在過去的二十年中，奈米孔吸引了越來越多學術研究的注意，其檢測屬於單分子實時檢測技術(Single molecule real time (SMRT) method)，相較於傳統的檢測方法，在體積、成本、通量、讀長和準確度等皆具有優勢，當檢測物濃度極低，甚至是需要精準計算溶液中所含之待測物分子數量時，傳統方法尚不具有足夠之檢測空間解析度來獲得上述所需數據，透過單個分子通過單個奈米孔洞，進而在離子電流中產生可檢測的臨時阻塞，奈米孔分析反映了它的簡單性，而可檢測的分析物範圍從核酸、肽鏈、蛋白質和生物分子復合物到有機聚合物。
目前可以從文獻中獲得大量奈米孔相關的知識基礎，包括材料的選擇、奈米孔的製程到奈米元件整合、感測器訊號量測，有別於早期的奈米孔發展是以生物性薄膜為主，因其結構穩定性、孔洞一致性等問題逐漸開始被固態奈米孔所取代，在過去十年中，已經發展許多於固態膜中製造奈米孔的技術。當中，為因應不同的感測需求且追求以更有效率、方便的方式製作奈米孔，奈米孔製程不再侷限使用高能離子束或者電子束，做為較新穎的電擊穿奈米孔加工製程在一些文獻中已展示顯著降低了奈米孔製造的複雜性和成本的優勢，為固態奈米孔元件的製造提供了新的選擇，但其製程所面臨的困境為無法如聚焦離子束或者穿隧式電子顯微鏡精準的將奈米孔定位。
本論文預計將兩種奈米孔製程-鑽孔及電擊穿整合，以聚焦離子束在奈米孔薄膜上人工製作缺陷，再通過多次介電擊穿於定位位置形成奈米孔洞，探討固態電子式奈米孔製作之精確性，最後將其應用在生物單分子，透過離子電流訊號希望能夠辨識出在低濃度環境下單一分子通過時的特徵訊號，以期待達到實時、精確和高通量之相關裝置發展目標。

Over the past two decades, nanopore, with the capability of single molecule real time (SMRT) technique, has drawn more and more scientific interests. Compared with traditional methods, nanopore-based diagnostic tool could offer various advantages, such as high sensitivity, small sizes, and low cost. The most attractive is nanopore is able to detect target molecules at very low concentrations from very small sample volumes, even if it is necessary to accurately calculate the number of molecules in the solution, that traditional methods are unable to reach the level with not high enough resolution. As a single molecule enters a single nanopore, it interrupts the ionic current through the pore, resulting in a detectable temporary current blockage. Nanoporebased sensing allows for the detection of a wide range of analytes from nucleic acids and peptide to protein and bio-molecular complexes in a simple way.
There is a large number of literature based around nanopore, including membrane material selection, nanopore fabrication, system integration, and sensor signal measurement. Most of the early nanopore-based sensors adopted biological nanopores, solid-state nanopores have been gradually growing in popularity due to structural stability and pore consistency. Many approaches for the fabrication of solid-state nanopores have been developed in these years. To date, solid-state nanopores have been fabricated primarily through a focused-electronic or ions beam, however, they have been tried to substitute by other nanofabrication strategies because of the need to generate nanopore in a more efficient and convenient way. As a novel method, multilevel pulse electrical breakdown has shown the potential in reducing the complexity and cost of nanopore fabrication.
In this paper, we report an accurate fabrication of solid-state nanopores with integrating two nanopore fabrications – ions beam drilling and electrical breakdown. Nanopore was fabricated through multilevel pulse voltage at the direct position of artificial defect which is previously manufactured on the membrane by focusing the ion beam. We envision this fabrication strategy would improve accuracy of nanopore fabrication but retain the advantage in simplicity and low-cost. Furthermore, may be used for further single-molecule experiments.

目錄
摘要 i
Abstract ii
誌謝 iv
目錄 v
圖目錄 viii
表目錄 xi
第一章 緒論 1
1.1 單分子檢測 1
1.1.1 生物單分子檢測需求 1
1.1.2 奈米孔生物分子感測器 3
1.1.3 傳統DNA定序檢測 5
1.2 奈米孔感測器 9
1.2.1 生物性奈米孔 10
1.2.2 固態式奈米孔 11
1.2.3 生物性及固態式奈米孔比較 13
1.3 固態式奈米孔製程 15
1.3.1 常見奈米孔製程 15
1.3.2 電擊穿奈米孔製程 21
1.3.3 奈米孔精確性之必要性 23
1.4 不確定性分析 24
1.5 研究動機 25
1.6 研究目的 26
1.7 論文架構 27
第二章 固態式奈米孔感測器 29
2.1 固態式奈米孔架構 29
2.1.1奈米孔晶片架構 29
2.1.2奈米孔流道架構 30
2.2 電擊穿製程穿孔機制 32
2.3 奈米孔離子流數學模型 34
2.4 奈米孔感測機制 35
2.5電性量測系統與訊號分析 36
2.5.1奈米孔晶片IV定性量測 37
2.5.2離子流訊號量測 37
2.6 DNA螢光標記 38
2.6.1 YOYO-1螢光標記機制 38
2.6.2 YOYO-1螢光溶液配置 39
第三章 定位電擊穿奈米孔製程 41
3.1 定位電擊穿奈米孔製程 41
3.1.1 聚焦離子束製程參數 41
3.1.2 電壓脈衝製程穿孔參數 44
3.1.3 奈米孔電流(I)-電壓(V) 定性量測 47
3.1.4 奈米孔孔徑估算 49
3.1.5 奈米孔觀測 52
3.2 電壓脈衝探討 58
3.2.1 擴孔 58
3.2.2 雕刻 59
3.3 奈米孔徑不確定性分析 61
3.3.1 未平衡之電流訊號 61
3.3.2 離子濃度 64
第四章 單分子檢測之易位螢光觀測及訊號量測 70
4.1奈米孔試片說明 70
4.2 DNA單分子檢測於奈米孔晶片之螢光觀測 72
4.2.1 螢光觀測實驗配置方式 72
4.2.2 DNA於奈米孔試片堵塞螢光觀測結果 73
4.2.3 DNA於奈米孔試片之易位行為螢光觀測結果 76
4.3 DNA單分子檢測於奈米孔晶片之訊號量測 79
4.3.1 訊號量測實驗配置方式 79
4.3.2 DNA於奈米孔試片之易位行為訊號量測結果 80
4.4 DNA單分子檢測於定位電擊穿製成之奈米孔晶片訊號量測 88
第五章 總結與未來發展 92
5.1 總結 92
5.2 研究成果 93
5.3 學術貢獻點 96
5.4 未來研究建議方向 102
附錄 105
附錄A：微訊號量測儀－Agilent B2912A 105
附錄B：膜片鉗放大器 - MultiClamp 700B Microelectrode Amplifier 106
附錄C：資料擷取器 - USB 6361 107
附錄D：聚焦離子束－FEI Helios Nanolab 600i System 108
附錄E：螢光顯微鏡－Zeiss Axioplan 2 109
附錄F：螢光顯微鏡－OPTIKA IM-3FL 110
附錄G：DNA螢光染料 - Invitrogen™ YOYO™-1 Iodide (491/509) 111
參考資料 112
著作發表 119
作者簡介 120

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