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UPenn Engineers Develop Instrument-Free Zika MDx, Immunoassay

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NEW YORK (GenomeWeb) – Researchers at the University of Pennsylvania have developed a microfluidics-based isothermal nucleic acid amplification device for low-cost Zika virus testing that runs without the need for electricity.

The research team — led by UPenn mechanical engineering professor Haim Bau — won $241,500 from the National Institute of Dental and Craniofacial Research earlier this month to build the test into a combined molecular diagnostic and immunoassay for Zika. The team also includes collaborators at the Instituto Conmemorativo Gorgas de Estudios de la Salud in Panama

The US Centers for Disease Control and Prevention recommends molecular testing of blood or plasma within the first week after a potential Zika infection and testing of urine for up to two weeks. After two weeks, the agency recommends immunoassays to detect host response to infection.

Of the 10 assays for Zika virus that have been granted Emergency Use Authorization from the US Food and Drug Administration, two are immunoassays and eight are molecular tests. The latter group includes six tests using typical real-time reverse-transcriptase PCR, a Hologic test that uses isothermal transcription-mediated amplification, and a Luminex test that uses the firm's bead-based multiplexing technology.

However, none of the molecular tests are instrument free, and there are no authorized assays that combine both molecular and immunoassay techniques.

The molecular component of Bau's assay uses a microfluidic chip combined with reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP). The group published a study in Analytical Chemistry earlier this year describing the device. In the study, the device used saliva samples, a chemically heated cup, and leuco crystal violet as the means for detecting reaction products by eye. Further, the reaction took less than 40 minutes to complete.

"We don't need to cycle the temperature, so the equipment that supports the test is much simpler than what is required for PCR," Bau explained in an interview with GenomeWeb. The device can also now support testing using any body fluid, such as saliva, blood, or urine, he said.

The assay has also been adapted to use fluorescent intercalating dye for quantitation, with a smartphone's flash used to excite the fluorescence and camera to monitor emission, Bau said.

One novel aspect of the test is its on-chip sample processing, which includes concentrating of the nucleic acids, which in turn leads to increased sensitivity.

"In our implementation the sample volume is decoupled from the reaction volume, and we can operate with sample volumes greater than the reaction volume," said Bau.

The microfluidic cassettes also contain four independent amplification reactors, which can potentially be used for positive and negative controls and co-detection of multiple targets, such as Zika and dengue viruses.

The heated cup uses an exothermic reaction commonly employed in ready-to-eat military rations, Bau said, which is essentially a magnesium alloy mixed with water to generate heat. And to regulate the temperature to the desired value, the system uses a phase-change material that melts at the incubation temperature.

"As an added bonus, you can also read the GPS coordinates where you carry out the test ... to tell you the density of Zika detection," Bau said.

In terms of the current grant, the test will now be developed to detect both RNA and antibodies. The immunoassay portion, which ultimately aims to distinguish between Zika and dengue viruses, will be more traditional, and that part of the project is currently ongoing.

At least three other groups have been developing combined molecular diagnostic and immunoassay systems to date. Rheonix recently received funding to develop its RT-LAMP and immunoassay system for Zika testing, Stat-Diagnostica is preparing to launch its PCR-based platform next year, and Fluxergy is adding assay modalities, including immunoassays, to a system that currently runs PCR. None of these systems is instrument free, however.

Meanwhile, groups such as PATH, Sandia National Labs, and AI Biosciences are developing low-cost, field-deployable molecular testing, but without the added immunoassay capabilities.

Antibody detection can be useful in part because it provides more of an historical record of exposure to Zika, Bau said, which can be particularly important to pregnant women.

The grant also includes collaboration with ICGES in Panama to verify the system's performance and ease of use with patient samples. Now that the funding has been awarded, the Panamanian research will require IRB approval at UPenn as the next step before moving forward.

The researchers are working out paths to commercialization that might include licensing, as various aspects of the device are subject to patent applications. "We, of course, are interested in commercializing the technology, otherwise there's no point in doing it," Bau said.

Future directions include a two-stage assay that uses isothermal recombinase polymerase amplification, or RPA, followed by LAMP, in order to further increase sensitivity and multiplex up to 16 targets from a single sample. This method also can be used for nested nucleic acid amplification, Bau said.

"That's where we're heading in the future ... to expand the Zika test to detect all mosquito-borne pathogens and maybe some other co-infections," he said.