臺灣博碩士論文加值系統

本研究中，我們利用BIAcore表面電漿共振(SPR)生物檢測器來研究人類血漿白蛋白(HSA)與藥物(ferulic acid、quercetin、chlorogenic acid、ibuprofen)之交互作用，得到其生成平衡常數(KA)、解離平衡常數(KD) 以及配位體(ligand)所能進行結合的最大數量(Rmax)值。
Ibuprofen為一解熱鎮痛劑，在體內主要有2個代謝途徑，分別為代謝為acyl glucuronide及CoA thioester。兩者皆會共價修飾內生性蛋白，而形成藥物-蛋白質合成物，導致藥效減少，而一些藥物與蛋白結合後，可能會引起副作用(肝毒性、過敏、免疫反應)。由於藥物共價修飾蛋白的重要性，故合成ibuprofen thioester及acyl glucuronide，並使用SPR檢測藥物共價修飾蛋白的過程。
另一方面，我們利用SPR相位量測的方法來即時量測HSA與藥物之交互作用，以提高SPR在生物檢測上之靈敏度。

In this study, Surface Plasmon Resonance (SPR) technology was used to measure the binding between human serum albumin and drugs (ferulic acid, quercetin, chlorogenic acid and ibuprofen). Equilibrium association rate constants (KA) and equilibrium dissociation rate constants (KD) and the maximal response in resonance unit (Rmax) were studied.
Ibuprofen is a popular analgesic drug. Its metabolites covalently modify proteins, and have been associated with some serious side effects (such as hepatotoxicity, immunocytopenias, hypersensitivity reactions) and decreasing efficacy in human body. These reactive metabolites can be acyl glucuronides or acyl-CoA thioesters. Ibuprofen thioester and acyl glucuronide were synthesized and SPR technology were employed to measure the process of drug covalently modified proteins.
In addition, for the sake of improving the sensitivity of SPR sensor, we studied the effects of SPR phase detection system to measure real time drug-protein interactions.

第一章 緒論
1.1 前言………………………………………………………….…1
1.2 細胞生物動力學理論理論分析…………………………….……4
1.2.1 細胞生物動力學理論……………………………...……...…4
1.2.2 Equilibrium Kinetics in Biacore System………………….......9
1.3自組裝單層膜(Self Assemble Monolayer, SAM)……...……..11
1.4量測藥物簡介……………………………………………...….…13
1.4.1 Ibuprofen………………………………………………....….13
1.4.2 Chlorogenic acid(綠原酸)…………………………...…….14
1.4.3 Ferulic acid(阿魏酸)……………………………………...15
1.4.4 Quercetin……………………………………………….........16
1.4.5 Acyl glucuronide……………………………………...….17
1.4.6 CoA thioester……………………………………...................19
第二章 表面電漿共振理論分析
2.1 表面電漿共振簡介………………….…………..………………21
2.2 表面電漿之傳播函數……………………………….…………..23
2.3 色散曲線之探討………………………………………………...26
2.4 激發表面電漿的方法…………………………………………...29
2.5 表面電漿多層系統的反射率及相位…………………………...30
2.5.1多層系統的反射係數……………………………………….31
2.5.2 多層系統的相位……………………………………………33
2.6 BIAcore X系統………….…………………………………….33
2.6.1 BIAcore X之光學原理-生化反應方面……………….….34
2.6.2 BIAcore X儀器之基本組成……………............................35
2.7 量測相位方法…………………………………………………...37
2.7.1外差干涉術………………………………………………….37
2.7.2 使用電光晶體調制的外差光源……………………………38
第三章 實驗步驟與架構
3.1實驗1-以BIAcore X探討藥物與蛋白質結合…………………41
3.1.1固定HSA於金表面……………………………...…………41
3.1.2 Ibuprofen thioester合成…………………………………...43
3.1.3 Allyl glucuronate合成…………………………………….44
3.2實驗2- SPR系統之即時量測……………………………………47
3.2.1 實驗架構…………………………………………………….47
第四章 結果與討論
4.1 實驗1-以BIAcore X探討藥物與蛋白質結合………………...53
4.1.1 典型感應圖( s e n s o r g r a m )之說明……………………53
4.1.2 實驗1-結果與討論…………………………………………54
4.2 實驗2- SPR系統之即時量測……………………………………64
4.2.1 實驗2-結果與討論…………………………………………64
第五章 結論…………………………………………………...……..72
參考文獻…………………………………………………………………73

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