With a freshly minted US patent in hand, the University of Utah Research Foundation has made available for licensing an ultra-fast PCR microchip technology that can perform real-time melt curve analysis in less time than existing commercial thermal cyclers.
The technology, which uses microfluidic channels to continuously flow a sample through different temperature zones, can theoretically complete 30 cycles of PCR in approximately six or seven minutes while measuring both bulk fluorescence and DNA melting after each cycle, according to its inventors.
In addition, the microchips are inexpensive and able to be manufactured in bulk, and as such may be particularly useful as the foundation for a point-of-care DNA testing system, Bruce Gale, an associate professor of mechanical engineering at the university and one of the technology's inventors, told PCR Insider this week.
Researchers have taken myriad approaches to reducing the cost and increasing the speed of thermal cycling and, by extension, quantitative real-time PCR or melt curve analysis. Innovative microfluidic schemes have fast become one of the most popular approaches because the small sample volumes enable rapid and precise heating and cooling.
Gale — along with Carl Wittwer, a professor of pathology at the University of Utah School of Medicine and co-founder and CSO of Idaho Technology; and Niel Crews, a former graduate student of Gale's who is now an assistant professor of mechanical engineering at Louisiana Tech University — originally set out to develop a microfluidic-based method for performing PCR using ultrasound.
"Niel had the idea that if we do it in this serpentine channel … from different sides, we can do it better … and we eventually figured out that if we just put heaters on two sides of these serpentine channels, we could do PCR without ultrasound, and it could potentially be very fast," Gale said.
The primary innovation that resulted is a microfluidic chip with a channel that zig-zags past heating elements maintaining different temperatures required for PCR. This essentially sets up a temperature gradient — from around 55° C for annealing to 95° C for denaturation — throughout the length of the channel, through which a sample and PCR reaction volume are pumped.
In addition, "we can make the fluid channel very small as it's coming down from the high temperature [zone], it gets down to the cooling temperature very quickly; and then widen the channel out a little as it goes back up to the hot zone," Gale said. As this happen, the sample moves through the extension phase temperature — around 72° C.
"Part of the idea here is that extension doesn't occur at just 72° C, but at all of those temperatures — it's just that 72° C is the best," Gale added. "So as it's moving through there, [the DNA] can actually extend on its way up to the hot side."
Changing the widths of the chip's channel also allows users to control, to an extent, the relative amount of time the sample spends in each temperature zones.
"We found that because the flow velocity can be relatively high, and the temperatures can be held relatively stable, that we can actually move the DNA or the whole PCR sample really quickly," Gale said. As such, "we can do about 30 cycles in six or seven minutes. That's not the fastest ever, but we were able to get that to work pretty consistently." That timeframe is also faster than any existing commercial instrument, although other academic research groups are developing methods to shave a few minutes more off of thermal cycling time (PCR Insider, 8/4/2011).
Another benefit of the setup is that users can monitor fluorescence intensity from fluorogenic probes as the sample moves between the different temperature zones. "It really is a fairly straight line, and if you take a picture of that, you can essentially get your melt curves just off of that data," Gale said.
Further, by monitoring fluorescence between channel segments, as the sample moves through the different cycles, users can obtain a real-time fluorescence curve, he added.
The current iteration of the chip uses a syringe pump to push the sample through, but, Gale noted, a more refined version of the technology could use a peristaltic pump or other automated method. Likewise, the group has been using CCD cameras to capture fluorescence data, "but at this point — and we haven't demonstrated it yet — there's no reason to think you couldn't grab something like a cell phone camera and somehow get your image with that fairly soon," Gale said.
Other potential embellishments could include multiple channels for highly parallel continuous flow PCR of multiple samples; or using a droplet generator such as those employed in technologies from RainDance Technologies or Bio-Rad to pump individual droplet-based PCR reaction volumes through the channels for digital PCR.
This week, the US Patent and Trademark Office awarded the University of Utah a patent covering the technology (see IP Watch, this issue), and the researchers are now seeking potential licensees and commercial partners to further develop the device for various applications.
"Maybe the most obvious application for me, because this is such a small device, is some sort of portable or field-based PCR testing," Gale said. "It would have relatively low power [consumption], just powering the heaters, which is not trivial. The pumping is relatively small. Reagent use could also be quite small."
Down the road, Gale added, the chip could be integrated into a larger instrument for an application such as field-based biothreat detection. "Most of our work has been done with simple things. For example, we've used it for viral detection of foot-and-mouth disease in India."