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Photograph of the optical
biosensor that is approximately 16 millimeters by 33 millimeters
in size. The horizontal purple lines are the channels on the
waveguide.
Photo
by Gary Meek |
A virulent strain of avian influenza (H5N1)
has begun to threaten not only birds but humans, with more than 300
infections and 200 deaths reported to the World Health Organization
since 2003. Looming is the threat of a pandemic, such as the 1918
Spanish flu that killed about 40 million people, health officials say.
�With so many different virus subtypes, our biosensor�s ability to
detect multiple strains of avian influenza at the same time is
critical,� noted Xu.
To test the biosensor, the researchers assessed its ability to detect
two avian influenza strains that previously infected poultry. The
results showed that a solution containing very few virus particles
could be detected by the sensor.
Xu tested a third strain of the virus as a control. When the sensor
coating was modified to collect only the other two strains, the
control strain was not detected even at high concentrations. Results
of this study were published online in August in the journal
Analytical and Bioanalytical Chemistry and will be included in
journal�s print edition on October 16. The work was funded by the U.S.
Department of Agriculture�s (USDA) Agricultural Research Service (ARS)
and the Georgia Research Alliance.
�The technology that Georgia Tech developed with our help has many
advantages over commercially available tests - improved sensitivity,
rapid testing and the ability to identify different strains of the
influenza virus simultaneously,� said David Suarez, a collaborator on
the project and research leader of exotic and emerging avian viral
diseases in ARS� Southeast Poultry Research Laboratory in Athens, Ga.
Suarez is providing antibodies and test samples for GTRI�s research.
The biosensor is coated with antibodies specifically designed to
capture a protein located on the surface of the viral particle. For
this study, the researchers evaluated the sensitivity of three unique
antibodies to detect avian influenza virus.
The sensor utilizes the interference of light waves, a concept called
interferometry, to precisely determine how many virus particles attach
to the sensor�s surface. More specifically, light from a laser diode
is coupled into an optical waveguide through a grating and travels
under one sensing channel and one reference channel.
Researchers coat the sensing channel with the specific antibodies and
coat the reference channel with non-specific antibodies. Having the
reference channel minimizes the impact of non-specific interactions,
as well as changes in temperature, pH and mechanical motion.
Non-specific binding should occur equally to both the test and
reference channels and thus not affect the test results.
An electromagnetic field associated with the light beams extends above
the waveguides and is very sensitive to the changes caused by
antibody-antigen interactions on the waveguide surface. When a liquid
sample passes over the waveguides, any binding that occurs on the top
of a waveguide because of viral particle attachment causes water
molecules to be displaced. This causes a change in the velocity of the
light traveling through the waveguide.
At the end of the waveguide, the light beams from the sensing and
reference channels are combined to create an interference pattern. The
pattern of alternating dark and light vertical stripes, or fringes, is
imaged on a simple detector. By doing a mathematical Fourier transform,
the researchers determine the degree to which the fringe patterns are
in or out of step with each other, known as phase shift. This phase
shift tells the amount of virus bound to the surface.
�The fringe pattern doesn�t look like it changes in the image, but
with math we find out the speed of the light in the test channel
changed creating this phase change,� explained Xu.
Current methods of identifying infected birds include virus isolation,
real-time reverse transcriptase polymerase chain reaction (RRT-PCR)
and antigen capture immunoassays. Virus isolation is a sensitive
technique, but typically requires five to seven days for testing.
RRT-PCR is commonly available in veterinary diagnostic laboratories,
but requires expensive equipment and appropriate laboratory facilities.
RT-PCR can take as little as three hours to get test results, but
routine surveillance samples are more often processed in 24 hours. The
antigen capture immunoassays can provide rapid test results, but
suffer from low sensitivity and high cost.
Beyond the waveguide sensor, the only additional external components
required for field-testing with GTRI�s biosensor include a
sample-delivery device (peristaltic pump), a data collection laptop
computer and a swab taken from a potentially infected bird. Power is
supplied by a nine volt battery and USB connection. The waveguides can
be cleaned and reused dozens of times, decreasing the per-test cost of
the chip fabrication.
Xu and Suarez are currently working together to test new unique
antibodies with the biosensor and to test different strains. In
addition, Xu is reducing the size of the prototype device to be about
the size of a lunchbox and making the computer analysis software more
user-friendly so that it can be field-tested in two years.
�We are continuing our collaboration and have provided additional
money to Georgia Tech to move the project along faster,� added Suarez.
�Since this technology is already set up so that you can use multiple
antibodies to detect different influenza subtypes, we are going to
extend the work to include the H5 subtype.�
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