腫瘤的轉移是癌症難以治療及高死亡率的重要原因，當腫瘤成長至一定大小後，便會釋放循環腫瘤細胞（Circulating Tumor Cell, CTC）進入循環系統，並藉由循環系統將癌細胞擴散至體內其他器官或組織，相比於傳統診斷出癌症的時間點，許多研究證明可以更早的在血液中發現循環腫瘤細胞的存在，並使癌症的能早期發現、預防與治療，而相較於單顆的CTC，由包含CTC與多種細胞組成的循環腫瘤細胞團簇（CTC cluster）被認為在血液中有更高的存活率及轉移優勢，是癌症研究中的重點趨勢。本研究開發之微流道系統包含兩大功能區域，第一功能區域為利用三段式確定性側向位移技術進行篩選，使CTC團簇與樣本中其他細胞分離，並直接沿著流到晶片的第二功能區域，此區域為細胞抓取暨培養之陷阱陣列，並於結構表面已膠原蛋白修飾，模擬細胞外基質環境，以利於進行癌細胞的貼附及培養。實驗結果顯示，確定性側向位移結構分選大型團簇的效率以及其回收率皆能達95%以上，小型團簇則在漫流速的情況下達80%以上，有鑑於篩選速度與效率的平衡，最終使用5μl/min的樣本體積流速進行全晶片的實驗，並成功的使用陷阱陣列在百顆團簇內達到98.5%的抓取效率，在為期三天的培養中，細胞也能夠穩定貼附與成長，且存活率維持在94.4%以上。

Metastasis is an important reason why cancer is challenging to be treated and has a high mortality. When the tumor grows to a certain size, it will release Circulating Tumor Cell (CTC) into the Circulatory system, and CTCs will spread in the body. Compared with the traditional time point of cancer diagnosis, many studies have proved that the existence of circulating tumor cells could be found in the blood earlier, and cancer can be detected, prevented and treated early. Compared with a single CTC, circulating tumor cell clusters (CTC clusters) composed of CTCs and a variety of cells are considered to have higher viability and metastasis potential in the blood, which is a key point in cancer research. The microfluidic system developed in this research contains two major functional areas. The first functional area is the use of three-level deterministic lateral displacement technology for sorting. CTC clusters are separated from other cells in the sample and flow directly to the second functional area of the chip. This area is a trap array for cell capturing and culturing. The surface has been modified with collagen to build the extracellular matrix environment to promote the attachment and culturing of cancer cells. Experimental results show that the deterministic lateral displacement sorting efficiency and recovery rate of large clusters can reach more than 95%. Both of small clusters can reach more than 80% under the condition of a lower flow rate. In view of the balance of sorting speed and efficiency, a sample volume flow rate of 5μl/min was used to perform a full-chip experiment, and the trap array was successfully used to achieve a capturing efficiency of 98.5% in a hundred clusters. The cells can also attach and grow stably during the three-day culture. And the cell viability remains above 94.4%.

Abstract 2
摘要 3
致謝 4
目錄 5
圖目錄 8
表目錄 11
第一章 緒論 12
1.1前言 12
1.2研究動機與目的 13
1.3研究背景 15
1.3.1生醫微機電與實驗室晶片 15
1.3.2癌症 16
1.3.2.1癌症簡介 16
1.3.2.2循環腫瘤細胞（Circulating Tumor Cell, CTC）及團簇 17
1.3.3居家定點照護檢驗（Point of care testing, POCT） 22
1.4文獻回顧 23
1.4.1循環腫瘤癌細胞團簇篩選技術 23
1.4.2細胞抓取暨培養平台 28
第二章 晶片設計與系統理論 31
2.1晶片設計概述 31
2.2功能理論與設計細節 33
2.2.1確定性側向位移（Deterministic lateral displacement, DLD） 33
2.2.1.1 理論介紹： 33
2.2.1.2 理論參數： 36
2.2.1.3 晶片參數設計： 38
2.2.2細胞抓取陷阱陣列 42
2.2.2.1 理論介紹 42
2.2.2.2 晶片參數設計 43
2.2.3 PDMS與表面修飾 46
2.2.3.1 Pluronic F-127 47
2.2.3.2 膠原蛋白（Type I Collagen） 48
2.3微流體系統基礎理論 49
2.3.1雷諾數（Reynolds number, Re） 49
2.3.2微流體流阻分析 50
第三章 微流體晶片製程 52
3.1製作流程 52
3.1.1微流體晶片母模製程 52
3.1.2微流體晶片製程 55
3.2製程結果 57
第四章 實驗材料與方法 58
4.1實驗材料製備 58
4.1.1人類癌症肺泡上皮細胞（Human lung epithelial carcinoma cell line, A549）培養 58
4.1.2人類T淋巴細胞 （Human T lymphocyte cell line, Jurkat）培養 60
4.1.3血液樣本製備 60
4.1.4 Pluronic F-127溶液製備 61
4.1.5膠原蛋白（Type I Collagen）溶液製備 61
4.1.6檢測螢光染劑製備及細胞染色 61
4.1.6.1 細胞追蹤螢光染色 61
4.1.6.2 細胞死活螢光染色 62
4.2實驗架設 63
4.3實驗操作流程 64
4.3.1晶片前處理 64
4.3.2晶片操作流程 64
第五章 實驗結果與討論 66
5.1腫瘤循環細胞及團簇的尺寸與型態分析 66
5.2腫瘤循環細胞團簇篩選分析 70
5.2.1 DLD陣列中粒子的運動表現 70
5.2.2 DLD陣列篩選效率 72
5.3腫瘤循環細胞團簇抓取分析 77
5.4腫瘤循環細胞團簇培養分析 81
第六章 結論 84
6.1結論 84
6.2未來展望 85
參考文獻 86

[1] F. R. Vogenberg, C. Isaacson Barash, and M. Pursel, "Personalized medicine: part 1: evolution and development into theranostics," (in eng), P t, vol. 35, no. 10, pp. 560-76, Oct 2010.
[2] G. C. o. D. Collaborators, "Global, regional, and national age-sex specific mortality for 264 causes of death, 1980–2016: a systematic analysis for the Global Burden of Disease Study 2016," in "Global Health Metrics," 2017, vol. 390.
[3] M. Ilie et al., ""Sentinel" circulating tumor cells allow early diagnosis of lung cancer in patients with chronic obstructive pulmonary disease," (in eng), PLoS One, vol. 9, no. 10, p. e111597, 2014.
[4] R. L. Siegel, K. D. Miller, H. E. Fuchs, and A. Jemal, "Cancer Statistics, 2021," CA: A Cancer Journal for Clinicians, vol. 71, no. 1, pp. 7-33, 2021.
[5] S. Meng et al., "Circulating Tumor Cells in Patients with Breast Cancer Dormancy," Clinical Cancer Research, vol. 10, no. 24, p. 8152, 2004.
[6] J.-M. Hou et al., "Clinical Significance and Molecular Characteristics of Circulating Tumor Cells and Circulating Tumor Microemboli in Patients With Small-Cell Lung Cancer," Journal of Clinical Oncology, vol. 30, no. 5, pp. 525-532, 2012.
[7] S. Amintas et al., "Circulating Tumor Cell Clusters: United We Stand Divided We Fall," (in eng), Int J Mol Sci, vol. 21, no. 7, Apr 10 2020.
[8] Y. Hong, F. Fang, and Q. Zhang, "Circulating tumor cell clusters: What we know and what we expect (Review)," (in eng), Int J Oncol, vol. 49, no. 6, pp. 2206-2216, Dec 2016.
[9] S. H. Au et al., "Clusters of circulating tumor cells traverse capillary-sized vessels," (in eng), Proc Natl Acad Sci U S A, vol. 113, no. 18, pp. 4947-4952, 2016.
[10] S. Heeke, B. Mograbi, C. Alix-Panabières, and P. Hofman, "Never Travel Alone: The Crosstalk of Circulating Tumor Cells and the Blood Microenvironment," (in eng), Cells, vol. 8, no. 7, Jul 13 2019.
[11] B. M. Szczerba et al., "Neutrophils escort circulating tumour cells to enable cell cycle progression," Nature, vol. 566, no. 7745, pp. 553-557, Feb 01 2019 .
[12] N. Aceto et al., "Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis," (in eng), Cell, vol. 158, no. 5, pp. 1110-1122, Aug 28 2014.
[13] L. Huang et al., "High Expression of Plakoglobin Promotes Metastasis in Invasive Micropapillary Carcinoma of the Breast via Tumor Cluster Formation," (in eng), J Cancer, vol. 10, no. 12, pp. 2800-2810, 2019.
[14] S. Gkountela et al., "Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding," (in eng), Cell, vol. 176, no. 1-2, pp. 98-112.e14, Jan 10 2019.
[15] M. Haemmerle, R. L. Stone, D. G. Menter, V. Afshar-Kharghan, and A. K. Sood, "The Platelet Lifeline to Cancer: Challenges and Opportunities," (in eng), Cancer Cell, vol. 33, no. 6, pp. 965-983, 2018.
[16] A. Kulasinghe, J. Zhou, L. Kenny, I. Papautsky, and C. Punyadeera, "Capture of Circulating Tumour Cell Clusters Using Straight Microfluidic Chips," (in eng), Cancers (Basel), vol. 11, no. 1, p. 89, 2019.
[17] H. W. Hou et al., "Isolation and retrieval of circulating tumor cells using centrifugal forces," Scientific Reports, vol. 3, no. 1, p. 1259, Feb 12 2013.
[18] A. F. Sarioglu et al., "A microfluidic device for label-free, physical capture of circulating tumor cell clusters," Nature Methods, vol. 12, no. 7, pp. 685-691, Jul 01 2015.
[19] N. Li, D. T. Kamei, and C. Ho, "On-Chip Continuous Blood Cell Subtype Separation by Deterministic Lateral Displacement," in 2007 2nd IEEE International Conference on Nano/Micro Engineered and Molecular Systems, , pp. 932-936, Jan 16-19 2007.
[20] K. Loutherback, J. D'Silva, L. Liu, A. Wu, R. H. Austin, and J. C. Sturm, "Deterministic separation of cancer cells from blood at 10 mL/min," (in eng), AIP Adv, vol. 2, no. 4, pp. 42107-42107, 2012.
[21] S. H. Au et al., "Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry," Scientific Reports, vol. 7, no. 1, p. 2433, May 26 2017.
[22] H. Song, T. Chen, B. Zhang, Y. Ma, and Z. Wang, "An integrated microfluidic cell array for apoptosis and proliferation analysis induction of breast cancer cells," Biomicrofluidics, vol. 4, no. 4, p. 044104, 2010.
[23] E. Moonen, R. Luttge, and J.-P. Frimat, "Single cell trapping by capillary pumping using NOA81 replica moulded stencils," Microelectronic Engineering, vol. 197, pp. 1-7, Oct 05 2018.
[24] C.-H. Lin et al., "A microfluidic dual-well device for high-throughput single-cell capture and culture," Lab on a Chip, 10.1039/C5LC00541H vol. 15, no. 14, pp. 2928-2938, 2015.
[25] 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, 2004.
[26] D. W. Inglis, J. A. Davis, R. H. Austin, and J. C. Sturm, "Critical particle size for fractionation by deterministic lateral displacement," (in eng), Lab Chip, vol. 6, no. 5, pp. 655-8, May 2006.
[27] J. McGrath, M. Jimenez, and H. Bridle, "Deterministic lateral displacement for particle separation: a review," Lab on a Chip, 10.1039/C4LC00939H vol. 14, no. 21, pp. 4139-4158, 2014.
[28] D. W. Inglis, "Efficient microfluidic particle separation arrays," Applied Physics Letters, vol. 94, no. 1, p. 013510, 2009.
[29] S. Ranjan, K. K. Zeming, R. Jureen, D. Fisher, and Y. Zhang, "DLD pillar shape design for efficient separation of spherical and non-spherical bioparticles," Lab on a Chip, 10.1039/C4LC00578C vol. 14, no. 21, pp. 4250-4262, 2014.
[30] J. P. Beech, "Microfluidics Separation and Analysis of Biological Particles," 2011.
[31] J. A. Davis, "Microfluidic Separation of Blood Components through Deterministic Lateral Displacement," 2008.
[32] D. Di Carlo, N. Aghdam, and L. P. Lee, "Single-Cell Enzyme Concentrations, Kinetics, and Inhibition Analysis Using High-Density Hydrodynamic Cell Isolation Arrays," Analytical Chemistry, vol. 78, no. 14, pp. 4925-4930, Jul 01 2006.
[33] D. Kim and A. E. Herr, "Protein immobilization techniques for microfluidic assays," (in eng), Biomicrofluidics, vol. 7, no. 4, pp. 41501-41501, 2013.
[34] A. Gokaltun, M. L. Yarmush, A. Asatekin, and O. B. Usta, "Recent advances in nonbiofouling PDMS surface modification strategies applicable to microfluidic technology," (in eng), Technology (Singap World Sci), vol. 5, no. 1, pp. 1-12, 2017.
[35] Z. Wu and K. Hjort, "Surface modification of PDMS by gradient-induced migration of embedded Pluronic," Lab on a Chip, 10.1039/B901651A vol. 9, no. 11, pp. 1500-1503, 2009.
[36] R. Kalluri and R. A. Weinberg, "The basics of epithelial-mesenchymal transition," (in eng), J Clin Invest, vol. 119, no. 6, pp. 1420-1428, 2009.