博碩士論文 107324023 詳細資訊




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姓名 賴欣瑩(Hsin-Ying 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 使用混合自組裝單層膜於矽奈米線場效電晶體檢測微小核醣核酸之研究
(Optimization of surface modification on silicon nanowire field effect transistor by mixed self-assembled monolayers for microRNA detection)
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摘要(中) 核酸檢測在現今已被廣泛運用於疾病的診斷及癒後,藉此改善病人之治療情形,而最常用於核酸檢測的方式為即時定量聚合酶反應,但其缺點為耗時、需加入螢光分子標記、需要昂貴的儀器設備與經過培訓的實驗人員。為了即時檢測分析,甚至於在醫療資源匱乏的環境條件下,也能提供定量檢測,致使許多團隊致力於尋求新的核酸檢測方式。
由於矽奈米線場效電晶體具有高靈敏度、高專一性、無須螢光標記、即時檢測、體積小且操作過程簡易等優點,因此被視為極具發展潛力之核酸檢測平台。矽奈米線場效電晶體的訊號大小取決於探針與目標分子(DNA, RNA, microRNA)雜交之數量,但由於探針密度會影響雜交效率及雜交數量,因此訊號大小亦會受到探針密度的影響。
由於實驗室先前已完成以3-氨基丙基三乙氧基矽烷((3-aminopropyl)triethoxysilane)形成之自組裝單層膜進行表面改質,而後再改質戊二醛(glutaraldehyde)及DNA探針於奈米線場效電晶體上來檢測人工合成之miR-21的研究。因此,本研究期望接續先前研究,嘗試對細胞培養樣品中萃取出之miR-21作檢驗,實驗結果顯示使用奈米線場效電晶體作為生物檢測平台能成功檢測萃取出之total RNA中的miR-21。為了更進一步探討探針密度的影響與系統性地研究表面改質的最適化,本研究選擇了silane-PEG-NH2及silane-PEG-OH形成之混合自組裝單層膜為研究方向,並先以人工合成之miR-21作為檢測樣品。期望以此能找出最佳自組裝單層膜的組成與最佳檢測條件,尤其是檢測溶液之鹽濃度,並提出檢測極限。使用聚乙二醇(poly(ethylene oxide))則是因為其具有抗非專一性吸附的效果,若將來以血清或血液作為檢測樣品,樣品內含的生物分子,如蛋白質及核酸等,可能會產生非專一性吸附,造成實驗結果的誤差。
首先,我們先以X射線光電子能譜學(X-ray photoelectron spectroscopy)作為驗證工具,確認已成功將探針改質於晶片上,之後探討探針密度對於電訊號之影響,而後再配合非專一性控制組實驗,來探討DNA探針在不同鹽離子濃度下的辨識能力。從實驗結果可以看到使用silane-PEG-NH2與silane-PEG-OH以莫耳比例為1比3之混合自組裝單層膜進行改質之晶片可提供最大之電訊號變化量及解析度。接著再以此比例進行更改鹽離子濃度之實驗,從實驗結果發現,於50mM bis-tris propane(BTP)的鹽溶液環境下,具有較好辨識能力。最後我們嘗試找出此元件對於核酸檢測之極限,從實驗結果發現,優化條件下所能檢測之核酸最低濃度可達0.1fM。
摘要(英) Currently, nucleic acid testing(NAT) has been widely applied for diagnosis and prognosis to improve patient care. However, quantitative real polymerase chain reaction(qPCR), the most popular technique for NAT is time-consuming, fluorescence labeling and requires expensive instrumentation with skillful personnel in lab. It is therefore essential to develop a new approach for NAT, especially in limited conditions, with applications in point-of-care(POC) testing. Silicon nanowire field effect transistor(SiNWFET) has been a promising candidate for NAT because of its high sensitivity, specificity, label-free detection, small size, and simple process, etc. In nucleic acid sensing by SiNWFET, the probe density is highly correlated to the signal because it determines the efficiency of target hybridization, which generates electrical variation.
In this thesis, the surface modification of SiNWFET biosensors with mixed self-assembled monolayers(SAMs), constituting of silane-PEG-NH2 and silane-PEG-OH at various ratio, is systematically investigated to optimize the probe density of synthetic miR-21 detection by SiNWFET modified with (3-aminopropyl)triethoxysilane(APTES) and exploit it for detection of miR-21 extracted from the cell cultures. Poly(ethylene oxide)(PEG) is employed to resist non-specific adsorption and modulate the probe density. The empirical data present that the nucleic sensors modified with mixed SAMs at the ratio of silane-PEG-NH2 : silane-PEG-OH = 1:3, verified by X-ray photoelectron spectroscopy(XPS), expressed the most significant electrical variation of DNA-microRNA hybridization recorded by SiNWFET. The calibration line produced from the SiNWFET DNA-sensors modified by mixed SAMs at that ratio also presented the highest resolution with the limit of detection(LOD) at 0.1fM. Another investigation at different ionic-strength condition also reveal that 50mM bis-tris propane(BTP) is better for discrimination ability of DNA probes.
關鍵字(中) ★ 矽奈米線場效電晶體
★ 微小核醣核酸
★ 混合自組裝單層膜
關鍵字(英) ★ silicon nanowire field effect transistor
★ microRNA
★ mixed self-assembled monolayers
論文目次 摘要 i
Abstract iii
誌謝 v
目錄 vii
圖目錄 x
表目錄 xiii
一、 緒論 1
二、 文獻回顧 4
2.1 核酸介紹 4
2.1.1 核酸分子 4
2.1.2 去氧核醣核酸 5
2.1.3 核醣核酸 7
2.1.4 微小核醣核酸 9
2.2 核酸類似物 11
2.2.1 鎖核酸 11
2.2.2 肽核酸 12
2.2.3 嗎啉基寡核苷酸 13
2.3 核酸檢測 14
2.4 矽奈米線場效電晶體 17
2.5 緩衝溶液鹽濃度 22
2.5.1 鹽離子濃度對於核酸雜交的影響 22
2.5.2 鹽離子濃度與Debye length的關係 23
2.6 晶片表面改質 26
2.6.1 自組裝單層膜 26
2.6.2 表面分子固定化 31
三、 實驗藥品、儀器及方法 33
3.1 實驗藥品 33
3.1.1 細胞培養 33
3.1.2 核酸萃取 33
3.1.3 晶片表面改質 33
3.2 儀器設備 36
3.3 實驗方法 37
3.3.1 細胞解凍 37
3.3.2 細胞培養 37
3.3.3 細胞冷凍保存 38
3.3.4 微小核醣核酸萃取 39
3.3.5 晶片表面改質 41
3.3.6 FET電性測量 43
四、 結果與討論 44
4.1 檢測從細胞萃取出之微小核醣核酸 44
4.2 XPS表面元素分析 47
4.3 探針密度對於電訊號影響 49
4.4 鹽濃度對於檢測microRNA之影響 55
4.5 元件靈敏度測試 60
五、 結論與未來展望 63
5.1 結論 63
5.2 未來展望 64
六、 參考文獻 66
七、 附件 70
7.1 COB(Chip on board)實驗 70
7.2 以AFM觀測矽表面於不同處理下之粗糙度 84
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指導教授 陳文逸(Wen-Yih Chen) 審核日期 2020-7-30
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