by Keyword: AFM

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Obiols-Rabasa, M., Oncins, G., Sanz, F., Tadros, T. F., Solans, C., Levecke, B., Booten, K., Esquena, J., (2017). Investigation of the elastic and adhesion properties of adsorbed hydrophobically modified inulin films on latex particles using Atomic Force Microscopy (AFM) Colloids and Surfaces A: Physicochemical and Engineering Aspects 524, 185-192

Graft polymer surfactants provide very good colloidal stability because of strong steric repulsions between adsorbed surfactant films. The elastic and adhesion properties of adsorbed hydrophobically modified inulin polymer surfactant (INUTEC NRA) have been directly measured using Atomic Force Microscopy (AFM) measurements. For this purpose, poly(methyl methacrylate/butyl acrylate), P(MMA/BuA), latexes prepared in the presence of the hydrophobically modified inulin (INUTEC NRA) were used. These latexes (diameter 118 nm and polydispersity index of 1.05) showed a very high colloidal stability in water and in the presence of electrolyte (up to 0.2 mol dm−3 KBr). The latexes were deposited on mica, which was silanated to enhance the adhesion of the latex particles to the surface. A silicon nitride tip with approximately 10 nm diameter that also contained an adsorbed layer of surfactant was used in the AFM apparatus. The tip was allowed to approach, contact thereafter the particles with an applied force of 12.5 nN, and finally detach from the film. Both elastic (Young’s) modulus of the film and adhesion force were studied. The results showed that the adsorbed surfactant films are highly elastic and their elastic modulus and adhesion force did not change significantly with the presence of Na2SO4 up to 0.05 mol dm−3. The high elastic contribution to the steric interaction ensures strong repulsion between the latex particles both in water and at high electrolyte concentrations. In addition, the lack of dependence of adhesion force on electrolyte concentration ensures uniform deposition of the latex particles on a flat substrate as for example in coating applications. These results show the advantages of using a graft polymer surfactant for enhancing the stability of particle suspensions, as illustrated in previous investigations.

Keywords: AFM, Colloidal stability, Interaction forces, Steric repulsion

Gumí-Audenis, Berta, Costa, Luca, Carlá, Francesco, Comin, Fabio, Sanz, Fausto, Giannotti, Marina, (2016). Structure and nanomechanics of model membranes by atomic force microscopy and spectroscopy: Insights into the role of cholesterol and sphingolipids Membranes 6, (4), 58

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information

Keywords: Atomic force microscopy, Force spectroscopy, Lipid membranes, Supported lipid bilayers, Nanomechanics, Cholesterol, Sphingolipids, Membrane structure, XR-AFM combination

Valero, C., Navarro, B., Navajas, D., García-Aznar, J. M., (2016). Finite element simulation for the mechanical characterization of soft biological materials by atomic force microscopy Journal of the Mechanical Behavior of Biomedical Materials 62, 222-235

The characterization of the mechanical properties of soft materials has been traditionally performed through uniaxial tensile tests. Nevertheless, this method cannot be applied to certain extremely soft materials, such as biological tissues or cells that cannot be properly subjected to these tests. Alternative non-destructive tests have been designed in recent years to determine the mechanical properties of soft biological tissues. One of these techniques is based on the use of atomic force microscopy (AFM) to perform nanoindentation tests. In this work, we investigated the mechanical response of soft biological materials to nanoindentation with spherical indenters using finite element simulations. We studied the responses of three different material constitutive laws (elastic, isotropic hyperelastic and anisotropic hyperelastic) under the same process and analyzed the differences thereof. Whereas linear elastic and isotropic hyperelastic materials can be studied using an axisymmetric simplification, anisotropic hyperelastic materials require three-dimensional analyses. Moreover, we established the limiting sample size required to determine the mechanical properties of soft materials while avoiding boundary effects. Finally, we compared the results obtained by simulation with an estimate obtained from Hertz theory. Hertz theory does not distinguish between the different material constitutive laws, and thus, we proposed corrections to improve the quantitative measurement of specific material properties by nanoindentation experiments.

Keywords: AFM, Cell mechanics, FEM, Nanoindentation, Soft-tissue

Van Der Hofstadt, M., Hüttener, M., Juárez, A., Gomila, G., (2015). Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope Ultramicroscopy 154, 29-36

Abstract With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates.

Keywords: Atomic Force Microscope (AFM), Living cell imaging, Bacteria division, Gelatine immobilization, Dynamic jumping mode

Botaya, Luis, Otero, Jorge, González, Laura, Coromina, Xavier, Gomila, Gabriel, Puig-Vidal, Manel, (2015). Quartz tuning fork-based conductive atomic force microscope with glue-free solid metallic tips Sensors and Actuators A: Physical 232, 259-266

Abstract Here, we devise a conductive Atomic Force Microscope (C-AFM) based on quartz tuning forks (QTFs) and metallic tips capable of simultaneously imaging the topography and conductance of a sample with nanoscale spatial resolution. The system is based on a header design which allows the metallic tip to be placed in tight and stable mechanical contact with the QTF without the need to use any glue. This allows electrical measurements to be taken with an electrically excited QTF with the two prongs free. The amplitude oscillation of the QTF is used to control the tip-sample distance and to acquire the topographic images. Meanwhile, the metallic tip is connected to a current–voltage amplifier circuit to measure the tip-sample field emission/tunneling current and to produce the conductive images. This method allows decoupled electrical measurement of the topography and electrical properties of the sample. The results we obtain from calibration samples demonstrate the feasibility of this measurement method and the adequacy of the performance of the system.

Keywords: AFM, Conductive AFM, Quartz tuning fork

Dols-Perez, A., Fumagalli, L., Gomila, G., (2014). Structural and nanomechanical effects of cholesterol in binary and ternary spin-coated single lipid bilayers in dry conditions Colloids and Surfaces B: Biointerfaces 116, 295-302

We investigate the effects of Cholesterol (Chol) in the structural and nanomechanical properties of binary and ternary spin-coated single lipid bilayers made of Dioleoylphosphatidylcholine (DOPC) and Sphingomyelin (SM) in dry conditions. We show that for the DOPC/Chol bilayers, Chol induces an initial increase of the bilayer thickness, followed by decrease for concentrations above 30% Chol. The mechanical properties, instead, appear practically insensitive to the Chol content. For the SM/Chol bilayers we have observed both the thinning of the bilayer and the decrease of the force necessary to break it for Chol content above 40. mol%. In both binary mixtures phase separation is not observed. For ternary single bilayers of DOPC/SM/Chol, Chol induces phase segregation and the formation of domains resembling lipid rafts. The domains show a thickness and mechanical response clearly distinct from the surrounding phase and dependent on the relative Chol content. Based on the results obtained for the binary mixtures, DOPC- and SM-enriched domains can be identified. We highlight that many of the effects of Chol reported here for the dry multicomponent single lipid bilayers resemble closely those observed in hydrated bilayers, thus offering an additional insight into their properties.

Keywords: AFM, Air-stable lipid layer, Force spectroscopy, Lipid raft, Spin-coating

Fumagalli, L., Edwards, Martin Andrew, Gomila, G., (2014). Quantitative electrostatic force microscopy with sharp silicon tips Nanotechnology 25, (49), 495701 (9)

Electrostatic force microscopy (EFM) probes are typically coated in either metal (radius ~ 30 nm) or highly-doped diamond (radius ~ 100 nm). Highly-doped silicon probes, which offer a sharpened and stable tip apex (radius ~ 1–10 nm) and are usually used only in standard atomic force microscopy, have been recently shown to allow enhanced lateral resolution in quantitative EFM and its application for dielectric constant measurement. Here we present the theoretical modelling required to quantitatively interpret the electrostatic force between these sharpened tips and samples. In contrast to a sphere-capped cone geometry used to describe metal/diamond-coated tips, modelling a sharpened silicon tip requires a geometry comprised of a cone with two different angles. Theoretical results are supported by experimental measurements of metallic substrates and ~10 nm radius dielectric nanoparticles. This work is equally applicable to EFM and other electrical scanned probe techniques, where it allows quantifying electrical properties of nanomaterials and 3D nano-objects with higher resolution.

Keywords: AFM, Dielectric constant, EFM, Dielectrics, Nanoparticles, Sharp tips

Birhane, Y., Otero, J., Pérez-Murano, F., Fumagalli, L., Gomila, G., Bausells, J., (2014). Batch fabrication of insulated conductive scanning probe microscopy probes with reduced capacitive coupling Microelectronic Engineering 119, 44-47

We report a novel fabrication process for the batch fabrication of insulated conductive scanning probe microscopy (SPM) probes for electrical and topographic characterization of soft samples in liquid media at the nanoscale. The whole SPM probe structure is insulated with a dielectric material except at the very tip end and at the contact pad area to minimize the leakage current in liquid. Additionally, the geometry of the conducting layer in the probe cantilever and substrate is engineered to reduce the parasitic capacitance coupling with the sample. The electrical characterization of the probes has shown that parasitic capacitances are significantly reduced as compared to fully metallized cantilevers.

Keywords: Conductive scanning probe microscopy (C-SPM), EFM, SECM, SECM-AFM, SIM

Barreto, S., Clausen, C. H., Perrault, C. M., Fletcher, D. A., Lacroix, D., (2013). A multi-structural single cell model of force-induced interactions of cytoskeletal components Biomaterials 34, (26), 6119-6126

Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks invitro. This illustrates that a combination ofcytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics.

Keywords: Actin bundles, Actin cortex, AFM (atomic force microscopy), Cytoskeleton, Finite element modeling, Microtubules

Dols-Perez, A., Sisquella, X., Fumagalli, L., Gomila, G., (2013). Optical visualization of ultrathin mica flakes on semitransparent gold substrates Nanoscale Research Letters 8, (1), 1-5

We show that optical visualization of ultrathin mica flakes on metallic substrates is viable using semitransparent gold as substrates. This enables to easily localize mica flakes and rapidly estimate their thickness directly on gold substrates by conventional optical reflection microscopy. We experimentally demonstrate it by comparing optical images with atomic force microscopy images of mica flakes on semitransparent gold. Present results open the possibility for simple and rapid characterization of thin mica flakes as well as other thin sheets directly on metallic substrates.

Keywords: Atomic force, Conductive AFM, Gold substrates, Metallic substrate, Optical image, Optical reflection, Optical visualization, Ultrathin layers, Atomic force microscopy, Geometrical optics, Gold, Mica, Optical microscopy, Substrates

Arimon, M., Sanz, F., Giralt, E., Carulla, N., (2012). Template-assisted lateral growth of amyloid- Bioconjugate Chemistry 23, (1), 27-32

Hoyo, J., Torrent-Burgués, J., Guaus, E., (2012). Biomimetic monolayer films of monogalactosyldiacylglycerol incorporating ubiquinone Journal of Colloid and Interface Science 384, (1), 189-197

Ubiquinone and plastoquinone are two of the main electron and proton shuttle molecules in biological systems, and monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in the thylakoid membrane of chloroplasts. Saturated MGDG, ubiquinone-10 (UQ) and MGDG:UQ mixed monolayers at the air/water interface have been studied using surface pressure-area isotherms and Brewster Angle Microscopy. Moreover, the transferred Langmuir-Blodgett films have been observed by Atomic Force Microscopy. The results show that MGDG:UQ mixtures present more fluid phase than pure MGDG, indicating a higher order degree for the later. It is also observed an important influence of UQ on the MGDG matrix before UQ collapse pressure and a low influence after this event, due to UQ expulsion from the MGDG matrix. This expulsion leads to a similar remaining UQ content for all the tested mixtures, indicating a limiting content of this molecule in the MGDG matrix at high surface pressures. The thermodynamic studies confirm the stability of the MGDG:UQ mixtures at low surface pressures, although presenting a non-ideal behaviour. Results point to consider UQ as a good candidate for studies of artificial photosynthesis.

Keywords: AFM, BAM, Biomimetic films, Langmuir-Blodgett film, Monogalactosyldiacylglycerol, Ubiquinone

Veeregowda, D. H., van der Mei, H. C., de Vries, J., Rutland, M. W., Valle-Delgado, J. J., Sharma, P. K., Busscher, H. J., (2012). Boundary lubrication by brushed salivary conditioning films and their degree of glycosylation Clinical Oral Investigations 16, (5), 1499-1506

Objectives: Toothbrushing, though aimed at biofilm removal, also affects the lubricative function of adsorbed salivary conditioning films (SCFs). Different modes of brushing (manual, powered, rotary-oscillatory or sonically driven) influence the SCF in different ways. Our objectives were to compare boundary lubrication of SCFs after different modes of brushing and to explain their lubrication on the basis of their roughness, dehydrated layer thickness, and degree of glycosylation. A pilot study was performed to relate in vitro lubrication with mouthfeel in human volunteers. Materials and methods: Coefficient of friction (COF) on 16-h-old SCFs after manual, rotary-oscillatory, and sonically driven brushing was measured using colloidal probe atomic force microscopy (AFM). AFM was also used to assess the roughness of SCFs prior to and after brushing. Dehydrated layer thicknesses and glycosylation of the SCFs were determined using X-ray photoelectron spectroscopy. Mouthfeel after manual and both modes of powered brushing were evaluated employing a split-mouth design. Results: Compared with unbrushed and manually or sonically driven brushed SCFs, powered rotary-oscillatory brushing leads to deglycosylation of the SCF, loss of thickness, and a rougher film. Concurrently, the COF of a powered rotary-oscillatory brushed SCF increased. Volunteers reported a slightly preferred mouthfeel after sonic brushing as compared to powered rotating-oscillating brushing. Conclusion: Deglycosylation and roughness increase the COF on SCFs. Clinical relevance: Powered rotary-oscillatory brushing can deglycosylate a SCF, leading to a rougher film surface as compared with manual and sonic brushing, decreasing the lubricative function of the SCF. This is consistent with clinical mouthfeel evaluation after different modes of brushing.

Keywords: AFM, Friction, Glycosylation, Salivary conditioning film, Toothbrushing, XPS

Redondo, L., Giannotti, M. I., Sanz, F., (2012). Stability of lipid bilayers as model membranes: Atomic force microscopy and spectroscopy approach Atomic force microscopy in liquid (ed. Baró, A. M., Reifenberger, R. G.), Wiley-VCH Verlag GmbH & Co.KGaA (Weinheim, Germany) Part I: General Atomic Force Microscopy, 259-284

Miranda Coelho, Nuno, Gonzalez-Garcia, Cristina, Salmeron-Sanchez, Manuel, Altankov, George, (2011). Arrangement of type IV collagen on NH(2) and COOH functionalized surfaces Biotechnology and Bioengineering 108, (12), 3009-3018

Apart from the paradigm that cell-biomaterials interaction depends on the adsorption of soluble adhesive proteins we anticipate that upon distinct conditions also other, less soluble ECM proteins such as collagens, associate with the biomaterials interface with consequences for cellular response that might be of significant bioengineering interest. Using atomic force microscopy (AFM) we seek to follow the nanoscale behavior of adsorbed type IV collagen (Col IV)-a unique multifunctional matrix protein involved in the organization of basement membranes (BMs) including vascular ones. We have previously shown that substratum wettability significantly affects Col IV adsorption pattern, and in turn alters endothelial cells interaction. Here we introduce two new model surfaces based on self-assembled monolayers (SAMs), a positively charged - NH(2), and negatively charged -COOH surface, to learn more about their particular effect on Col IV behavior. AFM studies revealed distinct pattern of Col IV assembly onto the two SAMs resembling different aspects of network-like structure or aggregates (suggesting altered protein conformation). Moreover, the amount of adsorbed FITC-labeled Col IV was quantified and showed about twice more protein on NH(2) substrata. Human umbilical vein endothelial cells attached less efficiently to Col IV adsorbed on negatively charged COOH surface judged by altered cell spreading, focal adhesions formation, and actin cytoskeleton development. Immunofluorescence studies also revealed better Col IV recognition by both alpha(1) and alpha(2) integrins on positively charged NH(2) substrata resulting in higher phosphorylated focal adhesion kinase recruitment in the focal adhesion complexes. On COOH surface, no integrin clustering was observed. Taken altogether these results, point to the possibility that combined NH(2) and Col IV functionalization may support endothelization of cardiovascular implants.

Keywords: Collagen type IV, SAMs, AFM, Surface-induced protein assembly, Endothelial cells, Vascular grafts

Roa, J. J., Oncins, G., Diaz, J., Sanz, F., Segarra, M., (2011). Calculation of young's modulus value by means of AFM Recent Patents on Nanotechnology 5, (1), 27-36

In the last years, Atomic Force Microscopy (AFM) has become a powerful tool not only to study the surface morphology but also the nanomechanics of all kind of samples. In this paper, the applicability of this technique is reviewed and its basic aspects of operation, advantages and drawbacks of using the AFM probe as a picoindenter (Force Spectroscopy mode, FS-AFM) are discussed. The patents concerning picoindentation measurements are discussed in the text and special attention is paid to measurements performed on hard materials as ceramics, as they have not been as thoroughly reviewed in the literature as in the case of soft matter. The possibilities of AFM in the nanomechanics field include the quantitative determination of the Young's modulus (E) and the transition force from elastic to plastic deformation regimes, the measurement of adhesion forces and deformation mechanisms while applying vertical forces in the range from tens of pN to mu N.

Keywords: Hard materials, Young's modulus, AFM-FS, Picoindentation technique

Toromanov, Georgi, González-García, Cristina, Altankov, George, Salmerón-Sánchez, Manuel, (2010). Vitronectin activity on polymer substrates with controlled -OH density Polymer 51, (11), 2329-2336

Vitronectin (VN) adsorption on a family of model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios (to obtain a controlled surface density of -OH groups) was investigated by Atomic Force Microscopy (AFM). It is shown that the fraction of the substrate covered by the protein depends strongly on the amount of hydroxyl groups in the sample and it monotonically decreases as the -OH density increases. Isolated globular-like VN molecules are observed on the surfaces with the higher OH density. As the fraction of hydroxyl groups decreases, aggregates of 3-5 VN molecules are observed on the sample. Overall cell morphology, focal adhesion formation and actin cytoskeleton development are investigated to assess the biological activity of the adsorbed VN on the different surfaces. Dermal fibroblast cells show excellent material interaction on the more hydrophobic samples (OH contents lower than 0.5), which reveals enhanced VN activity on this family of substrates as compared with other extracellular matrix proteins (e.g., fibronectin and fibrinogen).

Keywords: Copolymers, Vitronectin, AFM, Self-assembled monolayers, Cell-adhesion, Thermal transitions, Protein adsorption, Surfaces, Fibronectin, Biomaterials, Attachment, Fibrinogen

Gugutkov, D., Altankov, G., Hernandez, J. C. R., Pradas, M. M., Sanchez, M. S., (2010). Fibronectin activity on substrates with controlled -OH density Journal of Biomedical Materials Research - Part A 92A, (1), 322-331

Adhesion of human fibroblast to a family of fibronectin (FN) coated model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios to obtain a controlled surface density of -OH groups was investigated. Cell adhesion and spreading surprisingly decreased as the fraction of -OH groups on the Surface increased. AFM studies of FN conformation revealed formation of a protein network on the more hydrophobic surfaces. The density of this network diminished as the fraction of -OH groups in the sample increased, up to a maximal -OH concentration at which, instead of the network, only IN aggregates were observed. The kinetics of network development was followed at different adsorption times. Immunofluorescence for vinculin revealed the formation of well-developed focal adhesion complexes on the more hydrophobic surface (similar to the control glass), which became less defined as the fraction of -OH groups increased. Thus, the efficiency of cell adhesion is enhanced by the formation of FN networks on the substrate, directly revealing the importance of the adsorbed protein conformation for cell adhesion. However, cell-dependent reorganization of substrate-associated FN, which usually takes place on more hydrophilic substrates (as do at the control glass slides), was not observed in this system, suggesting the increased strength of protein-to-substrate interaction. Instead, the late FN matrix formation-after 3 days of culture-was again better pronounced on the more hydrophobic substrates and decreased as the fraction of -OH groups increase, which is in a good agreement with the results for overall cell morphology and focal adhesion formation.

Keywords: Cell adhesion, Fibronectin, Fibroblast, Extracellular matrix, AFM

Gugutkov, Dencho, Gonzalez-Garcia, Cristina, Rodriguez Hernandez, Jose Carlos, Altankov, George, Salmeron-Sanchez, Manuel, (2009). Biological activity of the substrate-induced fibronectin network: insight into the third dimension through electrospun fibers Langmuir 25, (18), 10893-10900

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network. Atomic force microscopy images of single FN molecules, at the early stages of adsorption on plane PEA, allow one to rationalize the process. Further, the role of the spatial organization of the FN network on the cellular response is investigated through its adsorption on electrospun fibers. Randomly oriented and aligned PEA fibers were prepared to mimic the three-dimensional organization of the extracellular matrix. The formation of the FN network on the PEA fibers but not on the supporting coverglass was confirmed. Fibroblasts aligned with oriented fibers, displayed extended morphology, developed linearly organized focal adhesion complexes, and matured actin filaments. Conversely, on random PEA fibers, cells acquired polygonal morphology with altered actin cytoskeleton but well-developed focal adhesions. Late FN matrix formation was also influenced: spatially organized FN matrix fibrils along the oriented PEA fibers and an altered arrangement on random ones.

Keywords: AFM, Cell-adhesion, Dependent conformations, Hydrophobic surfaces, Extracellular-matrix, Bound fibronectin, Polymer surfaces, Integrin binding, Biocompatibility, Adsorption

Nussio, M. R., Oncins, G., Ridelis, I., Szili, E., Shapter, J. G., Sanz, F., Voelcker, N. H., (2009). Nanomechanical characterization of phospholipid bilayer islands on flat and porous substrates: A force spectroscopy study Journal of Physical Chemistry B 113, (30), 10339-10347

In this study, we compare for the first time the nanomechanical properties of lipid bilayer islands on flat and porous surfaces. 1,2-Dimyzistoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers were deposited on flat (silicon and mica) and porous silicon (pSi) substrate surfaces and examined using atomic force spectroscopy and force volume imaging. Force spectroscopy measurements revealed the effects of the underlying substrate and of the lipid phase on the nanomechanical properties of bilayers islands. For mica and silicon, significant differences in breakthrough force between the center and the edges of bilayer islands were observed for both phospolipids. These differences were more pronounced for DMPC than for DPPC, presumably due to melting effects at the edges of DMPC bilayers. In contrast, bilayer islands deposited on pSi yielded similar breakthrough forces in the central region and along the perimeter of the islands, and those values in turn were similar to those measured along the perimeter of bilayer islands deposited on the flat substrates. The study also demonstrates that pSi is suitable solid support for the formation of pore-spanning phospholipid bilayers with potential applications in transmembrane protein studies, drug delivery, and biosensing.

Keywords: Black lipid-membranes, Gold surfaces, Supported bilayers, Channel activity, Micro-BLMS, Silicon, Proteins, Vesicles, AFM, Temperature measurement

Otero, J., Puig-Vidal, M., Frigola, M., Casals, A., (2009). Micro-to-nano optical resolution in a multirobot nanobiocharacterization station 2009 IEEE/RSJ International Conference on Intelligent Robots and Systems, IROS 2009 IEEE RSJ International Conference on Intelligent Robots and Systems , IEEE (St. Louis, USA) , 5357-5362

A multi-robot cooperation station for nano-bio characterization of biological specimens is presented. The station is composed of two long travel range and high resolution robots equipped with self-sensing nanoprobes that are able to cooperate with each other and with standard AFM systems, over a common sample. The robots are guided by the use of an upright high-depth-of-field optical microscope to perform complex nano-bio characterization experiments. To achieve the required precision between the two robots reference frames, specific image processing techniques are needed. One of the tips is dedicated to acquire the topography of the sample at nano scale while the second probe performs the biocharacterization experiments. The obtained results show that the two robots can cooperate within the required resolution in bacterial nanomechanical characterization while high resolution topographic images are acquired.

Keywords: AFM

Zazoua, A., Kherrat, R., Caballero, D., Errachid, A., Jaffrezic-Renault, N., Bessueille, F., Leonard, D., (2009). Characterisation of a Cr(VI) sensitive polysiloxane membrane by x-ray photoelectron spectrometry and atomic force microscopy Sensor Letters 6th Maghreb-Europe Meeting on Materials and Their Applications for Devices and Physical, Chemical and Biological Sensors , AMER SCIENTIFIC PUBLISHERS (Rabat, Morocco) 7, (5), 995-1000

Cr(VI) sensitive polysiloxane membranes containing tributylphosphate (TBP) or trioctylphosphine oxide (TOPO) were characterized in this study. TBP and TOPO as carriers, have a high selectivity for Cr(VI). The Potentiometric response of EMIS (Electrolyte/Membrane/Insulator/Semiconductor) sensors presents a quasi-nernstian response for Cr2O2-7 exchange. The ion exchange is shown by X-ray photoelectron spectrometry (XPS), the binding energy of the Cr 2p1/2 peak corresponding to Cr(VI) and the atomic composition after exposure to Cr(VI) shows a factor 1.7 higher for silopreneTBP membrane. The conformational topography of both polymeric membranes was characterized by Atomic Force Microscopy (AFM), the exchange of Cr(VI) leading to a heterogeneous topographic state. Adhesion force measurements are also performed to study the properties of adhesion of both selective membranes with a non-functionalized Si AFM tip and with an OTS functionalized one to study the interactions between the tip and the membrane, in liquid before and after the exposure of the membrane to ion chromium. The presence of the ionophores does not practically change the adhesion force compared to pure polysiloxane, showing a good solubility of the ionophore and the orientation of the alkyl chains towards the polysiloxane surface. After the exchange with Cr(VI), the adhesion force decreases drastically due to the hydrophilic character of the surface, complex of Cr(VI) with the P-O groups of both ionophore being oriented towards the surface.

Keywords: AFM, Electrolyte/membrane/insulator/semiconductor structures, Polysiloxane membrane, Xps

Sporer, C., Casal, L., Caballero, D., Samitier, J., Errachid, A., Perez-Garcia, L., (2009). Novel anionophores for biosensor applications: nano characterisation of SAMS based on amphiphilic imidazolium protophanes and cyclophanes on gold surfaces Sensor Letters 6th Maghreb-Europe Meeting on Materials and Their Applications for Devices and Physical, Chemical and Biological Sensors , AMER SCIENTIFIC PUBLISHERS (Rabat, Morocco) 7, (5), 757-764

Here we report on the results of surface deposition of the novel amphiphilic imidazolium heterocyclophanes and protophanes 1, 2, 3 onto gold electrodes by soft lithography and wet chemistry techniques. Depending on the specific functionalization conditions chosen, the surface properties and the pattern composition can vary widely. The formation of aggregates of monolayers or oligolayer structures and of rings with nano dimensioned wall widths has been investigated with Atomic Force Microscopy (AFM), Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) and Contact angle measurements.

Keywords: Afm, Imidazolium anionophores, Microcontact printing, Tof-sims

Bravo, R., Arimon, M., Valle-Delgado, J. J., Garcia, R., Durany, N., Castel, S., Cruz, M., Ventura, S., Fernàndez-Busquets, X., (2008). Sulfated polysaccharides promote the assembly of amyloid beta(1-42) peptide into stable fibrils of reduced cytotoxicity Journal of Biological Chemistry 283, (47), 32471-32483

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (A beta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, A beta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for A beta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different A beta(1-42) species on Ca2+ homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca2+ that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca2+ increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

Keywords: AFM, Alzheimers-disease, Chondroitin sulfate, Heparan-sulfate, Lipid-bilayers, Beta-peptide, In-vitro, Neurodegenerative diseases, Extracellular-matrix, Prion protein

Oncins, G., Torrent-Burgues, J., Sanz, F., (2008). Nanomechanical properties of arachidic acid Langmuir-Blodgett films Journal of Physical Chemistry C 112, (6), 1967-1974

The nanomechanical properties of Langmuir-Blodgett monolayers of arachidic acid extracted at surface pressures of 1, 15, and 35 mN/m and deposited on mica were investigated by atomic force microscopy, force spectroscopy, and lateral force microscopy. It was experimentally demonstrated that the arachidic acid molecular orientation depends on the extraction pressure. According to this, tilting angles of 50, 34, and 22 degrees with respect to the surface perpendicular were detected and identified as conformations that maximize van der Waals interactions between the arachidic acid alkyl chains. The vertical force needed to puncture the monolayers with the AFM tip strongly depends on the molecular tilting angles attained at different monolayer extraction surface pressures, obtaining values that range from 13.07 +/- 3.24 nN for 50 degrees to 22.94 +/- 5.49 nN for 22 degrees tilting angles. The different molecular interactions involved in the monolayer cohesion are discussed and quantitatively related to the experimental monolayer breakthrough forces. The friction measurements performed from low vertical forces up to monolayer disruption reveal the existence of three well-defined regimes: first, a low friction response due to the elastic deformation of the monolayer, which is followed by a sharp increase in the friction force due to the onset of a sudden plastic deformation. The last regime corresponds to the monolayer rupture and the contact between tip and substrate. The friction coefficient of the substrate is seen to depend on the monolayer extraction pressure, a fact that is discussed in terms of the relationship between the sample compactness and its rupture mechanism.

Keywords: AFM, SAM, Reflection-absortion spectroscopy, Lipid-bilayers, Frictional-properies, Molecular-structure, Thermal behavior, Nanometer-scale, Chain-length, LB films

Oncins, Gerard, Vericat, Carolina, Sanz, Fausto, (2008). Mechanical properties of alkanethiol monolayers studied by force spectroscopy Journal of Chemical Physics 128, (4), 044701

The mechanical properties of alkanethiol monolayers on Au(111) in KOH solution have been studied by force spectroscopy. The analysis of the vertical force versus penetration curves showed that monolayer penetration is a stepped process that combines elastic regions with sudden penetration events. The structural meaning of these events can be explained both by the creation of gauche defects on the hydrocarbon chains and by a cooperative molecular tilting model proposed by Barrena et al. [J. Chem. Phys. 113, 2413 (2000)]. The validity of these models for alkanethiol monolayers of different compactness and chain length has been discussed. The Young's modulus (E) of the monolayers has been calculated by using a recently developed model which considers the thickness of the monolayer as a parameter, thus allowing a decoupling of the mechanical properties of the thiol layer from those of the Au(111) substrate. As a result, the calculated E values are in the range of 50-150 Pa, which are remarkably lower than those previously reported in the literature.

Keywords: Adsorbed layers, AFM, Gold, Monolayers, Organic compounds, Self-assemblyYoung's modulus

Gomila, G., Toset, J., Fumagalli, L., (2008). Nanoscale capacitance microscopy of thin dielectric films Journal of Applied Physics 104, (2), 8

We present an analytical model to interpret nanoscale capacitance microscopy measurements on thin dielectric films. The model displays a logarithmic dependence on the tip-sample distance and on the film thickness-dielectric constant ratio and shows an excellent agreement with finite-element numerical simulations and experimental results on a broad range of values. Based on these results, we discuss the capabilities of nanoscale capacitance microscopy for the quantitative extraction of the dielectric constant and the thickness of thin dielectric films at the nanoscale.

Keywords: AFM, Thickness, Tip

Torrent-Burgues, J., Oncins, G., Sanz, F., (2008). Study of mixed Langmuir and Langmuir-Blodgett films of dissimilar components by AFM and force spectroscopy Colloids and Surfaces a-Physicochemical and Engineering Aspects 12th International Conference on Organized Molecular Films , Elsevier Science (Krakow, Poland) 321, (1-3), 70-75

In this study the structure of mixed Langmuir-Blodgett (LB) monolayers has been investigated using atomic force microscopy, lateral force microscopy and force spectroscopy, as well as the characteristics of the Langmuir monolayers by surface pressure-area isotherms and Brewster angle microscopy. Mixed films were of dissimilar compounds, a fatty acid such as arachidic acid and a macrocyclic compound. The mixture forms separated phases, but some degree of partial miscibility occurs, with domains at the micro-scale that have different nanomechanical and nanotribological properties. LB films transferred at the same surface pressure show different characteristics depending on the composition. The higher domains correspond to arachidic acid and some of these domains show the presence of two phases, which have been identified as phases with discrete molecular tilting angles.

Keywords: Mixed monolayers, Pressure-area isotherm, Langmuir-Blodgett, AFM, Force spectroscopy

Casuso, I., Pla, M., Gomila, G., Samitier, J., Minic, J., Persuy, M. A., Salesse, R., Pajot-Augy, E., (2008). Immobilization of olfactory receptors onto gold electrodes for electrical biosensor Materials Science & Engineering C 5th Maghreb-Europe Meeting on Materials and their Applicatons for Devices and Physical, Chemical and Biological Sensors , Elsevier Science (Mahdia, TUNISIA) 28, (5-6), 686-691

We investigate the immobilization of native nanovesicles containing functional olfactory receptors onto gold electrodes by means of atomic force microscopy in liquid. We show that nanovesicles can be adsorbed without disrupting them presenting sizes once immobilized ranging from 50 run to 200 nm in diameter. The size of the nanovesicles shows no dependence on the electrode hydrophobicity being constant in a height/width ratio close to 1:3. Nevertheless, electrode hydrophobicity does affect the surface coverage, the surface coverage is five times higher in hydrophilic electrodes than on hydrophobic ones. Surface coverage is also affected by nanovesicles dimensions in suspension, the size homogenization to around 50 nm yields a further five fold increment in surface coverage achieving a coverage of about 50% close to the hard spheres jamming limit (54.7%). A single layer of nanovesicles is always formed with no particle overlap. Present results provide insights into the immobilization on electrodes of olfactory receptors for further olfactory electrical biosensor development.

Keywords: AFM, Adsorption, Odorant, Taste

Rico, Félix, Roca-Cusachs, Pere, Sunyer, Raimon, Farré, Ramon, Navajas, Daniel, (2007). Cell dynamic adhesion and elastic properties probed with cylindrical atomic force microscopy cantilever tips Journal of Molecular Recognition John Wiley & Sons, Ltd. 20, (6), 459-466

Cell adhesion is required for essential biological functions such as migration, tissue formation and wound healing, and it is mediated by individual molecules that bind specifically to ligands on other cells or on the extracellular matrix. Atomic force microscopy (AFM) has been successfully used to measure cell adhesion at both single molecule and whole cell levels. However, the measurement of inherent cell adhesion properties requires a constant cell-probe contact area during indentation, a requirement which is not fulfilled in common pyramidal or spherical AFM tips. We developed a procedure using focused ion beam (FIB) technology by which we modified silicon pyramidal AFM cantilever tips to obtain flat-ended cylindrical tips with a constant and known area of contact. The tips were validated on elastic gels and living cells. Cylindrical tips showed a fairly linear force-indentation behaviour on both gels and cells for indentations > 200nm. Cylindrical tips coated with ligands were used to quantify inherent dynamic cell adhesion and elastic properties. Force, work of adhesion and elasticity showed a marked dynamic response. In contrast, the deformation applied to the cells before rupture was fairly constant within the probed dynamic range. Taken together, these results suggest that the dynamic adhesion strength is counterbalanced by the dynamic elastic response to keep a constant cell deformation regardless of the applied pulling rate.

Keywords: AFM, Cell adhesion, Cell mechanics, Cell stiffness

Domènech, Ò., Morros, A., Cabañas, M. E., Teresa Montero, M., Hernéndez-Borrell, J., (2007). Supported planar bilayers from hexagonal phases Biochimica et Biophysica Acta - Biomembranes 1768, (1), 100-106

In this work the presence of inverted hexagonal phases HII of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca2+ were observed via 31P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, HII phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being ∼ 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to HII phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (∼ 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from HII phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.

Keywords: 31P-NMR, AFM, ANS fluorescence, Cytochrome c (cyt c), Hexagonal phase (HII), Liposome, Supported planar bilayers (SPBs)