The chemistry of life is different from chemistry at large, in part because it takes place in tiny containers called cells.
So chemists at Stanford University, 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-Lòpez 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 http://www.stanford.edu/group/Zarelab/index.html
The above post is reprinted from materials provided by Stanford University. Note: Materials may be edited for content and length.
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