NEW YORK (GenomeWeb) – Since developing a microfluidic, continuous-flow, quantitative reverse transcriptase PCR system to detect viral RNA targets, researchers at Fraunhofer USA, a subsidiary of Germany's Fraunhofer Society, have been working to reduce manufacturing costs and identify potential partners for commercialization.
In a study published in November in Analytical and Bioanalytical Chemistry, the researchers used the qRT-PCR system to detect the L-gene, a biomarker for the Ebola virus, in a spiked liquid sample. The Boston-based group now aims to develop point-of-care (POC) devices based on the technology to diagnose infectious disease in low-resource areas.
The microfluidic chip contains a syringe pump, heaters, and an optical detection system. Researchers use the syringe pump to introduce the liquid sample and PCR reagents into the system through a microfluidic connector.
The purified RNA solution enters a mixing chamber and is then combined with the master mix, which contains the enzymes and other reagents required to conduct amplification.
The mixture travels through a reverse transcription zone, where reverse transcription enzymes convert RNA to complementary DNA.
The researchers tested two one-step reverse transcription PCR (qRT-PCR) mastermixes. The team first used the commercial Affymetrix (now a Thermo Fisher Scientific brand) VeriQuest master mix, which is composed of reverse transcriptase and DNA polymerase. The team also developed a custom-made one-step master mix that combines Affymetrix's VeriQuest master mix and the fast DNA polymerase included in Affymetrix's USB fast quantitative PCR probe master mix.
"We made our own [master mix] by mixing these two products because we weren't satisfied with the speed of the premade master mix, [and] when we tested the mixture in our system, it indeed worked better," explained Alexis Sauer-Budge, corresponding author and a former senior research scientist at Fraunhofer who is now employed at a private health consulting firm.
The newly formed complementary DNA and reagents travel through a 95° Czone, whereDNA polymerase activates and the double-stranded DNA is melted. The sample then travels to the thermal cycling area, moving through 40 repetitive cycles and between areas heated to 95° and 62° C. The DNA undergoes melting at the higher temperature zone portion of each loop, whereas annealing and polymerization occurs at the lower temperature zone. The total chip channel volume is 25 microliters, allowing low reagent consumption and therefore reducing consumable costs.
The optical detection system can then be used to measure fluorescence and real-time DNA amplification.
The team demonstrated that that the system performs qRT-PCR in 30 minutes, compared to 80 minutes for standard conventional benchtop thermocyclers. Although the researchers determined that their custom master mix generally delivered better results, they determined the assay's limit of detection using the commercial mix because it would be more readily translated to the market. Using the commercial mix and the Ebola virus RNA as the target, the team acheived a limit of detection of 10 RNA copies per microliter.
Christine McBeth, a coauthor on the study and senior research scientist at Fraunhofer, admitted certain limitations in the study, including challenges with the chip's flow speed. Bubble formation in the microfluidic channels caused elasticity and inhibited the flow of liquid.
"Because it's continuous flow PCR, the chip is based on syringe pumps," McBeth explained. "Getting the flow to be consistent through the entire device and not have bubble formation due to nucleation or temperature is one of the things we ran up against and had to optimize."
In the future, the authors plan to focus on sample pretreatment for RNA extraction from plasma, urine, and saliva, as the extraction of high-quality RNA plays a crucial role in producing successful qRT-PCR results.
The team aims to market the device at a price of $2,000, with disposable test chips for $0.50. Andre Sharon, director of Fraunhofer USA's Center for Manufacturing Innovation, explained that while Fraunhofer is a research and development organization, it is seeking both industrial and academic partners to field-test its device. With the device currently in proof-of-concept stage, the group is debating whether to license its IP to a partner company, or to spin off a company via a startup.
"As we bridge the gap between academics, basic research, and moving the device to market, we spoke to venture capitalists and found that … until the cost of cartridges are hitting pennies per chip… you're not going to get [the chip] to the market," McBeth said.
McBeth elaborated that her team is working on finding material thin enough to use in roll-to-roll manufacturing as well as improving elements such as bonding and ensuring a tight seal on the microfluidic chips. The team is also partnering with another Fraunhofer Society organization, Germany's Fraunhofer Institute for Surface Engineering and Thin Films to increase roll-to-roll manufacturing and cheapen the microchip cost.
Importantly, McBeth and her colleagues at Fraunhofer are aiming to apply isothermal amplification techniques into their platform, since that will allow reduced instrumentation and minimize usage of syringe pumps. While limitations such as a lack of quantification hinder isothermal reactions, McBeth believes that there is room for both types of techniques due to their advantages in the POC field. Her team is therefore pursuing opportunities to use both technologies in its platform.
The authorsbelieve that the platform holds promise for the detection of other RNA-based viruses such as Zika and chikungunya viruses, which comprise global health threats worldwide.