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

p-Phenylenediamine (PPD), a new substrate for peroxidase-mediated voltammet ric enzyme immunoassay, was investigated by electrochemical methods and use d 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-aminoph enyl)-2,5-cyclohexadiene-1,4-diimine in pH 3.0-7.0 Britton-Robinson (B-R) b uffer 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 vol tammetric 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-H2O 2-HRP voltammetric enzyme-linked immunoassay system with direct antigen coa ting (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 tha n that of the conventional spectrophotometric o-phenylenediamine (OPD) ELIS A method. The processes of the enzyme-catalyzed reaction and the electro-re duction of the product of the enzyme-catalyzed reaction have been investiga ted. (C) 2000 Elsevier Science B.V. All rights reserved.