The efficient and sensitive detection of mycotoxins is critical to ensure food safety and maintain public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensor based on an automated 3D DNA walker-enabled 'one-to-many' signal amplification was developed for sensitive detection of aflatoxin B1. Due to the efficient amplification effect of DNA walkers and the strong fluorescence properties of silver nanoclusters, a sensitive output of the amplified signal was produced by precisely regulating the activated trans-cleavage activity of the specific target DNA of Cas12a. In the established fluorescence biosensor, a small amount of aflatoxin B1 promoted the production of a large amount of activator by the established 3D DNA walker, which stimulated the trans-cleavage activity of Cas12a to degrade the single-stranded DNAs for synthesis of silver nanoclusters, leading to a decreased fluorescent signal. The established biosensor was able to achieve the sensitive detection of aflatoxin B1 under optimal conditions, obtaining a detection limit of 45.38 pg/mL in a linear range of 0.05 to 10 ng/mL. In addition, the developed biosensor showed good recoveries in spiked food samples (peanut milk and drinking water) at different concentrations. This work provided new insights for the applications of DNA walker and the development of a marker-free fluorescent biosensing platform based on CRISPR/Cas for the detection of mycotoxins.
