根據世界衛生組織的報告，在全球死亡人數裡，因為心血管疾病而死亡的人數佔了非常大的比例。在2015年超過1770萬人的死亡死於心血管疾病，所以心血管疾病被認定為是全球主要死因之一。在台灣，心血管疾病在十大死因中排名第二，造成的醫療支出對於全民健保系統也造成重大的負擔。然而傳統的檢測心血管疾病的方法包括斷層掃描、心電圖和心肌灌注成像掃描。雖然這些檢測的方法已被醫院廣泛應用，但是這些方法相對昂貴且不能在早期階段診斷出心血管疾病。另外，創新的分子生物標誌物如血液中的肌鈣蛋白I，C-反應蛋白，N端前腦利鈉肽以相對簡單，快速和非侵入式的方式對早期的心血管疾病可以檢測出徵兆。近幾年來，從細胞外囊泡釋放的微型核醣核酸已被認為是重要的心血管疾病生物標誌物用於心血管疾病的早期偵測及預後監控。在這項研究中，我們開發了一個整合型微流體系統搭載了高靈敏的場效應晶體管，且這微流體系統整合下列幾項功能:利用磁珠抓取檢體中的胞外囊泡、裂解胞外囊泡、純化出與心血管疾病相關的微型核醣核酸、以及場效電晶體檢測釋放的微型核醣核酸的濃度。整個偵測的流程可自動化地操作單一晶片在五小時內完成且對目標微型核醣核酸21和126的偵測濃度極限達到飛莫爾濃度範圍可符合人體生理濃度。在未來，這整合型微流體晶片將有非常大的潛力成為可以用於心血管疾病的早期診斷的工具。

According to World Health Organization’s reports, cardiovascular diseases (CVDs) are one of the major causes of death globally, responsible for over 17.7 million deaths in 2015. It is not only one of the leading causes of death worldwide but also second amongst the top ten leading causes of deaths in Taiwan. Traditional detecting methods include cardiac computerized tomography scan, electrocardiography and myocardial perfusion imaging scan. Although diagnosis of CVDs through bio-imaging techniques is common in hospitals, those methods are relatively costly and cannot diagnose CVDs at early stages. In contrast, the level of novel molecular biomarkers in blood, such as troponin I, C-reactive protein, or N-terminal pro-brain natriuretic peptide, provide an indication for early detection and prognosis monitoring of CVDs in a relatively simple, rapid and non-invasive manner. Recently, micro ribonucleic acids (miRNAs) extracted from extracellular vesicles (EVs) have been recognized as promising biomarkers for early detection of CVDs and prognosis monitoring. However, detection and quantification of miRNAs by using the existing methods are relatively labor-intensive and time-consuming. In this study, an integrated microfluidic system equipped with highly sensitive field-effect transistors which was capable of performing EVs extraction, EVs lysis, target miRNAs extraction and miRNAs detection has been reported. The entire detection process could be automated within five hours on a single chip and the detection limit of miRNAs was observed to be in a femtomolar range for two targeted miRNA, including miR-21 and miR-126, which meets the requirement of physiological concentrations. This integrated microfluidic system has great potential to be used as a tool for early detection of CVDs.

Table of contents
Abstract I
摘要 III
Table of contents IV
List of figures IX
List of tables XVI
Abbreviations and nomenclature XVII
Chapter 1 Introduction 1
1-1 Cardiovascular Diseases 1
1-2 Extracellular vesicles (EVs) and micro ribonucleic acids (miRNAs) 3
1-3 Literature survey 5
1-3-1 Microfluidics 5
1-3-2 Field-effect transistor 7
1-3-3 EVs detection by using microfluidic systems 8
1-4 Novelty and motivation 9
Chapter 2 Materials and methods 12
2-1 Cell culture and isolation of sample model of EVs 12
2-1-1 Quantification of EVs yields 13
2-1-2 Identification of EVs 14
2-1-3 EVs extraction on bench 16
2-2 Quantification methods about capture rate of EVs extraction 16
2-3 Quantification methods about capture rate of miRNAs extraction 17
2-4 Design and fabrication process of the microfluidic chip 20
2-4-1 Design of integrated microfluidic chip 21
2-4-2 Fabrication of integrated microfluidic chip 25
2-4-3 Working principle of microfluidic chip 26
2-5 Characterization of chip performances 28
2-5-1 Measurement of pumping rate 28
2-5-2 Measurement of mixing index 29
2-5-3 Measurement of wall shear force 30
2-6 Experimental setup 32
2-7 Experimental procedure 34
Chapter 3 Results and discussion 39
3-1 Performance of the integrated microfluidic system 39
3-1-1 Pumping rate of micropump 39
3-1-2 Mixing index of micromixer 41
3-1-3 Shear force of the micromixer 43
3-2 Image data of capturing EV by using CD63-coated magnetic beads 45
3-2-1 SEM images of captured EV on CD63-coated magnetic beads 47
3-2-2 Fluorescent image of captured EV on CD63-coated magnetic beads 49
3-3 Capture rate of EVs extraction 50
3-3-1 EVs extraction at different operation applied gauge pressure 51
3-3-2 EVs extraction at different time 52
3-4 Capture rate of miRNAs extraction 53
3-4-1 miRNAs extraction at different operation frequencies 53
3-4-2 miRNAs extraction at different time 55
3-5 FET detection 56
3-6 miRNAs detection by using the integrated microfluidic system 58
Chapter 4 Conclusions and future perspectives 61
4-1 Conclusions 61
4-2 Future perspectives 62
References 64

1. GBD 2013 Mortality and Causes of Death Collaborators, “Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013”, Lancet, vol. 385, pp. 117-171, Jan 2015.
2. V. Fuster, B.B. Kelly, A. Chockalingam, J.W. Farquhar, R.C. Hornik, F.B. Hu, P.R.Lamptey, J.C. Mbanya, A. Mills, J. Narula, R.A. Nugnet, J.W. Peabody, K. S. Reddy, S. Stachenko, D. Yach, C. Weinberger, R. Jackson, L. Jordan, K. Danforth, J. Wiltshire, and P. Kelley, “Promoting Cardiovascular Health in the Developing World: A Critical Challenge to Achieve Global Health”, Washington (DC), 2010.
3. B.V. Howard and J. Wylie-Rosett, “Sugar and cardiovascular disease: A statement for healthcare professionals from the Committee on Nutrition of the Council on Nutrition, Physical Activity, and Metabolism of the American Heart Association ”, Circulation, vol. 106, pp. 523-527, Jul 2002.
4. A. Al-Mawali, “Non-Communicable Diseases: Shining a Light on Cardiovascular Disease, Oman’s Biggest Killer”, Oman Medical Journal, vol. 30, pp. 227-228, Jul 2015.
5. I. Ikonomidis, K. Stamatelopoulos, and J. Lekakis, “Inflammatory and non-invasive vascular markers: The multimarker approach for risk stratification in coronary artery disease”, Elsevier, vol. 199, pp. 3-11, Jul 2008.
6. W. Wang, G. Sun, L. Zhang, Y. Zeng, “Circulating microRNAs as novel potential biomarkers for early diagnosis of acute stroke in humans”, European Heart Journal, vol. 31, pp.659-666, 2010.
7. A. J. Tijsen and Y. M. Pinto, “Non-cardiomyocyte microRNAs in heart failure”, Cardiovascular Research, vol. 93, pp. 573-582, Mar 2012.
8. J. Malda, J. Boere, C.H. van de Lest, P. van Weeren, and M.H. Wauben, “Extracellular vesicles - new tool for joint repair and regeneration”, Nat Rev Rheumatol, vol. 12 pp. 243-249, Apr 2016.
9. R.J. Berckmans, R. Nieuwland, P.P. Tak, A.N. Böing, F.P. Romijn, M.C. Kraan, F.C. Breedveld, C.E. Hack, and A. Sturk, “Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism”, Arthritis Rheum, vol. 46, pp. 2857-2866, Nov 2002.
10. K.W. Witwer, E.I. Buzás, L.T. Bemis, A. Bora, C. Lässer, J. Lötvall, E.N. Nolte-'t Hoen, M.G. Piper, S. Sivaraman, J. Skog, C. Théry, M.H. Wauben, and F. Hochberg, “Standardization of sample collection, isolation and analysis methods in extracellular vesicle research”, J Extracell Vesicles, vol. 2. p. 20360, May 2013.
11. V. Ambros, “The functions of animal microRNAs”, Nature, vol. 431, pp. 350-355, Sep 2004.
12. A. Esquela-Kerscher and F.J. Slack, “Oncomirs - microRNAs with a role in cancer”, Nat Rev Cancer, vol. 6, pp. 259-269, Apr 2006.
13. J. Li, B. Yao, H. Huang, Z. Wang, C. Sun, Y. Fan, Q. Chang, S. Li, X. Wang, and J. Xi, “Real-time polymerase chain reaction microRNA detection based on enzymatic stem-loop probes ligation”, Anal Chem, vol. 81, pp. 5446-5451, Jul 2009.
14. N. Nouraee and S. J. Mowla, “miRNA therapeutics in cardiovascular diseases: promises and problems”, Frontiers in Genetics, vol. 6, p. 232, Jun 2015.
15. B.A. Haider, A.S. Baras, M.N. McCall, J.A. Hertel, T.C. Cornish, and M.K. Halushka, “A critical evaluation of microRNA biomarkers in non-neoplastic disease”, PLoS One, vol 9, p. e89565, Feb 2014.
16. P.S. Mitchell, R.K. Parkin, E.M. Kroh, B.R. Fritz, S.K. Wyman, E.L. Pogosova-Agadjanyan, A. Peterson, J. Noteboom, K.C. O'Briant, A. Allen, D.W. Lin, N. Urban, C.W. Drescher, B.S. Knudsen, D.L. Stirewalt, R. Gentleman, R.L. Vessella, P.S. Nelson, D.B. Martin, and M.Tewari, “Circulating microRNAs as stable blood-based markers for cancer detection”, Proc Natl Acad Sci U S A, vol. 105, pp. 10513-10518, Jul 2008.
17. A.J. Tijsen, E.E. Creemers, P.D. Moerland, L.J. de Windt, A.C. van der Wal, W.E. Kok, and Y.M. Pinto, “MiR423-5p as a circulating biomarker for heart failure”, Circ Res, vol. 106, pp. 1035-1039, Apr 2010.
18. Y. Cheng, N. Tan, J. Yang, X. Liu, X. Cao, P. He, X. Dong, S. Qin, and C. Zhang, “A translational study of circulating cell-free microRNA-1 in acute myocardial infarction”, Clin Sci (Lond), vol. 119, pp. 87-95, Apr 2010.
19. Y. Devaux, M. Vausort, E. Goretti, P.V. Nazarov, F. Azuaje, G. Gilson, M.F. Corsten, B. Schroen, M.L. Lair, S. Heymans, and D.R. Wagner, “Use of irculating microRNAs to diagnose acute myocardial infarction”, Clin Chem, vol. 58, pp. 559-567, Mar 2012.
20. X. Ji, R. Takahashi, Y. Hiura, G. Hirokawa, Y. Fukushima, and N. Iwai, “ Plasma miR-208 as a biomarker of myocardial injury”, Clin Chem, vol. 55, pp. 1944-1949, Nov 2009.
21. E.E. Creemers, A.J. Tijsen, and Y.M. Pinto, “Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease?”, Circ Res, vol. 110, pp. 483-495, Feb 2012.
22. L.R. Volpatti, and A.K. Yetisen, “Commercialization of microfluidic devices”,Trends Biotechnol, vol. 32, pp. 347-350, Jul 2014.
23. W.B. Lee, C.Y. Fu, W.H. Chang, H.L. You, C.H. Wang, M.S. Lee, and G.B. Lee, “A microfluidic device for antimicrobial susceptibility testing based on a broth dilution method”, Biosensors and Bioelectronics, vol. 87, 669–678, Jan 2017.
24. C.C. Cheng, Y.Y. Tsai, K.W. Lin, H.I. Chen, W.H. Hsu, C.W. Hong, and W.C. Liu, “Characteristics of a Pd-oxide-In0.49Ga0.51P high electron mobility transistor (HEMT)-based hydrogen sensor”, Sensors and Actuators B-Chemical, vol. 113, pp. 29-35, Jan 2006.
25. B.H. Chu, B. S. Kang, C.Y. Chang, F. Ren, A. Goh, A. Sciullo, W. Wu, J. Lin, B.P. Gila, S.J. Pearton, J.W. Johnson, E.L. Piner, and K.J. Linthicum, “Wireless Detection System for Glucose and pH Sensing in Exhaled Breath Condensate Using AlGaN/GaN High Electron Mobility Transistors”, Ieee Sensors Journal, vol. 10 pp. 64-70, Jan 2010.
26. B.S. Kang, and S.J. Pearton, “Electrical detection of deoxyribonucleic acid hybridization with AlGaN/GaN high electron mobility transistors”, Applied Physics Letters, vol. 89, p. 122102, Sep 2006.
27. B.S. Kang, H.T. Wang, T.P. Lele, Y. Tseng, and F. Rena, “Prostate specific antigen detection using AlGaN/GaN high electron mobility transistors”, Applied Physics Letters, vol. 91, Sep 2007.
28. S.J. Pearton, B.S. Kang, S. Kim, F. Ren, B.P. Gila, C.R. Abernathy, J. Lin, and S.N.G. Chu, “GaN-based diodes and transistors for chemical, gas, biological and pressure sensing”, Journal of Physics-Condensed Matter, vol. 16, pp. R961-R994, Jul 2004.
29. F. Ren and S.J. Pearton, “Sensors using AlGaN/GaN based high electron mobility transistor for environmental and bio-applications, in Physica Status Solidi C: Current Topics in Solid State Physics”, Physica Status Solidi, Vol 9, pp. 393–398, Jan 2012.
30. A.P Turner, “ Biosensors: Fundamentals and applications - Historic book now open access”, Biosensors & Bioelectronics, vol. 65, p. A1, Mar 2015.
31. M. He, J. Crow, M. Roth, Y. Zeng, and A.K. Godwin, “Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology”, Lab on a Chip, vol. 14, pp. 3773-3780, Oct 2014.
32. T. Ueno and T. Funatsu, “Label-Free Quantification of MicroRNAs Using Ligase-Assisted Sandwich Hybridization on a DNA Microarray”, Plos One, vol. 9, p. e90920, Mar 2014.
33. Z. Zhao, Y. Yang, Y. Zeng, and M. He, “A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis”, Lab on a Chip, vol .16, pp. 489-496, Feb 2016.
34. P. Zhang, M. He, and Y. Zeng, “Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating”, Lab on a Chip, vol. 16, pp. 3033-3042, Aug 2016.
35. N.H. Heegaard, et al., “Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer”, Int J Cancer, vol. 130, pp. 1378-1386, Mar 2012.
36. S. Zhu, H. Wu, F. Wu, D. Nie, S. Sheng, and Y.Y. Mo, “MicroRNA-21 targets tumor suppressor genes in invasion and metastasis”, Cell Res, vol. 18, pp. 350-359, Mar 2008.
37. S.Y. Yang, J.L. Lin, and G.B. Lee, “A vortex-type micromixer utilizing pneumatically driven membranes”, Journal of Micromechanics and Microengineering, vol. 19, p. 035020, Feb 2009.
38. J. Fritz, A.G. Katopodis, F. Kolbinger, D. Anselmetti, “Force-mediated kinetics of single P-selectinyligand complexes observed by atomic force microscopy”, Biophysics, Vol. 95, pp. 12283–12288, Oct 1998
39. S.K. Patnaik, E.D. Kannisto, R. Mallick, A. Vachani, S. Yendamuri, “Whole blood microRNA expression may not be useful for screening non-small cell lung cancer”, PLOS ONE, vol. 12, p. e0181926, Apr 2017.
40. J.C. Akers, V. Ramakrishnan, R. Kim, J. Skog, I. Nakano, S. Pingle, J. Kalinina, W. Hua, S. Kesari, Y. Mao, X.O. Breakefield, F.H. Hochberg, E.G. Van Meir, B.S. Carter, C.C. Chen, “miR-21 in the Extracellular Vesicles (EVs) of Cerebrospinal Fluid (CSF): A Platform for Glioblastoma Biomarker Development”, PLOS ONE, vol. 8, e78115, Oct 2013.