Nanoscale mapping and affinity constant measurement of signal-transducing proteins by atomic force microscopy

Atomic force microscope (AFM) was used to measure the interaction force between two signal-transducing proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Ras homologue enriched in brain (Rheb), and to analyze the binding of glyceraldehyde-3-phosphate (Gly-3-P) to GAPDH. To enhance the recognition efficiency and avoid undesirable multiple interactions, the AFM probe and the substrate were each modified with a dendron, glutathione S-transferase (GST)-fused proteins were employed, and reduced glutathione (GSH) was conjugated at the apex of each immobilized dendron. The resulting median specific force between GAPDH and Rheb was 38 ± 1 pN at a loading rate of 3.7 × 10(3) pN/s. The measurements showed that the GAPDH-Rheb interaction was inhibited by binding of Gly-3-P. An adhesion force map showed individual GADPHs on the surface and that the number density of GAPDH decreased with the concentration of Gly-3-P. Maps obtained in the presence of various Gly-3-P concentrations provided information on the binding behavior, yielding a thermodynamic association constant of 2.7 × 10(5) M(-1).     

Mapping the elastic properties of granular Au films by contact resonance atomic force microscopy

Endowed with nanoscale spatial resolution, contact resonance atomic force microscopy (CR-AFM) provides extremely localized elastic property measurements. We advance here the applicability of CR-AFM on surfaces with nanosize features by considering the topography contribution to the CR-AFM signal. On nanosize granular Au films, the elastic modulus at the grain scale has been mapped out by considering a self-consistent deconvolution of the contact geometry effect in the CR-AFM image. Significant variation in the contact area over granular topography arises as the probe is either in single-?or multiple-asperity contact with the surface. Consequently, in extracting the elastic modulus from CR-AFM measurements on granular surfaces we considered both the normal and lateral couplings established through multiple-asperity contacts between the tip and the surface. Thus, by appropriately considering the change in the contact mechanics during CR-AFM imaging, variations in the elastic modulus have been revealed in the intergrain regions as well as across individual grains.     

Nanoscale detection of ionizing radiation damage to DNA by atomic force microscopy

The detection and quantification of ionizing radiation damage to DNA at a single-molecule level by atomic force microscopy (AFM) is reported. The DNA damage-detection technique combining supercoiled plasmid relaxation assay with AFM imaging is a direct and quantitative approach to detect gamma-ray-induced single- and double-strand breaks in DNA, and its accuracy and reliability are validated through a comparison with traditional agarose gel electrophoresis. In addition, the dependence of radiation-induced single-strand breaks on plasmid size and concentration at a single-molecule level in a low-dose (1 Gy) and low-concentration range (0.01 ng microL(-1)-10 ng microL(-1)) is investigated using the AFM-based damage-detection assay. The results clearly show that the number of single-strand breaks per DNA molecule is linearly proportional to the plasmid size and inversely correlated to the DNA concentration. This assay can also efficiently detect DNA damage in highly dilute samples (0.01 ng microL(-1)), which is beyond the capability of traditional techniques. AFM imaging can uniquely supplement traditional techniques for sensitive measurements of damage to DNA by ionizing radiation.     

Towards a better understanding of wood cell wall characterisation with contact resonance atomic force microscopy

Nowadays, the multi-scale modelling of wood has a great need for measurements of structural, chemical and mechanical properties at the lowest level. In this paper, the viscoelastic properties in the layers of a wood cell wall are investigated using the contact resonance mode of an atomic force microscope (CR-AFM). A detailed experimental protocol suitable for obtaining reproducible and quantifiable data is proposed. It is based on three main steps: sample preparation to obtain a good surface state, calibration of the contact modulus using reference samples, and image processing to produce the viscoelastic images. This protocol is applied on chestnut tension wood. The obtained topography and semi-quantitative viscoelastic maps are discussed with respect to the cell wall structure, sample preparation effects, and AFM measurement specificity compared with nanoindentation.     

Nanoscale periodic modulations on sodium chloride surface revealed by tuning fork atomic force microscopy

The sodium chloride surface is one of the most common platforms for the study of catalysts, thin film growth, and atmospheric aerosols. Here we report a nanoscale periodic modulation pattern on the surface of a cleaved NaCl single crystal, revealed by non-contact atomic force microscopy with a tuning fork sensor. The surface pattern shows two orthogonal domains, extending over the entire cleavage surface. The spatial modulations exhibit a characteristic period of 5.4 nm, along <110> crystallographic directions of the NaCl. The modulations are robust in vacuum, not affected by the tip-induced electric field or gentle annealing (<300?°C); however, they are eliminated after exposure to water and an atomically flat surface can be recovered by subsequent thermal annealing after water exposure. A strong electrostatic charging is revealed on the cleavage surface which may facilitate the formation of the observed metastable surface reconstruction.     

Nanoscale fracture analysis by atomic force microscopy of EPDM rubber due to high-pressure hydrogen decompression

The relationship between internal fracture due to high-pressure hydrogen decompression and microstructure of ethylene–propylene–diene–methylene linkage (EPDM) rubber was investigated by atomic force microscopy (AFM). Nanoscale line-like structures were observed in an unexposed specimen, and their number and length increased with hydrogen exposure. This result implies that the structure of the unfilled EPDM rubber is inhomogeneous at a nanoscale level, and nanoscale fracture caused by the bubbles that are formed from dissolved hydrogen molecules after decompression occurs even though no cracks are observed by optical microscopy. Since this nanoscale fracture occurred at a threshold tearing energy lower than that obtained from static crack growth tests of macroscopic cracks (T _s,th), it is supposed that nanoscale structures that fractured at a lower threshold tearing energy (T _nano,th) than T _s,th existed in the rubber matrix, and these low-strength structures were the origin of the nanoscale fracture. From these results, it is inferred that the fracture of the EPDM rubber by high-pressure hydrogen decompression consists of two fracture processes that differ in terms of size scale, i.e., bubble formation at a submicrometer level and crack initiation at a micrometer level. The hydrogen pressures at bubble formation and crack initiation were also estimated by assuming two threshold tearing energies, T _nano,th for the bubble formation and T _s,th for the crack initiation, in terms of fracture mechanics. As a result, the experimental hydrogen pressures were successfully estimated.     

Higher-order electromechanical response of thin films by contact resonance piezoresponse force microscopy

Piezoresponse scanning force microscopy (PFM) has turned into an established technique for imaging ferroelectric domains in ferroelectric thin films. At least for soft cantilevers, the piezoresponse signal is not only dependent on the elastic properties of the material under investigation but also on the elastic properties of the cantilever. Due to this dependency, the cantilever response and, therefore, the measured properties depend on the frequency of the small alternating current (AC) testing voltage. At the contact resonance, the cantilever response is maximum, and this increased sensitivity can be used to detect very small signals or to decrease the voltage applied to the sample. We have shown that by using the hysteretic ferroelectric switching, it is possible to separate the signal into its components (viz. electromechanical and electrostatic contributions). Additionally, the measurement frequency can be tuned such that the second and third harmonics of the electromechanical response can be detected at the cantilever resonance, providing information about the higher-order electromechanical coefficients. We assume that this nonlinear behavior seen in local and macroscopic measurements is rooted in the nonlinearity of the dielectric permittivity. Our results are of crucial importance for the study of ferroelectric and electromechanical properties of nanostructures.     

Contact force identification using the subharmonic resonance of a contact-mode atomic force microscopy

We propose a step-by-step experimental procedure for characterization of the nonlinear contact stiffness on surfaces using contact-mode atomic force microscopy. Our approach directly estimates the first-, second-, and third-order coefficients of the contact stiffness. It neither uses nor requires the underlying assumptions of the Hertzian contact theory. We use a primary resonance excitation of the probe to estimate the linear coefficient of the contact stiffness. We use the method of multiple scales to obtain closed-form expressions approximating the response of the probe to a subharmonic resonance excitation of order one-half. We utilize these expressions and higher-order spectral measurements to independently estimate the quadratic and cubic coefficients of the contact stiffness.     

Nanoscale capacitance imaging with attofarad resolution using ac current sensing atomic force microscopy

Nanoscale capacitance imaging with attofarad resolution (～1?aF) of a nano-structured oxide thin film, using ac current sensing atomic force microscopy, is reported. Capacitance images are shown to follow the topographic profile of the oxide closely, with nanometre vertical resolution. A comparison between experimental data and theoretical models shows that the capacitance variations observed in the measurements can be mainly associated with the capacitance probed by the tip apex and not with positional changes of stray capacitance contributions. Capacitance versus distance measurements further support this conclusion. The application of this technique to the characterization of samples with non-voltage-dependent capacitance, such as very thin dielectric films, self-assembled monolayers and biological membranes, can provide new insight into the dielectric properties at the nanoscale.     

Quantitative subsurface contact resonance force microscopy of model polymer nanocomposites

We present experimental results on the use of quantitative contact resonance force microscopy (CR-FM) for mapping the planar location and depth of 50?nm diameter silica nanoparticles buried beneath polystyrene films 30-165?nm thick. The presence of shallowly buried nanoparticles, with stiffness greater than that of the surrounding matrix, is shown to locally affect the surface contact stiffness of a material for all depths investigated. To achieve the necessary stiffness sensitivity, the CR-FM measurements are obtained utilizing the fifth contact eigenmode. Stiffness contrast is found to increase rapidly with initial increases in force, but plateaus at higher loads. Over the explored depth range, stiffness contrast spans roughly one order of magnitude, suggesting good depth differentiation. Scatter in the stiffness contrast for single images reveals nonuniformities in the model samples that can be explained by particle size dispersity. Finite element analysis is used to simulate the significant effect particle size can have on contact stiffness contrast. Finally, we show how measurements at a range of forces may be used to deconvolve particle size effects from depth effects.     

High-resolution and large dynamic range nanomechanical mapping in tapping-mode atomic force microscopy

High spatial resolution imaging of material properties is an important task for the continued development of nanomaterials and studies of biological systems. Time-varying interaction forces between the vibrating tip and the sample in a tapping-mode atomic force microscope contain detailed information about the elastic, adhesive, and dissipative response of the sample. We report real-time measurement and analysis of the time-varying tip-sample interaction forces with recently introduced torsional harmonic cantilevers. With these measurements, high-resolution maps of elastic modulus, adhesion force, energy dissipation, and topography are generated simultaneously in a single scan. With peak tapping forces as low as 0.6?nN, we demonstrate measurements on blended polymers and self-assembled molecular architectures with feature sizes at 1, 10, and 500?nm. We also observed an elastic modulus measurement range of four orders of magnitude (1?MPa to 10?GPa) for a single cantilever under identical feedback conditions, which can be particularly useful for analyzing heterogeneous samples with largely different material components.     

Precise control of nanofabrication by atomic force microscopy

With an aim of the precise control of the anodic oxidation process by atomic force microscopy, the technical improvement has been carried out based on the mechanism studies. The accuracy and reliability of the nanofabrication have been improved by the combination of ambient humidity control, improvement of instrumental performance and meniscus lifetime control. In parallel, the mechanism study has been proceeded through the detection of Faradaic current. The in situ Faradaic current detection of the nano-oxidation process can actually work as a sensitive monitor for the nano-oxidation process with a high reliability. From an engineering viewpoint with an eye to practical applications, controllable physical parameters which affect on the product size are enumerated to consider what we should do to raise the precision of nano-oxidation. Then the fast fabrication in a large area by a patchwork method, Faradaic current detection during oxidation-reduction reaction, and nanofabrication by current-control are shown as examples.     

压膜阻尼对原子力显微镜振动特性的影响研究

原子力显微镜(Atomic Force Microscope, AFM)在轻敲模式下工作时,随着探针针尖与样品距离的逐渐减小,空气压膜阻尼的作用随之增大。为研究压膜阻尼对原子力显微镜振动系统的影响,分别使用无针尖探针和微球针尖探针进行扫频实验,并基于振动理论将该过程简化,得到了两种不同的振动模型的系统刚度。在考虑压膜阻尼作用影响后,将微球针尖振动系统模型进一步简化为一维振子模型,并对压膜阻尼的影响进行讨论。实验表明空气压膜阻尼模型对于探针样品在微尺度下的作用过程是准确合理的。该结果对原子力显微镜轻敲模式研究具有重要意义。     

Nanofabrication with atomic force microscopy

Atomic force microscopy (AFM) was developed in 1986. It is an important and versatile surface technique, and is used in many research fields. In this review, we have summarized the methods and applications of AFM, with emphasis on nanofabrication. AFM is capable of visualizing surface properties at high spatial resolution and determining biomolecular interaction as well as fabricating nanostructures. Recently, AFM-based nanotechnologies such as nanomanipulation, force lithography, nanografting, nanooxidation and dip-pen nanolithography were developed rapidly. AFM tip (typical radius ranged from several nanometers to tens of nanometers) is used to modify the sample surface, either physically or chemically, at nanometer scale. Nanopatterns composed of semiconductors, metal, biomolecules, polymers, etc., were constructed with various AFM-based nanotechnologies, thus making AFM a promising technique for nanofabrication. AFM-based nanotechnologies have potential applications in nanoelectronics, bioanalysis, biosensors, actuators and high-density data storage devices.     

Dielectric constants by multifrequency non-contact atomic force microscopy

We present a method to obtain capacitive forces and dielectric constants of ultra-thin films on metallic substrates using multifrequency non-contact atomic force microscopy with amplitude feedback in air. Capacitive forces are measured via cantilever oscillations induced at the second bending mode and dielectric constants are calculated by fitting an analytic expression for the capacitance (Casuso et al 2007 Appl. Phys. Lett. 91 063111) to the experimental data. Dielectric constants for self-assembled monolayers of thiol molecules on gold (2.0±0.1) and sputtered SiO2 (3.6±0.07) were obtained under dry conditions, in good agreement with previous measurements. The high Q-factor of the second bending mode of the cantilever increases the accuracy of the capacitive measurements while the low applied potentials minimize the likelihood of variation of the dielectric constants at high field strength and of damage from dielectric breakdown of air.     

Specific aptamer-protein interaction studied by atomic force microscopy

Aptamers are a new class of synthetic DNA/RNA oligonucleotides generated from in vitro selection to selectively bind with various molecules. Due to their molecular recognition capability for proteins, aptamers are becoming promising reagents in protein detection and new drug development. In this study, the specific interaction between the protein immunoglobulin E (IgE) and its 37-nt aptamer has been measured directly by atomic force microscopy. The single-molecule unbinding force between IgE and the aptamer is determined using the Poisson statistical method. The individual unbinding force between IgE and its monoclonal antibody has also been obtained and compared to that between IgE and the aptamer. The results reveal the high affinity of the aptamer to protein, which could match or even surpass that of the antibody to its antigen.     

Nanodissection of single- and double-stranded DNA by atomic force microscopy

Nanodissection of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) has been investigated by atomic force microscopy (AFM). It is found that both ss- and dsDNA can be repeatedly dissected by an AFM tip. However, a comparison study indicates that ssDNA is a little bit more easily broken by the AFM tip than dsDNA. This is supported by the fact that the time requested to break ssDNA is shorter than that of dsDNA in the same dissection procedure under the same load. Our experiment also shows that dissection of the DNA strand is very sensitive to the load applied, and a small change of the load lead to different results.