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姓名	林弘庾(Hong-Yu Lin)  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	探討檢測微核醣核酸時核酸雜交反應誘發之反離子重新分布對矽奈米線場效電晶體電場效應的影響 (Counterion effects impact on microRNA detection by silicon nanowire field-effect transistors)
相關論文	★ 類澱粉胜肽聚集行為之電腦模擬 ★ 溶解度參數計算及量測於HPLC純化胜肽程序之最佳化研究 ★ 利用恆溫滴定微卡計量測蛋白質分子於溶液中之第二維里係數與自我聚集之行為 ★ 利用SPRi探討中性DNA探針相較於一般DNA探針在低鹽雜交環境下之優勢 ★ 矽奈米線場效電晶體多點之核酸檢測研究 ★ 使用不帶電中性核酸探針於矽奈米線場效電晶體檢測去氧核醣核酸與微核醣核酸之研究 ★ 運用nDNA 修飾引子於PCR及qPCR平台以提升專一性之研究 ★ 設計中性DNA引子及探針以提升PCR與qPCR專一性之研究 ★ 使用中性不帶電去氧核醣核酸探針於矽奈米線場效電晶體檢測微核醣核酸之研究 ★ 使用不帶電中性核酸探針於原位雜交技術檢測微核醣核酸之研究 ★ 設計不帶電中性核酸探針於矽奈米線場效電晶體來改善富含GC鹼基核醣核酸之檢測專一性 ★ 合成5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite以作為應用於原位合成之新穎性中性核苷酸之研究 ★ 立體紙基外泌體核酸萃取裝置應用於檢測不同微環境下癌細胞所釋放之外泌體與外泌體微小核醣核酸之表現量 ★ 利用抗原結合區段之抗體片段探針於矽奈米線場效電晶體來改善抗原檢測濃度極限之研究 ★ 利用表面電漿共振影像儀驗證最適化之抗非專一性吸附場效電晶體表面於血清環境下之免疫測定 ★ 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
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摘要(中)	由於矽奈米線場效電晶體 (Silicon nanowires field-effect transistors)同時具備超高靈敏度、即時檢測、免螢光標定、製程兼容、以及設備便宜等優勢，使其近年來廣泛受到科學家們的矚目，不僅被大幅應用於檢測液態切片 (Liquid biopsy)的相關研究中，也被視為極具發展潛力之生物分子檢測平台。 矽奈米線場效電晶體檢測核酸雜交反應的檢測機制 (Detection mechanism)，是直接以核酸互補股的電荷作為判斷標準，進一步分析核酸雜交反應前後元件電導 (Electrical conductance)的改變。以 n-type矽奈米線場效應電晶體為例，若是目標分子帶負電，其產生的電場將會排斥通道中的電子，導致元件電導下降；反之，若是目標分子帶正電，其產生的電場將會吸引通道中額外的電子，導致元件電導上升。 雖然目前大部分的文章皆使用上述概念做為判斷標準，但進一步探討後，我們將會發現實際的界面現象並非如此單純。因為核酸互補股具有電荷，所以電解質溶液中的反離子 (Counterions)將會被吸引，並被連帶導入至修飾上核酸探針的界面。被吸引的 Counterions不僅會產生狄拜遮蔽 (Debye screening)效應，還會對元件施加和目標物電荷相反的電場，使得元件無法直接量測到目標物的電荷，最終導致使用者難以利用現有的檢測機制判斷檢測結果。 為了探討 Counterion effects，我們首先使用 Mixed-SAMs (Silane-PEG-NH2：Silane-PEG-OH=1：3(mM/mM))以及戊二醛 (Glutaraldehyde)對元件進行表面改質，接著透過還原胺化反應將 miR-21核酸探針接枝至元件表面。表面改質完成後，本研究使用 X射線光電子能譜儀 (X-ray photoelectron spectroscopy)以及原子力顯微鏡 (Atomic force microscope)確認 Mixed-SAMs的表面特徵。此外，我們也使用界達電位粒徑分析儀確認核酸雜交反應前後元件界面電位 (Zeta potential)的變化。另一方面，我們更透過 COB (Chip on board)系統確認 Counterion effects對於 FETs電訊號的影響。 透過 COB系統的結果可發現，在50 mM BTP溶液中， FETs電訊號變化將會由 Counterions主導。反之，當溶液被更換為 DI water後， FETs電訊號變化將會由核酸雙股結構主導。不僅如此， Zeta potential的結果也展現出 Counterions將會主導界面電位。在核酸雜交反應後，雖然帶負電的互補股被引入至界面上，但Counterions也同時被吸引，才會使得界面電位不往負電偏移，反而往正電改變。 由本研究中的實驗結果可得知，目前對於矽奈米線場效電晶體檢測機制的闡述並不完全，甚至能夠發現其中有些不足之處。因此本研究將 Counterion effects和現有的檢測機制相互結合，並成功提出一項更加完整的方案解釋檢測低核酸互補股濃度時，矽奈米線場效電晶體的電訊號變化和核酸雜交反應誘發之 Ion redistribution間的關係。
摘要(英)	Recently, silicon nanowire field-effect transistors (SiNWFETs) have attracted the most because they have many indispensable advantages, such as, high sensitivity, label-free detection, real-time detection and mass-production because of relating to the semiconductor industry. Therefore, it has been not only widely used to detect liquid biopsy but also regarded as a detection platform with great development potential. The common SiNWFETs detection mechanism is based on the charge of the target and then analyzes the change of the electrical conductance before and after the hybridization, for example in the n-type FET, the charged carriers would accumulate on the surface of the channel and increase the drain current if positively charged target molecules were detected. On the other hand, when the receptors captured negatively charged target molecules, the charged carriers would decrease and hence reduce the electrical conductance. Although most researches agree with this concept, the actual interfacial phenomena would become extremely complicated during the hybridization. Counterions in the electrolyte solution would be attracted and move to the interface because RNA targets were charged. The counterions which were appealed not only screened the charges of the RNA targets but also exerted the electric field which was opposite to it exerted by the RNA/DNA duplexes. Thus, it is difficult to analyze the results based on the common detection mechanism. In order to verify our concept, we fabricated the biosensor by modifying the SiNWFETs with Mixed-SAMs of PEG including silane-PEG-NH2 and silane-PEG-OH at the ratio of 1:3 before treating with glutaraldehyde to immobilize the DNA probes which were complementary with miR-21 sequence. After that, X-ray photoelectron spectroscopy and atomic force microscope were used to confirm that Mixed-SAMs surface modification was successful. On the other hand, we confirmed the influence caused by counterion effects on the SiNWFETs electrical signal by using the chip on board system. Also, we verified the change of the zeta potential before and after the hybridization. According to chip on board results, we could realize that the SiNWFETs electrical signals would be dominated by counterions under higher ionic strength environment (50 mM BTP). On the other hand, the SiNWFETs electrical signals would be dominated by miR-21 under lower ionic strength environment (DI water). Furthermore, zeta potential results implied that counterions would be dominant. After hybridization, the counterions are also attracted simultaneously even though the negatively charged complementary strands were recognized and hybridized by the miR-21 probe. Thus, zeta potential didn’t become more negative but more relatively positive. Based on our results, we could realize that the common SiNWFETs detection mechanism won′t be able to elucidate the detection results. Therefore, we would like to integrate the counterion effects with the common SiNWFETs detection mechanism and provides a more precise mechanism perspective to describe the relationship between electrical signal changes of SiNWFETs and the ion redistribution caused by hybridization during the miRNA detection under ultra-low concentration.
關鍵字(中)	★ 反離子效應 ★ 狄拜遮蔽效應 ★ 微核糖核酸檢測 ★ 矽奈米線場效電晶體 ★ 生物感測器	關鍵字(英)	★ Counterion effects ★ Debye screening ★ miRNA detection ★ Silicon nanowires field-effect transistors ★ Biosensor
論文目次	摘要 VI Abstract VIII 致謝 X 目錄 XI 圖目錄 XIV 表目錄 XVII 第一章 緒論 1 第二章 文獻回顧 3 2.1 疾病檢測 3 2.1.1 疾病檢體 3 2.1.2 生物標示物 (Biomarker) 4 2.2 核酸介紹 6 2.2.1 核酸分子 6 2.2.2 去氧核醣核酸 7 2.2.3 核醣核酸 9 2.2.4 微小核醣核酸 11 2.3 核酸檢測 14 2.3.1 即時定量聚合酶鏈鎖反應 14 2.3.2 核酸檢測技術的瓶頸與轉機 17 2.4 矽奈米線場效電晶體 18 2.5 晶片表面改質(Surface modification) 23 2.5.1 自組裝單層膜 23 2.5.2 矽氧烷-聚乙二醇 (Silane-PEG)於自組裝單層膜之應用 26 2.6 緩衝溶液鹽離子濃度對於核酸雜交的影響 29 2.7 電解質溶液中離子的重新分佈 (Ion redistribution) 33 2.8 實驗動機 40 第三章 實驗藥品、儀器與方法 41 3.1 實驗藥品 41 3.2 儀器設備 43 3.3 實驗方法 44 3.3.1 實驗架構 44 3.3.2 溶液配置 45 3.3.3 以 Mixed-SAMs對 FETs做表面改質 46 3.3.4 Mixed-SAMs表面化學分析 49 3.3.5 界面電位 (Zeta potential)量測 50 3.3.6 FETs電性分析 51 第四章 結果與討論 53 4.1 Mixed-SAMs表面化學分析 53 4.1.1 原子力顯微鏡 53 4.1.2 化學分析電子能譜儀 55 4.2 Ion redistribution對於 FETs電訊號影響 58 4.2.1 離子濃度對於 Counterion effects之影響 58 4.2.2 探討Counterion effects對於 Liquid gate FETs以及 Back gate FETs之影響 63 4.3 Mixed-SAMs表面檢測 microRNA成效分析 68 第五章 結論 70 第六章 未來展望 71 第七章 參考文獻 72 第八章 附錄 79 8.1 藉由 ELISA以及 LSCM確認核酸雙股結構能否在電訊號量測的過程持續維持 79 8.2 不同 Wafer表面之界面電位量測 84 8.3 不同探針密度之 Mixed-SAMs表面檢測 microRNA成效分析 87
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指導教授	陳文逸(Wen-Yih Chen)	審核日期	2022-7-26
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