Competitive immunoassay using enzyme-regulated Fe3O4@COF/Fe3+ fluorescence probe for natural chloramphenicol detection

Abstract

Accurate and sensitive detection of chloramphenicol (CAP) in natural samples is essential for ensuring human health. Herein, an enzyme-regulated fluorescence sensor using Fe3O4@COF/Fe3+ probe, is developed for CAP determination. Fe3O4@COF, synthesized via hydrothermal method, exhibits dual functions as a magnetic carrier and signal probe. Bovine serum albumin conjugated-chloramphenicol, adsorbed on the surface of Fe3O4@COF, competes with CAP for antibody binding. The antibody interacts with alkaline phosphatase via the biotin-streptavidin system. Meanwhile, ascorbic acid, produced from the enzyme-catalyzed reaction dominated by alkaline phosphatase, effectively restores the fluorescence of Fe3O4@COF that is quenched by Fe3+. After experimental verification and gradual optimization, a logarithmic linear relationship between CAP concentration and fluorescence intensity is established in the range of 2 × 10−4∼10 μg mL−1, with a good limit of detection (9.2 × 10−5 μg mL−1). Proposed method exhibits excellent stability (15 days) and reusability (8 cycles), providing a sensitive and reliable method for accurate CAP detection. The readouts show good agreement with HPLC and recoveries during laboratory and natural CAP analysis.