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 <i>p</i>-aminophenyl phosphate (<i>p</i>APP) to produce a redox-active product, <i>p</i>-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 observed compared against a commercially available gold standard. This approach was used to develop the first electrochemical enzyme-linked immunosorbent assay (ELISA) to detect HIV in clinical samples, and excellent performance relative to a gold standard test was achieved