胞外囊泡是許多細胞所分泌大小介於40~100 nm膜狀結構，這類型的胞外囊泡含有蛋白質、mRNA、miRNA以及訊號分子。在生理上，這些胞外囊泡會在細胞之間協助蛋白質或RNA分子的運輸、在免疫學上具有抗原呈現(antigen presentation)的功能。近年研究發現，胞外囊泡與心臟病、腎臟病、腦部疾病的發生有關，此外，部分研究亦指出胞外囊泡在癌症形成中可能扮演重要角色。然而，因胞外囊泡大小極小且在生物樣本中含量極少，目前研究上常用的方法為超高速蔗糖密度梯度離心法(sucrose density gradient ultracentrifugation method)來收集，但超高速蔗糖密度梯度離心法不但耗時且消耗的生物樣品量多。因此，本文建立一具免疫親合性之微流道平台，透過結合高專一性免疫親合特性與微流道晶片之快速萃取、所消耗生物樣品量少等優點，來改善超高速離心法在胞外囊泡萃取收集步驟中耗時以及需較多生物樣品量之缺點。

Extracellular vesicles (EVs), secreted by cells, are membranous structures between 40 to 100 nm in size. EVs contain proteins, mRNA, miRNA, and signaling molecules. EVs facilitate the transport of these molecules between cells. Recently researchers found EVs may play an important role in not only heart, kidney diseases but cancerogenesis. However, EVs are quite small and their content in biological samples can be extremely low. Conventionally, the ultracentrifuge method is used to isolating EVs from serum or culture medium in which multiple steps from low speed to high speed centrifugations, are involved in order to collect EVs. In addition, a relatively large amount of sample is necessary for this method. Therefore, ultracentrifuge is time-consuming and not suitable in trace analysis. This thesis aims to establish a microfluidic immuno-affinity based approach to solve the disadvantages of ultracentrifuge methods.

1. Diercks, A. H., et al. (2009). "A microfluidic device for multiplexed protein detection in nano-liter volumes." Anal Biochem 386(1): 30-35.
2. Stone, H.A. et al. (2004) "Engineering flows in small devices: Microfluidics toward a lab-on-a-chip. Annual Review of Fluid Mechanics." 36: p. 381-411
3. Verpoorte, E. et al. (2003) "Microfluidics meets MEMS." Proceedings of the IEEE. 91(6): p. 930-953
4. Stoorvogel, W., et al. (2002). "The Biogenesis and Functions of Exosomes." Traffic 3(5): 321-330.
5. Johnstone, R. M. (2006). "Exosomes biological significance: A concise review." Blood Cells Mol Dis 36(2): 315-321.
6. Müller, (2012). "Novel tools for the study of cell type-specific exosomes and microvesicles" J Bioanal Biomed, 4(4): 046-060
7. Hadi, V., et al. (2007). "Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells." Nature Cell Biology 9(6): 654-659.
8. Taylor, D., et al. (2011). Exosome Isolation for Proteomic Analyses and RNA Profiling. Serum/Plasma Proteomics. R. J. Simpson and D. W. Greening, Humana Press. 728: 235-246.
9. Bobrie, A., et al. (2011). "Exosome secretion: molecular mechanisms and roles in immune responses." Traffic 12(12): 1659-1668
10. Fang, D. Y., King, H. W., Li, J. Y. and Gleadle, J. M. (2013), Exosomes and the kidney: Blaming the messenger. Nephrology, 18: 1–10.
11. Zhou, H., et al. (2006). "Exosomal Fetuin-A identified by proteomics: A novel urinary biomarker for detecting acute kidney injury." Kidney International 70(10): 1847-1857.
12. Miranda, K. C., et al. (2010). "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease." Kidney Int 78(2): 191-199.
13. Street, J., et al. (2012). "Identification and proteomic profiling of exosomes in human cerebrospinal fluid." Journal of Translational Medicine 10(1): 1-7.
14. Lai, R. C., et al. (2011). "Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease." Regen Med 6(4): 481-492.
15. Schorey, J. S. and S. Bhatnagar (2008). "Exosome function: from tumor immunology to pathogen biology." Traffic 9(6): 871-881.
16. Hurley, J. H. and G. Odorizzi (2012). "Get on the exosome bus with ALIX." Nat Cell Biol 14(7): 654-655
17. Chen, C., et al. (2010). "Microfluidic isolation and transcriptome analysis of serum microvesicles." Lab Chip 10(4): 505-511
18. Yoo, C. E., et al. (2012). "A direct extraction method for microRNAs from exosomes captured by immunoaffinity beads." Anal Biochem 431(2): 96-98
19. Davies, R. T., et al. (2012). "Microfluidic filtration system to isolate extracellular vesicles from blood." Lab Chip 12(24): 5202-5210.
20. Zhang, X., et al. (2006). "Continuous flow separation of particles within an asymmetric microfluidic device." Lab Chip 6(4): 561-566
21. Bhagat, A., et al. (2010). "Microfluidics for cell separation." Med Biol Eng Comput 48(10): 999-1014.
22. Zhao, C. and X. Cheng (2011). "Microfluidic separation of viruses from blood cells based on intrinsic transport processes." Biomicrofluidics 5(3): 32004-3200410.
23. Diercks, A. H., et al. (2009). "A microfluidic device for multiplexed protein detection in nano-liter volumes." Anal Biochem 386(1): 30-35.
24. Lin.C. M., et al. (2008). "Trapping of Bioparticles via Microvortices in a Microfluidic Device for Bioassay Applications." Analytical Chemistry 80(23): 8937-8945.
25. Xuan, X., et al. (2010). "Particle focusing in microfluidic devices." Microfluidics and Nanofluidics 9(1): 1-16.
26. Logozzi, M., et al. (2009). "High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients." PLoS One 4(4): e5219.
27. Théry, C., et al. (2001). "Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids." Current Protocols in Cell Biology, John Wiley & Sons, Inc.
28. Cheruvanky A., et al (2007). "Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator." Am J Physiol Renal Physiol, 292(5): 1657-1661.
29. Chris G., et al (2013). "EV isolation and Characterization" ISEV workshop abstract book. P. 19
30. Leonora B., et al (2013). "EV isolation and Characterization" ISEV workshop abstract book. P. 52
31. Prabhu R, (2001). "A novel method of preparation of small intestinal brush border membrane vesicles by polyethylene glycol precipitation." Analytical Biochemistry. 289(2):157-161
32. Momen-Heravi, F., et al. (2013). "Current methods for the isolation of extracellular vesicles." Biological Chemistry. 394(10): 1253-1262.
33. Emily, Z., et al. (2013). "Methods for the extraction and RNA profiling of exosomes." World Journal of Methodology 3(1): 11-18.
34. Rekker, K., et al. (2014). "Comparison of serum exosome isolation methods for microRNA profiling." Clinical Biochemistry 47(1–2): 135-138
35. C. Thery, et al.(2006). "Current Protocols in Cell Biology." UNIT 3.22