Application Of Surface Enhance Raman Spectroscopy For Assessment Of Metabolic Activity Of Microorganisms

The impact of microorganisms on various environmental phenomena is immeasurable;thus it is important to understand the characteristics of microorganisms including phenotypic and functional traits.However,conventional microbial cell techniques are mostly population-averaged methods that conceal the metabolic activity of single-cell in the community and most of the methods involve long and tedious cultivation.Raman spectroscopy is a label-free,non-invasive,and in-vivo method ideal for the characterization of microorganisms to obtain spatially resolved chemical information.Thus,this work focuses on analyzing the extent of the application of Raman spectroscopy-based technique to assess the metabolic activity of microorganisms.With the growing popularity of the non-destructive technique,surface enhanced Raman spectroscopy demands a highly sensitive and reproducible plasmonic nanoparticle substrate.Thus,novel bimetallic core-shell nanoparticles（Au@Ag@m SiO₂）that consist of a gold core,silver shell,and a mesoporous silica coating were synthesized.The Au@Ag@m SiO₂ nanoparticles were obtained by forming a coating of silica layer on Au@Ag nanoparticles and the mesoporous structure was created by employing template molecules such as surfactant and their subsequent removal.The mesoporous coating allowed selective screening based on the size of the analyte molecule.The Au@Ag@m SiO₂ nanoparticles were able to selectively enhance the small molecules by preventing the large macromolecules to reach the exciting zone of the core plasmonic substrate.Moreover,the mesoporous coating provided additional stability to the Au@Ag nanoparticles which was further proved by the reusability of the Au@Ag@m SiO₂ nanoparticles.Thus,this work offers a simple substrate,Au@Ag@m SiO₂ nanoparticles as a smart SERS substrate for effective detection of analytes.Detection of bioactive metabolites produced by bacteria is important for identifying biomarkers for infectious diseases.Thus,a surface-enhanced Raman spectroscopy-based technique was developed for the detection of bioactive metabolite indole produced by Escherichia coli in biological media.The use of highly sensitive Au@Ag@m SiO₂ core-shell nanoparticles resulted in the detection of indole concentration as low as 0.0886 m M in standard solution.The supplementation of growth media with 5 m M of exogenous tryptophan resulted in the production of a maximum yield of indole of 3.139 m M by E.coli O157:H7 at37°C.The growth of bacterial cells was reduced from 47.73×10⁸ to 1.033×10⁶ CFU/m L when the cells were grown in 0 and 10 m M exogenous tryptophan,respectively.The amount of indole in the Luria-Bertani（LB）media had an inverse correlation with the growth of cells,which resulted in a 3-log reduction in the colony-forming unit when the indole concentration in the media was 20 times higher than normal.This technique demonstrated effective detection of extracellular bioactive metabolites,however investigating the metabolic and molecular makeup of the cell can provide deep knowledge regarding the phenotypic and metabolic behavior of individual cells.Metabolic dynamics of bacterial cells are needed for understanding the correlation between changes in environmental conditions and cell metabolic activity.Hence,Raman spectroscopy combined with deuterium labelling was used to analyze the metabolic activity of a single E.coli O157:H7 cell.The incorporation of deuterium from heavy water into cellular biomolecules resulted in the formation of carbon-deuterium（CD）peaks in the Raman spectra,indicating cell metabolic activity.The broad vibrational peaks corresponding to CD and CH peaks encompassed different specific shifts of macromolecules such as protein,lipids,and nucleic acid.Moreover,the second derivative transformation of the obtained spectra showed that the cell lipid composition changed based on the utilization of tryptophan and oleic acid by the cell as the sole carbon source.Thus,the proposed method could semi-quantitatively determine total metabolic activity,macromolecule specific identification,and lipid and protein metabolism in a single cell.Rapid evaluation of the metabolic activity of microorganisms is crucial in the assessment of the disinfection ability of various antimicrobial agents in food industries.Thus,surface enhanced Raman spectroscopy combined with stable isotope probing was employed for the analysis of the disinfection of a single bacterial cell.The Raman signals from single E.coli O157:H7 was enhanced by the attachment of silver nanoparticles on the surface of bacterial cell obtained by in situ synthesis.The metabolic activity indicated by the formation of carbon-deuterium peak formation was observed as early as 2 h of incubation and showed no damage to cell integrity due to the presence of nanoparticles.Thus,the proposed SIP based SERS method was employed to measure the metabolic activity of E.coli cells in the chicken carcass wash water.The metabolic activity of treated cells did not recover even after extended incubation up to 6 h and the principal component analysis was able to distinguish metabolically active and inactive cells with great accuracy.The study proved that the proposed method has the potential to assess the metabolic activity of microorganisms in other food products and food contact surfaces and optimize disinfection and bacterial removal processes.The current work established that Raman spectroscopy is a novel method for the rapid assessment of the metabolic activity of microorganisms.Moreover,the study proved that the SERS method was capable of high-sensitive detection of extracellular bioactive metabolite produced by microorganisms.The SERS combined with stable isotope probing assists in single-cell metabolic activity measurement in food products under various environmental conditions.