A compression isotherm of a monolayer can be obtained by compressing a constant number of moles of the monolayer with the hydrophilic barriers at a constant temperature and atmospheric pressure. The surface pressure increases according to the nature of the monolayer of amphiphilic molecules. Compression isotherms can be used to observe phase transitions of the monolayer material in question. When ÂM is high, the molecules are sparsely distributed at the air-water interphase and only very few are interacting with each other. This is called the gas phase, in which the surface pressure is zero or very close to zero. When the barriers are compressed sufficiently, the surface pressure starts increasing and a phase transition first from gas (G) to a liquid expanded (LE) and finally to liquid condensed (LC) phase is observed. In the liquid phase the molecules are slightly more packed together. If the barriers are compressed even further, the liquid phase turns into a solid phase and the surface pressure increases dramatically. In this phase, the molecules are very closely packed together and the monolayer is said to be in a solid-like (S) phase. A typical compression isotherm of a membrane lipid (DPPC) is shown in figure 2-5.
At a certain point it is not possible to increase the surface pressure further – the ÂM has reached its minimum (Acoll), the forces acting on the film are high and the film collapses. At this point the molecules are forced out of the monolayer, forming micelles and layers on top of each other and dissolving into the subphase, causing the surface pressure to drop.
The point of collapse depends on variety of factors, including the history of the film, temperature, rate of compression and the monolayer material itself. The compressibility (Cs) of the film characterizes its rigidity and is given in equation 5 .$
Brewster angle microscopy (BAM)
Monolayers at air-water interface can be further analyzed with Brewster angle microscopy (BAM) and electric surface potential meter [183, 184]. BAM is a microscopic technique relying on the reflectivity differences of water and monolayer surfaces. P-polarized light at a Brewster’s angle of incidence (α) results in a minimum reflectivity from water surface and thus when monolayer is present, the optical properties of the interface are altered, resulting in increased reflectivity of the regions covered by the monolayer (figure 2-6). This allows a visual investigation of the monolayer structure, such as domain growth [122, 185]. Electric surface potential is used to study the difference in electrical potential above and below a monolayer at the air-water interface and is measured by observing the potential difference between a vibrating plate placed above the monolayer and a counter electrode immersed in the subphase .
Figure 2-6. A schematic figure of the Langmuir trough and the principle of Brewster angle microscope. a) No monolayer present at the air-water interface and therefore no reflection occurring from the p-polarized light beam. b) A monolayer is present at the air-water interface and a reflection of the p-polarized light occurs when the beam comes on the surface at a Brewster’s angle of incidence (α) (drawn based on ).
Preparation of Langmuir monolayers and compression isotherms
For Langmuir film studies, DPPC, DPPE, DOPC and equimolar mixtures of DPPC/DOPC and DPPE/DOPC phospholipids were dissolved in chloroform at a concentration of 1 mM. The solutions were further stored at 4 °C before use. The surface pressure (π) and electric surface potential (∆V) measurements were carried out with a KSV 2000 Langmuir balance (KSV Instruments, Ltd., Helsinki, Finland). Compression isotherms were determined for pure lipid monolayers as well as their various equimolar (1:1) mixtures with a Teflon® trough [6.5 cm (l) x 58 cm (w) x 1.0 cm (d)] holding two hydrophilic Delrin® barriers for symmetric compression. The system was equipped with an electrobalance holding a platinum Wilhelmy plate (perimeter 3.94 cm) as a surface pressure sensor and a surface potential measuring head with a vibrating electrode (KSV SPOT1). A stainless steel plate immersed 4 mm below the surface was used as a counter electrode. The apparatus was kept in a Plexiglas cabinet and temperature was kept constant at 20 ± 0.1 °C. Prior to each experiment, the trough and the barriers were washed by cotton soaked in chloroform and ethanol and then rinsed with Milli-Q water. The platinum plate was cleaned between each run by rinsing with Milli-Q water and ethanol, and finally heated to a red-hot glow in a propane flame to eliminate any organic contaminants. All solvents used for cleaning the trough and the barriers were of analytical grade. Any residual surface-active impurities were removed from subphase surface by sweeping and suction. The stability of the surface potential signal was checked before each experiment after cleaning the subphase surface. After the ∆V signal had stabilized and the surface pressure fluctuation was less than 0.2 mN.m-1 during compression stage, monolayers were spread from calibrated solutions using microsyringe (Hamilton Co., USA). After an equilibration time of 10 min, the films were compressed at the rate of 10 mm.min-1 by two symmetrically advancing barriers (5mm.min-1 per barrier). A computer and KSV software were used to control the experiments. Each compression isotherm was performed at least three times. Changes in the mechanical properties of the monolayers were studied through the values of compressibility modulus  and the collapse parameters, , and ∆Vcoll were determined directly from the compression isotherms.
During compression, the morphology of the films was imaged with a computer-interfaced Brewster angle microscope (KSV Optrel BAM 300, Helsinki, Finland) using p-polarized light from a class IIIb 10-mW laser at 632.8 nm, the lateral resolution of the instrument being 1 µm.
Atomic force microscopy (AFM)
Atomic force microscope (AFM) is a scanning probe microscope (SPM) that is used to form three dimensional images of surfaces with the aid of a physical probe with nano-scale dimensions. The surface of the sample is scanned while the interaction forces with the sharp cantilever tip are recorded. Apart from topography of the surface, AFM can be used to measure close-range interactions between the sample and the tip. AFM has been increasingly used in biological and medical applications in the 21st century and one of its major advantages indeed is its usability in aqueous and physiological conditions in real time .
Principle and basis of AFM technique
AFM is an interesting tool as it is able to provide images at atomic resolution with nanometer scale resolution height information. A typical AFM probe has multiple cantilevers each having their own tip made of silicon or silicon nitride, with a specific shape, usually triangular or rectangular. The probe scans the surface of the sample and the high precision movements of the sample positioning in x, y and z directions are adjusted by a piezoelectric scanner. This scanner controls the relative position of the probe and the sample surface and is located at the sample stage under the sample. A laser is directed on top of the AFM cantilever and a position sensitive photodetector is used to collect the laser beam reflected on top of the cantilever to record tip deflection while moving the sample surface. The top of the cantilever usually has a gold coating to improve reflection of the laser beam. The deflection signal is used to measure the forces resulting from the interactions between the AFM tip and the sample surface. Figure 2-7 illustrates the principle of AFM operation.
Figure 2-7. The principle of an atomic force microscope (AFM) (drawn based on [174, 189]).
When the AFM tip approaches the sample surface, it undergoes various different forces such as ionic repulsion, van der Waals forces, electrostatic and magnetic forces that act on the tip depending on the separation distance and physico-chemical nature of the tip and the sample. These forces are either attractive or repulsive and result in the deflection of the tip cantilever. At long separation distance the tip and the sample are not in contact and the forces acting between are attractive Van der Waals forces (shown as non-contact zone on figure 2-8) .
The cantilever deflects downwards. At short separation distance as the cantilever is brought closer to the surface, the tip makes contact with the sample and a repulsive force caused by electrostatic interactions between the sample and the tip increasingly takes over and causes the cantilever to deflect away from the surface (shown as contact zone on figure 2-8). These deflections lead to a change of the position of the laser spot reflected to a position sensitive photodetector with four sectors, and therefore changes the laser intensity received by each of these sectors. This light signal is then converted into an electric signal and sent to the piezoelectric scanner and thus a feedback loop to conserve either the height of the tip above the surface or the interaction forces between the tip and the surface can be created. The displacements of the piezoelectric scanner can then be used to construct a topographical height image of the sample surface. The resolution of the AFM image depends on the number of pixels of the image and the dimensions of the AFM tip (radius of curvature). The more pixels there are in an image, the more acquisition time the image recording requires. Generally an image resolution of 256 × 256 or 512 × 512 is sufficient in obtaining good quality images. The smaller the radius of curvature of the AFM tip, the better the precision of the sample topography is . The radius of curvature of an AFM tip is typically from 1 to 20 nm.
Depending on the level of contact between the sample and the tip, AFM can be operated in three different modes; contact mode, intermittent contact mode (TappingTM mode) and non-contact mode. In contact mode the tip and the sample are maintained in contact with each other and repulsive forces act on the tip and the cantilever is deflected with distance (d) proportionally to the force (F) according to Hooke’s law (equation 7) .
where kC is the spring constant of the cantilever. The contact mode can either be used by keeping the applied force (and therefore also the set-point value of the photodetector) constant in which case the measured tip-sample distance (z) changes due to the feedback loop, or by maintaining the z value constant and measuring the cantilever deflection. The contact mode provides images with high resolution and is the most frequently used of the three modes, however, it is not optimal for biological samples as the applied force can potentially damage fragile biological specimen and the tip can easily get contaminated by the sample.
The intermittent contact mode (TappingTM mode) is based on the oscillation of the tip at its resonance frequency (5-300 kHz) at defined amplitude. This causes the tip to periodically interact with the sample. When the tip comes into contact with the sample, the forces acting between the tip and the sample cause the pre-defined amplitude of the tip oscillation to change. Thus reduction of the oscillation amplitude is used as a feedback control signal . The oscillation amplitude is directly proportional to the average separation distance . Topographical height images can thus be obtained. In this mode the contact is only intermittent and therefore the frictional forces between the tip and the sample can be neglected and the damages to the sample are minimized . However, the acquisition time of an image with the intermittent contact mode is longer than the one with the contact mode.
In the non-contact mode the tip does not come into contact with the sample but oscillates around its resonance frequency at 1-10 nm above the sample surface, scanning the attractive forces between the tip and the surface. The oscillation amplitude is changed due to these forces. The non-contact mode does not cause any contamination of the tip by the sample, but the scanning time is very slow and the lateral resolution is weak.
A part from imaging, AFM can be used to measure mechanic forces between the AFM tip and the sample, such as the lipid bilayer or cell membrane, revealing structure-function relationships of single molecules at a pico Newton (pN) scale . These forces are obtained from the cantilever deflection according to Hooke’s law (equation 7) and are presented as force-distance curves (figure 2-9) showing the intensities of the forces acting between the tip and the sample as the tip approaches or retracts towards and from the sample surface.
Figure 2- 9. Schematic representation of a force curve obtained by AFM force spectroscopy indicating the different regions of the approach and retraction zones (drawn based on ).
When the tip approaches the sample surface (phase 1 in figure 2-9), it is far from the surface, at its equilibrium, and no interaction between the tip and the sample occurs. This is where the baseline of the force-distance curve (F = 0) is defined. Information on the long range interactions such as electrostatic effects at few micrometres above the surface can be obtained when the tip approaches the sample. These long-range interactions would cause a negative peak in the approach zone of the force-distance curve, finally leading to the detection of short-range forces, such as the Van der Waals forces, which are seen as a linear deflection of the tip (snap-in) shown as regions 2-5 in figure 2-9. At region 2 the tip is in contact with the bilayer sample, causing it to deform elastically, finally coming to the maximal deformation of the bilayer and, at region 4, penetrating into the membrane. At region 5 the tip continues to push the sample until the maximal force indicated by the user is reached and the tip is retracted from the sample, seen as a retraction curve, showing a region of adhesion and finally a point of rupture at which the tip and the sample are separated. The force difference between the pull-off and the baseline is referred to as the rupture force (fu). The approach zone of the force-distance curve, notably the elastic deformation region (region 2 of figure 2-9) allows the determination of the sample elasticity and rigidity such as the Young modulus or the stiffness, by different physical models. The larger the curvature of the contact point region is, the bigger is the sample deformation. The physical models of contact mechanics used for the determination of the sample elasticity include the Hertzian (sphere-on-flat) and Sneddon (cone-on-flat) theories, in which adhesion forces between the tip and the sample are neglected, and Derjaguin-Muller-Toporov (DMT) theory that takes the attractive interactions outside the contact area into account. The DMT model is readily integrated in the NanoScope Analysis software (Bruker, Palaiseau, France) but to gain more precision in elasticity calculations and in particularly in the case of the biofilms, we used the Sneddon model integrated in our automated algorithm .
Table of contents :
1. Scientific context
1.1 Biological membranes
1.1.2. Eukaryotic and prokaryotic cell membrane structures
126.96.36.199 Physical states of membrane phospholipids
1.2. From a single bacterium to bacterial biofilms
1.3. Antimicrobial peptides
1.3.2. Bovine Catestatin
1.4 Application of lipid membrane and bacterial biofilm models on peptide studies
1.4.1 Langmuir monolayers
1.4.2 Atomic force microscopy
1.4.3 Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR)
1.5 Aim of this work
2. Materials and methods
2.1. Preparation of phospholipid and lipopolysaccharide bilayers
2.2. Bacterial culture and formation of bacterial biofilms
2.2.1. E. coli bacterial strain
2.2.2 Determination of minimal inhibitory concentration
2.2.3. Bacterial culture
2.2.3. Bacterial growth in suspension
2.2.1. Formation of bacterial biofilms
2.2.4. Fluorescence microscopy and BacLight ™ staining
2.3. Langmuir method
2.3.1. Compression isotherms
2.3.2. Brewster angle microscopy (BAM)
2.3.3. Preparation of Langmuir monolayers and compression isotherms
2.4. Atomic force microscopy (AFM)
2.4.1. Principle and basis of AFM technique
2.4.2 Force spectroscopy
2.4.3. AFM for phospholipid and LPS bilayer experiments
2.4.4. AFM imaging and force spectra of biofilms
2.5. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)
2.5.1. Principles of ATR-FTIR
2.5.2. ATR-FTIR on phospholipid bilayers
2.5.3. ATR-FTIR on bacterial biofilms subjected to AMPs
3. Antimicrobial peptide action on model membranes
3.1 Phospholipid monolayer interaction with a cyclic antimicrobial peptide
3.2 Phospholipid bilayer interaction with antimicrobial peptides
3.2.1 Morphological and spectral characteristics of phospholipid bilayers
3.2.2 Phospholipid bilayer interaction with a cyclic antimicrobial peptide
3.2.3 Phospholipid bilayer interaction with a linear antimicrobial peptide
3.3 Lipopolysaccharide bilayer interaction with antimicrobial peptides
3.3.1 Morphological characteristics of LPS bilayers
3.3.2 Lipopolysaccharide bilayer interaction with a cyclic antimicrobial peptide
3.3.3 Lipopolysaccharide bilayer interaction with a linear antimicrobial peptide
3.4 Comparison of the action of a cyclic and a linear peptide on model membranes
4. Antimicrobial peptide action on bacterial biofilms
4.1 Bacterial growth in planktonic form
4.2 Formation of a bacterial biofilm
4.3 Cyclic antimicrobial peptide action on a bacterial biofilm
4.4 Linear antimicrobial peptide action on a bacterial biofilm
4.5 Comparison of the action of a cyclic and a linear peptide on biofilms