Label-Free G-Quadruplex Aptamer Fluorescence Assay for Ochratoxin A Using a Thioflavin T Probe

Ochratoxin A (OTA) is one of the most common mycotoxins contaminating feed and foodstuffs. Therefore, a great deal of concern is associated with AFB1 toxicity. In this work, a fast and sensitive fluorescence aptamer biosensor has been proposed for the OTA assay. In the absence of OTA, the OTA aptamer can form a G-quadruplex structure with thioflavin T (ThT) dye, which results in increased fluorescence. After joining OTA, OTA aptamer combines with OTA and the G-quadruplex can be formed. Only faint fluorescence was finally observed when ThT weakly reacts with the quadruplex. Through this test method, the entire reaction and analysis process of OTA can be completed in 10 min. Under optimal experimental conditions (600 nM OTA-APT, 7 μM ThT, and 3 min incubation time), this proposed assay has a good limit of detection (LOD) of 0.4 ng/mL and shows a good linear relationship within the range of 1.2⁻200 ng/mL under the best experimental conditions. This method has a high specificity for OTA relative to Ochratoxin B (23%) and Aflatoxin B₁ (13%). In addition, the quantitative determination of this method in real samples has been validated using a sample of red wine supplemented with a range of OTA concentrations (1.2 ng/mL, 12 ng/mL, and 40 ng/mL) with recoveries of 96.5% to 107%.