Polarity Switching and Stacking in Bioanalytical CE

描述（由申请人提供）：这里描述的项目涉及研究改善生化和临床实验室毛细管电泳（CE）方法的新方法。在毛细管或微芯片形式的CE方法实施的障碍之一，是低浓度灵敏度时，采用传统的紫外吸收检测。该项目将研究快速极性转换和反应产物堆积与电泳介导微分析（EMMA）的使用，着眼于提高这种相对较新的方法的临床应用的敏感性。该项目将分三个阶段进行：最初的实验将使用著名的Jaffe方法作为模型系统，这是一种广泛用于测定临床生物体液中肌酐的方法。Jaffe法涉及肌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中使用快速极性切换提高了方法的灵敏度。此外，由于柱上反应的产物是双阴离子，因此应该有可能引起产物的瞬时等色谱（tlTP）堆叠，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（即相对缓慢的）反应以及酶促反应，并进行快速极性切换和堆叠（如果可能的话）。总而言之，该项目将阐明快速极性切换的能力，以增强与EMMA一起使用时的产品形成，并可能导致临床实验室快速，小体积生物测定的新方法。

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.