We would like to acknowledge the use of the clean room facility SMIF at Duke University. We also want to thank Hao Qiang for his assistance in measuring the jet velocity. The authors thank Todd Rumbaugh of Hadland Imaging for providing the Shimadzu HPV-X camera used in this study.The work was funded in part by NIH through grants 5R03EB017886-02 and 4R37DK052985-20.

1.微细注:所有的微细加工过程在洁净室进行。 A镀铬掩模的微加工之前设计的， 见图2。 <img alt="图2" src="/files/ftp_upload/55106/55106fig2.jpg" /> 图2:图示在微流体芯片中的通道的设计和工作单位的尺寸。一个 ）掩模设计对准PDMS微通道（绿色）和玻璃基板（蓝色和红色）上的图案的。 二 ）在工作单元阵列的一个代表部分的掩模设计的放大图像。 c）一种工作单元的示意图示出了单元图案（蓝色）和一对金点的（红）。所有尺度显示在右下角。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55106/55106fig2large.jpg" target="_blank">请点击这里查看LAR</a>GER版本这个数字。 <ol><li> 金圆点图案 注:各金点的面积被设计为内25-30微米2，因此，它是足够大，以吸收激光能量用于生成气泡，但足够小以避免单个细胞附着到它。为金点制造的示意图示于图3a。 <ol><li> 载玻片清洁（在化学罩） <ol><li>冲洗用丙酮随后异丙醇（IPA）的载玻片，然后用N 2流干燥。 </li><li>浸泡在食人鱼溶液滑动（H 2 SO 4:2 O 2 = 4 H:1）10分钟。 </li><li>取出的幻灯片，用去离子水冲洗，然后用N 2流干燥。 </li><li>烘烤在电炉上的载玻片在120℃下进行10分钟。 </li><li>治疗在等离子体灰幻灯片在100瓦90秒。 </li></ol></li><li> 旋涂（旋涂罩） <ol><li>开启热板，并等待直至温度稳定在95℃。 </li><li>按照涂层过程: 1000转5秒以500转/ s斜坡; 那么3000转30秒以1000转/ s斜坡。 </li><li>通过施加真空固定清洁载玻片上旋涂机。 </li><li>覆盖P-20的滑动面，并开始该涂层配方。 </li><li>重复NFR-负性光刻胶涂覆过程。 </li><li>烘烤在95℃下滑动60秒和等待，直到它冷却到室温。 </li></ol></li><li> 光刻 <ol><li>安装铬掩模到掩模对准，并确保图案面朝下朝幻灯片。 </li><li>设置光刻配方​​硬曝光方式与9号紫外曝光的，并迅速对准掩模的玻璃基板。 </li><li>进行UV曝光之后，烘烤在95滑动6℃持续1分钟，然后让其冷却至室温。 </li></ol></li><li> 发展 <ol><li>制定对60号显影液幻灯片模式。 </li><li>从开发人员删除的幻灯片，它用去离子水冲洗，并用N 2流干燥。 </li><li>检查图案几何形状的显微镜下，并测量和记录特征尺寸。 </li></ol></li><li> 金沉积（电子束蒸发）和剥离 <ol><li>烘烤，在120℃下的滑动5分钟，并让其冷却到室温。 </li><li>在500乇和100W。清洁带中的反应性离子蚀刻（RIE）机等离子体滑动为90秒</li><li>钛的存款5纳米和Au的15nm的上使用电子束蒸发器17的滑动。 </li><li>浸泡过夜幻灯片中含光刻胶去除溶剂，以消除对NFR顶部的金色休息抵制玻璃烧杯中。 </li><li>检索幻灯片，我平齐吨，丙酮其次是IPA，并用N 2流干燥。 </li><li>在100瓦在清洁中的氧等离子体灰90年代以前干在115℃的热板的幻灯片。 </li></ol></li></ol></li><li> 通过剥离分子组装图案（MAPL） 注:各纤连蛋白涂覆的岛的面积被设定为内700-900微米2以促进适当的HeLa细胞在一个正方形的区域扩展，同时最小化多细胞聚集在岛上的机会。用于细胞图案化岛的制备示意图示于图3b。 <ol><li> 旋涂 <ol><li>重复步骤1.1.2，而是使用S1813阳性光致抗蚀剂和115℃的温度。 </li></ol></li><li> 对准光刻 <ol><li>装入的铬掩模到掩模对准器，并确保与图案面朝下朝着与所述滑动金色圆点。 </li><li>将光刻配方硬曝光模式，第9号的紫外线照射。 </li><li>对齐用的对准标记，周围设荫罩的边缘的底部滑动顶端掩模，作为空间参考。 </li><li>检查掩模的中心部分，并验证该图案特征正确对齐。 </li><li>不完整的烘烤后的紫外线照射。 </li></ol></li><li> 发展 <ol><li>重复步骤1.1.3，但有45秒的发展干燥加热板上，并在N 2存储保存之前。 </li></ol></li></ol></li><li> 化学处理 <ol><li>制备钝化溶液:0.5毫克/毫升的PLL-G-PEG在10mM HEPES缓冲液。 </li><li>检索N 2储存幻灯片并使用RIE的参数设置清洁:500托的压力，100瓦功率，90-S的持续时间。 </li><li>吸管钝化溶液到一张石蜡膜的一滴。&lt;/ LI&gt; <li>三明治电影和幻灯片的解决方案，确保与模式的端功能朝下向膜;避免气泡的形成。 </li><li>除去膜中的滑动之前等待45分钟。 </li><li>连续浸泡滑动在光致抗蚀剂去除剂，光致抗蚀剂去除剂和去离子水（1:1），和去离子水，并搅拌它在超声浴中90秒为每个浸泡。 </li><li>干在电炉上滑动在干燥器密封并将其储存在冰箱中之前以除去水分。 </li></ol></li><li> 芯片组装 <ol><li>重复步骤1.1.1-1.1.4，但与SU8-2025阴性光刻胶和参数设定所指称为MICROCHEM协议18，与光掩模制备的硅模具。 </li><li>制作出PDMS微通道（40毫米×25 x 5毫米，长×宽×高），并使用软光刻小PDMS板（800微米宽槽结构）。 </li><li>冲microchannel对于流体接入端口和清洁它用胶带，然后用IPA。 </li><li>屏蔽与PDMS板玻璃基板的图案化区域，并应用RIE（100瓦，500毫托，60秒），从周边区域去除的PLL-G-PEG。 </li><li>治疗所述微通道用RIE（25瓦特，500毫托，25秒）的降低的剂量，然后将其对准到所述图案化的玻璃基板（除去了小的PDMS板）;带来的微通道，并在一个立体镜下共形接触的图案化的玻璃基板上。 </li></ol></li></ol><img alt="图3" src="/files/ftp_upload/55106/55106fig3.jpg" /> 图3:微细加工和芯片组装的示意图。一 ）在玻璃基板上金色圆点格局。 b）在通过MAPL细胞图案的玻璃基板的制备。 c）与等离子体结合微流体通道的装配。请参阅材料清单的定义ÔF中的缩写。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55106/55106fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li> 细胞附着 注:HeLa细胞例行维持在DMEM补充有10％FBS和1％抗生素/抗有丝分裂溶液在细胞培养孵化器。为芯片组装和细胞附着的示意图示于图3c。 <ol><li>素，用PBS芯片在1微升/分钟30分钟，然后注入纤连蛋白溶液（50微克/毫升PBS中，1微升/分钟）45分钟。 </li><li>在等待时，trypsinize细胞培养物用0.25％胰蛋白酶-EDTA，把它们洗干净在培养基中，离心单个细胞，并在5×10 6个细胞/ mL重组在预热（37℃）培养液中的细胞悬浮液。 </li><li>替换用PBS纤连蛋白溶液和芯片在10微升/分钟5分钟冲洗。 </li><li>注入所制备的细胞悬浮液到芯片，停止流动，夹紧插座，并保持在细胞培养孵化器芯片30分钟。 </li><li>释放出口和10微升/分钟5分钟用细胞培养基芯片冲洗。降低培养液的灌注流量到0.75微升/分钟和维持细胞培养在培养箱2小时。 </li></ol></li></ol> 2.气泡产生和流量可视化注:实验装置的示意图示出在串列气泡生成，并与在微流体通道附近的生长的单细胞所得到的流相互作用图4a。 <ol><li> 串联的生成与两个激光器和定时控制气泡 <ol><li>放在倒置显微镜的阶段的微流体芯片和对齐两个脉冲Nd的焦点:在一对图案化的金点分隔的B YAG激光器（λ= 532纳米，5纳秒脉冲持续时间）Ÿ40微米。 </li><li>使用数字延迟发生器触发两个激光器大约2.5微秒的时间延迟产生的金色圆点发起的两个气泡。 </li><li>调整激光的输出能量（〜10μJ），以在50±2微米的产生气泡的最大直径。 </li><li>高速摄像头（200纳秒曝光和每秒200万次帧（fps））同步到激光器和捕捉泡沫膨胀，崩溃，泡沫泡互动，射流形成的动态。 </li></ol></li><li> 喷射速度的量化 <ol><li>记录喷射尖端（J 1）的位置在所述第一泡（B 1）和B 1的远端的近端，开始于第二气泡（B 2）的产生，并为J的触地结束1朝B1的远端;参见图5a的示意图。 </li><li>线性拟合的位置机器人的时程小时近端（喷射尖端）和远端端部。计算的前端位置与时间曲线的斜率为近端，以确定喷射速度。这两条线的交点表示喷射触地时间。更多的细节可以参考19中找到。 </li></ol></li><li> 串联气泡诱发的流场的可视化 <ol><li>制备1微米的聚苯乙烯（PS）珠粒在DI水中的悬浮液（2.6％重量/体积）和注入珠粒进入微流体芯片。 </li><li>产生串联气泡作为在步骤2.1.1-2.1.3所述。 </li><li>用一个高速摄影机在5M的帧率的用100纳秒的曝光时间组帧速率记录串联气泡动力学。 </li><li>载所获取的图像序列划分成一种商业PIV软件。 </li><li>将每个图像划分成16×16像素（PX）用75％的重叠较小询问窗口。 </li><li>应用多道次的迭代和地区的过滤器，以减少速度场，计算错误，并输出的结果中的速度矢量地图。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/55106/55106fig4.jpg" /> 图4:图示的实验装置和图像记录。一 ）实验装置为串联气泡产生。 b）用作不同类型的成像采集的时间序列。 FL:荧光显微镜，BF:明场，IFT:帧间的时间。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55106/55106fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.单细胞分析和生物效应注:串联气泡产生毗邻靶细胞，将所得的生物效应，研究在对峙距离依赖性的方式。对于不同类型的图像采集的时间序列在图4b中描绘。 <ol><li>膜穿孔和大分子的摄取 注:细胞外大分子的摄入到靶细胞的特征在于，膜不通透性碘化丙啶通过在细胞膜上的穿孔部位的渐进扩散（PI）。 <ol><li>以下步骤1.5，取代常规培养基（ 即，DMEM）中以在整个实验过程中的0.5微升/分钟的灌注流量运行PI溶液（100微克/毫升的DMEM）。 </li><li>编程显微镜控制软件来自动选择在转台的PI荧光立方体和同步CCD照相机通过PI荧光激发的定时捕获的荧光图像为200毫秒的曝光时间。 </li><li>后两个激光焦点对准到一对金点旁边的一个目标小区，记录都亮场（BF）和荧光（FL）图像串联泡处理之前。 </li><li>立即使用显微镜控制软件启动目标单元的时间推移的荧光图像记录不久步骤后2.1捕获PI摄取的历史。 </li></ol></li><li> 膜变形 <ol><li>制备1微米珠（1％重量/体积，用水溶性碳二亚胺活化的），并与肽2000（100微克/毫升PBS中）官能它们。 </li><li>以下步骤1.5，孵育贴壁细胞与官能化珠在1×10 9珠/ mL的密度在37℃下30分钟。 </li><li>重复步骤2.1以产生串联气泡旁边的靶细胞，并与超高速摄影机在500万fps的帧速率运行记录细胞变形。 </li><li>鉴定的3珠子留在成像平面上整个实验一三元组和编译它们作为位置（X 1，Y 1）（X 2，Y 2），和（x 3，y 3的）在实验中的坐标。 </li><li>之前和AF计算黑社会的面积用公式TER串联泡沫处理: <img alt="式（1）" src="/files/ftp_upload/55106/55106eq1.jpg" /></li><li>计算面积应变为: <img alt="公式（2）" src="/files/ftp_upload/55106/55106eq2.jpg" /></li><li>基于所述三个顶点的前和串列泡处理后的坐标，计算以下建立的协议20,21本地主应变和面积菌株。 </li></ol></li><li> 活力和细胞凋亡 注:FITC Annexin V的标记细胞，包括细胞凋亡的人，通过磷脂酰丝氨酸（绿色荧光）的外化。 PI标签坏死细胞（红色荧光）的细胞核。亮场成像用于记录细胞形态和协助细胞活力和凋亡的识别。 <ol><li>记录在微流体通道（通过在通道中的地址标签促进每个处理过的细胞的位置，参见图2b </str翁&gt;而重复）3.1步。 </li><li>灌流用FITC膜联蛋白V溶液在芯片15分钟，然后孵育另外2小时细胞培养液的处理过的细胞。阳性细胞显示绿色荧光。 </li><li>串联泡处理后24小时，重复步骤3.3.2研究在目标细胞中的长期生物效应。 </li></ol></li><li> 钙反应 <ol><li>用3 10微升％混合3微升的呋喃-2 AM原液（1mg / mL的在DMSO中）w / v的尼克F-127在低血清培养基中制备最终标记溶液（6微米）的500微升。 </li><li>以下步骤1.5，替换与标记溶液的细胞培养基和室温下孵育在黑暗中进行40分钟（1微升/分钟灌注率）。 </li><li>在开始细胞实验之前重新填充减少的血清培养基（0.75微升/分钟灌注率）的信道。 </li><li>启动比率成像（波长:三百八分之三百四纳米，50毫秒的曝光）使用商业PTI系统靶细胞。 </li><li>经过10秒的静息水平的细胞比例成像，产生气泡串联按照步骤2.1;用0.1秒，1分钟帧间时间（IFT）的第一个记录，然后增加IFT到1秒为另一分钟，2分钟后进一步增加至5秒。 </li><li>使用PTI软件计算在感兴趣的区域（ROI），其正比于细胞内钙离子浓度的比率R = F340 / F380。 </li></ol></li></ol>

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The authors have nothing to disclose.

单细胞分析，在用活细胞成像的组合，大大提高了在单个细胞，如表型的发展和免疫反应23动态和常变量过程的理解。在对比中菜或瓶的现有的细胞培养，微流体系统使微环境的精确控制，下至单细胞水平，在现实的时间。因此，先进的微流体技术和工艺在很大程度上改善了单细胞分析的吞吐量和重现性。通过整合软光刻和表面图案化，微流体系统可进一步设计成便于单个小区对spatiotemporally可变刺激的复杂模式响应的深入分析。例如，短暂的化学或机械的线索已成功交付到单个细胞，而他们的生物或机械反应被自动记录并分析24。尽管总的趋势，微流体在细胞水平上分析了空化引起的生物效应的应用已经非常有限。最值得注意的是，它仅被施加到悬浮液中的单个细胞通过micropillars 11捕集的气泡引起的膜穿孔。在大多数的贴壁细胞的研究中，控制细胞的形态保持在动态泡沫 - 细胞相互作用一致性的好处是不可用或在很大程度上被忽视。 我们已经开发出一种微流体系统以精确地控制的启动，后续扩展，和空化气泡塌陷动力学;与射流形成泡沫，泡沫的相互作用;和细胞形状，取向，粘合图案，并从气泡间隔距离，因此允许可再现喷射流以良好控制的实验条件下被应用到单个细胞。这种新颖的微流控系统非常适合开展基本ST对空化气泡（多个）udies -cell是相关的一个不同的范围的治疗性超声应用，如SWL，HIFU，和sonoporation相互作用。我们已经证明，我们的微流体系统可用于研究各种气穴引起的生物效应，包括细胞膜变形，膜穿孔，可行性和钙反应，以更可控的和可再现的方式。最值得注意的是，通过串联的气泡产生的定向喷射流使我们能够探测在在高应变速率的单个细胞，但没有很好的特点的机械性能和钙响应。由这样的喷射流所产生的局部的，脉冲的机械应力不能由常规的拉伸方法，如微吸管25和利用光26的与磁镊子27来实现。此外，与使用超声造影剂8,28-30或以前的研究激光诱导单蒲式耳bbles 31,32，我们的微流体系统提供了更好的控制的细胞几何形状和粘附条件，从而减少对细胞应答和生物学效应16的实质影响。 而我们的方法是可靠和可再现的，必须遵循协议谨慎以确保该实验系统忠实地再现。在微通道表面图案形成的质量是有气泡的气泡 - 细胞相互作用的重复性和下游生物效应的级联主要责任。例如，对于HeLa细胞，各金点的面积必须是25-30微米2，以确保稳定的气泡的产生，同时防止细胞附着。另一方面，各纤连蛋白涂覆的岛的区域具有被周围700-900微米2，以促进在方形的单个单元的适当扩频，同时减少形成在岛上聚集多个小区的机会。对准金点和纤连蛋白涂布的岛之间必须用掩模对准器的帮助下精确地执行，以保持1°和在0.5微米的轴向误差范围内的角度误差。此外，该实验系统提供的多功能性在监测的一系列事件，其时间尺度数量级变化（ 例如，气泡动力学:微秒，细胞变形:毫秒，膜穿孔:秒，凋亡:小时）。因此，通过微调触发信号（由数字延迟发生器控制）精确地控制图像获取和记录每个序列的定时是很重要的。单细胞培养物必须很好保持在所述微通道，以尽量减少在表型细胞至细胞的变化（大多称作形态学）。因此，需要至少2小时培养细胞附着并与下游细胞处理，然后再继续在岛上蔓延。建议始终保持流速在microchann下3微升/分钟埃尔，使得通过在小区中的循环培养基中产生的流动的剪切应力是在生理范围内。 我们的技术的一个电流限制是，串联气泡相互作用并将所得喷射流只能使用一次施加到靶细胞，因为金点将由激光照射消融。这个缺点限制了我们的实验系统来模拟通过超声治疗，其中细胞经常暴露在空化事件产生的生物学效应的能力。但是，这种时间上的限制可以通过将二氧化硅的另一薄层，以保护从直接暴露金点在液体15得到缓解。总的来说，我们的微流控系统提供了良好的控制实验条件，调查从空化泡（S）β细胞相互作用产生的生物学效应，并有几个独特的功能。首先，双方的尺寸和空化的位置的气泡在我们的电子xperimental系统可以通过调整由金点吸收入射激光能量被精确地控制。第二，单个细胞的几何形状和粘附是由细胞图案标准化，以尽量减少其对细胞应答和生物学效应的影响，从而导致更可再现的结果。第三，在微流体芯片设计的平行工作单元使其可行研究下游生物效应，如细胞生长，增殖，和潜在的基因表达，空化曝光后，由于每个单元可被寻址和单独地监测。总之，我们的微流体系统和方法，创建可靠的泡沫泡沫相互作用研究单个细胞，提高精度的生物效应。 我们当前的芯片设计用于个人HeLa细胞的分析。然而，对于其他的哺乳动物细胞系的图案岛屿的变形例也是可能的。一些改进可以提出，要进一步拓宽应用A芯片的纳秒。例如，锁相环-G-PEG分子代用品可以探讨从迁移甚至24小时后，停止单个图案化的细胞。此外，微流控阀新的芯片设计可以被探索，以控制细胞的局部微环境，从而使化学物质，如荧光标记或有毒的试剂将仅适用于特定的位置，而不影响芯片的其它区域的细胞。通过这些改进，这里提出的微流体系统可以提供机会来研究单个细胞，例如代谢，偏析，分化和基因表达，以下的空化曝光或由一个脉冲流产生的机械刺激的长期生物效应。

人们越来越认识到细胞的异质性，从基因，蛋白质和代谢物的随机产生的表达，存在较大的细胞群中，并在生物学作为一个基本原则，使细胞适应和进化1。因此，它常常是不精确和不可靠使用基于人口的散装测量理解各个细胞和它们之间的相互作用的功能。因此在生物和药理研究高息的单细胞分析开发新技术是，并且可以使用，例如，以更好地理解在干细胞生物学和癌症治疗2-4键信号通路和过程。在近年来，微流体平台的出现，极大地促进单细胞分析，其中将已与新颖的分析策略5进行定位，处理，并从单个细胞的反应的观察。 气穴起着各种范围的生物医学应用，包括高强度的癌症的治疗中具有重要作用聚焦超声（HIFU）6，由冲击波碎石肾结石的非侵入性碎片（SWL）7，药物或基因递送由sonoporation 8，并且通过水动力空化气泡9,10细胞或组织的最近报告破坏。尽管这样，空化气泡（多个）与生物组织和细胞相互作用的动态过程还没有被很好地理解。这是由于在通过超声，冲击波，和局部液压产生空化起始和气泡动力学的随机性;此外，还有一个缺有利的技术来解决的生物细胞本身固有的复杂和快的反应，特别是在单细胞水平的。 由于这些挑战，这并不奇怪，很少研究有蜂N报调查良好控制的实验条件下，泡沫细胞的相互作用。例如，单个细胞的膜穿孔被困在悬浮液11和人红细胞12的脉冲大变形已在微流体通道使用激光产生的单一气泡证明。后者的技术，但是，可以只在因核13的存在的真核细胞中产生非常小的变形。此外，很难在悬浮处理细胞时监视下游生物效应。在其它研究中，细胞结合的微泡（或超声造影剂）的超声激发用于生产单贴壁细胞的膜穿孔和/或细胞内钙离子的反应已经报道8。单贴壁细胞的膜穿孔，也可以通过使用在薄液层含有光吸收锥虫蓝溶液14激光产生的串联的气泡产生，或通过微秒激光脉冲通过在微容器15的光学吸收基板照射所产生的振荡气体气泡。时相比，光学吸收基底具有的优点在激光吸收台盼蓝溶液，因为后者是细胞毒性。更重要的是，激光产生的气泡是在比声学激发的气泡的气泡尺寸和位置方面更加可控。然而，在所有这些先前的研究中，细胞形状，取向，和粘附条件不加以控制，其可以基本上影响细胞响应和机械应力16产生生物效应。 为了克服在以前的研究这些缺点，我们最近开发了气泡的产生，细胞图案化，气泡的气泡 - 细胞相互作用，和实时通过使用微加工TECHN的一个独特的组合构成的微流控芯片细胞应答的生物测定法的实验系统iques。从其他外地区分我们的实验系统的三个主要功能有:1）在玻璃基板上的微米级金的点的图案形成，以使气泡生成17的局部激光吸收; 2）在同一基板上的细胞粘附的细胞外基质（ECM）的微米大小的岛的图案化，以控制这两个位置和各个单元的几何形状;和3）从3D气泡泡沫 - 细胞相互作用域的尺寸与准二维空间压缩，以促进在面内的可视化气泡气泡的相互作用，喷射流场，细胞变形，和生物效应，在所有捕获一个改进的成像序列（ 图1d）。 <img alt="图1" src="/files/ftp_upload/55106/55106fig1.jpg" /> 图1:微流体芯片和不同测定的示意图。 a）在频道的组装微流控芯片充满了蓝墨水<html>可视化。 b）在图案化的细胞和金的点在微流体芯片内的区域（在邻近的两金点之间的距离为40微米）。工作单位的许多对可以被布置在一个通道。 三）特写由一对金点的并粘附于细胞图案化区域中的HeLa细胞的单一工作单元的图像。 d）该设备的操作示意图。单个细胞附着和扩散上涂覆有纤连蛋白的"H"形岛。一对空化气泡（串联气泡）与抗相振荡的被照亮的金点的脉冲激光束（参见图4a），导致了快速和局部的喷射朝向目标小区移动附近的发生而产生。细胞可能会变形，中蕴含大分子摄入，和/或与钙反应的刺激，这取决于细胞的串联气泡的偏离距离（S D）。F ="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55106/55106fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 这个平台可以与荧光测定法和附着到细胞表面的气蚀引起的生物效应官能珠被进一步组合。特别是，该平台将打开在单细胞水平可靠和可定量测定的方式。到现在为止，我们已经使用了装置串联气泡诱导细胞膜变形，细胞穿孔和细胞内摄取，生存能力，细胞凋亡和细胞内钙响应的分析。在下面的协议中，我们描述了芯片制造的过程以及用于分析上述的各种生物效应的过程。此外，还描述了芯片的操作。

<table><tbody><tr><td>&lt;strong&gt;Reagent/Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>75 mm x 38 mm Plain Microscope Slides</td><td>Corning</td><td>2947-75X38</td><td></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>Sulfuric acid</td><td>Sigma Aldrich, Co.</td><td>320501</td><td>ACS reagent, 95.0-98.0%</td></tr><tr><td>Hydrogen peroxide</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>Primer P-20</td><td>Microchem</td><td>MCC Primer 80/20</td><td></td></tr><tr><td>NFR photoresist</td><td>JSR</td><td>NFR016D2</td><td></td></tr><tr><td>Photomask</td><td>Photoplotstore</td><td>N/A</td><td>4x4 Direct write mask</td></tr><tr><td>MF-319 Developer</td><td>Shipley (Rohm and Haas)</td><td>Microposit MF-319</td><td></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>S1813 photoresist</td><td>Shipley (Rohm and Haas)</td><td>S1813</td><td></td></tr><tr><td>PLL-g-PEG</td><td>SuSoS</td><td>PLL(20)-g[3.5]- PEG(2)</td><td></td></tr><tr><td>HEPES</td><td>ThermoFisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Paraffin film</td><td>HACH</td><td>251764</td><td></td></tr><tr><td>SU-2025 photoresist</td><td>Microchem</td><td>SU-2025</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>Microbore Tubing</td><td>Saint-Gobain PPL Corp.</td><td>S-54-HL</td><td></td></tr><tr><td>Metal pins</td><td>New England Small Tube</td><td>NE-1300-01</td><td>Cut Tube (straight), 0.025&amp;rdquo; OD x 0.017&amp;rdquo; ID x 0.50&amp;rdquo; Long</td></tr><tr><td>HeLa cells</td><td>Duke Cell Culture Facility</td><td>(307-CCL-2) HeLa, p.148</td><td></td></tr><tr><td>DPBS (1x) buffer</td><td>ThermoFisher Scientific</td><td>14190144</td><td></td></tr><tr><td>DMEM culture medium</td><td>ThermoFisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Fibronectin Bovine Protein, Plasma</td><td>ThermoFisher Scientific</td><td>33010018</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Propidium Iodide</td><td>ThermoFisher Scientific</td><td>P21493</td><td></td></tr><tr><td>Carboxylate Microspheres 1.00&amp;nbsp;&amp;mu;m</td><td>Polysciences, Inc</td><td>08226-15</td><td></td></tr><tr><td>Carboxylate Microspheres 2.00&amp;nbsp;&amp;mu;m</td><td>Polysciences, Inc</td><td>18327-10</td><td></td></tr><tr><td>EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)</td><td>ThermoFisher Scientific</td><td>22980</td><td></td></tr><tr><td>Sulfo-NHS</td><td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td><td>24510</td><td></td></tr><tr><td>Peptite-2000</td><td>Advanced BioMatrix</td><td>5020-5MG</td><td></td></tr><tr><td>FITC Annexin V</td><td>ThermoFisher Scientific</td><td>A13199</td><td></td></tr><tr><td>Fura-2, AM</td><td>ThermoFisher Scientific</td><td>F1221</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich, Co.</td><td>D2650</td><td></td></tr><tr><td>F-127</td><td>invitrogen</td><td>P6866</td><td>0.2 &amp;micro;m filtered (10% Solution in Water)</td></tr><tr><td>Reduced serum media&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11058021</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasma asher</td><td>Emitech</td><td>K-1050X</td><td>O&lt;sub&gt;2&lt;/sub&gt; / Ar plasma ashing of photoresist and other organic materials</td></tr><tr><td>Mask aligner</td><td>SUSS MicroTec&amp;nbsp;</td><td>Karl Suss MA6/BA6</td><td></td></tr><tr><td>E-beam evaporator</td><td>CHA Industries</td><td>CHA Industries Solution E-Beam</td><td></td></tr><tr><td>RIE</td><td>Trion Technology&amp;nbsp;</td><td>Trion Technology Phantom II</td><td>(oxide/ nitride/ polymer) etching</td></tr><tr><td>Stereoscope</td><td>AmScope</td><td>American Scope SM-4TZ-FRL</td><td>Stereo Microscope</td></tr><tr><td>Syringe pump</td><td>Chemyx Inc</td><td>NanoJet</td><td></td></tr><tr><td>Cell culture incubator</td><td>NuAire</td><td>AutoFlow NU-8500 Water Jacket CO&lt;sub&gt;2 &lt;/sub&gt;Incubator</td><td></td></tr><tr><td>Biological Safety Cabinets</td><td>NuAire</td><td>NU-425-400</td><td></td></tr><tr><td>Water bath</td><td>VWR</td><td>1122s</td><td></td></tr><tr><td>Centrifuge</td><td>IEC</td><td>Centra CL2</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio Observer Z1</td><td></td></tr><tr><td>Nd:YAG laser&amp;nbsp; (laser 1)</td><td>New Wave Research</td><td>Tempest</td><td></td></tr><tr><td>Nd:YAG laser&amp;nbsp; (laser 2)</td><td>New Wave Research</td><td>Orion</td><td></td></tr><tr><td>Delay generator</td><td>Berkeley Nucleonics&amp;nbsp;</td><td>BNC 565-8c</td><td></td></tr><tr><td>Flash lamp</td><td>Dyna-Lite</td><td>ML1000 fiber-coupled flashtube</td><td></td></tr><tr><td>high speed camera</td><td>DRS Hadland</td><td>&amp;nbsp;Imacon 200</td><td></td></tr><tr><td>high speed&amp;nbsp; camera</td><td>Shimadzu</td><td>HPV-X</td><td></td></tr><tr><td>high speed camera</td><td>Vision Research</td><td>Phantom V7.3</td><td></td></tr><tr><td>PIV software</td><td>LaVision</td><td>DaVis 7.2</td><td></td></tr><tr><td>camera</td><td>Zeiss</td><td>AxioCam MRc 5</td><td></td></tr><tr><td>software</td><td>Zeiss</td><td>AxioVision</td><td></td></tr><tr><td>PTI system</td><td>Horiba</td><td>S/N: 1705 RAM-X</td><td></td></tr><tr><td>EasyRatio software</td><td>Horiba</td><td>Easy Ratio Pro 2</td><td>version 2.3.125.86</td></tr><tr><td>63&amp;times; objective</td><td>Zeiss</td><td>LD Plan Neofluar</td><td></td></tr></tbody></table>

a microfluidic system with surface patterning for investigating cavitation bubble(s)&#8211;cell interaction and the resultant bioeffects at the single-cell level

在这个手稿，我们首先描述的微流体芯片的制造协议与金点和纤连蛋白涂布的区域中的相同的玻璃基板上，能精确控制串列式泡沫和具有良好定义的位置和形状的附近形成图案的单个细胞的产生上。然后，我们通过使用两个脉冲激光照射一对金点的有几微秒的时间延迟表明汇接气泡的产生。我们通过可视化的高速成像泡沫泡沫交互和射流形成，并使用粒子图像测速（PIV）描述所生成的流场。最后，我们提出这种技术单细胞分析，包括细胞膜上穿孔用高分子吸收，通过附加整联蛋白结合珠的位移确定的局部膜变形，并从比例成像细胞内钙离子反应的一些应用。我们的结果表明，一个快速和定向喷射流是亲由串联气泡相互作用，其可在接近生长的细胞的表面上施加一个高的局部剪切应力duced。此外，不同的生物效应可通过调节到汇接气泡从细胞相隔距离改变喷射流的强度来诱导。

在这项工作中所描述的微流体平台可用于调查气泡气泡的相互作用，并在单细胞水平分析各种气穴引起的生物效应。在这里，我们介绍几个例子来演示各种实验研究和生物测定可在我们的实验系统中进行的。我们将首先说明与射流形成，将得到的流场的可视化，和喷射速度（ 图5a）的计算串联气泡的瞬时相互作用。我们将串联然后本实施例泡沫诱发局部细胞膜变形（ 图5b），精确定位膜穿孔与PI摄取（ 图5c），和细胞内钙响应（ 图5d）。其他例子，如细胞活力和凋亡测定法，可以在参考22中找到。 图5a示出与射流形成，通过高速成像捕获，并且通过PIV表明所得到的流场的串联气泡相互作用的一个例子。具体来说，按照其极大的扩展，第一泡b 1分配（在0微秒生产）的崩溃不对称由第二泡B 2（2.5微秒生产）的快速扩张扭曲，导致一个"向上"的形成在3.4微秒喷射（J 1）和随后的喷射流，在5.4微秒所示。平均喷射速度由拟合线的对触地之前B1的近端（或杆）的位置的斜率确定（ 即，为B 1的前端接触）对时间。平均喷射速度被发现从20至58米/秒增加时B的最大直径为40 2增大到60微米。基于PIV分析，围绕t和定向喷射流的结果青霉气泡为10米/秒的数量级为10微米量级的宽度内被限制。它因此能够产生冲动和局部剪切应力和应力梯度到附近生长的靶细胞。 在图5b所示的另一实施例说明由串联产生的定向和局部喷射流引起的细胞膜变形气泡在僵局距离S D = 40微米。该膜的变形和恢复被附着到细胞膜的前缘的官能珠（由黄色虚线表示）的位移突出显示。当地菌株可从相邻的珠的三元的坐标来计算。在细胞表面上的不同位置计算出的最大面积的变化的示意图示出在中间。前缘主要拉伸（见该绿色圆），而后缘或纬度单元的全部擦除侧压缩（由红色圆圈表示），这表明在由串联气泡引起的喷射流所产生的细胞变形的异质性。在小区前缘区域应变的时间变化是在右侧示出。它包括在开始的几个快速振荡，随后大量持续拉伸约100微秒（FWHM），并在几毫秒的时间尺度随后逐渐恢复。 此外，大面积的应变和应变在小区积分前缘可以负责在小s的D所观察到的细胞膜穿孔，如图 图5c。在小S D（ 例如，10微米，或在一些情况下，20微米）和中间体s的D（ 即，大部分20和30微米的），局部膜破坏被诱导，导致细胞外的PI针尖摄取到细胞质。如果在细胞内的PI强度不断不饱和增加，发生坏死。在比较中，如果在PI强度是一个数量级降低并到达内继串联气泡处理10秒的高原，会发生与可能的细胞存活可修复穿孔，这是由在细胞形态的变化最小进一步支持。在大S D（ 即 40微米），可忽略不计PI摄取发现以下串联泡处理，表示可以忽略不计或不穿孔。电池可以与常规生长和增殖22生存。总的来说，结果表明在s的D≈20-40微米的范围内，从坏死细胞应答清晰过渡，通过修膜穿孔，非穿孔。 最后，我们表明在不同的S中的单个细胞通过串联的气泡引起的细胞内钙响应的比率成像结果D，尤其是S中D的亚致死范围= 30-50微米（ 图5d）。强度比正比于细胞内钙的量。在小S D（ 例如，30微米或在一些情况下，40微米），钙波从前缘行进到后缘显然以下串联气泡治疗在几秒钟内观察到。细胞内钙达到峰值浓度后，它衰变慢慢回在几分钟之内的静息水平。相反，在大S D（ 即，大部分为50微米，或在一些情况下，40微米），细胞内钙的增加是温和得多，也没有清晰可见的行进钙波可以被识别。这两种不同的钙反应，也可以从它们的强度比对时间的曲线进行区别，在强度比和上升时间的峰值幅度显著差异从静息水平达到峰值值。在一个更大的s的D（ 即，高于60微米），无钙的反应可以观察到。 HeLa细胞以各种s的D显示钙响应的百分比总结在图5d（右）。 总而言之，我们的结果表明，通过改变喷射流的强度（或改变s的D），各种生物效应可在类似的培养条件下从个人HeLa细胞，这是有可能的幅度和持续时间相关产的施加在细胞膜22的机械变形。 <img alt="图5" src="/files/ftp_upload/55106/55106fig5.jpg" /> 图5: 从串联气泡相互作用，细胞膜变形，膜穿孔，和钙反应测定结果。 A）流体运动和喷射通过串联泡沫相互作用引起的。左图:选择帧显示与射流形成串联泡沫相互作用和流场PIV所揭示。两个气泡的最大直径约50±2微米。中间:示意图喷射串联气泡，示出了第一气泡B1的中心轴，坐标（○）的起源，以及近端和远端（或杆）。右:测量杆位（含误差为±1微米）在近端和B 1的远端。 b）细胞的膜的变形。左:黄色虚线标签附着到细胞膜的前缘一个PS珠的位移，表示本地膜变形和恢复。中间:一个示意图，显示的峰面积菌株在左侧示出的细胞表面上的不同位置。右:在小区前缘的中间区域的菌株的时程。 Δε是面积应变计算浅教育署主应变和 ΔΔ是基于三重几何变化计算出的面积的应变。有关该地区的应变计算和误差分析的更多细节记录在参考22. C）细胞膜穿孔。左:亮场图像之前和PI摄取（0和120秒）的串联泡治疗和时间推移荧光图像后。白色箭头指示喷射流的方向。中东:平均PI强度与细胞内的时间的典型变化。右:在不同的Sð经历坏死（红色），可修穿孔（蓝色），和非穿孔（绿色）的细胞的百分比的统计结果。处理的细胞的数目:N，分别= 9 14，图14，和图8为s的D = 10，20，30，和40。统计误差为±1 /√N. D）心肌细胞钙响应。左:表示各个细胞的细胞内钙响应比率图像序列■在30微米和50微米s的D。红色箭头指示喷射方向。中东:在不同s的D典型响应曲线（比-时间曲线）。右:在不同的S D A钙响应的概率。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55106/55106fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>相隔距离（S D）（微米） </td><td>雷诺数（Re） </td><td>在YZ面平均剪切应力（PA） </td></tr><tr><td> 20 </td><td> 206.96 </td><td> 618.64 </td></tr><tr><td>三十</td><td> 354.8 </td><td> 709.69 </td></tr><tr><td> 40 </td><td> 378.78 </td><td> 657.22 </td></tr><tr><td> 50 </td><td> 163.85 </td><td> 323.61 </td></tr><tr><td> 60 </td><td> 105.08 </td><td> 42.21 </TD&gt; </tr></tbody></table> 表1:从PIV结果流的物理参数列表。估计雷诺数和平均剪切应力相对于不同偏离距离（S D）。 YZ平面指的是信道宽度x高度平面。平均剪切应力是从0至7.7微米的从通道底部的高度估计，因为在通道中的单元格的高度大约是6-7微米。

Watch this Scientific Journal Video about A Microfluidic System with Surface Patterning for Investigating Cavitation Bubblesâ€“Cell Interaction and the Resultant Bioeffects at the Single-cell Lev

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

a-microfluidic-system-with-surface-patterning-for-investigating

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

11.7K 次观看.  Duke University. A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

视频: A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)–Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

科研

生物学

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

生物工程

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

研究多孔介质中生物堵塞的微流体平台

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

环境科学

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. ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

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. ...

工程学

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

通过顺序毛细管辅助组装对微生物和微粒进行图案化

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant adsorption rates, evaluating equilibrium surface tensions as a function of bulk surfactant concentration, and relating how surface tension changes with changes in the interfacial area following equilibration. Simultaneous visualization of the interface using fluorescence imaging with a high-speed confocal microscope allows the direct evaluation of structure-function relationships. In the capillary pressure microtensiometer (CPM), a hemispherical air bubble is pinned at the end of the capillary in a 1 mL volume liquid reservoir. The capillary pressure across the bubble interface is controlled via a commercial microfluidic flow controller that allows for model-based pressure, bubble curvature, or bubble area control based on the Laplace equation. Compared to previous techniques such as the Langmuir trough and pendant drop, the measurement and control precision and response time are greatly enhanced; capillary pressure variations can be applied and controlled in milliseconds. The dynamic response of the bubble interface is visualized via a second optical lens as the bubble expands and contracts. The bubble contour is fit to a circular profile to determine the bubble curvature radius, R, as well as any deviations from circularity that would invalidate the results. The Laplace equation is used to determine the dynamic surface tension of the interface. Following equilibration, small pressure oscillations can be imposed by the computer-controlled microfluidic pump to oscillate the bubble radius (frequencies of 0.001-100 cycles/min) to determine the dilatational modulus The overall dimensions of the system are sufficiently small that the microtensiometer fits under the lens of a high-speed confocal microscope allowing fluorescently tagged chemical species to be quantitatively tracked with submicron lateral resolution.

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

用于动态界面共聚焦显微镜可视化的微量天体测量仪

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant ...

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

一种微流体方法，用于控制体力生成的信号振荡的功能研究

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and ...