Inner filter effect-based fluorescence immunoassay using nanobody-alkaline phosphatase fusion and gold nanoclusters for detecting ochratoxin A in pepper

As one of the main mycotoxins in food, ochratoxin A (OTA) poses a serious threat to food safety and human health. Herein, a sensitive inner filter effect-based fluorescence immunoassay (IFE-FLIA) using nanobody-alkaline phosphatase fusion (Nb-ALP) and gold nanoclusters (AuNCs) was developed for the detection of OTA in pepper. Because of the IFE of p-nitrophenyl phosphate (pNPP) against the fluorescence of AuNCs and the hydrolysis of pNPP catalyzed by ALP, a fluorescence sensing platform for “turn-on” detection of Nb-ALP was first constructed with pNPP, AuNCs, and Nb-ALP. Based on the good anti-OTA activity and enzyme activity of Nb-ALP, the integration of the fluorescence sensing platform for Nb-ALP with the Nb-ALP-mediated direct competitive enzyme immunoassay for OTA implements the construction of the IFE-FLIA. Under the optimal experimental conditions, the IFE-FLIA can be finished in 85 min and has a half maximal inhibition concentration (IC50) of 0.22 ng/mL with a limit of detection of 0.018 μg/kg and a linear range (IC20-IC80) of 0.11–0.53 ng/mL for OTA. The recovery study of the spiked pepper samples and the validation of high performance liquid chromatography demonstrated the good accuracy and precision of the developed method. In conclusion, the proposed IFE-FLIA was demonstrated to be a reliable tool for the analysis of trace OTA in pepper.