Name: fabrication and operation of acoustofluidic devices supporting bulk acoustic standing waves for sheathless focusing of particles
Uploaded: 2016-03-06T14:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles at JoVE.com

This work was supported by the National Science Foundation (through grants DMR-1121107, CMMI-1363483 and Graduate Research Fellowships (GRF-1106401) to C.W.S., D.F.C. and K.A.O.) and the National Institutes of Health (R21GM111584). The authors have no conflicts of interest.

1.光刻<ol><li>使用合适的软件包装设计的光罩，并提交设计为一个合格的光掩模打印机。21 </li><li>在无尘室设施，冲洗6"单面抛光硅片用丙酮源源不断（≥99.5％; 见表1），然后通过甲醇源源不断（99.8％， 见表1）。干燥晶片通过用N 2气喷涂和放置在晶片上的热板上在95℃下2分钟。 注:该晶片的掺杂分布和晶体取向不会影响下面的过程。 </li><li>通过用铝箔的片材覆盖保护旋涂机（以标准旋涂罩）的外槽及置于干净的Si晶片上的真空吸盘，在旋涂机的中心，以确保晶片。 </li><li>通过仔细倾倒，直到光致抗蚀剂覆盖了大部分沉积正性光刻胶直接在晶片的中心的晶片。请注意，以确保没有光致抗蚀剂无气泡。 注:在步骤确切程序1.5-1.10对应于表1所示的光致抗蚀剂;可能需要不同的光致抗蚀剂不同的程序。 </li><li>通过执行以下程序启动旋转循环: <ol><li>编程300rpm的速度，100转/秒的升温，和5秒的旋转时间开始旋转循环。 </li><li>程序1800转，1000转/秒的升温，和60秒的旋转时间，以均匀分布的光致抗蚀剂的速度。 </li><li>方案至0 rpm，1000转/秒的升温，以及0秒的旋转时间的速度结束旋转循环。 </li></ol></li><li>松开夹头的真空，并使用晶片镊子检索从卡盘的晶片。然后将晶片放置到热板上在110℃烘烤165秒。 注意:此步骤被称为"软烘焙"。 </li><li>加载掩模成掩模的夹持器对齐/光刻机。 </li><li>编辑光刻机的参数，以提供1400毫焦/厘米2（ 例如 ，对于13.5毫瓦/厘米2的输出强度，使用~103.7秒的曝光时间）的能量剂量。 </li><li>从支架上取下photopatterned晶片并将其放置在其相应的显影剂中的溶液（ 见表1）中5分钟。 </li><li>从开发商取出晶圆，以去离子水2 O源源不断清洗硅片，用氮气吹干。 注:过显影可能会导致图案膨胀，而下显影可能会导致不完全除去沿光图案化特征的光致抗蚀剂。 </li><li>检查在显微镜下晶片，确认印刷在光掩模被转移到光致抗蚀剂的图案。 </li></ol> 2.深层反应离子蚀刻<ol><li>加载光图案的硅晶片陷入了深深的室反应离子蚀刻仪器和蚀刻的流体通道到Si晶片到所需的深度以下标准的蚀刻过程。22 </li><li>小心卸载从室中的样本的蚀刻过程完成之后。 </li><li>从晶片除去过量光致抗蚀剂，准备一个大烧杯中与光致抗蚀剂去除剂（ 见表1）的专用于溶剂使用，并将其放置在热板上于65℃的通风良好的罩的溶液。 </li><li>浸没在光致抗蚀剂除去用溶液的晶片，并让它浸泡1小时。 注意:不同的解决方案可用于移除光致抗蚀剂（ 例如 ，丙酮中的溶液（≥99.5％） 见表1可以通过浸泡过夜除去光致抗蚀剂）。 </li><li>从烧杯中取出晶片，并用丙酮的交替流冲洗（≥99.5％; 见表1）和异丙醇（≥99.7％; 见表1）。干燥晶片W第i N 2气。 </li></ol> 3.清洁食人​​鱼<ol><li>在通风良好的罩（专用于使用的氨基酸），通过加入H 2 O 2制备食人鱼溶液（在水30.0重量％; 见表1）至H 2 SO 4（95.0-98.0％; 见表1）以1:3的比例在一个大的，干净的烧杯中。 注意:食人鱼的解决方案具有高度腐蚀性，是一种强氧化剂，而且非常危险。采取极端谨慎处理食人鱼解决方案，并佩戴合适的安全设备。 </li><li>淹没的蚀刻特性的离子蚀刻晶片面朝上和离开5分钟。小心取出晶片，并用去离子H 2 O.充分漂洗</li><li>再浸没在2分钟的食人鱼溶液的晶片。小心取出晶圆，并用大量的去离子H 2 O.充分漂洗</li><li>在专门为溶剂使用单独的通风罩，清洗晶圆与源源不断丙酮（≥99.5％; 见表1），随后加入甲醇（99.8％; 见表1）的稳定流，并干燥以N 2气体的晶片。按照相应的安全程序处置食人鱼的解决方案。 </li></ol> 4.准备硼硅玻璃盖<ol><li>使用划线工具，蚀刻直线入硼硅酸盐玻璃创建矩形段（ 例如 ，8×4 平方厘米）。仔细捕捉玻璃收回矩形段。 </li><li>把这些玻璃的细分市场之一，并将其​​放置在所需设计（实际尺寸）的打印副本的顶部，与黑色记号笔在玻璃纪念入口和出口的位置。 </li><li>钻入口和出口孔进入硼硅玻璃。 注意:正确的安全设备，在任何时候都可以穿。 <ol><li>修正了一个1/8"钻头进入钻床的嘴。把长方形的玻璃段之上的Al板钻孔，以便在玻璃上的标记是在Al板中的孔上方。固定在Al板用胶带的玻璃。 </li><li>小心地将饲料手柄开始小孔钻进玻璃和继续下去，直到孔穿过玻璃制成，以降低手柄。一旦孔完成后，取出磁带并慢慢抬起玻璃以除去玻璃粉末。放置玻璃粉末与水的烧杯中，并使用适当的安全程序丢弃。 </li><li>仔细擦干用非皮棉生产吸水布，玻璃，并按照同样的程序（步骤4.3.1-4.3.2）钻其他的入口和出口孔。 </li></ol></li><li>按照同样的步骤（第3节以上）清洁与食人鱼的解决方案矩形玻璃段。 注意:食人鱼的解决方案具有高度腐蚀性，是一种强氧化剂，而且非常危险。采取极端谨慎处理食人鱼解决方案，并佩戴合适的安全设备。 </li></ol> 5.阳极连接<ol><li>用划线工具，蚀刻直线到Si晶片围绕微流体芯片的周边，使得它比矩形玻璃段略小（ 例如 ，7×3厘米2）。小心管理单元沿着蚀刻线的晶片。 </li><li>冲洗硅段与丙酮源源不断（≥99.5％; 见表1），随后加入甲醇源源不断（99.8％; 见表1）。放置在热板上的晶片在95℃下进行2分钟干燥。 </li><li>随着硅段腐蚀的特性朝上，仔细加上SI段顶部的干净的玻璃，并确保孔正确对齐。 </li><li>仔细翻转段，同时确保了孔保持对准。由于玻璃段比所述Si段时，固定两个片段用双面胶带，其中胶带的一半固定在Si段的垂直边缘，而另一半带的固定悬垂玻璃。然后翻转段再次使得玻璃段是在上面，并放置在金属板的顶段的热板上。 </li><li>仔细的足够重的重量（ 即 ，至少5千克）的第二金属板（ 例如 ，钢）直接添加到组装的玻璃和Si段的顶部。 注意:此金属板不应该在与Si段或导电带接触。 </li><li>使用高压电源，一个引线（功率）连接到金属板上的装配玻璃和Si段和其它引线（地）的底部金属板的顶部。 </li><li>打开底层热板上的电压至1,000 V.检查通过使用万用表施加电压;按一次探测靠在底板和针对顶板其他探针。 注意:高电压是非常危险的;要小心，不要触摸金属板或连接线。 </li><li>离开热板在450℃下2小时，以使玻璃"盖子"来阳极键合到Si衬底。 2小时后返回到关闭加热板，关闭直流电源，并从金属板中删除该设备。 警告:金属板将是中和接合工艺之后极热，所以允许材料在关闭热板后，以冷却至少1小时。 </li></ol> 6.最终确定Acoustofluidic设备<ol><li>刮玻璃表面用剃刀以去除由阳极接合产生污垢和清洁用丙酮玻璃的表面上。 </li><li>制备聚二甲基硅氧烷（PDMS）的片材大约5毫米厚，并切成几个小，正方形板坯约10×10mm的2（ 见表1）。23 </li><li>使用3毫米活检穿孔切割一个孔在每个PDMS板坯的中心，以便通过它插入硅氧烷管。直接放置在板顶部的孔S中的玻璃基板上和胶水板坯用环氧树脂。 注意:要小心，不要使用太多的胶水，因为它会堵塞设备的孔。 </li><li>仔细胶锆钛酸铅（PZT）换能器，以在器件的背面上的硅段，居中-下方的微通道。 </li><li>焊接两条导线，以在PZT换能器的两个导电区域。要小心，接线牢固地附着在传感器PZT。通过PDMS的砖孔插入硅胶管，并添加额外的胶水周围砖和管材，以确保自己的依恋。 </li></ol> 7.操作Acoustofluidic设备<ol><li>安全设备直接与微客观下方安装到显微镜的阶段。 注:确保PZT传感器不放置一个小插件的设备下使与台接触。 </li><li>使用标准化的连接器，从OUTL连接硅胶管该装置固定在注射泵注射器ETS。 注:此结构是用于"撤回模式";注射泵可替代地用于注入样品到器件中。 </li><li>放置硅胶管通向装置的入口在含有流体样品（ 例如 ，悬浮液的聚苯乙烯珠或细胞）的小瓶。 </li><li>上放置装有一个搅拌盘的流体样品连续混合样品，并确保颗粒或细胞的恒定浓度保持在整个实验过程中的小瓶中。 </li><li>从PZT传感器的电线连接到从功率放大器的输出串联一个函数发生器。 地编程（ 例如 ，峰-峰电压和频率）上的功能发生器的设置，监视来自使用示波器所述放大器的输出信号。打开函数发生器和功率放大器以开始致动PZT换能器。6 <ol><li>来估计该装置的谐振频率，按照公式C =λ*ƒ， 其中 c是介质（ 即水）的音速，λ是声波的波长，ƒ是PZT换能器的频率。在一个半波长的谐波（我们展示了代表结果部分）的情况下，微通道的宽度应驻波长度的一半。 </li><li> 0-50五的范围内使用的峰 - 峰电压设定 注:在较高的压力振幅所施加的电压的结果的增加，因此，更加快速acoustophoresis。 </li></ol></li><li>打开显微镜，并确保在微流体通道显然是焦点。 </li><li>打开注射器泵应用流动和引入样品到器件中。监视流过与荧光模式的显微镜设备的实体。 </li><li>确保设备高效地集中partic莱斯通过调节供给到PZT换能器以修改的压力振幅的峰 - 峰值电压和通过执行预期谐振频率附近的一个频率扫描，以确定经验谐振频率。 </li></ol>

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The authors declare that they have no competing financial interests.

Acoustophoresis提供了一种简单，快速的方法来准确地安排流体微通道内的微观实体而不流体聚焦的方法使用的需要鞘流体。24这些设备对粒子或细胞操纵其他方法提供一些优点（ 例如 ，磁泳，25,26介27或惯性迫使28），由于其处理实体没有高磁化率，电动极化或窄尺寸分散度能力。此外，声驻波的聚焦节点可以远离激发的来源，这是这是不可能通过静磁场或电场为每恩绍定理定位29一个额外的优点是，声学设备可以跨越聚焦粒子广泛独立的流动方向，这是不可能的装置塔的施加流速和的吨依靠惯性力用于聚焦，28提供有效地输送增强探伤颗粒或细胞的应用，如流式细胞术和颗粒大小的装置。30,31的难易程度器件制造和操作可直接允许的类似的实施聚焦，集中，分级和排序悬浮于液体物品的设备。32 我们已经表明，在初级辐射力，这是由声驻波所产生的最强的力，1可以集中流过在流速超过1000毫升/小时为一个单一的孔设计的微流体通道的微粒。为100微升/分钟的固定的流量，我们表明，我们的设备可以集中颗粒进入一个窄流（ 即 ，50微米跨越），而不在电压任何鞘流体低至20 V峰值-峰值，实现了低对于间歇聚焦1000万汉邦-Power方法克莱斯/分钟处理密集浓缩的溶液（ 例如 ，6×10 8粒子/ ml），作为一个例子时。此外，该可以通过可以显着提高制造被致动以高次谐波产生组平行的节点的多孔acoustofluidic芯片或通道。33 虽然在此仅示出的设备需要在传统的微细加工中使用的材料和方法，我们强调，也有其他的技术少数可用于构造类似的设备。19,34,35这种方法的优点包括它的简单性以及最终器件的耐久性。 对这些设备的制造中的关键步骤包括光刻法来定义微通道，反应离子蚀刻的几何形状，以形成在硅和阳极接合通道熔合硅为透明"盖"，用于fluorescen观测CE显微镜。所有的这些步骤需要的洁净室设备，以避免在设备内的灰尘或碎屑的收集。一旦这些步骤完成后，然而，粘接PZT传感器和流体端口都比较简单，可以在洁净室的外部执行。 然而，该装置的适当的治疗为它的寿命是必不可少的。这包括:（1）培养将设备与钝化试剂（ 例如 ，聚（乙二醇）硅烷）之前，每个实验以防止残渣积聚;（2）冲洗设备中的信道与每个实验后的洗涤剂。碎片的积聚可能损害声波驻波的保真度，并且可以减少对装置内有效地集中颗粒或细胞的能力。我们还注意到，这些设备并不适合含接近一半的驻波大小的实体高度多分散样品或样品。 AcoustofluiDIC设备提供用于各种从胶体组件跨越到细胞分离和流式细胞术应用极大效用。处理生物样品与精度在高流速的能力可以允许通过这些微流体装置增加吞吐量的能力，同时减少从多余的试剂，大体积样品或笨重的设备成本用于分配鞘流体。使acoustofluidic设备所需的制造方法是直接的，并为它们的操作所需的程序是用户友好。我们希望这些程序将鼓励类似设备的广泛发展以促进新的研究领域跨越材料科学，生物技术和医学应用。

Acoustofluidic装置用于施加微观实体它们的浓度，对准，装配，禁闭或静态流体或层状flowstreams内的分离。1在这一大类的设备（ 例如 ，颗粒或细胞）的定向力，可以从散装产生的力声学驻波，表面声波的驻波（SSAWs）2或声行波3虽然我们专注于制造和配套体声波的驻波，支持SSAWs装置设备的运行近来备受关注，由于其精确操纵细胞的能力沿面4和快速排序中连续流动通道的细胞。5器件支撑体声波的驻波，但是，根据由压电换能器生成的装置，它激励在微流体驻波的壁的机械振动重新安排粒子腔在几何定义的谐振频率。这使得电势为相比SSAW设备产生更高的声压振幅，和微观实体从而，更快acoustophoretic运输6 这些驻波由一空间周期性设定压力节和波腹，当压力在时间振荡被固定在适当位置的。颗粒驻波响应通过迁移到压力节点或波腹，这取决于相对于流体的颗粒的机械性能，并且这是由声学造影因子描述: <img alt="公式1" src="/files/ftp_upload/53861/53861equation1.jpg" />其中变量ρ和β代表密度和可压缩性以及下标p和ƒ代表悬挂物体（ 例如 ，颗粒或细胞）和流体，分别7实体是拥有一个积极的原音对比系数（ 即 ɸ&gt; 0）迁移到压力点（S）;然而，这具有负面原音对比系数实体（ 即 ɸ&lt;0）迁移到压力波腹。7虽然大多数合成材料（ 如聚苯乙烯珠）及细胞表现出积极的声音对比，从基于硅树脂制成的弹性粒子材料，8脂肪分子9或其它高弹性的成分表现出水中负声的对比。在acoustofluidic装置的弹性体颗粒可用于小分子隔离10和装置，以限制合成颗粒11或单元12用于判别分拣的用途。13 Acoustofluidic设备通常从具有足够的刚性苏标准的材料（ 如硅和玻璃）制造支持度声学驻波。在许多acoustofluidic设备（包括本文所示的装置）中，机械波被设计为在最低的谐波模式，它由一个半波长驻波横跨所述微通道宽度的谐振。这种结构具有在沿通道的周围的信道和压力波腹的中心的压力节点。先前已表明，这些系统可用于基于芯片的流式细胞仪的应用程序14-16和应用范围从细胞到细胞的浓度的捕捉。17,18 我们描述制造，使用和支持体声波驻波的acoustofluidic装置的代表表现能力的方法的过程。该装置需要一个光刻步骤，一是蚀刻工序和一个熔合步骤，以永久地粘合玻璃"盖"的蚀刻硅衬底。我们注意到，其他acoustofluidi支持体声波驻波C器件可从结合到压电换能器的玻璃或石英毛细管，该别处描述来制造。19,20硅基器件提供鲁棒性和控制流动通道的几何形状的优点，它们一起允许众多类型的处理的对含有颗粒和细胞悬液样品。是可重复使用的提供的器件中，它们在使用之间适当清洗（ 即 ，由冲洗用缓冲液和洗涤剂的装置）。

<table><tbody><tr><td>Silicon wafers</td><td>Addison Engineering, Inc.</td><td>3P1</td><td>6&amp;rdquo; mechanical grade silicon wafer &amp;lt;111&gt;</td></tr><tr><td>AZ 9260 photoresist</td><td>MicroChemicals GmbH</td><td>AZ9260-Q</td><td>Positive photoresist</td></tr><tr><td>AZ 400K developer</td><td>MicroChemicals GmbH</td><td>AZ400K CONC-CS</td><td>Dilute 1 part AZ 400k in 4 parts deionized H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich, Co.</td><td>216763</td><td>30 wt.% in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich, Co.</td><td>320501</td><td>ACS reagent, 95.0-98.0%</td></tr><tr><td>1165 Photoresist Remover</td><td>Dow Chemical, Co.</td><td>DEM-10018073</td><td>1-methyl-2-pyrrolidinone based</td></tr><tr><td>Acetone</td><td>Sigma Aldrich, Co.</td><td>320110</td><td>ACS reagent, &amp;ge;99.5%</td></tr><tr><td>Isopropyl alcohol</td><td>Sigma Aldrich, Co.</td><td>W292907</td><td>&amp;ge;99.7%, FCC, FG</td></tr><tr><td>Methanol</td><td>Sigma Aldrich, Co.</td><td>322415</td><td>Anhydrous, 99.8%</td></tr><tr><td>Borosilicate glass&amp;nbsp; (Nexterion glass B)</td><td>Schott AG&amp;nbsp;</td><td>2098576</td><td>&lt;p&gt;Size: 120 x60 &amp;plusmn;0.1 mm Thickness: 1 &amp;plusmn;0.005 mm&lt;/p&gt;</td></tr><tr><td>Drill bit for glass and ceramic&amp;nbsp;</td><td>McMaster-Carr, Inc.</td><td>2954A1</td><td>Drill bit size: 1/8&amp;rdquo; Overall length: 2 3/16&amp;rdquo; Shank diameter: 7/64&amp;rdquo;</td></tr><tr><td>Polydimethylsiloxane (PDMS) kit</td><td>Sigma Aldrich, Co.</td><td>761036</td><td>Dow Corning, Co.; Sylgard 184&amp;reg;; 10 g clip-pack</td></tr><tr><td>Biopsy punch&amp;nbsp;&amp;nbsp;</td><td>Ted Pella, Inc.&amp;nbsp;</td><td>15078</td><td>Harris uni-core Tip ID: 3.0 mm&amp;nbsp;Tip OD: 3.40 mm</td></tr><tr><td>Lead zirconate titanate (PZT) transducer</td><td>APC International, Ltd.</td><td>Custom order, (841 WFB)</td><td>Length: 30.0 mm, Width: 5.0 mm, Freq.: 2.46 MHz, 2.0 mm end wrap for leads</td></tr><tr><td>Silicone tubing&amp;nbsp;</td><td>Cole Parmer Instrument, Co.</td><td>07625-22</td><td>0.6 mm I.D.</td></tr><tr><td>Polystyrene beads</td><td>Thermo Fischer Scientific, Inc.</td><td>F-8836</td><td>10 &amp;micro;m yellow-green fluorescence</td></tr></tbody></table>

fabrication and operation of acoustofluidic devices supporting bulk acoustic standing waves for sheathless focusing of particles

Acoustophoresis是指悬浮物体的位移响应定向军队从声音能量。鉴于该悬浮对象必须比声音的入射波长和流体通道的宽度通常为几十到几百微米的对面，acoustofluidic装置通常使用由压电换能器在高频脉动产生的超声波（在兆赫范围）。在依赖于装置的几何形状的特征频率，就有可能诱发驻波可以批量流中集中了沿期望的流体流线的颗粒的形成。在这里，我们描述了从常用的材料和洁净室设备acoustophoretic器件的制造方法。我们显示粒子的聚焦与正或负声学造影因素，其中朝向驻波的压力节点或波腹移动代表性结果，respectiv伊利。这些器件提供固定在精确定位大量微观实体（ 例如 ，细胞）或应用范围从仪来装配流动的流体巨大的实用价值。

我们设计的acoustofluidic设备包含一个trifurcating入口，具有300微米的宽度和trifurcating出口（ 图1A - B）中的主信道。我们注意到，我们仅在这项研究中使用的一个入口，用于所有的实验（ 即 ，能实现无鞘通过声辐射力聚焦粒子）通过阻断其他入口与可移除的塞子。按照以上描述的方法，我们构建了芯片具有313微米的沟道宽度，具有~4％的误差由于在微细加工（ 图1C - D）的缺陷。我们操作的设备在2.366兆赫的驱动频率以诱导半波长谐驻波。 我们使用连接到功率放大器的信号发生器，以产生高频正弦波形致动PZT TRansducer。我们使用示波器来测量从功率放大器产生验证信号形状和幅度的保真度的峰-峰输出电压（V PP）。使用注射泵，我们首先以100微升/分钟的速率注射的悬浮液中的绿色荧光聚苯乙烯珠没有PZT换能器作为阴性对照（ 图2A）的致动。接下来，我们致动装置在2.366兆赫，以形成横跨所述微通道宽度的半波长驻波（V pp的= 40伏; 图2B）。我们发现，这些颗粒，其具有正的声学造影因子，沿压力节点集中如预期6我们也注入红色荧光颗粒与负声学造影因子（ 即 ，ɸ≈-0.88，从先前描述的方法合成） 8，以验证我们的器件可诱导它们的浓度沿压力波腹（ <stroNG&gt;图2C）。 最后，我们研究以积极的声学造影因子粒子聚焦在一个流量范围内（ 即 ，0至1000微升/分钟通过注射器泵调节）和电压（ 即 ，0至50 Vpp）为的程度。由15帧的视频采集每个条件。的ImageJ软件用于样品的荧光强度轮廓五个横跨所述微通道的宽度。数值计算程序被用来平均化强度分布为每个条件和平滑使用内联的滤波程序中的平均数据。正如预期的，颗粒的程度聚焦（ 即 ，由荧光峰的宽度所限定，对应于颗粒流的宽度）与流速增加（ 图3A）降低。我们还发现，粒子的程度聚焦随着施加的电压增加（ 连接古尔3B）。 <img alt="图1" src="/files/ftp_upload/53861/53861fig1.jpg" /> 图1:Acoustofluidic装置支撑体声波驻波包含稠合到硼硅酸盐玻璃"盖"的蚀刻硅衬底的装置的顶部（A）和底部（B）的示意图，聚二甲基硅氧烷（PDMS）的块连接到硅氧烷油管和焊接到粘在装置的底部电线的压电换能器。该装置的顶部（C）和底部（D）的照片也显示。 <a href="https://www.jove.com/files/ftp_upload/53861/53861fig1large.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/53861/53861fig2.jpg" /> 图2:声聚焦粒子具有正负声学造影因素:（A）在此之前的锆钛酸铅（PZT）换能器的致动，在100微升流动的正面声学造影因子（10微米，黄绿色的聚苯乙烯珠）颗粒/分钟占据的所述微通道的宽度。 （ 二 ）PZT传感器后启动（V PP = 40 V和ƒ= 2.366兆赫），在（A）的粒子显示沿着驻波的压力节点集中。 （C），沿在没有应用流量的驻波的压力波腹集中负声学造影因素粒子（V PP = 40 V和ƒ= 2.366兆赫）。 <a href="https://www.jove.com/files/ftp_upload/53861/53861fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <im摹ALT ="图3"SRC ="/文件/ ftp_upload / 53861 / 53861fig3.jpg"/&gt; 图3:的聚苯乙烯珠的荧光强度曲线（ 图2A所示- B） 一种acoustofluidic装置的聚焦性能示为（A）的各种流速（范围从0到1000微升/分钟）的恒定峰到的40 V峰值电压和（B）各种应用的电压（从0到50 Vpp）为100微升/分钟的恒定流量。 <a href="https://www.jove.com/files/ftp_upload/53861/53861fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles at JoVE.com

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

制造和Acoustofluidic设备的运营支撑体声波驻波的聚焦鞘颗粒

Acoustofluidic设备使用微流体通道内的超声波处理，集中隔离悬浮微纳米级的实体。这个协议描述了这样支撑体声波驻波集中在中央流线型颗粒无护套的流体的辅助设备的制造和操作。

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

fabrication-operation-acoustofluidic-devices-supporting-bulk-acoustic

Research

JoVE Journal

Engineering

12.8K 次观看.  Duke University.  Acoustofluidic设备使用微流体通道内的超声波处理，集中隔离悬浮微纳米级的实体。这个协议描述了这样支撑体声波驻波集中在中央流线型颗粒无护套的流体的辅助设备的制造和操作。 

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

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

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 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. ...

生物工程

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

荧光显微镜对聚焦超声的刺激

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

Fabrication of Nanoheight Channels Incorporating Surface Acoustic Wave Actuation via Lithium Niobate for Acoustic Nanofluidics

Controlled nanoscale manipulation of fluids is known to be exceptionally difficult due to the dominance of surface and viscous forces. Megahertz-order surface acoustic wave (SAW) devices generate tremendous acceleration on their surface, up to 108 m/s2, in turn responsible for many of the observed effects that have come to define acoustofluidics: acoustic streaming and acoustic radiation forces. These effects have been used for particle, cell, and fluid manipulation at the microscale, although more recently SAW has been used to produce similar phenomena at the nanoscale through an entirely different set of mechanisms. Controllable nanoscale fluid manipulation offers a broad range of opportunities in ultrafast fluid pumping and biomacromolecule dynamics useful for physical and biological applications. Here, we demonstrate nanoscale-height channel fabrication via room-temperature lithium niobate (LN) bonding integrated with a SAW device. We describe the entire experimental process including nano-height channel fabrication via dry etching, plasma-activated bonding on lithium niobate, the appropriate optical setup for subsequent imaging, and SAW actuation. We show representative results for fluid capillary filling and fluid draining in a nanoscale channel induced by SAW. This procedure offers a practical protocol for nanoscale channel fabrication and integration with SAW devices useful to build upon for future nanofluidics applications.

We demonstrate fabrication of nanoheight channels with the integration of surface acoustic wave actuation devices upon lithium niobate for acoustic nanofluidics via liftoff photolithography, nano-depth reactive ion etching, and room-temperature plasma surface-activated multilayer bonding of single-crystal lithium niobate, a process similarly useful for bonding lithium niobate to oxides.

通过用于声学纳米流体的尼奥布精锂结合表面声波驱动纳米高通道的制造

Controlled nanoscale manipulation of fluids is known to be exceptionally difficult due to the dominance of surface and viscous forces. Megahertz-order ...

Fabrication of Surface Acoustic Wave Devices on Lithium Niobate

Manipulation of fluids and particles by acoustic actuation at small scale is aiding the rapid growth of lab-on-a-chip applications. Megahertz-order surface acoustic wave (SAW) devices generate enormous accelerations on their surface, up to 108 m/s2, in turn responsible for many of the observed effects that have come to define acoustofluidics: acoustic streaming and acoustic radiation forces. These effects have been used for particle, cell, and fluid handling at the microscale&#8212;and even at the nanoscale. In this paper we explicitly demonstrate two major fabrication methods of SAW devices on lithium niobate: the details of lift-off and wet etching techniques are described step-by-step. Representative results for the electrode pattern deposited on the substrate as well as the performance of SAW generated on the surface are displayed in detail. Fabrication tricks and troubleshooting are covered as well. This procedure offers a practical protocol for high frequency SAW device fabrication and integration for future microfluidics applications.

Two fabrication techniques, lift-off and wet etching, are described in producing interdigital electrode transducers upon a piezoelectric substrate, lithium niobate, widely used to generate surface acoustic waves now finding broad utility in micro to nanoscale fluidics. The as-produced electrodes are shown to efficiently induce megahertz order Rayleigh surface acoustic waves.&#160;

锂氮化锂表面声波设备的制造

Manipulation of fluids and particles by acoustic actuation at small scale is aiding the rapid growth of lab-on-a-chip applications. Megahertz-order ...

Fabrication and Characterization of Thickness Mode Piezoelectric Devices for Atomization and Acoustofluidics

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid more efficiently, in terms of &#64258;ow rate per power input, than those that rely on Rayleigh waves and other modes of vibration in LN or lead zirconate titanate (PZT). The complete device is composed of a transducer, a transducer holder, and a &#64258;uid supply system. The fundamentals of acoustic liquid atomization are not well known, so techniques to characterize the devices and to study the phenomena are also described. Laser Doppler vibrometry (LDV) provides vibration information essential in comparing acoustic transducers and, in this case, indicates whether a device will perform well in thickness vibration. It can also be used to &#64257;nd the resonance frequency of the device, though this information is obtained more quickly via impedance analysis. Continuous &#64258;uid atomization, as an example application, requires careful &#64258;uid flow control, and we present such a method with high-speed imaging and droplet size distribution measurements via laser scattering.

Fabrication of piezoelectric thickness mode transducers via direct current sputtering of plate electrodes on lithium niobate is described. Additionally, reliable operation is achieved with a transducer holder and &#64258;uid supply system and characterization is demonstrated via impedance analysis, laser doppler vibrometry, high-speed imaging, and droplet size distribution using laser scattering.

用于原子化和抗波流体的厚度模式压电器件的制造和特性

We present a technique to fabricate simple thickness mode piezoelectric devices using lithium niobate (LN). Such devices have been shown to atomize liquid ...

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

声学悬浮系统中非球形气泡振荡诱导微流

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid samples, such as chemical reagents and cell cultures, is predominantly accomplished through steady flows that are typically driven by syringe pumps, gravity, or capillary forces. The use of complementary oscillatory flows is seldom considered in applications despite its numerous advantages as recently demonstrated in the literature. The significant technical barrier to the implementation of oscillatory flows in microchannels is likely responsible for the lack of its widespread adoption. Advanced commercial syringe pumps that can produce oscillatory flow, are often more expensive and only work for frequencies less than 1 Hz. Here, the assembly and operation of a low-cost, plug-and-play type speaker-based apparatus that generates oscillatory flow in microchannels is demonstrated. High-fidelity harmonic oscillatory flows with frequencies ranging from 10-1000 Hz can be achieved along with independent amplitude control. Amplitudes ranging from 10-600 &#181;m can be achieved throughout the entire range of operation, including amplitudes &#62; 1 mm at the resonant frequency, in a typical microchannel. Although the oscillation frequency is determined by the speaker, we illustrate that the oscillation amplitude is sensitive to fluid properties and channel geometry. Specifically, the oscillation amplitude decreases with increasing channel circuit length and liquid viscosity, and in contrast, the amplitude increases with increasing speaker tube thickness and length. Additionally, the apparatus requires no prior features to be designed on the microchannel and is easily detachable. It can be used simultaneously with a steady flow created by a syringe pump to generate pulsatile flows.

The protocol demonstrates a convenient method to produce harmonic oscillatory flow from 10-1000 Hz in microchannels. This is performed by interfacing a computer-controlled speaker diaphragm to the microchannel in a modular manner.

用于在微通道中产生亚千赫兹振荡流的外部驱动器的组装和表征

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.