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

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

International audienceMicroglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice

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

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

Comparative Genomics and in vitro Infection of Field Clonal Isolates of Brucella melitensis Biovar 3 Did Not Identify Signature of Host Adaptation

Brucella spp. are responsible for brucellosis, a widespread zoonosis causing reproductive disorders in animals. Species-classification within this monophyletic genus is based on bacteriological and biochemical phenotyping. Traditionally, Brucella species are reported to have a preferential, but not exclusive mammalian host. However, this concept can be challenged since many Brucella species infect a wide range of animal species. Adaptation to a specific host can be a driver of pathogen variation. It is generally thought that Brucella species have highly stable and conserved genomes, however the degree of genomic variation during natural infection has not been documented. Here, we investigated potential genetic diversity and virulence of Brucella melitensis biovar 3 field isolates obtained from a single outbreak but from different host species (human, bovine, small ruminants). A unique MLVA-16 pattern suggested all isolates were clonal. Comparative genomic analyses showed an almost non-existent genetic diversity among isolates (only one SNP; no architectural rearrangements) and did not highlight any signature specific to host adaptation. Similarly, the strains showed identical capacities to enter and replicate in an in vitro model of macrophage infection. In our study, the absence of genomic variability and similar virulence underline that B. melitensis biovar 3 is a broad-host-range pathogen without the need to adapt to different hosts

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

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