Following the formation of supported lipid bilayers on mica: a study combining AFM, QCM-D, and ellipsometry - PubMed (original) (raw)
Following the formation of supported lipid bilayers on mica: a study combining AFM, QCM-D, and ellipsometry
Ralf P Richter et al. Biophys J. 2005 May.
Abstract
Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.
Figures
FIGURE 1
QCM-D response, i.e., changes in frequency (–○–) and dissipation (solid line), for the deposition of SUVs composed of DOPC/DOPS (molar ratio 4:1) on mica in 2 mM CaCl2. Lipid exposure starts at 0 min. The peak in dissipation and the minimum in frequency indicate the presence of intact vesicles bound at an intermediate state. The final frequency shift of −25 Hz and the low final dissipation shift indicate the presence of an SLB. A rinse with buffer (arrow) does not affect the SLB.
FIGURE 2
SLB formation from ∼10 μ_g/mL SUVs made of DOPC/DOPS (4:1) interrupted at an early stage. (A) QCM-D response (frequency, Δ_f (–○–), and dissipation, Δ_D_ (solid line), at 25 MHz); after the rinse (arrow), the adsorbed vesicles are not stable. (B and C) Two sequential images recorded after transfer of the mica-coated QCM-D sensor to the AFM; vesicles and bilayer patches, identified by their height and shape (cross section in inset, gray and black arrowheads, respectively) coexist. A few vesicles (asterisks) become ruptured by the influence of the AFM tip. Image size (_z_-scale), 1 mm (20 nm); the slow scan direction (contact mode) is indicated (arrows). The asymmetric and varying shape of individual vesicles is due to very low and slightly varying scanning forces (see Supplementary Material). (D) Changes in the ellipsometric angle, ΔΔ; after the rinse (solid line, arrow) at ΔΔ = −0.18°, corresponding to 30% of a complete SLB, the signal remains stable confirming that lipid material is not desorbing. For comparison, the response for the formation of a complete SLB is shown (dotted line), resulting in ΔΔ = −0.6°. Lipid deposition for the ellipsometry measurements was performed at concentrations of ∼5 _μ_g/mL (solid line) and ∼10 _μ_g/mL (dotted line), respectively.
FIGURE 3
Sequential AFM images (contact mode) before (A), during (B), and after (C) the merger of bilayer patches. Images A and C are scanned at lowest possible forces (∼100 pN). In image B the force was slightly increased (∼300 pN) inducing the merger (arrowheads) of several patches (asterisks). Before image acquisition, 25 _μ_g/mL SUVs of DOPC/DOPS (4:1) in 2 mM CaCl2 were incubated for ∼1 min and rinsed in buffer. Image size (_z_-scale), 1.25 mm (10 nm); the slow scan direction is indicated (arrows). Slight changes in the apparent size of individual patches and variations in the overall contrast (A and C) are due to slight instantaneous changes in the scanning force (see Supplementary Material).
FIGURE 4
Sequential AFM images (contact mode) of coalescence events of bilayer patches. After the tip-induced merger of patches 1–3 (A), the coalescence with patches 4 (B), 5–6 (C), and 7 (D) is induced by the movements of the reshaping patch. Before image acquisition, 25 _μ_g/mL SUVs of DOPC/DOPS (4:1) in 2 mM CaCl2 were incubated for ∼1 min and rinsed in buffer. Image size (_z_-scale), 1.75 mm (10 nm); the slow scan direction is indicated (arrows).
FIGURE 5
SLB formation from ∼50 μ_g/mL SUVs made of DOPC/DOPS (4:1) interrupted at a late stage. (A) QCM-D response (frequency, Δ_f (–○–), and dissipation, Δ_D_ (solid line), at 25 MHz); after the rinse (arrow) close to the minimum in frequency, surface-bound lipid material undergoes quick structural changes. (B) AFM image (tapping mode) taken after the transfer of the sample; extended patches coexist with a few vesicles (asterisks). The bilayer patches exhibit edges with small local radius of curvature (<150 nm; arrowheads). Image size (_z_-scale), 1.5 mm (20 nm). (C) Changes in the ellipsometric angle, ΔΔ; after rinsing (solid line, arrow) at ΔΔ = −0.41°, corresponding to 68% of a complete SLB, the signal remains stable confirming that lipid material is not desorbing. The response for the formation of a complete SLB is shown for comparison (dotted line). Lipid deposition for the ellipsometry measurements was performed at concentrations of ∼20 _μ_g/mL (solid line) and ∼10 _μ_g/mL (dotted line), respectively.
FIGURE 6
(A) AFM image of an SLB formed from a solution of 10 mg/mL SUVs made of DOPC/DOPS (4:1) and interrupted at a very late stage (contact mode). Numerous elongated defects persist. The ends of some of these defects (arrowheads) appear sharp. It is not clear whether these ends indeed represent a true bilayer boundary with a small but finite radius of curvature (∼15 nm). Alternatively, these points may mark the limits of an unresolved gap (dotted line) between two bilayer patches. (B) AFM image of an SLB formed from a solution of 100 mg/mL SUVs made of DOPC/DOPS (4:1) (tapping mode). The bilayer is ideal without visible defects, except for two protrusions (arrowheads), likely to be trapped vesicles. Image size (_z_-scale), 10 mm (10 nm).
FIGURE 7
QCM-D responses (frequency, Δ_f_ (–○–), and dissipation, Δ_D_ (solid line)) for the deposition of 0.1 mg/mL SUVs composed of different lipids on mica. Lipid exposure starts at 0 min. (A) DOPC in 2 mM CaCl2 (25 MHz, flow mode); after the rinse (black arrow) at an early stage of SLB formation, Δ_f_ and Δ_D_ remain stable, indicating that adsorbed vesicles are stably bound and neither desorb nor rupture; reincubation with SUVs (white arrow) leads to completion of the SLB formation. (B) DOPC in 2 mM EDTA (35 MHz, flow mode); formation of an SVL exhibiting high dissipation; a rinse with EDTA-containing buffer (black arrow) leads to partial desorption of the vesicles; a rinse with calcium-containing buffer (black dotted arrow) leads to the formation of an SLB, as characterized by a frequency shift of −26 Hz and a very low dissipation shift; subsequent addition of SUVs in 2 mM CaCl2 (white arrow) does not further affect the SLB. (C) DOTAP in 2 mM EDTA (35 MHz, flow mode); formation of an SLB; vesicles rupture instantaneously upon adsorption.
FIGURE 8
The local minimum in frequency, |Δ_f_min| (A and C), and SLB-formation time, _t_SLB (B and D), as a function of the fractional lipid charge, σ, (≡ average number of charges per lipid molecule). Data for mica (•) and silica (○; adapted from Richter et al., 2003) are shown in the presence of 2 mM EDTA (A and B) and 2 mM calcium (C and D), respectively. Data are given for lipid mixtures and conditions that lead to SLB formation; |Δ_f_min| is indicated only if the SLB formation exhibits a local minimum in frequency.
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