Application of p-phenylenediamine as an electrochemical substrate in peroxidase-mediated voltammetric enzyme immunoassay Original Research Article

p-Phenylenediamine (PPD), a new substrate for peroxidase-mediated voltammetric enzyme immunoassay, was investigated by electrochemical methods and used for the detection of plant virus. The product of PPD oxidation with H2O2 catalyzed by horseradish peroxidase (HRP) is 2,5-diamino-N,N′-di-(4-aminophenyl)-2,5-cyclohexadiene-1,4-diimine in pH 3.0–7.0 Britton–Robinson (B–R) buffer solution, which has a sensitive voltammetric peak at the potential of −0.97 V (versus Ag/AgCl) in pH 10.0 B–R buffer solution. By using this voltammetric peak current, HRP can be measured with a detection limit of 0.95 mU l−1 and a linear range of 1.75–750 mU l−1. Combined this new PPD–H2O2–HRP voltammetric enzyme-linked immunoassay system with direct antigen coating (DAC) enzyme-linked immunosorbent assay (ELISA), cucumber mosaic virus (CMV) can be detected as low as 0.5 ng ml−1, which is 10 times lower than that of the conventional spectrophotometric o-phenylenediamine (OPD) ELISA method. The processes of the enzyme-catalyzed reaction and the electro-reduction of the product of the enzyme-catalyzed reaction have been investigated.