Bubble cell for magnetic bead trapping in capillary electrophoresis

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. Figure Isovalues of the magnetic induction produced by two permanent magnets in attraction configuration with a capillary placed between the

Total serum IgE quantification by microfluidic ELISA using magnetic beads

The present work reports on the quantification of total IgE in human serum using a microanalytical device whose fluidics is driven by gravity and capillary forces only. Thanks to the eight parallel microchannels in each microchip, calibration and sample analysis are performed simultaneously. A mixture of magnetic bead/analyte/second antibody is incubated off-line and then percolated through the channels where magnetic beads are trapped, enabling the separation of the solid phase from the excess reagents. The entire assay is performed in less than 1h, and thanks to the miniaturized format, only a small volume of serum is required. Non-specific adsorption was first investigated and a blocking agent compatible with this allergy-based test was chosen. Then, the assay was optimized by determining the best magnetic bead and labelled antibody concentrations. After achievement of a calibration curve with a reference material, the protocol was applied to total IgE quantification of a patient serum sample that showed results in good accordance with those obtained by ImmunoCap® and Immunoaffinity capillary electrophoresis measurements. A detection limit of 17.5ngml−1 was achieved and good reproducibility (RSD < 10%) inter- and intra-chip was observed. Figure Off-line incubation of the patient sample with anti-IgE grafted magnetic beads and ALP-labelled anti-IgE is carried out in an Eppendorff. Detection is then performed with the GRAVI®-Cell device from DiagnoSwiss, where fluidics is driven by gravity and capillary forces onl

Magnetic track array for efficient bead capture in microchannels

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, 10mm) 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 magnet

Analysis of major milk whey proteins by immunoaffinity capillary electrophoresis coupled with MALDI-MS

Two major milk whey proteins, β-lactoglobulin and α-lactalbumin, are among the main cowmilk allergens and can cause allergy even at a very low concentrations. Therefore, these proteins are interesting targets in food analysis, not only for food quality control but also for highlighting the presence of allergens. Herein, a sensitive analysis for β-lactoglobulin and -lactalbumin was developed using immunoaffinity capillary electrophoresis hyphenated with MALDI-MS.Magnetic beads functionalized with appropriate antibodieswere used for β-lactoglobulin and α-lactalbumin immunocapture inside the capillary. After elution from the beads, analyte focusing and separation were performed by transient isotachophoresis followed by MALDI-MS analysis performed through an automated iontophoretic fraction collection interface. A LOD in the low nanomolar range was attained for both whey proteins. The method developed was further applied to the analysis of different milk samples including fortified soy milk

Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding

Total serum IgE quantification by microfluidic ELISA using magnetic beads

The present work reports on the quantification of total IgE in human serum using a microanalytical device whose fluidics is driven by gravity and capillary forces only. Thanks to the eight parallel microchannels in each microchip, calibration and sample analysis are performed simultaneously. A mixture of magnetic bead/analyte/second antibody is incubated off-line and then percolated through the channels where magnetic beads are trapped, enabling the separation of the solid phase from the excess reagents. The entire assay is performed in less than 1 h, and thanks to the miniaturized format, only a small volume of serum is required. Non-specific adsorption was first investigated and a blocking agent compatible with this allergy-based test was chosen. Then, the assay was optimized by determining the best magnetic bead and labelled antibody concentrations. After achievement of a calibration curve with a reference material, the protocol was applied to total IgE quantification of a patient serum sample that showed results in good accordance with those obtained by ImmunoCapA (R) and Immunoaffinity capillary electrophoresis measurements. A detection limit of 17.5 ng ml(-1) was achieved and good reproducibility (RSD < 10%) inter- and intra-chip was observed

Fish in lake ecological assessment in Europe: Quo Vadis?

It is well established that fish are sensitive indicators of environmental degradation and offer the major advantage of integrating the direct and indirect effects of stress over large scales of space and time. Nevertheless, the use of fish communities as indicators of environmental quality is highly challenging, therefore fish community has been one of the most neglected aspect of lake ecological monitoring. This paper gives an overview on fish-based assessment methods in Europe. By now, 15 Member States have finalised fish-based lake assessment systems, five of these assessment systems have been recently intercalibrated in the Alpine and Northern region, while Intercalibration is still ongoing in the Central-Baltic region. In contrary, several countries of the Mediterranean region have currently renounced the use of fish in lake assessment (mainly due to a low species richness, dominance of invasive taxa, and high costs of sampling), this opinion being strongly debated within region. This paper seeks to answer questions: How lake fish ecological assessment systems are built and used across Europe? Which pressures are assessed and are the pressure-response relationships tested? What are the main lessons and challenges of the lake fish methods` development and harmonization process

Capillary electrophoresis immunoassay using magnetic beads

Protein A-coated magnetic beads (0.3 μm) have been trapped in a small portion of a neutrally coated capillary (50 μm id). Anti-β-lactoglobulin (β-LG) antibodies have then been immobilized on the beads through strong affinity with protein A to subsequently capture β-LG from model or real samples. Once the immunocomplexes formed at physiological pH, a discontinuous buffer system has been used to release the partners and preconcentrate them by transient ITP. The antigens and antibodies have finally been separated by CZE and detected by UV absorbance. An LOQ of 55 nM has been achieved. This methodology has been applied to quantify native β-LG in pasteurized and ultra-high-temperature-treated bovine milk. All the described procedures, including immunosorbent preparation, sample extraction, cleanup, preconcentration, and separation are completely automated on a commercial CE instrument. As this CE immunoassay method is simple, rapid, selective, and sensitive, it should be a practical and attractive technology for the analysis of complicated biological samples

Magnetic Core Shell Nanoparticles Trapping in a Microdevice Generating High Magnetic Gradient

Magnetic core shell nanoparticles (MCSNPs) 30 nm diameter with a magnetic weight of 10% are usually much too small to be trapped in microfluidic systems using classical external magnets. Here, a simple microchip for efficient MCSNPs trapping and release is presented. It comprises a bed of micrometric iron beads (6–8 mm diameter) packed in a microchannel against a physical restriction and presenting a low dead volume of 0.8 nL. These beads of high magnetic permeability are used to focus magnetic field lines from an external permanent magnet and generate local high magnetic gradients. The nanoparticles magnetic trap has been characterised both by numerical simulations and fluorescent MCSNPs imaging. Numerical simulations have been performed to map both the magnetic flux density and the magnetic force, and showed that MCSNPs are preferentially trapped at the iron bead magnetic poles where the magnetic force is increased by 3 orders of magnitude. The trapping efficiency was experimentally determined using fluorescent MCSNPs for different flow rates, different iron beads and permanent magnet positions. At a flow rate of 100 mL h1, the nanoparticles trapping/release can be achieved within 20 s with a preconcentration factor of 4000