混合單分子薄膜應用於生物分子固定研究｜國立中央大學博碩士論文系統

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研究生：	蔡美儀 Mei-Yi Tsai
論文名稱：	混合單分子薄膜應用於生物分子固定研究 Preparation of mixed monolayer for biomolecule immobilization
指導教授：	陶雨台 Yu-Tai Tao 吳春桂 Chun-Guey Wu
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 化學學系 Department of Chemistry
畢業學年度：	92
語文別：	中文
論文頁數：	121
中文關鍵詞：	有機薄膜
外文關鍵詞：	self-assembled monolayer
相關次數：	點閱：23 下載：0
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本篇研究主要配製三組兩種成分之混合單層薄膜：對-巰基苯甲酸/對-甲氧基苯硫酚、對-羧基芐基硫醇/對-甲氧基芐基硫醇與對-羧基芐基硫醇/芐基硫醇，研究與分析混合單層薄膜之結構及分佈情形以製備均分散之羧酸官能基表面，並活化羧酸官能基，使單股DNA可經由醯胺鍵而固定在表面上，並用來偵測互補段DNA。混合分子薄膜之表面組成與分佈情形及末端官能基化學反應之進行，使用了反射式紅外線光譜(FT-IR)、橢圓儀(Ellipsometer)、與NEXAFS光譜觀測其結果與反應性。利用經動力學主導吸附之對-羧基芐基硫醇/芐基硫醇混合分子薄膜系統，以NHS/EDC活化羧酸官能基，使其能與修飾了胺基(amine)之單股DNA反應形成醯胺鍵(amide bond)而使DNA能固定於表面上。當固定於表面之探針(probe)DNA與互補段之目標(target)DNA反應，可由螢光光譜觀測到放光強度之變化。觀測到互補段DNA之最大偵測效率為對-羧基芐基硫醇/芐基硫醇混合分子薄膜比例為1: 9時為最佳比例。

In this study three kinds of binary mixed self-assembled monolayers were prepared on gold：p-mercaptobenzoic acid/4-methoxy-benzenethiol, 4-mercaptomethylbenzoic acid/4-mthoxybenzyl mercaptan, and 4-mercaptomethyl-benzoic acid/benzylmercaptan. The surface structure and distribution of the films were analyzed in order to prepare a randomly distributed carboxyl functional groups on surface. The carboxyl groups were used to immobilize ssDNA through amide linkage via chemical transformation. The ssDNA-attached surface was used to capture a complimentary DNA via hybridization. The compositions of mixed-monolayers and the chemical transformation of the surface functional groups were studied by reflection absorption infrared spectroscopy, ellipsometry, NEXAFS. The mixed SAMs prepared from 4-mercaptomethylbenzoic acid/benzylmercaptan by kinetical trapping method was activated by NHS/EDC to form the NHS-ester, which reacted with an amine-modified probe DNA to attach the single strand DNA in various concentrations on surface. Hybridization with a target DNA was measured by fluorimeter. Maximum loading of the target DNA was observed for a surface prepared from a solution containing 4-mercaptomethylbenzoic acid and benzylmercaptan in 1:9 ratio. II

總目錄
中文摘要………………………………………….. Ⅰ
英文摘要………………………………………….. Ⅱ
總目錄…………………………………………….. Ⅲ
圖目錄…………………………………………….. Ⅷ
壹、緒論……………………………………………
1 1-1有機自組裝分子薄膜導論…………………… 1
1-1-1烷基硫醇自組裝分子薄膜……………………... 2
1-1-2苯基硫醇自組裝分子薄膜……………………... 7
1-1-3混合單層分子薄膜的製備與結構……………... 9
1-1-4混合單層分子薄膜的鑑定及應用……………... 11
1-2生物感測器簡介……………………………… 12
1-2-1 生物感測器之類型…………………………….. 12
1-2-2有機分子薄膜應用於生物感測器之製備方式... 14
1-2-3 生物感測器之應用與發展…………………….. 18
貳、研究動機與方法……………………………… 19
參、實驗部分……………………………………… 21
3-1實驗用藥品…………………………………… 21
3-2合成部份……………………………………… 22 3
-2-1 對-羧基芐基硫醇的合成………………………. 22
3-2-2 對-甲氧基芐基硫醇的合成……………………. 23
3-2-3 對-巰甲基苯甲酸甲酯的合成…………………. 24 3
-2-4 重氮甲烷的製備……………………………….. 24
3-3實驗條件與步驟……………………………… 25 3
-3-1 實驗用基材的製備…………………………….. 25
3-3-2 硫醇分子薄膜的製備………………………….. 26
3-3-3 對-巰甲基苯甲酸甲酯分子薄膜的製備………. 27
3-3-4 混合單層薄膜的製備………………………….. 27
3-3-5 以模紋方式製備分子薄膜…………………….. 28
3-3-6 DNA固定於分子薄膜之表面反應…………... 28
3-3-6-1 DNA與緩衝溶液的配製與保存…………………. 28
3-3-6-2 表面活化步驟與探針DNA之固定……………… 29
3-3-6-3 目標DNA之偵測方式…………………………… 30
3-4實驗用儀器與技術…………………………… 31
3-4-1 核磁共振光譜儀……………………………….. 31
3-4-2真空蒸鍍儀……………………………………... 31
3-4-3旋轉塗佈機……………………………………... 31
3-4-4臭氧產生器……………………………………... 31
3-4-5 傅立葉紅外線光譜儀………………………….. 32
3-4-6橢圓儀…………………………………………... 33
3-4-7 掃描式穿隧顯微鏡…………………………….. 33
3-4-8原子力顯微鏡…………………………………... 35
3-4-9 掃描式電子顯微鏡…………………………….. 36
3-4-10 Near-Edge X-Ray Absorption Fine Structure …. 38
3-4-11 螢光光譜儀…………………………………… 38
肆、結果與討論…………………………………… 39
4-1 有機自組裝分子薄膜系統………...………… 39
4-1-1對-巰基苯甲酸………………………………….. 40
4-1-1.1紅外線光譜觀察結果……………………………… 40
4-1-2 對-羧基芐基硫醇………………………………. 45
4-1-2.1 紅外線光譜觀察結果…………………………….. 45
4-1-2.2 橢圓儀量測的結果……………………………….. 50
4-1-2.3 X光光電子能譜觀察結果………………………… 51
4-1-2.4 Near Edge X-Ray Absorption Fine Structure觀察結果…………………………………………………… 54
4-1-3 對-巰基苯甲酸/對-甲氧基苯硫酚混合單層薄膜………………………………………………... 57
4-1-4對-羧基芐基硫醇/對-甲氧基芐基硫醇混合單層薄膜………………………………………………. 61
4-1-5對-羧基芐基硫醇/芐基硫醇混合單層薄膜……. 64
4-1-5.1紅外線光譜觀察結果……………………………… 64
4-1-5.2掃描式穿隧顯微鏡觀察結果……………………… 68
4-2 探針DNA之固定……………………………. 69
4-2-1 關於P53基因…………………………………... 69
4-2-2 羧酸表面之活化與鑑定……………………….. 71
4-2-3 單股去氧核醣核酸之鍵結…………………….. 73
4-2-2.1紅外線光譜觀察結果……………………………… 73
4-2-2.2 原子力顯微鏡觀察結果………………………….. 76
4-3 目標DNA捕捉與釋放之偵測………………. 85
4-3-1 紅外線光譜觀察結果………………………….. 85
4-3-2螢光光譜之結果………………………………... 85
4-3-3 原子力顯微鏡觀察果………………………….. 89
4-4 微米轉印之結果……………………………... 95
4-4-1單股DNA固定於表面之掃描式電子顯微鏡觀察結果…………………………………………... 95
4-4-2單股DNA固定於表面之原子力顯微鏡觀察結果………………………………………………... 95
4-4-3雙股DNA之掃描式電子顯微鏡觀察結果……. 98
伍、結論與未來展望……………………………… 99
陸、參考文獻……………………………………… 102 VII

                                

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