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 H 2 O 2 . The Fenton reaction between H 2 O 2 and Fe 2+ generated hydroxyl radicals, which oxidized the o-phenylenediamine to 2,3-diamino-phenazine. A dualsignal 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 cutoff 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.

Current Use of Fenton Reaction in Drugs and Food

“…Abbas et al developed a simple and highly sensitive fluorometric method based on the Fenton reaction system to assess H 2 O 2 in foods [ 110 ]. Additionally, Wang et al developed a novel colorimetric and fluorescent ELISA based on the Fenton reaction triggered by glucose oxidation was constructed to quantitatively and qualitatively measure danofloxacin in milk [ 111 ].…”