Aptamer Probe-Assisted Strand Displacement-CRISPR/Cas12a Biosensor for Ultrasensitive Detection of AFB1.

The sensitive and accurate detection of aflatoxin B1 (AFB1) is crucial for public health. Herein, the aptamer (Apt)-lock-key-structure (A-LKS), composed of Apt capable of spontaneous amplification and its complementary ssDNA (cDNA), was designed. Based on the identification of AFB1 in A-LKS, an A-LKS-mediated-SDA-Cas12a signal cascade (ASCC) biosensor was developed for ultrasensitive AFB1 detection. In the absence of AFB1, the Apt initiates amplification using DNA hanging from the 5' end of cDNA as a template, thereby enhancing the stability of A-LKS and reducing nonspecific amplification. When AFB1 is present, Apt binds to it, initiating the SDA reaction and activating Cas12a to generate strong fluorescence signals. The proposed biosensor demonstrates excellent selectivity and high sensitivity, with a low LOD of 3.6 pg/mL and a linear range of 0.01-100 ng/mL. This biosensor was successfully applied in real samples with satisfactory recoveries (88.69-105.48%), indicating its potential application in real samples.