胞外囊泡是許多細胞所分泌大小介於40~100 nm膜狀結構，這類型的胞外囊泡含有蛋白質、mRNA、miRNA以及訊號分子。在生理上，這些胞外囊泡會在細胞之間協助蛋白質或RNA分子的運輸、在免疫學上具有抗原呈現(antigen presentation)的功能。近年研究發現，胞外囊泡與心臟病、腎臟病、腦部疾病的發生有關，此外，部分研究亦指出胞外囊泡在癌症形成中可能扮演重要角色。然而，因胞外囊泡大小極小且在生物樣本中含量極少，目前研究上常用的方法為超高速蔗糖密度梯度離心法(sucrose density gradient ultracentrifugation method)來收集，但超高速蔗糖密度梯度離心法不但耗時且消耗的生物樣品量多。因此，本文建立一具免疫親合性之微流道平台，透過結合高專一性免疫親合特性與微流道晶片之快速萃取、所消耗生物樣品量少等優點，來改善超高速離心法在胞外囊泡萃取收集步驟中耗時以及需較多生物樣品量之缺點。

Extracellular vesicles (EVs), secreted by cells, are membranous structures between 40 to 100 nm in size. EVs contain proteins, mRNA, miRNA, and signaling molecules. EVs facilitate the transport of these molecules between cells. Recently researchers found EVs may play an important role in not only heart, kidney diseases but cancerogenesis. However, EVs are quite small and their content in biological samples can be extremely low. Conventionally, the ultracentrifuge method is used to isolating EVs from serum or culture medium in which multiple steps from low speed to high speed centrifugations, are involved in order to collect EVs. In addition, a relatively large amount of sample is necessary for this method. Therefore, ultracentrifuge is time-consuming and not suitable in trace analysis. This thesis aims to establish a microfluidic immuno-affinity based approach to solve the disadvantages of ultracentrifuge methods.

摘要..…………………………………………………………………………………..1
致謝................................................................................................................................2
第一章 前言 6
1.1 研究背景 6
1.2 研究動機 6
第二章 文獻回顧 8
2.0 超高速蔗糖密度梯度離心法 8
2.1 過濾法 9
2.1.1超濾法(Ultrafiltration) 9
2.2 分子篩選液相層析法(SIZE-EXCLUSION LIQUID CHROMATOGRAPHY) 9
2.3 聚合沉澱法(PRECIPITATION) 9
2.3.1免疫親和性粒子 9
2.3.2肝素沉澱法(heparin affinity) 10
2.3.3聚乙二醇沉澱法(polyehtylenglycol, PEG) 10
2.4 微流道系統(MICROFLUIDIC SYSTEM) 10
2.4.1微流道過濾系統 10
2.4.2微流道層流分離系統 11
2.4.3 微流道免疫親和性系統 12
2.5 各系統方式比較 13
第三章 研究方法 14
3.1設計概念 14
3.2元件製程 14
3.2.1光罩設計 14
3.2.2黃光微影製程 15
3.2.3高分子材料聚二甲基矽氧烷翻模（PDMS molding） 16
3.2.4氧電漿接合處理與表面改質 17
3.2.5抗體接合修飾 17
3.3實驗設置 18
3.3.1不同類型微流道晶片純化實驗 18
3.3.2超高速離心純化實驗 19
3.3.3 圓柱陣列掃描式電子顯微鏡影像拍攝 20
3.3.4 RNA萃取純化 20
3.3.5 Agilent 2100 RNA濃度分析 20
3.3.6定量即時聚合酶連鎖反應 21
第四章 實驗結果 22
4.1 微流道晶片製作結果 22
4.2圓柱陣列微流道晶片之掃描式電子顯微鏡(SEM)結果圖 23
4.3 各類型方式之MIRNA萃取純化比較結果 24
4.3.1 Agilent 2100 RNA濃度分析 24
4.3.2定量即時聚合酶連鎖反應(Quantitative real time polymerase chain reaction, qPCR) 32
第五章 結論 33
第六章 參考文獻 34
附錄 38

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