Alkaline phosphatase triggered ratiometric fluorescence immunoassay for detection of zearalenone

Inspired by the super-binding capacity of pyrophosphate (ppi) and copper ion (Cu2+), an alkaline phosphatase (ALP) triggered ratiometric fluorescence immunoassay was established for detection of zearalenone (ZEN). In the assay, ALP-antibody acted as a bridge connecting ZEN concentration and ratiometric fluorescence signals. Due to the super-binding ability of ppi and Cu2+, Cu2+ was immobilized to form ppi-Cu2+ complex and lost its oxidation activity. The presence of ALP can hydrolyze ppi and release Cu2+ which can oxidize OPD to DAP. Since the excitation wavelength of DAP coincided with the emission wavelength of 2-aminoterephthalic acid (PTA-NH2), a ratiometric fluorescence immunoassay can be developed. The limit of detection (LOD) was 0.014 ng/mL and the linear detection range was 3.51–17.78 ng/mL. The ratiometric fluorescence immunoassay had high accuracy and reliability (recoveries, 101.10–107.20%; coefficient of variation (CV), 2.15–7.65%). This method provided new ideas for efficient and accurate detection of ZEN.