Toehold-Mediated Cascade Catalytic Assembly for Mycotoxin Detection and Its Logic Applications

In this work, an enzyme-free biosensor is reported for mycotoxin detection based on a toehold-mediated catalytic hairpin assembly (CHA) and a DNAzyme-cascaded hydrolysis reaction. In the presence of a mycotoxin, the recognition between an aptamer and the mycotoxin releases the trigger DNA. The trigger DNA initiates the toehold-mediated CHA, generating large amounts of partial duplex B/C with four toeholds, which can be used to assemble the DNAzyme-cascaded hydrolysis reaction. Furthermore, through a collaborative autoassembly reaction among the B/C duplex, DNA1, and DNA2, supramolecular nanostructures corresponding to Mg²⁺-dependent DNAzymes can be formed. With the incubation of Mg²⁺, the dual-modified (TAMRA/BHQ2) substrate strand DNA2 will be cleaved into two fragments, yielding a high TAMRA fluorescence signal for mycotoxin testing. Under optimal conditions, the sensing system was ultrasensitive and showed low detection limits of 0.2 pM for ochratoxin A (OTA), 0.13 pM for aflatoxin B1 (AFB1), and 0.17 pM for zearalenone (ZEN). The mycotoxin aptasensor also exhibited high selectivity and was successfully applied for the quantitative analysis of OTA, AFB1, and ZEN in wine samples. Due to the advantages of flexibility and versatility, this mycotoxin platform was used to fabricate several concatenated logic gates including "AND-INHIBIT", "INHIBIT-OR", "OR-AND", and "OR-INHIBIT" logic biocomputings. Such multiple functions of the logic system provided a universal sensing strategy for the intelligent detection of multiplex mycotoxins, demonstrating considerable potential in food safety and environmental monitoring.