水体细菌微生物多波长透射光谱特征分析研究（英文）

Spectral Features Analysis of Multi-Wavelength Transmission Spectra of Pathogenic Bacterial Microbes in Water

多波长透射光谱能够反映出样品细胞大小、形状、内部结构和化学组分等丰富而独特的信息,是微生物快速、实时、在线检测与识别的有利工具。将多波长透射光谱技术应用于水体致病性细菌微生物的快速有效检测对控制水体细菌微生物污染及保护饮用水源水质安全具有重要的现实意义。为了建立及发展基于多波长透射光谱技术的水体致病性细菌微生物快速有效的检测方法,采用紫外-可见分光光度计获取了多种水体致病性细菌微生物(如:肺炎克雷伯氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和大肠杆菌)在200～900nm波段的多波长透射光谱,对比分析了不同细菌及同种细菌在不同浓度时的多波长透射光谱特征。结果表明:对于同种细菌,当细菌浓度发生变化时,400～900nm波段透射光谱形状较为一致,并且在400,450,500和550nm波长处的光密度值与浓度具有很好的线性关系,该波段由细菌体的散射起主要作用;但在200～400nm波段范围内,细菌透射光谱的形状随细菌浓度的变化而变化,在200,258,300和350nm波长处的光密度值与细菌浓度分别具有很好的二次多项式关系。根据微粒的Mie散射理论,采用Levenberg-Marquardt非线性最小二乘方法对测得的四种细菌透射光谱进行了散射光谱和吸收光谱拟合,并对比分析了不同细菌散射光谱特征和吸收光谱特征,结果表明:四种细菌散射光谱的特征峰均在245nm波长处,但该波长处的光密度值具有明显差异性,这与不同细菌外部结构及内部结构细胞器的大小、形状等不同有关;而四种细菌吸收光谱特征峰均在260nm波长处,且不同细菌在240～400nm波段内吸收光谱也具有明显差异性,这与不同细菌细胞内的核酸、蛋白质等化学组分含量不同有关。该研究表明对于不同种细菌及具有不同浓度的同种细菌,测得的多波长透射光谱及计算出的散射光谱和吸收光谱特征都具有明显的不同,通过多波长透射光谱解析可以获得细菌多种特征参数,多波长透射光谱可以被用于快速有效检测水体中的致病性细菌微生物。该研究为发展水体细菌微生物快速在线监测仪提供了重要依据。

Multi-wavelength transmission spectrum can reflect the unique information on cell size,shape,internal structure and chemical composition of a sample,so multi-wavelength transmission spectroscopy is a powerful technique for real-time and in-site detection and identification of cells.And the development of multi-wavelength transmission spectroscopy for the rapid and effective detection ofbacterial microbes in water will be of great significance to the control of microbial contamination in water and the protection of the safety and health of water quality.In this paper,in order to develop the method of rapidly and accurately detecting the pathogenic bacterial microbes in water based on multi-wavelength transmission spectroscopy,the multi-wavelength transmission spectra in therange of 200~900 nm of various pathogenic bacterial microbes such as Klebsiella pneumoniae,Salmonella typhimurium,Staphylococcus aureus and Escherichiacoli were obtained adopting UV-Vis spectrophotometer for the first time,respectively.And the spectral features of different bacteria and the same bacteria with different concentrations were compared and analyzed.The results demonstrate thatfor different bacteria,when the concentration changes,the spectral shapes in the range of 400~900 nm are consistent,and the optical density value at 400,450,500 and 550 nm respectively has a very good linear relationship with bacterial concentration.But in the range of 200~400 nm,the spectral shapes change with the change of bacterial concentration,and the optical density value at 200,258,300 and 350 nm respectively has a very good quadratic polynomial relationship with bacterial concentration.According to Mie scattering theory,Levenberg-Marquardt nonlinear least square method was adopted to calculate the scattering spectra and absorption spectra based on the measured transmission spectra ofdifferent bacteria.And the spectral features of normalized scattering spectraand absorption spectra of different bacteria were compared and analyzed.The results demonstrate that for the four types of bacteria,all the characteristic spectral peaks of scattering spectra are at 245 nm.But for different bacteria,theoptical density values at characteristic peak are obviously different,which arises from the differences in cell size and shape of different bacteria.Furthermore,all the characteristic spectral peaks of absorption spectra of the four types of bacteria are at 260 nm.But for different bacteria,the spectral absorption band between 240~400 nm and spectral intensity at 260 nm are obvious different,which are attributed to the difference in content of chemical composition such as nucleic acid and protein in different bacteria.This study indicates that for the different bacteria and the same bacteria with different concentrations,the spectral features of multi-wavelength transmission spectra,calculated scattering spectra and absorption spectra are obviously different.And various parameters of bacteria can be obtained by the interpretation of multi-wavelength transmission spectra.So multi-wavelength transmission spectroscopy can be used torapidly and effectively detect pathogenic bacterial microbes in water.This study provides an important basis for the development of rapid and on-line monitoring instrument of bacteria in water.