Bioanalytical CE: Mixing, Reacting, Separating, Stacking

生物分析 CE：混合、反应、分离、堆叠

• 批准号: 7304414
• 负责人: TIMOTHY G STREIN
• 金额: $ 21.89万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7304414
• 关键词: Address; Affect; Antioxidants; Area; Biochemical; Biological; Biological Assay; Biological Models; Blood capillaries; Capillary Electrophoresis; Chemicals; Chemistry; Clinical; Collaborations; Condition; Convection; Creatinine; DNA analysis; Data; Detection; Development; Electrophoresis; Enzymes; Food; Human; Hydroxide Ion; Hydroxides; In Situ; Investigation; Ions; Laboratories; Lead; Life; Manuscripts; Mediating; Medical; Methodology; Methods; Micelles; Molecular; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Oxidation-Reduction; Phase; Picrates; Preparation; Process; Range; Rate; Reaction; Reagent; Sampling; Scheme; Scientist; Serum; Speed; System; Systems Analysis; Techniques; Technology; Time; Tube; Urine; Work; base; capillary; chemical reaction; design; dietary supplements; improved; microchip; novel; novel strategies; picrate; small molecule; success; technology/technique

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.

描述(申请人提供)：毛细管或微芯片形式的毛细管电泳法技术广泛应用的主要障碍之一是，当使用传统的紫外吸收检测时，浓度灵敏度较差。该项目涉及三种不同方法的研究，以提高生化和临床实验室毛细管电泳法(CE)的灵敏度和实用性。这项拟议的工作涉及对毛细管内局部条件的精确操纵，以影响CE分离过程中发生的分子水平过程。特别是，我们将研究使用快速极性切换(RP)和几种堆积方法来提高柱内反应(S)的速度和提高CE分析的浓度灵敏度。大多数拟议的工作涉及使用电泳中介微分析(EMMA)进行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是一种有用的方法，可以快速混合一个小分子反应的试剂，肌酸和苦味酸盐之间的Jaffe反应，我们将把这种方法扩展到其他小分子体系。对Jaffe反应产生的在线产物通过浓缩的氢氧化物塞进行瞬时等电性堆积(TITP)的补充工作似乎非常有效，并将继续进行，但也将探索更简单的TITP系统，以从根本上了解堆积动力学。这项工作的第三阶段将探索使用在线堆积和分离的胶束电动(MEKC)模式来进行毛细管内反应，形成不带电的产品。通过一项新的合作，我们将使用核磁共振光谱来研究和了解堆积和分离过程中胶束和分析物之间的相互作用。总而言之，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获得的灵敏度，并可能为临床实验室的快速、低容量生物分析带来新的方法。NARRITIVE这个项目涉及三种不同的方法来改进生化和临床实验室的毛细管电泳法。部分由于浓度灵敏度低的特点，毛细管或微芯片上基于CE的方法尚未得到充分开发，因此这种令人信服的分析模式没有得到充分利用。拟议的工作旨在提高基于CE的分析模式的灵敏度，方法是通过几种方法之一操纵毛细管中离散试剂区内离子的局部浓度，或通过增加作为分析方法一部分的柱内反应的速度。这项工作可能导致在临床实验室中开发快速、低容量和灵敏的生物检测的新方法。

项目成果

Investigating the effects of conductivity on zone overlap with EMMA: computer simulation and experiment.

• DOI: 10.1002/elps.201000451
• 发表时间: 2011-06
• 期刊: ELECTROPHORESIS
• 影响因子: 2.9
• 作者: Stahl, John W.;Catherman, Adam D.;Sampath, Ranasinghe K.;Seneviratne, C. Aravinda;Strein, Timothy G.
• 通讯作者: Strein, Timothy G.