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>

<ol>
	<li>Probstein, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physicochemical Hydrodynamics: An Introduction. , (2003).</li><li>Strathmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion-Exchange Membrane Separation Processes. , (2004).</li><li>Dydek, E. V., 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=Overlimiting+Current+in+a+Microchannel.">Overlimiting Current in a Microchannel.</a> Phys. Rev. Lett. 107 (11), 118301 (2011).</li><li>Kwak, R., Pham, V. S., Lim, K. M., Han, 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=Shear+flow+of+an+electrically+charged+fluid+by+ion+concentration+polarization:+scaling+laws+for+electroconvective+vortices.">Shear flow of an electrically charged fluid by ion concentration polarization: scaling laws for electroconvective vortices.</a> Phys. Rev. Lett. 110 (11), 114501 (2013).</li><li>Rubinstein, I., Zaltzman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-osmotically+induced+convection+at+a+permselective+membrane.">Electro-osmotically induced convection at a permselective membrane.</a> Phys. Rev. E. 62 (2), 2238-2251 (2000).</li><li>Kwak, R., Kim, S., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous-flow+biomolecule+and+cell+concentrator+by+ion+concentration+polarization.">Continuous-flow biomolecule and cell concentrator by ion concentration polarization.</a> Anal. Chem. 83 (19), 7348-7355 (2011).</li><li>Jeon, H., Lee, H., Kang, K. H., Lim, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+concentration+polarization-based+continuous+separation+device+using+electrical+repulsion+in+the+depletion+region.">Ion concentration polarization-based continuous separation device using electrical repulsion in the depletion region.</a> Sci.Rep. 3, 3483 (2013).</li><li>Kim, S. J., Ko, S. H., Kang, K. H., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+seawater+desalination+by+ion+concentration+polarization.">Direct seawater desalination by ion concentration polarization.</a> Nat. Nanotechnol. 5 (4), 297-301 (2010).</li><li>MacDonald, B. D., Gong, M. M., Zhang, P., Sinton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Out-of-plane+ion+concentration+polarizationfor+scalable+water+desalination.">Out-of-plane ion concentration polarizationfor scalable water desalination.</a> Lab Chip. 14 (4), 681-685 (2014).</li><li>Schoch, R. B., Han, J. Y., Renaud, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+phenomena+in+nanofluidics.">Transport phenomena in nanofluidics.</a> Rev.Mod. Phys. 80 (3), 839-883 (2008).</li><li>Kim, S. J., Song, Y. A., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofluidic+concentration+devices+for+biomolecules+utilizing+ion+concentration+polarization:+theory,+fabrication,+and+applications.">Nanofluidic concentration devices for biomolecules utilizing ion concentration polarization: theory, fabrication, and applications.</a> Chem. Soc. Rev. 39 (3), 912-922 (2010).</li><li>Mai, J. Y., Miller, H., Hatch, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+mapping+of+concentration+polarization+Induced+pH+changes+at+nanoconstrictions.">Spatiotemporal mapping of concentration polarization Induced pH changes at nanoconstrictions.</a> ACS Nano. 6 (11), 10206-10215 (2012).</li><li>Kim, B., 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=Tunable+ionic+transport+for+a+triangular+nanochannel+in+a+polymeric+nanofludic+system.">Tunable ionic transport for a triangular nanochannel in a polymeric nanofludic system.</a> ACS Nano. 7 (1), 740-747 (2013).</li><li>Mangano Syed, A., Mao, L., Han J, P., Song, Y. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+sub-50+nm+nanofluidic+junctions+in+a+PDMS+microchip+via+self-assembly+process+of+colloidal+silica+beads+for+electrokinetic+concentration+of+biomolecules.">Creating sub-50 nm nanofluidic junctions in a PDMS microchip via self-assembly process of colloidal silica beads for electrokinetic concentration of biomolecules.</a> Lab Chip. 14, 4455-4460 (2014).</li><li>Wang, Y. C., Stevens, A. L., Han, 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=Million-fold+preconcentration+of+proteins+and+peptides+by+nanofluidic+filter.">Million-fold preconcentration of proteins and peptides by nanofluidic filter.</a> Anal. Chem. 77 (14), 4293-4299 (2005).</li><li>Lee, J. H., Cosgrove, B. D., Lauffenburger, D. A., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfludic+concentration-enhanced+cellular+kinase+activity+assay.">Microfludic concentration-enhanced cellular kinase activity assay.</a> J. Am. Chem. Soc. 131 (30), 10340-10341 (2009).</li><li>Cheow, L. F., Han, 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=Continuous+signal+enhancement+for+sensitive+aptamer+affinity+probe+electrophoresis+assay+using+electrokinetic+concentration.">Continuous signal enhancement for sensitive aptamer affinity probe electrophoresis assay using electrokinetic concentration.</a> Anal. Chem. 83 (18), 7086-7093 (2011).</li><li>Ko, S. 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=Nanofluidic+preconcentration+device+in+a+straight+microchannel+using+ion+concentration+polarization.">Nanofluidic preconcentration device in a straight microchannel using ion concentration polarization.</a> Lab Chip. 12 (21), 4472-4482 (2012).</li><li>Gong, M. M., Nosrati, R., Gabriel, M. C. S., Zini, M., Sinton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+DNA+analysis+with+paper-based+ion+concentration+polarization.">Direct DNA analysis with paper-based ion concentration polarization.</a> J. Am. Chem. Soc. 137 (43), 13913-13919 (2015).</li><li>Hong, S., Kwak, R., Kim, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+flow+fractionation+system+applicable+to+preconcentration+and+field-flow+separation.">Paper-based flow fractionation system applicable to preconcentration and field-flow separation.</a> Anal. Chem. 88 (3), 1682-1687 (2016).</li><li>Han, S. I., Hwang, K. S., Kwak, R., Lee, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+paper-based+biomolecule+preconcentrator+based+on+ion+concentration+polarization.">Microfluidic paper-based biomolecule preconcentrator based on ion concentration polarization.</a> Lab Chip. 16, 2219-2227 (2016).</li><li>Zangle, T. A., Mani, A., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+experiments+of+concentration+polarization+and+ion+focusing+at+microchannel+and+nanochannel+interfaces.">Theory and experiments of concentration polarization and ion focusing at microchannel and nanochannel interfaces.</a> Chem. Soc. Rev. 39 (3), 1014-1035 (2010).</li><li>Mani, A., Bazant, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deionization+shocks+in+microstructures.">Deionization shocks in microstructures.</a> Phys. Rev. E. 84, 061504 (2011).</li><li>Slouka, Z., Senapati, S., Chang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+systems+with+ion-selective+membranes.">Microfluidic systems with ion-selective membranes.</a> Annu. Rev.Anal. Chem. 7, 317-335 (2014).</li><li>Kirby, B. J., Hasselbrink, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zeta+potential+of+microfluidic+substrates:+1.+Theory,+experimental+techniques,+and+effects+on+separations.">Zeta potential of microfluidic substrates: 1. Theory, experimental techniques, and effects on separations.</a> Electrophoresis. 25 (2), 187-202 (2004).</li><li>Kwak, R., Kang, J. Y., Kim, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+defining+biomolecule+preconcentration+by+merging+ion+concentration+polarization.">Spatiotemporally defining biomolecule preconcentration by merging ion concentration polarization.</a> Anal. Chem. 88 (1), 988-996 (2016).</li><li>Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+microfluidic+systems+in+poly(dimethylsiloxane).">Rapid prototyping of microfluidic systems in poly(dimethylsiloxane).</a> Anal. Chem. 70 (23), 4974-4984 (1998).</li><li>Campbell, D. J., 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=Replication+and+compression+of+surface+structures+with+polydimethylsiloxane+elastomer.">Replication and compression of surface structures with polydimethylsiloxane elastomer.</a> J. Chem. Educ. 76 (4), 537-541 (1999).</li><li>Lee, J. H., Song, Y. A., Han, 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=Multiplexed+proteomic+sample+preconcentration+device+using+surface-patterned+ion-selective+membrane.">Multiplexed proteomic sample preconcentration device using surface-patterned ion-selective membrane.</a> Lab Chip. 8 (4), 596-601 (2008).</li><li>Kwak, R., Guan, G., Peng, W. K., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+electrodialysis:+concentration+profiling+and+vortex+visualization.">Microscale electrodialysis: concentration profiling and vortex visualization.</a> Desalination. 308, 138-146 (2013).</li><li>Chambers, R. D., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantification+of+isotachophoresis+zones+using+nonfocusing+fluorescent+tracers.">Imaging and quantification of isotachophoresis zones using nonfocusing fluorescent tracers.</a> Anal. Chem. 81, 3022-3028 (2009).</li><li>Minerick, A. R., Ostafin, A. E., Chang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrokinetic+transport+of+red+blood+cells+in+microcapillaries.">Electrokinetic transport of red blood cells in microcapillaries.</a> Electrophoresis. 23 (14), 2165-2173 (2002).</li><li>Phan, D. -. T., Shaegh, S. A. M., Yang, C., Nguyen, N. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+concentration+in+a+microfluidic+paper-based+analytical+device+using+ion+concentration+polarization.">Sample concentration in a microfluidic paper-based analytical device using ion concentration polarization.</a> Sens. Actuators B. 222, 735-740 (2016).</li><li>Rubinstein, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+a+nonequilibrium+electro-osmotic+instability.">Direct observation of a nonequilibrium electro-osmotic instability.</a> Phys. Rev. Lett. 101, 236101 (2008).</li><li>Ouyang, 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=Microfluidic+platform+for+assessment+of+therapeutic+proteins+using+molecular+charge+modulation+enhanced+electrokinetic+concentration+assays.">Microfluidic platform for assessment of therapeutic proteins using molecular charge modulation enhanced electrokinetic concentration assays.</a> Anal. Chem. 88, 9669-9677 (2016).</li><li>Cheow, L. F., Sarkar, A., Kolitz, S., Lauffenburger, D., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+kinase+activities+from+single+cell+lysate+using+concentration-enhanced+mobility+shift+assay.">Detecting kinase activities from single cell lysate using concentration-enhanced mobility shift assay.</a> Anal. Chem. 86, 7455-7462 (2014).</li><li>Chen, C. -. 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=Enhancing+protease+activity+assay+in+droplet-based+microfluidics+using+a+biomolecule+concentrator.">Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator.</a> J. Am. Chem. Soc. 133, 10368-10371 (2011).</li><li>Kwak, R., Pham, V. S., Kim, B., Lan, C., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+salt+removal+by+unipolar+ion+conduction+in+ion+concentration+polarization+desalination.">Enhanced salt removal by unipolar ion conduction in ion concentration polarization desalination.</a> Sci. Rep. 6, 25349 (2016).</li></ol>

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<sup...

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><tbody><tr><td>Sylgard 184 Silicone Elastomer kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Trichlorosilane</td><td>Sigma Aldrich</td><td>175552</td><td>Highly toxic</td></tr><tr><td>Nafion perfluorinated resin, 20 wt%</td><td>Sigma Aldrich</td><td>527122</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>71394</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>60121</td><td></td></tr><tr><td>Alexa Fluor 488 carboxylic acid, succinimidyl ester</td><td>Invitrogen</td><td>A20000</td><td></td></tr><tr><td>Isothiocyanate-conjugated albumin</td><td>Sigma Aldrich</td><td>A9771</td><td></td></tr><tr><td>Phosphate buffer saline, 1x</td><td>Wengene</td><td>LB004-02</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>Epifluorescence microscope</td><td>Olympus</td><td>IX-71</td><td></td></tr><tr><td>Charged-coupled device camera</td><td>Hamamtsu Co.</td><td>ImageEM X2</td><td></td></tr><tr><td>Source measurement unit</td><td>Keithley Instruments</td><td>2635A</td><td></td></tr><tr><td>Covance-MP</td><td>Femto Science</td><td></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?...

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

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

The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.

Horizontal and vertical electrohydrodynamic liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields and polar dielectric liquids. The construction of basic apparatus and operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.

高压直流电桥从极地介电液的制备

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and ...

科研

工程学

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

生物工程

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

电生理实验人工双层膜的制备

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

生物学

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

无标记的分离和细胞的富集通过非接触式介电泳

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

医学

Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions

To drive electrohydrodynamic (EHD) flows in aqueous solutions, the separation of cation and anion transport pathways is essential because a directed electric body force has to be induced by ionic motions in liquid. On the other hand, positive and negative charges attract each other, and electroneutrality is maintained everywhere in equilibrium conditions. Furthermore, an increase in an applied voltage has to be suppressed to avoid water electrolysis, which causes the solutions to become unstable. Usually, EHD flows can be induced in non-aqueous solutions by applying extremely high voltages, such as tens of kV, to inject electrical charges. In this study, two methods are introduced to generate EHD flows induced by electrical charge separations in aqueous solutions, where two liquid phases are separated by an ion-exchange membrane. Due to a difference in the ionic mobility in the membrane, ion concentration polarization is induced between both sides of the membrane. In this study, we demonstrate two methods. (i) The relaxation of ion concentration gradients occurs via a flow channel that penetrates an ion-exchange membrane, where the transport of the slower species in the membrane selectively becomes dominant in the flow channel. This is a driving force to generate an EHD flow in the liquid. (ii) A long waiting time for the diffusion of ions passing through the ion-exchange membrane enables the generation of an ion-dragged flow by externally applying an electric field. Ions concentrated in a flow channel of a 1 x 1 mm2 cross-section determine the direction of the liquid flow, corresponding to the electrophoretic transport pathways. In both methods, the electric voltage difference required for an EHD flow generation is drastically reduced to near 2 V by rectifying the ion transport pathways.

The rectification of ion transport pathways is an effective method to generate one-directional ion-dragged electrohydrodynamic flows. By setting an ion-exchange membrane in a flow channel, an electrically polarized condition is generated and causes a liquid flow to be driven when an electric field is externally applied.

水电解质溶液中 Electrohydrodynamic 流的产生与控制

To drive electrohydrodynamic (EHD) flows in aqueous solutions, the separation of cation and anion transport pathways is essential because a directed ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

微量和外显子的纸基预集中和分离

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

生物化学

Fabrication of Carbon-Based Ionic Electromechanically Active Soft Actuators

Ionic electromechanically active capacitive laminates are a type of smart material that move in response to electrical stimulation. Due to the soft, compliant and biomimetic nature of this deformation, actuators made of the laminate have received increasing interest in soft robotics and (bio)medical applications. However, methods to easily fabricate the active material in large (even industrial) quantities and with a high batch-to-batch and within-batch repeatability are needed to transfer the knowledge from laboratory to industry. This protocol describes a simple, industrially scalable and reproducible method for the fabrication of ionic carbon-based electromechanically active capacitive laminates and the preparation of actuators made thereof. The inclusion of a passive and chemically inert (insoluble) middle layer (e.g., a textile-reinforced polymer network or microporous Teflon) distinguishes the method from others. The protocol is divided into five steps: membrane preparation, electrode preparation, current collector attachment, cutting and shaping, and actuation. Following the protocol results in an active material that can, for example, compliantly grasp and hold a randomly shaped object as demonstrated in the article.

This article describes a fast and simple manufacturing process of ionic electromechanically active composite materials for actuators in biomedical, biomimetic and soft robotics applications. The key fabrication steps, their importance for the actuators&#39; final properties, and some of the main characterization techniques are described in detail.

碳基离子机电活性软执行器的制造

Ionic electromechanically active capacitive laminates are a type of smart material that move in response to electrical stimulation. Due to the soft, ...

Ion-Exchange Membranes for the Fabrication of Reverse Electrodialysis Device

Reverse electrodialysis (RED) is an effective way to generate power by mixing two different salt concentrations in water using cation-exchange membranes (CEM) and anion-exchange membranes (AEM). The RED stack is composed of an alternating arrangement of the cation-exchange membrane and anion-exchange membrane. The RED device acts as a potential candidate for fulfilling the universal demand for future energy crises. Here, in this article, we demonstrate a procedure to fabricate a reverse electrodialysis device using laboratory-scale CEM and AEM for power production. The active area of the ion-exchange membrane is 49 cm2. In this article, we provide a step-by-step procedure for synthesizing the membrane, followed by the stack&#39;s assembly and power measurement. The measurement conditions and net power output calculation have also been explained. Furthermore, we describe the fundamental parameters that are taken into consideration for obtaining a reliable outcome. We also provide a theoretical parameter that affects the overall cell performance relating to the membrane and the feed solution. In short, this experiment describes how to assemble and measure RED cells on the same platform. It also contains the working principle and calculation used for estimating the net power output of the RED stack using CEM and AEM membranes.

We demonstrate the fabrication of a reverse electrodialysis device using a cation-exchange membrane (CEM) and anion-exchange membrane (AEM) for power generation.

用于制造反向电透析装置的离子交换膜

Reverse electrodialysis (RED) is an effective way to generate power by mixing two different salt concentrations in water using cation-exchange membranes ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

温度控制组件和液滴接口比莱耶的特征

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

电生理测量在中性转运体活性研究中的应用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...