作者要感谢以下研究理事会和赠款支持:EPSRC，HFSP，韩国国家研究基金会（项目编号R11 - 2009 - 044 - 1002 - 0K20904000004 - 09A050000410）。

1。 Microchip的制造<ol><li> SU8主制造。 要获得30秒40微米的高度，一个硅晶片上旋涂SU8 - 50（MicroChem公司）在3000转的微流体通道软烤热板在65 ° C时5分钟和95 ° C下30分钟蒸发溶剂和致密的薄膜。冷却后，暴露在300兆焦耳/ 2 SU8抵制360-440 nm的辐射下适当的口罩。曝光之后，晶圆烘烤在65 ° C时2分钟，4分钟，95℃。冷却后，制定并搅拌5分钟未曝光的地区（Micrpoposit EC溶剂，Chestech），（ 五）使用新鲜欧共体溶剂冲洗晶圆然后干下气体产生一个干净的表面。治疗正己烷和甲醇清洗晶圆。 SU8掌握制造过程，完成的最后一步（1,1,2,2 - perfluoocytl）蒸发三氯硅烷表面上（在40分钟的干燥器）。 </li><li> PDMS的curinG，切割和打孔。 要形成结构的微流体基板，我们在10:1（W / W）树脂交联剂的比例混合Sylgard 184（道康宁），然后倒在主。德加干燥器中，然后治设备（顶层）为一小时，在65 ° C。切了主治愈的PDMS和剥离。创建访问孔，流体管，用活组织检查打孔。固化的PDMS的另一层为20分钟（8克）均匀撒在一个10x10厘米的培养皿中的树脂，在65 ° C。将准备的顶层和底层，然后一夜之间治愈在65 ° C。剥离芯片从培养皿和使用的骰子。 </li></ol> 2。实验装置<ol><li>微流控设置<ol><li>注射器（塑料或玻璃从Beckton Dickinson公司或从上海黄金交易所分析科学）填写所需的解决方案，确保无气泡留在油管的任何部分。液滴生成​​两个阶段使用。水溶液（分散）PHASE包含感兴趣的试剂（例如在生长介质中的悬浮细胞或分子荧光团的解决方案）。在这种情况下行为作为连续相，油相，一般是形成从石油和表面活性剂的混合物，如飞雨表面活性剂在2％氟化油（飞雨技术）（3M Fluorinert电子液体FC - 40）;或2％在矿物油（Croda Singapore禾）跨度80。 </li><li>适合一端的针的注射器针尖一样的内部直径的管材（聚乙烯管，哈佛仪器）。应切断管子，以最短的长度，以尽量减少由于振动扰动流不稳定。 </li><li>连接油管的另一端，直接在PDMS基板砸出的洞。更复杂的解决方案，如使用石英毛细管端口或连接器（如厄普丘奇Nanoports），也可以实现一个更强大的界面之间的微流体装置和油管。连接插座港口的油管和直接到收藏家（废物收集或进一步分析物的处理）芯片。 </li><li>装入注射泵的注射器（如博士4400 Hpsi可编程的注射泵，哈佛仪器），并定义每个阶段（通常微升每分钟）所需的注入流量。一个建议，以避免水相，这将导致液滴的形成不稳定（取决于设备几何）的PDMS润湿的最低比例是1流油/水率。出于同样的原因，它也建议，先冲洗油相单独任何微流体通道，通过引进水相前，。用于本实验的最终设置是在图2a所示。 </li></ol></li><li>光学系统设置<ol><li>为了探测高时空分辨率的微流体通道内的小卷（几皮升）使用共聚焦显微镜（带自动贩卖机版次微米定位能力）。 </li><li>使用理想的高量子产率的水溶性荧光分子。激发分子使用的波长相匹配所涉及的荧光团吸收激光的操作。例如，可以有效地激发常见的荧光分子，如荧光素异硫氰酸（FITC）和Alexa Fluor公司488使用488纳米激光二极管（如PicoQuant有限公司，LDH - PC - 485）。在633 nm，荧光分子，如得克萨斯州红色或变藻蓝蛋白（APC），可以有效地退出使用他/ Ne激光（如新港的R - 31425）。 </li><li>使用脉冲激光在几MHz的重复率（几个ns脉冲分离）和足够的有区别的一生属性，荧光寿命成像（FLIM）实验的荧光团经营。 </li><li>使用光束控制反射镜和光学控制梁的高度，以及光束的方向。 </li><li>使用分色镜反映到客观升的激光束ENS。如果两束激光同时使用，可以安装双频段分色镜使反射的两束激光（如488和633 nm的光束从色度科技股份有限公司z488/633rdc镜是理想的）。 </li><li>使用无限远校正，高数值孔径（NA）显微镜物镜（即奥林巴斯60 × / 1.2 NA，水浸泡），使一个微流体通道内的激光光紧张的焦点。高大微（&gt; 100微米）的工作时使用的目标，必须适当选择，以确保其工作距离有效地覆盖全高度的微。 </li><li>收集的样品，使用相同的高NA目标，并通过相同的分色镜传输（微流体通道内）发出的荧光。 </li><li>使用单频段或双频段发射过滤器（例如，从色度科技股份有限公司z488/633过滤器），除去残留的激发光。 </li><li>聚焦荧光emissioN到75微米的针孔，使用一个平凸透镜（如50.2 F，新港有限公司）。共聚焦显微镜物镜平面位置的针孔。 </li><li>使用另一种分色镜（如630dcxr科技股份有限公司，色度）分离到两个探测器的荧光信号。 </li><li>过滤排放过滤器（如hq540/80米的色度滤波器技术公司）的分色镜反射的荧光和一个平凸透镜的焦点（例如，F = 30.0，内径25.4毫米，Thorlabs）到第（ &#39;绿色&#39;）探测器。对于涉及次要的红色荧光团的实验传输排放过滤器（例如，一个从色度科技股份有限公司hq640lp滤波器）与另一分色镜过滤的荧光，然后集中到第二个探测器（"红"）与另一平凸透镜。 </li><li>用于检测荧光光子雪崩光电二极管（如AQR - 141，EG＆G公司，在Perkin - Elmer）。最后的安装说明<strONG&gt;图2b。 </li></ol></li></ol> 3。液滴的生成和数据采集<ol><li>液滴的生成方法<ol><li>您可以使用两个主要的微配置产生水滴:注重一个流或丁字路口的格式（图3a和 3b）6,7两种方法都相当于只要形成液滴作为微本身。由于使用这些几何图形将两个不混溶流体和接口的后续发展毛细管的不稳定，是自发产生的单分散液滴（如一些femtoliters小）产生的剪切力的结果。 </li><li>您可以以不同的方式封装的试剂:试剂可以准备和混合所需的混合浓度/关闭芯片，或者他们可以被分开到芯片，然后结合在一起之前，液滴的形成（ 图 3c ）。这最后的方法提供了更高的灵活性，因为混合物/浓度，可以通过简单的调整相对流率修改。据悉注射器中的细胞悬液，细胞封装，应搅拌，以避免凝结的细胞在实验的时间表。 </li></ol></li><li>荧光数据采集。 <ol><li>夫妇的雪崩光电二极管检测器（2.2.12）的电子信号多功能DAQ设备的数据记录（例如，美国国家仪器公司的PCI 6602），在个人电脑上运行。 </li><li>对于FLIM的实验探测器连接到一个时间相关单光子计数（TCSPC）卡（如TimeHarp 100，PicoQuant有限责任公司），一个单独的PC上运行。此卡允许使用的TCSPC方法获得的荧光寿命数据:每个检测到的荧光光子相关的入射激光脉冲，因此每个发出的荧光光子被分配了一个延迟时间。此数据可以安装在一个单个或多个指数衰减曲线获得的荧光/ s的辐射率系数/ s的估计Deconvolute的原始数据，仪器响应函数（IRF）的探测器:这是使用低浓度金胺O解决方案（其中展品的几个皮秒荧光寿命）8 。 </li></ol></li></ol> 4。混合液滴内的细胞识别和映射<ol><li>混合液滴内的细胞识别和映射<ol><li>使用大肠杆菌十大应变检测的可行性实验。卢里亚- Bertani肉汤隔夜培养细胞实验前相匹配的光密度为0.5。 </li><li> 0.4μMSYTO9和1μM碘化丙啶用于检测细胞的可行性。 DNA插层染料与DNA结合后其荧光强度增加20倍以上。 SYTO9是一种绿色荧光染料，那就是我mbrane透水和碘化丙啶是一种红色荧光染料膜防渗。因此，活细胞荧光的"绿色"而死​​亡的细胞表现出"绿色"和"红"排放。 </li><li>使用绿色/红色荧光检测设置在2.2中引入）。 </li><li>使用便携，迷你磁力搅拌器（犹他州生物柴油的供应，犹他州）内挑起一个3ML BD plastipak注射器装的7mm的磁力搅拌棒，以防止细胞沉淀的细胞悬液。 </li></ol></li><li>使用FLIM的混合事件的映射<ol><li>光学探针量集中在一个微液滴沿都流淌着高度的一半。 </li><li>从两个水溶液（ 图3c），每个包含一个具有不同特点的荧光寿命（非交互）荧光团的表格滴。 </li><li>从通道一侧开始，开展沿整个宽度在1微米的间隔通道的每一个实验。渠道版GES可以很容易地确定荧光强度急剧下降，一旦激光束集中在其附近。 </li><li>实现算法，以区别于背景噪声信号连发（与水滴）油相，并建立每个突发时间。 </li><li>实施第二个算法提取实验已经进行了特定的宽度沿每个液滴的长度的延迟时间和强度值9 。 </li><li>然后使用最大似然估计（MLE）的算法，以评估为每个液滴在实验中的荧光寿命。8，在实验中的所有液滴的平均寿命值，降低最终的MLE计算错误（更多的水滴探测，误差越小）。 </li><li>一旦已获得各宽一生的轨迹，结合在2D地图上的所有轨迹。由于每个终身价值是与特定LAR的两个荧光基团的混合物，浓度（或混合）地图可以得到。 </li><li>另外，一个混合液滴的三维地图，可以很容易获得通过重复这个协议在不同的位置，沿微流体通道的高度。 </li></ol></li></ol> 5。数据分析<ol><li>重新诠释的原始实验数据文件到相应的计算语言，使他们能够得到进一步的分析（如文本文件，包含光子计数随着时间的推移变化，可以随时到Matlab中提取的数据处理和分析）。您可能已经编写特定的代码，以访问包含文件（如为FLIM）在更为复杂的实验，或为了一生的轨迹运行MLE的算法，或建立混合的格局2D地图数据。 </li></ol> 6。代表性的成果细胞识别典型的例子的VAR作为一个基于细胞的实验时间功能的光子计数iation显示在图 4a 和 C，超过两百毫秒内。重要的是，卢里亚- Bertani肉汤（LB培养基）确定水液滴边界的弱荧光背景的行为，而不同的光子阵阵，对应于单个细胞的存在，可在此背景下之上区分开来。 LB荧光背景的特点是由约矩形截面（红色和绿色的虚线标记）。 全宽半高（FWHM），荧光阵阵，所产生的大肠杆菌大肠杆菌细胞的30倍，比背景液滴的事件，这是液滴的相对长度（40微米，相当于30 picolitres量）和E.一致短大肠杆菌细胞（1.5-2.0微米）10。 双通道检测离子系统，活细胞或死细胞的存在，可以区别于绿色和红色通道分析图4 B和 D显示的概率分布来描述，每个液滴细胞的数量。 使用FLIM的混合事件的映射分子荧光寿命是个别荧光团，只能由它的化学环境的影响的内在属性。因此，它的测量可以用来获得卓越的分辨率和精度比综合荧光测量，这是依赖于实验因素（如浓度，激发强度和光学收集环境信息（如溶液的pH值，温度，极性和粘度）效率）。9,11 提出的其他地方， 有 8个是地图使用两个荧光基团与DI液滴内的混合模式fferent寿命值。包含两个荧光物种（非交互）的混合物的衰减将在双指数形式如公式1所示: <img src="/files/ftp_upload/3437/3437eq1.jpg" alt="公式1" />个人的寿命定义为τ1和τ2和每个组件的幅度分别是由α1和α2。的平均寿命可以被定义如下: <img src="/files/ftp_upload/3437/3437eq2.jpg" alt="公式（2）" />凡β1和β2的每个组件的一小部分，并定义如下: <img src="/files/ftp_upload/3437/3437eq3.jpg" alt="公式3" />由于探头体积是固定的，液滴的那个位置上的流动，一生的轨迹，沿着在那个特定的宽度液滴的长度值可以由此获得的。一个特定的宽度特征的轨迹，求均值6000液滴之间的ED，可以观察图 5a 。 贯穿整个通道宽度相结合的运动轨迹，导致形成一个二维的浓度（或混合）地图。一个典型的结果是在图5b。 平均之间的大量水滴的结果，所得到的结果的标准误差可以大大降低（1％与95％置信区间为图 5a的标准误差） 。假定所有的水滴几乎相同的一生轨迹的测定方法。这被证明是基本上正确的，主要有两个原因:如果液滴显著不同，获得的平均寿命轨迹将有平坦，少不同的配置文件（和沿长度平均液滴在一生的尖锐分歧始终检测）。此外，结果重现性好，无论个人液滴分析或液滴的数量计算中使用。 8 <img src="/files/ftp_upload/3437/3437fig1.jpg" alt="图1" /> 图1微流控芯片制造的流程。 <img src="/files/ftp_upload/3437/3437fig2.jpg" alt="图2" /> 图2）图代表的注射泵和注射器，连接到液滴的形成微流控芯片; B）一个典型的两个激光光学装置示意图-两个荧光基团（绿色和红色）的激发和检测 。 DC =分色镜，EM =排放过滤器，L为镜头，PH值=针孔，APD雪崩光电二极管探测器。 <img src="/files/ftp_upload/3437/3437fig3.jpg" alt="图3" /> 图3芯片上的液滴形成战略:一）流重点配置;二）丁字路口配置。 C）在医生的芯片封装oplets，两个原本单独的水溶液试剂。 <img src="/files/ftp_upload/3437/3437fig4.jpg" alt="图4" /> 图4（一）死亡记录的0.2秒的痕迹和光学读数（三）活体细胞（细胞悬液流速:1μL-1分钟，油:2μL 分钟 -1） 。每一个拱形的信号对应的弱荧光LB培养基，形成水液滴，液滴边界标有虚线。绿色信号代表超过633纳米的波长633纳米红色信号的读数。细胞的区别在于垂直插层染料的DNA产生的尖峰。 （二）死亡的细胞和（d）活细胞内单液滴的蜂窝入住的概率分布。 <img src="/files/ftp_upload/3437/3437fig5.jpg" alt="图5" /> 图5 a）典型沿在一个特定的宽度液滴的长度的平均寿命轨迹B ）;平均轨迹相结合的典型代表全宽沿液滴探测到一个二维的寿命（或浓度表示为％FITC /％STR - AF488）地图。

<ol>
	<li>Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+the+future+of+microfluidics.">The origins and the future of microfluidics.</a> Nature. 442, 368-368 (2006).</li><li>Zheng, B., Angew, I. s. m. a. g. i. l. o. v., F, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Microfluidic+Approach+for+Screening+Submicroliter+Volumes+against+Multiple+Reagents+by+Using+Preformed+Arrays+of+Nanoliter+Plugs+in+a+Three-Phase+Liquid/Liquid/Gas+Flow.">A Microfluidic Approach for Screening Submicroliter Volumes against Multiple Reagents by Using Preformed Arrays of Nanoliter Plugs in a Three-Phase Liquid/Liquid/Gas Flow.</a> Chem. Int. Ed. 44, 2520-2520 (2005).</li><li>Huebner, A., Sharma, S., Srisa-Art, M., Hollfelder, F., Edel, J. B., Demello, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdroplets:+A+sea+of+applications.">Microdroplets: A sea of applications.</a> Lab on a Chip. 8, 1244-1244 (2008).</li><li>deMello, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+detection+of+chemical+reactions+in+microfluidic+systems.">Control and detection of chemical reactions in microfluidic systems.</a> Nature. 442, 394-394 (2006).</li><li>Thorsen, T., Roberts, R. W., Arnold, F. H., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+pattern+formation+in+a+vesicle-generating+microfluidic+device.">Dynamic pattern formation in a vesicle-generating microfluidic device.</a> Phys. Rev. Lett. 86, 4163-4163 (2001).</li><li>Anna, S. L., Bontoux, N., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+dispersions+using+"flow+focusing"+in+microchannels.">Formation of dispersions using "flow focusing" in microchannels.</a> Appl. Phys. Lett. 82, 364-364 (2003).</li><li>Casadevall i Solvas, X., Srisa-Art, M., deMello, A. J., Edel, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+Fluidic+Mixing+in+Microdroplets+with+1+&#x03BC;s+Time+Resolution+Using+Fluorescence+Lifetime+Imaging.">Mapping of Fluidic Mixing in Microdroplets with 1 &#x03BC;s Time Resolution Using Fluorescence Lifetime Imaging.</a> Anal. Chem. 82, 3950-3950 (2010).</li><li>Robinson, T. A., Schaerli, Y., Wootton, R., Hollfelder, F., Dunsby, C., Baldwin, G., Neil, M., French, P., deMello, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+background+signals+from+fluorescence+thermometry+measurements+in+PDMS+microchannels+using+fluorescence+lifetime+imaging.">Removal of background signals from fluorescence thermometry measurements in PDMS microchannels using fluorescence lifetime imaging.</a> Lab. Chip. 9, 3437-3441 (2009).</li><li>Huebner, A., Srisa-Art, M., Holt, D., Abell, C., Hollfelder, F., deMello, A. J., Edel, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+protein+expression+in+single+cells+using+droplet+microfluidics.">Quantitative detection of protein expression in single cells using droplet microfluidics.</a> Chem. Commun. 1218, (2007).</li><li>Edel, J. B., Eid, J. S., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+single+molecule+FRET+efficiency+determination+for+surface+immobilized+DNA+using+maximum+likelihood+calculated+lifetimes.">Accurate single molecule FRET efficiency determination for surface immobilized DNA using maximum likelihood calculated lifetimes.</a> J. Phys. Chem. B. 73, 2986-2990 (2007).</li></ol>

我们描述了一个微流体装置的制造，实验装置和微滴形成和试剂封装和光学检测处理飞沫的相关协议。 （液滴检测单个细胞和流动的水滴内的混合过程的映射）的两个例子代表普遍的应用，目前正在与液滴微流体研究。 作为所提出的实验说明，目前使用的液滴微流体平台一定吸引力的特点，如高通量，高准完美的监禁，高重复性... ...大量的生产信息和正在生成该信息的高速，虽然，如果从这些小型化的平台得到充分利用，需要使用具有高时空分辨率的快速检测方法。在这种情况下，我们证明，这是可以使用高度精确的荧光光谱技术。特别是，FLIM的检测技术（这使得利用短脉冲激光与重复周期为几纳秒），揭示了能力，获得非常快的信息快速流动的水滴（约每秒200）。在这种情况下，检测到的事件的时间分辨率低至1微秒。液滴的大小和浓度限制，使用较大的数值孔径镜头和更大的功率激光器会很容易地允许高达5微米（液滴大小的限制被光刻决议所施加的小水滴内的纳米浓度检测制造过程中的亚微米级定位阶段）。 微流体液滴的理想人选，进行大规模的实验，涉及少量的试剂，单目标/事件。目前这项技术的局限性，涉及的困难，产生博士oplets从种类繁多，在高通量的方式不同来源（几十或几百）。此外，目标和操作难度，每个单液滴产生的规定，严重的限制，这些技术的实用性。因此，这些问题是重要的研究中心，旨在开发必要的技术解决方案，使微流体液滴的平台，成为一个高吞吐量应用的研究和分析标准的参考方法的努力。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>荧光素异硫氰酸（FITC） </td><td>试剂</td><td> Sigma - Aldrich公司</td><td> F3651 </td></tr><tr><td>链霉亲和的Alexa Fluor 488 </td><td>试剂</td><td>分子探针</td><td> S11223 </td></tr><tr><td>全氟萘烷</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> P9900 </td></tr><tr><td> 1H，1H，2H，2H - perfluorooctanol </td><td>试剂</td><td> Sigma - Aldrich公司</td><td> 370533 </td></tr><tr><td> Sylgard 184有机硅弹性体套件</td><td>试剂</td><td>英国Premier Farnell集团有限公司</td><td> 1673921 </td></tr><tr><td>金胺- O </td><td>试剂</td><td> Sigma - Aldrich公司</td><td> 861030 </td></tr><tr><td> 485纳米脉冲激光二极管</td><td>设备</td><td> PicoQuant </td><td> LDH - PC - 485 </td></tr><tr><td> TCSPC卡</td><td>设备</td><td> PicoQuant </td><td> TimeHarp 100 </td></tr><tr><td>分色镜</td><td>设备</td><td>色度科技股份有限公司</td><td> z488rdc， </td></tr><tr><td>显微镜物镜</td><td>设备</td><td>奥林巴斯</td><td> UPLSAPO 60XW </td></tr><tr><td>雪崩光电二极管</td><td>设备</td><td> AQR - 141，EG＆G公司，在Perkin - Elmer）。 </td><td> AQR - 141 </td></tr><tr><td> DMEM培养液</td><td>试剂</td><td> Invitrogen公司</td><td> ABCD1234 </td></tr><tr><td> SYTO9 </td><td>试剂</td><td> Invitrogen公司</td><td> S34854 </td></tr><tr><td>碘化丙啶</td><td>试剂</td><td> Invitrogen公司</td><td> P3566 </td></tr><tr><td>大肠埃希氏菌10株</td><td>细胞</td><td> Invitrogen公司</td><td> C4040 - 10 </td></tr></tbody></table>

fluorescence detection methods for microfluidic droplet platforms

微流体平台进行化学和生物学的发展在很大程度上带动一系列潜在的好处，伴随着系统的小型化。优点包括能够有效地处理纳米femoto升卷的样品，功能部件的简便集成，实现大规模复用，提高分析吞吐量，更好地控制和减少器乐足迹的内在倾向。 近年来，多大的兴趣集中在基于液滴微流体系统（或分段流动）和高通量实验平台的潜能的发展。2-4这里油包水型乳液是自发形成的微流体通道作为一个结果混溶两个阶段之间的毛细管不稳定。更重要的是，准确界定的体积和化学成分的微滴，可以产生频率几kHz。此外，封装在一个连续的混溶阶段，两个样本串扰和分散（扩散和泰勒）分隔的隔离舱的利益试剂可以消除，从而导致最小的交叉污染的能力，时间分析过程丝丝入扣。此外，因为没有液滴的内容和渠道墙（这是由连续相润湿）之间的接触，吸收和试剂的通道墙壁上的损失是可以避免的。 一旦这种液滴生成和处理，这是必要的，提取所需的分析信息。在这方面的检测方法的选择应迅速，提供高灵敏度和检测限低，适用于一系列分子物种，非破坏性的，并可以将微流体器件集成在一个浅显的方式。为了解决这个问题我们已经开发的需要uite实验工具和协议，使大量的小批量环境的光物理信息的提取，并适用于广泛的物理，化学和生物参数的分析。此中这些方法的两个例子，适用于检测单个细胞和内皮升体积的液滴的混合过程的映射。我们报告整个实验过程，包括微流控芯片制造，光学装置和液滴的生成和检测过程。

Watch this Scientific Journal Video about Fluorescence detection methods for microfluidic droplet platforms at JoVE.com

Fluorescence detection methods for microfluidic droplet platforms

基于液滴微流控平台的高通量实验的有希望的候选人，因为它们能产生廉价的皮升，自我隔离的船只在千赫率。通过整合，快速，灵敏，高分辨率荧光光谱方法，可以有效地提取这些系统内产生的大量信息，利用和利用。

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 ...

fluorescence-detection-methods-for-microfluidic-droplet-platforms

Research

JoVE Journal

Bioengineering

22.0K 次观看.  Imperial College London. 基于液滴微流控平台的高通量实验的有希望的候选人，因为它们能产生廉价的皮升，自我隔离的船只在千赫率。通过整合，快速，灵敏，高分辨率荧光光谱方法，可以有效地提取这些系统内产生的大量信息，利用和利用。

视频: Fluorescence detection methods for microfluidic droplet platforms

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

合并一个液滴后荧光恢复来衡量磷脂单层的二维扩散

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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 ...

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.

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 ...

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 ...