Investigation of mechanical properties of insulin crystals by atomic force microscopy

Mechanical properties of protein crystals and aggregates depend on the conformational and structural properties of individual protein molecules as well as on the packing density and structure within solid materials. An atomic force microscopy (AFM)-based approach is developed to measure the elastic modulus of small protein crystals by nanoindentation and is applied to measure the elasticity of insulin crystals. The top face of the crystals deposited on mica substrates is identified as the (001) face. Insulin crystals exhibit a nearly elastic response during the compression cycle. The elastic modulus measured on the top face has asymmetric distribution with a significant width. This width is related to the uncertainty in the deflection sensitivity. A model that takes into account the distribution of the sensitivity values is used to correct the elastic modulus. Measurements performed in aqueous buffer on several crystals at different locations with three different AFM probes give a mean elastic modulus of 164 +/- 10 MPa. This value is close to the static elastic moduli of other protein crystals measured by different techniques that are usually measured in the range from 100 MPa to 1 GPa. The measured modulus of insulin crystals falls between the elastic modulus values of insulin amyloid fibrils measured previously at two orthogonal directions (a modulus of 14 MPa was measured by compressing the fibril in the direction perpendicular to the fibril axis, and a modulus of 3.3 GPa was measured in the direction along the fibril axis). This comparison indicates the heterogeneous structure of fibrils in the direction perpendicular to the fibril axis, with a packing density of the amyloid fibril core that is higher than the average packing density in insulin crystals. The mechanical wear of insulin crystals is detected during AFM measurements. In nanoindentation experiments on insulin crystal, the compressive load by the AFM tip ( approximately 1 nN, corresponding to a pressure of around 5 MPa) occasionally removes protein molecules from the top or the second top layer of insulin crystal in a sequential manner. The molecular model of this surface damage is proposed. In addition, the removal of the multiple layers of molecules is observed during the AC-mode imaging in aqueous buffer. The number of removed layers depends on the scan size.     

Morphology and surface States of colloidal probucol nanoparticles evaluated by atomic force microscopy

Morphology and surface states of colloidal probucol nanoparticles after dispersion of probucol/polyvinylpyrrolidone (PVP)/sodium dodecyl sulphate (SDS) ternary ground mixture into water were investigated by atomic force microscopy (AFM). The observed particles had core-shell structure, i.e. drug nanocrystals were covered with PVP and SDS complex. The AFM phase image and the force curve analyses indicated that probucol nanoparticles with PVP K17 showed layer structure, compared to those with PVPK12. The structural difference was explainable in terms of the molecular states of PVP-SDS complex on the particle surface. These findings support not only the mechanism of drug nanoparticle formation but also the in vivo absorption results with the almost same particle size of ca. 40 nm.     

Observation of crystal growth of lysozyme protein crystals by atomic force microscopy

Surface structure and the propagation of elementary growth layers over the (010) face of orthorhombic lysozyme crystal is examined at a molecular‐scale resolution by the method of atomic force microscopy (AFM). The steps have a small number of kinks spaced by about 150 growth units. The step motion occurs via successive deposition of rows of growth units. The data obtained are discussed in terms of the model of one‐dimensional nucleation.     

Interaction forces between metal-chelating lipid monolayers measured by colloidal probe atomic force microscopy

Surface forces between LB films of metal-chelating lipids in water have been studied using colloidal probe atomic force microscopy. The LB films of an amphiphile functionalized by the iminodiacetic acid group were prepared on hydrophobic glass substrates. The electric double layer repulsion operated between these LB film surfaces changed depending on pH reflecting the different protonation states of the iminodiacetic acid groups. The titration curve of the iminodiacetic acid monolayer was obtained from the force profiles. The Cu²⁺ complexation process was also monitored by measuring the force profiles at various Cu²⁺ ion concentrations.     

Isoelectric point of fluorite by direct force measurements using atomic force microscopy

Interaction forces between a fluorite (CaF2) surface and colloidal silica were measured by atomic force microscopy (AFM) in 1 x 10(-3) M NaNO3 at different pH values. Forces between the silica colloid and fluorite flat were measured at a range of pH values above the isoelectric point (IEP) of silica so that the forces were mainly controlled by the fluorite surface charge. In this way, the IEP of the fluorite surface was deduced from AFM force curves at pH approximately 9.2. Experimental force versus separation distance curves were in good agreement with theoretical predictions based on long-range electrostatic interactions, allowing the potential of the fluorite surface to be estimated from the experimental force curves. AFM-deduced surface potentials were generally lower than the published zeta potentials obtained from electrokinetic methods for powdered samples. Differences in methodology, orientation of the fluorite, surface carbonation, and equilibration time all could have contributed to this difference.     

Nanometre resolution using high-resolution scanning electron microscopy corroborated by atomic force microscopy

The resolving power of high-resolution scanning electron microscopy was judged using topographical height data from atomic force microscopy in order to assess the technique as a tool for understanding nanoporous crystal growth.     

Video microscopy of colloidal suspensions and colloidal crystals

Colloidal suspensions are simple model systems for the study of phase transitions. Video microscopy is capable of directly imaging the structure and dynamics of colloidal suspensions in different phases. Recent results related to crystallization, glasses, and 2D systems complement and extend previous theoretical and experimental studies. Moreover, new techniques allow the details of interactions between individual colloidal particles to be carefully measured. Understanding these details will be crucial for designing novel colloidal phases and new materials, and for manipulating colloidal suspensions for industrial uses.     

Generation of amino-terminated surfaces by chemical lithography using atomic force microscopy

Self-assembled monolayers (SAMs) covered with nitroso end groups were reduced using an atomic force microscope. As the bias voltage become more negative (beyond -4 V), the surface potential of the scanned area become closer to that of the amino-terminated SAM. Following this chemical change, however, no change in topographic features was detected, implying retained stability of the underlying SAM layer. We then released carboxylate-modified polystyrene (PS) spheres into a pH 4 solution containing the sample. Subsequent imaging with atomic force microscopy (AFM) revealed that these PS spheres were only selectively immobilized on the regions that were originally scanned at -6 V to form amino termination. In summary, using AFM set to a specific voltage, we were able to selectively generate micropatterned regions of the SAM with amino termination.     

Nano-mechanical methods in biochemistry using atomic force microscopy

The atomic force microscope has been extensively used not only to image nanometer-sized biological samples but also to measure their mechanical properties by using the force curve mode of the instrument. When the analysis based on the Hertz model of indentation is applied to the approach part of the force curve, one obtains information on the stiffness of the sample in terms of Young's modulus. Mapping of local stiffness over a single living cell is possible by this method. The retraction part of the force curve provides information on the adhesive interaction between the sample and the AFM tip. It is possible to functionalize the AFM tip with specific ligands so that one can target the adhesive interaction to specific pairs of ligands and receptors. The presence of specific receptors on the living cell surface has been mapped by this method. The force to break the co-operative 3D structure of globular proteins or to separate a double stranded DNA into single strands has been measured. Extension of the method for harvesting functional molecules from the cytosol or the cell surface for biochemical analysis has been reported. There is a need for the development of biochemical nano-analysis based on AFM technology.     

Measuring the size dependence of Young's modulus using force modulation atomic force microscopy

The dependence of the local Young's modulus of organic thin films on the size of the domains at the nanometer scale is systematically investigated. Using atomic force microscopy (AFM) based imaging and lithography, nanostructures with designed size, shape, and functionality are preengineered, e.g., nanostructures of octadecanethiols inlaid in decanethiol self-assembled monolayers (SAMs). These nanostructures are characterized using AFM, followed by force modulation spectroscopy and microscopy measurements. Young's modulus is then extracted from these measurements using a continuum mechanics model. The apparent Young's modulus is found to decrease nonlinearly with the decreasing size of these nanostructures. This systematic study presents conclusive evidence of the size dependence of elasticity in the nanoregime. The approach utilized may be applied to study the size-dependent behavior of various materials and other mechanical properties.     

Capillary force in atomic force microscopy

Under ambient conditions, a water meniscus generally forms between a nanoscale atomic force microscope tip and a hydrophilic surface. Using a lattice gas model for water and thermodynamic integration methods, we calculate the capillary force due to the water meniscus for both hydrophobic and hydrophilic tips at various humidities. As humidity rises, the pull-off force rapidly reaches a plateau value for a hydrophobic tip but monotonically increases for a weakly hydrophilic tip. For a strongly hydrophilic tip, the force increases at low humidities (<30%) and then decreases. We show that mean-field density functional theory reproduces the simulated pull-off force very well.     

Single-photon atomic force microscopy

In the last few years, an array of novel technologies, especially the big family of scanning probe microscopy, now often integrated with other powerful imaging tools such as laser confocal microscopy and total internal reflection fluorescence microscopy, have been widely applied in the investigation of biomolecular interactions and dynamics. But it is still a great challenge to directly monitor the dynamics of biomolecular interactions with high spatial and temporal resolution in living cells. An innovative method termed “single-photon atomic force microscopy” (SP-AFM), superior to existing techniques in tracing biomolecular interactions and dynamics in vivo, was proposed on the basis of the combination of atomic force microscopy with the technologies of carbon nanotubes and single-photon detection. As a unique tool, SP-AFM, capable of simultaneous topography imaging and molecular identification at the subnanometer level by synchronous acquisitions and analyses of the surface topography and fluorescent optical signals while scanning the sample, could play a very important role in exploring biomolecular interactions and dynamics in living cells or in a complicated biomolecular background.     

Noncontact atomic force microscopy of perfect single crystals of pentacene prepared by crystallization from solution

Nearly perfect single crystals of pentacene were grown from trichlorobenzene solution. The surface structure of pentacene single crystals has been investigated by frequency modulation atomic force microscopy. Molecularly flat and extraordinarily wide terraces, extended over the width of more than a few micrometers with monomolecular steps, were consistently observed, suggesting that those pentacene crystals were nearly perfect single crystals. Molecular packing arrangements were revealed by FM-AFM for the first time.     

Single molecule charging by atomic force microscopy

AFM/KPM charging and charge mapping of polyamine charge carriers in a PMMA matrix is reported. Selective charging of the designed charge carrier is demonstrated at concentrations down to a single molecule. This works constitutes electrochemical charging and detection of single redox-active organic molecules in low dielectric matrices by probe microscopy.     

Halo-like structures studied by atomic force microscopy

Nanometer-sized clusters of copper have been produced in a hollow cathode sputtering source and deposited on SiO_x. Halo-like structures consisting of micrometer sized protrusions in the silicon oxide surface surrounded by thin rings of smaller particles are observed. The area in between seems to be depleted of particles. We propose that the halo-like structures are a result of electrostatic forces acting between the incoming charged clusters and charged regions on the surface. A simple computer simulation supports this suggestion.     

Measuring molecular weight by atomic force microscopy

Absolute-molecular-weight distribution of cylindrical brush molecules were determined using a combination of the Langmuir Blodget (LB) technique and Atomic Force Microscopy (AFM). The LB technique gives mass density of a monolayer, i.e., mass per unit area, whereas visualization of individual molecules by AFM enables accurate measurements of the molecular density, i.e., number of molecules per unit area. From the ratio of the mass density to the molecular density, one can determine the absolute value for the number average molecular weight. Assuming that the structure of brush molecules is uniform along the backbone, the length distribution should be virtually identical to the molecular weight distribution. Although we used only brush molecules for demonstration purpose, this approach can be applied for a large variety of molecular and colloidal species that can be visualized by a microscopic technique.     

Nanoparticle coding: size-based assays using atomic force microscopy

Described herein is a novel strategy for the construction and interrogation of an assay platform based on (1) the size encoding of labeled nanoparticles; (2) the high imaging resolution of atomic force microscopy; and (3) evaporatively driven self-assembly of dense nanoparticle layers. This strategy employs two different sized nanoparticles that couple in the presence of a target analyte. In this example, one set of particles is a few hundred nanometers in size and acts as a capture substrate, while a second set of smaller particles serve as the analyte label. Thus, by forming an evaporatively assembled layer from a mixture of the two particle dispersions, the imaged size of the smaller particles when bound to the larger capture particles identifies the presence of the analyte. This letter demonstrates the feasibility of our bar-code strategy by concept tests using the binding specificity of biotin-modified silica nanoparticles (300-nm diameter) with streptavidin-labeled gold nanoparticles (10-nm diameter). The potential to extensively multiplex this assay strategy is briefly discussed.     

Stretching single polysaccharides and proteins using atomic force microscopy

The past years have witnessed remarkable advances in our use of atomic force microscopy (AFM) for stretching single biomolecules, thereby contributing to answering many outstanding questions in biophysics and chemical biology. In these single-molecule force spectroscopy (SMFS) experiments, the AFM tip is continuously approached to and retracted from the biological sample, while monitoring the interaction force. The obtained force-extension curves provide key insight into the molecular elasticity and localization of single molecules, either on isolated systems or on cellular surfaces. In this tutorial review, we describe the principle of such SMFS experiments, and we survey remarkable breakthroughs made in manipulating single polysaccharides and proteins, including understanding the conformational properties of sugars and controlling them by force, measuring the molecular elasticity of mechanical proteins, unfolding and refolding individual proteins, probing protein-ligand interactions, and tuning enzymatic reactions by force. In addition, we show how SMFS with AFM tips bearing specific bioligands has enabled researchers to stretch and localize single molecules on live cells, in relation with cellular functions.     

Characterization of elastomeric blends by atomic force microscopy

The bulk mechanical properties of a blend of elastomers are found to depend on the micro and nano scale morphology of the phases of the materials in the blend. In this study, we examine the phase morphology of blends of incompatible elastomers using Atomic Force Microscopy (AFM). Specifically, nanoindentation and Tapping Mode AFM (TMAFM) imaging techniques are used as experimental tools for mapping the composition of unfilled elastomeric blends. Depending on the composition of the blend, either co‐continuous or discontinuous domain/matrix morphology is observed. To identify the different components in bromobutyl (BIIR)/natural rubber (NR) blends, nanoscale indentation measurements were made on the observed phase‐separated regions. Results from force mode AFM and mechanical measurements of bulk NR and BIIR are used to assist in the interpretation of the TMAFM results for the BIIR/NR blends. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 492–503, 2006