Synergistic Inhibition of Nonspecific Binding for Accurate Detection of <i>Escherichia coli</i> O157:H7 and Multilevel Signal Discrimination

Pathogenic <i>Escherichia coli</i> (<i>E. coli</i>), particularly <i>E. coli</i> O157:H7, is a major foodborne pathogen with significant clinical relevance, necessitating accurate and rapid subtype identification. However, the high genetic variability and biological similarity among <i>E. coli</i> strains pose challenges for conventional signal-strain detection methods, often resulting in false-positive outcomes. In this study, we developed a novel biosensing strategy based on plasmonic nanostructures functionalized with heterogeneous recognition elements that target two distinct epitopes of <i>E. coli</i> O157:H7. The sensor incorporates biological silent Raman tags for ratiometric signal output and magnetic enrichment to improve selectivity and minimize interference from nontarget bacteria. This design ensures excellent reproducibility and operational stability. The biosensor demonstrated an impressive limit of detection (LOD) of 1.2 CFU/mL, outperforming most existing methods. Furthermore, a cutoff value of 0.32 for the signal ratio yielded a positive predictive value of 98% and a negative predictive value of 94%, demonstrating a clear signal boundary and high accuracy for various types of signals. These results highlight the potential of our plasmonic biosensor as a rapid, ultrasensitive, and reliable point-of-care diagnostic tool for pathogen detection in complex food matrices.