The overall goal of this procedure is to design, fabricate, and utilize microfluidic chips capable of precisely and controllably injecting reagents into water in oil droplets, a process known as pico injection. This is achieved by first designing a device with an emulsion channel that narrows to a diameter smaller than that of the spherical droplets and spaces them evenly within inert carrier oil. As a second step, the water and oil droplets are introduced into the device and flow to the injection site.
A perpendicular channel containing the reagents to be injected. Next, a high voltage is applied to the injection fluid as droplets pass the injection site in order to cause the droplets and injection fluid to merge Results are obtained that show by tuning the magnitude of the applied voltage, the volume of injected reagent can be precisely controlled at picoliter resolutions. This technique was born outta the need to simplify the fabrication of our devices by eliminating the need for metal electrodes.
The advantages over existing techniques, so the ease and simplicity of device design, fabrication, and use The metal electrodes used in previous methods for prone to brake and sensitive to dust and debris in the channels during fabrication. By eliminating the electrodes, we've made our devices more robust and improved our fabrication efficiency. The first step is to design a microfluidic chip based on the needs of the experiment.
Use computer aided design software for this in order to make use of photo lithographic techniques later. This chip design has emulsion channels that are smaller than the spherical droplets to be used to allow for more effective pico injection. The pico injection site is based on a design found in the literature with the metal electrodes removed.
Additional channels serve as a Faraday moat to shield upstream droplets from the electric field. Once the design is completed, use soft photo lithographic techniques to fabricate the devices, then bond them to a glass microscope slide with the device ready for use. Prepare an air pressure control pump to pressurize a fluid containing reservoir.
Do this by modifying the pump output so that the pressurized air exits through a length of polyethylene tubing with an inner diameter of 2.7 millimeters. Terminate the tubing by fitting it over the nipple at the rear of a syringe tip. After sealing the space between the syringe and the tubing with epoxy, attach a 27.5 gauge needle using reagents appropriate to the experiment.
Prepare a uniform emulsion of aqueous droplets suspended in carrier oil. Load the emulsion into a one milliliter syringe equipped with a 27.5 gauge needle. Then secure the syringe in a syringe pump and orient it with a needle upward.
The next step is to prepare reagents for introduction into the microfluidic device and their reservoirs. Begin with a 15 milliliter centrifuge tube and cap. Use a biopsy punch or other convenient tool to punch three 0.5 millimeter holes into the cap.
Prepare a wire electrode with a diameter of 0.5 millimeters and a length of a few centimeters longer than the centrifuge tube. Insert this electrode into one hole in the cap. Make sure it reaches the bottom of the tube and extends a few centimeters beyond the top.
Next, prepare an approximately 20 centimeter length of polyethylene tubing. Insert this in a second hole. Again, it should reach the bottom of the tube in the remaining hole.
Thread about 2.5 centimeters of polyethylene tubing. This tube will rest above the fluid level. Apply UV curing epoxy to seal gaps on the top of the cap.
When the curing is complete, fill the tube with the desired pico injection fluid here, 100 millimolar sodium chloride and screw on the cap. Connect the output from the air pressure control pump to the shorter length of tubing in the cap. By inserting the needle into the lumen, the needle should fit snugly.
Place the centrifuge tube in its holder. Proceed by filling a one milliliter syringe with one molar sodium chloride. To serve as the Faraday moat fluid connect a 27.5 gauge needle and secure the syringe on a syringe pump.
Fill another one milliliter syringe with carrier spacer oil. Connect a 27.5 gauge needle and secure it in a syringe pump. At this point, connections can be made to the microfluidic chip.
First, ensure the chip is secured on a flat surface. Next, bring the open end of the long tube from the injection fluid container to the chip. Connect this tube to the inlet port of the pico injection fluid on the microfluidic chip.
Then attach polyethylene tubing to the syringe containing sodium chloride and extend it to the chip. Attach it to the inlet port for the Faraday moat. Also, use tubing to connect the syringe containing the carrier oil to its inlet port on the device.
Terminate a tube coming from the emulsion outlet port into a collection vessel. Finally, use tubing to connect the outlet port of the Faraday moat to a non conducting electrically isolated container. In the end, the microfluidic chip appears like this.
Now take a wire from the ground of a high voltage amplifier and extend it to the needle of the syringe with sodium chloride. Use an alligator clip to attach the wire to the needle. Extend another wire from the high voltage output of the amplifier to the electrode immersed in the pico injection fluid.
Use an alligator clip to make the connection as input to the amplifier. Use the computer output of a software controlled waveform generator. Begin infusion of the system by introducing the one molar sodium chloride faraday mote fluid to the device at 100 microliters per hour.
After that, start the droplet, emulsion and carrier oil to allow droplets to pass the pico injector at regular intervals, separated by a gap of carrier oil. Adjust the pressure applied to the pico injection fluid, such that the fluid at the pico injection orifice bulges into the droplet channel, but does not but off to form its own drops. Employ the waveform generator to produce a 10 kilohertz a C signal with an amplitude of zero to 10 volts to be amplified a thousand times by the high voltage amplifier.
To achieve pico injection. Apply this signal to the droplets as they pass the injection orifice and use the amplitude as a parameter. This movie shows the selective injection of droplets depending on the presence of a fluorescent dye.
In this instance, 200 drops per second passed by the injector, but rates of up to 10, 000 per second are possible with a right droplet detection mechanism. These figures show the injected volume fraction as a function of applied voltage for different molarity injection fluids. It is believed that higher voltages cause an earlier onset of coalescence leading to increased injection durations and volumes.
This two dimensional heat map shows the relationship between ionic solution molarity, and the voltage required to achieve injection volumes. Lower molarity solutions require higher voltages since they more readily reduce the electric field at the injection site. After watching this video, you should have a good understanding of how to quickly and robustly design and fabricate micro fluidic devices that allow for precise and controllable injection of reagents into your droplets.
Don't forget that working with high voltage can be extremely hazardous precautions, such as proper grounding, personal protective equipment, and built-in fail safes should always be employed.
