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標題: 應用酵素耦合NADH-Tetrazolium Salt反應於層析試紙上檢測乳酸之開發
Determination of L-lactate by Enzyme Coupled to NADH-Tetrazolium Salt Reaction on a Chromatographic Strip
作者: 藍敏綺
Lan, Min-Chi
關鍵字: 乳酸;L-lactate;乳酸脫氫酵素;NADH;Tetrazolium salt;層析試紙;L-lactate dehydrogenase;NADH;Tetrazolium salt;Chromatographic strip
出版社: 化學工程學系所
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隨著全球老年化的趨勢,未來醫療照護的需求將逐漸提升, 並且醫療照護的觀念會延伸至小型診所和一般家庭,因此有越來越多以定點照護 (Point-of-care, POC) 為訴求的生物感測器產生。由於人血清L-乳酸水平升高被認為是組織缺氧或代謝功能異常的指標,因此本研究希望能結合定點照護的概念,開發出檢測乳酸的生物感測器,並應用於臨床疾病的監護與救治。過去大部分研究多以酵素分析法結合液體試劑系統檢測乳酸,雖然酵素具有高度的受質專一性,非常適合分析不同光學構型的乳酸,但使用液體試劑不但耗時又無法提供即時即地的檢驗。因此本研究開發以L-乳酸脫氫酵素反應,結合生物化學常用的組織染色劑tetrazolium salt與NADH反應,在短時間內於層析試紙上產生肉眼可見的顏色變化,其值可對應到所欲檢測樣品之濃度。經實驗探討發現,其最佳分析條件為:將0.5 μL, 2-6 U/μL黃遞酵素固定化於試紙薄膜上,添加5 μL, 12 mM NBT、1 μL, 0.25 U/μL乳酸脫氫酵素和2 μL, 1.5×10-5 M NAD等試劑,以100 μL, pH 9, 0.1% Tween 20/0.01 M phosphate buffer做為移動相沖提十分鐘。透過最佳檢測條件,檢測乳酸的線性範圍在0.039 - 5 mM,偵測極限為0.053 mM。本研究顯示此分析系統應用於乳酸定量,具有操作簡易、穩定性佳、省時及方便等特性,倘若能再結合手機式分析儀進行數據分析,則極具開發潛力應用於醫護地點檢驗,提供即時即地的檢驗,節省樣本運送至分析中心的時間,減少血液分析的準確性隨運送時間而降低的問題,更可避免樣本在運送過程遭受污染。

With the global trend of an aging society, the demand for medical care will gradually rise in the future, and the concept of medical care will also be extended to small clinics and families, all of which have driven moe portable biosensors to be developed. Since high blood lactate levels are indicative of tissue hypoxia and abnormal metabolism, we hope to develop a biosensor for the detection of lactate. However, previous studies detect lactate by enzymatic method using liquid reagents. Although enzyme is highly specific to substrate and suitable for the analysis of different optical configuration of lactate, the use of liquid reagents are neither time-consuming nor inapplicable point-of-care test. Therefore, the aim of this study is to develop a dry reagent strip for measuring the lactate concentration with use of a scanner. Based on the enzymatic reaction coupled to NADH-tetrazolium salt on a chromatographic strip, the concentration of lactate can be directly determined by measuring the color intensity of formazan. The optimal conditions are found as follows: 0.5 μL, 2-6 U/ μl diaphorase immobilized on a strip; 5 μL, 12 mM nitrotetrazolium blue chloride, 1 μL, 0.25 U/μL L-lactate dehydrogenase and 2 μL, 1.5×10-5 M NAD as reaction reagents; 100 μL, pH 9, 0.1% Tween 20/0.01 M phosphate buffer as mobile phase, and reaction time of 10 minutes. Under these optimal conditions, a linear range from 0.039 to 5 mM with a detection limit of 0.053 mM is observed. The present work, shown to be a simple, stable, time-saving and convenient quantitative assay, provides a promising clinical testing method for lactate detection and has the potential for POCT when combining the hand-held analyzer.
其他識別: U0005-3007201218140400
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