合成電化學探針並應用在代謝物偵測｜國立清華大學博碩士論文庫

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研究生：	陳泉佑 Chen, Chuan-Yu
論文名稱：	合成電化學探針並應用在代謝物偵測 Synthesis and Application of Electrochemical Probe for the Metabolites Detection
指導教授：	陳貴通 Tan, Kui-Thong
口試委員:	洪嘉呈 Horng, Jia-Cherng 許馨云 Hsu, Hsin-Yun 陳貴通 Tan, Kui-Thong
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	122
中文關鍵詞：	代謝物、生物感測器、電化學、磺胺類藥物、二茂鐵、探針、循環伏安法、方波伏安法
外文關鍵詞：	metabolite, biosensor, electrochemistry, sulfa drug, ferrocene, probe, Cyclic Voltammetry, Square-wave Voltammetry
相關次數：	點閱：2 下載：0
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代謝物是任何在代謝過程中的反應物、中間體或是產物。目前已知的代謝物在生物體中有約有7900種，由於代謝物涉及生長、發育與生殖或能量產生及移轉，亦或是在生物上具有重要生物學功能，因此對代謝物的濃度變化進行分析，有助於了解生物的生理概況。
在代謝物中磺胺類藥物是最早拿來用在制菌的藥劑，目前大多數雖然已被抗生素所取代，但對於某些疾病、動植物用藥仍具有不可忽視的價值，故有其研究的重要性存在。
由於磺胺類藥物並無相對應的氧化還原酵素來偵測電流變化，所以我們運用結構轉換型電化學感測器的概念，設計出不需氧化還原酵素亦可偵測代謝物之模型。
電化學生物感測器之設計是利用雙硫化物鏈（Lip-BG），藉由自組裝單分子層在電極表面做修飾，BG負責標記SNAP-hCAⅡ蛋白中的SNAP-tag，則將蛋白修飾在電極表面。當Fc-Linker-SFA探針加入後，探針的SFA會與hCAⅡ蛋白的抑制區結合，使得探針距離電極表面較近，而產生電流訊號；當其他磺胺類分析物加入後，與hCAⅡ蛋白抑制區的探針做競爭，而被競爭游離走之探針因為距離電極表面較遠，造成電流訊號下降，借此達到分析磺胺類藥物。

Metabolites are the reactants, intermediates, and products of metabolism. It is estimated that metabolism of a biological cell comprises 7900 metabolites. Metabolites are important in many biological functions and involve in the normal cell growth, development, reproduction, energy generation and transfer. Hence, development of a selective and sensitive analytical method for the detection of metabolites can provide important insights for many cellular processes.
For the metabolites detection using electrochemical method, one of the fundamental limitations is the requirement of redox enzymes to generate electrochemical signals. However, there are only limited numbers of redox enzymes in nature which are applicable for metabolites detection by using electrochemical method. Many metabolites and drug molecules which do not have the corresponding redox enzymes are important in medical diagnosis and pharmaceutical industry. For example, sulfa drugs which are important for the treatment of many diseases and killing bacteria, have no corresponding redox enzymes to catalyze redox reaction to deliver the electrochemical signals.
The objective of this thesis is to develop an electrochemical sensor which does not require a redox enzyme to generate electrochemical signal to overcome the fundamental limitation of electrochemical method for the selective detection of metabolites and drug molecules.
The electrochemical sensor is constructed by using a recombinant protein, SNAP-hCAII and a dialkyldisulfide linker (Lip-BG) which consists of a disulfide moiety for the self-assembly on gold electrode and a benzylguanine (BG) for SNAP-tag protein labeling. The addition of electrochemical probe Ferrocene-Linker-Sulfonamide (Fc-Linker-SFA) will result in the binding of SFA to the hCAII protein in which the ferrocene will be in the close proximity to the gold electrode to enhance the electrochemical current. Upon addition of sulfonamide drug, the drug will displace the Fc-Linker-SFA from the hCAII binding and as a result, the electrochemical signals will decrease.

摘要......................................................i
Abstract.................................................ii
謝誌.....................................................iii
全目錄....................................................iv
圖目錄...................................................vii

第一章 緒論.................................................1
1-1 生物感測器..............................................1
1-1.1 簡介.................................................1
1-1.2 生物辨識元件...........................................2
1-1.3 訊號轉換元件...........................................4
1-1.4 生物辨識元件固定方法....................................4
1-2 代謝物..................................................6
1-2.1 磺胺類藥物............................................8
1-2.1.1 簡介...............................................8
1-2.1.2    碳酸酐酶............................................9
1-3 電化學.................................................10
1-3.1 原理................................................10
1-3.2 電化學感測器..........................................12
1-3.3 電化學測定法..........................................13
1-3.3.1 循環伏安法.........................................14
1-3.3.2    方波伏安法.........................................16

第二章 文獻回顧與研究動機.....................................18
2-1 研究目的...............................................18
2-2 文獻回顧...............................................18
2-2.1 液相層析儀...........................................18
2-2.2 質譜儀...............................................19
2-2.3 螢光感測器...........................................20
2-2.4 電化學感測器..........................................22
2-2.5 磺胺類藥物偵測........................................24

第三章 實驗構想設計與技術原理.................................27
3-1 實驗構想設計...........................................27
3-1.1 電化學生物感測器模型Ⅰ.................................29
3-2 實驗技術原理............................................31
3-2.1 自組裝單分子層薄膜技術.................................31
3-2.2 有機分子標記融合蛋白之技術..............................32
3-2.3 固相胜肽合成技術......................................34

第四章 實驗結果與討論........................................35
4-1 有機合成及蛋白製備.......................................35
4-2 早期電化學生物感測器模型..................................36
4-2.1 電化學生物感測器模型A設計與測試..........................36
4-2.2 電化學生物感測器模型B設計與測試..........................39
4-2.3 電化學生物感測器模型C設計與測試..........................43
4-2.3.1    模型C測試 （一）....................................44
4-2.3.2    模型C測試 （二）....................................45
4-2.3.3    模型C測試 （三）....................................47
4-3 電化學生物感測器模型Ⅰ測試................................49
4-3.1    模型Ⅰ初期測試......................................50
4-3.1.1    模型Ⅰ初測試........................................50
4-3.1.2    Fc活性穩定度之測試..................................52
4-3.1.3    不同長度之探針測試...................................53
4-3.1.4    不同濃度之探針測試...................................54
4-3.1.5    不同濃度之分析物測試.................................55
4-3.2    模型Ⅰ後期測試......................................57
4-3.2.1    模型Ⅰ改良測法之測試.................................57
4-3.2.2    不同蛋白之測試......................................58

第五章 結論................................................60
    
第六章 實驗部分.............................................61
6-1 實驗器材、藥品&溶劑......................................61
6-1.1 實驗器材.............................................61
6-1.2 實驗藥品.............................................63
6-1.3 溶劑................................................65
6-2 化合物合成實驗與光譜資料..................................66
6-2.1 有機合成部分..........................................66
6-2.2 固相合成部分..........................................77
6-2.2.1    實驗操作流程........................................77
6-2.2.2    化合物之實驗藥品與劑量...............................78
6-3 蛋白質表現與純化........................................80
6-3.1 製備儲備液&生物緩衝液..................................80
6-3.2 蛋白質表現...........................................83
6-3.3 蛋白質純化...........................................84
6-3.4 SDS-膠體電泳.........................................85
6-4 電化學測試.............................................86
6-4.1 製備儲備液&生物緩衝液..................................86
6-4.2 電化學測試前處理......................................87
6-4.3 電化學模型Ⅰ修飾流程...................................88
6-4.4 電化學模型Ⅰ初期測試流程................................89
6-4.5 電化學模型Ⅰ後期測試流程................................89

參考文獻...................................................90
附錄......................................................94

                                

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