A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B₁ by using enzyme-responsive just-in-time generation of a MnO₂ based nanocatalyst

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B₁ (AFB₁). It is based on the just-in-time generation of an MnO₂ nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO₄) is converted into manganese dioxide (MnO₂) which acts as an oxidase mimic that catalyzes the oxidation 3,3′,5,5′-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO₄ is reduced to Mn(II) ions. This results in a decrease in the amount of MnO₂ nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO₄. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB₁ and magnetic beads carrying bovine serum albumin conjugated to AFB₁ are used for the determination of AFB₁. In presence of AFB₁, it will compete with the BSA-conjugated AFB₁ (on the magnetic beads) for the labeled antibody against AFB₁ on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB₁ antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB₁ concentrations in the range from 0.1 to 100 ng mL⁻¹, with a 0.1 ng mL⁻¹ detection limit (at the 3S_blank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. .