|NEWS OF THE WEEK
Volume 77, Number 12
CENEAR 77 12 p.
|[Previous Story][Next Story]
A new form of microchemistry performed in individual phospholipid spheres with incredibly small volumes may allow chemists to probe biochemistry in the most realistic, controlled environment yet created for in vitro studies.
"These are the world's smallest test tubes," says Stanford University chemistry professor Richard N. Zare, who along with colleagues there and at Göteborg University, Sweden, and Pomona College in Claremont, Calif., designed the tiny reaction vessels and the means to study them individually [Science,283, 1892 (1999)].
Two phospholipid vesicles, one containing Ca2+ ions and the other containing the chelating agent fluo-3 (top left), show no fluorescence (bottom left) under ultraviolet irradiation while they're still separated. However, once the two vesicles fuse (top right), Ca2+ binds with fluo-3 and the resulting species is highly fluorescent (bottom right). [© Science]
Calling the work a "tour de force," Evan R. Williams, associate chemistry professor at the University of California, Berkeley, says it combines a number of different techniques, such as optical trapping and fluorescence microscopy, to create something "nobody's been able to do before, as far as looking at chemical reactions in a small frame."
In such tiny, enclosed volumes, much like those encountered in biochemical processes, surface interactions become increasingly significant. And important molecular collisions--involving enzymes, for example--occur more frequently. Consequently, studying the dynamics of such small-quantity interactions can't be done by extrapolating from reactions involving macroscopic quantities. And although research has been done on small-volume chemistry in micromachined wells, those containers are typically silica based, and behave differently from biological membranes.
This latest work solves those problems. Using a rotaevaporation technique, the group in just minutes can create spherical phospholipid vesicles between 50 nm and 50 m in diameter, and entrap reagents inside as the vesicles are forming. The vesicles encase volumes on the order of femtoliters on down to even zeptoliters (10-21 L). The process is so highly controlled that it can encapsulate only a few molecules--or even one--at a time. And by choosing different phospholipids, the group can change the hydrophobicity or hydrophilicity of the membranes.
What's special about this work, says University of Illinois, Urbana-Champaign, associate chemistry professor Jonathan V. Sweedler, is that the group can study single vesicles. "Unlike looking at populations, this is looking at individual vesicles, so you can follow the dynamics of the process without getting averaging effects," he says.
To perform the minuscule chemical reactions, the group first holds the reagent-filled spheres in place by either optically trapping them with an infrared laser or by sticking them to modified borosilicate glass surfaces. Then they use a technique called electroporation to deliver short, intense electrical pulses via tiny electrodes that open pores in the membranes, allowing reactants to get into the vesicles.
In an even more controlled experiment, they fuse two vesicles together with electrical pulses, allowing the contents to "spill their guts into each other," Zare says.
For example, they filled one vesicle with the chelating agent fluo-3, the other with Ca2+ solution. The chelating agent alone is weakly fluorescent, but when it binds to Ca2+, the fluorescence quantum yield increases up to 40 times. The group used Zare's hallmark laser fluorescence microscopy techniques to track the reaction. As expected, they initially observed little fluorescence in the vesicle containing fluo-3. When the vesicles were fused, the contents mixed and the fluorescence yield increased.
The group envisions using the technique as a biochemical delivery system, as well as for studying diffusional mixing and reaction times on a minuscule, or even on a single-atom scale. "The ability to work in things that mimic cells and the ability to deliver chemicals and genetic information on demand opens up lot a lot of possibilities," Zare says.[Previous Story][Next Story]
Chemical & Engineering News