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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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spatiotempor...

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 ="http://ecsource.jove.com/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 border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Reagent/Materials</td>
			<td style="width:171px;"></td>
			<td style="width:155px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">75x38mm Plain Microscope Slides</td>
			<td style="width:171px;">Corning</td>
			<td style="width:155px;">2947-75X38</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Acetone</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">320110</td>
			<td style="width:179px;">ACS reagent, &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Isopropyl alcohol</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">W292907</td>
			<td style="width:179px;">&#8805;99.7%, FCC, FG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Sulfuric acid</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">320501</td>
			<td style="width:179px;">ACS reagent, 95.0-98.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Hydrogen peroxide</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">216763</td>
			<td style="width:179px;">30 wt.% in H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Primer P-20</td>
			<td style="width:171px;">Microchem</td>
			<td style="width:155px;">MCC Primer 80/20</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">NFR photoresist</td>
			<td style="width:171px;">JSR</td>
			<td style="width:155px;">NFR016D2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Photomask</td>
			<td style="width:171px;">Photoplotstore</td>
			<td style="width:155px;">N/A</td>
			<td style="width:179px;">4x4 Direct write mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">MF-319 Developer</td>
			<td style="width:171px;">Shipley (Rohm and Haas)</td>
			<td style="width:155px;">Microposit MF-319</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">1165 Photoresist Remover</td>
			<td style="width:171px;">Dow Chemical, Co.</td>
			<td style="width:155px;">DEM-10018073</td>
			<td style="width:179px;">1-methyl-2-pyrrolidinone based</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">S1813 photoresist</td>
			<td style="width:171px;">Shipley (Rohm and Haas)</td>
			<td style="width:155px;">S1813</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PLL-g-PEG</td>
			<td style="width:171px;">SuSoS</td>
			<td style="width:155px;">PLL(20)-g[3.5]- PEG(2)</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">HEPES</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">15630080</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Paraffin film</td>
			<td style="width:171px;">HACH</td>
			<td style="width:155px;">251764</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SU-2025 photoresist</td>
			<td style="width:171px;">Microchem</td>
			<td style="width:155px;">SU-2025</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">PDMS</td>
			<td style="width:171px;">Dow Corning</td>
			<td style="width:155px;">184 SIL ELAST KIT 0.5KG</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microbore Tubing</td>
			<td style="width:171px;">Saint-Gobain PPL Corp.</td>
			<td style="width:155px;">S-54-HL</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Metal pins</td>
			<td style="width:171px;">New England Small Tube</td>
			<td style="width:155px;">NE-1300-01</td>
			<td style="width:179px;">&#160;Cut Tube (straight), 0.025&#8221; OD x 0.017&#8221; ID x 0.50&#8221; Long</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">HeLa cells</td>
			<td style="width:171px;">Duke Cell Culture Facility</td>
			<td style="width:155px;">(307-CCL-2) HeLa, p.148</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DPBS(1X) buffer</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">14190144</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DMEM culture medium</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">11995065</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Fibronectin Bovine Protein, Plasma</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">33010018</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">0.25% Trypsin-EDTA (1X)</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">25200056</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Propidium Iodide</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">P21493</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Carboxylate Microspheres 1.00&#956;m</td>
			<td style="width:171px;">Polysciences, Inc</td>
			<td style="width:155px;">08226-15</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Carboxylate Microspheres 2.00&#956;m</td>
			<td style="width:171px;">Polysciences, Inc</td>
			<td style="width:155px;">18327-10</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">22980</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Sulfo-NHS</td>
			<td style="width:171px;">Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td style="width:155px;">24510</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Peptite-2000</td>
			<td style="width:171px;">Advanced BioMatrix</td>
			<td style="width:155px;">5020-5MG</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">FITC Annexin V</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">A13199</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Fura-2, AM</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">F1221</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">DMSO</td>
			<td style="width:171px;">Sigma Aldrich, Co.</td>
			<td style="width:155px;">D2650</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">F-127</td>
			<td style="width:171px;">invitrogen</td>
			<td style="width:155px;">P6866</td>
			<td style="width:179px;">0.2 &#181;m filtered (10% Solution in Water)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Reduced serum media&#160;</td>
			<td style="width:171px;">ThermoFisher Scientific</td>
			<td style="width:155px;">11058021</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Name</td>
			<td style="width:171px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:175px;">Plasma asher</td>
			<td style="width:171px;">Emitech</td>
			<td style="width:155px;">K-1050X</td>
			<td style="width:179px;">O2 / Ar plasma ashing of photoresist and other organic materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Mask aligner</td>
			<td style="width:171px;">SUSS MicroTec&#160;</td>
			<td style="width:155px;">Karl Suss MA6/BA6</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">E-beam evaporator</td>
			<td style="width:171px;">CHA Industries</td>
			<td style="width:155px;">CHA Industries Solution E-Beam</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">RIE</td>
			<td style="width:171px;">Trion Technology&#160;</td>
			<td style="width:155px;">Trion Technology Phantom II</td>
			<td style="width:179px;">(oxide/ nitride/ polymer) etching</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Stereoscope</td>
			<td style="width:171px;">AmScope</td>
			<td style="width:155px;">American Scope SM-4TZ-FRL</td>
			<td style="width:179px;">Stereo Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Syringe pump</td>
			<td style="width:171px;">Chemyx Inc</td>
			<td style="width:155px;">NanoJet</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:175px;">Cell culture incubator</td>
			<td style="width:171px;">NuAire</td>
			<td style="width:155px;">AutoFlow NU-8500 Water Jacket CO2 Incubator</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Biological Safety Cabinets</td>
			<td style="width:171px;">NuAire</td>
			<td style="width:155px;">NU-425-400</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Water bath</td>
			<td style="width:171px;">VWR</td>
			<td style="width:155px;">1122s</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Centrifuge</td>
			<td style="width:171px;">IEC</td>
			<td style="width:155px;">Centra CL2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microscope</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">Axio Observer Z1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Nd:YAG laser&#160; (laser 1)</td>
			<td style="width:171px;">New Wave Research</td>
			<td style="width:155px;">Tempest</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Nd:YAG laser&#160; (laser 2)</td>
			<td style="width:171px;">New Wave Research</td>
			<td style="width:155px;">Orion</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Delay generator</td>
			<td style="width:171px;">Berkeley Nucleonics&#160;</td>
			<td style="width:155px;">BNC 565-8c</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Flash lamp</td>
			<td style="width:171px;">Dyna-Lite</td>
			<td style="width:155px;">ML1000 fiber-coupled flashtube</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed camera</td>
			<td style="width:171px;">DRS Hadland</td>
			<td style="width:155px;">&#160;Imacon 200</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed&#160; camera</td>
			<td style="width:171px;">Shimadzu</td>
			<td style="width:155px;">HPV-X</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">high speed camera</td>
			<td style="width:171px;">Vision Research</td>
			<td style="width:155px;">Phantom V7.3</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PIV software</td>
			<td style="width:171px;">LaVision</td>
			<td style="width:155px;">DaVis 7.2</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">camera</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">AxioCam MRc 5</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">software</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">AxioVision</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PTI system</td>
			<td style="width:171px;">Horiba</td>
			<td style="width:155px;">S/N: 1705 RAM-X</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">EasyRatio software</td>
			<td style="width:171px;">Horiba</td>
			<td style="width:155px;">Easy Ratio Pro 2</td>
			<td style="width:179px;">version 2.3.125.86</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">63&#215; objective</td>
			<td style="width:171px;">Zeiss</td>
			<td style="width:155px;">LD Plan Neofluar</td>
			<td style="width:179px;"></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?...

Watch this Scientific Journal Video about A Microfluidic System with Surface Patterning for Investigating Cavitation BubblesÃ¢â‚¬â€œCell 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 de Cartagena UDEC

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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

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 ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

氧微环境中的单个胰岛水平的定量和临时控制

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案:二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

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 ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...