(395f) AC Droplet Digital PCR

We have developed an AC electrospray droplet generation technology that generates tunable mono-dispersed micron-size water droplets in an immiscible oil at a much higher throughput than any existing droplet generation techniques. The technology also allows easy tuning of droplet size and generation rate to permit large dynamic range quantification of nucleic acids. It enables a novel droplet-digital PCR platform, with high sensitivity, high dynamic range and absolute quantification capability without resorting to Poisson statistics. The droplet generation unit consists of a bundle of micropipettes with conic tips. The sample and PCR mix is pumped through each micropipette and are pinched into droplets at the tips by AC Maxwell pressure without jet formation or Rayleigh instability. A co-flowing or cross-flowing oil phase then carries away the water droplets. To focus the electric field to produce sufficient Maxwell pressure to overcome the large capillary force of small droplets, conic micropipettes are pulled from glass capillaries with a tip diameter of about 10 μm¹. The frequency is tuned so that Taylor cones and tip-streaming droplets do not appear. The oil phase contains surfactants that stabilizes the emulsion but also allows droplets to pinch off from the micropipette. The droplet size can be easily tuned from 1pl to 1nl by adjusting the electric field and pressure.

This AC droplet digital PCR performs absolute quantification by distributing the templates into a far larger numbers of water droplets, such that each droplet will have only one or zero DNA templates. This binary distribution provides significantly more accuracy than the traditional Poisson distribution of templates in current ddPCR technologies, which typically yield multiple templates per droplet. The presence of a template in each droplet is registered by intercalating dye or Taqman optical reporters after PCR amplification. The thermal cycling is performed either in a batch format with a commercial thermal cycler or with an on-chip PCR unit. To achieve the goal of processing millions of droplets, an automated imaging and counting system with a moving stage and a control software is used. The software coordinates the camera action and stage movement to cover the whole area of the imaging chip. To facilitate the imaging, droplets are densely packed into a single chamber with on-chip pillars that do not allow droplet passage. Validation of this new technology is achieved by bench-marking against traditional qPCR with identical samples.

[1] Pan, Zehao, et al. "Universal Scaling of Robust Thermal Hot Spot and Ionic Current Enhancement by Focused Ohmic Heating in a Conic Nanopore." Physical Review Letters 117.13 (2016): 134301.