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.