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.

该提议的长期目标是发展敏感， 用于测量的选择性和可靠的电化学生物探针 生理相关性的代谢产物。在研究层面，那些 探针可用于研究周期性的关系 代谢物的波动和疾病的发展 人体。在应用程序层面，这些设备的开发 可以通过 为医生和门诊患者提供快速，可靠，轻松的 以及分析代谢物的低成本方式。在短期内，这个 提案将解决能量学和反应性问题 几种氧化还原介质具有各种脱氢酶。 氧化还原 介体为[ru（phendione）x（bpy）y] 2+，其中x = 3,2,1和y = 0,1,2; [ru（phendione）（phan-r，r'）l] 2+，其中l是bpy-r“或phan-r”；和 [CO（PEN-R，R'）3] 2+。 r'是-h，-ch3，-oh，-cl，-nh2，-nr3+，（r = h） 或r'= -phi，-phi -so3（r = r'）。 r“是-NH2或-COOH组。 酶是乳酸，苹果酸，酒精，甘油和lipoamide 脱氢酶。将探讨两种策略：首先，调解 NADH氧化在研究中的脱氢酶存在下， 其次，NADH氧化由lipoamide脱氢酶耦合的辅助 到其他脱氢酶。热力学和动力学参数将 使用均匀的标准电化学技术确定 解决方案。这些参数如何受到固定的影响 介体，辅因子和电极表面的酶将 也可以研究。表面固定的三种策略 最佳系统将根据其响应输出和 分析特征。这些是屏幕打印的电极， 从策略性地分层电极表面的薄聚合物膜 和液体膜探针。 pH，离子等变量的效果 反应上所有感应成分的强度和浓度 速率将在均质溶液中评估，并作为表面评估 密闭系统。这些知识将可以更好地控制 传感器的输出。