By Ben Butkus
A team led by scientists from the University of Alberta has developed and successfully tested a gel-based array platform for performing multiple microliter-scale PCR reactions in parallel using externally added nucleic acid template, according to a recently published research paper.
According to the researchers, the technology, dubbed In-Gel, is inexpensive and amenable to integration into point-of-care molecular testing devices for use by minimally trained staff in resource-poor environments.
In addition, the technology has been licensed by the University of Alberta to a startup company called Aquila Diagnostic Systems, which plans to integrate In-Gel into such diagnostic devices for blood-borne infectious diseases, PCR Insider has learned.
As described in a paper published online last week in Analytical Chemistry, In-Gel comprises a polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semi-solid posts that serve as reaction vessels for parallel PCR reactions.
The current version of the technology features a nine-by-nine array of gel posts each less than 1 µL in volume with a an intercalating dye added prior to polymerization to permit real-time PCR data acquisition and melting curve analysis, according to the paper.
In addition, the researchers have created a prototype analysis device consisting of a simple Peltier element for thermal cycling, and an inexpensive laser excitation source and CCD camera for product detection, they write in the paper.
The technology got its start when University of Alberta researchers were "trying to find ways to take what are really complicated molecular assays and put them in a form that can be used in a clinic — not just a large, city research hospital clinic, but any healthcare center," research team member Linda Pilarski, chair of biomedical nanotechnology and professor at the University of Alberta and Cross Cancer Institute, told PCR Insider this week.
"To do this, you have to have something that is automated, inexpensive, and simple," Pilarski said, adding that the researchers originally worked with layered silicon chips, but eventually realized that a polyacrylamide gel matrix had all of the desirable qualities they were looking for.
Performing PCR reactions in such a medium instead of liquid solution offers advantages such as the fact that the DNA and PCR reagents can be sequestered from surfaces and potential contaminants; and kept within close proximity to each other without the need for valves or other microfluidic components, which can be expensive to manufacture.
"It is ideal for low-resource settings, as well," Pilarski said. "It's a bit like Jell-o … and has a simple fabrication process. You could envision something that's desiccated, which means you can actually store it on the shelf; and has a water bladder full of sterile water, so you don't have to have clean water. And you can use a battery because it's not going to have big power requirements"
Other researchers have successfully performed PCR in a gel matrix, but all of these instances have used a defined chamber with relatively large volumes, and none has performed "simple, seamless post-PCR analysis of amplicons, such as melting curve analysis," the researchers wrote in their paper.
In order to demonstrate the viability of their technology, the University of Alberta researchers extracted DNA from BK virus to serve as an initial prototype template DNA. Using their platform, they were able to consistently detect as few as 34 virus templates in individual gel posts.
They also demonstrated that PCR was equally efficient and reproducible when template DNA was polymerized within the gel or when exogenous template was added onto pre-cast gel posts; and demonstrated that their technology produced equivalent results to traditional PCR using liquid medium and a Roche LightCycler instrument.
They also used the system to amplify HPA1 and FGFR2 genes in human genomic DNA, using as little as 2 to 5 nanograms of DNA template per gel post. This proof of principle was important because the researchers believe that, in addition to point-of-care diagnostic applications, the platform can be used to assess genes for personalized medicine and pharmacogenomic applications.
In addition, Pilarski noted, the arrayed nature of the new technology will allow users to run multiple tests on a single sample so as to test single clinical samples against several infectious diseases or measure gene expression from panels of genes involved in drug metabolism or disease progression.
The size of the gel array can also be increased by simply "ramping up the cameras and lasers," Pilarski said, which might make the platform attractive for research applications such as large-scale gene expression studies.
However, she added that it is unlikely that In-Gel would find utility as a discovery tool. "Technologies that have already been developed for multiplexing … will probably work as well or better" than the In-Gel technology for discovery purposes; "whereas they're not going to work very well in the clinic," Pilarski said. "They're too complicated. You don't need huge amounts of information in the clinic — that just complicates things. You really want the critical information to make a diagnosis or decide on a treatment strategy."
In order to exploit the commercial potential of the technology, the researchers are now looking into enclosing it and adding automated sample preparation and delivery to create a handheld or benchtop device.
So far, sample prep has been kept to a minimum as the researchers have analyzed both raw urine and blood in their experiments. "If you could do every test using raw sample, that would be ideal," Pilarski said. "But you're not going to be able to, because some agents are going to be present at low concentrations, and you'll need a means to concentrate those. That means you'll need some way of collecting the DNA, and then being able to put an amount from a large volume of blood or urine or whatever into a small volume that you need for the gel post."
In order to create a viable commercial device, the researchers have also enlisted the assistance of local startup Aquila Diagnostic Systems, which has taken a license to the technology from the University of Alberta in the area of blood-borne infectious disease.
Will Gibson, co-founder and president of Aquila, told PCR Insider that the company plans to develop a working prototype device over the next 18 months or so.
"There are some devices that have been built for use in the lab," Gibson said. "But we currently have people doing some feasibility work."
Aquila is currently seeking seed capital to support this development, but is not seeking investors until it is able to produce a viable prototype device, he added.
In the meantime, the University of Alberta team will continue to seek potential partners to exploit different applications for In-Gel. "It's a platform technology, so we've licensed this to Aquila only for the specified field of use," Pilarski said. "Other applications — for example, cancer-related testing — might require a different partnership, and we haven't identified any potential partners for that yet."
Pilarski added that the team has applied for provisional patents in the US surrounding the In-Gel technology and its applications.