近年來，由於包含了各種具疾病專一性的生物標的物，如蛋白質、信使核醣核酸、小分子核糖核酸等，大量存在於人體循環系統的胞外泌體已展現出用於臨床診斷的重大潛力。然而，現行用於胞外泌體的分離與定量之方法仍然仰賴於專門且昂貴的儀器以及訓練有素的專業人員，這可能無法支援一些臨床應用，例如定點即時檢測。因此，建立一個自動化的診斷平台能以從臨床樣品(例如，血漿或血液)中萃取出胞外泌體並定量其內和疾病相關的蛋白和核酸仍是一個急切的需要。為了滿足此需求，本研究開發了三個創新的整合型微流體平台，結合了多種技術，包括氣動式微流體、攪拌增強過濾、原位螢光增強薄膜免疫染色、與光誘發介電泳，用於胞外泌體的分離、濃縮、回收與特定胞外泌體的定量。首先，一個能夠從全血中分離並定量CD63+胞外泌體的整合型微流體系統首次被證實。此系統裝置一個創新的攪拌增強過濾模組，藉由同時產生連續擾動渦流與尺寸排除過濾，能顯著提升血液過濾之通量(22.3 μL/min，於8分鐘內回收178 μL的無血小板血漿)，並結合免疫磁珠抓取與酵素結合螢光增強技術以濃縮並定量CD63+胞外泌體。其次，一個使用光誘發介電泳操控、濃縮、並回收胞外泌體之全新的微流體平台被建立。胞外泌體於0.2 M的蔗糖溶液中之光誘發介電泳特性首次被探討，並能在32秒內快速濃縮272倍之胞外泌體。此外，一個光誘發胞外泌體分離平台被開發用於自動化分離、釋放(99.8%)、並回收(52.2%)胞外泌體。第三，一個結合濾膜之胞外泌體分離/計數平台，整合聚碳酸酯濾膜用於從全血中分離小胞外泌體(99.4%)，以及氧化鋁濾膜用於免疫染色與CD63+胞外泌體之數位計數，能達到81.1%的回收率、90.2%的染色效率、以及3 × 104 EVs/mL的偵測極限。外泌體蛋白於單個胞外泌體上之表現量可被探討及量化。此外，僅需2 μL之全血顯示了其用於指尖血檢測之潛在應用。總結來說，此三個整合型平台為自動化從血液處理到特定胞外泌體之分離與定量提供了巨大的潛力，期望能在不久的未來為胞外體疾病診做做出貢獻。

Extracellular vesicles (EVs), richly existing in body fluids, have shown a significant potential in clinical diagnosis since they contain various specific biomarkers, e.g., proteins, messenger ribonucleic acids (mRNA), and micro ribonucleic acids (miRNA), targeting to corresponding diseases. However, the existing approaches for EVs isolation and quantification still rely on specialized, expensive instruments and well-trained professionals, which may not support some clinical applications, such as point-of-care testing. Therefore, it is still in great need to establish an automatic diagnostic platform capable of extracting EVs from clinical samples (such as plasma or blood) and quantifying disease-associated proteins or nucleic acids. To address this need, three novel integrated microfluidic platforms were developed in this work for EVs isolation, enrichment, recovery and specific EVs quantification via combining multiple techniques, including pneumatic-driven microfluidics, stirring-enhanced filtration, membrane-based immunostaining with in situ fluorescence amplification, and optically-induced dielectrophoresis (ODEP). First, an integrated microfluidic system capable of isolating EVs from whole blood and quantifying CD63+ EVs in one single chip was first demonstrated. This system was equipped with a new stirring-enhanced filtration module by simultaneously generating continuous vortex flow and size-exclusion filtration to significantly enhance the filtering throughput (22.3 μL/min for retrieving 178-μL platelet-free plasma in 8 min) and combined with immunobead capture and enzyme-linked fluorescence amplification for enriching and quantifying CD63+ EVs. Secondly, a new microfluidic platform, using ODEP to manipulate, enrich and recover EVs, was established. The ODEP characterization of nanoscale EVs in 0.2 M sucrose was first explored and rapid EVs enrichment achieved a 272-fold increase in only 32 s. Moreover, an optically-induced EV isolation platform was developed to automate EVs isolation, releasing (99.8%) and recovery (52.2%). Thirdly, a membrane-based EV isolation/counting platform integrated with a polycarbonate (PC) membrane for small EVs isolation (99.4%) from blood and an aluminum oxide (AO) membrane for immunostaining and digital counting of CD63+ EVs in single EV level with a recovery rate of 81.1%, a staining efficiency ≥ 90.2%, and a limit of detection of 3 × 104 EVs/mL, was developed. The exosomal protein expression in single EV level was also investigated and quantified. Furthermore, only 2-μL whole blood was required, indicating a potential application for fingertip-based blood testing. To summarize, these three integrated platforms provide significant potentials for automating sample-to-answer processes from blood pretreatment to specific EVs isolation and quantification, thus contributing to EVs disease diagnosis in the near future.

Abstract I
中文摘要 III
誌謝 V
Table of contents VI
List of Tables X
List of Figures XI
Abbreviations and Nomenclature XX
Chapter 1 Introduction 1
1.1 Importance of circulating extracellular vesicles for disease diagnosis 1
1.2 EV isolation from body fluids 2
1.3 Microfluidics for EV isolation, detection and analysis 3
1.4 Motivation and novelty 4
1.4.1 An integrated microfluidic system for enrichment and quantification of circulating EVs from whole blood by utilizing stirring-enhanced filtration and magnetic bead-based immunoassay 4
1.4.2 Isolation and Recovery of Extracellular Vesicles Using Optically-Induced Dielectrophoresis on an Integrated Microfluidic Platform 7
1.4.3 Isolation and Digital Counting of Extracellular Vesicles from Blood via Membrane-integrated Microfluidics 9
1.5 Scope and structure of the dissertation 11
Chapter 2 An Integrated Microfluidic System for on-chip Enrichment and Quantification of Circulating Extracellular Vesicles from Whole Blood 13
2-1 Introduction 13
2.2 Materials and methods 14
2.2.1 Microfabrication and surface modification of microfluidic chip 14
2.2.2 Immunocapture and ELISA 15
2.2.3 Blood sample preparation and storage 17
2.2.4 Design of the integrated microfluidic chip 17
2.2.5 Procedure of EVs enrichment and quantification from whole blood 20
2.2.6 Scanning electron microscopy (SEM) measurement 21
2.2.7 Hemolysis analysis 23
2.2.8 N-structured illumination microscopy (N-SIM) 23
2.3 Results and discussion 31
2.3.1 Stirring-enhanced filtration 31
2.3.2 Hemolysis and clogging 32
2.3.3 Characterization of filtered PFP 34
2.3.4 EVs enrichment via immunocapture 36
2.3.5 Optimization of immunocapture and on-chip quantification of EVs 38
2.4 Summary 46
Chapter 3 Isolation and Recovery of Extracellular Vesicles Using Optically-Induced Dielectrophoresis on an Integrated Microfluidic Platform 47
3-1 Introduction 47
3.2 Materials and methods 48
3.2.1 PalmGRET cells and cell-derived EVs 48
3.2.2 ODEP for EVs manipulation and enrichment 49
3.2.3 Design and microfabrication of the OIEV platform 49
3.2.4 On-chip EVs isolation and recovery 51
3.2.5 Nanoparticles tracking analysis (NTA) 52
3.2.6 Transmission electron microscopy (TEM) 52
3.3 Results and discussion 58
3.3.1 EVs manipulation via ODEP 58
3.3.2 EVs enrichment via shrinking optical rings 60
3.3.3 Regulation of volumetric flow rate by pneumatic-driven microfluidics 61
3.3.4 EVs isolation and recovery via integration of ODEP and microfluidics 62
3.4 Summary 72
Chapter 4 Isolation and Digital Counting of Extracellular Vesicles from Blood via Membrane-integrated Microfluidics 73
4-1 Introduction 73
4.2 Materials and methods 74
4.2.1 Preparation of cell lines and cell-derived EVs 74
4.2.2 Preparation of blood samples 75
4.2.3 Experimental procedure for EVs isolation, staining, and counting 75
4.2.4 Membrane-based EVs isolation/counting (mEVic) microfluidic platform 77
4.2.5 Microfabrication of PDMS-based chip and package of membrane filters 78
4.2.6 Analysis tools 79
4.3 Results and discussion 84
4.3.1 EVs isolation from blood via stirring-enhanced filtration 84
4.3.2 Characterization of membrane-based EVs staining 85
4.3.3 CD63+ EVs staining/counting using PalmGRET EVs 88
4.3.4 Plasma CD63+ EVs staining/counting 90
4.4 Summary 100
Chapter 5 Conclusions and future perspectives 101
5.1 Conclusions 101
5.2 Future perspectives 103
5.2.1 Size-based EVs sorting via ODEP 103
5.2.2 Isolation of EVs from lipoproteins via ODEP 104
5.2.3 ODEP-based platform for disease diagnosis 104
5.2.4 Investigation of cancer biomarkers and clinical test 105
References 108
Publication list 129
A. Journal papers 129
B. Conference papers 131

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