Studies On SH-SY5Y Cell Activities Using Electrochemical Analysis Method Based On ITO Micro-pore Electrode

Each cell is an independent self-adjusting system, and it interacts and exchanges substance, energy, and message with the surrounding environment. Cell is both the basic constitution and functional unit of biological activities. Life is the external realization of cell's property. Cell embodies such characteristics as diversity, homogeneity and complexity of biological activities. For this reason, cell has long been the core of biosciences. Due to the variation of biological tissues, the same type of cells may differ in shape, size, biological activity, and physiological state. Statistical results obtained from experiments on cell colony often failed to provide the desired accurate message for biology and medicine because differences among individual cells are not under consideration. In contrast, studies on single cells could reveal the differences of cells in chemical components and physiological reaction, and are indispensable for early diagnosis of disease, prescription of drugs, and proper treatment.Cell is also the crosspoint between bioscience and chemistry study. Biological researches have progressed from biological body as a whole, over organs, tissues, into cells and subcellular fractions, while chemical researches into biological system have been pushed forward from atom, over molecule, macromolecule, molecular aggregate, to cell. Nowadays the interaction and overlapping between chemistry and biology propelled exploration into the mystery of biological activities, and promoted the fast development of analytical methods.In this dissertation, SH-SY5Y cells were chosen as the object of study, and electrochemical method was the fundamental approach. Indium Tin Oxide (ITO) micro-pore electrode and ITO array micro-pore electrode were fabricated, and improvements were made on traditional carbon fiber microelectrode. Then using the electrodes, the author made studies about SH-SY5Y single cell and multi-cell exocytoses under different conditions, and about the anoikis of SH-SY5Y cellsChapter1IntroductionThe significance and overall development of cell analysis, in particular, single cell analysis, were first introduced. In the matter of cellular communication, emphasis was put on studies related to cell exocytosis.Next, the author briefly reviewed the conventional means of single cell analysis, namely, image analysis, capillary electrophoresis, microelectrode electrochemical analysis, microfluidics. Finally, the author presented the purpose of study, object of study, research methods and devices.Chapter2The Fabrication and characterizing of the ITO micro-pore electrode and array micro-pore electrodeCompared with traditional electrodes, ITO micropore electrode was characterized by its good electrochemical property and fine photological property. The author manufactured a new type of ITO micro-pore electrochemical sensor and ITO array micro-pore electrode, using ITO and UV-light photolithography technology. When the electrode was applied to the assay of neurotransmitter, the desired result was achieved. Different from the conventional method, ITO micro-pore electrode can be used in monitoring cell exocytosis with electrochemical method. It can also collaborate with other methods like fluorescence and chemiluminescence and be used for studying cell's physiological activity.Chapter3Exocytosis of SH-SY5Y single cell with different shapes cultured on the ITO Micro-pore electrodeAmong the different forms of cell signal transmission, cells or neuronal communication, particularly through the release of specific biochemical or chemical messengers via exocytosis plays crucial roles in biological process. Using ITO micro-pore electrode, the author successfully conducted immediate and real-time monitoring of both single cell and multi-cell exocytosis. Then a comparison was made between different shapes of SH-SY5Y single cell exocytosis. The finding was that cells having clear process are more active than those having spherical shape. With regard to this phenomenon, discussion followed afterwards.Chapter4The study on response time and mechanism of SH-SY5Y cell exocytosis with various stimulantOne of the prerequisites for cell existence is reacting properly to external stimulation. The biological state of cells depends, to a large extent, on their procedural reaction to a series of ultra-cell signals. Exocytosis is the complicated physiological activity possessed by eukaryon. Researches into mechanism of exocytosis provide people with rationale for understanding life. When SH-SY5Y cells were experimented on, high potassium, nicotine and nitroso nornicotine were used respectively as stimulants. Experimental results showed that the timing for exocytosis caused by various stimulants differed greatly. In other words, exocytoses caused by different stimulants have to do with different mechanisms of exocytosis.Chapter5Fabrication of carbon fiber microelectrode and application to SECM and to the real-time monitoring of cell exocytosisCarbon fiber microelectrode, first employed by Wightman on testing single cell, has been widely used in experiments of single cell testing. The stability and sensitivity of carbon fiber microelectrode exerted immediate influence on experimental results, and its fabrication also played important roles in the experimental process. Traditional fabrication process of carbon fiber microelectrode had drawbacks like complex operation, low efficiency, operation under microscope, and possible leakage of electrode. In our experiments, new fabrication procedure has been employed, and the technology has been improved. In the process of fabrication, microscopy operation was no longer needed. Thus, the fabrication of carbon fiber electrode has turned out to be more convenient, economical and easier to handle.Characteristics of the carbon fiber microelectrode were confirmed electrochemically and when it was applied to SECM, desired results were obtained. When carbon fire microelectrode and ITO micro-pore electrode were used separately to monitor exocytosis, experimental data revealed that they had different advantages. ITO micro-pore electrode could closely approach the cell, so the larger interface enabled detection of more exocytotic events. Meanwhile, carbon fiber microelectrode could detect active exocytotic regions over the cell surface.Chapter6The real-time monitoring of the physiological activity of SH-SY5Y cells based on electrochemical impedance spectrumMost of the mammalian cells needed bind to certain substrate when growing in vitro and in vivo. This agglutination between cell and substrate will make cells extend gradually and grow into certain shapes. The growth of cells depended largely on their possible and early binding to the substrate. If they failed to immediately agglutinate the substrate or if their binding was interfered with, cells were unable to maintain normal physiological process and would die. Therefore, studies on cell binding were of significance for researches into physiological and pathological processes, such as wound healing, inflammation, cancerization. Arsenic trioxide, a type of toxic reagent, has been found having curative effects on the treatment of liver cancer, leukemia, and neuroblastoma. If external conditions like ingredients of culture medium, addition of antitumor medicine during incubation period can be changed, the conglutination and growth of tumor cells will be impeded, which will be of great significance to cancer treatment.Previous cell testing methods were all terminal test, unable to monitor the real-time alteration of cells'condition. A new type of ITO array micro-pore electrochemical sensor has been fabricated based on ITO and UV light photolithography technology and has been used in the label-free and real-time monitoring of cell's physiological function. Studies have also been made on the degree to which arsenic trioxide affected agglutination of SH-SY5Y cells. The experimental results showed that arsenic trioxide of a certain concentration had distinct influence on the conglutination of the SH-SY5Y cells and greatly inhibited the proliferation of the SH-SY5Y cells.ITO array micro-pore electrode used in our experiments did not contain any noble metal which was the component of traditional EIS method. Since ITO was characterized by good transmission of light, it was likely to combine electrochemical impedance measurement with other photological characterization measures, which throwed light on the development of cell sensor with compound functions.