Glucose oxidase-induced colorimetric immunoassay for qualitative detection of danofloxacin based on iron (Ⅱ) chelation reaction with phenanthroline

•Colorimetric ELISA could screen DAN with low LOD (2.5 ng mL−1) without instrument.•There was no matrix effect with four kinds of samples using the novel method.•Sharp color change decreased judgement mistakes by individual naked-eye observation. In this study, we developed a competitive colorimetric immunoassay for qualitative detection of DAN based on oxidation of iron (Ⅱ) (Fe2+) in the presence of glucose oxidase (GOx) and color change induced by Fe2+-phenanthroline (Phen) chromogenic system. Streptavidin (SA) acted as a linker between biotinylated anti-DAN-monoantibody (bio-mAb) and biotinylated GOx (bio-GOx) to form the immunocomplexes bio-mAb-SA-bio-GOx. In the absence of DAN, the immunocomplexes bio-mAb-SA-bio-GOx combining with coated DAN-ovalbumin (DAN-OVA) will be immobilized and catalyze glucose to produce H2O2. Fe2+ is oxidized to Fe3+ by H2O2, giving rise to a colorless result. In the presence of DAN, Fe2+ produces a chelation reaction with Phen, leading to orange-red color. Under optimal conditions, the detection limit (LOD) by naked eyes was 2.5 ng mL−1 in milk, chicken, beef, and pork samples. Low LOD, no matrix effect, and no signal reader requirement make it possibly applied to quickly screen DAN on site.