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.<...

<ol>
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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 int...

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

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

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 investiga...

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 ...

The overall goal of this experiment is to preconcentrate bioagents by confining the ion concentration polarization zone between two identical ion exchange membranes. This method can help answer key questions in the field such as how to detect low abundance biomolecule when its concentration is less than the sensor's limit of detection. The main advantage of this technique is that we can generate ICP zone and preconcentrated bioagent in a very specific collision regardless of operation conditions.To begin this procedure, use conventional photolithography or deep reactive ion etching to fabricate silicon masters for the polydimethylsiloxane microchannel and cation-selective membrane molds. Place the silicon masters in a vacuum desiccator with about 30 microliters of trichlorosilane. Close the desiccator and salinize the silicon masters for 30 minutes.Next, mix together a silicone elastomer base and a curing agent in a 10 to one by weight ratio to obtain uncured PDMS. Degas the mixture under vacuum for 30 minutes. Then pour the degassed uncured PDMS onto the silicon masters.Remove bubbles from the PDMS with a handheld blower and then cure the PDMS molds at 80 degrees Celsius for two hours. Separate the cured PDMS components from the silicon masters. Use a knife to shape each component into a rectangle.Next, cut two lines in the cation-selective membrane mold from the edge of the mold to the prefabricated microchannels. Use a two millimeter biopsy punch to punch a hole at the end of each L-shaped channel. Clean a glass slide in the micromachine surface of the cation exchange membrane mold with acrylic adhesive tape and a blower.Place the clean face of the mold on the glass slide to reversibly seal the mold to the slide. Place 10 microliters of cation exchange resin on the glass slide in contact with the ends of the L-shaped channels. Place the tip of a syringe over the microchannel holes and gently withdraw the plunger to pull the resin into the channels.Within one minute of filling the resin channels, carefully detach the mold without touching the patterned resin on the slide. Heat the slide at 95 degrees Celsius for five minutes to evaporate the solvent from the resin. Use a razor blade to remove unneeded sections of cation exchange resin from the slide to form L-shaped cation-selective membranes.Next, use the biopsy punch to punch a 2.0 millimeter reservoir at each end of the prefabricated microchannel in the PDMS microchannel component. Then punch two more holes in the PDMS corresponding to the ends of the L-shaped membranes. Treat the PDMS microchannel component and the membrane-patterned substrate with oxygen plasma for 40 seconds at 100 watts and 50 Place the treated face of the substrate on the treated face of the PDMS microchannel ensuring that the cation-selective membranes cross the microchannel and are in lined with the holes in the PDMS.Apply gentle pressure as needed to seal the PDMS component to the substrate to complete device assembly. To begin the experiment, obtain several test ionic solutions with varying concentrations and pHs. Prepare a buffer solution of one molar potassium chloride or sodium chloride.Add a small amount of negatively charged fluorescent dye to each test solution ensuring that the dye concentration is low enough that its contribution to an electrical current solution is negligible. Load the first test solution into one reservoir of the microchannel. Draw the solution into the microchannel by applying gentle negative pressure to the other microchannel reservoir.Place a large droplet of the test solution over both microchannel reservoirs to eliminate any pressure gradient in the channel. Then fill the cation-selective membrane reservoirs with the chosen buffer solution to compensate for the ICP effect. Load the ICP chip onto an inverted epifluorescence microscope equipped with a charge-coupled device camera.Connect an anode to the left membrane reservoir and a cathode to the right membrane reservoir. Connect the electrodes to a source measure unit. Use the source measure unit to apply a voltage to the device and measure the current response.Acquire fluorescent images of the chip during voltage application. After the experiment, analyze the fluorescence intensity with the appropriate imaging software. Using this method, a microfluidic preconcentrator was fabricated and the current voltage time responses in fluorescence intensity profiles corresponding to various test solutions were investigated.As in conventional single membrane preconcentrators, three different voltage regimes were observed. During the Ohmic and limiting regimes, the linear concentration gradient developed at the cation-selective membranes and merged after about a second. Unlike single membrane preconcentrators, in the over-limiting regime, the ICP zones merged in less than a second with depletion shock observable by fluorescence imaging.The corresponding drop in conductivity was reflected in the current time response. The current time then recovered. This was observed at several voltages in the over-limiting regime.The current recovery was attributed to connective transport by ICP zones combined between the cation-selective membranes which isolate the preconcentration plugs throughout the voltage application. This current recovery was not observed in conventional ICP preconcentrators as the depletion zone and preconcentration plug freely propagate along the microchannel. While increased ionic strength, acidity and basicity decreased the relative intensity of preconcentration plugs in the spatiotemporally-defined preconcentrator, the plugs were still confined.Additional tests approaching preconcentration indicated that wider cation exchange membranes in a narrower channel can facilitate ICP preconcentration in these unfavorable conditions. Once mastered, this technique can be done in 30 minutes if it is performed properly. After its development, this technique paved the way for researchers in the field of electrokinetics to explore applications in microfluidic systems.After watching this video, you should have a good understanding of how to integrate ion exchange materials between microfluidic systems and how to use this platform to preconcentrate bioagents.

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Research

JoVE Journal

Bioengineering

8.4K 조회수.  Korea Institute of Science and Technology. The overall goal of this experiment is to preconcentrate bioagents by confining the ion concentration polarization zone between two identical ion exchange membranes. This method can help answer key questions in the field such as how to detect low abundance biomolecule when its concentration is less than the sensor's limit of detection. The main advantage of this technique is that we can generate ICP zone and preconcentrated bioagent in a very specific collision regardless of operation conditi...