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研究生:鄭存孝
研究生(外文):Cun-Xiao Zheng
論文名稱:紫膜生物光電晶片檢測寡核苷酸與即時監測基因合成之應用
論文名稱(外文):Application of purple-membrane photoelectric biochip in detecting oligonucleotide and monitoring DNA synthesis
指導教授:陳秀美陳秀美引用關係
指導教授(外文):Hsiu-Mei Chen
口試委員:蔡伸隆曾文祺
口試委員(外文):Shen-Long TsaiWen-Chi Tseng
口試日期:2017-07-28
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:144
中文關鍵詞:基因合成奈米金紫膜
外文關鍵詞:DNA synthesisAuNPsPM
相關次數:
  • 被引用被引用:4
  • 點閱點閱:110
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古生噬鹽紫菌Halobacterium salinarum紫色細胞膜 (purple membrane, PM) 內存在有一跨膜蛋白-細菌視紫質 (bacteriorhodopsin, BR) ,為光驅動質子泵 (light-driven proton pump),受光後可吸收光能使質子從細胞膜內被推往細胞膜外,產生跨膜質子濃度梯度,可進而產生光電流訊號。本研究首先利用PM光電流訊號與入射光強度間成正關係及奈米金 (gold nanoparticle, AuNPs) 可遮光之事實,以固定化有單股探針基因DNA1之b-PM晶片 (ssDNA1-bPM晶片) 對不同濃度之標誌有AuNPs的單股目標基因DNA2 (ssDNA2-AuNPs) 分別進行注流式即時與靜態檢測,以光電流訊號變化差異找出最低檢測濃度,並探討AuNPs存在對檢測靈敏度之影響。在25 μL/min注流式即時檢測下,可檢測30 pM ssDNA2-AuNPs,而在靜態下檢測ssDNA2-AuNPs時比起對ssDNA2時之檢測具有有更佳靈敏度。其次利用固定化有單股引子基因DNA1'之ssDNA1'-bPM晶片對單股目標基因DNA3'進行雜合,並加入dNTP與Klenow Fragment (3' → 5' exo-) polymerase,靜態下進行基因合成之即時監測,結果發現,PM晶片可感應基因合成時所釋放之質子,而使其光電流下降。同一晶片至少可連續進行兩個鹼基之合成,證明PM晶片可應用於基因序列分析。
The purple membrane (PM) of halophilic archaeon Halobacterium salinarum contains a transmembrane protein, bacteriorhodopsin (BR), which is a light-driven proton pump capable of transporting a proton across the membrane upon illumination and subsequently generating photocurrents. Based on light scattering of gold nanoparticle (AuNPs) as well as the linear dependence of PM photocurrents on illumination intensities, a PM chip coated with single-stranded probe DNA1 was used to detect another AuNPs-conjugated single-stranded target DNA2 (ssDNA2-AuNPs) at different concentrations under both real-time flow-injection and static conditions, using PM photocurrents as the study parameter. The minimum concentration of ssDNA2-AuNPs to be detected at 25 μL/min was 30 pM, and the detection sensitivity for DNA2-AuNPs under a static condition was higher than that for ssDNA2. In addition, another PM chip coated with single-stranded primer DNA1' was used to capture single stranded target DNA3'; then the resulting PM-gene complex chip was used to real-time monitor DNA synthesis under a static condition, following the addition of a mixture containing dNTP and Klenow Fragment (3' → 5' exo-) polymerase. This complex chip sensed the protons produced during DNA synthesis and exhibited a reduction in photocurrent. At least two consecutive nucleotide incorporation cycles were detected by using a single PM-gene complex chip, demonstrating application feasibility of PM chips on gene sequencing.
中文摘要 I
英文摘要 II
目錄 III
表目錄 VI
圖目錄 IX
第一章 緒論 1
第二章 文獻回顧 2
2-1 Halobacterium salinarum與bacteriorhodopsin 2
2-1-1 BR結構 3
2-1-2 BR光循環與質子傳遞 4
2-1-3 BR光電響應 6
2-1-4 PM生物親和性單層固定化 9
2-1-5 PM晶片微生物檢測應用 11
2-2 金奈米粒子(gold nanoparticles,AuNPs)特性 13
2-2-1 AuNPs表面電漿共振吸收與消散特性 14
2-2-2 AuNPs於DNA檢測應用 18
2-2-2-1 以AuNPs為基礎的比色法 19
2-2-2-2 以AuNPs為基礎的電化學法 20
2-2-3 AuNPs應用於PM晶片 23
2-3 DNA定序方法比較 24
2-3-1 Roche 454 sequencing 26
2-3-2 Illumina (Solexa) sequencing 26
2-3-3 Ion-torrent sequencing 29
2-4 DNA定序時之重要參數 33
2-4-1 Klenow Fragment (3'→5' exo-) polymerase 33
2-4-2 pH值對Klenow fragment polymerase影響 34
2-4-3 Klenow fragment polymerase的短引子延伸 (short primer extension) 36
第三章 實驗 43
3-1 實驗目的與說明 43
3-2 實驗流程 46
3-3 量測 51
3-3-1 以三明治量測槽靜態下量測b-PM晶片光電流 51
3-3-2 以白金負電極微流道注流式即時量測b-PM光電流與ssDNA2-AuNPs檢測 52
3-3-3 以三明治量測槽靜態即時監測基因序列合成 53
第四章 結果討論 55
4-1 以模擬決定寡核苷酸固定化條件 55
4-1-1 固定Na+濃度下改變寡核苷酸固定化反應溫度 55
4-2 Tween 20添加對以ssDNA1-bPM晶片注流式即時檢測ssDNA2-AuNPs之影響 67
4-3 以ssDNA1-bPM晶片注流式即時檢測不同濃度之ssDNA2-AuNPs 70
4-4 以不同架橋製備ssDNA1-bPM晶片對ssDNA2-AuNPs靜態檢測靈敏度影響 82
4-5 探討ssDNA1-bPM晶片分別對AuNPs、ssDNA2與ssDNA2-AuNPs靜態檢測之差異 85
4-6 靜態即時監測基因合成時,反應溶液對b-PM晶片光電流密度之影響 89
4-6-1 ssDNA3'雜合於ssDNA1'-bPM晶片上之最適化時間探討 94
4-6-2 b-PM晶片在Buffer B、C、D溶液中進行靜態即時監測基因序列合成比較 95
4-6-3 b-PM晶片在Buffer D(pH 8.0)內進行靜態即時監測基因序列合成三循環 (三重複晶片實驗) 112
第五章 結論 120
第六章 參考文獻 122
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