An Amperometric Enzyme-linked Immunosensor Using Resveratrol as the Substrates for Horseradish Peroxidase for Brucella Melitensis Antibody Assay

As a natural product, resveratrol was evaluated as a potential substrate for horseradish peroxidase (HRP) and was applied to the amperometric enzyme-linked immunosensing assays. The sensors based on HRP-IgG were constructed by dispersing graphite, BrAg, and paraffin wax at room temperature. Optimal measurements of resveratrol substrate for HRP were investigated. The results of the comparison studies indicate that resveratrol is more feasible for HRP and more stable in the air than o-phenylenediamine, o-benzophenone aniline, and 3,3′, 5,5′-tetramethylbenzidine. In a Britton-Robinson buffer of pH 6.8, HRP-IgG could catalyze the oxidation reaction of resveratrol by H2O2, and the reductive current of the product of resveratrol at –376 mV increases in a certain concentration of HRP-IgG, binding to the Brucella melitensis antigen-modified electrode. The linear range of the Brucella melitensis antibody determination obtained with the proposed immunosensors is 3.0 × 10−4–1.65 × 10−2 g l−1 with a detection limit of 1 × 10−4 g l−1 (3σ). The immunosensor surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (relative standard deviation = 4.6%). The proposed method has been successfully used for the analysis of rabbit serum samples with satisfactory results.