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ASU Lab Develops Emulsion-based Continuous Flow Droplet qPCR for the Deep Sea and Beyond

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Arizona State University researchers recently published the details of an emulsion-based droplet qPCR platform believed to be the first ever to enable real-time analysis on a continuous-flow system.

Their technology is designed for applications such as repeated sampling and nucleic acid detection in extreme environments, and also provides researchers with somewhat of a blueprint for designing their own droplet-based real-time and digital PCR setups.

Although continuous-flow droplet-based PCR has been recently reported — by scientists at the University of Utah, for example— the ASU researchers believe their device is the first to accomplish real-time quantitative analysis in a continuous-flow system.

Their work, published in November in Lab on a Chip, was funded in part by the Monterey Bay Aquarium Research Institute as part of a larger project to build in situ underwater environmental analyzers which use PCR to detect microbes.

Chris Scholin, president of MBARI, told PCR Insider in an email that the group expects to have "the world's first autonomous, mobile, eco-genomic sensor in the water by the end of 2014." He said MBARI "will be using the dPCR module [developed at ASU]… to detect a wide range of genes indicative of particular microbes and the metabolic pathways they harbor and express."

Scholin also noted that they plan to install this PCR module on an entire fleet of long-range underwater vehicles, allowing "the sampler/analytical system to operate in the ocean for extended periods, from the surface to ~300m depth… in support of marine microbial investigations." The group also plans to make this system "hand portable for terrestrial applications," said Scholin.

Crafting the perfect PCR platform for extreme undersea conditions has been the job of Cody Youngbull and his team at ASU's School of Earth and Space Exploration. Youngbull said that the underwater element of this PCR platform resulted in "a lot of design constraints in terms of power consumption, how it has to be oriented during operations with gravity, and the extreme temperature ranges it has to operate in." However, the resulting device "is ultimately going to fit in a coffee can, and it really is autonomous and easy to set up."

The published results describe what Youngbull called a "half-step" to meeting these constraints, but he said the recent publication is about a year behind where the lab is now. In their current iteration, they have gone from droplet real-time PCR to digital, to further shrink size and power requirements. Their current platform is now "portable, low power, small, doesn't require cleaning, [and] much more autonomous," said Youngbull.

It is also quite adaptable to the needs of the researcher: "Because it's continuous flow, just as you can introduce sample continuously, you can introduce primers continuously, they come together in a coalescing event, so… how many primers do you want? I'm not limited; [if] you want 10,000 targets, I can do 10,000 targets," he said.

The process of building an extremes-withstanding PCR machine from scratch meant different approaches went head-to-head in the Youngbull lab. With the deep-sea constraints in mind — small, low power, able to be run autonomously in temperatures ranging from 0° to 50° C —"all options were on the table," Youngbull said. For example, the group considered loop-mediated isothermal amplification (LAMP). "But when we looked at it, the truth is there is a thermal step at the beginning and so there is heating in LAMP. Yes the actual amplification is done chemically, that's done isothermal, but there is a heating step in the actual assay, and when you look at all of that, people say LAMP can be done in 15 minutes — no, it can't. When your life is on the line to go from sample to data, LAMP takes a significant amount of time," he said.

Instead, his group chose standard enzyme-based PCR, but used a continuous flow thermocycler and an emulsion-based droplet methodology. With this system, "because your droplets are small and they respond quickly to thermal changes and there's so few reactions taking place in each droplet, they can be run much faster. So much faster that LAMP really doesn't become a huge benefit in this application," he said.

They also chose their cylindrical design to meet the power constraints of underwater PCR. "We don't want to oscillate the temperature of a thermal block, like would be done in an instrument you could plug into the wall, because it just wastes a lot of power," he said.

Thus, the published device pumps microliter-sized droplets through 40 turns of tubing wrapped around a cylindrical aluminum core which contains heating elements. For this particular experiment, they used TaqMan and a 1-minute cycling time, thus one wrap of tubing marched droplets past a 60°C annealing and extension phase, followed by a shorter voyage past a 95°C melt phase, repeated 40 times.

For fluorescence detection, they used a 64-channel photomultiplier tube and ball lenses such that "a little submicroliter droplet will whip by and we'll get ten photodetection events," said Youngbull. They chose PMT over CCD [cameras] because "when you're going to amplify, you want a good dynamic range in terms of the response of your detector. CCDs just don't give you the power of a photomultiplier tube, and they're power-hungry too, and we're running Linux on this system, these are the types of constraints I'm talking about," he said.

From the photodetection data, they then generate a Ct curve for each droplet. Youngbull claimed they can run PCR quite quickly with their device: "Especially because it's emulsion, droplet, fixed-zone stuff, we could pump the flow rates up, change those thermal profiles, and we could do much faster," he said. "I think 30 minutes is reasonable with that machine, maybe even faster."

However, because of the PMT and its controller, Youngbull estimated this beta version of their conveyor-belt design would cost about $50,000 to replicate. "The bleeding edge that we're on now is, we actually have a digital PCR instrument, so it's a digital RT-qPCR system," he said. In "the next-generation one, because we've gone to digital, you only have to detect at the endpoint, therefore you don't need a 64-channel PMT, you need one PMT, and then cost drops drastically."

Their platform contains a number of other innovative machining ideas. To get the droplets queued in a way that they can be tracked, their size was matched to the inner diameter of the tubing, so that they couldn't squeeze past each other. Droplets were sheared into the tubing by a T-channel droplet generator fabricated using soft lithography, although Youngbull's lab now uses a flow-focusing regime. In fact, their design team is flexible, and Youngbull said that co-author Tathagata Ray has continued to develop droplet generator designs to try out so that now, after much tinkering, "we don't do T-junctions, we do flow focusing" to create the droplets.

Another innovation was the emulsion-based aspect of the design. A continuous stream of PCR reagent was discretized into a segmented oil and water emulsion, with low-viscosity fluorinated oil and a pinch of fluorosurfactant. This oil was a bit of a conundrum for the team, said Youngbull. Emulsion-appropriate oils, available from commercial droplet-based PCR companies such as Bio-Rad and RainDance Technologies, are proprietary, claimed Youngbull.

"They don’t tell you what's in their fluorinated oil, and so it's hard to do research" with it, he said. "They’ve got the surfactants in those oils dialed in so well to match certain master mix so that their surface energies retain a very stable uniform droplet size, so if you're using your own master mix and their fluorinated oil, you're droplets aren't going to be very stable."

Andrew Hatch, a postdoc in Youngbull's lab and a co-author on the paper, had the requisite PCR expertise for this aspect of the project, complementing Youngbull's experience in physics and optics in particular.

The ASU researchers found the reagents they needed to craft their own combination. The fluorinated oil came from 3M, while the fluorosurfactant was from Cytonix (now owned by Life Technologies), according to their paper. Youngbull said other researchers have taken note of this and contacted his group to ask about the availability of the fluorinated oil.

Troubleshooting this emulsion oil was a key to their design, Youngbull emphasized. "That's what really allows you to isolate your droplets from the side wall… Now, using [the newest version of the] instrument, we use three phases, not just two, so not just the aqueous phase and the fluorinated oil of the surround, but we'll use a third phase to separate experiments, a mineral oil," he said. This three-phase system "allows you to make a machine that you could literally drive around in the back of a truck bouncing around, and your droplets might break up but … they're not going to mix across experiment or across primer," he said. "We think about droplets a lot," he added.

In Youngbull's estimation, digital droplet PCR in general is a major advance. "Droplet-based microfluidics are much less susceptible to cross-contamination and inhibition," he said. "They're the way of the future… for all kinds of analytics, not just biomedical PCR."

Although there may be limited biomedical applications, Youngbull envisions use of continuous flow PCR in sewage systems, to monitor wastewater for signs of dangerous pathogens. He also recently applied for funding to adapt the continuous-flow device for in situ monitoring of stratospheric microbiology.

"We are partnering with other people to put it on a balloon. We'll take the same instrument up to the stratosphere, because there's been a lot of excitement about biomes that exist permanently in the upper atmosphere. There's so much unknown about that, three quarters of it is unculturable," he said.

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