在本文研究中，提出以不同濃度的凝膠來製作一個平台，首先對粒徑60 nm與97 nm混合的螢光微珠進行雙向凝膠電泳實驗，以較強的垂直電場將所有微珠往出口驅動，再以較弱的水平電場將所有微珠往側向驅動，利用不同粒徑微珠造成的電泳速度差原理，將粒徑差距僅有37 nm的混合微珠在側向進行分離。也將此方法應用於分群胞外體，並分析在不同出口渠道的胞外體粒徑、數量分布，以及跨膜蛋白CD63標記的檢測，結果顯現在不同胞外體分群帶有不同的CD63表現量。

In this study, developing a platform with different concentrations of agarose gel to run orthogonal-field gel electrophoresis. In which orthogonal-field gel electrophoresis is applied to separate 60 nm and 97 nm fluorescent beads depends on their different electrophoresis mobility. A stronger vertical electric field drives all beads toward the outlet wells, while a weaker horizontal electric field is applied alternatively and used to separate the mixed beads that differ by only 37 nm in diameter. This method is also applied to separate extracellular vesicles (EVs) into different subgroups. The size profile and concentration of EVs harvested in different outlet wells have been characterized. It is found that, CD63 molecules are expressed differently in EV subgroups.

1. Taylor, D.D., S. Akyol, and C. Gercel-Taylor, Pregnancy-associated exosomes and their modulation of T cell signaling. The Journal of Immunology, 2006. 176(3): p. 1534-1542.
2. Pisitkun, T., R.-F. Shen, and M.A. Knepper, Identification and proteomic profiling of exosomes in human urine. Proceedings of the national academy of sciences of the United States of America, 2004. 101(36): p. 13368-13373.
3. Admyre, C., et al., Direct exosome stimulation of peripheral humanT cells detected by ELISPOT. European journal of immunology, 2006. 36(7): p. 1772-1781.
4. Vlassov, A.V., et al., Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochimica et Biophysica Acta (BBA)-General Subjects, 2012. 1820(7): p. 940-948.
5. Robbins, P.D. and A.E. Morelli, Regulation of immune responses by extracellular vesicles. Nature Reviews Immunology, 2014. 14(3): p. 195-208.
6. Del Conde, I., et al., Tissue-factor–bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood, 2005. 106(5): p. 1604-1611.
7. Gatti, S., et al., Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia–reperfusion-induced acute and chronic kidney injury. Nephrology Dialysis Transplantation, 2011. 26(5): p. 1474-1483.
8. Skog, J., et al., Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nature Cell Biology, 2008. 10(12): p. 1470-1476.
9. György, B., et al., Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and molecular life sciences, 2011. 68(16): p. 2667-2688.
10. Kowal, J., et al., Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proceedings of the National Academy of Sciences, 2016. 113(8): p. E968-E977.
11. Heijnen, H.F., et al., Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and-Granules. Blood, 1999. 94(11): p. 3791-3799.
12. Van Deun, J., et al., The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. Journal of extracellular vesicles, 2014. 3(1): p. 24858.
13. Colombo, M., et al., Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J Cell Sci, 2013: p. jcs. 128868.
14. Witwer, K.W., et al., Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. Journal of extracellular vesicles, 2013. 2(1): p. 20360.
15. Théry, C., et al., Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current protocols in cell biology, 2006: p. 3.22. 1-3.22. 29.
16. György, B., et al., Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters. Blood, 2011. 117(4): p. e39-e48.
17. Chen, C., et al., Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab on a chip, 2010. 10(4): p. 505-511.
18. Tang, Y.-T., et al., Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum. International journal of molecular medicine, 2017. 40(3): p. 834-844.
19. 太鼎生物科技公司. 什麼是中空纖維膜?TFF切向流過濾簡易說明. Available from: http://biopioneer.com.tw/?news=hollow_fiber_introduction.
20. Vergauwen, G., et al., Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Scientific Reports, 2017. 7.
21. Batrakova, E.V. and M.S. Kim, Using exosomes, naturally-equipped nanocarriers, for drug delivery. Journal of Controlled Release, 2015. 219: p. 396-405.
22. Shin, S., et al., Separation of extracellular nanovesicles and apoptotic bodies from cancer cell culture broth using tunable microfluidic systems. Scientific reports, 2017. 7(1): p. 9907.
23. 國家實驗研究院. Electron Beam Lithography. 2005; Available from: https://www.itrc.narl.org.tw/Research/Product/Nano/ebeam.php.
24. 莊榮輝. 電泳檢定法. 2000; Available from: http://juang.bst.ntu.edu.tw/ECX/Ana3.htm.
25. Jelínek, M. METHODS FOR STUDYING OF PROTEINS. Available from: http://slideplayer.com/slide/4366457/.
26. McMaster, G.K. and G.G. Carmichael, Analysis of single-and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proceedings of the National Academy of Sciences, 1977. 74(11): p. 4835-4838.
27. Holmes, D.L. and N.C. Stellwagen, The electric field dependence of DNA mobilities in agarose gels: A reinvestigation. Electrophoresis, 1990. 11(1): p. 5-15.
28. Rouser, G., S. Fleischer, and A. Yamamoto, Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids, 1970. 5(5): p. 494-496.
29. Maas, S.L., J. De Vrij, and M.L. Broekman, Quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing. Journal of visualized experiments: JoVE, 2014(92).
30. Schwartz, D.C. and C.R. Cantor, Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. cell, 1984. 37(1): p. 67-75.
31. 曾福生, 余俊欣, and 林金榮. 淺談脈衝式凝膠電泳技術. Available from: http://www.tfrin.gov.tw/dl.asp?fileName=20091020-111445_14%E6%B7%BA%E8%AB%87%E8%84%88%E8%A1%9D%E5%BC%8F%E5%87%9D%E8%86%A0%E9%9B%BB%E6%B3%B3%E6%8A%80%E8%A1%93.pdf.
32. Hsiao, Y.-H., et al., Continuous microfluidic assortment of interactive ligands (CMAIL). Scientific Reports, 2016. 6(1).
33. Dragovic, R.A., et al., Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine: Nanotechnology, Biology and Medicine, 2011. 7(6): p. 780-788.
34. Gardiner, C., et al., Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. Journal of extracellular vesicles, 2013. 2(1): p. 19671.
35. De Vrij, J., et al., Quantification of nanosized extracellular membrane vesicles with scanning ion occlusion sensing. Nanomedicine, 2013. 8(9): p. 1443-1458.
36. Nolte, E.N., et al., Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles. Nanomedicine: Nanotechnology, Biology and Medicine, 2012. 8(5): p. 712-720.
37. Nanoparticle Tracking Analysis. Available from: http://www.malvern.com/en/products/technology/nanoparticle-tracking-analysis/.
38. Maas, S.L., et al., Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. Journal of Controlled Release, 2015. 200: p. 87-96.
39. 「サイズ」「濃度」「表面電荷量」「粒子間相互作用」を計測. Available from: http://www.meiwafosis.com/products/nanoparticle/nanoparticle_tokucho.html#genri.
40. Pol, E., et al., Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. Journal of Thrombosis and Haemostasis, 2014. 12(7): p. 1182-1192.
41. Willmott, G., et al., Actuation of tunable elastomeric pores: Resistance measurements and finite element modelling. Experimental Mechanics, 2014. 54(2): p. 153-163.
42. Scientific, T.F., Overview of ELISA.
43. Kim, D.-k., et al., Chromatographically isolated CD63+ CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI. Proceedings of the National Academy of Sciences, 2016. 113(1): p. 170-175.
44. TSA Systems for Immunohistochemistry and In Situ Hybridization. Available from: http://www.blossombio.com/products/TSASystemsforImmunohistochemistryandInSituHybridization.html.