In situ single-molecule detection of antibody-antigen binding by tapping-mode atomic force microscopy

We performed in situ detection of specific and nonspecific binding during immunoreaction on surfaces at the same location before and after analyte was injected using tapping-mode atomic force microscopy (TM-AFM) in liquid and demonstrated the ability of TM-AFM to monitor the occurrence of single-molecule binding events and to distinguish nonspecific from specific binding by examining topographical change. Two antigen/antibody pairs were investigated: chorionic gonadotropin (hCG)/mouse monoclonal anti-hCG and goat IgG (anti-intact hCG)/ mouse monoclonal anti-goat IgG. Antibody (or antigen) molecules were covalently immobilized on uniform mixed self-assembled monolayers (SAMs) terminated with carboxylic acid and hydroxyl groups. Mixed SAMs allow the control of the density of immobilized antibody (or antigen) on surfaces to achieve the detection of individual antigens, antibodies, and antigen/antibody complexes. This in situ TM-AFM-based detection method allows the single-molecule detection of antigen/antibody binding under near-physiological environment and the distinction of nonspecific from specific binding. It could be extended into a microarray.     

In situ tensile testing of nanofibers by combining atomic force microscopy and scanning electron microscopy

A nanomechanical testing set-up is developed by integrating an atomic force microscope (AFM) for force measurements with a scanning electron microscope (SEM) to provide imaging capabilities. Electrospun nanofibers of polyvinyl alcohol (PVA), nylon-6 and biological mineralized collagen fibrils (MCFs) from antler bone were manipulated and tensile-tested using the AFM-SEM set-up. The complete stress-strain behavior to failure of individual nanofibers was recorded and a diversity of mechanical properties observed, highlighting how this technique is able to elucidate mechanical behavior due to structural composition at nanometer length scales.     

Monitoring DNA immobilization and hybridization on surfaces by atomic force microscopy force measurements

DNA immobilization and hybridization was carried out on Au substrates that were modified with mercaptopropanoic acid and then treated with aluminum(III) solution. The positively charged AI(III) film can be used to immobilize both ds-DNA and ss-DNA. Atomic force microscopy (AFM) was used to monitor the process by force measurements between a negatively charged silica tip and the substrates while immersed in dilute electrolyte. Surface hybridization of ss-DNA produces an increase in the surface charge and surface potential of the substrates, which is reflected by the increasing repulsive force as determined from AFM force-separation curves. A single-base mismatch was detectable in surface hybridization. The AFM force measuring technique was also employed to investigate the interaction of Ru(phen)3(2+) with ss-DNA and ds-DNA. The force measurement results showed that there is a small interaction between Ru(phen)3(2+) and ss-DNA, which was ascribed to the electrostatic binding of Ru(phen)3(2+) to the ss-DNA surface. For ds-DNA, there is a strong interaction which is believed to be due to the association or intercalation of Ru(phen)3(2+) with ds-DNA.     

An atomic force microscopy study of polyester surfaces

The surface properties of amorphous and crystalline polyester films, well below their glass transition temperature, have been studied with an atomic force microscope. For amorphous films a corrugated pattern develops on the surface as a result of scanning and the corrugations are always perpendicular to the scan direction. When scanning is stopped the pattern shows a slight relaxation; however, the surface is plastically deformed. When crystalline films are scanned, similar patterns are seen which are less pronounced and require a much longer scan time. These results suggest that the physical properties of a glassy polyester surface may be different from the bulk, and the freedom of macromolecules is reduced upon crystallization, thus suppressing molecular motion at the surface.     

Direct evidence of lipid rafts by in situ atomic force microscopy

Lipid rafts are membrane microdomains enriched with cholesterol, glycosphingolipids, and proteins. Although they are broadly presumed to play a pivotal role in various cellular functions, there are still fierce debates about the composition, functions, and even existence of lipid rafts. Here high-resolution and time-lapse in situ atomic force microscopy is used to directly confirm the existence of lipid rafts in native erythrocyte membranes. The results indicate some important aspects of lipid rafts: most of the lipid rafts are in the size range of 100-300 nm and have irregular shape; the detergent-resistant membranes consist of cholesterol microdomains and are not likely the same as the lipid rafts; cholesterol contributes significantly to the formation and stability of the protein domains; and Band III is an important protein of lipid rafts in the inner leaflet of erythrocyte membranes, indicating that lipid rafts are exactly the functional domains in plasma membrane. This work provides direct evidence of the presence, size, and main constitutive protein of lipid rafts at a resolution of a few nanometers, which will pave the way for studying their structure and functions in detail.     

In situ comparative studies of self-assembly adsorption of bovine serum albumin on nano films by atomic force microscopy

The self-assembly adsorption of function protein on crystal surfaces, as a common phenomenon, broadly takes place in many applications of biosensors, biocapsules and bioMEMS/bioNEMS. To systematically investigate the different adsorption characteristic of the same function protein on two different crystal surfaces under the identical environment, a hybrid surface composing silica and discontinuous Gold Nano Film (GNF) was fabricated by Physical Vapor Deposition (PVD) and ultrasonic cleaning method, where the dynamic process of the self-assembly adsorption of Bovine Serum Albumin (BSA) was in situ observed by Atomic Force Microscope (AFM). The variations on the junction area of the two different surfaces were studied in the aqueous solution before and after injecting BSA with 0.05 mg/ml concentration. It was found that silica, compared with same hydrophilic GNF, took on a fairly weak adsorption force. The results indicated that the adsorption strength of BSA on the hydrophilic crystal surface was determined not only by hydrophilic property, but also other interaction forces, like Van der Waals and so on. Moreover, observed under the contact mode of AFM, BSA adsorbed on GNF had great tendency to forming a ridge-like topography. These results may be helpful in the application of immunosensors and other areas.     

In situ atomic force microscopy tip-induced deformations and Raman spectroscopy characterization of single-wall carbon nanotubes

In this work, an atomic force microscope (AFM) is combined with a confocal Raman spectroscopy setup to follow in situ the evolution of the G-band feature of isolated single-wall carbon nanotubes (SWNTs) under transverse deformation. The SWNTs are pressed by a gold AFM tip against the substrate where they are sitting. From eight deformed SWNTs, five exhibit an overall decrease in the Raman signal intensity, while three exhibit vibrational changes related to the circumferential symmetry breaking. Our results reveal chirality dependent effects, which are averaged out in SWNT bundle measurements, including a previously elusive mode symmetry breaking that is here explored using molecular dynamics calculations.     

Direct observation of isolated ultrananodimensional diamond clusters using atomic force microscopy

Isolated ultrananodimensional diamond (UND) particles obtained by means of detonation synthesis have been studied using atomic force microscopy (AFM). The UND particles were deposited onto the surface of highly oriented pyrolytic graphite from a suspension based on organic compounds. The deposited UND particles were deaggregated using a two-stage treatment with ultrasound and high-dynamic-pressure pulses. The isolated UND particles were stabilized in suspension by a benzene additive. AFM images of individual UND particles have been obtained, and the phenomenon of their alignment along atomic steps on the substrate surface has been observed.     

Preparation and atomic force microscopy observation of lipid membranes on micro-patterned self-assembled monolayers

Using micro-contact printing (μCP) method, lipid membranes were deposited on the surface of micro-patterned self-assembled monolayers. Height and phase mode atomic force microscopy (AFM) images showed that the resulting deposited films were flat and that a micro-patterned composite bilayer system was constructed. It was also shown that the use of crystalline phase lipid membrane is effective for the preparation of the micro-patterned composite bilayer system of membranes with flat surface.     

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.     

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.     

Direct measurement of interaction force between hydrophilic silica surfaces in triblock copolymer solutions with salt by atomic force microscopy

Triblock copolymers composed of polyethylene oxide (PEO) and polypropylene oxide (PPO) are used in various fields as nonionic surfactants. In this study, we measured interaction forces between untreated hydrophilic silica surfaces in solutions with two typical triblock copolymers, Pluronic P123 (PEO₂₀PPO₇₀PEO₂₀) and F127 (PEO₉₉PPO₆₅PEO₉₉), in the presence of 1 mM and 500 mM NaCl using atomic force microscopy (AFM). In solutions at the copolymer concentration of 1 µM, which is below the critical micelle concentration (CMC), the measured interaction forces were monotonically repulsive in the presence of 1 mM NaCl, which suggested the brush-like conformation of copolymers on the surfaces. When the concentration of NaCl was increased to 500 mM, interaction forces became attractive, which indicated the bridging of adsorbed polymers onto surfaces, the strength of which varied depending on the affinity and adsorption density of copolymers. The interactions at the copolymer concentration of 1 mM, which were above the CMC of both copolymers, were steric repulsions between adsorbed micelles on the surfaces with 1 mM of NaCl. For 500 mM of NaCl, an attractive jump after a steric repulsion was observed only in the force curve for P123, which inferred that the displacement of micelles from the surfaces was presumably due to a decrease in the strength of adsorption caused by the dehydration of EO groups. These results indicated that the length of the EO group considerably affected the interactions.     

Study of the surfaces of CVD-WO3 films, by atomic force microscopy and spectroscopic ellipsometry

Atomic force microscopy imaging of chemical vapor deposition WO₃ films reveals the presence of domed crystallites that resemble the florets of cauliflowers with a rough surface texture. Annealing at 400 °C and above leads to further surface roughening, with estimated root mean square roughness values of 40–50 nm. Spectroscopic ellipsometry analysis shows that the surface layer becomes thicker with increasing oxygen flow rate during film deposition. This layer is predominantly amorphous for as-deposited films, and predominantly crystalline after annealing.     

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.     

Models of atomic scale contrast in dissipation images of binary ionic surfaces in non-contact atomic force microscopy

Using model ionic systems and the recently proposed theory of dynamical response at close approach (Kantorovich and Trevethan 2004 Phys.?Rev.?Lett. 93 236102) in non-contact atomic force microscopy (NC-AFM), we present the results of calculations performed to investigate the formation of atomic scale contrast in dissipation images. The accessible energy states and barriers of the microscopic tip-surface system are determined as a function of tip position above the surface. These are then used along with typical experimental parameters to investigate the dynamical response of the system and mechanisms of atomic scale contrast. We show how the damping signal contrast can appear either correlated or anti-correlated with the topography depending on the distance of closest approach and the system temperature. The dependence of the dissipated energy, and the reversibility of a structural change, on the tip frequency and system temperature is investigated and the relevance of this to single-atom manipulation with the NC-AFM is discussed.     

Non-contact atomic force microscopy study of atomic manipulation on an insulator surface by nanoindentation

Experimental results on vertical manipulation on an insulator surface using non-contact atomic force microscopy are presented. Cleaved ionic KCl(100) single crystal is used as an insulator surface. With the nanoindentation method used, the vertical manipulation of a single atom in an ionic crystal surface is more difficult than in a semiconductor surface. Therefore, in many cases, more than one surface atom is manipulated while, in rare cases, single-atom manipulation is successfully performed. Lateral manipulation of a vacancy has occasionally succeeded on the KCl(100) surface. We have presumed that the lateral manipulation was induced by pulling.