Stanford News



Researchers corral millions of microscopic membranes

Scientists at Stanford University have done the next best thing to packaging living cells in individual boxes for study. Borrowing microfabrication techniques from electrical engineering, the researchers have created a specially prepared surface that holds millions of cell-sized squares composed of an artificial membrane that closely mimics the surface of living cells.

According to the researchers, the ability to work with these independent membranes that are uniform in size and fixed in space makes many new experiments possible.

"We started out looking for better ways to study cell membranes. By applying micro-patterning to membranes, we've come up with something completely new and we're still coming to grips with the implications," said graduate student Jay T. Groves. The work is reported in a paper in the Jan. 31 issue of the journal Science, co-authored by Groves, chemistry Professor Steven G. Boxer and Ginzton Laboratory research associate Nick Ulman.

Steven G. Boxer

According to the scientists, not only are the micro-membranes likely to become an important research tool, but the system also could serve as the basis for improved cell and drug screening methods because it is ideally suited for automation.

The researchers believe this is the first time that microfabrication techniques have been applied to an entire biological system like a membrane. They were able to do this by adapting standard photolithography techniques from microelectronics to cover a surface of silica coated with silicon dioxide with a pattern of microscopic squares. Each square was about five microns (about five thousandths of a millimeter) on a side. Its surface was treated to be chemically compatible with the membrane. The surface of the borders that separate the squares, however, was made chemically repulsive. A square centimeter of this array contains 2.8 million of these microscopic "corrals."

Micropatches72dpi.GIF Jay T. Groves, left, and Nick Ulman inspecting a silica plate containing millions of microscopic membrane patches.

Next, the scientists dropped tiny bags, or vesicles, of membrane material extracted from chicken eggs into the corrals, which were submerged in saline water. They determined that the vesicles fused to form uniform membrane patches that completely covered the squares but stopped at the borders. These membranes are very thin, about 50 atoms thick.

Technically, the structures are called fluid bilayer membranes because they consist of two opposed layers of fatty acid molecules, or lipids. This is the basic structure of all living cell membranes.

Further, the researchers determined that the membrane patches were stable and retained their basic properties for several weeks. In addition, they demonstrated that each membrane patch was effectively isolated from its neighbors.

Stanford University has applied for a patent on the basic process.

This figure demonstrates microscopic membrane corrals, which are about five thousandths of a millimeter on a side. The membrane is formed of two layers of lipid molecules (the circles with wavy tails). These form over regions where the underlying silica surface has been made chemically compatible with them. The corral boundaries are treated so that they are chemically incompatible. Although there is no physical barrier, this process creates a chemical barrier that effectively isolates the membrane patches from each other.

One of the possible applications for these micro-membranes is as a tool for determining the structure of membrane proteins.

One of the most powerful methods for determining the structure of proteins is X-ray crystallography. For this method to work, however, researchers must purify and crystallize the material. This has proven very difficult for proteins associated with membranes because they cannot be easily separated from the membrane material.

"There is a revolution going on in structural biology. Each year the structures of hundreds to thousands of new proteins are solved. But so far only a handful of these have been membrane proteins," Boxer said.

He said that this is an area where the new method may help. Many associated proteins can move freely about on a membrane's surface. In previous work, the researchers have demonstrated that they can use electric fields to concentrate such proteins against two-dimensional membrane boundaries. They suggest that it should be possible to both concentrate and crystallize such proteins by applying an intense electrical field. If they are correct, the method could be used along with existing techniques for determining the two-dimensional structure of groups of membrane proteins and X-ray crystallography to identify the three-dimensional structure of a number of these compounds for the first time.

The black squares are corrals where the photoactive red dye added to the membrane has been bleached out. The fact that the effect is confined to individual corrals demonstrates that the membrane patches are isolated from each other.

A potential biomedical application is cell screening of the type required for leukemia patients. Doctors must closely monitor the different kinds of cells in a leukemia patient's blood. Using a small wafer holding millions of micro-patches that have been seeded with proteins that bind to different kinds of cells, it should be possible to obtain measurements of the relative numbers of different cell types by simply covering the wafer with blood, washing it off and counting the cells that remain stuck to different patches. Not only could this method identify and separate different kinds of cells like current methods, but it potentially could measure how well the cells are functioning, Groves suggested.

In a similar fashion, the technique might be used to screen for drugs such as channel blockers that interact with receptors on a cell's surface and interior membranes, the researchers said.

The color gradient in individual membrane corrals is caused by the application of an external electrical field. The field causes the red dye molecules to concentrate on one side of the membrane, demonstrating that it is possible to manipulate protein concentrations.

The starting point for the project was research that was completed in 1986 and 1987 by Stanford chemistry Professor Harden McConnell. He and his students figured out how to make two-dimensional membranes ­ technically termed "supported lipid bilayers." They also discovered that such membranes resembled the membranes in living cells so closely that they could trick cells into acting as if they were real. This enabled McConnell and his colleagues to perform key research regarding the nature of the interactions between cells.

Groves began working with supported bilayers in 1994. His first discovery was that scoring supported membranes with a pair of tweezers caused them to separate permanently. He took advantage of this characteristic to partition the membrane into triangles and squares. Next, he applied electrical fields to these large membrane patches and found that he could manipulate the concentration of certain types of proteins attached to the membranes.

Nick Ulman, an electrical engineer who works at Stanford's Ginzton Laboratory, joined the group in 1995. A postdoctoral student at the time, he approached Boxer looking for projects when David Bloom, the professor with whom he was working, decided to leave Stanford. Boxer got Ulman and Groves together and they came up with the idea of using microfabrication techniques to make smaller membrane corrals.

"The hardest part was deciding what to do," Ulman said. The actual processing involved was straightforward. But, because electrical engineers have never thought about deliberately exposing their microcircuits to salt water, there were few guidelines for them to follow, he said.

As a result, "we have developed something new at the interface between cell science, chemistry and electrical engineering that has widespread potential in many areas," Boxer said.


By David F. Salisbury
