This work was supported by a Research Award from the California Institute for Quantitative Biosciences (QB3), the Bridging the Gap Award from the Rogers Family Foundation, the UCSF/Sandler Foundation Program for Breakthrough Biomedical Research, a grant from BASF, and the NSF through the Faculty Early Career Development (CAREER) Program (DBI-1253293).

1. SU8 Master Fabrication
<ol>
	<li>Design the microfluidic structures for two layer fabrication using AutoCAD software and have the designs printed by a vendor on circuit board film with 10 &#181;m resolution. The details of device design are given in an attached reference11 and the channel geometries are shown in Figure 1. The layers should include alignment marks to help collocate features from each fabrication layer12.</li>
	<li>Place a pre-cleaned 3 inch diameter silicon wafer on a spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3035 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 2,000 rpm, providing a layer thickness of 50 &#181;m.</li>
	<li>Remove the wafer and bake on a 135 &#176;C hotplate for 30 min. Allow the wafer to cool to RT before moving on to the next step.</li>
	<li>Expose the coated wafer to the 1st layer mask (Figure 2A) under a collimated 190 mW, 365 nm LED for 90 sec. After exposure, place the wafer on a 135 &#176;C hotplate for 1 min, then cool to RT before proceeding to the next step.</li>
	<li>Place the wafer on the spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-2050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 1,375 rpm, resulting in a layer that provides an additional thickness of 135 &#181;m.</li>
	<li>Remove the wafer and bake on a 135 &#176;C hotplate for 30 min, then cool to RT before moving to the next step.</li>
	<li>Align the 2nd layer mask (Figure 2B) onto the geometry patterned in 1.3 and expose the coated wafer to a collimated 190 mW, 365 nm LED for 3 min. After exposure, place on a 135 &#176;C hotplate for 1 min, then cool to RT before proceeding to the next step.</li>
	<li>Develop the masks by immersing in a stirred bath of propylene glycol monomethyl ether acetate for 30 min. Wash the wafer in isopropanol and bake on a 135 &#176;C hotplate for 1 min. Place the developed master in a 100 mm Petri dish for PDMS molding.</li>
</ol>
2. PDMS Device Fabrication
<ol>
	<li>Prepare 10:1 PDMS by combining 50 g of silicone base with 5 g of curing agent in a plastic cup. Mix the contents with a rotary tool fitted with a stir stick. Degas the mixture inside a desiccator for 30 min, or until all air bubbles are removed.</li>
	<li>Pour the PDMS to give a thickness of 3 mm over the master and place back into the desiccator for further degassing. Once all bubbles are removed, bake the device at 60 &#176;C for 2 hr.</li>
	<li>Cut the device from the mold using a scalpel and place on a clean surface with the patterned side up. Cut the PDMS mold in half with a razor blade to separate Master 1 from Master 2 (Figure 3a). On the piece containing the 50 &#181;m fluid handling geometry imprinted by Master 1, punch the fluidic inlets and outlets with a 0.75 mm biopsy punch.</li>
	<li>Plasma treat the devices at 1 mbar O2 plasma for 60 sec in a 300 W plasma cleaner. Wet the surface of the unpunched piece of PDMS with a drop of DI water to temporarily retard PdMS-PDMS bonding and serve as a lubricant. While viewing through a stereo microscope, place Master 1 on Master 2 surface and slide the surfaces relatively until a mechanical lock is achieved when the recessed frames and protruding frames in Figure 3A mate.</li>
	<li>Place the device in a 60 &#176;C oven and bake the assembled device (Figure 3B) for two days at 60 &#176;C to evaporate the water and complete bonding.</li>
</ol>
3. Preparation of Reagents
<ol>
	<li>Fill 1 ml syringe with distilled water for the inner phase.</li>
	<li>Fill 1 ml syringe with HFE 7500 fluorinated oil with 1 wt. % biocompatible surfactant surfactant13 for the middle phase.</li>
	<li>Fill 10 ml syringe with 10 wt. % polyethylene glycol (PEG) in water solution containing 1 wt. % Tween 20 and 1 wt. % sodium dodecyl sulfate for the continuous phase.</li>
</ol>
4. System Preparation
<ol>
	<li>Place the microfluidic chip on the stage of an inverted microscope coupled with a digital camera capable of &#60; 100 &#181;sec shutter speeds.</li>
	<li>Mount all syringes on syringe pumps and attach 27 G needles. Attach ~ 30 cm lengths of PE-2 tubing on the needles and insert the loose ends into the appropriate punched holes in the device.</li>
	<li>Insert a 10 cm length of PE-2 into the exit port of the device and place the other end in a waste collection container.</li>
	<li>Prime the device by running the syringe pumps at high rates of speeds (2,000 &#181;l/min) until fluid in the tubing segments reaches the inlet ports of the device.</li>
</ol>
5. Emulsion Generation
<ol>
	<li>Focus the microscope on a region that contains the 50 &#181;m x 50 &#181;m orifice and the downstream exit channel.</li>
	<li>Set the syringe pumps to deliver fluid to the double emulsion generator at flow rates of 250 &#181;l/hr for the inner phase, 100 &#181;l/hr for the middle phase, and 700 &#181;l/hr for the continuous phase and wait 10 min for equilibration.</li>
	<li>Maintain the flow rates of the inner and middle phases at 250 &#181;l/hr and 100 &#181;l/hr, respectively. Set the flow rate of the outer phase at 1,050 &#181;l/hr. Wait 3-5 min for the double emulsions generation to stabilize under this set of flow conditions.</li>
	<li>Acquire 5 sec of video images at 30 Hz for offline processing via manual image analysis.</li>
	<li>Repeat 5.3 and 5.4 with the flow rates given in Table 1. The inner and middle phase flow rates are held constant and the carrier phase flow rate is varied by adjusting the setting of the syringe pump.</li>
</ol>

<ol>
	<li>Van Der Graaf, S., Schro&#235;n, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review.">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li><li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application:+comparison+with+simple+emulsions.">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li><li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-emulsion+drops+with+ultra-thin+shells+for+capsule+templates.">Double-emulsion drops with ultra-thin shells for capsule templates.</a> Lab Chip. 11 (18), 3162-3166 (2011).</li><li>Lim, S. W., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh-throughput+sorting+of+microfluidic+drops+with+flow+cytometry.">Ultrahigh-throughput sorting of microfluidic drops with flow cytometry.</a> Lab Chip. 13 (23), 4563-4572 (2013).</li><li>Bernath, K., Hai, M., Mastrobattista, E., Griffiths, A. D., Magdassi, S., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+compartmentalization+by+double+emulsions:+sorting+and+gene+enrichment+by+fluorescence+activated+cell+sorting.">In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.</a> Anal. Biochem. 325 (1), 151-157 (2004).</li><li>Seemann, R., Brinkmann, M., Pfohl, T., Herminghaus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+based+microfluidics.">Droplet based microfluidics.</a> Rep. Prog. Phys. 75 (1), 016601 (2012).</li><li>Bauer, W. A. C., Fischlechner, M., Abell, C., Huck, W. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+PDMS+microchannels+for+high-throughput+formation+of+oil-in-water+microdroplets+and+water-in-oil-in-water+double+emulsions.">Hydrophilic PDMS microchannels for high-throughput formation of oil-in-water microdroplets and water-in-oil-in-water double emulsions.</a> Lab Chip. 10 (14), 1814-1819 (2010).</li><li>Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., Stone, H. A., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monodisperse+double+emulsions+generated+from+a+microcapillary+device.">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308 (5721), 537-541 (2005).</li><li>Chang, F. C., Su, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+double+emulsification+utilizing+3D+PDMS+microchannels.">Controlled double emulsification utilizing 3D PDMS microchannels.</a> J. Micromech. Microeng. 18 (6), 065018 (2008).</li><li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device.">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802-807 (2012).</li><li>Tran, T. M., Cater, S., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coaxial+flow+focusing+in+poly(dimethylsiloxane)+microfluidic+devices.">Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices.</a> Biomicrofluidics. 8 (1), 016502 (2014).</li><li>. Lithography Available from: <a target="_blank" href="https://www.memsnet.org/mems/processes/lithography.html">https://www.memsnet.org/mems/processes/lithography.html</a> (2015)</li><li>O'Donovan, B., Eastburn, D. J., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrode-free+picoinjection+of+microfluidic+drops.">Electrode-free picoinjection of microfluidic drops.</a> Lab Chip. 12 (20), 4029-4032 (2012).</li><li>Chang, F. C., Lin, H. H., Su, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+W/O/W+double+emulsification+in+3-D+PDMS+micro-channels.">Controlled W/O/W double emulsification in 3-D PDMS micro-channels.</a> , 792-795 (2008).</li><li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device.">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802 (2012).</li></ol>

The authors have nothing to disclose.

The double emulsion generating geometry described here is designed to mimic the physics of glass capillary devices8. In these, aligned cylindrical glass capillaries are used to create a three phase coaxial jet that is sheared into uniform double emulsion droplets. The function of our 3D PDMS device is dependent on the central alignment of small features formed with 50 &#181;m tall fabrication with carrier phase channels that are 320 &#181;m in total height. There is a significant potential for to misaligning the taller features patterned by the 2nd layer mask in step 1.7 with respect to the 50 &#181;m tall geometry if masks are not accurately aligned. Proper alignment can be aided by designing alignment marks, such as concentric circles into masks to be co-located during photo patterning. The plasma bonding of the two PDMS halves of the device is a second process that can lead to significant misalignments of the final device. Plasma bonding of PDMS to PDMS is generally instantaneous, so in step 2.4 we describe the wetting of a device surface with DI water to retard bonding and allow manipulation so that the alignment frames shown in Figure 3A can be allowed to lock. If this is attempted without sufficient wetting, the PDMS surfaces will irreversibility bond before coming into proper alignment, and device must be disposed of and new PDMS molds made.
The double emulsion device is designed to take advantage of the fabrication techniques that that lead to uniformly hydrophobic surface properties. However, operation outside of the parameters described in the protocol requires some understanding of the required fluidic processes. A the junction of the inner and middle phases (Figure 1D, point &#34;1&#34;), a relatively high flow of the inner phase and low flow of the middle phase create a two phase jet, with the hydrophobic middle phase coating the channel walls. If the proportionate flow of the middle phase is increased, the generation of discrete water-in-oil droplets will begin to occur, eliminating the ability to form a coherent three-phase jet for drop formation in the orifice (Figure 1D, point &#34;3&#34;). After the channel expansion (Figure 1D, point &#34;2&#34;), a significant amount of carrier phase flow is required to create geometric separation between the middle phase and the hydrophobic channel walls. Reductions in carrier phase flow will eventually lead to the middle phase wetting the hydrophobic device walls. Significant reductions in the carrier phase flow may create flow conditions that are insufficient to shear the inner phases into a long, thin filament, thereby radically altering the physics of double emulsion droplet formation.
Once built, this device is designed to produce double emulsions from 14 to 50 &#181;m, a convenient size for sorting using commercial FACS instruments. If double emulsions outside this size range are desired, the orifice dimensions need to be scaled from the 50 &#181;m x 50 &#181;m size used here. Because the device is designed to produce water / oil / water double emulsions with uniformly hydrophobic surface properties, oil / water / oil double emulsions cannot created unless there were a surface treatment applied to make the device uniformly hydrophilic.
This work demonstrates an easy to fabricate PDMS device capable of the robust formation of water / oil / water double emulsions. Although previous investigators have reported the formation of double emulsions in devices created using 3D lithography14,15, the double emulsions formed in their devices had diameters that were measured in 100s of &#181;m. The device reported here is suited to producing double emulsions an order of magnitude smaller than this, providing volumes similar to mammalian cells and well suited to sorting by FACS.
Although these results may also be attained using glass capillary microfluidics, fabricating glass devices is laborious and requires many hands-on steps per device. For our all PDMS device, fabrication largely consists of molding, bonding, and baking PDMS slabs, processes that are simple, repeatable, and easy to scale to large numbers.
The utility of a lithographically fabricated device to generate double emulsions using co-axial flow focusing has been demonstrated. We hope that the straightforward fabrication and robust functionality of this double emulsion generator design should lead to its adaptation for scientific and industrial applications. Investigators previously deterred by the specialized skills required to work in glass capillary microfluidics, should be more comfortable using PDMS soft lithography, now a common laboratory technique. Furthermore, the small droplet sizes that can be produced is well suited to perform cell and biological assays in droplets, and quantification and sorting using FACS. For industrial applications, it has already been shown that these types of devices can be fabricated into arrays and parallelized10, enabling double emulsion generation rates to increase by orders of magnitudes as compared to single devices. In addition, the ability to form small double emulsions in large coaxial flow focusing channels should make the device resistant to fouling and clogging, which is critical when parallelizing the devices intended to run for long durations without intervention.

Double emulsions consist of droplets separated from a carrier phase by an intermediate, immiscible fluid layer, and are of particular interest due to their potential uses in industrial, pharmaceutical, and biological applications1. In some cases, the ability to encapsulate high value compounds in a double emulsion&#39;s core enables material to be protected and released in a controlled manner. For example, drugs may be encapsulated under solubility conditions not appropriate for the external carrier fluid2. Additionally, the intermediate oil layer can be used as a capsule template for the encapsulation and delivery of drugs, cosmetics, and nutrients3. In biology, double emulsions are also useful in high throughput screening because they allow a massive number of sub-nanoliter experiments to be carried out, then detected and sorted using a fluorescence-activated cell sorting (FACS) instrument4,5.
The design of double emulsions with desired performance characteristics requires the precise control of double emulsion size, composition, and uniformity. Although bulk emulsification processes, such as membrane emulsification, are used in industry, the resulting emulsions are highly polydisperse, exhibiting a wide variety of functional properties1. The field of droplet microfluidics is naturally suited the generation of monodisperse emulsions with carefully controlled composition6. Microfluidic double emulsion generation has been achieved with two main strategies, sequential drop making and glass capillary flow focusing. Double emulsions can be generated in planar PDMS devices using a two-step drop making process. First, aqueous-in-oil emulsions are created using a water-in-oil drop-making region of a device with hydrophobic channel walls. Next, the emulsion can be flowed or reinjected into a drop-making region with hydrophilic walls suited for oil in water drop-making4. However, hydrophilic surface treatment of PMDS requires an additional fabrication step and is often temporary7. The most controllable and repeatable method to form double emulsions is by co-axial flow focusing, a technique pioneered using glass capillary microfluidics, whereby a concentric jet containing the three phases is sheared through a small orifice to produce monodisperse droplets8. This technique allows for the production of droplets much smaller than the channel dimensions, with the precise size and composition of the double emulsion being a function of the flow rates of each phase. The large difference between droplet and channel size and the protective outer sheath flow prevents droplets from contacting the channel walls, rendering surface treatment unnecessary. However, such glass devices require custom fabrication of tapered capillary tips, along with careful assembly and sealing. Previous investigators have used 3D soft lithography to generate double emulsions using flow focusing physics, but these devices produced emulsions with diameters &#62; 150 &#181;m 9,10, roughly an order of magnitude larger than objects typically sorted with FACS. An attractive alternative would include the robust functionality and small droplet generation of glass capillary coaxial flow focusing with the ease of manufacture of PDMS soft lithography.
In this paper, we describe a double emulsion generator that uses co-axial flow focusing to produce &#8804; 50 &#181;m emulsions and is constructed entirely using 3D soft lithography11. Our device uses a clamshell approach to fabricate devices that includes a small shearing channel (Figure 1) to approximate the emulsion formation processes in a pulled glass capillary nozzle. More importantly, these devices require no specific surface treatment, and the all polymer construction provides easy and repeatable fabrication scalable to a large number of duplicate devices. Here, we outline the design, fabrication, and testing of the double emulsion generator. Double emulsion generation is shown to be robust and repeatable down to droplet diameters of 14 &#181;m. The coupling of functionality with ease of fabrication makes this device an appealing option for development of new double emulsion applications.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Photomasks</td>
			<td style="width:135px;">CadArt Servcies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:135px;">University Wafers</td>
			<td style="width:119px;">447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 3035</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y311074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 2050</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Krayden</td>
			<td style="width:119px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
			<td style="width:119px;">B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">P1369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
	</tbody>
</table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

The double emulsion generator consists of a co-axial flow focusing device created using 3D PDMS fabrication (Figure 1A). The geometry enables that formation of a three-phase co-axial jet to be sheared into a square, 50 &#181;m x 50 &#181;m orifice, allowing the formation of water / oil / water double emulsions (Figure 1B, Figure 1C). The inner aqueous phase and the middle oil phase are brought together at a junction with channel dimensions of 10 &#181;m x 50 &#181;m (Figure 1D, point &#34;1&#34;). Due to the hydrophobicity of PDMS, the fluorinated oil hugs the channel walls and the inner phase stays in the center of the channel as the fluids travel in a continuous jet, until a sudden expansion channel expansion is reached (Figure 1D, point &#34;2&#34;). At this location, the inner two phases are injected into the center of a 320 &#181;m tall junction that allows the relatively concentric introduction of the aqueous carrier phase. The three phases are forced into a 50 &#181;m x 50 &#181;m orifice (Figure 1D, point &#34;3&#34;), whereby the high flow rate of the carrier phase shears the inner two phases into a long, thin tendril that decomposes to from uniform droplets (Figure 1E).
The 3D PDMS fabrication requires the coupling of two unique PDMS molds in a clamshell configuration after molding on two layer lithographic masters. A 50 &#181;m tall layer is used to form the inner and middle fluid handling channels, along with the shearing orifice on Master 1 (Figure 2A), along with a complimentary protruding and recessed frames on opposing master. An additional 135 &#181;m tall layer is used to create the carrier fluid and the exit channels (Figure 2B). Assembly of the double emulsion generator utilizes the recessed and protruding frames (Figure 3A) for geometric alignment after plasma treatment (Figure 3B).
The double emulsion device was tested at a variety of flow conditions to demonstrate the formation of varied size, monodisperse double emulsions. For these experiments, the inner and middle phase flow rates were held constant and the carrier phase flow rate was modified to affect the shearing force during droplet generation. Experimental conditions are parameterized by the ratio of the carrier phase flow (Qc) to the sum of the inner two phases flows (Qsum). Images of droplet generation for experiments performed at Qc/Qsum from 3 to 57 are shown in Figure 4. An elongated region containing the inner two phases is observed to protrude into the 50 &#181;m x 50 &#181;m orifice and breaks into droplets that are convected downstream. Increasing the flow of the carrier phase (increasing Qc/Qsum) leads to the inner phases being sheared into progressively thinner regions that produce smaller droplets. Double emulsions produced by the device at different flow rates show an average diameter coefficient of variation of 5.2%. Histograms of droplet diameters for select values of Qc/Qsum also show the relative uniformity in the size of the droplets generated (Figure 5). The device demonstrates an ability to form double emulsions significantly smaller the orifice width, and shows a clear decreasing trend with increased Qc/Qsum (Figure 6). At highest carrier phase flow tested, 14 &#181;m double emulsions were formed using the 50 &#181;m x 50 &#181;m orifice.
<img alt="Figure 1" src="/files/ftp_upload/53516/53516fig1.jpg" /> 
Figure 1. Geometry of the double emulsion generator. (A) 3D model of the fabricated device. (B) Vertical cross section of the central channel showing introduction of the inner (gray), middle (red), and carrier (blue) phases. (C) Cross-section showing the jet containing the inner two phases entering the square orifice. (D) Top view of emulsion generation in the device. At junction (1) the injection of the hydrophobic middle phase is aided by the hydrophilic PDMS, which causes it to coat the channel walls. At junction (2) the channel expands and a jet of the inner two phases is sheared into the orifice (3) by the higher rate of flow of the continuous fluid to a point where the physics cause droplet formation. (E) A microscope image of double emulsion generation in the device. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53516/53516fig2.jpg" /> 
Figure 2. Lithographic production of the masters. (A) The mask used for preparation of 50 &#181;m features. Master 1 is used to mold the fluidic inlets, the inner / middle phase junction, the emulsion generation orifice, and a recessed trough for alignment. Master 2 contains a raised ridge used for alignment. (B) The mask used for preparation of 135 &#181;m features. The masters are mirror images that contain the carrier fluid routing channels and the exit channel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53516/53516fig3.jpg" /> 
Figure 3. Assembly of the PDMS device. (A) Fabricated PDMS slabs contain complimentary recessed and protruding frames. (B) Assembled, the frames interlock to provide optimum alignment of features. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53516/53516fig4.jpg" /> 
Figure 4. Images of generated double emulsions at different flow rates. The flow rate of the outer phase is changed to alter Qc/Qsum, which is given to the left of each image. Increasing Qc/Qsum narrows the jet of the inner fluids being sheared through the orifice, creating increasingly small droplets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53516/53516fig5.jpg" /> 
Figure 5. Histograms of double emulsion droplets sizes at different flow rates. The average coefficient of variation of the diameter of emulsion droplets produced at a given set of flow conditions is 5.2%. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53516/53516fig6.jpg" /> 
Figure 6. Droplet diameter versus normalized flow rate parameter. Adjusting the flow rate of the continuous phase allows production of double emulsions that are 30%-100% of the orifice diameter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Qi [&#956;l/hr]</td>
			<td>Qm [&#956;l/hr]</td>
			<td>Qsum [&#956;l/hr]</td>
			<td>Qc [&#956;l/hr]</td>
			<td>Qc<fon> / Qsum</fon></td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>1050</td>
			<td>3</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>2100</td>
			<td>6</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>3850</td>
			<td>11</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>5950</td>
			<td>17</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>8050</td>
			<td>23</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>10150</td>
			<td>29</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>11900</td>
			<td>34</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>17150</td>
			<td>49</td>
		</tr>
		<tr>
			<td>100</td>
			<td>250</td>
			<td>350</td>
			<td>19950</td>
			<td>57</td>
		</tr>
	</tbody>
</table>
Table 1. Flow rate parameters used for the experiments. The inner phase and middle phase flow rates (Qi, Qm) are held constant, giving a constant combined flow rate (Qsum). The carrier phase flow rate (Qc) is varied to produce double emulsions with different diameters. The ratio Qc/Qsum is the main nondimensional parameter describing experimental conditions.

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

double-emulsion-generation-using-polydimethylsiloxane-pdms-co-axial

Nanyang Technological University

Research

JoVE Journal

Bioengineering

16.0K Views.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

Video: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

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

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion co-flow device to generate uniform high-viscosity droplets in a low-viscosity fluid. The microfluidic capillary device has a common co-flow structure with its exit connecting to a wider tube. Elongated droplets of the low-viscosity fluid are first encapsulated by the high-viscosity fluid in the co-flow structure. As the elongated low-viscosity droplets flow through the exit, which is treated to be wetted by the low-viscosity fluid, phase inversion is then induced by the adhesion of the low viscosity droplets to the tip of the exit, which results in the subsequent inverse encapsulation of the high-viscosity fluid. The size of the resultant high-viscosity droplets can be adjusted by changing the flow rate ratio of the low-viscosity fluid to the high-viscosity fluid. We demonstrate several typical examples of the generation of high-viscosity droplets with a viscosity up to 11.9 Pas, such as glycerol, honey, starch, and polymer solution. The method provides a simple and straightforward approach to generate monodisperse high-viscosity droplets, which may be used in a variety of droplet-based applications, such as materials synthesis, drug delivery, cell assay, bioengineering, and food engineering.

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

Chemistry

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

Engineering

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Glass-Based Devices to Generate Drops and Emulsions

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A&#160;voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

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 ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...