A visual dual-signal paper biosensor based on CRISPR/Cas12a and nanozyme-responsive hydrogel for ultrasensitive detection of aflatoxin B1

Sensitive, rapid, and reliable detection of aflatoxin B1 (AFB1) in food is of great importance for ensuring food safety. In this study, a dual-mode paper-based biosensing platform was developed by integrating the CRISPR/Cas12a nucleic acid cleavage system with a nanozyme-responsive DNA hydrogel for highly sensitive and portable detection of AFB1. Upon the presence of the target, AFB1 binds specifically to the aptamer, thereby activating the CRISPR/Cas12a system and subsequently inducing the degradation of the hydrogel network, which leads to the release of the encapsulated Au@Pt nanozyme. The released nanozyme catalyzes the oxidation of TMB in the presence of H 2 O 2 to generate a blue-colored product, while simultaneously producing a quantifiable diffusion signal on the paper-based substrate, enabling dual-mode colorimetric and distance-based detection. The detection limit of naked-eye detection is 0.1 ng mL −1 , and it is 0.075 ng mL −1 in the distance detection mode. The proposed method can complete the signal output within 30 min and exhibits the advantages of operational simplicity, rapid response, and high sensitivity. In practical peanut samples, satisfactory recoveries and good stability were achieved, demonstrating its potential as a promising strategy for on-site detection of trace mycotoxins in food.