Researchers from the Center for Applied NanoBioscience and Medicine at the University of Arizona have published a paper describing a number of improvements to their fully integrated, microfluidic forensic device for rapid multiplex PCR-based DNA analysis.
The improvements include improved control systems for on-chip amplification, enhancements to the microfluidic chip design to improve sample movement through the device, and optimized on-chip PCR reagent storage.
With these developments, the researchers believe their system is now "close to commercialization" and may provide advantages in terms of workflow integration compared to competing platforms, research group member Frederic Zenhausern told PCR Insider this week.
However, the team is also searching for commercial partners or investors to help move the technology to market in the wake of the group's previous primary collaborator, the UK Forensic Science Service, dissolving earlier this year, Zenhausern said.
The University of Arizona team has for the past few years been working to fully integrate sample preparation, multiplex PCR, and capillary electrophoresis using microfluidics and electrochemical pumps into a handheld, disposable cartridge that can be inserted into a benchtop instrument to perform full short tandem repeat genotyping of human samples, from buccal swab to CODIS profile.
The platform shares several features in common, particularly the use of microfluidics, with competing platforms that are either on the market or in development from companies such as IntegenX, ZyGem and Lockheed Martin, or Network Biosystems. Each of these platforms also approaches multiplex PCR amplification and detection a little differently; for instance, the ZyGem/Lockheed Martin system uses infrared radiation for heating and cooling of a PCR cycle, while the Arizona team's system moves the reaction through different zones provided by a Peltier module.
The major difference, however, between the Arizona team's device and competing integrated microanalysis systems is the way the different stages of the forensic analysis workflow are integrated, Zenhausern said.
"The architecture is really focused on the sample-in/profile-out idea," he said. "We're not looking too much at the throughput here … but are really integrating the overall workflow process in a way that is completely self contained."
Zenhausern added that most competing platforms "have different modules for the different functions of the workflow, and then connect them in a way that is automated. In our system … there is still a lot of interface with the instrument … but our approach is really an integrated plastic cartridge in which you have all the fluidic management – the pump, valve, control center – and also the reagents. That's a little bit how our system is different from others — robust, simple, not as many moving parts."
However, while the system has performed well in previous proof-of-concept experiments, it still lacked several features that would make it truly automated and integrated — modifications that the team detailed in a paper published last week in the journal Analyst.
For instance, the group improved on-chip 17-plex PCR amplification of STR loci by improving control of the Peltier heating module, creating a more reliable microfluidic architecture, and, perhaps most importantly, integrating assay reagents into the PCR chamber.
To achieve this latter goal, Zenhausern and colleagues encapsulated PCR reagents into a solid phase material through a solid phase encapsulating assay mix, or SPEAM, bead-based hydrogel fabrication process. More specifically, they used a micro-SPEAM process from Cambridge, UK-based firm Q Chip to prepare large batches of reagent beads ranging in diameter from a few microns up to 2 millimeters that could be loaded into the PCR chamber of their device during cartridge assembly.
Commercially manufactured versions of the cartridge would theoretically already have these reagents encapsulated on board such that the cartridge could be stored in a refrigerator for up to three months without any degradation in quality or performance of the chemicals.
The researchers also improved the microfluidic circuitry of their system to address a previous problem they had encountered transferring sample between the plastic sample prep and PCR module and the glass CE module of their device, which would often cause a significant portion of the multiplexed amplicon to be lost.
The researchers also described changes to chip design to achieve "highly reliable metering and reduce evaporative loss during PCR thermal cycling," they wrote in their paper.
"Right now we're doing a lot of optimization," Zenhausern said. "The first step was, 'OK, can we do the workflow integration?' And the answer is yes, and we were among the first to demonstrate that. Now other people are also doing that."
"In the Analyst paper we are trying to address now beyond proof of concept: How do you integrate the system in terms of transferring that assay chemistry typically into a microfluidic platform … and how do you approach fabrication for making the device, et cetera?" he added.
Although much of the group's early work predated that of its competitors, Zenhausern admitted that "we have not been publishing much in the past year because of developments" with former collaborative partner, the UK Forensic Science Service, or FSS, which was UK government-owned company that provided forensic science services to UK police forces.
Under an EU Seventh Framework Program grant, the Arizona team and FSS were working with several other EU-based forensics groups to develop a prototype system for eventual deployment in various EU countries.
However, the FSS in March of this year underwent a reorganization that essentially broke the company into pieces, some of which were dispersed throughout various UK-based law enforcement agencies, Zenhausern said.
"That was a big deal," Zenhausern said. "They have been re-integrated in some capacity within the police forces … but they have in a way been dismantled from their operation the way it was before."
As such, the Arizona team was left to take over the development of its forensic analysis platform on its own, and is currently seeking corporate collaborators or investors in Europe or the US that might be able to help shepherd the system to market. The group is also considering building a startup company to commercialize the system.
In the meantime, they will continue to enhance the platform to keep up with current needs in forensic analysis, Zenhausern said. Chief among these improvements is the addition of real-time PCR capability, which would make the platform more conducive to dealing with casework samples such as blood-stained materials, as opposed to the buccal swab samples for which it is currently optimized.
"For the casework, we are integrating real-time PCR with the concept of still using CE as the endpoint, because you want to have a profile compatible with CODIS," Zenhausern said, adding that the group will publish its efforts in this area soon, and will present some early results at Select Biosciences' Lab-on-a-Chip World Congress later this month in San Diego.
The researchers have also been working with a group at Duke University and its Fitzpatrick Institute for Photonics to explore the use of surface-enhanced Raman spectroscopy for detection "so we can have a much more compact, portable device for things like pathogen detection," Zenhausern said.