Fluorescent and colorimetric detection of Norfloxacin with a bifunctional ligand and enzymatic signal amplification system

Abstract

We report a dual signal read-out enzyme-linked immunosorbent assay (ELISA) strategy for the sensitive detection of Norfloxacin (Nor) using a bifunctional ligand and enzymatic signal amplification system. In the proposed method, the immune response of an anti-Nor antibody against Nor-Biotin bifunctional ligands triggers the immobilization of streptavidin–alkaline phosphatase and the alkaline-phosphatase could continuous triggers the hydrolysis of L-ascorbic acid-phosphoric acid to produce abundant L-ascorbic acid (AA). Colorimetric signal is generated from the aggregation of azide and alkyne-based colloidal gold generated by a click-chemistry reaction with Cu+ catalysis (reduced by the AA), which changes its color from red to blue. A fluorescence signal is generated by the opening of the spirolactam ring caused by unreacted Cu2+ binding to the Cu2+ probe. Our method was successfully illustrated by detecting Nor in the range of3.18 × 10−2 to 6.88 × 103 pg/mL.The efficient detection of Nor was further confirmed using Nor added to commercially available milk, with a recovery in the range of 98.65%–104.01%. This work provides a promising method to detect Nor and other veterinary drug residues to guarantee food safety.