An automated microfluidic biosensor with bispecific rabbit monoclonal antibody and nanozyme-bioenzyme composite for dual antibiotic detection

Abstract

The detection of antibiotic residues in environmental water sources represents a critical ecological challenge with significant implications for public health. Conventional immunoassays for antibiotics often suffer from limitations in sensitivity and efficiency, primarily due to the lack of high-performance detection antibodies and the complexity of current operational procedures. In this study, we introduce a novel approach by bioengineering a florfenicol (FF) and thiamphenicol (THF) bispecific rabbit monoclonal antibody (rmAb) for the first time, which has been integrated into an automated microfluidic biosensor. This innovative biosensor employs an AuPtCu@HRP-hapten composite nanozyme-bioenzyme, demonstrating exceptional peroxidase-mimic catalytic activity. The automated immunosensor operates within a single microfluidic chip, enhancing both the simplicity and efficiency of the detection process. The rmAbs are non-destructively immobilized on protein A-functionalized agarose microspheres, serving as effective immunoreactive carriers. By introducing a smartphone sensing strategy, our biosensor achieved the detection of FF and THF residues in river water at remarkably low concentrations of 0.014 and 0.015 ng/mL within just 30 min, respectively. The method exhibits average recoveries between 96.58% and 104.50%, with standard deviations consistently below 5.34%. This sensing strategy not only significantly reduces detection time compared to traditional direct competitive immunoassays but also enhances sensitivity and accuracy. This user-friendly biosensor represents a promising advancement in antibiotic detection technology, making it well-suited for on-site applications and paving the way for future development of robust antibiotic sensing platforms.

Keywords

florfenicol / microfluidic chip / nanozyme-bioenzyme / rabbit monoclonal antibody / thiamphenicol

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2025 The Author(s). BMEMat published by John Wiley & Sons Australia, Ltd on behalf of Shandong University.