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: 8533716
• 负责人: Anup K. Singh
• 金额: $ 20.02万
• 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 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8533716
• 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; Health; 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; 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比例，并且可能在体内所有部位的生态学中发挥重要作用，因此开发新方法以获得这些微生物的样品用于基因组分析至关重要。在这项提案中，我们专注于一个解剖部位，口腔，并建议开发一种技术，从唾液中提取单个细菌细胞。为了实现这些目标，我们在Sandia（具有用于生物分析的集成微流控技术的专业知识），纽约大学牙科学院（具有口腔微生物生物学和口腔诊断学的专业知识）和联合基因组研究所（具有微生物生态学和测序的专业知识）之间建立了合作。该技术方法是建立一个集成的微流体细胞处理器，将识别，选择和分离成离散的微滴单一细菌从口腔细菌的混合物从人类唾液。微流控处理器将具有多个模块，以1）对细菌混合物进行荧光原位杂交，2）使用荧光激活的光子力偏转对单细胞进行分选，以及3）在将分选的细胞沉积在阵列上之前将其封装在微滴中。该设备的输入将是来自唾液的细菌细胞，输出将是含有不超过一个细菌的阵列液滴。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，我们将采取唾液样本，耗尽它们的丰富的细菌种类，并分离单个细胞，使用特定的16S探针。对唾液中整个细菌群体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探针来分离以前未表征的生物体进行基因组检测。分离的细胞将被表征为可培养的或不可培养的，以及已知的（测序的）或未知的。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，并可用作诊断以鉴定从健康受试者与疾病受试者中获得的细菌特征。公共卫生相关性：我们对我们体内发现的细菌知之甚少，这些细菌不能在实验室中生长，但却可能导致疾病。我们的目标是开发芯片实验室技术，用于从临床样本中分离生物体，并一次处理一个，以进行进一步的基因组分析。

项目成果

Single-cell protein analysis.

• DOI: 10.1016/j.copbio.2011.11.023
• 发表时间: 2012-02
• 期刊: CURRENT OPINION IN BIOTECHNOLOGY
• 影响因子: 7.7
• 作者: Wu, Meiye;Singh, Anup K.
• 通讯作者: Singh, Anup K.

Microfluidic Flow Cytometry for Single-Cell Protein Analysis.

• DOI: 10.1007/978-1-4939-2987-0_6
• 发表时间: 2015
• 期刊: Methods in molecular biology
• 作者: Meiye Wu;A. Singh

Single cell genome sequencing.

单细胞基因组测序。

• DOI: 10.1016/j.copbio.2011.11.018
• 发表时间: 2012-06
• 期刊: CURRENT OPINION IN BIOTECHNOLOGY
• 影响因子: 7.7
• 作者: Yilmaz, Suzan;Singh, Anup K.
• 通讯作者: Singh, Anup K.

Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH).

• DOI: 10.1039/c1lc20151d
• 发表时间: 2011-08-21
• 期刊: Lab on a chip
• 影响因子: 6.1
• 作者: Liu P;Meagher RJ;Light YK;Yilmaz S;Chakraborty R;Arkin AP;Hazen TC;Singh AK
• 通讯作者: Singh AK

Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples.

• DOI: 10.1371/journal.pone.0153699
• 发表时间: 2016
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Rhee M;Light YK;Meagher RJ;Singh AK
• 通讯作者: Singh AK