Microchip-Based Electrochemical Enzyme Immunoassays
In this chapter a microchip-based electrochemical enzyme immunoassay is developed and its performance is demonstrated for the determination of monoclonal mouse IgG as a model analyte. Such a direct homogeneous immunoassay requires the integration of electrokinetic mixing of alkaline phosphatase (ALP)-labeled anti-mouse IgG antibody (Ab-E) with the mouse IgG antigen (Ag) analyte in a precolumn reaction chamber, injection of immunochemical products into the separation channel, followed by rapid electrophoretic separation of enzyme-labeled free antibody and enzyme-labeled antibody-antigen complex. The separation is followed by a postcolumn reaction of enzyme tracer with p-aminophenyl phosphate (p-APP) substrate (S) and downstream amperometric detection of p-aminophenol (p-AP) product. Factors influencing the reaction, injection, separation, and detection processes are optimized. We have characterized the microchip-based immunoassay protocol. The resulting attractive analytical performance, along with distinct miniaturization and portability advantages of the electrochemical microsystem, offer considerable promise for designing self-contained and disposable chips for decentralized clinical diagnostics.
Key WordsMicrochip microfluidic electrochemical detection immunossay antibody amperometry alkaline phosphatase
- 14.Jenkins, S. H., Halsal, H. B., and Heineman, W. R. (1988) Subattomole immunoassay with electrochemical detection. Clin. Chem. 34, 1159–1159.Google Scholar
- 18.Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109, 129–135.Google Scholar