This work presents a multiplexed electrokinetically-controlled heterogeneous immunoassay that can process ten samples in parallel. The immunoassay microchip was soft-lithographically fabricated using poly(dimethylsiloxane) and glass. Controlling parameters of the electrokinetically-driven flow in the microfluidic network was determined by numerically simulating transport processes. Multiple passively adsorbed antigens captured antibodies present in samples, which then bound with TRITC-labeled detection antibodies to generate fluorescent signals. Antibodies against Escherichia coli O157:H7 and Helicobacter pylori were studied as model analytes. After conditions for antigen-coating were optimized, a 24-minute assay detected E. coli O157:H7 antibody in the concentration range of 0.02–10 μg/mL, and H. pylori antibody in the range of 0.1–50 μg/mL. In testing human serum samples, non-specific binding of serum components was effectively suppressed by using 10% (w/v) bovine serum albumin. An accuracy of 100% was achieved in detecting either E. coli O157:H7 antibody or H. pylori antibody from human serum samples. Simultaneous screening of both antibodies was also successfully demonstrated. The immunoassay chip shows an excellent potential for efficiently detecting multiple pathogenic infections in clinical environments.

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