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Peering Into Cell Membranes
April 1, 2015   
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Life processes depend fundamentally on phenomena occurring in membranes separating cells from their environment. Hitherto poorly understood, these phenomena will now be studied faster and more cheaply thanks to a microfluidic system developed at the Polish Academy of Sciences’ Institute of Physical Chemistry in Warsaw. The system enables the serial formation of cell membranes and measurement of the processes taking place in them.

Cell membranes separate the interior of the cell from its surroundings. Despite their fundamental role, many details of the mechanisms responsible for the functioning of cell membranes remain unknown. The main factor inhibiting progress in research is the difficulty in creating a nanometer-thick membrane for experimentation. But soon the study of cell membranes will become easier thanks to the microfluidic system developed at the Institute of Physical Chemistry, together with the Chemical Research Laboratory at Oxford University.

Typical cell membranes consist of two layers of phospholipids, with which various types of proteins bind in different ways. For several years, bilayer membranes have been prepared in laboratories by bringing into contact two droplets, each coated with a monolayer of lipids. If the process is performed skillfully, the drops do not merge and a lipid bilayer is formed spontaneously at the interface. This method is known as Droplet Interface Bilayers (DIB).

“Working with a group of Oxford University researchers headed by Prof. Hagan Bayley, we have constructed a microfluidic system that not only automates the process leading to the occurrence of highly stable contact at the interface of two microdroplets to form a bilayer, but also enables us to carry out electrophysiological measurements,” says Prof. Piotr Garstecki from the Institute of Physical Chemistry. “For example, we are able to follow the process of the incorporation of a specific protein in the cell membrane, in the presence of various inhibitors.”

In the new microfluidic system, there are two types of droplets coated with lipid monolayers formed inside microchannels filled with oil: one type of droplet contains a solution of a protein capable of incorporation into the cell membrane, and the other type contains a neutral liquid or inhibitors capable of binding with the protein in the first type of droplet. When two microdroplets, each of a different type, flow into a miniature measuring chamber, they are precisely positioned by special hydrodynamic traps developed by the Warsaw researchers together with technology company Scope Fluidics.

Magdalena Czekalska, a Ph.D. student at the institute, says, “The task is not a simple one. The membranes we are examining have a thickness of a few billionths of a meter, and they are easy to break. With the hydrodynamic traps we could not only stabilize the position of the droplets, but also prevent vibration of the membranes that occurs naturally during the flow.”

The biggest gain, however, is the ability to perform electrophysiological measurements. When it is formed, the new cell membrane is continuous and effectively prevents the flow of charge carriers between the two droplets. But if the protein dissolved in one of the droplets resembles a tube, just one molecule incorporated in the membrane is enough to form a pore through which ions can flow. In the system developed by the institute, these minute currents can be measured by micro-electrodes built into the measuring chamber.

Tomasz Kamiński, a Ph.D. student involved in the project and winner of the Foundation for Polish Science’s Ventures program, says, “In our tests we have observed currents appearing at the moment of incorporation in the membrane of a single nanopore of the alpha-haemolysin protein provided by the Oxford group. The spike was extremely small, only about 50 picoamps, but always very clear.”

Handmade bilayer membranes are very sensitive and usually last from a few minutes to a few hours. Artificial cell membranes in the new microfluidic system are much more stable: their life span is up to several days. At the same time, the system enables the detachment of one of the droplets leading to the destruction of the existing membrane, and the attachment of a new droplet, which involves the creation of a new membrane. The membrane protein dissolved in one droplet can therefore be tested with many droplets containing various concentrations of inhibitors blocking the nanopores. Importantly, the whole measurement cycle—the separation of the droplets, rinsing of the microelectrodes, the contact of new droplets, the formation of a membrane, and the measurement ending with the observation of the incorporation of the protein in the cell membrane—can take no more than three minutes.

Garstecki says, “The measurements we have carried out are proof that functional cell membranes are created in the new microfluidic system. We thus have fully automated measurements with minimum consumption of the reagents and samples necessary to carry out our experiments. The road to high-throughput studies of the mechanisms involved in cell membranes has been opened.”
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