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

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.