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研究生:張郁苓
研究生(外文):Yu-Ling Chang
論文名稱:化學修飾金表面電極於甲基胎兒蛋白之生物感測器
論文名稱(外文):A Modified Gold Electrode for a Portable Alpha-fetoprotein (AFP) Bio-sensor
指導教授:薛富盛薛富盛引用關係劉炯權
指導教授(外文):Fuh-Sheng ShieuChung-Chiun Liu
口試委員:林淑萍龔志謙
口試委員(外文):Shu-Ping LinChih-Chien Kung
口試日期:2016-07-21
學位類別:碩士
校院名稱:國立中興大學
系所名稱:材料科學與工程學系所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:58
中文關鍵詞:化學修飾電極胎兒甲基蛋白電化學分析抗體抗原生物檢測器
外文關鍵詞:chemical modified gold electrodeAFP biosensorelectrochemical biosensornon-sandwich assay
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此研究報告是使用化學修飾的金電極,接上probe胎兒甲基蛋白抗體用來檢測溶液中的胎兒甲基蛋白。胎兒甲基蛋白在懷孕母體血液中的濃度是重要的指標,通常在妊娠第二期的產前檢查伴隨其他項目來篩選是否有天生的腦部、脊隨開放的機率。
此實驗使用含硫醇基的分子為結合金電極與抗體蛋白的橋樑,並且添加雙硫醇基的分子來填補空隙,也降低蛋白的吸附產生的背景訊號。抗體與蛋白的反應結合時間、硫醇與雙硫醇的濃度比例,其最佳的條件將由循環伏安法、微分脈衝伏安法及開路電壓的電化學分析,得到定性定量的檢量線圖。
這個實驗設計是直接偵測在磷酸鹽緩衝水溶液中的胎兒甲基蛋白濃度,由於抗體和抗原有良好特異性的結合,沒有藉由生物觸媒或是染料的間接反應,不同濃度的蛋白與電極上的抗體有不等程度的結合。藉由分析疊合在特定電壓的電流訊號,檢量線得到有正相關濃度與電流的分布,也就是說直接測量抗體與抗原的結合在無其他蛋白的緩衝溶液是可行的。

We directly detect AFP (alpha-feto protein) with only anti-AFP immobilized with thiolated compounds to gold electrode sensor, with electrochemical analysis data to quantify the concentration levels of AFP in the phosphate buffer solution. After cleaning three-electrode sensor with alcohol and D.I. water, the electrode was immersed in the freshly prepared solution with certain ratio of co-assembling thiolated molecules of dithiothreitol (DTT) and 3-mercaptopropionic acid (3-MPA).
With the property of specific affinity for combination of antibody and antigen, this chemical modified sensor could be used to detect antigen, AFP, without enzyme, dyes or sandwich-assay design. For electrochemical analysis, the additional antigen was involved to form antibody-antigen complex which contributed to the signal of current.
In our interested potential, 0.2 V (vs. Ag/AgCl), the correlated current was noted to form the calibration curve for all the comparisons. Overlapping the differential pulse voltammetry of several concentrations, 0.01 ng/μl was successfully detected with a 1:20 DTT/thiol ratio. Addition of DTT to make a non-fouling surface did enhance the sensitivity of antibody-based AFP biosensor.


Acknowledgements i
Chinese Abstract ii
English Abstract iii
Contents iv
List of Figures and Tables vi
Chapter 1 Introduction 1
1.1 Motivation 2
1.2 Objectives 2
1.3 Overall Research Approach 3
Chapter 2 Background and Theory 6
2.1 Medical aspects 6
2.1.1 General Background for development of pregnant and disease 6
2.1.2 Screening Test 7
2.1.3 Statistic Data or History data 8
2.2 Bio-sensor 10
2.2.1 Self-Monitoring Blood Glucose Management 11
2.2.2 Home Pregnancy Test 13
2.3 Immuno-sensor 14
2.3.1 Structure of Antibody 14
2.3.2 Affinity of Antibody and Antigen 16
2.3.3 Self-Assembled Monolayer (SAM) 17
2.3.4 Mechanism of Immobilization of Antibody 19
2.4 Electrochemical Methods 20
2.4.1 Principle of Electrochemical Method 20
2.4.2 Cyclic Voltammetry and Differential Pulse Voltammetry 21
Chapter 3 Experimental 23
3.1 Chemicals and Electrode 24
3.2 Phosphate Buffer Solution 25
3.3 Aliquots of Antibody and Antigen 26
3.4 Protocol for Cross-Linking 27
3.5 System of Electrochemical Analyzer 31
Chapter 4 Results and Discussion 33
4.1 Factors of Protein Cross-Linking 33
4.2 Bonding between surface of gold electrode and the antibody 36
4.3 Reduction of Non-Fouling Surface 40
4.4 Optimization of Co-Assembled DTT/Thiol 42
4.5 Open Circuit Potential to Time of the Optimization Solution 48
Chapter 5 Conclusion 53
Reference 54


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