Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Summary

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Abstract

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

Introduction

Nanofluidics is an emerging research area of µTAS (Micro Total Analysis Systems) to study biological processes or transport phenomena of ions and molecules at the length scale of 10¹- 10² nm. With the advent of the nanofluidic tools such as nanochannels, transport processes of molecules and ions can be monitored with unprecedented precision and manipulated, if needed, by exploiting features that are available only at this length scale for separation and detection.^1,2 One of these characteristic nanoscale features is a high ratio of surface to bulk charge (or Dukhin number) in nanochannels that can cause a charge imbalance and initiate ion concentration polarization (ICP) between the nanochannel and microchannel.³

A common device platform for the study of nanofluidic phenomena consists of a two-microchannel system connected by an array of nanochannels as a junction.^4-6 The material of choice for building such a nanofluidic device is the silicon because of its high stiffness that prevents the channel from collapsing during bonding processes.⁷ However, silicon device fabrication requires expensive masks and substantial amount of processing in the cleanroom facility.^8-10 Due to the convenience of device fabrication through molding and plasma bonding, polydimethylsiloxane (PDMS) has widely been accepted as a building material for microfluidics and it would be an ideal material for nanofluidics as well. However, its low Young's modulus around 360-870 KPa, makes the PDMS channel easily collapsible during plasma bonding. The minimum aspect ratio of the nanochannel (width to depth) has to be less than 10:1 which means that the fabrication of PDMS devices via standard photolithography will become extremely challenging if the nanochannel depth has to be below 100 nm, requiring a channel width less than the current limit of photolithography at around 1 µm. To overcome this limitation, there have been attempts to create nanochannels in PDMS using non-lithographical methods such as stretching to initiate cracks with mean depth of 78 nm¹¹ or to form wrinkles after plasma treatment.¹² Collapsing a PDMS channel with mechanical pressure allowed a nanochannel height as low as 60 nm.¹³

Even though these highly inventive non-lithographic methods allowed building nanochannels below 100 nm in depth, the dimensional controllability of the nanochannel fabrication still poses an obstacle to a wide acceptance of PDMS as a building material for nanofluidic devices. Another critical problem of the nanochannels, whether in silicon or PDMS, is the surface functionalization in case there is a need to alter the surface charge on the channel wall for the manipulation of ions or molecules. After device assembly through bonding, the nanochannels are extremely difficult to reach for surface functionalization due to the diffusion-limited transport. To create a nanoscale channel with high dimensional fidelity and facile surface functionalization, the self-assembly method of colloidal particles induced by evaporation^14-16 in microfluidic devices can be one of the promising approaches. Besides the controllability of pore size and surface property, there is even a possibility to tune the size of the pore in-situ when using colloidal particles coated with polyelectrolytes by controlling temperature,¹⁷ pH,^18,19 and ionic strength.¹⁸ Because of these advantages, the self-assembly method of colloidal particles has already found applications for electrochromatography,²⁰ biosensors,²¹ protein concentration²² and separation of proteins and DNA in microfluidics.^14,23 In this study, we deployed this self-assembly method to build an electrokinetic preconcentration device in PDMS that requires a nanofluidic junction between two microchannels.²⁴ The fundamental mechanism behind the electrokinetic concentration is based on ion concentration polarization (ICP).²⁵ A detailed description of fabrication and assembly steps is included in the following protocol.

Protocol

1. Preparation of the Silica Colloidal Bead Suspensions

1. Preparation of 300 nm and 500 nm silica bead suspensions
   1. Vortex the silica bead stock suspension (10% w/v in water) for 30 sec. to obtain a homogeneous suspension. Pipette a total of 600 µl stock suspension into a 1.5 ml tube and centrifuge it at 2,600 x g for 1 min.
   2. Substitute the supernatant with 400 µl of 1 mM sodium phosphate buffer (PB, pH 7.0).
   3. Suspend the silica beads into a final concentration of 15% in 1 mM sodium phosphate solution at pH 7.0 through vortexing.
2. Surface functionalize 500 nm silica carboxyl beads with poly(allylamine hydrochloride, PAH), and with poly(sodium styrene sulfonate, PSS) polyelectrolytes
   1. Suspend 0.1 g of 500 nm silica beads with carboxyl group with 10 ml 1 M NaCl (pH 7.0) for 1 % (w/v) bead suspension.
   2. Prepare 0.4% PAH (MW 65K) in 1 M NaCl by dissolving 300 µl of the stock solution (20% w/v in water) in 15 ml of 1 M NaCl. Prepare 0.9% PSS (MW 70K) in 1 M NaCl solution by dissolving 0.18 g PSS in 20 ml 1 M NaCl solution. Vortex both solutions for 1 min. to dissolve the polyelectrolytes completely.
   3. Add 200 µl of PAH solution to 9.8 ml of 1% silica carboxyl beads in a 15 ml tube to deposit a positively charged polyelectrolyte layer on silica beads with carboxyl functional group. Vortex the bead suspension for 1 min. and incubate it on a tube rotator for 60 min. at RT.
   4. Centrifuge the bead suspension at 1801 x g for 1 min. and wash off the unbound PAH five times with 10 ml DI water. After each centrifuge and removal of the supernatant, the beads were densely packed at the bottom of the tube. Disrupt the bead clump by vigorous pipetting with 2 ml of DI water before adding 8 ml of DI water so that the beads can be re-suspended and washed off prior to the next centrifuge step.
   5. Follow the steps in 1.2.3 and 1.2.4 for PSS coating to deposit a negatively charged layer on the beads. Re-suspend the beads in 9.8 ml of 1 M NaCl prior to the PSS deposition after removing the DI water supernatant from the 5^th washing step of 1.2.4.
      1. Use the same vigorous pipetting step using 2 ml of 1 M NaCl to break up the bead clump at the bottom of the 15 ml tube and then add 8 ml of 1 M NaCl. Add 200 μl of PSS solution to 9.8 ml of the silica beads deposited with a single PAH layer. After vortexing for 1 min. and incubation for 60 min. on the tube rotator, repeat 5 washing steps with DI water.
      2. Measure the zeta potential of the beads before and after each polyelectrolyte coating using a dynamic light scattering system according to manufacturer's protocol to verify the polyelectrolyte deposition procedure has been performed correctly (see Table 1).
   6. Repeat five washing steps with DI water following the single PSS layer deposition and re-suspend the beads in 650 µl of 1 mM sodium phosphate buffer with 0.05% Tween 20 (15% w/v) prior to use in the microfluidic device to enhance its flowability.
3. Follow the procedure described above from 1.2.5 to 1.2.6 for 500 nm silica beads with amine functional group to deposit a single layer of PSS.

2. Fabrication of the PDMS Microfluidic Chip

1. Microfabrication of the silicon master
   1. Fabricate the silicon master for PDMS molding using microfabrication techniques as follows.
      1. Spin coat a 1 µm thin photoresist at 4,000 rpm on a silicon wafer. Pattern the layer using projection lithography (exposure time 170 msec.) and etch 700 nm deep and 2 µm wide planar nanochannels (acting as nanotraps for the silica beads) with reactive ion etching.
      2. Use the following etching parameters to achieve an etch rate of 3.5 nm/s: CHF₃ (45 sccm), CF₄ (15 sccm), Ar (100 sccm), pressure 100 mTorr, RF power 200 W.
   2. Spin coat the second 1 μm thick photoresist layer at 2,000 rpm and perform an alignment to the previously patterned nanotraps. Pattern the microchannels via contact lithography and by deep reactive ion etching (DRIE) of silicon. Use the DRIE parameters²⁶ in Table 2.
2. Fabrication of PDMS mold
   1. Silanize the silicon master with trichlorosilane (50 μl) in a vacuum jar O/N.
      CAUTION: Tricholorosilane is a toxic and corrosive material. Always use it in a chemical hood with proper personal protection equipment.
   2. Mix the base to the curing agent at 10:1 ratio and cast PDMS on the silanized silicon master and cure it at 70 °C for 2 hr in a convection oven.
   3. Remove the PDMS slab from the silicon master with a knife and plasma bond it on a blank wafer using a plasma cleaner after a plasma treatment in a plasma cleaner for 1 min. Attach tapes along the edge to mark a partition line for the following PDMS casting step.
   4. Silanize the PDMS mold in a vacuum jar with trichlorosilane (50 μl) O/N.
   5. Cast PDMS (base: curing agent at 10:1 ratio) on the silanized PDMS mold and cure it at 70 °C for 2 hr in a convection oven.
3. Fabrication of the PDMS device
   1. Peel off the cured PDMS slab from the PDMS mold along the partition line marked with the tape.
   2. Punch reservoir holes with 1.5 mm biopsy punch, clean with a tape, rinse with isopropyl alcohol (IPA) and dry with nitrogen.
   3. Plasma bond the PDMS device on a 25 mm x 75 mm microscope glass slide after a plasma treatment in a plasma cleaner for 1 min.
4. Ultrasonicate the bead suspension for 60 min. in an ultrasonic bath prior to filling. Pipette a 10 µl bead suspension (300 nm non-functionalized silica beads, or 500 nm silica carboxyl beads with PAH-PSS layers, or 500 nm silica amine beads with a PSS layer) into the inlets 4 and 6 each (see Figure 1 A, B) immediately after plasma bonding of the PDMS chip to a glass substrate. Tap gently on the PDMS chip with a pipette tip to enhance the bead packing.
   1. After filling the bead delivery channels, cover all the inlets except for 1 and 9 with tape. Air-dry the device for 3 hr and store at +4 °C prior to use. Figure 2 gives a step-by-step schematic of the colloidal self-assembly process.

3. Experiment for Electrokinetic Concentration of DNA

1. Fill the reservoirs 3, 7 with a buffer solution (10 μl of 1 mM PB) and reservoir 5 with a DNA sample (10 μl of 10 nM in 1 mM PB) and apply a gentle negative pressure with an inverted pipette tip on reservoirs 2, 8 and 10 to fill the channels with the solutions without bubbles (see Figure 1B).
2. Add 10 μl of 1 mM PB to reservoirs 2 and 8 and 10 μl of 10 nM DNA to reservoir 10 to balance the pressure and wait for 5 min. to reach equilibrium.
3. Insert the Pt wires into reservoirs 3, 5, 7, 10.
4. Apply voltage across the nanofluidic junction using a voltage divider connected to a source meter and Pt wires. First apply 30 V on reservoirs 5, 10 and GND on reservoirs 3, 7.
5. Decrease the voltage to 25 V on reservoir 10 after ~30 sec.
6. Use a mechanical shutter with a periodic opening in every 5 s to minimize photobleaching of the sample when recording the fluorescence signals from the DNA.

Results

An electrokinetic concentrator chip in PDMS that contains a self-assembled nanofluidic junction between two microchannels is shown in Figure 1A). The channel in the middle of the device is filled with a DNA sample solution and flanked by two buffer solution channels on each side via a 50 µm wide bead delivery channel (Figure 1B). The silica colloidal suspension is flown into the bead delivery channel immediately after plasma bonding to create a nanofluidi...

Discussion

Following the common device design scheme to study nanofluidics, we fabricated a nanofluidic junction between two microfluidic channels by using the evaporation-driven self-assembly of colloidal nanoparticles instead of lithographically patterning an array of nanochannels. When flowing the colloidal particles into the bead delivery channel, an array of nanotraps with a depth of 700 nm and a width of 2 µm on both sides of the bead delivery channel at a total width of 100 μm prevented the bead suspensio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly(Styrenesulfonic Acid) Sodium Salt	Polysciences	08772
Poly(allylamine) Solution	Sigma Aldrich	479144-5G
Silica Microsphere - 300 nm	Polysciences	24321
Silica Microsphere - 500 nm	Polysciences	24323
Silica Microsphere Carboxyl Functional - 500 nm	Polysciences	24753
Silica Microsphere Amine Functional - 500 nm	Polysciences	24756
Sylgard 184 Silicone Elastomer kit	Dow Corning
Trichlorosilane	Sigma Aldrich	175552
Ultrasonic Cleaner	Branson	3510
Tube Rotator	VWR	10136-084
Vortex Mixer	WiseMix	VM-10
Microcentrifuge	VWR	Micro 1207
Plasma Cleaner	Harrick Plasma	PDC-001-HP
PDMS Mixer	Thinky	ARE-250
Oven	Thermo Scientific	PR305220M
Epi-fluorescence Microscope	Nikon	Eclipse Ti
CCD Camera	Andor	Clara
Platinum Electrodes	Alfa Aesar	43014
Source Meter	Keithley	2400
Digital Multimeter	Extech	410
Microscopy Glass Slides	Thermo Scientific	2951-001
Tween 20	Merck Millipore	822184
Sodium chloride	Fisher Scientific	7646-14-5
Sodium phosphate monobasic	Sigma Aldrich	71505
Sodium phosphate dibasic	Sigma Aldrich	S3264
DNA	IDT		CAA CCG ATG CCA CAT CAT TAG CTA C
B-Phycoerythrin	Life Technologies	P-800
Dynamic light scattering system for Zeta Potential Measurement	Malvern	Zetasizer Nano S
Photoresist	Shipley	SPR700-1.0
Projection lithography	Nikon	NSR2005i9
Reactive Ion Etcher	Applied Materials	AME P5000
ICP deep reactive ion etcher	STS	STS-6"
Contact lithography	Electronic Visions	EV620
Photoresist Coater Developer	SSI	SSI 150
Non-contact surface profiler	Wyko	NT 9800