Multi-Dimensional Separation of Bacteria

描述（由申请人提供）：由于大量不可培养的生物体以及甚至在每个物种内的异质性，理解微生物及其宿主之间复杂关系的努力变得复杂。虽然现代宏基因组学方法令人钦佩地对微生物的身份进行了采样，但批量研究限制了可以从每种细菌中得出的其他推论。为了克服这个问题，同时保留原始种群多样性的线索，我们建议：基于结构（尺寸、形状和电泳）和功能（粘附性、趋化性）参数来建模、设计、构建和测试多维微流体分选机，以将复杂的细菌混合物分离到包含具有共同特性的细菌的箱中。将通过宏基因组研究获得分选的细菌的身份。该设备将能够确定复杂混合物中物种之间和物种内部的异质性。该微流体分离装置将利用1.根据大小和形状分离细菌的不对称箍缩流分级。2.基于电泳的流动分级，以基于表面电荷分离细菌。3.功能化磁珠，基于对细胞外基质（ECM）组分的粘附来分离细菌。4.根据细菌对化学刺激物的运动反应来分离细菌的趋化性，以及最后，5。多种分离模式，根据大小、形状、粘附性、对化学刺激的反应和表面电荷分离细菌。我们将使用微流控方法，因为（i）微流控系统的特征尺寸与细菌的大小兼容;（ii）复杂的流动路径可以轻松且低成本地加工;（iii）利用不同原理的许多分选模块可以集成到单个设备中。在每个特定的目标，我们在很大程度上依赖于直接数值模拟的粒子运动使用的代码，反映了二维几何。作为实验计划的一部分，我们将扩展我们的自定义粒子移动器程序的功能，以实现完整的3D模拟。一旦确定了设计参数，将使用已知细菌的颗粒混合物以及来自人类受试者的复杂混合物（对于3级和4级装置）来制造和严格测试装置。我们将利用模块化架构，促进模块的可互换性。长期目标是将其他分离模式添加到设备中，并将用于单细胞分离、DNA分离和芯片上扩增的模块集成到设备中，以允许对复杂混合物进行高通量分析。这些研究将导致设备，不仅捕捉复杂的混合物的多样性，但也允许直接分配的异质性的结构和功能特性，基因和基因产物内的每个单一物种的混合物，并帮助了解人类疾病。公共卫生相关性：这一提议代表了三位具有独特和互补兴趣的科学家之间的新的合作努力。通过专注于我们在流体动力学（Hu），微流体设计（Bau），临床和实验细菌感染（Worthen）的专业知识，我们建议：模型，设计，构建和测试基于结构（大小和形状和电泳）和功能（粘附性，趋化性）参数的多维微流体分选机，将复杂的细菌混合物分离到含有共享共同特性的细菌的箱中。将通过宏基因组研究获得分选的细菌的身份。该设备将能够确定复杂混合物中物种之间和物种内的异质性，例如在临床感染性疾病（我们对支气管扩张和坏死性小肠结肠炎特别感兴趣）中，其发病机制尚不清楚。我们也有兴趣帮助了解流体动力学（将向科学界提交一个新的数值模拟程序）和微流体学等工具如何与医学相交。

DESCRIPTION (provided by applicant): Efforts to understand the complex relationship between microbes and their hosts are complicated by the large number of nonculturable organisms, and the heterogeneity even within each species. While modern metagenomics approaches admirably sample the identity of microbes, bulk studies limit the other inferences that can be derived from each bacteria. In order to overcome this problem, yet retain clues to the diversity of the original population, we propose to: Model, design, build, and test a multidimensional, microfluidic sorter based on both structural (size-and shape and Electrophoresis) and functional (Adhesion, Chemotaxis) parameters to separate a complex bacterial mixture into bins containing bacteria that share common properties. The identities of the sorted bacteria will be obtained through metagenomic studies. The device will enable determination of the heterogeneity both between and within species in a complex mixture. This microfluidic separation device will utilize 1. Asymmetric pinched flow fractionation to separate bacteria based on size and shape. 2. Electrophoretic based flow fractionation to separate bacteria based on surface charge. 3. Functionalized magnetic beads to separate bacteria based on adhesion to extracellular matrix (ECM) components. 4. Chemotaxis to separate bacteria based on their motile response to chemical stimuli, and lastly, 5. Multi separation modalities to separate bacteria based on size, shape, adhesion, response to chemical stimuli, and surface charge. We will use microfluidic approaches since (i) the feature sizes of microfluidic systems are compatible with the size of the bacteria; (ii) complicated flow paths can be machined with ease and at low cost; (iii) many sorting modules utilizing diverse principles can be integrated into a single device. Within each specific aim we rely heavily on direct numerical simulation of particle movement using code that reflects 2-dimensional geometry. As part of the experimental plan, we will expand functionality of the our custom Particle Mover program to a full 3-D simulation. Once design parameters have been established, devices will be fabricated and tested rigorously using particles, mixtures of known bacteria, and for the 3 and 4-stage devices, complex mixtures from human subjects. We will make use of a modular architecture that facilitates interchangeability of modules. The long term goal is to add other separation modalities into the device and to integrate into the device modules for single cell isolation, DNA isolation and amplification on-chip, to permit high-throughput analysis of complex mixtures. These studies will lead to devices that not only capture the diversity of complex mixtures, but also permit direct assignment of the heterogeneity of structural and functional properties, genes and gene products within each single species in the mixture, and aid understanding of human disease. PUBLIC HEALTH RELEVANCE: This proposal represents a new collaborative effort between three established scientists with unique and complementary interests. By focusing our expertise in fluid dynamics (Hu), microfluidic design (Bau), and clinical and experimental bacterial infection (Worthen) we propose to: Model, design, build, and test a multidimensional, microfluidic sorter based on both structural (size-and shape and Electrophoresis) and functional (Adhesion, Chemotaxis) parameters to separate a complex bacterial mixture into bins containing bacteria that share common properties. The identities of the sorted bacteria will be obtained through metagenomic studies. The device will enable determination of the heterogeneity both between and within species in a complex mixture, such as in clinical infectious illnesses (we are particularly interested in Bronchiectasis and necrpotizing enterocolitis) whose pathogenesis is obscure. We also are interested in contributing to an understanding of how tools such as fluid dynamics (for which a new program of numerical simulation will be presented to the scientific community) and microfluidics intersect with medicine.