臺灣博碩士論文加值系統

本研究利用去氧核醣核酸之鹼基能夠與特定離子或分子產生作用力，產生非華生-克力克鹼基之配對形式。並結合訊號放大機制，設計能夠應用於不同領域之生化感測器。偵測上不僅具有良好的選擇性，也成功提升分析方法之靈敏度。
第一部分透過人工設計出一段對於偵測二價汞離子 (Hg2+) 具有高選擇性的 DNA 探針序列，並結合雜交連鎖反應 (hybridization chain reaction，HCR) 進一步放大電化學訊號以設計一個對於感測汞離子具有高度專一性與高靈敏度的電化學生物感測方法。由於胸腺嘧啶 (thymine，T) 能夠與汞離子產生具有專一性的相互作用力，在 DNA 探針上利用七對胸腺嘧啶與汞離子做結合，設計出一個可以有效偵測汞離子的生化感測器。在汞離子存在的情況下，探針序列會因為胸腺嘧啶與汞離子的專一性作用而彎折成髮夾型。反之，若溶液中不存在汞離子，探針序列與溶液中添加的兩段 DNA 片段將會進行 HCR 反應，在電極表面產生雙股 DNA 長鏈結構。而在雙股 DNA 長鏈上的大量負電會吸引溶液中帶正電的六銨合釕電化學訊號物質。透過偵測其氧化還原訊號的改變量，進而求出樣品中汞離子含量。
第二部分實驗為發展一個結合三段放大機制之免標記螢光偵測方法，用以偵測特定的核酸序列。首先，利用第一段髮夾型之核酸序列 (HP1) 用以辨認目標 DNA 序列。當 HP1 與目標 DNA 序列產生互補，產生的 3’ 端鈍端結構便會被水解酵素 exonuclease III (Exo III) 所辨認而將互補部分的 HP1 水解。除了將原本互補的目標序列釋放並觸發第一階段循環放大反應，同時也生成第一段殘基序列 (1st residue)。接著第一段殘基序列將互補於第二段髮夾型序列 (HP2)。HP2 結構中存在兩個胞嘧啶 (cytosine，C) 對位所組成之 C-C 錯誤配對結構。可與螢光分子 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) 作用並令其嵌入 HP2 的結構中，使 ATMND 的螢光焠熄。由 1st residue與 HP2 產生之互補將使HP2 的 3’ 端鈍端被 Exo III 辨認並使HP2被水解，釋放 1st residue 回到溶液中觸發第二段循環放大反應，同時並生成第二段殘基 (2nd residue) 序列。由於HP2 之結構被水解破壞，原本嵌在 HP2 結構中的 ATMND 也因此被釋出並恢復其螢光訊號。接著，在第二階段循環反應中被釋放的 2nd residue 會回頭與未反應的 HP2 產生互補，產生可被 Exo III 所辨認的末端結構並再次水解被互補的 HP2，產生更多的2nd residue，完成第三段的循環放大反應，並釋出大量的 ATMND。透過量測 ATMND 的螢光變化量，即可反推求出目標 DNA 序列的含量進行精確定量分析。
本研究主題結合訊號放大機制，開發了具有泛用性的分析方法，並不僅限於重金屬離子之檢測與特徵序列之分析，亦能為其他領域遇到之偵測問題提供可能的解決方案。

In this study, two biosensors based on non-Watson-Crick base pairings assisted with signal amplification strategies were presented. Interactions between mismatched oligonucleotide bases and certain molecules could specifically trap these molecules, which could be used as critical recognition mechanism in biosensor development.
In the first part, mercury (II) ion, as the analyte, was trapped in the thymine mismatch in the Hg probe to form the T-Hg-T complex. In the presence of mercury (II) ion, Hg probe would fold into a hairpin structure, which prevents the further progress of the hybridization chain reaction (HCR). Otherwise, the Hg probe would initiates the HCR and form a long double strand DNA sequence with hairpin 1 and hairpin 2 in the absence of mercury (II) ion. The proceeding of HCR produced a large amount of negative charges on electrode surface, resulting in the increase of the redox signal of hexaammineruthenium (III). The redox current was decreased with the increasing of the concentration of mercury (II) ion and could be used to estimate the concentration of mercury (II) ion in the sample.
In the second part, a DNA sequence biosensor is developed. The fluorophore, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), would be embedded in the cytosine mismatch at hybridization region of hairpin 2 and its fluorescence would be quenched. Upon the target DNA sequence was recognized by the hairpin 1, exonuclease III (Exo III) would digest the blunt terminus at 3’ end of hairpin 1, produced the residue 1 and release the target sequence for another cycle. Subsequently, the second amplification cycle was initiated by residue 1. The Exo III digested the 3’-blunt end at hairpin 2 upon the hybridization with residue 1, which led to the producing of a residue 2 and the releasing of residue 1 for more cycles. Again, the residue 2 would initiate the third cycle, making hairpin 2 digested by Exo III and releasing more residue 2. Upon being digested, the hairpin 2 was not only produced residue 2 but liberated the caged ATMND. Therefore, ATMND would be released and the fluorescence was restored. The restored fluorescence is related to the amount of the target DNA sequence and could be used for the used for the quantitation.
Both biosensors are possible to apply in different fields by replacing the recognition region in probe DNA. Such a platform design with good selectivity and versatile operation at room temperature showed the potential to be applied to practical analysis.

摘要 i
Abstract iv
誌謝 vi
目錄 vii
圖目錄 xi
表目錄 xiv
縮寫表 xv
一、 緒論 1
1.1 引言 1
1.2 去氧核醣核酸 (deoxyribonucleic acid，DNA) 2
1.3 非華生-克力克鹼基配對 (non-Watson-Crick base pair) 4
二、 利用雜交連鎖反應偵測汞離子之電化學生化感測器 6
2.1 簡介 6
2.1.1 汞 (mercury，Hg) 6
2.1.2 T−Hg2+−T 鍵結作用 7
2.1.3 電化學分析 7
2.1.3.1基本原理 8
2.1.3.2 循環伏安法 (cyclic voltammetry，CV) 10
2.1.3.3 方波伏安法 (square wave voltammetry，SWV) 12
2.1.3.4 計時庫倫法 (chronocoulometry，CC) 14
2.1.3.5 電化學阻抗頻譜 (electrochemistry impedance spectroscopy，EIS) 16
2.1.4 奈米材料簡介及其應用 18
2.2 研究動機 20
2.3 實驗設計 21
2.4 實驗部分 23
2.4.1 實驗儀器 23
2.4.2 藥品 24
2.4.3 實驗流程 26
2.4.3.1 膠電泳實驗 26
2.4.3.2 汞離子偵測電極之製備 27
2.4.3.3 以電極測定目標溶液中的汞離子含量 27
2.4.3.4 計時庫倫法測定 DNA 探針表面修飾率 28
2.5 結果與討論 28
2.5.1 網版電極逐層修飾定性結果 28
2.5.2 計時庫倫法測定表面修飾率 37
2.5.3 實驗參數最佳化 41
2.5.3.1 HCR 反應時間最佳化 41
2.5.3.2 汞離子反應時間最佳化 42
2.5.4 檢量線 43
2.5.5 干擾物與真實樣品 46
2.5.5.1 選擇性測試 46
2.5.5.2 真實樣品之回收率 47
2.6 結論 48
三、利用水解酵素輔助達成多重訊號放大之免標記核酸序列螢光分析方法 50
3.1 簡介 50
3.1.1 致病基因 (pathogenicity genes) 50
3.1.2 炭疽桿菌 (Bacillus anthracis) 50
3.1.3 核酸外切酶 III (exonuclease III) 51
3.1.4 ATMND 與胞嘧啶 53
3.1.5 螢光分析方法 54
3.2 研究動機 55
3.3 實驗設計 58
3.4 實驗 59
3.4.1 實驗儀器 59
3.4.2 藥品 59
3.4.3 實驗流程 61
3.4.3.1 膠電泳實驗 61
3.3.3.2 螢光分析流程 61
3.5 結果與討論 62
3.5.1 實驗設計反應定性 62
3.5.2 實驗參數最佳化 68
3.5.2.1 緩衝溶液最佳化 68
3.5.2.2 HP2 濃度最佳化 70
3.5.3 檢量線與於血清中檢測之測試 71
3.6 結論 76
四、總結 78
五、參考資料 79

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