本實驗利用一氧化二銅(Copper(I) oxide)催化NH3還原的特性，配合Urease (EC 3.5.1.5)可將尿素水解產生NH3，將酵素與一氧化二銅分別修飾在雙玻璃碳電極之上下游，結合流注系統發展低電位氧化模式偵測，並以恆電位儀控制電位及接收訊號，以達發展尿素生化感測器之目的。
    此尿素生化感測器之最佳化製備條件是用碳膏與20%一氧化二銅均勻混和後，修飾在下游電極表面，置於40 ℃烘箱30分鐘乾燥後在上游電極滴加0.5 unit Urease (EC 3.5.1.5)，靜置於4℃乾燥後再滴1 % 0.5 μl小牛血清蛋白，靜置於4 ℃乾燥後再1 % 0.5μl戊二醛，靜置於4 ℃乾燥即完成電極製備。在最佳化條件下，偵測環境為0.05 M pH 9 碳酸鹽緩衝溶液，偵測電位為200 mV (vs. Ag/AgCl)，載體流速為0.5 ml/min，樣品迴路體積為50 μl，進行尿素的量測，所得此感測器分析特性如下：線性範圍為1-10 mM(R=0.99)，電流密度為85.736 nA/mM，偵測極限(S/N=3)為85 μM，在精確度方面連續重覆偵測尿素20次操作下，所得到的相對標準差(RSD)為1.8%。且若在雙電極系統的上游電極修飾PbO2進行氧化前處理，可以避免環境中易氧化干擾物如多巴胺、腎上腺素、血清素、組織胺、乙醯苯酚、尿酸、抗壞血酸等。最後將此生化電極與標準方法進行相關性探討，其相關係數為0.995。

The copper(I) oxide based urea biosensor was fabricated in this experiment, and it possessed reduced overvoltage for ammonia determination. The enzyme Urease (EC 3.5.1.5) was drop-coated on the up-stream of dual electrode and subsequently
the NH3 was determination through copper(I) oxide modified down-stream
electrode.

    The preparation of this biosensor was as follows, carbon ink and 20% copper(I) oxide had been well mixed and drop-coated on the downsteam electrode ; the urease (0.5 unit) was dropped on the upstream and dried secondly;the 1% 0.5 μl bovine serum albumin drop-coated on the upstream and dried; the 1% 0.5 μl glutaraldehyde dropped on the upstream and dried finally. The optimized conditions in the 0.05M pH 9 carbonate buffer ; applied potential was 0.2 V (vs. Ag/AgCl) ; flow rate was 0.5 ml/min; sample loop was 50 μl. The analytical performances of biosensor equipped with linear range upto 10 mM(R=0.99) and sensitivity is 85.736 nA/mM, detection limit (S/N=3) was 85 μM, and precision is 1.8% by twenty successive measurement. In order to eliminate interference, PbO2 was used to pre-oxidize those easily oxidative compounds, such as ,dopamine, epinephrine, serotonin, histamine , acetaminophen ,uric acid, ascorbic acid. Compared with standard method (sigma640), the correlation coefficient was 0.995.

目錄 (Contents)
論文提要內容………………………………………………………I
Abstract…………………………………………………………II
目錄………………………………………………………………III
圖表目錄…………………………………………………………VI
第一章 序論………………………………………………………1
1-1 生化感測器的定義與組成……………………………………1
   1-1-1辨識元固定方法………………………………………2
    1-1-1-1 物理吸附法………………………………………2
    1-1-1-2 陷阱法……………………………………………2
    1-1-1-3 電沉積法…………………………………………3
    1-1-1-4 混合法……………………………………………4
    1-1-1-5 交聯法……………………………………………4
1-2化學修飾電極簡介……………………………………………5
    1-2-1 化學吸附法……………………………………………6
    1-2-2 共價鍵結法……………………………………………6
    1-2-3 高分子薄膜塗佈法……………………………………7
    1-2-4 非均相材料混合法……………………………………8
1-3 一氧化二銅性質與應用………………………………………10
    1-3-1 一氧化二銅性質………………………………………10
    1-3-2 一氧化二銅應用………………………………………11
1-4 流動分析系統…………………………………………………12
1-5 銨根之研究……………………………………………………15
    1-5-1光化學法………………………………………………15
    1-5-2 電化學法………………………………………………17
1-6 尿素之研究……………………………………………………19
    1-6-1 尿素形成與代謝………………………………………19
    1-6-2 尿素的臨床意義………………………………………22
    1-6-3 治療……………………………………………………22
1-7 尿素生化感測器………………………………………………25
    1-7-1 光化學法………………………………………………25
    1-7-2 電化學法………………………………………………27
1-8 本研究的目的………………………………………………34

第二章 實驗部份

2-1 儀器…………………………………………………………35
2-2 藥品…………………………………………………………35
2-3 實驗步驟……………………………………………………36
2-4 最佳化條件探討……………………………………………37
    2-4-1  辨識層組成最佳化探討……………………………37
     2-4-1-1 酵素修飾方法探討………………………………38
     2-4-1-2  酵素含量之探討…………………………………38
     2-4-1-3  小牛血清蛋白與戊二醛比例之探討……………38
     2-4-1-4  一氧化二銅與導電碳膠組成(催化劑含量)…39
    2-4-2  偵測操作條件探討…………………………………39
     2-4-2-1  偵測電位探討……………………………………39
     2-4-2-2  環境酸鹼度之探討………………………………39
     2-4-2-3  緩衝溶液種類之探討……………………………39
     2-4-2-4  緩衝溶液濃度之探討……………………………39
     2-4-2-5  電解質氯化鈉濃度之探討………………………40
     2-4-2-6  載體流速之探討…………………………………40
     2-4-2-7  載體迴路體積之探討……………………………40

2-5 尿素生化感測器特性探討…………………………………40

第三章 結果與討論

3-1 電化學偵測機制的探討……………………………………41
3-2 偵測條件之最適化…………………………………………45
    3-2-1  辨識層組成最佳化探討……………………………45
     3-2-1-1  酵素修飾方法探討………………………………45
     3-2-1-2  酵素含量之探討…………………………………47
     3-2-1-3  小牛血清蛋白與戊二醛比例之探討…………50
     3-2-1-4  一氧化二銅與導電碳膠組成(催化劑含量) … 50
    3-2-2  偵測操作條件探討…………………………………53
     3-2-2-1  偵測電位探討……………………………………53
     3-2-2-2  偵測環境之酸鹼度探討…………………………53
     3-2-2-3  緩衝溶液種類之探討……………………………55
     3-2-2-4  緩衝溶液濃度之探討……………………………57
     3-2-2-5  電解質氯化鈉濃度之探討………………………57
     3-2-2-6  載體流速之探討…………………………………60
     3-2-2-7  載體迴路體積之探討……………………………63

3-3 相關性探討………………………………………………………69

3-4 結論………………………………………………………………71 
圖表目錄
圖(一) ： Cu2O修飾電極之循環伏安圖………………………………42
   以循環伏安法檢視修飾50% Cu2O修飾電極之電化學行為，在0.05 M pH 7 碳酸鹽緩衝溶液中，掃描速率為50 mV/sec。
圖(二) ： 循環伏安法探討50% Cu2O修飾電極對於銨根偵測特性.44
   50% Cu2O修飾電極，在0.05 M pH 9 碳酸鹽緩衝溶液中，連續添加1 mM氯化銨之循環伏安法電化學行為，掃描速率為50 mV/sec。
圖(三)： 尿素生化感測器之電子傳遞機制示意圖…………………46
圖(四)： 酵素修飾方法之探討………………………………………48
圖(五)： 酵素修飾比例之探討………………………………………49
圖(六)： 小牛血清蛋白與戊二醛比例之探討………………………51
圖(七)： 一氧化二銅與導電碳膠組成 (催化劑含量)………………52
圖(八)： 偵測電位探討………………………………………………54
圖(九)： 環境酸鹼度之探討…………………………………………56
圖(十)： 緩衝溶液種類之探討………………………………………58
圖(十一)： 緩衝溶液濃度之探討……………………………………59
圖(十二)： 電解質氯化鈉濃度之探討………………………………61
圖(十三)： 載體流速之探討…………………………………………62
圖(十四)： 載體迴路體積之探討……………………………………64
圖(十五)： 校正曲線…………………………………………………66
圖(十六)： 尿素生化感測器再現性探討……………………………68
圖(十七)： 相關性探討………………………………………………70
表(一)     實驗最佳化條件與分析特性……………………………65
表(二)     尿素生化感測器之干擾物分析…………………………67
附圖 (A)   實驗裝置示意圖…………………………………………72
附圖 (B)   流體三電極反應槽裝置圖………………………………73

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