博碩士論文 92324042 詳細資訊




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姓名 王志豪(Chih-Hao Wang)  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 單股DNA於微奈米溝槽電極之基因晶片上固定化與雜交效率之最佳化
(Optimization of Immobilization and Hybridization Efficiency of Single-Strand DNA on Micro-Electrode Biochips)
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摘要(中) 本研究主要是利用表面改質與生物分子固定化來建立一個去氧核醣核酸分子檢測界面,並結合半導體製程及電路設計與量測,來研究一簡單新型的電子式基因晶片。整個研究的主題分為兩個部分,第一部分是探討去氧核醣核酸分子的固定化之驗證與最佳化,第二部分是探討以電性量測於去氧核醣核酸分子有雜交與無雜交的電性量測差異性之提升。藉由接觸角量測(contact angle measurement)、原子力顯微鏡(AFM)和表面化學元素分析儀(ESCA)來驗證去氧核醣核酸分子固定化,並利用HP4284量測不同條件下,有雜交與無雜交於微奈米溝槽內阻抗值之差異性。
在去氧核醣核酸分子固定化中,我們將二氧化矽表面經過適當的化學改質修飾後,固定去氧核醣核酸分子於表面上。實驗結果顯示,每一步化學改質與固定化反應均得驗證,並於pH為6.6,鹽濃度為0.3M的磷酸緩衝溶液中,固定化與雜交效率可達九成,且固定化與雜交反應時間僅需6小時與3小時即可達反應平衡。在電性測量測有無雜交於微奈米溝槽之差異性,實驗中選擇不同金奈米粒徑之標定、不同寬度之溝槽與量測頻率進行檢測。由電性量測訊號(阻抗值)可發現於頻率之影響較不明顯,但於金奈米粒徑為20nm與溝槽寬度為5
摘要(英) The research aimed at the optimizing the conditions for the immobilization of single-stranded DNA, hybridization of complementary ssDNA and detection of hybridization on the chip by electrical impedance. We used contact angle measurement, atomic force microscopy (AFM), electron spectroscopy for chemical analysis (ESCA) and UV-VIS to verify ssDNA immobilization and hybridization efficiency. Furthermore, gold nanoparticles were used to enhance the signal of the electrical impedance. In this study, we selected different frequency of the impedance measurement, different width of the gap and the sizes of the nanoparticles to reveal the optimum operation condition of the electrical gene chip. As a result, at the frequency of 100k Hz, particle size of 20nm and the gap width of 5mm, the chip has the difference of 16 times by electrical impedance in hybridization and non-hybridization.
關鍵字(中) ★ 金奈米
★ 去氧核醣核酸
★ 電性偵測
★ 基因晶片
關鍵字(英) ★ Gold nanoparticles
★ DNA
★ Electrical detection
★ Gene chip
論文目次 中文摘要................................ Ⅰ
Abstract................................ Ⅱ
圖目錄.................................. Ⅶ
表目錄.................................. Ⅹ
第一章 前言............................. 1
第二章 文獻回顧......................... 3
2.1. 生物晶片簡介....................... 3
2.1.1 去氧核醣核酸分子.................. 5
2.1.2 基因晶片檢測技術與發展............ 8
2.1.3 目前國內外基因晶片的產業現況...... 9
2.2. 表面改質與生物分子固定化........... 16
2.2.1. 分子自組單層膜................. 17
2.2.1.1. 硫醇類分子自組單層膜........... 19
2.2.1.2. 矽烷類或矽氧類分子自組單層膜... 21
2.2.2. 不同的生物分子之性質與固定化... 24
2.2.2.1. 去氧核醣核酸固定化............. 25
2.2.2.2. 蛋白質和抗體固定化............. 31
2.2.2.3. 生物膜的固定化................. 34
2.2.2.4. 去氧核醣核酸固定於蛋白質....... 36
2.3. 光學原理偵測生物分子交互作用....... 37
2.3.1. 金奈米粒子....................... 37
2.3.2. 表面電漿共振儀................... 41
2.3.3. Guided-Mode Resonance biosensor (GMR)43
2.4. 電性偵測原理....................... 45
第三章 實驗藥品與儀器設備............... 47
3.1 實驗藥品............................ 47
3.2 實驗儀器設備........................ 49
3.2.1表面化學分析電子能譜儀............. 49
3.3 實驗目的............................ 51
3.4 實驗方法............................ 52
3.4.1 實驗溶液配製...................... 52
3.4.1.1 緩衝液.......................... 52
3.4.2 實驗步驟.......................... 52
3.4.2.1 奈微米構槽基因晶片製備.......... 52
3.4.2.2 基材與晶片表面的清洗方法........ 54
3.4.2.2 去氧核醣核酸固定化與驗證........ 55
3.4.2.3 金奈米與不同官能基的吸附........ 56
3.4.2.4 固定化與雜交反應之最適化反應時間 57
3.4.2.5 電性量測實驗.................... 58
第四章 結果與討論....................... 59
4.1 Mfold模擬核酸序列之結果............. 59
4.2 去氧核醣核酸固定化之驗證............ 62
4.2.1 接觸角量測實驗.................... 62
4.2.2 原子力顯微鏡...................... 63
4.2.3 化學分析電子光譜儀................ 66
4.3.1 表面胺基之定量.................... 73
4.3.2 去氧核醣核酸固定量與雜交量之探討.. 75
4.3.3固定化及雜交時間之最適化........... 77
4.4 奈米粒子非專一性的吸附問題.......... 80
4.5 電性量測之探討...................... 85
4.5.1 背部電容值的影響................ 85
4.5.2 電性量測去氧核醣核酸雜交行為之結果 87
4.5.3 以不同頻率電性量測比較............ 88
4.5.4 以不同尺寸金奈米標定之比較........ 90
4.5.6 DNA雜交於不同寬度的微奈米溝槽之影響 95
4.6 Target DNA濃度對阻抗值之影響效應.... 97
第五章 結論與建議....................... 100
參考文獻................................ 103
圖目錄
圖2.1 (A)雙股DNA結構(B)不同種類去氧核醣核酸的鹼基 7
圖2.2 分子自組單層膜的組成........... 17
圖2.3 不同型種類的硫醇分子........... 20
圖2.4 硫醇分子吸附於金膜表面的機制... 20
圖2.5 不同種類的矽烷或矽氧化合物..... 21
圖2.6 Sagiv對於矽烷化合物水解機制的示意圖 22
圖2.7 Rye對於矽烷化合物水解機制的示意圖 23
圖2.8 硫醇修飾之去氧核醣核酸於金膜固定化 25
圖2.9 胺基修飾之去氧核醣核酸於金膜固定化 25
圖2.10 去氧核醣核酸分子於金膜上固定化. 26
圖2.11 三種具胺基官能基之矽氧化合物... 29
圖2.12 利用偶合劑將去氧核醣核酸分子固定化 29
圖2.13 二階段式的去氧核醣核酸分子固定化 31
圖2.14 抗體的結構圖................... 33
圖2.15 抗體之方位性的固定方法於二氧化矽 33
圖2.16 抗體之方位性的固定方法於金膜... 33
圖2.17 生物膜於金膜上之固定化......... 35
圖2.18 去氧核醣核酸分子於蛋白質上的固定化 36
圖2.19 金奈米奈子示意圖............... 39
圖2.20 以奈米金標定去氧核醣核酸雜交前後顏色的變化...................................... 40
圖2.21 SPR偵測示意圖.................. 42
圖2.22 GMR結構圖...................... 44
圖2.23. GMR的量測系統.................. 44
圖2.24. 並聯的電阻與電容結構............ 46
圖3.1. 表面化學分析電子能譜儀示意圖.... 50
圖3.2. 電子式基因晶片光罩圖............ 54
圖3.3. 表面改質與固定化流程圖.......... 56
圖4.1. 以Mfold預測核酸序列Target DNA之Hairpine構造及計算所得之.......................... 60
圖4.2. 以Mfold預測核酸序列Target DNA之Hairpine構造及計算所得之.......................... 61
圖4.3. 每一步改質晶片的表面形貌(A)經由酸洗後(B)經由矽氧化合物形成表面分子自組單層膜(C)接上SMPB Crosslinker(D)固定上Capture DNA (E)雜交Target DNA (F)雜交Probe DNA (G)有雜交以金奈米粒子標定(5mm×5mm) (H)無雜交以金奈米標定(5mm×5mm) (I)有雜交以金奈米粒子標定(1.2mm×1.2mm) (J)無雜交以金奈米標定(1.2mm×1.2mm)........................ 65
圖4.4. 二氧化矽基材上之化學分析(A)Si 2p3之分析 (B) P 2p3之分析 (C) N 1s之分析 (D)C 1s之分析 (E) O 2s之分析............................... 67
圖4.5. DETA於二氧化矽基材上形成分子自組單層膜之化學分析(A)Si 2p3之分析 (B) P 2p3之分析 (C) S 2p之分析 (D)C 1s之分析 (E) N 1s之分析 (F) O 2s之分析....................................... 68
圖4.6. SMPB於二氧化矽基材上形成分子自組單層膜改質之化學分析(A)Si 2p3之分析 (B) P 2p3之分析 (C) S 2p之分析 (D)C 1s之分析 (E) N 1s之分析 (F) O 2s之分析................................... 69
圖4.7. Capture DNA固定於於二氧化矽基材之化學分析(A)Si 2p3之分析 (B) P 2p3之分析 (C) S 2p之分析 (D)C 1s之分析 (E) N 1s之分析 (F) O 2s之分析 70
圖4.8. 比較表面改質與生物分子固定化四步驟的S 2p元素分析.................................. 71
圖4.9. 比較表面改質與生物分子固定化四步驟的P 2p3元素分析............................... 71
圖4.10. 比較表面改質與生物分子固定化四步驟的N 1s元素分析.................................. 72
圖4.11. Cibacron blue 檢量線............. 74
圖4.12. Cibacron blue結構式.............. 74
圖4.13. Capture DNA檢量線................ 76
圖4.14. Target DNA檢量線................. 76
圖4.15. Probe DNA 檢量線................. 77
圖4.16. 時間對上層溶液(Capture DNA)吸收值的關係圖........................................ 78
圖4.17. 時間對上層溶液(Target DNA)吸收值的關係圖........................................ 78
圖4.18. 時間對上層溶液(Probe DNA)吸收值的關係圖........................................ 79
圖4.19. 金奈米吸附情形於(A) 烷基官能基(B) 羥基官能基...................................... 82
圖4.20. 金奈米吸附情形(A)羧基(B)胺基..... 83
圖4.21. (A)Capture DNA 吸附金奈米情形(B)以鹽溶液沖提Capture DNA吸附之表面................. 84
圖4.22. (A)晶片示意圖(B)基因晶片之電路示意圖 86
圖4.23. 20nm金奈米標定頻率對有無阻抗值關係圖 89
圖4.24. 圖 2nm金奈米標定頻率對有無阻抗值關係圖......................................... 90
圖4.25. 以原子力顯微鏡觀察2nm金奈米於奈微米溝槽內分佈情形(A)無雜交(B)有雜交............... 92
圖4.26. 以原子力顯微鏡觀察20nm金奈米於分佈情形(A)有雜交(B)有雜交-溝槽內的分佈(C)無雜交(D)無雜交-溝槽內的分佈............................ 93
圖4.27. 100nm金奈米分佈情形(A)有雜交(B)無雜交......................................... 94
圖4.28. 2mm溝槽內金奈米粒子的分佈......... 96
圖4.29. 3mm溝槽內金奈米粒子的分佈......... 96
圖4.30. 5mm溝槽內金奈米粒子的分佈......... 97
圖4.31. 金奈米分佈情形(A)1pM(B)10pM(C)1nM. 99
圖4.32. Target DNA濃度對阻抗值關係圖...... 99
表目錄
表2.1. 微陣列晶片(microarray chip)及微處理晶片比較表..................................... 4
表2.2. 國內外主要基因晶片廠商與技術...... 10
表2.3. 不同方式生物分子固定化的比較...... 16
表2.4. 矽氧化合物於不同基材形成分子單層膜之活性測試....................................... 24
表2.5. 不同矽氧化合物官能基與去氧核醣核酸固定化比較....................................... 27
表2.6. 蛋白質固定化之比較................ 32
表2.7. 還原劑與還原之金奈米粒徑.......... 40
表3.1 實驗所使用去氧核醣核酸分子序列表... 48
表4.1 表面改質後接觸角之比較............ 63
表4.2 表面胺基固定量.................... 74
表4.3 雜交效率比較表.................... 76
表4.4 以金奈米20nm量測總阻抗值之差異性.. 86
表4.5 以電性量測未標定去氧核醣核酸於不同溝槽之差異性比較表............................... 87
表4.6 20nm金奈米標定頻率對有無阻抗值比較表 88
表4.7 2nm金奈米標定頻率對有無阻抗值比較表 89
表4.8 金奈米粒徑大小對有無雜交的影響比較表 91
表4.9 微奈米溝槽寬度對差異性的提升...... 95
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指導教授 陳文逸(Wen-Yih Chen) 審核日期 2005-6-23
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