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

Accurate and sensitive detection of chloramphenicol (CAP) in natural samples is essential for ensuring human health. Herein, an enzyme-regulated fluorescence sensor using Fe 3 O 4 @COF/Fe 3+ probe, is developed for CAP determination. Fe 3 O 4 @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 Fe 3 O 4 @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 Fe 3 O 4 @COF that is quenched by Fe 3+ . 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.