生物分子檢測是生醫領域重要的發展技術。該類技術的提昇可推動疾病預測、精準醫療等願景之實現。傳統生物分子檢測技術多被認為存在檢測時間太長、操作須由實驗專業人員進行、設備昂貴、受干擾物影響大等缺點。而一般生醫感測器雖然有檢測物免標記、靈敏性高、體積小等優勢，然而當檢測物濃度極低或是需要精準計算待測分子數量時，則已不具有足夠之空間解析度來獲取資訊。
　　固態式奈米孔感測器因具備接近單分子等級之空間解析能力，陸續在蛋白質檢測、細菌型態辨別、DNA定序等方面屢獲實驗驗證應用可行性。本研究以開發應用於生物分子檢測之固態式奈米孔微流體晶片為目標，延續本實驗室過去論文所建立之穩健固態式奈米孔元件製程，並彌補學長論文階段不足之面向，例如：微流體封裝方法優化、奈米孔試片流體狀態與孔徑之追蹤、本實驗室之精密電性量測系統建立與應用、以及他種生物分子易位螢光觀測與電流訊號量測。
　　本研究最終成功發展出可減少過去90 % 製程時間之多層模組化微流體封裝結構，並完全改善填充液體或空氣洩漏問題。利用微流體晶片之IV定性結果，確立奈米孔試片孔徑之追蹤方法。本研究建立了可應用於奈米孔感測器之精密電性量測系統，確認在受測試片電阻為50 MΩ下之電流雜訊為16 pA，也探討使用本系統進行奈米孔感測器量測時之雜訊變化模式，幫助本實驗室未來在精密電性量測相關研究之發展。細菌易位螢光觀測部分，本研究觀察大腸桿菌於1.5 μm ~ 5 μm微米孔周圍受電場驅動而表現出聚集、驅散、堆積、穿孔等不同的特徵行為，並在最後完成大腸桿菌微米孔易位電流訊號量測，單個訊號峰值屆於0.5~5 nA，時間長度約30 ms，並與螢光觀測之趨勢相互驗證。本論文是首篇描述了微流體在奈米孔試片中之場控導通及導通現象、以及描述細菌螢光觀測結果與電流訊號關係之研究。

Solid-state nanopore sensors have been experimentally validated in terms of protein detection, drug molecular detection, bacterial type discrimination, and DNA sequencing, due to their near-single-molecule-level spatial resolution. This research aims to develop solid-state nanopore microfluidic chips for biomolecule detection, and is expected to improve the shortcomings of the past work from our group, such as optimization of packaging methods, tracking of fluid state and pore size of nanopore test strips, establishment and application of precision electrical measurement systems in this laboratory, and biochemical molecular translocation fluorescence observation and current signal measurement.
Eventually, this study develops a multi-layer modular microfluidic package that reduced the 90% of the process time and completely solved the leakage issue of PDMS mocrofluidic chip. Also, the tracking method of the pore size of the nanopore test piece has been established. A precision electrical measurement system that can be applied to nanopore sensors has been established, helping the development of nanopore-related research in our group. This study observed the translocation behavior of Escherichia coli around the nanopore using fluorescence observation. It was found that the bacteria were driven by the electric field and showed different characteristic behaviors such as aggregation, dispersal, accumulation and perforation around the nanopore. At the end, translocation current signal of E.coli was measured and mutually verified with the result of fluorescence observation. This paper is the first to describe the open circuit and conduction phenomenon of nanopore-embedded microfluidic chip, and also to describe the relationship between fluorescence observations and translocation signals of E. coli.

中文摘要 i
Abstract ii
誌謝 iii
目錄 iv
圖目錄 viii
表目錄 xvi
第一章　緒論 1
1.1 生物分子檢測 1
1.1.1 傳統生物分子檢測技術 2
1.1.2 生醫感測器生物分子檢測技術 4
1.2 奈米孔生物分子感測器 5
1.2.1 生物性奈米孔 6
1.2.2 固態式奈米孔 7
1.2.3 生物性與固態式奈米孔特性比較 8
1.3 二維奈米材料 9
1.4 奈米孔電泳微流道封裝 10
1.5 研究動機 13
1.6 研究目的 15
1.7 論文架構 16
第二章 固態奈米孔分子檢測技術 19
2.1 固態式奈米孔感測器原理 19
2.2.1 感測器架構 19
2.2.2 感測器運作機制 22
2.2 奈米孔離子流數學模型 23
2.3 結合電性量測結構之固態奈米孔感測器 25
2.4.1 穿隧電流式電極 25
2.4.2 場效電晶體式電極 26
2.5 應用於細菌檢測之固態式奈米孔感測器 27
第三章　固態式奈米孔生物分子感測元件製程與封裝 30
3.1固態式奈米孔元件結構與製程 30
3.1.1奈米孔晶片結構 31
3.1.2客製化基材選擇與設計 33
3.1.3奈米孔元件製程步驟 34
3.1.4 二硫化鉬薄膜轉印製程 35
3.1.5 聚焦離子束奈米孔製程 36
3.2 奈米孔電泳微流道封裝 44
3.2.1 多層微流道封裝設計原理 44
3.2.2 微流道貼紙組裝方法 49
3.4 結論 51
第四章　奈米孔微流體封裝晶片之流體現象與基礎電性探討 53
4.1 奈米孔微流體封裝晶片之流體填充方法 53
4.2 奈米孔微流體封裝晶片流體等效電路 55
4.3 奈米孔理論孔徑估算探討 57
4.3.1 奈米孔徑理論估算值與SEM影像測量數值比較 57
4.3.2 理論公式估算孔徑與SEM影像測量孔徑之可能誤差來源探討 58
4.4 奈米孔微流體封裝晶片場控導通現象 62
4.5 奈米孔微流體導通實驗 63
4.5.1 可變偏壓下導通實驗 64
4.5.2 導通電壓與奈米孔孔徑關係 70
4.6 結論 73
第五章 應用於細菌奈米孔感測器之精密電性量測系統建立與訊號分析 75
5.1 精密電性量測系統與雜訊排除措施建立 75
5.1.1 精密電性量測系統 75
5.1.1.1系統硬體配置 75
5.1.1.2 LabVIEW使用者介面與資料格式 77
5.1.2 探針平台設置與奈米孔試片接線規劃 79
5.1.3 物理性雜訊排除措施 83
5.2 系統訊號雜訊分析 84
5.2.1 環境對訊號之影響 85
5.2.2 量測儀器端對訊號之影響 87
5.2.3 受測樣本特性對訊號之影響 92
5.3 奈米孔試片規格與雜訊關係 94
5.4 本系統應用於奈米孔感測器之量測評估 96
5.5 結論 98
第六章 固態式奈米孔之細菌易位螢光觀測與訊號量測 101
6.1 細菌於奈米孔微流道晶片中之螢光觀測 101
6.1.1 實驗樣本與奈米孔試片說明 102
6.1.2 實驗配置方式探討 104
6.1.3 細菌於不同孔徑奈米孔試片之易位行為螢光觀測結果 106
6.2 固態式奈米孔之生物分子易位訊號量測 116
6.2.1 易位訊號量測實驗配置 117
6.2.2 易位訊號量測實驗結果與討論 118
6.3 結論 128
第七章 總結與未來發展 130
7.1 總結 130
7.2 研究成果 131
7.3 本研究之學術貢獻點 133
7.4 未來研究建議 138
7.4.1 電晶體結構設計與製作 138
7.4.2 電晶體效應量測 139
7.4.3 奈米孔生物分子易位訊號量測與分析 140
7.4.4電晶體奈米孔元件訊號量測與分析 140
附錄 142
附錄8.1 : 蛋白質螢光觀測 142
附錄8.2 : 離子濃度對雜訊與生物分子易位訊號之影響 145
附錄8.3 : 5 μm Si3N4 微米孔細菌易位螢光觀測與易位訊號量測實驗 148
附錄8.4 : 蛋白質基本結構與尺寸特性 153
附錄8.5：微訊號量測儀－Agilent B2912A 155
附錄8.6：螢光顯微鏡－Zeiss Axioplan 2 156
附錄8.7：聚焦離子束－FEI Helios Nanolab 600i System 157
附錄8.8：Patch-Clamp放大器 158
附錄8.9：資料擷取器 – USB 6361 159
參考資料 160
著作發表 164
作者簡介 164

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