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.

这项提案的长期目标是发展敏感的，