Chapter CHAPTER CONCLUSIONS AND OUTLOOK In the first part of this work, separation conditions of Z and W genes were optimized with conventional with conventional capillary electrophoresis. The separation conditions developed with conventional CE were transferred to microfabricated CE. The separation of genes of interest was performed in less than minutes on glass chips compared to 25 minutes with conventional CE. Various coating materials were investigated: coating of the capillaries with PVP gave similar efficiencies compared to covalent coating with PA to reduce the EOF. This is advantageous for separations on microfabricated devices since acrylamide is toxic and covalent coatings are prone to deterioration. In addition, various materials were tested for on-chip separation, and separation with silicon microchips gave better results than with PDMS microchips. Microfabricated CE devices were integrated with microfabricated PCR devices for fast DNA amplification and separation. This system is extremely versatile, could be applied to other genes or samples of interest and could easily be combined with other microscale devices for the development of an integrated “lab-on-a-chip. In the second part of this work, array-based strategies for proteomics studies were developed. Two new methods for site-specific attachment of N-terminally modified peptides were reported for usage in peptide arrays. Biotinylated peptides were spotted onto glass slides functionalized with avidin to achieve instantaneous immobilization. Avidin serves as both an immobilizing agent and an agent to minimize non-specific absorption of proteins on glass surface. Alternatively, peptides containing an Nterminal cysteine were chemoselectively immobilized onto thioester-functionalized slides. Both methods show versatility in generating peptide-based microarrays suitable 115 Chapter for high-throughput screenings of kinase activities, and potentially other enzymatic activities. The intercalation of various PEGs between the slide surface and the immobilized peptides minimized non-specific binding of proteins to the glass surface, rendering it possible to eliminate the blocking step using BSA. In addition, a novel fluorescence-based approach for quantitative detection of peptide phosphorylation on chip using fluorescently-labeled anti-phosphoserine and antiphosphotyrosine antibodies was developed. Due to its sensitivity, high specificity and the short incubation time needed to detect phosphorylation, this antibody-based approach presents obvious advantages over other existing methods, and may find wider uses in high-throughput kinase screenings. The site-specific immobilization of proteins is even more critical since their active site needs to be accessible to the substrates. By arraying C-terminal biotinylated proteins on avidin functionalized slides, a very stable array of functionally active proteins was obtained. The only site-specific immobilization of proteins onto glass slide so far reported rely on the immobilization of His tag proteins onto Ni-NTA functionalized slides. This, however, is not very strong, nor stable. Intein-mediated biotinylation of proteins allows for their site-specific labeling at their C-termini, ensuring their sitespecific immobilization onto avidin functionalized slides. The advantage of avidin/biotin linkage over His-tag/Ni-NTA strategies for protein immobilization is highlighted by its ability to withstand a variety of chemical conditions, which may make this new protein array compatible with most biological assays. Microarrays of site-specifically attached proteins were developed. However, the expression of proteins still remain a lengthy procedure and proteins are stable only for 116 Chapter a short period of time. By using a cell-based array strategy, one could express proteins functionalized with a tag, which after cell lysis would bind to the functionalized slide surface thus creating arrays of functional and site-specifically attached proteins in a very short period of time. 117 . efficiencies compared to covalent coating with PA to reduce the EOF. This is advantageous for separations on microfabricated devices since acrylamide is toxic and covalent coatings are prone to deterioration chemoselectively immobilized onto thioester-functionalized slides. Both methods show versatility in generating peptide -based microarrays suitable 1 15 Chapter 5 for high-throughput screenings. anti-phosphoserine and anti- phosphotyrosine antibodies was developed. Due to its sensitivity, high specificity and the short incubation time needed to detect phosphorylation, this antibody -based approach