REACTIVITY AND ENERGETICS OF DEHYDROGENASE ENZYMES AND REDOX MEDIATORS

脱氢酶和氧化还原介质的反应性和能量

• 批准号: 6271545
• 负责人: Ana R. Guadalupe
• 金额: $ 10.9万
• 依托单位: UNIVERSITY OF PUERTO RICO RIO PIEDRAS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271545
• 关键词: NAD(P)H oxidoreductase; biosensor device; chemical transfer reaction; cobalt; coulometry; electrochemistry; enzyme activity; enzyme electrodes; immobilized enzymes; metal complex; oxidation reduction reaction; ruthenium; spectrometry; stop flow technique; thermodynamics

The long term goal of this proposal is the development of sensitive, selective and reliable electrochemical bioprobes for the measurement of metabolites of physiological relevance. At the research level, those probes can be used to study the relationship between periodic fluctuations of metabolites and the development of disorders in the human body. At an application level, the development of those devices can result in the decentralization of clinical laboratories by providing the physicians and ambulatory patients a fast, reliable, easy and low cost way of analyzing for metabolites. In a short term, this proposal will address the issues of energetics and reactivity of several redox mediators with various dehydrogenases. The redox mediators are [Ru(phendione)x(bpy)y]2+, where x= 3,2,1 and y= 0,1,2; [Ru(phendione)(phen-R,R')L]2+, where L is bpy-R" or phen-R"; and [Co(phen-R,R')3]2+. The R' is -H, -CH3, -OH, -Cl, -NH2, -NR3+, (R=H) or R' = -phi, -phi-SO3 (R =R'). R" is either a -NH2 or a -COOH group. The enzymes are lactate, malate, alcohol, glycerol and lipoamide dehydrogenases. Two strategies will be explored: first, the mediated NADH oxidation in the presence of the dehydrogenases under study and, secondly, the NADH oxidation aided by lipoamide dehydrogenase coupled to the other dehydrogenases. Thermodynamic and kinetic parameters will be determined using standard electrochemical techniques in homogeneous solutions. How those parameters are affected by the immobilization of the mediator, the cofactor and the enzyme at electrode surfaces will also be studied. Three strategies for the surface immobilization of the best systems will be evaluated based on their response output and analytical characteristics. Those are screen-printed electrodes, strategically layering of thin polymeric films at electrode surfaces and liquid membrane probes. The effect of variables such as pH, ionic strength and concentration of all sensing components on the reaction rate will be evaluated, in both homogeneous solutions and as surface confined systems. That knowledge will allow a better control of the sensor's output.

该提案的长期目标是开发敏感， 用于测量的选择性和可靠的电化学生物探针 生理相关的代谢物。在研究层面，这些 探针可用于研究周期性 代谢物的波动和疾病的发展， 人体在应用层面，这些设备的开发 可能导致临床实验室的分散化， 为医生和门诊患者提供一个快速、可靠、简单 和低成本的代谢物分析方法。在短期内，这 该提案将解决能源和反应性的问题， 几种氧化还原介体与各种脱氢酶。 氧化还原 介体为[Ru（phendione）x（bpy）y]2+，其中x= 3，2，1，y= 0，1，2; [Ru（苯二酮）（phen-R，R '）L]2+，其中L是bpy-R”或phen-R”;和 [Co（phen-R，R'）3]2+。R'为-H、-CH3、-OH、-Cl、-NH2、-NR3+，（R=H） 或R ′ = -φ、-φ-SO 3（R = R ′）。R”为-NH 2或-COOH基团。 这些酶是乳酸、苹果酸、酒精、甘油和硫辛酰胺 脱氢酶。将探讨两种策略：第一， 在所研究的脱氢酶存在下的NADH氧化， 其次，硫辛酰胺脱氢酶偶联的NADH氧化 到其他的沼泽。热力学和动力学参数将 使用标准电化学技术在均相 解决方案这些参数是如何受到固定化的影响的 电极表面的介体、辅因子和酶将 也被研究。表面固定化的三种策略 最佳系统将根据其响应输出进行评估， 分析特征。这些是丝网印刷电极 在电极表面上聚合物薄膜的策略性分层 和液膜探针。pH值、离子浓度等变量的影响 所有敏感组分的强度和浓度对反应的影响 将在均相溶液和表面溶液中评估速率 封闭系统这些知识将有助于更好地控制 传感器的输出。