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.

A Novel Inherently Radiopaque Bead for Transarterial Embolization to Treat Liver Cancer - A Pre-clinical Study.

PubMed

Duran, Rafael; Sharma, Karun; Dreher, Matthew R; Ashrafi, Koorosh; Mirpour, Sahar; Lin, MingDe; Schernthaner, Ruediger E; Schlachter, Todd R; Tacher, Vania; Lewis, Andrew L; Willis, Sean; den Hartog, Mark; Radaelli, Alessandro; Negussie, Ayele H; Wood, Bradford J; Geschwind, Jean-François H

2016-01-01

Embolotherapy using microshperes is currently performed with soluble contrast to aid in visualization. However, administered payload visibility dimishes soon after delivery due to soluble contrast washout, leaving the radiolucent bead's location unknown. The objective of our study was to characterize inherently radiopaque beads (RO Beads) in terms of physicomechanical properties, deliverability and imaging visibility in a rabbit VX2 liver tumor model. RO Beads, which are based on LC Bead® platform, were compared to LC Bead. Bead size (light microscopy), equilibrium water content (EWC), density, X-ray attenuation and iodine distribution (micro-CT), suspension (settling times), deliverability and in vitro penetration were investigated. Fifteen rabbits were embolized with either LC Bead or RO Beads + soluble contrast (iodixanol-320), or RO Beads+dextrose. Appearance was evaluated with fluoroscopy, X-ray single shot, cone-beam CT (CBCT). Both bead types had a similar size distribution. RO Beads had lower EWC (60-72%) and higher density (1.21-1.36 g/cc) with a homogeneous iodine distribution within the bead's interior. RO Beads suspension time was shorter than LC Bead, with durable suspension (>5 min) in 100% iodixanol. RO Beads ≤300 µm were deliverable through a 2.3-Fr microcatheter. Both bead types showed similar penetration. Soluble contrast could identify target and non-target embolization on fluoroscopy during administration. However, the imaging appearance vanished quickly for LC Bead as contrast washed-out. RO Beads+contrast significantly increased visibility on X-ray single shot compared to LC Bead+contrast in target and non-target arteries (P=0.0043). Similarly, RO beads demonstrated better visibility on CBCT in target arteries (P=0.0238) with a trend in non-target arteries (P=0.0519). RO Beads+dextrose were not sufficiently visible to monitor embolization using fluoroscopy. RO Beads provide better conspicuity to determine target and non-target embolization compared to LC Bead which may improve intra-procedural monitoring and post-procedural evaluation of transarterial embolization.

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.

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

[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.

Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

PubMed

Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

2015-12-01

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

Comparison of high pressure homogenization and stirred bead milling for the production of nano-crystalline suspensions.

PubMed

Nakach, Mostafa; Authelin, Jean-René; Perrin, Marc-Antoine; Lakkireddy, Harivardhan Reddy

2018-05-19

Currently, the two technologies primarily used for the manufacturing of nano-crystalline suspensions using top down process (i.e. wet milling) are high pressure homogenization (HPH) and stirred bead milling (SBM). These two technologies are based upon different mechanisms, i.e., cavitation forces for HPH and shear forces for stirred bead milling. In this article, the HPH and SBM technologies are compared in terms of the impact of the suspension composition the process parameters and the technological configuration on milling performances and physical quality of the suspensions produced. The data suggested that both HPH and SBM are suitable for producing nano-crystalline suspensions, although SBM appeared more efficient than HPH, since the limit of milling (d 50 ) for SBM was found to be lower than that obtained with HPH (100 nm vs 200 nm). For both these technologies, regardless of the process parameters used for milling and the scale of manufacturing, the relationship of d 90 versus d 50 could be described by a unique master curve (technology signature of milling pathway) outlining that the HPH leads to more uniform particle size distribution as compared to SBM. Copyright © 2018 Elsevier B.V. All rights reserved.

A microhydrodynamic rationale for selection of bead size in preparation of drug nanosuspensions via wet stirred media milling.

PubMed

Li, Meng; Alvarez, Paulina; Bilgili, Ecevit

2017-05-30

Although wet stirred media milling has proven to be a robust process for producing nanoparticle suspensions of poorly water-soluble drugs and thereby enhancing their bioavailability, selection of bead size has been largely empirical, lacking fundamental rationale. This study aims to establish such rationale by investigating the impact of bead size at various stirrer speeds on the drug breakage kinetics via a microhydrodynamic model. To this end, stable suspensions of griseofulvin, a model BCS Class II drug, were prepared using hydroxypropyl cellulose and sodium dodecyl sulfate. The suspensions were milled at four different stirrer speeds (1000-4000rpm) using various sizes (50-1500μm) of zirconia beads. Laser diffraction, SEM, and XRPD were used for characterization. Our results suggest that there is an optimal bead size that achieves fastest breakage at each stirrer speed and that it shifts to a smaller size at higher speed. Calculated microhydrodynamic parameters reveal two counteracting effects of bead size: more bead-bead collisions with less energy/force upon a decrease in bead size. The optimal bead size exhibits a negative power-law correlation with either specific energy consumption or the microhydrodynamic parameters. Overall, this study rationalizes the use of smaller beads for more energetic wet media milling. Copyright © 2017 Elsevier B.V. All rights reserved.

Suspension arrays based on nanoparticle-encoded microspheres for high-throughput multiplexed detection

PubMed Central

Leng, Yuankui

2017-01-01

Spectrometrically or optically encoded microsphere based suspension array technology (SAT) is applicable to the high-throughput, simultaneous detection of multiple analytes within a small, single sample volume. Thanks to the rapid development of nanotechnology, tremendous progress has been made in the multiplexed detecting capability, sensitivity, and photostability of suspension arrays. In this review, we first focus on the current stock of nanoparticle-based barcodes as well as the manufacturing technologies required for their production. We then move on to discuss all existing barcode-based bioanalysis patterns, including the various labels used in suspension arrays, label-free platforms, signal amplification methods, and fluorescence resonance energy transfer (FRET)-based platforms. We then introduce automatic platforms for suspension arrays that use superparamagnetic nanoparticle-based microspheres. Finally, we summarize the current challenges and their proposed solutions, which are centered on improving encoding capacities, alternative probe possibilities, nonspecificity suppression, directional immobilization, and “point of care” platforms. Throughout this review, we aim to provide a comprehensive guide for the design of suspension arrays, with the goal of improving their performance in areas such as multiplexing capacity, throughput, sensitivity, and cost effectiveness. We hope that our summary on the state-of-the-art development of these arrays, our commentary on future challenges, and some proposed avenues for further advances will help drive the development of suspension array technology and its related fields. PMID:26021602

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

USDA-ARS?s Scientific Manuscript database

Bead based multiplex assays (BBMA) also referred to as Luminex, MultiAnalyte Profiling or cytometric bead array (CBA) assays, are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several, up to 50-500 analytes within a single, small sample volume). Curren...

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.

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.

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.

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.

Aptamer-based microfluidic beads array sensor for simultaneous detection of multiple analytes employing multienzyme-linked nanoparticle amplification and quantum dots labels.

PubMed

Zhang, He; Hu, Xinjiang; Fu, Xin

2014-07-15

This study reports the development of an aptamer-mediated microfluidic beads-based sensor for multiple analytes detection and quantification using multienzyme-linked nanoparticle amplification and quantum dots labels. Adenosine and cocaine were selected as the model analytes to validate the assay design based on strand displacement induced by target-aptamer complex. Microbeads functionalized with the aptamers and modified electron rich proteins were arrayed within a microfluidic channel and were connected with the horseradish peroxidases (HRP) and capture DNA probe derivative gold nanoparticles (AuNPs) via hybridization. The conformational transition of aptamer induced by target-aptamer complex contributes to the displacement of functionalized AuNPs and decreases the fluorescence signal of microbeads. In this approach, increased binding events of HRP on each nanosphere and enhanced mass transport capability inherent from microfluidics are integrated for enhancing the detection sensitivity of analytes. Based on the dual signal amplification strategy, the developed aptamer-based microfluidic bead array sensor could discriminate as low as 0.1 pM of adenosine and 0.5 pM cocaine, and showed a 500-fold increase in detection limit of adenosine compared to the off-chip test. The results proved the microfluidic-based method was a rapid and efficient system for aptamer-based targets assays (adenosine (0.1 pM) and cocaine (0.5 pM)), requiring only minimal (microliter) reagent use. This work demonstrated the successful application of aptamer-based microfluidic beads array sensor for detection of important molecules in biomedical fields. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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.

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.

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.

Effect of particle size of parenteral suspensions on in vitro muscle damage.

PubMed

Brazeau, Gayle; Sauberan, Shauna L; Gatlin, Larry; Wisniecki, Peter; Shah, Jaymin

2011-01-01

Suspension particle size plays a key role in the release and stability of drugs for oral and parenteral formulations. However, the role of particle size in suspension formulations on tissue damage (myotoxicity) following intramuscular (IM) injection has not been systematically investigated. Myotoxicity was assessed by the release of cumulative creatine kinase (CCK) from the isolated extensor digitorium longus (EDL) and soleus (SOL) rat muscles for selected suspensions of phenytoin, bupivicane and diazepam. Particle size effects on myotoxicity, independent of any specific drug, were also investigated using characterized non-dissolving polystyrene beads. Myotoxicity was quantitated by the cumulative release of creatine kinase (CCK) from these isolated muscles over 90 or 120 min. The relationship between particle size and myotoxicity was dependent upon the drug in these suspensions. Diazepam and phenytoin suspensions were found to be less myotoxic than bupivicaine. Using unmodified and carboxy modified polystyrene beads, an optimal particle size for reduced myotoxicity following IM injection ranges from approx. 500 nm to 1 µM. The relationship between myotoxicity of IM suspensions and particle size is dependent upon the particular drug and suspension particle size.

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.

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.

Enhanced cell trapping throughput using DC-biased AC electric field in a dielectrophoresis-based fluidic device with densely packed silica beads.

PubMed

Lewpiriyawong, Nuttawut; Xu, Guolin; Yang, Chun

2018-03-01

This paper presents the use of DC-biased AC electric field for enhancing cell trapping throughput in an insulator-based dielectrophoretic (iDEP) fluidic device with densely packed silica beads. Cell suspension is carried through the iDEP device by a pressure-driven flow. Under an applied DC-biased AC electric field, DEP trapping force is produced as a result of non-uniform electric field induced by the gap of electrically insulating silica beads packed between two mesh electrodes that allow both fluid and cells to pass through. While the AC component is mainly to control the magnitude of DEP trapping force, the DC component generates local electroosmotic (EO) flow in the cavity between the beads and the EO flow can be set to move along or against the main pressure-driven flow. Our experimental and simulation results show that desirable trapping is achieved when the EO flow direction is along (not against) the main flow direction. Using our proposed DC-biased AC field, the device can enhance the trapping throughput (in terms of the flowrate of cell suspension) up to five times while yielding almost the same cell capture rates as compared to the pure AC field case. Additionally, the device was demonstrated to selectively trap dead yeast cells from a mixture of flowing live and dead yeast cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Ionene modified small polymeric beads

NASA Technical Reports Server (NTRS)

Rembaum, Alan (Inventor)

1977-01-01

Linear ionene polyquaternary cationic polymeric segments are bonded by means of the Menshutkin reaction (quaternization) to biocompatible, extremely small, porous particles containing halide or tertiary amine sites which are centers for attachment of the segments. The modified beads in the form of emulsions or suspensions offer a large, positively-charged surface area capable of irreversibly binding polyanions such as heparin, DNA, RNA or bile acids to remove them from solution or of reversibly binding monoanions such as penicillin, pesticides, sex attractants and the like for slow release from the suspension.

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.

Electric field directed assembly of high-density microbead arrays†

PubMed Central

Barbee, Kristopher D.; Hsiao, Alexander P.; Heller, Michael J.; Huang, Xiaohua

2010-01-01

We report a method for rapid, electric field directed assembly of high-density protein-conjugated microbead arrays. Photolithography is used to fabricate an array of micron to sub-micron-scale wells in an epoxy-based photoresist on a silicon wafer coated with a thin gold film, which serves as the primary electrode. A thin gasket is used to form a microfluidic chamber between the wafer and a glass coverslip coated with indium-tin oxide, which serves as the counter electrode. Streptavidin-conjugated microbeads suspended in a low conductance buffer are introduced into the chamber and directed into the wells via electrophoresis by applying a series of low voltage electrical pulses across the electrodes. Hundreds of millions of microbeads can be permanently assembled on these arrays in as little as 30 seconds and the process can be monitored in real time using epifluorescence microscopy. The binding of the microbeads to the gold film is robust and occurs through electrochemically induced gold-protein interactions, which allows excess beads to be washed away or recycled. The well and bead sizes are chosen such that only one bead can be captured in each well. Filling efficiencies greater than 99.9% have been demonstrated across wafer-scale arrays with densities as high as 69 million beads per cm2. Potential applications for this technology include the assembly of DNA arrays for high-throughput genome sequencing and antibody arrays for proteomic studies. Following array assembly, this device may also be used to enhance the concentration-dependent processes of various assays through the accelerated transport of molecules using electric fields. PMID:19865735

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.

Digital barcodes of suspension array using laser induced breakdown spectroscopy

PubMed Central

He, Qinghua; Liu, Yixi; He, Yonghong; Zhu, Liang; Zhang, Yilong; Shen, Zhiyuan

2016-01-01

We show a coding method of suspension array based on the laser induced breakdown spectroscopy (LIBS), which promotes the barcodes from analog to digital. As the foundation of digital optical barcodes, nanocrystals encoded microspheres are prepared with self-assembly encapsulation method. We confirm that digital multiplexing of LIBS-based coding method becomes feasible since the microsphere can be coded with direct read-out data of wavelengths, and the method can avoid fluorescence signal crosstalk between barcodes and analyte tags, which lead to overall advantages in accuracy and stability to current fluorescent multicolor coding method. This demonstration increases the capability of multiplexed detection and accurate filtrating, expanding more extensive applications of suspension array in life science. PMID:27808270

Pharmaceutical acrylic beads obtained by suspension polymerization containing cellulose nanowhiskers as excipient for drug delivery.

PubMed

Villanova, J C O; Ayres, E; Carvalho, S M; Patrício, P S; Pereira, F V; Oréfice, R L

2011-03-18

Direct compression is one of the most popular techniques to prepare tablets but only a few commercial excipients are well adapted for this process into controlled release formulations. In the last years, the introduction of new materials for drug delivery matrix tablets has become more important. This paper evaluated the physicochemical and flow properties of new polymeric excipient of ethyl acrylate, methyl methacrylate and butyl metacrylate, synthesized by suspension polymerization using cellulose nanowhiskers as co-stabilizer, to be used as direct compression for modified release tablets. Infrared spectroscopy (FTIR) confirmed the success of the copolymerization reaction. Scanning electron microscopy (SEM) showed that excipient was obtained how spherical beads. Thermal properties of the beads were characterized by thermogravimetric (TG) analysis. Particle size analysis of the beads with cellulose nanowhiskers (CNWB) indicated that the presence of the nanowhiskers led to a reduction of particle size and to a narrower size distribution. In vitro test showed that the nanowhiskers and beads produced are nontoxic. Parameters such as Hausner ratio, Carr's index and cotangent of angle α were employed to characterize the flow properties of CNWB beads. Furthermore, the beads are used to produce tablets by direct compression contained propranolol hydrochloride as model drug. Dissolution tests performed suggested that beads could be used as excipient in matrix tablets with a potential use in drug controlled release. Copyright © 2011 Elsevier B.V. All rights reserved.

Methods for functionalization of microsized polystyrene beads with titania nanoparticles for cathodic electrophoretic deposition.

PubMed

Radice, S; Kern, P; Dietsch, H; Mischler, S; Michler, J

2008-02-15

Functionalization of colloidal particles based on the use of polyelectrolytes and heterocoagulation was combined with electrophoretic deposition (EPD), with the aim of depositing titania-polystyrene (TiO(2)-PS) composite particles on Ti6Al4V substrates. The composite particles were obtained by heterocoagulation of TiO(2) nanoparticles on the surface of monosized polystyrene beads of 4.6 microm in diameter. Two alternative methods were developed for the preparation of the TiO(2)-PS suspensions in organic fluids for cathodic electrodeposition. The first method was carried out in alkaline aqueous medium with the use of polyelectrolytes and intermediate control measurements of zeta potential, conductivity, and pH; the second one was carried out directly in the organic solvent used for EPD, typically isopropanol. Examples of deposits obtained by EPD in both suspensions and a comparative analysis between the two methods are presented.

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.

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.

Suspension column for recovery and separation of substances using ultrasound-assisted retention of bead sorbents.

PubMed

Spivakov, Boris Ya; Shkinev, Valeriy M; Danilova, Tatiana V; Knyazkov, Nikolai N; Kurochkin, Vladimir E; Karandashev, Vasiliy K

2012-12-15

A novel approach to sorption recovery and separation of different substances is proposed which is based on the use of suspended bead sorbents instead of conventional packed beds of such sorbents. This makes it possible to employ small-sized beads which are trapped in a low-pressure column due to ultrasound-assisted retention, without any frits to hold the sorption material. A flow system including a separation mini-column, named herein a suspension column, has been developed and tested by the studies of solid phase extraction (SPE) of trace metals from bi-distilled water and sea water using a 150-μL column with a silica-based sorbent containing iminodiacetic groups (DIAPAK IDA) and having a grain size of 6 μm. The adsorption properties of DIAPAK IDA suspension (9.5mg) were evaluated through adsorption/desorption experiments, where the effect of solution pH and eluent on the SPE of trace metals were examined by ICP-MS or ICP-AES measurements. When sample solution was adjusted to pH 8.0 and 1 mol L(-1) nitric acid was used as eluent, very good recoveries of more than 90% were obtained for a number of elements in a single-step extraction. To demonstrate the versatility of the approach proposed and to show another advantage of ultrasonic field (acceleration of sorbate/sorbent interaction), a similar system was used for heterogeneous immunoassays of some antigens in ultrasonic field using agarose sorbents modified by corresponding antibodies. It has been shown that immunoglobulins, chlamidia, and brucellos bacteria can be quantitatively adsorbed on 15-μm sorbent (15 particles in 50 μL) and directly determined in a 50-μL mini-chamber using fluorescence detection. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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.

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

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 integrated open-cavity system for magnetic bead manipulation.

PubMed

Abu-Nimeh, F T; Salem, F M

2013-02-01

Superparamagnetic beads are increasingly used in biomedical assays to manipulate, transport, and maneuver biomaterials. We present a low-cost integrated system designed in bulk CMOS to manipulate and separate biomedical magnetic beads. The system consists of 8 × 8 coil-arrays suitable for single bead manipulation, or collaborative multi-bead manipulation, using pseudo-parallel executions. We demonstrate the flexibility of the design in terms of different coil sizes, DC current levels, and layout techniques. In one array module example, the size of a single coil is 30 μm × 30 μm and the full array occupies an area of 248 μm × 248 μm in 0.5 μm CMOS technology. The programmable DC current source supports 8 discrete levels up to 1.5 mA. The total power consumption of the entire module is 9 mW when running at full power.

Detection of magnetic microbeads and ferrofluid with giant magnetoresistance sensors

NASA Astrophysics Data System (ADS)

Feng, J.; Wang, Y. Q.; Li, F. Q.; Shi, H. P.; Chen, X.

2011-01-01

Giant magnetoresistance sensors based on multilayers [Cu/NiFeCo]×10/ Ta were fabricated by microfabrication technology. A GMR-bridge was used to detect the magnetic MyOne beads and Ferro fluid. The dependence of the GMR-bridge signals on the surface coverage of MyOne beads was studied. The results show that the GMR sensor is capable of detecting the magnetic beads. The detectable limit of MyOne beads is about 100, and the corresponding signal output is 8 μV. The GMR bridge signal is proportional to the surface coverage of the MyOne beads. The sensitivity of the GMR bridge is inversely proportional to the feature size of the GMR sensor. The GMR bridge integrated with microfludic channel was also used for dynamic detection of ferrofluid (suspension of Fe3O4 particles). The results show that the GMR bridge is capable of detecting the flow of ferrofluid, and the sensor signals are proportional to the concentration of the ferrofluid. The detection limit of concentration of the ferrofluid is 0.56 mg/ml, and the corresponding signal is 6.2 μV.

Effect of starting powders on the sintering of nanostructured ZrO2 ceramics by colloidal processing

NASA Astrophysics Data System (ADS)

Suárez, Gustavo; Sakka, Yoshio; Suzuki, Tohru S.; Uchikoshi, Tetsuo; Zhu, Xinwen; Aglietti, Esteban F.

2009-04-01

The effect of starting powders on the sintering of nanostructured tetragonal zirconia was evaluated. Suspensions were prepared with a concentration of 10 vol.% by mixing a bicomponent mixture of commercial powders (97 mol.% monoclinic zirconia with 3 mol.% yttria) and by dispersing commercially available tetragonal zirconia (3YTZ, Tosoh). The preparation of the slurry by bead-milling was optimized. Colloidal processing using 50 μm zirconia beads at 4000 rpm generated a fully deagglomerated suspension leading to the formation of high-density consolidated compacts (62% of the theoretical density (TD) for the bicomponent suspension). Optimum colloidal processing of the bicomponent suspension followed by the sintering of yttria and zirconia allowed us to obtain nanostructured tetragonal zirconia. Three different sintering techniques were investigated: normal sintering, two-step sintering and spark plasma sintering. The inhibition of grain growth in the bicomponent mixed powders in comparison with 3YTZ was demonstrated. The inhibition of the grain growth may have been caused by inter-diffusion of cations during the sintering.

On-Chip Magnetic Platform for Single-Particle Manipulation with Integrated Electrical Feedback.

PubMed

Monticelli, Marco; Torti, Andrea; Cantoni, Matteo; Petti, Daniela; Albisetti, Edoardo; Manzin, Alessandra; Guerriero, Erica; Sordan, Roman; Gervasoni, Giacomo; Carminati, Marco; Ferrari, Giorgio; Sampietro, Marco; Bertacco, Riccardo

2016-02-17

Methods for the manipulation of single magnetic particles have become very interesting, in particular for in vitro biological studies. Most of these studies require an external microscope to provide the operator with feedback for controlling the particle motion, thus preventing the use of magnetic particles in high-throughput experiments. In this paper, a simple and compact system with integrated electrical feedback is presented, implementing in the very same device both the manipulation and detection of the transit of single particles. The proposed platform is based on zig-zag shaped magnetic nanostructures, where transverse magnetic domain walls are pinned at the corners and attract magnetic particles in suspension. By applying suitable external magnetic fields, the domain walls move to the nearest corner, thus causing the step by step displacement of the particles along the nanostructure. The very same structure is also employed for detecting the bead transit. Indeed, the presence of the magnetic particle in suspension over the domain wall affects the depinning field required for its displacement. This characteristic field can be monitored through anisotropic magnetoresistance measurements, thus implementing an integrated electrical feedback of the bead transit. In particular, the individual manipulation and detection of single 1-μm sized beads is demonstrated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

PubMed

Yang, Yu; Wang, Jing; Wen, Haiyan; Liu, Hengchuan

2012-01-01

We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

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.

Image Decoding of Photonic Crystal Beads Array in the Microfluidic Chip for Multiplex Assays

PubMed Central

Yuan, Junjie; Zhao, Xiangwei; Wang, Xiaoxia; Gu, Zhongze

2014-01-01

Along with the miniaturization and intellectualization of biomedical instruments, the increasing demand of health monitoring at anywhere and anytime elevates the need for the development of point of care testing (POCT). Photonic crystal beads (PCBs) as one kind of good encoded microcarriers can be integrated with microfluidic chips in order to realize cost-effective and high sensitive multiplex bioassays. However, there are difficulties in analyzing them towards automated analysis due to the characters of the PCBs and the unique detection manner. In this paper, we propose a strategy to take advantage of automated image processing for the color decoding of the PCBs array in the microfluidic chip for multiplex assays. By processing and alignment of two modal images of epi-fluorescence and epi-white light, every intact bead in the image is accurately extracted and decoded by PC colors, which stand for the target species. This method, which shows high robustness and accuracy under various configurations, eliminates the high hardware requirement of spectroscopy analysis and user-interaction software, and provides adequate supports for the general automated analysis of POCT based on PCBs array. PMID:25341876

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.

The Use of Index-Matched Beads in Optical Particle Counters

PubMed Central

Hu, Zhishang; Ripple, Dean C

2014-01-01

In this paper, we demonstrate the use of 2-pyridinemethanol (2P) aqueous solutions as a refractive index matching liquid. The high refractive index and low viscosity of 2P-water mixtures enables refractive index matching of beads that cannot be index matched with glycerol-water or sucrose-water solutions, such as silica beads that have the refractive index of bulk fused silica or of polymethylmethacrylate beads. Suspensions of beads in a nearly index-matching liquid are a useful tool to understand the response of particle counting instruments to particles of low optical contrast, such as aggregated protein particles. Data from flow imaging and light obscuration instruments are presented for bead diameters ranging from 6 µm to 69 µm, in a matrix liquid spanning the point of matched refractive index. PMID:26601049

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.

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.

Renewable Surface Fluorescence Sandwich Immunoassay Biosensor for Rapid Sensitive Botulinum Toxin Detection in an Automated Fluidic Format

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

Grate, Jay W.; Warner, Marvin G.; Ozanich, Richard M.

2009-03-05

A renewable surface biosensor for rapid detection of botulinum toxin is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant fragment of the toxin heavy chain as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate epitopes of both this fragment and the holotoxin. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by the sequential injection flow system, creating a 3.6 microliter column. After perfusing the bead column with sample andmore » washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degree angle to one another delivered excitation light from a HeNe laser and collected fluorescent emission light for detection. After each measurement, the used sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes.« less

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis.

PubMed

Shrestha, B R; Tokuhara, K; Mii, M

2007-06-01

Protoplasts isolated from cell suspension culture of Phalaenopsis "Wataboushi" were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l L-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.

Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbead-based suspension array

USDA-ARS?s Scientific Manuscript database

Identification and serotyping of Shiga toxin-producing Escherichia coli during foodborne outbreaks can aid in matching clinical, food, and environmental isolates when trying to identify the sources of illness and ultimately food contamination. Herein we describe a Luminex microbead-based suspension ...

Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth.

PubMed

Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou

2006-09-01

An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.

Detection of bacteria from biological mixtures using immunomagnetic separation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

USGS Publications Warehouse

Madonna, A.J.; Basile, F.; Furlong, E.; Voorhees, K.J.

2001-01-01

A rapid method for identifying specific bacteria from complex biological mixtures using immunomagnetic separation coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been developed. The technique employs commercially available magnetic beads coated with polycolonal antibodies raised against specific bacteria and whole cell analysis by MALDI-MS. A suspension of a bacterial mixture is mixed with the immunomagnetic beads specific for the target microorganism. After a short incubation period (20 mins) the bacteria captured by the beads are washed, resuspended in deionized H2O and directly applied onto a MALDI probe. Liquid suspensions containing bacterial mixtures can be screened within 1 h total analysis time. Positive tests result in the production of a fingerprint mass spectrum primarily consisting of protein biomarkers characteristic of the targeted microorganism. Using this procedure, Salmonella choleraesuis was isolated and detected from standard bacterial mixtures and spiked samples of river water, human urine, and chicken blood. Copyright ?? 2001 John Wiley & Sons, Ltd.

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

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.

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

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.

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.

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.

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.

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.

Drug particle size influence on enteric beads produced by a droplet extrusion/precipitation method.

PubMed

Cerdeira, A M; Gouveia, L F; Goucha, P; Almeida, A J

2000-01-01

The influence of drug particle size on the production of enteric beads by a polymer precipitation technique was investigated. Drug particle dimensions are known to play an important role in most microencapsulation techniques. Bead morphology was greatly influenced by drug particle size, and spherical shaped beads could only be obtained after size reduction of nimesulide crystals. This is confirmed by the angle of repose measurements, which show a significant decrease in theta values when beads are formulated with smaller drug particles. Furthermore, results show that drug encapsulation efficiency and in vitro drug release rates are also greatly dependent on both drug particle size and drug/polymer ratio in the initial suspension. Preparations containing 10.2 microm drug particles show a two-fold increase in the release rates when compared to those prepared with 40 microm particles.

Impact of process parameters on the breakage kinetics of poorly water-soluble drugs during wet stirred media milling: a microhydrodynamic view.

PubMed

Afolabi, Afolawemi; Akinlabi, Olakemi; Bilgili, Ecevit

2014-01-23

Wet stirred media milling has proven to be a robust process for producing nanoparticle suspensions of poorly water-soluble drugs. As the process is expensive and energy-intensive, it is important to study the breakage kinetics, which determines the cycle time and production rate for a desired fineness. Although the impact of process parameters on the properties of final product suspensions has been investigated, scant information is available regarding their impact on the breakage kinetics. Here, we elucidate the impact of stirrer speed, bead concentration, and drug loading on the breakage kinetics via a microhydrodynamic model for the bead-bead collisions. Suspensions of griseofulvin, a model poorly water-soluble drug, were prepared in the presence of two stabilizers: hydroxypropyl cellulose and sodium dodecyl sulfate. Laser diffraction, scanning electron microscopy, and rheometry were used to characterize them. Various microhydrodynamic parameters including a newly defined milling intensity factor was calculated. An increase in either the stirrer speed or the bead concentration led to an increase in the specific energy and the milling intensity factor, consequently faster breakage. On the other hand, an increase in the drug loading led to a decrease in these parameters and consequently slower breakage. While all microhydrodynamic parameters provided significant physical insight, only the milling intensity factor was capable of explaining the influence of all parameters directly through its strong correlation with the process time constant. Besides guiding process optimization, the analysis rationalizes the preparation of a single high drug-loaded batch (20% or higher) instead of multiple dilute batches. Copyright © 2013 Elsevier B.V. All rights reserved.

Characteristics of dilute gas-solids suspensions in drag reducing flow

NASA Technical Reports Server (NTRS)

Kane, R. S.; Pfeffer, R.

1973-01-01

Measurements were performed on dilute flowing gas-solids suspensions and included data, with particles present, on gas friction factors, velocity profiles, turbulence intensity profiles, turbulent spectra, and particle velocity profiles. Glass beads of 10 to 60 micron diameter were suspended in air at Reynolds numbers of 10,000 to 25,000 and solids loading ratios from 0 to 4. Drag reduction was achieved for all particle sizes in vertical flow and for the smaller particle sizes in horizontal flow. The profile measurements in the vertical tube indicated that the presence of particles thickened the viscous sublayer. A quantitative theory based on particle-eddy interaction and viscous sublayer thickening has been proposed.

A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

NASA Astrophysics Data System (ADS)

Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

2016-02-01

Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

Microfabricated Renewable Beads-Trapping/Releasing Flow Cell for Rapid Antigen-Antibody Reaction in Chemiluminescent Immunoassay

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

Fu, Zhifeng; Shao, Guocheng; Wang, Jun

2011-04-01

A filter pillar-array microstructure was coupled with a pneumatic micro-valve to fabricate a reusable miniaturized beads-trapping/releasing flow cell, in which trapping and releasing beads can be conveniently realized by switching the micro-valve. This miniaturized device was suitable to construct automatic fluidic system for “renewable surface analysis”. The renewable surface strategy based on pneumatic micro-valve enabled capture of beads in beads chamber prior to each assay, and release of the used beads after the assay. Chemiluminescent competitive immunoassay of 3,5,6-trichloropyridinol (TCP) was performed as a model to demonstrate the application potential of this reusable miniaturized flow cell. The whole fluidic assaymore » process including beads trapping, immuno-binding, beads washing, beads releasing and signal collection could be completed in 10 min. Immunoassay of TCP using this miniaturized device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device had been successfully used for detection of TCP spiked in rat serum with average recovery of 97%. This investigation provides a rapid, sensitive, reusable, low-cost and automatic miniaturized device for solid-phase biochemical analysis for various purposes.« less

A simple device for measuring the minimum current velocity to maintain semi-buoyant fish eggs in suspension

USGS Publications Warehouse

Mueller, Julia S.; Cheek, Brandon D.; Chen, Qingman; Groeschel, Jillian R.; Brewer, Shannon K.; Grabowski, Timothy B.

2013-01-01

Pelagic broadcast spawning cyprinids are common to Great Plains rivers and streams. This reproductive guild produces non-adhesive semi-buoyant eggs that require sufficient current velocity to remain in suspension during development. Although studies have shown that there may be a minimum velocity needed to keep the eggs in suspension, this velocity has not been estimated directly nor has the influence of physicochemical factors on egg buoyancy been determined. We developed a simple, inexpensive flow chamber that allowed for evaluation of minimum current velocity needed to keep semi-buoyant eggs in suspension at any time frame during egg development. The device described here has the capability of testing the minimum current velocity needed to keep semi-buoyant eggs in suspension at a wide range of physicochemical conditions. We used gellan beads soaked in freshwater for 0, 24, and 48 hrs as egg surrogates and evaluated minimum current velocities necessary to keep them in suspension at different combinations of temperature (20.0 ± 1.0° C, 25.0 ± 1.0° C, and 28.0 ± 1.0° C) and total dissolved solids (TDS; 1,000 mg L-1, 3,000 mg L-1, and 6,000 mg L-1). We found that our methodology generated consistent, repeatable results within treatment groups. Current velocities ranging from 0.001–0.026 needed to keep the gellan beads in suspension were negatively correlated to soak times and TDS and positively correlated with temperature. The flow chamber is a viable approach for evaluating minimum current velocities needed to keep the eggs of pelagic broadcast spawning cyprinids in suspension during development.

Ingestion and regurgitation of living and inert materials by the estuarine copepod Eurytemora affinis (Poppe) and the influence of salinity

NASA Astrophysics Data System (ADS)

Powell, Mark D.; Berry, A. J.

1990-12-01

Eurytemora affinis (Poppe) fed on cultured Thalassiosira weissflogii (Grunnow) at rates of 200-34000 cells copepod -1 h -1. Feeding was delayed and diminished in bright light. In dim light, feeding was initially faster in 15‰ (27000-34000 copepod -1 h -1) than in 10‰ (23000-25000 copepod -1 h -1) and much faster than in 3‰ (6000 copepod -1 h -1). After 1-3 h, feeding continued more steadily in 3‰ (1200-6500 copepod -1 h -1) but slowed drastically in 10 and 15‰ to 200-5000 copepod -1 h -1). These patterns were maintained when copepods were first acclimated briefly to the test salinities. E. affinis fed at slightly higher rates on sterile latex beads of similar size to T. weissfloggi, fastest in 10‰ and slowest in 3‰. While the beads appeared in the guts, they did not appear in the faecal pellets and after 1 h (10, 15‰) or 3 h (3‰), their numbers in suspension recovered close to original counts. In contrast, beads infected with a marine bacterium were similarly eaten (at slightly higher rates than the sterile beads), and appeared in the guts and then in the faecal pellets, while numbers in suspension continued to fall or remained low. The contrasts between initial rapid feeding in 10-15‰ and slower steadier feeding in 3‰, and between regurgitation of swallowed sterile beads and passage through the gut of bacterially-contaminated beads, have significance for the biology of a copepod living in the upper reaches of an estuary.

Centrifugal sedimentation for selectively packing channels with silica microbeads in three-dimensional micro/nanofluidic devices.

PubMed

Gong, Maojun; Bohn, Paul W; Sweedler, Jonathan V

2009-03-01

Incorporation of nanofluidic elements into microfluidic channels is one approach for adding filtration and partition functionality to planar microfluidic devices, as well as providing enhanced biomolecular separations. Here we introduce a strategy to pack microfluidic channels with silica nanoparticles and microbeads, thereby indirectly producing functional nanostructures; the method allows selected channels to be packed, here demonstrated so that a separation channel is packed while keeping an injection channel unpacked. A nanocapillary array membrane is integrated between two patterned microfluidic channels that cross each other in vertically separated layers. The membrane serves both as a frit for bead packing and as a fluid communication conduit between microfluidic channels. Centrifugal force-assisted sedimentation is then used to selectively pack the microfluidic channels using an aqueous silica bead suspension loaded into the appropriate inlet reservoirs. This packing approach may be used to simultaneously pack multiple channels with silica microbeads having different sizes and surface properties. The chip design and packing method introduced here are suitable for packing silica particles in sizes ranging from nanometers to micrometers and allow rapid (approximately 10 min) packing with high quality. The liquid/analyte transport characteristics of these packed micro/nanofluidic devices have potential utility in a wide range of applications, including electroosmotic pumping, liquid chromatographic separations, and electrochromatography.

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.

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

Cultivation of cottontail rabbit epidermal (Sf1Ep) cells on microcarrier beads and their use for suspension cultivation of Treponema pallidum subsp. pallidum.

PubMed Central

Riley, B S; Cox, D L

1988-01-01

In vitro propagation of Treponema pallidum can be achieved by cocultivation with Sf1Ep cells. This study had two objectives: (i) to achieve suspension cultivation of Sf1Ep cells and (ii) to develop procedures for achieving the replication of T. pallidum in those cell cultures. Seven suspension cultures of Sf1Ep cells yielded an average of 7.2 x 10(8) T. pallidum (36-fold increase) after 12 days. Images PMID:3063209

Biotechnology

NASA Image and Video Library

2003-05-07

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. In this picture, the beads are trapped in the injection port shortly after injection. Swirls of beads indicate, event to the naked eye, the contents of the TCM are not fully mixed. The beads are similar in size and density to human lymphoid cells. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light

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.

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.

Immobilized OBOC combinatorial bead array to facilitate multiplicative screening.

PubMed

Xiao, Wenwu; Bononi, Fernanda C; Townsend, Jared; Li, Yuanpei; Liu, Ruiwu; Lam, Kit S

2013-07-01

One-bead-one-compound (OBOC) combinatorial library screening has been broadly utilized for the last two decades to identify small molecules, peptides or peptidomimetics targeting variable screening probes such as cell surface receptors, bacteria, protein kinases, phosphatases, proteases etc. In previous screening methods, library beads were suspended in solution and screened against one single probe. Only the positive beads were tracked and isolated for additional screens and finally selected for chemical decoding. During this process, the remaining negative beads were not tracked and discarded. Here we report a novel bead immobilization method such that a bead library array can be conveniently prepared and screened in its entirety, sequentially many times with a series of distinct probes. This method not only allows us to increase the screening efficiency but also permits us to determine the binding profile of each and every library bead against a large number of target receptors. As proof of concept, we serially screened a random OBOC disulfide containing cyclic heptapeptide library with three water soluble dyes as model probes: malachite green, bromocresol purple and indigo carmine. This multiplicative screening approach resulted in a rapid determination of the binding profile of each and every bead respective to each of the three dyes. Beads that interacted with malachite green only, bromocresol purple only, or both indigo carmine and bromocresol purple were isolated, and their peptide sequences were determined with microsequencer. Ultimately, the novel OBOC multiplicative screening approach could play a key role in the enhancement of existing on-bead assays such as whole cell binding, bacteria binding, protein binding, posttranslational modifications etc. with increased efficiency, capacity, and specificity.

Use of Magnetic Bead Resin and Automated Liquid Handler Extraction Methods to Robotically Isolate Nucleic Acids of Biological Agent Simulates

DTIC Science & Technology

2003-09-01

concentration, and Bacillus subtilis var. niger spores were detectable at 10,000 CFU/ml. When combined with bead beating, these spores were consistently...Bioloeical Aaent Simulants. Cell suspensions of Bacillus subtilis var. niger spores (BG spores ) and Erwinia herbicola vegetative cells were prepared for...use as biological simulants. BG spores were prepared by inoculating 1 g spores of Bacillus subtilis var. niger (Merck & Co., Inc., Whitehouse Station

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

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.

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.

Renewable surface fluorescence sandwich immunoassay biosensor for rapid sensitive botulinum toxin detection in an automated fluidic format.

PubMed

Grate, Jay W; Warner, Marvin G; Ozanich, Richard M; Miller, Keith D; Colburn, Heather A; Dockendorff, Brian; Antolick, Kathryn C; Anheier, Norman C; Lind, Michael A; Lou, Jianlong; Marks, James D; Bruckner-Lea, Cynthia J

2009-05-01

A renewable surface biosensor for rapid detection of botulinum neurotoxin serotype A is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant protein fragment of the toxin heavy chain ( approximately 50 kDa) as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate non-overlapping epitopes of the full botulinum holotoxin ( approximately 150 kDa). Both of the targeted epitopes are located on the recombinant fragment. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by a sequential injection flow system, creating a 3.6 microL column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degrees angle to one another delivered excitation light from a HeNe laser (633 nm) using one fiber and collected fluorescent emission light for detection with the other. After each measurement, the used Sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes using this system.

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.

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.

The role of silica colloids on facilitated cesium transport through glass bead columns and modeling

NASA Astrophysics Data System (ADS)

Noell, Alan L.; Thompson, Joseph L.; Corapcioglu, M. Yavuz; Triay, Inés R.

1998-05-01

Groundwater colloids can act as a vector which enhances the migration of contaminants. While sorbed to mobile colloids, contaminants can be held in the aqueous phase which prevents them from interacting with immobile aquifer surfaces. In this study, an idealized laboratory set-up was used to examine the influence of amorphous silica colloids on the transport of cesium. Synthetic groundwater and saturated glass bead columns were used to minimize the presence of natural colloidal material. The columns were assembled in replicate, some packed with 150-210 μm glass bead and others packed with 355-420 μm glass beads. The colloids used in these experiments were 100 nm amorphous silica colloids from Nissan Chemical Company. In the absence of these colloids, the retardation factor for cesium was 8.0 in the 150-210 μm glass bead columns and 3.6 in the 355-420 μm glass bead columns. The influence of anthropogenic colloids was tested by injecting 0.09 pore volume slugs of an equilibrated suspension of cesium and colloids into the colloid-free columns. Although there was little noticeable facilitation in the smaller glass bead columns, there was a slight reduction in the retardation of cesium in the larger glass bead columns. This was attributed to cesium having less of a retention time in the larger glass bead columns. When cesium was injected into columns with a constant flux of colloids, the retardation of cesium was reduced by 14-32% in the 150-210 μm glass bead columns and by 38-51% in the 355-420 μm glass bead columns. A model based on Corapcioglu and Jiang (1993) [Corapcioglu, M.Y., Jiang, S., 1993. Colloid-facilitated groundwater contaminant transport, Water Resour. Res., 29 (7) 2215-2226] was compared with the experimental elution data. When equilibrium sorption expressions were used and the flux of colloids through the glass bead columns was constant, the colloid facilitated transport of cesium was able to be described using an effective retardation coefficient. Fully kinetic simulations, however, more accurately described the colloid facilitated transport of cesium.

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

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.

Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes

PubMed Central

Hjelm, Barbara; Forsström, Björn; Löfblom, John; Rockberg, Johan; Uhlén, Mathias

2012-01-01

A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar. PMID:23284606

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

PubMed

Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

2018-04-01

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Static optical sorting in a laser interference field

NASA Astrophysics Data System (ADS)

Jákl, Petr; Čižmár, Tomáš; Šerý, Mojmír; Zemánek, Pavel

2008-04-01

We present a unique technique for optical sorting of heterogeneous suspensions of microparticles, which does not require the flow of the immersion medium. The method employs the size-dependent response of suspended dielectric particles to the optical field of three intersecting beams that form a fringelike interference pattern. We experimentally demonstrate sorting of a polydisperse suspension of polystyrene beads of diameters 1, 2, and 5.2μm and living yeast cells.

Biotechnology

NASA Image and Video Library

2003-05-05

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. The beads are similar in size and density to human lymphoid cells. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light. In this photograph, beads are trapped in the injection port, with bubbles forming shortly after injection.

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.

Dispersion of fine phosphor particles by newly developed beads mill

NASA Astrophysics Data System (ADS)

Joni, I. Made; Panatarani, C.; Maulana, Dwindra W.

2016-02-01

Fine phosphor Y2O3:Eu3+ particles has advanced properties compare to conventional particles applied for compact fluorescent lamp (CFL) as three band phosphor. However, suspension of fine particles easily agglomerated during preparation of spray coating of the CFL tube. Therefore, it is introduced newly developed beads mill system to disperse fine phosphor. The beads mill consist of glass beads, dispersing chamber (impellers), separator chamber, slurry pump and motors. The first important performance of beads mill is the performance of the designed on separating the beads with the suspended fine particles. We report the development of beads mill and its separation performance vary in flow rate and separator rotation speeds. The 27 kg of glass beads with 30 µm in size was poured into dispersing chamber and then water was pumped continuously through the slurry pump. The samples for the separation test was obtained every 1 hours vary in rotation speed and slurry flow rate. The results shows that the separation performance was 99.99 % obtained for the rotation speed of >1000 rpm and flow rate of 8 L/minute. The performances of the system was verified by dispersing fine phosphor Y2O3:Eu3+ particles with concentration 1 wt.%. From the observed size distribution of particles after beads mill, it is concluded that the current design of bead mill effectively dispersed fine phosphor Y2O3:Eu3+.

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.

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.

Volume fraction instability in an oscillating non-Brownian iso-dense suspension.

NASA Astrophysics Data System (ADS)

Roht, Y. L.; Gauthier, G.; Hulin, J. P.; Salin, D.; Chertcoff, R.; Auradou, H.; Ippolito, I.

2017-06-01

The instability of an iso-dense non-Brownian suspension of polystyrene beads of diameter 40 μm dispersed in a water-glycerol mixture submitted to a periodic square wave oscillating flow in a Hele-Shaw cell is studied experimentally. The instability gives rise to stationary bead concentration waves transverse to the flow. It has been observed for average particle volume fractions between 0.25 and 0.4, for periods of the square wave flow variation between 0.4 and 10 s and in finite intervals of the amplitude of the fluid displacement. The study shows that the wavelength λ increases roughly linearly with the amplitude of the oscillatory flow; on the other hand, λ is independent of the particle concentration and of the period of oscillation of the flow although the minimum threshold amplitude for observing the instability increases with the period.

Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI)

NASA Technical Reports Server (NTRS)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. In this picture, the beads are trapped in the injection port shortly after injection. Swirls of beads indicate, event to the naked eye, the contents of the TCM are not fully mixed. The beads are similar in size and density to human lymphoid cells. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light

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.

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.

Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation.

PubMed

Hoesli, Corinne A; Raghuram, Kamini; Kiang, Roger L J; Mocinecová, Dušana; Hu, Xiaoke; Johnson, James D; Lacík, Igor; Kieffer, Timothy J; Piret, James M

2011-02-01

Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo-islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion-based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale. © 2010 Wiley Periodicals, Inc.

The Influence of Crystal Size Distributions on the Rheology of Magmas: New Insights From Analog Experiments

NASA Astrophysics Data System (ADS)

Klein, Johannes; Mueller, Sebastian P.; Castro, Jonathan M.

2017-11-01

This study examines the influence of particle-size distributions on the rheology of particle suspensions by using analog experiments with spherical glass beads in silicone oil as a magma equivalent. The analyses of 274 individual particle-bearing suspensions of varying modality (unimodality, bimodality, trimodality, and tetramodality), as well as of polymodal suspensions with specific defined skewness and variance, are the first data set of its kind and provide important insights into the relationship between the solid particles of a suspension and its rheological behavior. Since the relationship between the rheology of particle-bearing suspensions and its maximum packing fraction ϕm is well established by several theoretical models, the results of the analog experiments of this study reveal that the polydispersity γ exerts the largest influence on ϕm. Consequently, the estimation of the polydispersity γ of a particle-size distribution is essential for estimating the viscosity of that given suspension.

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.

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

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.

Optical demodulation system for digitally encoded suspension array in fluoroimmunoassay

NASA Astrophysics Data System (ADS)

He, Qinghua; Li, Dongmei; He, Yonghong; Guan, Tian; Zhang, Yilong; Shen, Zhiyuan; Chen, Xuejing; Liu, Siyu; Lu, Bangrong; Ji, Yanhong

2017-09-01

A laser-induced breakdown spectroscopy and fluorescence spectroscopy-coupled optical system is reported to demodulate digitally encoded suspension array in fluoroimmunoassay. It takes advantage of the plasma emissions of assembled elemental materials to digitally decode the suspension array, providing a more stable and accurate recognition to target biomolecules. By separating the decoding procedure of suspension array and adsorption quantity calculation of biomolecules into two independent channels, the cross talk between decoding and label signals in traditional methods had been successfully avoided, which promoted the accuracy of both processes and realized more sensitive quantitative detection of target biomolecules. We carried a multiplexed detection of several types of anti-IgG to verify the quantitative analysis performance of the system. A limit of detection of 1.48×10-10 M was achieved, demonstrating the detection sensitivity of the optical demodulation system.

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.

The dispersion of fine chitosan particles by beads-milling

NASA Astrophysics Data System (ADS)

Rochima, Emma; Utami, Safira; Hamdani, Herman; Azhary, Sundoro Yoga; Praseptiangga, Danar; Joni, I. Made; Panatarani, Camellia

2018-02-01

This research aimed to produce fine chitosan particles from a crab shell waste by beads-milling method by two different concentration of PEG as dispersing agent (150 and 300 wt. %). The characterization was performed to obtain the size and size distribution, the characteristics of functional groups and the degree of deacetylation. The results showed that the chitosan fine particles was obtained with a milling time 120 minutes with the best concentration of PEG 400 150 wt. %. The average particle size of the as-prepared suspension is 584 nm after addition of acetic acid solution (1%, v/v). Beads milling process did not change the glucosamine and N-acetylglucosamine content on chitosan structure which is indicated by degree of deacetylation higher than 70%. It was concluded that beads milling process can be applied to prepare chitosan fineparticles by proper adjustment in the milling time, pH and dosage of dispersing agent.

Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI)

NASA Technical Reports Server (NTRS)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. The beads are similar in size and density to human lymphoid cells. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light. In this photograph, beads are trapped in the injection port, with bubbles forming shortly after injection.

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).

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.

CONTRIBUTIONS OF CHEMICAL AND DIFFUSIVE EXCHANGE TO T1ρ DISPERSION

PubMed Central

Cobb, Jared Guthrie; Xie, Jingping; Gore, John C.

2012-01-01

Variations in local magnetic susceptibility may induce magnetic field gradients that affect the signals acquired for MR imaging. Under appropriate diffusion conditions, such fields produce effects similar to slow chemical exchange. These effects may also be found in combination with other chemical exchange processes at multiple time scales. We investigate these effects with simulations and measurements to determine their contributions to rotating frame (R1ρ) relaxation in model systems. Simulations of diffusive and chemical exchange effects on R1ρ dispersion were performed using the Bloch equations. Additionally, R1ρ dispersion was measured in suspensions of Sephadex and latex beads with varying spin locking fields at 9.4T. A novel analysis method was used to iteratively fit for apparent chemical and diffusive exchange rates with a model by Chopra et al. Single- and double-inflection points in R1ρ dispersion profiles were observed, respectively, in simulations of slow diffusive exchange alone and when combined with rapid chemical exchange. These simulations were consistent with measurements of R1ρ in latex bead suspensions and small-diameter Sephadex beads that showed single- and double-inflection points, respectively. These observations, along with measurements following changes in temperature and pH, are consistent with the combined effects of slow diffusion and rapid −OH exchange processes. PMID:22791589

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.

Composite cryogels for lysozyme purification.

PubMed

Baydemir, Gözde; Türkoğlu, Emir Alper; Andaç, Müge; Perçin, Işık; Denizli, Adil

2015-01-01

Beads-embedded novel composite cryogel was synthesized to purify lysozyme (Lyz) from chicken egg white. The poly(hydroxyethyl methacrylate-N-methacryloyl-L-phenylalanine) (PHEMAPA) beads of smaller than 5 µm size were synthesized by suspension polymerization and then embedded into a poly(hydroxyethyl methacrylate) (PHEMA)-based cryogel column. The PHEMAPA bead-embedded cryogel (BEC) column was characterized by swelling tests, scanning electron microscopy (SEM), surface area measurements by the Brunauer-Emmett-Teller (BET) method, elemental analysis, and flow dynamics. The specific surface area of the PHEMAPA BEC was found as 41.2 m(2) /g using BET measurements. Lyz-binding experiments were performed using aqueous solutions in different conditions such as initial Lyz concentration, pH, flow rate, temperature, and NaCl concentration of an aqueous medium. The PHEMAPA BEC column could be used after 10 adsorption-desorption studies without any significant loss in adsorption capacity of Lyz. The PHEMAPA BEC column was used to purify Lyz from chicken egg white, and gel electrophoresis was used to estimate the purity of Lyz. The chromatographic application of the PHEMAPA BEC column was also performed using fast protein liquid chromatography. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

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.

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.

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.

Effective viscosity of a suspension of flagellar-beating microswimmers: Three-dimensional modeling

NASA Astrophysics Data System (ADS)

Jibuti, Levan; Zimmermann, Walter; Rafaï, Salima; Peyla, Philippe

2017-11-01

Micro-organisms usually can swim in their liquid environment by flagellar or ciliary beating. In this numerical work, we analyze the influence of flagellar beating on the orbits of a swimming cell in a shear flow. We also calculate the effect of the flagellar beating on the rheology of a dilute suspension of microswimmers. A three-dimensional model is proposed for Chlamydomonas Reinhardtii swimming with a breaststroke-like beating of two anterior flagella modeled by two counter-rotating fore beads. The active swimmer model reveals unusual angular orbits in a linear shear flow. Namely, the swimmer sustains orientations transiently across the flow. Such behavior is a result of the interplay between shear flow and the swimmer's periodic beating motion of flagella, which exert internal torques on the cell body. This peculiar behavior has some significant consequences on the rheological properties of the suspension. We calculate Einstein's viscosity of the suspension composed of such isolated modeled microswimmers (dilute case) in a shear flow. We use numerical simulations based on a Rotne-Prager-like approximation for hydrodynamic interaction between simplified flagella and the cell body. The results show an increased intrinsic viscosity for active swimmer suspensions in comparison to nonactive ones as well as a shear thinning behavior in accordance with previous experimental measurements [Phys. Rev. Lett. 104, 098102 (2010), 10.1103/PhysRevLett.104.098102].

A direct comparison of the performance of ground, beaded and silica-grafted MIPs in HPLC and turbulent flow chromatography applications.

PubMed

Fairhurst, Robert E; Chassaing, Christophe; Venn, Richard F; Mayes, Andrew G

2004-12-15

Spherical molecularly imprinted polymers (MIPs) specific to the beta-blocker propranolol have been synthesised using two different approaches and compared to traditional ground monolithic MIPs in HPLC and TFC applications. TFC is a LC technique used for rapid extraction of compounds directly from complex matrices. It can be easily coupled to HPLC and MS for automation of an extraction/analysis procedure. Spherical MIP beads were produced using a suspension polymerisation technique and silica/MIP composite beads by grafting MIP to spherical silica particles using a surface-bound initiator species. Synthesis of both beaded and silica-grafted MIPs was more practical than using the traditional grinding method and yields of spherical particles of the required size between 80 and 100% were routinely achieved. Under HPLC conditions, beaded and ground MIP materials showed a degree of chiral separation for all of the nine beta-blockers tested. The beaded MIP, however, showed much better flow properties and peak shape than the ground material. Silica-grafted MIP showed some separation in five of the drugs and a large improvement in peak shape and analysis times compared with both ground and beaded MIPs. The materials prepared were also used in extraction columns for Turbulent Flow Chromatography (TFC). Although no imprinting effect was observed under typical TFC conditions, beaded polymer materials showed promise for use as TFC extraction columns due to the good flow properties and clean extracts obtained.

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

Functional Silver-Silicone-Nanofilament-Composite Material for Water Disinfection.

PubMed

Meier, Margrith; Suppiger, Angela; Eberl, Leo; Seeger, Stefan

2017-01-01

The roughness of superhydrophobic silicone nanofilaments (SNFs) is exploited to enlarge the contact area of conventional filter material. As an efficient wetting of the filter material is crucial for water treatment, the wettability of SNFs is readily modified from superhydrophobic to hydrophilic during the functionalization process. SNFs are coated on glass beads and subsequently modified with biocidal silver nanoparticles (AgNPs). The enlarged surface area of SNFs allows a 30 times higher loading of AgNPs in comparison to glass beads without SNF coating. Thus, in column experiments, the AgNP-SNF-nanocomposite-modified glass beads exert superior antibacterial activity towards suspensions of E. coli K12 compared to AgNP functionalized glass beads without SNFs. Additionally, reusing the AgNP-SNF-nanocomposite-coated glass beads with fresh bacteria contaminated medium increases their efficacy and reduces the colony forming units by ≈6 log units. Thereby, the silver loss during percolation is below 0.1 μg mL -1 . These results highlight, first, the potential of AgNP-SNF-nanocomposite-modified glass beads as an effective filter substrate for water disinfection, and second, the efficiency of SNF coating in increasing the contact area of conventional filter material. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transport and retention of nanoscale C60 aggregates in water-saturated porous media.

PubMed

Wang, Yonggang; Li, Yusong; Fortner, John D; Hughes, Joseph B; Abriola, Linda M; Pennell, Kurt D

2008-05-15

Experimental and mathematical modeling studies were performed to investigate the transport and retention of nanoscale fullerene aggregates (nC60) in water-saturated porous media. Aqueous suspensions of nC60 aggregates (95 nm diameter, 1 to 3 mg/L) were introduced into columns packed with either glass beads or Ottawa sand at a Darcy velocity of 2.8 m/d. In the presence of 1.0 mM CaCl2, nC60 effluent breakthrough curves (BTCs) gradually increased to a maximum value and then declined sharply upon reintroduction of nC60-free solution. Retention of nC60 in glass bead columns ranged from 8 to 49% of the introduced mass, while up to 77% of the mass was retained in Ottawa sand columns. When nC60 suspensions were prepared in deionized water alone, effluent nC60 BTCs coincided with those of a nonreactive tracer (Br-), with minimal nC60 retention. Observed differences in nC60 transport and retention behavior in glass beads and Ottawa sand were consistent with independent batch retention data and theoretical calculations of electrostatic interactions between nC60 and the solid surfaces. Effluent concentration and retention profile data were accurately simulated using a numerical model that accounted for nC60 attachment kinetics and a limiting retention capacity.

Contributions of chemical and diffusive exchange to T1ρ dispersion.

PubMed

Cobb, Jared Guthrie; Xie, Jingping; Gore, John C

2013-05-01

Variations in local magnetic susceptibility may induce magnetic field gradients that affect the signals acquired for MR imaging. Under appropriate diffusion conditions, such fields produce effects similar to slow chemical exchange. These effects may also be found in combination with other chemical exchange processes at multiple time scales. We investigate these effects with simulations and measurements to determine their contributions to rotating frame (R1ρ ) relaxation in model systems. Simulations of diffusive and chemical exchange effects on R1ρ dispersion were performed using the Bloch equations. Additionally, R1ρ dispersion was measured in suspensions of Sephadex and latex beads with varying spin locking fields at 9.4 T. A novel analysis method was used to iteratively fit for apparent chemical and diffusive exchange rates with a model by Chopra et al. Single- and double-inflection points in R1ρ dispersion profiles were observed, respectively, in simulations of slow diffusive exchange alone and when combined with rapid chemical exchange. These simulations were consistent with measurements of R1ρ in latex bead suspensions and small-diameter Sephadex beads that showed single- and double-inflection points, respectively. These observations, along with measurements following changes in temperature and pH, are consistent with the combined effects of slow diffusion and rapid -OH exchange processes. Copyright © 2012 Wiley Periodicals, Inc.

Magnetic microstructures for regulating Brownian motion

NASA Astrophysics Data System (ADS)

Sooryakumar, Ratnasingham

2013-03-01

Nature has proven that it is possible to engineer complex nanoscale machines in the presence of thermal fluctuations. These biological complexes, which harness random thermal energy to provide functionality, yield a framework to develop related artificial, i.e., nonbiological, phenomena and devices. A major challenge to achieving positional control of fluid-borne submicron sized objects is regulating their Brownian fluctuations. In this talk a magnetic-field-based trap that regulates the thermal fluctuations of superparamagnetic beads in suspension will be presented. Local domain-wall fields originating from patterned magnetic wires, whose strength and profile are tuned by weak external fields, enable bead trajectories within the trap to be managed and easily varied between strong confinements and delocalized spatial excursions. Moreover, the frequency spectrum of the trapped bead responds to fields as a power-law function with a tunable, non-integer exponent. When extended to a cluster of particles, the trapping landscape preferentially stabilizes them into formations of 5-fold symmetry, while their Brownian fluctuations result in frequent transitions between different cluster configurations. The quantitative understanding of the Brownian dynamics together with the ability to tune the extent of the fluctuations enables the wire-based platform to serve as a model system to investigate the competition between random and deterministic forces. Funding from the U.S. Army Research Office under contract W911NF-10-1-0353 is acknowledged.

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).

Biotechnology

NASA Image and Video Library

2003-05-05

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. The beads are similar in size and density to human lymphoid cells. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light. In this photograph, a TCM is shown after mixing protocols, and bubbles of various sizes can be seen.

Implementation of a decoupled controller for a magnetic suspension system using electromagnets mounted in a planar array

NASA Technical Reports Server (NTRS)

Cox, D. E.; Groom, N. J.

1994-01-01

An implementation of a decoupled, single-input/single-output control approach for a large angle magnetic suspension test fixture is described. Numerical and experimental results are presented. The experimental system is a laboratory model large gap magnetic suspension system which provides five degree-of-freedom control of a cylindrical suspended element. The suspended element contains a core composed of permanent magnet material and is levitated above five electromagnets mounted in a planar array.

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

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.

A superconducting large-angle magnetic suspension

NASA Technical Reports Server (NTRS)

Downer, James; Goldie, James; Torti, Richard

1991-01-01

The component technologies were developed required for an advanced control moment gyro (CMG) type of slewing actuator for large payloads. The key component of the CMG is a large-angle magnetic suspension (LAMS). The LAMS combines the functions of the gimbal structure, torque motors, and rotor bearings of a CMG. The LAMS uses a single superconducting source coil and an array of cryoresistive control coils to produce a specific output torque more than an order of magnitude greater than conventional devices. The designed and tested LAMS system is based around an available superconducting solenoid, an array of twelve room-temperature normal control coils, and a multi-input, multi-output control system. The control laws were demonstrated for stabilizing and controlling the LAMS system.

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.

Cell specific, variable density, polymer microspheres

NASA Technical Reports Server (NTRS)

Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)

1977-01-01

Biocompatible polymeric microspheres having an average diameter below about 3 microns and having density at least 15% greater or lesser than organic cells and having covalent binding sites are provided in accordance with this invention. The microspheres are obtained by copolymerizing a hydroxy or amine substituted acrylic monomer such as hydroxyethylmethacrylate with a light or dense comonomer such as a fluoromonomer. A lectin or antibody is bound to the hydroxy or amine site of the bead to provide cell specificity. When added to a cell suspension the marked bead will specifically label the cell membrane by binding to specific receptor sites thereon. The labelled membrane can then be separated by density gradient centrifugation.

Porous cobalt spheres for high temperature gradient magnetically assisted fluidized beds

NASA Technical Reports Server (NTRS)

Atwater, James E.; Akse, James R.; Jovanovic, Goran N.; Wheeler, Richard R Jr; Sornchamni, Thana

2003-01-01

Porous metallic cobalt spheres have been prepared as high temperature capable media for employment in gradient magnetically assisted fluidization and filtration technologies. Cobalt impregnated alginate beads are first formed by extrusion of an aqueous suspension of Co3O4 into a Co(II) chloride solution. The organic polymer is thermally decomposed yielding cobalt oxide spheres, followed by reduction to the metallic state, and densification. Cobalt beads have been produced with porosities ranging between 10 and 50%, depending upon sintering conditions. The product media have been characterized by scanning electron microscopy (SEM), nitrogen adsorption porosimetry, and vibrating sample magnetometry. c2003 Elsevier Science Ltd. All rights reserved.

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.

Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI)

NASA Technical Reports Server (NTRS)

2003-01-01

Aboard the International Space Station (ISS), the Tissue Culture Module (TCM) is the stationary bioreactor vessel in which cell cultures grow. However, for the Cellular Biotechnology Operations Support Systems-Fluid Dynamics Investigation (CBOSS-FDI), color polystyrene beads are used to measure the effectiveness of various mixing procedures. The beads are similar in size and density to human lymphoid cells. Uniform mixing is a crucial component of CBOSS experiments involving the immune response of human lymphoid cell suspensions. The goal is to develop procedures that are both convenient for the flight crew and are optimal in providing uniform and reproducible mixing of all components, including cells. The average bead density in a well mixed TCM will be uniform, with no bubbles, and it will be measured using the absorption of light. In this photograph, a TCM is shown after mixing protocols, and bubbles of various sizes can be seen.

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

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

Magnetic bead based immuno-detection of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables using the Bio-Plex suspension array system.

PubMed

Day, J B; Basavanna, U

2015-04-01

Listeriosis, a disease contracted via the consumption of foods contaminated with pathogenic Listeria species, can produce severe symptoms and high mortality in susceptible people and animals. The development of molecular methods and immuno-based techniques for detection of pathogenic Listeria in foods has been challenging due to the presence of assay inhibiting food components. In this study, we utilize a macrophage cell culture system for the isolation and enrichment of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables for subsequent identification using the Luminex xMAP technique. Macrophage monolayers were exposed to infant formula, lettuce and celery contaminated with L. monocytogenes or L. ivanovii. Magnetic microspheres conjugated to Listeria specific antibody were used to capture Listeria from infected macrophages and then analyzed using the Bio-Plex 200 analyzer. As few as 10 CFU/mL or g of L. monocytogenes was detected in all foods tested. The detection limit for L. ivanovii was 10 CFU/mL in infant formula and 100 CFU/g in leafy greens. Microsphere bound Listeria obtained from infected macrophage lysates could also be isolated on selective media for subsequent confirmatory identification. This method presumptively identifies L. monocytogenes and L. ivanovii from infant formula, lettuce and celery in less than 28 h with confirmatory identifications completed in less than 48 h. Published by Elsevier Ltd.

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 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.

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.

Measurement of the average mass of proteins adsorbed to a nanoparticle by using a suspended microchannel resonator.

PubMed

Nejadnik, M Reza; Jiskoot, Wim

2015-02-01

We assessed the potential of a suspended microchannel resonator (SMR) to measure the adsorption of proteins to nanoparticles. Standard polystyrene beads suspended in buffer were weighed by a SMR system. Particle suspensions were mixed with solutions of bovine serum albumin (BSA) or monoclonal human antibody (IgG), incubated at room temperature for 3 h and weighed again with SMR. The difference in buoyant mass of the bare and protein-coated polystyrene beads was calculated into real mass of adsorbed proteins. The average surface area occupied per protein molecule was calculated, assuming a monolayer of adsorbed protein. In parallel, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and zeta potential measurements were performed. SMR revealed a statistically significant increase in the mass of beads because of adsorption of proteins (for BSA and IgG), whereas DLS and NTA did not show a difference between the size of bare and protein-coated beads. The change in the zeta potential of the beads was also measurable. The surface area occupied per protein molecule was in line with their known size. Presented results show that SMR can be used to measure the mass of adsorbed protein to nanoparticles with a high precision in the presence of free protein. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Shear and mixing effects on cells in agitated microcarrier tissue culture reactors

NASA Technical Reports Server (NTRS)

Cherry, Robert S.; Papoutsakis, E. Terry

1987-01-01

Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from: (1) direct interaction between microcarriers and turbulent eddies; (2) collisions between microcarriers in turbulent flow; and (3) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Impeller collisions occur when beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture reactors are discussed.

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.

Westinghouse modular grinding process - improvement for follow on processes

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

Fehrmann, Henning

2013-07-01

In nuclear power plants (NPP) ion exchange (IX) resins are used in several systems for water treatment. The resins can be in bead or powdered form. For waste treatment of spent IX resins, two methods are basically used: Direct immobilization (e.g. with cement, bitumen, polymer or High Integrity Container (HIC)); Thermal treatment (e.g. drying, oxidation or pyrolysis). Bead resins have some properties (e.g. particle size and density) that can have negative impacts on following waste treatment processes. Negative impacts could be: Floatation of bead resins in cementation process; Sedimentation in pipeline during transportation; Poor compaction properties for Hot Resin Supercompactionmore » (HRSC). Reducing the particle size of the bead resins can have beneficial effects enhancing further treatment processes and overcoming prior mentioned effects. Westinghouse Electric Company has developed a modular grinding process to crush/grind the bead resins. This modular process is designed for flexible use and enables a selective adjustment of particle size to tailor the grinding system to the customer needs. The system can be equipped with a crusher integrated in the process tank and if necessary a colloid mill. The crusher reduces the bead resins particle size and converts the bead resins to a pump able suspension with lower sedimentation properties. With the colloid mill the resins can be ground to a powder. Compared to existing grinding systems this equipment is designed to minimize radiation exposure of the worker during operation and maintenance. Using the crushed and/or ground bead resins has several beneficial effects like facilitating cementation process and recipe development, enhancing oxidation of resins, improving the Hot Resin Supercompaction volume reduction performance. (authors)« less

A SNP genotyping array for hexaploid oat

USDA-ARS?s Scientific Manuscript database

Recognizing a need in cultivated hexaploid oat (Avena sativa L.) for a reliable set of reference SNPs, we have developed a 6K BeadChip design containing 257 Infinium I and 5,486 Infinium II designs corresponding to 5,743 SNPs. Of those, 4,975 SNPs yielded successful assays after array manufacturing...

Cell specific, variable density, polymer microspheres

NASA Technical Reports Server (NTRS)

Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)

1978-01-01

Biocompatible polymeric microspheres having an average diameter below about 3 microns and having a density at least 15% greater or lesser than organic cells and having covalent binding sites are provided in accordance with this invention. The microspheres are obtained by copolymerizing a hydroxy or amine substituted acrylic monomer such as hydroxyethylmethacrylate with a light or dense comonomer such as a fluoromonomer. A lectin or antibody is bound to the hydroxy or amine site of the bead to provide cell specificity. When added to a cell suspension the marked bead will specifically label the cell membrane by binding to specific receptor sites thereon. The labelled membrane can then be separated by density gradient centrifugation.

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

Effective cluster model of dielectric enhancement in metal-insulator composites

NASA Astrophysics Data System (ADS)

Doyle, W. T.; Jacobs, I. S.

1990-11-01

The electrical permittivity of a suspension of conducting spheres at high volume loading exhibits a large enhancement above the value predicted by the Clausius-Mossotti approximation. The permittivity enhancement is a dielectric anomaly accompanying a metallization transition that occurs when conducting particles are close packed. In disordered suspensions, close encounters can cause a permittivity enhancement at any volume loading. We attribute the permittivity enhancements typically observed in monodisperse disordered suspensions of conducting spheres to local metallized regions of high density produced by density fluctuations. We model a disordered suspension as a mixture, or mesosuspension, of isolated spheres and random close-packed spherical clusters of arbitrary size. Multipole interactions within the clusters are treated exactly. External interactions between clusters and isolated spheres are treated in the dipole approximation. Model permittivities are compared with Guillien's experimental permittivity measurements [Ann. Phys. (Paris) Ser. 11, 16, 205 (1941)] on liquid suspensions of Hg droplets in oil and with Turner's conductivity measurements [Chem. Eng. Sci. 31, 487 (1976)] on fluidized bed suspensions of ion-exchange resin beads in aqueous solution. New permittivity measurements at 10 GHz on solid suspensions of monodisperse metal spheres in polyurethane are presented and compared with the model permittivities. The effective spherical cluster model is in excellent agreement with the experiments over the entire accessible range of volume loading.

The addition of nanochitosan suspension as filler in carrageenan-tapioca biocomposite film

NASA Astrophysics Data System (ADS)

Rochima, Emma; Fiyanih, Elisah; Afrianto, Eddy; Subhan, Ujang; Praseptiangga, Danar; Panatarani, Camellia; Joni, I. Made

2018-02-01

This research aimed to investigate the effect of nanochitosan (CSNPs) suspension by beads milling method as filler in carrageenan-tapioca biocomposite film. In addition, the antibacterial activity of CSNPs as filler with two food pathogenic bacteria, Staphylococcus aureus and Escherichia coli and then influence of nano fillers for appearance of films were observed. The incorporation of CSNPs suspension with 0.5, 1, 1.5 and 2 (%v/v) in carrageenan-tapioca film exhibited antibacterial activity againts both bacteria. CSNPs had slightly higher antimicrobial activity against E. coli aureus compared to S. aureus at all concentrations due to different mechanisms. Therefore, the best antimicrobial activity was obtained from 1 wt%. Furthermore the best antimicrobial activity was characterized by means of the thickness and transparency. The result showed that the thickness of film was 0.059 mm and the transparency was 87.88. It was concluded that the incorporation of CSNPs suspension 1 wt% in carrageenan-tapioca composite film is suitable for developing active packaging.

Photocatalytic degradation of pharmaceutical wastes by alginate supported TiO2 nanoparticles in packed bed photo reactor (PBPR).

PubMed

Sarkar, Santanu; Chakraborty, Sudip; Bhattacharjee, Chiranjib

2015-11-01

In recent years deposal of pharmaceutical wastes has become a major problem globally. Therefore, it is necessary to removes pharmaceutical waste from the municipal as well as industrial effluents before its discharge. The convectional wastewater and biological treatments are generally failed to separate different drugs from wastewater streams. Thus, heterogeneous photocatalysis process becomes lucrative method for reduction of detrimental effects of pharmaceutical compounds. The main disadvantage of the process is the reuse or recycle of photocatalysis is a tedious job. In this work, the degradation of aqueous solution of chlorhexidine digluconate (CHD), an antibiotic drug, by heterogeneous photocatalysis was study using supported TiO2 nanoparticle. The major concern of this study is to bring down the limitations of suspension mode heterogeneous photocatalysis by implementation of immobilized TiO2 with help of calcium alginate beads. The alginate supported catalyst beads was characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDAX) as well as the characteristic crystalline forms of TiO2 nanoparticle was confirmed by XRD. The degradation efficiency of TiO2 impregnated alginate beads (TIAB) was compared with the performance of free TiO2 suspension. Although, the degradation efficiency was reduced considerably using TIAB but the recycle and reuse of catalyst was increased quite appreciably. The kinetic parameters related to this work have also been measure. Moreover, to study the susceptibility of the present system photocatalysis of other three drugs ibuprofen (IBP), atenolol (ATL) and carbamazepine (CBZ) has been carried out using immobilized TiO2. The continuous mode operation in PBPR has ensured the applicability of alginate beads along with TiO2 in wastewater treatment. The variation of residence time has significant impact on the performance of PBPR. Copyright © 2015 Elsevier Inc. All rights reserved.

Nature of crystalline particle assembly in ring shaped colloidal stains from concentrated dispersions

NASA Astrophysics Data System (ADS)

Shao, Fenfen; Huynh, Trang; Somers, Anthony; Liu, Boyin; Fu, Jing; Muradoglu, Murat; Ng, Tuck Wah

2014-05-01

The drying of colloidal droplet suspensions is important in many realms of practical application and has sustained the interest of researchers over two decades. The arrangements of polystyrene and silica beads, both of diameter 1 μm, 10% by volume of solid deposited on normal glass (hydrophilic), and silicone (hydrophobic) surfaces evaporated from a suspension volume of 3 μL, were investigated. Doughnut shape depositions were found, imputing the influence of strong central circulation flows that resulted in three general regions. In the central region which had strong particle build-up, the top most layers of particle arrangement was confirmed to be disordered using power spectrum and radial distribution function analysis. On closer examination, this appeared more like frustrated attempts to crystallize into larger grains rather than beads arranging in a disordered fashion throughout the piling process. With an adapted micro-bulldozing operation to progressively remove layers of particles from the heap, we found that the later efforts to crystallize through lateral capillary inter-particle forces were liable to be undone once the particles contacted the disorganized particles underneath, which were formed out of the jamming of fast particles arriving at the surface.

Bioparticles assembled using low frequency vibration immune to evacuation drifts

NASA Astrophysics Data System (ADS)

Shao, Fenfen; Whitehill, James David; Ng, Tuck Wah

2012-08-01

The use of low frequency vibration on suspensions of glass beads in a droplet has been shown to develop a strong degree of patterning (to a ring) due to the manner with which the surface waves are modified. Functionalized glass beads that serve as bioparticles permit for sensitive readings when concentrated at specific locations. However, a time controlled exposure with analytes is desirable. The replacement of the liquid medium with analyte through extraction is needed to conserve time. Nevertheless, we show here that extraction with a porous media, which is simple and useable in the field, will strongly displace the patterned beads. The liquid removal was found to be dependent on two mechanisms that affect the shape of the droplet, one of contact hysteresis due to the outer edge pinning, and the other of liquid being drawn into the porous media. From this, we developed and demonstrated a modified well structure that prevented micro-bead displacement during evacuation. An added strong advantage with this approach lies with its ability to require only analytes to be dispensed at the location of aggregated particles, which minimizes analyte usage. This was analytically established here.

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

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.

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.

Intracellular protein determination using droplet-based immunoassays.

PubMed

Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E; Chanasakulniyom, Mayuree; Pitt, Andrew R; Cooper, Jonathan M

2011-07-01

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

Combined surface-enhanced Raman spectroscopy biotags and microfluidic platform for quantitative ratiometric discrimination between noncancerous and cancerous cells in flow

NASA Astrophysics Data System (ADS)

Pallaoro, Alessia; Hoonejani, Mehran R.; Braun, Gary B.; Meinhart, Carl; Moskovits, Martin

2013-01-01

Surface-enhanced Raman spectroscopy (SERS) biotags (SBTs) that carry peptides as cell recognition moieties were made from polymer-encapsulated silver nanoparticle dimers, infused with unique Raman reporter molecules. We previously demonstrated their potential use for identification of malignant cells, a central goal in cancer research, through a multiplexed, ratiometric method that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro based on receptor overexpression. Progress has been made toward the application of this quantitative methodology for the identification of cancer cells in a microfluidic flow-focusing device. Beads are used as cell mimics to evaluate the devices. Cells (and beads) are simultaneously incubated with two sets of SBTs while in suspension, then injected into the device for laser interrogation under flow. Each cell event is characterized by a composite Raman spectrum, deconvoluted into its single components to ultimately determine their relative contribution. We have found that using SBTs ratiometrically can provide cell identification in flow, insensitive to normal causes of uncertainty in optical measurements such as variations in focal plane, cell concentration, autofluorescence, and turbidity.

Preparation, characterization, and application of poly(vinyl alcohol)-graft-poly(ethylene glycol) resins: novel polymer matrices for solid-phase synthesis.

PubMed

Luo, Juntao; Pardin, Christophe; Zhu, X X; Lubell, William D

2007-01-01

Spherical crosslinked poly(vinyl alcohol) (PVA) beads with good mechanical stability were prepared by reverse-suspension polymerization, using dimethyl sulfoxide (DMSO) as a cosolvent in an aqueous phase. Poly(ethylene glycol)s with varying chain lengths were grafted onto the PVA beads by anionic polymerization of ethylene oxide. The thermal behavior, morphology, and swelling were evaluated for each of the new polymer matrices. High loading and good swelling in water and organic solvents were characteristic of the PEG-grafted PVA beads. The polymer beads also exhibited good mechanical and chemical stability and were unaffected by treatment with 6 N HCl and with 6 N NaOH. The hydroxyl groups of the PVA-PEG beads were converted into aldehyde, carboxylic acid, and isocyanate functions to provide scavenger resins and were extended by way of a benzyl alcohol in a Wang linker. The transglutaminase substrates dipeptides (Z-Gln-Gly) and heptapeptides (Pro-Asn-Pro-Gln-Leu-Pro-Phe) were synthesized on PVA-PEG_5, PVA-PEG_20, and the Wang linker-derivatized PVA-PEG resins. The cleavage of the peptides from the resins using MeOH/NH3 mixture at different temperatures (0 degrees C and room temp) and 50% TFA/DCM provided, respectively, peptide methyl esters, amides, and acids in good yields and purity as assessed by LC-MS analysis.

Poly(glycidyl methacrylate)-A soft template for the facile preparation of poly(glycidyl methacrylate) core-copper nanoparticle shell nanocomposite

NASA Astrophysics Data System (ADS)

Mohammed Safiullah, S.; Abdul Wasi, K.; Anver Basha, K.

2015-12-01

Poly(glycidyl methacrylate) core/copper nanoparticle shell nanocomposite (PGMA/Cu nanohybrid) was prepared by simple two step method (i) The synthesis of poly(glycidyl methacrylate) (PGMA) beads by free radical suspension polymerization followed by (ii) direct deposition of copper nanoparticles (CuNPs) on activated PGMA beads. The PGMA beads were used as a soft template to host the CuNPs without surface modification of it. In this method the CuNPs were formed by chemical reduction of copper salts using sodium borohydride in water medium and deposited directly on the activated PGMA. Two different concentrations of copper salts were employed to know the effect of concentration on the shape and size of nanoparticles. The results showed that, the different sizes and shapes of CuNPs were deposited on the PGMA matrix. The X-ray Diffraction study results showed that the CuNPs were embedded on the surface of the PGMA matrix. The scanning electron microscopic images revealed that the fabrication of CuNPs on the PGMA matrix possess different shapes and changes the morphology and nature of PGMA beads significantly. The fluorescent micrograph also confirmed that the CuNPs were doped on the PGMA surface. The thermal studies have demonstrated that the CuNPs deposition on the surface of PGMA beads had a significant effect.

A two-magnet strategy for improved mixing and capture from biofluids

PubMed Central

Doyle, Andrew B.; Haselton, Frederick R.

2016-01-01

Magnetic beads are a popular method for concentrating biomolecules from solution and have been more recently used in multistep pre-arrayed microfluidic cartridges. Typical processing strategies rely on a single magnet, resulting in a tight cluster of beads and requiring long incubation times to achieve high capture efficiencies, especially in highly viscous patient samples. This report describes a two-magnet strategy to improve the interaction of the bead surface with the surrounding fluid inside of a pre-arrayed, self-contained assay-in-a-tube. In the two-magnet system, target biomarker capture occurs at a rate three times faster than the single-magnet system. In clinically relevant biomatrices, we find a 2.5-fold improvement in biomarker capture at lower sample viscosities with the two-magnet system. In addition, we observe a 20% increase in the amount of protein captured at high viscosity for the two-magnet configuration relative to the single magnet approach. The two-magnet approach offers a means to achieve higher biomolecule extraction yields and shorter assay times in magnetic capture assays and in self-contained processor designs. PMID:27158286

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

PubMed

Pochechueva, Tatiana; Jacob, Francis; Goldstein, Darlene R; Huflejt, Margaret E; Chinarev, Alexander; Caduff, Rosemarie; Fink, Daniel; Hacker, Neville; Bovin, Nicolai V; Heinzelmann-Schwarz, Viola

2011-12-01

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection. © The Author(s) 2011. This article is published with open access at Springerlink.com

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.

Preparation of styrene-co-4-vinylpyridine magnetic polymer beads by microwave irradiation for analysis of trace 24-epibrassinolide in plant samples using high performance liquid chromatography.

PubMed

Zhang, Zhuomin; Zhang, Yi; Tan, Wei; Li, Gongke; Hu, Yuling

2010-10-15

In the study, a kind of novel styrene-co-4-vinylpyridine (St-co-4-VP) porous magnetic polymer beads was prepared by microwave irradiation using suspension polymerization. Microwave heating preparation greatly reduced the polymerization time to 1h. Physical characteristic tests suggested that these beads were cross-linking and possessed spherical shape, good magnetic response and porous morphologies with a narrow diameter distribution of 70-180 μm. Therefore, these beads displayed the long-term stability after undergoing 100-time extractions. Then, an analytical method for the determination of trace 24-epiBR in plant samples was developed by magnetic polymer bead extraction coupled with high performance liquid chromatography-fluorescence detection. St-co-4-VP magnetic polymer beads demonstrated the higher extraction selectivity for 24-epiBR than other reference compounds. Linear range was 10.00-100.0 μg/L with a relative standard deviation (RSD) of 6.7%, and the detection limit was 6.5 μg/kg. This analytical method was successfully applied to analyze the trace 24-epiBR in cole and breaking-wall rape pollen samples with recoveries of 77.2-90.0% and 72.3-83.4%, respectively, and RSDs were less than 4.1%. The amount of 24-epiBR in real breaking-wall rape pollen samples was found to be 26.2 μg/kg finally. This work proposed a sensitive, rapid, reliable and convenient analytical method for the determination of trace brassinosteroids in complicated plant samples by the use of St-co-4-VP magnetic polymer bead extraction coupled with chromatographic method. Copyright © 2010 Elsevier B.V. All rights reserved.

Critical Bursts in Filtration

NASA Astrophysics Data System (ADS)

Bianchi, Filippo; Thielmann, Marcel; de Arcangelis, Lucilla; Herrmann, Hans Jürgen

2018-01-01

Particle detachment bursts during the flow of suspensions through porous media are a phenomenon that can severely affect the efficiency of deep bed filters. Despite the relevance in several industrial fields, little is known about the statistical properties and the temporal organization of these events. We present experiments of suspensions of deionized water carrying quartz particles pushed with a peristaltic pump through a filter of glass beads measuring simultaneously the pressure drop, flux, and suspension solid fraction. We find that the burst size distribution scales consistently with a power law, suggesting that we are in the presence of a novel experimental realization of a self-organized critical system. Temporal correlations are present in the time series, like in other phenomena such as earthquakes or neuronal activity bursts, and also an analog to Omori's law can be shown. The understanding of burst statistics could provide novel insights in different fields, e.g., in the filter and petroleum industries.

Isolation, Identification, and Culture of Human Lymphatic Endothelial Cells.

PubMed

Lokmic, Zerina

2016-01-01

A protocol describing the isolation of foreskin lymphatic endothelial cells (LECs) and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented herein. To isolate LECs and LM LECs, tissues are mechanically disrupted to make a single-cell suspension, which is then enzymatically digested in dispase and collagenase type II. LECs and LM LECs, in the resulting single-cell suspension, are then sequentially labeled with antibodies recognizing fibroblast and endothelial cell surface antigens CD34 and CD31 and separated from the remaining components in the cell suspension by capture with magnetic beads. Viable LECs and LM LECs are then seeded and expanded on fibronectin-coated flasks. LEC and LM LEC purity is determined immunohistochemically using cell surface markers CD31, CD34, podoplanin, VEGFR-3 and nuclear marker PROX-1. Cells whose purity is >98 % are used for experiments between passage 4 and 6.

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.

Rule-Based Motion Coordination for the Adaptive Suspension Vehicle on Ternary-Type Terrain

DTIC Science & Technology

1990-12-01

robot-window-array* nil) (defvar *robot..window..width* nil) (defvar * rebot -.window..heig)ht* nil) (defvar *terrain-buffer* nil) (defvar *terrain...cond ((momrber leg lift-able-leg. -test #’equal) log) (t nil)) .(dafmethod (test-overlap- rebot ipltcable-leg) (log) (nond ((and (member leg place-able

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.

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.

Motion stability of the magnetic levitation and suspension with YBa2Cu3O7-x high-Tc superconducting bulks and NdFeB magnets

NASA Astrophysics Data System (ADS)

Li, Jipeng; Zheng, Jun; Huang, Huan; Li, Yanxing; Li, Haitao; Deng, Zigang

2017-10-01

The flux pinning effect of YBa2Cu3O7-x high temperature superconducting (HTS) bulk can achieve self-stable levitation over a permanent magnet or magnet array. Devices based on this phenomenon have been widely developed. However, the self-stable flux pinning effect is not unconditional, under disturbances, for example. To disclose the roots of this amazing self-stable levitation phenomenon in theory, mathematical and mechanical calculations using Lyapunov's stability theorem and the Hurwitz criterion were performed under the conditions of magnetic levitation and suspension of HTS bulk near permanent magnets in Halbach array. It is found that the whole dynamical system, in the case of levitation, has only one equilibrium solution, and the singular point is a stable focus. In the general case of suspension, the system has two singular points: one is a stable focus, and the other is an unstable saddle. With the variation of suspension force, the two first-order singular points mentioned earlier will get closer and closer, and finally degenerate to a high-order singular point, which means the stable region gets smaller and smaller, and finally vanishes. According to the center manifold theorem, the high-order singular point is unstable. With the interaction force varying, the HTS suspension dynamical system undergoes a saddle-node bifurcation. Moreover, a deficient damping can also decrease the stable region. These findings, together with existing experiments, could enlighten the improvement of HTS devices with strong anti-interference ability.

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

Ultra-senstitive magnesium oxide-based magnetic tunnel junctions for spintronic immunoassay

NASA Astrophysics Data System (ADS)

Shen, Weifeng

We systematically studied the spin-dependent tunnel properties of MgO-based magnetic tunnel junctions (MTJs). Utilizing the spin-coherent tunnel effects of the MgO (001) insulating layer, we have achieved large tunneling magnetoresistance (TMR) ratios (above 200%) at room temperature in optimized MTJ devices. We have shown that the MgO surface roughness, and therefore device magnetoresistance, depends strongly on the pressure of the Ar sputtering gas. We have investigated the characteristics of MgO-MTJs, including their dependence on barrier thickness and bias voltage, their thermal stability and resistance to electrostatic discharge (ESD). We have also fabricated MgO-MTJs with a synthetic antiferromagnetic (SAF) free layer, which exhibits a coherent, single-domain-like switching. Our data show that MgO-MTJs have superior properties for low-field magnetic field sensing applications as compared with conventional AlOx-based MTJs. Based on this giant TMR effect, we designed and developed ultra-sensitive magnetic tunnel junction (MTJ) sensors and sensor arrays for biomagnetic sensing applications. By integrating MTJ sensor arrays into microfluidic channels, we were able to detect the presence of moving, micron-size superparamagnetic beads in real time. We have obtained an average signal of 80 mV for a single Dynal M-280 bead, with a signal-to-noise ratio (SNR) of 24 dB. We also biologically treated the MTJ sensor array surfaces, and demonstrated the detection of 2.5 muM single strand target DNA labeled with 16-nm-diameter Fe3O 4 nanoparticles (NPs). Our measured signal of 72 muV indicates that the current system's detection limit for analyte DNA is better than 150 nM. We also demonstrated the detection of live HeLa cells labeled with Fe 3O4 nanoparticles, with an effective signal of 8 mV and a signal-to-noise ratio of 6 dB. These results represent an important milestone in the development of spintronics immunoassay technology: the detection of a single live cell labeled with magnetic nanoparticles. All the data show conclusively that MTJ sensors and sensor arrays are very promising candidates for future applications involving the accurate detection and identification of biomolecules tagged with magnetic labels.

Multiple pathogen biomarker detection using an encoded bead array in droplet PCR.

PubMed

Periyannan Rajeswari, Prem Kumar; Soderberg, Lovisa M; Yacoub, Alia; Leijon, Mikael; Andersson Svahn, Helene; Joensson, Haakan N

2017-08-01

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads. Copyright © 2017. Published by Elsevier B.V.

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.

Size exclusion deep bed filtration: Experimental and modelling uncertainties

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

Badalyan, Alexander, E-mail: alexander.badalyan@adelaide.edu.au; You, Zhenjiang; Aji, Kaiser

A detailed uncertainty analysis associated with carboxyl-modified latex particle capture in glass bead-formed porous media enabled verification of the two theoretical stochastic models for prediction of particle retention due to size exclusion. At the beginning of this analysis it is established that size exclusion is a dominant particle capture mechanism in the present study: calculated significant repulsive Derjaguin-Landau-Verwey-Overbeek potential between latex particles and glass beads is an indication of their mutual repulsion, thus, fulfilling the necessary condition for size exclusion. Applying linear uncertainty propagation method in the form of truncated Taylor's series expansion, combined standard uncertainties (CSUs) in normalised suspendedmore » particle concentrations are calculated using CSUs in experimentally determined parameters such as: an inlet volumetric flowrate of suspension, particle number in suspensions, particle concentrations in inlet and outlet streams, particle and pore throat size distributions. Weathering of glass beads in high alkaline solutions does not appreciably change particle size distribution, and, therefore, is not considered as an additional contributor to the weighted mean particle radius and corresponded weighted mean standard deviation. Weighted mean particle radius and LogNormal mean pore throat radius are characterised by the highest CSUs among all experimental parameters translating to high CSU in the jamming ratio factor (dimensionless particle size). Normalised suspended particle concentrations calculated via two theoretical models are characterised by higher CSUs than those for experimental data. The model accounting the fraction of inaccessible flow as a function of latex particle radius excellently predicts normalised suspended particle concentrations for the whole range of jamming ratios. The presented uncertainty analysis can be also used for comparison of intra- and inter-laboratory particle size exclusion data.« less

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.

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.

From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells.

PubMed

Tamagno, Gianluca; Vigolo, Simonetta; Olivieri, Massimiliano; Martini, Chiara; De Carlo, Eugenio

2014-01-01

Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.

Transport of nano-objects in narrow channels: influence of Brownian diffusion, confinement and particle nature

NASA Astrophysics Data System (ADS)

Liot, O.; Socol, M.; Garcia, L.; Thiéry, J.; Figarol, A.; Mingotaud, A. F.; Joseph, P.

2018-06-01

This paper presents experimental results about transport of dilute suspensions of nano-objects in silicon-glass micrometric and sub-micrometric channels. Two kinds of objects are used: solid, rigid latex beads and spherical capsule-shaped, soft polymersomes. They are tracked using fluorescence microscopy. Three aspects are studied: confinement (ratio between particle diameter and channel depth), Brownian diffusion and particle nature. The aim of this work is to understand how these different aspects affect the transport of suspensions in narrow channels and to understand the different mechanisms at play. Concerning the solid beads we observe the appearance of two regimes, one where the experimental mean velocity is close to the expected one and another where this velocity is lower. This is directly related to a competition between confinement, Brownian diffusion and advection. These two regimes are shown to be linked to the inhomogeneity of particles distribution in the channel depth, which we experimentally deduce from velocity distributions. This inhomogeneity appears during the entrance process into the sub-micrometric channels, as for hydrodynamic separation or deterministic lateral displacement. Concerning the nature of the particles we observed a shift of transition towards the second regime likely due to the relationships between shear stress and polymersomes mechanical properties which could reduce the inhomogeneity imposed by the geometry of our device.

Transport of nano-objects in narrow channels: influence of Brownian diffusion, confinement and particle nature.

PubMed

Liot, O; Socol, M; Garcia, L; Thiéry, J; Figarol, A; Mingotaud, A F; Joseph, P

2018-06-13

This paper presents experimental results about transport of dilute suspensions of nano-objects in silicon-glass micrometric and sub-micrometric channels. Two kinds of objects are used: solid, rigid latex beads and spherical capsule-shaped, soft polymersomes. They are tracked using fluorescence microscopy. Three aspects are studied: confinement (ratio between particle diameter and channel depth), Brownian diffusion and particle nature. The aim of this work is to understand how these different aspects affect the transport of suspensions in narrow channels and to understand the different mechanisms at play. Concerning the solid beads we observe the appearance of two regimes, one where the experimental mean velocity is close to the expected one and another where this velocity is lower. This is directly related to a competition between confinement, Brownian diffusion and advection. These two regimes are shown to be linked to the inhomogeneity of particles distribution in the channel depth, which we experimentally deduce from velocity distributions. This inhomogeneity appears during the entrance process into the sub-micrometric channels, as for hydrodynamic separation or deterministic lateral displacement. Concerning the nature of the particles we observed a shift of transition towards the second regime likely due to the relationships between shear stress and polymersomes mechanical properties which could reduce the inhomogeneity imposed by the geometry of our device.

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.

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.

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.

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.

Single particles accelerate final stages of capillary break-up

NASA Astrophysics Data System (ADS)

Lindner, Anke; Fiscina, Jorge Eduardo; Wagner, Christian

2015-06-01

Droplet formation of suspensions is present in many industrial and technological processes such as coating and food engineering. Whilst the finite-time singularity of the minimum neck diameter in capillary break-up of simple liquids can be described by well-known self-similarity solutions, the pinching of non-Brownian suspension depends in a complex way on the particle dynamics in the thinning thread. Here we focus on the very dilute regime where the filament contains only isolated beads to identify the physical mechanisms leading to the pronounced acceleration of the filament thinning observed. This accelerated regime is characterized by an asymmetric shape of the filament with an enhanced curvature that depends on the size and the spatial distribution of the particles within the capillary thread.

CELL SEPARATION ON ANTIGEN-COATED COLUMNS

PubMed Central

Wigzell, Hans; Andersson, Birger

1969-01-01

Glass and plastic bead columns coated with antigenic protein molecules were used as an immunological filter for cell populations containing immune cells of relevant specificity. A selective elimination of these immune cells from the passing cell suspension was regularly noted and it approached, in some experiments, complete abolition of the specific immune reactivity of the filtered cell population. This specific retention of immune cells by antigenic columns could be selectively blocked by the presence of free antigen molecules in the medium during filtration. The results obtained support the concept of a cell-associated antigen-specific receptor being present on the outer surface of immune cells, displaying the same antigen-binding specificity as the potential product of the cell, the humoral antibody. Using the present bead column system, results were obtained indicating that this receptor was an active product of the immune cells and not any passively adsorbed, cytophilic antibody. Antigenic bead columns may very well constitute a tool for the production in vitro of cell populations being specifically deprived of immune reactivity and allow detailed analysis of the characteristics of the cell-associated antibody of immune cells. PMID:5782770

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

A first-passage scheme for determination of overall rate constants for non-diffusion-limited suspensions

NASA Astrophysics Data System (ADS)

Lu, Shih-Yuan; Yen, Yi-Ming

2002-02-01

A first-passage scheme is devised to determine the overall rate constant of suspensions under the non-diffusion-limited condition. The original first-passage scheme developed for diffusion-limited processes is modified to account for the finite incorporation rate at the inclusion surface by using a concept of the nonzero survival probability of the diffusing entity at entity-inclusion encounters. This nonzero survival probability is obtained from solving a relevant boundary value problem. The new first-passage scheme is validated by an excellent agreement between overall rate constant results from the present development and from an accurate boundary collocation calculation for the three common spherical arrays [J. Chem. Phys. 109, 4985 (1998)], namely simple cubic, body-centered cubic, and face-centered cubic arrays, for a wide range of P and f. Here, P is a dimensionless quantity characterizing the relative rate of diffusion versus surface incorporation, and f is the volume fraction of the inclusion. The scheme is further applied to random spherical suspensions and to investigate the effect of inclusion coagulation on overall rate constants. It is found that randomness in inclusion arrangement tends to lower the overall rate constant for f up to the near close-packing value of the regular arrays because of the inclusion screening effect. This screening effect turns stronger for regular arrays when f is near and above the close-packing value of the regular arrays, and consequently the overall rate constant of the random array exceeds that of the regular array. Inclusion coagulation too induces the inclusion screening effect, and leads to lower overall rate constants.

Development of prototypes of bioactive packaging materials based on immobilized bacteriophages for control of growth of bacterial pathogens in foods.

PubMed

Lone, Ayesha; Anany, Hany; Hakeem, Mohammed; Aguis, Louise; Avdjian, Anne-Claire; Bouget, Marina; Atashi, Arash; Brovko, Luba; Rochefort, Dominic; Griffiths, Mansel W

2016-01-18

Due to lack of adequate control methods to prevent contamination in fresh produce and growing consumer demand for natural products, the use of bacteriophages has emerged as a promising approach to enhance safety of these foods. This study sought to control Listeria monocytogenes in cantaloupes and RTE meat and Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage conditions by using specific lytic bacteriophage cocktails applied either free or immobilized. Bacteriophage cocktails were introduced into prototypes of packaging materials using different techniques: i) immobilizing on positively charged modified cellulose membranes, ii) impregnating paper with bacteriophage suspension, and iii) encapsulating in alginate beads followed by application of beads onto the paper. Phage-treated and non-treated samples were stored for various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free phage cocktail was added, L. monocytogenes counts dropped below the detection limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C and 12°C. However, at 25°C, counts below the detection limit were observed after 3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria count was observed by the end of the storage period for all tested storage temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage application technique (spraying of free phage suspension, bringing in contact with bacteriophage-based materials (paper coated with encapsulated bacteriophage or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log cycle reduction in E. coli count was observed on the germinated sprouts by day 5. In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to significantly reduce the growth of L. monocytogenes at both storage temperatures, 4°C and 10°C, for 25 days regardless of bacteriophage application format (immobilized or non-immobilized (free)). In conclusion, the developed phage-based materials demonstrated significant antimicrobial effect, when applied to the artificially contaminated foods, and can be used as prototypes for developing bioactive antimicrobial packaging materials capable of enhancing the safety of fresh produce and RTE meat. Copyright © 2015 Elsevier B.V. All rights reserved.

Microfluidic device for acoustic cell lysis

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

Branch, Darren W.; Cooley, Erika Jane; Smith, Gennifer Tanabe

2015-08-04

A microfluidic acoustic-based cell lysing device that can be integrated with on-chip nucleic acid extraction. Using a bulk acoustic wave (BAW) transducer array, acoustic waves can be coupled into microfluidic cartridges resulting in the lysis of cells contained therein by localized acoustic pressure. Cellular materials can then be extracted from the lysed cells. For example, nucleic acids can be extracted from the lysate using silica-based sol-gel filled microchannels, nucleic acid binding magnetic beads, or Nafion-coated electrodes. Integration of cell lysis and nucleic acid extraction on-chip enables a small, portable system that allows for rapid analysis in the field.

Correcting the Relative Bias of Light Obscuration and Flow Imaging Particle Counters.

PubMed

Ripple, Dean C; Hu, Zhishang

2016-03-01

Industry and regulatory bodies desire more accurate methods for counting and characterizing particles. Measurements of proteinaceous-particle concentrations by light obscuration and flow imaging can differ by factors of ten or more. We propose methods to correct the diameters reported by light obscuration and flow imaging instruments. For light obscuration, diameters were rescaled based on characterization of the refractive index of typical particles and a light scattering model for the extinction efficiency factor. The light obscuration models are applicable for either homogeneous materials (e.g., silicone oil) or for chemically homogeneous, but spatially non-uniform aggregates (e.g., protein aggregates). For flow imaging, the method relied on calibration of the instrument with silica beads suspended in water-glycerol mixtures. These methods were applied to a silicone-oil droplet suspension and four particle suspensions containing particles produced from heat stressed and agitated human serum albumin, agitated polyclonal immunoglobulin, and abraded ethylene tetrafluoroethylene polymer. All suspensions were measured by two flow imaging and one light obscuration apparatus. Prior to correction, results from the three instruments disagreed by a factor ranging from 3.1 to 48 in particle concentration over the size range from 2 to 20 μm. Bias corrections reduced the disagreement from an average factor of 14 down to an average factor of 1.5. The methods presented show promise in reducing the relative bias between light obscuration and flow imaging.

Bioreactor Expansion of Skin-Derived Precursor Schwann Cells.

PubMed

Walsh, Tylor; Biernaskie, Jeff; Midha, Rajiv; Kallos, Michael S

2016-01-01

Scaling up the production of cells in a culture process is a critical step when trying to develop cell-based regenerative therapies. Static cultures often cannot be easily scaled up to clinically relevant cell numbers. Alternatively, bioreactors offer a highly valuable means to develop a clinical-ready process. To culture adherent cells in suspension, such as skin-derived precursor Schwann cells (SKP-SCs), microcarriers need to be used. Microcarriers are small spherical beads suspended within the vessel that allow for higher growth surface area to volume ratio. Here we describe the procedure of combining microcarriers with the controllability of bioreactors to generate higher cell densities in smaller reactor volumes leading to a more efficient and cost-effective cell production for applications in regenerative medicine.

Preparation of fluorescent nanodiamond suspensions using bead-assisted ultrasonic disintegration

NASA Astrophysics Data System (ADS)

Głowacki, Maciej J.; Gardas, Mateusz; Ficek, Mateusz; Sawczak, Mirosław; Bogdanowicz, Robert

2017-08-01

Nitrogen-vacancy (N-V) centers are the most widely studied crystallographic defect in the diamond lattice since their presence causes strong and stable fluorescence. The negative charge state of the defect (NV-) is especially desired because of its potential for quantum information processing. In this study, fluorescent suspensions of diamond particles have been produced by microbead-assisted ultrasonic disintegration of commercially obtained diamond powder containing N-V color centers. Zirconium dioxide ZrO2 was chosen as an abrasive and a mixture of deionized water and dimethyl sulfoxide (DMSO) was used as a solvent. Raman spectrum of the starting material has been obtained and the resulting liquids have been measured in terms of photoluminescence. Moreover, thin layer of the diamond particles has been deposited on a silicon substrate and examined using scanning electron microscopy (SEM). During the course of the experiment a new method, which uses sodium chloride NaCl as an abrasive, has been proposed. The results of fluorescence measurements of the suspension prepared using this technique are highly promising.

The Larger the Viscosity, the Higher the Bounce

NASA Astrophysics Data System (ADS)

Stern, Menachem; Klein Schaarsberg, Martin; Peters, Ivo; Dodge, Kevin; Zhang, Wendy; Jaeger, Heinrich

A low-viscosity liquid drop can bounce upon impact onto a solid. A high-viscosity drop typically just flattens, i.e., it splats. Surprisingly, our experiments with a droplet made of densely packed glass beads in silicone oil display the opposite behavior: the low-viscosity oil suspension drop splats. The high-viscosity oil suspension bounces. Increasing solvent viscosity increases the rebound energy. To gain insight into the underlying mechanism, we model the suspension as densely packed elastic spheres experiencing viscous lubrication drag between neighbors. The model reproduces the observed trends. Plots of elastic compression and drag experienced by the particles show that rebounds are made possible by (1) a fraction of the impact energy being stored during initial contact via elastic compression, (2) a rapid broadening of local lubrication drag interactions at the initial impact site into a spatially uniform upward force throughout the drop. Including finite wall drag due to the presence of ambient air into the numerical model diminishes and eventually cuts off the rebound.

Fabrication of Flexible Arrayed Lactate Biosensor Based on Immobilizing LDH-NAD⁺ on NiO Film Modified by GO and MBs.

PubMed

Chou, Jung-Chuan; Yan, Siao-Jie; Liao, Yi-Hung; Lai, Chih-Hsien; Wu, You-Xiang; Wu, Cian-Yi; Chen, Hsiang-Yi; Huang, Hong-Yu; Wu, Tong-Yu

2017-07-12

We proposed the flexible arrayed lactate biosensor based on immobilizing l-lactate dehydrogenase (LDH) and nicotinamide adenine dinucleotide ( NAD + ) on nickel oxide (NiO) film, and which the average sensitivity could be enhanced by using graphene oxide (GO) and magnetic beads (MBs). By using GO and MBs, it exhibits excellent sensitivity (45.397 mV/mM) with a linearity of 0.992 in a range of 0.2 mM to 3 mM. According to the results of electrochemical impedance spectroscopy (EIS), the electron transfer resistance of LDH- NAD + -MBs/GPTS/GO/NiO film was smaller than those of LDH-NAD⁺/GPTS/GO/NiO film and LDH- NAD + /GPTS/NiO film, and it presented the outstanding electron transfer ability. After that, the limit of detection, anti-interference effect and bending test were also investigated.

Fabrication of Flexible Arrayed Lactate Biosensor Based on Immobilizing LDH-NAD+ on NiO Film Modified by GO and MBs

PubMed Central

Yan, Siao-Jie; Liao, Yi-Hung; Lai, Chih-Hsien; Wu, You-Xiang; Wu, Cian-Yi; Chen, Hsiang-Yi; Huang, Hong-Yu; Wu, Tong-Yu

2017-01-01

We proposed the flexible arrayed lactate biosensor based on immobilizing l-lactate dehydrogenase (LDH) and nicotinamide adenine dinucleotide (NAD+) on nickel oxide (NiO) film, and which the average sensitivity could be enhanced by using graphene oxide (GO) and magnetic beads (MBs). By using GO and MBs, it exhibits excellent sensitivity (45.397 mV/mM) with a linearity of 0.992 in a range of 0.2 mM to 3 mM. According to the results of electrochemical impedance spectroscopy (EIS), the electron transfer resistance of LDH-NAD+-MBs/GPTS/GO/NiO film was smaller than those of LDH-NAD+/GPTS/GO/NiO film and LDH-NAD+/GPTS/NiO film, and it presented the outstanding electron transfer ability. After that, the limit of detection, anti-interference effect and bending test were also investigated. PMID:28704960

Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density.

PubMed

Byström, Sanna; Eklund, Martin; Hong, Mun-Gwan; Fredolini, Claudia; Eriksson, Mikael; Czene, Kamila; Hall, Per; Schwenk, Jochen M; Gabrielson, Marike

2018-02-14

Mammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density. Plasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI). Plasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p < 0.05). Plasma levels of seven proteins were positively associated and 13 proteins negatively associated with AD. For eleven of these proteins evidence for gene expression in breast tissue existed. Among these, ABCC11, TNFRSF10D, F11R and ERRF were positively associated with AD, and SHC1, CFLAR, ACOX2, ITGB6, RASSF1, FANCD2 and IRX5 were negatively associated with AD. Screening proteins in plasma indicates associations between breast density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.

A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

PubMed

Bordelon, Hali; Russ, Patricia K; Wright, David W; Haselton, Frederick R

2013-01-01

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3) to 5×10(8) copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6), 14×10(6), and 8×10(6) copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

A Magnetic Bead-Based Method for Concentrating DNA from Human Urine for Downstream Detection

PubMed Central

Bordelon, Hali; Russ, Patricia K.; Wright, David W.; Haselton, Frederick R.

2013-01-01

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×103 to 5×108 copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×106, 14×106, and 8×106 copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR. PMID:23861895

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

Dry Transfer Inoculation of Low-Moisture Spices Containing Antimicrobial Compounds.

PubMed

Hildebrandt, Ian M; Hu, Chuxuan; Grasso-Kelley, Elizabeth M; Ye, Peiran; Anderson, Nathan M; Keller, Susanne E

2017-02-01

Inoculation of a food product for use in subsequent validation studies typically makes use of a high concentration cocktail of microorganisms suspended in aqueous media. However, this inoculation method may prove difficult particularly when the food product is a low-moisture food containing antimicrobial compounds, such as some dried spices. In this study, a dry transfer method for inoculation of clove powder, oregano leaves, ginger powder, and ground black pepper with a five-serovar cocktail of Salmonella was developed and compared with a traditional aqueous inoculation procedure. Spices were inoculated at three levels, 10, 8, and 6 log CFU/g, by using both an aqueous suspension of Salmonella and a dry transfer of Salmonella from previously inoculated silica beads. At the highest inoculation level, the dry transfer method resulted in a significantly higher microbial load (P < 0.05) for ground cloves and oregano, but not for ginger and ground black pepper. At the intermediate inoculation level, differences were apparent only for ginger and black pepper. Inoculation levels of 6 log CFU/g resulted in recoveries below detection limits for both methods of inoculation. Additional examination on the survival of Salmonella on silica beads after inoculation and in clove powder after dry transfer from silica beads showed linear rates of decline, with a rate of -0.011 log CFU/g/day for beads and -0.015 log CFU/g/day for clove powder. The results suggest that dry transfer of Salmonella via inoculated silica beads is a viable alternative when traditional aqueous inoculation is not feasible.

Protecting chickens against coccidiosis in floor pens by administering Eimeria oocysts using gel beads or spray vaccination.

PubMed

Jenkins, Mark C; Parker, Carolyn; O'Brien, Celia; Persyn, Joseph; Barlow, Darren; Miska, Katarzyna; Fetterer, Raymond

2013-09-01

Control of avian coccidiosis is increasingly being achieved by the administration of low doses of Eimeria oocysts to newly hatched chicks. The purpose of this study was to test the efficacy of gel beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine to protect broilers raised in contact with litter. Newly hatched chicks were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel beads. Control, 1-day-old chicks were given an equivalent number of Eimeria oocysts (10(3) total) by oral gavage or received no vaccine (nonimmunized controls). All chicks were raised in floor-pen cages in direct contact with litter. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E. acervulina, E. maxima, and E. tenella challenge infection. Chickens immunized with Eimeria oocysts in gel beads or by spray vaccination displayed significantly (P < 0.05) greater weight gain (WG) compared to nonimmunized controls. Feed conversion ratio (FCR) also showed a significant (P < 0.05) improvement in both groups relative to nonimmunized controls. Moreover, WG and FCR in both groups was not significantly different (P > 0.05) from chickens immunized by oral gavage or from nonimmunized, noninfected controls. Oocyst excretion after Eimeria challenge by all immunized groups was about 10-fold less than in nonimmunized controls. These findings indicate that immunization efficacy of gel beads and spray vaccination is improved by raising immunized chicks in contact with litter.

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.

Simplified Analytical Model of a Six-Degree-of-Freedom Large-Gap Magnetic Suspension System

NASA Technical Reports Server (NTRS)

Groom, Nelson J.

1997-01-01

A simplified analytical model of a six-degree-of-freedom large-gap magnetic suspension system is presented. The suspended element is a cylindrical permanent magnet that is magnetized in a direction which is perpendicular to its axis of symmetry. The actuators are air core electromagnets mounted in a planar array. The analytical model consists of an open-loop representation of the magnetic suspension system with electromagnet currents as inputs.

Description of the Large Gap Magnetic Suspension System (LGMSS) ground-based experiment

NASA Technical Reports Server (NTRS)

Groom, Nelson J.

1991-01-01

A description of the Large Gap Magnetic Suspension System (LGMSS) ground-based experiment is presented. The LGMSS provides five degrees of freedom control of a cylindrical suspended element which is levitated above a floor-mounted array of air core electromagnets. The uncontrolled degree of freedom is rotation about the long axis of the cylinder (roll). Levitation and control forces are produced on a permanent magnet core which is embedded in the cylinder. The cylinder also contains light emitting diodes (LEDs), assorted electrons, and a power supply. The LEDs provide active targets for an optical position measurement system which is being developed in-house at the Langley Research Center. The optical position measurement system will provide six degrees of freedom position information for the LGMSS control system.

Red blood cell (RBC) suspensions in confined microflows: Pressure-flow relationship.

PubMed

Stauber, Hagit; Waisman, Dan; Korin, Netanel; Sznitman, Josué

2017-10-01

Microfluidic-based assays have become increasingly popular to explore microcirculation in vitro. In these experiments, blood is resuspended to a desired haematocrit level in a buffer solution, where frequent choices for preparing RBC suspensions comprise notably Dextran and physiological buffer. Yet, the rational for selecting one buffer versus another is often ill-defined and lacks detailed quantification, including ensuing changes in RBC flow characteristics. Here, we revisit RBC suspensions in microflows and attempt to quantify systematically some of the differences emanating between buffers. We measure bulk flow rate (Q) of RBC suspensions, using PBS- and Dextran-40, as a function of the applied pressure drop (ΔP) for two hematocrits (∼0% and 23%). Two distinct microfluidic designs of varying dimensions are employed: a straight channel larger than and a network array similar to the size of individual RBCs. Using the resulting pressure-flow curves, we extract the equivalent hydrodynamic resistances and estimate the relative viscosities. These efforts are a first step in rigorously quantifying the influence of the 'background' buffer on RBC flows within microfluidic devices and thereby underline the importance of purposefully selecting buffer suspensions for microfluidic in vitro assays. Copyright © 2017. Published by Elsevier Ltd.

Optimized suspension culture: the rotating-wall vessel

NASA Technical Reports Server (NTRS)

Hammond, T. G.; Hammond, J. M.

2001-01-01

Suspension culture remains a popular modality, which manipulates mechanical culture conditions to maintain the specialized features of cultured cells. The rotating-wall vessel is a suspension culture vessel optimized to produce laminar flow and minimize the mechanical stresses on cell aggregates in culture. This review summarizes the engineering principles, which allow optimal suspension culture conditions to be established, and the boundary conditions, which limit this process. We suggest that to minimize mechanical damage and optimize differentiation of cultured cells, suspension culture should be performed in a solid-body rotation Couette-flow, zero-headspace culture vessel such as the rotating-wall vessel. This provides fluid dynamic operating principles characterized by 1) solid body rotation about a horizontal axis, characterized by colocalization of cells and aggregates of different sedimentation rates, optimally reduced fluid shear and turbulence, and three-dimensional spatial freedom; and 2) oxygenation by diffusion. Optimization of suspension culture is achieved by applying three tradeoffs. First, terminal velocity should be minimized by choosing microcarrier beads and culture media as close in density as possible. Next, rotation in the rotating-wall vessel induces both Coriolis and centrifugal forces, directly dependent on terminal velocity and minimized as terminal velocity is minimized. Last, mass transport of nutrients to a cell in suspension culture depends on both terminal velocity and diffusion of nutrients. In the transduction of mechanical culture conditions into cellular effects, several lines of evidence support a role for multiple molecular mechanisms. These include effects of shear stress, changes in cell cycle and cell death pathways, and upstream regulation of secondary messengers such as protein kinase C. The discipline of suspension culture needs a systematic analysis of the relationship between mechanical culture conditions and biological effects, emphasizing cellular processes important for the industrial production of biological pharmaceuticals and devices.

Validation of a novel computer-assisted sperm analysis (CASA) system using multitarget-tracking algorithms.

PubMed

Tomlinson, Mathew James; Pooley, Karen; Simpson, Tracey; Newton, Thomas; Hopkisson, James; Jayaprakasan, Kannamanadias; Jayaprakasan, Rajisha; Naeem, Asad; Pridmore, Tony

2010-04-01

To determine the accuracy and precision of a novel computer-assisted sperm analysis (CASA) system by comparison with existing recommended manual methods. Prospective study using comparative measurements of sperm concentration and motility on latex beads and immotile and motile sperm. Tertiary referral fertility center with strong academic links. Sperm donors and male partners of couples attending for fertility investigations. None. Achievement of Accubead target value for high and low concentration suspensions. Repeatability as demonstrated by coefficients of variation and intraclass correlation coefficients. Correlation and limits of agreement between CASA and manual methods. The CASA measurements of latex beads and sperm concentrations demonstrated a high level of accuracy and repeatability. Repeated Accubead measurements attained the required target value (mean difference from target of 2.61% and 3.71% for high- and low-concentration suspensions, respectively) and were highly reproducible. Limits of agreement analysis suggested that manual and CASA counts compared directly could be deemed to be interchangeable. Manual and CASA motility measurements were highly correlated for grades a, b, and d but could not be deemed to be interchangeable, and manual motility estimates were consistently higher for motile sperm. The novel CASA system was able to provide semen quality measurements for sperm concentration and motility measurements which were at least as reliable as current manual methods. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

In vitro and in vivo evaluation of gastro-retentive carvedilol loaded chitosan beads using Gastroplus™.

PubMed

Praveen, Radhakrishnan; Prasad Verma, Priya Ranjan; Venkatesan, Jayachandran; Yoon, Dong-Han; Kim, Se-Kwon; Singh, Sandeep Kumar

2017-09-01

The objective of present investigation was to develop gastro-retentive controlled release system of carvedilol using biological macromolecule, chitosan. 3 2 full factorial design was adopted for optimization of tripolyphosphate (X 1 ) and curing time (X 2 ). Bead stability in 0.1N HCl, buoyancy duration, density, drug loading, dissolution efficiency and cumulative percentage release at 8th hour were evaluated as dependent variables. The levels of X 1 and X 2 of optimized formulation having maximum desirability was found to 2.0% w/v and 62.66min, respectively. The in silico predicted responses and observed response were found to be in good agreement (percent bias error: -13.295 to +13.269). SEM images showed numerous pores in the cross sectional image that renders buoyancy. AUC 0-∞ of optimized formulation was 1.47 times higher as compared to suspension corroborating enhanced extent of absorption. T max and mean residence time were significantly higher from optimized formulation vis a vis suspension. In silico study indicated maximum regional absorption from the duodenum (94.1%) followed by jejunum (5.6%). Wagner-Nelson and Loo-Reigelman method were the preferred deconvolution approach over numerical deconvolution to establish IVIVC. In conclusion, the study showed that gastro-retentive controlled release system prepared using chitosan could be a potential drug carrier of carvedilol with improved bioavailability. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting

PubMed Central

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-01-01

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496

Mild disintegration of the green microalgae Chlorella vulgaris using bead milling.

PubMed

Postma, P R; Miron, T L; Olivieri, G; Barbosa, M J; Wijffels, R H; Eppink, M H M

2015-05-01

In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25-145 gDW kg(-1)) over a range of agitator speeds (6-12 m s(-1)). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9-10 m s(-1) regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kgDW(-1). Copyright © 2014 Elsevier Ltd. All rights reserved.

Volatile dynamics in crystal-rich magma bodies, perspectives from laboratory experiments and theory

NASA Astrophysics Data System (ADS)

Faroughi, S.; Parmigiani, A.; Huber, C.

2013-12-01

The amount of volatiles and the dynamics of bubbles play a significant role on the transition between different volcanic eruption behaviors. The transport of exsolved volatiles through zoned magma chambers is complex and remains poorly constrained. Here we focus on the different transport of volatiles under two end member regimes: crystal-poor systems (bubbles form a suspension) versus crystal-rich reservoirs (multiphase porous media flow). We present a combination of multiphase flow laboratory experiments (using silicon oil and water) and a theoretical argument based on Stokes flow streamfunctions to contrast the differences between the transport of exsolved volatiles in both regimes. The first set of experiments involves the buoyant migration of water droplets in silicon oil in the absence of glass beads. We measure the non-linear hydrodynamic interaction between bubbles and its effect on slowing down the average flux of water droplets as the water volume fraction increases. Our experimental results are compared to a theoretical argument in which a streamfunction formulation is used to estimate the effect of a suspension on bubble migration. We find a good agreement between the new theory and our experimental results. The second set of experiments focuses on the transport of water (non-wetting fluid) in porous media saturated with viscous silicon oils. Contrary to suspension dynamics, in multiphase porous media, an increase in the saturation of non-wetting fluid leads to a non-linear increase in its volumetric flux. The steady-state migration of non-wetting fluid is controlled by the formation of viscous fingering instability that greatly enhances transport. We propose that the regime of energy dissipation during the migration of bubbles in heterogeneous magma reservoirs can change, leading to bubble accumulation in crystal-poor regions as fingering becomes unstable and volatiles form a disperse bubble suspension.

[Establishment and verification of detecting multiple biomarkers for ovarian cancer by suspension array technology].

PubMed

Zhao, B B; Yang, Z J; Wang, Q; Pan, Z M; Zhang, W; Li, D R; Li, L

2016-10-25

Objective: Establish and validation of combined detecting of CCL18, CXCL1, C1D, TM4SF1, FXR1, TIZ suspension array technology. Methods: (1)CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ protein were coupled with polyethylene microspheres. Biotinylated CCL18, CXCL1 polyclonal antibody and sheep anti-human IgG polyclonal antibody were prepared simultaneously. The best packaged concentrations of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigens were optimized. The best packaged concentrations of CCL18, CXCL1 polyclonal antibodys and C1D, TM4SF1, FXR1, TIZ sheep anti-human IgG polyclonal antibody were optimized to establish a stable detected suspension array.(2)Sixty patients confirmed by pathological examination with ovarian cancer(ovarian cancer group)which treated in Affiliated Tumor Hospital of Guangxi Medical University, 30 patients with ovarian benign tumor(benign group)and 30 cases of healthy women(control group)were chosen between September 2003 and December 2003. Suspension array technology and ELISA method were used to detect expression of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1 and TIZ IgG autoantibody contented in 3 groups of serum, then to compare the diagnostic efficiency and diagnostic accuracy of two methods(coefficient of variation between batch and batch). Results: (1)This research successfully established stable detecting system of CCL18, CXCL1, C1D, TM4SF1, FXR1 and TIZ IgG autoantibody. The best concentration of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigen package were 8, 8, 12, 8, 4 and 8 μg/ml; the best detection of CCL18, CXCL1 biotin polyclonal antibody and C1D, TM4SF1, FXR1, TIZ sheep anti-huamne IgG polyclonal antibody were respectively 4, 2, 2, 4, 4 and 2 μg/ml.(2)Suspension array technology and ELISA method were used to detect CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody of three groups in serum were similar( P >0.05).(3)The comparison of two methods in the diagnosis of efficiency: the diagnostic accuracy of two methods were 99.2%(119/120)and 94.2%(113/120), the difference was statistically significant( P =0.031). The sensitivity of the diagnosis of ovarian cancer of two methods were 100.0%(60/60)and 93.3%(56/60), specific degrees were 100.0%(59/59)and 93.4%(57/61), positive predictive value was 100.0%(60/60)and 93.3%(56/60), negative predictive value was 98.3%(59/60)and 95.0%(57/60), the difference was statistically significant( P <0.05).(4)The detected results of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody shown that the diagnostic accuracy of suspension array technology was superior to those of ELISA method(all P <0.05). Conclusion: The study has established the stable detection of suspension array technology, and the diagnostic efficiency and diagnostic accuracy was much better than that by ELISA.

Jllumina - A comprehensive Java-based API for statistical Illumina Infinium HumanMethylation450 and MethylationEPIC data processing.

PubMed

Almeida, Diogo; Skov, Ida; Lund, Jesper; Mohammadnejad, Afsaneh; Silva, Artur; Vandin, Fabio; Tan, Qihua; Baumbach, Jan; Röttger, Richard

2016-12-18

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.

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

Preparation of ionic-crosslinked chitosan-based gel beads and effect of reaction conditions on drug release behaviors.

PubMed

Chen, Shilan; Liu, Mingzhu; Jin, Shuping; Wang, Bin

2008-02-12

Drug-loaded chitosan (CS) beads were prepared under simple and mild condition using trisodium citrate as ionic crosslinker. The beads were further coated with poly(methacrylic acid) (PMAA) by dipping the beads in PMAA aqueous solution. The surface and cross-section morphology of these beads were observed by scanning electron microscopy and the observation showed that the coating beads had core-shell structure. In vitro release of model drug from these beads obtained under different reaction conditions was investigated in buffer medium (pH 1.8). The results showed that the rapid drug release was restrained by PMAA coating and the optimum conditions for preparing CS-based drug-loaded beads were decided through the effect of reaction conditions on the drug release behaviors. In addition, the drug release mechanism of CS-based drug-loaded beads was analyzed by Peppa's potential equation. According to this study, the ionic-crosslinked CS beads coated by PMAA could serve as suitable candidate for drug site-specific carrier in stomach.

Neonatal rat heart cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

Akins, Robert E.; Schroedl, Nancy A.; Gonda, Steve R.; Hartzell, Charles R.

1994-01-01

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

Neonatal rat heart cells cultured in simulated microgravity

NASA Technical Reports Server (NTRS)

Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

1997-01-01

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

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.

Formulation and characterization of a compacted multiparticulate system for modified release of water-soluble drugs--part 1--acetaminophen.

PubMed

Cantor, Stuart L; Hoag, Stephen W; Augsburger, Larry L

2009-03-01

The aim of this study was to characterize and evaluate a modified release, multiparticulate tablet formulation consisting of placebo beads and drug-loaded beads. Acetaminophen (APAP) bead formulations containing ethylcellulose (EC) from 40-60% and placebo beads containing 30% calcium silicate and prepared using 0-20% alcohol were developed using extrusion-spheronization and studied using a central composite experimental design. Particle size and true density of beads were measured. Segregation testing was performed using the novel ASTM D6940-04 method on a 50:50 blend of uncoated APAP beads (60%EC) : calcium silicate placebo beads (10% alcohol). Tablets were prepared using an instrumented Stokes-B2 rotary tablet press and evaluated for crushing strength and dissolution rate. Compared with drug beads (60%EC), placebo beads (10% alcohol) were smaller but had higher true densities: 864.8 mum and 1.27 g/cm(3), and 787.1 mum and 1.73 g/cm(3), respectively. Segregation testing revealed that there was approximately a 20% difference in drug content (as measured by the coefficient of variation) between initial and final blend samples. Although calcium silicate-based placebo beads were shown to be ineffective cushioning agents in blends with Surelease(R)-coated APAP beads, they were found to be very compactibile when used alone and gave tablet crushing strength values between 14 and 17 kP. The EC in the APAP bead matrix minimally suppressed the drug release from uncoated beads (t(100%) = 2 h). However, while tablets containing placebo beads reformulated with glycerol monostearate (GMS) showed a slower release rate (t(60%)= 5 h) compared with calcium silicate-based placebos, some coating damage ( approximately 30%) still occurred on compression as release was faster than coated APAP beads alone. While tablets containing coated drug beads can be produced with practical crushing strengths (>8 kP) and low compression pressures (10-35 MPa), dissolution studies revealed that calcium silicate-based placebos are ineffective as cushioning agents. Blend segregation was likely observed due to the particle size and the density differences between APAP beads and calcium silicate-based placebo beads; placebo bead percolation can perhaps be minimized by increasing their size during the extrusion-spheronization process. The GMS- based placebos offer greater promise as cushioning agents for compacted, coated drug beads; however, this requires an optimized compression pressure range and drug bead : placebo bead ratio (i.e., 50:50).

Anomalous yet Brownian.

PubMed

Wang, Bo; Anthony, Stephen M; Bae, Sung Chul; Granick, Steve

2009-09-08

We describe experiments using single-particle tracking in which mean-square displacement is simply proportional to time (Fickian), yet the distribution of displacement probability is not Gaussian as should be expected of a classical random walk but, instead, is decidedly exponential for large displacements, the decay length of the exponential being proportional to the square root of time. The first example is when colloidal beads diffuse along linear phospholipid bilayer tubes whose radius is the same as that of the beads. The second is when beads diffuse through entangled F-actin networks, bead radius being less than one-fifth of the actin network mesh size. We explore the relevance to dynamic heterogeneity in trajectory space, which has been extensively discussed regarding glassy systems. Data for the second system might suggest activated diffusion between pores in the entangled F-actin networks, in the same spirit as activated diffusion and exponential tails observed in glassy systems. But the first system shows exceptionally rapid diffusion, nearly as rapid as for identical colloids in free suspension, yet still displaying an exponential probability distribution as in the second system. Thus, although the exponential tail is reminiscent of glassy systems, in fact, these dynamics are exceptionally rapid. We also compare with particle trajectories that are at first subdiffusive but Fickian at the longest measurement times, finding that displacement probability distributions fall onto the same master curve in both regimes. The need is emphasized for experiments, theory, and computer simulation to allow definitive interpretation of this simple and clean exponential probability distribution.

Open-loop characteristics of magnetic suspension systems using electromagnets mounted in a planar array

NASA Technical Reports Server (NTRS)

Groom, Nelson J.; Britcher, Colin P.

1992-01-01

The open-loop characteristics of a Large-Gap Magnetic Suspension System (LGMSS) were studied and numerical results are presented. The LGMSS considered provides five-degree-of-freedom control. The suspended element is a cylinder that contains a core composed of permanent magnet material. The magnetic actuators are air core electromagnets mounted in a planar array. Configurations utilizing five, six, seven, and eight electromagnets were investigated and all configurations were found to be controllable from coil currents and observable from suspended element positions. Results indicate that increasing the number of coils has an insignificant effect on mode shapes and frequencies.

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

Stability considerations for magnetic suspension systems using electromagnets mounted in a planar array

NASA Technical Reports Server (NTRS)

Groom, Nelson J.; Britcher, Colin P.

1991-01-01

Mathematical models of a 5, 6, 7, and 8 coil large gap magnetic suspension system (MSDS) are presented. Some of the topics covered include: force and torque equations, reduction of state-space form, natural modes, origins of modes, effect of rotation in azimuth (yaw), future work, and n-coil ring conclusions.

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.

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

Automated preparation method for colloidal crystal arrays of monodisperse and binary colloid mixtures by contact printing with a pintool plotter.

PubMed

Burkert, Klaus; Neumann, Thomas; Wang, Jianjun; Jonas, Ulrich; Knoll, Wolfgang; Ottleben, Holger

2007-03-13

Photonic crystals and photonic band gap materials with periodic variation of the dielectric constant in the submicrometer range exhibit unique optical properties such as opalescence, optical stop bands, and photonic band gaps. As such, they represent attractive materials for the active elements in sensor arrays. Colloidal crystals, which are 3D gratings leading to Bragg diffraction, are one potential precursor of such optical materials. They have gained particular interest in many technological areas as a result of their specific properties and ease of fabrication. Although basic techniques for the preparation of regular patterns of colloidal crystals on structured substrates by self-assembly of mesoscopic particles are known, the efficient fabrication of colloidal crystal arrays by simple contact printing has not yet been reported. In this article, we present a spotting technique used to produce a microarray comprising up to 9600 single addressable sensor fields of colloidal crystal structures with dimensions down to 100 mum on a microfabricated substrate in different formats. Both monodisperse colloidal crystals and binary colloidal crystal systems were prepared by contact printing of polystyrene particles in aqueous suspension. The array morphology was characterized by optical light microscopy and scanning electron microscopy, which revealed regularly ordered crystalline structures for both systems. In the case of binary crystals, the influence of the concentration ratio of the large and small particles in the printing suspension on the obtained crystal structure was investigated. The optical properties of the colloidal crystal arrays were characterized by reflection spectroscopy. To examine the stop bands of the colloidal crystal arrays in a high-throughput fashion, an optical setup based on a CCD camera was realized that allowed the simultaneous readout of all of the reflection spectra of several thousand sensor fields per array in parallel. In agreement with Bragg's relation, the investigated arrays exhibited strong opalescence and stop bands in the expected wavelength range, confirming the successful formation of highly ordered colloidal crystals. Furthermore, a narrow distribution of wavelength-dependent stop bands across the sensor array was achieved, demonstrating the capability of producing highly reproducible crystal spots by the contact printing method with a pintool plotter.

Fundamentals and Application of Magnetic Particles in Cell Isolation and Enrichment

PubMed Central

Plouffe, Brian D.; Murthy, Shashi K.; Lewis, Laura H.

2014-01-01

Magnetic sorting using magnetic beads has become a routine methodology for the separation of key cell populations from biological suspensions. Due to the inherent ability of magnets to provide forces at a distance, magnetic cell manipulation is now a standardized process step in numerous processes in tissue engineering, medicine, and in fundamental biological research. Herein we review the current status of magnetic particles to enable isolation and separation of cells, with a strong focus on the fundamental governing physical phenomena, properties and syntheses of magnetic particles and on current applications of magnet-based cell separation in laboratory and clinical settings. We highlight the contribution of cell separation to biomedical research and medicine and detail modern cell separation methods (both magnetic and non-magnetic). In addition to a review of the current state-of-the-art in magnet-based cell sorting, we discuss current challenges and available opportunities for further research, development and commercialization of magnetic particle-based cell separation systems. PMID:25471081

Low-Cost Photolithographic Fabrication of Nanowires and Microfilters for Advanced Bioassay Devices

PubMed Central

Doan, Nhi M.; Qiang, Liangliang; Li, Zhe; Vaddiraju, Santhisagar; Bishop, Gregory W.; Rusling, James F.; Papadimitrakopoulos, Fotios

2015-01-01

Integrated microfluidic devices with nanosized array electrodes and microfiltration capabilities can greatly increase sensitivity and enhance automation in immunoassay devices. In this contribution, we utilize the edge-patterning method of thin aluminum (Al) films in order to form nano- to micron-sized gaps. Evaporation of high work-function metals (i.e., Au, Ag, etc.) on these gaps, followed by Al lift-off, enables the formation of electrical uniform nanowires from low-cost, plastic-based, photomasks. By replacing Al with chromium (Cr), the formation of high resolution, custom-made photomasks that are ideal for low-cost fabrication of a plurality of array devices were realized. To demonstrate the feasibility of such Cr photomasks, SU-8 micro-pillar masters were formed and replicated into PDMS to produce micron-sized filters with 3–4 µm gaps and an aspect ratio of 3. These microfilters were capable of retaining 6 µm beads within a localized site, while allowing solvent flow. The combination of nanowire arrays and micro-pillar filtration opens new perspectives for rapid R&D screening of various microfluidic-based immunoassay geometries, where analyte pre-concentration and highly sensitive, electrochemical detection can be readily co-localized. PMID:25774709

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.

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.

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-homogeneous flow profiles in sheared bacterial suspensions

NASA Astrophysics Data System (ADS)

Samanta, Devranjan; Cheng, Xiang

Bacterial suspensions under shear exhibit interesting rheological behaviors including the remarkable ``superfluidic'' state with vanishing viscosity at low shear rates. Theoretical studies have shown that such ``superfluidic'' state is linked with non-homogeneous shear flows, which are induced by coupling between nematic order of active fluids and hydrodynamics of shear flows. However, although bulk rheology of bacterial suspensions has been experimentally studied, shear profiles within bacterial suspensions have not been explored so far. Here, we experimentally investigate the flow behaviors of E. coli suspensions under planar oscillatory shear. Using confocal microscopy and PIV, we measure velocity profiles across gap between two shear plates. We find that with increasing shear rates, high-concentration bacterial suspensions exhibit an array of non-homogeneous flow behaviors like yield-stress flows and shear banding. We show that these non-homogeneous flows are due to collective motion of bacterial suspensions. The phase diagram of sheared bacterial suspensions is systematically mapped as functions of shear rates an bacterial concentrations. Our experiments provide new insights into rheology of bacterial suspensions and shed light on shear induced dynamics of active fluids. Chemical Engineering and Material Science department.

Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device

PubMed Central

Barbee, Kristopher D.; Hsiao, Alexander P.; Roller, Eric E.; Huang, Xiaohua

2011-01-01

We report the development of a microfabricated electrophoretic device for assembling high-density arrays of antibody-conjugated microbeads for chip-based protein detection. The device consists of a flow cell formed between a gold-coated silicon chip with an array of microwells etched in a silicon dioxide film and a glass coverslip with a series of thin gold counter electrode lines. We have demonstrated that 0.4 and 1 μm beads conjugated with antibodies can be rapidly assembled into the microwells by applying a pulsed electric field across the chamber. By assembling step-wise a mixture of fluorescently labeled antibody-conjugated microbeads, we incorporated both spatial and fluorescence encoding strategies to demonstrate significant multiplexing capabilities. We have shown that these antibody-conjugated microbead arrays can be used to perform on-chip sandwich immunoassays to detect test antigens at concentrations as low as 40 pM (6 ng/mL). A finite element model was also developed to examine the electric field distribution within the device for different counter electrode configurations over a range of line pitches and chamber heights. This device will be useful for assembling high-density, encoded antibody arrays for multiplexed detection of proteins and other types of protein-conjugated microbeads for applications such as the analysis of protein-protein interactions. PMID:20820631

Halbach arrays in precision motion control

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

Trumper, D.L.; Williams, M.E.

1995-02-01

The Halbach array was developed for use as an optical element in particle accelerators. Following up on a suggestion from Klaus Halbach, the authors have investigated the utility of such arrays as the permanent magnet structure for synchronous machines in cartesian, polar, and cylindrical geometries. Their work has focused on the design of a novel Halbach array linear motor for use in a magnetic suspension stage for photolithography. This paper presents the details of the motor design and its force and power characteristics.

High-density, microsphere-based fiber optic DNA microarrays.

PubMed

Epstein, Jason R; Leung, Amy P K; Lee, Kyong Hoon; Walt, David R

2003-05-01

A high-density fiber optic DNA microarray has been developed consisting of oligonucleotide-functionalized, 3.1-microm-diameter microspheres randomly distributed on the etched face of an imaging fiber bundle. The fiber bundles are comprised of 6000-50000 fused optical fibers and each fiber terminates with an etched well. The microwell array is capable of housing complementary-sized microspheres, each containing thousands of copies of a unique oligonucleotide probe sequence. The array fabrication process results in random microsphere placement. Determining the position of microspheres in the random array requires an optical encoding scheme. This array platform provides many advantages over other array formats. The microsphere-stock suspension concentration added to the etched fiber can be controlled to provide inherent sensor redundancy. Examining identical microspheres has a beneficial effect on the signal-to-noise ratio. As other sequences of interest are discovered, new microsphere sensing elements can be added to existing microsphere pools and new arrays can be fabricated incorporating the new sequences without altering the existing detection capabilities. These microarrays contain the smallest feature sizes (3 microm) of any DNA array, allowing interrogation of extremely small sample volumes. Reducing the feature size results in higher local target molecule concentrations, creating rapid and highly sensitive assays. The microsphere array platform is also flexible in its applications; research has included DNA-protein interaction profiles, microbial strain differentiation, and non-labeled target interrogation with molecular beacons. Fiber optic microsphere-based DNA microarrays have a simple fabrication protocol enabling their expansion into other applications, such as single cell-based assays.

Protein profiling in serum after traumatic brain injury in rats reveals potential injury markers.

PubMed

Thelin, Eric Peter; Just, David; Frostell, Arvid; Häggmark-Månberg, Anna; Risling, Mårten; Svensson, Mikael; Nilsson, Peter; Bellander, Bo-Michael

2018-03-15

The serum proteome following traumatic brain injury (TBI) could provide information for outcome prediction and injury monitoring. The aim with this affinity proteomic study was to identify serum proteins over time and between normoxic and hypoxic conditions in focal TBI. Sprague Dawley rats (n=73) received a 3mm deep controlled cortical impact ("severe injury"). Following injury, the rats inhaled either a normoxic (22% O 2 ) or hypoxic (11% O 2 ) air mixture for 30min before resuscitation. The rats were sacrificed at day 1, 3, 7, 14 and 28 after trauma. A total of 204 antibodies targeting 143 unique proteins of interest in TBI research, were selected. The sample proteome was analyzed in a suspension bead array set-up. Comparative statistics and factor analysis were used to detect differences as well as variance in the data. We found that complement factor 9 (C9), complement factor B (CFB) and aldolase c (ALDOC) were detected at higher levels the first days after trauma. In contrast, hypoxia inducing factor (HIF)1α, amyloid precursor protein (APP) and WBSCR17 increased over the subsequent weeks. S100A9 levels were higher in hypoxic-compared to normoxic rats, together with a majority of the analyzed proteins, albeit few reached statistical significance. The principal component analysis revealed a variance in the data, highlighting clusters of proteins. Protein profiling of serum following TBI using an antibody based microarray revealed temporal changes of several proteins over an extended period of up to four weeks. Further studies are warranted to confirm our findings. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

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.

Genetic diversity of K-antigen gene clusters of Escherichia coli and their molecular typing using a suspension array.

PubMed

Yang, Shuang; Xi, Daoyi; Jing, Fuyi; Kong, Deju; Wu, Junli; Feng, Lu; Cao, Boyang; Wang, Lei

2018-04-01

Capsular polysaccharides (CPSs), or K-antigens, are the major surface antigens of Escherichia coli. More than 80 serologically unique K-antigens are classified into 4 groups (Groups 1-4) of capsules. Groups 1 and 4 contain the Wzy-dependent polymerization pathway and the gene clusters are in the order galF to gnd; Groups 2 and 3 contain the ABC-transporter-dependent pathway and the gene clusters consist of 3 regions, regions 1, 2 and 3. Little is known about the variations among the gene clusters. In this study, 9 serotypes of K-antigen gene clusters (K2ab, K11, K20, K24, K38, K84, K92, K96, and K102) were sequenced and correlated with their CPS chemical structures. On the basis of sequence data, a K-antigen-specific suspension array that detects 10 distinct CPSs, including the above 9 CPSs plus K30, was developed. This is the first report to catalog the genetic features of E. coli K-antigen variations and to develop a suspension array for their molecular typing. The method has a number of advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for straightforward identification and detection of additional K-antigens in the future.

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.

Al2O3-ZrO2 Finely Structured Multilayer Architectures from Suspension Plasma Spraying

NASA Astrophysics Data System (ADS)

Tingaud, Olivier; Montavon, Ghislain; Denoirjean, Alain; Coudert, Jean-François; Rat, Vincent; Fauchais, Pierre

2010-01-01

Suspension plasma spraying (SPS) is an alternative to conventional atmospheric plasma spraying (APS) aiming at manufacturing thinner layers (i.e., 10-100 μm) due to the specific size of the feedstock particles, from a few tens of nanometers to a few micrometers. The staking of lamellae and particles, which present a diameter ranging from 0.1 to 2.0 μm and an average thickness from 20 to 300 nm, permits to manufacture finely structured layers. Moreover, it appears as a versatile process able to manufacture different coating architectures according to the operating parameters (suspension properties, injection configuration, plasma properties, spray distance, torch scan velocity, scanning step, etc.). However, the different parameters controlling the properties of the coating, and their interdependences, are not yet fully identified. Thus, the aim of this paper is, on the one hand, to better understand the influence of operating parameters on the coating manufacturing mechanisms (in particular, the plasma gas mixture effect) and, on the other hand, to produce Al2O3-ZrO2 finely structured layers with large varieties of architectures. For this purpose, a simple theoretical model was used to describe the plasma torch operating conditions at the nozzle exit, based on experimental data (mass enthalpy, arc current intensity, thermophysical properties of plasma forming gases, etc.) and the influences of the spray parameters were determined by mean of the study of sizes and shapes of spray beads. The results enabled then to reach a better understanding of involved phenomena and their interactions on the final coating architectures permitting to manufacture several types of microstructures.

Modeling shear-induced particle ordering and deformation in a dense soft particle suspension

NASA Astrophysics Data System (ADS)

Liao, Chih-Tang; Wu, Yi-Fan; Chien, Wei; Huang, Jung-Ren; Chen, Yeng-Long

2017-11-01

We apply the lattice Boltzmann method and the bead-spring network model of deformable particles (DPs) to study shear-induced particle ordering and deformation and the corresponding rheological behavior for dense DP suspensions confined in a narrow gap under steady external shear. The particle configuration is characterized with small-angle scattering intensity, the real-space 2D local order parameter, and the particle shape factors including deformation, stretching and tilt angles. We investigate how particle ordering and deformation vary with the particle volume fraction ϕ (=0.45-0.65) and the external shear rate characterized with the capillary number Ca (=0.003-0.191). The degree of particle deformation increases mildly with ϕ but significantly with Ca. Under moderate shear rate (Ca  =  0.105), the inter-particle structure evolves from string-like ordering to layered hexagonal close packing (HCP) as ϕ increases. A long wavelength particle slithering motion emerges for sufficiently large ϕ. For ϕ  =  0.61, the structure maintains layered HCP for Ca  =  0.031-0.143 but gradually becomes disordered for larger and smaller Ca. The correlation in particle zigzag movements depends sensitively on ϕ and particle ordering. Layer-by-layer analysis reveals how the non-slippery hard walls affect particle ordering and deformation. The shear-induced reconfiguration of DPs observed in the simulation agrees qualitatively with experimental results of sheared uniform emulsions. The apparent suspension viscosity increases with ϕ but exhibits much weaker dependence compared to hard-sphere suspensions, indicating that particle deformation and unjamming under shear can significantly reduce the viscous stress. Furthermore, the suspension shear-thins, corresponding to increased inter-DP ordering and particle deformation with Ca. This work provides useful insights into the microstructure-rheology relationship of concentrated deformable particle suspensions.

Modeling shear-induced particle ordering and deformation in a dense soft particle suspension.

PubMed

Liao, Chih-Tang; Wu, Yi-Fan; Chien, Wei; Huang, Jung-Ren; Chen, Yeng-Long

2017-11-01

We apply the lattice Boltzmann method and the bead-spring network model of deformable particles (DPs) to study shear-induced particle ordering and deformation and the corresponding rheological behavior for dense DP suspensions confined in a narrow gap under steady external shear. The particle configuration is characterized with small-angle scattering intensity, the real-space 2D local order parameter, and the particle shape factors including deformation, stretching and tilt angles. We investigate how particle ordering and deformation vary with the particle volume fraction ϕ (=0.45-0.65) and the external shear rate characterized with the capillary number Ca (=0.003-0.191). The degree of particle deformation increases mildly with ϕ but significantly with Ca. Under moderate shear rate (Ca  =  0.105), the inter-particle structure evolves from string-like ordering to layered hexagonal close packing (HCP) as ϕ increases. A long wavelength particle slithering motion emerges for sufficiently large ϕ. For ϕ  =  0.61, the structure maintains layered HCP for Ca  =  0.031-0.143 but gradually becomes disordered for larger and smaller Ca. The correlation in particle zigzag movements depends sensitively on ϕ and particle ordering. Layer-by-layer analysis reveals how the non-slippery hard walls affect particle ordering and deformation. The shear-induced reconfiguration of DPs observed in the simulation agrees qualitatively with experimental results of sheared uniform emulsions. The apparent suspension viscosity increases with ϕ but exhibits much weaker dependence compared to hard-sphere suspensions, indicating that particle deformation and unjamming under shear can significantly reduce the viscous stress. Furthermore, the suspension shear-thins, corresponding to increased inter-DP ordering and particle deformation with Ca. This work provides useful insights into the microstructure-rheology relationship of concentrated deformable particle suspensions.

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

A Laboratory Investigation of the Suspension, Transport, and Settling of Silver Carp Eggs Using Synthetic Surrogates

PubMed Central

Garcia, Tatiana; Zuniga Zamalloa, Carlo; Jackson, P. Ryan; Murphy, Elizabeth A.; Garcia, Marcelo H.

2015-01-01

Asian carp eggs are semi-buoyant and must remain suspended in the water to survive, supported by the turbulence of the flow, until they hatch and develop the ability to swim. Analysis of the transport and dispersal patterns of Asian carp eggs will facilitate the development and implementation of control strategies to target the early life stages. Experimenting with Asian carp eggs is complicated due to practical issues of obtaining eggs in close proximity to experimental facilities and extensive handling of eggs tends to damage them. Herein, we describe laboratory experiments using styrene beads (4.85 mm diameter) as synthetic surrogate eggs to mimic the physical properties of water-hardened silver carp eggs. The first set of experiments was completed in a rectangular vertical column filled with salt water. The salinity of the water was adjusted in an iterative fashion to obtain a close approximation of the fall velocity of the styrene beads to the mean fall velocity of silver carp water-hardened eggs. The terminal fall velocity of synthetic eggs was measured using an image processing method. The second set of experiments was performed in a temperature-controlled recirculatory flume with a sediment bed. The flume was filled with salt water, and synthetic eggs were allowed to drift under different flow conditions. Drifting behavior, suspension conditions, and settling characteristics of synthetic eggs were observed. At high velocities, eggs were suspended and distributed through the water column. Eggs that touched the sediment bed were re-entrained by the flow. Eggs saltated when they touched the bed, especially at moderate velocities and with a relatively flat bed. At lower velocities, some settling of the eggs was observed. With lower velocities and a flat bed, eggs were trapped near the walls of the flume. When bedforms were present, eggs were trapped in the lee of the bedforms in addition to being trapped near the flume walls. Results of this research study provide insights about transport, suspension, and dispersion of silver carp eggs. The knowledge gained from this study is useful to characterize the critical hydrodynamic conditions of the flow at which surrogates for silver carp water-hardened eggs settle out of suspension, and provides insight into how eggs may interact with riverbed sediments and morphology. PMID:26713855

A laboratory investigation of the suspension, transport, and settling of silver carp eggs using synthetic surrogates

USGS Publications Warehouse

Garcia, Tatiana; Zuniga Zamalloa, Carlo; Jackson, P. Ryan; Murphy, Elizabeth A.; Garcia, Marcelo H.

2015-01-01

Asian carp eggs are semi-buoyant and must remain suspended in the water to survive, supported by the turbulence of the flow, until they hatch and develop the ability to swim. Analysis of the transport and dispersal patterns of Asian carp eggs will facilitate the development and implementation of control strategies to target the early life stages. Experimenting with Asian carp eggs is complicated due to practical issues of obtaining eggs in close proximity to experimental facilities and extensive handling of eggs tends to damage them. Herein, we describe laboratory experiments using styrene beads (4.85 mm diameter) as synthetic surrogate eggs to mimic the physical properties of water-hardened silver carp eggs. The first set of experiments was completed in a rectangular vertical column filled with salt water. The salinity of the water was adjusted in an iterative fashion to obtain a close approximation of the fall velocity of the styrene beads to the mean fall velocity of silver carp water-hardened eggs. The terminal fall velocity of synthetic eggs was measured using an image processing method. The second set of experiments was performed in a temperature-controlled recirculatory flume with a sediment bed. The flume was filled with salt water, and synthetic eggs were allowed to drift under different flow conditions. Drifting behavior, suspension conditions, and settling characteristics of synthetic eggs were observed. At high velocities, eggs were suspended and distributed through the water column. Eggs that touched the sediment bed were re-entrained by the flow. Eggs saltated when they touched the bed, especially at moderate velocities and with a relatively flat bed. At lower velocities, some settling of the eggs was observed. With lower velocities and a flat bed, eggs were trapped near the walls of the flume. When bedforms were present, eggs were trapped in the lee of the bedforms in addition to being trapped near the flume walls. Results of this research study provide insights about transport, suspension, and dispersion of silver carp eggs. The knowledge gained from this study is useful to characterize the critical hydrodynamic conditions of the flow at which surrogates for silver carp water-hardened eggs settle out of suspension, and provides insight into how eggs may interact with riverbed sediments and morphology.

Novel method for high-throughput colony PCR screening in nanoliter-reactors

PubMed Central

Walser, Marcel; Pellaux, Rene; Meyer, Andreas; Bechtold, Matthias; Vanderschuren, Herve; Reinhardt, Richard; Magyar, Joseph; Panke, Sven; Held, Martin

2009-01-01

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies. PMID:19282448

Refractive multiple optical tweezers for parallel biochemical analysis in micro-fluidics

NASA Astrophysics Data System (ADS)

Merenda, Fabrice; Rohner, Johann; Pascoal, Pedro; Fournier, Jean-Marc; Vogel, Horst; Salathé, René-Paul

2007-02-01

We present a multiple laser tweezers system based on refractive optics. The system produces an array of 100 optical traps thanks to a refractive microlens array, whose focal plane is imaged into the focal plane of a high-NA microscope objective. This refractive multi-tweezers system is combined to micro-fluidics, aiming at performing simultaneous biochemical reactions on ensembles of free floating objects. Micro-fluidics allows both transporting the particles to the trapping area, and conveying biochemical reagents to the trapped particles. Parallel trapping in micro-fluidics is achieved with polystyrene beads as well as with native vesicles produced from mammalian cells. The traps can hold objects against fluid flows exceeding 100 micrometers per second. Parallel fluorescence excitation and detection on the ensemble of trapped particles is also demonstrated. Additionally, the system is capable of selectively and individually releasing particles from the tweezers array using a complementary steerable laser beam. Strategies for high-yield particle capture and individual particle release in a micro-fluidic environment are discussed. A comparison with diffractive optical tweezers enhances the pros and cons of refractive systems.

High angle of attack position sensing for the Southampton University magnetic suspension and balance system

NASA Technical Reports Server (NTRS)

Parker, David H.

1987-01-01

An all digital five channel position detection system is to be installed in the Southampton University Magnetic Suspension and Balance System (SUMSBS). The system is intended to monitor a much larger range of model pitch attitudes than has been possible hitherto, up to a maximum of a 90 degree angle of attack. It is based on the use of self-scanning photodiode arrays and illuminating laser light beams, together with purpose built processing electronics. The principles behind the design of the system are discussed, together with the results of testing one channel of the system which was used to control the axial position of a magnetically suspended model in SUMSBS. The removal of optically coupled heave position information from the axial position sensing channel is described.

Autoantibody targets in vaccine-associated narcolepsy.

PubMed

Häggmark-Månberg, Anna; Zandian, Arash; Forsström, Björn; Khademi, Mohsen; Lima Bomfim, Izaura; Hellström, Cecilia; Arnheim-Dahlström, Lisen; Hallböök, Tove; Darin, Niklas; Lundberg, Ingrid E; Uhlén, Mathias; Partinen, Markku; Schwenk, Jochen M; Olsson, Tomas; Nilsson, Peter

2016-09-01

Narcolepsy is a chronic sleep disorder with a yet unknown cause, but the specific loss of hypocretin-producing neurons together with a strong human leukocyte antigen (HLA) association has led to the hypothesis that autoimmune mechanisms might be involved. Here, we describe an extensive effort to profile autoimmunity repertoires in serum with the aim to find disease-related autoantigens. Initially, 57 serum samples from vaccine-associated and sporadic narcolepsy patients and controls were screened for IgG reactivity towards 10 846 fragments of human proteins using planar microarrays. The discovered differential reactivities were verified on suspension bead arrays in the same sample collection followed by further investigation of 14 antigens in 176 independent samples, including 57 narcolepsy patients. Among these 14 antigens, methyltransferase-like 22 (METTL22) and 5'-nucleotidase cytosolic IA (NT5C1A) were recognized at a higher frequency in narcolepsy patients of both sample sets. Upon sequence analysis of the 14 proteins, polymerase family, member 3 (PARP3), acyl-CoA-binding domain containing 7 (ARID4B), glutaminase 2 (GLS2) and cyclin-dependent kinase-like 1 (CDKL1) were found to contain amino acid sequences with homology to proteins found in the H1N1 vaccine. These findings could become useful elements of further clinical assays that aim towards a better phenotypic understanding of narcolepsy and its triggers.

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.

Dictyostelium discoideum mutants with conditional defects in phagocytosis

PubMed Central

1994-01-01

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s). PMID:7519624

Formulation of itraconazole nanococrystals and evaluation of their bioavailability in dogs.

PubMed

De Smet, Lieselotte; Saerens, Lien; De Beer, Thomas; Carleer, Robert; Adriaensens, Peter; Van Bocxlaer, Jan; Vervaet, Chris; Remon, Jean Paul

2014-05-01

The aim of the study is to increase the bioavailability of itraconazole (ITRA) using nanosized cocrystals prepared via wet milling of ITRA in combination with dicarboxylic acids. Wet milling was used in order to create a nanosuspension of ITRA in combination with dicarboxylic acids. After spray-drying and bead layering, solid state was characterized by MDSC, XRD, Raman and FT-IR. The release profiles and bioavailability of the nanococrystalline suspension, the spray-dried and bead layered formulation were evaluated. A monodisperse nanosuspension (549±51nm) of ITRA was developed using adipic acid and Tween®80. Solid state characterization indicated the formation of nanococrystals by hydrogen bounds between the triazole group of ITRA and the carboxyl group of adipic acid. A bioavailability study was performed in dogs. The faster drug release from the nanocrystal-based formulation was reflected in the in vivo results since Tmax of the formulations was obtained 3h after administration, while Tmax of the reference formulation was observed only 6h after administration. This fast release of ITRA was obtained by a dual concept: manufacturing of nanosized cocrystals of ITRA and adipic acid via wet milling. Formation of stable nanosized cocrystals via this approach seems a good alternative for amorphous systems to increase the solubility and obtain a fast drug release of BCS class II drugs. Copyright © 2013 Elsevier B.V. All rights reserved.

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...

Verification of performance specifications of a molecular test: cystic fibrosis carrier testing using the Luminex liquid bead array.

PubMed

Lacbawan, Felicitas L; Weck, Karen E; Kant, Jeffrey A; Feldman, Gerald L; Schrijver, Iris

2012-01-01

The number of clinical laboratories introducing various molecular tests to their existing test menu is continuously increasing. Prior to offering a US Food and Drug Administration-approved test, it is necessary that performance characteristics of the test, as claimed by the company, are verified before the assay is implemented in a clinical laboratory. To provide an example of the verification of a specific qualitative in vitro diagnostic test: cystic fibrosis carrier testing using the Luminex liquid bead array (Luminex Molecular Diagnostics, Inc, Toronto, Ontario). The approach used by an individual laboratory for verification of a US Food and Drug Administration-approved assay is described. Specific verification data are provided to highlight the stepwise verification approach undertaken by a clinical diagnostic laboratory. Protocols for verification of in vitro diagnostic assays may vary between laboratories. However, all laboratories must verify several specific performance specifications prior to implementation of such assays for clinical use. We provide an example of an approach used for verifying performance of an assay for cystic fibrosis carrier screening.

From the one-bead-one-compound concept to one-bead-one-reactor.

PubMed

Marani, Mariela M; Paradís-Bas, Marta; Tulla-Puche, Judit; Côté, Simón; Camperi, Silvia A; Cascone, Osvaldo; Albericio, Fernando

2007-01-01

The one-bead-one-compound method gives access to millions of compounds that can be screened directly on the bead. Although characterization techniques are increasingly potent and reliable, problems can still be encountered in deciphering the sequence of the active compound because of sensitiveness or manipulation of the bead. ChemMatrix, a totally PEG-based resin, has resolved the synthesis of peptides of outstanding difficulty. Like other PEG-based resins, it permits on-bead screening because of its compatibility in aqueous media and has the further advantage of having a high loading, comparable to polystyrene resins. ChemMatrix beads previously swelled in water can be nicely divided into four parts that can be characterized using different analytical techniques or just stored for safety or for further testing. The four bead parts show high homogeneity and can thus be considered to be replicas.

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.

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

PubMed Central

Ganz, Kyle R.; Clime, Liviu; Farber, Jeffrey M.; Corneau, Nathalie

2015-01-01

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-μm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy. PMID:25841016

Flexible corner cube retroreflector array for temperature and strain sensing† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra13284k

PubMed Central

Khalid, Muhammad Waqas; Ahmed, Rajib; Yetisen, Ali K.

2018-01-01

Optical sensors for detecting temperature and strain play a crucial role in the analysis of environmental conditions and real-time remote sensing. However, the development of a single optical device that can sense temperature and strain simultaneously remains a challenge. Here, a flexible corner cube retroreflector (CCR) array based on passive dual optical sensing (temperature and strain) is demonstrated. A mechanical embossing process was utilised to replicate a three-dimensional (3D) CCR array in a soft flexible polymer film. The fabricated flexible CCR array samples were experimentally characterised through reflection measurements followed by computational modelling. As fabricated samples were illuminated with a monochromatic laser beam (635, 532, and 450 nm), a triangular shape reflection was obtained at the far-field. The fabricated flexible CCR array samples tuned retroreflected light based on external stimuli (temperature and strain as an applied force). For strain and temperature sensing, an applied force and temperature, in the form of weight suspension, and heat flow was applied to alter the replicated CCR surface structure, which in turn changed its optical response. Directional reflection from the heated flexible CCR array surface was also measured with tilt angle variation (max. up to 10°). Soft polymer CCRs may have potential in remote sensing applications, including measuring the temperature in space and in nuclear power stations. PMID:29568510

Ferromagnetic nanowires: Field-induced self-assembly, magnetotransport and biological applications

NASA Astrophysics Data System (ADS)

Tanase, Monica

In this dissertation, a series of experiments on magnetic nanowires are described. Magnetic nanowires suspended in fluid solutions can be assembled and ordered by taking advantage of their large shape anisotropy. Magnetic manipulation and assembly techniques were developed, using electrodeposited Ni nanowires. Preorienting nanowires in a small magnetic field induced their self-assembly in continuous chains. A new technique of magnetic trapping allowed capture of single nanowires from fluid suspension on lithographically fabricated micromagnets. As described herein, the presence of an external magnetic field plays a fundamental role in all fluid assembly methods used. The dynamics of both chaining and trapping processes is described quantitatively in terms of the interplay of magnetic forces and fluid drag at low Reynolds number. Lithographic methods for addressing single nanowires for transport characterization were developed. Magnetotransport measurements were performed on individual straight and bent PtNiPt nanowires. The Pt end segments provided an oxide-free interface to the magnetic central segment. In straight nanowires, domain reversal was observed to occur via curling mode initiated in a small nucleation volume. Magnetotransport in bent nanowires allowed the investigation of a domain wall trapped at the bend. Magnetic trapping of nanowires on pre-fabricated electrodes was adapted as a successful alternative contacting technique to lithography. The self-assembly and manipulation techniques were adapted for manipulation of cells as nanowires were found to bind to cells through nonspecific adhesion mechanisms. Ni nanowires were found to outperform superparamagnetic beads in magnetic cell separations. Additionally, the large remnant magnetization of the nanowires allowed for low-field manipulation techniques. Self-assembled chains of cells were formed and single cells were localized on substrates patterned with micromagnets. A fluid flow method was developed to controllably introduce the cells in the proximity of arrays of micromagnets. Cells decorated the arrays forming patterns described well by dipolar interactions between the magnetic elements and the nanowires. Calculations of the locations favorable for trapping were performed by evaluating the energy of interaction between the array and the nanowires. A second-order mechanism of cell capture was also identified, i.e. chaining by wire-wire dipolar interaction.

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.

Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders.

PubMed

Sroka-Bartnicka, Anna; Karlsson, Isabella; Ndreu, Lorena; Quaranta, Alessandro; Pijnappel, Matthijs; Thorsén, Gunnar

2017-01-05

Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases. In this paper, we present a rapid and efficient method for glycosylation analysis of individual proteins focusing on changes in the degree of fucosylation or other alterations to the core structure of the glycans, such as the presence of bisecting N-acetylglucosamines and a modified degree of branching. Streptavidin-coated magnetic beads are used in combination with genetically engineered immunoaffinity binders, called VHH antibody fragments. A major advantage of the VHHs is that they are nonglycosylated; thus, enzymatic release of glycans from the targeted protein can be performed directly on the beads. After deglycosylation, the glycans are analyzed by MALDI-TOF-MS. The developed method was evaluated concerning its specificity, and thereafter implemented for studying the glycosylation pattern of two different proteins, alpha-1-antitrypsin and transferrin, in human serum and cerebrospinal fluid. To our knowledge, this is the first example of a protein array-type experiment that employs bead-based immunoaffinity purification in combination with mass spectrometry analysis for fast and efficient glycan analysis of individual proteins in biological fluid. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Optimization and qualification of an Fc Array assay for assessments of antibodies against HIV-1/SIV.

PubMed

Brown, Eric P; Weiner, Joshua A; Lin, Shu; Natarajan, Harini; Normandin, Erica; Barouch, Dan H; Alter, Galit; Sarzotti-Kelsoe, Marcella; Ackerman, Margaret E

2018-04-01

The Fc Array is a multiplexed assay that assesses the Fc domain characteristics of antigen-specific antibodies with the potential to evaluate up to 500 antigen specificities simultaneously. Antigen-specific antibodies are captured on antigen-conjugated beads and their functional capacity is probed via an array of Fc-binding proteins including antibody subclassing reagents, Fcγ receptors, complement proteins, and lectins. Here we present the results of the optimization and formal qualification of the Fc Array, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. Assay conditions were optimized for performance and reproducibility, and the final version of the assay was then evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Computational diffraction tomographic microscopy with transport of intensity equation using a light-emitting diode array

NASA Astrophysics Data System (ADS)

Li, Jiaji; Chen, Qian; Zhang, Jialin; Zuo, Chao

2017-10-01

Optical diffraction tomography (ODT) is an effective label-free technique for quantitatively refractive index imaging, which enables long-term monitoring of the internal three-dimensional (3D) structures and molecular composition of biological cells with minimal perturbation. However, existing optical tomographic methods generally rely on interferometric configuration for phase measurement and sophisticated mechanical systems for sample rotation or beam scanning. Thereby, the measurement is suspect to phase error coming from the coherent speckle, environmental vibrations, and mechanical error during data acquisition process. To overcome these limitations, we present a new ODT technique based on non-interferometric phase retrieval and programmable illumination emitting from a light-emitting diode (LED) array. The experimental system is built based on a traditional bright field microscope, with the light source replaced by a programmable LED array, which provides angle-variable quasi-monochromatic illumination with an angular coverage of +/-37 degrees in both x and y directions (corresponding to an illumination numerical aperture of ˜ 0.6). Transport of intensity equation (TIE) is utilized to recover the phase at different illumination angles, and the refractive index distribution is reconstructed based on the ODT framework under first Rytov approximation. The missing-cone problem in ODT is addressed by using the iterative non-negative constraint algorithm, and the misalignment of the LED array is further numerically corrected to improve the accuracy of refractive index quantification. Experiments on polystyrene beads and thick biological specimens show that the proposed approach allows accurate refractive index reconstruction while greatly reduced the system complexity and environmental sensitivity compared to conventional interferometric ODT approaches.

Optical diffraction tomography microscopy with transport of intensity equation using a light-emitting diode array

NASA Astrophysics Data System (ADS)

Li, Jiaji; Chen, Qian; Zhang, Jialin; Zhang, Zhao; Zhang, Yan; Zuo, Chao

2017-08-01

Optical diffraction tomography (ODT) is an effective label-free technique for quantitatively refractive index imaging, which enables long-term monitoring of the internal three-dimensional (3D) structures and molecular composition of biological cells with minimal perturbation. However, existing optical tomographic methods generally rely on interferometric configuration for phase measurement and sophisticated mechanical systems for sample rotation or beam scanning. Thereby, the measurement is suspect to phase error coming from the coherent speckle, environmental vibrations, and mechanical error during data acquisition process. To overcome these limitations, we present a new ODT technique based on non-interferometric phase retrieval and programmable illumination emitting from a light-emitting diode (LED) array. The experimental system is built based on a traditional bright field microscope, with the light source replaced by a programmable LED array, which provides angle-variable quasi-monochromatic illumination with an angular coverage of ±37 degrees in both x and y directions (corresponding to an illumination numerical aperture of ∼0.6). Transport of intensity equation (TIE) is utilized to recover the phase at different illumination angles, and the refractive index distribution is reconstructed based on the ODT framework under first Rytov approximation. The missing-cone problem in ODT is addressed by using the iterative non-negative constraint algorithm, and the misalignment of the LED array is further numerically corrected to improve the accuracy of refractive index quantification. Experiments on polystyrene beads and thick biological specimens show that the proposed approach allows accurate refractive index reconstruction while greatly reduced the system complexity and environmental sensitivity compared to conventional interferometric ODT approaches.

A high-density intraspecific SNP linkage map of pigeonpea (Cajanas cajan L. Millsp.)

PubMed Central

Mandal, Paritra; Bhutani, Shefali; Dutta, Sutapa; Kumawat, Giriraj; Singh, Bikram Pratap; Chaudhary, A. K.; Yadav, Rekha; Gaikwad, K.; Sevanthi, Amitha Mithra; Datta, Subhojit; Raje, Ranjeet S.; Sharma, Tilak R.; Singh, Nagendra Kumar

2017-01-01

Pigeonpea (Cajanus cajan (L.) Millsp.) is a major food legume cultivated in semi-arid tropical regions including the Indian subcontinent, Africa, and Southeast Asia. It is an important source of protein, minerals, and vitamins for nearly 20% of the world population. Due to high carbon sequestration and drought tolerance, pigeonpea is an important crop for the development of climate resilient agriculture and nutritional security. However, pigeonpea productivity has remained low for decades because of limited genetic and genomic resources, and sparse utilization of landraces and wild pigeonpea germplasm. Here, we present a dense intraspecific linkage map of pigeonpea comprising 932 markers that span a total adjusted map length of 1,411.83 cM. The consensus map is based on three different linkage maps that incorporate a large number of single nucleotide polymorphism (SNP) markers derived from next generation sequencing data, using Illumina GoldenGate bead arrays, and genotyping with restriction site associated DNA (RAD) sequencing. The genotyping-by-sequencing enhanced the marker density but was met with limited success due to lack of common markers across the genotypes of mapping population. The integrated map has 547 bead-array SNP, 319 RAD-SNP, and 65 simple sequence repeat (SSR) marker loci. We also show here correspondence between our linkage map and published genome pseudomolecules of pigeonpea. The availability of a high-density linkage map will help improve the anchoring of the pigeonpea genome to its chromosomes and the mapping of genes and quantitative trait loci associated with useful agronomic traits. PMID:28654689

Optical position measurement for a large gap magnetic suspension system: Design and performance analysis

NASA Technical Reports Server (NTRS)

Welch, Sharon S.; Clemmons, James I., Jr.; Shelton, Kevin J.; Duncan, Walter C.

1994-01-01

An optical measurement system (OMS) has been designed and tested for a large gap magnetic suspension system (LGMSS). The LGMSS will be used to study control laws for magnetic suspension systems for vibration isolation and pointing applications. The LGMSS features six degrees of freedom and consists of a planar array of electromagnets that levitate and position a cylindrical element containing a permanent magnet core. The OMS provides information on the location and orientation of the element to the LGMSS control system to stabilize suspension. The hardware design of this optical sensing system and the tracking algorithms are presented. The results of analyses and experiments are presented that define the accuracy limits of the optical sensing system and that quantify the errors in position estimation.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture Part 2: Image sequence analysis based evaluation and biological application.

PubMed

Járvás, Gábor; Varga, Tamás; Szigeti, Márton; Hajba, László; Fürjes, Péter; Rajta, István; Guttman, András

2018-02-01

As a continuation of our previously published work, this paper presents a detailed evaluation of a microfabricated cell capture device utilizing a doubly tilted micropillar array. The device was fabricated using a novel hybrid technology based on the combination of proton beam writing and conventional lithography techniques. Tilted pillars offer unique flow characteristics and support enhanced fluidic interaction for improved immunoaffinity based cell capture. The performance of the microdevice was evaluated by an image sequence analysis based in-house developed single-cell tracking system. Individual cell tracking allowed in-depth analysis of the cell-chip surface interaction mechanism from hydrodynamic point of view. Simulation results were validated by using the hybrid device and the optimized surface functionalization procedure. Finally, the cell capture capability of this new generation microdevice was demonstrated by efficiently arresting cells from a HT29 cell-line suspension. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optical position measurement for a Large Gap Magnetic Suspension System

NASA Technical Reports Server (NTRS)

Welch, Sharon S.; Shelton, Kevin J.; Clemmons, James I.

1991-01-01

This paper describes the design of an optical position measurement system which is being built as part of the NASA Langley Large Gap Magnetic Suspension System (LGMSS). The LGMSS is a five degree-of-freedom, large-gap magnetic suspension system which is being built for Langley Research Center as part of the Advanced Controls Test Facility (ACTF). The LGMSS consists of a planar array of electromagnets which levitate and position a cylindrically shaped model containing a permanent magnet core. The optical position measurement system provides information on the location and orientation of the model to the LGMSS control system to stabilize levitation of the model.

Construction of high-density bacterial colony arrays and patterns by the ink-jet method.

PubMed

Xu, Tao; Petridou, Sevastioni; Lee, Eric H; Roth, Elizabeth A; Vyavahare, Narendra R; Hickman, James J; Boland, Thomas

2004-01-05

We have developed a method for fabricating bacterial colony arrays and complex patterns using commercially available ink-jet printers. Bacterial colony arrays with a density of 100 colonies/cm(2) were obtained by directly ejecting Escherichia coli (E. coli) onto agar-coated substrates at a rapid arraying speed of 880 spots per second. Adjusting the concentration of bacterial suspensions allowed single colonies of viable bacteria to be obtained. In addition, complex patterns of viable bacteria as well as bacteria density gradients were constructed using desktop printers controlled by a simple software program. Copyright 2003 Wiley Periodicals, Inc.

Facile fabrication and characterization of a novel oral pH-sensitive drug delivery system based on CMC hydrogel and HNT-AT nanohybrid.

PubMed

Hossieni-Aghdam, Seyed Jamal; Foroughi-Nia, Behrouz; Zare-Akbari, Zhila; Mojarad-Jabali, Solmaz; Motasadizadeh, Hamidreza; Farhadnejad, Hassan

2018-02-01

The main aim of the present study was to design pH-sensitive bionanocomposite hydrogel beads based on CMC and HNT-AT nanohybrid and evaluate whether prepared bionanocomposite beads have the potential to be used in drug delivery applications. Atenolol (AT), as a model drug, was incorporated into the lumen of HA nanotubes via the co-precipitation technique. HNT/AT nanohybrid and CMC/HNT-AT beads were characterized via XRD, SEM, TGA, and FT-IR techniques. Drug loading and encapsulation efficiency was found to be high for CMC/HNT3 beads. Moreover, the swelling and drug release properties of the prepared CMC/HA-AT beads were investigated, and showed a pH sensitive swelling behavior with maximum its content at pH 6.8. Also, it was found that the swelling ratio of CMC/HNT beads was lower than that of pristine CMC beads. Drug release behavior of CMC/HNT-AT bionanocomposite hydrogel beads were investigated. A more sustained and controlled drug releases were observed for CMC/HNT-AT beads. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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

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

Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads

PubMed Central

Luciani, Mirella; Di Febo, Tiziana; Zilli, Katiuscia; Di Giannatale, Elisabetta; Armillotta, Gisella; Manna, Laura; Minelli, Fabio; Tittarelli, Manuela; Caprioli, Alfredo

2016-01-01

Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 103 E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 102 E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 101 E. coli O104:H4 initial load). The specificity was 100%. PMID:27379071

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.

CNV-WebStore: online CNV analysis, storage and interpretation.

PubMed

Vandeweyer, Geert; Reyniers, Edwin; Wuyts, Wim; Rooms, Liesbeth; Kooy, R Frank

2011-01-05

Microarray technology allows the analysis of genomic aberrations at an ever increasing resolution, making functional interpretation of these vast amounts of data the main bottleneck in routine implementation of high resolution array platforms, and emphasising the need for a centralised and easy to use CNV data management and interpretation system. We present CNV-WebStore, an online platform to streamline the processing and downstream interpretation of microarray data in a clinical context, tailored towards but not limited to the Illumina BeadArray platform. Provided analysis tools include CNV analsyis, parent of origin and uniparental disomy detection. Interpretation tools include data visualisation, gene prioritisation, automated PubMed searching, linking data to several genome browsers and annotation of CNVs based on several public databases. Finally a module is provided for uniform reporting of results. CNV-WebStore is able to present copy number data in an intuitive way to both lab technicians and clinicians, making it a useful tool in daily clinical practice.

Modeling of weld bead geometry for rapid manufacturing by robotic GMAW

NASA Astrophysics Data System (ADS)

Yang, Tao; Xiong, Jun; Chen, Hui; Chen, Yong

2015-03-01

Weld-based rapid prototyping (RP) has shown great promises for fabricating 3D complex parts. During the layered deposition of forming metallic parts with robotic gas metal arc welding, the geometry of a single weld bead has an important influence on surface finish quality, layer thickness and dimensional accuracy of the deposited layer. In order to obtain accurate, predictable and controllable bead geometry, it is essential to understand the relationships between the process variables with the bead geometry (bead width, bead height and ratio of bead width to bead height). This paper highlights an experimental study carried out to develop mathematical models to predict deposited bead geometry through the quadratic general rotary unitized design. The adequacy and significance of the models were verified via the analysis of variance. Complicated cause-effect relationships between the process parameters and the bead geometry were revealed. Results show that the developed models can be applied to predict the desired bead geometry with great accuracy in layered deposition with accordance to the slicing process of RP.

Single-cell barcoding and sequencing using droplet microfluidics.

PubMed

Zilionis, Rapolas; Nainys, Juozas; Veres, Adrian; Savova, Virginia; Zemmour, David; Klein, Allon M; Mazutis, Linas

2017-01-01

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

Single bead-based electrochemical biosensor.

PubMed

Liu, Changchun; Schrlau, Michael G; Bau, Haim H

2009-12-15

A simple, robust, single bead-based electrochemical biosensor was fabricated and characterized. The sensor's working electrode consists of an electrochemically etched platinum wire, with a nominal diameter of 25 microm, hermetically heat-fusion sealed in a pulled glass capillary (micropipette). The sealing process does not require any epoxy or glue. A commercially available, densely functionalized agarose bead was mounted on the tip of the etched platinum wire. The use of a pre-functionalized bead eliminates the tedious and complicated surface functionalization process that is often the bottleneck in the development of electrochemical biosensors. We report on the use of a biotin agarose bead-based, micropipette, electrochemical (Bio-BMP) biosensor to monitor H(2)O(2) concentration and the use of a streptavidin bead-based, micropipette, electrochemical (SA-BMP) biosensor to detect DNA amplicons. The Bio-BMP biosensor's response increased linearly as the H(2)O(2) concentration increased in the range from 1 x 10(-6) to 1.2 x10(-4)M with a detection limit of 5 x 10(-7)M. The SA-BMP was able to detect the amplicons of 1pg DNA template of B. Cereus bacteria, thus providing better detection sensitivity than conventional gel-based electropherograms.

Modeling and fabrication of scale-like cantilever for cell capturing

NASA Astrophysics Data System (ADS)

Liu, Boyin; Fu, Jing; Muradoglu, Murat

2013-12-01

The micro-domain provides excellent conditions for performing biological experiments on small populations of cells and has given rise to the proliferation of so-called lab-on-a-chip devices. In order to fully utilize the benefits of cell assays, means of retaining cells at defined locations over time are required. Here, the creation of scale-like cantilevers, inspired by biomimetics, on planar silicon nitride (Si3N4) film using focused ion beam machining is described. Using SEM imaging, regular tilting of the cantilever with almost no warping of the cantilever was uncovered. Finite element analysis showed that the scale-like cantilever was best at limiting stress concentration without difficulty in manufacture and having stresses more evenly distributed along the edge. It also had a major advantage in that the degree of deflection could be simply altered by changing the central angle. From a piling simulation conducted, it was found that a random delivery of simulated particles on to the scale-like obstacle should create a triangular collection. In the experimental trapping of polystyrene beads in suspension, the basic triangular piling structure was observed, but with extended tails and a fanning out around the obstacle. This was attributed to the aggregation tendency of polystyrene beads that acted on top of the piling behavior. In the experiment with bacterial cells, triangular pile up behind the cantilever was absent and the bacteria cells were able to slip inside the cantilever's opening despite the size of the bacteria being larger than the gap. Overall, the fabricated scale-like cantilever architectures offer a viable way to trap small populations of material in suspension.

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.

Dielectrophoresis-Assisted Integration of 1024 Carbon Nanotube Sensors into a CMOS Microsystem.

PubMed

Seichepine, Florent; Rothe, Jörg; Dudina, Alexandra; Hierlemann, Andreas; Frey, Urs

2017-05-01

Carbon-nanotube (CNT)-based sensors offer the potential to detect single-molecule events and picomolar analyte concentrations. An important step toward applications of such nanosensors is their integration in large arrays. The availability of large arrays would enable multiplexed and parallel sensing, and the simultaneously obtained sensor signals would facilitate statistical analysis. A reliable method to fabricate an array of 1024 CNT-based sensors on a fully processed complementary-metal-oxide-semiconductor microsystem is presented. A high-yield process for the deposition of CNTs from a suspension by means of liquid-coupled floating-electrode dielectrophoresis (DEP), which yielded 80% of the sensor devices featuring between one and five CNTs, is developed. The mechanism of floating-electrode DEP on full arrays and individual devices to understand its self-limiting behavior is studied. The resistance distributions across the array of CNT devices with respect to different DEP parameters are characterized. The CNT devices are then operated as liquid-gated CNT field-effect-transistors (LG-CNTFET) in liquid environment. Current dependency to the gate voltage of up to two orders of magnitude is recorded. Finally, the sensors are validated by studying the pH dependency of the LG-CNTFET conductance and it is demonstrated that 73% of the CNT sensors of a given microsystem show a resistance decrease upon increasing the pH value. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rare Earth Adsorption and Desorption with PEGDA Beads

DOE Data Explorer

Jiao, Yongqin; Brewer, Aaron; Park, Dan

2017-03-01

We synthesized PEGDA polymer hydrogel beads for cell embedding and compared REE biosorption with these beads via a gravity-driven flow through setup. One way to set up a flow through system is by cell encapsulation into polymer beads with a column setup similar to that used in the chromatography industry. To achieve this, we tested PEGDA for cell encapsulation, and tested REE biosorption under both batch mode and a follow through setup based on gravity . For making the cell embedded polymer beads, we used a fluidic device by which homogenous spherical particles of 0.5 to1 mm in diameter were synthesized. The beads are made relatively quickly, and the size of the beads can be controlled. PEGDA beads were polymerized by UV. Tb adsorption experiment was performed with beads with or without cells embedded.

Hot embossed polyethylene through-hole chips for bead-based microfluidic devices

PubMed Central

Chou, Jie; Du, Nan; Ou, Tina; Floriano, Pierre N.; Christodoulides, Nicolaos; McDevitt, John T.

2013-01-01

Over the past decade, there has been a growth of interest in the translation of microfluidic systems into real-world clinical practice, especially for use in point-of-care or near patient settings. While initial fabrication advances in microfluidics involved mainly the etching of silicon and glass, the economics of scaling of these materials is not amendable for point-of-care usage where single-test applications forces cost considerations to be kept low and throughput high. As such, a materials base more consistent with point-of-care needs is required. In this manuscript, the fabrication of a hot embossed, through-hole low-density polyethylene ensembles derived from an anisotropically etched silicon wafer is discussed. This semi-opaque polymer that can be easily sterilized and recycled provides low background noise for fluorescence measurements and yields more affordable cost than other thermoplastics commonly used for microfluidic applications such as cyclic olefin copolymer (COC). To fabrication through-hole microchips from this alternative material for microfluidics, a fabrication technique that uses a high-temperature, high-pressure resistant mold is described. This aluminum-based epoxy mold, serving as the positive master mold for embossing, is casted over etched arrays of pyramidal pits in a silicon wafer. Methods of surface treatment of the wafer prior to casting and PDMS casting of the epoxy are discussed to preserve the silicon wafer for future use. Changes in the thickness of polyethylene are observed for varying embossing temperatures. The methodology described herein can quickly fabricate 20 disposable, single use chips in less than 30 minutes with the ability to scale up 4x by using multiple molds simultaneously. When coupled as a platform supporting porous bead sensors, as in the recently developed Programmable Bio-Nano-Chip, this bead chip system can achieve limits of detection, for the cardiac biomarker C-reactive protein, of 0.3 ng/mL, thereby demonstrating the approach is compatible with high performance, real-world clinical measurements in the context of point-of-care testing. PMID:23183187

An ultra-fast EOD-based force-clamp detects rapid biomechanical transitions

NASA Astrophysics Data System (ADS)

Woody, Michael S.; Capitanio, Marco; Ostap, E. Michael; Goldman, Yale E.

2017-08-01

We assembled an ultra-fast infrared optical trapping system to detect mechanical events that occur less than a millisecond after a ligand binds to its filamentous substrate, such as myosin undergoing its 5 - 10 nm working stroke after actin binding. The instrument is based on the concept of Capitanio et al.1, in which a polymer bead-actin-bead dumbbell is held in two force-clamped optical traps. A force applied by the traps causes the filament to move at a constant velocity as hydrodynamic drag balances the applied load. When the ligand binds, the filament motion stops within 100 μs as the total force from the optical traps is transferred to the attachment. Subsequent translations signal active motions, such as the magnitude and timing of the motor's working stroke. In our instrument, the beads defining the dumbbell are held in independent force clamps utilizing a field-programmable gate array (FPGA) to update the trap beam positions at 250 kHz. We found that in our setup, acousto-optical deflectors (AODs) steering the beams were unsuitable for this purpose due to a slightly non-linear response in the beam intensity and deflection angle vs. the AOD ultra-sound wavelength, likely caused by low-amplitude standing acoustic waves in the deflectors. These aberrations caused instability in the force feedback loops leading to artefactual 20 nm jumps in position. This type of AOD non-linearity has been reported to be absent in electro-optical deflectors (EODs)2. We demonstrate that replacement of the AODs with EODs improves the performance of our instrument. Combining the superior beam-steering capability of the EODs, force acquisition via back-plane interferometry, and the dual high-speed FPGA-based feedback loops, we smoothly and precisely apply constant loads to study the dynamics of interactions between biological molecules such as actin and myosin.

Combined SERS biotags (SBTs) and microfluidic platform for the quantitative ratiometric discrimination between noncancerous and cancerous cells in flow

NASA Astrophysics Data System (ADS)

Pallaoro, Alessia; Hoonejani, Mehran R.; Braun, Gary B.; Meinhart, Carl; Moskovits, Martin

2012-10-01

SERS biotags are made from polymer-encapsulated silver nanoparticle dimers infused with unique Raman reporter molecules, and carry peptides as cell recognition moieties. We demonstrate their potential use for early and rapid identification of malignant cells, a central goal in cancer research. SERS biotags (SBTs) can be routinely synthesized and simultaneously excited with a single, low intensity laser source, making the determination of the relative contribution of the individual SBTs to the overall spectrum tractable. Importantly for biomedical applications, SERS employs tissuepenetrating lasers in the red to near-infrared range resulting in low autofluorescence. We have previously described a multiplexed, ratiometric method that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro based on receptor overexpression. Here we present the progress towards the application of this quantitative methodology for the identification of cancer cells in a microfluidic flow-focusing device. Beads are used as cell mimics to characterize the devices. Cells (and beads) are simultaneously incubated with two sets of SBTs while in suspension (simulating cells' capture from blood), then injected into the device for laser interrogation under flow. Each cell event is characterized by a composite Raman spectrum, deconvoluted into its single components to ultimately determine their relative contribution. We show that using SBTs ratiometrically can provide cell identification insensitive to normal causes of uncertainty in optical measurements such as variations in focal plane, cell concentration, autofluorescence, and turbidity.

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.

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.

MixHMM: Inferring Copy Number Variation and Allelic Imbalance Using SNP Arrays and Tumor Samples Mixed with Stromal Cells

PubMed Central

Schulz, Vincent; Chen, Min; Tuck, David

2010-01-01

Background Genotyping platforms such as single nucleotide polymorphism (SNP) arrays are powerful tools to study genomic aberrations in cancer samples. Allele specific information from SNP arrays provides valuable information for interpreting copy number variation (CNV) and allelic imbalance including loss-of-heterozygosity (LOH) beyond that obtained from the total DNA signal available from array comparative genomic hybridization (aCGH) platforms. Several algorithms based on hidden Markov models (HMMs) have been designed to detect copy number changes and copy-neutral LOH making use of the allele information on SNP arrays. However heterogeneity in clinical samples, due to stromal contamination and somatic alterations, complicates analysis and interpretation of these data. Methods We have developed MixHMM, a novel hidden Markov model using hidden states based on chromosomal structural aberrations. MixHMM allows CNV detection for copy numbers up to 7 and allows more complete and accurate description of other forms of allelic imbalance, such as increased copy number LOH or imbalanced amplifications. MixHMM also incorporates a novel sample mixing model that allows detection of tumor CNV events in heterogeneous tumor samples, where cancer cells are mixed with a proportion of stromal cells. Conclusions We validate MixHMM and demonstrate its advantages with simulated samples, clinical tumor samples and a dilution series of mixed samples. We have shown that the CNVs of cancer cells in a tumor sample contaminated with up to 80% of stromal cells can be detected accurately using Illumina BeadChip and MixHMM. Availability The MixHMM is available as a Python package provided with some other useful tools at http://genecube.med.yale.edu:8080/MixHMM. PMID:20532221

Metal-Containing Polystyrene Beads as Standards for Mass Cytometry

PubMed Central

Abdelrahman, Ahmed I.; Ornatsky, Olga; Bandura, Dmitry; Kinach, Robert; Dai, Sheng; Thickett, Stuart C.; Tanner, Scott

2010-01-01

We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells. PMID:20390041

Metal-Containing Polystyrene Beads as Standards for Mass Cytometry.

PubMed

Abdelrahman, Ahmed I; Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Kinach, Robert; Dai, Sheng; Thickett, Stuart C; Tanner, Scott; Winnik, Mitchell A

2010-01-01

We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells.

Evaluation of Bovine High-Density SNP Genotyping Array in Indigenous Dairy Cattle Breeds.

PubMed

Dash, S; Singh, A; Bhatia, A K; Jayakumar, S; Sharma, A; Singh, S; Ganguly, I; Dixit, S P

2018-04-03

In total 52 samples of Sahiwal ( 19 ), Tharparkar ( 17 ), and Gir ( 16 ) were genotyped by using BovineHD SNP chip to analyze minor allele frequency (MAF), genetic diversity, and linkage disequilibrium among these cattle. The common SNPs of BovineHD and 54K SNP Chips were also extracted and evaluated for their performance. Only 40%-50% SNPs of these arrays was found informative for genetic analysis in these cattle breeds. The overall mean of MAF for SNPs of BovineHD SNPChip was 0.248 ± 0.006, 0.241 ± 0.007, and 0.242 ± 0.009 in Sahiwal, Tharparkar and Gir, respectively, while that for 54K SNPs was on lower side. The average Reynold's genetic distance between breeds ranged from 0.042 to 0.055 based on BovineHD Beadchip, and from 0.052 to 0.084 based on 54K SNP Chip. The estimates of genetic diversity based on HD and 54K chips were almost same and, hence, low density chip seems to be good enough to decipher genetic diversity of these cattle breeds. The linkage disequilibrium started decaying (r 2  < 0.2) at 140 kb inter-marker distance and, hence, a 20K low density customized SNP array from HD chip could be designed for genomic selection in these cattle else the 54K Bead Chip as such will be useful.

Scaling, clustering and avalanches for steel beads in an external magnetic field

NASA Astrophysics Data System (ADS)

Marquinez, Alyse; Thvedt, Ingrid; Lehman, S. Y.; Jacobs, D. T.

2011-03-01

We investigated avalanches using uniform 3mm steel spheres (``beads'') dropped onto a conical bead pile within a uniform magnetic field. The bead pile is built by pouring beads onto a circular base where the bottom layer of beads had been glued randomly. Beads are then individually dropped from a fixed height after which the pile is massed. This process is repeated for thousands of bead drops. By measuring the number of avalanches of a given size that occurred during the experiment, the resulting avalanche size distribution was compared to a power law description as predicted by self-organized criticality. As the magnetic field intensity increased, the beads clustered to give a larger angle of repose and we measured the change in the avalanche size distribution. The moments of the distribution give a sensitive test of mean-field theory as the universality class for these bead piles. We acknowledge support from Research Corporation and NSF-REU grant DMR 0649112.

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

Microfluidic device for the assembly and transport of microparticles

DOEpatents

James, Conrad D [Albuquerque, NM; Kumar, Anil [Framingham, MA; Khusid, Boris [New Providence, NJ; Acrivos, Andreas [Stanford, CA

2010-06-29

A microfluidic device comprising independently addressable arrays of interdigitated electrodes can be used to assembly and transport large-scale microparticle structures. The device and method uses collective phenomena in a negatively polarized suspension exposed to a high-gradient strong ac electric field to assemble the particles into predetermined locations and then transport them collectively to a work area for final assembly by sequentially energizing the electrode arrays.

Mechanical Design of a 4-Stage ADR for the PIPER mission

NASA Technical Reports Server (NTRS)

James, Bryan L.; Kimball, Mark O.; Shirron, Peter J.; Sampson, Michael A.; Letmate, Richard V.; Jackson, Michael L.

2017-01-01

The four 1,280 bolometer detector arrays that will fly on the balloon borne PIPER mission will be cooled by a 4-stage adiabatic demagnetization refrigerator (ADR). Two of the three mechanically independent ADR assemblies provide thermal isolation to their salt pills through Kevlar suspensions while the other provides thermal isolation to its salt pill through the use of bellows and Vespel material. The ADR integrates with the detector arrays and it sits in a large bucket Dewar containing superfluid liquid helium. This paper will describe the complex mechanical design of the PIPER ADR, and summarize the mechanical analysis done to validate the design.The four 1,280 bolometer detector arrays that will fly on the balloon borne PIPER mission will be cooled by a 4-stage adiabatic demagnetization refrigerator (ADR). Two of the three mechanically independent ADR assemblies provide thermal isolation to their salt pills through Kevlar suspensions while the other provides thermal isolation to its salt pill through the use of bellows and Vespel material. The ADR integrates with the detector arrays and it sits in a large bucket Dewar containing superfluid liquid helium. This paper will describe the complex mechanical design of the PIPER ADR, and summarize the mechanical analysis done to validate the design.

Evaluation and application of an innovative method based on various chitosan composites and Lemna gibba for boron removal from drinking water.

PubMed

Türker, Onur Can; Baran, Talat

2017-06-15

Boron exists in various types of water environments, and it is difficult and costly to remove B with conventional treatment methods from drinking water. Clearly, alternative and cost effective treatment techniques are imperative. In the present study, an innovative and environment friendly method based on hybrid systems consisting of various chitosan composite beads and Lemna gibba were evaluated for removal of B from drinking water. Our results from batch adsorption experiment indicated that a plant-based chitosan composite bead has a higher capacity of B removal than mineral-based chitosan composite beads. Almost 50% of total B removal was achieved using the hybrid system based on dried Lemna-chitosan composite beads and Lemna gibba combination in 4 days. Even at the high B concentration (8mgBL -1 ), B in drinking water could be reduced to less than 2.4mgL -1 when 0.05g plant-based chitosan composite beads and 12 Lemna fronds were used for 50mL test solution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tunable Nanowire Patterning Using Standing Surface Acoustic Waves

PubMed Central

Chen, Yuchao; Ding, Xiaoyun; Lin, Sz-Chin Steven; Yang, Shikuan; Huang, Po-Hsun; Nama, Nitesh; Zhao, Yanhui; Nawaz, Ahmad Ahsan; Guo, Feng; Wang, Wei; Gu, Yeyi; Mallouk, Thomas E.; Huang, Tony Jun

2014-01-01

Patterning of nanowires in a controllable, tunable manner is important for the fabrication of functional nanodevices. Here we present a simple approach for tunable nanowire patterning using standing surface acoustic waves (SSAW). This technique allows for the construction of large-scale nanowire arrays with well-controlled patterning geometry and spacing within 5 seconds. In this approach, SSAWs were generated by interdigital transducers (IDTs), which induced a periodic alternating current (AC) electric field on the piezoelectric substrate and consequently patterned metallic nanowires in suspension. The patterns could be deposited onto the substrate after the liquid evaporated. By controlling the distribution of the SSAW field, metallic nanowires were assembled into different patterns including parallel and perpendicular arrays. The spacing of the nanowire arrays could be tuned by controlling the frequency of the surface acoustic waves. Additionally, we observed 3D spark-shape nanowire patterns in the SSAW field. The SSAW-based nanowire-patterning technique presented here possesses several advantages over alternative patterning approaches, including high versatility, tunability, and efficiency, making it promising for device applications. PMID:23540330

Formulation and characterization of a compacted multiparticulate system for modified release of water-soluble drugs--Part II theophylline and cimetidine.

PubMed

Cantor, Stuart L; Hoag, Stephen W; Augsburger, Larry L

2009-05-01

The purpose was to investigate the effectiveness of an ethylcellulose (EC) bead matrix and different film-coating polymers in delaying drug release from compacted multiparticulate systems. Formulations containing theophylline or cimetidine granulated with Eudragit RS 30D were developed and beads were produced by extrusion-spheronization. Drug beads were coated using 15% wt/wt Surelease or Eudragit NE 30D and were evaluated for true density, particle size, and sphericity. Lipid-based placebo beads and drug beads were blended together and compacted on an instrumented Stokes B2 rotary tablet press. Although placebo beads were significantly less spherical, their true density of 1.21 g/cm(3) and size of 855 mum were quite close to Surelease-coated drug beads. Curing improved the crushing strength and friability values for theophylline tablets containing Surelease-coated beads; 5.7 +/- 1.0 kP and 0.26 +/- 0.07%, respectively. Dissolution profiles showed that the EC matrix only provided 3 h of drug release. Although tablets containing Surelease-coated theophylline beads released drug fastest overall (t(44.2%) = 8 h), profiles showed that coating damage was still minimal. Size and density differences indicated a minimal segregation potential during tableting for blends containing Surelease-coated drug beads. Although modified release profiles >8 h were achievable in tablets for both drugs using either coating polymer, Surelease-coated theophylline beads released drug fastest overall. This is likely because of the increased solubility of theophylline and the intrinsic properties of the Surelease films. Furthermore, the lipid-based placebos served as effective cushioning agents by protecting coating integrity of drug beads under a number of different conditions while tableting.

EFFECT OF DIFFERENT REGIONS OF AMPLIFIED 16S RDNA ON A PERFORMANCE OF A MULTIPLEXED, BEAD-BASED METHOD FOR ANALYSIS OF DNA SEQUENCES IN ENVIRONMENTAL SAMPLES.

EPA Science Inventory

Using a bead-based method for multiplexed analysis of community DNA, the dynamics of aquatic microbial communities can be assessed. Capture probes, specific for a genus or species of bacteria, are attached to the surface of uniquely labeled, microscopic polystyrene beads. Primers...

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.

Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis.

PubMed

Parisi, C; Mastoraki, S; Markou, A; Strati, A; Chimonidou, M; Georgoulias, V; Lianidou, E S

2016-10-01

Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer. Copyright © 2016. Published by Elsevier B.V.

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

Towards Integrated Marmara Strong Motion Network

NASA Astrophysics Data System (ADS)

Durukal, E.; Erdik, M.; Safak, E.; Ansal, A.; Ozel, O.; Alcik, H.; Mert, A.; Kafadar, N.; Korkmaz, A.; Kurtulus, A.

2009-04-01

Istanbul has a 65% chance of having a magnitude 7 or above earthquake within the next 30 years. As part of the preparations for the future earthquake, strong motion networks have been installed in and around Istanbul. The Marmara Strong Motion Network, operated by the Department of Earthquake Engineering of Kandilli Observatory and Earthquake Research Institute, encompasses permanent systems outlined below. It is envisaged that the networks will be run by a single entity responsible for technical management and maintanence, as well as for data management, archiving and dissemination through dedicated web-based interfaces. • Istanbul Earthquake Rapid Response and Early Warning System - IERREWS (one hundred 18-bit accelerometers for rapid response; ten 24-bit accelerometers for early warning) • IGDAŞ Gas Shutoff Network (100 accelerometers to be installed in 2010 and integrated with IERREWS) • Structural Monitoring Arrays - Fatih Sultan Mehmet Suspension Bridge (1200m-long suspension bridge across the Bosphorus, five 3-component accelerometers + GPS sensors) - Hagia Sophia Array (1500-year-old historical edifice, 9 accelerometers) - Süleymaniye Mosque Array (450-year-old historical edifice,9 accelerometers) - Fatih Mosque Array (237-year-old historical edifice, 9 accelerometers) - Kanyon Building Array (high-rise office building, 5 accelerometers) - Isbank Tower Array (high-rise office building, 5 accelerometers) - ENRON Array (power generation facility, 4 acelerometers) - Mihrimah Sultan Mosque Array (450-year-old historical edifice,9 accelerometers + tiltmeters, to be installed in 2009) - Sultanahmet Mosque Array, (390-year-old historical edifice, 9 accelerometers + tiltmeters, to be installed in 2009) • Special Arrays - Atakoy Vertical Array (four 3-component accelerometers at 25, 50, 75, and 150 m depths) - Marmara Tube Tunnel (1400 m long submerged tunnel, 128 ch. accelerometric data, 24 ch. strain data, to be installed in 2010) - Air-Force Academy Array (72 ch. dense accelerometric array to be installed in 2010) - Gemlik Array (a dense basin array of 8 stations, to be installed in 2010) The objectives of these systems and networks are: (1) to produce rapid earthquake intensity, damage and loss assessment information after an earthquake (in the case of IERREWS), (2) to monitor conditions of structural systems, (3) to develop real-time data processing, analysis, and damage detection and location tools (in the case of structural networks) after an extreme event, (4) to assess spatial properties of strong ground motion and ground strain, and to characterise basin response (in the case of special arrays), (5) to investigate site response and wave propagation (in the case of vertical array). Ground motion data obtained from these strong motion networks have and are being used for investigations of attenuation, spatial variation (coherence), simulation benchmarking, source modeling, site response, seismic microzonation, system identification and structural model verification and structural health control. In addition to the systems and networks outlined above there are two temporary networks: KIMNET - a dense urban noise and microtremor network consisting of 50 broadband stations expected to be operational in mid 2009, and SOSEWIN - a 20-station, self-organizing structural integrated array at Ataköy in Istanbul.

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.

Response of Human Skin to Aesthetic Scarification

PubMed Central

Gabriel, Vincent A.; McClellan, Elizabeth A.; Scheuermann, Richard H.

2014-01-01

This study was undertaken to investigate changes in RNA expression in previously healthy adult human skin following thermal injury induced by contact with hot metal that was undertaken as part of aesthetic scarification, a body modification practice. Subjects were recruited to have pre-injury skin and serial wound biopsies performed. 4 mm punch biopsies were taken prior to branding and 1 hour, 1 week, and 1, 2 and 3 months post injury. RNA was extracted and quality assured prior to the use of a whole-genome based bead array platform to describe expression changes in the samples using the pre-injury skin as a comparator. Analysis of the array data was performed using k-means clustering and a hypergeometric probability distribution without replacement and corrections for multiple comparisons were done. Confirmatory q-PCR was performed. Using a k of 10, several clusters of genes were shown to co-cluster together based on Gene Ontology classification with probabilities unlikely to occur by chance alone. OF particular interest were clusters relating to cell cycle, proteinaceous extracellular matrix and keratinization. Given the consistent expression changes at one week following injury in the cell cycle cluster, there is an opportunity to intervene early following burn injury to influence scar development. PMID:24582755

Synthesis and characterization of nanoporous silica aerogel beads using cheap industrial grade sodium silacte precursor

NASA Astrophysics Data System (ADS)

Khan, Tasneem M. A.; Khan, Asiya; Sarawade, Pradip B.

2018-05-01

We report a method to synthesize low-density transparent mesoporous silica aerogel beads by ambient pressure drying (APD). The beads were prepared by acid-base sol-gel polymerization of sodium silicate in via the ball dropping method (BDM). To minimize shrinkage during drying, wet silica beads were initially prepared; their surfaces were then modified using trimethylchlorosilane (TMCS) via simultaneous solvent exchange and surface modification. The specific surface area and cumulative pore volume of the silica aerogel beads increased with an increase in the %V of TMCS. Silica aerogel beads with low packing bed density, high surface area, and large cumulative pore volume was obtained when TMCS was used. Properties of the final product were examined by BET, and TG-DT analyses. The hydrophobic silica aerogel beads were thermally stable up to 350°C. We discuss our results and compare our findings for modified versus unmodified silica beads.

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

Pooling of Immunomagnetic Separation Beads Does Not Affect Detection Sensitivity of Six Major Serogroups of Shiga Toxin-Producing Escherichia coli in Cattle Feces.

PubMed

Noll, Lance W; Baumgartner, William C; Shridhar, Pragathi B; Cull, Charley A; Dewsbury, Diana M; Shi, Xiaorong; Cernicchiaro, Natalia; Renter, David G; Nagaraja, T G

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) of the serogroups O26, O45, O103, O111, O121, and O145, often called non-O157 STEC, are foodborne pathogens. Cattle are asymptomatic reservoirs for STEC; the organisms reside in the hindgut and are shed in the feces, which serve as the source of food product contaminations. Culture-based detection of non-O157 STEC involves an immunomagnetic separation (IMS) step to capture the specific serogroups in complex matrices, such as feces. The IMS procedure is time consuming and labor intensive because of the need to subject each fecal sample to six individual beads. Therefore, our objective was to evaluate whether pooling of IMS beads affects sensitivity of non-O157 STEC detection compared with using individual IMS beads. The evaluation was done by comparing detection of serogroups in feces spiked with pure cultures (experiments 1 and 2) and from feces (n = 384) of naturally shedding cattle (experiment 3). In spiked fecal samples, detection with pools of three, four, six, or seven beads was similar to, or at times higher than, detection with individual IMS beads. In experiment 3, the proportions of fecal samples that tested positive for the six serogroups as detected by individual or pooled beads were similar. Based on noninferiority tests, detection with pooled beads was not substantially inferior to detection with individual beads (P > 0.05). In conclusion, the pooling of IMS beads is a better option for detection of STEC serogroups in fecal samples compared with individual beads because the procedure saves time and labor and has the prospect of a higher throughput.

Alginate and Algal-Based Beads for the Sorption of Metal Cations: Cu(II) and Pb(II)

PubMed Central

Wang, Shengye; Vincent, Thierry; Faur, Catherine; Guibal, Eric

2016-01-01

Alginate and algal-biomass (Laminaria digitata) beads were prepared by homogeneous Ca ionotropic gelation. In addition, glutaraldehyde-crosslinked poly (ethyleneimine) (PEI) was incorporated into algal beads. The three sorbents were characterized by scanning electron microscopy (SEM) coupled with energy dispersive X-ray analysis (EDX): the sorption occurs in the whole mass of the sorbents. Sorption experiments were conducted to evaluate the impact of pH, sorption isotherms, and uptake kinetics. A special attention was paid to the effect of drying (air-drying vs. freeze-drying) on the mass transfer properties. For alginate, freeze drying is required for maintaining the porosity of the hydrogel, while for algal-based sorbents the swelling of the material minimizes the impact of the drying procedure. The maximum sorption capacities observed from experiments were 415, 296 and 218 mg Pb g−1 and 112, 77 and 67 mg Cu g−1 for alginate, algal and algal/PEI beads respectively. Though the sorption capacities of algal-beads decreased slightly (compared to alginate beads), the greener and cheaper one-pot synthesis of algal beads makes this sorbent more competitive for environmental applications. PEI in algal beads decreases the sorption properties in the case of the sorption of metal cations under selected experimental conditions. PMID:27598128

On-bead antibody-small molecule conjugation using high-capacity magnetic beads.

PubMed

Nath, Nidhi; Godat, Becky; Benink, Hélène; Urh, Marjeta

2015-11-01

Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications. Copyright © 2015. Published by Elsevier B.V.

A highly efficient bead extraction technique with low bead number for digital microfluidic immunoassay

PubMed Central

Tsai, Po-Yen; Lee, I-Chin; Hsu, Hsin-Yun; Huang, Hong-Yuan; Fan, Shih-Kang; Liu, Cheng-Hsien

2016-01-01

Here, we describe a technique to manipulate a low number of beads to achieve high washing efficiency with zero bead loss in the washing process of a digital microfluidic (DMF) immunoassay. Previously, two magnetic bead extraction methods were reported in the DMF platform: (1) single-side electrowetting method and (2) double-side electrowetting method. The first approach could provide high washing efficiency, but it required a large number of beads. The second approach could reduce the required number of beads, but it was inefficient where multiple washes were required. More importantly, bead loss during the washing process was unavoidable in both methods. Here, an improved double-side electrowetting method is proposed for bead extraction by utilizing a series of unequal electrodes. It is shown that, with proper electrode size ratio, only one wash step is required to achieve 98% washing rate without any bead loss at bead number less than 100 in a droplet. It allows using only about 25 magnetic beads in DMF immunoassay to increase the number of captured analytes on each bead effectively. In our human soluble tumor necrosis factor receptor I (sTNF-RI) model immunoassay, the experimental results show that, comparing to our previous results without using the proposed bead extraction technique, the immunoassay with low bead number significantly enhances the fluorescence signal to provide a better limit of detection (3.14 pg/ml) with smaller reagent volumes (200 nl) and shorter analysis time (<1 h). This improved bead extraction technique not only can be used in the DMF immunoassay but also has great potential to be used in any other bead-based DMF systems for different applications. PMID:26858807

Induced movement of the magnetic beads and DNA-based dumbbell in a micro fluidic channel

NASA Astrophysics Data System (ADS)

Babić, B.; Ghai, R.; Dimitrov, K.

2007-12-01

We have explored controlled movement of magnetic beads and a dumbbell structure composed of DNA, a magnetic and a non-magnetic bead in a micro fluidic channel. Movement of the beads and dumbbells is simulated assuming that a net force is described as a superposition between the magnetic and hydrodynamic drag forces. Trajectories of beads and dumbbells are observed with optical light microscopy. The experimentally measured data show a good agreement with the simulations. This dynamical approach offers the prospect to stretch the DNA within the dumbbell and investigate its conformational changes. Further on, we demonstrate that short sonication can reduce multiple attachments of DNA to the beads.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

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.

A light sheet confocal microscope for image cytometry with a variable linear slit detector

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Khan, Foysal Z.; Powless, Amy J.; Benson, Devin; Hunter, Courtney; Fritsch, Ingrid; Muldoon, Timothy J.

2016-03-01

We present a light sheet confocal microscope (LSCM) capable of high-resolution imaging of cell suspensions in a microfluidic environment. In lieu of conventional pressure-driven flow or mechanical translation of the samples, we have employed a novel method of fluid transport, redox-magnetohydrodynamics (redox-MHD). This method achieves fluid motion by inducing a small current into the suspension in the presence of a magnetic field via electrodes patterned onto a silicon chip. This on-chip transportation requires no moving parts, and is coupled to the remainder of the imaging system. The microscopy system comprises a 450 nm diode 20 mW laser coupled to a single mode fiber and a cylindrical lens that converges the light sheet into the back aperture of a 10x, 0.3 NA objective lens in an epi-illumination configuration. The emission pathway contains a 150 mm tube lens that focuses the light onto the linear sensor at the conjugate image plane. The linear sensor (ELiiXA+ 8k/4k) has three lateral binning modes which enables variable detection aperture widths between 5, 10, or 20 μm, which can be used to vary axial resolution. We have demonstrated redox-MHD-enabled light sheet microscopy in suspension of fluorescent polystyrene beads. This approach has potential as a high-throughput image cytometer with myriad cellular diagnostic applications.

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.

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.

Immobilized magnetic beads-based multi-target affinity selection coupled with HPLC-MS for screening active compounds from traditional Chinese medicine and natural products.

PubMed

Chen, Yaqi; Chen, Zhui; Wang, Yi

2015-01-01

Screening and identifying active compounds from traditional Chinese medicine (TCM) and other natural products plays an important role in drug discovery. Here, we describe a magnetic beads-based multi-target affinity selection-mass spectrometry approach for screening bioactive compounds from natural products. Key steps and parameters including activation of magnetic beads, enzyme/protein immobilization, characterization of functional magnetic beads, screening and identifying active compounds from a complex mixture by LC/MS, are illustrated. The proposed approach is rapid and efficient in screening and identification of bioactive compounds from complex natural products.

Multiple scattering in chiral media: border effects, reduced depolarization, and sensitivity limit

NASA Astrophysics Data System (ADS)

Delplancke, Francoise; Badoz, Jacques P.; Boccara, A. Claude

1997-10-01

Suspensions of polystyrene latex beads in chiral solutions were investigated. The rotatory power, induced by solubilized sucrose, in near-forward scattering was measured via a method using polarization modulation by photo-elastic modulator. The sensitivity of the measurement was enhanced and optimized in order to measure sucrose concentrations as low as 5 mg/ml in a cell 5 mm thick only. Different concentrations and diameters of latex particles were used in combination with different sucrose concentrations going from 1 mg/ml up to saturation. The experiments showed that the apparent rotatory power is enhanced by multiple scattering, that depolarization effects are less important with highly concentrated sucrose solutions and that attention has to be paid to cell border effects in order to avoid important artifacts, in case of highly scattering suspensions. Qualitative and theoretical explanations of those observations are presented. One possible application of this method is to measure the sugar content in human blood, in vivo, non-invasively, through the skin. The concentration to be evaluated is at the sensitivity limit. So any artifact has to be removed carefully, e.g. skin cell birefringence or chirality.

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.

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads.

PubMed

Moore, J A; Nemat-Gorgani, M; Madison, A C; Sandahl, M A; Punnamaraju, S; Eckhardt, A E; Pollack, M G; Vigneault, F; Church, G M; Fair, R B; Horowitz, M A; Griffin, P B

2017-01-01

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols.

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads

PubMed Central

Moore, J. A.; Nemat-Gorgani, M.; Madison, A. C.; Punnamaraju, S.; Eckhardt, A. E.; Pollack, M. G.; Church, G. M.; Fair, R. B.; Horowitz, M. A.; Griffin, P. B.

2017-01-01

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols. PMID:28191268

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.

Evaluation of suspension array analysis for detection of egg yolk antibodies against Salmonella Enteritidis.

PubMed

Thomas, M E; Klinkenberg, D; Bergwerff, A A; van Eerden, E; Stegeman, J A; Bouma, A

2010-06-01

Salmonella enterica serovar Enteritidis (SE) is an important source of food-related diarrhoea in humans, and table eggs are considered the primordial source of contamination of the human food chain. Using eggs collected at egg-packing stations as samples could be a convenient strategy to detect colonization of layer flocks. The aim of this study was to evaluate egg yolk anti-Salmonella antibody detection using suspension array analysis. An egg yolk panel from contact-infected and non-colonized laying hens was used for the evaluation. Receiver Operating Characteristic (ROC) curves were generated to define a cut-off value and to assess the overall accuracy of the assay. The diagnostic sensitivity and specificity were estimated by maximum likelihood. Sensitivity was quantified on hen level and on sample level, and also quantified as a function of time since colonization. The area under the ROC curve was estimated at 0.984 (se 0.006, P<0.001). Of all colonized contact-infected hens, 67.6% [95% CI: 46.8, 100] developed an antibody response, which was detectable 17.4 days [14.3, 26.9] after colonization. In total, 98% [95.4, 99.4] of the 'immunopositive' hens had test positive eggs. The overall sensitivity of the immunological test was 66.7% [45.9, 98.7] and the specificity was 98.5% [97.8, 99.1]. This study provided essential parameters for optimizing surveillance programs based on detection of antibodies, and indicates that immunology based on examination of egg yolk gives important information about the Salmonella status of the flock. (c) 2010 Elsevier B.V. All rights reserved.

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.

Sub-grid drag models for horizontal cylinder arrays immersed in gas-particle multiphase flows

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

Sarkar, Avik; Sun, Xin; Sundaresan, Sankaran

2013-09-08

Immersed cylindrical tube arrays often are used as heat exchangers in gas-particle fluidized beds. In multiphase computational fluid dynamics (CFD) simulations of large fluidized beds, explicit resolution of small cylinders is computationally infeasible. Instead, the cylinder array may be viewed as an effective porous medium in coarse-grid simulations. The cylinders' influence on the suspension as a whole, manifested as an effective drag force, and on the relative motion between gas and particles, manifested as a correction to the gas-particle drag, must be modeled via suitable sub-grid constitutive relationships. In this work, highly resolved unit-cell simulations of flow around an arraymore » of horizontal cylinders, arranged in a staggered configuration, are filtered to construct sub-grid, or `filtered', drag models, which can be implemented in coarse-grid simulations. The force on the suspension exerted by the cylinders is comprised of, as expected, a buoyancy contribution, and a kinetic component analogous to fluid drag on a single cylinder. Furthermore, the introduction of tubes also is found to enhance segregation at the scale of the cylinder size, which, in turn, leads to a reduction in the filtered gas-particle drag.« less

A micro-reactor for preparing uniform molecularly imprinted polymer beads.

PubMed

Zourob, Mohammed; Mohr, Stephan; Mayes, Andrew G; Macaskill, Alexandra; Pérez-Moral, Natalia; Fielden, Peter R; Goddard, Nicholas J

2006-02-01

In this study, uniform spherical molecularly imprinted polymer beads were prepared via controlled suspension polymerization in a spiral-shaped microchannel using mineral oil and perfluorocarbon liquid as continuous phases. Monodisperse droplets containing the monomers, template, initiator, and porogenic solvent were introduced into the microchannel, and particles of uniform size were produced by subsequent UV polymerization, quickly and without wasting polymer materials. The droplet/particle size was varied by changing the flow conditions in the microfluidic device. The diameter of the resulting products typically had a coefficient of variation (CV) below 2%. The specific binding sites that were created during the imprinting process were analysed via radioligand binding analysis. The molecularly imprinted microspheres produced in the liquid perfluorocarbon continuous phase had a higher binding capacity compared with the particles produced in the mineral oil continuous phase, though it should be noted that the aim of this study was not to optimize or maximize imprinting performance, but rather to demonstrate broad applicability and compatibility with known MIP production methods. The successful imprinting against a model compound using two very different continuous phases (one requiring a surfactant to stabilize the droplets the other not) demonstrates the generality of this current simple approach.

Computer-aided diagnostic detection system of venous beading in retinal images

NASA Astrophysics Data System (ADS)

Yang, Ching-Wen; Ma, DyeJyun; Chao, ShuennChing; Wang, ChuinMu; Wen, Chia-Hsien; Lo, ChienShun; Chung, Pau-Choo; Chang, Chein-I.

2000-05-01

The detection of venous beading in retinal images provides an early sign of diabetic retinopathy and plays an important role as a preprocessing step in diagnosing ocular diseases. We present a computer-aided diagnostic system to automatically detect venous beading of blood vessels. It comprises of two modules, referred to as the blood vessel extraction module and the venus beading detection module. The former uses a bell-shaped Gaussian kernel with 12 azimuths to extract blood vessels while the latter applies a neural network-based shape cognitron to detect venous beading among the extracted blood vessels for diagnosis. Both modules are fully computer-automated. To evaluate the proposed system, 61 retinal images (32 beaded and 29 normal images) are used for performance evaluation.

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.

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

PubMed Central

Takita, Eiji; Kohda, Katsunori; Tomatsu, Hajime; Hanano, Shigeru; Moriya, Kanami; Hosouchi, Tsutomu; Sakurai, Nozomu; Suzuki, Hideyuki; Shinmyo, Atsuhiko; Shibata, Daisuke

2013-01-01

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation. PMID:23897972

Adhesion Molecule Expression and Function of Primary Endothelial Cells in Benign and Malignant Tissues Correlates with Proliferation

PubMed Central

Sievert, Wolfgang; Tapio, Soile; Breuninger, Stephanie; Gaipl, Udo; Andratschke, Nicolaus; Trott, Klaus-Rüdiger; Multhoff, Gabriele

2014-01-01

Background Comparative analysis of the cellular biology of the microvasculature in different tissues requires the availability of viable primary endothelial cells (ECs). This study describes a novel method to isolate primary ECs from healthy organs, repair blastemas and tumors as examples of non-proliferating and proliferating benign and malignant tissues and their functional characterization. Methodology/Principal Findings Single cell suspensions from hearts, lungs, repair blastemas and tumors were incubated consecutively with an anti-CD31 antibody and magnetic micro-beads, coupled to a derivative of biotin and streptavidin, respectively. Following magnetic bead separation, CD31-positive ECs were released by biotin-streptavidin competition. In the absence of micro-beads, ECs became adherent to plastic surfaces. ECs from proliferating repair blastemas and tumors were larger and exhibited higher expression densities of CD31, CD105 and CD102 compared to those from non-proliferating normal tissues such as heart and lung. The expression density of CD34 was particularly high in tumor-derived ECs, and that of CD54 and CD144 in ECs of repair blastemas. Functionally, ECs of non-proliferating and proliferating tissues differed in their capacity to form tubes in matrigel and to align under flow conditions. Conclusions/Significance This method provides a powerful tool to generate high yields of viable, primary ECs of different origins. The results suggest that an altered expression of adhesion molecules on ECs in proliferating tissues contribute to loss of EC function that might cause a chaotic tumor vasculature. PMID:24632811

Entropic depletion in colloidal suspensions and polymer liquids: Role of nanoparticle surface topography

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

Banerjee, Debapriya; Yang, Jian; Schweizer, Kenneth S.

2015-01-01

Here, we employ a hybrid Monte Carlo plus integral equation theory approach to study how dense fluids of small nanoparticles or polymer chains mediate entropic depletion interactions between topographically rough particles where all interaction potentials are hard core repulsion. The corrugated particle surfaces are composed of densely packed beads which present variable degrees of controlled topographic roughness and free volume associated with their geometric crevices. This pure entropy problem is characterized by competing ideal translational and (favorable and unfavorable) excess entropic contributions. Surface roughness generically reduces particle depletion aggregation relative to the smooth hard sphere case. However, the competition betweenmore » ideal and excess packing entropy effects in the bulk, near the particle surface and in the crevices, results in a non-monotonic variation of the particle-monomer packing correlation function as a function of the two dimensionless length scale ratios that quantify the effective surface roughness. As a result, the inter-particle potential of mean force (PMF), second virial coefficient, and spinodal miscibility volume fraction vary non-monotonically with the surface bead to monomer diameter and particle core to surface bead diameter ratios. A miscibility window is predicted corresponding to an optimum degree of surface roughness that completely destroys depletion attraction resulting in a repulsive PMF. Variation of the (dense) matrix packing fraction can enhance or suppress particle miscibility depending upon the amount of surface roughness. Connecting the monomers into polymer chains destabilizes the system via enhanced contact depletion attraction, but the non-monotonic variations with surface roughness metrics persist.« less

Two-dimensional patterning of colloidal crystals by means of lateral autocloning in edge-patterned cells

NASA Astrophysics Data System (ADS)

Emoto, Akira; Kamei, Tadayoshi; Shioda, Tatsutoshi; Kawatsuki, Nobuhiro; Ono, Hiroshi

2009-06-01

We report the experimental results of two-dimensional patterning of colloidal crystals using edge-patterned cells. Solvent evaporation of a colloidal suspension from the edge of the cell induces self-organized crystallization of spherical colloidal particles. From a reservoir of colloidal suspension in the cell, different colloidal suspensions are injected repetitively. An edge-patterned substrate is introduced into the cell as an upper substrate. As a result, different colloidal crystals are alternately stacked in the lateral direction according to the edge pattern. The characteristics of cloning formation are specifically showed including deformations from the original pattern. This two-dimensional patterning of three-dimensional colloidal crystals by means of lateral autocloning is promising for the development of photonic crystal arrays for use in optic and photonic devices.

An Attempt to Shorten Loading Time of Epirubicin into DC Beads® Using Vibration and a Sieve.

PubMed

Sonoda, Akinaga; Nitta, Norihisa; Yamamoto, Takefumi; Tomozawa, Yuki; Ohta, Shinichi; Watanabe, Shobu; Murata, Kiyoshi

2017-04-01

We investigated the possibility of shortening the time required for loading epirubicin into calibrated polyvinyl alcohol-based hydrogel beads (DC Beads ® ) to be used for transarterial chemoembolization. After separating the beads suspended in phosphate-buffered saline (PBS) solution by the use of a sieve (clearance 75 µm), epirubicin hydrochloride (EH) was loaded for 20, 30, or 60 s under vibration into DC beads. The EH loading rate into conventionally prepared (control) beads, i.e., beads loaded for 30 min without vibration, and vibration-loaded beads were calculated from the residual EH concentration in the bead-depleted EH solution. The amount of EH eluted from conventionally and vibration-loaded samples into a PBS solution (pH 7.0) was measured at 15 and 30 min and 1, 2, 6, 12, and 24 h. We also recorded the inhibitory effect of the PBS solution on the loading time. Using frozen sections, the EH load in the beads was evaluated visually under a fluorescence microscope. Spectrophotometry (495 nm) showed that the loading rate was 98.98 ± 0.34, 99.02 ± 0.32, and 99.50 ± 0.11 % with 20-, 30-, and 60-s vibration, respectively. The eluted rate was statistically similar between vibration- and statically loaded (control) beads. The PBS solution hampered EH loading into the beads. Visually, the distribution of EH in conventionally and vibration-loaded DC beads was similar. The use of vibration and the removal of PBS solution when epirubicin hydrochloride was loaded into DC beads dramatically shortened the loading time of epirubicin hydrochloride into DC beads.

Three-dimensional cultured glioma cell lines

NASA Technical Reports Server (NTRS)

Gonda, Steve R. (Inventor); Marley, Garry M. (Inventor)

1991-01-01

Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

Acoustic monitoring of a ball sinking in vibrated granular sediments

NASA Astrophysics Data System (ADS)

van den Wildenberg, Siet; Léopoldès, Julien; Tourin, Arnaud; Jia, Xiaoping

2017-06-01

We develop an ultrasound probing to investigate the dynamics of a high density ball sinking in 3D opaque dense granular suspensions under horizontal weak vibrations. We show that the motion of the ball in these horizontally vibrated glass bead packings saturated by water is consistent with the frictional rheology. The extracted stress-strain relation evidences an evolution of flow behaviour from frictional creep to inertial regimes. Our main finding is that weak external vibration primarily affects the yield stress and controls the depth of sinking via vibration-induced sliding at the grain contact. Also, we observe that the extracted rheological parameters depend on the size of the probing ball, suggesting thus a non-local rheology.

Sedimentation dynamics and equilibrium profiles in multicomponent mixtures of colloidal particles.

PubMed

Spruijt, E; Biesheuvel, P M

2014-02-19

In this paper we give a general theoretical framework that describes the sedimentation of multicomponent mixtures of particles with sizes ranging from molecules to macroscopic bodies. Both equilibrium sedimentation profiles and the dynamic process of settling, or its converse, creaming, are modeled. Equilibrium profiles are found to be in perfect agreement with experiments. Our model reconciles two apparently contradicting points of view about buoyancy, thereby resolving a long-lived paradox about the correct choice of the buoyant density. On the one hand, the buoyancy force follows necessarily from the suspension density, as it relates to the hydrostatic pressure gradient. On the other hand, sedimentation profiles of colloidal suspensions can be calculated directly using the fluid density as apparent buoyant density in colloidal systems in sedimentation-diffusion equilibrium (SDE) as a result of balancing gravitational and thermodynamic forces. Surprisingly, this balance also holds in multicomponent mixtures. This analysis resolves the ongoing debate of the correct choice of buoyant density (fluid or suspension): both approaches can be used in their own domain. We present calculations of equilibrium sedimentation profiles and dynamic sedimentation that show the consequences of these insights. In bidisperse mixtures of colloids, particles with a lower mass density than the homogeneous suspension will first cream and then settle, whereas particles with a suspension-matched mass density form transient, bimodal particle distributions during sedimentation, which disappear when equilibrium is reached. In all these cases, the centers of the distributions of the particles with the lowest mass density of the two, regardless of their actual mass, will be located in equilibrium above the so-called isopycnic point, a natural consequence of their hard-sphere interactions. We include these interactions using the Boublik-Mansoori-Carnahan-Starling-Leland (BMCSL) equation of state. Finally, we demonstrate that our model is not limited to hard spheres, by extending it to charged spherical particles, and to dumbbells, trimers and short chains of connected beads.

Multi-parameter analysis using photovoltaic cell-based optofluidic cytometer

PubMed Central

Yan, Chien-Shun; Wang, Yao-Nan

2016-01-01

A multi-parameter optofluidic cytometer based on two low-cost commercial photovoltaic cells and an avalanche photodetector is proposed. The optofluidic cytometer is fabricated on a polydimethylsiloxane (PDMS) substrate and is capable of detecting side scattered (SSC), extinction (EXT) and fluorescence (FL) signals simultaneously using a free-space light transmission technique without the need for on-chip optical waveguides. The feasibility of the proposed device is demonstrated by detecting fluorescent-labeled polystyrene beads with sizes of 3 μm, 5 μm and 10 μm, respectively, and label-free beads with a size of 7.26 μm. The detection experiments are performed using both single-bead population samples and mixed-bead population samples. The detection results obtained using the SSC/EXT, EXT/FL and SSC/FL signals are compared with those obtained using a commercial flow cytometer. It is shown that the optofluidic cytometer achieves a high detection accuracy for both single-bead population samples and mixed-bead population samples. Consequently, the proposed device provides a versatile, straightforward and low-cost solution for a wide variety of point-of-care (PoC) cytometry applications. PMID:27699122

A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules.

PubMed

Tsunehiro, Masaya; Meki, Yuma; Matsuoka, Kanako; Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2013-04-15

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 μL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Development of the rectal dosage form with silver-coated glass beads for local-action applications in lower sections of the gastrointestinal tract.

PubMed

Siczek, Krzysztof; Fichna, Jakub; Zatorski, Hubert; Karolewicz, Bożena; Klimek, Leszek; Owczarek, Artur

2018-03-01

Recent findings indicating the anti-inflammatory action of silver preparations through modulation of the gut microbiota and apoptosis of inflammatory cells predestine silver use in inflammatory bowel disease (IBD). The aim of our study was to validate the possibility of effective silver release from silver-coated glass beads for anti-inflammatory local application in the lower sections of the gastrointestinal (GI) tract. Silver-coated glass beads were prepared using magnetron method. Release of silver from the silver-coated glass bead surface was carried out in BIO-DIS reciprocating cylinder apparatus. Erosion of silver coating and indirect estimation of the silver release dynamics was assessed using scanning electron microscope. Rectal suppositories containing silver-coated glass beads were prepared using five different methods (M1-M5) and X-ray scanned for their composition. The XR microanalysis and the chemical composition analysis evidenced for a rapid (within 30 min) release of nearly 50% of silver from the coating of the glass beads, which remained stable up to 24 h of incubation. The most homogeneous distribution of beads in the entire volume of the suppository was obtained for formulation M5, where the molten base was poured into mold placed in an ice bath, and the beads were added after 10 s. Our study is the first to present the concept of enclosing silver-coated glass beads in the lipophilic suppository base to attenuate inflammation in the lower GI tract and promises efficient treatment with reduced side effects.

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.

Ergodicity, configurational entropy and free energy in pigment solutions and plant photosystems: influence of excited state lifetime.

PubMed

Jennings, Robert C; Zucchelli, Giuseppe

2014-01-01

We examine ergodicity and configurational entropy for a dilute pigment solution and for a suspension of plant photosystem particles in which both ground and excited state pigments are present. It is concluded that the pigment solution, due to the extreme brevity of the excited state lifetime, is non-ergodic and the configurational entropy approaches zero. Conversely, due to the rapid energy transfer among pigments, each photosystem is ergodic and the configurational entropy is positive. This decreases the free energy of the single photosystem pigment array by a small amount. On the other hand, the suspension of photosystems is non-ergodic and the configurational entropy approaches zero. The overall configurational entropy which, in principle, includes contributions from both the single excited photosystems and the suspension which contains excited photosystems, also approaches zero. Thus the configurational entropy upon photon absorption by either a pigment solution or a suspension of photosystem particles is approximately zero. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of Insulin Therapy using Hyper-insulinemic Normoglycemic Clamp on Inflammatory Response in Brain Dead Organ Donors.

PubMed

Aljiffry, M; Hassanain, M; Schricker, T; Shaheen, M; Nouh, T; Lattermann, R; Salman, A; Wykes, L; Metrakos, P

2016-05-01

Brain death is a major stress that is associated with a massive inflammatory response and systemic hyperglycemia. Severe inflammation leads to increased graft immunogenicity and risk of graft dysfunction; while acute hyperglycemia aggravates the inflammatory response and increases the risk of morbidity and mortality. Insulin therapy not only controls hyperglycemia but also suppresses inflammation. The present study is to investigate the anti-inflammatory properties and the normoglycemia maintenance of high dose insulin on brain dead organ donors. 15 brain dead organ donors were divided into 2 groups, insulin treated (n=6) and controls (n=9). Insulin was provided for a minimum of 6 h using the hyperinsulinemic normoglycemic clamp technique. The changes of serum cytokines, including IL-6, IL-10, IL-1β, IL-8, TNFα, TGFα and MCP-1, were measured by suspension bead array immunoassay and glucose by a glucose monitor. Compared to controls, insulin treated donors had a significant lower blood glucose 4.8 (4-6.9) vs. 9 (5.6-11.7) mmol/L, p<0.01); the net decreases of pro-inflammatory cytokines, such as IL-6 and MCP-1, and the net increase of anti-inflammatory cytokine, such as IL-10, reached significant level in insulin treated donors compared with those in controls. High dose insulin therapy decreases the concentrations of inflammatory cytokines in brain dead donors and preserves normoglycemia. High dose of insulin may have anti-inflammatory effects in brain dead organ donors and therefore, improve the quality of donor organs and potentially improve outcomes. © Georg Thieme Verlag KG Stuttgart · New York.

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

Size of the Dynamic Bead in Polymers

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

Agapov, Alexander L; Sokolov, Alexei P

2010-01-01

Presented analysis of neutron, mechanical, and MD simulation data available in the literature demonstrates that the dynamic bead size (the smallest subchain that still exhibits the Rouse-like dynamics) in most of the polymers is significantly larger than the traditionally defined Kuhn segment. Moreover, our analysis emphasizes that even the static bead size (e.g., chain statistics) disagrees with the Kuhn segment length. We demonstrate that the deficiency of the Kuhn segment definition is based on the assumption of a chain being completely extended inside a single bead. The analysis suggests that representation of a real polymer chain by the bead-and-spring modelmore » with a single parameter C cannot be correct. One needs more parameters to reflect correctly details of the chain structure in the bead-and-spring model.« less

The influence of gravity on the distribution of the deposit formed onto a substrate by sessile, hanging, and sandwiched hanging drop evaporation.

PubMed

Sandu, Ion; Fleaca, Claudiu Teodor

2011-06-15

The focus of the present article is the study of the influence of gravity on the particle deposition profiles on a solid substrate during the evaporation of sessile, hanging and sandwiched hanging drops of colloidal particle suspensions. For concentrations of nanoparticles in the colloidal solutions in the range 0.0001-1 wt.%, highly diluted suspensions will preferentially form rings while concentrated suspensions will preferentially form spots in both sessile and hanging drop evaporation. For intermediary concentrations, the particle deposition profiles will depend on the nanoparticle aggregation dynamics in the suspension during the evaporation process, gravity and on the detailed evaporation geometry. The evaporation of a drop of toluene/carbon nanoparticle suspension hanging from a pendant water drop will leave on the substrate a circular spot with no visible external ring. By contrast, a clear external ring is formed on the substrate by the sessile evaporation of a similar drop of suspension sandwiched between a water drop and the substrate. From the application viewpoint, these processes can be used to create preferential electrical conductive carbon networks and contacts for arrays of self-assembled nanostructures fabricated on solid substrates as well as on flexible polymeric substrates. Copyright © 2011 Elsevier Inc. All rights reserved.

Enhanced sub-micron colloidal particle separation with interdigitated microelectrode arrays using mixed AC/DC dielectrophoretic scheme.

PubMed

Swaminathan, Vikhram V; Shannon, Mark A; Bashir, Rashid

2015-04-01

Dielectrophoretic separation of particles finds a variety of applications in the capture of species such as cells, viruses, proteins, DNA from biological systems, as well as other organic and inorganic contaminants from water. The ability to capture particles is constrained by poor volumetric scaling of separation force with respect to particle diameter, as well as the weak penetration of electric fields in the media. In order to improve the separation of sub-micron colloids, we present a scheme based on multiple interdigitated electrode arrays under mixed AC/DC bias. The use of high frequency longitudinal AC bias breaks the shielding effects through electroosmotic micromixing to enhance electric fields through the electrolyte, while a transverse DC bias between the electrode arrays enables penetration of the separation force to capture particles from the bulk of the microchannel. We determine the favorable biasing conditions for field enhancement with the help of analytical models, and experimentally demonstrate the improved capture from sub-micron colloidal suspensions with the mixed AC/DC electrostatic excitation scheme over conventional AC-DEP methods.

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.

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

Sonoda, Akinaga, E-mail: akinagasonoda@yahoo.co.jp; Nitta, Norihisa; Yamamoto, Takefumi

PurposeWe investigated the possibility of shortening the time required for loading epirubicin into calibrated polyvinyl alcohol-based hydrogel beads (DC Beads{sup ®}) to be used for transarterial chemoembolization.MethodAfter separating the beads suspended in phosphate-buffered saline (PBS) solution by the use of a sieve (clearance 75 µm), epirubicin hydrochloride (EH) was loaded for 20, 30, or 60 s under vibration into DC beads. The EH loading rate into conventionally prepared (control) beads, i.e., beads loaded for 30 min without vibration, and vibration-loaded beads were calculated from the residual EH concentration in the bead-depleted EH solution. The amount of EH eluted from conventionally and vibration-loadedmore » samples into a PBS solution (pH 7.0) was measured at 15 and 30 min and 1, 2, 6, 12, and 24 h. We also recorded the inhibitory effect of the PBS solution on the loading time. Using frozen sections, the EH load in the beads was evaluated visually under a fluorescence microscope.ResultsSpectrophotometry (495 nm) showed that the loading rate was 98.98 ± 0.34, 99.02 ± 0.32, and 99.50 ± 0.11 % with 20-, 30-, and 60-s vibration, respectively. The eluted rate was statistically similar between vibration- and statically loaded (control) beads. The PBS solution hampered EH loading into the beads. Visually, the distribution of EH in conventionally and vibration-loaded DC beads was similar.DiscussionThe use of vibration and the removal of PBS solution when epirubicin hydrochloride was loaded into DC beads dramatically shortened the loading time of epirubicin hydrochloride into DC beads.« less

Phosphate uptake studies of cross-linked chitosan bead materials.

PubMed

Mahaninia, Mohammad H; Wilson, Lee D

2017-01-01

A systematic experimental study is reported that provides a molecular based understanding of cross-linked chitosan beads and their adsorption properties in aqueous solution containing phosphate dianion (HPO 4 2- ) species. Synthetically modified chitosan using epichlorohydrin and glutaraldehyde cross-linkers result in surface modified beads with variable hydrophile-lipophile character and tunable HPO 4 2- uptake properties. The kinetic and thermodynamic adsorption properties of cross-linked chitosan beads with HPO 4 2- species were studied in aqueous solution. Complementary structure and physicochemical characterization of chitosan beads via potentiometry, Raman spectroscopy, DSC, and dye adsorption measurements was carried out to establish structure-property relationships. The maximum uptake (Q m ) of bead systems with HPO 4 2- at equilibrium was 52.1mgg -1 ; whereas, kinetic uptake results for chitosan bead/phosphate systems are relatively rapid (0.111-0.113min -1 ) with an intraparticle diffusion rate-limiting step. The adsorption process follows a multi-step pathway involving inner- and outer-sphere complexes with significant changes in hydration. Phosphate uptake strongly depends on the composition and type of cross-linker used for preparation of chitosan beads. The adsorption isotherms and structural characterization of bead systems illustrate the role of surface charge, hydrophile-lipophile balance, adsorption site accessibility, and hydration properties of the chitosan bead surface. Copyright © 2016 Elsevier Inc. All rights reserved.

Hot embossed polyethylene through-hole chips for bead-based microfluidic devices.

PubMed

Chou, Jie; Du, Nan; Ou, Tina; Floriano, Pierre N; Christodoulides, Nicolaos; McDevitt, John T

2013-04-15

Over the past decade, there has been a growth of interest in the translation of microfluidic systems into real-world clinical practice, especially for use in point-of-care or near patient settings. While initial fabrication advances in microfluidics involved mainly the etching of silicon and glass, the economics of scaling of these materials is not amendable for point-of-care usage where single-test applications force cost considerations to be kept low and throughput high. As such, materials base more consistent with point-of-care needs is required. In this manuscript, the fabrication of a hot embossed, through-hole low-density polyethylene ensembles derived from an anisotropically etched silicon wafer is discussed. This semi-opaque polymer that can be easily sterilized and recycled provides low background noise for fluorescence measurements and yields more affordable cost than other thermoplastics commonly used for microfluidic applications such as cyclic olefin copolymer (COC). To fabrication through-hole microchips from this alternative material for microfluidics, a fabrication technique that uses a high-temperature, high-pressure resistant mold is described. This aluminum-based epoxy mold, serving as the positive master mold for embossing, is casted over etched arrays of pyramidal pits in a silicon wafer. Methods of surface treatment of the wafer prior to casting and PDMS casting of the epoxy are discussed to preserve the silicon wafer for future use. Changes in the thickness of polyethylene are observed for varying embossing temperatures. The methodology described herein can quickly fabricate 20 disposable, single use chips in less than 30 min with the ability to scale up 4 times by using multiple molds simultaneously. When coupled as a platform supporting porous bead sensors, as in the recently developed Programmable Bio-Nano-Chip, this bead chip system can achieve limits of detection, for the cardiac biomarker C-reactive protein, of 0.3 ng/mL, thereby demonstrating that the approach is compatible with high performance, real-world clinical measurements in the context of point-of-care testing. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Transparent arrays of silver nanowire rings driven by evaporation of sessile droplets

NASA Astrophysics Data System (ADS)

Wang, Xiaofeng; Kang, Giho; Seong, Baekhoon; Chae, Illkyeong; Teguh Yudistira, Hadi; Lee, Hyungdong; Kim, Hyunggun; Byun, Doyoung

2017-11-01

A coffee-ring pattern can be yielded on the three-phase contact line following evaporation of sessile droplets with suspended insoluble solutes, such as particles, DNA molecules, and mammalian cells. The formation of such coffee-ring, together with their suppression has been applied in printing and coating technologies. We present here an experimental study on the assembly of silver nanowires inside an evaporating droplet of a colloidal suspension. The effects of nanowire length and concentration on coffee-ring formation of the colloidal suspension were investigated. Several sizes of NWs with an aspect ratio between 50 and 1000 were systematically investigated to fabricate coffee-ring patterns. Larger droplets containing shorter nanowires formed clearer ring deposits after evaporation. An order-to-disorder transition of the nanowires’ alignment was found inside the rings. A printing technique with the evaporation process enabled fabrication of arrays of silver nanowire rings. We could manipulate the patterns silver nanowire rings, which might be applied to the transparent and flexible electrode.

Study of The Effect of Draw-bead Geometry on Stretch Flange Formability

NASA Astrophysics Data System (ADS)

Orlov, O. S.; Winkler, S. L.; Worswick, M. J.; Lloyd, D. J.; Finn, M. J.

2004-06-01

A fully instrumented stretch flange press equipped with a back-up punch and draw-beads near the specimen cutout area is simulated. The utilization of different draw-bead geometries is examined numerically to determine the restraining forces, strains and amount of damage generated in stretch flanges during forming. Simulations of the forming process are conducted for 1mm AA5182 sheets with circular cutouts. The damage evolution with the deformed specimens is investigated using the explicit dynamic finite element code, LS-DYNA, with a modified Gurson-based material model. It was found that double draw-beads can provide the same amount of restraining force as single draw-beads, but at reduced levels of damage.

Parental experiences with a hospital-based bead programme for children with congenital heart disease.

PubMed

Wilson, Valerie; Chando, Shingisai

2015-02-01

To present survey findings on parental experiences with a hospital-based bead programme for children with congenital heart disease. The Heart Beads programme commenced at a paediatric hospital in Australia in 2008. Children enrolled in the programme are awarded a distinctive bead for every procedure/treatment they have while in hospital. A previous evaluation study on the programme revealed that the beads are therapeutic for the child and parents; however, due to a small sample size, the results were representative of the experiences of a small number of families who participated in the programme. This was an evaluation study which employed a nonexperimental descriptive design. Surveys were mailed to all eligible families who enrolled in the programme. Data collection occurred between July-December 2012. Questions on parental experiences with the Heart Beads programme were divided into three categories: understanding, acknowledgement and quality. Descriptive statistics were obtained and analysed. One hundred and sixty-two mothers and 136 fathers responded to the survey. Heart Beads assisted mothers (83%) and fathers (80%) with understanding their child's condition and helped them with communication (mothers 80%, fathers 58%). The majority of fathers felt that their experience was acknowledged by nursing staff (64%) and medical staff (62%), while mothers indicated a higher response from nurses (76%) compared to medical staff (67%). Overall, parents rated the programme positively; however, there was some concern that children at times missed out on beads. Understanding how mothers and fathers experience the programme differently can guide staff in their communication with parents and inform future initiatives. The Heart Beads help nurses understand how parents are experiencing care and ways in which they can provide support and acknowledgement of the parent's experience. © 2014 John Wiley & Sons Ltd.

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

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.

Identification of Methylated Genes Associated with Aggressive Bladder Cancer

PubMed Central

Marsit, Carmen J.; Houseman, E. Andres; Christensen, Brock C.; Gagne, Luc; Wrensch, Margaret R.; Nelson, Heather H.; Wiemels, Joseph; Zheng, Shichun; Wiencke, John K.; Andrew, Angeline S.; Schned, Alan R.; Karagas, Margaret R.; Kelsey, Karl T.

2010-01-01

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment. PMID:20808801

Identification of methylated genes associated with aggressive bladder cancer.

PubMed

Marsit, Carmen J; Houseman, E Andres; Christensen, Brock C; Gagne, Luc; Wrensch, Margaret R; Nelson, Heather H; Wiemels, Joseph; Zheng, Shichun; Wiencke, John K; Andrew, Angeline S; Schned, Alan R; Karagas, Margaret R; Kelsey, Karl T

2010-08-23

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.

Using a laser source to measure the refractive index of glass beads and Debye theory analysis.

PubMed

Li, Shui-Yan; Qin, Shuang; Li, Da-Hai; Wang, Qiong-Hua

2015-11-20

Using a monochromatic laser beam to illuminate a homogeneous glass bead, some rainbows will appear around it. This paper concentrates on the study of the scattering intensity distribution and the method of measuring the refractive index for glass beads based on the Debye theory. It is found that the first rainbow due to the scattering superposition of backward light of the low-refractive-index glass beads can be explained approximately with the diffraction, the external reflection plus the one internal reflection, while the second rainbow of high-refractive-index glass beads is due to the contribution from the diffraction, the external reflection, the direct transmission, and the two internal reflections. The scattering intensity distribution is affected by the refractive index, the radius of the glass bead, and the incident beam width. The effects of the refractive index and the glass bead size on the first and second minimum deviation angle position are analyzed in this paper. The results of the measurements agree very well with the specifications.

Equalizer technology--Equal rights for disparate beads.

PubMed

Keidel, Eva-Maria; Ribitsch, Doris; Lottspeich, Friedrich

2010-06-01

One major limitation in proteomics is the detection and analysis of low-abundant proteins, i.e. in plasma. Several years ago, a technique to selectively enrich the relative concentration of low-abundant proteins was introduced by Boschetti and co-workers. It is based on a specific and saturable interaction of proteins to a high diversity of binding sites, realized by a hexapeptide library coupled to beads. This technology was commercialized as Equalizer beads or ProteoMiner. However, during application of ProteoMiner beads to plasma samples unexpected results questioned the proposed mode of action. Therefore, ProteoMiner beads were compared with chromatographic beads exhibiting completely different surface chemistry. Sepabeads FP-OD400 octadecyl, FP-DA400 diethylamine, FP-BU400 butyl, FP-HG400 hydroxyl and EXE056 epoxy were used. The results show that ProteoMiner or the different Sepabeads behave surprisingly similarly in the separation of complex protein mixtures. ProteoMiner beads interact with protein mixtures according to a general hydrophobic binding mechanism, where diversity in surface ligands plays only a negligible role.

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.

Note: A quartz cell with Pt single crystal bead electrode for electrochemical scanning tunneling microscope measurements.

PubMed

Xia, Zhigang; Wang, Jihao; Hou, Yubin; Lu, Qingyou

2014-09-01

In this paper, we provide and demonstrate a design of a unique cell with Pt single crystal bead electrode for electrochemical scanning tunneling microscope (ECSTM) measurements. The active metal Pt electrode can be protected from air contamination during the preparation process. The transparency of the cell allows the tip and bead to be aligned by direct observation. Based on this, a new and effective alignment method is introduced. The high-quality bead preparations through this new cell have been confirmed by the ECSTM images of Pt (111).

Single molecule actuation and detection on a lab-on-a-chip magnetoresistive platform

NASA Astrophysics Data System (ADS)

Chaves, R. C.; Bensimon, D.; Freitas, P. P.

2011-03-01

On-chip magnetic tweezers based on current loops were integrated with magnetoresistive sensors. Magnetic forces up to 1.0±0.3pN are produced to actuate on DNA anchored to the surface of a flow cell and labeled with micrometer-sized magnetic beads. The levitation of the beads stretches the immobilized DNA. The relative position of the magnetic beads is monitored using spin-valve sensors. A bead vertical displacement resolution of 60nm is derived for DNA molecular motor activity in a tweezer steady current regime.

Rhenium-coated glass beads for intracolonic administration attenuate TNBS-induced colitis in mice: Proof-of-Concept Study.

PubMed

Siczek, Krzysztof; Zatorski, Hubert; Pawlak, Wojciech; Fichna, Jakub

2015-01-01

In search for novel effective treatments in inflammatory bowel diseases, a new strategy employing glass beads coated with rhenium nanolayer has been developed and validated in the mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Briefly, mice were randomly divided into 5 experimental groups: control (vehicle alone, Group 1); control treated with rhenium-coated glass beads (Group 2); TNBS (Group 3); TNBS treated with rhenium-coated glass beads (Group 4); and TNBS treated with uncoated glass beads (Group 5). Mice from Group 2, 4 and 5 were treated with respective beads (once daily, 5 beads / animal, i.c.) between D3-D6 post-TNBS/vehicle and evaluation of colonic damage was performed on D7, based on macroscopic scoring and clinical parameters. Severe colonic inflammation developed in post-TNBS mice (Group 3) [P <0.001 vs. control (Group 1) for macroscopic score], which was significantly attenuated by treatment with rhenium-coated glass beads (Group 4) [P <0.01 vs. TNBS (Group 3), for macroscopic score]. Neither rhenium-coated glass beads had any effect in control animals (Group 2), nor uncoated glass beads influenced TNBS-induced colitis (Group 5). In conclusion, a novel and attractive strategy for the treatment of colonic inflammation has been proposed; therapy with rhenium-coated glass beads already proved effective in the mouse model of TNBS-induced colitis, now requires further characterization in clinical conditions.

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.

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.

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.

Optimal 3D culture of primary articular chondrocytes for use in the rotating wall vessel bioreactor.

PubMed

Mellor, Liliana F; Baker, Travis L; Brown, Raquel J; Catlin, Lindsey W; Oxford, Julia Thom

2014-08-01

Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology, but also maintain the gene expression characteristics of primary articular chondrocytes. Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 d. Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering.

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.

Design, characterisation and application of alginate-based encapsulated pig liver esterase.

PubMed

Pauly, Jan; Gröger, Harald; Patel, Anant V

2018-06-05

Encapsulation of hydrolases in biopolymer-based hydrogels often suffers from low activities and encapsulation efficiencies along with high leaching and unsatisfactory recycling properties. Exemplified for the encapsulation of pig liver esterase the coating of alginate and chitosan beads have been studied by creating various biopolymer hydrogel beads. Enzyme activity and encapsulation efficiency were notably enhanced by chitosan coating of alginate beads while leaching remained nearly unchanged. This was caused by the enzymatic reaction acidifying the matrix, which increased enzyme retention through enhanced electrostatic enzyme-alginate interaction but decreased activity through enzyme deactivation. A practical and ready-to-use method for visualising pH in beads during reaction by co-encapsulation of a conventional pH indicator was also found. Our method proves that pH control inside the beads can only be realised by buffering. The resulting beads provided a specific activity of 0.267 μmol ∙ min -1 ∙ mg -1 , effectiveness factor 0.88, encapsulation efficiency of 88%, 5% leaching and good recycling properties. This work will contribute towards better understanding and application of encapsulated hydrolases for enzymatic syntheses. Copyright © 2018 Elsevier B.V. All rights reserved.

Droplet-based microfluidic system to form and separate multicellular spheroids using magnetic nanoparticles.

PubMed

Yoon, Sungjun; Kim, Jeong Ah; Lee, Seung Hwan; Kim, Minsoo; Park, Tai Hyun

2013-04-21

The importance of creating a three-dimensional (3-D) multicellular spheroid has recently been gaining attention due to the limitations of monolayer cell culture to precisely mimic in vivo structure and cellular interactions. Due to this emerging interest, researchers have utilized new tools, such as microfluidic devices, that allow high-throughput and precise size control to produce multicellular spheroids. We have developed a droplet-based microfluidic system that can encapsulate both cells and magnetic nanoparticles within alginate beads to mimic the function of a multicellular tumor spheroid. Cells were entrapped within the alginate beads along with magnetic nanoparticles, and the beads of a relatively uniform size (diameters of 85% of the beads were 170-190 μm) were formed in the oil phase. These beads were passed through parallel streamlines of oil and culture medium, where the beads were magnetically transferred into the medium phase from the oil phase using an external magnetic force. This microfluidic chip eliminates additional steps for collecting the spheroids from the oil phase and transferring them to culture medium. Ultimately, the overall spheroid formation process can be achieved on a single microchip.

Liquid carry-over in an injection moulded all-polymer chip system for immiscible phase magnetic bead-based solid-phase extraction

NASA Astrophysics Data System (ADS)

Kistrup, Kasper; Skotte Sørensen, Karen; Wolff, Anders; Fougt Hansen, Mikkel

2015-04-01

We present an all-polymer, single-use microfluidic chip system produced by injection moulding and bonded by ultrasonic welding. Both techniques are compatible with low-cost industrial mass-production. The chip is produced for magnetic bead-based solid-phase extraction facilitated by immiscible phase filtration and features passive liquid filling and magnetic bead manipulation using an external magnet. In this work, we determine the system compatibility with various surfactants. Moreover, we quantify the volume of liquid co-transported with magnetic bead clusters from Milli-Q water or a lysis-binding buffer for nucleic acid extraction (0.1 (v/v)% Triton X-100 in 5 M guanidine hydrochloride). A linear relationship was found between the liquid carry-over and mass of magnetic beads used. Interestingly, similar average carry-overs of 1.74(8) nL/μg and 1.72(14) nL/μg were found for Milli-Q water and lysis-binding buffer, respectively.

To elute or not to elute in immunocapture bottom-up LC-MS.

PubMed

Levernæs, Maren Christin Stillesby; Broughton, Marianne Nordlund; Reubsaet, Léon; Halvorsen, Trine Grønhaug

2017-06-15

Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads. Two previously validated immunocapture based MS methods for the quantification of pro-gastrin releasing peptide (ProGRP) and human chorionic gonadotropin (hCG) were used as model systems. The tryptic peptide generation was shown to be protein dependent and influenced by protein folding and accessibility towards trypsin both on-beads and in the eluate. The elution of proteins bound to the beads was also shown to be incomplete. In addition, the on-beads digestion suffered from non-specific binding of the trypsin generated peptides. A combination of on-beads digestion and elution may be applied to improve both the quantitative (peak area of the signature peptides) and qualitative yield (number of missed cleavages, total number of identified peptides, coverage, signal intensity and number of zero missed cleavage peptides) of the target proteins. The quantitative yield of signature peptides was shown to be reproducible in all procedures tested. Copyright © 2017 Elsevier B.V. All rights reserved.

Electric-field-induced dielectrophoresis and heterogeneous aggregation in dilute suspensions of positively polarizable particles

NASA Astrophysics Data System (ADS)

Acrivos, Andreas; Qiu, Zhiyong; Markarian, Nikolai; Khusid, Boris

2002-11-01

We specified the conditions under which a dilute suspension of positively polarizable particles would undergo a heterogeneous aggregation in high-gradient strong AC fields and then examined experimentally and theoretically its kinetics [1]. Experiments were conducted on flowing dilute suspensions of heavy aluminum oxide spheres subjected to a high-gradient AC field (several kV/mm) such that the dielectrophoretic force acting on the particles was arranged in the plane perpendicular to the streamlines of the main flow. To reduce the gravitational settling of the particles, the electric chamber was kept slowly rotating around a horizontal axis. Following the application of a field, the particles were found to move towards both the high-voltage and grounded electrodes and to form arrays of "bristles" along their edges. The process was modeled by computing the motion of a single particle under the action of dielectrophoretic, viscous, and gravitational forces for negligibly small particle Reynolds numbers. The particle polarization required for the calculation of the dielectrophoretic force was measured in low-strength fields (several V/mm). The theoretical predictions for the kinetics of the particle accumulation on the electrodes were found to be in a reasonable agreement with experiment, although the interparticle interactions governed the formation of arrays of bristles. These bristles were formed in a two-step mechanism, which arose from the interplay of the dielectrophoretic force that confined the particles near the electrode edge and the dipolar interactions of nearby particles. The results of our studies provide the basic characteristics needed for the design and optimization of electro-hydrodynamic apparatuses. The work was supported by a NASA grant. The suspension characterization was conducted at the NJIT W.M. Keck Laboratory. 1. Z. Qiu, N. Markarian, B. Khusid, A. Acrivos, J. Apple. Phys., 92(5), 2002.

Observation and Kinematic Description of Long Actin Tracks Induced by Spherical Beads

PubMed Central

Kang, Hyeran; Perlmutter, David S.; Shenoy, Vivek B.; Tang, Jay X.

2010-01-01

We report an in vitro study comparing the growth of long actin tails induced by spherical beads coated with the verprolin central acidic domain of the polymerization enzyme N-WASP to that induced by Listeria monocytogenes in similar cellular extracts. The tracks behind the beads show characteristic differences in shape and curvature from those left by the bacteria, which have an elongated shape and a similar polymerization-inducing enzyme distributed only on the rear surface of the cell. The experimental tracks are simulated using a generalized kinematic model, which incorporates three modes of bead rotation with respect to the tail. The results show that the trajectories of spherical beads are mechanically deterministic rather than random, as suggested by stochastic models. Assessment of the bead rotation and its mechanistic basis offers insights into the biological function of actin-based motility. PMID:21044576

Hollow polydimethylsiloxane beads with a porous structure for cell encapsulation.

PubMed

Oh, Myeong-Jin; Ryu, Tae-Kyoung; Choi, S-W

2013-11-01

Based on a water-in-oil-in-water emulsion system, porous and hollow polydimethylsiloxane (PDMS) beads containing cells using a simple fluidic device with three flow channels are fabricated. Poly(ethylene glycol) (PEG) in the PDMS oil phase is served as a porogen for pore development. The feasibility of the porous PDMS beads prepared with different PEG concentrations (10, 20, and 30 wt%) for cell encapsulation in terms of pore size, protein diffusion, and cell proliferation inside the PDMS beads is evaluated. The PDMS beads prepared with PEG 30 wt% are exhibited a highly porous structure and facilitated fast diffusion of protein from the core domain to the outer phase, eventually leading to enhanced cell proliferation. The results clearly indicate that hollow PDMS beads with a porous structure could provide a favorable microenvironment for cell survival due to the large porous structure. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of diminished microbial contamination in handling of a novel daily disposable flat pack contact lens.

PubMed

Nomachi, Miya; Sakanishi, Kotaro; Ichijima, Hideji; Cavanagh, H Dwight

2013-05-01

To evaluate the efficacy of a novel daily disposable (DD) flat package in regard to microbial contamination on the anterior and posterior surfaces of a contact lens (CL) during handling of the lens for insertion. Four kinds of commercially available general blister-packed daily disposable contact lenses (DD CLs) as controls and a novel Magic 1-day Menicon Flat Pack as a test lens were used for this in vitro study. Lenses were removed from their packages using fingers coated with fluorescein 3 to 5 μm beads or an approximately 7×10(2) to 2×10(3) colony-forming unit (CFU)/mL Staphylococcus aureus suspension. The transfer of fluorescein beads to the surface of the lenses was then observed by fluorescence microscopy. Microbial contamination on the lenses was observed by light microscopy after a 2-day incubation period; and, the number of colonies isolated from the contaminated lenses was determined after 4 days of incubation. The number of fluorescein beads on the Magic lens was significantly less (p<0.05) than that of the general blister-packed control lenses. Adherence of microbial colonies was observed on both inner and outer surfaces of general blister-packed lenses, whereas no colony formation was found on the inner surface of the Magic lens, and the lowest bacterial adherence was observed for the Magic lens. The data demonstrated that placement of the Magic DD lens onto the eye is accompanied by diminished microbial contamination compared with general blister-packed DD CLs. Eye care professionals; however, should instruct patients to comply with intended use of DD CLs to prevent CL-associated microbial keratitis. In all cases, hand washing is mandated prelens insertion.

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

PubMed

Kubis, Hans-Peter; Scheibe, Renate J; Decker, Brigitte; Hufendiek, Karsten; Hanke, Nina; Gros, Gerolf; Meissner, Joachim D

2016-04-01

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation. © 2015 International Federation for Cell Biology.

Highly sensitive bacteria quantification using immunomagnetic separation and electrochemical detection of guanine-labeled secondary beads.

PubMed

Jayamohan, Harikrishnan; Gale, Bruce K; Minson, Bj; Lambert, Christopher J; Gordon, Neil; Sant, Himanshu J

2015-05-22

In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized secondary (polystyrene) beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead). While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 10⁸ guanine tags per secondary bead (7.5 x 10⁶ biotin-FITC per secondary bead, 20 guanines per oligonucleotide) bound to the target (E. coli). A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV) was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)3(2+)) as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3. We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in waste water effluent samples.

Oscillatory magnetic tweezers based on ferromagnetic beads and simple coaxial coils

NASA Astrophysics Data System (ADS)

Trepat, Xavier; Grabulosa, Mireia; Buscemi, Lara; Rico, Fèlix; Fabry, Ben; Fredberg, Jeffrey J.; Farré, Ramon

2003-09-01

We report the design and validation of simple magnetic tweezers for oscillating ferromagnetic beads in the piconewton and nanometer scales. The system is based on a single pair of coaxial coils operating in two sequential modes: permanent magnetization of the beads through a large and brief pulse of magnetic field and generation of magnetic gradients to produce uniaxial oscillatory forces. By using this two step method, the magnetic moment of the beads remains constant during measurements. Therefore, the applied force can be computed and varies linearly with the driving signal. No feedback control is required to produce well defined force oscillations over a wide bandwidth. The design of the coils was optimized to obtain high magnetic fields (280 mT) and gradients (2 T/m) with high homogeneity (5% variation) within the sample. The magnetic tweezers were implemented in an inverted optical microscope with a videomicroscopy-based multiparticle tracking system. The apparatus was validated with 4.5 μm magnetite beads obtaining forces up to ˜2 pN and subnanometer resolution. The applicability of the device includes microrheology of biopolymer and cell cytoplasm, molecular mechanics, and mechanotransduction in living cells.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

PubMed

Tibbe, Arjan G J; de Grooth, Bart G; Greve, Jan; Dolan, Gerald J; Rao, Chandra; Terstappen, Leon W M M

2002-03-01

Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension. Copyright 2002 Wiley-Liss, Inc.

Wax-incorporated emulsion gel beads of calcium pectinate for intragastric floating drug delivery.

PubMed

Sriamornsak, Pornsak; Asavapichayont, Panida; Nunthanid, Jurairat; Luangtana-Anan, Manee; Limmatvapirat, Sontaya; Piriyaprasarth, Suchada

2008-01-01

The purpose of this study was to prepare wax-incorporated pectin-based emulsion gel beads using a modified emulsion-gelation method. The waxes in pectin-olive oil mixtures containing a model drug, metronidazole, were hot-melted, homogenized and then extruded into calcium chloride solution. The beads formed were separated, washed with distilled water and dried for 12 h. The influence of various types and amounts of wax on floating and drug release behavior of emulsion gel beads of calcium pectinate was investigated. The drug-loaded gel beads were found to float on simulated gastric fluid if the sufficient amount of oil was used. Incorporation of wax into the emulsion gel beads affected the drug release. Water-soluble wax (i.e. polyethylene glycol) increased the drug release while other water-insoluble waxes (i.e. glyceryl monostearate, stearyl alcohol, carnauba wax, spermaceti wax and white wax) significantly retarded the drug release. Different waxes had a slight effect on the drug release. However, the increased amount of incorporated wax in the formulations significantly sustained the drug release while the beads remained floating. The results suggest that wax-incorporated emulsion gel beads could be used as a carrier for intragastric floating drug delivery.

Gas phase detection of explosives such as 2,4,6-trinitrotoluene by molecularly imprinted polymers.

PubMed

Bunte, Gudrun; Hürttlen, Jürgen; Pontius, Heike; Hartlieb, Kerstin; Krause, Horst

2007-05-15

Fast, reliable and inexpensive analytical techniques for trace detection of explosive components are in high demand. Our approach is to develop specific sensor coating materials based on molecularly imprinted polymers (MIPs). Despite the known inhibition of radical polymerisations by nitro groups and the known shrinkage of the polymer lattice during/after drying we were able to synthesize particulate MIPs by suspension polymerisation as well as thin MIP coatings by direct surface polymerisation on quartz crystal microbalances (QCM). The best method to purify the porous beads was Soxhlet extraction followed by supercritical carbon dioxide extraction (SFE with sc-CO2) at mild conditions (150 bar, 50 degrees C). At least a removal of >99.7% of the template was achieved. Performance tests of TNT imprinted polymer beads showed that acrylamide (AA) and more pronounced also methacrylic acid (MAA) possessed an enhanced adsorption tendency for gaseous TNT. An adsorption of 2,4-DNT, dinitrotoluene, by these MIPs was not detected. Using 2,4-DNT as template and methacrylamide, MAAM, a positive imprint effect for gaseous 2,4-DNT was achieved with no measurable cross-sensitivity for 2,4,6-TNT. The thin MIP coatings directly synthesized on the QCMs showed thicknesses of 20 to up to 500 nm. Preliminary screening experiments were performed for five different monomers and three different solvents (acetonitrile, chloroform and dimethylformamide). Best adsorption properties for TNT vapour until now showed a PAA-MIP synthesized with chloroform. Direct measurements of the mass attachment, respectively frequency decrease of the coated QCMs during vapour treatment showed a TNT-uptake of about 150 pg per microg MIP per hour. Results look worthy for further studies.

Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays.

PubMed

Tchou, Isabelle; Sabido, Odile; Lambert, Claude; Misery, Laurent; Garraud, Olivier; Genin, Christian

2003-03-03

Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a. These cells can capture a pathogen and then migrate and differentiate to a more mature stage. During this maturation process, dentritic cells express surface markers differentially. In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells. For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery. This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage. After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process. Both systems were equally efficient in terms of purification and yield. By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation. This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells. Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

Feasibility studies of oncornavirus production in microcarrier cultures.

PubMed

Manousos, M; Ahmed, M; Torchio, C; Wolff, J; Shibley, G; Stephens, R; Mayyasi, S

1980-06-01

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.

Trailing Shield For Welding On Pipes

NASA Technical Reports Server (NTRS)

Coby, John B., Jr.; Gangl, Kenneth J.

1991-01-01

Trailing shield ensures layer of inert gas covers hot, newly formed bead between two tubes or pipes joined by plasma arc welding. Inert gas protects weld bead from oxidation by air until cooler and less vulnerable to oxidation. Intended for use on nickel-base alloy pipes, on which weld beads remain hot enough to oxidize after primary inert-gas purge from welding-torch cup has passed.

Reproducibility, Reliability, and Validity of Fuchsin-Based Beads for the Evaluation of Masticatory Performance.

PubMed

Sánchez-Ayala, Alfonso; Farias-Neto, Arcelino; Vilanova, Larissa Soares Reis; Costa, Marina Abrantes; Paiva, Ana Clara Soares; Carreiro, Adriana da Fonte Porto; Mestriner-Junior, Wilson

2016-08-01

Rehabilitation of masticatory function is inherent to prosthodontics; however, despite the various techniques for evaluating oral comminution, the methodological suitability of these has not been completely studied. The aim of this study was to determine the reproducibility, reliability, and validity of a test food based on fuchsin beads for masticatory function assessment. Masticatory performance was evaluated in 20 dentate subjects (mean age, 23.3 years) using two kinds of test foods and methods: fuchsin beads and ultraviolet-visible spectrophotometry, and silicone cubes and multiple sieving as gold standard. Three examiners conducted five masticatory performance trials with each test food. Reproducibility of the results from both test foods was separately assessed using the intraclass correlation coefficient (ICC). Reliability and validity of fuchsin bead data were measured by comparing the average mean of absolute differences and the measurement means, respectively, regarding silicone cube data using the paired Student's t-test (α = 0.05). Intraexaminer and interexaminer ICC for the fuchsin bead values were 0.65 and 0.76 (p < 0.001), respectively; those for the silicone cubes values were 0.93 and 0.91 (p < 0.001), respectively. Reliability revealed intraexaminer (p < 0.001) and interexaminer (p < 0.05) differences between the average means of absolute differences of each test foods. Validity also showed differences between the measurement means of each test food (p < 0.001). Intra- and interexaminer reproducibility of the test food based on fuchsin beads for evaluation of masticatory performance were good and excellent, respectively; however, the reliability and validity were low, because fuchsin beads do not measure the grinding capacity of masticatory function as silicone cubes do; instead, this test food describes the crushing potential of teeth. Thus, the two kinds of test foods evaluate different properties of masticatory capacity, confirming fushsin beads as a useful tool for this purpose. © 2015 by the American College of Prosthodontists.

Hydrogel microparticles for biosensing

PubMed Central

Le Goff, Gaelle C.; Srinivas, Rathi L.; Hill, W. Adam; Doyle, Patrick S.

2015-01-01

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field. PMID:26594056

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.

Cellular manipulation and patterning using ferromagnetic nanowires

NASA Astrophysics Data System (ADS)

Hultgren, Anne

Ferromagnetic nanowires are demonstrated as an effective tool to apply forces to living cells. Both magnetic cell separations and the magnetic patterning of cells on a substrate will be accomplished through the use of cell-nanowire interactions as well as nanowire-magnetic field interactions. When introduced into cultures of NIH-3T3 cells, the nanowires are internalized by cells via the integrin-mediated adhesion pathway without inflicting any toxic effects on the cell cycle over the course of several days. In addition, the length of the nanowires was found to have an effect on the cell-nanowire interactions when the cells were dissociated from the tissue culture dish. To compare the effectiveness of the nanowires as a means of manipulating cells to the current technology which is based on superparamagnetic beads, magnetic cell separations were performed with electrodeposited Ni nanowires 350 nm in diameter and 5--35 mum long in field gradients of 80 T/m. Single-pass separations of NIH-3T3 cells bound to nanowires achieve up to 81% purity with 85% yield, a dramatic improvement over the 55% purity and 20% yield obtained with the beads. The yield for the separations were found to be dependent on the length of the nanowires, and was maximized when the length of the nanowires equaled the diameter of the cells. This dependence was exploited to perform a size-selective magnetic separation. Substrates containing arrays of micro-magnets, fabricated using photolithography, were placed in cell cultures. These micro-magnet arrays create regions of locally strong magnetic field gradients to trap nanowires in specific locations on the substrate. These substrates were used in conjunction with fluid flow and a weak, externally applied magnetic field to create and control patterns of cells bound to nanowires. Controlled isolation of heterogeneous pairs and groups of cells will enable the study of the biochemistry of cell-cell contacts.

Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing.

PubMed

Moon, Hui-Sung; Je, Kwanghwi; Min, Jae-Woong; Park, Donghyun; Han, Kyung-Yeon; Shin, Seung-Ho; Park, Woong-Yang; Yoo, Chang Eun; Kim, Shin-Hyun

2018-02-27

Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl -1 , which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.

Molecularly Imprinted Polymers Chitosan-Glutaraldehyde for Monosodium Glutamate

NASA Astrophysics Data System (ADS)

Mulyasuryani, Ani; Haryanto, Edi; Sulistyarti, Hermin; Rumhayati, Barlah

2018-01-01

Chitosan has been used as a functional monomer in the synthesis of molecularly imprinted polymers (MIP) for monosodium glutamate (MSG). MIP is made from a mixture of 5 g chitosan, 50 mg glutaraldehyde and 2 g MSG, MIP is formed as flakes and beads. MIPs are identified by the FTIR spectrum, SEM image and their adsorption capabilities. MIP flakes and beads have no structural differences if they are based on FTIR or SEM spectra, but MIP adsorption capacity of beads higher than flakes. Adsorption capacity of MIP flakes is 548 mg/g and MIP beads 627 mg/g.

Characterization of Buoyant Fluorescent Particles for Field Observations of Water Flows

PubMed Central

Tauro, Flavia; Aureli, Matteo; Porfiri, Maurizio; Grimaldi, Salvatore

2010-01-01

In this paper, the feasibility of off-the-shelf buoyant fluorescent microspheres as particle tracers in turbid water flows is investigated. Microspheres’ fluorescence intensity is experimentally measured and detected in placid aqueous suspensions of increasing concentrations of clay to simulate typical conditions occurring in natural drainage networks. Experiments are conducted in a broad range of clay concentrations and particle immersion depths by using photoconductive cells and image-based sensing technologies. Results obtained with both methodologies exhibit comparable trends and show that the considered particles are fairly detectable in critically turbid water flows. Further information on performance and integration of the studied microspheres in low-cost measurement instrumentation for field observations is obtained through experiments conducted in a custom built miniature water channel. This experimental characterization provides a first assessment of the feasibility of commercially available buoyant fluorescent beads in the analysis of high turbidity surface water flows. The proposed technology may serve as a minimally invasive sensing system for hazardous events, such as pollutant diffusion in natural streams and flash flooding due to extreme rainfall. PMID:22163540

Characterization of buoyant fluorescent particles for field observations of water flows.

PubMed

Tauro, Flavia; Aureli, Matteo; Porfiri, Maurizio; Grimaldi, Salvatore

2010-01-01

In this paper, the feasibility of off-the-shelf buoyant fluorescent microspheres as particle tracers in turbid water flows is investigated. Microspheres' fluorescence intensity is experimentally measured and detected in placid aqueous suspensions of increasing concentrations of clay to simulate typical conditions occurring in natural drainage networks. Experiments are conducted in a broad range of clay concentrations and particle immersion depths by using photoconductive cells and image-based sensing technologies. Results obtained with both methodologies exhibit comparable trends and show that the considered particles are fairly detectable in critically turbid water flows. Further information on performance and integration of the studied microspheres in low-cost measurement instrumentation for field observations is obtained through experiments conducted in a custom built miniature water channel. This experimental characterization provides a first assessment of the feasibility of commercially available buoyant fluorescent beads in the analysis of high turbidity surface water flows. The proposed technology may serve as a minimally invasive sensing system for hazardous events, such as pollutant diffusion in natural streams and flash flooding due to extreme rainfall.

Synthesis of a novel alginate-rubber joint immobilization strains H-1 and its application in removal of Pb (II) from aqueous solution

NASA Astrophysics Data System (ADS)

Li, Huidong; Huo, Kaili; Li, Xiaolei; Zhang, Lin; Yun, Yueqing; Song, Lei; Bai, Runying; Liu, Yuhong

2018-02-01

In this study, a novel alginate-rubber-strains immobilized beads (ARSIBs) was synthesized at the optimum conditions that the concentration of sodium alginate was 4%; the volume of bacterial suspension was 75%; the quality of rubber powder was 3.2%; the crosslinking time was 24 h by the orthogonal experiments. The optimum conditions for Pb (II) adsorption were 1.2% ARSIBs, 100 mg L-1 initial concentrations, pH 5 and 3 h contact time. The equilibrium data were well fitted by the Freundlich isotherm model. The biosorption process was nearly consistent with the pseudo-second-order model. Meanwhile, the biosorption mechanism could be that Pb (II) was adsorbed by the hydroxyl and carboxyl, finally precipitated with phosphate in the form of NaPb4(PO4)3, Pb5(PO4)3(OH) and Pb(H2PO4)2 based on the spectra of FTIR and XRD, respectively. In addition, the stability of ARSIBs was enhanced due to the addition to the rubber powder in the process of wastewater treatment.

A New 4D Imaging Method for Three-Phase Analogue Experiments in Volcanology (and Other Three-Phase Systems)

NASA Astrophysics Data System (ADS)

Oppenheimer, J.; Patel, K. B.; Lev, E.; Hillman, E. M. C.

2017-12-01

Bubbles and crystals suspended in magmas interact with each other on a small scale, which affects large-scale volcanic processes. Studying these interactions on relevant scales of time and space is a long-standing challenge. Therefore, the fundamental explanations for the behavior of bubble- and crystal-rich magmas are still largely speculative. Recent application of X-ray tomography to experiments with synthetic magmas has already improved our understanding of small-scale 4D (3D + time) phenomena. However, this technique has low imaging rates < 20 volumes per second (vps) and does not work well with analogues, making experiments costly and slow. We demonstrate a novel methodology for imaging bubble-particle interactions in analogue suspensions by utilizing Swept Confocally Aligned Planar Excitation (SCAPE) microscopy. This method based on laser-fluorescence has been used to image live biological processes at high speed and in 3D. It allows imaging rates of up to several hundred vps and image volumes up to 1 x 1 x 0.5 mm3, with a trade-off between speed and spatial resolution. We ran two sets of experiments with silicone oil and soda-lime glass beads of <50 µm diameter, contained within a vertical glass casing 50 x 5 x 4 mm3. We used two different bubble generation methods. In the first set of experiments, small air bubbles (< 1 mm) were introduced through a hole at the bottom of the sample and allowed to rise through a suspension with low-viscosity oil. We successfully imaged bubble rise and particle movements around the bubble. In the second set, bubbles were generated by mixing acetone into the suspension and decreasing the surface pressure to cause a phase change to gaseous acetone. This bubble generation method compared favorably with previous gum rosin-acetone experiments: they provided similar degassing behaviors, along with more control on suspension viscosity and optimal optical properties for laser transmission. Large volumes of suspended bubbles, however, interfered with the laser path. In this set, we were able to track bubble nucleation sites and nucleation rates in 4D. This promising technique allows the study of small-scale interactions in two- and three-phase systems, at high imaging rates and at low cost.

Manipulation of Superparamagnetic Beads on Patterned Exchange-Bias Layer Systems for Biosensing Applications.

PubMed

Ehresmann, Arno; Koch, Iris; Holzinger, Dennis

2015-11-13

A technology platform based on a remotely controlled and stepwise transport of an array arrangement of superparamagnetic beads (SPB) for efficient molecular uptake, delivery and accumulation in the context of highly specific and sensitive analyte molecule detection for the application in lab-on-a-chip devices is presented. The near-surface transport of SPBs is realized via the dynamic transformation of the SPBs' magnetic potential energy landscape above a magnetically stripe patterned Exchange-Bias (EB) thin film layer systems due to the application of sub-mT external magnetic field pulses. In this concept, the SPB velocity is dramatically influenced by the magnitude and gradient of the magnetic field landscape (MFL) above the magnetically stripe patterned EB substrate, the SPB to substrate distance, the magnetic properties of both the SPBs and the EB layer system, respectively, as well as by the properties of the external magnetic field pulses and the surrounding fluid. The focus of this review is laid on the specific MFL design in EB layer systems via light-ion bombardment induced magnetic patterning (IBMP). A numerical approach is introduced for the theoretical description of the MFL in comparison to experimental characterization via scanning Hall probe microscopy. The SPB transport mechanism will be outlined in terms of the dynamic interplay between the EB substrate's MFL and the pulse scheme of the external magnetic field.

Manipulation of Superparamagnetic Beads on Patterned Exchange-Bias Layer Systems for Biosensing Applications

PubMed Central

Ehresmann, Arno; Koch, Iris; Holzinger, Dennis

2015-01-01

A technology platform based on a remotely controlled and stepwise transport of an array arrangement of superparamagnetic beads (SPB) for efficient molecular uptake, delivery and accumulation in the context of highly specific and sensitive analyte molecule detection for the application in lab-on-a-chip devices is presented. The near-surface transport of SPBs is realized via the dynamic transformation of the SPBs’ magnetic potential energy landscape above a magnetically stripe patterned Exchange-Bias (EB) thin film layer systems due to the application of sub-mT external magnetic field pulses. In this concept, the SPB velocity is dramatically influenced by the magnitude and gradient of the magnetic field landscape (MFL) above the magnetically stripe patterned EB substrate, the SPB to substrate distance, the magnetic properties of both the SPBs and the EB layer system, respectively, as well as by the properties of the external magnetic field pulses and the surrounding fluid. The focus of this review is laid on the specific MFL design in EB layer systems via light-ion bombardment induced magnetic patterning (IBMP). A numerical approach is introduced for the theoretical description of the MFL in comparison to experimental characterization via scanning Hall probe microscopy. The SPB transport mechanism will be outlined in terms of the dynamic interplay between the EB substrate’s MFL and the pulse scheme of the external magnetic field. PMID:26580625

Analysis of particle in liquid using excitation-fluorescence spectral flow cytometer

NASA Astrophysics Data System (ADS)

Takenaka, Kei; Togashi, Shigenori

2018-01-01

We have developed a new flow cytometer that can measure the excitation-fluorescence spectra of a single particle. This system consists of a solution-transmitting unit and an optical unit. The solution-transmitting unit allows a sample containing particles to flow through the center of a flow cell by hydrodynamic focusing. The optical unit irradiates particles with dispersed white light (wavelength band: 400-650 nm) along the flow direction and measures their fluorescence spectra (wavelength band: 400-700 nm) using a spectroscopic photodetector array. The fluorescence spectrum of a particle changes with the shift of the wavelength of the excitation light. Using this system, the excitation-fluorescence spectra of a fluorescent particle were measured. Additionally, a homogenized tomato suspension and a homogenized spinach suspension were measured using the system. Measurement results show that it is possible to determine the components of vegetables by comparing measured fluorescence spectra of particles in a vegetable suspension.

Batch and continuous production of stable dense suspensions of drug nanoparticles in a wet stirred media mill

NASA Astrophysics Data System (ADS)

Afolabi, Afola we mi

One way to improve the bioavailability of poorly water-soluble drugs is to reduce particle size of drug crystals down to nanoscale via wet stirred media milling. An increase in total surface area per mass loading of the drug and specific surface area as well as reduced external mass transfer resistance allow a faster dissolution of the poorly-water soluble drug from nanocrystals. To prevent aggregation of nanoparticles, polymers and surfactants are dissolved in water acting as stabilizers via adsorption onto the drug crystals. In the last two decades, ample experimental data were generated in the area of wet stirred media milling for the production of drug nanoparticle suspensions. However, a fundamental scientific/engineering understanding of various aspects of this process is still lacking. These challenges include elucidation of the governing mechanism(s) during nanoparticle formation and physical stabilization of the nanosuspension with the use of polymers and surfactants (formulation parameters), understanding the impact of process parameters in the context of first-principle-based models, and production of truly nanosized drug particles (10-100 nm) with acceptable physical stability and minimal contamination with the media. Recirculation mode of milling operation, where the drug suspension in a holding tank continuously circulates through the stirred media mill, has been commonly used in lab, pilot, and commercial scales. Although the recirculation is continuous, the recirculation operation mode is overall a batch operation, requiring significant number of batches for a large-volume pharmaceutical product. Hence, development and investigation of a truly continuous process should offer significant advantages. To explain the impact of some of the processing parameters, stress intensity and stress number concepts were widely used in literature, which do not account for the effect of suspension viscosity explicitly. The impact of the processing parameters has not been explained in a predictive and reliable manner. In this dissertation, a comprehensive investigation of the production of Griseofulvin nanosuspensions in a wet stirred media mill operating in both the recirculation and continuous modes has been conducted to address the aforementioned fundamental challenges. Griseofulvin has been selected as a model poorly water-soluble BCS Class II drug. Impact of various formulation parameters such as stabilizer type and loading as well as processing parameters such as rotor speed, bead loading, bead size, suspension flow rate and drug loading was studied. A major novelty of the present contribution is that the impact of processing and formulation parameters has been analyzed and interpreted using a combined experimental-theoretical (microhydrodynamic model) approach. Such a comprehensive approach allowed us to intensify the process for the production of sub-100 nm drug particles, which could not be produced with top-down approaches in the literature so far. In addition, a multi-pass mode of continuous operation was developed and the so-called "Rehbinder effect", which has not been shown for the breakage of drug particles, was also elucidated. The dissertation work (1) indicated the need for a minimum polymeric stabilizer-to-drug ratio for proper stabilization of drug nanosuspensions as dictated by polymer adsorption and synergistic interactions between a polymeric stabilizer and a surfactant, (2) demonstrated the existence of an optimum polymer concentration from a breakage rate perspective in the presence of a surfactant, which results from the competing effects of viscous dampening and enhanced steric stabilization at higher polymer concentration, (3) developed fundamental understanding of the breakage dynamics-processing-formulation relationships and rationalized preparation of a single highly drug- loaded batch (20% or higher) instead of multiple dilute batches, (4) designed an intensified process for faster preparation of sub-100 nm particles with reduced specific energy consumption and media wear (i.e. minimal drug contamination), and (5) provided first evidence for the proof of Rehbinder effect during the milling of drugs. Not only do the polymers and surfactants allow proper physical stabilization of the nanoparticles in the suspensions, but they also do facilitate drug particle breakage. This dissertation also discusses applications of nanosuspensions and practical issues encountered during wet media milling.

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.

Two-bead polarizable water models combined with a two-bead multipole force field (TMFF) for coarse-grained simulation of proteins.

PubMed

Li, Min; Zhang, John Z H

2017-03-08

The development of polarizable water models at coarse-grained (CG) levels is of much importance to CG molecular dynamics simulations of large biomolecular systems. In this work, we combined the newly developed two-bead multipole force field (TMFF) for proteins with the two-bead polarizable water models to carry out CG molecular dynamics simulations for benchmark proteins. In our simulations, two different two-bead polarizable water models are employed, the RTPW model representing five water molecules by Riniker et al. and the LTPW model representing four water molecules. The LTPW model is developed in this study based on the Martini three-bead polarizable water model. Our simulation results showed that the combination of TMFF with the LTPW model significantly stabilizes the protein's native structure in CG simulations, while the use of the RTPW model gives better agreement with all-atom simulations in predicting the residue-level fluctuation dynamics. Overall, the TMFF coupled with the two-bead polarizable water models enables one to perform an efficient and reliable CG dynamics study of the structural and functional properties of large biomolecules.

Insights on novel particulate self-assembled drug delivery beads based on partial inclusion complexes between triglycerides and cyclodextrins.

PubMed

Aburahma, Mona Hassan

2016-09-01

Most of the newly designed drug molecules are lipophilic in nature and often encounter erratic absorption and low bioavailability after oral administration. Finding ways to enhance the absorption and bioavailability of these lipophilic drugs is one of the major challenges that face pharmaceutical industry nowadays. In view of that, the purpose of this review is to shed some light on a novel particulate self-assembling system named "beads" than can act as a safe carrier for delivering lipophilic drugs. The beads are prepared simply by mixing oils with cyclodextrin (CD) aqueous solution in mild conditions. A unique interaction between oil components and CD molecules occurs to form in situ surface-active complexes which are prerequisites for beads formation. This review mainly focuses on the fundamentals of beads preparation through reviewing present, yet scarce, literature. The key methods used for beads characterization are discussed in details. Also, the potential mechanisms by which beads increase the bioavailability of lipophilic drugs are illustrated. Finally, the related research areas that needs to be addressed in future for optimizing this promising delivery system are briefly outlined.

The correlation of methylation levels measured using Illumina 450K and EPIC BeadChips in blood samples

PubMed Central

Logue, Mark W; Smith, Alicia K; Wolf, Erika J; Maniates, Hannah; Stone, Annjanette; Schichman, Steven A; McGlinchey, Regina E; Milberg, William; Miller, Mark W

2017-01-01

Aim: We examined concordance of methylation levels across the Illumina Infinium HumanMethylation450 BeadChip and the Infinium MethylationEPIC BeadChip. Methods: We computed the correlation for 145 whole blood DNA samples at each of the 422,524 CpG sites measured by both chips. Results: The correlation at some sites was high (up to r = 0.95), but many sites had low correlation (55% had r < 0.20). The low correspondence between 450K and EPIC measured methylation values at many loci was largely due to the low variability in methylation values for the majority of the CpG sites in blood. Conclusion: Filtering out probes based on the observed correlation or low variability may increase reproducibility of BeadChip-based epidemiological studies. PMID:28809127

Control of microparticles packing density in a microfluidic channel for bead based immunoassays applications

NASA Astrophysics Data System (ADS)

Caballero-Robledo, Gabriel; Guevara-Pantoja, Pablo

2014-11-01

Bead based immunoassays in microfluidic devices have shown to greatly outperform conventional methods. But if functional point-of-care devices are to be developed, precise and reproducible control over the granulate packings inside microchannels is needed. In this work we study the efficiency of a nanoparticles magnetic trap previously developed by B. Teste et al. [Lab Chip 11, 4207 (2011)] when we vary the compaction of micrometric iron beads packed against a restriction inside a microfluidic channel. The packing density of the beads is finely and reproducibly changed by applying a vibrational protocol originally developed for macroscopic, dry granular systems. We find, counterintuitively, that the most compact and stable packings are up to four times less efficient in trapping nano particles than the loosest packings. This work has been supported by Conacyt, Mexico, under Grant No. 180873.

Construction of microscale structures in enclosed microfluidic networks by using a magnetic beads based method.

PubMed

Wang, Zhenyu; Zhang, Xiaojuan; Yang, Jun; Yang, Zhong; Wan, Xiaoping; Hu, Ning; Zheng, Xiaolin

2013-08-20

A large number of microscale structures have been used to elaborate flowing control or complex biological and chemical reaction on microfluidic chips. However, it is still inconvenient to fabricate microstructures with different heights (or depths) on the same substrate. These kinds of microstructures can be fabricated by using the photolithography and wet-etching method step by step, but involves time-consuming design and fabrication process, as well as complicated alignment of different masters. In addition, few existing methods can be used to perform fabrication within enclosed microfluidic networks. It is also difficult to change or remove existing microstructures within these networks. In this study, a magnetic-beads-based approach is presented to build microstructures in enclosed microfluidic networks. Electromagnetic field generated by microfabricated conducting wires (coils) is used to manipulate and trap magnetic beads on the bottom surface of a microchannel. These trapped beads are accumulated to form a microscale pile with desired shape, which can adjust liquid flow, dock cells, modify surface, and do some other things as those fabricated microstructures. Once the electromagnetic field is changed, trapped beads may form new shapes or be removed by a liquid flow. Besides being used in microfabrication, this magnetic-beads-based method can be used for novel microfluidic manipulation. It has been validated by forming microscale dam structure for cell docking and modified surface for cell patterning, as well as guiding the growth of neurons. Copyright © 2013 Elsevier B.V. All rights reserved.

Pre-packing of cost effective antibiotic cement beads for the treatment of traumatic osteomyelitis in the developing world - an in-vitro study based in Cambodia.

PubMed

Noor, S; Gilson, A; Kennedy, K; Swanson, A; Vanny, V; Mony, K; Chaudhry, T; Gollogly, J

2016-04-01

The developing world often lacks the resources to effectively treat the most serious injuries including osteomyelitis following open fractures or surgical fracture treatment. Antibiotic cement beads are a widely accepted method of delivering antibiotics locally to the infected area following trauma. This study is based in Cambodia, a low income country struggling to recover from a recent genocide. The study aims to test the effectiveness of locally made antibiotic beads and analyse their effectiveness after being gas sterilised, packaged and kept in storage Different antibiotic beads were manufactured locally using bone cement and tested against MRSA bacteria grown from a case of osteomyelitis. Each antibiotic was tested before and after a process of gas sterilisation as well as later being tested after storage in packaging up to 42 days. The gentamicin, vancomycin, amikacin and ceftriaxone beads all inhibited growth of the MRSA on the TSB and agar plates, both before and after gas sterilisation. All four antibiotics continued to show similar zones of inhibition after 42 days of storage. The results show significant promise to produce beads with locally obtainable ingredients in an austere environment and improve cost effectiveness by storing them in a sterilised condition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nanofibrous polymeric beads from aramid fibers for efficient bilirubin removal.

PubMed

Peng, Zihang; Yang, Ye; Luo, Jiyue; Nie, Chuanxiong; Ma, Lang; Cheng, Chong; Zhao, Changsheng

2016-08-16

Polymer based hemoperfusion has been developed as an effective therapy to remove the extra bilirubin from patients. However, the currently applied materials suffer from either low removal efficiency or poor blood compatibility. In this study, we report the development of a new class of nanofibrous absorbent that exhibited high bilirubin removal efficiency and good blood compatibility. The Kevlar nanofiber was prepared by dissolving micron-sized Kevlar fiber in proper solvent, and the beads were prepared by dropping Kevlar nanofiber solutions into ethanol. Owing to the nanofiborous structure of the Kevlar nanofiber, the beads displayed porous structures and large specific areas, which would facilitate the adsorption of toxins. In the adsorption test, it was noticed that the beads possessed an adsorption capacity higher than 40 mg g(-1) towards bilirubin. In plasma mimetic solutions, the beads still showed high bilirubin removal efficiency. Furthermore, after incorporating with carbon nanotubes, the beads were found to have increased adsorption capacity for human degradation waste. Moreover, the beads showed excellent blood compatibility in terms of a low hemolysis ratio, prolonged clotting times, suppressed coagulant activation, limited platelet activation, and inhibited blood related inflammatory activation. Additionally, the beads showed good compatibility with endothelial cells. In general, the Kevlar nanofiber beads, which integrated with high adsorption capacity, good blood compatibility and low cytotoxicity, may have great potential for hemoperfusion and some other applications in biomedical fields.

Genomic and transcriptomic predictors of triglyceride response to regular exercise

PubMed Central

Sarzynski, Mark A; Davidsen, Peter K; Sung, Yun Ju; Hesselink, Matthijs K C; Schrauwen, Patrick; Rice, Treva K; Rao, D C; Falciani, Francesco; Bouchard, Claude

2015-01-01

Aim We performed genome-wide and transcriptome-wide profiling to identify genes and single nucleotide polymorphisms (SNPs) associated with the response of triglycerides (TG) to exercise training. Methods Plasma TG levels were measured before and after a 20-week endurance training programme in 478 white participants from the HERITAGE Family Study. Illumina HumanCNV370-Quad v3.0 BeadChips were genotyped using the Illumina BeadStation 500GX platform. Affymetrix HG-U133+2 arrays were used to quantitate gene expression levels from baseline muscle biopsies of a subset of participants (N=52). Genome-wide association study (GWAS) analysis was performed using MERLIN, while transcriptomic predictor models were developed using the R-package GALGO. Results The GWAS results showed that eight SNPs were associated with TG training-response (ΔTG) at p<9.9×10−6, while another 31 SNPs showed p values <1×10−4. In multivariate regression models, the top 10 SNPs explained 32.0% of the variance in ΔTG, while conditional heritability analysis showed that four SNPs statistically accounted for all of the heritability of ΔTG. A molecular signature based on the baseline expression of 11 genes predicted 27% of ΔTG in HERITAGE, which was validated in an independent study. A composite SNP score based on the top four SNPs, each from the genomic and transcriptomic analyses, was the strongest predictor of ΔTG (R2=0.14, p=3.0×10−68). Conclusions Our results indicate that skeletal muscle transcript abundance at 11 genes and SNPs at a number of loci contribute to TG response to exercise training. Combining data from genomics and transcriptomics analyses identified a SNP-based gene signature that should be further tested in independent samples. PMID:26491034

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.

A method to track rotational motion for use in single-molecule biophysics.

PubMed

Lipfert, Jan; Kerssemakers, Jacob J W; Rojer, Maylon; Dekker, Nynke H

2011-10-01

The double helical nature of DNA links many cellular processes such as DNA replication, transcription, and repair to rotational motion and the accumulation of torsional strain. Magnetic tweezers (MTs) are a single-molecule technique that enables the application of precisely calibrated stretching forces to nucleic acid tethers and to control their rotational motion. However, conventional magnetic tweezers do not directly monitor rotation or measure torque. Here, we describe a method to directly measure rotational motion of particles in MT. The method relies on attaching small, non-magnetic beads to the magnetic beads to act as fiducial markers for rotational tracking. CCD images of the beads are analyzed with a tracking algorithm specifically designed to minimize crosstalk between translational and rotational motion: first, the in-plane center position of the magnetic bead is determined with a kernel-based tracker, while subsequently the height and rotation angle of the bead are determined via correlation-based algorithms. Evaluation of the tracking algorithm using both simulated images and recorded images of surface-immobilized beads demonstrates a rotational resolution of 0.1°, while maintaining a translational resolution of 1-2 nm. Example traces of the rotational fluctuations exhibited by DNA-tethered beads confined in magnetic potentials of varying stiffness demonstrate the robustness of the method and the potential for simultaneous tracking of multiple beads. Our rotation tracking algorithm enables the extension of MTs to magnetic torque tweezers (MTT) to directly measure the torque in single molecules. In addition, we envision uses of the algorithm in a range of biophysical measurements, including further extensions of MT, tethered particle motion, and optical trapping measurements.

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

Cell-based delivery of glucagon-like peptide-1 using encapsulated mesenchymal stem cells.

PubMed

Wallrapp, Christine; Thoenes, Eric; Thürmer, Frank; Jork, Anette; Kassem, Moustapha; Geigle, Peter

2013-01-01

Glucagon-like peptide-1 (GLP-1) CellBeads are cell-based implants for the sustained local delivery of bioactive factors. They consist of GLP-1 secreting mesenchymal stem cells encapsulated in a spherically shaped immuno-isolating alginate matrix. A highly standardized and reproducible encapsulation method is described for the manufacturing of homogeneous CellBeads. Viability and sustained secretion was shown for the recombinant GLP-1 and the cell endogenous bioactive factors like vascular endothelial growth factor, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor. Manufacturing and quality control is performed in compliance with good manufacturing practice and fulfils all regulatory requirements for human clinical use. GLP-1 CellBeads combine the neuro- and cardioprotective properties of both GLP-1 and mesenchymal stem cells. First promising results were obtained from preclinical studies and an ongoing safety trial in humans but further studies have to prove the overall potential of CellBead technology in cell-based regenerative medicine.

The effect of silver nanoparticles/graphene-coupled TiO2 beads photocatalyst on the photoconversion efficiency of photoelectrochemical hydrogen production

NASA Astrophysics Data System (ADS)

Ke, Chun-Ren; Guo, Jyun-Sheng; Su, Yen-Hsun; Ting, Jyh-Ming

2016-10-01

In this work, a novel configuration of the photoelectrochemical hydrogen production device is demonstrated. It is based on TiO2 beads as the primary photoanode material with the addition of a heterostructure of silver nanoparticles/graphene. The heterostructure not only caters to a great improvement in light harvesting efficiency (LHE) but also enhances the charge collection efficiency. For LHE, the optimized cell based on TiO2 beads/Ag/graphene shows a 47% gain as compared to the cell having a photoanode of commercial P25 TiO2 powders. For the charge collection efficiency, there is a pronounced improvement of an impressive value of 856%. The reason for the improvement in light absorption is attributed to either the light scattering of TiO2 beads or the surface plasmonic resonance on the Ag nanoparticles/graphene. The photoconversion efficiency (PCE) of the resulting cells is also presented and discussed. The PCE of the TiO2 beads/Ag/graphene cell is approximately 2.5 times than that of pure P25 cell.

Floating capsules containing alginate-based beads of salbutamol sulfate: In vitro-in vivo evaluations.

PubMed

Malakar, Jadupati; Datta, Prabir Kumar; Purakayastha, Saikat Das; Dey, Sanjay; Nayak, Amit Kumar

2014-03-01

The present study deals with the development and evaluations of stomach-specific floating capsules containing salbutamol sulfate-loaded oil-entrapped alginate-based beads. Salbutamol sulfate-loaded oil-entrapped beads were prepared and capsulated within hard gelatin capsules (size 1). The effects of HPMC K4M and potato starch weight masses on drug encapsulation efficiency (DEE) of beads and cumulative drug release at 10h (R10 h) from capsules was analyzed by 3(2) factorial design. The optimization results indicate increasing of DEE in the oil-entrapped beads and decreasing R10 h from capsules with increment of HPMC K4M and potato starch weight masses. The optimized formulation showed DEE of 70.02 ± 3.16% and R10 h of 56.96 ± 2.92%. These capsules showed floatation over 6h and sustained drug release over 10h in gastric pH (1.2). In vivo X-ray imaging study of optimized floating capsules in rabbits showed stomach-specific gastroretention over a prolonged period. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

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

In Situ Localized Growth of Ordered Metal Oxide Hollow Sphere Array on Microheater Platform for Sensitive, Ultra-Fast Gas Sensing.

PubMed

Rao, Ameya; Long, Hu; Harley-Trochimczyk, Anna; Pham, Thang; Zettl, Alex; Carraro, Carlo; Maboudian, Roya

2017-01-25

A simple and versatile strategy is presented for the localized on-chip synthesis of an ordered metal oxide hollow sphere array directly on a low power microheater platform to form a closely integrated miniaturized gas sensor. Selective microheater surface modification through fluorinated monolayer self-assembly and its subsequent microheater-induced thermal decomposition enables the position-controlled deposition of an ordered two-dimensional colloidal sphere array, which serves as a sacrificial template for metal oxide growth via homogeneous chemical precipitation; this strategy ensures control in both the morphology and placement of the sensing material on only the active heated area of the microheater platform, providing a major advantage over other methods of presynthesized nanomaterial integration via suspension coating or printing. A fabricated tin oxide hollow sphere-based sensor shows high sensitivity (6.5 ppb detection limit) and selectivity toward formaldehyde, and extremely fast response (1.8 s) and recovery (5.4 s) times. This flexible and scalable method can be used to fabricate high performance miniaturized gas sensors with a variety of hollow nanostructured metal oxides for a range of applications, including combining multiple metal oxides for superior sensitivity and tunable selectivity.

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).

Radar-based dynamic testing of the cable-suspended bridge crossing the Ebro River at Amposta, Spain

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

Gentile, Carmelo; Luzi, Guido

2014-05-27

Microwave remote sensing is the most recent experimental methodology suitable to the non-contact measurement of deflections on large structures, in static or dynamic conditions. After a brief description of the radar measurement system, the paper addresses the application of microwave remote sensing to ambient vibration testing of a cable-suspended bridge. The investigated bridge crosses the Ebro River at Amposta, Spain and consists of two steel stiffening trusses and a series of equally spaced steel floor beams; the main span is supported by inclined stay cables and two series of 8 suspension cables. The dynamic tests were performed in operational conditions,more » with the sensor being placed in two different positions so that the response of both the steel deck and the arrays of suspension elements was measured. The experimental investigation confirms the simplicity of use of the radar and the accuracy of the results provided by the microwave remote sensing as well as the issues often met in the clear localization of measurement points.« less

Development of a novel device to trap heavy metal cations: application of the specific interaction between heavy metal cation and mismatch DNA base pair.

PubMed

Torigoe, Hidetaka; Miyakawa, Yukako; Fukushi, Miyako; Ono, Akira; Kozasa, Tetsuo

2009-01-01

We have already found that Hg(II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4 degrees C. We have also found that Ag(I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4 degrees C. Using the specific interaction, we developed a novel device to trap each of Hg(II) and Ag(I) cation. The device is composed of 5'-biotinylated T-rich or C-rich DNA oligonucleotides, BIO-T20: 5'-Bio-T(20)-3' or BIO-C20: 5'-Bio-C(20)-3' (Bio is a biotin), immobilized on streptavidin-coated polystylene beads. When the BIO-T20-immobilized beads were added to a solution containing Hg(II) cation, and the beads trapping Hg(II) cation were collected by centrifugation, almost all of Hg(II) cation were removed from the solution. Also, when the BIO-C20-immobilized beads were added to a solution containing Ag(I) cation, and the beads trapping Ag(I) cation were collected by centrifugation, almost all of Ag(I) cation were removed from the solution. We conclude that, using the novel device developed in this study, Hg(II) and Ag(I) cation can be effectively removed from the solution.

Characterisation of bare and tannase-loaded calcium alginate beads by microscopic, thermogravimetric, FTIR and XRD analyses.

PubMed

Larosa, Claudio; Salerno, Marco; de Lima, Juliana Silva; Merijs Meri, Remo; da Silva, Milena Fernandes; de Carvalho, Luiz Bezerra; Converti, Attilio

2018-08-01

Incorporating enzymes into calcium alginate beads is an effective method to immobilise them and to preserve, at the same time, their catalytic activity. Sodium alginate was mixed with Aspergillus ficuum tannase in aqueous solution, and tannase-loaded calcium alginate beads were prepared using a simple droplet-based microfluidic system. Extensive experimental analysis was carried out to characterise the samples. Microscopic imaging revealed morphological differences between the surfaces of bare alginate matrix and tannase-loaded alginate beads. Thermal analysis allowed assessing the hydration contents of alginate and revealed the presence of tannase entrapped in the loaded beads, which was confirmed by vibrational spectroscopy. X-ray diffraction allowed us to conclude that alginate of tannase-loaded beads is not crystalline, which would make them suitable as carriers for possible controlled release. Moreover, they could be used in food applications to improve tea quality or clarify juices. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Wahyuni, Wulan Tri, E-mail: wulantriws@gmail.com; Department of Chemistry, FMIPA, Universitas Indonesia, Kampus UI Depok; Ivandini, Tribidasari A.

Biomolecule modified magnetic beads has been widely used in separation and sensing process. This study used streptavidin modified magnetic beads to immobilize biotin modified zanamivir. Biotin-streptavidin affinity facilitates immobilization of zanamivir on magnetic beads. Then interaction of zanamivir and neuraminidase was adopted as basic for enzyme detection. Detection of neuraminidase was performed at gold modified BDD using cyclic voltammetry technique. The measurement was carried out based on alteration of electrochemical signals of working electrode as neuraminidase response. The result showed that zanamivir was successfully immobilized on magnetic beads. The optimum amount of magnetic beads for zanamivir immobilization was 120 ug.more » Linear responses of neuraminidase were detected in concentration range of 0-15 mU. Detection limit (LOD) of measurement was 2.32 mU (R2 = 0.959) with precision as % RSD of 1.41%. Measurement of neuraminidase on magnetic beads could be also performed in the presence of mucin matrix. The linearity range was 0-8 mU with LOD of 0.64 mU (R2 = 0.950) and % RSD of 7.25%.« less

Automated Hybridization of X-ray Absorber Elements-A Path to Large Format Microcalorimeter Arrays

NASA Technical Reports Server (NTRS)

Moseley, S.; Kelley, R.; Allen, C.; Kilbourne, C.; Costen, N.; Miller, T.

2007-01-01

In the design of microcalorimeters, it is often desirable to produce the X-ray absorber separately from the detector element. In this case, the attachment of the absorber to the detector element with the required thermal and mechanical characteristics is a major challenge. In such arrays, the attachment has been done by hand. This process is not easily extended to the large format arrays required for future X- ray astronomy missions such as the New x-ray Telescope or NeXT. In this paper we present an automated process for attaching absorber tiles to the surface of a large-scale X-ray detector array. The absorbers are attached with stycast epoxy to a thermally isolating polymer structure made of SU-8. SU-8 is a negative epoxy based photo resist produced by Microchem. We describe the fabrication of the X-ray absorbers and their suspension on a handle die in an adhesive matrix. We describe the production process for the polymer isolators on the detector elements. We have developed a new process for the alignment, and simultaneous bonding of the absorber tiles to an entire detector array. This process uses equipment and techniques used in the flip-chip bonding industry and approaches developed in the fabrication of the XRS-2 instrument. XRS-2 was an X-ray spectrometer that was launched on the Suzaku telescope in July 10, 2005. We describe the process and show examples of sample arrays produced by this process. Arrays with up to 300 elements have been bonded. The present tests have used dummy absorbers made of Si. In future work, we will demonstrate bonding of HgTe absorbers.

Development of a novel colorimetric sensor based on alginate beads for monitoring rainbow trout spoilage.

PubMed

Majdinasab, Marjan; Hosseini, Seyed Mohammad Hashem; Sepidname, Marziyeh; Negahdarifar, Manizheh; Li, Peiwu

2018-05-01

Alginate is a non-toxic, renewable, and linear copolymer obtained from the brown algae Laminaria digitata that can be easily shaped into beads. Its good gel forming properties have made it useful for entrapping food and pharmaceutical ingredients. In this study, alginate beads were used in a novel application as a colorimetric sensor in food intelligent packaging. Colorimetric sensor was developed through entrapping red cabbage extract as a pH indicator in alginate beads. The pH indicator beads were used in rainbow trout packaging for monitoring fillets spoilage. Color change of beads during fish storage was measured using the CIELab method. The alginate bead colorimetric sensor is validated by measuring total volatile basic nitrogen (TVB-N) levels and microbial populations in fish samples. Moreover, peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) were evaluated during storage. Results indicated that increasing the bacterial population during storage and production of proteolytic enzymes resulted in protein degradation, accumulation of volatile amine compounds, increase in the pH and finally color change of alginate beads. The values of TVB-N, pH, PV and TBARS increased with time of storage. The results of TVB-N and microbial growth were in accordance with color change of beads and CIELab data. Therefore, the proposed system enjoys a high sensitivity to pH variations and is capable of monitoring the spoilage of fish or other protein-rich products through its wide range of color changes. The alginate beads containing the red cabbage extract can, thus, be used as a low-cost colorimetric sensor for intelligent packaging applications.

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.

Online measurement of bead geometry in GMAW-based additive manufacturing using passive vision

NASA Astrophysics Data System (ADS)

Xiong, Jun; Zhang, Guangjun

2013-11-01

Additive manufacturing based on gas metal arc welding is an advanced technique for depositing fully dense components with low cost. Despite this fact, techniques to achieve accurate control and automation of the process have not yet been perfectly developed. The online measurement of the deposited bead geometry is a key problem for reliable control. In this work a passive vision-sensing system, comprising two cameras and composite filtering techniques, was proposed for real-time detection of the bead height and width through deposition of thin walls. The nozzle to the top surface distance was monitored for eliminating accumulated height errors during the multi-layer deposition process. Various image processing algorithms were applied and discussed for extracting feature parameters. A calibration procedure was presented for the monitoring system. Validation experiments confirmed the effectiveness of the online measurement system for bead geometry in layered additive manufacturing.

Magnetic Enzymatic Platform for Organophosphate Pesticide Detection Using Boron-doped Diamond Electrodes.

PubMed

Pino, Flavio; Ivandini, Tribidasari A; Nakata, Kazuya; Fujishima, Akira; Merkoçi, Arben; Einaga, Yasuaki

2015-01-01

A simple and reliable enzymatic system for organophosporus pesticide detection was successfully developed, by exploiting the synergy between the magnetic beads collection capacity and the outstanding electrochemistry property of boron-doped diamond electrodes. The determination of an organophosphate pesticide, chlorpyrifos (CPF), was performed based on the inhibition system of the enzyme acetylcholinesterase bonded to magnetic beads through a biotin-streptavidin complex system. A better sensitivity was found for a system with magnetic beads in the concentration range of 10(-9) to 10(-5) M. The estimated limits of detection based on IC10 (10% acetylcholinesterase (AChE) inhibition) have been detected and optimized to be 5.7 × 10(-10) M CPF. Spiked samples of water of Yokohama (Japan) have been measured to validate the efficiency of the enzymatic system. The results suggested that the use of magnetic beads to immobilize biomolecules or biosensing agents is suitable to maintain the superiority of BDD electrodes.

Production of orthophosphate suspension fertilizers from wet-process acid

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

Jones, T.M.; Burnell, J.R.

1984-01-01

For many years, the Tennessee Valley Authority (TVA) has worked toward development of suspension fertilizers. TVA has two plants for production of base suspension fertilizers from wet-process orthophosphoric acid. One is a demonstration-scale plant where a 13-38-0 grade base suspension is produced by a three-stage ammoniation process. The other is a new batch-type pilot plant which is capable of producing high-grade base suspensions of various ratios and grades from wet-process acid. In this batch plant, suspensions and solutions can also be produced from solid intermediates.

Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes

PubMed Central

Oprenyeszk, Frederic; Sanchez, Christelle; Dubuc, Jean-Emile; Maquet, Véronique; Henrist, Catherine; Compère, Philippe; Henrotin, Yves

2015-01-01

This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects. PMID:26020773

Optical diamagnetic biosensor for immunocomplexes on beads

NASA Astrophysics Data System (ADS)

Norina, Svetlana B.

2000-12-01

In the present work, diamagnetic separation parameters for the porous beads are studied using optical video recording microscopy. The possible direct amount determination of single or double macromolecular layers immobilized in the meshes of the porous beads is demonstrated for the concentrations' range used in heterogenic immunotest and the affinity chromatography, where the direct rapid detection of ligands within sorbent particles is known to be the actual task. A gradient diamagnetic biosensor is described as suitable for rapid quantitative detection of single or double macromolecular layers in porous nonmagnetic beads. Measurements of capture traveling time or accumulation radius in gradient magnetic field have shown that it is possible to determine 0.20 mg/ml of macromolecular amount within several seconds. The portative devices were made on the base of the fabre optic technique to detect accumulation radius of collected beads in two gradient magnetic positions: diamagnetic and paramagnetic zones of magnetized wire with 55 μm in diameter and to registrate with a lot of fabre wires having 30 μm in diameters. The successive procedures of the present method can be described by: the obtaining of agarose immuno-beads, the incubation of beads with the ligand sample or the injection of sample through affinity mini-column, the submerging of the loaded beads into the glass cell containing Ni-wire or the narrow gap of magnetic poles; the computational obtaining of immuno- parameters; binding constants, accumulation radius. Several biotechnological applications of the biosensor are presented on sorbent beads, human lymphocytes.

Facile preparation and characterization of pH sensitive Mt/CMC nanocomposite hydrogel beads for propranolol controlled release.

PubMed

Farhadnejad, Hassan; Mortazavi, Seyed Alireza; Erfan, Mohammad; Darbasizadeh, Behzad; Motasadizadeh, Hamidreza; Fatahi, Yousef

2018-05-01

The main aim of the present study was to design pH-sensitive nanocomposite hydrogel beads, based on carboxymethyl cellulose (CMC) and montmorillonite (Mt)-propranolol (PPN) nanohybrid, and evaluate whether the prepared nanocomposite beads could potentially be used as oral drug delivery systems. PPN-as a model drug-was intercalated into the interlayer space of Mt clay mineral via the ion exchange procedure. The resultant nanohybrid (Mt-PPN) was applied to fabricate nanocomposite hydrogel beads by association with carboxymethyl cellulose. The characterization of test samples was performed using different techniques: X-Ray Diffraction (XRD), IR spectroscopy (FT-IR), thermal gravity analysis (TGA), and scanning electron microscopy (SEM). The drug encapsulation efficiency was evaluated by UV-vis spectroscopy, and was found to be high for Mt/CMC beads. In vitro drug release test was performed in the simulated gastrointestinal conditions to evaluate the efficiency of Mt-PPN/CMC nanocomposite beads as a controlled-release drug carrier. The drug release profiles indicated that the Mt-PPN/CMC nanocomposite beads had high stability against stomach acid and a sustained- and controlled-release profile for PPN under the simulated intestinal conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

Artocarpus heterophyllus L. seed starch-blended gellan gum mucoadhesive beads of metformin HCl.

PubMed

Nayak, Amit Kumar; Pal, Dilipkumar; Santra, Kousik

2014-04-01

Jackfruit (Artocarpus heterophyllus Lam., family: Moraceae) seed starch (JFSS)-gellan gum (GG) mucoadhesive beads containing metformin HCl were developed through ionotropic gelation technique. The effect of GG to JFSS ratio and CaCl2 concentration on the drug encapsulation efficiency (DEE, %) and cumulative drug release at 10h (R10h, %) was optimized and analyzed using response surface methodology based on 3(2) factorial design. The optimized JFSS-GG beads containing metformin HCl showed DEE of 92.67±4.46%, R10h of 61.30±2.37%, and mean diameter of 1.67±0.27 mm. The optimized beads showed pH-dependent swelling and mucoadhesivity with the goat intestinal mucosa. The in vitro drug release from all these JFSS-GG beads containing metformin HCl was followed zero-order pattern (R(2)=0.9907-0.9975) with super case-II transport mechanism over a period of 10 h. The beads were also characterized by SEM and FTIR. The optimized JFSS-GG beads containing metformin HCl exhibited significant hypoglycemic effect in alloxan-induced diabetic rats over prolonged period after oral administration. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions.

PubMed

Aoki, Kimiko; Tanaka, Hiroyuki; Kawahara, Takashi

2018-07-01

The standard method for personal identification and verification of urine samples in doping control is short tandem repeat (STR) analysis using nuclear DNA (nDNA). The DNA concentration of urine is very low and decreases under most conditions used for sample storage; therefore, the amount of DNA from cryopreserved urine samples may be insufficient for STR analysis. We aimed to establish a multiplexed assay for urine mitochondrial DNA typing containing only trace amounts of DNA, particularly for Japanese populations. A multiplexed suspension-array assay using oligo-tagged microspheres (Luminex MagPlex-TAG) was developed to measure C-stretch length in hypervariable region 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel. Based on these SNPs and the indel, the Japanese population can be classified into five major haplogroups (D4, B, M7a, A, D5). The assay was applied to DNA samples from urine cryopreserved for 1 - 1.5 years (n = 63) and fresh blood (n = 150). The assay with blood DNA enabled Japanese subjects to be categorized into 62 types, exhibiting a discriminatory power of 0.960. The detection limit for cryopreserved urine was 0.005 ng of nDNA. Profiling of blood and urine pairs revealed that 5 of 63 pairs showed different C-stretch patterns in HV1 or HV2. The assay described here yields valuable information in terms of the verification of urine sample sources employing only trace amounts of recovered DNA. However, blood cannot be used as a reference sample.

Controlled implant/soft tissue interaction by nanoscale surface modifications of 3D porous titanium implants.

PubMed

Rieger, Elisabeth; Dupret-Bories, Agnès; Salou, Laetitia; Metz-Boutigue, Marie-Helene; Layrolle, Pierre; Debry, Christian; Lavalle, Philippe; Vrana, Nihal Engin

2015-06-07

Porous titanium implants are widely employed in the orthopaedics field to ensure good bone fixation. Recently, the use of porous titanium implants has also been investigated in artificial larynx development in a clinical setting. Such uses necessitate a better understanding of the interaction of soft tissues with porous titanium structures. Moreover, surface treatments of titanium have been generally evaluated in planar structures, while the porous titanium implants have complex 3 dimensional (3D) architectures. In this study, the determining factors for soft tissue integration of 3D porous titanium implants were investigated as a function of surface treatments via quantification of the interaction of serum proteins and cells with single titanium microbeads (300-500 μm in diameter). Samples were either acid etched or nanostructured by anodization. When the samples are used in 3D configuration (porous titanium discs of 2 mm thickness) in vivo (in subcutis of rats for 2 weeks), a better integration was observed for both anodized and acid etched samples compared to the non-treated implants. If the implants were also pre-treated with rat serum before implantation, the integration was further facilitated. In order to understand the underlying reasons for this effect, human fibroblast cell culture tests under several conditions (directly on beads, beads in suspension, beads encapsulated in gelatin hydrogels) were conducted to mimic the different interactions of cells with Ti implants in vivo. Physical characterization showed that surface treatments increased hydrophilicity, protein adsorption and roughness. Surface treatments also resulted in improved adsorption of serum albumin which in turn facilitated the adsorption of other proteins such as apolipoprotein as quantified by protein sequencing. The cellular response to the beads showed considerable difference with respect to the cell culture configuration. When the titanium microbeads were entrapped in cell-laden gelatin hydrogels, significantly more cells migrated towards the acid etched beads. In conclusion, the nanoscale surface treatment of 3D porous titanium structures can modulate in vivo integration by the accumulative effect of the surface treatment on several physical factors such as protein adsorption, surface hydrophilicity and surface roughness. The improved protein adsorption capacity of the treated implants can be further exploited by a pre-treatment with autologous serum to render the implant surface more bioactive. Titanium microbeads are a good model system to observe these effects in a 3D microenvironment and provide a better representation of cellular responses in 3D.

Study the Effect of SiO2 Based Flux on Dilution in Submerged Arc Welding

NASA Astrophysics Data System (ADS)

kumar, Aditya; Maheshwari, Sachin

2017-08-01

This paper highlights the method for prediction of dilution in submerged arc welding (SAW). The most important factors of weld bead geometry are governed by the weld dilution which controls the chemical and mechanical properties. Submerged arc welding process is used generally due to its very easy control of process variables, good penetration, high weld quality, and smooth finish. Machining parameters, with suitable weld quality can be achieved with the different composition of the flux in the weld. In the present study Si02-Al2O3-CaO flux system was used. In SiO2 based flux NiO, MnO, MgO were mixed in various proportions. The paper investigates the relationship between the process parameters like voltage, % of flux constituents and dilution with the help of Taguchi’s method. The experiments were designed according to Taguchi L9 orthogonal array, while varying the voltage at two different levels in addition to alloying elements. Then the optimal results conditions were verified by confirmatory experiments.

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.

Carbohydrate gel beads as model probes for quantifying non-ionic and ionic contributions behind the swelling of delignified plant fibers.

PubMed

Karlsson, Rose-Marie Pernilla; Larsson, Per Tomas; Yu, Shun; Pendergraph, Samuel Allen; Pettersson, Torbjörn; Hellwig, Johannes; Wågberg, Lars

2018-06-01

Macroscopic beads of water-based gels consisting of uncharged and partially charged β-(1,4)-d-glucan polymers were developed to be used as a novel model material for studying the water induced swelling of the delignified plant fiber walls. The gel beads were prepared by drop-wise precipitation of solutions of dissolving grade fibers carboxymethylated to different degrees. The internal structure was analyzed using Solid State Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance and Small Angle X-ray Scattering showing that the internal structure could be considered a homogeneous, non-crystalline and molecularly dispersed polymer network. When beads with different charge densities were equilibrated with aqueous solutions of different ionic strengths and/or pH, the change in water uptake followed the trends expected for weak polyelectrolyte gels and the trends found for cellulose-rich fibers. When dried and subsequently immersed in water the beads also showed an irreversible loss of swelling depending on the charge and type of counter-ion which is commonly also found for cellulose-rich fibers. Taken all these results together it is clear that the model cellulose-based beads constitute an excellent tool for studying the fundamentals of swelling of cellulose rich plant fibers, aiding in the elucidation of the different molecular and supramolecular contributions to the swelling. Copyright © 2018 Elsevier Inc. All rights reserved.

Quercetin-imprinted chromatographic sorbents revisited: optimization of synthesis and rebinding protocols for application to natural resources.

PubMed

Pardo, Antonelle; Mespouille, Laetitia; Blankert, Bertrand; Trouillas, Patrick; Surin, Mathieu; Dubois, Philippe; Duez, Pierre

2014-10-17

Molecularly imprinted polymers (MIPs) based on quercetin and synthesized by either bulk, precipitation or suspension polymerization were characterized in terms of size and shape by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After a study of rebinding protocols, the optimal materials were evaluated as sorbents for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to confirm the presence of imprinted cavities and to assess their selectivity. Besides quercetin, other structurally related natural compounds, naringenin, daidzein and curcumin, were employed for selectivity tests of MIPs. Although rebinding protocols previously described for such MIPs are typically based on binding, washing and eluting methanol-based solutions, we show that this highly polar solvent leads to weak specific interactions (imprinting factor<1) and poor sorbent properties, most probably because of hydrogen-bonding interferences between the MIP and MeOH. Similar experiments performed in tetrahydrofuran yield to much more improved properties (imprinting factor>2.4). This calls for reviewing most of previously published data on quercetin-MIPs; in proper binding conditions, published MIPs may prove more performing than initially assessed. As expected, tested MIPs exhibited the highest selective rebinding towards quercetin template (imprinting effect, quercetin, 3.41; naringenin, 1.54; daidzein, 1.38; curcumin, 1.67); the differences in selectivity between quercetin analogues were explained by the ligand geometries and H-bonding patterns obtained from quantum-chemical calculations. The evaluation of MIPs under identical analytical conditions allowed investigating the effect of the production method on chromatographic performances. The MIPs in bead materials (for quercetin, peak width, 0.69; number of theoretical plates, 143; symmetry factor, 2.22) provided a significant improvement in chromatographic efficiency over the bulk materials (for quercetin, peak width, 1.25; number of theoretical plates, 115; symmetry factor, 2.92). Using the quercetin-beaded MIP as SPE sorbent, quercetin was selectively extracted from Allium cepa L. extract. The MIP developed in this work therefore appears highly promising for the enrichment and determination of quercetin in natural products. Copyright © 2014 Elsevier B.V. All rights reserved.

Single-Walled Carbon Nanotubes Probed with Insulator-Based Dielectrophoresis

PubMed Central

2017-01-01

Single-walled carbon nanotubes (SWNTs) offer unique electrical and optical properties. Common synthesis processes yield SWNTs with large length polydispersity (several tens of nanometers up to centimeters) and heterogeneous electrical and optical properties. Applications often require suitable selection and purification. Dielectrophoresis is one manipulation method for separating SWNTs based on dielectric properties and geometry. Here, we present a study of surfactant and single-stranded DNA-wrapped SWNTs suspended in aqueous solutions manipulated by insulator-based dielectrophoresis (iDEP). This method allows us to manipulate SWNTs with the help of arrays of insulating posts in a microfluidic device around which electric field gradients are created by the application of an electric potential to the extremities of the device. Semiconducting SWNTs were imaged during dielectrophoretic manipulation with fluorescence microscopy making use of their fluorescence emission in the near IR. We demonstrate SWNT trapping at low-frequency alternating current (AC) electric fields with applied potentials not exceeding 1000 V. Interestingly, suspended SWNTs showed both positive and negative dielectrophoresis, which we attribute to their ζ potential and the suspension properties. Such behavior agrees with common theoretical models for nanoparticle dielectrophoresis. We further show that the measured ζ potentials and suspension properties are in excellent agreement with a numerical model predicting the trapping locations in the iDEP device. This study is fundamental for the future application of low-frequency AC iDEP for technological applications of SWNTs. PMID:29131586

Mechanics of biomimetic systems propelled by actin comet tails

NASA Astrophysics Data System (ADS)

Kang, Hyeran; Tambe, Dhananjay; Shenoy, Vivek; Tang, Jay

2009-03-01

The motility of intracellular bacterial pathogens such as Listeria monocytogenes is driven by filamentous actin comet tails in a variety of trajectories. Here, we present the in vitro study on the actin-based movements using spherical beads of different sizes coated with VCA protein, a partial domain of N-Wasp, in platelet extracts. Long term two-dimensional trajectories of the spherical beads motility show characteristic difference than those observed for bacteria, which have both elongated shape and asymmetric expression of the polymerization inducing enzyme. The trajectories also vary sensitively with the bead size and shape. These results provide a useful test to our new analytical model including the rotation of the bead relative to the tail.

Dynamics of magnetic particles in cylindrical Halbach array: implications for magnetic cell separation and drug targeting.

PubMed

Babinec, Peter; Krafcík, Andrej; Babincová, Melánia; Rosenecker, Joseph

2010-08-01

Magnetic nanoparticles for therapy and diagnosis are at the leading edge of the rapidly developing field of bionanotechnology. In this study, we have theoretically studied motion of magnetic nano- as well as micro-particles in the field of cylindrical Halbach array of permanent magnets. Magnetic flux density was modeled as magnetostatic problem by finite element method and particle motion was described using system of ordinary differential equations--Newton law. Computations were done for nanoparticles Nanomag-D with radius 65 nm, which are often used in magnetic drug targeting, as well as microparticles DynaBeads-M280 with radius 1.4 microm, which can be used for magnetic separation. Analyzing snapshots of trajectories of hundred magnetite particles of each size in the water as well as in the air, we have found that optimally designed magnetic circuits of permanent magnets in quadrupolar Halbach array have substantially shorter capture time than simple blocks of permanent magnets commonly used in experiments, therefore, such a Halbach array may be useful as a potential source of magnetic field for magnetic separation and targeting of magnetic nanoparticles as well as microparticles for delivery of drugs, genes, and cells in various biomedical applications.

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.

77 FR 57112 - Notice of Inventory Completion: U.S. Department of Defense, Army, Fort Sill Museum, Lawton, OK

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-17

... beads, 1 bone bead, 2 fragments of animal bone, and 1 leather/cloth fragment. Based on examination, the burial dates to between 1869 and 1890. The skeleton was determined to be Native American based on... located in the bed of a ravine and covered with large flat stones. The archaeological evidence, including...

Dermal miconazole nitrate nanocrystals - formulation development, increased antifungal efficacy & skin penetration.

PubMed

Pyo, Sung Min; Hespeler, David; Keck, Cornelia M; Müller, Rainer H

2017-10-05

Miconazole nitrate nanosuspension was developed to increase its antifungal activity and dermal penetration. In addition, the nanosuspension was combined with the synergistic additive chlorhexidine digluconate. The production was performed by wet bead milling and both production and formulation parameters were optimized. A stabilizer screening revealed poloxamer 407 and Tween 80 both at 0.15% as the most effective stabilizers for miconazole nanosuspensions at 1.0%. The nanocrystals were incorporated into a hydroxypropyl cellulose gel base. Short-term stability (3months) of the nanocrystal bulk population could be shown at room temperature and fridge. Besides the stable bulk nanocrystals, some longitudinal crystal growth to needle like crystals occurred. The addition of ionic compounds as the chlorhexidine digluconate often destabilizes suspensions. Surprisingly here, the addition minimized the crystal growth. An underlying mechanism is proposed. An inhibition zone assay was performed using Candida albicans (ATCC ® 10231™). When comparing the nanocrystals in suspension and in gel to μm-sized miconazole nitrate formulations and two market products, the increase in inhibition zone diameter for the nanosuspension formulations was most pronounced in the chlorhexidine digluconate free formulations. These nanocrystal formulations were closely or similarly effective as the microsuspensions and the market products containing the synergistic chlorhexidine digluconate, showing the potential of the nanosuspension formulation. Nanosuspension performance was even further increased when chlorhexidine digluconate was added. Ex-vivo skin penetration studies on porcine ears revealed distinctly less remaining miconazole nitrate on the skin surface for nanocrystals (e.g., 76-86%) compared to market products (e.g. 94%). Also, penetration was increased e.g. in skin depth of 5-10μm from <1.0/1.7% to e.g. 3.3-6.2% for nanocrystals. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploiting automatic on-line renewable molecularly imprinted solid-phase extraction in lab-on-valve format as front end to liquid chromatography: application to the determination of riboflavin in foodstuffs.

PubMed

Oliveira, Hugo M; Segundo, Marcela A; Lima, José L F C; Miró, Manuel; Cerdà, Victor

2010-05-01

In the present work, it is proposed, for the first time, an on-line automatic renewable molecularly imprinted solid-phase extraction (MISPE) protocol for sample preparation prior to liquid chromatographic analysis. The automatic microscale procedure was based on the bead injection (BI) concept under the lab-on-valve (LOV) format, using a multisyringe burette as propulsion unit for handling solutions and suspensions. A high precision on handling the suspensions containing irregularly shaped molecularly imprinted polymer (MIP) particles was attained, enabling the use of commercial MIP as renewable sorbent. The features of the proposed BI-LOV manifold also allowed a strict control of the different steps within the extraction protocol, which are essential for promoting selective interactions in the cavities of the MIP. By using this on-line method, it was possible to extract and quantify riboflavin from different foodstuff samples in the range between 0.450 and 5.00 mg L(-1) after processing 1,000 microL of sample (infant milk, pig liver extract, and energy drink) without any prior treatment. For milk samples, LOD and LOQ values were 0.05 and 0.17 mg L(-1), respectively. The method was successfully applied to the analysis of two certified reference materials (NIST 1846 and BCR 487) with high precision (RSD < 5.5%). Considering the downscale and simplification of the sample preparation protocol and the simultaneous performance of extraction and chromatographic assays, a cost-effective and enhanced throughput (six determinations per hour) methodology for determination of riboflavin in foodstuff samples is deployed here.

Decoupling suspension controller based on magnetic flux feedback.

PubMed

Zhang, Wenqing; Li, Jie; Zhang, Kun; Cui, Peng

2013-01-01

The suspension module control system model has been established based on MIMO (multiple input and multiple output) state feedback linearization. We have completed decoupling between double suspension points, and the new decoupling method has been applied to CMS04 magnetic suspension vehicle in national mid-low-speed maglev experiment field of Tangshan city in China. Double suspension system model is very accurate for investigating stability property of maglev control system. When magnetic flux signal is taken back to the suspension control system, the suspension module's antijamming capacity for resisting suspension load variety has been proved. Also, the external force interference has been enhanced. As a result, the robustness and stability properties of double-electromagnet suspension control system have been enhanced.