胞外體已經在生物研究領域方面獲得的極大的關注，由於胞外體擁有許多獨特的地方，像是做為細胞間傳遞訊息的橋樑，以及具有做為預防與診斷疾病的生物標誌。我們不只對於胞外體傳遞訊息的特徵感到好奇，也想透過胞外體所表現的生物特徵去判斷我們器官的健康狀態。在我們想要了解身體器官間的溝通前，我們首先必須知道如何將胞外體成功的分離出來。
目前為止也已經有許多分離胞外體的方法，像是差速高速離心法(differential ultracentrifigation)，是廣為被使用的分離胞外體方式，然而分離過程十分的耗時、操作的器材也相對比較笨重且昂貴，回收率也相對較低，而且在極高的轉速之下，也容易造成胞外體降解，並使蛋白質群聚等不好的現象。因此在分離胞外體的系統上，我們選擇使用紙基裝置做為我們分離胞外體的平台。目標是希望可以打造擁有以下優點的紙基裝置: 方便、快速、低成本，以及藉由它高篩選率的特性，將我們的目標胞外體成功的從溶液中分離出來。除了做出更簡便的實驗步驟外，我們最後希望可以透過我們的紙基裝置利用不同的抗體同時分離出帶有不同表面抗原的胞外體，將整體分離的效率達到最大化。

Extracellular vesicles (EVs) have been caught a lot of attention in several biological fields recently. Due to its variable characteristics such as intercellular communication, signal transporting and potentials as diagnostic and prognostic biomarkers, we are curious about not only the messages EVs carry, but also our organs are in a good situation or not. To know the inside world of communication in our body, working on isolating EVs is the first and priority step.
Currently, there are several methods in isolating EVs. Differential ultracentrifugation is a method that has been widely used to isolate EVs. However, the process is time-costly, cumbersome, and required expensive instruments. Additionally, due to the high acceleration in centrifugation, it may cause degradation in EVs and the co-precipitation of protein aggregates. Therefore, we chose to use paper-based devices as our isolation platform. Our goal is to build up a device, which has the following advantages: convenient, quick, low budget, and high selectivity in capturing our target EVs. Despite the simple protocol, we tend to isolate different types of EVs simultaneously using one paper-based device.

摘要 1
Abstract 2
致謝詞 3
Table of Contents 4
List of Figures 6
List of Tables 8
List of Abbreviations 9
Chapter 1 Introduction 10
1-1 Background 10
1-2 Introduction of Extracellular Vesicles 10
1-3 Current isolation techniques 13
1-3-1 EV Enrichment Method 13
1-4 EV Characterization Techniques 16
Chapter 2 Aim of study 19
Chapter 3 Experiment design and method 21
3-1 Experiment design 21
3-2 Extracellular vesicles nurture & collection method 22
3-2-1 HEK293T cell nurture method 23
3-2-2 HEK293T cell collection method 23
3-3 Paper-based preparation 24
3-3-1 Paper-based materials and chemicals : 24
3-3-2 Fabrication of the paper-based devices 24
3-3-3 Surface modification 25
3-4 Eppendorf Container design 26
3-5 Isolation of extracellular vesicles 28
3-5-1 Testing samples 28
3-5-2 Paper-based immunoaffinity devices 29
3-6 Microscopy Characterization 29
3-6-1 PE-biotin Examination 30
3-6-2 EV Characterization 30
3-7 Resistive Pulse Sensing (RPS) – Qnano Characterization 31
3-7-1 Instrument Setup and Prepare Calibration 31
3-7-2 Calibration, Washing Upper Fluid Cell and Sample Measurement 33
3-8 Luminescence Characterization 34
3-8-1 EV Capacity Measurement 34
3-8-2 ELISA Detection on Different Antibodies for EV specificity 35
Chapter 4 Results and Discussion 37
4-1 Result of reagents in different proportion 37
4-1-1 PE-biotin Fluorescence Intensity 37
4-1-2 EV Sample Fluorescence Intensity Result 38
4-2 Fluorescence Intensity in different blocking component 39
4-3 Comparison of different surface preparation method 41
4-3-1 PE-biotin Fluorescence Intensity Result 41
4-3-2 EV sample Fluorescence Intensity Result 42
4-4 Comparison of different reagent mixing method 43
4-4-1 PE-biotin Fluorescence Intensity 43
4-4-2 EV sample Fluorescence Intensity Result 44
4-5 Applying Lid Design to Eppendorf Mixing Process 46
4-5-1 EV standard curve by Fluorescence Intensity 46
4-5-2 EV Luminescence curve by Luminescence plate reader 47
4-6 EV Capture rate Characterized by microscope 50
4-7 EV Capture rate Characterized by Qnano 51
Chapter 5 Conclusions 53
Chapter 6 Future Works 54
Chapter 7 References 55

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