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論文中文名稱:利用酵素法建立檢測輔酶A平台應用在乙醯轉化酶的檢測分析 [以論文名稱查詢館藏系統]
論文英文名稱:New Coenzyme A Fluorescence Detecting Platform : Applicable to
Monitoring Acetyl Transferases [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:101
出版年度:102
中文姓名:王茗卉
英文姓名:Ming-Hui Wang
研究生學號:100738051
學位類別:碩士
語文別:中文
口試日期:2013-06-20
論文頁數:63
指導教授中文名:黃聲東
指導教授英文名:Sheng-Tung Huang
口試委員中文名:郭憲壽;林俊茂
口試委員英文名:Hsien-Shou Kuo;Chun-Mao Lin
中文關鍵詞:輔酶A乙醯基轉移酶肉鹼氯黴素
英文關鍵詞:Coenzyme AAcetyl transferaseCarnitineChloramphenicol
論文中文摘要:硫醇是生物體內扮演著維持細胞的正常氧化還原。在此之前,實驗室開發過香豆素衍生物的硫醇探針BCC,本篇論文研究即想將BCC 硫醇探針應用在檢測人體循環系統產出的輔酶A (Coenzyme A, CoA)上。我們期望用BCC 測定輔酶A和建立一個系統監控乙醯基轉移酶 (Acetyl transferase)。我們證明了利用長波長螢光探針BCC 釋放螢光訊號來檢測輔酶A 且這個訊號是有特定性的。此外,我們亦利用此酵素法來證明CrAT (carnitine acetyltransferase) 和CAT(chloramphenicol acetyltransferase) 的酵素動力學。BCC 的靈敏性也讓我們成功地運用範圍在微莫爾量下檢測到肉鹼,此法也將可以被廣泛用於臨床診斷的生理分析物測量上。雖然,在氯黴素使用方面無法突破目前最靈敏的偵測極限,但我們提供了一個在食品檢測上的便利方法。
論文英文摘要:The thiols are part of endogenous antioxidant system that maintains the cells’ normal redox potential and prominent biomarkers. Previously our group had prepared and demonstrated a new long-wavelength latent florescent probe, BCC, to detect Cys and GSH. We expect to extend the application of BCC for assaying CoA and establishing platform applicable to monitor acetyl transferases. We have successful implemented the first long-wavelength latent fluorogenic substrate BCC as the off-on flourimetric indicators for detecting CoA; the fluorescence signal generated by this assay is specific. Furthermore, BCC, acetyl-CoA, and CAT or CrAT with the presence of corresponding substrate are easy assay platform for real time monitoring either CAT or CrAT enzymatic activities. In-retrospect, BCC, acetyl-CoA, and CrAT is a sensitive fluorimetric indicator for quantitatively measuring carnitine in the nanomolar ranges. This CoA platform for carnitine is expected to be applicable to measure broad ranges of important physiological analyte for the clinical diagnostic.
論文目次:目錄

英文摘要…………………………………………………………………………………………………………………………………………i
中文摘要………………………………………………………………………………………………………………………………………ii
致謝……………………………………………………………………………………………………………………………………………iii
目錄………………………………………………………………………………………………………………………………………………iv
表目錄…………………………………………………………………………………………………………………………………………vii
圖目錄………………………………………………………………………………………………………………………………………viii
附錄目錄………………………………………………………………………………………………………………………………………ix
前言…………………………………………………………………………………………………………………………………………………1
第一章 介紹……………………………………………………………………………………………………………………………2
1.1 硫醇Thiol………………………………………………………………………………………………………………………2
1.2 肉鹼ʟ-Carnitine………………………………………………………………………………………………………5
1.3 氯黴素Chloramphenicol…………………………………………………………………………………………7
1.4 研究動機與目的………………………………………………………………………………………………………………8
第二章 實驗方法與設備………………………………………………………………………………………………………9
2.1 實驗儀器……………………………………………………………………………………………………………………………9
2.2 實驗試藥…………………………………………………………………………………………………………………………10
2.3 合成方法…………………………………………………………………………………………………………………………12
2.3.1 Compound 1的合成………………………………………………………………………………………………12
2.3.2 Compound 2的合成………………………………………………………………………………………………12
2.3.3 Compound 3的合成………………………………………………………………………………………………13
2.3.4 Compound 4的合成………………………………………………………………………………………………14
2.3.5 Compound 5的合成………………………………………………………………………………………………15
2.3.6 BCC的合成…………………………………………………………………………………………………………………16
2.3.7 螢光劑Coumarin step1的合成………………………………………………………………………16
2.3.8 螢光劑Coumarin step2的合成………………………………………………………………………17
2.3.9 螢光劑Coumarin的合成………………………………………………………………………………………17
2.4 實驗檢測…………………………………………………………………………………………………………………………18
2.4.1 BCC對acetyl translation反應條件實驗方法………………………………………18
2.4.1.1 不同pH之buffer測試……………………………………………………………………………………18
2.4.1.2 Acetyl CoA濃度測試……………………………………………………………………………………19
2.4.1.3 DMSO在反應中體積百分比測試………………………………………………………………………20
2.4.1.4 BCC濃度測試………………………………………………………………………………………………………21
2.4.2 檢測CoA檢量線之方式……………………………………………………………………………………………22
2.4.3 檢測肉鹼檢量線之方式……………………………………………………………………………………………23
2.4.4 檢測氯黴素檢量線之方式………………………………………………………………………………………24
2.4.5 Carnitine acetyltransferase (CrAT) –酵素動力學…………………25
2.4.6 Chloramphenicol acetyltransferase (CAT) –酵素動力學…26
2.4.7 BCC檢測食品內肉鹼含量………………………………………………………………………………………27
2.4.7.1 全脂牛奶………………………………………………………………………………………………………………27
2.4.7.2 全脂奶粉………………………………………………………………………………………………………………28
2.4.8 ABD-F檢測食品內肉鹼含量…………………………………………………………………………………29
2.4.8.1 全脂牛奶………………………………………………………………………………………………………………29
2.4.8.2 全脂奶粉………………………………………………………………………………………………………………30
2.4.9 BCC檢測食品內氯黴素含量…………………………………………………………………………………31
2.4.9.1 全脂牛奶………………………………………………………………………………………………………………31
2.4.9.2 全指奶粉Whole milk powder…………………………………………………………………32
2.4.9.3 蜂蜜………………………………………………………………………………………………………………………33
第三章 結果與討論……………………………………………………………………………………………………………34
3.1 分子鑑定…………………………………………………………………………………………………………………………34
3.2 最佳條件測試…………………………………………………………………………………………………………………35
3.2.1 BCC對CoA和acetyl translation測試……………………………………………………35
3.2.2 不同pH之buffer測試……………………………………………………………………………………………37
3.2.3 Acetyl CoA濃度測試…………………………………………………………………………………………38
3.2.4 DMSO在反應中體積百分比測試……………………………………………………………………………39
3.2.5 BCC濃度測試……………………………………………………………………………………………………………40
3.3 檢測極限LOD…………………………………………………………………………………………………………………41
3.3.1 CoA的檢測極限…………………………………………………………………………………………………………41
3.3.2 肉鹼的檢測極限…………………………………………………………………………………………………………42
3.3.3 氯黴素的檢測極限……………………………………………………………………………………………………43
3.4 酵素動力學分析………………………………………………………………………………………………………………44
3.4.1 Carnitine acetyltransferase (CrAT)……………………………………………44
3.4.2 Chloramphenicol acetyltransferase (CAT)………………………………46
3.5 食品檢測…………………………………………………………………………………………………………………………49
第四章 結論………………………………………………………………………………………………………………………………51
參考文獻………………………………………………………………………………………………………………………………………52
附錄………………………………………………………………………………………………………………………………………………56
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