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组的很大比例，并且可能在体内所有部位的生态中发挥重要作用，因此开发获取这些微生物样本用于基因组分析的新方法至关重要。在这个建议中，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，我们与桑迪亚大学（拥有用于生物分析的集成微流体技术的专业知识）、纽约大学牙科学院（拥有口腔微生物组学和口腔诊断的专业知识）和联合基因组研究所（拥有微生物生态学和测序的专业知识）建立了合作关系。该技术方法是建立一个集成的微流控细胞处理器，该处理器将从人类唾液中的口腔细菌混合物中识别、选择并分离成离散的微滴。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)使用荧光激活的光子力偏转对单个细胞进行分选，以及3)将分选的细胞封装在微滴中，然后将其沉积在阵列上。该设备的输入将是唾液中的细菌细胞，输出将是不超过一种细菌的排列液滴。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，我们将采集唾液样本，耗尽其中丰富的细菌种类，并使用特定的16S探针分离单个细胞。对唾液中整个细菌种群的宏基因组分析将用于鉴定新的细菌序列。利用新的序列信息，我们将设计16S探针来分离以前未被鉴定的生物体进行基因组检测。分离的细胞将被定性为可培养或不可培养，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，并可用于诊断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，但仍可能导致疾病。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，并逐一处理，以进一步进行基因组分析。

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.