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Proof-of-Concept C. Diff MDx Device Uses Isothermal Amplification, SlipChip Microfluidics


NEW YORK (GenomeWeb) – Immunoassays and toxin tests for Clostridium difficile can be non-specific and insensitive, while culturing the bacteria requires special equipment, expertise, and has a slow turnaround time. Molecular tests of stool samples are the gold standard diagnostic for deadly Clostridium difficile infections, but are too costly for low-resource, point-of-care settings.

Researchers at the University of Southampton have now developed an isothermal microfluidics-based assay that doesn't require a power source, or a degree in biochemistry, to run. Described last month in Analyst, the proof-of-concept device uses SlipChip microfluidics and recombinase polymerase amplification, or RPA, to detect a gene of C. diff bacteria.

Infection with C. diff causes the death of about 14,000 people in the US each year, according to the US Centers for Disease Control and Prevention. Broad-spectrum antibiotics can sometimes lead this bacterium to overpopulate the colon, outcompeting natural gut flora, producing toxins, and causing infectious diarrhea. A low-cost device to quickly diagnose infection could save lives, particularly in locations without access to a clinical lab.

In an interview, Nefeli Tsaloglou, author of the Analyst report and now a Marie Curie fellow in the lab of George Whitesides at Harvard, said the prototype device was created using laser cutting and computer numerical control micromachining of poly-methyl methacryalte. Compared to the etched glass usually used for SlipChip, this high-throughput, low-cost production was novel, as was the fact that the device gives a real-time, quantitative read-out, Tsaloglou said.

The SlipChip method was introduced in 2009 by the lab of Rustem Ismagalov, then at the University of Chicago. Tsaloglou said the Southampton group had no affiliation with that lab. "We were just fans and admirers, so I tried to do something using that concept," she said.

The device was vetted on C. diff samples cultured by Public Health England, which Tsaloglou described as the UK-equivalent of the US Centers for Disease Control and Prevention. Development was funded by the National Institute of Health Research, part of the National Health System in the UK.

Interestingly, this work also spun off from a larger project which had funding from Sharp labs of Europe, an arm of Sharp Electronics. The Southampton group is currently developing digital microfluidics using electrowetting on dielectrics, working to miniaturize biological detection of proteins and nucleic acids on this platform, Tsaloglou said.

The RPA SlipChip was intended to run a quantitative assay, so the proof-of-concept model requires a custom confocal imaging setup. However, the device might be made fieldable with commercially available portable confocal systems, Tsaloglou said.

The assay also used a heater for the RPA reaction, but Tsaloglou noted that it did work at room temperature. "The quantification became more complicated, and my aim was to do the real-time quantification, so if you aim for [a qualitative] endpoint it can work at room temperature and you don't have to have heaters, you can just have insulators as layers on your device," she said.

TwistDx, a subsidiary of Alere, holds the intellectual property rights to the RPA technology.

Tsaloglou's future work in the Whitesides lab will focus on paper-based microfluidics, which she said might be the solution to the conundrum of commercializing miniaturized nucleic acid testing. Similar work is also being done by Catherine Klapperich and colleagues at Boston University, who are working on combining paper and blister packaging of PCR reagents.

"I think paper is a material that people are familiar with, so it might have wider impact in the public," Tsaloglou said. "It's based on the lateral flow concept like the pregnancy test that people already know how to use. And everything is there [for commercialization], the companies know how to market it, the more modern tests [are] quantitative, they're no longer qualitative, and that had been the issue with lateral flow."

The Whitesides group is currently developing paper-based microfluidics using micropads, or multilayer paper devices that have incorporated electronics, Tsaloglou said. "They're much more complicated than a simple piece of paper and they can have multiple flows," she said.