Digital droplet PCR affords enhanced accuracy and sensitivity compared to quantitative PCR. This is a simple method for digital droplet PCR requiring minimal equipment and no expertise. Particle templated emulsification is a microfluidic-free method of emulsification that does not require any specialized equipment.
Additionally, emulsification occurs over a period of seconds and scales to container volume. For commercial beads, add 0.5 grams of dried polyacrylamide particles compatible with particle template emulsification, or PTE, to 30 milliliters of sterile water in a 50-milliliter conical tube, and mix well. Incubate at room temperature for 30 minutes.
For the microfluidic fabrication of beads, pour one milliliter of photoresist onto the center of a three-inch silicon wafer, and then spin it at 500 RPM for 30 seconds, followed by 1250 RPM for 30 seconds. Place the wafer onto a hotplate set to 95 degrees Celsius for 15 minutes to evaporate the solvent. Secure the photomask onto the silicon wafer with a cover glass slide, and expose the wafer under a collimated UV LED for 2.5 minutes.
Place the wafer on a hotplate set to 95 degrees Celsius for five minutes for post exposure baking. Develop the photoresist silicon wafer by immersing it in a bath of 100%PGMEA for up to 15 minutes. Rinse the wafer with fresh 100%PGMEA, followed by 100%isopropanol.
Air-dry the wafer, then dry it on a hotplate set to 95 degrees Celsius for one minute, and place it into a clean three-inch Petri dish. Mix the PDMS silicon base and curing reagent in a 10:1 ratio, Then degas the mixed PDMS using a desiccator under vacuum until no air bubbles are observable. Pour the degassed PDMS in the Petri dish, ensuring that the silicon wafer is completely submerged.
Degas the silicon wafer and PDMS, and then cure the PDMS by placing the silicon wafer into an oven set to 65 degrees Celsius for at least 60 minutes. Excise a block of PDMS containing the microfluidic features from the Petri dish, using a scalpel. Punch the inlets and the outlets into the PDMS block using a 0.75-millimeter biopsy punch.
Then remove any dust and particulates with the repetitive application and removal of packaging tape to the block surface. Clean a glass slide by rinsing it with 100%isopropanol, and subsequently air-drying the surface. Plasma treat both the glass slide and the PDMS with one millibar of oxygen plasma for one minute, using a plasma bonder.
Affix the PDMS to the glass slide by placing the plasma-treated PDMS with features facing down onto the glass slide, plasma-treated side facing up. Place the slide into an oven set to 65 degrees Celsius for at least 30 minutes, to complete the bonding. Treat all microfluidic channels with a fluorinated surface treatment to ensure surface hydrophobicity, then bake the device at 65 degrees Celsius for at least 10 minutes.
Load both polyacrylamide, or PAA, and hydrofluoroether, or HFE, solution-containing syringes into syringe pumps. Connect both syringes to the microfluidic device using polyethylene tubing. Run the drop generation device and collect one milliliter of the droplets in a 15-milliliter collection tube, then incubate for three hours at room temperature for polymerization.
After the incubation, remove the lower layer of oil by pipetting. Add one milliliter of 20%PFO in HFE oil to the 15-milliliter collection tube as a chemical demulsifier. After mixing, spin down the 15-milliliter collection tube at 2000 x g, for two minutes.
Remove the bottom layer by pipetting, and repeat the process one more time. Add two milliliters of 2%sorbitan monooleate in hexane to the 15-milliliter collection tube and vortex it. Spin the tube at 3000 x g for three minutes, and remove the supernatant by pipetting to remove the surfactant solution.
Repeat this twice. Add five milliliters of TEBST buffer and mix well, then spin down at 3000 x g for three minutes and remove the supernatant by pipetting. Repeat this three times, then resuspend in five milliliters of TEBST.
Prepare the polyacrylamide particles for particle templated emulsification by centrifuging at 6000 x g for one minute to pellet the particles. Then remove the supernatant by pipetting, and resuspend the pellet in sterile water. Repeat three times to ensure removal of any residual TEBST.
Prepare the disperse phase in a fresh 15-milliliter centrifuge tube, using a PCR master mix, the appropriate primers, and a fluorescein hydrolysis probe, as described in the text manuscript. Incubate at room temperature for five minutes under gentle agitation, using a tube rotator. Centrifuge the disperse phase at 6000 x g for one minute and remove the supernatant.
Add one microliter of Saccharomyces cerevisiae genomic DNA to the pellet, and mix thoroughly by pipetting or vigorous tapping. Add 200 microliters of 2%fluorosurfactant in HFE oil to the the tube, for emulsification. Dislodge the pellet by pipetting or tapping the tube, and then vortex at 3000 RPM for 30 seconds.
Allow for the emulsions to settle for one minute, then remove 100 microliters of the bottom oil phase, and replace this volume with fresh 2%fluorosurfactant in HFE oil. Gently invert the tube several times to mix. Repeat this three times or more, until small satellite droplets are removed.
After two to five minutes of settling, remove the bottom oil phase. Replace this volume with 5%fluorosurfactant in fluorocarbon oil. Pipette 100 microliters of sample into 200-microliter PCR tubes, and place the PCR tubes into a thermocycler.
Pipette the sample onto a counting slide for fluorescent imaging, and image the sample. Particle templated emulsification affords superb monodispersity. As the excess supernatant is removed stabilizing surfactant is added, and the sample is vortexed to generate the emulsion.
The resultant droplets consist of particle core and aqueous sample containing reagents and sample molecules or cells necessary for the reaction. Polydispersed droplets with templating particles indicate insufficient vortex. Another issue is generation of excessive satellites, which lower encapsulation efficiency.
Satellites should comprise no more than 10%of the total encapsulated sample volume. They can be cleared from the emulsion by washing with fresh oil. The utility of PTE can be demonstrated in digital PCR when droplets containing amplified targets become fluorescent, allowing direct quantitation of targets.
The fluorescent droplets yield few positives when the target is rare, and many when it is abundant. These results are repeatable using commercially available polyacrylamide particles, achieving accurate measurements over the same range. The removal of supernatant from the disperse phase is important for sample encapsulation.
Knowing the volume of the pellet and supernatant can help identify the interface between the two. When in doubt, err on the side of removing the supernatant.
