作者非常感谢汤姆罗宾逊的手稿，C.Bärtschi和H.奔驰的定制压力控制系统建设的校对。我们还要承认使用洁净室设施第一和光学显微镜中心（LMC），无论是在ETH苏黎世。这项工作是在第七框架计划（ERC开始格兰特，项目编号203428，nμLIPIDs）资助下，由默克雪兰诺公司和欧洲研究委员会（ERC）。

1。 SU-8制作大师准备两个主模具的通道（流体和控制，原理图及尺寸见图2）以下协议，但不同的掩模图案。该过程示于图3a。 <ol><li>开始通过10分钟在180℃下加热4英寸硅晶片装载于旋涂机脱水晶片，并使用下列协议旋涂SU-8 2015: <ol><li>旋转晶片，在100 rpm离心20秒（开放旋涂机的盖子，分配后在该步骤中，盖上盖子分配的SU-8）。 </li><li>旋转晶片，在500 rpm离心10秒（这会扩散抗蚀剂在整个晶片）。 </li><li>旋转晶片在1750 rpm离心30秒（定义的抗蚀剂，以20微米的高度）。 </li></ol></li><li>除去从用丙酮（用棉签）在晶片的边缘抵抗，因为这将否则粘到加热板的下一个步骤。晶圆转移到啊otplate在95℃和热4分钟。的软烘烤之后，暴露在抗蚀剂通过光掩模贴在钠钙玻璃中的掩模对准器，用紫外灯（150毫焦耳/厘米2，在365nm处测量）。 </li><li>在曝光后，再次加热晶片在95℃下进行5分钟的加热板上。冷却晶圆至室温，然后沉浸入SU-8开发者4分钟洗澡。轻轻摇动除去未曝光的SU-8。与洁净室等级异丙醇冲洗晶圆并用氮气吹干。检查在显微镜下，如果开发成功（尤其是电池的陷阱）。如果小区的陷阱被完全开发的，在硅晶片的反射应该是可见的。如果有必要，再开发晶片几分钟。 </li><li>烤晶片在烘箱中2小时，在180℃以除去所有残余的溶剂。检查渠道，台阶仪的高度。如果高度不同于所希望的高度，重复此协议开始于步骤1.1与新的晶片和改变旋转速度（步骤1.1.3）。通过硅烷化的晶片与1H，1H，2H，2H-全氟癸基dimethylchloro硅烷在干燥器中过夜完成母模的制造。 </li></ol> 2。主用于制作微接触印刷<ol><li>为了制备主模具的微接触印刷，脱水的硅晶片10分钟，在180℃下旋涂1毫升HDMS到该晶片在7500 rpm离心30秒（抗蚀剂更好地粘合到硅烷化的表面）。旋涂2毫升AZ1518积极抵制这一协议: <ol><li>静态分配的抗蚀剂上的晶片。 </li><li>旋转晶片为5秒，在500转每分钟。 </li><li>晶圆旋转60秒，4000转。 </li></ol></li><li>在100℃下50秒的软烘烤的加热板上后，暴露出通过光掩模和掩模对准抗蚀于UV光（21毫焦/厘米2在365nm处）。开发抗拒AZ 726的浴75秒。冲洗晶圆开发与去离子水和吹塑ðRy的氮气。通过加热所述晶片，在115℃下进行50秒的去除残留的溶剂。 Silanize晶圆在真空下过夜。 </li></ol> 3。微流控芯片的制作一个两层的设备设计用于这些实验。将两层分开制备和粘接在一起之前，在芯片最终结合到载玻片上（ 图3b）。此步骤描述了PDMS层的生产和微接触印刷的PDMS压模。 <ol><li>制备10:1混合物的PDMS和固化剂。总共制备约80g的PDMS（使用我们的设计中，这将导致在10个芯片）。混合两部分大力和脱气的PDMS，直到混合物是无气泡的（〜30分钟）。 </li><li>放置晶片的培养皿，并用胶带内的控制层上的底部。接着，倒〜50克PDMS的顶部，并把该晶片为至少2小时的烘箱中，在80℃以确保完全固化的PDMS。重复同样的步骤对微接触印刷晶圆，这个加入约20 g的PDMS是必要的。 </li><li>旋涂在晶片上的PDMS与流体层以2,000 rpm的旋转速度，以形成一个约40微米高的PDMS层。在80℃下固化的PDMS在烘箱中1小时以上</li><li>当这两个部分被固化，从晶圆去除控制层，切成用剃刀刀片件。通过使用1毫米活检冲床冲压力连接孔。对于存储，最好是把一块胶布在渠道。 </li><li>粘合两层，取旋涂晶片和顶端部分，并将其放置在等离子体清洁器23。等离子体处理后，迅速配合大的工作距离显微镜下两个部分。添加围绕置于顶部的部分一些备用的PDMS。这个步骤不是必须的，但是它有利于从晶片除去的PDMS。将晶片在烘箱中在80℃下至少1小时。 </li><li>用手术刀小心地从晶片剥离PDMS和削减芯片。冲头进入孔为用1.5毫米的活检穿孔机的流体连接。 </li><li>将PDMS芯片现在已完成并且可以存储数月。可选的:如果芯片被用于一个水库，切200μl的移液管尖的上端部，并使用一些备用的，半固化的PDMS粘上它上面。把芯片在烘箱中再次在80℃下放置至少1小时。 </li></ol> 4。粘接到玻璃幻灯片使用该设备进行直接的酶测定法，贴合后所需的唯一步骤是表面的封闭。对于这个协议，进行A。但是，使用微流控芯片免疫分析或酶联免疫吸附，请参阅协议B，限制结合位点只在载玻片（ 即全内反射荧光显微镜或SPR）。如果这种限制并不重要，是指A，但用生物素标记的结合物在步骤4.3，并继续执行步骤4.8在协议B来创建一个功能齐全的表面。 在此之前进行协议A和B:这是明智的过滤所有用过的蛋白质溶液介绍他们到芯片之前的。碎片和蛋白聚集体可以以其他方式阻止细胞的陷阱，从而减少了室可用于分析的次数。为此目的，可使用非蛋白吸附过滤器单元把它们引入通道之前过滤所有的解决方案。 协议A（酶测定） <ol><li>要完成微流体装置，粘接部位的PDMS到载玻片。要做到这一点，首先清洗用肥皂水，蒸馏水和乙醇的载玻片上。用氮气流干燥的幻灯片。用透明胶带清洁PDMS的一部分。 </li><li>将PDMS部分和清洁载玻片到等离子体清洁器45秒在18 W·为了保证紧密粘接，把芯片的热板上（100℃）5分钟。 </li><li>粘接后，填写与f芯片luids。一个简单的方法来实现这一目标是使用离心力。切为200μl移液管尖端的下部。用于流体通道，填充它们用封闭溶液（牛血清白蛋白（4％重量/体积）或聚-L-赖氨酸）接枝的聚乙二醇（0.05％重量/体积），并将提示进入入口。填充控制层入口用水或用等渗溶液， 例如 PBS中。将芯片放入离心机（800×g离心）5分钟。该频道现在应该充满液体完全。如果不是，请重复此步骤。 </li><li>孵育至少30分钟，在室温下封闭溶液。以10微升/分钟使用注射泵的流速洗涤液从流体层的PBS。该设备已准备好进行细胞实验。 </li></ol>协议B（免疫） 对于从步骤4.7起表面改性的完整概述，请参考表1。 <ol start="5"><li>在cubate PDMS印章的微接触印刷与BBSA / BSA溶液（ 如 1-100）。 30分钟后，用蒸馏水彻底清洗邮票和干燥在氮气流中的时间戳。迅速将邮票上的清洁玻璃板滑动（ 图4）。 </li><li>暴露与邮票载玻片用在等离​​子体清洁器的微流体芯片，以氧等离子体45秒在最大功率（18瓦）的顶端部分一起。等离子体处理之后，除去印模和对齐印刷表面的设备的微容器的下方。放置在芯片上的热板上30分钟，在50℃下</li><li>以封闭剩余的表面上，引入无菌过滤的牛血清白蛋白溶液（4％（重量/ PBS中V））与一个离心分离机芯片（参见步骤4.2）。孵育1小时，洗涤液从流体层，用PBS（参见步骤4.3）中。该芯片可以被直接使用或贮存长达2周，在4℃下在潮湿的箱。 </li><li>对于一个功能齐全的结合表面活性E，采用抗生物素蛋白（0.025％（W /在PBS V））推入储。使用5微升/分钟的流速进行30分钟通过流体通道撤回该抗生物素蛋白溶液。随后，冲洗的PBS 10分钟。彻底清洗油箱。 </li><li>随后，加入蛋白G（0.0025％（W /在PBS V））和另外30分钟平齐。用PBS再10分钟后洗净。接着，10分钟，添加目标抗体（0.0001％（重量/ PBS中五））。 10分钟后，停止流动15分钟，以允许绑定。接着，洗净，用PBS 10分钟。 </li></ol> 5。细胞实验该协议是由于各种可能的试验以通用的术语。作为一个例子所需要的G6PDH毒性测定用试剂中给出。该设备的初始细胞捕集效率是大约2.5％， 即 5超过200个细胞将被困住。细胞陷阱的最终占用强烈依赖于所使用的细胞系，该协议中止细胞系和时间将细胞通过信道进行冲洗。对于细胞自然生长于悬浮液（如U937），单细胞可以在大约75％的几分钟后腔室被发现。其它腔要么不填写，或者由两个或更多个细胞占据。在一般情况下，单个单元可容纳于贴壁的细胞系更小。当使用这里给出的，而温和的助悬协议的百分比降低至约30％（HEK，MLT细胞）。单细胞占用可以通过细​​胞的胰蛋白酶处理得到改善，但是，这也可能改变的实验的结果。 取决于目标分子，吸附或吸收到PDMS会影响结果。可吸附通过阻断与BSA或PLL-G-PEG表面减少。吸收是不可能的大部分亲水性的生物分子，但它应该检查（小）的亲脂性的细胞成分。 <ol><li>根据特定的协议准备的细胞（ 如 </EM&gt;药品加成）。对于悬浮细胞，直接增加细胞芯片。为粘合剂的细胞系中，使用无酶的解离缓冲液，以产生细胞悬浮液。它们引入到芯片上，以减少细胞团的量之前，筛选细胞。 </li><li>使用注射泵和前向气流加载单元上的芯片，而不是撤回细胞悬浮液。使用5微升/分钟的悬浮细胞系和10-20微升/分钟流速较高粘接细胞的流速，因为减少了细胞的非特异性附着到通道壁上。 </li><li>当陷阱被填充后，关闭室。如果许多细胞非特异性粘附于表面，尽量在10-30微升/分钟的流量与细胞分离液清洗他们离开。 </li></ol>协议A（酶测定） <ol start="4"><li>通过芯片撤回裂解液。在这个例子中，该缓冲液是添加吐温20（10 mM的Tris-盐酸，10低渗缓冲毫米氯化钾，1.5毫摩尔MgCl 2，1％（V / V）吐温20）。对于裂解，快速启闭室（ 即 500毫秒为10-50微升/分钟7,21流速）。为G6PDH测定中，加入2毫摩尔葡萄糖-6 - 磷酸，0.5mM的NADP，0.5 U / ml的心肌黄酶和0.3mM的刃天青的裂解缓冲液。开始监控溶解后，立即动力学反应。 </li></ol> 注 :选择，适用于测定任何缓冲，但是裸记住，强烈的清洁剂（如海卫，SDS）裂解细胞立即会导致分析物室开放期间的损失。 协议B（免疫） 对于ELISA细胞实验（流速，室状态等 ）的简短说明，请参考表1。 <ol start="5"><li>用于ELISA，所需的检测试剂（ 例如第二抗体，标记抗原）直接添加到裂解缓冲液中。此处添加矫饰N 20降低了非特异性吸附。根据步骤5.4裂解细胞。孵育混合物10-15分钟，以允许结合（培养时间取决于所使用的抗原和抗体）。 </li><li>抗体酶结合物吸附到非特异性的通道壁可导致检测试剂贮存器和信道已经里面的一个转换。为减少这种吸附问题，通过芯片在孵化时间刷新用于检测酶的永久抑制剂（室封闭，加入检测试剂之前）。对HRP，用20mM的HCl的水以永久失活的非特异性吸附的酶。 </li><li>后的培养时间，介绍的检测试剂（ 例如 Amplex Red试和过氧化氢 ​​为HRP）的芯片。不久再次打开室洗未结合的第二抗体路程，添加检测试剂。立即开始监视动力学反应。 </li><li>校准:固定该抗体对感兴趣的目标。关闭室。在缓冲液中或在细胞裂解物中的片制备不同浓度的分析物。适用浓度的芯片和快速交换内室（500毫秒开标时间）的音量。这个开口时间足够短，以确保没有额外累积发生从分析物冲洗，并检测分子从微室的体积只有一个特定的号码。从溶液内抗原的已知浓度和所述腔室的体积（625 PL），计算出室内的量。此后，继续执行步骤5.5并介绍了检测部分。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration.">Microfluidic large-scale integration.</a> Science. 298 (5593), 580-584 (2002).</li><li>Gao, Y., Majumdar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+valve-enabled+microfluidic+cell+co-culture+platform+and+demonstration+of+its+applications+to+neurobiology+and+cancer+biology.">A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology.</a> Biomed. Microdevices. 13 (3), 539-548 (2011).</li><li>White, A. K., VanInsberghe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+microfluidic+single-cell+RT-qPCR.">High-throughput microfluidic single-cell RT-qPCR.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (34), 13999-14004 (2011).</li><li>Schwanh&#228;usser, B., Busse, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7437), 337-342 (2011).</li><li>Kortmann, H., Kurth, F., Blank, L. M., Dittrich, P. S., Schmid, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+real+time+analysis+of+protein+secretion+from+single+cells.">Towards real time analysis of protein secretion from single cells.</a> Lab Chip. 9 (21), 3047-3049 (2009).</li><li>Huang, Y., Cai, D., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-+and+Nanotechnologies+for+Study+of+Cell+Secretion.">Micro- and Nanotechnologies for Study of Cell Secretion.</a> Anal. Chem. 83 (12), 4393-4406 (2011).</li><li>Huang, N. T., Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+microfluidic+platform+for+in+situ+cellular+cytokine+secretion+immunophenotyping.">An integrated microfluidic platform for in situ cellular cytokine secretion immunophenotyping.</a> Lab Chip. 12 (20), 4093-4101 (2012).</li><li>Eyer, K., Stratz, S., Kuhn, P., K&#252;ster, S. K., Dittrich, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementing+enzyme-linked+immunosorbent+assays+on+a+microfluidic+chip+to+quantify+intracellular+molecules+in+single+cells.">Implementing enzyme-linked immunosorbent assays on a microfluidic chip to quantify intracellular molecules in single cells.</a> Anal. Chem. 85 (6), 3280-3287 .</li><li>Aebi, U., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glow+discharge+unit+to+render+electron+microscope+grids+and+other+surfaces+hydrophilic.">A glow discharge unit to render electron microscope grids and other surfaces hydrophilic.</a> J. Electron Microsc. Tech. 7 (1), 29-33 (1987).</li><li>Pandolfi, P. P., Sonati, F., Rivi, R., Mason, P., Grosveld, F., Luzzatto, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+housekeeping+gene+encoding+glucose+6-phosphate+dehydrogenase+(G6PD):+G6PD+is+dispensable+for+pentose+synthesis+but+essential+for+defense+against+oxidative+stress.">Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress.</a> EMBO J. 14 (21), 5209-5215 (1995).</li></ol>

作者宣称，他们有没有竞争的财务权益。

微流体技术开辟为单细胞分析的新的和引人入胜的可能性。特别是，可能性陷阱和微流控工具分别固定细胞已经允许在单细胞特性和响应系统的短期和长期的研究。此外，细胞在高频率的微滴，在微型芯片上所产生的封装性，使单个细胞分泌的研究，这不能用传统的流式细胞仪设备来执行。微滴的方法，但是，当有需要的分析协议孵化和洗涤步骤一定的局限性。本方法结合了细胞俘获和隔离的两?...

在过去的许多研究已经揭示了大量的细胞群1-3中的细胞-细胞间的差异，特别是信号处理4，或细胞内的生物分子如蛋白质5,6，代谢物，辅因子和7,8的量。这些非均匀性被认为是对细胞的适应和进化9极其重要，而且在癌症等疾病63-82的产生和治疗中发挥关键作用。因此，在单细胞水平的研究是在生物和药理研究兴趣高的，尤其是当这些研究揭示了不同的细胞反应与生物活性的化学物质，处理后。 近年来，许多分析平台已经开发了促进单个活细胞的分析或细胞含量的化学组成。而荧光激活细胞分选（FACS）是金圣andard对单个活细胞的非常高通量分析，该方法不能被用于细胞内或分泌化合物的定量。微流体平台的出现已经答应为定位，治疗和观察单个细胞的新的分析策略。微流控芯片中的一个里程碑达成与由地震和同事14,15实现灵活的PDMS阀门的整合。这些阀门是有用的，因为它们可以隔离区域上的芯片， 例如分开两种文化16。此外，它们特别适用于单细胞分析，因此有助于减少分析物的稀释问题。这种方法对单细胞分析的力量已经由汉森和他的同事，谁从几百单细胞在17平行分析的基因表达最近展示。 当靶向蛋白质和代谢物的分析是非常困难的，由于缺乏合适的一mplification方法中，大量不同的化合物存在，和它们的化学性质的变化。此外，大多数胞内生物分子预计将存在于几万种18的次序低的拷贝数，因此所用的分析方法必须具有高灵敏度。更强大的测定例如免疫测定和酶联免疫分析（ELISA）是很难集成到微流控装置，因为它们需要几个洗涤和温育步骤，以及表面固定。 由于这些挑战，这并不奇怪，只有几个例子被报道了，其中蛋白质或代谢物进行定量的单细胞水平。例如，关于荧光化合物的分泌的研究已有报道19,20。最近，用ELISA法的实施，提出了从细胞培养分泌（非荧光）蛋白（THP-1细胞）21和单（我的分析mmune）细胞10。靶向细胞内的蛋白质，Shi 等人开发了促进细胞内蛋白质的鉴定通过免疫测定11的装置的信令在肿瘤细胞途径的分析的微流体装置。然而，蛋白质的唯一的相对量进行了测定，并没有酶促扩增被用来增加对低丰度蛋白的信号。 最近，我们能够以单细胞俘获微型装置用荧光测定法8和免疫22（ 图1）相结合。细胞是被动被困在微米级关卡结构，它允许供应和（快速）交换媒介和其他化学试剂无细胞的任何运动。围绕每个陷阱的环形阀使细胞的分离在一个非常小的量（以下简称"微室"）。该阀引入细胞裂解（低渗）缓冲，他后立即启动NCE防止细胞内分子或分泌的分子扩散而去。最重要的是，由于量（625 PL）的小尺寸，避免大量稀释的分子。此外，由于溶解和分析都在芯片的同一位置进行的，不存在损失，由于运输分析物。这里所描述的芯片设计包括8交替的行，7或8微容器的，共计60微容器。腔被致动以行，从而使交叉污染沿一条线被排除。 该平台可以与荧光测定法，以及免疫学测定法（ 图1d）组合使用。对于后者，我们建立了协议的抗体，这是与芯片的生产和装配过程兼容的固定。因此，该平台开辟了道路敏感，可靠和可计量的测定在单细胞水平。到现在为止，我们已经使用该设备为i的分析ntracellular和分泌的酶（通过酶测定法相对定量），细胞内辅助因子，蛋白质和小分子（绝对定量通过终点测定法或ELISA）。在下文中，我们描述了由多层软光刻和抗体的形成图案的协议通过微接触印刷和表面化学的手段的手段芯片制造的过程。此外，芯片的使用和操作的一些例子给出。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Name of the Reagent</td><td>Company</td><td>Catalogue Number</td><td>Comments (optional)</td></tr><tr><td>SU-8 2015</td><td>MicroChem Corp. (Netwon, MA)</td><td>n.a.</td><td></td></tr><tr><td>1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane</td><td>ABCR (Karlsruhe, Germany)</td><td>AB103608</td><td></td></tr><tr><td>4-Methylumbelliferyl &amp;beta;-D-N,N&amp;prime;-diacetylchitobioside hydrate</td><td>Sigma Aldrich</td><td>M9763</td><td></td></tr><tr><td>Acetone</td><td>Merck VWR (Darmstadt, Germany)</td><td>100014</td><td></td></tr><tr><td>Avidin</td><td>AppliChem (Axon Lab AG)</td><td>A-2568</td><td></td></tr><tr><td>AZ 1518</td><td>AZ Electronic Materials (Wiesbaden, Germany)</td><td>n.a.</td><td></td></tr><tr><td>AZ 726 developer</td><td>AZ Electronic Materials (Wiesbaden, Germany)</td><td>n.a.</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A-4503</td><td></td></tr><tr><td>Bovine serum albumin, biotin</td><td>Sigma Aldrich</td><td>A-8549</td><td></td></tr><tr><td>Cell dissociation buffer</td><td>Invitrogen</td><td>13151-014</td><td></td></tr><tr><td>Hexamethyldisilazane (HDMS)</td><td>Sigma Aldrich</td><td>40215</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fluka</td><td>84422</td><td></td></tr><tr><td>Isopropanol</td><td>Merck VWR (Darmstadt, Germany)</td><td>109634</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Fluka</td><td>63068</td><td></td></tr><tr><td>MR developer 600</td><td>Microresist technology GmbH (Berlin, Germany)</td><td>n.a.</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>10010-031</td><td></td></tr><tr><td>PLL-g-PEG grafted</td><td>SuSoS, (D&amp;uuml;bendorf, Switzerland)</td><td>n.a.</td><td></td></tr><tr><td>PLL-g-PEG grafted biotin</td><td>SuSoS, (D&amp;uuml;bendorf, Switzerland)</td><td>n.a.</td><td></td></tr><tr><td>Potassium chloride</td><td>Fluka</td><td>60132</td><td></td></tr><tr><td>Protein G, biotin</td><td>Sigma Fine Chemicals</td><td>41624</td><td></td></tr><tr><td>Silicon wafer</td><td>Si-Mat (Kaufering, Germany)</td><td>n.a.</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit (PDMS)</td><td>Dow Corning</td><td>39100000</td><td></td></tr><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Biorad</td><td>1610716</td><td></td></tr><tr><td>Tween 20</td><td>Biorad</td><td>1706531</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Material Name</td><td>Company</td><td>Catalogue Number</td><td>Comments (optional)</td></tr><tr><td>0.22 &amp;micro;m PES syringe filter</td><td>TRP</td><td>99722</td><td></td></tr><tr><td>1/1.5 mm biopsy puncher</td><td>Miltex, York PA</td><td>33-31AA/33-31A</td><td></td></tr><tr><td>Cell Trics filter 20 &amp;micro;m</td><td>Partec</td><td>04-004-2325</td><td></td></tr><tr><td>Centrifuge Sigma 3-18K</td><td>Kuehner</td><td>n.a.</td><td></td></tr><tr><td>Hotplate HP 160 III BM</td><td>Sawatec, Sax, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>MA-6 mask aligner</td><td>Karl Suess</td><td>n.a.</td><td></td></tr><tr><td>Multizoom AZ100M microscope</td><td>Nikon Corporation</td><td>n.a.</td><td></td></tr><tr><td>Photomask</td><td>Microlitho, Essex, U.K.</td><td>n.a</td><td></td></tr><tr><td>Plasma Cleaner PDC-32G</td><td>Harrick</td><td>n.a.</td><td></td></tr><tr><td>Spin coater Modell WS-400 BZ-6NPP/LITE</td><td>Laurell</td><td>n.a.</td><td></td></tr><tr><td>Spin Modules SM 180 BM</td><td>Sawatec, Sax, Switzerland</td><td>n.a.</td><td></td></tr><tr><td>Step profiler Dektak XT Advanced</td><td>Bruker</td><td>n.a.</td><td></td></tr><tr><td>Syringe pump neMESYS</td><td>Cetoni</td><td>n.a.</td><td></td></tr></tbody></table>

a microfluidic chip for the versatile chemical analysis of single cells

我们提出了一个微流体装置，使多个单体电池并联定量测定细胞内生物分子。为此目的，将细胞被动被困在一个微容器的中间。在激活时，控制层，所述细胞是从一个小的腔室周围的容积隔离。周围的体积然后可以在不影响分离的细胞进行交换。然而，在该腔室的短的开闭，在该腔室中的溶液可在几百毫秒取代。由于腔室的可逆性，可以将细胞暴露于不同溶液依次以高度可控的方式， 例如 ，用于温育，洗涤，最后，细胞裂解。该密封的微容器使裂解液的潴留，尽量减少和细胞裂解后，控制稀释。自体溶解和分析出现在同一位置，高灵敏度被保持，因为没有进一步ðilution分析物的损失或运输过程中发生。因此，微室的设计使之从单个细胞释放的细胞内分子（阿托摩尔，zeptomoles）非常小的拷贝数的可靠和可重复的分析。此外，许多微容器可以被布置成阵列格式，允许在一次许多细胞的分析，考虑到合适的光学仪器被用于监视。我们已经使用该平台的概念验证研究，分析细胞内蛋白质，酶，辅助因子和相对或绝对量化的方式第二信使。

我们的平台能够分析存在于或由单个细胞产生的多种细胞内的以及所分泌的分子。在这里，我们想呈现不同的实例研究强调了各种可能的分析。我们将举一个例子，对于分泌的酶（ 图5a），以及细胞内的酶（ 图5b）和蛋白质（ 图5c和d）所示 。更多的例子，如辅助因子或小分子，请参阅Eyer 等（2012）和Eyer 等人 （2013）。 <p class="jo...

Watch this Scientific Journal Video about A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells at JoVE.com

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

在这篇文章中，我们提出了一个微流控芯片单细胞分析。它使细胞内的蛋白质，酶，辅因子，以及第二信使通过荧光测定法或免疫测定装置的量化。

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

a-microfluidic-chip-for-versatile-chemical-analysis-single

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

14.8K 次观看.  ETH Zurich, Switzerland. 在这篇文章中，我们提出了一个微流控芯片单细胞分析。它使细胞内的蛋白质，酶，辅因子，以及第二信使通过荧光测定法或免疫测定装置的量化。 

视频: A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

用可寻址滴微阵列计算单细胞中的蛋白质

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

科研

癌症研究

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

生物工程

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Millifluidics的化学合成和时间分辨机理研究

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

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 ...

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 ...

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 ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...