NEW YORK (GenomeWeb) ― Manipulating miniature droplets and subjecting them to high temperatures on an open digital microfluidic device leads to evaporation that can seriously disrupt concentration-dependent enzymatic reactions, like PCR.
Humidifying the device helps, as does enveloping droplets with oil, but the ability to have droplets open to the air would be a big advantage for some workflows.
In a recent proof-of-principle study in Lab on a Chip, a group at Sandia National Laboratories in Livermore, Calif., has described a "just-in-time" method for replenishment that uses pre-heated droplets.
Results from experiments with the replenishable chip were indistinguishable from bench-scale methods in three representative biochemical reactions ― RNA fragmentation, first strand cDNA synthesis, and PCR. The technique was stable over a range of temperatures and incubation times, obviating the need for oil, humidifying chambers, and off-chip heating. Meanwhile, the method's use of inexpensive disposable chips makes biofouling less of a concern.
In an interview this week, Steven Branda, a senior member of the technical staff at Sandia and corresponding author on the study, said that the new method would likely be used in a fieldable digital microfluidic device that purifies and preps samples for multiple analyses, such as PCR followed by sequencing. A prototype of Sandia's automated library prep and next-generation sequencing-enabling microfluidic technology was described last year in In Sequence.
The key to the new replenishment method was pre-heating the replenishment droplets. These are manipulated on a six-layer printed circuit board, or PCB, digital microfluidic device.
The method now furthers one of the group's aims ― adapting the PCB microfluidic device to become a hub for freestanding genomics platforms.
"It's very versatile, you can link together a lot of different kinds of reactions, and you can then siphon off pieces of those reactions and do diagnostics ... or you can send them down a separate workflow," Branda said.
"In our case, we're doing a lot of sample prep for high-throughput sequencing. That's a really complicated workflow, and we want to be able to start with a dirty sample ― usually blood ― and go all the way through to a library that you'd put on a sequencer," he said.
Branda cited a microfluidic device developed by Advanced Liquid Logic, a company acquired by Illumina last year, as an example of commercialization of this sort of technology.
PCB-based microfluidics use actuation electrodes buried under the surface of the chip to direct droplets, "almost like chess pieces on a chess board," Branda said. Application of voltage to a square will cause a droplet to shuttle over. Miniature reactions can be collided to mix them, or kept separate by adjusting the spacing of the electrodes. "When you bring in a replenishment droplet, you just have to make sure the path that it takes doesn't come into contact with any other paths that have droplets," Branda said.
"PCB is incredibly cheap ... you can do many different designs and print out lots of chips at very low cost. We're trying to do something that's fieldable; we want it disposable and we want the assays done at a buck or less," he said.
In the PCR world, commercially available digital PCR devices also use a microscale approach, but require oil to keep droplets separated.
"In some cases [oil] makes all the downstream processing sloppier; you have to deal with the oil in some way, you often have to use valves and gaskets to handle it," Branda said. Oil also extracts some reagents from reaction droplets, and is incompatible with others. "We've been focusing on air for simplicity, [but] also because in a lot of our bioassays we lose things to the oil," he said.
Digital microfluidics for PCR has things in common with droplet PCR as well as with other microfluidics lab-on-chip methodologies. At the moment, Branda said the PCB device runs traditional PCR in large droplets danced around the chip. For his group, the PCR step is not necessarily for detection, but rather for amplification prior to high-throughput sequencing.
Theoretically, a digital microfluidic device could be used for digital PCR — where nucleic acids are diluted to one per droplet and quantification comes from running Poisson statistics on positive droplets — but this would likely require retooling.
The just-in-time replenishment method currently needs a user to eyeball shrinking droplets. However, there is precedent for detecting concentration of salts or imaging fluorescent markers that could be added to future iterations of the device to trigger droplet rehydration, Branda suggested.
The Sandia lab continues to work with the US Department of Defense on microfluidic hub workflows, and the group has some IP on that aspect, Branda said. The group is also interested in cultivating commercial partners, particularly to develop a microfluidic diagnostics platform that can perform PCR as well as sequencing sample prep from whole blood.