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: 6240023
• 负责人: Ana R. Guadalupe
• 金额: $ 10.83万
• 依托单位: UNIVERSITY OF PUERTO RICO RIO PIEDRAS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240023
• 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发展敏感、 用于测量的选择性且可靠的电化学生物探针 具有生理相关性的代谢物。在研究层面，那些 探针可用于研究周期之间的关系 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 可以通过以下方式导致临床实验室的分散化 为医生和门诊患者提供快速、可靠、简单的服务 以及代谢物分析的低成本方法。短期内，这 该提案将解决能量学和反应性问题 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 且 y= 0,1,2； [Ru(phendione)(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' = -phi, -phi-SO3 (R =R')。 R”是-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。将探索两种策略：第一，中介 所研究的脱氢酶存在下的 NADH 氧化， 其次，硫辛酰胺脱氢酶辅助的 NADH 氧化 与其他脱氢酶有关。热力学和动力学参数将 使用标准电化学技术在均相中测定 解决方案。这些参数如何受到固定化的影响 电极表面的介体、辅因子和酶将 也有待研究。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。这些是丝网印刷的电极， 在电极表面策略性地分层聚合物薄膜 和液膜探针。 pH、离子等变量的影响 所有传感成分对反应的强度和浓度 将在均质溶液和表面形式中评估速率 受限系统。这些知识将有助于更好地控制 传感器的输出。