Multi-Dimensional Separation of Bacteria

描述(由申请人提供)：由于大量的非可培养生物，甚至在每个物种内的异质性，理解微生物和它们的宿主之间的复杂关系的努力是复杂的。虽然现代元基因组学方法对微生物的身份进行了令人钦佩的采样，但大量研究限制了可以从每种细菌中得出的其他推断。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：基于结构参数(大小、形状和电泳)和功能参数(粘附性、趋化性)，对多维微流控分选器进行建模、设计、建造和测试，以将复杂的细菌混合物分离到包含具有共同属性的细菌的垃圾箱中。分类细菌的身份将通过元基因组学研究获得。该装置将能够确定复杂混合物中物种之间和物种内的异质性。这种微流控分离装置将利用1.不对称夹带流动分离技术，根据细菌的大小和形状进行分离。2.基于表面电荷的分离细菌的电泳法。3.功能化磁珠，基于对细胞外基质(ECM)成分的黏附来分离细菌。4.根据细菌对化学刺激的运动反应来分离细菌的趋化性，最后是5.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来分离细菌的多种分离方式。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相适应；(Ii)复杂的流道可以轻松且低成本地加工；(Iii)使用不同原理的许多分拣模块可以集成到一个设备中。在每个特定的目标中，我们很大程度上依赖于使用反映二维几何的代码对粒子运动的直接数值模拟。作为实验计划的一部分，我们将扩展我们的定制粒子移动程序的功能，以实现完整的3-D模拟。一旦设计参数确定，装置将使用已知细菌的颗粒混合物进行严格制造和测试，对于3级和4级装置，将使用来自人体的复杂混合物。我们将使用便于模块互换的模块化体系结构。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，用于芯片上的单细胞分离、DNA分离和扩增，以实现对复杂混合物的高通量分析。这些研究将导致设备不仅捕捉复杂混合物的多样性，而且允许直接分配混合物中每个单一物种的结构和功能特性、基因和基因产物的异质性，并有助于了解人类疾病。公共卫生相关性：这项提议代表了三位具有独特和互补兴趣的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：建模、设计、建造和测试基于结构(大小、形状和电泳)和功能(粘附性、趋化性)参数的多维微流体分选器，以将复杂的细菌混合物分离到装有具有共同属性的细菌的垃圾箱中。分类细菌的身份将通过元基因组学研究获得。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，例如在发病机制不明的临床感染性疾病(我们对支气管扩张症和坏死性小肠结肠炎特别感兴趣)中。我们也有兴趣帮助理解流体动力学(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结合。

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.