Characterization Of Bacteria With Surface Enhanced Raman Spectroscopy

Bacterial infections have become a massive threat to human health,with hundreds of millions of intestinal infections caused by bacteria worldwide each year.Moreover,they are a significant cause of increased morbidity and mortality worldwide.Therefore,identifying microorganisms is essential in biology,biotechnology,and medicine.In recent years,great attention has been paid to the SERS research of bacteria.The SERS technique provides excellent convenience for studying the bacterial response to the external environment because of its high sensitivity,the amount of spectral fingerprint information provided,the speed of analysis,and the ease of operation.SERS is widely used in bacterial research,and although there are many reports on bacterial SERS spectra,no uniform SERS scheme for characterizing bacteria has been established.Moreover,not all bacteria in the same medium can obtain SERS spectra of representative bacteria.Meanwhile,equally crucial for SERS spectroscopy results are some measurement parameters in addition to the medium: exposure time to laser irradiation,excitation wavelength and irradiation time,etc.Therefore,this paper aims to obtain representative SERS spectra of specific bacteria and investigate the factors affecting the SERS spectral signal of selected bacteria to obtain reproducible SERS spectra of particular bacteria with a high number of characteristic peaks.The specific work is as follows:(1)The effects of the type of bacterial medium,incubation time,excitation wavelength of the detected bacteria,and irradiation time of the bacterial samples under the laser on the SERS spectra of bacteria were studied,and optimization experiments selected the most suitable standardized conditions.The experiments showed that S.dysenteriae 1 had a better spectral reproducibility and a high number of characteristic peaks when the bacteria were incubated in an LB medium for 48 h with an excitation wavelength of 532 nm.While the remaining three bacteria were cultured in LB medium for 24 h with an excitation wavelength of 633 nm,the SERS spectra of the bacteria were better reproduced,and the number of characteristic peaks was higher.A first step was taken to obtain representative culture methods and detection conditions for the SERS spectra of specific bacteria.(2)The effects of physical means frequently used in experiments on bacterial SERS spectra are discussed.The bacteria were treated by ultrasound,UV irradiation,and temperature.The conditions were optimized to show more number,intensity,and spectral reproducibility of the characteristic peaks of the bacteria by optimizing the ultrasound time,UV irradiation time,and temperature.The results showed that the SERS spectra of S.dysenteriae 1 were better reproduced.The characteristic peaks were higher when the bacteria were treated with ultrasound for 1 min and UV irradiation for 10 min at 40 ℃.The SERS spectra of S.dysenteriae 2 and E.faecalis were better reproduced,and the number of characteristic peaks was higher when the bacteria were treated with no ultrasound and UV irradiation at room temperature;the SERS spectra of E.faecalis were better reproduced,and the number of characteristic peaks was higher when the bacteria were treated with no ultrasound and UV irradiation at room temperature.The number of characteristic peaks was higher for E.coli when treated with ultrasound for 30 s,UV irradiation for 10 min,and 70 ℃.It provides a reference for the experimental process to obtain the representative SERS spectra of specific bacteria in terms of sonication time,UV irradiation time,and temperature.(3)Bacteria were treated with ethanol and p H buffer solution,and conditions such as ethanol concentration,p H of buffer solution,and type of buffer fraction were optimized.The changes in bacterial spectra were reflected by the changes in PCA scatter and load plots,which provided a basis for selecting ethanol and buffer solutions for obtaining highly reproducible SERS spectra of specific bacteria.The experimental results showed that S.dysenteriae 2 and E.faecalis were not treated with ethanol.The SERS spectra of bacteria were more reproducible and had a higher number of characteristic peaks when S.dysenteriae 1 and E.coli were treated with 25% and 100% ethanol,respectively;compared with the other three bacteria,the SERS spectra of E.faecalis varied more significantly with p H;moreover,more characteristic peaks and better reproducibility could be obtained for bacteria treated with BR buffer solution compared with phosphate buffer solution and carbonate buffer solution,but slightly lower than acetate buffer solution.The selection of ethanol and buffer solutions for obtaining highly reproducible SERS spectra of specific bacteria provided the basis for the selection.