这项工作是由研究奖由美国加州理工学院定量生​​物科学（QB3），在缩小差距奖由罗杰斯家族基金会，加州大学旧金山分校/桑德勒基金会计划为突破生物医学研究，从巴斯夫赠款和美国国家科学基金会通过支持学院早期职业发展（CAREER）计划（DBI-1253293）。

1. SU8主制造<ol><li>设计微流体结构中使用AutoCAD软件两层制作，并与10微米的分辨率由印刷电路板上胶片供应商的设计。器件设计的细节在一个附加的基准11和通道几何形状示于图1中 。这些层应该包括对准标记，以帮助从每个制造层 12并置的特征。 </li><li>放置一个预净化3英寸直径的硅晶片上旋涂机和打开真空将其粘贴到卡盘。申请1 ml的SU8-3035在晶片和旋转的中心，持续20秒在500rpm，然后在30秒以2000rpm，提供50μm的层厚度。 </li><li>取出晶片并烘烤上的135℃热板上30分钟。允许晶片移动到下一步骤之前，冷却至室温。 </li><li>揭露涂晶圆到1层掩膜（ 图2A </strong&gt;的）下一个准直190毫瓦，365纳米LED 90秒。曝光后，放置在一个135℃热板上的晶片1分钟，然后冷却至RT在进行下一步骤之前。 </li><li>放置在晶片上旋涂机和打开真空将其粘贴到卡盘。申请1 ml的SU8-2050在晶片和旋转的中心，持续20秒在500rpm，然后在30秒在1375转，从而产生层，可提供135微米的附加厚度。 </li><li>移动到下一步骤之前，取出晶片并烘烤上的135℃热板上进行30分钟，然后冷却至室温。 </li><li> 第 2次层掩模（图2B）对齐到图案化在1.3的几何形状和涂布的晶片暴露于准直的190毫瓦，365纳米LED 3分钟。曝光后，放置在一个135℃热板上进行1分钟，然后冷却至RT在进行下一步骤之前。 </li><li>通过浸入丙二醇单甲基醚乙酸酯的搅拌浴中30分钟，开发口罩。洗片在异丙醇和烘烤在135℃热板上进行1分钟。将发达师傅在100毫米的培养皿的PDMS成型。 </li></ol> 2. PDMS设备制造<ol><li>通过组合50个克硅酮基料而言，用5克在塑料杯中的固化剂1的PDMS:准备10。混合内容物用装有搅拌棒的旋转工具。脱气30分钟，或直至所有的气泡除去的干燥器内的混合物。 </li><li>倒入PDMS，得到厚度为3毫米以上的主站和放置放回干燥器进行进一步脱气。一旦所有的气泡被除去，烘烤设备在60℃下2小时。 </li><li>从使用上清洁的表面与图案的一面了手术刀和地点的模切设备。切PDMS模具一半用刀片从硕士2人（ 图3a）单独的主1。在含有50微米流体处理几何大师1印片，冲流体入口和出口有0.75毫米活检穿孔。 </li><li>等离子治疗装置在1毫巴O 2等离子体60秒，一个300瓦等离子清洗。润湿未打孔片的PDMS的表面上一滴去离子水，以暂时延缓PDMS-PDMS键合，并作为润滑剂。同时通过立体显微镜，地点硕士1人观看的主2面相对滑动面，直至机械锁，实现当凹入框架和图 3A队友伸出帧。 </li><li>将设备放置在一个60℃的烘箱烤的组装的装置（图3B）两天，在60℃以蒸发水和完全粘结。 </li></ol> 3.制备试剂<ol><li>填充1ml注射器用蒸馏水内相。 </li><li>填写1毫升注射器与HFE 7500氟油与1重量。 ％生物相容性表面活性剂的表面活性剂13的中间阶段。 </li><li>填充10毫升注射器用10重量％polyethylenë乙二醇（PEG）在含有1重量水溶液。 ％吐温20和1重量％十二烷基硫酸钠为连续相。 </li></ol> 4.系统准备<ol><li>放置在微流体芯片上的倒置显微镜加上能够&lt;100微秒的快门速度的数字照相机的阶段。 </li><li>所有装入注射器上的注射泵和连接27G的针头。附加〜PE-2管30厘米长的针头插入有始有终到适当的打孔设备中的漏洞。 </li><li>插入PE-2的10cm长到设备的出口，并放置在一个垃圾收集容器的另一端。 </li><li>素的装置，通过运行速度的高速率的注射泵（2000微升/分钟），直到流体在管段到达装置的入口端口。 </li></ol> 5.乳液代<ol><li>聚焦显微镜对包含50微米×50微米孔和一个区域下游出口通道。 </li><li>设定注射器泵将流体输送到双乳剂发生器在250微升/小时的内相，加入100μl/小时为中间相，和700微升/小时的连续相的流速，并等待10分钟以达到平衡。 </li><li>保持内和中间相的流速，在250微升/小时和100微升/小时，分别。在1050微升/小时设定外相的流率。等待3-5分钟为双乳液一代在这组流状况稳定。 </li><li>采集5秒的视频图像时，透过手动图像分析30赫兹脱机处理。 </li><li>重复5.3和5.4 与表1中给出的流速，内和中间相的流速保持恒定和载波相位的流速是通过调节注射器泵的设置来改变。 </li></ol>

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The authors have nothing to disclose.

这里所描述的双乳液生成几何形状设计模仿的玻璃毛细管装置8的物理过程。在这些，对齐圆筒形玻璃毛细管用于创建被剪切成均匀的双乳液液滴三相同轴射流。我们的3D PDMS装置的功能是依赖于用50微米高的制造具有载波相位通道是320微米的总高度形成小的特征的中心对准。有一个显著潜力以错开的第二层掩模，在步骤1.7图案相对于50μm的高大几何的高功能，如果掩模不能准确地对准。适当的对准可以通过设计对准标记，例如同心圆入口罩被共同定位过程中的照片图案化来辅助。的装置的两个半体的PDMS等离子体键合是可能导致最终器件的显著不对准的第二过程。等离子焊接PDMS至PDMS的一般是瞬间的，所以在步骤2.4，我们描述了装置表面的润湿用DI水以延迟接合并允许处理，使得在图3A中所示的对准帧可被允许以锁定。如果这是试图在没有足够的湿润，在PDMS表面将不可逆转键进入适当的调整之前，和设备必须加以处置和新的PDMS模具制作。 双乳液装置设计成采取的制造技术优点，即该铅均匀疏水表面性质。然而，在该协议中描述的参数之外的操作要求所要求的流体方法的一些了解。内层和中间相的交界处（图1D，点"1"），内相和中间相的低流量的相对高的流创建的两相喷射，与疏水性中间相涂敷的通道壁。如果中间相的比例流量增加，离散水包油微滴的产生将开始发生，消除以形成相干的三相喷射在孔（图1D滴形成的能力，点"3" ）。后的信道的扩展（图1D，点"2"），一个显著量的载体相流的需要来创建中间相和疏水通道壁之间的几何距离。在载波相位流量减少，最终将导致中间相润湿的疏水性设备的壁。显著减少载波相位流动可产生流动条件不足以剪切的内相为长，细丝，从而从根本上改变的双乳液液滴形成的物理过程。 一旦建成，该装置的设计，生产从14双乳液到50微米，一个方便的大小商业FACS分拣使用仪器。如果超出该尺寸范围双乳液期望，孔尺寸需要从这里使用的50微米×50微米大小缩放。因为该设备被设计为产生水/油/水双乳液有均匀的疏水表面性质，油/水/油双乳液不能创建除非有施加到使设备均匀地亲水表面处理。 这项工作表明一个简单的制造PDMS设备能够健壮形成水/油/水双重乳液。虽然以前的研究报告双乳液的使用3D光刻14,15创建的设备的形成，形成了他们的设备的双乳液有直径分别测量微米100S的。这里所报告的设备适合于生产双乳液幅度比这个更小的顺序，提供卷类似于哺乳动物细胞和适合于由FA排序CS。 虽然这些结果也可以用玻璃毛细管微流控技术，制造玻璃的设备来实现是费力的，需要很多动手的每个设备的步骤。对于我们所有的PDMS设备制造主要包括铸造，焊接，烘烤PDMS板，这是简单的，重复的，并且易于扩展到大量进程。 一个光刻制造的器件的效用来生成使用共轴流聚焦已证明双乳液。我们希望简单的制造和这双乳液发生器的设计强大的功能应该导致其适应的科学和工业应用。以前在玻璃毛细管微流控芯片工作所需的专业技能阻止调查人员，使用硅橡胶软光刻，现在常见的实验室技术应该会更舒适。此外，可以产生小的液滴尺寸很适合于perfor米细胞并以小滴的生物测定法，定量和使用FACS分选。对于工业应用，它已经被证明，这些类型的设备可被制造成阵列和并行化10，使双乳剂产生率相比，单个设备，以提高到几个量级。此外，能够形成大的同轴流量聚焦通道小双乳液应使设备抵抗结垢和堵塞，并行旨在在不干预运行较长的时间的设备时，这是至关重要的。

<html>双乳液包括由一中间，不混溶流体层从载体相分离的液滴的，并且是特别令人感兴趣的，因为它们在工业，制药潜在用途，以及生物应用1。在某些情况下，以封装高价值的化合物在双乳剂的核心的能力使材料受到保护和释放以控制的方式。例如，药物可能不适合于外部载体流体 2的溶解度的条件下被封装。此外，中间层的油可以被用作用于药物，化妆品，和营养物质3的封装和递送的胶囊模板。在生物学中，双乳液，可以在高通量筛选有用的，因为它们允许进行子纳升实验数量庞大出来，然后检测和分选使用荧光激活细胞分选（FACS）仪器4,5。 ENT"&gt;双乳液具有所需的性能特性的设计需要的双乳液的尺寸，组合物和均匀性的精确控制。虽然散装乳化过程，如膜乳化，在工业中使用，所得到的乳液是高度多分散的，显示出各种各样的功能特性1。液滴的微流体的字段被自然地适合单分散乳剂的产生与仔细控制组合物6。微流体双乳液生成已经实现了两个主要策略，顺序降制作和玻璃毛细管流聚集。双乳剂可以在使用两步骤降决策过程的平面PDMS的设备来产生。首先，含水包油乳液用的是水包油滴制作具有疏水通道壁的装置的区域。接着创建的，该乳液可以是流入或重新注入与适用于水包油型亲水墙壁下降，使得区域落制作4。然而，PMDS的亲水性表面处理需要额外的制造步骤和通常是暂时的7。最可控和可重复的方法，以形成双乳液是通过共轴流聚焦，一种技术使用玻璃毛细管微流体，由此形成含有三个阶段的同心射流通过一个小孔剪切以产生单分散的液滴8首创。这种技术允许用于生产液滴比通道尺寸小得多的，与双乳液是各相的流速的函数的精确大小和组成。液滴和信道的大小和保护性外鞘流之间的大的差异防止液滴从接触通道壁，使表面处理不必要。然而，这种玻璃设备需要锥形毛细管提示定制加工，以及精心的组装和密封。以前的研究已经用3D软光刻造影生成使用流聚集物理双乳液，但这些设备生产的乳剂，直径&gt;150μm的9,10，要比一般分拣用FACS较大的物体大约一个数量级。一个有吸引力的替代方案将包括强大的功能的玻璃毛细管同轴流量和小液滴产生聚焦与易于制造的硅橡胶软光刻技术。 在本文中，我们描述了使用同轴流动聚焦以产生≤50μm的乳剂和完全使用三维软光刻11构成的双乳液生成器。我们的设备采用了翻盖的方法来制造的设备，其中包括一个小剪切通道（图1）来近似所述乳液形成过程中拉玻璃毛细管喷嘴。更重要的是，这些设备不需要特定的表面处理，并且所有的聚合物结构提供简单和可重复的制造SCalable到大量的重复装置。在这里，我们概述了设计，制造和测试的双乳液生成器。双乳液生成被证明是健壮的，可重复至14微米的微滴直径。功能轻松制作的耦合使得该器件一个有吸引力的选项，新的双乳液的应用开发。

<table><tbody><tr><td>Photomasks</td><td>CadArt Servcies</td><td></td><td></td></tr><tr><td>3&quot; silicon wafers, P type, virgin test grade</td><td>University Wafers</td><td>447</td><td></td></tr><tr><td>SU-8 3035</td><td>Microchem</td><td>Y311074</td><td></td></tr><tr><td>SU-8 2050</td><td>Microchem</td><td>Y111072</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>Krayden</td><td>4019862</td><td></td></tr><tr><td>1 ml syringes</td><td>BD</td><td>309628</td><td></td></tr><tr><td>10 ml syringes</td><td>BD</td><td>309604</td><td></td></tr><tr><td>27 gaugue needles</td><td>BD</td><td>305109</td><td></td></tr><tr><td>PE 2 polyethylene tubing</td><td>Scientific Commodities, Inc.</td><td>B31695-PE/2</td><td></td></tr><tr><td>Novec 7500</td><td>Fisher Scientific</td><td>98-0212-2928-5</td><td>Commonly knowns as HFE 7500</td></tr><tr><td>Biocompatable surfactant</td><td>Ran Biotechnologies</td><td>008-FluoroSurfactant</td><td></td></tr><tr><td>35,000 MW PEG</td><td>Sigma Aldrich</td><td>1546660</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1369</td><td></td></tr><tr><td>Sodium dodecyl sulfate&amp;nbsp;</td><td>Sigma Aldrich</td><td>L3771</td><td></td></tr></tbody></table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

双乳剂发生器由使用三维PDMS制造（图1A）产生的共轴流聚焦装置的。几何图形，可允许形成一个三相同轴射流被剪切成一个正方形，50微米×50微米的孔，允许水/油/水双乳液（图1B， 图1C）的形成。的内水相和中间油相都汇集于连接处为10微米的通道尺寸x 50微米（图1D，点"1"）。由于PDMS的疏水性，氟化油拥抱通道壁和内相停留在通道作为流体的中心在连续喷射行进，直到突然膨胀通道扩展达到（图1D，点"2" ）。在这个位置中，内部两相被注入到一个320微米的高大JUNC的中心针对这样可以使相对同心引进水载波相位。这三个阶段被强制进入一个50微米×50微米的孔（图1D，点"3"），从而使载波相位剪的高流速的内两相到细长卷须而分解，从均匀的液滴（ 图1E）。 三维PDMS制造需要的两个独特的硅橡胶模具在翻盖配置成型两种层光刻大师之后的耦合。甲50微米高的层被用于形成所述内和中间流体处理通道，连同上万事达1（图2A）的剪切孔，以及免费凸出和凹进帧上相对的主站。一个附加的135微米高的层被用于创建载体流体和出口通道（图2B）。双乳液发电机组件利用牛逼他凹和等离子体处理（图3B）经过几何对准突出帧（ 图3A）。 双乳液装置在各种流量条件下进行测试以证明不同大小的，单分散的双乳液的形成。对于这些实验中，内和中间相的流速保持恒定和载波相位流速改性液滴生成期间影响剪切力。实验条件是由载波相位流量（Q c）在所述内两相的总和的比率参数流（Q 总和）。产生液滴的以Q C / 3至57 之和q进行的实验图像示于图4中 。观察到含有内两相的细长区域伸入50微米×50微米孔和断裂成液滴被对流下游。一世D.加强载波相位的流量（提高Q C / Q 总和 ）导致所述内相被剪切成能产生更小的液滴逐渐变薄的区域。由该装置在不同的流速产生双乳液显示出5.2％的变化的平均直径系数。液滴直径为q的C / Q 总和选择值的直方图也显示中所产生的微滴（图5）的大小相对均匀性。该装置展示了，以形成双乳液显著较小孔口宽度的能力，并显示出具有增加Q C / Q 总和 （图6）的明确减少的趋势。在最高载波相位流量测试，使用50微米×50微米的小孔形成14微米的双乳液。 <img alt="图1" src="/files/ftp_upload/53516/53516fig1.jpg" /> 图1.几何域金字塔之戒的Ë乳液发电机。在制造的器件的（A）3D模型。 （B）的中心通道表示引入的内（灰色）的垂直横截面，中间（红色），以及载体（蓝色）相。 （C）的横截面示出包含内部两个阶段进入方孔中射流。 （D）的乳液产生在设备顶视图。交界（1）在注入疏水中间相是由亲水性的PDMS，这会导致它的外套通道壁辅助。交界（2）的信道扩展和内两相的射流被剪切成孔（3）由较高的速率流动的连续流体的一个点，其中物理原因液滴形成。 （ 五）复乳一代器件的显微图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53516/53516fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。&lt;</a>/ P&gt; <img alt="图2" src="/files/ftp_upload/53516/53516fig2.jpg" /> 图2.平版印刷生产的主人 。 （A）的用于制备50μm的特征的掩模。主站1是用来模制流体入口，所述内/中间相交界，该乳液产生孔口，以及一个凹槽对准。硕士2包含用于对准凸起的脊。 （二）用于制备135微米功能的面膜。主人是包含载液布线通道和出口通道镜像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53516/53516fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53516/53516fig3.jpg" /> 图3.大会PDMS设备 。 （ <s仲&gt; A）捏造PDMS板包含免费的凹陷和凸出的框架。 （B）组装，联锁，提供最佳的功能定位框架。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53516/53516fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53516/53516fig4.jpg" /> 图生成双乳液4.图像以不同的流速。外相的流速被改变，以改变Q C / Q 总和 ，这是考虑到每个图像的左侧。增加Q C / Q 总和变窄通过节流孔被剪切内液体的喷射，产生越来越小的液滴。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53516/53516fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5.直方图双乳液液滴的尺寸在不同的流速 。在给定的流动条件下生产的乳滴的直径平均变异系数为5.2％。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53516/53516fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53516/53516fig6.jpg" /> 图6.雾滴直径与归一化的流量参数 。调整连续相的流速可以生产双乳液是30％的孔径-100％的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53516/53516fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </P&gt; <table fo:keep-together.within-page="1"><tbody><tr><td> 数 Q i [微升/小时] </td><td> Q·M·[微升/小时] </td><td> 之和q [微升/小时] </td><td> Q C [ 微升/小时] </td><td> QÇ <fon> / Q 总和 </fon></td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 1050 </td><td> 3 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 2100 </td><td> 6 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 3850 </td><td> 11 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 5950 </td><td> 17 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 8050 </td><td> 23 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 10150 </td><td> 29 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 11900 </td><td> 34 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 17150 </td><td> 49 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 19950 </td><td> 57 </td></tr></tbody></table> 用于实验表1流速的参数。内相和中间相的流速（Q I，Q 米 ）被保持不变，从而以恒定的组合流速（Q 总和）。载波相位流动速率（QC）是变化的，以产生双乳液具有不同的直径。的比例Q C / Q 总和是主无量纲参数描述的实验条件。

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

double-emulsion-generation-using-polydimethylsiloxane-pdms-co-axial

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

16.0K 次观看.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

视频: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

微流控芯片控制，弹性微阀阵列

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

科研

生物学

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

高吞吐量的单细胞和多细胞微胶囊

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

生物工程

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion co-flow device to generate uniform high-viscosity droplets in a low-viscosity fluid. The microfluidic capillary device has a common co-flow structure with its exit connecting to a wider tube. Elongated droplets of the low-viscosity fluid are first encapsulated by the high-viscosity fluid in the co-flow structure. As the elongated low-viscosity droplets flow through the exit, which is treated to be wetted by the low-viscosity fluid, phase inversion is then induced by the adhesion of the low viscosity droplets to the tip of the exit, which results in the subsequent inverse encapsulation of the high-viscosity fluid. The size of the resultant high-viscosity droplets can be adjusted by changing the flow rate ratio of the low-viscosity fluid to the high-viscosity fluid. We demonstrate several typical examples of the generation of high-viscosity droplets with a viscosity up to 11.9 Pas, such as glycerol, honey, starch, and polymer solution. The method provides a simple and straightforward approach to generate monodisperse high-viscosity droplets, which may be used in a variety of droplet-based applications, such as materials synthesis, drug delivery, cell assay, bioengineering, and food engineering.

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

反相共流微流控毛细管装置制备高粘度水滴的方法

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

化学

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Semi-3-Dimensional 流聚焦微流控装置产生尺寸控制的聚乙二醇二丙烯酸酯液滴

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

工程学

Glass-Based Devices to Generate Drops and Emulsions

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A&#160;voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

基于玻璃的设备，用于产生液滴和乳液

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. ...

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 ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

用于组合插头生产的微流体装置的设置

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

作者聚焦：在精准肿瘤学中整合计算和实验方法

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

用于组合塞生产的双层微流控装置

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

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微流控芯片控制，弹性微阀阵列

高吞吐量的单细胞和多细胞微胶囊

微型涡辅助电穿孔的顺序传递分子

反相共流微流控毛细管装置制备高粘度水滴的方法

Semi-3-Dimensional 流聚焦微流控装置产生尺寸控制的聚乙二醇二丙烯酸酯液滴

基于玻璃的设备，用于产生液滴和乳液

多级可变高度的光刻多层带瓣微流体装置

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

用于组合塞生产的双层微流控装置

用于组合塞生产的双层微流控装置