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How to Detect Hydrogen Peroxide of Cells by Raman Spectroscopy
Hydrogen peroxide(H202)is a
key part of reactive oxygen species(ROS)and generated
as a
by·product of a wide range of biological processes. It has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location. Such as stimulating cell proliferation, intracellular signal transduction, Excess hydrogen peroxide can lead to structural and functional derangements of cell lipids, proteins, DNA and other molecular. Because of its easy access and relative stability,it has become an important tool for cellular oxidative damage research. The mechanisms of cellular damage induced by Hydrogen peroxide are concern of research．
Up to now, micro-Raman spectroscopy has become an important analytical tool for in vivo bioanalysis. As the Raman technique does not require staining or fixed cells, you can get information of cells under physiological conditions. Moreover, it enables rapid, non-invasive, abel-free analysis of individual cells. Compared with fluorescent methods, it has an unparalleled advantage. An increasing number of Raman and SERS studies on single cells describe numerous applications, including discrimination between normal and transformed cells, detection of glutamate in single synaptosomes, diagnosis of cancer，sorting of single living microorganisms, detection of intracellular pH, detection of biological changes related to cell cycle and cell death, and so on.
In this paper, we used micro.Raman Spectroscopy, and obtained Raman spectroscopy of individual cellsunder different hydrogen peroxide concentrations, to analyze its composition changes; Then，we fabricated ahydrogen peroxide-sensitive nano-probe, and applied it into cells, observing the SERS changes intracellular.
The details are as follows:
1. We used micro-Raman Spectroscopy to obtain the spectroscopy of individual cells．Through mixing hydrogen peroxide with cell culture, we made micro-environmet of 0mM, 0.1mM，0.5mM, lmM, 3mM H202．The individual cells' raman spectroscopy changed, reflecting the chemical component changes inside the cells. With the increasing of the concentration of H202, Raman signal of protein and nucleic acid weakened, reflecting the damages to its structure and decreasing of content. Principal component analysis shows that with the increase of H2O2 concentration, the difference between normal cells and damaged cells were widening, and the cells could be divided into twO groups obviously.
2. We successfully fabricated a kind of hydrogen peroxide-sensitive nano-probe．Ag／DTNB@Si02 SERS probe, with 5,5' dithio bis(2-nitrobenzoic acid)(DTNB)as Raman signal molecules, 40nm silver nanopanicle8 as the core, 2nm silica as the shell. This nano-probe is very stable with a strong SERS signal. The 1326cm-1 Raman peak is an remarkable signal. As the silver nanoparticles can be corroded by hydrogen peroxide, and its diameter is directly related to its enhancement, hydrogen peroxide can greatly affect the Raman signal intensity. The result showed that with increasing H202 concentration, the cells are damaged and the Raman signals are significantly reduced.
3. We successfully fabricated a kind of hydrogen peroxide-sensitive nano-probe．Ag/DTNB@Si02 SERS probe, with 5,5' dithio bis(2-nitrobenzoic acid)(DTNB)as Raman signal molecules, 40nm silver nanopanicle8 as the core, 2nm silica as the shell. This nano-probe is very stable with a strong SERS signal. The 1326cm-1 Raman peak is an remarkable signal. As the silver nanoparticles can be corroded by hydrogen peroxide, and its diameter is directly related to its enhancement, hydrogen peroxide can greatly affect the Raman signal intensity. The result showed that with increasing H202 concentration, the cells are damaged and the Raman signals are significantly reduced.
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