This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.

1.设计和MDF芯片的制备<ol><li>绘制使用商业软件，如AutoCAD单个丙烯酸系层的图案，并保存该图形。 <ol><li>回顾在图1A中的四层压克力板图案的设计和确认层间的连接。 </li></ol></li><li>制造所有的丙烯酸类片材，并通过激光烧蚀使用CO 2激光划线器（图1B，2A 和 2B）的双面胶带。16 <ol><li>打开电池激光划线和机器连接到控制笔记本。 </li><li>运行的商业软件，通过点击打开设计步骤1.1模式“设计图案的文件。” </li><li>将一块空白丙烯酸系片材或双面胶带上的激光划线器的XYZ台。 </li><li>设置激光束的焦点的丙烯酸系片材或双面胶带具有自动alignmen的表面上通过激光划片的制造商提供吨棒。 </li><li>发送所设计的图案，以该激光划线器的丙烯酸系片材或双面胶带的直接加工。 </li></ol></li><li>使用镊子从丙烯酸系片材除去保护纸和吹表面的清洁氮气。 </li><li>堆叠的丙烯酸系片材，并在一个热粘合机在2kg / cm 2的压力结合在一起在110 45分钟 °C至形成流/电刺激通道组件。 </li><li>准备在显微镜盖玻璃作为在芯片中的细胞培养基材。 <ol><li>把防护玻璃成染色缸和填充罐用稀释10倍的物质列表中列出的洗涤剂。 </li><li>把盖玻璃和染色缸中的超声波免缝滤清器并清洁罩玻璃15分钟。 </li><li>倒入稀释的洗涤剂出来的染色缸中，加注蒸馏水罐子，并重复步骤1.5。2 3次。 </li><li>通过将其附着在双面胶带前用氮气吹干燥清洗玻璃盖。 </li></ol></li><li>附着的清洗玻璃盖与双面​​胶带构图步骤1.2的流量/电刺激通道组件。 </li><li>坚持在中纤板芯片组装与超级胶水的第1层13件亚克力适配器连接到单独的开口。密度板芯片组装就完成了（ 图2D）。 </li><li>与使用前紫外线照射30分钟消毒完整的中纤板芯片组装。 </li></ol> 2.设置密度板芯片的盐桥网络<ol><li>在使用之前，消毒所有的塑料管，用手指拧紧螺母， 并且在图3B中所示的离心管通过设置的15分钟的保持时间在121 °下在高压釜中。 </li><li>经由介质入口连接氟塑管（ 图3B-ⅰ）到MDF芯片组件并且，如图1A所示插座适配器。 </li><li>连接介质入口的Luer锥形和出口塑料管在图3B-i到所述旋塞3路。 </li><li>取2.5 ml的CO 2的-equilibrated磷酸盐缓冲盐水（PBS）的使用3毫升注射器和注射器连接到入口塑料管的活塞3路。空3毫升注射器连接到插座的塑料管的活塞3路。这两个注射器通过MDF芯片从而互连。 注：孵育PBS在37 °C 5％-CO 2细胞培养孵化器O / N来获得二氧化碳 -equilibrated PBS。 </li><li>封蓝色的开口和绿色适配器（图1A）上的第1层PMMA板用白色固体手指拧紧螺母（图3B-ⅲ）。 </li><li>与CO 2填充盐桥通道和培养室中设置步骤2.4中所述的注射器-equilibrated PBS。避免形成气泡。 </li><li>接下来，把CO 2 -equilibrated PBS含在37℃，5％-CO 2细胞培养孵化器的O / N培养MDF芯片。这允许在双面胶带的溶解空气，以形成在所述腔室的泡沫。 </li><li>冲洗掉在通道中的气泡，通过使用两个注射器快速PBS流动的通道。泵的PBS来回如果必要的。 </li><li>漏的PBS中通过3路活塞连接到所述介质出口管的通道。 </li><li>使用一个新的3-ml注射器，取2.5毫升的 CO 2 -equilibrated Dulbecco氏改良的Eagle培养基（DMEM）（见步骤3.5），并更换连接到用新的入口的注射器。笔芯渠道与网上平台。 </li><li>制备盐桥网络。 <ol><li>时间上去除固体螺母（ 图3B-III）绿色适配器（ 图1A），注入3％热（&gt; 70℃）的琼脂糖放入盐BRI通过开口中的绿色的适配器（图1A）DGE通道。 注意：热琼脂糖制备：将1.5克琼脂糖粉末在50毫升PBS中。通过在高压釜设定20分钟的保持时间在121℃灭菌。 </li><li>停止注射琼脂糖当液体充满四分之三的盐桥通道的长度的。 </li><li>通过琼脂糖注射后拧固螺母（图3B-ⅲ）密封在绿色适配器（图1A）的孔。 </li><li>替换在蓝色适配器（图1A）与半透明管状手指拧紧螺母（图3B）的固体坚果。 </li><li>加载3％预热琼脂糖入管状螺母（图3B-ⅳ）。 </li><li>嵌入前的银/氯化银电极（图3B-v）的入管状螺母（图3B-ⅳ）凝固的琼脂糖。 </li></ol></li><li>完成盐桥网络的设置完成后，注入CL1-5肺癌细胞到培养腔室，一个腔室的时间。 </li></ol> 3.准备癌细胞，并设立Electrotactic实验<ol><li>肺癌细胞株CL1-5经常文化。 <ol><li>培养的CL1-5肺癌细胞，从教授杨泮池，17中的完全培养基中，在37中得到一个75T细胞培养烧瓶 °下在5％CO 2的气氛中。完全培养基由DMEM和10％牛胎儿血清（FBS）的。子培养细胞每3至4天。用于执行electrotaxis实验的细胞是从原始来源小于25代。 </li></ol></li><li>加入2毫升0.25％胰蛋白酶缓冲器向指数生长的细胞CL1-5并孵育在37 2分钟 °C 5％-CO 2细胞培养孵化器电池detachmen吨。 </li><li>终止该拆卸过程用6ml 10％FBS的DMEM，并离心将细胞在300克5分钟，室温。然后丢弃培养基和悬浮细胞沉淀用5毫升PBS中。 </li><li>计数细胞，在PBS中的数目。然后取1×10 6个细胞，并离心以300g为5分钟，在室温。 </li><li>丢弃的PBS。悬浮细胞用预热的 CO 2 -equilibrated的DMEM，并调整细胞密度为1×10 6个细胞/ ml。 注：在孵育37完全培养基 °C 5％-CO 2细胞培养孵化器O / N来获得二氧化碳 -equilibrated DMEM。 </li></ol> 4.设立Electrotactic实验<ol><li>从密度板芯片的出口处，注入0.3毫升的细胞，1×10 6 /毫升，进入芯片。 </li><li>孵育在细胞培养孵化器的细胞接种密度纤维板芯片（设置在37 ℃和5％的CO 2）为2-4小时。 </li><li> MDF微设置流体系统。 <ol><li>安装MDF微流体系统在透明的氧化铟锡玻璃（ITO），加热器。测量密度板芯片用K型热电偶密度板芯片与ITO玻璃之间夹住的温度。控制在ITO加热器与一个比例 - 积分 - 微分（PID）控制器。设置MDF微流体系统孵化温度为37 °下与PID控制器。 注：在ITO加热器的制造和细胞培养加热系统的安装是在以往的报告18,19说明。 </li><li>安装在倒置显微镜上的计算机控制的XYZ电动载物台的温度控制的中密度纤维板芯片组件。电动载物台的使用方法在步骤5.1中描述。通过使用四通道注射泵入口泵完全培养基向培养腔室。 </li></ol></li><li>泵完全培养基到MDF芯片以20微升/小时的流速。从出的文化浪费让被收集在离心管（示出为图 3A中的“废物”）。 </li><li>在MDF中芯片孵育将细胞再16-18小时，在37℃。 </li><li>要更换介质，泵含有50，25，第5和0μM激酶抑制剂，分别培养基，向每个培养室在20μL/分钟，持续10分钟的流速。 注：加入3毫升无菌水到10.6毫克激酶抑制剂准备Rho相关蛋白激酶抑制剂Y27632的10mM储备溶液。稀释10mM储备溶液至50，25和5μM，分别使用10％FBS的DMEM培养基中。 </li><li>孵育细胞中的激酶抑制剂另外60分钟，在37℃。设置介质流速至20微升/小时。使用相同的培养温度和流量在后面的步骤上述率。 </li><li>使用电线到直流电源连接到经由芯片上的银/氯化银电极密度板芯片。 </li><li>串接电流表我n中的电路。使用电流表监测中的电流密度纤维板芯片。 </li><li>接通直流电源和通过调节供电电压以15至19五设置电流表86.94μA的电流 注意：考虑欧姆定律E = I /（σAEFF），其中I是电流流过块状材料， 即 ，在electrotactic室中的培养基，σ（= 1.38Ω-1米- 1）是培养基中，甲EFF（= 0.21毫米2为3毫米宽×0.07毫米高）的导电率是electrotactic腔的有效截面面积，并且电流（I）中，以产生一个300毫伏/毫米所需EF中密度板芯片是86.94μA。 </li></ol> 5.采集和细胞图像分析<ol><li>控制使用MATLAB GUI程序的机动阶段。通过安装在microsco密度板微流体系统PE，移动芯片采用电动舞台的观察区域。 </li><li>安装在显微镜用数字单镜头反光（SLR）相机记录细胞图像。 </li><li>以使用4X物镜以15分钟的间隔2小时的显微图像。 </li><li>细胞迁移分析。 <ol><li>运行NIH ImageJ的1.47 V. </li><li>从初始的距离进行分析，以细胞的质心的最终位置。 </li><li> 进入 “文件”→“导入”→“图像序列”，并导入九张图像细胞迁移分析。第一图像是零时间的结果，最后图象是120分钟的结果。 </li><li>进入“ 分析”→“设置测量”，并勾选旁边的“ 重心” </li><li>点击“ 写意选择”图标。 </li><li>描绘了选择的细胞的边​​缘中的第一图像。 </li><li>进入“编辑”→ </em&gt;“选择”→“ 添加到管理器” </li><li>转到第九图像和描绘在第一图像上所选择的同一细胞的边缘。细胞位置可以使用第二到第八图像进行追踪。 </li><li>进入“编辑”→“ 选项”→“ 添加到管理器” </li><li>重复步骤5.4.5至5.4.9，以90至100个细胞采集数据。 </li><li>进入“ 分析”→“测量”，以获得所选单元格的最初和最后的位置的结果。 注：迁移速度被定义为每小时的平均细胞运动的长度。的指向性被定义为余弦，其中是dcEF（从阳极到阴极）的矢量和从一个单元的起点到其最终位置的向量之间的角度。的指向性是-1细胞向阳极迁移和1对细胞向阴极迁移。对于一组随机心肌梗死光栅单元，所述指向性为0 2 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

我们发现秉承丙烯酸适配器到MDF芯片的1层是棘手的过程。仅1至2微升的强力胶的应用是足够坚定奉行适配器到MDF芯片。更大量的胶水导致强力胶和不遵守的一个不完整的聚合。一旦丙烯酸适配器牢固地附着在芯片中密度纤维板，液体泄漏的微流体系统很少发生。此外，O / N培养在真空室内有助于去除双面胶带/盖玻璃或双面胶带/丙烯酸片接口之间截留空气。因此，该过程增强了培养室中密度板芯片...

贴壁细胞下的直流电场（dcEF）朝向阳极或阴极移动的行为被称为electrotaxis。细胞的行为electrotactic起着胚胎发生，神经再生，和伤口愈合一个显著作用。1肿瘤细胞如鼠前列腺癌细胞，2-乳腺癌细胞，3和肺腺癌细胞4-8已根据所施加的dcEF所示electrotactic运动。生理EF已在腺组织进行了测量。9,10- Electrotaxis也有报道在腺相关的肿瘤细胞。2,3两者合计，癌细胞的electrotaxis被认为是一个转移因子11控制的电指导dcEF下的癌细胞可以是用于癌症的未来治疗的潜在的方法。然而，今天，electrotaxis的具体分子机制仍存在争议。因此，INF进行调查对癌症细胞迁移的电刺激的luence可以方便的策略的发展为癌症的治疗。 最近，生物微流体装置已被制造用于研究的细胞响应流动的剪切力，12化学梯度，13和电刺激4 体外 。使用聚二甲基硅氧烷（PDMS）或聚甲基丙烯酸甲酯（PMMA，也被称为丙烯酸类）的生物微流体装置的制造已成功地减少了此类实验的故障率。此外，使用基于丙烯酸的微流体装置作为原型调查生物受试者比使用PDMS芯片简单。在丙烯酸​​类装置的各种功能已经开发了用于electrotaxis研究。然而，没有任何先前的设计都能够同时测试的各种化学条件对细胞的影响，并在电场为electrotaxis研究。因此，我们开发了一种微流体装置，所述亩ltichannel双电场（MDF）四个独立培养通道和八个不同的实验条件在一个芯片包含芯片。 丙烯酸基密度板芯片，首先报道了侯等人，8集成电刺激和几个化学隔离通道。这些化学隔离通道可以用来培养在一个实验中不同类型的细胞。所述dcEF在通道是由一电源产生的。两个独立的电场，一用施加的电场强度（EFS）和另一用0 EFS，在各化学分离的信道进行的。以这种方式，在芯片提供了更好的控制的共存EF和化学刺激。此外，从MDF芯片内部的化学扩散数值模拟结果表明，无交叉污染，24小时的试验期后，通道之间发生。8 相比于叠维策报道李等人，14密度板芯片提供了一个更大的文化区域，其允许电刺激的细胞的进一步的生化分析。此外，与中密度纤维板芯片的较大的观察区域，更多的细胞可以在测试中观察，所以迁移速度的电刺激的细胞的或指向性的分析更准确。报告由Huang等人4和Tsai等人。15以前的研究的单通道芯片设计允许被测试仅一种类型的细胞或化学的。然而，中密度纤维板芯片可用于研究的各种化学品上electrotaxis的影响，以及电刺激对不同类型的细胞的作用。换句话说，中密度纤维板芯片允许对化学剂量依赖性的高效学习。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >12800-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >16000-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >25200-072</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Basic Life</td>
			<td >BL2651</td>
			<td></td>
		</tr>
		<tr >
			<td >Y-27632 (hydrochloride)</td>
			<td >Cayman Chemical Co</td>
			<td >10005583</td>
			<td></td>
		</tr>
		<tr >
			<td >agarose</td>
			<td >LONZO, USA</td>
			<td >SeaKem LE AGAROSE</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe</td>
			<td>Terumo</td>
			<td>3 ml with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td>Nipro</td>
			<td >with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >PMMA (acrylic)</td>
			<td >HiShiRon Industries CO., Ltd, Taiwan</td>
			<td ></td>
			<td >thickness 1mm, 2mm</td>
		</tr>
		<tr >
			<td >acrylic adaptor</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td >1/4-28 port, 10x10x6 mm</td>
			<td>customized</td>
		</tr>
		<tr >
			<td >nut</td>
			<td >Thermo Fisher Scientific Inc.</td>
			<td >UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope cover glass</td>
			<td >Deckgl&#228;ser, Germany</td>
			<td >24x60 mm</td>
			<td></td>
		</tr>
		<tr >
			<td >double-sided tape</td>
			<td >3M</td>
			<td >PET 8018</td>
			<td></td>
		</tr>
		<tr >
			<td >super glue</td>
			<td >3M</td>
			<td >Scotch Liquid Plus Super Glue</td>
			<td></td>
		</tr>
		<tr >
			<td >Teflon tube</td>
			<td >HENG YI ENTERPRISE CO., LTD., Taiwan</td>
			<td >UPTB_06, DUPONT TEFLON BRAND RESIN FEP TUBING</td>
			<td>outer diameter 1/16 in., inner diameter 0.03 in.; Upchurch Scienti&#64257;c</td>
		</tr>
		<tr >
			<td >TFD4 detergent</td>
			<td >Franklab, France</td>
			<td >TFD4</td>
			<td></td>
		</tr>
		<tr >
			<td >ultrasonic steri cleaner</td>
			<td >LEO ULTRASONIC CO., LTD., Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo bonder</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2 laser scriber</td>
			<td >LTT group, Taiwan</td>
			<td >ISL-II</td>
			<td></td>
		</tr>
		<tr >
			<td >indium tin oxide glass (ITO glass)</td>
			<td >AimCore Technology Co., Ltd</td>
			<td >TN/STN, &#8806;10&#937;</td>
			<td></td>
		</tr>
		<tr >
			<td >proportional-integral-derivative (PID) controller</td>
			<td>JETEC Electronics Co., Japen</td>
			<td >TTM-J40-R-AB,</td>
			<td></td>
		</tr>
		<tr >
			<td >K-type thermocouple</td>
			<td >TECPEL</td>
			<td >TPK-02A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-channel syringe pump</td>
			<td >KdScientific, USA</td>
			<td >250P</td>
			<td></td>
		</tr>
		<tr >
			<td >DC power supply</td>
			<td >GWInstek, Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >X-Y-Z motor stage</td>
			<td >TanLian, E-O Co. Ltd., Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >inverted microscope</td>
			<td >Olympus, Japan</td>
			<td >CKX41</td>
			<td></td>
		</tr>
		<tr >
			<td >digital SLR camera</td>
			<td >Canon, Japan</td>
			<td >60D</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip

定向细胞迁移的下一个直流电场（dcEF）的行为被称为electrotaxis。生理dcEF在胚胎发育，细胞分化和伤口愈合过程中引导细胞运动的显著的作用已被证明在许多研究。通过将微流体芯片的electrotaxis测定中，调查过程缩短和实验误差最小化。近年来，微流体装置制成的聚合物质（例如，甲基丙烯酸甲酯，聚甲基丙烯酸甲酯，或丙烯酸）或聚二甲基硅氧烷（PDMS）已被广泛用于研究细胞的响应电刺激。但是，与制造一个PDMS装置所需的许多步骤中，丙烯酸类微FL uidic芯片的简单而快速的结构使得它适合于设备的原型和生产。然而，没有报道的设备，这有助于该组分化学DCE的效率研究对细胞˚F影响。在这份报告中，我们描述我们的设计和丙烯酸系多信道双电场（MDF）芯片调查化学和电刺激对肺癌细胞的效果并发的制造。中密度纤维板芯片提供了一个测试的电气/化学刺激八种组合。该芯片不仅大大缩短了实验所需的时间，但在electrotaxis研究也提高了准确性。

制造中密度纤维板设备和组装 丙烯酸基密度板芯片的示意图示于图1A。四压克力板，一个覆盖玻璃，13丙烯酸适配器，和一块双面胶带在已完成的中密度纤维板芯片（图2D）的组件被使用。只有四个独立的文化通道中密度纤维板设备。然而，芯片上的盐桥网络创建八个不同的?...

Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

electrotaxis-studies-lung-cancer-cells-using-multichannel-dual

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8.6K 次观看.  Academia Sinica. Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

视频: Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Fluorescence detection methods for microfluidic droplet platforms

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

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

荧光检测方法，为微流体液滴平台

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

科研

生物工程

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

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 Microfluidic Chip for ICPMS Sample Introduction

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

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

微流体芯片ICPMS进样

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

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

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

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

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

使用微流体芯片以确定性方式建立基于单细胞的共培养物

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