An improved ELISA for the determination of tobacco mosaic virus with linear sweep voltammetry detection based on a new system of PAP-H2O2-HRP

An improved ELISA for the determination of tobacco mosaic virus (TMV) with linear sweep voltammetry based on a new system of p-aminophenol (PAP)- H2O2 -horseradish peroxidase (HRP) has been developed. The enzymatic product 3-[ (4-hydroxyphenyl)amino]-4-(2-amino-5-hydroxyphenyl)-6-[(4-hydroxyphenyl)imi no]-2,4-cyclohexadiene-l-one, produced from HRP catalyzing the oxidation of PAP with H2O2, yields a sensitive linear sweep voltammetric response at a potential of -0.45 V (vs. SCE) in Britton-Robinson (BR) buffer solution. By using this voltammetric peak, HRP can be measured with a detection limit o f 0.4 mU/L and a linear range of 1.0 similar to 1.0 x 10(2) mU/L. The detec tion limit for the clarified TMV is 4.0 ng/mL and the highest dilution rati o detected for the infected leaf sap is 1:3.9 x 10(6). The processes of the enzyme-catalyzed reaction and the electro-reduction of the product of the enzyme-catalyzed reaction have been investigated.