The overall goal of this procedure is to generate monodispersed water and oil microdroplets for biological studies using a capillary based centrifugal axi-symmetric co-flowing microfluidic device. This method can help produce monodispersed microdroplet for the biochemical field to advance the study of work in synthesis, single-molecule PCR, single encapsultation, and the simple modeling of living cells. The main advantages of this technique are that it is easy and adjustable.
It requires less than a microliter of sample solution and it can produce monodispersed microdroplets of the required size. Demonstrating the procedure will be Morita, a postdoc from my laboratory. The capillary based microfluidic assembly begins with preparing the capillary holder.
Begin with milling four discs from two millimeter thick polyacetyl plastic plates using a milling machine. The discs are used to form a column supported by rods and screws. Using M.2 screws, assemble the plates.
First, from discs one and two, assemble the bottom of the holder. Then, shorten the screws by cutting them in the threaded sections. Connect three of the four holes to secure the two discs making a 0.9 centimeter stack.
Similarly, assemble the top part of the holder from discs three and four. Use two of the holes in each plate to connect the plates. Make the stack 0.7 centimeters tall.
Now, use a long screw to join the two empty holes in the top part of the holder with the two empty holes in the bottom part of the holder. The long screw should put a gap between the holders that is about two to three millimeters and the head of the screw should overhang the completed holder by about 2.2 centimeters. Each experiment uses a new set of capillaries fabricated from two capillaries.
From a 90 millimeter capillary, make two inner capillaries using a glass cutter. Make two equally-sized sections. From a 150 millimeter capillary, make three inner capillary sections.
Next, using a glass puller, sharpen each capillary section. Set the puller weight to its maximum and set the heat level to 60-70 degrees for the outer capillary or 70-80 for the inner capillary. Then, carefully sharpen the glass.
Keep the length of the constricted parts of the capillaries within the advised limits. If the length of the tips is too long or short, adjust the heating element until the correct length is achieved. Now, prepare to cut the tips.
First, use some clear tape to secure a capillary to a microscope stage. Overhang the tip under the objective. Second, using a platinum wire covered by glass beads, cut the tip off in three steps.
First, put the capillary where the break should occur in contact with a cold bead. Second, heat the wire for one to two seconds. Then third, cut the tip off by cooling the platinum wire.
This operation should be performed using a foot pedal control to heat the wire. Now, using a microforge, adjust the diameters of the capillary orifices. For the two outer glass capillaries, make their orifices 60 microns.
For the three inner capillaries make orifices having diameters of five, 10 and 20 microns. To generate the microdroplets, first fill an outer glass capillary with an oil-containing surfactant such as hexadecane containing 2%sorbitin monooleate by weight. Load 10 microliters of the solution into the capillary.
Then, secure the loaded copillary to the bottom part of the holder. Next, using capillary action, load about 0.1 microliters of aqueous solution into an inner glass capillary. Position the loaded inner capillary in the upper part of the holder.
Now, insert the inner capillary across the holder and into the outer capillary. While observing the tip through a microscope, turn the long screw of the holder to slowly and carefully advance in the inner capillary into the outer capillary. Advance the inner capillary to where the outer capillary's diameter is between 100 and 150 microns.
Now, introduce 100 microliters of the same oil with surfactant into the bottom of a 1.5 milliliter microtube. This will serve as a microdoplet collection tube. Carefully position the holder and capillaries into the loaded microtube.
Make sure the outer capillary is not in contact with the solution in the microtube. Then, load the tube in to a tabletop swing-out type centrifuge. To generate the microdroplets, run the centrifuge at 1, 600 times gravity for one or two seconds.
Then, remove the capillaries and holder from the tube and take an aliquot of the water-oil microdroplets collected in the tube. Eject the microdroplets onto a glass slide and image them for analysis. Using this protocol, various water-oil microdroplets where made from hexadecane containing 2%sorbitin monooleate.
Droplets made using an inner pipette with an orifice diameter of five microns were measured to be 8.3 microns in diameter on average. Droplets made using an inner pipette with an orifice diameter of 10 microns were measured to be 12.7 microns in diameter on average. Droplets made using an inner pipette with a 20 microns orifice were about 17.9 microns in diameter.
Thus, this method produces monodispersed microdroplets that have a diameter close to the size of the inner capillary orifice. After watching this video, you should have a good understanding of how to produce and control the size of monodispersed water and oil microdroplets for quantitative chemical experiments. Once mastered, this technique can be done in five to 10 minutes, if it is performed properly.
