This work was financially supported by the Ministry of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026 (K. Y. Lo), No. MOST 104-2112-M-030-002 (Y. S. Sun), and National Taiwan University Career Development Project (103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material and Advanced Devices, National Taiwan University, for the use of the cell culture room.

1.芯片设计和制造<ol><li>绘制图案被使用商业软件24的PMMA基板和双面胶带消融。 <ol><li>研究化学浓度和剪切应力的影响，在其每五个培养区（ 图1A和1B）的端绘制具有变化宽度的"圣诞树"的图案。 </li><li>研究化学浓度和电场的影响，绘制了"圣诞树"的图案为盐桥两个流体通道（ 图2A和2B）。 </li></ol></li><li>划线上的聚甲基丙烯酸甲酯片材或通过加载相应文件到的 CO 2激光划线双面胶带单个图案。 <ol><li>打开激光划片机，并检查其与电脑连接。打开模式使用商业软件进行消融。 </li><li>将有机玻璃片或双面胶带Ø■首页划片的舞台。如有必要，使用校准杆和可见的He-Ne激光调节的 CO 2激光的焦点。 </li><li>加载模式进入划片上PMMA板材或带消融。 </li><li>拿起图案片或胶带，删除不需要的部分，并清洗用氮气吹面。 注:聚甲基丙烯酸甲酯片的厚度为1毫米，并且该双面胶带为0.07毫米或0.22毫米。 </li></ol></li></ol> 2.芯片组装<ol><li>与超级胶水，通过对准螺纹和孔在顶部附丙烯酸适配器（长度×宽度×高度= 10毫米×10毫米×5毫米，在中间螺纹）连接到最顶层的芯片 - 大多数层。这些适配器作为介质入口/出口和盐桥连接器。 </li><li>组装层流罩内的微流体芯片。 </li><li>组装化学剪切应力的微流体芯片（CSS芯片）。 <ol><li>一个ttach使用2个刻划双面胶带的划线的PMMA片3张。 （ 图1A和1B）。 </li><li>多一个片0.07毫米厚的双面胶添加到芯片的底部和芯片连接到一个直径10厘米的培养皿。 </li><li>把组件芯片在真空室中过夜。 </li></ol></li><li>为了组装化学电场芯片（CEF芯片），聚甲基丙烯酸甲酯片附着到厚的双面胶带= 0.22毫米（ 图2A和2B）。 <ol><li>附加划线PMMA板和双面胶带，使用此相同的片带的直径10厘米的培养皿。 </li></ol></li><li>离开罩内组装芯片和其暴露在紫外线下消毒30分钟。 </li><li>通过塑料管，并用手指拧紧螺母连接入口，以两个3毫升的注射器。通过塑料管和一个手指拧紧螺母连接插座到废液管。 注:蒸压对于使用前15分钟所有的管子和螺母在121℃。 </li><li>慢慢地压低在注射器素用PBS流体通道的柱塞。推注射器来回去除气泡。 </li><li>把芯片培养箱内过夜，在37℃下，5％CO 2。 注意:这两个步骤的目的是在芯片内洗芯片和除去任何剩余的气泡。 </li><li>就拿芯片出来的孵化器。 </li><li>准备琼脂盐桥在CEF芯片产生的电场。 <ol><li>溶解使用微波在1×磷酸盐缓冲盐水（PBS）缓冲液3％低熔点琼脂糖。 </li><li>注射液分阶段琼脂糖放入盐桥通道和插入电极液凝固之前。 </li><li>放置银/氯化银电极到管状螺母。 </li></ol></li><li>注射器连接到注射泵，不断流动的培养基（Dulbecco改进的EagleR17;培养基，DMEM）中成20微升的流速为10分钟的流体通道/分钟更换PBS中。 </li></ol> 3.细胞制备及实验装置注意:预暖1×PBS中，培养培养基（DMEM加FBS）和胰蛋白酶在使用前37℃水浴中。 <ol><li>板2×10 5个肺癌CL1-5细胞25,26中用DMEM加10％胎牛血清（FBS）的供给的10cm培养皿。孵育培养箱内的细胞在5％ 的 CO 2，在37℃直至90％汇合。 </li><li>吸出培养基并用预热1×PBS中洗一次细胞。 2毫升0.05％胰蛋白酶缓冲液添加至细胞中并等待2至3分钟，在37℃下分离的细胞。 </li><li>转移细胞到15毫升的无菌离心管中，加6 ml培养介质的进入管。轻轻颠倒试管混合并取出5微升含细胞培养基用于在hemocytomete计数细胞数河</li><li>离心300×管 克5分钟。暂停10 6个细胞于1ml培养基中，然后将含有细胞的培养基中，在1毫升注射器。 </li><li>注入含有细胞的介质进入从出口的微流控芯片，并确保该溶液通过五个培养区域分配的所有。孵育培养箱内的芯片下，5％CO 2在37℃下2小时。 </li></ol> 4.实验装置<ol><li>取芯片出保育箱，将其放在一个透明的氧化铟锡（ITO）的玻璃加热器的顶部。 注:ITO玻璃被连接到比例 - 积分 - 微分（PID）控制器，用于通过反馈从热耦合器37±0.5℃，将温度保持在ITO加热器和芯片之间紧紧夹住。 </li><li>把芯片加热器组件上倒置显微镜的电动XY载物台的顶部，用于跟踪细胞迁移或固定XY台倒置荧光显微镜测定ROS的产生。 </li><li>产生不同的化学物质浓度，填充两个注射器用5ml相对浓度0和1（溶解于培养基中）的化学溶液和它们泵入芯片在所需流率:在CSS芯片，以0.3ml的流速/分钟1小时;在CEF芯片，以20微升/分钟的流速在第20分钟，20微升/小时的另一个120分钟（总时间= 2小时）的流速。 </li><li>在CEF芯片，用于跟踪细胞迁移，程序显微镜的XY阶段重复拍照，通过数字单镜头反光文化领域，每15分钟2内（视场）的意见某个字段（DSLR）相机，小时。 </li><li>为了测量产生活性氧的，使用基于荧光的指示器2&#39;- -7&#39;- dichlorodihydrofluoresce二乙酸酯（2&#39;- 7&#39;- DCFDA）。 <ol><li>准备2&#39;-7&#39;- DCFDA的股票在10毫米分子生物学级迪甲基亚砜（DMSO）中。稀DCFDA在DMEM只无血清（5微米的DMEM）。潜在的脱乙酰酶可以增加背景信号，并在细胞减少的信号。 </li><li>在CSS芯片或之后的CEF的芯片的EF刺激的2小时的剪切应力刺激1小时后，泵2&#39;- -7&#39;- DCFDA（5μM在DMEM）到芯片在20μL/分钟的流速为前20分钟和20微升/小时的另外20分钟的流速。用于洗涤，泵的DMEM到芯片在20微升/小时的流速进行20分钟。 </li><li>拍照，通过电荷耦合器件（CCD）照相机，培养区域内的某些视场的，用于分析荧光强度。 </li></ol></li></ol> 5.化学品浓度，剪切应力和电场的计算<ol><li>在这两个CSS芯片和芯片CEF，计算在五个养殖区域的化学浓度。例如，通过注入精矿的H 2 O 2的ations 0并从两个入口200μM，0，25，则产生100，175和200μM的浓度。 注意:通过假定所有的液体分流顺畅，同样左右叉，相对浓度中的五个培养区域分别是0，1/8，1/2，7/8和1。 </li><li>在CSS芯片，采用在每个培养区域的计算的剪切应力（τ） <img alt="式（1）" src="/files/ftp_upload/54397/54397eq1.jpg" /> 27，其中Q是体积流量，η是流体粘度，h是信道的高度，w是所述信道的宽度。 注:通过设定Q = 0.3ml /分钟在每个入口（Q f = 0.12毫升/分钟在各培养区） 中，η= 0.0008帕·秒为培养基中，h = 1毫米，以及w = 1〜4毫米，在剪切应力的计算从0.0048帕（4毫米宽的区域），以0.0192帕（1毫米宽的区域）范围内。 </li><li>在在CEF芯片，计算EF强度中的每个使用E = I /（σAEFF）（欧姆定律），其中I是穿过流体通道中流动的电流的文化区域，σ是培养基的电导率，和A eff是信道的有效横截面面积。 注意:使用σ= 1.38Ω-1 M -1为培养基和A EFF = 0.22毫米2（宽= 1毫米，高度= 0.22毫米）时，EF强度计算为E（毫伏/毫米）= I（ A）×3.3×10 6。 <ol><li> 如图2D所示，治疗的等效电路五C形截面电路用四个（8 + 35 + 8）段和一个（5 + 35 + 5）段。 注:通过分析，根据基尔霍夫电压定律和欧姆定律这一并联电路，在五个文化流动的电流如，I 1到I 5从底部到顶部，被计算为围绕0.49 I（区域1），0.25 I（区域2），0.13 I（区域3），0.08 I（区域4），和0.05 我 （区5），分别，其中I是总的直流（dc）。与0.157毫安，254的EF，130，67，41，和26毫伏/毫米的施加直流被五个培养领域内产生。 注:对于微流体网络的简化电分析中，所有的流体段被认为是与电阻正比于其长度电阻。 </li></ol></li></ol> 6.数据分析注意:使用ImageJ的软件进行数据分析。 <ol><li>分析ROS的生产。 <ol><li>运行该软件的ImageJ。进入"文件"→打开加载一个荧光图像进行分析。 </li><li>转到"图像"→"类型"→"16位"改变了IM年龄为灰度。 </li><li>绘制多边形附上一个单元格。去"分析"→"测量"来测量细胞的平均荧光强度。 </li><li>重复6.1.3从三个独立实验的至少50个细胞收集强度，并计算与平均值的标准误差（SEM）的平均强度。 </li><li>重复6.1.1-6.1.4每个实验条件。 </li></ol></li><li>分析细胞迁移。 <ol><li>运行该软件的ImageJ。进入"文件"→打开加载时间= 0时拍摄的图像进行分析。 </li><li>绘制多边形附上一个单元格。去"分析"→"测量"，测量细胞的质量中心为（x 1，Y 1）。 </li><li>重复6.2.1- 6.2.2测量相同的小区的质量的其它图像的中心为（X 2，Y 2）取在时间= 吨 。 </li><li>计算的迁移速率（以微米/小时）该小区作为<img alt="公式（2）" src="/files/ftp_upload/54397/54397eq2.jpg" /> 。 </li><li>重复6.2.1-6.2.4收集来自三个独立实验的至少50个细胞的迁移率，并计算与平均值的标准误差（SEM）的平均迁移率。 </li><li>从6.2.2和6.2.3，计算该细胞的迁移指向性余弦或θ <img alt="公式3" src="/files/ftp_upload/54397/54397eq3.jpg" /> ，其中θ是施加的EF的矢量之间，并从开始到小区（ 图2G）的端部位置的矢量的角度（从正到负）。 </li><li>重复6.2.6从三个独立实验的至少50个细胞收集迁移指向性，并计算与平均值的标准误差（SEM）的平均迁移指向性。 </li><li>计算用SEM的平均迁移率和用SEM的平均迁移指向性为每个实验条件。 注:指向性+1表示所有细胞向阴极迁移，和-1值表示所有单元向阳极迁移。一组随机移动的细胞的指向性是接近0。 </li></ol></li><li>分析单元格对齐。 <ol><li>运行该软件的ImageJ。进入"文件"→"打开"加载图像进行分析。 </li><li>治疗细胞为椭圆和画一条线，以指示一个单元的长轴线。转到"分析"→"办法"来衡量β线和水平EF方向之间的角度。 </li><li>重复6.3.2从三个独立实验的至少50个细胞收集β，计算平均余弦β与（SEM）平均值的标准误差。 </li><li>重复6.3.1-6.3.3每个实验条件。 注:余弦为+1β表示所有的细胞在平行于所施加的EF对齐，和一个0值指示所有单元对齐perpendicularly以施加EF。 </li></ol></li></ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+chemical+and+electrical+stimulation+on+lung+cancer+cells+using+a+multichannel-dual-electric-field+chip.">Simultaneous chemical and electrical stimulation on lung cancer cells using a multichannel-dual-electric-field chip.</a> Biomicrofluidics. 8, 052007 (2014).</li><li>Lo, K. Y., Wu, S. Y., Sun, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+device+for+studying+the+production+of+reactive+oxygen+species+and+the+migration+in+lung+cancer+cells+under+single+or+coexisting+chemical/electrical+stimulation.">A microfluidic device for studying the production of reactive oxygen species and the migration in lung cancer cells under single or coexisting chemical/electrical stimulation.</a> Microfluid Nanofluidics. 20, 15 (2016).</li><li>Lo, K. Y., Zhu, Y., Tsai, H. F., Sun, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+shear+stresses+and+antioxidant+concentrations+on+the+production+of+reactive+oxygen+species+in+lung+cancer+cells.">Effects of shear stresses and antioxidant concentrations on the production of reactive oxygen species in lung cancer cells.</a> Biomicrofluidics. 7, 64108 (2013).</li><li>Cheng, J. Y., Wei, C. W., Hsu, K. H., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-write+laser+micromachining+and+universal+surface+modification+of+PMMA+for+device+development.">Direct-write laser micromachining and universal surface modification of PMMA for device development.</a> Sensor Actuat B-Chem. 99, 186-196 (2004).</li><li>Chen, J. J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+gene+expression+in+invasion+by+a+lung+cancer+model.">Global analysis of gene expression in invasion by a lung cancer model.</a> Cancer Res. 61, 5223-5230 (2001).</li><li>Shih, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapsin+response+mediator+protein-1+and+the+invasion+and+metastasis+of+cancer+cells.">Collapsin response mediator protein-1 and the invasion and metastasis of cancer cells.</a> J Natl Cancer I. 93, 1392-1400 (2001).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+shear+devices+for+quantitative+analysis+of+cell+adhesion.">Microfluidic shear devices for quantitative analysis of cell adhesion.</a> Anal Chem. 76, 5257-5264 (2004).</li><li>Fried, L. E., Arbiser, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honokiol,+a+Multifunctional+Antiangiogenic+and+Antitumor+Agent.">Honokiol, a Multifunctional Antiangiogenic and Antitumor Agent.</a> Antioxid Redox Sign. 11, 1139-1148 (2009).</li><li>Chisti, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+damage+to+animal+cells.">Hydrodynamic damage to animal cells.</a> Crit Rev Biotechnol. 21, 67-110 (2001).</li><li>Davies, P. F., Remuzzi, A., Gordon, E. J., Dewey, C. F., Gimbrone, M. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+reactive+oxygen+species+in+endothelial+cells+under+different+pulsatile+shear+stresses+and+glucose+concentrations.">Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations.</a> Lab Chip. 11, 1856-1863 (2011).</li><li>Tsai, H. F., Peng, S. W., Wu, C. Y., Chang, H. F., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+oral+squamous+cell+carcinoma+cells+in+a+multiple-electric-field+chip+with+uniform+flow+field.">Electrotaxis of oral squamous cell carcinoma cells in a multiple-electric-field chip with uniform flow field.</a> Biomicrofluidics. 6, 34116 (2012).</li></ol>

The authors have nothing to disclose.

PMMA基芯片使用激光烧蚀和写入与基于PDMS芯片，从而需要更复杂的软光刻当它们是便宜和更容易的方法制造。设计的微流体芯片之后，在制造和装配可以只在5分钟内完成。有迹象表明，应注意在执行实验中支付给一些关键的步骤。首先是"组装"的问题。适配器应适当粘在最上面的层的芯片。如果施加太大胶可能泄漏进入流体通道，并且如果使用太少的胶液可能泄漏出来的芯片。另外，在组装的PMMA...

体内细胞通过各种生物分子，包括细胞外基质（ECM），碳水化合物，脂质，细胞和其它细胞的包围。它们官能通过响应微环境刺激如使用ECM相互作用和反应的各种生长因子的化学梯度。传统上， 在体外细胞研究是在细胞培养皿进行，其中细胞和试剂的消耗大，细胞生长在静态（非循环）环境。最近，随着流体部件集成的微制造的器件中的更可控的方式提供了用于细胞的研究的替代的平台。这样的装置能够同时最大限度地减少细胞和试剂的消耗产生的化学和物理刺激的精确微环境。这些微流体芯片可以由玻璃基板，硅晶片，聚二甲基硅氧烷（PDMS）聚合物，聚甲基丙烯酸甲酯（PMMA）基片，或polyethylenetere邻苯二甲酸酯（PET）的基板1-3。基于PDMS的设备是透明的，生物相容的，并能透过气体，使它们适合于长期的细胞培养和研究。 PMMA和PET基材是廉价和易于使用激光烧蚀和写作进行处理。 微流体装置应提供细胞与稳定可控的微环境，其中细胞受到不同的化学和物理刺激。例如，微流体芯片被用来研究细胞的趋化性。而不是雇用Boyden小室和毛细管4,5这些微型流体装置能产生精确的化学梯度用于研究细胞的行为1,6,7传统的方法。另一例子是研究细胞的下电场功率（EFs），一个electrotaxis现象名为定向迁移。据报道细胞Electrotactic行为可能与神经再生8，胚胎发育9和伤口愈合10,11。许多研究已经进行了调查各种细胞类型，包括癌细胞12,13的electrotaxis，淋巴细胞14,15，白血病细胞11，和干细胞16。按照惯例，培养皿和盖玻片用于构建electrotactic室产生的EF 17。这种简单的设置姿势介质蒸发和不精确EF的问题，但是它们可以通过封闭的，明确定义的流体通道12,18,19的微流体装置来克服。 为了系统地研究下精确，可控化学和电刺激细胞的反应，这将是很有用处的发展能够同时提供多个刺激细胞的微流体装置。例如，Li 等人 。报告基于​​PDMS-微流体装置，用于创建单个或共存化学梯度和EFS 20。 Kao 等 。开发私奔类似微流体芯片由EF文件6调节肺癌细胞的趋化性。此外，为了提高吞吐量，侯等人 。设计和制作基于PMMA-多声道的双电场芯片，提供细胞与8种不同组合的刺激，是（2 EF优势×4化学物浓度）21。为了进一步增加整个并添加剪切应力刺激，我们开发了两个PMMA基微流体装置根据单个或共存化学/电/剪切应力刺激研究细胞应答。 由Lo 等 22,23报道，这些设备包括五个独立的细胞培养渠道受到持续流体流动，模仿体内循环系统。在第一芯片（化学剪切应力芯片或CSS的芯片），五个相对浓度为0，1/8，1/2，7/8，和1在培养区域生成，并剪切应力梯度是机生产线土木工程署内各五个文化领域。在第二芯片（该化学电场芯片或CEF芯片），通过使用一个单组电极和第2注射泵中，除了这些培养区域内五个不同的化学浓度时，产生五个EF强度。数值计算和模拟执行，以便更好地设计和操作这些芯片，而这些设备内培养肺癌细胞受到单个或共存刺激用于相对于观察到的活性氧物种的它们的响应（ROS），迁移率，和运移方向。这些芯片被证明是节省时间，高吞吐量和可靠的器件用于研究细胞对各种微环境刺激的反应。

<table border="0" cellpadding="0" cellspacing="0" width="1070">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:460px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Trypsin</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td>detach cell from the dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Fetal bovine serum (FBS)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">10082147</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10-cm cell culture Petri dish</td>
			<td style="width:213px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Bright-Line&#160;Hemacytometer</td>
			<td style="width:213px;">Sigma</td>
			<td>Z359629</td>
			<td>Cell Counting Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PMMA</td>
			<td style="width:213px;">Customized</td>
			<td style="width:119px;">Customized</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Adaptor</td>
			<td style="width:213px;">Customized</td>
			<td style="width:119px;">Customized</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.07/0.22 mm double-sided tape&#160;</td>
			<td>3M</td>
			<td>8018/9088</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Low melting point agarose</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:119px;">A9414</td>
			<td>Salt bridge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#39;-7&#39;-dichlorodihydrofluoresce diacetate</td>
			<td>Sigma</td>
			<td style="width:119px;">D6883</td>
			<td>Intracellular ROS measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Indium tin oxide (ITO) glass</td>
			<td style="width:213px;">Merck</td>
			<td>300739</td>
			<td>Heater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proportional-integral-derivative controller&#160;</td>
			<td>JETEC Electronics Co.</td>
			<td>TTM-J4-R-AB</td>
			<td>Temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal coupler</td>
			<td>TECPEL</td>
			<td style="width:119px;">TPK-02A</td>
			<td>Temperature controller</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CO2 laser scriber</td>
			<td>Laser Tools &#38; Technics Corp.</td>
			<td style="width:119px;">ILS2</td>
			<td>Microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pumps</td>
			<td>New Era</td>
			<td style="width:119px;">NE-300</td>
			<td>Pumping medium and chemicals into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power supply</td>
			<td>Major Science&#160;</td>
			<td style="width:119px;">MP-300V</td>
			<td>Supplying direct currents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Monitoring cell migration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted fluorescent microscope</td>
			<td style="width:213px;">Nikon</td>
			<td style="width:119px;">TS-100</td>
			<td>Monitoring cell migartion and fluorescencent signals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DSLR camera</td>
			<td>Canon</td>
			<td>60D</td>
			<td>Recording bright-field images&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCD camera</td>
			<td>Nikon</td>
			<td style="width:119px;">DS-Qi1</td>
			<td>Recording fluorescent images&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">super glue</td>
			<td style="width:213px;">3M Scotch</td>
			<td style="width:119px;">7004</td>
			<td>Attaching adaptors to PMMA substrates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoCAD</td>
			<td style="width:213px;">Autodesk Inc.</td>
			<td style="width:119px;"></td>
			<td>Designing microfluidic chips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMSO</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Dissolving DCFDA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ImgeJ</td>
			<td style="width:213px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td>Quantifying fluorescent intensities and cell migration</td>
		</tr>
	</tbody>
</table>

designing microfluidic devices for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli

微流体设备能够创建pH值，温度，盐浓度，和其它物理或化学刺激的精确和可控的细胞微环境。他们已经通过像环境在体内提供了通常用于体外细胞研究。尤其，细胞如何响应于化学梯度，电场，应力和剪应力引起了许多利益，因为这些现象对理解细胞特性和功能非常重要。这些微流体芯片可以由玻璃基板，硅晶片，聚二甲基硅氧烷（PDMS）聚合物，聚甲基丙烯酸甲酯（PMMA）基片，或聚对苯二甲酸乙酯（PET）衬底。出这些材料，PMMA基底是便宜的，并且可以使用激光烧蚀和书写容易加工。虽然一些微流体装置已被设计和制造用于产生多个共存化学和电刺激，其中没有一个被认为是够在减少实验重复序列，特别是用于筛选目的有效。在这份报告中，我们描述了我们的两个基于PMMA微流控芯片设计和制造用于研究细胞反应，在生产活性氧和迁移，下单或共存化学/电气/剪切应力刺激。第一芯片产生五个相对浓度0，1/8，1/2，7/8，和1在培养的区域，与内部这些领域中产生的剪切应力梯度在一起。第二芯片产生相同的相对浓度，但与各培养区中创建的五个不同的电场强度。这些装置不仅提供细胞与精确，可控的微环境，也大大增加了实验的吞吐量。

化学 - 剪切应力（CSS）芯片 CSS的芯片进行了三次PMMA板，每个的厚度为1毫米，经由两个双面胶带连接在一起，每一个的厚度0.07毫米（ 图1A和1B）的。的"圣诞树"结构产生的0 5相对浓度，1/8，1/2，7/8和1中的五个培养领域。由培养面积设计为三角形，剪切应力梯度，与相关的体积流速，流体粘度和流体通?...

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Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

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Bioengineering

8.8K 次观看.  National Taiwan University. Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

视频: Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

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

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microfluidic Mixers for Studying Protein Folding

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

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

研究蛋白质折叠的微流体混合器

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

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

一种意志的运动反应的及时性和准确性，以振动触觉刺激评价方法

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...