An Enhanced RatiometricFluorescence Immunosensorfor Rapid Detection of AFB₁ Based on the Dual-Enzyme SynergisticCo-Catalytic Effect of Natural Enzymes and Nanozymes

Aflatoxin B₁ (AFB₁) is considered a critical analyte in food safety testing due to its widespread contamination and high toxicity. In this study, a ratiometric fluorescent immunosensor with synergistic catalysis by natural enzymes and nanozymes (N-N-RF-immunosensor) was constructed for the rapid detection of AFB₁. Copper oxide and zeolitic imidazolate framework composite nanomaterials (CuO/ZIF-8 NCM) with peroxidase and oxidase activities were prepared. A ratiometric fluorescence signal was generated through the synergistic cocatalytic effect of the CuO/ZIF-8 NCM and alkaline phosphatase, in response to the competition interaction between the anti-idiotypic nanobody-alkaline phosphatase fusion protein (Nb2-5-AP) and AFB₁. The assay could detect aflatoxins within 140 min, exhibiting a half maximal inhibitory concentration (IC₅₀) of 0.032 ng/mL, a limit of detection (LOD) of 2.4 pg/mL, and a linear range of 0.0024–1.043 ng/mL. Compared with the traditional enzyme-linked immunosorbent assay (ELISA), the sensitivity was enhanced 25-fold, while the detection parameter was shortened by 1/3. Furthermore, the immunosensor was successfully applied in the actual samples, with recoveries ranging from 81 to 127%. The N-N-RF-immunosensor shows great potential for rapid and sensitive detection of aflatoxins.