博碩士論文 103324040 詳細資訊




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姓名 蔡致勤(Chih-Chin Tsai)  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 使用不帶電中性核酸探針於矽奈米線場效電晶體檢測去氧核醣核酸與微核醣核酸之研究
(Silicon nanowire field-effect biosensor for DNA and microRNA detection using neutralized-DNA (nDNA) probes)
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摘要(中) 在最近幾年,奈米場效電晶體已成為一個在生物醫學與檢測應用上面的強力工具。因為奈米場效電晶體有快速反應時間、高靈敏性、高專一性、體積小、即時偵測等優點。加上由於現行傳統的生物辨識技術如qPCR 及微陣列檢測基因序列,都須要加入螢光分子來標記這些生物分子,而奈米場效電晶體的非標定直接量測就可以避免因螢光標定等所造成之困擾。另外奈米場效電晶體的最低感測極限可以達到fM等級,因此已有相當多的研究團隊想利用此工具應用在感測基因序列上面並且希望藉由此能建立起一種較簡單的方法在重大疾病診斷上。然而這個方法在基因檢測上還是存在幾個難題。主要為溶液的離子強度會影響德拜長度(Debye length) ,並直接來影響奈米場效電晶體的感測。在本研究中我們專注在使用不帶電磷酸甲基化DNA(nDNA)探針來提升場效電晶體對DNA的感測還有探討相關的感測機制。不帶電磷酸甲基化DNA探針與目標核酸序列之間不存在靜電排斥力,因此造成不帶電磷酸甲基化DNA探針與目標核酸序列在低鹽濃度下,比帶電DNA探針容易地雜交形成雙股螺旋結構。我們也會設計部分修飾nDNA的探針來進一步增進奈米場效電晶體的檢測。從感測在不同鹽離子強度下、不同濃度的目標序列下還有非專一性控制組實驗,可以發現部分修飾nDNA的探針不僅能提高感測的靈敏度,還能增加與目標序列彼此之間的專一性。另外我們也會利用nDNA探針來檢測microRNA並探討與檢測DNA之間的不同。最後我們成功地架設奈米場效電晶體微陣列系統來檢測microRNA,並且從同一片晶片檢測不同序列的microRNA,我們可以看到奈米場效電晶體微陣列系統於檢測基因序列的可行性。
摘要(英) In recent years, nanowire field-effect transistor (NWFET) has been a powerful tool for the application of biomedical science, because its fast response time, high sensitivity and specificity, small size, real time detection, etc. And nowadays, conventional technics like qPCR microarray detects gene sequences, they need to add fluorescence to label the biomolecules. However,there is no use of labelling biomolecules in NWFET ,so it doesn’t have the side effect of labelling. In addition, the limit of detection (LOD) of NWFET can reach to femtomolar regime. So researchers want to apply it to gene sequencing and hope to find an easy way to disease diagnosis. While there are still some difficulties in this method, especially for DNA and microRNA detecting. In general, the main factor called Debye length mainly affect the sensing. In this study, we focus on using neutralized DNA (nDNA) probes which is uncharged to improve DNA detecting using FET biosensors and study the related mechanism. Neutralized DNA (nDNA) probes which is uncharged can form duplex with target sequence more easily than DNA probes in low salt concentration buffer, because there is less repulse force between the two sequences. We also designed some partially modified nDNA probes to improve our sensing. From the sensing in different ionic strength, different target concentrations sensing and non-specific control tests show that partially modified nDNA probes can not only enhance sensitivity but also improve specificity. In addition, we detect microRNAs using nDNA probes and find the difference between DNAs and microRNAs detecting. Finally, we successfully set up a SiNWFET microarray system to detect microRNAs. And from the experiments of sensing different microRNAs in one chip, we see the feasibility of detecting different microRNAs using SiNWFET microarray system.
關鍵字(中) ★ 奈米場效電晶體
★ 核醣核酸
★ 微核醣核酸
關鍵字(英) ★ Nano-wire Field effect transistor
★ DNA
★ micro-RNA
論文目次 摘要 i
Abstract ii
誌謝 iv
目錄 v
圖目錄 ix
表目錄 xiv
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機 2
1.3 論文架構 4
第二章 文獻回顧 7
2.1 生物檢測器量測核酸的發展 7
2.2 矽奈米線場效電晶體運作原理 8
2.3 DNA與microRNA之檢測 13
2.3.1 DNA的結構 13
2.3.2 DNA之檢測於奈米場效電晶體 17
2.3.3 RNA之結構 19
2.3.4 microRNA的介紹 20
2.3.5 microRNA之檢測於奈米場效電晶體 21
2.4 量測核酸之探針種類 23
2.4.1 肽核酸 24
2.4.2 鎖核酸 25
2.4.3 嗎啉基寡核苷酸 27
2.4.4 磷酸甲基化去氧核醣核酸 28
2.5 緩衝溶液鹽離子濃度與Debye–Hückel theory 31
2.5.1 溶液鹽離子濃度對於核酸雜交的影響 31
2.5.2 Debye length與溶液鹽離子濃度的關係 32
2.5.3 鹽濃度與Debye length於場效電晶體量測的影響 34
第三章 實驗藥品、儀器設備與方法 40
3.1 實驗藥品 40
3.2 晶片製造過程 41
3.3 晶片表面化學修飾 43
3.3.1 晶片表面清洗 43
3.3.2 氧電漿處理 43
3.3.3 修飾 APTES (3-Aminopropyltriethoxysilane) 44
3.3.4 修飾Glutaraldehyde 44
3.3.5 核酸探針固定化 44
3.4 X射線光電子能譜學 (XPS) 分析 45
3.5 圓二色光譜儀測定構型 46
3.6 微流道系統 47
3.7 晶片量測系統 48
3.8 量測流程 51
3.9 數據處理 52
3.10 點片機系統 55
3.11 萃取total RNA 57
第四章 結果與討論 59
4.1 溶液中SiNWFET量測曲線 59
4.2 XPS表面分析 60
4.3 部分修飾nDNA以圓二色光譜儀測定構型 61
4.4 鹽離子濃度對於量測DNA的影響 65
4.4.1 比較nDNA 與DNA探針訊號上的差異 65
4.4.2 比較nDNA 與DNA探針訊雜比的差異 71
4.4.3 nDNA-p4與nDNA-p5探針設計 74
4.5 針對DNA樣品濃度的檢測 75
4.5.1 不同探針之間的訊號差別 75
4.5.2 極低DNA樣品濃度的檢測 77
4.6 鹽離子濃度對於量測microRNA的影響 81
4.6.1 不同鹽濃度對於DNA-microRNA雜交上的影響 81
4.6.2 不同探針檢測microRNA的影響 88
4.7 奈米線微陣列晶片檢測microRNA 90
4.7.1 3X3微陣列檢測microRNA 90
4.7.2 同一晶片檢測不同microRNA與濃度檢量線 95
4.7.3 檢測T24 cell total RNA 101
4.7.4 實驗晶片整體良率 102
第五章 結論與未來展望 104
5.1 結論 104
5.2 未來展望 105
第六章 參考文獻 107
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指導教授 陳文逸(Wen-Yih Chen) 審核日期 2016-7-25
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