臺灣博碩士論文加值系統

本研究一開始用不同濃度以及粒徑的奈米金粒子濃度加入 NIH 3T3 細胞去觀察細胞的生長曲線，發現粒徑較小的奈米金粒子對於細胞的影響較為顯著，因此選定較小粒徑的奈米金粒子加入 NIH 3T3 細胞後去做檢測，在光學顯微鏡(Optical microscope)下觀測發現加入奈米金的 NIH 3T3 細胞外觀型態較腫脹，觸手也較短，接著將 NIH 3T3 細胞在液態下用原子力顯微鏡(Atomic force microscope)去做掃描，觀察隨著加入奈米金粒子的時間不同，是否有影響，發現有添加奈米金粒子的細胞高度隨著天數上升，表面的顆粒及掃描被拖曳的痕跡都較為明顯。
第二部分觀測的螢光有細胞的骨架肌動蛋白以及細胞的內質網，加入奈米金粒子後螢光顯示出 NIH 3T3 細胞骨架的絲狀結構有被破壞掉，變為顆粒狀，而內質網也變得不完整，此結果顯示肌動蛋白被破壞造成觸手無法正常伸展。
第三部分力學量測，發現在細胞核的以及細胞觸手的部分黏滯力有隨著加入奈米金粒子的時間增長而上升，而且細胞核的黏滯力大於觸手的黏滯力，此結果顯示結構有遭到破壞造成黏滯力的上升。
最後一個部分將基板導線層製作出來後在光學顯微鏡下觀測，再去使用掃描電子顯微鏡(Scanning electrical microscopy)對沉積的氮化矽絕緣層以及沉積的電極柱做觀測，觀察表面以及參數下的厚度是否正確，並運用 PDMS 製作出培養細胞水槽的模具，放在製作好的基板後，測試是否可以在基板上成功培養細胞。

This study started with different concentrations and particle sizes of gold. The gold nanoparticle concentrations were added into NIH 3T3 cells to observe cell growth curve. The cells of a smaller diameter of gold nanoparticles had a more significant impact, so the smaller particles sizes were selected.
The gold nanoparticles that were added into NIH 3T3 cells were found to have more swelling and dendritic became shorting observed by Optical microscope. The NIH 3T3 cells in the liquid were used to measure with the AFM (Atomic force microscope), observed the topography of NIH 3T3 cells with the nanoparticles at different times.
The second part of this study is by fluorescence microscope, the fluorescence observation have actin skeleton of cells and the endoplasmic reticulum of cells. After adding gold nanoparticle to the actin filaments of NIH 3T3 are broken and appeared as an aspiration of dots and endoplasmic reticulum of cells becomes incomplete.
The third part of the mechanical measurements is by AFM. After adding gold nanoparticles, the nucleus and dendritic adhesion force of cell became bigger than the
control with increased time. The adhesion force of nucleus is greater than dendritic.
The last part, observed the substrate after the conductor layer produced by optical microscope. The SEM (Scanning electrical microscopy) measured the thickness of the parameters of the deposition of silicon nitride insulating layer made and observed the surface. PDMS made for using cultured cells, after on the substrate
to test cells can be successfully cultured on the substrate.

致謝 ......... I
中文摘要 .... II
Abstract ..... III
圖目錄 ......VIII
第一章 緒論 ...... 1
1.1 奈米金粒子 ........ 1
1.2 微機電系統 ........... 2
1.3 研究動機 .............. 3
1.4 小鼠胚胎纖維母細胞(NIH 3T3) .......... 4
第二章 實驗儀器原理 ....... 5
2.1 原子力顯微鏡簡介與基本原理 ...... 5
2.1.1 原子力顯微鏡簡介 ......... 5
2.1.2 原子力顯微鏡原理 ........ 5
2.1.3 原子力顯微鏡硬體架構 .......... 6
2.1.4 原子力顯微鏡的操作模式 ....... 8
2.1.5 作用力-距離曲線(Force-Distance curve) ............... 10
2.2 多電極陣列 ................. 12
2.2.1 細胞貼附 ............. 12
2.2.2 多電極陣列基板 ............. 13
2.3 細胞培養所需之設備 ............ 14
2.3.1 二氧化碳培養箱(CO2 incubator)............. 14
2.3.2 液態氮細胞儲存桶(Liquid nitrogen cell storage) .................................. 14
2.3.3 無菌操作台(Laminar flow hood)............... 15
2.3.4 離心機(Centrifuges) .............. 15
2.3.5 恆溫水浴槽(Water baths) .......... 16
2.3.6 倒立式螢光顯微鏡(Inverted fluorescence microscope).................... 16
2.3.7 掃描探針顯微鏡(Atomic Force Microscope)................... 17
2.3.8 場發射式掃描電子顯微鏡(Field-Emission Scanning Electron Microscope ) ...... 17
3.1 光罩設計以及製作 ................... 18
3.2 基板作業 ................. 19
3.2.1 清洗基板 ........ 19
3.2.2 光阻塗佈 ......... 19
3.2.3 軟烤 .............. 19
3.2.4 曝光 .............. 19
3.2.5 顯影 ................. 19
3.2.6 熱蒸鍍 ............. 20
3.2.7 舉離 ................. 20
3.2.8 硬烤 ................. 20
3.2.9 化學鍍金 .......... 20
3.2.10 絕緣層沉積 ............ 21
3.2.11 蝕刻 ............. 21
3.3 基板製作流程 .............. 22
3.4 生物樣品製備 .......... 24
3.4.1 細胞培養 ......... 24
3.4.2 細胞繼代 ......... 24
3.4.3 細胞冷凍保存 ............. 24
3.4.5 細胞計數 ........ 24
3.4.6 細胞染色 ......... 25
3.4.7 封片 ..................... 27
第四章 結果與討論 ................ 28
4.1 奈米金粒子檢測 .................. 28
4.2 NIH 3T3 細胞生長曲線.......... 30
4.4 原子力顯微鏡在液態下掃描 NIH 3T3 細胞.......................... 33
4.5 NIH 3T3 細胞加入奈米金粒子螢光檢測............................ 38
4.5.1 肌動蛋白-NIH 3T3 正常培養 1 到 5 天 ........................... 38
4.5.2 肌動蛋白-NIH 3T3 加入奈米金粒子培養 1 到 5 天 .............................. 40
4.5.3 內質網-NIH 3T3 正常培養 1 到 5 天 ............................... 43
4.5.4 內質網-NIH 3T3 加入奈米金粒子培養 1 到 5 天 ................................. 45
4.6 原子力顯微鏡在液態下對 NIH 3T3 細胞進行奈米機械量測 ................................ 48
4.6.1 QNM 模式下對 NIH 3T3 細胞進行奈米機械量測 ............................. 48
4.6.1 Tapping 模式下對 NIH 3T3 細胞進行奈米機械量測 ......................... 50
4.7 多電極陣列基板 .............. 52
4.7.1 導線層 .................... 52
4.7.2 絕緣層沉積 ...................... 52
4.7.3 三維電極柱 ........................ 54
4.7.4 電性量測架構 ..................... 56
4.7.5 電性量測結果 ..................... 58
第五章 結論及未來展望 ...................... 59
參考文獻 .......................... 60

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