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研究生:郭寶財
研究生(外文):Kuo,Pao-Tsai
論文名稱:以因子實驗設計分析以二環戊二烯亞鐵修飾碳糊電極之反應參數對偵測過氧化氫的應答電流之影響及其應用於葡萄糖生醫感測器之研究
論文名稱(外文):A Factorial Design for Analysis of the Effect of Reaction Parameters on the Responding Current of Detection of Hydrogen Peroxide for the Carbon Paste Electrode Modified with the Ferrocene and Its Application to the Glucose Biosensor
指導教授:林浩林浩引用關係
指導教授(外文):Lin,Hau
學位類別:碩士
校院名稱:南台科技大學
系所名稱:化學工程與材枓工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:117
中文關鍵詞:二環戊二烯亞鐵生醫感測器碳糊電極
外文關鍵詞:ferroceneBiosensorcarbon paste electrode
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摘要

現今之食品工業有時會添加過氧化氫作為防腐劑,故發展出快速且方便使用的過氧化氫感測器為重要之研究主題。近年來,糖尿病已成為國人十大死亡原因之一,所以發展出簡便快速的葡萄糖生醫感測器亦為重要的研究主題。葡萄糖和氧氣藉由葡萄糖氧化酵素的催化,可產生葡萄糖酸和過氧化氫。本研究運用二環戊二烯亞鐵來修飾碳糊電極,二環戊二烯亞鐵本身具有強催化性能,與具有導電性能的碳粉製作成碳糊電極,藉以提升過氧化氫之應答電流,在磷酸鹽緩衝溶液中偵測過氧化氫之應答電流,計算過氧化氫之濃度,藉以推算葡萄糖之濃度。
實驗設計法普遍應用於許多領域,對於科學試驗甚至工業製程改進更是非常重要的工具,本研究實施三個因子〔操作電位、攪拌速率及磷酸鹽緩衝溶液(PBS)之pH值〕及二個水準之因子實驗設計來分析以二環戊二烯亞鐵修飾碳糊電極之反應參數對偵測過氧化氫的靈敏度及平均應答電流的影響,由因子實驗設計的效果值顯示操作電位在-200 mV之靈敏度及平均應答電流高於操作電位在-50 mV之靈敏度及平均應答電流,攪拌速率在500 rpm之靈敏度及平均應答電流高於攪拌速率在300 rpm之靈敏度及平均應答電流,pH = 7.4之靈敏度及平均應答電流高於pH = 4.0之靈敏度及平均應答電流,本研究運用SPSS(Statistical Package for the Social Science)統計分析軟體進行分析,對靈敏度之變異數分析的結果顯示操作電位的主效果檢定(F = 151.494,顯著性<0.05)、攪拌速率的主效果檢定(F = 7.288,顯著性<0.05)與pH值之主效果檢定(F = 94.461,顯著性<0.05)皆達顯著水準。對平均應答電流之變異數分析的結果顯示操作電位的主效果檢定(F = 141.515,顯著性<0.05)、攪拌速率的主效果檢定(F = 7.487,顯著性<0.05)與pH值之主效果檢定(F = 89.901,顯著性<0.05)也皆達顯著水準,結果顯示本研究之最適操作條件為操作電位= -200 mV,攪拌速率=500 rpm,pH = 7.4。
在30℃下,以二環戊二烯亞鐵修飾碳糊電極[二環戊二烯亞鐵:碳粉=3:7(重量比)] ,當操作電位為-200 mV ,攪拌速率為500 rpm,以0.05 M 磷酸鹽緩衝溶液(pH=7.4)偵測過氧化氫之應答電流,可得偵測極限為0.02 mM H2O2,線性範圍為 0.02~1.2 mM H2O2,R2為0.9998,靈敏度為161.43 µA/cm2.mM H2O2。
ABSTRACT
Nowadays, sometimes the hydrogen peroxide is used in the food industry for the purpose of preservation and therefore, developing a hydrogen peroxide sensor is an important research subject. In recent years, the diabetes has become one of the top ten causes of death for the people in our country. Therefore, developing a glucose biosensor which can detect the glucose rapidly and conveniently is also an important research subject. The glucose and oxygen can be catalyzed by the glucose oxidase to produce the gluconic acid and hydrogen peroxide. A study was conducted to use the ferrocene to modify the carbon paste electrode. Because the ferrocene(Fe(C5H5)2) possessed the excellent catalytic characteristic, it can be used with the graphite carbon powders which possessed the excellent conductivity to make the carbon paste electrode and to elevate the responding current of hydrogen peroxide. The responding current of hydrogen peroxide is detected in the phosphate buffer solution(PBS) and then the concentration of the hydrogen peroxide can be obtained and consequently, the concentration of the glucose can be determined.
The experimental design is used widely in many fields and it is an important tool for the test and the improvement of the process of industrial production. A study of factorial design involving three factors (operating potential, stirring rate, and pH value of PBS) and two levels was performed to analyze the effect of reaction parameters on the sensitivity and average responding current of detection of hydrogen peroxide for the carbon paste electrode modified with the ferrocene. The results of the calculating the effects showed that the sensitivity and average responding current at -200 mV operating potential were higher than those at -50 mV operating potential, the sensitivity and average responding current at 500 rpm stirring rate were higher than those at 300 rpm stirring rate, and the sensitivity and average responding current at pH=7.4 were higher than those at pH=4.0. The SPSS(Statistical Package for the Social Science) was used to perform the analysis of variance. The results showed that the main effect of operating potential ( F = 151.494, p value < 0.05 ) , the main effect of stirring rate ( F = 7.288, p value < 0.05 ) and the main effect of pH value ( F = 94.461, p value < 0.05 ) were significant on the sensitivity. The main effect of operating potential ( F = 141.515, p value < 0.05 ) , the main effect of stirring rate ( F = 7.487, p value < 0.05 ) and the main effect of pH value ( F = 89.901, p value < 0.05 ) were also significant on the average responding current. The results showed that the optimum operating condition for this research is operating potential = -200 mV , stirring rate = 500 rpm and pH = 7.4.
At 30℃, -200 mV operating potential, 500 rpm stirring rate and in 0.05M PBS buffer solution ( pH = 7.4 ) , when the carbon paste electrode was modified with the ferrocene [ferrocene : graphite carbon powder = 3 : 7 ( weight ratio )] , the detection limit was 0.02 mM H2O2 , the linear
range was 0.02~1.2 mM H2O2 , R2 = 0.9998 and the sensitivity was 161.43µA/cm2.mM H2O2.
目 次
摘要…………………………………………………………………………………...iv
英文摘要………………………………………………………………………………v誌謝…………………………………………………………………………………...vi
目次…………………………………………………………………………………..vii
表目錄………………………………………………………………………………...ix
圖目錄………………………………………………………………..……………......x
第一章 緒論…………………………………………………………………………..1
1.1 研究動機與目的……………………………………………………………1
1.2 糖尿病概述…………………………………………………………………2
1.2.1血糖………………………………………………………….…………3
1.2.2尿糖………………………...…………………………………………..3
1.3 生醫感測器概述…………….……………………………………………...4
1.3.1生物辨識元件……………………………………………………….…6
1.3.2傳感元件………………………...……………………………………..8
1.4 電流式生醫感測器偵測生理物質之原理………………………………..12
1.5 電子傳媒(Mediator)…..………………………………………………..14
1.6 電化學法原理……………………………………………………………..16
1.7 電化學分析方法…………………………………………………………..17
1.7.1 循環伏安法( Cyclic Voltammetry, CV ) …………………………….17
1.7.2 定電位法( Constant-Potential Method ) …………………………….21
1.8 過氧化氫之概述…………………………………………………………..21
1.9 固定辨識元件的目的及方法…………………………………........……..22
1.10 Nafion 之介紹……………………………………………………………23
第二章 實驗部份..…………………………………………………………………..26
2.1 藥品與儀器………………………………………………………………..26
2.1.1 實驗藥品…...…………………………………………………..…….26
2.1.2 實驗儀器……………………………………………………….…….27
2.2 藥品配製…………………………………………………………………..29
2.2.1 磷酸鹽緩衝溶液(PBS)……………………………………………29
2.2.2 1 M鹽酸………………………………………………………….…29
2.2.3 1 M氫氧化鈉...................................................................…………..29
2.2.4 過氧化氫溶液(100 mM)……………………….…………………30
2.2.5 葡萄糖溶液(100 mM)………………………………………………30
2.2.6 葡萄糖氧化酵素溶液……….…….…………………………………30
2.2.7 Nafion 酒精溶液……………………………………………….…..31
2.3 電極之製備………………………………………………………………..31
2.3.1 工作電極材料之前處理…………………………………………......31
2.3.2 碳糊電極之製備……………………………………………………..31
2.3.3 酵素電極之製備……………………………………………………..33
第三章 因子實驗設計之因子的高低水準之決定…………………………………34
3.1 實驗內容概述……………………………………………………………..34
3.2 偵測過氧化氫之操作電位的高低水準之決定…………………………..34
3.3 偵測過氧化氫之攪拌速率的高低水準之決定…………………………..42
3.4 偵測過氧化氫之緩衝溶液之pH值的高低水準之決定……………..…..46
第四章 因子實驗設計之解釋名詞與實驗之決定…………………………………50
4.1 解釋名詞…………………………………………………………………..50
4.2 決定因子實驗設計之實驗………………………………………………..54
第五章 結果與討論…………………………………………………………………57
5.1 因子實驗設計之結果………………………………………………….....57
5.2 最適條件下碳糊電極偵測過氧化氫之偵測極限及線性範圍…….........97
5.3 酵素碳糊電極對葡萄糖之感測結果……...............................................104
第六章 結論…………………………………………………………………..........107
參考文獻……………………………………………………………..……………..109
附錄A 運用SPSS統計軟體對以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之靈敏度作變異數分析的結果…………....……………..……………..…..116
附錄B 運用SPSS統計軟體對以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之平均應答電流作變異數分析的結果………………....…..………...…….117










表目錄
表 3.1 不同操作電位下之碳糊電極偵測過氧化氫之靈敏度、平均應答電流及R2比較表..............................................................................................................41
表 3.2 不同攪拌速率下之碳糊電極偵測過氧化氫之靈敏度、平均應答電流及R2比較表..............................................................................................................45
表 3.3 碳糊電極在不同pH值之PBS緩衝溶液中偵測過氧化氫之靈敏度、平均應答電流及R2比較表....................................................................................49
表 4.1 因子實驗設計之三個因子和二個水準.........................................................52
表 4.2 因子實驗設計之符號表.................................................................................53
表 4.3 因子實驗設計之實驗的反應條件.................................................................55
表 4.4 計算23因子實驗設計之效果值之符號表....................................................56
表 5.1 以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之靈敏度之因子實驗設計
的十六個實驗之實驗結果..............................................................................91
表 5.2 以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之平均應答電流之因子實驗設計的十六個實驗之實驗結果..................................................................92
表 5.3以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之靈敏度之因子實驗設計的效果值..........................................................................................................93
表 5.4以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之平均應答電流之因子實驗設計的效果值..............................................................................................94
表 5.5以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之靈敏度之變異數分析結果......................................................................................................................95
表 5.6以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之平均應答電流之變異數分析結果..........................................................................................................96
表 5.7以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫的偵測極限、線性範圍、靈敏度與R2值...................................................................................................100
表 5.8未經修飾之碳糊電極偵測過氧化氫的偵測極限、線性範圍、靈敏度與R2值..................................................................................................................103
表5.9酵素碳糊電極偵測葡萄糖之靈敏度、平均應答電流與R2值..................................................................................................................106
圖目錄
圖 1.1 生醫感測器的基本架構圖……………………………………………….…..5
圖 1.2 電化學電極示意圖……………………………………………………….…..9
圖 1.3 電流式生醫感測器偵測葡萄糖之原理………………………………….…13
圖 1.4 二環戊二烯亞鐵(ferrocene)的化學結構圖…………………………….…15
圖 1.5 對流、擴散、泳動時物質的傳輸狀態………………………………….….16
圖 1.6 循環伏安法的示意圖…………………………………………………….…19
圖 1.7 循環伏安法之電位掃瞄與相對應之電流……………………………….…19
圖 1.8 循環伏安圖之電位掃瞄與相對應之電流……………………………….…20
圖 1.9 Nafion之化學結構式……………………………………………………...24
圖 1.10 Nafion薄膜之微結構圖………..………………………………………...25
圖 2.1 三電極系統……………………………………….…………………………28
圖 2.2 所選用之單芯銅電線尺寸示意圖…………………………………….……31
圖 2.3 碳糊電極製作流程示意圖………………………………………….………32
圖 2.4 碳糊電極示意圖.............................................................................................32
圖 2.5 酵素電極示意圖…………………………………………………………….33
圖 3.1經二環戊二烯亞鐵修飾之碳糊電極的 CV圖 …..………………………..36
圖 3.2未經修飾之碳糊電極的 CV 圖…………………………………………….37
圖 3.3 經二環戊二烯亞鐵修飾之碳糊電極與未經修飾之碳糊電極的CV圖…..38
圖 3.4 不同操作電位下之碳糊電極偵測過氧化氫的應答電流之
TB圖……………………………………………………..……………..…..39
圖 3.5 不同操作電位下之碳糊電極偵測過氧化氫的應答電流之
檢量線圖........................................................................................................40
圖 3.6 不同攪拌速率下之碳糊電極偵測過氧化氫的應答電流之
TB圖……………………………………………………..……………..…..43
圖 3.7 不同攪拌速率下之碳糊電極偵測過氧化氫的應答電流之
檢量線圖........................................................................................................44
圖 3.8 碳糊電極在不同pH值之PBS緩衝溶液中偵測過氧化氫之應答電流之TB圖…………………………………………………………………….….......47
圖 3.9 碳糊電極在不同pH值之PBS緩衝溶液中偵測過氧化氫之應答電流之檢量線圖............................................................................................................48
圖 5.1以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之(l)實驗].......................................................................................59
圖 5.2以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之(l)實驗]...................................................................................60
圖 5.3以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之a實驗]..........................................................................................61
圖 5.4以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之a實驗]......................................................................................62
圖 5.5以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之b實驗]..........................................................................................63
圖 5.6以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之b實驗]......................................................................................64
圖 5.7以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之ab實驗]........................................................................................65
圖 5.8以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之ab實驗]....................................................................................66
圖 5.9以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之c實驗]..........................................................................................67
圖 5.10以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之c實驗]............................................................................68
圖 5.11以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之ac實驗]......................................................................................69
圖 5.12以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之ac實驗]...........................................................................70
圖 5.13以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之bc實驗]......................................................................................71
圖 5.14以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之bc實驗]..........................................................................72

圖 5.15以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之abc實驗]....................................................................................73
圖 5.16以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之abc實驗].........................................................................74
圖 5.17以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之(l)重複實驗].............................................................................75
圖 5.18以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之(l)重複實驗]..................................................................76
圖 5.19以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之a重複實驗]................................................................................77
圖 5.20以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之a重複實驗]....................................................................78
圖 5.21以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之b重複實驗]................................................................................79
圖 5.22以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之b重複實驗]....................................................................80
圖 5.23以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之ab重複實驗]..............................................................................81
圖 5.24以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之ab重複實驗]..................................................................82
圖 5.25以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之c重複實驗]................................................................................83
圖 5.26以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之c重複實驗]....................................................................84
圖 5.27以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之ac重複實驗]..............................................................................85
圖 5.28以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之ac重複實驗]...................................................................86
圖 5.29以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之bc重複實驗]..............................................................................87

圖 5.30以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之bc重複實驗]..................................................................88
圖 5.31以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之TB圖[因子實驗設計之abc重複實驗]............................................................................89
圖 5.32以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之應答電流之檢量線圖[因子實驗設計之abc重複實驗].................................................................90
圖 5.33以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之偵測極限的 TB 圖..................................................................................................................98
圖 5.34以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之線性範圍的TB 圖..................................................................................................................99
圖5.35以二環戊二烯亞鐵修飾碳糊電極偵測過氧化氫之檢量線圖................................................................................................................100
圖5.36未經修飾之碳糊電極偵測過氧化氫之偵測極限的 TB 圖................................................................................................................101
圖5.37未經修飾之碳糊電極偵測過氧化氫之線性範圍的TB 圖................................................................................................................102
圖5.38未經修飾之碳糊電極偵測過氧化氫之檢量線圖................................................................................................................103
圖 5.39酵素碳糊電極偵測葡萄糖之TB圖............................................................105
圖 5.40酵素碳糊電極偵測葡萄糖之檢量線圖......................................................106
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