陳林祈Chen, Lin-chi臺灣大學:生物產業機電工程學研究所余宗穆Yu, Chung-MuChung-MuYu2010-05-052018-07-102010-05-052018-07-102009U0001-2907200916301700http://ntur.lib.ntu.edu.tw//handle/246246/180317本研究以生物材料為基材開發高穩定性與生物相容性之酵素陽極並加入奈米碳管有效提高其對葡萄糖催化之靈敏度，製程簡便快速且節省成本。研究中生物燃料電池陰陽極組成分別選用葡萄糖氧化酵素與漆氧化酵素，陽極媒介分子為BZQ（1, 4-benzoquinone）或DHB（2, 5-dihydroxybenzaldehyde），在不同部分的實驗中有所選擇，陰極為ABTS。為瞭解酵素型生物燃料電池基本運作，本研究先以較簡便的滴乾吸附法進行酵素與媒介分子固定，吸附式酵素電極陽極與陰極分別能對葡萄糖與氧氣進行催化產生催化電流，且分別在有隔膜與無隔膜的生物燃料電池系統中以葡萄糖溶液為燃料發電。然而此電極於水溶液時酵素與媒介分子快速脫附使穩定性極差。SPCE/CNT/BSA-GOx-DHB奈米構裝酵素陽極上成功的以牛血清蛋白為基團共價鍵結帶有醛基的DHB，並以交聯反應固定葡萄糖氧化酵素，完成了高穩定性的酵素電極。利用奈米碳管修飾電極表面提高其電化學特性使其催化葡萄糖氧化的響應電流增加近100倍，在循環伏安掃描法0.5 V處的催化電流由0.5 μA/cm2上升至45 μA/cm2。而在最佳化電極製程後，酵素電極對葡萄糖的催化電流更上升至263 μA/cm2。由循環伏安掃描得到電極上有3.82×10-9 mole/cm2 DHB固定量。以流動注射式分析對葡萄糖進行感測可得到連續穩定的響應，是酵素電極穩定的證據，由不同濃度葡萄糖的催化電流回歸得KM值130.1 ± 23.60 mM，證明固定後的酵素活性依然被保存沒有被改變。而浸泡緩衝溶液保存到第七天仍可正常工作，保有85 %的催化活性。以SPCE/CNT/BSA-GOx-DHB酵素電極配合漆氧化酵素溶液陰極，組成的葡萄糖生物燃料電池在線性掃描伏安法0.25 V時有最大功率24.33 μW/cm2，開環電位0.68 V。以定電位放電測量得到的功率最大值為16.13 μW/cm2。在製備電極的交聯聚合反應中，分別加入奈米碳管與奈米金完成SPCE/CNT/CNT-BSA-GOx-DHB以及SPCE/CNT/AuNP-BSA-GOx-DHB酵素電極，因為增加了酵素電極反應層中的導電性，酵素電極在流動注射式分析的靈敏度約可增加40 %的效果。而在組成生物燃料電池時增加了大約10 %最大功率。In this study, we developed a highly stable and biocompatible bioanode based on biomaterials with a simple, fast and cost-effective process, and enhanced we its catalytic activity for glucose by carbon nanotubes (CNT). In our research, glucose oxidase (GOx) and laccase were chosen as biocatalysts for the oxidation of glucose and the reduction of oxygen, respectively, in biofuel cells. The chosen mediators for the bioanodes were 1,4-benzoquinone or 2,5-dihydroxybenzaldehyde (DHB) depending on the immobilizing methods, and ABTS was the mediator for cathode. To develop a reliable enzymatic biofuel cell, we prepared enzymatic electrodes by adsorption method first, which is an easier and faster way. A biofuel cell assembled by such a bioelectrode can generate energy with a maximum power density of ca. 16 μW/cm2 for a two- compartment system, and ca. 9 μW/cm2 for a membraneless system. However, there is a serious leaching problem of enzymes and mediators, and it caused the unstability of bioelectrodes. A highly stable nano-assembled bioanode, SPCE/CNT/BSA-GOx-DHB, was developed by covalent immobilization of GOx and DHB with a cross-linked bovine serum albumin matrix. The bioanode showed reversible redox activity of DHB, and the amount of immobilized DHB on the electrode was evaluated ca. 3.82×10-9 mole/cm2. The catalytic response of glucose increased more than 100 times when we modified the bioanode with CNT, and it’s further improved by optimizing the crosslinking process. In flow inject mode, the bioanode showed steady and reproducible responses to glucose oxidation under continuous detection. The KM of bioanode was determined to be 130.1 ± 23.60 mM, which indicates that the activity of glucose oxidase is kept after crosslinking. And the bioanode also showed high storage stability, where more than 85% of activity after storage in PBS solution for 7 days. To construct the glucose/O2 biofuel cell, we assembled the SPCE/CNT/BSA-GOx-DHB anode with a laccase solution cathode. The cell’s open circuit voltage was 0.68 V and its maximum power density was 16.13 μW/cm2 at 0.2 V in pH 7 PBS at 25 ℃ with 100 mM glucose. CNT and gold nanoparticles (Au-NP) were added in the crosslinking mixture, respectively, to improve the conductivity, and it increased 10 % of the maximum power density.致謝 i要 iibstract iii錄 iv目錄 viii目錄 xii一章 前言與研究目的 1-1 前言 1-2 研究目的 3-3 研究架構 4二章 文獻探討 5-1 生物燃料電池簡介 5-2 生物燃料電池之原理 6-3 生物燃料電池之酵素電極技術發展 10三章 研究方法 13-1 實驗儀器與設備 14-2 實驗藥品 15-3 電化學分析方法 17-3-1 三極式電化學分析系統 17-3-2 二極式電化學分析系統 18-3-2-1 開環電位測量 19-3-2-2 線性掃描伏安法 19-4 以吸附法製備之酵素電極 20-4-1 網印碳電極製備 20-4-2 以PEDOT-PSS吸附之酵素電極製備 21-4-3 吸附式酵素電極單極測試 21-4-4 吸附式酵素燃料電池測試 21-5 奈米構裝酵素電極之開發 22-5-1 奈米碳管-網印碳電極之製備 22-5-2 奈米構裝酵素陽極之製備 23-5-3 奈米構裝酵素陽極之分析 24-5-3-1 酵素陽極之循環伏安分析 24-5-3-2 酵素陽極之線性掃描伏安分析 24-5-3-3 流動注射式系統之電流時間法分析 24-5-3-4 酵素陽極之材料特性分析 25-5-4 奈米構裝酵素陽極效能提升評估 25-5-5 奈米構裝酵素陽極於生物燃料電池之應用 25-5-5-1 奈米構裝酵素陽極組成之生物燃料電池 25-5-5-2 生物燃料電池操作條件對其效能之影響 25四章 結果與討論 26-1 以吸附法製備之生物燃料電池效能分析 26-1-1 吸附式酵素電極工作原理 26-1-2 酵素電極循環伏安法分析 27-1-3 酵素電極對葡萄糖與氧氣之催化反應 28-1-4 酵素電極組成之生物燃料電池效能測試 28-2 奈米構裝酵素陽極特性分析 35-2-1 奈米構裝酵素陽極之工作原理 35-2-2 電極製備清洗條件決定 37-2-3 奈米構裝酵素陽極之電化學特性 42-2-4 奈米碳管對奈米構裝酵素陽極效能增進 50-2-5 葡萄糖氧化酵素固定方法分析 53-2-6 媒介分子DHB固定方法之分析 58-2-7 以BZQ為媒介分子之奈米構裝酵素電極 59-3 奈米構裝酵素陽極之效能改善 64-3-1 交聯環境對酵素電極之影響 64-3-2 酵素包覆量對電極效能之影響 67-3-3 DHB用量對電極效能之影響 67-3-4 牛血清蛋白用量對電極效能之影響 68-4 流動注射式分析法對奈米構裝酵素陽極之分析 72-5 奈米構裝酵素陽極於生物燃料電池應用 80-5-1 奈米酵素陽極葡萄糖燃料電池組成與功率分析 80-5-2 定電位放電法之功率測量 86-5-3 陽極環境酸鹼值對生物燃料電池之影響 89-6 以奈米材料進行奈米構裝酵素陽極之改善 91五章 結論與建議 95-1 結論 95-2 建議 98考文獻 99application/pdf2174881 bytesapplication/pdfen-US生物燃料電池酵素電極葡萄糖氧化酵素奈米碳管奈米材料2, 5-dihydroxybenzaldehyde牛血清蛋白biofuel cellenzyme electrodeglucose oxidasecarbon nanotubenano-materialsbovine serum albuminthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/180317/1/ntu-98-R96631016-1.pdf