By Ben Butkus
A University of Chicago research group has published a pair of scientific papers demonstrating proof of concept for a multivolume digital PCR method; a microfluidic chip technology for performing it; and its potential use in HIV or hepatitis C viral load monitoring.
According to the researchers, their method has a higher dynamic range than other digital PCR approaches, and the enabling technology — called SlipChip — is easier to use and less expensive than comparable digital PCR platforms.
In addition, some of the researchers have launched a startup company called SlipChip LLC in order to commercialize the technology for point-of-care molecular diagnostic and genomics research applications, one of its co-founders said this week.
At the core of the new research papers and startup company is the SlipChip technology, developed in the laboratory of University of Chicago chemistry professor Rustem Ismagilov, now a professor at the California Institute of Technology.
SlipChip is a microfluidics-based consumable device that actually comprises two glass slides in close contact: A bottom slide containing wells preloaded with reagents, and a top slide that acts as a lid for the reagent wells. The device also features fluidic paths that connect to each other only when the top and bottom slide are aligned in a specific configuration, thus allowing samples and reagents to mix in discrete volumes.
Last year, Ismagilov and colleagues published a paper in Lab on a Chip demonstrating how SlipChip could be used to partition samples into the type of uniform, nanoscale reaction volumes essential to performing digital PCR (PCR Insider, 10/7/2010).
The researchers said at the time that they were seeking industrial collaborators to help commercialize SlipChip not only for PCR-related applications, but also for techniques such as protein crystallization, cell culture, and immunoassays.
Since then, however, the group has "made a lot of progress with the individual pieces" of an entire platform surrounding the disposable SlipChip technology, so much so that they felt confident in starting a company of the same name to prototype and commercialize it, Feng Shen, co-founder and director of R&D at SlipChip, told PCR Insider.
"SlipChip is a pretty general technology, and we have lots of different applications, so we haven't finalized the platform yet because we need to pick the most important one," Shen said.
However, digital PCR — in particular a variation called multivolume digital PCR — seems to be one of the most promising applications for SlipChip, as demonstrated in a pair of papers published by the group this month.
In the first paper, published online Oct. 7 in Analytical Chemistry, Shen, Ismagilov, and colleagues describe a protocol for performing multivolume digital PCR, as well as a theoretical radial SlipChip design that allows a user to isolate and mix sample and PCR chemicals in any of 160 wells with volumes of 125, 25, 5, or 1 nanoliter.
With only 160 wells — as opposed to several thousand or more for commercial microfluidic digital PCR platforms; or millions for nanodroplet-based instruments — the SlipChip device is, according to Shen, much easier and faster to use and can be manufactured for a much lower cost than its comparators, but with a comparable or better dynamic range.
"For some viral load tests … or some gene expression analyses … you want to achieve a really large dynamic range, anywhere from 30 to 40 particles to 10 million particles per milliliter," Shen said. "That's a huge dynamic range. If you want to address this with [other] digital PCR platforms, which use only one size well [or] partition, it requires a huge number of partitions to achieve that goal.
"Basically we use wells with different volumes to achieve the same dynamic range with a much smaller total number of wells," Shen added. "In this case, we can spread the wells out so it's easy to image, and the chip itself is easy to fabricate."
Shen admitted that nanodroplet-based digital PCR platforms, such as those offered by freshly minted Bio-Rad subsidiary QuantaLife, can achieve a great dynamic range and can even generate discrete reaction volumes in a very short amount of time.
"But the analysis of the droplets, which use a flow cytometry imaging system, takes something like three to four hours," Shen said. "Also, the cost and complexity of the instrument are important, since we want to develop a platform for a resource-limited setting."
In the Analytical Chemistry paper, the researchers also describe software that can be used to design and analyze dilution series in digital PCR, making the multivolume technique adaptable to other digital PCR technologies currently on the market or under development — "anything where you can generate different volumes of droplets," Shen said.
In their second paper, published online Oct. 13 in the Journal of the American Chemical Society, the researchers demonstrate the use of their multivolume approach and rotational SlipChip to determine viral HIV and hepatitis C loads.
In particular, the researchers introduced another modified SlipChip design containing two additional well volumes of 0.2 and 625 nanoliters to further expand its dynamic range; and used it to quantify HIV viral RNA purified from clinical samples.
They found that SlipChip could be used to analyze a single sample at a dynamic range of 1.7 x 102 to 2.0 x 107 molecules per milliliter at a three-fold resolution with a limit of detection of 40 molecules per milliliter. Further, they showed that SlipChip results were self-consistent and in good agreement with results determined using Roche's Cobas AmpliPrep/Cobas TaqMan HIV-1 test.
The low end of SlipChip's dynamic range might be useful for HIV detection or viral load monitoring, Shen said; whereas the high end might be more useful for hepatitis, which often has viral loads of up to 1 million particles per milliliter.
The upshot, according to Shen, is that SlipChip steps in where there are "hardly any quantitative analysis methods with a three-fold resolution that are ideal for a resource-limited setting," where diseases like HIV and HCV are particularly endemic.
Having demonstrated the potential of their technology, Shen and colleagues a few months ago founded SlipChip LLC in an attempt to commercialize it.
The SlipChip technology as described in these papers still requires a thermal cycler and a basic imaging system to detect and analyze the positive PCR reactions, thus adding to the potential cost of an integrated point-of-care molecular testing platform.
However, the researchers have also published research showing how digital PCR on SlipChip could be combined with isothermal amplification methods, which would eliminate the need for a thermal cycler. In particular, in a paper published in April in Analytical Chemistry, they demonstrated combining SlipChip with TwistDx's recombinase polymerase amplification chemistry to count single molecules of DNA from methicillin-resistant Staphylococcus aureus.
"Also we are currently working … on a [much simpler] readout method, [where] you don't have to use a really fancy imager to do the readout," Shen said. "It would basically be like a color change or black and white dots. In addition, in the papers the device is fabricated from glass, but we have … a working prototype for a plastic SlipChip [that] can be mass produced at a much lower cost."
SlipChip, the company, is only a few months old, Shen said, and is currently located in the Incubator Laboratory Facility in the Chicago Technology Park. The company has thus far received an undisclosed amount of angel funding, a pair of Small Business Innovation Research grants from the National Institutes of Health, and some government contracts to support research and development.
Shen said that the University of Chicago has filed for patents on all aspects of the SlipChip design and the multivolume digital PCR method.
Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.