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New biosensor illuminates search for brain chemicals

STANFORD -- * Speeding efforts to identify important new brain chemicals.

These are just two of the potential applications for a new biological sensor system that has been developed by Stnaford researchers.

The system uses a single cell or a group of cells as a sensor that can be tailored to respond to specific types of biologically active molecules. The biosensor is combined with a well-known chemical separation process, called capillary electrophoresis, which separates complex chemical mixtures into their individual components and delivers them to the biosensor one by one.

"Using whole cells as sensors has had one major drawback. A cell can respond to many different biological compounds. So, when a cell reacts to something in a mixture, it is difficult to identify just what the cell is responding to. We are able to get around this problem by combining whole-cell biosensors with capillary electrophoresis which allows us to identify the specific compounds that are triggering the reaction," says chemistry Professor Richard N. Zare, who headed the research.

As a result, the researchers have been able to benefit from the major advantage of using cells as biosensors - their selectivity. Chemical sensors generally react to a wide range of chemicals, whether they are biologically active or not. Cells, on the other hand, respond only to biologically important compounds. This is particularly beneficial when studying complex biological fluids that can contain thousands of different chemicals.

The new system is described in the Jan. 6 issue of the journal Science. Collaborating on its development were Jason B. Shear, now a postdoctoral student at Cornell University; Stanford chemistry graduate student Harvey A. Fishman; undergraduate Delia Garigan; Nancy L. Allbritton, now an assistant professor at the University of California at Irvine; and Richard H. Scheller, a Howard Hughes Medical Research Institute associate investigator in Stanford's department of physiology and biophysics.

The electrophoresis system uses an electrical field to separate mixtures of biological molecules. Each type of molecule moves through a liquid mixture at a different rate depending on its electrical charge, size, shape and other factors. The compounds reach one end of a capillary, a tube of fused quartz with an inside diameter smaller than a human hair. They form distinct bands as they move along the tiny tube. When they reach the other end of the capillary, which has been carefully centered over a cell sitting on a microscope stage, the compounds are sprayed onto the tiny biosensor.

"In this way we can deliver about a millionth of a drop of a given chemical to an individual cell," says Fishman.

By measuring the time it takes different compounds to reach the end of the capillary (a process that takes between five and 30 minutes) and comparing that with the time it takes for known samples to travel the same distance, the researchers can identify known compounds. An alternate method is to treat the biosensor with an antagonist that knocks out the cell's ability to detect a known compound. Then, if the cell stops reacting to one of the compounds in a mixture, the researchers know it must be the same compound.

The scientists have developed two different ways to determine whether the cell responds to a given compound.

One approach, demonstrated with rat brain cells, employs fluorescence. When one or more of the receptors on the cell's surface binds with a compound, it triggers what is known as the "G-protein cascade." This is a fundamental cellular process that causes major changes within the cell, including a change in the concentration of calcium ions. By introducing a fluorescent dye into the cells that is turned on by the change in ion concentration, the researchers can see the cells light up in the microscope eye piece.

"Theoretically, this amplification of the cell receptor recognition event allows us to detect the presence of a single molecule of a given compound," Zare says.

The second approach, used with unfertilized frog eggs, is to measure the change in electrical conductance that occurs in the cell's membrane when the shape of the channels that allow specific ions to move into and out of the cell changes.

The researchers have tested the system with three biologically important compounds: the neurotransmitter acetylcholine; bradykinin, a peptide found in blood that is associated with inflammation; and adenosine triphosphate, a compound that carries chemical energy in all living organisms. The scientists report that they could detect these compounds with a sensitivity that ranged from a few million molecules down to a few thousand - levels comparable to those achieved by the best detection techniques used previously.

The researchers also used frog eggs to demonstrate that it is possible to use this approach to tailor-make biosensors to detect specific compounds. They injected some of the eggs with a form of messenger-RNA that included instructions for making receptors on the egg's surface sensitive to seratonin, a neurotransmitter thought to influence mood. As a result, the altered eggs reacted to the presence of seratonin, whereas the unaltered ones did not.

"This opens the possibility of using all the neat tools of genetic engineering to make biosensors for a large number of specific compounds. I can imagine building a kind of 'mega-detector' that consists of a large array of cells, each cell altered to detect a different compound. The capillary could be scanned over the array, identifying unknown substances by identifying which of the biosensors it triggers," says Fishman.

Another way in which the researchers would like to use the new system is to study the chemical interactions between different parts of the brain. By siphoning compounds off the neurons in one part of the brain, then running them through the capillary electrophoresis system, spraying them on another part of the brain, and seeing how the neurons there react, they might be able to garner valuable insights on the nature of chemical communications within the brain.

Yet another natural application for the system is to screen the compounds that are created when a drug breaks down in the body to determine which are biologically active. This could help drug company researchers more easily identify those metabolites that are potentially harmful, the scientists say.

The research was funded by the National Institute of Mental Health and Beckman Instruments, Inc. Stanford University has applied for a patent on the process.



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