A novel colorimetric aptasensor for ultrasensitive detection of aflatoxin M based on the combination of CRISPR-Cas12a, rolling circle amplification and catalytic activity of gold nanoparticles.

Colorimetric approaches have received noticeable attention among sensing methods in view of simplicity and watching the color change of sample by the naked eyes. However, developing colorimetric sensing methods which show high sensitivity is still problematic. Herein, based on CRISPR-Cas12a, rolling circle amplification (RCA) and catalytic activity of gold nanoparticles (AuNPs), a colorimetric aptasensor was introduced for highly sensitive detection of aflatoxin M (AFM). In the presence of AFM, the CRISPR-Cas12a is inactivated and large single-stranded DNA (ssDNA) structures are formed on the surface of AuNPs following the addition of T4 DNA ligase and phi29 DNA polymerase. So, the sample color remains yellow after addition of 4-nitrophenol. However, no huge DNA structure is observed on the surface of AuNPs in the absence of target because of activation of CRISPR-Cas12a and digestion of primer. So, the color of sample switches to colorless. The results indicated that the biosensor had high selectivity toward AFM and the approach achieved a detection limit as low as 0.05 ng/L. In addition, it could sensitively identify AFM in the spiked milk samples. Overall, this approach is highly sensitive and does not require sophisticated equipment. Therefore, it maintains promising potential for other mycotoxins detection in real samples by simply replacing the applied sequences.