OLA is a versatile microfluidic platform useful to the synthetic biology community. in particular, to bioengineer artificial cells. OLA-based liposomes can help us understand the self-organization principles in biology and construct cell-mimicking assemblies.
OLA produces monodispersed, unilaminar, cell-size liposomes in a high-throughput manner and with an excellent encapsulation efficiency. It requires very small sample volumes, in the order of 50 microliters, and the formed liposomes are immediately available for further on-chip experimentation. OLA is useful for studying biological reactions within microconfinements, vesicle dynamics, and biomolecular condensates and their interactions with membranes.
OLA has also been applied as a high-throughput platform for drug screening, for example, to test the permeability and membranic activity of antimicrobials. Surface functionalization, which is PV treatment, is a critical step for the protocol and needs to be carefully performed to prevent the PV solution for entering the inner aquea and lipid-carrying organic channels. Also, the post-production channel should be just long enough for the separation of octanol pockets to form liposomes.
Ketan Ganar, a PhD student from my laboratory, will help Chang Chen demonstrate the procedure. To begin, take a four-inch-diameter clean silicon wafer and clean it further using pressurized air to remove any dust particles. Mount the wafer on a spin coater and gently dispense around five milliliters of a negative photoresist in the center of the wafer.
To obtain a 10-micrometer-thick photoresist layer, set the spin coat settings at 500 RPM for 30 seconds with an acceleration of 100 RRP per second for initial spreading, followed by a 60-second spin at 3, 000 RPM with an acceleration of 500 RPM per second. Then, spin coat the wafer. Bake the wafer on a heating plate for two minutes at 65 degrees Celsius, and then for five minutes at 95 degree Celsius.
Once the wafer cools down, mount the wafer in the printing chamber of the direct right optical lithography machine and feed the octanol-assisted liposome assembly or OLA design, into the software. Once the design is printed, bake the wafer at 65 degrees Celsius for one minute, followed by 95 degrees Celsius for three minutes. To wash off the uncured photoresist, dip the wafer in a glass beaker containing the developer solution until the uncured photoresist is fully removed.
Hard-bake the wafer at 150 degrees Celsius for 30 minutes to ensure that the printed design is firmly attached to the wafer surface and does not come off in the downstream fabrication process. To prepare the microfluidic device, place the master wafer on a square piece of aluminum and wrap the aluminum foil around the wafer, forming a well-like structure. Gently pour the PDMS mixture on the master wafer.
Incubate the assembly in an oven at 70 degrees Celsius for at least two hours. Take the master wafer out of the oven and let it cool down. To remove the solidified polydimethylsiloxane, or PDMS block, remove the aluminum foil and then carefully peel off the PDMS block from the edge of the wafer.
Take a transparent glass cover slip, pour approximately 0.5 milliliters of PDMS onto the center of the glass slide, and spread it across the cover slip by gently tilting the glass slide, ensuring total coverage of the glass slide with the PDMS. Mount the glass slide on the spin coater. Ensure it is centrally placed so that the middle of the slide overlaps with the center of the pressure shaft.
Then spin the glass slide at 500 RPM for 15 seconds at an increment of 100 RPM per second and 1, 000 RPM for 30 seconds at an increment of 500 RPM per second. Place the PDM-coated glass slide with the coated side facing upward on a raised platform like a block of PDMS in a covered Petri dish. Bake it at 70 degrees Celsius for two hours.
Place the PDMS block with the engraved channels facing upward and the PDMs-coated glass slide with the coated side facing upward in the vacuum chamber of the plasma cleaner. Switch on the vacuum and expose the contents to air plasma at a radio frequency of 12 megahertz for 15 seconds to activate the surfaces. Oxygen plasma can be seen in the form of a pinkish hue.
Immediately after the plasma treatment, place the PDMS-coated glass slide on a clean surface with the PDMS side facing upward. Gently place the PDMS block with the microfluidic pattern now facing toward the PDMS-coated glass slide, allowing them to bond. Bake the bonded devices at 70 degrees Celsius for two hours.
Dispense 200 microliters of 5%polyvinyl alcohol, or OVA solution, into a 1.5-milliliter tube and connect it to the microfluidic reservoir stand. Insert the tubing such that one end is submerged in the PVA solution and the other end is connected to the inlet of the outer aqueous channel of the microfluidic device. Increase the pressure of the outer aqueous phase to 100 millibars to flow the PVA solution in the outer aqueous channels.
Increase the pressures of the inner aqueous and lipid-carrying organic phases to 120 millibars to prevent the backflow of the PVA solution inside these channels. Flow the PVA solution in this manner for approximately five minutes, ensuring complete functionalization of the exit channel. Increase the pressure in the lipid-carrying organic and inner aqueous channels to two bars to remove the PVA solution, and immediately detach the tubing from the outer aqueous inlet.
Simultaneously, use a tubing connected to a negative pressure channel to remove excess PVA from the exit channel. Bake the device at 120 degrees Celsius for 15 minutes and let it cool down before use. The device can be stored under ambient conditions for at least one month.
Dispense the solutions into three 1.5-milliliter tubes and assemble them. Connect them to the PVA-treated microfluidic chip and apply positive pressure on the three channels, around 100 millibars on the inner aqueous and lipid-carrying organic channels and around 200 millibars on the outer aqueous channel. Once all three phases start co-flowing at the junction, ensure double emulsion production begins and adjust the pressure according to its quality.
As the double emulsion droplets flow, the octanol all pockets become more and more prominent and finally get pinched off, forming liposomes. A bright-field image of the rapid generation of double emulsion droplets is shown here. The fluorescent lipid channel shows the formation of an octanol all pocket due to partial de-wetting.
The inner aqueous channel shows the encapsulation of yellow fluorescent protein. The dewetting of the octanol pocket in the exit channel which forms a liposome is shown in this figure. This image represents the schematic of the pH-dependent transitioning of the homogenous solution of polylysine and ATP encapsulated within the liposome to phase-separated polylysine ATP coacervates.
The initial acidic environment in the liposome renders the molecular charge of ATP to be neutral, inhibiting coacervation. When the pH inside the liposomes equilibrates with the externally applied pH increase, ATP gains a negative charge, triggering coacervation. The spatial distribution of polylysine and the membrane and the time lapse images of the formation of polylysine ATP coacervates within the liposomes are shown in this figure.
The external edition of a basic buffer raises the pH level inside the liposomes over the course of minutes and initiates coacervation. T equals zero minutes refers to the time just before the occurrence of the first coacervation event. While demonstrating procedure, it's crucial to patiently adjust the channel pressure in order to generate a steady double emulsion production.
OLA and its variations have been employed in diverse studies, for example, growth and division of liposomes understanding the dynamics of biomolecular condensates, cell-free expression of protein, as well as encapsulation of bacteria. So in contusion, we believe that OLA is a versatile platform for synthetic biology.
