Nanometer scale organization of mixed surfactin/phosphatidylcholine monolayers.

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and of dipalmitoyl phosphatidylcholine (DPPC) were deposited on mica and their nanometer scale organization was investigated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). AFM topographic images revealed phase separation for mixed monolayers prepared at 0.1, 0.25, and 0.5 surfactin molar ratios. This was in agreement with the monolayer properties at the air-water interface indicating a tendency of the two compounds to form bidimensional domains in the mixed systems. The step height measured between the surfactin and the DPPC domains was 1.2 +/- 0.1 nm, pointing to a difference in molecular orientation: while DPPC had a vertical orientation, the large peptide ring of surfactin was lying on the mica surface. The N/C atom concentration ratios obtained by XPS for pure monolayers were compatible with two distinct geometric models: a random layer for surfactin and for DPPC, a layer of vertically-oriented molecules in which the polar headgroups are in contact with mica. XPS data for mixed systems were accounted for by a combination of the two pure monolayers, considering respective surface coverages that were in excellent agreement with those measured by AFM. These results illustrate the complementarity of AFM and XPS to directly probe the molecular organization of multicomponent monolayers.     

Molecular organization of surfactin-phospholipid monolayers: effect of phospholipid chain length and polar head

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC>DPPC>DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC>DPPE>DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.     

Molecular organization of surfactin-phospholipid monolayers: Effect of phospholipid chain length and polar head

Mixed monolayers of the surface-active lipopeptide surfactin-C₁₅ and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC > DPPC > DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC > DPPE > DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.     

Ca2+-Induced Effect on Mechanical Properties of Sulfatide-Incorporated Vesicles

The Ca(2+)-induced effect on the nanomechanical properties of vesicles prepared at a different ratio of dipalmitoylphosphatidylcholine (DPPC)/sulfatide was studied using atomic force microscope (AFM) on a mica surface. Vesicles were prepared by extrusion and adsorbed on the mica surface. The forces, measured between an AFM tip and the vesicle, showed that the breakthrough of the tip into the vesicles occurred two times. Force data prior to the first breakthrough were fitted well with the Hertzian model to estimate Young's modulus and bending modulus of the vesicles. Sulfatide incorporation led to a decrease of around 90% in Young's modulus and bending modulus of the vesicles due to the hydration of the headgroups, while the addition of Ca(2+) induced dehydration to recover the properties. The change of the physical properties seems to be attributed to the headgroup packing order of the vesicles, which is determined by the interference with the hydration shell.     

Structural alterations of phospholipid film domain morphology induced by cholesterol

Structures of the monolayer films of dipalmitoylphosphatidylcholine (DPPC) mixed with different amounts of cholesterol were studied at air-water interface using surface pressure-area measurements, epifluorescence microscopy and atomic force microscopy (AFM). Pure DPPC, cholesterol or DPPC-cholesterol mixtures were dissolved in organic solvents with a small amount of fluorescently labeled phospholipid probe (NBD-PC) and spread onto the air-water interface. Surface pressure-area isotherms and epifluorescence microscopy of such films at the air-water interface suggested that DPPC undergoes a gas to fluid to condensed phase transition, while cholesterol undergoes a gas to solid-like transition. A shift of the surface pressure-area curve to lower area per molecule was observed when cholesterol was mixed with DPPC. Epifluorescence microscopy showed the formation of spiral shaped domains for mixed monolayers. Increase in cholesterol content abolished domain characteristics possibly due to fluidizing property of cholesterol. AFM measurements of monolayers, transferred onto freshly cleaved mica by Langmuir-Blodgett technique, revealed the alterations caused by cholesterol on the gel and fluid domains of such films. AFM measurements re-established similar trend in domain characteristics as evidenced in epifluorescence microscopy.     

Nanoscale membrane activity of surfactins: Influence of geometry, charge and hydrophobicity

We used real-time atomic force microscopy (AFM) to visualize the interactions between supported lipid membranes and well-defined surfactin analogs, with the aim to understand the influence of geometry, charge and hydrophobicity. AFM images of mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers recorded after injection of cyclic surfactin at 1 mM, i.e. well-above the critical micelle concentration, revealed a complete solubilization of the bilayers within 30 min. A linear analog having the same charge and acyl chains was able to solubilize DOPC, but not DPPC, and to promote redeposition leading eventually to a new bilayer. Increasing the charge of the polar head or the length of the acyl chains of the analogs lead to the complete solubilization of both DOPC and DPPC, thus to a stronger membrane activity. Lastly, we found that at low surfactin concentrations (40 µM), DPPC domains were always resistant to solubilization. These data demonstrate the crucial role played by geometry, charge and hydrophobicity in modulating the membrane activity (solubilization, redeposition) of surfactin. Also, this study suggests that synthetic analogs are excellent candidates for developing new surfactants with tunable, well-defined properties for medical and biotechnological applications.     

Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope.

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.     

Imaging soft samples with the atomic force microscope: gelatin in water and propanol.

We have imaged mica coated with thin gelatin films in water, propanol, and mixtures of these two liquids by atomic force microscopy (AFM). The elastic modulus (Young's modulus) can be tuned from 20 kPa to more than 0.1 GPa depending on the ratio of propanol to water. The resolution is best in pure propanol, on the order of 20 nm, and becomes worse for the softer samples. The degradation in resolution can be understood by considering the elastic indentation of the gelatin caused by the AFM tip. This indentation becomes larger and thus the contact area becomes larger the softer the sample is. Therefore this study may be used to estimate the resolution to be expected with an AFM on other soft samples, such as cells. Nondestructive imaging was possible only by imaging at forces < 1 nN. This was difficult to achieve in contact mode because of drift in the zero load deflection of the cantilever, supposedly caused by temperature drift, but straightforward in tapping mode.     

Sickle cell trait human erythrocytes are significantly stiffer than normal

Atomic force microscopy (AFM) allows for high-resolution topography studies of biological cells and measurement of their mechanical properties in physiological conditions. In this work, AFM was employed to measure the stiffness of abnormal human red blood cells from human subjects with the genotype for sickle cell trait. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that Young's modulus of pathological erythrocytes was approximately three times higher than in normal cells. Observed differences indicate the effect of the polymerization of sickle hemoglobin as well as possible changes in the organization of the cell cytoskeleton associated with the sickle cell trait.     

Distribution of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine/cholesterol monolayers: a model for lipid rafts

The distribution of low concentrations of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol monolayers supported on mica has been studied using atomic force microscopy (AFM). The monolayers studied correspond to a pure gel phase and a mixture of liquid-expanded (LE) and liquid-condensed (LC) phases for DPPC and to a single homogeneous liquid-ordered phase for 2:1 DPPC/cholesterol. The addition of 2.5-5% GM1 to phase-separated DPPC monolayers resulted in small round ganglioside-rich microdomains in the center and at the edges of the LC domains. Higher amounts of GM1 (10%) give numerous filaments in the center of the LC domains and larger patches at the edges. A gel phase DPPC monolayer containing GM1 showed large domains containing a network of GM1-rich filaments. The addition of GM1 to a liquid-ordered 2:1 DPPC/cholesterol monolayer gives small, round domains that vary in size from 50 to 150 nm for a range of surface pressures. Larger amounts of GM1 lead to coalescence of the small, round domains to give longer filaments that cover 30-40% of the monolayer surface for 10 mol % GM1. The results indicate that biologically relevant GM1 concentrations lead to submicron-sized domains in a cholesterol-rich liquid-ordered phase that is analogous to that found in detergent-insoluble membrane fractions, and are thought to be important in membrane microdomains or rafts. This demonstrates that AFM studies of model monolayers and bilayers provide a powerful method for the direct detection of microdomains that are too small for study with most other techniques.     

Interactions of surfactin with membrane models.

Surfactin, an acidic cyclic lipopeptide produced by strains of Bacillus subtilis, is a powerful biosurfactant possessing biological activities. Interactions of ionized surfactin (two negative charges) with lecithin vesicles have been monitored by changes in its CD spectra. These changes are more important in the presence of Ca2+ ions. We have studied the penetration of ionized surfactin into lipid monolayers. Using dimyristoyl phospholipids, the surfactin penetration is more important in DMPC than in DMPE monolayers and is greatly reduced in DMPA monolayers because of electrostatic repulsion. The surfactin penetration is lowered when the acyl chain length of the phospholipids increases. The exclusion pressure varies from 40 mN m-1 for DMPC to 30 mN m-1 for DPPC and 18 mN m-1 for egg lecithin. The presence of Ca2+ ions, which neutralize the charges of both surfactin and lipids in the subphase, leads to an important change of the penetration process that is enhanced in the case of acidic, but also of long chain (higher than C14) zwitterionic phospholipids (DPPC and lecithin). From compression isotherms of mixed surfactin/phospholipid monolayers, it appears that surfactin is completely miscible with phospholipids. The present study shows that surfactin penetrates spontaneously into lipid membranes by means of hydrophobic interactions. The insertion in the lipid membrane is accompanied by a conformation change of the peptide cycle.     

Atomic force microscope image contrast mechanisms on supported lipid bilayers

This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures onto a monolayer of DSPE show an abrupt breakthrough event at a repeatable, material-dependent force. The breakthrough force for DSPE and MGDG is sizable, whereas the breakthrough force for DOPE is too small to measure accurately. Contact-mode AFM images on 1:1 mixed monolayers of DSPE/DOPE and MGDG/DOPE have a high topographic contrast at loads between the breakthrough force of each phase, and a low topographic contrast at loads above the breakthrough force of both phases. Frictional contrast is inverted and magnified at loads above the breakthrough force of both phases. These results emphasize the important role that surface forces and mechanics can play in imaging multicomponent biomembranes with AFM.     

Application of atomic force microscopy to the study of micromechanical properties of biological materials

Atomic force microscopy (AFM) has been used to study the micromechanical properties of biological systems. Its unique ability to function both as an imaging device and force sensor with nanometer resolution in both gaseous and liquid environments has meant that AFM has provided unique insights into the mechanical behaviour of tissues, cells and single molecules. As a surface scanning device, AFM can map properties such as adhesion and the Young's modulus of surfaces. As a force sensor and nanoindentor AFM can directly measure properties such as the Young's modulus of surfaces or the binding forces of cells. As a stress-strain gauge AFM can study the stretching of single molecules or fibres and as a nanomanipulator it can dissect biological particles such as viruses or DNA strands. The present paper reviews key research that has demonstrated the versatility of AFM and how it can be exploited to study the micromechanical behaviour of biological materials.     

Change in rigidity in the activated form of the glucose/galactose receptor from Escherichia coli: a phenomenon that will be key to the development of biosensors

Recently a periplasmic glucose/galactose binding protein, GGRQ26C, immobilized on a gold surface has been used as an active part of a glucose biosensor based on quartz microbalance technique. However the nature of the glucose detection was not clear. Here we have found that the receptor protein film immobilized on the gold surface increases its rigidity when glucose is added, which explains the unexpected detection signal. To study the rigidity change, we developed a new fast and simple method based on using atomic force microscopy (AFM) in tapping mode. The method was verified by explicit measurements of the Young's modulus of the protein film by conventional AFM methods. Since there are a host of receptors that undergo structural change when activated by ligand, AFM can play a key role in the development and/or optimization of biosensors based on rigidity changes in biomolecules.     

Study in mono and bilayers of the interaction of hepatitis G virus (GBV-C/HGV) synthetic antigen E2(99-118) with cell membrane phospholipids

The interaction of the hepatitis G synthetic peptide E2(99-118) with cell membrane phospholipids of different characteristics such as dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) was studied by Langmuir isotherms. Epifluorescence microscopy and Atomic force microscopy (AFM) was also used to study interactions with DPPC. Compression isotherms of DPPC/E2(99-118) and DPPG/E2(99-118) mixed monolayers showed negative deviation from ideallity consistent with the existence of attractive interactions. The incorporation of the peptide in DPPC monolayer was also confirmed in epifluorescence microscopy and AFM studies. The peptide retarded the formation of DPPC domains and did not let the phospholipid get organized. No important differences in the interactions with DPPC (neutral) or DPPG (anionic) were found, thus suggesting that electrostatics forces do not have a predominant influence in these interactions.     

Simple display system of mechanical properties of cells and their dispersion

The mechanical properties of cells are unique indicators of their states and functions. Though, it is difficult to recognize the degrees of mechanical properties, due to small size of the cell and broad distribution of the mechanical properties. Here, we developed a simple virtual reality system for presenting the mechanical properties of cells and their dispersion using a haptic device and a PC. This system simulates atomic force microscopy (AFM) nanoindentation experiments for floating cells in virtual environments. An operator can virtually position the AFM spherical probe over a round cell with the haptic handle on the PC monitor and feel the force interaction. The Young's modulus of mesenchymal stem cells and HEK293 cells in the floating state was measured by AFM. The distribution of the Young's modulus of these cells was broad, and the distribution complied with a log-normal pattern. To represent the mechanical properties together with the cell variance, we used log-normal distribution-dependent random number determined by the mode and variance values of the Young's modulus of these cells. The represented Young's modulus was determined for each touching event of the probe surface and the cell object, and the haptic device-generating force was calculated using a Hertz model corresponding to the indentation depth and the fixed Young's modulus value. Using this system, we can feel the mechanical properties and their dispersion in each cell type in real time. This system will help us not only recognize the degrees of mechanical properties of diverse cells but also share them with others.     

Aggregation of gramicidin A in phospholipid Langmuir-Blodgett monolayers

The aggregation of Gramicidin A (gA) in dipalmitoylphosphatidylcoline (DPPC) monolayers is investigated by both thermodynamic and structural methods. Compression isotherm analysis and atomic force microscopy (AFM) observations are performed. Our experimental results indicate that gA aggregation does occur in DPPC monolayers even at very low gA concentration (about 8 x 10(-4) mol%). At the low gA concentration limit, the aggregation process seems to be mainly horizontal (i.e., side-by-side, into the monolayer plane), following a fractal pattern growth producing the formation of typical, flat (0.5 nm height) "doughnut" structures, with a diameter of approximately 150 nm. These structures appear to be composed of smaller subunits (about 70 nm diameter) showing the same doughnut structure. At a molar fraction of approximately 3.8 mol%, the big doughnuts start to disaggregate and only small doughnuts appear. Above a gA concentration of approximately 4.4 mol%, all doughnuts (large and small) disappear, and the morphology assumes the appearance of a patchwork of two distinct phases: one that, being very flat, can be associated with a gA-free or gA-poor DPPC phase, and a second one, characterized by a more corrugated surface, associated with a gA-rich DPPC phase. At gA concentration of approximately 5 mol%, a percolation transition in the gA-rich DPPC phase occurs. Thermodynamic data indicate that the maximum of miscibility between gA and DPPC molecules occurs at approximately 28 mol%, suggesting that gA could aggregate in hexamers that are, on average, bound to 16 DPPC molecules. At the same concentration, AFM images show a network of small gA aggregation units of a size compatible with gA hexamers.     

Lateral organization of GM1 in phase-separated monolayers visualized by scanning force microscopy

Phase separation of glycolipids in lipid mono- and bilayers is of great interest for the understanding of membrane function. The distribution of the ganglioside GM1 in sphingomyelin (SM)/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), SM/1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DOPC) and SM/cholesterol/POPC Langmuir-Blodgett (LB) monolayers transferred at 36 mN/m has been studied by scanning force microscopy. Besides lateral organization of the glycolipid in LB monolayers as deduced from topography, material properties have been investigated by phase imaging, pulsed force mode and force modulation microscopy. It was shown that GM1 preferentially clusters in an ordered lipid matrix, i.e. the SM phase in the case of the SM/POPC and SM/DOPC mixture or in the ordered phase of POPC/SM/cholesterol monolayers. At higher local concentrations, three-dimensional protrusions enriched in GM1 occur, which may represent a precursor for the formation of micelles budding into the aqueous subphase. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-002-0232-4.     

An AFM study on mechanical properties of native and dimethyl suberimidate cross-linked pericardium tissue

Changes in the stiffness of hog pericardium tissue, native and treated with dimethyl suberimidate (DMS), are investigated by atomic force microscopy (AFM). Young's modulus is calculated on the basis of the Hertz-Sneddon model. The cross-linking process increases the stiffness of the tissue. The values of Young's modulus are higher for the DMS stabilized pericardium than for the native one. We also observe that the Young's modulus of native tissue increases when the time between getting the biological material and performing the measurements is longer. This process is probably connected with natural degradation of the biological samples.     

Supramolecular organization of heteroxylan-dehydrogenation polymers (synthetic lignin) nanoparticles

The supramolecular organization of particles composed of heteroxylans (HX) and synthetic lignin (dehydrogenation polymer, DHPs) was studied by light scattering (LS), atomic force microscopy (AFM), and fluorescent probes. Results from static and quasi-elastic light scattering indicate a dense core surrounded by a soft corona. Such organization is also supported by AFM images of the particles that display Gaussian height profiles when a low tapping force is applied, whereas the shape of the profile obtained at a higher mechanical solicitation is irregular and sharp due to deformation of the particles resulting from the tip indentation. This suggests a difference in mechanical behavior between the inner and outer parts of the particles. The formation of local chemical heterogeneities was demonstrated by use of two fluorescent polarity probes (pyrene and methyl-amino-pyrene) to be induced by the core-corona organization.