Label-Free Detections of GST-fused Protein and the Interaction of Proteins via Silicon Nanowire Field-Effect Transistor

奈米感測器至今已發展了數年的時間，由於其具有高靈敏度與不需標定的特性，在感測帶電荷分子上具有相當的優勢。這篇論文主要利用矽奈米線場效應電晶體來感測蛋白質與蛋白質間的交互作用力；在細胞內，蛋白質間往往藉由交互作用力來進行維持生裡作用的機制，因此若可以大量且快速檢測未知的蛋白質間作用力，可以了解蛋白質的功能並幫助其相關的疾病控制方面的研究。 藉由固定於矽奈米線表面上的帶電荷蛋白質，其電場會影響奈米線的導電度，我們觀察蛋白質結合時所帶來的導電度變化，從而確認蛋白質間作用力的存在。於攜鈣素方面，已成功感測到和心肌肌鈣蛋白在特定鈣離子濃度範圍下的結合，更進而在生理條件下成功感測到攜鈣素與N-型電壓閘門鈣離子通道的結合，證實了矽奈米線場效應電晶體作為生物感測器的可行性與優勢。於三磷酸鳥苷結合蛋白-3A方面我們明顯觀察到其與9-β-D-呋喃核糖鳥嘌呤-5''-二磷酸的結合態在純磷酸鹽緩衝液中的解離狀況，這可逆的結合-解離說明了解離速率在往後的實驗中是需要考慮的。 藉由穀胱甘肽硫轉移酶的親和力純化方式，我們成功製作出一個可重複使用的、高靈敏度、免標定的生物感測晶片，並可於生理條件下快速地偵測蛋白質間作用力，對了解蛋白質功能上可提供實質的作用。Nanosensors have been developed for recent several years, and have potential on sensing charged molecules because the properties of high-sensitivity and label-free. This thesis is about sensing the protein-protein interactions via silicon nanowire field-effect transistor. Proteins often participate in the machanisms for the maintaining of physiology in cells with such interactions. If the detections of the interactions which is not confirmed could be rapid and quantitative, that will be much helpful to realize the functions of the proteins and the realative treatments of diseases caused from the abnormal functions of proteins. By the immobilization of proteins to the surface of the silicon nanowires, the conductance of nanowires will be affected by the electric field of the charged proteins.And we could observe the changes of the conductance caused by the association of proteins and comfirm the interaction between proteins. About calmodulin, the association of troponin I and calmodulin had been detected succefully in specific range of calcium concentration. Furthermore, we detected the association of N-type voltage-gated calcium channel and calmodulin in physiological condition. That proves the possibility and advantages of the silicon nanowire field-effect transistor as a biosensor. About Rab3A, we observed the dissociation of GDP and Rab3A in pure phosphate buffer solution. The reversible association-dissociation of GDP and Rab3A illustrates that dissociation rate should be taked in consideration in experiments about Rab3A activated by GTP. With the advantages of GST pull-dwon assay, we have developed a reusable, label-free and high sensitivity biosensor chip which could detect the protein-protein interactions in physiological conditions, provides us to understand the function of the proteins