Jan. 12, 2004 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, 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.
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