Microfluidic Systems with Monoliths Enabling Sample Preparation in Proteomics

该方案旨在设计、制备和测试包含多孔的微流控器件 将在“样品制备”中提供无与伦比的性能的聚合物整体，以及 鉴定复杂混合物中的蛋白质，并使其丰富的潜力得以充分开发 蛋白质组学研究。它将提供一系列增强的工具，用于处理生物 样品。由玻璃或塑料制成的设备将包含原位制备的功能整体 液体前驱体通过简单的自由基聚合过程。这将负担得起仿制药 具有针对所需应用优化的多孔性的大孔整体式聚合物， 随后可以通过紫外光引发的光接枝来功能化。光嫁接使 放置几种不同的功能(如疏水性、亲水性、电离性或亲和性) 以连续或分层或梯度化学的方式放置在精确的位置。这项提议还旨在 在开发酶和配体的共价固定化的新方法时 将能够在同一通道中独立固定多个生物分子。vbl.使用 连续光嫁接的好处，集成了样品采集、酶促 将准备蛋白质的消化和多肽的分离。困难的蛋白质混合物将是 使用具有精心设计的选择性的分离模块将其分离成不太复杂的样品。 整体结构性能的优化有望提供接近100%的回收率 样本中存在的所有蛋白质。具体的设计也将受到关注 以及控制所有的制备过程，以确保两者的高批次重现性 构建块和复合设备。这些设备与质谱仪的直接耦合 将简化和加速蛋白质组学研究。我们期待这个项目将产生实际的 用于样品制备的一系列全新的通用组件和设备的实施 以及扩展微量分析的蛋白质组或其组成的质谱学研究 能力远远超过目前的技术水平。

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.