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Stevens Institute Team Using $320K NSF Grant to Advance Microgel Molecular Beacon MDx System

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NEW YORK (GenomeWeb) – Researchers from the Stevens Institute of Technology have received $320,000 from the National Science Foundation to support the development of a multiplex molecular diagnostic system based on microgel-tethered molecular beacon hybridization probes.

The group, led by professor Matthew Libera, believes it has developed a way to retain the sensitivity of molecular beacon probes on a solid surface, something that has been difficult to achieve. In doing so, the team also hopes to eventually create a molecular platform to detect and diagnose infectious diseases with greater throughput than current technologies, Libera told BioArray News.

According to Libera, current non-molecular methods for diagnosing infectious pathogens can take up to 72 hours or more, resulting in inaccurate, delayed, or ineffective treatment. New molecular technologies have reduced the time required for diagnosis, in some cases down to just hours or less. However, the complexity and lack of throughput of many of these technologies has hindered widespread clinical use, particularly in large hospital settings.

Under their new grant, Libera and his colleagues intend to complete work they have already begun to couple a nucleic acid amplification method called nucleic acid sequence-based amplification, or NASBA, with a real-time, self-reporting array-based detection method that relies on molecular beacon hybridization probes tethered to highly hydrated electron-beam-patterned polyethylene glycol microgels. The researchers believe that diagnostic devices based on this approach could have less complexity and higher throughput than existing technologies for molecular diagnosis of infectious diseases.

According to the group’s grant abstract, the microscopic hydrogel and molecular beacon approach allows for the use of a very small amount of target DNA for each test, meaning throughput can be increased significantly.

The team described earlier development of its system of surface patterned microgel-tethered molecular beacons in a 2012 paper in Soft Matter.

In that study, Libera and his coauthors described how they used focused electron beams to create surface-patterned microgels to which molecular beacon probes could be immobilized via biotin–streptavidin binding, with the aim of maintaining the high performance typical of untethered molecular beacon probes free in solution.

Unlike typical fluorescent tagging detection methods, molecular beacon probes are self-reporting, becoming fluorescent upon binding with their target. Because of this ability, they have become an attractive tool, and in solution-based hybridization assays, they have shown high sensitivity and specificity and a high fluorescent signal relative to their background.

However, this signal-to-background ratio has broken down when researchers have attempted to immobilize these probes onto solid substrates in order to create a microarray format for more multiplex detection.

Libera and his colleagues believed they could enhance the performance of surface-bound molecular beacons by making the surface they are bound to as wet as possible using microgels.

"People have tried to use [molecular beacons] in array format, binding them to solid surfaces like spotting microarrays, but they haven’t worked very well because of interactions between the molecular beacons and the solid surface," Libera said. "We have been able to avoid that by grafting microgels to a solid surface."

He added that the microgels are "fixed like a microarray, but they are almost like microscopic soft contact lenses, and they swell and absorb a lot of water so the molecular beacons remain in a very hydrated water-like environment. [The sensitivity] is not as good as when they are free-floating in solution, but it’s much better than covalently binding them to a solid surface."

In its 2012 report, the Libera lab team assessed the performance of its microgel-tethered molecular beacons using beacons designed to distinguish between methicillin-sensitive and methicillin-resistant Staphylococcus aureus. The group measured a signal-to-background ratio between 40 and 50, lower than typical for beacons in solution, but "substantially higher than many other surface-tethering approaches," according to the authors.

The team also reported in the 2012 paper that the platform exhibited both low non-specific background and high specific fluorescence when the microgel-tethered molecular beacons were exposed to multiple targets, suggesting that the system would "lend itself well to high-sensitivity, self-reporting oligonucleotide-based assays."

Moving forward, Libera said he and his colleagues plan to use the new NSF funding to demonstrate that they can successfully couple their microgel-tethered molecular beacons with target amplification, using NASBA to sensitively detect pathogen targets.

He said the team also plans to do additional work to try to tether the NASBA process itself to the microgel surface, essentially binding the NASBA primers to the gel surface in close proximity to the molecular beacons so that amplification and detection both take place on the microgel.

The group is in conversation with clinicians at several centers to plan an evaluation of the platform using clinical samples with known infection status, and is seeking other collaborators, particularly those who are interested in commercialization opportunities.

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