Sensitive label-free bacterial detection and rapid antibiotic susceptibility testing via gold nanolayer coating on bacterial surfaces in multi-well SERS plates

Rapid and accurate bacterial identification, along with antibiotic susceptibility testing (AST), is critical in clinical and public health settings, particularly for preventing severe complications like sepsis. In this study, we introduce a multi-well Surface-Enhanced Raman Spectroscopy (SERS) platform combined with a gold nanolayer coating strategy on the bacterial surface for sensitive, label-free detection of uropathogenic bacteria. A simple electroless chemical Au plating technique was used to fabricate plasmonic multi-well SERS plates functionalized with high-density, sponge-like gold nanostructures. Furthermore, an additional modified in situ electroless chemical thin Au plating process was applied directly to bacterial surfaces in the multi-well SERS plates, enabling efficient trapping and enhanced SERS detection of bacterial macromolecules. This approach significantly amplified the Raman signals of bacteria within 30 min of the gold nanolayer coating reaction, enabling the identification of bacterial species at concentrations as low as 10 3 –10 4 CFU/mL without the need for prior culturing. Chemometric analysis of SERS spectra demonstrated 100% accuracy in differentiating five clinically relevant uropathogenic bacterial species at the low detectable concentrations. Additionally, the platform facilitated rapid AST within 1–2 h of minimal culturing. The proposed platform demonstrated high sensitivity and specificity, with detection limits comparable to clinical diagnostic thresholds and rapid susceptibility assessment, showing its potential for rapid and direct bacterial diagnostics in urinary tract infections (UTIs) and other infectious diseases. This innovative approach represents a significant advancement in microbial diagnostics, enabling point-of-care testing for bacterial infections, improving patient care, and reducing the spread of antibiotic resistance.