Anti-Idiotypic Nanobody Alkaline Phosphatase Fusion Protein-Triggered On-Off-On Fluorescence Immunosensor for Aflatoxin in Cereals

文献类型： 外文期刊

第一作者： Yu, Gege

作者： Yu, Gege;Wang, Jiamin;Zhang, Yao;Wu, Haofen;Wang, Yueqi;Cui, Yan;Yang, Yuefan;Wang, Jianlong;Wang, Yanru;Tang, Xiaoqian;Zhang, Qi;Li, Peiwu;Sun, Jing;Chen, Ran

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关键词： Aflatoxin; Anti-idiotypic Nanobody; Alkalinephosphatase; Fluorescence immunosensor; Cereals

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.1； 五年影响因子：6.3 ）

ISSN： 0021-8561

年卷期： 2023 年 71 卷 45 期

页码：

收录情况： SCI

摘要： Nanobodies (Nbs) are widely used in immunoassays with the advantages of small size and high stability. Here, the nanobody employed as the surrogate of aflatoxin antigen and the recognition mechanism of antiaflatoxin mAb with nanobody was studied by molecular modeling, which verified the feasibility of Nbs as antigen substitutes. On this basis, a nanobody-alkaline phosphatase fusion protein (Nb-AP) was constructed, and a highly sensitive "on-off-on" fluorescent immunosensor (OFO-FL immunosensor) based on the calcein/Ce3+ system was developed for aflatoxin quantification. Briefly, calcein serves as a signal transducer, and its fluorescence can be quenched after it is bound with Ce3+. In the presence of Nb-AP, AP catalyzed p-nitrophenyl phosphate to generate orthophosphate, which competes in binding with Ce3+, leading to fluorescence recovery. The method has a linearity range of 0.005-100 ng/mL, and the IC50 of the OFO-FL immunosensor was 0.063 ng/mL, which was 18-fold lower than that of conventional enzyme-linked immunosorbent assay. The assay was successfully applied in food samples with a recovery of 88-121%.

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