Rapid On-Site Detection of Clarithromycin in Water Using an Evanescent Wave Fluorescence Immunosensor

Clarithromycin (CLA), a widely used macrolide antibiotic and contaminant of emerging concern (CEC), is frequently detected in aquatic environments. Its residues can induce bacterial antibiotic resistance, threatening public health and ecosystems. In this study, we constructed an evanescent wave fluorescence immunoassay for the rapid detection of clarithromycin. It detects CLA in water quickly, sensitively, and in a portable format. The sensor combines evanescent wave fluorescence with an indirect competitive immunoassay. It uses a CLA–bovine serum albumin (BSA) conjugate on an optical fiber bioprobe, along with a Cy5.5-labeled anti-clarithromycin monoclonal antibody (Ka = 7.40 × 109 L/mol) as the signal reporter. After optimization, the assay gave a linear range of 7.50–77.45 μg/L. The LOD was 1.67 μg/L, and the LOQ was 7.50 μg/L. The functionalized fiber bioprobe also held up well in repeated use. It stayed stable for over 200 regeneration cycles, with an RSD of 5.63%. The entire automated detection workflow was completed within 20 min without manual intervention. Real-sample validation was performed using tap water and river water spiked at 10, 30, and 60 μg/L, yielding recoveries ranging from 87.13% to 104.84%, with relative standard deviations below 13.08%. These results were corroborated by ELISA. These attributes, combined with the instrument’s portability and the probe’s regenerability, render this biosensor a promising analytical platform for on-site environmental monitoring of clarithromycin residues.