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

Summary

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.

Abstract

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).

Introduction

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 gradients^1,2. 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 boundary^1,3. 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 current^3,4,5. Interestingly, the flat depletion zone serves as an electrostatic barrier, which filters out^6,7,8,9 and/or preconcentrates targets^10,11. 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 membranes^{10,11,12,13,14} and geometries^{6,15,16,17,18,19,20,21}; this is why researchers have been able to develop various types of filtration^6,7,8,9 and preconcentration^10,11 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.

Protocol

1. Fabrication of Cation Exchange Membrane-integrated Microfluidic Chips

1. Preparation of silicon masters
   1. 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.
   2. Fabricate the silicon masters by using either conventional photolithography or deep reactive ion etching²⁷.
   3. Silanize the micropatterned silicon masters with trichlorosilane (~30 μL) in a vacuum jar for 30 min.
      CAUTION: Trichlorosilane is a pyrophoric liquid that is flammable and has an acute toxicity (inhalation, oral ingestion).
2. Preparation of PDMS molds
   1. 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.
   2. Pour the uncured PDMS on the silicon masters, remove the bubbles with a blower, and cure the PDMS at 80 °C for 2 h in a convection oven.
   3. 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).
3. Patterning the cation exchange membranes
   1. 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 µm; height: 50 µm; interchannel distance: 100 µm; 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.
   2. 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.
   3. According to the microflow patterning technique²⁹, release ~10 µL 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 µm, 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 µm, because the patterned ion selective membrane will become thicker than 1 µm; such a thick membrane may create a gap between the membrane and the PDMS channel¹³.
   4. Detach the PDMS mold without touching the patterned resin and place the glass slide on the heater at 95 °C for 5 min to evaporate the solvent in the resin.
      NOTE: The thickness of the patterned membrane is usually less than < 1 µm. 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.
   5. 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.
4. Integration of the microchannel and the membrane-patterned substrate
   1. 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 µm; height: 10 µm), but it is bonded to the ends of the neighboring channel (Figure 1d).
   2. 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.

2. ICP Preconcentration

1. Preparation for the experiment
   1. 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.
   2. Add a negatively charged fluorescent dye (~1.55 µM) to the test solutions.
      NOTE: The concentration of the added dye should be much lower than that of the salt ions (< 10 µM) so that the charged dyes do not contribute to an electrical current^30,31.
   3. 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).
   4. 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.
   5. 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).
2. Visualization of the ICP phenomenon and ICP preconcentration
   1. 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.
   2. Capture fluorescent images with a charge-coupled device camera and analyze the fluorescent intensity using imaging software³².

Results

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...

Discussion

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...

Materials

List of materials used in this article

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