An electrokinetically-controlled immunoassay for simultaneous detection of multiple microbial antigens.

An electrokinetically-controlled heterogeneous immunoassay microchip for multiple analyte detection was developed in this study. Numerical simulation was employed to study the transport process in a microfluidic network (microFN). The operation parameters obtained from numerical simulation was then applied to immunoassay experiment. The effectiveness of the automatic electrokinetic control was demonstrated in a separate experiment using fluorescein dye. The immunoassay microchip was made of poly(dimethylsiloxane)(PDMS)/PDMS-coated glass using soft lithography and replica molding. Multi-antigen immobilization was accomplished by adsorbing the antigen molecules onto a PDMS-coated glass slide and by using a microFN. Immobilized lysate antigen of Escherichia coli O157: H7 at different concentrations was assayed and the lower detect limit was 3 microg/mL. The assay also displayed very good specificity, when different microbial lysate antigens were immobilized, including Escherichia coli and Helicobacter pylori, and the primary and secondary antibodies were mixtures of different species. The time required for the immunoassay, from antigen coating to signal detection, was only one hour. While still an un-optimized prototype, this automatic-operating, high-throughput immunoassay microchip shows a great potential in detecting multiple pathogenic infections efficiently for clinical applications.