Multidimensional Electrofocusing on Gradient Monoliths

梯度巨石的多维电聚焦

• 批准号: 7939904
• 负责人: CORNELIUS F IVORY
• 金额: $ 30.06万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-24 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7939904
• 关键词: Acrylamides; Adopted; Automation; Back; Biomedical Engineering; Blood capillaries; Cell Line; Charge; Chemical Engineering; Chromatography; Collaborations; Complement; Development; Dimensions; Electrophoresis; Emerging Technologies; Engineering; Equilibrium; Exclusion; Fluorescent Dyes; High Pressure Liquid Chromatography; In Situ; Individual; Isoelectric Focusing; Isoelectric Point; Knowledge; Label; Laboratories; Location; Mammalian Cell; Measures; Mechanics; Methods; Microfluidic Microchips; Modeling; Molecular; Molecular Sieve Chromatography; Molecular Weight; Pattern; Peptides; Performance; Phase; Physiologic pulse; Plastics; Process; Property; Proteins; Protocols documentation; Research; Resolution; Running; Sampling; Schools; Silicon Dioxide; Solutions; Techniques; Technology; Testing; Two-Dimensional Polyacrylamide Gel Electrophoresis; Width; Work; base; capillary; chemotactic factor inactivator; design; electric field; experience; functional group; instrument; meetings; microchip; novel; operation; pH gradient; performance tests; polymerization; protein complex; public health relevance; simulation; solute; tool

DESCRIPTION (provided by applicant): Conventional 2D-PAGE is cumbersome, slow and difficult to automate. Although, multidimensional, high performance capillary chromatography, electrophoresis and electrochromatography are robust and fully automatable, they are bottlenecked by their inherently serial column operation. These shortcomings can be circumvented by adopting a 2D platform which utilizes several new electrofocusing methods that are based on a novel gradient-monolith technology. The use of monolithic chromatography packings allows us to design a platform which has many of the advantages of chromatographic operation, e.g., automation, in a highly parallelized platform while the use of novel in situ fixed gradients will permit implementation of a new set of equilibrium gradient methods on this platform, specifically, electrofocusing based on conductivity gradients, size-exclusion gradients and pH gradients. The ability to do this in a microchip format is enabled by the recent emergence of technologies for photoinitiated polymerization and gradient functionalization of monolithic packings combined with the development of alternative electrofocusing methods for protein separation. Some key implications of 2D electrofocusing are (1) that the final location and width of protein peaks are independent of their initial distribution, (2) that multiple sample pulses may be loaded onto the platform to increase the amount of low-abundance proteins, and (3) that portions of the sample load can be temporarily sequestered (upstream) or eluted (downstream) off-chip to reduce the high-abundance protein burden during processing. This application seeks to demonstrate two or more of these novel 1D electrofocusing techniques in gradient monolithic packings and then combine them into a 2D microchip. Public Health Relevance Statement: The ability to build a 2D electrofocusing platform in a microchip format is enabled by the recent emergence of photoinitiated polymerization and gradient functionalization of monolithic packings combined with the development of alternative electrofocusing methods for protein separation. This project seeks to demonstrate two or more of these novel 1D electrofocusing techniques in gradient monolithic packings and then combine them into a 2D microchip. It will also develop many of the engineering fundamentals needed for multidimensional platform simulations, microscale process integration and chip design.

描述（由申请人提供）：常规2D-PAGE繁琐、缓慢且难以自动化。尽管多维高效毛细管色谱、电泳和电色谱是稳健的且可完全自动化的，但它们被其固有的串联柱操作所阻碍。这些缺点可以通过采用2D平台来规避，该平台利用基于新型梯度整料技术的几种新的电聚焦方法。整体色谱填料的使用允许我们设计具有色谱操作的许多优点的平台，例如，在高度并行化的平台中，自动化，而使用新的原位固定梯度将允许在该平台上实施一组新的平衡梯度方法，具体地，基于电导率梯度、尺寸排阻梯度和pH梯度的电聚焦。 最近出现的光引发聚合和整体填料的梯度官能化技术，结合蛋白质分离的替代电聚焦方法的发展，使得能够以微芯片的形式实现这一点。2D电聚焦的一些关键含义是（1）蛋白质峰的最终位置和宽度与其初始分布无关，（2）可以将多个样品脉冲加载到平台上以增加低丰度蛋白质的量，以及（3）样品负载的部分可以被暂时隔离在一个实施方案中，可以在芯片外（上游）或洗脱（下游）以减少处理期间的高丰度蛋白质负荷。本申请试图在梯度单片填料中展示这些新颖的1D电聚焦技术中的两种或更多种，然后将它们联合收割机组合成2D微芯片。 公共卫生相关性声明：通过最近出现的光引发聚合和整体填料的梯度官能化以及用于蛋白质分离的替代电聚焦方法的发展，能够以微芯片形式构建2D电聚焦平台。该项目旨在展示两个或更多的这些新的一维电聚焦技术在梯度单片包装，然后将它们联合收割机到一个二维微芯片。它还将开发多维平台仿真，微尺度工艺集成和芯片设计所需的许多工程基础。