添加甲殼素寡糖對電流式葡萄糖生物感測器性能影響之研究

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研究生：	吳班尼 Benny - Gosali
論文名稱：	添加甲殼素寡糖對電流式葡萄糖生物感測器性能影響之研究 Behavior of Chitosan Oligosacharides Addition on Amperometric Glucose Biosensor Performance
指導教授：	李嘉平 Chiapyng Lee 郭俞麟 Yu-lin Kuo 王文 Wen Wang
口試委員:	李振綱 Cheng-Kang Lee 王詩涵 Shih-han Wang
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	115
中文關鍵詞：	葡萄糖生物感測器、甲殼素寡糖、網印技術、碳電極
外文關鍵詞：	Chitosan Oligosaccharides
相關次數：	點閱：274 下載：3
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摘要
本實驗成功利用網印技術製備電流式葡萄糖生物感測器。利用網印技術可成功將碳電極以及絕緣層印製在PVC基板上。含蓋赤血鹽和甲殼素寡糖的試劑層也同樣利用網印技術印製在感測試片上的固定區域，之後再將葡萄糖氧化酶溶液滴覆在試劑層上，則成功製備出酵素修飾電極。
在黏結層部分，則是利用鋼板印刷技術，印製在感測試片特定區域，並結合親水層即可成功在感測試片上製備出樣品流道，這樣就完成葡萄糖感測器的製備動作。
網印碳電極葡萄糖生物感測器的表面型態分析，則是運用光學顯微鏡和掃描式電子顯微鏡來分析，電化學性質分析則是利用循環伏安法和時間電流分析法來分析。
本實驗也有進行干擾以及穩定性的測試，此葡萄糖生物感測器具有相當快的訊號回應時間(8秒以下)，並且樣品需求量只需要1.5 μl，靈敏度可達0.027 μA/mg/dL，感測線性範圍為50~600 mg/dL，平均變異係數為5.7%，並且不受氧氣和維他命C的感擾，穩定性方面，在過了55天之後，感測器性能仍然有93%的起始活性，並且發現酵素修飾層中添加了甲殼素寡糖，能夠成功提高葡萄糖生物感測器的靈敏度以及穩定性。

ABSTRACT

An amperometric glucose biosensor based on screen printed carbon electrode (SPCE) was successfully developed in this research. Carbon electrode and insulation layer was screen-printed onto PVC substrate. Reagent layer which contained ferricyanide and chitosan oligosaccharides was also screen-printed on the defined area. The glucose oxidase solution was applied to the reagent layer by drop-coating technique. Adhesive layer was stencil-printed onto the SPCE to form the spacer and define the sample chamber. Finally application of the hydrophilic layer to the glucose biosensor completed the whole fabrication process.
The physical characteristics of the SPCE and glucose biosensor were studied by optical microscope (OM), and scanning electron microscope (SEM) technique, while their electrochemical characteristics were studied by cyclic voltammetry and amperometry technique. Interference and stability tests were also done in this research.
The fabricated glucose biosensor exhibited a relatively fast response (below 10s), and only 1.5μL sample needed. Its sensitivity was 0.027μA/mg/dL, and had linear detection range from 50-600mg/dL. The average coefficient of variation was 5.7%. No interference effects from oxygen and ascorbic acid (1.5mg/dL) were observed during the interference tests. After 55 days, glucose biosensor retained 93% of its initial activity. Chitosan oligosaccharides addition into the glucose biosensor’s reagent layer successfully increased its sensitivity and stability.

TABLE OF CONTENTS

Abstract................................................................................................................................i
Table of contents.................................................................................................................ii
List of figures.....................................................................................................................iii
List of tables.......................................................................................................................iv
Acknowledgements.............................................................................................................v

Chapter I. Introduction.........................................................................................................1
	I.1. Chemical sensors and biosensors......................................................................1
		I.1.1. Chemical sensors and biosensors definition.......................................1
	I.2. Importance of biosensors...................................................................................2
		I.2.1. Applications of biosensors..................................................................2
			I.2.1.1. Health care application of biosensors..................................3
			I.2.1.2. Environmental monitoring application of biosensors..........3
			I.2.1.3. Food and drink analysis application of biosensors..............4
			I.2.1.4. Defense and military applications of biosensors.................5
	I.3. Diabetes status...................................................................................................5
	I.4. Objective and scope of this research.................................................................8
	I.5. Significance of this research.............................................................................9

Chapter II. Literature review..............................................................................................11
	II.1. Transduction method of biosensors................................................................12
		II.1.1. Electrochemical based biosensors...................................................12
			II.1.1.1. Conductimetric biosensors...............................................13
			II.1.1.2. Amperometric biosensors.................................................14
			II.1.1.3. Potentiometric biosensors................................................17
			II.1.1.4. FET-based biosensors......................................................18
		II.1.2. Optical based biosensors.................................................................20
		II.1.3. Quartz crystal microbalance (QCM) based biosensors...................22
		II.1.4. Thermal or calorimetric based biosensors......................................23
	II.2. Immobilization methods................................................................................24
		II.2.1. Physical adsorption.........................................................................24
		II.2.2. Entrapment......................................................................................25
		II.2.3. Cross-linking...................................................................................25
		II.2.4. Covalent bonding............................................................................26
	II.3. Biosensor fabrication technology...................................................................28
		II.3.1. Thick film technology.....................................................................28
		II.3.2. Thin film technology.......................................................................31
	II.4. Performance factors of biosensors.................................................................35
	II.5. Chitin and chitosan........................................................................................36
		II.5.1. Chitin and chitosan properties.........................................................37
		II.5.2. Chitosan based materials.................................................................39
		II.5.3. Chemical modifications of chitosan................................................40
		II.5.4. Chitosan oligomers..........................................................................41
		II.5.5. Chitosan applications.......................................................................42



	II.6. Glucose biosensor technology........................................................................45
		II.6.1. Evolution of glucose biosensor.......................................................45
			II.6.1.1. First generation glucose biosensor...................................47
			II.6.1.2. Second generation glucose biosensor..............................48
			II.6.1.3. Third generation glucose biosensor.................................51

Chapter III. Experimental..................................................................................................53
	III.1. Instruments....................................................................................................55
		III.1.1. Semi-automatic screen printer, screens and stencil.......................55
		III.1.2. Sonicator........................................................................................57
		III.1.3. Potentiostat....................................................................................58
		III.1.4. Other instruments..........................................................................58
	III.2. Materials and reagents..................................................................................59
		III.2.1. Substrate and inks..........................................................................59
		III.2.2. Buffer and solvents........................................................................59
		III.2.3. Reagent ink and enzyme................................................................60
		III.2.4. Other materials...............................................................................61
	III.3. Preparation of buffer solutions......................................................................61
	III.4. Preparation of glucose solutions...................................................................61
	III.5. Preparation of reagent ink.............................................................................61
	III.6. Preparation of enzyme solution....................................................................62
	III.7. Substrate handling and preconditioning........................................................62
	III.8. Carbon electrodes screen printing.................................................................64
	III.9. Insulation layer screen printing.....................................................................65
	III.10. Reagent layer screen printing......................................................................66
	III.11. Adhesive layer stencil printing...................................................................67
	III.12. Enzyme solution drop-coating....................................................................68
	III.13. Sensor strip finishing..................................................................................68
	III.14. Glucose biosensor characterization............................................................69
		III.14.1. Physical characterization.............................................................69
		III.14.2. Electrochemical characterization.................................................70
			III.14.2.1. Cyclic voltammetry.......................................................70
			III.14.2.2. Amperometry................................................................70

Chapter IV. Results and discussion...................................................................................71
	IV.1. Glucose biosensor.........................................................................................71
		IV.1.1. Mediated glucose biosensor mechanism.......................................71
		IV.1.2. Important features..........................................................................76
	IV.2. Glucose biosensor design.............................................................................78
	IV.3. Glucose biosensor fabrication process.........................................................80
		IV.3.1. Substrate selection.........................................................................80
		IV.3.2. Carbon electrodes..........................................................................81
		IV.3.3. Insulation layer..............................................................................82
		IV.3.4. Reagent layer.................................................................................82
		IV.3.5. Adhesive layer...............................................................................86
		IV.3.6. Hydrophilic layer...........................................................................86



	IV.4. Characterization of screen printed carbon electrode (SPCE).......................87
		IV.4.1. Physical characterization of SPCE................................................87
		IV.4.2. Electrochemical characterization of SPCE....................................88
			IV.4.2.1. Cyclic voltammetry analysis...........................................89
			IV.4.2.2. Effect of scan rate...........................................................90
			IV.4.2.3. Electron transfer rate.......................................................92
	IV.5. Characterization of glucose biosensor..........................................................93
		IV.5.1. Physical characterization of glucose biosensor.............................93
		IV.5.2. Electrochemical characterization of glucose biosensor.................94
			IV.5.2.1. Cyclic voltammetry analysis...........................................94
			IV.5.2.2. Amperometry analysis....................................................97
				IV.5.2.2.1. Applied potential effect...................................97
				IV.5.2.2.2. Construction of calibration curves...................99
			IV.5.3. Interference study on glucose biosensor..........................101
			IV.5.4. Stability of glucose biosensor..........................................103

Chapter V. Conclusions and future work.........................................................................105

References........................................................................................................................106

Author’s information.......................................................................................................115

                                

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