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>

<ol><li>Van Der Graaf, S., Schroën, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review%5BTitle%5D)+AND+%22J.+Membrane+Sci%22%5BJournal%5D)">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li>
<li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application%3A+comparison+with+simple+emulsions%5BTitle%5D)+AND+%22Int.+J.+Pharm%22%5BJournal%5D)">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li>
<li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Double-emulsion+drops+with+ultra-thin+shells+for+capsule+templates%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Double-emulsion drops with ultra-thin shells for capsule templates.</a> Lab Chip. 11 (18), 3162-3166 (2011).</li>
<li>Lim, S. W., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrahigh-throughput+sorting+of+microfluidic+drops+with+flow+cytometry%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Ultrahigh-throughput sorting of microfluidic drops with flow cytometry.</a> Lab Chip. 13 (23), 4563-4572 (2013).</li>
<li>Bernath, K., Hai, M., Mastrobattista, E., Griffiths, A. D., Magdassi, S., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+compartmentalization+by+double+emulsions%3A+sorting+and+gene+enrichment+by+fluorescence+activated+cell+sorting%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.</a> Anal. Biochem. 325 (1), 151-157 (2004).</li>
<li>Seemann, R., Brinkmann, M., Pfohl, T., Herminghaus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Droplet+based+microfluidics%5BTitle%5D)+AND+%22Rep.+Prog.+Phys%22%5BJournal%5D)">Droplet based microfluidics.</a> Rep. Prog. Phys. 75 (1), 016601(2012).</li>
<li>Bauer, W. A. C., Fischlechner, M., Abell, C., Huck, W. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrophilic+PDMS+microchannels+for+high-throughput+formation+of+oil-in-water+microdroplets+and+water-in-oil-in-water+double+emulsions%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Hydrophilic PDMS microchannels for high-throughput formation of oil-in-water microdroplets and water-in-oil-in-water double emulsions.</a> Lab Chip. 10 (14), 1814-1819 (2010).</li>
<li>Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., Stone, H. A., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monodisperse+double+emulsions+generated+from+a+microcapillary+device%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308 (5721), 537-541 (2005).</li>
<li>Chang, F. C., Su, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlled+double+emulsification+utilizing+3D+PDMS+microchannels%5BTitle%5D)+AND+%22J.+Micromech.+Microeng%22%5BJournal%5D)">Controlled double emulsification utilizing 3D PDMS microchannels.</a> J. Micromech. Microeng. 18 (6), 065018(2008).</li>
<li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802-807 (2012).</li>
<li>Tran, T. M., Cater, S., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coaxial+flow+focusing+in+poly%28dimethylsiloxane%29+microfluidic+devices%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices.</a> Biomicrofluidics. 8 (1), 016502(2014).</li>
<li>Lithography. , Available from: 
 	
 <a target="_blank" href="https://www.memsnet.org/mems/processes/lithography.html">https://www.memsnet.org/mems/processes/lithography.html</a> (2015).</li>
<li>O'Donovan, B., Eastburn, D. J., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrode-free+picoinjection+of+microfluidic+drops%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Electrode-free picoinjection of microfluidic drops.</a> Lab Chip. 12 (20), 4029-4032 (2012).</li>
<li>Chang, F. C., Lin, H. H., Su, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlled+W%2FO%2FW+double+emulsification+in+3-D+PDMS+micro-channels%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Controlled W/O/W double emulsification in 3-D PDMS micro-channels.</a> 3rd IEEE Int. Conf. Nano/Micro Eng. Mol. Syst. NEMS, , 792-795 (2008).</li>
<li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802(2012).</li>
</ol>

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...

<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?...

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

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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

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

通过胶体粒子自组装过程创建在PDMS微流控芯片分50纳米的纳米流体结

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

工程学

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 ...

生物工程

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. ...

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (&#181;PIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between &#181;PIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

仿生的微流体模型肺呼吸腺泡航空公司

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

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 ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

神经科学

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

基本的有丝分裂纺锤体的重组在球形乳液液滴

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...