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研究生： 范丞緯
Fan, Chen-Wei
論文名稱： 利用抗體修飾凝膠分選胞外體
Sorting Extracellular Vesicles Using Antibody-Functionalized Agarose Gels
指導教授： 陳致真
Chen, Chih-Chen
口試委員: 賴品光
Lai, Pin-Kuang
北森武彥
Kitamori, Takehiko
學位類別： 碩士
Master
系所名稱： 工學院 - 動力機械工程學系
Department of Power Mechanical Engineering
論文出版年： 2020
畢業學年度： 108
語文別： 英文
論文頁數： 61
中文關鍵詞： 胞外體 、電泳 、洋菜膠 、免疫親合法 、抗體
外文關鍵詞： Extracellular vesicles, Electrophoresis, Agarose gel, Immunoaffinity, Antibody
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    • 近年來胞外體（EVs）受到到廣泛研究，因為胞外體在細胞間的溝通扮演了很重要的角色，如調節免疫反應、腫瘤生長等，另外在臨床醫學中也有很多應用，可用於檢測臨床狀態、疾病進展等等。然而目前主流分選胞外體的方式多是透過大小及密度進行分離，這些方法的缺點為處理時間長，胞外體變形破裂等問題。 因此我們開發了一種基於免疫親和法的分離技術，藉由NHS活化微珠將抗體修飾在凝膠內部，並進行凝膠電泳，使得CD63+胞外體在電泳時會被抗體抓取，再使用水平強電場釋放被抓取CD63+的胞外體，最終達到分選CD63+胞外體的目的。
    目前共製作並測試三種裝置雛型，分別為(1)濾紙凝膠、(2)sulfo凝膠、(3)NHS微珠凝膠，使用三種裝置對胞外體進行分選，並使用冷光、螢光、ELISA及qNano分析，證明三種裝置都能成功抓取胞外體，其中NHS微珠凝膠性能最為優異，其抗體連接效率可達95%，抓取效率可達76%。相信此裝置在未來對胞外體分離上，能夠提供一項創新的選擇，並促進胞外體研究的發展。

    In recent years, extracellular vesicles (EVs) have been widely studied, because EVs play an important role in cell-to-cell communication, such as regulating immune responses and tumor growth. In addition, they have many clinical applications, which can be used to detect clinical status, disease progression and more. However, the current mainstream methods for sorting EVs have disadvantages include long processing time, deformation and rupture of EVs. Therefore, we have demonstrated an easy-to-approach immunoaffinity-based isolation technique, using NHS activated beads to immobilize antibodies within the agarose gel. By gel electrophoresis, CD63+ EVs will be captured by the antibody during electrophoresis, and a strong electric field will be applied to release the captured CD63+ EVs, thereby achieving the purpose of sorting EVs with different affinity.
    At present, three types of gel prototypes have been tested, namely (1) filter paper gel, (2) Sulfo gel, and (3) NHS bead gel. By chemiluminescence, fluorescent, ELISA, and qNano analysis proved that all three devices can successfully capture extracellular vesicles, especially the performance of NHS gel is the best among three gels. The antibody conjugation efficiency can reach up to 95%, and the capture rate is 76%. We believed that this device can provide a novel option for EV isolation and facilitating EVs research.

    Table of Contents 摘要 2 Abstract 3 List of Figures 8 List of Tables 11 1.1 Background 12 1.2 Introduction of extracellular vesicles (EVs) 12 1.3 EV isolation methods 13 1.4 Gel electrophoresis 16 1.5 Continuous microfluidic assortment of interactive ligands, CMAIL 17 1.6 Detection of EVs by ZnO-nanowires-coated three-dimensional scaffold chip device__________________________________________________ 18 1.7 Tunable resistive pulse sensing, tRPS 20 1.8 Enzyme-linked immunosorbent assay, ELISA 23 1.9 Research motivation and aim 24 Chapter .2 Experimental Design and Methods 26 2.1 Experimental flow chart 26 2.2 Device working principle 27 2.3 Instrument setup 28 2.4 Antibody-functionalized gel preparation 29 2.4.1 Agarose gel mold design 29 2.4.2 Paper gel preparation 32 2.4.3 Sulfo-SANPAH gel preparation 33 2.4.4 NHS beads gel 34 2.5 Cell culture and EV collection 35 2.5.1 Human embryonic kidney cell (HEK 293T) culture 35 2.5.2 HEK 293T EV collection 35 2.6 Experimental analysis tools and methods 37 2.6.1 Inverted optical microscope (DMIL LED, Leica, Major, Germany) 37 2.6.2 Inverted electronically controlled microscope system (DMI6000 B, Leica, Major, Germany)________________________________________________________37 2.6.3 Tunable resistive pulse sensing, qNano (Q5013, IZON Science, New Zealand) 37 2.6.4 Luminescence Imaging System (Amersham™ Imager 600, AI600) 38 2.6.5 Multilabel Plate Reader (POLARstar Omega, BMG LABTECH) 38 2.6.6 Image J biological-image analysis software 39 Chapter .3 Results and Discussions 40 3.1 tRPS analysis of HEK-293T extracellular vesicles 40 3.2 Sample injection and collection 41 3.3 Analysis of paper gel samples 42 3.3.1 Paper gel feasibility test 42 3.3.2 ELISA CD63 protein expression analysis of paper gel samples 42 3.3.3 qNano analysis of filter paper gel samples 43 3.4 Optimization of Sulfo-SANPAH crosslink efficiency 45 3.5 Sulfo-SANPAH gel sample analysis 46 3.5.1 Fluorescence analysis of antibody crosslinking efficiency 46 3.5.2 qNano analysis of Sulfo-SANPAH gel sample 47 3.6 NHS beads gel optimization 49 3.7 NHS beads gel characterization 50 3.7.1 Fluorescence analysis of antibody conjugate efficiency 50 3.7.2 NHS beads gel feasibility test 51 3.7.3 qNano analysis of NHS beads gel samples 52 3.7.4 NHS beads gel two-dimensional electrophoresis 54 3.7.5 Two-dimensional electrophoresis NHS beads gel samples characterization 55 3.7.6 Zeta potential analysis of collected samples 56 Chapter .4 Conclusions 58 References 59

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