This method can help to answer the key questions in the microfluidic droplet formation field, including fabricating the semi-three dimensional flow focusing PDMS chip, and producing the pure PGD droplets. The main advantage of this tactic is that the semi-3D flow focusing device provides larger testing grounds for flow focusing mold. Pure PGD makes sure that the ratio of PGD particles is lower than 10%The implications of this technique can be extended to many biochemical implications due to the greater surface area to volume ratio.

Nozzle, large-scale applications will be benefit for this technique with consuming a few microliters of sample. Now this method can provide insight into PGD droplet formation. It can also be applied to other systems such as gas-equipped flow focusing analyzing.

Generally, individuals new to this method will struggle because of clogging in the orifice and the instability of tip streaming mold. Of this method is critical, as semi-3d flow focusing chip fabrication is difficult to learn because the alignment of top and bottom PDMS layers to fabricate semifluidic chip by hands is not reviewed. Design the two photo masks using standard techniques.

Use two separate layers for mask one and two in the same drawing file to ensure that all connections line up between the different channels. Mask one contains the dispersed phased inlet channel and an orifice, while mask two contains the continuous phase inlet channel, the filter, and the outlet. Use the two alignment marks and print the different layers independently to a chrome plate on glass.

Next, in a designated photolithography lab, place a clean, 3-inch diameter silicon wafer on a spin coater and turn on the vacuum to affix the wafer to the spin chuck. Spin coat two to three milliliters of SU-8 2025 negative photoresist onto the wafer for 10 seconds at 1, 000 rpm. Then change the speed to 3, 000 rpms for 30 seconds.

This will provide a layer thickness of 20 microns for the first layer. Soft bake the first layer by placing it on a 95 degrees celsius hot plate for six minutes. And then, remove the wafer and let it cool back to room temperature.

Expose the soft-baked wafer to mask number one under a collimated UV light for 18 seconds. Following UV exposure, post bake the wafer on a 95 degrees celsius hot plate for six minutes. Afterwards, allow the wafer to cool back to room temperature.

Repeat the spincoating process a second time using two to three milliliters of SU-8 2100 negative photoresist. Spin the wafer for 10 seconds at 1, 000 rpm, and then at 2, 000 rpms for 30 seconds. This provides a second layer thickness of 130 microns.

After soft baking the wafer on a 95 degrees celsius hot plate for 35 minutes, line up mask number two on the second layer photoresist, and expose it to UV light for 30 seconds. Then, post bake it on a 95 degree celsius hot plate for seven minutes. Once cooled back to room temperature, develop the wafer by immersing in a stirred bath of 50 milliliters of propylene glycol methyl ether acetate until features become clear on the wafer.

Then, wash the wafer with ethyl alcohol. Finally, place the wafer on the thermostatic platform and hard bake it at 95 degrees celsius for two hours. Mix a PDMS monomer and its curing agent for four minutes at a 10:1 ratio for the top layer and an 8:1 ratio for the bottom layer.

Using an automatic ointment agitator, place the silicon wafer into a 90 millimeter Petri dish, and then pour the 8:1 ratio PDMS mixture over the mold to a thickness of two to three millimeters. Then, degas the PDMS in a vacuum chamber until all the bubbles disappear. Place the Petri dish in an oven and cure the PDMS at 80 degrees celsius for one hour.

Once cooled back to room temperature, use a scalpel to cut the device at least three millimeters away from the features, and slowly peel the PDMS layer from the silicon wafer. Then, punch the dispersed phase inlet, the continuous phase inlet, and the outlet in the top PDMS layer using a 0.75 millimeter diameter punch. Clean the PDMS surface with adhesive tape to remove any dust particles.

Then, place both the top and bottom PDMS layers simultaneously into a plasma cleaner, and expose to plasma for two minutes at 300 watts. Immediately following plasma exposure, place the top layer on the bottom layer and slide the surfaces relatively until features are aligned viewing through a stereo microscope. Cure the device in an oven at 120 degrees celsius for one day to enhance its strength and complete the bonding.

To begin, prepare the continuous phase solution by dissolving 18%of a silicone-based non-ionic surfactant in hexadecane. Next, prepare the solution of the dispersed phase by starting with a hydrophilic peg diacrylate, one millimole per liter of a fluorescent dye, and adding five milligrams per milliliter of a photo initiator. Fill the one milliliter reservoirs of a pneumatic pressure controller with the continuous phase.

Then, fill a 200 microliter gel-loading tip with the dispersed phase. Place the semi-3D microfluidic device on the stage of an inverted optical microscope equipped with a high speed camera. Next, connect a fluorinated ethylene propylene tube to the punched hole of the continuous phase by first attaching it to a short, stainless steel tube, inserting the tube into the end of a gel loading tip, and placing the tip into the punched hole of the dispersed phase.

Then, insert a 20 cm long FEP tube into the outlet of the device, and place the end into a Petri dish. Focus the inverted microscope on a region that contains the two phase intersection, the orifice region, and the downstream channel. Then, set pressures of the two phases using a connected pneumatic pressure controller.

Set the dispersed phase at 15 millibar, and the continuous phase at 30 millibar. Wait three minutes for the pressure to stabilize, and stable fluid flow is reached, carrying no bubbles or PDMS residue. Next, set the pressure of the dispersed phase as the base level pressure of the system to 45 millibar.

Increase the pressure of the continuous phase until the emulsion breakup mode is changed from jetting to the tip streaming mode. Then, wait five minutes for it to stabilize. Once set up, place the tube end connecting the outlet hole to Petri dish, and collect the droplets.

Expose them to UV light where they will rapidly solidify into spheres. Because of the difference of depth of the dispersed phase channel, and the continuous phase channel, the dispersed phase flow is squeezed on all directions by the continuous phase flow. As a result, symmetric conical liquid tip forms to produce droplets continuously.

The size of droplets is changed by the pressure ratio of dispersed and continuous phases flow. Here, the pressure of the continuous phase is modified to affect the sheering force so that the droplet breakup processes change from the jetting mode to the tip streaming mode. The droplets are solidified by photopolymerization forming particles upon exiting the chip.

The droplets shown here were formed with different pressure ratios. The resulting droplet radius has an inverse relationship with the continuous phase to dispersed phase pressure ratio. This technique paves the way for researchers in the field of biological applications and provides a powerful tool for drug discovery, synthesis of biomolecules and the diagnostic testing.

After watching this video, you should have a good understanding of how to fabricate semi-three dimensional microfluidic chips, and how to produce a few micro PGD droplets. Don't forget that working with photoresist PGMEA has a can be extremely hazardous and precautions such as wearing disposable gloves and a mask should always be taken while performing this procedure.