A nanozyme-based cascade colorimetric aptasensor for amplified detection of ochratoxin A

Abstract

Colorimetric assays have been widely developed for the detection of toxin ochratoxin A (OTA), but most of them suffer from moderate sensitivity when they are adopted for the detection of trace OTA in a complicated food matrix. For the purpose of overcoming this issue, an innovative cascade reaction-based colorimetric aptasensor was developed for the achievement of high sensitivity. The biotin-labelled OTA aptamer was immobilized onto streptavidin magnetic beads by means of the biotin–streptavidin reaction. With OTA binding to its aptamer, the structural switching of the aptamer results in the release of the alkaline phosphatase-labelled oligonucleotide, which is partially complementary to the aptamer. Following the magnetic separation, the cascade reaction is initiated through the enzymatic conversion of ascorbic acid-2-phosphate into ascorbic acid. Subsequent to that, the generated ascorbic acid reduces MnO₂ nanosheets to Mn²⁺ ions, accordingly destroying the oxidase-mimicking activity of MnO₂ nanosheets. In consequence, it is not possible to oxidize 3,3′,5,5′-tetramethylbenzidine (TMB), a substrate for oxidase, with Mn²⁺ for the production of the blue colour product (TMB Ox). With the increasing amount of OTA, a colour change occurs from blue to colourless. The cascade reaction has the potential of greatly amplifying the detection signal, together with remarkably improving the sensitivity, making this colorimetric sensor a universal and promising platform for the highly sensitive detection of mycotoxins in the field of public food safety monitoring.