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.     

Chemical force microscopy of microcontact-printed self-assembled monolayers by pulsed-force-mode atomic force microscopy

A novel chemically sensitive imaging mode based on adhesive force detection by previously developed pulsed-force-mode atomic force microscopy (PFM-AFM) is presented. PFM-AFM enables simultaneous imaging of surface topography and adhesive force between tip and sample surfaces. Since the adhesive forces are directly related to interaction between chemical functional groups on tip and sample surfaces, we combined the adhesive force mapping by PFM-AFM with chemically modified tips to accomplish imaging of a sample surface with chemical sensitivity. The adhesive force mapping by PFM-AFM both in air and pure water with CH3- and COOH-modified tips clearly discriminated the chemical functional groups on the patterned self-assembled monolayers (SAMs) consisting of COOH- and CH3-terminated regions prepared by microcontact printing (microCP). These results indicate that the adhesive force mapping by PFM-AFM can be used to image distribution of different chemical functional groups on a sample surface. The discrimination mechanism based upon adhesive forces measured by PFM-AFM was compared with that based upon friction forces measured by friction force microscopy. The former is related to observed difference in interactions between tip and sample surfaces when the different interfaces are detached, while the latter depends on difference in periodic corrugated interfacial potentials due to Pauli repulsive forces between the outermost functional groups facing each other and also difference in shear moduli of elasticities between different SAMs.     

Chemical force microscopy of self-assembled monolayers on sputtered gold films patterned by phase separation

Patterned self-assembled monolayers (SAMs) were formed on gold films and observed by friction force microscopy (FFM) and adhesive force mapping with pulsed-force mode atomic force microscopy (PFM-AFM). The substrate gold films were prepared by sputtering gold on flat surfaces of osmium-coated cover glass with surface roughness, Ra, of 0.3 nm. The patterned samples with the CH3 and COOH terminated regions were prepared using the Langmuir-Blodgett (LB) method, partial removal of the LB film by ultrasonication, and SAM formation. The CH3 and COOH terminated regions of the patterned SAMs in air and in water were observed by mapping friction and adhesive forces with FFM and PFM-AFM, respectively, using gold-coated AFM tips chemically modified with a thiol compound terminating in CH3 or COOH. The adhesive forces measured in air increased in the order of CH3/CH3, CH3/COOH (or COOH/CH3) and COOH/COOH, while those in water increased in reverse order. The enormous high adhesive force observed in water for CH3/CH3 was attributed to hydrophobic interaction between the CH3 tip and the CH3 terminated sample surface. With CH3 tip, the lower friction force was observed, however, in water on the CH3 terminated region than on the COOH terminated region. This experimental finding raises a question as to what is the effective normal load in friction measurements in water.     

Immobilization of DNA on 11-mercaptoundecanoic acid-modified gold (111) surface for atomic force microscopy imaging

Immobilized DNA on preformed 11-mercaptoundecanoic acids (MUDA) self-assembled monolayers (SAMs) on a gold (111) surface was bound by a divalent cation bridges was imaged by atomic force microscopy (AFM). The DNA immobilization was attributed to the formation of ionic bridges between the carboxylate groups of MUDA and the phosphate groups of DNA. AFM images revealed that DNA molecules could be immobilized strongly enough to permit stable and reproducible imaging. The effect of different bridge cations, such as Mg(2+), Zn(2+) and Cu(2+), and the pH of DNA assembled solution on immobilization and conformation of DNA was studied. Plasmid DNA pBR 322/Pst I molecules were straightened by using a molecular combing technique on the MUDA surface.     

Hybridisation of short DNA molecules investigated with in situ atomic force microscopy

By introducing the complementary DNA (cDNA) strand to a molecular layer of short single stranded DNA (ssDNA), immobilised on a gold surface, we have investigated hybridisation between the two DNA strands through the technique of in situ atomic force microscopy (AFM). Before introduction of cDNA, the ssDNA molecular layer was modulated with the spacer molecule mercaptohexanol (MCH), which makes the ssDNA molecules more accessible for hybridisation.With in situ AFM, we have monitored the formation of a smooth, mixed molecular layer containing ssDNA and MCH. Furthermore, the hybridisation between the two DNA strands has been studied. Introduction of the cDNA strand resulted in an increase in smoothness and thickness of the molecular layer. Both the increase in order and thickness of the molecular layer can be expected if hybridisation occurs, since double stranded DNA molecules have a more rigid and elongated structure than ssDNA molecules.     

AFM imaging in solution of protein-DNA complexes formed on DNA anchored to a gold surface

A chemical procedure for anchoring DNA molecules to gold surfaces was used to facilitate the imaging of DNA and DNA-protein complexes in buffer solution by tapping mode atomic force microscopy (TMAFM). For preparing flat gold surfaces, a novel approach was employed by evaporating small amounts of gold onto freshly cleaved mica to give flat films that were stable under aqueous buffer conditions. The thickness of the investigated films ranged from 1 to 10 nm. For typical films of 4-6 nm, which were stable under aqueous buffer conditions, the root mean square (RMS) roughness ranged between 0.25 and 0.5 nm, as measured by atomic force microscopy (AFM). This roughness is comparable to that of obtained by the template stripped gold (TSG) technique, which is widely used in scanning probe microscopy but involves more preparation steps. In order to visualize DNA and DNA-protein complexes by TMAFM, the DNA was chemisorbed to the gold surface through a linker carrying a terminal thiol group at the 5'-end of each of the DNA strands. The modified DNA fragments were bound to the gold films and imaged in buffer solution, while unmodified DNA could not be visualized. Since the DNA was not dried during the process, it can be assumed that its native conformation was retained. This mode of anchoring did not prevent interaction with proteins, as confirmed by the observation that the topology of a complex formed by adding the protein to a surface-anchored DNA was the same as that obtained by anchoring a pre-formed complex to the gold surface. We attribute this observation to the fact that the DNA is anchored to the gold surfaces only through its ends, therefore the DNA-support interaction is minimized but imaging is still possible.     

The control of the configuration of nucleic acid molecules deposited for electron microscopy, by ionic bombardment of carbon films

DNA molecules bind to carbon films placed on the surface of aqueous solutions and are extended and oriented by the flow forces on removal from the surface. The number of molecules bound increases with ionic strength suggesting that the bonding is hydrophobic. If carbon films are subjected to ionic bombardment in a flow discharge, they can be modified so that molecules bind strongly, and are deposited in a tangled, random-coil configuration, uninfluenced by flow forces. The strength of binding is decreased by reducing the discharge time and current, and by addition of salt (NaCl), suggesting that the bonding is ionic. When weakly bound, the molecules are condensed by ethanol. The strength of binding can be adjusted so that the molecules are largely untangled by flow, and in a form suitable for the mapping of bound enzymes, or other identifiable markers. Aggregation and extension of molecules, that occur on unmodified films, and the binding of cytochrome c, that occurs in methods based on that of Kleinschmidt & Zahn (1959) are avoided. Both double and single-stranded molecules can be deposited and seen after rotary shadowing with Pt. The discharge can be performed in a vacuum produced by a standard oil-filled rotary pump.     

Atomic force microscopy tips (cantilevers) as molecular nucleic acid sensors

A model single strand DNA (ssDNA) was covalently immobilized onto AFM tips (cantilevers) as specific ligand. These tips were interacted with the buffer solutions with or without free ssDNA molecules as the target strands to be detected. Immobilization and hybridization onto the cantilever surfaces were observed by optical and fluorescence microscopies. Interactions between the AFM tip (cantilever) and the aqueous medium (therefore with the target ssDNAs) were quantified by obtaining the "percent separation distance" ("PSD") as the main variable. The PSD values obtained for the buffer solutions were between -2.07 and +4.91%. There were slight increases in the negative values when non-complementary ssDNA molecules were introduced into the buffer. However, after hybridization with its complementary ssDNA, the PSD values were significantly increased (between -32.24 and -43.47%). There was a correlation between the concentration of the complementary target ssDNA in the medium and the PSD value. As a result of these promising results it was concluded that this approach may be further developed to create AFM-based molecular sensors for diverse applications.     

Electron microscopic visualization of complementary labeled DNA with platinum‐containing guanine derivative

The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast‐enhanced heavy element. The stretched single‐chain DNA was obtained by modifying double‐stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single‐stranded DNA pieces on the support film with platinum containing guanine derivative to form base‐specific hydrogen bond; and (iii) producing a magnified image of the base‐specific labeled DNA. Stretched single‐stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum‐containing guanine derivative serves as a high‐dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom‐by‐atom analysis and it is promising way toward future DNA‐sequencing or molecular diagnostics of nucleic acids by electron microscopic observation. Microsc. Res. Tech. 79:280–284, 2016. © 2016 Wiley Periodicals, Inc.     

Adsorption of DNA molecules to different support films.

Protein-free adsorption of the DNA of the Escherichia coli bacteriophage T7 to carbon, collodion, aluminium-beryllium and aluminium films was studied. It was found that the appearance of DNA strands depended greatly upon the kind of support film used. Direct adsorption of DNA to aluminium-beryllium or aluminium films yielded specimens with 'thin and long' and 'thick and short' regions along the strand. Well extended, uncoiled and unaggregated DNA molecules were obtained only when DNA was adsorbed to carbon, collodion or mica in the presence of intercalating dyes such as ethidium bromide. Adsorption properties of the different films are well correlated with their surface charge. Aluminium-beryllium films carry a strong positive surface charge, aluminium films a weak positive charge and carbon films a weak negative charge. It is suggested that for the preparation of specimens by spontaneous adsorption of well extended and unaggregated strands it is necessary that the DNA molecule is stiffened by a ligand such as an intercalating dye, and that the charge on the surface of the support film is opposite to the charge of the macromolecule.     

Identifying sequence similarities between DNA molecules

An atomic force microscope (AFM) imaging technique is described to compare sequences between two different DNA molecules and precisely locate nonhomologies in DNA strands. Sequence comparisons are made by forming heteroduplexes between the two molecules and, by AFM imaging the intact molecules formed, identifying both homologous and nonhomologous regions. By forming heteroduplexes between linearized wildtype pSV-beta-galactosidase plasmid (6821 bp) and a series of deletion mutants we have identified nonhomologies (deletions) as small as 22 bp and as large as 418 bp. Furthermore, by incorporating our technique for AFM-mediated restriction mapping of DNA these mutations can be positioned relative to EcoRI restriction sites. These results suggest AFM can be useful in identifying molecular level similarities and differences in DNA.     

Molecular order affecting electron transport through ssDNA

DNA is considered to be the ideal model for studies of electron transport in molecule/conductor systems due to its stability, easily controlled structure and the presumed electrical properties. Scanning tunnelling microscope (STM) studies of single-stranded DNA bound to Au (1 1 1) or Au nanodots with a thiol linker were carried out under ambient conditions. The results show that the electron transfer between the STM tip and the gold is governed by the serial resistance of the oligomer strands and a water film. Electron transfer properties also depend on the alignment of the DNA strands. Measurements show that well-ordered parallel arrangement of the molecules protruding from flat crystalline surfaces is favourable for electron transport compared with unordered arrangements of molecules on spherical nanodots. Nanodots are good candidates for effective charge production by absorption of light allowing chemical reactions to happen at the dots, which can be used for storing the light energy. Understanding electron transport through molecular structures is of crucial importance for the development of such novel photovoltaic devices.     

Monitoring of glass derivatization with pulsed force mode atomic force microscopy

Non-specific adsorption of proteins at solid/liquid interfaces is a major problem in the use of synthetic biomaterials and in ultrasensitive detection methods. Grafting surfaces with a dense layer of poly(ethylene glycol) (PEG) or other polymers is a most widely used strategy to solve this task. While such modified surfaces have been characterized by their ability to resist protein adsorption, the polymer layers themselves have rarely been studied in fine detail. Atomic force microscopy (AFM) using the pulsed force mode (PFM), is an ideal technique to investigate structural features and physiochemical properties of surfaces because topology and adhesion are simultaneously detected with high lateral resolution. In the present study, PFM-AFM was applied to thoroughly characterize different stages of glass derivatization, up to the formation of a dense PEG layer. Lateral inhomogeneities in topology and/or adhesion were observed at all stages before PEG attachment. The covalently bound PEG, however, was seen to form a densely packed monolayer with maximal thickness, smooth surface, and weak adhesion. Thus, PFM-AFM appears to be a valuable tool for the characterization of protein-repelling surfaces in solution.     

304不锈钢表面预处理对沉积CrMoN涂层性能的影响

使用砂纸将对304不锈钢基体依次打磨至1200^#(1^#工艺)、2000^#(2^#工艺),以及打磨磨至2000^#并经粒度0.5μm金刚石抛光膏抛光(3^#工艺)后,在其表面沉积CrMoN涂层,研究了涂层的物相组成、表面与截面形貌、硬度、表面疏水性、耐腐蚀性能和导电性。结果表明:1^#工艺预处理基体表面沉积涂层的表面粗糙度最大,2^#工艺预处理后的次之,3^#工艺预处理后的最小;涂层均由CrN,Cr₂N,Mo₂N等物相组成;随着基体表面粗糙度的降低,涂层的显微硬度、自腐蚀电位和水接触角增大,自腐蚀电流密度、极化后的界面接触电阻降低;2^#工艺预处理基体表面沉积CrMoN涂层的综合性能优异,与3^#工艺预处理的接近。     

Effect of Water Swelling on the Tribological Properties of PMMA Spin-Cast Film and Brush in Aqueous Environment

Due to their light weight, low corrosion and good tribological properties, polymer films have been widely studied in dry condition as well as recently in aqueous environment. Though the presence of water can further reduce the friction, it promotes the wear rate of the polymer films. As a remedy to decrease the wear rate of polymer films under aqueous condition, in this study, we used PMMA brush which is chemically anchored to a substrate and compared its friction and wear properties with those of conventional PMMA spin-cast film. Ellipsometry, contact angle measurements and atomic force microscopy are used to study the surface properties, e.g., wear mechanisms and wear depths of PMMA films. Under different sliding speeds and applied loads, PMMA brush showed lower friction than PMMA spin-cast film in aqueous. Moreover, it was shown that the swelling of water molecules is a dominant factor in determining the wear durability of PMMA films in which PMMA brush showed better wear performance than PMMA spin-cast film.     

Effect of Surface Roughening of Substrate Steel on the Improvement of Delamination Strength and Tribological Behavior of Hydrogenated Amorphous Carbon Coating Under Lubricated Conditions

The adhesion strength of diamond-like carbon (DLC) coatings is an obstacle in efforts to improve the reliability of coated products. It is generally believed that the roughening of the substrate surface improves the adhesion between a substrate and coating. The effect of surface roughening of the substrate on the delamination strength of DLC coating and the tribological behavior under lubrication were studied. Five types of roughened substrates were prepared by a wet blast device with differing materials, shapes, and sizes of the shot particles. A hydrogenated DLC film was deposited using plasma-enhanced chemical vapor deposition on the roughened substrates. The tribological properties were investigated under air and lubrication with pure water or n-decane. It was found that the delamination strength of the DLC coating could be improved by roughening the substrate surface, especially by spherical particles. It was also found that slight polishing of either the DLC surface deposited on the rough substrate or the roughened substrate before deposition significantly reduced the wear of the counter surface. The remaining chemical element of alumina particles on the roughened surface affected the delamination strength of the DLC coating.     

Conformational dynamics of single molecules visualized in real time by scanning force microscopy: macromolecular mobility on a substrate surface in different vapours

We describe a technique to visualize and effect in real time motion and conformational transitions of single macromolecules. Two steps are involved. First, scanning force microscopy (SFM) was applied to detect in situ conformational transitions of single polymer molecules adsorbed on a substrate surface. Secondly, these changes were induced by controlled variations of environmental conditions in a microscope environmental chamber. In particular, we have revealed that exposure of a substrate with adsorbed macromolecules to vapours of different nature was able to increase molecular mobility and to stimulate conformational transitions of the polymer chains on the surface. Realization of SFM observation in a variable vapour environment was not as difficult as in liquid media. Variations of the vapour composition affected the oscillation dynamics of the cantilever with the scanning probe only to a small extent, and did not impede continuation of the scanning procedure. In fact, the characteristic times of the observed conformational changes were large enough (minutes to dozens of minutes) for sampling images repeatedly. Although recording of an SFM image was slow and required several minutes, we were able to visualize step‐by‐step the successive stages of the slow conformational transformation of the macromolecules adhering to the substrate, i.e. to investigate a molecular response to the environment changes in real time. Here, we studied the reversible collapse–decollapse transitions of cylindrical poly(methacrylate)‐graft‐poly(n‐butyl acrylate) brush‐like macromolecules exposed to different vapours. Single macromolecules on mica tended to assume a compacted globular conformation when exposed to the vapour of compounds, which due to their amphiphilic nature adsorb on mica and lower the surface energy of the substrate (e.g. alcohols). By contrast, the macromolecules adopted extended two‐dimensional worm‐like conformations in the vapours of compounds having high values of surface tension (such as water). In our opinion, the reason for the observed tendency was a competition in spreading on the substrate surface between the macromolecules and the co‐adsorbed vapour molecules. If the brush‐like macromolecules succeeded in the spreading, they acquired an extended conformation. Otherwise they collapsed to globuli in order to reduce the surface area per macromolecule. Thus, the enhanced mobility of synthetic macromolecules on a substrate observed in a vapour environment in combination with the possibility to manipulate the macromolecular conformation via changes in a vapour phase and the ability to visualize the transitions of the macromolecules individually, provides challenging prospects for SFM studies on the dynamics of single molecules under applied external stimuli.     

Patterning DNA on microm scale on mica

Double-stranded DNA molecules were patterned by selective adsorption to aminosilane patterns on mica surfaces. Line patterns with 10 microm spacing were made by photolithography and transferred to a polymer stamp. The stamp was then used for applying aminosilane molecules by microcontact-printing technique on mica substrates. We applied DNA in Tris-EDTA (TE) buffer solution on the patterned substrate, and incubated it for 5 min at room temperature. The sample was then rinsed with pure water, and dried with nitrogen gas. Tapping mode force microscopy showed that DNA was adsorbed selectively on the aminosilanized parts of the mica substrate. We also tried to bridge two aluminum electrodes with DNA using AC electrophoresis.     

Measurements of thickness dispersion in biolayers by scanning force microscopy and comparison with spectroscopic ellipsometry analysis

Measuring the thickness of biological films remains a difficult task when using differential measurements by atomic force microscopy (AFM). The use of microstructured substrates combined with a selective adsorption constitutes an alternative to tribological measurements. The statistical thickness analysis of biological layers, especially via the dispersion measurements, can provide a way to quantify the molecular orientation. AFM thicknesses were then compared with those obtained optically by spectroscopic ellipsometry (SE) and surface plasmon resonance enhanced ellipsometry (SPREE). The biolayers could then be modeled using a vertical gradient of optical index, which reflects height dispersions. Thiol-modified DNA strands of various lengths account for a good biological model for the study of the strand motion in air and in liquid.     

Density Variation in the Ultrathin Liquid Perfluoropolyether Films on Solid Surfaces

Variation of density in the ultrathin liquid perfluoropolyether films on solid surfaces has been studied by Monte Carlo simulations of Lennard-Jones systems. The liquid film is 8.75 nm thin film consisting of polymer molecules assuming the structure of perfluoropolyether Z having molecular weights of 3840, 2500 and 1700 g/mol and interacting among themselves by Lennard-Jones potential. The substrate is assumed to be continuous without atomic structure and exerting Lennard-Jones potential on liquid molecules in the ultrathin film. The system temperature is considered to be 25 °C and the liquid molecules also have the gravitational potential. It is found that the bead density decreases towards the surface in a thinner sublayer in the ultrathin liquid film above the surface and the thickness of this sublayer just above the surface may increase with the increase of molecular weight of the of polymers in the film. Repulsive potential of the surface further decreases the bead density near the surface. The results are compared with the experimental results of the pefluoropolyether lurbricants by X-ray reflectivity (Shouji, et al., 1998).