| It is particularly important to acquire precise and specific information about cellular physiological processes for research of life science. Traditional methods for analyzing cellular physiological processes are indirect and require numerous cells to extract some specific components. However, the averaging data of numerous cells might actually obscure the true mechanism of cellular process. Therefore, in-situ techniques for analyzing a single cell or cell aggregates can help to know more about the dynamic individual difference and interactions between cells. Raman spectrum is a non-intrusive optical analysis technique without any ionizing radiation. Compared with infrared and fluorescent spectrum, Raman spectrum has advantages of narrow spectral band width, low interference of water, difficult to quench, can be excited with near-infrared laser. It is very suitable for the biological system research in solution. Nonetheless, due to the tiny size of cell, very low concentration of the component and the weak intensity of Raman scattering, it is very difficult to acquire the Raman signal from a single cell. Therefore, surface plasmon resonance of metal nanoparticles and nanostructures is often applied to acquire the surface enhanced Raman spectrum (SERS) of cells. As a type of SERS-active substrate, metal-coated micro-tips can be prepared simply, which may play an important role in label-free analysis of a single cell or cell aggregates. In this dissertation, by combining the studies on structure, preparation technique and enhancement effect of micro-tip, the whole system for cell culture, monitoring and Raman spectra detection in-situ were developed to meet the trends and needs of cell analysis.The work mainly consisted of four parts as follows:Firstly, structure and preparation technique of stabilizer-free SERS-active micro-tips were designed.Aiming at cell analysis, the structure of micro-tips included substrate, coupling layer and cover layer. Based on the comparison of different preparation techniques, it was proved better to employ the method of stretching in high temperature and magnetron sputtering. Compared with the traditional methods like self-assembly, the cleanliness of the surfaces of the metal Nps is beneficial for absorbing biomolecules on their surfaces.Secondly, systematic study about the parameter variation which affect the morphology and Raman enhancement of micro-tips were achieved to optimize the preparation.The parameter variations which decide the cone height and the tip size were studied during the preparation of substrate. Meanwhile, the parameter variations which decide the Raman enhancement was studied during the preparation of coupling layer and cover layer. The mutual restriction relationship between the parameters were illuminated by explaining the mechanism of metal film formation.Thirdly, a small informationalized device for cell culture and monitoring in-situ was developed.This equipment was used on inverted biological microscope which can realize monitoring and feedback control of the internal environment by using SCM system. It can also display and store the environmental data in computer. Meanwhile, it can accomplish the remote interaction with users by GSM module. Finally, the performance of the device was verified through controlled experiments with H08910 cell culturing, which demonstrated the cells kept the good shape after 24 h culture.Finally, Raman spectra detection system for cell analysis was established. Combined with the SERS effect studies of micro-tips in solution, Raman spectra of A549 cell were detected.Through the optimization of the optical path, the overall structure design, assembly and commissioning of the Raman spectra detection system for cell analysis were accomplished. Based on this system, SERS effect studies of micro-tips in solution were easy to achieve. The performance of the system was evaluated by detecting the Raman spectra of A549 cell with micro-tips. |
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