3.2.1 Preparation of unilamellar vesicles
Vesicles are well suited to investigate protein-membrane interactions. They differ in size and application. The radii vary from 20 nm (small unilamellar vesicles, SUVs), 100-200 nm (large unilamellar vesicles, LUVs) up to 100 µm (giant unilamellar vesi-cles, GUVs).61–63
Small unilamellar vesicles
For the preparation of small unilamellar vesicles (SUVs) lipid films with 0.4 mg of lipid material were used. These films were obtained by merging different lipid stock solu-tions (dissolved in chloroform; c = 2-10 mg/mL) in a test tube. This enabled to pre-pare lipid films with defined lipid compositions. After removing the chloroform in a nitrogen flush, the films were dried under vacuum at 30°C. The films were then stored at 4 °C until use.
The films were rehydrated with citrate buffer (30 min) and subsequently the test tubes were vortexed three times for 30 s in a five-minute interval, resulting in the formation of multilamellar vesicles (MLVs). The MLVs were then treated in an ultra-sonic bath for 30 min at RT to obtain SUVs.
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Giant unilamellar vesicles (GUVs) with a diameter of ≦ 1 µm were prepared by an electro-formation process, first described by Angelova et al.64 A mixture of lipids dis-solved in chloroform (c = 0.5 mg/mL) were added on two indium tin oxide (ITO) co-vered glass slides. The chloroform was removed under reduced pressure for at least 30 min, resulting in a lipid film on top of the ITO. These ITO slides were then assem-bled to a chamber and sealed with a silicon ring and two Teflon spacers. Afterwards a sucrose solution (298 mOsmol/kg) was filled into the chamber. The connection to the generator was achieved with copper stripes, so each ITO slide was linked to one pole (Figure 3.1). A sinusoidal alternating current voltage of 1.6 V (peak-to-peak) and 10 Hz was applied for 3 h, resulting in GUV formation. After collection of the GUVs they were stored at room temperature for a maximum 3 days.
Figure 3.1: Schematic drawing of a GUV electro-formation chamber. On each ITO slide a self-adhesive copper stripe is placed via a Teflon spacer. The Teflon spacers connect the chamber with a voltage source, which results in the formation of GUVs.
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3.2.2 Substrate surface preparation
Various experimental methods require different substrates, which vary in material or size. Due to this, surface functionalization strategies can be used to obtain stable membrane models. In this work two different functionalized substrate types were employed to generate supported lipid membranes and adhered giant unilamellar ves-icles.
Lipid bilayer and monolayer on silicon dioxide wafers
Silicon wafers coated with silicon dioxide (SiO2) from Silicon Materials, Inc. (PA, USA) were cut into 1.9 cm x 0.8 cm rectangles.For reflectometric interference spectroscopy (RIfS) experiments wafers with 5000 nm SiO2 layer thickness and for atomic force microscopy (AFM) measurements wafers with 100 nm SiO2 were used.
For both techniques the substrates were hydrophilized with an aqueous ammonia hy-drogen peroxide solution (H2O/NH3 (25%)/H2O2 (30%) 5:1:1 (v/v/v)) for 30 min at 70 °C. Hydrophilized substrates were rinsed with ultrapure water and stored in ul-trapure water. Before use the substrates were dried in a nitrogen flush and treated with oxygen plasma (30 s, 0.2 mbar, 60 % power). Afterwards, SUVs were spread on the hydrophilic surfaces, resulting in a lipid bilayer.
Lipid monolayers were prepared on hydrophobic silicon wafers. Therefore, the wa-fers were first cleaned with Hellmanex (15 min) and ultrapure water (2 x 15 min) in an ultrasonic bath. Then they were treated with oxygen plasma (30 s, 0.2 mbar, 60%
power) and subsequently incubated with 1,1,1,3,3,3-Hexamethyldisilazane (HMDS).
Incubation was performed overnight in a sealed chamber at 120°C and under reduced pressure, yielding hydrophobic surfaces (Figure 3.2).
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Figure 3.2: Hydrophobic functionalization of silicon dioxide wafers with HMDS.
Silanization and PEGylation to generate adhered GUVs
First, glass slides had to be functionalized with NeutrAvidin to obtain immobilized biotinylated giant unilamellar vesicles (GUVs). The glass slides (24 x 50 mm, Ther-moFisher Scientific Gerhard Menzel, Brunswick, Germany) were cut into 1.0 cm x 1.0 cm substrates, cleaned with ethanol p.a. and ultrapure water. After treat-ment with oxygen plasma (30 s, 0.2 mbar, 60 % power) pure 3-glycidyloxi-propyltri-methoxysilane (GOPTS, stored in an argon atmosphere) was added between two sub-strates and incubated for one hour at 80 °C in a glass weighing bottle with an argon atmosphere. In that time, a mixture of methoxy- and biotin-functionalized PEGs (1:1, 2:1 and 3:1) was heated in a thermomixer at 85-95 °C (dependent on the PEG-length) and 1400 rpm. The substrates were separated from each other and then rinsed with acetone. Drying in a nitrogen flush and placing the substrates on a preheated alumi-num block (80°C) enabled the addition of 200 µL of the molten PEG-mixture between two substrates without solidification. Afterwards, the substrates were placed in the weighing bottle and incubated at 85-95 °C for 4 h. The substrates were separated, in-tensely rinsed with ultrapure water and dried in a nitrogen flush. If the molten PEG mixture was still present on the substrates (impure surface), the rinsing step was re-peated. Prior to use the substrates were stored under argon at 4 °C. The silanization and PEGylation is illustrated in Figure 3.3.
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Figure 3.3: Silanization and PEGylation of glass substrates, resulting in methoxy-PEG and biotin-PEG surfaces.
To obtain adhered GUVs, the functionalized substrates were first incubated with Neu-trAvidin (50 µg/mL in PBS) for 30 min. Then the NeuNeu-trAvidin-functionalized sub-strates were rinsed six times with PBS and further six times with sucrose buffer. 10-50 µL of a biotin-containing GUV suspension (in sucrose) was added and incubated for 15 min in a humidity chamber to avoid osmolar changes. Via biotin-NeutrAvidin interactions the GUVs adhered to the surface.
To verify a complete coverage of the substrates with NeutrAvidin, the surface was incubated with DyLight® 594 labeled Neutravidin/unlabeled NeutrAvidin in a ratio of 99:1.