Ann
Arbor, MI (3/5/98)- Two new sensor technologies allow hitherto unimaginable
access to the inside workings of the cell. One, called PEBBLEs, represent
the smallest biosensors ever developed. The other combines nanoliter-volume
separation technique with spectroscopy to provide quick detection
of compounds inside cells.
University of Michigan researchers have created PEBBLEs (Probes Encapsulated
By BioListic Embedding), an innovative method for monitoring the biochemistry
of living cells. These polymer-based sensors work inside mammalian
cells where they can detect subtle changes in concentrations of ions and
small molecules.
"PEBBLEs are self-contained sensors powerful enough to detect even slight
changes in cell biochemistry, but small enough to avoid damaging the cell,"
said Heather Clark, a U-M graduate student in chemistry who helped develop
the sensor.
Clark will present results from her research this week in a presentation
at PITTCON '98, the Pittsburgh Conference on Analytical Chemistry and Applied
Spectroscopy, taking place in New Orleans.
"For the first time, we can observe real-time chemical processes inside
a living cell. The goal of the project is to learn what happens inside
the cell when it is exposed to neurotoxic agents. If we can learn exactly
how these toxins trigger a flood of ions in and out of cells, we may be
able to speed up development of antidotes or countermeasures for lethal
biological warfare agents," says Raoul Kopelman, the U-M's Kasimir Fajans
Professor of Chemistry, Physics and Applied Physics.
This ability to "watch what's happening inside a cell" has a wide variety
of potential applications in other fields---including cancer therapy, glucose
monitoring, drug or chemical toxicity testing, and all areas of biosensing,
he noted
The researchers have created PEBBLEs as small as 20 nanometers in diameter.
The tiny polymer spheres contain many surface pores. When Clark adds dyes
to a polymer microemulsion suspension during pebble fabrication, the dyes
are naturally taken up by the PEBBLE's pores. Each dye will bind to, just
one type of ion or molecule.
Clark has produced PEBBLEs specific for calcium, oxygen, magnesium and
pH---the acidity level in a solution.
When a PEBBLE is exposed to even very small quantities of its target substance,
the dye in the PEBBLE glows when activated by a specific wavelength of
light. As the concentration of the targeted substance changes, the intensity
of the PEBBLE's fluorescence increases or decreases.
Clark uses pico-injection techniques or a gene gun to fire them randomly
into human or mouse cells in a culture dish. "The PEBBLEs blast through
the cell membrane like bullets," Clark explains, "but because they are
so small, they rarely do any damage. Mortality of cells shot with PEBBLEs
is only 2 percent higher than in control cells."
Measurement technique provides snapshot of cell physiology
A team of chemists and physiologists at the University of Illinois has
created a related sensing method, a new measurement technique that simultaneously
can identify and measure more than 30 compounds found in a single cell.
The method combines nanoliter sampling, capillary electrophoresis and fluorescence
spectroscopy for direct, convenient and highly sensitive measurements.
"By combining a nanoliter-volume separation technique with an information-rich
spectroscopic detection scheme, we can obtain both qualitative and quantitative
chemical information about the target species," said Jonathan Sweedler,
a professor of chemistry and a researcher at the university's Beckman Institute
for Advanced Science and Technology. "We can therefore more completely
identify and measure biologically important compounds in individual cells
without performing any chemical reactions to make the compounds detectable."
"We begin by placing a freshly isolated cell in a microvial where it
is homogenized and then drawn into a capillary tube. The chemicals then
separate in the capillary by electrophoresis and move into a flow cell
where they are stimulated by a laser. The laser-induced fluorescence is
then collected by a CCD/spectrograph and analyzed by a computer," said
Robert Fuller of UI.
"We can identify compounds not only by the separation time, but also
by the spectral fingerprints in the fluorescence emission. This means we
are able to distinguish between compounds that migrate at the same time,
thereby avoiding potential misidentification," he explained.
Since the detection scheme is based upon the native fluorescence of
individual molecules, the researchers need not perform any additional chemistry
in order to identify or quantify compounds. "Since we are not relying on
any chemical reactions, we are able to measure the true amount of chemicals
that are contained in the cell," Fuller said.
While most measurement techniques can identify or measure only a few
compounds at the same time, the new technique can handle up to 30 compounds.
"By looking at so many compounds simultaneously, we really get a nice
snapshot of the cell's physiology," Fuller said. "The concentrations of
the various chemicals can indicate both the general health and the metabolic
state of the cell. Such measurements also can aid in the identification
of neurotransmitters and the mechanisms of their regulation."
The researchers describe the technique in the February 1998 issue of
the journal Neuron.
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