Electrochemical Enzyme-Linked Immunosorbent Assay Featuring Proximal Reagent Generation: Detection of Human Immunodeficiency Virus Antibodies in Clinical Samples

We describe a simple electrochemical immunoassay for human immunodeficiency virus (HIV) antibody detection that localizes capture and detection reagents in close proximity to a microelectrode. Antigenic peptides from HIV-1 gp41 or HIV-2 gp36 were covalently attached to a SU-8 substrate that also presented a template for the deposition of three-dimensional microelectrodes. The detection of HIV antibodies was achieved with an electrochemical immunoassay where an alkaline phosphatase conjugated secondary antibody reacts with p-aminophenyl phosphate (pAPP) to produce a redox-active product, p-aminophenol. The current derived from the oxidation of the reporter group increased linearly over a wide antibody concentration range (0.001–1 μg mL–1), with a detection limit of 1 ng mL–1 (6.7 pM) for both HIV-1 and HIV-2. This level of sensitivity is clinically relevant, and the feasibility of this approach for clinical sample testing was also evaluated with HIV clinical patient samples, with excellent performance obse...