The authors would like to thank Professor Howard Ceri from the Biological Sciences Department of the University of Calgary for providing bacterial strains. A. Kumar acknowledges support from NSERC. T. Thundat acknowledges financial support from the Canada Excellence Research Chair (CERC) program. The authors would also like to acknowledge help from Ms. Zahra Nikakhtari for help with videography.

这里进行的实验方案在下面描述的顺序。微细加工的协议，用于创建微流体平台在步骤讨论1。步骤2中记载的细菌培养协议（ 图2），和第三步涉及到的实验配置（ 图3）的装配。最后，实际的实验步骤是在步骤4中描述。 1，芯片制造过程注意:适当的安全程序必须遵循下面描述的过程。咨询机构安全官员的详细资料。 <ol><li>设计与适当的软件（ 例如 ，L-EDIT）面具。该通道的设计由一个主微通道宽度625微米。的信道的中心区域包含的微柱体的阵列50微米的直径，间距25μm的间隔（参见参考文件）。 </li><li>打印当前设计上的玻璃（5"×5"的钠钙玻璃），它的厚度为0.09"，并且由约70纳米厚的一层铬（Cr）的涂布，使用以制备光掩模掩蔽。使用AZ400K显影剂，持续1分钟，然后，蚀刻使用铬蚀刻剂Cr层约1分钟用丙酮剥离光刻胶，用冷食人鱼溶液清洗。（H 2 SO 4和H 2 O 2的比例为3:1）。 </li><li>光刻<ol><li>化学清洁标准4"硅片20分钟食人鱼的解决方案。 </li><li>用去离子水清洗晶圆，并擦干。 </li><li>加热晶片的热板上（200℃，15分钟）。 </li><li>大衣的硅片光致抗蚀剂。这里，在正性光刻胶AZ4620旋涂在硅晶片上以2000rpm，持续25秒，得到12.5微米厚的层。 </li><li>由浮动的晶片90秒的在100℃氮气流下除去通过在热板上的晶片的软烘烤所有的溶剂。然后，保持在真空中在t他相同的温度，持续60秒。 </li><li>将晶圆在黑暗中24小时进行脱水。 </li><li>暴露晶片于UV光以所设计的图案转移到光致抗蚀剂。 </li><li>沉浸在光致抗蚀剂显影剂溶液（AZ400K）为240秒的晶片。然后，用异丙醇冲洗晶片并通过放置在氮气流中干燥。 </li></ol></li><li> ICP-DRIE（电感耦合等离子体 - 深反应离子蚀刻）过程<ol><li>应用DRIE蚀刻。根据所需的设备最终深度（50微米在本次调查）选择适当的蚀刻深度。光致抗蚀剂作为在此过程中的掩模层。 </li><li>除去丙酮的残留光致抗蚀剂和清洗晶片。 </li></ol></li><li> PDMS（聚二甲基硅氧烷）铸造<ol><li>对于硅烷化硅模具师傅使用三氯甲基硅烷（TCMS）。倒入2〜3滴三氯甲基硅烷的小瓶，并将其放置在旁边的干燥器硅模具师傅。允许2-3小时的硅烷化过程完成。 </li><li>在一个单独的容器中，混合的Sylgard 184聚硅氧烷基与固化剂以重量比10:1，制备PDMS。通过将其在真空条件下（约2小时）脱气PDMS。 </li><li>把硅母模的持有人。然后，倒在硅原型模具的PDMS形成PDMS压模。确保气泡不会在PDMS在此过程中形成。 </li><li>固化的PDMS 2小时，在80℃。 </li><li>剥去从母模的PDMS印章。然后，切PDMS压模成单独的芯片。最后，使用切割核心钻洞的入口（S）和出口（S）。 </li></ol></li><li>硅橡胶玻璃的粘接<ol><li>暴露盖玻片和PDMS压模，以氧等离子体进行30秒。债券的PDMS印章的盖玻片。 </li><li>实现PDMS压模和盖玻片之间的适当密封，通过将其在烘箱中在70℃下进行10分钟退火设备。 </LI&gt; </ol></li></ol> 2，细菌培养注意:正确的生物安全协议必须遵循的步骤2-4。咨询机构安全官员的详细资料。 <ol><li>准备LB琼脂平板<ol><li>添加20克的Luria-Bertani（LB）琼脂（米勒）粉末和500ml超纯水至1升烧瓶中。搅拌使之溶解的粉末。 </li><li>通过高压灭菌在15磅，121℃下进行15分钟杀菌。 </li><li>让烧瓶冷却至50-55℃的长椅上，或在水浴中的生物安全罩。 </li><li>添加抗生素四环素达到50微克/毫升的最终浓度。纷飞拌匀。 </li><li>将该混合物倒入平板上。填写每块板，直到1月2日 - 2月3日已满。 </li><li>火焰气泡简单地弹出他们，如果他们形成。凝固气泡难以普及细菌培养过。 </li><li>将平板在室温下冷却过夜。 </li><li>当他们冷静，放盘放回他们的袖子，封袋，标签（抗生素及日期），并且在4℃商店。 注:盖板用锡箔的股票，轻停用许多抗生素。 </li></ol></li><li> LB培养基的制备<ol><li>添加20克的Luria-Bertani（LB）肉汤（米勒）粉末和1升超纯水至烧瓶中。搅拌使之溶解的粉末。 </li><li>通过高压灭菌在15磅，121℃下进行15分钟杀菌。 </li><li>让烧瓶冷却至50-55℃的长椅上，或在水浴中的生物安全罩。 </li><li>添加抗生素四环素达到50微克/毫升的最终浓度。纷飞拌匀。 </li><li>当冷，可将标记的瓶子在4℃。 注:盖上瓶锡箔，轻停用许多抗生素。 </li></ol></li><li>在LB琼脂平板上培养细菌（该协议使用荧光假单胞菌 ） <ol><li>以细菌股价从冷冻（-80℃℃），并放置在冰上。 </li><li>放置在-80℃的细菌股票和生物安全罩内的LB琼脂平板上。 </li><li>条纹的细菌菌株上的LB琼脂平板上以Z字形图案。覆盖琼脂平板上，并培育其在30℃过夜。最后，存储盘在冰箱4℃。 </li></ol></li><li>准备细菌溶液（S1） <ol><li>倒入50毫升LB培养基介质高压灭菌瓶中。生物安全罩内执行此操作。 </li><li>从LB琼脂板烧瓶传输​​单个菌落。此操作也应生物安全罩内进行。 </li><li>将烧瓶在摇床培养箱中在30℃和150rpm下进行足够时间（4小时）。 </li></ol></li><li>制备的稀细菌溶液（S2）的<ol><li>倒入5ml的LB肉汤培养基经消毒的塑料管。 </li><li>稀释S1用的LB肉汤培养基混合。接着，旋涡振荡溶液。稀的溶液达到所需的光学人的密度值（OD测定在600nm = 0.1）。 注:生物膜的实验通常采用在这附近的OD值。 </li></ol></li></ol> 3，准备好实验装置<ol><li>使用镊子连接柔性塑料管（0.20"ID）到微芯片的入口（S）和（多个）出口的入口和出口进行了预先钻入微芯片（步骤1.5.5）的PDMS部，在该调查，微芯片包括两个入口和一个出口。 </li><li>填充注射器（S）与细菌溶液（S2解决方案），并删除所有气泡注射器（S）。 </li><li>连接注射器头（S）（30克0.5"钝针）到入口管（S），然后连接出口管（S）废容器。 </li></ol> 4，运行实验<ol><li>连接的注射器（S）到注射器尖端（次）。 </li><li>地方和固定注射器（S）到注射泵。然后将光学显微镜的合乎需求的物镜下的微芯片编倍率（ 例如 40倍）。盖上一个活细胞腔室装置，以保持恒定的温度环境细菌生长（30℃， 荧光假单胞菌 ）的微芯片。 </li><li>设置泵以所需流速水平（例如10微升/小时），并启动流体泵送。 </li><li>一旦细菌被引入到室中，生物膜的形成也被启动。生物膜的形成和成熟，通常会发生在一段几小时甚至几天。观察并通过显微镜利用生物膜生长的图像。 </li></ol>

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The authors have nothing to disclose.

我们证明了模拟多孔介质的生物膜研究发展的生境复杂的简单的微流体装置。它有决定了实验的结果，几个关键步骤。它们包括设备的几何形状。而后期的几何形状可以改变，充分的孔隙空间幡形成是必要的。此外，Valiei 等 。1表明，流光的形成只发生在一定的流量范围。在流率低于阈值的情况下，变形的生物膜成缆可能不会被观察到。但是，超过一定的阈另一个流量值，生物膜骨折可以支配，不允许形成幡。可困扰着这些实验的另一个问题是气泡，可以成为被困在微柱阵列中。通常这些泡沫必须通过增加流速最初，然后逐渐减小它为所需的值被删除。 微流控平台如这些提供了一些优势和一些限制。的平台使我们有小培养体积的工作，并具有结合用户定义的功能的灵活性。例如，不同的多孔结构可通过改变微柱阵列的几何参数进行模拟。即使是模仿实际多孔介质的随机结构结构可以制作在微流体平台18。此外，一些这样的信道可在单个设备允许收集显著相关数据进行准确的统计分析来实现。然而，微流体系统通常模仿多孔介质的二维结构。设备，可以模拟多孔介质的立体性通常是相当具有挑战性的制作。 幡的形成和演化都不能很好地理解的是，进一步的研究需要在这个方向。了解飘带是如何形成的，并导致格式成熟生物膜结构的离子将是相关的，以各种各样的方案，包括生物医学装置，如心脏支架，在土壤中的生物膜，和过滤系统的堵塞。我们的微流体平台是朝这个方向迈出的一步。

最近，我们展示了模仿多孔介质1的微流体器件细菌生物膜形成动态。细菌生物膜基本上是由胞外聚合物（EPS）2-4包裹表面聚集的细菌菌落。这些薄膜的细菌可以形成在几乎每一个可以想象小生从光滑表面的更复杂的生境的多孔介质。 Valiei 等人 1所用的微流体装置与微柱阵列来模拟多孔介质结构和研究生物膜形成在该装置的流体流速的函数。他们发现，在一定的水流流态，被称为幡丝状生物膜开始不同柱子之间出现。缆可在同一被拴或两端固体表面上，但是该结构的其余部分被悬浮在液体中。流光形成通常开始后生物膜的初始层已经形成，它的格式离子可支配生物膜的长期演变在这样复杂的栖息地。最近，一些研究人员调查了流光形成的过程中。亚兹迪等 5表明，幡可以在旋涡流从一个振荡的泡沫起源形成。在另一个实验中，RUSCONI 等 6研究了拖缆的形成信道的曲率和通道几何形状的影响。他们发现，该拖缆可在微通道的弯曲部分构成，并流光形态涉及蠕动。最近的研究已经表明，拖缆可以有各种天然的和人造的场景广泛影响，因为它们可以作为前体到成熟的结构中的多孔界面的形成，导致迅速和灾难性的生物膜增殖的生物医学系统，并且还造成实质性的流量 - 结构相互作用等 1,7-9。 生物膜幡常常表格IN复合栖息地，如多孔介质。理解生物膜生长在多孔介质中的环境相关的几个环境和工业过程，如污水生物处理10，保持在情况井筒的完整性，如CO 2的捕获11和在土壤中12堵孔。观察在如此复杂的栖息地生物被膜的形成往往是具有挑战性的，由于多孔介质的透明度。在这种情况下，基于微流体的多孔介质的平台可以证明非常有利的，因为它们允许实时和原位监测。微流体的另一个优点是，建立多个生物反应器在一个单一的生物微流体平台，同时允许在线监测和/或传感器的结合的能力。为执行在一个设备和收集显著相关数据进行准确的统计分析的能力，多个实验室实验的灵活性是重要的副词antage微流体系统13,14。 在上述讨论的范围内，在多孔介质环境理解流光形成动力学是将多个应用程序是有益的。在这项研究中，我们开发的协议，用于在设备模拟多孔介质调查流光形成。微流体平台的制造中，描述了用于细胞培养和实验所需的步骤。在我们的实验中， 荧光假单胞菌的野生型细菌菌株被使用。P.荧光假单胞菌，在土壤中自然存在，对维持土壤生态15了关键作用。该菌株采用已被基因工程改造以表达绿色荧光蛋白（GFP）组成。

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Flourescent Microscope</td>
			<td style="width:255px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB agar</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1425-500</td>
			<td>suspend 40 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB broth</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td>suspend 20 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Biosafety hood</td>
			<td style="width:255px;">Microzone corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Petri-dish</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">875712</td>
			<td>sterile 100mmx15mm polystyrene petri dish</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:325px;">Incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:339px;">Excella E24incubator shaker series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL sterilized centrifuge tube</td>
			<td style="width:255px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td>Polypropylene Rnase-/Dnase-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Tetracycline free base</td>
			<td style="width:255px;">MP Biomedicals</td>
			<td style="width:119px;">103012</td>
			<td>50 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">SYLGARD 184 silicone</td>
			<td style="width:255px;">Dow Corning Corporation</td>
			<td style="width:119px;">68037-59-2</td>
			<td>Elastomer Base and curing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Positive photoresist (AZ4620)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Plastic tube</td>
			<td style="width:255px;">Cole- Parmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for biofilm streamer formation in a microfluidic device with micro-pillars

几个细菌种类具有附着到表面并殖它们在薄膜中称为生物膜的形式的能力。生长在多孔介质生物膜有关的几个工业和环境过程，如污水处理和CO 2封存。我们使用荧光假单胞菌 ，革兰氏阴性好氧性细菌，调查生物膜形成中的微流体装置，模仿多孔介质。微流体装置由微型职位的阵列，它是用软光刻技术制造的。接着，生物膜形成中这些设备具有流量进行了调查，我们证明被称为我们的设备幡丝状生物膜的形成。此处提供的制作与微流体器件组装的详细协议以及细菌培养协议。是为实验与微流体器件的详细过程还伴随着代表介绍结果。

使用上述微细加工协议，PDMS微流体器件构成。 图1示出了扫描型电子显微镜（SEM）的PDMS的图像设备。 图1a示出了该装置的入口部分。甲叉状的入口被创建，以均衡压头跨器件。进一步SEM成像还表明，支柱壁几乎是垂直的（ 图1b）。将培养的细菌溶液（ 图2）中的稀释和其光密度调节至0.1的值。我们研究了生物膜的形成在微流体装置的输入流率的函数。当P。荧光假单胞菌注入设备在低流率的0.8微升/小时，细菌附着和生物膜的形成发生在该装置的壁上。即使经过较长时间（&gt; 20小时），比面拥抱生物膜其它没有其他细菌的结构我们再观察。接着，将同样的实验重复的8微升/小时的流速。在这种情况下，生物膜形成输液稀释的细菌培养物在几分钟后再次启动。然而，几个小时后，出现微柱之间延伸的丝状结构的附近观察到的装置（ 图4）的中间部分。这些丝状结构可以通过不动细菌的存在可视化。这些结构被称为拖缆和它们丝状生物膜那些只拴在一个或两个端部到表面上。该结构的其余部分通常是悬浮在液体介质（如在这种情况下）， 图4示出了生物膜流光结构随时间的演化。缆通常形成由于在粘弹性生物膜的流体剪切的效果， 图5示出了流线和速度轮廓为流过一系列支柱。仿真结果表明，该横幅的在我们的微流体系统的形式本质上是沿着流体流动的流线对齐。流动的结构和形成生物膜缆之间的相关性还没有得到很好的理解。然而，Das和库马尔16最近提出，这些幡形成的固有的粘弹性生物膜的高粘度液体状态。他们根据自己的猜想上的时间尺度生物膜流光形成通常远远超过生物膜的粘弹性松弛时间尺度的观察。生物膜是已知表现为粘弹性体的液体，因此在时间尺度比粘弹性松弛时间尺度要大得多，所以它们基本上表现为高粘性液体17。根据该配方，拖缆可以预计到起源于高剪切应力的位置， 图5示出了高的速度在通道中的位置，并且这些位置用的高剪切应力的位置重合。在最初的PHA<html>生长本身，拖缆附近观察到这些位置（ 图4）来发起。 <img alt="图1" src="/files/ftp_upload/51732/51732fig1highres.jpg" /> 图1的扫描电子显微镜（SEM）图像，所述微流体通道（顶视图）的。 A）入口段，含微柱B）地区。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/51732/51732fig2highres.jpg" /> 图涉及细菌培养2连续的步骤。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> NT"FO:保together.within页="总是"&gt; <img alt="图3" src="/files/ftp_upload/51732/51732fig3highres.jpg" /> 图3设置为微流体实验。1光学显微镜（倒置），2注射器泵，3 -图像及数据采集，4注射器含有染料（可选），5 -注射器含有细菌，6 -废物储存器。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/51732/51732fig4highres.jpg" /> 幡进化图4定时聚焦成像，图像平面对应于Z =设备25微米，即中间。虚线椭圆证明生物膜的飘带。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig4large.jpg" target="_blank">请点击</a>这里查看这个数字的放大版本。 <img alt="图5" src="/files/ftp_upload/51732/51732fig5highres.jpg" /> 图5计算流体力学模拟出流过微柱的流线和速度轮廓。流体流动是由上到下，速度规模为米/秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51732/51732fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

protocol-for-biofilm-streamer-formation-microfluidic-device-with

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.1K 次观看.  University of Alberta. Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. 

视频: Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

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 ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...