Bead Populations Research Articles

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

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

Single liquid crystalline dendrimer under smectic-like mean field

ABSTRACTMonte Carlo Simulation studies of different generation liquid crystalline dendrimers (LCDrs) () under smectic-like mean field have been presented. Mean field-induced configurational and alignment properties of several LCDrs at different field strengths and layer spacing values were investigated. Tractable coarse-grained force fields to model both monomer–monomer and monomer–field interaction potentials have been formulated. Particularly, the effect of mean field on the shape, size and orientation of LCDrs was addressed. Depending on the generation of an LCDr, strength of the field and the layer spacing, on average spherically symmetric LCDrs were changed to either rod- or disc-like shapes. At a relatively strong field, smaller-generation LCDrs stretched to their maximum size at bigger values of layer spacing, whereas larger-generation LCDrs compressed to attain short-cylinder-/disc-like shape at smaller values of layer spacing. In addition to stretching and compression, sub-molecular partitioning has also been observed. Two layers of mesogenic units separated by populations of spherical beads were formed along the field direction. Those layers of mesogens are oriented along the field direction with intensity of orientability highly depending on the field strength.

Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis

This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the latter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 μL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth).

False-Positive Reactions Against HLA Class II Molecules Detected in Luminex Single-Antigen Bead Assays

Anti-donor HLA-specific antibodies (DSA) are associated with poor graft outcomes in renal transplantations [1, 2]. Panel reactive antibody (PRA) assays using the Luminex platform are commonly used to detect DSA. PRA assays are performed by using three different panels: 1) pooled antigen panels that use bead populations coated in affinity-purified HLA molecules obtained from multiple cell lines, which are used as screening tests; 2) phenotype panels that use bead populations bearing HLA proteins from a cell line derived from a single individual; and 3) single-antigen bead (SAB) assays that use beads coated in molecules representing a single cloned allelic HLA antigen [3]. Each bead population in phenotype panels features more than one type of HLA molecule, thus requiring expertise to interpret the results, whereas SAB assays provide accurate identification of HLA antibodies [3]. However, cloned HLA antigens are not in their native state. Purification and bead coating can lead to conformational changes that could cause binding of clinically irrelevant antibodies [4, 5, 6, 7, 8, 9]. Here, we report a case with suspected false reactions against denatured HLA class II molecules in SAB assays.

Particle Tracking and Multispectral Collocation Method for Particle-to-Particle Binding Assays

We present a simple-to-implement method for analyzing images of randomly distributed particles transported through a fluidic channel. We term this method particle imaging, tracking and collocation (PITC). Our method uses off-the-shelf optics including a CCD camera, epifluorescence microscope, and a dual-view color separator to image freely suspended particles in a wide variety of microchannels (with optical access for image collection). The particles can be transported via electrophoresis and/or pressure driven flow to increase throughput of analysis. We here describe the implementation of the algorithm and demonstrate and validate three of its capabilities: (1) identification of particle coordinates, (2) tracking of particle motion, and (3) monitoring of particle interaction via collocation analysis. We use Monte Carlo simulations for validation and optimization of the input parameters. We also present an experimental demonstration of the analysis on challenging image data, including a flow of two, interacting Brownian particle populations. In the latter example, we use PITC to detect the presence of target DNA by monitoring the hybridization-induced binding between the two populations of beads, each functionalized with DNA probes complementary to the target molecule.

1013-LB

Aim LIFECODES Class II IDv2 (LM2Q) is a bead-based immunoassay for the detection of class II panel reactive antibodies (IgG). Purified Class II antigens are covalently attached to Luminex beads to create a target for antibodies that may be found in sera. Compared to its predecessor product (LM2), the number of beads found in LM2Q was increased to include beads conjugated with purified antigens from the DQ locus. The aim of the current study was to compare the detection of antibodies when using the expanded panel (LM2Q) vs. the original panel alone (LM2). A second aim of the study was to compare the detection of DR-reactive antibodies when using LM2Q vs. LM2. The third and final aim of the study was to compare the detection of antibodies reactive to DQ antigens when using the expanded panel (LM2Q) vs. the pool of DQ antigens conjugated to a single bead population in LIFECODES LifeScreen Deluxe (LMX). Methods Six hundred (600) serum samples were identified from libraries of excess samples. The sera were tested with LM2Q, LM2, and LMX. Briefly, and separately with each product, an aliquot of beads was diluted into wash buffer to which the serum was then added. Following an incubation and a series of wash steps, the treated beads were then exposed to a anti-human IgG-conjugated to phycoerythrin. After an additional incubation, the bead/conjugate blend was diluted and the fluorescence measured in a Luminex instrument. The observed Median Fluorescence Intensity (MFI) values of each reaction were analyzed to determine a reportable result of positive or negative. An individual bead reaction was considered positive when the observed MFI (normalized to the observed background) was greater than the lot-specific cutoff value and negative when the normalized MFI value was less than the lot-specific value. The presence of positive bead reactions which created a pattern of antibody reactivity was deemed to be a positive reportable result. The final reportable results were analyzed with 2x2 table analysis to determine the % co-positivity (sensitivity), % co-negativity (specificity), and % agreement between LM2Q, LM2, and LMX. Results In the comparison of the detection of class II reactive antibody, the LIFECODES Class II IDv2 kit showed 99.7% co-positivity (98.3-99.9% [95%CI]), 72.2% co-negativity (66.6-77.1% [95%CI]) and 87.2% agreement (84.3-89.6% [95%CI]) when compared to results obtained with LIFECODES Class II ID. In the comparison of the detection of DR-reactive antibody, the LIFECODES Class II IDv2 kit showed 97.5% co-positivity (95.2-98.7% [95%CI]), 98.2% co-negativity (95.9-99.2% [95%CI]) and 97.8% agreement (96.3-98.7% [95%CI]) when compared to results obtained with LIFECODES Class II ID. In the comparison of the detection of DQ-reactive antibody, the LIFECODES Class II IDv2 kit showed 99.6% co-positivity (98.0-99.9% [95%CI]), 81.9% co-negativity (77.3-85.8%[95%CI]) and 90.3% agreement (87.7-92.4% [95%CI]) when compared to results obtained with LIFECODES LifeScreen Deluxe (LMX). The results of the study indicated that the expanded panel of DQ antigen-conjugated beads found in LIFECODES Class II IDv2 was associated with an increase in antibody detection; that the presence of the DQ-conjugated beads did not alter the ability to detect DR-reactive antibodies; and that the panel of DQ-antigen-conjugated beads was associated with an increase in antibody detection beyond that which was detected using a pool of DQ antigens conjugated to a single bead population.

High-Frequency Sound-Speed, Attenuation, and Reflection Measurements Using Water-Saturated Glass Beads of Different Sizes

Sound propagation in water-saturated glass beads was studied under controlled laboratory conditions over a frequency range from 200 to 900 kHz for sound-speed measurements and from 200 to 800 kHz for attenuation measurements. The influence of the grain size on both p-wave speed and attenuation was studied using three kinds of glass beads. A strong negative dispersion of the sound speed over the whole frequency range studied was observed with the three different populations of beads used in this experiment (mean grain size: 272 μm, 520 μm, and 1.33 mm). In addition, it was found that not only does the attenuation increase nonlinearly with frequency, but it also depends on the size of the beads. These results are consistent with earlier measurements performed by other researchers over the same frequency range with glass beads of about the same sizes. In this paper, we also discuss the results of the measurement (over a wide frequency range: 200 kHz-1.5 MHz) of the reflection coefficient of a thick slab of glass beads whose surface was smoothed, as well as the correlation between the behavior of this reflection coefficient and the negative dispersion of the sound speed. For this experiment, five populations of beads (91 μm, 272 μm, 520 μm, 133 mm, and 2.98 mm) were used.

Serodiagnosis of Lyme borreliosis with bead based immunoassays using multiplex technology

The serological diagnosis of Lyme borreliosis is accomplished by the detection of IgG and IgM antibodies specific for relevant antigens of the spirochetal pathogen Borrelia burgdorferi. Instead of the usual enzyme immune assay for screening and the Western blot technique for confirmation, bead based multiplex assays with multiple simultaneously performed distinct reactions can provide quick, automatically derived and reliable results in a single run by flow cytometer technology. The broad analytical dynamic range of assay signals and the high sensitivity and specificity of the multiplex formats allow even for a reliable use in CSF based analyses for antibody specificity index in supposed neuroborreliosis. Fluorescence intensity of the bead based reactions can be transformed into quantified values as U/ml, either for each single antigen or summed up for a group of relevant key antigens. Additionally or alternatively distinct reactions of single bead populations can be transformed to Western blot band equivalents. Internal and external quality controls with the multiplex systems show characteristic data equivalent to the conventional assay formats, so that the advantages of the multiplex assays are ready for use in the routine diagnostic laboratory.

Simultaneous high speed optical and impedance analysis of single particles with a microfluidic cytometer

We describe a microfluidic cytometer that performs simultaneous optical and electrical characterisation of particles. The microfluidic chip measures side scattered light, signal extinction and fluorescence using integrated optical fibres coupled to photomultiplier tubes. The channel is 80 μm high and 200 μm wide, and made from SU-8 patterned and sandwiched between glass substrates. Particles were focused into the analysis region using 1-D hydrodynamic focusing and typical particle velocities were 0.1 ms(-1). Excitation light is coupled into the detection channel with an optical fibre and focused into the channel using an integrated compound air lens. The electrical impedance of particles is measured at 1 MHz using micro-electrodes fabricated on the channel top and bottom. This data is used to accurately size the particles. The system is characterised using a range of different sized polystyrene beads (fluorescent and non-fluorescent). Single and mixed populations of beads were measured and the data compared with a conventional flow cytometer.

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

Whole Blood Immunoassay Based on Centrifugal Bead Sedimentation

Centrifugal "lab on a disk" microfluidics is a promising avenue for developing portable, low-cost, automated immunoassays. However, the necessity of incorporating multiple wash steps results in complicated designs that increase the time and sample/reagent volumes needed to run assays and raises the probability of errors. We present proof of principle for a disk-based microfluidic immunoassay technique that processes blood samples without conventional wash steps. Microfluidic disks were fabricated from layers of patterned, double-sided tape and polymer sheets. Sample was mixed on-disk with assay capture beads and labeling antibodies. Following incubation, the assay beads were physically separated from the blood cells, plasma, and unbound label by centrifugation through a density medium. A signal-laden pellet formed at the periphery of the disk was analyzed to quantify concentration of the target analyte. To demonstrate this technique, the inflammation biomarkers C-reactive protein and interleukin-6 were measured from spiked mouse plasma and human whole blood samples. On-disk processing (mixing, labeling, and separation) facilitated direct assays on 1-μL samples with a 15-min sample-to-answer time, <100 pmol/L limit of detection, and 10% CV. We also used a unique single-channel multiplexing technique based on the sedimentation rate of different size or density bead populations. This portable microfluidic system is a promising method for rapid, inexpensive, and automated detection of multiple analytes directly from a drop of blood in a point-of-care setting.

Target-wave to spiral-wave pattern transition in a discrete Belousov-Zhabotinsky reaction driven by inactive resin beads

Wave pattern formation and transition in chemical and biochemical reaction systems can reveal the system properties. We investigate the pattern transition from target waves to spiral waves upon the increment of inactive beads in a discrete system model, where ion-exchange resin loaded with Belousov-Zhabotinsky catalyst corresponds to the active beads. The results show that inactive beads slow the propagation speed of target waves and increase the wave frequency. As the population of inactive beads increases, clusters are formed, which then break waves into segments where bounded spiral pairs are generated and separated into individual spirals. From this observation, we conclude that the population of inactive resin beads acts as the bifurcation parameter controlling the wave pattern transition from targets to spirals.

Size-based separation and collection of mouse pancreatic islets for functional analysis

Islet size has recently been demonstrated to be an important factor in determining human islet transplantation outcomes. In this study, a multi-layered microfluidic device was developed and quantified for size-based separation of a heterogeneous population of mouse islets. The device was fabricated using standard soft lithography and polydimethylsiloxane (PDMS). Size-based separation was first demonstrated via injection of a heterogeneous population of glass beads between 50-300 microm in diameter which were separated into five sub-populations based on their diameter. Next, a heterogeneous population of mouse pancreatic islets, between 50-250 microm in diameter was separated into four sub-populations. Throughout this process the islets remained intact without any signs of damage, as indicated by cell viability staining. Islet glucose-stimulated insulin secretion of each sub-population of islets was also evaluated demonstrating that islets smaller than 150 microm have superior stimulation indexes (SI) compared to islets larger than 150 microm. In this study, we found that islets between 100 microm and 150 microm in diameter had the greatest SI value in a heterogeneous population of islets.

Abstract C25: Enabling in situ quantitative biomarker analysis in the context of tissue morphology

Abstract Modern personalized medicine makes use of individual molecular biomarkers to subdivide tumors into subsets that behave differently from each other. A large number of clinical trials are evaluating several novel therapeutic targets and relevant biomarkers. Tissue biomarkers are gaining prominent position in drug discovery and development programs and are commonly included in clinical trial testing to identify patients that would profit from particular treatments, measure drug efficacy and/or monitor the success of therapeutic interventions. The demands on accuracy and reliability of tests are very high. The quality of tissue biomarker testing can be affected by various pre-analytic factors (time to fixation, time of fixation, type of fixation, method of tissue processing), analytic factors (assay validation, equipment validation, test reagents, standardized control materials) and post-analytic factors (interpretation criteria, use of image analysis, quality assurance procedures). In this poster we will focus on analytic and post-analytic aspects, and more specifically - imaging cytometry as a method of choice in automated analysis of tissue and tissue microarray specimens. Materials and Methods: 1. Standard cytometric QC materials - for within-run precision of fluorescence measurements, Flow Check beads (Beckman-Coulter), for run-to-run precision of fluorescence measurements, CaliBrite Beads (B.D. Biosciences), for run-to-run precision of chromatic staining, negative and positive punch cores of breast tissue labeled for HER2/Neu with CB11 antibody developed with DAB and counterstained with Hematoxylin. 2. Tissue microarrays - whole mouse, rat and major human organ TMAs (Invitrogen).Arrays stained with dual IHC protocols for CD31/CD34, cytokeratin 7/CD31, S100/GFAP, kappa/lambda (in situ hybridization) and Ki67/CD68. 3. Specimens were analyzed on an iCys® Imaging Cytometer (CompuCyte Corporation, Westwood, MA). Quality control measures and Results: a) within-run precision of fluorescence measurement - a Coefficient of Variation (CV) of fluorescence intensity of less than 3% measured over a large bead population; b) run-torun precision of fluorescence measurements, the mean of fluorescence intensity measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs; c) run-to-run precision of chromatic staining - the mean DAB laser light loss measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs. TMA analysis: instrument automatically identified core elements, performed spectral deconvolution to isolate markers of interest. Chromatic measurements by laser-light-loss and fluorescence measurements were obtained simultaneously and combined to quantify the expression of the markers of interest. High resolution images ofthe cores were obtained simultaneously with the quantitative analytical data. Quantitative end-points on a per-core basis included: percent positive marker expression, mean expression level, total marker expression level and co-localization of markers. The technique of “image masking” effectively reduced the background levels in tissue elements. Summary: LSC technology provides accurate and reproducible measurements of tissue biomarkers in unbiased, highly quantitative manner. It is well suited for tissue-based biomarker discovery and clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C25.

Removal of Inhibitory Effects in a Serum Cardiac Troponin I Immunoassay

The measurement of cardiac troponin I (cTnI)1 has become the gold standard for the clinical diagnosis of myocardial infarction (1). Varieties of commercial sandwich-type immunoassays are used for the measurement of cTnI concentration in human serum or plasma. Several of these assays use pairs of monoclonal antihuman cTnI antibodies with high specificities for the invariant part of the cTnI molecule (amino acid residues 30–110), in accordance with the recommended guidelines (2). These assays are often based on magnetic beads or latex particles to take advantage of the ease of bead/particle washing, which can minimize assay interference by matrix proteins. Signal amplification by means of chemiluminescence or electrochemiluminescence allows the detection of serum cTnI at concentrations <0.05 μg/L. As a part of the recent effort of the IFCC Working Group for Standardization of Troponin I to develop a cTnI reference measurement system (1), we have used various measurement techniques to investigate binding affinities between 6 monoclonal antibodies (mAbs) obtained from HyTest (each serving as either the capture or the detection antibody) and cTnI, in the form of either a reference material (NIST SRM 2921) or a cTnI-positive serum pool (PS). One of the techniques is the multiplexed bead array (3), which resembles the commercial bead- or particle-based immunoassays. The capture mAb molecules are covalently immobilized on different bead populations to capture cTnI. The bound cTnI …

Large‐Area, Nanoimprint‐Assisted Microcontact Stripping for the Fabrication of Microarrays of Fouling/Nonfouling Nanostructures

Methods for the accurate positioning of nanometric beads on a substrate have been developed over a number of years, and range from serial atomic force microscopy (AFM) techniques for single-bead positioning to parallel techniques for the positioning of large populations of beads in monolayer or multilayer architectures, typically from a liquid suspension. For example, topographic cues have been used for bead-based protein array production, although in this case, there is a random distribution of beads within the topography. Bead patterning has also been achieved in capillaries using a micromolding in capillaries (MIMIC) technique. Line patterns with micrometer widths are possible with this technique, achieving good multilayer organization. For monolayer bead patterning at micrometer dimensions, electrostatic forces and similar electrostatic assemblies using nanoxerography, as well as patterning by selective chemical functionalization, by transfer of particles from a liquid–liquid interface, and by subtracting top–down processes, are possible. Recently, approaches to the micropatterning of nanodimensioned beads based on contact printing have appeared. Single-particle resolution has been achieved by directed assembly of the nanobeads on structured poly(dimethylsiloxane) (PDMS) templates. The beads are then transferred from the filled PDMS template to a substrate by microcontact printing. The beads’ surface composition during assembly must be tuned tomaximize the bead–surface interaction forces and to avoid strong adherence to the PDMS. Printing of dry nanobeads is, however, hard to realize. On the other hand, selective removal of beads from a surface using PDMSstructured molds has been achieved by lift-off and microcontact stripping. While the former needs a 3 h contact heat treatment to pattern close-packed silica nanobeads, the latter reports on a resolution limitation due to the crystal packing of the beads. Normally, in the reported contact-printing-based methods, the beads have to be loosely attached so that bead transfer or removal will not be inhibited. Here we present a contact-stripping method for micropatterning nanobead arrays, based on structured poly(methyl methacrylate) (PMMA) stamps and using a nanoimprinter apparatus, which allows careful control of the temperature and pressure during the contact stripping. Scheme 1 represents the patterning method. PMMA stamps, produced by nanoimprint lithography, are placed on the top of the nanobead-coated substrate with the structured parts of the stamp in contact with the beads inside the nanoimprinter chamber. Then pressure and temperature are applied for a few minutes. Afterwards, the stamps are carefully separated from the substrate. As a result, the particles in contact with the stamp are removed from the substrate, producing nanobead-patterned regions that reproduce the structure of the stamp. Microcontact

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

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

An integrated multimodal acoustic particle manipulator and optical evanescent field waveguide

A new acoustic/optical/microfluidic system is presented for the manipulation of bead‐tagged DNA molecules. Acoustic radiation forces are used to manipulate microspheres into and away from the evanescent field of a laser coupled waveguide that is integrated into the reflector of the acoustic chamber. With suitable fluorophores the presence of the target DNA can be detected with a fluorescence microscope enabling large populations of beads to be examined simultaneously. The integrated waveguide and multimodal acoustic chamber are presented here, with results showing that the microspheres can be successfully detected as they are brought into the evanescent field using a quarter‐wave acoustic configuration. It is also shown that by measuring the time of flight of a microsphere between the half‐ and quarter‐wave nodal planes the bead size can be determined, providing a means of multiplexing the detection (detecting a range of different target DNA sequences).

A dual-purpose synthetic colloidal platform for protease mapping: substrate profiling for Dengue and West Nile virus proteases

In a proof of concept study, we created a small focused fluorescent hexapeptide library onto 14 multiplexed barcoded sets of silica particles to probe the substrate recognition specificity of West Nile and Dengue virus proteases. A flow cytometric analysis demonstrated that the optical signature of each bead population remained distinguishable throughout the solid-phase peptide synthesis and proteolytic assay. As expected, both proteases displayed a narrow specificity for lysine and arginine residues in the P1 and P2 substrate positions. This open-ended platform enables the fast and simultaneous identification of peptide substrates and is applicable to other proteases.

Microvortex for focusing, guiding and sorting of particles

We report a microvortex manipulator (MVM) that is a passive, scalable system with great potential for the manipulation and separation of particulate samples in microfluidic environments. The movement of particles is determined by a unique combination of helical flow, buoyant, and gravitational forces. Helical flows are induced by topographically patterned microchannel surfaces, which have previously been used for molecular mixing in microfluidic devices. We illustrate the mechanism of MVM and its applications in passive focusing of beads and cells into parallel streams and guiding of particles and cells. We also explore the application of the unique density-selectivity of microvortex focusing and successfully sort a mixture of two bead populations whose density difference is as small as 0.1 g cm(-3).