Nano Scientists Reach Holy Grail in Label-Free Cancer Marker Detection: Single Molecules

Nano Scientists Reach Holy Grail in
Label-Free Cancer Marker
Detection: Single Molecules

July 24, 2013 — 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 , a
publication of the American Chemical
Society.
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.