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University of Missouri Team Seeks to Outlicense Reusable, Microscale PCR Device

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Scientists from the University of Missouri have developed a microfluidic chip and high-conductivity agarose gel that could enable a highly sensitive, portable, and reusable nucleic acid testing device for point-of-care and field applications.

Having recently secured a key US patent covering the device, MU's Office of Technology Management and Industry Relations is now offering the technology for license, claiming it has broad applicability in the medical, agricultural, veterinary, and environmental testing markets.

In addition, one of the technology's inventors, Shantanu Bhattacharya, now employed by the Indian Institute of Technology, Kanpur, is attempting to develop the technology as a low-cost, portable nucleic acid detection system to test food for contaminants such as Escherichia coli and Listeria monocytogenes.

In an interview with PCR Insider this week, Bhattacharya said that he and colleagues at MU originally began developing their micro-PCR device as a potential tool for the agricultural industry to diagnose diseases in the field, particularly porcine reproductive and respiratory syndrome and infectious bovine rhinotracheitis.

The group started with the idea of doing PCR on a smaller scale and detecting end products using capillary electrophoresis, which is "not so uncommon," Bhattacharya said, "but we added the development of a micropump … and a microchip that we fabricated … in order to test all these assays in the diagnostic lab" in the animal hospital of the University of Missouri-Columbia College of Veterinary Medicine.

Eventually the researchers, several of whom had engineering and microelectromechanical systems backgrounds, added several innovations from that field, the first of which was a heater design for thermal cycling that relied on a 2D heat transfer equation model and enabled precise temperature control of +/- 1° C.

Then, the team developed what may have been the most important component of the device: a biofunctionalized thin film coating for the surface of the chip comprising layers of polydimethylsiloxane, silicon, and a coating called "spin-on glass" commercially available from semiconductor manufacturing firm Filmtronics.

More specifically, the PDMS layer, which contains at least one microfluidic path, is irreversibly bonded to the spin-on glass layer of the silicon substrate using oxygen plasma treatment. The end result is a miniaturized DNA amplification system with a highly hydrophobic interior that confers several advantages over other chip-based DNA amplification schemes.

First, the device is able to amplify extremely small amounts of genetic material, down to as little as 1 picogram per 5 microliters of sample. In addition, the chip is reusable: After a first DNA amplification procedure is performed, the device can be easily washed using a specially formulated buffer because the chip does not suffer from non-specific binding of DNA to its surface.

"Simply by washing we don't experience any cross-contamination [between samples], even though a separate PCR reaction was just done on the device," Bhattacharya said.

Filmtronic's spin-on glass material is a key enabling technology, and although it has been used in various MEMS systems, "we developed our own method for making it biofriendly and fabricating it in the device, and it has never been used for this purpose," Bhattacharya said.

Currently, the temperature of the thermally variable layer of the chip is controlled with a proportional-integral-derivative controller using a thermocouple temperature sensor, and the scientists have been able to use this technique to reduce temperature ramp-up and ramp-down times to nearly one-tenth that of conventional thermal cyclers.

Because the system conducts endpoint PCR, the researchers developed yet another enabling technology in the form of an agarose gel formulation containing platinum nanoparticles that serve to greatly increase the mobility of DNA during capillary electrophoresis-based detection of PCR products.

The researchers believe that this innovation, coupled with the DNA amplification chip and optical waveguide technology, could form the basis of a complete and highly compact DNA testing platform for true field or point-of-care use.

This week, MU received US Patent No. 8,173,077 covering the reusable PCR amplification device (see IP Watch, this issue). Another patent covering the platinum nanoparticle-enhanced agarose gel formulation is still pending.

In an e-mail to PCR Insider, UM researcher and technology co-inventor Keshab Gangopadhyay said that the new patent "will distinguish us from competitors" due to its "unique innovations through unique heater design, novel use of [spin-on glass], reliability, reusability, et cetera. We also believe the other utility application of enhancing the DNA mobility through [platinum nanoparticles] will add a value proposition, which many companies may need to license as they may use [nanoparticles] for their application[s]."

UM's tech-transfer office has made the technology available for licensing, marketing it as enabling the application of PCR to "on-site analysis of samples with minimum sample input" with commercial implications for health, medicine, agriculture, veterinary, and environmental uses.

In the meantime, IIT-Kanpur's Bhattacharya has obtained permissions from UM and the technology's inventors to explore using versions of the device for various nucleic acid testing applications, primarily in the food safety testing market, but also in the area of detecting antibiotic resistance in diseases such as urinary tract infections.

In addition, Bhattacharya and colleagues are working on developing a real-time PCR version of the device using SYBR Green chemistry; as well as methods to multiplex the device.

"We have tried to do PCR using pathogenic bacteria such as E. coli using specific primer design and various [sample] concentrations" to gauge the specificity of the method," Bhattacharya said. "Using a mix of three or four strains of bacteria, we were able to detect one of them, so the specificity is pretty good." If multiple samples and primers are used, he added, "then the multiplexing potential is there."

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