Aflatoxin B1 (AFB1) is a highly toxic mycotoxin and strongly carcinogenic and teratogenic to humans. Achieving CRISPR-based sensing system for highly sensitive detection of AFB1 remains a significant challenge. A CRISPR/Cas12a-powered self-sustaining catalytic nucleic acid circuit (CPSCN) is proposed for the sensitive and accurate detection of aflatoxin B1 (AFB1). In the CPSCN sensing system, CRISPR/Cas12a/gRNA1 indirectly recognizes AFB1 by aptamer and effectively triggers trans-cleavage of a pair of self-reporting single-stranded DNA-locked RNA2 fluorescence probes (named as dual-winged fluorescence probe). The released RNA2 then initiates an autocatalytic positive feedback loop, resulting in continuous probe cleavage. The CPSCN system achieves highly sensitive detection in a single-enzyme one-step process. The method exhibits excellent quantitative capability for AFB1 detection with a detection limit of 7.5 pg/mL. In real samples, the detection results of the proposed biosensor show high consistency with those obtained by enzyme-linked immunosorbent assay (ELISA). This work not only provides a novel strategy for AFB1 detection with high sensitivity, robustness, and operational simplicity, but also offers a new insight for designing signal amplification strategies of CRISPR-based biosensors.
