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Researchers Developing Plasmonic Patch to Increase Sensitivity of Fluorescence Tests

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NEW YORK (360Dx) – Researchers at Washington University, St. Louis, and Wright-Patterson Air Force Base have developed a plasmonic patch that they believe could be applied to improve the sensitivity of a broad range of research and clinical diagnostic tests.

Although fluorescence-based detection and imaging techniques are convenient to use, the weakness of the fluorescence signal remains a challenge in detecting and quantifying analytes that have low abundance in biological species, Srikanth Singamaneni, a WUSTL professor and one of the developers of the technology, said in an interview.

By applying a plastic film integrated with metal nanostructures as a final step in assay development, diagnostic test developers could use the plasmonic patch to increase fluorescence intensity a hundredfold in existing instruments and assays, Singamaneni said, improving sensitivity and the limit of detection by two to three orders of magnitude over existing tests.

The plasmonic patch intensifies the fluorescent signal used for detecting antibodies, molecular biomarkers, and analytes in diagnostic tests. It would be particularly useful when patients carry low levels of antigens in the blood or urine and the fluorescent signal is feeble, making diagnosis difficult, the developers of the technology said.

Improving the signal-to-noise ratio of the assays without deviating from existing assay protocols has a range of potential benefits for diagnostic tests, the researchers said. It will lower the cost of implementing tests, "eliminate cross-laboratory and cross-platform inconsistency, and potentially propel these technologies to use in point-of-care, in-field, and resource-limited settings," they said in a recent paper published in Light: Science & Applications.

In it, they describe a proof-of-concept technique that developers can add to various fluorescent surfaces. Their work has primarily focused on the introduction of the plasmonic patch concept and on demonstrating its application in enhancing fluoroimmunoassays implemented in microtiter plates and microarrays. However, the technique has implications in bioimaging, blotting, histology, and virtually any application involving fluorescence, Singamaneni said. It can be readily adapted to a number of different fluorescence-based technologies and has potential for use at the point of care and in resource-limited settings.

Brian Cunningham, director of the micro and nanotechnology laboratory at the University of Illinois at Urbana-Champaign said in an interview that "the plasmonic patch demonstrated by the Singamaneni group represents a simple, elegant, and highly effective means for amplifying the output of any surface-based fluorescence assay, which is broadly used for diagnostics and life science research applications."

The paper demonstrates the ability to design plasmonic metal nanoparticles with resonant properties for a wavelength that assures strong coupling interactions with a fluorophore, said Cunningham, who is not involved in the development of the plasmonic patch.

Change is not good

The problem with existing methods that seek to enhance signal properties is that "you are always asking people to change what they are already using," Singamaneni said. With existing techniques, people can't purchase a 96-well polystyrene plate with antibodies and easily enhance its fluorescence intensity for diagnostic testing, and developers looking to significantly enhance fluorescence, he noted, must use a special type of surface — a metal nano-island or photonic crystal surface, for example.

"Naturally, there is some reluctance to use that, and with good reason," he said, adding, "Once you change the plate surface chemistry, optimizing antibody immobilization becomes a challenge, and sometimes the antibody will not perform as well as it would on a polystyrene plate."

To solve this problem, the new technique enables developers to improve fluorescence sensitivity by placing the thin elastomeric film with integrated metal nanostructures on top of the plate.

"That leads to a large enhancement of fluorescence," Singamaneni said. "The most important thing here is that we are not asking people to change what they are doing."

He and his colleagues have launched a company, Auragent Bioscience, which has licensed the technology from Washington University, and it is working to scale up the proof of concept and start commercialization.

Their first product will be optimized for use with protein microarrays, and it should be available within nine months for research use. "We want to sell the first product for research use and convince the research community that it is a convenient and easy tool," he said. When that has been accomplished, the group anticipates developing products that can be applied in clinical diagnostics, but that could take at least three years to accomplish and require regulatory approvals.

"This plastic film doesn't care about what's underneath it," Singamaneni said. "It can be a DNA, RNA, or protein microarray, for example. If there is fluorescence and your nanoparticles are properly designed, you'll see 100-times enhancement."

In addition to scaling up their existing proof of concept, the researchers are looking into testing applications other than microtiter plates and microarrays, including flow cytometry and Western blotting.

In applying the plastic film, some processing of the polymer is required, "but nothing too difficult," and that lends to the technology's scalability, Singamaneni said. The “ready-to-use” technique can be integrated with current biomedical research and clinical infrastructure to generate immediate results and impact, the researchers said.

Cunningham noted that the patches can be "reproducibly and uniformly fabricated over large surface areas, cut to any desired size, and easily manipulated to place on glass slides, into the wells of microplates, or any other assay surface." The publication further describes how metal nanoparticles can be passivated with very thin dielectric thin films to prevent metal quenching of fluorophores. "It represents a simple, inexpensive, and robust way to improve the detection limits of broad classes of assays," Cunningham added.