Microfluidic system for studying cell membrane mechanisms


Wednesday, 22 April, 2015


Microfluidic system for studying cell membrane mechanisms

Researchers have constructed a microfluidic system with which to study the mechanisms responsible for transport through the cell membrane - a structure which has previously been difficult to examine due to the difficulty in creating a nanometre-thick membrane for experimentation. The system was developed at the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw, in cooperation with the Chemical Research Laboratory at Oxford University and reported in the journal Lab on a Chip.

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 the laboratory 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).

As explained by IPC PAS Professor Piotr Garstecki, the corresponding author on the paper, the new microfluidic system “not only automates the process leading to the formation of highly stable contact at the interface of two microdroplets to form a bilayer, but also enables us to carry out electrophysiological measurements. We are able to follow the process of, for example, the incorporation of a specific protein in the cell membrane, in the presence of various inhibitors.”

The system features two types of droplets coated with lipid monolayers formed inside microchannels filled with oil: one containing a solution of a protein capable of incorporation into the cell membrane; the other containing a neutral liquid or inhibitors capable of binding with the protein in the other type of droplet. When two microdroplets, each of a different type, flow into a miniature measuring chamber, they are precisely positioned by hydrodynamic traps. These were developed by IPC PAS together its spin-off company, Scope Fluidics.

“The task is not a simple one,” noted PhD student and paper co-author Magdalena Czekalska. “The membranes we are examining have a thickness of a few billionths of a metre, and they are easy to break. With the hydrodynamic traps we can not only stabilise the position of the droplets, but also prevent vibration of the membranes that occurs naturally during flow.”

The system also provides the ability to perform electrophysiological measurements. At the moment of formation, 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 will form a pore through which ions may flow. The system enables these minute currents to be measured by microelectrodes built into the measuring chamber.

“In our tests we have observed currents appearing at the moment of incorporation in the membrane of a single nanopore of alpha-haemolysin protein, provided by the Oxford group,” said PhD student and co-author Tomasz Kaminski. “The spike was extremely small, only about 50 picoamps (10-12 amps), but always very clear.”

Handmade bilayer membranes are very sensitive and usually persist from a few minutes to a few hours. Artificial cell membranes in the new system, however, have a life span of up to several days. At the same time, the system allows for 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. Furthermore, the whole measurement cycle can take as little as three minutes.

“The measurements we have carried out are proof that functional cell membranes are created in the new microfluidic system,” said Professor Garstecki. “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.”

Image caption: A bilayer lipid membrane is formed at the junction of two droplets. The phenomena occurring on it can be analysed by the appropriate electrodes. The idea of constructing a microfluidic system for serial studies of cell membranes is presented by PhD student Tomasz Kaminski. Image credit: Polish Academy of Sciences, Grzegorz Krzylewski.

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