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

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B (AFB). It is based on the just-in-time generation of an MnO 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 (KMnO) is converted into manganese dioxide (MnO) 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), KMnO is reduced to Mn(II) ions. This results in a decrease in the amount of MnO nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB and magnetic beads carrying bovine serum albumin conjugated to AFB are used for the determination of AFB. In presence of AFB, it will compete with the BSA-conjugated AFB (on the magnetic beads) for the labeled antibody against AFB on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB 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 AFB concentrations in the range from 0.1 to 100 ng mL, with a 0.1 ng mL detection limit (at the 3S 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. Graphical abstract Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO)-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B (AFB) by coupling with hydrolytic reaction of AOx toward AA and the KMnO-Mn(II)-TMB system [note: 3,3',5,5'-tetramethylbenzidine: TMB].