人體血清白蛋白構形改變之電化學及表面電漿共振分析研究

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吳政豐(Cheng-Feng Wu) 查詢紙本館藏

畢業系所

機械工程學系

論文名稱

人體血清白蛋白構形改變之電化學及表面電漿共振分析研究
(On the Conformational Changein Human Serum Albumin UsingElectrochemical and Surface PlasmonResonance Techniques)

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摘要(中)

在本研究當中利用電化學與表面電漿共振技術來偵測人體血清白蛋白受鉻(VI)後導致構形的改變，本研究採用載玻片濺鍍47nm金膜，以自我聚集方式(Self-assemble monolayer)吸附二胺基硫代乙醇(2-aminoethanethiol)，再吸附人體血清白蛋白(HSA)後作為偵測電極，再將含有鉻(VI)溶液流過吸附HSA的電極，利用電化學與表面電漿共振技術能即時偵測的優點，來偵測人體血清白蛋白在受鉻吸附後所產生的構形變化，在表面電漿共振技術方面，利用人體血清蛋白(HSA)在含鉻(VI)溶液中產生的共振角偏移，判斷人體血清白蛋白之構形變化程度。在電化學技術方面利用以下三種:1.比較此系統開路電壓OCP之高低以判斷人體血清蛋白遭受鉻中毒之構形改變程度。2.另外，CV圖譜中（電流對電壓圖）藉由電流訊號的改變會影響電壓之測量，電壓峰值出現位置及振幅高低，顯示人體血清白蛋白受鉻毒害後之構形改變大小。3.交流阻抗頻譜法可用來探討電化學系統電容、電阻等。由人體血清白蛋白(HSA)與鉻(VI)反應前後電容、電阻等變化，量測此與蛋白質構形改變之關聯性。除了即時偵測人體血清白蛋白與鉻所造成的構形變化外，也分析表面電漿共振與電化學數據的正確性與靈敏度提升。在表面分析方面則以原子力顯微鏡(AFM)探測蛋白質遭鉻離子(VI)毒化前後表面之粗糙度變化。FT-IR光譜儀則用於檢測蛋白質在遭鉻(VI)離子構形改變前後之鍵結震動模式變化。

摘要(英)

The conformational change in human serum albumin (HSA) has been investigated using dc-cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) technique in this work. A slice of glass was sputtered a thin film of gold (47 nm), undergone self-assembly of 2-aminoethanethiol (AET) in ethanol solution and coated with the HAS solution to act as a specimen for this study. A stream of dichromate (Cr2O72-) solution flowed on the immobilized HAS caused a bio-interaction between the HAS and Cr (VI) thus resulting conformational change of the HAS. SPR and electrochemical techniques were used in the following to monitor the in-situ conformational change of the HAS due to its bio-interaction with Cr (VI).1. Monitoring using SPR technique:
The interfacial change resulted from conformational change of immobilized HAS caused by Cr (VI) could be detected using SPR through the change of resonance angle.2. Monitoring using electrochemical techniques: (1) Shift in the open circuit potential (OCP) of the system reflected the conformational change of the HAS resulted from its toxic reaction with Cr (VI) ion. (2) Presence of current peaks in cyclic voltammograms (CV) at certain potentials were responsible for specific toxic reactions between the HAS and Cr (VI) ion. (3) Analysis of the resistive and capacitive data of the EIS provided a check on the conformational change of the HAS arisen from its toxic reaction with Cr (VI) ion. In additional to monitor the toxic reaction between the HAS and the hexavalent chromium. The validity and sensitivity of the SPR and electrochemical signals are discussed. The surface roughness of the system was estimated by atomic force microscope (AFM). The conformational change of the HAS was confirmed via analysis of vibration mode from the FT-IR spectroscopy.

關鍵字(中)

★ 蛋白質構形改變
★ 人體血清白蛋白
★ 電化學
★ 表面電漿共振

關鍵字(英)

★ surface plasmon resonance
★ electrochemical
★ human serum albumin
★ conformational change

論文目次

Abstract (Chinese) I
Abstract (English) III
Acknowledgement V
Contents VI
List of Tables X
List of Figures XIII
Chapter I Introduction 1
1.1 Conformational change in the structure of protein 1
1.2 Property of human serum albumin 1
1.3 Structure of human serum albumin 2
1.4 The goals of this work 4
Chapter II Literature Review and Theoretical Aspects 5
2.1. Toxicity of Chromium (VI) 5
2.1.1 Characteristic of hexavalent chromium 5
2.1.2 Application of chromium 5
2.1.3 Harm of Chromium to Human’s health 6
2.2. Adsorbed of HSA on a SAMs-gold electrode 7
2.2.1 Introduction of self-assembled monolayers (SAMs) 7
2.2.2 SAMs of 2-aminoethanethiol on the gold electrode 8
2.3.Theoretical aspects for Electrochemical and Surface Plasmon Resonance detection 8
2.3.1 Cyclic voltammetry (CV) theory 8
2.3.2 Electrochemical impedance spectroscopy theory 10
2.3.3 Electron-transfer characteristics of hexacyanoferrates 13
2.3.4 Surface plasmon resonance (SPR) theory 15
Chapter III Experimental Procedures 20
3.1 Reagents 20
3.2 Sample preparation 20
3.2.1 Gold film substrate 20
3.2.2 Self-assembled monolayers formation 21
3.2.3 Protein adsorption 21
3.3 Experimental instruments 22
3.3.1 Electrochemical experiment 22
3.3.2 SPR spectrometer 23
3.3.3 AFM surface morphology 23
3.3.4 ESCA surface oxide analysis 24
3.3.5 FT-IR spectrometer 24
Chapter IV Results 25
4.1 Examination of surface roughness using AFM 25
4.1.1 Roughness of the AET-assembled on gold 25
4.1.2 Roughness on the HSA having reacted with CrO3 solution 25
4.2 FT-IR and XPS analysis for AET assembled and HSA adsorbed film 26
4.3 Detection of conformational change of the HSA structure using SPR 27
4.3.1 SPR curves for the HSA-adsorbed system 27
4.3.2 SPR curves for the HSA-adsorbed film reacted with a stream of chromate solution 28
4.4 Measurement of open circuit potential in a system 29
4.4.1 OCP for AET assembled-gold electrode 29
4.4.2 OCP for HSA adsorbed-system 30
4.4.3 OCP of HSA having reacted with in CrO3 solution 30
4.5 Cyclic voltammetric study on system 31
4.5.1 Effect of scan rate 31
4.5.2 Effect of numbers of cycles 32
4.5.3 CV for the electrolyte containing PBS and Fe(CN)64- 32
4.5.4 CV for the electrolyte containing PBS, Fe(CN)64- and CrO3 34
4.5.5 CV for the HSA having reacted with CrO3 to cause conformational change 36
4.6 EIS study on the system 37
4.6.1 Concentration effect of Fe(CN)63-/ 4- on the EIS spectra 38
4.6.2 Concentration effect of CrO3 on the EIS spectra 38
4.6.3 EIS for the electrolyte containing PBS and CrO3 39
4.6.4 EIS for the electrolyte contained PBS and Fe(CN)63-/4 39
4.6.5 EIS for the electrolyte contained PBS, Fe(CN)63-/4-and CrO3 40
4.7 Time-dependence of the EIS in various systems 41
4.7.1 Detection of Cr(VI) using single Au electrode and AET/Au electrode system 41
4.7.2 EIS for the electrolyte containing PBS and CrO3 42
4.7.3 EIS for the electrolyte containing PBS, Fe(CN)63-/4-and CrO3 44
Chapter V Discussion 47
5.1 Conformational change in HSA by SPR detection 47
5.2 Conformational change in HSA by electrochemical detection 48
5.2.1 Cyclic voltammogram analyze 48
5.2.2 Electrochemical impedance spectroscopy analyze 49
Chapter VI Conclusions 53
Chapter VII Future Work 55
References 57

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指導教授

陳顯禎、林景崎
(Shean-jen Chen、Jing-Chie Lin)

審核日期

2003-7-15

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