臺灣博碩士論文加值系統

本研究利用微機電系統製程技術之灌注成形法以及微壓印技術，完成聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)微流體濃度梯度晶片之製備，以應用於內皮細胞對葡萄糖最適濃度之研究。研究策略為利用微混合器及多層分流微結構以於PDMS微流道產生一濃度梯度效應，且比較不同流速對於濃度梯度效應之影響；另外，再透過細胞貼附區寬度為300 μm且間距為200 μm之壓印圖形，以探討在不同葡萄糖濃度梯度(0~10%、0~20%、5~15%、6~17%)下，對於內皮細胞之最適濃度。經實驗驗證在不同流速(20、30、40、50 μL/min)下濃度梯度皆呈現穩定分佈，流速超過20 μL/min進行實驗時，因流速過快會造成細胞脫落之現象，且內皮細胞對於葡萄糖之最適濃度為7~15%，而當葡萄糖濃度過高或過低於此最適濃度值時皆會造成內皮細胞萎縮或凋亡之現象。故本研究所建立的微流體濃度梯度晶片系統，不但可解決傳統藥物濃度調配上相當費時且無法於同一實驗區內進行多種濃度測試的問題，在未來將有助於藥物濃度測試及藥物過敏測試等應用領域的發展

In this study, the Polydimethylsilcoxane (PDMS) concentration gradient microfluidic chip and PDMS stamp are fabricated by MEMS, casting molding, and microimprinting technology. The chip is used to research optimizing concentration of endothelial cells on glucose concentration gradient. The experiment strategy is based on a microfluidic network to generate a concentration gradient in solution, and observes the effect of concentration gradient in different flow rates. Besides, it combines micropatterns that cell adhesion space width with 300 μm and gap with 200 μm to discuss the optimizing glucose concentration (0~10%, 0~20%, 5~15%, 6~17%) of endothelial cells. Through the experiment data, we can prove that the concentration gradients are all stable under different flow rates (20, 30, 40, 50 μL/min), however, when flow rates are over 20 μL/min, cell leaves substrate surface. The result shows that the glucose concentration gradient in the 7~15% is the optimizing concentration of endothelial cells, nevertheless, too high and low concentration will lead cell to leave or die. This study can solve the problems of the different drug concentrations prepare and test. It will apply drug concentration test and hypersensitive test in the future.

摘要 ..................................................I
ABSTRACT ..............................................II
誌謝 ..................................................III
目錄 ..................................................IV
圖目錄 ................................................VIII
表目錄 ................................................XII
第一章 緒論 .........................................1
1-1 前言 .........................................1
1-2 微流體晶片的發展與應用 .......................2
1-2-1 微機電系統技術與微流體晶片 ...................2
1-2-2 微流體晶片之製程技術 .........................4
1-2-3 微流體晶片之應用 .............................7
1-3 微圖案於生物培養之技術 .......................12
1-4 研究動機與目的 ...............................18
1-5 研究架構 .....................................19
第二章 晶片之設計與製作 .............................21
2-1 光罩設計與製作 ...............................21
2-1-1 濃度梯度微流體晶片光罩設計與製作 .............21
2-1-2 微壓印頭光罩設計與製作 .......................23
2-2 SU-8製程技術與微結構製作 .....................24
2-2-1 SU-8製程技術 .................................24
2-2-2 微結構製作 ...................................25
2-3 PDMS灌注成形技術及翻製流程 ...................32
2-3-1 EPOXY二次翻模 ................................36
2-4 PDMS晶片接合與組裝技術 .......................37
2-4-1 玻璃基板清洗及滅菌 ...........................37
2-4-2 電漿接合原理 .................................38
2-4-3 PDMS氧電漿接合步驟 ...........................39
2-5 PDMS壓印頭製作 ...............................40
第三章 實驗與研究方法 ...............................41
3-1 實驗藥品及其配製 .............................41
3-1-1 實驗藥品 .....................................41
3-1-2 藥品配製 .....................................42
3-2 細胞培養與收集計數 ...........................43
3-2-1 細胞繼代培養 .................................45
3-2-1-1 細胞解凍 .....................................46
3-2-2 細胞計數 .....................................47
3-3 實驗儀器 .....................................50
3-3-1 倒立式螢光光學顯微鏡 .........................50
3-3-2 場放射-掃描式電子顯微鏡 ......................51
3-3-3 表面粗度儀 ...................................52
3-3-4 真空抽氣系統 .................................53
3-3-5 微量注射幫浦 .................................54
3-4 實驗方法 .....................................55
3-4-1 微流體濃度梯度之形成方法及實驗 ...............55
3-4-2 微壓印實驗 ...................................57
3-4-3 微壓印圖案與微流濃度梯度晶片結合方法之實驗 ...58
3-4-4 內皮細胞葡萄糖最適濃度之實驗 .................62
第四章 結果與討論 ...................................64
4-1 微結構晶片製程結果 ...........................64
4-2 濃度梯度實驗結果 .............................68
4-2-1 濃度梯度實驗結果 .............................68
4-2-2 流體剪應力之計算 .............................71
4-3 微壓印實驗結果 ...............................72
4-4 晶片接合結果比較 .............................75
4-5 內皮細胞葡萄糖最適濃度實驗結果 ...............77
第五章 結論與建議 ...................................91
5-1 結論 .........................................91
5-2 建議 .........................................93
參考文獻 .............................................94
自述 ..................................................103

[1]D. A. Stenger, G. W. Gross, E. W. Keefer, K. M. Shaffer, J. D. Andreadis, W. Ma, and J. J. Pancrazio, “Detection of physiologically active compounds using cell-based biosensors,” Trends in Biotechnology, 19, pp. 304-309, 2001.
[2]L. G. Griffith and G. Naughton, “Tissue engineering-current challenges and expanding opportunities,” Science, 295, pp. 1009-1014, 2000.
[3]D. Kleinfeld, K. H. Kahler, and P. E. Hockberger, “Controlled outgrowth of dissociated neurons on patterned substrates,” Journal of Neuroscience, 8, pp. 4098-4120, 1988.
[4]S. N. Bhatia, M. L. Yarmush, and M. Toner, “Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts,” Journal of Biomedical Materials Research, 34, pp. 189-199, 1997.
[5]D. Lehnert, B. Wehrle-Haller, C. David, U. Weiland, C. Ballestrem, B. A. Imhof, and M. Bastmeyer, “Cell behaviour on micropatterned substrata: limits of extracellular matrix geometry for spreading and adhesion,” Journal of Cell Science, 117, pp. 41-52, 2004.
[6]K. Seiler, D. J. Harrison and A. Manz, “Planar chips technology for miniaturization and integration of separation techniques into monition systems,” Journal of Chromatography, 593, pp. 253-258, 1992.
[7]Y. N. Xia and G. M. Whitesides, “Soft lithography,” Angewandte Chemie-International Edition, 37, pp. 551-575, 1998.
[8]L. Martynova, L. E. Locascio, M. Gaitan, G. W. Kramer, R. G. Christensen, and W. A. MacCrehan, "Fabrication of plastic microfluid channels by imprinting methods," Analytical Chemistry, 69, pp. 4783-4789, 1997.
[9]H. Becker and U. Heim, "Polymer hot embossing with silicon master structures," Sensors and Materials, 11, pp. 297-304, 1999.
[10]M. Heckele, W. Bacher, and K. D. Muller, "Hot embossing - The molding technique for plastic microstructures," Microsystem Technologies, 4, pp. 122-124, 1998.
[11]H. Becker, and U. Heim, "Hot embossing as a method for the fabrication of polymer high aspect ratio structures," Sensors and Actuators a-Physical, 83, pp. 130-135, 2000.
[12]S. Y. Chou, P. R. Krauss, and P. J. Renstrom, "Imprint lithography with 25-nanometer resolution," Science, 272, pp. 85-87, 1996.
[13]R. W. Jaszewski, and P. Smith, "Hot embossing in polymers as a direct way to pattern resist," Microelectronic Engineering, 42, pp. 575-578, 1998.
[14]L. E. Locascio, C. E. Perso, and C. S. Lee, "Measurement of electroosmotic flow in plastic imprinted microfluid devices and the effect of protein adsorption on flow rate," Journal of Chromatography A, 857, pp. 275-284, 1999.
[15]M. Rode, and B. Hillerich, "Self-aligned positioning of microoptical components by precision prismatic grooves impressed into metals," Journal of Microelectromechanical Systems, 8, pp. 58-64, 1999.
[16]D. Snakenborg, H. Klank, and J. P. Kutter, “Microstructure fabrication with a CO2 laser system,” Journal of Micromechanics and Microengineering, 14, pp. 182-189, 2004.
[17]K. S. Huang, T. H. Lai, and Y. C. Lin, “Manipulating the generation of Ca-alginate microspheres using microfluidic channels as a carrier of gold nanoparticles,” Lab on a Chip, 6, pp. 954-957, 2006.
[18]K. S. Huang, T. H. Lai, and Y. C. Lin, “Using a microfluidic chip and internal gelation reaction for monodisperse calcium alginate microparticles generation,” Frontiers in Bioscience, 12, pp. 3061-3067, 2006.
[19]R. M. McCormick, R. J. Nelson, M. G. AlonsoAmigo, J. Benvegnu, and H. H. Hooper, “Microchannel electrophoretic separations of DNA in injection-molded plastic substrates,” Analytical Chemistry, 69, pp. 2626-2630, 1997.
[20]P. J. Crosland-Taylor, “A device for counting small particles suspended in a fluid through a tube,” Nature, 171, pp. 37-38, 1953.
[21]D. A. Drew, and R. T. Lahey, “Phase-distribution mechanisms in turbulent low-quality 2-phase flow in a circular pipe,” Journal of Fluid Mechanics, 117, pp. 91-106, 1982.
[22]J. Takagi, M. Yamada, M. Yasuda, and M. Seki, “Continuous particle separation in a microchannel having asymmetrically arranged multiple branches,” Lab on a Chip, 5, pp. 778-784, 2005.
[23]S. L. Anna, N. Bontoux, and H. A. Stone, “Formation of dispersions using "flow focusing" in microchannels,” Applied Physics Letters, 82, pp. 364-366, 2003.
[24]I. Kobayashi, S. Mukataka, and M. Nakajima, “Novel asymmetric through-hole array microfabricated on a silicon plate for formulating monodisperse emulsions,” Langmuir, 21, pp. 7629-7632, 2005.
[25]K. Liu, H. J. Ding, J. Liu, Y. Chen, and X. Z. Zhao, “Shape-controlled production of biodegradable calcium alginate gel microparticles using a novel microfluidic device,” Langmuir, 22, pp. 9453-9457, 2006.
[26]H. Zhang, E. Tumarkin, R. Peerani, Z. Nie, R. M. A. Sullan, G. C. Walker, and E. Kumacheva, “Microfluidic production of biopolymer microcapsules with controlled morphology,” Journal of the American Chemical Society, 128, pp. 12205-12210, 2006.
[27]X. Jiang, Q. Xu, S. K. W. Dertinger, A. D. Stroock, T. Fu, and G. M. Whitesides, “A general method for patterning gradients of biomolecules on surfaces using microfluidic networks,” Analytical Chemistry, 77, pp. 2338-2347, 2005.
[28]R. M. Lorenz, G. S. Fiorini, G. D. M. Jeffries, D. S. W. Lim, M. He, and D. T. Chiu, “Simultaneous generation of multiple aqueous droplets in a microfluidic device,” Analytica Chimica Acta, 630, pp. 124-130, 2008.
[29]X. Zhu, L. Y. Chu, B. Chueh, M. Shen, B. Hazarika, N. Phadke, and S. Takayama, “Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation,” Analyst, 129, pp. 1026-1031, 2004.
[30]S. K. W. Dertinger, D. T. Chiu, N. L. Jeon, and G. M. Whitesides, “Generation of gradients having complex shapes using microfluidic networks,” Analytical Chemistry, 73, pp. 1240-1246, 2001.
[31]T. H. Park, and M. L. Shuler, “Integration of cell culture and microfabrication technology,” Biotechnology Progress, 19, pp. 243-253, 2003.
[32]A. Folch, and M. Toner, “Microengineering of cellular interactions,” Annual Review of Biomedical Engineering, 2, pp. 227-256, 2000.
[33]C. Wyart, C. Ybert, L. Bourdieu, C. Herr, C. Prinz, and D. Chatenay, “Constrained synaptic connectivity in functional mammalian neuronal networks grown on patterned surfaces,” Journal of Neuroscience Methods, 117, pp. 123-131, 2002.
[34]Y. Katanosaka, J. H. Bao, T. Komatsu, T. Suemori, A. Yamada, S. Mohri, and K. Naruse, “Analysis of cyclic-stretching responses using cell-adhesion-patterned cell,” Journal of Biotechnology, 133, pp. 82-89, 2008.
[35]T. Das, S. K. Mallick, D. Paul, and T. K. Maiti, “Microcontact printing of concanavalin a and its effect on mammalian cell morphology,” Journal of Colloid and Interface Science, 314, pp. 71-79, 2007.
[36]S. Takayama, J. C. McDonald, E. Ostuni, M. N. Liang, P. J. A. Kenis, R. F. Ismagilov, and G. M. Whitesides, “Patterning cells and their environments using multiple laminar fluid flows in capillary networks,” Proceedings of the National Academy of Sciences of the United States of America, 96, pp. 5545-5548, 1999.
[37]S. Takayama, E. Ostuni, X. P. Qian, J. C. McDonald, X. Y. Jiang, P. LeDuc, M. H. Wu, D. E. Ingber, and G. M. Whitesides, “Topographical micropatterning of poly(dimethylsiloxane) using laminar flows of liquids in capillaries,” Advanced Materials, 13, pp. 570-574, 2001.
[38]S. Takayama, E. Ostuni, P. LeDuc, K. Naruse, D. E. Ingber, and G. M. Whitesides, “Laminar flows - subcellular positioning of small molecules,” Nature, 411, pp. 1016, 2001.
[39]Y. D. Kim, C. B. Park, and D. S. Clark, “Stable sol-gel microstructured and microfluidic networks for protein patterning,” Biotechnology and Bioengineering, 73, pp. 331-337, 2001.
[40]Y. Li, B. Yuan, H. Ji, D. Han, S. Chen, F. Tian, and X. Jiang, “A method for patterning multiple types of cells by using electrochemical desorption of self-assembled monolayers within microfluidic channels,” Angewandte Chemie-International Edition, 46, pp. 1094-1096, 2007.
[41]A. Folch, B. H. Jo, O. Hurtado, D. J. Beebe, and M. Toner, “Microfabiricated elastomeric stencils for micropatterning cell cultures,” Journal of Biomedical Materials Research, 52, pp. 346-353, 2000.
[42]N. E. Sanjana, and S. B. Fuller, “A fast flexible ink-jet printing method for patterning dissociated neurons in culture,” Journal of Neuroscience Methods, 136, pp. 151-163, 2004.
[43]M. Bani-Yaghoub, R. Tremblay, R. Voicu, G. Mealing, R. Monette, C. Py, K. Faid, and M. Sikorska, “Neurogenesis and neuronal communication on micropatterned neurochips,” Biotechnology and Bioengineering, 92, pp. 336-345, 2005.
[44]http://www.microchem.com/, Specialty electronic materials for emerging and niche lithographic processes, 2005.
[45]H. Andersson, C. Jönsson, C. Moberg, and G. Stemme, “Consecutive microcontact printing-ligands for asymmetric catalysis in silicon channels,” Sensors and Actuators B, 79, pp. 78-84, 2001.
[46]B. H. Jo, L. M. V. Lerberghe, K. M. Motsegood, and D. J. Beebe, “Three-Dimensional Micro-Channel Fabrication in PDMS Elastomer,” Journal of Microelectromechanical Systems, 9, pp. 76-81, 2000.