我们感谢Kenis集团在伊利诺伊大学厄巴纳 - 香槟分校的有益讨论，并慷慨地提供洁净室设施的使用。 这项工作是由美国国立卫生研究院的途径，以独立的PI奖，根据批准号4R00HG004183 - 03（查尔斯M施罗德和Melikhan Tanyeri）。 这项工作是支持由美国国家科学基金会通过一个研究生研究奖学金埃里克M.约翰逊查瓦里亚。

由两层混合（聚二甲基硅氧烷（PDMS）/玻璃）粒子约束微流体装置的水动力的陷阱。步骤1-2描述的微流体装置的制造和步骤3-4讨论设备的设计和操作。 1。 SU - 8模具制作（视频没有显示） <ol><li>用丙酮和异丙醇（IPA）清洁两个硅片（直径3“）。 </li><li>与N 2，放在烤盘上在65℃1分钟以去除残留的水分干晶圆。 </li><li>自旋Coat晶圆＃1 SU - 8 2050光阻（PR），在4000转30秒创建〜40微米厚的流体层的模具。旋涂晶圆PR＃2 1500 RPM，以创建一个控制层〜150微米厚的模具30秒。 </li><li>软烤晶圆＃1在65 ° 3分钟，然后在95 ° C为6分​​钟，软烤晶圆在65＃2℃，5分钟，然后在95 ° C下20分钟。 </li><li>使晶圆，以紫外线各自的面具（晶圆＃1：港口和流体通道，晶圆＃2：端口和控制层）和适当的曝光力度（〜150兆焦耳/平方厘米〜260兆焦耳/厘米2 ）。 </li><li>后烘晶圆＃1在65 °为1分钟，然后在95 ° C为6分​​钟C。后烘晶圆在65＃2 ° C为5分钟，然后在95 ° C为10分钟。 </li><li>开发与丙二醇甲醚醋酸酯（PGMEA）晶圆直到固化PR被删除。 IPA和干燥与N 2冲洗晶圆。 </li></ol> 2。微流控设备制造<ol><li> Silanize的SU - 8模具表面含有三氯氢硅几滴用玻璃盘〜10分钟，置于真空干燥器中的晶圆。表面硅烷化助攻剥落的SU - 8模具（PDMS）的副本。 </li><li>混合和脱气PDMS在基地：交联剂比例的流体和控制层分别为15:1和5:1。 </li><li>旋涂到流体层模具（晶圆＃1）为30秒，在750转15:1的PDMS混合，然后放入培养皿中的晶圆。放入培养皿中的控制层模具和5:1 PDMS的混合物倒到模具〜4毫米的厚度。 </li><li>烤晶圆/ PDMS的30分钟，在70 ° C至部分固化硅橡胶层。 </li><li>晶圆/ PDMS室温冷却后，切的PDMS的翻版，这将成为控制层（晶圆＃2），从培养皿用手术刀剥离和关闭的SU - 8模具。冲孔一个接入端口将作为21号针头与芯片上的膜阀的微。 </li><li>控制层放置到晶圆＃1（旋涂PDMS的流体层）的PDMS副本。仔细调整和控制层密封流体层使用立体显微镜。请一定要清除所有气泡层与层之间，烘烤70 ° C过夜完全治愈这两个层次。这烘烤的步骤将导致PDMS两层铁板一块板坯。 </li><li>后冷却到室温，剪切和剥离PDMS副本，包含控制和关闭SU - 8模具的流体层，用手术刀。除去多余PDMS和单独用刀片，每个设备单元。打孔接入端口，以21号针头与流体层的微。 </li><li>债券的硅橡胶板盖玻片，以获得一个完整的设备。首先，与丙酮和异丙醇清洁盖玻片（编号：1.5，24 × 45毫米）。接下来，500毫托下盖玻片和副本的PDMS氧等离子体表面处理为30秒，并立即把两个表面接触，形成一个不可逆转的密封。 </li><li>烘烤过夜，以增加粘接的硅橡胶层和盖玻片之间的设备。 </li></ol>步骤3-4描述实施使用上文所述的微流体装置的流体力学陷阱。 3。流体力学陷阱实验装置<ol><li>倒置显微镜阶段的微流体装置，并放置到安全的阶段片段。 </li><li>填充缓冲区和样品溶液分别与两个气密注射器和他们放在一个哈佛器械注射泵（博士2000可编程）。缓冲区和提供的样品溶液分别通过1毫升和250μL注射器，微流体装置。通常情况下，50毫米的Tris / TRIS盐酸缓冲溶液（pH 8.0）中含0.02％V / V Triton X - 100的的缓冲液使用。示例解决方案包括一个颗粒悬浮在缓冲液（如荧光聚苯乙烯珠）。 </li><li>注射器（提供的样品和缓冲）和微流体装置之间建立流体连接。 1 / 16“外径（OD值）x 0.020”内径（ID），perfluoroalkoxy（PFA）管使用鲁尔锁适配器连接注射器。 PFA管的另一端连接到微流体装置的进气道与24号元升管。 T -阀可放置之间的微流体装置，以控制送样样品注射器和取样口。 </li><li>在微流体装置的出口渠道的建立流体连接。 PFA管（1 / 16“外径× 0.020”编号），使用24号金属管连接的两个插座渠道。 PFA管网点应的长度相等。浸既充满缓冲溶液中，服务之间的注射器和出口渠道保持恒定的压力降成1.5 mL离心管出口管。 </li><li>填写氟化载体油3毫升鲁尔锁的塑料注射器，以防止空气泄漏到在操作过程中的流体层中的芯片上的阀门。通过PDMS膜阀腔中的空气被压到微流体层，后来通过插座端口的流体流动装置中删除。 </li><li>连接端口加压惰性气体（氮气）在控制层供片上的阀门操作。为此，我们使用一个氮气罐（2200磅）和一个电子压力调节器的阀片上的微流体装置供应0-30 PSI。氮气罐连接到压力调节器，使用6.3“外径× 0.170”内径管。通过1 / 16“外径× 0.020”编号24号在它的终点的金属管的PFA管，压力调节器连接到微流体装置。 </li><li>流体连接和微流体装置冲洗用0.5 mL缓冲液中，以确保所有的气泡从包括出口渠道的系统中删除。用于2000-5000微升/小时之间的结算气泡范围的典型流量率。气泡冲洗后的微流体通道，降低流速50-100μL/小时，这是一个典型的体积流量颗粒捕集。 </li><li>此时，流体连接建立，样品和缓冲的解决方案是在一个固定的流速（50-100μL/小时）的微流体装置，设备准备流体力学诱捕。 </li></ol> 4。液力补漏程序<ol><li>运行定制的LabVIEW代码，自动颗粒捕集（见LabVIEW代码的使用下面的说明）。 </li><li>使用显微镜XY平移台，位置在相机视图的中心诱捕地区（十字槽）。诱捕地区带入物镜的焦点，并调整相机设置以优化成像条件。 </li><li>在摄像机的角度等中心的投资回报率将是陷阱中心的地位领域，选择有兴趣的矩形区域（ROI）。 </li><li>初始化偏移应用到芯片上的阀门的压力。的出口渠道之一，宽100-200微米收缩，是为了提供一个芯片上的阀门抵消压力。恒定关断设定压力使芯片上的阀门来调整通道十字槽中心附近的停滞点的位置。对于大多数实验，抵消压力PSI之间设置0-12通道的尺寸（高度和宽度），收缩宽度，芯片上的阀门（阀门尺寸，膜的厚度等）的规格而定。 </li><li>启动反馈控制器和调整比例增益，以优化陷阱的反应。反馈控制器将应用到芯片上的阀门，以推动停滞点的位置，最大限度地减少错误或粒子的位置和设定点（陷阱中心）之间的距离调整的压力。根据流速和芯片上的阀门的位置，有一个最佳的比例增益值，从而增加了陷阱稳定，消除不必要的粒子振荡。 </li><li>捕获的粒子。 LabVIEW代码会自动捕获颗粒进入诱捕地区之一。所需的粒子被捕获后，它可能关闭样品流和缓冲液中分离出被困粒子，如果需要的话。 </li><li>监视器捕获粒子，并保持使用手动对焦或自动聚焦显微镜设置在图像平面的粒子重点。它可能需要稍微调整，以确保在很长一段时间，规模诱捕事件（几分钟到几小时）过程中的陷阱稳定的反馈控制器的比例增益。 </li></ol> LabVIEW代码：反馈控制器的使用注意事项自动颗粒捕集是使用线性反馈控制算法实现使用一个自定义的LabVIEW代码实现。 LabVIEW代码从CCD相机捕获的图像，并发送一个电势（电压）压力调节器，积极调制的动态气动阀片上的位置（部分开放/封闭状态）。由于阀门位置的变化，我在一个插座线的流体流量的调整后，从而重新定位驻点，使水动力诱捕。在反馈环路中的步骤顺序，并在图像捕获率（10-60赫兹）反复执行。 LabVIEW代码在每一个反馈循环周期执行以下步骤： <ul><li> 图像拍摄图像获得一个“目标”粒子诱捕地区使用10倍物镜荧光显微镜的微设备（NA：0.4）和CCD相机。 </li><li> 粒子跟踪。粒子的质心位置是确定的，是发起和粒子跟踪算法。颗粒本地化粒子的排放强度分布拟合到一个点扩散函数（PSF），从中心位置确定。 </li><li>更新的压力，片上的动态阀流场的控制权。使用一个比例控制器的反馈控制算法计算。这样，阀的动作是重新定位的停滞点，这对粒子水动力，以引导粒子朝着陷阱中心。 </li></ul> LabVIEW代码在诱捕粒子捕捉每一个图像记录以下数据：1）时间过去了，2）质心（X，Y），3被困粒子的位置）的陷阱中心的地位，4）粒子的距离，从陷阱中心，5）施加压力，以片上的阀门。此外，代码还记录被困粒子在AVI文件格式的电影。 5。代表性的成果我们被困了各种大小（100，540，830纳米，直径为2.2微米）的荧光聚苯乙烯微球，采用液力陷阱。图1（a）显示了一个被困在一个微流体装置的十字槽交界处一个粒子的形象。直接从数据记录的LabVIEW代码的过程中捕获的事件或从录制的电影文件中捕获粒子通过跟踪和本地化的中心位置，可确定一个被困粒子的轨迹。图1（b）显示了一个被困沿出口通道方向的粒子（2.2微米的荧光聚苯乙烯珠）的轨迹。珠最初是被困（正方形）为3分钟，然后沿的出口通道（圆圈）之一的陷阱和逃脱公布。沿纵流轴（进气口通道的方向;数据未显示）的粒子轨迹是相似的，如在图1（b）所示沿伸展流轴（流出方向）的粒子轨迹。一个粒子的位移沿出口通道指示为被困珠（直径2.2微米）的陷阱中心的直方图是在图1（c）所示。在这项工作中所描述的反馈控制算法，俘获粒子局限于内± 1微米，沿入口和出口通道方向的陷阱中心。 流体力学诱捕使用的微流体装置的原理图如图2所示。集成微流体装置由一个流体层和控制层，并采用标准的多层软光刻技术，如本文所述。流体层包含的缓冲区和采样通道，以及跨槽通道的几何形状，以方便流体力学诱捕。控制层由定位在流体层的出口渠道之一以上的气动阀，控制和流体层是由一个薄的弹性膜分离。在设备的运行，在控制层的阀门是用氮气加压，迫使到流体层薄膜，从而诱导在出口通道的收缩。动态气动阀收缩可变数量的出口通道，通过改变应用到控制层，在出口渠道调整相对流速，使精细控制驻点压力。 <img src="/files/ftp_upload/2517/2517fig1.jpg" alt="图1" /> 图1：粒子补漏。 （一）在流体力学的陷阱局限于一个单一珠的形象。除了 ​​珠陷阱中心，几个未捕获的珠子显示在捕获区域。（二）沿被困的出口渠道（正方形）粒子的轨迹。当粒子从陷阱（箭头）发布，它逃脱。沿的出口通道（圆圈）之一（三）一个被困珠（直径2.2微米）沿出口渠道的陷阱中心的位移直方图。 <img src="/files/ftp_upload/2517/2517fig2.jpg" alt="图2" /> 图2：流体力学诱捕的微流体装置示意图，水动力的陷阱构造使用一个两层的微流体装置。流体层由一个样品入口，F我们的缓冲区的进气口，和两个废物网点。控制层由一个坐落在流体层的出口渠道之一上的气动薄膜阀。在反对插座通道收缩抵消压力提供了一个气动阀。典型的通道尺寸介于100-500微米。 （一）在区域，样品入口是由两个缓冲区的进气口集中的流。 （二）在区域，反对入口流交汇的十字槽交界发生诱捕。气动阀（C）是定位于顶级的出口渠道之一。停滞点的位置是调节阀的压力，这调制。

<ol>
	<li>Tanyeri, M., Johnson-Chavarria, E. M., Schroeder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Trap+for+Single+Particles+and+Cells.">Hydrodynamic Trap for Single Particles and Cells.</a> Applied Physics Letters. 96, 224101-224101 (2010).</li><li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+Single-Beam+Gradient+Force+Optical+Trap+for+Dielectric+Particles.">Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles.</a> Optics Letters. 11, 288-290 (1986).</li><li>Neuman, K. C., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+trapping.">Optical trapping.</a> Review of Scientific Instruments. 75, 2787-2809 (2004).</li><li>Gosse, C., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+tweezers:+Micromanipulation+and+force+measurement+at+the+molecular+level.">Magnetic tweezers: Micromanipulation and force measurement at the molecular level.</a> Biophysical Journal. 82, 3314-3329 (2002).</li><li>Chiou, P. Y., Ohta, A. T., Wu, M. C., C, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+manipulation+of+single+cells+and+microparticles+using+optical+images.">Massively parallel manipulation of single cells and microparticles using optical images.</a> Nature. 436, 370-372 (2005).</li><li>Cohen, A. E., Moerner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+trapping+and+manipulating+nanoscale+objects+in+solution.">Method for trapping and manipulating nanoscale objects in solution.</a> Applied Physics Letters. 86, 093109-09 (2005).</li><li>Evander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+acoustic+cell+trapping+in+a+microfluidic+perfusion+system+for+online+bioassays&amp;quot;,+Analytical+Chemistry+79.">Noninvasive acoustic cell trapping in a microfluidic perfusion system for online bioassays&amp;quot;, Analytical Chemistry 79.</a> , 2984-2991 (2007).</li><li>Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolithic+microfabricated+valves+and+pumps+by+multilayer+soft+lithography.">Monolithic microfabricated valves and pumps by multilayer soft lithography.</a> Science. 288, 113-116 (2000).</li><li>Kim, M. C., Wang, Z. H., Lam, R. H. W., Thorsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+better+cell+trap:+Applying+Lagrangian+modeling+to+the+design+of+microfluidic+devices+for+cell+biology.">Building a better cell trap: Applying Lagrangian modeling to the design of microfluidic devices for cell biology.</a> Journal of Applied Physics. 103, (2008).</li><li>Lutz, B. R., Chen, J., Schwartz, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+tweezers:+1.+Noncontact+trapping+of+single+cells+using+steady+streaming+microeddies.">Hydrodynamic tweezers: 1. Noncontact trapping of single cells using steady streaming microeddies.</a> Analytical Chemistry. 78, 5429-5435 (2006).</li></ol>

目前基于流体力学流动的粒子操纵微流体的方法可以被定性为接触式或非接触的方法。接触式的方法使用流体流动的物理限制和固定颗粒对微通道的墙壁 9，非接触式方法，而依靠循环流量或microeddies 10 。在这项工作中，我们提出一个自由的解决方案颗粒流体流动的唯一行动诱捕方法。在一个微十字槽设备的流体驻点流体力学的陷阱，使小颗粒的约束和操纵。在这种设备中，自动?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>21 gauge blunt needle</td>
<td>&nbsp;</td>
<td> Zephyrtronics</td>
<td> ZT-5-021-1-L</td>
<td>For punching port holes in PDMS</td>
</tr>
<tr>
<td>3 ml plastic syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309585</td>
<td>For filling valve with oil</td>
</tr>
<tr>
<td>Si wafers</td>
<td>&nbsp;</td>
<td>University Wafer</td>
<td>&nbsp;</td>
<td> 3&#x201D; P(100) single side polished 380 &mu;m test grade</td>
</tr>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48404-428</td>
<td>24 x 40 mm #1.5</td>
</tr>
<tr>
<td>DAQ card</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI 6229</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent beads</td>
<td>&nbsp;</td>
<td>Spherotech</td>
<td>FP-2056-2</td>
<td>2.2 &mu;m Nile red</td>
</tr>
<tr>
<td>Fluorinert </td>
<td>&nbsp;</td>
<td>3M</td>
<td>FC 40</td>
<td>Fluorinated carrier oil</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>IX-71</td>
<td>&nbsp;</td>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Version 9.0f3 (32bit)</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>MZ6</td>
<td>For aligning PDMS control layer to fluidic layer.</td>
</tr>
<tr>
<td>Mechanical Convection Oven</td>
<td>&nbsp;</td>
<td> VWR</td>
<td>1300U</td>
<td>For baking devices to create monolithic PDMS slabs with two layers.</td>
</tr>
<tr>
<td>Microfluidic tubing and connectors</td>
<td>&nbsp;</td>
<td>Upchurch Scientific</td>
<td>&nbsp;</td>
<td>1/16 x .020 PFA tubing and super flangeless fittings</td>
</tr>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>GE Silicones</td>
<td>RTV 615 A&amp;B</td>
<td>&nbsp;</td>
<tr>
<td>Plasma Chamber</td>
<td>&nbsp;</td>
<td>Harrick</td>
<td>PDC-001</td>
<td>&nbsp;</td>
<tr>
<td>Pressure Transducer</td>
<td>&nbsp;</td>
<td>Proportion Air</td>
<td>DQPV1</td>
<td>&nbsp;</td>
<tr>
<td>Spin Coater</td>
<td>&nbsp;</td>
<td>Specialty Coating Systems</td>
<td>G3P-8 Spin Coat</td>
<td>&nbsp;</td>
<tr>
<td>Photoresist</td>
<td>&nbsp;</td>
<td>MicroChem</td>
<td>SU 8 2050</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000 Programmable</td>
<td>&nbsp;</td>
<tr>
<td>Terminal Block</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>BNC 2110</td>
<td>For analog output to pressure regulator and read out.</td>
</tr>
<tr>
<td>UV Collimated Light Source and Exposure System</td>
<td>&nbsp;</td>
<td>OAI</td>
<td>Model 30 Enhanced Light Source</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a microfluidic-based hydrodynamic trap for single particles

限制和操纵单个粒子在免费的解决方案的能力是一个关键的有利基础和​​应用科学技术。导致在物理学和生物学的重大进展，包括从分子细胞水平上的光，磁，电动，声等技术为基础的粒子诱捕方法。在这篇文章中，我们将介绍一个基于新的微流体技术，完全基于流体力学流体流动的颗粒捕集和操纵。使用这种方法，我们展示了诱捕的微观和纳米尺度粒子在水溶液中长时间尺度。水力陷阱，包括一个综合的跨槽通道的几何形状，两个对立的层流的融合，从而产生了一种平面流体停滞点（零速度点）伸展流动的微流体装置。在这种设备中，粒子只限于在流场主动控制的陷阱中心，保持在流体驻点粒子的位置。在这种方式下，粒子有效地被困在免费的解决方案，使用一个定制的LabVIEW代码实现反馈控制算法。控制算法由一个粒子在微流体装置，粒子跟踪，颗粒质心位置的决心，积极调整，通过调节应用到一个芯片上的气动阀压力调节器的压力和流体流动的图像采集。这样，芯片上的动态计量阀功能调节的出口渠道相对流速，从而使停滞点的位置和颗粒捕集细规模控制。基于微流体的流体力学陷阱展品颗粒捕集方法的几个优点。未经被困对象的物理或化学性质的具体要求任意粒子的流体力学诱捕。此外，水动力诱捕可以集中或拥挤的粒子悬浮在“单一”的目标对象，这是很难用替代的力量基于现场的诱捕方法的约束。水力陷阱是用户友好，直接实现，并可能被添加到现有的微流体装置，以方便长时间粒子的捕获和分析。总体而言，水动力陷阱是一个新的平台，和无颗粒表面固定的禁闭，显微观察和消除潜在的微扰光，磁，电领域的解决方案的小颗粒的自由诱捕的需要。

Watch this Scientific Journal Video about A Microfluidic-based Hydrodynamic Trap for Single Particles at JoVE.com

A Microfluidic-based Hydrodynamic Trap for Single Particles

在这篇文章中，我们提出一个基于微流体的方法，根据流体力学流动的粒子约束。我们证明使用的反馈控制机制，在流体停滞点稳定的粒子诱捕，从而使分娩和任意颗粒显微集成微装置。

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

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16.6K 次观看.  University of Illinois at Urbana-Champaign. 在这篇文章中，我们提出一个基于微流体的方法，根据流体力学流动的粒子约束。我们证明使用的反馈控制机制，在流体停滞点稳定的粒子诱捕，从而使分娩和任意颗粒显微集成微装置。

视频: A Microfluidic-based Hydrodynamic Trap for Single Particles

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

科研

生物工程

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

培养哺乳动物细胞使用一次性使用气动生物反应器系统

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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一个单粒子为基础的微流控液力陷阱