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BU Team Demos Microfluidic 'Flu Chip' as Multiple Firms Seek to Commercialize Sample Prep Component


This article has been updated from a previous version to clarify that Wave 80 is evaluating the SPE technology but has not licensed it.

By Ben Butkus

Boston University researchers have published research demonstrating the ability of their single-use microfluidic chip to extract, PCR amplify, and detect RNA from influenza A virus with sensitivity comparable to or better than conventional reverse transcriptase PCR or direct immunoassay.

The work is an important step toward developing a chip-based point-of-care molecular diagnostic technology for the developing world, an endeavor that the research group is now undertaking, team leader Catherine Klapperich told PCR Insider this week.

In addition, the study provides important proof of principle for the nucleic acid sample prep portion of the platform — a solid-phase extraction technology being used by companies such as BioHelix and NobleGen for various molecular diagnostics and molecular biology research applications.

In the study, published in March in PLoS One, the BU scientists developed a single-use microfluidic chip integrating the SPE and continuous-flow reverse transcription PCR, and demonstrated its ability to amplify influenza A RNA from 146 human nasopharyngeal aspirate and nasopharyngeal swab specimens collected at two clinical sites between 2008 and 2010.

Their technology achieved 96 percent sensitivity and 100 percent specificity compared with conventional benchtop RT-PCR; and 98 percent sensitivity and 96 percent specificity compared with direct immunofluorescence assays.

The microfluidic-based test, nicknamed the "flu chip," was also able to extract and amplify influenza A RNA directly from the clinical specimens with viral loads down to 103 copies per milliliter in three hours or less, the researchers reported.

PCR Insider first reported on the technology's development in January 2010, following publication of an earlier proof-of-concept paper demonstrating a marriage of the microfluidic chip and an isothermal amplification method called helicase-dependent amplification, or HDA, to detect DNA from Escherichia coli (PCR Insider, 1/28/2010).

However, for its most recent study, the BU group moved away from the HDA method and used TaqMan-based PCR for amplification and capillary electrophoresis for endpoint detection, mostly as a way to demonstrate that its integrated microfluidic sample prep and amplification technology was up to snuff.

"We actually used the [Agilent] 2100 Bioanalyzer because we didn't want to reinvent the wheel," Klapperich said. "Our main concern is sample prep, so when we did this we said, 'OK, we are going to continuous flow RT-PCR, and we wanted to make sure that we had a number of different, independent ways of measuring what we were amplifying on the chip to make sure we were amplifying the product that we were intending to."

The main difference between the microfluidic chip and conventional PCR is that the chip contains a pair of stationary heaters at a constant temperature and a pump that moves sample through a "serpentine path" for thermal cycling.

"Not having to thermocycle the entire chip cuts down on quite a bit of the feedback control that you need," and thus cuts cost, an important consideration for a potential point-of-care technology, Klapperich said. "But if you were going to make this into something that would truly work at the point of care, an engineering company would have to make a box that can do those two things, [pumping the sample and providing different temperature zones] … which are much simpler and faster than thermocycling," she added.

Klapperich's lab still works with BioHelix on the sample prep front, and the company, which owns the isothermal HDA technology, has been developing its own integrated point-of-care molecular device based on SPE, HDA, and lateral flow detection for detecting sexually transmitted infectious agents such as Chlamydia trachomatis and Neisseria gonorrhoeae (PCR Insider, 10/13/2011).

As such, the BU team's most recent study lends additional credibility to the SPE sample prep technology that BioHelix is using. Klapperich noted that the group was particularly pleased with the technology's ability to extract and purify RNA samples for downstream amplification and analysis, which has in the past proven tricky.

BU has submitted patent applications for the technology that underlies the SPE columns being used in its microfluidic chip, and the school's tech-transfer office is working with other companies besides BioHelix to commercialize it.

For instance, early-stage next-generation sequencing company NobleGen has licensed some of the technology from Klapperich's lab to aid in the development of DNA sample prep in front of its nanopore-based sequencing technology for potential point-of-care clinical use, NobleGen CEO Frank Feist told PCR Insider this week.

NobleGen's sequencing approach uses a process called circular DNA conversion, or CDC, "which takes DNA and converts it into an expanded synthetic representation [in] a cyclical enzyme-driven process," Feist said.

The company currently automates that process using an "off-the-shelf liquid handler," Feist said. However, Klapperich is a co-principal investigator along with NobleGen co-founder Amit Meller on a four-year, $4.2 million grant from the National Human Genome Research Institute exploring single-molecule sequencing with the company's nanopore-induced photon emission technology (see PCR Insider sister publication In Sequence, 9/14/2010).

Klapperich's role in that grant is "development of a microfluidic chip-based device that essentially carries out that CDC chemistry process," Feist said. "That would enable us to basically do this whole sequencing approach of ours for a point-of-care solution. That's the nature of why we took a license to Catherine's work."

Another potential licensee is Bay Area molecular diagnostics startup Wave 80 Biosciences, which is evaluating the SPE technology as part of a point-of-care HIV viral load test. "We are extracting RNA from whole blood for [Wave 80], which is really close to field testing at this time," Klapperich said. "That's the closest the technology has gotten to a prospective field study with patients."

Wave 80 officials could not be reached for comment.

As far as the BU team's work goes, it would like to develop a simpler readout technique for its microfluidic chip as part of its goal of developing a true handheld, point-of-care molecular testing device that could be CLIA waived for use in the developed world and could be used by non-trained medical personnel in the developing world.

"Our next step would be the pretty simple task of how we read this out without taking it out of the chip," Klapperich said. "There are several ways to do that. Since we did a TaqMan assay, if you had a sensitive-enough optical reader, which exists, you just take a picture of the chip after so many cycles and you could quantify the fluorescence. You could also tack on a CE unit."

However, Klapperich added, "what we really want to do, since our goal is for global health applications, is to spit out the amplicons onto a paper readout and do a visible read. As a lab we'll probably go in that direction, but other people are interested in doing more of the real-time fluorescent reads. You could theoretically get really nice quantitative information from that."

Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.