Using a microfluidic device called SlipChip, a cell phone camera, and cloud computing, a California Institute of Technology team has shown the superiority of digital over real-time isothermal PCR under perturbed experimental conditions.
The work, published in November in Analytical Chemistry, specifically demonstrates the potential of the SlipChip device running digital loop-mediated isothermal amplification, or dRT-LAMP, as a method for precise quantification of nucleic acids in resource-limited settings.
The SlipChip technology was originally developed at the University of Chicago in the laboratory of Rustem Ismagilov, now a professor at Caltech. The technology gets its name from the way the reactions are performed: On a credit card-sized plate, nanoliter volumes can be loaded with reagents, while parallel wells are filled with sample. Upon "slipping" the cover chip, the volumes are combined, initiating the PCR reaction.
Ismagilov and colleagues have previously published a series of peer-reviewed publications showing the utility of SlipChip for various digital PCR applications, including the use of isothermal amplification methods. They also founded a startup company called SlipChip LLC to commercialize the device.
The most recent study was funded in part by an award of more than $15 million from the Defense Advanced Research Projects Agency of the US Department of Defense. The award is part of the agency's "Autonomous Diagnostics to Enable Prevention and Therapeutics: Diagnostics on Demand," or ADEPT: DxOD, program, and is scheduled to expire in August 2014, according to a defense department press release.
Ismagilov, the corresponding author on the recent paper, explained to PCR Insider that this funding has guided the recent development of his group's technology.
"The effort [at DARPA] has been on very simple, easy-to-operate devices, so we've really been focusing on that quite extensively," he said. The award is specifically for perfecting a platform that has analytical parity with typical reference laboratory equipment but can be used to do molecular analysis in limited resource settings such as the developing world, the battlefield, and the home. According to the DARPA release, the finalized SlipChip technology would be eligible for a CLIA waiver.
Amplifying nucleic acids in these resource-limited locales requires a platform free from expensive machinery, and one that can be operated by non-experts. The ideal device would also tolerate fluctuations in temperature and reaction times, and be able to be read and interpreted quickly. Running digital RT-LAMP on SlipChip fits that bill, but vetting the procedure has also yielded insights more broadly applicable to PCR science.
"We’re finding that real-time is not robust to all kinds of perturbations — experimental imaging, processing, and so forth — but digital is surprisingly robust," Ismagilov said. "You can change the temperature all over the place, you can change reaction time all over the place … you can use [low-quality] imaging, and very simplified analysis, and you still get the right answer."
He added that "if we want to take quantitative measurements out of complex instruments and into the field or into homes, robustness is going to be of paramount importance, and we would be able to use digital approaches more effectively than real-time, kinetic approaches."
In the Analytical Chemistry paper, Ismagilov's group specifically showed improved robustness in amplifying HIV-1 RNA using digital RT-LAMP on SlipChip as compared to real-time RT-LAMP. For example, they varied the temperature over a 6°C temperature range and found their digital method could still detect a two-fold change in HIV-1 concentration, while the real-time method could not. They also found the digital chemistry more robust to a 20-minute change in reaction time.
The ease of use of their device is particularly evident in a short video on the Ismagilov lab YouTube channel. Here, a five-year-old boy demonstrates the other innovation shown in the latest paper — that SlipChip data, essentially derived from fluorescent spots on the chip, can be quantified with a cell phone camera in about a minute and processed on a remote server.
Ismagilov said that the video is "trying to convey the fact that it's not some complicated thing that requires five PhDs and 17 lasers." As he explained it, the five-year-old in the video understood the gist of the experiment thusly: "There are a lot of molecules that we need to count — some of them reflect disease, some of them reflect the state of the environment — and the way to count them all is by using a chip and a cell phone."
The lab's next steps are to validate these results by measuring HIV viral load using patient blood samples. They also plan to adapt the device to measure other viruses, such as hepatitis C.