A self-assembled DNA double-crossover-based fluorescent aptasensor for highly sensitivity and selectivity in the simultaneous detection of aflatoxin M1 and aflatoxin B1

Realizing the simultaneous speedy detection of multiple mycotoxins in contaminated food and feed is of great practical importance in the domain of food manufacturing and security. Herein, a fluorescent aptamer sensor based on self-assembled DNA double-crossover was developed and used for effective simultaneous quantitative detection of aflatoxins M1 and B1 by fluorescence resonance energy transfer (FRET). Fluorescent dye-modified aflatoxin M1 and B1 aptamers are selected as recognition elements and signal probes, and DNA double crosses are consistently locked by the aflatoxin aptamers, which results in a "turn-off" of the fluorescent signal. In the presence of AFM1 and AFB1, the aptamer sequences are more inclined to form Apt-AFM1 and Apt-AFB1 complexes, and the fluorescent probes are released from the DNA double-crossing platform, leading to an enhanced fluorescent signal (Cy3: 568 nm; Cy5: 660 nm). Under the optimal conditions, the signal response of the constructed fluorescent aptamer sensor showed good linearity with the logarithm of AFM1 and AFB1 concentrations, with detection limits of 6.24 pg/mL and 9.0 pg/mL, and a wide linear range of 0.01-200 ng/mL and 0.01-150 ng/mL, respectively. In addition, the effect of potential interfering substances in real samples was analyzed, and the aptasensor presented a good interference immunity. Moreover, by modifying and designing aptamer probes, the sensor can be applied to high-throughput simultaneous screening of other analytes, providing a new approach for the development of fluorescent aptamer sensors.