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Researchers strive for next dimension

With completion of the human genome project, researchers know a lot about our genetic makeup. How that translates into action in our bodies in the form of proteins is the next frontier of understanding.

In the field of proteomics, however, researchers are limited by separation techniques. Blood, for instance, is thought to contain more than 50,000 proteins. Researchers would like to better understand the function of these proteins, which may be indicators of disease known as biomarkers, but they can only separate out about 2,500 proteins at a time using gel electrophoresis.

Neil Ivory and Prashanta Dutta recently received an initial $200,000 National Science Foundation grant to develop a new platform for separating out a much larger number of proteins.

The current method, developed in the mid-1970s, uses two different separation techniques in series. Each method allows for the separation of about 50 protein peaks. Taken together, they can separate about 2500 proteins.

In the first technique, isoelectric focusing, the researchers use a polymer gel with a specified pore size to separate proteins by adding chemicals called “ampholytes” to the protein sample and then putting an electric current through it. The proteins migrate to the part of the gel where they have no charge, which is called their isoelectric point. They can be separated and isolated, even if they are only present in tiny concentrations.

A second method, called SDS-PAGE, further separates the proteins according to their molecular weights. A negatively charged surfactant is added to the gel to coat the proteins. Because of their different sizes, the proteins will travel through the pores in the gel at different rates, separating out by molecular weight.

Using a lab-on-a-chip developed at WSU, Ivory and Dutta are hoping to add two or more steps to greatly increase the number of proteins that can be separated. They believe the microscale unit operations and requisite design tools needed to carry out more complex separations on a microchip have already been developed or are close to development and can be integrated into their design.

Dutta’s microchip uses a novel technique that puts the proteins into tiny channels built on a substrate called poly-di-methyl siloxane (PDMS). While the eye of an average-sized needle is 1,230 microns wide, Dutta’s channel is only 10 microns. Reducing the size of the process allows the researchers to do separations 10 to 100 times faster than traditional laboratory methods like the gel described above.
The biggest challenge, says Ivory, is getting though the intersection without losing resolution as you transfer from one technique to the next. Ivory thinks the choice of techniques and design of the intersection on the microchip is the key to a solution.

”If you can solve this problem, there are a lot of people who will be interested,’’ he said. “While this may appear to be a daunting task at this time, it is probably not very different in scale or complexity than evolving to a modern computer microprocessor from the concept of a single transistor.’’

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