Enzyme-controlled Dissolution of MnO Nanoflakes with Enzyme Cascade Amplification for Colorimetric Immunoassay

A new colorimetric immunosensing platform accompanying enzyme cascade amplification strategy was fabricated for quantitative screening of small-molecular mycotoxins (aflatoxin B, AFB used in this case) coupling with enzyme-controlled dissolution of MnO nanoflakes. The visual colored assay was executed by high-efficient MnO-3,3',5,5'-tetramethylbenzidine (TMB) system (blue). In the presence of ascorbic acid, MnO nanoflakes were dissolved into Mn ions, thus resulting in a perceptible color change from blue to colorless. The reaction could be weakened through ascorbate oxidase to catalyze ascorbic acid into dehydroascorbic acid, which indirectly depended on the concentration of ascorbate oxidase. By using ascorbate oxidase/ anti-AFB antibody-labeled gold nanoparticles, a novel competitive-type colorimetric enzyme immunoassay was developed for detection of AFB on AFB-bovine serum albumin (BSA)-conjugated magnetic beads. Upon addition of target AFB, the analyte competed with the conjugated AFB-BSA on the magnetic beads for the labeled anti-AFB antibody on the gold nanoparticles. Under optimal conditions, the absorbance decreased with increasing target AFB within the dynamic range of 0.05-150ngmL with a detection limit of 6.5pgmL at the 3S level. The precision and specificity of the MnO-TMB-based immunosensing system were acceptable. In addition, method accuracy was further validated for monitoring spiked peanut samples, giving results matched well with those obtained from commercialized AFB ELISA kit.