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<p>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&#xa0;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&#xa0;min with a visual detection limit of 9.37&#xa0;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.</p> Graphical abstract <p></p>

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

  • 期刊论文

  • 发表日期：

    2026年3月26日

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  • Zhenxian Lin,
  • Jingbo Liu,
  • Hui Liu,
  • Jie Zhang,
  • Jianmei Zhang,
  • Guiliang Zheng

摘要

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.

Graphical abstract