This work was supported by the internal fund of the Korea Institute of Science and Technology (2E26180) and by the Next Generation Biomedical Device Platform program, funded by the National Research Foundation of Korea (NRF-2015M3A9E202888).

1.阳离子交换膜制造集成微流控芯片<ol><li> 主人硅的制备 <ol><li>设计2种硅主人:一种用于图案化的阳离子交换树脂和其他建设有聚二甲基硅氧烷（PDMS）微通道。 注:几何细节将在步骤1.3.1和1.4.1的描述。 </li><li>通过使用常规的光刻和深反应离子蚀刻27制造硅主人。 </li><li>在真空罐30分钟Silanize的微图案硅主人与三氯硅烷（〜30μL）。 注意:三氯氢硅是发火液体是易燃，有急性毒性（吸入，口服）。 </li></ol></li><li> PDMS模具的研制 <ol><li>在一个10混合与固化剂的有机硅弹性体基体:1的比例和放置杯与此未固化的PDMS（30-40毫升为一个4英寸的硅晶片上复制微结构）在真空罐30分钟以除去气泡。 注:聚硅氧烷基中含有的硅氧烷低聚物与乙烯基和基于铂的催化剂终止。固化剂含有交联具有三个与硅氢化键28的低聚物。 </li><li>倒在硅主人未固化的PDMS，用鼓风机除去气泡，并在80℃固化的PDMS用于在对流烘箱2小时。 </li><li>从硅主人分离固化的PDMS和适当形状用刀子的PDMS（平方的形状，如在图2a-b所示，ⅳ）。 </li></ol></li><li> 图案化该阳离子交换膜 <ol><li>削减一半的PDMS模具的垂直于两个平行的微通道，并在配备2.0毫米的活检穿孔的PDMS通道的两端打孔。 注:将PDMS模具用于图案阳离子选择性膜具有两个参数等位基因微通道（宽:100微米;高度:50微米;道间距离:100微米; 图1a）。模具的原始形状可通过镜像沿着切割线切片的模具是可想而知的。 L形的微通道推荐用于冲压的两个孔不重叠。 </li><li>清洁载玻片和PDMS模具用胶带和鼓风机，把模具上的玻璃载片，以创建它们之间的可逆附着。 </li><li>根据微流图形化技术29，释放〜10微升在通道开口端的阳离子交换树脂已在步骤切片1.3.1（ 图1b）。放置在冲压孔的注射器头并拉动柱塞（ 图1b中的黑色箭头）;温和的负压将拉动阳离子交换树脂，该树脂将填补两个通道。 注:建议的微通道的高度是大于1581峰; m，由于树脂的粘度高，需要高的压力来填充通道。另一方面，最好是，该高度不超过100微米，由于图案化的离子选择性膜将变得厚于1微米;这种厚的膜可以创建在膜和PDMS通道13之间的间隙。 </li><li>分离与PDMS模具不接触有图案的树脂，并放置在加热器的载玻片在95℃下5分钟，以蒸发在树脂中的溶剂。 注:图案化膜的厚度通常比&lt;1微米以下。模具被轻轻铰接模具的开放式侧分离的（虚线和图1b中的箭头）。最好是填充树脂后，拆下模不到1分钟。如果模具分离的几分钟后，可以得到更厚的膜，但它们将有一个凹形由于毛细作用。 </li><li>剥离不必要的用刀片图案化膜的一部分，使得两个分离的线图案（ 图1c）。 注:此处使用的阳离子交换材料具有全氟化基团，这意味着图案不牢固地结合到玻璃上。因此，简单的旱冰方法可以很容易地除去该膜的不必要的部分。 </li></ol></li><li> 所述微通道和膜图形化衬底的集成 <ol><li>冲在微通道的端部的两个孔，另外两个孔，其中该膜的图案将位于接合的PDMS通道到在步骤1.3制造的膜图形化衬底之后。 注意:将PDMS微通道具有一个信道（宽度:50-100微米;高:10微米），但它被结合到相邻的信道（ 图1d）的端部。 </li><li>在100瓦和50毫乇粘结的PDMS微通道的膜图案化衬底氧等离子体处理后，立即对40秒。 注意:垂直放置在图案化膜的微通道的中间。 </li></ol></li></ol> 2. ICP富集<ol><li> 为实验准备 <ol><li>制备各种测试方案，包括1-100毫米氯化钾，1mM的氯化钠（pH约7），1毫摩尔NaCl和0.2mM的盐酸（pH值〜3.7），1毫摩尔NaCl和0.2mM的氢氧化钠（pH值的混合物的混合物〜 10.3），和1x磷酸盐缓冲的盐水。 </li><li>带负电的荧光染料（〜1.55微米）添加到试验溶液。 注:加入的染料的浓度应比该盐离子（&lt;10微米）的低得多，以使带电的染料不到的电流30，31作出贡献。 </li><li>装载在通道中的一个储存器的样品溶液，并应用负压至另一贮存器，以填补与溶液中的信道。由R流体动力连接两个水库eleasing一个大墨滴，以消除沿通道（ 图2a）中的压力梯度。 </li><li>填充两个水库，其被连接到阳离子交换模式，使用注射器或移液管，以补偿在油藏的ICP效果缓冲溶液（1M，氯化钾或1M NaCl）中。 </li><li>放置导线在水库，跨越两个图案化的膜（在左侧储存器和阴极右侧阳极），并将它们与一个源测量单元（ 图2a）进行连接。 </li></ol></li><li> 的ICP现象和ICP富集可视化 <ol><li>在加载一个倒置落射荧光显微镜的ICP设备。施加电压（0.5100 V）和测量与源测量单元中的电流响应。 </li><li>捕获荧光图像与电荷耦合器件照相机和分析利用成像软件32的荧光强度。 </li></ol></li></ol>

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The authors have nothing to disclose.

我们所描述的制造协议和spatiotemporally限定预浓缩的在一个范围内施加电压（0.5-100 V）离子强度（1-100毫米）的性能，和pH值（3.7-10.3），实现了10,000倍在10分钟内的染料与蛋白质的富集。像以前的ICP设备的富集性能变得更高更好的电压和更低的离子强度。我们可以考虑在此一附加参数在两个阳离子交换膜之间的距离。如果我们增加膜间的距离，电场施加相同的电压下降低，从而导致在预浓缩速度26的下降。 在这项工作中使用的微流图形化技术29是用于图案化的阳离子交换树脂健壮的方法，因此它一直为离子交换材料结合进微流体系统的金标准方法之一。 ñevertheless，有必要制造2并列阳离子交换膜具有短膜间距离（小于几百微米）。在步骤1.3.3-1.3.4，所述阳离子交换树脂是在液相中。因此，在这两个微通道的树脂可以折叠，并且在通道的开口端的剩余树脂滴也可以在模具脱离期间淹没（步骤1.3.4）。构建具有高图案保真度2的阳离子交换膜，我们所用的树脂具有相对高的粘度（在溶剂中的阳离子交换材料的20％），并仔细设置以指定的分离方向上的拆卸过程。 尽管这一平台的高经营灵活证实，读者可能会担心从大范围确定操作窗口内的最佳条件。一位代表权衡是富集速度与ICP的效果稳定性之间。如CAN在图5中郭等人可以看到。 26，高外加电压（&gt; 50 V），可以快速凝聚的目标;然而，这也导致在耗尽区（1毫米/ 图7a pH 7）中，这降低了样品富集的稳定性强涡流。因此，富集速度变得很难预测33。在目前阶段，我们建议用一个稳定的，可预测的，并且spatiotemporally固定富集相对低的电压（&lt;30 V）和离子强度（&lt;10毫米）试验条件。预浓缩速度和预浓缩塞的稳定性之间的这种折衷还涉及非线性ICP的来源（表面导电，EOF和电渗透不稳定）。非线性ICP的以相对小的电压（&lt;50 V）的主要来源是EOF，创造在耗尽区中的相干涡旋对（ 图3b），该乐广告一个稳定的富集。以相对高的电压（&gt; 50 V），非线性ICP的主要来源被改变到电渗透不稳定，导致混乱的多个旋涡，这降低了富集的稳定性。 最近，基于纸张的ICP平台已经由藩等人开发的。 34，龚等人。 19，汉等人。 21。与微孔结构，这些纸装置可以抑制电渗不稳定4，35，缓解稳定问题。然而，纸通道的尺寸一般是约0.5-5毫米，这是比常规微流体通道大得多。随机光纤网络这个更宽广的渠道纸导致在预浓缩塞不规则运动。这已经在纸质ICP preconcentrators不可避免的，因为在最小特征蜡图案和剪纸大小 （ 即制造方法打造纸通道）大约是几百微米。 的ICP预浓缩已在广泛的用于预浓缩各种生物剂biomicrofluidic平台被使用;扩增各种测定的信号;并检测目标，如治疗性蛋白质36，肽37，适体17和酶38。这些以前的作品有针对性的荧光标记的生物分子。这是因为我们不能指定确切的操作条件（即电压和流速），以维持现场富集，所以我们首先需要找到的预浓缩目标的适当条件。从以前的工作出发，合并后的ICP现象使我们可以总是在一个宽范围的操作条件修正预浓缩塞，同时保持高度的灵活性ICP设备。例如，我们可以调节与切向流体流的合并的ICP系统，并在连续流模式39操作。这表明，我们现在可以扩展ICP preconcentrators的应用无标记检测技术，在不使用的可视化工具和示踪剂。时空可控的这种独特的优势提供了与普通台式平台，如聚合酶链反应机器和质谱仪的ICP设备集成一个强大的商业机会。

离子浓度极化（ICP）是指在一个选择性渗透膜离子富集和离子耗尽期间发生，导致与离子浓度梯度1,2额外电位降的现象。该浓度梯度是线性的，并作为一个较高电压被施加（欧姆制度），直到在膜上的离子浓度变得更陡接近零（限制性制度）。在此扩散限制条件，梯度（和相应的离子通量）已被公知的最大化/饱和1。超出了这个常规理解，当电压（或电流）进一步增大时，一个overlimiting电流观察到的，与平坦耗尽区和非常尖锐的浓度梯度在区域边界1,3。平坦区具有一个非常低的离子浓度，但表面导电，电osmoti<html> Ç渗流（EOF），和/或电渗透不稳定促进离子通量和诱导overlimiting电流的3，4，5。有趣的是，平耗尽区用作静电屏障，该滤波器滤除6，7，8，9和/或预浓缩目标10，11。由于存在不足量的离子以筛选带电粒子的表面电荷（对于满意的电中性），所述颗粒不能穿过该耗尽区，因此，在其边界排队。该非线性ICP效应是在不同类型的膜10，11，12，13的一个通用的现象，&gt; 14和几何形状6，15，16，17，18，19，20，21;这就是为什么研究人员已经能够开发各类过滤6，7，8，9 富集采用非线性ICP 10，11台设备。 即使有这样的高度的灵活性和健壮性，它仍然是澄清的非线性ICP设备的操作条件的实用的挑战。比较方案的非线性区快速通过阳离子交换膜，这导致阴离子朝向阳极移动的位移去除阳离子。作为一个结果，平耗尽区传播快，这使人想起休克传播22。摩尼等。称这种动态的去离子（或耗竭）冲击23。到在指定的检测位置预浓缩的目标，防止离子耗尽区的扩展是必要的，例如，通过施加EOF或压力驱动流对区扩张24。 Zangle 等。 22澄清ICP传播的标准中的一维模型，它高度依赖于电泳迁移17，离子强度18，pH值25，等等。这表明，适当的操作条件将根据样品条件被改变。 在这里，我们提出详细的设计和实验协议，一个spatiotemp内预浓缩目标的新的ICP平台口服定义的26位。离子耗尽区的扩展是由离子富集区阻断，在一个指定的位置离开静止富集塞，不论工作时间，施加电压，离子强度和pH值。该详细视频协议是为了表明，以阳离子交换膜整合到微流体装置，并表明相比于以往的新ICP平台的富集性能的最简单的方法。

<table border="0" cellpadding="0" cellspacing="0" width="720">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Sylgard 184 Silicone Elastomer kit</td>
			<td style="width:195px;">Dow Corning</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Trichlorosilane</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td style="width:119px;">175552</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nafion perfluorinated resin, 20 wt%</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">527122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">71394</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">60121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td style="width:119px;">A20000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isothiocyanate-conjugated albumin</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">A9771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffer saline, 1X</td>
			<td>Wengene</td>
			<td style="width:119px;">LB004-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epifluorescence microscope</td>
			<td>Olympus</td>
			<td style="width:119px;">IX-71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Charged-coupled device camera</td>
			<td>Hamamtsu Co.</td>
			<td style="width:119px;">ImageEM X2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Source measurement unit</td>
			<td>Keithley Instruments</td>
			<td style="width:119px;">2635A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Covance-MP</td>
			<td style="width:195px;">Femto Science</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

merging ion concentration polarization between juxtaposed ion exchange membranes to block the propagation of the polarization zone

离子浓度极化（ICP）的现象是最普遍的方法来预浓缩低丰度的生物样品之一。该ICP诱导带电生物分子（即离子耗尽区）无创性区域和目标可以在此区域的边界上预浓缩。尽管高富集表演使用ICP，它是很难找到的非传播离子耗尽区中的操作条件。为了克服这个狭窄的操作窗口，我们最近开发了spatiotemporally固定富集的新平台。不像前述仅使用离子耗尽的方法，该平台还使用ICP（ 即，离子富集）的极性相反，停止离子耗尽区的传播。通过与耗尽区的富集区面临的两区合并在一起，并停止。在本文中，我们描述了详细的实验协议来建立这个spatiotemporally定义ICP platfORM和它们与常规设备的比较表征新平台的富集动态。定性离子浓度分布和电流 - 时间响应成功捕获合并的ICP和单机ICP之间的不同的动态。在与传统的一个可以仅〜5伏固定富集的位置，新的平台可以在操作条件的宽范围的特定位置产生一个目标冷凝插头:电压（0.5-100 V）中，离子强度（1-100毫摩尔）和pH（3.7-10.3）。

膜集成微流体预浓缩的概略的制造步骤示于图1。的制造的详细描述的协议中给出。在设计和spatiotemporally限定预浓缩26的设备中的图像进行了对比与那些常规的预浓缩11（ 图2）的。在spatiotemporally定义预浓缩的ICP现象的电流-电压-时间响应和荧光强度分布（ 图3-4）而言进行了研究。类似于用单膜预浓缩3,11，三种不同的机制的ICP现象（欧姆，限制和overlimiting）中的电流-电压曲线中观察到:5 V 0.5-1 V（欧姆和限制性的）和（overlimiting） 。然而，目前的非常规回收率为在电流 - 时间曲线检测作为离子富集和离子耗尽区合并。接着，将ICP富集在不同的时间和电压与spatiotemporally定义预浓缩（ 图5）和常规的单膜装置（ 图6）进行了测试。预浓缩动力学通过荧光图像，电流 - 时间响应，并在不同的距离和时间的荧光强度曲线定量。当比较两个平台，新ICP平台示出了总是收集两个阳离子选择性隔膜图案之间的目标（荧光染料）的优点。此外，可以确认该预浓缩插头保持在不同离子强度（1-100 mM氯化钠）和pH值（3.7-10.3）相同，核实的操作条件很宽的范围（ 图合流ICP预浓缩的高可用性7）。在图8中 ，10,000倍蛋白preconcentr通货膨胀也证明。 <img alt="图1" src="/files/ftp_upload/55313/55313fig1.jpg" /> 图1.阳离子交换膜集成的微流体芯片的制作步骤。 29，膜图案化的玻璃基板贴合与用氧等离子体处理（d）将PDMS微通道-后PDMS模具使用微流构图技术（ 三 一 ）填充有阳离子交换树脂。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55313/55313fig2.jpg" /> 图2. spatiotemporally限定预浓缩（a）和常规预浓缩的示意图（二）。 （a）在第ê新的平台，两个薄膜图案（i）中，离子耗尽之间/富集区的开发，并与线性合并在一起（欧姆和限制性制度; ii）或非线性（overlimiting制度;ⅲ）浓度分布。在所有三个电流制度，离子富集区阻止耗尽区和目标的传播（空心圆圈; i）的在离子耗尽和富集区（;我弯曲，虚线）的接口预浓缩。与PDMS通道的壁是带负电荷的，并且这产生电场下的两个阳离子交换膜之间电渗流（EOF）。该EOF不断提供对目标的枯竭和富集区的接口。 （b）在常规平台，只有离子耗尽区是发达的线性膜附近（欧姆和限制性制度;ⅱ）和非线性（overlimiting制度;ⅲ）浓度梯度。由于EOF提供了指标，富集人所以发生在耗尽区边界的，但此区域（和预浓缩插头）移动从阳离子交换膜远（黑色箭头;ⅰ）。值得注意的是，没有在离子浓度没有增加此处没有离子富集区（II-III）。在（AB），该装置的图像显示在（ⅳ）。 C 0表示初始离子浓度。 V +和G表示在阳极和阴极，分别。从参考26吨转载来自美国化学学会的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55313/55313fig3.jpg" /> 图3. 合并2的阳离子交换膜之间ICP现象。 （ 一 ）的电流-电压曲线显示了三个不同regimES（欧姆，限制和overlimiting）。电流响应由斜坡向上以0.25 V中的每个40秒，这是重复三次离散间隔的电压测量。误差条指示电流响应的标准偏差。 （B，C）在这三个制度，得到荧光图像（b）和沿AA&#39;在通道（c）的中间强度分布。黄色，虚线方框表示的阳离子选择膜的位置。使用1用1.55微米（1微克/毫升）带负电荷的荧光染料毫米氯化钾溶液。从参考26翻印获得美国化学学会的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55313/55313fig4.jpg" /> 图4。 <stroNG&gt;合并离子耗竭和富集区的瞬态动力学。 （A，B）在欧姆限制性制度，线性浓度梯度&lt;从阳离子交换膜（1秒，然后重叠一起成长）（&gt; 1秒）。 （c）在overlimiting制度，两个ICP区域被合并更快地（&lt;0.6秒）与耗尽休克（在0.2秒的黑色箭头）。 （ 四 - 六 ）的电流-时间响应表明当前最初下降是由于低浓度耗尽区的生长，其对应于低的导电性。那么目前的下跌是由于回收由两层膜之间的密闭涡旋对流运输。从参考的26翻印获得美国化学学会的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;p系列="jove_content"FO: - together.within页保留="1"&gt; <img alt="图5" src="/files/ftp_upload/55313/55313fig5.jpg" /> 图5. 在5，10 Spatiotemporally固定富集，和20 V（ 一 - 三 ）合并的ICP的荧光图像和当前时间的响应（ 四 - 六 ）随时间（0-100多个）。黄色，虚线表示的阳离子交换膜的位置。 （G）时间推移荧光强度分布沿着微绘（AA&#39;）。峰强度随着时间的推移，有固定的位置。 （H）的峰强度倍（ 即，有多少次大于初始荧光强度越大）。在更高的电压下，更快的EOF提供目标朝向离子耗尽和富集区的界面，所以在预浓缩速度增加。在20 V A尖峰被耗竭引起的震动（ <s仲&gt;图4c）。该荧光染料震荡边界上积累，因为它推动染料。这初步积累，当消耗震荡符合离子富集区，创造了峰值强度曲线的峰值，然后有所分散。如在图4c中可以看出，在0.8秒，峰宽度大于它在0.4秒。这可能是因为左侧的Nafion模式（ 图2a）的左侧是电浮动，累计染料可能会传播出去。从参考26翻印获得美国化学学会的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55313/55313fig6.jpg" /> 在5，10和20伏在常规ICP预浓缩 图6. ICP现象。 （ 一 - 三 ）离子耗尽区的荧光图像和当前时间响应（ 四 - 六 ）随时间（0-100多个）。耗尽区和富集插头的传播在荧光图像被清楚地显示。因此，该旋涡不仅限于，所以当前的恢复不发生，即使是在overlimiting制度。黄，虚线标出的阳离子交换膜的位置。 （G）时间推移荧光强度分布沿着微绘（AA&#39;）。峰强度随着时间的推移，但位置从膜上移开。传统的ICP装置（H）峰强度折。与此相反的合并的ICP装置（ 图5H），有无ICP区域的限制没有强度尖峰，因为荧光强度增加作为染料预浓缩。 T的增加他峰值强度折叠类似于同时合并的ICP装置的（在给定电压）。这表明，该预浓缩插头保持就位的时间长度是将富集的性能是至关重要的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/55313/55313fig7.jpg" /> 图7. 在不同的离子强度Spatiotemporally定义富集（1-100毫米氯化钠）和pH值（3.7-10.3）。 （a）于50伏作为后100秒的操作，获得荧光图像可以看出，在预浓缩塞的位置仍然两个阳离子交换膜（黄色，虚线）之间，即使强度下高离子减弱强度和在强酸性或碱性解。 （B，C）的峰强度的位置和其强度倍（ 即 ，有多少次大于初始强度），在10映射，20，50，和100 V.对于一个单一的条件（1，10， 100mM的和/或pH 3.7，图7和10），有对应于四个电压条件四个数据点。在更高的电压，有在所有的情况下更高的峰值加强折。 100 V在1毫摩尔NaCl（pH 7）中，因为峰值强度已经触及的最高值（由于摄像机的饱和度），在50五从峰强度分布没有测试，峰区域还确定，1 ％的峰强度，这是由误差条（b，C）表示如下。更高的电压和更强大的巅峰位置EOF向右移动，具有较高的强度倍和更清晰的富集插头。灰色框表示的阳离子交换膜的位置。 0的距离（a）表示x轴的原点（B，C），这是在左侧的阳离子交换膜的右边缘。距离的原点是左膜的右边缘。从参考26翻印获得美国化学学会的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/55313/55313fig8.jpg" /> 图8.示范spatiotemporally固定蛋白质富集的。使用在1×磷酸盐缓冲盐水溶液的FITC-白蛋白（1微克/毫升）。还加入0.1％吐温20，以防止非特异性结合。由于富集在较高离子强度（ 图7）几乎不实现，我们一倍的Nafion图案（200微米）的宽度和所使用的较窄的PDMS通道（50微米）。以这种方式，ICP富集的性能被提高加宽离子通路和减少在沟道的离子的绝对量。在100伏时，峰值和平均荧光强度的施加电压在白色的，虚线框，这是两个阳离子交换膜之间的区域进行跟踪。内操作的10分钟，将蛋白质预浓缩至10毫克/毫升（峰值）和〜0.1毫克/毫升（平均），指示10,000-和100倍preconcentrations分别。在0，10和20分钟，得到的插图荧光图像。在这项工作中，有20分钟的操作就足以预浓缩目标分子，所以我们没有覆盖更长的时间。从参考26翻印获得美国化学学会的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55313/55313fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone at JoVE.com

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

为一个新的离子浓度极化（ICP）平台的协议，可以停止ICP区的传播，不管操作条件进行说明。这一独特的平台能力，在于所使用的合并离子耗竭和充实，这是的ICP现象的两个极性。

并列的合并离子交换膜之间的离子浓差极化，以块极化区的传播

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

merging-ion-concentration-polarization-between-juxtaposed-ion

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.4K 次观看.  Korea Institute of Science and Technology. 为一个新的离子浓度极化（ICP）平台的协议，可以停止ICP区的传播，不管操作条件进行说明。这一独特的平台能力，在于所使用的合并离子耗竭和充实，这是的ICP现象的两个极性。 

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

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

多分析物生物芯片（MAB），基于全固态离子选择性电极（ASSISE）的生理研究

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

合并一个液滴后荧光恢复来衡量磷脂单层的二维扩散

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

聚焦 Ion 光束平版印刷到蚀刻纳米架构到微电极

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

硅纳米线与光学刺激研究细胞内和细胞间电耦合

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

牙齿和呼吸细胞及器官在微重力下的繁殖

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...