A nanotechnology holy grail in label-free cancer marker detection: Single molecules

QDOTS imagesCAKXSY1K 8(Nanowerk News) Just months after setting a record for  detecting the smallest single virus in solution, researchers at the Polytechnic  Institute of New York University (NYU-Poly) have announced a new breakthrough:  They used a nano-enhanced version of their patented microcavity biosensor to  detect a single cancer marker protein, which is one-sixth the size of the  smallest virus, and even smaller molecules below the mass of all known markers.  This achievement shatters the previous record, setting a new benchmark for the  most sensitive limit of detection, and may significantly advance early disease  diagnostics.  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.
Stephen Arnold, university professor of applied  physics and member of the Othmer-Jacobs Department of Chemical and Biomolecular  Engineering, published details of the achievement in Nano Letters (“Label-Free Detection of Single Protein Using a  Nanoplasmonic-Photonic Hybrid Microcavity”), a publication of the American  Chemical Society.
The  detection of single thyroid cancer marker (Thyroglobulin, Tg) and bovine serum  albumin (BSA) proteins with masses of only 1 ag and 0.11 ag (66 kDa),  respectively.
In 2012, Arnold and his team were able to detect in solution the  smallest known RNA virus, MS2, with a mass of 6 attograms. Now, with  experimental work by postdoctoral fellow Venkata Dantham and former student  David Keng, two proteins have been detected: 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.”
This latest milestone builds on a technique pioneered by Arnold  and collaborators from NYU-Poly and Fordham University.  In 2012, the  researchers set the first sizing record by treating a novel biosensor with  plasmonic gold nano-receptors, enhancing the electric field of the sensor and  allowing even the smallest shifts in resonant frequency to be detected. Their  plan was to design a medical diagnostic device capable of identifying a single  virus particle in a point-of-care setting, without the use of special assay  preparations.
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. “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.”
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.  Among other advances,  Arnold and his colleagues posit that 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.
Arnold named the novel method of label-free detection  “whispering gallery-mode biosensing” because light waves 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 that the detector receives. When a molecule like a cancer marker clings to  a gold nanoshell attached to the microsphere, the microsphere’s resonant  frequency shifts by a measureable amount.
The research has been supported by a grant from the National  Science Foundation (NSF). This summer, Arnold will begin the next stage of  expanding the capacity for these biosensors. The NSF has awarded a new $200,000  grant to him in collaboration with University of Michigan professor Xudong Fan.  The grant will support the construction of a multiplexed array of plasmonically  enhanced resonators, which should allow a variety of protein to be identified in  blood serum within minutes.
Source: Polytechnic Institute of New York  University

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