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論文中文名稱:合成新隱藏型長波長螢光探針用於偵測還原酵素,並搭配脫氫酵素成為生化分析之應用 [以論文名稱查詢館藏系統]
論文英文名稱:Synthesis of a New Long-Wavelength Latent Fluorimetric Indicator for Analytes Determination in the DT-Diaphorase Coupling Dehydrogenase Assay System [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:化學工程研究所
出版年度:97
中文姓名:彭逸翔
英文姓名:Yi-Xiang Peng
研究生學號:95738047
學位類別:碩士
語文別:中文
口試日期:2008-06-18
論文頁數:69
指導教授中文名:黃聲東
口試委員中文名:郭憲壽;林俊茂;吳瑞裕
中文關鍵詞:醌隱藏型螢光分子探針還原酵素三甲基鎖
英文關鍵詞:QuinoneLatent fluorophoreDT DiaphoraseTrimethyl lock.
論文中文摘要:設計合成對於還原酵素(DT Diaphorase)具有專一作用之新隱藏型長波長螢光探針BQC。新隱藏型長波長螢光探針BQC是利用還原酵素(DT Diaphorase)氧化NADH而還原Quinone,使三甲基鎖關環並且造成分子內環氨之形成,最後會釋放出螢光分子,而螢光分子釋放在生理溫度的緩衝溶液中是自發且不可逆之過程。螢光分子所釋放出來的長波長訊號(595nm)具有獨特的識別性質並且靠近紅外光之區域範圍,而螢光分子的釋放會被維他命K所抑制。對生理環境中所存在的各種不同硫化物做還原的測試,隱藏型螢光探針不會發生氧化還原反應,依然保持專一的性質。新隱藏型長波長螢光探針對於偵測還原酵素(DT Diaphorase)有很高的靈敏性,並搭配脫氫酵素(dehydrogenase)氧化分析物,造成NAD+還原成NADH成為分析系統之應用。新的螢光生化分析系統對ethanol、3-hydroxybutyrate與glucose-1-phosphate的偵測效果範圍在0.2~10 μM,在未來應用於生化檢測系統開發與新抗癌藥物設計深具價值。
論文英文摘要:We synthesized a new long-wavelength latent fluorogenic probe BQC (1) to monitor DT Diaphorase activity. The fluorogenic chemical transformation of BQC triggered by DT Diaphorase in the presence of NADH is through a series of tandem reactions, DT Diaphorase -catalyzed benzoquinone reduction, trimethyllocks cyclization and intramolecular urea formation, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the near red spectrum region with emission maxima at 595nm, and it could be competitively inhibited by menadione. The fluorescent response of BQC is insensitive to various biological thiol reductants. Furthermore, pro-fluorophore BQC is a sensitive fluorimetric indicator for analytes determination in the oxygen-insensitive DT Diaphorase-coupled dehydrogenases assay by including NAD+ which will convert to NADH by reaction in the presence of analytes. This novel oxygen-insensitive assay demonstrates a good relationship in detecting ethanol, 3-hydroxybutyrate and glucose-1-phosphate in 0.2 to 10 μM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clinical diagnostic.
論文目次:摘 要...................................................I
ABSTRACT................................................II
誌謝....................................................IV
目錄.....................................................V
圖目錄..................................................IX
第一章 前言..............................................1
第二章 文獻回顧..........................................2
2.1 生化檢測器(Biosensor)簡介.......................2
2.2 生化檢測器(Biosensor)之應用.....................2
2.2.1 電化學法........................................2
2.2.2 UV吸收光譜法....................................3
2.2.3 螢光光譜法......................................4
2.2.3.1 螢光原理........................................4
2.2.3.2 生物晶片(Chip)..................................5
2.2.3.3 螢光免疫分析法(Immunofluorescence).............6
2.2.3.4 流式細胞儀(Flow sorter cytometry)...............7
2.2.3.5 短波長螢光分子探針..............................8
2.2.3.6 長波長螢光分子探針.............................10
2.3 還原酵素(DT Diaphorase)的特性..................12
2.4 三甲基鎖(trimethyl lock).......................14
第三章 研究動機與目的...................................15
第四章 材料與方法.......................................18
4.1 實驗儀器.......................................18
4.2 實驗試藥.......................................19
4.3 實驗方法與流程.................................21
4.3.1 Synthesis of amide (8)合成方法.................21
4.3.2 Preparation of amine (9)合成方法...............22
4.3.3 Synthesis of latent fluorophore (1)合成方法....23
4.3.4 螢光光譜 (Fluorescence spectra)檢測............25
4.3.4.1 BQC之螢光隱藏性測試............................25
4.3.4.2 BQC與DT Diaphorase釋放螢光測試.................25
4.3.4.3 酵素動力學觀察.................................25
4.3.4.4 BQC隱藏螢光穩定性測試..........................26
4.3.4.5 螢光分子(3)螢光穩定性測試......................28
4.3.4.6 化學還原劑還原BQC..............................28
4.3.5 HPLC高效液相層析檢測條件.......................29
4.3.5.1 HPLC觀察BQC釋放機制............................29
4.3.5.2 HPLC觀察BQC與還原劑的作用......................30
4.3.6 新生化檢測系統檢測 .............................31
4.3.6.1 不同Ethanol的濃度檢測..........................31
4.3.6.2 不同Hydroxybutyrate的濃度檢測..................32
4.3.6.3 不同Glucose-1-phosphate的濃度檢測..............33
第五章 結果與討論.......................................35
5.1 全合成實驗步驟.................................35
5.2 螢光訊號 (Fluorescence spectra)檢測............36
5.3 BQC用於偵測還原酵素(DT Diaphorase)之活性.......37
5.4 BQC釋放機制....................................38
5.5 酵素動力學.....................................39
5.5.1. 即時觀察BQC與DT Diaphorase作用情形.............39
5.5.2. 螢光釋放速率對BQC濃度做圖......................40
5.5.3. 酵素動力學雙倒數作圖...........................41
5.6 穩定性測試.....................................43
5.6.1 使用螢光光譜儀觀察BQC與不同還原劑之測試........43
5.6.2 螢光分子(3)與不同還原劑之測試..................44
5.6.3 BQC與化學還原劑之測試..........................45
5.6.4 使用HPLC觀察BQC與不同還原劑之測試..............46
5.7 BQC對DT Diaphorase之活性平台建立...............47
5.8 新型生化檢測系統的建立.........................48
5.8.1 Ethanol的濃度檢測..............................49
5.8.2 Hydroxybutyrate的濃度檢測......................50
5.8.3 Glucose-1-phosphate的濃度檢測..................51
第六章 結論.............................................52
參考文獻................................................53
附錄....................................................57
1.1 amide (8)之IR圖譜...................................57
1.2 amide (8)之1H-NMR圖譜...............................58
1.3 amide (8)之13C-NMR圖譜..............................59
1.4 amide (8)之Mass圖譜.................................60
2.1 amine (9)之IR圖譜...................................61
2.2 amine (9)之1H-NMR圖譜...............................62
2.3 amine (9)之13C-NMR圖譜..............................63
2.4 amine (9)之Mass圖譜.................................64
3.1 Latent fluorophore (1) 之IR圖譜.....................65
3.2 Latent fluorophore (1) 1H-NMR圖譜...................66
3.3 Latent fluorophore (1)之13C-NMR圖譜.................67
3.4 Latent fluorophore (1) )之Mass圖譜..................68
4. 螢光分子(3)檢量線建立................................69
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論文全文使用權限:同意授權於2010-07-15起公開