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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊台鴻(Tai-Horng Young) | |
| dc.contributor.author | Meng-Hua Yen | en |
| dc.contributor.author | 顏孟華 | zh_TW |
| dc.date.accessioned | 2021-06-15T05:58:11Z | - |
| dc.date.available | 2012-08-19 | |
| dc.date.copyright | 2010-08-19 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-16 | |
| dc.identifier.citation | [1] J. B. Edel, et al., Microfluidic routes to the controlled production of nanoparticles. Chemical Communications 10 (2002) 1136-7.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47401 | - |
| dc.description.abstract | 透明生物微流體晶片可利用即時的細胞生長觀察提供必要的資訊以助於細胞研究；本研究發展透明生物微流體晶片來研究細胞在微控制環境下的行為，本研究利用二氧化碳雷射機器加工及LIBWE等，直寫式雷射方法來發展透明微流體晶片；接著本論文成功利用這些方法發展一個具有驅電性或驅化性的透明生物微流體晶來研究細胞行為；此外，本研究亦發展透明導電玻璃圖樣形成技術及細胞圖樣形成技術，此為可應用於細胞行為研究的兩種圖樣形成技術。
透明微流體晶片包含玻璃及壓克力微流體晶片；製作玻璃微流體晶片的四種加工方法，包含二氧化碳雷射機器、使用有機吸收劑的紫外光LIBWE、使用有機吸收劑的可見光LIBWE、使用金屬吸收劑的可見光LIBWE等，皆可以產生無碎裂且高品質的微流道；關於製作壓克力微流體晶片，使用金屬吸收劑的紫外光LIBWE亦可被利用來蝕刻壓克力並且產生高品質的微流道；上述之方法都是直寫式雷射加工，因此整個微流體晶片的發展時間可少於二十四小時；此外，因LIBWE為本研究重要的加工方法，其加工原理也將被深入討論。 本研究提出一個在顯微鏡上可長時間且即時觀察細胞遷移的自主微流體晶片；結合微流道及透明導電玻璃電極，形成一個具有化學濃度梯度且可培養及觀察細胞的相連微腔室。關於和許小姐合作的細胞趨化性研究，我們以超解析率明視野光學顯微術觀測癌細胞絲狀偽足在表皮生長因子濃度梯度下的動態變化，發現在濃度高的表皮生長因子周圍，癌細胞CL1-0的絲狀偽足活動較高。關於和黃慶文小姐合作的細胞趨電性研究，我們利用架在顯微鏡上可長時間做趨電性研究的晶片來觀察肺癌細胞的趨電反應，並以肺癌細胞的高轉移能力（CL1-5）及低轉移能力（CL1-0）來驗證此趨電性晶片的功能。 最後，本論文發展了經由可見光LIBWE的透明導電玻璃圖樣形成技術及經由二氧化碳或紫外光雷射機器加工的細胞圖樣形成技術；經由可見光雷射引發背面蝕刻方法的透明導電玻璃圖樣形成技術所得的結果超越其他正面雷射蝕刻的方法；另外透明導電玻璃圖樣形成技術被應用在製作透明氣體流量計。細胞圖樣形成技術是一種簡單且有效率的技術；此技術是利用在玻璃基材上一層阻隔層來使細胞不貼附，再利用雷射加工移除設定圖案區域，細胞便可貼附及生長在此區域。 | zh_TW |
| dc.description.abstract | Experiments with transparent bio-microfluidic chip can provide essential real-time observation for cell growth and cell studies. Therefore, this study developed a transparent bio-microfluidic chip for study of cell behavior under microcontrolled environment. This study developed transparent microfluidic chips by laser direct-write methods (LDW), which are the laser-induced backside wet etching (LIBWE) systems and CO2 laser machining. Then, this study successfully used these methods to develop a transparent bio-microfluidic chip with chemotaxis or electrotaxis for study of cell behavior. In addition, this study develops two patterning techniques, ITO patterning and cell patterning, to apply in study of cell behaviors.
Transparent microfluidic chips include glass and PMMA microfluidic chip. For glass microfluidic chip, four fabrication methods are used for fabricating. These methods, which include modified CO2 laser machining, UV LIBWE using organic absorber, visible LIBWE using organic absorbers and visible LIBWE using metallic absorbers, can produce crack-free and high-quality microchannels. For PMMA microfluidic chip, visible LIBWE using the metallic absorber is used to fabricate and also produces high-quality microchannels. These four fabrication methods are LDW methods, so the overall development time of glass or PMMA microfluidic chip is shortened to less than 24 h. In addition, the mechanism of LIBWE is discussed in depth because the LIBWE in this dissertation is an important fabrication method. This study presents an autonomous microfluidic chip for long-term and real-time observation of cell migration by a microscope. It was combined the microchannels and ITO electrodes to form a closed microchamber with chemical gradient for cell culturing and observation. For chemotaxis collaborated with the Miss Hsu, we used a super-resolution microscopy technique, non-interferometric wide-field optical profilometry (NIWOP), to observe filopodium activity of cells under the EGF gradient. Higher filopodium activity is observed at the side facing higher EGF concentration for single CL1-0 cell subjecting to the EGF gradient. For electrotaxis collaborated with the Miss Huang, we enabled observing the electrotactic response of lung cancer cells by a sealed culture chamber that is suitable for long-term electrotaxis study with a microscope. We used lung cancer cell lines with high and low metastasis potential, CL1–5 and CL1–0, respectively, to demonstrate the function of the electrotactic chip. Finally, this study developed the ITO patterning by visible LIBWE and the cell patterning by CO2 or UV laser machining. The obtained ablation result from ITO patterning by visible LIBWE excels that from front-side laser ablation. The ITO patterning was then utilized in fabricating a transparent gas flow meter. The cell patterning was a simple and effective method for patterning cells on a glass substrate. A passivation layer that is capable of preventing cell adhesion was first coated onto glass surface and then cells adhere and grow cleanly in the laser defined pattern. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T05:58:11Z (GMT). No. of bitstreams: 1 ntu-99-D92548010-1.pdf: 12207557 bytes, checksum: 81deb65ea687dd98d249765e0d5da8ad (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 摘要 i
Abstract iii Chapter 1 Introduction 1 1.1 Background and literature review 1 1.1.1 Development of transparent bio-microfluidic chip 2 1.1.1.1 Overview of transparent microfluidic chip 2 1.1.1.2 Selection of substrate material and fabrication methods 5 1.1.2 Study of cell behavior under microcontrolled environment 10 1.1.3 ITO patterning and cell patterning 11 1.2 Purpose and skeleton of this dissertation 12 Chapter 2 Transparent glass and PMMA microfluidic chip 15 2.1 Fabrication methods of transparent microfluidic chip 15 2.1.1 Glass fabrication methods 20 2.1.1.1 LIBWE 24 2.1.2 PMMA fabrication methods 29 2.2 Previous preparation for fabrication of glass and PMMA microfluidic chip 32 2.2.1 Common measurement instruments 32 2.2.2 Common chemicals and substrates 34 2.3 Transparent glass microfluidic chip 35 2.3.1 Modified CO2 laser machining 37 2.3.1.1 Experiment 38 2.3.1.2 Results and discussion 40 2.3.2 LIBWE system 61 2.3.2.1 Similar experimental setup and common calculation formulas 61 2.3.2.1.1 Similar experimental setup 61 2.3.2.1.2 Common calculation formulas 64 2.3.2.2 UV LIBWE system (266 nm laser) 65 2.3.2.2.1 Experiment 66 2.3.2.2.2 Results and discussion 67 2.3.2.3 Visible LIBWE system (532nm laser) 88 2.3.2.3.1 Organic absorber (Rose Bengal dissolved in acetone or water) 89 2.3.2.3.2 Metallic absorber (gallium and eutectic indium/gallium) 100 2.3.3 Brief summary 117 2.4 Fabrication of transparent PMMA microfluidic chip and M-LIBWE mechanism 121 2.4.1 Experiment 123 2.4.2 Results 125 2.4.3 Discussion 134 2.4.3.1 Comparison of PMMA fabrication techniques 134 2.4.3.2 Comparison of dot etching and trench etching 136 2.4.3.3 LIBWE mechanism 139 2.4.4 Brief summary 144 2.5 Summary 145 Chapter 3 Cell culturing and migration in microcontrolled environment – chemotaxis and electrotaxis of lung cancer cell in microfluidic chip 149 3.1 Transparent cell-culture chip with chemical-gradient 149 3.1.1 Chemical gradient generation by conventional and microfluidic devices 149 3.1.2 Chip design and fabrication 157 3.1.2.1 First generation and second generation cell culturing chip using O-ring spacer 157 3.1.2.2 Third generation chip with double-sided tape (3M) 161 3.1.2.3 The fourth generation chip that utilizes culture dish as substrate 163 3.1.2.4 Chip temperature homogeneity 164 3.1.2.4.1 Temperature distribution measurement by IR thermometry 165 3.1.2.4.2 Temperature measurement by thermal couple 166 3.1.2.5 Brief summary 169 3.1.3 Simulation and observation of flow field and chemical gradient 170 3.1.3.1 Image-based flow field and chemical gradient measurement 171 3.1.3.2 Medium flow and replacement 174 3.1.3.3 Chemical gradient buildup and control 180 3.1.4 Cell culture in microchamber 187 3.1.4.1 Cell lines, reagents and cell imaging 188 3.1.4.2 Cytotoxicity test for double-sided tape 190 3.1.4.3 Cell cultured in the second and third generation chip. 193 3.1.4.4 Cell growth rate in the forth generation chip 196 3.2 Application of Chemotaxis: Label-free quantification of cancer-cell filopodium activities in chemical gradient 197 3.2.1 Microscopy for chemotaxis 198 3.2.2 Super-resolution bright-field microscopy for chemotaxis study 200 3.2.2.1 Super-resolution bright-field microscopy 200 3.2.2.2 Design and fabrication of the micro cell culture chamber 202 3.2.2.3 Cell line and chemicals 202 3.2.3 Filopodium activity of cancer cells 203 3.3 Application of electrotaxis: Electrotaxis of lung cancer cells in a multiple-electric-field chip 206 3.3.1 Electrotaxis for conventional and microfluidic devices 207 3.3.2 Materials and methods 210 3.3.2.1 Electrotactic Chip Design and Fabrication 210 3.3.2.2 System for electrotaxis study 210 3.3.2.3 SFC and MFC design and fabrication 211 3.3.2.4 Electric field simulation and measurement 214 3.3.2.5 Cell preparation 215 3.3.2.6 Electrotaxis experiment 215 3.3.2.7 Cell imaging and cell migration measurement 217 3.3.3 Results and discussion 218 3.3.3.1 EF simulation and measurement 218 3.3.3.2 Cell response in single field chip (SFC) and multi-field chip (MFC) 219 3.3.3.3 Cell migration in EF 220 3.4 Summary 222 Chapter 4 ITO patterning and cell patterning 224 4.1 ITO glass patterning by visible LIBWE using organic absorber 224 4.1.1 Introduction 224 4.1.2 Experiment 229 4.1.2.1 ITO patterning by visible-LIBWE 229 4.1.2.2 Transparent gas flow meter 231 4.1.3 Results and discussion 233 4.1.3.1 ITO ablation by visible-LIBWE 233 4.1.3.2 TCR of ITO 241 4.1.3.3 Electrical property of ablated ITO strips 242 4.1.3.4 Flow-induced resistance change 244 4.2 Cell patterning by CO2 laser or UV laser machining 248 4.2.1 Introduction 248 4.2.2 Experiment 250 4.2.2.1 Glass surface modification 251 4.2.2.2 Chip surface pattering by laser ablation of coated glass slides 252 4.2.2.3 Cell culture and fluoresce dye staining 254 4.2.2.4 EGFR (Epidermal Growth Factor Receptor) detection on patterned cell array 255 4.2.2.5 SEM and EDS scanning 255 4.2.3 Results and discussion 255 4.3 Summary 264 Chapter 5 Conclusion and further work 266 5.1 Conclusion 266 5.1.1 Fabrication of glass and PMMA microfluidic chip 266 5.1.2 Study of cell behavior under microcontrolled environment 268 5.1.3 ITO patterning and cell patterning 270 5.2 Further work 270 5.2.1 LIBWE system 270 5.2.2 Study of cell behavior under microcontrolled environment 271 REFERENCE 273 Appendix A: Symbol and abbreviated Index 289 Appendix B: Temperature calculation for laser irradiating 292 | |
| dc.language.iso | en | |
| dc.subject | 二氧化碳雷射機器 | zh_TW |
| dc.subject | 雷射引發背面蝕刻(LIBWE) | zh_TW |
| dc.subject | 趨化性 | zh_TW |
| dc.subject | 趨電性 | zh_TW |
| dc.subject | 細胞培養系統 | zh_TW |
| dc.subject | 細胞圖樣形成技術 | zh_TW |
| dc.subject | 透明導電玻璃圖樣形成技術 | zh_TW |
| dc.subject | Laser-induced backside wet etching(LIBWE) | en |
| dc.subject | chemotaxis | en |
| dc.subject | electrotaxis | en |
| dc.subject | cell culture system | en |
| dc.subject | cell patterning | en |
| dc.subject | ITO patterning | en |
| dc.subject | CO2 laser machining | en |
| dc.title | 發展透明生物微流體晶片以研究微控制環境下之細胞行為 | zh_TW |
| dc.title | Development of Transparent Bio-Microfluidic Chip for Study of Cell Behavior Under Microcontrolled Environment | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 鄭郅言(Ji-Yen Cheng) | |
| dc.contributor.oralexamcommittee | 魏培坤(Pei-Kuen Wei),郭志禹(Chih-Yu Kuo),沈家寧(Chia-Ning Shen),白果能(Konan Peck),邱顯泰(Hsien-Tai Chiu) | |
| dc.subject.keyword | 雷射引發背面蝕刻(LIBWE),二氧化碳雷射機器,透明導電玻璃圖樣形成技術,細胞圖樣形成技術,細胞培養系統,趨電性,趨化性, | zh_TW |
| dc.subject.keyword | Laser-induced backside wet etching(LIBWE),CO2 laser machining,ITO patterning,cell patterning,cell culture system,electrotaxis,chemotaxis, | en |
| dc.relation.page | 299 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-17 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
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