コロイド粒子の自己組織化プロセスを介して、PDMSマイクロ流体チップ内のサブ50 Nmのナノ流体ジャンクショ​​ンを作成します

This protocol demonstrates a novel and simple self-assembly technique of colloidal silica nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane. The bead membrane with a pore size of approximately 45 nanometers, acting as a cation-selective membrane, can be used for the electrokinetic concentration of DNA and protein samples. Overview:In the first step, the PDMS device with nanotraps is cast through a double-molding process.

After plasma bonding the device to a glass substrate, surface functionalized silica beads are injected into the microchannels to form the self-assembly colloidal membrane. Lastly, a voltage is applied across the membrane using a source meter and voltage divider. The ion depletion and concentration increase are observed using an inverted epifluorescence microscope, with a CCD camera.

Preparation of the Silica Colloidal Particle Suspensions:Starting with the 300 nanometer silica beads;vortex bead stock for 30 seconds;withdraw 600 microliter suspension into an Eppendorf tube, and centrifuge for one minute at 2, 600g. After centrifugation, carefully remove the supernatant. Resuspend the beads with 400 microliters of one-millimolar sodium phosphate buffer, to reach a 15%working concentration.

Vortex to reach a homogeneous suspension. Repeat the same steps to prepare 500-nanometer silica beads. For 500-nanometer carboxylic silica beads, weigh 100 milligrams in a 15-milliliter Falcon tube.

Then transfer 9.8 milliliters of one-molar sodium chloride to resuspend the beads. Vortex the tube vigorously. Invert the tube several times to displace the beads impacted at the bottom.

To modify surface charge on the beads, we prepare two polyelectrolytes:PAH and PSS. The 0.9%PSS is prepared by dissolving 180 milligrams of PSS with 200 milliliters, one-molar sodium chloride. The 0.4%PAH is prepared by deluding 300 microliters of the stock solution into 15 milliliters of one-molar sodium chloride.

Vortex both solutions to ensure they're fully dissolved. To deposit a positively charged PAH layer, add 200 microliters of the PAH solution to the bead suspension. Vortex for one minute and incubate the suspension in a rotator for one hour at room temperature.

To wash away the unbound PAH, centrifuge the suspension at 1, 801g for one minute, to precipitate the beads. Remove the supernatant. Pipette one to two milliliters of water into the tube to disrupt the beads.

Add the remaining eight milliliters of water and then vortex. Repeat the washing procedure four more times. After the fifth wash and centrifuge, discard the supernatant and restore the beads with 9.8 milliliters one-molar sodium chloride.

For depositing the negatively charged PSS layer, add 200 microliters of the PSS solution to the bead suspension. Vortex for one minute and incubate the suspension in the rotator for one hour at room temperature. Wash the beads five times with DI water as done for the PAH coating.

After the last wash, discard the supernatant, and resuspend the beads with 650 microliters of one-millimolar sodium phosphate buffer with 0.05%Tween 20. Transfer the bead suspension to an eppendorf tube. For preparation of the 500-nanometer amine silica beads, repeat the same procedure to deposit a single negatively-charged PSS layer.

Fabrication of the PDMS Microfluidic Chip:To fabricate the silicon master, first spin coat a one-micrometer-thin photoresist at 4, 000 RPM on a silicon wafer. Then use projection lithography and reactive ion etching to create 700-nanometer-deep and two-micrometer-wide planar nanochannels as nanotraps for the beads. Then spin coat the 2nd photoresist layer at 2, 000 RPM and perform an alignment to the previously-patterned nanotraps.

Add in the microchannels via contact lithography and by deep-reactive ion etching of silicon. To create the release layer, silanate the silicon master in a vacuum jar with 50 microliters trichlorosilane, overnight. To cast the PDMS mold, mix the PDMS base with curing agent at a 10:1 ratio.

Pour the mix on top of the silicon master. Place the master with PDMS in a vacuum jar to remove the air bubbles and bake it in the oven at 70 degrees C to cross link the PDMS. After baking for two hours, carefully remove the PDMS slab from the silicon master.

Treat the surface with plasma to create a permanent bonding to a blank silicon wafer. Attach tape along the edge to mark a partition line for the following PDMS casting step. To create a mold-release layer, silanate the PDMS mold in a vacuum jar overnight.

Cast PDMS on top of the PDMS mold and transfer it into a vacuum jar for degassing. Then bake it in the oven. Peel off the cured PDMS slab from the PDMS mold along the partition line marked with tape.

Cut off the devices carefully and punch holes with a 1.5 millimeter biopsy punch in the reservoirs. After surface cleaning with tape and IPA, plasma bond the PDMS device on a 25-millimeter by 75-millimeter microscope glass slide. Immediately after plasma bonding, inject 10 microliters of the suspension into inlets four and six of the bead delivery channels.

After filling the bead delivery channels, cover all the reservoirs except outlets one and nine, of the bead delivery channels. Air dry the device for three hours at room temperature and store at four degrees C prior to use. Micrographs of the device show the colloidal particles assemble inside the upper and lower bead delivery channels.

The SEM image shows self-assembled 300-nanometer silica colloidal particles trapped with the nanotrap arrays, between the sample and the buffer channel. Fill inlets three and seven of the buffer channels with 10 microliters of one-millimolar sodium phosphate buffer. Fill sample channel inlet five with 10 microliters of 10-nanomolar DNA sample.

Apply a gentle negative pressure with an inverted pipette tip on all the outlets to fill the channels with the solutions without air bubbles. Add 10 microliters of sodium phosphate buffer to reservoirs two and eight, and 10 microliters of DNA sample to reservoir 10, to stabilize the pressure for five minutes. Experiment for Electrokinetic Concentration of DNA:Connect the microfluidic chip with a voltage divider connected to a source meter.

Insert the PT wires into reservoirs three, seven, five and 10. Apply voltage across a nanofluidic junction. First apply 30 volts on reservoirs five and 10, and ground on reservoirs three and seven.

Decrease the voltage to 25 volts on reservoir 10 after approximately 30 seconds. Results of Electrokinetic Preconcentration:The microfluidic channel is filled with DNA, with initial concentration at 10 nanomolar. Time-lapse micrographs show the formation of an ion depletion region, either nanofluidic colloidal junctions.

The ion depletion region was initiated in 10 seconds. A concentrated DNA plug was generated when the voltage is applied the sample channel, while the buffer channels were grounded. The dotted lines have been used to highlight the channel walls.

A concentration factor of about 1, 700 fold is achieved within 15 minutes using 300-nanometer colloidal membrane. The average fluorescence intensity of DNA is increasing as a function of time in a nanoporous membrabe. The dotted lines represent the signal level for 10-nanomolar, 17-micromolar, two-micromolar and 10-micromolar DNA.

The concentrated plug is generated at the nanoporous membrane of the 300-nanometer silica beads;the 500-nanometer silica beads;the 500-nanometer silica amine beads, coated with a single layer of PSS;and the 500-nanometer silica carboxyl beads, coated with two layers of PAH and PSS. Conclusions:Using the self-assembled colloidal silica membrane with an estimated pore size of 45 nanometers, we demonstrated ion concentration polarization near the nanofluidic junction, and concentrate a DNA sample by 1700 times within 15 minutes. Compared to the silicon-based nanochannels, the major advantage of self-assembled colloidal membrane, is a possibility of performing surface functionalization completely outside of the sealed microfluidic device, in a separate vial.

We believe that this approach opens up a possibility to fabricate a micro nanofluidic platform rapidly with easily tunable pore sizes, by controlling the bead size, the ionic strength or the pH value of the buffer solution to study the ionic and molecular transport in the 10 to 100 nanometer range.