By Ben Butkus
Scientists from the University of Hull in the UK have developed a prototype microfluidic device for rapid PCR amplification using integrated microwave heating and air impingement cooling.
The technology can perform PCR amplification using heating rates and reagent quantities an order of magnitude faster and smaller than commercial instruments; and its small size may make it applicable to a wide range of point-of-testing applications, according to the researchers.
The group is now seeking potential investors or industrial collaborators to further vet the technology and help achieve the next steps in potentially commercializing a product, one of the researchers said this week.
The scientists from the University of Hull's department of chemistry and Centre for Biomedical Research, and a small communications technology company called Exxel Amplifiers, described the prototype device and demonstrated its utility in a paper published online in advance of print last month in Lab on a Chip.
Stephen Haswell, a professor of chemistry at the university and corresponding author on the paper, told PCR Insider this week that the research group designed the PCR technology as part of a larger project to develop a microfluidic lab on a chip that integrates DNA extraction, amplification, and fingerprinting.
"On the sidelines of this was looking more closely at the PCR process," Haswell said. Conventional PCR instrumentation, he said, often produces a data curve caused by a thermal mass transfer effect as the instrument tries to hit optimal temperatures.
"What was obvious from the chemist's point of view was that this is an incredibly inefficient process, because it leads to mispriming and a lack of real control of the amplification mechanism, which is purely a biochemical reaction," Haswell said. He added that many commercial PCR assay kits have actually been modified to work with this so-called thermal lag.
To address this issue, the group designed a device that would eliminate the thermal transfer process. The device consists of a tuned microwave cavity for direct substrate heating coupled with compressed air delivery for air impingement cooling.
"The microwaves heat the glass directly by an RF excitation," Haswell said. "So you're heating the entire thing homogeneously at the same time. You're not heating something that is then heating the glass; you're just heating the glass."
Because the sample volumes are very small — less than a microliter — "it effectively has no thermal mass in that process, and thermal transfer is instantaneous, so you're not seeing these thermal lags," Haswell said, adding that the cooling process is equally fast.
To test their device, the researchers performed amplification of the Amelogenin locus from saliva samples collected from volunteers. They extracted DNA from the sample using Qiagen's QIAamp DNA micro kit, and conducted PCR using hot-start GoTaq DNA polymerase from Promega.
They analyzed PCR products by capillary electrophoresis and ran PCR control samples on a TC-312 thermal cycler from Techne. The on-chip amplification reaction involved thermal cycling between temperatures for DNA denaturation (94°C), primer annealing (59°C), and DNA extension (72°C) for 28 cycles.
They were able to amplify Amelogenin DNA from both male and female donors. Further, the microwave heating system varied no less than plus or minus 0.1°C at each temperature target; and was found to have a response speed "orders of magnitude" faster than that of current commercial systems, including the Techne and other undisclosed platforms, the researchers wrote in their paper.
Specifically, the microwave system enabled 28 cycles to be performed in 42 minutes, which bested previous reports of 33 cycles in 127 minutes for microwave PCR systems operated with larger reagent volumes, according to the paper.
"Now that we have that 0.1-degree control, we would like to be able to look at the way things like hybridization reactions work, where … you can really fine tune into the number of bases you have present, and control that efficiently," Haswell said.
One application envisioned by the researchers is point-of-testing forensics analysis. However, other applications that could benefit from portable, fast, and inexpensive PCR, such as point-of-care diagnostics and environmental testing, are also possible.
"We can make this into a small device with relatively low power needs," Haswell said. "An aspect of this is the capability to do stuff like real-time PCR and hybridization reactions in a medical setting. And you could multiplex this to look at multiple infectious diseases. And there would be no problem putting a fiber optic laser probe or something like that in it. But we've got a lot more development to go."
The group has applied for PCT patents on the microwave method and more broadly on the lab on a chip. In the meantime, the researchers are exploring potential commercialization routes for the technology, "which is still at a very early stage," Haswell said.
"If we could find someone with a real need [for] either the portability, speed, or selectivity …we engineer our own boxes, make our own chips, and if you wanted a lot of them, we could find a way to do it," Haswell said. "But we don't have any need to do that. So we need people that would want to pull this technology from us so we could pass it over to them."