This work was supported by the US Army Medical Research and Materiel Command and by the Small Business Innovation Research and Small Business Technology Transfer program; Contract No. W81XWH-13-C-0093. We would like to thank Dr. Lucy Lee at the University of Fraser for being our RTgill-W1 cell culture mentor, and to acknowledge Dr. Niels Bols of Waterloo University for the development of the RTgill-W1cell line.

1.测试材料的制备注意:为了制备生物芯片进行测试，RTgill-W1细胞几种汇合烧瓶需要准备。需要瓶的数量的一个很好的估计一个合流T175烧瓶中接种16生物芯片。 <ol><li>执行使用无菌技术II级生物安全柜（biohood）以下步骤。用70％乙醇消毒biohood并放置在罩的任何材料。 <ol><li>通过解冻纤连蛋白的一个为1mg小瓶中并在100毫升无菌的L-15培养基稀释为10微克/ ml的浓度制备为流体生物芯片纤连蛋白底物。冻结在-20℃下在无菌的50ml锥形聚丙烯管40,42毫升分装。在解冻到播种前生物芯片室温下几个小时。一旦解冻，不要再次冷冻。 </li><li>通过加入50ml胎牛血清，5毫升的200mM的L-丙氨酰-L-谷氨酰胺SUP制备细胞培养基plement和5ml青霉素/链霉素溶液（10,000单位青霉素/毫升和10000微克链霉素/ ml）的库存至500ml的L-15介质。这将产生560毫升含9％的血清的细胞培养介质。冷藏。 注意:这将是用于培养瓶和生物芯片的完整的细胞培养基。 </li></ol></li><li>粉状媒体样​​品瓶<ol><li>提前准备用自动粉末分配器含有60毫克±L-15ex粉0.5％，0.1 DRAM单元口小瓶。标签的小瓶粉末配发的日期和冷藏瓶（以50只），在再封金属化塑料袋;各含有了三片1克硅胶干燥包。 注释:L-15ex粉状介质小瓶可制成并存储了预先测试的长达9个月。 </li></ol></li><li>通过稀释家用漂白用去离子（DI）水达到100ml 20％的漂白剂的溶液中。估计有5毫升漂白溶液将需要对每个双向ochip。 </li><li>通过切割高压灭菌生物相容性管（待接种2个部分的每个生物芯片）为27毫米的部分使生物芯片管组件和适合与聚碳酸酯滑移Luer配件的管的两端。放置管组件中的耐高压加热的袋和高压釜在134℃8分钟。此外釜生物芯片插头（2％生物芯片）在一个单独的包在相同的设置。 </li><li>高压釜DI水30分钟at121℃。 注意:此水将用于播种后漂洗生物芯片。估计采用10ml将需要对每个生物芯片。 注:各生物芯片通道的实际体积为2毫升，但溶于5ml无菌水通过除去漂白溶液后的各信道漂洗。 </li><li>通过切割生物相容性管27毫米的部分和附接阳滑鲁尔装配件到管道的两端使注射器注入管道组件。发生在纸热封袋消毒和高压灭菌管组件8分钟，134＃176℃。 </li></ol> 2.生物芯片射流播种过程注意:请那里的生物芯片或媒体使用无菌技术II级生物安全柜进行处理的所有程序。 <ol><li>原定播种前二十四小时，从biohood制造商的包装和无菌塑料仪表到位的情况下删除该生物芯片。 </li><li>使用20％的漂白剂溶液如下消毒生物芯片 注意:从历史上看，此漂白过程已可防止在生物芯片真菌生长长期储存过程中的事件由生产商所做的生物芯片的等离子体灭菌是无效的。 <ol><li>用20毫升无菌注射器用注射器注射管组件连接并在biohood工作，注入将2ml 20％的漂白剂溶液的成生物芯片的各通道。让生物芯片坐1小时的漂白粉溶液。 </li><li>一小时真空asp的后愤怒的从两个通道使用连接到真空抽吸管路中的无菌雄性滑路厄组件的漂白溶液中。漂洗该生物芯片时，使用生物芯片管组件中的一个为漏极。 </li><li>使用附连到无菌注射器注射组件无菌20毫升注射器，冲洗芯片的每个通道用5ml无菌水，使多余的水排入在biohood的容器。然后真空吸掉作为漂白刚才描述和放置生物芯片放回塑料仪表案件，并在biohood离开，直到与细胞接种第二天的水。 </li></ol></li><li>六十分钟，接种生物芯片，注入将2ml10μg/ ml的纤连蛋白溶液加入到生物芯片的各通道之前。留在biohood 60分钟的生物芯片，然后用细胞接种在生物芯片之前真空抽吸掉纤连蛋白（如在步骤2.2.3描述）。放置无菌生物芯片管组件的两个部分（参照在生物芯片的端口1.4节）。 </li><li> Trypsinize使用在美国典型培养物保藏中心（ATCC）的产品说明片16所概述的程序每16生物芯片1汇合RTgill-W1 T175烧瓶中。 <ol><li>吸掉来自细胞的汇合烧瓶（多个）媒体。 </li><li>冲洗用15ml PBS的细胞层，然后吸关闭。 </li><li>加6 ml胰蛋白酶/ EDTA至每个T175烧瓶中的细胞层和允许细胞trypsinize〜5分钟。 </li><li> 15 ml完全L-15细胞培养基添加到每个烧瓶中以停止胰蛋白酶消化。 </li><li>结合细胞悬液在无菌的一次性容器。 注:容器尺寸可从150-500毫升，取决于被接种的生物芯片的数目而变化。有5 ml的细胞悬浮液将每生物芯片需要估计。 </li></ol></li><li>除去约1毫升细胞悬浮液，并放入用于计数一个离心管中。使用显微镜明用10X objective和血球，计数细胞的10微升等分试样，并计算达到2.5×10 5个细胞/ ml的细胞悬浮液所需要的完整的L-15细胞培养基的体积。 注意:如果使用的细胞悬浮液的种子烧瓶继续培养，这将是这样做的点。调整用完整的L-15细胞培养基的细胞悬浮液的浓度。 </li><li>使用无菌20毫升syringeattached到无菌注射器注入管道组件（见1.6节），注入2.5细胞悬液到生物芯片的各通道的外部端口（ 即 ，不具有连接管的端口），允许一些额外的细胞悬浮液的流出管到废物容器中的biohood。 注意:这将保证整个通道和连接的管道将充满的细胞悬浮液。 <ol><li>通过与路厄件和泵系列插入软管的自由端为每个生物芯片通道的封闭环纳克成用于每个通道的外端口。 </li><li>擦去多余的介质了与用70％乙醇浸湿的纸巾闭环，并放置在生物芯片放回塑料盒在20℃的孵化器。给每个生物芯片的唯一标识号。 </li></ol></li><li>天4和7，请从20℃培养箱生物芯片和补充媒体在所有温度平衡完整的L-15细胞培养基的生物芯片。按照相同的步骤，如步骤2.6除刚刚使用L-15细胞培养基代替（无细胞悬液）。将生物芯片早在20℃培养箱中4天进食后。 <ol><li>当天7喂奶后，删除和丢弃从芯片的软管，并在生物芯片插入蒸压排水塞。 </li><li>放置生物芯片中有6℃培养箱一个框，直到用于测试。 注意:该芯片可以储存在冷藏温度下长达9个月，但仍存活的测试我n个ECIS的读者。 </li></ol></li></ol> 3. ECIS测试与生物芯片<ol><li>如果使用准备测试的化学物质。 （参见布伦南等人于2012年7制备的试验化学品）。 </li><li>从手提箱中取出ECIS阅读器和测试设备。从6℃培养箱中取出事先准备好的生物芯片。放置在生物芯片上的纸巾。打开ECIS读者。 </li><li>用10ml注射器，分配10毫升控制水的成标记0.5盎司透明塑料控制罐和10ml试验样品放入一个标记0.5盎司透明塑料试验瓶。 注意事项:1）一定要去除气泡的精确测量。 2）注射器和罐子颜色编码;蓝色用于控制和红色测试。 </li><li>从铝箔袋删除两个粉状媒体小瓶。粉状媒体瓶打开其中一个（使用多功能的工具，如果需要的话）和一小瓶的全部内容倒入与控制水的罐子，下探整个小瓶成解决方案也是如此。重复与测试罐子此过程。盖上盖子并摇晃罐子，以确保粉末溶解。 </li><li>用9ml要么控制（蓝色注射器）或测试（红色注射器）从各自的小瓶解决方案填充每个彩色10毫升注射器。从注射器去除气泡。 </li><li>从生物芯片端口​​取下插头连接的流失。 </li><li>将生物芯片到ECIS的读者可移动塑料托盘，并盖上盖子。 </li><li>附加充式注射器外生物芯片的端口，并附加注射器活塞。 </li><li>从主屏幕上，选择"下一步"开始前测试。 注:读者软件会检查阻抗。如果阻抗范围内由用户（通常在1000和3000欧姆）的屏幕将寄存器设定为"墨盒合格"， 如图3所示，并指示用户选择"下一步"，该软件将进行到步骤3.10）。如果阻抗不内因为有缺陷的生物芯片或读者（通常是由于电极的错位）生物芯片的连接故障的设定范围，然后屏幕将注册"墨盒故障"，用户将不得不采取"取消"的选项或"验证"测试。 <ol><li>选择"取消"回到步骤3.9）。选择"确认"继续接收"柱体"消息，然后选择"下一步"以后的步骤3.10）。 注意:最好是把生物芯片并目视检查其是否有缺陷或泄漏，如果一个"墨盒故障"以新生物芯片在继续之前收到的消息。 </li></ol></li></ol><img alt="图3" src="/files/ftp_upload/53555/53555fig3.jpg" /> 图3:用可接受的阻抗读数生物芯片的ECIS阅读器截图的截图显示了在每个4个共同的欧姆阻抗初始读数ntrol电极（CE）和流体生物芯片内的4试样电极（SE）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53555/53555fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="10"><li>当读者提示，使用软键盘输入样品信息，然后选择"接受"完成时。 注意:ECIS阅读器软件将自动加盖与日期，时间和由用户输入的其他信息的各数据集;例如生物芯片号码，以及化学和浓度的类型。阻抗数据的两个分将从插入生物芯片被收集和一个屏幕上的定时器将倒计时时间。 </li><li>当被一个红色框内闪动屏幕上的说明提示2分钟后，点击"下一步"。当闪烁的绿框"现在注入样品"提示，同时注入从所附的注射器的控制和测试介质装入生物芯片陈荫罴平。完成后离开注射器在生物芯片的地方。 注意:阻抗值将为60分钟收集每分钟一次。如果ECIS阅读器软件确定该处理通道是从控制信道在试验开始后的10分钟和60分钟之间的任何点统计学差异，则在屏幕上显示将表明，样品"污染"。如果治疗不从控制通道不同，那么屏幕上会显示"没有检测到污染"在试运行的结束。 </li><li>记录的测试结果被污染或不污染每个样品。 </li><li>在试运行结束后，删除和丢弃的生物芯片。冲洗并空气干燥该注射器，测试小瓶和可拆卸的塑料托盘其中容纳在测试期间的生物芯片。 注:ECIS读者有4 GB用于操作软件的板载存储，运行测试产生的控制模型和测试文件。这使得若干个ousand测试文件被存储在阅读器。文件可以用USB跳跃驱动器进行检索，并且如果需要传送到计算机，以便进一步分析用于研究目的。 </li></ol>

<ol>
	<li>Pancrazio, J. J., Whelan, J. P., Borkholder, D. A., Ma, A., Stenger, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+cell-based+biosensors.">Development and application of cell-based biosensors.</a> Ann. Biomed. Eng. 27, 697-711 (1999).</li><li>States, S., Scheuring, M., Kuchta, J., Newberry, J., Casson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility-based+analytical+methods+to+ensure+public+water+supply+security.">Utility-based analytical methods to ensure public water supply security.</a> J. Am. Water Works Assoc. 95, 103-115 (2003).</li><li>Kelly, T., Baxter, W., McCauley, M., Koglin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+of+Screening+Technologies+for+Detection+of+Toxic+Industrial+Chemicals+in+All+Hazards+Receipt+Facilities.">Testing of Screening Technologies for Detection of Toxic Industrial Chemicals in All Hazards Receipt Facilities.</a> EPA/600/R-08/034. , 1-25 (2008).</li><li>van der Schalie, W. H., James, R. R., Gargan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+a+battery+of+rapid+toxicity+sensors+for+drinking+water+evaluation.">Selection of a battery of rapid toxicity sensors for drinking water evaluation.</a> Biosens. Bioelectron. 22, 18-27 (2006).</li><li>Iuga, A., Lerner, E., Shedd, T. R., van der Schalie, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+responses+of+a+melanophore+cell+line+to+chemical+contaminants+in+water.">Rapid responses of a melanophore cell line to chemical contaminants in water.</a> J. Appl. Toxicol. 29, 346-349 (2009).</li><li>Curtis, T. M., 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=Suitability+of+invertebrate+and+vertebrate+cells+in+a+portable+impedance-based+toxicity+sensor:+temperature+mediated+impacts+on+long-term+survival.">Suitability of invertebrate and vertebrate cells in a portable impedance-based toxicity sensor: temperature mediated impacts on long-term survival.</a> Toxicol In Vitro. 27, 2016-2066 (2013).</li><li>Brennan, L. M., Widder, M. W., Lee, L. E. J., van der Schalie, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+storage+and+impedence-based+water+toxicity+testing+capabilities+of+fluidic+biochips+seeded+with+RTgill-W1+cells.">Long-term storage and impedence-based water toxicity testing capabilities of fluidic biochips seeded with RTgill-W1 cells.</a> Toxicol In Vitro. 26, 736-745 (2012).</li><li>Widder, M. W., Brennan, L. M., Hanft, E. A., Schrock, M. E., James, R. R., van der Schalie, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+refinement+of+a+field-portable+drinking+water+toxicity+sensor+and+a+fluidic+biochip.">Evaluation and refinement of a field-portable drinking water toxicity sensor and a fluidic biochip.</a> J. Appl. Toxicol. , (2014).</li><li>O'Shaughnessy, T. J., Gray, S. A., Pancrazio, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+neuronal+networks+as+environmental+biosensors.">Cultured neuronal networks as environmental biosensors.</a> J. Appl.Toxicol. 24, 379-385 (2004).</li><li>Eltzov, E., Marks, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+aquatic+biosensors.">Whole-cell aquatic biosensors.</a> Anal. Bioanal. Chem. 400, 895-913 (2011).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morphological+biosensor+for+mammalian+cells.">A morphological biosensor for mammalian cells.</a> Nature. 366, 591-592 (1993).</li><li>Curtis, T. M., 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=A+portable+cell-based+impedance+sensor+for+toxicity+testing+of+drinking+water.">A portable cell-based impedance sensor for toxicity testing of drinking water.</a> Lab on a Chip. 9, 2176-2183 (2009).</li><li>Xiao, C., Luong, J. H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cytotoxicity+by+emerging+impedance+spectroscopy.">Assessment of cytotoxicity by emerging impedance spectroscopy.</a> Toxicol. .Appl. Pharmacol. 206 (2), 102-112 (2005).</li><li>Xing, J. Z., Zhu, L., Gabos, S., Xie, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectronic+cell+sensor+assay+for+detection+of+cytotoxicity+and+prediction+of+acute+toxicity.">Microelectronic cell sensor assay for detection of cytotoxicity and prediction of acute toxicity.</a> Toxicol. In Vitro. 20, 995-1004 (2006).</li><li>Lee, L. E. J., Dayeh, V. R., Schirmer, K., Bols, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+potential+uses+of+fish+gill+cell+lines:+examples+with+RTgill-W1.">Applications and potential uses of fish gill cell lines: examples with RTgill-W1.</a> In Vitro Cell. Develop. Biol.- Animal. 45, 127-134 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RT-gill-W1+(ATCC+CRL-2523)+Culture+Method.">RT-gill-W1 (ATCC CRL-2523) Culture Method.</a> American Type Culture Collection (ATCC). , (2015).</li></ol>

Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Citations of commercial organizations or trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations. Authors Linda M. Brennan and Mark W. Widder, both of the US Army Center for Environmental Health Research, declare that they have no competing financial interests. Michael Mayo, Michael McAleer and Alex Greis are employees of Nanohmics that produces the disposable biochips and instruments used in this Article.10251980

ECIS的技术在实验室环境下表现良好，并能在浓度是相关的人体健康检测潜在的水污染物。该技术的便携和包装使得它有利于现场使用。 在该协议的技术成功的关键步骤如下:1）培养，播种期间保持无菌条件下，与生物芯片的馈送，2）保持种子生物芯片在冷藏条件下，直到准备用于自RTgill-W1细胞测试很长，一旦它们进行温度高于25℃时，将无法生存，3）准确称L形15ex在粉状介质小瓶和精确地测量水样品，以避免产生假阳性，其可通过在移位引起的媒体而不是样品毒性渗透压，4）按照ECIS屏幕上的用户说明运行测试。在阅读器软件将提醒如果双向用户ochip是用于测试（基于初始阻抗读数）不可接受时生物芯片首先插入读卡器。如果阻抗水平测试不能接受的，该软件将不允许用户与测试继续进行，直到一个新的生物芯片时使用。为不可接受的阻抗读数的原因通常是由于与ECIS读者销或沿着所述生物芯片的胶合边缘之一流体泄漏的生物芯片的电极的轻微未对准。 有一些限制这种技术，因为ECIS传感器只被用饮用水测试和不与表面水。所述RTgill-W1细胞是在生物芯片上不能较长时间（时间帧可以是从数小时至数天取决于温度耐受冷冻或温度大大高于25℃。在生物芯片起作用的温度范围最好从冷冻以室温7，他们准备立即使用，然而，作为后R权从冷库emoved。便携式冷藏容器目前在该领域使用的军队人员对温度敏感的用品。这些相同的容器可以用于种子生物芯片运输。 这种技术的另一个限制是，即使它是一个宽波段毒性传感器，它不，如果在所有反应良好，以抑制胆碱酯酶的化合物，如某些杀虫剂。为了填补这一能力差距，ECIS的传感器被设计以提供具有更广泛的毒性测试的用户测试水样时，在结合使用市售的快速农药试验测定。该试剂盒是一种快速酶法测定设计为在30分钟内检测有机磷和氨基甲酸酯杀虫剂。 ECIS的传感器补充WQAS-PM（水质分析系统 - 预防医学）域水测试系统，目前使用的军事预防医学人员发现，阿尔森IC，铅，氰化物或饮用水样本。虽然ECIS传感器将不能识别污染物是什么，这将表明，如果某些金属或有机化合物的存在，这表明水可能不适合人食用。 ECIS的测试结果可用一个小时之内。然后将水样可发出用于进一步分析的污染物的鉴定是否有阳性测试结果。 如上所述，ECIS的读者被设计为，其包括为了覆盖污染物检测的广谱单独酶ACE试剂盒的系统的一部分。这两个读者都被包装在运输领域为士兵野外使用一个坚固的情况下。

总体目标是开发用于细胞接种，储存和流体生物芯片的测试中的ECIS生物传感器的方法。对于该生物传感器的开发目标是，以满足美国陆军规格，可以检测饮用水供应的可能的污染正在由士兵一个字段便携式装置。对于毒性传感器的要求是，它可以在有关对人类健康浓度迅速检测有毒工业化合物的广谱（一个小时内），该装置是现场便携式，和生物组分将具有一个货架寿命至少九个月。冷藏，但不冻结，易腐成分是可以接受的。 从历史上看，与生物成分将它们现场便携式水检测技术（如抗体，酶，或核酸）已经被分析物特异性1-3。这些类型的生物传感器的缺点在于，它们将ONLŸ同时检测一种类型的化学品。如果怀疑该多个化学是本需要多个传感器。如果特定的传感器是不是在测试曲目，在水中化学污染物很容易被忽视。 广泛的毒性传感器，另一方面，必须填写此项技术差距的潜力。这些通常具有细胞成分对他们4-8。广泛的毒性生物传感器的优点是它们可以检测各种各样的化学污染物，其中包括它们的混合物和未知数，在时间5,9,10相对较短的存在。 使用细胞单层的电阻抗作为一个可能的毒性传感器，它也被称为电细胞-基底阻抗感测（ECIS）的测量的概念，首先由Giaever和Keese酒店11所述。在过去的二十年中，已经证明是细胞VIAB的敏感指标ility和细胞毒性。基本上，即附着在电极上的生物芯片的细胞单层暴露于高频率和低的电压和电流强度交流电流信号。细胞的汇合单层阻碍电子的流动。当细胞单层的完整性受到损害（当被引入一种有毒化学物质，如），ECIS的传感器记录在电阻抗的变化11-14。 图1示出ECIS的原则关系到细胞单层上的生物芯片。 <img alt="图1" src="/files/ftp_upload/53555/53555fig1.jpg" /> 图1:关于与简化ECIS读者电气原理生物芯片细胞单层ECIS插图原理 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53555/53555fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 12所述的ECIS传感器技术被使用。这些细胞不适合现场使用实际的，然而，因为它们需要频繁更换培养基，只在有限的保质期，并且需要人工的 CO 2的环境和37℃温育温度。它被发现从虹鳟鱼鳃上皮细胞（RTgill W-1细胞）衍生的市售的细胞系可以在在环境CO 2室温进行测试，形成在生物芯片融合单层，可以存储在冷藏温度，和不得不在有关对人类健康的12个浓度一快速反应（1小时或更少）的化学品的广谱。 RTgill-W1细胞毒理学应用，以及在基础研究，是由Lee 等人 15审查。 对于播种，存储和容纳流体生物芯片检测方法就在ECIS传感器流体生物芯片RTgill-W1细胞单层如下所述。流体生物芯片可以保存长达9个月在冷藏状态，并且可以在一个冷藏集装箱运，对于饮用水supplies.The伴随ECIS阅读器，或测试单元的测试，被单独提供。生物芯片有两个组成部分的人;上部的聚碳酸酯层，用两个分开的流体通道，以及包含每第四频道电极焊盘用于阻抗传感低级电子层。有每个垫10工作电极;每个电极是250微米的直径。组装的生物芯片具有用于当插入ECIS测试单元获取阻抗读数金电极连接。每两个封闭流体的U形通道将持有2毫升RTgill-W1细胞悬浮液; 图2示出了在ECIS读者汇合的细胞上的单个感测电极的倍率的流体的生物芯片。 图2:在ECIS读者射流生物芯片的放大区域显示RTgill-W1细胞的单一感应电极覆盖单层<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53555/53555fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Fetal bovine serum</td><td>Life Technologies, Inc. www.lifetechnologies.com</td><td>16000-085</td><td>Store @ -20 &amp;deg;C. Thaw @ room temperature before use. Ingredient for complete L-15 cell culture media (10%).</td></tr><tr><td>Fibronectin, bovine plasma</td><td>EMD Millipore Corp.&amp;nbsp;&amp;nbsp; www.emdmillipore.com&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>341631-1 mg</td><td>Store @ -20 &amp;deg;C. Thaw @ room temperature before use. Mix with L-15 media for a concentration of 10 &amp;#956;g/ml and freeze @ -20 &amp;deg;C in aliquots. Use as substrate for biochips.</td></tr><tr><td>L-15 media without L-glutamine</td><td>Lonza&amp;nbsp; www.lonzabioscience.com</td><td>12-700F</td><td>Basal media for cell culture and feeding biochips. Store at 6 &amp;deg;C.</td></tr><tr><td>L-15ex powdered media with phenol red</td><td>US Biological&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.usbio.net</td><td>L1501</td><td>Media is weighed out in 60 mg aliquots in 0.1 dram vials and stored at 6 &amp;deg;C in foil pouches with dessicant packs. Nine month shelf-life. Mixed with 10 ml of water sample for testing in biochips.</td></tr><tr><td>PBS, w/o Ca&lt;sup&gt;2+&lt;/sup&gt; or Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Lonza&amp;nbsp; www.lonzabioscience.com</td><td>17-516F</td><td>Store at room temperature. Used for rinsing media when trypsinizing cell culture flasks.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Trypsin, EDTA</td><td>Lonza&amp;nbsp; www.lonzabioscience.com</td><td>CC-5012</td><td>Store @ -20 &amp;deg;C. Thaw at room temperature and use to trypsinize cell culture flasks.&amp;nbsp;</td></tr><tr><td>T175 culture flasks</td><td>Fisher Scientific&lt;br/&gt; www.fishersci.com</td><td>12-565-30</td><td>Used for culturing RTgill-W1 cells.</td></tr><tr><td>Bleach</td><td>Chlorox&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.chlorox.com</td><td></td><td>Diluted to 20% with millique or distilled water for cleaning ECIS chips. Any household bleach is acceptable.</td></tr><tr><td>70% ethyl alcohol</td><td></td><td></td><td>For disinfecting biohood surfaces and any materials being placed in biohood.</td></tr><tr><td>Rainbow trout gill cells (RTgill-W1)&amp;nbsp;</td><td>American Type Tissue Culture Collection&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.atcc.org</td><td>CRL-2523</td><td>Cells cultured and used for biosensor (seeding biochips).</td></tr><tr><td>GlutaMAX-1 Supplement, 200 mM</td><td>Lonza&amp;nbsp; www.lonzabioscience.com</td><td>35050-061</td><td>Store at room temperature.&amp;nbsp; Ingredient for complete L-15 cell culture media (1%).</td></tr><tr><td>Penn/Strep Stock 10K/10K</td><td>Lonza&amp;nbsp; www.lonzabioscience.com</td><td>17-602E</td><td>Store @ -20 &amp;deg;C. Thaw @ room temperature before use. Ingredient for complete L-15 cell culture media (1%).</td></tr><tr><td>Pharmed BPT tubing</td><td>U.S. Plastic Corp.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.usplastic.com</td><td>57317</td><td>Cut in 27 mm sections and autoclaved. Used for seeding biochips with cells and as a closed loop between media changes.</td></tr><tr><td>Polycarbonate luer fittings for Pharmed tubing assemblies</td><td>Value Plastics&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>MTLS210-9</td><td>Secured to each end of cut Pharmed tubing for insertion into bichips.</td></tr><tr><td>20 ml syringes, slip-tip</td><td>VWR Scientific&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; us.vwr.com</td><td>BD302831</td><td>Used for injection of cell suspension for seeding ECIS chips, as well as for feeding chips.</td></tr><tr><td>0.1 dram snap-cap polypropylene microvials</td><td>Bottles Jars and Tubes, Inc.&amp;nbsp;&amp;nbsp; www.bottlesjarsandtubes.com</td><td>30600</td><td>Used to store 60 mg aliquots of L-15ex powdered media.</td></tr><tr><td>60 mil Lexan fluidic ECIS biochips</td><td>Nanohmics, Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp; www.nanohmics.com</td><td></td><td>Custom-made by Nanohmics, Inc. RTgill-W1 cells will be injected into the biochips and seeded chips will be placed in ECIS reader for testing.</td></tr><tr><td>Autoclavable Plastic Instrument Box&lt;br/&gt; 17 1/2&quot; x 7 3/4&quot; x 2 3/8&quot;</td><td>Medi-Dose EPS&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; medidose.com</td><td>IB701</td><td>Used to store the following; autoclaved plugs, biochips that have been cleaned, seeded biochips.&amp;nbsp;</td></tr><tr><td>Paper heat-seal sterilization pouches, 7 &amp;frac12;&amp;rdquo; x 13&amp;rdquo;</td><td>CardinalHealth&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.cardinalhealth.com</td><td>90713</td><td>Used for autoclaving tubing and fittings and plugs.</td></tr><tr><td>Quantos automated powder dispenser</td><td>Mettler Toledo&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.mt.com</td><td>QB5</td><td>Automated dispension of 60 mg aliquots of powdered L-15ex into 0.1 dram vials.</td></tr><tr><td>ECIS reader</td><td>Nanohmics, Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp; www.nanohmics.com</td><td></td><td>Custom-made by Nanohmics, Inc. Seeded biochip is inserted into the reader for conducting water toxicity testing.</td></tr><tr><td>3 x 5 metalized 2.5 mil polypropylene reclosable bags</td><td>Uline&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.uline.com</td><td>S-16893</td><td>Packaging and storage for both seeded biochips and powdered L-15ex media vials.</td></tr><tr><td>Leatherman squirt ps4</td><td>Amazon&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.Amazon.com</td><td></td><td>Used to open powdered media vials.</td></tr><tr><td>1 g silica gel desiccant packets&amp;nbsp;</td><td>Uline&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.uline.com</td><td>S-3902</td><td>Put in polypropylene bags with L-15ex powdered media vials to prevent the powder from picking up moisture.</td></tr><tr><td>Sterile 250 or 500 ml Nalgene bottles</td><td>Fisher Scientific&lt;br/&gt; www.fishersci.com</td><td>09-740-25C or E</td><td>Hold cell suspensions for seeding ECIS chips in biohood.</td></tr><tr><td>Plugs for biochips</td><td>Nanohmics, Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp; www.nanohmics.com</td><td></td><td>Custom-made by Nanohmics, Inc. Used to seal ports on biochips before storage at 6 &amp;deg;C.</td></tr><tr><td>Drains for ECIS biochips</td><td>Nanohmics, Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp; www.nanohmics.com</td><td></td><td>Custom-made by Nanohmics, Inc. Placed on 2 inner ports on biochips prior to insertion in ECIS reader.&amp;nbsp; Allows for excess media to drain from channels during test injections.&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific&lt;br/&gt; www.fishersci.com</td><td>S17040</td><td>Needed for counting cells prior to adjusting cell suspension for injection into biochips.</td></tr><tr><td>Brightfield microscope w/ 10X objective</td><td>Leitz Labovert</td><td></td><td>Any brightfield microscope is acceptable.</td></tr><tr><td>Class II biological safety cabinet</td><td></td><td></td><td>Any class II biological safety cabinet where cell culture can be performed under sterile conditions is acceptable.</td></tr><tr><td>Microcentrifuge tubes, 0.6 ml</td><td>Fisher Scientific&lt;br/&gt; www.fishersci.com</td><td>02-681-311</td><td>Holds 1 ml of cell suspension prior to counting cells.</td></tr><tr><td>Slip 10 cc red syringes</td><td>Procedure Products, Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.procedureproducts.com</td><td>S/49S 30-R</td><td>Withdraws 9 ml of test water sample and used to inject sample into biochip.</td></tr><tr><td>Slip 10 cc blue syringes</td><td>Procedure Products, Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.procedureproducts.com</td><td>S/49S 30-B</td><td>Withdraws 9 ml of control water sample and used to inject sample into biochip.</td></tr><tr><td>&amp;frac12; oz. clear pet plastic jar w/ white ribbed lined caps</td><td>SKS Bottle &amp;amp; Packaging, Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.sks-bottle.com</td><td>0605-30</td><td>Sample vials used for mixing L-15ex powder and 10 ml of water sample for testing.</td></tr><tr><td>50 ml sterile conical polypropylene centrifuge tubes</td><td>Fisher Scientific&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; www.fishersci.com</td><td>12-565-269</td><td>Used to hold 40 ml aliquots of 10 &amp;#956;g/ml fibronectin at -20 &amp;deg;C.</td></tr><tr><td>NIDS ACE Acetylcholinesterase Inhibitor Detection Test</td><td>ANP Technologies, Inc.&lt;br/&gt;www.anptinc.com</td><td>ACE-400</td><td>Sensor designed for the rapid detection of pesticides in drinking water</td></tr></tbody></table>

preparation and testing of impedance-based fluidic biochips with rtgill-w1 cells for rapid evaluation of drinking water samples for toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

在本文中描述的技术ECIS在美国环保署（USEPA）进行测试赞助的技术测试和评估计划（TTEP）。被选定为测试作为有毒工业化合物，可能是饮用水的可能的污染物的广谱的代表十三化学品。在测试中，13 9被ECIS一个小时内的浓度是相关的人类健康检测8。 表1示出了这种污染物测试的结果; 图4是代表什么一个"被污染的"结果将类似于上ECIS的读者屏幕。在大多数情况下，蜂窝阻抗为被污染的样品与对照相比减少。偶尔，某些化合物可能引起的阻抗的增加。 也总结在表1中 </s仲&gt;是干净的水测试。第四清洁水的样品运行，并在任何样品中没有检测到污染（参见图5）为"无污染检测到"的代表性屏幕截图。 <img alt="图4" src="/files/ftp_upload/53555/53555fig4.jpg" /> 图4:ECIS读卡器截图一个"被污染的"水样的从被污染的水样归一化阻抗的图形和结果的一个例子。蓝线代表每个控制电极的归一化的阻抗;红线代表每个试样电极中的标准化阻抗。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53555/53555fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53555/53555fig5.jpg" /> 图5:ECIS读卡器截图一个"不污染检测到"水样的从已不被污染的水样归一化阻抗的图形和结果的一个例子。蓝线代表每个控制电极的归一化的阻抗;红线代表每个试样电极中的标准化阻抗。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53555/53555fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>类别</td><td>污染物</td><td colspan="3">浓度测试（毫克/升）1 </td><td colspan="2">检测≤1小时 N = 4/4芯片</td></tr><tr><td rowspan="8">农药</td><td>涕灭威</td><td colspan="3"> 0.17 </td><td colspan="2">没有</td></tr><tr><td>阿尔森<html> IC（亚砷酸钠） </td><td colspan="3"> 4.5 </td><td colspan="2">是</td></tr><tr><td>叠氮化物（叠氮化钠） </td><td colspan="3"> 46.​​7 </td><td colspan="2">是</td></tr><tr><td>苯线磷</td><td colspan="3"> 0.56 </td><td colspan="2">没有</td></tr><tr><td>甲胺磷</td><td colspan="3"> 1.4 </td><td colspan="2">没有</td></tr><tr><td>甲基对硫磷</td><td colspan="3"> 33.6 </td><td colspan="2">是</td></tr><tr><td>百草枯（二） </td><td colspan="3"> 4.6 </td><td colspan="2">没有</td></tr><tr><td>五氯酚（钠） </td><td colspan="3"> 71.9 </td><td colspan="2">是</td></tr><tr><td rowspan="5">工业化学品</td><td>氨</td><td colspan="3"> 924 </td><td colspan="2">是</td></tr><tr><td>铜（铜II硫酸盐） </td><td colspan="3"> 103 </td><td colspan="2">是</td></tr><tr><td>氰化物（钠） </td>3"&gt; 14 </td><td colspan="2">是</td></tr><tr><td>汞（氯化物） </td><td colspan="3"> 24.7 </td><td colspan="2">是</td></tr><tr><td>甲苯</td><td colspan="3"> 444 </td><td colspan="2">是</td></tr><tr><td>清洁水2 </td><td>没有</td><td colspan="3"> NA </td><td colspan="2">没有</td></tr><tr><td colspan="7"> 1浓度测试是相同的由的Widder 等人的手稿。 （2014年）。 </td></tr><tr><td colspan="7"> 2 40清洁水样品用没有污染运行。 </td></tr></tbody></table> 表1:通过检测ECIS水样污染物。

Watch this Scientific Journal Video about Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity at JoVE.com

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

准备和测试阻抗为基础与RTgill-W1细胞饮用水进行快速评估水样进行毒性射流生物芯片

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

preparation-testing-impedance-based-fluidic-biochips-with-rtgill-w1

Nanyang Technological University

Research

JoVE Journal

Environment

9.0K 次观看.  U.S. Army Center for Environmental Health Research. This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described. 

视频: Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

用于实时细胞分析仪的阵列金电极再生

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

科研

生物化学

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

NRCC的膜片钳芯片上细胞的培养和电

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

神经科学

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

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

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

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

生物工程

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

芯片上的压力诱导抗生素易感性测试

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

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

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

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 ...

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 ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

通过电电池-衬底传感评估 MOF 诱导的电池行为变化

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

作者聚焦:评估人肺上皮细胞对金属有机框架反应的进展 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

用于实时评估金属有机框架毒理学特征的电池-基底传感

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

准备和测试阻抗为基础与RTgill-W1细胞饮用水进行快速评估水样进行毒性射流生物芯片

摘要

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微流体为基础的电化学生物芯片的无标记的DNA杂交分析

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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

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

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