觀察單一分子進入細胞內之行為表現是科學家很想研究的課題，但缺乏有效之量測平台。要觀測其方式有兩種主要方式：Patch clamp微注入法與電穿孔法，利用Patch clamp微注入法可以精準從局部地方注射分子進入細胞內，但容易因為內外壓差導致細胞脹破，且無法控制單一且定量分子進入細胞內。傳統電穿孔法可以針對有效單一細胞電擊送藥，但無法從特定小區域送入，使定量的工作變為困難。有鑑於此，本實驗室發展可以控制單一分子進入細胞之觀測系統。此系統其中最重要就是單一細胞局部電穿孔平台，改良傳統電穿孔針對單一細胞整體電擊，此方式更能有效控制送藥部位並減低對細胞傷害。
本文提出新型單一細胞局部電穿孔晶片，利用1μm間距之電極陣列達到單一細胞並且在細胞特定極小區內完成電穿孔步驟。利用ITO透明導電金屬製作電極，改良傳統上金屬電極造成觀測視野遮蔽的困擾且可運用於高倍油鏡觀察。
本實驗製作出全透明局部電穿孔晶片，成功使電穿孔影響範圍侷限至單一細胞0.9μm的間距內。利用不同電擊時間降低對細胞之傷害且控制進入之螢光量，並使因電場而打開之洞能再合上。最後利用分子擴散之模型反推局部電穿孔所影響之範圍。此外，本文也針對調控ITO製程參數，電阻和均勻度已達商用之水準。

Tracking a single molecule in the cell plays a significant role in biology. Currently, there is a need for a method to deliver molecules to a cell while tracking such delivery.
We develop a new platform for tracking a single molecule delivered by localized eletropoation. In this work, a single cell is elecroporated locally at a specific membrane position on a transparent electrode chip. The whole eletroporation process was recorded by CCD camera by tracking PI dye entering the HeLa cell. Transparent ITO electrodes were employed to apply a confine E-field in a sub-micron range for localized cell electroporation.
In conventional single cell electroporation, electric field are applied to the whole cell between tow electrode. As a result many pores will be generated with different permeability and size, resulting in difficulties in tracking molecules delivered into the cell. In this paper, very localized electroporation at a specified region on a single cell surface by transparent ITO electrodes is realized, which can not only generate well-controlled nano-pores allowing for rapid recovery of the cell surface, but also provide a clear optical path for cell monitoring and molecular tracking.
In this work we demonstrate very localized electroporation to decrease the damage to the cell membrane. We can also observe resealing of the electroporation hole. We use a diffusion model to confirm localized electroportion is confined in 0.9μm region. We can control electroporation region down to 0.9μm and control the electroporation time to increase the survivability.

誌謝 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 vii
表目錄 xi
第一章 緒論 1
1.1 前言 1
1.2 目前細胞單分子送藥、觀測之技術與挑戰 2
1.3 研究動機和目標 5
第二章 文獻回顧 8
2.1 電穿孔原理 8
2.2 細胞電穿孔晶片文獻回顧 11
2.2.1 流動式晶片(Flow device) 11
2.2.2 定點式晶片 14
2.2.3 膜片箝制(Patch clamp) 18
2.3 細胞電穿孔晶片之比較 21
第三章 實驗設計 23
3.1 設計流程 23
3.2 設計理念 24
3.3 第一代局部電穿孔晶片設計 26
3.4 第二代局部電穿孔晶片設計 28
3.5 實驗系統之架設 31
3.6 實驗材料與化學藥品 32
3.6.1 所選細胞與細胞培養 32
3.6.2 實驗用化學學藥品 33
3.7 實驗儀器原理介紹 34
3.7.1 射頻濺鍍機(sputter) 34
3.7.2 電子槍蒸鍍機(e-gun) 35
3.7.3 聚焦式離子束 (Focused Ion Beam, FIB) 36
3.7.4 反應式離子蝕刻機(Reactive Ion Etcher ,RIE) 37
3.7.5 電漿輔助化學氣相沈積(PECVD) 38
3.7.6 紫外線光譜儀(UV-VIS spectrophotometer) 39
3.7.7 X-射線繞射儀(X-Ray Diffraction Metrology) 39
3.7.8 四點探針(Four-Point Probe) 40
第四章 結果與討論 41
4.1 晶片製程參數調控 41
4.1.1 ITO 電阻與均勻度 41
4.1.2 ITO蝕刻速率之調控 45
4.2 實驗對照組分析和細胞濃度控制 47
4.3 第一代電穿孔晶片測試結果 49
4.4 第二代局部電穿孔晶片測試結果與分析 52
4.4.1 局部電穿孔晶片:電穿孔時間一秒 52
4.4.2 局部電穿孔晶片:施加單一脈衝 54
4.5 細胞內擴散模型分析 57
第五章 結論 62
第六章 參考文獻 63

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