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Results for “Atomic force microscopy (AFM)”

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PubMed2001

Adsorption and Bioactivity of Protein A on Silicon Surfaces Studied by AFM and XPS.

The adsorption of protein A on silicon surfaces was studied by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy. The deposition was made statically from various concentrations of protein A in water solution. The biological activity was checked by the immobilization of rabbit immunoglobulin G. The protein adsorption occurs in least two different phases and leads to a multilayer film. The first monolayer of proteins is rapidly adsorbed on the surface. The adsorption of the second layer of proteins occurs much more slowly (a thousand times slower) and also involves the third monolayer. The protein A of the first monolayer is denaturated and biologically inactive. On the contrary, the proteins of the second monolayer keep their natural diameter and remain biologically active. AFM artifacts such as the convolution with small objects and the resulting estimation of the coverage ratio are discussed. Copyright 2001 Academic Press.

Journal Article↗

PubMed1999

Direct measurement of DNA by means of AFM.

We have demonstrated that the electrostatic stretch-and-positioning method is useful for the analysis of a long DNA molecule by means of atomic force microscopy (AFM). DNA molecules were stretched parallel to the field line, and immobilized onto the aluminum electrodes patterned on a glass plate. Through AFM observation, we confirmed the immobilization of individual DNA molecules, not aggregate.

Bacteriophage lambda↗

PubMed2001

Investigation by atomic force microscopy of forces at the origin of cement cohesion.

In cement paste, the cohesion results of the interactions between calcium silicate hydrate (CSH) surfaces in an interstitial ionic solution. (N, V, T) Monte Carlo simulations show that the interactions are due to the ion correlation forces influenced by the surface charge density, the ionic concentration and the ion valence. This paper deals with the direct measurement in solutions by atomic force microscopy (AFM) of the forces and the interaction ranges between a probe and an atomically smooth substrate covered by CSH nanoparticles. Different electrolytic solutions (Ca(OH)2, CaCl2, NaCl, NaOH) have been used in order to determine influent parameters permitting to identify the nature of acting forces. Investigations have been rendered possible by selecting appropriate experimental setup and solutions. The selected probe and substrate on which CSH nanoparticles have previously grown are neutral regarding the reactivity during experiments permitting the exchange of solutions. Results show that a force originates from electrostatic nature and differs from Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Agreement is found between experiments and (N,V,T) Monte Carlo simulations of ionic correlation forces. These forces are at the origin of the cohesion of cement paste.

Journal Article↗

PubMed2006

Direct and real-time visualization of the disassembly of a single RecA-DNA-ATPgammaS complex using AFM imaging in fluid.

RecA disassembly from circular double-stranded DNA (dsDNA) was studied by atomic force microscopy (AFM) imaging in fluid on a single molecule scale. The RecA/DNA complex was formed in the presence of ATPgammaS, and the disassembly was then initiated by buffer exchange to rinse off ATPgammaS. Performing AFM imaging in fluid allowed direct and real-time visualization of the disassembly of RecA from dsDNA in solution. It was found that RecA disassembly commenced from multiple sites both in deionized water and in buffer; the areas where RecA dissociated showed the appearance of "gaps" in the filamentous structure. RecA further disassembled either through the already existing "gaps" or by generation of new gaps. The disassembly was slower in buffer than in deionized water, suggesting that ions also contribute to the stabilization of the complex. RecA hexamers and monomers were observed in deionized water and in buffer, respectively, during the disassembly process.

Adenosine Triphosphate↗

PubMed2005

Study on the interaction in single antigen-antibody molecules by AFM.

The interaction between single synthesized antigen and its monoclonal antibody was directly monitored by Atomic Force Microscopy (AFM) with antigen-labeled AFM tip. The details about tip modification and antibody immobilization were described. The interactions were monitored by the change of adhesive force in separating the bound antigen-antibody. The results showed that two processes could be observed in the combination of antigen-antibody system. Possible mechanisms of rupture process as independent unbinding and cooperative unbinding were discussed. At slow separation process, the antigen-antibody linkage appeared to rupture sequentially. Increasing the separation rate from 50 nm/s to 2000 nm/s led to an increase in the possibility of cooperative unbinding. The pH influenced the affinity constants greatly, but had little effect on the magnitude of unbinding forces. The obtained results were compared with affinity constants between complete antigen and antibody obtained by immunoassay method (ELISA). The observed single rupture force between individual antibody and antigen molecules provided pertinent information relating to the manner how the antibody molecule binds to its specific antigen.

Antibodies, Monoclonal↗

PubMed2005

The extracellular matrix of the human aortic wall: ultrastructural observations by FEG-SEM and by tapping-mode AFM.

Fragments of human ascending aorta harvested during heart surgery were cryofractured and observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Elastic fibers appear as irregular, undulated laminae of variable size and shape. Their surface shows an evident fibrous texture suggestive of a criss-crossed, delicate filamentous scaffold and is marked by a number of features such as ridges, holes and protruding ribs. At higher magnification, both SEM and AFM show the surface composed of a finely granular material, with a bead size of approximately 20 nm. However, the thickness of the metal coating in one case, and the tip convolution effect on the other, may equally result in an artifactual enlargement of the structures, so that the beads may be significantly smaller. The surfaces created by the fracture always appear smooth and compact and with this technique do not reveal significant detail. The collagen component is mostly represented by small, uniform fibrils gathered in flexuous bundles and following a wavy course not unlike that of the elastic laminae. An orthogonal lattice of small proteoglycans is readily evident even without a specific treatment. Occasionally, the fibrils appear encrusted or engulfed in a grainy matrix reminiscent of the elastic fiber surface. Fluid Tapping-Mode Atomic Force Microscopy simultaneously reveals the surface-bound proteoglycans and the inner architecture of the fibrils, composed of smaller subunits following a spiral course with a winding angle of approximately 17 degrees.

Aged↗

PubMed2004

Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM.

The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tapping-mode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.

Erythrocyte Membrane↗

PubMed2006

Structure and identity of 4,4'-thiobisbenzenethiol self-assembled monolayers.

Self-assembled monolayers (SAMs) of 4,4'-thiobisbenzenethiol (TBBT) can be formed on Au surface spontaneously. The structural characteristics and adsorption behavior of TBBT SAMs on Au have been investigated by surface enhanced Raman scattering (SERS), electrochemical cyclic voltammetry (CV), ac impedance spectroscopy (EIS), and atomic force microscopy (AFM). It is demonstrated that TBBT adsorbed on Au by losing a H atom, forming one Au-S bond, and the other mercapto group is free at the surface of the monolayer owing to the presence of the nu(S-H) at 2513 cm(-1) and the delta(C-S-H) at 910 cm(-1) in SERS. The enhancement of the vibration of C-S (1064 cm(-1)), the aromatic C-H vibration (3044 cm(-1)), and the absence of the vibration of S-S illustrate TBBT adsorbed on Au forming a monolayer with one benzene ring tilted with respect to the Au surface. The interpretation of the observed frequencies is aided by ab initio molecular orbital (MO) calculations at the HF/6-31G level of theory. Electrochemical CV and EIS indicate TBBT monolayers can passivate the Au effectively for its low ratio of pinhole defects (theta = 99.6%). AFM studies give details about the surface morphology. The applications of TBBT SAMs have been extensively investigated by exposure of Cu2+ ion to TBBT SAMs on Au and covalent adsorption of metal nanoparticles. Electrochemical, X-ray photoelectron spectroscopic, and SERS results indicate that Cu2+ can react with TBBT SAMs and present on TBBT SAMs as Cu(I). A scanning electron microscopic image of Ag nanoparticles on TBBT/Au and the Raman spectrum of TBBT in smooth macroscopic Au/TBBT SAMs/Ag nanoparticle sandwich structure indicate that metal nanoparticles can be adsorbed on TBBT SAMs effectively through covalent linkage.

Journal Article↗

PubMed2004

Direct visualization of straw cell walls by AFM.

The structural relationship of cellulose, hemicellulose, and lignin in plant cell walls is still a mystery needing to be explored. By using atomic force microscopy (AFM) the surface of straw at different layers was directly observed, and the structural characteristics were analyzed by topographic analysis and FT-IR spectra. It was found that a compact layer of wax covered the outside of the straw, which protects the straw from insects and microorganisms. At the boundary of the primary and second wall there appears a network structure of cellulose and hemicellulose, with some lignin localised on the surface of the network. It is consistent with the model of a cell wall suggested by Vincent. Inside the second cell wall, there is a layer mainly composed of a cellulose crystalline region. High-resolution AFM observation reveals that the crystalline structure consists of both triclinic and monoclinic unit cells. An AFM phase image showing the structural relation between cellulose microfibrils, hemicellulose, and lignin in the straw cell wall.

Cell Wall↗

PubMed1999

Graphical method for force analysis: macromolecular mechanics with atomic force microscopy.

We present a graphical method for a unifying, quantitative analysis of molecular bonding-force measurements by atomic force microscopy (AFM). The method is applied to interpreting a range of phenomena commonly observed in the experimental AFM measurements of noncovalent, weak bonds between biological macromolecules. The analysis suggests an energy landscape underlying the intermolecular force and demonstrates that many observations, such as "snaps-on," "jumps-off," and hysteresis loops, are different manifestations of a double-well energy landscape. The analysis gives concrete definitions for the operationally defined "attractive" and "adhesive" forces in terms of molecular parameters. It is shown that these operationally defined quantities are usually functions of the experimental setup, such as the stiffness of the force probe and the rate of its movement. The analysis reveals a mechanical instability due to the multistate nature of molecular interactions and provides new insight into macromolecular viscosity. The graphical method can equally be applied to a quantitative analysis of multiple unfolding of subunits of the giant muscle protein titin under AFM.

Connectin↗

PubMed2004

Correlation of AFM and SFA measurements concerning the stability of supported lipid bilayers.

Phospholipid bilayers were studied by means of atomic force microscopy (AFM) and a surface force apparatus (SFA). The stability of the supported bilayers was described by the amount of irregularities in the topography of the membrane by means of AFM and by the occurrence of hemifusion in the SFA, which is an indicator of defective bilayers. The bilayers, composed of lipids having the same headgroup but different chain lengths in the two leaflets, were prepared by Langmuir-Blodgett deposition and transferred at different surface pressures. The topography of the supported bilayers in aqueous solution, as imaged by AFM, revealed an increasing number of defects in the supported lipid membranes with decreased deposition pressure of the outer lipid layer. These defects, which appeared in the form of monolayer and bilayer (self-assembled) thick holes within the membrane, were energetically favorable over an evenly depleted bilayer. We found that the quantity of these defects (holes of </=0.5 micro m diameter and covering up to 30% of the surface area) correlated well with the stability of the bilayers as measured by SFA, a truly complementary instrument.

Elasticity↗

PubMed2000

AFM characterization of single strand-specific endonuclease activity on linear DNA.

The specificity of nucleases for nicked and un-nicked double-stranded DNA has been characterized using atomic force microscopy (AFM). We have found that AFM has advantages over the usual macroscopic analyses, such as sucrose gradient centrifugation or electrophoresis, in characterizing nuclease digestion. In particular, short DNA fragments resulting from non-specific digestion were detected and, thus, the true length distribution of digested DNA was revealed. A simple numerical method is proposed to estimate the number of nicked sites per DNA molecule based on AFM images.

DNA↗

PubMed2004

Alternative method for fabricating chemically functionalized AFM tips: silane modification of HF-treated Si3N4 probes.

An alternative method for fabricating functionalized, atomic force microscopy (AFM) tips is presented. This technique is simple and requires only minimal preparation and tip modification to generate chemically sensitive probes that have a robust organic monolayer of flexible terminal chemistry attached to the surface. Specifically, commercially microfabricated Si3N4 AFM tips were modified with self-assembled monolayers (SAMs) of octadecyltrichlorosilane and (11-bromoundecyl)trichlorosilane after removing the native silicon oxide surface layer with concentrated hydrofluoric acid. The structure of these SAM films on solid silicon nitride surfaces was studied using contact angle goniometry and Fourier transform infrared spectroscopy. Pull-off force measurements on various bare (mica, graphite, and silicon) and SAM-functionalized substrates confirm that mechanically robust, long-chain organic silane SAMs can be formed on HF-treated Si3N4 tips without the presence of an intervening oxide layer. Adhesion experiments show that the integrity of the organic film on the chemically modified tips is maintained over repeated measurements and that the functionalized tips can be used for chemical sensing experiments since strong discrimination between different surface chemistries is possible.

Journal Article↗

PubMed2006

Ligands on the string: single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells.

Atomic force microscopy (AFM) was used to probe topology, conformational changes and initial substrate-carrier interactions of Na+-glucose co-transporter (SGLT1) in living cells on a single-molecule level. By scanning SGLT1-transfected Chinese hamster ovary (CHO) cells with AFM tips carrying an epitope-specific antibody directed against the extramembranous C-terminal loop 13, significant recognition events could be detected. Specificity was confirmed by the absence of events in nontransfected CHO cells and by the use of free antigen and free antibody superfusion. Thus, contrary to computer predictions on SGLT1 topology, loop 13 seems to be part of the extracellular surface of the transporter. Binding probability of the antibody decreased upon addition of phlorizin, a specific inhibitor of SGLT1, suggesting a considerable conformational change of loop 13 when the inhibitor occludes the sugar translocation pathway. Using an AFM tip carrying 1-thio-D-glucose, direct evidence could be obtained that in the presence of Na+ a sugar-binding site appears on the transporter surface. The binding site accepts the sugar residue of the glucoside phlorizin, free D-glucose, and D-galactose, but not free L-glucose and probably represents the first of several selectivity filters of the transporter. This work demonstrates the potential of AFM to study the presence and dynamics of plasma membrane transporters in intact cells on the single molecule level.

Animals↗

PubMed2006

High resolution non-contact AFM imaging of liquids condensed onto chemically nanopatterned surfaces.

The wetting of ethanol and octane on chemically nanopatterned surfaces has been investigated using Atomic Force Microscopy (AFM) under controlled environmental conditions. The patterns were generated on a methyl-terminated, organic monolayer using an AFM electro-oxidation process. The subsequent wetting of the organic liquids was studied using non-contact mode AFM under equilibrium conditions with the vapor. This study of condensed nanoliquids provides the first reliable measurements of sub 100 nm liquid profile shapes. The derived contact angles give an estimate of the line tension.

Ethanol↗

PubMed2000

Atomic force microscopy-based detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices.

Type II tropocollagen molecules were reacted with matrix metalloproteinase 8 (MMP-8) and the binding sites as well as the cleavage site of MMP-8 were detected on individual molecules using atomic force microscopy (AFM). Approximately 300-nm-long coiled-coil tropocollagen molecules were straightened and immobilized on an atomically flat surface for detection by AFM. The direct visualization of individual collagen molecules revealed heterogeneous characteristics of MMP-8:collagen complexes. We observed that there existed multiple MMP-8 nonspecific binding sites on the collagen molecules, but cleavage always took place at a unique site. When collagen molecules, straightened and immobilized on the surface, were reacted with MMP-8, a site of cleavage appeared as a gap in stretched molecules. This is the first report to visually show direct collagenase:collagen interactions using AFM. The described AFM-based analysis has potential as a protein analysis tool for understanding a complex mechanism of enzyme:substrate interactions.

Animals↗

PubMed2006

Characteristic features of Bacillus cereus cell surfaces with biosorption of Pb(II) ions by AFM and FT-IR.

In order to elucidate the potential mechanisms involved in the biosorption of metal ions, atomic force microscopy (AFM) and Fourier transform infrared (FT-IR) spectroscopy were used to characterize the interaction between Pb2+ and Bacillus cereus. AFM imaging of the biomass surfaces exposed to different concentrations of lead ions solution showed a major morphological change occurred after Pb2+ biosorption. The FT-IR spectra indicated the binding characteristics of the lead ions involved the carboxyl, hydroxyl and amino groups in the biomass. Equilibrium biosorption experiments of Pb2+ were carried out to investigate the effects of pH values and the initial metal concentrations. The experimental isotherm data were then modeled using Langmuir, Freundlich, and Redlich-Peterson isotherm equations. As a result, the Redlich-Peterson model yielded the best fit of experimental data. Kinetics experiments showed the biosorption was a rapid process and the pseudo-second-order model was successfully applied to predict the rate constant of biosorption.

Adsorption↗

PubMed2003

The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae, Nematomorpha): observations by TEM, SEM and AFM.

The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cuticle is composed of 30-50 compact layers. The number of the layers is higher in the central part of the animal's body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately 640 nm in diameter and of indefinite length. The fibres run strictly parallel within each layer, while in adjoining layers they run at a variable angle from 45 degrees in the central body to 90 degrees in the extremities. Each fibre shows a barely detectable filamentous inner structure and is enveloped in a thin highly regular net formed by hexagonal meshes. Our results suggested that these fibres should be proteinaceous although non-collagenous. Thinner radial fibres run among the large fibres and across all the layers and span the whole thickness of the cuticle from the epithelial layer located deep underneath the large fibres up to the epicuticle on the external surface of the animal.

Animals↗