Thermus thermophilus Argonaute-Assisted Nanopore-Encoding Strategy for Multiplexed Mycotoxin Detection

Multiplexed and portable detection of small molecular food contaminants in complex food matrices remains a challenge in analytical chemistry. Herein, inspired by the multiplexed detection, nucleic-acid-targeting capability, programmability of Thermus thermophilus argonaute (TtAgo), and fingerprint recognition of nanopore technology, we proposed a TtAgo-assisted nanopore encoding strategy to detect three mycotoxins simultaneously (AFB₁, OTA, and ZEN). In this method, the effective signal conversion strategy turns mycotoxins into the DNA encoding reporters that were observed via nanopore technology . In general, guide DNAs (gDNAs) were released in the presence of mycotoxins, followed by specific recognition and multiplexed cleavage mediated by TtAgo and the release of encoding reporters. The translocation of encoding reporters produces highly distinguishable fingerprint information due to favorable signal resolution of nanopore. Owing to the encoding strategy mediated by TtAgo and the high resolution of nanopore, this method displayed low LODs (AFB₁ 67.18 fM, OTA 13.32 fM, and ZEN 11.69 fM) with wide linear ranges. Furthermore, the specificity and practical application of this method were also verified and showed satisfactory results. In conclusion, this strategy offers a novel avenue for the multiplexed and portable detection of small molecules.