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研究生： 林哲瑩
Lin, Che-Ying
論文名稱： 高敏度表面與高解析技術診斷具疾病表現之單一核苷酸多型性:運用SAMs技術固定寡核苷酸
High-sensetive Surfaces and High-resolution Techniques to Diagnose the Single Nucleotide Polymorphism: Use SAMs technique to immobilize oligonucleotides.
指導教授： 廖峻德
Liao, Jiunn-Der
學位類別： 碩士
Master
系所名稱： 工學院 - 材料科學及工程學系
Department of Materials Science and Engineering
論文出版年： 2005
畢業學年度： 93
語文別： 中文
論文頁數： 62
中文關鍵詞： 自我組裝單分子層 、高解析式X光光電子能譜儀 、單一核苷酸多形性 、核酸雜合反應 、75°低掠角反射傅立葉轉換式紅外線光譜儀
外文關鍵詞： 75 degrees grazing angle FTIR, HRXPS, Self-assembled monolayers, Single nucleotide polymorphism, DNA hybridization
相關次數： 點閱：71  下載：1 
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    • 　　組裝單分子層(Self-Assembled Monolayers，SAMs)具有穩定性高、製程簡單、以及末端官能基的可變異性，目前廣被運用在基礎科學研究所需的理想的表面、具功能性的生物感測表面等。SAMs可以化學鍵結方式固定生物單體物種，以用來進行生物分子辨識的應用，如：單一核苷酸多型性(Single Nucleotide Polymorphism，SNP)。本研究中，運用SAMs技術在Au（111）面上化學吸附硫端，並外露末端官能基O=C-OH，緊接著以EDC、NHS作為表面活化劑與媒合劑，結合在寡核苷酸分子5’端修飾的胺基（-NH2），利用O=C-OH與-NH2在SAMs表面形成醯胺鍵，達到DNA序列片段固定在Au表面的目的。
    　　
    　　實驗中針對不同溫度、pH值以及媒合劑濃度的變化，建立固定寡核苷酸引子的成效較佳化條件。分析方面則採用同步輻射中心之高解析式X光光電子能譜儀以及75°低掠角反射傅立葉轉換式紅外線光譜儀，針對不同過程的表面做原子鍵結及元素分佈的分析，並由螢光顯微鏡的觀察，建立在Au面上進行核酸雜合反應的效能評估。在表面型態分析部分則以原子力顯微鏡針對表面掃描分析，觀察試片表面經過固定寡核苷酸分子與核酸雜合反應後的表面型態變化。
    　　
    　　研究結果顯示，在本研究所採用的不同條件(溫度、pH值)中，以37℃且在酸鹼度為pH值4.5的緩衝溶液的條件下進行寡核苷酸引子固定在晶片上，其固定結果較好；在進行核酸雜合反應時，效能也較佳。當媒合劑濃度為100 mM條件下，固定寡核苷酸引子比媒合劑濃度為1 mM或10 mM的效果要好。對於寡核苷酸引子固定在SAMs上，各階段新的化學結構變化也給予驗證。

    　Self-Assembled Monolayers (SAMs) provide a highly stable structure, easily prepared process and flexible functional group. Nowadays SAMs is widely used as an ideal surface on basic science research and for functional surface of Biosensors. Bio-monomer can be immobilized onto SAMs by chemical bonding and applied for molecular recognition, e.g. Single Nucleotide Polymorphism, SNP. The objective of this study is to immobilize DNA primers onto gold surface by introducting SAMs. In the experiment, SAMs was chemically adsorbed on Au(111) and created a tailor functional group of O-C=OH, which was then activated by coupling EDC and NHS, and subsequent to link with 5’ modified Amino group Oligonucleotides by amide bond forming with O-C=OH functional group of SAMs. The factors of different temperatures, pH values, and the concentration of coupling reagents for oligonucleotides immobilization were investigated. The optimized recombination was build using HRXPS (High Resolution X-ray Photoelectron Spectroscopy) of NSRRC (National Synchortron Radiation Research Center) and 75 degrees grazing angle FTIR (Fourier Transform Infrared Spectrometer). The instruments were used to analyze atomic binding energies, elements distribution, and absorbance of functional groups. DNA hybridization was observed using Fluorescence Microscope, and their surface morphologies were mapped by AFM (Atomic Force Microscope). Experimental results demonstrated that the optimized condition for DNA hybridization was determined at 37℃, pH value of 4.5 and the concentration of coupling agent around 100 mM. In accordance with oligonucleotides immobilization, the presences of new chemical structures at different stages were also identified in this study.

    目錄 第一章 序論 1-1前言.................................................................1 1-2研究動機與目的.............................................2 1-3文獻回顧.........................................................3 1-3.1自我組裝單分子層......................................3 1-3.2 SAMs固定生醫單體....................................4 1-3.3固定寡核苷酸之表面分析..........................6 1-3.4 DNA雜合反應檢測.....................................7 1-4 研究架構........................................................9 第二章 理論基礎 2-1自我組裝單分子層(SAMs)原理....................10 2-2生物分子固定理論........................................14 2-2-1 固定理論....................................................14 2-2-2 物理吸附法................................................14 2-2-3 膠體包埋法................................................14 2-2-4 共價鍵結法................................................15 2-2-5 架橋連結法................................................15 2-2-6 共價鍵接合機制........................................16 2-3 接觸角原理...................................................21 2-4 同步輻射光源...............................................23 2-5 XPS................................................................24 2-6 原子力顯微鏡原理與應用...........................25 第三章 材料與方法 3-1 Dot Hybridization 雜合反應實驗..................28 3-2 寡核苷酸固定與雜合反應實驗材料...........30 3-3自我組裝單分子層製備................................30 3-3-1玻璃器皿清洗.............................................30 3-3-2 基材製備....................................................30 3-4 寡核苷酸固定在SAMs基材與雜合反應......31 3-4-1 固定Sense primer1在SAMs基材上.............31 3-4-2 固定Sense primer2在SAMs基材上，及試片上之雜合反應........................31 3-5 接觸角量測....................................................32 3-6 XPS分析.........................................................33 3-7 FTIR分析........................................................34 3-8 掃描式探針顯微鏡-AFM mold(Scanning Probe Mic)roscope, SPM)................35 3-9 螢光顯微鏡觀察...............................................................................................35 第四章 結果與討論 4-1 冷光實驗評估pH值對寡核苷酸之固定與雜合成效之影響..........................37 4-2 接觸角量測與表面親疏水性之分析...............................................................39 4-3 紅外線吸收光譜與表面官能基之分析評估寡核苷酸固定成效...................43 4-4 光電子能譜儀分析寡核苷酸固定與雜合成效及表面之元素分布...............49 4-5螢光顯微鏡觀察核酸雜合反應成效................................................................55 4-6 原子力顯微鏡觀察寡核苷酸固定與核酸雜合反應後之表面型態變化.......58 結論.........................................................................................................................62 附錄 參考文獻

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