Bioanalytical CE: Mixing, Reacting, Separating, Stacking

描述（由申请人提供）：毛细管或微芯片形式的CE技术广泛实施的主要障碍之一是，当采用传统的紫外吸收检测时，浓度灵敏度较差。本项目涉及研究三种不同的方法，以提高生化和临床实验室毛细管电泳（CE）方法的敏感性和实用性。提出的工作包括精确操纵毛细管内的局部条件，以影响CE分离过程中发生的分子水平过程。特别是，我们将研究快速极性切换（RPS）和几种堆叠方法的使用，以提高柱内反应的速率，并提高基于ce的分析的浓度敏感性。大多数提出的工作涉及使用电泳介导的微分析（EMMA）的小分子的在线化学反应。在之前和正在进行的工作中，我们已经证明RPS是一种有效的方法，可以快速混合一种小分子反应的试剂，肌酐和苦克酸之间的Jaffe反应，我们将把这种方法扩展到其他小分子系统中。与Jaffe反应通过浓缩的氢氧化物堵塞在线生成的产物的瞬态等速电泳堆积（tITP）的补充工作似乎非常有效，并将继续进行，但更简单的tITP系统也将被探索，以获得对堆积动力学的基本理解。这项工作的第三阶段将探索在线堆积和分离的胶束电动（MEKC）模式在毛细反应中形成不带电产物的使用。通过新的合作，我们将使用核磁共振（NMR）光谱来研究和理解胶束和分析物在堆积和分离过程中的相互作用。总之，该项目将阐明各种动态在线技术的一般能力，以提高小分子EMMA可达到的灵敏度，并可能导致临床实验室快速，小体积生物测定的新方法。本项目涉及研究三种不同的方法来改进生化和临床实验室的毛细管电泳（CE）方法。部分由于浓度敏感性差的特点，毛细管或微芯片中基于ce的方法尚未得到充分发展，因此这种令人信服的分析模式未得到充分利用。所提出的工作旨在通过几种方法中的一种来操纵毛细管中离散试剂区域内离子的局部浓度，或通过增加作为分析方法一部分的柱内反应的速率，从而提高基于CE的分析模式的灵敏度。这项工作可能会导致临床实验室快速，小容量和敏感的生物测定新方法的发展。

DESCRIPTION (provided by applicant): One of the major barriers to the wider implementation of CE technology, either in capillary or microchip form, is poor concentration sensitivity when conventional UV absorbance detection is employed. This project involves the investigation of three separate approaches to improve the sensitivity and utility of capillary electrophoresis (CE) methodologies for the biochemical and clinical laboratories. The proposed work involves precise manipulation of local conditions within capillary tubes to affect molecular-level processes that occur during CE separation processes. In particular, we will investigate the use of rapid polarity switching (RPS) and also several stacking methodologies to enhance the rate(s) of in-column reaction(s) and to improve concentration sensitivity of CE-based analyses. Most of the proposed work involves in-line chemical reactions of small molecules using electrophoretically mediated micro- analysis (EMMA). In prior and on-going work, we have shown that RPS has proven to be a useful methodology to quickly mix the reagents of one small molecule reaction, the Jaffe reaction between creatinine and picrate, and we will extend the use of this approach to other small molecule systems. Complementary work with transient isotachophoretic stacking (tITP) of the in-line generated product of the Jaffe reaction via a concentrated plug of hydroxide appears to be quite effective, and will continue, but simpler tITP systems will also be explored to gain fundamental understanding of the stacking dynamics. The third phase of this work will explore the use of both micellar electrokinetic (MEKC) modes of on-line stacking and separation for in-capillary reactions forming uncharged products. Through a new collaboration, we will use nuclear magnetic resonance (NMR) spectroscopy to investigate and understand the interactions between micelles and analytes during stacking and separation processes. In all, this project will elucidate the general capability of various dynamic in-line techniques to enhance the sensitivity attainable with small molecule EMMA, and may lead to new approaches for rapid, low-volume bioassays in the clinical laboratory. NARRITIVE This project involves the investigation of three separate approaches to improve capillary electrophoresis (CE) methodology for the biochemical and clinical laboratories. Partly because of the characteristically poor concentration sensitivity, CE-based methods, in either capillaries or on microchips, have not been fully developed and this compelling mode of analysis has therefore been underutilized. The proposed work is aimed at increasing the sensitivity of CE- based modes of analysis either by manipulating, through one of several means, the local concentration of ions within discrete reagent zones in a capillary, or by increasing the rate of in-column reactions that are part of the assay method. This work may lead to development of new approaches for rapid, low-volume and sensitive bioassays in the clinical laboratory.