"Applications of D2O labelling combined with Raman Microscopy for Mycobacteria Analysis and Application of SERS for the investigation of Photocatalytic Reaction"
Abstract
In this study, D2O labelling combined with single-cell Raman imaging (SCRI) was applied for the investigation of the metabolic mechanism of mycobacteria. Bacterial cells were fixed on a hydrophobic glass slide surface and measured in an aqueous environment. Numbers of single cell were randomly selected for Raman imaging. Multiple linear regression (MLR) was used for quantitative evaluation. The bacterial metabolic activity was evaluated by calculating the ratio between the deuterium-labeled macromolecules and the non-labeled macromolecules which is defined as the intensity ratio between newly appeared carbon-deuterium (C-D) band and the original carbon-hydrogen (C-H) band. Through the data evaluation, the C-D content distribution in individual cells can be visualized along the longest cell length. The metabolic heterogeneity in individual mycobacterial cells and the differential responses of mycobacteria to different antibiotics at single-cell level were determined. In addition, asymmetric growth of mycobacteria was observed through Raman imaging. In a second part, the reaction kinetics and mechanism of hydrogenation of 4-nitrothiophenol (4-NTP) to 4-aminothiophenol (4-ATP) were systematically investigated via surface-enhanced Raman microscopy (SERS). The SERS measurements were performed using a new type of simple and cheap silver plasmonic film, which enables monitoring of the reaction under difference conditions. The reaction was observed as the concentration of H+ and Cl- were carefully controlled, and the formation of the aromatic azo specie 4, 4-dimercaptoazobenzene (DMAB) and other intermediates of the 4-NTP to 4-ATP reduction was detected only under conditions of low H+-concentration or limited Cl--concentration. The reaction rate constant describing the disappearance of 4-NTP was quantitatively determined and was found to vary linearly with the H+-concentration. Based on the kinetic traces of the intermediates, a reaction mechanism for the 4-NTP to 4-ATP reduction is suggested, and rate constants for the individual reactions are presented that fit the measured kinetic traces.