Uncovering Lipid Membranes

Dual Polarisation Interferometry (DPI) is an important tool that enables drug discovery, as well as an important enabling tool for biochemistry and cell biology. The AnaLight 4D scientific instruments by Farfield represent analytical methods that provides high resolution measurements of density, dimensions and quantitative mass for biomolecules and their complexes. There is a clear link between changes in these physical parameters and the interactions of biological membranes and proteins or peptides — at least, their functional and structural elements.

The experiment

In this case, an AnaLight 4D instrument was used to perform DPI experiments. This involved utilising an unmodified AnaChip, with temperature being maintained at lower than 20 degrees Celsius at all times. The experiment used analytical-grade buggers and reagents, while a Farfield Aries degasser was used to degas all solutions before being used. The running buffer for the experiment was 10 mM HEPES, 150mM sodium chloride.

Step 1: Cleaning of the AnaChip

For 15 minutes, the AnaChip was cleaned with a Proclean UV-ozone cleaner. It was then left until the next morning. The UV-ozone cleaner helps get rid of contaminants at the molecular level. The AnaChip was then placed in the instrument, with the usual calibration procedures taking place with 80% ethanol/water for linearisation and chip calibration steps. For the bulk calibration, HPLC-grade water was utilised.

Step 2: The lipids extruded

DOPC lipid thin films were then made ready, being dissolved in HPLC-grade chloroform, as well as having solvent removed via high vacuum, before being placed under nitrogen to extract the leftover solvent. DOPC lipids were then taken up in HPLC grade water and extruded with 100nm polycarbonate membranes. A minimum of 19 lipid passes then took place to ensure that particle distribution was uniform.

Step 3: Deposition and removal of lipid bilayer

Next, a good coverage of lipid bilayer on the surface was sought, with very little non-specific binding. In combination with the birefringence and lipid mass DPI measurements, it was confirmed that the lipid bilayer surface was excellent. The process was then mimicked for DOPC/DOPG lipid bilayer membranes, albeit with a different lipid deposition buffer. Finally, the lipid bilayer membranes were removed with sodium dodecyl sulphate.

The lipid bilayer established

The critical components of lipid bilayer membranes are phospholipids. They consist of a polar, water-soluble head group, as well as two non-polar hydrocarbon tails, and they form on their own into liposomes. When liposomes get injected onto the AnaChip surface, the head groups find themselves attracted to the hydrophilic chip surface through the means of electrostatic interaction. With the surface populated by liposomes, they then deform and maximise surface area on the chip. Once at a critical coverage, they burst and form a surface lipid bilayer membrane.

Any alterations in the birefringence and mass during interactions are related to shifts in alignment order within the bilayer, which distinguishes disruption of the bilayer from direct measurement of association to head group.

Conclusion

Overall, it can be seen that DPI can be utilised to study lipid bilayer membranes behaviour when deposited on the AnaChip surface. The AnaLight 4D analytical instruments afford researchers a unique combo of real-time, high-res data on mass, dimension, density and birefringence.