Rigidity-Responsive Fluorescence Polarization Detection of Aflatoxin B ₁ via Programmable RCA-Coupled CRISPR/Cas12a and a Conformation-Restricted Depolarization Reporter

detection. In this system, target binding induces the release of a complementary DNA strand from an immobilized aptamer duplex, initiating padlock probe circularization and RCA to generate abundant Cas12a-activating amplicons. A rationally engineered conformation-restricted depolarization reporter (CRD-Reporter), in which the fluorophore is confined within a rigid duplex framework, provides an intrinsically high FP signal. Upon Cas12a activation, collateral cleavage disrupts the rigid architecture, releasing freely rotating fragments and producing a pronounced FP decrease. Unlike intensity-based CRISPR assays, the FP readout effectively suppresses background interference and signal fluctuations. The proposed assay exhibits a wide linear range from 0.003 to 300 ng/mL with a low detection limit of 0.00113 ng/mL, high specificity, and excellent accuracy in grains, peanuts, and tea samples. This work establishes a robust FP-based CRISPR sensing paradigm for reliable mycotoxin monitoring in complex food systems.