Quartz crystal microbalance and atomic force microscopy to characterize mimetic systems based on supported lipids bilayer

Abstract

Quartz Crystal Microbalance (QCM) with dissipation and Atomic Force Microscopy (AFM) are two characterization techniques that allow describing processes taking place at solid-liquid interfaces. Both are label-free and, when used in combination, provide kinetic, thermodynamic and structural information at the nanometer scale of events taking place at surfaces. Here we describe the basic operation principles of both techniques, addressing a non-specialized audience, and provide some examples of their use for describing biological events taking place at supported lipid bilayers (SLBs). The aim is to illustrate current strengths and limitations of the techniques and to show their potential as biophysical characterization techniques.

Keywords: atomic force microscopy; biomimetic membranes; label-free detection; quartz crystal microbalance; supported lipid bilayers.

FIGURE 1

Events that can be studied using Supported Lipids Bilayers: (A) Formation by vesicle fusion. (B) Formation of lipid domains (C) Formation of tethered bilayers (D) Insertion of proteins (E) Fusion of proteoliposomes (F) Following biomolecular interactions such as that between soluble ligands and membrane proteins.

FIGURE 2

Description of QCM operation principle. (A) Oscillation of the quartz crystal (of thickness d) driven by the electrodes without (left) and with (right) material deposited on top illustrating how the wavelength λ of the resonance frequency changes to λ ’ for different overtones n. (B) Illustration of the energy dissipation (D_n) extracted from the bandwith (Γ_n) and frequency (f_n) changes of the loaded crystal. (C) Illustration of the penetration length (L_p) that depends on the frequency (ω), density (ρ) and viscosity (η) of the solution.

FIGURE 3

Modes of detecting resonant phenomena. (A), ring-down method, where the driving signal is abruptly shut off the decay as a current trace on an oscilloscope is observed. (B), impedance analysis in which one may either sweep the frequency of an AC excitation across the resonance.

FIGURE 4

(A) Top and side view of a quartz crystal showing the gold electrodes on top and bottom that allow the connection to electrically drive the crystal oscillation. (B) Lateral view of a diagram of the QCM measuring chamber (left). The crystal is inserted into a sealed chamber that allows contact with the electrodes from the bottom and exposure of the active upper region to liquid that can be exchanged through a pumping system. Right, example of a typical data acquisition set where the frequency drop (red line) registered follows the adsorption of material to the crystal surface and the dissipation in real time (gray line) (Figure 5 for further details).

FIGURE 5

Sensogram. The change in frequency Δf/n (left axis) and dissipation ΔD (right axis) as a supported lipid bilayer (SLB) is formed from vesicle fusion. The regions depicted (I, II and III) illustrate the configurations of the sample that can account for the changes observed in sensogram signal. (I) the chamber is filled with the desired buffer and frequency and dissipation shifts are taken as reference for the rest of the experiment. (II) intact liposomes adsorbed on the surface shift both Δf/n and ΔD. (III) liposome rupture releases water, reducing both the frequency change and the dissipation, as the lipids spread on the surface to form the SLB.

FIGURE 6

Description of AFM principle. (A) A laser reflected from the back of a cantilever impinges on a photodiode and reports on the vertical displacement of the tip as it scans the Surface. (B) In contact mode, the tip touches the sample and deflects vertically following surface topography. (C) In tapping mode, a tip oscillating at or near its resonant frequency scans over the surface while maintaining its amplitude constant. The piezo vertical displacements reflect the surface topography.

FIGURE 7

AFM topographic image of an SLB made of a mixture of lipids. (A) SLB composed of DPPC 45% DLPC 18% B sitosterol 8% PIP 24% that segregate into liquid ordered and liquid disordered domains (Velez et al., unpublished results). The colors represent differences in height: lighter regions protrude from the surface more than darker regions (B) The profile under the black line in (A) shows the 1 nm height of the ordered domains. (C) Three dimensional representation of the image in (A). (D) illustration of the lipid arrangement originating the height difference between ordered and disordered domains.

FIGURE 8

AFM topographic image of protein seggregation in an SLB. (A) The protein is bacterial cytoskeletal protein FtsZ covalently attached to a lipid bilayer as described in (Márquez et al., 2019). FtsZ protein forms filaments in the presence of GTP (B) Topographic image that shows the filaments formed on top of the SLB upon GTP addition. The colors represent differences in height: lighter regions protrude from the surface more than darker regions (unpublished image from Velez’s lab.) (C) Profile under the blue line in (B) that shows the height of the filaments.