本論文是以原子力顯微鏡(atomic force microscope, AFM)作為研究工具，對生化分子進行表面形貌掃瞄以及測量分子間作用力。利用AFM的輕敲式模式觀察普昂(prion)胜肽片段SHPrP109-122、沙門氏菌(salmonella)及微脂球(liposome)等生化分子在表面上的形貌。結果顯示，普昂會形成大範圍的類澱粉樣纖維沉澱，其長寛高分別為> 400 nm、11.2 ± 1.3 nm及0.9 ± 0.1 nm。實驗發現探針的力道會影響所觀測得的高度，因此必須以不使普昂高度變化的力道來掃瞄表面形貌。配合固態核磁共振儀(solid state NMR)的實驗結果，我們推測類澱粉樣纖維是以反向平行(anti-parallel)的方式排列而成，並組成兩條互相同向平行的原纖維細絲。
對於微脂球及沙門氏菌表面形貌掃瞄結果顯示，微脂球的平均直徑及高度為299 ± 70 nm及52.9 ± 17.1 nm；沙門氏菌呈桿狀，其長寛高分別為2.37 ± 0.44 □m、1.29 ± 0.20 □m及0.74 ± 0.22 □m。實驗中發現，沙門氏菌的表面有大小似微脂球的小隆起，推測可能是細胞膜表面的皺摺。在觀察微脂球形貌時發現，微脂球在雲母(mica)及石墨(HOPG)的表面上會因為不穩定，而崩解形成脂雙層(lipid bilayer)及單層脂質層，造成微脂球不穩定的原因，推測與微脂球的組成、大小、接觸的表面性質以及探針的作用等有關聯。利用此現象我們將修飾了沙門氏菌抗體的微脂球置於HOPG表面上，待微脂球崩解即可在HOPG表面上形成具有沙門氏菌抗體的脂質層，藉此修飾方法，我們以抗原–抗體間的作用力將沙門氏菌固定在表面上。
對於分子間作用力的測量，我們利用AFM測量15c5對鹼金族、鹼土族及Pb2+的斷鍵力(unbinding force)，並且觀察溶劑極性及鄰近分子對於斷鍵力與錯合機率的影響。結果顯示15c5對Na+與Mg2+不會發生2:1錯合，而對K+與Pb2+會形成2:1的錯合，且斷鍵力各別為41 ± 10 pN及70 ± 17 pN，而。我們利用改變探針載荷速率(loading rate)實驗，發現在室溫下測量到的斷鍵力不受探針載荷速率的影響，暗示15c5與K+錯合物的錯合/解離速率快過於探針的載荷速率，錯合物沒有由探針獲得能量幫助解離，因此測量的斷鍵力為熱力學平衡狀態下的作用力。

This study is focus on using atomic force microscope (AFM) as the tool to scan the biomolecular surface morphology and measure molecular interactions. For the biomolecules, we use tapping mode AFM to scan the surface morphology of liposome, salmonella, and peptide segment of prion. According to the surface morphology experiment of prion, the prion will form a large domain fibril, and its length is greater than 400 nm, its width is 11.2 ± 1.3 nm, and its height is 0.9 ± 0.1 nm. The experiment also shows that the probe as the applied force increasing, the height of the prion will decrease. Therefore the experiment will be based on using the force that can not cause the change of prion height to perform the scan of surface morphology. Accroding with the data of the solid-state NMR can conduct that fibril is arranged in the anti-parallel order and and form by two parallel fibrils.
The surface morphology scans of the Semonella and liposome shows that salmonella is with a stick-like structure and its dimension are 2.37 (± 0.44) □m x 1.29 (± 0.20) □m x 0.74 (± 0.22) □m. The average diameter and height of liposome is 299 ± 70 nm and 52.9 ± 17.1 nm. The experiment also shows that there are several humps about the size of liposome on the surface of the salmonella, and it is conjecture to be the wrinkles of the cell membrane.In the other hand, the observing of liposome shows that liposome will collapse into lipid bilayer and monolayer due to its instability on mica and HOPG surface. According to the phenomenon, we put the Salmonella antibody functionalized liposome on HOPG. We successfully fix the salmonella on the surface by using antigen–antibody interactions.
For the molecular interactions measurement, we use AFM to measure the unbinding forces of 15-crown-5 ether (15c5) to alkali metal ion, alkaline earth metal ion, and Pb2+. On the other side, we observe the effects of solvent polarity and neighboring molecules of the unbinding force and binding probability. The result shows that 15c5 will form the 2:1 complexation to the larger ion, such as, K+ and Pb2+. The smaller ions will not form the complex, such as Na+ and Mg2+. Under the temperature experiment and loading rate experiment, we find that the unbinding force is independent to the loading rate when it probes under room temperature. This means that the unbinding process is under thermodynamic equilibrium state. Neverless, the tempeture decrease to 262 K, the unbinding force is dependent to the loading rate. This means that under the lower temperature the molecular dynamics decrease.

1 第一章 緒論 1
1-1 前言 1
1-2 原子力顯微鏡介紹 2
2 第二章 以原子力顯微鏡觀察生物分子表面形貌 6
2-1 實驗目的 6
2-2 利用原子力顯微鏡觀察普昂蛋白的胜肽片段在表面上的形貌 7
2-2-1 普昂簡介 7
2-2-2 樣品製備 8
2-2-3 實驗結果與討論 9
2-2-4 結論 13
2-3 利用原子力顯微鏡觀察微脂球及沙門氏菌的形貌 14
2-3-1 微脂球及沙門氏菌簡介 14
2-3-2 樣品製備 18
2-3-3 實驗結果與討論 18
2-3-4 結論 24
3 第三章 以原子力顯微鏡測量皇冠醚分子與金屬陽離子的斷鍵力 25
3-1 實驗動機與目的 25
3-2 測量分子間作用力的方法 27
3-2-1 生物膜力學探針 28
3-2-2 光學鑷子 29
3-2-3 原子力顯微術 30
3-3 表面修飾方法的介紹 32
3-3-1 牛血清白蛋白吸附 33
3-3-2 化學修飾法 33
3-4 探針載荷速率 36
3-5 研究動機 39
3-6 實驗部分 41
3-6-1 藥品、耗材與儀器 41
3-6-2 實驗步驟 44
3-6-3 實驗儀器 53
3-7 結果與討論 56
3-7-1 定量十五皇冠五醚與鉀及鉛離子的斷鍵力 56
3-7-2 環境因素對於斷鍵力的影響 60
3-7-3 斷鍵力與能量之間的關係 70
3-8 結論 86
4 第四章 參考文獻及附錄 88

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