The authors have no acknowledgments.

1. EHD Flow Induced by Rectified Ion Transport
<ol>
	<li>Development of a flow channel device to rectify ion transport pathways
	<ol>
		<li>Make a PTFE mold of the reservoir:
		<ol>
			<li>Cut a 13 x 30 x 10 mm3 mold from a polytetrafluoroethylene (PTFE) block using a milling machine (see Figure 2). Alternatively, purchase a custom-made product.</li>
			<li>Adhere acrylic plates of 15 x 18 x 1 mm3 at both ends of the PTFE mold with a plastic adhesive, which will make slits in the reservoir to settle the bias electrodes. These parts can be cut out from a large plate or purchased.</li>
			<li>Adhere the acrylic plates of 13 x 30 x 1 mm3 at the top and bottom surfaces of the PTFE mold with a plastic adhesive to make planar surfaces for clear observation.</li>
		</ol>
		</li>
		<li>Mix a silicone elastomer base and curing agent in the ratio of 10:1 in a 50 mL tube and shake the tube by hand.</li>
		<li>Settle the liquid PDMS in a vacuum vessel and degas it by using a rotary pump.</li>
		<li>Remove the tube from the vessel. Pour the PDMS into a 40 x 50 x 24 mm3 plastic vessel to mold the outer shape of the reservoir and place the reservoir mold (see step 1.1.1) in it.</li>
		<li>Bake the whole body of the liquid PDMS on a hotplate at 80 &#176;C for about 4 h.</li>
		<li>After the bake, isolate the PDMS reservoir from the PTFE mold and the outer vessel by hand. Make a slit across the center of the reservoir by using a surgical knife. This will be used to put the edges of an anion-exchange membrane (prepared in step 1.1.16) into it using tweezers. 
		NOTE: The PDMS reservoir is filled with electrolyte solutions later, as shown in Figure 2.</li>
		<li>Obtain glass plates (made by special order) with a circular shape of 18 mm in diameter or in a square with 18 mm edges.</li>
		<li>Wash the glass plates by soaking them in acetone, ethanol, and pure water (in that order) in an ultrasonication bath for 15 min each.</li>
		<li>Blow any residual liquids away with an air gun or heat the glass plates with a hotplate for 5 min at about 473 K.</li>
		<li>Using radio frequency sputtering, coat the glass surface with Cr or Ti exposed to Ar plasma for 1 min at 75 W and successively, deposit an Au thin film for 5 min at 75 W, setting the thickness at approximately 100 nm. 
		NOTE: Before coating the glass surface with the target metals, the samples were set in a vacuum chamber that was evacuated with a rotary pump and a molecular diffusion pump until the pressure decreased to 1 x 10&#8722;2 Pa.</li>
		<li>Solder a lead on the Au electrode surface using a soldering iron. 
		NOTE: The shape of the Au electrode may possibly be replaced by squares and helical wires, maintaining surface areas large enough to generate ionic currents.</li>
		<li>With tweezers, set the glass plates coated with an Au thin film at both ends of the reservoir. These are the bias electrodes.</li>
		<li>Cut an anion-exchange membrane into a rectangular shape of 20 x 18 mm2 by using scissors. A surface area of 13 mm in width and 10 mm in height is exposed to a liquid. Here, a box cutter or surgical knife may also be used to cut the membrane.</li>
		<li>Cut out a rectangular piece of 3 x 5.5 mm2 from one edge of the membrane with scissors. 
		NOTE: The thickness of the anion-exchange membrane is 220 &#181;m. The membrane is easily cut with scissors or a box cutter. The edges of the membrane are partly fixed with the slits in the chamber.</li>
		<li>Solidify a PDMS block with a stainless rod of a 1 x 1 mm2 cross-section in the same way as in steps 1.1.4 - 1.1.5, to create a flow channel that penetrates the membrane. Leave the construction overnight and then pull the stainless rod out of the PDMS block.</li>
		<li>Cut the PDMS block with a square flow channel into a 3 x 6 x 4.5 mm piece (see Figure 2) using a surgical knife. Make slits along the outer edges, then attach it to the membrane within the rectangular cutout. 
		NOTE: The top face of the channel has to be set horizontally for a clear observation of the particles in the flow channel via the transparent wall.</li>
	</ol>
	</li>
	<li>Preparation of solutions and pretreatments for experiments
	<ol>
		<li>Prepare NaOH aqueous solutions at concentrations of 1 x 10&#8722;1, 1 x 10&#8722;2, and 1 x 10&#8722;3 mol/L by diluting the stock solution.</li>
		<li>Make a dispersion of polystyrene particles of 2.93 &#181;m on average in diameter in each of the NaOH solutions prepared in step 1.2.1 by setting the concentration to 4.2 x 10&#8722;3 vol%. 
		NOTE: The size of the tracer particles may be changed appropriately to improve observability.</li>
		<li>Ultrasonicate the formatted anion-exchange membrane of 20 x 18 mm2 with a slit of 3 x 5.5 mm2 2x for 10 min in pure water at a power of 100 W.</li>
		<li>With tweezers, set the anion-exchange membrane with the PDMS flow channel into the PDMS reservoir. Fill the reservoir with 4 mL of NaOH solution using a micropipette. 
		NOTE: The membrane surface and flow channel are immersed in the solution, where the membrane surface exposed to the solution is at least 100x larger than the cross-section of the flow channel.</li>
		<li>Apply an electric potential of 2.2 V by using a DC power source in forward and backward directions for 2 h each in series, to improve the conductivity of the membrane before observation.</li>
		<li>Pull the Au electrodes out with tweezers. Remove the solution from the reservoirs using a micropipette.</li>
		<li>Set new Au electrodes in the reservoirs with tweezers. Fill the reservoirs with 4 mL of NaOH solution using a micropipette. Start observations when the solution is equilibrated. 
		NOTE: It may take a few minutes of waiting time until the natural convection settles down, which can be judged by observing the behavior of tracer particles.</li>
	</ol>
	</li>
	<li>Experimental setup and measurement systems
	<ol>
		<li>Set the frame rate and the exposure time of a high-speed complementary metal-oxide-semiconductor (CMOS) camera to 500 fps and 1 ms, respectively. 
		NOTE: As shown in Figure 1, the experimental device is set on the stage of a microscope connected to a high-speed CMOS camera to record the particle motions. The view is magnified in a 15 in monitor with a 100X lens.</li>
		<li>Remove any bubbles from the channel by inserting the tip of a micropipette into the channel end to push or pull out them, before applying an electric potential.</li>
		<li>Externally apply an electric potential of 2.2 V to the Au bias electrodes. Simultaneously monitor the electrical responses by using a potentiostat or a DC power source with a digital multimeter. 
		NOTE: The voltage value is determined to be the upper limit, avoiding the water electrolysis that generates O2 and H2 bubbles in the solution.</li>
		<li>Record the behavior of the tracer particles on the computer.</li>
		<li>Measure an electric potential difference between both ends of the flow channel by using Au probe electrodes and a digital multimeter to confirm that the concentration gradient of ions triggers an EHD flow38,39.</li>
		<li>Determine the origin of the Cartesian coordinate system at the center of the channel. 
		NOTE: The x-axis is along the longitudinal direction of the flow channel, and the y- and z-axes are in the horizontal and vertical directions in the cross-section of the channel, respectively, as shown in Figure 2. The transparent PDMS channel allows liquid flows to be visualized along the x-axis. The view is focused on the xy plane at z = 0 by controlling the depth of focus. The flow data are independent of x in the test section except just near the inlet and outlet of the channel, and the observation point is set at approximately 0.75 mm downstream from the origin, such that x = 0.75, y = 0, and z = 0 mm.</li>
		<li>After a single measurement (of 15 s), short-circuit the electrodes by connecting them to each other with a lead for 20 min until the solution is equilibrated.</li>
		<li>Next, move the entirety of the solution to another vessel (e.g., a 10 mL sample bottle) and stir it with a micropipette.</li>
		<li>Pour the stirred solution into the chamber again using a micropipette when iteratively performing the experiment. 
		NOTE: After observation, the EHD flow velocity is evaluated by using the particle image velocimetry (PIV) method39, which can be done by using the appropriate software to trace the displacement of particles and numerically evaluate the velocity. A detailed explanation of PIV methods and how to use them is omitted here because PIV analyses have been widely used and the procedures of computations depend on the software and operating system that is being used.</li>
	</ol>
	</li>
</ol>
2. Observation of Cation-induced EHD Flows
<ol>
	<li>Development of experimental device
	<ol>
		<li>Form Au bias electrodes with a 26 x 10 mm2 surface on the bottom glass plate according to procedures similar to those previously described in steps 1.1.5 - 1.1.7.</li>
		<li>Using radio frequency sputtering, coat a glass surface with Cr or Ti exposed to Ar plasma for 2 min at 75 W and deposit an Au thin film for 5 min at 75 W. 
		NOTE: This shape of electrode is determined in order to highly concentrate electric fields in the narrowest channel region. The ratio of the electrode surface, whose area of 10 x 10 mm2 is exposed to a liquid, to the cross-section of the channel is ideally 100:1; this ratio is predicted to be sufficient to drop the electric potential at the channel by a large amount16.</li>
		<li>Solder a lead line at an edge of the electrodes by using a soldering iron.</li>
		<li>From a large silicone rubber sheet, cut out 2 chambers, each made of a 1 x 1 x 1 mm3 flow channel placed between two 10 x 10 x 1 mm3 reservoirs, using a surgical knife (see Figure 3). These parts may be replaced by PDMS.</li>
		<li>Cut out a cation-exchange membrane with an average thickness of 127 &#181;m to 20 x 30 mm using a box cutter or surgical knife, as shown in Figure 3.</li>
		<li>Ultrasonicate each part in pure water for 15 min by applying 100 W.</li>
		<li>Insert a cation-exchange membrane between the chambers using tweezers, as shown in Figure 3. This will separate 2 electrolyte solutions of different concentrations.</li>
		<li>Press and seal the stack of the chambers and cation-exchange membrane with glass plates whose dimensions are 26 mm in width and 38 mm long.</li>
	</ol>
	</li>
	<li>Preparation of solutions
	<ol>
		<li>Prepare a dispersion of polystyrene particles of an average diameter of 1.01 &#181;m in a 1 x 10&#8722;2 mol/L tris(hydroxymethyl)aminomethane ethylenediaminetetraacetic acid (Tris-EDTA) buffer solution, where the volume ratio is adjusted to 1 x 10&#8722;2 vol%.</li>
		<li>Prepare a mixture of 1 mol/L of KCl and 1 x 10&#8722;2 mol/L of Tris-EDTA.</li>
		<li>Inject the Tris-EDTA/polystyrene particle and the Tris-EDTA/KCl solutions into the lower and upper chambers, respectively, via syringe needles inserted from the side walls of the chambers. 
		NOTE: The quantity of the solutions injected into each chamber is about 210 &#181;L.</li>
		<li>Wait for about 18 h until the solution is equilibrated as a result of a diffusion of the ions to relax the ion concentration difference between the upper and lower layers. 
		NOTE: In the diffusion process, K+ in the upper solution and H+ in the membrane are expected to penetrate the membrane first, and Cl&#8722; is expected to follow them.</li>
	</ol>
	</li>
	<li>Experimental setup and measurement systems
	<ol>
		<li>Set the experimental device developed in step 2.1 on the stage of the inverted microscope by hand, as shown in Figure 3. Connect the microscope to a high-speed CMOS camera to monitor the trajectories of the particle motions and record the observation data on a computer.</li>
		<li>Apply an electric potential difference of 2 V for 6 s between the two electrodes by using a function generator as a power source.</li>
		<li>To confirm that EHD flows are induced by ion transport, measure the ionic currents simultaneously by using an ammeter40.</li>
		<li>Analyze the recorded trajectories of the particles by the particle tracking velocimetry (PTV) method39. 
		NOTE: After the observations, the EHD flow velocity is evaluated by the PTV method, which is possible by using appropriate software, to trace the displacement of particles and numerically evaluate the velocity. A detailed explanation of PTV methods and how to use them is omitted here because PTV analyses have been widely used and the procedures of the computations depend on the software and operating system that is being used.</li>
	</ol>
	</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+insulating+walls+on+the+structure+of+electrodynamic+flows+in+a+channel.">Effect of insulating walls on the structure of electrodynamic flows in a channel.</a> Technical Physics. 57, 1181-1187 (2012).</li><li>Yano, A., Doi, K., Kawano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+electrohydrodynamic+flow+through+a+pore+in+ion-exchange+membrane.">Observation of electrohydrodynamic flow through a pore in ion-exchange membrane.</a> International Journal of Chemical Engineering and Applications. 6, 254-257 (2015).</li><li>Doi, K., Yano, A., Kawano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrohydrodynamic+flow+through+a+1+mm2+cross-section+pore+placed+in+an+ion-exchange+membrane.">Electrohydrodynamic flow through a 1 mm2 cross-section pore placed in an ion-exchange membrane.</a> The Journal of Physical Chemistry B. 119, 228-237 (2015).</li><li>Yano, A., Shirai, H., Imoto, M., Doi, K., Kawano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+dependence+of+cation-induced+electrohydrodynamic+flow+passing+through+an+anion+exchange+membrane.">Concentration dependence of cation-induced electrohydrodynamic flow passing through an anion exchange membrane.</a> Japanese Journal of Applied Physics. 56, 097201 (2017).</li><li>Nagura, R., Doi, K., Kawano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+microparticle+transport+driven+by+ionic+current+conditions+in+electrically+polarized+aqueous+solutions.">Characterisation of microparticle transport driven by ionic current conditions in electrically polarized aqueous solutions.</a> Micro &amp; Nano Letters. 12, 526-531 (2017).</li><li>Doi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonequilibrium+ionic+response+of+biased+mechanically+controllable+break+junction+(MCBJ)+electrodes.">Nonequilibrium ionic response of biased mechanically controllable break junction (MCBJ) electrodes.</a> The Journal of Physical Chemistry C. 118, 3758-3765 (2014).</li></ol>

The authors have nothing to disclose.

The purpose of this study was to separate cations and anions in aqueous solutions in terms of spatial distributions and transport numbers. Using an anion-exchange membrane, the transport of anions and cations could be rectified in the membrane and in a flow channel that penetrates the membrane, respectively. Alternatively, a cation-exchange membrane that separated high and low concentration solutions worked to generate electrically polarized solutions after a considerable waiting time. As a result, rectified ionic curren...

Recently, liquid flow control techniques have attracted much attention because of interest in the applications of micro- and nanofluidic devices1,2,3,4,5,6,7,8,9,10,11,12,13,14,15. In polar solutions, such as aqueous solutions and ionic liquids, ions and electrically charged particles usually bring about electrical charges in liquid flows. The transport of such polarized particles provides an expansion of various applications, such as single-molecule manipulation6,10,11,13,14,15,16,17, ion diode devices12,18, and liquid flow control19,20,21,22. EHD flow has been an applicable phenomenon for liquid flow control systems since Stuetzer1,2 invented the ion drag pump. Melcher and Taylor3 published an important article in which the theoretical framework of EHD flow was well reviewed and some outstanding experiments were also demonstrated. Saville4 and his coworkers23,24 contributed to the following expansion of EHD technologies in liquids. However, there were some limitations to inducing liquid flows driven by electric forces, because tens of kV have to be applied in liquids to inject electrical charges in non-polar solutions, such as oils, to polarize them1,2,3. This is a disadvantage for aqueous solutions because the water electrolysis that is induced by an electric potential higher than 1.23 V changes the characteristics of solutions and makes the solutions unstable.
In micro- and nanofluidic channels, surface charges of channel walls cause the concentration of counterions that effectively induce electroosmotic flows (EOFs) under externally applied electric fields25,26,27,28,29. Using EOFs, some liquid pumping techniques have been applied in aqueous solutions, reducing the electric voltages30,31,32. On the other hand, EOFs are limited to being generated in micro- and nanospaces in which surface areas become more dominant than liquid volumes. Furthermore, depending on the transport of highly concentrated ions very near the wall surfaces, such as in electric double layers, the slip boundary only causes the liquid flow, which may not be sufficient to make pressure gradients7,8,22,26,27. Fine tuning, such as to channel dimensions and salt concentrations, is required for the applications of EOF. In contrast, EHD flows driven by body forces seem to be available to transport masses and energies if the application voltages can be reduced to avoid degrading solvents. Recently, some researchers have suggested applications of EHD flows with low voltages33,34,35,36. Although these technologies have not yet been implemented, the frontiers are expected to expand.
In previous studies, we also conducted experimental and theoretical work on EHD flows in aqueous solutions37,38,39,40. It was supposed that the rectification of ion transport pathways was effective to generate electrically charged solutions that cause electric body forces under electric fields. By using an ion-exchange membrane and a flow channel crossing the membrane, we were able to rectify ionic currents. When applying an anion-exchange membrane, cations concentrated in the flow channel dragged the solvents and developed an EHD flow37,38,39. A difference in the mobility of ion species was an important factor when separating the cationic and anionic currents. Ion-exchange membranes effectively worked to modulate the mobility due to the ion selectivity. Ion transport phenomena were also investigated from the viewpoint of ionic current density influenced by applied electric fields41. These studies have been fruitful for developing manipulation techniques for single molecules, namely, micro- and nanoparticles, whose motions are strongly affected by thermal fluctuations11,16,17. EOFs and EHD flows are expected to expand the variety of precise flow control methods as well as pressure gradients.
In this study, we demonstrate two methods to drive EHD flows in aqueous solutions. First, an NaOH solution is used for a working fluid to drive an EHD flow37,38,39. An anion-exchange membrane separates the liquid into two parts. A polydimethylsiloxane (PDMS) flow channel with a cross-section of 1 x 1 mm and a length of 3 mm penetrates the membrane. By applying an electric potential of 2.2 V, the electrophoretic transport of Na+, H+, and OH&#8722; ions is induced along the electric fields. An anion-exchange membrane and a flow channel effectively work to separate the ion transport pathways, where anions dominantly pass through the membrane and cations concentrate in the flow channel, although both species usually move in opposite directions, maintaining the electroneutrality. Thus, such a condition does not cause a driving force for liquid flows. This structure is crucial to generating an EHD flow whose flow speed reaches on the order of 1 mm/s in the channel because highly concentrated cations accelerated by external electric fields drag solvent molecules. EHD flows are observed and recorded by using a microscope and a high-speed camera as shown in Figure 1. Second, a concentration difference between two liquid phases separated by an ion-exchange membrane causes an electrically polarized condition to be generated crossing an ion-exchange membrane40. In this study, we find the importance of a considerable waiting time to equilibrate ion distributions and a corresponding electric potential, which cause preferable conditions to apply to a body force in a liquid. Crossing the ion-exchange membrane, a weakly polarized condition is achieved. In such a condition, an externally applied electric field induces directional ion transport that generates a body force in a liquid, and as a result, the momentum transfer from the ions to the solvent develops an EHD flow.
As mentioned above, the present devices succeed in drastically decreasing the applied voltage difference to a few volts, and thus this method is can be used for aqueous solutions, although the conventional electrical charge injection methods required tens of kV and are limited to an application to non-aqueous solutions.

<table><tbody><tr><td>Sylgard 184</td><td>Dow Corning Corp.</td><td>3097366-0516, 3097358-1004</td><td>PDMS</td></tr><tr><td>Acetone</td><td>Wako Pure Chemical Industries, Ltd.</td><td>012-00343</td><td></td></tr><tr><td>Ethanol</td><td>Wako Pure Chemical Industries, Ltd.</td><td>054-00461</td><td></td></tr><tr><td>0.1 mol/L Sodium Hydroxide Solution</td><td>Wako Pure Chemical Industries, Ltd.</td><td>196-02195</td><td></td></tr><tr><td>Pottasium Chloride</td><td>Wako Pure Chemical Industries, Ltd.</td><td>163-03545</td><td></td></tr><tr><td>Tris-EDTA buffer 100x concentrate</td><td>Sigma-Aldrich Co. LLC.</td><td>T9285-10014L</td><td></td></tr><tr><td>2.93 &amp;mu;m polystyrene particle</td><td>Merck KGaA</td><td>L300 Rouge</td><td>Tracer particle</td></tr><tr><td>1.01 &amp;mu;m polystyrene particle</td><td>Merck KGaA</td><td>K100(23716)</td><td>Tracer particle</td></tr><tr><td>Anion exchange membrane</td><td>ASTOM Corp.</td><td>Neosepta AHA</td><td></td></tr><tr><td>Gold (Au)</td><td>Furuuchi Chemical Corp.</td><td>AUT-13301X</td><td>Sputtering target metal</td></tr><tr><td>Titanium</td><td>Furuuchi Chemical Corp.</td><td>TIT-72301X</td><td>Sputtering target metal</td></tr><tr><td>Chromium</td><td>Furuuchi Chemical Corp.</td><td>CRT-24301X</td><td>Sputtering target metal</td></tr><tr><td>Hight-speed CMOS camera</td><td>Keyence Corp.</td><td>VW-600M</td><td></td></tr><tr><td>Microscope</td><td>Keyence Corp.</td><td>VW-9000</td><td></td></tr><tr><td>Data logger</td><td>Keyence Corp.</td><td>NR-500, NR-HA08</td><td></td></tr><tr><td>Laser displacement meter</td><td>Keyence Corp.</td><td>LK-G5000, LK-H008W</td><td></td></tr><tr><td>PIV and PTV software</td><td>DITECT Co. Ltd.</td><td>Flownizer 2D</td><td></td></tr><tr><td>Potentiostat</td><td>AMTEK Inc.&amp;nbsp;</td><td>VersaSTAT4</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus Corp.</td><td>IX73</td><td></td></tr><tr><td>High-speed CMOS camera</td><td>Andor Technology Ltd.</td><td>Zyla 5.5 sCMOS</td><td></td></tr><tr><td>Function generator</td><td>NF Corp.&amp;nbsp;</td><td>WF1945B</td><td></td></tr><tr><td>Function generator</td><td>NF Corp.&amp;nbsp;</td><td>WF1973</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>AS ONE Corp.</td><td>AS22GTU</td><td></td></tr><tr><td>Rotary pump</td><td>ULVAC, Inc.</td><td>G-100S</td><td>Degas liquid PDMS</td></tr><tr><td>Rotary pump</td><td>ULVAC, Inc.</td><td>GLD-201A</td><td>Sputtering&amp;nbsp;</td></tr><tr><td>Molecular diffusion pump</td><td>ULVAC, Inc.</td><td>VPC-400</td><td>Sputtering</td></tr></tbody></table>

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.

Figure 4 (video figure) presents a representative result of an EHD flow generation, resulting from the rectification of ion transport pathways and highly concentrated cations that induced a liquid flow in the channel, according to step 1 of the protocol. Figure 5 shows a result of the PIV analysis, where 20 data points near the center of the channel (y = z = 0 mm) were averaged. In the case of the 1 x 10&#8...

Watch this Scientific Journal Video about Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions at JoVE.com

Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions

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.

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

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8.8K Views.  Osaka University. 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.

Video: Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

Environment

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.

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

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

Biology

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Bioengineering

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4&#8211;8 &#181;m/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.

The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid samples, such as chemical reagents and cell cultures, is predominantly accomplished through steady flows that are typically driven by syringe pumps, gravity, or capillary forces. The use of complementary oscillatory flows is seldom considered in applications despite its numerous advantages as recently demonstrated in the literature. The significant technical barrier to the implementation of oscillatory flows in microchannels is likely responsible for the lack of its widespread adoption. Advanced commercial syringe pumps that can produce oscillatory flow, are often more expensive and only work for frequencies less than 1 Hz. Here, the assembly and operation of a low-cost, plug-and-play type speaker-based apparatus that generates oscillatory flow in microchannels is demonstrated. High-fidelity harmonic oscillatory flows with frequencies ranging from 10-1000 Hz can be achieved along with independent amplitude control. Amplitudes ranging from 10-600 &#181;m can be achieved throughout the entire range of operation, including amplitudes &#62; 1 mm at the resonant frequency, in a typical microchannel. Although the oscillation frequency is determined by the speaker, we illustrate that the oscillation amplitude is sensitive to fluid properties and channel geometry. Specifically, the oscillation amplitude decreases with increasing channel circuit length and liquid viscosity, and in contrast, the amplitude increases with increasing speaker tube thickness and length. Additionally, the apparatus requires no prior features to be designed on the microchannel and is easily detachable. It can be used simultaneously with a steady flow created by a syringe pump to generate pulsatile flows.

The protocol demonstrates a convenient method to produce harmonic oscillatory flow from 10-1000 Hz in microchannels. This is performed by interfacing a computer-controlled speaker diaphragm to the microchannel in a modular manner.

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid ...

Glass-Based Devices to Generate Drops and Emulsions

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A&#160;voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. ...

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

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

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