支持这项工作已部分由下批准号EFRI 0938047美国国家科学基金会，以及由弗吉尼亚理工大学的关键技术和应用科学（ICTAS）的支持。作者想表达感谢伊娃施梅尔茨博士和保罗·罗伯茨博士为他们的那种MOSE-L细胞的礼物。作者承认安吉拉·安德森为她协助细胞培养，Caitlan Swaffar她帮助编辑这个文件，并准备实验，以及所有Bioelectromechanical系统实验室成员。

1。制造原型CDEP微流体设备:概述<ol><li>使用光致抗蚀剂和深反应离子蚀刻（DRIE）蚀刻所需的信道设计（ 图1）到硅片（ 图2）按照以前报道的程序22。沉积薄薄的一层聚四氟乙烯的改善的微型装置从晶片上剥离。 </li></ol><img alt="图1" src="/files/ftp_upload/50634/50634fig1.jpg" /> 图1。低频连续分选装置的示意图，样品通道运行左至右为500μm宽的带锯齿收缩至100μm。两对电极的流体通道构成的源和汇的电极，分别和由20μm厚的PDMS阻挡从样品通道是分开的。 <img alt="图2" src="/files/ftp_upload/50634/50634fig2.jpg" /> 图2。该布标一个CDEP装置ication过程（a）对于60秒，UV光选择性地与光致抗蚀剂通过CDEP设备的几何形状的掩模图案进行曝光（B）光致抗蚀剂是使用显影剂除去。（三）深反应离子蚀刻（DRIE）来蚀刻50微米深的特点，和5分钟的湿法蚀刻以四甲基氢氧化铵（TMAH）的25％，在70℃是用于减少在侧壁上的粗糙度（d）一个特氟隆涂层被添加以改善从设备释放晶片（E）的PDMS倒入可重复使用的晶片邮票脱气后，在100℃F.（F）的PDMS从晶片除去固化45分钟。 PDMS和干净的载玻片暴露于空气等离子处理2分钟，粘合在一起。 <ol start="2"><li>用铝箔包装的晶圆，以防止溢出。在10:1至固化剂，脱气约20分钟，比弹性体的混合聚二甲基硅氧烷（PDMS），倒到晶片上。热在100℃下45分钟，以避免损坏邮票的特富龙涂层。让设备冷却下来。 </li><li>冷却后，除去铝箔，装饰用手术刀片PDMS，并用钝冲床适合于管子的尺寸打孔进入孔在通道的入口/出口。在这里，1.5毫米的钝冲床使用。 </li><li>用漂洗肥皂和水，乙醇，去离子水，和干燥用的空气，分别清洗的玻璃显微镜载片上。用苏格兰魔术胶带清洁PDMS设备。显微镜下检查，以确保通道是干净的。暴露在滑动和PDMS装置空气等离子体2​​分钟。用力按下设备的通道侧到载玻片上，从而避免形成装置和滑动（ 图3）之间的气泡。 </li></ol><img alt="图3" src="/files/ftp_upload/50634/50634fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig3highres.jpg"/> 图3。 </st荣&gt;（a）该硅晶片制造邮票，以及（b）与所述样品和填充有分别的绿色和红色食用色素，流体通道电极成品PDMS-玻璃装置中使用的光刻掩模。参见图1尺寸。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50634/50634fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 2。制备细胞悬液中的DEP缓冲<ol><li>制备低导电率（100μS/厘米）的缓冲，这将被称为"介电泳缓冲液"（8.5％蔗糖[重量/体积]，0.3％葡萄糖[重量/体积]，0.725％的RPMI [重量/体积]） 16 </li><li>暂停细胞中的DEP缓冲器在3×10 6个细胞/ ml。这里，癌晚期小鼠卵巢表面上皮细胞（MOSE-L）细胞的使用。 </li></ol> 注:由台盼蓝染色排除法细胞活力分析显示活力略有下降早期MOSE（MOSE-E）的细胞在室温下5小时后，悬浮于DEP缓冲液（由95.1-91.6％）。据预测，在可行性类似轻微下降发生于MOSE-L细胞，表明细胞不应该被存储在缓冲DEP长期。 <ol start="3"><li>使用荧光膜渗透性染料，如酶促活化的钙黄绿素AM染细胞。 </li><li>测量染色的细胞悬浮液的导电性。电导率应约100μS/ cm的。如果电导率测量太高，旋转样品下来，重悬在DEP缓冲器以3×10 6个细胞/ ml，并重复电导率测量。 </li></ol> 3。加载CDEP微流控芯片<ol><li>放置在真空下，微流控芯片30分钟。 </li><li>同时，切下一块的柔性管，以跨越从注射泵的距离，显微镜载物台，其中所述微流体芯片将位于。这里，管道的6英寸长是必需的。飞度油管针尖上的注射器。 </li><li>通过将目标收集样品体积通过管的横截面面积估计所需的长度为出口管路。切需要管的长度。 </li><li>绘制细胞悬液注入注射器。检查无气泡存在于管或注射器。 </li><li>在从真空除去芯片，插入出口管路和素样品通道迅速，以避免被截留的空气气泡在通道。吸，以避免破裂的薄（20微米）时，绝缘壁轻轻拍打注射器，虽然它已经观察到，在阻挡可承受近5毫升/小时的恒定的高通量而不破裂。 </li><li>以最优惠的电极渠道，快速地将空的枪头在每个电极流体通道的一个出口。吸取200μl的PBS中含有少量罗丹明B和轻轻拍打到流体引入到电极通道的另一端。保持温和的压力直到PBS与罗丹明B明显进步了空的枪头的尖端。重复每个电极通道。罗丹明B仅用于荧光可视化的流体电极通道。 </li><li>吸后，填充每个枪头储用PBS罗丹明B。避免气泡形成在移液管尖水库，并通过轻轻地上下吹打除去任何可见的气泡。拆下出口管路和适当地丢弃，然后插入一个新的出口管路水库，收集目标卷。 </li></ol> 4。使用CDEP芯片细胞的特征分频点<ol><li>定位在倒置显微镜配备有数码相机（ 图4）的阶段的芯片。 </li></ol><img alt="图4" src="/files/ftp_upload/50634/50634fig4.jpg" fo:content-width="5in" fo:src="/files/ftp_upload/50634/50634fig4highres.jpg"/> 图4。 </stroNG&gt;（a）整体实验装置CDEP实验。该CDEP芯片位于倒置显微镜的平台上。一个摄像头的图像发送到计算机进行录制。注射泵保持恒定的入口流。函数发生器产生被加强了由高电压放大器和连接到所述CDEP芯片通过导线电极的信号。示波器监视发送到芯片的信号。（二）CDEP芯片和流体和电子连接的详细视图。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50634/50634fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 <ol start="2"><li>安装注射器注射泵和插入线电极插入电极流体通道水库。 </li><li>通过以1毫升/小时的流体泵，以确保注射泵和注射器之间的良好接触。目视检查沟道长度，以保证细胞的畅通和没有气泡或泄漏。红UCE流量0.005毫升/小时。 注:吞吐量高达100毫升/小时31,35已经在其他几何形状的设备已经实现。 </li><li>为了安全起见，由开机函数发生器，高压放大器和示波器，并调整到所需的电压和频率测试电子产品。这里这些参数是200 V和20千赫。经核实可接受的性能，关闭电源。电极连接到高电压放大器。 注:在用于这些实验的高电压下工作的电子时，应遵守相应的电气安全规程。 </li><li>在达到稳流，应用所需的电压在所希望的频率。在该实验中，电压为200伏RMS在5-60千赫之间的频率。 </li><li>与图像处理技术（步骤5）来量化细胞分布在通道和表征交叉频率的细胞应答的记录的视频文件。要做到这一点，保持一个恒定的电压和系统地改变频率。在该实验中，以及随机实验运行之间的开始和结束时，记录控制单元的分布，该细胞的不施加电场的分布。估计的时间让细胞从入口流到监控位置（此处，5分钟）所需的量。设置一个新的频率后，等待该段时间，然后开始录制（这里是2分钟的视频细胞分布的）。 </li></ol> 5。为表征分频点图像处理<ol><li>利用一种计算机脚本，分析每个视频帧由帧来确定相对的y位置（z方向是该信道的垂直深度，y方向是沟道宽度，而x-方向是沟道长度）在每一发荧光的细胞或颗粒的指定的x坐标从在沟道高介电泳力的区域的下游。建立相关显示的图tribution。 </li><li>在每个电压和频率与所述控制分布的中心线比较分布的中心线。对于给定的电压和频率的变化，确定交叉频率，这里的中心线相匹配的控制的y位置。 </li></ol> 6。不同的实验，以进行细胞分选富集或<ol><li>暂停在DEP缓冲器所需的混合物在3×10 6个颗粒/毫升的总浓度。这里，该混合物是MOSE-L细胞中的DEP缓冲器4微米的荧光珠。执行先前描述的步骤，以制备CDEP设备。 </li><li>从混合物进行排序或富集的细胞，确定了靶细胞的交叉频率和如先前所描述的背景颗粒。操作实验的靶细胞和背景粒子的2分频的频率之间的频率，使得粒子的两个群体经历的DEP力在相反ð从根本上来说。要MOSE-L细胞和4微米微珠进行排序，操作上11.90±0.63 kHz的微型装置。 MOSE-L细胞的第一交叉频率最近报道Salmanzadeh 等人 33 </li><li>收集的有序样品中的内容，在收集对象的卷取出出口管路，通过将一个手套的手指在所述出口开口并轻轻拉着管。倒入收集目标体积成储以供进一步分析注:其他样品的回收方法可以包括附加的永久贮存到芯片和由简单的移液除去样品。 </li></ol>

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拉斐尔·达瓦洛斯博士有一个正在申请专利的非接触式介。

DEP是一个强大的技术来确定粒子的介电性能和引导粒子运动对分拣，隔离，或富集的应用。由于与样品直接接触电极的不利影响，其他人也采取了类似先前提出的方法，以避免接触的方法。例如，巴希尔等人，通过使用由一个薄的玻璃盖玻片从印刷电路板的电极分离的微流体设备的PDMS开发的无接触式的DEP装置，以及该技术也取得了在视频格式。23,36 这里，我们已经表明一个CDEP芯片和流电极使用单一的PDMS衬底的制造中，并在实验方案从细胞和荧光珠的混合物中分离卵巢癌细胞。所提出的技术已成功地用于各种更复杂的和生理学相关的应用程序，包括分选的LIVe和死细胞，28肿瘤启动从前列腺癌细胞的细胞中，30癌细胞从稀红细胞，31,32和乳腺癌37和卵巢癌的阶段中分化29 CDEP也被用于混合颗粒。38这些应用表明，通过使用简单的技术提出，不同的目的，可以简单地通过改变所述通道的几何形状的设计来实现的。 。DEP上的微尺度操纵粒子13对于球形颗粒是有用的，平移的介电电泳力取决于粒子和其悬浮介质的尺寸和电气性能，以及电场梯度的平方: <img alt="式（1）" src="/files/ftp_upload/50634/50634eq1.jpg" /> 式中，εm是悬浮介质的介电常数，r是半径颗粒和Rc [K（ω）]是克劳修斯- Mossotti（CM）因子的实部。在CM的因素是粒子的相对极化率的量度相比，悬浮介质，并确定介电电泳力的方向。它被描述为<img alt="公式2" src="/files/ftp_upload/50634/50634eq2.jpg" /> 哪里<img alt="式（3）" src="/files/ftp_upload/50634/50634eq3.jpg" />和<img alt="公式4" src="/files/ftp_upload/50634/50634eq4.jpg" />是粒子和介质，分别的复数介电常数。复介电常数， <img alt="方程（5）" src="/files/ftp_upload/50634/50634eq5.jpg" /> ，取决于电导率（σ）和频率（ω）。球形颗粒的CM因子-0.5和1之间的理论上的约束。如果CM因子为阴性，所述颗粒经历NDEP因为介质是比粒子更极化，使颗粒从区域移开高电场梯度。如果CM因子为正，则粒子比介质更极化和他们经历pDEP它们走向的高电场梯度的区域。 用于生物颗粒是不均匀的结构，如细胞，克劳修斯 - Mossotti因子可以从该粒子的介电常数的有效值来确定: <img alt="公式6" src="/files/ftp_upload/50634/50634eq6.jpg" /> 哪里<img alt="公式7" src="/files/ftp_upload/50634/50634eq7.jpg" />和<img alt="公式8" src="/files/ftp_upload/50634/50634eq8.jpg" />是细胞的分别的内部，例如在细胞质和细胞膜，有效的复介电常数的复介电常数; r是小区的半径，d为质膜的厚度26 当DEP发生颗粒悬浮在液，相对于流体的粒子的运动将产生在颗粒曳力。确定整体受力作用在粒子时，这个阻力必须加以考虑。对于这里关注的情况下，粘性力占主导地位和粒子被假设球形，体积小，相对较低的速度运动，所以斯托克斯拖动法律提供了一个很好的近似的阻力: <img alt="公式9" src="/files/ftp_upload/50634/50634eq9.jpg" /> 其中η是流体的粘度，U p是粒子的速度， 和 u f是流体，其也可被移动的速度。给定的介电电泳力，已知流体和颗粒性质，以及已知的流速，拖曳力和介电电泳力之间的平衡就可以解决估计粒子速度。剪切速率的细胞经历应低于该阈值，在其中CELL裂解可能发生。 粒子的电气性能特性是必要的预测和控制它们将如何DEP下作出回应。在这项工作中，我们特别利用低频CDEP与MOSE-L细胞，以证明该协议用于确定细胞的第一交叉频率，然后显示的聚苯乙烯珠和MOSE-L细胞连续分选基于它们的相对的DEP的响应。 改变CDEP设备的几何形状会改变电场的空间梯度，允许设备被设计用于高频或低频的操作，并为高选择性和对于一个指定的信元类型排序的效率。此外，高通量设备可以通过制造更宽的信道进行开发，30个信道并行，30或由多层制造35，其中的电极通道的垂直堆叠的上方和下方相对深样品通道。薄膜形成层之间的势垒。初步测试已制成的聚甲基丙烯酸甲酯（PMMA）和聚碳酸酯的设备（PC）薄膜已经证明MOSE-L细胞中的DEP响应。目前正在努力完善多层次的高通量设备和改进，迈向最终的插件和播放平台的外围系统。从基本实验技术呈现扩张，该装置的应用程序和规格可以定制，以适应特殊的需求，如排序与表征，或添加试样槽和半自动化系统的装置。

生物样品中富集和分选粒子往往是必要的用于随后的分析。1比如，从体液中分离稀有细胞在癌症的检测和个体化医学重要的应用。2,3最常用的富集技术是荧光激活细胞分选术（FACS ）4，磁激活细胞分选（MACS），5依赖于表达的表面标志来区分细胞。其他策略包括液力6或7,8惯性分选，光学镊子，9 acoustophoresis，10和介电电泳。11,12介电电泳是在非均匀电场的存在下，偏振粒子的运动。13 DEP已被用于了广泛的应用，2,14包括基于生存能力，15表征细胞的生物电性能分拣单元，16个和排序ING时引起的变化在细胞的生物物理特性。17,18传统的DEP利用内的微流体通道施加电压，并诱导非均匀的电场。13虽然这是一个功能强大的技术进行图案化的平面电极，挑战可能出现，如电极脱层和电解。绝缘体为基础的介电电泳（影像介电泳）19已经解决积垢，电极剥离，电极字段通过诱导的非均匀性在直流电场图案形成绝缘结构空间降解的挑战。影像介电泳已被用于选择性地分离活的和死的细菌细胞，19隔离细菌孢子，20和操纵的DNA，在其他应用21。焦耳加热可能是一个挑战，因为它可以发生作为经常需要高的直流电压的结果。为了改善这些问题，无接触的DEP的微型器件已被开发。22-24 。22独此策略是金属电极更换为充满液体电极通道高导电性的解决方案。这些流体电极两端的薄绝缘屏障，通过AC电压样品通道电容耦合。消除电极与样品接触减少了与DEP为基础的方法，如电解，形成气泡，样品污染和电极脱层的相关问题。其结果是，CDEP因为它支持细胞的样品中的存活率为生物样品特别有用。重要的是，CDEP能保持样品不育。一个芯片就可以在一个细胞培养罩准备和实验可以，而不需要样品接触到金属电极或要求该样本是开放的环境为进行NT。一个简单的贮能固定到芯片插座，以便无菌样品回收。此外，流体的电极由相同的生物相容的聚合物材料（PDMS）的制造作为样品通道可减少产生与金属电极的自定义图案化，需要为制造时的成本高，而且限制了需要专门的洁净室设备的初始图案形成可重复使用的硅片邮票。 由于DEP粒子的运动依赖于粒子和介质，以及该电场的空间梯度的特征。一种粒子和频率依赖因子，称为克劳修斯 - Mossotti（CM）因子，取值范围为-0.5〜1，并确定该介电泳力的方向。的频率，所述CM因子正好为零称为交叉频率。这是在该无介电电泳力作用在粒子和CM因子变化标志点。一个S英格尔分频点惰性固体微球时出现CM因素的变化由负转正25对于哺乳动物细胞中低电导率缓冲液为0.01 S / m的，第一分频点表示从NDEP到pDEP存在近10过渡的顺序- 100千赫，并通过大小，形状，细胞骨架和细胞的膜性能的影响。26,27的第二交叉频率从pDEP转向NDEP制度是10MHz的量级，并且是由影响核-细胞质比率，细胞质电导率和内质网。27 DEP力可以在不流体流动的存在下被应用，但这里我们利用流动流体来实现的悬浮颗粒的连续的分拣。介电电泳力和斯托克斯阻力的综合影响决定了粒子的平移运动。 我们在两个频率范围为开发运行的设备。高FREQUENCŸ设备（100-600千赫）已使用pDEP和细胞实现批量分拣操作，如前列腺肿瘤起始细胞（国际信托），鼠卵巢表面上皮细胞（MOSE）细胞，MDA-MB-231乳腺癌细胞，或住THP -1细胞通过选择性地对绝缘位于样品通道职位俘获所关注的细胞。28-31下限频率（5-100千赫）器件连续工作，并且当在一个频率操作在其中一个人口经历pDEP而背景人群的经验NDEP，可以重定向粒子轨迹来实现排序。32-34在这些低频器件已被用于癌症细胞从红血细胞进行排序，确定变化的渐进MOSE细胞系的介电性能，并阐明的非作用有毒鞘脂处理对倒车积极MOSE细胞的攻击性特点。此外，CDEP微型器件可被设计成在增加的吞吐量进行操作，目前最多为1毫升/小时河31,35 如描述的那样，灵活性和制造工艺的成本低使定制设计的设备的几何形状，其允许呈现的实验步骤是相关于广泛的应用范围。 CDEP的长期目标是实现在临床水平无标记细胞分选和富集，对后续的培养或处理样品回收。这里介绍的方法是一种简单，廉价的方法，从制造到实验，这增加了DEP的可访问性。我们展示了一个准备CDEP芯片和实验协议，实现定性和卵巢癌细胞荧光微球的富集。

<table><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning, Midland, MI,USA</td><td>Sylgard 184</td><td></td></tr><tr><td>Glass slides </td><td>The Microscope Depot</td><td>76079</td><td>2x3 inch-ground edges</td></tr><tr><td>Microbore PTFE Tubing</td><td>Cole-Parmer Instrument Co, Vernon Hills, IL, USA</td><td>EW-06417-31</td><td>Thin walled 20 gauge, 0.032&quot;ID x 0.056&quot;OD, 100 ft/roll</td></tr><tr><td>Luer-slip plastic syringes</td><td>National Scientific company</td><td>S7510-1</td><td></td></tr><tr><td>Needle tip</td><td>Howard Electronic instruments</td><td>JG20-1.0</td><td>20 Gauge 1.0&quot;, ID=0.025&quot; OD=0.036&quot;</td></tr><tr><td>D(+)-Sucrose</td><td>Fisher Scientific, Fair Lawn, NJ</td><td>S3-500</td><td></td></tr><tr><td>D(+)-glucose, reagent ACS, anhydrous</td><td>Acros Organics N.V., Fair Lawn, NJ</td><td>AC410955000</td><td></td></tr><tr><td>RPMI-1640 Medium</td><td>Quality Biological Inc.</td><td>112-025-101</td><td></td></tr><tr><td>Calcein AM, Molecular Probes</td><td>Invitrogen Corp. (life technologies), Carlsbad, CA, USA</td><td>C3100MP</td><td>excitation wavelength 488/emission wavelength 516</td></tr><tr><td>Rhodamine B, O</td><td>Science Lab</td><td>SLR1465-100G</td><td>excitation wavelength 540/emission wavelength 625</td></tr><tr><td>Phosphate buffered saline (10X)</td><td>Gbiosciences, St. Louis, MO</td><td>RC-147</td><td></td></tr><tr><td>Leica, inverted light microscope </td><td>Leica Microsystems, Bannockburn, IL, USA</td><td>Leica DMI 6000B</td><td></td></tr><tr><td>Leica DFC420, color camera</td><td>Leica Microsystems, Bannockburn, IL, USA</td><td>Leica DFC420</td><td></td></tr><tr><td>Function generator </td><td>GW Instek, Taipei, Taiwan</td><td>GFG-3015</td><td></td></tr><tr><td>Wideband power amplifier </td><td>Amp-Line Corp., Oakland Gardens, NY, USA</td><td>AL-50HF-A</td><td></td></tr><tr><td>HFHV Output Transformer</td><td></td><td>AL-T50-V25/300-F100K-600K</td><td></td></tr><tr><td>High voltage amplifier</td><td>Trek</td><td>Model 2205</td><td></td></tr><tr><td>USB Modular Oscilloscope, 100 MHz</td><td>AgilentTechnologies</td><td>U2701A</td><td></td></tr><tr><td>Expanded Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC-001/002 (115/230V)</td><td>air plasma</td></tr><tr><td>Scotch Magic tape</td><td>3M</td><td></td><td>any available width is sufficient</td></tr><tr><td>1.5 mm puncher </td><td>Harris Uni-Core</td><td>Z708836-25EA</td><td></td></tr><tr><td>.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>FluoSpheres Sulfate Microspheres</td><td>Invitrogen</td><td>F8858</td><td>4.0 &amp;mu;m, red fluorescent (excitation wavelength 580/emission wavelength 605)</td></tr><tr><td>AZ 9260 photoresist </td><td>AZ Electronic Materials</td><td></td><td></td></tr><tr><td>AZ 400 K developer</td><td>AZ Electronic Materials</td><td></td><td></td></tr><tr><td>Tetramethylammonium hydroxide (TMAH) 25% </td><td></td><td></td><td>provided by Virginia Tech cleanroom</td></tr><tr><td>Teflon coating</td><td></td><td></td><td>applied using DRIE machine</td></tr><tr><td>Silicon wafer</td><td>University Wafer</td><td>452</td><td>100 mm diameter, 500 &amp;mu;m thickness, one side polished (SSP) </td></tr><tr><td>Deep Reactive Ion Etching (DRIE)</td><td>Alcatrel</td><td>AMS SDE 100</td><td></td></tr></tbody></table>

label-free isolation and enrichment of cells through contactless dielectrophoresis

介电电泳（DEP）是由偏振光在非均匀电场粒子经受平移运动的现象，并且可以被用来指示微粒的表面标志物独立的方式运动。传统上，DEP设备包括图案的样品通道的平面金属电极。这种方法可以是昂贵的，并且需要专门的洁净室环境。最近，已开发出一种称为非接触式介电电泳（CDEP）无接触的方式。该方法利用DEP的经典原则，同时避免了流体图案电极和电极和样品之间的直接接触从一个单一的聚二甲基硅氧烷（PDMS）基板的样品通道，并具有应用程序设计进行排序和丰富微粒快速微流体策略。独有的方法，是在电场经由含有高导电性的流体，它从流体中分离电极通道产生的通过薄绝缘阻挡样品通道。因为金属电极不直接接触样品，电解，电极剥离，和样品污染是可以避免的。此外，这使得一种廉价和简单的制造工艺。 CDEP因此非常适合用于操作敏感的生物粒子。因粒子作用在介电电泳力不仅取决于由装置的几何形状可定制的设计中产生的电场的空间梯度，但细胞的内在生物物理特性。因此，CDEP是取决于表面表达，可能在一个人口被可变表达，同时还允许生物粒子的表征，富集，分选和分子生物标志物，避免了无标记技术。 在这里，我们展示了制造和试验用CDEP的基本知识。我们采用软版画解释CDEP芯片的制备简单Ÿ技术。我们将讨论的实验步骤，用于表征颗粒或细胞的频率在该介电电泳力是零的交叉频率。最后，我们证明了使用该技术进行分类的卵巢癌细胞的混合物与荧光微球（珠）。

DEP应定性观察在样品通道，以确认该设备是可操作的，当在电场存在。一种方法来测试DEP的存在是为了远离交​​叉频率是强劲pDEP和NDEP进行了预测（约&gt; 40 kHz到观察pDEP和&lt;10 kHz到观察NDEP对大多数哺乳动物癌细胞在调整频率值与100μS/ cm的电导率缓冲液）。应当指出的是惰性的微球表现出pDEP低于交叉频率和NDEP以上的交叉频率。为锯齿特征几何这里提出，pDEP应在更高的测试频率可以看出对于细胞，由珍珠链和聚焦在该信道的锯齿边缘特征的细胞或颗粒的发生表示，而在较低的测试频率，NDEP应该是明显的，因为细胞被限制在该区域更近的通道的直边。在这些低频率，细胞可能LYS缘于电穿孔，因此样品可能会出现包含更少的细胞，并且那些是可见的，如果使用酶促活化的荧光染料可能会放大或模糊。验证在哪个pDEP和NDEP发生频率允许交叉频率在协议中描述的决心。用于哺乳动物癌细胞，如MOSE-L用在这里，我们观察到NDEP在5千赫（ 图5a）和强pDEP在60千赫（ 图5b）。 MOSE-L细胞的直径为17.7±3.3微米（N = 268个细胞）和珠粒具有4微米的标称直径。相比MOSE细胞在早期发展阶段MOSE-L细胞的介电性能的完整的分析可以在最近的工作由Salmanzadeh 等 33中找到如果pDEP和NDEP不会在这些极端观察到，各种问题可能会发生。该样品的电导率可能过高由于污染物或细胞裂解。 INSUFFICIEnt的电场可能会由于气泡被困在流体通道电极，其防止对接壤的样品通道的通道的一部分的电流传导产生。非定常流可通过截留在注射器或进口管道的气泡引起的，从气泡不保留该设备在真空条件下保持足够的时间，或在从真空，泄漏是由于粘结不良的PDMS的剥离除去不迅速填充所述装置产生的载玻片上，由于用力过猛眼泪在隔膜在泵工作范围的极端填充时的通道，或流速发挥。 除了确定细胞的交叉频率，该技术可用于混合物中，通过MOSE-L细胞和珠的混合物，此处所示的排序。此示例采用负介（NDEP）由4微米微珠频率，其中MOSE-L细胞有正面的介电电泳（pDEP）表现出的优势。我们Øperated设备在200V RMS和10千赫（ 图6a），并观察到两种细胞和珠进行混合，因为他们经历NDEP。在50千赫我们观察MOSE-L细胞的运动到通道的顶部，而珠被限制在通道的下部，使混合物分离成两个部分（ 图6b）。 <img alt="图5" src="/files/ftp_upload/50634/50634fig5.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig5highres.jpg"/> 图5。 MOSE-L细胞的位置，通过改变所施加的电场的频率进行操作。该照片是在最后的锯齿特征在样品通道和所述流体电极分别位于右边的角落。它们被填充有含有PBS若丹明B，其中荧光可视化的频道（一个）MOSE-L细胞经历NDEP在200 V RMS<html>和5千赫。（二）MOSE-L细胞感受珍珠链和pDEP在200 V RMS和60千赫。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50634/50634fig5highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" src="/files/ftp_upload/50634/50634fig6.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig6highres.jpg"/> 图6。通过最终的锯齿特征MOSE-L细胞（绿色）和4微米的小珠（红）的混合物流入一个低频CDEP装置的样品通道（a）于 200 V和10千赫的细胞和小珠混合。箭头指向隐约出现了可能已经死亡，由于低频率条件下的细胞（B）在200 V和50千赫的细胞经历pDEP，并移动到该信道的特征的边缘，而珠遇到NDEP并保持局限在该区域更靠近直边。p_upload/50634/50634fig6highres.jpg"目标="_blank"&gt;点击这里查看大图。

Watch this Scientific Journal Video about Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis at JoVE.com

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

非接触式介电电泳（CDEP）达到通过其固有的介电性能和分拣颗粒的富集。流体通道的电极取代金属电极的传统，以DEP，适合CDEP非破坏无菌表征和生物粒子进行排序。我们将演示如何编写一个CDEP微型设备，并进行细胞鉴定和分选实验。

无标记的分离和细胞的富集通过非接触式介电泳

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

label-free-isolation-enrichment-cells-through-contactless

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

15.9K 次观看.  Virginia Tech. 非接触式介电电泳（CDEP）达到通过其固有的介电性能和分拣颗粒的富集。流体通道的电极取代金属电极的传统，以DEP，适合CDEP非破坏无菌表征和生物粒子进行排序。我们将演示如何编写一个CDEP微型设备，并进行细胞鉴定和分选实验。 

视频: Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

通过玻璃表面功能靶细胞提取方法

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

科研

癌症研究

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 ...

生物工程

Development of a 3D Graphene Electrode Dielectrophoretic Device

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The protocol details the procedures to construct a versatile, reusable, multiple layer, laminated dielectrophoresis chamber. Specifically, six layers of 50 &#181;m x&#160;0.7 cm x&#160;2 cm graphene paper and five layers of double sided tape were alternately stacked together, then clamped to a glass slide. Then a 700 &#956;m diameter micro-well was drilled through the laminated structure using a computer-controlled micro drilling machine. Insulating properties of the tape layer between adjacent graphene layers were assured by resistance tests. Silver conductive epoxy connected alternate layers of graphene paper and formed stable connections between the graphene paper and external copper wire electrodes. The finished device was then clamped and sealed to a glass slide. The electric field gradient was modeled within the multi-layer device. Dielectrophoretic behaviors of 6 &#956;m polystyrene beads were demonstrated in the 1 mm deep micro-well, with medium conductivities ranging from 0.0001 S/m to 1.3 S/m, and applied signal frequencies from 100 Hz to 10 MHz. Negative dielectrophoretic responses were observed in three dimensions over most of the conductivity-frequency space and cross-over frequency values are consistent with previously reported literature values. The device did not prevent AC electroosmosis and electrothermal flows, which occurred in the low and high frequency regions, respectively. The graphene paper utilized in this device is versatile and could subsequently function as a biosensor after dielectrophoretic characterizations are complete.

A microdevice with high throughput potential is used to demonstrate three-dimensional (3D) dielectrophoresis (DEP) with novel materials. Graphene nanoplatelet paper and double sided tape were alternately stacked; a 700 &#956;m micro-well was drilled transverse to the layers. DEP behavior of polystyrene beads was demonstrated in the micro-well.

三维石墨电极介电泳装置的研制

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The ...

工程学

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

发育生物学

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 ...

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

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

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 Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using the principle of the polarization of the cancer cells in the sample volume by applying an external electric field. This article demonstrates how a microfluidic platform can be utilized for high-throughput continuous sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using hydrodynamic dielectrophoresis (HDEP) from the cell mixture. By generating an electric field between two electrodes placed side-by-side with a micron-sized gap between them in an HDEP microfluidic chip, non-tumor breast epithelial cells (MCF-10A) can be pushed away, exhibiting negative DEP inside the main channel, while the non-metastatic breast cancer cells follow their course unaffected when suspended in cell medium due to having conductivity higher than the membrane conductivity. To demonstrate this concept, simulations were performed for different values of medium conductivity, and the sorting of cells was studied. A parametric study was carried out, and a suitable cell mixture conductivity was found to be 0.4 S/m. By keeping the medium conductivity fixed, an adequate AC frequency of 0.8 MHz was established, giving maximum sorting efficiency, by varying the electric field frequency. Using the demonstrated method, after choosing the appropriate cell mixture suspension medium conductivity and frequency of the applied AC, maximum sorting efficiency can be achieved.

Microfluidic Device for the Separation of Non-Metastatic MCF-7 and Non-Tumor MCF-10A Breast Cancer Cells Using AC Dielectrophoresis

Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells.

使用交流介电泳分离非转移性（MCF-7）和非肿瘤（MCF-10A）乳腺癌细胞的微流体装置

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

免疫与感染

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

用于细菌筛选、选择和分离的可光降解水凝胶接口

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) offer a patient-derived cell source for conducting mechanistic studies of diseases or for several therapeutic applications. Understanding hMSC properties, such as their electrical behavior at various maturation stages, has become more important in recent years. Dielectrophoresis (DEP) is a method that can manipulate cells in a nonuniform electric field, through which information can be obtained about the electrical properties of the cells, such as the cell membrane capacitance and permittivity. Traditional modes of DEP use metal electrodes, such as three-dimensional electrodes, to characterize the response of cells to DEP. In this paper, we present a microfluidic device built with a photoconductive layer capable of manipulating cells through light projections that act as in situ virtual electrodes with readily conformable geometries. A protocol is presented here that demonstrates this phenomenon, called light-induced DEP (LiDEP), for characterizing hMSCs. We show that LiDEP-induced cell responses, measured as cell velocities, can be optimized by varying parameters such as the input voltage, the wavelength ranges of the light projections, and the intensity of the light source. In the future, we envision that this platform could pave the way for technologies that are label-free and perform real-time characterization of heterogeneous populations of hMSCs or other stem cell lines.

Here, we present light-induced dielectrophoresis as a label-free approach for characterizing heterogeneous cell lines, specifically human mesenchymal stem cells (hMSCs). This paper describes a protocol for using and optimizing a microfluidic device with a photoconductive layer to characterize the electrical behavior of hMSCs without altering their native state.