Electrochemical detection for horseradish peroxidase-based enzyme immunoassay using p-aminophenol as substrate and its application in detection of plant virus Original Research Article

A sensitive electrochemical method for horseradish peroxidase (HRP)-based enzyme immunoassay using p-aminophenol (PAP) as substrate is described. The enzymatic product of PAP oxidation with H2O2 catalyzed by HRP in Britton–Robinson (B–R) buffer solution of pH 4.7 is 3,4-di-[(4-hydroxyphenyl)amino]-6-[(4-hydroxyphenyl) imino]-2,4-cyclohexadiene-1-one, which yields a sensitive second order derivative linear-sweep voltammetric peak at potential of −0.56 V (versus Ag/AgCl) in B–R buffer solution of pH 7. The processes of the enzymatic catalyzed reaction and the electrode reduction of the enzymatic product have been carefully investigated. By using this voltammetric response, HRP can be measured with a detection limit of 0.4 mU l−1 and a linear range of 1–100 mU l−1. So it was further applied to immunoassay and cucumber mosaic virus (CMV) was detected as a model. The detection limit of the purified CMV is 0.5 ng ml−1, which is 10 times lower than that of traditional colorimetric o-phenylenediamine (OPD) enzyme-linked immunosorbent assay (ELISA) method. The results show greatly increased sensitivity by electrochemical enzyme immunoassay.