Investigation of voltammetric enzyme-linked immunoassay system based on N-heterocyclic substrate of 2,3-diaminopyridine

A new voltammetric enzyme-linked immunoassay system using the electrochemical substrate 2,3-diaminopyridine (DAP) and horseradish peroxidase (HRP) system has been developed. DAP is oxidized with H2O2 catalyzed by HRP, and the resulting electroactive product produces a sensitive voltammetric peak at potential of −0.72 V (vs. saturated calomel electrode (SCE)) in Britton-Robinson (BR) buffer solutions. The enzyme-catalyzed reaction conditions and voltammetric detection conditions have been investigated in detail. Under the selected optimum conditions, the linear range for detection of free HRP is from 6.0 × 10−11 to 1.0 × 10−8 g mL−1 with a detection limit of 1.0 × 10−12 g mL−1. The new voltammetric detection system has been successfully applied for the assay of prostate specific antigen (PSA) in human serum ranging from 0.4 to 100 ng mL−1 with a detection limit of 0.1 ng mL−1, which is five times lower than that of traditional o-phenylenediamine (OPD) spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. The proposed N-heterocyclic electrochemical detection system of DAP-H2O2-HRP has provided a new and much improved immunoassay method.