In silico simulation-guided engineering of multifunctional bivalent light up aptamer for sensitive and on-site detection of AFB1 using label-free ratiometric fluorescent aptasensor

Aflatoxin B1 (AFB1) is considered the most toxic mycotoxin and particularly prevalent in cereals, posing great threat to human health. Therefore, rapid, sensitive and easy-to-use strategies for on-site screening of AFB1 are urgently needed. Here, we established a in silico simulation-guided engineering (ISSGE) strategy for rational and systematic optimization of an AFB1 light up aptamer, further facilitated the construction of a novel label-free ratiometric fluorescent aptasensor. Guided by the in silico simulation, the original 80 nt AFB1 aptamer was first truncated to obtain a 13 nt minimum active structure, and identified a 9 nt critical binding domain in the top stem-loop structure, with hydrogen bond and charge force for target interaction. Subsequently, a multifunctional bivalent aptamer (BiApt) with increased binding affinity, improved fluorescence enhancement ability and methanol tolerance was obtained. Benefiting from the above optimization, a novel label-free ratiometric fluorescent aptasensor was designed based on the competitive binding between AFB1 and Thioflavin T in the BiApt. Through this approach, the detection of AFB1 was achieved within 3 min, with a low LOD of 0.036 ng·mL−1 and a broad linear range of 0.1–800 ng·mL−1. The proposed ISSGE strategy offers a universal and systematic strategy for aptamer engineering, significantly promoting their practical applications. The developed ratiometric fluorescent aptasensor is promising in realizing large-scale and on-site screening of AFB1 in cereal products.