FISH "N" Chips: A Microfluidic Processor for Isolating and Analyzing Microbes

FISH“N”芯片：用于分离和分析微生物的微流体处理器

• 批准号: 7914248
• 负责人: Anup K. Singh
• 金额: $ 54.05万
• 依托单位: SANDIA CORP-SANDIA NATIONAL LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-17 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7914248
• 关键词: Anatomic Sites; Archives; Bacteria; Bacterial Infections; Biological; Biological Assay; Cell Separation; Cells; Clinical; Collaborations; Complex; Complex Mixtures; Coupled; DNA; DNA Probes; DNA Sequence; Dentistry; Deposition; Devices; Diagnostic; Diagnostic tests; Disease; Ecology; Effectiveness; Encapsulated; Escherichia coli; Flow Cytometry; Fluorescence; Fluorescent in Situ Hybridization; Genes; Genome; Genomics; Goals; Human; Individual; Institutes; Joints; Laboratories; Lactobacillus acidophilus; Lactobacillus fermentum; Measures; Metagenomics; Methods; Microbe; Microfluidics; Optics; Oral; Oral cavity; Organism; Output; Phylogenetic Analysis; Play; Population; Process; Ribosomal RNA; Role; Saliva; Salivary; Sampling; Sensitivity and Specificity; Site; Sorting - Cell Movement; Specificity; Specimen; Streptococcus mutans; Techniques; Technology; Testing; Therapeutic Agents; Time; base; college; design; genome sequencing; human disease; micro-total analysis system; microbial; microbiome; microorganism; new technology; novel; novel strategies; oral bacteria; photonics; public health relevance; rRNA Genes; single cell analysis

DESCRIPTION (provided by applicant): Since uncultivable microorganisms comprise a large percentage of the microbiome, and are likely to play a major role in the ecology at all sites within the body, it is critical to develop new approaches to obtain samples of these microorganisms for genomic analysis. In this proposal we focus on one anatomic site, the mouth, and propose to develop a technology to extract single bacterial cells from saliva. To attain these goals, we have formed a collaboration between Sandia (with expertise in integrated microfluidic technology for biological analysis), NYU College of Dentistry (with expertise in oral microbiomics and oral-based diagnostics), and the Joint Genome Institute (with expertise in microbial ecology and sequencing). The technological approach is to build an integrated microfluidic cell processor that will identify, select, and isolate into discrete microdroplets single bacteria from a mixture of oral bacteria from human saliva. The microfluidic processor will have multiple modules to 1) perform fluorescence in situ hybridization on a mixture of bacteria, 2) sort single cells using fluorescence activated photonic-force deflection, and 3) encapsulate sorted cells in microdroplets before depositing them on an array. The input to the device will be bacterial cells from saliva and the output will be arrayed droplets containing no more than one bacterium. We will first characterize and validate this processor using a mixture of pure bacterial cultures. Subsequently we will take salivary samples, deplete them of abundant bacterial species, and isolate individual cells, using specific 16S probes. Metagenomic analysis on the entire population of bacteria in saliva will be used to identify new bacterial sequences. With new sequence information, we will design 16S probes to isolate previously uncharacterized organisms for genomic testing. Isolated cells will be characterized as cultivable or non-cultivable, and known (sequenced) or unknown. Ultimately this technique will be used to extract sequence-quality genomic DNA from individual microorganism and can be used as a diagnostic to identify bacterial signatures obtained from healthy versus diseases subjects. PUBLIC HEALTH RELEVANCE: We know little about bacteria found in our bodies that can not be grown in the laboratory, but which may nevertheless cause disease. Our goal is to develop lab-on-a-chip technologies for isolating organisms from clinical samples and processing them, one at a time, for further genomic analysis.

描述（由申请人提供）：由于不可培养的微生物占微生物组的很大一部分，并且很可能在体内所有部位的生态学中都起着重要作用，因此开发新方法以获取这些微生物的样品进行基因组分析。在此提案中，我们专注于一个解剖部位，即口，并提出开发一种从唾液中提取单个细菌细胞的技术。为了实现这些目标，我们已经建立了桑迪亚（具有综合微流体技术的专业知识，用于生物分析），纽约大学牙科学院（具有口腔微生物学和基于口服的诊断方面的专业知识）和联合基因组研究所（具有微生物生态学和测序方面的专业知识）。技术方法是建立一个集成的微流体细胞处理器，该处理器将从人类唾液中的口服细菌的混合物中识别，选择和分离为离散的微虫单细菌。微流体处理器将具有多个模块至1）在细菌的混合物上进行荧光原位杂交，2）使用荧光激活的光子强度偏转和3）在将它们沉积在阵列之前，将单个细胞分类。设备的输入将是来自唾液的细菌细胞，输出将是含不超过一个细菌的液滴。我们将首先使用纯细菌培养物的混合物来表征和验证该处理器。随后，我们将使用特定的16S探针采集唾液样本，耗尽丰富的细菌物种，并分离单个细胞。对唾液中整个细菌种群的宏基因组分析将用于鉴定新的细菌序列。有了新的序列信息，我们将设计16S探针，以分离以前未表征的生物以进行基因组测试。分离的细胞将被表征为可栽培或不可培养的，并且已知（测序）或未知。最终，该技术将用于从单个微生物中提取序列质量的基因组DNA，并可以用作诊断，以鉴定从健康与疾病受试者中获得的细菌特征。公共卫生相关性：我们对在实验室无法种植的身体中发现的细菌知之甚少，但可能会引起疾病。我们的目标是开发实验室芯片技术，以将生物与临床样本隔离，并一次处理它们，以进行进一步的基因组分析。