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"So far we have been lucky that terrorists have
used well-known biological agents like anthrax and sarin gas," says
David Cliffel, assistant professor of chemistry at Vanderbilt
University, who led the development group working under the auspices
of the Vanderbilt Institute for Integrative Biosystems Research and
Education. "But how will we respond if one of these groups uses recent
advances in genetic engineering to produce an agent that is new and
unknown?"
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Close-up of four-channel microphysiometer;
Photo
by Daniel Dubois, Vanderbilt University.
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Part of the answer, Cliffel says, is the device he
and his colleagues have developed, called a four-channel
microphysiometer. It is a modification of a 10-year-old commercial
device called the Cytosensor made by the company Molecular Devices
that measures changes in acidity in a small chamber holding between
100,000 to 1,000,000 individual cells. Cliffel's research team has
added three additional sensors so that the machine can simultaneously
chart minute-by-minute variations in the concentrations of oxygen,
glucose, and lactic acid, in addition to pH.
The added capability – reported in the Feb. 1 issue
of the journal Analytical Chemistry and now available online – is
important because the basic metabolism of a cell involves consuming
oxygen and glucose and producing lactic and carbonic acid. As a result,
monitoring variations in these four chemicals allows researchers to
quickly assess the impact that exposure to different chemicals have on
the activity and health of relatively small groups of cells.
"I envision having a microphysiometer with an array
of chambers," says Cliffel. "One of them contains heart cells, another
contains kidney cells, another nerve cells and so on. Then, when an
unknown agent is pumped into all these chambers, we quickly will be
able to determine exactly which part of the body it attacks and the
response of the affected cells will provide us with important clues
about the manner of its attack."
Because of its potential application for
bioterrorism and chemical and biological warfare, the development of
the device has been funded by the Defense Advance Research Projects
Agency. But the microphysiometer also has important potential
applications in detecting and assessing the toxicity of environmental
pollutants. It also has many possible uses in basic biological
research, its developers point out. The microphysiometer consists of a
series of reservoirs, switches, rotary pumps and tiny chambers made
from two thin membrane sheets that contain the cell colonies. The
original unit also included a single sensor that measured changes in
acidity (pH) in the extracellular liquid.
"Over the years, the Cytosensor has been used in a
number of studies involving changes in pH," says Cliffel. "But its
usefulness was limited because it could only measure a single
variable. We realized that analytical chemists had recently developed
new techniques that would allow us to simultaneously measure
variations in several different key compounds."
Using these techniques, Cliffel's interdisciplinary
research team – chemistry post-doctoral assistants Sven Eklund and
Dale Taylor working with senior research associate Eugene Kozlov and
research professor Ales Prokop from chemical engineering – developed
the three additional sensors out of specially coated electrodes. They
attached these to another commercial device that has recently come on
the market, called a multipotentiostat, that allowed them to take
simultaneous readings from the sensors.
One of the biggest problems they had with these
modifications was due to the fact that one of the devices was designed
to be controlled by a Windows computer and the other by a Macintosh.
"In the beginning, there was a tremendous amount of cross talk between
the two computers that we had to eliminate," Eklund says.
The researchers tested the modified device with
several different toxic agents and two cell types.
In one test they added fluoride to Chinese hamster
ovary cells. Fluoride blocks cells' ability to convert glucose into
ATP, the chemical that cells use as an energy source. Their
measurements showed that the lactate concentration and acidification
rate dropped rapidly as the cell slowed its production while oxygen
and glucose concentrations rose as the cell consumption slowed.
"We could see the cells basically go into
hibernation," says Cliffel. "Then, when we flushed out the fluoride,
we could see them start up again."
They ran similar tests with two other known
metabolic poisons, antimycin A and 2,4-dinitrophenol, and a type of
cell that produces connective tissue called a fibroblast and got
similar results.
Last year, the Vanderbilt researchers upgraded a
Cytosensor at the Edgewood Chemical Biological Center at the Aberdeen
Proving Ground in Maryland. Since then their ECBC collaborators have
been using the device to study cell response to a number of different
chemical and biological agents.
Since submitting the recent paper, Cliffel's group
has also successfully tested the device with two pesticides, parathion
and paraoxon, and two common pollutants, the gas additive MTBE and
hexachromium, the pollutant that Erin Brochovich made famous.
A paper that provides detailed instructions on how
to modify the Cytosensor and multipotentiostat to make a four-channel
microphysiometer has been accepted for publication by Humana Press and
is scheduled to appear later in the year. |
