An ultrasensitive electrochemical immunosensor constructed via Ag-GO-Nf nanocomposites for the detection of pefloxacin.

Pefloxacin (PEF), a third-generation quinolone antimicrobial drug, is widely used in fisheries and animal husbandry. However, overuse poses food safety hazards and serious threats to human health. In this study, a label-free electrochemical immunosensor based on Ag-GO-Nf nanocomposites was developed for the rapid detection of PEF. Although electrochemical immunoassays are well-established for quinolone detection, no specific method has been reported for PEF. We synthesized silver nanoparticle/graphene oxide/nafion nanocomposites (Ag-GO-Nf) via in situ reduction, where the synergistic effect of GO (high conductivity) and AgNPs (redox-active) significantly enhanced electron transfer efficiency. The nanocomposites were characterized by UV-Vis, XRD, SEM, and TEM, while electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to monitor the stepwise immunosensor assembly. Key experimental parameters were optimized systematically. The developed sensor exhibited a wide linear range (0.50-100 ng/mL), an ultra-low limit of detection (0.27 ng/mL, S/N = 3), and high specificity toward structural analogues. Practical assays in spiked pork and milk samples yielded recoveries of 97.0-100.4%, while the current signal retained 90.77% of its initial value after 28 days, demonstrating excellent long-term stability. This work provides a robust technical strategy for rapid, sensitive, and efficient PEF detection.