Following aptamer-thrombin binding by force measurements

The rupture forces between an aptamer (1)-functionalized AFM tip and a thrombin-modified Au surface are analyzed. The rupture force for a single aptamer/thrombin complex is determined as approximately 4.45 pN. The analysis of the system reveals that the rupture forces correspond to the melting of the G-quadruplex structure of the aptamer bound to the thrombin. This melting of the G-quadruplex leads to the dissociation of the aptamer/thrombin complex.     

Cellular unbinding forces of initial adhesion processes on nanopatterned surfaces probed with magnetic tweezers

To study the dependence of unbinding forces on the distance of molecularly defined and integrin specific c(-RGDfK-) ligand patches in initial cellular adhesion processes, we developed a magnetic tweezers setup for applying vertical forces of up to 200 pN to rat embryonic fibroblasts. The ligand patch distance is controlled with a hexagonally close packed pattern of biofunctionalized gold nanoparticles prepared by block-copolymer micelle nanolithography. Each gold nanoparticle potentially activates up to one alpha(v)beta(3)-integrin. The distances between the gold nanoparticles determine the separation of individual integrins and thus the assembly of integrin clusters. The results show an increase in cellular unbinding forces from approximately 6 to more than 200 pN for a decreasing ligand distance of 145 to 58 nm after 5 min of cell adhesion. Furthermore, we observe a strong dependence on adhesion time during the first 10 min of cell surface contact suggesting an active, cooperative cell response that is controlled by the spacing between individually activated integrins.     

Contact mechanics modeling of pull-off measurements: effect of solvent,probe radius,and chemical binding probability on the detection of single-bond rupture forces by atomic force microscopy

Pull-off forces for chemically modified atomic force microscopy tips in contact with flat substrates coated with receptor molecules are calculated using a Johnson, Kendall, and Roberts contact mechanics model. The expression for the work of adhesion is modified to account for the formation of discrete numbers of chemical bonds (nBonds) between the tip and substrate. The model predicts that the pull-off force scales as nBonds(1/2), which differs from a common assumption that the pull-off force scales linearly with nBonds. Periodic peak progressions are observed in histograms generated from hundreds of computed pull-off forces. The histogram periodicity is the signature of discrete chemical interactions between the tip and substrate and allows estimation of single-bond rupture forces. The effects of solvent, probe tip radius, and chemical binding probability on the detection of single-bond forces are examined systematically. A dimensionless parameter, the effective force resolution, is introduced that serves as a quantitative predictor for determining when periodicity in force histograms can occur. The output of model is compared to recent experimental results involving tips and substrates modified with self-assembled monolayers. An advantage of this contact mechanics approach is that it allows straightforward estimation of solvent effects on pull-off forces.     

The assessment of particle friction of a drug substance and a drug carrier substance

The centrifuge technique, which has been previously used in adhesion experiments, has been modified for use in single particle friction studies. Both flat compacted surfaces and large single particles were used as substrate surfaces to allow assessment of drug-drug, drug-drug carrier and drug carrier-drug carrier friction forces. Particle size, particle shape and surface roughness were identified as main factors influencing the change from a static into a dynamic friction process and the division between friction due to adhesion and ploughing. The forces of adhesion and friction were found to be proportional to the reversible energy of adhesion. The ratio between the force of adhesion and the press-on force applied and the ratio between the force of friction and the press-on force can be related to the yield stress and the reduced Young's modulus of the materials in contact.     

Adhesion force approximation at varying consolidation stresses for fine powder under humid conditions

Interparticle adhesion forces in fine powders are greatly influenced by varying relative humidity (RH) conditions. The present study estimated the interparticle adhesion forces developed in corn starch powder under humid conditions at varying applied consolidation stresses using tensile strength determination approach. Shear test was used to determine tensile strength of powder at 1–9 kPa consolidation pressures and extrapolated values of tensile strength at zero stress were used for force estimation in non-consolidated powders. A strong dependence of interparticle adhesion force on consolidation and RH conditions was observed, mainly due to alteration in the number of adhesive contacts and contact area. The results indicated that, at low consolidation and high RH, capillary force is the prevailing force contributing to the total interparticle adhesion in contrast to higher consolidation conditions where load induced contact force plays a dominant role. Furthermore, for nonconsolidated samples, the adhesion forces registered a steep jump above 60% RH which was primarily attributed to dominance of the liquid bridge forces. Also, forces determined from tensile strength approach and those predicted theoretically, as a summation of individual forces, yielded a similar trend. Overall, a simple and effective approach for interparticle force estimation of consolidated as well as loosely packed powders under varying humidity conditions is presented here.     

Piconewton‐Scale Analysis of Ras‐BRaf Signal Transduction with Single‐Molecule Force Spectroscopy

Intermolecular interactions dominate the behavior of signal transduction in various physiological and pathological cell processes, yet assessing these interactions remains a challenging task. Here, this study reports a single‐molecule force spectroscopic method that enables functional delineation of two interaction sites (≈35 pN and ≈90 pN) between signaling effectors Ras and BRaf in the canonical mitogen‐activated protein kinase (MAPK) pathway. This analysis reveals mutations on BRaf at Q257 and A246, two sites frequently linked to cardio‐faciocutaneous syndrome, result in ≈10?30 pN alterations in Ras? BRaf intermolecular binding force. The magnitude of changes in Ras? BRaf binding force correlates with the size of alterations in protein affinity and in α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐sensitive glutamate receptor (‐R)‐mediated synaptic transmission in neurons expressing replacement BRaf mutants, and predicts the extent of learning impairments in animals expressing replacement BRaf mutants. These results establish single‐molecule force spectroscopy as an effective platform for evaluating the piconewton‐level interaction of signaling molecules and predicting the behavior outcome of signal transduction.     

Surface deflection of a microtubule loaded by a concentrated radial force

Microtubules are hollow cylindrical filaments of a eukaryotic cytoskeleton which are sensitive to externally applied radial forces due to their low circumferential elastic modulus. In this work, an orthotropic elastic shell model for microtubules is used to study the surface radial deflection of a microtubule loaded by a concentrated radial force generated by either a single molecular motor or a radial indentation tip. Our results show that the maximum surface radial deflection of a microtubule generated by a concentrated radial force of a few pN can be as large as a few nanometers (a significant fraction of the radius of microtubules), which could cause significant surface morphological non-uniformity of the microtubule. In contrast, radial indentation under a much larger compressive force, which can be as large as a few hundreds of pN, will cause hardening of the circumferential elastic modulus almost equal to the longitudinal modulus of microtubules. In this case, our results show that a microtubule can withstand a concentrated radial compressive force as large as a few hundreds of pN, with a maximum radial deflection not more than a few nanometers, in good agreement with recent experiments on radial indentation of microtubules. These results offer useful data and new insights into the basic understanding of elastic interaction between microtubules and molecular motors and radial indentation of microtubules.     

Biocompatible force sensor with optical readout and dimensions of 6 nm3

We have developed a nanoscopic force sensor with optical readout. The sensor consists of a single-stranded DNA oligomer flanked by two dyes. The DNA acts as a nonlinear spring: when the spring is stretched, the distance between the two dyes increases, resulting in reduced F?rster resonance energy transfer. The sensor was calibrated between 0 and 20 pN using a combined magnetic tweezers/single-molecule fluorescence microscope. We show that it is possible to tune the sensor's force response by varying the interdye spacing and that the FRET efficiency of the sensors decreases with increasing force. We demonstrate the usefulness of these sensors by using them to measure the forces internal to a single polymer molecule, a small DNA loop. Partial conversion of the single-stranded DNA loop to a double-stranded form results in the accumulation of strain: a force of approximately 6 pN was measured in the loop upon hybridization. The sensors should allow measurement of forces internal to various materials, including programmable DNA self-assemblies, polymer meshes, and DNA-based machines.     

Evaluation of interaction between histidine binding Cu2+ ion and histidine by atomic force microscopy

This paper presents a direct interaction force measurement between histidine molecules using AFM force-distance curve measurement. AFM force-distance curves between the histidine-modified cantilever and substrate in the different conditions with or without intercalating Cu2+ ion were measured and interpreted via Gaussian curve fitting analyses. The adhesion force between histidine molecules was shown to be 110 pN under the presence of Cu2+. The result was compareable to the measured adhesion force about 0 pN, which was measured by the removal of Cu2+ ion with the addition of EDTA. The result indicated the direct histidine-histidie interaction was difficult without the role of the bridigible ionic component. From the results, the possibility of direct measurement on chemical affinities between biomolecules was suggested by using AFM force-distance curve analyses. Especially, the current approach showed the possible affinity measurement techniques that elucidate the role of bridge ions.     

Whole-cell sensing for a harmful bloom-forming microscopic alga by measuring antibody--antigen forces

Aureococcus anophagefferens, a harmful bloom-forming alga responsible for brown tides in estuaries of the Middle Atlantic U.S., has been investigated by atomic force microscopy for the first time, using probes functionalized with a monoclonal antibody specific for the alga. The rupture force between a single monoclonal antibody and the surface of A. anophagefferens was experimentally found to be 246$pm$11 pN at the load rate of 12 nN/s. Force histograms for A. anophagefferens and other similarly-sized algae are presented and analyzed. The results illustrate the effects of load rates, and demonstrate that force-distance measurements can be used to build biosensors with high signal-to-noise ratios for A. anophagefferens. The methods described in this paper can be used, in principle, to construct sensors with single-cell resolution for arbitrary cells for which monoclonal antibodies are available.     

Quantitative modeling of forces in electromagnetic tweezers

This paper discusses numerical simulations of the magnetic field produced by an electromagnet for generation of forces on superparamagnetic microspheres used in manipulation of single molecules or cells. Single molecule force spectroscopy based on magnetic tweezers can be used in applications that require parallel readout of biopolymer stretching or biomolecular binding. The magnetic tweezers exert forces on the surface-immobilized macromolecule by pulling a magnetic bead attached to the free end of the molecule in the direction of the field gradient. In a typical force spectroscopy experiment, the pulling forces can range between subpiconewton to tens of piconewtons. In order to effectively provide such forces, an understanding of the source of the magnetic field is required as the first step in the design of force spectroscopy systems. In this study, we use a numerical technique, the method of auxiliary sources, to investigate the influence of electromagnet geometry and material parameters of the magnetic core on the magnetic forces pulling the target beads in the area of interest. The close proximity of the area of interest to the magnet body results in deviations from intuitive relations between magnet size and pulling force, as well as in the force decay with distance. We discuss the benefits and drawbacks of various geometric modifications affecting the magnitude and spatial distribution of forces achievable with an electromagnet.     

Stability of thin solid films

A small amplitude perturbation analysis is used to determine the conditions under which a solid film several hundred ångströms thick on a substrate will rupture. If the perturbation grows with time the film is unstable and rupture may occur, whereas if the perturbation decays the film is stable. Film rupture is caused essentially by diffusion of atoms along the free interface of the film which can, under certain conditions, amplify a perturbation applied to the film-gas interface. This surface diffusion is generated by a gradient of the chemical potential along the free interface. The chemical potential is affected by the curvature of the interface, by the pre-existing internal stresses normally found in thin films (they generate a strain energy term in the chemical potential) and by interaction forces between the atoms at the gas-solid interface with those of the film and substrate. The thin film is assumed to behave like an elastic body. The difference in the forces which act on a volume element in a film thinner than the range of interaction forces between the atoms of the film and substrate and the forces in a bulk solid is accounted for by introducing a body force into the equations of displacement of an elastic solid. Because of the difficulties in writing boundary conditions at the film-substrate interface, two limiting situations are considered: (1) a thin film on a rigid substrate and (2) a thin free film. A critical internal stress necessary for rupture is identified. The time of rupture is estimated from the inverse of the maximum growth coefficient of the perturbation. The dominant wavelength corresponding to the maximum growth coefficient gives an idea as to the size of the islands formed through rupture.     

Probing polydopamine adhesion to protein and polymer films: microscopic and spectroscopic evaluation

Polydopamine has been found to be a biocompatible polymer capable of supporting cell growth and attachment, and to have antibacterial and antifouling properties. Together with its ease of manufacture and application, it ought to make an ideal biomaterial and function well as a coating for implants. In this paper, atomic force microscope was used to measure the adhesive forces between polymer-, protein- or polydopamine-coated surfaces and a silicon nitride or polydopamine-functionalised probes. Surfaces were further characterised by contact angle goniometry, and solutions by circular dichroism. Polydopamine was further characterised with infrared spectroscopy and Raman spectroscopy. It was found that polydopamine functionalisation of the atomic force microscope probe significantly reduced adhesion to all tested surfaces. For example, adhesion to mica fell from 0.27 ± 0.7 to 0.05 ± 0.01 nN nm^?1. The results suggest that polydopamine coatings are suitable to be used for a variety of biomedical applications.     

Molecular handles for the mechanical manipulation of single-membrane proteins in living cells

We have developed a procedure to selectively biotinylate a specific membrane protein, enabling its attachment to external force probes and thus allowing its mechanical manipulation within its native environment. Using potassium channels as model membrane proteins in oocytes, we have found that Maleimide-PEG3400-biotin is the crosslinker with highest conjugation selectivity and accessibility to external probes. Neutravidin-coated beads provide for directed attachment while avoiding nonspecific interactions with the cell. The technology was successfully tested by mechanical manipulation of biotinylated extracellular residues of channels in oocytes using an atomic force microscope under conditions which preserve function of the channels. Binding forces of /spl sim/80 pN at 100 nN/s were measured.     

AFM‐Untersuchungen zu adhäsiven Wechselwirkungen an einkristallinem Si und voroxidiertem SiC in Abhängigkeit von der relativen Luftfeuchte

AFM investigations of the adhesive interactions of single‐crystal Si and preoxidized SiC as a function of the relative humidity The chemical structure of untreated and thermally oxidized single‐crystal 6H‐SiC surfaces was analysed using Auger electron spectroscopy. The wetting behaviour of the surfaces by water was studied using “sessile drop” method, and measurement of the adhesion force between Si tips and sample surfaces was conducted using an atomic force microscope. At a low humidity the adhesion force depended on the annealing temperature used for preoxidation. This was primarily attributed to different Si₄C₄O₄ volume fractions in the amorphous, stoichiometric SiO₂ surface layers, which varied as a function of the preoxidation treatment. The decrease of the adhesion force with increasing humidity varied owing to the different wetting properties of the samples.     

Mechanics and chemistry: single molecule bond rupture forces correlate with molecular backbone structure

We simultaneously measure conductance and force across nanoscale junctions. A new, two-dimensional histogram technique is introduced to statistically extract bond rupture forces from a large data set of individual junction elongation traces. For the case of Au point contacts, we find a rupture force of 1.4 ± 0.2 nN, which is in good agreement with previous measurements. We then study systematic trends for single gold metal-molecule-metal junctions for a series of molecules terminated with amine and pyridine linkers. For all molecules studied, single molecule junctions rupture at the Au-N bond. Selective binding of the linker group allows us to correlate the N-Au bond-rupture force to the molecular backbone. We find that the rupture force ranges from 0.8 nN for 4,4' bipyridine to 0.5 nN in 1,4 diaminobenzene. These experimental results are in excellent quantitative agreement with density functional theory based adiabatic molecular junction elongation and rupture calculations.     

High density single-molecule-bead arrays for parallel single molecule force spectroscopy

The assembly of a highly parallel force spectroscopy tool requires careful placement of single-molecule targets on the substrate and the deliberate manipulation of a multitude of force probes. Since the probe must approach the target biomolecule for covalent attachment, while avoiding irreversible adhesion to the substrate, the use of polymer microspheres as force probes to create the tethered bead array poses a problem. Therefore, the interactions between the force probe and the surface must be repulsive at very short distances (<5 nm) and attractive at long distances. To achieve this balance, the chemistry of the substrate, force probe, and solution must be tailored to control the probe-surface interactions. In addition to an appropriately designed chemistry, it is necessary to control the surface density of the target molecule in order to ensure that only one molecule is interrogated by a single force probe. We used gold-thiol chemistry to control both the substrate's surface chemistry and the spacing of the studied molecules, through binding of the thiol-terminated DNA and an inert thiol forming a blocking layer. For our single molecule array, we modeled the forces between the probe and the substrate using DLVO theory and measured their magnitude and direction with colloidal probe microscopy. The practicality of each system was tested using a probe binding assay to evaluate the proportion of the beads remaining adhered to the surface after application of force. We have translated the results specific for our system to general guiding principles for preparation of tethered bead arrays and demonstrated the ability of this system to produce a high yield of active force spectroscopy probes in a microwell substrate. This study outlines the characteristics of the chemistry needed to create such a force spectroscopy array.     

Nanoscale Mechanosensing of Natural Killer Cells is Revealed by Antigen‐Functionalized Nanowires

Cells sense their environment by transducing mechanical stimuli into biochemical signals. Commonly used tools to study cell mechanosensing provide limited spatial and force resolution. Here, a novel nanowire‐based platform for monitoring cell forces is reported. Nanowires are functionalized with ligands for cell immunoreceptors, and they are used to explore the mechanosensitivity of natural killer (NK) cells. In particular, it is found that NK cells apply centripetal forces to nanowires, and that the nanowires stimulate cell contraction. Based on the nanowire deformation, it is calculated that cells apply forces of down to 10 pN, which is the smallest value demonstrated so far by microstructured platforms for cell spreading. Furthermore, the roles of: i) nanowire topography and ii) activating ligands in the cell immune function are studied and it is found that only their combination produces enhanced population of activated NK cells. Thus, a mechanosensing mechanism of NK cells is proposed, by which they integrate biochemical and mechanical stimuli into a decision‐making machinery analogous to the AND logic gate, whose output is the immune activation. This work reveals unprecedented mechanical aspects of NK cell immune function and introduces an innovative nanomaterial for studying cellular mechanics with unparalleled spatial and mechanical resolution.     

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.     

Nanofork for single cells adhesion measurement via ESEM-nanomanipulator system

In this paper, single cells adhesion force was measured using a nanofork. The nanofork was used to pick up a single cell on a line array substrate inside an environmental scanning electron microscope (ESEM). The line array substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam (FIB) etching process while the line array substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line array substrate were used, i.e., 1 μm and 2 μm. Results showed that cells attached on the 1 μm gap line array substrate required more force to be released as compared to the cells attached on the 1 μm gap line array substrate.