Structural insights into the AFB aptamer coupled with a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB detection.

Aflatoxin B (AFB) is a typical food contaminant. A truncated DNA aptamer of AFB was reported by our team in previous work. However, the recognition mechanism between aptamer and AFB was lacking, which was crucial for the design of related aptasensor. Herein, the binding of aptamer to AFB was systematically studied and found that it was an exothermic process and the conformation of aptamer changed during the recognition process. Loop bases in the secondary structure of aptamer formed a special binding pocket to recognize AFB. Van der Waals and electrostatic interaction were the main driving forces. By blocking the stem bases guided by the structural investigation, a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB detection was constructed, and the sensitivity was improved by target recycling. Under optimal conditions, the linear detection range for AFB was 0.01-20 ng/mL, and AFB was accurately determined in corn and wheat samples. This work laid a theoretical foundation for the design of AFB aptasensor, and the developed detection model came up with new ideas for the development of CRISPR/Cas12a-based aptasensor.