Towards High Throughput Proteomics: A Micro/nanofluidic Framework for Blotless W

描述（由申请人提供） 摘要：在传统的实验室生物学中，通常使用劳动密集型、耗时的测定，特别是通过蛋白质印迹的蛋白质分析。虽然蛋白质印迹在识别复杂液体中的靶蛋白质方面提供了卓越的特异性，但多步骤蛋白质印迹仍然充满了需要训练有素的用户干预的缓慢过程。此外，蛋白质印迹结果充其量是半定量的，并且需要可观的样品质量用于检测。在一个旨在改变实验室生物学面貌的范式转变中，首席研究员建议使用源于她实验室研究的集成微纳米技术的新概念来简化和加快Western印迹分析。具体来说，主要研究者提出了自动化多步测定，包括主力免疫印迹，以产生与单细胞分析兼容的系统。我们利用有利的微米和纳米级的现象，显着提高三个运输机制的核心蛋白质印迹分析：蛋白质样品的电泳分析，选择分离的蛋白质组分的转移，并在完全集成的纳米多孔膜上的分离物种的印迹。在短期内，主要研究人员将解决两个具有挑战性的问题，一个直接来自基础研究，另一个具有直接转化潜力。即，1）新技术将用于监测病原性损伤下单个宿主细胞（巨噬细胞）的自分泌和旁分泌信号传导，作为表征“第一应答者”先天免疫应答的手段。此外，2）主要研究者试图通过使用新引入的技术来分析通过激光显微切割前列腺活检样品捕获的小的均质细胞群中的MAO-A水平，来验证单胺氧化酶-A（MAO-A）在侵袭性前列腺癌表型中的重要性或使其无效。这里概述的工作是变革性的，也是转化性的-由此产生的创新将成为重塑疾病机制基础科学研究的基础，并为新的医学诊断和治疗策略提供信息。

DESCRIPTION (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.