Dual-mode immunoassay system based on glucose oxidase-triggered Fenton reaction for qualitative and quantitative detection of danofloxacin in milk

Abstract

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.