A team of researchers at Boston University is developing a microfluidic chip using a PCR alternative, claiming it could serve as the basis for low-cost, handheld molecular diagnostics for use in global health environments.
The key to the system is helicase-dependent amplification, an isothermal amplification method that is similar to PCR in many ways but does not require a thermal cycler, and is therefore more suitable for in-field use.
While a handful of studies have demonstrated the use of HDA on microfluidic chips, the BU team recently published a study that it believes is the first to couple on-chip sample preparation with on-chip amplification.
A proof-of-concept paper published earlier this month in Biomedical Microdevices describes the integrated chip, which includes a micro-solid-phase extraction column to isolate DNA from whole bacteria, along with a series of valves, vents, channels, and 25-milcroliter reaction chambers for performing the HDA.
The researchers demonstrated in the study that they could use the chip to detect as few as 10 colony-forming units of Escherichia coli in about 50 minutes, and concluded that "the low cost of materials, isothermal amplification, and integrated nature of this device make it a good candidate for further development for point-of-care testing."
Team leader Catherine Klapperich told PCR Insider that the project's primary goal is to develop simple, point-of-care assays for use in the global healthcare setting, which is why the BU team opted for HDA as the amplification method.
"At the bench, HDA doesn't have that many advantages over PCR in a lot of people's opinions, which is why it hasn't really been a breakout technique," Klapperich said.
For example, she noted that HDA often exhibits non-specific amplification toward the end of the reaction, which can lead to problems if the readout is not performed quickly.
The key benefit for HDA, Klapperich said, is the fact that the entire amplification process can take place at a single temperature. Rather than using heat to denature the DNA as PCR does, HDA uses a heat-stable helicase that enzymatically unravels the two complementary strands. As a result, it can be performed directly on extracted DNA and requires only one primer set.
"With HDA, all we have to do is go from room temperature to the temperature where the reaction is happening at 65 degrees C," Klapperich said. "We get amplification if we have the operator read the assay at a particular point in time, and then we avoid the saturation effects that can happen at the end of the reaction when you start to get non-specific reactions happening."
In the type of field-based applications that the BU team envisions for its device, "if you can get rid of the thermal cycling, you can get rid of a lot of engineering," she said.
Other HDA Efforts
The BU team is not alone in its pursuit of miniaturized point-of-care devices based on HDA.
Qiagen, for example, announced earlier this month that it had acquired ESE, a privately held developer of compact optical measurement devices, for $19 million. Qiagen said that it plans to use the ESE technology to develop portable nucleic acid-based point-of-care tests and that it has already demonstrated the use of ESE’s technology with HDA assays that it licensed from BioHelix in 2008.
BioHelix, a spinout of New England Biolabs that was founded in 2004, holds the key intellectual property surrounding HDA.
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Qiagen said that it has verified the ESE systems with HDA-based assays for Salmonella, E. coli, and influenza viruses. The company said that it plans to develop portable tests for use in acute care and critical-care settings in the US, and for infectious-disease testing in the developing world.
Tamara Ranalli, director of business development for BioHelix, told PCR Insider that the company has outlicensed the HDA technology to another company that is developing a "similar system" to Qiagen's, though she could not disclose the licensee.
Ranalli said that BioHelix is also developing its own integrated HDA-based testing system that relies on a cassette-based, lateral flow approach rather than microfluidics.
She said the company is targeting infectious-disease testing and genetic testing for the platform, and that it plans to seek approval from the US Food and Drug Administration for the system next year.
"The whole molecular field is looking to go into point of care," Ranalli noted, adding that the company's isothermal technology is "well suited" for healthcare environments where low-cost and low-complexity systems are a necessity.
BU's Klapperich said that the cost of materials for her team's chip was in the range of around $10 to $20, but she said she is confident that they would be able to get that within the $1 to $2 range required for global health applications.
First of all, she noted, the researchers bought small amounts of specialty thermal plastics, "so once you start buying them in bulk the price can go down dramatically — 10 or 100 fold."
In addition, she noted, "we don't have to use the material we used in the paper to do HDA because we don't have to go up to a very high temperature." For the study described in the paper, the team used cyclic olefin polymers, which have a glass transition temperature of around 135 degrees C.
"We used it because we use it for PCR," Klapperich said, "but since the HDA reaction happens at 65 degrees C, you can use an acrylic material, which is basically pennies for the same amount of material."
Another way the team plans to reduce costs is to eliminate the layered structure that it used for the proof of principle in favor of injection molding, "which drives down costs significantly," she said.
With those modifications, the materials cost for the chip "could be easily less than a dollar," said Klapperich.
There would also be room to cut reagent costs by shrinking the assay volume, she said. "We did sort of a standard assay volume for HDA where we went as small as we could go in the tube and then we did that on chip, but I think we can go at least a factor of two smaller, maybe more."
The system as envisioned would not be entirely self-contained: it would still require an external heater and an external reader.
Klapperich said her team is currently looking at low-cost alternatives for the heater, such as the phase-change materials used in air-activated hand and toe warmers.
"You can get a few of those for a buck," she said. "It turns out that we can get 65 degrees C for at least an hour using those things in a really simple embodiment of the chip, and we can get amplification. So eliminating the battery for the heater is what we're working on right now."
As for the fluorescence reader, "there's nothing active on the chip that's reading the fluorescence right now," Klapperich said, adding that a "next step" would be to "team up with folks who are optics people and put something together that would make it totally untethered from the laboratory."
Klapperich's team is collaborating with several research groups to develop assays for the platform. In one project, with Satish Singh at Boston Medical Center, the researchers are developing a C. difficile assay.
"We're working on extending that to other infectious diarrheas so we can hopefully have a multiple infectious diarrhea chip," Klapperich said.
In another collaboration, with researchers at the BU School of Medicine, Klapperich and her team are looking to develop assays for influenzas A, B, and novel H1N1.
"We're still in the tube phase of development for the flu work, but it seems to work nicely for C. difficile," she said.