (clone 183-H12-5C). Fifty-six isolates that belonged to six HIV-1 subtypes and CRFs were successfully detected with p-values below 0.021; limits of detection ranging from 3.7 to 3 × 104 pg/ml. The intra- and inter-assay variation gave coefficient of variations below 6 and 14%, respectively. The 183-bead Luminex assay also displayed higher sensitivity of 91% and 98% compared to commercial p24 ELISA and a previously described Luminex assay. The p24 concentrations measured by the 183-bead Luminex assay showed a significant correlation (R=0.92, p<0.0001) with the data obtained from quantitative real time PCR. This newly developed p24 assay leverages the advantages of the Luminex platform, which include smaller sample volume and simultaneous detection of up to 500 analytes in a single sample, and delivers a valuable tool for the field. Copyright © 2016 Elsevier B.V. All rights reserved.

Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology.

PubMed

Hoare, R; Thompson, K D; Herath, T; Collet, B; Bron, J E; Adams, A

2016-01-01

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.

Multiplex analysis of pro-inflammatory cytokines in serum of Actinobacillus pleuropneumoniae-infected pigs.

PubMed

Wyns, H; Croubels, S; Vandekerckhove, M; Demeyere, K; De Backer, P; Goddeeris, B M; Meyer, E

2015-10-01

Porcine pleuropneumonia is a severe respiratory disease caused by Actinobacillus (A.) pleuropneumoniae. The aim of the present study was to analyze serum samples of A. pleuropneumoniae-infected pigs for TNF-α, IL-1β and IL-6 using a cytometric bead array (CBA) 3-plex assay and additionally for IL-6 using ELISA. The CBA 3-plex assay was successfully validated for use in serum. The limits of detection varied between 0.012 and 0.333 ng/mL, and the inter- and inter-assay coefficients of variation were <5% and <10%, respectively. Increased levels were observed for all 3 cytokines following experimental infection with A. pleuropneumoniae. Mean peak concentrations of TNF-α and IL-6 were recorded at 12h and at 10h p.i., respectively. For IL-6, similar concentration-time profiles were observed with CBA and ELISA. It is proposed that this immuno-assay can be applied for the screening of immunomodulatory properties of drugs and vaccine adjuvants in infection, inflammation and vaccination. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branch, Darren W.; Vreeland, Erika C.; McClain, Jamie L.

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP andmore » the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.« less

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

DOE PAGES

Branch, Darren W.; Vreeland, Erika C.; McClain, Jamie L.; ...

2017-07-21

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP andmore » the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.« less

Detection of Salmonella enterica serovar Enteritidis (SE) Antibodies in Serum Using A Polystyrene Bead/SE Flagella Agglutination Assay

USDA-ARS?s Scientific Manuscript database

Serologic screening of flocks can be an important method to detect Salmonella enteritidis (SE) infections but can be labor intensive or lack specificity. Our goal was to develop a rapid agglutination assay using SE flagella adsorbed to polystyrene beads as a simple, relatively specific test to dete...

Analysis of differential gene expression by bead-based fiber-optic array in nonfunctioning pituitary adenomas.

PubMed

Jiang, Z; Gui, S; Zhang, Y

2011-05-01

Nonfunctioning pituitary adenomas (NFPAs) are relatively common, accounting for 30% of all pituitary adenomas; however, their pathogenesis remains enigmatic. To explore the possible pathogenesis of NFPAs, we used fiber-optic BeadArray to examine gene expression in 5 NFPAs compared with 3 normal pituitaries. 4 differentially expressed genes were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG). The array analysis indentified significant increases in the expression of 1,402 genes and 383 expressed sequence tags (ESTs), and decreases in 1,697 genes and 113 ESTs in the NFPAs. Bioinformatic and pathway analysis showed that the genes HIGD1B, FAM5C, PMAIP1 and the pathway cell-cycle regulation may play an important role in tumorigenesis and progression of NFPAs. Our data suggest fiber-optic BeadArray combined with pathway analysis of differential gene expression profile appears to be a valid approach for investigating the pathogenesis of tumors. © Georg Thieme Verlag KG Stuttgart · New York.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

PubMed

Berger, Sanne Schou; Lauritsen, Klara Tølbøll; Boas, Ulrik; Lind, Peter; Andresen, Lars Ole

2017-11-01

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Fabrication and optical characterization of imaging fiber-based nanoarrays.

PubMed

Tam, Jenny M; Song, Linan; Walt, David R

2005-09-15

In this paper, we present a technique for fabricating arrays containing a density at least 90 times higher than previously published. Specifically, we discuss the fabrication of two imaging fiber-based nanoarrays, one with 700nm features, another with 300nm features. With arrays containing up to 4.5x10(6) array elements/mm(2), these nanoarrays have an ultra-high packing density. A straightforward etching protocol is used to create nanowells into which beads can be deposited. These beads comprise the sensing elements of the nanoarray. Deposition of the nanobeads into the nanowells using two techniques is described. The surface characteristics of the etched arrays are examined with atomic force microscopy and scanning electron microscopy. Fluorescence microscopy was used to observe the arrays. The 300nm array features and the 500nm center-to-center distance approach the minimum feature sizes viewable using conventional light microscopy.

Incorporation of beads into oral films for buccal and oral delivery of bioactive molecules.

PubMed

Castro, Pedro M; Sousa, Flávia; Magalhães, Rui; Ruiz-Henestrosa, Victor Manuel Pizones; Pilosof, Ana M R; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Manuela E

2018-08-15

The association of alginate beads and guar-gum films in a single delivery system was idealized to promote a more effective buccal and oral delivery of bioactive molecules. A response surface method (experimental design approach) was performed to obtain optimal formulations of alginate beads to be incorporated into guar gum oral films as combined buccal and oral delivery systems for caffeine delivery. The combined formulation was further characterized regarding physicochemical properties, drug release, cell viability and buccal permeability. Beads average size, determined by dynamic light scattering (DLS), was of 3.37 ± 6.36 μm. Film thickness was set to 62 μm. Scanning electron microscopy micrographs revealed that beads were evenly distributed onto the film matrix and beads size was in accordance to data obtained from DLS analysis. Evaluation of Fourier-transform infrared spectra did not indicate the formation of new covalent bonds between the matrix of guar-gum films, alginate beads and caffeine. In vitro release assays by dialysis membrane allowed understanding that the combination of guar-gum films and alginate beads assure a slower release of caffeine when compared with the delivery profile of free caffeine from alginate beads or guar-gum films alone. MTT assay, performed on human buccal carcinoma TR146 cell line, allowed concluding that neither guar-gum film, alginate beads nor guar-gum film incorporated into alginate beads significantly compromised cell viability after 12 h of exposure. As demonstrated by in vitro permeability assay using TR146 human buccal carcinoma cell lines, combination of guar-gum films and alginate beads also promoted a slower release and, thus, lower apparent permeability (1.15E-05 ± 3.50E-06) than for caffeine solution (2.68E-05 ± 7.30E-06), guar-gum film (3.12E-05 ± 4.70E-06) or alginate beads (2.01E-05 ± 3.90E-06). The conjugation of alginate beads within an orodispersible film matrix represents an effective oral/buccal delivery system that induces a controlled release along with an enhanced intimate contact with cell layers that may promote higher in vivo bioavailability of carried drugs. Copyright © 2018. Published by Elsevier Ltd.

Improving efficiency of a small forensic DNA laboratory: validation of robotic assays and evaluation of microcapillary array device.

PubMed

Crouse, Cecelia A; Yeung, Stephanie; Greenspoon, Susan; McGuckian, Amy; Sikorsky, Julie; Ban, Jeff; Mathies, Richard

2005-08-01

To present validation studies performed for the implementation of existing and new technologies to increase the efficiency in the forensic DNA Section of the Palm Beach County Sheriff's Office (PBSO) Crime Laboratory. Using federally funded grants, internal support, and an external Process Mapping Team, the PBSO collaborated with forensic vendors, universities, and other forensic laboratories to enhance DNA testing procedures, including validation of the DNA IQ magnetic bead extraction system, robotic DNA extraction using the BioMek2000, the ABI7000 Sequence Detection System, and is currently evaluating a micro Capillary Array Electrophoresis device. The PBSO successfully validated and implemented both manual and automated Promega DNA IQ magnetic bead extractions system, which have increased DNA profile results from samples with low DNA template concentrations. The Beckman BioMek2000 DNA robotic workstation has been validated for blood, tissue, bone, hair, epithelial cells (touch evidence), and mixed stains such as semen. There has been a dramatic increase in the number of samples tested per case since implementation of the robotic extraction protocols. The validation of the ABI7000 real-time quantitative polymerase chain reaction (qPCR) technology and the single multiplex short tandem repeat (STR) PowerPlex16 BIO amplification system has provided both a time and a financial benefit. In addition, the qPCR system allows more accurate DNA concentration data and the PowerPlex 16 BIO multiplex generates DNA profiles data in half the time when compared to PowerPlex1.1 and PowerPlex2.1 STR systems. The PBSO's future efficiency requirements are being addressed through collaboration with the University of California at Berkeley and the Virginia Division of Forensic Science to validate microcapillary array electrophoresis instrumentation. Initial data demonstrated the electrophoresis of 96 samples in less than twenty minutes. The PBSO demonstrated, through the validation of more efficient extraction and quantification technology, an increase in the number of evidence samples tested using robotic/DNA IQ magnetic bead DNA extraction, a decrease in the number of negative samples amplified due to qPCR and implementation of a single multiplex amplification system. In addition, initial studies show the microcapillary array electrophoresis device (microCAE) evaluation results provide greater sensitivity and faster STR analysis output than current platforms.

Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.

PubMed

Deng, Lili; Qi, Zongtai; Zou, Binjie; Wu, Haiping; Huang, Huan; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

2012-07-03

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

PubMed Central

Baker, Harold N.; Murphy, Robin; Lopez, Erica; Garcia, Carlos

2012-01-01

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results. In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample. Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.) In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders. We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity. PMID:22806215

Magnetic bead-quantum dot assay for detection of a biomarker for traumatic brain injury

NASA Astrophysics Data System (ADS)

Kim, Chloe; Searson, Peter C.

2015-10-01

Current diagnostic methods for traumatic brain injury (TBI), which accounts for 15% of all emergency room visits, are limited to neuroimaging modalities. The challenges of accurate diagnosis and monitoring of TBI have created the need for a simple and sensitive blood test to detect brain-specific biomarkers. Here we report on an assay for detection of S100B, a putative biomarker for TBI, using antibody-conjugated magnetic beads for capture of the protein, and antibody-conjugated quantum dots for optical detection. From Western Blot, we show efficient antigen capture and concentration by the magnetic beads. Using magnetic bead capture and quantum dot detection in serum samples, we show a wide detection range and detection limit below the clinical cut-off level.Current diagnostic methods for traumatic brain injury (TBI), which accounts for 15% of all emergency room visits, are limited to neuroimaging modalities. The challenges of accurate diagnosis and monitoring of TBI have created the need for a simple and sensitive blood test to detect brain-specific biomarkers. Here we report on an assay for detection of S100B, a putative biomarker for TBI, using antibody-conjugated magnetic beads for capture of the protein, and antibody-conjugated quantum dots for optical detection. From Western Blot, we show efficient antigen capture and concentration by the magnetic beads. Using magnetic bead capture and quantum dot detection in serum samples, we show a wide detection range and detection limit below the clinical cut-off level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05608j

Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis.

PubMed

Li, Huiyan; Leulmi, Rym Feriel; Juncker, David

2011-02-07

Antibody microarrays are a powerful tool for rapid, multiplexed profiling of proteins. 3D microarray substrates have been developed to improve binding capacity, assay sensitivity, and mass transport, however, they often rely on photopolymers which are difficult to manufacture and have a small pore size that limits mass transport and demands long incubation time. Here, we present a novel 3D antibody microarray format based on the entrapment of antibody-coated microbeads within alginate droplets that were spotted onto a glass slide using an inkjet. Owing to the low concentration of alginate used, the gels were highly porous to proteins, and together with the 3D architecture helped enhance mass transport during the assays. The spotting parameters were optimized for the attachment of the alginate to the substrate. Beads with 0.2 µm, 0.5 µm and 1 µm diameter were tested and 1 µm beads were selected based on their superior retention within the hydrogel. The beads were found to be distributed within the entire volume of the gel droplet using confocal microscopy. The assay time and the concentration of beads in the gels were investigated for maximal binding signal using one-step immunoassays. As a proof of concept, six proteins including cytokines (TNFα, IL-8 and MIP/CCL4), breast cancer biomarkers (CEA and HER2) and one cancer-related protein (ENG) were profiled in multiplex using sandwich assays down to pg mL(-1) concentrations with 1 h incubation without agitation in both buffer solutions and 10% serum. These results illustrate the potential of beads-in-gel microarrays for highly sensitive and multiplexed protein analysis.

Bead-based microfluidic immunoassay for diagnosis of Johne's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wadhwa, Ashutosh; Foote, Robert; Shaw, Robert W

2012-01-01

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne s disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types ofmore » SAB, and types of magnetic beads) were optimized for a great degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested by commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads by laser-induced fluorescence. Antigen-coated magnetic beads treated with bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples obtained with the system were compared to those obtained with the flow cytometer, a high level of correlation (linear regression, r2 = 0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD. Key Words: Mycobacterium avium ssp. paratuberculosis, Johne s disease, microfluidics, lab-on-a-chip.« less

Ultrasensitive Immunosensor for Cancer Biomarker Proteins using Gold Nanoparticle Film Electrodes and Multienzyme-Particle Amplification

PubMed Central

Mani, Vigneshwaran; Chikkaveeraiah, Bhaskara V.; Patel, Vyomesh; Gutkind, J. Silvio; Rusling, James F.

2009-01-01

A densely packed gold nanoparticle platform combined with a multiple-enzyme labeled detection antibody-magnetic bead bioconjugate was used as the basis for an ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum. Sensitivity was greatly amplified by synthesizing magnetic bioconjugates particles containing 7500 horseradish peroxidase (HRP) labels along with detection antibodies (Ab2) attached to activated carboxyl groups on 1 µm diameter magnetic beads. These sensors had sensitivity of 31.5 µA mL ng−1 and detection limit (DL) of 0.5 pg mL−1 for prostate specific antigen (PSA) in 10 µL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, eight fold better than a previously reported carbon nanotube (CNT) forest immunosensor featuring multiple labels on carbon nanotubes, and near or below the normal serum levels of most cancer biomarkers. Measurements of PSA in cell lysates and human serum of cancer patients gave excellent correlations with standard ELISA assays. These easily fabricated AuNP immunosensors show excellent promise for future fabrication of bioelectronic arrays. PMID:19216571

Development of hydrogel TentaGel shell-core beads for ultrahigh throughput solution-phase screening of encoded OBOC combinatorial small molecule libraries.

PubMed

Baek, Hyoung Gee; Liu, Ruiwu; Lam, Kit S

2009-01-01

The one-bead one-compound (OBOC) combinatorial library method enables the rapid generation and screening of millions of discrete chemical compounds on beads. Most of the OBOC screening methods require the library compounds to remain tethered to the bead during screening process. Methods have also been developed to release library compounds from immobilized beads for in situ solution phase or "lawn" assays. However, this latter approach, while extremely powerful, is severely limited by the lack of suitable solid supports for such assays. Here, we report on the development of a novel hydrogel TentaGel shell-core (HTSC) bead in which hydrogel is grafted onto the polystyrene-based TentaGel (TG) bead as an outer shell (5-80 mum thick) via free radical surface-initiated polymerization. This novel shell-core bilayer resin enables the preparation of encoded OBOC combinatorial small molecule libraries, such that the library compounds reside on the highly hydrophilic outer layer and the coding tags reside in the polystyrene-based TG core. Using fluorescein as a model small molecule compound, we have demonstrated that fluorescein molecules that have been linked covalently to the hydrogel shell via a disulfide bond could readily diffuse out of the hydrogel layer into the bead surrounding after reduction with dithiothreitol. In contrast, under identical condition, the released fluorescein molecules remained bound to unmodified TG bead. We have prepared an encoded OBOC small molecule library on the novel shell-core beads and demonstrated that the beads can be readily decoded.

Trapping and Collection of Lymphocytes Using Microspot Array Chip and Magnetic Beads

NASA Astrophysics Data System (ADS)

Hashioka, Shingi; Obata, Tsutomu; Tokimitsu, Yoshiharu; Fujiki, Satoshi; Nakazato, Hiroyoshi; Muraguchi, Atsushi; Kishi, Hiroyuki; Tanino, Katsumi

2006-04-01

A microspot array chip, which has microspots of a magnetic thin film patterned on a glass substrate, was fabricated for trapping individual cells and for measuring their cellular response. The chip was easily fabricated by conventional semiconductor fabrication techniques on a mass production level as a disposable medical device. When a solution of lymphocyte-bound-magnetic beads was poured into the magnetized chip, each lymphocyte was trapped on each microspot of the magnetic thin film. The trapped cells were easily recovered from the chip using a micromanipulator. The micro-spot array chip can be utilized for arraying live cells and for measuring the response of each cell. The chip will be useful for preparing on array of different kinds of cells and for analyzing cellular response at the single cell level. The chip will be particularly useful for detecting antigen-specific B-lymphocytes and antigen-specific antibody complementary deoxyribonucleic acid (cDNA).

Localized transfection on arrays of magnetic beads coated with PCR products.

PubMed

Isalan, Mark; Santori, Maria Isabel; Gonzalez, Cayetano; Serrano, Luis

2005-02-01

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Optimization of bead milling parameters for the cell disruption of microalgae: process modeling and application to Porphyridium cruentum and Nannochloropsis oculata.

PubMed

Montalescot, V; Rinaldi, T; Touchard, R; Jubeau, S; Frappart, M; Jaouen, P; Bourseau, P; Marchal, L

2015-11-01

A study of cell disruption by bead milling for two microalgae, Nannochloropsis oculata and Porphyridium cruentum, was performed. Strains robustness was quantified by high-pressure disruption assays. The hydrodynamics in the bead mill grinding chamber was studied by Residence Time Distribution modeling. Operating parameters effects were analyzed and modeled in terms of stress intensities and stress number. RTD corresponded to a 2 CSTR in series model. First order kinetics cell disruption was modeled in consequence. Continuous bead milling was efficient for both strains disruption. SI-SN modeling was successfully adapted to microalgae. As predicted by high pressure assays, N. oculata was more resistant than P. cruentum. The critical stress intensity was twice more important for N. oculata than for P. cruentum. SI-SN modeling allows the determination of operating parameters minimizing energy consumption and gives a scalable approach to develop and optimize microalgal disruption by bead milling. Copyright © 2015 Elsevier Ltd. All rights reserved.

Toxin activity assays, devices, methods and systems therefor

DOEpatents

Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

2016-04-05

Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels.

PubMed

Zhang, He; Liu, Lian; Li, Cheuk-Wing; Fu, Huayang; Chen, Yao; Yang, Mengsu

2011-11-15

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid Detection of Enterobacter Sakazakii in milk Powder using amino modified chitosan immunomagnetic beads.

PubMed

Zhu, Yinglian; Wang, Dongfeng

2016-12-01

Chitosan immunomagnetic beads (CIBs) were first prepared through converting hydroxyl groups of natural polymer material-chitosan into amino groups using epichlorohydrin and ethylenediamine as modification agent and then coupling with polyclonal antibodies of Enterobacter sakazakii using glutaraldehyde as cross-linking agent. The beads before coupling with antibodies were characterized by magnetic property measurement, FTIR, SEM and XRD technologies. In the assay a natural polysaccharide-chitosan, which has good biological and chemical properties such as non-toxicity, biocompatibility and high chemical reactivity was first used for synthesis of immunomagnetic beads. The detection method first established in this paper that combined the beads with chromogenic medium together to rapid detect E. sakazakii in milk powder could greatly improve the detection specificity and working efficiency. The beads exhibited a maximum capturing capacity of 1×10 6 cfu/g with the detection sensitivity of 4cfu/g. The results demonstrate that the assay is a straightforward, specific and sensitive alternative for rapid detection of E.sakazakii in food matrix. The total analysis time was as little as about 25h, which greatly shorten the detection time. The method can provides new ideas not only to preparation technique of immunomagnetic beads but to imunne detection technique in food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

A Review of Current Methods for Analysis of Mycotoxins in Herbal Medicines

PubMed Central

Zhang, Lei; Dou, Xiao-Wen; Zhang, Cheng; Logrieco, Antonio F.; Yang, Mei-Hua

2018-01-01

The presence of mycotoxins in herbal medicines is an established problem throughout the entire world. The sensitive and accurate analysis of mycotoxin in complicated matrices (e.g., herbs) typically involves challenging sample pretreatment procedures and an efficient detection instrument. However, although numerous reviews have been published regarding the occurrence of mycotoxins in herbal medicines, few of them provided a detailed summary of related analytical methods for mycotoxin determination. This review focuses on analytical techniques including sampling, extraction, cleanup, and detection for mycotoxin determination in herbal medicines established within the past ten years. Dedicated sections of this article address the significant developments in sample preparation, and highlight the importance of this procedure in the analytical technology. This review also summarizes conventional chromatographic techniques for mycotoxin qualification or quantitation, as well as recent studies regarding the development and application of screening assays such as enzyme-linked immunosorbent assays, lateral flow immunoassays, aptamer-based lateral flow assays, and cytometric bead arrays. The present work provides a good insight regarding the advanced research that has been done and closes with an indication of future demand for the emerging technologies. PMID:29393905

Self-organizing magnetic beads for biomedical applications

NASA Astrophysics Data System (ADS)

Gusenbauer, Markus; Kovacs, Alexander; Reichel, Franz; Exl, Lukas; Bance, Simon; Özelt, Harald; Schrefl, Thomas

2012-03-01

In the field of biomedicine magnetic beads are used for drug delivery and to treat hyperthermia. Here we propose to use self-organized bead structures to isolate circulating tumor cells using lab-on-chip technologies. Typically blood flows past microposts functionalized with antibodies for circulating tumor cells. Creating these microposts with interacting magnetic beads makes it possible to tune the geometry in size, position and shape. We developed a simulation tool that combines micromagnetics and discrete particle dynamics, in order to design micropost arrays made of interacting beads. The simulation takes into account the viscous drag of the blood flow, magnetostatic interactions between the magnetic beads and gradient forces from external aligned magnets. We developed a particle-particle particle-mesh method for effective computation of the magnetic force and torque acting on the particles.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

Imaging optical sensor arrays.

PubMed

Walt, David R

2002-10-01

Imaging optical fibres have been etched to prepare microwell arrays. These microwells have been loaded with sensing materials such as bead-based sensors and living cells to create high-density sensor arrays. The extremely small sizes and volumes of the wells enable high sensitivity and high information content sensing capabilities.

A Multiplex Microsphere-Based Immunoassay Increases the Sensitivity of SIV-Specific Antibody Detection in Serum Samples and Mucosal Specimens Collected from Rhesus Macaques Infected with SIVmac239.

PubMed

Powell, Rebecca L R; Ouellette, Ian; Lindsay, Ross W; Parks, Christopher L; King, C Richter; McDermott, Adrian B; Morrow, Gavin

2013-06-01

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex(®) Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%-100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.

Devices, systems, and methods for conducting sandwich assays using sedimentation

DOEpatents

Schaff, Ulrich Y; Sommer, Gregory J; Singh, Anup K; Hatch, Anson V

2015-02-03

Embodiments of the present invention are directed toward devices, systems, and method for conducting sandwich assays using sedimentation. In one example, a method includes generating complexes on a plurality of beads in a fluid sample, individual ones of the complexes comprising a capture agent, a target analyte, and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

An Automated High-throughput Array Microscope for Cancer Cell Mechanics

NASA Astrophysics Data System (ADS)

Cribb, Jeremy A.; Osborne, Lukas D.; Beicker, Kellie; Psioda, Matthew; Chen, Jian; O'Brien, E. Timothy; Taylor, Russell M., II; Vicci, Leandra; Hsiao, Joe Ping-Lin; Shao, Chong; Falvo, Michael; Ibrahim, Joseph G.; Wood, Kris C.; Blobe, Gerard C.; Superfine, Richard

2016-06-01

Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.

Poisson Statistics of Combinatorial Library Sampling Predict False Discovery Rates of Screening

PubMed Central

2017-01-01

Microfluidic droplet-based screening of DNA-encoded one-bead-one-compound combinatorial libraries is a miniaturized, potentially widely distributable approach to small molecule discovery. In these screens, a microfluidic circuit distributes library beads into droplets of activity assay reagent, photochemically cleaves the compound from the bead, then incubates and sorts the droplets based on assay result for subsequent DNA sequencing-based hit compound structure elucidation. Pilot experimental studies revealed that Poisson statistics describe nearly all aspects of such screens, prompting the development of simulations to understand system behavior. Monte Carlo screening simulation data showed that increasing mean library sampling (ε), mean droplet occupancy, or library hit rate all increase the false discovery rate (FDR). Compounds identified as hits on k > 1 beads (the replicate k class) were much more likely to be authentic hits than singletons (k = 1), in agreement with previous findings. Here, we explain this observation by deriving an equation for authenticity, which reduces to the product of a library sampling bias term (exponential in k) and a sampling saturation term (exponential in ε) setting a threshold that the k-dependent bias must overcome. The equation thus quantitatively describes why each hit structure’s FDR is based on its k class, and further predicts the feasibility of intentionally populating droplets with multiple library beads, assaying the micromixtures for function, and identifying the active members by statistical deconvolution. PMID:28682059

Flow-orthogonal bead oscillation in a microfluidic chip with a magnetic anisotropic flux-guide array.

PubMed

van Pelt, Stijn; Derks, Roy; Matteucci, Marco; Hansen, Mikkel Fougt; Dietzel, Andreas

2011-04-01

A new concept for the manipulation of superparamagnetic beads inside a microfluidic chip is presented in this paper. The concept allows for bead actuation orthogonal to the flow direction inside a microchannel. Basic manipulation functionalities were studied by means of finite element simulations and results were oval-shaped steady state oscillations with bead velocities up to 500 μm/s. The width of the trajectory could be controlled by prescribing external field rotation. Successful verification experiments were performed on a prototype chip fabricated with excimer laser ablation in polycarbonate and electroforming of nickel flux-guides. Bead velocities up to 450 μm/s were measured in a 75 μm wide channel. By prescribing the currents in the external quadrupole magnet, the shape of the bead trajectory could be controlled.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads. PMID:22688388

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas.

PubMed

Jiang, Zhiquan; Gui, Songbo; Zhang, Yazhuo

2010-09-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors.

Analysis of differential gene expression by bead-based fiber-optic array in growth-hormone-secreting pituitary adenomas

PubMed Central

JIANG, ZHIQUAN; GUI, SONGBO; ZHANG, YAZHUO

2010-01-01

Growth-hormone-secreting pituitary adenomas (GHomas) account for approximately 20% of all pituitary neoplasms. However, the pathogenesis of GHomas remains to be elucidated. To explore the possible pathogenesis of GHomas, we used bead-based fiber-optic arrays to examine the gene expression in five GHomas and compared them to three healthy pituitaries. Four differentially expressed genes were chosen randomly for validation by quantitative real-time reverse transcription-polymerase chain reaction. We then performed pathway analysis on the identified differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes. Array analysis showed significant increases in the expression of 353 genes and 206 expressed sequence tags (ESTs) and decreases in 565 genes and 29 ESTs. Bioinformatic analysis showed that the genes HIGD1B, HOXB2, ANGPT2, HPGD and BTG2 may play an important role in the tumorigenesis and progression of GHomas. Pathway analysis showed that the wingless-type signaling pathway and extracellular-matrix receptor interactions may play a key role in the tumorigenesis and progression of GHomas. Our data suggested that there are numerous aberrantly expressed genes and pathways involved in the pathogenesis of GHomas. Bead-based fiber-optic arrays combined with pathway analysis of differentially expressed genes appear to be a valid method for investigating the pathogenesis of tumors. PMID:22993617

Direct observation of intermediate states in model membrane fusion.

PubMed

Keidel, Andrea; Bartsch, Tobias F; Florin, Ernst-Ludwig

2016-03-31

We introduce a novel assay for membrane fusion of solid supported membranes on silica beads and on coverslips. Fusion of the lipid bilayers is induced by bringing an optically trapped bead in contact with the coverslip surface while observing the bead's thermal motion with microsecond temporal and nanometer spatial resolution using a three-dimensional position detector. The probability of fusion is controlled by the membrane tension on the particle. We show that the progression of fusion can be monitored by changes in the three-dimensional position histograms of the bead and in its rate of diffusion. We were able to observe all fusion intermediates including transient fusion, formation of a stalk, hemifusion and the completion of a fusion pore. Fusion intermediates are characterized by axial but not lateral confinement of the motion of the bead and independently by the change of its rate of diffusion due to the additional drag from the stalk-like connection between the two membranes. The detailed information provided by this assay makes it ideally suited for studies of early events in pure lipid bilayer fusion or fusion assisted by fusogenic molecules.

Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads.

PubMed

Centi, Sonia; Tombelli, Sara; Minunni, Maria; Mascini, Marco

2007-02-15

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.

Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System.

PubMed

Akama, Kenji; Shirai, Kentaro; Suzuki, Seigo

2016-07-19

Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay.

PubMed

Schnabel, Christiane L; Babasyan, Susanna; Freer, Heather; Wagner, Bettina

2017-06-01

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses. Copyright © 2017 Elsevier B.V. All rights reserved.

Charging of multiple interacting particles by contact electrification.

PubMed

Soh, Siowling; Liu, Helena; Cademartiri, Rebecca; Yoon, Hyo Jae; Whitesides, George M

2014-09-24

Many processes involve the movement of a disordered collection of small particles (e.g., powders, grain, dust, and granular foods). These particles move chaotically, interact randomly among themselves, and gain electrical charge by contact electrification. Understanding the mechanisms of contact electrification of multiple interacting particles has been challenging, in part due to the complex movement and interactions of the particles. To examine the processes contributing to contact electrification at the level of single particles, a system was constructed in which an array of millimeter-sized polymeric beads of different materials were agitated on a dish. The dish was filled almost completely with beads, such that beads did not exchange positions. At the same time, during agitation, there was sufficient space for collisions with neighboring beads. The charge of the beads was measured individually after agitation. Results of systematic variations in the organization and composition of the interacting beads showed that three mechanisms determined the steady-state charge of the beads: (i) contact electrification (charging of beads of different materials), (ii) contact de-electrification (discharging of beads of the same charge polarity to the atmosphere), and (iii) a long-range influence across beads not in contact with one another (occurring, plausibly, by diffusion of charge from a bead with a higher charge to a bead with a lower charge of the same polarity).

Measurements of the Rotation of the Flagellar Motor by Bead Assay.

PubMed

Kasai, Taishi; Sowa, Yoshiyuki

2017-01-01

The bacterial flagellar motor is a reversible rotary nano-machine powered by the ion flux across the cytoplasmic membrane. Each motor rotates a long helical filament that extends from the cell body at several hundreds revolutions per second. The output of the motor is characterized by its generated torque and rotational speed. The torque can be calculated as the rotational frictional drag coefficient multiplied by the angular velocity. Varieties of methods, including a bead assay, have been developed to measure the flagellar rotation rate under various load conditions on the motor. In this chapter, we describe a method to monitor the motor rotation through a position of a 1 μm bead attached to a truncated flagellar filament.

Location of Biomarkers and Reagents within Agarose Beads of a Programmable Bio-nano-chip

PubMed Central

Jokerst, Jesse V.; Chou, Jie; Camp, James P.; Wong, Jorge; Lennart, Alexis; Pollard, Amanda A.; Floriano, Pierre N.; Christodoulides, Nicolaos; Simmons, Glennon W.; Zhou, Yanjie; Ali, Mehnaaz F.

2012-01-01

The slow development of cost-effective medical microdevices with strong analytical performance characteristics is due to a lack of selective and efficient analyte capture and signaling. The recently developed programmable bio-nano-chip (PBNC) is a flexible detection device with analytical behavior rivaling established macroscopic methods. The PBNC system employs ≈300 μm-diameter bead sensors composed of agarose “nanonets” that populate a microelectromechanical support structure with integrated microfluidic elements. The beads are an efficient and selective protein-capture medium suitable for the analysis of complex fluid samples. Microscopy and computational studies probe the 3D interior of the beads. The relative contributions that the capture and detection of moieties, analyte size, and bead porosity make to signal distribution and intensity are reported. Agarose pore sizes ranging from 45 to 620 nm are examined and those near 140 nm provide optimal transport characteristics for rapid (<15 min) tests. The system exhibits efficient (99.5%) detection of bead-bound analyte along with low (≈2%) nonspecific immobilization of the detection probe for carcinoembryonic antigen assay. Furthermore, the role analyte dimensions play in signal distribution is explored, and enhanced methods for assay building that consider the unique features of biomarker size are offered. PMID:21290601

The Use of AlphaScreen Technology in HTS: Current Status

PubMed Central

Eglen, Richard M; Reisine, Terry; Roby, Philippe; Rouleau, Nathalie; Illy, Chantal; Bossé, Roger; Bielefeld, Martina

2008-01-01

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal. In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions. Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems. Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular. PMID:20161822

Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays.

PubMed

D'Souza, Ashwin; Sanghavi, Paulomi; Rai, Ashim; Pathak, Divya; Mallik, Roop

2016-12-05

We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance.

Evaluation of in vitro serial antibiotic elution from meropenem-impregnated polymethylmethacrylate beads after ethylene oxide gas and autoclave sterilization.

PubMed

Báez, L A; Langston, C; Givaruangsawat, S; McLaughlin, R

2011-01-01

To determine the elution properties of meropenem and to compare the elutions of meropenem-impregnated polymethylmethacrylate (PMMA) beads without sterilization (P-M-C) to those sterilized with steam (P-M-A) and ethylene oxide gas (P-M-EO). A commercial bead mould was used to produce four groups of beads: one group without antibiotic (negative control), and three groups of meropenem-impregnated beads: P-M-C, P-M-A, and P-M-EO. The beads were placed in a phosphate buffered solution and eluent samples were collected. Concentrations of the antibiotic in eluent samples from the two sterilized groups and the control beads were determined using a microbiological assay at 1, 3, 6 and 12 hours and at 1, 2, 3, 6, 9, 12, 15, 18, 22, 26, and 30 days. The microbiological assay resulted in no zone of inhibition at all time periods for the P-M-A samples and the samples of PMMA without antimicrobial. The meropenem concentration on the eluent remained above 4 mcg/ml for 15 days in the P-M-C group and until day 18 for P-M-EO group. There was no statistical difference in AUC0-∞ (p<0.318), however significance did occur for MRT (p<0.005) when comparing P-M-C and P-M-EO with the later being higher. The meropenem incorporated in the PMMA beads eluted effectively and gradually decreased after the 24 hour peak, but remained above the concentration level of 4 mcg/ml for 15 days in the P-M-C group and until day 18 for P-M-EO group. Ethylene oxide does not adversely affect meropenem's elution from PMMA beads.

In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uchida, Aya; Kishi, Kasane; Aiyama, Yoshimi

In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. Themore » bead-derived GDNF signals were able to induce the stratified aggregate formation of GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted A{sub single} and marginal A{sub paired}–A{sub aligned} GFRα1-positive spermatogonia and was surrounded by A{sub aligned} GFRα1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFRα1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis. - Highlights: • A novel bead transplantation assay was developed to examine the in vivo effects of growth factors on spermatogonia. • A rapid aggregation of GFRα1-positive spermatogonia was induced by the transplanted GDNF-soaked beads. • Tightly-compacted A{sub single} and marginal A{sub paired}–A{sub aligned} spermatogonia were formed in each GFRα1-positive aggregate.« less

Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform.

PubMed

Sista, Ramakrishna S; Eckhardt, Allen E; Srinivasan, Vijay; Pollack, Michael G; Palanki, Srinivas; Pamula, Vamsee K

2008-12-01

A digital microfluidic platform for performing heterogeneous sandwich immunoassays based on efficient handling of magnetic beads is presented in this paper. This approach is based on manipulation of discrete droplets of samples and reagents using electrowetting without the need for channels where the droplets are free to move laterally. Droplet-based manipulation of magnetic beads therefore does not suffer from clogging of channels. Immunoassays on a digital microfluidic platform require the following basic operations: bead attraction, bead washing, bead retention, and bead resuspension. Several parameters such as magnetic field strength, pull force, position, and buffer composition were studied for effective bead operations. Dilution-based washing of magnetic beads was demonstrated by immobilizing the magnetic beads using a permanent magnet and splitting the excess supernatant using electrowetting. Almost 100% bead retention was achieved after 7776-fold dilution-based washing of the supernatant. Efficient resuspension of magnetic beads was achieved by transporting a droplet with magnetic beads across five electrodes on the platform and exploiting the flow patterns within the droplet to resuspend the beads. All the magnetic-bead droplet operations were integrated together to generate standard curves for sandwich heterogeneous immunoassays on human insulin and interleukin-6 (IL-6) with a total time to result of 7 min for each assay.

Heterogeneous Immunoassays Using Magnetic beads On a Digital Microfluidic Platform

PubMed Central

Sista, Ramakrishna S.; Eckhardt, Allen E.; Srinivasan, Vijay; Pollack, Michael G.; Palanki, Srinivas; Pamula, Vamsee K.

2009-01-01

A digital microfluidic platform for performing heterogeneous sandwich immunoassays based on efficient handling of magnetic beads is presented in this paper. This approach is based on manipulation of discrete droplets of samples and reagents using electrowetting without the need for channels where the droplets are free to move laterally. Droplet-based manipulation of magnetic beads therefore does not suffer from clogging of channels. Immunoassays on a digital microfluidic platform require the following basic operations: bead attraction, bead washing, bead retention, and bead resuspension. Several parameters such as magnetic field strength, pull force, position, and buffer composition were studied for effective bead operations. Dilution-based washing of magnetic beads was demonstrated by immobilizing the magnetic beads using a permanent magnet and splitting the excess supernatant using electrowetting. Almost 100% bead retention was achieved after 7776 fold dilution-based washing of the supernatant. Efficient resuspension of magnetic beads was achieved by transporting a droplet with magnetic beads across five electrodes on the platform and exploiting the flow patterns within the droplet to resuspend the beads. All the magnetic-bead droplet operations were integrated together to generate standard curves for sandwich heterogeneous immunoassays on Human Insulin and Interleukin-6 (IL-6) with a total time to result of seven minutes for each assay. PMID:19023486

Quantum Dot-Based Luminescent Oxygen Channeling Assay for Potential Application in Homogeneous Bioassays.

PubMed

Zhuang, Si-Hui; Guo, Xin-Xin; Wu, Ying-Song; Chen, Zhen-Hua; Chen, Yao; Ren, Zhi-Qi; Liu, Tian-Cai

2016-01-01

The unique photoproperties of quantum dots are promising for potential application in bioassays. In the present study, quantum dots were applied to a luminescent oxygen channeling assay. The reaction system developed in this study was based on interaction of biotin with streptavidin. Carboxyl-modified polystyrene microspheres doped with quantum dots were biotinylated and used as acceptors. Photosensitizer-doped carboxyl-modified polystyrene microspheres were conjugated with streptavidin and used as donors. The results indicated that the singlet oxygen that was released from the donor beads diffused into the acceptor beads. The acceptor beads were then exited via thioxene, and were subsequently fluoresced. To avoid generating false positives, a high concentration (0.01 mg/mL) of quantum dots is required for application in homogeneous immunoassays. Compared to a conventional luminescent oxygen channeling assay, this quantum dots-based technique requires less time, and would be easier to automate and miniaturize because it requires no washing to remove excess labels.

Controlled transport of latex beads through vertically aligned carbon nanofiber membranes

NASA Astrophysics Data System (ADS)

Zhang, L.; Melechko, A. V.; Merkulov, V. I.; Guillorn, M. A.; Simpson, M. L.; Lowndes, D. H.; Doktycz, M. J.

2002-07-01

Stripes of vertically aligned carbon nanofibers (VACNFs) have been used to form membranes for size selectively controlling the transport of latex beads. Fluidic structures were created in poly(dimethylsiloxane) (PDMS) and interfaced to the VACNF structures for characterization of the membrane pore size. Solutions of fluorescently labeled latex beads were introduced into the PDMS channels and characterized by fluorescence and scanning electron microscopy. Results show that the beads size selectively pass through the nanofiber barriers and the size restriction limit correlates with the interfiber spacing. The results suggest that altering VACNF array density can alter fractionation properties of the membrane. Such membranes may be useful for molecular sorting and for mimicking the properties of natural membranes.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

PubMed Central

Wiklander, Oscar P. B.; Bostancioglu, R. Beklem; Welsh, Joshua A.; Zickler, Antje M.; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W.; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O.; Mohammad, Dara K.; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z.; Jones, Jennifer C.; EL Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

PubMed

Wiklander, Oscar P B; Bostancioglu, R Beklem; Welsh, Joshua A; Zickler, Antje M; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O; Mohammad, Dara K; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z; Jones, Jennifer C; El Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Model-based variance-stabilizing transformation for Illumina microarray data.

PubMed

Lin, Simon M; Du, Pan; Huber, Wolfgang; Kibbe, Warren A

2008-02-01

Variance stabilization is a step in the preprocessing of microarray data that can greatly benefit the performance of subsequent statistical modeling and inference. Due to the often limited number of technical replicates for Affymetrix and cDNA arrays, achieving variance stabilization can be difficult. Although the Illumina microarray platform provides a larger number of technical replicates on each array (usually over 30 randomly distributed beads per probe), these replicates have not been leveraged in the current log2 data transformation process. We devised a variance-stabilizing transformation (VST) method that takes advantage of the technical replicates available on an Illumina microarray. We have compared VST with log2 and Variance-stabilizing normalization (VSN) by using the Kruglyak bead-level data (2006) and Barnes titration data (2005). The results of the Kruglyak data suggest that VST stabilizes variances of bead-replicates within an array. The results of the Barnes data show that VST can improve the detection of differentially expressed genes and reduce false-positive identifications. We conclude that although both VST and VSN are built upon the same model of measurement noise, VST stabilizes the variance better and more efficiently for the Illumina platform by leveraging the availability of a larger number of within-array replicates. The algorithms and Supplementary Data are included in the lumi package of Bioconductor, available at: www.bioconductor.org.

Establishment and evaluation of a bead-based luminex assay allowing simultaneous quantification of equine IL-12 and IFN-γ.

PubMed

Duran, Maria Carolina; Willenbrock, Saskia; Müller, Jessika-M V; Nolte, Ingo; Feige, Karsten; Murua Escobar, Hugo

2013-04-01

Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are key cytokines in immunemediated equine melanoma therapy. Currently, a method for accurate simultaneous quantification of these equine cytokines is lacking. Therefore, we sought to establish an assay that allows for accurate and simultaneous quantification of equine IL-12 (eIL-12) and IFN-γ (eIFN-γ). Several antibodies were evaluated for cross-reactivity to eIL-12 and eIFN-γ and were used to establish a bead-based Luminex assay, which was subsequently applied to quantify cytokine concentrations in biological samples. Cytokine detection ranged from 31.5-5,000 pg/ml and 15-10,000 pg/ml for eIL-12 and eIFN-γ, respectively. eIL-12 was detected in supernatants of stimulated peripheral blood mononuclear cells (PBMCs) and supernatants/cell lysates of eIL-12 expression plasmid-transfected cells. Low or undetectable cytokine concentrations were measured in negative controls. In equine serum samples, the mean measured eIL-12 concentration was 1,374 ± 8 pg/ml. The bead-based assay and ELISA for eIFN-γ used to measure eIFN-γ concentrations, showed similar concentrations. Results demonstrate, to our knowledge for the first time, that cross-reactive antibody pairs to eIL-12 and eIFN-γ and Luminex bead-based technology allow for accurate, simultaneous and multiplexed quantification of these key cytokines in biological samples.

An Electrochemical Genosensing Assay Based on Magnetic Beads and Gold Nanoparticle-Loaded Latex Microspheres for Vibrio cholerae Detection.

PubMed

Low, Kim-Fatt; Rijiravanich, Patsamon; Singh, Kirnpal Kaur Banga; Surareungchai, Werasak; Yean, Chan Yean

2015-04-01

An ultrasensitive electrochemical genosensing assay was developed for the sequence-specific detection of Vibrio cholerae DNA using magnetic beads as the biorecognition surface and gold nanoparticle-loaded latex microspheres (latex-AuNPs) as a signal-amplified hybridization tag. This biorecognition surface was prepared by immobilizing specific biotinylated capturing probes onto the streptavidin-coupled magnetic beads. Fabricating a hybridization tag capable of amplifying the electrochemical signal involved loading multiple AuNPs onto polyelectrolyte multilayer film-coated poly(styrene-co-acrylic acid) latex microspheres as carrier particles. The detection targets, single-stranded 224-bp asymmetric PCR amplicons of the V. cholerae lolB gene, were sandwich-hybridized to magnetic bead-functionalized capturing probes and fluorescein-labeled detection probes and tagged with latex-AuNPs. The subsequent electrochemical stripping analysis of chemically dissolved AuNPs loaded onto the latex microspheres allowed for the quantification of the target amplicons. The high-loading capacity of the AuNPs on the latex microspheres for sandwich-type dual-hybridization genosensing provided eminent signal amplification. The genosensing variables were optimized, and the assay specificity was demonstrated. The clinical applicability of the assay was evaluated using spiked stool specimens. The current signal responded linearly to the different V. cholerae concentrations spiked into stool specimens with a detection limit of 2 colony-forming units (CFU)/ml. The proposed latex-AuNP-based magnetogenosensing platform is promising, exhibits an effective amplification performance, and offers new opportunities for the ultrasensitive detection of other microbial pathogens.

A dynamic sandwich assay on magnetic beads for selective detection of single-nucleotide mutations at room temperature.

PubMed

Wang, Junxiu; Xiong, Guoliang; Ma, Liang; Wang, Shihui; Zhou, Xu; Wang, Lei; Xiao, Lehui; Su, Xin; Yu, Changyuan

2017-08-15

Single-nucleotide mutation (SNM) has proven to be associated with a variety of human diseases. Development of reliable methods for the detection of SNM is crucial for molecular diagnosis and personalized medicine. The sandwich assays are widely used tools for detecting nucleic acid biomarkers due to their low cost and rapid signaling. However, the poor hybridization specificity of signal probe at room temperature hampers the discrimination of mutant and wild type. Here, we demonstrate a dynamic sandwich assay on magnetic beads for SNM detection based on the transient binding between signal probe and target. By taking the advantage of mismatch sensitive thermodynamics of transient DNA binding, the dynamic sandwich assay exhibits high discrimination factor for mutant with a broad range of salt concentration at room temperature. The beads used in this assay serve as a tool for separation, and might be helpful to enhance SNM selectivity. Flexible design of signal probe and facile magnetic separation allow multiple-mode downstream analysis including colorimetric detection and isothermal amplification. With this method, BRAF mutations in the genomic DNA extracted from cancer cell lines were tested, allowing sensitive detection of SNM at very low abundances (0.1-0.5% mutant/wild type). Copyright © 2017 Elsevier B.V. All rights reserved.

Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens.

PubMed Central

Perera, V Y; Creasy, M T; Winter, A J

1983-01-01

An indirect sandwich enzyme-linked immunosorbent assay, using antibody covalently coupled to nylon beads, has been adapted for the detection of Brucella antigens. Optimum conditions were achieved by incubation of 1 ml of reaction mixture with a single bead, and by minimizing nonspecific interactions through the use of beads coated with purified bovine antibodies, preabsorption of third layer rabbit antibodies with normal bovine serum, and treatment of beads with normal goat serum before addition of the goat anti-rabbit enzyme conjugate. Beta-galactosidase was selected for use with clinical samples primarily because of low levels of endogenous enzyme in bovine leukocytes. Use of a fluorogenic substrate enhanced sensitivity 20-fold. Under these conditions, 100 fg of solubilized crude lipopolysaccharide or 8 to 10 Brucella cells was detectable in a fixed volume of 1 ml. A system was also devised for concentrating antigen which permitted ready detection of 2 pg of lipopolysaccharide in a volume of 50 ml (40 fg/ml). Attempts to detect lipopolysaccharide in the presence of concentrated serum or plasma were unsuccessful, but 10 brucellae added to a suspension of leukocytes from 100 ml of normal bovine blood were easily measured. PMID:6415094

Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2

PubMed Central

2014-01-01

Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections. PMID:25328501

Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2

NASA Astrophysics Data System (ADS)

Zhu, Min; Hu, Yonghong; Li, Guirong; Ou, Weijun; Mao, Panyong; Xin, Shaojie; Wan, Yakun

2014-09-01

Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.

Ultrasensitive Detection of Ricin Toxin in Multiple Sample Matrixes Using Single-Domain Antibodies.

PubMed

Gaylord, Shonda T; Dinh, Trinh L; Goldman, Ellen R; Anderson, George P; Ngan, Kevin C; Walt, David R

2015-07-07

Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although ricin intoxication is not transmittable from person to person, even a single ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for ricin intoxication, ultrasensitive analytical assays capable of detecting ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of ricin toxin. The Simoa was able to detect ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using immuno-polymerase chain reaction (IPCR). Using a fully automated state-of-the-art platform, the Simoa HD-1 analyzer, the assay time was reduced to 64 min.

RELIC: a novel dye-bias correction method for Illumina Methylation BeadChip.

PubMed

Xu, Zongli; Langie, Sabine A S; De Boever, Patrick; Taylor, Jack A; Niu, Liang

2017-01-03

The Illumina Infinium HumanMethylation450 BeadChip and its successor, Infinium MethylationEPIC BeadChip, have been extensively utilized in epigenome-wide association studies. Both arrays use two fluorescent dyes (Cy3-green/Cy5-red) to measure methylation level at CpG sites. However, performance difference between dyes can result in biased estimates of methylation levels. Here we describe a novel method, called REgression on Logarithm of Internal Control probes (RELIC) to correct for dye bias on whole array by utilizing the intensity values of paired internal control probes that monitor the two color channels. We evaluate the method in several datasets against other widely used dye-bias correction methods. Results on data quality improvement showed that RELIC correction statistically significantly outperforms alternative dye-bias correction methods. We incorporated the method into the R package ENmix, which is freely available from the Bioconductor website ( https://www.bioconductor.org/packages/release/bioc/html/ENmix.html ). RELIC is an efficient and robust method to correct for dye-bias in Illumina Methylation BeadChip data. It outperforms other alternative methods and conveniently implemented in R package ENmix to facilitate DNA methylation studies.

Bimodal imprint chips for peptide screening: integration of high-throughput sequencing by MS and affinity analyses by surface plasmon resonance imaging.

PubMed

Wang, Weizhi; Li, Menglin; Wei, Zewen; Wang, Zihua; Bu, Xiangli; Lai, Wenjia; Yang, Shu; Gong, He; Zheng, Hui; Wang, Yuqiao; Liu, Ying; Li, Qin; Fang, Qiaojun; Hu, Zhiyuan

2014-04-15

Peptide probes and drugs have widespread applications in disease diagnostics and therapy. The demand for peptides ligands with high affinity and high specificity toward various targets has surged in the biomedical field in recent years. The traditional peptide screening procedure involves selection, sequencing, and characterization steps, and each step is manual and tedious. Herein, we developed a bimodal imprint microarray system to embrace the whole peptide screening process. Silver-sputtered silicon chip fabricated with microwell array can trap and pattern the candidate peptide beads in a one-well-one-bead manner. Peptides on beads were photocleaved in situ. A portion of the peptide in each well was transferred to a gold-coated chip to print the peptide array for high-throughput affinity analyses by surface plasmon resonance imaging (SPRi), and the peptide left in the silver-sputtered chip was ready for in situ single bead sequencing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the bimodal imprint chip system, affinity peptides toward AHA were efficiently screened out from the 7 × 10(4) peptide library. The method provides a solution for high efficiency peptide screening.

One-step detection of pathogens and cancer biomarkers by the naked eye based on aggregation of immunomagnetic beads

NASA Astrophysics Data System (ADS)

Chen, Yiping; Xianyu, Yunlei; Sun, Jiashu; Niu, Yajing; Wang, Yu; Jiang, Xingyu

2015-12-01

This report shows that immunomagnetic beads (IMBs) can act as the optical readout for assays, in addition to serving as the carrier for purification/separation. Under the influence of an external magnet, IMBs are attracted to coat one side of a test tube. IMBs specifically bound to targets can form a narrow brown stripe, whereas free IMBs will form a diffuse, yellow coating on the side of the test tube. Target analytes can aggregate initially dispersed IMBs in a sample concentration-dependent manner, yielding a color change from yellow to brown that can be seen with the naked eye. This assay combines the convenience of a lateral flow assay, allowing a one-step assay to finish within 15 min, with the sensitivity of an enzyme-linked immonosorbent assay.This report shows that immunomagnetic beads (IMBs) can act as the optical readout for assays, in addition to serving as the carrier for purification/separation. Under the influence of an external magnet, IMBs are attracted to coat one side of a test tube. IMBs specifically bound to targets can form a narrow brown stripe, whereas free IMBs will form a diffuse, yellow coating on the side of the test tube. Target analytes can aggregate initially dispersed IMBs in a sample concentration-dependent manner, yielding a color change from yellow to brown that can be seen with the naked eye. This assay combines the convenience of a lateral flow assay, allowing a one-step assay to finish within 15 min, with the sensitivity of an enzyme-linked immonosorbent assay. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07044a

Holographic optical tweezers for object manipulations at an air-liquid surface.

PubMed

Jesacher, Alexander; Fürhapter, Severin; Maurer, Christian; Bernet, Stefan; Ritsch-Marte, Monika

2006-06-26

We investigate holographic optical tweezers manipulating micro-beads at a suspended air-liquid interface. Axial confinement of the particles in the two-dimensional interface is maintained by the interplay between surface tension and gravity. Therefore, optical trapping of the micro-beads is possible even with a long distance air objective. Efficient micro-circulation of the liquid can be induced by fast rotating beads, driven by the orbital angular momentum transfer of incident Laguerre-Gaussian (doughnut) laser modes. Our setup allows various ways of creating a tailored dynamic flow of particles and liquid within the surface. We demonstrate examples of surface manipulations like efficient vortex pumps and mixers, interactive particle flow steering by arrays of vortex pumps, the feasibility of achieving a "clocked" traffic of micro beads, and size-selective guiding of beads along optical "conveyor belts".

FRET detection of Octamer-4 on a protein nanoarray made by size-dependent self-assembly

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; You, David J.

2010-01-01

An alternative approach for fabricating a protein array at nanoscale is suggested with a capability of characterization and/or localization of multiple components on a nanoarray. Fluorescent micro- and nanobeads each conjugated with different antibodies are assembled by size-dependent self-assembly (SDSA) onto nanometer wells that were created on a polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL). Antibody-conjugated beads of different diameters are added serially and electrostatically attached to corresponding wells through electrostatic attraction between the charged beads (confirmed by zeta potential analysis) and exposed p-doped silicon substrate underneath the PMMA layer. This SDSA method is enhanced by vibrated-wire-guide manipulation of droplets on the PMMA surface containing nanometer wells. Saturation rates of antibody-conjugated beads to the nanometer patterns are up to 97% under one component and 58–70% under two components nanoarrays. High-density arrays (up to 40,000 wells) could be fabricated, which can also be multi-component. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to fluorescent-tagged secondary antibodies to Octamer-4 (Oct4), which eliminates the need for multiple steps of rinsing. The 100 nm green beads are covalently conjugated with anti-Oct4 to capture Oct4 peptides (39 kDa); where the secondary anti-Oct4 and F(ab)2 fragment of anti-gIgG tagged with phycoerythrin are then added to function as an indicator of Oct4 detection. FRET signals are detected through confocal microscopes, and further confirmed by Fluorolog3 spectrofluorometer. The success rates of detecting Oct4 are 32% and 14% of the beads in right place under one and two component nanoarrays, respectively. Ratiometric FRET is used to quantify the amount of Oct4 peptides per each bead, which is estimated about 2 molecules per bead. PMID:20652550

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-09-19

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-07-11

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

Bead mediated separation of microparticles in droplets.

PubMed

Wang, Sida; Sung, Ki-Joo; Lin, Xiaoxia Nina; Burns, Mark A

2017-01-01

Exchange of components such as particles and cells in droplets is important and highly desired in droplet microfluidic assays, and many current technologies use electrical or magnetic fields to accomplish this process. Bead-based microfluidic techniques offer an alternative approach that uses the bead's solid surface to immobilize targets like particles or biological material. In this paper, we demonstrate a bead-based technique for exchanging droplet content by separating fluorescent microparticles in a microfluidic device. The device uses posts to filter surface-functionalized beads from a droplet and re-capture the filtered beads in a new droplet. With post spacing of 7 μm, beads above 10 μm had 100% capture efficiency. We demonstrate the efficacy of this system using targeted particles that bind onto the functionalized beads and are, therefore, transferred from one solution to another in the device. Binding capacity tests performed in the bulk phase showed an average binding capacity of 5 particles to each bead. The microfluidic device successfully separated the targeted particles from the non-targeted particles with up to 98% purity and 100% yield.

Integration of Magnetic Bead-Based Cell Selection into Complex Isolations

PubMed Central

2018-01-01

Magnetic bead-based analyte capture has emerged as a ubiquitous method in cell isolation, enabling the highly specific capture of target populations through simple magnetic manipulation. To date, no “one-size fits all” magnetic bead has been widely adopted leading to an overwhelming number of commercial beads. Ultimately, the ideal bead is one that not only facilitates cell isolation but also proves compatible with the widest range of downstream applications and analytic endpoints. Despite the diverse offering of sizes, coatings, and conjugation chemistries, few studies exist to benchmark the performance characteristics of different commercially available beads; importantly, these bead characteristics ultimately determine the ability of a bead to integrate into the user’s assay. In this report, we evaluate bead-based cell isolation considerations, approaches, and results across a subset of commercially available magnetic beads (Dynabeads FlowComps, Dynabeads CELLection, GE Healthcare Sera-Mag SpeedBeads streptavidin-blocked magnetic particles, Dynabeads M-270s, Dynabeads M-280s) to compare and contrast both capture-specific traits (i.e., purity, capture efficacy, and contaminant isolations) and endpoint compatibility (i.e., protein localization, fluorescence imaging, and nucleic acid extraction). We identify specific advantages and contexts of use in which distinct bead products may facilitate experimental goals and integrate into downstream applications. PMID:29732449

Comprehensive Analysis of DNA Methylation Data with RnBeads

PubMed Central

Walter, Jörn; Lengauer, Thomas; Bock, Christoph

2014-01-01

RnBeads is a software tool for large-scale analysis and interpretation of DNA methylation data, providing a user-friendly analysis workflow that yields detailed hypertext reports (http://rnbeads.mpi-inf.mpg.de). Supported assays include whole genome bisulfite sequencing, reduced representation bisulfite sequencing, Infinium microarrays, and any other protocol that produces high-resolution DNA methylation data. Important applications of RnBeads include the analysis of epigenome-wide association studies and epigenetic biomarker discovery in cancer cohorts. PMID:25262207

On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay.

PubMed

Petrovic, Voin; Olaisen, Camilla; Sharma, Animesh; Nepal, Anala; Bugge, Steffen; Sundby, Eirik; Hoff, Bård Helge; Slupphaug, Geir; Otterlei, Marit

2017-04-15

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Superparamagnetic microbead transport induced by a magnetic field on large-area magnetic antidot arrays

NASA Astrophysics Data System (ADS)

Ouk, Minae; Beach, Geoffrey S. D.

2017-12-01

A method is presented for directed transport of superparamagnetic microbeads (SPBs) on magnetic antidot patterned substrates by applying a rotating elliptical magnetic field. We find a critical frequency for transport, beyond which the bead dynamics transitions from stepwise locomotion to local oscillation. We also find that the out-of-plane (HOOP) and in-plane (HIP) field magnitudes play crucial roles in triggering bead motion. Namely, we find threshold values in HOOP and HIP that depend on bead size, which can be used to independently and remotely address specific bead populations in a multi-bead mixture. These behaviors are explained in terms of the dynamic potential energy lansdscapes computed from micromagnetic simulations of the substrate magnetization configuration. Finally, we show that large-area magnetic patterns suitable for particle transport and sorting can be fabricated through a self-assembly lithography technique, which provides a simple, cost-effective means to integrate magnetic actuation into microfluidic systems.

Sensitive electrochemical immunoassay for 2,4,6-trinitrotoluene based on functionalized silica nanoparticle labels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jun; Liu, Guodong; Wu, Hong

2008-03-03

We present a poly(guanine)-functionalized silica nanoparticle (NP) label-based electrochemical immunoassay for sensitively detecting 2,4,6-trinitrotoluene (TNT). This immunoassay takes advantage of magnetic bead–based platform for competitive displacement immunoreactions and separation, and use electroactive nanoparticles as labels for signal amplification. For this assay, anti-TNT-coated magnetic beads interacted with TNT analog-conjugated poly(guanine)-silica NPs and formed analog-anti-TNT immunocomplexes on magnetic beads. The immunocomplexes coated magnetic beads were exposed to TNT samples, which resulted in displacing the analog conjugated poly(guanine) silica NPs into solution by TNT. In contrast, there are no guanine residues releasing into the solution in the absence of TNT. The reaction solutionmore » was then separated from the magnetic beads and transferred to the electrode surface for electrochemical measurements of guanine oxidation with Ru(bpy)32+ as mediator. The sensitivity of this TNT assay was greatly enhanced through dual signal amplifications: 1) a large amount of guanine residues on silica nanoparticles is introduced into the test solution by displacement immunoreactions and 2) a Ru(bpy)32+-induced guanine catalytic oxidation further enhances the electrochemical signal. Some experimental parameters for the nanoparticle label-based electrochemical immunoassay were studied and the performance of this assay was evaluated. The method is found to be very sensitive and the detection limit of this assay is ~ 0.1 ng mL-1 TNT. The electrochemical immunoassay based on the poly[guanine]-functionalized silica NP label offers a new approach for sensitive detection of explosives.« less

Development of a bead-based multiplexed assay for simultaneous quantification of five bovine cytokines by flow cytometry.

PubMed

Rodrigues, Valérie; Baudier, Jean Baptiste; Chantal, Isabelle

2017-09-01

Quantifying cytokines is extremely important in studies of host-pathogen interactions. Multiplex assays are commercially available but only for human and mouse cytokines. Here a method for the simultaneous quantification of five important bovine cytokines IFNγ, IL-4, IL-10, IL-12, and TNFα in cell culture supernatants, using flow cytometry was reported. Functional beads from BD Biosciences expressing specific APC intensity were used. Commercially available antibodies against bovine cytokines were covalently coupled to beads as capture antibodies. Fixed recombinant cytokines were revealed with a second monoclonal antibody coupled with biotin, then revealed with streptavidin-PE. This complex was analyzed using a standard flow cytometer. Experiments were performed to check no cross reactions had occurred. The limits of detection ranged between 0.08 and 0.4 ng/ml depending on the cytokine, and the linearity between the lower and higher limits was remarkable (R 2  > 99.8%). Finally, native cytokines from cell culture supernatants were tested. Results were compared using the standard ELISA test and showed that concentrations of native cytokine in cell culture supernatants were comparable with the two methods, with a wider dynamic range using beads and flow cytometry than with ELISA assays. Bovine IFNγ, IL-4, IL-10, IL-12, and TNFα in culture supernatants can be now simultaneously detected in a single assay, using a standard flow cytometer for both basic and high-throughput analyses. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

PubMed

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Parallel RNA extraction using magnetic beads and a droplet array.

PubMed

Shi, Xu; Chen, Chun-Hong; Gao, Weimin; Chao, Shih-Hui; Meldrum, Deirdre R

2015-02-21

Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device. Here, we present a simple, effective configuration for rapidly obtaining purified RNA from low concentration cell medium. This Total RNA Extraction Droplet Array (TREDA) utilizes an array of surface-adhering droplets to facilitate the transportation of magnetic purification beads seamlessly through individual buffer solutions without solid structures. The fabrication of TREDA chips is rapid and does not require a microfabrication facility or expertise. The process takes less than 5 minutes. When purifying mRNA from bulk marine diatom samples, its repeatability and extraction efficiency are comparable to conventional tube-based operations. We demonstrate that TREDA can extract the total mRNA of about 10 marine diatom cells, indicating that the sensitivity of TREDA approaches single-digit cell numbers.

Parallel RNA extraction using magnetic beads and a droplet array

PubMed Central

Shi, Xu; Chen, Chun-Hong; Gao, Weimin; Meldrum, Deirdre R.

2015-01-01

Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device. Here, we present a simple, effective configuration for rapidly obtaining purified RNA from low concentration cell medium. This Total RNA Extraction Droplet Array (TREDA) utilizes an array of surface-adhering droplets to facilitate the transportation of magnetic purification beads seamlessly through individual buffer solutions without solid structures. The fabrication of TREDA chips is rapid and does not require a microfabrication facility or expertise. The process takes less than 5 minutes. When purifying mRNA from bulk marine diatom samples, its repeatability and extraction efficiency are comparable to conventional tube-based operations. We demonstrate that TREDA can extract the total mRNA of about 10 marine diatom cells, indicating that the sensitivity of TREDA approaches single-digit cell numbers. PMID:25519439

Direct observation of intermediate states in model membrane fusion

PubMed Central

Keidel, Andrea; Bartsch, Tobias F.; Florin, Ernst-Ludwig

2016-01-01

We introduce a novel assay for membrane fusion of solid supported membranes on silica beads and on coverslips. Fusion of the lipid bilayers is induced by bringing an optically trapped bead in contact with the coverslip surface while observing the bead’s thermal motion with microsecond temporal and nanometer spatial resolution using a three-dimensional position detector. The probability of fusion is controlled by the membrane tension on the particle. We show that the progression of fusion can be monitored by changes in the three-dimensional position histograms of the bead and in its rate of diffusion. We were able to observe all fusion intermediates including transient fusion, formation of a stalk, hemifusion and the completion of a fusion pore. Fusion intermediates are characterized by axial but not lateral confinement of the motion of the bead and independently by the change of its rate of diffusion due to the additional drag from the stalk-like connection between the two membranes. The detailed information provided by this assay makes it ideally suited for studies of early events in pure lipid bilayer fusion or fusion assisted by fusogenic molecules. PMID:27029285

Magnetic Levitation as a Platform for Competitive Protein-Ligand Binding Assays

PubMed Central

Shapiro, Nathan D.; Soh, Siowling; Mirica, Katherine A.; Whitesides, George M.

2012-01-01

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (Kd’s within the range of ~ 10 nM to 100 µM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 minutes – 2 hours). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater than approximately 65 kDa. PMID:22686324

Magnetic levitation as a platform for competitive protein-ligand binding assays.

PubMed

Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

2012-07-17

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater than approximately 65 kDa.

Generalization of the normal-exponential model: exploration of a more accurate parametrisation for the signal distribution on Illumina BeadArrays.

PubMed

Plancade, Sandra; Rozenholc, Yves; Lund, Eiliv

2012-12-11

Illumina BeadArray technology includes non specific negative control features that allow a precise estimation of the background noise. As an alternative to the background subtraction proposed in BeadStudio which leads to an important loss of information by generating negative values, a background correction method modeling the observed intensities as the sum of the exponentially distributed signal and normally distributed noise has been developed. Nevertheless, Wang and Ye (2012) display a kernel-based estimator of the signal distribution on Illumina BeadArrays and suggest that a gamma distribution would represent a better modeling of the signal density. Hence, the normal-exponential modeling may not be appropriate for Illumina data and background corrections derived from this model may lead to wrong estimation. We propose a more flexible modeling based on a gamma distributed signal and a normal distributed background noise and develop the associated background correction, implemented in the R-package NormalGamma. Our model proves to be markedly more accurate to model Illumina BeadArrays: on the one hand, it is shown on two types of Illumina BeadChips that this model offers a more correct fit of the observed intensities. On the other hand, the comparison of the operating characteristics of several background correction procedures on spike-in and on normal-gamma simulated data shows high similarities, reinforcing the validation of the normal-gamma modeling. The performance of the background corrections based on the normal-gamma and normal-exponential models are compared on two dilution data sets, through testing procedures which represent various experimental designs. Surprisingly, we observe that the implementation of a more accurate parametrisation in the model-based background correction does not increase the sensitivity. These results may be explained by the operating characteristics of the estimators: the normal-gamma background correction offers an improvement in terms of bias, but at the cost of a loss in precision. This paper addresses the lack of fit of the usual normal-exponential model by proposing a more flexible parametrisation of the signal distribution as well as the associated background correction. This new model proves to be considerably more accurate for Illumina microarrays, but the improvement in terms of modeling does not lead to a higher sensitivity in differential analysis. Nevertheless, this realistic modeling makes way for future investigations, in particular to examine the characteristics of pre-processing strategies.

Ionic liquid as a potential solvent for preparation of collagen-alginate-hydroxyapatite beads as bone filler.

PubMed

Iqbal, Bushra; Sarfaraz, Zenab; Muhammad, Nawshad; Ahmad, Pervaiz; Iqbal, Jibran; Khan, Zia Ul Haq; Gonfa, Girma; Iqbal, Farasat; Jamal, Arshad; Rahim, Abdur

2018-07-01

In this study, collagen/alginate/hydroxyapatite beads having different proportions were prepared as bone fillers for the restoration of osteological defects. Ionic liquid was used to dissolve the collagen and subsequently the solution was mixed with sodium alginate solution. Hydroxyapatite was added in different proportions, with the rationale to enhance mechanical as well as biological properties. The prepared solutions were given characteristic bead shapes by dropwise addition into calcium chloride solution. The prepared beads were characterized using FTIR, XRD, TGA and SEM analysis. Microhardness testing was used to evaluate the mechanical properties. The prepared beads were investigated for water adsorption behavior to ascertain its ability for body fluid uptake and adjusted accordingly to the bone cavity. Drug loading and subsequently the antibacterial activity was investigated for the prepared beads. The biocompatibility was assessed using the hemolysis testing and cell proliferation assay. The prepared collagen-alginate-HA beads, having biocompatibility and good mechanical properties, have showed an option of promising biologically active bone fillers for bone regeneration.

Impact of Compounding Error on Strategies for Subtyping Pathogenic Bacteria

PubMed Central

Orfe, Lisa; Davis, Margaret A.; Lafrentz, Stacey; Kang, Min-Su

2008-01-01

Abstract Comparative-omics will identify a multitude of markers that can be used for intraspecific discrimination between strains of bacteria. It seems intuitive that with this plethora of markers we can construct higher resolution subtyping assays using discrete markers to define strain “barcodes.” Unfortunately, with each new marker added to an assay, overall assay robustness declines because errors are compounded exponentially. For example, the difference in accuracy of strain classification for an assay with 60 markers will change from 99.9% to 54.7% when average probe accuracy declines from 99.999% to 99.0%. To illustrate this effect empirically, we constructed a 19 probe bead-array for subtyping Listeria monocytogenes and showed that despite seemingly reliable individual probe accuracy (>97%), our best classification results at the strain level were <75%. A more robust strategy would use as few markers as possible to achieve strain discrimination. Consequently, we developed two variable number of tandem repeat (VNTR) assays (Vibrio parahaemolyticus and L. monocytogenes) and demonstrate that these assays along with a published assay (Salmonella enterica) produce robust results when products were machine scored. The discriminatory ability with four to seven VNTR loci was comparable to pulsed-field gel electrophoresis. Passage experiments showed some instability with ca. 5% of passaged lines showing evidence for new alleles within 30 days (V. parahaemolyticus and S. enterica). Changes were limited to a single locus and allele so conservative rules can be used to determine strain matching. Most importantly, VNTRs appear robust and portable and can clearly discriminate between strains with relatively few loci thereby limiting effects of compounding error. PMID:18713065

Leukocyte production of inflammatory mediators is inhibited by the antioxidants phloretin, silymarin, hesperetin, and resveratrol.

PubMed

Fordham, Jezrom B; Naqvi, Afsar Raza; Nares, Salvador

2014-01-01

Antioxidants possess significant therapeutic potential for the treatment of inflammatory disorders. One such disorder is periodontitis characterised by an antimicrobial immune response, inflammation, and irreversible changes to the supporting structures of the teeth. Recognition of conserved pathogen-associated molecular patterns is a crucial component of innate immunity to Gram-negative bacteria such as Escherichia coli, as well as the periodontal pathogen Aggregatibacter actinomycetemcomitans. In this study, we investigated the antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol to ascertain whether they altered the production of inflammatory mediators by innately-activated leukocytes. Peripheral blood mononuclear cells were stimulated with lipopolysaccharide purified from Aggregatibacter actinomycetemcomitans, and the production of cytokines, chemokines, and differentiation factors was assayed by enzyme-linked immunosorbent assay, cytometric bead array, and RT-PCR. Significant inhibition of these factors was achieved upon treatment with Phloretin, Silymarin, Hesperetin, and Resveratrol. These data further characterise the potent anti-inflammatory properties of antioxidants. Their ability to inhibit the production of inflammatory cytokines, chemokines, and differentiation factors by a heterogeneous population of leukocytes has clear implications for their therapeutic potential in vivo.

Leukocyte Production of Inflammatory Mediators Is Inhibited by the Antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol

PubMed Central

Fordham, Jezrom B.; Raza Naqvi, Afsar

2014-01-01

Antioxidants possess significant therapeutic potential for the treatment of inflammatory disorders. One such disorder is periodontitis characterised by an antimicrobial immune response, inflammation, and irreversible changes to the supporting structures of the teeth. Recognition of conserved pathogen-associated molecular patterns is a crucial component of innate immunity to Gram-negative bacteria such as Escherichia coli, as well as the periodontal pathogen Aggregatibacter actinomycetemcomitans. In this study, we investigated the antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol to ascertain whether they altered the production of inflammatory mediators by innately-activated leukocytes. Peripheral blood mononuclear cells were stimulated with lipopolysaccharide purified from Aggregatibacter actinomycetemcomitans, and the production of cytokines, chemokines, and differentiation factors was assayed by enzyme-linked immunosorbent assay, cytometric bead array, and RT-PCR. Significant inhibition of these factors was achieved upon treatment with Phloretin, Silymarin, Hesperetin, and Resveratrol. These data further characterise the potent anti-inflammatory properties of antioxidants. Their ability to inhibit the production of inflammatory cytokines, chemokines, and differentiation factors by a heterogeneous population of leukocytes has clear implications for their therapeutic potential in vivo. PMID:24707119

Dual-force aggregation of magnetic particles enhances label-free quantification of DNA at the sub-single cell level.

PubMed

Nelson, Daniel A; Strachan, Briony C; Sloane, Hillary S; Li, Jingyi; Landers, James P

2014-03-28

We recently reported the 'pinwheel effect' as the foundation for a DNA assay based on a DNA concentration-dependent aggregation of silica-coated magnetic beads in a rotating magnetic field (RMF). Using a rotating magnet that generated a 5 cm magnetic field that impinged on a circular array of 5mm microwells, aggregation was found to only be effective in a single well at the center of the field. As a result, when multiple samples needed to be analyzed, the single-plex (single well) analysis was tedious, time-consuming and labor-intensive, as each well needed to be exposed to the center of the RMF in a serial manner for consistent well-to-well aggregation. For more effective multiplexing (simultaneous aggregation in 12 wells), we used a circular array of microwells and incorporated 'agitation' as a second force that worked in concert with the RMF to provide effective multiplexed aggregation-based DNA quantitation. The dual-force aggregation (DFA) approach allows for effective simultaneous aggregation in multiple wells (12 demonstrated) of the multi-well microdevice, allowing for 12 samples to be interrogated for DNA content in 140 s, providing a ∼35-fold improvement in time compared to single-plex approach (80 min) and ∼4-fold improvement over conventional fluorospectrometric methods. Furthermore, the increased interaction between DNA and beads provided by DFA improved the limit of detection to 250 fg μL(-1). The correlation between the DFA results and those from a fluorospectrometer, demonstrate DFA as an inexpensive and rapid alternative to more conventional methods (fluorescent and spectrophotometric). Copyright © 2014 Elsevier B.V. All rights reserved.

Microfluidic Bead Suspension Hopper

PubMed Central

2014-01-01

Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. We developed a bead suspension hopper that exploits sedimentation to load beads into a microfluidic droplet generator. A suspension hopper continuously delivered synthesis resin beads (17 μm diameter, 112,000 over 2.67 h) functionalized with a photolabile linker and pepstatin A into picoliter-scale droplets of an HIV-1 protease activity assay to model ultraminiaturized compound screening. Likewise, trypsinogen template DNA-coated magnetic beads (2.8 μm diameter, 176,000 over 5.5 h) were loaded into droplets of an in vitro transcription/translation system to model a protein evolution experiment. The suspension hopper should effectively remove any barriers to using suspensions as sample inputs, paving the way for microfluidic automation to replace robotic library distribution. PMID:24761972

Bead mediated separation of microparticles in droplets

PubMed Central

Sung, Ki-Joo; Lin, Xiaoxia Nina; Burns, Mark A.

2017-01-01

Exchange of components such as particles and cells in droplets is important and highly desired in droplet microfluidic assays, and many current technologies use electrical or magnetic fields to accomplish this process. Bead-based microfluidic techniques offer an alternative approach that uses the bead’s solid surface to immobilize targets like particles or biological material. In this paper, we demonstrate a bead-based technique for exchanging droplet content by separating fluorescent microparticles in a microfluidic device. The device uses posts to filter surface-functionalized beads from a droplet and re-capture the filtered beads in a new droplet. With post spacing of 7 μm, beads above 10 μm had 100% capture efficiency. We demonstrate the efficacy of this system using targeted particles that bind onto the functionalized beads and are, therefore, transferred from one solution to another in the device. Binding capacity tests performed in the bulk phase showed an average binding capacity of 5 particles to each bead. The microfluidic device successfully separated the targeted particles from the non-targeted particles with up to 98% purity and 100% yield. PMID:28282412

Bead-based competitive fluorescence immunoassay for sensitive and rapid diagnosis of cyanotoxin risk in drinking water.

PubMed

Yu, Hye-Weon; Jang, Am; Kim, Lan Hee; Kim, Sung-Jo; Kim, In S

2011-09-15

Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 μg/L, with a limit of detection of 0.03 μg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.

Soluble Protein Analysis using a Compact Bench-top Flow Cytometer

NASA Technical Reports Server (NTRS)

Pappas, Dimitri; Kao, Shib-Hsin; Cyr, Johnathan

2004-01-01

Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health (blood cell count, leukocyte differential, etc.) and a wide array of biochemistry and immunology assays. research settings, the application of this technique to soluble protein analysis is also possible. Proteomic beads using fluorescent dyes for optical encoding were used to monitor six cytokines simultaneously in cell medium of cell cultures in stationary and rotating cell culture systems. The results of this work demonstrate that a compact flow cytometer, such as a system proposed for space flight, can detect a variety of soluble proteins for crew health and biotechnology experiments during long-term missions.

Analytical validation of a psychiatric pharmacogenomic test.

PubMed

Jablonski, Michael R; King, Nina; Wang, Yongbao; Winner, Joel G; Watterson, Lucas R; Gunselman, Sandra; Dechairo, Bryan M

2018-05-01

The aim of this study was to validate the analytical performance of a combinatorial pharmacogenomics test designed to aid in the appropriate medication selection for neuropsychiatric conditions. Genomic DNA was isolated from buccal swabs. Twelve genes (65 variants/alleles) associated with psychotropic medication metabolism, side effects, and mechanisms of actions were evaluated by bead array, MALDI-TOF mass spectrometry, and/or capillary electrophoresis methods (GeneSight Psychotropic, Assurex Health, Inc.). The combinatorial pharmacogenomics test has a dynamic range of 2.5-20 ng/μl of input genomic DNA, with comparable performance for all assays included in the test. Both the precision and accuracy of the test were >99.9%, with individual gene components between 99.4 and 100%. This study demonstrates that the combinatorial pharmacogenomics test is robust and reproducible, making it suitable for clinical use.

The elution of colistimethate sodium from polymethylmethacrylate and calcium phosphate cement beads.

PubMed

Waterman, Paige; Barber, Melissa; Weintrob, Amy C; VanBrakle, Regina; Howard, Robin; Kozar, Michael P; Andersen, Romney; Wortmann, Glenn

2012-06-01

Gram-negative bacilli resistance to all antibiotics, except for colistimethate sodium (CMS), is an emerging healthcare concern. Incorporating CMS into orthopedic cement to treat bone and soft-tissue infections due to these bacteria is attractive, but the data regarding the elution of CMS from cement are conflicting. The in vitro analysis of the elution of CMS from polymethylmethacrylate (PMMA) and calcium phosphate (CP) cement beads is reported. PMMA and CP beads containing CMS were incubated in phosphate-buffered saline and the eluate sampled at sequential time points. The inhibition of the growth of a strain of Acinetobacter baumannii complex by the eluate was measured by disk diffusion and microbroth dilution assays, and the presence of CMS in the eluate was measured by mass spectroscopy. Bacterial growth was inhibited by the eluate from both PMMA and CP beads. Mass spectroscopy demonstrated greater elution of CMS from CP beads than PMMA beads. The dose of CMS in PMMA beads was limited by failure of bead integrity. CMS elutes from both CP and PMMA beads in amounts sufficient to inhibit bacterial growth in vitro. The clinical implications of these findings require further study.

Confocal nanoscanning, bead picking (CONA): PickoScreen microscopes for automated and quantitative screening of one-bead one-compound libraries.

PubMed

Hintersteiner, Martin; Buehler, Christof; Uhl, Volker; Schmied, Mario; Müller, Jürgen; Kottig, Karsten; Auer, Manfred

2009-01-01

Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.

Bead-based immunoassay allows sub-picogram detection of histidine-rich protein 2 from Plasmodium falciparum and estimates reliability of malaria rapid diagnostic tests

PubMed Central

Rogier, Eric; Plucinski, Mateusz; Lucchi, Naomi; Mace, Kimberly; Chang, Michelle; Lemoine, Jean Frantz; Candrinho, Baltazar; Colborn, James; Dimbu, Rafael; Fortes, Filomeno; Udhayakumar, Venkatachalam; Barnwell, John

2017-01-01

Detection of histidine-rich protein 2 (HRP2) from the malaria parasite Plasmodium falciparum provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different P. falciparum transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate P. falciparum transmission intensity and measure the impact of interventions. PMID:28192523

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis.

PubMed

Li, Xingrui; Zhang, Dongfeng; Zhang, Huimin; Guan, Zhichao; Song, Yanling; Liu, Ruochen; Zhu, Zhi; Yang, Chaoyong

2018-02-20

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.

A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

PubMed

Heusermann, Wolf; Ludin, Beat; Pham, Nhan T; Auer, Manfred; Weidemann, Thomas; Hintersteiner, Martin

2016-05-09

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.

Quantum dots encoded Au coated polystyrene bead arranged micro-channel for multiplex arrays.

PubMed

Cao, Yuan-Cheng; Wang, Zhan; Yang, Runyu; Zou, Linling; Zhou, Zhen; Mi, Tie; Shi, Hong

2016-01-01

This paper describes a promising micro-channel multiplex immunoassay method based on the quantum dots encoded beads which requires micro-volume sample. Briefly, Au nanoparticles coated polystyrene (PS) beads were prepared and Quantum dots (QDs) were employed to encode 4 types of the PS beads by different emission wavelength QDs and various intensities. Different coding types of the beads were immobilized with different antibodies on the surface and BSA was used to block the unsatisfied sites. The antibody linked beads were then arranged in the 150 µm diameter optical capillary where the specific reactions took place before the detections. Results showed that the antibody on the Au coated surface maintains the bioactivity for the immunoreactions. Using this system, the fluorescent intensity was linear with analyte concentration in the range of 1×10(-7)-1×10(-5) mg/mL (RSD<5%, 4 repeats) and the lower detection limit reached 5×10(-8) mg/mL. It was proved to be a promising approach for the future miniaturization analytical devices. Copyright © 2015 Elsevier B.V. All rights reserved.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

PubMed

Yazdi, Soroush H; Giles, Kristen L; White, Ian M

2013-11-05

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive displacement assay is applicable to detection of a variety of biological macromolecules, including proteins and proteolytic enzymes.

A magnetic nanobead-based bioassay provides sensitive detection of single- and biplex bacterial DNA using a portable AC susceptometer

PubMed Central

Strömberg, Mattias; Zardán Gómez de la Torre, Teresa; Nilsson, Mats; Svedlindh, Peter; Strømme, Maria

2014-01-01

Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin–biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM–NDA further towards implementation in point-of-care and outpatient settings. PMID:24174315

Quantum dots and duplex-specific nuclease enabled ultrasensitive detection and serotyping of Dengue viruses in one step in a single tube.

PubMed

Shen, Wei; Gao, Zhiqiang

2015-03-15

Leveraging on the enzymatic processing of Dengue virus (DV) RNA hybridized quantum dot-capped DNA capture probes (QD-CPs), an ultrasensitive assay for the detection and serotyping of DVs is described in the report. Briefly, DV-specific DNA CPs are first capped by QDs and then conjugated to magnetic beads. In a sample solution, strands of DV RNA form heteroduplexes with the QD-CPs on the magnetic beads. The CPs together with the QDs in the heteroduplexes are subsequently cleaved off the magnetic beads by a duplex-specific nuclease (DSN), releasing the QDs to the solution, freeing the target RNA strands, and availing them for another around of hybridization with the remaining QD-CPs. After removing the magnetic beads along with unreacted (uncleaved) QD-CPs by using a permanent magnet, ultrasensitive fluorescent detection of DV is realized through the cleaved QDs. Serotyping of DV is accomplished by a judicious design of the QD-CPs. The assay combines excellent signal generation by the highly fluorescent QDs and the effortlessness of utilizing magnetic beads in the removal of the unreacted QD-CPs. The highly efficient DSN cleavage in conjunction with its excellent mismatch discrimination ability permits serotyping of DVs in one tube with excellent sensitivity and selectivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Self-Assembled Polystyrene Beads for Templated Covalent Functionalization of Graphitic Substrates Using Diazonium Chemistry.

PubMed

Van Gorp, Hans; Walke, Peter; Bragança, Ana M; Greenwood, John; Ivasenko, Oleksandr; Hirsch, Brandon E; De Feyter, Steven

2018-04-11

A network of self-assembled polystyrene beads was employed as a lithographic mask during covalent functionalization reactions on graphitic surfaces to create nanocorrals for confined molecular self-assembly studies. The beads were initially assembled into hexagonal arrays at the air-liquid interface and then transferred to the substrate surface. Subsequent electrochemical grafting reactions involving aryl diazonium molecules created covalently bound molecular units that were localized in the void space between the nanospheres. Removal of the bead template exposed hexagonally arranged circular nanocorrals separated by regions of chemisorbed molecules. Small molecule self-assembly was then investigated inside the resultant nanocorrals using scanning tunneling microscopy to highlight localized confinement effects. Overall, this work illustrates the utility of self-assembly principles to transcend length scale gaps in the development of hierarchically patterned molecular materials.

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array.

PubMed

Bist, Itti; Bhakta, Snehasis; Jiang, Di; Keyes, Tia E; Martin, Aaron; Forster, Robert J; Rusling, James F

2017-11-21

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S N 2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu 2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10 6 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD 50 and Comet assay results.

Quality control, analysis and secure sharing of Luminex® immunoassay data using the open source LabKey Server platform

PubMed Central

2013-01-01

Background Immunoassays that employ multiplexed bead arrays produce high information content per sample. Such assays are now frequently used to evaluate humoral responses in clinical trials. Integrated software is needed for the analysis, quality control, and secure sharing of the high volume of data produced by such multiplexed assays. Software that facilitates data exchange and provides flexibility to perform customized analyses (including multiple curve fits and visualizations of assay performance over time) could increase scientists’ capacity to use these immunoassays to evaluate human clinical trials. Results The HIV Vaccine Trials Network and the Statistical Center for HIV/AIDS Research and Prevention collaborated with LabKey Software to enhance the open source LabKey Server platform to facilitate workflows for multiplexed bead assays. This system now supports the management, analysis, quality control, and secure sharing of data from multiplexed immunoassays that leverage Luminex xMAP® technology. These assays may be custom or kit-based. Newly added features enable labs to: (i) import run data from spreadsheets output by Bio-Plex Manager™ software; (ii) customize data processing, curve fits, and algorithms through scripts written in common languages, such as R; (iii) select script-defined calculation options through a graphical user interface; (iv) collect custom metadata for each titration, analyte, run and batch of runs; (v) calculate dose–response curves for titrations; (vi) interpolate unknown concentrations from curves for titrated standards; (vii) flag run data for exclusion from analysis; (viii) track quality control metrics across runs using Levey-Jennings plots; and (ix) automatically flag outliers based on expected values. Existing system features allow researchers to analyze, integrate, visualize, export and securely share their data, as well as to construct custom user interfaces and workflows. Conclusions Unlike other tools tailored for Luminex immunoassays, LabKey Server allows labs to customize their Luminex analyses using scripting while still presenting users with a single, graphical interface for processing and analyzing data. The LabKey Server system also stands out among Luminex tools for enabling smooth, secure transfer of data, quality control information, and analyses between collaborators. LabKey Server and its Luminex features are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. PMID:23631706

Quality control, analysis and secure sharing of Luminex® immunoassay data using the open source LabKey Server platform.

PubMed

Eckels, Josh; Nathe, Cory; Nelson, Elizabeth K; Shoemaker, Sara G; Nostrand, Elizabeth Van; Yates, Nicole L; Ashley, Vicki C; Harris, Linda J; Bollenbeck, Mark; Fong, Youyi; Tomaras, Georgia D; Piehler, Britt

2013-04-30

Immunoassays that employ multiplexed bead arrays produce high information content per sample. Such assays are now frequently used to evaluate humoral responses in clinical trials. Integrated software is needed for the analysis, quality control, and secure sharing of the high volume of data produced by such multiplexed assays. Software that facilitates data exchange and provides flexibility to perform customized analyses (including multiple curve fits and visualizations of assay performance over time) could increase scientists' capacity to use these immunoassays to evaluate human clinical trials. The HIV Vaccine Trials Network and the Statistical Center for HIV/AIDS Research and Prevention collaborated with LabKey Software to enhance the open source LabKey Server platform to facilitate workflows for multiplexed bead assays. This system now supports the management, analysis, quality control, and secure sharing of data from multiplexed immunoassays that leverage Luminex xMAP® technology. These assays may be custom or kit-based. Newly added features enable labs to: (i) import run data from spreadsheets output by Bio-Plex Manager™ software; (ii) customize data processing, curve fits, and algorithms through scripts written in common languages, such as R; (iii) select script-defined calculation options through a graphical user interface; (iv) collect custom metadata for each titration, analyte, run and batch of runs; (v) calculate dose-response curves for titrations; (vi) interpolate unknown concentrations from curves for titrated standards; (vii) flag run data for exclusion from analysis; (viii) track quality control metrics across runs using Levey-Jennings plots; and (ix) automatically flag outliers based on expected values. Existing system features allow researchers to analyze, integrate, visualize, export and securely share their data, as well as to construct custom user interfaces and workflows. Unlike other tools tailored for Luminex immunoassays, LabKey Server allows labs to customize their Luminex analyses using scripting while still presenting users with a single, graphical interface for processing and analyzing data. The LabKey Server system also stands out among Luminex tools for enabling smooth, secure transfer of data, quality control information, and analyses between collaborators. LabKey Server and its Luminex features are freely available as open source software at http://www.labkey.com under the Apache 2.0 license.

Parallel manipulation of individual magnetic microbeads for lab-on-a-chip applications

NASA Astrophysics Data System (ADS)

Peng, Zhengchun

Many scientists and engineers are turning to lab-on-a-chip systems for faster and cheaper analysis of chemical reactions and biomolecular interactions. A common approach that facilitates the handling of reagents and biomolecules in these systems utilizes micro/nano beads as the solid carrier. Physical manipulation, such as assembly, transport, sorting, and tweezing, of beads on a chip represents an essential step for fully utilizing their potentials in a wide spectrum of bead-based analysis. Previous work demonstrated manipulation of either an ensemble of beads without individual control, or single beads but lacks the capability for parallel operation. Parallel manipulation of individual beads is required to meet the demand for high-throughput and location-specific analysis. In this work, we introduced two methods for parallel manipulation of individual magnetic microbeads, which can serve as effective lab-on-a-chip platforms and/or efficient analytic tools. The first method employs arrays of soft ferromagnetic patterns fabricated inside a microfluidic channel and subjected to an external magnetic field. We demonstrated that the system can be used to assemble individual beads (1-3 mum) from a flow of suspended beads into a regular array on the chip, hence improving the integrated electrochemical detection of biomolecules bound to the bead surface. By rotating the external field, the assembled microbeads can be remotely controlled with synchronized, high-speed circular motion around individual soft magnets on the chip. We employed this manipulation mode for efficient sample mixing in continuous microflow. Furthermore, we discovered a simple but effective way of transporting the microbeads on the chip by varying the strength of the local bias field within a revolution of the external field. In addition, selective transport of microbeads with different size was realized, providing a platform for effective on-chip sample separation and offering the potential for multiplexing capability. The second method integrates magnetic and dielectrophoretic manipulations of the same microbeads. The device combines tapered conducting wires and fingered electrodes to generate desirable magnetic and electric fields, respectively. By externally programming the magnetic attraction and dielectrophoretic repulsion forces, out-of-plane oscillation of the microbeads across the channel height was realized. This manipulation mode can facilitate the interaction between the beads with multiple layers of sample fluid inside the channel. We further demonstrated the tweezing of microbeads in liquid with high spatial resolutions, i.e., from submicrometer to nanometer range, by fine-tuning the net force from magnetic attraction and dielectrophoretic repulsion of the beads. The highresolution control of the out-of-plane motion of the microbeads led to the invention of massively parallel biomolecular tweezers. We believe the maturation of bead-based microtweezers will revolutionize the state-of-art tools currently used for single cell and single molecule studies.

Development and evaluation of spherical molecularly imprinted polymer beads.

PubMed

Kempe, Henrik; Kempe, Maria

2006-06-01

The majority of studies on molecularly imprinted polymers has until now been carried out on irregularly shaped particles prepared by grinding of polymer monoliths. The preparation procedures are time- and labor-consuming and produce particles of wide size distributions. To answer the need for fast and straightforward routes to spherical molecularly imprinted polymer beads, we have developed a method comprising the formation of droplets of pre-polymerization solution directly in mineral oil by vigorous mixing followed by transformation of the droplets into solid spherical beads by photoinduced free-radical polymerization. No detergents or stabilizers were required for the droplet formation. Factors influencing the bead synthesis have been investigated and are detailed here. The beads were evaluated in parallel with corresponding irregularly shaped particles prepared from polymer monoliths. Conditions for the synthesis of propranolol-imprinted poly(methacrylic acid-co-trimethylolpropane trimethacrylate) beads in the size range of 1-100 microm in almost quantitative yield are described. The beads were applied as the recognition element in a 96-well plate format radioligand assay of propranolol in human serum.

Characterization of Plasmodium Lactate Dehydrogenase and Histidine-Rich Protein 2 Clearance Patterns via Rapid On-Bead Detection from a Single Dried Blood Spot

PubMed Central

Markwalter, Christine F.; Gibson, Lauren E.; Mudenda, Lwiindi; Kimmel, Danielle W.; Mbambara, Saidon; Thuma, Philip E.; Wright, David W.

2018-01-01

Abstract. A rapid, on-bead enzyme-linked immunosorbent assay for Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein 2 (HRP2) was adapted for use with dried blood spot (DBS) samples. This assay detected both biomarkers from a single DBS sample with only 45 minutes of total incubation time and detection limits of 600 ± 500 pM (pLDH) and 69 ± 30 pM (HRP2), corresponding to 150 and 24 parasites/μL, respectively. This sensitive and reproducible on-bead detection method was used to quantify pLDH and HRP2 in patient DBS samples from rural Zambia collected at multiple time points after treatment. Biomarker clearance patterns relative to parasite clearance were determined; pLDH clearance followed closely with parasite clearance, whereas most patients maintained detectable levels of HRP2 for 35–52 days after treatment. Furthermore, weak-to-moderate correlations between biomarker concentration and parasite densities were found for both biomarkers. This work demonstrates the utility of the developed assay for epidemiological study and surveillance of malaria. PMID:29557342

Force measurements of a magnetic micro actuator proposed for a microvalve array

NASA Astrophysics Data System (ADS)

Chang, Pauline J.; Chang, Frank W.; Yuen, Michelle C.; Otillar, Robert; Horsley, David A.

2014-03-01

Low-cost, easily-fabricated and power-efficient microvalves are necessary for many microfluidic lab-on-a-chip applications. In this study, we present a simple, low-power, scalable, CMOS-compatible magnetic actuator for microvalve applications composed of a paramagnetic bead as the ball valve over a picoliter reaction well etched into a silicon substrate. The paramagnetic bead, composed of either pure FeSi or magnetite in a SiO2 matrix, is actuated by the local magnetic field gradient generated by a microcoil in an aqueous environment, and the reaction well is situated at the microcoil center. A permanent magnet beneath the microvalve device provides an external magnetic biasing field that magnetizes the bead, enabling bidirectional actuation and reducing the current required to actuate the bead to a level below 10 mA. The vertical and radial magnetic forces exerted on the bead by the microcoil were measured for both pure FeSi and composite beads and agree well with the predictions of 2D axisymmetric finite element method models. Vertical forces were within a range of 13-80 nN, and radial forces were 11-60 nN depending on the bead type. The threshold current required to initiate bead actuation was measured as a function of bead diameter and is found to scale inversely with volume for small beads, as expected based on the magnetic force model. To provide an estimate of the stiction force acting between the bead and the passivation layer on the substrate, repeated actuation trials were used to study the bead throw distance for substrates coated with silicon dioxide, Parylene-C, and photoresist. The stiction observed was lowest for a photoresist-coated substrate, while silicon dioxide and Parylene-C coated substrates exhibited similar levels of stiction.

Growth and morphology of thermophilic dairy starters in alginate beads.

PubMed

Lamboley, Laurence; St-Gelais, Daniel; Champagne, Claude P; Lamoureux, Maryse

2003-06-01

The aim of this research was to produce concentrated biomasses of thermophilic lactic starters using immobilized cell technology (ICT). Fermentations were carried out in milk using pH control with cells microentrapped in alginate beads. In the ICT fermentations, beads represented 17% of the weight. Some assays were carried out with free cells without pH control, in order to compare the ICT populations with those of classical starters. With Streptococcus thermophilus, overall populations in the fermentor were similar, but maximum bead population for (8.2 x 10(9) cfu/g beads) was 13 times higher than that obtained in a traditional starter (4.9 x 10(8) cfu/ml). For both Lactobacillus helveticus strains studied, immobilized-cell populations were about 3 x 10(9) cfu/g beads. Production of immobilized Lb. bulgaricus 210R strain was not possible, since no increases in viable counts occurred in beads. Therefore, production of concentrated cell suspension in alginate beads was more effective for S. thermophilus. Photomicrographs of cells in alginate beads demonstrated that, while the morphology of S. thermophilus remained unchanged during the ICT fermentation, immobilized cells of Lb. helveticus appeared wider. In addition, cells of Lb. bulgaricus were curved and elongated. These morphological changes would also impair the growth of immobilized lactobacilli.

Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.

PubMed

Mang, Samuel; Bucher, Hannes; Nickolaus, Peter

2016-01-01

The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.

Detection of Her2-overexpressing cancer cells using keyhole shaped chamber array employing a magnetic droplet-handling system.

PubMed

Okochi, Mina; Koike, Shinji; Tanaka, Masayoshi; Honda, Hiroyuki

2017-07-15

An on-chip gene expression analysis compartmentalized in droplets was developed for detection of cancer cells at a single-cell level. The chip consists of a keyhole-shaped reaction chamber with hydrophobic modification employing a magnetic bead-droplet-handling system with a gate for bead separation. Using three kinds of water-based droplets in oil, a droplet with sample cells, a lysis buffer with magnetic beads, and RT-PCR buffer, parallel magnetic manipulation and fusion of droplets were performed using a magnet-handling device containing small external magnet patterns in an array. The actuation with the magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads. After reverse transcription and amplification by thermal cycling, fluorescence was obtained for detection of overexpressing genes. For clinical detection of gastric cancer cells in peritoneal washing, the Her2-overexpressing gastric cancer cells spiked within normal cells was detected by gene expression analysis of droplets containing an average of 2.5 cells. Our developed droplet-based cancer detection system manipulated by external magnetic force without pumps or valves offers a simple and flexible set-up for transcriptional detection of cancer cells, and will be greatly advantageous for less-invasive clinical diagnosis and prognostic prediction. Copyright © 2016 Elsevier B.V. All rights reserved.

High temperature structural insulating material

DOEpatents

Chen, Wayne Y.

1987-01-06

A high temperature structural insulating material useful as a liner for cylinders of high temperature engines through the favorable combination of high service temperature (above about 800.degree. C.), low thermal conductivity (below about 0.2 W/m.degree. C.), and high compressive strength (above about 250 psi). The insulating material is produced by selecting hollow ceramic beads with a softening temperature above about 800.degree. C., a diameter within the range of 20-200 .mu.m, and a wall thickness in the range of about 2-4 .mu.m; compacting the beads and a compatible silicate binder composition under pressure and sintering conditions to provide the desired structural form with the structure having a closed-cell, compact array of bonded beads.

High temperature structural insulating material

DOEpatents

Chen, Wayne Y.

1987-01-01

A high temperature structural insulating material useful as a liner for cylinders of high temperature engines through the favorable combination of high service temperature (above about 800.degree. C.), low thermal conductivity (below about 0.2 W/m.degree. C.), and high compressive strength (above about 250 psi). The insulating material is produced by selecting hollow ceramic beads with a softening temperature above about 800.degree. C., a diameter within the range of 20-200 .mu.m, and a wall thickness in the range of about 2-4 .mu.m; compacting the beads and a compatible silicate binder composition under pressure and sintering conditions to provide the desired structural form with the structure having a closed-cell, compact array of bonded beads.

Serotype determination of Salmonella by xTAG assay.

PubMed

Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying

2017-10-01

Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin.

PubMed

Skaik, Younis; Battermann, Anja; Hiller, Oliver; Meyer, Oliver; Figueiredo, Constanca; Salama, Abdulgabar; Blasczyk, Rainer

2013-05-31

Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Application of upconversion luminescent-magnetic microbeads with weak background noise and facile separation in ochratoxin A detection

NASA Astrophysics Data System (ADS)

Liao, Zhenyu; Zhang, Ying; Su, Lin; Chang, Jin; Wang, Hanjie

2017-02-01

Ochratoxin A (OTA), the most harmful and abundant ochratoxin, is chemically stable and commonly existed in foodstuffs. In this work, upconversion luminescent-magnetic microbeads (UCLMMs) -based cytometric bead array for OTA detection with a less reagent consumption and high sensitivity has been established and optimized. In UCLMMs, upconversion nanocrystals (UCNs) for optical code present a weak background noise and no spectral cross talk between the encoding signals and target labels under two excitation conditions to improve detection sensitivity. While the superparamagnetic Fe3O4 nanoparticles (Fe3O4 NPs) aim for rapid analysis. The results show that the developed method has a sensitivity of 9.553 ppt below HPLC with a 50-μL sample and can be completed in <2 h with good accuracy and high reproducibility. Therefore, different colors of UCLMMs will become a promising assay platform for multiple mycotoxins after further improvement.

Infinium HumanMethylation450 BeadChip

Cancer.gov

The HumanMethylation450 BeadChip offers a unique combination of comprehensive, expert-selected coverage and high throughput at a low price, making it ideal for screening large sample populations such as those used in genome-wide association study cohorts. By providing quantitative methylation measurement at the single-CpG–site level for normal and FFPE samples, this assay offers powerful resolution for understanding epigenetic changes.

Comparison of three multiplex cytokine analysis systems: Luminex, SearchLight and FAST Quant.

PubMed

Lash, Gendie E; Scaife, Paula J; Innes, Barbara A; Otun, Harry A; Robson, Steven C; Searle, Roger F; Bulmer, Judith N

2006-02-20

Multiplex cytokine analysis technologies have become readily available in the last five years. Two main formats exist: multiplex sandwich ELISA and bead based assays. While these have each been compared to individual ELISAs, there has been no direct comparison between the two formats. We report here the comparison of two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex). All three kits differed from each other for different analytes and there was no clear pattern of one system giving systematically different results than another for any analyte studied. We suggest that each system has merits and several factors including range of analytes available, prospect of development of new analytes, dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use. We also suggest that results obtained from different systems cannot be combined.

Separation and dual detection of prostate cancer cells and protein biomarkers using a microchip device.

PubMed

Huang, Wanfeng; Chang, Chun-Li; Brault, Norman D; Gur, Onur; Wang, Zhe; Jalal, Shadia I; Low, Philip S; Ratliff, Timothy L; Pili, Roberto; Savran, Cagri A

2017-01-31

Current efforts for the detection of prostate cancer using only prostate specific antigen are not ideal and indicate a need to develop new assays - using multiple targets - that can more accurately stratify disease states. We previously introduced a device capable of the concurrent detection of cellular and molecular markers from a single sample fluid. Here, an improved design, which achieves affinity as well as size-based separation of captured targets using antibody-conjugated magnetic beads and a silicon chip containing micro-apertures, is presented. Upon injection of the sample, the integration of magnetic attraction with the micro-aperture chip permits larger cell-bead complexes to be isolated in an upper chamber with the smaller protein-bead complexes and remaining beads passing through the micro-apertures into the lower chamber. This enhances captured cell purity for on chip quantification, allows the separate retrieval of captured cells and proteins for downstream analysis, and enables higher bead concentrations for improved multiplexed ligand targeting. Using LNCaP cells and prostate specific membrane antigen (PSMA) to model prostate cancer, the device was able to detect 34 pM of spiked PSMA and achieve a cell capture efficiency of 93% from culture media. LNCaP cells and PSMA were then spiked into diluted healthy human blood to mimic a cancer patient. The device enabled the detection of spiked PSMA (relative to endogenous PSMA) while recovering 85-90% of LNCaP cells which illustrated the potential of new assays for the diagnosis of prostate cancer.

Cell density related H2 consumption in relation to anoxic Fe(0) corrosion and precipitation of corrosion products by Shewanella oneidensis MR-1.

PubMed

De Windt, Wim; Boon, Nico; Siciliano, Steven D; Verstraete, Willy

2003-11-01

In the absence of oxygen, a protective H2 film is formed around an Fe(0) surface, inhibiting the electron flow from this surface. Our study of anoxic corrosion of Fe(0) beads revealed that, in the presence of Shewanella oneidensis MR-1, H2 removal and precipitation of Fe mineral particles on the cell surface are determining processes for corrosion. These two biologically mediated processes were governed by cell density. H2 removal by Shewanella oneidensis was detected at cell concentrations of 1.0 x 10(6) live cells ml-1 and higher and H2 was electron donor for denitrification of NO3-. The removal of the protective H2 layer from Fe(0) beads by Shewanella oneidensis, resulted in an increase of Fe release out of the Fe(0) beads from 153 +/- 25 mg l(-1) to 196 +/- 7 mg l-1 after 20 h. When the cell concentration exceeded 1.0 x 10(8) live cells ml-1, precipitation of iron minerals on the cell surface was characteristic for the greatest percentage of MR-1 cells, whereas micrometre-scale iron precipitates not associated with culturable cell biomass significantly decreased in number. Addition of supernatant of a corrosion assay with high cell concentration induced metabolic activity in a corrosion assay with low cell concentration, resulting in increased H2 consumption and Fe release from Fe(0) beads. Homoserine lactone-like molecules were detected in the supernatant by a bio-assay, suggesting the involvement of a quorum-sensing regulatory mechanism.

An Accurate Scatter Measurement and Correction Technique for Cone Beam Breast CT Imaging Using Scanning Sampled Measurement (SSM) Technique.

PubMed

Liu, Xinming; Shaw, Chris C; Wang, Tianpeng; Chen, Lingyun; Altunbas, Mustafa C; Kappadath, S Cheenu

2006-02-28

We developed and investigated a scanning sampled measurement (SSM) technique for scatter measurement and correction in cone beam breast CT imaging. A cylindrical polypropylene phantom (water equivalent) was mounted on a rotating table in a stationary gantry experimental cone beam breast CT imaging system. A 2-D array of lead beads, with the beads set apart about ~1 cm from each other and slightly tilted vertically, was placed between the object and x-ray source. A series of projection images were acquired as the phantom is rotated 1 degree per projection view and the lead beads array shifted vertically from one projection view to the next. A series of lead bars were also placed at the phantom edge to produce better scatter estimation across the phantom edges. Image signals in the lead beads/bars shadow were used to obtain sampled scatter measurements which were then interpolated to form an estimated scatter distribution across the projection images. The image data behind the lead bead/bar shadows were restored by interpolating image data from two adjacent projection views to form beam-block free projection images. The estimated scatter distribution was then subtracted from the corresponding restored projection image to obtain the scatter removed projection images.Our preliminary experiment has demonstrated that it is feasible to implement SSM technique for scatter estimation and correction for cone beam breast CT imaging. Scatter correction was successfully performed on all projection images using scatter distribution interpolated from SSM and restored projection image data. The resultant scatter corrected projection image data resulted in elevated CT number and largely reduced the cupping effects.

Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

NASA Astrophysics Data System (ADS)

Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

2002-06-01

Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koh, Chung-Yan; Light, Yooli Kim; Piccini, Matthew Ernest

Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required formore » PCR.« less

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

PubMed Central

Hilton, Hugo G; Parham, Peter

2013-01-01

Monoclonal antibodies with specificity for HLA class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing 3-6 HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with one of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1μg/ml) PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50μg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23, and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. PMID:23510417

Antibody-Coupled Magnetic Beads Can Be Reused in Immuno-MRM Assays To Reduce Cost and Extend Antibody Supply.

PubMed

Zhao, Lei; Whiteaker, Jeffrey R; Voytovich, Uliana J; Ivey, Richard G; Paulovich, Amanda G

2015-10-02

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring mass spectrometry (immuno-MRM) enables precise quantification of peptides. Affinity-purified polyclonal antibodies are routinely used as affinity reagents in immuno-MRM assays, but they are not renewable, limiting the number of experiments that can be performed. In this technical note, we describe a workflow to regenerate anti-peptide polyclonal antibodies coupled to magnetic beads for enrichments in multiplex immuno-MRM assays. A multiplexed panel of 44 antibodies (targeting 60 peptides) is used to show that peptide analytes can be effectively stripped off of antibodies using acid washing without compromising assay performance. The performance of the multiplexed panel (determined by correlation, agreement, and precision of reused assays) is reproducible (R(2) between 0.81 and 0.99) and consistent (median CVs 8-15%) for at least 10 times of washing and reuse. Application of this workflow to immuno-MRM studies greatly reduces per sample assay cost and increases the number of samples that can be interrogated with a limited supply of polyclonal antibody reagent. This allows more characterization for promising and desirable targets prior to committing funds and efforts to conversion to a renewable monoclonal antibody.

High temperature structural insulating material

DOEpatents

Chen, W.Y.

1984-07-27

A high temperature structural insulating material useful as a liner for cylinders of high temperature engines through the favorable combination of high service temperature (above about 800/sup 0/C), low thermal conductivity (below about 0.2 W/m/sup 0/C), and high compressive strength (above about 250 psi). The insulating material is produced by selecting hollow ceramic beads with a softening temperature above about 800/sup 0/C, a diameter within the range of 20-200 ..mu..m, and a wall thickness in the range of about 2 to 4 ..mu..m; compacting the beads and a compatible silicate binder composition under pressure and sintering conditions to provide the desired structural form with the structure having a closed-cell, compact array of bonded beads.

Capturing and concentrating adenovirus using magnetic anionic nanobeads

PubMed Central

Sakudo, Akikazu; Baba, Koichi; Ikuta, Kazuyoshi

2016-01-01

We recently demonstrated how various enveloped viruses can be efficiently concentrated using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). However, the exact mechanism of interaction between the virus particles and anionic beads remains unclear. To further investigate whether these magnetic anionic beads specifically bind to the viral envelope, we examined their potential interaction with a nonenveloped virus (adenovirus). The beads were incubated with either adenovirus-infected cell culture medium or nasal aspirates from adenovirus-infected individuals and then separated from the supernatant by applying a magnetic field. After thoroughly washing the beads, adsorption of adenovirus was confirmed by a variety of techniques, including immunochromatography, polymerase chain reaction, Western blotting, and cell culture infection assays. These detection methods positively identified the hexon and penton capsid proteins of adenovirus along with the viral genome on the magnetic beads. Furthermore, various types of adenovirus including Types 5, 6, 11, 19, and 41 were captured using the magnetic bead procedure. Our bead capture method was also found to increase the sensitivity of viral detection. Adenovirus below the detectable limit for immunochromatography was efficiently concentrated using the magnetic bead procedure, allowing the virus to be successfully detected using this methodology. Moreover, these findings clearly demonstrate that a viral envelope is not required for binding to the anionic magnetic beads. Taken together, our results show that this capture procedure increases the sensitivity of detection of adenovirus and would, therefore, be a valuable tool for analyzing both clinical and experimental samples. PMID:27274228

Anionic polymer, poly(methyl vinyl ether-maleic anhydride)-coated beads-based capture of human influenza A and B virus.

PubMed

Sakudo, Akikazu; Baba, Koichi; Tsukamoto, Megumi; Sugimoto, Atsuko; Okada, Takashi; Kobayashi, Takanori; Kawashita, Norihito; Takagi, Tatsuya; Ikuta, Kazuyoshi

2009-01-15

An anionic magnetic beads-based method was developed for the capture of human influenza A and B viruses from nasal aspirates, allantoic fluid and culture medium. A polymer, poly(methyl vinyl ether-maleic anhydride) [poly(MVE-MA)], was used to endow magnetic beads with a negative charge and bioadhesive properties. After incubation with samples containing human influenza virus, the beads were separated from supernatants by applying a magnetic field. The adsorption [corrected] of the virus by the beads was confirmed by hemagglutinin assay, immunochromatography, Western blotting, egg infection, and cell infection. Successful capture was proved using 5 H1N1 influenza A viruses, 10 H3N2 influenza A viruses, and 6 influenza B viruses. Furthermore, the infectivity in chicken embryonated eggs and Madin-Darby canine kidney (MDCK) cells of the captured human influenza virus was similar to that of the total viral quantity of starting materials. Therefore, this method of capture using magnetic beads coated with poly(MVE-MA) can be broadly used for the recovery of infectious human influenza viruses.

Dysprosium sorption by polymeric composite bead: robust parametric optimization using Taguchi method.

PubMed

Yadav, Kartikey K; Dasgupta, Kinshuk; Singh, Dhruva K; Varshney, Lalit; Singh, Harvinderpal

2015-03-06

Polyethersulfone-based beads encapsulating di-2-ethylhexyl phosphoric acid have been synthesized and evaluated for the recovery of rare earth values from the aqueous media. Percentage recovery and the sorption behavior of Dy(III) have been investigated under wide range of experimental parameters using these beads. Taguchi method utilizing L-18 orthogonal array has been adopted to identify the most influential process parameters responsible for higher degree of recovery with enhanced sorption of Dy(III) from chloride medium. Analysis of variance indicated that the feed concentration of Dy(III) is the most influential factor for equilibrium sorption capacity, whereas aqueous phase acidity influences the percentage recovery most. The presence of polyvinyl alcohol and multiwalled carbon nanotube modified the internal structure of the composite beads and resulted in uniform distribution of organic extractant inside polymeric matrix. The experiment performed under optimum process conditions as predicted by Taguchi method resulted in enhanced Dy(III) recovery and sorption capacity by polymeric beads with minimum standard deviation. Copyright © 2015 Elsevier B.V. All rights reserved.

Adsorption of Nanoplastics on Algal Photosynthesis

NASA Astrophysics Data System (ADS)

Turner, James; Bhattacharya, Priyanka; Lin, Sijie; Ke, Pu Chun

2010-03-01

The rapid accumulation of disposed plastics in the environment, especially in the Pacific Ocean, has become a global concern in recent years. Photo, chemical and physical degradations constantly fragment these plastics into a wide array of macroscopic to microscopic particles. As a result, marine organisms such as algae may be exposed to plastic particles through ingestion, adsorption and other forms of uptake. Such interactions, currently little understood, could potentially impact on the health state of the entire food chain. Here we report on polystyrene-algae interaction and its impact on algal photosynthesis. We first investigated the adsorption of polystyrene beads (20 nm) on a cellulose film coated on a 96-well plate. We derived a supralinear increase of the adsorption with the beads concentration for both positively and negatively charged polystyrene beads, with a saturation observed for the negatively charged polystyrene beads of concentration above 1.6 mg/mL. Using a bicarbonate indicator we discovered decreased carbon dioxide depletion due to polystyrene-algae binding. Since polystyrene beads also mediated algae aggregation, nanoplastics may alternatively be harnessed for waste water treatment.

Gallic acid reduces cell growth by induction of apoptosis and reduction of IL-8 in HepG2 cells.

PubMed

Lima, Kelly Goulart; Krause, Gabriele Catyana; Schuster, Aline Daniele; Catarina, Anderson Velasque; Basso, Bruno Souza; De Mesquita, Fernanda Cristina; Pedrazza, Leonardo; Marczak, Elisa Simon; Martha, Bianca Andrade; Nunes, Fernanda Bordignon; Chiela, Eduardo Cremonese Filippi; Jaeger, Natália; Thomé, Marcos Paulo; Haute, Gabriela Viegas; Dias, Henrique Bregolin; Donadio, Márcio Vinícius Fagundes; De Oliveira, Jarbas Rodrigues

2016-12-01

Hepatocellular carcinoma is the most prevalent primary liver tumor and is among the top ten cancer that affect the world population. Its development is related, in most cases, to the existence of chronic liver injury, such as in cirrhosis. The knowledge about the correlation between chronic inflammation and cancer has driven new researches with anti-inflammatory agents that have potential for the development of antitumor drugs. Gallic acid is a phenolic acid found in many natural products and have shown anti-inflammatory, anti-tumor, anti-mutagenic and antioxidant actions. The purpose of this study was to investigate the effect of gallic acid on acute and chronic cell proliferation and inflammatory parameters of hepatocellular carcinoma cells (HepG2), as well as to investigate the mechanisms involved. Results showed that the gallic acid decreased the proliferation of HepG2 cells in a dose-dependent manner (Trypan blue exclusion assay), without causing necrosis (LDH assay). We observed a significant increase in the percentage of small and regular nuclei (Nuclear Morphometric Analysis assay), a significant induction of apoptosis by Annexin V-FITC and PI assay and no interference with the cell cycle using the FITC BrdU Flow Kit. We observed a significant reduction in the levels of IL-8 and increased levels of IL-10 and IL-12 (Cytometric Bead Array Human Inflammation Assay). Furthermore, gallic acid caused no cancer cells regrowth at a long term (Cumulative Population Doubling assay). According to these results, gallic acid showed a strong potential as an anti-tumor agent in hepatocellular carcinoma cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Microfluidic bead-based diodes with targeted circular microchannels for low Reynolds number applications.

PubMed

Sochol, Ryan D; Lu, Albert; Lei, Jonathan; Iwai, Kosuke; Lee, Luke P; Lin, Liwei

2014-05-07

Self-regulating fluidic components are critical to the advancement of microfluidic processors for chemical and biological applications, such as sample preparation on chip, point-of-care molecular diagnostics, and implantable drug delivery devices. Although researchers have developed a wide range of components to enable flow rectification in fluidic systems, engineering microfluidic diodes that function at the low Reynolds number (Re) flows and smaller scales of emerging micro/nanofluidic platforms has remained a considerable challenge. Recently, researchers have demonstrated microfluidic diodes that utilize high numbers of suspended microbeads as dynamic resistive elements; however, using spherical particles to block fluid flow through rectangular microchannels is inherently limited. To overcome this issue, here we present a single-layer microfluidic bead-based diode (18 μm in height) that uses a targeted circular-shaped microchannel for the docking of a single microbead (15 μm in diameter) to rectify fluid flow under low Re conditions. Three-dimensional simulations and experimental results revealed that adjusting the docking channel geometry and size to better match the suspended microbead greatly increased the diodicity (Di) performance. Arraying multiple bead-based diodes in parallel was found to adversely affect system efficacy, while arraying multiple diodes in series was observed to enhance device performance. In particular, systems consisting of four microfluidic bead-based diodes with targeted circular-shaped docking channels in series revealed average Di's ranging from 2.72 ± 0.41 to 10.21 ± 1.53 corresponding to Re varying from 0.1 to 0.6.

Development of a human-specific B. thetaiotaomicron IMS/ATP assay for measuring viable human contamination in surface waters in Baja California, Mexico

EPA Science Inventory

Immunomagnetic separation/adenosine triphosphate (IMS/ATP) assays utilize paramagnetic beads and target-specific antibodies to isolate target organisms. Following isolation, adenosine tri-phosphate (ATP) is extracted from the target population and quantified. An inversely-couple...

A novel magnet focusing plate for matrix-assisted laser desorption/ionization analysis of magnetic bead-bound analytes.

PubMed

Gode, David; Volmer, Dietrich A

2013-05-15

Magnetic beads are often used for serum profiling of peptide and protein biomarkers. In these assays, the bead-bound analytes are eluted from the beads prior to mass spectrometric analysis. This study describes a novel matrix-assisted laser desorption/ionization (MALDI) technique for direct application and focusing of magnetic beads to MALDI plates by means of dedicated micro-magnets as sample spots. Custom-made MALDI plates with magnetic focusing spots were made using small nickel-coated neodymium micro-magnets integrated into a stainless steel plate in a 16 × 24 (384) pattern. For demonstrating the proof-of-concept, commercial C-18 magnetic beads were used for the extraction of a test compound (reserpine) from aqueous solution. Experiments were conducted to study focusing abilities, the required laser energies, the influence of a matrix compound, dispensing techniques, solvent choice and the amount of magnetic beads. Dispensing the magnetic beads onto the micro-magnet sample spots resulted in immediate and strong binding to the magnetic surface. Light microscope images illustrated the homogeneous distribution of beads across the surfaces of the magnets, when the entire sample volume containing the beads was pipetted onto the surface. Subsequent MALDI analysis of the bead-bound analyte demonstrated excellent and reproducible ionization yields. The surface-assisted laser desorption/ionization (SALDI) properties of the strongly light-absorbing γ-Fe2O3-based beads resulted in similar ionization efficiencies to those obtained from experiments with an additional MALDI matrix compound. This feasibility study successfully demonstrated the magnetic focusing abilities for magnetic bead-bound analytes on a novel MALDI plate containing small micro-magnets as sample spots. One of the key advantages of this integrated approach is that no elution steps from magnetic beads were required during analyses compared with conventional bead experiments. Copyright © 2013 John Wiley & Sons, Ltd.

Strand Displacement Amplification Reaction on Quantum Dot-Encoded Silica Bead for Visual Detection of Multiplex MicroRNAs.

PubMed

Qu, Xiaojun; Jin, Haojun; Liu, Yuqian; Sun, Qingjiang

2018-03-06

The combination of microbead array, isothermal amplification, and molecular signaling enables the continuous development of next-generation molecular diagnostic techniques. Herein we reported the implementation of nicking endonuclease-assisted strand displacement amplification reaction on quantum dots-encoded microbead (Qbead), and demonstrated its feasibility for multiplexed miRNA assay in real sample. The Qbead featured with well-defined core-shell superstructure with dual-colored quantum dots loaded in silica core and shell, respectively, exhibiting remarkably high optical encoding stability. Specially designed stem-loop-structured probes were immobilized onto the Qbead for specific target recognition and amplification. In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-quadruplexes, which could be selectively signaled by a fluorescent G-quadruplex intercalator. In one-step operation, the Qbead-based isothermal amplification and signaling generated emissive "core-shell-satellite" superstructure, changing the Qbead emission-color. The target abundance-dependent emission-color changes of the Qbead allowed direct, visual detection of specific miRNA target. This visualization method achieved limit of detection at the subfemtomolar level with a linear dynamic range of 4.5 logs, and point-mutation discrimination capability for precise miRNA analyses. The array of three encoded Qbeads could simultaneously quantify three miRNA biomarkers in ∼500 human hepatoma carcinoma cells. With the advancements in ease of operation, multiplexing, and visualization capabilities, the isothermal amplification-on-Qbead assay could potentially enable the development of point-of-care diagnostics.

Autoantibody Profiles in Collagen Disease Patients with Interstitial Lung Disease (ILD): Antibodies to Major Histocompatibility Complex Class I-Related Chain A (MICA) as Markers of ILD

PubMed Central

Furukawa, Hiroshi; Oka, Shomi; Shimada, Kota; Masuo, Kiyoe; Nakajima, Fumiaki; Funano, Shunichi; Tanaka, Yuki; Komiya, Akiko; Fukui, Naoshi; Sawasaki, Tatsuya; Tadokoro, Kenji; Nose, Masato; Tsuchiya, Naoyuki; Tohma, Shigeto

2015-01-01

Interstitial lung disease (ILD) is frequently associated with collagen disease. It is then designated as collagen vascular disease-associated ILD (CVD-ILD), and influences patients’ prognosis. The prognosis of acute-onset diffuse ILD (AoDILD) occurring in patients with collagen disease is quite poor. Here, we report our investigation of auto-antibody (Ab) profiles to determine whether they may be useful in diagnosing CVD-ILD or AoDILD in collagen disease. Auto-Ab profiles were analyzed using the Lambda Array Beads Multi-Analyte System, granulocyte immunofluorescence test, Proto-Array Human Protein Microarray, AlphaScreen assay, and glutathione S-transferase capture enzyme-linked immunosorbent assay in 34 patients with rheumatoid arthritis (RA) with or without CVD-ILD and in 15 patients with collagen disease with AoDILD. The average anti-major histocompatibility complex class I-related chain A (MICA) Ab levels were higher in RA patients with CVD-ILD than in those without (P = 0.0013). The ratio of the average anti-MICA Ab level to the average anti-human leukocyte antigen class I Ab level (ie, MICA/Class I) was significantly higher in RA patients with CVD-ILD compared with those without (P = 4.47 × 10−5). To the best of our knowledge, this is the first report of auto-Ab profiles in CVD-ILD. The MICA/Class I ratio could be a better marker for diagnosing CVD-ILD than KL-6 (Krebs von den lungen-6). PMID:26327779

Guar gum succinate-sodium alginate beads as a pH-sensitive carrier for colon-specific drug delivery.

PubMed

Seeli, D Sathya; Dhivya, S; Selvamurugan, N; Prabaharan, M

2016-10-01

Guar gum succinate - sodium alginate (GGS-SA) beads cross-linked with barium ions were prepared and characterized as a pH sensitive carrier for colon-specific drug delivery. The structure of GGS-SA beads was confirmed by FT-IR spectroscopy. Scanning Electron Microscope (SEM) studies revealed that the drug loaded GGS-SA beads prepared using 2:2 (w/v) weight percent of GGS and SA had a diameter about 1.4mm and roughly spherical in shape. X-ray diffraction (XRD) studies showed that the peaks corresponding to GGS and SA at 13.5°, 17.5°, 20.2° and 13.5°, 22°, 24.1°, respectively were destroyed in GGS-SA beads which show that these beads are more amorphous in nature. Swelling studies demonstrated the pH-dependent swelling behavior of GGS-SA beads. The beads showed higher swelling degrees in pH 7.4 than that in pH 1.2 due to the existence of anionic groups in the polymer chains. The drug release study showed that the amount of model drug, ibuprofen, released from the GGS-SA beads was higher in pH 7.4 than that in pH 1.2 due to the pH-dependent swelling behavior of the beads. MTT assay revealed that GGS-SA beads at a concentration range of 0-30μg/ml had no cytotoxic effect on the cultured mouse mesenchymal stem cells (C3H10T1/2). These results suggest that GGS-SA beads can be used as effective colon-specific drug delivery system with pH-dependent drug release ability. Copyright © 2016 Elsevier B.V. All rights reserved.

Magneto-mechanical detection of nucleic acids and telomerase activity in cancer cells.

PubMed

Weizmann, Yossi; Patolsky, Fernando; Lioubashevski, Oleg; Willner, Itamar

2004-02-04

The ultra-sensitive magneto-mechanical detection of DNA, single-base-mismatches in nucleic acids, and the assay of telomerase activity are accomplished by monitoring the magnetically induced deflection of a cantilever functionalized with magnetic beads associated with the biosensing interface. The analyzed M13phi DNA hybridized with the nucleic acid-functionalized magnetic beads is replicated in the presence of dNTPs that include biotin-labeled dUTP. The resulting beads are attached to an avidin-coated cantilever, and the modified cantilever is deflected by an external magnetic field. Similarly, telomerization of nucleic acid-modified magnetic beads in the presence of dNTPs, biotin-labeled dUTP, and telomerase from cancer cell extracts and the subsequent association of the magnetic beads to the cantilever surface results in the lever deflection by an external magnetic field. M13phi DNA is sensed with a sensitivity limit of 7.1 x 10(-20) M by the magneto-mechanical detection method.

Peptide ligands targeting integrin alpha3beta1 in non-small cell lung cancer.

PubMed

Lau, Derick; Guo, Linlang; Liu, Ruiwu; Marik, Jan; Lam, Kit

2006-06-01

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.

Enhanced identification and biological validation of differential gene expression via Illumina whole-genome expression arrays through the use of the model-based background correction methodology

PubMed Central

Ding, Liang-Hao; Xie, Yang; Park, Seongmi; Xiao, Guanghua; Story, Michael D.

2008-01-01

Despite the tremendous growth of microarray usage in scientific studies, there is a lack of standards for background correction methodologies, especially in single-color microarray platforms. Traditional background subtraction methods often generate negative signals and thus cause large amounts of data loss. Hence, some researchers prefer to avoid background corrections, which typically result in the underestimation of differential expression. Here, by utilizing nonspecific negative control features integrated into Illumina whole genome expression arrays, we have developed a method of model-based background correction for BeadArrays (MBCB). We compared the MBCB with a method adapted from the Affymetrix robust multi-array analysis algorithm and with no background subtraction, using a mouse acute myeloid leukemia (AML) dataset. We demonstrated that differential expression ratios obtained by using the MBCB had the best correlation with quantitative RT–PCR. MBCB also achieved better sensitivity in detecting differentially expressed genes with biological significance. For example, we demonstrated that the differential regulation of Tnfr2, Ikk and NF-kappaB, the death receptor pathway, in the AML samples, could only be detected by using data after MBCB implementation. We conclude that MBCB is a robust background correction method that will lead to more precise determination of gene expression and better biological interpretation of Illumina BeadArray data. PMID:18450815

A powerful tool for genome analysis in maize: development and evaluation of the high density 600 k SNP genotyping array.

PubMed

Unterseer, Sandra; Bauer, Eva; Haberer, Georg; Seidel, Michael; Knaak, Carsten; Ouzunova, Milena; Meitinger, Thomas; Strom, Tim M; Fries, Ruedi; Pausch, Hubert; Bertani, Christofer; Davassi, Alessandro; Mayer, Klaus Fx; Schön, Chris-Carolin

2014-09-29

High density genotyping data are indispensable for genomic analyses of complex traits in animal and crop species. Maize is one of the most important crop plants worldwide, however a high density SNP genotyping array for analysis of its large and highly dynamic genome was not available so far. We developed a high density maize SNP array composed of 616,201 variants (SNPs and small indels). Initially, 57 M variants were discovered by sequencing 30 representative temperate maize lines and then stringently filtered for sequence quality scores and predicted conversion performance on the array resulting in the selection of 1.2 M polymorphic variants assayed on two screening arrays. To identify high-confidence variants, 285 DNA samples from a broad genetic diversity panel of worldwide maize lines including the samples used for sequencing, important founder lines for European maize breeding, hybrids, and proprietary samples with European, US, semi-tropical, and tropical origin were used for experimental validation. We selected 616 k variants according to their performance during validation, support of genotype calls through sequencing data, and physical distribution for further analysis and for the design of the commercially available Affymetrix® Axiom® Maize Genotyping Array. This array is composed of 609,442 SNPs and 6,759 indels. Among these are 116,224 variants in coding regions and 45,655 SNPs of the Illumina® MaizeSNP50 BeadChip for study comparison. In a subset of 45,974 variants, apart from the target SNP additional off-target variants are detected, which show only a minor bias towards intermediate allele frequencies. We performed principal coordinate and admixture analyses to determine the ability of the array to detect and resolve population structure and investigated the extent of LD within a worldwide validation panel. The high density Affymetrix® Axiom® Maize Genotyping Array is optimized for European and American temperate maize and was developed based on a diverse sample panel by applying stringent quality filter criteria to ensure its suitability for a broad range of applications. With 600 k variants it is the largest currently publically available genotyping array in crop species.

Development and potential applications of microarrays based on fluorescent nanocrystal-encoded beads for multiplexed cancer diagnostics

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Grinevich, Regina; Efimov, Anton E.; Nabiev, Igor; Sukhanova, Alyona

2014-05-01

Advanced multiplexed assays have recently become an indispensable tool for clinical diagnostics. These techniques provide simultaneous quantitative determination of multiple biomolecules in a single sample quickly and accurately. The development of multiplex suspension arrays is currently of particular interest for clinical applications. Optical encoding of microparticles is the most available and easy-to-use technique. This technology uses fluorophores incorporated into microbeads to obtain individual optical codes. Fluorophore-encoded beads can be rapidly analyzed using classical flow cytometry or microfluidic techniques. We have developed a new generation of highly sensitive and specific diagnostic systems for detection of cancer antigens in human serum samples based on microbeads encoded with fluorescent quantum dots (QDs). The designed suspension microarray system was validated for quantitative detection of (1) free and total prostate specific antigen (PSA) in the serum of patients with prostate cancer and (2) carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) in the serum of patients with breast cancer. The serum samples from healthy donors were used as a control. The antigen detection is based on the formation of an immune complex of a specific capture antibody (Ab), a target antigen (Ag), and a detector Ab on the surface of the encoded particles. The capture Ab is bound to the polymer shell of microbeads via an adapter molecule, for example, protein A. Protein A binds a monoclonal Ab in a highly oriented manner due to specific interaction with the Fc-region of the Ab molecule. Each antigen can be recognized and detected due to a specific microbead population carrying the unique fluorescent code. 100 and 231 serum samples from patients with different stages of prostate cancer and breast cancer, respectively, and those from healthy donors were examined using the designed suspension system. The data were validated by comparing with the results of the "gold standard" enzyme-linked immunosorbent assay (ELISA). They have shown that our approach is a good alternative to the diagnostics of cancer markers using conventional assays, especially in early diagnostic applications.

Sensor Fusion to Estimate the Depth and Width of the Weld Bead in Real Time in GMAW Processes

PubMed Central

Sampaio, Renato Coral; Vargas, José A. R.

2018-01-01

The arc welding process is widely used in industry but its automatic control is limited by the difficulty in measuring the weld bead geometry and closing the control loop on the arc, which has adverse environmental conditions. To address this problem, this work proposes a system to capture the welding variables and send stimuli to the Gas Metal Arc Welding (GMAW) conventional process with a constant voltage power source, which allows weld bead geometry estimation with an open-loop control. Dynamic models of depth and width estimators of the weld bead are implemented based on the fusion of thermographic data, welding current and welding voltage in a multilayer perceptron neural network. The estimators were trained and validated off-line with data from a novel algorithm developed to extract the features of the infrared image, a laser profilometer was implemented to measure the bead dimensions and an image processing algorithm that measures depth by making a longitudinal cut in the weld bead. These estimators are optimized for embedded devices and real-time processing and were implemented on a Field-Programmable Gate Array (FPGA) device. Experiments to collect data, train and validate the estimators are presented and discussed. The results show that the proposed method is useful in industrial and research environments. PMID:29570698

Sensor Fusion to Estimate the Depth and Width of the Weld Bead in Real Time in GMAW Processes.

PubMed

Bestard, Guillermo Alvarez; Sampaio, Renato Coral; Vargas, José A R; Alfaro, Sadek C Absi

2018-03-23

The arc welding process is widely used in industry but its automatic control is limited by the difficulty in measuring the weld bead geometry and closing the control loop on the arc, which has adverse environmental conditions. To address this problem, this work proposes a system to capture the welding variables and send stimuli to the Gas Metal Arc Welding (GMAW) conventional process with a constant voltage power source, which allows weld bead geometry estimation with an open-loop control. Dynamic models of depth and width estimators of the weld bead are implemented based on the fusion of thermographic data, welding current and welding voltage in a multilayer perceptron neural network. The estimators were trained and validated off-line with data from a novel algorithm developed to extract the features of the infrared image, a laser profilometer was implemented to measure the bead dimensions and an image processing algorithm that measures depth by making a longitudinal cut in the weld bead. These estimators are optimized for embedded devices and real-time processing and were implemented on a Field-Programmable Gate Array (FPGA) device. Experiments to collect data, train and validate the estimators are presented and discussed. The results show that the proposed method is useful in industrial and research environments.

Molecular and immunological tools for the evaluation of the cellular immune response in the neotropical monkey Saimiri sciureus, a non-human primate model for malaria research.

PubMed

Riccio, Evelyn K P; Pratt-Riccio, Lilian R; Bianco-Júnior, Cesare; Sanchez, Violette; Totino, Paulo R R; Carvalho, Leonardo J M; Daniel-Ribeiro, Cláudio Tadeu

2015-04-18

The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus. Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences. The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-γ. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-β, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed after 6-12 hrs of culture, except for LTA in PBMC, whose expression was best analysed after 24 hrs of culture. Real-time PCR facilitates the analysis of a large number of cytokines altered during malaria infection, and this technique is considered the best tool for the evaluation of the cellular immune response in S. sciureus.

Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

PubMed

Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø; Sikorski, Pawel; Strand, Berit L; Standal, Therese

2015-01-01

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

In vitro evaluation of antibiotic diffusion from antibiotic-impregnated biodegradable beads and polymethylmethacrylate beads.

PubMed Central

Mader, J T; Calhoun, J; Cobos, J

1997-01-01

Antibiotic-impregnated beads are used in the dead bone space following debridement surgery to deliver local, high concentrations of antibiotics. Polymethylmethacrylate (PMMA), 2,000-molecular-weight (MW) polylactic acid (PLA), Poly(DL-lactide)-coglycolide (PL:CG; 90:10, 80:20, and 70:30), and the combination 2,000-MW PLA-70:20 PL:CG were individually mixed with clindamycin, tobramycin, or vancomycin. Beads were placed in 1 ml of phosphate-buffered saline (PBS) and incubated at 37 degrees C. The PBS was changed daily, and the removed PBS samples were stored at -70 degrees C until the antibiotic in each sample was determined by microbiological disk diffusion assay. Nondissolving PMMA beads with tobramycin and clindamycin had concentrations well above breakpoint sensitivity concentrations (i.e., the antibiotic concentrations at the transition point between bacterial killing and resistance to the antibiotic) for more than 90 days, but vancomycin concentrations dropped by day 12. ALl PLA, PL:CG, and the 2,000-MW PLA-70:30 PL:CG biodegradable beads release high concentrations of all the antibiotics in vitro for the period of time needed to treat bone infections (i.e., 4 to 8 weeks). Antibiotic-loaded PLA and PL:CG beads have the advantage of better antibiotic elution and the ability to biodegradable (thereby averting the need for secondary surgery for bead removal) compared to the PMMA beads presently used in the clinical setting. PMID:9021200

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

PubMed Central

Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø.; Sikorski, Pawel

2015-01-01

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering. PMID:25769043

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

PubMed

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-04-03

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

Shape Evolution of Highly Lattice-Mismatched InN/InGaN Nanowire Heterostructures

NASA Astrophysics Data System (ADS)

Yan, Lifan; Hazari, Arnab; Bhattacharya, Pallab; Millunchick, Joanna M.

2018-02-01

We have investigated the structure and shape of GaN-based nanowires grown on (001) Si substrates for optoelectronic device applications. The nanowire heterostructures contained InN disks and In0.4Ga0.6N barrier layers in the active region. The resulting nanowire array comprised two differently shaped nanowires: shorter pencil-like nanowires and longer bead-like nanowires. The two different nanowire shapes evolve due to a variation in the In incorporation rate, which was faster for the bead-like nanowires. Both types of nanowires exhibited evidence of significant migration of both Ga and In during growth. Ga tended to diffuse away and down along the sidewalls, resulting in a Ga-rich shell for all nanowires. Despite the complex structure and great variability in the In composition, the optical properties of the nanowire arrays were very good, with strong luminescence peaking at ˜ 1.63 μm.

Multi-colored immunochromatography using nanobeads for rapid and sensitive typing of seasonal influenza viruses.

PubMed

Sakurai, Akira; Takayama, Katsuyoshi; Nomura, Namiko; Yamamoto, Naoki; Sakoda, Yoshihiro; Kobayashi, Yukuharu; Kida, Hiroshi; Shibasaki, Futoshi

2014-12-01

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza viruses because of its rapid turnaround and ease of use. Despite the usefulness of IC, the limit of detection of common IC kits is as high as 10(3)-10(4) plaque forming units (pfu) per reaction, resulting in their limited sensitivities. Early diagnosis within 24h would provide more appropriate timing of treatment. In this study, a multi-colored NanoAct™ bead IC was established to detect seasonal influenza viruses. This method has approximately 10-fold higher sensitivity than that of colloidal gold or colored latex bead IC assays, and does not require specific instruments. More notably, NanoAct™ bead IC can distinguish influenza A and B viruses from clinical samples with a straightforward readout composed of colored lines. Our results will provide new strategies for the diagnosis, treatment, and a chance to survey of influenza viruses in developing countries and in the field research. Copyright © 2014 Elsevier B.V. All rights reserved.

The impact of methylation quantitative trait loci (mQTLs) on active smoking-related DNA methylation changes.

PubMed

Gao, Xu; Thomsen, Hauke; Zhang, Yan; Breitling, Lutz Philipp; Brenner, Hermann

2017-01-01

Methylation quantitative trait loci (mQTLs) are the genetic variants that may affect the DNA methylation patterns of CpG sites. However, their roles in influencing the disturbances of smoking-related epigenetic changes have not been well established. This study was conducted to address whether mQTLs exist in the vicinity of smoking-related CpG sites (± 50 kb) and to examine their associations with smoking exposure and all-cause mortality in older adults. We obtained DNA methylation profiles in whole blood samples by Illumina Infinium Human Methylation 450 BeadChip array of two independent subsamples of the ESTHER study (discovery set, n  = 581; validation set, n  = 368) and their corresponding genotyping data using the Illumina Infinium OncoArray BeadChip. After correction for multiple testing (FDR), we successfully identified that 70 out of 151 previously reported smoking-related CpG sites were significantly associated with 192 SNPs within the 50 kb search window of each locus. The 192 mQTLs significantly influenced the active smoking-related DNA methylation changes, with percentage changes ranging from 0.01 to 18.96%, especially for the weakly/moderately smoking-related CpG sites. However, these identified mQTLs were not directly associated with active smoking exposure or all-cause mortality. Our findings clearly demonstrated that if not dealt with properly, the mQTLs might impair the power of epigenetic-based models of smoking exposure to a certain extent. In addition, such genetic variants could be the key factor to distinguish between the heritable and smoking-induced impact on epigenome disparities. These mQTLs are of special importance when DNA methylation markers measured by Illumina Infinium assay are used for any comparative population studies related to smoking-related cancers and chronic diseases.

A new dimension in retrograde flow: centripetal movement of engulfed particles.

PubMed Central

Caspi, A; Yeger, O; Grosheva, I; Bershadsky, A D; Elbaum, M

2001-01-01

Centripetal motion of surface-adherent particles is a classic experimental system for studying surface dynamics on a eukaryotic cell. To investigate bead migration over the entire cell surface, we have developed an experimental assay using multinuclear giant fibroblasts, which provide expanded length scales and an unambiguous frame of reference. Beads coated by adhesion ligands concanavalin A or fibronectin are placed in specific locations on the cell using optical tweezers, and their subsequent motion is tracked over time. The adhesion, as well as velocity and directionality of their movement, expose distinct regions of the cytoplasm and membrane. Beads placed on the peripheral lamella initiate centripetal motion, whereas beads placed on the central part of the cell attach to a stationary cortex and do not move. Careful examination by complementary three-dimensional methods shows that the motion of a bead placed on the cell periphery takes place after engulfment into the cytoplasm, whereas stationary beads, placed near the cell center, are not engulfed. These results demonstrate that centripetal motion of adhering particles may occur inside as well as outside the cell. Inhibition of actomyosin activity is used to explore requirements for engulfment and aspects of the bead movement. Centripetal movement of adherent particles seems to depend on mechanisms distinct from those driving overall cell contractility. PMID:11566772

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

PubMed

Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

2014-07-01

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Testing implications of varying targets for Bordetella pertussis: comparison of the FilmArray Respiratory Panel and the Focus B. pertussis PCR assay

PubMed Central

Jerris, Robert C; Williams, Sally R; MacDonald, Heather J; Ingebrigtsen, Danielle R; Westblade, Lars F; Rogers, Beverly B

2015-01-01

Background The FilmArray Respiratory Panel (RP) detects multiple pathogens, including Bordetella pertussis. The multiplex PCR system is appropriate for a core laboratory or point of care due to ease of use. The purpose of this study is to compare the analytical sensitivity of the FilmArray RP, which targets the promoter region of the B. pertussis toxin gene, with the Focus real-time PCR assay, which targets the insertion sequence IS481. Methods Seventy-one specimens from patients aged 1 month to 18 years, which had tested positive for B. pertussis using the Focus assay, were analysed using the FilmArray RP. Results Forty-six specimens were positive for B. pertussis by both the Focus and the FilmArray RP assays. Twenty-five specimens were negative for B. pertussis using the FilmArray RP assay, but positive using the Focus assay. Conclusions The FilmArray RP assays will detect approximately 1/3 less cases of B. pertussis than the Focus assay. PMID:25742911

Fluorescent detection of C-reactive protein using polyamide beads

NASA Astrophysics Data System (ADS)

Jagadeesh, Shreesha; Chen, Lu; Aitchison, Stewart

2016-03-01

Bacterial infection causes Sepsis which is one of the leading cause of mortality in hospitals. This infection can be quantified from blood plasma using C - reactive protein (CRP). A quick diagnosis at the patient's location through Point-of- Care (POC) testing could give doctors the confidence to prescribe antibiotics. In this paper, the development and testing of a bead-based procedure for CRP quantification is described. The size of the beads enable them to be trapped in wells without the need for magnetic methods of immobilization. Large (1.5 mm diameter) Polyamide nylon beads were used as the substrate for capturing CRP from pure analyte samples. The beads captured CRP either directly through adsorption or indirectly by having specific capture antibodies on their surface. Both methods used fluorescent imaging techniques to quantify the protein. The amount of CRP needed to give a sufficient fluorescent signal through direct capture method was found suitable for identifying bacterial causes of infection. Similarly, viral infections could be quantified by the more sensitive indirect capture method. This bead-based assay can be potentially integrated as a disposable cartridge in a POC device due to its passive nature and the small quantities needed.

Force-velocity relationship of single actin filament interacting with immobilised myosin measured by electromagnetic technique.

PubMed

Holohan, S-J P; Marston, S B

2005-06-01

The effect of applying an external load to actin filaments moving in the in vitro motility assay is studied. Bead-tailed actin filaments were made by polymerising actin onto 2.8 microm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 microg/ml rabbit fast skeletal myosin in the presence of ATP and 0.5% methylcellulose. The motility cell was inserted between the pole-pieces of an electromagnet and the fluorescent beads and filaments were observed. The force-current relationship of the electromagnet was determined from the velocity of free beads in viscous solution and Stokes' equation. The magnet produced up to 6 pN force on the Dynabeads at 1 A. Many bead-tailed actin filaments stuck to the surface, but the beads that did move moved at the same speed as unloaded f-actin in the same cell. Bead-tailed filaments slowed down under an increasing magnetic load, eventually stalled and then slid backward under increasing load before detaching from the surface. Single-filament force-velocity curves were constructed and a stalling force of about 0.6 pN/mm of actin filament estimated.

High content analysis of phagocytic activity and cell morphology with PuntoMorph.

PubMed

Al-Ali, Hassan; Gao, Han; Dalby-Hansen, Camilla; Peters, Vanessa Ann; Shi, Yan; Brambilla, Roberta

2017-11-01

Phagocytosis is essential for maintenance of normal homeostasis and healthy tissue. As such, it is a therapeutic target for a wide range of clinical applications. The development of phenotypic screens targeting phagocytosis has lagged behind, however, due to the difficulties associated with image-based quantification of phagocytic activity. We present a robust algorithm and cell-based assay system for high content analysis of phagocytic activity. The method utilizes fluorescently labeled beads as a phagocytic substrate with defined physical properties. The algorithm employs statistical modeling to determine the mean fluorescence of individual beads within each image, and uses the information to conduct an accurate count of phagocytosed beads. In addition, the algorithm conducts detailed and sophisticated analysis of cellular morphology, making it a standalone tool for high content screening. We tested our assay system using microglial cultures. Our results recapitulated previous findings on the effects of microglial stimulation on cell morphology and phagocytic activity. Moreover, our cell-level analysis revealed that the two phenotypes associated with microglial activation, specifically cell body hypertrophy and increased phagocytic activity, are not highly correlated. This novel finding suggests the two phenotypes may be under the control of distinct signaling pathways. We demonstrate that our assay system outperforms preexisting methods for quantifying phagocytic activity in multiple dimensions including speed, accuracy, and resolution. We provide a framework to facilitate the development of high content assays suitable for drug screening. For convenience, we implemented our algorithm in a standalone software package, PuntoMorph. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis and screening of one-bead-one-compound cyclic peptide libraries.

PubMed

Qian, Ziqing; Upadhyaya, Punit; Pei, Dehua

2015-01-01

Cyclic peptides have been a rich source of biologically active molecules. Herein we present a method for the combinatorial synthesis and screening of large one-bead-one-compound (OBOC) libraries of cyclic peptides against biological targets such as proteins. Up to ten million different cyclic peptides are rapidly synthesized on TentaGel microbeads by the split-and-pool synthesis method and subjected to a multistage screening protocol which includes magnetic sorting, on-bead enzyme-linked and fluorescence-based assays, and in-solution binding analysis of cyclic peptides selectively released from single beads by fluorescence anisotropy. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. This method allows a single researcher to synthesize and screen up to ten million cyclic peptides and identify the most active ligand(s) in ~1 month, without the time-consuming and expensive hit resynthesis or the use of any special equipment.

Bead-based screening in chemical biology and drug discovery.

PubMed

Komnatnyy, Vitaly V; Nielsen, Thomas E; Qvortrup, Katrine

2018-06-11

High-throughput screening is an important component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds requires assays amenable to miniaturisation and automization. Combinatorial chemistry holds a unique promise to deliver structurally diverse libraries for early drug discovery. Among the various library forms, the one-bead-one-compound (OBOC) library, where each bead carries many copies of a single compound, holds the greatest potential for the rapid identification of novel hits against emerging drug targets. However, this potential has not yet been fully realized due to a number of technical obstacles. In this feature article, we review the progress that has been made in bead-based library screening and its application to the discovery of bioactive compounds. We identify the key challenges of this approach and highlight key steps needed for making a greater impact in the field.

Potentiometric Aptasensing of Vibrio alginolyticus Based on DNA Nanostructure-Modified Magnetic Beads.

PubMed

Zhao, Guangtao; Ding, Jiawang; Yu, Han; Yin, Tanji; Qin, Wei

2016-12-02

A potentiometric aptasensing assay that couples the DNA nanostructure-modified magnetic beads with a solid-contact polycation-sensitive membrane electrode for the detection of Vibrio alginolyticus is herein described. The DNA nanostructure-modified magnetic beads are used for amplification of the potential response and elimination of the interfering effect from a complex sample matrix. The solid-contact polycation-sensitive membrane electrode using protamine as an indicator is employed to chronopotentiometrically detect the change in the charge or DNA concentration on the magnetic beads, which is induced by the interaction between Vibrio alginolyticus and the aptamer on the DNA nanostructures. The present potentiometric aptasensing method shows a linear range of 10-100 CFU mL -1 with a detection limit of 10 CFU mL -1 , and a good specificity for the detection of Vibrio alginolyticus . This proposed strategy can be used for the detection of other microorganisms by changing the aptamers in the DNA nanostructures.

A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

PubMed

Niu, Hongmei; Klem, Thomas; Yang, Jinsong; Qiu, Yongchang; Pan, Luying

2017-07-01

Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors. Copyright © 2017 Elsevier B.V. All rights reserved.

A high-throughput liquid bead array-based screening technology for Bt presence in GMO manipulation.

PubMed

Fu, Wei; Wang, Huiyu; Wang, Chenguang; Mei, Lin; Lin, Xiangmei; Han, Xueqing; Zhu, Shuifang

2016-03-15

The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants. Copyright © 2015 Elsevier B.V. All rights reserved.

Cavity-induced microstreaming for simultaneous on-chip pumping and size-based separation of cells and particles.

PubMed

Patel, Maulik V; Nanayakkara, Imaly A; Simon, Melinda G; Lee, Abraham P

2014-10-07

We present a microfluidic platform for simultaneous on-chip pumping and size-based separation of cells and particles without external fluidic control systems required for most existing platforms. The device utilizes an array of acoustically actuated air/liquid interfaces generated using dead-end side channels termed Lateral Cavity Acoustic Transducers (LCATs). The oscillating interfaces generate local streaming flow while the angle of the LCATs relative to the main channel generates a global bulk flow from the inlet to the outlet. The interaction of these two competing velocity fields (i.e. global bulk velocity vs. local streaming velocity) is responsible for the observed separation. It is shown that the separation of 5 μm and 10 μm polystyrene beads is dependent on the ratio of these two competing velocity fields. The experimental and simulation results suggest that particle trajectories based only on Stokes drag force cannot fully explain the separation behavior and that the impact of additional forces due to the oscillating flow field must be considered to determine the trajectory of the beads and ultimately the separation behavior of the device. To demonstrate an application of this separation platform with cellular components, smaller red blood cells (7.5 ± 0.8 μm) are separated from larger K562 cells (16.3 ± 2.0 μm) with viabilities comparable to those of controls based on a trypan blue exclusion assay.

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

PubMed

Hilton, H G; Parham, P

2013-04-01

Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. © 2013 John Wiley & Sons A/S.

Microcinematographic analysis of tethered Leptospira illini.

PubMed Central

Charon, N W; Daughtry, G R; McCuskey, R S; Franz, G N

1984-01-01

A model of Leptospira motility was recently proposed. One element of the model states that in translating cells the anterior spiral-shaped end gyrates counterclockwise and the posterior hook-shaped end gyrates clockwise. We tested these predictions by analyzing cells tethered to a glass surface. Leptospira illini was incubated with antibody-coated latex beads (Ab-beads). These beads adhered to the cells, and subsequently some cells became attached to either the slide or the cover glass via the Ab-beads. As previously reported, these cells rapidly moved back and forth across the surface of the beads. In addition, a general trend was observed: cells tethered to the cover glass rotated clockwise around the Ab-bead; cells tethered to the slide rotated counterclockwise around the Ab-bead. A computer-aided microcinematographic analysis of tethered cells indicated that the direction of rotation of cells around the Ab-bead was a function of both the surface of attachment and the shape of the cell ends. The results can best be explained by assuming that the gyrating ends interact with the glass surface to cause rotation around the Ab-beads. The analysis obtained indicates that the hook- and spiral-shaped ends rotate in the directions predicted by the model. In addition, the tethered cell assay permitted detection of rapid, coordinated reversals of the cell ends, e.g., cells rapidly switched from a hook-spiral configuration to a spiral-hook configuration. These results suggest the existance of a mechanism which coordinates the shape of the cell ends of L. illini. Images PMID:6501226

A magnetic nanobead-based bioassay provides sensitive detection of single- and biplex bacterial DNA using a portable AC susceptometer.

PubMed

Strömberg, Mattias; Zardán Gómez de la Torre, Teresa; Nilsson, Mats; Svedlindh, Peter; Strømme, Maria

2014-01-01

Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin-biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM-NDA further towards implementation in point-of-care and outpatient settings. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-License, which permits use and distribution in any medium, provided the original work is properly cited.

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

PubMed

Simon, Stéphanie; Fiebig, Uwe; Liu, Yvonne; Tierney, Rob; Dano, Julie; Worbs, Sylvia; Endermann, Tanja; Nevers, Marie-Claire; Volland, Hervé; Sesardic, Dorothea; Dorner, Martin B

2015-11-26

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples

PubMed Central

Simon, Stéphanie; Fiebig, Uwe; Liu, Yvonne; Tierney, Rob; Dano, Julie; Worbs, Sylvia; Endermann, Tanja; Nevers, Marie-Claire; Volland, Hervé; Sesardic, Dorothea; Dorner, Martin B.

2015-01-01

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future. PMID:26703727

Rapid and field-deployable biological and chemical Raman-based identification

NASA Astrophysics Data System (ADS)

Botonjic-Sehic, Edita; Paxon, Tracy L.; Boudries, Hacene

2011-06-01

Pathogen detection using Raman spectroscopy is achieved through the use of a sandwich immunoassay. Antibody-modified magnetic beads are used to capture and concentrate target analytes in solution and surface-enhanced Raman spectroscopy (SERS) tags are conjugated with antibodies and act as labels to enable specific detection of biological pathogens. The rapid detection of biological pathogens is critical to first responders, thus assays to detect E.Coli and Anthrax have been developed and will be reported. The problems associated with pathogen detection resulting from the spectral complexity and variability of microorganisms are overcome through the use of SERS tags, which provide an intense, easily recognizable, and spectrally consistent Raman signal. The developed E. coli assay has been tested with 5 strains of E. coli and shows a low limit of detection, on the order of 10 and 100 c.f.u. per assay. Additionally, the SERS assay utilizes magnetic beads to collect the labeled pathogens into the focal point of the detection laser beam, making the assay robust to commonly encountered white powder interferants such as flour, baking powder, and corn starch. The reagents were also found to be stable at room temperature over extended periods of time with testing conducted over a one year period. Finally, through a specialized software algorithm, the assays are interfaced to the Raman instrument, StreetLab Mobile, for rapid-field-deployable biological identification.

High-volume extraction of nucleic acids by magnetic bead technology for ultrasensitive detection of bacteria in blood components.

PubMed

Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

2007-01-01

Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid-binding matrices. We adapted this technology with a broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresis-derived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primer-probe system and locked nucleic acid technology. Co-amplification of human beta(2)-microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. For automated magnetic bead-based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 x 10(3) colony-forming units (CFU)/L for S. epidermidis and 22 x 10(3) CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid contamination of reagents or externally during testing of 1030 PCs. High-volume nucleic acid extraction improved the detection limit of the assay. The improvement of the primer-probe system and the integration of an IC make the RT-PCR assay appropriate for bacteria screening of platelets.

A new bead-based human platelet antigen antibodies detection assay versus the monoclonal antibody immobilization of platelet antigens assay.

PubMed

Porcelijn, Leendert; Huiskes, Elly; Comijs-van Osselen, Ilona; Chhatta, Aniska; Rathore, Vipul; Meyers, Matthew; de Haas, Masja

2014-06-01

The performance of a newly developed Luminex bead-based platelet (PLT) antibody detection method (PAKLx) was compared with the monoclonal antibody immobilization of PLT antigens (MAIPA) assay and the LifeScreen Deluxe Luminex bead-based HLA Class I antibody detection method (LMX). Six sera containing anti-human PLT antigen (HPA)-1a (n=2), HPA-1b, HPA-2b, HPA-3a, or HPA-5b were tested in titration. A total of 194 sera, including HPA-1a, -1b, -2a, -2b, -3a, -5a, and -5b antibodies with or without HLA antibodies (n=63); glycoprotein (GP) IV antibodies (n=1); PLT autoantibodies (n=3); HLA antibodies (n=45); and samples with no PLT-reactive antibodies (n=82), were tested in both assays. Comparable levels of sensitivity were obtained for the MAIPA and PAKLx. The PAKLx showed four (6%) false-negative results in 67 sera with HPA or GP-reactive antibodies: anti-HPA-3a (n=1) or anti-HPA-5b (n=3). The PAKLx showed in 10 of the total 194 samples (5%) the presence of antibodies not detected by the MAIPA. This concerned broadly GP-reactive antibodies (n=7), anti-GPIIb/IIIa combined with anti-HPA-3a (n=1), anti-HPA-1a (borderline, n=1), and anti-GPIV (n=1). Testing 175 sera for anti-HLA Class I antibodies in the PAKLx and LMX showed four discrepant results: PAKLx negative and LMX positive, n=3 and n=1, respectively. For the vast majority of the specimens tested (93%) the results of the PAKLx were in concordance with the MAIPA. The PAKLx is a fast, easy to perform, and sensitive PLT antibody screening method. © 2013 AABB.

Seroepidemiology of Toxoplasma in a coastal region of Haiti; Multiplex bead assay detection of immunoglobulin G antibodies that recognize the SAG2A antigen

PubMed Central

Priest, J. W.; Moss, D. M.; Arnold, B. F.; Hamlin, K.; Jones, C. C.; Lammie, P. J.

2018-01-01

Summary Toxoplasma gondii is a globally distributed parasitic protozoan that infects most warm blooded animals. We incorporated a bead coupled with recombinant SAG2A protein into our Neglected Tropical Disease (NTD) multiplex bead assay (MBA) panel and used it to determine Toxoplasma infection rates in two studies in Haiti. In a longitudinal cohort study of children 0–11 years old, the infection rate varied with age reaching a maximum of 0.131 infections/ year in children 3 years of age (95% CI = 0.065, 0.204). The median time to seroconversion was estimated to be 9.7 years (95% CI = 7.6, ∞). In a cross-sectional, community-wide survey of residents of all ages, we determined an overall seroprevalence of 28.2%. The seroprevalence age curve from the cross-sectional study also suggested that the force of infection varied with age and peaked at 0.057 infections/ year (95% CI = 0.033, 0.080) at 2.6 years of age. Integration of the Toxoplasma MBA into NTD surveys may allow for better estimates of the potential burden of congenital toxoplasmosis in underserved regions. PMID:25600668

Cavitands Endow All-Dielectric Beads With Selectivity for Plasmon-Free Enhanced Raman Detection of Nε-Methylated Lysine.

PubMed

Alessandri, Ivano; Biavardi, Elisa; Gianoncelli, Alessandra; Bergese, Paolo; Dalcanale, Enrico

2016-06-22

SiO2/TiO2 microbeads (T-rex) are promising materials for plasmon-free surface-enhanced Raman scattering (SERS), offering several key advantages in biodiagnostics. In this paper we report the combination of T-rex beads with tetraphosphonate cavitands (Tiiii), which imparts selectivity toward Nε-methylated lysine. SERS experiments demonstrated the efficiency and selectivity of the T-rex-Tiiii assays in detecting methylated lysine hydrochloride (Nε-Me-Lys-Fmoc) from aqueous solutions, even in the presence of the parent Lys-Fmoc hydrochloride as interferent. The negative results obtained in control experiments using TSiiii ruled out any other form of surface recognition or preferential physisorption. MALDI-TOF analyses on the beads exposed to Nε-Me-Lys-Fmoc revealed the presence of the Tiiii•Nε-Me-Lys-Fmoc complex. Raman analyses based on the intensity ratio of Nε-Me-Lys-Fmoc and cavitand-specific modes resulted in a dose-response plot, which allowed for estimating the concentration of Nε-methylated lysine from initial solutions in the 1 × 10(-3) to 1 × 10(-5) M range. These results can set the basis for the development of new Raman assays for epigenetic diagnostics.

Protein aggregation induced during glass bead lysis of yeast

PubMed Central

Papanayotou, Irene; Sun, Beimeng; Roth, Amy F.; Davis, Nicholas G.

2013-01-01

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein–protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation. PMID:20641011

Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains

DTIC Science & Technology

2012-06-01

trifluoroacetic acid; Tm: melting temperature; TMB, 3,3′,5,5′-tetramethylbenzidine; UPLC , ultra performance liquid chromatography; VAMP, vesicle...activity determination by UPLC . Alternately, in large-scale preparations, phosphoryla- tion reaction was stopped by removing the Src with sepharose beads...peptides. ENZYMATIC ACTIVITY ASSAYS Activity assays were based on UPLC separation and measurement of the cleaved products from a 17-residue SNAP-25

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads

PubMed Central

Wang, Lin; Acosta, Miguel A.; Leach, Jennie B.; Carrier, Rebecca L.

2013-01-01

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems. PMID:23443975

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads.

PubMed

Wang, Lin; Acosta, Miguel A; Leach, Jennie B; Carrier, Rebecca L

2013-04-21

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.

Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

PubMed

Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

2013-03-07

Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

Detection of avian influenza antigens in proximity fiber, droplet, and optical waveguide microfluidics

NASA Astrophysics Data System (ADS)

Yoon, Jeong-Yeol; Heinze, Brian C.; Gamboa, Jessica; You, David J.

2009-05-01

Virus antigens of avian influenza subtype H3N2 were detected on two different microfluidic platforms: microchannel and droplet. Latex immunoagglutination assays were performed using 920-nm highly carboxylated polystyrene beads that are conjugated with antibody to avian influenza virus. The bead suspension was merged with the solutions of avian influenza virus antigens in a Y-junction of a microchannel made by polydimethylsiloxane soft lithography. The resulting latex immunoagglutinations were measured with two optical fibers in proximity setup to detect 45° forward light scattering. Alternatively, 10 μL droplets of a bead suspension and an antigen solution were merged on a superhydrophobic surface (water contact angle = 155°), whose movement was guided by a metal wire, and 180° back light scattering is measured with a backscattering optical probe. Detection limits were 0.1 pg mL-1 for both microchannel with proximity fibers and droplet microfluidics, thanks to the use of micro-positioning stages to help generate reproducible optical signals. Additionally, optical waveguide was tested by constructing optical waveguide channels (filled with mineral oil) within a microfluidic device to detect the same light scattering. Detection limit was 0.1 ng mL-1 for an optical waveguide device, with a strong potential of improvement in the near future. The use of optical waveguide enabled smaller device setup, easier operation, smaller standard deviations and broader linear range of assay than proximity fiber microchannel and droplet microfluidics. Total assay time was less than 10 min.

Magnetic nanobeads present during enzymatic amplification and labeling for a simplified DNA detection protocol based on AC susceptometry

NASA Astrophysics Data System (ADS)

Bejhed, Rebecca S.; Strømme, Maria; Svedlindh, Peter; Ahlford, Annika; Strömberg, Mattias

2015-12-01

Magnetic biosensors are promising candidates for low-cost point-of-care biodiagnostic devices. For optimal efficiency it is crucial to minimize the time and complexity of the assay protocol including target recognition, amplification, labeling and read-out. In this work, possibilities for protocol simplifications for a DNA biodetection principle relying on hybridization of magnetic nanobeads to rolling circle amplification (RCA) products are investigated. The target DNA is recognized through a padlock ligation assay resulting in DNA circles serving as templates for the RCA process. It is found that beads can be present during amplification without noticeably interfering with the enzyme used for RCA (phi29 polymerase). As a result, the bead-coil hybridization can be performed immediately after amplification in a one-step manner at elevated temperature within a few minutes prior to read-out in an AC susceptometer setup, i.e. a combined protocol approach. Moreover, by recording the phase angle ξ = arctan(χ″/χ'), where χ and χ″ are the in-phase and out-of-phase components of the AC susceptibility, respectively, at one single frequency the total assay time for the optimized combined protocol would be no more than 1.5 hours, often a relevant time frame for diagnosis of cancer and infectious disease. Also, applying the phase angle method normalization of AC susceptibility data is not needed. These findings are useful for the development of point-of-care biodiagnostic devices relying on bead-coil binding and magnetic AC susceptometry.

Trapping and dynamic manipulation of polystyrene beads mimicking circulating tumor cells using targeted magnetic/photoacoustic contrast agents

NASA Astrophysics Data System (ADS)

Wei, Chen-Wei; Xia, Jinjun; Pelivanov, Ivan; Hu, Xiaoge; Gao, Xiaohu; O'Donnell, Matthew

2012-10-01

Results on magnetically trapping and manipulating micro-scale beads circulating in a flow field mimicking metastatic cancer cells in human peripheral vessels are presented. Composite contrast agents combining magneto-sensitive nanospheres and highly optical absorptive gold nanorods were conjugated to micro-scale polystyrene beads. To efficiently trap the targeted objects in a fast stream, a dual magnet system consisting of two flat magnets to magnetize (polarize) the contrast agent and an array of cone magnets producing a sharp gradient field to trap the magnetized contrast agent was designed and constructed. A water-ink solution with an optical absorption coefficient of 10 cm-1 was used to mimic the optical absorption of blood. Magnetomotive photoacoustic imaging helped visualize bead trapping, dynamic manipulation of trapped beads in a flow field, and the subtraction of stationary background signals insensitive to the magnetic field. The results show that trafficking micro-scale objects can be effectively trapped in a stream with a flow rate up to 12 ml/min and the background can be significantly (greater than 15 dB) suppressed. It makes the proposed method very promising for sensitive detection of rare circulating tumor cells within high flow vessels with a highly absorptive optical background.

A Novel Method for Rapid Hybridization of DNA to a Solid Support

PubMed Central

Pettersson, Erik; Ahmadian, Afshin; Ståhl, Patrik L.

2013-01-01

Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself. PMID:23950946

Potentiometric Aptasensing of Vibrio alginolyticus Based on DNA Nanostructure-Modified Magnetic Beads

PubMed Central

Zhao, Guangtao; Ding, Jiawang; Yu, Han; Yin, Tanji; Qin, Wei

2016-01-01

A potentiometric aptasensing assay that couples the DNA nanostructure-modified magnetic beads with a solid-contact polycation-sensitive membrane electrode for the detection of Vibrio alginolyticus is herein described. The DNA nanostructure-modified magnetic beads are used for amplification of the potential response and elimination of the interfering effect from a complex sample matrix. The solid-contact polycation-sensitive membrane electrode using protamine as an indicator is employed to chronopotentiometrically detect the change in the charge or DNA concentration on the magnetic beads, which is induced by the interaction between Vibrio alginolyticus and the aptamer on the DNA nanostructures. The present potentiometric aptasensing method shows a linear range of 10–100 CFU mL−1 with a detection limit of 10 CFU mL−1, and a good specificity for the detection of Vibrio alginolyticus. This proposed strategy can be used for the detection of other microorganisms by changing the aptamers in the DNA nanostructures. PMID:27918423

Using electrical and optical tweezers to facilitate studies of molecular motors.

PubMed

Arsenault, Mark E; Sun, Yujie; Bau, Haim H; Goldman, Yale E

2009-06-28

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells.

Using electrical and optical tweezers to facilitate studies of molecular motors†

PubMed Central

Arsenault, Mark E.; Sun, Yujie; Bau, Haim H.; Goldman, Yale E.

2013-01-01

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells. PMID:19506758

Integration of minisolenoids in microfluidic device for magnetic bead-based immunoassays

NASA Astrophysics Data System (ADS)

Liu, Yan-Jun; Guo, Shi-Shang; Zhang, Zhi-Ling; Huang, Wei-Hua; Baigl, Damien; Chen, Yong; Pang, Dai-Wen

2007-10-01

Microfluidic devices with integrated minisolenoids, microvalves, and channels have been fabricated for fast and low-volume immunoassay using superparamagnetic beads and well-known surface bioengineering protocols. A magnetic reaction area can be formed in the microchannel, featuring a high surface-to-volume ratio and low diffusion distances for the reagents to the bead surface. Such a method has the obvious advantage of easy implementation at low cost. Moreover, the minisolenoids can be switched on or off and the magnetic field intensity can be tuned on demand. Fluids can be manipulated by controlling the integrated air-pressure-actuated microvalves. Accordingly, magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic device automatically in longitudinal mode. With a sample consumption of 0.5μl and a total assay time of less than 15min, goat immunoglobulin G was detected and the method exhibited a detection limit of 4.7ng/ml.

Droplet microfluidics with magnetic beads: a new tool to investigate drug-protein interactions.

PubMed

Lombardi, Dario; Dittrich, Petra S

2011-01-01

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug-protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.

Multiplex Immunoassay Profiling.

PubMed

Stephen, Laurie

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex ® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.

Chicken IgY-Fc antibody elicited in a rabbit as first coat on frosted glass beads in immunoassays.

PubMed

Polson, A

1993-07-01

The observation of Esteves and Binaghi that the Fc moieties of mammalian IgG are antigenically similar but antigenically unrelated to the Fc fraction of chicken IgG (IgY) was usefully applied in enzyme linked immuno-sorbent assay (ELISA). In this communication its use in ELISA of two plant viruses viz. tobacco mosaic virus (common strain) and grape vine A virus is illustrated. Frosted glass beads were used as reagent carriers and the reactions were conducted in the wells of multi-titre plates.

Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

DTIC Science & Technology

2017-01-01

regulated by Pim and what is the outcome of treating animals with Pim inhibitors. To explore these tasks we first stained human tumor and normal samples ...Sepharose beads. The beads were incubated with 100 ng of purified Pim-1 proteins at 37 °C for 30 minutes. Samples were analyzed by immunoblot assays using...secreted by these cells using novel technology, develop potential urine biomarkers of treatment success. 1R21 CA178324-01A1 (Kraft, PI) 06/01/15

An integrated centrifugo-opto-microfluidic platform for arraying, analysis, identification and manipulation of individual cells.

PubMed

Burger, R; Kurzbuch, D; Gorkin, R; Kijanka, G; Glynn, M; McDonagh, C; Ducrée, J

2015-01-21

In this work we present a centrifugal microfluidic system enabling highly efficient collective trapping and alignment of particles such as microbeads and cells, their multi-colour fluorescent detection and subsequent manipulation by optical tweezers. We demonstrate array-based capture and imaging followed by "cherry-picking" of individual particles, first for fluorescently labelled polystyrene (PS) beads and then for cells. Different cell lines are discriminated based on intracellular as well as surface-based markers.

Pharmacokinetics and analgesic effect of ketorolac floating delivery system.

PubMed

Radwan, Mahasen A; Abou El Ela, Amal El Sayeh F; Hassan, Maha A; El-Maraghy, Dalia A

2015-05-01

The efficacy of ketorolac tromethamine (KT) floating alginate beads as a drug delivery system for better control of KT release was investigated. The formulation with the highest drug loading, entrapment efficiency, swelling, buoyancy, and in vitro release would be selected for further in vivo analgesic effect in the mice and pharmacokinetics study in rats compared to the tablet dosage form. KT floating alginate beads were prepared by extrusion congealing technique. KT in plasma samples was analyzed using a UPLC MS/MS assay. The percentage yield, drug loading and encapsulation efficiency were increased proportionally with the hydroxypropylmethyl cellulose (HPMC) polymer amount in the KT floating beads. A reverse relationship was observed between HPMC amount in the beads and the KT in vitro release rate. F3-floating beads were selected, due to its better in vitro results (continued floating for >8 h) than others. A longer analgesic effect was observed for F3 in fed mice as compared to the tablets. After F3 administration to rats, the Cmax (2.2 ± 0.3 µg/ml) was achieved at ∼2 h and the decline in KT concentration was slower. F3 showed a significant increase in the AUC (1.89 fold) in rats as compared to the tablets. KT was successfully formulated as floating beads with prolonged in vitro release extended to a better in vivo characteristic with higher bioavailability in rats. KT in floating beads shows a superior analgesic effect over tablets, especially in fed mice.

Nominal effective immunoreaction volume of magnetic beads at single bead level.

PubMed

Wang, Rui; Chen, Yuan; Fan, Kai; Ji, Feng; Wu, Jian; Yu, Yong-Hua

Immunomagnetic bead (IMB)-based enzyme-linked immunosorbent assay (ELISA) has been the tool frequently used for protein detection in research and clinical laboratories. For most ELISA reactions the recommended dosage of IMBs is usually according to their weight (mg) or mass fraction (w/v) instead of the bead number. Consequently, the processes occurring in the immediate vicinity of the IMBs have always been ignored by researchers and they cannot be revealed in detail during the ELISA reaction. In this paper, we established the relationship between number of IMBs and colorimetric results, and further proposed a new concept of "nominal effective immunoreaction volume (NEIV)" to characterize a single IMB during ELISA reaction. Results showed that the NEIV of a single IMB has a constant value, which is unrelated to the amount of beads and the concentration of antigen. Optimal results of the colorimetric ELISA are achieved when the incubation volume meets each IMB's NEIV and is no longer enhanced by increasing the incubation volume. Thus, the reliable and relatively precise number of IMBs for ELISA detection during practical application could be determined. Most importantly, a study using IMB's NEIV would lay the foundation for a kinetics analysis of IMBs and antigens for future study.

Development of an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay specific for Enterocytozoon bieneusi in water samples.

PubMed

Sorel, N; Guillot, E; Thellier, M; Accoceberry, I; Datry, A; Mesnard-Rouiller, L; Miégeville, M

2003-01-01

Microsporidia have become widely recognized as important human pathogens. Among Microsporidia, Enterocytozoon bieneusi is responsible for severe gastrointestinal disease. To date, no current therapy has been proven effective. Their mode of transmission and environmental occurrence are poorly documented because of the lack of detection methods that are both species-specific and sensitive. In this study, we developed a sensitive and specific molecular method to detect E. bieneusi spores in water samples. The molecular assay combined immunomagnetic separation (IMS) and polymerase chain reaction (PCR) amplification to detect E. bieneusi spores. A comparison was made of IMS magnetic beads coated with two different monoclonal antibodies, one specific for the Encephalitozoon genus that cross-reacts with E. bieneusi and the other specific only for the E. bieneusi species itself. Immunotech beads coated with the antibody specific for E. bieneusi were found to be the most effective combination. The highly specific IMS-PCR assay developed in this study provides a rapid and sensitive means of screening water samples for the presence of E. bieneusi spores.

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY.

PubMed

Edupuganti, Soujanya Ratna; Edupuganti, Om Prakash; O'Kennedy, Richard; Defrancq, Eric; Boullanger, Stéphanie

2013-04-01

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

Sorting white blood cells in microfabricated arrays

NASA Astrophysics Data System (ADS)

Castelino, Judith Andrea Rose

Fractionating white cells in microfabricated arrays presents the potential for detecting cells with abnormal adhesive or deformation properties. A possible application is separating nucleated fetal red blood cells from maternal blood. Since fetal cells are nucleated, it is possible to extract genetic information about the fetus from them. Separating fetal cells from maternal blood would provide a low cost noninvasive prenatal diagnosis for genetic defects, which is not currently available. We present results showing that fetal cells penetrate further into our microfabricated arrays than adult cells, and that it is possible to enrich the fetal cell fraction using the arrays. We discuss modifications to the array which would result in further enrichment. Fetal cells are less adhesive and more deformable than adult white cells. To determine which properties limit penetration, we compared the penetration of granulocytes and lymphocytes in arrays with different etch depths, constriction size, constriction frequency, and with different amounts of metabolic activity. The penetration of lymphocytes and granulocytes into constrained and unconstrained arrays differed qualitatively. In constrained arrays, the cells were activated by repeated shearing, and the number of cells stuck as a function of distance fell superexponentially. In unconstrained arrays the number of cells stuck fell slower than an exponential. We attribute this result to different subpopulations of cells with different sticking parameters. We determined that penetration in unconstrained arrays was limited by metabolic processes, and that when metabolic activity was reduced penetration was limited by deformability. Fetal cells also contain a different form of hemoglobin with a higher oxygen affinity than adult hemoglobin. Deoxygenated cells are paramagnetic and are attracted to high magnetic field gradients. We describe a device which can separate cells using 10 μm magnetic wires to deflect the paramagnetic cells. We present preliminary results from a test system that separates paramagnetic beads from latex beads. The separation is limited by our ability to produce the high field gradients which are necessary to separate cells according to their hemoglobin content, and we present estimates of the magnetic gradients we achieved.

A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads.

PubMed

He, Hong-qiu; Ma, Xiao-hui; Liu, Bin; Chen, Wei-zu; Wang, Cun-xin; Cheng, Shao-hui

2008-03-01

To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9. The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.

A rapid diagnostic test and mobile "lab in a suitcase" platform for detecting Ceratocystis spp. responsible for Rapid ‘Ōhi‘a Death

USGS Publications Warehouse

Atkinson, Carter T.; Watcher-Weatherwax, William; Roy, Kylle; Heller, Wade P; Keith, Lisa

2017-01-01

We describe a field compatible molecular diagnostic test for two new species of Ceratocystis that infect `ōhi`a (Metrosideros polymorpha) and cause the disease commonly known as Rapid `Ōhi`a Death. The diagnostic is based on amplification of a DNA locus within the internal transcribed spacer region that separates fungal 5.8S ribosomal genes. The assay uses forward and reverse primers, recombinase polymerase, and a fluorescent probe that allows isothermal (40oC) amplification and simultaneous quantification of a 115 base pair product with a battery operated fluorometer. DNA extractions are field compatible and can be done by heating wood drill shavings to 100oC in Instagene® solution containing Chelex® resin to bind potential amplification inhibitors. The initial heat treatment is followed by a short bead beating step with steel ball bearings and zirconium beads to release DNA. DNA is subsequently purified with a magnetic bead based extraction method that does not require silica columns or centrifugation. The assay is designed around a portable “lab-in-a-suitcase” platform that includes a portable fluorometer, miniature centrifuge, and heat block that operate off either 120V AC power sources or a 12 volt battery with a portable inverter, a magnetic rack designed for 1.5 ml tubes and magnetic bead DNA purification, pipettes and consumable reagents and tubes. The entire assay from DNA extraction to results can be performed in less than 90 minutes on up to six independent samples plus a positive and negative control. Sensitivity based on suspensions of Ceratocystis endoconidia (spores) that were added to wood shavings and processed under field conditions by Instagene® magnetic bead DNA extraction was up to 163 spores/mg wood for Species A and 55 spores/mg wood for Species B in 95% of replicates as determined by probit analysis. Sensitivity increased 5–10 fold to 19 spores/mg wood for Species A and 9 spores/mg wood for Species B when extractions were performed with a commercial, silica column based DNA purification kit. The test did not cross react with other common fungi that have been isolated from `ōhi`a.

A Functional Assay for GPR55: Envision Protocol.

PubMed

Anavi-Goffer, Sharon; Ross, Ruth A

2016-01-01

AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.

3D Printing and Assay Development for Point-of-Care Applications

NASA Astrophysics Data System (ADS)

Jagadeesh, Shreesha

Existing centralized labs do not serve patients adequately in remote areas. To enable universal timely healthcare, there is a need to develop low cost, portable systems that can diagnose multiple disease (Point-of-Care (POC) devices). Future POC diagnostics can be more multi-functional if medical device vendors can develop interoperability standards. This thesis developed the following medical diagnostic modules: Plasma from 25 microl blood was extracted through a filter membrane to demonstrate a 3D printed sample preparation module. Sepsis biomarker, C - reactive protein, was quantified through adsorption on nylon beads to demonstrate bead-based assay suitable for 3D printed disposable cartridge module. Finally, a modular fluorescent detection kit was built using 3D printed parts to detect CD4 cells in a disposable cartridge from ChipCare Corp. Due to the modularity enabled by 3D printing technique, the developed units can be easily adapted to detect other diseases.

Determination of palladium, platinum and rhodium in geologic materials by fire assay and emission spectrography

USGS Publications Warehouse

Hapfty, J.; Riley, L.B.

1968-01-01

A method is described for the determination of palladium down to 4ppb (parts per billion, 109), platinum down to 10 ppb and rhodium down to 5 ppb in 15 g of sample. Fire-assay techniques are used to preconcentrate the platinum metals into a gold bead, then the bead is dissolved in aqua regia and diluted to volume with 1M hydrochloric acid. The solution is analysed by optical emission spectrography of the residue from 200 ??l of it evaporated on a pair of flat-top graphite electrodes. This method requires much less sample handling than most published methods for these elements. Data are presented for G-1, W-1, and six new standard rocks of the U.S. Geological Survey. The values for palladium in W-1 are in reasonable agreement with previously published data. ?? 1968.

Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.

PubMed

Hommatsu, Manami; Okahashi, Hisamitsu; Ohta, Keisuke; Tamai, Yusuke; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

2013-01-01

A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e. LDR product) to the probe DNA. Simple fluid manipulations enabled both allele-specific hybridization and the removal of non-specifically bound DNA in the wash step. Furthermore, the use of CL detection greatly simplified the detection scheme, since CL does not require a light source for excitation of the fluorescent dye tags on the LDR products. Preliminary results demonstrated that this analytical system could detect both homozygous and heterozygous mutations, without the expensive instrumentation and cumbersome procedures required by conventional DNA microarray-based methods.

Absolute counting of neutrophils in whole blood using flow cytometry.

PubMed

Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

2014-12-01

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry.

Comparing PMMA and calcium sulfate as carriers for the local delivery of antibiotics to infected surgical sites.

PubMed

McConoughey, Stephen J; Howlin, Robert P; Wiseman, Jessica; Stoodley, Paul; Calhoun, Jason H

2015-05-01

Antibiotic-loaded bone cement is a primary option for treatment of orthopedic infections. Poly(methyl methacrylate) (PMMA) is a widely used cement that, when loaded with antibiotics in spacer or bead form, has been shown to reduce infection rates. However, PMMA is not resorbable and requires a second surgery for removal, while also acting as a potential foreign body for bacterial colonization. Alternatively, resorbable bone cements, such as calcium sulfate, have been proposed and present the advantage of being completely reabsorbed. It is unknown whether the antibiotic elution characteristics of absorbable bone cements are similar to PMMA. This study (1) characterized antibiotic elution from synthetic, highly purified calcium sulfate cement beads of varying sizes against pathogenic bacteria both in liquid culture and seeded on agar plates, (2) tested calcium sulfate beads against PMMA beads loaded with the same antibiotics, and (3) analyzed the structural differences between how PMMA and calcium sulfate bind to antibiotics. In every assay, the calcium sulfate beads performed as well as, or better than, the PMMA beads in inhibition of bacterial growth and elution of vancomycin in vitro with complete elution observed from calcium sulfate within three days. These data suggest that calcium sulfate, functions, as well as PMMA in the patient setting for infection control. © 2014 Wiley Periodicals, Inc.

A portable optical reader and wall projector towards enumeration of bio-conjugated beads or cells

PubMed Central

McArdle, Niamh A.; Kendlin, Jane L.; O’Connell, Triona M.; Ducrée, Jens

2017-01-01

Measurement of the height of a packed column of cells or beads, which can be direclty related to the number of cells or beads present in a chamber, is an important step in a number of diagnostic assays. For example, haematocrit measurements may rapidly identify anemia or polycthemia. Recently, user-friendly and cost-efficient Lab-on-a-Chip devices have been developed towards isolating and counting cell sub-populations for diagnostic purposes. In this work, we present a low-cost optical module for estimating the filling level of packed magnetic beads within a Lab-on-a-Chip device. The module is compatible with a previously introduced, disposable microfluidic chip for rapid determination of CD4+ cell counts. The device is a simple optical microscope module is manufactured by 3D printing. An objective lens directly interrogates the height of packed beads which are efficiently isolated on the finger-actuated chip. Optionally, an inexpensive, battery-powered Light Emitting Diode may project a shadow of the microfluidic chip at approximately 50-fold magnification onto a nearby surface. The reader is calibrated with the filling levels of known concentrations of paramagnetic beads within the finger actuated chip. Results in direct and projector mode are compared to measurements from a conventional, inverted white-light microscope. All three read-out methods indicate a maximum variation of 6.5% between methods. PMID:29267367

SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Qijian; Jia, Gaofeng; Hyten, David L.

A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of largemore » scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.« less

SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean

DOE PAGES

Song, Qijian; Jia, Gaofeng; Hyten, David L.; ...

2015-08-28

A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of largemore » scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.« less

SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean.

PubMed

Song, Qijian; Jia, Gaofeng; Hyten, David L; Jenkins, Jerry; Hwang, Eun-Young; Schroeder, Steven G; Osorno, Juan M; Schmutz, Jeremy; Jackson, Scott A; McClean, Phillip E; Cregan, Perry B

2015-08-28

A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of large scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad. Copyright © 2015 Song et al.

Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode.

PubMed

Hernández-Neuta, Iván; Pereiro, Iago; Ahlford, Annika; Ferraro, Davide; Zhang, Qiongdi; Viovy, Jean-Louis; Descroix, Stéphanie; Nilsson, Mats

2018-04-15

Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120μL of DNA dilution at flow rates ranging from 1 to 5μL/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

PubMed

Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

2017-01-01

Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

PubMed Central

Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2011-01-01

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules.

PubMed

Han, M; Gao, X; Su, J Z; Nie, S

2001-07-01

Multicolor optical coding for biological assays has been achieved by embedding different-sized quantum dots (zinc sulfide-capped cadmium selenide nanocrystals) into polymeric microbeads at precisely controlled ratios. Their novel optical properties (e.g., size-tunable emission and simultaneous excitation) render these highly luminescent quantum dots (QDs) ideal fluorophores for wavelength-and-intensity multiplexing. The use of 10 intensity levels and 6 colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic measurements indicate that the QD-tagged beads are highly uniform and reproducible, yielding bead identification accuracies as high as 99.99% under favorable conditions. DNA hybridization studies demonstrate that the coding and target signals can be simultaneously read at the single-bead level. This spectral coding technology is expected to open new opportunities in gene expression studies, high-throughput screening, and medical diagnostics.

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens.

PubMed

Ondigo, Bartholomew N; Park, Gregory S; Gose, Severin O; Ho, Benjamin M; Ochola, Lyticia A; Ayodo, George O; Ofulla, Ayub V; John, Chandy C

2012-12-21

Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.

Real-time monitoring of lactate extrusion and glucose consumption of cultured cells using a lab-on-valve system.

PubMed

Schulz, Craig M; Scampavia, Louis; Ruzicka, Jaromir

2002-12-01

Microsequential injection (microST) provides microfluidic operations that are ideally suited for cellular function studies and as a means of validating targets for drug discovery. MicroSI carried out within the lab-on-valve (LOV) manifold, is an ideal platform for spectroscopic studies on living cells that are grown on microcarrier beads and kept thermostated while their metabolism is probed in real-time. In this paper a microbioreactor is integrated into the LOV manifold allowing measurement of cellular lactate extrusion and glucose consumption rates of a cell culture that is automatically renewed prior to each measurement. Glucose consumption and lactate extrusion are monitored using NAD-linked enzymatic assays. The microSI-LOV setup has demonstrated a linear analysis range of 0.05-1.00 mM for lactate and 0.1-5.6 mM for glucose. These assays were conducted in a serial fashion requiring 3 microL of cellular perfusate and 10 s for glucose determination and 30 s for the lactate assay. Overall waste generated per lactate/glucose assay is < 200 microL. This work was performed using two different transfected hepatocyte cell lines, which adhere to Cytopore microcarrier beads. This novel approach to metabolic screening allows for the rapid evaluation of the effects of dosing cells with chemical agents.

A Multiplexed Assay That Monitors Effects of Multiple Compound Treatment Times Reveals Candidate Immune-Enhancing Compounds.

PubMed

Zhao, Ziyan; Henowitz, Liza; Zweifach, Adam

2018-05-01

We previously developed a flow cytometry assay that monitored lytic granule exocytosis in cytotoxic T lymphocytes stimulated by contacting beads coated with activating anti-CD3 antibodies. That assay was multiplexed in that responses of cells that did or did not receive the activating stimulus were distinguished via changes in light scatter accompanying binding of cells to beads, allowing us to discriminate compounds that activate responses on their own from compounds that enhance responses in cells that received the activating stimulus, all within a single sample. Here we add a second dimension of multiplexing by developing means to assess in a single sample the effects of treating cells with test compounds for different times. Bar-coding cells before adding them to test wells lets us determine compound treatment time while also monitoring activation status and response amplitude at the point of interrogation. This multiplexed assay is suitable for screening 96-well plates. We used it to screen compounds from the National Cancer Institute, identifying several compounds that enhance anti-LAMP1 responses. Multiple-treatment-time (MTT) screening enabled by bar-coding and read via high-throughput flow cytometry may be a generally useful method for facilitating the discovery of compounds of interest.

Comparison of the FilmArray Respiratory Panel and Prodesse Real-Time PCR Assays for Detection of Respiratory Pathogens ▿ †

PubMed Central

Loeffelholz, M. J.; Pong, D. L.; Pyles, R. B.; Xiong, Y.; Miller, A. L.; Bufton, K. K.; Chonmaitree, T.

2011-01-01

We compared the diagnostic performance and overall respiratory pathogen detection rate of the premarket version of the FilmArray Respiratory Panel (RP) multiplex PCR assay (Idaho Technology, Inc., Salt Lake City, UT) with those of the Food and Drug Administration (FDA)-cleared Prodesse ProFlu+, ProFAST+, ProParaflu+, Pro hMPV+, and ProAdeno+ real-time PCR assays (Gen-Probe, San Diego, CA). The assays were performed on a panel of 192 nasopharyngeal-secretion specimens collected from 81 children under 1 year of age with upper respiratory tract symptoms. To resolve discordant results and confirm pathogens detected only by the larger FilmArray panel, we performed laboratory-developed real-time PCR assays. Among viruses detectable by both commercial assays (adenovirus, human metapneumovirus, influenza A virus, influenza B virus, parainfluenza viruses 1 to 3, and respiratory syncytial virus), the FilmArray and Prodesse assays showed good overall agreement (181/192 [94.3%]; kappa = 0.87; 95% CI, 0.79 to 0.94). FilmArray RP detected more parainfluenza viruses 1 and 3 than ProParaflu+ (18 versus 13) while ProAdeno+ detected more adenoviruses (11 versus 6), but these differences were not statistically significant. Additionally, FilmArray RP detected 138 pathogens (confirmed as true positives) not included in the Prodesse assays (rhinovirus [RV]/enterovirus [EV], 118; bocavirus, 8; coronavirus, 7; parainfluenza virus 4, 4; Mycoplasma pneumoniae, 1). FilmArray RP was cleared by the FDA following the completion of this study. The FDA-cleared version includes the following targets: adenovirus, coronaviruses HKU1 and NL63, human metapneumovirus (hMPV), influenza A virus (to type level only), influenza A H1 seasonal virus, influenza A H3 seasonal virus, influenza A virus H1-2009, influenza B virus, parainfluenza viruses 1 to 4, respiratory syncytial virus (RSV), and RV/EV (no differentiation). The larger panel in the FilmArray RP assay allowed the detection of additional respiratory pathogens compared to the Prodesse assays. In this population of young children with upper respiratory tract infection, RV/EV accounted for the majority of the additional pathogens detected by FilmArray RP. PMID:21998418

Multiplexing detection of IgG against Plasmodium falciparum pregnancy-specific antigens

PubMed Central

Fonseca, Ana Maria; Quinto, Llorenç; Jiménez, Alfons; González, Raquel; Bardají, Azucena; Maculuve, Sonia; Dobaño, Carlota; Rupérez, Maria; Vala, Anifa; Aponte, John J.; Sevene, Esperanza; Macete, Eusebio; Menéndez, Clara

2017-01-01

Background Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens. Methods and findings We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ) and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87). IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106) than in Mozambican men (n = 102) and Spanish individuals (n = 101; p<0.05). Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003–2005 than during 2010–2012, in accordance with the levels of malaria transmission reported for these years in Mozambique. Conclusions The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among pregnant women and dependent on the level of exposure to malaria. This platform provides a high-throughput approach to investigating correlates of protection and identifying serological markers of exposure for malaria in pregnancy. PMID:28715465

Molecular signatures distinguishing active from latent tuberculosis in peripheral blood mononuclear cells, after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD) or Candida: a preliminary report.

PubMed

Stern, Joel N H; Keskin, Derin B; Romero, Viviana; Zuniga, Joaquin; Encinales, Liliana; Li, Changlin; Awad, Carlos; Yunis, Edmond J

2009-01-01

Purified protein derivative (PPD) or tuberculin skin testing is used to identify infected individuals with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immunity to Mtb. In the present study, we compared PBMC cultures in the presence of tuberculin or Candida antigens using cytokine bead arrays and RNA microarrays. Measurements of different cytokines and chemokines in supernatants of PMBC cultures in the presence of PPD showed increased levels of interferon (IFN)-gamma in active tuberculosis infection (ATBI) and latent TB infected (LTBI) compared to controls, and increased levels of TNF-alpha in ATBI compared with LTBI. Also, we found increase of IL-6 in cultures of PPD positive and controls but not in the cultures with Candida. We also report the molecular signature of tuberculosis infection, in ATBI patients, the following genes were found to be up-regulated and absent in LTBI individuals: two kinases (JAK3 and p38MAPK), four interleukins (IL-7, IL-2, IL-6, and IFNbeta1), a chemokine (HCC-4) a chemokine receptor (CxCR5), two interleukin receptors (IL-1R2 and IL-18R1), and three additional ones (TRAF5, Smad2, CIITA, and NOS2A). By contrast, IL-17 and IGFBP3 were significantly up-regulated in LTBI. And, STAT4, GATA3, Fra-1, and ICOS were down-regulated in ATBI but absent in LTBI. Conversely, TLR-10, IL-15, DORA, and IKK-beta were down-regulated in LTBI but not in ATBI. Interestingly, the majority of the up-regulated genes found in ATBI were found in cultures stimulated with tuberculin (PPD) or Candida antigens, suggesting that these pathogens stimulate similar immunological pathways. We believe that the molecular signature distinguishing active from latent tuberculosis infection may require using cytokine bead arrays along with RNA microarrays testing cell cultures at different times following in vitro proliferation assays using several bacterial antigens and PPD.

Encapsulated human hepatocellular carcinoma cells by alginate gel beads as an in vitro metastasis model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Xiao-xi; Liu, Chang; University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049

2013-08-15

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and often forms metastases, which are the most important prognostic factors. For further elucidation of the mechanism underlying the progression and metastasis of HCC, a culture system mimicking the in vivo tumor microenvironment is needed. In this study, we investigated the metastatic ability of HCC cells cultured within alginate gel (ALG) beads. In the culture system, HCC cells formed spheroids by proliferation and maintained in nuclear abnormalities. The gene and protein expression of metastasis-related molecules was increased in ALG beads, compared with the traditional adhesion culture. Furthermore, several gene expressionmore » levels in ALG bead culture system were even closer to liver cancer tissues. More importantly, in vitro invasion assay showed that the invasion cells derived from ALG beads was 7.8-fold higher than adhesion cells. Our results indicated that the in vitro three-dimensional (3D) model based on ALG beads increased metastatic ability compared with adhesion culture, even partly mimicked the in vivo tumor tissues. Moreover, due to the controllable preparation conditions, steady characteristics and production at large-scale, the 3D ALG bead model would become an important tool used in the high-throughput screening of anti-metastasis drugs and the metastatic mechanism research. -- Highlights: •We established a 3D metastasis model mimicking the metastatic ability in vivo. •The invasion ability of cells derived from our model was increased significantly. •The model is easy to reproduce, convenient to handle, and amenable for large-scale.« less

Simultaneous flow cytometric measurement of antigen attachment to phagocytes and phagocytosis.

PubMed

Laopajon, Witida; Takheaw, Nuchjira; Kasinrerk, Watchara; Pata, Supansa

2016-01-01

The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

PubMed

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

PubMed

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-08-17

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

PubMed Central

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-01-01

We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

A replaceable dual-enzyme capillary microreactor using magnetic beads and its application for simultaneous detection of acetaldehyde and pyruvate.

PubMed

Shi, Jing; Zhao, Wenwen; Chen, Yuanfang; Guo, Liping; Yang, Li

2012-07-01

A novel replaceable dual-enzyme capillary microreactor was developed and evaluated using magnetic fields to immobilize the alcohol dehydrogenase (ADH)- and lactate dehydrogenase (LDH)-coated magnetic beads at desired positions in the capillary. The dual-enzyme assay was achieved by measuring the two consumption peaks of the coenzyme β-nicotinamide adenine dinucleotide (NADH), which were related to the ADH reaction and LDH reaction. The dual-enzyme capillary microreactor was constructed using magnetic beads without any modification of the inner surface of the capillary, and showed great stability and reproducibility. The electrophoretic resolution for different analytes can be easily controlled by altering the relative distance of different enzyme-coated magnetic beads. The apparent K(m) values for acetaldehyde with ADH-catalyzed reaction and for pyruvate with LDH-catalyzed reaction were determined. The detection limits for acetaldehyde and pyruvate determination are 0.01 and 0.016 mM (S/N = 3), respectively. The proposed method was successfully applied to simultaneously determine the acetaldehyde and pyruvate contents in beer samples. The results indicated that combing magnetic beads with CE is of great value to perform replaceable and controllable multienzyme capillary microreactor for investigation of a series of enzyme reactions and determination of multisubstrates. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Developing a Salivary Antibody Multiplex Immunoassay to ...

EPA Pesticide Factsheets

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. The principal objective of this work is to develop an immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using the Luminex xMAP solution-phase assay. Beads were coupled to antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary detection antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen coupled and control beads were then incubated with prospectively-collected human saliva samples, analyzed on a Luminex 100 platform, and the presence

Design of a bovine low-density SNP array optimized for imputation

USDA-ARS?s Scientific Manuscript database

The Illumina BovineLD BeadChip was designed to support imputation to higher density genotypes in dairy and beef breeds by including single-nucleotide polymorphisms (SNPs) that had a high minor allele frequency as well as uniform spacing across the genome except at the ends of the chromosome where de...

Stationary microfluidics: molecular diagnostic assays by moving magnetic beads through non-moving liquids

NASA Astrophysics Data System (ADS)

Becker, Holger; Carstens, Cornelia; Kuhlmeier, Dirk; Sandetskaya, Natalia; Schröter, Nicole; Zilch, Christian; Gärtner, Claudia

2013-03-01

Commonly, microfluidic devices are based on the movement of fluids. For molecular diagnostics assays which often include steps like PCR, this practically always involves a more or less complicated set of external pumps, valves and liquid controls. In the presented paper, we follow a different approach in which the fluid after sample introduction remains stationary and the main bioactive sample molecules are moved through a chain of reaction compartments which contain the different reagents necessary for the assay. The big advantage of this concept is the lack of any external fluid actuation/control. Results on sample carry-over experiments and complete assays will be given.

Polymers mediate a one-pot route for functionalized quantum dot barcodes with a large encoding capacity.

PubMed

Zhang, Ding Sheng-Zi; Jiang, Yang; Wei, Dan; Wei, Xunbin; Xu, Hong; Gu, Hongchen

2018-06-21

With the increasing demands for high-throughput multiplexed bioassays, quantum dot (QD)-encoded microbeads as biocarriers for various bioreactions have attracted considerable attention. However, three key requirements for these biocarriers are still longstanding issues: a stable fluorescence intensity, a large encoding capacity and abundant surface functional groups. Here, a novel one-pot strategy is developed, generating functionalized QD-encoded microspheres with a strong fluorescence intensity and optical stability. With poly(styrene-co-maleic anhydride) (PSMA) molecules as mediators, the encapsulation of QDs and carboxylation of the bead surface are integrated together, greatly improving the preparation efficiency and guaranteeing their potential application in biodetection. Moreover, the mechanism for preparing QD-doped beads is further proposed, which helps to precisely manipulate the preparation process and accurately encode the beads. Through this approach, a single- and dual-color barcode library of QD-encoded microspheres has been successfully established, which demonstrates their great potential in suspension arrays.

Fabrication of a microfluidic device for studying the in situ drug-loading/release behavior of graphene oxide-encapsulated hydrogel beads.

PubMed

Veerla, Sarath Chandra; Kim, Da Reum; Yang, Sung Yun

2018-01-01

Controlled drug delivery system is highly important for not only prolonged the efficacy of drug but also cellular development for tissue engineering. A number of biopolymer composites and nanostructured carriers behave been used for the controlled drug delivery of therapeutics. Recently, in vitro microfluidic devices that mimic the human body have been developed for drug-delivery applications. A microfluidic channel was fabricated via a two-step process: (i) polydimethyl siloxane (PDMS) and curing agent were poured with a 10:2 mass ratio onto an acrylic mold with two steel pipes, and (ii) calcium alginate beads were synthesized using sodium alginate and calcium chloride solutions. Different amounts (10, 25, 50 μg) of graphene oxide (GO) were then added by Hummers method, and studies on the encapsulation and release of the model drug, risedronate (Ris), were performed using control hydrogel beads (pH 6.3), GO-containing beads (10GO, 25GO and 50GO), and different pH conditions. MC3T3 osteoblastic cells were cultured in a microchannel with Ris-loaded GO-hydrogel beads, and their proliferation, viability, attachment and spreading were assessed for a week. The spongy and textured morphology of pristine hydrogel beads was converted to flowery and rod-shaped structures in drug-loaded hydrogel beads at reduced pH (6.3) and at a lower concentration (10 μg) of GO. These latter 10GO drug-loaded beads rapidly released their cargo owing to the calcium phosphate deposited on the surface. Notably, beads containing a higher amount of GO (50GO) exhibited an extended drug-release profile. We further found that MC3T3 cells proliferated continuously in vitro in the microfluidic channel containing the GO-hydrogel system. MTT and live/dead assays showed similar proliferative potential of MC3T3 cells. Therefore, a microfluidic device with microchannels containing hydrogel beads formulated with different amounts of GO and tested under various pH conditions could be a promising system for controlled drug release. The GO and drug (risedronate, Rig) were directed loaded into a hydrogel placed in a microchannel. Through interactions such as hydrogen bonding between Go and the Rig-loaded GO-hydrogel beads, the bead-loaded microfluidic device supported MC3T3 proliferation and development as osteoblast without additional osteogenic differentiation supplements.

Report on the Infinium 450k methylation array analysis workshop: April 20, 2012 UCL, London, UK.

PubMed

Morris, Tiffany; Lowe, Robert

2012-08-01

A new platform for DNA methylome analysis is Illumina's Infinium HumanMethylation450. This technology is an extension of the previous HumanMethylation27 BeadChip and allows the methylation status of 12 samples per chip and 4 to 8 chips (total of 48 to 96 samples) to be assessed simultaneously for more than 480,000 cytosines across the genome. The platform incorporates two different probe types using different assay designs (InfiniumI and InfiniumII). Although this has allowed the assessment of more CpG sites, it has also introduced technical variation between the two probe types, which has complicated the analysis process. Many groups are working on normalization methods and analysis pipelines while many others are struggling to make sense of their new data sets. This motivated the organization of a meeting held at University College London that focused solely on the analysis methods and problems related to this new platform. The meeting was attended by 125 computational and bench scientists from 11 countries. There were 10 speakers, a small poster session and a discussion session.

On-chip Magnetic Separation and Cell Encapsulation in Droplets

NASA Astrophysics Data System (ADS)

Chen, A.; Byvank, T.; Bharde, A.; Miller, B. L.; Chalmers, J. J.; Sooryakumar, R.; Chang, W.-J.; Bashir, R.

2012-02-01

The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment could prevent cross-contamination, provide high recovery yield and ability to study biological traits at a single cell level These advantages of on-chip biological experiments contrast to conventional methods, which require bulk samples that provide only averaged information on cell metabolism. We report on a device that integrates microfluidic technology with a magnetic tweezers array to combine the functionality of separation and encapsulation of objects such as immunomagnetically labeled cells or magnetic beads into pico-liter droplets on the same chip. The ability to control the separation throughput that is independent of the hydrodynamic droplet generation rate allows the encapsulation efficiency to be optimized. The device can potentially be integrated with on-chip labeling and/or bio-detection to become a powerful single-cell analysis device.

Biomarkers in the Diagnosis and Prognosis of Alzheimer's Disease.

PubMed

Schaffer, Cole; Sarad, Nakia; DeCrumpe, Ashton; Goswami, Disha; Herrmann, Sara; Morales, Jose; Patel, Parth; Osborne, Jim

2015-10-01

Alzheimer's disease (AD) is a neurodegenerative disease that inhibits cognitive functions and has no cure. This report reviews the current diagnostic standards for AD with an emphasis on early diagnosis using the cerebrospinal fluid (CSF) biomarkers amyloid-beta, t-tau, and p-tau and fluorodeoxyglucose positron emission tomography imaging. Abnormal levels of these CSF biomarkers and decreased cerebral uptake of glucose have recently been used in the early diagnosis of AD in experimental studies. These promising biomarkers can be measured using immunoassays performed in singleplex or multiplex formats. Although presently, there are no Food and Drug Administration-approved in vitro diagnostics (IVDs) for early detection of AD, a multiplex immunoassay measuring a panel of promising AD biomarkers in CSF may be a likely IVD candidate for the clinical AD diagnostic market. Specifically, the INNO-BIA AlzBio3 immunoassay kit, performed using bead arrays on the xMAP Luminex analyzer, allows simultaneous quantification of amyloid-beta, t-tau, and p-tau biomarkers. AD biomarkers can also be screened using enzyme-linked immunosorbent assays that are offered as laboratory-developed tests. © 2014 Society for Laboratory Automation and Screening.

Removal of toxic metals from leachates from hazardous solid wastes and reduction of toxicity to microtox by the use of calcium alginate beads containing humic acid.

PubMed

Pandey, Ashok K; Pandey, Shri Dhar; Misra, Virendra

2002-06-01

Improper disposal of hazardous wastes can lead to release of potentially harmful substances through leaching such as heavy metals, which ultimately contaminate soil, sediment surface water, and groundwater through runoff. To remove these toxic metals and avoid any adverse effect on the ecosystem, a novel approach involving calcium alginate (CA) beads containing humic acid (HA) was used. For this, 10% leachates of the waste obtained from two major industrial units with electroplating processess were prepared at neutral pH and analyzed by atomic absorption spectrophotometry (AAS). Both leachates contained Cd, Cu, Cr, Ni, Mn, Fe, and Zn. The concentrations of Ni, Mn, Fe, and Zn in the waste were found to be significant. The leachates analyzed were passed through columns packed with calcium alginate beads with or without humic acid. The concentrations of various metals in beads and in different fractions collected after adsorption were measured. Data recorded indicate that calcium alginate beads containing humic acids are more efficient in removal of all metals in substantial amounts from the two leachates. Along with removal of metals, this process led to considerable detoxification of the leachates as tested by Microtox assay, indicated by earlier protection and higher EC(50). The significance of the results in relation to removal of toxic metals by beads containing humic acid is discussed. (c) 2002 Elsevier Science (USA).

Acetylcholinesterase immobilized capillary reactors coupled to protein coated magnetic beads: A new tool for plant extract ligand screening

PubMed Central

Vanzolini, Kenia Lourenço; Jiang, Zhengjin; Zhang, Xiaoqi; Vieira, Lucas Campos Curcino; Corrêa, Arlene Gonçalvez; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Moaddel, Ruin

2013-01-01

The use of immobilized capillary enzyme reactors (ICERs) and enzymes coated to magnetic beads ((NT or CT)-MB) for ligand screening has been adopted as a new technique of high throughput screening (HTS). In this work the selected target was the enzyme acetylcholinesterase (AChE), which acts on the central nervous system and is a validated target for the treatment of Alzheimer’s disease, as well as for new insecticides. A new approach for the screening of plant extracts was developed based on the ligand fishing experiments and zonal chromatography. For that, the magnetic beads were used for the ligand fishing experiments and capillary bioreactors for the activity assays. The latter was employed also under non-linear conditions to determine the affinity constants of known ligands, for the first time, as well as for the active fished ligand. PMID:24148457

Electrochemical Detection of E. coli O157:H7 in Water after Electrocatalytic and Ultraviolet Treatments Using a Polyguanine-Labeled Secondary Bead Sensor.

PubMed

Beeman, Michael G; Nze, Ugochukwu C; Sant, Himanshu J; Malik, Hammad; Mohanty, Swomitra; Gale, Bruce K; Carlson, Krista

2018-05-10

The availability of clean drinking water is a significant problem worldwide. Many technologies exist for purifying drinking water, however, many of these methods require chemicals or use simple methods, such as boiling and filtering, which may or may not be effective in removing waterborne pathogens. Present methods for detecting pathogens in point-of-use (POU) sterilized water are typically time prohibitive or have limited ability differentiating between active and inactive cells. This work describes a rapid electrochemical sensor to differentially detect the presence of active Escherichia coli (E. coli) O157:H7 in samples that have been partially or completely sterilized using a new POU electrocatalytic water purification technology based on superradicals generated by defect laden titania (TiO₂) nanotubes. The sensor was also used to detect pathogens sterilized by UV-C radiation for a comparison of different modes of cell death. The sensor utilizes immunomagnetic bead separation to isolate active bacteria by forming a sandwich assay comprised of antibody functionalized secondary magnetic beads, E. coli O157:H7, and polyguanine (polyG) oligonucleotide functionalized secondary polystyrene beads as an electrochemical tag. The assay is formed by the attachment of antibodies to active receptors on the membrane of E. coli , allowing the sensor to differentially detect viable cells. Ultravioloet (UV)-C radiation and an electrocatalytic reactor (ER) with integrated defect-laden titania nanotubes were used to examine the sensors’ performance in detecting sterilized cells under different modes of cell death. Plate counts and flow cytometry were used to quantify disinfection efficacy and cell damage. It was found that the ER treatments shredded the bacteria into multiple fragments, while UV-C treatments inactivated the bacteria but left the cell membrane mostly intact.

Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

NASA Astrophysics Data System (ADS)

Zhou, Huijuan; Wu, Baoyan

2008-12-01

The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

Ultrasensitive and rapid detection of β-conglutin combining aptamers and isothermal recombinase polymerase amplification.

PubMed

Jauset-Rubio, Miriam; Sabaté Del Río, Jonathan; Mairal, Teresa; Svobodová, Markéta; El-Shahawi, Mohammad S; Bashammakh, Abdulaziz S; Alyoubi, Abdulrahman O; O'Sullivan, Ciara K

2017-01-01

Lupin is increasingly being used in a variety of food products due to its nutritional, functional and nutraceutical properties. However, several examples of severe and even fatal food-associated anaphylaxis due to lupin inhalation or ingestion have been reported, resulting in the lupin subunit β-conglutin, being defined as the Lup an 1 allergen by the International Union of Immunological Societies (IUIS) in 2008. Here, we report an innovative method termed aptamer-recombinase polymerase amplification (Apta-RPA) exploiting the affinity and specificity of a DNA aptamer selected against the anaphylactic β-conglutin allergen termed β-conglutin binding aptamer II (β-CBA II), facilitating ultrasensitive detection via isothermal amplification. Combining magnetic beads as the solid phase with Apta-RPA detection, the total assay time was reduced from 210 min to just 25 min, with a limit of detection of 3.5 × 10 -11  M, demonstrating a rapid and ultrasensitive generic methodology that can be used with any aptamer. Future work will focus on further simplification of the assay to a lateral flow format. Graphical Abstract Schematic representation of the rapid and novel bead-based Apta-RPA assay.

Incorporating Aptamers in the Multiple Analyte Profiling Assays (xMAP): Detection of C-Reactive Protein.

PubMed

Bernard, Elyse D; Nguyen, Kathy C; DeRosa, Maria C; Tayabali, Azam F; Aranda-Rodriguez, Rocio

2017-01-01

Aptamers are short oligonucleotide sequences used in detection systems because of their high affinity binding to a variety of macromolecules. With the introduction of aptamers over 25 years ago came the exploration of their use in many different applications as a substitute for antibodies. Aptamers have several advantages; they are easy to synthesize, can bind to analytes for which it is difficult to obtain antibodies, and in some cases bind better than antibodies. As such, aptamer applications have significantly expanded as an adjunct to a variety of different immunoassay designs. The Multiple-Analyte Profiling (xMAP) technology developed by Luminex Corporation commonly uses antibodies for the detection of analytes in small sample volumes through the use of fluorescently coded microbeads. This technology permits the simultaneous detection of multiple analytes in each sample tested and hence could be applied in many research fields. Although little work has been performed adapting this technology for use with apatmers, optimizing aptamer-based xMAP assays would dramatically increase the versatility of analyte detection. We report herein on the development of an xMAP bead-based aptamer/antibody sandwich assay for a biomarker of inflammation (C-reactive protein or CRP). Protocols for the coupling of aptamers to xMAP beads, validation of coupling, and for an aptamer/antibody sandwich-type assay for CRP are detailed. The optimized conditions, protocols and findings described in this research could serve as a starting point for the development of new aptamer-based xMAP assays.

A novel pooled-sample multiplex luminex assay for high-throughput measurement of relative telomere length.

PubMed

Jasmine, Farzana; Shinkle, Justin; Sabarinathan, Mekala; Ahsan, Habibul; Pierce, Brandon L; Kibriya, Muhammad G

2018-03-12

Relative telomere length (RTL) is a potential biomarker of aging and risk for chronic disease. Previously, we developed a probe-based RTL assay on Luminex platform, where probes for Telomere (T) and reference gene (R) for a given DNA sample were tested in a single well. Here, we describe a method of pooling multiple samples in one well to increase the throughput and cost-effectiveness. We used four different microbeads for the same T-probe and four different microbeads for the same R-probe. Each pair of probe sets were hybridized to DNA in separate plates and then pooled in a single plate for all the subsequent steps. We used DNA samples from 60 independent individuals and repeated in multiple batches to test the precision. The precision was good to excellent with Intraclass correlation coefficient (ICC) of 0.908 (95% CI 0.856-0.942). More than 67% of the variation in the RTL could be explained by sample-to-sample variation; less than 0.1% variation was due to batch-to-batch variation and 0.3% variation was explained by bead-to-bead variation. We increased the throughput of RTL Luminex assay from 60 to 240 samples per run. The new assay was validated against the original Luminex assay without pooling (r = 0.79, P = 1.44 × 10 -15 ). In an independent set of samples (n = 550), the new assay showed a negative correlation of RTL with age (r = -0.41), a result providing external validation for the method. We describe a novel high throughput pooled-sample multiplex Luminex assay for RTL with good to excellent precision suitable for large-scale studies. © 2018 Wiley Periodicals, Inc.

Novel multiplex bead-based assay for detection of IDH1 and IDH2 mutations in myeloid malignancies.

PubMed

Shivarov, Velizar; Ivanova, Milena; Hadjiev, Evgueniy; Naumova, Elissaveta

2013-01-01

Isocitrate dehydrogenase 1 and 2 (IDH) mutations are frequently found in various cancer types such as gliomas, chondrosarcomas and myeloid malignancies. Their molecular detection has recently gained wide recognition in the diagnosis and prognosis of these neoplasms. For that purpose various molecular approaches have been used but a universally accepted method is still lacking. In this study we aimed to develop a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the most frequent IDH1 (p.R132C, p.R132G, p.R132H, p.R132L, p.R132S) and IDH2 (p.R140Q, p.R172K) mutations. The method includes four steps: 1) PCR amplification of the targeted fragments with biotinylated primers; 2) Direct hybridization to barcoded microbeads with specific LNA-modified oligonucleotide probes; 3) Incubation with phycoerythrin coupled streptavidin; 4) Acquisition of fluorescent intensities of each set of beads on a flow platform (LuminexCorp., USA). We tested the performance of the assay on both artificial plasmid constructs and on clinical samples from 114 patients with known or suspected myeloid malignancies. The method appeared to be superior to direct sequencing having a much higher sensitivity of 2.5% mutant alleles. Applying this method to patients' samples we identified a total of 9 mutations (one IDH1 p.R132C, seven IDH2 p.R140Q and one IDH2 p.R172K). In conclusion, this method could be successfully implemented in the diagnostic work-up for various tumors known to harbor IDH1/2 mutations (e.g. myeloid malignancies, gliomas, etc.). International initiatives are needed to validate the different existing methods for detection of IDH1/2 mutations in clinical settings.

Hydrodynamic metamaterials: Microfabricated arrays to steer, refract, and focus streams of biomaterials

PubMed Central

Morton, Keith J.; Loutherback, Kevin; Inglis, David W.; Tsui, Ophelia K.; Sturm, James C.; Chou, Stephen Y.; Austin, Robert H.

2008-01-01

We show that it is possible to direct particles entrained in a fluid along trajectories much like rays of light in classical optics. A microstructured, asymmetric post array forms the core hydrodynamic element and is used as a building block to construct microfluidic metamaterials and to demonstrate refractive, focusing, and dispersive pathways for flowing beads and cells. The core element is based on the concept of deterministic lateral displacement where particles choose different paths through the asymmetric array based on their size: Particles larger than a critical size are displaced laterally at each row by a post and move along the asymmetric axis at an angle to the flow, while smaller particles move along streamline paths. We create compound elements with complex particle handling modes by tiling this core element using multiple transformation operations; we show that particle trajectories can be bent at an interface between two elements and that particles can be focused into hydrodynamic jets by using a single inlet port. Although particles propagate through these elements in a way that strongly resembles light rays propagating through optical elements, there are unique differences in the paths of our particles as compared with photons. The unusual aspects of these modular, microfluidic metamaterials form a rich design toolkit for mixing, separating, and analyzing cells and functional beads on-chip. PMID:18495920

Two-dimensional tracking of ncd motility by back focal plane interferometry.

PubMed Central

Allersma, M W; Gittes, F; deCastro, M J; Stewart, R J; Schmidt, C F

1998-01-01

A technique for detecting the displacement of micron-sized optically trapped probes using far-field interference is introduced, theoretically explained, and used to study the motility of the ncd motor protein. Bead motions in the focal plane relative to the optical trap were detected by measuring laser intensity shifts in the back-focal plane of the microscope condenser by projection on a quadrant diode. This detection method is two-dimensional, largely independent of the position of the trap in the field of view and has approximately 10-micros time resolution. The high resolution makes it possible to apply spectral analysis to measure dynamic parameters such as local viscosity and attachment compliance. A simple quantitative theory for back-focal-plane detection was derived that shows that the laser intensity shifts are caused primarily by a far-field interference effect. The theory predicts the detector response to bead displacement, without adjustable parameters, with good accuracy. To demonstrate the potential of the method, the ATP-dependent motility of ncd, a kinesin-related motor protein, was observed with an in vitro bead assay. A fusion protein consisting of truncated ncd (amino acids 195-685) fused with glutathione-S-transferase was adsorbed to silica beads, and the axial and lateral motions of the beads along the microtubule surface were observed with high spatial and temporal resolution. The average axial velocity of the ncd-coated beads was 230 +/- 30 nm/s (average +/- SD). Spectral analysis of bead motion showed the increase in viscous drag near the surface; we also found that any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the nonhydrolyzable nucleotide adenylylimidodiphosphate. PMID:9533719

Measuring binding of protein to gel-bound ligands using magnetic levitation.

PubMed

Shapiro, Nathan D; Mirica, Katherine A; Soh, Siowling; Phillips, Scott T; Taran, Olga; Mace, Charles R; Shevkoplyas, Sergey S; Whitesides, George M

2012-03-28

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.

Measuring Binding of Protein to Gel-Bound Ligands Using Magnetic Levitation

PubMed Central

Shapiro, Nathan D.; Mirica, Katherine A.; Soh, Siowling; Phillips, Scott T.; Taran, Olga; Mace, Charles R.; Shevkoplyas, Sergey S.; Whitesides, George M.

2012-01-01

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (> 70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems. PMID:22364170

Magnetic beads (Dynabead) toxicity to endothelial cells at high bead concentration: implication for tissue engineering of vascular prosthesis.

PubMed

Tiwari, A; Punshon, G; Kidane, A; Hamilton, G; Seifalian, A M

2003-10-01

Magnetic beads (Dynabeads) have been used for the purification of endothelial cells. One application for this procedure may be for single-stage seeding of bypass grafts. The number of endothelial cells (EC) isolated is crucial and therefore to increase the number of cells extracted, a higher number of Dynabeads per cell may need to be used. The effect of large numbers of CD31 Dynabeads on cell proliferation/metabolism is unknown. We undertook this study using CD31-coated Dynabeads and EC from human umbilical vein. EC were coated at concentrations of 4, 10, or 50 beads per cell. The cells were cultured for 6 days with control being normal EC. Cellular proliferation was assessed by trypsinization of cells and metabolism assessed with an Alamar blue viability assay. In a further experiment a compliant polyurethane graft was single-stage seeded with both coated Dynabeads and normal EC. The results showed that using a higher number of beads per cell resulted in a reduction in cell proliferation and a reduction in cell metabolism. The total number of Dynabeads-coated cells in culture compared to controls (%) by day 6 were 30.7 +/- 2.56, 41.3 +/- 9.8 and 59.2 +/- 7.3 for 50, 10, and 4 beads per cell, respectively. The corresponding results for Alamar blue were 43.7 +/- 1.2, 61.8 +/- 1.4, and 72.1 +/- 4.3. The seeded grafts showed reduced metabolism with the Dynabeads-coated EC. In conclusion, high numbers of beads per cell have a late detrimental effect on cell proliferation and metabolism. Therefore for single-stage seeding lower numbers of Dynabeads will need to be used with resultant reduction in the number of available EC.

Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor-α

NASA Astrophysics Data System (ADS)

Rush, Michael D.; Walker, Elisabeth M.; Prehna, Gerd; Burton, Tristesse; van Breemen, Richard B.

2017-03-01

To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts.

Bubble-driven mixer integrated with a microfluidic bead-based ELISA for rapid bladder cancer biomarker detection.

PubMed

Lin, Yen-Heng; Wang, Chia-Chu; Lei, Kin Fong

2014-04-01

In this study, fine bubbles were successfully generated and used as a simple, low-cost driving force for mixing fluids in an integrated microfluidic bead-based enzyme-linked immunosorbent assay (ELISA) to rapidly and quantitatively detect apolipoprotein A1 (APOA1), a biomarker highly correlated with bladder cancer. A wooden gas diffuser was embedded underneath a microfluidic chip to refine injected air and generate bubbles of less than 0.3 mm. The rising bubbles caused disturbances and convection in the fluid, increasing the probability of analyte interaction. This setup not only simplifies the micromixer design but also achieves rapid mixing with a small airflow as a force. We used this bubble-driven micromixer in a bead-based ELISA that targeted APOA1. The results indicate that this micromixer reduced the time for each incubation from 60 min in the conventional assay to 8 min with the chip, resulting in a reduction of total ELISA reaction time from 3-4 h to 30-40 min. Furthermore, the concentration detection limit was 9.16 ng/mL, which was lower than the detection cut-off value (11.16 ng/mL) for bladder cancer diagnosis reported in the literature. Therefore, this chip can be used to achieve rapid low-cost bladder cancer detection and may be used in point-of-care cancer monitoring.

Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins

DTIC Science & Technology

2016-04-01

streptavidin-phycoerythrin (PE) similar to sandwich enzyme-linked immunosorbent assays ( ELISAs ). The 3 fluorescent markers (2 beads plus PE) allow for...within the kit, this worked out to a set of expensive, problematic, and subjective ELISA . The space on the black-96 well plate was split between cell...ARL US Army Research Laboratory BBB blood–brain barrier CSF cerebral spinal fluid DOD US Department of Defense ELISA enzyme-linked immunosorbent

Identification of biological agents using surface enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Paxon, Tracy L.; Duthie, R. Scott; Renko, Casey; Burns, Andrew A.; Lesaicherre, Marie L.; Mondello, Frank J.

2011-05-01

GE Global Research Center, in collaboration with Morpho Detection, Inc. has developed an assay scheme for the identification of biological agents using Surface Enhanced Raman Scattering (SERS). Specifically, unique spectroscopic signatures are generated using SERS tags consisting of individual glass-encapsulated gold nanoparticles and surfacebound reporter molecules. These SERS tags are modified with a capture moiety specific to the antigen of interest, and serve as a spectroscopic label in a bead-based sandwich assay. Assays are being developed for a variety of pathogens and this paper will focus on aspects of assay development, optimization, stabilization and validation. Results on the development of an assay to detect Ricin toxin will be presented, and preliminary feasibility studies for the detection of additional pathogens will be discussed.

Quantification of DNA using the luminescent oxygen channeling assay.

PubMed

Patel, R; Pollner, R; de Keczer, S; Pease, J; Pirio, M; DeChene, N; Dafforn, A; Rose, S

2000-09-01

Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics. Luminescent oxygen channeling assay (LOCI(TM)) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types. In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets. This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification.

PubMed

Guan, Weihua; Chen, Liben; Rane, Tushar D; Wang, Tza-Huei

2015-09-03

We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

PubMed Central

Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

2015-01-01

We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples. PMID:26333806

Use of Bead-Based Serologic Assay to Evaluate Chikungunya Virus Epidemic, Haiti.

PubMed

Rogier, Eric W; Moss, Delynn M; Mace, Kimberly E; Chang, Michelle; Jean, Samuel E; Bullard, Stevan M; Lammie, Patrick J; Lemoine, Jean Frantz; Udhayakumar, Venkatachalam

2018-06-01

The index case of chikungunya virus (CHIKV) in Haiti was reported during early 2014; the vector, the pervasive Aedes aegypti mosquito, promoted rapid spread throughout the country. During December 2014-February 2015, we collected blood samples from 4,438 persons at 154 sites (62 urban, 92 rural) throughout Haiti and measured CHIKV IgG by using a multiplex bead assay. Overall CHIKV seroprevalence was 57.9%; differences between rural (mean 44.9%) and urban (mean 78.4%) areas were pronounced. Logistic modeling identified the urban environment as a strong predictor of CHIKV exposure (adjusted odds ratio 3.34, 95% CI 2.38-4.69), and geographic elevation provided a strong negative correlation. We observed no correlation between age and antibody positivity or titer. Our findings demonstrated through serologic testing the recent and rapid dissemination of the arbovirus throughout the country. These results show the utility of serologic data to conduct epidemiologic studies of quickly spreading mosquitoborne arboviruses.

Reconfigurable acquisition system with integrated optics for a portable flow cytometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirleis, Matthew A., E-mail: matthew.kirleis@nrl.navy.mil; Mathews, Scott A.; Verbarg, Jasenka

2013-11-15

Portable and inexpensive scientific instruments that are capable of performing point of care diagnostics are needed for applications such as disease detection and diagnosis in resource-poor settings, for water quality and food supply monitoring, and for biosurveillance activities in autonomous vehicles. In this paper, we describe the development of a compact flow cytometer built from three separate, customizable, and interchangeable modules. The instrument as configured in this work is being developed specifically for the detection of selected Centers for Disease Control (CDC) category B biothreat agents through a bead-based assay: E. coli O157:H7, Salmonella, Listeria, and Shigella. It has two-colormore » excitation, three-color fluorescence and light scattering detection, embedded electronics, and capillary based flow. However, these attributes can be easily modified for other applications such as cluster of differentiation 4 (CD4) counting. Proof of concept is demonstrated through a 6-plex bead assay with the results compared to a commercially available benchtop-sized instrument.« less

Reconfigurable acquisition system with integrated optics for a portable flow cytometer.

PubMed

Kirleis, Matthew A; Mathews, Scott A; Verbarg, Jasenka; Erickson, Jeffrey S; Piqué, Alberto

2013-11-01

Portable and inexpensive scientific instruments that are capable of performing point of care diagnostics are needed for applications such as disease detection and diagnosis in resource-poor settings, for water quality and food supply monitoring, and for biosurveillance activities in autonomous vehicles. In this paper, we describe the development of a compact flow cytometer built from three separate, customizable, and interchangeable modules. The instrument as configured in this work is being developed specifically for the detection of selected Centers for Disease Control (CDC) category B biothreat agents through a bead-based assay: E. coli O157:H7, Salmonella, Listeria, and Shigella. It has two-color excitation, three-color fluorescence and light scattering detection, embedded electronics, and capillary based flow. However, these attributes can be easily modified for other applications such as cluster of differentiation 4 (CD4) counting. Proof of concept is demonstrated through a 6-plex bead assay with the results compared to a commercially available benchtop-sized instrument.

Reconfigurable acquisition system with integrated optics for a portable flow cytometer

NASA Astrophysics Data System (ADS)

Kirleis, Matthew A.; Mathews, Scott A.; Verbarg, Jasenka; Erickson, Jeffrey S.; Piqué, Alberto

2013-11-01

Portable and inexpensive scientific instruments that are capable of performing point of care diagnostics are needed for applications such as disease detection and diagnosis in resource-poor settings, for water quality and food supply monitoring, and for biosurveillance activities in autonomous vehicles. In this paper, we describe the development of a compact flow cytometer built from three separate, customizable, and interchangeable modules. The instrument as configured in this work is being developed specifically for the detection of selected Centers for Disease Control (CDC) category B biothreat agents through a bead-based assay: E. coli O157:H7, Salmonella, Listeria, and Shigella. It has two-color excitation, three-color fluorescence and light scattering detection, embedded electronics, and capillary based flow. However, these attributes can be easily modified for other applications such as cluster of differentiation 4 (CD4) counting. Proof of concept is demonstrated through a 6-plex bead assay with the results compared to a commercially available benchtop-sized instrument.

Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

NASA Astrophysics Data System (ADS)

Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

2015-09-01

We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

Utility of HLA Antibody Testing in Kidney Transplantation

PubMed Central

Konvalinka, Ana

2015-01-01

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase.

PubMed

Brodelius, P; Nilsson, K; Mosbach, K

1981-12-01

Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca(2+)-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a " trickle-bed " reactor.

Heterodyne holographic microscopy of gold particles.

PubMed

Atlan, Michael; Gross, Michel; Desbiolles, Pierre; Absil, Emilie; Tessier, Gilles; Coppey-Moisan, Maïté

2008-03-01

We report experimental results on heterodyne holographic microscopy of subwavelength-size gold particles. The apparatus uses continuous green-laser illumination of the metal beads in a total internal reflection configuration for dark-field operation. Detection of the scattered light at the illumination wavelength on a charge-coupled-device array detector enables 3D localization of brownian particles in water.

DNA methylome profiling of maternal peripheral blood and placentas reveal potential fetal DNA markers for non-invasive prenatal testing.

PubMed

Xiang, Yuqian; Zhang, Junyu; Li, Qiaoli; Zhou, Xinyao; Wang, Teng; Xu, Mingqing; Xia, Shihui; Xing, Qinghe; Wang, Lei; He, Lin; Zhao, Xinzhi

2014-09-01

Utilizing epigenetic (DNA methylation) differences to differentiate between maternal peripheral blood (PBL) and fetal (placental) DNA has been a promising strategy for non-invasive prenatal testing (NIPT). However, the differentially methylated regions (DMRs) have yet to be fully ascertained. In the present study, we performed genome-wide comparative methylome analysis between maternal PBL and placental DNA from pregnancies of first trimester by methylated DNA immunoprecipitation-sequencing (MeDIP-Seq) and Infinium HumanMethylation450 BeadChip assays. A total of 36 931 DMRs and 45 804 differentially methylated sites (DMSs) covering the whole genome, exclusive of the Y chromosome, were identified via MeDIP-Seq and Infinium 450k array, respectively, of which 3759 sites in 2188 regions were confirmed by both methods. Not only did we find the previously reported potential fetal DNA markers in our identified DMRs/DMSs but also we verified fully the identified DMRs/DMSs in the validation round by MassARRAY EpiTYPER. The screened potential fetal DNA markers may be used for NIPT on aneuploidies and other chromosomal diseases, such as cri du chat syndrome and velo-cardio-facial syndrome. In addition, these potential markers may have application in the early diagnosis of placental dysfunction, such as pre-eclampsia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Two decades of warming increases diversity of a potentially lignolytic bacterial community

PubMed Central

Pold, Grace; Melillo, Jerry M.; DeAngelis, Kristen M.

2015-01-01

As Earth's climate warms, the massive stores of carbon found in soil are predicted to become depleted, and leave behind a smaller carbon pool that is less accessible to microbes. At a long-term forest soil-warming experiment in central Massachusetts, soil respiration and bacterial diversity have increased, while fungal biomass and microbially-accessible soil carbon have decreased. Here, we evaluate how warming has affected the microbial community's capability to degrade chemically-complex soil carbon using lignin-amended BioSep beads. We profiled the bacterial and fungal communities using PCR-based methods and completed extracellular enzyme assays as a proxy for potential community function. We found that lignin-amended beads selected for a distinct community containing bacterial taxa closely related to known lignin degraders, as well as members of many genera not previously noted as capable of degrading lignin. Warming tended to drive bacterial community structure more strongly in the lignin beads, while the effect on the fungal community was limited to unamended beads. Of those bacterial operational taxonomic units (OTUs) enriched by the warming treatment, many were enriched uniquely on lignin-amended beads. These taxa may be contributing to enhanced soil respiration under warming despite reduced readily available C availability. In aggregate, these results suggest that there is genetic potential for chemically complex soil carbon degradation that may lead to extended elevated soil respiration with long-term warming. PMID:26042112

Co-delivery of cisplatin and doxorubicin from calcium phosphate beads/matrix scaffolds for osteosarcoma therapy.

PubMed

Hess, Ulrike; Shahabi, Shakiba; Treccani, Laura; Streckbein, Philipp; Heiss, Christian; Rezwan, Kurosch

2017-08-01

Bone substitute materials with a controlled drug release ability can fill cavities caused by the resection of bone tumours and thereby combat any leftover bone cancer cells. The combined release of different cytostatics seems to enhance their toxicity. In this study, calcium phosphate beads and matrix scaffolds are combined for a long-term co-delivery of cis-diamminedichloroplatinum (cisplatin, CDDP) and doxorubicin hydrochloride (DOX) as clinical relevant model drugs. Tricalcium phosphate/alginate beads as additional drug carrier are produced by droplet extrusion with ionotropic gelation and incorporated in scaffold matrix by freeze gelation without sintering. CDDP shows a short burst release while DOX has a continuous release measurable over the entire study period of 40days. Drug release from matrix is decreased by ~30% compared to release from beads. Nevertheless, all formulations follow the Korsmeyer-Peppas release kinetic model and show Fickian diffusion. Cytotoxic activity was conducted on MG-63 osteosarcoma cells after 1, 4, and 7days with WST-1 cell viability assay. Co-loaded composites enhance activity towards MG-63 cells up to ~75% toxicity while reducing the released drug quantity. The results suggest that co-loaded beads/matrix scaffolds are highly promising for osteosarcoma therapy due to synergistic effects over a long period of more than a month. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection.

PubMed

Martel, Ralph R; Botros, Ihab W; Rounseville, Matthew P; Hinton, James P; Staples, Robin R; Morales, David A; Farmer, John B; Seligmann, Bruce E

2002-11-01

The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.

Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

NASA Astrophysics Data System (ADS)

Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

2017-02-01

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

Biocide-mediated corrosion of coiled tubing.

PubMed

Sharma, Mohita; An, Dongshan; Liu, Tao; Pinnock, Tijan; Cheng, Frank; Voordouw, Gerrit

2017-01-01

Coiled tubing corrosion was investigated for 16 field water samples (S5 to S20) from a Canadian shale gas field. Weight loss corrosion rates of carbon steel beads incubated with these field water samples averaged 0.2 mm/yr, but injection water sample S19 had 1.25±0.07 mm/yr. S19 had a most probable number of zero acid-producing bacteria and incubation of S19 with carbon steel beads or coupons did not lead to big changes in microbial community composition. In contrast other field water samples had most probable numbers of APB of 102/mL to 107/mL and incubation of these field water samples with carbon steel beads or coupons often gave large changes in microbial community composition. HPLC analysis indicated that all field water samples had elevated concentrations of bromide (average 1.6 mM), which may be derived from bronopol, which was used as a biocide. S19 had the highest bromide concentration (4.2 mM) and was the only water sample with a high concentration of active bronopol (13.8 mM, 2760 ppm). Corrosion rates increased linearly with bronopol concentration, as determined by weight loss of carbon steel beads, for experiments with S19, with filtered S19 and with bronopol dissolved in defined medium. This indicated that the high corrosion rate found for S19 was due to its high bronopol concentration. The corrosion rate of coiled tubing coupons also increased linearly with bronopol concentration as determined by electrochemical methods. Profilometry measurements also showed formation of multiple pits on the surface of coiled tubing coupon with an average pit depth of 60 μm after 1 week of incubation with 1 mM bronopol. At the recommended dosage of 100 ppm the corrosiveness of bronopol towards carbon steel beads was modest (0.011 mm/yr). Higher concentrations, resulting if biocide is added repeatedly as commonly done in shale gas operations, are more corrosive and should be avoided. Overdosing may be avoided by assaying the presence of residual biocide by HPLC, rather than by assaying the presence of residual surviving bacteria.

Biocide-mediated corrosion of coiled tubing

PubMed Central

An, Dongshan; Liu, Tao; Pinnock, Tijan; Cheng, Frank; Voordouw, Gerrit

2017-01-01

Coiled tubing corrosion was investigated for 16 field water samples (S5 to S20) from a Canadian shale gas field. Weight loss corrosion rates of carbon steel beads incubated with these field water samples averaged 0.2 mm/yr, but injection water sample S19 had 1.25±0.07 mm/yr. S19 had a most probable number of zero acid-producing bacteria and incubation of S19 with carbon steel beads or coupons did not lead to big changes in microbial community composition. In contrast other field water samples had most probable numbers of APB of 102/mL to 107/mL and incubation of these field water samples with carbon steel beads or coupons often gave large changes in microbial community composition. HPLC analysis indicated that all field water samples had elevated concentrations of bromide (average 1.6 mM), which may be derived from bronopol, which was used as a biocide. S19 had the highest bromide concentration (4.2 mM) and was the only water sample with a high concentration of active bronopol (13.8 mM, 2760 ppm). Corrosion rates increased linearly with bronopol concentration, as determined by weight loss of carbon steel beads, for experiments with S19, with filtered S19 and with bronopol dissolved in defined medium. This indicated that the high corrosion rate found for S19 was due to its high bronopol concentration. The corrosion rate of coiled tubing coupons also increased linearly with bronopol concentration as determined by electrochemical methods. Profilometry measurements also showed formation of multiple pits on the surface of coiled tubing coupon with an average pit depth of 60 μm after 1 week of incubation with 1 mM bronopol. At the recommended dosage of 100 ppm the corrosiveness of bronopol towards carbon steel beads was modest (0.011 mm/yr). Higher concentrations, resulting if biocide is added repeatedly as commonly done in shale gas operations, are more corrosive and should be avoided. Overdosing may be avoided by assaying the presence of residual biocide by HPLC, rather than by assaying the presence of residual surviving bacteria. PMID:28746397

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

PubMed Central

2012-01-01

Background Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Methods Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Results Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. Conclusion With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA. PMID:23259607

Deficiency of IL-18 Aggravates Esophageal Carcinoma Through Inhibiting IFN-γ Production by CD8+T Cells and NK Cells.

PubMed

Li, Jiantao; Qiu, Gang; Fang, Baoshuan; Dai, Xiaohui; Cai, Jianhui

2018-03-01

To investigate the potential role of interleukin-18 (IL-18) in immunomodulation during tumorigenesis of esophageal carcinoma and elucidate the underlying molecular mechanism, we employed IL-18 knockout mice for this purpose. Carcinogen 4-nitroquinoline 1-oxide (4NQO) was administrated in drinking water to induce occurrence of esophageal squamous cell carcinoma (ESCC). T cell activation as indicated by the surface CD molecules was analyzed with flow cytometry. The serous content of interferon-γ (IFN-γ) along with other cytokines was determined by inflammatory human cytokine cytometric bead array. The cytotoxicity assay was performed by co-culture of tumor cells with immune cells and relative cell viability was determined by lactate dehydrogenase (LDH) assay. Apoptotic cells were stained with Annexin-V/propidium iodide (PI) and analyzed by flow cytometry. Cell proliferation was measured with Cell Counting Kit-8 (CCK-8) assay. Our data demonstrated that deficiency of IL-18 promoted the progression and development of 4NQO-induced ESCC. Loss of IL-18 suppressed the activation of T cells in the esophagus. Deficiency of IL-18 inhibited the IFN-γ production by CD8 + T cells and natural killer (NK) cells. Absence of IL-18 inhibited the cytotoxicity of CD8 + T cells and NK cell in vitro. Moreover, deficiency of IL-18 promoted the apoptosis of CD8 + T cells and inhibited the proliferation of CD8 + T cells in vitro. Our data elucidated the immunomodulatory role of IL-18 during tumorigenesis of ESCC, whose deficiency compromised antitumor immunity and contributed to immune escape of esophageal carcinoma. Our results also indicated the therapeutic potential of exogenous IL-18 against ESCC, which warrants further investigations.

BRSCW Reference Set Application: Karen Abbott -University of Arkansas (2014) — EDRN Public Portal

Cancer.gov

Our earlier glycoproteomic studies have identified bisecting glycoslyation and core fucosylation changes on particular glycoproteins in endometrioid ovarian cancer tissues and plasma (Abbott et al, 2010, Proteomics). We have validated that these glycan changes occur on the same glycoproteins in serous ovarian cancer plasma using a lectin-pull down western blot assays. We would like to used pooled reference samples to develop a sensitive magnetic bead-based assay to detect these glycoproteins with bisecting and core fucosylation changes.

Targeting the UPR to Circumvent Endocrine Resistance in Breast Cancer

DTIC Science & Technology

2015-10-01

were submitted for the screen. Kinase assay protocol (DiscoverX): For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well...blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of...unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test

Functional Characterization of CENP-A Post-Translational Modifications in Chromosome Segregation

DTIC Science & Technology

2014-07-01

methods: In vitro methylation assays: 6XHis tagged Human NRMT1 (Gift from Ian Maccara) was purified from E - coli and used for the methylation assays...expression vector in BL21 E . coli and purified on Ni-NTA beads and then cleaved using Factor X (Sigma-Aldrich). The cleaved proteins were then negatively...AUTHOR(S) 5d. PROJECT NUMBER Sathyan Kizhakke Mattada 5e. TASK NUMBER E -Mail: sk8fz@virginia.edu 5f. WORK UNIT NUMBER 7. PERFORMING

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance ▿ †

PubMed Central

Jang, Hyunjung; Kim, Jihyun; Choi, Jae-jin; Son, Yeojin; Park, Heekyung

2010-01-01

The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 102 copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 104 copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms. PMID:20573874

Demonstration of four immunoassay formats using the array biosensor

NASA Technical Reports Server (NTRS)

Sapsford, Kim E.; Charles, Paul T.; Patterson, Charles H Jr; Ligler, Frances S.

2002-01-01

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

Miniaturization of High-Throughput Epigenetic Methyltransferase Assays with Acoustic Liquid Handling.

PubMed

Edwards, Bonnie; Lesnick, John; Wang, Jing; Tang, Nga; Peters, Carl

2016-02-01

Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter volumes to high-density assay formats using only acoustic energy-no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio. © 2015 Society for Laboratory Automation and Screening.

High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis.

PubMed

Lee, Hwanyong; Jung, Jinhee; Han, Song-I; Han, Ki-Ho

2010-10-21

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from peripheral blood lysate using magnetic oligo-dT beads. The extraction is achieved through lateral magnetophoresis, generated by a ferromagnetic wire array inlaid on a glass substrate. This RNA microextractor separated more than 80% of magnetic beads with a flow rate up to 20 ml h(-1), and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein (A(260)/A(280)) was >1.7, indicating that the extraction technology yielded nearly pure RNA. The feasibility of this technique was evaluated further for its applicability to reverse transcription polymerase chain reaction (RT-PCR) procedures by performing cDNA synthesis and PCR. The analysis verified that the RNA microextractor is a practical method for easy, rapid, and high-precision RT-PCR using minimal reagent volumes without requiring highly trained personnel. In addition, it can be readily incorporated into genetic analysis procedures for realizing automated on-chip genetic platforms in a micro format.

Increased apoptosis and peripheral blood mononuclear cell suppression of bone marrow mesenchymal stem cells in severe aplastic anemia.

PubMed

Chao, Yu-Hua; Lin, Chiao-Wen; Pan, Hui-Hsien; Yang, Shun-Fa; Weng, Te-Fu; Peng, Ching-Tien; Wu, Kang-Hsi

2018-06-05

Although immune-mediated pathogenesis is considered an important aspect of severe aplastic anemia (SAA), its underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs) are essential to the formation of specialized microenvironments in the bone marrow (BM), and MSC insufficiency can trigger the development of SAA. To find MSC alterations in the SAA BM, we compared BM MSCs from five children with SAA and five controls. Peripheral blood mononuclear cells (PBMCs) were cocultured with MSCs to evaluate the supportive effects of MSCs on hematopoiesis. Cytometric bead array immunoassay was used to determine cytokine excretion by MSCs. The immune functions of MSCs and their conditioned medium (CM) were evaluated by PBMC proliferation assays. SAA MSCs were characterized by a high percentage of cells in the abnormal sub-G1 phase of the cell cycle, which suggests an increased rate of apoptosis in SAA MSCs. In comparison with control MSCs, PBMCs cocultured with SAA MSCs displayed significantly reduced PBMC proliferation (P = 0.009). Aberrant cytokine profiles were secreted by SAA MSCs, with increased concentrations of interleukin-6, interferon-γ, tumor necrosis factor-α, and interleukin-1β in the CM. PBMC proliferation assays demonstrated additional immunosuppressive effects of SAA MSCs (P = 0.016) and their CM (P = 0.013). Our data revealed increased apoptosis and PBMC suppression of SAA MSCs. The alterations of MSCs may contribute to the formation of functionally abnormal microenvironments in SAA BM. © 2018 Wiley Periodicals, Inc.

Optimal use of tandem biotin and V5 tags in ChIP assays

PubMed Central

Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

2009-01-01

Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. PMID:19196479

On-chip Magnetic Separation and Cell Encapsulation in Droplets†

PubMed Central

Chen, Aaron; Byvank, Tom; Chang, Woo-Jin; Bharde, Atul; Vieira, Greg; Miller, Brandon; Chalmers, Jeffrey J.; Bashir, Rashid; Sooryakumar, Ratnasingham

2014-01-01

The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment would prevent cross-contamination, provide high recovery yield, and enable study of biological traits at a single cell level. These advantages of on-chip biological experiments is a significant improvement for myriad of cell analyses over conventional methods, which require bulk samples providing only averaged information on cell metabolism. We report on a device that integrates mobile magnetic trap array with microfluidic technology to provide, combined functionality of separation of immunomagnetically labeled cells or magnetic beads and their encapsulation with reagents into pico-liter droplets. This scheme of simultaneous reagent delivery and compartmentalization of the cells immediately after sorting, all performed seamlessly within the same chip, offers unique advantages such as the ability to capture cell traits as originated from its native environment, reduced chance of contamination, minimal use and freshness of the reagent solution that reacts only with separated objects, and tunable encapsulation characteristics independent of the input flow. In addition to the demonstrated preliminary cell viability assay, the device can potentially be integrated with other up- or downstream on-chip modules to become a powerful single-cell analysis tool. PMID:23370785

Simultaneous capture and sequential detection of two malarial biomarkers on magnetic microparticles.

PubMed

Markwalter, Christine F; Ricks, Keersten M; Bitting, Anna L; Mudenda, Lwiindi; Wright, David W

2016-12-01

We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5-8h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Comparison of the Roche Cobas(®) 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia.

PubMed

Phillips, Samuel; Garland, Suzanne M; Tan, Jeffery H; Quinn, Michael A; Tabrizi, Sepehr N

2015-01-01

The recently FDA (U.S. food and drug administration) approved Roche Cobas(®) 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions. Evaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia. A selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array. Overall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array. The Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia. Copyright © 2014 Elsevier B.V. All rights reserved.

Reduced signal crosstalk multi neurotransmitter image sensor by microhole array structure

NASA Astrophysics Data System (ADS)

Ogaeri, Yuta; Lee, You-Na; Mitsudome, Masato; Iwata, Tatsuya; Takahashi, Kazuhiro; Sawada, Kazuaki

2018-06-01

A microhole array structure combined with an enzyme immobilization method using magnetic beads can enhance the target discernment capability of a multi neurotransmitter image sensor. Here we report the fabrication and evaluation of the H+-diffusion-preventing capability of the sensor with the array structure. The structure with an SU-8 photoresist has holes with a size of 24.5 × 31.6 µm2. Sensors were prepared with the array structure of three different heights: 0, 15, and 60 µm. When the sensor has the structure of 60 µm height, 48% reduced output voltage is measured at a H+-sensitive null pixel that is located 75 µm from the acetylcholinesterase (AChE)-immobilized pixel, which is the starting point of H+ diffusion. The suppressed H+ immigration is shown in a two-dimensional (2D) image in real time. The sensor parameters, such as height of the array structure and measuring time, are optimized experimentally. The sensor is expected to effectively distinguish various neurotransmitters in biological samples.

Peamaclein--a new peach allergenic protein: similarities, differences and misleading features compared to Pru p 3.

PubMed

Tuppo, L; Alessandri, C; Pomponi, D; Picone, D; Tamburrini, M; Ferrara, R; Petriccione, M; Mangone, I; Palazzo, P; Liso, M; Giangrieco, I; Crescenzo, R; Bernardi, M L; Zennaro, D; Helmer-Citterich, M; Mari, A; Ciardiello, M A

2013-01-01

Among the peach-derived allergens which are already known, the lipid transfer protein (Pru p 3) seems to be the one to exert severe allergic reactions. To identify and characterize a new peach allergen causing a clinical picture similar to that of Pru p 3. Patients were selected on the basis of their severe clinical reactivity and negative results to a panel of peach allergens available on the ISAC103 microarray. Several in-house and commercial preparations were compared. Several methods were used to characterize the newly identified molecule. Specific IgE and inhibition assays were performed using the Allergen micro-Beads Array (ABA) assay. Negative ISAC results to Pru p 3 were confirmed by additional testing in contrast with the positive results obtained by commercial Pru p 3-enriched peach peel extracts. The analyses of one of these preparations led to the identification of Peamaclein, a new allergenic protein. It is a small, basic, cysteine-rich, heat-stable, digestion-resistant protein, homologous to a potato antimicrobial peptide. Peamaclein was able to trigger positive skin test reactions and to bind IgE in the ABA assay. It displays an electrophoretic mobility and chromatographic behaviour similar to that of Pru p 3; therefore, it can be hidden in Pru p 3 preparations. In fact, Pru p 3-enriched peach peel extracts were found to contain both Pru p 3 and Peamaclein by means of comparative in vivo testing, and by biochemical and immunochemical assays. Commercially available anti-Pru p 3 polyclonal antibodies were found to have a double specificity for the two molecules. A new allergen from peach belonging to a new family of allergenic proteins has been identified and characterized. This knowledge on Peamaclein will improve our understanding on the clinical aspects of the peach allergy and the quality of diagnostic reagents. © 2012 Blackwell Publishing Ltd.

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

PubMed

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

ChAMP: updated methylation analysis pipeline for Illumina BeadChips.

PubMed

Tian, Yuan; Morris, Tiffany J; Webster, Amy P; Yang, Zhen; Beck, Stephan; Feber, Andrew; Teschendorff, Andrew E

2017-12-15

The Illumina Infinium HumanMethylationEPIC BeadChip is the new platform for high-throughput DNA methylation analysis, effectively doubling the coverage compared to the older 450 K array. Here we present a significantly updated and improved version of the Bioconductor package ChAMP, which can be used to analyze EPIC and 450k data. Many enhanced functionalities have been added, including correction for cell-type heterogeneity, network analysis and a series of interactive graphical user interfaces. ChAMP is a BioC package available from https://bioconductor.org/packages/release/bioc/html/ChAMP.html. a.teschendorff@ucl.ac.uk or s.beck@ucl.ac.uk or a.feber@ucl.ac.uk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

A negative-pressure-driven microfluidic chip for the rapid detection of a bladder cancer biomarker in urine using bead-based enzyme-linked immunosorbent assay.

PubMed

Lin, Yen-Heng; Chen, Ying-Ju; Lai, Chao-Sung; Chen, Yi-Ting; Chen, Chien-Lun; Yu, Jau-Song; Chang, Yu-Sun

2013-01-01

This paper describes an integrated microfluidic chip that is capable of rapidly and quantitatively measuring the concentration of a bladder cancer biomarker, apolipoprotein A1, in urine samples. All of the microfluidic components, including the fluid transport system, the micro-valve, and the micro-mixer, were driven by negative pressure, which simplifies the use of the chip and facilitates commercialization. Magnetic beads were used as a solid support for the primary antibody, which captured apolipoprotein A1 in patients' urine. Because of the three-dimensional structure of the magnetic beads, the concentration range of the target that could be detected was as high as 2000 ng ml(-1). Because this concentration is 100 times higher than that quantifiable using a 96-well plate with the same enzyme-linked immunosorbent assay (ELISA) kit, the dilution of the patient's urine can be avoided or greatly reduced. The limit of detection was determined to be approximately 10 ng ml(-1), which is lower than the cutoff value for diagnosing bladder cancer (11.16 ng ml(-1)). When the values measured using the microfluidic chip were compared with those measured using conventional ELISA using a 96-well plate for five patients, the deviations were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8%. The entire measurement time is 6-fold faster than that of conventional ELISA. This microfluidic device shows significant potential for point-of-care applications.

Integrated analyses of proteins and their glycans in a magnetic bead-based multiplex assay format.

PubMed

Li, Danni; Chiu, Hanching; Chen, Jing; Zhang, Hui; Chan, Daniel W

2013-01-01

Well-annotated clinical samples are valuable resources for biomarker discovery and validation. Multiplex and integrated methods that simultaneously measure multiple analytes and generate integrated information about these analytes from a single measurement are desirable because these methods help conserve precious samples. We developed a magnetic bead-based system for multiplex and integrated glycoprotein quantification by immunoassays and glycan detection by lectin immunosorbent assays (LISAs). Magnetic beads coupled with antibodies were used for capturing proteins of interest. Biotinylated antibodies in combination with streptavidin-labeled phycoerythrin were used for protein quantification. In the LISAs, biotinylated detection antibodies were replaced by biotinylated lectins for glycan detection. Using tissue inhibitor of metallopeptidase 1 (TIMP-1), tissue plasminogen activator, membrane metallo-endopeptidase, and dipeptidyl peptidase-IV (DPP-4) as models, we found that the multiplex integrated system was comparable to single immunoassays in protein quantification and LISAs in glycan detection. The merits of this system were demonstrated when applied to well-annotated prostate cancer tissues for validation of biomarkers in aggressive prostate cancer. Because of the system's multiplex ability, we used only 300 ng of tissue protein for the integrated detection of glycans in these proteins. Fucosylated TIMP-1 and DPP-4 offered improved performance over the proteins in distinguishing aggressive and nonaggressive prostate cancer. The multiplex and integrated system conserves samples and is a useful tool for validation of glycoproteins and their glycoforms as biomarkers. © 2012 American Association for Clinical Chemistry

Integrated air stream micromixer for performing bioanalytical assays on a plastic chip.

PubMed

Geissler, Matthias; Li, Kebin; Zhang, Xuefeng; Clime, Liviu; Robideau, Gregg P; Bilodeau, Guillaume J; Veres, Teodor

2014-10-07

This paper describes the design, functioning and use of an integrated mixer that relies on air flux to agitate microliter entities of fluid in an embedded microfluidic cavity. The system was fabricated from multiple layers of a thermoplastic elastomer and features circuits for both liquid and air supply along with pneumatic valves for process control. Internally-dyed polymer particles have been used to visualize flow within the fluid phase during agitation. Numerical modelling of the micromixer revealed an overall efficacy of 10(-1) to 10(-2) for momentum transfer at the air-water interface. Simulation of air vortex dynamics showed dependency of the flow pattern on the velocity of the flux entering the cavity. Three bioanalytical assays have been performed as proof-of-concept demonstrations. In a first assay, cells of Listeria monocytogenes were combined with magnetic nanoparticles (NPs), resulting in high-density coverage of the bacteria's surface with NPs after 1 min of agitation. This finding is contrasted by a control experiment without agitation for which interaction between bacteria and NPs remains low. In a second one, capture and release of genomic DNA from fungi through adsorption onto magnetic beads was tested and shown to be improved by agitation compared to non-agitated controls. A third assay finally involved fluorescently-labelled target oligonucleotide strands and polystyrene particles modified with DNA capture probes to perform detection of nucleic acids on beads. Excellent selectivity was obtained in a competitive hybridization process using a multiplexed micromixer chip design.

Development and Validation of a Novel Diagnostic Test for Human Brucellosis Using a Glyco-engineered Antigen Coupled to Magnetic Beads

PubMed Central

Ciocchini, Andrés E.; Rey Serantes, Diego A.; Melli, Luciano J.; Iwashkiw, Jeremy A.; Deodato, Bettina; Wallach, Jorge; Feldman, Mario F.; Ugalde, Juan E.; Comerci, Diego J.

2013-01-01

Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values, a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited laboratory infrastructure and/or minimally trained community health workers. PMID:23459192

Temperature-controlled microintaglio printing for high-resolution micropatterning of RNA molecules.

PubMed

Kobayashi, Ryo; Biyani, Manish; Ueno, Shingo; Kumal, Subhashini Raj; Kuramochi, Hiromi; Ichiki, Takanori

2015-05-15

We have developed an advanced microintaglio printing method for fabricating fine and high-density micropatterns and applied it to the microarraying of RNA molecules. The microintaglio printing of RNA reported here is based on the hybridization of RNA with immobilized complementary DNA probes. The hybridization was controlled by switching the RNA conformation via the temperature, and an RNA microarray with a diameter of 1.5 µm and a density of 40,000 spots/mm(2) with high contrast was successfully fabricated. Specifically, no size effects were observed in the uniformity of patterned signals over a range of microarray feature sizes spanning one order of magnitude. Additionally, we have developed a microintaglio printing method for transcribed RNA microarrays on demand using DNA-immobilized magnetic beads. The beads were arrayed on wells fabricated on a printing mold and the wells were filled with in vitro transcription reagent and sealed with a DNA-immobilized glass substrate. Subsequently, RNA was in situ synthesized using the bead-immobilized DNA as a template and printed onto the substrate via hybridization. Since the microintaglio printing of RNA using DNA-immobilized beads enables the fabrication of a microarray of spots composed of multiple RNA sequences, it will be possible to screen or analyze RNA functions using an RNA microarray fabricated by temperature-controlled microintaglio printing (TC-µIP). Copyright © 2014 Elsevier B.V. All rights reserved.

Generic Features of Tertiary Chromatin Structure as Detected in Natural Chromosomes

PubMed Central

Müller, Waltraud G.; Rieder, Dietmar; Kreth, Gregor; Cremer, Christoph; Trajanoski, Zlatko; McNally, James G.

2004-01-01

Knowledge of tertiary chromatin structure in mammalian interphase chromosomes is largely derived from artificial tandem arrays. In these model systems, light microscope images reveal fibers or beaded fibers after high-density targeting of transactivators to insertional domains spanning several megabases. These images of fibers have lent support to chromonema fiber models of tertiary structure. To assess the relevance of these studies to natural mammalian chromatin, we identified two different ∼400-kb regions on human chromosomes 6 and 22 and then examined light microscope images of interphase tertiary chromatin structure when the regions were transcriptionally active and inactive. When transcriptionally active, these natural chromosomal regions elongated, yielding images characterized by a series of adjacent puncta or “beads”, referred to hereafter as beaded images. These elongated structures required transcription for their maintenance. Thus, despite marked differences in the density and the mode of transactivation, the natural and artificial systems showed similarities, suggesting that beaded images are generic features of transcriptionally active tertiary chromatin. We show here, however, that these images do not necessarily favor chromonema fiber models but can also be explained by a radial-loop model or even a simple nucleosome affinity, random-chain model. Thus, light microscope images of tertiary structure cannot distinguish among competing models, although they do impose key constraints: chromatin must be clustered to yield beaded images and then packaged within each cluster to enable decondensation into adjacent clusters. PMID:15485905

Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3.

PubMed

Li, Xue-Bo; Wang, Qing-Shan; Feng, Yu; Ning, Shu-Hua; Miao, Yuan-Ying; Wang, Ye-Quan; Li, Hong-Wei

2014-11-01

Forensic DNA analysis of sexual assault evidence requires unambiguous differentiation of DNA profiles in mixed samples. To investigate the feasibility of magnetic bead-based separation of sperm from cell mixtures using a monoclonal antibody against MOSPD3 (motile sperm domain-containing protein 3), 30 cell samples were prepared by mixing 10(4) female buccal epithelial cells with sperm cells of varying densities (10(3), 10(4), or 10(5) cells/mL). Western blot and immunofluorescence assays showed that MOSPD3 was detectable on the membrane of sperm cells, but not in buccal epithelial cells. After biotinylated MOSPD3 antibody was incubated successively with the prepared cell mixtures and avidin-coated magnetic beads, microscopic observation revealed that each sperm cell was bound by two or more magnetic beads, in the head, neck, mid-piece, or flagellum. A full single-source short tandem repeat profile could be obtained in 80% of mixed samples containing 10(3) sperm cells/mL and in all samples containing ≥10(4) sperm cells/mL. For dried vaginal swab specimens, the rate of successful detection was 100% in both flocked and cotton swabs preserved for 1 day, 87.5% in flocked swabs and 40% in cotton swabs preserved for 3 days, and 40% in flocked swabs and 16.67% in cotton swabs preserved for 10 days. Our findings suggest that immunomagnetic bead-based separation is potentially a promising alternative to conventional methods for isolating sperm cells from mixed forensic samples.

Development of silicon photonic microring resonator biosensors for multiplexed cytokine assays and in vitro diagnostics

NASA Astrophysics Data System (ADS)

Luchansky, Matthew Sam

In order to guide critical care therapies that are personalized to a patient's unique disease state, a diagnostic or theranostic medical device must quickly provide a detailed biomolecular understanding of disease onset and progression. This detailed molecular understanding of cellular processes and pathways requires the ability to measure multiple analytes in parallel. Though many traditional sensing technologies for biomarker analysis and fundamental biological studies (i.e. enzyme-linked immunosorbent assays, real-time polymerase chain reaction, etc.) rely on single-parameter measurements, it has become increasingly clear that the inherent complexity of many human illnesses and pathways necessitates quantitative and multiparameter analysis of biological samples. Currently used analytical methods are deficient in that they often provide either highly quantitative data for a single biomarker or qualitative data for many targets, but methods that simultaneously provide highly quantitative analysis of many targets have yet to be adequately developed. Fields such as medical diagnostics and cellular biology would benefit greatly from a technology that enables rapid, quantitative and reproducible assays for many targets within a single sample. In an effort to fill this unmet need, this doctoral dissertation describes the development of a clinically translational biosensing technology based on silicon photonics and developed in the chemistry research laboratory of Ryan C. Bailey. Silicon photonic microring resonators, a class of high-Q optical sensors, represent a promising platform for rapid, multiparameter in vitro measurements. The original device design utilizes 32-ring arrays for real-time biomolecular sensing without fluorescent labels, and these optical biosensors display great potential for more highly multiplexed (100s-1000s) measurements based on the impressive scalability of silicon device fabrication. Though this technology can be used to detect a variety of molecules, this dissertation establishes the utility of microring resonator chips for multiparameter analysis of several challenging protein targets in cell cultures, human blood sera, and other clinical samples such as cerebrospinal fluid. Various sandwich immunoassay formats for diverse protein analytes are described herein, but the bulk of this dissertation focuses on applying the technology to cytokine analysis. Cytokines are small signaling proteins that are present in serum and cell secretomes at concentrations in the pg/mL or ng/mL range. Cytokines are very challenging to quantitate due to their low abundance and small size, but play important roles in a variety of immune response and inflammatory pathways; cytokine quantitation is thus important in fundamental biological studies and diagnostics, and complex and overlapping cytokine roles make multiplexed measurements especially vital. In a typical experiment, microfluidics are used to spatially control chip functionalization by directing capture antibodies against a variety of protein targets to groups of microring sensors. In each case, binding of analytes to the rings causes a change in the local refractive index that is transduced into a real-time, quantitative optical signal. This photonic sensing modality is based on the interaction of the propagating evanescent field with molecules near the ring surface. Since each microring sensor in the array is monitored independently, this technology allows multiple proteins to be quantified in parallel from a single sample. This dissertation describes the fabrication, characterization, development, and application of silicon photonic microring resonator technology to multiplexed protein measurements in a variety of biological systems. Chapter 1 introduces the field of high-Q optical sensors and places microring resonator technology within the broader context of related whispering gallery mode devices. The final stages of cleanroom device fabrication, in which 8" silicon wafers that contain hundreds of ring resonator arrays are transformed into individual functional chips, are described in Chapter 2. Chapter 3 characterizes the physical and optical properties of the microring resonator arrays, especially focusing on the evanescent field profile and mass sensitivity metrics. Chapter 4 demonstrates the ability to apply ring resonator technology to cytokine detection and T cell secretion analysis. Chapter 5 builds on the initial cytokine work to demonstrate the simultaneous detection of multiple cytokines with higher throughput to enable studies of T cell differentiation. In preparation for reaching the goal of cytokine analysis in clinical samples, Chapter 6 describes magnetic bead-based signal enhancement of sandwich immunoassays for serum analysis. Additional examples of the utility of nanoparticles and sub-micron beads for signal amplification are described in Chapter 7, also demonstrating the ability to monitor single bead binding events. Chapter 8 describes an alternative cytokine signal enhancement strategy based on enzymatic amplification for human cerebrospinal fluid (CSF) analysis. Chapter 9 adds work with other CSF protein targets that are relevant to the continuing development of a multiparameter Alzheimer's Disease diagnostic chip. Future directions for multiplexed protein analysis as it pertains to important immunological studies and in vitro diagnostic applications are defined in Chapter 10. (Abstract shortened by UMI.).

Large-Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria.

PubMed

Iino, Ryota; Sakakihara, Shouichi; Matsumoto, Yoshimi; Nishino, Kunihiko

2018-01-01

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

PubMed

Silbereisen, Angelika; Tamborrini, Marco; Wittwer, Matthias; Schürch, Nadia; Pluschke, Gerd

2015-10-05

Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural outbreak and bio-threat situations.

Nanoscale Surface Plasmonics Sensor With Nanofluidic Control

NASA Technical Reports Server (NTRS)

Wei, Jianjun; Singhal, Sameer; Waldeck, David H.; Kofke, Matthew

2013-01-01

Conventional quantitative protein assays of bodily fluids typically involve multiple steps to obtain desired measurements. Such methods are not well suited for fast and accurate assay measurements in austere environments such as spaceflight and in the aftermath of disasters. Consequently, there is a need for a protein assay technology capable of routinely monitoring proteins in austere environments. For example, there is an immediate need for a urine protein assay to assess astronaut renal health during spaceflight. The disclosed nanoscale surface plasmonics sensor provides a core detection method that can be integrated to a lab-on-chip device that satisfies the unmet need for such a protein assay technology. Assays based upon combinations of nanoholes, nanorings, and nanoslits with transmission surface plasmon resonance (SPR) are used for assays requiring extreme sensitivity, and are capable of detecting specific analytes at concentrations as low as picomole to femtomole level in well-controlled environments. The device operates in a transmission mode configuration in which light is directed at one planar surface of the array, which functions as an optical aperture. The incident light induces surface plasmon light transmission from the opposite surface of the array. The presence of a target analyte is detected by changes in the spectrum of light transmitted by the array when a target analyte induces a change in the refractive index of the fluid within the nanochannels. This occurs, for example, when a target analyte binds to a receptor fixed to the walls of the nanochannels in the array. Independent fluid handling capability for individual nanoarrays on a nanofluidic chip containing a plurality of nanochannel arrays allows each array to be used to sense a different target analyte and/or for paired arrays to analyze control and test samples simultaneously in parallel. The present invention incorporates transmission mode nanoplasmonics and nanofluidics into a single, microfluidically controlled device. The device comprises one or more arrays of aligned nanochannels that are in fluid communication with inflowing and outflowing fluid handling manifolds that control the flow of fluid through the arrays. The array acts as an aperture in a plasmonic sensor. Fluid, in the form of a liquid or a gas and comprising a sample for analysis, is moved from an inlet manifold through the nanochannel array, and out through an exit manifold. The fluid may also contain a reagent used to modify the interior surfaces of the nanochannels, and/or a reagent required for the detection of an analyte.

Detection of Clonorchis sinensis circulating antigen in sera from Chinese patients by immunomagnetic bead ELISA based on IgY.

PubMed

Nie, Ge; Wang, Ting; Lu, Shengjun; Liu, Wenqi; Li, Yonglong; Lei, Jiahui

2014-01-01

Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools. We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000-9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000-4999) and 75.0% (9 of 12) in cases of light infection (EPG <1000) of clonorchiasis. Together 36 of total 42 (85.7%) serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG) with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10) or cysticercosis (n = 10). Cross-reactivity was 6.7% (2 of 30) with schistosomiasis japonica and 10.0% (3 of 30) with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons. Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of circulating antigen in human clonorchiasis.

Electrochemical biotin detection based on magnetic beads and a new magnetic flow cell for screen printed electrode.

PubMed

Biscay, Julien; González García, María Begoña; Costa García, Agustín

2015-01-01

The use of the first flow-cell for magnetic assays with an integrated magnet is reported here. The flow injection analysis system (FIA) is used for biotin determination. The reaction scheme is based on a one step competitive assay between free biotin and biotin labeled with horseradish peroxidase (B-HRP). The mixture of magnetic beads modified with streptavidin (Strep-MB), biotin and B-HRP is left 15 min under stirring and then a washing step is performed. After that, 100 μL of the mixture is injected and after 30s 100 μL of 3,3',5,5'-Tetramethylbenzidine (TMB) is injected and the FIAgram is recorded applying a potential of -0.2V. The linear range obtained is from 0.01 to 1 nM of biotin and the sensitivity is 758 nA/nM. The modification and cleaning of the electrode are performed in an easy way due to the internal magnet of the flow cell. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of Immunoglobulin G Antibodies to Taenia solium Cysticercosis Antigen Glutathione-S-Transferase-rT24H in Malian Children Using Multiplex Bead Assay.

PubMed

Moss, Delynn M; Handali, Sukwan; Chard, Anna N; Trinies, Victoria; Bullard, Stevan; Wiegand, Ryan E; Doumbia, Seydou; Freeman, Matthew C; Lammie, Patrick J

2018-05-01

Blood samples from 805 students attending 42 elementary schools in Mopti, Sikasso, and Koulikoro regions, and Bamako district in Mali participated in a school water, sanitation, and hygiene intervention. Immunoglobulin (Ig) G responses to several antigens/pathogens were assessed by a multiplex bead assay (MBA), and the recombinant Taenia solium T24H antigen was included. Of all students tested, 8.0% were positive to rT24H, but in some schools 25-30%. A cluster of 12 widespread school locations showed not only a relative risk of 3.23 for T. solium exposure and significantly higher IgG responses ( P < 0.001) but also significantly lower elevation ( P = 0.04) (m, above sea level) compared with schools outside the cluster. All schools at elevations < 425 m showed significantly higher IgG responses ( P = 0.017) than schools at elevations ≥ 425 m. The MBA is an excellent serological platform that provides cost-effective opportunities to expand testing in serosurveys.

Controlled Lateral Positioning of Microparticles Inside Droplets Using Acoustophoresis.

PubMed

Fornell, Anna; Nilsson, Johan; Jonsson, Linus; Periyannan Rajeswari, Prem Kumar; Joensson, Haakan N; Tenje, Maria

2015-10-20

In this paper, we utilize bulk acoustic waves to control the position of microparticles inside droplets in two-phase microfluidic systems and demonstrate a method to enrich the microparticles. In droplet microfluidics, different unit operations are combined and integrated on-chip to miniaturize complex biochemical assays. We present a droplet unit operation capable of controlling the position of microparticles during a trident shaped droplet split. An acoustic standing wave field is generated in the microchannel, and the acoustic forces direct the encapsulated microparticles to the center of the droplets. The method is generic, requires no labeling of the microparticles, and is operated in a noncontact fashion. It was possible to achieve 2+-fold enrichment of polystyrene beads (5 μm in diameter) in the center daughter droplet with an average recovery of 89% of the beads. Red blood cells were also successfully manipulated inside droplets. These results show the possibility to use acoustophoresis in two-phase systems to enrich microparticles and open up the possibility for new droplet-based assays that are not performed today.

Detection of bacteria from a cecal anaerobic competitive exclusion culture with an immunoassay electrochemiluminescence sensor

NASA Astrophysics Data System (ADS)

Beier, Ross C.; Young, Colin R.; Stanker, Larry H.

1999-01-01

A competitive exclusion (CE) culture of chicken cecal anaerobes has been developed and used in this laboratory for control of Salmonella typhimurium in chickens. The CE culture consists of 29 different species of micro-organisms, and is known as CF3. Detection of one of the CF3 bacteria, Eubacteria, and S. typhimurium were demonstrated using a commercial immunomagnetic (IM) electrochemiluminescence (ECL) sensor, the ORIGENR Analyzer. Analysis was achieved using a sandwich immunoassay. Bacteria were captured on antibody- conjugated 280 micron sized magnetic beads followed by binding of reporter antibodies labelled with ruthenium (II) tris(dipyridyl) chelate [Ru(bpy)32+]. The magnetic beads were then trapped on an electrode in the reaction cell of the ORIGENR Analyzer by a magnet, and the ECL was evoked from Ru(bpy)32+ on the tagged reporter antibodies by an electrical potential at the electrode. Preliminary IM-ECL assays with Eubacteria yielded a detection limit of 105 cfu/mL. Preliminary IM-ECL assays with S. typhimurium yielded a similar detection limit of 105 cfu/mL.

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

PubMed

Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

2014-11-26

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

Regulation of Leukocyte Infiltration into Ovarian Cancer by Tumour-Stroma Interactions; A Microarray View of Cancer Microenvironment

DTIC Science & Technology

2005-03-01

and EpCAM-linked magnetic beads to separate the cells. Success is assessed on flow cytometry using 2G3, Laminin, FAPa and CK7 markers. On the array...that are >90% enriched for CK7 in the epithelial component, and >80% FAPa for the non-epithelial component. At this moment, however, we have not got

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

PubMed

Du, Xiaohui; Li, Wanming; Du, Guansheng; Cho, Hansang; Yu, Min; Fang, Qun; Lee, Luke P; Fang, Jin

2018-03-06

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

Interleukin-6, interleukin-8, and soluble tumor necrosis factor receptor-I in the cord blood as predictors of chronic lung disease in premature infants.

PubMed

An, Hiromi; Nishimaki, Shigeru; Ohyama, Makiko; Haruki, Atsushi; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Kobayashi, Yoshinori; Mori, Masaaki; Seki, Kazuo; Yokota, Shumpei

2004-11-01

In order to predict the late-development of chronic lung disease of prematurity (CLD), cytokines in the cord blood were assessed in this study. Eighteen premature infants with CLD were enrolled. Cord blood plasma levels of cytokines of these infants and 12 control infants without CLD were measured including interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, soluble TNF receptor-I, and soluble IL-6 receptor using a cytometric bead array and an enzyme-linked immunosorbent assay. The cord blood IL-6, IL-8, and sTNFR-I levels were significantly elevated in CLD infants compared with those in control (P < .05). IL-1beta, IL-2, IL-4, IL-10, and IFN-gamma were undetectable in both groups. CLD infants with maternal chorioamnionitis had higher IL-6 than those without chorioamnionitis (P < .01). In CLD infants, IL-6 was higher in the infants who required prolonged oxygen therapy (P < .05). Elevated inflammatory cytokines in the cord blood are associated with the progression to CLD.

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

NASA Technical Reports Server (NTRS)

Chen, J.; Fabry, B.; Schiffrin, E. L.; Wang, N.; Ingber, D. E. (Principal Investigator)

2001-01-01

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Quantitative measurements and modeling of cargo–motor interactions during fast transport in the living axon

PubMed Central

Seamster, Pamela E; Loewenberg, Michael; Pascal, Jennifer; Chauviere, Arnaud; Gonzales, Aaron; Cristini, Vittorio; Bearer, Elaine L

2013-01-01

The kinesins have long been known to drive microtubule-based transport of sub-cellular components, yet the mechanisms of their attachment to cargo remain a mystery. Several different cargo-receptors have been proposed based on their in vitro binding affinities to kinesin-1. Only two of these—phosphatidyl inositol, a negatively charged lipid, and the carboxyl terminus of the amyloid precursor protein (APP-C), a trans-membrane protein—have been reported to mediate motility in living systems. A major question is how these many different cargo, receptors and motors interact to produce the complex choreography of vesicular transport within living cells. Here we describe an experimental assay that identifies cargo–motor receptors by their ability to recruit active motors and drive transport of exogenous cargo towards the synapse in living axons. Cargo is engineered by derivatizing the surface of polystyrene fluorescent nanospheres (100 nm diameter) with charged residues or with synthetic peptides derived from candidate motor receptor proteins, all designed to display a terminal COOH group. After injection into the squid giant axon, particle movements are imaged by laser-scanning confocal time-lapse microscopy. In this report we compare the motility of negatively charged beads with APP-C beads in the presence of glycine-conjugated non-motile beads using new strategies to measure bead movements. The ensuing quantitative analysis of time-lapse digital sequences reveals detailed information about bead movements: instantaneous and maximum velocities, run lengths, pause frequencies and pause durations. These measurements provide parameters for a mathematical model that predicts the spatiotemporal evolution of distribution of the two different types of bead cargo in the axon. The results reveal that negatively charged beads differ from APP-C beads in velocity and dispersion, and predict that at long time points APP-C will achieve greater progress towards the presynaptic terminal. The significance of this data and accompanying model pertains to the role transport plays in neuronal function, connectivity, and survival, and has implications in the pathogenesis of neurological disorders, such as Alzheimer’s, Huntington and Parkinson’s diseases. PMID:23011729

Quantitative measurements and modeling of cargo-motor interactions during fast transport in the living axon

NASA Astrophysics Data System (ADS)

Seamster, Pamela E.; Loewenberg, Michael; Pascal, Jennifer; Chauviere, Arnaud; Gonzales, Aaron; Cristini, Vittorio; Bearer, Elaine L.

2012-10-01

The kinesins have long been known to drive microtubule-based transport of sub-cellular components, yet the mechanisms of their attachment to cargo remain a mystery. Several different cargo-receptors have been proposed based on their in vitro binding affinities to kinesin-1. Only two of these—phosphatidyl inositol, a negatively charged lipid, and the carboxyl terminus of the amyloid precursor protein (APP-C), a trans-membrane protein—have been reported to mediate motility in living systems. A major question is how these many different cargo, receptors and motors interact to produce the complex choreography of vesicular transport within living cells. Here we describe an experimental assay that identifies cargo-motor receptors by their ability to recruit active motors and drive transport of exogenous cargo towards the synapse in living axons. Cargo is engineered by derivatizing the surface of polystyrene fluorescent nanospheres (100 nm diameter) with charged residues or with synthetic peptides derived from candidate motor receptor proteins, all designed to display a terminal COOH group. After injection into the squid giant axon, particle movements are imaged by laser-scanning confocal time-lapse microscopy. In this report we compare the motility of negatively charged beads with APP-C beads in the presence of glycine-conjugated non-motile beads using new strategies to measure bead movements. The ensuing quantitative analysis of time-lapse digital sequences reveals detailed information about bead movements: instantaneous and maximum velocities, run lengths, pause frequencies and pause durations. These measurements provide parameters for a mathematical model that predicts the spatiotemporal evolution of distribution of the two different types of bead cargo in the axon. The results reveal that negatively charged beads differ from APP-C beads in velocity and dispersion, and predict that at long time points APP-C will achieve greater progress towards the presynaptic terminal. The significance of this data and accompanying model pertains to the role transport plays in neuronal function, connectivity, and survival, and has implications in the pathogenesis of neurological disorders, such as Alzheimer’s, Huntington and Parkinson's diseases.

Rapid and quantitative detection of zoonotic influenza A virus infection utilizing coumarin-derived dendrimer-based fluorescent immunochromatographic strip test (FICT).

PubMed

Yeo, Seon-Ju; Huong, Dinh Thi; Hong, Nguyen Ngoc; Li, Chun-Ying; Choi, Kyunghan; Yu, Kyoungsik; Choi, Du-Young; Chong, Chom-Kyu; Choi, Hak Soo; Mallik, Shyam Kumar; Kim, Hak Sung; Sung, Haan Woo; Park, Hyun

2014-01-01

Great efforts have been made to develop robust signal-generating fluorescence materials which will help in improving the rapid diagnostic test (RDT) in terms of sensitivity and quantification. In this study, we developed coumarin-derived dendrimer-based fluorescent immunochromatographic strip test (FICT) assay with enhanced sensitivity as a quantitative diagnostic tool in typical RDT environments. The accuracy of the proposed FICT was compared with that of dot blot immunoassay techniques and conventional RDTs. Through conjugation of coumarin-derived dendrimers with latex beads, fluorescent emission covering broad output spectral ranges was obtained which provided a distinct advantage of easy discrimination of the fluorescent emission of the latex beads with a simple insertion of a long-pass optical filter away from the excitation wavelength. The newly developed FICT assay was able to detect 100 ng/10 μL of influenza A nucleoprotein (NP) antigen within 5 minutes, which corresponded to 2.5-fold higher sensitivity than that of the dot blot immunoassay or conventional RDTs. Moreover, the FICT assay was confirmed to detect at least four avian influenza A subtypes (H5N3, H7N1, H7N7, and H9N2). On applying the FICT to the clinical swab samples infected with respiratory viruses, our FICT assay was confirmed to differentiate influenza H1N1 infection from other respiratory viral diseases. These data demonstrate that the proposed FICT assay is able to detect zoonotic influenza A viruses with a high sensitivity, and it enables the quantitation of the infection intensity by providing the numerical diagnostic values; thus demonstrating enhanced detectability of influenza A viruses.

Magnetic Bead Based Immunoassay for Autonomous Detection of Toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Y; Hara, C A; Knize, M G

2008-05-01

As a step towards toward the development of a rapid, reliable analyzer for bioagents in the environment, we are developing an automated system for the simultaneous detection of a group of select agents and toxins. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag{trademark} reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag{trademark} through a cleavable linkage. Aqueous samples are incubatedmore » with the mixture of antibodies along with streptavidin-coated magnetic beads coupled to a photo-activatable porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactivable group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags{trademark}. Released eTags{trademark} are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 ng/mL (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated on a flow-through format with higher LODs of 125 ng/mL (or 2.5 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.« less

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

PubMed

Wan, Zhi; Ostendorff, Heather P; Liu, Ziying; Schneider, Lynda C; Rothschild, Kenneth J; Lim, Mark J

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children's Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease.

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing

PubMed Central

Wan, Zhi; Ostendorff, Heather P.; Liu, Ziying; Schneider, Lynda C.; Rothschild, Kenneth J.

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the “matrix effect” caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children’s Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease. PMID:29389948

Fluorescent polymer sensor array for detection and discrimination of explosives in water.

PubMed

Woodka, Marc D; Schnee, Vincent P; Polcha, Michael P

2010-12-01

A fluorescent polymer sensor array (FPSA) was made from commercially available fluorescent polymers coated onto glass beads and was tested to assess the ability of the array to discriminate between different analytes in aqueous solution. The array was challenged with exposures to 17 different analytes, including the explosives trinitrotoluene (TNT), tetryl, and RDX, various explosive-related compounds (ERCs), and nonexplosive electron-withdrawing compounds (EWCs). The array exhibited a natural selectivity toward EWCs, while the non-electron-withdrawing explosive 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) produced no response. Response signatures were visualized by principal component analysis (PCA), and classified by linear discriminant analysis (LDA). RDX produced the same response signature as the sampled blanks and was classified accordingly. The array exhibited excellent discrimination toward all other compounds, with the exception of the isomers of nitrotoluene and aminodinitrotoluene. Of particular note was the ability of the array to discriminate between the three isomers of dinitrobenzene. The natural selectivity of the FPSA toward EWCs, plus the ability of the FPSA to discriminate between different EWCs, could be used to design a sensor with a low false alarm rate and an excellent ability to discriminate between explosives and explosive-related compounds.

A portable array biosensor for food safety

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Ngundi, Miriam M.; Shriver-Lake, Lisa C.; Taitt, Chris R.; Ligler, Frances S.

2004-11-01

An array biosensor developed for simultaneous analysis of multiple samples has been utilized to develop assays for toxins and pathogens in a variety of foods. The biochemical component of the multi-analyte biosensor consists of a patterned array of biological recognition elements immobilized on the surface of a planar waveguide. A fluorescence assay is performed on the patterned surface, yielding an array of fluorescent spots, the locations of which are used to identify what analyte is present. Signal transduction is accomplished by means of a diode laser for fluorescence excitation, optical filters and a CCD camera for image capture. A laptop computer controls the miniaturized fluidics system and image capture. Results for four mycotoxin competition assays in buffer and food samples are presented.

Microfluidic production of bioactive fibrin micro-beads embedded in crosslinked collagen used as an injectable bulking agent for urinary incontinence treatment.

PubMed

Vardar, E; Larsson, H M; Allazetta, S; Engelhardt, E M; Pinnagoda, K; Vythilingam, G; Hubbell, J A; Lutolf, M P; Frey, P

2018-02-01

Endoscopic injection of bulking agents has been widely used to treat urinary incontinence, often due to urethral sphincter complex insufficiency. The aim of the study was to develop a novel injectable bioactive collagen-fibrin bulking agent restoring long-term continence by functional muscle tissue regeneration. Fibrin micro-beads were engineered using a droplet microfluidic system. They had an average diameter of 140 μm and recombinant fibrin-binding insulin-like growth factor-1 (α 2 PI 1-8 -MMP-IGF-1) was covalently conjugated to the beads. A plasmin fibrin degradation assay showed that 72.5% of the initial amount of α 2 PI 1-8 -MMP-IGF-1 loaded into the micro-beads was retained within the fibrin micro-beads. In vitro, the growth factor modified fibrin micro-beads enhanced cell attachment and the migration of human urinary tract smooth muscle cells, however, no change of the cellular metabolic activity was seen. These bioactive micro-beads were mixed with genipin-crosslinked homogenized collagen, acting as a carrier. The collagen concentration, the degree of crosslinking, and the mechanical behavior of this bioactive collagen-fibrin injectable were comparable to reference samples. This novel injectable showed no burst release of the growth factor, had a positive effect on cell behavior and may therefore induce smooth muscle regeneration in vivo, necessary for the functional treatment of stress and other urinary incontinences. Urinary incontinence is involuntary urine leakage, resulting from a deficient function of the sphincter muscle complex. Yet there is no functional cure for this devastating condition using current treatment options. Applied physical and surgical therapies have limited success. In this study, a novel bioactive injectable bulking agent, triggering new muscle regeneration at the injection site, has been evaluated. This injectable consists of cross-linked collagen and fibrin micro-beads, functionalized with bound insulin-like growth factor-1 (α 2 PI 1-8 -MMP-IGF-1). These bioactive fibrin micro-beads induced human smooth muscle cell migration in vitro. Thus, this injectable bulking agent is apt to be a good candidate for regeneration of urethral sphincter muscle, ensuring a long-lasting treatment for urinary incontinence. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Photonic crystal beads from gravity-driven microfluidics.

PubMed

Gu, Hongcheng; Rong, Fei; Tang, Baocheng; Zhao, Yuanjin; Fu, Degang; Gu, Zhongze

2013-06-25

This Letter reports a simple method for the mass production of 3D colloidal photonic crystal beads (PCBs) by using a gravity-driven microfluidic device and online droplet drying method. Compared to traditional methods, the droplet templates of the PCBs are generated by using the ultrastable gravity as the driving force for the microfluidics, thus the PCBs are formed with minimal polydispersity. Moreover, drying of the droplet templates is integrated into the production process, and the nanoparticles in the droplets self-assemble online. Overall, this process results in PCBs with good morphology, low polydispersity, brilliant structural colors, and narrow stop bands. PCBs could be bulk generated by this process for many practical applications, such as multiplex-encoded assays and the construction of novel optical materials.

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

NASA Astrophysics Data System (ADS)

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

2011-09-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

PubMed Central

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

2011-01-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. PMID:21974603

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

PubMed

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

2011-09-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics

Designing a multiroute synthesis scheme in combinatorial chemistry.

PubMed

Akavia, Adi; Senderowitz, Hanoch; Lerner, Alon; Shamir, Ron

2004-01-01

Solid-phase mix-and-split combinatorial synthesis is often used to produce large arrays of compounds to be tested during the various stages of the drug development process. This method can be represented by a synthesis graph in which nodes correspond to grow operations and arcs to beads transferred among the different reaction vessels. In this work, we address the problem of designing such a graph which maximizes the number of produced target compounds (namely, compounds out of an input library of desired molecules), given constraints on the number of beads used for library synthesis and on the number of reaction vessels available for concurrent grow steps. We present a heuristic based on a discrete search for solving this problem, test our solution on several data sets, explore its behavior, and show that it achieves good performance.

A dynamic bead-based microarray for parallel DNA detection

NASA Astrophysics Data System (ADS)

Sochol, R. D.; Casavant, B. P.; Dueck, M. E.; Lee, L. P.; Lin, L.

2011-05-01

A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening.

GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

1994-09-01

GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by amore » sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.« less

Lab-on-a-disc agglutination assay for protein detection by optomagnetic readout and optical imaging using nano- and micro-sized magnetic beads.

PubMed

Uddin, Rokon; Burger, Robert; Donolato, Marco; Fock, Jeppe; Creagh, Michael; Hansen, Mikkel Fougt; Boisen, Anja

2016-11-15

We present a biosensing platform for the detection of proteins based on agglutination of aptamer coated magnetic nano- or microbeads. The assay, from sample to answer, is integrated on an automated, low-cost microfluidic disc platform. This ensures fast and reliable results due to a minimum of manual steps involved. The detection of the target protein was achieved in two ways: (1) optomagnetic readout using magnetic nanobeads (MNBs); (2) optical imaging using magnetic microbeads (MMBs). The optomagnetic readout of agglutination is based on optical measurement of the dynamics of MNB aggregates whereas the imaging method is based on direct visualization and quantification of the average size of MMB aggregates. By enhancing magnetic particle agglutination via application of strong magnetic field pulses, we obtained identical limits of detection of 25pM with the same sample-to-answer time (15min 30s) using the two differently sized beads for the two detection methods. In both cases a sample volume of only 10µl is required. The demonstrated automation, low sample-to-answer time and portability of both detection instruments as well as integration of the assay on a low-cost disc are important steps for the implementation of these as portable tools in an out-of-lab setting. Copyright © 2016 Elsevier B.V. All rights reserved.

Methods and devices for protein assays

DOEpatents

Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

2009-11-03

Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

DNA strand displacement reaction for programmable release of biomolecules.

PubMed

Ramezani, Hamid; Jed Harrison, D

2015-05-14

Sample cleanup is a major processing step in many analytical assays. Here, we propose an approach to capture-and-release of analytes based on the DNA strand displacement reaction (SDR) and demonstrate its application to a fluoroimmunoassay on beads for a thyroid cancer biomarker, thyroglobulin. The SDR-based cleanup showed no interference from matrix molecules in serum.

Evaluation of chlorine treatment levels on inactivation of human norovirus and MS2 bacteriophage during sewage treatment

USDA-ARS?s Scientific Manuscript database

This study examined the inactivation of human norovirus (HuNoV) GI.1 and GII.4 by chlorine under conditions that mimic sewage treatment. Using a porcine gastric mucin-magnetic bead (PGM-MB) assay, no statistically significant loss in HuNoV binding (inactivation) was observed for secondary effluent ...

Bacteriophage-based rapid and sensitive detection of Escherichia coli O157:H7 isolates from ground beef

USDA-ARS?s Scientific Manuscript database

We investigated efficacy of bacteriophage-based detection technology to detect Escherichia coli O157:H7 from ground beef. The assay involved 8 h enrichment of cold stressed beef samples in presence of antimicrobials followed by capture of the pathogen on O157:H7-specific immunomagnetic beads and sp...

Best Practices and Joint Calling of the HumanExome BeadChip: The CHARGE Consortium

PubMed Central

Grove, Megan L.; Yu, Bing; Cochran, Barbara J.; Haritunians, Talin; Bis, Joshua C.; Taylor, Kent D.; Hansen, Mark; Borecki, Ingrid B.; Cupples, L. Adrienne; Fornage, Myriam; Gudnason, Vilmundur; Harris, Tamara B.; Kathiresan, Sekar; Kraaij, Robert; Launer, Lenore J.; Levy, Daniel; Liu, Yongmei; Mosley, Thomas; Peloso, Gina M.; Psaty, Bruce M.; Rich, Stephen S.; Rivadeneira, Fernando; Siscovick, David S.; Smith, Albert V.; Uitterlinden, Andre; van Duijn, Cornelia M.; Wilson, James G.; O’Donnell, Christopher J.; Rotter, Jerome I.; Boerwinkle, Eric

2013-01-01

Genotyping arrays are a cost effective approach when typing previously-identified genetic polymorphisms in large numbers of samples. One limitation of genotyping arrays with rare variants (e.g., minor allele frequency [MAF] <0.01) is the difficulty that automated clustering algorithms have to accurately detect and assign genotype calls. Combining intensity data from large numbers of samples may increase the ability to accurately call the genotypes of rare variants. Approximately 62,000 ethnically diverse samples from eleven Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium cohorts were genotyped with the Illumina HumanExome BeadChip across seven genotyping centers. The raw data files for the samples were assembled into a single project for joint calling. To assess the quality of the joint calling, concordance of genotypes in a subset of individuals having both exome chip and exome sequence data was analyzed. After exclusion of low performing SNPs on the exome chip and non-overlap of SNPs derived from sequence data, genotypes of 185,119 variants (11,356 were monomorphic) were compared in 530 individuals that had whole exome sequence data. A total of 98,113,070 pairs of genotypes were tested and 99.77% were concordant, 0.14% had missing data, and 0.09% were discordant. We report that joint calling allows the ability to accurately genotype rare variation using array technology when large sample sizes are available and best practices are followed. The cluster file from this experiment is available at www.chargeconsortium.com/main/exomechip. PMID:23874508

Relaxation dynamics of internal segments of DNA chains in nanochannels

NASA Astrophysics Data System (ADS)

Jain, Aashish; Muralidhar, Abhiram; Dorfman, Kevin; Dorfman Group Team

We will present relaxation dynamics of internal segments of a DNA chain confined in nanochannel. The results have direct application in genome mapping technology, where long DNA molecules containing sequence-specific fluorescent probes are passed through an array of nanochannels to linearize them, and then the distances between these probes (the so-called ``DNA barcode'') are measured. The relaxation dynamics of internal segments set the experimental error due to dynamic fluctuations. We developed a multi-scale simulation algorithm, combining a Pruned-Enriched Rosenbluth Method (PERM) simulation of a discrete wormlike chain model with hard spheres with Brownian dynamics (BD) simulations of a bead-spring chain. Realistic parameters such as the bead friction coefficient and spring force law parameters are obtained from PERM simulations and then mapped onto the bead-spring model. The BD simulations are carried out to obtain the extension autocorrelation functions of various segments, which furnish their relaxation times. Interestingly, we find that (i) corner segments relax faster than the center segments and (ii) relaxation times of corner segments do not depend on the contour length of DNA chain, whereas the relaxation times of center segments increase linearly with DNA chain size.

Application of a Halbach magnetic array for long-range cell and particle separations in biological samples

NASA Astrophysics Data System (ADS)

Kang, Joo H.; Driscoll, Harry; Super, Michael; Ingber, Donald E.

2016-05-01

Here, we describe a versatile application of a planar Halbach permanent magnet array for an efficient long-range magnetic separation of living cells and microparticles over distances up to 30 mm. A Halbach array was constructed from rectangular bar magnets using 3D-printed holders and compared to a conventional alternating array of identical magnets. We theoretically predicted the superiority of the Halbach array for a long-range magnetic separation and then experimentally validated that the Halbach configuration outperforms the alternating array for isolating magnetic microparticles or microparticle-bound bacterial cells at longer distances. Magnetophoretic velocities (ymag) of magnetic particles (7.9 μm diameter) induced by the Halbach array in a microfluidic device were significantly higher and extended over a larger area than those induced by the alternating magnet array (ymag = 178 versus 0 μm/s at 10 mm, respectively). When applied to 50 ml tubes (˜30 mm diameter), the Halbach array removed >95% of Staphylococcus aureus bacterial cells bound with 1 μm magnetic particles compared to ˜70% removed using the alternating array. In addition, the Halbach array enabled manipulation of 1 μm magnetic beads in a deep 96-well plate for ELISA applications, which was not possible with the conventional magnet arrays. Our analysis demonstrates the utility of the Halbach array for the future design of devices for high-throughput magnetic separations of cells, molecules, and toxins.

Flexible Teflon nanocone array surfaces with tunable superhydrophobicity for self-cleaning and aqueous droplet patterning.

PubMed

Toma, Mana; Loget, Gabriel; Corn, Robert M

2014-07-23

Tunable hydrophobic/hydrophilic flexible Teflon nanocone array surfaces were fabricated over large areas (cm(2)) by a simple two-step method involving the oxygen plasma etching of a colloidal monolayer of polystyrene beads on a Teflon film. The wettability of the nanocone array surfaces was controlled by the nanocone array dimensions and various additional surface modifications. The resultant Teflon nanocone array surfaces were hydrophobic and adhesive (a "gecko" type of surface on which a water droplet has a high contact angle but stays in place) with a contact angle that correlated with the aspect ratio/sharpness of the nanocones. The surfaces switched to a superhydrophobic or "lotus" type of surface when hierarchical nanostructures were created on Teflon nanocones by modifying them with a gold nanoparticle (AuNPs) film. The nanocone array surfaces could be made superhydrophobic with a maximum contact angle of 160° by the further modification of the AuNPs with an octadecanethiol (C18SH) monolayer. Additionally, these nanocone array surfaces became hydrophilic when the nanocone surfaces were sequentially modified with AuNPs and hydrophilic polydopamine (PDA) layers. The nanocone array surfaces were tested for two potential applications: self-cleaning superhydrophobic surfaces and for the passive dispensing of aqueous droplets onto hybrid superhydrophobic/hydrophilic microarrays.

Biomechanics of P-selectin PSGL-1 bonds: Shear threshold and integrin-independent cell adhesion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Zhihua; Goldsmith, Harry L.; MacIntosh, Fiona A.

2006-03-01

Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP) stimulated platelets or P-selectin bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14-3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that while blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by ~60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though inmore » synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 dissociated previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a subtle shear threshold for P-selectin PSGL-1 binding was also noted at shear rates<100/s and at force loading rates of ~300pN/sec. Results are discussed in light of biophysical computations that characterize the collision between unequal size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion that may be physiologically relevant.« less

Electrostatic mechanism of nucleosomal array folding revealed by computer simulation

PubMed Central

Sun, Jian; Zhang, Qing; Schlick, Tamar

2005-01-01

Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended “beads-on-a-string” conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA–nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt. PMID:15919827

Electrostatic mechanism of nucleosomal array folding revealed by computer simulation.

PubMed

Sun, Jian; Zhang, Qing; Schlick, Tamar

2005-06-07

Although numerous experiments indicate that the chromatin fiber displays salt-dependent conformations, the associated molecular mechanism remains unclear. Here, we apply an irregular Discrete Surface Charge Optimization (DiSCO) model of the nucleosome with all histone tails incorporated to describe by Monte Carlo simulations salt-dependent rearrangements of a nucleosomal array with 12 nucleosomes. The ensemble of nucleosomal array conformations display salt-dependent condensation in good agreement with hydrodynamic measurements and suggest that the array adopts highly irregular 3D zig-zag conformations at high (physiological) salt concentrations and transitions into the extended "beads-on-a-string" conformation at low salt. Energy analyses indicate that the repulsion among linker DNA leads to this extended form, whereas internucleosome attraction drives the folding at high salt. The balance between these two contributions determines the salt-dependent condensation. Importantly, the internucleosome and linker DNA-nucleosome attractions require histone tails; we find that the H3 tails, in particular, are crucial for stabilizing the moderately folded fiber at physiological monovalent salt.

Jllumina - A comprehensive Java-based API for statistical Illumina Infinium HumanMethylation450 and Infinium MethylationEPIC BeadChip data processing.

PubMed

Almeida, Diogo; Skov, Ida; Lund, Jesper; Mohammadnejad, Afsaneh; Silva, Artur; Vandin, Fabio; Tan, Qihua; Baumbach, Jan; Röttger, Richard

2016-10-01

Measuring differential methylation of the DNA is the nowadays most common approach to linking epigenetic modifications to diseases (called epigenome-wide association studies, EWAS). For its low cost, its efficiency and easy handling, the Illumina HumanMethylation450 BeadChip and its successor, the Infinium MethylationEPIC BeadChip, is the by far most popular techniques for conduction EWAS in large patient cohorts. Despite the popularity of this chip technology, raw data processing and statistical analysis of the array data remains far from trivial and still lacks dedicated software libraries enabling high quality and statistically sound downstream analyses. As of yet, only R-based solutions are freely available for low-level processing of the Illumina chip data. However, the lack of alternative libraries poses a hurdle for the development of new bioinformatic tools, in particular when it comes to web services or applications where run time and memory consumption matter, or EWAS data analysis is an integrative part of a bigger framework or data analysis pipeline. We have therefore developed and implemented Jllumina, an open-source Java library for raw data manipulation of Illumina Infinium HumanMethylation450 and Infinium MethylationEPIC BeadChip data, supporting the developer with Java functions covering reading and preprocessing the raw data, down to statistical assessment, permutation tests, and identification of differentially methylated loci. Jllumina is fully parallelizable and publicly available at http://dimmer.compbio.sdu.dk/download.html.

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

PubMed

Teste, Bruno; Ali-Cherif, Anaïs; Viovy, Jean Louis; Malaquin, Laurent

2013-06-21

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

Improving T Cell Expansion with a Soft Touch

PubMed Central

Lambert, Lester H.; Goebrecht, Geraldine K.E.; De Leo, Sarah E.; O’Connor, Roddy S.; Nunez-Cruz, Selene; Li, Tai-De; Yuan, Jinglun; Milone, Michael C.; Kam, Lance C.

2017-01-01

Protein-coated microbeads provide a consistent approach for activating and expanding populations of T cells for immunotherapy, but don’t fully capture the properties of antigen presenting cells. In this report, we enhance T cell expansion by replacing the conventional, rigid bead with a mechanically soft elastomer. Polydimethylsiloxane (PDMS) was prepared in a microbead format and modified with activating antibodies to CD3 and CD28. Three different formulations of PDMS provided an extended proliferative phase in both CD4+-only and mixed CD4+/CD8+ T cell preparations. CD8+ T cells retained cytotoxic function, as measured by a set of biomarkers (perforin production, LAMP2 mobilization, and IFN-γ secretion) and an in vivo assay of targeted cell killing. Notably, PDMS beads presented a nanoscale polymer structure and higher rigidity than associated with conventional bulk material. These data suggest T cells respond to this higher rigidity, indicating an unexpected effect of curing conditions. Together, these studies demonstrate that adopting mechanobiology ideas into the bead platform can provide new tools for T cell-based immunotherapy. PMID:28122453

Long-term clinical and immunological effects of p53-SLP® vaccine in patients with ovarian cancer.

PubMed

Leffers, Ninke; Vermeij, Renee; Hoogeboom, Baukje-Nynke; Schulze, Ute R; Wolf, Rinze; Hamming, Ineke E; van der Zee, Ate G; Melief, Kees J; van der Burg, Sjoerd H; Daemen, Toos; Nijman, Hans W

2012-01-01

Vaccine-induced p53-specific immune responses were previously reported to be associated with improved response to secondary chemotherapy in patients with small cell lung cancer. We investigated long-term clinical and immunological effects of the p53-synthetic long peptide (p53-SLP®) vaccine in patients with recurrent ovarian cancer. Twenty patients were immunized with the p53-SLP® vaccine between July 2006 and August 2007. Follow-up information on patients was obtained. Clinical responses to secondary chemotherapy after p53-SLP® immunizations were determined by computerized tomography and/or tumor marker levels (CA125). Disease-specific survival was compared to a matched historical control group. Immune responses were analyzed by flow cytometry, proliferation assay, interferon gamma (IFN-γ) ELISPOT and/or cytokine bead array. Lymphocytes cultured from skin biopsy were analyzed by flow cytometry and proliferation assay. Of 20 patients treated with the p53-SLP® vaccine, 17 were subsequently treated with chemotherapy. Eight of these patients volunteered another blood sample. No differences in clinical response rates to secondary chemotherapy or disease-specific survival were observed between immunized patients and historical controls (p = 0.925, resp. p = 0.601). p53-specific proliferative responses were observed in 5/8 patients and IFN-γ production in 2/7 patients. Lymphocytes cultured from a prior injection site showing inflammation during chemotherapy did not recognize p53-SLP®. Thus, treatment with the p53-SLP® vaccine does not affect responses to secondary chemotherapy or survival, although p53-specific T-cells do survive chemotherapy. Copyright © 2011 UICC.

Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.

2015-08-07

Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extractionmore » improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.« less

Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

PubMed

Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

2015-03-01

The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

PubMed

Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

2004-01-30

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYS

EPA Science Inventory

AbstractTITLE: A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYSABSTRACT BODY: Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characte...

Hydrodynamic chromatography of polystyrene microparticles in micropillar array columns.

PubMed

Op de Beeck, Jeff; De Malsche, Wim; Vangelooven, Joris; Gardeniers, Han; Desmet, Gert

2010-09-24

We report on the possibility to perform HDC in micropillar array columns and the potential advantages of such a system. The HDC performance of a pillar array column with pillar diameter = 5 microm and an interpillar distance of 2.5 microm has been characterized using both a low MW tracer (FITC) and differently sized polystyrene bead samples (100, 200 and 500 nm). The reduced plate height curves that were obtained for the different investigated markers all overlapped very well, and attained a minimum value of about h(min)=0.3 (reduction based on the pillar diameter), corresponding to 1.6 microm in absolute value and giving good prospects for high efficiency separations. The obtained reduced retention time values were in fair agreement with that predicted by the Di Marzio and Guttman model for a flow between flat plates, using the minimal interpillar distance as characteristic interplate distance. Copyright 2010 Elsevier B.V. All rights reserved.

Determination of gold in geologic materials by solvent extraction and atomic-absorption spectrometry

USGS Publications Warehouse

Huffman, Claude; Mensik, J.D.; Riley, L.B.

1967-01-01

The two methods presented for the determination of traces of gold in geologic materials are the cyanide atomic-absorption method and the fire-assay atomic-absorption method. In the cyanide method gold is leached with a sodium-cyanide solution. The monovalent gold is then oxidized to the trivalent state and concentrated by extracting into methyl isobutyl ketone prior to estimation by atomic absorption. In the fire-assay atomic-absorption method, the gold-silver bead obtained from fire assay is dissolved in nitric and hydrochloric acids. Gold is then concentrated by extracting into methyl isobutyl ketone prior to determination by atomic absorption. By either method concentrations as low as 50 parts per billion of gold can be determined in a 15-gram sample.

Rapid Engineering of Three-Dimensional, Multicellular Tissues With Polymeric Scaffolds

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Jordan, Jacqueline; Fraga, Denise N.

2007-01-01

A process has been developed for the rapid tissue engineering of multicellular-tissue-equivalent assemblies by the controlled enzymatic degradation of polymeric beads in a low-fluid-shear bioreactor. In this process, the porous polymeric beads serve as temporary scaffolds to support the assemblies of cells in a tissuelike 3D configuration during the critical initial growth phases of attachment of anchorage-dependent cells, aggregation of the cells, and formation of a 3D extracellular matrix. Once the cells are assembled into a 3D array and enmeshed in a structural supportive 3D extracellular matrix (ECM), the polymeric scaffolds can be degraded in the low-fluid-shear environment of the NASA-designed bioreactor. The natural 3D tissuelike assembly, devoid of any artificial support structure, is maintained in the low-shear bioreactor environment by the newly formed natural cellular/ECM. The elimination of the artificial scaffold allows normal tissue structure and function.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients.

PubMed

Schlick, Bettina; Massoner, Petra; Lueking, Angelika; Charoentong, Pornpimol; Blattner, Mirjam; Schaefer, Georg; Marquart, Klaus; Theek, Carmen; Amersdorfer, Peter; Zielinski, Dirk; Kirchner, Matthias; Trajanoski, Zlatko; Rubin, Mark A; Müllner, Stefan; Schulz-Knappe, Peter; Klocker, Helmut

2016-01-01

Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients

PubMed Central

Schlick, Bettina; Massoner, Petra; Lueking, Angelika; Charoentong, Pornpimol; Blattner, Mirjam; Schaefer, Georg; Marquart, Klaus; Theek, Carmen; Amersdorfer, Peter; Zielinski, Dirk; Kirchner, Matthias; Trajanoski, Zlatko; Rubin, Mark A.; Müllner, Stefan; Schulz-Knappe, Peter; Klocker, Helmut

2016-01-01

Background Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens. Methods Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology. Results Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076). Conclusions We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation. PMID:26863016

[Studies on a sequential injection renewable surface reflectance spectrophotometric system using a microchip flow cell].

PubMed

Wang, Jian-ya; Fang, Zhao-lun

2002-02-01

A microchip flow cell was developed for flow injection renewable surface assay by reflectance spectrophotometry. The flow cell was coupled to a sequential injection system and optical fiber photometric detection system. The flow cell featured a three-layer structure. The flow channel was cut into a silicone rubber membrance which formed the middle layer, and a porous filter was inlayed across a widened section of the channel to trap microbeads introduced into the flow cell. The area of the detection window of the flow cell was approximately 3.6 mm2, the volume of the bead trapped in the flow cell was 2.2 microL, the depth of the bead layer was 600 microns. A multistrand bifurcated optical fiber was coupled with incident light, detector and flow cell. The chromogenic reaction of Cr(VI) with 1,5-diphenylcarbohydrazide (DPC) which was adsorbed on trapped Polysorb C-18 beads was used as a model reaction to optimize the flow cell design and the experimental system. The reflectance of the renewable reaction surface was monitored at 540 nm. With 100 microL sample loaded and 1.0 mL.min-1 carrier flow rate, the linear response range was 0-0.6 microgram.mL-1 Cr(VI). A detection limit (3 sigma) of 6 ng.mL-1, precision of 1.5% RSD(n = 11), and a throughput of 64 samples per hour were achieved. Considerations in system and flow cell design, the influence of depth of the bead layer, weight of beads used, and the flow rates of carrier stream on the performance were discussed.

A lysozyme and magnetic bead based method of separating intact bacteria.

PubMed

Diler, Ebru; Obst, Ursula; Schmitz, Katja; Schwartz, Thomas

2011-07-01

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Detection Techniques for Biomolecules using Semi-Conductor Nanocrystals and Magnetic Beads as Labels

NASA Astrophysics Data System (ADS)

Chatterjee, Esha

Continued interest in the development of miniaturized and portable analytical platforms necessitates the exploration of sensitive methods for the detection of trace analytes. Nanomaterials, on account of their unique physical and chemical properties, are not only able to overcome many limitations of traditional detection reagents but also enable the exploration of many new signal transduction technologies. This dissertation presents a series of investigations of alternative detection techniques for biomolecules, involving the use of semi-conductor nanocrystals and magnetic beads as labels. Initial research focused on the development of quantum dot-encapsulating liposomes as a novel fluorescent label for immunoassays. This hybrid nanomaterial was anticipated to overcome the drawbacks presented by traditional fluorophores as well as provide significant signal amplification. Quantum dot-encapsulating liposomes were synthesized by the method of thin film hydration and characterized. The utility of these composite nanostructures for bioanalysis was demonstrated. However, the longterm instability of the liposomes hampered quantitative development. A second approach for assay development exploited the ability of gold nanoparticles to quench the optical signals obtained from quantum dots. The goal of this study was to demonstrate the feasibility of using aptamer-linked nanostructures in FRET-based quenching for the detection of proteins. Thrombin was used as the model analyte in this study. Experimental parameters for the assay were optimized. The assay simply required the mixing of the sample with the reagents and could be completed in less than an hour. The limit of detection for thrombin by this method was 5 nM. This homogeneous assay can be easily adapted for the detection of a wide variety of biochemicals. The novel technique of ferromagnetic resonance generated in magnetic bead labels was explored for signal transduction. This inductive detection technique lends itself to miniaturization, is capable of mass production and is inexpensive to fabricate. The device consisted of a microwave circuit in which a slotline and a coplanar waveguide were integrated with a biochemically activated sensor area. The magnetic beads were immobilized at the sensor area by bio-specific reactions. Experiments conducted on this prototype show promising results for using ferromagnetic resonance -based detection of magnetic labels for fabrication of portable and inexpensive sensor devices. The next stage of work addresses the issue of patterning of sensing surfaces with biomolecules. The ability to selectively immobilize biomolecules on surfaces has far-reaching applications, including sensor development. A simple and widely applicable method for the photopatterning of chitosan films with biotin was presented. Chitosan is a biocompatible and biodegradable polymer. The proposed method was capable of forming spatially defined biotin features on the order of tens of microns, together with a significant reduction of non-specific protein binding and increase in hydrophilicity of the sensor surface. The entire patterning process, inclusive of the blocking step, could be completed in under an hour. This straightforward method for the selective patterning of the biocompatible polymer chitosan is expected to be widely useful in the field of bioanalysis.

Estimation of actomyosin active force maintained by tropomyosin and troponin complex under vertical forces in the in vitro motility assay system

PubMed Central

Ishii, Shuya; Kawai, Masataka; Ishiwata, Shin'ichi

2018-01-01

The interaction between actin filaments and myosin molecular motors is a power source of a variety of cellular functions including cell division, cell motility, and muscular contraction. In vitro motility assay examines actin filaments interacting with myosin molecules that are adhered to a substrate (e.g., glass surface). This assay has been the standard method of studying the molecular mechanisms of contraction under an optical microscope. While the force generation has been measured through an optically trapped bead to which an actin filament is attached, a force vector vertical to the glass surface has been largely ignored with the in vitro motility assay. The vertical vector is created by the gap (distance) between the trapped bead and the glass surface. In this report, we propose a method to estimate the angle between the actin filament and the glass surface by optically determining the gap size. This determination requires a motorized stage in a standard epi-fluorescence microscope equipped with optical tweezers. This facile method is applied to force measurements using both pure actin filaments, and thin filaments reconstituted from actin, tropomyosin and troponin. We find that the angle-corrected force per unit filament length in the active condition (pCa = 5.0) decreases as the angle between the filament and the glass surface increases; i.e. as the force in the vertical direction increases. At the same time, we demonstrate that the force on reconstituted thin filaments is approximately 1.5 times larger than that on pure actin filaments. The range of angles we tested was between 11° and 36° with the estimated measurement error less than 6°. These results suggest the ability of cytoplasmic tropomyosin isoforms maintaining actomyosin active force to stabilize cytoskeletal architecture. PMID:29420610

A novel high-throughput screening format to identify inhibitors of secreted acid sphingomyelinase.

PubMed

Mintzer, Robert J; Appell, Kenneth C; Cole, Andrew; Johns, Anthony; Pagila, Rene; Polokoff, Mark A; Tabas, Ira; Snider, R Michael; Meurer-Ogden, Janet A

2005-04-01

Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.

PubMed

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-08-18

Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20-85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications.

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins

PubMed Central

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-01-01

Background Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. Results We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20–85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. Conclusion This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications. PMID:16109166

A sensitive and innovative detection method for rapid C-reactive proteins analysis based on a micro-fluxgate sensor system

PubMed Central

Yang, Zhen; Zhi, Shaotao; Feng, Zhu; Lei, Chong; Zhou, Yong

2018-01-01

A sensitive and innovative assay system based on a micro-MEMS-fluxgate sensor and immunomagnetic beads-labels was developed for the rapid analysis of C-reactive proteins (CRP). The fluxgate sensor presented in this study was fabricated through standard micro-electro-mechanical system technology. A multi-loop magnetic core made of Fe-based amorphous ribbon was employed as the sensing element, and 3-D solenoid copper coils were used to control the sensing core. Antibody-conjugated immunomagnetic microbeads were strategically utilized as signal tags to label the CRP via the specific conjugation of CRP to polyclonal CRP antibodies. Separate Au film substrates were applied as immunoplatforms to immobilize CRP-beads labels through classical sandwich assays. Detection and quantification of the CRP at different concentrations were implemented by detecting the stray field of CRP labeled magnetic beads using the newly-developed micro-fluxgate sensor. The resulting system exhibited the required sensitivity, stability, reproducibility, and selectivity. A detection limit as low as 0.002 μg/mL CRP with a linearity range from 0.002 μg/mL to 10 μg/mL was achieved, and this suggested that the proposed biosystem possesses high sensitivity. In addition to the extremely low detection limit, the proposed method can be easily manipulated and possesses a quick response time. The response time of our sensor was less than 5 s, and the entire detection period for CRP analysis can be completed in less than 30 min using the current method. Given the detection performance and other advantages such as miniaturization, excellent stability and specificity, the proposed biosensor can be considered as a potential candidate for the rapid analysis of CRP, especially for point-of-care platforms. PMID:29601593

The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

PubMed

Nantakomol, Duangdao; Imwong, Malika; Soontarawirat, Ingfar; Kotjanya, Duangporn; Khakhai, Chulalak; Ohashi, Jun; Nuchnoi, Pornlada

2009-05-01

Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas. (c) 2008 Clinical Cytometry Society.

An enzyme-free flow cytometric bead assay for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification.

PubMed

Qi, Yan; Qiu, Liying; Fan, Wenjiao; Liu, Chenghui; Li, Zhengping

2017-08-07

A versatile flow cytometric bead assay (FCBA) coupled with a completely enzyme-free signal amplification mechanism is developed for the sensitive detection of microRNAs (miRNAs). This new strategy integrates click chemistry-mediated ligation chain reaction (CLCR) with hybridization chain reaction (HCR) for enzyme-free signal amplification on magnetic beads (MBs), and a flow cytometer for the robust fluorescence readout of the MBs. Firstly, target miRNA can initiate CLCR on the surface of MBs based on the click chemical ligation between dibenzocyclooctyne (DBCO)- and azide-modified single-stranded DNA (ssDNA) probes, and the amount of ligated ssDNA sequences on the MBs will be proportional to the dosage of target miRNA. Afterward, each of the ligated ssDNA products can trigger a cascade chain reaction of hybridization events between two alternating fluorophore-tagged hairpin probes, resulting in another signal amplification pathway with an amplified accumulation of fluorophores on the MBs. Finally, the fluorophore-anchored MBs are directly and rapidly analyzed by using a flow cytometer without any separation or elution processes. Herein, the click nucleic acid ligation only occurs on the surface of MBs, so the nonspecific ligations are greatly inhibited compared with that of ligation reaction performed in homogeneous solution. Furthermore, the signal amplification by CLCR-HCR is highly efficient but totally enzyme-free, which may overcome the potential drawbacks of conventional enzyme-catalyzed signal amplification protocols and lead to a high sensitivity. The CLCR-HCR-based FCBA has pushed the detection limit of let-7a miRNA down to the femtomolar (fM) level, showing great potential in miRNA-related biological studies and disease diagnosis.

Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

PubMed

Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

2012-01-01

We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.

Purifying Nucleic Acids from Samples of Extremely Low Biomass

NASA Technical Reports Server (NTRS)

La Duc, Myron; Osman, Shariff; Venkateswaran, Kasthuri

2008-01-01

A new method is able to circumvent the bias to which one commercial DNA extraction method falls prey with regard to the lysing of certain types of microbial cells, resulting in a truncated spectrum of microbial diversity. By prefacing the protocol with glass-bead-beating agitation (mechanically lysing a much more encompassing array of cell types and spores), the resulting microbial diversity detection is greatly enhanced. In preliminary studies, a commercially available automated DNA extraction method is effective at delivering total DNA yield, but only the non-hardy members of the bacterial bisque were represented in clone libraries, suggesting that this method was ineffective at lysing the hardier cell types. To circumvent such a bias in cells, yet another extraction method was devised. In this technique, samples are first subjected to a stringent bead-beating step, and then are processed via standard protocols. Prior to being loaded into extraction vials, samples are placed in micro-centrifuge bead tubes containing 50 micro-L of commercially produced lysis solution. After inverting several times, tubes are agitated at maximum speed for two minutes. Following agitation, tubes are centrifuged at 10,000 x g for one minute. At this time, the aqueous volumes are removed from the bead tubes and are loaded into extraction vials to be further processed via extraction regime. The new method couples two independent methodologies in such as way as to yield the highest concentration of PCR-amplifiable DNA with consistent and reproducible results and with the most accurate and encompassing report of species richness.

DNA methylation array analysis identifies breast cancer associated RPTOR, MGRN1 and RAPSN hypomethylation in peripheral blood DNA.

PubMed

Tang, Qiuqiong; Holland-Letz, Tim; Slynko, Alla; Cuk, Katarina; Marme, Frederik; Schott, Sarah; Heil, Jörg; Qu, Bin; Golatta, Michael; Bewerunge-Hudler, Melanie; Sutter, Christian; Surowy, Harald; Wappenschmidt, Barbara; Schmutzler, Rita; Hoth, Markus; Bugert, Peter; Bartram, Claus R; Sohn, Christof; Schneeweiss, Andreas; Yang, Rongxi; Burwinkel, Barbara

2016-09-27

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 x 10-6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

Portable point-of-care blood analysis system for global health (Conference Presentation)

NASA Astrophysics Data System (ADS)

Dou, James J.; Aitchison, James Stewart; Chen, Lu; Nayyar, Rakesh

2016-03-01

In this paper we present a portable blood analysis system based on a disposable cartridge and hand-held reader. The platform can perform all the sample preparation, detection and waste collection required to complete a clinical test. In order to demonstrate the utility of this approach a CD4 T cell enumeration was carried out. A handheld, point-of-care CD4 T cell system was developed based on this system. In particular we will describe a pneumatic, active pumping method to control the on-chip fluidic actuation. Reagents for the CD4 T cell counting assay were dried on a reagent plug to eliminate the need for cold chain storage when used in the field. A micromixer based on the active fluidic actuation was designed to complete sample staining with fluorescent dyes that was dried on the reagent plugs. A novel image detection and analysis algorithm was developed to detect and track the flight of target particles and cells during each analysis. The handheld, point-of-care CD4 testing system was benchmarked against clinical cytometer. The experimental results demonstrated experimental results were closely matched with the flow cytometry. The same platform can be further expanded into a bead-array detection system where other types of biomolecules such as proteins can be detected using the same detection system.

Allergy and allergic mediators in tears.

PubMed

Leonardi, Andrea

2013-12-01

The identification of inflammatory mediators in the tear fluid have been extensively used in ocular allergy to find either a 'disease marker', to better understand the immune mechanisms involved in the ocular surface inflammation, or to identify potential targets for therapeutic interventions. While the clinical characteristics allow a relatively convincing diagnosis of ocular allergic diseases, in the initial, non active phases, or in the chronic stages, the diagnosis may not be clear. Although not highly specific, total tear IgE can be measured with local tests by inserting a paper strip in the lower meniscus. The measurement of tear specific inflammatory markers, such as histamine, tryptase, ECP, IL-4, IL-5 and eotaxin, may be useful for the diagnosis or monitoring ocular allergy. New technologies such as multiplex bead assays, membrane-bound antibody array and proteomic techniques can characterize the distribution of a wide range of bioactive trace proteins in tears. Dozens of mediators, cytokines, chemokines, growth factors, angiogenic modulators, enzymes and inhibitors were thus identified in small tear samples using these techniques, providing the possible identification of specific biomarker for either specific disease or disease activity. However, to date, there is no a single specific laboratory test suitable for the diagnosis and monitoring of allergic conjunctivitis. Copyright © 2013 Elsevier Ltd. All rights reserved.

RCP: a novel probe design bias correction method for Illumina Methylation BeadChip.

PubMed

Niu, Liang; Xu, Zongli; Taylor, Jack A

2016-09-01

The Illumina HumanMethylation450 BeadChip has been extensively utilized in epigenome-wide association studies. This array and its successor, the MethylationEPIC array, use two types of probes-Infinium I (type I) and Infinium II (type II)-in order to increase genome coverage but differences in probe chemistries result in different type I and II distributions of methylation values. Ignoring the difference in distributions between the two probe types may bias downstream analysis. Here, we developed a novel method, called Regression on Correlated Probes (RCP), which uses the existing correlation between pairs of nearby type I and II probes to adjust the beta values of all type II probes. We evaluate the effect of this adjustment on reducing probe design type bias, reducing technical variation in duplicate samples, improving accuracy of measurements against known standards, and retention of biological signal. We find that RCP is statistically significantly better than unadjusted data or adjustment with alternative methods including SWAN and BMIQ. We incorporated the method into the R package ENmix, which is freely available from the Bioconductor website (https://www.bioconductor.org/packages/release/bioc/html/ENmix.html). niulg@ucmail.uc.edu Supplementary data are available at Bioinformatics online. Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the US.

Trapping and dynamic manipulation with magnetomotive photoacoustic imaging of targeted microspheres mimicking metastatic cancer cells trafficking in the vasculature

NASA Astrophysics Data System (ADS)

Wei, Chenwei; Xia, Jinjun; Pelivanov, Ivan; Hu, Xiaoge; Gao, Xiaohu; O'Donnell, Matthew

2012-02-01

Trapping and manipulation of micro-scale objects mimicking metastatic cancer cells in a flow field have been demonstrated with magnetomotive photoacoustic (mmPA) imaging. Coupled contrast agents combining gold nanorods (15 nm × 50 nm; absorption peak around 730 nm) with 15 nm diameter magnetic nanospheres were targeted to 10 μm polystyrene beads recirculating in a 1.6 mm diameter tube mimicking a human peripheral vessel. Targeted objects were then trapped by an external magnetic field produced by a dual magnet system consisting of two disc magnets separated by 6 cm to form a polarizing field (0.04 Tesla in the tube region) to magnetize the magnetic contrast agents, and a custom designed cone magnet array with a high magnetic field gradient (about 0.044 Tesla/mm in the tube region) producing a strong trapping force to magnetized contrast agents. Results show that polystyrene beads linked to nanocomposites can be trapped at flow rates up to 12 ml/min. It is shown that unwanted background in a photoacoustic image can be significantly suppressed by changing the position of the cone magnet array with respect to the tube, thus creating coherent movement of the trapped objects. This study makes mmPA imaging very promising for differential visualization of metastatic cells trafficking in the vasculature.

Massively parallel haplotyping on microscopic beads for the high-throughput phase analysis of single molecules.

PubMed

Boulanger, Jérôme; Muresan, Leila; Tiemann-Boege, Irene

2012-01-01

In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of less than 1:10,000. We also accurately quantified chimera formation during the exponential phase of PCR by different DNA polymerases.

A colorimetric indicator-displacement assay array for selective detection and identification of biological thiols.

PubMed

Qian, Sihua; Lin, Hengwei

2014-03-01

A simple, inexpensive yet highly selective colorimetric indicator-displacement assay array for the simultaneous detection and identification of three important biothiols at micromolar concentrations under physiological conditions and in real samples has been developed in this work. With use of an array composed of metal indicators and metal ions, clear differentiation among cysteine, homocysteine and glutathione was achieved. On the basis of the colour change of the array, quantification of each analyte was accomplished easily, and different biothiols were identified readily using standard chemometric approaches (hierarchical clustering analysis). Moreover, the colorimetric sensor array was not responsive to changes with 19 other natural amino acids, and it showed excellent reproducibility. Importantly, the sensor array developed was successfully applied to the determination and identification of the three biothiols in a real biological sample.

Integrated Optic Chemical-Biological Sensors

DTIC Science & Technology

1999-02-26

response. In this process, an enzyme ( urease ) acts as a catalyst, converting a specific substrate (urea) to a specific product (ammonia). Implementing...a sandwich assay, a urease labeled antibody is introduced to a surface bound antigen. This complex is exposed to urea, generating ammonia. Using a...containing suspected agents. After agent binding to the antibody-coated beads, an appropriate enzyme labeled antibody (an antibody with a urease label

Application of a receptor-binding-capture qRTPCR assay to concentrate human norovirus from sewage and to study the distribution and stability of the virus

USDA-ARS?s Scientific Manuscript database

Human noroviruses (HuNoVs) are major agents of gastroenteritis and water is an important route of transmission. Using magnetic beads conjugated with blood group-like antigens previously reported as receptors for HuNoV, we developed a simple and rapid receptor-binding capture and magnetic sequestra...

Highly specific fiber optic immunosensor coupled with immunomagnetic separation for detection of low levels of Listeria monocytogenes and L. ivanovii

PubMed Central

2012-01-01

Background Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection. Results Anti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (103–108 CFU/mL) tested in the IMS assay; the 1-μm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-μm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dynabeads anti-Listeria antibody (9 %). Furthermore, capture efficiency for MyOne-2D12 was highly specific for L. monocytogenes and L. ivanovii. Subsequently, we captured L. monocytogenes by MyOne-2D12 and MyOne-3F8 from hotdogs inoculated with mono- or co-cultures of L. monocytogenes and L. innocua (10–40 CFU/g), enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qPCR assays. The detection limit for L. monocytogenes and L. ivanovii by the fiber-optic immunosensor was 3 × 102 CFU/mL using MAb-2D12 as capture and reporter antibody. Selective media plating, light-scattering, and qPCR assays confirmed the IMS and fiber-optic results. Conclusions IMS coupled with a fiber-optic sensor using anti-InlA MAb is highly specific for L. monocytogenes and L. ivanovii and enabled detection of these pathogens at low levels from buffer or food. PMID:23176167

DNA: Polymer and molecular code

NASA Astrophysics Data System (ADS)

Shivashankar, G. V.

1999-10-01

The thesis work focusses upon two aspects of DNA, the polymer and the molecular code. Our approach was to bring single molecule micromanipulation methods to the study of DNA. It included a home built optical microscope combined with an atomic force microscope and an optical tweezer. This combined approach led to a novel method to graft a single DNA molecule onto a force cantilever using the optical tweezer and local heating. With this method, a force versus extension assay of double stranded DNA was realized. The resolution was about 10 picoN. To improve on this force measurement resolution, a simple light backscattering technique was developed and used to probe the DNA polymer flexibility and its fluctuations. It combined the optical tweezer to trap a DNA tethered bead and the laser backscattering to detect the beads Brownian fluctuations. With this technique the resolution was about 0.1 picoN with a millisecond access time, and the whole entropic part of the DNA force-extension was measured. With this experimental strategy, we measured the polymerization of the protein RecA on an isolated double stranded DNA. We observed the progressive decoration of RecA on the l DNA molecule, which results in the extension of l , due to unwinding of the double helix. The dynamics of polymerization, the resulting change in the DNA entropic elasticity and the role of ATP hydrolysis were the main parts of the study. A simple model for RecA assembly on DNA was proposed. This work presents a first step in the study of genetic recombination. Recently we have started a study of equilibrium binding which utilizes fluorescence polarization methods to probe the polymerization of RecA on single stranded DNA. In addition to the study of material properties of DNA and DNA-RecA, we have developed experiments for which the code of the DNA is central. We studied one aspect of DNA as a molecular code, using different techniques. In particular the programmatic use of template specificity makes gene expression a prime example of a biological code. We developed a novel method of making DNA micro- arrays, the so-called DNA chip. Using the optical tweezer concept, we were able to pattern biomolecules on a solid substrate, developing a new type of sub-micron laser lithography. A laser beam is focused onto a thin gold film on a glass substrate. Laser ablation of gold results in local aggregation of nanometer scale beads conjugated with small DNA oligonucleotides, with sub-micron resolution. This leads to specific detection of cDNA and RNA molecules. We built a simple micro-array fabrication and detection in the laboratory, based on this method, to probe addressable pools (genes, proteins or antibodies). We have lately used molecular beacons (single stranded DNA with a stem-loop structure containing a fluorophore and quencher), for the direct detection of unlabelled mRNA. As a first step towards a study of the dynamics of the biological code, we have begun to examine the patterns of gene expression during virus (T7 phage) infection of E-coli bacteria.

Comprehensive Astronaut Immune Assessment Following a Short-Duration Space Flight

NASA Technical Reports Server (NTRS)

Crucian, Brian; Stowe, Raymond; Yetman, Deborah; Pierson, Duane; Sams, Clarence

2006-01-01

Immune system dysregulation has been demonstrated to occur during spaceflight and has the potential to cause serious health risks to crewmembers participating in exploration class missions. As a part of an ongoing NASA flight experiment assessing viral immunity (DSO-500), a generalized immune assessment was performed on 3 crewmembers who participated in the recent STS-114 Space Shuttle mission. The following assays were performed: (1) comprehensive immunophenotype analysis; (2) T cell function/intracellular cytokine profiles; (4) secreted Th1/Th2 cytokine profiles via cytometric bead array. Immunophenotype analysis included a leukocyte differential, lymphocyte subsets, T cell subsets, cytotoxic/effector CD8+ T cells, memory/naive T cell subsets and constitutively activated T cells. Study timepoints were L-180, L-65, L-10, R+0, R+3 and R+14. Detailed data are presented in the poster text. As expected from a limited number of human subjects, data tended to vary with respect to most parameters. Specific post-flight alterations were as follows (subject number in parentheses): Granulocytosis (2/3), reduced NK cells (3/3), elevated CD4/CD8 ratio (3/3), general CD8+ phenotype shift to a less differentiated phenotype (3/3), elevated levels of memory CD4+ T cells (3/3), loss of L-selectin on T cell subsets (3/3), increased levels of activated T cells (2/3), reduced IL-2 producing T cell subsets (3/3), levels of IFNg producing T cells were unchanged. CD8+ T cell expression of the CD69 activation markers following whole blood stimulation with SEA+SEB were dramatically reduced postflight (3/3), whereas other T cell function assessments were largely unchanged. Cytometric bead array assessment of secreted T cell cytokines was performed, following whole blood stimulation with either CD3/CD28 antibodies or PMA+ionomycin for 48 hours. Specific cytokines assessed were IFNg, TNFa, IL-2, IL-4, IL-5, IL-10. Following CD3/CD28 stimulation, all three crewmembers had a mission-associated reduction in the levels of secreted IFNg. One crewmember had a post-flight inversion in the IFNg/IL-10 ratio postflight, which trended back to baseline by R+14. Detailed cytokine data are presented in the poster text. This testing regimen was designed to correlate immunophenotype changes (thought to correspond to specific in-vivo immune responses or pathogenesis), against altered leukocyte function and cytokine profiles. In-flight studies are required to determine if post-flight alterations are reflective of the in-flight condition, or are a response to landing and readaptation.

Optimized MOL-PCR for Characterization of Microbial Pathogens.

PubMed

Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

2016-01-06

Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium. Copyright © 2016 John Wiley & Sons, Inc.

Single-cell analysis and sorting using droplet-based microfluidics.

PubMed

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2013-05-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

Single-cell analysis and sorting using droplet-based microfluidics

PubMed Central

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2014-01-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. as an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. the beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ~200 Hz as well as cell enrichment. the microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ~1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d. PMID:23558786

Evolution of the Immune Response to Chronic Airway Colonization with Aspergillus fumigatus Hyphae.

PubMed

Urb, Mirjam; Snarr, Brendan D; Wojewodka, Gabriella; Lehoux, Mélanie; Lee, Mark J; Ralph, Benjamin; Divangahi, Maziar; King, Irah L; McGovern, Toby K; Martin, James G; Fraser, Richard; Radzioch, Danuta; Sheppard, Donald C

2015-09-01

Airway colonization by the mold Aspergillus fumigatus is common in patients with underlying lung disease and is associated with chronic airway inflammation. Studies probing the inflammatory response to colonization with A. fumigatus hyphae have been hampered by the lack of a model of chronic colonization in immunocompetent mice. By infecting mice intratracheally with conidia embedded in agar beads (Af beads), we have established an in vivo model to study the natural history of airway colonization with live A. fumigatus hyphae. Histopathological examination and galactomannan assay of lung homogenates demonstrated that hyphae exited beads and persisted in the lungs of mice up to 28 days postinfection without invasive disease. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammatory reaction and peribronchial infiltration of lymphocytes. Whole-lung cytokine analysis from Af bead-infected mice revealed an increase in proinflammatory cytokines and chemokines early in infection. Evidence of a Th2 type response was observed only early in the course of colonization, including increased levels of interleukin-4 (IL-4), elevated IgE levels in serum, and a mild increase in airway responsiveness. Pulmonary T cell subset analysis during infection mirrored these results with an initial transient increase in IL-4-producing CD4(+) T cells, followed by a rise in IL-17 and Foxp3(+) cells by day 14. These results provide the first report of the evolution of the immune response to A. fumigatus hyphal colonization. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Three-dimensional transgenic cell model to quantify genotoxic effects of space environment

NASA Astrophysics Data System (ADS)

Gonda, S. R.; Wu, H.; Pingerelli, P. L.; Glickman, B. W.

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of multiple copies of defined target genes for genotoxic assessment. Rat 2λ fibroblasts, genetically engineered to contain high-density target genes for mutagenesis (Stratagene, Inc., Austin, TX), were cocultured with human epithelial cells on Cytodex beads in the High Aspect Ratio Bioreactor (Synthecon, Inc, Houston, TX). Multi-bead aggregates were formed by day 5 following the complete covering of the beads by fibroblasts. Cellular retraction occurred 8-14 days after coculture initiation culminating in spheroids retaining few or no beads. Analysis of the resulting tissue assemblies revealed: multicellular spheroids, fibroblasts synthesized collagen, and cell viability was retained for the 30-day test period after removal from the bioreactor. Quantification of mutation at the LacI gene in Rat 2λ fibroblasts in spheroids exposed to 0-2 Gy neon using the Big Blue color assay (Stratagene, Inc.), revealed a linear dose-response for mutation induction. Limited sequencing analysis of mutant clones from 0.25 or 1 Gy exposures revealed a higher frequency of deletions and multiple base sequencing changes with increasing dose. These results suggest that the three-dimensional, multicellular tissue assembly model produced in NASA bioreactors are applicable to a wide variety of studies involving the quantification and identification of genotocity including measurement of the inherent damage incurred in Space.

Pectin-cysteine conjugate: synthesis and in-vitro evaluation of its potential for drug delivery.

PubMed

Majzoob, Sayeh; Atyabi, Fatemeh; Dorkoosh, Farid; Kafedjiiski, Krum; Loretz, Brigitta; Bernkop-Schnürch, Andreas

2006-12-01

This study was aimed at improving certain properties of pectin by introduction of thiol moieties on the polymer. Thiolated pectin was synthesized by covalent attachment of cysteine. Pectin-cysteine conjugate was evaluated for its ability to be degraded by pectinolytic enzyme. The toxicity profile of the thiolated polymer in Caco-2-cells, its permeation enhancing effect and its mucoadhesive and swelling properties were studied. Moreover insulin-loaded hydrogel beads of the new polymer were examined for their stability in simulated gastrointestinal conditions and their drug release profile. The new polymer displayed 892.27 +/- 68.68 micromol thiol groups immobilized per g polymer, and proved to have retained its biodegradability, upon addition of Pectinex Ultra SPL in-vitro, determined by viscosity measurements and titration method. Pectin-cysteine showed no severe toxicity in Caco-2 cells, as tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Moreover, the synthesized polymer exhibited a relative permeation enhancement ratio of 1.61 for sodium fluorescein, compared to unmodified pectin. Pectin-cysteine conjugate exhibited approximately 5-fold increased in in-vitro adhesion duration and significantly improved cohesive properties. Zinc pectin-cysteine beads showed improved stability in simulated gastrointestinal media; however, insulin release from these beads followed the same profile as unmodified zinc pectinate beads. Due to favourable safety and biodegradability profile, and improved cohesive and permeation-enhancing properties, pectin-cysteine might be a promising excipient in various transmucosal drug delivery systems.

Cytokine and chemokine profiles in fibromyalgia, rheumatoid arthritis and systemic lupus erythematosus: a potentially useful tool in differential diagnosis.

PubMed

Wallace, Daniel J; Gavin, Igor M; Karpenko, Oleksly; Barkhordar, Farnaz; Gillis, Bruce S

2015-06-01

Making a correct diagnosis is pivotal in the practice of clinical rheumatology. Occasionally, the consultation fails to provide desired clarity in making labeling an individual as having fibromyalgia (FM), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). A chemokine and cytokine multiplex assay was developed and tested with the goal of improving and achieving an accurate differential diagnosis. 160 patients with FM, 98 with RA and 100 with SLE fulfilling accepted criteria were recruited and compared to 119 controls. Supernatant cytokine concentrations for IL-6, IL-8, MIP-1 alpha and MIP-1 beta were determined using the Luminex multiplex immunoassay bead array technology after mitogenic stimulation of cultured peripheral blood mononuclear cells. Each patient's profile was scored using a logistical regression model to achieve statistically determined weighting for each chemokine and cytokine. Among the 477 patients evaluated, the mean scores for FM (1.7 ± 1.2; 1.52-1.89), controls (-3.56 ± 5.7; -4.59 to -2.54), RA (-0.68 ± 2.26; -1.12 to -0.23) and SLE (-1.45 ± 3.34, -2.1 to -0.79). Ninety-three percent with FM scored positive compared to only 11% of healthy controls, 69% RA or 71% SLE patients had negative scores. The sensitivity, specificity, positive predictive and negative predictive value for having FM compared to controls was 93, 89, 92 and 91%, respectively (p < 2.2 × 10(-16)). Evaluating cytokine and chemokine profiles in stimulated cells reveals patterns that are uniquely present in patients with FM. This assay can be a useful tool in assisting clinicians in differentiating systemic inflammatory autoimmune processes from FM and its related syndromes and healthy individuals.

SNPs in stress-responsive rice genes: validation, genotyping, functional relevance and population structure

PubMed Central

2012-01-01

Background Single nucleotide polymorphism (SNP) validation and large-scale genotyping are required to maximize the use of DNA sequence variation and determine the functional relevance of candidate genes for complex stress tolerance traits through genetic association in rice. We used the bead array platform-based Illumina GoldenGate assay to validate and genotype SNPs in a select set of stress-responsive genes to understand their functional relevance and study the population structure in rice. Results Of the 384 putative SNPs assayed, we successfully validated and genotyped 362 (94.3%). Of these 325 (84.6%) showed polymorphism among the 91 rice genotypes examined. Physical distribution, degree of allele sharing, admixtures and introgression, and amino acid replacement of SNPs in 263 abiotic and 62 biotic stress-responsive genes provided clues for identification and targeted mapping of trait-associated genomic regions. We assessed the functional and adaptive significance of validated SNPs in a set of contrasting drought tolerant upland and sensitive lowland rice genotypes by correlating their allelic variation with amino acid sequence alterations in catalytic domains and three-dimensional secondary protein structure encoded by stress-responsive genes. We found a strong genetic association among SNPs in the nine stress-responsive genes with upland and lowland ecological adaptation. Higher nucleotide diversity was observed in indica accessions compared with other rice sub-populations based on different population genetic parameters. The inferred ancestry of 16% among rice genotypes was derived from admixed populations with the maximum between upland aus and wild Oryza species. Conclusions SNPs validated in biotic and abiotic stress-responsive rice genes can be used in association analyses to identify candidate genes and develop functional markers for stress tolerance in rice. PMID:22921105

Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India.

PubMed

Kaushal, H; Bras-Gonçalves, R; Avishek, K; Kumar Deep, D; Petitdidier, E; Lemesre, J-L; Papierok, G; Kumar, S; Ramesh, V; Salotra, P

2016-07-01

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL. © 2016 British Society for Immunology.

Type I IFN-related NETosis in ataxia telangiectasia and Artemis deficiency.

PubMed

Gul, Ersin; Sayar, Esra Hazar; Gungor, Bilgi; Eroglu, Fehime Kara; Surucu, Naz; Keles, Sevgi; Guner, Sukru Nail; Findik, Siddika; Alpdundar, Esin; Ayanoglu, Ihsan Cihan; Kayaoglu, Basak; Geckin, Busra Nur; Sanli, Hatice Asena; Kahraman, Tamer; Yakicier, Cengiz; Muftuoglu, Meltem; Oguz, Berna; Cagdas Ayvaz, Deniz Nazire; Gursel, Ihsan; Ozen, Seza; Reisli, Ismail; Gursel, Mayda

2017-11-16

Pathological inflammatory syndromes of unknown etiology are commonly observed in ataxia telangiectasia (AT) and Artemis deficiency. Similar inflammatory manifestations also exist in patients with STING-associated vasculopathy in infancy (SAVI). We sought to test the hypothesis that the inflammation-associated manifestations observed in patients with AT and Artemis deficiency stem from increased type I IFN signature leading to neutrophil-mediated pathological damage. Cytokine/protein signatures were determined by ELISA, cytometric bead array, or quantitative PCR. Stat1 phosphorylation levels were determined by flow cytometry. DNA species accumulating in the cytosol of patients' cells were quantified microscopically and flow cytometrically. Propensity of isolated polymorhonuclear granulocytes to form neutrophil extracellular traps (NETs) was determined using fluorescence microscopy and picogreen assay. Neutrophil reactive oxygen species levels and mitochondrial stress were assayed using fluorogenic probes, microscopy, and flow cytometry. Type I and III IFN signatures were elevated in plasma and peripheral blood cells of patients with AT, Artemis deficiency, and SAVI. Chronic IFN production stemmed from the accumulation of DNA in the cytoplasm of AT and Artemis-deficient cells. Neutrophils isolated from patients spontaneously produced NETs and displayed indicators of oxidative and mitochondrial stress, supportive of their NETotic tendencies. A similar phenomenon was also observed in neutrophils from healthy controls exposed to patient plasma samples or exogeneous IFN-α. Type I IFN-mediated neutrophil activation and NET formation may contribute to inflammatory manifestations observed in patients with AT, Artemis deficiency, and SAVI. Thus, neutrophils represent a promising target to manage inflammatory syndromes in diseases with active type I IFN signature. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput.

PubMed

Gierahn, Todd M; Wadsworth, Marc H; Hughes, Travis K; Bryson, Bryan D; Butler, Andrew; Satija, Rahul; Fortune, Sarah; Love, J Christopher; Shalek, Alex K

2017-04-01

Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

PubMed Central

Euler, Milena; Wang, Yongjie; Heidenreich, Doris; Patel, Pranav; Strohmeier, Oliver; Hakenberg, Sydney; Niedrig, Matthias; Hufert, Frank T.

2013-01-01

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. PMID:23345286

Acetylcholinesterase affinity-based screening assay on Lippia gracilis Schauer extracts.

PubMed

Vanzolini, K L; da F Sprenger, R; Leme, G M; de S Moraes, V R; Vilela, A F L; Cardoso, C L; Cass, Q B

2018-05-10

The use of affinity-based protein assay produced by covalently linking acetylcholinesterase to magnetic beads, followed by chemical characterization of the selective binders using Liquid Chromatography with tandem High-Resolution Mass Spectrometry (LC-HRMS) is herein described for profiling crude aqueous natural product extracts. The fishing assay was first modulated using galanthamine as a reference ligand and then, the assay condition was adjusted for the aqueous leaves extracts obtained from Lippia gracilis Schauer (genotype 201) that was used as the natural combinatory library. From the experiments, a selective binder has been undisclosed with an accurate mass of 449.1131 m/z and identified as eriodictyol 2'-O-glucoside or eriodictyol 3'-O-glucoside. The selectivity of the binding assay was demonstrated, as much as, that erydictiol 7-O-glucoside was not fished, although it was present in the crude aqueous extract. The binding assay platform exhibited high specificity and did not require any sample pretreatment, making it appropriate for profiling binders at natural libraries. Copyright © 2018 Elsevier B.V. All rights reserved.

A capillary-based multiplexed isothermal nucleic acid-based test for sexually transmitted diseases in patients.

PubMed

Xu, Gaolian; Zhao, Hang; Cooper, Jonathan M; Reboud, Julien

2016-10-06

We demonstrate a multiplexed loop mediated isothermal amplification (LAMP) assay for infectious disease diagnostics, where the analytical process flow of target pathogens genomic DNA is performed manually by moving magnetic beads through a series of plugs in a capillary. Heat is provided by a water bath and the results are read by the naked eye, enabling applications in low resource settings.

Integration of Multiplex Bead Assays for Parasitic Diseases into a National, Population-Based Serosurvey of Women 15-39 Years of Age in Cambodia

PubMed Central

Priest, Jeffrey W.; Jenks, M. Harley; Moss, Delynn M.; Mao, Bunsoth; Buth, Sokhal; Wannemuehler, Kathleen; Soeung, Sann Chan; Lucchi, Naomi W.; Udhayakumar, Venkatachalam; Gregory, Christopher J.; Huy, Rekol; Muth, Sinuon; Lammie, Patrick J.

2016-01-01

Collection of surveillance data is essential for monitoring and evaluation of public health programs. Integrated collection of household-based health data, now routinely carried out in many countries through demographic health surveys and multiple indicator surveys, provides critical measures of progress in health delivery. In contrast, biomarker surveys typically focus on single or related measures of malaria infection, HIV status, vaccination coverage, or immunity status for vaccine-preventable diseases (VPD). Here we describe an integrated biomarker survey based on use of a multiplex bead assay (MBA) to simultaneously measure antibody responses to multiple parasitic diseases of public health importance as part of a VPD serological survey in Cambodia. A nationally-representative cluster-based survey was used to collect serum samples from women of child-bearing age. Samples were tested by MBA for immunoglobulin G antibodies recognizing recombinant antigens from Plasmodium falciparum and P. vivax, Wuchereria bancrofti, Toxoplasma gondii, Taenia solium, and Strongyloides stercoralis. Serologic IgG antibody results were useful both for generating national prevalence estimates for the parasitic diseases of interest and for confirming the highly focal distributions of some of these infections. Integrated surveys offer an opportunity to systematically assess the status of multiple public health programs and measure progress toward Millennium Development Goals. PMID:27136913

Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation

PubMed Central

2016-01-01

Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. During drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing “fit-for-purpose” bioanalytical approaches. Enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ADA detection due to their high sensitivity and throughput. During the past decade, LC/MS has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. In fully taking these advantages, we describe here an immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADA in human plasma. Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or anti-drug Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ADA is then released from the beads and subjected to trypsin digestion followed by LC/MS detection of specific universal peptides for each ADA isotype. The LC/MS data are analyzed using cut-point and calibration curve. The proof-of-concept of this methodology is demonstrated by detecting preexisting ADA in human plasma. PMID:27034966

Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation.

PubMed

Chen, Lin-Zhi; Roos, David; Philip, Elsy

2016-01-01

Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-drug antibodies that can pose problems for both patient safety and product efficacy. During drug development immunogenicity is usually examined by risk-based approach along with specific strategies for developing "fit-for-purpose" bioanalytical approaches. Enzyme-linked immunosorbent assays and electrochemiluminescence immunoassays are the most widely used platform for ADA detection due to their high sensitivity and throughput. During the past decade, LC/MS has emerged as a promising technology for quantitation of biotherapeutics and protein biomarkers in biological matrices, mainly owing to its high specificity, selectivity, multiplexing, and wide dynamic range. In fully taking these advantages, we describe here an immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADA in human plasma. Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or anti-drug Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a magnet. ADA is then released from the beads and subjected to trypsin digestion followed by LC/MS detection of specific universal peptides for each ADA isotype. The LC/MS data are analyzed using cut-point and calibration curve. The proof-of-concept of this methodology is demonstrated by detecting preexisting ADA in human plasma.

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.

PubMed

Estes, Matthew D; Yang, Jianing; Duane, Brett; Smith, Stan; Brooks, Carla; Nordquist, Alan; Zenhausern, Frederic

2012-12-07

This study reports the design, prototyping, and assay development of multiplexed polymerase chain reaction (PCR) on a plastic microfluidic device. Amplification of 17 DNA loci is carried out directly on-chip as part of a system for continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE). For enhanced performance of on-chip PCR amplification, improved control systems have been developed making use of customized Peltier assemblies, valve actuators, software, and amplification chemistry protocols. Multiple enhancements to the microfluidic chip design have been enacted to improve the reliability of sample delivery through the various on-chip modules. This work has been enabled by the encapsulation of PCR reagents into a solid phase material through an optimized Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process. SPEAM bead technology is reliably coupled with precise microfluidic metering and dispensing for efficient amplification and subsequent DNA short tandem repeat (STR) fragment analysis. This provides a means of on-chip reagent storage suitable for microfluidic automation, with the long shelf-life necessary for point-of-care (POC) or field deployable applications. This paper reports the first high quality 17-plex forensic STR amplification from a reference sample in a microfluidic chip with preloaded solid phase reagents, that is designed for integration with up and downstream processing.

Dual chronoamperometric detection of enzymatic biomarkers using magnetic beads and a low-cost flow cell.

PubMed

Moral-Vico, Javier; Barallat, Jaume; Abad, Llibertat; Olivé-Monllau, Rosa; Muñoz-Pascual, Francesc Xavier; Galán Ortega, Amparo; del Campo, F Javier; Baldrich, Eva

2015-07-15

In this work we report on the production of a low cost microfluidic device for the multiplexed electrochemical detection of magneto bioassays. As a proof of concept, the device has been used to detect myeloperoxidase (MPO), a cardiovascular biomarker. With this purpose, two bioassays have been optimized in parallel onto magnetic beads (MBs) for the simultaneous detection of MPO endogenous peroxidase activity and quantification of total MPO. Since the two bioassays produced signals of different magnitude for each concentration of MPO tested, two detection strategies have been compared, which entailed registering steady state currents (Iss) under substrate flow, and measuring the peak currents (Ip) produced in a stopped flow approach. As it will be shown, appropriate tuning of the detection and flow conditions can provide extremely sensitive detection, but also allow simultaneous detection of assays or parameters that would produce signals of different orders of magnitude when measured by a single detection strategy. In order to demonstrate the feasibility of the detection strategy reported, a dual MPO mass and activity assay has been finally applied to the study of 10 real plasma samples, allowing patient classification according to the risk of suffering a cardiovascular event. Copyright © 2015 Elsevier B.V. All rights reserved.

Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

PubMed

Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

2010-02-15

A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

LVAD Implant as a Bridge to Heart Transplantation is Associated with Allosensitization as Measured by Single Antigen Bead Assay

PubMed Central

Shankar, Nisha; Daly, Richard; Geske, Jennifer; Kushwaha, Sudhir K; Timmons, Michael; Joyce, Lyle; Stulak, John; Gandhi, Manish; Kremers, Walter; Park, Soon; Pereira, Naveen L

2013-01-01

Background Left ventricular assist devices (LVAD) as a bridge (BTT) to heart transplantation (HTX) may be limited by the formation of anti-HLA antibodies. Whether sensitization occurs with continuous axial flow LVAD implant as assessed by Single Antigen Bead (SAB) assay is unknown. Methods Cytotoxic panel reactive antibody (PRA) and SAB assays were analyzed in HTX recipients undergoing LVAD implant as a BTT. Sensitization was defined as peak anti-HLA antibody values of >2000 mean fluorescent intensity as these values have been found to correlate with flow cytometric crossmatch results. Results LVADs were implanted as BTT in 30 patients. There were 7% (2/30) of patients prior to and no patients after LVAD implant with PRA >10%. However, 20% (6/30) of patients prior to and 53% (16/30) after LVAD were sensitized as measured by SAB (p=0.024). At HTX, 47% (14/30) of patients remained sensitized. A positive virtual crossmatch was observed in 28% (4/14) of the sensitized patients at HTX. There was no difference between the sensitized and non-sensitized groups (p>0.4 for all) in usage of blood products (64 11 vs. 63 39 units), time to HTX (286 63 vs. 257 48 days) and 1 year after HTX, there were no differences in rejection (total rejection score 0.30 vs. 0.37) and survival (93% vs. 88%). Conclusion Allosensitization after LVAD is common despite cytotoxic PRA being negative. One year after HTX, this sensitization does not translate into increased acute cellular or antibody mediated rejection or reduced survival. PMID:23743727

Next-Generation Autoantibody Testing by Combination of Screening and Confirmation-the CytoBead® Technology.

PubMed

Sowa, Mandy; Hiemann, Rico; Schierack, Peter; Reinhold, Dirk; Conrad, Karsten; Roggenbuck, Dirk

2017-08-01

Occurrence of autoantibodies (autoAbs) is a hallmark of autoimmune diseases, and the analysis thereof is an essential part in the diagnosis of organ-specific autoimmune and systemic autoimmune rheumatic diseases (SARD), especially connective tissue diseases (CTDs). Due to the appearance of autoAb profiles in SARD patients and the complexity of the corresponding serological diagnosis, different diagnostic strategies have been suggested for appropriate autoAb testing. Thus, evolving assay techniques and the continuous discovery of novel autoantigens have greatly influenced the development of these strategies. Antinuclear antibody (ANA) analysis by indirect immunofluorescence (IIF) on tissue and later cellular substrates was one of the first tests introduced into clinical routine and is still an indispensable tool for CTD serology. Thus, screening for ANA by IIF is recommended to be followed by confirmatory testing of positive findings employing different assay techniques. Given the continuous growth in the demand for autoAb testing, IIF has been challenged as the standard method for ANA and other autoAb analyses due to lacking automation, standardization, modern data management, and human bias in IIF pattern interpretation. To address these limitations of autoAb testing, the CytoBead® technique has been introduced recently which enables automated interpretation of cell-based IIF and quantitative autoAb multiplexing by addressable microbead immunoassays in one reaction environment. Thus, autoAb screening and confirmatory testing can be combined for the first time. The present review discusses the history of autoAb assay techniques in this context and gives an overview and outlook of the recent progress in emerging technologies.

Femtosecond laser modification of an array of vertically aligned carbon nanotubes intercalated with Fe phase nanoparticles

PubMed Central

2013-01-01

Femtosecond lasers (FSL) are playing an increasingly important role in materials research, characterization, and modification. Due to an extremely short pulse width, interactions of FSL irradiation with solid surfaces attract special interest, and a number of unusual phenomena resulted in the formation of new materials are expected. Here, we report on a new nanostructure observed after the interaction of FSL irradiation with arrays of vertically aligned carbon nanotubes (CNTs) intercalated with iron phase catalyst nanoparticles. It was revealed that the FSL laser ablation transforms the topmost layer of CNT array into iron phase nanospheres (40 to 680 nm in diameter) located at the tip of the CNT bundles of conical shape. Besides, the smaller nanospheres (10 to 30 nm in diameter) are found to be beaded at the sides of these bundles. Some of the larger nanospheres are encapsulated into carbon shells, which sometime are found to contain CNTs. The mechanism of creation of such nanostructures is proposed. PMID:24004518

Femtosecond laser modification of an array of vertically aligned carbon nanotubes intercalated with Fe phase nanoparticles.

PubMed

Labunov, Vladimir; Prudnikava, Alena; Bushuk, Serguei; Filatov, Serguei; Shulitski, Boris; Tay, Beng Kang; Shaman, Yury; Basaev, Alexander

2013-09-03

Femtosecond lasers (FSL) are playing an increasingly important role in materials research, characterization, and modification. Due to an extremely short pulse width, interactions of FSL irradiation with solid surfaces attract special interest, and a number of unusual phenomena resulted in the formation of new materials are expected. Here, we report on a new nanostructure observed after the interaction of FSL irradiation with arrays of vertically aligned carbon nanotubes (CNTs) intercalated with iron phase catalyst nanoparticles. It was revealed that the FSL laser ablation transforms the topmost layer of CNT array into iron phase nanospheres (40 to 680 nm in diameter) located at the tip of the CNT bundles of conical shape. Besides, the smaller nanospheres (10 to 30 nm in diameter) are found to be beaded at the sides of these bundles. Some of the larger nanospheres are encapsulated into carbon shells, which sometime are found to contain CNTs. The mechanism of creation of such nanostructures is proposed.

Enzymatic hydrolysate-induced displacement reaction with multifunctional silica beads doped with horseradish peroxidase-thionine conjugate for ultrasensitive electrochemical immunoassay.

PubMed

Lin, Youxiu; Zhou, Qian; Lin, Yuping; Tang, Dianping; Niessner, Reinhard; Knopp, Dietmar

2015-08-18

A novel (invertase) enzymatic hydrolysate-triggered displacement reaction strategy with multifunctional silica beads, doped with horseradish peroxidase-thionine (HRP-Thi) conjugate, was developed for competitive-type electrochemical immunoassay of small molecular aflatoxin B1 (AFB1). The competitive-type displacement reaction was carried out on the basis of the affinity difference between enzymatic hydrolysate (glucose) and its analogue (dextran) for concanavalin A (Con A) binding sites. Initially, thionine-HRP conjugates were doped into nanometer-sized silica beads using the reverse micelle method. Then monoclonal anti-AFB1 antibody and Con A were covalently conjugated to the silica beads. The immunosensor was prepared by means of immobilizing the multifunctional silica beads on a dextran-modified sensing interface via the dextran-Con A binding reaction. Gold nanoparticles functionalized with AFB1-bovine serum albumin conjugate (AFB1-BSA) and invertase were utilized as the trace tag. Upon target AFB1 introduction, a competitive-type immunoreaction was implemented between the analyte and the labeled AFB1-BSA on the nanogold particles for the immobilized anti-AFB1 antibody on the electrode. The invertase followed by gold nanoparticles hydrolyzed sucrose into glucose and fructose. The produced glucose displaced the multifunctional silica beads from the electrode based on the classical dextran-Con A-glucose system, thus decreasing the catalytic efficiency of the immobilized HRP on the electrode relative to that of the H2O2-thionine system. Under optimal conditions, the detectable electrochemical signal increased with the increasing target AFB1 in a dynamic working range from 3.0 pg mL(-1) to 20 ng mL(-1) with a detection limit of 2.7 pg mL(-1). The strong bioconjugation with two nanostructures also resulted in a good repeatability and interassay precision down to 9.3%. Finally, the methodology was further validated for analysis of naturally contaminated or spiked AFB1 peanut samples, giving results matched well with those from a commercialized AFB1 enzyme-linked immunosorbent assay kit. Importantly, the system provides a signal-on competitive-type immunosensing platform for ultrasensitive detection of small molecules.

Compatibility of epirubicin-loaded DC bead™ with different non-ionic contrast media.

PubMed

Sarakbi, Iman; Krämer, Irene

2016-12-01

The aim of this study was to determine the compatibility of epirubicin-loaded DC bead™ with different non-ionic contrast media over a period of seven days when stored light protected under refrigerated conditions. DC bead™ (2 ml) (Biocompatibles UK Ltd) of the bead size 70-150 µm ( = DC bead M1) or bead size 100-300 µm were loaded with 75 mg epirubicin powder formulation (Farmorubicin® dissolved in 3 ml water for injection to a concentration of 25 mg/ml) or 76 mg epirubicin injection solution (Epimedac® 2 mg/ml) within 2 h or 6 h, respectively. After removal of the excess solution, the epirubicin-loaded beads were mixed in polypropylene syringes with an equal volume (∼1.5 ml) of contrast media, i.e. Accupaque™ 300 (Nycomed Inc.), Imeron® 300 (Bracco S.p.A), Ultravist® 300 (Bayer Pharma AG), Visipaque™ 320 (GE Healthcare) and agitated in a controlled manner to get a homogenous suspension. Syringes with loaded beads in contrast media were stored protected from light under refrigeration (2-8℃). Compatibility was determined by measuring epirubicin concentrations in the suspensions in triplicate on day 0, 1, and 7. A reversed phase high-performance liquid chromatography assay with ultraviolet detection was utilized to analyze the concentration and purity of epirubicin. Mixing of epirubicin-loaded beads with different non-ionic contrast media released 0.1-0.5% of epirubicin over a period of 24 h, irrespectively, of the DC bead™ size or type of contrast media. No further elution or degradation was observed after seven days when the admixtures were stored protected from light under refrigeration. Compatibility of epirubicin-loaded DC bead™ with an equal volume of different contrast media in polypropylene syringes is given over a period of seven days. Due to a maximum elution of 0.1-0.5% of epirubicin from loaded DC bead™, admixtures with contrast media can be prepared in advance in centralized cytotoxic preparation units. Microbiological aspects have to be considered when determining the expiration date of the product. © The Author(s) 2015.

Optimization of Gas Metal Arc Welding Process Parameters

NASA Astrophysics Data System (ADS)

Kumar, Amit; Khurana, M. K.; Yadav, Pradeep K.

2016-09-01

This study presents the application of Taguchi method combined with grey relational analysis to optimize the process parameters of gas metal arc welding (GMAW) of AISI 1020 carbon steels for multiple quality characteristics (bead width, bead height, weld penetration and heat affected zone). An orthogonal array of L9 has been implemented to fabrication of joints. The experiments have been conducted according to the combination of voltage (V), current (A) and welding speed (Ws). The results revealed that the welding speed is most significant process parameter. By analyzing the grey relational grades, optimal parameters are obtained and significant factors are known using ANOVA analysis. The welding parameters such as speed, welding current and voltage have been optimized for material AISI 1020 using GMAW process. To fortify the robustness of experimental design, a confirmation test was performed at selected optimal process parameter setting. Observations from this method may be useful for automotive sub-assemblies, shipbuilding and vessel fabricators and operators to obtain optimal welding conditions.

Directed Assembly of Cells with Magnetic Nanowires

NASA Astrophysics Data System (ADS)

Tanase, M.; Hultgren, A.; Chen, C. S.; Reich, D. H.

2003-03-01

We demonstrate the use of magnetic nanowires for assembly and manipulation of mammalian cells. Currently, superparamagnetic beads are used for manipulations of cells, but large field strengths and gradients are required for these to be effective. Unlike the beads, the large remnant magnetization of the nanowires offers the prospect of a variety of low-field manipulation techniques. Ferromagnetic nanowires suspended in fluids can be easily manipulated and assembled using small magnetic field [1]. The wires can be bound to cells, and the dipolar interaction between the nanowires can be used to create self-assembled cell chains. Microfabricated arrays of Py magnets were used to trap single cells or chains of cells bound to Ni nanowires. Possible applications of these techniques include controlled initiation of cell cultures, as well as isolation of individual cells. This work was supported by DARPA/AFOSR Grant No. F49620-02-1-0307 and by the David and Lucile Packard Foundation Grant No. 2001-17715. [1] M. Tanase et.al., Nanoletters 1, 155 (2001), J. Appl. Phys. 91, 8549 (2002).

Encapsulation of single cells on a microfluidic device integrating droplet generation with fluorescence-activated droplet sorting.

PubMed

Wu, Liang; Chen, Pu; Dong, Yingsong; Feng, Xiaojun; Liu, Bi-Feng

2013-06-01

Encapsulation of single cells is a challenging task in droplet microfluidics due to the random compartmentalization of cells dictated by Poisson statistics. In this paper, a microfluidic device was developed to improve the single-cell encapsulation rate by integrating droplet generation with fluorescence-activated droplet sorting. After cells were loaded into aqueous droplets by hydrodynamic focusing, an on-flight fluorescence-activated sorting process was conducted to isolate droplets containing one cell. Encapsulation of fluorescent polystyrene beads was investigated to evaluate the developed method. A single-bead encapsulation rate of more than 98 % was achieved under the optimized conditions. Application to encapsulate single HeLa cells was further demonstrated with a single-cell encapsulation rate of 94.1 %, which is about 200 % higher than those obtained by random compartmentalization. We expect this new method to provide a useful platform for encapsulating single cells, facilitating the development of high-throughput cell-based assays.

Nanowire liquid pumps

NASA Astrophysics Data System (ADS)

Huang, Jian Yu; Lo, Yu-Chieh; Niu, Jun Jie; Kushima, Akihiro; Qian, Xiaofeng; Zhong, Li; Mao, Scott X.; Li, Ju

2013-04-01

The ability to form tiny droplets of liquids and control their movements is important in printing or patterning, chemical reactions and biological assays. So far, such nanofluidic capabilities have principally used components such as channels, nozzles or tubes, where a solid encloses the transported liquid. Here, we show that liquids can flow along the outer surface of solid nanowires at a scale of attolitres per second and the process can be directly imaged with in situ transmission electron microscopy. Microscopy videos show that an ionic liquid can be pumped along tin dioxide, silicon or zinc oxide nanowires as a thin precursor film or as beads riding on the precursor film. Theoretical analysis suggests there is a critical film thickness of ~10 nm below which the liquid flows as a flat film and above which it flows as discrete beads. This critical thickness is the result of intermolecular forces between solid and liquid, which compete with liquid surface energy and Rayleigh-Plateau instability.

Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry.

PubMed

Zhao, Yong-Qiang; Song, Jian; Wang, Hong-Li; Xu, Bin; Liu, Feng; He, Kun; Wang, Na

2016-04-01

The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity. In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice. Graphical Abstract ᅟ.

Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhao, Yong-Qiang; Song, Jian; Wang, Hong-Li; Xu, Bin; Liu, Feng; He, Kun; Wang, Na

2016-04-01

The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity. In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice.