Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH).
Microfluidic fluorescence in situ hybridization and flow cytometry (μFlowFISH).
复制标题
DOI：
10.1039/c1lc20151d
复制
发表时间：
2011-08-21
期刊：
影响因子：
6.1
通讯作者：
Singh AK
中科院分区：
文献类型：
--
作者：
Liu P;Meagher RJ;Light YK;Yilmaz S;Chakraborty R;Arkin AP;Hazen TC;Singh AK
我们描述了一种集成的微流控装置（µFlowFISH），能够进行16 S rRNA荧光原位杂交（FISH），然后进行流式细胞术检测，以识别天然微生物群落中的细菌。该设备被用于检测的物种参与生物修复的铬（VI）和其他金属的地下水样品中的高度污染的环境网站（汉福德，华盛顿州，美国）。µFlowFISH无缝集成了两个组件：在两个光聚合膜之间形成的杂交室，其中细胞和探针被电泳加载，孵育和洗涤;以及下游交叉结构，用于将细胞电动聚焦成单行流，用于流式细胞术分析。该装置能够分析各种细菌，包括需氧菌、兼性菌和厌氧菌，并使用培养的微生物（包括大肠杆菌）以及从Hanford研究中心分离的两种菌株（脱硫弧菌菌株RCH 1和假单胞菌菌株RCH 2）进行了初步测试和验证，这两种菌株参与了Cr（VI）的还原和固定。结合标记和检测效率的74-97%的实验中观察到的培养细胞的简单混合物确认特异性标记。获得的结果与常规流式细胞术获得的结果非常一致，证实了µFlowFISH的准确性。最后，该设备被用于分析从汉福德现场不同日期收集的水样。我们能够监测假单胞菌的数量。只有100-200个细胞加载到微芯片中。µFlowFISH方法为复杂样品中微生物细胞的定量检测提供了一个自动化平台，非常适合分析细胞数量较少的珍贵样品，例如在极端环境小生境、生物修复场所和人类微生物组中发现的样品。
We describe an integrated microfluidic device (µFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The µFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed; and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74–97% were observed in experiments with simple mixtures of cultured cells confirmed specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of µFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford Site. We were able to monitor the numbers of Pseudomonas sp. with only 100–200 cells loaded into the microchip. The µFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome.
登录
查看更多内容
影响因子：
6.1
作者：
Meagher, Robert J.;Hatch, Anson V.;Singh, Anup K.
通讯作者：
Singh, Anup K.
DOI：
10.1002/cyto.990140205
发表时间：
1993-01-01
期刊：
CYTOMETRY
影响因子：
--
作者：
WALLNER, G;AMANN, R;BEISKER, W
通讯作者：
BEISKER, W
影响因子：
14.9
作者：
Loy A;Maixner F;Wagner M;Horn M
通讯作者：
Horn M
影响因子：
2.8
作者：
Gadd, Geoffrey Michael
通讯作者：
Gadd, Geoffrey Michael
影响因子：
2.2
作者：
Gerdts, G;Luedke, G
通讯作者：
Luedke, G