Dual-competition mediated ratiometric fluorescent immunoassay for detection of ochratoxin A

Ochratoxin A (OTA) presents a significant threat to global food safety, necessitating the development of sensitive and accurate detection. Herein, a novel dual-competition mediated ratiometric fluorescent immunoassay was developed for OTA detection. The initial immune-competitive reaction was operated through coated antigen (OTA-bovine serum albumin, OTA-BSA), OTA with monoclonal antibody (McAb). Subsequently, ascorbic acid 2-phosphate was dephosphorylated to ascorbic acid by alkaline phosphatase-conjugated second antibody (ALP-IgG) combined with McAb, which was further oxidized by cerium ions (Ce⁴⁺) to dehydroascorbic acid (DHAA). Under the second competition reaction between DHAA and Ce⁴⁺ with O-phenylenediamine, the generated quinoxaline derivative and 2,3-diaminophenazine produced ratiometric fluorescence to achieve accurate detection. The immunoassay showed the limit of detection of 1.35 ng/g and signal response time of 40 min. The corn spike experiments revealed the recovery rates of 91.65 %-105.44 %. In conclusion, the proposed immunoassay highlights a reliable and innovative solution for OTA detection, which has great application potential.