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研究生:王可瑄
研究生(外文):Ke-HsuanWang
論文名稱:葡萄糖氧化酵素在氣/液界面的吸附行為及其對葡萄糖生物感測器特性影響的研究
論文名稱(外文):The studies on the adsorption behavior of glucose oxidase and its effect on the characteristics of glucose sensors
指導教授:李玉郎
指導教授(外文):Yuh-Lang Lee
學位類別:博士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
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:2012
畢業學年度:100
語文別:中文
論文頁數:157
中文關鍵詞:吸附行為葡萄糖氧化酵素Langmuir-Blodgett沉積技術蛋白質二級結構單分子膜模板葡萄糖感測器
外文關鍵詞:adsorptionglucose oxidaseLangmuir-Blodgett techniquesecondary structuretemplate monolayerglucose biosensor
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本研究探討葡萄糖氧化酵素(GOx)由水溶液中吸附至氣/液界面的吸附行為,且分析GOx的吸附行為對固定酵素薄膜感測特性的影響。藉由GOx的表面壓-吸附時間曲線(π-t)、表面壓-每分子佔據面積等溫線(π-A)、遲滯曲線及布魯斯特角顯微鏡(BAM),了解GOx的吸附行為及其與Langmuir單分子層的交互作用。以原子力顯微鏡(AFM)觀察GOx LB膜表面形態。GOx二級結構及GOx含量分別由圓二色光譜儀(CD)及紅外線光譜儀(FTIR)量測。葡萄糖感測器特性分析則採用安培法進行討論。
GOx由溶液吸附至氣/液界面,根據其表面壓變化可以分成二個階段。於第一吸附階段,GOx以單分子膜形式存在於氣/液界面上,由於界面-分子間作用力,GOx構形以β-sheet結構為主。持續吸附至界面的GOx促使第二吸附階段的發生,原先吸附於界面的GOx被推擠出氣/液界面,形成多層GOx分子膜,引起GOx構形轉換成α-helix結構。
由葡萄糖感測實驗結果得知,α-helix構形的GOx具有較高的電化學活性,藉由硬酯胺(ODA)單分子膜為模板吸附GOx,除了可以增加GOx的吸附量,利用分子之間的靜電吸引力,GO分子能分布均勻地併入ODA單分子膜,避免GOx聚集堆疊的行為而降低酵素催化能力。具有緊密均相的固定化酵素薄膜也能藉由阻隔棒的壓縮控制來製備。利用逐層沉積於白金電極基板上修飾緊密規則排列的金奈米粒子分子膜,及GOx/金奈米粒子分子膜,可製備一高靈敏度(0.521 μA•cm-2•mM-1)及短應答時間(12 s)之生物感測器。

The adsorption behavior of glucose oxidase (GOx) from aqueous solution to the air/liquid interface was studied. The GOx monolayer at the air/liquid was transferred on the surstrate for application to glucose sensor. With the variation of surface pressure in the adsorption stage (π-t), pressure-area (π-A) isotherms, hysteresis curves, and the Brewster angle microscopy (BAM), the GOx adsorption and the interaction between GOx and Langmuir monolayer were elucidated. The morphology of GOx LB films was also examined by the observation of atomic force microscopy (AFM). The GOx secondary structure and the GOx amount were examined by a circular dichroism (CD) spectroscopy and Fourier transform infrared spectroscopy (FTIR). Amperometric measurements were made for determining the performance of assembled glucose sensors.
The adsorption of GOx from aqueous solution to an air/liquid interface can be classified into two stages according to the surface pressure variation. In the first adsorption stage, GOx adsorb as a single-molecular layer, extended by the interface-molecule interaction, and organize as a mainly β-sheet conformation. The second adsorption stage is initiated by further adsorption of GOx into the interface, pushing up the pre-adsorbed proteins away from the interface, forming GOx multilayer, and inducing a conformational transition into α-helix.
The glucose sensing experiments demonstrate that GOx with α-helix conformation has a much higher electroacivity than the β-sheet. The electrostatic attractive interaction between octadecylamine (ODA) and GOx not only enchance the amount of adsorbed GOx, but also promote the uniformity of ODA and GOx to avoid the reduction in GOx catalytic ability. It is possible to prepare an immobilized enzyme film with condensed and homogeneous phase by surface-film compression. Overall, the {AuNPs-1/GOxAuNPs-2} sensor prepared by layer-by-layer deposition with a close-packed AuNP monolayer and the GOx/AuNPs monolayer exhibited the highest current sensitivity (0.521 μA•cm-2•mM-1) and the fastest response time (within 12 s).
第1章 緒論 1
1-1 前言 1
1-2 研究動機及目的 2
第2章 文獻回顧 3
2-1 Langmuir-Blodgett單分子膜簡介 3
2-1-1 Langmuir-Blodgett單分子膜 3
2-1-2 Langmuir-Blodgett薄膜製備 14
2-2 蛋白質簡介 17
2-2-1 蛋白質結構與特性 17
2-2-2 穩定蛋白質結構之因素 21
2-2-3 牛血清蛋白,溶菌酶及葡萄糖氧化酵素簡介 24
2-2-4 蛋白質於氣/液界面的吸附行為 28
2-3 感測器簡介 31
2-3-1 生物感測器的發展及反應機制 32
2-3-2 Langmuir-Blodgett法製備生物感測器 36
第3章 實驗材料與方法 39
3-1 實驗材料與藥品 39
3-2 實驗裝置及方法 41
3-2-1 Langmuir-Boldgett沉積裝置 41
3-2-2 布魯斯特角顯微鏡 45
3-2-3 原子力顯微鏡 47
3-2-4 圓二色光譜儀 48
3-2-5 傅立葉轉換紅外光光譜儀 50
3-2-6 奈米金粒子合成及粒徑測定 52
3-2-7 穿透式電子顯微鏡 53
3-2-8 恆電位儀 54
第4章 實驗結果與討論 56
4-1 葡萄糖氧化酵素於氣/液界面的吸附行為 56
4-1-1 葡萄糖氧化酵素之濃度效應對其吸附行為的影響 56
4-1-2 單分子膜頭基效應對葡萄糖氧化酵素吸附行為的影響 63
4-1-3 濃度效應對表面壓-時間吸附曲線的影響 91
4-2 蛋白質於氣/液界面的構形轉換機制 96
4-2-1 牛血清蛋白與溶菌酶於氣/液界面之吸附行為 96
4-2-2 蛋白質於氣/液界面之構形變化 99
4-3 LB沉積技術製備電流式葡萄糖感測器 106
4-3-1 葡萄糖氧化酵素構形對於感測性能的影響 109
4-3-2 單分子膜模板對於感測性能的影響 114
4-3-3 固定化酵素薄膜形態對感測性能的影響 120
4-3-4 LB膜層數對於感測性能的影響 128
4-4 金奈米粒子修飾電流式葡萄糖感測器 132
4-4-1 葡萄糖氧化酵素與金奈米粒子於氣/液界面的吸附行為 133
4-4-2 金奈米粒子的組裝方式對於感測性能的影響 139
第5章 結論與後續工作建議 147
參考文獻 150

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