Investigation of voltammetric enzyme-linked immunoassay based on a new system of HAP-H2O2-HRP

By introducing heterocyclic compound to immunoassay system as an electrochemical substrate for the fist time, a new voltammetric enzyme-linked immunoassay system of 3-hydroxyl-2-aminopyridine (HAP)-H2O2-horseradish peroxidase (HRP) has been developed. HAP was oxidized with H2O2 catalyzed by HRP, and the resulting electroactive product produced a sensitive voltammetric peak at potential of −0.36 V (vs. SCE) in Britton–Robinson (BR) buffer solution. The process of the enzyme-catalyzed reaction and the electro-reduction of the product have been investigated in detail. The linear range for detection of free HRP was from 4.0 × 10−13 to 1.0 × 10−9 g/mL with a detection limit of 1.2 × 10−13 g/mL. The new system has been successfully applied for the assay of α-fetoprotein (αFP) in human serum ranging from 0.1 to 200 ng/mL with a detection limit of 0.1 ng/mL, which was 10 times lower than that of traditional spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. HAP-H2O2-HRP voltammetric enzyme-linked immunoassay showed a promising alternative approach in the detection of αFP in clinical diagnosis.