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Mechanical forces play a vital role in biological processes at molecular and cellular levels, significantly impacting various diseases such as cancer, cardiovascular disease, and COVID-19. Recent advancements in dynamic force spectroscopy (DFS) techniques have enabled the application and measurement of forces and displacements with high resolutions, providing crucial insights into the mechanical pathways underlying these diseases. Among DFS techniques, the biomembrane force probe (BFP) stands out for its ability to measure bond kinetics and cellular mechanosensing with pico-newton and nano-meter resolutions. Here, a comprehensive overview of the classical BFP-DFS setup is presented and key advancements are emphasized, including the development of dual biomembrane force probe (dBFP) and fluorescence biomembrane force probe (fBFP). BFP-DFS allows us to investigate dynamic bond behaviors on living cells and significantly enhances the understanding of specific ligand-receptor axes mediated cell mechanosensing. The contributions of BFP-DFS to the fields of cancer biology, thrombosis, and inflammation are delved into, exploring its potential to elucidate novel therapeutic discoveries. Furthermore, future BFP upgrades aimed at improving output and feasibility are anticipated, emphasizing its growing importance in the field of cell mechanobiology. Although BFP-DFS remains a niche research modality, its impact on the expanding field of cell mechanobiology is immense. [ABSTRACT FROM AUTHOR]

Abstract Mechanical forces play a vital role in biological processes at molecular and cellular levels, significantly impacting various diseases such as cancer, cardiovascular disease, and COVID‐19. Recent advancements in dynamic force spectroscopy (DFS) techniques have enabled the application and measurement of forces and displacements with high resolutions, providing crucial insights into the mechanical pathways underlying these diseases. Among DFS techniques, the biomembrane force probe (BFP) stands out for its ability to measure bond kinetics and cellular mechanosensing with pico‐newton and nano‐meter resolutions. Here, a comprehensive overview of the classical BFP‐DFS setup is presented and key advancements are emphasized, including the development of dual biomembrane force probe (dBFP) and fluorescence biomembrane force probe (fBFP). BFP‐DFS allows us to investigate dynamic bond behaviors on living cells and significantly enhances the understanding of specific ligand‐receptor axes mediated cell mechanosensing. The contributions of BFP‐DFS to the fields of cancer biology, thrombosis, and inflammation are delved into, exploring its potential to elucidate novel therapeutic discoveries. Furthermore, future BFP upgrades aimed at improving output and feasibility are anticipated, emphasizing its growing importance in the field of cell mechanobiology. Although BFP‐DFS remains a niche research modality, its impact on the expanding field of cell mechanobiology is immense.

The dark generation of reactive oxygen species (ROS), particularly hydroxyl radicals (·OH), is crucial in the oxidative transformation of various pollutants. However, the mechanisms behind this process are predominantly linked to direct O 2 activation by reduced substances such as Fe(II) and natural organic matter. In this study, we introduce a previously overlooked dual-ligand mechanism that significantly amplifies ·OH generation on iron oxyhydroxides, facilitated by cysteine and pyrophosphate. Our findings reveal that these ligands collaboratively boost ·OH generation by 99.5-125.7% compared to Fe(II) alone. This enhancement occurs through a two-step electron transfer (ET) process, where cysteine transfers electrons to O 2 through iron oxyhydroxides. The complexation of pyrophosphate with iron oxyhydroxides further reduces the thermodynamic barriers, notably promoting this ET process and significantly improving the electron utilization efficiency for O 2 activation by the electron donor cysteine. Such a process has shown its great potential for effectively driving the oxidative transformation of various pollutants, including As(III), dichlorophenol, and carbamazepine. These findings offer valuable insights for nature-based pollutant mitigation in soil and subsurface environments.

Oriented attachment of nanocrystalline subunits is recognized as a common crystallization pathway that is closely related to formation of nanoparticle superlattices, mesocrystals, and other kinetically stabilized structures. Approaching particles have been observed to rotate to achieve coalignment while separated by nanometer-scale solvent layers. Little is known about the forces that drive coalignment, particularly in this "solvent-separated" regime. To obtain a mechanistic understanding of this process, we used atomic-force-microscopy-based dynamic force spectroscopy with tips fabricated from oriented mica to measure the adhesion forces between mica (001) surfaces in electrolyte solutions as a function of orientation, temperature, electrolyte type, and electrolyte concentration. The results reveal an ∼60° periodicity as well as a complex dependence on electrolyte concentration and temperature. A continuum model that considers the competition between electrostatic repulsion and van der Waals attraction, augmented by microscopic details that include surface separation, water structure, ion hydration, and charge regulation at the interface, qualitatively reproduces the observed trends and implies that dispersion forces are responsible for establishing coalignment in the solvent-separated state.

Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand–receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability. [ABSTRACT FROM AUTHOR]

Cells sense and respond to mechanical stimuli for activation, proliferation, migration, and differentiation. The associated mechanosensing and biomechanical properties of cells and tissues are significantly implicated in the context of cancer, fibrosis, dementia, and cardiovascular diseases. To gain more mechanobiology insights, dynamic force spectroscopies (DFSs), particularly optical tweezers (OT), have been further advanced to enable in situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside of a cell. In this review, we first explain the classic OT-DFS rationales and discuss their applications to protein biophysics, extracellular biomechanics, and receptor-mediated cell mechanosensing. As a non-invasive technique, optical tweezers’s unique advantages in probing cytoplasmic protein behaviors and manipulating organelles inside living cells have been increasingly explored in recent years. Hereby, we then introduce and highlight the emerging OT rationales for intracellular force measurement including refractive index matching, active–passive calibration, and change of light momentum. These new approaches enable intracellular OT-DFS and mechanical measurements with respect to intracellular motor stepping, cytosolic micro-rheology, and biomechanics of irregularly shaped nuclei and vesicles. Last but not least, we foresee future OT upgrades with respect to overcoming phototoxicity and system drifting for longer duration live-cell measurements; multimodal integration with advanced imaging and nanotechnology to obtain higher spatiotemporal resolution; and developing simultaneous, automated, and artificial intelligence–inspired multi-trap systems to achieve high throughput. These further developments will grant unprecedented accessibility of OT-DFS and force measurement nanotools to a wider biomedical research community, ultimately opening the floodgates for exciting live-cell mechanobiology and novel therapeutic discoveries.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry. [ABSTRACT FROM AUTHOR]

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force–distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell–Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme.

Heterogeneous nucleation induced by natural organic matter (NOM) can lower the energy barrier for calcium arsenate (Ca-As) precipitation, which aids in immobilizing arsenate (AsⅤ). However, it remains unclear how certain chemical functionalities of NOM affect Ca-As nucleation at the molecular scale. By analyzing changes in the local supersaturation and/or interfacial energy, the present work investigates the Ca-As heterogeneous nucleation kinetics and mechanisms on functional-group–modified model surfaces. Mica surfaces modified by functional groups of amine (NH 2), hydroxyl (OH), or carboxyl (COOH) through self-assembled monolayers were used to investigate how chemical functionalities affect the Ca-As heterogeneous nucleation, in which the distributions of formation kinetics and size (as measured by the change in particle height) of nucleated Ca-As particles were measured by using in situ atomic force microscopy. In a parallel analysis, a quartz-crystal microbalance with dissipation was used to detect the buildup of Ca2+ and/or HAsO 4 2− ions at the solid–fluid interface. PeakForce quantitative nanomechanical mapping and dynamic force spectroscopy using functional-group–modified tips made it possible to calculate the binding energies holding functional groups to Ca-As particles. Nucleated Ca-As particles were characterized by using Raman spectroscopy and high-resolution transmission electron microscopy. The results indicate that the height of amorphous Ca-As particles formed on a modified mica surface may be ranked in descending order as NH 2 > OH > bare mica > COOH, as determined by the buildup of Ca2+ and HAsO 4 2− ions at the solid–fluid interface and the decrease of interfacial energy due to the functional groups. These nanoscale observations and molecular-scale determinations improve our understanding of the roles played by chemical functionalities on NOM in immobilizing dissolved As through heterogeneous nucleation in soil and water. [ABSTRACT FROM AUTHOR]

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF165 was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF165 was dependent on the concentration of VEGF165. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF165 complexes was revealed, which was similar to that of the intact aptamer/VEGF165. Besides, the dissociation rate constant (koff) of split aptamer/VEGF165 was close to that of intact aptamer/VEGF165, and the interaction force of split aptamer/VEGF165 was higher than the force of intact aptamer/VEGF165. It indicated that split aptamer also possessed high affinity with VEGF165. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level. [ABSTRACT FROM AUTHOR]

Experimental and computational approaches are utilized to investigate the influence of electrostatic fields on the binding force between human coagulation protein thrombin and its DNA aptamer. The thiolated aptamer was deposited onto gold substrate located in a liquid cell filled with binding buffer, then the thrombin-functionalized atomic force microscopy (AFM) probe was repeatedly brought into contact with the aptamer-coated surface under applied electrical potentials of −100, 0, and 100 mV respectively. Force drops during the pull-off process were measured to determine the unbinding forces between thrombin and aptamer in a range of loading rates spanning from ~3 × 102 to ~1 × 104 pN/s. The results from experiments showed that both of the binding strength and propensity of the complex are drastically diminished under positive electrode potential, whereas there is no influence on the molecular binding from negative electrode potential. We also used a theoretical analysis to explain the nature of electrostatic potential and field inside the aptamer-thrombin layer, which in turn could quantify the influence of the electrostatically repulsive force on a thrombin molecule that promotes dissociation from the aptamer due to positive electrode potential, and achieve good agreement with the experimental results. The study confirms the feasibility of electrostatic modulation upon the binding interaction between thrombin and aptamer, and implicates an underlying application perspective upon nanoscale manipulation of the stimuli responsive biointerface. [ABSTRACT FROM AUTHOR]

Xun Huang,1,2 Jiexiang He,2,3 Huan-tian Zhang,1 Kai Sun,2,3 Jie Yang,1 Huajun Wang,1 Hongxin Zhang,2,3 Zhenzhao Guo,2,3 Zhen-gang Zha,1 Changren Zhou2,31Department of Bone and Joint Surgery, The First Affiliated Hospital of Jinan University, 2Department of Materials Science and Engineering, Jinan University, 3Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou, ChinaAbstract: CD44 ligand–receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand–receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 µg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand–receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process.Keywords: atomic force microscopy, tumor, dynamic force spectroscopy, kinetic and thermodynamic interactions, affinity, nanoindentation

Cell surface receptors, often called transmembrane receptors, are key cellular components as they control and mediate cell communication and signalling, converting extracellular signals into intracellular signals. Elucidating the molecular details of ligand binding (cytokine, growth factors, hormones, pathogens,...) to cell surface receptors and how this binding triggers conformational changes that initiate intracellular signalling is needed to improve our understanding of cellular processes and for rational drug design. Unfortunately, the molecular complexity and high hydrophobicity of membrane proteins significantly hamper their structural and functional characterization in conditions mimicking their native environment. With its piconewton force sensitivity and (sub)nanometer spatial resolution, together with the capability of operating in liquid environment and at physiological temperature, atomic force microscopy (AFM) has proven to be one of the most powerful tools to image and quantify receptor-ligand bonds in situ under physiologically relevant conditions. In this article, a brief overview of the rapid evolution of AFM towards quantitative biological mapping will be given, followed by selected examples highlighting the main advances that AFM-based ligand-receptor studies have brought to the fields of cell biology, immunology, microbiology, and virology, along with future prospects and challenges. [ABSTRACT FROM AUTHOR]

Force plays critical roles in cell adhesion and mechano-signaling, partially by regulating the dissociation rate, i.e., off-rate, of receptor-ligand bonds. However, the mechanism of such regulation still remains elusive. As a controversial topic of the field, when measuring the "off-rate vs. force" relation of the same molecular system, different dynamic force spectroscopy (DFS) assays (namely, force-clamp and force-ramp assays) often yield contradictive results. Such discrepancies hurdled our further understanding of molecular binding, and casted doubt on the existing theoretical models. In this work, we used a live-cell DFS technique, biomembrane force probe, to measure the single-bond dissociation in three receptor-ligand systems which respectively have important functions in vascular and immune systems: human platelet GPIbα-VWF, mouse T cell receptor-OVA peptide:MHC, and mouse platelet integrin αIIbβ3-fibrinogen. Using force-clamp and force-ramp assays in parallel, we identified that the force loading disrupted the stability of molecular bonds in a rate-dependent manner. This disruptive effect was achieved by the transitioning of bonds between two dissociation states: faster force loading induces more bonds to adopt the fast-dissociating state (and less to adopt the slow-dissociating state). Based on this mechanism, a new biophysical model of bond dissociation was established which took into account the effects of both force magnitude and loading rate. Remarkably, this model reconciled the results from the two assays in all three molecular systems under study. Our discoveries provided a new paradigm for understanding how force regulates receptor-ligand interactions and a guideline for the proper use of DFS technologies. Furthermore, our work highlighted the opportunity of using different DFS assays to answer specific biological questions in the field of cell adhesion and mechano-signaling. [ABSTRACT FROM AUTHOR]

Receptor-mediated cell mechanosensing plays critical roles in cell spreading, migration, growth, and survival. Dynamic force spectroscopy (DFS) techniques have recently been advanced to visualize such processes, which allow the concurrent examination of molecular binding dynamics and cellular response to mechanical stimuli on single living cells. Notably, the live-cell DFS is able to manipulate the force "waveforms" such as tensile versus compressive, ramped versus clamped, static versus dynamic, and short versus long lasting forces, thereby deriving correlations of cellular responses with ligand binding kinetics and mechanical stimulation profiles. Here, by differentiating extracellular mechanical stimulations into two major categories, tensile force and compressive force, we review the latest findings on receptor-mediated mechanosensing mechanisms that are discovered by the state-of-the-art live-cell DFS technologies. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • DFS experiment and MD simulation are combined to study thrombin-aptamer binding. • Multiple factors affecting force measurement are comprehensively elucidated. • Enhanced analytical techniques are employed to determine dissociation force quanta. • Dissociation force of the complex is lower than that of G-quadruplex breakdown. Abstract Previous literature on dynamic force spectroscopy of the thrombin-aptamer complex has shown significantly different force magnitudes even at similar AFM tip velocities, implying the need for a more elaborate evaluation of the force interaction. In this study, we employ a combination of experimental and computational methods to reveal comprehensively how the unbinding force can possibly depend on the loading rate, coverage density, binding rate, linker stiffness and binding buffer composition. In addition, we utilize enhanced analytical techniques that include autocorrelation function, probability density convolution, and Poisson distribution to determine the dissociation force quanta of multiple bond dissociation from force spectroscopy experiments. We then predict the unbinding force measured in the experiments by using atomistic simulation data with significantly broader loading rate range following a continuum model. The dissociation forces of thrombin-aptamer complex are found to be lower than forces associated with the breakdown of G-quadruplex on aptamer, while comparable to forces associated with DNA melting, which indicates the complex dissociation could be more likely attributed to the bond breakage between thrombin and aptamer, rather than the collapse of G-quadruplex on aptamer. [ABSTRACT FROM AUTHOR]

Food safety is vital in everyday life. Food toxins are small peptides or proteins that can cause disease by disrupting biological macromolecules such as enzymes or cellular receptors. Toxins are introduced either externally or from internal, spoilage-related pathogens. To keep these unsafe foods off the market, detection techniques for various toxins have been devised. Although many techniques serve this purpose well, challenges remain regarding sensitivity, specificity, and cost, especially for trace amounts of highly lethal proteinaceous toxins. Atomic force microscopy (AFM) is a widely used nanotechnology for imaging and force measurement in biophysics and biology. Its high sensitivity in probing specific, single-molecule binding between molecules is applicable to food toxin detection. In this review, after a summary of the development of AFM, different detection operation modes are introduced along with examples of their applications. Cantilever sensing and recognition imaging are found to be the appropriate AFM techniques for detection. Their shared functionalization approaches are outlined for two categories of surfaces: silicon and gold. Recent progress in AFM biosensors and their applications to food toxin detection are discussed. Single-molecule sensitivity and ease of designing sensing schemes make these AFM techniques excellent candidates for real-world application. Existing challenges in designing sensing molecules and preventing the food matrix from confounding signals are not only applicable to AFM techniques but to most current biosensors. Through the collaboration among materials science, chemistry, and molecular biology, solving these issues will promote significant advancement in AFM-based food toxin detection. Besides different detection operation modes of AFM, a recognition element can be coupled to an AFM to produce an instrument that is capable of providing extraordinarily high sensitivity, high selectivity, and a cost-effective path toward recognizing these contaminants and securing the safety of the food supply. Image 1 • Accurate, trace detection of food toxins is critical for securing the food supply. • AFM's biosensing capabilities can sensitively and reliably detect food toxins. • Chemical functionalization of the AFM probe tip is straight-forward and flexible. [ABSTRACT FROM AUTHOR]

Magnetic tape heads are ubiquitously used to read and record on magnetic tapes in technologies as diverse as old VHS tapes, modern hard-drive disks, or magnetic bands on credit cards. Their design highlights the ability to convert electric signals into fluctuations of the magnetic field at very high frequencies, which is essential for the high-density storage demanded nowadays. Here, we twist this conventional use of tape heads to implement one in a magnetic tweezers design, which offers the unique capability of changing the force with a bandwidth of -10 kHz. We calibrate our instrument by developing an analytical expression that predicts the magnetic force acting on a superparamagnetic bead based on the Karlqvist approximation of the magnetic field created by a tape head. This theory is validated by measuring the force dependence of protein L unfolding/folding step sizes and the folding properties of the R3 talin domain. We demonstrate the potential of our instrument by carrying out millisecond-long quenches to capture the formation of the ephemeral molten globule state in protein L, which has never been observed before. Our instrument provides the capability of interrogating individual molecules under fast-changing forces with a control and resolution below a fraction of a piconewton, opening a range of force spectroscopy protocols to study protein dynamics under force. [ABSTRACT FROM AUTHOR]

A dream in powder technology is to predict the structure and flow of a powder from precise knowledge of the interactions between the particles. The most important interaction is adhesion. In this paper several aspects of adhesion are discussed. First, the influence of humidity and capillary forces is analyzed. To calculate capillary forces the structure of the microcontact needs to be known with an accuracy much better than 1 nm. Determining the structure of a microcontact with such resolution is demanding, if possible at all. Considering that wear can lead to a change of the atomic structure such knowledge is practically impossible. Second, the work to break an adhesive contact depends on the effective spring constant by which the force is applied. Third, adhesion forces depend on the separation speed and not only the surface chemistry and the structure of the particles in the contact region. Fourth, we suggest to distinguish between contact and bridging adhesion.

Oriented attachment of nanocrystalline subunits is recognized as a common crystallization pathway that is closely related to formation of nanoparticle superlattices, mesocrystals, and other kinetically stabilized structures. Approaching particles have been observed to rotate to achieve coalignment while separated by nanometer-scale solvent layers. Little is known about the forces that drive coalignment, particularly in this "solvent-separated" regime. To obtain a mechanistic understanding of this process, we used atomic-forcemicroscopy-based dynamic force spectroscopy with tips fabricated from oriented mica to measure the adhesion forces between mica (001) surfaces in electrolyte solutions as a function of orientation, temperature, electrolyte type, and electrolyte concentration. The results reveal an -60° periodicity as well as a complex dependence on electrolyte concentration and temperature. A continuum model that considers the competition between electrostatic repulsion and van der Waals attraction, augmented by microscopic details that include surface separation, water structure, ion hydration, and charge regulation at the interface, qualitatively reproduces the observed trends and implies that dispersion forces are responsible for establishing coalignment in the solvent-separated state. [ABSTRACT FROM AUTHOR]

Free-energy landscapes govern the behavior of all interactions in the presence of thermal fluctuations in the fields of physical chemistry, materials sciences, and the biological sciences. From the energy landscape, critical information about an interaction, such as the reaction kinetic rates, bond lifetimes, and the presence of intermediate states, can be determined. Despite the importance of energy landscapes to understanding reaction mechanisms, most experiments do not directly measure energy landscapes, particularly for interactions with steep force gradients that lead to premature jump to contact of the probe and insufficient sampling of transition regions. Here we present an atomic force microscopy (AFM) approach for measuring energy landscapes that increases sampling of strongly adhesive interactions by using white-noise excitation to enhance the cantilever’s thermal fluctuations. The enhanced fluctuations enable the recording of subtle deviations from a harmonic potential to accurately reconstruct interfacial energy landscapes with steep gradients. Comparing the measured energy landscape with adhesive force measurements reveals the existence of an optimal excitation voltage that enables the cantilever fluctuations to fully sample the shape and depth of the energy surface. [ABSTRACT FROM AUTHOR]

The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc–GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galβ1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galβ1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and β-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups. [ABSTRACT FROM AUTHOR]

In this paper, the strength and kinetics of two charge-transfer complexes, naphthol-methylviologen and pyrene-methylviologen, are studied using dynamic force spectroscopy. The dissociation rates indicate an enhanced stability of the pyrene-methylviologen complex, which agrees with its higher thermodynamic stability compared to naphthol-methylviologen complex.

Dynamic force spectroscopy (DFS) makes it possible to investigate specific interactions between two molecules such as ligand-receptor pairs at the single-molecule level. In the DFS method based on the Bell-Evans model, the unbinding force applied to a molecular bond is increased at a constant rate, and the force required to rupture the molecular bond is measured. By analyzing the relationship between the modal rupture force and the logarithm of the loading rate, microscopic potential barrier landscapes and the lifetimes of bonds can be obtained. However, the results obtained, for example, in the case of streptavidin/biotin complexes, have differed among previous studies and some results have been inconsistent with theoretical predictions. In this study, using an atomic force microscopy technique that enables the precise analysis of molecular interactions on the basis of DFS, we investigated the effect of the sampling rate on DFS analysis. The shape of rupture force histograms, for example, was significantly deformed at a sampling rate of 1 kHz in comparison with that of histograms obtained at 100 kHz, indicating the fundamental importance of ensuring suitable experimental conditions for further advances in the DFS method.

To understand and design molecular functions on the basis of molecular recognition processes, the microscopic probing of the energy landscapes of individual interactions in a molecular complex and their dependence on the surrounding conditions is of great importance. Dynamic force spectroscopy (DFS) is a technique that enables us to study the interaction between molecules at the single-molecule level. However, the obtained results differ among previous studies, which is considered to be caused by the differences in the measurement conditions. We have developed an atomic force microscopy technique that enables the precise analysis of molecular interactions on the basis of DFS. After verifying the performance of this technique, we carried out measurements to determine the landscapes of streptavidin-biotin interactions. The obtained results showed good agreement with theoretical predictions. Lifetimes were also well analyzed. Using a combination of cross-linkers and the atomic force microscope that we developed, site-selective measurement was carried out, and the steps involved in bonding due to microscopic interactions are discussed using the results obtained by site-selective analysis.

Viscoelastic fluids exhibit rheological nonlinearity at a high shear rate. Although typical nonlinear effects, shear thinning and shear thickening, have been usually understood by variation of intrinsic quantities such as viscosity, one still requires a better understanding of the microscopic origins, currently under debate, especially on the shear-thickening mechanism. We present accurate measurements of shear stress in the bound hydration water layer using noncontact dynamic force microscopy. We find shear thickening occurs above ~106 s-1 shear rate beyond 0.3-nm layer thickness, which is attributed to the nonviscous, elasticityassociated fluidic instability via fluctuation correlation. Such a nonlinear fluidic transition is observed due to the long relaxation time (~10-6 s) of water available in the nanoconfined hydration layer, which indicates the onset of elastic turbulence at nanoscale, elucidating the interplay between relaxation and shear motion, which also indicates the onset of elastic turbulence at nanoscale above a universal shear velocity of ~1mm/s. This extensive layer-by-layer control paves the way for fundamental studies of nonlinear nanorheology and nanoscale hydrodynamics, as well as provides novel insights on viscoelastic dynamics of interfacial water. [ABSTRACT FROM AUTHOR]

Multifrequency atomic force microscopy is a powerful nanoscale imaging and characterization technique that involves excitation of the atomic force microscope (AFM) probe and measurement of its response at multiple frequencies. This paper reports the design, fabrication, and evaluation of AFM probes with a specified set of torsional eigen-frequencies that facilitate enhancement of sensitivity in multifrequency AFM. A general approach is proposed to design the probes, which includes the design of their generic geometry, adoption of a simple lumped-parameter model, guidelines for determination of the initial dimensions, and an iterative scheme to obtain a probe with the specified eigen-frequencies. The proposed approach is employed to design a harmonic probe wherein the second and the third eigen-frequencies are the corresponding harmonics of the first eigen-frequency. The probe is subsequently fabricated and evaluated. The experimentally evaluated eigen-frequencies and associated mode shapes are shown to closely match the theoretical results. Finally, a simulation study is performed to demonstrate significant improvements in sensitivity to the second- and the third-harmonic spectral components of the tip–sample interaction force with the harmonic probe compared to that of a conventional probe. [ABSTRACT FROM PUBLISHER]

Mucins are linear, heavily O-glycosylated proteins with physiological roles that include cell signaling, cell adhesion, inflammation, immune response and tumorgenesis. Cancer-associated mucins often differ from normal mucins by presenting truncated carbohydrate chains. Characterization of the binding properties of mucins with truncated carbohydrate side chains could thus prove relevant for understanding their role in cancer mechanisms such as metastasis and recognition by the immune system. In this work, heterotypic interactions of model mucins that possess the Tn (GalNAcαThr/ Ser) and T (Galβ1-3GalNAcαThr/Ser) cancer antigens derived from porcine submaxillary mucin (PSM) were studied using atomic force microscopy. PSM possessing only the Tn antigen (Tn-PSM) was found to bind to PSM analogs possessing a combination of T, Tn and STn antigens as well as biosynthetic analogs of the core 1 blood group A tetrasaccharide (GalNAcα1-3[Fucα1-2] Galβ1-3GalNAcαSer/Thr). The rupture forces for the heterotypic interactions ranged from 18- to 31 pN at a force-loading rate of -0.5 nN/s. The thermally averaged distance from the bound complex to the transition state (xβ) was estimated to be in the range 0.37-0.87 nm for the first barrier of the Bell Evans analysis and within 0.34-0.64 nm based on a lifetime analysis. These findings reveal that the binding strength and energy landscape for heterotypic interactions of Tn-PSM with the above mucins, resemble homotypic interactions of Tn-PSM. This suggests common carbohydrate epitope interactions for the Tn cancer antigen with the above mucin analogs, a finding thatmay be important to the role of the Tn antigen in cancer cells. [ABSTRACT FROM AUTHOR]

The remarkable properties of bone derive from a highly organized arrangement of coaligned nanometer-scale apatite platelets within a fibrillar collagen matrix. The origin of this arrangement is poorly understood and the crystal structures of hydroxyapatite (HAP) and the nonmineralized collagen fibrils alone do not provide an explanation. Moreover, little is known about collagen-apatite interaction energies, which should strongly influence both the molecular-scale organization and the resulting mechanical properties of the composite.We investigated collagen-mineral interactions by combining dynamic force spectroscopy (DFS) measurements of binding energies with molecular dynamics (MD) simulations of binding and atomic force microscopy (AFM) observations of collagen adsorption on single crystals of calcium phosphate for four mineral phases of potential importance in bone formation. In all cases, we observe a strong preferential orientation of collagen binding, but comparison between the observed orientations and transmission electron microscopy (TEM) analyses of native tissues shows that only calcium-deficient apatite (CDAP) provides an interface with collagen that is consistent with both. MD simulations predict preferred collagen orientations that agree with observations, and results from both MD and DFS reveal large values for the binding energy due to multiple binding sites. These findings reconcile apparent contradictions inherent in a hydroxyapatite or carbonated apatite (CAP) model of bone mineral and provide an energetic rationale for the molecular-scale organization of bone. [ABSTRACT FROM AUTHOR]

Atomic force imaging and spectroscopy provide unique tools for investigating molecular interactions and dynamics in biomolecular and biomineral systems in situ. Herein, three recent examples of methods used to gain mechanistic insights into the self-assembly of protein matrices and biomolecular controls over mineral formation are reviewed. Studies of S-layer protein assembly reveal the complex nature of the nucleation and growth pathway, demonstrate the importance of kinetic traps in determining that pathway and provide quantification of the energy barriers controlling formation rates. Investigations of citrate and polypeptide modification of calcium oxalate monohydrate growth combined with molecular dynamics simulations (MD) demonstrate the importance of stereochemical matching at atomic steps on the crystal surface and establish a direct relationship between the step edge binding energies and shape modification. Measurements of step kinetics lead to detailed atomic-scale models that include both thermodynamic and kinetic effects, including time-dependent phenomena related to the multi-stage binding dynamics of polypeptide chains. Dynamic force spectroscopy measurements of binding between amelogenin peptide segments and hydroxyapatite (HAP) crystal faces, again combined with MD simulations, establish an energetic rationale for the observed c-axis elongation characteristic of HAP in tooth enamel, based on determinations of the peptide-HAP binding free energy. These examples demonstrate the deep level of understanding that can be obtained by applying in situ AFM imaging and force spectroscopy to biomolecular and biomineral systems. [ABSTRACT FROM AUTHOR]