Investigation of the Influence of Stress on Label-Free Bacterial Surface-Enhanced Raman Spectra

Label-free surface-enhanced Raman spectroscopy (SERS) has been proposed as a promising bacterial detection technique. However, the quality of the collected bacterial spectra can be affected by the time between sample acquisition and the SERS measurement. This study evaluated how storage stress stimuli influence the label-free SERS spectra of Pseudomonas syringae samples stored in phosphate buffered saline. The results indicate that when faced with nutrient limitations and changes in osmatic pressure, samples at room temperature (25 °C) exhibit more significant spectral changes than those stored at cold temperature (4 °C). At higher temperatures, bacterial communities secrete extracellular biomolecules that induce programmed cell death and result in increases in the supernatant SERS signals. Surviving cells consume cellular components to support their metabolism, thus leading to measurable declines in cell SERS intensity. Two-dimensional correlation spectroscopy analysis suggests that cellular component signatures decline sequentially in the following order: proteins, nucleic acids, and lipids. Extracellular nucleic acids, proteins, and carbohydrates are secreted in turn. After subtracting the SERS changes resulting from storage, we evaluated bacterial response to viral infection. P. syringae SERS profile changes enable accurate bacteriophage Phi6 quantification over the range of 10⁴-10¹⁰ PFU/mL. The results indicate that storage conditions impact bacterial label-free SERS signals and that such influences need to be accounted for and if possible avoided when detecting bacteria or evaluating bacterial response to stress stimuli.