Investigation the Binding Mechanism of Aptamers to Ochratoxin A and Development of Competitive Colorimetric Sensing Platforms

Ochratoxin A (OTA) is widely present in various products such as grains, legumes and their products, coffee, wine, grape juice and dried fruits. It is highly carcinogenic and pathogenic and is classified as a Group 2B human carcinogen, posing a significant threat to human health. Therefore, the development of rapid, accurate and easy-to-operate new detection methods is particularly important. In this study, a long-chain aptamer (Apt) was truncated and optimized to obtain a short aptamer Apt-8 with significantly improved affinity and specificity. Further, microscale thermophoresis (MST), isothermal titration calorimetry (ITC), circular dichroism (CD), molecular dynamics simulation (MD) and molecular docking techniques were used to systematically analyze the binding affinity, heat changes during the binding process, conformational changes, binding mode, driving energy and key binding sites of Apt-8 and OTA, providing a solid structural basis for sensor design. In addition, a straightforward and efficient method for the detection of OTA has been developed by integrating terminal deoxynucleotidyl transferase (TdT) with aptamer-based colorimetry. The sensor showed a good linear relationship with the concentration of OTA, with a detection limit as low as 0.026 ng/mL and a spiked recovery rate of 98.33% to 106.3%, indicating high accuracy of the method. This detection method is simple to operate, rapid and efficient, with high sensitivity, strong stability and good repeatability, and is suitable for rapid visual detection of OTA, showing great potential in on-site point-of-care testing.