本研究利用靜電力顯微鏡的高區域解析度性質，可以鎖定數奈米範圍內，直接同時觀察單一纖維的表面形貌及靜電力特性，配合界達電位、圓二色光譜、傅立葉轉換紅外線光譜，對胰島素纖維表面靜電力性質直接進行觀察。透過控制孵育時間，加入的鹽類濃度來了解表面電荷分佈與結構變化的關連性。結果顯示胰島素纖維在聚集生長時，表面電荷量會隨時間增加而變大。鹽類濃度增加會造成纖維分段變短，表面電荷分佈差異變大。此外，本實驗也藉由不同種類的胰島素，以原子力顯微鏡、圓二色光譜儀探討胺基酸序列差異對胰島素纖維生長速度及結構的影響。實驗結果顯示親水性官能基的數量會影響纖維生成的速度，而其位置會影響纖維對抗冷凍造成的斷裂的能力。

In this report, electrostatic force microscopy (EFM), zeta-potential analyzer, circular dichroism Spectrophotometer and Fourier transform infrared spectroscopy are used to study the electrostatic property of surface on insulin fibril. EFM provides a simultaneous probe of topography and electrostatic property with highly local resolution in nanometer-scale. To understand the correlation between charge distribution on fibril surface and structure transformation, we controlled the incubation time and salt concentration. The results show that the surface charge increases with incubation time, and with the salt concentration increased, the pitch is found to decrease, variation of charge distribution are increased. In addition, we also have evaluated influence of amino acid sequence on growth rate of fibril associated with bovine, porcine and human insulin by atomic force microscopy and circular dichroism Spectrophotometer. The experimental results show that the number of hydrophilic groups on the peptide sequence will affect the speed of fibril generated, and the location of hydrophilic groups on the peptide sequence will affect the stability of the fibril under the frozen environment.

中文摘要 iii
Abstract iv
目錄 v
圖次 viii
表次 x
第壹章 緒論 1
1-1 前言 1
1-2 胰島素與疾病 1
1-3 研究動機 2
第貳章 儀器與實驗 4
2-1 原子力顯微鏡 (Atomic Force Microscopy, AFM) 4
2-1-1 簡介 4
2-1-2 儀器原理 4
2-1-3 工作方式 5
2-1-4 使用儀器及參數 7
2-2 靜電力顯微鏡 (Electrostatic Force Microscopy, EFM) 9
2-2-1 簡介 9
2-2-2 儀器原理 9
2-2-3 工作方式 11
2-2-4 使用儀器及參數 11
2-3 界達電位儀 (Zeta potential analyzer) 12
2-3-1 簡介 12
2-3-2 儀器原理 13
2-3-3 使用儀器及參數 13
2-4 圓二色光譜儀 (Circular Dichroism, CD) 14
2-4-1 儀器原理 14
2-4-2 使用儀器及參數 16
2-5 傅立葉轉換紅外線光譜儀 (Fourier transform infrared spectroscopy) 17
2-5-1 儀器原理 17
2-5-2 使用儀器及參數 17
2-6 實驗材料 18
2-6-1 實驗材料 18
2-6-2 實驗藥品 18
2-7 樣品製備 19
2-7-1 基板準備 19
2-7-2 胰島素纖維溶液製備 19
2-7-3 含鹽胰島素纖維溶液製備 20
2-7-4 AFM及EFM樣品製備 20
2-7-5 界達電位樣品製備 20
2-7-6 CD樣品製備 20
2-7-7 冷凍樣品製備 21
第參章 結果與討論 23
3-1 二級結構變化與纖維表面電荷的相關性 23
3-1-1 前言 23
3-1-2 CD及FTIR觀察二級結構變化 24
3-1-3 界達電位 25
3-1-4 EFM掃描 26
3-1-5 討論 28
3-1-6 結論 31
3-2 加入鹽類改變胰島素纖維的表面形貌與電荷分佈 32
3-2-1 前言 32
3-2-2 CD觀察二級結構變化 32
3-2-3 AFM觀察纖維形貌變化 33
3-2-4 EFM觀察電荷分佈變化 35
3-2-5 討論 39
3-2-6 結論 40
3-3 胰島素序列差異對其類澱粉蛋白纖維生長速率影響 42
3-3-1 前言 42
3-3-2 牛、豬、人三種胰島素胺基酸序列差異 43
3-3-3 AFM觀察纖維數量差異 44
3-3-4 CD觀察二級結構變化 45
3-3-5 討論 46
3-3-6 結論 47
3-4 胰島素官能基位置對纖維冷凍的穩定性影響 49
3-4-1 前言 49
3-4-2 AFM觀察纖維形貌變化 49
3-4-3 CD及FTIR觀察二級結構變化 53
3-4-4 討論 53
3-4-5 結論 54
第肆章 結論 56
第伍章 未來展望 58
第陸章 參考文獻 59

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