State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China.
J Dairy Sci. 2020 Sep;103(9):7826-7833. doi: 10.3168/jds.2020-18256. Epub 2020 Jun 26.
In this study, a novel colorimetric and fluorescent dual-mode ELISA based on glucose oxidase (GOx)-triggered Fenton reaction was developed for the qualitative and quantitative detection of danofloxacin (DAN). In this system, streptavidin-linked biotinylated anti-DAN-monoclonal antibody (SA-Bio-mAb) and biotinylated GOx (Bio-GOx) form the immune complex mAb-Bio-SA-Bio-GOx. In the absence of DAN, the mAb-Bio-SA-Bio-GOx would be immobilized by combining with coated DAN-BSA and catalyzed glucose to generate HO. The Fenton reaction between HO and Fe generated hydroxyl radicals, which oxidized the o-phenylenediamine to 2,3-diamino-phenazine. A dual-signal immunoassay with colorimetry and fluorescence as the signal readout was established. In the presence of DAN, DAN and DAN-BSA competed with Bio-mAb, decreasing the connection between immune complexes and DAN-BSA and finally resulting in lower signal of colorimetry and fluorescence. Under optimal conditions, the limit of detection of the fluorescence immunoassay was 0.337 ng/mL and was 5.24-fold lower than that of traditional ELISA. The colorimetric immunoassay cut-off value was 30 ng/mL in milk. The average recoveries of the method for milk samples that are spiked with different concentrations of DAN were 91.1 to 128.3%, with a coefficient of variation of 0.7 to 8.2%. These results of the method exhibited good agreement with those of liquid chromatography-tandem mass spectrometry system (LC-MS/MS) method. In brief, this work provides an improved screening strategy with high sensitivity and accuracy for the qualitative or quantitative detection of DAN in milk monitoring.
在这项研究中，开发了一种基于葡萄糖氧化酶（GOx）触发芬顿反应的新型比色和荧光双模式 ELISA，用于定性和定量检测达氟沙星（DAN）。在该系统中，链霉亲和素连接的生物素化抗 DAN-单克隆抗体（SA-Bio-mAb）和生物素化 GOx（Bio-GOx）形成免疫复合物 mAb-Bio-SA-Bio-GOx。在没有 DAN 的情况下，mAb-Bio-SA-Bio-GOx 会通过与包被的 DAN-BSA 结合而被固定，并催化葡萄糖生成 HO。HO 和 Fe 之间的芬顿反应生成羟基自由基，将邻苯二胺氧化为 2,3-二氨基吩嗪。建立了以比色法和荧光法作为信号读出的双信号免疫分析。在 DAN 存在的情况下，DAN 和 DAN-BSA 与 Bio-mAb 竞争，减少免疫复合物与 DAN-BSA 的连接，最终导致比色法和荧光法的信号降低。在最佳条件下，荧光免疫分析的检测限为 0.337 ng/mL，比传统 ELISA 低 5.24 倍。牛奶中比色免疫分析的截断值为 30 ng/mL。该方法用于牛奶样品中不同浓度 DAN 加标回收的平均回收率为 91.1%至 128.3%，变异系数为 0.7%至 8.2%。该方法的结果与液相色谱-串联质谱系统（LC-MS/MS）方法的结果具有良好的一致性。总之，这项工作为牛奶监测中 DAN 的定性或定量检测提供了一种改进的高灵敏度和准确性的筛选策略。
