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Poly-NYU Team's Whispering Gallery Resonance Mode Biosensor Achieves Detection of Single Proteins

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Researchers at the Polytechnic Institute of New York University have developed a biosensor capable of label-free detection of single proteins.

In a study published in the online edition of Nano Letters in June, the researchers used the device to detect single molecules of the thyroid cancer marker thyroglobulin and bovine serum albumin proteins.

According to Stephen Arnold, a professor of physics and chemistry at Poly-NYU and leader of the projects, based on the results of the study, the researchers "conservatively project that we could measure [single analytes as small as] 5 kilodalton."

The sensor relies on whispering gallery mode resonance, a phenomenon first observed in the whispering gallery of London's St. Paul's Cathedral where whispers can be heard throughout the room due to the travel of sound waves along its circular walls. In their device, the Poly-NYU team made use of the same principle, only in their case using light waves circulating throughout a microscopic sphere.

As with the sound waves at St. Paul's, "the optical wave comes around the sphere in phase, and you get a resonance of the sphere," Arnold told ProteoMonitor. And, if something – a protein, for instance – falls on the sphere, it changes the wavelength of that resonance, indicating its presence.

Such devices in and of themselves, however, are not sensitive enough to detect single protein molecules, Arnold said, noting that the resonance shift caused by such a target would fall well below the level of noise in the system.

To enhance the shift, then, the researchers attached gold nanoshells to the microspheres. These nanoshells act as a sort of amplifier, increasing the resonance shift observed upon the capture of a target molecule.

Using this approach, Arnold and his colleagues last year detected the 6 attogram RNA bacteriophage MS2 in a study published in Applied Physics Letters. When they went to apply the technique to proteins, though, they found it was even more sensitive than they had anticipated.

This, Arnold said, was due to the fact that the gold nanoshells used in the sensor aren't completely smooth surfaces, as the researchers had presumed, but, in fact, contain a number of irregularities inherent in their fabrication. These irregularities – small bumps on the surface, essentially – further enhance the signal generated by the target molecule.

And, because the enhancement is a function of how close to these bumps the target can get, the smaller the molecule, the larger the effect. For instance, for thyroglobulin, with mass of 1 attogram, the result was a roughly three-fold enhancement of signal, while for BSA, with a mass of 0.11 attograms, the effect was around 15-fold.

At that level of sensitivity, the device could detect single molecules "of any cancer marker that we could find," Arnold said.

He noted that the researchers had not tested the platform in a complex biological sample like plasma, but said that he believed "it wouldn't have a problem with that."

"There will always be problems if the surface is not prepared well or the antibodies [used to functionalize the device] are not appropriate," Arnold said.

Moving forward, he said he and his colleagues aim to develop systems to draw target analytes toward the sensors to speed the assays, likely via optical means.

"It's very difficult in a solution with salt in it to create an electrostatic field that can pull these [targets], but in a light field, something like optical tweezers, [which use lasers to attract or repel objects] has the ability to pull them in," Arnold said. He added that he had recently received a National Science Foundation grant for investigating this idea.

Arnold said that he is not interested in commercializing the technology himself, but he noted that San Diego, Calif.-based biotech firm Genalyte, which uses whispering gallery mode resonance in its Maverick Detection System, has enquired about his lab's gold nanoshell work.

Martin Gleeson, chief scientific officer at Genalyte, told ProteoMonitor that the company was not "pursuing any specific IP with respect to [Arnold's] research at this point," but that it is "certainly interested in incorporating as many technologies as possible to improve [its] systems, whether in-house or through licensing."

He said that he had not personally reviewed the Nano Letters study, but that achieving detection of single proteins "certainly is getting down to a great level of sensitivity, so that would be a significant step" for the technology.

Genalyte's Maverick device uses ring resonators as opposed to the spheres used by Arnold and his colleagues. The company offers chips consisting of 128 ring resonators functionalized in groups of four, providing researchers the ability to simultaneously monitor up to 32 different proteins while obtaining four data points for each analyte.

The system, Gleeson said, works in whole plasma, and has demonstrated more than eight orders of dynamic range with sensitivity extending into the pictogram per mL range.

The Maverick is currently available as a research-use-only instrument, and the company is putting together a 510(k) submission for a next-generation clinical instrument that it plans to submit to the US Food and Drug Administration in the first half of 2014.

The company has focused its internal clinical work with the platform on autoimmune disease, Gleeson said, but "this is fundamentally a platform technology and can be applied in many different areas."

The company has received "a lot of interest in using the technology in nanoparticle analysis, in looking at drug autoimmune responses, and a lot of interest where people have their own specific assays and analytes that they want to test on our system," he said.

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