The microcavity biosensor that set a record by detecting the smallest single virus in solution has reached a new breakthrough: detecting a single label-free cancer marker protein. The achievement, which shatters the previous record and sets a new benchmark for the most sensitive limit of detection, may significantly advance early disease diagnostics. In 2012, Stephen Arnold of Polytechnic Institute of New York University (NYU-Poly) and colleagues at Fordham University and New York City College of Technology developed a novel microcavity biosensor treated with plasmonic gold nanoreceptors, which enhance the electric field of the sensor and allow even the smallest shifts in resonant frequency to be detected. Using this biosensor, they detected in solution the smallest known RNA virus, named MS2, which has a mass of 6 attograms (See: Whispering gallery sensor detects single viruses). At the time, the notion of detecting a single protein — phenomenally smaller than a virus — was set forth as the ultimate goal. “Proteins run the body,” explained Arnold, an applied physics professor at NYU-Poly and member of the Othmer-Jacobs Department of Chemical and Biomolecular Engineering. “When the immune system encounters virus, it pumps out huge quantities of antibody proteins, and all cancers generate protein markers. A test capable of detecting a single protein would be the most sensitive diagnostic test imaginable.” Researchers at the Polytechnic Institute of New York University illustrate the novel way they detected the BSA protein found in blood — even smaller than a single cancer marker. As the BSA protein lands on the gold nanoshell that is attached to a microcavity, the bumpy gold sphere acts as a nanoamplifier of the interaction, leading to an enhanced shift in the cavity’s resonance frequency. The charted waves show how the light wavelength shifts (red) once the BSA molecule lands on the nanoshell. Courtesy of NYU-Poly. Using a nanoenhanced version of the biosensor and experimental results by postdoctoral fellow Venkata Dantham and former student David Keng, the team detected two proteins: a human cancer marker protein called thyroglobulin, with a mass of just 1 attogram, and the bovine form of a common plasma protein, serum albumin, with a far smaller mass of 0.11 attogram. “An attogram is a millionth of a millionth of a millionth of a gram,” said Arnold, “and we believe that our new limit of detection may be smaller than 0.01 attogram.” To the surprise of the researchers, examination of their nanoreceptor under a transmission electron microscope revealed that its gold shell surface was covered with random bumps roughly the size of a protein. Computer mapping and simulations created by Stephen Holler, once Arnold’s student and now assistant professor of physics at Fordham University, showed that these irregularities generate their own highly reactive local sensitivity field extending out several nanometers, amplifying the capabilities of the sensor far beyond original predictions. “A virus is far too large to be aided in detection by this field,” Arnold said. “Proteins are just a few nanometers across — exactly the right size to register in this space.” The implications of single protein detection are significant and may lay the foundation for improved medical therapeutics. Arnold and his colleagues posit that, among other advances, the ability to follow a signal in real time — to actually witness the detection of a single disease marker protein and track its movement — may yield new understanding of how proteins attach to antibodies. Unlike current technology, which attaches a fluorescent molecule, or label, to the antigen to allow it to be seen, the new process detects the antigen without an interfering label. Arnold named the novel method of label-free detection “whispering gallery-mode biosensing” because lightwaves in the system reminded him of the way that voices bounce around the whispering gallery under the dome of St. Paul’s Cathedral in London. A laser sends light through a glass fiber to a detector. When a microsphere is placed against the fiber, certain wavelengths of light detour into the sphere and bounce around inside, creating a dip in the light received by the detector. When a molecule such as a cancer marker clings to a gold nanoshell attached to the microsphere, the microsphere’s resonant frequency shifts by a measurable amount. This summer, Arnold and University of Michigan professor Xudong Fan will collaborate under a $200,000 National Science Foundation grant to expand the capacity of these biosensors. The grant will support the construction of a multiplexed array of plasmonically enhanced resonators, which should allow a variety of proteins to be identified in blood serum within minutes. The research appears in Nano Letters (doi: 10.1021/nl401633y). For more information, visit: www.poly.edu