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研究生:王子寧
研究生(外文):Zih-Ning Wang
論文名稱:寡核酸鏈探針固定化於金箔載體與檢測分析之研究
論文名稱(外文):Study of the Immobilization and Detection of Oligonucleotides on Gold
指導教授:靳宗玫
指導教授(外文):Tsung-Mei Chin
學位類別:碩士
校院名稱:中國文化大學
系所名稱:應用化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:90
中文關鍵詞:寡核酸鏈探針硫金鍵石英晶體振盪天平
外文關鍵詞:QCMoligonucleotide probe
相關次數:
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近年來運用微小化、固相載體的方式進行生物分子的分析與檢測,其相關研究日益受到重視。本論文研究主要目標是:發展廣用固定化寡核酸鏈探針策略。現今固相感測器發展,增加有效固定化探針率是感測器製備工作上的一大挑戰。我們的策略是發展廣用探針載體,提供功能性探針附著於固相載體上的一個平台。選用序列組成單純、化學性質穩定之寡核酸鏈固定在載體上作為廣用探針,探針的化學固定化方法及操作過程將會比較容易掌握。本論文採用靈敏度高的石英晶體振盪天平作為偵測廣用寡核酸鏈探針於金箔表面的化學性修飾情形,以及探針與待測物之檢測反應。
實驗設計係以覆蓋金箔之石英晶體作為固相載體,已成功地找出固定化之化學操作程序,透過硫金鍵將寡核酸鏈探針植栽在金箔載體上,並進行功能性探針導入載體,及其生化反應檢測。
於本論文中亦探討二種清洗金箔載體的方法:SPM(sulfate peroxide mixture)法與APM(ammonium peroxide mixture)法。由實驗結果顯示SPM法清洗後之金箔載體穩定性優於APM法。另依據實驗結果顯示,於生化樣品檢測過程,僅需微量的蛋白質Av-Ap(低於2.5 × 10-8 g/mL)即可檢測出攜有Biotin之廣用寡核酸鏈探針,可以大量節省經濟成本。經與呈色反應偵測對照比較,石英晶體振盪天平的偵測靈敏度高於一般生化呈色反應。
The use of miniaturized solid-phase surfaces for the biomolecule detection and analysis has become increasingly more attractive. The aim of this thesis is to develop a strategy of an immobilized oligonucleotide probe as a platform. The architecture of immobilized molecular layer is an important research area in the field of biosensing. The challenge connecting probes on the solid surface is to reduce as many as possible the side reactions during the procedures of chemical immobilization. Our approach is to anchor on the solid surface a simple and stable oligonucleotide which provides a platform for the attachment and molecular recognition of versatile probes. In this study, we selected the highly sensitive equipment, quartz crystal microbalance (QCM), to detect the immobilization of oligonucleotides and the target-probe hybridization.
We have developed a simple, convenient and reliable procedure to anchor oligonucleotide probes on a gold-coated quartz crystal through formation of the S-Au bond, and then a complementary DNA fragment carrying a functional group successfully hybridized with the immobilized probe, and performed the desired bioanalysis.
Two cleaning methods, SPM (sulfate peroxide mixture) and APM (ammonium peroxide mixture), was compared. The SPM method gave a better result in term of the reproducibility. According to the experimental results, only a little amount of the protein Av-Ap (lower than 2.5x10-8 g/mL) was needed to detect the biotin connected to an oligonucleotide probe. In addition, the QCM technology was more sensitive than the colormetric reaction.
目錄
==============================================
表目錄------------------------------------------------------------------V
圖目錄----------------------------------------------------------------VII
中文摘要--------------------------------------------------------------IX
英文摘要--------------------------------------------------------------XI
第一章緒論-------------------------------------------------------------1
1-1 前言----------------------------------------------------------------1
1-2 生物感測器的簡介----------------------------------------------1
1-2-1 生物感測器的發展--------------------------------------------2
1-2-2 生物感測器的種類--------------------------------------------4
1-2-2-1 固定化的生物感測元件與待測物的結合方式--------4
1-2-2-2 固定化的生物感測元件分類-----------------------------6
1-2-2-3 訊號換能器分類--------------------------------------------6
1-3 壓電晶體免疫感測器------------------------------------------12




1-3-1 壓電效應------------------------------------------------------12
1-3-2 壓電材料------------------------------------------------------13
1-3-3 偵測原理------------------------------------------------------15
1-3-4 壓電晶體的表面修飾---------------------------------------17
1-4 研究動機---------------------------------------------------------19
第二章 材料與方法-----------------------------------------------20
2-1 實驗試劑---------------------------------------------------------20
2-2 儀器設備---------------------------------------------------------21
2-3 實驗內容---------------------------------------------------------24
2-3-1 配製廣用寡核酸鏈探針溶液------------------------------24
2-3-2金箔載體的表面清洗-----------------------------------------25
2-3-2-1 SPM法-------------------------------------------------------26
2-3-2-2 APM法-------------------------------------------------------27
2-3-3 測試組廣用寡核酸鏈探針實驗---------------------------28
2-3-4 試驗組廣用寡核酸鏈探針實驗---------------------------31




第三章 結果與討論-----------------------------------------------33
3-1 廣用寡核酸鏈探針設計概念---------------------------------33
3-2 金箔載體的表面清洗效果------------------------------------37
3-2-1 SPM法----------------------------------------------------------37
3-2-2 APM法---------------------------------------------------------37
3-2-3 SPM法與APM法於金箔載體上的反應性比較--------41
3-2-4 SPM法與APM法於金箔載體反應時間比較-------------42
3-3 廣用寡核酸鏈探針設計---------------------------------------43
3-4 廣用寡核酸鏈探針檢測方式---------------------------------43
3-4-1 增加待測物分子量------------------------------------------46
3-4-2 呈色反應------------------------------------------------------46
3-5廣用寡核酸鏈探針的實驗設計-------------------------------48
3-6廣用寡核酸鏈探針的測試組實驗與結果討論-------------54
3-6-1 雙股探針固定化及其與蛋白質之反應------------------54
3-6-2 呈色反應實驗------------------------------------------------61




3-7廣用寡核酸鏈探針的試驗組實驗與結果討論-------------62
3-7-1 單股探針固定化、與互補序列雜合及其與
蛋白質之反應-------------------------------------------------62
第四章 結論----------------------------------------------------------71
結論--------------------------------------------------------------------71
參考文獻--------------------------------------------------------------73
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