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

High-sensitive protein analysis by FESI-CEMALDI-MS

Capillary zone electrophoresis (CZE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) are two techniques highly suitable for the separation and detection of intact proteins. Herein, based on the use of a recently introduced iontophoretic fraction collection interface for the coupling of CE and MALDI-MS, the potential of the combination of both techniques for the analysis of intact proteins is assessed. To further provide a bioanalytical platform with high-sensitivity capabilities, field-enhanced sample injection is integrated as on online preconcentration strategy upstream from the electrokinetic separation. Under optimized conditions, more than 3200- and 4800-fold improvement, respectively in terms of peak height and peak area, as well as LODs ranging from 5 to 10 nM, has been achieved

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

Evaluation of carrier ampholyte-based capillary electrophoresis for separation of

Carrier ampholyte-based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4-9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline-rich di- to decapeptides, and phosphinic pseudopeptides - tetrapeptide mimetics synthesized as a mixture of four diastereomers having the -P(O)(OH)-CH2- moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility

Fountain pen for scanning electrochemical microscopy

A fountain pen probe has been developed to extend the scope of scanning electrochemical microscopy (SECM) experiments to dry surfaces. The fountain pen is fabricated by UV-photoablation of a polyethylene terephthalate (PET) film and consists on one side of one microchannel for flowing an electrolyte solution to the open tip, integrating a reference/counter electrode and on the other side a carbon track. The exposed tip of the track forms the working electrode located close to the microchannel outlet. The fountain pen can operate in a pointillist mode where a nanolitre droplet at the bottom of the probe connects it to a well-defined surface area to study locally the substrate, but can also operate in a scanning mode leaving a linear wet track of solution behind it to monitor the surface activity. The electrochemical characterization of the proposed fountain pen probe was performed by cyclic voltammetry, approach curves and lateral line scans over insulating and conductive substrates, showing that the flow rate and the probe-substrate distance have a major influence on its electrochemical behavior. An SECM image of a gold on glass EPFL logo is presented as a proof-ofconcept that fountain pen probes can be employed for the detection of surface activity when scanning in a contact regime

Compatible Buffer for Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A compatible buffer system for the coupling of capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed. The employed interface consists of a robot to drive a sliver-covered separation capillary and an AnchorChip MALDI-MS target. The outlet of the capillary is grounded and connected to the pre-deposited buffer droplet on the MALDI target to make the electric connection and allow sample crystallization for MALDI-MS. The possibility of using only one buffer already containing the matrix for MALDI-MS for the separation and the ionization was investigated and tested on protein and peptide samples. The results show that the proposed buffer system is suitable for CE-MALDI-MS coupling, simplifies the traditional buffer mixing steps in off-line CE-MALDI-MS protocols, and is therefore highly promising for on-line analysis

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