DETECTION OF BIOMOLECULES BY CHEMILUMINESCENCE

化学发光法检测生物分子

• 批准号: 3439006
• 负责人: NEIL D DANIELSON
• 金额: $ 10.03万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-05-01 至 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3439006
• 关键词: collagen; coulometry; electrochemistry; erythromycin; high performance liquid chromatography; hydrazines; hydroxyproline; luminescence; oxalates; pyridine; ruthenium

We have reported that the electrochemiluminescence reaction of a reducing agent with tris (bipyridine) ruthenium (III) (Ru(bpy)3+3) is more broad in scope and not just seen for a few compounds such as hydrazines or oxalate. In particular, mono-, di-, and tri-alkyl amines can be detected in the order indicated at nmole to pmole levels. To allow facile study of this fast (<1 sec) reaction, we have modified an HPLC fluorometer for post- column mixing. It is capable of detecting tripropylamine at 0.04 pmoles. Pharmaceuticals difficult to detect in the UV such as erythromycin and clindamycin-2-phosphate have been assayed easily at the ppb level. Three areas of study are proposed building on these completed projects. First, to permit faster start-up and more versatile operation, we plan to do on- line oxidation of Ru(bpy)3+2 to Ru(byp)3+3 using the ESA coulometric flow cell. Electrochemical pretreatment of the mobile phase to lower detection limits can also be done with another coulometric guard cell. To enhance chemiluminescence lifetime, cationic, anionic, zwitterionic, and nonionic surfactants will also be used to produce a micellar solution. Secondly, fundamental studies of Ru(bpy)3+3 chemiluminescence involving the mechanism of this reaction with both amines and sulfur compounds. Wide bore capillary GC will be used to quantify the suspected products. Finally, analytical applications using HPLC for the determination of biomolecules such as amino acids, peptides, and proteins are spelled out. In particular, secondary amino acids such as proline and hydroxyproline in proteins such as collagen will be focused on.

本文报道了一种还原性的电化学发光反应， 具有三（联吡啶）钌（III）（Ru（bpy）3+3）的试剂在 范围，而不仅仅是几种化合物，如肼或草酸盐。 特别地，单-、二-和三-烷基胺可以在聚合物中检测到。 以nmole至pmole水平表示的顺序。 为了便于研究 快速（<1秒）反应，我们已经修改了HPLC荧光计后， 柱混合 它能够检测0.04 pmol的三丙胺。 在紫外线下难以检测的药物，如红霉素和 克林霉素-2-磷酸酯在ppb水平下容易测定。 三 在这些已完成的项目的基础上提出了研究领域。 第一、 为了加快启动速度和实现更灵活的操作，我们计划- 使用ESA库仑流将Ru（bpy）3+2线性氧化成Ru（byp）3+3 cell. 移动的相的电化学预处理以降低检测 也可以用另一个库仑保护电池进行限制。 加强 化学发光寿命、阳离子、阴离子、两性离子和非离子 表面活性剂也将用于产生胶束溶液。 第二、 Ru（bpy）_3 ~（3+）化学发光机理的基础研究 与胺和硫化合物的反应。 大口径 将使用毛细管GC对可疑产物进行定量。 最后，使用HPLC测定 生物分子如氨基酸、肽和蛋白质被拼出。 特别地，在哺乳动物中的二级氨基酸如脯氨酸和羟脯氨酸是必需的。 蛋白质如胶原蛋白将被关注。