A label-free CRISPR/Cas12a-dimeric G-quadruplex/thioflavin T aptasensor for the sensitive detection of deoxynivalenol

Abstract

Rapid detection of deoxynivalenol (DON) is crucial for food safety. Herein, a highly sensitive method integrating recombinase polymerase amplification (RPA)-CRISPR/Cas12a with dimeric G-quadruplex (G₄) was developed for DON detection. In this method, DON was specifically recognized by an aptamer, resulting in the release of a complementary DNA (cDNA) which initially hybridized with the aptamer on the well of a microplate. The released cDNA in the supernatant was sucked out of the microplate, amplified through RPA and activated Cas12a for the trans-cleavage of G₄. The disruption of the dimeric G₄/thioflavin T (ThT) structures resulted in a significant fluorescence decrease for DON detection. The CRISPR/Cas12a system with excellent specificities, the RPA with efficient amplification capabilities and the dimeric G₄/ThT with admirable fluorescent properties induced synergistic effects and remarkably enhanced the detection performance with a low detection limit of 0.0539 ng/mL. The whole detection time of the assay was approximately 1.5 h. Moreover, the minimal content of DON that could be detected in rice flour and corn flour was 2.5 μg/kg. This method offers a promising alternative for DON detection and broadens the application of the CRISPR technique for non-nucleic acid target detection, satisfying the urgent demand in the field of grain analysis.