Sensor chip L1 - lipophilic modification

The sensor surface is dextran coated and modified with lipophilic substances (alkyl chains; (1)). Liposomes diffuse to the dextran surface and are attached directly to the sensor surface without the need to incorporate anchor molecules within the liposomes. Protocols for liposome formation do not need to be modified. The lipid surface mimics biological membranes and can be used in studies of membrane systems. Either before or after liposome binding, the vesicles can be modified by incorporating membrane bound molecules. The sensor surface can be regenerated to allow attachment of various types of liposomes, but care must be taken to remove non-specific bound proteins to the sensor surface.

Not all publications agree on the type of lipid surface on the sensor chip after binding of the liposomes. It was shown that the vesicles can be either intact (1),(2) or fused to a lipid bilayer (3),(4).

The experimental setup for the L1 chip is the same as for the HPA chip. Capturing of liposomes is done after a short pulse of 40 mM Octyl-D-glucoside or 20 mM CHAPS (3-[(3-cholamidopropyl)-dimethylammonia]-1-propan-sulfonate) at 5 μl/min to clean the sensor chip (1). Liposome concentration of 0.5–2 mM with respect to phospholipids is commonly sufficient. If compatible with the user-defined ligands, one short pulse of 10-100 mM NaOH followed by the regeneration solution should be injected after liposome binding to stabilize the base line.

Possible areas of non-specific binding should be tested with a pulse of 0.1 mg/ml BSA or other suitable protein. A fully covered sensor chip has only a minor (40 ± 25 RU) binding of BSA whereas an uncovered surface binds up to 430 RU of BSA. This pulse could also serve as a blocking step to avoid interference from non-specific binding (5).

Detergents and organic solvents may alter or destabilize liposome binding or even cause liposomes to disintegrate. Such agents can be used in attempts to strip the liposomes from Sensor chip L1 in preparation for binding new liposomes. The validity of such a treatment must be fully tested for every new condition.

Lipid samples isolated from for instance blood plasma contain many proteins. These proteins tend to bind to the L1-chip in a non-specific manner and are hard to remove. Incubation with a solution of Trypsin/EDTA (e.g. from cell culture) will remove most of the bound proteins but leave the hydrophobic binding anchor intact.

Because many interactions with lipids depend on calcium or magnesium ions, several alterations to the standard flow buffer can be made.

Capturing protocols

Short protocol 1: The flow buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 3 mM CaCl₂, 0.6 mM MgCl₂) is 0.22 μm filtered and degassed before use (6). The sensor chip surface is cleaned with an injection of 10 μl of 40 mM octyl glucoside, followed by a wash period of two minutes. Then 5 μl of phospholipids (76 μg/ml) is injected at 10 μl/min resulting in a response increase of 2000 RU. A surface without phospholipids is used as a control (7),(8).

Short protocol 2: The sensor chip is cleaned by injecting 40 μl 20 mM Chaps with a flow rate of 20 μl/min followed by an extraclean command. The lipids (1 mM) are loaded by injecting 80 μl at a flow rate of 2 μl/min. The surface is stabilized by an injection of 50 μl 10 mM NaOH with a flow rate of 100 μl/min (1).

Short Protocol 3: After cleaning the sensor surface, the lipids are captured with a 2-25 μl injection of 3 mM liposomes at a flow rate of 2-5 μl/min. Stabilization is done by two injections of buffer which results in a stable surface of about 7000 RU captured liposomes. After experimentation the surface is stripped with a 10 μl injection of 50/50 (v/v) 100 mM HCl/isopropanol solution (8).

The amount of captured lipids depends on the type of lipid composition of the vesicle, vesicle size and contact time.

After using the sensor chip, the surface must be cleaned before storage. Cleaning is done with an injection of 40 μl 20 mM CHAPS at 20 μl/min. The sensor chip is undocked and thoroughly washed with clean water, blown dry and stored over dry silica gel under nitrogen at ± 4°C (1).

More information about the protein-membrane interactions can be found in the publication of Besenicar (9).