癌症研究應用之指壓致動式細胞分選微系統晶片研發

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研究生：	李宇雙 Lee, Yu-Shuang
論文名稱：	癌症研究應用之指壓致動式細胞分選微系統晶片研發 A Finger-powered Cell-sorting Microsystem Chip for Cancer-study Applications
指導教授：	劉承賢 Liu, Cheng-Hsien
口試委員:	徐琅 Hsu, Long 陳皇銘 Chen, Huang-Ming
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2019
畢業學年度：	107
語文別：	中文
論文頁數：	91
中文關鍵詞：	微系統晶片、癌症、指壓微幫浦、細胞分選、確定性側向位移
外文關鍵詞：	Microfluidics, Cancer, Cell-sorting, Finger-powered, DLD
相關次數：	點閱：4 下載：0
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癌症為全球第二大主要死亡原因，死亡人數佔總死亡人數的1/6之多，而其大部分屬於實性瘤——異常增生組織塊，內含的腫瘤浸潤免疫細胞數量與比例已被證明與癌症類型、階段、甚至是預後相關，因此對其的追蹤、觀察、監測更有著極大的需求，但對於設備不均、專業人員不足之國家，及往後個人化醫療的需求，笨重、昂貴、操作複雜、高功耗的設備便成了一大阻礙。本研究所研發的整合化微系統晶片，以功能上區分，包含指壓微型幫浦自給動力供應、微型混合器染劑染色細胞、確定性側向位移目標細胞分選、低倍率顯微鏡拍攝計數。實驗結果顯示，能透過指壓幫浦與附加微型閥模組，間接推送定量不同流體並有效防止逆流，並平均推送體積為2.37 µL（致動室R = 2.00 mm）及3.72 µL（致動室R = 2.50 mm）。微型混合器能於推送下以設計比例混合染劑及樣本，並染色完成之細胞溶液連同緩衝溶液由同一幫浦模組推送入確定性測性位移處，從四區微柱臨界直徑漸小之連續分選區域，分散完成非目標細胞之偏移。最後，以小鼠直腸癌實性瘤單細胞懸液為樣本下，以注射幫浦推送，可達平均98.63%的純率，回收率則為90.18%；以指壓幫浦推送也可達成純率97.23%以及回收率88.74%之高效率，並完整晶片運作能於一分鐘內完成。期望利用此系統能對於未來癌症實性瘤中腫瘤浸潤免疫細胞之追蹤、分析的達成更加簡易、快速、便捷。

Cancer is the second leading cause of death globally, and almost 1 in 6 deaths is due to cancer. The most common type of cancer is solid tumor, an abnormal mass of tissue. The immune cells in it are called tumor-infiltrating immune cells, which were proved that the amount of them related to tumor type, stage, even prognosis. Thus, there is a great demand for these cells-counting, -monitoring, and -detecting. However, for the either technologically or professionally disadvantaged regions, and the need of personalized medicine in the future, the bulky, complex, expensive, and high-power medical instruments become obstacles. In this study, we reported an integrated microsystem chip with the functions of self-powered supply by a finger pump, stain cells by micromixers, DLD cell-sorting, and cell-counting by microscope. The experimental results show that the different fluid can be separately, indirectly pushed through the finger pump and the additional valve module. The backflow can be effectively prevented via our design. The average pushed volume is 2.37 μL (for actuation chamber with R = 2.00 mm) and 3.72 μL (for actuation chamber with R = 2.50 mm). The micromixer can be used to effectively mix the stain and cell sample together in a design ratio. Then, the stained cell solution and the buffer solution are pushed into the deterministic displacement region by the same pump module. From the four-stage continuous sorting area where the critical diameters are gradually getting smaller, the non-target cells can be dispersedly shifted. Finally, we used suspension of solid tumor from CT26-bearing BALB/c mouse as a sample. With the syringe pump supplying the driving force, the purity and recovery rate can be achieved, 98.63% and 90.18% respectively. By using the finger pump instead, it can also achieve a high efficiency. The purity is 97.23%, and the recovery rate is 88.74%, respectively. The whole function operation on our proposed microsystem chip can be done in less than a minute. Our developed microsystem chip is expected to support an easier, faster and more convenient way for the tracking and analysis of tumor infiltrating immune cells in solid tumors.

Abstract----------I
摘要----------II
致謝----------III
目錄----------1
圖目錄----------3
第一章    序論----------7
1    前言----------7
2    研究動機與目的----------8
3    研究背景----------10
3.1    生醫微機電與實驗室晶片----------10
3.2    癌症----------11
3.3    電腦視覺----------14
4    文獻回顧----------15
4.1    微型幫浦----------15
4.2    微型閥----------16
4.3    微型混合器----------20
4.4    白血球分離技術----------25
第二章    系統理論與晶片設計----------27
1    系統理論----------27
1.1    微流體晶片設計理論----------27
1.2    微型指壓幫浦理論----------31
1.3    微型混合理論----------33
1.4    確定性側向位移理論（Deterministic lateral displacement）----------35
2    微系統晶片設計----------38
2.1    詳細設計及功能介紹----------38
2.2    運作流程----------46
第三章    微系統晶片製程----------48
1    製作流程----------48
1.1    微流道母模製程----------48
1.2    微幫浦壓力腔製程----------50
1.3    薄膜製程----------51
1.4    系統晶片製程----------52
2    製程結果----------55
第四章    實驗材料與方法----------57
1    實驗材料----------57
1.1    BALB/c白鼠脾細胞（BALB/c Mouse Splenocytes）----------57
1.2    BALB/c白鼠結腸癌細胞（BALB/c Mouse carcinoma cell line, CT26）培養----------58
1.3    BALB/c白鼠結腸癌實性瘤懸液（Suspension of solid tumor from CT26-bearing BALB/c Mouse）----------59
1.4    劉氏染色劑（Liu’s stain）----------60
2    實驗設備----------62
第五章    實驗結果與討論----------63
1    晶片一：指壓幫浦致動與微混合器樣本染色晶片----------63
1.1    指壓微幫浦、主被動閥----------63
1.2    微型混合器----------70
1.3    實際操作晶片測試----------74
2    晶片二：DLD細胞分選與影像辨識計數晶片----------76
2.1    細胞大小與頻率分佈分析----------76
2.2    確定性側向位移測試----------77
3    整體微系統晶片檢測----------83
3.1    運作概況測試----------83
3.2    整體晶片細胞分選效率探討----------84
第六章    結論與未來展望----------86
1    結論----------86
2    未來展望----------87
參考文獻----------88


                                

[1] World Health Organization, "Global Health Estimates 2016: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2016," Geneva2018.
[2] F. Bray and I. Soerjomataram, "The Changing Global Burden of Cancer: Transitions in Human Development and Implications for Cancer Prevention and Control," in Cancer: Disease Control Priorities, Third Edition (Volume 3), H. Gelband, P. Jha, R. Sankaranarayanan, and S. Horton, Eds. Washington (DC), 2015.
[3] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D. M. Parkin, D. Forman, and F. Bray, "Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012," International Journal of Cancer, vol. 136, no. 5, pp. E359-E386, Mar 1 2015.
[4] Christopher Mader, "The Biology of Cancer," The Yale Journal of Biology and Medicine, vol. 80, no. 2, pp. 91-91, 12/ 2007.
[5] A. C. Chiang and J. Massague, "Molecular Origins of Cancer Molecular Basis of Metastasis," New England Journal of Medicine, vol. 359, no. 26, pp. 2814-2823, Dec 25 2008.
[6] C. A. Klein, "Cancer - The metastasis cascade," Science, vol. 321, no. 5897, pp. 1785-1787, Sep 26 2008.
[7] L. M. Coussens, L. Zitvogel, and A. K. Palucka, "Neutralizing Tumor-Promoting Chronic Inflammation: A Magic Bullet?," Science, vol. 339, no. 6117, pp. 286-291, Jan 18 2013.
[8] A. J. Gentles, A. M. Newman, C. L. Liu, S. V. Bratman, W. G. Feng, D. Kim, V. S. Nair, X. Yue, A. Khuong, C. D. Hoang, M. Diehn, R. B. West, S. K. Plevritis, and A. A. Alizadeh, "The prognostic landscape of genes and infiltrating immune cells across human cancers," Cancer Research, vol. 75, no. 22, Nov 15 2015.
[9] D. Hanahan and L. M. Coussens, "Accessories to the Crime: Functions of Cells Recruited to the Tumor Microenvironment," Cancer Cell, vol. 21, no. 3, pp. 309-322, Mar 20 2012.
[10] L. A. Torre, F. Bray, R. L. Siegel, J. Ferlay, J. Lortet-Tieulent, and A. Jemal, "Global Cancer Statistics, 2012," Ca-a Cancer Journal for Clinicians, vol. 65, no. 2, pp. 87-108, Mar-Apr 2015.
[11] A. Elkhattouti, M. Hassan, and C. R. Gomez, "Stromal fibroblast in age-related cancer: role in tumorigenesis and potential as novel therapeutic target," Frontiers in Oncology, vol. 5, Jul 27 2015.
[12] C. Liu, C. J. Workman, and D. A. A. Vignali, "Targeting regulatory T cells in tumors," Febs Journal, vol. 283, no. 14, pp. 2731-2748, Jul 2016.
[13] F. Veglia, M. Perego, and D. Gabrilovich, "Myeloid-derived suppressor cells coming of age," Nature Immunology, vol. 19, no. 2, pp. 108-119, Feb 2018.
[14] J. Galon, A. Costes, F. Sanchez-Cabo, A. Kirilovsky, B. Mlecnik, C. Lagorce-Pages, M. Tosolini, M. Camus, A. Berger, P. Wind, F. Zinzindohoue, P. Bruneval, P. H. Cugnenc, Z. Trajanoski, W. H. Fridman, and F. Pages, "Type, density, and location of immune cells within human colorectal tumors predict clinical outcome," Science, vol. 313, no. 5795, pp. 1960-1964, Sep 29 2006.
[15] R. D. Schreiber, L. J. Old, and M. J. Smyth, "Cancer Immunoediting: Integrating Immunity's Roles in Cancer Suppression and Promotion," Science, vol. 331, no. 6024, pp. 1565-1570, Mar 25 2011.
[16] A. Guyton and J. Hall, Textbook of Medical Physiology, 11th ed. Philadelphia, PA, USA: Elsevier Inc., 2006, pp. 446-448.
[17] R. Dijkink and C. D. Ohl, "Laser-induced cavitation based micropump," Lab on a Chip, vol. 8, no. 10, pp. 1676-1681, Oct 2008.
[18] B. T. Chia, H. H. Liao, and Y. J. Yang, "A novel thermo-pneumatic peristaltic micropump with low temperature elevation on working fluid," Sensors and Actuators a-Physical, vol. 165, no. 1, pp. 86-93, Jan 2011.
[19] K. Iwai, K. C. Shih, X. Lin, T. A. Brubaker, R. D. Sochol, and L. W. Lin, "Finger-powered microfluidic systems using multilayer soft lithography and injection molding processes," Lab on a Chip, vol. 14, no. 19, pp. 3790-3799, Oct 7 2014.
[20] L. Klintberg, M. Karlsson, L. Stenmark, J. A. Schweitz, and G. Thornell, "A large stroke, high force paraffin phase transition actuator," Sensors and Actuators a-Physical, vol. 96, no. 2-3, pp. 189-195, Feb 28 2002.
[21] D. J. Beebe, J. S. Moore, J. M. Bauer, Q. Yu, R. H. Liu, C. Devadoss, and B. H. Jo, "Functional hydrogel structures for autonomous flow control inside microfluidic channels," Nature, vol. 404, no. 6778, pp. 588-+, Apr 6 2000.
[22] K. Yoshida, M. Kikuchi, J. H. Park, and S. Yokota, "Fabrication of micro electro-rheological valves (ER valves) by micromachining and experiments," Sensors and Actuators a-Physical, vol. 95, no. 2-3, pp. 227-233, Jan 1 2002.
[23] H. Hartshorne, Y. B. Ning, W. E. Lee, and C. Backhouse, "Development of microfabricated valves for mu TAS," Micro Total Analysis Systems '98, pp. 379-381, 2000.
[24] M. A. Unger, H. P. Chou, T. Thorsen, A. Scherer, and S. R. Quake, "Monolithic microfabricated valves and pumps by multilayer soft lithography," Science, vol. 288, no. 5463, pp. 113-116, Apr 7 2000.
[25] A. Y. Nobakht, M. Shahsavan, and A. Paykani, "Numerical Study of Diodicity Mechanism in Different Tesla-Type Microvalves," Journal of Applied Research and Technology, vol. 11, pp. 876-885, Dec 2013.
[26] B. Mosadegh, C. H. Kuo, Y. C. Tung, Y. S. Torisawa, T. Bersano-Begey, H. Tavana, and S. Takayama, "Integrated elastomeric components for autonomous regulation of sequential and oscillatory flow switching in microfluidic devices," Nature Physics, vol. 6, no. 6, pp. 433-437, Jun 2010.
[27] E. Choi, B. Kim, and J. Park, "High-throughput microparticle separation using gradient traveling wave dielectrophoresis," Journal of Micromechanics and Microengineering, vol. 19, no. 12, Dec 2009.
[28] Z. Yang, S. Matsumoto, H. Goto, M. Matsumoto, and R. Maeda, "Ultrasonic micromixer for microfluidic systems," Sensors and Actuators a-Physical, vol. 93, no. 3, pp. 266-272, Oct 15 2001.
[29] R. H. Liu, R. Lenigk, R. L. Druyor-Sanchez, J. N. Yang, and P. Grodzinski, "Hybridization enhancement using cavitation microstreaming," Analytical Chemistry, vol. 75, no. 8, pp. 1911-1917, Apr 15 2003.
[30] X. Z. Niu and Y. K. Lee, "Efficient spatial-temporal chaotic mixing in microchannels," Journal of Micromechanics and Microengineering, vol. 13, no. 3, pp. 454-462, May 2003.
[31] J. H. Tsai and L. W. Lin, "Active microfluidic mixer and gas bubble filter driven by thermal bubble micropump," Sensors and Actuators a-Physical, vol. 97-8, pp. 665-671, Apr 1 2002.
[32] B. Xu, T. N. Wong, N. T. Nguyen, Z. Z. Che, and J. C. K. Chai, "Thermal mixing of two miscible fluids in a T-shaped microchannel," Biomicrofluidics, vol. 4, no. 4, Dec 2010.
[33] H. H. Bau, J. H. Zhong, and M. Q. Yi, "A minute magneto hydro dynamic (MHD) mixer," Sensors and Actuators B-Chemical, vol. 79, no. 2-3, pp. 207-215, Oct 15 2001.
[34] B. He, B. J. Burke, X. Zhang, R. Zhang, and F. E. Regnier, "A picoliter-volume mixer for microfluidic analytical systems," Analytical Chemistry, vol. 73, no. 9, pp. 1942-1947, May 1 2001.
[35] V. Mengeaud, J. Josserand, and H. H. Girault, "Mixing processes in a zigzag microchannel: Finite element simulations and optical study," Analytical Chemistry, vol. 74, no. 16, pp. 4279-4286, Aug 15 2002.
[36] L. R. Huang, E. C. Cox, R. H. Austin, and J. C. Sturm, "Continuous particle separation through deterministic lateral displacement," Science, vol. 304, no. 5673, pp. 987-990, May 14 2004.
[37] F. Khodaee, S. Movahed, N. Fatouraee, and F. Daneshmand, "Numerical Simulation of Separation of Circulating Tumor Cells from Blood Stream in Deterministic Lateral Displacement (Dld) Microfluidic Channel," Journal of Mechanics, vol. 32, no. 4, pp. 463-471, Aug 2016.
[38] C. I. Civin, T. Ward, A. M. Skelley, K. Gandhi, Z. P. Lee, C. R. Dosier, J. L. D'Silva, Y. Chen, M. Kim, J. Moynihan, X. C. Chen, L. Aurich, S. Gulnik, G. C. Brittain, D. J. Recktenwald, R. H. Austin, and J. C. Sturm, "Automated Leukocyte Processing by Microfluidic Deterministic Lateral Displacement," Cytometry Part A, vol. 89a, no. 12, pp. 1073-1083, Dec 2016.
[39] National Aeronautics and Space Administration, "Man-Systems Integration Standards," vol. 1.
[40] H. Sakamoto and H. Haniu, "A Study on Vortex Shedding from Spheres in a Uniform-Flow," Journal of Fluids Engineering-Transactions of the Asme, vol. 112, no. 4, pp. 386-392, Dec 1990.
[41] D. W. Inglis, J. A. Davis, R. H. Austin, and J. C. Sturm, "Critical particle size for fractionation by deterministic lateral displacement," Lab on a Chip, vol. 6, no. 5, pp. 655-658, 2006.
[42] J. McGrath, M. Jimenez, and H. Bridle, "Deterministic lateral displacement for particle separation: a review," Lab on a Chip, vol. 14, no. 21, pp. 4139-4158, 2014.
[43] R. Vernekar, T. Kruger, K. Loutherback, K. Morton, and D. W. Inglis, "Anisotropic permeability in deterministic lateral displacement arrays," Lab on a Chip, vol. 17, no. 19, pp. 3318-3330, Oct 7 2017.
[44] J. A. Davis, "Microfluidic separation of blood components through deterministic lateral displacement," Doctoral thesis, Princeton University, 2008.

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