Antigen-binding-enhanced divalent nanobody-based immunomagnetic separation coupled with ATP bioluminescence for...

Current Salmonella detection methods are time-consuming or incapable of effectively discriminating viable bacteria from dead cells, restricting their practical applications in food safety monitoring. Thus, in this study, an antigen-binding-enhanced Salmonella divalent nanobody (diVHH) was optimally expressed and chemically conjugated with magnetic beads (MBs). Combined with ATP bioluminescence, a novel strategy was developed for the rapid detection of viable Salmonella. Optimization of prokaryotic expression significantly improved the diVHH yield, with an approximately 2.96-fold enhancement. Then, the diVHH-functionalized MBs achieved capture efficiencies exceeding 90% for multiple tested Salmonella serotypes. By integrating immunomagnetic separation with ATP bioluminescence, the established assay exhibited a linear detection range from 10² to 10⁶ CFU/mL, with a limit of detection of 10 CFU/mL for S. Enteritidis and 12 CFU/mL for S. Typhimurium. The entire detection procedure could be completed within 2 h. In spiked food samples, the method achieved favorable recovery rates of 96.00-106.89% with low coefficients of variation (0.78-7.54%). Notably, this method could be successfully applied to the semi-quantitative analysis of multiple Salmonella serotypes in environmental and food samples, and the obtained results were highly consistent with plate counting results. This study demonstrates that engineered diVHHs exhibit enhanced antigen‑binding capacity and can serve as excellent recognition reagents for selective Salmonella detection to enable a reliable and rapid assay for viable Salmonella with promising application potential.