Development of Enzymatic Microreactors for the Analysis of Proteins via Mass Spectrometry
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Many techniques exist for protein analysis including gel electrophoresis, immunoassays and mass spectrometry to name a few. Although each of these techniques offers different advantages there are challenges associated with protein analysis that present limitations and drawbacks for each technique. These include limited sample volumes, incompatible matrices, complex mixtures, limit of detection, and complicated and time consuming analysis techniques. The research described in this thesis deals with methods which address the above mentioned challenges. The fabrication of enzymatic microreactors and the development of an extraction technique to remove proteins from incompatible matrices are presented. A low temperature solvent bonding technique was developed that can easily be modified to accommodate pH and temperature sensitive enzymes such as pepsin. Microfluidic chips fabricated with poly(methyl methacrylate) were constructed using this bonding method. The low temperature bonding method allows enzymes to be patterned on microfluidic devices prior to bonding with no negative impact on enzyme activity. Both peptic and tryptic reactors were fabricated using this technique. These devices have a lifetime of one month and can perform protein digestions in as little as 2.4 seconds. Alternatively, enzymatic microreactors were developed using a novel enzyme immobilization method on porous polymer monolith columns. These columns have the advantage of having the dual functionality of an enzymatic microreactor and an electrospray emitter permitting on-line digestions to be performed. Furthermore, the new immobilization method allows columns to have a longer lifetime as this method permitted the regeneration of the columns with fresh enzyme once enzyme activity was lost. To overcome the issues associated with incompatible matrices, an extraction technique using acetonitrile and octyl-β-D-glycopyranoside was developed to remove proteins from organic matrices. This was developed to detect the presence of prion proteins in biodiesel. Afterwards, the prion proteins were enzymatically digested and detected by mass spectrometry. Finally both types of enzyme microreactors were applied to proteins that are difficult to digest with traditional in-solution digestions. The digestion of prion proteins and α-1-protease inhibitor were found to be more efficient and conducted in significantly less time demonstrating the potential use of these devices in clinical research.