The overall goal of this experiment is to synthesize janus microhydrogels that are composed of the same base material and have an isotropic thermo-responsiveness, as well as organophilic and hydrophilic loading capability using the liquid-liquid phase separation of super-saturated N-isopropylacrylamide monomer. Masote can help answer key questions in the field of drop delivery systems, specifically for the development of advanced trocharia, which requires hydrophilic/organophilic fuel holding capability inside a single particle. The main advantage of this patho is to synthesize mono dispered janus microhydrogels with the clearly compartmentalized intomorphology and to modulate the size of the janus microhydrogels with the help of microphilic device.
Demonstrating the procedure will be done by Kyoung Duck Seo and Andrew Choi who are my PhD students. Use the patterned wafer obtained as described in the text protocol as the master mold for PDMS casting. Mix the PDMS prepolymer and a curing agent homogeneously at a weight ratio of 10 to one.
For example, use one gram of curing agent for 10 grams of PDMS prepolymer. Pour the PDMS prepolymer into the master mold and degas it for one hour in a vacuum chamber. Then, place the master mold with the PDMS prepolymer into an oven at 65 degrees Celsius for three hours.
Cut the cured PDMS into the size of a single chip using a sharp scalpel. Carefully peel off the cured PDMS replica from the master mold by hand. Punch inlet and outlet holes into one of the replicas using a hole puncher with a slightly smaller diameter than the outer diameter of the connecting tubing.
Then, apply air plasma treatment to the bonding area of each replica using a Corona treater. Drop five microliters of methanol onto the air plasma treated areas. Finally align two identical PDMS replicas to fabricate the HFMD by hand manipulation.
And check alignment via a microscope. Place the HFMD in an oven set to 65 degrees Celsius overnight to strengthen the bond between the two PDMS replicas. Dissolve NIPAAm monomer in de-ionized water at a weight to weight ratio of one to one using a vortex mixer.
For example, dissolve 10 grams of NIPAAm in 10 milliliters of de-ionized water. Allow the monomer solution to rest in a vertical position at room temperature for at least 15 minutes. When the interface separating the two phases becomes clear, carefully extract two milliliters of monomer solution from the N-rich and N-poor phases without disturbing this interface by using a pipette.
Next, add four milligrams of MBAAm to the extracted N-rich and N-poor monomer solutions as a crosslinker. Then, add four milligrams of the photoinitiator to the solutions to prepare core fluids one and two for the low crosslinker concentration sample. Finally, dissolve 10 weight percentage of oil surfactant into mineral oil to prepare the sheath fluid.
Load two milliliters of core fluids one and two and the sheath fluid into three separate three millileter syringes. Mount the syringes into the syringe pumps and connect each syringe to the appropriate fluid inlet of the HFMD using tubing. Use the tubing to connect the fluid outlet of the HFMD to a collection reservoir.
Set the syringe pumps to infuse core fluids one and two at flow rates of two microliters per minute. And to infuse the sheath fluid at 10 microliters per minute. Then, position the UV light source perpendicularly about one centimeter away from the collection reservoir.
Switch on the UV light source and visually monitor the continuous production of janus microhydrogels. Collect the fabricated janus microhydrogels into a conical tube and wash them using isopropyl alcohol. Then, centrifuge the conical tube to settle the microhydrogels.
Repeat the centrifugation step using de-ionized water with the water surfactant to remove the leftover isopropyl alcohol around the janus microhydrogels. Store completely washed janus microhydrogels in a 10 milliliter vial containing de-ionized water. Use a pipette to place the synthesized janus microhydrogels into a 24 well plate.
Allow the hydrogels to settle for 15 seconds until a mono layer is formed at the bottom surface of the well. Obtain an image of the janus microhydrogel at 24 degrees Celsius using an upright optical microscope with a 5x objective lens. Set a thermoelectric module under the well plate and control the voltage of this module to increase the temperature of the solution containing janus microhydrogels to 32 degrees Celsius.
Obtain an image of the janus microhydrogel at 32 degrees Celsius once more by using an upright optical microscope with a 5x objective lens. From the 25 images of different janus microhydrogels at 24 and 32 degrees Celsius, measure the radius of the polymerized phases pN-rich and pN-poor using image analysis software according to the manufacturer's instructions. Shown here is the schematic diagram of the HFMD for generating janus microdroplets.
The N-rich and N-poor solutions are precisely injected into the HFMD and broken up into microdroplets at the orifice by the sheath fluid of mineral oil. Janus microdroplets obtained through HFMD have different volume ratios of N-rich and N-poor solutions as shown here. The volume ratio of the N-rich and N-poor solution composing microhydrogels is controlled by altering the flow rate of each solution.
Here, schematic diagrams and optical micrographs of the janus microdroplets and microhydrogels in response to environmental and temperature changes are shown. To quantify thermo-responsiveness, the radius of the janus microdroplets and microhydrogels was measured and showed in anisotropic, thermo-responsive behavior which is caused by the differences in NIPAAm monomer concentration between pN-rich and pN-poor. The organophilic/hydrophilic loading capability of these microhydrogels is observed as fat-soluble dyes strongly prefer to dissolve in the N-rich solution.
While water soluble dyes prefer to dissolve in the N-poor solution. Janus microhydrogels with hydrophilic/organophilic dual loading capability inside a single particle are finally fabricated. Once mastered, this technique can be done in 40 minutes if it is prepared properly.
While attempting this procedure, it is important to remember to maintain the ambient condition, especially the temperature, because highly concentrated NIPAAm solution crystallizes below 25 degrees Celsius. After its development, this technique will pave the way for researchers in the field of drug delivery systems to develop functional microhydrogels that can control the drug releasing rates and encapsulate multi-drugs without cross-contamination. After watching this video you should have a good understanding of how to synthesize janus microhydrogels composed of base material poly NIPAAm.
