Polarity Switching and Stacking in Bioanalytical CE

生物分析 CE 中的极性切换和堆叠

• 批准号: 6804324
• 负责人: TIMOTHY G STREIN
• 金额: $ 13.9万
• 依托单位: BUCKNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6804324
• 关键词: capillary electrophoresis; chemical kinetics; creatinine; enzyme activity; method development; molecular polarity; nitrophenol; small molecule

DESCRIPTION (provided by applicant): The project described here involves the investigation of new approaches to improve capillary electrophoresis (CE) methodology for the biochemical and clinical laboratories. One of the barriers to the implementation of CE methodology, either in capillary or microchip form, is poor concentration sensitivity when conventional UV absorbance detection is employed. This project will investigate the use of both rapid polarity switching and reaction product stacking with etectrophoretically mediated microanalysis (EMMA), with an eye towards enhancing the sensitivity of clinical applications of this relatively new methodology. The project will be in three phases: The initial experiments will be carried out using the well-known Jaffe method, a widely used methodology for the determination of creatinine in clinical biofluids, as a model system. The Jaffe method involves the reaction of creatinine with alkaline picrate. Preliminary experiments have indicated that the use of rapid polarity switching during the on-column mixing step of an EMMA analysis improves the sensitivity of the method. In addition, since the product of the on-column reaction is doubly anionic, it should be possible to evoke transient isotachophoretic (tlTP) stacking of the product to further enhance the analytical signal (phase 2). The third phase of this project will involve the application of the EMMA methodology with rapid polarity switching -- and stacking, if possible -- to other chemical systems including kinetically challenged (i.e. relatively slow) reactions involving small molecules as well as enzymatic reactions. In all, this project will elucidate the capability of rapid polarity switching to enhance product formation when employed with EMMA, and may lead to new approaches for rapid, low-volume bioassays in the clinical laboratory.

描述（由申请人提供）：这里描述的项目涉及新方法的研究，以改善生物化学和临床实验室的毛细管电泳（CE）方法。无论是毛细管还是微芯片形式的CE方法的实施的障碍之一是当采用常规UV吸光度检测时，浓度灵敏度差。该项目将研究使用快速极性转换和反应产物堆积与电泳介导的微量分析（EMMA），着眼于提高这种相对较新的方法的临床应用的灵敏度。该项目将分为三个阶段：最初的实验将使用众所周知的Jaffe方法进行，这是一种广泛使用的方法，用于测定临床生物流体中的肌酐，作为模型系统。Jaffe方法涉及肌酸酐与碱性苦味酸盐的反应。初步实验表明，在EMMA分析的柱上混合步骤期间使用快速极性切换提高了该方法的灵敏度。此外，由于柱上反应的产物是双阴离子的，因此应该可以引起产物的瞬时等速电泳（tlTP）堆积，以进一步增强分析信号（阶段2）。该项目的第三阶段将涉及将EMMA方法与快速极性转换-如有可能，还包括叠加-应用于其他化学系统，包括涉及小分子的动力学挑战（即相对缓慢）反应以及酶促反应。总之，该项目将阐明与EMMA一起使用时快速极性转换以增强产物形成的能力，并可能导致临床实验室中快速，低容量生物测定的新方法。