Enzyme cascade-amplified immunoassay based on the nanobody–alkaline phosphatase fusion and MnO₂ nanosheets for the detection of ochratoxin A in coffee

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Ochratoxin A (OTA) is a common food contaminant with multiple toxicities and thus rapid and accurate detection of OTA is indispensable to minimize the threat of OTA to public health. Herein a novel enzyme cascade-amplified immunoassay (ECAIA) based on the mutated nanobody-alkaline phosphatase fusion (mNb-AP) and MnO₂ nanosheets was established for detecting OTA in coffee. The detection principle is that the dual functional mNb-AP could specifically recognize OTA and dephosphorylate the ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and the MnO₂ nanosheets mimicking the oxidase could be reduced by AA into Mn²⁺ and catalyze the 3,3',5,5'-tetramethyl benzidine into blue oxidized product for quantification. Using the optimal conditions, the ECAIA could be finished within 132.5 min and shows a limit of detection of 3.38 ng mL⁻¹ (IC₁₀) with an IC₅₀ of 7.65 ng mL⁻¹ and a linear range (IC₂₀-IC₈₀) of 4.55-12.85 ng mL⁻¹. The ECAIA is highly selective for OTA. Good recovery rates (84.3-113%) with a relative standard deviation of 1.3-3% were obtained and confirmed by high performance liquid chromatography with a fluorescence detector. The developed ECAIA was demonstrated to be a useful tool for the detection of OTA in coffee which provides a reference for the analysis of other toxic small molecules.