? The emergence of effective therapies for modulating disordered signal transduction in human disease has brought about a crucial need to assay the activity of signaling proteins in patients and model organisms. To meet this need, the current grant application proposes to design, build, and test an analytical platform for the performance of single-cell kinase assays on primary patient cells. State-of-the art microfabrication technology will be employed to engineer a microfluidic system that integrates electronics and photonics for the performance of microanalytical chemical separations. The microfluidic system will be coupled to a meso-scale incubator for sample preparation. The components of the hybrid device will be designed and optimized individually for their given function, and then integrated into a multifunctional platform. This platform will be used to carry out a newly developed method for assaying the catalytic activity of specific kinases in individual cells. In the final phase of the work assays of kinases known to be essential for the development and maintenance of an exemplary disease will be demonstrated in patient samples. The activity of the tyrosine kinase Bcr-Abl and the serine/threonine kinase protein kinase B in the cells of patients with chronic myelogenous leukemia will be assessed after exposure to varying doses of the recently approved kinase inhibitor imatinib mesylate. The direct assay of kinases before and after ex vivo exposure of the patient's cells to this pharmaceutical compound will provide unique and clinically valuable data regarding the drug's efficacy and dosing for personalized tailoring of treatment options. The single-cell data will also enable subpopulations of cells within a given patient to be assessed to address cell-to-cell heterogeneity in response to therapy. A number of kinase inhibitors are now in development or clinical trials for a broad range of diseases making the development of this novel instrumentation of widespread value for biomedical research and clinical application. ? ? ?
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