Sensitive quantification of aflatoxin B1 in peanuts using DNAzyme-aptamer probe and advanced multivariate calibration

Aflatoxin B1 (AFB1) is a hazardous mycotoxin commonly found in agricultural products, necessitating sensitive and reliable detection methods. In this work, a novel aptamer-based fluorescence sensing strategy was developed, in which DNAzyme acts as both signal generator and amplifier. In the absence of AFB1, a blocking strand hybridizes with the DNAzyme arm, inhibiting its catalytic activity. When AFB1 is present, the aptamer specifically binds the target, releasing the blocking strand and restoring DNAzyme activity. The activated DNAzyme cleaves a fluorophore–quencher labeled substrate, leading to enhanced fluorescence. To achieve accurate quantification, a probe technique-based generalized multivariate standard addition strategy (GMSAprobe) was employed. The method enabled reliable determination of AFB1 in peanut samples with recoveries of 96–102% and a detection limit of 0.56 μg/kg, consistent with results from a commercial ELISA kit and well below regulatory limits.