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New 'Inverse Sensitivity' Nanosensor Generates Highest Signal at Lowest Protein Concentrations

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Researchers at Imperial College London and the University of Vigo have developed a new plasmonic nanosensor that produces signal in inverse proportion to the amount of target molecule present.

In a study published last month in Nature Materials the scientists described how they used the sensor to detect prostate specific antigen in whole human serum at concentrations as low as 10-18 grams per mL roughly an order of magnitude lower than existing sensor technologies.

According to Molly Stevens, professor of biomedical materials and regenerative medicine at Imperial College and one of the study leaders, the device is the first to utilize the "phenomenon of inverse sensitivity" and is, in principle, applicable to "detecting any protein as long as antibodies directed against it are commercially available."

The sensor achieves its inverse sensitivity by using an enzyme to control the level of signal generated by a gold nanoparticle-based detection platform. In such a sensor, biomolecule binding events trigger shifts in the nanoparticles' localized surface plasmon resonance that can be measured, allowing detection of the target molecule. Additionally, past work has shown that these LSPR shifts are increased when another metallic nanostructure is present near the original sensor nanoparticles.

Taking advantage of this phenomenon, Stevens and her colleagues developed an approach wherein they generated silver nanostructures in situ in order to heighten the LSPR shifts exhibited by the gold nanoparticles in response to a binding event. By devising the system to generate more silver nanostructures when the target molecule was in low concentration and fewer when it was at high concentration, they were able to build a nanosensor that generated higher levels of signal the lower the concentration of the target analyte.

The key to this was their use of the enzyme glucose oxidase, GOx, to modulate growth of the silver nanocrystals. When GOx is present in high concentrations, it leads to the development of freestanding silver nanocrystals instead of deposits of silver on the gold nanoparticles. When, on the other hand, it is present in low concentrations, it leads to the growth of a silver coating atop the nanoparticles, which increases their subsequent LSPR shifts during analyte binding.

The researchers used this phenomenon for biosensing by linking GOx to detection antibodies and incorporating it into a classic sandwich ELISA. The less target analyte present, the less GOx present and thus the more silver deposits and the more dramatic an LSPR shift.

Applying the system to the detection of PSA, the Vigo and Imperial College teams detected the protein at levels as low as 10-18 grams per mL – roughly one order of magnitude lower, they noted, than previously published ultrasensitive ELISA technologies, including commercial products like Quanterix's Single Molecule Array system.

The researchers have yet to test the system with additional proteins, but Stevens told ProteoMonitor via e-mail this week that they expect it will exhibit similar sensitivity for any analytes.

"We demonstrated that inverse sensitivity depends on the concentration of [GOx] enzyme regardless of the method used to immobilize it on the surface," she said. "Therefore, the method can be applied to any protein as long as antibodies are available."

The researchers intend to commercialize the technology and "are currently talking to potential investors or licensing partners," Stevens said, although she declined to identify any by name.

The technology should be fairly straightforward to manufacture, she added. "The fabrication of the gold [nanoparticles] is not complicated, and the chemicals needed are not very different from the ones utilized for staining an electrophoresis gel with silver. It should be fairly easy to commercialize."

Ultra-sensitive protein detection is an attractive technology given the low abundance of many important protein biomarkers. It is also potentially a route for expanding and improving use of existing protein markers – as in the case of Quanterix's AccuPSA test (PM 10/21/2011), for instance, or Singulex's efforts to use troponin – an established marker for acute heart damage – for long-term cardiac health monitoring (PM 12/17/2010). Ultra-sensitive devices could also prove useful for the study and diagnosis of neurological diseases, particularly for developing tests in plasma where markers linked to disorders like Alzheimer's are likely to exist in very low concentrations.

Quanterix and Singulex, both of which employ digital ELISA approaches, are currently the leaders in ultra-sensitive protein detection and, Stevens acknowledged, digital ELISA formats do have some potential advantages over the plasmonic system she and her colleagues developed.

"Digital ELISA utilizes a common ELISA format to achieve extreme sensitivity, and therefore it may be more user friendly than our method as it currently stands," she said, although she added that the researchers "are working on adapting it to the conventional ELISA procedure."

The technique also requires a longer time – roughly three hours – for signal generation, the authors noted.

However, Stevens said, the device's unique design makes it an interesting potential route for detecting extremely low-abundance proteins.

"The advantage of sensors working with inverse sensitivity is that the signal originated by the target analyte at ultralow concentrations is the highest, and therefore it is detected with the highest confidence," she said. "Therefore, these sensors are particularly suitable for detecting proteins at ultralow concentrations."

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