Development of a Novel Probe-Based Dual-Detection platform System for Flexible Detection of Staphylococcus aureus across Transmission Pathways

Immunodetection plays a crucial role in the prevention and treatment of Staphylococcus aureus infections. However, existing methods often suffer from limited performance, poor quantification, and applicability due to the use of low-sensitivity, nonspecific, and nontargeted probes. To address these challenges, we identified high-affinity, specific phage-displayed nanobody probes and established a dual-detection platform that enabled accurate and flexible screening of S. aureus across diverse settings. High-affinity phage-displayed nanobodies targeting S. aureus were selected to replace the conventional probes and were randomly paired with five monoclonal antibodies to demonstrate their universality. To further enhance detection sensitivity, we synthesized and optimized a core–shell magnetic–fluorescent nanolabel (Fe3O4@PS@TQD), which outperformed traditional colorimetric and fluorescent tags. By integrating these advanced components, we established a dual-detection platform capable of qualitative detection within 10 min and quantitative analysis within 30 min, achieving superior sensitivity and applicability compared with recently reported methods and commercial kits. The system exhibited excellent specificity, reproducibility, accuracy, and stability, with a detection limit as low as 5 CFU/mL after 4 h of pre-enrichment. In summary, this study presents a novel probe-driven dual-detection platform that meets the urgent need for rapid S. aureus on-site detection in complex environments.