Cyanidin-horseradish peroxidase-hydroperoxide reaction system and its application in enzymelinked immunosensing assays

Abstract

A cyanidin-based horseradish peroxidase (HRP)-catalyzed reaction system was established in this work. In B-R buffer solutions (pH 6.8), a UV-visible absorbance peak of cyanidin (CAG) at 540 nm (A p1) appeared. After the oxidation reaction of CAG catalyzed by HRP in the presence of H2O2, a significant absorbance peak at 482 nm (A p2) occurred. The ratio R(A P2/A P1) was proportional to the HRP concentration. The application of CAG in the enzyme-linked immunosensing assays was investigated using food and mouth disease virus antigen (FMDVAg) as a model analyte. In sandwich immunoreaction, the analyte FMDVAg and food and mouth disease virus antibody (FMDVAb)-modified magnetic nanoparticles bound the supported conconvalina (Con A) bound with HRP-FMDVAb. After de-absorbing and separating, the HRP-FMDVAb-FMDVAg-FMDVAb-magnetic nanoparticles complexes were subject to enzymatic reaction and UV-visible absorbance measurements. The HRP moiety of the immunoreaction complexes can catalyze the oxidation reaction of CAG by H2O2, and the substrate CAG is converted to products. Based on this principle, a sandwich assay model has been employed to determine FMDVAg in rabbit serum samples with the aid of FMDVAb-Fe3O4 magnetic nanoparticles. The linear range of the FMDVAg determination is 1.5×10−8−2.7×10−6 g/mL with the relatively standard deviation of 3.7% (n = 11). The detection limit is 3.1×10−9 g/mL. Additional advantages of the typical substrate such as OPD, OAP and TMB are good water-solubility and stability.