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論文中文名稱:新型雙酵素系統生化檢測器:偵測水楊酸酶新型隱藏式螢光探針合成 [以論文名稱查詢館藏系統]
論文英文名稱:A novel bienzyme-platform biosensor:the synthesis of new latent fluorophore for salicylate hydroxylase. [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
畢業學年度:97
出版年度:98
中文姓名:鄧竣任
英文姓名:Chun-Jen Teng
研究生學號:96738047
學位類別:碩士
語文別:中文
口試日期:2009-06-22
論文頁數:70
指導教授中文名:黃聲東
口試委員中文名:郭憲壽;林俊茂
中文關鍵詞:水楊酸酶隱藏型式螢光探針雙酵素系統
英文關鍵詞:Salicylate hydroxylaseLatent fluorophoreBienzyme system
論文中文摘要:隱藏式螢光探針(Latent fluorophore)主要運用化學反應的方式將螢光分子作改質,使螢光分子的螢光消失或螢光的強度下降,並能藉由化學、催化反應將螢光分子釋放;設計三種不同程序方式合成對於羥化酵素水楊酸酶( Salicylate hydroxylase)具有專一作用之新型隱藏型式螢光探針SF。新型隱藏式螢光探針螢光顯色主要運用水楊酸酶( Salicylate hydroxylase)及NADH在有氧的情況下,使得水楊酸(Salicylate acid)進行去碳酸基反應(Decarboxylation)和羥化反應(Hydroxylation reaction)得到Catechol,再經由quinone-methide-rearrangement reaction釋放螢光分子。新隱藏式螢光探針對於偵測水楊酸酶(Salicylate hydroxylase)有很高的靈敏性,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訊號(597nm)具有獨特的識別性質。本實驗結合雙酵素系統搭配脫氫酵素(dehydrogenase)氧化分析物,造成NAD+還原成NADH應用於偵測3-hydroxybutyrate(0.2~2 μM),具有線性關係,其未來應用於生化檢測系統開發深具價值。
論文英文摘要:We synthesized a new latent fluorogenic probe SF to detect salicylate hydroxylase. Salicylate hydroxylase catalyzes a stoichiometric conversion of salicylate acid to catechol in the presence of NADH and oxygen. The fluorogenic chemical transformation of SF triggered by SHL is through a tandem reaction, decarboxylation, hydroxylation, and quinine-methide-rearrangement reaction, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the spectrum region with emission maxima at 597nm. Furthermore, we established a bienzyme system composed a dehydrogenase (3-hydroxybutyrate dehydrogenase) and Salicylate hydroxylase (SHL) to detect 3-hydroxybutyrate. The novel fluorimetric indicator SF demonstrated a good linear relationship in detecting 3 –hydroxybutyrate in 0.2 to 2.0 µM range, which presents to the applicability for the construction of biosensors in the future.
論文目次:中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 viii
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)之發展 2
2.2.1 電化學上的應用 2
2.2.2 光譜法上的應用 3
2.2.2.1 吸收光譜法上的應用 3
2.2.2.2 螢光光譜法上的應用 4
2.2.2.2.1 螢光原理 4
2.2.2.2.2 螢光感測分析技術 5
2.2.2.2.3隱藏式螢光分子探針用於偵測DT-Diaphorase 6
2.2.2.2.4隱藏式螢光分子探針用於偵測penicillin G acylase 7
2.3 Salicylate hydroxylase(SHL) 8
2.4 Salicylate hydroxylase的應用 9
2.5 The quinone-methide-rearrangment-elimination 10
2.6螢光分子 11
第三章 研究動機與目的 13
3.1 隱藏式螢光分子 14
3.2 釋放機制 14
3.2.1目標隱藏式螢光分子SQC的釋放機制 14
3.2.2目標隱藏式螢光分子SNBD及SF的釋放機制 15
3.3 前驅物 17
第四章 合成 18
4.1 實驗儀器 18
4.2 實驗藥品 19
4.3 實驗方法 20
4.3.1合成化合物6 20
4.3.2合成化合物9 21
4.3.3合成化合物11 22
4.3.4合成化合物13 22
4.3.5合成化合物14 23
4.3.6合成化合物15 23
4.3.7合成化合物18 24
4.3.8合成化合物19 24
4.3.9合成化合物20 25
4.4 螢光光譜 (Fluorescence spectra)檢測 26
4.4.1 SF 之螢光隱藏性測試 26
4.4.2 SF 與Salicylate hydroxylase 釋放螢光測試 26
4.4.3 Salicylate hydroxylase動力學觀察 26
4.4.4 新生化檢測系統檢測 27
4.4.4.1不同Hydroxybutyrate 的濃度檢測 27
第五章 結果與討論 30
5.1 目標產物L1 30
5.1.1 Retrosynthesis of L1 30
5.1.2 步驟A合成L1結果 31
5.1.3 步驟B合成L1結果 33
5.2 目標產物L2 35
5.2.1 步驟A合成L2之結果 36
5.2.1.1 化合物L2.2合成結果 36
5.2.1.2 化合物26合成L2之結果 38
5.2.2 步驟B合成L2之結果 40
5.3 目標產物L3 42
5.3.1 步驟A合成隱藏式螢光分子SF之結果 43
5.3.2 步驟B合成隱藏式螢光分子SF之結果 43
5.3.3 新型隱藏式螢光分子SF全合成流程 44
5.4 目標產物合成總結 45
5.5 螢光訊號 (Fluorescence spectra)檢測 46
5.6 SF用於偵測羥化酵素Salicylate hydroxylase(SHL) 47
5.7即時觀察SF與Salicylate hydroxylase作用情形 48
5.8新型生化檢測系統的建立 49
5.9 Hydroxybutyrate 的濃度檢測 50
第六章 結論 51
參考文獻 52
附錄
圖1 化合物6之1H-NMR 圖譜 55
圖2 化合物11 1H-NMR 圖譜 56
圖3 化合物13 1H-NMR 圖譜 57
圖4 化合物13 13C-NMR 圖譜 58
圖5 化合物6之雙聚物 1H-NMR 圖譜 59
圖6 螢光劑NBD-NH2之雙聚物 1H-NMR 圖譜 60
圖7 化合物14 1H-NMR 圖譜 61
圖8 化合物15 1H-NMR 圖譜 62
圖9 化合物19 IR 圖譜 63
圖10 化合物19 1H-NMR 圖譜 64
圖11 化合物19 Mass EI 圖譜 65
圖12 化合物20 IR 圖譜 66
圖13 化合物20 1H-NMR 圖譜 67
圖14 化合物20 Mass ESI- 圖譜 68
圖15 化合物19 DTA 圖譜 69
圖16 化合物20 DTA 圖譜 70
表目錄
表1 化合物9合成至目標產物L1反應情形與結果 32
表2 化合物13合成隱藏式螢光劑濃度情況及反應結果 37
表3 Triphosgene與化合物6之濃度情形與反應結果 39
表4 Triphosgene與螢光劑NBD-NH2濃度情況...................................... 41
圖目錄
圖2-1 Clark electrode 3
圖2-2 結合bienzyme system做另一基質(substrate)的檢測.......... 3
圖2-3 Ellman`s reagent ................................................ 4
圖2-4 Jablonski模型......................................... 5
圖2-5 醣類的螢光感測器示意圖......................... 6
圖2-6 隱藏式螢光分子探針偵測DT-Diaphorase....................................... 7
圖2-7 隱藏式螢光分子探針偵測penicillin G acylase................................ 8
圖2-8 Hydroxylation reaction......................................... 8
圖2-9 電化學雙酵素系統(bienzyme system) 檢測示意圖................... 9
圖2-10 電化學三酵素系統檢測示意圖......................................... 9
圖2-11 標靶藥物釋放機制......................................... 10
圖2-12 隱藏式螢光劑BCC釋放機制......................................... 10
圖2-13 Latent fluorophore螢光釋放示意圖......................................... 11
圖2-14 coumarin衍生物之螢光劑......................................... 11
圖2-15 螢光分子NBD-NH2......................................... 12
圖3-1 隱藏式螢光探針偵測SHL酵素示意圖......................................... 14
圖5-1 目標產物......................................... 26
圖5-2 目標隱藏式螢光分子SQC螢光釋放機制...... 27
圖5-3 目標隱藏式螢光分子SNBD螢光釋放機制...... 28
圖5-4 目標隱藏式螢光分子SF螢光釋放機制........ 28
圖5-5 前驅物分子......................................... 29
圖4-1 化合物6合成......................................... 16
圖4-2 化合物18合成......................................... 20
圖5-6 Retrosynthesis目標產物L1......................................... 30
圖5-7 以步驟A合成目標產物L1......................................... 31
圖5-8 以步驟B合成目標產物L1預測反應流程................................... 34
圖5-9 Retrosynthesis目標產物L2......................................... 35
圖5-10 起始物之離去基......................................... 36
圖5-11 螢光劑接離去基......................................... 40
圖5-12 以步驟B合成目標物L2......................................... 41
圖5-13 Retrosynthesis目標物L3......................................... 42
圖5-14 新型隱藏式螢光分子探針SF 的流程圖...................................... 44
圖5-15 不同型式目標產物....................................... 45
圖5-16 螢光分子隱藏效果......................................... 46
圖5-17 SF用於偵測羥化酵素Salicylate hydroxylase(SHL)....................... 47
圖5-18 即時觀察SF與Salicylate hydroxylase作用情形...................... 48
圖5-19 雙酵素(Bienzyme)檢測系統......................................... 49
圖5-20 Hydroxybutyrate 的濃度檢測......................................... 50
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