應用於生醫的微製作與網印電化學感測器

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蘇虹娜(Kalpana Settu) 查詢紙本館藏

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論文名稱

應用於生醫的微製作與網印電化學感測器
(Microfabricated and Screen-printed Electrochemical Sensors for Biomedical Applications)

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摘要(中)

本研究發展電化學生物感測器，所使用的技術包括微製造和網印這兩種製造方法。這些感測器將應用在生物醫學上，如監控致病菌與檢測疾病的生物標記。
本研究的微製造感測器是在玻璃基板上製造的指叉金微電極感測器，將用以檢測人體尿液中與牛奶樣品中的大腸桿菌。在細菌生長的期間，頻率範圍1 Hz到1 MHz的電化學阻抗譜測量，據以進行等效電路分析，從而推論出大腸桿菌的生長所引起電極表面之電雙層電容值的改變是阻抗變化的主因。在人體尿液檢測上，細菌生長期間由於會形成細菌生物膜累積在感測器表面上而產生阻抗變化。尿液中大腸桿菌的濃度可以靠著在細菌開始成長後的某一時間點測量在某一指定頻率之阻抗值然後對照在該時間點的校正曲線而得知。在牛奶檢測上，阻抗譜上可以觀察到阻抗值因大腸桿菌增生而降低的現象，這是由於牛奶中細菌代謝引起離子的濃度變化所致。建立阻抗降低10%所需的時間相對於細菌濃度的校正曲線，牛奶中大腸桿菌的濃度可以靠著測量10%阻抗變化的時間而得知。
本研究的網印感測器是以碳為電極材料，將用於白蛋白與EN2蛋白的檢測。若用於檢測尿液中微量白蛋白，首先使用共價鍵固定法將人體白蛋白抗體固定在網印感測器表面，然後採用免疫反應測定原理，以計時電流法的電化學技術來對尿液中的白蛋白做定量檢測。為了要檢測尿液中的EN2蛋白，石墨烯被加入網印碳電極中以提高靈敏度；首先在網印石墨烯碳電極表面修飾接上能與EN2蛋白做特異性結合的單股DNA，然後採用循環伏安法來定量EN2蛋白的濃度。

摘要(英)

This study focused on the development of electrochemical biosensors for biomedical applications, such as pathogenic bacteria monitoring and disease biomarker detection. Two methods were utilized for the fabrication of these sensors, namely the microfabrication and screen-printing techniques.
The microfabricated biosensors were interdigitated gold microelectrode sensors designed and microfabricated on glass substrate for E. coli bacterial detection in human urine and cow milk samples. Electrochemical impedance spectroscopic measurements were carried out during bacterial growth over a frequency range of 1 Hz to 1 MHz. Based on equivalent circuit analysis, it was inferred that double layer capacitance was responsible for the impedance change caused by the E. coli growth. In human urine, the change in impedance during bacterial growth was due to formation and accumulation of bacterial biofilms on the sensor surface. The urinary E. coli concentration could be determined from the calibration curve at a specific frequency with a selected growth time. In cow milk, during E. coli growth, a decrease in impedance was observed due to the ionic concentration change in the milk caused by bacterial metabolism. The E. coli concentration in milk could be determined from the calibration curve of the bacterial concentration with respect to the time when the impedance changed by 10%.
The screen-printed biosensors were carbon-based sensors for albumin and EN2 protein detection. For the detection of microalbumin in urine, anti-human albumin antibodies were immobilized on the screen-printed sensor surface by the covalent immobilization method. Then the immunoassay was achieved by quantitating the albumin in urine with the chronoamperometric (CA) electrochemical measurement technique. For the detection of EN2 protein in urine, graphene was incorporated in the screen-printed carbon electrode to enhance the detection sensitivity. The screen-printed carbongraphene sensor surface was modified with DNA strand which is specific to EN2 protein binding. Cyclic voltammetry measurement was employed for the quantitative detection of EN2 protein concentration.

關鍵字(中)

★ 大腸桿菌
★ 生物傳感器
★ 微細加工
★ 絲網印刷
★ 電化學
★ 蛋白
★ 免疫反應測定

關鍵字(英)

★ Immunoassay
★ Biosensors
★ Microfabrication
★ Screen-printing
★ Electrochemical
★ Escherichia coli
★ Protein

論文目次

ABSTRACT I
摘要 II
ACKNOWLEDGEMENT III
Contents IV
List of Figures VII
List of Tables IX
CHAPTER 1: Introduction 1
1.1 Biosensors 1
1.1.1 Electrochemical biosensors 6
1.1.2 Electrochemical techniques 7
1.2 Sensor fabrication techniques 17
1.2.1 Microfabrication 17
1.2.2 Screen-printing 23
1.3 Summary and Objectives of this Study 25
1.4 Organization of this dissertation 26
CHAPTER 2: Impedimetric Method for Measuring Ultra-low E. coli Concentrations in Human Urine 27
2.1 Abstract 27
2.2 Introduction 27
2.3 Materials and methods 30
2.3.1 Chemicals and reagents 30
2.3.2 Sensor fabrication and experimental setup 30
2.3.3 Bacterial culture and impedance measurement 32
2.3.4 Bacterial urine sample preparation 32
2.3.4 Scanning electron microscopy (SEM) 33
2.4 Results and discussion 33
2.4.1 Impedance response of E. coli in urine 33
2.4.2 Monitoring of E. coli growth in urine 34
2.4.3 Equivalent circuit analysis for impedance measurement system 37
2.4.4 Detection of E. coli in urine samples 43
2.5 Summary 46
Chapter 3: Impedance Sensor for Rapid Enumeration of E. coli in Milk Samples 48
3.1 Abstract 48
3.2 Introduction 48
3.3 Materials and methods 52
3.3.1 Chemicals and reagents 52
3.3.2 Fabrication of gold interdigitated microelectrode on glass substrate 52
3.3.3 Bacterial culture 53
3.3.4 Spiked milk sample preparation 55
3.3.5 Impedance measurement 55
3.4 Results and discussion 55
3.4.1 Characterization of interdigitated microelectrode sensor 55
3.4.2 Impedance analysis of E. coli growth in milk samples 57
3.4.3 Quantification of E. coli in milk samples 62
3.5 Summary 66
Chapter 4: Screen-Printed Carbon Electrode-based Electrochemical Immunosensor for Rapid Detection of Microalbuminuria 67
4.1 Abstract 67
4.2 Introduction 67
4.3 Materials and methods 70
4.3.1 Reagents 70
4.3.2 Fabrication of screen-printed carbon paste electrodes (SPCE) 70
4.3.3 Immobilization of anti-HSA antibody on the SPCE 71
4.3.4 Surface morphology and composition analysis 71
4.3.5 Electrochemical measurements 71
4.3.6 Human urine samples preparation and analysis 72
4.4 Results and discussion 72
4.4.1 Characterization of SPCE electrodes 72
4.4.2 Characterization of SPCE electrodes 74
4.4.3 Optimization of antibody immobilization and antigen binding times 78
4.4.4 Interference study 80
4.4.5 Detection of HSA 81
4.4.6 Application of optimum immunosensor for the determination of microalbuminuria in human 83
4.5 Summary 85
Chapter 5: Development of Screen-printed Carbon/Graphene-based DNA Biosensor for EN2 Protein Detection 86
5.1 Abstract 86
5.2 Introduction 86
5.3 Materials and methods 88
5.3.1 Reagents 88
5.3.2 Fabrication of screen-printed carbon graphene electrodes (SPCGE) 89
5.3.3 Immobilization of DNA on the SPCGE 89
5.3.4 Surface morphology and composition analysis 90
5.3.5 Electrochemical measurements 90
5.3.6 Electrochemical detection of EN2 protein 90
5.4 Results and discussion 90
5.4.1 Characterization of DNA immobilization 91
5.4.2 Optimization of sensing conditions 94
5.4.2 EN2 protein detection 99
5.5 Summary 101
CHAPTER 6: Conclusion and Future Work 102
6.1 Conclusion 102
6.1. Future work 103
References 104
Appendix 114

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指導教授

蔡章仁、劉仁材、陳靖容(Jang-Zern Tsai Jen-Tsai Liu Ching-Jung Chen)

審核日期

2016-8-29

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