癌細胞透過入侵循環系統而達到轉移之過程，與細胞的脫離以及貼附機制有關，為了探討此一現象，本研究分為靜態實驗與動態實驗兩部分。靜態實驗利用電子細胞基質阻抗判斷技術(Electric Cell-substrate Impedance Sensing, ECIS)製作阻抗感測晶片，使用戊二醛交聯之明膠微圖案，吸引細胞貼附於電極上方，並監測HepG2細胞之形態與貼附變化。而藉由每隔1小時所得到的細胞貼附面積與阻抗變化之關係，可成為動態實驗參考之依據。
動態實驗藉由PDMS微流道內壁之設計，改變細胞的貼附條件，觀察癌細胞欲入侵之前的貼附行為以及阻塞之可能，並將HepG2肝癌細胞注入微流道內，模擬癌細胞經過微血管之情形。利用RIE以電漿轟擊PDMS，改變微流道內壁之粗糙度，進而改變細胞貼附之能力。癌細胞進入微流道時所表現之不同狀況，可反應該環境對癌細胞透過循環系統達到轉移的成功與否，也表示微血管於某種狀況下，阻塞不通的情形，並探討癌細胞透過肝門靜脈進入肝臟時，癌細胞貼附以及阻塞之情形，進而協助癌症之預防及治療，如飲食、藥物治療、基因治療等研究。

For metastasis of tumor cell invade into the circulatory system, it have to do with tumor-cell of detachment and attachment. This work presents the first part of a new framework for preventing the tumor-cell of carcinoma in situ transition from one organ to others. Using an ECIS (electric cell-substrate impedance sensing) chip coated with glutaraldehyde (GA)-crosslinked gelatin patterns suitable for cell attachment, the author monitor the cell adhesion situation not only by optical microscope but also by electrical means. This cell-culture experiment with 1-hour time resolution so far demonstrates that the attachment moment for HepG2 on gelatin surface is no longer than 4 hours after the cell dosing and these tumor cells cannot stay on GA-crosslinked gelatin surface for more than 7 hours.
The second part of experiment is dynamic. The author design an experiment and a microfluidic chip for investigating the relationship between the metastasis and the surface morphology of blood vessels. Using RIE (reactive ion etch) to change the inner wall morphology of PDMS. To different inner wall of PDMS roughness, the ability of cell adhesion is different. When tumor-cell through the microchannel, it may pass or get stuck. It’s mean that capillary may plugged by tumor-cell, or still smooth. Finally, to explore the  adhesion of tumor-cell and blocking when tumor-cell into the liver through the hepatic portal vein. It can assist in the prevention and treatment of cancer, such as diet, drug, gene therapy research.

目錄
中文摘要..................................................................................................... I
英文摘要....................................................................................................II
目錄...........................................................................................................III
圖目錄........................................................................................................V
表目錄...................................................................................................... IX
第一章 緒論...............................................................................................1
1-1 微機電系統...........................................................................1
1-2 研究動機...............................................................................2
1-3 文獻回顧...............................................................................6
1-3-1 癌細胞於循環系統之轉移.........................................6
1-3-2 肝硬化與癌細胞及循環系統的關係.........................8
1-3 研究目的.............................................................................13
1-4 研究架構.............................................................................15
第二章 肝癌細胞貼附之靜態實驗........................................................16
2-1 實驗設計.............................................................................16
2-2 電阻抗式感測晶片製作與改良.........................................18
2-3 實驗量測與結果.................................................................23
第三章 肝癌細胞貼附之動態實驗........................................................29
3-1 量測實驗設備架設說明.....................................................29
3-2 仿微血管之微流道設計.....................................................30
3-3 流動實驗設計.....................................................................32
3-4 微流道製作.........................................................................35
3-5 PDMS 微流道之表面粗糙化..............................................42
3-6 實驗量測結果.....................................................................63
第四章 結論與未來建議........................................................................67
4-1 本文貢獻之彙整..................................................................67
4-2 未來工作與建議..................................................................69
參考文獻...................................................................................................70
附錄A RIE 以氧氣電漿蝕刻Parylene N 之蝕刻率..............................76
附錄 B RIE 以氧氣電漿蝕刻Parylene C 之蝕刻率.............................77
附錄C 個人著作 “THE MINIUMUM TIME ESTIMATION FOR
INITIATING TUMOR-CELL ATTACHMENT”發表於國際傳感器會議
之全文。...................................................................................................79

圖目錄
圖1-1 癌細胞的轉移過程。....................................................................5
圖1-2 循環性癌細胞的生產過程與血管的SEM 圖。..........................7
圖1-3 共軛焦顯微鏡拍攝貼附於肺臟微血管之腫瘤細胞。................7
圖1-4 腫瘤細胞貼附於肝血竇(左圖)以及入侵至組織(右圖)。..........8
圖1-5 肝臟示意圖。................................................................................9
圖1-6 肝小葉示意圖。..........................................................................10
圖1-7 肝纖維化示意。(A)正常的肝血竇，藍色為星狀細胞，紫色為
Kupffer 細胞；(B)肝臟損傷後，星狀細胞增生以及週遭產生膠原組織
(黃色區域)。............................................................................................12
圖1-8 不同表面粗糙度對於親疏水性及細胞貼附的影響。..............14
圖1-9 細胞隨時間增加而增加貼附區域示意。..................................14
圖1-10 論文架構。................................................................................15
圖2-1 單一HeLa cell 細胞在不同接種時間貼附情形之量測結果。17
圖2-2 本實驗之感測電極與實體。......................................................18
圖2-3 感測晶片製作流程。..................................................................19
圖2-4 感測晶片製作流程(續)。...........................................................20
圖2-5 parylene C 包覆之電路板與晶片。.............................................22
圖2-6 細胞量測後電路板銹蝕之比較。..............................................22
圖2-7 感測電極編號。..........................................................................24
圖2-8 CH3 電極上方HepG2 細胞貼附之情形。.................................24
圖2-9 細胞貼附於CH3 電極上的電訊號。........................................25
圖2-10 CH3 電極上方HepG2 細胞貼附之情形。...............................25
圖2-11 細胞貼附於CH3 電極上的電訊號。.......................................26
圖2-12 CH3 電極上細胞貼附面積與阻抗變化量之關係。................27
圖2-13 CH5 電極上細胞貼附面積與阻抗變化量之關係。................28
圖3-1 實驗架設示意。..........................................................................29
圖3-2 實驗架設。..................................................................................30
圖3-3 微流道3D 模型。.......................................................................31
圖3-4 流道中間的觀察區 (單位：μm)。............................................32
圖3-5 SU-8 光阻厚度相對曝光劑量之對照表。.................................36
圖3-6 本實驗室真空烘箱(型號:DOV-30)。........................................37
圖3-7 PDMS 氧氣電漿表面處理化學狀態表示圖。...........................37
圖3-8 微流道製作流程圖。..................................................................39
圖3-9 完成顯影的SU-8 母模。............................................................40
圖3-10 OM 拍攝之SU-8 微流道。.......................................................40
圖3-11 SU-8 微流道觀察區之膜厚(量測區域為圖3-8 的A-A 線段)。
...................................................................................................................41
圖3-12 SEM 拍攝之PDMS 微流道。...................................................41
圖3-13 PDMS 微流道與Glass 接合。..................................................41
圖3-14 倒立式顯微鏡拍攝之微流道觀察區。....................................42
圖3-15 電漿蝕刻PDMS 之SEM 圖。.................................................43
圖3-16 本實驗室之反應離子蝕刻機(型號：SAMCO RIE1-C)。.....44
圖3-17 氟氧混合電漿蝕刻PDMS 13 min 之結果(參數為O2: 13
sccm；CF4: 37 sccm；80W)。...............................................................45
圖3-18 氧氣電漿蝕刻PDMS 14 min 之結果(參數為O2: 50 sccm；
80W)。.....................................................................................................46
圖3-19 Ra 與蝕刻時間的關係(左圖為CF4 O2 之混合氣體，右圖為O2
氣體)。.....................................................................................................47
圖3-20 表面接觸角量測儀。................................................................58
圖3-21 混合氣體電漿蝕刻PDMS 之時間與表面接觸角的關係。...59
圖3-22 CF4 與O2 混合氣體電漿蝕刻PDMS 之接觸角量測。...........60
圖3-23 O2 電漿蝕刻PDMS 之時間與表面接觸角的關係。...............61
圖3-24 O2 電漿蝕刻PDMS 之接觸角量測。.......................................62
圖3-25 微流道實驗影像擷取位置。....................................................63
圖3-26 微流道實驗影像擷取。............................................................64
圖A-1 載玻片上Parylene N 之量測點。.............................................76
圖A-2 氧氣電漿蝕刻Parylene N 之關係圖。.....................................76
圖B-1 載玻片上Parylene C 之量測點。..............................................77
圖B-2 氧氣電漿蝕刻Parylene C 之關係圖。......................................77
圖B-3 氧氣電漿蝕刻Parylene C 之關係圖。......................................78

表目錄
表1-1 癌細胞轉移前與轉移後之五年相對生存率。............................4
表2-1 CH3 電極之細胞貼附面積估算與阻抗變化量。......................27
表2-2 CH5 電極之細胞貼附面積估算與阻抗變化量。......................27
表3-1 電漿蝕刻PDMS 之參數。.........................................................43
表3-2 PDMS 表面粗糙度之參數。.......................................................47
表3-3 PDMS 以混合氣體電漿蝕刻後之表面形貌。...........................48
表3-4 PDMS 氧氣電漿蝕刻後之表面形貌。.......................................53
表3-5 CF4 與O2 混合氣體電漿蝕刻PDMS 之表面接觸角。.............59
表3-6 O2 電漿蝕刻PDMS 之表面接觸角。.........................................61
表A-1 以氧氣電漿蝕刻之剩餘膜厚量測與蝕刻率。.........................76
表B-1 以氧氣電漿蝕刻之剩餘膜厚量測與蝕刻率(100W，50sccm)。
...................................................................................................................77
表B-2 以氧氣電漿蝕刻之剩餘膜厚量測與蝕刻率(80W，30sccm)。
...................................................................................................................78

[1]J.M. Bustillo, R.T. Howe, and R.S. Muller, “Surface micromachining for microelectromechanical systems,” Proceedings of the IEEE, 86 (8), pp. 1552-1573, 1998.
[2]G.T.A. Kovacs, N.I. Maluf, and K.E. Petersen, “Bulk micromachining of silicon,” Proceedings of the IEEE, 86 (8), pp. 1536-1551, 1998.
[3]楊嘉麗，“新興科技介紹-微機電技術”，資策會資訊市場情報中心產業研究報告，頁1-16，2003 年。
[4]R.H. Liu, J. Yang, R. Lenigk, J. Bonanno, and P. Grodzinski, “Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection,” Analytical Chemistry, 76 (7), pp. 1824-1831, 2004.
[5]Rean-Der Chien, “Micromolding of biochip devices designed with microchannels,” Sensors and Actuators, A: Physical, 128 (2), pp. 238-247, 2006.
[6]J. Schroers, Q. Pham, and A. Desai, “Thermoplastic forming of bulk netallic glass - A technology for MEMS and microstructure fabrication,” Journal of Microelectromechanical Systems, 16 (2), pp. 240-247, 2007.
[7]A. Mata, E.J. Kim, C.A. Boehm, and A.J. Fleischman, G.F. Muschler, and S.A.  Roy, “three-dimensional scaffold with precise micro-architecture and surface micro-textures,” Biomaterials, 30 (27), pp. 4610-4617, 2009.
[8]H. Lorenz, M. Despont, N. Fahrni, N. LaBianca, P. Renaud, and P. Vettiger, “SU-8: a low-cost negative resist for MEMS,” Journal of Micromechanics and Microengineering, 7 (3), pp. 121-124, 1997.
[9]C. Liu, J.-B. Huang, Z. Zhu, F. Jiang, S. Tung, Y.-C. Tai, and C.-M. Ho, “A micromachined flow shear-stress sensor based on thermal transfer principles,” Journal of Microelectromechanical Systems, 8 (1), pp. 90-98, 1999.
[10]C. Liu, T. Tsao, G.-B. Lee, J.T.S. Leu, Y.W. Yi, Y.-C. Tai, and C.-M. Ho, “Out-of-plane magnetic actuators with electroplated permalloy for fluid dynamics control,” Sensors and Actuators, A: Physical, 78 (2), pp. 190-197, 1999.
[11]P. Yao, G.J. Schneider, and D.W. Prather, “Three-dimensional lithographical fabrication of microchannels,” Journal of Microelectromechanical Systems, 14 (4), pp. 799-805, 2005
[12]Jiun-Min Wang and Lung-Jieh Yang, “Electro-hydro-dynamic (EHD) micropumps with electrode protection by parylene and gelatin,” Tamkang Journal of Science and Engineering, 8 (3), pp. 231-236, 2005.
[13]李國賓，“下一波生物晶片－微流體生醫晶片”，科學發展月刊，385期, pp. 72-77, 2005.
[14]Cancer Facts & Figures, American Cancer Society, pp. 9-22, 2010.
[15]J. Yang, S.A. Mani, J.L. Donaher, S. Ramaswamy, R.A. Itzykson, C. Come, P. Savagner, I. Gitelman, A. Richardson, and R.A. Weinberg, “Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis,” Cell, 117 (7), pp. 927-939, 2004.
[16]P.S. Steeg, “Metastasis suppressors alter the signal transduction of cancer cells,” Nature Reviews Cancer, 3 (1), pp. 55-63, 2003.
[17]P. Carmeliet and R. K. Jain, “Angiogenesis in cancer and other diseases,” Nature, 407, 249-257, 2000.
[18]A. B. Al-Mehdi, K. Tozawa, A. B. Fisher, L. Shientag, A. Lee, and R. J. Muschel, “Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: A new model for metastasis,” Nature Medicine, 6 (1), pp. 100-102, 2000.
[19]P. Gassmann, M.-L. Kang, S.T. Mees, J. Haier, “In vivo tumor cell adhesion in the pulmonary microvasculature is exclusively mediated by tumor cell - endothelial cell interaction,” BMC Cancer, 10 (177), 2010.
[20]J. Haier, T. Korb, B. Hotz, H.-U. Spiegel, N. Senninger, M.G. Sarr, M.G. Stelzner, K.E. Behrns and R.B. Adams, “An intravital model to monitor steps of metastatic tumor cell adhesion within the hepatic microcirculation,” Journal of Gastrointestinal Surgery, 7 (4), pp. 507-515, 2003.
[21]T. Bogenrieder and M. Herlyn, “Axis of evil: Molecular mechanisms of cancer metastasis,” Oncogene, 22 (43), pp. 6524-6536, 2003.
[22]T. Bogenrieder and M. Herlyn, “Effects of tamoxifen on pulmonary fibrosis after cobalt-60 radiotherapy in breast cancer patients,” Oncogene, 22 (43), pp. 6524-6536, 2003.
[23]A.J. Witkamp, E. De Bree, R. Van Goethem, and F.A.N. Zoetmulder, “Rationale and techniques of intra-operative hyperthermic intraperitoneal chemotherapy,” Cancer Treatment Reviews, 27 (6), pp. 365-374, 2001.
[24]“The World Book Encyclopedia L,” Vol. 12, pp. 393-394, World Book Inc., Chicago, 2006.
[25]C.C. Cunningham, and C.G. Van Horn, “Energy availability and alcohol-related liver pathology,” Alcohol Research and Health, 27 (4), pp. 291-299, 2003.
[26]M. Yuen, E. Sablon, H. Yuan, D. Wong, C. Hui, B. Wong, A. Chan, and C. Lai, “Significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis- related complications, and hepatocellular carcinoma,” Hepatology, 37 (3), pp. 562-567, 2003.
[27]M.A. O'Connell and S.A. Rushworth, “Curcumin: Potential for hepatic fibrosis therapy? ,” British Journal of Pharmacology, 153 (3), pp. 403-405, 2008.
[28]D. Gospodarowicz, D. Delgado, and I. Vlodavsky, “Permissive effect of the extracellular matrix on cell proliferation in vitro,” Proceedings of the National Academy of Sciences of the United States of America, 77 (7 II), pp. 4094-4098, 1980.
[29]D.V. Sakharov, A.F.H. Jie, M.E.A. Bekkers, J.J. Emeis, and D.C. Rijken, “Polylysine as a vehicle for extracellular matrix–targeted local drug delivery, providing high accumulation and long-term retention within the vascular wall,” Arteriosclerosis, Thrombosis, and Vascular Biology, 21 (6), pp. 943-948, 2001.
[30]Y.-C Ou, C.-W. Hsu, L.-J. Yang, H.-C. Han, Y.-W. Liu, and C.-Y. Chen, “The micropatterns of glutaraldehyde-crosslinked gelatin as ECM for attachment of tumor cells,” 4th IEEE International Conference on Nano/Micro Engineered and Molecular Systems, NEMS 2009, art. no. 5068585, pp. 314-318, 2009.
[31]A. Ranella, M. Barberoglou, S. Bakogianni, C. Fotakis, and E. Stratakis, “Tuning cell adhesion by controlling the roughness and wettability of 3D micro/nano silicon structures,” Acta Biomaterialia, 6 (7), pp. 2711-2720, 2010.
[32]F. Gentile, L. Tirinato, E. Battista, F. Causa, C. Liberale, E.M. di Fabrizio, and P. Decuzzi, “Cells preferentially grow on rough substrates,” Biomaterials, 31 (28), pp. 7205-7212, 2010.
[33]L.-J. Yang, W.-Z. Lin, T.-J. Yao and Y.-C. Tai, “Photo-patternable gelatin as protection layers in low-temperature surface micromachinings,” Sensors and Actuators, A: Physical, 103 (1-2), pp. 284-290, 2003.
[34]W.-C. Lin and L.-J. Yang, “The patterning of glutaraldehyde-crosslinked gelatin,” Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS), pp. 173-176, 2004.
[35]Lung-Jieh Yang and Yu-Cheng Ou, “The micro patterning of glutaraldehyde (GA)-crosslinked gelatin and its application to cell-culture,” Lab on a Chip, 5(9), pp. 979-984, 2005.
[36]Y. C. Ou, C. W. Hsu, L. J. Yang, H. C. Han, Y. W. Liu and C. Y. Chen, “Attachment of tumor cells to the micropatterns of glutaraldehyde (GA)-crosslinked gelatin,” Sensors and Materials, 20 (8), pp. 435-446, 2008.
[37]歐育誠，“以明膠微圖案進行細胞之操控貼附、培養與監控”，博士論文，淡江大學機械與機電工程學系，指導教授楊龍杰，民國98年7月。
[38]I. Giaever and C. R. Keese, “Monitoring fibroblast behavior in tissue culture with an applied electric field,” Proceedings of the National Academy of Sciences of the United States of America, 81 (12), pp. 3761-3764, 1984.
[39]I. Giaever and C. R. Keese, “A morphological biosensor for mammalian cells,” Nature, 366 (6455), pp. 591-592, 1993.
[40]A. Rothermel, R. Kurz, M. Rüffer, , W. Weigel, , H. G. Jahnke, A. K. Sedello, H. Stepan, , R. Faber , K. Schulze-Forster and A. A. Robitzki, “Cells on a chip - The use of electric properties for highly sensitive monitoring of blood-derived factors involved in angiotensin II type 1 receptor signalling,” Cellular Physiology and Biochemistry, 16 (1-3), pp. 51-58, 2005.
[41]M. Brischwein, S. Herrmann, W. Vonau, F. Berthold, H. Grothe and E. R. Motrescu, “Electric cell-substrate impedance sensing with screen printed electrode structures,” Lab on a Chip, 6 (6), pp. 819-822, 2006.
[42]P. Seriburi, S. McGuire, A. Shastry, K. F. Böhringer and D. R. Meldrum, “Measurement of the cell-substrate separation and the projected area of an individual adherent cell using electric cell-substrate impedance sensing,” Analytical Chemistry, 80 (10), pp. 3677-3683, 2008.
[43]Y. Qiu, R. Liao and X. Zhang, “Real-time monitoring primary cardiomyocyte adhesion based on electrochemical impedance spectroscopy and electrical cell-substrate impedance sensing,” Analytical Chemistry, 80 (4), pp. 990-996, 2008.
[44]T.S. Hug, “Biophysical methods for monitoring cell-substrate interactions in drug discovery,” Assay Drug Dev Technol 1 (3), pp. 479-488, 2003.
[45]P.E. Gates, W.D. Strain, and A.C. Shore, “Human endothelial function and microvascular ageing,” Experimental Physiology, 94 (3), pp. 311-316, 2009.
[46]M.-C. Bélanger and Y. Marois, “Hemocompatibility, biocompatibility, inflammatory and in vivo studies of primary reference materials low-density polyethylene and polydimethylsiloxane: A review,” Journal of Biomedical Materials Research, 58 (5), pp. 467-477, 2001.
[47]C.R. Tamanaha, L.J. Whitman, and R.J. Colton, “Hybrid macro-micro fluidics system for a chip-based biosensor,” Journal of Micromechanics and Microengineering, 12 (2), pp. N7-N17, 2002.
[48]K.S. Ryu, X. Wang, K. Shaikh, and C. Liu, “A method for precision patterning of silicone elastomer and its applications,” Journal of Microelectromechanical Systems, 13 (4), pp. 568-575, 2004.
[49]江良規 譯，「運動生理學」，台灣商務印書館股份有限公司，pp. 104，中華民國78年5月。
[50]S. Bhattacharya, A. Datta, J.M. Berg, and S. Gangopadhyay, “Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength,” Journal of Microelectromechanical Systems, 14 (3), pp. 590-597, 2005.
[51]Rodney Boyer，生物化學，第三版，歐亞書局，pp. 54，2006年。
[52]O. Schlager, A. Giurgea, C. Margeta, D. Seidinger, S. Steiner-Boeker, B. Van Der Loo and R. Koppensteiner, “Wall shear stress in the superficial femoral artery of healthy adults and its response to postural changes and exercise,” European Journal of Vascular and Endovascular Surgery, 41 (6), pp. 821-827, 2011.
[53]R.L. Street, G.Z. Watters, J.K. Vennard, “Elementary fluid mechanics,” 7ed, ISBN: 0-471-01310-2, pp. 327, 1996.
[54]J.R. Levick, C.C. Michel, “Microvascular fluid exchange and the revised Starling principle,” Cardiovascular Research 87 (2), pp. 198-210, 2010.
[55]Gersteltec Sarl, “SU-8 negative tone photoresist thickness 15-200 μm,” GM 1070 technical datasheet, Swizerland, 2005.
[56]J. Garra, T. Long, J. Currie, T. Schneider, R. White and M. Paranjape, “Dry etching of polydimethylsiloxane for microfluidic systems,” Journal of Vacuum Science and Technology, Part A: Vacuum, Surfaces and Films, 20 (3), pp. 975-982, 2002.