Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.

Bloodstream infections are among the most common causes of hospitalizations and are often life-threatening. To establish an effective therapy for a patient, the timely identification of the causative agent and characterization of its antimicrobial susceptibility and resistance profiles are essential.

This pilot study investigated the metabolic responses of five selected bacteria to physiological stress. Surface-enhanced Raman spectroscopy was used to analyze spectral changes associated with the release of adenine, a key metabolite indicative of stress conditions. Laboratory-synthesized spherical silver and gold nanoparticles, which remained stable over an extended period, were employed as enhanced surfaces. Bacterial cultures were analyzed under standard conditions and in the presence of a selected stressor—demineralized water—inducing osmotic stress. The results showed that the adenine signal originated from metabolites released into the surrounding environment rather than directly from the bacterial cell wall. The study confirms the suitability of these cost-effective and easily synthesized stable nanoparticles for the qualitative detection of bacterial metabolites using a commercially available Raman instrument.

Rapid and accurate detection of bacterial physiological states, including viability and stress responses, is essential for both microbiology research and clinical diagnostics. Conventional culture-based methods are slow, while molecular approaches often fail to capture dynamic metabolic responses. In this study, we present a proof-of-concept strategy that employs surface-enhanced Raman scattering (SERS) to monitor metabolites released by Staphylococcus aureus and Escherichia coli subjected to controlled osmotic stress. Our results demonstrate that bacterial stress responses may be monitored by changes in purine metabolism, indicated by SERS detection of adenine and hypoxanthine at concentrations far below the detection limits of conventional Raman spectroscopy. The long-term monitoring of metabolite excretion highlights the robustness of this approach for assessing bacterial stress dynamics. Taken together, these findings establish the feasibility of near-real-time metabolic fingerprinting of bacterial activity, offering a pathway toward rapid viability assessment and potentially enabling antimicrobial sensitivity on clinically relevant timescales.