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研究生:楊振宇
研究生(外文):Chen-Yu Yang
論文名稱:電流式葡萄糖、酮體以及膽固醇感測基材之研究
論文名稱(外文):Studies of matrices used for glucose, ketone bodies and cholesterol amperometric biosensors
指導教授:王盈錦
指導教授(外文):Yng-Jiin Wang
學位類別:博士
校院名稱:國立陽明大學
系所名稱:醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:215
中文關鍵詞:葡萄糖葡萄糖感測器膽固醇膽固醇感測試片酮體酮體感測試片明膠褐藻酸濕紡纖維
外文關鍵詞:glucoseglucose sensorcholesterolcholesterol stripketon bodiesketone bodies stripgelatinalginatewet spinning fiber
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糖尿病、酮酸症以及膽固醇過高分別為現代常見之文明病。通常為檢測此類之慢性疾病,皆須經過繁雜之檢測儀器以及過程以獲取血糖、酮體或者是膽固醇於血液中之含量。
本研究中試以不同之基材、電子傳遞媒介物以及酵素分別做為一次式血糖、酮體、膽固醇試片以及長時效性檢測之血糖感測電極。在血糖檢測試片中,以調配不同濃度之PVP(polyvinylpyrrolidone)並控制濃度為2.5%(w/w)時,與0.42M ferricyanide混合可完成偵測敏感度最高之感測試片,並且其反應時間僅需約10秒即可達到敏感度0.67 μA/mM,並且符合理想電極在偵測的葡萄糖濃度範圍中,符合酵素動力學中,反應電流與受測物質濃度的一次正比關係。另外,酮體感測試片中,親水性基材PEO(polyethylene oxide)以及酮體去氫酶酵素系統的搭配,在利用ferricyanide為電子傳遞媒介物的環境下,以β-hydroxybutyrate溶液進行檢測,發現在濃度範圍0~5.5mM範圍中,反應時間僅需30秒反應電流與受測物濃度間即可有一正比關係,其線性回歸曲線可以達0.99,且敏感度約為0.67uA/mM。而再利用全血作為測試溶液時,由於全血中干擾物質較多,因此雖30秒時,線性回歸曲線也可以達0.99,但是敏感度則僅為0.46uA/mM。
而在膽固醇試片中,由於膽固醇酯本身為非水溶性物質,因此需要一可以提高大量酵素與受質之間接觸面積之基材,因而選用以第一型膠原蛋白為材料進行電紡所製備出之電紡不織布纖維,搭配膽固醇酯酶以及膽固醇氧化酵素,以ferricyanide為電子傳遞媒介物製備出一次式膽固醇試片。相較於以PEO為基材,電紡基材更能提供試片敏感性,且於在試片滴入膽固醇溶液後約20秒,反應電流與受質濃度間開始有一線性變化,且線性回歸曲線可達0.97,當反應進行90秒以後,線性回歸曲線更可以達到0.999,說明以膠原蛋白電紡而成之基材,對於疏水性受質於試片的偵測敏感度,具有一定之幫助。
第二部分為長時效性之葡萄糖感測電極的研究。首先以HEMA (2-hydroxyethyl methacrylate)以及vinylferrocene(VFc)作為單體(monomer),以AIBN作為起始劑進行共聚反應,並且測得其單體共聚比分別為VFc為0.01以及HEMA為1.96。在合成了不同VFc在共聚物中成分比為10%、16%以及20%並標定為VFc-HEMA(10),VFc-HEMA(16) 以及 VFc-HEMA(20)。分別以上述基材做為葡萄糖感測電極之基材,發現VFc-HEMA(16)其對於葡萄糖濃度的變化,有最佳之反應性,其半大速濃度(Michalis-Menten constants, Km)以及該電極最大反應電流(Jmax)分別為22.6mM, 893 �嫀/cm2 ,比較單純以VFc作為電極基材之電極的14.1mM, 495 uA/cm2,皆高上許多。而在長時效的檢測上,將感測電極置入磷酸鹽緩衝溶液(phosphate buffer)中於40C下保存,經過27小時後VFc-HEMA(16)電流殘留率約有45%,但單純VFc電極電流殘留率則僅剩23%,說明利用共聚反應將VFc固定於高分子鏈中,對於感測電極的敏感度以及保存能力,皆有一定提升之效果。
另外一方面,與VFc的聚合反應中,選擇了另一種水膠單體acrylamide進行共聚反應,同樣在進行共聚反應後,推算出單體共聚比分別為VFc為0.02以及HEMA為1.35。在分別聚合出不同VFc在共聚物中莫耳成分比為10%、15%以及20%並標定為VFc-acrylamide(10)、VFc-acrylamide(15) 以及VFc-acrylamide(20)後,分別將上述高分子作為葡萄糖感測電極基材。在反應電流密度的表現上VFc-acrylamide(10) ≒VFc-acrylamide(15) >VFc-acrylamide(20),於葡萄糖濃度為14.52mM時,約在700�嫀/cm2,高於VFc-HEMA(16)同測定濃度時兩倍之多。在長時效的測定試驗中,相同的VFc-acrylamide(10)於40C PB 27小時後,電流殘留約仍有52.14%左右高於單純VFc為基材之感測電極許多。
除了以人造合成之高分子做為電極包埋基材外,另外也應用濕式紡織技術所生成之明膠(gelatin)線材,作為葡萄糖感測電極之基材。
研究中所用濕紡機將射出速度定義為V0,捲取切線速度則定義為V1。而其之間的比例V1/V0則為此一纖維織拉伸比例。研究中明膠/褐藻酸濕式紡織纖維利用拉伸試驗機測定纖維的機械強度研究中,明膠於混合溶液中的濃度由2.45mg/ml增加至6mg/ml,褐藻酸的混合重量百分比為1.5%(w/w),改變拉伸比0、1、2、3,則纖維之楊氏系數將皆由0.2上升至約0.4 N/mm2,說明改變明膠濃度並不會對於纖維本身的機械強度造成多大之改變影響;反觀隨著拉伸比的上升卻會使的纖維變細、變較為堅韌。
明膠/褐藻酸線材中,完成後的濕式紡織的纖維將以PB於40C下溶液清洗3天,約可以洗去98%褐藻酸,並且明膠與褐藻酸的比例量由一開始的重量比例1:7.5(w/w)開始,慢慢增加明膠之相對含量,發現當明膠與褐藻酸的相對比例達到1:1.67(w/w)或是更高比例褐藻酸時,皆可生成具有孔洞的纖維且現其孔洞約為0.027~0.224um2之間。
研究中,因為纖維的親水性佳以及具備高孔隙度,因此將洗去褐藻酸的纖維,應用於葡萄糖感測電極的開發製備當中。在葡萄糖感測電極研究中,電極基材分別以單純的碳粉以及混有明膠纖維的碳粉,與葡萄糖氧化酵素(Glucose oxidase)以及氧化電子傳遞媒介物(ferrocene)混合後,完成葡萄糖氧化電極。
在葡萄糖氧化電極實驗,藉以調控葡萄糖溶液的添加量,增加葡萄糖的濃度,並以300mV操作電位,進行定電壓的測量。結果顯示,在沒有添加明膠纖維單純以碳粉固定住酵素、電子傳遞媒介物的電極,其敏感度僅為0.26�嫀/cm2mM,反觀在有添加明膠纖維之感測電極,其敏感度可高達0.85 �嫀/cm2mM,約單純碳膠電極之4倍。因此多孔性的明膠纖維材料,對於葡萄糖感測器具有提高其反應性的能力。
Diabetes and cardiovascular disorder are the common diseases of human beings. Tests for blood sugar, ketone bodies and cholesterol are the major components for home care systems. In this study, we have fabricated and tested both test strips and enzyme-based electrodes for these metabolites.
For the disposable glucose testing, the strip constructed from glucose oxidase, ferricyanide(0.42M) and PVP (polyvinylpyrrolidone, 2.5% w/w) has sensitivity of about 0.67 μA/mM after 10 seconds of responding time. For the disposable ketone bodies sensing, the strip composed of β-hydroxybutyrate dehydrogenase enzyme, NAD+, ferricyanide and PEO (polyethyleneoxide) has a linear regression line with β-hydroxybutyrate ranged from 0 to 5.5mM and sensing sensitivity of 0.67uA/mM. For the disposable cholesterol sensing, the strip was constructed from cholesterol oxidase, cholester esterase, ferricyanide and type I collagen e-spun fibers. A linear regression of 0.99 was obtained with cholesterol ranging from 0 to 7.75mM with the sensitivity of 0.08uA/mM.
In the study of long-term detectable glucose enzyme electrode, a series of copolymers comprised of two monomer units: vinylferrocene (VFc) and hydroxyethyl methacrylate (HEMA) were synthesized using AIBN as the initiator for free radical polymerization. The relative polymerization reactivities of these two monomers were 0.01 and 1.96 for VFc and HEMA, respectively. Copolymers with three different VFc molar fractions, [Vfc-HEMA(10), Vfc-HEMA(16), Vfc-HEMA(20)], were synthesized and the VFc-HEMA(16) copolymer was used to construct a glucose enzyme electrode.
The apparent Michalis-Menten constant (Km) and maximal current density (Jmax) of the VFc-HEMA(16) glucose sensor (22.6mM, 893 �嫀/cm2 ) were higher than the that of VFc glucose enzyme electrode (14.1mM, 495 uA/cm2). The VFc-HEMA(16) based glucose electrode has higher stability than VFc-based electrode when stored at 40C or room temperature.
Alternatively, VFc and acrylamide(reactivity of 0.02 and 1.35, respectively) were synthesized when nominal VFc mole contents of 10%, 15% and 20%, designed as VFc-acrylamide(10), VFc-acrylamide(15) and VFc-acrylamide(20).
The glucose sensor based on VFc-acrylamide(15) has a higher reaction current with better stability as compared with VFc-acrylamide(10), VFc-acrylamide(20) and VFc glucose enzyme electrodes, with approximately two times reaction current as compared with VFc-HEMA(15) at the glucose concentration of 14.52mM.
Other than synthetic polymers, gelatin fibers containing interconnected porous structure can be used as the matrix of enzyme electrode for glucose sensing. In this study, we have fabricated porous gelatin fibers by wet-spinning process, and the application of these porous gelatin fibers for enzyme immobilization was demonstrated. The glucose oxidase and ferrocencarboxaldehyde were immobilized on gelatin wet spinning fiber with immobilization ratio of glucose oxidase of 14.9%. The gelatin fiber-glucose oxidase amperometric sensor has responding current of about 15 uA/cm2 with 43% of current retained after stored at 250C PB solution for 20 hours. This compares with the gelatin fiber-ferrocene amperometric sensors of 100 uA/cm2 with 43% of current retained after 48 hours at 250C.
第一章 背景介紹
1.1糖尿病、酮酸症以及膽固醇介紹 1
1.2生物感測器 9
1.3電化學理論 11
1.3.1循環伏安法(Cyclic voltammetry , CV) 11
1.3.2酵素催化系統 14
1.3.3 Apparent Michaelis-Menten Constant 15
1.4葡萄糖、酮體以及膽固醇酵素感測器原理 21
1.4.1葡萄糖酵素感測電極原理 21
1.4.2酮體感測電極原理 23
1.4.3膽固醇酵素感測電極原理 25
1.5電子媒介物(Electron Mediator)簡介 27
1.5.1電子媒介物之功用 27
1.5.2電子媒介物固定方式 28
1.5.3 Ferrocene簡介 29
1.5.4 Potassium Ferricyanide簡介 32
1.6甲基丙烯酸-2-羥基乙酯(HEMA)以及丙烯醯胺 (acrylamide)簡介 32
1.6.1 Poly(HEMA) [Poly(2-hydroxy ethylmethacrylate) ] 34
1.6.2 Poly(Acrylamide) 36
1.7共聚單體之反應性比理論 38
1.8第一型膠原蛋白(Type I collagen)及明膠(Gelatin)簡介 40
1.8.1 第一型膠原蛋白(Type I collagen) 40
1.8.2 明膠(Gelatin)簡介 41
1.9 膠原蛋白細絲以及明膠線材(threads)之應用 44
1.10一次式感測電極試片以及長時效感測電極之設計 49

第二章 實驗設備與藥品 52
2.1實驗藥品 52
2.2實驗儀器 53

第三章葡萄糖感測試片之研究
3.1研究動機 55
3.2實驗方法 55
3.2.1葡萄糖感測試片之製作 55
3.2.2葡萄糖感測試片循環伏安儀測定 56
3.2.3葡萄糖感測試片定電位測試 57
3.3結果與討論 57

第四章 酮體感測試片之研究
4.1研究動機 63
4.2實驗方法 63
4.2.1酮體感測試片之製作 63
4.2.2酮體感測試片循環伏安儀測定 65
4.2.3酮體感測試片定電位測試 66
4.3結果與討論 66

第五章 膽固醇感測試片之研究
5.1研究動機 73
5.2實驗方法 73
5.2.1第一型膠原蛋白(Type I collagen)的製備 73
5.2.2膠原蛋白純度鑑定 75
5.2.3第一型膠原蛋白之濃度測定 78
5.2.4膽固醇感測試片製作 79
5.2.5膽固醇感測試片循環伏安儀測試 80
5.2.6膽固醇感測試片定電位測試 81
5.3結果與討論 82

第六章 Poly(VFc-co-HEMA)及poly(VFc-co-Acrylamide)為基材之長時效葡萄糖感測電極
6.1研究動機 88
6.2基材合成實驗 88
6.2.1合成poly(vinylferrocene-co-2-hydroxyethyl methacrylate) 88
6.2.2合成poly(vinylferrocene-co-Acrylamide) 90
6.2.3聚合物中二茂鐵(vinylferrocene)含量測試 91
6.2.4共聚單體的反應性比(reactivity ratios)測定 91
6.2.5分子量之測定 93
6.3電極之表面改質 93
6.3.1碳膠電極之製成(葡萄糖電極) 93
6.4電化學測試
6.4.1循環伏安儀測試 95
6.4.2定電位測試(Stationary potential measurement) 96
6.4.3葡萄糖碳膠電極之保存安定性 97
6.5結果與討論 98
6.5.1 Poly(vinylferrocene-co-HEMA)之合成 98
6.5.2共聚單體之反應性比 100
6.5.3分子量分佈 103
6.5.4電極之電化學分析及葡萄糖分析 104
6.5.4.1固定電壓之測量 104
6.5.4.2酵素電極的反應動力常數 109
6.5.4.3酵素電極之保存安定性測定 111
6.5.5 Poly(vinylferrocene-co-acrylamide)之合成 115
6.5.6共聚單體之反應性比 117
6.5.7電極之電化學分析及葡萄糖分析 119
6.5.7.1定電壓之測定分析 119
6.5.7.2酵素電極之保存安定性測定 121

第七章 以明膠濕紡纖維為基材之長時效炮萄糖感測電極
7.1研究動機 126
7.2 Gelatin 濕式紡織(Gelatin wet spinning fiber)之製備 126
7.2.1 Gelating濕紡纖維製作方法 126
7.2.2 Gelatin wet spinning fiber表面孔洞生成條件測試 127
7.2.3機械強度測試 128
7.2.4 Alginate洗出測試 129
7.2.5細絲吸水率測定 129
7.2.6掃描式電子顯微鏡(SEM)樣本之製備 130
7.2.7葡萄糖氧化酵素與ferrocencarboxaldehyde於
gelatin細絲上的固定 130
7.3電極之表面改質 133
7.4電化學測試 134
7.4.1循環伏安儀測試 134
7.4.2定電位測試 135
7.4.3葡萄糖碳膠電極之保存安定性 135
7.5結果與討論 136
7.5.1Gelatin/alginate 濕式紡織纖維製備 136
7.5.2 Gelatin/alginate濕紡纖維含水率測定 140
7.5.3 Gelatin/alginate濕紡纖維機械性質測試 142
7.5.4 Gelatin/alginate濕式紡織纖維alginate洗出試驗 145
7.5.5 Gelatin wet spinning fiber表面孔洞生成條件測試 148
7.5.6 Gelatin濕式紡織纖維於葡萄糖感測電極上之應用 160
7.5.6.1 Gelatin纖維/碳膠電極與單純碳膠電極之比較 160
7.5.6.2 Gelatin纖維與葡萄糖氧化酵素化學鍵結固定實驗 162
7.5.6.3化學固定以及物理吸附酵素之感測電極長時效性檢測比較 166
7.5.6.4 Gelatin纖維與ferrocencarboxaldehyde化學鍵結固定實驗 170
7.5.6.5化學固定以及物理吸附ferrocencarboxaldehyde之感測電極長時效性檢測比較 176

第八章 結論及未來方向 180
第九章 參考文獻 183
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