拉曼光谱测定单个细菌芽孢吡啶二羧酸浓度及机理研究

Probing the Machanism and Ca-DPA Concentration of Individual Bacillus Spores Using Trapping and Raman Spectroscopy

应用激光镊子拉曼光谱技术(LTRS)测定19株芽孢杆菌的单个细菌芽孢吡啶二羧酸(DPA)浓度,并验证了系统的重现性。一束30mW,785nm的近红外激光导入倒置显微镜,形成光镊,随机俘获水溶液中的单个芽孢,同时收集其拉曼光谱信号。细菌芽孢的DPA是以与钙离子形成的鳌合物(Ca-DPA)形式存在,收集到的芽孢拉曼光谱信号反映的是Ca-DPA信息,选用信号最强、与Ca-DPA浓度成线性关系的1017cm-1作为其定量的谱峰。通过1017cm-1的峰强,推算出Ca-DPA的浓度。结果显示,单个芽孢的拉曼光谱能够很好地反映出单个芽孢的特征信息,不同种、同种不同菌株之间的芽孢的DPA浓度有所不同,同一菌株的不同芽孢个体之间DPA浓度也存在差异。该方法不需要复杂的分离、纯化过程,可在水溶液中直接俘获单个芽孢并收集其拉曼光谱信号,便可得到单个芽孢的信息,读出DPA的浓度水平。

Measuring the levels of 2,6-pyridine dicarboxylic acid （DPA） in bacteria spores could provide the information about the DPA function,resistance mechanism and the mechanism of spore germination.The authors have measured levels of Ca-DPA of individual spores of different 19 kinds of Bacillus which from different sources,species,and strains by using laser tweezers Raman spectroscopy （LTRS）.Also we have verified the reproducibility of the system simultaneously.To investigate the biochemical components and structure in single spore,a Raman tweezers setup was used to record the Raman spectrum of single spore.A NIR laser beam （30 mW,785 nm） was introduced into an inverted microscope to form a tweezers for trapping the spore suspended in water,and the Raman scatter was excited by the same beam.Raman spectra of 30 spores of 19 bacilus strains which collected from different area in China were recorded,and 100 spores of B.subtilis ACCC10243 were measured.A spore of the same strain was probed 100 times for verifying the reproducibility of the LTRS system.A Matlab 7.0 edited program and Origin 8.0 were used to process the spectral data.Because Ca-DPA is the chelate of DPA and the calcium ion,and the strongest Raman bands at 1 017 cm-1 was from Ca-DPA component of the spore,its intensity was linearly with the Ca-DPA concentration.Therefore,the 1 017 cm-1 bands of Ca-DPA could be used as the quantitative standard peak,and then calculated the concentration of Ca-DPA could be calculated according the intensity of 1 017 cm-1 peak.The results showed that Raman spectra of single spore can reflect the characteristics information of it.The diversity of Ca-DPA levels not only happened between different species and strains of bacillus,but also happened between different individual spores in the same strains of bacillus.Conclusion from these measurements is that there is heterogeneity in different individual spores.It is convenient to trapping and collecting its Raman spectrum in water directly,and then get the information of the level of DPA,without the complex preparation of separating,purifying spores and Abstracting DPA,so we predict LTRS as a high sensitivity,high accuracy,rapid and effective method in the research of individual spores.