A highly sensitive and dual-readout immunoassay for norfloxacin in milk based on QDs-FM@ALP-SA and click chemistry

A dual-readout immunoassay based on QDs-FM@ALP-SA and click chemistry was developed for quick and sensitive detection of norfloxacin (NOR), which is an important fluoroquinolone antibiotic. In the system, the NOR-biotin conjugate (NOR-Biotin) was synthesized by click chemistry for signal transformation, and alkaline phosphatase-labeled streptavidin (ALP-SA) was attached to quantum dot fluorescence microspheres (QDs-FM) by an activated ester method to form QDs-FM@ALP-SA for signal amplification. Here, QDs-FM was a dual-functional carrier: it was used not only as a chemiluminescence signal amplification carrier but also as a fluorescent signal due to its fluorescence character. The NOR antibody was coated on a 96-well chemiluminescence microtiter plate, and NOR-Biotin was bound to the antibody specifically. Then, QDs-FM@ALP-SA was combined with NOR-Biotin to develop a direct competition chemiluminescence/fluorescence immunoassay (dc-CLIA/FIA). The IC₅₀ values were 0.345 and 1.206 ng/mL for dc-CLIA/FIA, respectively. The linear range was 0.013-12.48 ng/mL and 0.042-39.86 ng/mL, respectively. The recovery from the standard fortified blank milk samples was in the range of 86.44%-101.3%. Therefore, this method could be a useful tool for routine screening of NOR residues in milk.