臺灣博碩士論文加值系統

本研究是藉由原子力顯微鏡(AFM)研究MerP蛋白與Hg2+離子間的交互作用力，並探討影響影像的兩個因素：「觀測環境」與「樣品基板」。
主要的工作重點可分為三個部份，第一部份，我們將Si和Si3N4探針，置於三種不同的探針座，在大氣、DI water和五種生物常用溶液：PBS、TE buffer、DMEM+FBS、Home made及K3PO4溶液間進行振盪，觀測環境對於懸臂振盪的影響，並進一步藉由Q值的計算，量化環境對於AFM解析度的影響。另外使用JOEL的超高真空系統進行真空環境下懸臂振盪的研究。由掃頻量測結果可知，在不同環境中，懸臂的共振頻率與Q值會隨著環境中的阻尼增加而變小。其Q值大小為：真空>大氣>DI water>其他溶液。
第二部份，為了了解探針在掃描樣品時與樣品表面間的交互作用關係，我們使用三種探針(Si、Si+Au、Si+Au+Hg2+)，四種基板 (mica、mica+Au、Si、Si+Au)，以量測分子間作用力的方式，測量探針針尖和基板間的黏滯關係。發現三支探針與mica基板間的黏滯現象最小，即探針在掃描過程中受到mica基板的干擾最小，證明了mica是四種基板中最適合做為觀測平台的材質。
第三部份，我們利用原子力顯微鏡對汞離子結合MerP蛋白進行研究，首先我們將蛋白均勻的分散在雲母基板表面，並運用原子力顯微鏡在大氣與液態環境下觀測MerP蛋白奈米級的結構。接著利用原子力顯微鏡量測分子間作用力的功能對MerP蛋白和Hg2+離子間的引力作一個研究，發現MerP蛋白和Hg2+離子間有一個~219.03的交互作用力，推測這個引力來自於MerP蛋白半胱氨酸中硫醇基的部位與汞離子間的鍵結。

In this work, the cantilever effect in various environments and the interaction measurements between AFM modified tips and substrates are discussed with Atomic force microscope (AFM). The MerP-Hg2+ system on mica surface is then chosen for the proposed studies.

Ⅰ：Environment Effect
The resonance frequencies of cantilevers driven by different modes are measured in vacuum, atmosphere, and solutions respectively. The Q factors are estimated to understand the performance of AFM scanning images. The damping forces affecting the resonance frequency and Q factor are discussed.

Ⅱ：Substrate Effect
To study substrate effect with AFM, force-distance curves are taken with various tips and substrates. Si、Si+Au、Si+Au+Hg2+ tips are considered. Si、 Au/Si 、Mica and Au/Mica surfaces are selected for AFM observation. It is found that Mica surface is a suitable material for our studies.

Ⅲ：MerP-Hg2+ system investigation with AFM
The topography with nano-structue of MerP in atmosphere and liquid environments is investigated. The force distance curve is studied to understand the interaction between MerP protein and Hg2+ ion. The force is about 400nN. That interaction is proposed to correspond to the Binding between Hg2+ and thiol group of cysteine of MerP.

目錄
摘要 Ⅰ
Abstract Ⅲ
目錄 Ⅴ
圖目錄 ⅤⅢ
表目錄 ⅩⅡ

第一章 序論 1
1-1 前言 1
1-2 研究動機 2
1-3 各式生物溶液 4
1-4 各式基板 7
1-5 汞 7
1-6 MerP蛋白 9
1-7 論文導讀 10

第二章 儀器介紹與工作原理 11
2-1 原子力顯微鏡 11
2-1-1 成像原理 12
2-1-2 操作模式 16
2-1-3 探針 18
2-1-4 Q值(Quality factor) 23
2-1-5 機械應力量測-作用力-距離曲線(Force-Distance
curve)29
2-2 蛋白純化之管柱層析法(column chromatography) 33
2-2-1 離子交換法(ion exchange) 35
2-2-2 疏水性層析法(hydrophobic interaction
chromatography；HIC) 37
2-2-3 凝膠過濾法(gel filtration) 39
2-3 蛋白純化之膠體電泳法 41

第三章 「觀測環境」對AFM影像的影響 45
3-1 實驗目的 45
3-2 實驗設計 46
3-3 實驗結果與討論 47
3-3-1 「驅動振幅」對於懸臂振盪的影響 47
3-3-2 大氣用探針座(DEA holder) 50
3-3-3 scanner驅動的液態用探針座(SDL holder) 53
3-3-4 含激振壓電陶瓷的液態用探針座(DEL holder) 62
3-3-5 真空下的頻譜圖量測 69
3-4 小結 72

第四章 「樣品基板」對AFM影像的影響 74
4-1 實驗目的 74
4-2 實驗設計 74
4-3 實驗結果與討論 76

第五章 MerP蛋白觀測與Hg2+離子間的交互作用力量測 86
5-1 實驗目的 86
5-2 實驗設計 86
5-2-1 MerP蛋白的複製與純化 86
5-2-2 MerP表面形貌觀測 91
5-2-3 MerP-Hg2+系統交互作用力量測 91
5-3 實驗結果與討論 92
5-3-1 MerP表面形貌觀測 92
5-3-2 MerP蛋白與Hg2+引力量測 97

第六章 結論 100
第七章 參考資料 102

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