The overall goal of this procedure is to build and test an all poly dimethyl suboxane microfluidic device that utilizes coaxial flow focusing to generate double emulsions. This method can help answer key questions in chemistry and biology that require ultra high throughput screening, such as the directed evolution of enzymes and the identification of rare phenotypes. The main advantage of this technique is that it enables the rapid production of double emulsion generators using soft lithography fabrication with no specialized surface treatments.
To Begin design the microfluidic structures for two layer fabrication. Using AutoCAD software, have the designs printed on circuit board film with 10 micron resolution as described in the reference shown here. Apply one to two milliliters of SU 8 30 35 in the center of a pre-cleaned three inch diameter silicon wafer place on a spin coder and affix it to the chuck.
Spin for 20 seconds at 500 RPMs followed by 30 seconds at 2000 RPMs. Remove the wafer and bake it on a 135 degree Celsius hot plate for 30 minutes. Allow the wafer to cool to room temperature before moving on to the next step.
Now expose the coated wafer to the mask for the first layer under a collated LED light for 90 seconds. After exposure, place the wafer back onto the 135 degrees Celsius hot plate for one minute. Once cooled, apply one to two milliliters of SUH 2050 in the center of the wafer.
Place the wafer back onto the spin coder and spin for 20 seconds at 500 RPMs. Then spin for 30 seconds at 1, 375 RPMs. When the spin coder stops spinning, remove the wafer and place it back onto the 135 degrees Celsius hot plate for 30 minutes.
Then cool the wafer back down to room temperature before moving to the next step. Align the mask for the second layer onto the geometry that was patterned in the previous steps, and expose the coated wafer to the UV light once again for three minutes after exposure, place the wafer onto the 135 degrees Celsius hot plate for one minute. Once cooled, developed the masks by immersing the wafer into a stirred bath of propylene glycol, monomethyl ether acetate.
After 30 minutes in the bath, remove the wafer and wash it in isopropanol. Next, bake the wafer on the 135 degrees Celsius hot plate for one minute. Cool the wafer, and then place the developed master into a 100 millimeter Petri dish.
Prepare a batch of PDMS by combining 50 grams of silicone base with five grams of curing agent in a plastic cup. Use a rotary tool fitted with a stir stick to mix the components and then degas the mixture inside a desiccate for about 30 minutes or until all the air bubbles are removed. Next, pour the PDMS over the master to a thickness of three millimeters.
Then place the Petri dish into the desiccate for further degassing. Once all bubbles are removed, bake the device at 60 degrees Celsius for two hours. Cut the PDMS device from the mold using a scalpel and place it onto a clean surface with the pattern side facing up.
Then cut the PDMS in half with a razor blade to separate the two sides of the device on the piece containing the 50 micrometer fluid handling geometry imprinted by master one. Punch the fluidic inlets and outlets with a 0.75 millimeter biopsy punch. Next, place the PDMS pieces with their patterned sides up into a plasma chamber plasma.
Treat the device with oxygen plasma at one millibar for 60 seconds in a 300 watt plasma cleaner. Then remove the samples and wet the surface of the unquenched piece of PDMS with a drop of DI water to serve as a temporary lubricant while viewing the device through a stereo microscope. Place the plasma treated surfaces together and slide the surfaces until a mechanical lock is achieved.
When the recessed frames and protruding frames mate, once assembled, place the device in a 60 degree Celsius oven and bake it for two days to evaporate the water and complete the bonding. Then affix the assembled device to a glass slide using several drops of uncured PDMS, bake the construct in a 60 degree Celsius oven for one hour. To cure the PDMS place, the microfluidic chip on the stage of an inverted microscope coupled with a digital camera that is capable of shutter speeds of at least 100 microseconds.
Next, mount three 10 milliliter syringes onto syringe pumps and attach 27 gauge needles to each of them. Attach about 30 centimeters of PE two tubing to each of the needles and insert the loose ends of the tubes into the appropriate punched holes in the device. Then insert a 10 centimeter length of PE two into the exit port of the device and place the other end in a waste collection container.
Once set up, prime the device by running the syringe pumps at high rates of speed. Stop the pumps. Once the fluid in the tubing segments reaches the inlet ports of the device, focus the microscope on the region of the chip that contains a 50 by 50 micrometer orifice and adjust the frame so it includes the downstream exit channel.
Set the syringe pumps to deliver the fluid to the double emulsion generator at a flow rate of 250 microliters per hour for the inner phase 100 microliters per hour for the middle phase and 700 microliters per hour. For the continuous phase. Wait 10 minutes for the system to equilibrate.
Next, increase the flow rate of the outer phase to 1050 microliters per hour. Wait an additional three to five minutes for the double emulsions generation to stabilize under this set of flow conditions. Once stabilized, acquire five seconds of video images at 30 hertz.
Save the videos for offline processing via manual image analysis. Repeat this process while varying the flow rate of the outer phase using the values found in table one of the accompanying text protocol. Maintain the rates of the inner and middle phases.
During this process, the double emulsion device was tested at a variety of flow conditions. To demonstrate the formation of varied size mono dispersed double emulsions, the ratio of the continuous phase to the sum of the inner and middle phase is shown above each image. Histograms of droplet diameters for select flow ratios show the relative uniformity in the size of the droplets generated.
The resulting double emulsions produce an average diameter coefficient variation of 5.2%The device demonstrates an ability to form double emulsions significantly smaller than the orifice width, and shows a clear decreasing trend with increased flow ratios. At the highest carrier phase flow tested 14 micrometer double emulsions were formed using the 50 by 50 micrometer orifice After its development. This technique paves the way for chemical and biological investigators to perform droplet microfluidics experiments that are compatible with standard flow cytometry detection platforms.
After watching this video, you should have a good understanding of how to fabricate and test microfluidic, double emulsion generators that use simple all PDMS construction techniques.
