The authors thank Scott Longwell for helpful comments and edits to the manuscript and Robert Puccinelli for device photography. The authors acknowledge generous support from a Beckman Institute Technology Development Grant. K.B. is supported by a NSF GFRP fellowship and the TLI component of the Stanford Clinical and Translational Science Award to Spectrum (NIH TL1 TR 001084); P.F. acknowledges a McCormick and Gabilan Faculty Fellowship.

1.多层器件设计注:不同的高度和/或光致抗蚀剂的特性必须按顺序在晶片中不同的制造步骤来创建最终复合功能被加入。因此，被包括为每个单独的高度和光致抗蚀剂图案在晶片必须在其自身的掩模（ 图4）进行打印。 <ol><li>下载计算机辅助设计（CAD）绘图程序（ 如 AutoCAD的教育版）。 </li><li> "通过绘制4晶圆面积"圈子定义4。晶圆设计（ 图4，其他资源）是作为一个例子。 </li><li> 4"晶圆轮廓内，发生设备使用接壤300微米折线矩形。光刻过程中使用对齐这些设备的边界。 </li><li> 为每个不同的高度或光致抗蚀剂所需的最终设计（ 即流轮不同的层，流量低，流量大，使用图层面板的设计和控制）。 <ol><li>相应的层上所需的特定高度的设计特点。该示例的设计示出了4种不同的活性层，每个都有其自己的颜色（ 图4）。 注:设备国界，全球性的文本，并且晶片轮廓应自己的层上进行（即 1负的设计），其中后，将出现在所有层全球对齐。不同的光刻胶的特性（如完全密封的阀门必须与正制造抗拒）都必须出现在不同的层，无论高度。 </li></ol></li><li> 使用封闭装置境内零宽度的折线设计，设备功能。 <ol><li>考虑表1的设计参数，以增加成功制造的机会。 </li><li>对于每一个高度，选择在图层面板层，并添加了高度的所有功能。 </li></ol></li><li> 准备使用每个4"晶圆圈插入一个5之内"矩形边框的基本模板文件（其他资源）的透明薄膜印刷设计。每一层将在为顺序加入各光致抗蚀剂层的一个单独的透明薄膜进行印刷。 注:本基本遮盖文件表示用于打印的最终设计。 <ol><li>为了完成设计，将所有图层关闭除1负，AZ50 XT阀层。整个晶片与有源层（ 即 ，阀）和全局特征（ 即 ，设备的边界）进行复制。 </li><li>打开基本模板文件并粘贴到设计矩形有权AZ50 XT阀门。使用外部晶圆边缘对齐，并随后粘贴后删除。 </li><li>重复层的其余部分（ 例如，在设计实例:流动方形低，流动方高，控制）。例如透明性文件中提供（其他资源）。 </li><li>发送文件到一个COM商用印刷公司（ 例如，FINELINE成像）的投影胶片上打印。使用32000 DPI进行打印&gt; 10微米的功能和高达50,000 DPI较小的特点。如果需要的功能小于7微米，责令铬掩模而不是透明胶片。 </li></ol></li></ol> 表1:设计参数与建议。设计考虑在微流体装置的CAD设计过程中避免常见的陷阱。 <a href="http://ecsource.jove.com/files/ftp_upload/55276/Jove_Table_1.xlsx" target="_blank">请点击这里查看此表。 （右键点击下载）。</a> 2.准备一个晶圆光刻注意:这些步骤还出现在表格式见表2。 <ol><li> 在洁净室或指定洁净区，洁净和脱水4"测试级硅晶片（单边Polished）。 <ol><li>用甲醇冲洗晶片良好。 注意:没有进一步清洗步骤需要如果使用下述的SU-8的粘附层。从这个协议偏离其它粘结层（ 例如 ，HMDS）往往需要更彻底的清洁，如食人鱼蚀刻。 </li><li>吹干用N 2或压缩空气。 </li><li>在95℃下烘烤上的铝加热板上10分钟，以完全蒸发溶剂。 </li></ol></li><li> 制造SU-8 2005年的均匀5微米厚层，提高后续光阻剂层的附着力。 <ol><li>放置在清洁晶片上旋涂机，开启真空以它固定到旋转卡盘，和吹走灰尘用N 2或压缩空气。 </li><li>适用于1-2毫升SU-8 2005负性光刻胶的晶圆和自旋中心如下:蔓延:500转，10秒，133转/秒 加速;投:3000转，40秒，266转/秒的加速度。 </li><li>移除晶圆和软烘烤通过切换设置在65℃下保存2电炉和95℃按照下述程序C的晶片:65℃:2分钟，95℃:3分钟，65℃:2分钟。 </li><li>允许晶片冷却至室温。 </li><li>放置晶片在紫外掩模对准的卡盘和无掩模（&#39;整片曝光&#39;）暴露于124毫焦耳的总能量沉积（这里，在〜6.2毫瓦/厘米2灯强度20秒）。如果可能，选择难接触模式，实现了300微米晶圆:面具的分离。 </li><li> 65℃:2分钟，95℃4分钟，65℃:2分钟通过切换设置在65℃下保存2电炉和95℃如下之间的晶片去除晶片和曝光后烘烤。 </li></ol></li></ol> 制造圆形阀门 <ol start="3"><li>使用在线AZ50 XT阀预测资源26计划的自旋速度为所需阀门的尺寸和高度。 注:以下步骤将DEPO坐阀定义和回流舍入正光刻胶55微米层。 </li><li>放置在晶片上旋涂机，开启真空以它固定到旋转卡盘，和吹走灰尘用N 2或压缩空气。 </li><li>申请AZ50 XT正光致抗蚀剂的2-3毫升到晶片的中心。旋转如下:蔓延:200转，10秒，133转/秒的加速度;投:1200转，40秒，266转/秒的加速度;快旋删除边珠:3400转，1秒3400转/秒的加速度。 </li><li>在5"培养皿中，仔细地放下晶圆，让20分钟放松。 </li><li>软烘烤在电炉上在晶片65℃ - 112℃，22分钟，450℃/ h的变化速度。 </li><li>取出晶片，并让在培养皿中用于环境补液在RT休息过夜。 </li><li>磁带流圆形透明面罩5"玻璃板打印面朝下（最接近晶圆），并装入UV掩模对准面罩定位。暴露晶片UV的930兆焦耳在6个周期（ <eM&gt;例如，在6.2〜毫瓦/平方厘米2灯强度25秒的6个周期，30秒等待风险之间的时间）。 </li><li>在25毫升AZ500k 1的搅拌浴中立即开发晶片:6"玻璃盘3开发3-5分钟或直到浴变成紫色和功能出现。 <ol><li>取出晶圆，并用去离子水冲洗干净。 </li><li>评估采用轮廓（10.5毫克手写笔力）回流焊前的高度。 注:根据厂家说明书进行操作的轮廓，仔细定位力手写笔旁边剖析之前所期望的层上的功能频道。在本协议所使用的设置有以下几条:手写笔力10.5毫克，长度1000微米，速度200微米/秒，政权下了。 </li></ol></li><li>回流硬烤晶片以熔融和圆形阀特性如下:65℃ - 190 ℃，15小时，10℃/小时斜坡速度。 </li><li>让晶片冷却至室温。评估后回流的高度使用PROFIlometer（10.5毫克手写笔力）。 55微米±2微米的高度应该预计该设备的几何形状。 </li></ol> 3.编造在串联可变高度功能<ol><li>继续与发达晶圆与珠合成器设计流量低，流量高和人字搅拌机透明可变高度制作。 </li><li>要为设计调整方案，使用制造商的数据表25来确定曝光能量，旋转速度和烘烤时间参数，允许±5％的容差。 注:此协议使用编造纺在阀功能SU-8 2050负性光刻胶55微米高的流量低层。 </li><li> 放置在旋涂机的清洗晶片，开启真空以它固定到旋转卡盘，和吹走灰尘用N 2或压缩空气。 <ol><li>应用1-2毫升的SU-8 2050负光致抗蚀剂的晶片和自旋的中心为:传播:500转，10秒，133转/秒的加速度;投:3000转，40秒，266转/秒的加速度。旋光刻胶在开发阀的功能。 </li></ol></li><li>小心地将纺晶片5"培养皿并让在平坦的表面，或直至任何纹路消失20分钟放松。 </li><li> 65℃:2分钟，95℃:8分钟，65℃:2分钟由两个电炉设定在65℃，95℃如下放置卸下晶片和软烘烤。 </li><li>允许晶片冷却至室温。 </li><li>胶带流量低透明度掩模的石英5"玻璃板打印面朝下（最靠近晶片）并装入紫外线掩模对准的掩模定位器。 </li><li>放在紫外线光刻卡盘的晶片上，用显微镜目镜或相机，小心地将新的流量低层功能，流量阀轮层功能。通过调整设备的边界水平，垂直和倾斜轴的装置边境的重点面具开始。接下来，对准十字功能赌注吐温层。最后，确认阀门特点相交流量低配适用。 </li><li>暴露在170毫焦耳UV沉积（在6.2〜毫瓦/ 平方厘米28秒）。 </li><li> 65℃:2分钟，95℃:9分钟，65℃:2分钟通过设定在65℃下保存2电炉和95℃如下之间切换取出晶片和后曝光烘烤。 </li><li>不发展，允许晶片冷却至室温并继续流动高层的制造。这个流程高层将光刻胶30微米添加到未开发的55微米的光致抗蚀层产生先前未曝光的位置85微米的特点。 </li><li> 传播:3.3〜3.10使用SU-8 2025年用这些修改的旋涂设置流量高图层蒙版，重复步骤500转，10秒，133转/秒的加速度;投:3500 rpm时，40秒，266转/秒的加速度。 <ol><li>暴露在198毫焦耳UV沉积（32号在6.2〜毫瓦/ 平方厘米）。 </li></ol></li><li>如果没有开发板的平，让晶圆冷却至室温，然后继续混沌混合器人字形层的制造。在该层的最终特征将具有125微米的总高度:55微米，从流最低层，从流广场层30微米，并从该混沌混合器人字纹层40微米（参见图3），并包括35微米的人字形槽。 </li><li> 采用SU-8 2025人字形图层蒙版进行了以下修改，以确保字槽完全内流动高通道概述重复步骤3.3至3.10。 <ol><li>使用下面的软烘烤程序:65℃:2分钟，95℃:7分钟，65℃:2分钟。 </li><li>暴露在148毫焦耳UV沉积（第24号在6.2〜毫瓦/ 平方厘米）。 </li></ol></li><li>所有层被完成之后，浸渍晶片在25毫升6"玻璃皿的SU-8显影剂的搅拌浴3.5分钟或直至特征清楚地出现发展，检查featurES可以选择使用立体镜清晰，定义要素边界。 </li><li>硬烤晶片，以稳定的热板上所有光致抗蚀剂特征如下:65℃ - 165℃，2小时30分钟，120℃/ h的变化速度。 </li><li>评估功能高度使用轮廓（10.5毫克手写笔力）的所有层。 </li></ol> 4.控制晶圆制造<ol><li>干净，脱水，并制造新的4"硅晶片上的5微米的粘合层如第4。 </li><li>制造使用SU-8 2025负性光刻胶25微米控制层。 </li><li>放置在晶片上旋涂机，开启真空以它固定到旋转卡盘，和吹走灰尘用N 2或压缩空气。 </li><li>应用1-2毫升的SU-8 2025负光致抗蚀剂中的晶片和自旋的中心为:传播:500rpm下，10秒，133转/秒的加速度;投:3500 rpm时，40秒，266转/秒的加速度。 </li><li>通过之间切换取出晶圆和烘软2电炉设定在65℃，95℃如下:65℃:2分钟，95℃:5分钟，65℃:2分钟。 </li><li>允许晶片冷却至室温。 </li><li>对齐控制透明度面膜5"玻璃板装入紫外线光刻。 </li><li>将晶圆紫外线光刻机的卡盘和暴露在155毫焦耳UV沉积（在6.2〜毫瓦/平方厘米2灯强度25秒）。 </li><li> 65℃:2分钟，95℃:6分钟，65℃:2分钟通过设定在65℃下保存2电炉和95℃如下之间切换取出晶片和后曝光烘烤。 </li><li>在6"玻璃盘浸泡在晶圆25ml的SU-8开发的搅拌浴中1分钟或直至出现功能开发检查功能使用立体镜。 </li><li>硬烤晶片，以稳定光致抗蚀剂特征如下:65℃ - 165 ℃，2小时30分钟，120℃/小时斜坡速度。 </li></ol> 5.硅烷晶圆处理，便于PDMS电梯-OFF <ol><li>放置在晶片架完成的晶片自由水或水溶性试剂的通风橱内的钟罩真空干燥器内。 </li><li>引擎盖下，使用滴管三氯1滴（1H，1H，2H，2H -perfluorooctyl）硅烷（PFOTS）向干燥器内部载玻片和地点。 </li><li>关闭干燥器盖和1分钟施加真空。 </li><li> 1分钟后，关掉真空，而无需重新加压或排空钟罩。 </li><li>让混合物坐而雾化PFOTS大衣晶圆表面10分钟。 </li><li>打开钟罩盖，并用镊子取出晶圆。放入一个培养皿PDMS的副本成型。在处置适当危险废物硅烷涂层幻灯片。 注:涂氟化硅烷晶片可用于成百上千倍，而无需重新处理。 1:10 PDMS牺牲层可以被投射在晶圆，治愈，并丢弃第一硅烷处理去除后Ëxcess硅烷基从晶圆表面。 </li></ol> 6. PDMS副本成型<ol><li>根据现有的开放式访问协议16在玻璃上的"推"几何制造多层微流体装置。 注:详细的协议，还可以在网站上27发现。 </li><li>通过目测，确保所有阀门正确对齐控制线和所有入口（在流和控制层两者）都在继续之前完全打孔。 </li></ol> 7.从生产水凝胶液滴的珠<ol><li>连接管（ 如聚乙烯）装有水流量控制系统（ 例如，注射泵，流体控制器，或者与水库28一个开源的电磁阀阵列）。 </li><li>金属插脚连接管，并连接到设备端口的控制线入口。通过设置流动续加压设备控制线选择的rol系统25psi的每一行。确保阀关闭并重新打开在显微镜下检查。 注:按照所选择的流控制系统制造商的说明。在这项工作中，定制的软件控制的气动系统使用的25磅的压缩空气（压力）和大气压力（减压）之间进行切换的电磁阀施加压力到每一行。此系统上的细节可以在讨论中找到。 </li><li> 准备试剂和装油的定制微流体压力容器。 <ol><li>使用推针，冲两个孔在低温小瓶管的顶部，插入毛细管PEEK管成一个孔，并插入连接于管道进入第二孔的金属销。 </li><li>在用环氧树脂密封到位油管。让干燥1小时。 </li></ol></li><li>在等待时，在微量离心管中，暂停3.9毫克LAP光引发剂的入100微升去离子水的（[LAP] = 39毫克/毫升）以制备光引发剂用于聚合液滴水凝胶珠粒溶液。避光。 </li><li>在第二离心管中，加入132微升去离子水，172微升PEG二丙烯酸酯，12微升LAP溶液，和85微升HEPES缓冲液，使水凝胶微滴溶液。 </li><li>转移的水凝胶微滴溶液到完成低温管容器中。 注:添加剂用于其它应用，如纳米晶体，磁性粒子或生物分子可以包含在HEPES组件内。 </li><li>低温管容器的管路连接到一个可控制的压力源和将PEEK管连接到该设备试剂入口。 </li><li>用2％体积/体积离子表面活性剂制备的轻质矿物油的10毫升（ 例如 ，司盘80）和0.05％EM90为油滴乳液。滤波器使用0.22微米的注射器过滤器和负载1毫升进入第二低温管容器。 </li><li>将PEEK管在用于液滴收集设备的出口。 </li><li>去除空气bubbl通过加压油，水，或PEG混合物入口（4磅操作压力）从设备ES。打开所有阀门。 1分钟后，或直至气泡都通过PDMS设备渗透到每个阀门相继关闭的流体通路。例如，脱泡搅拌机人字，打开阀门进1，混合1出来，混合垃圾。然后减压进1，将1出来，混合垃圾，直到所有的泡沫都不见了。 </li><li>当该装置脱泡后加压，减压RO1油阀，并设置油压10psi的。 </li><li>设置的PEG混合物的压力到9磅，depressure上游阀（入口1，滴眼液1），并根据需要调整，以产生所需尺寸的液滴。液滴的大小可以通过显微镜用摄像头，支持50 fps或较高者确定。 </li><li>当液滴稳定后，从UV光源定位5mm的点（ 例如，一个UV点固化系统与液体光导（LLG）或聚焦的UV LED）在D的聚合区域evice和从UV源应用100毫瓦/厘米2紫外光（365纳米）。 </li><li>珠加压阀筛收看聚合珠收集并确保液滴硬化成珠。 LLG调整需要达到充分聚合。 </li><li>珠卸压阀筛并通过出口PEEK管收集到珠管。 </li></ol>

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The authors declare that they have no competing financial interests.

这项工作表明用于与阀和可变高度的几何形状的多层微流体装置可被调整为具有简单的修改根据我们的在线工具26和制造商的说明书25工艺参数的任何应用程序的完整多步光刻协议。该协议旨在神秘面纱多层光刻为希望构建微流体器件超越了简单的，被动层模具的研究人员。 多层微流体装置使单个软聚合物装置中追求先进功能的应用范围从?...

<html>在过去的15年里，微流体作为一个领域经历了快速的增长，随着新技术使流体的操控在微米尺度1的爆炸。微流体系统是湿实验室功能吸引力平台，因为小的体积必须实现更快的速度和灵敏度的潜力，而在同一时间急剧增加的吞吐量和通过利用比例2,3的经济降低成本。多层微流体系统已在高通量生化分析应用中尤其显著的影响，如单细胞分析4，5，6，单分子分析（ 例如，数字PCR 7），蛋白质晶体8，转录因子结合测定F"&gt; 9，10，和细胞筛选11。 微流体的一个核心目标是能够在单个设备中执行复杂的流体操作总生化分析12设备"芯片上的实验室"的发展。的多层软光刻技术的发展已帮助实现通过使芯片上阀，混合器的创作这一目标，和泵小体积13，14，15内主动地控制流体。尽管他们的优势和应用证明，许多微流体技术仍然由非专业的用户主要是unharnessed。广泛采用已经在部分具有挑战性，由于难以获得微细加工设施，也由于制造技术沟通不足。这是FO更是如此- [R多层微流体装置设有阀门或复杂的几何结构:关于重要的设计参数和制造技术的详细，实用的信息的缺乏往往在开始涉及这些设备的设计和创作项目阻止新的研究人员。 本文旨在通过展示制作多层微流体装置与阀和高度可变的特点，从设计参数，并开始通过各种制造步骤移动一个完整的协议，以解决这方面的知识差距。通过专注于制造的最初的光刻工序，该协议其他补充协议的微流控芯片16，描述从模具铸造设备和运行具体实验下游步骤。 与单片芯片上阀的微流体设备是由两层组成:一个"流"层，其中，所关注的流体在微操纵通道，和一个"控制"层，其中含有空气或水的微通道可以选择性地调节在流动层14的流体流动。这两层分别制造在一个单独的硅成型的主人，其随后在称为方法用于聚二甲基硅氧烷（PDMS）复制品成型"软光刻17"。以形成多层器件中，每个的PDMS层都浇铸在它们各自的成型主人，然后对准彼此，从而形成具有在每一层信道的复合PDMS设备。在那里流和控制信道彼此交叉并仅通过一个薄膜被分开的位置形成阀门;控制信道的加压挠曲该膜以阻塞流动通道，并在本地移动所述流体（ 图1）。 活性片上阀可以以多种方式来制造，这取决于所需的最终应用。阀门可以在任何一个"下推"或"推"几何构造，这取决于控制层是否高于或流层的下方（ 图1）15。 "向上推"的几何形状允许较低关闭压力和防止脱层更高的器件稳定性，而"下推"几何结构允许流动通道是在与键合衬底直接接触，赋予基板表面的选择性官能化或图案化的优点供以后功能18，19。 阀也可以是故意泄漏"筛子"阀或完全密封的，这取决于流动通道的横截面轮廓。筛阀门是用于捕获珠，细胞或其它macroanalytes 1是有用的，并通过使用典型的负光致抗蚀剂（ 即，SU-8系列），其公顷被制造已经矩形轮廓。当一个控制信道被加压超过这些阀的区域中，控制和流层之间的PDMS膜各向同性偏转到阀的矩形轮廓，而不密封所述角落，允许流体流动，但是捕集宏观尺度颗粒（ 图1）。相反，完全密封的微流体阀由包括阀位置的圆形光致抗蚀剂的一小片制成。与此几何，控制信道的加压偏转膜对圆形流层以完全密封通道，停止流体流动。在流动层圆形轮廓通过典型的光刻步骤后熔化和正性光刻胶（ 例如，AZ50 XT或SPR 220）的回流产生的。之前我们已经证实，阀地区的回流焊后高度取决于所选特征尺寸21。此协议演示了这两种阀的几何形状与制造在一个胎圈合成装置。 <img alt="图1" src="/files/ftp_upload/55276/55276fig1.jpg" /> 图1:多层微流体阀的几何形状。为筛典型的"推"器件结构和前（上）和后（底）加压完全密封的阀。 <a href="http://ecsource.jove.com/files/ftp_upload/55276/55276fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 设备也可以包括复杂的被动功能，例如混沌混合器13和片上需要一个单一的流动层内的多个不同高度的特征电阻20。实现可变高度流动层，不同的组已经采用许多方法，包括印刷电路板蚀刻22，多层的PDMS浮雕对准23，或多工序Photolithography 24。我们小组发现在单个模制主多步光刻是一种有效的和可再现的方法。要做到这一点，采用建设层负性光刻胶（例如 SU-8系列光刻胶）厚渠道没有发展的每一层的应用程序之间的一个简单的光刻技术。每一层是根据在硅基主其使用厚度制造商的说明25在负光致抗蚀剂纺丝。这种高度的特征然后被图案化到用固定到玻璃掩膜板和对准曝光之前的先前纺丝层的特定透明掩模（ 图2）的层。在多步光刻，各层之间的精确对准是在形成完整的可变高度流动通道的关键。对准之后，各层经受依赖厚度曝光后烘烤。没有发展，下一层是卡ilarly图案。以这种方式，高大特征可以通过使用多个掩模来建立一个单一的流动晶片上层 - 层。跳过每个步骤之间的发展，以前光致抗蚀剂层可以被用于产生复合高度特征（ 即两个25微米的层可以使一个50微米的功能）24。此外，通道地面的功能，如乱搅拌机字槽13可使用与先前曝光的功能层进行。最后显影步骤结束处理，创建具有可变高度（ 图3）的功能的单个流晶片。 这里，提供了一种用于多步光刻完整的协议，其中包括的必要制造芯片上阀和具有多个高度流动通道的所有过程的例子。该制造协议在多层微流体珠合成需要阀门和variab的上下文中提出的乐高为特点的功能。该装置包括T连接在油鞘生成水滴，片电阻通过控制泊肃性，混沌混合器均化液滴组件以调节流速，并且两个完全密封和筛阀，以使涉及多个试剂自动化工作流输入。使用多步光刻，这些特征各自不同的层上，根据高度或光致抗蚀剂制成;下面层在这个协议来构建:（1）流量圆形阀层（55微米，AZ50 XT）（2）流动的低层（55微米，SU-8 2050）（3）流动的高层（85微米，SU- 8 2025年，30微米添加剂高度），和（4）人字槽（125微米，SU-8 2025年，40微米添加剂高度）（ 图3）。 水凝胶珠可被用于各种应用，包括选择表面官能用于下游分析，药物包封，拉迪otracing和成像测定法，和细胞掺入;我们以前使用的这些装置的更加复杂的版本以产生含有镧系纳米发光材料20光谱方面经编码的PEG水凝胶珠。这里讨论的设计都包含在其他资源任何实验室在他们的研究工作如果需要使用。我们预计，该协议将提供专家和非专家在制造多层微流体装置阀门或复杂的几何形状，以降低壁垒微流体进入和提高制造成功的机会都感兴趣的一个开放的资源。

<table border="0" cellpadding="0" cellspacing="0" width="1709">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Materials</td>
			<td style="width:332px;"></td>
			<td style="width:259px;"></td>
			<td style="width:659px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mylar Transparency Masks, 5&#34;</td>
			<td>FineLine Plotting</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5&#34; Quartz Plates</td>
			<td>United Silica&#160;</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#34; Silicon Wafers, Test Grade</td>
			<td>University Wafer</td>
			<td>452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU8 2005, 2025, 2050 photoresist</td>
			<td>Microchem</td>
			<td>Y111045, Y111069, Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Az50XT&#160;</td>
			<td>Integrated Micromaterials</td>
			<td>AZ50XT-Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AZ400K 1:3 Developer</td>
			<td>Integrated Micromaterials</td>
			<td>AZ400K1:3-CS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyrex 150 mm glass dish</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3140150-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wafer Petri Dishes, 150 mm</td>
			<td>VWR</td>
			<td>25384-326</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wafer Tweezers&#160;</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>78410-2W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOTS)</td>
			<td>Sigma-Aldrich</td>
			<td>448931-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#34; x 3&#34; glass slides</td>
			<td>Thomas Scientific&#160;</td>
			<td>6686K20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RTV 615 elastomeric base and curing agent PDMS set</td>
			<td>Momentive&#160;</td>
			<td>RTV615-1P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing, 0.02&#34; O.D.&#160;</td>
			<td>Fischer Scientific&#160;</td>
			<td>14-171-284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary PEEK tubing,&#160; 510 um OD, 125 um ID</td>
			<td>Zeus</td>
			<td>Custom</td>
			<td>360 um PEEK is readily available by Idex (catalog number: 1571)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyro 4 mL tube</td>
			<td>Greiner Bio-One</td>
			<td>127279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epoxy, 30-minute</td>
			<td>Permatex</td>
			<td>84107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal Pins, 0.025&#34; OD, .013&#34; ID</td>
			<td>New England Small Tube</td>
			<td>NE-1310-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol) diacrylate, Mn 700</td>
			<td>Sigma-Aldrich</td>
			<td>455008-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate photoinitator&#160;</td>
			<td>Tokyo Chemical Industry Co.</td>
			<td>L0290</td>
			<td>We typically synthesize LAP in-house.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>330779-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Span-80</td>
			<td>Sigma-Aldrich</td>
			<td>85548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ABIL EM 90</td>
			<td>UPI Chem</td>
			<td>420095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Equipment</td>
			<td>Equivalent equiptment or homebuilt setups will work equally as well</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mask Aligner</td>
			<td>Karl Suss</td>
			<td>MA6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Profilometer</td>
			<td>KLA-Tencor</td>
			<td>Alpha-Step D500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spin Coater</td>
			<td>Laurell Technologies</td>
			<td>WS-650-23</td>
			<td>Any spincoater can be used that accepts 100 mm wafers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum Dessicator, Bell-Jar Style</td>
			<td>Bel-Art</td>
			<td>420100000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oven</td>
			<td>Cole-Palmer</td>
			<td>WU-52120-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Spot Curing System with 3 mm LLG option</td>
			<td>Dymax</td>
			<td>41015</td>
			<td>UV LEDs, Xenon Arc Lamps, or other UV sources of the same intensity work equally as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MFCS Microfluidic Fluid Control System</td>
			<td>Fluidgent</td>
			<td>MFCS-EZ</td>
			<td>Syringe pumps, custom pneumatics or other control systems can also be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automated control scripting</td>
			<td>MATLAB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hotplate</td>
			<td>Tory Pines Scientific</td>
			<td>HP30</td>
			<td>Any hotplate with uniform heating (i.e. aluminum or ceramic plates) will suffice.&#160;</td>
		</tr>
	</tbody>
</table>

multi-step variable height photolithography for valved multilayer microfluidic devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

这里，我们通过使能够从液滴生成的聚乙二醇（PEG）水凝胶珠的设备展示阀，可变高度的多层微流体模具的制造 （ 图2）。完整的制造过程的概要包括在图3.使用从以前的工作的设计元素，珠合成采用4的高度在其流动层包括:（1）倒圆为层流调制（55微米）（2）流动AZ50 XT阀低信道为在较高电阻（55微米）将试剂（3）流动高通道在到出...

Watch this Scientific Journal Video about Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices at JoVE.com

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

multi-step-variable-height-photolithography-for-valved-multilayer

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

14.2K 次观看.  Stanford University. Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads. 

视频: Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes.&#160;Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 &#181;l. &#160;Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest.

多步制备技术，以恢复多个代谢化合物的种类进行深入和信息化代谢组学分析

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...