本研究的重心是探討生長環境對胰島素(insulin)構型變化的影響，實驗主要是觀察: (ⅰ)孵育時間、(ⅱ)奈米粒子 (ⅲ) 離子效應，這些因素對胰島素聚集現象以及二級結構變化的影響。我們利用圓二色光譜儀 (Circular dichroism spectroscopy, CD) 及傅氏轉換紅外光譜 ( Fourier Transform Infrared Spectrometry, FT-IR)，觀察胰島素二級結構的變化，並輔以原子力顯微鏡 (Atomic Force Microscope, AFM) 以及穿透式電子顯微鏡 (Transmission Electron Microscope, TEM)得到其纖維的表面形貌。實驗結果顯示，胰島素纖維構型隨著孵育時間的增加由α-helix 轉變為 β-sheet。加入Au 奈米粒子會與胰島素形成配位基鍵結，孵育後的胰島素纖維較短且粗。另一方面，Fe3O4奈米粒子與胰島素之間僅存在凡得瓦爾作用力，且可用於分離溶液中的胰島素纖維。此外，加入鹽類會使胰島素纖維構型轉變速率加速至少10倍，且胰島素纖維長度較原先短約5~10倍，高度則多2~3倍，尤其在高鹽類濃度下有非晶性的不定型(amorphous)聚集形成。這些現象是因為胰島素的表面電荷被鹽類陰離子遮蔽，降低了胰島素分子間的靜電作用力所致。

We study the conformational change of insulin fibril growth from three aspects: the impact of (i) incubation time; (ⅱ) nano-particles; (iii) and ion added. We used circular dichroism (CD) spectroscopy and fourier transform infrared spectroscopy (FT-IR) to obtain the structural transition of the insulin, and gain the morphology information of fibril by atomic force microscopy (AFM) and transmission electron microscopy (TEM). We show that the insulin transform from α-helix to β-sheet structure as increased incubated time. The addition of Au nanoparticles (NPs) caused the formation of coordination bond with insulin fiber and produced shorter and thicker insulin fibril . The Fe3O4 NPs, on the other hand, offered only van der Waals interaction toward insulin fibril. Hence they could be used to separate insulin fibril from solution. Finally, addition of salts can induce the conformation changes of insulin fibril ten times faster than that without salts. And the insulin fibril fragment was two or three times shorter than that produced without salts. At high salt concentration, insulin formed amorphous aggregates. This phenomenon was attribute to anions from salt: covering the surface charge of insulin fibril, they weaken the original electrostatic repulsion among insulin fibrils and result in their aggregation.

論文審定書 i
中文摘要 iv
Abstract v
第壹章 緒論 1
1-1 前言 1
1-2 研究背景 2
1-2-1 胰島素與大腦 2
1-2-2 胰島素異常與疾病的關聯 2
1-3胰島素類澱粉纖維 3
1-4 研究動機 4
第貳章 儀器與實驗 5
2-1 原子力顯微鏡 ( Atomic Force Microscopy, AFM ) 5
2-1-1 簡介 5
2-1-2 基本原理 6
2-1-3 工作模式 7
2-1-4 力學測量模式-力距離曲線 9
2-1-5 實驗使用的AFM儀器 10
2-2 圓二色光譜儀 (Circular Dichroism, CD) 11
2-2-1 測量原理 11
2-2-2 蛋白質二級結構的圓二色光譜 12
2-2-3 CD參數設定 13
2-3傅氏轉換紅外線光譜儀(Fourier transform infrared spectroscopy) 14
2-3-1 原理 14
2-3-2實驗儀器 15
2-3-3 實驗參數 15
2-4界達電位粒徑分析儀 ( Zeta potential analyzer ) 16
2-4-1 儀器原理 16
2-4-2 實驗儀器 17
2-5 X-ray光譜繞射儀 ( X-ray diffraction) 17
2-5-1 繞射原理 17
2-5-2 應用及用途 18
2-5-3 實驗儀器 18
2-6 低溫性穿透式電子顯微鏡 (Cryo-TEM) 18
2-6-1儀器原理 18
2-6-2應用 18
2-6-3實驗儀器 19
2-7 實驗材料 19
2-7-1 基板 19
2-7-2 實驗藥品 20
2-8 實驗操作方法 20
2-8-1 基板清洗 20
2-8-2 生物樣品配製 20
2-8-3 粒子效應單元中Insulin (1mg/ml) 溶液的配置 21
2-8-4 離子效應單元中Insulin (1mg/ml) 溶液的配置 21
第參章 結果與討論 23
3-1胰島素於水溶液中生長機制的探討 23
3-1-1 利用CD與FT-IR光譜分析胰島素隨孵育時間二級結構的變化 24
3-1-2 藉由AFM及TEM觀察胰島素隨孵育時間之纖維形貌變化 26
3-1-3 室溫下孵育時間對已纖維化胰島素二級結構變化之光譜分析 30
3-1-4 室溫下孵育時間對已纖維化胰島素表面結構變化的分析 31
3-2 Fe3O4 Nanoparticles (NPs)對胰島素纖維生長的影響 40
3-2-1 前言 40
3-2-2 Fe3O4的特性分析 41
3-2-3 Fe3O4對胰島素纖維二級結構改變的影響 42
3-2-4 結論 48
3-3 Gold NPs(Nanoparticles, NPs)對胰島素纖維生長的影響3-3-1 前言 49
3-3-2 加入Au NPs對胰島素二級結構的影響 50
3-3-3 總結Au NPs對胰島素生長的影響 60
3-4 鹽濃度對胰島素類澱粉纖維形成的影響 61
3-4-1 前言 61
3-4-2 添加鹽類對胰島素二級結構變化的影響 62
3-4-3 添加Cu2+、Zn2+、Fe2+氯化鹽類對胰島素二級結構變化的影響 67
3-4-4 添加Fe3+氯化鹽類對胰島素二級結構變化的影響 70
3-4-5 討論 72
3-4-6 結論 77
第肆章 結論 78
第伍章 未來展望 79
第陸章 參考文獻 81

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