以奈米片狀四氧化三鈷修飾電極搭配流注分析系統偵測NADH

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Title:	以奈米片狀四氧化三鈷修飾電極搭配流注分析系統偵測NADH
Other Titles:	Detection of NADH with nanosheet of Cobalt(II,III) oxide based modified in flow injection analysis system
Authors:	陳映慈;Chen, Ying-Cih
Contributors:	淡江大學化學學系碩士班林孟山;Lin, Meng-shan
Keywords:	四氧化三鈷;流注分析系統;NADH;Cobalt(II,III) oxide;Flow injection analysis system(FIA)
Date:	2012
Issue Date:	2012-06-21 06:37:53 (UTC+8)
Abstract:	NAD+/NADH是相當重要的輔酶，超過300種的酵素會利用此對分子以進行氧化還原作用，因此NADH的量測可發展大量的生化感測器。而在歷史文獻中，已經知道在石墨電極上偵測NADH的氧化時，需使用相當高的過電壓，且在NADH氧化過程中會產生自由基陽離子中間體的氧化物種吸附在電極表面上，造成電極產生毒化現象造成直傳的阻礙以及靈敏度下降。因此，本研究傾向發展低電位偵測NADH的方式。本研究中進一步探討流注分析系統(FIA)以評估偵測NADH時的最佳化條件：70% Co3O4修飾電極，偵測環境為0.2M pH 6.00 磷酸鹽類緩衝溶液，偵測電位為100 mV (vs. Ag/AgCl)，載體流速0.25 mL/min，樣品迴路體為50μL。偵測NADH之分析特性分別為：線性範圍為10-100μM (R=0.99878)，電流密度為0.35899 nA/μM，偵測極限(S/N=3)為1μM，連續重覆二十次偵測NADH，所得到相對標準偏差(RSD)為1.4%，反應時間(t90%)為12.26s；在0.1V的電位下，大部分的易氧化物質如dopamine、Urea、 (±)-epinephrine、serotonin、histamine、4-acetaminophen等，均不會有明顯干擾本系統之偵測，僅有抗壞血酸(AA)造成干擾，因此在偵測前先加入抗壞血酸氧化酶進行前處理，加入3U的抗壞血酸氧化酶處理五分鐘之後進行偵測，可避免AA之干擾。 NAD+/NADH is an important cofactors for a class of apozymes in several dehydrogenase, which involves in more than 300 biological reactions. Therefore, a NADH based sensor possesses a great potentiality in development of other biosensors. However, the prior studies indicate that direct oxidation of NADH requires a high overvoltage over 0.7 V, which would be suffered from most of all common biological antioxidants. Besides, a significant surface fouling effect due to the strong adsorbability of the oxidized intermediate also causes a bad reproducibility and low sensitivity. In this work, we develop a simple, sensitive and reliable NADH sensor based upon the Cobalt (II, III) oxide modified electrode. Owning to the redox property of passive layer of the cobalt oxide, the oxidation potential of the NADH can be shifted from 700 mV to 100 mV. In order to develop a high sensitive scheme for NADH determination, this sesor is held by using a high efficient flow injection analysis system. After optimization, 70% cobalt (II, III) oxide modified carbon ink electrode with a constant operating potential of 100 mV (vs. Ag/AgCl) in 0.2 M phosphate buffer, pH 6.00, and flow rate of 0.25 mL/min were chosen as the optimal conditions. A suitable linear range 10 to 100 μM (R=0.999) with a sensitivity of 0.36 nA/μM is achieved. The detection limit base on 21 successive blank injections is 4.25 μM (S/N=3).the relative standard deviation (RSD) for 20 successive measurements of 75 μM NADH is only 1.4%, which indicates the high stability of this method. Besides, most antioxidants such as dopamine, uric acid, (±)-epinephrine, serotonin, histamine, and 4-acetaminophen do not affect the NADH determination. However, ascorbic acid (AA) causes a significant interference to this scheme. Here, an ascorbate oxidase (EC 1.10.3.3) was utilized to oxidize the ascorbic acid before sample injected into the flow injection analysis system, and the interference from ascorbic acid was eliminated, successfully.
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