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.
