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Molecular roller coaster analyzes compounds

Visualize a pack of various molecules in a compound, all mingling in a solution. Then imagine molecules being whisked away from the crowd, one at a time, into a tiny tube by an invisible force. About halfway through this molecular roller coaster, the molecules are flashed with ultraviolet light beamed through a small window as they scream by.

Although the molecules may or may not be having fun, the ride is quite revealing from a chemical viewpoint. The type and quantity of molecules in the compound can be determined from the time it takes various molecules to take their ride and from the amount of ultraviolet light they absorb. Imagine, then, the vast quantity of information that can be gathered by running 96 of these roller coasters at the same time.

That, in short, describes the operation of a new instrument created by Ed Yeung and his team of researchers at the Ames Laboratory. Called multiplexed capillary electrophoresis using absorption detection, this method promises to be faster, better and cheaper than alternative technologies.

This breakthrough is only the latest from Yeung, director of Ames Lab's Chemical and Biological Sciences Program and a distinguished professor of chemistry at Iowa State University, who has a long history of innovations in analytical chemistry. However, Yeung is particularly excited about this technology, as it has wide-reaching potential.

"Any kind of chemical measurements that involve separation can, in principle, be fitted to use this technology," Yeung says. Indeed, such technology has applications in several burgeoning fields, including combinatorial chemistry, drug discovery, genomics and proteomics (the study of protein expression and function). Such instruments are finding increasing utility in pharmaceutical, genetics, medical and forensics laboratories, and may someday be used in doctors offices.

By size and by charge

Capillary electrophoresis works on the same principles as gel electrophoresis, commonly used in DNA fingerprinting. An electrical current (the invisible force) causes molecules to migrate at different speeds, according to size and charge. The capillaries are silica tubes about 2 feet long, with an inside diameter of 75 microns (about the diameter of a human hair). Because these capillaries can disperse heat so well, an electrical charge of up to 20,000 volts can be used. The high voltage means separations can be done in as little as 15 minutes. "Absorbance detection" describes how the system detects molecules. Ultraviolet light is focused through a tiny window to illuminate the capillaries, and the amount of light absorbed by the migrating molecules is detected by an array of photodiodes. Absorbance detection makes Yeung's approach more widely applicable than other forms of capillary electrophoresis.

The ultraviolet absorption data, along with the time it takes the molecules to migrate, are sent to a standard personal computer. Custom software processes the data to generate charts called electropherograms, revealing the type and quantity of the separated molecules. The data can also be exported in tabular form to standard software packages.

"Multiplexed" means that the machine runs several samples at a time by using multiple capillaries. The machine works with 96-well titer plates to fit standard laboratory equipment. The result is automated, high-throughput analysis, a much-sought-after technology in today's biotech industries.

Faster, better, cheapter

Alternatives to multiplexed capillary electrophoresis with absorption detection include high-performance liquid chromatography and capillary electrophoresis with fluorescence detection.

"HPLC is a widely accepted practice," says Shelley Coldiron, president and co-founder of CombiSep, Inc., a new company formed to turn Yeung's technology into a commercial instrument. "We have to educate people and let them know we can operate comparable to what they're getting with HPLC."

Another factor working in CombiSep's favor is that its technology uses less solvent than HPLC. "We use 1,000 times less solvent than HPLC," says Coldiron, noting that this results in lower environmental hazards and related costs. Although HPLC systems cost much less than CombiSep's machine, they are limited to one analysis at a time. "We can take 96 of those individual instruments and do the same analyses with one of ours," says Coldiron.

Capillary electrophoresis systems that use laser-excited fluorescence detection are also made in multiplexed versions that can run 96 analyses at a time. These machines play an essential role in DNA sequencing; in fact, Yeung won an R&D 100 Award for developing just such an instrument in the 1990s.

Yeung's latest technology, however, can handle a wider range of compounds, thanks to its ability to detect molecules using ultraviolet light. "Only about 10 percent of all compounds fluoresce naturally," says Coldiron, "so you have to put optical tags on those compounds that don't fluoresce naturally." The "optical tags" are fluorescent dyes that are expensive and may be toxic.

What's more, multiplexed systems can be slow at processing all the data that results from running 96 simultaneous analyses. "It's kind of self-defeating. You're generating all this data but you get bottlenecked in data processing," Coldiron says. To sidestep this hurdle, CombiSep has written software that can process the data in approximately 15-30 minutes, according to Coldiron.

"Right here, and quickly"

Yeung began his Ames Lab research on the technology in 1998 when he realized there were limitations on existing capillary electrophoresis technology. Things progressed quickly from there. "At the point of publication, we realized it would be commercially viable, so we filed for a patent," says Yeung. Yeung soon helped start what became CombiSep to develop and sell instruments based on the technology. "I realized we needed to move extremely fast," he says. "We had to do something right here, and quickly."

In addition to Yeung and Coldiron, the company's founders include Marc Porter, an ISU chemistry professor and director of ISU's Microanalytical Instrumentation Center, serving as vice-president; Steve Ringlee, an area entrepreneur; and Roy Strasburg, director of R&D.

The market for high-throughput analysis technologies such as multiplexed capillary electrophoresis is "hot" right now, according to Coldiron. CombiSep is striving to get into this market as quickly as possible, especially as it is competing with several large companies in the fast-growing, multibillion-dollar market for these instruments. "There's always someone out there developing similar ideas," Coldiron says.

Prototype to product, pronto

For such a complex technology, CombiSep has moved extremely fast. Although officially founded in December 1999, the company really did not get rolling until April 2000. Since then, the company has made and tested several prototypes of its MCE 2000 multiplexed capillary electrophoresis instrument. CombiSep shipped its first machine to Procter & Gamble Pharmaceuticals for evaluation in late February 2001. Five instruments are in various states of assembly. Moveover, the company raised $1 million in start-up funds.

CombiSep has been able to move quickly for several reasons, according to Coldiron. First, Yeung had already proved that the technology would work. Second, CombiSep has been able to hire top-notch technologists. These include the technology's co-inventor, Ho-Ming Pang, as well as founder Strasburg and Jeremy Kenseth, both Ph.D. chemistry graduates from ISU. Third, the company has contracted resources and expertise from Ames Lab and ISU to accelerate its development rather than hiring its own staff. ISU's Center for Advanced Technology Development and Center for Industrial Research and Service have both assisted the company.

CombiSep also contracted with Ames Lab's Engineering Services Group to fabricate various components and to design and build the electronics that go into the machine. Jerry Hand, supervisor of the Lab's mechanical development shop, says the CombiSep project is "pretty much" like the others his group tackles. Much of the effort consists of fabricating components made of aluminum and other materials using the shop's lathes, mills and wire electrical discharge machining system. Hand says his entire group has worked on the CombiSep project. In addition, the electronics tech shop of the Lab helped design the electronics for the prototype.


For more information:
Ed Yeung, (515) 294-8062

Research funded by:
DOE Office of Basic Energy Sciences


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