Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3098
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
引用: 1. Wee, Y.J., Kim, J. N., and Ryu, H. W., Biotechnological production of lactic acid and its recent applications. Food Technol Biotech, 2006. 44(2): p. 163-172. 2. Benninga, H., A history of lactic acid making : a chapter in the history of biotechnology. Kluwer Academic Publishers, Dordrecht, Netherlands, 1990. p. 1-61. 3. 李繼煌, 聚乳酸之開發與應用綜論. 朝陽科技大學應用化學系碩士論文, 2006. 4. Passarella, S., de Bari, L., Valenti, D., Pizzuto, R., Paventi, G., and Atlante, A., Mitochondria and L-lactate metabolism. Febs Lett, 2008. 582(25-26): p. 3569-3576. 5. Boldt, J., Kumle, B., Suttner, S., and Haisch, G., Point-of-care (POC) testing of lactate in the intensive care patient - Accuracy, reliability, and costs of different measurement systems. Acta Anaesth Scand, 2001. 45(2): p. 194-199. 6. Ozkan, M., Erhan, E., Terzi, O., Tan, I., and Ozoner, S. K., Thermostable amperometric lactate biosensor with Clostridium thermocellum L-LDH for the measurement of blood lactate. Talanta, 2009. 79(5): p. 1412-1417. 7. Talasniemi, J.P., Pennanen, S., Savolainen, H., Niskanen, L., and Llesivuori, J., Analytical investigation: assay of D-lactate in diabetic plasma and urine. Clin Biochem, 2008. 41(13): p. 1099-1103. 8. 陳永展, 西藏紅景天對運動能力與生理心理的作用. 國立屏東教育大學體育學系教學碩士論文, 1996. 9. 王玲玲、郭振輝, D-乳酸的生化特性及其臨床研究進展. 廣東醫學期刊, 2011. 32(23): p. 3144-3147. 10. 張祐銘、楊垂勳, D乳酸中毒. 內科學誌, 2010. 21: p. 192-196. 11. 洪永璋, 聚乳酸二氧化矽奈米複合材料的製備與特性之研究. 國立成功大學化學研究所碩士論文, 2008. 12. 林卓儀, 以Lactobacillus amylovorus 利用澱粉醱酵製造乳酸. 逢甲大學化學工程學系碩士論文, 2009. 13. Campbell, M.K., Biochemistry, 3rd edition,林順富、陳師瑩、顏瑞鴻、蕭慧美編譯. 偉明圖書有限公司, 臺北, 2001. p. 476-496. 14. Hofvendahl, K., and Hahn-Hagerdal, B., Factors affecting the fermentative lactic acid production from renewable resources. Enzyme Microb Tech, 2000. 26(2-4): p. 87-107. 15. Basaran, M., Sever, K., Kafali, E., Ugurlucan, M., Sayin, O. A., Tansel, T., Alpagut, U., Dayioglu, E., and Onursal, E., Serum lactate level has prognostic significance after pediatric cardiac surgery. J Cardiothor Vasc An, 2006. 20(1): p. 43-47. 16. Cheung, P.-Y. and Finer, N.N., Plasma lactate concentration as a predictor of death in neonates with severe hypoxemia requiring extracorporeal membrane oxygenation. The Journal of Pediatrics, 1994. 125(5, Part 1): p. 763-768. 17. Broder, G. and Weil, M.H., Excess Lactate: An Index of Reversibility of Shock in Human Patients. Science, 1964. 143(3613): p. 1457-1459. 18. Demers, P., Elkouri, S., Martineau, R., Couturier, A., and Cartier, R., Outcome with high blood lactate levels during cardiopulmonary bypass in adult cardiac operation. The Annals of Thoracic Surgery, 2000. 70(6): p. 2082-2086. 19. Munoz, R., Laussen, P.C., Palacio, G., Zienko, L., Piercey, G., and Wessel, D.L., Changes in whole blood lactate levels during cardiopulmonary bypass for surgery for congenital cardiac disease: An early indicator of morbidity and mortality. The Journal of Thoracic and Cardiovascular Surgery, 2000. 119(1): p. 155-162. 20. Skoog, D.A., Principles of instrumental analysis. Harcourt Brace Jovanovich College Publishers, New York, 2004. p. 714-716. 21. Emmert, J., Analytical requirements for the measurement of lactate. Workshop Report Accusport, Zurich, 1994. p. 26-28. 22. Shimojo, N., Naka, K., Nakajima, C., Yoshikawa, C., Okuda, K., and Okada, K., Test-strip method for measuring lactate in whole blood. Clin Chem, 1989. 35(9): p. 1992-4. 23. Shimojo, N., Naka, K., Uenoyama, H., Hamamoto, K., Yoshioka, K., and Okuda, K., Electrochemical assay system with single-use electrode strip for measuring lactate in whole blood. Clin Chem, 1993. 39(11 Pt 1): p. 2312-4. 24. Sprules, S.D., Hart, J. P., Pittson, R., and Wring, S. A., Evaluation of a new disposable screen-printed sensor strip for the measurement of NADH and its modification to produce a lactate biosensor employing microliter volumes. Electroanal, 1996. 8(6): p. 539-543. 25. Silanikove, N., and Shapiro, F., Rapid and accurate determination of D- and L-lactate, lactose and galactose by enzymatic reactions coupled to formation of a fluorochromophore: Applications in food quality control. Food Chem, 2010. 119(2): p. 829-833. 26. Ballesta-Claver, J., Valencia-Miron, M. C., and Capitan-Vallvey, L. F., One-shot lactate chemiluminescent biosensor. Anal Chim Acta, 2008. 629(1-2): p. 136-144. 27. Marti, R., Varela, E., Segura, R. M., Alegre, J., Surinach, J. M., and Pascual, C., Determination of D-lactate by enzymatic methods in biological fluids: Study of interferences. Clin Chem, 1997. 43(6): p. 1010-1015. 28. Gleitz, J., Tosch, C., and Peters, T., Continuous enzyme-linked fluorometric detection of L-(+)-lactate released from rat brain vesicles under anoxic conditions. J Neurosci Meth, 1996. 67(2): p. 97-102. 29. Ishiyama, M., Miyazono, Y., Sasamoto, K., Ohkura, Y., and Ueno, K., A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta, 1997. 44(7): p. 1299-1305. 30. Seidler, E., New nitro-monotetrazolium salts and their use in histochemistry. Histochemical Journal, 1980. 12(6): p. 619-630. 31. Eun, A.J.C., Huang, L., Chew, F. T., Li, S. F. Y., and Wong, S. M., Detection of two orchid viruses using quartz crystal microbalance (QCM) immunosensors. J Virol Methods, 2002. 99(1–2): p. 71-79. 32. 黃長榮, 免疫層析檢驗法在登革病毒檢測上之應用及開發. 國立中興大學昆蟲學系碩士論文, 2003. 33. 陳燕惠, 奈米碳管與膠體金於電化學生物感測器上之應用. 國立中興大學化學工程學系碩士論文, 2006. 34. 黃俐芳, 免疫生物感測器在登革熱早期檢測上之應用. 國立東華大學生物技術研究所碩士論文, 2000. 35. Lam, S.K., and Devine, P. L., Evaluation of capture ELISA and rapid immunochromatographic test for the determination of IgM and IgG antibodies produced during dengue infection. Clinical and Diagnostic Virology, 1998. 10(1): p. 75-81. 36. 李佳霖, 染色介質poly-L-lysine搭配不同反應性染料對染料免疫分析的影響及多重待測物分析檢測的開發. 國立中興大學化學工程學系碩士論文, 2007. 37. Jones, K.D., Troubleshooting protein binding in nitrocellulose membranes. Part 1: Principles. IDV Technology Magazine, 1999. 5(2): p. 32-41. 38. Beford, M.A., Millipore''s short guide for developing immunochromatographic test strips, 2nd edition. Millipore Corp., 1999. 39. Davis, D.J., and Pietro, A. San, Evidence for the role of sulfhydryl groups in a pH-dependent transition of ferredoxin:NADP oxidoreductase. Arch Biochem Biophys, 1977. 184(2): p. 572-577. 40. 呂峰洲、林仁混, 基礎酵素學. 聯經出版社, 臺北, 1991. p. 317-385. 41. Schwert, G.W., Miller, B. R., and Peanasky, R. J., Lactic dehydrogenase. The Journal of Biological Chemistry, 1967. 242: p. 3245-3252. 42. Silverstein, E., and Boyer, P. D., Equilibrium reaction rates and the mechanisms of bovine heart and rabbit muscle lactate dehydrogenases. The Journal of Biological Chemistry, 1964. 239: p. 3901-3907. 43. 蕭芳雯, 酵素催化合成4''-OH乙醯化白藜蘆醇-最適化與動力學探討. 國立中興大學化學工程學系碩士論文, 2011. 44. Gutfreund, H., Cantwell, R., McMurray, C. H., Criddle, R. S., and Hathaway, G. , The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound. Biochem J, 1968. 106(3): p. 683-687. 45. Uribarri, J., Oh, M. S., and Carroll, H. J., D-lactic acidosis: a review of clinical presentation, biochemical features, and pathophysiologic mechanisms. Med Sci Sport Exer, 1998. 77(2): p. 73-82. 46. Galban, J., de Marcos, S., and Castillo, J.R., Fluorometric-enzymatic lactate determination based on enzyme cytochrome b2 fluorescence. Anal Chem, 1993. 65(21): p. 3076-3080. 47. Martínez-Olmos, A., Ballesta-Claver, J., Palma, A., Valencia-Mirón, M.d.C., and Capitán-Vallvey, L., A Portable Luminometer with a Disposable Electrochemiluminescent Biosensor for Lactate Determination. Sensors-Basel, 2009. 9(10): p. 7694-7710.
摘要: 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.
URI: http://hdl.handle.net/11455/3098
其他識別: U0005-3007201218140400
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3007201218140400
Appears in Collections:化學工程學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.