支持这项研究是从通过结合生物研究和教育（VIIBRE）范德比尔特研究所，美国国家科学基金会和塞尔系统生物学和生物工程本科研究经验（塞尔SyBBURE）授予＃0649883。

一，微流体设备制造<ol><li>在创建一个微流体装置的第一步是绘制两个设备的立体布局，在电脑辅助绘图（CAD）程序。我们已经使用AutoCAD，但也可用其他绘图程序，如CleWin或CorelDRAW。 </li><li>下一步是要编造光刻用掩模。器件尺寸的不同，所需的分辨率和预算，这些面具可能是捏造的铬（分辨率最高的，成本高），在感光胶片上创建，甚至开销的透明度，使用高分辨率的打印机上直接打印。在这里，我们使用了由高级复制品公司，北安多弗，马生产的铬口罩。 </li><li>下一步是将其转移到一个光敏环氧创建一个提出救济模具从面具的格局。 <ol><li>首先，负SU8光刻胶层剥离到一个硅晶片所需的厚度，和烤挥发多余的溶剂。光致抗蚀剂的配方和转速控制的沉积层的厚度。 </li><li>然后，该模式是接触到一个预先确定的剂量紫外线光通过掩膜的。烘烤后曝光交叉连接的光致抗蚀剂涂层的光暴露的地区，使其不溶性。 </li><li>接下来是一步的发展，其中未曝光的抵制是一种化学品开发中删除，留下一个积极筹集救灾的结构称为主。 </li><li>第三烘焙步骤，称为硬烤步骤是可选的进一步修复的结构。 </li></ol></li><li>硅橡胶成型<ol><li>我们使用有机硅弹性体聚（二甲基硅氧烷）微流体装置1副本成型。首先，基体材料是在与固化剂，在模具中倒入一个浅的容器，如培养皿的重量比10:1混合，真空脱气，直到所有可见的气泡都没有了。 </li><li>固化硅橡胶的主4H至少在一个精心夷为平地的烤箱，然后放置在65 ° C交叉连接的聚合物发生。 </li><li> PDMS固化后，模压部分切出的培养皿。孔打孔通过设备的访问在完成设备的微流体空间的顶部。 </li></ol></li><li>附件玻璃<ol><li>清洁PDMS造成不可挽回的保税接触氧等离子体清洁玻璃。首先，的成型和冲压的PDMS和一个干净的载玻片置于粘接面成等离子清洁。我们使用的PDC - 32G Harrick等离子清洁。 </li><li>后室是封闭的，运营商将在真空泵，然后无线电频率或射频源。等离子将自行点燃室，一个略带紫色的光芒，在会议厅内证明。 </li><li>曝光后30秒，等离子，射频信号源，然后真空泵关闭。 </li><li>快速，载玻片和PDMS设备是从会议厅中删除，并带来直接接触（成型的硅橡胶表面的一面朝下）。这将形成一个不可逆转的债券，还将使亲水性的表面性质。 </li><li>如果需要，可实现不同的表面化学，通过固定的蛋白质表面吸附或共价键与硅橡胶。 </li><li>该设备可以被充满液体为去离子水，生长介质，或由毛细管力或用注射器使用温和的压力地下水人工等。 </li></ol></li></ol>二。流微流控芯片的量化重分析<ol><li>为了校准设备中的压力驱动流，微结构的流动细胞首先预装离子水。 </li><li>一个液体含有水库，如塑料注射器或流量控制模块，（如图1所示）是连接到上游入口好，水库中的流体高度以上的高度以及在下游保持。 </li><li>样品废物收集和定期分析天平权衡。 </li><li>绘图总量与总时间曲线的斜率给出的平均体积流率（图2）。我们发现，重现性好，广泛的压头和不同的压力，维护系统的非线性平均速度。 </li></ol>三。流程可视化，速度映射和亲水/疏水<ol><li>模型校准，3微米的两个羧基永贵和平原永贵表面荧光乳胶珠被收购公司Polysciences，DI水稀释至0.01％的固体浓度。 </li><li>珠流经的栖息地，一个ZEISS AXIOVERT 25荧光显微镜配备一个Mightex公元前- B013 - U单色相机上的可视化。 </li><li>单个帧快速连续被抓获，并组装成电影与ImageJ软件程序包（图3）。 </li></ol>四。建模细菌的生长和运输的微流体装置（EcoChip） <ol><li> 弧菌 。 GFP侃，一个非致病性的绿色荧光蛋白表达生物体生长到约10 9细胞/毫升的浓度为14小时卢里亚- Bertani培养基。 </li><li>细菌在培养基中被引入EcoChip设备，并允许殖民化设备，建立19小时通宵的絮体和电影。 </li><li>生长介质从水井中删除所有的栖息地被刷新人工海水（ASW的配方，请参阅王等。），使不同密度的细菌在不同的栖息地的剩余。 </li><li>一个栖息地保持密封，无流量，而在其他栖息地维持血流缓慢的条件。 </li><li>细菌生长的荧光和白光照片拍摄了数天（图4）期间每隔15-20小时。 </li></ol>五，代表性的成果流量控制模块提供了简单的方法，通过多种栖息地的管理和分发流动。重量分析被证明是一个简单易懂的方法来确定的流速通过栖息地的结构，它的栖息地和输入和输出口井之间的压力差的液压阻力取决于。对于珠流实验中，我们所观察到uncarboxylated珠（图2）的设备表面珠积累大得多。此外，直径较大的珠子，6日和10毫米，更可能成为较​​小的孔开口夹带设备，并开始积累。更快的流速降低颗粒保留和夹带。 在细菌的生长实验，水流条件的影响是显而易见的。连续剪切力会导致细菌聚集在一起，形成絮体，而不是作为单个细胞中发现。大量细菌絮凝物的运输是一个重要的环境的过程，是极难在一个宏观的系统研究。 <img src="/files/ftp_upload/1741/1741fig1.jpg" alt="图1" /> 图1原理图的显示功能和操作流模块。 <img src="/files/ftp_upload/1741/1741fig2.jpg" alt="图2" /> 图2：通过结构化的栖息地。流率，重量不同的列的高度分析确定。柱高度的比例：60 / 40 = 1.5，确定流量比率：1.04 / 0.71 = 1.46。为H = 40毫米，致使流速为V = 0.71微升/分钟，为H = 60毫米为V = 1.04μL / min的。估计平均线速度3.1 mm / s和4.6毫米/秒。 <img src="/files/ftp_upload/1741/1741fig3.jpg" alt="图3" /> 图3。运输与不通过结构化的栖息地流淌羧3um乳胶珠。 <img src="/files/ftp_upload/1741/1741fig4.jpg" alt="图4" /> 图4。细菌生长和生物膜形成的种子密度函数在一个缓慢的流动存在的变化。

<ol>
	<li>Whitesides, G., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annual Review of Biomedical Engineering. 3, 335-373 (2001).</li><li>Wang, W., Shor, L. M., LeBoeuf, E. J., Wikswo, J. P., Kosson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+protozoa+through+narrow+channels.">Mobility of protozoa through narrow channels.</a> Applied and Environmental Microbiology. 71, 4628-4637 (2005).</li></ol>

EcoChip系统是适应个别实验的需要。可以比较容易地创建新主人，并且一次主人是捏造的，可以转换为需要额外的精确复制的设备。流量控制模块是使用简单，不需要特殊的设备或复杂的连接，并可以作为一个简单的下降头部压力驱动流系统建模。额外的扩展这项工作正在进行中，包括创建腐殖酸涂层的渠道，系统不同的流体流动的水化学。使用这种方法，细菌的微观尺度的表面和多孔介质中的?...

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window on a microworld: simple microfluidic systems for studying microbial transport in porous media

多孔介质中微生物的生长和运输地下水和地表水，营养物质在环境中的回收利用，以及直接用于饮用水供应的病原体的传播质量有重要影响。天然多孔介质是一个复杂的物理拓扑结构，各种不同的表面化学，营养物质和电子受体的动态梯度，和片状分布的微生物组成。超过一个微米尺度，这些功能有很大的差别，使微生物的运输难以解释的宏观尺度的调查结果，并具有挑战性的机理模型验证。在这里，我们演示了如何简单的微流体装置，可用于可视化微生物的相互作用与微结构的栖息地，影响观察到的现象，以确定关键工序，和系统验证预测模型。构造简单，易于使用的流动细胞透明，生物相容性和氧气的渗透性材料聚（二甲基硅氧烷）。标准的光刻方法被用来制造微结构的主人，和副本成型用于铸造微结构的流动细胞的主人。流动池室的物理设计是适应实验的要求：微可以有所不同，从简单的线性连接复杂的拓扑结构与功能的大小为2微米的小。我们的模块化EcoChip流单元阵列，几十种功能相同的商会和流量控制由重力驱动流模块。我们展示通过EcoChip设备的使用，物理结构和压头可保持不变或更改系统而影响表面化学，流体性质，或微生物群的特点是调查。通过运输，使用非致病性的实验，绿色荧光蛋白表达<em&gt;弧菌</em&gt;菌株，说明我们的栖息地结构，流量条件下，接种上的基本运输现象的规模的重要性，并实时粒子尺度观测，证明微流体提供了一个隐藏的世界的一个令人信服的观点。

Watch this Scientific Journal Video about Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media at JoVE.com

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

微流体装置，可用于可视化实时和复杂的自然过程，在适当的物理尺度。我们已经开发出一种简单的微流体装置，模仿天然多孔介质的主要特点，为研究细菌的生长和运输在地下。

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

window-on-microworld-simple-microfluidic-systems-for-studying

Research

JoVE Journal

Biology

10.7K 次观看.  Vanderbilt University. 微流体装置，可用于可视化实时和复杂的自然过程，在适当的物理尺度。我们已经开发出一种简单的微流体装置，模仿天然多孔介质的主要特点，为研究细菌的生长和运输在地下。

视频: Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

科研

生物学

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

测量线虫固体介质的寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

一个微流体装置量化细菌趋稳定的浓度梯度

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

进行阴道灌洗，结晶紫染色法和阴道细胞学评价小鼠发情周期分期鉴定

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.