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.
