Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.

间充质干细胞和单核细胞的纳米1.（NPS）标签<ol><li>在Dulbecco改良的Eagle培养基（DMEM）培养间充质干细胞（MSCs）补充有10％胎牛血清（FBS）和抗生素，以≥80％汇合标记之前。类似地，培养的THP-1细胞（ATCC）在罗斯韦尔园区纪念研究所（RPMI）1640培养基中补充有10％FBS的至〜10 6个细胞/ ml的密度。 </li><li>负载二氧化硅纳米粒子（〜500微米）用搅拌一夜钙黄绿素染料溶液（200微米）。制造使用前述协议PLGA-AM钙黄绿素（CAM）。22 <ol><li>溶解250微克的CAM和100mg PLGA（50:50）的氯仿，在4℃。 </li><li>生成在室温下使用高速均化器（13600 XG，60秒）的单乳剂纳米粒。使用离心分离（3400×g离心5分钟），洗涤（双分销商蒸发在收集之前的化学罩（≥3小时）的氯仿LLED水），冷冻干燥和储存于-20℃。 </li></ol></li><li>孵育CAM-PLGA纳米颗粒（1毫克），或钙黄绿素的二氧化硅纳米粒子（150微克）在0.01％聚-L-赖氨酸（PLL），在室温下溶液15 - 20分钟。 </li><li>离心机在3400×g离心5分钟以在1毫升各自培养基的纳米颗粒再悬浮之前除去过量的PLL上清液。 </li><li>孵育细胞（MSC或THP-1，〜1 - 2×10 6个细胞在总）的纳米颗粒约24小时（0.1毫克·毫升-1贴标浓度）。 </li><li>分离并收获使用2毫升0.25％胰蛋白酶（5分钟，37℃）的标记的粘附MSC和用6ml DMEM中淬灭。在细胞（1000 xg离心，4分钟），并悬浮降速至10 5的浓度- 10 6个细胞/毫升的微流体的处理。使用标记的THP-1细胞（10月5日至10月6日细胞/ ml）直接用于微流体的净化。 </li></ol> 2.微流体器件的制备<ol><li> 器件制造 <ol><li>制造微流体螺旋装置（500微米（W）×115微米（h））进行与来自使用标准软光刻步骤的商业试剂盒聚二甲基硅氧烷（PDMS）。23 </li><li>拌30 G碱基预聚物和3克在称量舟彻底固化剂。解气体中60分钟的干燥器将混合物以除去任何气泡。 </li><li>倒入PDMS混合到与螺旋通道设计图案的硅片模具师傅细心的〜5的高度 - 10mm以下。 </li><li>解气体在干燥器真空60分钟将混合物再次以除去任何气泡。重复这个过程，直到所有的气泡被淘汰。 </li><li>固化在80℃的炉中的PDMS混合物2小时，直到PDMS被设置。确保在晶片的固化具有恒定装置高度期间不倾斜。 </li><li>剪下用手术刀PDMS螺旋装置并小心剥离从母模的PDMS板。 </li><li> TR即时通讯装置的边缘用手术刀以确保表面平滑以便键合。 </li><li>冲床入口两个孔（1.5毫米），两洞（1.5 mm）适用使用1.5毫米活检冲床的PDMS设备上的插座。 </li><li>用异丙醇（IPA）冲洗设备以除去任何碎片和干燥设备在80℃的烘箱中5分钟。 </li><li>清洁使用胶带将PDMS设备（与通道功能）的底面。 </li><li>用遮蔽胶带的载玻片（2"3"）的清洁一侧。 </li><li>小心放置和暴露PDMS设备和载玻片的清理过的表面在等离子体清洁器的腔室，并使其受到真空60秒。接下来，打开等离子最大功率，直到室变成了粉红色的颜色降低室内的压力。 <ol><li>暴露表面以空气等离子体60秒。当进入物理等离子体创建的PDMS和玻璃使紧粘结的暴露表面上反应性物质人接触。关闭等离子体功率和释放来自等离子体清洁器的压力来检索设备和载玻片。 </li></ol></li><li>通过紧紧按压暴露于等离子体的表面，并确保没有气泡被困在两个表面之间粘合的PDMS设备和载玻片在一起。 </li><li>加热使用加热板2小时设定在80℃，以加强键合的键合设备。 </li></ol></li><li> 设备操作 <ol><li>油管切割两片（1.52毫米外径）的约15 - 20厘米对进口注射器和每个管子的一端固定一个注射器尖端（表23）。 </li><li>切管两片（1.52毫米外径）的〜5 - - 10厘米为网点和附加到PDMS设备的出口孔。 </li><li>事先，直到其流出的出口管的样品运行，手动素用含70％乙醇的注射器设备。允许乙醇坐30秒至1分钟来灭菌装置。 </li><li>负载30毫升过滤（0.2微米孔径）鞘缓冲液（磷酸盐缓冲盐水（PBS）中含有0.1％牛血清白蛋白（BSA））转换成60毫升注射器和固定在注射器泵的注射器。将泵的正确设置（注射器尺寸:60毫升，容量60,000微升）。 注:加入的BSA的PBS中是最小化的细胞 - 细胞的结合和非特异性细胞和PDMS设备之间的结合。 </li><li>负载3毫升标记细胞进3毫升注射器并固定在一个单独的注射泵的注射器。将泵的正确设置（注射器尺寸:3毫升，体积:3000微升）。 </li><li>检查没有气泡被截留在注射器以确保稳定的流动。轻轻喷射液体滴出，喷嘴去除气泡。 </li><li>入口注射器提示和管路连接到注射器，并把它们插入到设备（鞘和样品入口）各自的入口。保证有沿管子无气泡。 </li><li>安装设备到一个反转在细胞分选过程中编相位差显微镜即时成像。 </li><li>安全小烧杯浪费和两个15毫升管靠近设备上使用粘合剂的显微镜阶段。 </li><li>放置出口管路进入废物烧杯中。 </li><li>对样品鞘缓冲流动比率设置为1:10并同时启动注射泵启动排序过程（例如:120微升/ min的样品注射器和1200微升/ min的护套的注射器;渠道流速〜0.38米/秒）。 </li><li>运行设备1.5分钟的流量稳定。这可以通过在明场通道内壁附近惯性聚焦细胞存在相衬使用高速摄像机进行确认（5000〜 - 每10,000个帧（fps），曝光时间:10-50微秒）。 </li><li>定位的出口管路成不同的管，以从细胞出口和废弃物出口收集洗脱液。 </li></ol></li></ol>

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本文所描述的DFF细胞纯化技术使以高通量的方式标记的细胞的快速和连续分离。这种分离方法非常适合于大样本容量或高浓度的细胞样本处理，比传统的基于膜过滤是容易长时间使用后堵塞更好。同样，基于亲和力的磁选需要这是费力又昂贵的附加细胞标记的步骤。纯化的细胞被示为在通道内保持它们的标记，由于停留时间短剂和细胞形态以及（ 图3）以最小流量/剪切诱导的作用（剪切应力τ，〜200-250达因/厘米2）（&lt; 1秒）20,24。此外，用户可以通过简单地代与所需介质或缓冲溶液鞘缓冲液（盐水）选择排序标记的细胞的洗脱溶液中。 这是至关重要的有一个在微通道之前使用它可以细胞集中行为产生不利的影响没有灰尘或碎屑。这可以通过彻底用IPA清洗入口孔，以及使用掩蔽胶带清洁等离子体接合之前与PDMS表面来实现。由于微型高流量条件下运行（〜1 - 1.5毫升/分钟），它也是重要的加热在电炉上键合的PDMS装置，确保与PDMS和载玻片之间的强粘接。在器件操作中，如果细胞不聚焦良好，这意味着流率是不充分的。人们可以通过检查设备的入口区域和注射器提示，以确保没有泄漏故障排除。 从标记细胞绑定纳米粒子的去除是不是因为常见的实验室分离技术（如离心）琐碎不能单独颗粒/不同大小（ 例如，未结合的颗粒和标记细胞）细胞。尽管人们可以争辩说，反复清洗和培养基的变化可以去除从贴壁细胞游离纳米颗粒在一定程度上，这个过程是费力的和无效的悬浮细胞培养物。该DFF微流体装置利用基于大小的排序和是高度通用的，因为它可以同时用于悬浮和贴壁细胞具有大尺寸范围，以及去除不同的材料的NP的。如果靶细胞是不同的尺寸，所述DFF设备可以通过改变流动条件或通道几何结构（ 例如，通道高度以及内和外通道宽度的比率），以实现有效的分离进行优化。先前已表明，相比于在纳米粒子除去具有最小细胞损失常规离心DFF优越。22此外，细胞恢复性能仍然很高（&gt; 90％）时的样品浓度增加到10 7个细胞/ ml。这是在现有的惯性微流体细胞分离方法这是一般的关键改进LY限于10 5个细胞/毫升容量由于细胞-细胞相互作用和细胞在平衡位置过分拥挤。25 而DFF技术实现基于大小差异的分离，它不能被用于其它细胞工程方法如标记和未标记的细胞的分离，由于在小区大小可忽略的变化。对于基于亲和力的排序和其它常用的分离方式（磁化率和导电率）的细胞表面标记物，可考虑这样的潜在的应用。 DFF技术也并不适合基于荧光多路复用细胞分选。一个更好的选择是使用荧光激活细胞为标记的细胞与在高吞吐量操作不同的荧光的单步排序分选（FACS），但需要昂贵和笨重的设备。 最后，该协议是用于工程细胞时的纯化是非常有用的含变革剂（生长因子，药物，皮质类固醇等 ）纳米粒子被用于生物成像和/或细胞疗法。这是因为残留的NP自由能发挥上， 在体内注射时对这些外来颗粒响应邻近细胞或免疫细胞脱靶变革效应。它也可能会无意中产生信号生物成像过程中可能会产生误导。之前使用这些游离纳米粒子的除去从而风险降低到非靶细胞，最小化在免疫系统汇编，并减少细胞的连续标记/工程中的步骤之间的污染的问题。其成功的实施大大方便细胞标记，使在治疗基于粒子细胞工程的无干扰的使用情况。

由代理加载微/纳米颗粒（纳米颗粒）的工程细胞是一种简单的，基因组整合-自由，和通用的方法，以提高生物成像能力和增加/再生医学补充其天然的治疗性质。1-3蜂窝修改都通过标记所获得的质膜或细胞质剂加载纳米粒的过量浓度以饱和结合位点。然而，这种方法的主要缺点是留在溶液中后细胞标记过程未结合颗粒的显著数量时，其可以潜在地混淆颗粒工程细胞的精确识别或4,5-复杂化的治疗效果。此外，接触含变革纳米粒在浓度过高剂（生长因子，皮质类固醇等 ）可导致细胞毒性和误导暴露可引起对非靶细胞意想不到的后果。甚至包括o颗粒载体F"生物相容性"的材料[ 例如 ，聚（乳酸共乙醇酸），PLGA]可以煽动在某些条件下，以及有效的免疫细胞反应。6这与免疫功能受损（ 例如 ，类风湿性关节炎），其潜在的延迟个体特别危险全身纳米间隙。7因此，现有的引入颗粒的工程细胞的高效去除游离颗粒是非常重要的，以减少毒性和降低误导暴露于体内剂加载颗粒。 常规梯度离心常被用来改造的细胞从游离颗粒中分离，但是费力的和在批处理模式操作。此外，通过高速离心期间细胞和密度梯度介质可能损害细胞的完整性和/或影响细胞行为的组分经历剪切应力。8微流体是与SEV一个有吸引力的替代方案ERAL分离技术，包括确定性的横向位移（DLD）9，dieletrophoresis 10,11和acoustophoresis为小颗粒分离和缓冲交换应用开发12。然而，这些方法从低通量遭受（1-10微升·分钟- 1），并容易堵塞的问题。活性分离如基于介电电泳的方法还需要在固有的介电电泳细胞表型或其他细胞标记的步骤来实现的分离的差异。一个更有前景的方法包括惯性微流体-颗粒或细胞的横向迁移跨越简化集中在由于在高雷诺数（Re）13显性升力（F L）不同的位置由于其高流动条件和优越的尺寸分辨率。 ，它经常被利用的规模为基础的细胞分离14,15和缓冲交换应用。16-18 HoweveR，缓冲液交换性能仍然很差与~10-30％污染物的溶液，作为分离的细胞通常保持接近原始和新的缓冲液之间的边界。16-18更重要的是，靶细胞的大小分布是相似的实现精确惯性聚焦和分离从其中提出了一个问题特别是在异质大小的细胞类型，如间充质干细胞（MSC）的处理的原缓冲溶液19中我们先前已开发出一种新的惯性微流体细胞分选技术称为迪安流分离（DFF），用于使用一个2入口，2-插座螺旋微器件隔离循环肿瘤细胞（的CTC）从全血20和细菌21。在此视频协议中，我们将描述标签的THP-1（人急性单核细胞白血病细胞系）的悬浮液单核细胞（〜15微米）和干细胞（10-30微米）用钙黄绿素-L的方法oaded纳米粒，其次是DFF螺旋微型为标记的细胞和除去未纳米粒的有效回收的制造和操作。22这种单一步骤纯化策略使标记的悬浮液，悬浮于新鲜缓冲液中没有离心粘附细胞连续恢复。此外，它可以处理高达10万个细胞·毫升-1，细胞密度为顺应再生医学应用。

<table><tbody><tr><td>&lt;strong&gt;Cell lines &amp;amp; Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mesenchymal Stem Cells (MSCs)</td><td>Lonza</td><td>PT-2501</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>THP-1 monocyte cells (THP-1)</td><td>ATCC</td><td>TIB-202</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 media</td><td>Lonza</td><td>12-702F</td><td></td></tr><tr><td>&lt;strong&gt;Reagents &amp;amp; Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.01% poly-L-lysine (PLL)</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>302113</td><td>Syringe 3 ml Luer-Lock</td></tr><tr><td>60 ml Syringe</td><td>BD</td><td>309653</td><td>Syringe 60 ml Luer-Lock</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Biowest</td><td>P6154-100GR</td><td></td></tr><tr><td>Calcein, AM (CAM)</td><td>Life Technologies</td><td>C1430</td><td></td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>#P/7507/17</td><td>HPLC Grade 2.5 L</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Lonza</td><td>17-516Q/12</td><td></td></tr><tr><td>Plain Microscope Slides</td><td>Fisher Scientific</td><td>FIS#12-550D</td><td>75 x 25 x 1 mm&lt;sup&gt;3&lt;/sup&gt;</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>SYLGARD&amp;reg; 184</td><td></td></tr><tr><td>Scotch tape</td><td>3M</td><td>21200702044</td><td>18 mm x 25 m</td></tr><tr><td>Silica NPs (&amp;sim;200 &amp;mu;m)</td><td>Sigma-Aldrich</td><td>748161</td><td>Pore size 4 nm</td></tr><tr><td>Syringe Tip</td><td>JEC Technology</td><td>7018302</td><td>23 G 0.013 x 0.25</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Tygon Tubing</td><td>Spectra-Teknik</td><td>06419-01</td><td>0.02 x 0.06&quot; 100</td></tr><tr><td>Poly (D,L-lactide-co-glycolide) (PLGA; 50:50)</td><td>Sigma-Aldrich</td><td>P2191</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biopsy punch</td><td>Harris Uni-Core</td><td>69036-15</td><td>1.50 mm</td></tr><tr><td>Dessicator</td><td>Scienceware</td><td>111/4 IN OD</td><td></td></tr><tr><td>High-speed Camera</td><td>Phantom&amp;nbsp;</td><td>V9.1</td><td></td></tr><tr><td>Inverted phase-contrast microscope&amp;nbsp;</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-002</td><td></td></tr><tr><td>Syringe Pump</td><td>Chemyx</td><td>CX Fusion 200</td><td></td></tr></tbody></table>

microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering

与活性成分载微/纳米工程细胞（NPS）正在成为一个日益流行的方法，以提高本地治疗特性，使生物成像和控制细胞表型。一个关键尚未充分解决的问题是，仍无法通过常规的离心容易地除去细胞标记后未结合颗粒的显著数目。这导致增加的生物成像的背景噪声，并且可以赋予变革效应到相邻的非靶细胞。在这个协议中，我们提出称为院长流分离（DFF）惯性微流体为基础的缓冲区交换策略，以高通量的方式免费纳米粒子标记细胞有效地分离。发达螺旋微型装置便于悬浮在新的缓冲溶液纯化的细胞（THP-1和干细胞）的连续集合（&gt; 90％细胞回收），同时实现未结合的荧光染料或染料加载纳米粒（SIL的&gt; 95％的耗尽ICA或PLGA）。这个单步，基于大小的细胞纯化策略使高细胞处理吞吐量（10 6个细胞/分），并且是微/纳米工程细胞大容积细胞纯化以实现无干扰的临床应用是非常有用的。

标记与生物显像剂装载纳米粒过夜细胞后，将标记的细胞（含有自由粒子）收获并通过DFF螺旋微型纯化以除去游离的NP在单一步骤的过程（ 图1A）。该2入口，2出口螺旋微通道是由工程软件设计和使用微制造SU-8光刻胶。然后将图案化的硅晶片被用作用于使用软光刻技术（ 图1B）的PDMS复制品成型的模板。来执行细胞分选，而内壁入口运行在一个较高的流速（1:10）的新鲜缓冲溶液捏细胞样品流动细胞样品泵入螺旋微型装置的外壁的入口。分离是通过惯性聚焦较大标记的细胞在内壁和朝向外壁（ 图2A）小未结合的纳米粒子的迪安-引起的回流来实现。该器件是第一APPLI编辑从装有荧光钙黄绿素纳米颗粒二氧化硅悬浮标记细胞的单核细胞（THP-1）进行排序（〜大小500纳米）。通过高速摄像（ 图2A），为确认和流式细胞术分析（ 图2B），获得较高的分离效率。从设备细胞出口收集洗脱液的图像还表明有效地除去二氧化硅和PLGA纳米颗粒 - 从THP-1细胞（〜1 5微米）。此外，荧光成像显示，在标记的THP-1细胞内化的NP DFF纯化（插图）（ 图3）中被很好地保留。因为MSC大小异质性，螺旋微通道的几何形状随后通过改变通道的高度和分岔设计，以使干细胞（ 图4）的有效收集改性。 <img alt="图1" src="/files/ftp_upload/54327/54327fig1.jpg" /> 图1（A）OVerall工作流程，利用院长流分离（DFF）微流控技术的悬架和贴壁细胞后快速标签单步​​微/纳米粒子（NPS）的去除。 （B）中的DFF螺旋装置由工程软件设计并使用微加工的硅晶片上形成图案。 PDMS设备由软光刻方法以后产生。实验装置由2个注射泵注入标记细胞在不同的流量转化为持续的净化和标记细胞的采集设备的新鲜生理盐水缓冲液中。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54327/54327fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54327/54327fig2.jpg" /> 图2. 纳米粉体通过DFF标记THP-1细胞去除。（A）的插图DFF分离原理。较大的标记细胞出现强劲的惯性升力（F L，红色箭头）和Dean阻力（F D，黄色箭头），并专注于微通道内壁附近。较小的纳米粒和缓冲溶液仅受迪安阻力和再循环到外壁达到分离。代表性的高速和表示THP-1细胞（红色箭头）和二氧化硅纳米粒子在出口区域装载有钙黄绿素染料的平衡位置的荧光图像。基于流式细胞术通过门控大小（FSC-A）和粒度（SSC-A）特性所决定的分析（B）的分离性能。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54327/54327fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54327/54327fig3.jpg" /> 图3。 <strong<html> &gt;的纯化的标记使用DFF。插图显示相应的相位合同（上图）和单个细胞的荧光显微镜图像THP-1细胞（红色箭头）和NPS去除率代表亮场图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54327/54327fig3large.jpg" target="_blank">请点击此处查看这个大版本数字。</a> <img alt="图4" src="/files/ftp_upload/54327/54327fig4.jpg" /> 图4.从纳米粒子间充质干细胞（MSCs）通过DFF。（A）代表高速图像说明干细胞的有效分离（红色箭头）和PLGA纳米粒（蓝色箭头）到不同网点的去除 。黄色虚线指示微通道壁的大致位置。 （B）分离干细胞从二氧化硅和PLGA纳米粒性能。PG"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

这个协议描述了使用惯性基于微流体缓冲交换策略的纯化微/纳米工程细胞与未结合的颗粒的有效消耗。

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

microfluidic-buffer-exchange-for-interference-free-micronanoparticle

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.0K 次观看.  Nanyang Technological University. 这个协议描述了使用惯性基于微流体缓冲交换策略的纯化微/纳米工程细胞与未结合的颗粒的有效消耗。 

视频: Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

科研

生物工程

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 ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

通过胶体粒子自组装过程创建在PDMS微流控芯片分50纳米的纳米流体结

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

工程学

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

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 Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

胶体半导体纳米晶系统研究的模块化微流控技术

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

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 ...

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. This platform has a three-dimensional (3D) network with curved fluid channels and three pneumatic valves, which create networks, channels, and spaces through duplex replication with polydimethylsiloxane (PDMS). The device operates based on the transient response of a fluid flow rate controlled by a pneumatic valve in the following order: (1) sample loading, (2) sample blocking, (3) sample concentration, and (4) sample release. The particles are blocked by thin diaphragm layer deformation of the sieve valve (Vs) plate and accumulate in the curved microfluidic channel. The working fluid is discharged by the actuation of two on/off valves. As a result of the operation, all particles of various magnifications were successfully intercepted and disengaged. When this technology is applied, the operating pressure, the time required for concentration, and the concentration rate may vary depending on the device dimensions and particle size magnification.

The present protocol describes a pneumatic microfluidic platform that can be used for efficient microparticle concentration.

气动驱动的微流体平台，用于微颗粒浓缩

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

并列的合并离子交换膜之间的离子浓差极化，以块极化区的传播

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.