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UPenn Researcher Developing Non-Instrumented Sample-to-Answer HIV Viral Load Test

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University of Pennsylvania engineer Changchun Liu is planning to use a mentored career development grant from the National Institute of Allergy and Infectious Diseases to develop a non-instrumented, isothermal, microfluidic sample-to-answer assay to detect HIV virus.

Liu received $128,358 from the NIAID for his project, which will involve fully integrating an exothermic heating cartridge with a microfluidic single-chamber nucleic acid amplification cassette that uses a porous membrane to isolate and concentrate target molecules — both of which he has developed previously. He will also work to integrate sample preparation including HIV virus lysis and RNA isolation and concentration into the microfluidic system.

Coming from a background in chemistry and mechanical engineering, Liu told PCR Insider that the mentorship structure of his project, including relationships with several microbiology and infectious disease experts, will provide important support for the further development of the assay.

"I think it's very important to develop a low-cost [point-of-care] device for developing and developed countries for HIV, so I proposed this project." Liu said.

Liu said that he plans to continue refining his single-chamber device, which uses loop-mediated isothermal amplification, or LAMP. He described the cartridge as a multi-function enzymatic amplification reactor that relies on a porous membrane to allow nucleic acid isolation, purification, and concentration without the need for an elution step.

Liu and his colleagues have published several papers demonstrating aspects of the device and its ability to detect not only HIV, but also Escherichia coli in saliva. The group has also used the system to detect E. coli in stool; as well as to detect the infectious agents that cause herpes, tuberculosis, and malaria — all work that has not yet been published.

"I think for this platform, HIV detection is only one [of many] applications," he said.

Liu published a report last year in the journal Analyst that described using the membrane cassette to amplify HIV nucleic acids and detect HIV-1 in oral fluids with a detection limit of less than 10 HIV particles per sample.

In the study, Liu's group also tested the membrane-containing amplification cassette against a LAMP process without the membrane, showing that the membrane did not adversely impact the efficiency of LAMP, and even improved the detection limit about a thousand-fold by allowing the removal of potential inhibitors from the saliva sample.

In another paper, published in Lab on a Chip last July, Liu and his colleagues described the self-heating element of the device, a "thermal battery" that uses a wicking filter paper to control the flow of water into an exothermic reactor where magnesium reacts with water in the presence of iron. This is housed in a paraffin frame that acts as a phase-change material, regulating the chamber's temperature.

In this report, Liu showed that the reactor was able to maintain the 65° C reaction temperature necessary for LAMP reactions in external temperatures between 20° C and 40° C. He and his group also demonstrated that they could modify the reactor temperature by using wicking filter paper strips of different widths, suggesting the heater could be adapted to operate at different temperatures to support different methods of isothermal amplification.

Liu said that under the NIAID grant, he plans to more fully integrate the isothermal amplification chip and the exothermic heating mechanism. He will also work to integrate more sample preparation into the system, including lysis and RNA isolation, by incorporating buffer-containing pouches with thermally actuated valves.

"In my prior work, I have developed thermally actuated PDMS valves for sealing the amplification reaction chamber," Liu wrote in an e-mail. "Here, I will use [the] thermal battery in place of electric heaters to actuate the pouches and valves. For fluid pumping, the actuation chambers (or pump chamber) underneath the pouches will comprise expandable, Expancel microspheres embedded in paraffin."

Of the early tests of the system described in the Analyst and Lab on a Chip papers, Liu and his colleagues wrote that they believe the device could achieve an even lower detection limit than ten molecules per sample — possibly down to a single molecule — but that demonstrating this will require a large number of future experiments. In the meantime, the group wrote, "the performance of the device appears to be comparable to that of the state-of-the-art benchtop PCR machines."

In the studies, Liu and his colleagues used a basic visual fluorescence detection method using a mini reusable UV light-emitting diode keychain. But this method can only provide qualitative results, they wrote.

Liu said that, moving forward, he plans to work on a camera-based detection system, developing a software application for smart phones to analyze and quantify fluorescence. While this is still in the works, Liu said preliminary experiments have indicated that this is feasible.

Additionally, the group reported that by building more flow-control capabilities into the chip to discharge the amplification products, the system could be used with lateral flow strips to provide semi-quantitative detection.

Liu's is not the only group working on self-heated nucleic acid amplification assays for HIV and other pathogen detection. A group from PATH, led by Paul LaBarre, demonstrated another "equipment-free" nucleic acid assay in a paper in PLoS One last year (PCR Insider 6/9/2011).

The PATH group's device is much larger than Liu's microfluidic system, and unlike Liu, the PATH team has not yet reported on specific plans or methods to integrate chemical heating-driven sample-preparation steps, though they told PCR Insider at the time that separate teams are working on removing the requirements for electricity for sample preparation and detection.

Liu estimated that the full device would end up costing below $10 and could "be minimized by mass production through simple plastic manufacturing methods such as injection molding."

According to Liu, while the work is still early, his group has been approached by some companies interested in working with him to commercialize the device. He did not detail any of these potential partners.

But generally, he said, if the team can demonstrate the reliability of its microfluidic chip, the device could be commercialized "in the near future."

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