Sensitive detection of aflatoxin B1 using a dual-mode fluorescent aptasensor based on cascade signal amplification

Aflatoxin B1 (AFB1) is considered a potent carcinogenic mycotoxin and is commonly found in cereals and food products; therefore, its ultrasensitive detection is of great importance to public health. In this study, we developed an innovative dual-mode fluorescent aptasensor based on the entropy-driven strand displacement reaction (ESDR), rolling circle amplification (RCA), and DNAzymes for cascade signal amplification. In this aptasensor, AFB1 competes with aptamer AF29 for binding, releasing the previously bound antisense strands, thereby converting AFB1 abundance into a nucleic acid signal. The released antisense oligonucleotides trigger the subsequent ESDR, liberating P1-G4 and P2-Dz, respectively. P1-G4 contains a G-quadruplex sequence, and after the RCA reaction, it converts the nucleic acid signal into a readable fluorescence signal through the specific binding between the G-quadruplex and thioflavin T. On the other hand, P2-Dz is an exceptional DNAzyme that can specifically cleave fluorophore/quencher-labeled DNA probes and release fluorophores, thereby significantly enhancing the fluorescence signal. Under optimized conditions, the method shows high sensitivity, with a detection limit of 2.9 pmol L^-1 for the RCA-G-quadruplex channel and 28.2 nmol L^-1 for the DNAzyme channel, which are better than those reported in most previous studies. Moreover, it exhibits satisfactory accuracy (recoveries, 92.96-108.91%; RSD, 1.80-9.11%). Importantly, the flexibly designed nucleic acid primer allows this analytical method to be adapted for monitoring various targets according to specific requirements. Thus, this system provides a novel approach for accurate and ultrasensitive detection of AFB1 and other analytes.