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>
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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 t...

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 dimension...

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

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Bioengineering

15.9K 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.