The overall goal of this procedure is to generate phospholipid bilayer vesicles with increased control over membrane, unla, molarity, mono dispersity, and internal contents. Using cad, a chamber is manufactured from acrylic sheets and natural rubber. A suspended lipid bilayer is produced by loading the chamber with lipids and solution.
Next, an inkjet containing the solution to be encapsulated is set up with the jetting apparatus. Once the appropriate settings have been established, jetted fluid pulses will produce lipid bilayer vesicles. Ultimately, microfluidic jetting yields repeatable mono dispersed bilayer vesicles.
The main advantage of this technique over existing methods like electro formation, emulsion, or droplet generation, is increased level of control governing membrane, unilateral molarity, mono dispersity, and internal contents. Demonstrating the procedure will be Johanna Harrow, a graduate student from a laboratory and assisting will be Kara Patel, an undergraduate student Using CAD software, draw a shape for the chamber that can be made from acrylic using a laser cutter first drill, one 16th inch through holes on both sides of the assembled acrylic chamber. That will drain to the infinity shaped well.
Second, cut a small rectangle with scissors from a 0.2 millimeter acrylic sheet to serve as the bottom of the infinity shaped. Well secure this in place with quick drying adhesive, making sure not to get glue in the viewing areas. The acrylic sheet should be flushed to the chamber wall and secure to the bottom of the chamber.
Third, use quick drying adhesive to glue a thin piece of natural rubber sheet over the drilled holes. Press them in place with tweezers. Test all of the glued seams for leaks.
There shouldn't be any. Lastly, using a 23 gauge needle, poke holes into the rubber through the drilled holes, the piezoelectric inkjet tip will be inserted through these holes. Stock phospholipids should be dissolved in chloroform and stored at minus 20 degrees Celsius.
D-P-H-P-C or DOPC. Lipids are used in this demonstration. Lipids dissolved in chloroform are transferred to a glass vial while holding the vial at an angle, gently dry the lipids under argon or nitrogen gas.
Then place the vial with its cap slightly loose under vacuum for one to two hours to remove residual solvent. A desiccate can also be used for this step. Now solubilize the lipids in end decking for a final lipid concentration of 25 milligrams per milliliter.
Vortex the solution. Briefly seal the cap with param and then sonicate for 15 minutes. Store this solution at minus 20 degrees Celsius.
Next, prepare a stock sucrose solution with physiological like osmolarity. This is the jetting solution. Consider adding a drop of dark food dye or fluorescent beads to the solution for added contrast or fluorescence.
Load the sucrose solution into a one milliliter syringe to remove trapped air. Flick the syringe so any bubbles will rise to the lure opening and can be expelled. Next, attach a 33 millimeter 0.22 micron filter to the syringe and push out one droplet through the filter.
Now unscrew the female lure adapter of the inkjet and press it onto the end of the filter. Push out some fluid to remove any trapped air. Lastly, attach the syringe to the top of the inkjet fluid.
Should travel to the inkjet tip if the syringe has been correctly attached. First, mount the syringe assembly to the microscope stage and wire up the inkjet controller for this experiment. The stage and syringe need independent control with the stage and syringe both moving in three dimensions for visualization.
Set up a high speed camera capable of at least 1000 frames per second. Set it to use image-based auto triggering to initiate recordings. Now, secure the chamber to the stage with tape and carefully align the inkjet tip with the hole made in the rubber sheet on the side of the chamber.
First, adjust by eye and then make fine adjustments using the microscope. Once aligned, back out the syringe along one dimension to load the infinity shaped. Well then apply gentle pressure to the plunger, so a droplet forms on the nozzle.
This provides some initial back pressure. The jetting parameters need to be set. Next specific values for a trapezoidal bipolar wave are presented on screen applied voltage and the number of jet pulses per trigger control vesicle size.
Now load the chamber with 25 to 30 microliters of the lipid solution in end decade, covering the full surface of the infinity shaped. Well then to each, well add 25 microliters of glucose solution to the outermost edge pipetting slowly and smoothly, the glucose should not mix into the lipid solution. Within 10 minutes, a lipid bilayer will form at the center of the chamber.
Setting up the bilayer is the most critical step since the bilayer is unstable and can break depending on preparation. Be patient. It will work out once you practice filling the chamber a few times.
Now, carefully insert the inkjet tip back into the chamber when the tip is within 200 microns of the droplet interface. Bilayer, start the jetting process and record the data using the inkjet provided insight into the vesicle generation process. For instance, varying the distance between the inkjet nozzle and lipid bilayer affected the force that deforms the membrane.Close.
Proximity to the bilayer focused the jet stream and prevented the membrane from dispersing energy away from the point of vesicle generation. Also, the vortex travel increased with the voltage applied to the pizo electric actuator. Vesicle formation was visualized as a sequence of events following their formation.
Vesicle stability tended to vary with vesicle diameter, with smaller vesicles being more stable. After watching this video, you should have a good understanding of how to generate lipid violator vesicles using microfluidic jetting.
