我们感谢果蝇股，Tarjani阿加瓦尔博士Krishanu雷为保持果蝇笼，彼得·若为nuIs25和CGC为C.的菌株。 SPK取得jsIs609在迈克尔·九重实验室。我们感谢他的帮助下，在微流体装置jsIs609动物的线粒体运输时间的推移成像Arpan Agnihotri（BITS Pilani的）。我们非常感谢博士瓦沙拉Thirumalai和Surya普拉卡什为我们提供了与斑马鱼的胚胎。我们感谢博士奎师那和CIFF NCBS支持部科学和技术中心的纳米技术（第SR/55/NM-36-2005）的旋转盘共聚焦显微镜的使用。我们也感谢Kaustubh劳，V. Venkatraman和雀塔那Sachidanand的进行讨论。这项工作是由DBT博士后研究（SM），DST快车道计划（SM）和DBT授予（SPK）。 SA的支持由DST和CSIR SPK补助金。

1。 SU8主制造<ol><li>设计的微流体的结构使用Clewin软件和打印它使用65,024 DPI激光绘图仪与最小特征尺寸为8μm的对电路板膜。 </li><li>清洁2厘米×2厘米的硅晶片，与在20％的KOH，持续1分钟，并在去离子水中冲洗，每一个晶片的流动层和其相应的控制层的原生氧化层。 </li><li>件用氮气吹干，4小时，脱水，在120℃的热板上。允许的作品冷却至室温，然后再进行下一个步骤。 </li><li>将硅块在时间上的微调夹头，打开真空。量的硅表面与〜20微升的六甲基二硅氮烷（HMDS）和涂层使用SPIN150喷丝在500rpm下5秒，持续30秒，然后3,000 rpm下。 </li><li>完全覆盖硅晶片〜1.5毫升SU8-2025（ <a href="http://www.microchem.com/Prod-SU82000.htm" target="_blank">包含http://</a>www.microchem.com/Prod-SU82000.htm）和大衣晶片使用SPIN150喷丝在500rpm下，持续5秒，30秒，然后2,000 rpm转速。该纺丝的协议产生〜40微米的光致抗蚀剂的厚度是适合的C.早期幼虫阶段的控制和流量层线虫 。 </li><li>自旋〜1.5毫升的SU8-2050使用SPIN150喷丝在500rpm下5秒，然后2,000 rpm转速，持续30秒，得到的光致抗蚀剂的厚度约为80微米，后期幼体阶段C 的适合用于流层制造线虫 ， 果蝇 /斑马鱼幼虫及其相应的控制层的基流层。 </li><li>放置在硅基片上热板与SU8涂布层的顶部上。软烘烤涂覆的硅片在65℃下进行1分钟，然后95℃10分钟。允许的作品冷却至室温，然后再进行下一个步骤。 </li><li>将曝光舞台上SU8-2025涂层表面的软出炉的硅块面对面临的UV灯的顶部。流层和控制层模式使用的光掩膜设计I（L1， 图1B）和设计（二）（L2， 图1B）分别为早期幼虫阶段C.线虫 。 SU8层的顶部上，并放置在光掩模，确保掩模对涂布层是平坦的。打开快门的UV灯和200瓦特的UV灯，持续15秒，通过光掩模暴露软烘烤SU8晶片。 </li><li>使用的光掩膜设计I（L1， 图1B）和设计（二）（L2， 图1B）上涂SU8-2050件（准备步骤1.6）制作的流程层和控制层C.为后期幼体阶段线虫 。使用的光掩膜的设计I（L1， 图1C）和设计（二）（L2， 图1C）为基础的流层和控制层分别为果蝇 /斑马鱼幼虫的晶圆上涂SU8-2050（准备我n步1.6）。揭露SU8表面，通过光掩模，以200瓦，持续15秒的UV灯。 </li><li>将暴露的硅片与涂覆层的顶部上的热板上。发布为1分钟，然后95℃下10分钟，在65℃下烘烤晶片。允许件冷却下来，然后再进行下一个步骤。 </li><li>开发件使用的SU8显影剂溶液20分钟。清洗件在异丙醇（IPA），并用氮气吹干气体。 </li><li>放置在干燥器中的硅片与的SU8图案上顶部。大衣件tricholoro（1H，1H，2H，2H-全氟辛基）硅烷2小时，在干燥器中的蒸气用50μl。 </li></ol> 注意事项：在KOH治疗期间，光阻涂布和发展，采取安全预防措施，对化学品的处理。硅烷的蒸气涂料应在通风良好的地方干燥器内进行。保护SU8抵制过度暴露在光线下。使用防护眼镜UV韧带羟色胺的来源。 2。 PDMS模具制造<ol><li>准备10:1 PDMS通过组合用5克在塑料杯中的固化剂50克Sylgard的184碱。手动混合的内容〜3分钟。德加的干燥器内，以除去所有的气泡混合过程中形成的混合物。 </li><li>以上的硅晶片用SU8图案设计II，倒入5毫米厚的聚二甲基硅氧烷的混合物，用于控制层，轻轻地避免形成气泡。如果气泡形成过程中浇注，脱气PDMS SU8在低真空模式，以除去所有的气泡。 </li><li> SU8-2025模式的设计，我将在硅片，流层，微调夹头和，打开真空保持。量晶片以〜1毫升的PDMS混合物和涂覆的晶片使用在500rpm喷丝SPIN150 5秒后跟1,500 rpm下，持续30秒。 </li><li>大衣〜1毫升PDMS与80微米的SU8-2050设计I（L1， 图1B）的图案，对于硅晶片上的混合流层C.为后期幼体阶段线虫使用SPIN150喷丝在500rpm下，持续5秒，随后以1000rpm，持续30秒。涂上一层厚厚的聚二甲基硅氧烷混合硅块SU8-2050的流层设计I（L1， 图1C）， 果蝇 /斑马鱼幼虫的模式，在500转每分钟为35秒SPIN150微调。 </li><li>烘烤PDMS涂硅块的设计和相应的设计（二）控制层的晶圆含有PDMS为C.线虫和/或果蝇 /斑马鱼幼虫在热空气对流烘箱中在50℃下进行6小时。 </li><li>切出含有SU8图案II 用于 C从硅衬底的PDMS片线虫和/或果蝇 /斑马鱼幼虫用锋利的刀片。 〜1毫米直径的储层的顶部连接的主要陷阱在PDMS模具使用哈里斯打孔器打一个小孔。 </li><li>将打孔PDMS块（含40微米厚的模具控制层L2）上的塑料托盘，与成型体的控制层侧的面朝上。将PDMS涂层的硅晶片载有40微米厚的SU8的设计，我在同一个托盘，PDMS涂层表面朝上。插入托盘等离子体清洁器腔室的内部，开启真空，持续2分钟。随后，打开等离子电源，降低腔室压力，直到将光粉红色。两个表面暴露至18瓦特空气等离子体，在低真空下，持续2分钟。 </li><li> C.为后期幼体阶段，将打孔的PDMS块从80微米厚的含SU8主模式II 线虫或果蝇 /斑马鱼幼虫上的塑料托盘，与成型体的控制层侧的面朝上。将相应的焙烤PDMS层旋涂在硅晶片上，含有80微米厚的设计，我购买C.线虫阶段或果蝇的 /斑马鱼幼虫同时在同一托盘上的平板PDMS涂层的小号urface朝上。上述使用相同的协议，将其暴露在空气等离子切割。 </li><li>将两个等离子体处理后的表面为C.线虫和/或果蝇 /斑马鱼幼虫连同温和的压力和烘烤它们在热空气对流烘箱中在50℃下2小时。 </li><li>切出的硅衬底C. SU8的设计，我为保税设备线虫和/或果蝇 /斑马鱼幼虫。打孔水库的流路的入口和出口的通道孔。 </li><li>将保税PDMS模具在塑料托盘C.线虫 ，与模制侧的面朝上的流动层设计。清洁玻璃盖片（22 X 22毫米，厚度1号），并把它放在同样的塑料托盘。将PDMS模具和玻璃盖片的等离子腔体内部装有。公开的底部的PDMS表面的装置和盖玻片至18瓦特空气等离子在低的压力，持续2分钟。将两个等离子体处理的表面，与根幔压力和烤它们在热空气对流烘箱中在50℃下2小时。 </li><li>将装置存放在一个清洁的环境，以备将来使用。 </li></ol> 3。其他果蝇的 /斑马设备的步骤<ol><li>公开清洁的玻璃表面的尺寸，2厘米×2厘米用tricholoro50μl的（1H，1H，2H，2H-全氟辛基）硅烷2小时，在干燥器中的蒸气。 </li><li>自旋〜1毫升在步骤2.1制备使用SPIN150喷丝在500rpm，35秒的硅烷化的玻璃表面上的10:1的PDMS组合。烘烤的PDMS涂覆的玻璃基板在热空气烘箱中，在50℃6小时，与涂覆的表面朝上。允许PDMS层冷却至室温，然后再进行下一个步骤。 </li><li>打孔机使用一个定制的尖锐的金属打孔器的PDMS的中间，以形成隔离物的PDMS层的涂敷层。类似果蝇 /斑马鱼（L1， 图1C中的流程设计的形状设计的金属打孔器</strONG&gt;）。 </li><li>公开的PDMS间隔层和单键合的嵌段（流动层和控制层， 果蝇 /斑马鱼幼虫在步骤2.9）至18瓦特空气等离子体，使用在低真空度的等离子体清洁器。一起放置两个等离子体处理的表面，他们在一个热空气烘箱中于50℃下2小时和烘烤。 </li><li>从玻璃剪切键合设备，冲头进入孔的入口和出口的水库和债券使用的等离子体清洁器，提到在步骤2.11，玻璃盖片（22×22毫米，厚度1号）。这将产生一个固定装置为一龄果蝇幼虫〜500微米的通道高度。斑马鱼幼虫，重复的的PDMS间隔层制造工艺与一个额外的键合工序。双层的PDMS-S生产900微米的高度为30 HFP斑马鱼幼虫的渠道。 </li></ol> 注意事项：避免设备制造过程中的粉尘颗粒。两个等离子清洗的表面的需要是完全无尘适当的结合。存储设备，在干燥器中在一个特殊的洁净室的情况下是不可用的，以尽量减少设备中的灰尘颗粒的积累。 4。使用硅橡胶膜<ol><li>通过18号针（外径〜1.25毫米的微柔性管（内径〜1.6毫米，外径为〜4.8毫米）的一端连接到一个加压的氮气气体供给调节器和另一端连接到主陷阱储层）粘到管。 A 3路活塞用于在中间管连接允许应用程序或在膜上的压力释放。 </li><li>填充管的PDMS的移动设备连接到蒸馏水中，用10厘米柱，然后再连接到储存器的主要的C.陷阱线虫和/或果蝇 /斑马鱼幼虫的移动设备。 </li><li>转动阀的供氮读取14 psi和监察次偏转朝向主陷阱膜Ë流道倒置显微镜在低倍。的水柱在微柔性管的长度的3路活塞打开时，被压缩。偏转膜的边缘是在透射光中（ 图1I和1J）可见。膜偏转导致的PDMS膜下的尘埃颗粒/气泡的位移，并推压它们的信道边界。 </li><li>等待，直到填补了微通道液体前，完全没有任何气体。 </li><li>释放压力，通过使用的3路活塞放松到其静止位置的膜。 </li></ol> 5。插入C.线虫 ， 果蝇和斑马鱼幼虫侵入设备，固定在灵活的PDMS膜<ol><li>填充M9的缓冲液的流路与[3克KH 2 PO 4，6克Na 2 HPO 4，5克NaCl，1毫升1M的用MgSO 4，H 2 O至1升，由引渡消毒oclaving] C.线虫 18或1X PBS [8克NaCl，0.2克KCl，1.44克Na 2 HPO 4，0.24克KH 2 PO 4，H 2 O至1 L，灭菌果蝇 /斑马鱼幼虫19之前的10分钟的高压灭菌]实验。 </li><li>找到一个单一的C.线虫所需的阶段NGM板18上一个（或一龄果蝇幼虫琼脂鸡蛋板或手动dechorionated的30斑马鱼斑马鱼幼虫在液体培养基中20），使用低倍率体视显微镜。选择一个单一的动物，使用小体积的缓冲溶液成一个微小费。使用微尖，其最终削减，以适应更大的果蝇或斑马鱼幼虫在缓冲。 </li><li>推入单一的有机体通过流道的入口处。调整移液压力定位下的动物个体的主要陷阱。 </li><li>慢慢地增加压力的PDMS膜定位议员动物对的NEL边界和固定用14 psi的压力C.线虫 ， 果蝇 ，斑马鱼幼虫和3磅7磅。 </li><li>固定的动物放置在显微镜的视场的中心。使用倒置显微镜，高分辨率明场或荧光成像所需的设置。获得单或时间推移荧光图像在预定义的帧速率。 </li><li>释放陷阱的压力和监管的动物在低放大倍率为5-10分钟的运动。 </li><li>冲洗动物，并插入一个新的动物，以重复上述步骤。 </li><li>清洗所有的动物从废液池，用蒸馏水采用注射器。干的推动空气通道的使用注射器以备将来使用。 </li></ol> 注意事项：动物移液压力要求较高的内流的主要通道从陷阱中释放后，往往表现出的运动/健康欠佳，应避免为IMA晋。清洁的流路的任何痕量的缓冲液，以防止堵塞的通道晶体。如果液压油用于高分辨率成像，清洁玻璃盖片是随后的实验中，能保持良好的信噪比。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+bidirectional+mitochondrial+transport+is+coordinated+with+axonal+outgrowth.">The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth.</a> J. Cell. Sci. 104, 917-927 (1993).</li><li>Louie, K., Russo, G. J., Salkoff, D. B., Wellington, A., Zinsmaier, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+imaging+conditions+on+mitochondrial+transport+and+length+in+larval+motor+axons+of+Drosophila.">Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila.</a> Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 151, 159-172 (2008).</li><li>Pilling, A. D., Horiuchi, D., Lively, C. M., Saxton, W. 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没有利益冲突的声明。

PDMS微流体装置是光学透明的，因此，可用于任何透明/半透明的模型生物体在体内成像的高分辨率。我们的设计是适合高倍率完整的活的动物细胞和亚细胞事件的时空成像。微细加工用软光刻技术，可轻松操作的模式生物的各种尺寸的设备尺寸。 C.不同阶段制造各种尺寸的移动设备线虫 ， 果蝇和斑马鱼幼虫。移动设备具有不同的高度为40μm和80μm，在其流层通道?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>硅晶片</td><td>大学晶圆</td><td> 150毫米（100）机械级SSP硅</td><td></td></tr><tr><td> Clewin软件</td><td> WieWeb软件</td><td>版本2.90 </td><td></td></tr><tr><td>激光光绘机</td><td>精细线成像</td><td> 65,024 DPI </td><td></td></tr><tr><td> HMDS </td><td> Sigma-Aldrich公司</td><td> 440191-100ML </td><td></td></tr><tr><td> SU8 </td><td> Microchem </td><td> SU8-2025年，SU8-2050 </td><td></td></tr><tr><td>开发人员</td><td> Microchem </td><td> SU8开发</td><td></td></tr><tr><td>硅烷</td><td> Sigma-Aldrich公司</td><td> 448931-10G </td><td></td></tr><tr><td> PDMS </td><td>道康宁</td><td> SYLGARD 184 </td><td></td></tr><tr><td> UV灯</td><td>奥丽尔</td><td> 66943 </td><td> 200W汞奥列尔灯</td></tr><tr><td>热空气炉中</td><td>超仪器</td><td>订制</td><td>设置在50℃下</td></tr><tr><td>热板</td><td> IKA实验室设备</td><td> 3810000 </td><td> <a href="http://www.ika.com" target="_blank">http://www.ika.com</a> </td></tr><tr><td>等离子清洗机</td><td> Harrick旅游等离子</td><td> PDC-32G </td><td></td></tr><tr><td>微调</td><td>半导体制造装置</td><td> SPIN150-NPP </td><td> <a href="http://www.SPS-Europe.com" target="_blank">www.SPS-Europe.com</a> </td></tr><tr><td>盖玻片</td><td>金印</td><td> 22 X 22毫米，第1号的厚度</td><td></td></tr><tr><td>线虫</td><td> 秀丽隐杆线虫遗传学中心（CGC） </td><td> e1265，ayIs4 </td><td></td></tr><tr><td> 果蝇 </td><td>布卢明顿</td><td>的P {chaGAL4} / CYO，UAS-syt.eGFP </td><td></td></tr><tr><td>斑马鱼</td><td>印度野生型</td><td>野生型</td><td></td></tr><tr><td>聚乙烯管</td><td>西格玛</td><td> Z279803 </td><td></td></tr><tr><td>微针</td><td>西格玛</td><td> Z118044 </td><td>切成1厘米的片</td></tr><tr><td> 3路活塞</td><td>西格玛</td><td> S7521 </td><td></td></tr><tr><td>哈里斯冲床</td><td>西格玛</td><td> Z708631 </td><td></td></tr><tr><td>压缩氮气</td><td>当地燃气供应商</td><td></td><td>使用的压力调节器来控制</td></tr><tr><td>体视显微镜&lt;/ TD&gt; <td>尼康</td><td> SMZ645 </td><td></td></tr><tr><td>共聚焦显微镜</td><td>安道​​尔与奥林巴斯</td><td>横河电机的旋转圆盘激光共聚焦显微镜</td><td></td></tr><tr><td> ImageJ的</td><td>美国国立卫生研究院</td><td> <a href="http://www.rsbweb.nih.gov/ij" target="_blank">www.rsbweb.nih.gov / IJ</a> </td><td>基于Java的图像处理程序</td></tr></tbody></table>

simple microfluidic devices for in vivo imaging of c. elegans, drosophila and zebrafish

微机电制流体设备提供一个可访问的微环境中的小型生物体内研究。简单的制造工艺可用于微流体装置使用软光刻技术1-3。亚细胞成像4,5，微流体器件已被用于在体内激光显微2,6和细胞成像4,7 在体内成像需要固定的生物体。取得这样的成绩使用吸力5,8，锥形通道6,7,9，变形膜2-4,10，吸气额外的冷却5，麻醉气体，温度敏感凝胶12，氰基丙烯酸酯胶13和麻醉剂如左旋咪唑14 ，15。常用麻醉药影响突触传递16,17和是已知的亚细胞神经传输4上有不利影响。在这种ST乌德我们展示了一个基于聚二甲基硅氧烷（PDMS）的装置，可以让完整的遗传模式生物，如线虫线虫 ， 果蝇和斑马鱼幼虫的麻醉剂无固定膜。这些模式生物在体内研究微流体装置，因为他们小的直径和光学透明或半透明的机构适合。体直径范围从〜10微米〜800微米的早期幼虫阶段C.线虫和斑马鱼幼虫和要求的不同尺寸的微流体装置，实现完整的固定的高分辨率时间的推移成像。这些生物体被固定使用施加的压力由压缩氮气通过液体柱和成像使用倒置显微镜。从陷阱释放的动物在10分钟之内返回到正常运动。 我们演示了四个应用程序时移成像C.线虫即，成像线粒体运输中的神经元，突触前囊泡运输中的运输缺陷的突变体，谷氨酸受体运输和Q神经母细胞分裂。从这样的电影中获得的数据，表明微流的固定化是一个有用的和准确的方法，麻醉动物（ 图1J和3C-F 4）相比时， 在体内细胞和亚细胞事件的数据的获取。 设备外形尺寸进行修改，以允许时间推移成像的不同阶段C.的一龄果蝇和斑马鱼幼虫。运输标记GFP（syt.eGFP）在感觉神经元的突触小泡的基本完整的一龄果蝇幼虫表达的胆碱能神经的感觉神经元中的突触囊泡的标记显示定向运动。一个类似的装置已经被用来进行时间推移成像的心跳在受精后30小时（HPF）斑马鱼幼虫。这些数据表明，我们已经开发的简单的设备可以适用于各种各样的模型系统，以研究几个细胞在体内的生物和发育的现象。

固定装置是一个通过粘接两层制成的双层PDMS块：的流动层（第1层）和一个控制层（第2层），如在图1中所示。的主要的陷阱被连接到氮气瓶通过一个调节器和一个三路停止旋塞应用必要的（3-14磅）的压力通过液体柱（ 图1A）到膜上。偏转膜固定C。线虫 ， 果蝇或斑马鱼幼虫在具有不同尺寸（ 图1B和1C）的流动通道设计。 C.不?...

Watch this Scientific Journal Video about Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish at JoVE.com

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

一个简单的微流体装置已经发展到进行麻醉免费<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;完整<em&gt;果蝇</em幼虫和斑马鱼幼虫。该设备采用可变形的PDMS膜固定的模式生物，以执行时间的推移成像的许多过程，如心脏的跳动，细胞分裂和亚细胞神经传输。我们证明了使用本设备，并从不同的模型系统收集的数据的不同类型的显示例子。

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

simple-microfluidic-devices-for-vivo-imaging-c-elegans-drosophila

Research

JoVE Journal

Bioengineering

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

17.0K 次观看.  NCBS-TIFR. 一个简单的微流体装置已经发展到进行麻醉免费

视频: Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

关于纺织品有机电子设备的简单和可扩展的制作方法

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...