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系統識別號 U0026-0812200914325604
論文名稱(中文) 結合銀增強技術與奈米金-鏈黴卵白素複合體搭配光學與阻抗式晶片於DNA雜交分析
論文名稱(英文) Study of a DNA Hybridization Assay Based on Grayscale and Impedance Measurements Used Silver Enhancement and Gold-streptavidin
校院名稱 成功大學
系所名稱(中) 奈米科技暨微系統工程研究所
系所名稱(英) Institute of Nanotechnology and Microsystems Engineering
學年度 96
學期 2
出版年 97
研究生(中文) 張譽懷
研究生(英文) Yu-Huai Chang
學號 q2695408
學位類別 碩士
語文別 中文
論文頁數 138頁
口試委員 口試委員-張長泉
口試委員-林弘萍
指導教授-林裕城
中文關鍵字 奈米金-鏈黴卵白素  生物晶片  微機電技術  DNA 雜交技術  銀析出現象 
英文關鍵字 MEMS technology  silver enhancement reaction  DNA hybridization  biochip  gold-streptavidin 
學科別分類
中文摘要 本研究提出新型DNA雜交反應訊號分析方法,結合微機電技術與生物晶片科技,同時利用奈米金-鏈黴卵白素複合體(gold-streptavidin)與銀析出增強溶液放大反應偵測訊號,並配合金屬銀析出沉澱之反應,以光學式與阻抗式偵測系統來量測訊號,有別於傳統以酵素、螢光或放射性同位素標識呈色方法,建立一新型DNA雜交反應分析系統,提供另一種有效且方便的DNA分析檢測方式。在DNA樣品選擇上,選擇以鮑氏不動桿菌(Acinetobacter baumannii)作為DNA樣品來源,主要是因為臨床上鮑氏不動桿菌發生院內感染率很高。

此外,在微晶片上執行DNA雜交反應具有改良多樣性、低樣品用量與高靈敏度等特性,且可以縮短偵測反應的時間。實驗架構上,同時利用生物素-鏈黴卵白素(biotin-streptavidin)具有專一性鍵結能力,且奈米粒子具有催化銀析出增強溶液特性,作為放大訊號理論基礎,並採用3-環氧丙三甲基矽氧烷(3-glycidoxypropyl-trimethoxysilane, 3-GPS)進行晶片表面化學修飾,並將具有專一性序列探針固定在晶片表面,對待測DNA樣品作定性與定量之分析,加入銀析出增強溶液,來增顯雜交反應的偵測訊號,同時利用光學式灰階掃描與阻抗式電阻量測二種方法,來擷取實驗結果的影像及數據。

本研究方法已可完成DNA雜交的步驟與目標,光學式與阻抗式對DNA樣品之偵測靈敏度分別達到8.25 ng/mL與8.25×10-1 ng/mL,且使用的偵測方式也可搭配銀析出現象產生可供檢測之訊號,大幅縮短檢驗時間。此外,在實驗中將討論DNA雜交參數與靈敏度等問題,同時利用生物分子特性、化學性修飾、奈米粒子技術與銀訊號增強等方法,建立一個全新、快速且靈敏之DNA雜交訊號分析系統,未來將提供DNA分析檢測一個全新的方向與思維。
英文摘要 This study reports a novel DNA hybridization assay method which binding micro-electro mechanical systems (MEMS) and biochip technology. Using gold-streptavidin and silver enhancement solution, as a catalyst for silver precipitation, magnify hybridization detection signals. Finally, using optical and electric system detect signals. Compared with traditional labeling methods such as enzyme, fluorescence or radioisotope, we supply a new DNA hybridization assay system that is simpler, cheaper and more convenient. We choose Acinetobacter baumannii as DNA sample source, because the occurrence of bacteremia by Acinetobacter baumannii for glucose nonfermenters on clinical diagnosis is maximum.

Furthermore, DNA hybridization reacted on micro-chip having advantage of low sample waste, high sensitivity and short detection time. In experiment scheme, we combine the specificity binding ability of biotin-streptavidin and catalyst for silver precipitation ability of nanoparticles as the foundation theory. At the same time, used 3-glycidoxypropyl-trimethoxysilane (3-GPS) to modify the surface of slide chip and immobilize DNA probes with specificity sequence on slide chip. DNA sample are used to quantify and qualify test rely on silver enhancement method with optical and electric system to get images and data of experiment results.

The relationship between sample concentration and detection signal is discussed and the detection limit for the DNA sample are 8.25 ng/mL and 8.25×10-1 ng/mL in optical and electric system respectively. In addition, the parameter, specificity and sensitivity of the DNA hybridization assay chip are discussed. We combine bio-molecule, chemical modification, nano-technology and silver enhancement to achieve a novel DNA hybridization system. The high applicability and biochemical efficiency of this study can provide an alternative for rapid, sensitive and convenient DNA hybridization assay method.
論文目次 中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖目錄 X
表目錄 XXII
第一章 序論 1
1-1 研究背景 1
1-2 文獻回顧 4
1-2-1 DNA雜交分析基本理論 4
1-2-2 奈米粒子訊號放大研究之發展 6
1-3 研究目的與動機 16
1-4 實驗架構 19
1-5 研究策略 20
第二章 材料與方法 22
2-1 實驗藥品介紹 22
2-2 實驗材料製備與方法 24
2-2-1 質體DNA萃取技術(DNA extraction) 24
2-2-2 聚合酶鍊反應(Polymerase Chain Reaction, PCR) 27
2-3 銀增強溶液析出之反應原理 29
2-4 實驗偵測儀器介紹與原理 31
2-4-1 光學式DNA雜交偵測平台之建立與操作歩驟 31
2-4-2 阻抗式DNA雜交偵測平台之建立與操作歩驟 36
第三章 DNA雜交反應晶片之設計與製作 38
3-1 晶片結構與設計 38
3-1-1 光學掃描式晶片 38
3-1-2 電性阻抗式晶片 39
3-2 光罩與設計 42
3-3 晶片製程 45
3-3-1 基材清洗 46
3-3-2 金屬蒸鍍 49
3-3-3 黃光微影製程 51
3-3-4 金屬蝕刻 55
3-3-5 表面修飾 58
3-3-6 雜交反應區製作 59
第四章 銀析出DNA雜交反應之結果與討論 63
4-1 基材選擇 64
4-2 晶片表面修飾 65
4-3 質體DNA萃取之結果與濃度換算 66
4-4 光學式DNA雜交反應分析實驗 68
4-4-1 探針濃度對應灰階值之實驗與結果 70
4-4-2 奈米金-鏈黴卵白素複合體反應時間與濃度之實驗與結果 72
4-4-3 奈米金-鏈黴卵白素反應溫度對應灰階值之實驗與結果 86
4-4-4 DNA雜交反應時間對應灰階值之實驗與結果 89
4-4-5 DNA雜交反應溫度對應灰階值之實驗與結果 90
4-5 DNA雜交樣品濃度對應灰階值之實驗與結果 94
4-6 不同DNA雜交樣品對應灰階值之探討與驗證 98
4-6-1 不同DNA樣品對灰階值之影響 98
4-6-2 DNA樣品特異性之探討 100
4-6-3 同種不同株與同屬不同種特異性之探討 105
4-7 電性阻抗式DNA雜交反應分析實驗 108
4-7-1 微電極DNA雜交分析晶片 108
4-7-2 奈米金-鏈黴卵白素濃度對應阻抗值之實驗與結果 109
4-8 DNA雜交樣品濃度對應阻抗值之實驗與結果 113
4-9 不同DNA雜交樣品對應阻抗值之探討與驗證 116
4-9-1不同DNA樣品對阻抗值之影響 116
4-9-2 DNA樣品特異性之探討 118
4-9-3同種不同株與同屬不同種特異性之探討 120
第五章 結論與建議 123
5-1 結論 123
5-2 建議 127
5-3 未來與展望 128
參考文獻 129
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