金奈米粒子與DNA一對一鍵結及其在檢測單一核苷酸變異的應用

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姓名

賴怡穎(I-ying Lai) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

金奈米粒子與DNA一對一鍵結及其在檢測單一核苷酸變異的應用
(One to One Linkage between Gold Nanoparticle and DNA and Its Application in Detecting Single Nucleotide Mutation)

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摘要(中)

本研究利用金奈米粒子ㄧ對ㄧ鍵結單一條聚核苷酸，來檢測36個鹼基之DNA片段中的單一核苷酸變異（single nucleotide mutation）。首先，我們使用氫硫基化合物Mercaptoundecanoic acid包覆金奈米粒子表面使其親水化；改質後金奈米粒子於高溫80℃與200 mM鹽溶液中有很好的穩定性，並可穩定保存約十個月。此外，利用固相合成方式使金奈米粒子鍵結單一條聚核苷酸，兩條互補且與奈米金形成一對一鍵結的探針進行雜合反應，會形成二聚體結構，可經由穿透式顯微鏡與動態粒徑分析儀證實此結構的存在。我們以一對一鍵結的金奈米探針來偵測單一核苷酸變異的ssDNA，並與一對多鍵結的金奈米探針進行比較。實驗結果發現，一對一與一對多鍵結的金奈米探針均可清楚辨別單一核苷酸變異，但經多次操作後，也發現一對一鍵結的金奈米探針造成的背景干擾遠比一對多鍵結的金奈米探針來得低。

摘要(英)

Water-soluble gold nanoparticles were stabily dispersed in 200mM NaCl solution for ten months by surface modification with mercaptoundecanoic acid. We developed an efficient method to link nanogold with single strain DNA (ssDNA). One to one conjugates of the Au/ssDNA(+) were dimerized with the complimentary Au/ssDNA(-) and imaged by TEM and nanoparticle tracking analysis. Both one to one conjugates of Au/ssDNA and randomly linked Au/ssDNA were able to detect single nucleotide mutation by absorb intensity. The lower background absorb intensity of one to one conjugates can be estimated by the detection results.

關鍵字(中)

★ 金奈米粒子
★ DNA
★ 單一核苷酸變異

關鍵字(英)

★ Single Nucleotide Mutation
★ Gold nanoparticle
★ DNA

論文目次

中文摘要 I
英文摘要 II
總目錄 III
圖目錄 V
表目錄 VII
第一章緒論 1
1.1 研究動機 1
1.2 研究目的與進行策略 2
第二章文獻回顧 4
2.1 奈米材料簡介 4
2.2 金奈米粒子的特性 5
2.3 金奈米粒子的製備方法 7
2.4 金奈米粒子親水化改質 9
2.4.1 氫硫基化合物(Mercaptocarbonic acid)改質 9
2.4.2 高分子披覆(Polymer-coating)改質 11
2.5 金奈米粒子與生物分子鍵結 12
2.5.1 奈米粒子與蛋白質接合 14
2.5.2 奈米粒子與聚核苷酸 (DNA)接合 16
2.6 奈米粒子鍵結DNA數量 18
2.7 奈米粒子在生物學上應用 22
第三章實驗藥品、設備與方法 25
3.1實驗藥品 25
3.2實驗設備 28
3.3 實驗方法 30
3.3.1 金奈米粒子的製備 30
3.3.2 金奈米粒子表面親水化改質 32
3.3.3 金奈米粒子穩定性試驗 33
3.3.4金奈米粒子鍵結單根ssDNA 34
3.3.5 吸附基材表面處理 36
第四章結果與討論 39
4.1 金奈米粒子合成 39
4.1.1 金奈米粒子於甲苯溶液中的粒徑分布 41
4.1.2 金奈米粒子於甲苯溶液的吸收光光譜 42
4.2 金奈米粒子表面親水化改質 43
4.2.1 利用氫硫基化合物MUA修飾金奈米粒子表面 45
4.2.2 室溫下鹽穩定性試驗 49
4.2.3 鹽、熱穩定性試驗 52
4.2.4 長時間觀察改質後金奈米粒子粒徑變化 54
4.3 金奈米粒子與ssDNA的一對一鍵結 55
4.3.1 脫附一對一鍵結的金奈米粒子 56
4.3.2 鑑定與ssDNA一對一鍵結的金奈米粒子 57
4.3.3 金奈米粒子二聚體結構 58
4.4 檢測單一核苷酸變異之DNA 62
4.4.1 金奈米粒子表面ssDNA與石英片表面ssDNA固相雜合 63
4.4.2 金奈米粒子表面ssDNA與石英片表面single mismatch ssDNA進行固相雜合 66
4.4.3 脫附夾具內雜合反應的金奈米粒子 69
第五章結論與結論與未來研究方向 71
5.1 結論 71
5.2 未來研究方向 73
參考文獻 74

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指導教授

阮若屈(Ruoh-chyu Ruaan)

審核日期

2008-7-8

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