Strong hydrophobic interaction between graphene oxide and supported lipid bilayers revealed by AFM

Understanding the interaction between graphene oxide (GO) and lipid membranes is of great importance for its various applications in biotechnology. Here, we investigated the interaction between GO and charged supported lipid bilayers (SLBs) by in situ atomic force microscope (AFM) imaging. It was found that GO could peel off a single layer of positively charged SLBs and deposited on the hydrophobic part of the remaining sublayer. Then free lipid molecules would assemble on GO surface and formed 1.5 bilayers in a lipid‐GO‐lipid manner. For negatively charged lipid bilayers, however, GO deposited to the SLBs only when its concentration was very high. These results indicate that, in addition to electrostatic interaction, the hydrophobic interaction plays an important role when GO sheets deposit onto the charged lipid bilayers, and should be helpful to understand possible cytotoxicity and antibiosis of graphene‐related nanomaterials. Microsc. Res. Tech. 79:721–726, 2016. © 2016 Wiley Periodicals, Inc.     

Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM

The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5 x 10(-18)J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1 x 10(4) under the assumption that the area of the cell surface was about 5,000 microm(2). These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM.     

Optimisation of sample preparation methods for air imaging of ocular mucins by AFM

The addition of cations to the imaging buffer for AFM has been previously shown to improve the binding of biological molecules to mica. Investigations were carried out to find the concentration of NiCl(2) required to immobilize mucin molecules on a freshly cleaved mica surface, for imaging using intermittent contact in air. Drop-deposition of samples prepared in HEPES buffer with 1, 2 and 5mM NiCl(2) revealed the sensitivity of the mucin molecules to salt. Dialysis of the mucin solutions dramatically reduced the amount of salt present and allowed single molecules to be imaged, revealing a variation in thickness along their length. Spray deposition of the same mucin solutions produced single molecules that, although less affected by co-adsorbed salt, showed a degree of self-folding. This shows the sensitive balance between HEPES and NiCl(2) required for successful imaging of the sub-molecular features of individual mucin molecules.     

Visualizing filamentous actin on lipid bilayers by atomic force microscopy in solution

The surface structure of actin filaments (F-actin) was visualized at high resolution, by atomic force microscopy (AFM) in aqueous solution, in large paracrystals prepared on positively charged lipid monolayers. The increased stability of these closely packed specimens allowed us to show that both the long pitch (38 nm) and the monomer (5.8 nm) can be directly resolved by AFM in the contact mode. The right-handed helical surface, distinguishable in high resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was 10 ± 1 nm, whereas the smaller inter-filament distance, 8 ± 1 nm, was consistent with interdigitation of the filaments. The 10 ± 1 nm F-actin diameter is in good agreement with the results of fibre X-ray diffraction. As such specimens are relatively easy to prepare without specialized equipment, this method may allow the study of the thin filaments in which F-actin-associated proteins are also present.     

The crack growth behavior of Incoloy 800H under fatigue and dwell-fatigue conditions at elevated temperature

Fatigue crack growth rate (FCGR) tests with different load ratios and dwell-fatigue crack growth rate (DFCGR) tests with different dwell times were conducted at 750°C for Incoloy 800H. As the load ratio increases from 0.1 to 0.5, the crack growth rate increased and the transition ??K value from region I to region II (Paris regime) shifted leftward. In DFCGR tests with dwell time of 10 and 30 seconds, the Paris regime started at relatively lower ??K level and the crack grew much faster than in FCGR tests. However, the crack growth rates between the 10 sec and 30 sec dwell times were relatively similar. The higher crack growth rates in the DFCGR tests compared to FCGR tests was associated with the reduction of the M₂₃C₆ precipitates in the vicinity of the advancing crack by the Cr depletion, suggesting the crack propagation in DFCGR conditions was environmentally assisted. The crack growth rate was controlled by trans-granular mode regardless of the dwell time because the dwell time was not enough to cause creep damage.     

Molecular asperity theory of boundary friction

A computer investigation of the hypothesis that boundary friction is caused by molecular forces between the tails of long-chain molecules attached vertically to the sliding surfaces is reported. It has previously been shown that the interaction statements which apply to this system are the Slater and Bartell scattering centre potentials. The Slater scattering centre potential was less accurate but was considered to see how much it affects the calculated friction.The frictional force was calculated and shown to be a two-term relation. The first term is due to the interaction energy barrier encountered when moving one surface from one equilibrium position to the next, together with the work done against internal rotation barriers. The second term is due to the lifting of one end group over the opposite one as the chains move, a molecular “asperity” friction. The calculations show that the friction rises with normal load in a manner similar to that found experimentally. As with almost all theoretical predictions of strength calculated from intermolecular potentials, these forces are some ten times too large, a discrepancy which is usually attributed to dislocations.     

Analysis of force curves obtained on the live cell membrane using chemically modified AFM probes

Force curves were obtained on the live cell surface using an atomic force microscope mounted with a modified tip with the bifunctional covalent crosslinker, disuccinimidyl suberate, which forms a covalent bond with amino-bearing molecules on the cell surface. A ramp delay time of 1.0 s was introduced before the start of the retraction regime of the force curve to increase the stationary reaction time between the crosslinkers on the tip and the amino groups on the cell surface. While live cell surface responses to forced contact with a non-functionalized tip rarely showed evidence of tip–cell interaction, those obtained with modified tips gave clear indication of prolonged adhesion which was terminated by a single step release of the tip to its neutral position. Under the given experimental conditions of this work, 58% of a total of 198 force curves gave only one jump and 70% of those with one jump gave the final rupture force of 4.5±0.22 nN. The result emphasized the uniqueness of the observed mechanical response of the cell surface when probed with chemically modified tips.     

Nanometre localization of single ReAsH molecules

ReAsH is a red‐emitting dye that binds to the unique sequence Cys‐Cys‐Xaa‐Xaa‐Cys‐Cys (where Xaa is a noncysteine amino acid) in the protein. We attached a single ReAsH to a calmodulin with an inserted tetracysteine motif and immobilized individual calmodulins to a glass surface at low density. Total internal reflection fluorescence microscopy was used to image individual ReAsH molecules. We determined the centre of the distribution of photons in the image of a single molecule in order to determine the position of the dye within 5 nm precision and with an image integration time of 0.5 s. The photostability of ReAsH was also characterized and observation times ranging from several seconds to over a minute were observed. We found that 2‐mercaptoethanesulphonic acid increased the number of collected photons from ReAsH molecules by a factor of two. Individual ReAsH molecules were then moved via a nanometric stage in 25 or 40 nm steps, either at a constant rate or at a Poisson‐distributed rate. Individual steps were clearly seen, indicating that the observation of translational motion on this scale, which is relevant for many biomolecular motors, is possible with ReAsH.     

Topographic effects on adhesive force mapping of stretched DNA molecules by pulsed-force-mode atomic force microscopy

Adhesive interaction between a tip and a sample surface was examined on a microscopic scale by pulsed-force-mode atomic force microscopy (PFM-AFM). The signal measured by monitoring pull-off force is influenced by various factors such as topography, elasticity, electrostatic charges, and adsorbed water on surfaces. Here, we focus on the topographic effects on the adhesive interaction. To clarify the topographic influence, the adhesive force measurement of a stretched DNA molecule with a smaller radius of curvature than that of a tip was carried out at low relative humidity (RH) with an alkanethiol-modified tip. The experimental conditions such as low RH and the use of the alkanethiol-modified tip were required to minimise the influence of water capillary force on hydrated DNA strands. The hydrophobic modification of a substrate surface was also important to minimise the adsorbed water effect. The DNA molecules were stretched on the substrate surfaces by an immobilisation process called a dynamic molecular combing method. The two-component vapour-phase surface modification with an alkylsilane mixed with a silane derivative containing an amino end group enhanced the DNA adsorption due to the electrostatic interaction. The experimental results for the topographic effects on the adhesive force mapping were reproducible.     

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica

Multivalent ions in solution are known to mediate attraction between two like‐charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either ‘salt bridging’ or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re‐explore current theories of like‐charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.     

Positively charged nanogold label allows the observation of fine cell filopodia and flagella in solution by atmospheric scanning electron microscopy

Optical microscopy is generally the first choice to observe microbes and cells. However, its resolution is not always sufficient to reveal specific target structures, such as flagella and pili, which are only nanometers wide. ASEM is an attractive higher resolution alternative, as the sample is observed in aqueous solution at atmospheric pressure. Sample pretreatment for ASEM only comprises simple tasks including fixation, gold labeling, and reagent exchange, taking less than 1 h in total. The lengthy sample pretreatments often required for more classical electron microscopies, such as embedding and dehydration, are unnecessary, and native morphology is preserved. In this study, positively charged nanogold particles were used to label the surfaces of bacteria and cultured animal cells, exploiting their net negative surface charge. After gold enhancement to increase the size of the nanogold particles, ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella. This natural shape contrasts starkly with images of dried flagella recorded by standard SEM. Positively charged nanogold labeled the plasma membrane of cultured COS7 cells, and after enhancement allowed filopodia as thin as 100 nm in diameter to be clearly visualized. Based on these studies, ASEM combined with positively charged nanogold labeling promises to become an important tool for the study of cell morphology and dynamics in the near future. Microsc. Res. Tech. 77:153–160, 2014. © 2013 Wiley Periodicals, Inc.     

Static and Dynamic Properties of Oil Films Containing Organic Polar Compounds on Metal Surfaces,Using Electric Measurements

Using electric measurements, an oil film of liquid paraffin solutions of some surface active compounds was investigated in static, transitional, and dynamic states.

In the static state, the film strength for mechanical deformation depended on the surface active compound in the oil. The adsorbed molecules in the oil film formed a multilayered film causing a mesomorphic state in the oil film. The oil film thickness in the transitional state was reduced by about 30 percent, in comparison with that in the static state. This reduction depended on which surface active compound was added. The film thickness decreased depending on the sliding velocity when it was below 30 cm/sec. The structure of an oil film below 30 cm/sec sliding velocity was considered to be in a dynamic equilibrium between the destruction or disturbance by a mechanical force and the molecular rearrangement or recovery of the structure.     

Effect of surface conjugation chemistry on the sensitivity of microcantilever sensors

Microcantilever sensors are an offshoot of atomic force microscopy and are useful tools for effectively detecting a target biomolecule. The recognition of the target molecule on the biosensor is based on the physical bending of the microcantilever, which is driven by a specific molecular interaction between the target molecule and the sensor surface. In this study, to enhance the sensitivity of the microcantilever sensor, the sensor surface was modified through a surface conjugation method using self-assembled monolayers (SAMs) and heterobifunctional cross-linkers. After the surface modification of the microcantilever sensor, the sensitivity for L-cysteine was recorded. The detection of L-cysteine was influenced by the active site and the molecular size of the cross-linked compound attached onto the surface of the microcantilever.     

New direct observations of asphalts and asphalt binders by scanning electron microscopy and atomic force microscopy

Observations made using AFM and SEM have been combined in order to study the structure of asphalts. Fluorescence microscopy was used to aid in understanding the structural changes occurring when polymer is added to the asphalts.   With the atomic force microscope we are able to study the structure of the asphalts without any pre-preparation. Despite very low resolution, our study reveal ed a network of asphaltene molecules with regard to asphalt gel. The same result is obtained by SEM observation but with a much better resolution. SEM observation, however, needs an adequate preparation method.   In the presence of polymer we observed a rearrangement of the initial asphaltene association which leads to the assumption that polymer can aggregate the asphaltene phase.     

Data analysis of interaction forces measured with the atomic force microscope

The force-distance cycle mode of the atomic force microscope (AFM) allows for detection of interaction forces between the AFM-tip and a substrate (probe). This can either be a direct tip-sample interaction or an interaction between molecules coupled to the tip and probe, respectively. The interaction forces are typically in the range of a few pN to some hundred pN. In this article we describe algorithms for the analysis of force-distance cycles, to quantify interaction forces between tip and probe. Both, the direct tip-probe interaction as well as the interaction between specifically bound molecules are analyzed. The molecules bound to tip and probe have to be either long and flexible or have to be bound via a flexible cross linker. The algorithms are exemplified on direct tip-probe interactions and on unbinding events of cadherins which are bound via PEG-spacers to the AFM-tip and to the probe.     

Exploring the molecular forces within and between CbsA S-layer proteins using single molecule force spectroscopy

We used single molecule atomic force microscopy (AFM) to gain insight into the molecular forces driving the folding and assembly of the S-layer protein CbsA. Force curves recorded between tips and supports modified with CbsA proteins showed sawtooth patterns with multiple force peaks of 58+/-26pN that we attribute to the unfolding of alpha-helices, in agreement with earlier secondary structure predictions. The average unfolding force increased with the pulling speed but was independent on the interaction time. Force curves obtained for CbsA peptides truncated in their C-terminal region showed similar periodic features, except that fewer force peaks were seen. Furthermore, the average unfolding force was 83+/-45pN, suggesting the domains were more stable. By contrast, cationic peptides truncated in their N-terminal region showed single force peaks of 366+/-149pN, presumably reflecting intermolecular electrostatic bridges rather than unfolding events. Interestingly, these large intermolecular forces increased not only with pulling speed but also with interaction time. We expect that the intra- and intermolecular forces measured here may play a significant role in controlling the stability and assembly of the CbsA protein.     

Optimized sample preparation for high-resolution AFM characterization of fixed human cells

Atomic force microscopy enables the simultaneous acquisition of high-resolution topographical and biophysical data allowing integrated analysis of cell surfaces during development and pathogenesis, and, critically, can link molecular and biophysical events. Here we used atomic force microscopy to analyse endometrial epithelial cells and neuronally differentiated P19 cells. Optimized reproducible sample preparation techniques enabled micro- and nanoscale multi-parameter analysis. Comparative analysis using atomic force microscopy and scanning electron microscopy demonstrated the utility of atomic force microscopy for examining tissue morphology, and its ability to generate data allowing differentiation of cells from different origins to be monitored. At low resolution atomic force microscopy produced topographic data complementary to scanning electron microscopy images, whilst at high resolution atomic force microscopy captured novel cell surface structural detail for both epithelial and neuronal cell types. Analysis of surface roughness provided biophysical data which enabled qualitative and quantitative differences between samples to be measured. This study provides an important optimization of sample preparation enabling more generalized atomic force microscopy utilization for cellular analysis required for advanced cell surface morphological studies.     

液体润滑剂的黏度对边界滑移影响的实验研究

为了了解微纳米间隙中流体的流动特性,采用原子力显微镜对微纳米间隙中的固体和液体边界滑移进行了实验研究,主要研究了液体润滑剂的黏度对边界滑移的影响。实验中采用的固体样品为SiO2,液体样品为两种不同黏度的季戊四醇油酸酯,分子式为C77H140O8,黏度分别为32mm2/s和150mm2/s。采用相对速度法对实验数据进行了处理,结果表明,不同黏度的季戊四醇油酸酯和SiO2表面作用时都会发生边界滑移,黏度大,产生的滑移大。其原因是,随着黏度升高,邻近固体表面的液体分子与和固体表面相接触的液体分子之间的剪切力增大,可更加容易地克服固液界面间的作用力,更加容易产生边界滑移。