Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

A sensitive voltammetric enzyme immunoanalytical method is described for assay of horseradish peroxidase (HRP), labelled-HRP and antigen:antibody:labelled-HRP conjugate (Ag : AB : IgG-HRP) using 1,5-dihydroxynaphthalene (1,5-DHN) as substrate. 1,5-DHN is an electrochemical inactive substance, but it may be oxidized by O-2 in air to 5-hydroxy-1,4-naphthoquinone in basic solution, which produces a well-defined voltammetric reduction peak. After adding H2O2 and HRP, HRP catalyzes H2O2 oxidizing 5-hydroxy-1,4-naphthoquinone to 3-hydroxyphthalic acid, leading to the decrease of the voltammetric reduction peak, with which the activity of HRP and the labelled-HRP, further the antigen and antibody may be determined. The mechanism of this voltammetric enzyme-linked immunoassay system is different from the previous reports, in which there are no voltammetric reduction peaks before the addition of H2O2 and HRP, and the reduction peaks appear after the addition of H2O2 and HRP. The height of the voltammetric peak is directly proportional to the concentration of HRP in the previous systems, while the decrease of the voltammetric peak is linear with the concentration of HRP in this system. This new electrochemical method is combined with an indirect enzyme immunoassay using direct antigen-coating format for the detection of the purified tobacco mosaic virus (TMV) with a linear range from 25.0 to 340.0 ng/mL, and a detection limit 25.0 ng/mL.