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Q&A: Sandia’s Ken Patel on Developing a Microfluidics-based DNA Forensics System

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patel.jpgNAME: Kamlesh (Ken) Patel

POSITION: Manager and lead for the Advance Systems Engineering and Development Group, Sandia National Laboratories, Livermore, Calif.

As molecular diagnostics and DNA testing move ever closer to the point of care, microfluidics have become a key enabling technology. At the forefront of this movement is Sandia National Laboratories, where researchers from the Department of Advance Systems Engineering and Development and the Department of Biotechnology and Bioengineering have collaborated over the last several years on a number of microfluidics-based systems for molecular biology research and diagnostics.

For instance, a group led by Kamlesh Patel has been developing an automated, microfluidics-based sample preparation process for next-generation sequencing of unknown pathogens, part of Sandia's Rapid Threat Organism Recognition, or RapTOR, project, which is geared toward developing rapid sequencing for bioterrorism applications (PCR Insider, 5/12/11).

In addition, PCR Insider sister publication ProteoMonitor last year covered a new spinning disc-based immunoassay system under development at Sandia spinout company SpinDx.

More recently, Patel and colleagues Michael Bartsch and Ron Renzi at Sandia have been working on integrating some technology from the aforementioned sequencing sample prep platform with other cutting-edge microfluidics and PCR technologies into a field-based platform for human DNA testing and forensics.

PCR Insider recently caught up with Patel to discuss this effort. Following is an edited transcript of the conversation.


Give me an overview of how your lab is developing microfluidics technology for molecular biology applications.

We do work heavily in the protein space, but also the nucleic acid space, RNA or DNA, depending on what we're trying to do, all in the grand context of infectious disease.

The reason Sandia is involved in a lot of this ... is for public health – on the domestic level, but we also have a national security mission that revolves around trying to protect against the potential of new [and emerging] diseases ... or [a terrorist] developing the next superbug.

These are far-out scenarios, but the focus is on having technology that is integrated, multi-functional, and [doesn't] need a full laboratory to do the analysis – you might have it in a compact hand-held unit. Or something that is mobile … for rapid decision making.

We've done some things in nucleic acids particularly around the area of sample prep and marrying those different analytical tools on the back end … to try and make a system whole, basically going from blood all the way to a stable cDNA intermediate for either PCR or eventually sequencing.

One technology that we’ve been working on that I’ve been quite involved in is a digital microfluidics hub -- basically library prep and sample prep for sequencers. We’ve taken the basis of that technology and applied it to DNA forensics -- not in the traditional sense that you’d find in [a crime scene investigation], but to support battlefield forensics, which is better known as expeditionary forensics. The idea is that when you’re in the heat of a battle and using all your resources to investigate special ops and different scenarios, you have to have your forensics capabilities out in front in the field. There is a tremendous push within the intelligence community to have that capability very immediately available, rather than collecting samples, bagging them, and having them shipped back to a full laboratory.

Based on some of the technology that was in the digital microfluidics hub, we’ve developed a flexible, integrated forensics platform to do human DNA profiling. Some of the goals we’re looking at are trying to do the DNA forensics in less than an hour, [and] have it all be integrated and sit in a backpack kind of format.

This would be integrating microfluidic sample prep and STR analysis?

Yes. We do cheek swabs. You’d have a port to put it into, you press a button, and it automatically extracts the nucleic acid from your cheek cell lysate. [The platform] then introduces [nucleic acid] into a magnetic bead collection assay for normalization, and then mixes in some of the assay cocktail, the PCR primers, and then [performs] amplification. We have a pretty slick way of doing amplification that is pretty different from a standard thermocycler. Then we basically do an electrophoretic separation. It’s very similar to what you’d do on the benchtop, but we’ve got it integrated into a package that can be moved around.

Does this use the spinning disc technology that SpinDx is using?

It’s not. It’s a different platform. The way it works is that usually engineers like myself spend an enormous amount of time to create a kit to carry out all these processes, and it’s a very simple kind of linear process of going from sample in, sample extracted, something added to it, then it goes to analysis.

We’ve kind of interrupted that linear flow in that we have a central hub that helps manage your flow of sample and processing much like a robot would in a clinical laboratory, moving titer plates from one instrument to another. The hub is kind of an automated droplet platform that can move these droplets around basically using electrical voltages. Then we can interface these droplets to the outside world, meaning the instrument or function of choice, through a capillary interface. For example, if we wanted to do PCR, we would have a droplet with the DNA and mix it with the PCR enzymes, and rather than doing PCR in the droplet, I would take it off to a unique thermocycler and do the thermal cycling there. Once it’s done, we bring it back to the hub and then send it off to the electrophoretic separation or whatever else needs to be done.

It’s this modular approach connecting a lot of different functionalities together with this droplet router.

And the microfluidic hub right now is in sort of a bread box, tabletop format?

Yes. We’re eventually trying to get it into a package that we can take out into the field, that fits in a backpack. At this point it takes up about two or three feet.

You mentioned a “really slick” way of doing amplification. Can you detail that?

Most PCR is done in a microcentrifuge tube that you put into a block that goes up and down in temperature. But it still requires somebody to pipette in all these reagents. Alternatively, if you go into the fluidic chip world, a strategy is to have picoliter-volume droplets in an oil stream. And alternatively, you could skip the oil thing and just have it all flow past a series of heaters at different temperatures.

But what we find out is there are limitations to how you can use those various approaches because of the oil phase or the various surface areas involved. Because we need to be portable, we need to be really conscious about power. But we didn’t want to go too far away from how traditional PCR works. STR amplification is pretty finicky to messing around. We wanted to do as much as we could to mimic thermal cycling but reduce the power constraints.

We’ve made a PCR wheel, and we call it rotary zone PCR. Imagine a wheel divided into wedges, and this wheel contains wedges that all have different temperatures -- 95, 65, and 72 [degrees Celsius], and one at room temperature. On the walls of this cylinder, with the wedges that are separate from one another, there are grooves cut into it through which we can basically wrap a tube. And if we have this all connected to the digital microfluidics hub, we can use it to pull a small bolus of off that and into a tube, which is nested inside this wheel. And it’s just held there, and the wheel basically spins to different temperature zones to do the amplification. You’re not moving the sample, and it’s just nested inside these little hot and cold regions, where instead of thermocycling, we change the temperature by actually spinning the different temperature zones in contact with the tube.

This allows you to keep those wedges at the same temperature. You don’t need all these sophisticated feedback loops and Peltier [heaters] to ramp temperature up and down. These temperature zones can be rotated using a simple stepper motor.

And when it’s done, we push that four- or five-microliter bolus back onto our device and carry on the readout part of the assay -- an electrophoretic separation.

In doing this, we avoid a lot of the complications of going down to extremely low volumes. We need a sizeable amount of DNA to run the separation, anyway. We don’t have to mess with the stoichiometric ratios of all the things in there, because we can just kind of scale everything by 10 and it works just fine. And you still have a useful amount of DNA left to do something else if you need to. And, there are no manual steps of taking the sample and doing PCR in a tube, then taking the contents of the tube onto another device.

Also, these things are meant to be disposable, and I call them tubes, but they’re basically fluidic cartridges.

Have you published or patented any of these particular technologies?

Yes, we applied for a patent on the rotary zone technology, and we’re in the process of writing a manuscript to highlight the forensics platform. And we’ve presented it at various conferences. It’s at that stage where proof of principle is done, and we’re comfortable with the performance in that it works, but now we need to push all the wrinkles out of it.

And this is all being done through your lab at Sandia, not through the SpinDx startup?

Yes, this is with funding from within Sandia and also from the United States Army Criminal Investigation Laboratory. We’re basically at a pause right now before a Phase II program. It’s in a prototype phase. At Sandia, we’re really good at taking things to a proof-of-principle stage, but our goal is to then partner with companies and other institutions to drive it into the commercial sector. That’s just not our forte -- we’re not connected to the customer enough to understand the finer points and needs. I think this needs another year or two to finalize the packaging and then we’ll be at that point.

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