紙基微流道升降閥門裝置應用於液態切片中外泌體微小核醣核酸萃取

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姓名

賴藝芳(Yi-Fang Lai) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

紙基微流道升降閥門裝置應用於液態切片中外泌體微小核醣核酸萃取
(Microvalve-controlled fluidic system for extracting exosomal nucleic acids from bio-samples by paper-based devices)

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摘要(中)

外泌體能穩定存在人體體液（如:血液、尿液、唾液、腦脊液）且因其內部含有特異性蛋白質，脂質和核酸（包括mRNA，miRNA，DNA）可被攜帶，作為傳輸和細胞間交換訊號的生物標誌分子。因為外泌體有此特性使其在液體活檢中常作為病理治療或癒後追蹤，也可避免對患者進行重複的侵入性檢查，且可即時監測治療效果、耐藥性和疾病的演變。而紙質分析設備在一次性診斷測試方面具有巨大潛力，與傳統的microchip相比，紙張價格低廉可通過毛細作用吸收流體，無需外部泵或電源，而試劑可以通過打印器在紙上進行微流道圖案化，用過的紙設備可以通過焚化處理以避免污染，使這些紙質設備價格實惠且易於使用。但如何控制液體的流動方向及精準分配用以達到多步驟分析及減少人力、儀器、藥品及檢測成本也備受重視。
為此本實驗室欲發展一簡易紙質流體系統，系統中具有升降閥門來控制流體方向及反應時間等。微流體通道是洗削在熱塑性材料 (PMMA) 確保所有液體樣品吸附在親水紙條上，並以 10:1 的比例將疏水性 PDMS 膜夾在兩PMMA 之間作為緩衝層鎖上螺絲形成一封閉空間減少汙染。相關的表面性質也利用表面輪廓儀及AFM確認粗糙度後為後續實驗所用。螺絲閥門作為主動控制閥來減緩液體流速。而紙的孔隙率則用以調節流體的速度並增加延遲時間(10分鐘，最大壓力10MPa)優化核酸吸收的操作時間。結合實驗室對於外泌體純化分離及萃取微小核醣核酸研究，節省市面上對於外泌體純化的昂貴儀器及繁瑣程序人力時間。我們改質抗體於紙基上用以純化外泌體，且在紙基上化學改質二氧化矽藉此吸附裂解外泌體後微小核糖核酸，藉由SEM、NTA及paper-based ELISA確認exosome尺寸大小及其免疫特徵，用最適化條件進行裂解、吸附、清洗及脫附微小核糖核酸。由逆轉錄定量聚合酶鏈反應 (RT-qPCR) 結果得知，相較於無閥門控制的流體系統，使用控制閥門延緩流速，能將萃取到外泌體微小核糖核酸產量提高約 2.47 倍，洗脫萃取效能約為65%，檢測極限落在10PM-100fM之間。最後將紙基升降閥門微流道裝置應用於臨床慢性傷口成功分離純化外泌體和萃取其miR21得到初步慢性傷口和microRNA間意義，期望能以此裝置更貼近POCT的應用。

摘要(英)

Paper-based devices, first introduced by Whitesides group in 2007, have been widely developed for infectious conditions, disease status, and medical treatment efficiency because of satisfying the ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid and robust, Equipment-free, and Deliverable to end-users) established by the World Health Organization (WHO). Rerouting fluidic flow is important for lab-on-chip to achieve complex multiple-steps assay. Valves can enable the accurate and timed delivery of fluid by varying the direction and modulating the rate of the flow.
Herein, our lab developed a cost-effective, rapid, and easy-to-use microfluidic system to extract exosome and exosomal nucleic acids by fabricating PMMA microfluidic channel to control the fluidic direction, covered by a 10:1 with hydrophobic PDMS membrane to ensure the adsorption of all liquid sample onto the hydrophilic paper strip. The flow rate was modulated by screw valves as active controllers of the paper porosity for optimal operation time of nucleic acid absorption (10 minutes, maximum pressure 10MPa). Exosome properties were characterized by SEM, NTA and paper-based ELISA whereas miRNA-21 levels were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), which reported a 65% of miRNA contents of standard samples retained and presented an increase of 2.47-fold efficiency in extracting exosomal miRNA-21, compared to the similar procedure without the microvalve system. Finally, the designed system was successfully applied for fluidic clinical chronic wounds to capture exosomes and miR21, encouraging an adoption of Point of Care Testing.

關鍵字(中)

★ 微小核醣核酸
★ 紙基微流道
★ 外泌體
★ 閥門控制

關鍵字(英)

★ microRNA
★ Paper-based devices
★ exosome
★ microvalve-controlled

論文目次

摘要 i
ABSTRACT iii
誌謝 iv
目錄 v
圖目錄 x
表目錄 xiv
一、緒論 1
1.1 研究背景 1
1.2 論文架構 2
二、文獻回顧 5
2.1 核酸介紹 5
2.1.1 核酸分子 5
2.1.2 去氧核醣核酸 6
2.1.3 核醣核酸 8
2.1.4 微小核醣核酸 9
2.2核酸萃取 11
2.2.1液相核酸萃取 11
2.2.2固相核酸萃取 12
2.2.3核酸吸附二氧化矽機制 12
2.2.4 Langmuir adsorption isotherm model 13
2.3分子生物檢測平台 14
2.3.1聚合酶鏈鎖反應(Polymerase chain reaction， PCR) 14
2.3.2即時定量聚合酶鏈鎖反應（Quantitative real time polymerase chain reaction，qPCR) 15
2.4細胞外囊泡(Extracellular vesicles) 介紹 18
2.4.1外泌體(Exosome)介紹 18
2.4.2外泌體與癌症 20
2.4.3外泌體應用 21
2.5 Paper-based device 23
2.5.1 paper-based immunoaffinity device 23
2.5.2 paper-based sol-gel modification 23
2.5.3 paper-based biosensors for exoxome and exosomal nucleic acids extraction 24
2.6 Controlling the flow rate in paper-based devices 30
2.6.1 controlling the flow control without valves 30
2.6.2 controlling the flow control with valves 32
三、實驗藥品、儀器及方法 35
3.1實驗藥品 35
3.1.1等溫吸附實驗 35
3.1.2升降閥門微流道裝置材料 35
3.1.3標準品外泌體 35
3.1.4 Paper-based device 36
3.1.5 Paper-based ELISA 36
3.1.6 Paper-based nucleic acid extraction device 37
3.1.7 Nucleic acid extraction kit 37
3.1.8即時聚合酶鏈式反應 37
3.2儀器設備 38
3.3實驗方法 38
3.3.1 恆溫吸附實驗 38
3.3.2微流道裝置來源 39
3.3.3 Sol-gel silica coated method 39
3.3.4細胞培養來源 40
3.3.5外泌體純化濃縮 40
3.3.6 Paper-based immunoaffinity method 40
3.3.7 Paper-based ELISA 42
3.3.8 Paper-based nucleic acid extraction device 43
3.3.9 Microvalve-controlled fluidic system 45
3.3.10反轉錄及時聚合酶鍊式反應(qRT-PCR) 46
四、結果與討論 48
4.1升降閥門裝置材料鑑定 48
4.1.1 PMMA表面型態以及塗佈PDMS膜前後表面粗糙度變化……….. 48
4.1.2 升降閥門裝置施壓力檢測 52
4.2 paper-based device分離純化外泌體 55
4.2.1 Ultrafiltration方法濃縮HCT116大腸癌細胞培養液中外泌體 55
4.2.2 paper-based immunoaffinity method捕獲外泌體表面型態與粒徑大小分佈 56
4.2.3 P-ELISA檢測cell culture medium HCT116 exosome免疫特徵…….. 59
4.3 paper-based device萃取外泌體微小核醣核酸 61
4.3.1 paper based sol-gel 改質方法鑑定 61
4.3.2 pH值、鹽類、鹽濃度對核糖核酸吸附於二氧化矽之影響 64
4.4升降閥門裝置應用於純化外泌體以及萃取外泌體內微小核醣核酸 73
4.4.1升降閥門裝置應用於萃取 HCT116癌細胞之exosome 74
4.5升降閥門微流道裝置應用於慢性傷口(chronic wound)檢體 83
五、結論 86
六、未來展望 88
七、參考文獻 89
八、附錄 95

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2021-8-20

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