系統識別號 U0026-2408201118173600
論文名稱(中文) 以電化學法量化分析骨髓過氧化酶
論文名稱(英文) An electrochemical detection method to quantify myeloperoxidase
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 盧佩眉
研究生(英文) Pei-Mei Lu
學號 P86984120
學位類別 碩士
語文別 英文
論文頁數 47頁
口試委員 指導教授-張憲彰
中文關鍵字 急性心肌梗塞  骨髓過氧化酶  四甲基聯苯胺 
英文關鍵字 acute myocardial infarction  myeloperoxidase  tetramethylbenzidine 
中文摘要 骨髓過氧化酶(Myeloperoxidase, MPO)是存在於嗜中性白血球和單核球中的一種酵素,其功能主要是在人體內可催化生成次氯酸(HOCl)以破壞侵入人體的病原體。過去的研究顯示,急性心肌梗塞(acute myocardial infarction, AMI)病人(>91.7 ng/ml)在血漿中MPO的含量明顯高於正常人(20~60 ng/ml),且在動脈粥狀硬化斑形成的過程亦證實HOCl最先造成低密度脂蛋白(low density lipoprotein, LDL)氧化,進而啟動動脈粥狀硬化的產生。因此,血漿中MPO的含量可用來評估AMI發病的危險性。本研究將MPO修飾於金電極的表面,並以氧化還原活性物質四甲基聯苯胺(3,3’,5,5’-tetra-methylbenzidine, TMB)作為電子傳遞物,利用電化學分析法,量測TMB的氧化還原訊號,以討論MPO酵素與過氧化氫(hydrogen peroxide, H2O2)之間的反應。TMB於修飾MPO酵素的金電極表面,依然具有其氧化還原的特性。當溶液中含有H2O2時,可促使MPO酵素催化TMB氧化態的生成,因此,藉由TMB還原電流的變化,可以成功地推知MPO酵素與溶液中的H2O2反應之關係。相較於傳統檢測方法,我們將MPO與TMB催化反應的時間縮短至4分鐘。於本研究中,所量測的MPO線性範圍為3.3 ~ 330 ng/ml(線性為R2=0.9866),檢測極限為3.3 ng/ml。由結果可知,我們成功利用電化學法了解MPO酵素催化反應,藉由此方法未來將有機會能夠發展出一套簡單且快速預測AMI的晶片。
英文摘要 Myeloperoxidase (MPO) is a protein which is present in neutrophils and monocytes; its function mainly catalyzes H2O2 and Cl− to generate hypochlorite (HOCl), destroying the pathogen in human body. Previous researches have shown that the plasma MPO concentration is significantly higher in acute myocardial infarction (AMI) patients (>91.7 ng/ml) than healthy people (20 ~ 60 ng/ml). The formation of atherosclerotic plaque also confirmed that the process of low-density lipid (LDL) oxidation is caused by HOCl first, and followed activated the production of atherosclerosis. Thus, MPO levels in plasma can be served as a marker to assess the risk of AMI onset. In this work, we modified MPO on the gold electrode surface by self-assembled monolayer. 3,3’,5,5’-tetra-methylbenzidine (TMB) was used as redox mediator to analyze the reaction between MPO and hydrogen peroxide (H2O2). It showed that the redox signal of TMB on modified MPO electrode exhibited good electrochemical response. When the solution contains H2O2, MPO can catalyze TMB to produce TMB(ox). Therefore, the change of TMB reduction current can reveal the reaction between MPO and H2O. Compared to traditional detection methods, we shortened the response time of MPO-catalyzed TMB to 4 mins. In this study, the linear regression of MPO was 3.3 ~ 330 ng/ml (R2=0.9866), and the limit of detection was 3.3 ng/ml. Based on the results, we successfully detected the MPO catalyzed reaction by electrochemical method. In the future, we can develop a simply and rapid MPO detection chip by using electrochemical method for the risk prediction of AMI.
論文目次 Abstract I
中文摘要 II
致謝 III
Content IV
Table List VI
Figure List VII
Chapter 1 Introduction 1
1.1 Acute Myocardial Infarction 1
1.2 Atherosclerosis 4
1.2.1 Initiation Factor 4
1.2.2 Foam Cell Formation 5
1.2.3 Stability of Plaque 6
1.3 Myeloperoxidase and Acute Myocardial Infarction 8
1.4 Myeloperoxidase 10
1.5 Myeloperoxidase Detection Method 12
1.6 3, 3’, 5, 5’-Tetramethlbenzidine 14
1.7 Aim of This Study 16
Chapter 2 Materials and Experiments 17
2.1 Equipments 17
2.2 Materials 17
2.2.1 Chemicals for Microfabrication 17
2.2.2 Chemicals for Electrochemical Analysis 17
2.3 Test Solutions 18
2.4 Chip Fabrication 18
2.5 Fabrication of the Sensing Interface 20
2.6 The Experiment of Electrochemical Characteristic 21
Chapter 3 Results and Discussion 22
3.1 The Reaction Mechanism of HRP and MPO 22
3.2 Characteristic of the Modified Au Electrode 24
3.3 The Selection of Electron Transfer Mediator 26
3.4 The Electrochemical Characteristic of TMB and H2O2 28
3.5 The Reaction of HRP/TMB/H2O2 29
3.5.1 The Incubation Time of HRP Reaction 30
3.5.2 Calibration Curve of HRP 32
3.6 The Relationship between MPO, H2O2 and TMB 34
3.7 The Effect of Incubation Time 36
3.8 Calibration of MPO Concentration 38
Chapter 4 Conclusion and Prospect 40
Reference 41
自述 47
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