Air-liquid microfluidics-integrated surface-enhanced Raman spectroscopy for selective molecular adsorption and detection to achieve bacterial discrimination

Bacterial discrimination is crucial for accurate microbiological diagnosis and timely antibiotic treatment. Surface-enhanced Raman spectroscopy (SERS) is an ideal technique due to its non-invasive, label-free molecular sensing capabilities. By analyzing bacterial supernatants, containing various purine derivatives, SERS can differentiate bacterial species based on their unique spectral distributions. However, the same bacterial species with different antibiotic resistance may secrete similar purine derivatives, differing only slightly in composition. Furthermore, each purine derivative may have a different molecular affinity to the SERS substrate, making it difficult to distinguish the exact molecular ratio. To improve SERS-based bacterial discrimination, we propose an air-liquid microfluidics-integrated SERS system (ALM-SERS) that selectively adsorbs and detects bacterial secretions. By taking features of precise microdroplet manipulation and contact area from microfluidics and features of selective molecular adsorption and fingerprints characterization from the SERS technique, we successfully perform a "sequential molecular adsorption" strategy to address the signal interference of complicated molecular mixtures in existing SERS methods. As a proof of concept, we first evaluate the molecular affinity of purine derivatives and then demonstrate the competitive analyte adsorption using adenine/cytosine and hypoxanthine/uracil sample mixtures. Finally, we tested six bacterial supernatants, including two Gram types and four strains with identical taxonomy but differing antibiotic resistance. Another six clinically isolated bacterial samples with different antibiotic resistance were also applied. Our results showed that a successful bacterial discrimination between similar species was not possible using a single SERS spectrum. In summary, the ALM-SERS system offers a powerful approach for bacterial discrimination, even when spectral differences are subtle. Beyond microbiology, this technique holds potential for analyzing complex molecular mixtures in drug development, food safety, and environmental hazard detection.