臺灣博碩士論文加值系統

近年來由於生物感測技術的進步，藉由表面電漿共振技術可以即時的檢視分子間的交互作用，提高了生物分子交互作用分析(Biomolecular interaction analysis; BIA)的發展；而DNA與小分子物質間的交互作用，為篩選具有潛力之藥物的重要步驟之ㄧ。本研究的目的是以SPR 技術探討固定於SPR金片上之經Biotin 標記的DNA分子與小分子物質間之交互作用。本研究可以提供一個快速篩選具有生理活性小分子物質的平台。
本研究先將感測金片以食人魚溶液(Piranha solution)清洗金片上雜質。再使用16-mercaptohexadecanoic acid (16-MHDA)與6-mercapto-1
-hexanol(6-MCH)，兩種尾端分別帶有不同官能基之硫醇(Thiol)，以1：0、1：3、1：10與1：20四種不同比例之混合硫醇，於金片表面形成自組單層膜(self-assembled monolayer；SAM)，找出對金片表面具最佳修飾性的SAM。結果顯示，比例1：10之混合式自組單層膜，可得到最高的角度變化量。接著比較Avidin與Streptavidin於不同離子強度、pH值之環境下，對於Biotin-NF-κB固定量之影響。結果顯示，兩者在I=0.16、pH=5之環境下皆具有最好的Biotin-NF-κB固定量。隨後分別確定了Avidin、Biotin-NF-κB與Complementary NF-κB之最適濃度分別為250 μg/ml、10μg/ml與5μg/ml。在專一性測試中，使用Non-biotinylated DNA與Non-complementary DNA進行非專一性吸附測試。結果顯示，Biotin-NF-κB的部分所形成的角度變化量確為Avidin-Biotin之專一性吸附所造成；雜交反應時所造成之角度變化量確為Biotin-NF-κB與Complementary NF-κB之間的專一性雜交反應所造成。而再生性測試中，0.01 M HCl可達到不傷害金片表面其他的親和性分子，並且可以把Complementary NF-κB於雙股螺旋結構中分離，以0.01 M HCl做第一次再生可還原94.4 %的原偵測力，第二次再生可達到91.6 %。最後以Sanguinarine進行 DNA 與小分子間動力學分析，求得Sanguinarine與NF-κB DNA的親和常數 KA= 1.25 × 105 M-1。

The recent development of biosensor technologies for biomolecular interaction analysis enables monitoring of a variety of molecular reactions in real-time by surface Plasmon resonance (SPR). In this study, we aim to demonstrate the molecular interactions between small molecules and biotinylated target DNA probe immobilized on sensor chips is detectable by SPR technology. This work demonstrates the possibility of applying the biosensor approach in the search for bioactive constituents from small molecules.
The experiments start with cleansing the gold surface with Piranha solution. After the surface being cleaned, self-assembled monolayer (SAM) is adsorbed onto the surface. SAM with COOH functional group 16-mercaptohexadecanoic (16-MHDA) was mixed with SAM possessing OH functional group 6-mercapto-1-hexanol (6-MCH) in different ratios such as 1:0, 1:3, 1:10 and 1:20 to find the best ratio which can cause the maximum angle shift. The result show that the best mixed SAM for Avidin and double helix DNA immobilization is 16-MHDA/6-MCH in 1:10 ratio. Afterwards, compare with the angle shift caused by Biotin-NF-κB immobilization onto Avidin/Streptavidin modified surface in different pH and ion strength. The result show that both of Biotin-NF-κB immobilization on avidin/streptavidin cause the maximum angle shift in pH=5, 0.16 of the ion strength. As the follow experiments can decide that the best concentration of avidin, Biotin-NF-κB and Complementary NF-κB was 250μg/ml, 10μg/ml and 5μg/ml. In this study used Non-biotinylated DNA and Non-complementary DNA to decide the angle shift was caused by the specific interaction. The result prove that the angle shift was caused by the specific interaction between avidin-biotin and DNA hybridization. Using 0.01 M HCl as the regeneration solution for the first time leads to 94.4 % of the original detection ability; the second time result in 91.6 % of the original detection ability. The association constant (KA) of Sanguinarine binding to NF-κB DNA was 1.25 × 105 M-1.

目錄 i
表目錄 iv
圖目錄 v
縮寫表 vii
摘要 viii
Abstract x
第一章 緒論 - 1 -
1-1 研究動機與目的 - 1 -
1-2 小分子物質與DNA之作用機制 - 2 -
1-2-1 小分子物質與DNA 交互作用之前人研究 - 3 -
1-3 生物感測器 - 5 -
1-3-1 生物感測器之類型 - 7 -
1-3-2 表面電漿共振技術簡介 - 11 -
1-3-2-1 表面電漿共振技術原理 - 14 -
1-4 金片表面修飾 - 19 -
1-4-1 自組單層膜 - 21 -
1-4-2 生物親和性固定化 - 25 -
1-5 動力學參數分析 - 28 -
第二章 材料與方法 - 30 -
2-1 藥品試劑 - 30 -
2-2 儀器設備 - 32 -
2-3 實驗流程 - 35 -
2-4 SPR操作流程 - 36 -
2-5 金片前處理 - 37 -
2-6 金片表面修飾 - 37 -
2-6-1最佳SAMs比例探討 - 38 -
2-7 生物親和性分子固定 - 38 -
2-7-1 實驗用DNA序列 - 39 -
2-7-2 最佳固定化條件探討 - 40 -
2-8 DNA雜交反應 - 40 -
2-9 專一性試驗 - 40 -
2-10 再生性試驗 - 41 -
2-11 小分子動力學分析 - 41 -
第三章 結果與討論 - 43 -
3-1 最佳SAMs比例探討 - 43 -
3-2 生物親和性分子固定 - 47 -
3-2-1 Avidin/Streptavidin之比較 - 47 -
3-3 DNA雜交反應 - 55 -
3-4 專一性測試 - 58 -
3-5 再生性測試 - 61 -
3-6 DNA與小分子之動力學分析 - 63 -
第四章 結論與未來展望 - 66 -
4-1 結論 - 66 -
4-2 未來展望 - 68 -
參考文獻 - 69 -

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