This work was supported by the PRESTO "Design and Control of Cellular Functions" research area of the Japan Science and Technology Agency (JST), a Grant-in-Aid for Scientific Research of Innovative Areas "Molecular Robotics" (Project No. 24104002) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, Grant-in-Aid for Young Scientists (A) (Project No. 24680033) and Scientific Research (B) (Project No. 26280097) from the Japan Society for the Promotion of Science (JSPS), and the Creative Design for Bioscience and Biotechnology course of the School of Bioscience and Biotechnology at Tokyo Tech.

1.基于毛细管的微流控芯片的制备<ol><li> 将持有最多 注意:保持器设计示于图2A。 <ol><li>切出的每个人的四个盘（图2A（ⅰ） - （ⅳ）），从使用铣床2毫米厚的聚缩醛塑料板。使用下面的尺寸为每个保持器的四个圆盘的:（ⅰ）盘1直径8.5毫米，毛细管孔（CH）直径1.3毫米，螺丝孔（SH）直径1.8毫米; （ⅱ）盘2直径8.7毫米，CH直径为2.0毫米，SH直径1.8毫米; （三）盘3直径8.7毫米，CH直径为0.5毫米，SH直径1.8毫米;和（iv）的光盘4的直径9.1毫米，CH 1.0mm直径，SH直径1.8毫米。 </li><li>组装使用M2×40螺钉（图2B）的持有者。保持器（图2B）的底部部分由盘1和盘2在图2A（i）中，（ii）和一个上部（ 图支架的URE 2B）由盘3和图 2A中的盘4（ⅲ），（ⅳ）。 <ol><li>构造支架的底部，插入螺丝在三个SH每个盘1和2通过夹持下一块的螺纹部的缩短的螺钉。保持螺杆的长度为0.9厘米（长度相同的夹持器的底部部分）。 </li><li>构造支架的上部，插入螺钉插入两SH每个盘3和4通过夹持下一块的螺纹部的缩短的螺钉。保持螺杆的长度为0.7厘米（长度相同的夹持器的上半部分）。 </li><li>为了组装支架，加入使用长螺钉夹持器的底部和上部。 注意:使保持器确切的每个部分的长度:底部为0.9厘米;上部为0.7厘米（ 图2B）。 </li></ol></li></ol></li><li> 玻璃毛细管的制作 <ol><li>使用两种类型的玻璃毛细管的:一个内部玻璃毛细管（外径（OD）/内径（ID）:1.0 / 0.6 MM），和一个外玻璃毛细管（外径/内径:2.0 /1.12毫米）。 </li><li>使用玻璃切割器的外玻璃毛细管分成三个相等的部分，然后用玻璃刀到内玻璃毛细管分成两个相等的部分。 </li><li>锐化用玻璃毛细管拉出器（图3A）的每个分割内层和外层玻璃毛细管。设置在最大的车夫的重量。在用于外玻璃毛细管60度和70度的内毛细管设置拉出器的热水平。精心磨砺的玻璃毛细管。 <ol><li>保持玻璃毛细管的收缩部中的尖端的长度:内毛细管是1.5-1.8厘米;外毛细管是0.8-1.0厘米（图3C）。如果此长度比所描述的长度更短或更长，请调整拉出器的热水平。 </li></ol></li><li> F九，内部或外部的玻璃毛细管的microforge站使用磁带（图3B）。 </li><li>切断用microforge在三个步骤 （图3B）的玻璃毛细管的尖端:（ⅰ）触摸玻璃毛细管的尖端上的铂丝的玻璃珠，（ii）由踩踏脚踏加热铂线切换为1-2秒，和（iii）1-2秒后，切断该玻璃毛细管的尖端通过冷却铂丝。 <ol><li>调整内（D I）和外部（D 2 O）毛细小孔的直径分别。内层玻璃毛细管的小孔直径为5，10和20微米（D I = 5,10，20微米）和所述外层玻璃毛细管（D 2 O）是在该实验中为60μm（D 2 O = 60微米）。 注意:玻璃毛细管是一次性的。重复玻璃capillar的制造IES。 </li></ol></li></ol></li></ol> 2.程序生成W / O型微滴<ol><li>填用含有油的表面活性剂的外玻璃毛细管。的油和表面活性剂的混合物是含有十六烷2％（W / W）在该实验中（图4A）脱水山梨醇单油酸酯。 注意:有油和表面活性剂的许多组合（例如，油可以是氟化的或碳酸;表面活性剂可以是离子的，非离子的，或含氟化合物）。 <ol><li>引入10ul含有十六烷山梨糖醇单油酸酯为外玻璃毛细管。 在图4A中，外层玻璃毛细管（D 2 O）的孔直径为60微米（D 2 O = 60微米）。要调整玻璃毛细管的孔口，回到步骤1.2.4-1.2.5。 </li></ol></li><li>在保持器（图4B）的底部设置的外毛细管。 </li><li> DRA瓦特大约0.1微升的水溶液成内玻璃毛细管（图4C）通过毛细作用。在图4C中，内层玻璃毛细管（D I）的孔直径为10微米（D I = 10微米）。要调整玻璃毛细管的孔口，回到步骤1.2.4-1.2.5。 </li><li>在保持器（图4D-a）中的上部设置的内毛细管中。插入的内毛细管导入外毛细管（ 图4D-a）中。望着白点圈，如图4D-一个 ，观察内部毛细管的外表毛细管内的位置（外毛细管（w）的 = 130微米的内径）（图4D-B，C）使用数字显微镜。在外部的毛细管的内毛细管的位置必须设置为 w = 100-150微米。 注意:要改变内的位置毛细管中的外毛细管，请转动螺丝在保持器的上部。从而， 距离 w可以精确地控制。 </li><li>引入100μl包含十六烷山梨醇单油酸酯（2％W / W）的至1.5 ml的样品微管的底部。安装保持器，具有内，外毛细管，在样品微管（图4E-a）中。一定要检查外毛细管从空气-油界面（ 图4E-B）保持了。 </li><li>离心机用台式摆动出型微型离心机在1600 XG的比重为1-2秒，产生微滴（ 图4F）样品微管。开展在室温下所有的实验。 注意:使用摆动出式离心机。液滴可以与样品微管的侧壁碰撞而当使用一个固定角型离心机崩解。 </li><li>慢慢地绘制出W / O液滴吸管，然后，把它们放在一个玻璃小号立德。 </li><li>使用数字显微镜（倍率，200X）中产生的微滴的捕获图像。 </li></ol>

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No conflicts of interest are declared.

使用该装置中，单分散的W / O微滴被喷射流17的高原-Rayleigh不稳定性产生的。显微镜检查没有发现卫星液滴的存在。在器件的制造中，三个关键步骤是必不可少的，成功地产生单分散的W / O微滴。首先，供给含有油表面活性剂和水溶液的直进流，四个盘毛细管孔必须设置在同心图案。第二，内毛细血管小心地插入到外毛细管，因为如果它接触到上部支架毛细管的尖端破裂容易。这个操作是很困难的，所以我们推荐使用放大镜。最后，为了使水溶液17的喷射流，内毛细管导入外毛细管的位置必须设置为 w = 100-150微米。如果离心麦克风产生的W / O型微滴的大小分布rofluidic设备不是单分散，检查外毛细管内毛细血管的位置。来改变在所述外毛细管的内毛细管的位置，请转动螺丝在保持器的上部。从而， 距离 w可以精确地控制。 为了使单分散W / O型微滴，有电流限制。有困难的增加，因为在研究的所有实验均在台式离心机的最大离心速度进行离心速率（如果需要的话）。此外，液滴生成是由各种不同的样品溶液的困难，限制依赖于离心机的设计。多管毛细管作为内毛细管可以由材料和解决方案18的各种组合提供包封的微滴。 微流体器件具有比传统微法的两个主要优点:ⅰ）电子ASY和健壮制造，以及ii）将样品溶液的仅一小体积（0.1微升要求）。第一，基于毛细管的离心轴对称共同流入微流体装置的制造是简单的和鲁棒性。只有薄的毛细管，毛细管保持器，和一个样品微管是必需的。的制造时间是该器件5-10分钟。相比的其它的微流体装置的制造设备的制造需要更少的时间。此外，毛细管保持器是健壮和可重复使用。因此，唯一的消耗品的玻璃毛细管和样品微管中的装置，这使得它比其它微流体系统更便宜。最后，由于油和水流量是由离心力产生，没有浪费的体积。 1-2秒，设备生成大量微滴的。 微滴的理想人选，进行涉及少量样本搜索解决方案的高通量实验ñ。用该装置，在理论上是可能产生从0.1微升样品溶液的成百上千的每秒10微米大小的微滴。因此，该装置可容纳通过最小化所需要的快速定量分析样品的体积与珍贵生物样品工作。这种装置可用于分析的生化反应6-9和单细胞酶促反应10。

W / O微滴1-5对生物化学和生物工程的研究中许多重要的应用，包括蛋白质合成6，蛋白质结晶7，乳液PCR 8,9，电池封装10和人造细胞样系统5,6的建设。以产生用于这些应用W / O型的微滴，重要的标准是大小和W / O微滴的单分散性的控制。用于制造微流体装置的单分散，大小可控的W / O微滴11是基于共同流动方法12,13，流动聚焦方法14,15，而在微通道丁字路口方法16。虽然这些方法产生高度单分散的W / O微滴，该微制造过程需要微通道的制备复杂处理和专门的技术，并且还需要大量的样品溶液（至少几百81，因为在注射器泵和管即进行试样溶液的微通道的必然死体积的1）。因此，W被需要/ O微滴易于使用和低死体积的方法来产生单分散的。 本文用的实验程序的视频沿，描述了一种离心基于毛细管的轴对称共同流入微流体用于产生细胞大小的设备17中，单分散的W / O微滴（ 图1）。这个简单的方法实现大小单分散性和可控性的大小。它仅需要一个台式微型离心机并固定在一个采样微型管基于毛细管轴对称共同流入微流体装置。我们的方法只需要非常小的体积（0.1微升），和不浪费的样品的任何显著体积。

<table><tbody><tr><td>2-mm-thick polyacetal plastic plate</td><td>Tool</td><td>Nikkyo Technos, Co., Ltd. (Japan)</td><td>244-6432-08</td></tr><tr><td>Milling machine</td><td>Tool</td><td>Roland DG Co., Ltd. (Japan)</td><td>MDX-40A</td></tr><tr><td>End Mill RSE230-0.5*2.5</td><td>Tool</td><td>NS Tool Co., Ltd. (Japan)</td><td>01-00644-00501</td></tr><tr><td>M2*40 screws</td><td>Tool</td><td>Jujo Synthetic Chemistry Labo. (Japan)</td><td>0001-024</td></tr><tr><td>Glass Capillry Puller</td><td>Tool</td><td>Narishige (Japan)</td><td>PC-10</td></tr><tr><td>Microforge</td><td>Tool</td><td>Narishige (Japan)</td><td>MF-900</td></tr><tr><td>Inner Glass Capillary</td><td>Tool</td><td>Narishige (Japan)</td><td>G-1</td></tr><tr><td>Outer Glass Capillary</td><td>Tool</td><td>World Precision Instruments Inc. (USA)</td><td>1B200-6</td></tr><tr><td>1.5 ml Sample tube</td><td>Tool</td><td>INA OPTIKA CO.,LTD (Japan)</td><td>ST-0150F</td></tr><tr><td>Hexadecane</td><td>Reagent</td><td>Wako Pure Chemical Industries Ltd. (Japan)</td><td>080-03685&amp;nbsp;</td></tr><tr><td>Sorbitan monooleate (Span 80)</td><td>Reagent</td><td>Tokyo Chemical Industry Co., Ltd. (Japan)</td><td>S0060</td></tr><tr><td>Milli Q system</td><td>Reagent</td><td>Merck Millipore Corporation (Germany)</td><td>ZRQSVP030</td></tr><tr><td>Swinging-out-type Mini-centrifuge</td><td>Tool</td><td>Hitech Co., Ltd. (Japan)</td><td>ATT101</td></tr><tr><td>Digital Microscope</td><td>Tool</td><td>KEYENCE Corporation (Japan)</td><td>VHX-2001</td></tr></tbody></table>

capillary-based centrifugal microfluidic device for size-controllable formation of monodisperse microdroplets

在这里，我们证明了快速生产尺寸可控，单分散，W / O型使用基于毛细管离心微流体装置的微滴的一个简单方法。的W / O微滴最近已在，使小型化的化学实验有力方法中。因此，W是需要/ O微滴开发一种通用的方法来产生单分散的。我们已经开发了用于生成单分散W¯¯基于基于毛细管离心轴对称共同流入微流体设备/ O微滴的方法。我们成功地通过调整毛细管孔口控制微滴的大小。我们的方法需要的设备比用其它微流体技术更容易使用的，只需要对封装样品溶液小体积（0.1-1微升），并能生产数十万的每秒的W / O微滴的。我们预计这种方法将有助于生物学研究需要的宝贵的生物小号通过保护该样品进行快速定量分析生化和生物学研究的体积amples。

在这项研究中，我们通过使用基于毛细管离心微流体装置（图1）呈现为细胞大小的W / O微滴的产生的简单方法。微流体装置是由一个毛细管保持器（图2B）的，两个玻璃毛细管（ 如图 3C内和外玻璃 ​​毛细管），和含有含表面活性剂的油微管。我们注射0.1微升样品溶液到内玻璃毛细管，并置于所述内玻璃毛细管到外玻璃 ​​毛细管（图4D）。通过样品溶液17（图1B）的喷射流的高原-Rayleigh不稳定性生成并且是至少2小时，17的稳定细胞大小的W / O微滴。 从毛细管巴不同尺寸的W的典型例子/Ø产生的微滴SED离心微流体装置示于图5中 。 图5A-F示出了W / O微滴的数字显微镜图像和大小分布直方图（N = 200）。使用内部毛细管用一个D的生成W / O型微滴I = 5（A，B），10（C，D），或20微米（E，F）口，同时保持D 2 O 和 W恒定在60微米直径的和115微米，分别。所生成的W / O微滴的大小的测量获得的显微图像的分析获得的。 对于 d I = 5，10，和20微米的孔，微滴的平均直径为8.3微米（标准偏差（SD）为0.9μm，变异系数（CV）为10.8％），12.7微米（SD 1.1微米，CV分别为8.6％），和17.9微米（SD 1.4微米，CV为7.8％）。这些结果表明，我们成功获得单分散W / O microdrople通过该方法TS。此外，在W / O微滴直径为几乎相同的内毛细管孔（图5G）。因此，W / O微滴的平均大小可以很容易地在宽范围内调整，通常为5至20微米，利用微型器件。 <img alt="图1" src="/files/ftp_upload/53860/53860fig1.jpg" /> 图1的离心毛细管基于轴对称共同流入微流体装置和方法生成的W / O微滴（ 离心基于毛细管的轴对称共同流入微流体装置和形成使用该装置的W / O微滴的（A）图的概述圈），（B）W，通过水溶液17的喷射流的高原瑞利不稳定性产生/ O微滴中，D i是内在的玻璃毛细管孔直径，D 2 O是内部玻璃毛细管孔直径，w是外毛细管内径，制造的器件的（C）照片。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53860/53860fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53860/53860fig2.jpg" /> 图2.设置毛细管保持器（A）的制造的聚缩醛塑料毛细管保持器的设计 :直径在保持器的单位为毫米。 （二）持有人的照片组成的上部和下部的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53860/53860fig2large.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/53860/53860fig3.jpg" /> <str翁&gt;图3.制备玻璃毛细管的（A）锐化用玻璃毛细管拉出器，（B）的毛细管的由microforge切出的尖端（黑点的圆圈）和方案切的玻璃毛细管出来的尖端，所制作的内，外毛细管（C）的照片。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53860/53860fig3large.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/53860/53860fig4.jpg" /> 图4. W / O微滴的微流体装置设置和生成的流程图（A）外的毛细管的制备。油导入外毛细管，（B）的外毛细管设置到保持器的底部，所述内毛细管（C）的制备表面活性剂:水溶液博士芒成通过毛细管的内毛细管，（D）的内毛细管设置到保持器（A）的上部。检查的内毛细管使用数字显微镜（B，C），（E）的座与安装在该样品管（一）内和外毛细管是在外侧毛细管。检查外毛细管保持从空气-油界面了，（F）最后，样品微管是由桌面摆动出型微型离心机离心。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53860/53860fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53860/53860fig5.jpg" /> 使用不同直径的毛细血管微滴生成图 5. 形成单分散的单元格大小W / O微滴的数码显微镜图像和粒度分布直方图（N = 200）米，D I = 5（A，B），10（C，D），和20微米（E，F），（G）的玻璃毛细管的孔口直径和所产生的W / O微滴的直径的相关性。误差线显示生成的W / O微滴的直径的标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53860/53860fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets at JoVE.com

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

基于毛细管的离心微流体装置单分散微滴的大小可控的形成

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

capillary-based-centrifugal-microfluidic-device-for-size-controllable

Nanyang Technological University

Research

JoVE Journal

Engineering

10.3K 次观看.  Tokyo Institute of Technology. Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

视频: Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

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

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

基于液滴在微流体装置的被动抽水高速输送系统

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

生物学

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

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

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

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

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

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

用于从单一DNA分子大规模平行蛋白质合成的Femtoliter滴体阵列

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

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. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

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 ...