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

描述(由申请人提供)：由于不可培养的微生物占微生物群的很大比例，并且可能在人体内所有部位的生态中发挥重要作用，因此开发新的方法来获取这些微生物的样本用于基因组分析是至关重要的。在这项提案中，我们将重点放在一个解剖部位，即口腔，并建议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，我们在Sandia(擅长生物分析的集成微流控技术)、纽约大学牙科学院(擅长口腔微生物学和口腔诊断学)和联合基因组研究所(擅长微生物生态学和测序)之间建立了合作关系。技术方法是建立一个集成的微流控细胞处理器，它将识别、选择并分离出来自人类唾液的口腔细菌混合物中的单个细菌。微流控处理器将有多个模块，用于1)对细菌混合物进行荧光原位杂交，2)使用荧光激活的光学力偏转对单个细胞进行分类，以及3)将分类后的细胞封装在微滴中，然后将它们存放在阵列上。该设备的输入将是唾液中的细菌细胞，输出将是排列的液滴，其中包含不超过一种细菌。我们将首先使用纯细菌培养的混合物来表征和验证这个处理器。随后，我们将采集唾液样本，清除其中丰富的细菌种类，并使用特定的16S探针分离单个细胞。对唾液中细菌的整个种群进行元基因组分析将被用来识别新的细菌序列。有了新的序列信息，我们将设计16S探针来分离以前未鉴定的生物进行基因组测试。分离的细胞将被描述为可培养或不可培养，以及已知(测序)或未知。最终，这项技术将被用于从单个微生物中提取序列质量的基因组DNA，并可用于诊断从健康受试者和疾病受试者获得的细菌特征。与公共卫生相关：我们对在我们体内发现的细菌知之甚少，这些细菌不能在实验室中培养，但可能会导致疾病。我们的目标是开发芯片实验室技术，从临床样本中分离生物体，并逐一进行处理，以进行进一步的基因组分析。

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.