Electrochemical detection for horseradish peroxidase-based enzyme immunoassay using p-aminophenol as substrate and its application in detection of plant virus
W. Sun et al., Electrochemical detection for horseradish peroxidase-based enzyme immunoassay using p-aminophenol as substrate and its application in detection of plant virus, ANALYT CHIM, 434(1), 2001, pp. 43-50
A sensitive electrochemical method for horseradish peroxidase (HRP)-based e
nzyme immunoassay using p-aminophenol (PAP) as substrate is described. The
enzymatic product of PAP oxidation with H2O2 catalyzed by HRP in Britton-Ro
binson (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 sensiti
ve 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 th
e enzymatic catalyzed reaction and the electrode reduction of the enzymatic
product have been carefully investigated. By using this voltammetric respo
nse, HRP can be measured with a detection Limit of 0.4 mUI(-1) and a linear
range of 1-100 mUI(-1). So it was further applied to immunoassay and cucum
ber 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 traditi
onal colorimetric o-phenylenediamine (OPD) enzyme-linked immunosorbent assa
y (ELISA) method. The results show greatly increased sensitivity by electro
chemical enzyme immunoassay. (C) 2001 Elsevier Science B.V. AU rights reser
ved.