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.
Chatrathi
Madhu Prakash
MP
Chemistry Division, Naval Research Laboratory, Washington DC, USA.
Collins
Greg E
GE
Wang
Joseph
J
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D004563:Electrochemistry / Q000379:methods*; D007124:Immunoenzyme Techniques / Q000379:methods*; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D051379:Mice; D046228:Microarray Analysis / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000379:methods
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D000469:Alkaline Phosphatase