胞外體（Extracellular vesicles）是細胞分泌和釋放的一種生物顆粒為細胞溝通的過程中的傳遞物質之一，因此，胞外體和免疫反應、血管生成等生理調節息息相關。近幾年來由於檢測技術的發展，使得胞外體逐漸成為研究的焦點，不僅能夠作為疾病發展、治療效果的指標物，更具有預測疾病以及發展成藥物載體的潛力。 然而目前利用大小差異分離胞外體的方式皆有時間過長、一次樣品數量少等缺點。因此我們希望開發出一套能夠快速分離不同大小的胞外體，且能夠普遍使用於各個實驗室或是研究場所的系統。為了能夠達到廣為使用的目的，我們選擇凝膠電泳的方式，使胞外體在電泳的過程中因泳動率異而分離。
我們利用微米製程的方式，製作出垂直回收凝膠中樣品的裝置。該裝置預計可將回收胞外體的時間縮短至一小時內，並利用syringe pump定量且定速的方法，取代手動回收樣品，降低人工操作時帶來的誤差，提高實驗結果的穩定性。期盼本研究中新穎的凝膠電泳裝置能成為分離方法的新選擇，為胞外體研究的發展做出貢獻。

Extracellular vesicles are biological particles secreted and released by cells. They are one of the substances in the process of cell communication. Therefore, extracellular vesicles are technology detection physiological regulation such as immune response and angiogenesis, etc. Due to detection technology development, extracellular vesicles have gradually become the focus of research in recent years. They can be used as indicators of disease development and treatment effects and have the potential to predict diseases and develop into drug carriers. However, the current methods of separating extracellular bodies using size differences have disadvantages such as too long time and a small number of samples at a time. Therefore, we hope to develop a system that can quickly separate extracellular vesicles of different sizes and is widely used in various laboratories or research sites. In order to be widely used, we choose gel electrophoresis to separate the extracellular bodies due to size differences during the electrophoresis process.
We used microfabrication to make a device for the vertical collection of samples in the gel. The device is expected to shorten the collection time of extracellular vesicles to less than one hour. It uses a quantitative and fixed-rate method of syringe pump to replace manual sample recovery, reduce errors caused by manual operation, and improve stability of experimental results. We hope that the novel gel electrophoresis device in this study can become a new choice of separation method and contribute to the development of extracellular vesicles research.

摘要 1
ABSTRACT 2
CHAPTER 1 INTRODUCTION 10
1.1 BACKGROUND 10
1.2 EXTRACELLULAR VESICLES (EVS) 11
1.3 EVS ISOLATION METHODS 13
1.4 GEL ELECTROPHORESIS 15
1.5 MICROFLUIDICS 17
1.6 TUNABLE RESISTIVE PULSE SENSING (TRPS) 18
1.7 MOTIVATION AND PURPOSE 19
CHAPTER 2 MATERIALS AND METHODS 20
2.1 HEK-293T PALMGRET CELL CULTURE AND EVS COLLECTION 20
2.1.1 Cell culture 21
2.1.2 EVs collection 21
2.2 ANALYSIS TOOLS AND METHODS 23
2.2.1 qNano (Tunable resistive pulse sensing, Q5013, IZON Science) 23
2.2.2 Luminescence Imaging System (Amersham™ Imager 600, AI600) 23
2.3 SYSTEM PRELIMINARY TEST 23
2.3.1 Gel mold design 24
2.3.2 Agarose gel preparation and electrophoresis 25
2.4 2D ELECTROPHORESIS 27
2.4.1 Gold electrodes 29
2.4.2 PDMS bonding 30
2.5 DEVICE IMPROVEMENT 30
2.5.1 PDMS film 31
2.5.2 Syringe pump to collect samples 31
CHAPTER 3 RESULTS AND DISCUSSIONS 33
3.1 PRELIMINARY TEST 33
3.1.1 Tracking EVs by bioluminescence 33
3.1.2 ImageJ analysis 34
3.1.3 qNano analysis 36
3.2 NARROWER INLET 38
3.3 2D ELECTROPHORESIS 40
3.4 DEVICE IMPROVEMENT 43
3.5 VERIFY THAT THE DEVICE CAN GENERATE A VERTICAL ELECTRIC FIELD 44
3.6 GEL TEST 46
CHAPTER 4 CONCLUSIONS 50
CHAPTER 5 REFERENCES 51

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