Force Spectroscopy Methods Research Articles

Foldamer-Based Mechanoresponsive Materials: Molecular Nanoarchitectonics to Advanced Functions.

Artificial molecules that respond to external stimuli such as light, heat, chemical signals, and mechanical force have garnered significant interest due to their tunable functions, variable optical properties, and mechanical responses. Particularly, mechanoresponsive materials featuring molecules that respond to mechanical stress or show force-induced optical changes have been intriguing due to their extraordinary functions. Despite the promising potential of many such materials reported in the past, practical applications have remained limited, primarily because their functions often depend on irreversible covalent bond rupture. Foldamers, oligomers that fold into well-defined secondary structures, offer an alternative class of mechanoactive motifs. These molecules can reversibly sustain mechanical stress and efficiently dissipate energy by transitioning between folded and unfolded states. This review focuses on the emerging properties of foldamer-based mechanoresponsive materials. We begin by highlighting the mechanical responses of foldamers in their molecular form, which have been primarily investigated using single-molecule force spectroscopy and other analytical methods. Following this, we provide a detailed survey of the current trends in foldamer-appended polymers, emphasizing their emerging mechanical and mechanochromic properties. Subsequently, we present an overview of the state-of-the-art advancements in foldamer-appended polymers, showcasing significant reports in this field. This review covers some of the most recent advances in this direction and draws a perspective for further development.

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.

Получение частиц диоксида титана (рутила) на поверхности восстановленного оксида графена в сверхкритическом изопропаноле

Sequential synthesis of rutile modification titanium oxide particles on reduced graphene oxide in supercritical isopropanol is described. In this case, only graphene oxide was reduced to reduced graphene oxide. A one-stage method (one-pot) was also developed for the preparation of rutile particles on reduced graphene oxide, where supercritical isopropanol was the graphene oxide reducing agent and the reaction medium. The resulting composites were studied using X-ray phase analysis, transmission electron microscopy, and atomic force spectroscopy methods.

Получение наночастиц оксида титана на поверхности восстановленного оксида графена в сверхкритическом изопропаноле

Sequential synthesis of anatase modification titanium oxide nanoparticles on reduced graphene oxide in supercritical isopropanol is described. In this case, only graphene oxide was reduced to reduced graphene oxide. A one-stage method (one-pot) was also developed for the preparation of titanium oxide nanoparticles on reduced graphene oxide, where supercritical isopropanol was the graphene oxide reducing agent and the reaction medium. The resulting nanocomposites were studied using X-ray phase analysis, transmission electron microscopy, and atomic force spectroscopy methods.

Factors That Control the Force Needed to Unfold a Membrane Protein in Silico Depend on the Mode of Denaturation.

Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane proteins. To identify factors that determine the force rupture levels in force-induced membrane protein unfolding, we applied our near-atomic-level Upside molecular dynamics package to study the vertical and lateral pulling of bacteriorhodopsin (bR) and GlpG, respectively. With our algorithm, we were able to selectively alter the magnitudes of individual interaction terms and identify that, for vertical pulling, hydrogen bond strength had the strongest effect, whereas other non-bonded protein and membrane-protein interactions had only moderate influences, except for the extraction of the last helix where the membrane-protein interactions had a stronger influence. The up-down topology of the transmembrane helices caused helices to be pulled out as pairs. The rate-limiting rupture event often was the loss of H-bonds and the ejection of the first helix, which then propagated tension to the second helix, which rapidly exited the bilayer. The pulling of the charged linkers across the membrane had minimal influence, as did changing the bilayer thickness. For the lateral pulling of GlpG, the rate-limiting rupture corresponded to the separation of the helices within the membrane, with the H-bonds generally being broken only afterward. Beyond providing a detailed picture of the rupture events, our study emphasizes that the pulling mode greatly affects the factors that determine the forces needed to unfold a membrane protein.

High-low Kelvin probe force spectroscopy for measuring the interface state density.

The recently proposed high-low Kelvin probe force microscopy (KPFM) enables evaluation of the effects of semiconductor interface states with high spatial resolution using high and low AC bias frequencies compared with the cutoff frequency of the carrier transfer between the interface and bulk states. Information on the energy spectrum of the interface state density is important for actual semiconductor device evaluation, and there is a need to develop a method for obtaining such physical quantities. Here, we propose high-low Kelvin probe force spectroscopy (high-low KPFS), an electrostatic force spectroscopy method using high- and low-frequency AC bias voltages to measure the interface state density inside semiconductors. We derive an analytical expression for the electrostatic forces between a tip and a semiconductor sample in the accumulation, depletion, and inversion regions, taking into account the charge transfer between the bulk and interface states in semiconductors. We show that the analysis of electrostatic forces in the depletion region at high- and low-frequency AC bias voltages provides information about the interface state density in the semiconductor bandgap. As a preliminary experiment, high-low KPFS measurements were performed on ion-implanted silicon surfaces to confirm the dependence of the electrostatic force on the frequency of the AC bias voltage and obtain the interface state density.

Microfluidics-Based Force Spectroscopy Enables High-Throughput Force Experiments with Sub-Nanometer Resolution and Sub-Piconewton Sensitivity.

Several techniques have been established to quantify the mechanicals of single molecules. However, most of them show only limited capabilities of parallelizing the measurement by performing many individual measurements simultaneously. Herein, a microfluidics-based single-molecule force spectroscopy method, which achieves sub-nanometer spatial resolution and sub-piconewton sensitivity and is capable of simultaneously quantifying hundreds of single-molecule targets in parallel, is presented. It relies on a combination of total internal reflection microscopy and microfluidics, in which monodisperse fluorescent beads are immobilized on the bottom of a microfluidic channel by macromolecular linkers. Application of a flow generates a well-defined shear force acting on the beads, whereas the nanomechanical linker response is quantified based on the force-induced displacement of individual beads. To handle the high amount of data generated, a cluster analysis which is capable of a semi-automatic identification of measurement artifacts and molecular populations is implemented. The method is validated by probing the mechanical response polyethylene glycol linkers and binding strength of biotin-NeutrAvidin complexes. Two energy barriers (at 3 and 5.7 Å, respectively) in the biotin-NeutrAvidin interaction are resolved and the unfolding behavior of talin's rod domain R3 in the force range between 1 to ≈10 pN is probed.

Enthalpic and entropic contributions to the activation free energy of single noncovalent bonds in molecular systems: A computational methodology

Insight into enthalpic and entropic contributions to the activation free energy of noncovalent bonds is crucial for understanding various phenomena in molecular systems, from formation of different molecular conformers to complex biophysical and biochemical processes. Standard experimental and computational methods provide only the average strength of noncovalent bonds. In this paper, we present a simple, formal, computational framework to determine both energy contributions for a single bond. Our approach is based on steered molecular dynamics simulations, the dynamic force spectroscopy method, and a modified theoretical model of force-induced bond rupture. To demonstrate the methodology, the enthalpic and entropic components for a single intramolecular hydrogen bond of a peptide helix were determined. The sum of the contributions is in agreement with the previously reported Gibbs energy for intramolecular hydrogen bonds. The proposed methodological framework is universal and can be applied to other noncovalent interactions in various molecular systems.

Single-Molecule Force Probing of RGD-Binding Integrins on Pancreatic Cancer Cells.

Integrin-targeting arginine-glycine-aspartic acid (RGD)-based nanocarriers have been widely used for tumor imaging, monitoring of tumor development, and delivery of anticancer drugs. However, the thermodynamics of an RGD-integrin formation and dissociation associated with binding dynamics, affinity, and stability remains unclear. Here, we probed the binding strength of the binary complex to live pancreatic cancer cells using single-molecule binding force spectroscopy methods, in which RGD peptides were functionalized on a force probe tip through poly(ethylene glycol) (PEG)-based bifunctional linker molecules. While the density of integrin αV receptors on the cell surface varies more than twofold from cell line to cell line, the individual RGD-integrin complexes exhibited a cell type-independent, monovalent bond strength. The load-dependent bond strength of multivalent RGD-integrin interactions scaled sublinearly with increasing bond number, consistent with the noncooperative, parallel bond model. Furthermore, the multivalent bonds ruptured sequentially either by one or in multiples, and the force strength was comparable to the synchronous rupture force. Comparison of energy landscapes of the bond number revealed a substantial decrease of kinetic off-rates for multivalent bonds, along with the increased width of the potential well and the increased potential barrier height between bound and unbound states, enhancing the stability of the multivalent bonds between them.

A nanoscale DNA force spectrometer capable of applying tension and compression on biomolecules.

Single molecule force spectroscopy is a powerful approach to probe the structure, conformational changes, and kinetic properties of biological and synthetic macromolecules. However, common approaches to apply forces to biomolecules require expensive and cumbersome equipment and relatively large probes such as beads or cantilevers, which limits their use for many environments and makes integrating with other methods challenging. Furthermore, existing methods have key limitations such as an inability to apply compressive forces on single molecules. We report a nanoscale DNA force spectrometer (nDFS), which is based on a DNA origami hinge with tunable mechanical and dynamic properties. The angular free energy landscape of the nDFS can be engineered across a wide range through substitution of less than 5% of the strand components. We further incorporate a removable strut that enables reversible toggling of the nDFS between open and closed states to allow for actuated application of tensile and compressive forces. We demonstrate the ability to apply compressive forces by inducing a large bend in a 249bp DNA molecule, and tensile forces by inducing DNA unwrapping of a nucleosome sample. These results establish a versatile tool for force spectroscopy and robust methods for designing nanoscale mechanical devices with tunable force application.

AFM vs. Nanoindentation: Nanomechanical properties of organic-rich Shale

Obtaining elastic properties of organic-rich shales through conventional geomechanical testing could be challenging due to availability of good quality core plugs and significant heterogeneous nature of the samples. In this regard, force spectroscopy methods, nanoindentation and atomic force microscopy (AFM) are two main powerful techniques to characterize elastic properties in nano/microscale. In this study, we investigate the applicability of these two methods on the same samples, by quantifying elastic modulus from the Bakken Shale. AFM provided us with modulus maps of higher resolution compared to the modulus maps from the nanoindentation that were created via geostatistical methods. Moreover, results from these methods were compared to demonstrate the advantages and shortcomings of each and discripancy in the outcome. To do so, multi-cluster deconvolution approach was adopted in the statistical analysis on the nanoindentation data, demonstrating 3 separate clusters and mechanical phases. AFM technique, similarly, distinguished three separate (mineral and organic) phases based on the corresponding modulus values, though with higher accuracy compared to nanoindentation and better distinction and less tolerance. It was found that nanoindentation, because it collects discrete datapoints that are farther apart from each other when thermal maturity is increased in the samples, would have difficulty to separate organic matter from intermediary phases. Overall, the range of modulus for each phase was larger in the data that was obtained by nanoindentation compared to the AFM which can be interpreted to the size of the tip and general higher resolution in the later one which is expected to probe a single particle rather than an aggregate of particles.

Abstract 33: Characterizing the Changes in Mechanical Properties of the Actin Cortex in Highly Metastatic Cancers

Abstract Purpose: Cancer is the second leading cause of death globally with approximately 9.5 million cancer related deaths worldwide in 2018. Many of these deaths are the result of complications due to metastatic progression. Metastasis is a complex, dynamic process that requires cancerous cells to undergo major cytoskeletal reorganization prior to migration and intravasation. Previous research studying the biophysical properties of the cytoskeleton utilize techniques that require the cells be adhered to a hard substrate, causing modifications to the organization of the cytoskeleton that are not physiologically relevant. To better understand the mechanics of cancer cells and metastasis, we have developed an alternative approach to studying the cell cortex. Methods: Utilizing a novel, Atomic Force Microscopy-based, quasi-static force spectroscopy method pioneered by our lab, we are able to measure cortical tension and intracellular pressure by gently compressing non-adherent cells with a tipless cantilever, which results in a more physiologically relevant cortical morphology. We utilized this method to compare changes in cortical mechanics between healthy and cancerous cells, as well during metastatic progression of cancer cells in vitro for melanoma, breast, and ovarian cancers. Results: Previous research that utilized adherent cells indicated a decrease in stiffness after tumorigenesis, but our method indicates an increase in stiffness by determining the cortical tension and intracellular pressure in the case of melanoma. Additionally, a non-linear, overall decrease in tension and pressure can be seen in both the melanoma and ovarian cell lines that model increasing metastatic potential. Conclusion: Due to the important role invasiveness plays in the severity of cancer, categorizing the mechanical properties of cancer cells may prove to be a critical diagnostic and prognostic indicator for oncologists. Our novel force microscopy method better captures physiologically relevant cortical morphology and may enable the capture of clinically significant information on tumor invasiveness and progression. Citation Format: Jamie Neal, Chynna Smith, Michelle Baird, Alexander Cartagena-Rivera. Characterizing the Changes in Mechanical Properties of the Actin Cortex in Highly Metastatic Cancers [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 33.

Endothelial glycocalyx shields the interaction of SARS-CoV-2 spike protein with ACE2 receptors

Endothelial cells (ECs) play a crucial role in the development and propagation of the severe COVID-19 stage as well as multiorgan dysfunction. It remains, however, controversial whether COVID-19-induced endothelial injury is caused directly by the infection of ECs with SARS-CoV-2 or via indirect mechanisms. One of the major concerns is raised by the contradictory data supporting or denying the presence of ACE2, the SARS-CoV-2 binding receptor, on the EC surface. Here, we show that primary human pulmonary artery ECs possess ACE2 capable of interaction with the viral Spike protein (S-protein) and demonstrate the crucial role of the endothelial glycocalyx in the regulation of the S-protein binding to ACE2 on ECs. Using force spectroscopy method, we directly measured ACE2- and glycocalyx-dependent adhesive forces between S-protein and ECs and characterized the nanomechanical parameters of the cells exposed to S-protein. We revealed that the intact glycocalyx strongly binds S-protein but screens its interaction with ACE2. Reduction of glycocalyx layer exposes ACE2 receptors and promotes their interaction with S-protein. These results indicate that the susceptibility of ECs to COVID-19 infection may depend on the glycocalyx condition.

Dynamic cellular biomechanics in responses to chemotherapeutic drug in hypoxia probed by atomic force spectroscopy.

The changes in cellular structure play an important role in cancer cell development, progression, and metastasis. By exploiting single-cell, force spectroscopy methods, we probed biophysical and biomechanical kinetics (stiffness, morphology, roughness, adhesion) of brain, breast, prostate, and pancreatic cancer cells with standard chemotherapeutic drugs in normoxia and hypoxia over 12–24 hours. After exposure to the drugs, we found that brain, breast, and pancreatic cancer cells became approximately 55–75% less stiff, while prostate cancer cells became more stiff, due to either drug-induced disruption or reinforcement of cytoskeletal structure. However, the rate of the stiffness change decreased up to 2-folds in hypoxia, suggesting a correlation between cellular stiffness and drug resistance of cancer cells in hypoxic tumor microenvironment. Also, we observed significant changes in the cell body height, surface roughness, and cytoadhesion of cancer cells after exposure to drugs, which followed the trend of stiffness. Our results show that a degree of chemotherapeutic drug effects on biomechanical and biophysical properties of cancer cells is distinguishable in normoxia and hypoxia, which are correlated with alteration of cytoskeletal structure and integrity during drug-induced apoptotic process.

Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations

Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies.

Sugar-Pucker Force-Induced Transition in Single-Stranded DNA.

The accurate knowledge of the elastic properties of single-stranded DNA (ssDNA) is key to characterize the thermodynamics of molecular reactions that are studied by force spectroscopy methods where DNA is mechanically unfolded. Examples range from DNA hybridization, DNA ligand binding, DNA unwinding by helicases, etc. To date, ssDNA elasticity has been studied with different methods in molecules of varying sequence and contour length. A dispersion of results has been reported and the value of the persistence length has been found to be larger for shorter ssDNA molecules. We carried out pulling experiments with optical tweezers to characterize the elastic response of ssDNA over three orders of magnitude in length (60–14 k bases). By fitting the force-extension curves (FECs) to the Worm-Like Chain model we confirmed the above trend:the persistence length nearly doubles for the shortest molecule (60 b) with respect to the longest one (14 kb). We demonstrate that the observed trend is due to the different force regimes fitted for long and short molecules, which translates into two distinct elastic regimes at low and high forces. We interpret this behavior in terms of a force-induced sugar pucker conformational transition (C3′-endo to C2′-endo) upon pulling ssDNA.

Influence of fluoroethylene carbonate on the solid electrolyte interphase of silicon anode for Li-ion batteries: A scanning force spectroscopy study**Project supported by the State Grid Technology Project, China (Grant No. DG71-17-010).

Silicon is an important high capacity anode material for the next generation Li-ion batteries. The electrochemical performances of the Si anode are influenced strongly by the properties of the solid electrolyte interphase (SEI). It is well known that the addition of flouroethylene carbonate (FEC) in the carbonate electrolyte is helpful to improve the cyclic performance of the Si anode. The possible origin is suggested to relate to the modification of the SEI. However, detailed information is still absent. In this work, the structural and mechanical properties of the SEI on Si thin film anode in the ethylene-carbonate-based (EC-based) and FEC-based electrolytes at different discharging and charging states have been investigated using a scanning atomic force microscopy force spectroscopy (AFMFS) method. Single-layered, double-layered, and multi-layered SEI structures with various Young’s moduli have been visualized three dimensionally at nanoscale based on the hundreds of force curves in certain scanned area. The coverage of the SEI can be obtained quantitatively from the two-dimensional (2D) project plots. The related analysis indicates that more soft SEI layers are covered on the Si anode, and this could explain the benefits of the FEC additive.

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping.

Single-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors to the mechanical response of soft materials within the cell. To expand the single-molecule toolbox, we have developed a surface-free force spectroscopy assay based on a high-speed hydrodynamic trap capable of applying extremely high tensions for long periods of time. High-speed single-molecule trapping is enabled by a rigid and gas-impermeable microfluidic chip, rapidly and inexpensively fabricated out of glass, double-sided tape and UV-curable adhesive. Our approach does not require difficult covalent attachment chemistries, and enables simultaneous force application and single-molecule fluorescence. Using this approach, we have induced a highly extended state with twice the contour length of B-DNA in regions of partially intercalated double-stranded (dsDNA) by applying forces up to 250 pN. This highly extended state resembles the hyperstretched state of dsDNA, which was initially discovered as a structure fully intercalated by dyes under high tension. It has been hypothesized that hyperstretched DNA could also be induced without the aid of intercalators if high-enough forces were applied, which matches our observation. Combining force application with single-molecule fluorescence imaging is critical for distinguishing hyperstretched DNA from single-stranded DNA that can result from peeling. High-speed hydrodynamic trapping is a powerful yet accessible force spectroscopy method that enables the mechanics of biomolecules to be probed in previously difficult to access regimes.

Thromboresistant properties of nanostructured tantalum coatings on the stainless steel surfaces

The nanostructured coatings of tantalum and its compounds on the stainless steel surfaces were investigated by atomic force microscopy. Their hydrophilic properties were studied by sessile drop and atomic force spectroscopy methods. The thromboresistant properties of samples were analysed in vitro by incubation of the samples in the rich platelets plasma of patients with acute coronary syndrome. The effect of surface properties on the thromboresistant properties was analysed.

Investigation of the Effect of Electron Radiation on the Structure of Polypropylene Using Optical and Atomic Force Spectroscopy Methods

Using optical and atomic-force microscopy methods, it has been established that exposure to electron radiation causes a change in the supramolecular structure of polypropylene – in films obtained by recrystallization of irradiated polypropylene, spherulites have a less perfect shape than films obtained from unirradiated polypropylene. Also, the films obtained by recrystallization of irradiated polypropylene surface roughness is greater.