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Lab on a chip used for protein studies

Ten years ago, ORNL's Mike Ramsey built the first lab on a chip. Now, improved versions of this miniature chemistry lab are being shipped all over the world. A toaster-sized, computerized device containing four matchbox-sized protein identification chips modeled after Ramsey's invention is being mass-produced by Caliper Technologies, Inc., in California. It is being sold to biotechnology firms by Agilent Technologies.

"Proteins, like DNA, pose a massive chemical measurement problem," Ramsey says. "But we have learned how to use a lab on a chip to measure molecular weights of proteins in much smaller samples and in much shorter times than are required by conventional methods."

Caliper Technologies is developing microchips for drug discovery. Such a device would help pharmaceutical firms rapidly identify compounds effective in inhibiting the activity of disease-causing proteins. "We believe that an automated device with massively parallel microfluidic chips can work with sample volumes that are 1/10,000th the volumes analyzed in conventional benchtop devices, at 10 to 100 times the speed or more," Ramsey says.

The current drug discovery chip contains four channels—thinner than human hair—that connect reservoirs, all of which are carved into a rectangular glass plate, using microfabrication technologies. A disease-related enzyme is introduced into a chip channel. Because of pressure differences, the enzyme and a modified substrate flow through the channel network, mix, and react. The reaction product is fluorescent when exposed to a laser beam. The amount of fluorescence is a measure of the reaction rate.

When a test compound is introduced into the chip through another channel, it typically reacts with the enzyme, blocking out the substrate so less of the fluorescent product is produced at a time. The reduced fluorescence signal indicates the effectiveness of the test inhibitor compound. By introducing different test compounds to the device every 5 seconds, it is possible to rapidly compare reaction rates to identify potentially effective drugs. In a recent demonstration at Caliper, nearly a million compounds were screened while using less than 1 microgram of enzyme (usually a very valuable material).

In ORNL's Chemical and Analytical Sciences Division, considerable research is being conducted by Ramsey's group on developing improved lab-on-a-chip technologies for biological, environmental, forensic, and defense applications. The lab on a chip has been honored by R&D magazine as one of the 40 top innovations that have come about since the magazine began its R&D 100 competition in 1963. It also has been recognized by a panel of citizens as one of the top 23 technologies developed using Department of Energy funding.

In 1998 at Caliper Technologies, Rose Ramsey (Mike's wife) and a colleague there first demonstrated that the lab on a chip could complete a two-dimensional (2D) separation of peptides in under 10 minutes. The 2D chip separation uses two types of separations to resolve the peptides. In one channel, they are separated by differences in migration speed and in another channel by differences in peptide charge and size in response to an electric field (capillary electrophoresis). By contrast, it takes 24 to 48 hours to do this separation using conventional 2D gel electrophoresis. More recently, Stephen Jacobson, Chris Culbertson, and Norbert Gottschlich contributed to a newly designed 2D chip that works even faster.

Mike, Rose, and Robert Foote of ORNL then received funding from the Laboratory Directed Research and Development Program at ORNL to analyze proteins by combining the lab on a chip with an electrospray ionization time-of-flight mass spectrometer (ESI/MS). Rose, who conceived the idea, and Iulia Lazar, a post-doctoral fellow at ORNL, showed that the procedure could be used to rapidly analyze hemoglobin, the protein that makes blood cells red and transports oxygen from the lungs to the body tissues.

In the chip, the hemoglobin from a drop of blood is reacted with the enzyme trypsin, which cleaves the blood protein at the scattered sites of two amino acids. The fragments of the hemoglobin are electrosprayed directly from the chip as ions into the mass spectrometer.

"The pattern of the fragments gives a fingerprint for the protein that can be compared to amino-acid sequences in the protein database, allowing us to identify the protein," Rose says. She used this technique to sequence over 70% of human hemoglobin from a drop of blood. She demonstrated that the technique can rapidly distinguish sickle-cell hemoglobin, whose fingerprint is different from that of normal hemoglobin. This work was recently published in Analytical Chemistry. This technique could also be used to rapidly screen for other hemoglobin variants.

The lab on a chip may be small but its potential for advancing medical diagnosis and treatment is quite large.

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