Application of the Dimeric G-Quadruplex and toehold-mediated strand displacement reaction for fluorescence biosensing of ochratoxin A

Abstract

Ochratoxin A (OTA) is one of the most toxic mycotoxins that exists in various agro-products and foods. Here, a non-label and enzyme-free fluorescence biosensor for highly specific detection of OTA has been developed by the combination of toehold-mediated strand displacement reaction (TMSD) and G-quadruplex dimer/ThT (G-dimer/ThT). The DNA duplex (aptamer-IP) is composed of the anti-OTA aptamer and a single stranded initiation probe (IP). In the presence of OTA, the attachment of target to aptamer leads to the liberation of the IP, which activates the cycle TMSD amplifications of two hairpin probes (H1 and H2) accompanied by the production of numerous H1-H2 assemblies. This double-stranded H1-H2 structure results in the proximity between the 5'-end overhang tail of H1 and the 3'-end stem of H2 to liberate the pre-blocked G-dimer sequence for lighting up ThT. In addition, the method displayed a stable fluorescence emission in the high-salt media. It was successfully applied to analyze OTA in real food samples. Hence, the constructed fluorescence biosensing platform might provide a new way for OTA and other toxin analysis detection.

Keywords: Aptamer; Fluorescence biosensor; G-quadruplex dimer/ThT; OTA; Toehold-mediated strand displacement reaction.