癌症(亦稱為惡性腫瘤)是一種細胞增生所引起的嚴重疾病。這種細胞增殖可以透過轉移進一步轉移到人體的其他正常器官，癌症細胞的轉移大多從原發腫瘤部位通過循環系統或淋巴系統轉移到其他正常器官。癌症對人類的健康生活有很大的影響是由於其偵測困難和治療費用昂貴以及大多數侵入性治療有害人體；對於沒有特異性診斷生物標記物的癌症，尤其是膽管癌（CCA），更加劇其診斷困難度。CCA是上皮細胞病變所引起的致死性疾病，好發於膽管中。由於缺乏特定的生物標誌物，並且由於膽管的位置而難以診斷，因此常常在晚期被發現進而錯過治療黃金時期。為了提高CCA早期診斷的準確性，一種有前瞻性的方法是使用CCA特異性親和試劑通過液態生物檢體來檢測CCA細胞。在這項研究中，膽汁（一種接近CCA病灶的體液）被使用於臨床上檢測CCA的液態生物檢體。此外我們設計並製造了一種新的微流體晶片，可以通過使用三種不同類型的親和試劑(anti-EpCAM antibody,多醣體以及適體)自動檢測膽汁中的CCA細胞。實驗結果顯示這三種親和試劑可以在僅僅3 mL 中的膽汁成功抓取CCA腫瘤細胞。這種微流體系統可以作為在臨床中檢測CCA的有前瞻性的工具。我們使用的微流體晶片可以在80分鐘內從3 mL的膽汁中分離出膽管癌病人的癌細胞，大型機台實驗結果顯示使用三種不同親和性試劑均可以從81位病人中有效的分離出膽管癌細胞，且準確率高達96.2%，靈敏度為100%，特異度為72.7%。微流體晶片實驗顯示準確率、靈敏度、特異度皆高達100%。新的微流體晶片將有潛力成為臨床上膽管癌的預後以及早期診斷是否遠端轉移的工具。

Cancer, also called malignant tumor, is a serious disease that causes hyperplasia. This cell proliferation may migrate to normal organs in the human body by a process called metastasis, which is initiated when cancer cells from the primary tumor site migrate to a secondary site through the circulatory or lymphatic system. Cancer has had a great impact on human lives, due to its difficult and expensive management, invasive treatment progress and harmful health deterioration; it is difficult for cancer early diagnosis, especially cholangiocarcinoma (CCA), which has no specific diagnostic biomarkers. CCA is a lethal disease of epithelial origin which occurs in the bile ducts. It is often diagnosed at advanced stages due to the lack of specific biomarkers and difficulty in diagnosis due to the location of the bile ducts. To improve the accuracy of early diagnosis of CCA, one of the promising approaches is to use CCA-specific affinity reagents to detect CCA cells by liquid biopsy. In this study, bile, a body fluid close to CCA lesions, has been used as a clinical sample for CCA cell isolation and detection. Moreover, a new microfluidic chip was developed to automatically detect CCA cells in the bile by using three types of affinity reagents, including epithelial cell adhesion molecule, a saccharide and an aptamer. It was reported the first instance of isolating CCA tumor cells in patients’ bile (3 mL) within 80 min using the integrated microfluidic chip. On-bench experimental results showed that the three affinity reagents could successfully and specifically isolate tumor cells from 81 CCA patients with an accuracy of 96.2%, a sensitivity of 100% and a specificity of 72.7%. Meanwhile, on-chip experimental results showed that the accuracy, sensitivity and specificity were all 100% using the developed microfluidic system. The new assay proved to be highly significant in clinical prognosis of patients and this microfluidic system may serve as a promising tool for early detection of distant CCA metastasis in clinical settings.

Abstract 2
中文摘要 4
誌謝 6
Table of contents 8
List of figures 11
List of tables 15
Nomenclature and abbreviations 16
Chapter 1 Introduction 18
1-1 Cholangiocarcinoma (CCA) 18
1-2 Microfluidics and Lab on a chip (LOC) 19
1-3 Bile and bile cytology 20
1-4 Affinity reagents 21
1-4.1 Epithelial cell adhesion molecule (anti-EpCAM antibody) 21
1-4.2 Aptamers 22
1-4.3 Glycosaminoglycan 22
1-5 Literature survey 23
1-5.1 Screening of aptamers 23
1-5.2 Glycosaminoglycan 24
1-5.3 Micropumps and micromixers 25
1-6 Motivation and novelty 26
Chapter 2 Materials and Methods 29
2-1 Experimental procedure 29
2-2 Enrichment of CCA cells from bile samples using magnetic beads coated with three affinity reagents 31
2-2.1 Conjugation of affinity reagents on magnetic beads 31
2-2.2 Sample and bile pretreatment 32
2-3 Detection of CCA cells using immunostaining 33
2-4 Microfluidic chip and pneumatically controlled microdevices 34
2-5 Characterization of micro components on the microfluidic chip 43
Chapter 3 Results and Discussion 46
3-1 CCA cells isolated from clinical samples 46
3-2 Relationship between number of cancer cells isolated and tumor size measurements for patients undergoing chemotherapy 54
3-3 Characterization of micropumps and micromixers 57
3-4 Discussion 68
4-1 Conclusion 71
4-2 Future perspectives 72
References 74

1. B. Blechacz, "Cholangiocarcinoma: current knowledge and new developments," Gut and Liver, vol. 11, pp. 13-26, 2017.
2. G. Lendvai, T. Szekerczs, I. Illys, R. Dra, E. Kontsek, A. Ggl, A. Kiss, K. Werling, I. Kovalszky, Z. Schaff and K. Borka, "Cholangiocarcinoma: classification, histopathology and molecular carcinogenesis," Pathology & Oncology Research, vol. 26, pp. 3-15, 2020.
3. M. M. Grimsrud and T. Folseraas, "Pathogenesis, diagnosis and treatment of premalignant and malignant stages of cholangiocarcinoma in primary sclerosing cholangitis," Liver International, vol. 39, pp. 2230-2237, 2019.
4. P. Pereira, S. Santos, R. Morais, R. Gaspar, E. Rodrigues-Pinto, F. Vilas-Boas and G. Macedo, "Role of peroral cholangioscopy for diagnosis and staging of biliary tumors," Digestive Diseases, vol. 38, pp. 431-439, 2020.
5. C. Chelakkot, J. Ryu, M. Y. Kim, J. S. Kim, D. Kim, J. Hwang, S. H. Park, S. B. Ko, J. W. Park, M. Y. Jung, R. N. Kim, K. Song, Y. J. Kim, Y. L. Choi, H. S. Lee and Y. K. Shin, "An immune-magnetophoretic device for the selective and precise enrichment of circulating tumor cells from whole blood," Micromachines, vol. 11, pp.12-25, 2020.
6. G. M. Whitesides, "The origins and the future of microfluidics," Nature, vol. 442, pp. 368-373, 2006.
7. S. K. Sia and G. M. Whitesides, "Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies," Electrophoresis, vol. 24, pp. 3563-3576, 2003.
8. M. Schönberger and M. Hoffstetter, "Emerging Trends in Medical Plastic Engineering and Manufacturing", 2016.
9. A. M. Foudeh, T. F. Didar, T. Veres and M. Tabrizian, "Microfluidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics," Lab on-chip, vol. 12, pp. 3249-3266, 2012.
10. C. Wu, G. Z. Gao, K. F. Zhai, L. S. Xu and D. G. Zhang, "A visual Hg2+ detection strategy based on distance as readout by G-quadruplex DNAzyme on microfluidic paper," Food Chemistry, vol. 331, pp. 22-37, 2020.
11. T. Hu, M. Zhang, Z. Wang, K. Chen, X. Li and Z. H. Ni, "Layer-by-layer self-assembly of MoS2/PDDA hybrid film in microfluidic chips for ultrasensitive electrochemical immunosensing of alpha-fetoprotein," Microchemical Journal, vol. 158, pp. 78-91, 2020.
12. D. C. Duffy, J. C. McDonald, O. J. A. Schueller and G. M. Whitesides, "Rapid prototyping of microfluidic systems in poly(dimethylsiloxane)," Analytical Chemistry, vol. 70, pp. 4974-4984, 1998.
13. P. Gopinathan, N. J. Chiang, A. Bandaru, A. Sinha, W. Y. Huang, S. C. Hung, Y. S. Shan and G. B. Lee, "Exploring circulating tumor cells in cholangiocarcinoma using a novel glycosaminoglycan probe on a microfluidic platform," Advanced Healthcare Materials, vol. 9, pp.12-25, 2020.
14. L. Y. Hung, N. J. Chiang, W. C. Tsai, C. Y. Fu, Y. C. Wang, Y. S. Shan and G. B. Lee, "A microfluidic chip for detecting cholangiocarcinoma cells in human bile," Scientific Reports, vol. 7, pp. 22-40, 2017.
15. H. Govil, V. Reddy, L. Kluskens, D. Treaba, R. Massarani-Wafai, S. Selvaggi and P. Gattuso, "Brush cytology of the biliary tract: Retrospective study of 278 cases with histopathologic correlation," Diagnostic Cytopathology, vol. 26, pp. 273-277, 2002.
16. S. L. Topalian, F. S. Hodi, J. R. Brahmer, S. N. Gettinger, D. C. Smith, D. F. McDermott, J. D. Powderly, R. D. Carvajal, J. A. Sosman, M. B. Atkins, P. D. Leming, D. R. Spigel, S. J. Antonia, L. Horn, C. G. Drake, D. M. Pardoll, L. P. Chen, W. H. Sharfman, R. A. Anders, J. M. Taube, T. L. McMiller, H. Y. Xu, A. J. Korman, M. Jure-Kunkel, S. Agrawal, D. McDonald, G. D. Kollia, A. Gupta, J. M. Wigginton and M. Sznol, "Safety, Activity, and Immune correlates of anti-PD-1 antibody in cancer," New England Journal of Medicine, vol. 366, pp. 2443-2454, 2012.
17. K. Groff, J. Brown and A. J. Clippinger, "Modern affinity reagents: Recombinant antibodies and aptamers," Biotechnology Advances, vol. 33, pp. 1787-1798, 2015.
18. W. C. Tsai, L. Y. Hung, T. Y. Huang, Y. S. Shan, S. C. Hung, G. B. Lee, “A microfluidic system for detection of cholangiocarcinoma cells by using heparan sulfate octasaccharides,”, in 2017 IEE 12th International Conference on Nano/Micro Engineered and Molecular Systems, vol.22, pp. 489-493, 2017.
19. D. D. Taylor and C. Gercel-Taylor, "MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer," Gynecologic Oncology, vol. 110, pp. 13-21, 2008.
20. T. Yamashita, J. F. Ji, A. Budhu, M. Forgues, W. Yang, H. Y. Wang, H. L. Jia, Q. H. Ye, L. X. Qin, E. Wauthier, L. M. Reid, H. Minato, M. Honda, S. Kaneko, Z. Y. Tang and X. W. Wang, "EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features," Gastroenterology, vol. 136, pp. 1012-1024, 2009.
21. L. L. Wang, "Early diagnosis of breast cancer," Sensors, vol. 17, pp. 222-231, 2017.
22. S. Padthaisong, M. Thanee, N. Namwat, J. Phetcharaburanin, P. Klanrit, N. Khuntikeo, A. Titapun, S. Sungkhamanon, H. Saya and W. Loilome, "Overexpression of a panel of cancer stem cell markers enhances the predictive capability of the progression and recurrence in the early stage cholangiocarcinoma," Journal of Translational Medicine, vol. 18, pp. 221-230, 2020.
23. R. A. Potyrailo, R. C. Conrad, A. D. Ellington and G. M. Hieftje, "Adapting selected nucleic acid ligands (aptamers) to biosensors," Analytical Chemistry, vol. 70, pp. 3419-3425, 1998.
24. S. P. Song, L. H. Wang, J. Li, J. L. Zhao and C. H. Fan, "Aptamer-based biosensors,",TracTrends in Analytical Chemistry, vol. 27, pp. 108-117, 2008.
25. D. E. Huizenga and J. W. Szostak, "A DNA aptamer that binds adenosine and atp," Biochemistry, vol. 34, pp. 656-665, 1995.
26. R. Kirby, E. J. Cho, B. Gehrke, T. Bayer, Y. S. Park, D. P. Neikirk, J. T. McDevitt and A. D. Ellington, "Aptamer-based sensor arrays for the detection and quantitation of proteins," Analytical Chemistry, vol. 76, pp. 4066-4075, 2004.
27. H. A. Ho and M. Leclerc, "Optical sensors based on hybrid aptamer/conjugated polymer complexes," Journal of the American Chemical Society, vol. 126, pp. 1384-1387, 2004.
28. G. D. Liu, X. Mao, J. A. Phillips, H. Xu, W. H. Tan and L. W. Zeng, "Aptamer-nanoparticle strip biosensor for sensitive detection of cancer cells," Analytical Chemistry, vol. 81, pp. 10013-10018, 2009.
29. A. D. Keefe, S. Pai and A. Ellington, "Aptamers as therapeutics," Nature Reviews Drug Discovery, vol. 9, pp. 537-550, 2010.
30. A. Nairn and K. Moremen, "Handbook of Glycomics," 2009.
31. M. Hook, L. Kjellen, S. Johansson and J. Robinson, "Cell-surface glycosaminoglycans," Annual Review of Biochemistry, vol. 53, pp. 847-869, 1984.
32. Y. Kanke, R. I. Bashey and Y. Mori, "Biochemistry of glycosaminoglycans - present knowledge," New York State Journal of Medicine, vol. 81, pp. 1322-1327, 1981.
33. J. Pedlar, "Biochemistry of glycosaminoglycans in the skin and oral-mucosa of the rat," Archives of Oral Biology, vol. 29, pp. 591-597, 1984.
34. S. Sugimoto, H. Matsubayashi, H. Kimura, K. Sasaki, K. Nagata, S. Ohno, K. Uesaka, K. Mori, K. Imai and K. Hotta, "Diagnosis of bile duct cancer by bile cytology: usefulness of post-brushing biliary lavage fluid," Endoscopy international open, vol. 3, pp. E323-356, 2015.
35. L. Y. Feng, Y. Chen, J. S. Ren and X. G. Qu, "A graphene functionalized electrochemical aptasensor for selective label-free detection of cancer cells," Biomaterials, vol. 32, pp. 2930-2937, 2011.
36. P. Gopinathan, L. Y. Hung, C. H. Wang, N. J. Chiang, Y. C. Wang, Y. S. Shan and G. B. Lee, "Automated selection of aptamers against cholangiocarcinoma cells on an integrated microfluidic platform," Biomicrofluidics, vol. 11, pp. 78-92, 2017.
37. M. Mende, C. Bednarek, M. Wawryszyn, P. Sauter, M. B. Biskup, U. Schepers and S. Bräse, "Chemical synthesis of glycosaminoglycans," Chemical Reviews, vol. 116, pp. 8193-8255, 2016.
38. E. Iwase and I. Shimoyama, "Multistep sequential batch assembly of three-dimensional ferromagnetic microstructures with elastic hinges," Journal of Microelectromechanical Systems, vol. 14, pp. 1265-1271, 2005.
39. C. S. Zhang, D. Xing and Y. Y. Li, "Micropumps, microvalves, and micromixers within PCR microfluidic chips: Advances and trends," Biotechnology Advances, vol. 25, pp. 483-514, 2007.
40. C. Hayakawa, M. Hoshikawa, J. Imura, T. Ueno and J. Koike, "Bile cytology: A new scoring system for improving diagnostic accuracy," Diagnostic Cytopathology, vol. 47, pp. 641-647, 2019.
41. R. Stoyanova, F. Lomoschitz, W. Schima and A. Klaus, "Minimally invasive approach for complicated choledocholithiasis in an elderly patient after roux-y gastric bypass", Obes. Surg; vol. 31, pp. 3896-3898, 2021.
42. M. A. M. Gijs, "Magnetic bead handling on-chip: new opportunities for analytical applications," Microfluidics and Nanofluidics, vol. 1, pp. 22-40, 2004.
43. M. F. Y, S.fukahori, I. SAKUMA, K. Hagiwara, Y. china, H. saito, T. oiwa, "Cell concentration method using YM fixative solution for cytodiagnosis of lung cancer," Japanese Society of Clinical Cytology, vol. 26, pp. 77-82, 1987.
44. D. Balci, E. O. Kirimker, E. Ustuner, A. A. Yilmaz and A. Azap, "Stage I-laparoscopy partial ALPPS procedure for perihilar cholangiocarcinoma," Journal of Surgical Oncology, vol. 22, pp.77-90, 2021.
45. Nehls O, Gregor M, Klump B ,"Serum and bile markers for cholangiocarcinoma. Semin Liver Dis", Semin Liver Dis, vol. 24, pp. 139-54, 2004.
46. T. H. Lu, P. Gopinathan, N. J. Chiang, C. J. Huang, H. C. Tu, S. C. Hung, Y. S. Shan, G. B. Lee, "Exfoliated tumor cells in bile as a promising indicator of disease status in cholangiocarcinoma." Sensors and Actuators B: Chemical, 130526, 2021.