Investigation Of Surface-enhanced Raman Spectroscopy Of Extracellular Environment

Cell is the basic fundamental structure of life activitIes.Cell controls all the metabolism of living organisms.In multicellular organisms,physiological status decides secretion of neurotransmitters,hormones and cytokines.Then these molecules are released to extracellular environment and affect target cells.The influence of cells will change the physiological process of the whole organism.Therefore,monitoring these molecules can help us better understand cell division,differentiation and apoptosis.Raman spectroscopy is very suitable for in-situ study of living cells since it is a non-invasive and non-photobleaching method which can reflect the inherent fingerprint information of samples and avoid auto-fluorescence and Raman signal of water and carbon dioxide.Surface-enhanced Raman spectroscopy(SERS)can overcome the limitation of low sensitivity of normal Raman.Nowadays,Raman spectroscopy has been a important technology in the research of biological system.However,it is still challenging to get reliable signal without influence of cell activity and normal cellular functions.What's more,because concentration and distribution of cells are complex,and the spectra of mixture are not the linear combination of spectra of pure substances,we need better mathematical methods to extract significant information from the complex data.In this thesis,we combine in-situ cell culture instruments and SERS substrate.We monitor the apoptosis process by indirect detection and the adhesion process by direct detection.The main content and result are listed as follows:1.With modification of 4-Mercaptopyridine(4-MPy)on gold nanoparticle assembled substrate,we prepared high sensitive pH sensors and characterized pH response, sensitivity,stability,cycle performance,and reliability of this pH sensitive substrate systematically,discussed the fabrication and service conditions of these sensors in living cell detection.We analysed pH distribution of different cell and found that the pHe of single cancer cells is lower than the normal cells but higher than the pHe of high density cancer cells in cancer tissues.We also traced the spatial and temporal change of pH distribution of human tongue squamous cells(TCA-8113 and SCC-4)during apoptosis stimulated by TGF-?.This experiments showed the decrease of pHe at early stage of apoptosis was fast and slowed down at middle and late stage.2.To reduce the interference of impurities,we used iodide to modify the SERS substrate and test CaSki cells in culture media without FBS.We got a series of spectra with high signal-to-noise ratio and reproducibility.Then we analysed the imaging result with PCA and K-means methods and got the principal component and spatial difference.We found that the main contributions of SERS signals are from the chromophores of tryptophan and phenylalanine With the comparison of analysis results and spectra of some pure substances,we obtained the distribution of protein on the cell membrane.