An Enhanced Ratiometric Fluorescence Immunosensor for Rapid Detection of AFB 1 Based on the Dual-Enzyme Synergistic Co-Catalytic Effect of Natural Enzymes and Nanozymes

作者：Yinnan Jing, Jiamin Wang, Xuan Liu, Shurong Li, Qinkai Yang, Mengnan Wang, Xueyi Fan, Guangyuan Ge, Kai Zheng, Jianlong Wang, Xiaoqian Tang, Qi Zhang, Yanru Wang · 发表于：Journal of Agricultural and Food Chemistry · 年份：2025 · DOI：10.1021/acs.jafc.5c01127 · 被引用次数：15 · 研究领域：Advanced Nanomaterials in Catalysis、Advanced biosensing and bioanalysis techniques、Biosensors and Analytical Detection

Aflatoxin B 1 (AFB 1 ) 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 AFB 1 . 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 AFB 1 . The assay could detect aflatoxins within 140 min, exhibiting a half maximal inhibitory concentration (IC 50 ) 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.