Enzyme-controlled dissolution of MnO₂ 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 B₁, AFB₁ used in this case) coupling with enzyme-controlled dissolution of MnO₂ nanoflakes. The visual colored assay was executed by high-efficient MnO₂-3,3',5,5'-tetramethylbenzidine (TMB) system (blue). In the presence of ascorbic acid, MnO₂ nanoflakes were dissolved into Mn²⁺ 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-AFB₁ antibody-labeled gold nanoparticles, a novel competitive-type colorimetric enzyme immunoassay was developed for detection of AFB₁ on AFB₁-bovine serum albumin (BSA)-conjugated magnetic beads. Upon addition of target AFB₁, the analyte competed with the conjugated AFB₁-BSA on the magnetic beads for the labeled anti-AFB₁ antibody on the gold nanoparticles. Under optimal conditions, the absorbance decreased with increasing target AFB₁ within the dynamic range of 0.05-150ngmL^-1 with a detection limit of 6.5pgmL^-1 at the 3S_blank level. The precision and specificity of the MnO₂-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 AFB₁ ELISA kit.