(Provided by the applicant) Abstract: In conventional laboratory biology, labor-intensive, time-consuming assays are routinely used, in particular protein analysis by Western blot. While Western blots provide exceptional specificity in identifying targeted proteins in complex fluids, the multi-step Western is nevertheless fraught with slow processes requiring trained user intervention. Further, Western blot results are semi-quantitative at best and require appreciable sample mass for detection. In a paradigm shift that aims to change the face of laboratory biology, the principal investigator proposes to streamline and expedite Western blot analysis using a new concept for integrated micro and nanotechnology stemming from research in her laboratory. Specifically, the principal investigator proposes to automate multi-step assays, including workhorse immunoblots, in a format that yields systems compatible with single cell analysis. We harness advantageous micro and nanoscale phenomena to significantly improve three transport mechanisms core to Western blot analysis: electrophoretic analysis of protein samples, transfer of select separated protein fractions, and blotting of separated species on fully integrated nanoporous membranes. In the immediate term, the principle investigator will tackle two challenging questions, one directly from basic research and one with immediate translational potential. Namely,1) the new technology will be employed to monitor autocrine and paracrine signaling of a single host cell (macrophage) under pathogenic insult as a means to characterize 'first responder'innate immune response. Further, 2) the principal investigator seeks to validate or invalidate the importance of the enzyme monoamine oxidase-A (MAO-A) in aggressive prostate cancer phenotypes, by using the newly introduced technology to analyze MAO-A levels in small, homogeneous cell populations captured by laser microdissection of prostate biopsy samples. The work outlined here is transformative as well as translational - the resulting innovation will serve as a foundation for reshaping basic scientific study of disease mechanisms as well as inform novel medical diagnostic and therapeutic strategies.
The proposed work addresses aging-related tissue degeneration and pathologies by introducing new point-of care diagnostic technology to monitor tissue degeneration and response to therapy. We see the new tools as critical for assessing response to therapy, a difficult task without biochemical information as is currently the case. Communities such as aged individuals, those suffering from cancer cachexia, and muscle degeneration illness or injury (muscular dystrophy, ALS, burns) will benefit from the new assessment approach.
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