臺灣博碩士論文加值系統

本論文中以導電性聚苯胺(PANI)為電極表面修飾高分子，以循環伏安法(CV)聚合PANI於Pt/Al2O3電極表面製備成PANI/Pt/Al2O3電極，再使用電吸附法固定化HRP酵素並以戊二醛(GA)為交聯劑，製備GA-HRP/PANI/Pt/Al2O3電極。另外，在PANI聚合過程中添加牛血清蛋白(BSA)，分別製備為PANI(BSA)/Al2O3與GA-HRP/PANI(BSA)/Al2O3兩種電極。此外，研究過程中亦合成中孔矽材料SBA-15，作為HRP固定化的載體，應用於GA/SBA-15(HRP)/PANI/Pt/Al2O3電極的製備。將上述製備所得的電極使用CV法感測過氧化氫，探討電極之感測特性。另外，探討了溶氧對於PANI/Pt/Al2O3電極感測過氧化氫的影響，並嘗試添加除氧劑於感測系統中，去除溶氧的影響。
分別以GA-HRP/PANI/Pt/Al2O3與GA-HRP/PANI(BSA)/Pt/Al2O3電極於0.1 M磷酸鹽緩衝溶液(pH 6.2)中感測過氧化氫，其感測濃度線性範圍為10 μM ~ 23.9 mM與10 μM ~ 35.2 mM，感測靈敏度由37.55提升至44.31 μA mM-1。在穩定性測試，分別使用PANI/Pt/Al2O3與PANI(BSA)/Pt/Al2O3電極於17天中21次感測2.39 mM的過氧化氫，可發現在第17天的剩餘感測活性由PANI/Pt/Al2O3電極的40.8 %提升至PANI(BSA)/Pt/Al2O3電極的80.5 %，顯示PANI中包埋BSA對於感測的靈敏度與穩定性有顯著的提升。在干擾測試方面，分別添加濃度為0.5 mM的尿酸 、尿素與維它命C於含有2.39 mM過氧化氫的感測系統中，對於GA-HRP/PANI/Pt/Al2O3電極的感測靈敏度與還原電流的影響皆小10 %。
在GA-HRP/PANI/Pt/Al2O3與GA/SBA-15(HRP)/PANI/Pt/Al2O3電極比較部份，於0.1 M磷酸鹽緩衝溶液(pH 6.2)中感測過氧化氫，感測濃度線性範圍皆為10 μM ~ 23.9 mM，GA/SBA-15(HRP)/PANI/Pt/Al2O3電極的靈敏度由不含SBA-15的37.55 μA mM-1下降至25.05 μA mM-1，於16天中15次感測1.96 mM的過氧化氫，在第16天的感測剩餘感測活性由GA-HRP/PANI/Pt/Al2O3電極的40.5 %提升至PANI(BSA)/Pt/Al2O3電極的75.2 %，顯示HRP酵素固定於SBA-15中對於活性的穩定有明顯的幫助。
使用除氧劑去除溶液中溶氧的影響部份，實驗結果顯示添加硫代硫酸鈉可有效去除溶液中溶氧，當添加濃度小於1 mM時，可有效去除溶液中的溶氧並不會影響過氧化氫的感測濃度。

In this study, the PANI or PANI(BSA) films was electrochemically synthesized on the Pt/Al2O3 base to form the PANI/Pt/Al2O3 or PANI(BSA)/Pt/Al2O3 electrode, and horseradish peroxidase (HRP) was then immobilized in PANI or PANI(BSA) films to construct the GA-HRP/PANI/Pt/Al2O3 or GA-HRP/PANI(BSA)/Pt/Al2O3 biosensor. Furthermore, the mesoporous silica SBA-15 was prepared and was employed to entrap HRP in order to construct a GA/SBA-15(HRP)/PANI/Pt/Al2O3 electrode. The properties and the performances of those bio-electrodes were fully investigated. Moreover, the influence of the dissolved oxygen on sensing of hydrogen peroxide with those PANI modified electrode was discussed and the possible elimination was proposed by using oxygen scavengers.
For electrodes, such as GA-HRP/PANI/Pt/Al2O3 and GA-HRP/PANI(BSA)/Pt/Al2O3, the linear correlation for sensing hydrogen peroxide in a 0.1 M phosphate buffer were obtained in the range of 10 μM ~ 23.9 mM and 10 μM ~ 35.2 mM, respectively, while the sensitivities were 37.55 and 44.31μA mM-1, respectively. By comparing the stability after sensing 2.39 mM hydrogen peroxide for twenty-one times in a 17-day period, the percentage of residual response current were 40.8 % for PANI/Pt/Al2O3 and 80.5 % for PANI(BSA)/Pt/Al2O3, indicating that BSA was useful for improving the sensitivity and stability of PANI modified hydrogen peroxide biosensor. In addition, 0.5 mM of uric acid, urea, or ascorbic acid would cause less than 10% reduction of cyclic voltammetry (CV) current on sensing 2.39 mM hydrogen peroxide for GA-HRP/PANI/Pt/Al2O3.
For the electrode GA/SBA-15(HRP)/PANI/Pt/Al2O3, the linear correlation and the sensitivity for sensing hydrogen peroxide sensor in a 0.1 M phosphate buffer were obtained in the range of 10 ~ 23.9 mM and 25.05 μA mM-1, respectively. After sensing 1.96 mM hydrogen peroxide for fifteen times in a 16-day period, the percentage of residual response current were 75.2 %, which was better than that of GA-HRP/PANI/Pt/Al2O3 (40.5%), implicating that SBA-15 was also able to stabilize the entrapped HRP and therefore improved the performance of the constructed biosensor.
Finally, we demonstrated the influence of the dissolved oxygen on sensing hydrogen peroxide with PANI modified electrodes, and we proposed strategies to eliminate the influences. Our results inindicated that the oxygen scavenger such as sodium thiosulfate was able to effectively remove the dissolved oxygen, thereby reduce its effect on the performance of electrode, Meanwhile, sodium thiosulfate displayed the negligible effect on hydrogen peroxide measurements while its applied concentration was below 1 mM.

中文摘要..................................................................................................Ⅲ
Abstract.....................................................................................................Ⅴ
誌謝..........................................................................................................Ⅶ
目錄..........................................................................................................Ⅸ
圖目錄.................................................................................................. .XVI
表目錄..................................................................................................XXVI

第一章 緒論............................................................................................1
1.1 過氧化氫之簡介...........................................................................1
1.2 生物感測器之簡介.......................................................................4
1.2.1 生物感測器之定義與發展歷史回顧....................................4
1.2.2 生物感測器的分類..............................................................10
1.2.3 過氧化氫生物感測器之文獻回顧.................... .................15
1.3 酵素之簡介.................................................................................18
1.3.1 酵素之命名..........................................................................18
1.3.2 酵素的分類..........................................................................19
1.3.3 酵素的反應特性..................................................................20
1.3.4 辣根過氧化酵素(HRP)之簡介............................................21
1.3.4.1 HRP之結構介紹...........................................................22
1.3.4.2 HRP酵素催化過氧化氫反應機制...............................24
1.3.5 酵素固定化方法..................................................................24
1.4 導電性高分子簡介.....................................................................29
1.4.1 發展史..................................................................................29
1.4.2 導電性高分子的掺雜..........................................................32
1.4.3 導電性高分子之能帶理論與導電原理..............................32
1.4.4 導電性高分子的應用..........................................................36
1.4.5 導電性高分子聚苯胺..........................................................38
1.4.5.1 聚苯胺之結構型態與顏色變化...................................38
1.4.5.2 聚苯胺之合成...............................................................42
1.4.5.3 聚苯胺之紅外線光譜分析...........................................43
1.5 中孔矽材料簡介.........................................................................45
1.5.1 中孔矽材料之分類與發展歷史回顧..................................45
1.5.2 非離子界面活性劑介紹......................................................48
1.5.3 SBA-15之孔洞性質分析....................................................50
1.5.4 中孔矽材料之應用..............................................................60
1.6 研究動機.....................................................................................62

第二章 研究架構簡介..........................................................................64

第三章 研究設備與藥品......................................................................70
3.1 實驗儀器.....................................................................................70
3.2 實驗藥品.....................................................................................71
3.3 研究中所使用之貴重儀器簡介.................................................73

第四章 過氧化氫感測電極製備與感測性質分析..............................79
4.1 研究目的.....................................................................................79
4.2 實驗方法與程序.........................................................................80
4.2.1 溶液配製..............................................................................80
4.2.2 過氧化氫濃度標定..............................................................82
4.2.3 蛋白質定量分析..................................................................84
4.2.4 HRP酵素活性測定.............................................................86
4.2.5 電極之製備..........................................................................88
4.2.5.1 Ag/AgCl(3M NaCl)參考電極之製備...........................88
4.2.5.2 Pt/Al2O3電極之製備.....................................................89
4.2.5.3 PANI/Pt/Al2O3電極之製備...........................................91
4.2.5.4 GA-HRP/PANI/Pt/Al2O3酵素電極之製備...................93
4.2.6 過氧化氫感測性質測定......................................................95
4.3 實驗結果與討論.........................................................................97
4.3.1 過氧化氫濃度標定..............................................................97
4.3.2 電極之製備與感測性質測定..............................................99
4.3.2.1 PANI/Pt/Al2O3 電極之製備..........................................99
4.3.2.2 PANI/Pt/Al2O3 電極之表面型態觀察........................103
4.3.2.3 Pt/Al2O3 與PANI/Pt/Al2O3電極之性質分析.............105
4.3.2.4 GA-HRP/PANI/Pt/Al2O3酵素電極之PANI
聚合圈數對過氧化氫感測性質的影響....................108
4.3.2.5 戊二醛(GA)使用體積與濃度對於GA-HRP/PANI/Pt/Al2O3電極感測之影響................113
4.3.2.6 感測溶液pH值對於GA-HRP/PANI/Pt/Al2O3電
極感測之影響..............................................................119
4.3.2.7 GA-HRP/PANI/Pt/Al2O3電極感測之選擇性............121
4.3.2.8 使用GA-HRP/PANI/Pt/Al2O3電極感測尿液樣品…127
4.3.2.9 PANI/Pt/Al2O3與GA-HRP/PANI/Pt/Al2O3電
極之穩定性探討..........................................................130
4.4 小結..........................................................................................132

第五章 探討添加牛血清蛋白對於聚苯胺高分子膜的影響............133
5.1 研究目的...................................................................................133
5.2 實驗方法與程序.......................................................................134
5.2.1 PANI(BSA)/Pt/Al2O3電極之製備.....................................134
5.2.2 GA-HRP/PANI(BSA)/Pt/Al2O3酵素電極之製備.............135
5.3 實驗結果與討論.......................................................................137
5.3.1 PANI(BSA)/Pt/Al2O3 複合膜電極之製備與性質分析...137
5.3.1.1 PANI(BSA)/Pt/Al2O3 電極之製備.............................138
5.3.1.2 BSA最適添加量探討................................................140
5.3.1.3 FTIR 分析..................................................................144
5.3.1.4 TGA 分析...................................................................146
5.3.1.5 探討添加BSA所製備的電極對於過氧化氫
感測穩定性的影響......................................................148
5.4 小結..........................................................................................156

第六章 中孔矽材料SBA-15之合成與製備為電極之
感測性質分析........................................................................157
6.1 研究目的...................................................................................157
6.2 實驗方法與程序.......................................................................159
6.2.1 SBA-15 之合成.................................................................159
6.2.2 以SBA-15為載體固定HRP酵素探討...........................161
6.2.3 GA/SBA-15(HRP)/PANI/Pt/Al2O3酵素電極之製備........164
6.3 實驗結果與討論.......................................................................166
6.3.1 SBA-15之製備條件探討..................................................166
6.3.1.1 TGA分析....................................................................167
6.3.1.2 SEM與TEM分析......................................................169
6.3.1.3 XRD分析....................................................................175
6.3.1.4 BET分析.....................................................................181
6.3.2 以SBA-15為載體固定HRP酵素探討...............................195
6.3.2.1 探討不同含浸方法對於HRP酵素固定量的影響....195
6.3.2.2 探討吸附時間及pH值對於HRP酵素固定量的
影響............................................................................201
6.3.2.3 GA-/SBA-15(HRP)/PANI/Pt/Al2O3電極之性質
分析............................................................................208
6.3.2.4 GA-/SBA-15(HRP)/PANI/Pt/Al2O3電極之穩
定性探討....................................................................213
6.4 小結..........................................................................................215

第七章 探討過氧化氫感測過程中溶氧的影響................................216
7.1 研究目的...................................................................................216
7.2 實驗方法與程序.......................................................................217
7.2.1 溶氧感測性質測定............................................................217
7.3 實驗結果與討論.......................................................................218
7.3.1 探討過氧化氫感測過程中溶氧的影響............................218
7.3.1.1 Pt/Al2O3電極部份.......................................................218
7.3.1.2 PANI/Pt/Al2O3電極部份.............................................223
7.3.2 探討添加除氧劑之除氧效果及對於過氧化氫
感測的影響........................................................................227
7.4 小結..........................................................................................232

第八章 綜合討論................................................................................233
8.1 探討HRP酵素對於過氧化氫感測電流的貢獻......................233
8.2 比較擴孔劑添加量對於SBA-15吸附HRP酵素的影響…...239
8.3 探討HRP酵素是否固定於SBA-15孔洞中............................243
8.4 本研究中自製之過氧化氫感測電極的感測性質並
與文獻比較..............................................................................246

第九章 結論與建議............................................................................249

參考文獻................................................................................................252

個人簡歷................................................................................................264

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