Triple-readout immunoassay based on copper ion trigger for the detection of ochratoxin A

Background: Ochratoxin A (OTA) is a widespread mycotoxin with nephrotoxic, neurotoxic and immunotoxic properties. It exhibits elevated thermal stability and a prolonged half-life and is resistant to removal. Long-term exposure to OTA increases the risk of carcinogenesis and influences human health. The detection methods of single signal mode had poor anti-interference ability and were prone to false positive results, while the detection method of multi-signal mode has higher adaptability to conditions and can offset environmental interference through self-correction. Therefore, proposing multi-signal and high-sensitivity strategies to detect OTA levels in various foods is all-important. Results: A triple-readout immunoassay based on copper ions trigger was developed for detecting Ochratoxin A. The ascorbic acid 2-phosphate (AAP) was hydrolyzed to ascorbic acid (AA) by alkaline phosphatase (ALP). The unreacted AAP rapidly coordinated with Cu2+ to produce green Cu2+-AAP complexes which emitted blue/green (B/G) signals. Meanwhile, Cu2+ oxidized AA to produce dehydroascorbic acid (DHAA) which reacted with ophenylenediamine (OPD) to form the blue-fluorescence quinoxaline derivative (DFQ). Simultaneously, Cu2+ oxidized the unreacted OPD to form yellow 2,3-diaminophenazine (DAP) which produced yellow signals (Y). The B/G and Y was read by a smartphone. The limits of detection (LOD) of B/G, fluorescence and Y value readout models were 0.13 ng/mL, 0.16 ng/mL and 0.15 ng/mL. Their linear ranges were 6.25-100 ng/mL, 6.25-200 ng/ mL and 3.13-12.5 ng/mL. Besides, all three readout models showed excellent specificity for OTA, and the recovery rates were satisfactory from 94.79 % to 112.68 %. Significance: A triple-readout immunoassay based on copper ions trigger for the detection of ochratoxin A was constructed. The materials for detection are accessible and economical. Triple signal modes not only complement and verify each other to improve the reliability of the results but also can be selected according to the detection scenes to increase the freedom of detection. It can be used to detect other targets by altering the coated antigen and its corresponding antibody.