Hotspot-enhanced SERS Biosensor: Triplex noble nanoparticle-aptamer supramolecules on micropatterned silicon for simultaneous detection of foodborne pathogens.

Foodborne pathogens significantly impact public health and cause substantial economic losses globally, necessitating rapid, sensitive, and multiplex detection techniques. This study presents a novel surface-enhanced Raman spectroscopy (SERS) biosensor enabling highly sensitive and simultaneous detection of Salmonella enteritidis (S. enteritidis) and Pseudomonas aeruginosa (P. aeruginosa). The biosensor utilizes sculpted triangular silicon substrates (stSi) decorated uniformly with silver (Ag) and gold (Au) nanoparticles (stSi@Ag-Au), fabricated through electrochemical reduction and galvanic displacement methods, creating robust electromagnetic hotspots. Functional gold nanostars synthesized via a seed-mediated growth method were deposited onto stSi@Ag-Au, forming a Raman-active substrate, which was further modified with pathogen-specific aptamers and distinct Raman probes-5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for S. enteritidis and 4-(mercaptomethyl)benzonitrile (MMBN) for P. aeruginosa. These components assemble into supramolecular complexes capable of specifically recognizing target pathogens, generating intense, unique Raman signals in a concentration-dependent manner, enabling concurrent quantitative analysis of both pathogens. The developed method achieves rapid detection within 45 min, demonstrates a broad linear detection range (10°-10 CFU mL) with detection limits of 1.33 CFU mL (Salmonella enteritidis) and 1.12 CFU mL (Pseudomonas aeruginosa), and has been successfully validated in practical applications in diverse matrices, including drinking water, orange juice, and Dendrobium nobile Lindl. juice samples, underscoring its effectiveness for rapid food safety assessments. Overall, this supramolecular-based SERS biosensing strategy provides a highly sensitive, rapid, and reliable solution for simultaneous pathogen detection, significantly advancing capabilities in foodborne disease prevention and control.