Multiple Isothermal Amplification Coupled with CRISPR–Cas14a for the Naked-eye and Colorimetric Detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is highly toxic and challenging to remove, posing significant risks to both human health and economic development. Therefore, there is an urgent need to develop rapid, simple, and sensitive detection technologies. In this study, we introduce a naked-eye and colorimetric method based on multiple isothermal amplifications coupled with CRISPR–Cas14a and investigate its biosensing properties. This technique utilizes composite nanoprobes (MAPs) comprising magnetic nanoparticles and gold nanoparticles. AFB1 is efficiently identified through an aptamer competition process facilitated by magnetic nanoparticles, which triggers multiple isothermal amplification. This converts trace amounts of the toxin into a large quantity of DNA signal. Upon specific activation of the CRISPR–Cas14a complex, the MAPs are cleaved, resulting in significant changes in both color and colorimetric signal. The method demonstrates acceptable sensitivity, with a detection limit of 31.90 pg mL –1 and a wide detection range from 0.05 to 10 ng mL –1 . Furthermore, the assay exhibits satisfactory specificity and high accuracy when it is applied to practical samples. Our approach offers a universal sensing platform with potential applications in food safety, environmental monitoring, and clinical diagnostics.