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Warfare Agent Proof-of-Concept Platform Being Explored for Clinical Detection

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NEW YORK (360Dx) – UK-based University of Hertfordshire and the Defense Science and Technology Laboratory (DSTL) have developed a proof-of-concept digital microfluidic platform that detects four classes of simulated chemical and biological warfare agents on a chip — toxins, vegetative bacteria, bacterial spores, and viruses. Specifically, the system detected the human serum albumin protein toxin, Escherichia coli bacteria, the Bacillus atrophaeus spore, and the MS2 bacteriophage virus.

The research team built what it believes is among the first uses of an electrowetting on dielectric (EWOD) chip that enabled rapid and specific detection of the four common types of biomolecules and organisms.

EWOD sampling collects highly concentrated droplet samples that can now be measured with high levels of accuracy using the fully automated digital microfluidic research prototype, Loïc Coudron, one of the platform developers and a research fellow in digital microfluidics and biodetection at the University of Hertfordshire, said in an interview.

The developers are just starting to explore clinical applications for the platform but they believe that with appropriate funding, the platform could be made available in a few years for testing at the point of care in remote locations where central laboratories are not available, Coudron said.

In the current issue of Biosensors and Bioelectronics, the researchers described the development of a fully integrated portable platform compatible with potential for future use in-field deployable biodetection systems.

The digital microfluidic platform and its enzyme-linked immunosorbent assay (ELISA) selectively captures antigens using antibody-coated magnetic beads. A secondary detection antibody measures the emission of light from a chemiluminescent reaction. The process requires a sequence of movements associated with the effective extraction, re-suspension, and mixing of the magnetic beads within 2.5 µl droplets, the developers said.

The same type of technology has already been used in remote settings for clinical diagnostics by other research groups, Coudron noted. He pointed to the work of an international group which published the results of a study last year in which they used an optimized inkjet-printed microfluidic cartridge and a portable control system to perform serological immunoassays. They tested for vaccine-preventable measles and rubella from blood obtained from adults and children in Kakuma, a refugee camp located in remote northwestern Kenya.

Their digital microfluidic devices used electrostatic forces to mix and separate reagents and samples in small droplets of fluids and measured IgG antibodies for measles and rubella, enabling simultaneous testing of four samples in 35 minutes. The IgG assays demonstrated sensitivities of 86 percent and specificities of 80 percent when the researchers compared their results with those of reference tests conducted in a central lab, according to the study. 

Detection using the microfluidic device was less sensitive and specific than laboratory-based ELISA testing in matched serum samples, but the results nonetheless demonstrated the potential for use of digital microfluidics in serosurveillance, particularly in areas where centralized laboratories are unavailable, the researchers said.

Ryan Fobel, cofounder and CEO of Sci-Bots, who participated in the Kenya field study, said in an interview that the instrument and methods described in the paper written by Coudron and his colleagues "are similar to those used in our research — a magnetic bead-based sandwich ELISA." The main difference between the two platforms, he said, is the new study's application to the detection of airborne pathogens.

"I'm confident that the detection scheme can work," Fobel said. "The tricky part is finding the right trade-off between assay time, sensitivity, and specificity for the given application."

Digital microfluidics adoption

To reduce costs for lab-on-a-chip microfluidic tests, researchers generally are seeking to either improve the performance of paper-based lateral flow assays or to develop inexpensive methods for manufacturing high-performance microchannels and related components. These methods are usually limited to measuring one patient sample at a time, which can present a bottleneck when large populations of people need screening.

Among the advantages of the digital microfluidic platform, according to the developers of these systems, is that it enables concurrent droplet operations — including merging, mixing, splitting, and metering from reservoirs­ — allowing processing of several samples in parallel.

"Digital microfluidics is one of the most versatile techniques for automated liquid handling out there," Fobel said. "What makes it unique is that the relatively generic chips can be reconfigured through software. This gives digital microfluidics the potential to be a universal instrument, a fact nicely demonstrated by [the University of Hertfordshire] paper showing the detection of four distinct classes of pathogens on a single chip."

Fobel said that the field's coming of age is evident not only in recent field studies but also in commercially available diagnostic systems cleared by the US Food and Drug Administration, such as GenMark Diagnostics' ePlex system for diagnosing respiratory and blood stream pathogens, and Baebies’ newborn screening platform.

In the area of warfare agent detection, the US government has been funding projects based on alternate technologies.

The US Department of Health and Human Services' Biomedical Advanced Research and Development Authority (BARDA) approved a two-year, $10.7 million extension of a multiyear contract with First Light Biosciences (now called First Light Diagnostics) for development of its automated MultiPath Platform, which uses digital imaging and rapid tests to detect anthrax poisoning.

Under a three-year, $8.1 million contract sponsored by the HHS Biomedical Advanced Research and Development Authority, InBios International is developing a point-of-care diagnostic test that could determine within minutes whether a patient has been infected with the Bacillus anthracis bacterium that causes anthrax. 

And, as part of a contract with the US Department of Defense, researchers at the University of Arizona and University of Nevada are developing a point-of-care immunoassay — in which a fluid sample is diffused through a nitrocellulose membrane — as an alternative to a standard method involving the culturing of samples to identify threatening biological agents.

Commercial aspirations

The University of Hertfordshire researchers have made good progress with the development of the platform for detecting biowarfare agents and are continuing their collaboration with DSTL, part of the UK Ministry of Defense, but more extensive development work is needed to develop it for clinical applications, Coudron said.

Their platform is achieving levels of sensitivity required for clinical diagnostics, but they need to refine its interface so that it can be easier to use, he said. For diagnostic applications, the group is also looking to engage with clinical entities that can start to test the platform using patient samples, and it needs to conduct an analysis of potential routes to market.

It is also seeking knowledge transfer agreements or collaborative R&D initiatives to advance the platform's development, and it is looking to apply methods it developed in earlier projects to improve the user interface.

Further, the researchers are exploring supplying their technology as a brick, or system building block, to entities doing lab-on-chip system development who would then use it to develop applications. Companies that offer this type of service are beginning to pop up, Coudron said.

For example, Fobel's Sci-Bots already provides an open, digital microfluidics platform — including hardware, software, and consumables — to enable rapid development of portable, automated lab-based applications.

The firm is seeing "growing interest within the academic research community, but an increasing share of our sales are going to startups and more established industry players who are interested in developing new products using digital microfluidics technology," Fobel said.

Coudron said that because his group developed its platform as a proof of concept for defense applications, keeping down overall cost had not been a design constraint. However, in developing the technology for applications in clinical diagnostics, where it can in theory detect any disease for which a suitable antibody is available, reducing costs and thus the overall price is important, he added.

Consequently, the group is looking to identify options to mass produce inexpensive, disposable microfluidic chips for the diagnostic platform. The researchers are exploring the fabrication of disposable inkjet-printed microfluidic substrates and microelectronic components such as electrodes.

Reduced cost can lead to less functionality, so the system configuration also depends on the level of integration and functionality that a user requires, Coudron noted.

"Based on my own experience, it takes a huge team of people and a great deal of resources to go from a lab-based prototype like the one shown [by Coudron and his colleagues] to something that can operate in a difficult field environment at a scale appropriate for a field demonstration involving hundreds to thousands of samples," Fobel said. "And there are plenty of challenges in translating this type of research into a real-world product. However, if there is enough demand and if someone has the resources to throw at it, these are all solvable problems."