Vesicle chemistry: A
new way to get life-like reactions
BY DAVID F. SALISBURY
In the beginning there were living cells. Then
scientists created test tubes. Now a team of Stanford
chemists, working with researchers at the University of
Göteborg in Sweden and Pomona College in Claremont,
Calif., have found a way to make tiny, cell-sized
containers, called vesicles, and use them to study the
chemical reactions of biological molecules in an
environment that closely mimics the interior of a living
cell.
"We now have the world's smallest test
tubes," says Richard N. Zare, the Marguerite Blake
Wilbur Professor of Chemistry at Stanford, who headed the
research effort reported in the March 19 issue of the
journal Science.
The ability to study chemical reactions in cell-like
containers has a number of possible applications. Among
them are:
- Investigating important parts of the cell's
metabolism including accumulation and release of
neurotransmitters and synthesis of proteins;
- Examining the biochemistry of cells infected with
pathogens
- Delivering drugs and genes to single cells.
"In the past, when studying the chemistry of
life, we had two basic choices: to experiment 'in vivo'
in living cells or 'in vitro' in glass
containers. Now we have a third choice, that is
in-between the two, but much closer to 'in vivo,'"
Zare says of the vesicle approach.
Producing this new form of micro-chemistry begins by
creating tiny vesicles that contain a single chemical
compound. The researchers found that they can reliably
create these membrane sacs in a few minutes. They do so
by floating a layer of artificial membrane on the surface
of a mixture of a desired chemical and a suitable
solvent, such as a mixture of alcohol and water, and then
causing the solvent to boil away by lowering the air
pressure above the membrane. As the water evaporates, it
leaves behind vesicles filled with the desired chemical
that range in size from 50 microns to 50 nanometers in
diameter, from roughly the width of a human hair to one
hundredth that size.
The chemists used a type of artificial membrane called
a phospholipid bilayer that is organic and closely
resembles the membranes of living cells. By using
different types of phospholipids, they can vary the
physical and chemical characteristics of the membranes in
a way that mimics the variations found in nature, Zare
says.
The researchers found two ways to use the vesicles to
produce minuscule chemical reactions.
The first approach involves immersing the vesicles in
a liquid containing a second chemical that will react
with the chemical that they contain. A laser-based tool
called optical tweezers allows them to manipulate these
delicate, microscopic objects easily. Using the optical
tweezers, the researchers position a vesicle between two
electrodes. Zapping the membrane sac with a mild
electrical pulse causes pores to open in the membrane
wall, allowing the chemical inside the vesicle to mix and
react with the chemical outside.
The second approach allows the reaction of controlled
amounts of chemical reagents. The researchers create two
sets of vesicles each filled with a different chemical.
They then use the optical tweezers to position a pair of
vesicles, each containing one of the two chemicals that
they want to react, between the electrodes. By zapping
the two vesicles with a slightly stronger electrical
pulse, the researchers can cause the two membrane sacs to
break down and then recombine into a single, larger
vesicle. The recombination happens so fast that very
little of the chemicals trapped inside the original
vesicles escapes.
In their initial experiments, the researchers used
different fluorescent dyes to study the chemical
reactions that resulted. But, according to Zare, they can
use a variety of other instruments to measure these
micro-chemical reactions.
Many of the reactions that take place within a cell do
not work in the same way at larger volumes. That is
because the molecules inside the cell driven by
thermal energy are continuously careening off each
other and bouncing off the cell's membrane wall. The
researchers estimate that a single enzyme and a single
substrate a molecule that the enzyme reacts with
will bounce off each other about 300,000 times per second
in a moderately sized vesicle and the substrate will
bounce off the membrane about 200 million times per
second.
There are other approaches to creating extremely small
volumes to study chemical reactions. But most of these
use methods such as micromachining small wells in silicon
wafers that create environments with physical
characteristics closer to that of glass than that of
living cells. So they are not as effective for studying
biochemical reactions, Zare says.
Other contributors to the research were: Stanford
graduate students Daniel T. Chiu, Clyde F. Wilson and
Alexander Moscho; Stanford undergraduates Anuj Gaggar and
Biren P. Modi; University of Göteborg assistant
professor Owe Orwar and his students Frida Ryttsén,
Anette Strömberg, Cecilia Farre, Anders Karlsson and
Sture Nordholm. Professor Roberto A. Garza-López from
Pomona College also participated in the research..
The project was funded by the National Institute on
Drug Abuse, the Swedish Foundation for Strategic
Research; and the Swedish Natural Science Research
Council.
Other relevant material: Zarelab web page at http://www.stanford.edu/group/Zarelab/index.html
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