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

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姓名

周韋成(Wei-Cheng Chou) 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

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
(Improvement of GC-rich RNA detection specificity by methylated phosphate DNA probe design on nanowire field effect transistor)

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摘要(中)

在近幾年來，核酸檢測的發展對於精準醫療的進展扮演很重要的角色，由於多晶矽奈米線場效電晶體具高靈敏度、迅速的反應時間及即時檢測，因此在核酸檢測應用端上已經成為一有力工具在。
在核酸檢測中，富含GC鹼基的核酸序列容易具有非專一性的結合，常常造成精準醫療發展上的阻礙。造成非專一性的結合是因為富含GC鹼基之序列容易形成二級結構，形成hairpin或是loop結構，在核酸檢測的訊號上帶來不準確性及可能在醫療上錯誤的執行。
為了能夠解決非專一性的核酸檢測，我們嘗試利用甲基化核酸DNA作為我們的檢測探針，甲基化核酸探針因為其核苷酸間磷酸根骨幹之官能基被甲基化，所以成為不帶電的DNA類似物。在我們實驗室先前的研究中，因為其不帶電的特性，在低鹽環境下能夠使DNA雙股在雜交時其靜電排斥效應減弱，形成穩定雙股螺旋結構，而一般DNA則因為靜電排斥力，其雙股螺旋結構較為不穩定，因此本研究設計nDNA於探針中，進一步針對基因檢測更困難的富含GC鹼基序列，嘗試得到較佳專一性，確實且有效率地提升DNA探針在與另一股DNA雜交時的辨識力。
在本研究中，選擇(3-aminopropyl)triethoxysilane(APTES)化合物為固定DNA探針之偶聯劑，為建立穩定且較佳的檢測訊號，所以先嘗試去找到APTES固定DNA探針的最佳化學條件，來使固定在多晶矽奈米線場效電晶體上之DNA探針具一致性，實驗結果發現，APTES溶於99％及95％酒精，在環境通入氮氣後比起沒有通入氮氣，其表面型態更為均勻，且固定上DNA探針經由FET量測，所得到之每一次量測訊號穩定許多。而後利用甲基化核酸DNA作為探針，檢測富含GC鹼基之序列，從實驗結果中，我們在低鹽濃度的情況下，確實有效地提升檢測富含GC鹼基的辨識力，且我們把在低鹽濃度之雜交環境溫度提升至40℃，更發現到nDNA探針辨識單一鹼基錯誤配對的能力進一步提升，因此從結果上來看，利用甲基化核酸DNA探針在多晶矽奈米線場效電晶體的核酸檢測上，與一般DNA探針比較下，甲基化核酸DNA具更大的潛力，配合雜交環境的選擇，可以創造更佳的檢測專一性。

摘要(英)

In recent years, researches on detection of nucleic acid have played an important role in the development of precision medicine. Nanowire field effect transistor(NWFET) has been a powerful tool for DNA or protein sensor due to their high sensitivity, fast response time and can be applied in real-time detection.
Nonspecific biosensing signal of these GC-rich nucleic acids will hamper the progress of the research into gene sequences. It is believed that the main cause of nonspecific biosensing of GC-rich sequences is the formation of a secondary structure such as hairpins or loops and result in self-complementary conformation.
To solve the nonspecific biosensing, we try to make good use of phosphate methylated DNA(nDNA) as a detection probe on the sensor surface. It is an uncharged DNA analogue due to the backbone phosphate groups changed by methylphosphate groups, making no electrostatic repulsion during hybridization between nDNA and regular DNA. From the previous study of our laboratory, the perfect match sequences will become more stable due to the reduction of electrostatic repulsion between the complementary DNA. As the result, we can indeed increase the efficiency of mismatch discrimination.
In our studies, we tried to find a optimized condition of incubating FET substrate with (3-aminopropyl)triethoxysilane(APTES) to achieve uniform ligand density. Followed by immobilized nDNA as probe for possible better binding/hybridization specificity of GC-rich target miRNA. By detecting HCV-3b RNA on the NWFET, nDNA probe can successfully improve the mismatch discrimination in GC-rich nucleic acids in a lower hybridization salt concentration. We conclude that nDNA probe can effectively increase specificity on detecting GC-rich nucleic acids in NWFET. With the advantages of lower salt concentration condition, we believe that designed nDNA probes provide tremendous potential in biosensing on NWFET with higher specificity comparing with that of natural DNA probe.

關鍵字(中)

★ 矽奈米線場效電晶體
★ 核酸
★ GC 含量
★ 單一核苷酸多形性

關鍵字(英)

★ silicon nanowire field effect transistor
★ nucleic acid
★ GC content
★ single nucleotide polymorph

論文目次

摘要 i
Abstract iii
誌謝 v
目錄 vii
圖目錄 xi
表目錄 xvii
第一章緒論 1
第二章文獻回顧 4
2.1 基因檢測 4
2.1.1 基因檢測平台 5
2.2 矽奈米線場效電晶體生物感測器 9
2.3 核酸分子 15
2.3.1 核酸分子概論 15
2.3.2 去氧核醣核酸結構 15
2.3.3核醣核酸結構 19
2.3.4 微小核糖核酸結構 20
2.3.5 量測核酸探針種類 21
2.3.6 序列之G(Guanine)、C(Cytosine)鹼基含量影響 27
2.4 單一核苷酸多型性 30
2.4.1 SNP概論 30
2.4.2 SNP錯誤配對辨識能力之研究 31
2.5 晶片表面改質 33
2.5.1 自組裝單層膜表面改質技術 33
2.5.2 表面分子固定化 39
2.5.3 3-氨基丙基三乙氧基矽烷(APTES)於自組裝單層膜之優化 40
第三章實驗藥品、儀器設備與方法 45
3.1 實驗藥品 45
3.2 儀器設備 46
3.3 晶片表面改質 47
3.3.1 晶片表面光阻去除及氧電漿處理 47
3.3.2 修飾APTES((3-aminopropyl)triethoxysilane) 47
3.3.3 修飾GA(glutaraldehyde) 47
3.3.4 探針固定化 48
3.4 FET電性測量 49
3.5 Circular Dichroism (CD)實驗 50
3.6 SYBR Green螢光熔點測量實驗 50
3.7 nDNA質譜分析 51
第四章結果與討論 53
4.1 表面改質優化 53
4.1.1改變溶劑之水含量修飾APTES 53
4.1.2 溶劑中加入弱鹼修飾APTES 58
4.1.3 修飾戊二醛(Glutaraldehyde) 59
4.2 部分修飾nDNA以圓二色光譜儀測定構型 62
4.3 XPS表面元素分析 65
4.4 探針於辨識單一核甘酸錯誤配對之能力探討 67
4.4.1 HCV-3b序列之Tm(melting temperature)量測 68
4.4.2 一般DNA探針辨識在富含GC鹼基單一核苷酸錯位能力探討 72
4.4.3 nDNA探針辨識在富含GC鹼基單一核苷酸錯位能力探討 73
4.4.4 於低鹽環境下nDNA/DNA探針辨識在富含GC鹼基單一核苷酸錯位能力探討 76
4.4.5 提升雜交溫度以提升nDNA/DNA探針辨識效果 82
第五章結論與未來展望 86
5.1 結論 86
5.2 未來展望 87
第六章參考文獻 89

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2018-7-12

推文