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. Fabrication of Cation Exchange Membrane-integrated Microfluidic Chips
<ol>
	<li>Preparation of silicon masters
	<ol>
		<li>Design two kinds of silicon masters: one for patterning a cation exchange resin and the other for building a microchannel with polydimethylsiloxane (PDMS). 
		NOTE: The detail geometry will be described in the steps 1.3.1 and 1.4.1.</li>
		<li>Fabricate the silicon masters by using either conventional photolithography or deep reactive ion etching27.</li>
		<li>Silanize the micropatterned silicon masters with trichlorosilane (~30 &#956;L) in a vacuum jar for 30 min. 
		CAUTION: Trichlorosilane is a pyrophoric liquid that is flammable and has an acute toxicity (inhalation, oral ingestion).</li>
	</ol>
	</li>
	<li>Preparation of PDMS molds
	<ol>
		<li>Mix a silicone elastomer base with a curing agent at a 10:1 ratio and place the cup with this uncured PDMS (30-40 mL for replicating microstructures on a 4-in silicon wafer) in a vacuum jar for 30 min to remove the bubbles. 
		NOTE: The silicone base contains siloxane oligomers terminating with vinyl groups and a platinum-based catalyst. The curing agent contains crosslinking oligomers that have three silicon-hydride bonds28.</li>
		<li>Pour the uncured PDMS on the silicon masters, remove the bubbles with a blower, and cure the PDMS at 80 &#176;C for 2 h in a convection oven.</li>
		<li>Detach the cured PDMS from the silicon masters and properly shape the PDMS with a knife (squared shapes, as shown in Figure 2a-b, iv).</li>
	</ol>
	</li>
	<li>Patterning the cation exchange membranes
	<ol>
		<li>Cut half of the PDMS mold perpendicularly to the two parallel microchannels and punch holes at the ends of the PDMS channels with a 2.0-mm biopsy punch. 
		NOTE: The PDMS mold for patterning the cation selective membrane has two parallel microchannels (width: 100 &#181;m; height: 50 &#181;m; interchannel distance: 100 &#181;m; Figure 1a). The original shape of the mold can be imagined by mirroring the sliced mold along the cutting line. L-shaped microchannels are recommended for punching the two holes without overlapping.</li>
		<li>Clean a glass slide and the PDMS mold with tape and a blower and put the mold onto the glass slide to create reversible attachment between them.</li>
		<li>According to the microflow patterning technique29, release ~10 &#181;L of a cation exchange resin at the open end of the channel that was sliced in step 1.3.1 (Figure 1b). Place the syringe head on the punched holes and pull the plunger (black arrows in Figure 1b); a gentle negative pressure will pull the cation exchange resin, and the resin will fill the two channels. 
		NOTE: It is recommended that the height of the microchannel is greater than 15 &#181;m, because the high viscosity of the resin requires high pressure to fill the channels. On the other hand, it is better that the height does not exceed 100 &#181;m, because the patterned ion selective membrane will become thicker than 1 &#181;m; such a thick membrane may create a gap between the membrane and the PDMS channel13.</li>
		<li>Detach the PDMS mold without touching the patterned resin and place the glass slide on the heater at 95 &#176;C for 5 min to evaporate the solvent in the resin. 
		NOTE: The thickness of the patterned membrane is usually less than &#60; 1 &#181;m. The mold is gently detached by hinging the mold to the open-ended side (dotted line and arrow in Figure 1b). It is best to detach the mold less than 1 min after filling the resin. If the mold is detached a few minutes later, thicker membranes could be obtained, but they would have a concave shape due to the capillary effect.</li>
		<li>Peel off the unnecessary part of the patterned membrane with a razor blade, making two separated line-patterns (Figure 1c). 
		NOTE: The cation exchange material used here has perfluorinated groups, meaning the pattern is not strongly bonded to the glass. Therefore, the simple blading method can easily remove the unnecessary part of the membrane.</li>
	</ol>
	</li>
	<li>Integration of the microchannel and the membrane-patterned substrate
	<ol>
		<li>Punch two holes at the ends of microchannels and another two holes where the membrane patterns will be located after bonding the PDMS channel to the membrane-patterned substrate fabricated in step 1.3. 
		Note: The PDMS microchannel has one channel (width: 50-100 &#181;m; height: 10 &#181;m), but it is bonded to the ends of the neighboring channel (Figure 1d).</li>
		<li>Bond the PDMS microchannel to the membrane-patterned substrate immediately after oxygen plasma treatment for 40 s at 100 W and 50 mTorr. 
		NOTE: Place the patterned membrane perpendicularly on the middle of the microchannel.</li>
	</ol>
	</li>
</ol>
2. ICP Preconcentration
<ol>
	<li>Preparation for the experiment
	<ol>
		<li>Prepare various test solutions, including 1-100 mM KCl, 1 mM NaCl (pH ~7), the mixture of 1 mM NaCl and 0.2 mM HCl (pH ~3.7), the mixture of 1 mM NaCl and 0.2 mM NaOH (pH ~10.3), and 1x phosphate-buffered saline.</li>
		<li>Add a negatively charged fluorescent dye (~1.55 &#181;M) to the test solutions. 
		NOTE: The concentration of the added dye should be much lower than that of the salt ions (&#60; 10 &#181;M) so that the charged dyes do not contribute to an electrical current30,31.</li>
		<li>Load the sample solution in one reservoir of the channel and apply negative pressure to the other reservoir to fill the channel with the solution. Connect the two reservoirs hydrodynamically by releasing a large droplet to eliminate the pressure gradient along the channel (Figure 2a).</li>
		<li>Fill the two reservoirs, which are connected to the cation exchange patterns, with buffer solutions (1 M KCl or 1 M NaCl) using a syringe or a pipet to compensate for the ICP effect in the reservoirs.</li>
		<li>Place the wires at the reservoirs, across the two patterned membranes (anode on the left reservoir and cathode on the right), and connect them with a source measurement unit (Figure 2a).</li>
	</ol>
	</li>
	<li>Visualization of the ICP phenomenon and ICP preconcentration
	<ol>
		<li>Load the ICP device on an inverted epifluorescence microscope. Apply a voltage (0.5100 V) and measure the current response with a source measurement unit.</li>
		<li>Capture fluorescent images with a charge-coupled device camera and analyze the fluorescent intensity using imaging software32.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We have described the fabrication protocol and the performance of a spatiotemporally defined preconcentrator in a range of the applied voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3), achieving a 10,000-fold preconcentration of dyes and protein within 10 min. As like previous ICP devices, the preconcentration performance becomes better at higher voltage and at lower ionic strength. One additional parameter we can consider here is the distance between two cation exchange membranes. If we increase the inter-membrane distance, the electric field decreases under the same applied voltage, resulting in the decrease of the preconcentration speed26.
The microflow patterning technique29 used in this work is a robust method for patterning cation exchange resins, so it has been one of the gold-standard methods for integrating the ion exchange materials into microfluidic systems. Nevertheless, it is necessary to fabricate two juxtaposed cation exchange membranes with a short intermembrane distance (smaller than few hundred micrometers). In steps 1.3.3-1.3.4, the cation exchange resin is in a liquid phase. Therefore, the resin in the two microchannels can be collapsed, and the remaining resin drop at the open end of the channels can also flood during the mold detachment (step 1.3.4.). To build two cation exchange membranes with high pattern fidelity, we used the resin with a relatively high viscosity (20% of the cation exchange material in the solvents) and carefully set the detachment process with a designated detaching direction.
Even though the high operating flexibility of this platform was demonstrated, the reader might be concerned about determining the optimal conditions from the wide range within the operating window. One representative trade-off is between the preconcentration speed and the stability of the ICP effect. As can be seen in Figure 5 in Kwak et al.26, a high applied voltage (&#62; 50 V) can condense targets quickly; however, this also induces strong vortices in the depletion zone (1 mM / pH 7 in Figure 7a), which decreases the stability of the sample preconcentration. Accordingly, the preconcentration speed becomes difficult to predict33. In the current stage, we recommend experimental conditions with a relatively low voltage (&#60; 30 V) and ionic strength (&#60; 10 mM) for a stable, predictable, and spatiotemporally fixed preconcentration. This trade-off between the preconcentration speed and the stability of the preconcentrated plug is also related to the sources of the nonlinear ICP (surface conduction, EOF, and electro-osmotic instability). The main source of the nonlinear ICP at a relatively small voltage (&#60; 50 V) is EOF, creating a coherent vortex pair in the depletion zone (Figure 3b), which leads to a stable preconcentration. At a relatively high voltage (&#62; 50 V), the main source of the nonlinear ICP is changed to electro-osmotic instability, resulting chaotic multiple vortexes, which decrease the stability of the preconcentration.
Recently, paper-based ICP platforms have been developed by Phan et al.34, Gong et al.19, and Han et al.21. These paper devices with microporous structures can suppress electro-osmotic instability4,35 and alleviate the stability issue. However, the sizes of the paper channels are generally about 0.5-5 mm, which is much bigger than a conventional microfluidic channel. This wider paper channel with random fiber networks causes irregular motions in the preconcentrated plugs. This has been inevitable in paper-based ICP preconcentrators, because the minimum feature size of wax patterning and paper cutting (i.e., fabrication methods to build paper channels) is about few hundred micrometers.
The ICP preconcentrator has been used in a wide range of biomicrofluidic platforms for preconcentrating various bio-agents; amplifying the signals of various assays; and detecting targets, such as therapeutic proteins36, peptides37, aptamers17, and enzymes38. These previous works targeted fluorescence-labeled biomolecules. This is because we cannot specify the exact operating conditions (i.e., voltage and flow rate) to maintain the preconcentration site, so we first need to find the proper conditions for the preconcentrator targets. Departing from previous work, the merged ICP phenomenon allows us to always fix the preconcentrated plugs at a broad range of operating conditions while maintaining the high flexibility of the ICP devices. For example, we can modulate the merged ICP system with a tangential fluid flow, and operate it in the continuous-flow mode39. This indicates that we can now extend the applications of ICP preconcentrators to label-free detection techniques without using visualization instruments and tracers. This unique advantage of the spatiotemporal controllability provides a strong commercial opportunity to integrate the ICP device with generic benchtop platforms, such as polymerase chain reaction machines and mass spectrometers.

Ion concentration polarization (ICP) refers to a phenomenon that occurs during ion enrichment and ion depletion on a permselective membrane, resulting in an additional potential drop with ion concentration gradients1,2. This concentration gradient is linear, and it becomes steeper as a higher voltage is applied (Ohmic regime) until the ion concentration on the membrane approaches zero (limiting regime). At this diffusion-limited condition, the gradient (and corresponding ion flux) has been known to be maximized/saturated1. Beyond this conventional understanding, when the voltage (or current) is increased further, an overlimiting current is observed, with flat depletion zones and very sharp concentration gradients at the zone boundary1,3. The flat zone has a very low ion concentration, but surface conduction, electro-osmotic flow (EOF), and/or electro-osmotic instability promote ion flux and induce an overlimiting current3,4,5. Interestingly, the flat depletion zone serves as an electrostatic barrier, which filters out6,7,8,9 and/or preconcentrates targets10,11. Since there is an insufficient amount of ions to screen the surface charges of charged particles (for satisfying electroneutrality), the particles cannot pass through this depletion zone and therefore line up at its boundary. This nonlinear ICP effect is a generic phenomenon in various types of membranes10,11,12,13,14 and geometries6,15,16,17,18,19,20,21; this is why researchers have been able to develop various types of filtration6,7,8,9 and preconcentration10,11 devices using the nonlinear ICP.
Even with such high flexibility and robustness, it is still a practical challenge to clarify the operating conditions for the nonlinear ICP devices. The nonlinear regime of the ICP quickly removes cations through a cation exchange membrane, which causes the displacement of anions moving towards the anode. As a result, the flat depletion zone propagates quickly, which is reminiscent of shock propagation22. Mani et al. called this dynamic the deionization (or depletion) shock23. To preconcentrate targets at a designated sensing position, preventing the expansion of the ion depletion zone is necessary, for example, by applying EOF or pressure-driven flow against the zone expansion24. Zangle et al.22 clarified the criteria for ICP propagation in a one-dimensional model, and it highly depends on electrophoretic mobility17, ionic strength18, pH25, and so on. This indicates that proper operating conditions will be altered according to the sample conditions.
Here, we present detailed design and experimental protocols for a novel ICP platform that preconcentrates targets within a spatiotemporally defined position26. The expansion of the ion depletion zone is blocked by the ion enrichment zone, leaving a stationary preconcentration plug at an assigned position, regardless of the operating time, applied voltage, ionic strength, and pH. This detailed video protocol is intended to show the simplest method to integrate cation exchange membranes into microfluidic devices and to demonstrate the preconcentration performance of the new ICP platform compared to the conventional one.

<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

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 schematic fabrication steps of a membrane-integrated microfluidic preconcentrator are shown in Figure 1. A detailed description of the fabrication is given in the Protocol. The designs and device images of the spatiotemporally defined preconcentrator26 are contrasted with those of a conventional preconcentrator11 (Figure 2). The ICP phenomenon in the spatiotemporally defined preconcentrator was investigated in terms of current-voltage-time responses and fluorescent intensity profiles (Figure 3-4). Similar to the ICP phenomenon with a single-membrane preconcentrator3,11, three different regimes (Ohmic, limiting, and overlimiting) were observed in the current-voltage curve: 0.5-1 V (Ohmic and limiting) and 5 V (overlimiting). However, a nonconventional current recovery was detected in the current-time curve as the ion enrichment and the ion depletion zones merged. Next, the ICP preconcentration was tested at different times and voltages with the spatiotemporally defined preconcentrator (Figure 5) and the conventional one-membrane device (Figure 6). The preconcentration dynamics were quantified by fluorescence images, current-time responses, and fluorescent intensity graphs over different distances and times. When comparing the two platforms, the new ICP platform shows an advantage in always collecting targets (fluorescent dyes) between the two cation selective membrane patterns. In addition, it was confirmed that the preconcentration plug remains the same in different ionic strengths (1-100 mM NaCl) and pH values (3.7-10.3), verifying the high availability of the merging ICP preconcentrator in wide ranges of operating conditions (Figure 7). In Figure 8, a 10,000-fold protein preconcentration was also demonstrated.
<img alt="Figure 1" src="/files/ftp_upload/55313/55313fig1.jpg" /> 
Figure 1. Fabrication steps of a cation exchange membrane-integrated microfluidic chip. After a PDMS mold is filled with a cation exchange resin using the microflow patterning technique (a-c)29, the membrane-patterned glass substrate is bonded with a PDMS microchannel by oxygen plasma treatment (d). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55313/55313fig2.jpg" /> 
Figure 2. Schematics of the spatiotemporally defined preconcentrator (a) and conventional preconcentrator (b). (a) In the new platform, between two membrane patterns (i), ion depletion/enrichment zones are developed and merged together with linear (Ohmic and limiting regime; ii) or nonlinear (overlimiting regime; iii) concentration profiles. In all three current regimes, the ion enrichment zone blocks the propagation of the depletion zone and targets (hollow circles; i) are preconcentrated at the interface of the ion depletion and enrichment zones (curved, dotted line; i). The wall of the PDMS channel is negatively charged, and this generates electro-osmotic flow (EOF) between the two cation exchange membranes under an electric field. The EOF continuously delivers targets towards the interface of the depletion and enrichment zones. (b) In the conventional platform, only the ion depletion zone is developed near the membrane with linear (Ohmic and limiting regime; ii) and nonlinear (overlimiting regime; iii) concentration gradients. As the EOF delivers the targets, the preconcentration also occurs at the depletion zone boundary, but this zone (and the preconcentrated plug) moves away from the cation exchange membrane (black arrow; i). It is noted that there is no increase in the ion concentration here without the ion enrichment zone (ii-iii). In (a-b), the device images are shown in (iv). C0 represents the initial ion concentration. V+ and G indicate the anode and the cathode, respectively. Reprinted from Reference 26t with permission from The American Chemical Society. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55313/55313fig3.jpg" /> 
Figure 3. Merged ICP phenomenon between two cation exchange membranes. (a) The current-voltage curve shows three distinct regimes (Ohmic, limiting, and overlimiting). The current response is measured by ramping up the voltage at discrete intervals of 0.25 V every 40 s, which is repeated three times. The error bar indicates the standard deviation of the current responses. (b, c) In the three regimes, fluorescence images (b) and intensity profiles along A-A&#8242; at the middle of the channel (c) were obtained. Yellow, dotted boxes indicate the locations of the cation selective membranes. 1 mM KCl solution with a 1.55 &#181;M (1 &#181;g/mL) negatively charged fluorescent dye was used. Reprinted from Reference 26 with permission from The American Chemical Society. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55313/55313fig4.jpg" /> 
Figure 4. Transient dynamics of merging ion depletion and enrichment zones. (a, b) In the Ohmic-limiting regimes, the linear concentration gradients grow (&#60; 1 s) from the cation exchange membrane and then overlap together (&#62; 1 s). (c) In the overlimiting regime, the two ICP zones are merged more quickly (&#60; 0.6 s) with the depletion shock (black arrow at 0.2 s). (d-f) The current-time responses show that the current is initially dropped due to the growth of the low-concentration depletion zone, which corresponds to low electrical conductivity. The current drop is then recovered due to a convective transport by vortices confined between two membranes. Reprinted from Referenced 26 with permission from The American Chemical Society. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55313/55313fig5.jpg" /> 
Figure 5. Spatiotemporally fixed preconcentration at 5, 10, and 20 V. (a-c) Fluorescence images of the merged ICP and the current-time responses (d-f) over time (0-100 s). The yellow, dotted lines indicate the location of the cation exchange membranes. (g) Time-lapse fluorescent intensity profiles are plotted along the microchannel (A-A&#8242;). The peak intensities increase as time passes, with fixed locations. (h) The peak intensity fold (i.e., how many times greater than the initial fluorescent intensity). At higher voltages, the faster EOF delivers targets towards the interface of the ion depletion and enrichment zones, so the preconcentration speed increases. A spike at 20 V is induced by the depletion shock (Figure 4c). The fluorescent dyes accumulate on the shock boundary as it pushes the dyes. This initial accumulation is then somewhat dispersed when the depletion shock meets the ion enrichment zone, creating the spike in the peak intensity curve. As can be seen in Figure 4c, at 0.8 s, the peak was wider than it was at 0.4 s. This is probably because the left side of the left Nafion pattern (Figure 2a) was electrically floated, and the accumulated dyes could spread out. Reprinted from Reference 26 with permission from The American Chemical Society. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55313/55313fig6.jpg" /> 
Figure 6. ICP phenomenon in the conventional ICP preconcentrator at 5, 10, and 20 V. (a-c) Fluorescence images of the ion depletion zone and the current-time response (d-f) over time (0-100 s). The propagation of the depletion zone and the preconcentration plug is clearly visualized in the fluorescence images. Accordingly, the vortices are not confined, so the current recovery does not occur, even in the overlimiting regime. Yellow, dotted lines mark the location of the cation exchange membranes. (g) Time-lapse fluorescent intensity profiles are plotted along the microchannel (A-A&#8242;). The peak intensities increase as time passes, but the location moves away from the membrane. (h) Peak intensity fold of the conventional ICP device. In contrast to the merged ICP device (Figure 5h), there is no intensity spike without the confinement of ICP zones, because the fluorescent intensity increased as the dye was preconcentrated. The increase of the peak intensity fold is similar to that of the merged ICP device at the same time (at a given voltage). This indicates that the length of time that the preconcentrated plug is held in place is crucial to the preconcentration performance. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/55313/55313fig7.jpg" /> 
Figure 7. Spatiotemporally defined preconcentration at various ionic strengths (1-100 mM NaCl) and pH values (3.7-10.3). (a) Fluorescence images obtained after 100 s of operation at 50 V. As can be seen, the locations of the preconcentration plugs are still between the two cation exchange membranes (yellow, dotted lines), even though the intensity is weakened under high ionic strength and in a strong acidic or basic solution. (b, c) The location of the peak intensity and its intensity fold (i.e., how many times greater than the initial intensity), mapped under 10, 20, 50, and 100 V. For a single condition (1, 10, 100 mM and/or pH 3.7, 7, and 10), there are four data points corresponding to the four voltage conditions. At higher voltages, there is a higher peak intensify fold in all cases. 100 V was not tested in 1 mM NaCl (pH 7) because the peak intensity already touched the highest values (due to the saturation of the camera) at 50 V. From the peak intensity profile, the peak region is also identified, with 1% below the peak intensity, which is represented by error bars (b, c). A higher voltage and a stronger EOF shift the peak location to the right, with a higher intensity fold and a sharper preconcentration plug. Gray boxes indicate the locations of the cation exchange membranes. The 0 distance (a) represents the origin of the x-axis (b, c), which is on the right edge of the left cation exchange membrane. The origin of the distance is the right edge of the left membrane. Reprinted from Reference 26 with permission from The American Chemical Society. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55313/55313fig8.jpg" /> 
Figure 8. Demonstration of spatiotemporally fixed protein preconcentration. FITC-albumin (1 &#181;g/mL) in 1x phosphate-buffered saline solution was used. 0.1% Tween 20 was also added to prevent nonspecific binding. Since the preconcentration is hardly achieved at a higher ionic strength (Figure 7), we doubled the width of the Nafion pattern (200 &#181;m) and used a narrower PDMS channel (50 &#181;m). In this way, the performance of ICP preconcentration was enhanced by broadening the ion pathway and reducing the absolute amount of ions in the channel. At an applied voltage of 100 V, the peak and averaged fluorescent intensities were traced in the white, dotted box, which is the region between the two cation exchange membranes. Within 10 min of operation, the proteins were preconcentrated up to 10 mg/mL (peak) and ~0.1 mg/mL (averaged), indicating 10,000- and 100-fold preconcentrations, respectively. The inset fluorescence images were obtained at 0, 10, and 20 min. In this work, a 20-min operation was enough to preconcentrate the target molecules, so we did not cover longer operating times. Reprinted from Reference 26 with permission from The American Chemical Society. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55313/55313fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</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

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.4K Views.  Korea Institute of Science and Technology. 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.

Video: 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.

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

Engineering

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.

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

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.

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

Biology

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.

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

Medicine

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.

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.

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

Biochemistry

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.

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.

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.

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.

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. ...