By Ben Butkus
Thermal Gradient, an upstate New York molecular diagnostics startup, said this week that it has begun manufacturing prototype disposable test cartridges for performing rapid, inexpensive PCR assays as part of a future portable HIV detection and monitoring system for use in developing countries.
The company, which claims its technology enables users to perform PCR "as fast as nature will allow," is now integrating sample preparation technology into its devices and developing a companion readout instrument with the hope of conducting pilot studies for a complete 30-minute HIV blood test by the end of next year, President and CEO Joel Grover told PCR Insider this week.
In the meantime, Thermal Gradient, which is developing its platform under a three-year, $3 million Phase II Small Business Innovation Research grant from the National Institute of Allergy and Infectious Diseases, is seeking private investors and applying for additional grants to help commercialize the technology, Grover said.
Founded in 2004 by Grover and colleague Robert Juncosa, Rochester-based Thermal Gradient started life as a biodefense company seeking to exploit its founders' flow-based, lab-on-a-chip technology for detecting biothreat agents.
According to Grover, the company had intended to use its technology for molecular diagnostics from the start, but after raising some seed money from a local investor, Thermal Gradient applied for and won a grant from the US Department of Homeland Security to develop the biodefense applications.
"We found ourselves in the biodefense business for about the first three years of our existence," Grover said. "We followed that string, and then we had a mutual parting of the ways." Concurrently, the company was moving farther away from its intended molecular diagnostics goal, Grover said, so it applied for a Phase I SBIR grant to develop its technology for rapid point-of-care HIV testing.
"That allowed us to show that the technology could do detection," Grover said. "At the conclusion of that grant, we applied for the Phase II, which we're working on right now." Thermal Gradient's current grant term began in February 2010 and is set to expire at the end of January 2013.
Thermal Gradient's core technology, for which Grover and Juncosa have been awarded a US patent, is based on a concept known as continuous flow PCR, in which a sample and PCR reaction components flow through a lab-on-a-chip device with different temperature zones to achieve rapid amplification.
First developed by researchers at Imperial College London, the technique has since been replicated many times, Grover said. However, "the problem with most of the devices that people were coming up with was that they were curiosities — glass slides on top of silicon heaters laid out in kind of a planar way. They were interesting, but they weren't particularly fast."
Grover and Juncosa invented a way to fold the flow path into a layered structure that doesn't require valves, pumps, or other moving components, thereby reducing the complexity and cost of the technique.
According to Grover, Thermal Gradient's device comprises three conducting layers separated by two insulating layers sandwiched between two temperature reservoirs — one somewhat warmer than the denaturing temperature of PCR, and the other slightly cooler than the annealing temperature. This establishes in the three conducting regions of the device appropriate temperatures for PCR denaturing, annealing, and extension. "You can fiddle with the insulating layers a little to get the extension temperature wherever you want it," Grover said.
The device then has a continuous path etched through the conducting layers and insulating layers, such that fluid moving through the path will pass through the denaturing, annealing, and extension temperature zones.
"Since all the fluid has to do is cross from one layer into the next to go through one temperature region to the next, you can get thermal gradients in the range of hundreds of degrees per second, so that you're taking tenths of a second to go from denaturing to annealing," Grover explained.
"Now you have a device that can do PCR basically as fast as the kinetics allow you to," he added. "But you're doing it in a device that, among other things, is dead simple, works at constant temperature, and works at constant flow. You simply pass the fluid through the device, and when it comes out the other end the PCR is done."
According to Thermal Gradient, the technique can perform 8.5 to 9 logs of amplification (greater than 300 million times) in approximately eight minutes.
When Thermal Gradient first developed its technology for homeland security applications, it needed to make it durable for field deployment, Grover said. "But obviously for diagnostics you want something that is a throwaway," he said. "And so our recent breakthrough is that we've found materials that can be injection molded for all three layers, including the conducting layers; and we've made such devices."
Meantime, under its NIAID grant, Thermal Gradient is working with researchers at the University of Massachusetts Medical School to develop an effective HIV PCR assay for use on the company's device. The UMass researchers are also interested in adapting the technology for detecting mother-to-child HIV transmission.
Thermal Gradient has also developed proprietary sample preparation technology, which it is currently integrating into its cartridges; and is working on developing a readout instrument. The end goal is a system that can perform an HIV test "blood in to answer out" in 30 minutes, Grover said.
"We're moving ahead to the mass-produced version of the endpoint device, and we'll follow shortly after that with a real-time [version]," Grover said. "Hopefully by the end of the year we'll have put all of the pieces of the cartridge together and have demonstrated the full test. And next year we wrap the instrument around it. We want to be doing a pilot study by the end of next year."
Thermal Gradient will be entering a rapidly crowding field for PCR-based point-of-care molecular diagnostics, although HIV viral load testing has not yet been a huge area of development for companies. Most notably, Cepheid, whose GeneXpert molecular diagnostics platform is probably the most advanced disposable cartridge-based platform on the market, just last week announced that it is receiving $5 million from the Foundation for Innovative New Diagnostics to help accelerate development of a GeneXpert test for HIV viral load that will serve as a companion test to its much-ballyhooed tuberculosis test on the same platform (PCR Insider, 2/3/2011).
But Thermal Gradient is betting on the idea that in the developing world, the market for rapid HIV viral detection and monitoring will continue to expand.
"In low-resource settings, they're rapidly bringing anti-retroviral therapy to people," Grover said. "But because of the cost of doing viral load monitoring, almost none of the people who are getting ART are having their viral loads checked, as would be the case in the developed world."
These patients are increasingly susceptible to developing mutated HIV infections or coming out of remission, and thus possibly developing full-blown AIDS or transmitting HIV to others without realizing it.
"What's really needed is a solution to bring viral load monitoring out into the field and to make it very inexpensive," Grover said.
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