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Cellular Secrets: New microchip charts cell chatter

Gillis' research team consists of, left to right, Jia Yao, Jaya Ghosh, Gillis, Xin (Alice) Liu and Syed Barizuddin. All are graduate students, with the exception of Liu, who is a research scientist.

Story By: Anita Neal Harrison

Eavesdropping is usually seen as a bad thing, but University of Missouri Professor Kevin Gillis has earned great respect for gathering information using advanced eavesdropping techniques.

Gillis listens in on some of the world’s quietest “cellular conversations,” not whispered secrets exchanged over mobile phones, but information transfer between biological cells. An expert in the study of electrical properties of biological cells and tissues, electrophysiology, Gillis has been eavesdropping on cells since his graduate days. Currently, this professor in MU’s Biological Engineering Department is serving as the principal investigator on a project aimed at revolutionizing research on how cells communicate.

The National Institutes of Health (NIH) is funding Gillis’ project to develop a microchip for studying exocytosis, a process in which cells secrete signaling molecules. Exocytosis is a basic biological process covered in Bio 101, “but it has applications in understanding all things that are related to transmitter release, such as information transfer in the brain,” Gillis said.

Uniquely qualified to lead the research, Gillis’ background is strong in both electrical and biological engineering. His bachelor’s, master’s and doctorate degrees, all from Washington University in St. Louis, are in electrical engineering, and he completed his post-doctorate training at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, under the direction of Erwin Neher, who won a Nobel Prize in 1991 for his contributions to electrophysiology research.

Gillis’ former adviser, Stan Misler, an associate professor of internal medicine, cell biology/physiology at the Washington University School of Medicine, describes Gillis as “eminently respected in his field.”

Misler added that the “lab on a chip” Gillis is developing should open up new research on exocytosis, the biological process Gillis has dedicated his career to studying.

How exocytosis works

Cells have within them membranous sacs, or vesicles, that hold signaling molecules. These molecules can be hormones, such as adrenaline, or neurotransmitters, such as serotonin. Upon stimulation, a cell’s vesicles fuse with the cell’s outer membrane and then release the signaling molecules to the outside, where they act upon and cause reactions in other cells.

“The same process is also used to release insulin,” Gillis said, “so if we can understand how this process is regulated, we can potentially develop therapeutic approaches to regulate secretion of insulin.”

There are already drugs that act on this pathway to increase insulin secretion to treat Type 2 diabetes. There also is a drug, L-Dopa, that treats Parkinson’s disease by affecting how much of the neurotransmitter dopamine is packaged in a vesicle. The cosmetic drug Botox is another example.

“The way Botox works is it inhibits the release of the neurotransmitter acetylcholine from motor nerves onto muscle,” Gillis says. “So in other words, when you inject this into your face, the muscles in your face relax because the motor nerves don’t release the transmitter anymore. It inhibits fusion, so the muscle relaxes, and that’s what gets rid of your wrinkles.”

Drugs can affect exocytosis in different ways with similar results. To determine the exact effect of the drug, researchers measure the rate and amount of transmitter being released using electrochemistry; the transmitter molecules contain electrons that emit a tiny, but measurable, current. Microelectrodes can pick up that current and send it to an amplifier, which then feeds the information into a computer. There, researchers can see a graph tracking the current’s spikes.

“Each spike of current corresponds with a fusion of these individual vesicles that are, say, 300 nanometers in diameter, and then we can analyze these spikes of current to tell us how much transmitter was in that vesicle and the fusion kinetics, the time course of how that vesicle fused, so we can understand how this process occurs,” Gillis said.

Currently, researchers studying exocytosis, as well as drugs’ effects on exocytosis, use carbon fiber microelectrodes to pick up the current. Carbon fiber microelectrodes are just a few microns across, similar to the size of the cell, and researchers must place them adjacent to cells that will be stimulated to undergo exocytosis.

“That is a slow, labor-intensive process,” Gillis said. “You have to do this under a microscope with micromanipulators, bringing this carbon fiber to a cell. You do it one cell at a time, and it takes a number of minutes between each recording.”

Microchip technology

The microchip Gillis is developing with the NIH grant would take the same measurements but do it much more quickly.

“With a microchip, the idea is we can pattern a whole array of electrodes on a chip so that someday, we can do many recordings in parallel, or even if we do them one at a time, we can move from one to the next very quickly,” Gillis said.

“The NIH is always interested in technology development and engineering that will directly increase patient care,” he continued. “They’re also interested in developing tools that will mean we can get more bang for the buck. We can get research done more quickly on how things work, which leads to the next generation of therapies.”

Gillis began the project in 2000 with a $708,160 grant from the National Science Foundation (NSF); the NIH grant, which totaled $2,737,923, started in 2004 and will last through next July. Some of the accomplishments of the last nine years include developing ways to stimulate exocytosis on the chips, figuring out how to direct cells to the electrodes and determining the best design for the electrodes — size, shape and materials — to get the most signal without too much extra noise.

Gillis conducts his research at MU’s Dalton Cardiovascular Research Center. The center’s mission is “to advance knowledge of the causes, diagnostics, therapies and cures for cardiovascular disease through research and teaching that merges discipline and technologies.” Researchers at Dalton come from the fields of biochemistry, biological engineering, biological sciences, biomedical sciences, electrical engineering, medicine, pharmacology, physiology, physics, and veterinary medicine and surgery; research programs at the center include investigations into cardiac functions, cystic fibrosis, exercise, kidney failure, membrane transport, muscular dystrophy, neurohumoral control of circulation, and tumor angiogenesis.

A key collaborator with Gillis on this research project is Shubhra Gangopadhyay, a C.W. LaPierre professor in electrical and computer engineering. Her lab in Engineering Building West has microfabrication facilities so she can deposit the metal films onto the microchips the to make the electrodes and the insulating material between electrodes. Transparent metals are preferred for both because a transparent chip allows researchers to make optical measurements along with electrochemical ones, “so you can get more pieces of information,” Gillis explained.

One metal showing great promise for use on the electrode is diamond-like carbon. Carbon is conducive to electricity, and in its diamond form, is transparent. Diamond-like carbon is also hard, so it doesn’t easily scratch off the chip, and it’s biocompatible, meaning cells don’t react to it. And it’s not as expensive as it might sound because it’s not gem-quality diamond.

The chips are approximately one-by-three inches, and the electrodes are on the order of 10 microns, or cell-sized. The electrodes have to be small enough that just one cell will fit on each one; smaller is also better because it lessens the extra “noise,” or unwanted currents, the electrode picks up.

“Our favorite approach most recently is to make little wells that are cell-sized, and the cells fall into the wells,” Gillis said, adding one of his lab’s next goals is to make the electrode even smaller than the well, so just the middle of the well measures current.

Looking ahead

Gillis has experimented with some advanced, automated approaches for directing the cells to the electrodes, but with the well design, researchers can just drop solution containing cells onto the chip and the cells will settle in place. Meanwhile, areas of the chip between the electrodes are coated with materials to keep the cells from sticking where they shouldn’t (Teflon works great for this). The perimeter of the chip holds connection pads for hook up to amplifiers.

So far, researchers have recorded from one electrode at a time, with results in line with measurements from carbon fiber microelectrodes. Even measuring from one electrode at a time is five to 10 times faster than using carbon fiber because chips require much less labor, but the goal is to get up to 1,000 electrodes on the chip and do simultaneous measurements, so thousands of cells could be tested in the time it now takes to do one.

Right now, one of the main design limitations is the amplifiers, which are too big and too expensive to connect more than a few to each chip. Collaborators at Cornell University are working to develop new amplifier technology that will make it possible to put the amplifiers on the chip itself.

For Gillis, who has seen and made some incredible advances in exocytosis research over the last two decades, the opportunity to revolutionize the technology is thrilling work.

“As an engineer, I like developing advanced technology,” he said, “but also, now, I’ve been a biologist for a number of years, and by developing your own tools, you’re always on the cutting edge of being able to do the most informative scientific experiments.”

Already, Gillis is working on an application to renew the NIH grant when it ends next summer.

“Integration is really the next big step, integrating these various pieces we’ve developed — new electrode materials, targeting cells to electrodes, new amplifiers,” Gillis said. “We want to put it all together and ask biological questions at the cutting edge of exocytosis research. That’s the direction we’re going.”