Multidimensional Electrofocusing on Gradient Monoliths

描述(申请人提供)：传统的2D页面繁琐，速度慢，难以实现自动化。尽管多维、高性能毛细管色谱、电色谱和电色谱具有很强的稳定性和全自动化，但它们固有的串联柱操作是瓶颈。这些缺点可以通过采用2D平台来避免，该平台利用了几种基于新的梯度整体技术的新的电聚焦方法。整体色谱柱的使用使我们能够在高度并行化的平台中设计出具有许多色层操作优点的平台，例如，自动化，同时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，特别是基于电导梯度、尺寸排除梯度和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功能化技术，以及用于蛋白质分离的替代电聚焦方法的开发，使得在微芯片格式中实现这一点成为可能。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数量，以及(3)可以将部分样品负载临时隔离(上游)或洗脱(下游)芯片外，以减少处理过程中高丰度蛋白质的负担。本申请寻求在梯度单片封装中演示两种或两种以上这种新型1D电聚焦技术，然后将它们组合到2D微芯片中。 公共卫生相关声明：最近出现的光引发聚合和整体填料的梯度功能化，以及用于蛋白质分离的替代电聚焦方法的开发，使以微芯片格式建立2D电聚焦平台的能力成为可能。该项目旨在展示两种或两种以上这种新型1D电聚焦技术在梯度单片封装中的应用，然后将它们组合成2D微芯片。它还将开发多维平台模拟、微型工艺集成和芯片设计所需的许多工程基础。

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.