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研究生:藍允君
研究生(外文):Yun-Chun Lan
論文名稱:表面電漿共振系統應用於Methyleneviolet與核酸交互作用之研究
論文名稱(外文):Study on the interaction between Methylene violet and DNA using a surface plasmon resonance based biosensor
指導教授:蔡文琦蔡文琦引用關係
指導教授(外文):Wen-Chi Tsai
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:96
語文別:中文
論文頁數:88
中文關鍵詞:生物感測器表面電漿共振生物感測器小牛胸線雙股核酸甲基紫道諾黴素交互作用藥物
外文關鍵詞:biosensorsurface plasmon resonancecalf thymus DNAmethylene violetdaunomycininteractiondrug
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在基因定序技術成熟的今日,我們可以輕易的取得動、植物,甚至是微生物的核酸序列或結構,這有利於應用在治療疾病的投藥機制上。而有別於以往儀器的缺點,目前以親合性生物感測器最能及時且迅速地取得生化分子之間相互作用之分析資訊。
  表面電漿共振 (surface plasmon resonance,SPR) 生物感測器為測量生物分子間在固體與液體或固體與氣體介面發生交互作用時,所造成介面之介電常數的微小改變,可在無須對生物分子做任何的標記下,即時地分析生物分子間交互作用的變化。
  本研究以探討methylene violet (MV) 與小牛胸腺雙股DNA (calf thymus DNA) 的互動關係為模式系統,利用表面電漿共振生物感測器偵測Methylene Violet與核酸之間的交互作用以了解雙股DNA與另一生化小分子之間的相關聯性,且分析其動力學參數來了解其兩者間的親合關係。最後,再以市售之抗癌藥物:道諾黴素(Daunorubicin或Daunomycin)做實驗,算出其與DNA之動力常數,並與文獻中的實驗結果做比較。利用親合性感測器分析探討小分子與DNA交互作用,藉此建立藥物篩選的平台,並根據製藥的原理及流程,以期日後能藉此使開發藥物的過程快速且提高藥物的治療效能。
  最後結果顯示DNA與methylene violet的KA=150 1/M。DNA與daunomycin的KA=780 1/M,與文獻中的值KA=81000 1/M,低了約100倍左右,可能原因是儀器不同所產生的結果誤差,或結合在金片上的DNA量不夠飽和。而methylene violet 的再生性實驗結果顯示:0.001 M的NaOH有最好的再生效果,可將methylene violet與DNA間的鍵結完全打斷,為實驗中最好的再生試劑。
The efficient and rapid detection on molecular interactions between small molecules and target DNA is an important step in the discovery of drugs intended for use in therapy. Traditional analysis of the binding between DNA and DNA binding molecules were performed by footprinting and filter binding assay. These assays did not allow a real time study of association and dissociation with DNA.
In this study, an optical biosensor, based on surface plasmon resonance was used as a new analytical device to investigate the interaction between bio-molecules. The device provides a fast and label free detection platform.
To examine the interaction between small molecule and DNA, calf thymus DNA and methylene violet was used as a model system. The immobilization of DNA onto gold surface was optimized. Covalent binding via ZrO2 showed the best results. Binding affinity constant can be estimated by dynamics evaluation. The association constant (KA) of methylene violet binding to calf thymus DNA was 150 1/M. The KA of daunomycin binding to ctDNA was 780 1/M. Regeneration process of methylene violet was advantageous in terms of convenience and cost saving. HCl and NaOH was used to break up the binding between DNA and methylene violet. The optimum result was obtained with 0.001 M NaOH as the regeneration solution.
目錄
目錄--------------------------------------------------------------------------ⅰ
圖目錄-----------------------------------------------------------------------ⅳ
表目錄-----------------------------------------------------------------------ⅶ
中文摘要--------------------------------------------------------------------ⅷ
英文摘要--------------------------------------------------------------------ⅹ
壹、緒論-----------------------------------------------------------------1~33
1-1前言-------------------------------------------------------------------1
1-2前人研究回顧--------------------------------------------------------3
1-3生物感測器之簡介--------------------------------------------------8
1-4表面電漿共振生物感測器(Surface Plasmon
Resonance ; SPR)之原理------------------------------------10
1-5 Methylene violet(MV)之簡介---------------------------------19
1-6 Daunomycin之介紹-----------------------------------------------21
1-7 DNA於晶片上之固定法介紹-------------------------------------23
1-8親和性分析簡介----------------------------------------------------32
貳、 研究材料與方法----------------------------------------------34~47
2-1實驗試劑------------------------------------------------------------34
2-2實驗儀器------------------------------------------------------------36
2-3 SPR儀器基本介紹------------------------------------------------37
2-4實驗流程------------------------------------------------------------42
2-5新金片清洗法----------------------------------------------------------43
2-6 Calf Thymus DNA濃度的確定-------------------------------------43
2-7實驗步驟----------------------------------------------------------------44
参、實驗結果與討論-----------------------------------------------48~80
3-1實驗的主要流程---------------------------------------------------48
3-2洗含有SAM的金片-----------------------------------------------50
3-3 Fluorescence Spectrophotometer實驗結果------------------57
3-4反應槽溫度與SPR共振角度變化之關係----------------------60
3-5以通電法直接固定DNA於金片表面----------------------------61
3-6比較cystamine與ZrO2固定DNA之角度變化量-----------63
3-7比較不同濃度MV的SPR角度變化量並求得與DNA間之動力
學常數-------------------------------------------------------------------70
3-8 Methylene violet再生性實驗----------------------------------------72
 3-9金片再生性實驗---------------------------------------------------------76
3-10比較不同濃度daunomycin的SPR角度變化量並求得與DNA
間的動力學常數-------------------------------------------------------79
肆、結論與未來展望------------------------------------------------81~82
4-1結論-----------------------------------------------------------------------81
4-2未來展望------------------------------------------------------------------82
伍、參考文獻---------------------------------------------------------83~88
林瑤冷,陳子江,蘇文寬,王建鈞。2005。丙酮及異丙酮蒸氣對聚苯胺薄膜激發表面電漿共振特性影響之研究。中正嶺學報,第三十三卷,第二期:1-11。
林義傑。2003。壓電晶體免疫感測器應用於甲型胎兒蛋白之檢測。中國文化大學生物科技所碩士論文。
侯明宏,甘魯生。1999。生物分子交互作用分析系統。科儀新知,第二十卷,第六期:88-97。
陳子江,蘇文寬,林瑤冷。2004。奈米銀粒子溶液激發表面電漿波特性之研究。中正嶺學報,第三十三卷,第一期:1-10。
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