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.
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