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 shatters the previous record and sets a new benchmark for the most sensitive limit of detection – and could 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. They used it to detect, in solution, the smallest known RNA virus, MS2, which has a mass of 6 attograms. At the time, detecting a single protein – phenomenally smaller than a virus – was the ultimate goal. “Proteins run the body,” explained Arnold, a professor at NYU-Poly. “When the immune system encounters [a] 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.” 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. Whispering-gallery-mode biosensing enables detection of bovine serum albumin (BSA) protein found in blood – even smaller than a single cancer marker. As the BSA protein lands on the gold nanoshell attached to a microcavity, the bumpy gold sphere acts as a nanoamplifier, enhancing the shift in the cavity’s resonance frequency. The charted waves show how the wavelength shifts (red) once the BSA molecule lands on the nanoshell. ΔλR = the change in the whispering-gallery mode resonance wavelength; λR = the whispering-gallery-mode resonance wavelength; E0 = the electric field associated with the wave circulating in the whispering-gallery mode; I = the intensity of the laser signal transmitted through the tapered coupling fiber. Courtesy of NYU-Poly. “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.” When they examined their nanoreceptor under a transmission electron microscope, the researchers were surprised to find 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 an assistant professor at Fordham University, showed that these irregularities generate their own highly reactive local sensitivity field extending out several nanometers, amplifying the sensor’s capabilities 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. Current technology attaches a fluorescent label to the antigen, but the new process is label free. Arnold named the new technique “whispering-gallery-mode biosensing” because the behavior of the system’s lightwaves reminded him of a whispering gallery: A laser sends light through a glass fiber to a detector; when a microsphere is placed against the fiber, certain wavelengths 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. Arnold and University of Michigan professor Xudong Fan planned to collaborate this summer under a $200,000 National Science Foundation grant to construct 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 information on the previous record, see “Whispering Gallery Sensor Detects Single Viruses” at www.photonics.com/a52315.