Microfluidic Systems with Monoliths Enabling Sample Preparation in Proteomics

该提案旨在设计、制备和测试含有多孔纳米颗粒的微流体装置。 聚合物整料，将提供无与伦比的性能，在"样品制备"和 鉴定复杂混合物中的蛋白质，并充分利用 蛋白质组学研究它将提供一系列增强的工具， 由玻璃或塑料制成的装置将包含由以下物质原位制备的功能性整料： 液体前体通过简单的自由基聚合过程。这将提供一个通用的 具有针对所需应用优化的多孔性质的大孔整体聚合物， 随后可以通过UV引发的光接枝来官能化。光接枝使得 几种不同功能（如疏水性、亲水性、可离子化或亲和性）的放置 在串联放置或作为分层或梯度化学品放置的精确位置中。该提案还旨在 在共价固定酶和配体的新方法的发展， 将能够在同一通道中独立固定多个生物分子。使用 顺序光接枝、集成样品收集的单片装置、酶促 准备蛋白质的消化和肽的分离。蛋白质混合物将是 使用具有精心设计的选择性的分离模块分离成较不复杂的样品。 优化整料的结构性能，预计回收率接近100 所有存在于样本中的蛋白质。具体的设计也将给予关注 并控制所有制备过程，以确保两者的高批次间重现性， 积木和复合器件。将这些设备直接耦合到质谱仪 将简化和加速蛋白质组学研究。我们希望，该项目将导致实际的 实施一系列全新的样品制备通用组件和设备 以及蛋白质组或其组分的质谱研究， 能力远远超出了目前的技术水平。

This proposal aims at the design, preparation, and testing of microfluidic devices containing porous polymer monoliths that will provide unmatched performance in "sample preparation" and the identification of proteins in complex mixtures and enable full exploitation of the rich potential of proteomic research. It will provide access to an enhanced array of tools for handling biological samples.The devices made of glass or plastic will contain functional monoliths prepared in situ from liquid precursors via a simple free radical polymerization process. This will afford a generic macroporous monolithic polymer with porous properties optimized for the desired application that can subsequently be functionalized by UV intiated photografting. The photografting enables the placement of sevral different functionalities (such as hydrophobic, hydrophilic, ionizable, or affinity) in precise locations placed in series or as layered or gradient chemistries. This proposal also aims at the development of new approaches to the covalent immobilization of enzymes and ligands that will enable independent immobilization of multiple biological molecules in the same channel. Using the benefits of sequential photografting, monolithic devices integrating sample collection, enzymatic digestion of proteins, and separation of peptides will be prepared. Difficult protein mixtures will be separated into less complex samples using separation modules with carefully designed selectivities. Optimization of structural properties of the monoliths is expected to afford recoveries close to 100% for all of the proteins present in the sample. Specifics attention will also will be paid to designing and controling all preparation processes to ensure high batch-to-batch reproducibility of both building blocks and composite devices. Direct coupling of these devices to a mass spectrometer will simplify and accelerate proteomic research. We expect that this project will result in the practical implementation of a totally new array of versatile components and devices for sample preparation and the mass spectroscopic study of proteomes or their components extending microanalytical capabilities well beyond the current state of the art.