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 H₂O₂. The Fenton reaction between H₂O₂ and Fe²⁺ 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）触发的Fenton反应的比色和荧光双模式ELISA试剂盒，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）形成了免疫复合物mAb-Bio-SA-Bio-GOx。在没有DAN的情况下，可通过与包被的DAN-BSA和催化的葡萄糖结合产生H ₂ O ₂来固定mAb-Bio-SA-Bio-GOx 。h的芬顿反应₂ ö ₂和Fe ²⁺产生的羟基自由基，其氧化的Ò-苯二胺成2，3-二氨基吩嗪。建立了以比色法和荧光作为信号读数的双信号免疫测定法。在存在DAN的情况下，DAN和DAN-BSA与Bio-mAb竞争，从而降低了免疫复合物与DAN-BSA之间的联系，最终导致色度和荧光信号降低。在最佳条件下，荧光免疫分析的检出限为0.337 ng / mL，比传统ELISA的检测限低5.24倍。牛奶中比色免疫分析的临界值为30 ng / mL。该方法对加标了不同浓度DAN的牛奶样品的平均回收率为91.1至128.3％，变异系数为0.7至8.2％。该方法的这些结果与液相色谱-串联质谱系统（LC-MS / MS）的方法具有很好的一致性。简而言之，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筛选策略，具有较高的灵敏度和准确性。