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.

目錄 (Contents)
謝誌	I
中文摘要	II
英文摘要	III
第一章  緒論	1
1.1 感測器的種類	1
1.1.1 生化感測器的組成	2
1.1.2 辨識元件的固定方式	3
1.2化學修飾電極	5
1.2.1 吸附法(adsorption) 	6
1.2.2 共價鍵修飾法(covalently bonded)	6
1.2.3 氧化還原聚合物(Redox polymer coating)	7
1.2.4 非均相材料混合法	7
1.3 流注分析系統 (Flow Injection Analysis, FIA)	8
1.3.1 流注分析系統之四大部分	8
1.3.2 分散的主要原因	9
1.4 四氧化三鈷(Cobalt(II,III) oxide,Co3O4 )簡介與製備	12
1.5	 NAD+/NADH的簡介	14
1.6	不同類型偵測NADH的修飾方式	17
1.6.1 有機物的修飾	18
1.6.2 無機物的修飾	26
1.7 研究目的	28
第二章 實驗	29
2.1 實驗儀器	29
2.1.1 電化學分析	29
2.1.2 流注分析系統	29
2.1.3 其他	30
2.2 藥品與藥品配製	31
2.2.1 藥品	31
2.2.2 藥品配製	32
2.3 四氧化三鈷的製備	32
2.4 電極的製備	33
2.4.1 電極的前處理	33
2.4.2 四氧化三鈷的修飾	33
2.5 實驗設計	33
2.5.1 電化學偵測機制的探討	33
2.5.2 偵測NADH最佳化的探討	34
2.5.2.1 電位的最佳化	34
2.5.2.2 緩衝溶液pH值的最佳化	34
2.5.2.3 修飾比例的最佳化	35
2.5.2.4 緩衝溶液的最佳化	35
2.5.2.5 緩衝溶液濃度的最佳化	35
2.5.2.6 載流液體流速的最佳化	35
2.5.2.7 載體迴路體積的最佳化	36
第三章 結果與討論	37
3.1 電化學偵測機制討論	37
3.2 偵測NADH最佳化的探討	48
3.2.1. 電位最佳化的探討	48
3.2.2 緩衝溶液pH的探討	49
3.2.3 緩衝溶液種類的探討	50
3.2.4 緩衝溶液濃度的探討	52
3.2.5 修飾比例的探討	53
3.2.6 載流液體流速的探討	55
3.2.7 載體迴路體積的探討	56
3.3 四氧化三鈷對氧化NADH特性分析	58
第四章 結論	65
參考資料	69

圖表目錄
圖(1) 樣品擴散波峰的種類	10
圖(2) 分散度的定義	11
圖(3) Co3O4結構	13
圖(4) NAD+/NADH的結構	16
圖(5) 高溫反應釜	31
圖(6) Co3O4 XRD圖譜	39
圖(7) Co3O4 ESCA圖譜	39
圖(8) Co3O4 FE-SEM的影像	40
圖(9) 自製與市售Co3O4背景電流衰退情形	41
圖(10) 氧氣對NADH反應訊號的影響	42
圖(11) Co3O4與NADH的反應機構	43
圖(12) 修飾Co3O4與純導電碳膠電極偵測NADH的探討	45
圖(13) 旋轉速度的探討	47
圖(14) 電位最佳化的探討	49
圖(15) 緩衝溶液pH的探討	50
圖(16) 緩衝溶液種類的探討	51
圖(17)緩衝溶液濃度的探討	53
圖(18) 修飾比例的探討	54
圖(19) 載流液體流速的探討	56
圖(20) 載體迴路體積的探討	57
圖(21) NADH分析校正曲線	59
圖(22) NADH分析校正曲線	62
圖(23) 偵測NADH之再現性探討	62

表(1) 不同去氫酶對受質的反應	15
表(2) 常見的有機修飾物	17
表(3) 偵測NADH最佳化偵測條件	58
表(4) 分析特性	59
表(5) 干擾物的探討	60
表(6) 分析特性	61
表(7) 干擾物的探討	63
表(8) 干擾物的處理	64
表(9) NADH偵測方法的比較	65
表(10)  NADH偵測方法的比較	66

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