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The surface-enhanced Raman spectroscopy (SERS)-based analysis of bacteria suffers from the lack of a standard SERS detection protocol (type of substrates, excitation frequencies, and sampling methodologies) that could be employed throughout laboratories to produce repeatable and valuable spectral information. In this work, we have examined several factors influencing the spectrum and signal enhancement during SERS studies conducted on both Gram-negative and Gram-positive bacterial species: Escherichia coli and Bacillus subtilis, respectively. These factors can be grouped into those which are related to the structure and types of plasmonic systems used during SERS measurements and those that are associated with the culturing conditions, types of culture media, and method of biological sample preparation.

Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures