個人化醫療被視為「大數據」、「物聯網」之後下一個即將快速發展的新興產
業，而DNA 定序則被視為其技術核心。奈米孔定序技術被視為第四代的定序技
術，量測機制為將具有奈米孔洞之薄膜置於離子溶液中，並在其中一端加入欲檢
測之DNA 分子。若在其兩端施加一微小電場，溶液中之離子及表面帶負電荷之
DNA 在電泳力的驅使之下通過奈米孔到達另外一個流體腔室。而當DNA 通過奈
米孔時根據其上之不同結構之鹼基會形成不同的DNA 易位訊號。目前發展較成
熟的技術為生物性奈米孔，然而因為使用壽命短、製程複雜以及空間解析度低等
缺點，而本研究以固態式奈米孔的技術將其克服。
本研究採用二維奈米材料－二硫化鉬製作為奈米孔，透過其0.8 nm 的薄膜
厚度成功地將理論空間解析度從目前的29 bp 提升至2 bp。此外，關於奈米孔的
製程方面，本研究使用聚焦離子束製作，並針對了聚焦離子束製程的兩種參數(轟
擊時間和聚焦圖形)進行了深度的探討，成功地將奈米孔孔徑從10 nm 縮小至7
nm。在易位行為的監控方面，本研究在使用YOYO-1 對DH5α DNA 進行螢光修
飾之後，直接使用螢光顯微鏡觀察YOYO-1 的易位行為，降低易位訊號的誤判
機會本研究整合上述奈米孔元件於PDMS 電泳微流道晶片，觀察DNA 在不同奈
米孔孔徑下的易位速度可達12 μm/sec 以上(60 nm nanopore)。
本論文為首篇整合微流體、奈米孔與螢光技術觀測於一平台上之研究，所開
發之技術未來可應用於下一階段電晶體式DNA 奈米孔定序。

Personalized medical considered the next generation industries fast growing up after "big data" and "Internet of things". The nanopore sequencing is the core of it. It is regarded as the technology of 4th generation. The mechanism of nanopore sequencing is to set up the chip with nanopore membrane in ion solution, and add DNA molecules in one side of the fluidic cell. The ion in the solution and the DNA molecules will pass through the nanopore by electrophoretic force if we apply the electric field in both side of fluidic cell. When DNA pass through the nanopore, the ionic current will being blocked and causing the characteristic signal from different DNA base. The development of biological nanopore is more mature. However, there are some disadvantages like low translocation velocity, difficulty of fabrication, and low spatial resolution.
In this study, we fabricated the nanopore on the membrane of MoS2. We raised the spatial resolution from 29 bp to 2 bp by the thickness of nanopore shrinking from 10 nm to 0.8 nm. In fabrication respect, we adopt the focused ion beam. Moreover, shrink the diameter of nanopore from 10 nm to 7 nm by tuning the dwell time and the pattern size. In the observation respect, we observed and recorded the genomic DNA of DH5α translocation directly throw fluorescence microscopy by modifying DNA with YOYO-1. This method will reduce the error rate. In addition, we have designed an integrated PDMS chip for CE manipulation. The maximum velocity of translocation that we can traced is 12 μm/sec.
This paper is the first study integrated by microfluidic, nanopore, and fluorescence observation technology. It can apply to the study of nanopore transistor in the future.

中文摘要 i
Abstract ii
圖目錄 vii
表目錄 xii
第一章 緒論 1
1.1 DNA定序檢測技術 1
1.1.1 第一代定序技術 3
1.1.2 第二代定序技術 5
1.1.3 新一代定序技術比較 6
1.2 奈米孔檢測機制 7
1.3 固態電子二維奈米材料 9
1.3.1 二維奈米材料之種類 10
1.3.2 二維奈米材料之性質比較 10
1.3.3 二維奈米材料製程與組裝 12
1.3.4 二維奈米材料之發展與應用 19
1.4 研究動機 23
1.5 研究目的與方法 25
1.6 論文架構 27
第二章 固態電子奈米孔DNA定序技術之原理 29
2.1 DNA易位行為探討 29
2.1.1 電泳力探討 31
2.1.2 緩衝液性質探討 32
2.1.3 薄膜表面沾黏現象 35
2.1.4 其他緩速機制探討 37
2.2 奈米孔薄膜厚度及孔徑大小探討 39
2.2.1 奈米孔大小對於易位訊號之影響 39
2.2.2 奈米孔穿孔機制 41
2.2.3 奈米孔薄膜厚度對空間解析度之影響 43
2.3 結論 44
第三章 二硫化鉬半導體薄膜之特性探討 46
3.1 基本性質探討 46
3.1.1 化學穩定性與溫度穩定性探討 47
3.1.2 退火製程探討 48
3.1.3 尺寸型態特性探討 49
3.2 光學性質探討 51
3.2.1 拉曼光譜 51
3.2.2 吸收光譜 52
3.2.3 光致螢光光譜 53
3.3半導體特性探討 54
3.3.1 直接能隙 54
3.3.2 電子遷移率 55
3.4 結論 56
第四章 二硫化鉬固態電子奈米孔製程研究成果 58
4.1 系統架設及客製化氮化矽窗口基材選用 58
4.1.1 奈米材料組裝平台架設 58
4.1.2 微電流精密訊號量測平台架設 60
4.1.3 氮化矽窗格基材 61
4.2 二硫化鉬二維奈米材料組裝與材料分析 62
4.2.1 二硫化鉬薄膜組裝 62
4.2.2 原子力顯微鏡分析二硫化鉬 64
4.2.3 拉曼光譜圖分析二硫化鉬 65
4.3 電泳行為觀測及緩速機制參數探討 70
4.3.1 流體封裝結構設計 70
4.3.2 參考電極製作 73
4.3.3 YOYO-1螢光標記機制探討 74
4.3.4 SEM影像及AFM影像觀測DNA 75
4.4 結論 77
第五章 二硫化鉬固態電子奈米孔用於DNA奈米孔易位行為觀測 78
5.1 聚焦離子束奈米孔製程探討 78
5.1.1 聚焦圖形對奈米孔孔徑之影響 81
5.1.2 轟擊時間對奈米孔孔徑之影響 83
5.2 低雜訊電流檢測系統設計與驗證 86
5.2.1 系統配置與設計 86
5.2.2 環境雜訊量測與抑制 87
5.3 DNA易位行為螢光觀測 89
5.3.1 螢光顯微鏡觀測螢光標記DNA 89
5.3.2 不同偏壓對DNA緩速效果之探討 93
5.3.3 不同奈米孔徑對DNA緩速效果之探討 95
5.4 結論 100
第六章 總結 101
6.1 總結 101
6.2 研究成果 102
6.3 學術貢獻 104
6.4 未來研究建議 107
6.4.1全整合手持式奈米孔檢測機台 107
6.4.2 流道貼紙晶片製作 108
6.4.3 Multilevel Pulse-voltage Injection奈米孔製程開發 110
附錄 111
附錄A：微訊號量測儀－Agilent B2912A 111
附錄B： 螢光顯微鏡－Zeiss Axioplan 2 112
12.5x-1,000x 112
附錄C： 聚焦離子束－FEI Helios Nanolab 600i System 113
附錄D：Patch-Clamp放大器 114
附錄E：資料擷取器 – USB 6361 115
附錄F：奈米孔電晶體Layout與製程設計 116
F.1 設計原理 116
F.2 晶片布局與製程設計 119
F.3 晶片封裝設計 121
參考資料 122
著作發表 131
作者簡介 132

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