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Sample records for magnetic bead microarray

  1. An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis

    PubMed Central

    Wang, Shuaiqin; Sun, Yujia; Liu, Yan; Xiang, Guangxin; Wang, Lei; Cheng, Jing; Liu, Peng

    2015-01-01

    We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-μl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, “amplicon-in-answer-out” operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis. PMID:25825617

  2. 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.

  3. 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.

  4. 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

  5. Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels.

    PubMed

    Liu, Lifen; Wu, Simin; Jing, Fengxiang; Zhou, Hongbo; Jia, Chunping; Li, Gang; Cong, Hui; Jin, Qinghui; Zhao, Jianlong

    2016-06-15

    In this study, we developed a multiplex immunoassay system that combines the suspension and planar microarray formats within a single layer of polydimethylsiloxane (PDMS) using soft lithography technology. The suspension format was based on the target proteins forming a sandwich structure between the magnetic beads and the quantum dot (QD) probes through specific antibody-antigen interactions. The planar microarray format was produced by fabricating an array of micro-wells in PDMS. Each micro-well was designed to trap a single microbead and eventually generated a microbead array within the PDMS chamber. The resultant bead-based on-chip assay could be used for simultaneously detecting three lung cancer biomarkers-carcinoembryonic antigen (CEA), fragments of cytokeratin 19 (CYFRA21-1) and neuron-specific enolase (NSE)-in 10 μl of human serum, with a wide linear dynamic range (1.03-111 ng/mL for CEA and CYFRA21-1; 9.26-1000 ng/ml for NSE) and a low detection limit (CEA: 0.19 ng/ml; CYFRA21-1: 0.97 ng/ml; NSE: 0.37 ng/ml; S/N=3). Our micro-well chip does not require complex e-beam lithography or the reactive ion etching process as with existing micro-well systems, which rely on expensive focused ion beam (FIB) milling or optical fiber bundles. Furthermore, the current approach is easy to operate without extra driving equipment such as pumps, and can make parallel detection for multiplexing with rapid binding kinetics, small reagent consumption and low cost. This work has demonstrated the importance of the successful application of on-chip multiplexing sandwich assays for the detection of biomarker proteins.

  6. Fully automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micro-polystyrene bead composites, 'Beads on Beads'.

    PubMed

    Matsunaga, Tadashi; Maeda, Yoshiaki; Yoshino, Tomoko; Takeyama, Haruko; Takahashi, Masaaki; Ginya, Harumi; Aasahina, Junko; Tajima, Hideji

    2007-08-06

    Magnetic beads have served as a conventional bioassay platform in biotechnology. In this study, a fully automated immunoassay was performed using novel nano- and microbead-composites constructed by assembling nano-magnetic beads onto polystyrene microbeads, designated 'Beads on Beads'. Nano-sized bacterial magnetic particles (BacMPs) displaying the immunoglobulin G (IgG)-binding domain of protein A (ZZ domain) were used for the construction of 'Beads on Beads' via the interaction of biotin-streptavidin. The efficient assembly of 'Beads on Beads' was performed by gradual addition of biotin-labeled BacMPs onto streptavidin-coated polystyrene microbeads. Approximately 2000 BacMPs were uniformly assembled on a single microbead without aggregation. The constructed 'Beads on Beads' were magnetized and separated from the suspension by using an automated magnetic separation system with a higher efficiency than BacMPs alone. Furthermore, fully automated detection of prostate-specific antigens was performed with the detection limit of 1.48 ng mL(-1). From this preliminary assay, it can be seen that 'Beads on Beads' could be a powerful tool in the development of high-throughput, fully automated multiplexed bioassays.

  7. SISCAPA peptide enrichment on magnetic beads using an in-line bead trap device.

    PubMed

    Anderson, N Leigh; Jackson, Angela; Smith, Derek; Hardie, Darryl; Borchers, Christoph; Pearson, Terry W

    2009-05-01

    A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-microm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of alpha(1)-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces.

  8. SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device*S⃞

    PubMed Central

    Anderson, N. Leigh; Jackson, Angela; Smith, Derek; Hardie, Darryl; Borchers, Christoph; Pearson, Terry W.

    2009-01-01

    A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-μm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of α1-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces. PMID:19196707

  9. Bubble cell for magnetic bead trapping in capillary electrophoresis.

    PubMed

    Gassner, Anne-Laure; Proczek, Gaëlle; Girault, Hubert H

    2011-12-01

    A bubble cell capillary classically used to extend the optical path length for UV-vis detection is employed here to trap magnetic beads. With this system, a large amount of beads can be captured without inducing a strong pressure drop, as it is the case with magnetic beads trapped in a standard capillary, thereby having less effect on the experimental conditions. Using numerical simulations and microscopic visualizations, the capture of beads inside a bubble cell was investigated with two magnet configurations. Pressure-driven and electro-osmotic flow velocities were measured for different amounts of protein-A-coated beads or C18-functionalized beads (RPC-18). Solid-phase extraction of a model antibody on protein-A beads and preconcentration of fluorescein on RPC-18 beads were performed as proof of concept experiments.

  10. Magnet polepiece design for uniform magnetic force on superparamagnetic beads.

    PubMed

    Fallesen, Todd; Hill, David B; Steen, Matthew; Macosko, Jed C; Bonin, Keith; Holzwarth, George

    2010-07-01

    Here we report construction of a simple electromagnet with novel polepieces which apply a spatially uniform force to superparamagnetic beads in an optical microscope. The wedge-shaped gap was designed to keep partial differential B(x)/ partial differential y constant and B large enough to saturate the bead. We achieved fields of 300-600 mT and constant gradients of 67 T/m over a sample space of 0.5x4 mm(2) in the focal plane of the microscope and 0.05 mm along the microscope optic axis. Within this space the maximum force on a 2.8 microm diameter Dynabead was 12 pN with a spatial variation of approximately 10%. Use of the magnet in a biophysical experiment is illustrated by showing that gliding microtubules propelled by the molecular motor kinesin can be stopped by the force of an attached magnetic bead.

  11. Biodesulfurization using Pseudomonas delafieldii in magnetic polyvinyl alcohol beads.

    PubMed

    Guobin, S; Jianmin, X; Chen, G; Huizhou, L; Jiayong, C

    2005-01-01

    To immobilize Pseudomonas delafieldii R-8 cells in magnetic polyvinyl alcohol (PVA) beads for biodesulfurization. Magnetic PVA beads were prepared by a freezing-thawing technique under liquid nitrogen. The beads have distinct super-paramagnetic properties and their saturation magnetization is 8.02 emu g(-1). The desulfurization rate of the immobilized cells could reach 40.2 mmol kg(-1) h(-1). Desulfurization patterns of dibenzothiophene in model oil with the immobilized and free cells were represented by the Michaelis-Menten equation. The Michaelis constant for both immobilized and free cells was 1.3 mmol l(-1). The cells immobilized in magnetic PVA beads could be stably stored and be repeatedly used over 12 times for biodesulfurization. The immobilized cells could be easily separated by magnetic field. Magnetic PVA beads are easy to prepare. The immobilization process in the paper is to increase the efficiency of cells and to decrease the cost of operations.

  12. Attomolar protein detection using a magnetic bead surface coverage assay.

    PubMed

    Tekin, H Cumhur; Cornaglia, Matteo; Gijs, Martin A M

    2013-03-21

    We demonstrate a microfluidic method for ultra-sensitive protein detection in serum. First, 'large' (2.8 μm) antibody-functionalized magnetic beads specifically capture antigen from a serum matrix under active microfluidic mixing. Subsequently, the large beads loaded with the antigens are gently exposed to a surface pattern of fixed 'small' (1.0 μm) antibody-coated magnetic beads. During the exposure, attractive magnetic bead dipole-dipole interactions improve the contact between the two bead types and help the antigen-antibody immunocomplex formation, while non-specific large bead adsorption is limited by exploiting viscous drag forces in the microfluidic channel on the small-bead pattern. This efficient antigen-antibody recognition and binding mechanism mimics a biological process of selective recognition of tissue molecules, like is the case when leukocytes roll and slow down on blood vessel walls by selectin-mediated adhesion. After exposure of the large beads to the pattern of small beads, the antigen concentration is detected by simply counting the number of surface pattern-bound large magnetic beads. The new technique allows detection of proteins down to the sub-zeptomole range. In particular, we demonstrate detection of only 200 molecules of Tumor Necrosis Factor-α (TNF-α) in a serum sample volume of 5 μL, corresponding to a concentration of 60 attomolar or 1 fg mL(-1).

  13. Biological magnetometry: torque on superparamagnetic beads in magnetic fields.

    PubMed

    van Oene, Maarten M; Dickinson, Laura E; Pedaci, Francesco; Köber, Mariana; Dulin, David; Lipfert, Jan; Dekker, Nynke H

    2015-05-29

    Superparamagnetic beads are widely used in biochemistry and single-molecule biophysics, but the nature of the anisotropy that enables the application of torques remains controversial. To quantitatively investigate the torques experienced by superparamagnetic particles, we use a biological motor to rotate beads in a magnetic field and demonstrate that the underlying potential is π periodic. In addition, we tether a bead to a single DNA molecule and show that the angular trap stiffness increases nonlinearly with magnetic field strength. Our results indicate that the superparamagnetic beads' anisotropy derives from a nonuniform intrabead distribution of superparamagnetic nanoparticles.

  14. Guided self-assembly of magnetic beads for biomedical applications

    NASA Astrophysics Data System (ADS)

    Gusenbauer, Markus; Nguyen, Ha; Reichel, Franz; Exl, Lukas; Bance, Simon; Fischbacher, Johann; Özelt, Harald; Kovacs, Alexander; Brandl, Martin; Schrefl, Thomas

    2014-02-01

    Micromagnetic beads are widely used in biomedical applications for cell separation, drug delivery, and hyperthermia cancer treatment. Here we propose to use self-organized magnetic bead structures which accumulate on fixed magnetic seeding points to isolate circulating tumor cells. The analysis of circulating tumor cells is an emerging tool for cancer biology research and clinical cancer management including the detection, diagnosis and monitoring of cancer. Microfluidic chips for isolating circulating tumor cells use either affinity, size or density capturing methods. We combine multiphysics simulation techniques to understand the microscopic behavior of magnetic beads interacting with soft magnetic accumulation points used in lab-on-chip technologies. Our proposed chip technology offers the possibility to combine affinity and size capturing with special antibody-coated bead arrangements using a magnetic gradient field created by Neodymium Iron Boron permanent magnets. The multiscale simulation environment combines magnetic field computation, fluid dynamics and discrete particle dynamics.

  15. Magnetic track array for efficient bead capture in microchannels.

    PubMed

    Abonnenc, Mélanie; Gassner, Anne-Laure; Morandini, Jacques; Josserand, Jacques; Girault, Hubert H

    2009-10-01

    Magnetism-based microsystems, as those dedicated to immunoaffinity separations or (bio)chemical reactions, take benefit of the large surface area-to-volume ratio provided by the immobilized magnetic beads, thus increasing the sensitivity of the analysis. As the sensitivity is directly linked to the efficiency of the magnetic bead capture, this paper presents a simple method to enhance the capture in a microchannel. Considering a microchannel surrounded by two rectangular permanent magnets of different length (L (m) = 2, 5, 10 mm) placed in attraction, it is shown that the amount of trapped beads is limited by the magnetic forces mainly located at the magnet edges. To overcome this limitation, a polyethylene terephthalate (PET) microchip with an integrated magnetic track array has been prototyped by laser photo-ablation. The magnetic force is therefore distributed all along the magnet length. It results in a multi-plug bead capture, observed by microscope imaging, with a magnetic force value locally enhanced. The relative amount of beads, and so the specific binding surface for further immunoassays, presents a significant increase of 300% for the largest magnets. The influence of the track geometry and relative permeability on the magnetic force was studied by numerical simulations, for the microchip operating with 2-mm-long magnets.

  16. Incorporation of pyrene in polypyrrole/polystyrene magnetic beads.

    PubMed

    Głowala, Paulina; Budniak, Adam; Krug, Pamela; Wysocka, Barbara; Berbeć, Sylwia; Dec, Robert; Dołęga, Izabela; Kacprzak, Kamil; Wojciechowski, Jarosław; Kawałko, Jakub; Kępka, Paweł; Kępińska, Daria; Kijewska, Krystyna; Mazur, Maciej

    2014-10-15

    Pyrene, a fluorescent dye, was incorporated into polystyrene particles coated with polypyrrole. The incorporation was achieved by treating the polypyrrole/polystyrene (PPy/PS) beads in a tetrahydrofuran (THF) solution of the pyrene fluorophore followed by rinsing with methanol. The polystyrene cores of the beads swell in THF, allowing penetration of pyrene molecules into the polystyrene structure. The addition of methanol causes contraction of the swollen polystyrene, which encapsulates the dye molecules inside the beads. It is shown that the polypyrrole coating is permeable with respect to both the dye and the solvent, allowing the transport of molecules between the polystyrene cores and the contacting solution. The polypyrrole adlayer can be used as a matrix for the incorporation of magnetic nanoparticles. Embedded particles provide magnetic functionality to the PPy/PS beads. It is demonstrated that the pyrene-loaded beads can be manipulated with an external magnetic field. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Plasma membrane isolation using immobilized concanavalin A magnetic beads.

    PubMed

    Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

    2012-01-01

    Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

  18. Configurational Statistics of Magnetic Bead Detection with Magnetoresistive Sensors.

    PubMed

    Henriksen, Anders Dahl; Ley, Mikkel Wennemoes Hvitfeld; Flyvbjerg, Henrik; Hansen, Mikkel Fougt

    2015-01-01

    Magnetic biosensors detect magnetic beads that, mediated by a target, have bound to a functionalized area. This area is often larger than the area of the sensor. Both the sign and magnitude of the average magnetic field experienced by the sensor from a magnetic bead depends on the location of the bead relative to the sensor. Consequently, the signal from multiple beads also depends on their locations. Thus, a given coverage of the functionalized area with magnetic beads does not result in a given detector response, except on the average, over many realizations of the same coverage. We present a systematic theoretical analysis of how this location-dependence affects the sensor response. The analysis is done for beads magnetized by a homogeneous in-plane magnetic field. We determine the expected value and standard deviation of the sensor response for a given coverage, as well as the accuracy and precision with which the coverage can be determined from a single sensor measurement. We show that statistical fluctuations between samples may reduce the sensitivity and dynamic range of a sensor significantly when the functionalized area is larger than the sensor area. Hence, the statistics of sampling is essential to sensor design. For illustration, we analyze three important published cases for which statistical fluctuations are dominant, significant, and insignificant, respectively.

  19. Configurational Statistics of Magnetic Bead Detection with Magnetoresistive Sensors

    PubMed Central

    Henriksen, Anders Dahl; Ley, Mikkel Wennemoes Hvitfeld; Flyvbjerg, Henrik; Hansen, Mikkel Fougt

    2015-01-01

    Magnetic biosensors detect magnetic beads that, mediated by a target, have bound to a functionalized area. This area is often larger than the area of the sensor. Both the sign and magnitude of the average magnetic field experienced by the sensor from a magnetic bead depends on the location of the bead relative to the sensor. Consequently, the signal from multiple beads also depends on their locations. Thus, a given coverage of the functionalized area with magnetic beads does not result in a given detector response, except on the average, over many realizations of the same coverage. We present a systematic theoretical analysis of how this location-dependence affects the sensor response. The analysis is done for beads magnetized by a homogeneous in-plane magnetic field. We determine the expected value and standard deviation of the sensor response for a given coverage, as well as the accuracy and precision with which the coverage can be determined from a single sensor measurement. We show that statistical fluctuations between samples may reduce the sensitivity and dynamic range of a sensor significantly when the functionalized area is larger than the sensor area. Hence, the statistics of sampling is essential to sensor design. For illustration, we analyze three important published cases for which statistical fluctuations are dominant, significant, and insignificant, respectively. PMID:26496495

  20. Programmable and automated bead-based microfluidics for versatile DNA microarrays under isothermal conditions.

    PubMed

    Penchovsky, Robert

    2013-06-21

    Advances in modern genomic research depend heavily on applications of various devices for automated high- or ultra-throughput arrays. Micro- and nanofluidics offer possibilities for miniaturization and integration of many different arrays onto a single device. Therefore, such devices are becoming a platform of choice for developing analytical instruments for modern biotechnology. This paper presents an implementation of a bead-based microfluidic platform for fully automated and programmable DNA microarrays. The devices are designed to work under isothermal conditions as DNA immobilization and hybridization transfer are performed under steady temperature using reversible pH alterations of reaction solutions. This offers the possibility for integration of more selection modules onto a single chip compared to maintaining a temperature gradient. This novel technology allows integration of many modules on a single reusable chip reducing the application cost. The method takes advantage of demonstrated high-speed DNA hybridization kinetics and denaturation on beads under flow conditions, high-fidelity of DNA hybridization, and small sample volumes are needed. The microfluidic devices are applied for a single nucleotide polymorphism analysis and DNA sequencing by synthesis without the need for fluorescent removal step. Apart from that, the microfluidic platform presented is applicable to many areas of modern biotechnology, including biosensor devices, DNA hybridization microarrays, molecular computation, on-chip nucleic acid selection, high-throughput screening of chemical libraries for drug discovery.

  1. 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.

  2. Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers.

    PubMed

    Blumenschein, Nicholas; Han, Daewoo; Steckl, Andrew J

    2015-09-04

    Magnetic beads with ~1.9 µm average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for the purpose of investigating phase change of those liquid segments. The magnetic beads were externally controlled using a magnet, allowing for the beads to bridge the air valve between the adjacent liquid segments. A hydrophobic coating was applied to the inner surface of the tube to enhance the separation between two liquid segments. The applied magnetic field formed an aggregate cluster of magnetic beads, capturing a certain liquid amount within the cluster that is referred to as carry-over volume. A fluorescent dye was added to one liquid segment, followed by a series of liquid transfers, which then changed the fluorescence intensity in the neighboring liquid segment. Based on the numerical analysis of the measured fluorescence intensity change, the carry-over volume per mass of magnetic beads has been found to be ~2 to 3 µl/mg. This small amount of liquid allowed for the use of comparatively small liquid segments of a couple hundred microliters, enhancing the feasibility of the device for a lab-in-tube approach. This technique of applying small compositional variation in a liquid volume was applied to analyzing the binary phase diagram between water and the surfactant C12E5 (pentaethylene glycol monododecyl ether), leading to quicker analysis with smaller sample volumes than conventional methods.

  3. Thermophysical and Magnetic Properties of Carbon Beads Containing Cobalt Nanocrystallites

    NASA Astrophysics Data System (ADS)

    Izydorzak, M.; Skumiel, A.; Leonowicz, M.; Kaczmarek-Klinowska, M.; Pomogailo, A. D.; Dzhardimalieva, G. I.

    2012-04-01

    Magnetic Co-beads were fabricated in the course of a three-step procedure comprising preparation of a metal-acrylamide complex, followed by frontal polymerization and finally pyrolysis of the polymer. The composites obtained were composed of cobalt nanocrystallites stabilized in a carbon matrix built of disordered graphite. The crystallite size, material morphology, fraction of the magnetic component, and thus the magnetic properties can be tailored by a proper choice of the processing variables. The samples were subjected to an alternating magnetic field of different strengths ( H = 0 to 5 kA · m-1) at a frequency of f = 500 kHz. From the calorimetric measurements, we concluded that the relaxation processes dominate in the heat generation mechanism for the beads pyrolyzed at 773 K. For the beads pyrolyzed at 1073 K, significant values of magnetic properties, such as the coercive force and remanence give substantial contribution to the energy losses for hysteresis. The specific absorption coefficient ( SAR) related to the cobalt mass unit for the 1073 K pyrolyzed beads {({SAR} = 1340 W \\cdot g^{-1 }_cobalt)} is in very good conformity with the results obtained by other authors. The effective density power loss, caused by eddy currents, can be neglected for heating processes applied in magnetic hyperthermia. The Co-beads can potentially be applied for hyperthermia treatment.

  4. Magnetically promoted rapid immunoreactions using functionalized fluorescent magnetic beads: a proof of principle.

    PubMed

    Sakamoto, Satoshi; Omagari, Kenshi; Kita, Yoshinori; Mochizuki, Yusuke; Naito, Yasuyuki; Kawata, Shintaro; Matsuda, Sachiko; Itano, Osamu; Jinno, Hiromitsu; Takeuchi, Hiroya; Yamaguchi, Yuki; Kitagawa, Yuko; Handa, Hiroshi

    2014-04-01

    Accurate detection and monitoring of disease-related biomarkers is important in understanding pathophysiology. We devised a rapid immunoreaction system that uses submicrometer polymer-coated fluorescent ferrite (FF) beads containing both ferrites (magnetic iron oxide) and fluorescent europium complexes. FF beads were prepared by encapsulation of hydrophobic europium complexes into the polymer layers of affinity magnetic beads using organic solvent. A sandwich immunoassay using magnetic collection of antibody-coated FF beads to a specific place was performed. Brain natriuretic peptide and prostate-specific antigen were selected as target detection antigens to demonstrate the feasibility of this approach. An immunohistochemical staining using magnetic collection of antibody-coated FF beads onto carcinoma cell samples was also performed. The sandwich immunoassays, taking advantage of the magnetic collection of antibody-coated FF beads, detected target antigens within 5 min of sample addition. Without magnetic collection, the sandwich immunoassay using antibody-coated FF beads required long times, similar to conventional immunoassays. Using the magnetic collection of antibody-coated FF beads, immunohistochemical staining enabled discrimination of carcinoma cells within 20 min. This proof of principle system demonstrates that immunoreactions involving the magnetic collection of antibody-coated FF beads allow acceleration of the antigen-antibody reaction. The simple magnetic collection of antibody-coated FF beads to a specific space enables rapid detection of disease-related biomarkers and identification of carcinoma cells.

  5. Magnetically-actuated, bead-enhanced silicon photonic immunosensor

    PubMed Central

    Valera, Enrique; McClellan, Melinda S.; Bailey, Ryan C.

    2015-01-01

    Magnetic actuation has been introduced to an optical immunosensor technology resulting in improvements in both rapidity and limit of detection for an assay quantitating low concentrations of a representative protein biomarker. For purposes of demonstration, an assay was designed for monocyte chemotactic protein 1 (MCP-1), a small cytokine which regulates migration and infiltration of monocytes and macrophages, and is an emerging biomarker for several diseases. The immunosensor is based on arrays of highly multiplexed silicon photonic microring resonators. A one-step sandwich immunoassay was performed and the signal was further enhanced through a tertiary recognition event between biotinylated tracer antibodies and streptavidin-coated magnetic beads. By integrating a magnet under the sensor chip, magnetic beads were rapidly directed towards the sensor surface resulting in improved assay performance metrics. Notably, the time required in the bead binding step was reduced by a factor of 11 (4 vs 45 min), leading to an overall decrease in assay time from 73 min to 32 min. The magnetically-actuated assay also lowered the limit of detection (LOD) for MCP-1 from 124 pg mL−1 down to 57 pg mL−1. In sum, the addition of magnetic actuation into bead-enhanced sandwich assays on a silicon photonic biosensor platform might facilitate improved detection of biomarkers in point-of-care diagnostics settings. PMID:26528374

  6. Fabrication of a microfluidic enzyme reactor utilizing magnetic beads.

    PubMed

    Liu, Xiaojun; Lo, Roger C; Gomez, Frank A

    2009-06-01

    An enzyme-catalyzed microfluidic assay using magnetic micro-beads is described. Here, diaphorase (DI) (E.C. 1.6.99) is covalently attached to the magnetic micro-beads (2.7 mum) and integrated into a short section of a microchip fabricated from PDMS. DI converts non-fluorescent resazurin to fluorescent resorufin in the presence of nicotinamide adenine dinucleotide phosphate (NADH). In this work, an embedded magnet holds the micro-beads in place within the microchannel while a solution of resazurin and NADH in buffer is flowed through the beads. Incorporation of the micro-beads into the microchannel requires only a few minutes and offers well-defined spatial resolution and reproducibility. At a flow rate of 41.2 microL/h, a stable state for the enzyme reaction in the microfluidic format was achieved within 50 s. The maximum conversion of the reaction was obtained at a concentration of 1.25 mM NADH. The reaction yield is affected by ZnCl(2) and at concentrations in excess of 90.0 mM, the activity of DI was almost double without ZnCl(2). At 5.2 mM potassium chloride, the activity of DI reached its maximum value. Overall, the conversion of resazurin in microfluidic format was more than twice than that in a batch assay.

  7. A rapid automatic processing platform for bead label-assisted microarray analysis: application for genetic hearing-loss mutation detection.

    PubMed

    Zhu, Jiang; Song, Xiumei; Xiang, Guangxin; Feng, Zhengde; Guo, Hongju; Mei, Danyang; Zhang, Guohao; Wang, Dong; Mitchelson, Keith; Xing, Wanli; Cheng, Jing

    2014-04-01

    Molecular diagnostics using microarrays are increasingly being used in clinical diagnosis because of their high throughput, sensitivity, and accuracy. However, standard microarray processing takes several hours and involves manual steps during hybridization, slide clean up, and imaging. Here we describe the development of an integrated platform that automates these individual steps as well as significantly shortens the processing time and improves reproducibility. The platform integrates such key elements as a microfluidic chip, flow control system, temperature control system, imaging system, and automated analysis of clinical results. Bead labeling of microarray signals required a simple imaging system and allowed continuous monitoring of the microarray processing. To demonstrate utility, the automated platform was used to genotype hereditary hearing-loss gene mutations. Compared with conventional microarray processing procedures, the platform increases the efficiency and reproducibility of hybridization, speeding microarray processing through to result analysis. The platform also continuously monitors the microarray signals, which can be used to facilitate optimization of microarray processing conditions. In addition, the modular design of the platform lends itself to development of simultaneous processing of multiple microfluidic chips. We believe the novel features of the platform will benefit its use in clinical settings in which fast, low-complexity molecular genetic testing is required.

  8. Magnetic Bead Actuation of Saccular Hair Cells

    NASA Astrophysics Data System (ADS)

    Rowland, David; Ramunno-Johnson, Damien; Lee, Jae-Hyun; Cheon, Jinwoo; Bozovic, Dolores

    2011-11-01

    When decoupled from the overlying membrane, hair bundles of the amphibian sacculus exhibit spontaneous oscillation. To explore the dynamics of this innate motility without an imposed external load, we recorded their oscillations with a high-speed CMOS camera, and applied mechanical manipulation that minimally alters the geometry of an individual hair bundle. We present a technique that utilizes micron-sized magnetic particles to actuate the stereociliary bundle with a magnetized probe. Quasi-steady-state displacements were imposed on freely oscillating bundles. Our data indicate that deflection of the bundle affects both the frequency and the amplitude of the oscillations, with a crossing of the bifurcation that is dependent on the direction and speed of the applied offset.

  9. Analysis of liquid bead microarray antibody assay data for epidemiologic studies of pathogen-cancer associations.

    PubMed

    Colombara, Danny V; Hughes, James P; Burnett-Hartman, Andrea N; Hawes, Stephen E; Galloway, Denise A; Schwartz, Stephen M; Bostick, Roberd M; Potter, John D; Manhart, Lisa E

    2015-10-01

    Liquid bead microarray antibody (LBMA) assays are used to assess pathogen-cancer associations. However, studies analyze LBMA data differently, limiting comparability. We generated 10,000 Monte Carlo-type simulations of log-normal antibody distributions (exposure) with 200 cases and 200 controls (outcome). We estimated type I error rates, statistical power, and bias associated with t-tests, logistic regression with a linear exposure and with the exposure dichotomized at 200 units, 400 units, the mean among controls plus two standard deviations, and the value corresponding to the optimal sensitivity and specificity. We also applied these models, and data visualizations (kernel density plots, receiver operating characteristic (ROC) curves, predicted probability plots, and Q-Q plots), to two empirical datasets to assess the consistency of the exposure-outcome relationship. All strategies had acceptable type I error rates (0.03 ≤ P ≤ 0.048), except for the dichotomization according to optimal sensitivity and specificity, which had a type I error rate of 0.27. Among the remaining methods, logistic regression with a linear predictor (Power=1.00) and t-tests (Power=1.00) had the highest power to detect a mean difference of 1.0 MFI (median fluorescence intensity) on the log scale and were unbiased. Dichotomization methods upwardly biased the risk estimates. These results indicate that logistic regression with linear predictors and unpaired t-tests are superior to logistic regression with dichotomized predictors for assessing disease associations with LBMA data. Logistic regression with continuous linear predictors and t-tests are preferable to commonly used LBMA dichotomization methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Polymerase chain reaction system using magnetic beads for analyzing a sample that includes nucleic acid

    DOEpatents

    Nasarabadi, Shanavaz [Livermore, CA

    2011-01-11

    A polymerase chain reaction system for analyzing a sample containing nucleic acid includes providing magnetic beads; providing a flow channel having a polymerase chain reaction chamber, a pre polymerase chain reaction magnet position adjacent the polymerase chain reaction chamber, and a post pre polymerase magnet position adjacent the polymerase chain reaction chamber. The nucleic acid is bound to the magnetic beads. The magnetic beads with the nucleic acid flow to the pre polymerase chain reaction magnet position in the flow channel. The magnetic beads and the nucleic acid are washed with ethanol. The nucleic acid in the polymerase chain reaction chamber is amplified. The magnetic beads and the nucleic acid are separated into a waste stream containing the magnetic beads and a post polymerase chain reaction mix containing the nucleic acid. The reaction mix containing the nucleic acid flows to an analysis unit in the channel for analysis.

  11. Magnetic bead based assays for complement component C5.

    PubMed

    DiScipio, Richard G; Schraufstatter, Ingrid U

    2017-11-01

    Two novel magnetic agarose bead based assays have been developed to measure complement component C5 interaction with C3b and the Factor I Modules (FIMs) of C7. One innovation was to couple C3b onto the magnetic agarose bead using the alternative pathway C3 convertase, which resulted in a linkage of the ligand by a covalent ester bond. A second innovation was to employ nickel ion charged N,N,N'-tris(carboxymethyl)ethylene-diamine-magnetic agarose to capture recombinantly prepared C7 FIMs that were expressed with an oligo-histidine linker followed by an acidic domain that provided a spacer enabling the C7 modules exposure to C5. Detection was brought about by peroxidase coupled to C5. Both assays exhibited adequate statistics suitable for screening. As examples of the utility of these new methods, we chose to examine influence of natural products on C5 interaction. Fucoidan and β-glucans were observed to inhibit C3b-C5 interaction, and dextran sulfate was similarly active; however, rosmarinic acid had no measurable effect. In contrast only β-glucans from two species of macrofungi were able to interfere with interaction of C5 with the FIMs of C7. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. [Enrichment of giant panda microsatellite markers using dynal magnet beads].

    PubMed

    Shen, Fu-Jun; Watts, Phill; Zhang, Zhi-He; Zhang, An-Ju; Sanderson, Stephanie; Kemp, Steve J; Yue, Bi-Song

    2005-05-01

    The 400 -600 bp DNA fractions of giant panda containing STR sequences were captured by hybridization with the oligonucleotide probes attached to streptavadin coated magnetic beads (Dynal). The enriched DNA were ligated into pGEM-T and then transformed into E. coil JM109 competent cells. In total 260 positive clones were identified from 2 880 transformants in the libraries which were screened by gamma-32 P radiolabelled probes. Finally, we got 54 sequences and successfully designed 37 pairs of STR primers for giant panda. The results showed that this method is very efficient to isolate microsatellite markers.

  13. In situ single cell detection via microfluidic magnetic bead assay

    PubMed Central

    KC, Pawan; Zhang, Ge; Zhe, Jiang

    2017-01-01

    We present a single cell detection device based on magnetic bead assay and micro Coulter counters. This device consists of two successive micro Coulter counters, coupled with a high gradient magnetic field generated by an external magnet. The device can identify single cells in terms of the transit time difference of the cell through the two micro Coulter counters. Target cells are conjugated with magnetic beads via specific antibody and antigen binding. A target cell traveling through the two Coulter counters interacts with the magnetic field, and have a longer transit time at the 1st counter than that at the 2nd counter. In comparison, a non-target cell has no interaction with the magnetic field, and hence has nearly the same transit times through the two counters. Each cell passing through the two counters generates two consecutive voltage pulses one after the other; the pulse widths and magnitudes indicating the cell’s transit times through the counters and the cell’s size respectively. Thus, by measuring the pulse widths (transit times) of each cell through the two counters, each single target cell can be differentiated from non-target cells even if they have similar sizes. We experimentally proved that the target human umbilical vein endothelial cells (HUVECs) and non-target rat adipose-derived stem cells (rASCs) have significant different transit time distribution, from which we can determine the recognition regions for both cell groups quantitatively. We further demonstrated that within a mixed cell population of rASCs and HUVECs, HUVECs can be detected in situ and the measured HUVECs ratios agree well with the pre-set ratios. With the simple device structure and easy sample preparation, this method is expected to enable single cell detection in a continuous flow and can be applied to facilitate general cell detection applications such as stem cell identification and enumeration. PMID:28222140

  14. Thermophysical and Magnetic Properties of Carbon Beads Containing Nickel Nanocrystallites

    NASA Astrophysics Data System (ADS)

    Skumiel, A.; Izydorzak, M.; Leonowicz, M.; Pomogailo, A. D.; Dzhardimalieva, G. I.

    2011-09-01

    Ferromagnetic and superparamagnetic nickel nanocrystallites, stabilized in a carbon matrix, were prepared by a three-step procedure including formation of a Ni acrylamide complex, followed by frontal polymerization and pyrolysis of the polymer at various temperatures. It was found that the procedure applied enables fabrication of magnetic beads containing metallic nanocrystallites embedded in a carbon matrix. The size of the crystallites, their morphology, volume fraction, and magnetic properties can be tailored by the pyrolysis temperature. The size of the crystallites affects their behavior in an external magnetic field, i.e., a heating process is the most effective for a sample pyrolyzed at 873 K. The revealed H n-type dependence of the temperature increase rate, (d T/d t) t=0, on the amplitude of the magnetic field indicates the presence of both superparamagnetic and ferromagnetic particles in all the samples studied since n > 2. For the superparamagnetic particles, the heating mechanism is associated with Néel relaxation. For the lower values of the magnetic field amplitude, H < H 0, the relaxation losses dominate whereas for the opposite case, H > H 0, the magnetic hysteresis is the main source of thermal energy losses. The composites containing magnetic Ni nanocrystallites entrapped in a carbon matrix can be potentially applied for hyperthermia treatment.

  15. Dose-response curve of a microfluidic magnetic bead-based surface coverage sandwich assay.

    PubMed

    Cornaglia, Matteo; Trouillon, Raphaël; Tekin, H Cumhur; Lehnert, Thomas; Gijs, Martin A M

    2015-09-25

    Magnetic micro- and nanoparticles ('magnetic beads') have been used to advantage in many microfluidic devices for sensitive antigen (Ag) detection. Today, assays that use as read-out of the signal the number count of immobilized beads on a surface for quantification of a sample's analyte concentration have been among the most sensitive and have allowed protein detection lower than the fgmL(-1) concentration range. Recently, we have proposed in this category a magnetic bead surface coverage assay (Tekin et al., 2013 [1]), in which 'large' (2.8μm) antibody (Ab)-functionalized magnetic beads captured their Ag from a serum and these Ag-carrying beads were subsequently exposed to a surface pattern of fixed 'small' (1.0μm) Ab-coated magnetic beads. When the system was exposed to a magnetic induction field, the magnet dipole attractive interactions between the two bead types were used as a handle to approach both bead surfaces and assist with Ag-Ab immunocomplex formation, while unspecific binding (in absence of an Ag) of a large bead was reduced by exploiting viscous drag flow. The dose-response curve of this type of assay had two remarkable features: (i) its ability to detect an output signal (i.e. bead number count) for very low Ag concentrations, and (ii) an output signal of the assay that was non-linear with respect to Ag concentration. We explain here the observed dose-response curves and show that the type of interactions and the concept of our assay are in favour of detecting the lowest analyte concentrations (where typically either zero or one Ag is carried per large bead), while higher concentrations are less efficiently detected. We propose a random walk process for the Ag-carrying bead over the magnetic landscape of small beads and this model description explains the enhanced overall capture probability of this assay and its particular non-linear dose response curves.

  16. Magnetic bead detection using domain wall-based nanosensor

    SciTech Connect

    Corte-León, H.; Krzysteczko, P.; Schumacher, H. W.; Manzin, A.; Cox, D.; Antonov, V.; Kazakova, O.

    2015-05-07

    We investigate the effect of a single magnetic bead (MB) on the domain wall (DW) pinning/depinning fields of a DW trapped at the corner of an L-shaped magnetic nanodevice. DW propagation across the device is investigated using magnetoresistance measurements. DW pinning/depinning fields are characterized in as-prepared devices and after placement of a 1 μm-sized MB (Dynabeads{sup ®} MyOne{sup ™}) at the corner. The effect of the MB on the DW dynamics is seen as an increase in the depinning field for specific orientations of the device with respect to the external magnetic field. The shift of the depinning field, ΔB{sub dep} = 4.5–27.0 mT, is highly stable and reproducible, being significantly above the stochastic deviation which is about 0.5 mT. The shift in the deppinning field is inversely proportional to the device width and larger for small negative angles between the device and the external magnetic field. Thus, we demonstrate that DW-based devices can be successfully used for detection of single micron size MB.

  17. Use of magnetic beads for Gram staining of bacteria in aqueous suspension.

    PubMed

    Yazdankhah, S P; Sørum, H; Larsen, H J; Gogstad, G

    2001-12-01

    A Gram staining technique was developed using monodisperse magnetic beads in concentrating bacteria in suspension for downstream application. The technique does not require heat fixation of organisms, electrical power, or a microscope. Gram-negative and Gram-positive bacteria were identified macroscopically based on the colour of the suspension. The bacteria concentrated on magnetic beads may also be identified microscopically.

  18. Using magnets and magnetic beads to dissect signaling pathways activated by mechanical tension applied to cells.

    PubMed

    Marjoram, R J; Guilluy, C; Burridge, K

    2016-02-01

    Cellular tension has implications in normal biology and pathology. Membrane adhesion receptors serve as conduits for mechanotransduction that lead to cellular responses. Ligand-conjugated magnetic beads are a useful tool in the study of how cells sense and respond to tension. Here we detail methods for their use in applying tension to cells and strategies for analyzing the results. We demonstrate the methods by analyzing mechanotransduction through VE-cadherin on endothelial cells using both permanent magnets and magnetic tweezers.

  19. Using Magnets and Magnetic Beads to Dissect Signaling Pathways Activated by Mechanical Tension Applied to Cells

    PubMed Central

    Marjoram, R.J.; Guilluy, C; Burridge, K.

    2015-01-01

    Cellular tension has implications in normal biology and pathology. Membrane adhesion receptors serve as conduits for mechanotransduction that lead to cellular responses. Ligand-conjugated magnetic beads are a useful tool in the study of how cells sense and respond to tension. Here we detail methods for their use in applying tension to cells and strategies for analyzing the results. We demonstrate the methods by analyzing mechanotransduction through VE-cadherin on endothelial cells using both permanent magnets and magnetic tweezers. PMID:26427549

  20. Lectin-Magnetic Beads for Plasma Membrane Isolation.

    PubMed

    Lee, Yu-Chen; Liu, Hsuan-Chen; Chuang, Carol; Lin, Sue-Hwa

    2015-07-01

    Plasma membrane proteins mainly function to transmit external signals into the cell. Many plasma membrane receptor tyrosine kinases (e.g., HER2 and EGFR) are known to mediate oncogenic progression, making them prime targets for cancer therapy. Recently, it has become important to identify plasma membrane proteins that are differentially expressed in normal versus cancer cells, in drug-sensitive versus drug-resistant cells, or among tumor cells that metastasize to different organ sites because these differentially expressed membrane proteins may lead to the identification of therapeutic targets or diagnostic markers. In addition, there is an increased interest in identifying cell-surface proteins that could serve as markers for stem cells, progenitor cells, or cells of different lineages. Traditionally, membrane isolation requires multiple centrifugation steps to isolate different organelles based on their density. With the advent of affinity matrix technology, it is possible to separate organelles based on their molecular differences. A defining characteristic of the plasma membrane is that plasma membrane proteins are more extensively glycosylated than are intracellular membrane proteins. As a result, affinity chromatography employing lectin, a carbohydrate-binding protein, is commonly used to isolate plasma membrane proteins. We have extended this concept for plasma membrane isolation by using concanavalin A (ConA), a lectin with mannose specificity. Here we describe a protocol that uses immobilized ConA bound to magnetic beads to isolate plasma membranes from homogenized cell lysates. The captured plasma membrane proteins are then solubilized from the ConA-magnetic beads by detergents in the presence of a competing sugar, methyl α-mannopyranoside.

  1. Three-dimensional pattern formation of magnetically labeled microgel beads for biological tissue engineering

    NASA Astrophysics Data System (ADS)

    Kawamoto, H.; Inoue, H.; Nakamura, M.

    2009-03-01

    We commenced basic research on the three-dimensional (3D) pattern formation of microgel beads for applications in biological tissue engineering. In this new technique, microgel beads are premagnetized by doping them with magnetic nanoparticles. Living cells will be included in the beads for actual use. If a nonuniform magnetic field is applied to a solution containing these magnetized beads, the beads will align, contact, and form a 3D structure. The structure is controlled by the seed pattern of the magnetic particles plugged in a substrate and the profile of the magnetic field distribution. We constructed tubes, which imitate blood vessels, for demonstration using gel beads whose diameters are of the order of several tens of micrometers. The diameter of the demonstrated tube was less than 0.5 mm and its length was 6.6 mm, although living cells were not included in the beads. Numerical calculations by using the discrete element method were conducted to confirm the formation of the tube and to predict the effect of centrifugal force, which will be applied to fill cells in the space between magnetically patterned beads. Although this unique technology is in the nascent stage, this 3D pattern formation technique by the control of the magnetic field has potential to be one of the effective engineering technologies for manufacturing 3D patterned biological tissues in the future.

  2. Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons

    SciTech Connect

    Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

    2007-07-01

    A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3, 3´, 5, 5´- tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

  3. Magnetic tweezers with high permeability electromagnets for fast actuation of magnetic beads

    SciTech Connect

    Chen, La; Offenhäusser, Andreas; Krause, Hans-Joachim

    2015-04-15

    As a powerful and versatile scientific instrument, magnetic tweezers have been widely used in biophysical research areas, such as mechanical cell properties and single molecule manipulation. If one wants to steer bead position, the nonlinearity of magnetic properties and the strong position dependence of the magnetic field in most magnetic tweezers lead to quite a challenge in their control. In this article, we report multi-pole electromagnetic tweezers with high permeability cores yielding high force output, good maneuverability, and flexible design. For modeling, we adopted a piece-wise linear dependence of magnetization on field to characterize the magnetic beads. We implemented a bi-linear interpolation of magnetic field in the work space, based on a lookup table obtained from finite element simulation. The electronics and software were custom-made to achieve high performance. In addition, the effects of dimension and defect on structure of magnetic tips also were inspected. In a workspace with size of 0.1 × 0.1 mm{sup 2}, a force of up to 400 pN can be applied on a 2.8 μm superparamagnetic bead in any direction within the plane. Because the magnetic particle is always pulled towards a tip, the pulling forces from the pole tips have to be well balanced in order to achieve control of the particle’s position. Active video tracking based feedback control is implemented, which is able to work at a speed of up to 1 kHz, yielding good maneuverability of the magnetic beads.

  4. Magnetic tweezers with high permeability electromagnets for fast actuation of magnetic beads.

    PubMed

    Chen, La; Offenhäusser, Andreas; Krause, Hans-Joachim

    2015-04-01

    As a powerful and versatile scientific instrument, magnetic tweezers have been widely used in biophysical research areas, such as mechanical cell properties and single molecule manipulation. If one wants to steer bead position, the nonlinearity of magnetic properties and the strong position dependence of the magnetic field in most magnetic tweezers lead to quite a challenge in their control. In this article, we report multi-pole electromagnetic tweezers with high permeability cores yielding high force output, good maneuverability, and flexible design. For modeling, we adopted a piece-wise linear dependence of magnetization on field to characterize the magnetic beads. We implemented a bi-linear interpolation of magnetic field in the work space, based on a lookup table obtained from finite element simulation. The electronics and software were custom-made to achieve high performance. In addition, the effects of dimension and defect on structure of magnetic tips also were inspected. In a workspace with size of 0.1 × 0.1 mm(2), a force of up to 400 pN can be applied on a 2.8 μm superparamagnetic bead in any direction within the plane. Because the magnetic particle is always pulled towards a tip, the pulling forces from the pole tips have to be well balanced in order to achieve control of the particle's position. Active video tracking based feedback control is implemented, which is able to work at a speed of up to 1 kHz, yielding good maneuverability of the magnetic beads.

  5. Rapid screening of peptide probes through in situ single-bead sequencing microarray.

    PubMed

    Wang, Weizhi; Wei, Zewen; Zhang, Di; Ma, Huailei; Wang, Zihua; Bu, Xiangli; Li, Menglin; Geng, Lingling; Lausted, Christopher; Hood, Leroy; Fang, Qiaojun; Wang, Hao; Hu, Zhiyuan

    2014-12-02

    Peptide ligands as targeting probes for in vivo imaging and drug delivery have attracted great interest in the biomedical community. However, high affinity and specificity screening of large peptide libraries remains a tedious process. Here, we report a continuous-flow microfluidic method for one-bead-one-compound (OBOC) combinatorial peptide library screening. We screened a library with 2 × 10(5) peptide beads within 4 h and discovered 140 noncanonical peptide hits targeting the tumor marker, aminopeptidase N (APN). Using the Clustal algorithm, we identified the conserved sequence Tyr-XX-Tyr in the N terminal. We demonstrated that the novel sequence YVEYHLC peptides have both nanomolar affinity and high specificity for APN in ex vivo and in vivo models. We envision that the successful demonstration of this integrated novel nanotechnology for peptide screening and identification open a new avenue for rapid discovery of new peptide-based reagents for disease diagnostics and therapeutics.

  6. Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array.

    PubMed

    Wang, Bin; Howel, Paul; Bruheim, Skjalg; Ju, Jingfang; Owen, Laurie B; Fodstad, Oystein; Xi, Yaguang

    2011-02-11

    A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA). The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment. Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.

  7. A finite element model of cell deformation during magnetic bead twisting.

    PubMed

    Mijailovich, Srboljub M; Kojic, Milos; Zivkovic, Miroslav; Fabry, Ben; Fredberg, Jeffrey J

    2002-10-01

    Magnetic twisting cytometry probes mechanical properties of an adherent cell by applying a torque to a magnetic bead that is tightly bound to the cell surface. Here we have used a three-dimensional finite element model of cell deformation to compute the relationships between the applied torque and resulting bead rotation and lateral bead translation. From the analysis, we computed two coefficients that allow the cell elastic modulus to be estimated from measurements of either bead rotation or lateral bead translation, respectively, if the degree of bead embedding and the cell height are known. Although computed strains in proximity of the bead can be large, the relationships between applied torque and bead rotation or translation remain virtually linear up to bead rotations of 15 degrees, above which geometrical nonlinearities become significant. This appreciable linear range stands in contrast to the intrinsically nonlinear force-displacement relationship that is observed when cells are indented during atomic force microscopy. Finally, these computations support the idea that adhesive forces are sufficient to keep the bead firmly attached to the cell surface throughout the range of working torques.

  8. [Optimization of genomic DNA extraction with magnetic bead- based semi-automatic system].

    PubMed

    Ling, Jie; Wang, Hao; Zhang, Shuai; Zhang, Dan-dan; Lai, Mao-de; Zhu, Yi-min

    2012-05-01

    To develop a rapid and effective method for genomic DNA extraction with magnetic bead-based semi-automatic system. DNA was extracted from whole blood samples semi-automatically with nucleic acid automatic extraction system.The concentration and purity of samples was determined by UV-spectrophotometer. Orthogonal design was used to analyze the main effect of lysis time, blood volume, magnetic bead quantity and ethanol concentration on the DNA yield; also the 2-way interaction of these factors. Lysis time, blood volume, magnetic bead quantity and ethanol concentration were associated with DNA yield (P<0.05), but no interaction existed. DNA yield was higher under the condition with 15 min of lysis time, 100 μl of blood volume, 80 μl of magnetic beads and 80 % of ethanol. A significant association was found between the magnetic bead quantity and DNA purity OD260/OD280 (P=0.008). Interaction of blood volume and lysis time also existed (P=0.013). DNA purity was better when the extracting condition was 40 μl of magnetic beads, 15 min of lysis time and 100 μl of blood volume. Magnetic beads and ethanol concentration were associated with DNA purity OD260/OD230 (P=0.017 and P<0.05), the result was better when magnetic beads was 40 μl and ethanol concentration was 80 %. The results indicate that the optimized conditions with 40 μl magnetic beads will generate higher quality of genomic DNA from the whole blood samples.

  9. Magnetic bead counter using a micro-Hall sensor for biological applications

    SciTech Connect

    Lee, W.; Kim, K.; Joo, S.; Kim, S.U.; Rhie, K.; Hong, J.; Shin, K-H.; and Kim, K.H.

    2009-04-13

    Micro-Hall sensors have been fabricated, and various numbers of micron-size magnetic beads have been placed within the sensor area. The Hall resistances measured at room temperature are found to be proportional to the number of the beads, and are in good agreement with the numerically simulated results presented in this study. Our sensors are designed to measure the number of beads between zero and full-scale signals for a given number range of interest. The effects of miniaturizing the beads and sensors to nanoscale are also discussed.

  10. Colloidally Assembled Zinc Ferrite Magnetic Beads: Superparamagnetic Labels with High Magnetic Moments for MR Sensors.

    PubMed

    Park, Jooneon; Porter, Marc D; Granger, Michael C

    2017-06-14

    Magnetic particles are widely used as labels in magnetoresistive sensors. To use magnetic particles as labels, several important characteristics should be considered, such as superparamagnetism, a high magnetic moment per particle (m), facile surface functionalization and biomolecule immobilization, colloidal stability, and analyte specificity. In this paper, we describe the preparation of magnetic labels with a high m, using colloidal assemblies of superparamagnetic zinc ferrite nanoparticles (ZFNPs, ∼9 nm). Also, several properties of these particles are compared with those of commercially available magnetic beads, Dynabeads and TurboBeads. The colloidally assembled zinc ferrite magnetic beads (ZFMBs, ∼160 nm) were synthesized by assembling ZFNPs via an emulsion-based assembly approach. While retaining superparamagnetism at room temperature, the m of ZFMBs is ∼4000× higher than that of the constituent ZFNPs. Surface functionalization with a layer of polyacrylic acid stabilized the ZFMBs in aqueous solution and enabled conjugation with streptavidin via carbodiimide linking chemistry. The streptavidinated ZFMBs can be suspended in aqueous buffer for ≥24 h, whereas 1.05 μm Dynabeads and 30 nm TurboBeads undergo ballistic deposition and instantaneous aggregation in solution, respectively. Finally, the streptavidinated ZFMBs were employed as labels in an immunoassay for the detection of osteopontin, a potential pancreatic cancer marker, proving superior to the commercial particles in terms of limit of detection and dynamic range. We expect that the work presented in this article can be extended to other biological applications, especially where superparamagnetic particles with a high m and colloidal stability are needed.

  11. On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis

    NASA Astrophysics Data System (ADS)

    Florescu, Octavian; Wang, Kevan; Au, Patrick; Tang, Jimmy; Harris, Eva; Beatty, P. Robert; Boser, Bernhard E.

    2010-03-01

    We have demonstrated a postprocessed complementary metal oxide semiconductor (CMOS) integrated circuit (IC) capable of on-chip magnetic separation, i.e., removing via magnetic forces the nonspecifically bound magnetic beads from the detection area on the surface of the chip. Initially, 4.5 μm wide superparamagnetic beads sedimenting out of solution due to gravity were attracted to the detection area by a magnetic concentration force generated by flowing current through a conductor embedded in the IC. After sedimentation, the magnetic beads that did not bind strongly to the functionalized surface of the IC through a specific biochemical complex were removed by a magnetic separation force generated by flowing current through another conductor placed laterally to the detection area. As the spherical bead pivoted on the surface of the chip, the lateral magnetic force was further amplified by mechanical leveraging, and 50 mA of current flowing through the separation conductor placed 18 μm away from the bead resulted in 7.5 pN of tensile force on the biomolecular tether immobilizing the bead. This force proved high enough to break nonspecific interactions while leaving specific antibody-antigen bonds intact. A sandwich capture immunoassay on purified human immunoglobulin G showed strong correlation with a control enzyme linked immunosorbent assay and a detection limit of 10 ng/ml or 70 pM. The beads bound to the detection area after on-chip magnetic separation were detected optically. To implement a fully integrated molecular diagnostics platform, the on-chip magnetic separation functionality presented in this work can be readily combine with state-of-the art CMOS-based magnetic bead detection technology.

  12. Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

    PubMed

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

    2014-07-01

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

  13. Conditions for efficient on-chip magnetic bead detection via magnetoresistive sensors.

    PubMed

    Albisetti, E; Petti, D; Cantoni, M; Damin, F; Torti, A; Chiari, M; Bertacco, R

    2013-09-15

    A commonly used figure of merit of magnetoresistive sensors employed to detect magnetic beads labeling biomolecules in lab-on-chip applications is the sensor sensitivity (S0) to external magnetic fields in the linear region of the sensor. In this paper we show that, in case of lock-in detection and bead excitation by a small AC magnetic field, S0 is not the good figure of merit to optimize. Indeed, the highest sensitivity to the magnetic beads is achieved biasing the sensor in the region of its characteristics where the product between the DC bias field and the second derivative of the resistance with respect to the magnetic field is maximum. The validity of this criterion, derived from a phenomenological model of bead detection, is proved in case of magnetic tunneling junction sensors detecting magnetic beads with 250nm diameter. This work paves the way to the development of a new generation of sensors properly designed to maximize the bead sensitivity.

  14. On-chip magnetic bead-based DNA melting curve analysis using a magnetoresistive sensor

    NASA Astrophysics Data System (ADS)

    Rizzi, Giovanni; Østerberg, Frederik W.; Henriksen, Anders D.; Dufva, Martin; Hansen, Mikkel F.

    2015-04-01

    We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches of the differential sensor geometry. The sensor surfaces are functionalized with wild type (WT) and mutant type (MT) capture probes, differing by a single base insertion (a single nucleotide polymorphism, SNP). Complementary biotinylated targets in suspension couple streptavidin magnetic beads to the sensor surface. The beads are magnetized by the field arising from the bias current passed through the sensors. We demonstrate the first on-chip measurements of the melting of DNA hybrids upon a ramping of the temperature. This overcomes the limitation of using a single washing condition at constant temperature. Moreover, we demonstrate that a single sensor bridge can be used to genotype a SNP.

  15. Magnetic Bead Based Immunoassay for Autonomous Detection of Toxins

    SciTech Connect

    Kwon, Y; Hara, C A; Knize, M G; Hwang, M H; Venkatesteswaran, K S; Wheeler, E K; Bell, P M; Renzi, R F; Fruetel, J A; Bailey, C G

    2008-05-01

    As a step towards toward the development of a rapid, reliable analyzer for bioagents in the environment, we are developing an automated system for the simultaneous detection of a group of select agents and toxins. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag{trademark} reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag{trademark} through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads coupled to a photo-activatable porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactivable group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags{trademark}. Released eTags{trademark} are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 ng/mL (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated on a flow-through format with higher LODs of 125 ng/mL (or 2.5 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

  16. Magnetic bead based immunoassay for autonomous detection of toxins.

    PubMed

    Kwon, Youngeun; Hara, Christine A; Knize, Mark G; Hwang, Mona H; Venkateswaran, Kodumudi S; Wheeler, Elizabeth K; Bell, Perry M; Renzi, Ronald F; Fruetel, Julie A; Bailey, Christopher G

    2008-11-15

    We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

  17. Magnetic actuator for the control and mixing of magnetic bead-based reactions on-chip.

    PubMed

    Berenguel-Alonso, Miguel; Granados, Xavier; Faraudo, Jordi; Alonso-Chamarro, Julián; Puyol, Mar

    2014-10-01

    While magnetic bead (MB)-based bioassays have been implemented in integrated devices, their handling on-chip is normally either not optimal--i.e. only trapping is achieved, with aggregation of the beads--or requires complex actuator systems. Herein, we describe a simple and low-cost magnetic actuator to trap and move MBs within a microfluidic chamber in order to enhance the mixing of a MB-based reaction. The magnetic actuator consists of a CD-shaped plastic unit with an arrangement of embedded magnets which, when rotating, generate the mixing. The magnetic actuator has been used to enhance the amplification reaction of an enzyme-linked fluorescence immunoassay to detect Escherichia coli O157:H7 whole cells, an enterohemorrhagic strain, which have caused several outbreaks in food and water samples. A 2.7-fold sensitivity enhancement was attained with a detection limit of 603 colony-forming units (CFU) /mL, when employing the magnetic actuator.

  18. Magnetic Pycnoporus sanguineus-loaded alginate composite beads for removing dye from aqueous solutions.

    PubMed

    Yang, Chih-Hui; Shih, Ming-Cheng; Chiu, Han-Chen; Huang, Keng-Shiang

    2014-06-18

    Dye pollution in wastewater is a severe environmental problem because treating water containing dyes using conventional physical, chemical, and biological treatments is difficult. A conventional process is used to adsorb dyes and filter wastewater. Magnetic filtration is an emerging technology. In this study, magnetic Pycnoporus sanguineus-loaded alginate composite beads were employed to remove a dye solution. A white rot fungus, P. sanguineus, immobilized in alginate beads were used as a biosorbent to remove the dye solution. An alginate polymer could protect P. sanguineus in acidic environments. Superparamagnetic nanomaterials, iron oxide nanoparticles, were combined with alginate gels to form magnetic alginate composites. The magnetic guidability of alginate composites and biocompatibility of iron oxide nanoparticles facilitated the magnetic filtration and separation processes. The fungus cells were immobilized in loaded alginate composites to study the influence of the initial dye concentration and pH on the biosorption capacity. The composite beads could be removed easily post-adsorption by using a magnetic filtration process. When the amount of composite beads was varied, the results of kinetic studies of malachite green adsorption by immobilized cells of P. sanguineus fitted well with the pseudo-second-order model. The results indicated that the magnetic composite beads effectively adsorbed the dye solution from wastewater and were environmentally friendly.

  19. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  20. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  1. Use of a magnetic field to modify and detect avalanche behavior on a conical bead pile

    NASA Astrophysics Data System (ADS)

    Johnson, Nathan; Lehman, Susan

    2015-03-01

    A conical bead pile subject to slow driving and an external magnetic field is used to test the effects of drop height and cohesion on avalanche statistics. Magnetically susceptible beads were dropped onto a pile from different heights and into different strengths of magnetic field. Avalanches were recorded by the change in mass as beads fall off the pile. For beads dropped from a low drop height with no cohesion, the avalanche size distribution follows a power law. As cohesion increases, we observe an increase in the probability of very large avalanches and decreases in the mid-size avalanches. The resulting bump in the avalanche distribution moves to larger avalanche size as the cohesion in the system is increased, matching the prediction by an analytic theory from a mean-field model of slip avalanches. The model also makes predictions for avalanche duration, which is not measurable with our current system. Since the steel beads are magnetized while in the applied magnetic field, their motion during an avalanche creates a change in magnetic flux. To detect this motion, we have placed a large-diameter pick-up coil around the pile. Results of the testing and calibration of this coil to measure avalanche duration are presented.

  2. Magnetic steering of arc and bead characteristics in submerged arc strip cladding

    SciTech Connect

    Mallya, U.D.; Srinivas, H.S. )

    1993-11-01

    Submerged arc strip cladding has been widely used when larger thicknesses of clad materials are required. Use of wider strips and higher current have all been attempted to achieve higher productivity and these have been associated with arc blow phenomena and unacceptable bead characteristics. Investigations were carried out to study the effect of various magnetizing parameters (flux density, dwell time) and welding current on the bead characteristics, using 60-mm-wide strips of 309L stainless steel. Results of the investigation indicate very encouraging trends in increasing the melt-off rate/bead height and decreasing penetration and hence dilution by 23%. The magnetizing flux density individually did not indicate significant influence on any of the bead characteristics studied.

  3. Development of a Magnetic Beads Quantitative Detection System for Fast Diagnosis

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yujiro; Morishita, Tomohiro; Matsuyama, Kenji; Takasa, Kenji; Shibasaki, Ichiro

    This paper reports the development and performance of a detection system for magnetic beads. The system consists of a semiconductor based magneto-resistance sensor for beads detection and a lateral flow kit. Detection of anti-gen of H.Influenza at concentration of 0.1ng/ml was performed with satisfactory sensitivity, showing the system to be a promising for immunoassay.

  4. Manipulation of superparamagnetic beads on patterned Au/Co/Au multilayers with perpendicular magnetic anisotropy

    NASA Astrophysics Data System (ADS)

    Jarosz, A.; Holzinger, D.; Urbaniak, M.; Ehresmann, A.; Stobiecki, F.

    2016-08-01

    The magnetophoresis of water-suspended 4 μm-diameter superparamagnetic beads above topographically patterned, sputter deposited Ti(4 nm)/Au(60 nm)/[Co(0.7 nm)/Au(1 nm)] × 3 multilayers with perpendicular magnetic anisotropy was investigated. The results impressively demonstrate that the magnetic stray field landscape above the stripe structure when superimposed with an external, slowly rotating, field enables the directed transport of magnetic beads across the stripe panel with velocities up to 12 μm s-1.

  5. Optimization of the Magnetic Recovery of Hits from One-Bead-One-Compound Library Screens.

    PubMed

    Mendes, Kimberly; Ndungu, J M; Clark, Lorraine F; Kodadek, Thomas

    2015-09-14

    On-bead screening of one-bead-one-compound (OBOC) libraries is a useful procedure for the identification of protein ligands. An important aspect of this experiment is the method by which beads that bind the target protein are separated from those that do not. Ideally, such a method would be rapid and convenient and result in the isolation of 100% of the "hits" with no false positives (beads that display compounds that are not good ligands for the target). We introduced a technique in which beads that have bound a labeled target protein can be magnetized, thus allowing their convenient isolation ( Astle et al. Chem. Biol. 2010 , 17 , 38 - 45 ). However, recent work in our laboratory and others has shown that magnetic hit recovery can result in the isolation of large numbers of false positives and has also suggested that many true hit beads are missed. In this study, we employ a well-defined model system to examine the efficiency of various magnetic hit isolation protocols. We show that the choice of reagents and the particular operations employed are critical for optimal results.

  6. Simple enrichment of thiol-containing biomolecules by using zinc(II)-cyclen-functionalized magnetic beads.

    PubMed

    Fujioka, Haruto; Tsunehiro, Masaya; Kawaguchi, Maho; Kuramoto, Yasuhiro; Kurosaki, Hiromasa; Hieda, Yuhzo; Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

    2014-07-01

    A simple and efficient method based on magnetic-bead technology has been developed for the enrichment of thiol-containing biomolecules, such as l-glutathione and cysteine-containing peptides. The thiol-binding site on the bead is a mononuclear complex of zinc(II) with 1,4,7,10-tetraazacyclododecane (cyclen); this is linked to a hydrophilic cross-linked agarose coating on a particle that has a magnetic core. All steps for the thiol-affinity separation are conducted in aqueous buffers with 0.10 mL of the magnetic beads in a 1.5 mL microtube. The entire separation protocol for thiol-containing compounds, from addition to elution, requires less than one hour per sample, provided the buffers and the zinc(II)-cyclen-functionalized magnetic beads have been prepared in advance. The thiol-affinity magnetic beads are reusable at least 15 times without a decrease in their thiol-binding ability, and they are stable for six months at room temperature.

  7. Dye removal from aqueous solution by magnetic alginate beads crosslinked with epichlorohydrin.

    PubMed

    Rocher, Vincent; Bee, Agnès; Siaugue, Jean-Michel; Cabuil, Valérie

    2010-06-15

    Innovative magnetic alginate beads are used to remove organic pollutants from aqueous solution under different experimental conditions. These alginate beads (EpiMAB) are prepared by an extrusion technique and crosslinked with epichlorohydrin. They contain both magnetic nanoparticles and activated carbon (AC). With the addition of magnetic properties, the beads can be easily recovered or manipulated with an external magnetic field. Their capacity to adsorb pollutants is linked to encapsulated AC and to active sites coming from both magnetic nanoparticles and alginate. The efficiency of the beads as biosorbent for the removal of dyes is assessed using methyl orange (MO) and methylene blue (MB) as model molecules. The dye uptake is found to vary with the initial concentration and the charge of the adsorbed molecule. The Langmuir equation fits well the adsorption data with maximum adsorption capacities of 0.02 mmol/g for MO and 0.7 mmol/g for MB. Kinetics experiments are performed to evaluate the equilibrium time; the pseudo-second-order kinetic model adequately describes the experimental data. The influence of the pH of the solution on adsorption is also investigated and a comparison with alginate beads crosslinked by calcium ions is made.

  8. A novel magnetic bead bioassay platform using a microchip-based sensor for infectious disease diagnosis.

    PubMed

    Aytur, Turgut; Foley, Jonathan; Anwar, Mekhail; Boser, Bernhard; Harris, Eva; Beatty, P Robert

    2006-07-31

    New technologies are greatly needed to improve laboratory tests that can be used in point-of-care clinical settings. Here, a biosensor was used to detect micron-scale paramagnetic beads in order to replace the conventional enzymatic label used in ELISAs. This novel biosensor was fabricated through standard complementary metal oxide semiconductor (CMOS) manufacturing and was used to quantify magnetic beads bound to the sensor surface by immunological recognition, analogous to ELISA. CMOS technology can integrate multiple laboratory functions into the sensor chip, potentially enabling inexpensive, compact and sophisticated diagnostic systems for a number of diseases. We present results for two immunological assays: antigen capture of purified mouse IgG and detection of human anti-dengue virus IgG in clinical serum samples. The sensitivity of detecting purified protein with magnetic beads was comparable to ELISA. We found a high correlation between the ELISA optical density and the biosensor output in the clinical assay. We also demonstrate the use of a controlled magnetic field to remove non-specifically bound magnetic beads from the sensor surface, effectively washing the sensor surface. This novel sensor can be mass-produced at low cost and can detect magnetic beads bound to the surface through specific antibody-antigen interactions, making it a potential platform for new simplified and rapid point-of-care diagnostic tests.

  9. Design of a Microfluidic Chip for Magnetic-Activated Sorting of One-Bead-One-Compound Libraries.

    PubMed

    Cho, Choi-Fong; Lee, Kyungheon; Speranza, Maria-Carmela; Bononi, Fernanda C; Viapiano, Mariano S; Luyt, Leonard G; Weissleder, Ralph; Chiocca, E Antonio; Lee, Hakho; Lawler, Sean E

    2016-06-13

    Molecular targeting using ligands specific to disease markers has shown great promise for early detection and directed therapy. Bead-based combinatorial libraries have served as powerful tools for the discovery of novel targeting agents. Screening platforms employing magnetic capture have been used to achieve rapid and efficient identification of high-affinity ligands from one-bead-one-compound (OBOC) libraries. Traditional manual methodologies to isolate magnetized "hit" beads are tedious and lack accuracy, and existing instruments to expedite bead sorting tend to be costly and complex. Here, we describe the design and construction of a simple and inexpensive microfluidic magnetic sorting device using standard photolithography and soft lithography approaches to facilitate high-throughput isolation of magnetized positive hit beads from combinatorial libraries. We have demonstrated that the device is able to sort magnetized beads with superior accuracy compared to conventional manual sorting approaches. This chip offers a very convenient yet inexpensive alternative for screening OBOC libraries.

  10. Moment Selective Digital Detection of Single Magnetic Beads for Multiplexed Bioassays

    NASA Astrophysics Data System (ADS)

    Llandro, J.; Hayward, T. J.; Bland, J. A. C.; Morecroft, D.; Castaño, F. J.; Colin, I. A.; Ross, C. A.

    2008-06-01

    Research into lab-on-a-chip multiplexed bioassays has focused on libraries of biochemical probes, indexed by optically encoded micron-sized labels. However, few current methods have reconciled large multiplexing capability with a rapid detection system amenable to miniaturization. Magnetic identification of labels provides a strong candidate solution to this problem, yet no proposed single-label magnetic detection system can both read and encode magnetic labels. We present a magnetic multiplexed assay in lab-on-a-chip format which identifies target biomolecules from the hybridization results by reading encoded magnetic beads. We show that a microfabricated magnetoresistive ring-shaped sensor can read the magnetic moments of individual commercially available paramagnetic beads using an active digital technique. This work provides proof of principle for a new approach to magnetic labeling of biomolecules for high-throughput bioassays.

  11. Magnetic bead-based label-free electrochemical detection of DNA hybridization.

    PubMed

    Wang, J; Kawde, A N; Erdem, A; Salazar, M

    2001-11-01

    Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.

  12. Adsorption of a cationic surfactant by a magsorbent based on magnetic alginate beads.

    PubMed

    Obeid, Layaly; El Kolli, Nadia; Dali, Noëlle; Talbot, Delphine; Abramson, Sébastien; Welschbillig, Mathias; Cabuil, Valérie; Bée, Agnès

    2014-10-15

    Adsorption of cetylpyridinium chloride (CPC), a cationic surfactant, by magnetic alginate beads (MagAlgbeads) was investigated. The magnetic adsorbent (called magsorbent) was prepared by encapsulation of magnetic functionalized nanoparticles in an alginate gel. The influence on CPC adsorption of several parameters such as contact time, pH and initial surfactant concentration was studied. The equilibrium isotherm shows that adsorption occurs through both electrostatic interactions with charge neutralization of the carboxylate groups of the beads and hydrophobic interactions inducing the formation of surfactant aggregates in the beads. The dosage of calcium ions released in the solution turns out to be a useful tool for understanding the adsorption mechanisms. Adsorption is accompanied by a shrinking of the beads that corresponds to a 45% reduction of the volume. Adsorption kinetic experiments show that equilibrium time is strongly dependent on the surfactant concentration, which monitors the nature of the interactions. On the other hand, since the pH affects the ionization state of adsorption sites, adsorption depends on the pH solution, maximum adsorption being obtained in a large pH range (3.2-12) in agreement with the pKa value of alginate (pKa=3.4-4.2). Finally, due to the formation of micelle-like surfactants aggregates in the magnetic alginate beads, they could be used as a new efficient magsorbent for hydrophobic pollutants.

  13. Magnetic chitosan/clay beads: A magsorbent for the removal of cationic dye from water

    NASA Astrophysics Data System (ADS)

    Bée, Agnès; Obeid, Layaly; Mbolantenaina, Rakotomalala; Welschbillig, Mathias; Talbot, Delphine

    2017-01-01

    A magnetic composite material composed of magnetic nanoparticles and clay encapsulated in cross-linked chitosan beads was prepared, characterized and used as a magsorbent for the removal of a cationic dye, methylene blue (MB), from aqueous solutions. The magnetic properties of these beads represent an advantage to recover them at the end of the depollution process. The optimal weight ratio R=clay:chitosan for the removal of MB in a large range of pH was determined. For beads without clay, the maximal adsorption capacity of MB occurs in the pH range [9-12], while for beads with clay, the pH range extends by increasing the amount of clay to reach [3-12] for R>0.5. Adsorption isotherms show that the adsorption capacity of magnetic beads is equal to 82 mg/g. Moreover, the kinetics of dye adsorption is relatively fast since 50% of the dye is removed in the first 13 min for an initial MB concentration equal to 100 mg/L. The estimation of the number of adsorption sites at a given pH shows that the main driving force for adsorption of MB in a large range of pH is the electrostatic interaction between the positively charged dye and the permanent negative charges of clay.

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

    PubMed

    Kim, Chloe; Searson, Peter C

    2015-11-14

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

  15. An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities.

    PubMed

    Choi, Jin-Woo; Oh, Kwang W; Thomas, Jennifer H; Heineman, William R; Halsall, H Brian; Nevin, Joseph H; Helmicki, Arthur J; Henderson, H Thurman; Ahn, Chong H

    2002-02-01

    This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems.

  16. Asynchronous Magnetic Bead Rotation (AMBR) Microviscometer for Label-Free DNA Analysis

    PubMed Central

    Li, Yunzi; Burke, David T.; Kopelman, Raoul; Burns, Mark A.

    2014-01-01

    We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR) microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes. PMID:25587411

  17. Fine-tuning of magnetic and microfluidic viscous forces for specific magnetic bead-based immunocomplex formation

    NASA Astrophysics Data System (ADS)

    Cornaglia, M.; Tekin, H. C.; Lehnert, T.; Gijs, M. A. M.

    2013-08-01

    We investigate the working principle of a novel type of microfluidic sandwich immunoassay, as used for the detection of biomarkers. The heterogeneous assay is based on the specific interactions between an array of functionalized superparamagnetic beads and a flow of secondary superparamagnetic beads that carry the antigens and are simultaneously used as detection labels. We identify the main forces governing the immunoassay performance and develop a combined finite element method/analytical model to predict and control these forces. The clue for the improved assay specificity is in the fine-tuning of inter-bead magnetic dipolar and microfluidic viscous forces, which allows strongly reducing non-specific interactions, while enhancing the specific formation of immunocomplexes. We exploit our theoretical model to explain the enhanced sensitivity of magnetic bead-based immunoassay experiments performed in microfluidic chips.

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

    NASA Astrophysics Data System (ADS)

    Kim, Chloe; Searson, Peter C.

    2015-10-01

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

  19. 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.

  20. Increased case finding of tuberculosis from sputum and sputum deposits after magnetic bead concentration of mycobacteria.

    PubMed

    Liu, Jun; Sun, Zhan-Qiang; Pei, Hao; Zhang, Shi-Liang; Zhang, Shu-Lin; Wilson, Stuart; De Smet, Koen; Cerullakandiyil, Noushad; Thayyullathil, Thanveer; Al-Suwaidi, Zubaida; Song, Yan-Zheng

    2013-05-01

    Concentration of mycobacteria from sputum by centrifugation prior to acid-fast microscopy increases case finding compared to direct microscopy of the sputum (direct smear). However, centrifugation has to be performed outside the safety cabinet and many laboratories do not have access to a centrifuge. Magnetic bead extraction of the mycobacteria is an alternative method that can be performed in a cabinet with just a magnet. Magnetic TB-Bead (Microsens Medtech Ltd) extraction of mycobacteria from sputum prior to microscopy was compared to direct smear on 78 sputum samples. Microscopy of the TB-Bead extracts identified all of 26 of the direct smear positive samples either with the same microscopy score or, in 19/27 of samples, with an increased microscopy score which aided microscopy detection. In addition, microscopy of the TB-Bead extracts identified 10 additional positive samples compared to direct smear; which represents a statistically significant increase in case finding of 38% (p = 0.002) compared to direct smear. In a separate study, TB-Beads enabled further 4 positive samples to be detected from 30 centrifuged pellets that were originally smear negative; two of these were subsequently found to be positive when the original deposits were reinvestigated by smear microscopy. By concentrating mycobacteria from sputum and sputum deposits, TB-Beads have been demonstrated to increase the number of positive sputum samples which could increase case-finding. The TB-Bead method is simple and rapid and compatible with use within a safety cabinet. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Alginate/magnetite hybrid beads for magnetically stimulated release of dopamine.

    PubMed

    Kondaveeti, Stalin; Cornejo, Daniel R; Petri, Denise Freitas Siqueira

    2016-02-01

    Hybrid beads composed of magnetite nanoparticles (MNP) and alginate (Alg) were synthesized and coded as Alg-MNP. They were incubated in dopamine (DOPA) solution (5 g/L), at pH 7.4 and 8 °C, during 12 h, promoting the DOPA loaded magnetic beads, coded as Alg-MNP/DOPA. The release of DOPA was further evaluated in the absence and the presence of external magnetic field (EMF) of 0.4 T. The products Alg-MNP and Alg-MNP/DOPA were characterized by scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS), Fourier transform infrared vibrational spectroscopy (FTIR), UV spectrophotometry, thermogravimetric analyses (TGA), inductively coupled plasma atomic emission spectroscopy (ICP-AES) analyses and superconducting quantum interference device (SQUID) magnetometer. The magnetic and chemical properties of Alg-MNP beads were not affected by DOPA loading. The incorporation of DOPA into the beads depended on the pH and on the negative charge density. At pH 7.4 38% of DOPA were loaded into Alg-MNP beads, whereas at pH 2 or using neat Alg beads (lower charge density than Alg-MNP) the loading efficiency decreased to one third or less. In the absence of EMF, 24% of the loaded DOPA was released from Alg-MNP at pH 7.4 over a period of 26 h. The released amount increased to 33% under the stimulus of EMF. A model was proposed to explain the loading efficiency of charged drugs, as DOPA, into hybrid beads and the role played by EMF on delivery systems, where drug and matrix are oppositely charged. The results suggest that the alginate combined with magnetite nanoparticles is a promising system for release of DOPA in the presence of EMF. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. 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.

  3. Magnetically-assisted impedimetric detection of bacteria using phage-modified carbon microarrays.

    PubMed

    Shabani, Arghavan; Marquette, Christophe A; Mandeville, Rosemonde; Lawrence, Marcus F

    2013-11-15

    This study presents an investigation on the possibility of improving the detection limit of bacteria with an inexpensive electrochemical, impedimetric sensor platform, by integrating the sensor with magnetic manipulation. The approach uses T4 bacteriophage coated Dynabeads to selectively capture and concentrate E. coli K12 cells from samples, to increase the sensitivity of detection at the surface of functionalized screen-printed carbon microarrays. Fluorescence and flow cytometry measurements indicate that the surface modification of the magnetic beads, with phages, and binding with the bacteria, were successful. Integration of the screen-printed carbon-based impedimetric sensor, with a magnetic manipulation system, was found to improve the sensitivity of the device, decreasing the limit of detection of E. coli K12 from 10(4) to 10(3) cfu/mL. We have also demonstrated that this approach provides for more specific detection of bacteria, enabling the operator to account for non-specific adsorption, and detection of bacteria in more complex (real) samples (milk).

  4. 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

  5. Design Considerations for CMOS-Integrated Hall-Effect Magnetic Bead Detectors for Biosensor Applications.

    PubMed

    Skucha, K; Gambini, S; Liu, P; Megens, M; Kim, J; Boser, Be

    2013-06-05

    We describe a design methodology for on-chip magnetic bead label detectors based on Hall-effect sensors. Signal errors caused by the label-binding process and other factors that limit the minimum detection area are quantified and adjusted to meet typical assay accuracy standards. The methodology is demonstrated by designing an 8192 element Hall sensor array, implemented in a commercial 0.18 μm CMOS process with single-mask postprocessing. The array can quantify a 1% surface coverage of 2.8 μm beads in 30 seconds with a coefficient of variation of 7.4%. This combination of accuracy and speed makes this technology a suitable detection platform for biological assays based on magnetic bead labels.

  6. Design Considerations for CMOS-Integrated Hall-Effect Magnetic Bead Detectors for Biosensor Applications

    PubMed Central

    Skucha, K.; Gambini, S.; Liu, P.; Megens, M.; Kim, J.; Boser, BE

    2014-01-01

    We describe a design methodology for on-chip magnetic bead label detectors based on Hall-effect sensors. Signal errors caused by the label-binding process and other factors that limit the minimum detection area are quantified and adjusted to meet typical assay accuracy standards. The methodology is demonstrated by designing an 8192 element Hall sensor array, implemented in a commercial 0.18 μm CMOS process with single-mask postprocessing. The array can quantify a 1% surface coverage of 2.8 μm beads in 30 seconds with a coefficient of variation of 7.4%. This combination of accuracy and speed makes this technology a suitable detection platform for biological assays based on magnetic bead labels. PMID:25031503

  7. Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed

    PubMed Central

    Yavuz, Handan; Say, Ridvan; Andaç, Müge; Bayraktar, Necmi; Denizli, Adil

    2004-01-01

    Background Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders. Methods Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed. Results Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity. Conclusions Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium. PMID:15329149

  8. Assesment of dimethyl phthalate removal from aqueous phase using barium hexaferrite containing magnetic beads.

    PubMed

    Osman, Bilgen; Özer, Elif Tümay; Kara, Ali; Güçer, Şeref; Beşirli, Necati

    2012-07-15

    The barium hexaferrite (BaFe(12)O(19)) containing magnetic poly (ethylene glycol dimethacrylate-vinyl pyridine; mag-poly [EGDMA-VP]) beads (average diameter=53-212 μm) were synthesized and characterized. Their use as an adsorbent in the removal of dimethyl phthalate (DMP) from an aqueous solution was investigated. The mag-poly (EGDMA-VP) beads were prepared by copolymerizing of 4-vinyl pyridine (VP) with ethylene glycol dimethacrylate (EGDMA). The mag-poly (EGDMA-VP) beads were characterized by N(2) adsorption/desorption isotherms (BET), vibrating sample magnetometer (VSM), X-ray powder diffraction (XRD), elemental analysis, scanning electron microscope (SEM), and swelling studies. At a fixed solid/solution ratio, the various factors affecting the adsorption of DMP from aqueous solutions such as pH, initial concentration, contact time, and temperature were analyzed. The maximum DMP adsorption capacity of the mag-poly (EGDMA-VP) beads was determined as 96.2 mg/g at pH 3.0, 25 °C. All the isotherm data can be fitted with both the Langmuir and the Dubinin-Radushkevich isotherm models. The pseudo-first-order, pseudo-second-order, Ritch-second-order, and intraparticle diffusion models were used to describe the adsorption kinetics. The thermodynamic parameters obtained indicated the exothermic nature of the adsorption. The DMP adsorption capacity did not change after 10 batch successive reactions, demonstrating the usefulness of the magnetic beads in applications.

  9. Removal of diethyl phthalate from aqueous phase using magnetic poly(EGDMA-VP) beads.

    PubMed

    Özer, Elif Tümay; Osman, Bilgen; Kara, Ali; Beşirli, Necati; Gücer, Seref; Sözeri, Hüseyin

    2012-08-30

    The barium hexaferrite (BaFe(12)O(19)) containing magnetic poly(ethylene glycol dimethacrylate-vinyl pyridine), (mag-poly(EGDMA-VP)) beads (average diameter=53-212 μm) were synthesized and characterized. Their use as an adsorbent in the removal of diethyl phthalate (DEP) from an aqueous solution was investigated. The mag-poly(EGDMA-VP) beads were prepared by copolymerizing of 4-vinyl pyridine (VP) with ethylene glycol dimethacrylate (EGDMA). The mag-poly(EGDMA-VP) beads were characterized by N(2) adsorption/desorption isotherms (BET), vibrating sample magnetometer (VSM), X-ray powder diffraction (XRD), elemental analysis, scanning electron microscope (SEM) and swelling studies. At a fixed solid/solution ratio, the various factors affecting the adsorption of DEP from aqueous solutions such as pH, initial concentration, contact time and temperature were analyzed. The maximum DEP adsorption capacity of the mag-poly(EGDMA-VP) beads was determined as 98.9 mg/g at pH 3.0, 25°C. All the isotherm data can be fitted with both the Langmuir and the Dubinin-Radushkevich isotherm models. The pseudo first-order, pseudo-second-order, Ritch-second-order and intraparticle diffusion models were used to describe the adsorption kinetics. The thermodynamic parameters obtained indicated the exothermic nature of the adsorption. The DEP adsorption capacity did not change after 10 batch successive reactions, demonstrating the usefulness of the magnetic beads in applications.

  10. Specificity and kinetics of norovirus binding to magnetic bead- conjugated histo-blood group antigens

    USDA-ARS?s Scientific Manuscript database

    Histo-blood group antigens (HBGA) have been identified as candidate receptors for human norovirus (NOR). Type A, type H1, and Lewis histo-blood group antigens (HBGAs) in humans have been identified as major targets for NOR binding. Pig HBGA-conjugated magnetic beads have been utilized as a means ...

  11. Attempt to remove peanut allergens from peanut extracts, using IgE-attached magnetic beads.

    USDA-ARS?s Scientific Manuscript database

    Immunoglobulin E (IgE) antibodies from sera of peanut-allergic individuals are known to bind specifically to major peanut allergens, Ara h 1 and Ara h 2. The objective of this study was to determine the efficiency of magnetic beads (Dynabeads) attached with IgE antibodies in the removal of major pea...

  12. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections.

    PubMed

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-03-23

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals.

  13. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

    PubMed Central

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-01-01

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  14. On-line protein capture on magnetic beads for ultrasensitive microfluidic immunoassays of cancer biomarkers.

    PubMed

    Otieno, Brunah A; Krause, Colleen E; Latus, Alina; Chikkaveeraiah, Bhaskara V; Faria, Ronaldo C; Rusling, James F

    2014-03-15

    Accurate, sensitive, multiplexed detection of biomarker proteins holds significant promise for personalized cancer diagnostics. Here we describe the incorporation of a novel on-line chamber to capture cancer biomarker proteins on magnetic beads derivatized with 300,000 enzyme labels and 40,000 antibodies into a modular microfluidic immunoarray. Capture and detection chambers are produced from PDMS on machined molds and do not require lithography. Protein analytes are captured from serum or other biological samples in the stirred capture chamber on the beads held in place magnetically. The beads are subsequently washed free of sample components, and wash solutions sent to waste. Removal of the magnet and valve switching sends the magnetic bead-protein bioconjugates into a detection chamber where they are captured on 8 antibody-decorated gold nanoparticle-film sensors and detected amperometrically. Most steps in the immunoassay including protein capture, washing and measurement are incorporated into the device. In simultaneous assays, the microfluidic system gave ultralow detection limits of 5 fg mL(-1) for interleukin-6 (IL-6) and 7 fg mL(-1) for IL-8 in serum. Accuracy was demonstrated by measuring IL-6 and IL-8 in conditioned media from oral cancer cell lines and showing good correlations with standard ELISAs. The on-line capture chamber facilitates rapid, sensitive, repetitive protein separation and measurement in 30 min in a semi-automated system adaptable to multiplexed protein detection. © 2013 Elsevier B.V. All rights reserved.

  15. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A1

    PubMed Central

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen; Folch-Puy, Emma; Foronjy, Robert; Jalili, Roxana; Jendresen, Christian Bille; Kimura, Masashi; Kraft, Edward; Lindemose, Søren; Lu, Jin; McLain, Teri; Nutt, Leta; Ramon-Garcia, Santiago; Smith, Joseph; Spivak, Aaron; Wang, Michael L.; Zanic, Marija; Lin, Sue-Hwa

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic-bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers. PMID:18765283

  16. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.

    PubMed

    Yeung, Yik A; Wittrup, K Dane

    2002-01-01

    Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.

  17. Antibody-integrated and functionalized graphite-encapsulated magnetic beads, produced using ammonia gas plasma technology, for capturing Salmonella.

    PubMed

    Sakudo, Akikazu; Chou, Han; Nagatsu, Masaaki

    2015-03-01

    Salmonella spp. is the single and most important causative agent of foodborne infections, especially involving foods such as eggs, milk and meat. To prevent infection, a reliable surveillance system is required that can quickly and sensitively detect Salmonella. Here, we describe the development of antibody-integrated magnetic beads that are functionalized by a novel strategy using ammonia gas plasma. Ammonia plasma, produced by a radio frequency (RF) power supply, was allowed to react with the surface of graphite-encapsulated magnetic beads, resulting in the introduction of amino groups. An anti-Salmonella antibody was then anchored by sulfide groups present on the protein surface to the amino groups of the magnetic beads via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The potential usefulness of these magnetic beads for capturing Salmonella was examined as follows. The beads were incubated with Salmonella in liquid medium and then separated from the supernatant by applying a magnetic field. After thorough washing, adsorption of Salmonella to the beads was confirmed by immunochromatography, polymerase chain reaction and a direct culture assay. Our findings indicate that the capture and concentration of Salmonella using the antibody-integrated magnetic beads was more efficient than commercial Dynabeads® anti-Salmonella, which are conventionally used for concentrating Salmonella from liquid cultures. We believe this novel bead technology will contribute to the enhanced detection of Salmonella.

  18. Magnetic bead toy ingestion: uses and disuses in children.

    PubMed

    Adikibi, Boma T; Arnold, Marion; van Niekerk, Gertruida; Alexander, Angus; Numanoglu, Alp; Millar, Alastair J W

    2013-07-01

    A 2-year-old female presented acutely with peritonitis and small bowel obstruction. An abdominal radiograph demonstrated a radiopaque foreign body. At laparotomy she was found to have bowel perforations with entero-enteric fistulae caused by four magnets. The magnets were removed, and debridement and closure of the perforations performed. We review our case and highlight this problem to other medical practitioners as a potential cause of significant morbidity and mortality in the paediatric population.

  19. A Criterion for the Complete Deposition of Magnetic Beads on the Walls of Microchannels.

    PubMed

    Pallares, Jordi

    2016-01-01

    This paper analyzes numerical simulations of the trajectories of magnetic beads in a microchannel, with a nearby permanent cubical magnet, under different flow and magnetic conditions. Analytically derived local fluid velocities and local magnetic forces have been used to track the particles. A centered position and a lateral position of the magnet above the microchannel are considered. The computed fractions of deposited particles on the walls are compared successfully with a new theoretically derived criterion that imposes a relation between the sizes of the magnet and the microchannel and the particle Stokes and Alfvén numbers to obtain the complete deposition of the flowing particles on the wall. In the cases in which all the particles, initially distributed uniformly across the section of the microchannel, are deposited on the walls, the simulations predict the accumulation of the major part of particles on the wall closest to the magnet and near the first half of the streamwise length of the magnet.

  20. Magnetic alginate beads for Pb(II) ions removal from wastewater.

    PubMed

    Bée, Agnès; Talbot, Delphine; Abramson, Sébastien; Dupuis, Vincent

    2011-10-15

    A magnetic adsorbent (called magsorbent) was developed by encapsulation of magnetic functionalized nanoparticles in calcium-alginate beads. The adsorption of Pb(II) ions by these magnetic beads was studied and the effect of different parameters, such as initial concentration, contact time and solution pH value on the adsorption of Pb(II) ions was investigated. Our magsorbent was found to be efficient to adsorb Pb(II) ions and maximal adsorption capacity occurred at pH 2.3-6. The classical Langmuir model used to fit the experimental adsorption data showed a maximum sorption capacity close to 100 mg g(-1). The experimental kinetic data were well correlated with a pseudo second-order model, 50% of the Pb(II) ions were removed within 20 min and the equilibrium was attained around 100 min. Moreover our magsorbent was easily collected from aqueous media by using an external magnetic field. These results permitted to conclude that magnetic alginate beads could be efficiently used to remove heavy metals in a water treatment process.

  1. Capture and Concentration of Waterborne Pathogens Using Lectin and Antibody Coupled Magnetic Beads

    SciTech Connect

    Bennett, Alena M.; Ozanich, Rich M.

    2005-01-01

    Capture and Concentration of Waterborne Pathogens Using Lectin and Antibody Coupled Magnetic Beads. ALENA BENNETT (University of Puget Sound, Tacoma, WA, 98416) RICHARD M. OZANICH, JR. (Pacific Northwest National Laboratory, Richland, WA 99352). The primary challenge of the surveillance of natural and introduced biological threats in large water samples is the purification and concentration process. A method for simultaneously capturing many types of biological pathogens is desired. Lectins coupled with magnetic beads were studied due to their ability to bind to the carbohydrates on the surfaces of cells. With lectin coupled beads we attempted to trap Escherichia coli, Bacillus subtilis, and Brevundimonas diminuta. Also E. coli antibody coupled beads were tested for their effectiveness at concentrating E. coli cells. Bench top indirect and direct cell capture methods were studied for both lectins and antibodies. The indirect method was found to be more effective for cell concentration. Experiments are underway to understand the differences in the two approaches and improve the direct capture method for implementation on an online automated system.

  2. Use of self assembled magnetic beads for on-chip protein digestion.

    PubMed

    Slovakova, Marcela; Minc, Nicolas; Bilkova, Zuzana; Smadja, Claire; Faigle, Wolfgang; Fütterer, Claus; Taverna, Myriam; Viovy, Jean-Louis

    2005-09-01

    The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.

  3. DNA-magnetic bead detection using disposable cards and the anisotropic magnetoresistive sensor

    NASA Astrophysics Data System (ADS)

    Hien, L. T.; Quynh, L. K.; Huyen, V. T.; Tu, B. D.; Hien, N. T.; Phuong, D. M.; Nhung, P. H.; Giang, D. T. H.; Duc, N. H.

    2016-12-01

    A disposable card incorporating specific DNA probes targeting the 16 S rRNA gene of Streptococcus suis was developed for magnetically labeled target DNA detection. A single-stranded target DNA was hybridized with the DNA probe on the SPA/APTES/PDMS/Si as-prepared card, which was subsequently magnetically labeled with superparamagnetic beads for detection using an anisotropic magnetoresistive (AMR) sensor. An almost linear response between the output signal of the AMR sensor and amount of single-stranded target DNA varied from 4.5 to 18 pmol was identified. From the sensor output signal response towards the mass of magnetic beads which were directly immobilized on the disposable card surface, the limit of detection was estimated about 312 ng ferrites, which corresponds to 3.8 μemu. In comparison with DNA detection by conventional biosensor based on magnetic bead labeling, disposable cards are featured with higher efficiency and performances, ease of use and less running cost with respects to consumables for biosensor in biomedical analysis systems operating with immobilized bioreceptor.

  4. Concentric Magnetic Structures for Magnetophoretic Bead Collection, Cell Trapping and Analysis of Cell Morphological Changes Caused by Local Magnetic Forces

    PubMed Central

    Huang, Chen-Yu; Wei, Zung-Hang

    2015-01-01

    Concentric magnetic structures (ring and square) with domain wall (DW) pinning geometry are designed for biological manipulation. Magnetic beads collection was firstly demonstrated to analyse the local magnetic field generated by DWs and the effective regions to capture magnetic targets of size 1 μm. Primary mouse embryonic fibroblasts (MEFs) are magnetically labeled by internalizing poly (styrene sulfonic acid) stabilized magnetic nanoparticles (PSS-MNPs) and then are selectively trapped by head-to-tail DWs (HH DWs) or tail-to-tail DWs (TT DWs) to be arranged into linear shape or cross shape. The morphologies and the nuclear geometry of the cells growing on two kinds of concentric magnetic structures are shown to be distinctive. The intracellular magnetic forces generated by the local magnetic field of DWs are found to influence the behaviour of cells. PMID:26270332

  5. Magnetomechanics of superparamagnetic beads on a magnetic merry-go-round: from micromagnetics to radial looping

    NASA Astrophysics Data System (ADS)

    Sajjad, Umer; Bahne Holländer, Rasmus; Klingbeil, Finn; McCord, Jeffrey

    2017-04-01

    The motion of functionalized superparamagnetic beads provides the foundation for the manipulation of labelled chemical and biological species in microfluidic environments, where patterned ferromagnetic thin films serve as a versatile and reconfigurable platform for biomedical applications. A recurring release and capture of superparamagnetic microbeads is achieved by moving stray magnetic field gradients generated by a circulating micromagnetic state of a soft magnetic disc. The full micromagnetic analysis of the transport dynamics fully describes the fundamental alternating behaviour of microsphere motion. An excellent level of agreement is obtained between experiment and modelling for all stages of motion, confirming the validity of describing particle motion dynamics in the applied quasi ab initio modelling approach. The demonstrated comprehension of the non-linear microbead displacement opens the way towards the modelling of various alternative dynamic excitation schemes, even for complicated integrated micromagnetic platforms, for controlled biological analyte–superparamagnetic bead manipulation.

  6. Efficient capture and simple quantification of circulating tumor cells using quantum dots and magnetic beads.

    PubMed

    Min, Hyegeun; Jo, Seong-Min; Kim, Hak-Sung

    2015-06-03

    Circulating tumor cells (CTCs) are valuable biomarkers for monitoring the status of cancer patients and drug efficacy. However, the number of CTCs in the blood is extremely low, and the isolation and detection of CTCs with high efficiency and sensitivity remain a challenge. Here, we present an approach to the efficient capturing and simple quantification of CTCs using quantum dots and magnetic beads. Anti-EpCAM antibody-conjugated quantum dots are used for the targeting and quantification of CTCs, and quantum-dot-attached CTCs are isolated using anti-IgG-modified magnetic beads. Our approach is shown to result in a capture efficiency of about 70%-80%, enabling the simple quantification of captured CTCs based on the fluorescence intensity of the quantum dots. The present method can be used effectively in the capturing and simple quantification of CTCs with high efficiency for cancer diagnosis and monitoring.

  7. Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

    PubMed

    Hudson, Elton P; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

    2012-01-01

    In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

  8. Magnetic bead assisted labeling of antibodies at nanogram scale.

    PubMed

    Dezfouli, Mahya; Vickovic, Sanja; Iglesias, Maria Jesus; Nilsson, Peter; Schwenk, Jochen M; Ahmadian, Afshin

    2014-01-01

    There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 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.

  10. Salmonella detection in a microfluidic channel using orbiting magnetic beads

    NASA Astrophysics Data System (ADS)

    Ballard, Matt; Mills, Zachary; Owen, Drew; Hanasoge, Srinivas; Hesketh, Peter; Alexeev, Alexander

    2015-03-01

    We use three-dimensional simulations to model the detection of salmonella in a complex fluid sample in a microfluidic channel. Salmonella is captured using magnetic microbeads orbiting around soft ferromagnetic discs at the microchannel bottom subjected to a rotating external magnetic field. Numerical simulations are used to model the dynamics of salmonella and microbeads throughout the detection process. We examine the effect of the channel geometry on the salmonella capture, and the forces applied to the salmonella as it is dragged through the fluid after capture. Our findings guide the design of a lab-on-a-chip device to be used for detection of salmonella in food samples in a way that ensures that salmonella captured by orbiting microbeads are preserved until they can be extracted from the system for testing, and are not washed away by the fluid flow or damaged due to the experience of excessive stresses. Such a device is needed to detect bacteria at the food source and prevention of consumption of contaminated food, and also can be used for the detection of a variety of biomaterials of interest from complex fluid samples. Support from USDA and NSF is gratefully acknowledged.

  11. Specific capture of the hydrolysate on magnetic beads for sensitive detecting plant vacuolar processing enzyme activity.

    PubMed

    Zhou, Jun; Cheng, Meng; Zeng, Lizhang; Liu, Weipeng; Zhang, Tao; Xing, Da

    2016-05-15

    Conventional plant protease detection always suffers from high background interference caused by the complex coloring metabolites in plant cells. In this study, a bio-modified magnetic beads-based strategy was developed for sensitive and quantitative detection of plant vacuolar processing enzyme (VPE) activity. Cleavage of the peptide substrate (ESENCRK-FITC) after asparagine residue by VPE resulted in the 2-cyano-6-amino-benzothiazole (CABT)-functionalized magnetic beads capture of the severed substrate CRK-FITC via a condensation reaction between CABT and cysteine (Cys). The catalytic activity was subsequently obtained by the confocal microscopy imaging and flow cytometry quantitative analysis. The sensor system integrated advantages of (i) the high efficient enrichment and separation capabilities of magnetic beads and (ii) the catalyst-free properties of the CABT-Cys condensation reaction. It exhibited a linear relationship between the fluorescence signal and the concentration of severed substrate in the range of 10-600 pM. The practical results showed that, compared with normal growth conditions, VPE activity was increased by 2.7-fold (307.2 ± 25.3 μM min(-1)g(-1)) upon cadmium toxicity stress. This platform effectively overcame the coloring metabolites-caused background interference, showing fine applicability for the detection of VPE activity in real samples. The strategy offers great sensitivity and may be further extended to other protease activity detection.

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

    PubMed Central

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

    2016-01-01

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

  13. A Magnetic Bead-Based Protein Kinase Assay with Dual Detection Techniques

    PubMed Central

    Zhou, Guangchang; Sylvester, Juliesta E.; Wu, Ding; Veach, Darren R.; Kron, Stephen J.

    2010-01-01

    A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immuno-chemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via non-covalent biotin-streptavidin interactions. This non-covalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immuno-chemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC50 (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immuno-chemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable and inexpensive route to the discovery of small molecule drug leads. PMID:20807497

  14. Cetylpyridinium chloride/magnetic alginate beads: an efficient system to remove p-nitrophenol from wastewater

    NASA Astrophysics Data System (ADS)

    Obeid, Layaly; Bee, Agnes; Talbot, Delphine; Abramson, Sebastien; Welschbillig, Mathias

    2014-05-01

    The adsorption process is one of the most efficient methods to remove pollutants from wastewater provided that suitable adsorbents are used. In order to produce environmentally safe adsorbents, natural polymers have received increasing attention in recent years. Thus, alginate, a polysaccharide extracted from brown seaweeds, is extensively used as inexpensive, non-toxic and efficient biosorbent. Furthermore, it has been shown that the encapsulation of magnetic materials in alginate beads facilitates their recovery from wastewater after the adsorption step, by the use of an external magnetic field gradient, obtained with a magnet or an electromagnet [1, 2]. In the present work, we have studied the adsorption affinity of magnetic alginate beads (called magsorbents)for p-nitrophenol (PNP), used as a hydrophobic pollutant, in presence of cetylpyridinium chloride (CPC), a cationic surfactant. First, the effect of different parameters (pH solution, contact time, surfactant initial concentration…) on the adsorption of CPC on the alginate beads was investigated. Adsorption of the surfactant occurs due to electrostatic attractions between its cationic head groups and negative carboxylate functions of the alginate beads. At larger surfactant concentrations, adsorption is also due to the interaction between the hydrocarbon chains of CPC forming aggregated structures capable of solubilizing hydrophobic solutes. In a second step, we showed that PNP can reach up to 95% of adsorption in the beads in presence of CPC, although the pollutant is poorly adsorbed by alginate in absence of the surfactant. At highest CPC concentrations, desorption occurs as micellar solubilization is preferred over coadsorption. Our magsorbents appear to efficiently remove both cationic surfactant and hydrophobic pollutants and we hope that this fundamental research will be helpful for the future development of magnetically assisted processes in water treatment plants. 1. A.Bee, D.Talbot, S.Abramson, V

  15. Bioinspired methodology for preparing magnetic responsive chitosan beads to be integrated in a tubular bioreactor for biomedical applications.

    PubMed

    Song, Wenlong; Oliveira, Mariana B; Sher, Praveen; Gil, Sara; Nóbrega, J Miguel; Mano, João F

    2013-08-01

    Magnetic responsive chitosan beads were prepared using a methodology inspired by the rolling of water droplets over lotus leaves. Liquid precursors containing chitosan and magnetic microparticles were dispensed in the form of spherical droplets and crosslinked with genipin over synthetic superhydrophobic surfaces. Scanning electronic microscopy, histology and micro-computed tomography were employed to characterize the structure of the prepared composite beads and the inner distribution of the magnetic particles. Cellular metabolic activity tests showed that fibroblasts-like (L929 cell line) can adhere and proliferate on the prepared chitosan beads. We hypothesize that such spherical biomaterials could be integrated in a new concept of tubular bioreactor. The magnetic beads can be immobilized by an external magnetic field at specific positions and may be transported along the bioreactor by the drag of the culture medium flow. The system behavior was also studied through numerical modeling, which allowed to identify the relative importance of the main parameters, and to conclude that the distance between carrier beads plays a major role on their interaction with the culture medium and, consequently, on the overall system performance. In an up-scaled version of this bioreactor, the herein presented system may comprise different chambers in serial or parallel configurations. This constitutes a simple way of preparing magnetic responsive beads combined with a new design of bioreactor, which may find application in biomedicine and biotechnology, including in cell expansion for tissue engineering or for the production of therapeutic proteins to be used in cell therapies.

  16. Zanamivir immobilized magnetic beads for voltammetric measurement of neuraminidase at gold-modified boron doped diamond electrode

    SciTech Connect

    Wahyuni, Wulan Tri; Ivandini, Tribidasari A.; Saepudin, Endang; Einaga, Yasuaki

    2016-04-19

    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. 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%.

  17. Acetylcholinesterase Immobilized on Magnetic Beads for Pesticides Detection: Application to Olive Oil Analysis

    PubMed Central

    Ben Oujji, Najwa; Bakas, Idriss; Istamboulié, Georges; Ait-Ichou, Ihya; Ait-Addi, Elhabib; Rouillon, Régis; Noguer, Thierry

    2012-01-01

    This work presents the development of bioassays and biosensors for the detection of insecticides widely used in the treatment of olive trees. The systems are based on the covalent immobilisation of acetylcholinesterase on magnetic microbeads using either colorimetry or amperometry as detection technique. The magnetic beads were immobilised on screen-printed electrodes or microtitration plates and tested using standard solutions and real samples. The developed devices showed good analytical performances with limits of detection much lower than the maximum residue limit tolerated by international regulations, as well as a good reproducibility and stability. PMID:22969377

  18. Acetylcholinesterase immobilized on magnetic beads for pesticides detection: application to olive oil analysis.

    PubMed

    Ben Oujji, Najwa; Bakas, Idriss; Istamboulié, Georges; Ait-Ichou, Ihya; Ait-Addi, Elhabib; Rouillon, Régis; Noguer, Thierry

    2012-01-01

    This work presents the development of bioassays and biosensors for the detection of insecticides widely used in the treatment of olive trees. The systems are based on the covalent immobilisation of acetylcholinesterase on magnetic microbeads using either colorimetry or amperometry as detection technique. The magnetic beads were immobilised on screen-printed electrodes or microtitration plates and tested using standard solutions and real samples. The developed devices showed good analytical performances with limits of detection much lower than the maximum residue limit tolerated by international regulations, as well as a good reproducibility and stability.

  19. Co-immobilized enzymes in magnetic chitosan beads for improved hydrolysis of macromolecular substrates under a time-varying magnetic field.

    PubMed

    Yang, Kun; Xu, Ning-Shou; Su, Wei Wen

    2010-07-20

    Glucoamylase and alpha-amylase co-immobilized with gamma ferric oxide powders in chitosan beads for consecutive starch liquefaction and saccharification under different magnetic fields was investigated. The chitosan concentration in the beads was found to greatly affect the immobilized enzyme performance. Superior immobilization efficiency and enzyme stability were noted when 2% instead of 4% chitosan was utilized. Using confocal microscopy and scanning electron microscopy, the beads with 2% chitosan were seen to exhibit a more rugged surface topology with more macropores and accommodate more protein near the external surface than with the 4% chitosan beads. An optimum loading ratio between alpha-amylase and glucoamylase exists that gives the highest glucose production, and this ratio varies with the size of the beads. The inclusion of the gamma ferric oxide powders renders the beads magnetically anisotropic and causes them to tumble under a single-phase alternating magnetic field, resulted in increased overall reaction rates. When exposed to a three-phase alternating magnetic field, these beads were stirred vigorously, also leading to enhanced reaction rates. The use of multi-enzyme co-immobilization in magnetic anisotropic chitosan beads may be extended to other practical applications that involve coordinated enzymatic reactions of macromolecular substrates. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  20. Prion protein-coated magnetic beads: synthesis, characterization and development of a new ligands screening method.

    PubMed

    de Moraes, Marcela Cristina; Santos, Juliana Bosco; Dos Anjos, Daniel Meira; Rangel, Luciana Pereira; Vieira, Tuane Cristine Ramos Gonçalves; Moaddel, Ruin; da Silva, Jerson Lima

    2015-01-30

    Prion diseases are characterized by protein aggregation and neurodegeneration. Conversion of the native prion protein (PrP(C)) into the abnormal scrapie PrP isoform (PrP(Sc)), which undergoes aggregation and can eventually form amyloid fibrils, is a critical step leading to the characteristic path morphological hallmark of these diseases. However, the mechanism of conversion remains unclear. It is known that ligands can act as cofactors or inhibitors in the conversion mechanism of PrP(C) into PrP(Sc). Within this context, herein, we describe the immobilization of PrP(C) onto the surface of magnetic beads and the morphological characterization of PrP(C)-coated beads by fluorescence confocal microscopy. PrP(C)-coated magnetic beads were used to identify ligands from a mixture of compounds, which were monitored by UHPLC-ESI-MS/MS. This affinity-based method allowed the isolation of the anti-prion compound quinacrine, an inhibitor of PrP aggregation. The results indicate that this approach can be applied to not only "fish" for anti-prion compounds from complex matrixes, but also to screening for and identify possible cellular cofactors involved in the deflagration of prion diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Prion protein-coated magnetic beads: Synthesis, characterization and development of a new ligands screening method☆

    PubMed Central

    de Moraes, Marcela Cristina; Santos, Juliana Bosco; dos Anjos, Daniel Meira; Rangel, Luciana Pereira; Vieira, Tuane Cristine Ramos Gonçalves; Moaddel, Ruin; da Silva, Jerson Lima

    2016-01-01

    Prion diseases are characterized by protein aggregation and neurodegeneration. Conversion of the native prion protein (PrPC) into the abnormal scrapie PrP isoform (PrPSc), which undergoes aggregation and can eventually form amyloid fibrils, is a critical step leading to the characteristic path morphological hallmark of these diseases. However, the mechanism of conversion remains unclear. It is known that ligands can act as cofactors or inhibitors in the conversion mechanism of PrPC into PrPSc. Within this context, herein, we describe the immobilization of PrPC onto the surface of magnetic beads and the morphological characterization of PrPC-coated beads by fluorescence confocal microscopy. PrPC-coated magnetic beads were used to identify ligands from a mixture of compounds, which were monitored by UHPLC–ESI-MS/MS. This affinity-based method allowed the isolation of the anti-prion compound quinacrine, an inhibitor of PrP aggregation. The results indicate that this approach can be applied to not only “fish” for anti-prion compounds from complex matrixes, but also to screening for and identify possible cellular cofactors involved in the deflagration of prion diseases. PMID:25576041

  2. A novel and rapid assay for HIV-1 protease detection using magnetic bead mediation.

    PubMed

    Esseghaier, Chiheb; Ng, Andy; Zourob, Mohammed

    2013-03-15

    A simple sensing assay was established for label-free detection of HIV-1 protease. HIV-1 protease peptide substrate conjugated to magnetic beads via its N-terminus is directly fixed onto the sensor gold surface through the sulphur atom of cysteine. Surface plasmon resonance (SPR) was used to study the peptide substrate cleavage efficiency of the protease with magnetic beads of different sizes (1 μm and 30 nm). Cyclic voltammetry and faradic impedance spectroscopy were employed in order to characterize the functionalized gold electrode. It was found that the nano-sized beads are a more efficient sensing probe for the protease. Electrochemical biosensing showed a gradual decrease in charge transfer resistance after injection of the HIV-1 protease. The experimental data established a detection limit of 10 pg/ml, as well as demonstrated a drug screening assay. This HIV-1 protease biosensor represents a new detection approach which will lead to low-cost point-of-care devices for sensitive HIV-1 diagnosis, as well as high-throughput drug screening platforms.

  3. Development of an enzymatic reaction device using magnetic bead-cluster handling

    NASA Astrophysics Data System (ADS)

    Shikida, Mitsuhiro; Takayanagi, Kentaro; Honda, Hiroyuki; Ito, Hiroshi; Sato, Kazuo

    2006-09-01

    We previously proposed a magnetic bead-cluster handling device for Micro-total analysis systems (Micro-TAS) and investigated its operation principle. The device does not need mechanical fluidic devices, such as pumps and valves, for handling solutions. We further developed the biochemical reaction unit chip, which is a key component in Micro-TAS, by applying a bead-cluster handling mechanism. We were able to do the enzymatic reaction by using hydrolysis between alkaline phosphatase and p-nitrophenyl phosphate. We also confirmed that the obtained calibration curves were linear during the enzymatic reaction. We had 70% reaction efficiency on the reaction chip by performing a comparative experiment. From these results, we concluded that our developed reaction chip is applicable to enzymatic immuno-assay systems.

  4. 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.

  5. Paramagnetic Beads and Magnetically Mediated Strain Enhance Cardiomyogenesis in Mouse Embryoid Bodies

    PubMed Central

    Geuss, Laura R.; Wu, Douglas C.; Ramamoorthy, Divya; Alford, Corinne D.; Suggs, Laura J.

    2014-01-01

    Mechanical forces play an important role in proper embryologic development, and similarly such forces can directly impact pluripotency and differentiation of mouse embryonic stem cells (mESC) in vitro. In addition, manipulation of the embryoid body (EB) microenvironment, such as by incorporation of microspheres or microparticles, can similarly influence fate determination. In this study, we developed a mechanical stimulation regimen using permanent neodymium magnets to magnetically attract cells within an EB. Arginine-Glycine-Aspartic Acid (RGD)-conjugated paramagnetic beads were incorporated into the interior of the EBs during aggregation, allowing us to exert force on individual cells using short-term magnetization. EBs were stimulated for one hour at different magnetic field strengths, subsequently exerting a range of force intensity on the cells at different stages of early EB development. Our results demonstrated that following exposure to a 0.2 Tesla magnetic field, ESCs respond to magnetically mediated strain by activating Protein Kinase A (PKA) and increasing phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression. The timing of stimulation can also be tailored to guide ESC differentiation: the combination of bone morphogenetic protein 4 (BMP4) supplementation with one hour of magnetic attraction on Day 3 enhances cardiomyogenesis by increasing contractile activity and the percentage of sarcomeric α-actin-expressing cells compared to control samples with BMP4 alone. Interestingly, we also observed that the beads alone had some impact on differentiation by increasingly slightly, albeit not significantly, the percentage of cardiomyocytes. Together these results suggest that magnetically mediated strain can be used to enhance the percentage of mouse ESC-derived cardiomyocytes over current differentiation protocols. PMID:25501004

  6. Resistive pulse sensing of magnetic beads and supraparticle structures using tunable pores

    PubMed Central

    Willmott, Geoff R.; Platt, Mark; Lee, Gil U.

    2012-01-01

    Tunable pores (TPs) have been used for resistive pulse sensing of 1 μm superparamagnetic beads, both dispersed and within a magnetic field. Upon application of this field, magnetic supraparticle structures (SPSs) were observed. Onset of aggregation was most effectively indicated by an increase in the mean event magnitude, with data collected using an automated thresholding method. Simulations enabled discrimination between resistive pulses caused by dimers and individual particles. Distinct but time-correlated peaks were often observed, suggesting that SPSs became separated in pressure-driven flow focused at the pore constriction. The distinct properties of magnetophoretic and pressure-driven transport mechanisms can explain variations in the event rate when particles move through an asymmetric pore in either direction, with or without a magnetic field applied. Use of TPs for resistive pulse sensing holds potential for efficient, versatile analysis and measurement of nano- and microparticles, while magnetic beads and particle aggregation play important roles in many prospective biosensing applications. PMID:22662090

  7. Detection of a magnetic bead by hybrid nanodevices using scanning gate microscopy

    NASA Astrophysics Data System (ADS)

    Corte-León, H.; Krzysteczko, P.; Marchi, F.; Motte, J.-F.; Manzin, A.; Schumacher, H. W.; Antonov, V.; Kazakova, O.

    2016-05-01

    Hybrid ferromagnetic(Py)/non-magnetic metal(Au) junctions with a width of 400 nm are studied by magnetotransport measurements, magnetic scanning gate microscopy (SGM) with a magnetic bead (MB) attached to the probe, and micromagnetic simulations. In the transverse geometry, the devices demonstrate a characteristic magnetoresistive behavior that depends on the direction of the in plane magnetic field, with minimum/maximum variation when the field is applied parallel/perpendicular to the Py wire. The SGM is performed with a NdFeB bead of 1.6 μm diameter attached to the scanning probe. Our results demonstrate that the hybrid junction can be used to detect this type of MB. A rough approximation of the sensing volume of the junction has the shape of elliptical cylinder with the volume of ˜1.51 μm3. Micromagnetic simulations coupled to a magnetotransport model including anisotropic magnetoresistance and planar Hall effects are in good agreement with the experimental findings, enabling the interpretation of the SGM images.

  8. Evidence of protein-free homology recognition in magnetic bead force-extension experiments

    NASA Astrophysics Data System (ADS)

    O'Lee, D. J.; Danilowicz, C.; Rochester, C.; Kornyshev, A. A.; Prentiss, M.

    2016-07-01

    Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data.

  9. Evidence of protein-free homology recognition in magnetic bead force–extension experiments

    PubMed Central

    (O’) Lee, D. J.; Danilowicz, C.; Rochester, C.; Prentiss, M.

    2016-01-01

    Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data. PMID:27493568

  10. Magnetic chitosan beads for covalent immobilization of nucleoside 2'-deoxyribosyltransferase: application in nucleoside analogues synthesis.

    PubMed

    Fernández-Lucas, Jesús; Harris, Ruth; Mata-Casar, Iria; Heras, Angeles; de la Mata, Isabel; Arroyo, Miguel

    2013-09-01

    Cross-linked magnetic chitosan beads were prepared in presence of epichlorohydrin under alkaline conditions, and subsequently incubated with glutaraldehyde in order to obtain an activated support for covalent attachment of nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT). Changing the amount of magnetite (Fe(3)O(4)) and epichlorohydrin (EPI) led to different macroscopic beads to be used as supports for enzyme immobilization, whose morphology and properties were characterized by scanning electron microscopy, spin electron resonance (ESR), and vibrating sample magnetometry (VSM). Once activated with glutaraldehyde, the best support was chosen after evaluation of immobilization yield and product yield in the synthesis of thymidine from 2'-deoxyuridine and thymine. In addition, optimal conditions for highest activity of immobilized LrNDT on magnetic chitosan were determined by response surface methodology (RSM). Immobilized biocatalyst retained 50 % of its maximal activity after 56.3 h at 60 °C, whereas 100 % activity was observed after storage at 40 °C for 144 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of 2'-deoxyribonucleoside analogues as well as arabinosyl-nucleosides such as vidarabine (ara-A) and cytarabine (ara-C). Furthermore, this is the first report which describes the enzymatic synthesis of these arabinosyl-nucleosides catalyzed by an immobilized nucleoside 2'-deoxyribosyltransferase. Finally, the attached enzyme to magnetic chitosan beads could be easily recovered and recycled for 30 consecutive batch reactions with negligible loss of catalytic activity in the synthesis of 2,6-diaminopurine-2'-deoxyriboside and 5-trifluorothymidine.

  11. A Criterion for the Complete Deposition of Magnetic Beads on the Walls of Microchannels

    PubMed Central

    Pallares, Jordi

    2016-01-01

    This paper analyzes numerical simulations of the trajectories of magnetic beads in a microchannel, with a nearby permanent cubical magnet, under different flow and magnetic conditions. Analytically derived local fluid velocities and local magnetic forces have been used to track the particles. A centered position and a lateral position of the magnet above the microchannel are considered. The computed fractions of deposited particles on the walls are compared successfully with a new theoretically derived criterion that imposes a relation between the sizes of the magnet and the microchannel and the particle Stokes and Alfvén numbers to obtain the complete deposition of the flowing particles on the wall. In the cases in which all the particles, initially distributed uniformly across the section of the microchannel, are deposited on the walls, the simulations predict the accumulation of the major part of particles on the wall closest to the magnet and near the first half of the streamwise length of the magnet. PMID:27007336

  12. Magnetic Scanometric DNA Microarray Detection of Methyl Tertiary Butyl Ether Degrading Bacteria for Environmental Monitoring

    PubMed Central

    Chan, Mei-Lin; Jaramillo, Gerardo; Hristova, Krassimira R.; Horsley, David A.

    2010-01-01

    A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary-butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications. PMID:20889328

  13. Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium: The use of magnetic beads conjugated with multiple capture antibodies

    USDA-ARS?s Scientific Manuscript database

    Streptavidin-coated magnetic beads were conjugated with biotinylated capture antibodies to both Escherichia coli O157:H7 and Samonella Typhimurium to form multi-pathogen capture immunomagnetic beads (IMB-M). The efficacy of these beads was investigated and compared to the use of a mixture of IMB ag...

  14. Discrimination of clostridium species using a magnetic bead based hybridization assay

    NASA Astrophysics Data System (ADS)

    Pahlow, Susanne; Seise, Barbara; Pollok, Sibyll; Seyboldt, Christian; Weber, Karina; Popp, Jürgen

    2014-05-01

    Clostridium chauvoei is the causative agent of blackleg, which is an endogenous bacterial infection. Mainly cattle and other ruminants are affected. The symptoms of blackleg are very similar to those of malignant edema, an infection caused by Clostridium septicum. [1, 2] Therefore a reliable differentiation of Clostridium chauvoei from other Clostridium species is required. Traditional microbiological detection methods are time consuming and laborious. Additionally, the unique identification is hindered by the overgrowing tendency of swarming Clostridium septicum colonies when both species are present. [1, 3, 4] Thus, there is a crucial need to improve and simplify the specific detection of Clostridium chauvoei and Clostridium septicum. Here we present an easy and fast Clostridium species discrimination method combining magnetic beads and fluorescence spectroscopy. Functionalized magnetic particles exhibit plentiful advantages, like their simple manipulation in combination with a large binding capacity of biomolecules. A specific region of the pathogenic DNA is amplified and labelled with biotin by polymerase chain reaction (PCR). These PCR products were then immobilized on magnetic beads exploiting the strong biotin-streptavidin interaction. The specific detection of different Clostridium species is achieved by using fluorescence dye labeled probe DNA for the hybridization with the immobilized PCR products. Finally, the samples were investigated by fluorescence spectroscopy. [5

  15. Specific detection of DNA using quantum dots and magnetic beads for large volume samples

    SciTech Connect

    Kim, Yeon S.; Kim, Byoung CHAN; Lee, Jin Hyung; Kim, Jungbae; Gu, Man Bock

    2006-10-01

    Here we present a sensitive DNA detection protocol using quantum dots (QDs) and magnetic beads (MBs) for large volume samples. In this study, QDs, conjugated with streptavidin, were used to produce fluorescent signals while magnetic beads (MBs) were used to isolate and concentrate the signals. The presence of target DNAs lead to the sandwich hybridization between the functionalized QDs, the target DNAs and the MBs. In fact, the QDs-MBs complex, which is bound using the target DNA, can be isolated and then concentrated. The binding of the QDs to the surface of the MBs was confirmed by confocal microscopy and Cd elemental analysis. It was found that the fluorescent intensity was proportional to concentration of the target DNA, while the presence of noncomplementary DNA produced no significant fluorescent signal. In addition, the presence of low copies of target DNAs such as 0.5 pM in large volume samples up to 40 ml were successfully detected by using a magnet-assisted concentration protocol which consequently results in the enhancement of the sensitivity more than 100-fold.

  16. Electrochemical magnetic beads-based immunosensing platform for the determination of α-lactalbumin in milk.

    PubMed

    Ruiz-Valdepeñas Montiel, Víctor; Campuzano, Susana; Torrente-Rodríguez, Rebeca M; Reviejo, A Julio; Pingarrón, José M

    2016-12-15

    Alpha-lactalbumin (α-LA) is one of the whey proteins in cows' milk that has been identified as allergenic. In this work, we present, for the first time, a very sensitive magnetic beads (MBs)-based immunosensor for the determination of α-LA. A sandwich configuration involving selective capture and horseradish peroxidase-labeled detector antibodies was implemented on carboxylic acid-modified magnetic beads, captured magnetically under the surface of a disposable screen-printed carbon electrode for amperometric detection using the hydroquinone (HQ)/H2O2 system. The α-LA immunosensor exhibited a wide linear range (37.0-5000pg/ml), a low limit of detection (LOD, 11.0pg/ml) and noteworthy selectivity against other non-target proteins. The MBs-based immunosensing platform was applied successfully for the determination of α-LA in several varieties of milk (raw and UHT cows' milk as well as human milk) and infant formulations. The results were corroborated with those obtained using a commercial ELISA method, thereby substantiating the analytical merits of this unique method.

  17. Structure and Properties of Magnetic (Co, Fe, Fe{sub 3}C and Ni) Carbon Beads

    SciTech Connect

    Leonowicz, Marcin; Izydorzak, Marta; Pomogailo, Anatolii D.; Dzhardimalieva, Gulzhian I.

    2010-12-02

    Nanoparticles exhibit unique physical properties due to the surface or quantum-size effects. Particular attention has been focused on magnetic nanoparticles and substantial progress has been done in this field. In this work magnetic composites, consisting of elementary metals or carbides nanocrystallites, stabilized in carbon matrix, were prepared by the procedure comprising formation of appropriate metal acrylamide complexes, followed by frontal polymerization and pyrolysis of the polymer at various temperatures. Application of frontal polymerization and further pyrolysis enables formation of composite beads consisting of Co, Fe, Fe{sub 3}C or Ni nanocrystallites stabilized in carbon matrix. It was found that the lowest pyrolysis temperature, which enables the production of metallic nanocrystallites, was 673 K for Co and Ni, and 773 K for Fe. The magnetic properties of the beads, percentage of the metallic component, their composition and shape depended on the pyrolysis temperature. Extracts on the basis of composites containing Fe{sub 3}C showed no cytotoxicity, whereas those containing Co and Ni exhibited negligible cytotoxicity up to concentrations of 6.25 mg/ml.

  18. 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

  19. Self-assembled magnetic bead biosensor for measuring bacterial growth and antimicrobial susceptibility testing.

    PubMed

    Kinnunen, Paivo; McNaughton, Brandon H; Albertson, Theodore; Sinn, Irene; Mofakham, Sima; Elbez, Remy; Newton, Duane W; Hunt, Alan; Kopelman, Raoul

    2012-08-20

    Bacterial antibiotic resistance is one of the major concerns of modern healthcare worldwide, and the development of rapid, growth-based, antimicrobial susceptibility tests is key for addressing it. The cover image shows a self-assembled asynchronous magnetic bead rotation (AMBR) biosensor developed for rapid detection of bacterial growth. Using the biosensors, the minimum inhibitory concentration of a clinical E. coli isolate can be measured within two hours, where currently tests take 6-24 hours. A 16-well prototype is also constructed for simple and robust observation of the self-assembled AMBR biosensors.

  20. Determination of cyclosporin A in 20% ethanol by a magnetic beads-based immunofluorescence assay.

    PubMed

    Kiselev, M V; Gladilin, A K; Melik-Nubarov, N S; Sveshnikov, P G; Miethe, P; Levashov, A V

    1999-05-01

    A rapid magnetic beads-based immunoassay for the immunodepressant drug cyclosporin A (CsA) has been developed. The method allows CsA determination in medium with a higher content of ethanol compared to conventional immunochemical techniques due to increased antibody stability. Monitoring of the drug in ethanol extracts from patient's whole blood without many-fold dilution with aqueous buffer is possible. The assay has adequate specificity and sensitivity for CsA to be suitable for the routine monitoring of therapy. Copyright 1999 Academic Press.

  1. Fabrication of a pen-shaped portable biochemical reaction system based on magnetic bead manipulation

    NASA Astrophysics Data System (ADS)

    Shikida, Mitsuhiro; Inagaki, Noriyuki; Okochi, Mina; Honda, Hiroyuki; Sato, Kazuo

    2011-06-01

    A pen-shaped platform that is similar to a mechanical pencil is proposed for producing a portable reaction system. A reaction unit, as the key component in the system, was produced by using a heat shrinkable tube. A mechanical pencil supplied by Mitsubishi Pencil Co. Ltd was used as the pen-shaped platform for driving the reaction cylinder. It was actuated using an inchworm motion. We confirmed that the magnetic beads were successfully manipulated in the droplet in the cylinder-shaped reaction units.

  2. Fibrous polymer grafted magnetic chitosan beads with strong poly(cation-exchange) groups for single step purification of lysozyme.

    PubMed

    Bayramoglu, Gulay; Tekinay, Turgay; Ozalp, V Cengiz; Arica, M Yakup

    2015-05-15

    Lysozyme is an important polypetide used in medical and food applications. We report a novel magnetic strong cation exchange beads for efficient purification of lysozyme from chicken egg white. Magnetic chitosan (MCHT) beads were synthesized via phase inversion method, and then grafted with poly(glycidyl methacrylate) (p(GMA)) via the surface-initiated atom transfer radical polymerization (SI-ATRP). Epoxy groups of the grafted polymer, were modified into strong cation-exchange groups (i.e., sulfonate groups) in the presence of sodium sulfite. The MCTH and MCTH-g-p(GMA)-SO3H beads were characterized by ATR-FTIR, SEM, and VSM. The sulphonate groups content of the modified MCTH-g-p(GMA)-4 beads was found to be 0.53mmolg(-1) of beads by the potentiometric titration method. The MCTH-g-p(GMA)-SO3H beads were first used as an ion-exchange support for adsorption of lysozyme from aqueous solution. The influence of different experimental parameters such as pH, contact time, and temperature on the adsorption process was evaluated. The maximum adsorption capacity was found to be 208.7mgg(-1) beads. Adsorption of lysozyme on the MCTH-g-p(GMA)-SO3H beads fitted to Langmuir isotherm model and followed the pseudo second-order kinetic. More than 93% of the adsorbed lysozyme was desorbed using Na2CO3 solution (pH 11.0). The purity of the lysozyme was checked by HPLC and SDS gel electrophoresis. In addition, the MCTH-g-p(GMA)-SO3H beads prepared in this work showed promising potential for separation of various anionic molecules.

  3. Identification of serum biomarkers for lung cancer using magnetic bead-based SELDI-TOF-MS

    PubMed Central

    Song, Qi-bin; Hu, Wei-guo; Wang, Peng; Yao, Yi; Zeng, Hua-zong

    2011-01-01

    Aim: To identify novel serum biomarkers for lung cancer diagnosis using magnetic bead-based surface-enhanced laser desorption/ionization time-of-flight mass spectrum (SELDI-TOF-MS). Methods: The protein fractions of 121 serum specimens from 30 lung cancer patients, 30 pulmonary tuberculosis patients and 33 healthy controls were enriched using WCX magnetic beads and subjected to SELDI-TOF-MS. The spectra were analyzed using Bio-marker Wizard version 3.1.0 and Biomarker Patterns Software version 5.0. A diagnostic model was constructed with the marker proteins using a linear discrimination analysis method. The validity of this model was tested in a blind test set consisted of 8 randomly selected lung cancer patients, 10 pulmonary tuberculosis patients and 10 healthy volunteers. Results: Seventeen m/z peaks were identified, which were significantly different between the lung cancer group and the control (tuberculosis and healthy control) groups. Among these peaks, the 6445, 9725, 11705, and 15126 m/z peaks were selected by the Biomarker Pattern Software to construct a diagnostic model for lung cancer. This four-peak model established in the training set could discriminate lung cancer patients from non-cancer patients with a sensitivity of 93.3% (28/30) and a specificity of 90.5% (57/63). The diagnostic model showed a high sensitivity (75.0%) and a high specificity (95%) in the blind test validation. Database searching and literature mining indicated that the featured 4 peaks represented chaperonin (M9725), hemoglobin subunit beta (M15335), serum amyloid A (M11548), and an unknown protein. Conclusion: A lung cancer diagnostic model based on bead-based SELDI-TOF-MS has been established for the early diagnosis or differential diagnosis of lung cancers. PMID:22019958

  4. Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads.

    PubMed

    Duarte, Gabriela R M; Price, Carol W; Littlewood, Janice L; Haverstick, Doris M; Ferrance, Jerome P; Carrilho, Emanuel; Landers, James P

    2010-03-01

    A novel solid phase extraction technique is described where DNA is bound and eluted from magnetic silica beads in a manner where efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the beta-globin gene. By effectively controlling the movement of the solid phase in the presence of a static sample, the issues associated with reproducibly packing a solid phase in a microchannel and maintaining consistent flow rates are eliminated. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 0.6 microL of blood at a concentration which is suitable for PCR amplification. In addition, the technique presented here requires inexpensive, common laboratory equipment, making it easily adopted for both clinical point-of-care applications and on-site forensic sample analysis.

  5. Application of mass transfer theory to biomarker capture by surface functionalized magnetic beads in microcentrifuge tubes.

    PubMed

    Scherr, Thomas F; Markwalter, Christine F; Bauer, Westley S; Gasperino, David; Wright, David W; Haselton, Frederick R

    2017-08-01

    In many diagnostic assays, specific biomarker extraction and purification from a patient sample is performed in microcentrifuge tubes using surface-functionalized magnetic beads. Although assay binding times are known to be highly dependent on sample viscosity, sample volume, capture reagent, and fluid mixing, the theoretical mass transport framework that has been developed and validated in engineering has yet to be applied in this context. In this work, we adapt this existing framework for simultaneous mass transfer and surface reaction and apply it to the binding of biomarkers in clinical samples to surface-functionalized magnetic beads. We discuss the fundamental fluid dynamics of vortex mixing within microcentrifuge tubes as well as describe how particles and biomolecules interact with the fluid. The model is solved over a wide range of parameters, and we present scenarios when a simplified analytical expression would be most accurate. Next, we review of some relevant techniques for model parameter estimation. Finally, we apply the mass transfer theory to practical use-case scenarios of immediate use to clinicians and assay developers. Throughout, we highlight where further characterization is necessary to bridge the gap between theory and practical application. Copyright © 2017. Published by Elsevier B.V.

  6. Viscoelastic Properties Measurement of Human Lymphocytes by Atomic Force Microscopy Based on Magnetic Beads Cell Isolation.

    PubMed

    Li, Mi; Liu, Lianqing; Xiao, Xiubin; Xi, Ning; Wang, Yuechao

    2016-03-28

    Cell mechanics has been proved to be an effective biomarker for indicating cellular states. The advent of atomic force microscopy (AFM) provides an exciting instrument for measuring the mechanical properties of single cells. However, current AFM single-cell mechanical measurements are commonly performed on cell lines cultured in vitro which are quite different from the primary cells in the human body. Investigating the mechanical properties of primary cells from clinical environments can help us to better understand cell behaviors. Here, by combining AFM with magnetic beads cell isolation, the viscoelastic properties of human primary B lymphocytes were quantitatively measured. B lymphocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and CD19 magnetic beads cell isolation. The activity and specificity of the isolated cells were confirmed by fluorescence microscopy. AFM imaging revealed the surface topography and geometric parameters of B lymphocytes. The instantaneous modulus and relaxation time of living B lymphocytes were measured by AFM indenting technique, showing that the instantaneous modulus of human normal B lymphocytes was 2~3 kPa and the relaxation times were 0.03~0.06 s and 0.35~0.55 s. The differences in cellular visocoelastic properties between primary B lymphocytes and cell lines cultured in vitro were analyzed. The study proves the capability of AFM in quantifying the viscoelastic properties of individual specific primary cells from the blood sample of clinical patients, which will improve our understanding of the behaviors of cells in the human body.

  7. Degradation of synthetic pollutants in real wastewater using laccase encapsulated in core-shell magnetic copper alginate beads.

    PubMed

    Le, Thao Thanh; Murugesan, Kumarasamy; Lee, Chung-Seop; Vu, Chi Huong; Chang, Yoon-Seok; Jeon, Jong-Rok

    2016-09-01

    Immobilization of laccase has been highlighted to enhance their stability and reusability in bioremediation. In this study, we provide a novel immobilization technique that is very suitable to real wastewater treatment. A perfect core-shell system composing copper alginate for the immobilization of laccase (Lac-beads) was produced. Additionally, nFe2O3 was incorporated for the bead recycling through magnetic force. The beads were proven to immobilize 85.5% of total laccase treated and also to be structurally stable in water, acetate buffer, and real wastewater. To test the Lac-beads reactivity, triclosan (TCS) and Remazol Brilliant Blue R (RBBR) were employed. The Lac-beads showed a high percentage of TCS removal (89.6%) after 8h and RBBR decolonization at a range from 54.2% to 75.8% after 4h. Remarkably, the pollutants removal efficacy of the Lac-beads was significantly maintained in real wastewater with the bead recyclability, whereas that of the corresponding free laccase was severely deteriorated.

  8. Low-Temperature Magnetic Force Microscopy on Single Molecule Magnet-Based Microarrays.

    PubMed

    Serri, Michele; Mannini, Matteo; Poggini, Lorenzo; Vélez-Fort, Emilio; Cortigiani, Brunetto; Sainctavit, Philippe; Rovai, Donella; Caneschi, Andrea; Sessoli, Roberta

    2017-03-08

    The magnetic properties of some single molecule magnets (SMM) on surfaces can be strongly modified by the molecular packing in nanometric films/aggregates or by interactions with the substrate, which affect the molecular orientation and geometry. Detailed investigations of the magnetism of thin SMM films and nanostructures are necessary for the development of spin-based molecular devices, however this task is challenged by the limited sensitivity of laboratory-based magnetometric techniques and often requires access to synchrotron light sources to perform surface sensitive X-ray magnetic circular dichroism (XMCD) investigations. Here we show that low-temperature magnetic force microscopy is an alternative powerful laboratory tool able to extract the field dependence of the magnetization and to identify areas of in-plane and perpendicular magnetic anisotropy in microarrays of the SMM terbium(III) bis-phthalocyaninato (TbPc2) neutral complex grown as nanosized films on SiO2 and perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA), and this is in agreement with data extracted from nonlocal XMCD measurements performed on homogeneous TbPc2/PTCDA films.

  9. 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.

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

    PubMed Central

    Wei, Chen-Wei; Xia, Jinjun; Hu, Xiaoge; Gao, Xiaohu; O’Donnell, Matthew

    2012-01-01

    Abstract. 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. PMID:23223993

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

    PubMed

    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.

  12. Improvement of extraction capability of magnetic molecularly imprinted polymer beads in aqueous media via dual-phase solvent system.

    PubMed

    Hu, Yuling; Liu, Ruijin; Zhang, Yi; Li, Gongke

    2009-08-15

    In this study, a novel and simple dual-phase solvent system for the improvement of extraction capability of magnetic molecularly imprinted polymer (MIP) beads in aqueous sample was proposed. The method integrated MIP extraction and micro-liquid-liquid extraction (micro-LLE) into only one step. A magnetic MIP beads using atrazine as template was synthesized, and was applied to aqueous media by adding micro-volume of n-hexane to form a co-extraction system. The magnetic MIP beads preferred to suspend in the organic phase, which shielded them from the disturbance of water molecule. The target analytes in the water sample was extracted into the organic phase by micro-LLE and then further bound to the solid-phase of magnetic MIP beads. The beads specificity was significantly improved with the imprinting efficiency of template increasing from 0.5 to 4.4, as compared with that in pure aqueous media. The extraction capacity, equilibration process and cross-selectivity of the MIP dual-phase solvent extraction system were investigated. The proposed method coupled with high-performance liquid chromatography was applied to the analysis of atrazine, simazine, propazine, simetryn, prometryne, ametryn and terbutryn in complicated sample such as tomato, strawberry juice and milk. The method is selective, sensitive and low organic solvent-consuming, and has potential to broaden the range of MIP application in biological and environmental sample.

  13. Flexible programming of cell-free protein synthesis using magnetic bead-immobilized plasmids.

    PubMed

    Lee, Ka-Young; Lee, Kyung-Ho; Park, Ji-Woong; Kim, Dong-Myung

    2012-01-01

    The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different genes in a same reaction mixture. Since the incubation periods of individual template plasmids are freely controllable, relative expression levels of multiple proteins can be tuned to desired levels. We expect that the presented results will find wide application to the flexible design and execution of synthetic pathways in cell-free chassis.

  14. MicroRNA Sensor Based on Magnetic Beads and Enzymatic Probes

    NASA Astrophysics Data System (ADS)

    Zhang, Yue; Zhou, Dejian; He, Junhui

    2014-12-01

    MicroRNAs are associated with multiple cellular processes and diseases. Here, we designed a highly sensitive, magnetically retrievable biosensor using magnetic beads (MBs) as a model RNA sensor. The assay utilized two biotinylated probes, which were hybridized to the complementary target miRNA in a sandwich assay format. One of the biotinylated ends of the hybridization complex was immobilized onto the surface of a NeutrAvidin (NAV) coated MB and the other biotinylated end was conjugated to HRP via NAV-biotin interaction. The results were presented by colorimetric absorbance of the resorufin product from amplex red oxidation. We show that by combining the use of MBs as well as bio-specific immobilization, the sensitivity of miRNA detection is down to 100 pM. This model HRP-MBs system can be used for simple, rapid colorimetric quantification of low level DNA/RNA or other small molecules.

  15. Towards a programmable microfluidic valve: Formation dynamics of two-dimensional magnetic bead arrays in transient magnetic fields

    NASA Astrophysics Data System (ADS)

    Wittbracht, F.; Eickenberg, B.; Weddemann, A.; Hütten, A.

    2011-06-01

    The induction of dipolar coupling has proven to allow for the initiation of self-assembled, reconfigurable particle clusters of superparamagnetic microbeads suspended in a carrier liquid. The adjustment of the interplay between magnetic and hydrodynamic forces opens various possibilities for guiding strategies of these superstructures within microfluidic devices. In this work, the formation dynamics of such particle clusters under the influence of a rotating magnetic field are studied. Different agglomeration regimes are characterized by the dimensionality of the confined objects. The growth dynamics of the obtained agglomerates are analyzed quantitatively in order to deduce the microscopic growth mechanisms. The growth of two-dimensional clusters is governed by the addition of bead chains to previously formed agglomerates. Time scales for the cluster growth are characterized by the chain dissociation rate. Based on the experimental findings, we may conclude to a linear dependence of the chain dissociation rate on the rotation frequency of the applied magnetic field.

  16. Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments.

    PubMed

    Yeh, Chia-Hao; Chen, Shu-Hui; Li, Ding-Tzai; Lin, Hong-Ping; Huang, Hung-Jen; Chang, Chi-I; Shih, Wen-Ling; Chern, Chi-Liang; Shi, Fong-Ku; Hsu, Jue-Liang

    2012-02-10

    Purification of glycopeptides prior to the analysis by mass spectrometry (MS) is demanded due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among various purification methods, hydrophilic interaction liquid chromatography (HILIC) has become a popular method in recent years. In this work, we reported a novel magnetic bead-based zwitterionic HILIC (ZIC-HILIC) material which was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on iron oxide magnetic nanoparticles. The resulting magnetic ZIC-HILIC nanoparticles were shown to provide high specificity and high recovery yield (95-100%) for the enrichment of glycopeptides from a standard glycoprotein, fetuin, using a simple magnetic bar. In addition, we proposed a two-step HILIC enrichment strategy using magnetic ZIC-HILIC nanoparticles for a large scale analysis of glycoproteins in complex biological samples. Using this approach, we identified 85 N-glycosylation sites in 53 glycoproteins from urine samples. Two novel glycosylation sites on N513 of uromodulin and N470 of lysosomal alpha-glucosidase which have not yet been reported were identified by two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies conducted using PNGase F deglycosylation and 18O enzymatic labeling. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. A Magnetic Bead-Based Sensor for the Quantification of Multiple Prostate Cancer Biomarkers

    PubMed Central

    Jokerst, Jesse V.; Chen, Zuxiong; Xu, Lingyun; Nolley, Rosalie; Chang, Edwin; Mitchell, Breeana; Brooks, James D.; Gambhir, Sanjiv S.

    2015-01-01

    Novel biomarker assays and upgraded analytical tools are urgently needed to accurately discriminate benign prostatic hypertrophy (BPH) from prostate cancer (CaP). To address this unmet clinical need, we report a piezeoelectric/magnetic bead-based assay to quantitate prostate specific antigen (PSA; free and total), prostatic acid phosphatase, carbonic anhydrase 1 (CA1), osteonectin, IL-6 soluble receptor (IL-6sr), and spondin-2. We used the sensor to measure these seven proteins in serum samples from 120 benign prostate hypertrophy patients and 100 Gleason score 6 and 7 CaP using serum samples previously collected and banked. The results were analyzed with receiver operator characteristic curve analysis. There were significant differences between BPH and CaP patients in the PSA, CA1, and spondin-2 assays. The highest AUC discrimination was achieved with a spondin-2 OR free/total PSA operation—the area under the curve was 0.84 with a p value below 10−6. Some of these data seem to contradict previous reports and highlight the importance of sample selection and proper assay building in the development of biomarker measurement schemes. This bead-based system offers important advantages in assay building including low cost, high throughput, and rapid identification of an optimal matched antibody pair. PMID:26421725

  18. Comparison between magnetic bead and qPCR library normalisation methods for forensic MPS genotyping.

    PubMed

    Mehta, Bhavik; Venables, Samantha; Roffey, Paul

    2017-04-18

    Massively parallel sequencing (MPS) is fast approaching operational use in forensic science, with the capability to analyse hundreds of DNA identity and DNA intelligence markers in multiple samples simultaneously. The ForenSeq™ DNA Signature Kit on MiSeq FGx™ (Illumina) workflow can provide profiles for autosomal short tandem repeats (STRs), X chromosome and Y chromosome STRs, identity single nucleotide polymorphisms (SNPs), biogeographical ancestry SNPs and phenotype (eye and hair colour) SNPs from a sample. The library preparation procedure involves a series of steps including target amplification, library purification and library normalisation. This study highlights the comparison between the manufacturer recommended magnetic bead normalisation and quantitative polymerase chain reaction (qPCR) methods. Furthermore, two qPCR chemistries, KAPA® (KAPA Biosystems) and NEBNext® (New England Bio Inc.), have also been compared. The qPCR outperformed the bead normalisation method, while the NEBNext® kit obtained higher genotype concordance than KAPA®. The study also established an MPS workflow that can be utilised in any operational forensic laboratory.

  19. Reliable noninvasive genotyping based on excremental PCR of nuclear DNA purified with a magnetic bead protocol.

    PubMed

    Flagstad, O; Røed, K; Stacy, J E; Jakobsen, K S

    1999-05-01

    A new protocol for extraction of DNA from faeces is presented. The protocol involves gentle washing of the surface of the faeces followed by a very simple DNA extraction utilizing the wash supernatant as the source of DNA. Unlike most other protocols, it does not involve the use of proteinase K and/or organic extraction, but is instead based on adsorption of the DNA to magnetic beads. The protocol was tested by microsatellite genotyping across six loci for sheep and reindeer faeces. Comparison with DNA extracted from blood demonstrated that the protocol was very reliable, even when used on material stored for a long time. The protocol was compared with another simple, solid-phase DNA-binding protocol, with the result that the bead-based protocol gave a slightly better amplification success and a lower frequency of allelic drop-outs. Furthermore, our experiments showed that the surface wash prior to DNA extraction is a crucial step, not only for our protocol, but for other solid-phase protocols as well.

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

    PubMed

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

    2012-07-01

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

  1. Magnetic bead-liposome hybrids enable sensitive and portable detection of DNA methyltransferase activity using personal glucose meter.

    PubMed

    Zhang, Youna; Xue, Qingwang; Liu, Jifeng; Wang, Huaisheng

    2017-01-15

    DNA methyltransferase (MTase) plays a critical role in maintaining genome methylation patterns, which has a close relationship to cancer and bacterial diseases. This encouraged the need to develop highly sensitive, simple, and robust assays for DNA MTase detection and inhibitor screening. Herein, a simple, sensitive, and specific DNA MTase activity assay was developed based on magnetic beads-liposome hybrids combined with personal glucose meter (PGM) for quantitative detection of DNA MTase and inhibitor screening. First, a magnetic beads-liposome hybrid probe is designed by the hybridization of p1DNA-functionalized magnetic bead with p2DNA-functionalized glucoamylase-encapsulated liposome (GEL). It integrates target recognition, magnetic separation and signal amplification within one multifunctional design. Then, in the presence of Dam MTase, the hybrids probe was methylated, and cleaved by methylation-sensitive restriction endonuclease Dpn I, making liposome separated from magnetic bead by magnetic separation. Finally, the separated liposome was decomposed, liberating the encapsulated glucoamylase to catalyze the hydrolysis of the signal substrate amylose with multiple turnovers, producing a large amount of glucose for quantitative readout by the PGM. In the proposed assay, the magnetic beads-liposome hybrids offered excellent sensitivity due to primary amplification via releasing numerous glucoamylase from a liposome followed by a secondary enzymatic amplification. The use of portable quantitative device PGM bypasses the requirement of complicated instruments and sophisticated operations, making the method simple and feasible for on-site detection. Moreover, the proposed assay was successfully applied in complex biological matrix and screen suitable inhibitor drugs for DAM for disease(s) treatment. The results reveal that the approach provides a simple, sensitive, and robust platform for DNA MTases detection and screening potential drugs in medical research and

  2. Separation of magnetic beads in a hybrid continuous flow microfluidic device

    NASA Astrophysics Data System (ADS)

    Samanta, Abhishek; Ganguly, Ranjan; Datta, Amitava; Modak, Nipu

    2017-04-01

    Magnetic separation of biological entities in microfluidic environment is a key task for a large number of bio-analytical protocols. In magnetophoretic separation, biochemically functionalized magnetic beads are allowed to bind selectively to target analytes, which are then separated from the background stream using a suitably imposed magnetic field. Here we present a numerical study, characterizing the performance of a magnetophoretic hybrid microfluidic device having two inlets and three outlets for immunomagnetic isolation of three different species from a continuous flow. The hybrid device works on the principle of split-flow thin (SPLITT) fractionation and field flow fractionation (FFF) mechanisms. Transport of the magnetic particles in the microchannel has been predicted following an Eulerian-Lagrangian model and using an in-house numerical code. Influence of the salient geometrical parameters on the performance of the separator is studied by characterizing the particle trajectories and their capture and separation indices. Finally, optimum channel geometry is identified that yields the maximum capture efficiency and separation index.

  3. Frustrated crystallization of a monolayer of magnetized beads under geometrical confinement

    NASA Astrophysics Data System (ADS)

    Schockmel, J.; Vandewalle, N.; Opsomer, E.; Lumay, G.

    2017-06-01

    We present a systematic experimental study of the confinement effect on the crystallization of a monolayer of magnetized beads. The particles are millimeter-scale grains interacting through the short range magnetic dipole-dipole potential induced by an external magnetic field. The grains are confined by repulsing walls and are homogeneously distributed inside the cell. A two-dimensional (2d) Brownian motion is induced by horizontal mechanical vibrations. Therefore, the balance between magnetic interaction and agitation allows investigating 2d phases through direct visualization. The effect of both confinement size and shape on the grains' organization in the low-energy state has been investigated. Concerning the confinement shape, triangular, square, pentagonal, hexagonal, heptagonal, and circular geometries have been considered. The grain organization was analyzed after a slow cooling process. Through the measurement of the averaged bond order parameter for the different confinement geometries, it has been shown that cell geometry strongly affects the ordering of the system. Moreover, many kinds of defects, whose observation rate is linked to the geometry, have been observed: disclinations, dislocations, defects chain, and also more exotic defects such as a rosette. Finally, the influence of confinement size has been investigated and we point out that no finite-size effect occurs for a hexagonal cell, but the finite-size effect changes from one geometry to another.

  4. A method for the rapid discovery of naturally occurring products by proteins immobilized on magnetic beads and reverse affinity chromatography.

    PubMed

    Arai, Midori A; Kobatake, Eiji; Koyano, Takashi; Kowithayakorn, Thaworn; Kato, Shigeaki; Ishibashi, Masami

    2009-12-01

    A highly efficient screening method for naturally occurring products that bind to a specific target protein was demonstrated by using hVDR magnetic beads. The native ligand 1alpha,25(OH)2 VD3 (1) was selectively bound by hVDR magnetic beads when present in a mixture of natural compounds. Furthermore, this method was shown to be applicable to the identification of natural products that interact with a specific protein immobilized on the beads from an extract of a natural resource. Two new natural compounds were isolated by this method. This approach will be helpful for the discovery of novel, naturally occurring products that bind to specific target proteins. This method has the further advantages that it can identify the HPLC peak corresponding to the target compound for isolation, as well as provide important UV, CD, or MS profile information.

  5. The influence of flexible dye-sensitized solar cell modified by different magnetic bead contents

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Jen; Chou, Jung-Chuan; Liao, Yi-Hung; Huang, Chin-Hui; Chu, Chia-Ming

    2017-03-01

    In this study, the influence of different magnetic bead (MB) contents on photovoltaic efficiency of flexible dye-sensitized solar cell (DSSC) was investigated. MB was incorporated into TiO2 film by spin coating method. The short-circuit current density (Jsc), open-circuit voltage (Voc), fill factor (F.F.), and the photovoltaic conversion efficiency (η) of DSSC were measured by digital source meter and solar light simulator, which provided by a 1000 W Xenon (Xe) lamp to simulate sunlight under one sun AM1.5 G (100 mW/cm2) illumination. According to the experimental results, the DSSC modified by MB could improve many photovoltaic characteristics than modified by pure TiO2 working electrode.

  6. Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhao, Yong-Qiang; Song, Jian; Wang, Hong-Li; Xu, Bin; Liu, Feng; He, Kun; Wang, Na

    2016-04-01

    The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity. In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice.

  7. Aptamer capturing of enzymes on magnetic beads to enhance assay specificity and sensitivity.

    PubMed

    Zhao, Qiang; Li, Xing-Fang; Le, X Chris

    2011-12-15

    Activity and specificity of enzyme molecules are important to enzymatic reactions and enzyme assays. We describe an aptamer capturing approach that improves the specificity and the sensitivity of enzyme detection. An aptamer recognizing the target enzyme molecule is conjugated on a magnetic bead, increasing the local concentration, and serves as an affinity probe to capture and separate minute amounts of the enzyme. The captured enzymes catalyze the subsequent conversion of fluorogenic substrate to fluorescent products, enabling a sensitive measure of the active enzyme. The feasibility of this technique is demonstrated through assays for human alpha thrombin and human neutrophil elastase (HNE), two important enzymes. Thrombin (2 fM) and 100 fM HNE can be detected. The incorporation of two binding events, substrate recognition and aptamer binding, greatly improves assay specificity. With its simplicity, this approach is applicable to biosensing and detection of disease biomarkers.

  8. Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment.

    PubMed

    Ficarro, Scott B; Adelmant, Guillaume; Tomar, Maria N; Zhang, Yi; Cheng, Vincent J; Marto, Jarrod A

    2009-06-01

    Qualitative and quantitative analysis of phosphorylation continues to be both an important and a challenging experimental paradigm in proteomics-based research. Unfortunately researchers face difficulties inherent to the optimization of complex, multivariable methods and their application to the analysis of rare and often experimentally intractable phosphorylated peptides. Here we describe a platform based on manipulation of magnetic beads in a 96-well format that facilitates rapid evaluation of experimental parameters required for enrichment of phosphopeptides. Optimized methods provided for automated enrichment and subsequent LC-MS/MS detection of over 1000 unique phosphopeptides (approximately 1% FDR) from 50 microg of cell lysates. In addition we demonstrate use of this platform for identification of phosphopeptides derived from proteins separated by SDS-PAGE and visualized near the detection limit of silver staining.

  9. Magnetic bead-based nucleic acid purification kit: Clinical application and performance evaluation in stool specimens.

    PubMed

    Yoon, Jihoon G; Kang, Jin Seok; Hwang, Seung Yong; Song, Jaewoo; Jeong, Seok Hoon

    2016-05-01

    Two different methods - the semi-automated magnetic bead-based kit (SK, Stool DNA/RNA Purification kit®) and the manual membrane column-based kit (QS, QIAamp® DNA Stool Mini kit) - for purifying nucleic acids from clinical stool samples were compared and evaluated. The SK kit was more user-friendly than QS due to the reduced manual processing, partial automation, and short turnaround time with half cost. Furthermore, SK produced high yields in both DNA and RNA extractions but poor purity in RNA extraction. In the assessment of rotavirus and Clostridium difficile infection, both kits had equivalent or more sensitive performance compared with the standard method. Although SK showed some interference and inhibition in nucleic acid extraction, the performance, including the repeatability, linearity, analytical sensitivity, and matrix effect, was sufficient for routine clinical use. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Magnetic bead purification of labeled DNA fragments forhigh-throughput capillary electrophoresis sequencing

    SciTech Connect

    Elkin, Christopher; Kapur, Hitesh; Smith, Troy; Humphries, David; Pollard, Martin; Hammon, Nancy; Hawkins, Trevor

    2001-09-15

    We have developed an automated purification method for terminator sequencing products based on a magnetic bead technology. This 384-well protocol generates labeled DNA fragments that are essentially free of contaminates for less than $0.005 per reaction. In comparison to laborious ethanol precipitation protocols, this method increases the phred20 read length by forty bases with various DNA templates such as PCR fragments, Plasmids, Cosmids and RCA products. Our method eliminates centrifugation and is compatible with both the MegaBACE 1000 and ABIPrism 3700 capillary instruments. As of September 2001, this method has produced over 1.6 million samples with 93 percent averaging 620 phred20 bases as part of Joint Genome Institutes Production Process.

  11. Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry.

    PubMed

    Zhao, Yong-Qiang; Song, Jian; Wang, Hong-Li; Xu, Bin; Liu, Feng; He, Kun; Wang, Na

    2016-04-01

    The protein toxin ricin obtained from castor bean plant (Ricinus communis) seeds is a potent biological warfare agent due to its ease of availability and acute toxicity. In this study, we demonstrated a rapid and simple method to detect ricin in serum in vitro. The ricin was mixed with serum and digested by trypsin, then all the peptides were efficiently extracted using Cu-chelated magnetic beads and were detected with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The specific ricin peptides were identified by Nanoscale Ultra Performance liquid chromatography coupled to tandem mass spectrometry according to their sequences. The assay required 2.5 hours, and a characteristic peptide could be detected down to 4 ng/μl and used as a biomarker to detect ricin in serum. The high sensitivity and simplicity of the procedure makes it valuable in clinical practice. Graphical Abstract ᅟ.

  12. The Application of Magnetic Bead Selection to Investigate Interactions between the Oral Microbiota and Salivary Immunoglobulins

    PubMed Central

    Madhwani, Tejal

    2016-01-01

    The effect of humoral immunity on the composition of the oral microbiota is less intensively investigated than hygiene and diet, in part due to a lack of simple and robust systems for investigating interactions between salivary immunoglobulins and oral bacteria. Here we report the application of an ex situ method to investigate the specificity of salivary immunoglobulins for salivary bacteria. Saliva collected from six volunteers was separated into immunoglobulin and microbial fractions, and the microbial fractions were then directly exposed to salivary immunoglobulins of “self” and “non-self” origin. Antibody-selected bacteria were separated from their congeners using a magnetic bead system, selective for IgA or IgG isotypes. The positively selected fractions were then characterized using gel-based eubacterial-specific DNA profiling. The eubacterial profiles of positively selected fractions diverged significantly from profiles of whole salivary consortia based on volunteer (P≤ 0.001%) and immunoglobulin origin (P≤ 0.001%), but not immunoglobulin isotype (P = 0.2). DNA profiles of separated microbial fractions were significantly (p≤ 0.05) less diverse than whole salivary consortia and included oral and environmental bacteria. Consortia selected using self immunoglobulins were generally less diverse than those selected with immunoglobulins of non-self origin. Magnetic bead separation facilitated the testing of interactions between salivary antibodies and oral bacteria, showing that these interactions are specific and may reflect differences in recognition by self and non-self immunoglobulins. Further development of this system could improve understanding of the relationship between the oral microbiota and the host immune system and of mechanisms underlying the compositional stability of the oral microbiota. PMID:27483159

  13. Multiplexed electrochemical immunoassay of biomarkers using metal sulfide quantum dot nanolabels and trifunctionalized magnetic beads.

    PubMed

    Tang, Dianping; Hou, Li; Niessner, Reinhard; Xu, Mingdi; Gao, Zhuangqiang; Knopp, Dietmar

    2013-08-15

    A novel multiplexed stripping voltammetric immunoassay protocol was designed for the simultaneous detection of multiple biomarkers (CA 125, CA 15-3, and CA 19-9 used as models) using PAMAM dendrimer-metal sulfide quantum dot (QD) nanolabels as distinguishable signal tags and trifunctionalized magnetic beads as an immunosensing probe. The probe was prepared by means of co-immobilization of primary monoclonal anti-CA 125, anti-CA 15-3 and anti-CA 19-9 antibodies on a single magnetic bead. The PAMAM dendrimer-metal sulfide QD nanolabels containing CdS, ZnS and PbS were synthesized by using in situ synthesis method, which were utilized for the labeling of polyclonal rabbit anti-CA 125, anti-CA 15-3 and anti-CA 19-9 detection antibodies, respectively. A sandwich-type immunoassay format was adopted for the simultaneous determination of target biomarkers in a low-binding microtiter plate. The subsequent anodic stripping voltammetric analysis of cadmium, zinc, and lead components released by acid from the corresponding QD nanolabels was conducted at an in situ prepared mercury film electrode based on the difference of peak potentials. Experimental results indicated that the multiplexed immunoassay enabled the simultaneous detection of three cancer biomarkers in a single run with wide dynamic ranges of 0.01-50 U mL(-1) and detection limits (LODs) of 0.005 U mL(-1). Intra-assay and inter-assay coefficients of variation (CVs) were less than 7.2% and 10.4%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum specimens between the multiplexed immunoassay and a commercially available enzyme-linked immunosorbent assay (ELISA). Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Rapid Isolation of Staphylococcus aureus Pathogens from Infected Clinical Samples Using Magnetic Beads Coated with Fc-Mannose Binding Lectin

    PubMed Central

    Seiler, B.; Gamini, N.; Rodas, M.; Penary, M.; Giordano, G.; Oswald, E.; Super, M.; Ingber, D. E.

    2016-01-01

    Here we describe how Staphylococcus aureus bacteria can be rapidly isolated from clinical samples of articular fluid and synovial tissue using magnetic beads coated with the engineered chimeric human opsonin protein, Fc-mannose-binding lectin (FcMBL). The FcMBL-beads were used to capture and magnetically remove bacteria from purified cultures of 12 S. aureus strains, and from 8 articular fluid samples and 4 synovial tissue samples collected from patients with osteoarthritis or periprosthetic infections previously documented by positive S. aureus cultures. While the capture efficiency was high (85%) with purified S. aureus strains grown in vitro, direct FcMBL-bead capture from the clinical samples was initially disappointing (< 5% efficiency). Further analysis revealed that inhibition of FcMBL binding was due to coating of the bacteria by immunoglobulins and immune cells that masked FcMBL binding sites, and to the high viscosity of these complex biological samples. Importantly, capture of pathogens using the FcMBL-beads was increased to 76% efficiency by pretreating clinical specimens with hypotonic washes, hyaluronidase and a protease cocktail. Using this approach, S. aureus bacteria could be isolated from infected osteoarthritic tissues within 2 hours after sample collection. This FcMBL-enabled magnetic method for rapid capture and concentration of pathogens from clinical samples could be integrated upstream of current processes used in clinical microbiology laboratories to identify pathogens and perform antibiotic sensitivity testing when bacterial culture is not possible or before colonies can be detected. PMID:27275840

  15. Construction of hydrodynamic bead models from high-resolution X-ray crystallographic or nuclear magnetic resonance data.

    PubMed Central

    Byron, O

    1997-01-01

    Computer software such as HYDRO, based upon a comprehensive body of theoretical work, permits the hydrodynamic modeling of macromolecules in solution, which are represented to the computer interface as an assembly of spheres. The uniqueness of any satisfactory resultant model is optimized by incorporating into the modeling procedure the maximal possible number of criteria to which the bead model must conform. An algorithm (AtoB, for atoms to beads) that permits the direct construction of bead models from high resolution x-ray crystallographic or nuclear magnetic resonance data has now been formulated and tested. Models so generated then act as informed starting estimates for the subsequent iterative modeling procedure, thereby hastening the convergence to reasonable representations of solution conformation. Successful application of this algorithm to several proteins shows that predictions of hydrodynamic parameters, including those concerning solvation, can be confirmed. PMID:8994627

  16. Detection Techniques for Biomolecules using Semi-Conductor Nanocrystals and Magnetic Beads as Labels

    NASA Astrophysics Data System (ADS)

    Chatterjee, Esha

    Continued interest in the development of miniaturized and portable analytical platforms necessitates the exploration of sensitive methods for the detection of trace analytes. Nanomaterials, on account of their unique physical and chemical properties, are not only able to overcome many limitations of traditional detection reagents but also enable the exploration of many new signal transduction technologies. This dissertation presents a series of investigations of alternative detection techniques for biomolecules, involving the use of semi-conductor nanocrystals and magnetic beads as labels. Initial research focused on the development of quantum dot-encapsulating liposomes as a novel fluorescent label for immunoassays. This hybrid nanomaterial was anticipated to overcome the drawbacks presented by traditional fluorophores as well as provide significant signal amplification. Quantum dot-encapsulating liposomes were synthesized by the method of thin film hydration and characterized. The utility of these composite nanostructures for bioanalysis was demonstrated. However, the longterm instability of the liposomes hampered quantitative development. A second approach for assay development exploited the ability of gold nanoparticles to quench the optical signals obtained from quantum dots. The goal of this study was to demonstrate the feasibility of using aptamer-linked nanostructures in FRET-based quenching for the detection of proteins. Thrombin was used as the model analyte in this study. Experimental parameters for the assay were optimized. The assay simply required the mixing of the sample with the reagents and could be completed in less than an hour. The limit of detection for thrombin by this method was 5 nM. This homogeneous assay can be easily adapted for the detection of a wide variety of biochemicals. The novel technique of ferromagnetic resonance generated in magnetic bead labels was explored for signal transduction. This inductive detection technique lends

  17. Electrochemical biotin detection based on magnetic beads and a new magnetic flow cell for screen printed electrode.

    PubMed

    Biscay, Julien; González García, María Begoña; Costa García, Agustín

    2015-01-01

    The use of the first flow-cell for magnetic assays with an integrated magnet is reported here. The flow injection analysis system (FIA) is used for biotin determination. The reaction scheme is based on a one step competitive assay between free biotin and biotin labeled with horseradish peroxidase (B-HRP). The mixture of magnetic beads modified with streptavidin (Strep-MB), biotin and B-HRP is left 15 min under stirring and then a washing step is performed. After that, 100 μL of the mixture is injected and after 30s 100 μL of 3,3',5,5'-Tetramethylbenzidine (TMB) is injected and the FIAgram is recorded applying a potential of -0.2V. The linear range obtained is from 0.01 to 1 nM of biotin and the sensitivity is 758 nA/nM. The modification and cleaning of the electrode are performed in an easy way due to the internal magnet of the flow cell.

  18. Rotation-Driven Microfluidic Disc for White Blood Cell Enumeration Using Magnetic Bead Aggregation.

    PubMed

    Ouyang, Yiwen; Li, Jingyi; Haverstick, Doris M; Landers, James P

    2016-11-15

    We recently defined a magnetic bead-based assay that exploited an agglutination-like response for DNA and applied it to DNA-containing cell enumeration using inexpensive benchtop hardware [ J. Am. Chem. Soc. 2012 , 134 ( 12 ), 5689 - 96 ]. Although cost-efficient, the open-well format assay required numerous manual steps, and the magnetic field actuation scheme was not readily adaptable for integration. Here, we demonstrate a low-cost (<$2 in-lab), higher-throughput "pinwheel assay" platform that relies on a combination of a disposable rotation-driven microdisc (RDM), and a simple bidirectional rotating magnetic field (bi-RMF). The assay was transformed into an integrated microfluidic system using a multilayered polyester microfluidic disc created through laser print, cut and laminate fabrication, with fluid flow controlled by rotation speed without any mechanical valves. The RDM accepts four samples that undergo on-chip dilution to five different concentrations that cover the effective concentration range needed for downstream cell counting by pinwheel assay. We show that a bi-RMF is effective for the simultaneous actuation of pinwheel assays in 20 detection chambers. The optimization of the bi-RMF frequencies allows the RDM-based pinwheel assay detect human genomic DNA down to a mass of human genomic DNA (5.5 picograms) that is roughly equal to the mass in a single cell. For proof of principle, enumeration of the white blood cells in human blood samples on the RDM provided data correlating well (C.V. of 10%) with those obtained in a clinical lab. Fusing the cost-effective RDM with a simple bi-RMF provides a promising strategy for automation and multiplexing of magnetic particle-based agglutination assays.

  19. Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics.

    PubMed

    Ngo, Hoan T; Gandra, Naveen; Fales, Andrew M; Taylor, Steve M; Vo-Dinh, Tuan

    2016-07-15

    One of the major obstacles to implement nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is the lack of sensitive and practical DNA detection methods that can be seamlessly integrated into portable platforms. Herein we present a sensitive yet simple DNA detection method using a surface-enhanced Raman scattering (SERS) nanoplatform: the ultrabright SERS nanorattle. The method, referred to as the nanorattle-based method, involves sandwich hybridization of magnetic beads that are loaded with capture probes, target sequences, and ultrabright SERS nanorattles that are loaded with reporter probes. Upon hybridization, a magnet was applied to concentrate the hybridization sandwiches at a detection spot for SERS measurements. The ultrabright SERS nanorattles, composed of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for signal detection. Using this method, a specific DNA sequence of the malaria parasite Plasmodium falciparum could be detected with a detection limit of approximately 100 attomoles. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. These test models demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. Furthermore, the method's simplicity makes it a suitable candidate for integration into portable platforms for POC and in resource-limited settings applications.

  20. Magnetic bead technology for viral RNA extraction from serum in blood bank screening.

    PubMed

    Albertoni, Guilherme Ambrozio; Arnoni, Carine Prisco; Araujo, Patricia Regina Barboza; Andrade, Sheila Siqueira; Carvalho, Fabrício Oliveira; Girão, Manoel João Batista Castello; Schor, Nestor; Barreto, José Augusto

    2011-01-01

    Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.

  1. Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics

    PubMed Central

    Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

    2016-01-01

    One of the major obstacles to implement nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is the lack of sensitive and practical DNA detection methods that can be seamlessly integrated into portable platforms. Herein we present a sensitive yet simple DNA detection method using a surface-enhanced Raman scattering (SERS) nanoplatform: the ultrabright SERS nanorattle. The method, referred to as the nanorattle-based method, involves sandwich hybridization of magnetic beads that are loaded with capture probes, target sequences, and ultrabright SERS nanorattles that are loaded with reporter probes. Upon hybridization, a magnet was applied to concentrate the hybridization sandwiches at a detection spot for SERS measurements. The ultrabright SERS nanorattles, composed of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for signal detection. Using this method, a specific DNA sequence of the malaria parasite Plasmodium falciparum could be detected with a detection limit of approximately 100 attomoles. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. These test models demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. Furthermore, the method’s simplicity makes it a suitable candidate for integration into portable platforms for POC and in resource-limited settings applications. PMID:26913502

  2. Magnetic Bead-Based Colorimetric Immunoassay for Aflatoxin B1 Using Gold Nanoparticles

    PubMed Central

    Wang, Xu; Niessner, Reinhard; Knopp, Dietmar

    2014-01-01

    A competitive colorimetric immunoassay for the detection of aflatoxin B1 (AFB) has been established using biofunctionalized magnetic beads (MBs) and gold nanoparticles (GNPs). Aflatoxin B1-bovine serum albumin conjugates (AFB-BSA) modified MBs were employed as capture probe, which could specifically bind with GNP-labeled anti-AFB antibodies through immunoreaction, while such specific binding was competitively inhibited by the addition of AFB. After magnetic separation, the supernatant solution containing unbound GNPs was directly tested by UV-Vis spectroscopy. The absorption intensity was directly proportional to the AFB concentration. The influence of GNP size, incubation time and pH was investigated in detail. After optimization, the developed method could detect AFB in a linear range from 20 to 800 ng/L, with the limit of detection at 12 ng/L. The recoveries for spiked maize samples ranged from 92.8% to 122.0%. The proposed immunoassay provides a promising approach for simple, rapid, specific and cost-effective detection of toxins in the field of food safety. PMID:25405511

  3. High-Throughput N-Glycan Analysis with Rapid Magnetic Bead-Based Sample Preparation.

    PubMed

    Szigeti, Marton; Guttman, Andras

    2017-01-01

    N-glycan profiling of therapeutic glycoproteins is essential to ensure the activity and efficacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than half of the biopharmaceuticals are glycoproteins representing multibillion dollar worldwide business, further emphasizing the importance of their analysis. In the biomedical field, on the other hand, revealing disease-related glycan structure alterations holds the promise of the discovery of new biomarkers for early diagnostics. Therefore, there is a great demand for widely applicable, high-throughput sample preparation and analysis methods for N-glycan profiling of glycoproteins. One of the newest exciting developments of the field is the magnetic bead based glycoprotein sample preparation technique. A detailed protocol of this method is given in this chapter in conjunction with rapid capillary electrophoresis analysis of the prepared samples by laser induced fluorescence detection (CE-LIF). N-glycans are digested by the endoglycosidase PNGase F and the released carbohydrates are labeled with the charged fluorophore dye of aminopyrenetrisulfonate (APTS). Effective glycan capture by magnetic microparticles enabled fast, easily automated sample preparation both in individual (single vial) and 96-well plate formats, including excess dye removal. Rapid separation of APTS labeled IgG glycans is also shown utilizing an optimized CE-LIF protocol.

  4. An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads.

    PubMed

    Tao, Xiaoqi; Jiang, Haiyang; Yu, Xuezhi; Zhu, Jinghui; Wang, Xia; Wang, Zhanhui; Niu, Lanlan; Wu, Xiaoping; Shen, Jianzhong

    2013-05-01

    A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. Copyright © 2013 John Wiley & Sons, Ltd.

  5. Theory and simulation of the dynamics, deformation, and breakup of a chain of superparamagnetic beads under a rotating magnetic field

    NASA Astrophysics Data System (ADS)

    Vázquez-Quesada, A.; Franke, T.; Ellero, M.

    2017-03-01

    In this work, an analytical model for the behavior of superparamagnetic chains under the effect of a rotating magnetic field is presented. It is postulated that the relevant mechanisms for describing the shape and breakup of the chains into smaller fragments are the induced dipole-dipole magnetic force on the external beads, their translational and rotational drag forces, and the tangential lubrication between particles. Under this assumption, the characteristic S-shape of the chain can be qualitatively understood. Furthermore, based on a straight chain approximation, a novel analytical expression for the critical frequency for the chain breakup is obtained. In order to validate the model, the analytical expressions are compared with full three-dimensional smoothed particle hydrodynamics simulations of magnetic beads showing excellent agreement. Comparison with previous theoretical results and experimental data is also reported.

  6. Lab-on-a-chip for ultrasensitive detection of carbofuran by enzymatic inhibition with replacement of enzyme using magnetic beads.

    PubMed

    Llopis, Xavier; Pumera, Martin; Alegret, Salvador; Merkoçi, Arben

    2009-01-21

    In this paper an ultrasensitive method to determine toxicity due to pesticides in a glass lab-on-a-chip by means of enzymatic inhibition of acetylcholinesterase immobilised on magnetic beads is described. The reproducible insertion of a controlled amount of enzyme-coupled magnetic beads inside the chip channel and their immobilisation in a capture region with the aid of a magnetic field has been optimised. This procedure enables the easy renewal of the biosensing material after each determination in a highly reproducible manner. Several operational parameters such as the working potential for the selective detection of thiocholine (TCh) on a platinum disc electrode, the TCh detection reproducibility and sensitivity, the electroosmotic flow driving voltage and the inhibition time were also evaluated or optimised. The detection of carbofuran (one of the most toxic carbamate pesticides) has been achieved down to the nanomolar level.

  7. Ion exchange kinetics of magnetic alginate ferrogel beads produced by external gelation.

    PubMed

    Teixeira, Vânea Ferreira Torres; Pereira, Nádia Rosa; Waldman, Walter Ruggeri; Ávila, Ana Luiza Cassiano Dias; Pérez, Victor Haber; Rodríguez, Rubén Jesus Sánchez

    2014-10-13

    This paper reports on a study of the influence of sodium alginate concentration and iron addition on the ion exchange kinetics of calcium alginate ferrogel beads produced by external gelation. The calcium absorption and sodium release of the beads were fitted to Fick's second law for unsteady state diffusion in order to obtain the effective diffusion coefficients of Na(+) and Ca(2+). The dried beads were characterized concerning their thermal stability, particle size distribution and morphology. The gelation kinetics showed that an increase in alginate concentration from 1% to 2% increased the Ca(2+) equilibrium concentration, but presented no effect on Ca(2+) effective diffusion coefficient. Alginate concentration higher than 2% promoted saturation of binding sites at the bead surfaces. The addition of iron promoted faster diffusion of Ca(2+) inside the gel beads and reduced the Ca(2+) equilibrium concentration. Also, iron particles entrapped in the alginate gel beads promoted greater absorption of water compared to pure alginate gel and lower thermal stability of the beads. The main diffusion of Ca(2+) into and Na(+) out from the bead took place during the first 60 min, during which almost 85-90% of the Ca(2+) equilibrium concentration is achieved, indicating that this period is sufficient to produce a Ca-alginate bead with high crosslinking of the polymer network. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Preparation of magnetic multi-functional molecularly imprinted polymer beads for determining environmental estrogens in water samples.

    PubMed

    Lin, Zhenkun; He, Qiaoya; Wang, Lituo; Wang, Xuedong; Dong, Qiaoxiang; Huang, Changjiang

    2013-05-15

    Magnetic multi-functional molecularly imprinted polymer (mag-MFMIP) beads were prepared for determining environmental estrogens in water samples. This novel material adopted a superparamagnetic microparticle as the supporter and used 17β-estradiol (E2) as the imprinting template, aiming for recognizing many kinds of environmental estrogens simultaneously. Characterization analysis and binding experiments revealed that mag-MFMIP beads had outstanding magnetic property, large adsorption capacity and high competitive selectivity to most of the commonly seen environmental estrogens. When mag-MFMIP beads were used as dispersed solid-phase extraction (SPE) adsorbents in water samples, the recoveries of estriol (E3), bisphenol A (BPA), E2 and ethynylestradiol (EE) were 72.2 - 92.1%, 89.3 - 96.0%, 93.3 - 102% and 89.7 - 95.9%, respectively with relative standard deviation (RSD) lower than 7.0%. These results indicated that mag-MFMIP beads had the potential to be the common adsorbents for many kinds of environmental estrogens, representing a novel application of MIPs in the field of environmental chemistry. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

    NASA Astrophysics Data System (ADS)

    Xie, Xin; Zhang, Xu; Yu, Bingbin; Gao, Huafang; Zhang, Huan; Fei, Weiyang

    2004-09-01

    A series of simplified protocols are developed for extracting genomic DNA from saliva by using the magnetic nanobeads as absorbents. In these protocols, both the enrichment of the target cells and the adsorption of DNA can be achieved simultaneously by our functionally modified magnetic beads in one step, and the DNA-nanobeads complex can be used as PCR templates. HLA typing based on an oligonucleotide array was conducted by hybridization with the PCR products. The result shows that the protocols are robust and sensitive.

  10. Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions.

    PubMed Central

    Lund, V; Schmid, R; Rickwood, D; Hornes, E

    1988-01-01

    Dynabeads are magnetic monosized beads with high stability, high uniformity, unique paramagnetic properties, low particle-particle interaction, and high dispersibility. Different reactive groups; hydroxyl, carboxyl and amino groups can be attached to the surface. Several methods for covalent attachment of DNA or oligonucleotides to the beads were investigated. Best coupling yields were obtained by carbodiimide-mediated end-attachment of 5'-phosphate and 5'-NH2 modified nucleic acids to respectively amino and carboxyl beads. The carboxyl beads showed a low degree of non-specific binding, while a better yield of end-attached nucleic acids was obtained using the amino beads. The DNA-beads worked efficiently in hybridization experiments, and the kinetics of hybridization approach those of solution hybridization. PMID:3205723

  11. A sandwich immunoassay for brucellosis diagnosis based on immune magnetic beads and quantum dots.

    PubMed

    Li, Li; Yin, Dehui; Xu, Kun; Liu, Yushen; Song, Dandan; Wang, Juan; Zhao, Chao; Song, Xiuling; Li, Juan

    2017-07-15

    Brucellosis is a serious zoonosis with a rapid increase in incidence across epidemic regions. Currently, there are numerous methods for diagnosing brucellosis. However, these studies often have a few defects, such as low sensitivity, poor specificity, time consuming, laborious, and even potential biological risk. To overcome these shortcomings, we have developed a rapid, sensitive and accurate diagnosis procedure for brucellosis based on the immune magnetic beads (IMB) probe and quantum dots (QDs) - staphylococcal protein A (SPA) probe. With the presence of Brucella antibody in the tested serum, the QDs-SPA probe links to the IMB probe and an immune-sandwich complex is formed. As a result, the fluorescence intensity from QDs increased significantly and was correlated with the amount of Brucella antibody. Under the optimized conditions, 248 blood samples were detected and the diagnosis effect was evaluated. The area under the receiver-operating characteristic (ROC) curve was 0.970 (95% confidence interval (CI), 0.9479-0.9920). The diagnostic sensitivity was 96.15% (95% CI, 91.82-98.58%), the diagnostic specificity was 94.12% (95% CI, 87.64-97.81%) with a fluorescence intensity cutoff value of 150.4 and the detection time was only 100min. This diagnostic procedure can be satisfactorily applied to the diagnosis of brucellosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing.

    PubMed

    de Almeida, Fernando G; Vanzolini, Kenia L; Cass, Quezia B

    2017-01-05

    Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors have been prospected. In order to endorse this research field we have developed a new tool for ACE ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs was inhibited by lisinopril with a half maximal inhibitory concentration (IC50) of 10nM. At the fishing assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE kinetics determination, inhibition and binder screening.

  13. Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays.

    PubMed

    Caragata, Michael; Shah, Alok K; Schulz, Benjamin L; Hill, Michelle M; Punyadeera, Chamindie

    2016-03-15

    Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the sample collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.

  14. Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: an anticipated analytical tool for food safety.

    PubMed

    Hervás, Miriam; López, Miguel Angel; Escarpa, Alberto

    2009-10-27

    In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 microg L(-1) and EC(50) 0.079 microg L(-1) were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

  15. Single functional magnetic-bead as universal biosensing platform for trace analyte detection using SERS-nanobioprobe.

    PubMed

    Xiao, R; Wang, C W; Zhu, A N; Long, F

    2016-05-15

    SERS biosensor has demonstrated remarkable potential to analyze various bio/chemical targets with ultrahigh sensitivity. However, the development of universal SERS biosensing platforms with a uniform and reproducible structure that can quantitatively detect a broad range of trace analytes remains a significant challenge. The production of SERS nanotags with abundant Raman reporters and rational structure to conjugate with detection biomolecules is another key to design SERS-nanobioprobes. Here, we introduce a facile single magnetic-bead biosensing platform, formed by combining the captured antibodies/antigens conjugated magnetic-beads and the Au@Raman-Reporters@Ag sandwich-based nanorod tags labeled nanobioprobes. The advantage of the robust sandwich-structure-based nanotags is attributed not only to the high density Raman reporters contained inside, with high EF value because of enhanced electromagnetic field density, but also to the flexibility for bioconjugation of the detection biomolecules. The 3-D structure of the functional magnetic-bead provides a perfect platform to rapidly capture and enrich biomolecules. Ultrasensitive detection of two small molecules and a protein was achieved in samples, respectively.

  16. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology.

    PubMed

    Sakudo, Akikazu; Viswan, Anchu; Chou, Han; Sasaki, Tadahiro; Ikuta, Kazuyoshi; Nagatsu, Masaaki

    2016-07-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito‑borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave‑excited inductively‑coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody‑integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV‑infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1‑4 onto the beads was confirmed using reverse transcription‑polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody‑integration, represents a significant improvement in the detection of DENV.

  17. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology

    PubMed Central

    SAKUDO, AKIKAZU; VISWAN, ANCHU; CHOU, HAN; SASAKI, TADAHIRO; IKUTA, KAZUYOSHI; NAGATSU, MASAAKI

    2016-01-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito-borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave-excited inductively-coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody-integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV-infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1–4 onto the beads was confirmed using reverse transcription-polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody-integration, represents a significant improvement in the detection of DENV. PMID:27221214

  18. Rapid detection of Clostridium difficile via magnetic bead aggregation in cost-effective polyester microdevices with cell phone image analysis.

    PubMed

    DuVall, Jacquelyn A; Cabaniss, Scott T; Angotti, Morgan L; Moore, John H; Abhyankar, Mayuresh; Shukla, Nishant; Mills, Daniel L; Kessel, Bryan G; Garner, Gavin T; Swami, Nathan S; Landers, James P

    2016-10-07

    Pathogen detection has traditionally been accomplished by utilizing methods such as cell culture, immunoassays, and nucleic acid amplification tests; however, these methods are not easily implemented in resource-limited settings because special equipment for detection and thermal cycling is often required. In this study, we present a magnetic bead aggregation assay coupled to an inexpensive microfluidic fabrication technique that allows for cell phone detection and analysis of a notable pathogen in less than one hour. Detection is achieved through the use of a custom-built system that allows for fluid flow control via centrifugal force, as well as manipulation of magnetic beads with an adjustable rotating magnetic field. Cell phone image capture and analysis is housed in a 3D-printed case with LED backlighting and a lid-mounted Android phone. A custom-written application (app.) is employed to interrogate images for the extent of aggregation present following loop-mediated isothermal amplification (LAMP) coupled to product-inhibited bead aggregation (PiBA) for detection of target sequences. Clostridium difficile is a pathogen of increasing interest due to its causative role in intestinal infections following antibiotic treatment, and was therefore chosen as the pathogen of interest in the present study to demonstrate the rapid, cost-effective, and sequence-specific detection capabilities of the microfluidic platform described herein.

  19. A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads.

    PubMed

    Yamaguchi, Akemi; Matsuda, Kazuyuki; Uehara, Masayuki; Honda, Takayuki; Saito, Yasunori

    2016-02-04

    We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure. We have already developed the droplet-PCR machine, which can amplify and detect specific nucleic acids rapidly and automatically. Connecting the droplet-PCR machine to our novel automated extraction device enables PCR analysis within 15 min, and this system can be made available as a point-of-care testing in clinics as well as general hospitals.

  20. Screening inhibitors of xanthine oxidase from natural products using enzyme immobilized magnetic beads by high-performance liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Wang, Ting; Li, Dapeng; Yu, Boyang; Qi, Jin

    2017-03-06

    In this study, high-performance liquid chromatography coupled with tandem mass spectrometry was used to assess the results of bioactive compound screening from natural products using immobilized enzyme magnetic beads. We compared three commercial magnetic beads with modified amino, carboxy and N-hydroxysuccinimide groups, respectively. Amino magnetic beads performed best for immobilization and were selected for further experiments. Xanthine oxidase was immobilized on amino magnetic beads and applied to screen potential inhibitors in fresh Zingiber officinale Roscoe, extracts of Scutellaria baicalensis Georgi and Pueraria lobata Ohwi. In total, 12 potential xanthine oxidase ligands were identified from fresh Zingiber root and Scutellaria root extracts, of which eight were characterized and the concentration required for 50% inhibition was determined. Preliminary structure-function relationships were discussed based on these results. A convenient and effective method was therefore developed for the identification of active compounds from complex natural product mixtures. This article is protected by copyright. All rights reserved.

  1. Direct transfer of HRPII-magnetic bead complexes to malaria rapid diagnostic tests significantly improves test sensitivity.

    PubMed

    Ricks, Keersten M; Adams, Nicholas M; Scherr, Thomas F; Haselton, Frederick R; Wright, David W

    2016-08-05

    The characteristic ease of use, rapid time to result, and low cost of malaria rapid diagnostic tests (RDTs) promote their widespread use at the point-of-care for malaria detection and surveillance. However, in many settings, the success of malaria elimination campaigns depends on point-of-care diagnostics with greater sensitivity than currently available RDTs. To address this need, a sample preparation method was developed to deliver more biomarkers onto a malaria RDT by concentrating the biomarker from blood sample volumes that are too large to be directly applied to a lateral flow strip. In this design, Ni-NTA-functionalized magnetic beads captured the Plasmodium falciparum biomarker HRPII from a P. falciparum D6 culture spiked blood sample. This transfer of magnetic beads to the RDT was facilitated by an inexpensive 3D-printed apparatus that aligned the sample tube with the sample deposition pad and a magnet beneath the RDT. Biomarkers were released from the bead surface onto the lateral flow strip using imidazole-spiked running buffer. Kinetics of HRPII binding to the Ni-NTA beads as a function of blood sample volume were explored prior to determining the effect of the proposed method on the limit of detection of Paracheck RDTs. More than 80 % of HRPII biomarkers were extracted from blood sample volumes ranging from 25 to 250 µL. The time required to reach 80 % binding ranged from 5 to 60 min, depending on sample volume. Using 250 μL of blood and a 30-min biomarker binding time, the limit of detection of the Paracheck Pf RDT brand was improved by 21-fold, resulting in a limit of detection below 1 parasite/μL. This approach has the sensitivity and simplicity required to assist in malaria elimination campaigns in settings with limited access to clinical and laboratory resources.

  2. Ultrasonic alignment of bio-functionalized magnetic beads and live cells in PDMS micro-fluidic channel.

    PubMed

    Islam, Afroja T; Siddique, Ariful H; Ramulu, T S; Reddy, Venu; Eu, Young-Jae; Cho, Seung Hyun; Kim, CheolGi

    2012-12-01

    In this work, we demonstrated the alignment of polystyrene latex microspheres (diameter of 1 ~45 μm), bio-functionalized superparamagnetic beads (diameter 2.8 μm), and live cells (average diameter 1 ~2 μm) using an ultrasonic standing wave (USW) in a PDMS microfluidic channel (330 μm width) attached on a Si substrate for bio-medical applications. To generate a standing wave inside the channel, ultrasound of 2.25 MHz resonance frequency (for the channel width) was applied by two ultrasound transducers installed at both sides of the channel which caused the radiation force to concentrate the micro-particles at the single pressure nodal plane of USW. By increasing the frequency to the next resonance condition of the channel, the particles were concentrated in dual nodal planes. Migration time of the micro-particles towards the single nodal plane was recorded as 108 s, 17 s, and 115 s for polystyrene particles of 2 μm diameter, bio-functionalized magnetic beads, and live cells, respectively. These successful alignments of the bio-functionalized magnetic beads along the desired part of the channel can enhance the performance of a sensor which is applicable for the bio-hybrid system and the alignment of live cells without any damage can be used for sample pre-treatment for the application of lab-on-a-chip type bioassays.

  3. Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads for magnetic affinity separation of histidine-tagged proteins.

    PubMed

    Vereshchagina, T A; Fedorchak, M A; Sharonova, O M; Fomenko, E V; Shishkina, N N; Zhizhaev, A M; Kudryavtsev, A N; Frank, L A; Anshits, A G

    2016-01-28

    Magnetic Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads (Ni-ferrosphere beads - NFB) of a core-shell structure were synthesized starting from coal fly ash ferrospheres having diameters in the range of 0.063-0.050 mm. The strategy of NFB fabrication is an oriented chemical modification of the outer surface preserving the magnetic core of parent beads with the formation of micro-mesoporous coverings. Two routes of ferrosphere modification were realized, such as (i) hydrothermal treatment in an alkaline medium resulting in a NaP zeolite layer and (ii) synthesis of micro-mesoporous silica on the glass surface using conventional methods. Immobilization of Ni(2+) ions in the siliceous porous shell of the magnetic beads was carried out via (i) the ion exchange of Na(+) for Ni(2+) in the zeolite layer or (ii) deposition of NiO clusters in the zeolite and silica pores. The final NFB were tested for affinity in magnetic separation of the histidine-tagged green fluorescent protein (GFP) directly from a cell lysate. Results pointed to the high affinity of the magnetic beads towards the protein in the presence of 10 mM EDTA. The sorption capacity of the ferrosphere-based Ni-beads with respect to GFP was in the range 1.5-5.7 mg cm(-3).

  4. Optimization of magnetoresistive sensor current for on-chip magnetic bead detection using the sensor self-field

    NASA Astrophysics Data System (ADS)

    Henriksen, Anders Dahl; Rizzi, Giovanni; Østerberg, Frederik Westergaard; Hansen, Mikkel Fougt

    2015-04-01

    We investigate the self-heating of magnetoresistive sensors used for measurements on magnetic beads in magnetic biosensors. The signal from magnetic beads magnetized by the field due to the sensor bias current is proportional to the bias current squared. Therefore, we aim to maximize the bias current while limiting the sensor self-heating. We systematically characterize and model the Joule heating of magnetoresistive sensors with different sensor geometries and stack compositions. The sensor heating is determined using the increase of the sensor resistance as function of the bias current. The measured temperature increase is in good agreement with a finite element model and a simple analytical thermal model. The heat conductance of our system is limited by the 1 μm thick electrically insulating silicon dioxide layer between the sensor stack and the underlying silicon wafer, thus the heat conductance is proportional to the sensor area and inversely proportional to the oxide thickness. This simple heat conductance determines the relationship between bias current and sensor temperature, and we show that 25 μm wide sensor on a 1 μm oxide can sustain a bias current of 30 mA for an allowed temperature increase of 5 °C. The method and models used are generally applicable for thin film sensor systems. Further, the consequences for biosensor applications of the present sensor designs and the impact on future sensor designs are discussed.

  5. Speed improvement of a pathogenic micro-organism population detection with LAPS system by a magnetic bead separation and a pH detection.

    PubMed

    Moon, H S; Ryu, S; Yum, D; Kim, H

    2004-01-01

    In this paper, a magnetic bead based immobilization method and pH detection method is applied to the LAPS (light addressable potentiometric sensor) system to detect a pathogenic micro-organism population. Magnetic beads are very small, superparamagnetic particles (0.8 approximately 5.0 microm in diameter) that are able to sustain a magnetic domain under excitation and do not exhibit residual magnetization when the external field is removed. By using magnetic beads as an immobilization method, other bulky and complex method can be alternated. To verify the method, an urease labeled anti-salmonella typhimurium antibody is used to detect a pathogenic micro-organism( S. typhimurium ) population by a bias voltage maximum slope detection.

  6. Optimization of the virus concentration method using polyethyleneimine-conjugated magnetic beads and its application to the detection of human hepatitis A, B and C viruses.

    PubMed

    Uchida, Eriko; Kogi, Mieko; Oshizawa, Tadashi; Furuta, Birei; Satoh, Koei; Iwata, Akiko; Murata, Mitsuhiro; Hikata, Mikio; Yamaguchi, Teruhide

    2007-07-01

    To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads was developed in our previous study. However, several viruses could not be concentrated by this method. In this paper, the conditions of virus concentration were optimized to concentrate a wide range of viruses more efficiently. The PEI beads adsorbed viruses more efficiently than other cationic polymers, and the optimum virus concentration was obtained under weak acidic conditions. Mass spectrometric analysis revealed that several serum proteins, such as complement type 3, complement type 4 and immunoglobulin M (IgM), were co-adsorbed by the PEI beads, suggesting that the beads may adsorb viruses not only by direct adsorption, but also via immune complex formation. This hypothesis was confirmed by the result that poliovirus, which PEI beads could not adsorb directly, could be concentrated by the beads via immune complex formation. On the other hand, hepatitis A (HAV) and hepatitis C (HCV) viruses were adsorbed directly by PEI beads almost completely. Like poliovirus, hepatitis B virus (HBV) was concentrated efficiently by the addition of anti-HBV IgM. In conclusion, virus concentration using PEI beads is a useful method to concentrate a wide range of viruses and can be used to enhance the sensitivity of detection of HAV, HBV and HCV.

  7. Integration of antibody by surface functionalization of graphite-encapsulated magnetic beads using ammonia gas plasma technology for capturing influenza A virus.

    PubMed

    Sakudo, Akikazu; Chou, Han; Ikuta, Kazuyoshi; Nagatsu, Masaaki

    2015-05-01

    Antibody-integrated magnetic beads have been functionalized for influenza A virus capture. First, ammonia plasma produced by a radio frequency power source was reacted with the surface of graphite-encapsulated magnetic beads to introduce amino groups. Anti-influenza A virus hemagglutinin antibody was then anchored by its surface sulfide groups to the amino groups on the beads via N-succinimidyl 3-(2-pyridyldithio) propionate. After incubation with influenza A virus, adsorption of the virus to the beads was confirmed by immunochromatography, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and inoculation of chicken embryonated eggs, indicating that virus infectivity is maintained and that the proposed method is useful for the enhanced detection and isolation of influenza A virus.

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

    PubMed

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

    2015-04-01

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

  9. An immune sandwich assay of carcinoembryonic antigen based on the joint use of upconversion phosphors and magnetic beads.

    PubMed

    Li, Yaohua; Wu, Zhengjun; Liu, Zhihong

    2015-06-21

    We herein report a sensitive and selective immunosensor for carcinoembryonic antigen (CEA) based on the joint use of upconversion phosphors (UCPs) and magnetic beads (MBs). UCPs as the signal probe were designed with a core-shell structure which provided a 40-fold enhancement of the luminescence intensity. Poly(acrylic acid) (PAA)-modified UCPs were covalently conjugated with the anti-CEA antibody (coating), and streptavidin functionalized magnetic beads were combined with another biotin-tagged anti-CEA antibody. With the assistance of a magnet, the as-formed immune sandwich in the presence of CEA can be readily separated from the assay matrix. The immunosensor showed a linear dynamic range for CEA within 0.05-20 ng mL(-1) in a buffered aqueous solution, and 0.1-20 ng mL(-1) in a human serum sample. The sensor was highly specific to CEA. Our results have suggested the potential application of the UCP-MB based immunoassay for CEA in clinical analysis.

  10. Identification of c-di-AMP-Binding Proteins Using Magnetic Beads.

    PubMed

    Kampf, Jan; Gundlach, Jan; Herzberg, Christina; Treffon, Katrin; Stülke, Jörg

    2017-01-01

    To identify cytosolic proteins that bind to cyclic di-AMP, a biotinylated analog of the nucleotide is used for protein pull-down experiments. In this approach, biotinylated c-di-AMP is coupled to Streptactin-covered beads. After protein separation using standard SDS-PAGE, the protein(s) of interest are identified by mass spectrometric analyses.

  11. Centrifugal gas-phase transition magnetophoresis (GTM)--a generic method for automation of magnetic bead based assays on the centrifugal microfluidic platform and application to DNA purification.

    PubMed

    Strohmeier, Oliver; Emperle, Alexander; Roth, Günter; Mark, Daniel; Zengerle, Roland; von Stetten, Felix

    2013-01-07

    Transportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge "LabDisk". The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ± 3.6%. Bead based DNA purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ± 24% and for lambda phage DNA 43% ± 10% compared to manual reference purification in test tubes. Complete purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity purification, solely requiring the adjustment of number and volumes of the fluidic chambers.

  12. Effects of a Strong Static Magnetic Field on Bacterium Shewanellaoneidensis: An Assessment by Using Whole Genome Microarray.

    SciTech Connect

    Gao, W.; Liu, Y.; Zhou, J.-Z.; Hongjun, P.

    2007-04-02

    The effect of a strong static 14.1 T magnetic field on logphase cells of bacterial strain Shewanella oneidensis MR-1 was evaluatedby using whole genome microarray of this bacterium. Although differenceswere not observed between the treatment and control by measuring theoptical density (OD), colony forming unit (CFU), as well as post-exposuregrowth of cells, transcriptional expression levels of 65 genes werealtered according to our microarray data. Among these genes, 21 wereupregulated while other 44were downregulated, compared withcontrol.

  13. Peptidomic analysis of Chinese shrimp ( Fenneropenaeus chinensis) hemolymph by magnetic bead-based MALDI-TOF MS

    NASA Astrophysics Data System (ADS)

    Wang, Baojie; Liu, Mei; Jiang, Keyong; Zhang, Guofan; Wang, Lei

    2013-03-01

    Peptides in shrimp hemolymph play an important role in the innate immune response. Analysis of hemolymph will help to detect and identify potential novel biomarkers of microbial infection. We used magnetic bead-based purification (ClinProt system) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to characterize shrimp hemolymph peptides. Shrimp serum and plasma were used as the source of samples for comparative analysis, and it was found that serum was more suitable for shrimp hemolymph peptidomic analysis. To screen potential specific biomarkers in serum of immune-challenged shrimps, we applied magnetic bead-based MALDI-TOF MS to serum samples from 10 immune-challenged and 10 healthy shrimps. The spectra were analyzed using FlexAnalysis 3.0 and ClinProTools 2.1 software. Thirteen peptide peaks significantly different between the two groups were selected as candidate biomarkers of lipopolysaccharide (LPS)-infection. The diagnostic model established by genetic algorithm using five of these peaks was able to discriminate LPS-challenged shrimps from healthy control shrimps with a sensitivity of 90% and a specificity of 100%. Our approach in MALDITOF MS-based peptidomics is a powerful tool for screening bioactive peptides or biomarkers derived from hemolymph, and will help to enable a better understanding of the innate immune response of shrimps.

  14. 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.

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

    PubMed

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

    2017-08-15

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

  16. Ultra-fast synthesis of magnetic and luminescent silica beads for versatile bioanalytical applications

    NASA Astrophysics Data System (ADS)

    Müller-Schulte, Detlef; Schmitz-Rode, T.; Borm, Paul

    2005-05-01

    A method for the synthesis of both spherically shaped micro/nano silica particles and silica hybrid particles using a novel inverse sol-gel suspension technique was developed. The technique enables the synthesis of beads within seconds and provides a simple basis for quantum dot and biosubstances encapsulation. The carriers can be used as DNA adsorbents, individually addressable optical codes for bioassays and biomolecule library screening as well as photonic crystals.

  17. 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.

  18. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    PubMed

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

    Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

    2007-01-01

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

  20. Switch on or switch off: an optical DNA sensor based on poly(p-phenylenevinylene) grafted magnetic beads.

    PubMed

    Srinivas, Anupama R Gulur; Peng, Hui; Barker, David; Travas-Sejdic, Jadranka

    2012-05-15

    There has been an enormous demand for commercial label-free DNA sensors in a diverse range of fields including pre-emptive medicine, diagnostics, environmental monitoring, and food industry. Addressing the need for sensitive, selective and facile DNA sensors, we demonstrate a novel switch on/off sensor design that utilizes sandwich hybridization between photoluminescent anionic conjugated polyelectrolyte (CPE) bound captureprobe coated onto magnetic beads, target and the signaling probe. The hybridization-readout in our sensor was monitored by either fluorescence resonance energy transfer (FRET, switch-on) or superquenching (switch-off) depending on the type of signaling probe used. Moreover recent designs that utilize beads for sensing DNA have been limited towards using electrostatic interactions or intercalation of dyes to observe FRET. To our knowledge this is the first report of a switch on/off sensor utilizing either FRET or superquenching thus providing flexibility for future development of such rapid, facile and sensitive DNA sensors. The FRET-based sensor was investigated by optimizing the reaction parameters and selectivity. A low detection limit of 240 fmol in 2 mL of SSC buffer was achieved.

  1. A Rapid Detection Method of Brucella with Quantum Dots and Magnetic Beads Conjugated with Different Polyclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Song, Dandan; Qu, Xiaofeng; Liu, Yushen; Li, Li; Yin, Dehui; Li, Juan; Xu, Kun; Xie, Renguo; Zhai, Yue; Zhang, Huiwen; Bao, Hao; Zhao, Chao; Wang, Juan; Song, Xiuling; Song, Wenzhi

    2017-03-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Traditional methods for detection of Brucella spp. take 48-72 h that does not meet the need of rapid detection. Herein, a new rapid detection method of Brucella was developed based on polyclonal antibody-conjugating quantum dots and antibody-modified magnetic beads. First, polyclonal antibodies IgG and IgY were prepared and then the antibody conjugated with quantum dots (QDs) and immunomagnetic beads (IMB), respectively, which were activated by N-(3-dimethylaminopropyl)- N'-ethylcar-bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form probes. We used the IMB probe to separate the Brucella and labeled by the QD probe, and then detected the fluorescence intensity with a fluorescence spectrometer. The detection method takes 105 min with a limit of detection of 103 CFU/mL and ranges from 10 to 105 CFU/mL ( R 2 = 0.9983), and it can be well used in real samples.

  2. The on-bead digestion of protein corona on nanoparticles by trypsin immobilized on the magnetic nanoparticle.

    PubMed

    Hu, Zhengyan; Zhao, Liang; Zhang, Hongyan; Zhang, Yi; Wu, Ren'an; Zou, Hanfa

    2014-03-21

    Proteins interacting with nanoparticles would form the protein coronas on the surface of nanoparticles in biological systems, which would critically impact the biological identities of nanoparticles and/or result in the physiological and pathological consequences. The enzymatic digestion of protein corona was the primary step to achieve the identification of protein components of the protein corona for the bottom-up proteomic approaches. In this study, the investigation on the tryptic digestion of protein corona by the immobilized trypsin on a magnetic nanoparticle was carried out for the first time. As a comparison with the usual overnight long-time digestion and the severe self-digestion of free trypsin, the on-bead digestion of protein corona by the immobilized trypsin could be accomplished within 1h, along with the significantly reduced self-digestion of trypsin and the improved reproducibility on the identification of proteins by the mass spectrometry-based proteomic approach. It showed that the number of identified bovine serum (BS) proteins on the commercial Fe3O4 nanoparticles was increased by 13% for the immobilized trypsin with 1h digestion as compared to that of using free trypsin with even overnight digestion. In addition, the on-bead digestion of using the immobilized trypsin was further applied on the identification of human plasma protein corona on the commercial Fe3O4 nanoparticles, which leads the efficient digestion of the human plasma proteins and the identification of 149 human plasma proteins corresponding to putative critical pathways and biological processes.

  3. 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.

  4. Rapid screening and identification of α-glucosidase inhibitors from mulberry leaves using enzyme-immobilized magnetic beads coupled with HPLC/MS and NMR.

    PubMed

    Tao, Yi; Zhang, Yufeng; Cheng, Yiyu; Wang, Yi

    2013-02-01

    α-Glucosidase plays important roles in the digestion and absorption of carbohydrates in the small intestine. The inhibition of α-glucosidase is regarded as a potential way to treat diabetes. We established an approach to screening α-glucosidase inhibitors from medicinal plants using enzyme-coated magnetic bead. Using 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide as reaction reagents, α-glucosidase was immobilized on the magnetic beads by covalent linkage. The conjugation of α-glucosidase to the magnetic beads was characterized using scanning electron microscope and X-ray diffractometer. The proposed approach was applied in fishing potential α-glucosidase inhibitors from extract of Morus alba, a Chinese medicinal plant. The structures of potential active compounds were identified via liquid chromatography-mass spectrometry and nuclear magnetic resonance. The results demonstrated that two flavonoids (isoquercitrin and astragalin) could bind to α-glucosidase, which was confirmed via conventional α-glucosidase inhibitory assay. Our findings suggested that enzyme-coated magnetic beads may be suitable for discovering active compounds from medicinal plants.

  5. Heat generation ability in AC magnetic field of nano MgFe2O4-based ferrite powder prepared by bead milling

    NASA Astrophysics Data System (ADS)

    Hirazawa, Hideyuki; Aono, Hiromichi; Naohara, Takashi; Maehara, Tsunehiro; Sato, Mitsunori; Watanabe, Yuji

    2011-03-01

    Nanosized MgFe2O4-based ferrite powder having heat generation ability in an AC magnetic field was prepared by bead milling and studied for thermal coagulation therapy applications. The crystal size and the particle size significantly decreased by bead milling. The heat generation ability in an AC magnetic field improved with the milling time, i.e. a decrease in crystal size. However, the heat generation ability decreased for excessively milled samples with crystal sizes of less than 5.5 nm. The highest heat ability (ΔT=34 °C) in the AC magnetic field (370 kHz, 1.77 kA/m) was obtained for fine MgFe2O4 powder having a ca. 6 nm crystal size (the samples were milled for 6-8 h using 0.1 mm ϕ beads). The heat generation of the samples was closely related to hysteresis loss, a B-H magnetic property. The reason for the high heat generation properties of the samples milled for 6-8 h using 0.1 mm ϕ beads was ascribed to the increase in hysteresis loss by the formation of a single domain. Moreover, the improvement in heating ability was obtained by calcination of the bead-milled sample at low temperature. In this case, the maximum heat generation (ΔT=41 °C) ability was obtained for a ca. 11 nm crystal size sample was prepared by crystal growth during the sample calcination. On the other hand, the ΔT value for Mg0.5Ca0.5Fe2O4 was synthesized using a reverse precipitation method decreased by bead milling.

  6. Aptamer-Magnetic Bead Quantum Dot Sandwich Assays for Foodborne Pathogen Detection: Pros, Cons, and Lessons Learned.

    PubMed

    Bruno, John G; Sivils, Jeffrey C; Phillips, Taylor

    2017-07-01

    DNA and RNA aptamers have been extensively investigated as potential competitors for antibodies for a variety of applications including food safety testing. Ultrasensitive fluorescence detection of foodborne pathogenic bacteria as low as 1-10 cells/mL has been achieved using aptamers coupled to quantum dots in clear pristine buffers for environmental sample detection. Quantum dots offer other advantages, including single UV or blue light source multiplex (multicolored) detection. However, quantum dots can exhibit decreased fluorescence in some food matrixes and even completely fail to fluoresce in some fatty matrixes, as documented in this report. Given the need to detect substances in complex food matrixes (and from data reported elsewhere), aptamer-magnetic bead pull-down methods followed by enzymatic/fluorometric- or PCR-based detection methods may be more robust methods for testing in foods or enrichment cultures. Other lessons learned, including the initial choice of aptamer targets to enhance assay specificity, are also discussed.

  7. Microgels at the Water/Oil Interface: In Situ Observation of Structural Aging and Two-Dimensional Magnetic Bead Microrheology.

    PubMed

    Huang, Shilin; Gawlitza, Kornelia; von Klitzing, Regine; Gilson, Laurent; Nowak, Johannes; Odenbach, Stefan; Steffen, Werner; Auernhammer, Günter K

    2016-01-26

    Stimuli-responsive microgels can be used as stabilizers for emulsions. However, the details of structure and the viscoelastic property of the microgel-laden interface are still not well-known. We synthesized fluorescently labeled microgels and used confocal microscopy to observe their arrangement at the water/oil interface. The microgels aggregated spontaneously at the interface, and the aggregated structure reorganized due to thermal motion. The structure of the interfacial layer formed by microgels depended on the microgel concentration at the interface. We suggest that the structure was controlled by the aggregation and adsorption of microgels at the interface. The interparticle separation between microgels at the interface decreased over time, implying a slow aging process of the microgels at the interface. Magnetic beads were introduced at the interface and used to trigger deformation of the microgel layer. Under compression and shear the microgels in the aggregated structure rearranged, leading to plastic deformation, and some elastic responses were also observed.

  8. Microwave synthesis of gibberellin acid 3 magnetic molecularly imprinted polymer beads for the trace analysis of gibberellin acids in plant samples by liquid chromatography-mass spectrometry detection.

    PubMed

    Zhang, Zhuomin; Tan, Wei; Hu, Yuling; Li, Gongke; Zan, Song

    2012-02-21

    In this study, novel GA3 magnetic molecularly imprinted polymer (mag-MIP) beads were synthesized by a microwave irradiation method, and the beads were applied for the trace analysis of gibberellin acids (GAs) in plant samples including rice and cucumber coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS). The microwave synthetic procedure was optimized in detail. In particular, the interaction between GA3 and functional monomers was further studied for the selection of the optimal functional monomers during synthesis. It can be seen that the interaction between GA3 and acrylamide (AM) finally selected was stronger than that between GA3 and other functional monomers. GA3 mag-MIP beads were characterized by a series of physical tests. GA3 mag-MIP beads had a porous and homogeneous surface morphology with stable chemical, thermal and magnetic properties. Moreover, GA3 mag-MIP beads demonstrated selective and specific absorption behavior for the target compounds during unsaturated extraction, which resulted in a higher extraction capacity (∼708.4 pmol for GA3) and selectivity than GA3 mag-non-imprinted polymer beads. Finally, an analytical method of GA3 mag-AM-MIP bead extraction coupled with HPLC-MS detection was established and applied for the determination of trace GA1, GA3, GA4 and GA7 in rice and cucumber samples. It was satisfactory that GA4 could be actually found to be 121.5 ± 1.4 μg kg(-1) in real rice samples by this novel analytical method. The recoveries of spiked rice and cucumber samples were found to be 76.0-109.1% and 79.9-93.6% with RSDs of 2.8-8.8% and 3.1-7.7% (n = 3), respectively. The proposed method is efficient and applicable for the trace analysis of GAs in complicated plant samples.

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

    PubMed

    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 10(3) E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 10(2) E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 10(1) E. coli O104:H4 initial load). The specificity was 100%.

  10. 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

  11. Application of porcine gastric mucin-conjugated magnetic beads and polyethylene glycol goncentration and detection of human noroviruses from green onion and grape

    USDA-ARS?s Scientific Manuscript database

    Objective: To set up detection methods for norovirus in fruits and vegetables by using porcine gastric mucin-conjugated magnetic beads (PGM-MB) and polyethylene glycol 8000 (PEG8000) concentrating and detecting the norovirus in green onion and grape. Methods: The highest virus dilution given a posit...

  12. Planar Hall effect bridge sensors with NiFe/Cu/IrMn stack optimized for self-field magnetic bead detection

    NASA Astrophysics Data System (ADS)

    Henriksen, Anders Dahl; Rizzi, Giovanni; Hansen, Mikkel Fougt

    2016-03-01

    The stack composition in trilayer Planar Hall effect bridge sensors is investigated experimentally to identify the optimal stack for magnetic bead detection using the sensor self-field. The sensors were fabricated using exchange-biased stacks Ni80Fe20(tFM)/Cu(tCu)/Mn80Ir20(10 nm) with tFM = 10, 20, and 30 nm, and 0 ≤ tCu ≤ 0.6 nm. The sensors were characterized by magnetic hysteresis measurements, by measurements of the sensor response vs. applied field, and by measurements of the sensor response to a suspension of magnetic beads magnetized by the sensor self-field due to the sensor bias current. The exchange bias field was found to decay exponentially with tCu and inversely with tFM. The reduced exchange field for larger values of tFM and tCu resulted in higher sensitivities to both magnetic fields and magnetic beads. We argue that the maximum magnetic bead signal is limited by Joule heating of the sensors and, thus, that the magnetic stacks should be compared at constant power consumption. For a fixed sensor geometry, the figure of merit for this comparison is the magnetic field sensitivity normalized by the sensor bias voltage. In this regard, we found that sensors with tFM = 20 nm or 30 nm outperformed those with tFM = 10 nm by a factor of approximately two, because the latter have a reduced AMR ratio. Further, the optimum layer thicknesses, tCu ≈ 0.6 nm and tFM = 20-30 nm, gave a 90% higher signal compared to the corresponding sensors with tCu = 0 nm.

  13. Improving of catalase stability properties by encapsulation in alginate/Fe3O4 magnetic composite beads for enzymatic removal of H2O2.

    PubMed

    Doğaç, Yasemin Ispirli; Çinar, Mürvet; Teke, Mustafa

    2015-01-01

    The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10-100 mg) and enzyme concentrations (0.25-1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25-50°C), optimum pH (3.0-8.0), kinetic parameters, thermal stability (20-70°C), pH stability (4.0-9.0) operational stability (0-390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.

  14. Online magnetic bead based dynamic protein affinity selection coupled to LC-MS for the screening of acetylcholine binding protein ligands.

    PubMed

    Pochet, Lionel; Heus, Ferry; Jonker, Niels; Lingeman, Henk; Smit, August B; Niessen, Wilfried M A; Kool, Jeroen

    2011-06-15

    A magnetic beads based affinity-selection methodology towards the screening of acetylcholine binding protein (AChBP) binders in mixtures and pure compound libraries was developed. The methodology works as follows: after in solution incubation of His-tagged AChBP with potential ligands, and subsequent addition of cobalt (II)-coated paramagnetic beads, the formed bead-AChBP-ligand complexes are fetched out of solution by injection and trapping in LC tubing with an external adjustable magnet. Non binders are then washed to the waste followed by elution of ligands to a SPE cartridge by flushing with denaturing solution. Finally, SPE-LC-MS analysis is performed to identify the ligands. The advantage of the current methodology is the in solution incubation followed by immobilized AChBP ligand trapping and the capability of using the magnetic beads system as mobile/online transportable affinity SPE material. The system was optimized and then successfully demonstrated for the identification of AChBP ligands injected as pure compounds and for the fishing of ligands in mixtures. The results obtained with AChBP as target protein demonstrated reliable discrimination between binders with pK(i) values ranging from at least 6.26 to 8.46 and non-binders.

  15. Detection of Schistosoma mansoni Eggs in Feces through their Interaction with Paramagnetic Beads in a Magnetic Field

    PubMed Central

    Fagundes Teixeira, Candida; Neuhauss, Erli; Ben, Renata; Romanzini, Juliano; Graeff-Teixeira, Carlos

    2007-01-01

    Background Diagnosis of intestinal schistosomiasis in low endemic areas is a problem because often control measures have reduced egg burdens in feces to below the detection limits of classical coproparasitological methods. Evaluation of molecular methods is hindered by the absence of an established standard with maximum sensitivity and specificity. One strategy to optimize method performance, where eggs are rare events, is to examine large amounts of feces. A novel diagnostic method for isolation of Schistosoma mansoni eggs in feces, and an initial evaluation of its performance is reported here. Methodology/Principal Findings Known amounts of S. mansoni eggs were seeded into 30 g of normal human feces and subjected to a sequence of spontaneous sedimentation, sieving, Ritchie method, incubation and isolation through interaction with paramagnetic beads. Preliminary tests demonstrated the efficacy of lectins as ligands, but they also indicated that the paramagnetic beads alone were sufficient to isolate the eggs under a magnetic field through an unknown mechanism. Eggs were identified by microscopic inspection, with a sensitivity of 100% at 1.3 eggs per gram of feces (epg). Sensitivity gradually decreased to 25% at a concentration of 0.1 epg. In a preliminary application of the new method to the investigation of a recently established focus in southern Brazil, approximately 3 times more eggs were detected than with the thick-smear Kato-Katz method. Conclusions/Significance The novel S. mansoni detection method may significantly improve diagnosis of infections with low burdens in areas of recent introduction of the parasite, areas under successful control of transmission, or in infected travelers. It may also improve the evaluation of new treatments and vaccines. PMID:18060086

  16. Magneto-optical biosensing platform based on light scattering from self-assembled chains of functionalized rotating magnetic beads.

    PubMed

    Park, Sang Yoon; Handa, Hiroshi; Sandhu, Adarsh

    2010-02-10

    We describe a simple protocol for the rapid, highly sensitive, and quantitative measurement of the concentration of biomolecules in a solution by monitoring light scattered by self-assembled chains of functionalized superparamagnetic beads (SBs) rotating in the solution. A rotating external field (H(ex)) applied to an aqueous solution containing 250 nm diameter biotinylated SBs produced linear chains of SBs rotating in phase with Hex due to magnetically induced self-assembly. At constant Hex, the addition of avidin to the solution led to the formation of longer SB-chains than without the presence of avidin. The generation of longer SB-chains was revealed by increases in the amplitude of the oscillating optical transmittance signal of the magnetic colloid solution. Monitoring changes in the amplitude of the optical transmittance of the solution enabled quantitative determination of the concentration of avidin added to the solution with a sensitivity of 100 pM (6.7 ng/mL) and a dynamic range of at least 3 orders of magnitude. The rotating chains acted as biomolecule probes and micromagnetic mixers, enabling detection of biomolecular recognition in less than 30 s. This approach offers a rapid, highly sensitive, inexpensive, and homogeneous means for detecting biorecognition processes.

  17. Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip.

    PubMed

    Zhang, Lei; Deraney, Rachel N; Tripathi, Anubhav

    2015-11-01

    While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 10(1) to 5 × 10(6) copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs.

  18. Using annexin V-coated magnetic beads to capture active tissue factor-bearing microparticles from body fluids.

    PubMed

    Gieseler, Frank; Gamperl, Hans; Theophil, Frederike; Stenzel, Inga; Quecke, Tabea; Ungefroren, Hendrik; Lehnert, Hendrik

    2014-02-01

    Microparticles, found in all body fluids including peripheral blood, are important elements that regulate cellular interactions under both physiological and pathological conditions. They play an important role in blood clot formation and increased cell aggregation. However, little is known about the components of the microparticles and their mechanism of action. A method to quantify and assess the underlying mechanism of action of microparticles in pathologies is therefore desirable. We present a specific method to isolate cell-derived microparticles from malignant effusions using annexin V-coated magnetic microbeads. The microparticles can be detected by flow cytometry. Our results show that the microparticles can be isolated with >80% specificity when bound to annexin V-coated magnetic beads, which was originally developed for the detection of apoptotic cells. We also show that the isolated microparticles were still functionally active and can be used for further analysis. Thus, our method enables isolation as well as structural and functional characterisation of the microparticles which are produced in numerous patho-physiological situations. This should help gain a deeper insight into various disease situations, which in turn should pave the way for the development of novel drugs and specific therapy strategies. © 2013 International Federation for Cell Biology.

  19. A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection.

    PubMed

    Laschi, Serena; Miranda-Castro, Rebeca; González-Fernández, Eva; Palchetti, Ilaria; Reymond, Frédéric; Rossier, Joël S; Marrazza, Giovanna

    2010-11-01

    In this work, the characterisation and the optimisation of hybridisation assays based on a novel, rapid and sensitive micro-analytical, gravity-driven, flow device is reported. This device combines a special chip containing eight polymer microchannels, with a portable, computer-controlled instrument. The device is used as a platform for affinity experiments using oligonucleotide-modified paramagnetic particles. In our approach, both hybridisation and labelling events are performed on streptavidin-coated paramagnetic microparticles functionalized with a biotinylated capture probe. Modified particles, introduced in the microchannel inlet of the chip, accumulate near the electrode surface by virtue of a magnetic holder. After hybridisation with the complementary sequence, the hybrid is labelled with an alkaline phosphatase conjugate. The electrochemical substrate for alkaline phosphatase revelation is p-aminophenyl phosphate. Solutions and reagents are sequentially passed through the microchannels, until enzyme substrate is added for in situ signal detection. Upon readout, the magnet array is flipped away, beads are removed by addition of regeneration buffer, and the so-regenerated chip is ready for further analysis. This protocol has been applied to the analytical detection of specific DNA sequences of Legionella pneumophila, with an RSD=8.5% and a detection limit of 0.33 nM.

  20. Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip

    PubMed Central

    Zhang, Lei; Deraney, Rachel N.; Tripathi, Anubhav

    2015-01-01

    While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 101 to 5 × 106 copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs. PMID:26734116

  1. Water dispersible cross-linked magnetic chitosan beads for increasing the antimicrobial efficiency of aminoglycoside antibiotics.

    PubMed

    Grumezescu, Alexandru Mihai; Andronescu, Ecaterina; Holban, Alina Maria; Ficai, Anton; Ficai, Denisa; Voicu, Georgeta; Grumezescu, Valentina; Balaure, Paul Cătălin; Chifiriuc, Carmen Mariana

    2013-09-15

    The aim of this study was to obtain a nano-active system to improve antibiotic activity of certain drugs by controlling their release. Magnetic composite nanomaterials based on magnetite core and cross-linked chitosan shell were synthesized via the co-precipitation method and characterized by Fourier transform infrared spectroscopy (FT-IR), infrared microscopy (IRM), scanning electron microscopy (SEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The prepared magnetic composite nanomaterials exhibit a significant potentiating effect on the activity of two cationic (kanamycin and neomycin) drugs, reducing the amount of antibiotics necessary for the antimicrobial effect. The increase in the antimicrobial activity was explained by the fact that the obtained nanosystems provide higher surface area to volume ratio, resulting into higher surface charge density thus increasing affinity to microbial cell and also by controlling their release. In addition to the nano-effect, the positive zeta potential of the synthesized magnetite/cross-linked chitosan core/shell magnetic nanoparticles allows for a more favorable interaction with the usually negatively charged cell wall of bacteria. The novelty of the present contribution is just the revealing of this synergistic effect exhibited by the synthesized water dispersible magnetic nanocomposites on the activity of different antibiotics against Gram-positive and Gram-negative bacterial strains. The results obtained in this study recommend these magnetic water dispersible nanocomposite materials for applications in the prevention and treatment of infectious diseases.

  2. Magnet-Bead Based MicroRNA Delivery System to Modify CD133+ Stem Cells

    PubMed Central

    Wiekhorst, Frank; Steinhoff, Gustav

    2016-01-01

    Aim. CD133+ stem cells bear huge potential for regenerative medicine. However, low retention in the injured tissue and massive cell death reduce beneficial effects. In order to address these issues, we intended to develop a nonviral system for appropriate cell engineering. Materials and Methods. Modification of human CD133+ stem cells with magnetic polyplexes carrying microRNA was studied in terms of efficiency, safety, and targeting potential. Results. High microRNA uptake rates (~80–90%) were achieved without affecting CD133+ stem cell properties. Modified cells can be magnetically guided. Conclusion. We developed a safe and efficient protocol for CD133+ stem cell modification. Our work may become a basis to improve stem cell therapeutical effects as well as their monitoring with magnetic resonance imaging. PMID:27795713

  3. Mesoporous silica beads embedded with semiconductor quantum dots and iron oxide nanocrystals: dual-function microcarriers for optical encoding and magnetic separation.

    PubMed

    Sathe, Tushar R; Agrawal, Amit; Nie, Shuming

    2006-08-15

    Mesoporous beads are promising materials for embedding functional nanoparticles because of their nanometer-sized pores and large surface areas. Here we report the development of silica microbeads embedded with both semiconductor quantum dots (QD) and iron oxide (Fe3O4) nanocrystals as a new class of dual-function carriers for optical encoding and magnetic separation. The embedding (doping) process is carried out by either simultaneous or sequential addition of quantum dots and iron oxide (Fe3O4) nanocrystals in solution. The doping process is fast and quantitative, but the incorporated iron oxide strongly attenuates the signal intensity of QD fluorescence. We find that this attenuation is not due to conventional fluorescence quenching but is caused by the broad optical absorption spectrum of mixed-valence Fe3O4. For improved biocompatibility and reduced nonspecific binding, the encoded beads are further coated with amphiphilic polymers such as octylamine poly(acrylic acid). The results indicate that the polymer-coated beads are well suited for target capturing and enrichment, yielding magnetic separation efficiencies higher than 99%. By combining the multiplexing capability of QDs with the superparamagnetic properties of iron oxide nanocrystals, this class of encoded beads is expected to find broad applications in high-throughput and multiplexed biomolecular assays.

  4. Fabrication of magnetic alginate beads with uniform dispersion of CoFe2O4 by the polydopamine surface functionalization for organic pollutants removal

    NASA Astrophysics Data System (ADS)

    Li, Xiaoli; Lu, Haijun; Zhang, Yun; He, Fu; Jing, Lingyun; He, Xinghua

    2016-12-01

    A simple and efficient method for production of magnetic composites by decorating CoFe2O4 with polydopamine (PDA) through oxidative polymerization of dopamine was conducted. Further, magnetic alginate beads with porous structure containing well-dispersed CoFe2O4-PDA were fabricated by ionic crosslinking technology. The resulting SA@CoFe2O4-PDA beads were characterized using scanning electron microscopy, Fourier transform infrared spectrometry, X-ray diffractometer, vibrating sample magnetometer and X-ray photoelectron spectroscopy. Adsorption potential of SA@CoFe2O4-PDA beads for organic dyes including Methylene Blue (MB), Crystal Violet (CV) and Malachite Green (MG) was evaluated. SA@CoFe2O4-PDA beads exhibited excellent adsorption performances due to the composite effect, large surface area and porous structure. Organic dyes could be removed from water solution with high efficiency in a wide pH range of 4.0-9.0. Moreover, it exhibited much higher adsorptivity towards MB and CV with the maximum adsorption capacities of 466.60 and 456.52 mg/g, respectively, which were much higher than that of MG (248.78 mg/g). Ca-electrolyte had obvious adverse effects on MB and CV adsorption than MG. FTIR and XPS demonstrated that carboxylate, catechol, hydroxyl and amine groups might be involved in adsorption of organic dyes. The characteristics of wide pH range, high adsorption capacity and convenient magnetic separation would make SA@CoFe2O4-PDA beads as effective adsorbent for removal of organic dyes from wastewater.

  5. Turn-on chemiluminescent sensing platform for label-free protease detection using streptavidin-modified magnetic beads.

    PubMed

    Zhang, Huanhuan; Yu, Dalong; Zhao, Yanjun; Fan, Aiping

    2014-11-15

    We report a label-free streptavidin-modified magnetic beads (SA-MBs)-based sensing platform for turn-on chemiluminescent (CL) detection of protease using trypsin as model analyte. In the assay, a biotinylated peptide containing an arginine and a terminal cysteine was used as the substrate of trypsin. Upon adding the peptide into a basic luminol-NaIO4 solution, the terminal cysteine induced a strong CL signal. Surprisingly a much lower CL was emitted when the peptide was immobilized on the surface of SA-MBs. Based on this phenomenon, we designed a turn-on CL sensing system for protease using trypsin as model and its inhibitors screening. In the absence of trypsin, the peptide was coupled to the SA-MBs surface, resulting in a low CL background. Upon the addition of trypsin, the peptide can be catalytically hydrolyzed at the C-terminus of arginine, resulting in the formation of free cysteine-containing residues and subsequent CL recovery with the addition of luminol and NaIO4. The simple method does not require washing or separating procedures. Trypsin at a concentration as low as 10 pM can be assayed using this new CL sensing system. Additionally, the proposed method can be employed for screening the inhibitors of trypsin. This new sensing strategy could be easily extended to assay other proteases by simply changing the peptide substrate.

  6. Rapid removal of copper with magnetic poly-acrylic weak acid resin: quantitative role of bead radius on ion exchange.

    PubMed

    Fu, Lichun; Shuang, Chendong; Liu, Fuqiang; Li, Aimin; Li, Yan; Zhou, Yang; Song, Haiou

    2014-05-15

    A novel magnetic weak acid resin NDMC was self-synthesized for the removal of Cu(2+) from aqueous solutions. NDMC showed superior properties on the removal of Cu(2+) compared to commercial resins C106 and IRC-748, which was deeply investigated by adsorption isotherms and kinetic tests. The equilibrium adsorption amount of Cu(2+) onto NDMC (267.2mg/g) was almost twice as large as that onto IRC-748 (120.0mg/g). The adsorption kinetics of Cu(2+) onto the three resins fitted well with the pseudo-second-order equation. The initial adsorption rate h of NDMC was about 4 times that of C106 and nearly 8 times that of IRC-748 at the initial concentration of 500mg/L. External surface area was determined to be the key factor in rate-controlling by further analyzing the adsorption thermodynamics, kinetics parameters and physicochemical properties of the resins. NDMC resin with the smallest bead radius possessed the largest external surface and therefore exhibited the fastest kinetics. The adsorption amount of Cu(2+) onto NDMC was not influenced as the concentration of Na(+) increased from 1.0 to 10.0mM/L. Dilute HCl solution could effectively desorb Cu(2+). NDMC demonstrated high stability during 10 adsorption/desorption cycles, showing great potential in the rapid removal of Cu(2+) from wastewater.

  7. A novel assay for screening inhibitors targeting HIV-1 integrase dimerization based on Ni-NTA magnetic agarose beads

    PubMed Central

    Zhang, Dawei; He, Hongqiu; Liu, Mengmeng; Meng, Zhixia; Guo, Shunxing

    2016-01-01

    Human immunodeficiency virus (HIV)-1 integrase (IN), which mediates integration of viral cDNA into the cellular chromosome, is a validated antiviral drug target. Three IN inhibitors, raltegravir, elvitegravir and dolutegravir, have been clinically approved since 2008. However, drug resistance have emerged in infected patients receiving treatment using these drugs which share the same mechanism of action and have a low genetic barrier for resistance. Therefore, there is an urgent need to develop drugs with novel mechanism. IN requires a precise and dynamic equilibrium between several oligomeric species for its activities. The modulation of the process which is termed as IN oligomerization, presents an interesting allosteric target for drug development. In this research, we developed a magnetic beads based approach to assay the IN dimerization. Then, using the assay we screened a library of 1000 Food and Drug Administration (FDA)-approved drugs for IN dimerization inhibitors and identified dexlansoprazole as a potential IN dimerization inhibitor. In conclusion, the assay presented here has been proven to be sensitive and specific for the detection of IN dimerization as well as for the identification of antiviral drugs targeting IN dimerization. Moreover, a FDA-approved proton-pump inhibitors, dexlansoprazole, was identified as a potential inhibitor for IN dimerization. PMID:27137477

  8. Development of an in situ magnetic beads based RT-PCR method for electrochemiluminescent detection of rotavirus

    NASA Astrophysics Data System (ADS)

    Zhan, Fangfang; Zhou, Xiaoming

    2012-12-01

    Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.

  9. Facile fabrication of an electrochemical aptasensor based on magnetic electrode by using streptavidin modified magnetic beads for sensitive and specific detection of Hg(2.).

    PubMed

    Wu, Dan; Wang, Yaoguang; Zhang, Yong; Ma, Hongmin; Pang, Xuehui; Hu, Lihua; Du, Bin; Wei, Qin

    2016-08-15

    In this work, a novel electrochemical aptasensor was developed for sensitive and specific detection of Hg(2+) based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure via application of thionine (Th) as indicator signal. For the fabrication of the aptasensor, streptavidin modified magnetic beads (Fe3O4-SA) was firmly immobilized onto the magnetic glassy carbon electrode (MGCE) benefited from its magnetic character. Then biotin labeled T-riched single stranded DNA (Bio-ssDNA) connected with Fe3O4-SA specifically and steadily because of the specific binding capacity between streptavidin and biotin. The stable structure of T-Hg(2+)-T formed in the present of Hg(2+) provided convenience for the intercalation of Th. The detection of Hg(2+) was achieved by recording the differential pulse voltammetry (DPV) signal of Th. Under optimal experimental conditions, the linear range of the fabricated electrochemical aptasensor was 1-200nmol/L, with a detection limit of 0.33nmol/L. Furthermore, the proposed aptasensor may find a potential application for the detection of Hg(2+) in real water sample analysis.

  10. Repetitive Immunosensor with a Fiber-Optic Device and Antibody-Coated Magnetic Beads for Semi-Continuous Monitoring of Escherichia coli O157:H7.

    PubMed

    Taniguchi, Midori; Saito, Hirokazu; Mitsubayashi, Kohji

    2017-09-19

    A rapid and reproducible fiber-optic immunosensor for Escherichia coli O157:H7 (E. coli O157:H7) was described. The biosensor consisted of a flow cell, an optical fiber with a thin Ni layer, and a PC linked fluorometer. First, the samples with E. coli O157:H7 were incubated with magnetic beads coated with anti-E. coli O157:H7 antibodies and anti-E. coli O157:H7 antibodies labeled cyanine 5 (Cy5) to make sandwich complexes. Then the Cy5-(E. coli O157:H7)-beads were injected into a flow cell and pulled to the magnetized Ni layer on the optical fiber set in the flow cell. An excitation light (λ = 635 nm) was used to illuminate the optical fiber, and the Cy5 florescent molecules facing the optical fiber were exposed to an evanescent wave from the optical fiber. The 670 nm fluorescent light was measured using a photodiode. Finally, the magnetic intensity of the Ni layer was removed and the Cy5-E. coli O157:H7-beads were washed out for the next immunoassay. E. coli O157:H7, diluted with phosphate buffer (PB), was measured from 1 × 10⁵ to 1 × 10⁷ cells/mL. The total time required for an assay was less than 15 min (except for the pretreatment process) and repeating immunoassay on one optical fiber was made possible.

  11. Comperative study of catalase immobilization on chitosan, magnetic chitosan and chitosan-clay composite beads.

    PubMed

    Başak, Esra; Aydemir, Tülin; Dinçer, Ayşe; Becerik, Seda Çınar

    2013-12-01

    Catalase was immobilized on chitosan and modified chitosan. Studies were carried out on free-immobilized catalase concerning the determination of optimum temperature, pH, thermal, storage stability, reusability, and kinetic parameters. Optimum temperature and pH for free catalase and catalase immobilized were found as 35°C and 7.0, respectively. After 100 times of repeated tests, the immobilized catalases on chitosan-clay and magnetic chitosan maintain over 50% and 60% of the original activity, respectively. The ease of catalase immobilization on low-cost matrices and good stability upon immobilization in the present study make it a suitable product for further use in the food industry.

  12. On-Chip Magnetic Bead Manipulation and Detection Using a Magnetoresistive Sensor-Based Micro-Chip: Design Considerations and Experimental Characterization.

    PubMed

    Gooneratne, Chinthaka P; Kodzius, Rimantas; Li, Fuquan; Foulds, Ian G; Kosel, Jürgen

    2016-08-26

    The remarkable advantages micro-chip platforms offer over cumbersome, time-consuming equipment currently in use for bio-analysis are well documented. In this research, a micro-chip that includes a unique magnetic actuator (MA) for the manipulation of superparamagnetic beads (SPBs), and a magnetoresistive sensor for the detection of SPBs is presented. A design methodology, which takes into account the magnetic volume of SPBs, diffusion and heat transfer phenomena, is presented with the aid of numerical analysis to optimize the parameters of the MA. The MA was employed as a magnetic flux generator and experimental analysis with commercially available COMPEL™ and Dynabeads(®) demonstrated the ability of the MA to precisely transport a small number of SPBs over long distances and concentrate SPBs to a sensing site for detection. Moreover, the velocities of COMPEL™ and Dynabead(®) SPBs were correlated to their magnetic volumes and were in good agreement with numerical model predictions. We found that 2.8 μm Dynabeads(®) travel faster, and can be attracted to a magnetic source from a longer distance, than 6.2 μm COMPEL™ beads at magnetic flux magnitudes of less than 10 mT. The micro-chip system could easily be integrated with electronic circuitry and microfluidic functions, paving the way for an on-chip biomolecule quantification device.

  13. On-Chip Magnetic Bead Manipulation and Detection Using a Magnetoresistive Sensor-Based Micro-Chip: Design Considerations and Experimental Characterization

    PubMed Central

    Gooneratne, Chinthaka P.; Kodzius, Rimantas; Li, Fuquan; Foulds, Ian G.; Kosel, Jürgen

    2016-01-01

    The remarkable advantages micro-chip platforms offer over cumbersome, time-consuming equipment currently in use for bio-analysis are well documented. In this research, a micro-chip that includes a unique magnetic actuator (MA) for the manipulation of superparamagnetic beads (SPBs), and a magnetoresistive sensor for the detection of SPBs is presented. A design methodology, which takes into account the magnetic volume of SPBs, diffusion and heat transfer phenomena, is presented with the aid of numerical analysis to optimize the parameters of the MA. The MA was employed as a magnetic flux generator and experimental analysis with commercially available COMPEL™ and Dynabeads® demonstrated the ability of the MA to precisely transport a small number of SPBs over long distances and concentrate SPBs to a sensing site for detection. Moreover, the velocities of COMPEL™ and Dynabead® SPBs were correlated to their magnetic volumes and were in good agreement with numerical model predictions. We found that 2.8 μm Dynabeads® travel faster, and can be attracted to a magnetic source from a longer distance, than 6.2 μm COMPEL™ beads at magnetic flux magnitudes of less than 10 mT. The micro-chip system could easily be integrated with electronic circuitry and microfluidic functions, paving the way for an on-chip biomolecule quantification device. PMID:27571084

  14. Magnetic-resonance imaging and simplified Kozeny-Carman-model analysis of glass-bead packs as a frame of reference to study permeability of reservoir rocks

    NASA Astrophysics Data System (ADS)

    Wang, Dayong; Han, Dongyan; Li, Wenqiang; Zheng, Zhanpeng; Song, Yongchen

    2017-08-01

    Permeability variation in reservoir rocks results from the combined effects of various factors, and makes porosity-permeability ( ϕ- k) relationships more complex, or, in some cases, non-existent. In this work, the ϕ- k relationship of macroscopically homogeneous glass-bead packs is deduced based on magnetic resonance imaging (MRI) measurement and Kozeny-Carman (K-C) model analysis; these are used as a frame of reference to study permeability of reservoir rocks. The results indicate: (1) most of the commonly used simplified K-C models (e.g. the simplified traditional (omitting specific surface area), high-order, threshold, and fractal models) are suitable for estimating permeability of glass-bead packs. The simplified traditional model does not present obvious dependence on rock samples. Whether for the glass-bead packs or clean natural sandstones, the sample coefficients almost remain invariant. Comparably, the high-order, the fractal, and the threshold models are strongly sample-specific and cannot be extrapolated from the glass-bead packs to natural sandstones; (2) the ϕ- k relationships of quartz sands and silty sandstones resemble those of the glass-bead packs, but they significantly deviate from the K-C models at low porosities due to small pore entry radius; (3) a small amount of intergranular cements (<10%v) does not affect the general variation trend of permeability with porosity but can potentially increase predictive errors of the K-C models, whereas in the case of more cements, the ϕ- k relationships of sandstones become uncertain and cannot be described by any of these K-C models.

  15. Magnetic-resonance imaging and simplified Kozeny-Carman-model analysis of glass-bead packs as a frame of reference to study permeability of reservoir rocks

    NASA Astrophysics Data System (ADS)

    Wang, Dayong; Han, Dongyan; Li, Wenqiang; Zheng, Zhanpeng; Song, Yongchen

    2017-03-01

    Permeability variation in reservoir rocks results from the combined effects of various factors, and makes porosity-permeability (ϕ-k) relationships more complex, or, in some cases, non-existent. In this work, the ϕ-k relationship of macroscopically homogeneous glass-bead packs is deduced based on magnetic resonance imaging (MRI) measurement and Kozeny-Carman (K-C) model analysis; these are used as a frame of reference to study permeability of reservoir rocks. The results indicate: (1) most of the commonly used simplified K-C models (e.g. the simplified traditional (omitting specific surface area), high-order, threshold, and fractal models) are suitable for estimating permeability of glass-bead packs. The simplified traditional model does not present obvious dependence on rock samples. Whether for the glass-bead packs or clean natural sandstones, the sample coefficients almost remain invariant. Comparably, the high-order, the fractal, and the threshold models are strongly sample-specific and cannot be extrapolated from the glass-bead packs to natural sandstones; (2) the ϕ-k relationships of quartz sands and silty sandstones resemble those of the glass-bead packs, but they significantly deviate from the K-C models at low porosities due to small pore entry radius; (3) a small amount of intergranular cements (<10%v) does not affect the general variation trend of permeability with porosity but can potentially increase predictive errors of the K-C models, whereas in the case of more cements, the ϕ-k relationships of sandstones become uncertain and cannot be described by any of these K-C models.

  16. Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

    PubMed

    Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

    2015-04-15

    In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples. Copyright © 2014. Published by Elsevier B.V.

  17. Specific detection of foot-and-mouth disease serotype Asia 1 virus by carboxyl-magnetic beads conjugated with single-domain antibody.

    PubMed

    Yang, Shunli; Yin, Shuanghui; Shang, Youjun; Wang, Di; Ma, Weimin; He, Jijun; Guo, Jianhong; Cai, Jianping; Liu, Xiangtao

    2015-09-15

    Immunomagnetic nanobead (IMNB) labeled with specific antibody, has been demonstrated to be useful for the capturing and detection of viruses. In this study, we developed an imunomagnetic bead based on carboxyl-magnetic beads (MNB) labeled with a single-domain antibody (sdAb) for capturing foot-and-mouth disease (FMD) Asia 1 virus. After magnetic separation, complexes of MNB-sdAb-virus were detected with either a sandwich ELISA or QDs-C5 probe under a fluorescence microscope, and the complexes were used as templates for extraction of total RNA for amplification of the VP1 or 3D gene fragments using RT-PCR and real-time RT-PCR. The Asia 1 VLPs were efficiently captured through IMNB with a high binding rate of 5.09 μg of antigen/μl of bead suspension. Moreover, this method has been successfully used to capture Asia 1 antigen in synthetic samples. Ultimately, a specific and highly sensitive capture FMDV Asia 1 tool has been established that has the potential to enhance the sensitivity and reliability when diagnosing FMDV Asia 1.

  18. 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.

  19. 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

  20. Analysis of Glycoproteins in Human Serum by Means of Glycospecific Magnetic Bead Separation and LC-MALDI-TOF/TOF Analysis with Automated Glycopeptide Detection

    PubMed Central

    Sparbier, Katrin; Asperger, Arndt; Resemann, Anja; Kessler, Irina; Koch, Sonja; Wenzel, Thomas; Stein, Günter; Vorwerg, Lars; Suckau, Detlev; Kostrzewa, Markus

    2007-01-01

    Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively. PMID:17916798

  1. A Highly Selective and Sensitive Fluorescence Detection Method of Glyphosate Based on an Immune Reaction Strategy of Carbon Dot Labeled Antibody and Antigen Magnetic Beads.

    PubMed

    Wang, Duo; Lin, Bixia; Cao, Yujuan; Guo, Manli; Yu, Ying

    2016-08-03

    A sensitive fluorescence detection method for glyphosate (GLY) was established based on immune reaction. First, carbon dot labeled antibodies (lgG-CDs) which were able to specifically identify glyphosate were prepared with the environmentally friendly carbon dots (CDs) and glyphosate antibody (lgG). lgG-CDs could be used to in situ visualize the distribution of glyphosate in plant tissues. In order to eliminate the effects of excess lgG-CDs on the determination of GLY, antigen magnetic beads Fe3O4-GLY based on magnetic nanoparticles Fe3O4 and glyphosate were constructed and utilized to couple with the excess lgG-CDs. After magnetic separation to remove antigen magnetic beads, there was a linear relationship between the fluorescence intensity of lgG-CDs and the logarithmic concentration of glyphosate in the range of 0.01-80 μg/mL with a detection limit of 8 ng/mL. The method was used for the detection of glyphosate in Pearl River water, tea, and soil samples with satisfactory recovery ratio between 87.4% and 103.7%.

  2. Magnetic beads-based DNA hybridization chain reaction amplification and DNAzyme recognition for colorimetric detection of uranyl ion in seafood.

    PubMed

    Zhang, Hongyan; Cheng, Xian; Chen, Lian; Mo, Fan; Xu, LiangJun; Fu, FengFu

    2017-03-01

    A novel colorimetric biosensor, which employs DNAzyme-functionalized magnetic beads (MBs) as recognition probe, enzyme-assisted catalytic oxidation of TMB (3,3',5,5'-tetramethylbenzidine sulfate) as signal and DNA hybridization chain reaction as amplification strategy, has been developed for detecting trace uranyl ion (UO2(2+)) in seafood and aqueous environment with high sensitivity and specificity. We demonstrated that UO2(2+) can specifically cleave DNAzyme immobilized on MBs surface to release a short single-strand DNA (primer), and the released primer trigger DNA hybridization chain reaction to form a long one dimensional DNA concatamer on the MBs surface. The resulting long DNA concatamer could capture a large amount of HRP to generate the one UO2(2+)-to-multiple HRP amplification effect. Upon the addition of TMB-H2O2 solution, the HRP-tagged DNA concatamer-MBs conjugates could catalyze the H2O2-mediated oxidation of TMB, and thus results in a color change from colorless to blue in solution. This provided a sensitive and selective sensing platform for the visual or colorimetric detection of UO2(2+). The proposed biosensor has high sensitivity and strong anti-interference capability, it can be used to detect as low as 2.5 ppb (9.25 nM) of UO2(2+) by naked-eye observation and 0.09 ppb (0.33 nM) of UO2(2+) by UV-visible spectrometry with no interference of other ions and a RSD ≤ 6% (n = 5). With the help of this method, we have successfully determined trace UO2(2+) in fish muscle and river water with a recovery of 93-106%. High sensitivity and specificity, as well operation convenience, low cost and strong resistibility to the matrix, which makes our method a potential approach for the on-site detection of UO2(2+) in seafood and aqueous environment.

  3. Phagocytosis of mycobacteria by U937 cells: a rapid method for monitoring uptake and separating phagocytosed and free bacteria by magnetic beads.

    PubMed

    Whyte, J; Roberts, A D; Morley, K A; Sharp, R J; Marsh, P D

    2000-01-01

    A human-derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50-60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence-activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h.

  4. Magnetic bead fluorescent immunoassay for the rapid detection of the novel inflammation marker YKL40 at the point-of-care.

    PubMed

    Schmalenberg, Michael; Beaudoin, Christopher; Bulst, Ludwig; Steubl, Dominik; Luppa, Peter B

    2015-12-01

    Pneumonia is one of the leading causes of death worldwide.We present a magnetic bead fluorescent sandwich immunoassay that allows rapid serum measurement of the novel inflammation marker YKL40 (CHI3L1) at the point of care (POC) that could aid pneumonia diagnosis. The magnetic beads serve as the solid phase for separation of YKL40 from serum. The readout is performed using a small and robust fluorescence reader,which detects the turnover of a fluorescent substrate. The assay procedure, from sample addition to data retrieval, consists of three steps and is performed in less than 20 min. The presented assay has a linear range from 3 to 111 ng/mL, with a limit of detection of 2.9 ng/mL. The average recoveries were found between 101 and 111%. The developed method was applied in sera from healthy subjects (n= 14; c(YKL40)= 50 ± 49 ng/mL) and from pneumonia patients (n = 14; c(YKL40) = 333.6 ± 225 ng/mL). The elevated YKL40 concentrations in pneumonia-diseased patients are in good agreement with previously published data. The POC-ready device provides a simple immunoassay that could help to optimize pneumonia inflammation diagnostics in low-resource settings.

  5. [Non-modified magnetic beads coupled with multiple real-time PCR for detection and quantification of mycotoxigenic fungi in paprika samples].

    PubMed

    Jin, Yan; Zhang, Wei-Wei; Wang, Su-Yuan; Ye, Zheng-Mao; Zhang, Li-Shi; Pei, Xiao-Fang

    2015-01-01

    To establish a method for detecting 3 common toxigenic molds (Aspergillus, Penicillium, and Fusarium) based on non-modified magnetic beads coupled with multiple real-time PCR (NMB-multiple qPCR). The primers and genus-specific probes were designed based on the rDNA sequences to develop a multiple real-time PCR using non-modified magnetic bead to enrichment of fungal spores. The sensitivity, specificity and repeatability of this assay were evaluated. The detection limit of this assay for spiked samples was 10(4) CFU/g, demonstrating a 10-fold greater detection sensitivity of this assay than that of real-time PCR. The NMB-multiple qPCR assay also showed good specificity and reproducibility and yielded comparable results with those by traditional colony counting method for spiked samples (P>0.05). NMB-multiple qPCR assay we established allows rapid and sensitive detection of common mycotoxigenic fungi in paprika.

  6. Effects of pretreatment protocols on human amniotic fluid protein profiling with SELDI-TOF MS using protein chips and magnetic beads.

    PubMed

    Deng, Biping; Dong, Zhaogang; Liu, Yanguo; Wang, Chune; Liu, Jia; Wang, Chuanxin; Qu, Xun

    2010-08-05

    There is increasing interest in the use of human amniotic fluid (AF) proteomics with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for diagnosing pregnancy-associated abnormalities. A critical parameter of diagnostic biomarkers is the accuracy and reproducibility of protein patterns. We evaluated the effects of common pretreatment protocols on protein patterns generated using SELDI mass spectrometry with two different protein capture strategies (including functional protein chips and functionalized magnetic beads prior to MS analysis) in AF. Various extrinsic factors involved in processing and storing amniotic fluid, including matrix composition, sample storage time, temperature and freeze-thaw cycles, were analyzed regarding their impact on AF protein patterns using SELDI mass spectrometry with 2 different protein capture strategies. Three extrinsic factors (sample storage for 3days at either room temperature or 4 degrees C or freeze-thawing the sample 5 times) significantly decreased the number or intensities of protein peaks detected in AF. Matrix dilutions also changed the spectra of AF, with more peaks and higher intensities observed with 50% alpha-cyano-4-hydroxycinnamic acid (CHCA). Moreover, protein chips captured more proteins or peptides than magnetic beads on SELDI-TOF MS profiling of AF. These results suggest that extrinsic factors must be taken into account for valid data interpretation to ensure good reproducibility of AF profiling by SELDI mass spectrometry. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins.

    PubMed

    Chang, Peter S; Absood, Afaf; Linderman, Jennifer J; Omann, Geneva M

    2004-02-15

    A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G(beta2) in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10(6). These methods were coupled with immunodetection methods and an adenosine 5(')-diphosphate-ribosylation assay to measure the amount of membrane-associated G(ialpha) proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.

  8. Lab-on-a-disc agglutination assay for protein detection by optomagnetic readout and optical imaging using nano- and micro-sized magnetic beads.

    PubMed

    Uddin, Rokon; Burger, Robert; Donolato, Marco; Fock, Jeppe; Creagh, Michael; Hansen, Mikkel Fougt; Boisen, Anja

    2016-11-15

    We present a biosensing platform for the detection of proteins based on agglutination of aptamer coated magnetic nano- or microbeads. The assay, from sample to answer, is integrated on an automated, low-cost microfluidic disc platform. This ensures fast and reliable results due to a minimum of manual steps involved. The detection of the target protein was achieved in two ways: (1) optomagnetic readout using magnetic nanobeads (MNBs); (2) optical imaging using magnetic microbeads (MMBs). The optomagnetic readout of agglutination is based on optical measurement of the dynamics of MNB aggregates whereas the imaging method is based on direct visualization and quantification of the average size of MMB aggregates. By enhancing magnetic particle agglutination via application of strong magnetic field pulses, we obtained identical limits of detection of 25pM with the same sample-to-answer time (15min 30s) using the two differently sized beads for the two detection methods. In both cases a sample volume of only 10µl is required. The demonstrated automation, low sample-to-answer time and portability of both detection instruments as well as integration of the assay on a low-cost disc are important steps for the implementation of these as portable tools in an out-of-lab setting.

  9. Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

    PubMed

    Barizuddin, Syed; Balakrishnan, Baskar; Stringer, R Cody; Dweik, Majed

    2015-08-01

    A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and

  10. Magnetic bead-based phage anti-immunocomplex assay (PHAIA) for the detection of the urinary biomarker 3-phenoxybenzoic acid to assess human exposure to pyrethroid insecticides

    PubMed Central

    Kim, Hee-Joo; Ahn, Ki Chang; González-Techera, Andrés; González-Sapienza, Gualberto G.; Gee, Shirley J.; Hammock, Bruce D.

    2010-01-01

    Noncompetitive immunoassays are advantageous over competitive assays for the detection of small molecular weight compounds. We recently demonstrated that phage peptide libraries can be an excellent source of immunoreagents that facilitate the development of sandwich-type noncompetitive immunoassays for the detection of small analytes, avoiding the technical challenges of producing anti-immunocomplex antibody. In this work we explore a new format that may help to optimize the performance of the phage anti-immunocomplex assay (PHAIA) technology. As a model system we used a polyclonal antibody to 3-phenoxybenzoic acid (3-PBA) and an anti-immunocomplex phage clone bearing the cyclic peptide CFNGKDWLYC. The assay setup with the biotinylated antibody immobilized onto streptavidin-coated magnetic beads significantly reduced the amount of coating antibody giving identical sensitivity (50% saturation of the signal (SC50) = 0.2–0.4 ng/ml) to the best result obtained with direct coating of the antibody on ELISA plates. The bead-based assay tolerated up to 10 and 5% of methanol and urine matrix, respectively. This assay system accurately determined the level of spiked 3-PBA in different urine samples prepared by direct dilution or clean-up with solid-phase extraction after acidic hydrolysis with overall recovery of 80–120%. PMID:19101498

  11. Entrapment of DyP-type peroxidase from Pseudomonas fluorescens Pf-5 into Ca-alginate magnetic beads.

    PubMed

    Wasak, Agata; Drozd, Radosław; Struk, Łukasz; Grygorcewicz, Bartłomiej

    2017-03-21

    The aim of this study was to investigate the optimal conditions for the immobilization and stabilization of DyP1B dye decolorizing peroxidases type B (DyP1B) from Pseudomonas fluorescens Pf-5 immobilized in Ca-alginate ferromagnetic beads. The immobilized DyP1B was used in the degradation of the Reactive Blue 5 (RB5) synthetic dye. The enzyme was successfully entrapped in a Ca-alginate matrix and showed an encapsulation efficiency of 94%. The concentration of DyP1B (0.8 mg mL(-1) ), 2% of alginate and magnetite (10.0 mg mL(-1) ) was optimal for immobilization. The immobilized DyP1B showed optimum activity at pH 7.0 and 40 °C compared with pH 5.5 and 30 °C for free peroxidase. Reusability studies showed that after five cycles, the immobilized DyP1B system retained more than 58% of its initial activity. The immobilized DyP1B was able to decolorize RB5 at concentrations of 0.1, 0.05, and 0.01% (w v(-1) ) with efficiency rates of about 20, 29, and 45%, respectively. The immobilization of DyP1B in alginate beads with the addition of Fe3 O4 increased its catalytic and applicative potential. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  12. Removal of Hg(II) from aqueous solution by resin loaded magnetic β-cyclodextrin bead and graphene oxide sheet: Synthesis, adsorption mechanism and separation properties.

    PubMed

    Cui, Limei; Wang, Yaoguang; Gao, Liang; Hu, Lihua; Wei, Qin; Du, Bin

    2015-10-15

    Resin loaded magnetic β-cyclodextrin bead and graphene oxide sheet (MCD-GO-R) was synthesized successfully and found to be an excellent adsorbent for Hg(II) removal. The as-prepared adsorbent was characterized by SEM, FTIR, BET, magnetization curve and zeta potential analysis respectively. Good magnetic performance made MCD-GO-R simply recover from aqueous solution at low magnetic field within 30s. And also, the rich functional groups and outstanding dispersity play an important role in the adsorption process. The maximum adsorption capacity was 88.43 mg g(-1) at 323 K and pH 7.1. The as-prepared adsorbent could perform well in a wide pH range from 4.0 to 10.0. Static adsorption experimental data showed good correlation with pseudo-second-order model and Freundlich isotherm models. It was found that the contaminant adsorption was accomplished mainly via chelation or ion exchange and come to equilibrium in only 30 min. All experimental results, especially the excellent reproducibility and resistance to ion interference, suggest that MCD-GO-R has promising applications in water treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Plastic protein microarray to investigate the molecular pathways of magnetic nanoparticle-induced nanotoxicity

    NASA Astrophysics Data System (ADS)

    Liu, Yingshuai; Li, Xuelian; Bao, Shujuan; Lu, Zhisong; Li, Qing; Li, Chang Ming

    2013-05-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 μg ml-1 or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors.

  14. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manufacturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthesized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microarrays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

  15. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manu facturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthe-sized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microar-rays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

  16. Solid-state sensor incorporated in microfluidic chip and magnetic-bead enzyme immobilization approach for creatinine and glucose detection in serum

    NASA Astrophysics Data System (ADS)

    Lin, Yen-Heng; Chiang, Chien-Hung; Wu, Min-Hsien; Pan, Tung-Ming; Luo, Ji-Dung; Chiou, Chiuan-Chian

    2011-12-01

    Solid-state sensors are stable and inexpensive electric transducers for biomedical measurement. This study proposes a microfluidic chip incorporated with a solid-state sensor for measuring glucose and creatinine in blood serum. Magnetic beads are employed to immobilize enzymes and deliver them in a micro-channel. Glucose and creatinine can be measured at 2-8 mM and 10-2 to 10 mM, respectively, which is a meaningful range in human blood. The immobilization approach also addresses the issue of the long-term preservation of enzymes in microfluidic devices. The proposed device is suitable for multi-target measurement in a point-of-care system.

  17. Asynchronous magnetic bead rotation (AMBR) micro-viscometer for rapid, sensitive and label-free studies of bacterial growth and drug sensitivity

    PubMed Central

    Sinn, Irene; Albertson, Theodore; Kinnunen, Paivo; Breslauer, David N.; McNaughton, Brandon H.; Burns, Mark A.; Kopelman, Raoul

    2012-01-01

    The long turnaround time in antimicrobial susceptibility testing (AST) endangers patients and encourages the administration of wide spectrum antibiotics, thus resulting in alarming increases of multi-drug resistant pathogens. A method for faster detection of bacterial proliferation presents one avenue towards addressing this global concern. We report on a label-free asynchronous magnetic bead rotation (AMBR) based viscometry method that rapidly detects bacterial growth and determines drug sensitivity by measuring changes in the suspension’s viscosity. With this platform, we observed the growth of a uropathogenic Escherichia coli isolate, with an initial concentration of 50 cells per drop, within 20 minutes; in addition, we determined the gentamicin minimum inhibitory concentration (MIC) of the E. coli isolate within 100 minutes. We thus demonstrated a label-free, micro-viscometer platform that can measure bacterial growth and drug susceptibility more rapidly, with lower initial bacterial counts than existing commercial systems, and potentially with any microbial strains. PMID:22507307

  18. Novel electrochemical immunoassay for human IgG1 using metal sulfide quantum dot-doped bovine serum albumin microspheres on antibody-functionalized magnetic beads.

    PubMed

    Chen, Zongbao; Lu, Minghua

    2017-08-01

    A new magneto-controlled electrochemical immunosensing system was developed for the sensitive detection of low-abundance protein (IgG1 used in this case) with a sandwich-type assay format on monoclonal mouse anti-human Fab-specific IgG1-functionalized magnetic bead. Metal sulfide (CdS) quantum dot-doped bovine serum albumin (QD-BSA) was synthesized and functionalized with monoclonal Fc-specific anti-human antibody. In the presence of IgG1, the immobilized antibody on magnetic bead was selective to capture the Fab region of the analyte, followed to be sandwiched by the conjugated antibody onto QD-BSA. The subsequent anodic stripping voltammetric analysis of cadmium ion, released by acid from quantum dot, was conducted at an in situ prepared mercury film electrode. Under optimal conditions, the voltammetric current increased with the increasing of target IgG1 within a dynamic working range from 10 pg mL(-1) to 100 ng mL(-1). The limit of detection of this immunosensor was evaluated to 3.4 pg mL(-1) at 3sblank criterion. The precision, selectivity and method accuracy were acceptable. Analysis of human serum samples revealed good accordance with the results obtained by commercial enzyme-linked immunosorbent assay method. Importantly, this concept offers promise for cost-effective analysis of low-abundance cancer biomarkers without the need of natural enzymes. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation

    PubMed Central

    Fugier, Emilie; Dumont, Audrey; Malleron, Annie; Poquet, Enora; Mas Pons, Jordi; Baron, Aurélie; Vauzeilles, Boris; Dukan, Sam

    2015-01-01

    Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope. PMID:26061695

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

    PubMed

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

    2013-05-31

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

  1. Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis.

    PubMed

    Zhao, Xing-Chun; Wang, Le; Sun, Jing; Jiang, Bo-Wei; Zhang, Er-Li; Ye, Jian

    2016-01-01

    Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB-sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases.

  2. Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis

    PubMed Central

    Zhao, Xing-Chun; Wang, Le; Sun, Jing; Jiang, Bo-Wei; Zhang, Er-Li; Ye, Jian

    2016-01-01

    Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB–sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases. PMID:27442128

  3. Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads.

    PubMed

    Laschi, Serena; Palchetti, Ilaria; Marrazza, Giovanna; Mascini, Marco

    2009-09-01

    In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin-enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0-2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.

  4. Generation of memory T cells for adoptive transfer using clinical-grade anti-CD62L magnetic beads.

    PubMed

    Verfuerth, S; Sousa, P S E; Beloki, L; Murray, M; Peters, M D; O'Neill, A T; Mackinnon, S; Lowdell, M W; Chakraverty, R; Samuel, E R

    2015-10-01

    Pre-clinical studies of allogeneic stem cell transplantation suggest that depletion of naive T cells from donor lymphocytes will reduce the risk of GvHD but preserve immunity to infectious pathogens. In this study, we have established a clinical-grade protocol under good manufacturing practice conditions for purging CD62L(+) naive T cells from steady-state leukapheresis products using the CliniMACS system. The efficacy of immunomagnetic CD62L depletion was assessed by analysis of cell composition and functional immune responses. A median 2.9 log CD62L depletion was achieved with no evidence of CD62L shedding during the procedure and a mean T-cell yield of 47%. CD62L(-) cells comprised an equal mix of CD4(+) and CD8(+) T cells, with elimination of B cells but maintenance of regulatory T cells and natural killer cell populations. CD62L-depleted T cells were predominantly CD45RA(-) and CD45RA(+) effector memory (>90%) and contained the bulk of pentamer-staining antivirus-specific T cells. Functional assessment of CD62L(-) cells revealed the maintenance of antiviral T-cell reactivity and a reduction in the alloreactive immune response compared with unmanipulated cells. Clinical-grade depletion of naive T cells using immunomagnetic CD62L beads from steady-state leukapheresis products is highly efficient and generates cells suitable for adoptive transfer in the context of clinical trials.

  5. Electrochemical immunosensor for carcinoembryonic antigen based on nanosilver-coated magnetic beads and gold-graphene nanolabels.

    PubMed

    Chen, Huafeng; Tang, Dianping; Zhang, Bing; Liu, Bingqian; Cui, Yuling; Chen, Guonan

    2012-03-15

    A novel redox-active magnetic nanostructure was synthesized by using a wet chemical method for high-efficiency electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte). The nanostructures based on the combination of a magnetic nanocore, a layer of electroactive poly(o-phenylenediamine) (PPD), and a silver metallic shell displayed good adsorption properties for the attachment of anti-CEA antibody selective to CEA. The magnetic nanostructure presented good redox behaviors to facilitate and modulate the way it was integrated into a magnetic carbon paste electrode. The assay was based on a sandwich-type immunoassay protocol by using nanogold-patterned graphene oxide nanoscales (AuNP-GO), conjugated with horseradish peroxidase-labeled anti-CEA, as secondary antibodies and biofunctionalized magnetic nanostructures as immunosensing probes. Under optimal conditions, the nanoparticle-based immunocomposites exhibited good electrochemical responses for the determination of CEA, and allowed the detection of CEA at a concentration as low as 1.0 pg mL(-1) at a signal-to-noise ratio of 3. In addition, the magnetic immunosensing had good reproducibility, and acceptable accuracy, and could be successfully applied for the detection of CEA in the clinical serum specimens. Significantly, by controlling the target biomolecules, this assay can be easily extended for use with other immunosensings, and thus represents a versatile design routine. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Preparation of magnetic indole-3-acetic acid imprinted polymer beads with 4-vinylpyridine and β-cyclodextrin as binary monomer via microwave heating initiated polymerization and their application to trace analysis of auxins in plant tissues.

    PubMed

    Zhang, Yi; Li, Yuanwen; Hu, Yuling; Li, Gongke; Chen, Yueqin

    2010-11-19

    Auxin is a crucial phytohormone for precise control of growth and development of plants. Due to its low concentration in plant tissues which are rich in interfering substances, the accurate determination of auxins remains a challenge. In this paper, a new strategy for isolation and enrichment of auxins from plant tissues was obtained by the magnetic molecularly imprinted polymer (mag-MIP) beads, which were prepared by microwave heating initiated suspension polymerization using indole-3-acetic acid (IAA) as template. In order to obtain higher selective recognition cavities, an enhanced imprinting method based on binary functional monomers, 4-vinylpyridine (4-VP) and β-cyclodextrin (β-CD), was adopted for IAA imprinting. The morphological and magnetic characteristics of the mag-MIP beads were characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy and vibrating sample magnetometry. A majority of resultant beads were within the size range of 80-150μm. Porous surface morphology and good magnetic property were observed. Furthermore, the mag-MIP beads fabricated with 4-VP and β-CD as binary functional monomers exhibited improved recognition ability to IAA, as compared with the mag-MIP beads prepared with the individual monomer separately. Competitive rebinding experiment results revealed that the mag-MIP beads exhibited a higher specific recognition for the template than the non-imprinted polymer (mag-NIP) beads. An extraction method by mag-MIP beads coupled with high performance liquid chromatography (HPLC) was developed for determination of IAA and indole-3-butyric acid (IBA) in plant tissues. Linear ranges for IAA and IBA were in the range of 7.00-100.0μgL(-1) and 10.0-100.0μgL(-1), and the detection limits were 3.9 and 7.4μgL(-1), respectively. The analytical performance was also estimated by seedlings or immature embryos samples from three different plant tissues, pea, rice and wheat. Recoveries were in the range of 70

  7. In vivo labeling and specific magnetic bead separation of RNA for biofilm characterization and stress-induced gene expression analysis in bacteria.

    PubMed

    Stankiewicz, Nikolai; Gold, Andrea; Yüksel, Yousra; Berensmeier, Sonja; Schwartz, Thomas

    2009-12-01

    The method of in vivo labeling and separation of bacterial RNA was developed as an approach to elucidating the stress response of natural bacterial populations. This technique is based on the incorporation of digoxigenin-11-uridine-5'-triphosphate (DIG-11-UTP) in the RNA of active bacteria. The digoxigenin fulfills a dual role as a label of de novo synthesized RNA and a target for magnetic bead separation from a total RNA extract. Depending on the growth conditions and the population's composition, the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total RNA in gram-positive and gram-negative reference bacteria as well as in natural biofilms from drinking water, surface water, and lake sediment. Separation of DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average separation yield from total RNA extracts was about 95% of labeled RNA. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, as was evaluated by spiking experiments with an unmarked DNA amplicon. Applicability of the method developed was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress-induced genes (vanA and rpoS) in reference bacteria.

  8. Bead magnetorelaxometry with an on-chip magnetoresistive sensor.

    PubMed

    Dalslet, Bjarke Thomas; Damsgaard, Christian Danvad; Donolato, Marco; Strømme, Maria; Strömberg, Mattias; Svedlindh, Peter; Hansen, Mikkel Fougt

    2011-01-21

    Magnetorelaxometry measurements on suspensions of magnetic beads are demonstrated using a planar Hall effect sensor chip embedded in a microfluidic system. The alternating magnetic field used for magnetizing the beads is provided by the sensor bias current and the complex magnetic susceptibility spectra are recorded as the 2nd harmonic of the sensor response. The complex magnetic susceptibility signal appears when a magnetic bead suspension is injected, it scales with the bead concentration, and it follows the Cole-Cole expression for Brownian relaxation. The complex magnetic susceptibility signal resembles that from conventional magnetorelaxometry done on the same samples apart from an offset in Brownian relaxation frequency. The time dependence of the signal can be rationalized as originating from sedimented beads.

  9. Development and Validation of a Novel Diagnostic Test for Human Brucellosis Using a Glyco-engineered Antigen Coupled to Magnetic Beads

    PubMed Central

    Ciocchini, Andrés E.; Rey Serantes, Diego A.; Melli, Luciano J.; Iwashkiw, Jeremy A.; Deodato, Bettina; Wallach, Jorge; Feldman, Mario F.; Ugalde, Juan E.; Comerci, Diego J.

    2013-01-01

    Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values, a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited

  10. Development and validation of a novel diagnostic test for human brucellosis using a glyco-engineered antigen coupled to magnetic beads.

    PubMed

    Ciocchini, Andrés E; Rey Serantes, Diego A; Melli, Luciano J; Iwashkiw, Jeremy A; Deodato, Bettina; Wallach, Jorge; Feldman, Mario F; Ugalde, Juan E; Comerci, Diego J

    2013-01-01

    Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values, a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited

  11. Dual Recognition Strategy for Specific and Sensitive Detection of Bacteria Using Aptamer-Coated Magnetic Beads and Antibiotic-Capped Gold Nanoclusters.

    PubMed

    Cheng, Dan; Yu, Mengqun; Fu, Fei; Han, Weiye; Li, Gan; Xie, Jianping; Song, Yang; Swihart, Mark T; Song, Erqun

    2016-01-05

    Food poisoning and infectious diseases caused by pathogenic bacteria such as Staphylococcus aureus (SA) are serious public health concerns. A method of specific, sensitive, and rapid detection of such bacteria is essential and important. This study presents a strategy that combines aptamer and antibiotic-based dual recognition units with magnetic enrichment and fluorescent detection to achieve specific and sensitive quantification of SA in authentic specimens and in the presence of much higher concentrations of other bacteria. Aptamer-coated magnetic beads (Apt-MB) were employed for specific capture of SA. Vancomycin-stabilized fluorescent gold nanoclusters (AuNCs@Van) were prepared by a simple one-step process and used for sensitive quantification of SA in the range of 32-10(8) cfu/mL with the detection limit of 16 cfu/mL via a fluorescence intensity measurement. And using this strategy, about 70 cfu/mL of SA in complex samples (containing 3 × 10(8) cfu/mL of other different contaminated bacteria) could be successfully detected. In comparison to prior studies, the developed strategy here not only simplifies the preparation procedure of the fluorescent probes (AuNCs@Van) to a great extent but also could sensitively quantify SA in the presence of much higher concentrations of other bacteria directly with good accuracy. Moreover, the aptamer and antibiotic used in this strategy are much less expensive and widely available compared to common-used antibodies, making it cost-effective. This general aptamer- and antibiotic-based dual recognition strategy, combined with magnetic enrichment and fluorescent detection of trace bacteria, shows great potential application in monitoring bacterial food contamination and infectious diseases.

  12. Controlled torque on superparamagnetic beads for functional biosensors.

    PubMed

    Janssen, X J A; Schellekens, A J; van Ommering, K; van Ijzendoorn, L J; Prins, M W J

    2009-03-15

    We demonstrate that a rotating magnetic field can be used to apply a controlled torque on superparamagnetic beads which leads to a tunable bead rotation frequency in fluid. Smooth rotation is obtained for field rotation frequencies many orders of magnitude higher than the bead rotation frequency. A quantitative model is developed, based on results from a comprehensive set of experiments at different field strengths and frequencies. At low frequencies (<10Hz), rotation is due to a small permanent magnetic moment in the bead. At high frequencies (kHz-MHz), the torque results from a phase lag between the applied field and the induced magnetic moment, caused by the non-zero relaxation time of magnetic nanoparticles in the bead. The control of torque and rotation will enable novel functional assays in bead-based biosensors.

  13. Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.

    PubMed

    Li, Yunfang; Xu, Jia; Chen, Yu; Mei, Zhinan; Xiao, Yuxiu

    2015-12-18

    Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.

  14. A Novel Assay for Screening Inhibitors Targeting HIV Integrase LEDGF/p75 Interaction Based on Ni2+ Coated Magnetic Agarose Beads

    PubMed Central

    Dawei, Zhang; Hongqiu, He; Mengmeng, Liu; Zhixia, Meng; Shunxing, Guo

    2016-01-01

    HIV-1 integrase (IN) plays an essential role in viral replication and thus serves as an important target for chemotherapeutic intervention against HIV-1 infection. However, the current three clinical IN inhibitors, raltegravir, elvitegravir and dolutegravir share the same inhibitory mechanism, resulting in a common clinical resistance profile which have emerged in infected patients receiving treatment. Therefore, it is important to develop small molecule inhibitors that impair IN function with distinct mechanisms of action. In this work, a magnetic-beads based biochemical assay targeting the protein-protein interaction (PPI) between HIV IN and the cellular cofactor LEDGF/p75 was developed for identification of HIV-1 IN inhibitors. Furthermore, a library containing 1000 US. Food and Drug Administration (FDA)-approved drugs currently used for human medication was screened to identify inhibitors targeting the PPI. The assay was proved to be quite robust and with the novel assay we successfully identified dexlansoprazole (IC50 of 4.8 μM), a FDA-approved proton pump inhibitor, as a potential inhibitor for the PPI between IN and LEDGF/p75, which bound to the LEDGF/p75 partner with a kinetic dissociation (Kd) constant of 330 nM ± 2.6 nM. PMID:27633629

  15. Selective collection and detection of MCF-7 breast cancer cells using aptamer-functionalized magnetic beads and quantum dots based nano-bio-probes.

    PubMed

    Hua, Xin; Zhou, Zhenxian; Yuan, Liang; Liu, Songqin

    2013-07-25

    A novel strategy for selective collection and detection of breast cancer cells (MCF-7) based on aptamer-cell interaction was developed. Mucin 1 protein (MUC1) aptamer (Apt1) was covalently conjugated to magnetic beads to capture MCF-7 cell through affinity interaction between Apt1 and MUC1 protein that overexpressed on the surface of MCF-7 cells. Meanwhile, a nano-bio-probe was constructed by coupling of nucleolin aptamer AS1411 (Apt2) to CdTe quantum dots (QDs) which were homogeneously coated on the surfaces of monodispersed silica nanoparticles (SiO2 NPs). The nano-bio-probe displayed similar optical and electrochemical performances to free CdTe QDs, and remained high affinity to nucleolin overexpressed cells through the interaction between AS1411 and nucleolin protein. Photoluminescence (PL) and square-wave voltammetric (SWV) assays were used to quantitatively detect MCF-7 cells. Improved selectivity was obtained by using these two aptamers together as recognition elements simultaneously, compared to using any single aptamer. Based on the signal amplification of QDs coated silica nanoparticles (QDs/SiO2), the detection sensitivity was enhanced and a detection limit of 201 and 85 cells mL(-1) by PL and SWV method were achieved, respectively. The proposed strategy could be extended to detect other cells, and showed potential applications in cell imaging and drug delivery.

  16. Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

    PubMed Central

    Pengpumkiat, Sumate; Koesdjojo, Myra; Rowley, Erik R.; Mockler, Todd C.; Remcho, Vincent T.

    2016-01-01

    Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA. PMID:26930667

  17. Assessment of suitability of magnetic beads for purification of rat plasma in proteomic analyses by matrix-assisted laser desorption ionization-time-of-flight MS.

    PubMed

    Mohottalage, Susantha; Vincent, Renaud; Kumarathasan, Prem

    2009-01-01

    Plasma is a complex matrix and has to be clarified or fractionated to obtain informative MS data. Although there are a number of prefractionation methods to clean up complex biological matrixes before proteomic analysis, these methods require large sample volumes and are costly and time-consuming. Alternatively, recently introduced magnetic beads (MB) appear to be attractive in overcoming these difficulties. Therefore, we were interested in investigating the applicability of MB in the clarification of rat plasma samples for proteome analyses. For this purpose, we used complementary supports, such as hydrophobic interaction chromatography-based MB (MB-C18) and weak cation-exchange chromatography-based MB (MB-WCX). MB-based fractionated samples were either spotted directly or underwent tryptic digestion before matrix-assisted laser desorption ionization (MALDI) spotting. Samples from both MB separation techniques gave clean and well-resolved MALDI-time-of-flight MS spectra in the low molecular mass range of 1-10 kDa with alpha-cyano-4-hydroxycinnamic acid as the matrix. Both techniques gave approximately 300 analyte peaks in this mass range. Our results showed that both MB-based separation procedures gave complementary mass spectral information. This approach provided information on the identity of a number of less-abundant and more-abundant proteins in plasma. Our findings suggest that this MB-based proteomic approach can be valuable in conducting faster screening of plasma samples for protein profiling.

  18. Use of anti-CD3/CD28 mAb coupled magnetic beads permitting subsequent phenotypic analysis of activated human T cells by indirect immunofluorescence.

    PubMed

    Pène, Jérôme; Rahmoun, Massilva; Temmerman, Stéphane; Yssel, Hans

    2003-12-01

    Functional analysis of T lymphocytes requires in vitro stimulation of these cells under experimental conditions that mimic as closely as possible physiological in vivo stimulation and that involve antigen/T cell receptor (TCR)-mediated activation. Because of the low frequency of antigen-specific T cells in human clinical samples, stimulation with a combination of anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) is a preferred method. Interaction of these mAbs with their ligand results in modulation of the mAb-ligand complex from the cell surface. However, as a result of incomplete modulation, CD3/CD28 mAb complexes often remain at the cell surface, thereby precluding subsequent indirect immunofluorescence and flow cytometry analysis using mouse immunoglobulin (Ig)-specific antibodies. This is of importance in situations in which no specific fluorochrome-conjugated mAbs are available, such as in screening procedures of Ig-containing hybridoma culture supernatants. We propose here the use of CD3/CD28 mAbs, linked to magnetic beads allowing standardization of the activation conditions, optimal activation of T cells and complete modulation of antigen-antibody complexes from the cell surface.

  19. Long term stability of Oligo (dT) 25 magnetic beads for the expression analysis of Euglena gracilis for long term space projects

    NASA Astrophysics Data System (ADS)

    Becker, Ina; Strauch, Sebastian M.; Hauslage, Jens; Lebert, Michael

    2017-05-01

    The unicellular freshwater flagellate Euglena gracilis has a highly developed sensory system. The cells use different stimuli such as light and gravity to orient themselves in the surrounding medium to find areas for optimal growth. Due to the ability to produce oxygen and consume carbon dioxide, Euglena is a suitable candidate for life support systems. Participation in a long-term space experiment would allow for the analysis of changes and adaptations to the new environment, and this could bring new insights into the mechanism of perception of gravity and the associated signal transduction chain. For a molecular analysis of transcription patterns, an automated system is necessary, capable of performing all steps from taking a sample, processing it and generating data. One of the developmental steps is to find long-term stable reagents and materials and test them for stability at higher-than-recommended temperature conditions during extended storage time. We investigated the usability of magnetic beads in an Euglena specific lysis buffer after addition of the RNA stabilizer Dithiothreitol over 360 days and the lysis buffer with the stabilizer alone over 455 days at the expected storage temperature of 19 °C. We can claim that the stability is not impaired at all after an incubation period of over one year. This might be an interesting result for researchers who have to work under non-standard lab conditions, as in biological or medicinal fieldwork.

  20. Magnetic beads-based DNAzyme recognition and AuNPs-based enzymatic catalysis amplification for visual detection of trace uranyl ion in aqueous environment.

    PubMed

    Zhang, Hongyan; Lin, Ling; Zeng, Xiaoxue; Ruan, Yajuan; Wu, Yongning; Lin, Minggui; He, Ye; Fu, FengFu

    2016-04-15

    We herein developed a novel biosensor for the visual detection of trace uranyl ion (UO2(2+)) in aqueous environment with high sensitivity and specificity by using DNAzyme-functionalized magnetic beads (MBs) for UO2(2+) recognition and gold nano-particles (AuNPs)-based enzymatic catalysis oxidation of TMB (3,3',5,5'-tetramethylbenzidine sulfate) for signal generation. The utilization of MBs facilitates the magnetic separation and collection of sensing system from complex sample solution, which leads to more convenient experimental operation and more strong resistibility of the biosensor to the matrix of sample, and the utilization of AuNPs-based enzymatic catalysis amplification greatly improved the sensitivity of the biosensor. Compared with the previous DNAzyme-based UO2(2+) sensors, the proposed biosensor has outstanding advantages such as relative high sensitivity and specificity, operation convenience, low cost and more strong resistibility to the matrix of sample. It can be used to detect as low as 0.02 ppb (74 pM) of UO2(2+) in aqueous environment by only naked-eye observation and 1.89 ppt (7.0 pM) of UO2(2+) by UV-visible spectrophotometer with a recovery of 93-99% and a RSD ≤ 5.0% (n=6) within 3h. Especially, the visual detection limit of 0.02 ppb (74 pM) is much lower than the maximum allowable level of UO2(2+) (130 nM) in the drinking water defined by the U.S. Environmental Protection Agency (EPA), indicating that our method meets the requirement of rapid and on-site detection of UO2(2+) in the aqueous environment by only naked-eye observation. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Analysis of antibody profiles in symptomatic malaria in three sentinel sites of Ivory Coast by using multiplex, fluorescent, magnetic, bead-based serological assay (MAGPIX™).

    PubMed

    Koffi, David; Touré, André Offianan; Varela, Marie-Louise; Vigan-Womas, Inès; Béourou, Sylvain; Brou, Somela; Ehouman, Marie-France; Gnamien, Laeticia; Richard, Vincent; Djaman, Joseph Allico; Perraut, Ronald

    2015-12-21

    Advances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead-based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast. Recombinant proteins or peptides derived from liver or blood stage antigens of Plasmodium falciparum (CSP, LSA141, LSA3, SALSA, PF13-DBL1α1, GLURP, AMA1, MSP1p19, MSP4p20), the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour-coded microsphere (Luminex™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings (rural and periurban) were analysed. Highly variable seroprevalence of antibody responses against parasite antigens was found ranging from 3 (gSG6) to 97% (MSP4p20). A marked prevalence and significantly higher level of antibodies was found in patients from the rural site (Korhogo), those harbouring the lowest level of parasitaemia. The use of whole schizont extract could not discriminate immunity level, contrary to parasite-derived recombinant proteins or peptides. Prevalence of responders to LSA141 and levels of antibodies to PF13 were significantly different between the three settings. Moreover, the post-treatment clearance of parasites was clearly associated with a significantly higher level of antibody response for almost 50% of the parasite antigens tested. The multiplex MBA-Magpix technology assay provides an accurate high throughput monitoring of parasite-specific antibodies during symptomatic malaria. The levels of antibody responses may provide a risk criterion with respect to the degree of parasitic infection

  2. New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag".

    PubMed

    Okada, Yoshiaki; Takano, Tomoko Y; Kobayashi, Nozomi; Hayashi, Arisa; Yonekura, Masaaki; Nishiyama, Yuji; Abe, Tomohiro; Yoshida, Takuya; Yamamoto, Takao A; Seino, Satoshi; Doi, Takefumi

    2011-05-18

    Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.

  3. PCR and magnetic bead-mediated target capture for the isolation of short interspersed nucleotide elements in fishes.

    PubMed

    Liu, Dong; Zhu, Guoli; Tang, Wenqiao; Yang, Jinquan; Guo, Hongyi

    2012-01-01

    Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180-360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNA(Leu)-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs.

  4. PCR and Magnetic Bead-Mediated Target Capture for the Isolation of Short Interspersed Nucleotide Elements in Fishes

    PubMed Central

    Liu, Dong; Zhu, Guoli; Tang, Wenqiao; Yang, Jinquan; Guo, Hongyi

    2012-01-01

    Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180–360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNALeu-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs. PMID:22408437

  5. Highly Avid Magnetic Bead Capture: An Efficient Selection Method for de novo Protein Engineering Utilizing Yeast Surface Display

    PubMed Central

    Ackerman, Margaret; Levary, David; Tobon, Gabriel; Hackel, Benjamin; Davis Orcutt, Kelly; Wittrup, K. Dane

    2010-01-01

    Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million-fold improvements in binding affinity by alternating rounds of diversification and flow cytometry-based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads—allowing isolation of extremely weak binders from billions of non-binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000-fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin-biotin that do not cross-react to streptavidin alone. PMID:19363813

  6. Direct Numerical Simulation of Pore-Scale Flow in a Bead Pack: Comparison with Magnetic Resonance Imaging Observations

    SciTech Connect

    Yang, Xiaofan; Scheibe, Timothy D.; Richmond, Marshall C.; Perkins, William A.; Vogt, Sarah J.; Codd, Sarah L.; Seymour, Joseph D.; Mckinley, Matthew I.

    2013-04-01

    A significant body of current research is aimed at developing methods for numerical simulation of flow and transport in porous media that explicitly resolve complex pore and solid geometries, and at utilizing such models to study the relationships between fundamental pore-scale processes and macroscopic manifestations at larger (i.e., Darcy) scales. A number of different numerical methods for pore-scale simulation have been developed, and have been extensively tested and validated for simplified geometries. However, validation of pore-scale simulations of fluid velocity for complex, three-dimensional (3D) pore geometries that are representative of natural porous media is challenging due to our limited ability to measure pore-scale velocity in such systems. Recent advances in magnetic resonance imaging (MRI) offer the opportunity to measure not only the pore geometry, but also local fluid velocities under steady-state flow conditions in 3D and with high spatial resolution. In this paper, we present a 3D velocity field measured at sub-pore resolution (tens of micrometers) over a centimeter-scale 3D domain using MRI methods. We have utilized the measured pore geometry to perform 3D simulations of Navier-Stokes flow over the same domain using direct numerical simulation techniques. We present a comparison of the numerical simulation results with the measured velocity field. It is shown that the numerical results match the observed velocity patterns well overall except for a variance and small systematic scaling which can be attributed to the known experimental error in the MRI measurements. The comparisons presented here provide strong validation of the pore-scale simulation methods and new insights for interpretation of uncertainty in MRI measurements of pore-scale velocity. This study also provides a potential benchmark for future comparison of other pore-scale simulation methods.

  7. Direct numerical simulation of pore-scale flow in a bead pack: Comparison with magnetic resonance imaging observations

    NASA Astrophysics Data System (ADS)

    Yang, Xiaofan; Scheibe, Timothy D.; Richmond, Marshall C.; Perkins, William A.; Vogt, Sarah J.; Codd, Sarah L.; Seymour, Joseph D.; McKinley, Matthew I.

    2013-04-01

    A significant body of current research is aimed at developing methods for numerical simulation of flow and transport in porous media that explicitly resolve complex pore and solid geometries, and at utilizing such models to study the relationships between fundamental pore-scale processes and macroscopic manifestations at larger (i.e., Darcy) scales. A number of different numerical methods for pore-scale simulation have been developed, and have been extensively tested and validated for simplified geometries. However, validation of pore-scale simulations of fluid velocity for complex, three-dimensional (3D) pore geometries that are representative of natural porous media is challenging due to our limited ability to measure pore-scale velocity in such systems. Recent advances in magnetic resonance imaging (MRI) offer the opportunity to measure not only the pore geometry, but also local fluid velocities under steady-state flow conditions in 3D and with high spatial resolution. In this paper, we present a 3D velocity field measured at sub-pore resolution (tens of micrometers) over a centimeter-scale 3D domain using MRI methods. We have utilized the measured pore geometry to perform 3D simulations of Navier-Stokes flow over the same domain using direct numerical simulation techniques. We present a comparison of the numerical simulation results with the measured velocity field. It is shown that the numerical results match the observed velocity patterns well overall except for a variance and small systematic scaling which can be attributed to the known experimental uncertainty in the MRI measurements. The comparisons presented here provide strong validation of the pore-scale simulation methods and new insights for interpretation of uncertainty in MRI measurements of pore-scale velocity. This study also provides a potential benchmark for future comparison of other pore-scale simulation methods. 2012 Elsevier Science.

  8. Magnetic beads as an extraction medium for simultaneous quantification of acetaminophen and structurally related compounds in human serum.

    PubMed

    Bylda, Caroline; Velichkova, Vanya; Bolle, Jens; Thiele, Roland; Kobold, Uwe; Volmer, Dietrich A

    2015-06-01

    This paper describes a sample preparation method that complements a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for acetaminophen and eight structurally-related compounds in human serum (C. Bylda, R. Thiele, U. Kobold, D.A. Volmer. Drug Test. Anal. 2014, 6, 451). The analytes (acetaminophen [APAP] + metabolites acetaminophen-glucuronide [APG], -cysteine [APC], -mercapturate [APM] and -cysteine [APC], structurally similar analogues phenacetin and p-phenetidine, as well as tricyclic antidepressants imipramine and amitryptiline) were extracted from serum using magnetized hyper-crosslinked polystyrene particles. The sample preparation protocol was developed by means of a design of experiments (DoE) statistical approach. Using three representative compounds from the analyte panel with different polarities (high, medium, and low), two screening designs were used to identify factors that exhibited significant impact on recovery of the analytes. These parameters were then optimized to permit extraction of the complete target panel exhibiting a broad range of chemical polarities. Liquid chromatographic separations were achieved by gradient elution using a pentafluorphenyl column with subsequent detection by electrospray ionization-triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. The method was linear over the range 0.1-100 µg/mL for APAP, APG, p-phenetidine and phenacetin, 0.03-50 µg/mL for APS, and 0.01-10 µg/mL for APM, APC, imipramine and amitriptyline, with R(2)  > 0.99. The assay exhibited good precision with CVs ranging from 2 to 9% for all analytes; the accuracy was assessed by comparing two LC-MS/MS methods using a set of 68 patient samples. Copyright © 2014 John Wiley & Sons, Ltd.

  9. 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

  10. Rapid and sensitive detection of Escherichia coli O157:H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification.

    PubMed

    Aydin, Muhsin; Herzig, Gene P D; Jeong, Kwang Cheol; Dunigan, Samantha; Shah, Parth; Ahn, Soohyoun

    2014-01-01

    Escherichia coli O157:H7 is a major foodborne pathogen that has posed serious problems for food safety and public health. Recent outbreaks and recalls associated with various foods contaminated by E. coli O157:H7 clearly indicate its deleterious effect on food safety. A rapid and sensitive detection assay is needed for this harmful organism to prevent foodborne illnesses and control outbreaks in a timely manner. We developed a magnetic bead-based immunoassay for detection of E. coli O157:H7 (the most well-known Shiga toxigenic E. coli strain) with a 96-well microplate as an assay platform. Immunomagnetic separation (IMS) and tyramide signal amplification were coupled to the assay to increase its sensitivity and specificity. This immunoassay was able to detect E. coli O157:H7 in pure culture with a detection limit of 50 CFU/ml in less than 3 h without an enrichment step. The detection limit was decreased 10-fold to 5 CFU/ml with addition of a 3-h enrichment step. When this assay was tested with other nontarget foodborne pathogens and common enteric bacteria, no cross-reactivity was found. When tested with artificially contaminated ground beef and milk samples, the assay sensitivity decreased two- to fivefold, with detection limits of 250 and 100 CFU/ml, respectively, probably because of the food matrix effect. The assay results also were compared with those of a sandwich-type enzyme-linked immunosorbent assay (ELISA) and an ELISA coupled with IMS; the developed assay was 25 times and 4 times more sensitive than the standard ELISA and the IMS-ELISA, respectively. Tyramide signal amplification combined with IMS can improve sensitivity and specificity for detection of E. coli O157:H7. The developed assay could be easily adapted for other foodborne pathogens and will contribute to improved food safety and public health.

  11. Small, porous polyacrylate beads

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Shiao-Ping Siao (Inventor); Dreyer, William J. (Inventor)

    1976-01-01

    Uniformly-shaped, porous, round beads are prepared by the co-polymerization of an acrylic monomer and a cross-linking agent in the presence of 0.05 to 5% by weight of an aqueous soluble polymer such as polyethylene oxide. Cross-linking proceeds at high temperature above about 50.degree.C or at a lower temperature with irradiation. Beads of even shape and even size distribution of less than 2 micron diameter are formed. The beads will find use as adsorbents in chromatography and as markers for studies of cell surface receptors.

  12. Crosslinked, porous, polyacrylate beads

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Shiao-Ping S. (Inventor); Dreyer, William J. (Inventor)

    1977-01-01

    Uniformly-shaped, porous, round beads are prepared by the co-polymerization of an acrylic monomer and a cross-linking agent in the presence of 0.05 to 5% by weight of an aqueous soluble polymer such as polyethylene oxide. Cross-linking proceeds at high temperature above about 50.degree. C or at a lower temperature with irradiation. Beads of even shape and even size distribution of less than 2 micron diameter are formed. The beads will find use as adsorbents in chromatography and as markers for studies of cell surface receptors.

  13. Crosslinked, porous, polyacrylate beads

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Shiao-Ping Siao (Inventor); Dreyer, William J. (Inventor)

    1976-01-01

    Uniformly-shaped, porous, round beads are prepared by the co-polymerization of an acrylic monomer and a cross-linking agent in the presence of 0.05 to 5% by weight of an aqueous soluble polymer such as polyethylene oxide. Cross-linking proceeds at high temperature above about 50.degree.C or at a lower temperature with irradiation. Beads of even shape and even size distribution of less than 2 micron diameter are formed. The beads will find use as adsorbents in chromatography and as markers for studies of cell surface receptors.

  14. Acupressure Bead in the Eustachian Tube.

    PubMed

    Igarashi, Kazunori; Matsumoto, Yu; Kakigi, Akinobu

    2015-08-01

    In this article, we aim to enlighten practitioners and patients involved with acupressure beads and to contribute to their safer use by reporting a unique case of insidious intrusion of an acupressure bead into the eustachian tube. A metallic object was found in the eustachian tube of a patient while conducting a magnetic resonance imaging (MRI) examination. The object was later confirmed to be an auricular acupressure bead, and was successfully removed by performing a tympanoplasty and a canal wall down mastoidectomy. The bead was assumed to have passed through an existing perforation of the tympanic membrane. According to previously published literature, tympanic membrane perforations exist in ∼1% of the population. Therefore, middle-ear foreign bodies are relatively common occurrences for otolaryngologists. However, metallic objects such as acupressure beads are especially important in the sense that they can cause severe burns during MRI. To avoid potential complications, acupressure-bead practitioners should be aware of the possibility that intrusions through the tympanic membrane could go unnoticed.

  15. Chromosome Microarray.

    PubMed

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed.

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

    SciTech Connect

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

    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 of 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

  17. Automated Immunomagnetic Separation and Microarray Detection of E. coli O157:H7 from Poultry Carcass Rinse

    SciTech Connect

    Chandler, Darrell P. ); Brown, Jeremy D.; Call, Douglas R. ); Wunschel, Sharon C. ); Grate, Jay W. ); Holman, David A.; Olson, Lydia G.; Stottlemyer, Mark S.; Bruckner-Lea, Cindy J. )

    2001-09-01

    We describe the development and application of a novel electromagnetic flow cell and fluidics system for automated immunomagnetic separation of E. coli directly from unprocessed poultry carcass rinse, and the biochemical coupling of automated sample preparation with nucleic acid microarrays without cell growth. Highly porous nickel foam was used as a magnetic flux conductor. Up to 32% recovery efficiency of 'total' E. coli was achieved within the automated system with 6 sec contact times and 15 minute protocol (from sample injection through elution), statistically similar to cell recovery efficiencies in > 1 hour 'batch' captures. The electromagnet flow cell allowed complete recovery of 2.8 mm particles directly from unprocessed poultry carcass rinse whereas the batch system did not. O157:H7 cells were reproducibly isolated directly from unprocessed poultry rinse with 39% recovery efficiency at 103 cells ml-1 inoculum. Direct plating of washed beads showed positive recovery of O 157:H7 directly from carcass rinse at an inoculum of 10 cells ml-1. Recovered beads were used for direct PCR amplification and microarray detection, with a process-level detection limit (automated cell concentration through microarray detection) of < 103 cells ml-1 carcass rinse. The fluidic system and analytical approach described here are generally applicable to most microbial detection problems and applications.

  18. Bead-Dazzled Baskets.

    ERIC Educational Resources Information Center

    St. Clair, Sharon

    2002-01-01

    Presents an art lesson used when teaching about North American Indians to fourth- and fifth-grade students. Explains that the students learn how to make baskets using a coil-wrap technique with colored yarns and beads. Provides a step-by-step explanation of how to create the baskets. (CMK)

  19. Weld-Bead Shaver

    NASA Technical Reports Server (NTRS)

    Guirguis, Kamal; Price, Daniel S.

    1990-01-01

    Hand-held power tool shaves excess metal from inside circumference of welded duct. Removes excess metal deposited by penetration of tungsten/inert-gas weld or by spatter from electron-beam weld. Produces smooth transition across joint. Easier to use and not prone to overshaving. Also cuts faster, removing 35 in. (89 cm) of weld bead per hour.

  20. Fluorescent beads disintegrate actin networks.

    PubMed

    Golde, Tom; Schuldt, Carsten; Schnauß, Jörg; Strehle, Dan; Glaser, Martin; Käs, Josef

    2013-10-01

    We studied the influence of fluorescent polystyrene beads on both entangled and cross-linked actin networks. Thermal bead fluctuations were observed via video particle tracking and analyzed with one-point microrheology. Illumination of fluorescent beads with their appropriate excitation wavelength leads to a drastic softening of actin gels. Other wavelengths and bright field microscopy do not increase thermal bead fluctuations. This effect cannot be significantly reduced by adding common oxygen scavengers. We conclude that the usage of fluorescent beads impairs results when studying the microrheology of actin networks.

  1. Coated Aerogel Beads

    NASA Technical Reports Server (NTRS)

    Littman, Howard (Inventor); Plawsky, Joel L. (Inventor); Paccione, John D. (Inventor)

    2014-01-01

    Methods and apparatus for coating particulate material are provided. The apparatus includes a vessel having a top and a bottom, a vertically extending conduit having an inlet in the vessel and an outlet outside of the vessel, a first fluid inlet in the bottom of the vessel for introducing a transfer fluid, a second fluid inlet in the bottom of the vessel for introducing a coating fluid, and a fluid outlet from the vessel. The method includes steps of agitating a material, contacting the material with a coating material, and drying the coating material to produce a coated material. The invention may be adapted to coat aerogel beads, among other materials. A coated aerogel bead and an aerogel-based insulation material are also disclosed.

  2. Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase-coated magnetic beads: A key step toward codon emancipation.

    PubMed

    Salehi, Amin S M; Smith, Mark T; Schinn, Song-Min; Hunt, Jeremy M; Muhlestein, Christina; Diray-Arce, Joann; Nielsen, Brent L; Bundy, Bradley C

    2017-06-08

    Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate-based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase-coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real-time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  3. A simple method to recover Norovirus from fresh produce with large sample size by using histo-blood group antigen-conjugated to magnetic beads in a recirculating affinity magnetic separation system (RCAMS).

    PubMed

    Tian, Peng; Yang, David; Mandrell, Robert

    2011-06-30

    Human norovirus (NoV) outbreaks are major food safety concerns. The virus has to be concentrated from food samples in order to be detected. PEG precipitation is the most common method to recover the virus. Recently, histo-blood group antigens (HBGA) have been recognized as receptors for human NoV, and have been utilized as an alternative method to concentrate human NoV for samples up to 40 mL in volume. However, to wash off the virus from contaminated fresh food samples, at least 250 mL of wash volume is required. Recirculating affinity magnetic separation system (RCAMS) has been tried by others to concentrate human NoV from large-volume samples and failed to yield consistent results with the standard procedure of 30 min of recirculation at the default flow rate. Our work here demonstrates that proper recirculation time and flow rate are key factors for success in using the RCAMS. The bead recovery rate was increased from 28% to 47%, 67% and 90% when recirculation times were extended from 30 min to 60 min, 120 min and 180 min, respectively. The kinetics study suggests that at least 120 min recirculation is required to obtain a good recovery of NoV. In addition, different binding and elution conditions were compared for releasing NoV from inoculated lettuce. Phosphate-buffered saline (PBS) and water results in similar efficacy for virus release, but the released virus does not bind to RCAMS effectively unless pH was adjusted to acidic. Either citrate-buffered saline (CBS) wash, or water wash followed by CBS adjustment, resulted in an enhanced recovery of virus. We also demonstrated that the standard curve generated from viral RNA extracted from serially-diluted virus samples is more accurate for quantitative analysis than standard curves generated from serially-diluted plasmid DNA or transcribed-RNA templates, both of which tend to overestimate the concentration power. The efficacy of recovery of NoV from produce using RCAMS was directly compared with that of the

  4. Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation.

    PubMed

    Tong, Chaobo; Guo, Baocheng; He, Shunping

    2009-02-19

    Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 x 105 and 1.7 x 105 per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the

  5. Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

    PubMed Central

    Tong, Chaobo; Guo, Baocheng; He, Shunping

    2009-01-01

    Background Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 × 105 and 1.7 × 105 per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened

  6. A superparamagnetic bead driven fluidic device (Invited Paper)

    NASA Astrophysics Data System (ADS)

    Husband, Benjamin; Melvin, Tracy; Evans, Alan G. R.

    2005-07-01

    Injection strategies have been employed in the field of fluidic MEMS using piezo electric or thermal actuators. A very popular application for such technology is inkjet printing. Largely this technology is used to produce droplets of fluid in air; the aim of this investigation is to produce an injection device for the precise dispensing of nanolitre volumes of fluid. A novel technique for dispensing fluid using superparamagnetic beads has been investigated. The beads used (Dynal Biotech) contain a homogeneous dispersion of Fe2O3, allowing for easy control with a magnet. This magnetic property is exploited, by a plug of approximately 60 000 beads within a micro channel. This is accomplished by applying a non-uniform magnetic field from a bullet magnet within close proximity of the bead plug. Once the plug is formed it can be moved along the micro channel by moving the magnet and thus, provide a plunger-like action. Previous work has demonstrated a bead plug device is able to dispense fluid from a micro channel at rates up to 7.2μlmin-1. This is an investigation using silicon and Pyrex fabricated micro channels with smaller dimensions, such that the dimensions will be similar to those which will be used to produce a pipette device. Here results are presented using these fabricated micro channels, where the effects of using differently sized bead plugs and varying velocities are examined. The results follow our proposed theory; further analysis is required to determine the operation of a bead plug during all states of movement.

  7. Efficient, validated method for detection of mycobacterial growth in liquid culture media by use of bead beating, magnetic-particle-based nucleic acid isolation, and quantitative PCR.

    PubMed

    Plain, Karren M; Waldron, Anna M; Begg, Douglas J; de Silva, Kumudika; Purdie, Auriol C; Whittington, Richard J

    2015-04-01

    Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10(4)-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n=54) and sheep fecal and tissue (n=90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis.

  8. Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

    PubMed Central

    Waldron, Anna M.; Begg, Douglas J.; de Silva, Kumudika; Purdie, Auriol C.; Whittington, Richard J.

    2015-01-01

    Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 104-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n = 54) and sheep fecal and tissue (n = 90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis. PMID:25609725

  9. Switchable cell trapping using superparamagnetic beads

    SciTech Connect

    Bryan, M. T.; Smith, K. H.; Real, M. E.; Bashir, M. A.; Fry, P. W.; Fischer, P.; Im, M.-Y.; Schrefl, T.; Allwood, D. A.; Haycock, J. W.

    2010-04-30

    Ni{sub 81}Fe{sub 19} microwires are investigated as the basis of a switchable template for positioning magnetically-labeled neural Schwann cells. Magnetic transmission X-ray microscopy and micromagnetic modeling show that magnetic domain walls can be created or removed in zigzagged structures by an applied magnetic field. Schwann cells containing superparamagnetic beads are trapped by the field emanating from the domain walls. The design allows Schwann cells to be organized on a surface to form a connected network and then released from the surface if required. As aligned Schwann cells can guide nerve regeneration, this technique is of value for developing glial-neuronal co-culture models in the future treatment of peripheral nerve injuries.

  10. Dynamic micro-Hall detection of superparamagnetic beads in a microfluidic channel.

    PubMed

    Aledealat, K; Mihajlović, G; Chen, K; Field, M; Sullivan, G J; Xiong, P; Chase, P B; von Molnár, S

    2010-12-01

    We report integration of an InAs quantum well micro-Hall magnetic sensor with microfluidics and real-time detection of moving superparamagnetic beads. Beads moving within and around the Hall cross area result in positive and negative Hall voltage signals respectively. Relative magnitudes and polarities of the signals measured for a random distribution of immobilized beads over the sensor are in good agreement with calculated values and explain consistently the shape of the dynamic signal.

  11. Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology

    PubMed Central

    Miller, Melissa B.; Tang, Yi-Wei

    2009-01-01

    Summary: The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles. PMID:19822891

  12. 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.

  13. A microfluidic chip-based fluorescent biosensor for the sensitive and specific detection of label-free single-base mismatch via magnetic beads-based "sandwich" hybridization strategy.

    PubMed

    Wang, ZongWen; Fan, YingWei; Chen, JinFa; Guo, Ying; Wu, WeiHua; He, Ye; Xu, LiangJun; Fu, FengFu

    2013-08-01

    A novel microfluidic chip-based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads-based "sandwich" hybridization strategy, was developed for the sensitive and ultra-specific detection of single-base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single-base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer-related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10(-11) M, a RSD (n = 5) < 5% and a discrimination factor of 3.58-4.54 for one-base mismatch.

  14. A universal SERS aptasensor based on DTNB labeled GNTs/Ag core-shell nanotriangle and CS-Fe3O4 magnetic-bead trace detection of Aflatoxin B1.

    PubMed

    Yang, Mingxiu; Liu, Guokun; Mehedi, Hassan Md; Ouyang, Qin; Chen, Quansheng

    2017-09-15

    A novel universal Surface-enhanced Raman Spectroscopy (SERS) based aptasensor platform for the trace detection of Aflatoxin B1 (AFB1), a common food contaminating mycotoxin, has been constructed, with the aid of the specific interaction between AFB1 and aptamers. The amino-terminal aptamer conjugated magnetic-bead (CS-Fe3O4) and the gold nanotriangles (GNTs)-DTNB@Ag-DTNB nanotriangles (GDADNTs) were used as the capturer and the reporter of AFB1, respectively. Under the optimized assay condition, the platform shows a distinguished sensitivity with the LOD as low as 0.54 pg/mL and the linear range from 0.001 to 10 ng/mL, a high stability of the SERS substrate activity remained three months at least, a decent reproducibility with RSD of ca. 5%, and a good selectivity to the general coexisted interferences. The distinguished sensitivity and selectivity for trace AFB1 detection has been achieved mainly due to the strong Raman enhancement effect of GNTs as the kernel for GDADNTs from the double-layer of the reporter molecules, the specificity of aptamer and superparamagnetic CS-Fe3O4 respectively. Furthermore, the proposed SERS aptasensor is universal to other trace molecules detection with the specific aptamers. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Chemiluminescent determination of H2O2 using 4-(1,2,4-triazol-1-yl)phenol as an enhancer based on the immobilization of horseradish peroxidase onto magnetic beads.

    PubMed

    Yang, Xiaoyan; Guo, Yingshu; Mei, Zhenhua

    2009-10-01

    This article describes the employment of a novel p-phenol derivative, 4-(1,2,4-triazol-1-yl)phenol (TRP), as a highly potent signal enhancer of the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) chemiluminescence (CL) system. The CL reaction conditions were optimized, and the enhancement characteristics of TRP were compared with those of p-iodophenol (PIP). TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The developed system was then applied to the determination of H(2)O(2) with immobilized HRP using magnetic beads as a solid support. The linear range for H(2)O(2) was 2.0x10(-6) to 1.0x10(-3) M. The detection limit for H(2)O(2) was 2.0x10(-6) M. The proposed sensor was applied successfully to the determination of H(2)O(2) in rainwater.

  16. Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads

    PubMed Central

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

    2015-01-01

    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 × 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)32+) 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

  17. 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

  18. "Micro-robots" pick up a glass bead

    SciTech Connect

    2011-01-01

    "Micro-robots", which are really collections of particles animated by magnetic fields, pick up a glass bead and move it around the screen. Each movement is precisely controlled. The "asters" were designed by Alexey Snezkho and Igor Aronson at Argonne National Laboratory. Video courtesy Nature Materials. Read the full story at http://go.usa.gov/KAT

  19. Acceleration of microwave-assisted enzymatic digestion reactions by magnetite beads.

    PubMed

    Chen, Wei-Yu; Chen, Yu-Chie

    2007-03-15

    In this study, we demonstrated that microwave-assisted enzymatic digestion could be greatly accelerated by multifunctional magnetite beads. The acceleration of microwave-assisted enzymatic digestion by the presence of the magnetite beads was attributable to several features of the beads. Their capacity to absorb microwave radiation leads to rapid heating of the beads. Furthermore, their negatively charged functionalities cause adsorption of proteins with opposite charges onto their surfaces by electrostatic interactions, leading to a concentration on the surfaces of the beads of proteins present in trace amounts in the solution. The adsorbed proteins are denatured and hence rendered vulnerable to enzymatic digestion and are digested on the beads. For microwave heating, 30 s was sufficient for carrying out the tryptic digestion of cytochrome c, in the presence of magnetite beads, while 1 min was adequate for tryptic digestion of myoglobin. The digestion products were characterized by MALDI-MS. This rapid enzymatic digestion allowed the entire time for identification of proteins to be greatly reduced. Furthermore, specific proteins present in trace quantities were enriched from the sample on the magnetite beads and could be rapidly isolated from the sample by employing an external magnetic field. These multiple roles of magnetite beads, as the absorber for microwave irradiation, the concentrating probe, and the agent for unfolding proteins, contributed to their capability of accelerating microwave-assisted enzymatic digestion. We also demonstrated that trypsin immobilized magnetite beads were suitable for use in microwave-assisted enzymatic digestion.

  20. 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.

  1. Stability optimization of microbial surface-enhanced Raman spectroscopy detection with immunomagnetic separation beads

    NASA Astrophysics Data System (ADS)

    Uusitalo, Sanna; Kögler, Martin; Välimaa, Anna-Liisa; Petäjä, Jarno; Kontturi, Ville; Siitonen, Samuli; Laitinen, Riitta; Kinnunen, Matti; Viitala, Tapani; Hiltunen, Jussi

    2017-03-01

    Immunomagnetic separation (IMS) beads with antibody coating are an interesting option for biosensing applications for the identification of biomolecules and biological cells, such as bacteria. The paramagnetic properties of the beads can be utilized with optical sensing by migrating and accumulating the beads and the bound analytes toward the focus depth of the detection system by an external magnetic field. The stability of microbial detection with IMS beads was studied by combining a flexible, inexpensive, and mass producible surface-enhanced Raman spectroscopy (SERS) platform with gold nanoparticle detection and antibody recognition by the IMS beads. Listeria innocua ATCC 33090 was used as a model sample and the effect of the IMS beads on the detected Raman signal was studied. The IMS beads were deposited into a hydrophobic sample well and accumulated toward the detection plane by a neodymium magnet. For the first time, it was shown that the spatial stability of the detection could be improved up to 35% by using IMS bead capture and sample well placing. The effect of a neodymium magnet under the SERS chip improved the temporal detection and significantly reduced the necessary time for sample stabilization for advanced laboratory testing.

  2. A Bead-Based Method for Multiplexed Identification and Quantitation of DNA Sequences Using Flow Cytometry

    PubMed Central

    Spiro, Alexander; Lowe, Mary; Brown, Drew

    2000-01-01

    A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-μm diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and flow cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte. In this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation. PMID:11010868

  3. Extended release of vitamins from magnetite loaded polyanionic polymeric beads.

    PubMed

    Sonmez, Maria; Verisan, Cristina; Voicu, Georgeta; Ficai, Denisa; Ficai, Anton; Oprea, Alexandra Elena; Vlad, Mihaela; Andronescu, Ecaterina

    2016-08-30

    Here we explore a novel approach of increasing the release duration of folic and ascorbic acid from magnetite entrapped into calcium-alginate beads. Synthesis and characterization of magnetite-vitamins complexes are reported. The magnetite-vitamins complexes were characterized by FT-IR, XRD, SEM, BET and DTA-TG. Also calcium-alginate magnetic beads were prepared by dripping a mixture of sodium alginate with magnetite-vitamins complexes into calcium chloride solution. Extended release profile of the two experimental models was evaluated and quantified by UV-vis.

  4. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  5. Magnetic Levitation of MC3T3 Osteoblast Cells as a Ground-Based Simulation of Microgravity

    PubMed Central

    Kidder, Louis S.; Williams, Philip C.; Xu, Wayne Wenzhong

    2009-01-01

    Diamagnetic samples placed in a strong magnetic field and a magnetic field gradient experience a magnetic force. Stable magnetic levitation occurs when the magnetic force exactly counter balances the gravitational force. Under this condition, a diamagnetic sample is in a simulated microgravity environment. The purpose of this study is to explore if MC3T3-E1 osteoblastic cells can be grown in magnetically simulated hypo-g and hyper-g environments and determine if gene expression is differentially expressed under these conditions. The murine calvarial osteoblastic cell line, MC3T3-E1, grown on Cytodex-3 beads, were subjected to a net gravitational force of 0, 1 and 2 g in a 17 T superconducting magnet for 2 days. Microarray analysis of these cells indicated that gravitational stress leads to up and down regulation of hundreds of genes. The methodology of sustaining long-term magnetic levitation of biological systems are discussed. PMID:20052306

  6. Magnetic Levitation of MC3T3 Osteoblast Cells as a Ground-Based Simulation of Microgravity.

    PubMed

    Hammer, Bruce E; Kidder, Louis S; Williams, Philip C; Xu, Wayne Wenzhong

    2009-11-01

    Diamagnetic samples placed in a strong magnetic field and a magnetic field gradient experience a magnetic force. Stable magnetic levitation occurs when the magnetic force exactly counter balances the gravitational force. Under this condition, a diamagnetic sample is in a simulated microgravity environment. The purpose of this study is to explore if MC3T3-E1 osteoblastic cells can be grown in magnetically simulated hypo-g and hyper-g environments and determine if gene expression is differentially expressed under these conditions. The murine calvarial osteoblastic cell line, MC3T3-E1, grown on Cytodex-3 beads, were subjected to a net gravitational force of 0, 1 and 2 g in a 17 T superconducting magnet for 2 days. Microarray analysis of these cells indicated that gravitational stress leads to up and down regulation of hundreds of genes. The methodology of sustaining long-term magnetic levitation of biological systems are discussed.

  7. 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.

  8. Glass-bead peen plating

    NASA Technical Reports Server (NTRS)

    Graves, J. R.

    1974-01-01

    Peen plating of aluminum, copper, and nickel powders was investigated. Only aluminum was plated successfully within the range of peen plating conditions studied. Optimum plating conditions for aluminum were found to be: (1) bead/powder mixture containing 25 to 35% powder by weight, (2) peening intensity of 0.007A as measured by Almen strip, and (3) glass impact bead diameter of at least 297 microns (0.0117 inches) for depositing-100 mesh aluminum powder. No extensive cleaning or substrate preparation is required beyond removing loose dirt or heavy oil.

  9. Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency

    PubMed Central

    Rosenberg, Jacob M.; Price, Jordan V.; Barcenas-Morales, Gabriela; Ceron-Gutierrez, Lourdes; Davies, Sophie; Kumararatne, Dinakantha S.; Döffinger, Rainer; Utz, Paul J.

    2015-01-01

    Background Anti-cytokine autoantibodies (ACAAs) are pathogenic in a handful of rare immunodeficiencies. However, the prevalence and significance of other ACAAs across immunodeficiencies have not yet been described. Objective We sought to profile ACAAs in a diverse cohort of serum samples from patients with immunodeficiency and assess the sensitivity and specificity of protein microarrays for ACAA identification and discovery. Methods Highly multiplexed protein microarrays were designed and fabricated. Blinded serum samples from a cohort of 58 patients with immunodeficiency and healthy control subjects were used to probe microarrays. Unsupervised hierarchical clustering was used to identify clusters of reactivity, and after unblinding, significance analysis of microarrays was used to identify disease-specific autoantibodies. A bead-based assay was used to validate protein microarray results. Blocking activity of serum containing ACAAs was measured in vitro. Results Protein microarrays were highly sensitive and specific for the detection of ACAAs in patients with autoimmune polyendocrine syndrome type I and pulmonary alveolar proteinosis, detecting ACAA levels consistent with those in the published literature. Protein microarray results were validated by using an independent bead-based assay. To confirm the functional significance of these ACAAs, we tested and confirmed the blocking activity of select ACAAs in vitro. Conclusion Protein microarrays are a powerful tool for ACAA detection and discovery, and they hold promise as a diagnostic for the evaluation and monitoring of clinical immunodeficiency. PMID:26365387

  10. Random glycopeptide bead libraries for seromic biomarker discovery.

    PubMed

    Kracun, Stjepan K; Cló, Emiliano; Clausen, Henrik; Levery, Steven B; Jensen, Knud J; Blixt, Ola

    2010-12-03

    Identification of disease-specific biomarkers is important to address early diagnosis and management of disease. Aberrant post-translational modifications (PTM) of proteins such as O-glycosylations (O-PTMs) are emerging as triggers of autoantibodies that can serve as sensitive biomarkers. Here we have developed a random glycopeptide bead library screening platform for detection of autoantibodies and other binding proteins. Libraries were build on biocompatible PEGA beads including a safety-catch C-terminal amide linker (SCAL) that allowed mild cleavage conditions (I(2)/NaBH(4) and TFA) for release of glycopeptides and sequence determination by ESI-Orbitrap-MS(n). As proof-of-principle, tumor -specific glycopeptide reporter epitopes were built-in into the libraries and were detected by tumor-specific monoclonal antibodies and autoantibodies from cancer patients. Sequenced and identified glycopeptides were resynthesized at the preparative scale by automated parallel peptide synthesis and printed on microarrays for validation and broader analysis with larger sets of sera. We further showed that chemical synthesis of the monosaccharide O-glycopeptide library (Tn-glycoform) could be diversified to other tumor glycoforms by on-bead enzymatic glycosylation reactions with recombinant glycosyltransferases. Hence, we have developed a high-throughput flexible platform for rapid discovery of O-glycopeptide biomarkers and the method has applicability in other types of assays such as lectin/antibody/enzyme specificity studies as well as investigation of other PTMs.

  11. 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.

  12. Juxta-clavicular beaded lines.

    PubMed

    Franco, Gennaro; Donati, Pietro; Muscardin, Luca; Maini, Antonio; Morrone, Aldo

    2006-08-01

    We present a case series of 63 patients diagnosed with juxta-clavicular beaded lines. This condition is more frequent in dark-skinned people and corresponds to an anatomical variant of simple sebaceous hyperplasia. In view of the strong reactivity of the melanocytes in dark-skinned people, and of the possible hypochromic results, no treatment is advised.

  13. Improving detection of avalanches on a conical bead pile

    NASA Astrophysics Data System (ADS)

    Vajpeyi, Avi; Lehman, Susan; Dahmen, Karin; Leblanc, Michael; Uhl, Jonathan

    A conical bead pile subject to slow driving and an external magnetic field is used as a simple system to investigate the variations in the avalanche size probability distribution function. Steel beads are dropped onto the pile from different heights and at different strengths of applied magnetic field. Avalanches are recorded by the change in mass as beads fall off the pile. Experimentally we observe an increasing deviation from power law behavior as the field and thus cohesion between the beads increases. We compare our experimental results for the probability distribution function to the results of an analytic theory from a mean-field model of slip avalanches [Dahmen, Nat Phys 7, 554 (2011)]. The model also makes predictions for avalanche duration, which is not measurable with the existing system. To more fully characterize the avalanching behavior of the pile over time, a high-speed camera has been added to the system to record the largest avalanches and allow more detailed analysis. The conical pile geometry presents a challenge for observation and particle tracking over the full pile. Our implementation scheme and preliminary results from the video analysis are presented. Research supported by NSF CBET 1336116 and 1336634.

  14. Calibration beads containing luminescent lanthanide ion complexes

    EPA Science Inventory

    The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, will be enhanced by the availability of narrow-band emitting lanthanide calibration beads. These beads can also be used to characterize spectrographic instruments, including mi...

  15. Calibration beads containing luminescent lanthanide ion complexes

    EPA Science Inventory

    The reliability of lanthanide luminescence measurements, by both flow cytometry and digital microscopy, will be enhanced by the availability of narrow-band emitting lanthanide calibration beads. These beads can also be used to characterize spectrographic instruments, including mi...

  16. Seeds used for Bodhi beads in China

    PubMed Central

    2014-01-01

    Background Bodhi beads are a Buddhist prayer item made from seeds. Bodhi beads have a large and emerging market in China, and demand for the beads has particularly increased in Buddhism regions, especially Tibet. Many people have started to focus on and collect Bodhi beads and to develop a Bodhi bead culture. But no research has examined the source plants of Bodhi beads. Therefore, ethnobotanical surveys were conducted in six provinces of China to investigate and document Bodhi bead plants. Reasons for the development of Bodhi bead culture were also discussed. Methods Six provinces of China were selected for market surveys. Information was collected using semi-structured interviews, key informant interviews, and participatory observation with traders, tourists, and local residents. Barkhor Street in Lhasa was focused on during market surveys because it is one of the most popular streets in China. Results Forty-seven species (including 2 varieties) in 19 families and 39 genera represented 52 types of Bodhi beads that were collected. The most popular Bodhi bead plants have a long history and religious significance. Most Bodhi bead plants can be used as medicine or food, and their seeds or fruits are the main elements in these uses. ‘Bodhi seeds’ have been historically used in other countries for making ornaments, especially seeds of the legume family. Many factors helped form Bodhi bead culture in China, but its foundation was in Indian Buddhist culture. Conclusions As one of the earliest adornment materials, seeds played an important role for human production and life. Complex sources of Bodhi beads have different cultural and historical significance. People bought and collected Bodhi beads to reflect their love and admiration for the plants. Thus, the documentation of Bodhi bead plants can serve as a basis for future investigation of Bodhi bead culture and modern Buddhist culture. PMID:24479788

  17. Seeds used for Bodhi beads in China.

    PubMed

    Li, Feifei; Li, Jianqin; Liu, Bo; Zhuo, Jingxian; Long, Chunlin

    2014-01-30

    Bodhi beads are a Buddhist prayer item made from seeds. Bodhi beads have a large and emerging market in China, and demand for the beads has particularly increased in Buddhism regions, especially Tibet. Many people have started to focus on and collect Bodhi beads and to develop a Bodhi bead culture. But no research has examined the source plants of Bodhi beads. Therefore, ethnobotanical surveys were conducted in six provinces of China to investigate and document Bodhi bead plants. Reasons for the development of Bodhi bead culture were also discussed. Six provinces of China were selected for market surveys. Information was collected using semi-structured interviews, key informant interviews, and participatory observation with traders, tourists, and local residents. Barkhor Street in Lhasa was focused on during market surveys because it is one of the most popular streets in China. Forty-seven species (including 2 varieties) in 19 families and 39 genera represented 52 types of Bodhi beads that were collected. The most popular Bodhi bead plants have a long history and religious significance. Most Bodhi bead plants can be used as medicine or food, and their seeds or fruits are the main elements in these uses. 'Bodhi seeds' have been historically used in other countries for making ornaments, especially seeds of the legume family. Many factors helped form Bodhi bead culture in China, but its foundation was in Indian Buddhist culture. As one of the earliest adornment materials, seeds played an important role for human production and life. Complex sources of Bodhi beads have different cultural and historical significance. People bought and collected Bodhi beads to reflect their love and admiration for the plants. Thus, the documentation of Bodhi bead plants can serve as a basis for future investigation of Bodhi bead culture and modern Buddhist culture.

  18. Microarray in parasitic infections

    PubMed Central

    Sehgal, Rakesh; Misra, Shubham; Anand, Namrata; Sharma, Monika

    2012-01-01

    Modern biology and genomic sciences are rooted in parasitic disease research. Genome sequencing efforts have provided a wealth of new biological information that promises to have a major impact on our understanding of parasites. Microarrays provide one of the major high-throughput platforms by which this information can be exploited in the laboratory. Many excellent reviews and technique articles have recently been published on applying microarrays to organisms for which fully annotated genomes are at hand. However, many parasitologists work on organisms whose genomes have been only partially sequenced. This review is mainly focused on how to use microarray in these situations. PMID:23508469

  19. Gel bead composition for metal adsorption

    SciTech Connect

    Scott, C.D.; Woodward, C.A.; Byers, C.H.

    1990-12-18

    This patent describes a gel bead consisting essentially of a sufficient amount of water and propylene glycol alginate to allow for bead formation and a sufficient amount of bone gelatin to allow for metal absorption and chemically crosslinked in an alkaline medium to form a stable structure. A gel bead contained therein a biological absorbent capable of removing metals from solution.

  20. Thermal Profile of Metallic Beads in a Bead Sterilizer,

    DTIC Science & Technology

    1986-08-01

    another commonly used chairside sterilization method, principally for endodontic instruments. The bead sterilizer consists of a heated chamber containing...sterilization of endodontic instruments. However, several investigators have shown sterilization times ranging from 3 seconds to 14 minutes, are...et.al. The effect of autoclave sterilization on endodontic files. Oral Surg 55(2): 204-207, 1983. 3. Parkes, R. B. and Kolstad, R. A. Effects of

  1. DNA Microarray Technology

    SciTech Connect

    WERNER-WASHBURNE, MARGARET; DAVIDSON, GEORGE S.

    2002-01-01

    Collaboration between Sandia National Laboratories and the University of New Mexico Biology Department resulted in the capability to train students in microarray techniques and the interpretation of data from microarray experiments. These studies provide for a better understanding of the role of stationary phase and the gene regulation involved in exit from stationary phase, which may eventually have important clinical implications. Importantly, this research trained numerous students and is the basis for three new Ph.D. projects.

  2. Molecular diagnostics using magnetic nanobeads

    NASA Astrophysics Data System (ADS)

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

    2010-01-01

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

  3. Dynamic trajectory analysis of superparamagnetic beads driven by on-chip micromagnets

    PubMed Central

    Abedini-Nassab, Roozbeh; Lim, Byeonghwa; Yang, Ye; Howdyshell, Marci; Sooryakumar, Ratnasingham; Yellen, Benjamin B.

    2015-01-01

    We investigate the non-linear dynamics of superparamagnetic beads moving around the periphery of patterned magnetic disks in the presence of an in-plane rotating magnetic field. Three different dynamical regimes are observed in experiments, including (1) phase-locked motion at low driving frequencies, (2) phase-slipping motion above the first critical frequency fc1, and (3) phase-insulated motion above the second critical frequency fc2. Experiments with Janus particles were used to confirm that the beads move by sliding rather than rolling. The rest of the experiments were conducted on spherical, isotropic magnetic beads, in which automated particle position tracking algorithms were used to analyze the bead dynamics. Experimental results in the phase-locked and phase-slipping regimes correlate well with numerical simulations. Additional assumptions are required to predict the onset of the phase-insulated regime, in which the beads are trapped in closed orbits; however, the origin of the phase-insulated state appears to result from local magnetization defects. These results indicate that these three dynamical states are universal properties of bead motion in non-uniform oscillators. PMID:26648596

  4. Pulling on super paramagnetic beads with micro cantilevers: single molecule mechanical assay application.

    PubMed

    Muñoz, Romina; Aguilar Sandoval, Felipe; Wilson, Christian A M; Melo, Francisco

    2015-07-22

    This paper demonstrates that it is possible to trap and release a super paramagnetic micro bead by fixing three super paramagnetic micro beads in a triangular array at the sensitive end of a micro cantilever, and by simply switching on/off an external magnetic field. To provide evidence of this principle we trap a micro bead that is attached to the free end of single DNA molecule and that has been previously fixed at the other end to a glass surface, using the standard sample preparation protocol of magnetic tweezers assays. The switching process is reversible which preserves the integrity of the tethered molecule, and a local force applied over the tethered bead excludes the neighbouring beads from the magnetic trap. We have developed a quadrature phase interferometer which is able to perform under fluid environments to accurately measure small deflections, which permits the exploration of DNA elasticity. Our results agree with measurements from magnetic tweezer assays performed under similar conditions. Furthermore, compared to the magnetic tweezer methodology, the combination of the magnetic trap with a suitable measurement system for cantilever deflection, allows for the exploration of a wide range of forces using a local method that has an improved temporal resolution.

  5. Photocleavage Based Affinity Purification and Printing of Cell-free Expressed Proteins: Application to Proteome Microarrays

    PubMed Central

    Lim, Mark; Rothschild, Kenneth J.

    2008-01-01

    Proteome microarrays hold great promise for various biotechnological and biomedical applications including mapping protein-protein interactions, drug discovery and biomarker discovery. However, the need to express, purify and print thousands of functional proteins at high density on a microarray substrate presents challenges which limit their wide-spread availability and use. We report the development of new methods, based on photocleavage, for the purification and printing of nascent proteins. Photocleavable biotin (PC-biotin) is incorporated into nascent proteins by misaminoacylated tRNAs used in a coupled transcription/translation rabbit reticulocyte cell-free expression system. Proteins were affinity isolated onto (strept)avidin coated beads and then photo-released (PC-SNAG). Compared to polyhistidine tag based affinity purification, PC-SNAG provided a higher purity yet comparable yield using a GST test protein. Antibody mediated PC-SNAG is also demonstrated. PC-SNAG proteins were found to exhibit native enzymatic activity and were suitable for the printing of ordered protein microarrays used in protein-protein interaction assays. Alternatively, when beads carrying photocleavably tethered proteins were placed in close proximity to an activated planar surface and then illuminated, proteins were transferred directly to the surface (PC-PRINT) to form discrete spots whose dimensions match that of the beads. PC-PRINT can provide an inexpensive method to fabricate very large scale, high density proteome microarrays. Moreover, transferring the proteins off the beads significantly reduces background auto-fluorescence observed with common bead types. In order to decode nascent proteins which are deposited by PC-PRINT from individual beads, the feasibility of using photocleavable quantum dot codes is demonstrated. PMID:18762158

  6. Single- and dual-bead microrheology of semiflexiblefd virus solutions

    NASA Astrophysics Data System (ADS)

    Addas, Karim M.

    this well-defined system of monodisperse virus solutions. The results are also compared with an active microrheology method. In the active method, an oscillatory magnetic force is applied to single micron-sized magnetic beads and the complex shear modulus is derived from the response of the bead. Measurements are also shown for a rotating disk macrorheology technique. The results from the three methods agree within experimental errors.

  7. Analysis of tear inflammatory mediators: A comparison between the microarray and Luminex methods.

    PubMed

    Dionne, Karen; Redfern, Rachel L; Nichols, Jason J; Nichols, Kelly K

    2016-01-01

    Inflammatory mediators have been shown to modulate dry eye (DE) disease and may correlate with disease severity, yet the methods used and the associated findings vary significantly in the literature. The goal of this research was to compare two methods, the quantitative microarray and the magnetic bead assay, for detecting cytokine levels in extracted tear samples across three subject groups. Tears were collected from Schirmer strips of the right and left eyes of 20 soft contact lens wearers (CL), 20 normal non-contact lens wearers (NOR), and 20 DE subjects and stored at -80 °C. Tear proteins were eluted and precipitated using ammonium bicarbonate and acetone. The right and left eye samples were combined for each subject. Following the Bradford protein quantitation method, 10 µg of total protein was used for each of the two analyses, Quantibody® Human Inflammation Array 3 (RayBiotech) and High Sensitivity Human Cytokine Magnetic Bead Kit (Millipore). The assays were run using the GenePix® 4000B Scanner (Molecular Devices) or the Luminex MagPix® plate reader (Luminex), respectively. The data were then compared between the two instruments and the three subject groups. Of the 40 proteins on the Quantibody® microarray, seven had average expression levels above the lower limit of detection: ICAM-1, MCP-1, MIG, MCSF, TIMP-1, TIMP-2, and TNF-RI. Significant differences in expression levels (p<0.05) were detected between the CL and DE groups for MCSF, TIMP-1, and TNF R1, between the NOR and DE groups for ICAM-1, and between the CL and NOR groups for ICAM-1, MCP-1, MCSF, TIMP-1, TIMP-2, and TNF-R1 when using the Student t test. Of the 13 proteins tested with Luminex, IL-1β, IL-4, IL-6, IL-7, and IL-8 had expression levels above the minimum detectable level, and these were most often detected using the Luminex assay compared to the Quantibody® microarray. Contrarily, IL-2, IL-12, IL-13, INF-g, and GM-CSF were detected more frequently using the Quantibody® microarray

  8. Validation of a mobile phone-assisted microarray decoding platform for signal-enhanced mutation detection.

    PubMed

    Zhang, Guanbin; Li, Caixia; Lu, Yuan; Hu, Hua; Xiang, Guangxin; Liang, Zhiqing; Liao, Pu; Dai, Pu; Xing, Wanli; Cheng, Jing

    2011-08-15

    We have established a mobile phone-assisted microarray decoding platform for signal-enhanced mutation detection. A large amount of single-stranded DNA (ssDNA) was obtained by combining symmetric PCR and magnetic isolation, and ssDNA prepared with magnetic bead as label was further allowed to hybridize against the tag-array for decoding purpose. High sensitivity and specificity was achieved with the detection of genomic DNA. When simultaneously genotyping nine common mutations associated with hereditary hearing loss, the detection limit of 1 ng genomic DNA was achieved. Significantly, a mobile phone was also used to record and decode the genotyping results through a custom-designed imaging adaptor and a dedicated mobile phone software. A total of 51 buccal swabs from patients probably with deafness-related mutations were collected and analyzed. The genotyping results were all confirmed by fluorescence-based laser confocal scanning and direct DNA sequencing. This mobile phone-assisted decoding platform provides an effective but economic mutation detection alternative for the future quicker and sensitive detection of virtually any mutation-related diseases in developing and underdeveloped countries.

  9. Functional GPCR microarrays.

    PubMed

    Hong, Yulong; Webb, Brian L; Su, Hui; Mozdy, Eric J; Fang, Ye; Wu, Qi; Liu, Li; Beck, Jonathan; Ferrie, Ann M; Raghavan, Srikanth; Mauro, John; Carre, Alain; Müeller, Dirk; Lai, Fang; Rasnow, Brian; Johnson, Michael; Min, Hosung; Salon, John; Lahiri, Joydeep

    2005-11-09

    This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.

  10. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.

  11. Multievidence microarray mining.

    PubMed

    Seifert, Martin; Scherf, Matthias; Epple, Anton; Werner, Thomas

    2005-10-01

    Microarray mining is a challenging task because of the superposition of several processes in the data. We believe that the combination of microarray data-based analyses (statistical significance analysis of gene expression) with array-independent analyses (literature-mining and promoter analysis) enables some of the problems of traditional array analysis to be overcome. As a proof-of-principle, we revisited publicly available microarray data derived from an experiment with platelet-derived growth factor (PDGF)-stimulated fibroblasts. Our strategy revealed results beyond the detection of the major metabolic pathway known to be linked to the PDGF response: we were able to identify the crosstalking regulatory networks underlying the metabolic pathway without using a priori knowledge about the experiment.

  12. 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.

  13. Structure and superparamagnetic behaviour of magnetite nanoparticles in cellulose beads

    SciTech Connect

    Correa, Jose R.; Bordallo, Eduardo; Canetti, Dora; Leon, Vivian; Otero-Diaz, Luis C.; Negro, Carlos; Gomez, Adrian; Saez-Puche, Regino

    2010-08-15

    Superparamagnetic magnetite nanoparticles were obtained starting from a mixture of iron(II) and iron(III) solutions in a preset total iron concentration from 0.04 to 0.8 mol l{sup -1} with ammonia at 25 and 70 {sup o}C. The regeneration of cellulose from viscose produces micrometrical spherical cellulose beads in which synthetic magnetite were embedded. The characterization of cellulose-magnetite beads by X-ray diffraction, Scanning and Transmission Electron Microscopy and magnetic measurement is reported. X-ray diffraction patterns indicate that the higher is the total iron concentration and temperature the higher is the crystal size of the magnetite obtained. Transmission Electron Microscopy studies of cellulose-magnetite beads revealed the distribution of magnetite nanoparticles inside pores of hundred nanometers. Magnetite as well as the cellulose-magnetite composites exhibit superparamagnetic characteristics. Field cooling and zero field cooling magnetic susceptibility measurements confirm the superparamagnetic behaviour and the blocking temperature for the magnetite with a mean size of 12.5 nm, which is 200 K.

  14. BEADS: A Realistic Approach to Elementary Statistics.

    ERIC Educational Resources Information Center

    Gamble, Andy

    1983-01-01

    Having students gather their own statistics is promoted. The BEADS program provides an alternative; it simulated sampling from a binomial distribution. Illustrations from the program are included. (MNS)

  15. Microarrays for Undergraduate Classes

    ERIC Educational Resources Information Center

    Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

    2006-01-01

    A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by…

  16. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  17. Microarrays for Undergraduate Classes

    ERIC Educational Resources Information Center

    Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

    2006-01-01

    A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by…

  18. Beads, beaded-fibres and fibres: Tailoring the morphology of poly(caprolactone) using pressurised gyration.

    PubMed

    Hong, Xianze; Edirisinghe, Mohan; Mahalingam, Suntharavathanan

    2016-12-01

    This work focuses on forming bead on string poly(caprolactone) (PCL) by using gyration under pressure. The fibre morphology of bead on string is an interesting feature that falls between bead-free fibres and droplets, and it could be effectively controlled by the rheological properties of spinning dopes and the major processing parameters of the pressurised gyration system which are working pressure and rotating speed. Bead products were not always spherical in shape and tended to be more elliptical, therefore both their width and length were measured. The average bead width and length produced spanned a range 145-660μm and 140-1060μm, respectively. The average distance between two adjacent beads (i.e. inter-bead distance) and the bead size (width and length) are shown to be a function of processing parameters and polymer concentration. An interesting morphology i.e. beads with short fibre was observed when using a high polymer concentration. Bead on string structure agglomeration was promoted by a low polymer concentration. Formation of droplets or agglomerated bead on string is promoted below 5wt% polymer concentration, and beads with short fibre were present in the microstructure beyond a polymer concentration of 20wt%. Copyright © 2016. Published by Elsevier B.V.

  19. Correlated matrix-assisted laser desorption/ionization mass spectrometry and fluorescent imaging of photocleavable peptide-coded random bead-arrays

    PubMed Central

    Lim, Mark J; Liu, Ziying; Braunschweiger, Karen I; Awad, Amany; Rothschild, Kenneth J

    2013-01-01

    RATIONALE Rapidly performing global proteomic screens is an important goal in the post-genomic era. Correlated matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and fluorescent imaging of photocleavable peptide-coded random bead-arrays was evaluated as a critical step in a new method for proteomic screening that combines many of the advantages of MS with fluorescence-based microarrays. METHODS Small peptide-coded model bead libraries containing up to 20 different bead species were constructed by attaching peptides to 30–34 µm diameter glass, agarose or TentaGel® beads using photocleavable biotin or a custom-designed photocleavable linker. The peptide-coded bead libraries were randomly arrayed into custom gold-coated micro-well plates with 45 µm diameter wells and subjected to fluorescence and MALDI mass spectrometric imaging (MALDI-MSI). RESULTS Photocleavable mass-tags from individual beads in these libraries were spatially localized as ∼65 µm spots using MALDI-MSI with high sensitivity and mass resolution. Fluorescently tagged beads were identified and correlated with their matching photocleavable mass-tags by comparing the fluorescence and MALDI-MS images of the same bead-array. Post-translational modification of the peptide Kemptide was also detected on individual beads in a photocleavable peptide-coded bead-array by MALDI-MSI alone, after exposure of the beads to protein kinase A (PKA). CONCLUSIONS Correlated MALDI-MS and fluorescent imaging of photocleavable peptide-coded random bead-arrays can provide a basis for performing global proteomic screening. © 2013 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons, Ltd. PMID:24285390

  20. Adsorption of ochratoxin A from grape juice by yeast cells immobilised in calcium alginate beads.

    PubMed

    Farbo, Maria Grazia; Urgeghe, Pietro Paolo; Fiori, Stefano; Marceddu, Salvatore; Jaoua, Samir; Migheli, Quirico

    2016-01-18

    Grape juice can be easily contaminated with ochratoxin A (OTA), one of the known mycotoxins with the greatest public health significance. Among the different approaches to decontaminate juice from this mycotoxin, microbiological methods proved efficient, inexpensive and safe, particularly the use of yeast or yeast products. To ascertain whether immobilisation of the yeast biomass would lead to successful decontamination, alginate beads encapsulating Candida intermedia yeast cells were used in our experiments to evaluate their OTA-biosorption efficacy. Magnetic calcium alginate beads were also prepared by adding magnetite in the formulation to allow fast removal from the aqueous solution with a magnet. Calcium alginate beads were added to commercial grape juice spiked with 20 μg/kg OTA and after 48 h of incubation a significant reduction (>80%), of the total OTA content was achieved, while in the subsequent phases (72-120 h) OTA was slowly released into the grape juice by alginate beads. Biosorption properties of alginate-yeast beads were tested in a prototype bioreactor consisting in a glass chromatography column packed with beads, where juice amended with OTA was slowly flowed downstream. The adoption of an interconnected scaled-up bioreactor as an efficient and safe tool to remove traces of OTA from liquid matrices is discussed.

  1. New Analysis Techniques for Avalanches in a Conical Bead Pile with Cohesion

    NASA Astrophysics Data System (ADS)

    Tieman, Catherine; Lehman, Susan

    2015-03-01

    Avalanche statistics and pile geometry for 3 mm steel spheres dropped on a conical bead pile were studied at different drop heights and different cohesion strengths. The pile is initially built on a circular base and is subsequently slowly driven by adding one bead at a time to the apex of the pile. We investigate the dynamic response of the pile by recording avalanches off the pile over the course of tens of thousands of bead drops. The level of cohesion is tuned through use of an applied uniform magnetic field. Changes in the pile mass and geometry were investigated to determine the effect of cohesion and drop height on the angle of repose. The angle of repose increased with cohesion strength, and decreased somewhat for higher drop heights. The packing density of beads is expected to decrease as magnetic cohesion increases, but for our 20 000-bead pile, this effect has not been observed. The proportion of beads removed from the pile by different avalanche sizes was also calculated. Although larger avalanches are much rarer occurrences, they carry away a larger fraction of the total avalanched mass than small avalanches. As the pile cohesion increases, the number of small and medium avalanches decreases so that this mass loss distribution shifts more strongly to large sizes.

  2. Bead Assembly Magnetorotation as a Signal Transduction Method for Protein Detection

    PubMed Central

    Hecht, Ariel; Commiskey, Patrick; Shah, Nicholas; Kopelman, Raoul

    2013-01-01

    This paper demonstrates a proof-of-principle for a new signal transduction method for protein detection called Bead Assembly Magnetorotation (BAM). In this paper, we chose to focus on the protein thrombin, a popular choice for proof-of-principle work in this field. BAM is based on using the protein target to mediate the formation of aptamer-coated 1 μm magnetic beads into a bead assembly, formed at the bottom of a 1 μL hanging droplet. The size, shape and fractal dimension of this bead assembly all depend on the protein concentration. The protein concentration can be measured in two ways: by magnetorotation, in which the rotational period of the assembly correlates with the protein concentration, or by fractal analysis. Additionally, a microscope-free magnetorotation detection method is introduced, based on a simple laser apparatus built from standard laboratory components. PMID:23639345

  3. Porous bead packings for gas chromatography

    NASA Technical Reports Server (NTRS)

    Pollock, G. E.; Woeller, F. H.

    1979-01-01

    Porous polyaromatic packing beads have low polarity, high efficiency, short retention time, and may be synthesized in size range of 50 to 150 micrometers (100 to 270 mesh). Mechanically strong beads may be produced using various materials depending on elements and compounds to be identified.

  4. Expanded polylactide bead foaming - A new technology

    NASA Astrophysics Data System (ADS)

    Nofar, M.; Ameli, A.; Park, C. B.

    2015-05-01

    Bead foaming technology with double crystal melting peak structure has been recognized as a promising method to produce low-density foams with complex geometries. During the molding stage of the bead foams, the double peak structure generates a strong bead-to-bead sintering and maintains the overall foam structure. During recent years, polylactide (PLA) bead foaming has been of the great interest of researchers due to its origin from renewable resources and biodegradability. However, due to the PLA's low melt strength and slow crystallization kinetics, the attempts have been limited to the manufacturing methods used for expanded polystyrene. In this study, for the first time, we developed microcellular PLA bead foams with double crystal melting peak structure. Microcellular PLA bead foams were produced with expansion ratios and average cell sizes ranging from 3 to 30-times and 350 nm to 15 µm, respectively. The generated high melting temperature crystals during the saturation significantly affected the expansion ratio and cell density of the PLA bead foams by enhancing the PLA's poor melt strength and promoting heterogeneous cell nucleation around the crystals.

  5. Ultrasonic Characterization of Glass Beads

    NASA Astrophysics Data System (ADS)

    Lassila, I.; Siiriä, S.; Gates, F. K.; Hæggström, E.

    2008-02-01

    We report on the progress in developing a method for an in-line granule size measurement using ultrasonic through transmission method. The knowledge of granule size is important in the production of pharmaceutical dosage forms where the current optical and rheological methods have limitations such as fouling of the optical windows. The phase velocity of a wave propagated through interstitial air between glass balls of 1, 2 and 10 mm in diameter was 254±5 m/s, 261±3 m/s and 320±9 m/s, respectively. The power spectral density of the received signals showed that high frequencies were attenuated more in case of smaller beads due to increased scattering.

  6. Biolog phenotype microarrays.

    PubMed

    Shea, April; Wolcott, Mark; Daefler, Simon; Rozak, David A

    2012-01-01

    Phenotype microarrays nicely complement traditional genomic, transcriptomic, and proteomic analysis by offering opportunities for researchers to ground microbial systems analysis and modeling in a broad yet quantitative assessment of the organism's physiological response to different metabolites and environments. Biolog phenotype assays achieve this by coupling tetrazolium dyes with minimally defined nutrients to measure the impact of hundreds of carbon, nitrogen, phosphorous, and sulfur sources on redox reactions that result from compound-induced effects on the electron transport chain. Over the years, we have used Biolog's reproducible and highly sensitive assays to distinguish closely related bacterial isolates, to understand their metabolic differences, and to model their metabolic behavior using flux balance analysis. This chapter describes Biolog phenotype microarray system components, reagents, and methods, particularly as they apply to bacterial identification, characterization, and metabolic analysis.

  7. Application of magnetic immobilized microorganisms. Ethanol production by saccharomyces cerevisiae

    SciTech Connect

    Birnbaum, S.; Larsson, P.O.

    1982-01-01

    Magnetic Ca alginate yeast beads, made by incorporation of magnetite or the colloidal magnetic liquid ferrofluid, exhibited catalytic behavior similar to that of their nonmagnetic counterparts. The magnetic immobilized preparations short-term performance, long-term operational stability, and capacity for in-situ activation were unaffected by the inclusion of magnetic material. The magnetic quality of the alginate beads provides manipulatory advantages.

  8. Analyzing Microarray Data.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2017-03-01

    Because there is no widely used software for analyzing RNA-seq data that has a graphical user interface, this protocol provides an example of analyzing microarray data using Babelomics. This analysis entails performing quantile normalization and then detecting differentially expressed genes associated with the transgenesis of a human oncogene c-Myc in mice. Finally, hierarchical clustering is performed on the differentially expressed genes using the Cluster program, and the results are visualized using TreeView.

  9. Membrane-based microarrays

    NASA Astrophysics Data System (ADS)

    Dawson, Elliott P.; Hudson, James; Steward, John; Donnell, Philip A.; Chan, Wing W.; Taylor, Richard F.

    1999-11-01

    Microarrays represent a new approach to the rapid detection and identification of analytes. Studies to date have shown that the immobilization of receptor molecules (such as DNA, oligonucleotides, antibodies, enzymes and binding proteins) onto silicon and polymeric substrates can result in arrays able to detect hundreds of analytes in a single step. The formation of the receptor/analyte complex can, itself, lead to detection, or the complex can be interrogated through the use of fluorescent, chemiluminescent or radioactive probes and ligands.

  10. Fused Bead Analysis of Diogenite Meteorites

    NASA Technical Reports Server (NTRS)

    Mittlefehldt, D.W.; Beck, B.W.; McSween, H.Y.; Lee, C.T. A.

    2009-01-01

    Bulk rock chemistry is an essential dataset in meteoritics and planetary science [1]. A common method used to obtain the bulk chemistry of meteorites is ICP-MS. While the accuracy, precision and low detection limits of this process are advantageous [2], the sample size used for analysis (approx.70 mg) can be a problem in a field where small and finite samples are the norm. Fused bead analysis is another bulk rock analytical technique that has been used in meteoritics [3]. This technique involves forming a glass bead from 10 mg of sample and measuring its chemistry using a defocused beam on a microprobe. Though the ICP-MS has lower detection limits than the microprobe, the fused bead method destroys a much smaller sample of the meteorite. Fused bead analysis was initially designed for samples with near-eutectic compositions and low viscosities. Melts generated of this type homogenize at relatively low temperatures and produce primary melts near the sample s bulk composition [3]. The application of fused bead analysis to samples with noneutectic melt compositions has not been validated. The purpose of this study is to test if fused bead analysis can accurately determine the bulk rock chemistry of non-eutectic melt composition meteorites. To determine this, we conduct two examinations of the fused bead. First, we compare ICP-MS and fused bead results of the same samples using statistical analysis. Secondly, we inspect the beads for the presence of crystals and chemical heterogeneity. The presence of either of these would indicate incomplete melting and quenching of the bead.

  11. Illumina WG-6 BeadChip strips should be normalized separately.

    PubMed

    Shi, Wei; Banerjee, Ashish; Ritchie, Matthew E; Gerondakis, Steve; Smyth, Gordon K

    2009-11-11

    Illumina Sentrix-6 Whole-Genome Expression BeadChips are relatively new microarray platforms which have been used in many microarray studies in the past few years. These Chips have a unique design in which each Chip contains six microarrays and each microarray consists of two separate physical strips, posing special challenges for precise between-array normalization of expression values. None of the normalization strategies proposed so far for this microarray platform allow for the possibility of systematic variation between the two strips comprising each array. That this variation can be substantial is illustrated by a data example. We demonstrate that normalizing at the strip-level rather than at the array-level can effectively remove this between-strip variation, improve the precision of gene expression measurements and discover more differentially expressed genes. The gain is substantial, yielding a 20% increase in statistical information and doubling the number of genes detected at a 5% false discovery rate. Functional analysis reveals that the extra genes found tend to have interesting biological meanings, dramatically strengthening the biological conclusions from the experiment. Strip-level normalization still outperforms array-level normalization when non-expressed probes are filtered out. Plots are proposed which demonstrate how the need for strip-level normalization relates to inconsistent intensity range variation between the strips. Strip-level normalization is recommended for the preprocessing of Illumina Sentrix-6 BeadChips whenever the intensity range is seen to be inconsistent between the strips. R code is provided to implement the recommended plots and normalization algorithms.

  12. Ultrasensitive carbohydrate-peptide SPR imaging microarray for diagnosing IgE mediated peanut allergy.

    PubMed

    Joshi, Amit A; Peczuh, Mark W; Kumar, Challa V; Rusling, James F

    2014-11-21

    Severity of peanut allergies is linked to allergen-specific immunoglobulin E (IgE) antibodies in blood, but diagnostics from assays using glycoprotein allergen mixtures may be inaccurate. Measuring IgEs specific to individual peptide and carbohydrate epitopes of allergenic proteins is promising. We report here the first immunoarray for IgEs utilizing both peptide and carbohydrate epitopes. A surface plasmon resonance imaging (SPRi) microarray was equipped with peptide and β-xylosyl glycoside (BXG) epitopes from major peanut allergen glycoprotein Arachis hypogaea h2 (Ara-h2). A monoclonal anti-IgE antibody was included as positive control. IgEs were precaptured onto magnetic beads loaded with polyclonal anti-IgE antibodies to enhance sensitivity and minimize non-specific binding. As little as 0.1 attomole (0.5 pg mL(-1)) IgE was detected from dilute serum in 45 min. IgEs binding to Ara-h2 peptide and BXG were quantified in 10 μL of patient serum and correlated with standard ImmunoCAP values.

  13. Comparison of normalization methods for Illumina BeadChip HumanHT-12 v3

    PubMed Central

    2010-01-01

    Background Normalization of microarrays is a standard practice to account for and minimize effects which are not due to the controlled factors in an experiment. There is an overwhelming number of different methods that can be applied, none of which is ideally suited for all experimental designs. Thus, it is important to identify a normalization method appropriate for the experimental setup under consideration that is neither too negligent nor too stringent. Major aim is to derive optimal results from the underlying experiment. Comparisons of different normalization methods have already been conducted, none of which, to our knowledge, comparing more than a handful of methods. Results In the present study, 25 different ways of pre-processing Illumina Sentrix BeadChip array data are compared. Among others, methods provided by the BeadStudio software are taken into account. Looking at different statistical measures, we point out the ideal versus the actual observations. Additionally, we compare qRT-PCR measurements of transcripts from different ranges of expression intensities to the respective normalized values of the microarray data. Taking together all different kinds of measures, the ideal method for our dataset is identified. Conclusions Pre-processing of microarray gene expression experiments has been shown to influence further downstream analysis to a great extent and thus has to be carefully chosen based on the design of the experiment. This study provides a recommendation for deciding which normalization method is best suited for a particular experimental setup. PMID:20525181

  14. A Novel Approach for Purification and Selective Capture of Membrane Vesicles of the Periodontopathic Bacterium, Porphyromonas gingivalis: Membrane Vesicles Bind to Magnetic Beads Coated with Epoxy Groups in a Noncovalent, Species-Specific Manner

    PubMed Central

    Nakao, Ryoma; Kikushima, Kenji; Higuchi, Hideo; Obana, Nozomu; Nomura, Nobuhiko; Bai, Dongying; Ohnishi, Makoto; Senpuku, Hidenobu

    2014-01-01

    Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs–their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs. PMID:24830438

  15. Stability of gold bead tissue markers.

    PubMed

    Miller, Joel M; Rossi, Ethan A; Wiesmair, Martin; Alexander, Danielle E; Gallo, Orazio

    2006-04-27

    Significant soft tissue features in the orbit and elsewhere are not resolved by MRI or any other imaging method. We describe a new method that uses tiny ( approximately 0.1 mm diameter) gold beads as markers to visualize movements of such tissues with high spatial resolution ( approximately 100 microm) and moderate temporal resolution ( approximately 100 ms). We describe bead fabrication, implantation, imaging, and image processing to extract three-dimensional bead coordinates. We then present results of an experiment to determine the stability of gold bead tissue markers (GBTMs) over time in normally moving orbital tissues. Most beads (76%) implanted in sclera, muscle, tendon, and connective tissue were highly stable over the 6-month measurement period. Beads that were judged unstable drifted only a few 100 microm. Bead flows with gaze suggested that posterior Tenon's capsule moves with the globe, that the lateral rectus belly may sideslip, producing "bridle forces," and that the posterior medial rectus pulley sling moves freely anteriorly and posteriorly, but hardly vertically, as required by the "coordinated active pulley" hypothesis. The GBTM method seems applicable to study such short time course phenomena as extraocular muscle (EOM) and connective tissue movement as a function of gaze and such long time course phenomena as myopic eye growth.

  16. Surface chemistries for antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

  17. Some aspects of the host response to methacrylic acid containing beads in a mouse air pouch.

    PubMed

    Patel, Riteshkumar J; Sefton, Michael V

    2012-08-01

    Exudate analysis indicated that both hydroxamated methacrylic acid-based (HX-MAA) and methacrylic acid-based (MAA) beads without hydroxamation, implanted in an air pouch with gelatin discs, elicited similar biological responses, with both resulting in high cell counts and chemokine (MCP3) levels, relative to control, poly(methyl methacrylate) (PMMA) beads. HX-MAA beads had been used elsewhere to restore homeostatic levels of extracellular matrix degrading enzymes, matrix metalloproteases, whereas the MAA beads (without hydroxamation) promoted angiogenesis in wound healing models. Furthermore, both beads appeared to activate exudate macrophages in the classical manner (high IL12/IL10 ratio) at day 4. Exudate macrophages, which were isolated by a magnetic bead protocol and analyzed by RT-PCR, showed a mixed response: elevated levels of an alternative marker (mannose receptor C type lectin) and a classical marker (IL6) were seen, in macrophages from the HX-MAA and MAA group compared to the PMMA group at day 4. A partial shift to an alternatively activated phenotype was seen by day 10. Copyright © 2012 Wiley Periodicals, Inc.

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

    PubMed

    Zhu, Yinglian; Wang, Dongfeng

    2016-12-01

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

  19. Estimating the proportion of microarray probes expressed in an RNA sample

    PubMed Central

    Shi, Wei; de Graaf, Carolyn A.; Kinkel, Sarah A.; Achtman, Ariel H.; Baldwin, Tracey; Schofield, Louis; Scott, Hamish S.; Hilton, Douglas J.; Smyth, Gordon K.

    2010-01-01

    A fundamental question in microarray analysis is the estimation of the number of expressed probes in different RNA samples. Negative control probes available in the latest microarray platforms, such as Illumina whole genome expression BeadChips, provide a unique opportunity to estimate the number of expressed probes without setting a threshold. A novel algorithm was proposed in this study to estimate the number of expressed probes in an RNA sample by utilizing these negative controls to measure background noise. The performance of the algorithm was demonstrated by comparing different generations of Illumina BeadChips, comparing the set of probes targeting well-characterized RefSeq NM transcripts with other probes on the array and comparing pure samples with heterogenous samples. Furthermore, hematopoietic stem cells were found to have a larger transcriptome than progenitor cells. Aire knockout medullary thymic epithelial cells were shown to have significantly less expressed probes than matched wild-type cells. PMID:20056656

  20. Plating by glass-bead peening

    NASA Technical Reports Server (NTRS)

    Babecki, A. J.; Haehner, C. L.

    1971-01-01

    Technique permits plating of primarily metallic substrates with either metals or nonmetals at normal temperature. Peening uses compressed air to apply concurrent streams of small glass beads and powdered plating material to the substrate.

  1. Beads + String = Atoms You Can See.

    ERIC Educational Resources Information Center

    Hermann, Christine K. F.

    1998-01-01

    Presents hands-on activities that give students a head start in learning the vocabulary and basic theory involved in understanding atomic structure. Uses beads to represent protons, neutrons, and electrons and string to represent orbitals. (DDR)

  2. Beads + String = Atoms You Can See.

    ERIC Educational Resources Information Center

    Hermann, Christine K. F.

    1998-01-01

    Presents hands-on activities that give students a head start in learning the vocabulary and basic theory involved in understanding atomic structure. Uses beads to represent protons, neutrons, and electrons and string to represent orbitals. (DDR)

  3. Ecotoxicogenomics: Microarray interlaboratory comparability.

    PubMed

    Vidal-Dorsch, Doris E; Bay, Steven M; Moore, Shelly; Layton, Blythe; Mehinto, Alvine C; Vulpe, Chris D; Brown-Augustine, Marianna; Loguinov, Alex; Poynton, Helen; Garcia-Reyero, Natàlia; Perkins, Edward J; Escalon, Lynn; Denslow, Nancy D; Cristina, Colli-Dula R; Doan, Tri; Shukradas, Shweta; Bruno, Joy; Brown, Lorraine; Van Agglen, Graham; Jackman, Paula; Bauer, Megan

    2016-02-01

    Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training. Published by Elsevier Ltd.

  4. Synthesis and studies of water-soluble Prussian Blue-type nanoparticles into chitosan beads.

    PubMed

    Folch, Benjamin; Larionova, Joulia; Guari, Yannick; Molvinger, Karine; Luna, Carlos; Sangregorio, Claudio; Innocenti, Claudia; Caneschi, Andrea; Guérin, Christian

    2010-10-21

    A new approach to the synthesis of highly stable aqueous colloids of coordination polymer nanoparticles was developed by using water-soluble chitosan beads as template and as stabilizing agent. The method consists in the synthesis of nanocomposite beads containing cyano-bridged coordination polymer nanoparticles via step-by-step coordination of the metal ions and the hexacyanometallate precursors into the chitosan pores and then water solubilization of these as-obtained nanocomposite beads. We obtain a large range of M(2+)/[M'(CN)(6)](3-)/chitosan (where M(2+) = Ni(2+), Cu(2+), Fe(2+), Co(2+), Mn(2+) and M' = Fe(3+) and Cr(3+)) nanocomposite beads and their respective aqueous colloids containing coordination polymer core/chitosan shell nanoparticles. The nanocomposite beads and the corresponding aqueous colloids were studied by Infrared (IR) and UV-Vis spectroscopy, nitrogen sorption (BET), Transmission Electron Microscopy (TEM), High Resolution Transmission Electron Microscopy (HRTEM) and magnetic analyses, which reveal the presence of homogeneously dispersed uniformly-sized cyano-bridged coordination polymer nanoparticles. The detailed studies of the static and dynamic magnetic properties of these nanoparticles show the occurrence of a spin-glass like behavior presumably produced by intra-particle spin disorder due to the low spin exchange energy characterizing these materials.

  5. Modeling Aspects of Two-Bead Microrheology

    NASA Astrophysics Data System (ADS)

    Hohenegger, Christel; Forest, M. Gregory

    2008-07-01

    We revisit the Mason and Weitz (Phys. Rev. Lett., 74, 1995) and Levine and Lubensky (Phys. Rev. Lett., 85, 2000) analysis for one- and two-bead microrheology. Our first motivation is the possibility of drawing inferences from experimental data about local diffusive properties of individual beads and nonlocal dynamic moduli of the medium separating the two beads. Our second motivation is the ability to perform direct numerical simulations of hydrodynamically coupled Brownian beads in soft matter. For both goals, we first must have a model for the coupling between these two transport properties. We reformulate the coupled generalized Langevin equations (GLE) following the scalar GLE analysis of Fricks et al. (J. Appl. Math., 2008), assuming an exponential series parametrization of both local and nonlocal memory kernels. We then show the two-bead GLE model can be represented as a vector Ornstein-Uhlenbeck process, which allows for a fast and statistically accurate numerical simulation of coupled bead paths (time series) and of ensemble-averaged statistics of the process. In this proceedings, we announce the framework to accomplish these two goals of inversion and direct simulation.

  6. Microarrays in cancer research.

    PubMed

    Grant, Geraldine M; Fortney, Amanda; Gorreta, Francesco; Estep, Michael; Del Giacco, Luca; Van Meter, Amy; Christensen, Alan; Appalla, Lakshmi; Naouar, Chahla; Jamison, Curtis; Al-Timimi, Ali; Donovan, Jean; Cooper, James; Garrett, Carleton; Chandhoke, Vikas

    2004-01-01

    Microarray technology has presented the scientific community with a compelling approach that allows for simultaneous evaluation of all cellular processes at once. Cancer, being one of the most challenging diseases due to its polygenic nature, presents itself as a perfect candidate for evaluation by this approach. Several recent articles have provided significant insight into the strengths and limitations of microarrays. Nevertheless, there are strong indications that this approach will provide new molecular markers that could be used in diagnosis and prognosis of cancers. To achieve these goals it is essential that there is a seamless integration of clinical and molecular biological data that allows us to elucidate genes and pathways involved in various cancers. To this effect we are currently evaluating gene expression profiles in human brain, ovarian, breast and hematopoetic, lung, colorectal, head and neck and biliary tract cancers. To address the issues we have a joint team of scientists, doctors and computer scientists from two Virginia Universities and a major healthcare provider. The study has been divided into several focus groups that include; Tissue Bank Clinical & Pathology Laboratory Data, Chip Fabrication, QA/QC, Tissue Devitalization, Database Design and Data Analysis, using multiple microarray platforms. Currently over 300 consenting patients have been enrolled in the study with the largest number being that of breast cancer patients. Clinical data on each patient is being compiled into a secure and interactive relational database and integration of these data elements will be accomplished by a common programming interface. This clinical database contains several key parameters on each patient including demographic (risk factors, nutrition, co-morbidity, familial history), histopathology (non genetic predictors), tumor, treatment and follow-up information. Gene expression data derived from the tissue samples will be linked to this database, which

  7. The Genopolis Microarray Database

    PubMed Central

    Splendiani, Andrea; Brandizi, Marco; Even, Gael; Beretta, Ottavio; Pavelka, Norman; Pelizzola, Mattia; Mayhaus, Manuel; Foti, Maria; Mauri, Giancarlo; Ricciardi-Castagnoli, Paola

    2007-01-01

    Background Gene expression databases are key resources for microarray data management and analysis and the importance of a proper annotation of their content is well understood. Public repositories as well as microarray database systems that can be implemented by single laboratories exist. However, there is not yet a tool that can easily support a collaborative environment where different users with different rights of access to data can interact to define a common highly coherent content. The scope of the Genopolis database is to provide a resource that allows different groups performing microarray experiments related to a common subject to create a common coherent knowledge base and to analyse it. The Genopolis database has been implemented as a dedicated system for the scientific community studying dendritic and macrophage cells functions and host-parasite interactions. Results The Genopolis Database system allows the community to build an object based MIAME compliant annotation of their experiments and to store images, raw and processed data from the Affymetrix GeneChip® platform. It supports dynamical definition of controlled vocabularies and provides automated and supervised steps to control the coherence of data and annotations. It allows a precise control of the visibility of the database content to different sub groups in the community and facilitates exports of its content to public repositories. It provides an interactive users interface for data analysis: this allows users to visualize data matrices based on functional lists and sample characterization, and to navigate to other data matrices defined by similarity of expression values as well as functional characterizations of genes involved. A collaborative environment is also provided for the definition and sharing of functional annotation by users. Conclusion The Genopolis Database supports a community in building a common coherent knowledge base and analyse it. This fills a gap between a local

  8. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

  9. NIST/ISAC standardization study: variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads.

    PubMed

    Hoffman, Robert A; Wang, Lili; Bigos, Martin; Nolan, John P

    2012-09-01

    Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue™), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned

  10. Living-cell microarrays.

    PubMed

    Yarmush, Martin L; King, Kevin R

    2009-01-01

    Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment.

  11. The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

    ERIC Educational Resources Information Center

    Dunlap, Dacey; Patrick, Patricia

    2012-01-01

    During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

  12. The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

    ERIC Educational Resources Information Center

    Dunlap, Dacey; Patrick, Patricia

    2012-01-01

    During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

  13. Beaded streams of Arctic permafrost landscapes

    NASA Astrophysics Data System (ADS)

    Arp, C. D.; Whitman, M. S.; Jones, B. M.; Grosse, G.; Gaglioti, B. V.; Heim, K. C.

    2014-07-01

    Beaded streams are widespread in permafrost regions and are considered a common thermokarst landform. However, little is known about their distribution, how and under what conditions they form, and how their intriguing morphology translates to ecosystem functions and habitat. Here we report on a Circum-Arctic inventory of beaded streams and a watershed-scale analysis in northern Alaska using remote sensing and field studies. We mapped over 400 channel networks with beaded morphology throughout the continuous permafrost zone of northern Alaska, Canada, and Russia and found the highest abundance associated with medium- to high-ice content permafrost in moderately sloping terrain. In the Fish Creek watershed, beaded streams accounted for half of the drainage density, occurring primarily as low-order channels initiating from lakes and drained lake basins. Beaded streams predictably transition to alluvial channels with increasing drainage area and decreasing channel slope, although this transition is modified by local controls on water and sediment delivery. Comparison of one beaded channel using repeat photography between 1948 and 2013 indicate relatively stable form and 14C dating of basal sediments suggest channel formation may be as early as the Pleistocene-Holocene transition. Contemporary processes, such as deep snow accumulation in stream gulches effectively insulates river ice and allows for perennial liquid water below most beaded stream pools. Because of this, mean annual temperatures in pool beds are greater than 2 °C, leading to the development of perennial thaw bulbs or taliks underlying these thermokarst features. In the summer, some pools stratify thermally, which reduces permafrost thaw and maintains coldwater habitats. Snowmelt generated peak-flows decrease rapidly by two or more orders of magnitude to summer low flows with slow reach-scale velocity distributions ranging from 0.1 to 0.01 m s-1, yet channel runs still move water rapidly between pools

  14. Bead-Fourier path integral molecular dynamics.

    PubMed

    Ivanov, Sergei D; Lyubartsev, Alexander P; Laaksonen, Aatto

    2003-06-01

    Molecular dynamics formulation of Bead-Fourier path integral method for simulation of quantum systems at finite temperatures is presented. Within this scheme, both the bead coordinates and Fourier coefficients, defining the path representing the quantum particle, are treated as generalized coordinates with corresponding generalized momenta and masses. Introduction of the Fourier harmonics together with the center-of-mass thermostating scheme is shown to remove the ergodicity problem, known to pose serious difficulties in standard path integral molecular dynamics simulations. The method is tested for quantum harmonic oscillator and hydrogen atom (Coulombic potential). The simulation results are compared with the exact analytical solutions available for both these systems. Convergence of the results with respect to the number of beads and Fourier harmonics is analyzed. It was shown that addition of a few Fourier harmonics already improves the simulation results substantially, even for a relatively small number of beads. The proposed Bead-Fourier path integral molecular dynamics is a reliable and efficient alternative to simulations of quantum systems.

  15. Aerogel Beads as Cryogenic Thermal Insulation System

    NASA Technical Reports Server (NTRS)

    Fesmire, J. E.; Augustynowicz, S. D.; Rouanet, S.; Thompson, Karen (Technical Monitor)

    2001-01-01

    An investigation of the use of aerogel beads as thermal insulation for cryogenic applications was conducted at the Cryogenics Test Laboratory of NASA Kennedy Space Center. Steady-state liquid nitrogen boiloff methods were used to characterize the thermal performance of aerogel beads in comparison with conventional insulation products such as perlite powder and multilayer insulation (MLI). Aerogel beads produced by Cabot Corporation have a bulk density below 100 kilograms per cubic meter (kg/cubic m) and a mean particle diameter of 1 millimeter (mm). The apparent thermal conductivity values of the bulk material have been determined under steady-state conditions at boundary temperatures of approximately 293 and 77 kelvin (K) and at various cold vacuum pressures (CVP). Vacuum levels ranged from 10(exp -5) torr to 760 torr. All test articles were made in a cylindrical configuration with a typical insulation thickness of 25 mm. Temperature profiles through the thickness of the test specimens were also measured. The results showed the performance of the aerogel beads was significantly better than the conventional materials in both soft-vacuum (1 to 10 torr) and no-vacuum (760 torr) ranges. Opacified aerogel beads performed better than perlite powder under high-vacuum conditions. Further studies for material optimization and system application are in progress.

  16. Single bead-based electrochemical biosensor

    PubMed Central

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

    2009-01-01

    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 μm, 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 H2O2 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 H2O2 concentration increased in the range from 1×10−6 to 1.2×10−4 M with a detection limit of 5×10−7 M. The SA-BMP was able to detect the amplicons of 1 pg DNA template of B. Cereus bacteria, thus providing better detection sensitivity than conventional gel-based electropherograms. PMID:19767195

  17. Bead-Fourier path integral molecular dynamics

    NASA Astrophysics Data System (ADS)

    Ivanov, Sergei D.; Lyubartsev, Alexander P.; Laaksonen, Aatto

    2003-06-01

    Molecular dynamics formulation of Bead-Fourier path integral method for simulation of quantum systems at finite temperatures is presented. Within this scheme, both the bead coordinates and Fourier coefficients, defining the path representing the quantum particle, are treated as generalized coordinates with corresponding generalized momenta and masses. Introduction of the Fourier harmonics together with the center-of-mass thermostating scheme is shown to remove the ergodicity problem, known to pose serious difficulties in standard path integral molecular dynamics simulations. The method is tested for quantum harmonic oscillator and hydrogen atom (Coulombic potential). The simulation results are compared with the exact analytical solutions available for both these systems. Convergence of the results with respect to the number of beads and Fourier harmonics is analyzed. It was shown that addition of a few Fourier harmonics already improves the simulation results substantially, even for a relatively small number of beads. The proposed Bead-Fourier path integral molecular dynamics is a reliable and efficient alternative to simulations of quantum systems.

  18. Alternate polyelectrolyte coating of chitosan beads for extending drug release.

    PubMed

    Srinatha, A; Pandit, Jayanta K

    2008-01-01

    In the present study, we addressed the factors modifying ciprofloxacin release from multiple coated beads. Beads were prepared by simple ionic cross-linking with sodium tripolyphoshate and coated with alginate and/or chitosan to prepare single, double, or multilayered beads. The water uptake capacity depended on the nature of beads (coated or uncoated) and pH of test medium. The number of coatings given to the beads influenced ciprofloxacin release rate. The coating significantly decreased the drug release from the beads in comparison to uncoated beads (p < 0.001). When the beads were given three coatings, viz., alginate, chitosan, and again alginate, the drug release appeared to follow the pattern exhibited by colon-targeted drug delivery systems with time dependent release behavior. The increase in coating formed a barrier for easy ingress of dissolution medium into the bead matrix, reducing the diffusion of drug.

  19. 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.

  20. Microarray simulator as educational tool.

    PubMed

    Ruusuvuori, Pekka; Nykter, Matti; Mäkiraatikka, Eeva; Lehmussola, Antti; Korpelainen, Tomi; Erkkilä, Timo; Yli-Harja, Olli

    2007-01-01

    As many real-world applications, microarray measurements are inapplicable for large-scale teaching purposes due to their laborious preparation process and expense. Fortunately, many phases of the array preparation process can be efficiently demonstrated by using a software simulator tool. Here we propose the use of microarray simulator as an aiding tool in teaching of computational biology. Three case studies on educational use of the simulator are presented, which demonstrate the effect of gene knock-out, synthetic time series, and effect of noise sources. We conclude that the simulator, used for teaching the principles of microarray measurement technology, proved to be a useful tool in education.

  1. Cooling rates of lunar volcanic glass beads

    NASA Astrophysics Data System (ADS)

    Hui, H.; Hess, K. U.; Zhang, Y.; Peslier, A. H.; Lange, R. A.; Dingwell, D. B.; Neal, C. R.

    2016-12-01

    It is widely accepted that the Apollo 15 green and Apollo 17 orange glass beads are of volcanic origin. The diffusion profiles of volatiles in these glass beads are believed to be due to degassing during eruption (Saal et al., 2008). The degree of degassing depends on the initial temperature and cooling rate. Therefore, the estimations of volatiles in parental magmas of lunar pyroclastic deposits depend on melt cooling rates. Furthermore, lunar glass beads may have cooled in volcanic environments on the moon. Therefore, the cooling rates may be used to assess the atmospheric condition in an early moon, when volcanic activities were common. The cooling rates of glasses can be inferred from direct heat capacity measurements on the glasses themselves (Wilding et al., 1995, 1996a,b). This method does not require knowledge of glass cooling environments and has been applied to calculate the cooling rates of natural silicate glasses formed in different terrestrial environments. We have carried out heat capacity measurements on hand-picked lunar glass beads using a Netzsch DSC 404C Pegasus differential scanning calorimeter at University of Munich. Our preliminary results suggest that the cooling rate of Apollo 17 orange glass beads may be 12 K/min, based on the correlation between temperature of the heat capacity curve peak in the glass transition range and glass cooling rate. The results imply that the parental magmas of lunar pyroclastic deposits may have contained more water initially than the early estimations (Saal et al., 2008), which used higher cooling rates, 60-180 K/min in the modeling. Furthermore, lunar volcanic glass beads could have been cooled in a hot gaseous medium released from volcanic eruptions, not during free flight. Therefore, our results may shed light on atmospheric condition in an early moon.

  2. Cooling Rates of Lunar Volcanic Glass Beads

    NASA Technical Reports Server (NTRS)

    Hui, Hejiu; Hess, Kai-Uwe; Zhang, Youxue; Peslier, Anne; Lange, Rebecca; Dingwell, Donald; Neal, Clive

    2016-01-01

    It is widely accepted that the Apollo 15 green and Apollo 17 orange glass beads are of volcanic origin. The diffusion profiles of volatiles in these glass beads are believed to be due to degassing during eruption (Saal et al., 2008). The degree of degassing depends on the initial temperature and cooling rate. Therefore, the estimations of volatiles in parental magmas of lunar pyroclastic deposits depend on melt cooling rates. Furthermore, lunar glass beads may have cooled in volcanic environments on the moon. Therefore, the cooling rates may be used to assess the atmospheric condition in an early moon, when volcanic activities were common. The cooling rates of glasses can be inferred from direct heat capacity measurements on the glasses themselves (Wilding et al., 1995, 1996a,b). This method does not require knowledge of glass cooling environments and has been applied to calculate the cooling rates of natural silicate glasses formed in different terrestrial environments. We have carried out heat capacity measurements on hand-picked lunar glass beads using a Netzsch DSC 404C Pegasus differential scanning calorimeter at University of Munich. Our preliminary results suggest that the cooling rate of Apollo 17 orange glass beads may be 12 K/min, based on the correlation between temperature of the heat capacity curve peak in the glass transition range and glass cooling rate. The results imply that the parental magmas of lunar pyroclastic deposits may have contained more water initially than the early estimations (Saal et al., 2008), which used higher cooling rates, 60-180 K/min in the modeling. Furthermore, lunar volcanic glass beads could have been cooled in a hot gaseous medium released from volcanic eruptions, not during free flight. Therefore, our results may shed light on atmospheric condition in an early moon.

  3. Digital magnetic tagging for multiplexed suspension-based biochemical assays

    NASA Astrophysics Data System (ADS)

    Mitrelias, T.; Trypiniotis, T.; Palfreyman, J. J.; Hong, B.; Vyas, K.; Hayward, T. J.; Llandro, J.; Kopper, K. P.; Bland, J. A. C.; Robertson, P. A.; Barnes, C. H. W.

    2009-04-01

    Microarrays and suspension (or bead)-based technologies have attracted significant interest for their broad applications in high throughput molecular biology. However, the throughput of microarrays will always be limited by the array density and the slow diffusion of molecules to their binding sites. Suspension-based technologies, in which all the reactions take place directly on the surface of microcarriers functionalized with molecular probes, could offer true multiplexing due to the possibility of extending their detection capability by a straightforward expansion of the size of the chemical library of probes. To fully exploit their potential, the microcarriers must be tagged, but the number of distinct codes available from spectrometric/graphical/physical encoding methods is currently fairly limited. A digital magnetic tagging method based on magnetic microtags, which have been anisotropy engineered to provide stable magnetization directions which correspond to digital codes, is reported. The tags can be suspended in solution and functionalized with a variety of biological molecular probes. Magnetic tagging offers several benefits compared to the traditional optical encoding techniques currently employed. It offers minimal background signals, potential for a large number of distinct codes, miniaturization of devices, and the ability to write a code in situ. Experimental data showing the reading of individual magnetic microbars from samples comprising 50×20 μm2 Ni elements, as well as micromagnetic simulations that show the feasibility of stray field detection, are presented. The stray fields of the magnetic microbars spanning a range of 60 mOe were detected by a microfabricated fluxgate sensor scanned in a raster fashion over the sample that was placed about 70 μm away. Free floating tags have also been fabricated for use in microfluidic systems. A magnetic lab-on-a-chip device could be used for tagging biomolecular probes for applications in genome

  4. Label-free method for cell counting in crude biological samples via paramagnetic bead aggregation.

    PubMed

    Li, Jingyi; Liu, Qian; Xiao, Li; Haverstick, Doris M; Dewald, Alison; Columbus, Linda; Kelly, Kimberly; Landers, James P

    2013-12-03

    Under chaotropic conditions, DNA released from lysed cells causes the aggregation of paramagnetic beads in a rotating magnetic field in a manner that is independent of the presence of other cellular components. The extent of aggregation correlates with the mass of DNA in a quantitative manner (Leslie, D. C. et al., J. Am. Chem. Soc. 2012, 134, 5689-96), and from this, the number of DNA-containing cells in the sample can be enumerated. Microbial growth testing is demonstrated by monitoring bead aggregation with E. coli in the presence of ampicillin. Without the need for fluorescent labeling or Coulter counting, the white blood cell count can be defined directly from a microliter of crude whole blood. Specificity is brought to the process by coupling bead-based immunocapture with DNA-bead aggregation allowing for the enumeration of CD4+ T cells from human blood samples. The results of DNA-induced bead aggregation had a 95% correlation with those generated by flow cytometry. With the process requiring only inexpensive, widely available benchtop laboratory hardware, a digital camera, and a simple algorithm, this provided a highly accessible alternative to more expensive cell-counting techniques.

  5. Effects of Cohesion On the Dynamic Response of A Conical Bead Pile

    NASA Astrophysics Data System (ADS)

    Palchoudhuri, Paroma; Lehman, Susan; Jacobs, D. T.

    2014-03-01

    We investigate the critical behavior of a 3D conical bead pile built from uniform 3 mm steel spheres. The pile is initially built on a circular base and is subsequently slowly driven through the addition of one bead at a time to the apex of the pile. We investigate the dynamic response of the pile by recording avalanches from the pile over the course of tens of thousands of bead drops, and determining the resulting distribution of avalanche size. In previous work, we have shown that dropping the beads onto the pile from a greater height causes the distribution to deviate from a simple power law due to a stark reduction in number of the largest avalanches. By placing the pile in a uniform magnetic field, we are now observing changes in the avalanche size distribution due to cohesion. When there is cohesion between beads, we find an increase in probability for the largest avalanches and a strong decrease in the probability of medium-sized avalanches. We also observe an increase in the time between avalanches as the cohesion of the system increases. Preliminary results on the effect of simultaneously increasing cohesion, which tends to make large avalanches more probable, and increasing drop height, which tends to make large avalanches less probable, will also be presented.

  6. Reverse Phase Protein Microarrays.

    PubMed

    Baldelli, Elisa; Calvert, Valerie; Hodge, Alex; VanMeter, Amy; Petricoin, Emanuel F; Pierobon, Mariaelena

    2017-01-01

    While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive all cellular functions including proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool for understanding cellular functions.Alteration of the cellular homeostasis driven by elaborate intra- and extracellular interactions has become one of the most studied fields in the era of personalized medicine and targeted therapy. Increasing interest has been focused on developing and improving proteomic technologies that are suitable for analysis of clinical samples. In this context, reverse-phase protein microarrays (RPPA) is a sensitive, quantitative, high-throughput immunoassay for protein analyses of tissue samples, cells, and body fluids.RPPA is well suited for broad proteomic profiling and is capable of capturing protein activation as well as biochemical reactions such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations across hundreds of samples using a limited amount of biological material. For these reasons, RPPA represents a valid tool for protein analyses and generates data that help elucidate the functional signaling architecture through protein-protein interaction and protein activation mapping for the identification of critical nodes for individualized or combinatorial targeted therapy.

  7. Multiplex profiling of glycoproteins using a novel bead-based lectin array.

    PubMed

    Wang, Hong; Li, Hong; Zhang, Wei; Wei, Liming; Yu, Hongxiu; Yang, Pengyuan

    2014-01-01

    Lectin array is becoming important in profiling targeted glycan/glycoprotein, but weak interaction between lectin and glycan causes low sensitivity of the approach. This study aims to develop a bead-based lectin array for improving the sensitivity of glycosylation profiling. Lectins are chemically coupled to fluorescent dye coated microbeads, and glycan-lectin recognition is carried out three dimensionally. The performance of this platform was evaluated, and the LOD of lectin Ricinus communis agglutinin 120 (RCA120) was 50 pg/mL (1 pM) of asialofetuin, providing the bead-based lectin microarray with the highest sensitivity among the reported lectin microarrays. Furthermore, multiplexed assay was performed, which allowed the simultaneous detection of multiple carbohydrate epitopes in a single reaction vessel. The glycosylation patterns of hepatocellular carcinoma associated immunoglobulin G were analyzed, and increased (α-1,6) core fucosylation and (α-2,6) sialylation patterns were observed, which may provide significant clinical evidence for disease diagnosis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Two Year Old With Water Bead Ingestion.

    PubMed

    Jackson, Jami; Randell, Kimberly A; Knapp, Jane F

    2015-08-01

    Foreign body ingestion is a common pediatric complaint. Two case reports describe intestinal obstruction in children from an ingestion of a single superabsorbent water ball, requiring surgical removal. We describe nonsurgical management of an asymptomatic child who ingested approximately 100 superabsorbent water beads.Because of the risk for subsequent intestinal obstruction, the patient was admitted for whole bowel irrigation. This case report is the first describing use of whole bowel irrigation in the management of an asymptomatic patient with multiple water beads ingestion.

  9. Cytokine measurement using cytometric bead arrays.

    PubMed

    Castillo, Luis; MacCallum, Donna M

    2012-01-01

    Cytokines can be measured by enzyme-linked immunosorbent assay (ELISA) or multiplex assay. Both techniques are commonly used in immunology to detect the presence of antibody or antigen in a sample. However, multiplex bead array technology provides the means to simultaneously measure multiple analytes in a single reaction, thereby saving time and resources. This method can detect up to 30 proteins at once, using a relatively small sample volume, without losing sensitivity, accuracy, or reproducibility. In this chapter, we describe the cytometric bead array (CBA) approach to simultaneously measure multiple cytokines in biological samples such as spleen, kidney, or serum from mice infected with the human fungal pathogen Candida albicans.

  10. Anisotropic bead models for molecular hydrodynamics

    NASA Astrophysics Data System (ADS)

    Pastor, Richard W.; Zwanzig, Robert

    1989-05-01

    Some qualitative effects of slip hydrodynamic boundary conditions can be incorporated into bead models by replacing the scalar friction constant of a bead with a friction tensor. Translational and rotational diffusion coefficients are calculated analytically and exactly for polygons, analytically but approximately for spherical shells, and numerically by the method of Bloomfield and de la Torre. The calculated diffusion constants of benzene agree with experiment; such agreement is not possible using scalar friction constants. Some comments are made about the transition from overall slip to stick hydrodynamic behavior when tensor friction constants are used.

  11. Bead and Process for Removing Dissolved Metal Contaminants

    SciTech Connect

    Summers, Bobby L., Jr.; Bennett, Karen L.; Foster, Scott A.

    2005-01-18

    A bead is provided which comprises or consists essentially of activated carbon immobilized by crosslinked poly (carboxylic acid) binder, sodium silicate binder, or polyamine binder. The bead is effective to remove metal and other ionic contaminants from dilute aqueous solutions. A method of making metal-ion sorbing beads is provided, comprising combining activated carbon, and binder solution (preferably in a pin mixer where it is whipped), forming wet beads, and heating and drying the beads. The binder solution is preferably poly(acrylic acid) and glycerol dissolved in water and the wet beads formed from such binder solution are preferably heated and crosslinked in a convection oven.

  12. Chemistry of Natural Glycan Microarray

    PubMed Central

    Song, Xuezheng; Heimburg-Molinaro, Jamie; Cummings, Richard D.; Smith, David F.

    2014-01-01

    Glycan microarrays have become indispensable tools for studying protein-glycan interactions. Along with chemo-enzymatic synthesis, glycans isolated from natural sources have played important roles in array development and will continue to be a major source of glycans. N- and O-glycans from glycoproteins, and glycans from glycosphingolipids can be released from corresponding glycoconjugates with relatively mature methods, although isolation of large numbers and quantities of glycans are still very challenging. Glycosylphosphatidylinositol (GPI)-anchors and glycosaminoglycans (GAGs) are less represented on current glycan microarrays. Glycan microarray development has been greatly facilitated by bifunctional fluorescent linkers, which can be applied in a “Shotgun Glycomics” approach to incorporate isolated natural glycans. Glycan presentation on microarrays may affect glycan binding by GBPs, often through multivalent recognition by the GBP. PMID:24487062

  13. Latex beads as probes of a neural crest pathway: effects of laminin, collagen, and surface charge on bead translocation

    PubMed Central

    1984-01-01

    In the trunk region of avian embryos, neural crest cells migrate along two pathways: dorsally just under the ectoderm, and ventrally between the neural tube and the somites. Previous work from this laboratory has shown that uncoated latex beads are able to translocate along the ventral neural crest pathway after injection into young embryos; however, beads coated with fibronectin are restricted from the ventral route ( Bronner -Fraser, M.E., 1982, Dev. Biol., 91: 50-63). Here, we extend these observations to determine the effects of other macromolecules on bead distribution. The data show that laminin-coated beads, like fibronectin-coated beads, are restricted from the ventral pathway. In contrast, beads coated with type I collagen translocate ventrally after injection. Because macromolecules have characteristic charge properties, changes in surface charge caused by coating the beads may confound interpretation of the results. Electrostatic effects on bead movement were examined by coating the latex beads with polyamino acids in order to predictably alter the initial surface charge. The surface charge before injection was measured for beads coated with amino acid polymers or with various biologically important macromolecules; the subsequent translocation ability of these beads was then monitored in the embryo. Polylysine-coated beads (positively charged) were restricted from the ventral pathway as were fibronectin and laminin-coated beads, even though fibronectin and laminin beads were both negatively charged. In contrast, polytyrosine -coated beads ( neutrally charged) translocated ventrally as did negatively charged collagen-coated or uncoated beads. The results demonstrate that no correlation exists between the charge properties on the latex bead surface and their subsequent ability to translocate along the ventral pathway. Therefore, an adhesion mechanism independent of surface charge effects must explain the restriction or translocation of latex beads on a

  14. Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare

    PubMed Central

    Chou, Jie; Wong, Jorge; Christodoulides, Nicolaos; Floriano, Pierre N.; Sanchez, Ximena; McDevitt, John

    2012-01-01

    Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications. PMID:23202219

  15. Liquid bead array technology in the detection of common translocations in acute and chronic leukemias.

    PubMed

    Shackelford, Rodney E; Jackson, Keith D; Hafez, Michael J; Gocke, Christopher D

    2013-01-01

    Hematologic malignancies often have specific chromosomal translocations that promote cancer initiation and progression. Translocation identification is often vital in the diagnosis, prognosis, and treatment of malignancies. A variety of methods including metaphase cytogenetics, in situ hybridization, microarray techniques, Southern blotting, and many variations of PCR are used to identify translocations. While all these techniques have utility, many have drawbacks limiting their clinical usefulness: high cost, slow turnaround time, low density, large sample requirements, high complexity, and difficult validation and standardization. Multiplexed RT-PCR combined with liquid bead array detection overcomes many of these limitations, allowing simultaneous amplification and detection of multiple translocations within one patient sample. This system has high reliability, reproducibility, and flexibility; low cost and low complexity; rapid turnaround time; and appropriate analyte density. Recently, Asuragen Inc. has developed a multiplexed RT-PCR liquid bead array panel that simultaneously analyzes 12 fusion transcripts found in four major types of hematologic malignancies, allowing rapid and efficient diagnosis. In this chapter, we review liquid bead array technology in relation to the specific hematologic translocations analyzed in the Signature LTx panel.

  16. BACs-on-beads: a new robust and rapid detection method for prenatal diagnosis.

    PubMed

    Choy, Richard Kwong Wai; Chen, Ying; Sun, Xiao-Fang; Kwok, Yvonne Ka Yin; Leung, Tak Yeung

    2014-04-01

    Karyotyping, the gold standard used for diagnosis of chromosomal abnormalities, is being progressively replaced by rapid aneuploidy testing (RAT) techniques such as quantitative fluorescence-PCR, FISH and multiplex ligation-dependent probe amplification for diagnosing the common aneuploidies or chromosomal microarray analysis for comprehensive genome-wide testing. However, due to technical limitations, current RATs are confined to the detection of common aneuploidies 13, 18, 21 and sex chromosomes. To overcome the limitations of RATs, a bacterial artificial chromosomes-on-beads (BoBs™) assay technology has been introduced for the detection of the common aneuploidies as well as specific microdeletion syndromes. The BoBs assay is a bead-based multiplex assay using polystyrene beads impregnated with two spectrally distinct infrared fluorochromes to create a liquid array of up to 100 unique spectral signatures that supports the analysis of that scale of simultaneous hybridization assays on a minute DNA sample. This review gives an overview on the collective experiences of BoBs applications in prenatal diagnosis.

  17. Capturing and concentrating adenovirus using magnetic anionic nanobeads

    PubMed Central

    Sakudo, Akikazu; Baba, Koichi; Ikuta, Kazuyoshi

    2016-01-01

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

  18. Gel bead composition for metal adsorption

    DOEpatents

    Scott, Charles D.; Woodward, Charlene A.; Byers, Charles H.

    1991-01-01

    The invention is a gel bead comprising propylene glycol alginate and bone gelatin and is capable of removing metals such as Sr and Cs from solution without adding other adsorbents. The invention could have application to the nuclear industry's waste removal activities.

  19. Gel bead composition for metal adsorption

    DOEpatents

    Scott, Charles D.; Woodward, Charlene A.; Byers, Charles H.

    1990-01-01

    The invention is a gel bead comprising propylene glycol alginate and bone gelatin and is capable of removing metals such as Sr and Cs from solution without adding other adsorbents. The invention could have application to the nuclear industry's waste removal activities.

  20. Gel bead composition for metal adsorption

    SciTech Connect

    Scott, C.D.; Woodward, C.A.; Byers, C.H.

    1989-04-04

    The invention is a gel bead comprising propylene glycol alginate and bone gelatin and is capable of removing metals such as Sr and Cs from solution without adding other adsorbents. The invention could have application to the nuclear industry's waste removal activities. 4 tabs.

  1. Gel bead composition for metal adsorption

    SciTech Connect

    Scott, C.D.; Woodward, C.A.; Byers, C.H.

    1991-02-26

    This patent describes a gel bead comprising propylene glycol alginate and bone gelatin and is capable of removing metals such as Sr and Cs from solution without adding other adsorbents. The invention could have application to the nuclear industry's waste removal activities.

  2. Glass-Bead Blasting Alters Antenna Surface

    NASA Technical Reports Server (NTRS)

    Fortenberry, James W.; Jilka, Richard L.; Kimmel, Boyce; Garcia, Ramon D.; Cofield, Richard E.; Klose, Gerhardt J.; O'Toole, Thomas

    1987-01-01

    Thermal-emissivity properties improved, and focal length adjusted. Experiments show gentle blasting with glass beads produces beneficial changes in macroscopic surface shapes and in microscopic surface features of lightweight microwave reflectors made of thin metal reflective surfaces on deformable substrates of aluminum honeycomb.

  3. Alginate Beads Containing Lactase: Stability and Microstructure.

    PubMed

    Traffano-Schiffo, Maria Victoria; Aguirre Calvo, Tatiana R; Castro-Giraldez, Marta; Fito, Pedro J; Santagapita, Patricio R

    2017-06-12

    β-Galactosidase (lactase) is a widely used enzyme in the food industry; however, it has low stability against thermal and mechanical treatments. Due to this, the purpose of the present research was to analyze the encapsulation of lactase in alginate-Ca(II) beads in order to maintain its enzymatic activity toward freezing, freezing/thawing, and storage. Also, the effect of the addition of trehalose, and arabic and guar gums and their influence on the microstructure as well as on thermal properties and molecular mobility were studied. Lactase was successfully encapsulated in alginate-Ca(II) beads, and the inclusion of trehalose was critical for activity preservation toward treatments, being improved in guar gum-containing systems. The gums increased the Tm' values, which represents a valuable technological improvement. Finally, the presence of secondary excipients affected the microstructure, showing rods with smaller outer diameter and with lower compactness than alginate-Ca(II) beads. Also, bead composition greatly affects the size, shape, and relaxation times.

  4. Wood mimetic hydrogel beads for enzyme immobilization.

    PubMed

    Park, Saerom; Kim, Sung Hee; Won, Keehoon; Choi, Joon Weon; Kim, Yong Hwan; Kim, Hyung Joo; Yang, Yung-Hun; Lee, Sang Hyun

    2015-01-22

    Wood component-based composite hydrogels have potential applications in biomedical fields owing to their low cost, biodegradability, and biocompatibility. The controllable properties of wood mimetic composites containing three major wood components are useful for enzyme immobilization. Here, lipase from Candida rugosa was entrapped in wood mimetic beads containing cellulose, xylan, and lignin by dissolving wood components with lipase in [Emim][Ac], followed by reconstitution. Lipase entrapped in cellulose/xylan/lignin beads in a 5:3:2 ratio showed the highest activity; this ratio is very similar to that in natural wood. The lipase entrapped in various wood mimetic beads showed increased thermal and pH stability. The half-life times of lipase entrapped in cellulose/alkali lignin hydrogel were 31- and 82-times higher than those of free lipase during incubation under denaturing conditions of high temperature and low pH, respectively. Owing to their biocompatibility, biodegradability, and controllable properties, wood mimetic hydrogel beads can be used to immobilize various enzymes for applications in the biomedical, bioelectronic, and biocatalytic fields.

  5. Extraction and labeling methods for microarrays using small amounts of plant tissue.

    PubMed

    Stimpson, Alexander J; Pereira, Rhea S; Kiss, John Z; Correll, Melanie J

    2009-03-01

    Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5-30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng-7 microg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).

  6. Detection of Escherichia coli O157:H7 using immuno beads

    NASA Astrophysics Data System (ADS)

    Tu, Shu-I.; Gehring, Andrew

    2005-11-01

    A new fluorescent sandwich method for the detection of Escherichia coli O157:H7 was developed. Strepavidin coated magnetic beads and fluorescence beads reacted with biotinylated anti E. coli O157 antibodies to form the immuno magnetic beads (IMB) and immuno fluorescence beads (IFB), respectively. The E. coli bacteria captured by IMB were further labeled with IFB to form IMBM-(E. coliO157:H7)N-IFBO sandwich complexes where the subscripts M, N and O were integral numbers. Using broth cultured E. coli O157:H7, the sandwich method was able to detect the bacteria at the level of ~ 103to 104 CFU/mL. Known quantity of freshly cultured E. coli O157:H7 cells were added to ground beef obtained from local markets. The bacteria in inoculated beef patties were enriched in EC broth containing novobiocin. After enriched for 4 h at 40 °C, the developed IMB-IFB method was applied to detect the presence of E. coli O157:H7. The results demonstrated that the developed method could detect the presence of 1 CFU of E. coli O157:H7 per gram of ground beef.

  7. Effect of Cellulose Acetate Beads on Interleukin-23 Release.

    PubMed

    Nishise, Shoichi; Abe, Yasuhiko; Nomura, Eiki; Sato, Takeshi; Sasaki, Yu; Iwano, Daisuke; Yoshizawa, Kazuya; Yagi, Makoto; Sakuta, Kazuhiro; Ueno, Yoshiyuki

    2016-08-01

    Interleukin (IL)-23, which is released by activated monocytes and neutrophils, promotes production of high levels of IL-17 by T-helper 17 cells. Cellulose acetate (CA) beads are used as carriers for granulocyte and monocyte (GM) adsorptive apheresis using Adacolumn. Contact between blood and CA beads induces cytokine release; however, their inflammatory effects on IL-23 release are unclear. We aimed to clarify the effect of CA beads on IL-23 release in vitro. We incubated peripheral blood with and without CA beads and measured IL-23. Compared to blood samples incubated without CA beads, blood samples incubated with CA beads had significantly decreased amounts of IL-23. In conclusion, CA beads inhibited IL-23 release from adsorbed GMs. The biological effects of this decrease in IL-23 release during GM adsorption to CA beads need further clarification.

  8. Cell membrane deformations under magnetic force modulation characterized by optical tracking and non-interferometric widefield profilometry.

    PubMed

    Wang, Chun-Chieh; Jian, Hung-Jhang; Wu, Chih-Wei; Lee, Chau-Hwang

    2008-08-01

    We measured cell membrane deformations under the modulation of piconewton magnetic force by using optical tracking and noninterferometric widefield optical profilometry. The magnetic force was applied to fibronectin-coated paramagnetic beads that bound to transmembrane protein integrins. At an image-acquisition rate of 20 frame/min, optical tracking provided positioning accuracy better than 70 nm for bead displacements on cell membranes, and optical profilometry obtained membrane topography with 20 nm depth resolution. We elucidated the correlation between the bead movements and membrane deformations. When the magnetic force dominated the bead movements, the membrane arose in front of the bead and the height increased with the bead velocity. On the other hand, when the bead was mainly driven by the cytoskeletons, the membrane profiles showed no relevance to the motion of the bead. In this case, the bead moved faster on smooth membranes. A model based on the dynamics of actin cytoskeletons is proposed to explain these observation results.

  9. A Controlled Drug-Delivery Experiment Using Alginate Beads

    ERIC Educational Resources Information Center

    Farrell, Stephanie; Vernengo, Jennifer

    2012-01-01

    This paper describes a simple, cost-effective experiment which introduces students to drug delivery and modeling using alginate beads. Students produce calcium alginate beads loaded with drug and measure the rate of release from the beads for systems having different stir rates, geometries, extents of cross-linking, and drug molecular weight.…

  10. 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

  11. A Controlled Drug-Delivery Experiment Using Alginate Beads

    ERIC Educational Resources Information Center

    Farrell, Stephanie; Vernengo, Jennifer

    2012-01-01

    This paper describes a simple, cost-effective experiment which introduces students to drug delivery and modeling using alginate beads. Students produce calcium alginate beads loaded with drug and measure the rate of release from the beads for systems having different stir rates, geometries, extents of cross-linking, and drug molecular weight.…

  12. 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.

  13. Comparing Bacterial DNA Microarray Fingerprints

    SciTech Connect

    Willse, Alan R.; Chandler, Darrell P.; White, Amanda M.; Protic, Miroslava; Daly, Don S.; Wunschel, Sharon C.

    2005-08-15

    Detecting subtle genetic differences between microorganisms is an important problem in molecular epidemiology and microbial forensics. In a typical investigation, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial strains, where the patterns of DNA fragment sizes are proxies for a microbe's genotype. The limited genomic sample captured on a gel is often insufficient to discriminate nearly identical strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if the number of probes on the microarray is sufficiently large, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate the statistical fingerprinting problem for 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

  14. Microarray Technologies in Fungal Diagnostics.

    PubMed

    Rupp, Steffen

    2017-01-01

    Microarray technologies have been a major research tool in the last decades. In addition they have been introduced into several fields of diagnostics including diagnostics of infectious diseases. Microarrays are highly parallelized assay systems that initially were developed for multiparametric nucleic acid detection. From there on they rapidly developed towards a tool for the detection of all kind of biological compounds (DNA, RNA, proteins, cells, nucleic acids, carbohydrates, etc.) or their modifications (methylation, phosphorylation, etc.). The combination of closed-tube systems and lab on chip devices with microarrays further enabled a higher automation degree with a reduced contamination risk. Microarray-based diagnostic applications currently complement and may in the future replace classical methods in clinical microbiology like blood cultures, resistance determination, microscopic and metabolic analyses as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel nucleic acid based biomarkers. Here I focus an microarray technologies in diagnostics and as research tools, based on nucleic acid-based arrays.

  15. Monoclonal antibody selection for interleukin-4 quantification using suspension arrays and forward-phase protein microarrays.

    PubMed

    Wang, L; Cole, K D; Peterson, A; He, Hua-Jun; Gaigalas, A K; Zong, Y

    2007-12-01

    A recombinant mouse interleukin-4 (IL-4) and three different purified rat antimouse IL-4 monoclonal antibodies (Mab) with different clonalities were employed as a model system. This system was used to examine monoclonal antibody effectiveness using both conventional and high-throughput measurement techniques to select antibodies for attaining the most sensitive detection of the recombinant IL-4 through the "sandwich-type" immunoassays. Surface plasmon resonance (SPR) measurements and two high-throughput methods, suspension arrays (also called multiplexed bead arrays) and forward-phase protein microarrays, predicted the same capture (BVD4-1D11) and detection (BVD6-24G2) antibody pair for the most sensitive detection of the recombinant cytokine. By using this antibody pair, we were able to detect as low as 2 pg/mL of IL-4 in buffer solution and 13.5 pg/mL of IL-4 spiked in 100% normal mouse serum with the multiplexed bead arrays. Due to the large amount of material required for SPR measurements, the study suggests that the multiplexed bead arrays and protein microarrays are both suited for the selection of numerous antibodies against the same analyte of interest to meet the need in the areas of systems biology and reproducible clinical diagnostics for better patient care.

  16. SNP Microarray in FISH Negative Clinically Suspected 22q11.2 Microdeletion Syndrome

    PubMed Central

    Jain, Manish; Kalsi, Amanpreet Kaur

    2016-01-01

    The present study evaluated the role of SNP microarray in 101 cases of clinically suspected FISH negative (noninformative/normal) 22q11.2 microdeletion syndrome. SNP microarray was carried out using 300 K HumanCytoSNP-12 BeadChip array or CytoScan 750 K array. SNP microarray identified 8 cases of 22q11.2 microdeletions and/or microduplications in addition to cases of chromosomal abnormalities and other pathogenic/likely pathogenic CNVs. Clinically suspected specific deletions (22q11.2) were detectable in approximately 8% of cases by SNP microarray, mostly from FISH noninformative cases. This study also identified several LOH/AOH loci with known and well-defined UPD (uniparental disomy) disorders. In conclusion, this study suggests more strict clinical criteria for FISH analysis. However, if clinical criteria are few or doubtful, in particular newborn/neonate in intensive care, SNP microarray should be the first screening test to be ordered. FISH is ideal test for detecting mosaicism, screening family members, and prenatal diagnosis in proven families. PMID:27051557

  17. SNP Microarray in FISH Negative Clinically Suspected 22q11.2 Microdeletion Syndrome.

    PubMed

    Halder, Ashutosh; Jain, Manish; Kalsi, Amanpreet Kaur

    2016-01-01

    The present study evaluated the role of SNP microarray in 101 cases of clinically suspected FISH negative (noninformative/normal) 22q11.2 microdeletion syndrome. SNP microarray was carried out using 300 K HumanCytoSNP-12 BeadChip array or CytoScan 750 K array. SNP microarray identified 8 cases of 22q11.2 microdeletions and/or microduplications in addition to cases of chromosomal abnormalities and other pathogenic/likely pathogenic CNVs. Clinically suspected specific deletions (22q11.2) were detectable in approximately 8% of cases by SNP microarray, mostly from FISH noninformative cases. This study also identified several LOH/AOH loci with known and well-defined UPD (uniparental disomy) disorders. In conclusion, this study suggests more strict clinical criteria for FISH analysis. However, if clinical criteria are few or doubtful, in particular newborn/neonate in intensive care, SNP microarray should be the first screening test to be ordered. FISH is ideal test for detecting mosaicism, screening family members, and prenatal diagnosis in proven families.

  18. Disordered spherical bead packs are anisotropic

    NASA Astrophysics Data System (ADS)

    Schröder-Turk, G. E.; Mickel, W.; Schröter, M.; Delaney, G. W.; Saadatfar, M.; Senden, T. J.; Mecke, K.; Aste, T.

    2010-05-01

    Investigating how tightly objects pack space is a long-standing problem, with relevance for many disciplines from discrete mathematics to the theory of glasses. Here we report on the fundamental yet so far overlooked geometric property that disordered mono-disperse spherical bead packs have significant local structural anisotropy manifest in the shape of the free space associated with each bead. Jammed disordered packings from several types of experiments and simulations reveal very similar values of the cell anisotropy, showing a linear decrease with packing fraction. Strong deviations from this trend are observed for unjammed configurations and for partially crystalline packings above 64%. These findings suggest an inherent geometrical reason why, in disordered packings, anisotropic shapes can fill space more efficiently than spheres, and have implications for packing effects in non-spherical liquid crystals, foams and structural glasses.

  19. Liquid crystalline gel beads of curdlan.

    PubMed

    Dobashi, Toshiaki; Yoshihara, Hiromi; Nobe, Masahiro; Koike, Michiru; Yamamoto, Takao; Konno, Akira

    2005-01-04

    Curdlan beads consisting of liquid crystalline gel (LCG) and amorphous gel (AG) in alternating layers in a wide range of diameters were newly prepared by interfacial insolubilization reactions using calcium chloride as the setting reagent. The thickness of the liquid crystalline layer was proportional to the diameter of the gel bead, and the proportional constant agreed with that determined for the cylindrical gel prepared by a dialysis method. The proportional constant initially increased with increasing calcium concentration of the dispersing medium and saturated at a high concentration limit. These results suggest that the mechanisms for forming the alternating LCG/AG structures prepared with different boundary conditions are the same. The LCG/AG structure could be controlled by calcium concentration.

  20. Discrete dipole approximation simulation of bead enhanced diffraction grating biosensor

    NASA Astrophysics Data System (ADS)

    Arif, Khalid Mahmood

    2016-08-01

    We present the discrete dipole approximation simulation of light scattering from bead enhanced diffraction biosensor and report the effect of bead material, number of beads forming the grating and spatial randomness on the diffraction intensities of 1st and 0th orders. The dipole models of gratings are formed by volume slicing and image processing while the spatial locations of the beads on the substrate surface are randomly computed using discrete probability distribution. The effect of beads reduction on far-field scattering of 632.8 nm incident field, from fully occupied gratings to very coarse gratings, is studied for various bead materials. Our findings give insight into many difficult or experimentally impossible aspects of this genre of biosensors and establish that bead enhanced grating may be used for rapid and precise detection of small amounts of biomolecules. The results of simulations also show excellent qualitative similarities with experimental observations.

  1. Investigation of factors controlling GTA weld bead geometry

    SciTech Connect

    Smartt, H.B.; Key, J.F.

    1981-01-01

    In welding processes employing a consumable electrode, the input of heat and mass to the fusion zone is coupled. In contrast, in the gas tungsten arc welding (GTAW) process, which uses a nonconsumable tungsten electrode, the input of heat and mass to the fusion zone are not coupled. As a result, the relationships between process parameters (current, arc voltage, welding speed, and filler wire speed) and weld bead geometry (bead width, penetration, and reinforcement) for GTA welding are complex. This work presents an experimental study of the process-parameter/weld-bead-geometry relationships for constant parameter GTAW of Type 304 stainless steel. Bead-on-plate results for partial-penetration welds in 12.5-mm thick plate, using an automatic GTAW machine, are presented. Measurements of bead width, penetration, and bead transverse cross-sectional area are given for autogenous welding; measurements of bead width, penetration, reinforcement, cross-sectional area, and dilution are given for nonautogenous welding.

  2. Green stone beads at the dawn of agriculture

    PubMed Central

    Bar-Yosef Mayer, Daniella E.; Porat, Naomi

    2008-01-01

    The use of beads and other personal ornaments is a trait of modern human behavior. During the Middle and Upper Paleolithic periods, beads were made out of shell, bone, ivory, egg shell, and occasionally of minerals. During the transition to agriculture in the Near East, stone, in particular green stone, was used for the first time to make beads and pendants. We observed that a large variety of minerals of green colors were sought, including apatite, several copper-bearing minerals, amazonite and serpentinite. There seems to be an increase with time of distance from which the green minerals were sought. Because beads in white, red, yellow, brown, and black colors had been used previously, we suggest that the occurrence of green beads is directly related to the onset of agriculture. Green beads and bead blanks were used as amulets to ward off the evil eye and as fertility charms. PMID:18559861

  3. Noncovalent hydrogel beads as microcarriers for cell culture.

    PubMed

    Wieduwild, Robert; Krishnan, Swati; Chwalek, Karolina; Boden, Annett; Nowak, Mirko; Drechsel, David; Werner, Carsten; Zhang, Yixin

    2015-03-23

    Hydrogel beads as microcarriers could have many applications in biotechnology. However, bead formation by noncovalent cross-linking to achieve high cell compatibility by avoiding chemical reactions remains challenging because of rapid gelation rates and/or low stability. Here we report the preparation of homogeneous, tunable, and robust hydrogel beads from peptide-polyethylene glycol conjugates and oligosaccharides under mild, cell-compatible conditions using a noncovalent crosslinking mechanism. Large proteins can be released from beads easily. Further noncovalent modification allows for bead labeling and functionalization with various compounds. High survival rates of embedded cells were achieved under standard cell culture conditions and after freezing the beads, demonstrating its suitability for encapsulating and conserving cells. Hydrogel beads as functional system have been realized by generating protein-producing microcarriers with embedded eGFP-secreting insect cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Green stone beads at the dawn of agriculture.

    PubMed

    Bar-Yosef Mayer, Daniella E; Porat, Naomi

    2008-06-24

    The use of beads and other personal ornaments is a trait of modern human behavior. During the Middle and Upper Paleolithic periods, beads were made out of shell, bone, ivory, egg shell, and occasionally of minerals. During the transition to agriculture in the Near East, stone, in particular green stone, was used for the first time to make beads and pendants. We observed that a large variety of minerals of green colors were sought, including apatite, several copper-bearing minerals, amazonite and serpentinite. There seems to be an increase with time of distance from which the green minerals were sought. Because beads in white, red, yellow, brown, and black colors had been used previously, we suggest that the occurrence of green beads is directly related to the onset of agriculture. Green beads and bead blanks were used as amulets to ward off the evil eye and as fertility charms.

  5. Ceramic Spheres From Cation Exchange Beads

    NASA Technical Reports Server (NTRS)

    Dynys, F. W.

    2003-01-01

    Porous ZrO2 and hollow TiO2 spheres were synthesized from a strong acid cation exchange resin. Spherical cation exchange beads, polystyrene based polymer, were used as a morphological-directing template. Aqueous ion exchange reaction was used to chemically bind (ZrO)(2+) ions to the polystyrene structure. The pyrolysis of the polystyrene at 600 C produces porous ZrO2 spheres with a surface area of 24 sq m/g with a mean sphere size of 42 microns. Hollow TiO2 spheres were synthesized by using the beads as a micro-reactor. A direct surface reaction - between titanium isopropoxide and the resin beads forms a hydrous TiO2 shell around the polystyrene core. The pyrolysis of the polystyrene core at 600 C produces hollow anatase spheres with a surface area of 42 sq m/g with a mean sphere size of 38 microns. The formation of ceramic spheres was studied by XRD, SEM and B.E.T. nitrogen adsorption measurements.

  6. Giant Magnetoresistive Sensors for DNA Microarray

    PubMed Central

    Xu, Liang; Yu, Heng; Han, Shu-Jen; Osterfeld, Sebastian; White, Robert L.; Pourmand, Nader; Wang, Shan X.

    2009-01-01

    Giant magnetoresistive (GMR) sensors are developed for a DNA microarray. Compared with the conventional fluorescent sensors, GMR sensors are cheaper, more sensitive, can generate fully electronic signals, and can be easily integrated with electronics and microfluidics. The GMR sensor used in this work has a bottom spin valve structure with an MR ratio of 12%. The single-strand target DNA detected has a length of 20 bases. Assays with DNA concentrations down to 10 pM were performed, with a dynamic range of 3 logs. A double modulation technique was used in signal detection to reduce the 1/f noise in the sensor while circumventing electromagnetic interference. The logarithmic relationship between the magnetic signal and the target DNA concentration can be described by the Temkin isotherm. Furthermore, GMR sensors integrated with microfluidics has great potential of improving the sensitivity to 1 pM or below, and the total assay time can be reduced to less than 1 hour. PMID:20824116

  7. The control of beads diameter of bead-on-string electrospun nanofibers and the corresponding release behaviors of embedded drugs.

    PubMed

    Li, Tingxiao; Ding, Xin; Tian, Lingling; Hu, Jiyong; Yang, Xudong; Ramakrishna, Seeram

    2017-05-01

    Bead-on-string nanofibers, with appropriate control of the beads diameter, are potential fibrous structures for efficient encapsulation of particle drugs in micron scales and could achieve controlled drug release for tissue engineering applications. In this study, the beads diameter of electrospun bead-on-string nanofibers was controlled by adjusting the concentration of spinning polymer, poly (lactic-co-glycolic acid) (PLGA), and the solvent ratio of chloroform to acetone. The images of the scanning electron microscopy (SEM) suggested that bead-on-string nanofibers could be successfully obtained only with a certain range of PLGA solution concentration. Moreover, with the decrease in the solvent ratio of chloroform to acetone, the range was left-shifted towards a smaller concentration. In addition, increase in the PLGA solution concentration within the range the beads diameter became greater and the shape of the beads changed from oval to slender when increasing the PLGA concentration within the range. The bead-on-string nanofibers with different beads diameter were further used to load micro-particle drugs of tetracycline hydrochloride, as a model drug, to examine the release behavior of nanofibers scaffold. The release profiles of drug loaded bead-on-string nanofibers demonstrated the possibility to alleviate the burst drug release by means of beads diameter control. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. 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

  9. Sensitivity Enhancement of Bead-based Electrochemical Impedance Spectroscopy (BEIS) biosensor by electric field-focusing in microwells.

    PubMed

    Shin, Kyeong-Sik; Ji, Jae Hoon; Hwang, Kyo Seon; Jun, Seong Chan; Kang, Ji Yoon

    2016-11-15

    This paper reports a novel electrochemical impedance spectroscopy (EIS) biosensors that uses magnetic beads trapped in a microwell array to improve the sensitivity of conventional bead-based EIS (BEIS) biosensors. Unloading the previously measured beads by removing the magnetic bar enables the BEIS sensor to be used repeatedly by reloading it with new beads. Despite its recyclability, the sensitivity of conventional BEIS biosensors is so low that it has not attracted much attentions from the biosensor industry. We significantly improved the sensitivity of the BEIS system by introducing of a microwell array that contains two electrodes (a working electrode and a counter electrode) to concentrate the electric field on the surfaces of the beads. We confirmed that the performance of the BEIS sensor in a microwell array using an immunoassay of prostate specific antigen (PSA) in PBS buffer and human plasma. The experimental results showed that a low concentration of PSA (a few tens or hundreds of fg/mL) were detectable as a ratio of the changes in the impedance of the PBS buffer or in human plasma. Therefore, our BEIS sensor with a microwell array could be a promising platform for low cost, high-performance biosensors for applications that require high sensitivity and recyclability.

  10. Analysis of inter-event times for avalanches on a conical bead pile with cohesion

    NASA Astrophysics Data System (ADS)

    Lehman, Susan; Johnson, Nathan; Tieman, Catherine; Wainwright, Elliot

    2015-03-01

    We investigate the critical behavior of a 3D conical bead pile built from uniform 3 mm steel spheres. Beads are added to the pile by dropping them onto the apex one at a time; avalanches are measured through changes in pile mass. We investigate the dynamic response of the pile by recording avalanches from the pile over tens of thousands of bead drops. We have previously shown that the avalanche size distribution follows a power law for beads dropped onto the pile apex from a low drop height. We are now tuning the critical behavior of the system by adding cohesion from a uniform magnetic field and find an increase in both size and number for very large avalanches and decreases in the mid-size avalanches. The resulting bump in the avalanche distribution moves to larger avalanche size as the cohesion in the system is increased. We compare the experimental inter-event time distribution to both the Brownian passage-time and Weibull distributions, and observe a shift from the Weibull to Brownian passage-time as we raise the threshold from measuring time between events of all sizes to time between only the largest system-spanning events. These results are both consistent with those from a mean-field model of slip avalanches in a shear system [Dahmen, Nat Phys 7, 554 (2011)].

  11. Scaling Analysis And Tuning Parameters For Avalanches On A Slowly-Driven Conical Bead Pile

    NASA Astrophysics Data System (ADS)

    Lehman, Susan; Christman, Lilianna; Palchoudhuri, Paroma; Jacobs, D. T.

    2014-03-01

    We report the results of our investigation of the dynamic behavior of a 3D conical beadpile composed of 3 mm steel beads. Beads are added to the pile by dropping them onto the apex one at a time; avalanches are measured through changes in pile mass. We have previously shown that the avalanche size distribution generally follows a power law relation for beads dropped onto the pile apex from a low drop height; for higher drop heights or beads dropped over a larger region, the distribution deviates from a power law due to a reduction in the number of larger avalanches. We are now tuning the critical behavior of the system through the addition of cohesion from a uniform magnetic field, and we find an increase in the probability of very large avalanches and decreases in the mid-size avalanches. Similar distributions have been observed previously by other researchers in conical piles of sand, suggesting a possibility that cohesion may have been a factor. All our distributions without cohesion show universality by collapsing onto a common curve in a scaling analysis; so far no scaling has been found in the system with cohesion. The distribution of the time between avalanche events of various size has also been analyzed and shown to depend on both drop height and cohesion strength.

  12. Changes in the Distribution of Avalanches on a Conical Bead Pile with Cohesion

    NASA Astrophysics Data System (ADS)

    Walker, Justine; Lehman, Susan; Dahmen, Karin; Leblanc, Michael; Uhl, Jonathan

    The probability distributions for avalanches of varying size are experimentally determined for a slowly driven, conical bead pile. The pile is composed of roughly 20 000 steel spheres, 3 mm in diameter, atop a circular base; it is driven by adding one bead at a time to the apex of the pile. We investigate the dynamic response of the pile by recording avalanches off the pile over the course of tens of thousands of bead drops. The avalanching behavior is studied at different drop heights and different amounts of cohesion between the beads. The level of cohesion is tuned through use of an applied uniform magnetic field. Smaller, local avalanches are distinguished from larger, non-local avalanches and the moments of the avalanche distribution are calculated separately for these different populations. The resulting moments scale with cohesion differently, and the results are compared to the scaling predictions from an analytic mean-field model and corresponding simulation of slip avalanches in a shear system [Dahmen, Nat Phys 7, 554 (2011)]. Research supported by NSF CBET 1336116 and 1336634.

  13. Tuning Parameters and Scaling For Avalanches On A Slowly-Driven Conical Bead Pile with Cohesion

    NASA Astrophysics Data System (ADS)

    Lehman, Susan; Jacobs, D. T.; Palchoudhuri, Paroma; Vajpeyi, Avi; Walker, Justine; Dahmen, Karin; Leblanc, Michael; Uhl, Jonathan

    Slip avalanches on a slowly driven pile are investigated experimentally using a 3D conical pile built from uniform 3 mm steel beads. Beads are added to the pile by dropping them onto the apex one at a time; avalanches are measured through changes in pile mass. We investigate the dynamic response of the pile by recording avalanches from the pile over the course of tens of thousands of bead drops. The statistical properties of the avalanches, including probability of particular avalanche sizes and the time between avalanches of given size, are well-characterized by universal power laws and scaling functions. By adding a uniform magnetic field, we may systematically vary the cohesion between the beads and tune the critical behavior of the system. As the cohesion increases we observe an increase in both size and number for very large avalanches and decreases in the mid-size avalanches, causing a deviation from the power law. A full study of the effect of cohesion on the size and time distributions is in process, combining the experimental results with predictions from an analytical mean-field model [Dahmen, Nat Phys 7, 554 (2011)]. Research supported by NSF CBET 1336116 and 1336634.

  14. Demonstrations of the Action and Reaction Law and the Energy Conservation Law Using Fine Spherical Plastic Beads

    ERIC Educational Resources Information Center

    Khumaeni, A.; Tanaka, S.; Kobayashi, A.; Lee, Y. I.; Kurniawan, K. H.; Ishii, K.; Kagawa, K.

    2008-01-01

    Equipment for demonstrating Newton's third law and the energy conservation law in mechanics have successfully been constructed utilizing fine spherical plastic beads in place of metal ball bearings. To demonstrate Newton's third law, special magnetized Petri dishes were employed as objects, while to examine the energy conservation law, a…

  15. Lab-on-a-Chip Magneto-Immunoassays: How to Ensure Contact between Superparamagnetic Beads and the Sensor Surface

    PubMed Central

    Eickenberg, Bernhard; Meyer, Judith; Helmich, Lars; Kappe, Daniel; Auge, Alexander; Weddemann, Alexander; Wittbracht, Frank; Hütten, Andreas

    2013-01-01

    Lab-on-a-chip immuno assays utilizing superparamagnetic beads as labels suffer from the fact that the majority of beads pass the sensing area without contacting the sensor surface. Different solutions, employing magnetic forces, ultrasonic standing waves, or hydrodynamic effects have been found over the past decades. The first category uses magnetic forces, created by on-chip conducting lines to attract beads towards the sensor surface. Modifications of the magnetic landscape allow for additional transport and separation of different bead species. The hydrodynamic approach uses changes in the channel geometry to enhance the capture volume. In acoustofluidics, ultrasonic standing waves force µm-sized particles onto a surface through radiation forces. As these approaches have their disadvantages, a new sensor concept that circumvents these problems is suggested. This concept is based on the granular giant magnetoresistance (GMR) effect that can be found in gels containing magnetic nanoparticles. The proposed design could be realized in the shape of paper-based test strips printed with gel-based GMR sensors. PMID:25586262

  16. Lab-on-a-Chip Magneto-Immunoassays: How to Ensure Contact between Superparamagnetic Beads and the Sensor Surface.

    PubMed

    Eickenberg, Bernhard; Meyer, Judith; Helmich, Lars; Kappe, Daniel; Auge, Alexander; Weddemann, Alexander; Wittbracht, Frank; Hütten, Andreas

    2013-09-17

    Lab-on-a-chip immuno assays utilizing superparamagnetic beads as labels suffer from the fact that the majority of beads pass the sensing area without contacting the sensor surface. Different solutions, employing magnetic forces, ultrasonic standing waves, or hydrodynamic effects have been found over the past decades. The first category uses magnetic forces, created by on-chip conducting lines to attract beads towards the sensor surface. Modifications of the magnetic landscape allow for additional transport and separation of different bead species. The hydrodynamic approach uses changes in the channel geometry to enhance the capture volume. In acoustofluidics, ultrasonic standing waves force µm-sized particles onto a surface through radiation forces. As these approaches have their disadvantages, a new sensor concept that circumvents these problems is suggested. This concept is based on the granular giant magnetoresistance (GMR) effect that can be found in gels containing magnetic nanoparticles. The proposed design could be realized in the shape of paper-based test strips printed with gel-based GMR sensors.

  17. Demonstrations of the Action and Reaction Law and the Energy Conservation Law Using Fine Spherical Plastic Beads

    ERIC Educational Resources Information Center

    Khumaeni, A.; Tanaka, S.; Kobayashi, A.; Lee, Y. I.; Kurniawan, K. H.; Ishii, K.; Kagawa, K.

    2008-01-01

    Equipment for demonstrating Newton's third law and the energy conservation law in mechanics have successfully been constructed utilizing fine spherical plastic beads in place of metal ball bearings. To demonstrate Newton's third law, special magnetized Petri dishes were employed as objects, while to examine the energy conservation law, a…

  18. DNA microarray integromics analysis platform.

    PubMed

    Waller, Tomasz; Gubała, Tomasz; Sarapata, Krzysztof; Piwowar, Monika; Jurkowski, Wiktor

    2015-01-01

    The study of interactions between molecules belonging to different biochemical families (such as lipids and nucleic acids) requires specialized data analysis methods. This article describes the DNA Microarray Integromics Analysis Platform, a unique web application that focuses on computational integration and analysis of "multi-omics" data. Our tool supports a range of complex analyses, including - among others - low- and high-level analyses of DNA microarray data, integrated analysis of transcriptomics and lipidomics data and the ability to infer miRNA-mRNA interactions. We demonstrate the characteristics and benefits of the DNA Microarray Integromics Analysis Platform using two different test cases. The first test case involves the analysis of the nutrimouse dataset, which contains measurements of the expression of genes involved in nutritional problems and the concentrations of hepatic fatty acids. The second test case involves the analysis of miRNA-mRNA interactions in polysaccharide-stimulated human dermal fibroblasts infected with porcine endogenous retroviruses. The DNA Microarray Integromics Analysis Platform is a web-based graphical user interface for "multi-omics" data management and analysis. Its intuitive nature and wide range of available workflows make it an effective tool for molecular biology research. The platform is hosted at https://lifescience.plgrid.pl/.

  19. Signal enhancement using a switchable magnetic trap

    DOEpatents

    Beer, Neil Reginald [Pleasanton, CA

    2012-05-29

    A system for analyzing a sample including providing a microchannel flow channel; associating the sample with magnetic nanoparticles or magnetic polystyrene-coated beads; moving the sample with said magnetic nanoparticles or magnetic polystyrene-coated beads in the microchannel flow channel; holding the sample with the magnetic nanoparticles or magnetic polystyrene-coated beads in a magnetic trap in the microchannel flow channel; and analyzing the sample obtaining an enhanced analysis signal. An apparatus for analysis of a sample includes magnetic particles connected to the sample, a microchip, a flow channel in the microchip, a source of carrier fluid connected to the flow channel for moving the sample in the flow channel, an electromagnet trap connected to the flow line for selectively magnetically trapping the sample and the magnetic particles, and an analyzer for analyzing the sample.

  20. Microfluidic microarray systems and methods thereof

    DOEpatents

    West, Jay A. A. [Castro Valley, CA; Hukari, Kyle W [San Ramon, CA; Hux, Gary A [Tracy, CA

    2009-04-28

    Disclosed are systems that include a manifold in fluid communication with a microfluidic chip having a microarray, an illuminator, and a detector in optical communication with the microarray. Methods for using these systems for biological detection are also disclosed.

  1. Drug-eluting beads for liver embolization: concentration of doxorubicin in tissue and in beads in a pig model.

    PubMed

    Namur, Julien; Wassef, Michel; Millot, Jean-Marc; Lewis, Andrew L; Manfait, Michel; Laurent, Alexandre

    2010-02-01

    To evaluate the local tissue concentrations of the antineoplastic agent doxorubicin and the amount of drug still present inside drug delivery embolization beads at different time points after embolization and to compare doxorubicin levels with histologic modifications around the beads in a pig liver model. It was hypothesized that doxorubicin-eluting beads maintain cytotoxic concentrations of drug locally over a period of several weeks, as suggested by in vitro elution tests. Left lobe hepatic artery embolization was performed in 10 pigs with 100-300-microm or 700-900-microm beads loaded with 37.5 mg doxorubicin/mL. Control unloaded 100-300-microm beads were injected in five pigs. Livers were sampled 28 days or 90 days after embolization. The amount of drug retained inside the beads was assessed with infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue around the beads were determined with microspectrofluorimetry and compared with tissue modifications on hematein eosin saffron-stained sections. Doxorubicin-eluting beads eluted 43% of their initial drug load after 28 days and 89% after 90 days. Doxorubicin was present in tissues around the beads at both time points, with a significant decrease over time (P = .0004). The drug was detected at distances as far as 600 microm from the bead edge. Doxorubicin tissue concentrations ranged from 0.55 microM to 6.80 microM, [corrected] which are cytotoxic levels in hepatocyte cell cultures. High concentrations of drug were associated with coagulative necrosis of liver parenchyma. Doxorubicin-eluting beads 100-300 microm in size induced more necrosis than 700-900-microm beads (P = .0036). Doxorubicin-eluting beads deliver high concentrations of the drug over a period of at least 3 months at several hundred micrometers from the bead, leading to significant cytotoxic effects. Copyright (c) 2010 SIR. Published by Elsevier Inc. All rights reserved.

  2. Discovery of novel integrin ligands from combinatorial libraries using a multiplex "beads on a bead" approach.

    PubMed

    Cho, Choi-Fong; Amadei, Giulio A; Breadner, Daniel; Luyt, Leonard G; Lewis, John D

    2012-11-14

    The development of screening approaches to identify novel affinity ligands has paved the way for a new generation of molecular targeted nanomedicines. Conventional methods typically bias the display of the target protein to ligands during the screening process. We have developed an unbiased multiplex "beads on a bead" strategy to isolate, characterize, and validate high affinity ligands from OBOC libraries. Novel non-RGD peptides that target α(v)β(3) integrin were discovered that do not affect cancer or endothelial cell biology. The peptides identified here represent novel integrin-targeted agents that can be used to develop targeted nanomedicines without the risk of increased tumor invasion and metastasis.

  3. The Microarray Revolution: Perspectives from Educators

    ERIC Educational Resources Information Center

    Brewster, Jay L.; Beason, K. Beth; Eckdahl, Todd T.; Evans, Irene M.

    2004-01-01

    In recent years, microarray anal