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

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姓名

王逸軒(Yi-Hsueh Wang) 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
(Using neutralized-DNA on silicon nanowire field-effect transistor for microRNA detection)

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★ 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
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★ 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	★ 利用核適體作為訊號放大器於矽奈米線場效電晶體免疫感測器對生物標記物進行定量分析

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摘要(中)

近年來各種生物標記物( biomarkers )如DNA、RNA、蛋白質等生物分子的檢測方法被發展與推廣並期待與臨床上診斷跟治療結合。在眾多檢測平台中由於奈米線場效電晶體具備高靈敏度、快速反應、高專一性等優點，加上現行傳統的生物辨識技術如 qPCR 及微陣列檢測基因序列，都須要加入螢光分子來標記這些生物分子，而奈米線場效電晶體是直接量測電訊號變化，能免去螢光標定造成的不便，因此是極具發展潛力的檢測平台。目前有相當多研究團隊致力於運用奈米線場效電晶體檢測與重大疾病相關之基因序列，然而在基因檢測上還是受到核酸雜交溶液中鹽離子濃度決定的德拜長度(debye length)限制，影響了檢測的靈敏度。本研究中我們使用不帶電磷酸根甲基化 DNA(nDNA)探針來提升場效電晶體對miRNA的檢測效果。不帶電磷酸根甲基化 DNA 探針與目標核酸序列之間靜電排斥力較小，因此使不帶電磷酸甲基化 DNA 探針與目標核酸序列雜交時在低鹽濃度下，比傳統DNA 探針更容易雜交形成雙股螺旋結構。我們嘗試在不同鹽離子濃度下比較nDNA探針及DNA探針對miRNA的檢測能力，並建立檢量線及檢測敏感度(sensitivity)的量測，從實驗結果可以看到，檢測同樣濃度的miRNA，nDNA探針的電訊號變化量較DNA探針更大，在低鹽濃度下進行檢測兩種探針的電訊號變化量差異更為明顯。nDNA探針對miRNA的檢測，於10mM鹽濃度溶液中實驗最低檢測極限可以達到0.1fM。而且針對不同預期含量的檢測標的物，也能用nDNA探針搭配不同的鹽濃度溶液使檢測能有更佳的效果。若標的物只有fM等級，可以在10mM鹽濃度溶液中進行量測，確保能有最好的檢測靈敏度。若標的物預期含量較高，濃度約在10fM到1pM(元件最低定量極限為10fM)間之標的物，可以選擇nDNA探針搭配50mM BTP緩衝液進行檢測，得到的數據能進行較精確的定量分析。

摘要(英)

In recent years, many biomarkers detection methods have been studied and look forward to combine with clinical diagnosis and treatments. Due to it’s high sensitivity, fast response and specificity, silicon nanowire field effect transistor(SiNWFET) has been a powerful tool for gene sensing. Other conventional decetion methods like microarray or qPCR need to label fluorescence marker on biomolecules. SiNWFET is a label-free detection method, because the signal is come from electricity changing. Lots of group have been studying on the gene sequence related with cancer. While there are still some difficulties in this method, especially for DNA and microRNA detecting. The sensitivity of SiNWFET is limited by debye length, which is depending on the sensing buffer salt concentration. In this study, we focus on using neutralized phosphate-methylated DNA(nDNA) to enhance SiNWFET performance. Because of the lower electrostatic repulsion between nDNA and target sequence, nDNA can form duplex more easily in low salt concentration buffer. We sensing miR-21 sequence in different salt concentration buffer, set up the calibration curve. We also figure out the limit of detection(LOD) and limit of quantitation(LOQ). Base on the experiment results, we found that nDNA probe has larger signal change compare to DNA probe, especially sensing in low salt concentration buffer. The device’s limit of detection can reach to 0.1fM in 10mM BTP. We also found that by using nDNA probe with different salt concentration sensing buffer can improve the device’s performance. If the sample target concentration only has fM level, we can use 10mM BTP sensing buffer to perform the highest sensitivity; if the sample target concentration is around 10fM to 1pM (our device’s limit of quantitation is 10fM), we can use 50mM salt concentration sensing buffer to get higher resolution datas for quantitative analysis.

關鍵字(中)

★ 矽奈米線場效電晶體
★ 微核醣核酸

關鍵字(英)

★ silicon nano-wire field effect transistor
★ micro-RNA

論文目次

中文摘要 i
Abstract iii
誌謝 v
目錄 vi
圖目錄 viii
表目錄 xi
第一章緒論 1
第二章文獻回顧 4
2.1 核酸簡介 4
2.1.1 去氧核醣核酸 4
2.1.2 核醣核酸 7
2.1.3 微小核醣核酸 8
2.2 核酸類似物 9
2.2.1 肽核酸 9
2.2.2 鎖核酸 10
2.2.3 磷酸根甲基化去氧核醣核酸 12
2.3 場效電晶體生物感測器 15
2.4 德拜長度 22
2.4.1 Debye length 與溶液鹽離子濃度的關係 22
2.4.2 鹽濃度與 Debye length 於場效電晶體量測的影響 23
2.5 探針密度 25
第三章實驗藥品、儀器設備與方法 27
3.1 實驗藥品 27
3.2 儀器設備 28
3.3 nDNA序列合成 29
3.4 nDNA水解純化 30
3.5 晶片表面改質 31
3.5.1 晶片表面光阻去除及氧電漿處理 31
3.5.2 修飾APTES (3-Aminopropyltriethoxysilane) 31
3.5.3 修飾GA(glutaraldehyde) 32
3.5.4 探針固定化 32
3.6 FET電性測量 34
第四章結果與討論 35
4.1 鹽濃度對於 DNA-miRNA 雜交的影響 35
4.2 不同探針檢測miRNA的影響 40
4.3 不同miRNA樣品濃度檢測 44
4.4 極低濃度miRNA樣品檢測 49
第五章結論與未來展望 53
5.1 結論 53
5.2 未來展望 54
第六章參考文獻 55

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2018-1-23

推文