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研究生:楊佳勳
研究生(外文):Jia-Xun Yang
論文名稱:利用矽奈米線場效應電晶體偵測蛋白質以及蛋白質交互作用
論文名稱(外文):Label-Free Detections of GST-fused Protein and the Interaction of Proteins via Silicon Nanowire Field-Effect Transistor
指導教授:陳逸聰陳逸聰引用關係
指導教授(外文):Yit-Tsong Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:67
中文關鍵詞:矽奈米線場效電晶體攜鈣素鈣離子穀胱甘肽硫轉移酶N型電壓閘門鈣離子通道三磷酸鳥苷結合蛋白-3A9-β-D-呋
外文關鍵詞:silicon nanowire field-effect transistorcalmodulincalcium ionglutathione-S-transferaseN-type voltage-gated calcium channelRab3AGDP
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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.

論文口試委員審定書 i
誌謝 ii
目錄 iii
圖目錄 vi
表目錄 ix
摘要 x
Abstract xi
中英文名詞對照 xiii
第一章 序論 1
1-1 場效應電晶體簡介 1
1-1.1 發展簡史 1
1-1.2 矽奈米線場效應電晶體 2
1-1.3 矽奈米線場效應電晶體應用於蛋白質觀測上 4
1-2 攜鈣素(calmodulin)與三磷酸鳥苷結合蛋白-3A(Rab3A) 7
1-2.1 攜鈣素(calmodulin) 7
1-2.2 三磷酸鳥苷結合蛋白-3A(Rab3A) 9
1-2.3 可重複使用的連接分子系統 13
1-3 實驗目標 15
第二章 實驗方法與製程 17
2-1 矽奈米線場效應電晶體製作 17
2-1.1 外層電極製作 17
2-1.2 矽奈米線合成 19
2-1.3 氣體-液體-固體成長機制 20
2-1.4 內層電極製作 23
2-2 元件的電學性質 25
2-3 緩衝液配製 26
2-4 晶片修飾方式 28
2-4.1 晶片清潔與修飾連接分子 28
2-4.2 修飾探測蛋白質 30
2-5 電性量測系統 31
2-6 Zeta電位(zeta potential) 32
第三章 實驗結果與討論 35
3-1 攜鈣素Calmodulin 35
3-1.1 表面修飾證明 35
3-1.1.1 免疫螢光表面修飾 35
3-1.1.2 原子力顯微鏡表面掃描 36
3-1.1.3 矽奈米線場效應電晶體偵測結果 38
3-1.2 攜鈣素偵測結果 41
3-1.3 攜鈣素與心肌肌鈣蛋白的蛋白質間作用力偵測 44
3-1.4 攜鈣素與N-型電壓閘門鈣離子通道的偵測 47
3-1.5 討論 50
3-2 Rab3A... 51
3-2.1 Rab3A的螢光免疫實驗 51
3-2.2 Rab3A的偵測結果 52
3-2.3 偵測Rab3A與其作用蛋白之結合 54
3-2.4 討論 57
第四章 結論 61

參考文獻 62
文獻發表 67

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