CAREER: microMOSAIC Frameworks for Next-Generation Proteomic Technology

这项研究将引入新的光学图案微室和流动控制技术，以克服集成障碍，这些障碍阻碍了二十年来的微流体研究提供令人信服的替代方案。Western blotting和二维电泳（2DE）。蛋白质组学技术的突破至关重要：虽然基因组学革命对理解生命过程产生了广泛的影响，但可以说更重要的是？蛋白质组学革命仍未实现。蛋白质与功能有更直接的联系，具有更复杂的生物化学性质，数量比基因多10-100倍。因此，几乎所有的生命科学研究都需要多步骤分析，如Western blot和2DE。然而，台式多步骤分离需要大量的人力、时间投入和宝贵的样品来产生半定量；这些分析将从整合中受益匪浅。独特的ìMosaic集成策略-微室区域光模式与离散纳米结构分离材料-有可能彻底改变21世纪紧迫的蛋白质组学挑战的多步骤分离。多步骤蛋白质组学分析测量的不是一种，而是多种物理化学和功能特性。迄今为止，多步骤微分离的性能一直受到现状设计策略的影响，即将第一个分析步骤（单个微通道）耦合到第二个分析步骤（微通道阵列）。遗憾的是，这种方法会离散化？通过将第一级分离读数映射到第二级的离散隔间来读出它们。为了克服集成损失，PI提出了一种彻底改变现状的方法，即引入用离散纳米材料进行区域光模式的微室。支持ìMosaic集成技术的基础研究将包括：1)开发和定量评估微/纳米制造技术和设备，用于聚合物的电荷，化学和光图像化，以实现流体网络中的局部生化/物理功能。在图案纳米结构材料内的反应将使用荧光成像进行研究，在控制实验中使用受控的均质试剂流来确定特异性、动力学和总体亲和力。2)分离集成与分散控制策略的高效多步分离。PI将研究电迁移，扩散和反应之间的相互作用经验，分析，并与3D多物理场求解。PI将识别和控制运输和反应机制；即空间非均匀表面反应的能带展宽。虽然在均匀的微通道网络中可以理解耦合的分散和反应，但3D几何形状的情况并非如此，3D非均相反应模式支撑着本文提出的ìMosaic多步骤分析。总之，这些研究将提供新的知识：制造；纳米结构材料、分离装置和分析工程；实验方法。虽然可用的台式蛋白质组学工具无处不在，但在吞吐量，定量，动态范围和自动化方面存在显着限制。所提出的新颖和广泛适用的工具将具体推进每一个？影响系统生物学、合成生物学和生物燃料。可能的结果包括：基于模拟的治疗剂筛选，新型纤维素生物燃料的高通量鉴定，以及细菌生物修复的合理选择算法。PI将通过两门正在开发的课程中的新型多媒体讲座材料，以及在新的K-12合作伙伴关系（劳伦斯科学创新实验室大厅）中作为实践模块，将研究与教学结合起来。PI提出了一项全面的战略，以招募和留住代表性不足的学生，并在三个NSF REU中担任领导职务。作为伯克利大学的指导老师？她将支持美国女工程师协会（SWE）和伯克利大学(Berkeley？在美国国家科学基金会的支持下，成立了一个新成立的工程女性研究生小组。通过担任会议主席、技术项目委员会服务和社区建设（例如，MEMS中的女性），她是更大的技术社区的领导者。

This research will introduce novel optically patterned microchamber and flow control technology to overcome integration barriers that have prevented two decades of microfluidics research from offering compelling alternatives to the work-horse applications? Western blotting and two-dimensional electrophoresis (2DE). Breakthroughs in proteomic technology are essential: while the genomic revolution has had sweeping impact on understanding of life processes, the arguably more important ?proteomic revolution remains unrealized. Proteins are more directly linked with function, more biochemically complex, and 10-100x more numerous than genes. Thus, nearly all life science studies require multi-step assays such as Western blot and 2DE. Yet, benchtop multi-step separations demand huge labor, time investment, and precious sample to yield semi-quantitation; these assays would benefit immensely from integration. The unique ìMosaic integration strategy--microchambers regionally photopatterned with discrete nanostructured separation materials--has the potential to revolutionize multi-step separations for the pressing proteomic challenges of the 21st century.Multi-step proteomic assays measure not one, but multiple physicochemical and functional properties. To date, the performance of multi-step microseparations has suffered from status quo design strategies that couple a first assay step (a single microchannel) to a second assay step that is an array of microchannels. Regrettably this approach ?discretizes? first-stage separation readouts by mapping them to discrete compartments in a second-stage. To overcome integration losses, the PI proposes a radical departure from status quo by introducing microchambers regionally photopatterned with discrete nanomaterials. Fundamental studies underpinning ìMosaic integration technology will include: 1) Development & quantitative evaluation of micro/nanofabrication techniques and devices for charge-, chemical-, and photo-patterning of polymers to achieve localized biochemical/physical function within fluidic networks. Reactions within the patterned nanostructured materials will be studied using fluorescence imaging, determining specificity, kinetics, and overall affinity using controlled, homogenous reagent flows in control experiments. 2) Separation integration with dispersion control strategies for high performance multi-step separations. PI will investigate the interplay between electromigration, diffusion, and reaction empirically, analytically, and with 3D multiphysics solvers. The PI will identify and control transport and reaction mechanisms; i.e., band broadening with spatially non-uniform surface reactions. Although coupled dispersion and reaction are understood in homogeneous microchannel networks, such is not the case with 3D geometries, 3D heterogeneous reaction patterns underpinning the ìMosaic multi-step assays proposed here. Together, these studies will provide new knowledge in: fabrication; nanostructured materials, separations device and assay engineering; experimental methods. While available bench-top proteomic tools are ubiquitous, significant limitations in throughput, quantitation, dynamic range, and automation exist. The proposed novel and widely applicable tools will specifically advance each ? impacting systems biology, synthetic biology, and biofuels. Likely outcomes include: simulation-based screening of therapeutic agents, high-throughput identification of new cellulosic biofuels, and rational selection algorithms for bacterial bioremediation. The PI will integrate research with teaching through new multi-media lecture material in two developing courses and as hands-on modules in a new K-12 partnership (Lawrence Hall of Science Ingenuity Labs). The PI proposes a comprehensive strategy to recruit and retain underrepresented students and holds leadership positions on three NSF REU?s. As faculty advisor to Berkeley?s Society of Women Engineers (SWE), she will bolster SWE and Berkeley?s nascent Graduate Women in Engineering group through NSF support. She is a leader in the larger technical community through conference chair-ship, technical program committee service, and community building (e.g., Women in MEMS).