Membrane-based actuation for high-speed single molecule force spectroscopy studies using AFM

Atomic force microscopy (AFM)-based dynamic force spectroscopy of single molecular interactions involves characterizing unbinding/unfolding force distributions over a range of pulling speeds. Owing to their size and stiffness, AFM cantilevers are adversely affected by hydrodynamic forces, especially at pulling speeds >10 μm/s, when the viscous drag becomes comparable to the unbinding/unfolding forces. To circumvent these adverse effects, we have fabricated polymer-based membranes capable of actuating commercial AFM cantilevers at speeds ≥100 μm/s with minimal viscous drag effects. We have used FLUENT^®, a computational fluid dynamics (CFD) software, to simulate high-speed pulling and fast actuation of AFM cantilevers and membranes in different experimental configurations. The simulation results support the experimental findings on a variety of commercial AFM cantilevers and predict significant reduction in drag forces when membrane actuators are used. Unbinding force experiments involving human antibodies using these membranes demonstrate that it is possible to achieve bond loading rates ≥10⁶ pN/s, an order of magnitude greater than that reported with commercial AFM cantilevers and systems.     

Thermo-mechanical responses of a surface-coupled AFM cantilever

Atomic force microscopy (AFM) has been widely used for measuring mechanical properties of biological specimens such as cells, DNA, and proteins. This is usually done by monitoring deformations in response to controlled applied forces, which have to be at ultralow levels due to the extreme softness of the specimens. Consequently, such experiments may be susceptible to thermal excitations, manifested as force and displacement fluctuations that could reduce the measurement accuracy. To take advantage of, rather than to be limited by, such fluctuations, we have characterized the thermomechanical responses of an arbitrarily shaped AFM cantilever with the tip coupled to an elastic spring. Our analysis shows that the cantilever and the specimen behave as springs in parallel. This provides a method for determining the elasticity of the specimen by measuring the change in the tip fluctuations in the presence and absence of coupling. For rectangular and V-shaped cantilevers, we have derived a relationship between the mean-square deflection and the mean-square inclination and an approximate expression for the specimen spring constant in terms of contributions to the mean-square inclination from the first few vibration modes.     

Large colloidal probes for atomic force microscopy: Fabrication and calibration issues

Atomic force microscopy (AFM) is a powerful tool to investigate interaction forces at the micro and nanoscale. Cantilever stiffness, dimensions and geometry of the tip can be chosen according to the requirements of the specific application, in terms of spatial resolution and force sensitivity. Colloidal probes (CPs), obtained by attaching a spherical particle to a tipless (TL) cantilever, offer several advantages for accurate force measurements: tunable and well‐characterisable radius; higher averaging capabilities (at the expense of spatial resolution) and sensitivity to weak interactions; a well‐defined interaction geometry (sphere on flat), which allows accurate and reliable data fitting by means of analytical models. The dynamics of standard AFM probes has been widely investigated, and protocols have been developed for the calibration of the cantilever spring constant. Nevertheless, the dynamics of CPs, and in particular of large CPs, with radius well above 10 μm and mass comparable, or larger, than the cantilever mass, is at present still poorly characterized. Here we describe the fabrication and calibration of (large) CPs. We describe and discuss the peculiar dynamical behaviour of CPs, and present an alternative protocol for the accurate calibration of the spring constant.     

A general and efficient cantilever functionalization technique for AFM molecular recognition studies

Atomic force microscopy (AFM) is a versatile technique for the investigation of noncovalent molecular associations between ligand–substrate pairs. Surface modification of silicon nitride AFM cantilevers is most commonly achieved using organic trialkoxysilanes. However, susceptibility of the Si? O bond to hydrolysis and formation of polymeric aggregates diminishes attractiveness of this method for AFM studies. Attachment techniques that facilitate immobilization of a wide variety of organic and biological molecules via the stable Si? C bond on silicon nitride cantilevers would be of great value to the field of molecular recognition force spectroscopy. Here, we report (1) the formation of stable, highly oriented monolayers on the tip of silicon nitride cantilevers and (2) demonstrate their utility in the investigation of noncovalent protein–ligand interactions using molecular recognition force spectroscopy. The monolayers are formed through hydrosilylation of hydrogen‐terminated silicon nitride AFM probes using a protected α‐amino‐ω‐alkene. This approach facilitates the subsequent conjugation of biomolecules. The resulting biomolecules are bound to the tip by a strong Si? C bond, completely uniform with regard to both epitope density and substrate orientation, and highly suitable for force microscopy studies. We show that this attachment technique can be used to measure the unbinding profiles of tip‐immobilized lactose and surface‐immobilized galectin‐3. Overall, the proposed technique is general, operationally simple, and can be expanded to anchor a wide variety of epitopes to a silicon nitride cantilever using a stable Si? C bond. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 761–765, 2012.     

Surface viscoelasticity of individual gram-negative bacterial cells measured using atomic force microscopy

The cell envelope of gram-negative bacteria is responsible for many important biological functions: it plays a structural role, it accommodates the selective transfer of material across the cell wall, it undergoes changes made necessary by growth and division, and it transfers information about the environment into the cell. Thus, an accurate quantification of cell mechanical properties is required not only to understand physiological processes but also to help elucidate the relationship between cell surface structure and function. We have used a novel, atomic force microscopy (AFM)-based approach to probe the mechanical properties of single bacterial cells by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of the AFM tip due to the viscoelastic properties of the cell. For these experiments, we chose a representative gram-negative bacterium, Pseudomonas aeruginosa PAO1, and we used regular V-shaped AFM cantilevers with pyramid-shaped and colloidal tips. We find that the cell response is well described by a three-element mechanical model which describes an effective cell spring constant, k(1), and an effective time constant, tau, for the creep deformation. Adding glutaraldehyde, an agent that increases the covalent bonding of the cell surface, produced a significant increase in k(1) together with a significant decrease in tau. This work represents a new attempt toward the understanding of the nanomechanical properties of single bacteria while they are under fluid conditions, which could be of practical value for elucidating, for instance, the biomechanical effects of drugs (such as antibiotics) on pathogens.     

The distribution of sugar chains on the vomeronasal epithelium observed with an atomic force microscope

The distribution of sugar chains on tissue sections of the rat vomeronasal epithelium, and the adhesive force between the sugar and its specific lectin were examined with an atomic force microscope (AFM). AFM tips were modified with a lectin, Vicia villosa agglutinin, which recognizes terminal N-acetyl-D-galactosamine (GalNAc). When a modified tip scanned the luminal surface of the sensory epithelium, adhesive interactions between the tip and the sample surface were observed. The final rupture force was calculated to be approximately 50 pN based on the spring constant of the AFM cantilever. Distribution patterns of sugar chains obtained from the force mapping image were very similar to those observed using fluorescence-labeled lectin staining. AFM also revealed distribution patterns of sugar chains at a higher resolution than those obtained with fluorescence microscopy. Most of the adhesive interactions disappeared when the scanning solution contained 1 mM GaINAc. The adhesive interactions were restored by removing the sugar from the solution. Findings suggest that the adhesion force observed are related to the binding force between the lectin and the sugars distributed across the vomeronasal epithelium.     

Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp

To understand the role of physical forces at a cellular level, it is necessary to track mechanical properties during cellular processes. Here we present a protocol that uses flat atomic force microscopy (AFM) cantilevers clamped at constant height, and light microscopy to measure the resistance force, mechanical stress and volume of globular animal cells under compression. We describe the AFM and cantilever setup, live cell culture in the AFM, how to ensure stability of AFM measurements during medium perfusion, integration of optical microscopy to measure parameters such as volume and track intracellular dynamics, and interpretation of the physical parameters measured. Although we use this protocol on trypsinized interphase and mitotic HeLa cells, it can also be applied to other cells with a relatively globular shape, especially animal cells in a low-adhesive environment. After a short setup phase, the protocol can be used to investigate approximately one cell per hour.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods

The measurement of viscoelasticity of cells in physiological environments with high spatio-temporal resolution is a key goal in cell mechanobiology. Traditionally only the elastic properties have been measured from quasi-static force-distance curves using the atomic force microscope (AFM). Recently, dynamic AFM-based methods have been proposed to map the local in vitro viscoelastic properties of living cells with nanoscale resolution. However, the differences in viscoelastic properties estimated from such dynamic and traditional quasi-static techniques are poorly understood. In this work we quantitatively reconstruct the local force and dissipation gradients (viscoelasticity) on live fibroblast cells in buffer solutions using Lorentz force excited cantilevers and present a careful comparison between mechanical properties (local stiffness and damping) extracted using dynamic and quasi-static force spectroscopy methods. The results highlight the dependence of measured viscoelastic properties on both the frequency at which the chosen technique operates as well as the interactions with subcellular components beyond certain indentation depth, both of which are responsible for differences between the viscoelasticity property maps acquired using the dynamic AFM method against the quasi-static measurements.     

Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods

The measurement of viscoelasticity of cells in physiological environments with high spatio-temporal resolution is a key goal in cell mechanobiology. Traditionally only the elastic properties have been measured from quasi-static force-distance curves using the atomic force microscope (AFM). Recently, dynamic AFM-based methods have been proposed to map the local in vitro viscoelastic properties of living cells with nanoscale resolution. However, the differences in viscoelastic properties estimated from such dynamic and traditional quasi-static techniques are poorly understood. In this work we quantitatively reconstruct the local force and dissipation gradients (viscoelasticity) on live fibroblast cells in buffer solutions using Lorentz force excited cantilevers and present a careful comparison between mechanical properties (local stiffness and damping) extracted using dynamic and quasi-static force spectroscopy methods. The results highlight the dependence of measured viscoelastic properties on both the frequency at which the chosen technique operates as well as the interactions with subcellular components beyond certain indentation depth, both of which are responsible for differences between the viscoelasticity property maps acquired using the dynamic AFM method against the quasi-static measurements.     

Nonspecific interactions in AFM force spectroscopy measurements

Sample-probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand-receptor interaction. A prolonged contact time may be required to initiate ligand-receptor binding as a result of slow on-rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate-probe contact, we investigated the interaction of bovine serum albumin (BSA)-functionalized cantilever with BSA-coated glass, polyethylene glycol (PEG)-functionalized glass, Pluronic-treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA-functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion.     

Application of atomic force microscopy for characteristics of single intermolecular interactions

Atomic force microscopy (AFM) can be used to make measurements in vacuum, air, and water. The method is able to gather information about intermolecular interaction forces at the level of single molecules. This review encompasses experimental and theoretical data on the characterization of ligand-receptor interactions by AFM. The advantage of AFM in comparison with other methods developed for the characterization of single molecular interactions is its ability to estimate not only rupture forces, but also thermodynamic and kinetic parameters of the rupture of a complex. The specific features of force spectroscopy applied to ligand-receptor interactions are examined in this review from the stage of the modification of the substrate and the cantilever up to the processing and interpretation of the data. We show the specificities of the statistical analysis of the array of data based on the results of AFM measurements, and we discuss transformation of data into thermodynamic and kinetic parameters (kinetic dissociation constant, Gibbs free energy, enthalpy, and entropy). Particular attention is paid to the study of polyvalent interactions, where the definition of the constants is hampered due to the complex stoichiometry of the reactions.     

Nanoscale investigation of pathogenic microbial adhesion to a biomaterial

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate. The two model microbes used in this study, Candida parapsilosis (ATCC 90018) and Pseudomonas aeruginosa (ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting. Attractive interactions exist between C. parapsilosis and both unmodified silicone rubber and P. aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 +/- 0.25 nN in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force decreases to 2.0 +/- 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface. These data suggest that C. parapsilosis may adhere to both silicone rubber and P. aeruginosa biofilms, possibly contributing to patient morbidity and mortality. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection.     

The NTA-His6 bond is strong enough for AFM single-molecular recognition studies

There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site-directed nitrilotriacetic acid (NTA)-polyhistidine (Hisn) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA-Hisn bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA-terminated alkanethiols and solid supports functionalized with His6-Gly-Cys peptides in the presence of Ni2+. The force histogram obtained at a loading rate of 6600 pN s(-1) showed three maxima at rupture forces of 153 +/- 57 pN, 316 +/- 50 pN and 468 +/- 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50-100 pN unbinding forces typically observed by AFM for receptor-ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off-rate constant of dissociation, k(off) approximately 0.07 s(-1). This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA-His6 bond. We conclude that the NTA-His6 system is a powerful, well-suited platform for the stable, oriented immobilization of proteins in AFM single-molecule studies.     

Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding

Single-molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has emerged as an important tool for probing biomolecular interaction and exploring the forces, dynamics, and energy landscapes that underlie function and specificity of molecular interaction. These studies require attaching biomolecules on solid supports and AFM tips to measure unbinding forces between individual binding partners. Herein we describe efficient and robust protocols for probing RNA interaction by AFM and show their value on two well-known RNA regulators, the Rev-responsive element (RRE) from the HIV-1 genome and an adenine-sensing riboswitch. The results show the great potential of AFM–SMFS in the investigation of RNA molecular interactions, which will contribute to the development of bionanodevices sensing single RNA molecules.     

Lateral mechanical coupling of stereocilia in cochlear hair bundles

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).     

Mechanical force analysis of peptide interactions using atomic force microscopy

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.     

Biomolecular force measurements and the atomic force microscope

The atomic force microscope (AFM) is a surface-sensitive instrument capable of imaging biological samples at nanometer resolution in all environments including liquids. The sensitivity of the AFM cantilever, to forces in the pico Newton range, has been exploited to measure breakaway forces between biomolecules and to measure folding-unfolding forces within single proteins. By attaching specific antibodies to cantilevers the simultaneous imaging of target antigens and identification of antigen-antibody interactions have been demonstrated.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.     

Unfolding and extraction of a transmembrane alpha-helical peptide: dynamic force spectroscopy and molecular dynamics simulations

An atomic force microscope (AFM) was used to visualize CWALP(19)23 peptides ((+)H(3)N-ACAGAWWLALALALALALALWWA-COO(-)) inserted in gel-phase DPPC and DSPC bilayers. The peptides assemble in stable linear structures and domains. A model for the organization of the peptides is given from AFM images and a 20 ns molecular dynamics (MD) simulation. Gold-coated AFM cantilevers were used to extract single peptides from the bilayer through covalent bonding to the cystein residue. Experimental and simulated force curves show two distinct force maxima. In the simulations these two maxima correspond to the extraction of the two pairs of tryptophan residues from the membrane. Unfolding of the peptide precedes extraction of the second distal set of tryptophans. To probe the energies involved, AFM force curves were obtained from 10 to 10(4) nm/s and MD force curves were simulated with 10(8)-10(11) nm/s pulling velocities (V). The velocity relationship with the force, F, was fitted to two fluctuation adhesive potential models. The first assumes the pulling produces a constant bias in the potential and predicts an F approximately ln (V) relationship. The second takes into account the ramped bias that the linker feels as it is being driven out of the adhesion complex and scales as F approximately (ln V)2/3.