Multidimensional Electrofocusing on Gradient Monoliths

描述（申请人提供）：传统2D-PAGE操作繁琐，速度慢，难以自动化。尽管多维、高效的毛细管色谱、电泳和电色谱是强大的和完全自动化的，但它们固有的串行柱操作是瓶颈。这些缺点可以通过采用二维平台来克服，该平台利用了几种基于新型梯度单块技术的新型电聚焦方法。单片色谱填料的使用使我们能够设计一个具有色谱操作许多优点的平台，例如，在高度并行化的平台上实现自动化，而使用新颖的原位固定梯度将允许在该平台上实施一套新的平衡梯度方法，特别是基于电导率梯度，尺寸排除梯度和pH梯度的电聚焦。

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.