Download high-resolution photos of Sven Elklund working on microphysiometer or close-up of four-channel microphysiometer.
NASHVILLE, Tenn. – The ability to analyze and defend against novel
biological agents has been strengthened by the development of a new
device that can monitor the metabolism of living cells in near real
time.
"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?"
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, which 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 for control 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.
For more news about Vanderbilt research, visit Exploration, Vanderbilt’s online research magazine, at www.exploration.vanderbilt.edu.
Media contact: David Salisbury, (615) 322-NEWS
David.Salisbury@vanderbilt.edu