Development of a highly sensitive electrochemical immunosensor and lateral flow immunoassay for enrofloxacin using...

Owing to the rapid expansion of the aquaculture industry, the veterinary antibiotic enrofloxacin (ENR) has become a widespread contaminant of concern in environmental and food safety fields. To enable effective monitoring and control of ENR residues, there is an urgent need to develop accurate and user-friendly immunoassays. The preparation of high-quality antibodies relies critically on rationally designed haptens. In this study, we optimized the hapten structure of ENR using computer-aided molecular modeling and the rigid spacer arm strategy. It was found that the antibody generated from the ENR-2 hapten, which features a benzene ring as the spacer, displayed superior affinity (titer 1:248,000) and specificity. Through systematic screening of coating antigens, ENR-3 was identified as the optimal heterologous antigen, leading to significantly improved assay performance. Based on this selected antibody-antigen pair, an electrochemical immunosensor was constructed by integrating gold nanocages (Au NCs) and manganese-zinc ferrite nanoparticles (Mn-Zn-Fe NPs), which achieved a broad detection range from 0.0003 to 300 ng/mL, outperforming most existing ENR immunosensors. In addition, a lateral flow immunoassay (LFIA) was developed for rapid on-site analysis, delivering results within 15 min with a visual detection limit of 9.37 ng/mL. This study not only enriches hapten design theory but also establishes a robust dual-format detection system for ENR, offering reliable tools for residue monitoring.