Sensitive Microscale Thermophoresis Assay for Rapid Ochratoxin A Detection with Fluorescently Labeled Engineered Aptamer

Ochratoxin A (OTA) is a widespread mycotoxin that causes contamination in a variety of foodstuffs and environments, inducing great health risks to humans and animals. Rapid and sensitive detection of OTA is necessary for food safety, environmental health, and risk assessment. Herein, we report an aptamer microscale thermophoresis (MST) assay for OTA, with the unique merits of ratiometric analysis, rapid measurement, simple operation, high sensitivity, low sample consumption, and high throughput. A fluorescein (FAM)-labeled high-affinity DNA aptamer with a G-quadruplex and duplex structure was used as the recognition element for OTA, and MST, which measures the fluorescence responses of the sample solution inside capillaries to a mild temperature increase generated by infrared laser heating, was employed for signal generation. Upon OTA binding, the FAM-labeled aptamer probe underwent changes in conformation and stability, and the bound and unbound aptamer probes showed significant differences in their MST signals. To achieve sensitive detection of OTA with a large signal change, we systematically characterized aptamers with different stem lengths, which had large effects on the MST responses of the aptamer probes to OTA. We found that a 32-mer aptamer with FAM label at the 3' end gave a sensitive MST response to OTA, allowing OTA detection within seconds with a detection limit of 0.98 nM under optimal experimental conditions. This aptamer MST assay shows potential in real sample analysis and broad applications.