This work was supported by NIH R21 EB008177-01A2 and New York University Abu Dhabi (NYUAD) Research Enhancement Fund 2013. We express our thanks to the technical staff of MIT MTL for their support during microfabrication and James Weston and Nikolas Giakoumidis of NYUAD for their support in taking SEM pictures and building a voltage divider, respectively.&#160;The device fabrication in PDMS was conducted in the microfabrication core facility of NYUAD.&#160;Lastly, we would like to thank Rebecca Pittam from the NYUAD Center for Digital Scholarship for video shooting and editing.

1. Preparation of the Silica Colloidal Bead Suspensions
<ol>
	<li>Preparation of 300 nm and 500 nm silica bead suspensions
	<ol>
		<li>Vortex the silica bead stock suspension (10% w/v in water) for 30 sec. to obtain a homogeneous suspension. Pipette a total of 600 &#181;l stock suspension into a 1.5 ml tube and centrifuge it at 2,600 x g for 1 min.</li>
		<li>Substitute the supernatant with 400 &#181;l of 1 mM sodium phosphate buffer (PB, pH 7.0).</li>
		<li>Suspend the silica beads into a final concentration of 15% in 1 mM sodium phosphate solution at pH 7.0 through vortexing.</li>
	</ol>
	</li>
	<li>Surface functionalize 500 nm silica carboxyl beads with poly(allylamine hydrochloride, PAH), and with poly(sodium styrene sulfonate, PSS) polyelectrolytes
	<ol>
		<li>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.</li>
		<li>Prepare 0.4% PAH (MW 65K) in 1 M NaCl by dissolving 300 &#181;l 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.</li>
		<li>Add 200 &#181;l 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.</li>
		<li>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.</li>
		<li>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 5th washing step of 1.2.4.
		<ol>
			<li>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 &#956;l 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.</li>
			<li>Measure the zeta potential of the beads before and after each polyelectrolyte coating using a dynamic light scattering system according to manufacturer&#39;s protocol to verify the polyelectrolyte deposition procedure has been performed correctly (see Table 1).</li>
		</ol>
		</li>
		<li>Repeat five washing steps with DI water following the single PSS layer deposition and re-suspend the beads in 650 &#181;l 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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
2. Fabrication of the PDMS Microfluidic Chip
<ol>
	<li>Microfabrication of the silicon master
	<ol>
		<li>Fabricate the silicon master for PDMS molding using microfabrication techniques as follows.
		<ol>
			<li>Spin coat a 1 &#181;m 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 &#181;m wide planar nanochannels (acting as nanotraps for the silica beads) with reactive ion etching.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Spin coat the second 1 &#956;m 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.</li>
	</ol>
	</li>
	<li>Fabrication of PDMS mold
	<ol>
		<li>Silanize the silicon master with trichlorosilane (50 &#956;l) 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.</li>
		<li>Mix the base to the curing agent at 10:1 ratio and cast PDMS on the silanized silicon master and cure it at 70 &#176;C for 2 hr&#160;in a convection oven.</li>
		<li>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.</li>
		<li>Silanize the PDMS mold in a vacuum jar with trichlorosilane (50 &#956;l) O/N.</li>
		<li>Cast PDMS (base: curing agent at 10:1 ratio) on the silanized PDMS mold and cure it at 70 &#176;C for 2 hr in a convection oven.</li>
	</ol>
	</li>
	<li>Fabrication of the PDMS device
	<ol>
		<li>Peel off the cured PDMS slab from the PDMS mold along the partition line marked with the tape.</li>
		<li>Punch reservoir holes with 1.5 mm biopsy punch, clean with a tape, rinse with isopropyl alcohol (IPA) and dry with nitrogen.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Ultrasonicate the bead suspension for 60 min. in an ultrasonic bath prior to filling. Pipette a 10 &#181;l 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.
	<ol>
		<li>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 &#176;C prior to use. Figure 2 gives a step-by-step schematic of the colloidal self-assembly process.</li>
	</ol>
	</li>
</ol>
3. Experiment for Electrokinetic Concentration of DNA
<ol>
	<li>Fill the reservoirs 3, 7 with a buffer solution (10 &#956;l of 1 mM PB) and reservoir 5 with a DNA sample (10 &#956;l 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).</li>
	<li>Add 10 &#956;l of 1 mM PB to reservoirs 2 and 8 and 10 &#956;l of 10 nM DNA to reservoir 10 to balance the pressure and wait for 5 min. to reach equilibrium.</li>
	<li>Insert the Pt wires into reservoirs 3, 5, 7, 10.</li>
	<li>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.</li>
	<li>Decrease the voltage to 25 V on reservoir 10 after ~30 sec.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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&#160;&#181;m on both sides of the bead delivery channel at a total width of 100&#160;&#956;m prevented the bead suspension from flowing into the buffer and sample channel due to the surface tension at the nanotraps. Once trapped, the colloidal particles packed in the bead delivery channel rapidly and formed a nanoporous junction between the sample and buffer channel.
It is important to load the bead suspension immediately after plasma bonding so that the capillary force drives the silica bead suspension up to the entrance of the outlet reservoirs in the temporarily hydrophilic bead delivery channel. In order to prevent an air bubble blocking the flow in the inlet reservoir, it is highly recommended to reach the bottom of the reservoir with a pipette tip and then release the bead suspension into the reservoir. In the case of the surface-functionalized beads with polyelectrolytes, their flowability was drastically reduced compared to the silica beads without surface functionalization and tended to aggregate more easily and adhere to the channel surface during the filling process. In order to prevent a clogging of the channel with the polyelectrolyte-coated beads, we added a surfactant, 0.05% Tween 20, to the bead suspension. In case there was still a clogging problem during filling, a gentle tapping on the PDMS chip with a pipette tip generally helped to resolve it.
Also, it is important that the bead suspension was not completely dried out after evaporation since it would be difficult to infiltrate the bead membrane with the sodium phosphate buffer solution again. Therefore, after 3 hr of partial evaporation, all in- and outlets of the PDMS device were taped and kept at 4 &#176;C for storage prior to use so the bead packing stays moist. During the preconcentration experiments, the self-assembled bead maintained its structural stability for the most part. However, in few instances, we observed a dislocation of the beads which indicated a defective packing of the beads in microchannels. The self-assembled silica beads ranging from a diameter of 900 nm down to 300 nm after the self-assembly can be seen in Figure 4. The theoretical pore size of the bead packing was ~45 nm, approximately ~15% of the colloidal particle diameter. We could confirm the pore size using a SEM analysis and measured a pore size approximately 20% of the bead diameter after packing.
Using the self-assembled 300 nm and 500 nm colloidal particle membranes as an ion-selective nanoporous junction, we could initiate ion depletion region at 30 V and concentrate 10 nM Cy5 tagged DNA (CAA CCG ATG CCA CAT CAT TAG CTA C) in 1 mM sodium phosphate buffer (Figure 5). By continuously flowing the DNA sample towards the ion depletion zone with an electroosmotic flow at a voltage difference V2-V1 =5 V, we could increase the initial DNA concentration by ~1,700 folds within 15 min. (Figure 6a, b). 500 nm beads allowed more robust DNA concentration than 300 nm beads, as shown in Figure 6b). Since the electrokinetic concentration is based on a force balance between the electroosmotic force and the highly nonlinear electrophoretic forces, the resulting concentration factor is determined by the degree to which this force balance can be maintained during electrokinetic concentration.27
Another significant advantage of deploying the colloidal particles for building a nanofluidic junction is the ease with which its surface functionalization can be performed. Instead of creating a nanochannel through bonding first and then performing a surface functionalization on it, we can simply surface functionalize the colloidal particles in a vial outside of the device first and then flow them into the channel for self-assembly. Based on this approach, we could initiate ICP using the 500 nm silica amine particles coated with a single layer of PSS and 500 nm silica carboxyl particles coated with a layer of PAH and PSS (Figure 6 c and d), at lower voltages (8 V and 10 V, respectively) than the colloidal particles without surface functionalization (30 V). This result shows that the surface functionalization of the colloidal particles prior to the self-assembly was effective to increase the surface charge of colloidal particles and resulted in higher ICP. However, in terms of the concentrator factor obtained, the nanofluidic junction of the surface functionalized beads was less effective than the non-functionalized silica beads. The amine/PSS-coated beads enabled a factor of ~200, while the carboxyl/PAH/PSS bead membrane showed a 1,000-fold increase after 15 min. (Figure 6d). This result can be explained by a higher surface charge of the surface functionalized nanopores that led to an increased length of the ion depletion region pushing the sample concentration plug farther away from the bead membrane and therefore, to less stable concentration. We believe that shortening the total width of the nanoporous bead membrane from currently 1 mm (the section of the bead membrane parallel to the sample channel) could mitigate this instability issue. According to our previous study, the width of the nanoporous junction determines the amount of ionic current passing through it.28 As the width increases, the ionic current increases and since more cations can migrate through the membrane, the depletion length increases and the concentration plug is further pushed away from the nanoporous junction. Therefore, the accumulation occurs in a less confined manner and the sample plug becomes less stable. Empirically, the nanoporous junction should be ~100-400 &#181;m in width. Another feature to improve was an insufficient thickness of the PDMS wall of 15&#160;&#181;m between the sample channel and the bead delivery. This thin PDMS section led to an insufficient bonding that enabled an ionic current between the buffer and sample channel. Therefore, the entire bead membrane section parallel to the sample channel (1 mm in width) was acting as a nanoporous junction, even though only 100&#160;&#956;m of the bead was intended as a nanoporous junction membrane according to the total width of the nanotrap array. The PDMS wall thickness should be at least 25 &#181;m or higher.

Nanofluidics is an emerging research area of &#181;TAS (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&#39;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 &#181;m. 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 evaporation14-16 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.

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

creating sub-50 nm nanofluidic junctions in pdms microfluidic chip via self-assembly process of colloidal particles

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.

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 &#181;m wide bead delivery channel (Figure 1B). The silica colloidal suspension is flown into the bead delivery channel immediately after plasma bonding to create a nanofluidic junction between the sample and the buffer solution channel. The nanotrap array consisting of 700 nm deep and 2&#160;&#956;m wide nanochannels is used to trap the colloidal particles. Its scanned image obtained with a non-contact surface profiler is shown in Figure 1C). The colloidal bead membranes after evaporation are shown in Figure 1D). The SEM in Figure 1E) shows the silica beads trapped at the planar nanotrap array separating the sample channel from the bead delivery channel. The 300 nm silica bead packing shows highly ordered hexagonal packing with some minor defects that could cause a variation in the concentration behavior (Figure 1F). The design of the PDMS concentrator chip with its dimensions can be found <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54145/Device_design_new.pptx">here</a> and in the Supplemental Files.
<img alt="Figure 1" src="/files/ftp_upload/54145/54145fig1.jpg" /> 
Figure 1. Microfluidic concentrator in PDMS with an integrated sub-50 nm nanoporous junction. (A) Photo of the PDMS concentrator device. (B) Schematic of the micro-nanofluidic device with a bead delivery channel between the sample and buffer solution channel. The voltage is applied across the bead membranes between the sample channel and the buffer solution channels. (C) Surface profile of the nanotrap array in PDMS with a width of 2&#160;&#956;m and a depth of 700 nm. (D) Micrograph of the device with a colloidal particle assembly inside the bead delivery channel after evaporation. (E) Scanning electron micrograph of the self-assembled 300 nm silica colloidal particles with the nanotrap arrays between the sample and buffer channel. The 300 nm beads are trapped at the entrance of the nanotraps due to surface tension. (F) Hexagonally packed 300 nm silica beads inside the bead delivery microchannel after evaporation. (Adapted from Ref. 25 with permission from The Royal Society of Chemistry) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54145/54145fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A schematic of the microfabrication steps for the PDMS concentrator device is shown in Figure 2. To make a PDMS device, a double PDMS casting is required. The bead filling process in the PDMS concentrator is shown in Figure 3. The details for the microfabrication and the filling process can be found in the protocol. The zeta potential of the silica beads without and with polyelectrolyte coating is shown in Table 1.
<img alt="Figure 2" src="/files/ftp_upload/54145/54145fig2.jpg" /> 
Figure 2. Schematic of the fabrication process for the silicon master, the PDMS master and the PDMS concentrator device. After two photolithographic and etching steps, the silicon master is cast with PDMS. After a double-molding, the PDMS device is assembled via plasma bonding and filled with a bead suspension. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54145/54145fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54145/54145fig3.jpg" /> 
Figure 3. Step-by-step schematic for self-assembly of colloidal silica beads. 10 &#181;l of the bead suspension was pipetted in to the bead delivery channels immediately after plasma treatment. Once the bead delivery channel was filled, all but two inlets 1 and 9 were covered with tape and the devices air dried for 3 hr prior to use. (Reproduced from Ref. 25 with permission from The Royal Society of Chemistry) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54145/54145fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table>
	<tbody>
		<tr>
			<td>Colloidal particles (500 nm)</td>
			<td>Zeta potential (mV)</td>
		</tr>
		<tr>
			<td>Silica</td>
			<td>-2.04</td>
		</tr>
		<tr>
			<td>Silica amine</td>
			<td>19.6</td>
		</tr>
		<tr>
			<td>Silica carboxyl</td>
			<td>-19.73</td>
		</tr>
		<tr>
			<td>Silica carboxyl, PAH coated</td>
			<td>31.8</td>
		</tr>
		<tr>
			<td>Silica carboxyl, PAH, PSS coated</td>
			<td>-28.5</td>
		</tr>
		<tr>
			<td>Silica amine, PSS coated</td>
			<td>-31.2</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Zeta potential of silica beads at 25 &#176;C. 0.1% (w/v) colloidal solutions were used for the measurements (n=3).
The SEM images taken from the bead packing channel after drying out show a pore size ranging between 60 nm, 91 nm and 170 nm, as shown in Figure 4. The pore size corresponds to approximately 20% of the bead size, 300 nm, 500 nm and 900 nm, respectively (15% of the bead diameter is the theoretical pore size).
<img alt="Figure 4" src="/files/ftp_upload/54145/54145fig4.jpg" /> 
Figure 4. SEM images of self-assembled 300 nm (A), 500 nm (B) and 900 nm (C) silica colloidal bead packing. PDMS devices were reversibly bonded to glass slides and beads flown into the channel using negative pressure. After air-drying the devices O/N, the PDMS devices were peeled of the glass carefully and imaged. This pore sizes were estimated to be 60&#177;2, 91&#177;5 and 170&#177;7 nm for 300 nm, 500 nm and 900 nm beads respectively (n=9). These pore sizes were close to the theoretical size, ~15% of the bead diameter. (Adapted from Ref. 25 with permission from The Royal Society of Chemistry) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54145/54145fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
When applying voltage of 30 V across the 300 nm bead membrane, an ion depletion zone was observed near the colloidal membrane inside a microchannel filled with a fluorescent labeled DNA (Figure&#160;5 A, B). When lowering the voltage to 25 V on the left side, the DNA molecules got accumulated in the form of a plug and its concentration increased due to electroosmotic flow driven by a voltage difference of 30 V- 25 V across the sample channel (Figure&#160;5 C, D).
<img alt="Figure 5" src="/files/ftp_upload/54145/54145fig5.jpg" /> 
Figure 5.&#160;Time-lapse micrographs show the formation of an ion depletion region near the nanofluidic colloidal junctions in the channel filled with DNA (initial concentration of 10 nM). The ion depletion region was initiated at t=10 s and a concentrated DNA plug was generated at V2 = 30 V and V1 = 25 V across the sample channel while the buffer channels were grounded. The dotted lines have been used to highlight the channels walls. A concentration factor of ~1,700 folds was achieved within 15 min. using a 300 nm colloidal membrane. (Reproduced from Ref. 25 with permission from The Royal Society of Chemistry) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54145/54145fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The silica membranes with a bead size of 300 nm and 500 nm showed the highest concentration factor at ~1,700 times for the Cy 5 tagged DNA (CAA CCG ATG CCA CAT CAT TAG CTA C) within 15 min. (Figure&#160;6 A, B). The polyelectrolyte-coated silica bead membranes led to a 200- to 1,000-fold increase in the DNA concentration after 15 min. (Figure&#160;6 C, D).

<img alt="Figure 6" src="/files/ftp_upload/54145/54145fig6.jpg" /> 
Figure 6.&#160;Fluorescence intensity of DNA as a function of time for (A) 300 nm silica beads (B) 500 nm silica beads and (C) 500 nm PSS-coated silica amine beads and (D) 500 nm PAH/PSS coated silica carboxyl beads. The dotted lines represent the fluorescence signal intensity level for 10 nM (A, B , C, D), 17 &#181;M (A, B), 2 &#181;M (C) and 10 &#181;M (D) DNA. The results have been normalized against background fluorescence. (Reproduced from Ref. 25 with permission from The Royal Society of Chemistry) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54145/54145fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>Process time&#160;</td>
			<td>Etch mode</td>
			<td>Passivation mode&#160;</td>
		</tr>
		<tr>
			<td>Process time&#160;</td>
			<td>6 s</td>
			<td>4.5 s</td>
		</tr>
		<tr>
			<td>Overrun&#160;</td>
			<td>0.5 s</td>
			<td>0 s</td>
		</tr>
		<tr>
			<td>Platen generator Power&#160;</td>
			<td>80 W</td>
			<td>60 W</td>
		</tr>
		<tr>
			<td>Coil generator Power&#160;</td>
			<td>600 W</td>
			<td>600 W</td>
		</tr>
		<tr>
			<td>Gas</td>
			<td>SF6 70 sccm&#160;</td>
			<td>C4F8 35 sccm</td>
		</tr>
		<tr>
			<td>Etch rate</td>
			<td colspan="2">1.47 &#181;m/min</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;DRIE parameters.

Watch this Scientific Journal Video about Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles at JoVE.com

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

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.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

creating-sub-50-nm-nanofluidic-junctions-pdms-microfluidic-chip-via

Nanyang Technological University

Research

JoVE Journal

Engineering

10.6K Views.  New York University Abu Dhabi (NYUAD). 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.

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

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

Biology

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Chemistry

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. This platform has a three-dimensional (3D) network with curved fluid channels and three pneumatic valves, which create networks, channels, and spaces through duplex replication with polydimethylsiloxane (PDMS). The device operates based on the transient response of a fluid flow rate controlled by a pneumatic valve in the following order: (1) sample loading, (2) sample blocking, (3) sample concentration, and (4) sample release. The particles are blocked by thin diaphragm layer deformation of the sieve valve (Vs) plate and accumulate in the curved microfluidic channel. The working fluid is discharged by the actuation of two on/off valves. As a result of the operation, all particles of various magnifications were successfully intercepted and disengaged. When this technology is applied, the operating pressure, the time required for concentration, and the concentration rate may vary depending on the device dimensions and particle size magnification.

The present protocol describes a pneumatic microfluidic platform that can be used for efficient microparticle concentration.

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. ...

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...