Multi-Dimensional Separation of Bacteria

描述（由申请人提供）：由于大量不可培养的生物以及每个物种内部的异质性，理解微生物与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，但大量研究限制了从每种细菌中得出的其他推论。为了克服这一问题，同时保留原始种群多样性的线索，我们建议：基于结构（大小和形状以及电泳）和功能（粘附性，化学亲和性）参数建模，设计，构建和测试一个多维微流控分选器，将复杂的细菌混合物分离到含有具有共同特性的细菌的容器中。将通过宏基因组学研究获得被分类细菌的特性。该装置将能够测定复杂混合物中物种之间和物种内部的异质性。这种微流控分离装置将利用1。根据大小和形状分离细菌的非对称捏流分馏。2. 基于电泳的流动分馏，根据表面电荷分离细菌。3. 功能化磁珠分离细菌基于黏附细胞外基质（ECM）成分。4. 化学趋向性：根据细菌对化学刺激的运动反应来分离细菌。根据细菌的大小、形状、粘附性、对化学刺激的反应和表面电荷，采用多种分离方式分离细菌。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尺寸兼容；（ii）复杂的流道可以容易而低成本地加工；(3)多种原理的分选模块可集成到一个设备中。在每个特定目标中，我们严重依赖于使用反映二维几何形状的代码的粒子运动的直接数值模拟。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程序的功能到一个完整的三维模拟。一旦设计参数确定，设备将被制造和严格测试，使用颗粒，已知细菌的混合物，对于第三和第四阶段的设备，来自人类受试者的复杂混合物。我们将使用模块化体系结构来促进模块的互换性。长期目标是将其他分离模式添加到设备中，并集成到设备模块中，用于单细胞分离，DNA分离和片上扩增，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混合物的多样性，而且还允许直接分配结构和功能特性的异质性，基因和基因产物在混合物中的每一个单一物种，并有助于了解人类疾病。公共卫生相关性：本提案代表了具有独特和互补兴趣的三位知名科学家之间的新合作努力。通过集中我们在流体动力学（Hu），微流体设计（Bau）以及临床和实验细菌感染（Worthen）方面的专业知识，我们建议：基于结构（尺寸和形状以及电泳）和功能（粘附性，Chemotaxis）参数建模，设计，构建和测试多维微流体分选器，将复杂的细菌混合物分离到含有具有共同特性的细菌的容器中。将通过宏基因组学研究获得被分类细菌的特性。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，例如临床感染性疾病（我们对发病机制不明的支气管扩张和坏死性小肠结肠炎特别感兴趣）。我们也有兴趣帮助理解流体动力学（为此将向科学界提出一个新的数值模拟程序）和微流体学等工具如何与医学交叉。

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.