Name: a microfluidic platform for precision small-volume sample processing and its use to size separate biological particles with an acoustic microdevice
Uploaded: 2015-11-23T15:00:00.000+00:00
Duration: 11 min 32 s
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice at JoVE.co

This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344, and partially supported by LLNL&#8217;s Laboratory Directed Research and Development (LDRD) program, 14-LW-077. The authors thank Michael Wilson, Mark Stenglein and Joe DeRisi at the Univeristy of California, San Francisco for generously providing GGV and Boa cell samples. EJF acknowledges support from the LLNL Lawrence Scholar Graduate Program. MS acknowledges support from the UC Office of the President Lab Fees Research Program. LLNL-JRNL-665235

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The authors have nothing to disclose.

该协议提出了微流控设备的系统级集成，以宏观的设备进行自动化生物样品处理。这个平台的模块化允许它为适用于任何连续流动装置中，和，作为一个例子，所提出的协议的重点是表征和优化acoustofluidic粒子分离装置的性能。这个协议的三个主要优点突出:（ⅰ）模块化和芯片到世界的接口，（ⅱ）鲁棒表征设备的性能，以及精确计量样品体积为粒子分离（ⅲ）自动处理。 一世。模块化和芯片到世界的接口 如图2所示，微流体芯片被安装在一个自定义的实验板以很容易地装配到一台显微镜直接观察。线路板含有＃6-40 UNF螺纹在一个5毫米间距网格，ENA孔金光闪闪芯片被固定，并且被制成流体连接。流体连接PEEK管与机械加工的端面，该密封件对流体芯片用橡胶面密封垫圈和一个不锈钢的衣领。这个接口方案，可以方便片替换和快速装置的重新设计，需要很少或没有改变其他系统组件，提供芯片脚印符合网格格式。例如，我们已经使用该平台的微流控芯片为连续流动电泳，热裂解细胞，29快速试剂化学合成的混合，和单细胞捕获和询问。 二。设备性能强大的表征 为了优化任何微流体分离装置的性能，它的操作，必须首先充分表征。此处所描述的系统支持的快速和自动化协议来做到这一点的发展。对于具体examp声学聚焦设备中，聚焦质量，工作频率，并在微流体通道集中颗粒的位置的文件，必须进行测量为每个单独的设备。这些测量需要通过一系列的压电陶瓷驱动器的频率，电压和流速扫，以确定最佳参数的组合为高质量的分离。所提出的协议自动改变这些可调参数和捕捉相关数据- 即 ，颗粒中流动的信道进行后处理，以产生颗粒的聚焦质量，频率和位置 （图3）所需的测量的荧光图像。 的声学装置的性能全面表征要求重复步骤4.4和4.5在需要不同的实验条件下。例如，一个芯片的绝对聚焦位置被以相对低的流率和高的电压运行频率扫描发现s至确保完全迁移到节点位置。另外，这样的频率扫描可以评估装置组件（具有已知尺寸的聚苯乙烯珠上运行时）的质量，或者，以确定如何先前未知粒子类型将表现在系统（后一个芯片的特点与珠）。与来自压电陶瓷至微流体通道良好能量转移的芯片将导致紧聚焦在高流速（&gt; 1毫升/分钟）和低电压（12〜15 V pp的），而那些与能量转移差不会集中连在低流速下（&lt;100微升/分钟）和高电压（&gt; 20 V峰）。我们已经发现，微流体芯片和压电陶瓷之间的紧密接触为有效的能量转移到流体临界。粘结微流体芯片和压电陶瓷将使可靠的生产的高性能设备的最佳方法的进一步调查。 最后，一​​个完整可以通过结合步骤4（ 和图3）与来自SPO和LPO作为相关操作参数的功能收集的颗粒数的基于图像的频率扫描测量来获得的acoustophoretic装置的操作的画面，从进行了微球分离实验如在步骤5中描述的。如图6所示，这样一系列自动化实验可以迅速表征单个设备的性能和可调谐性，通知最优参数空间的用户来操作设备，用于颗粒分离。 三。对于粒子分离自动化小样本处理 对于成功的和精确的微流控芯片基于样本的处理，这是至关重要的，以可靠地和精确地计，负载，提供和收集的流体的体积，因为他们通过。这个精度是特别重要的，当样本量小（〜0.1-1毫升），这是在临床或研究实验室环境常见。30精确样品处理是在采用样品手工撤回到注射器和输注到装置与样品时的无反馈的传统微流体实验有挑战性已被分离和当它应该被收集。所提出的协议采用自动化样品线圈装载和配药加上从流量传感器的实时反馈，以使小体积的样品重复分离。 图5示出了在SPO和LPO从一个典型的分离试验测得的气流文件。首先，为至少35微升导致缓冲器被加载，以确保稳定的流量之前样品达到声学芯片。样品体积小于100微升不推荐用于此系统的配置，因为样品稀释由于领先缓冲器变得过量。空气的插头使用在日的开始Ë注射龙头缓冲区之前向样品塞从以下，防止混合和稀释试样和作为一个指标的流量传感器的流体中分离出来。后的初始瞬变流体开始通过系统移动，在两个出口尖峰信号指示第一气泡的通过。这些瞬态之后是稳定的流动的一个长周期作为样品流过系统，那么当第二气泡穿过另一个尖峰，最后最终降低流速为零注射器泵停止之后。 通过流量传感器的空气塞的通道被用作触发点切换阀来启动和停止样品采集，从而由非样本流体体积最小丢失的样品和稀释。处理的样品体积的闭环计量无需重新编程这些值输入样值被改变每次实验开始之前。此功能尤其重要的，当样品量是有限的，例如在许多临床样品的情况下。实时流量监测还有助于排除故障;差的运行（例如，在出口中的一个的堵塞形成）是从所得到的气流文件立即明显的， 如在图5b中。 为了演示的灵活性和acoustofluidic分离的使用提出了系统架构的有效性，纯化登革病毒及纪源病毒的股票是通过微流控芯片掺入细胞储存和处理分开。 图7a显示，Raji细胞以及分离病毒，为97 ％Raji细胞离开所述芯片被发现是在过氧化脂质，从而留下DENV在SPO高度富集的样品。相比较而言，DENV分离的效率较低，与DENV的70％退出在SPO找到的芯片。这可以归因于轻微对流混合引起的separati的匝上信道，但更可能在一定DENV颗粒连同Raji细胞进入LPO迁移。细胞在整个流线横向迁移拖动一些流体与它们，即使在低雷诺数。通过这个机制，以及非特异性表面吸附，病毒颗粒转移到LPO。尽管如此，DENV在SPO的高度富集的样品是显著益处，例如当从头测序被用于检测和鉴定病毒。 图7b显示，在一次试验运行中，只有约70％的Boa细胞退出芯片中发现了过氧化脂质，有近100％的分离效率Raji细胞进行比较。相比，Raji细胞，因此导致更小的声学力，在两种细胞类型之间的分离性能上的差异可以归因于一个更小的平均尺寸或博阿细胞的密度较低。为了证实或驳斥这些推测，宝儿的大小，密度和形态细胞悬浮宝儿细胞（其正常生长贴壁）必须被准确测量，努力作进一步调查。在相同的实验中，类似于与DENV实验中，大部分的回收GGV从SPO退出，指示病毒级分的富集。 所提出的数据突出的工程技术广泛适用的平台所固有的挑战，处理各种生物样品。重要的是，生物相互作用可以开始玩同样大的作用，在物理和机械效应。然而，这些初步的实验也证明了权力和使用该系统的架构来样加工的临床和研究应用的承诺。作为一个强大的，良好的特点设计的系统，这个平台提供了寻求答案，新的科学问题的能力。

样品分离和分级分离是应用微流体技术的最有前途的领域之一。这样的样品处理步骤是不可分割的有效的临床诊断，治疗的发展，生物监测工作，并在生命科学研究和技术进步。一个无数的微流体分离策略已经被证明为流体悬浮颗粒和胶体，以及化学和生物物种;一些评论提供的最新研究进展和发展概述在这些领域1 - 9。虽然许多这些微流体分离技术（以下简称为"芯设备"）已经被广泛表征，少数报告已经考虑在系统级样品分离问题。核心设备是典型的个人厘米尺度芯片，接口到含氟聚合物管，流体通过一位移或压力泵递送。然而，如果微流体的承诺 - 包括增加自动化，可靠性和减少样品体积 - 是成为现实，至少相当于必须努力致力于一个完全分离的系统到其中的核心设备是集成设计。 此外，对于微流体方法生物检测的一个主要挑战是在宏观到微观界面。这不仅是指一个微流体装置，宏观组​​件的物理"世界到芯片"连接，并以典型的临床或分析样品体积（〜0.1-10毫升）和微流控芯片的内部容积之间的失配（〜 0.01-10微升），而且还从桥接这些大小尺度所产生的统计抽样限制。这些问题有助于人们认为样品前处理和制剂是生物检测中的"薄弱环节"。10在这项工作た描述的平台对解决这些挑战KES主要步骤。 服用一个系统级视图，该协议细节精确计量的分析规模的体积（范围从0.15至1.5毫升）于〜10分钟的时间尺度的可靠的处理。这是一个"一键"操作:一旦包含样本和目的地小瓶馏分收集源小瓶放置到系统中，"运行"命令启动的程序，并且所有步骤都是计算机控制的。在一个运行结束时，收集瓶可以从系统的分离级分的下游分析去除。 在这个系统中的核心设备是一个acoustophoresis芯片，其提取哺乳动物细胞大小（5-20​​微米）的粒子从样品。 Acoustophoretic分离这里选择主要是因为它是高通量（高达μl/ min的100秒），无标记，和非接触，从而提供在分离存活维鲁优点SES从细胞中，很少有其他微流控技术无法比拟的。声粒子的物理学聚焦已被广泛描述，11 - 13，不是本协议的重点，但基本概念的简要概述如下理解应用到微流体分离，以帮助。 超声波的驻波谐振在流体填充微通道产生压力场，这引起力量驱动颗粒朝向低压的节点。的力的大小取决于粒子的体积上，并从相对密度和颗粒的压缩性衍生的声学造影因子和悬浮流体。14同样地，声学聚焦非常适合的细胞大小（〜7-15分离微米）的病毒大小（〜50〜200纳米）的粒子。较大的颗粒迁移向压力节点;然而，由于力量级是非常小的颗粒小于2-3微米，这些小颗粒或溶解的物质几乎不移动。我们的特异性声学分离实施，如前所述，15包括一个薄壁细分流体通道，并允许可调谐，聚焦位置的非对称安置。这增加了灵活性在设备的设计，和性能优势，包括增加的分离质量和速度，都充分别处描述。16,17 然而，在这项工作中所描述的系统级设计方法的一大优点是，它是适用于种类繁多的微流体核心设备。例如，大多数其它连续流分离模式，包括惯性，流场分馏，确定性横向位移（DLD），和各种类型的电动装置的可容易地并入，以适当调整的要算为改变入口/出口结构，流速和样本量。与各种类型的片场（电场，磁场）或梯度（热，化学）设备可能需要到芯片，或额外的硬件集成，该平台可容纳该附加的连接。 这个协议提供了设计的微流体分离装置，并通过深反应离子蚀刻来制造含硅玻璃芯片（DRIE，等离子蚀刻工艺，可以在许多微制造设施，它使用交流蚀刻和钝化的循环以获得深所需的步骤功能与垂直侧壁18）。接着，我们描述了acoustofluidic设备的表征，以确定分离的最佳运行参数，最后细节的完全集成的分离系统和用于处理生物样品的过程。典型的器件特性分析结果和样本处理数据，然后介绍和讨论，这Appro公司的主要优势ACH被突出显示，包括模块化，鲁棒性，精度和自动化。

<table><tbody><tr><td colspan="4">Materials required for Steps 1-3: Device Design, Fabrication and Assembly</td></tr><tr><td>Double Sided Polished Silicon Wafer</td><td>Silicon Quest International, Inc. San Jose, CA, USA</td><td>100 mm &amp;lt;100&gt;&amp;nbsp; prime wafer</td><td>1-20 ohm-cm, 495 &amp;#177; 25&amp;#160;&amp;micro;m Double-side polished</td></tr><tr><td>Glass Wafer</td><td>Bullen Ultrasonics, Eaton, OH, USA</td><td>100 mmx0.5 mm Boro</td><td></td></tr><tr><td>Photoresist, AZ 1518</td><td>MicroChemicals GmbH, Ulm, Germany</td><td>AZ 1518</td><td>Photoresist used to adhere wafer to blank wafer for DRIE etching</td></tr><tr><td>Photoresist, AZ 4620</td><td>MicroChemicals GmbH, Ulm, Germany</td><td>AZ 4620</td><td>Photoresist to define fluidic and via mask patterns</td></tr><tr><td>DRIE plasma etcher</td><td>STPS, Newport, NP, United Kingdom</td><td>Multiplex Advance Oxide Etch (AOE) ICP system</td><td></td></tr><tr><td>Wafer Bonder</td><td>Electronic Visions Group,&amp;nbsp; St.Florian am Inn, Austria</td><td>EVG 501</td><td></td></tr><tr><td>Dicing saw</td><td>Kulicke &amp;amp; Soffa Industries, Singapore</td><td>K&amp;amp;S 982</td><td></td></tr><tr><td>Epoxy kit</td><td>Epoxy Technology, Billerica, MA, USA</td><td>EPO-TEK 301</td><td>Epoxy used to couple piezo and microfluidic chip</td></tr><tr><td>PZT piezoceramic&amp;nbsp;</td><td>Piezo Systems, Woburn, MA, USA</td><td>PSI-5A4E</td><td>37.5 &amp;times; 10 &amp;times; 0.5 mm</td></tr><tr><td>28 AWG Kynar-insulated solid wire</td><td>Squires Electronics, Cornelius, OR, USA</td><td>UL1422</td><td></td></tr><tr><td>2-part silver epoxy&amp;nbsp;</td><td>MG Chemicals, Surrey, BC, Canada</td><td>8331</td><td>Conductive adhesive for attaching wire leads to PZT</td></tr><tr><td>L-edit</td><td>Tanner EDA, Monrovia, CA, USA</td><td>Ledit v15.1 64-bit</td><td>CAD software for mask layout</td></tr><tr><td colspan="4">Materials required for Step 4: Characterizaiton of Acoustic Focusing Performance</td></tr><tr><td>Dual Pump</td><td>Harvard Apparatus, Holliston, MA, USA</td><td>PHD ULTRA Series, 703007</td><td></td></tr><tr><td>5&amp;#160;ml syringes</td><td>Becton-Dickinson, Franklin Lakes, NJ, USA</td><td>309646</td><td></td></tr><tr><td>Luer to Threaded port adapter</td><td>IDEX, Oak Harbor, WA, USA</td><td>P-659</td><td>Connects syringe to tubing</td></tr><tr><td>Union</td><td>IDEX, Oak Harbor, WA, USA</td><td>P-623</td><td>Connects world to chip connections to fluoropolymer tubing.&amp;nbsp; Can also use webbed&amp;nbsp;</td></tr><tr><td>Ferrule 1/4-28 flat bottom</td><td>IDEX, Oak Harbor, WA, USA</td><td>P-200</td><td>Used with nut to make connections between tubing and syringe, flow sensors and world to chip hardware</td></tr><tr><td>Nut&amp;nbsp; 1/4-28 flat bottom</td><td>IDEX, Oak Harbor, WA, USA</td><td>P-202</td><td>Used with ferrule to make connections between tubing and syringe, flow sesnsors and world to chip hardware</td></tr><tr><td>1/16&quot; OD Fluoropolymer tubing</td><td>IDEX, Oak Harbor, WA, USA</td><td>1912L</td><td>Tubing to connect syringe pumps to world to chip connections.&amp;nbsp; Tubing size is not critical during claibration steps (.01-.03&quot; ID typically used, other suitable part numbers: 1907L, 1902L).</td></tr><tr><td>Small ID fluoropolymer tubing</td><td>IDEX, Oak Harbor, WA, USA</td><td>1476-20</td><td>Used for Flow restrictors: 0.006&quot; ID, 1/16&quot; OD FEP&amp;nbsp;</td></tr><tr><td>PEEK tubing</td><td></td><td></td><td>Connects from chip to fluoropolymer tubing.</td></tr><tr><td>Cooling fan</td><td>Multicomp, Leeds, England</td><td>MC19663</td><td></td></tr><tr><td>Function Generator</td><td>Agilent, Santa Clara, CA, USA</td><td>33220A</td><td></td></tr><tr><td>RF amplifier</td><td>ENI, Rochester, NY</td><td>325 LA</td><td>Must be able to amplify signals from &amp;lt;1&amp;#160;V in the range of 1-2&amp;#160;MHz to 25&amp;#160;V&lt;sub&gt;pp&lt;/sub&gt; to the piezo.</td></tr><tr><td>CCD Camera</td><td>Photometrics, Tucscon, AZ, USA</td><td>CoolSnap HQ</td><td></td></tr><tr><td>Inverted Microscope</td><td>Zeiss, Oberkochen, Germany</td><td>Axiovert S100</td><td></td></tr><tr><td>FITC filter set</td><td>Chroma Tech, VT, USA</td><td>SP101</td><td></td></tr><tr><td>Objective, 10X</td><td>Zeiss, Oberkochen, Germany</td><td>ACHROPLAN</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronix, Beaverton, OR, USA</td><td>TDS3014B</td><td>To monitor voltage output by RF amplifier</td></tr><tr><td>MatLab</td><td>Mathworks, Natick, MA, USA</td><td>R2014a</td><td></td></tr><tr><td>Driver interface software to integrate Photometrics camera with LabVIEW</td><td>R Cubed Software, Lawrenceville, NJ, USA</td><td>SITK</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich, St. Lousi, MO, USA</td><td>P9416</td><td></td></tr><tr><td>Dragon Green Fluorescent 5.76 or 7.32&amp;#160;&amp;micro;m Beads</td><td>Bangs Laboratory, IN, USA</td><td>FS06F</td><td></td></tr><tr><td colspan="4">Additional materials required for Step 5: Automated Separation</td></tr><tr><td>Multiport valves</td><td>VICI, Houston, TX, USA</td><td>C25Z-3180EUHA</td><td>In the current configuration 4 valves are needed</td></tr><tr><td>Flow Sensors</td><td>Sensirion, Westlake Village, CA, USA</td><td>SLI-1000</td><td></td></tr><tr><td>Fluoropolymer tubing, .01 and .03&quot; ID</td><td>IDEX, Oak Harbor, WA, USA</td><td>1902L and 1912L</td><td>High purity PFA preferred</td></tr><tr><td>Nut</td><td>VICI, Houston, TX, USA</td><td>ZN1PK-10</td><td>Used with ferrule to make connections between tubing and valves. Alternative part numbers: MZN1PK-10, LZN1PK-10</td></tr><tr><td>Ferrule</td><td>VICI, Houston, TX, USA</td><td>ZGF1PK-10</td><td>Used with nut to make connections between tubing and valves.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>LabVIEW</td><td>National Instruments, Austin, TX, USA</td><td>LabVIEW Professional Development system</td><td>Laboratory Automation Software</td></tr><tr><td>PBS</td><td>Teknova, Hollister, CA, USA</td><td>P0200</td><td></td></tr><tr><td>Raji Cells</td><td>ATTC, Manassas, VA, USA</td><td>CCL86</td><td></td></tr><tr><td>Boa Cells</td><td></td><td></td><td>Kindly provided by the DeRisi Laboratory at UCSF</td></tr><tr><td>GGV</td><td></td><td></td><td>Kindly provided by the DeRisi Laboratory at UCSF</td></tr><tr><td>DENV</td><td></td><td></td><td>Kindly provided by Jose Pena at LLNL</td></tr><tr><td>Coulter Counter Z2</td><td>Beckman Coulter, Brea, CA, USA</td><td>Z2</td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific, Waltman, MA, USA</td><td>0267151B</td><td></td></tr></tbody></table>

a microfluidic platform for precision small-volume sample processing and its use to size separate biological particles with an acoustic microdevice

微流体装置的一个主要优点是操纵小样品体积，从而减少了浪费试剂和保护珍贵样品的能力。然而，为了实现鲁棒的样品操作，有必要解决设备集成与宏观环境。为了实现可重复的，敏感的颗粒分离与微流体装置，该协议提供了一个完整的自动化和集成的微流控平台，使采用微流体装置0.15-1.5毫升样品精密加工。该系统的重要方面包括模块化设备的布局，并导致可靠和灵活的全球芯片连接强大的固定装置，并完成闭环样品采集，系统清理和涂刷步骤，以确保可重复的操作完全自动化液体处理。不同的微流体装置可以互换使用这种架构。在这里，我们将一个acoustofluidic设备，详细阐述其characteriz通货膨胀，性能优化，并证明其用于大小分离的生物样品。通过在分离实验使用的实时反馈，样品采集进行了优化，节约和浓缩样品。尽管要求的多台设备的集成，这种结构的优点包括处理未知样品，没有额外的系统优化，便于装置的更换，和精确，鲁棒样加工的能力。

该声微流体装置设计的主要特性在图1中被突出显示，并描述充分别处。15简言之，两种流体流侧由端在分离信道，由薄的硅壁从并行旁通通道分离。由于初级辐射力的大小可随着颗粒的体积，大颗粒迁移出来朝向位于相邻的回收流体流中的声压节点混合样品输入流，同时小颗粒停留在原来的流（图1B 中的C）。该细分双通道架构提高颗粒分离17，并允许调整节点的位置使用不同的旁路通道的流体。16流体线路板及固定装置提供了一个强大的平台，为世界芯片的连接，和模块化设计使流体芯片之间的快速变化（图2）。这个Configuration同样允许可逆流体连接，这密封高达1,000 psi的，也可以快速和可靠地（图2B 中的E）制成。 一个芯片的谐振频率 f RES可使用简单的一维分析计算来估计（参见步骤1.1.1）。从我们的设备的横截面，16的更完整的2D有限元模型为2节点谐振预期聚焦位置为225微米的壁和预期的频率为1.68兆赫。然而，实际的设备˚F 清晰度可以通过±100千赫变化，这取决于操作温度及纵向和横向的共振耦合。因此，装置组件以后中 ，f 水库必须凭经验证实在相 ​​关流速和压电驱动电压，在协议中的步骤4中所述。 图3显示了以200μl/分钟取代表频率扫描，<html>与水的主要和旁通通道，和20 V pp的供给到压电。当压电和微流体装置之间耦合良好，颗粒将紧紧聚焦在共振频率，从而在荧光强度和迁移到预期的聚焦位置（图3A）一个明确的峰。与此相反，当存在差耦合，颗粒将不会聚焦良好，频率扫描的结果将是类似于图3B。在这种装置中，压电换能器可能需要被重新安装，如果可逆粘合剂已被使用;否则，这种装置不适合时高质量聚焦或快流是用于需要的应用程序的关键。 图 3中的数据通知操作频率后续实验的选择，且电压和流可用于高效颗粒分离率范围。 文件/ ftp_upload / 53051 / 53051fig5.jpg"/&gt; 图5.自动化来样加工。样本中样品盘装载（ 一）示意图 。 （B）的一个成功的分离实验的代表性的流动剖面。从运行间隔期间，SPO出口堵塞在约220秒（ 三）流动剖面。 <a href="https://www.jove.com/files/ftp_upload/53051/53051fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 识别芯片，是有效的分离器之后，它们被结合在图4中所描绘的系统。系统总容积为最小通过使用管道用0.01"沿主流动通道的内径（蓝线）。 图5A示出的化妆通过系统输注的典型样品塞。需要少量的"龙头缓冲区"（〜35微升），以prece德样品中的流动，以消除流量波动，而样品通过分离芯片移动。 图5B示出了从一个成功的自动声学分离试验所得的数据流。一个成功运行的主要特点包括:（1）在流量都LPO和SPO瞬加压力建立和流体开始流过系统，（2）尖峰表示气泡的通道（中插图示出了单个气泡），它应达到LPO前SPO的放大剖面由于来自两个出口的不平等流，（3）通过两个气泡作为样本的移动之间两个出口通过系统稳定的流动，和（4）逐渐减少流量在两个出口的全部样品体积之后被输送到系统中。有问题的运行是从流动曲线类似于图5C，它出现了SPO 220余秒后成为阻塞立即显现出来。在THI<html>案，一个类似于步骤5.3中描述的清洁程序应运行以疏通通道。 <img alt="图6" src="/files/ftp_upload/53051/53051fig6.jpg" /> 图6.设备可调性:粒度和电压的影响 。在过氧化脂质萃取聚苯乙烯微球的百分数取决于颗粒尺寸和电压供给到压电陶瓷。每一行对应于一个不同的工作电压在共振频率，通过该装置在步骤4中的总流量描述确定为200微升/分钟，用1.62和1.64兆赫之间施加的驱动频率。误差棒代表至少三个独立实验的标准偏差。转载和http://pubs.rsc.org / EN /内容/ ArticleLanding / 2014 / AN / c4an00034j修改的化学皇家学会的许可。目标="_空白"&gt;点击此处查看该图的放大版本。 图6示出了编译使用各种聚苯乙烯颗粒尺寸和压电驱动电压，演示所需有效分离的工作参数分离结果。在一般情况下，更高的电压（即，大于声学力）所需的提取更小的颗粒，如预期。驱动电压不能无限期但是增加，由于更大的散热及声流的增加的效果。13情节用作该显示显著不同的恢复在特定的驱动电压（例如，10-粒子间分离粒径一般的指导和2微米粒子在8.8 V峰峰值）将分开好。通常，颗粒种群具有大尺寸差，如病毒（〜100nm）的和细胞（〜10微米）能迅速且有效地分离，因为可以差异的细胞erent尺寸（例如，6-8微米盘状红细胞和8-15微米白细胞）。与其他细胞类型的工作所需的特定条件必须根据经验确定，如细胞形状，密度和可压缩性影响其声学造影，除了细胞大小。为此，在步骤4中的程序是用于确定可用分离条件为任何新的小区或颗粒型是有用的，而不仅用于评估特定acoustophoresis设备的质量。 <img alt="图7" src="/files/ftp_upload/53051/53051fig7.jpg" /> 细胞病毒加标样品图7的分离效率的影响。从（A）的Raji细胞掺入登革热病毒（DENV） 和 （B）Raji细胞和博阿肾细胞掺有金门病毒（纪源）分离的结果。收集的百分比被定义为病毒或细胞来自特定排出馏分出口相比总量离开芯片。误差线（A）的3个试验的标准差，而在（B）中的样品只进行了由于低量获得处理一次。的1×PBS用作样品缓冲液和回收液，用水在所有实验的旁通流路。芯片的工作频率1.60和1.64兆赫之间变化，在16〜20 V pp的驱动电压。 <a href="https://www.jove.com/files/ftp_upload/53051/53051fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 为了证明这一点平台生物粒子分离的实用性，我们首先用它来 ​​处理充分表征生物样品:人类Raji细胞（10 5个细胞/ ml，平均粒径8-10微米25）掺有登革热病毒（DENV，10 5 PFU /毫升，直径约50纳米的26），图7A显示Raji细胞和DENV收集在两个SPO和LPO的百分比（定义为从与从芯片收集各颗粒的总量比较每个出口收集的每种颗粒类型的级分）。该实验重复三次，并用Coulter计数器Raji细胞进行定量，而DENV是使用逆转录定量聚合酶链反应（RT-qPCR的）定量。 接着，该系统的性能在一个场景，是更现实的，用于处理的临床样品，在其中颗粒的确切物理特性可以是未知的进行了测试，并可用样品量较低。在这里，我们评估了标题最近金门病毒（纪源），蛇病原体感染蟒蛇肾细胞27的纪源病毒颗粒的尺寸分离尚未测定，但由于纪源属于沙粒病毒科 ，它很可能的相似的大小，在100至150纳米。28我们处理的样本与纪源尖刺（10 4 PFU / ml）的插入的Raji人细胞（未感染对象为病毒），和蟒肾细胞（感染目标纪源，近似尺寸10微米27），具有在10 4个细胞/ ml的两种类型的细胞。由于这些样品在低量可用，仅来自一个实验运行的分离结果列于这项工作。 图7B显示 Raji细胞，博阿细胞的百分比，和纪源从SPO和LPO收集。细胞进行定量在这些实验中通过计数上血细胞计数器和病毒用RT-qPCR定量。

Watch this Scientific Journal Video about A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice at JoVE.co

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

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-microfluidic-platform-for-precision-small-volume-sample-processing

Research

JoVE Journal

Bioengineering

13.7K 次观看.  Lawrence Livermore National Laboratory. This protocol describes a system architecture for performing automated small volume (0.15–1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

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

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

制造，操作和表面声波驱动声逆流微流体流动可视化

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

科研

工程学

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 ...

生物工程

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

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

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 ...

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

制造和Acoustofluidic设备的运营支撑体声波驻波的聚焦鞘颗粒

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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 ...

Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip (LOC) system. The simultaneous excitation of the fundamental frequency and its third harmonic, which is termed as dual-frequency excitation, to a pair of interdigital transducers (IDTs) could generate a new type of standing acoustic waves in a microfluidic channel. Varying the power and the phase in the dual-frequency excitation signals results in a reconfigurable field of the acoustic radiation force applied to the microparticles across the microchannel (e.g., the number and location of the pressure nodes and the microparticle concentrations at the corresponding pressure nodes). This article demonstrates that the motion time of the microparticle to only one pressure node can be reduced ~2-fold at the power ratio of the fundamental frequency greater than ~90%. In contrast, there are three pressure nodes in the microchannel if less than this threshold. Furthermore, adjusting the initial phase between the fundamental frequency and the third harmonic results in different motion rates of the three SSAW pressure nodes, as well as in the percentage of microparticles at each pressure node in the microchannel. There is a good agreement between the experimental observation and the numerical predictions. This novel excitation method can easily and non-invasively integrate into the LOC system, with a wide tenability and only a few changes to the experimental set-up.

A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

双频激励下驻面声波的微粒操纵

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip ...

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

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

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

高通量单细胞的分离和文化的微流体平台

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

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. This platform has a three-dimensional (3D) network with curved fluid channels and three pneumatic valves, which create networks, channels, and spaces through duplex replication with polydimethylsiloxane (PDMS). The device operates based on the transient response of a fluid flow rate controlled by a pneumatic valve in the following order: (1) sample loading, (2) sample blocking, (3) sample concentration, and (4) sample release. The particles are blocked by thin diaphragm layer deformation of the sieve valve (Vs) plate and accumulate in the curved microfluidic channel. The working fluid is discharged by the actuation of two on/off valves. As a result of the operation, all particles of various magnifications were successfully intercepted and disengaged. When this technology is applied, the operating pressure, the time required for concentration, and the concentration rate may vary depending on the device dimensions and particle size magnification.

The present protocol describes a pneumatic microfluidic platform that can be used for efficient microparticle concentration.

气动驱动的微流体平台，用于微颗粒浓缩

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.