Combination of density-clustering and supervised classification for event identification in single-molecule force spectroscopy data

Single-molecule force spectroscopy(SMFS)measurements of the dynamics of biomolecules typically require identifying massive events and states from large data sets,such as extracting rupture forces from force-extension curves(FECs)in pulling experiments and identifying states from extension-time trajectories(ETTs)in force-clamp experiments.The former is often accomplished manually and hence is time-consuming and laborious while the latter is always impeded by the presence of baseline drift.In this study,we attempt to accurately and automatically identify the events and states from SMFS experiments with a machine learning approach,which combines clustering and classification for event identification of SMFS(ACCESS).As demonstrated by analysis of a series of data sets,ACCESS can extract the rupture forces from FECs containing multiple unfolding steps and classify the rupture forces into the corresponding conformational transitions.Moreover,ACCESS successfully identifies the unfolded and folded states even though the ETTs display severe nonmonotonic baseline drift.Besides,ACCESS is straightforward in use as it requires only three easy-to-interpret parameters.As such,we anticipate that ACCESS will be a useful,easy-to-implement and high-performance tool for event and state identification across a range of single-molecule experiments.

Investigation on the Folding Mode of a Polymer Chain in a Spiral Crystal by Single Molecule Force Spectroscopy

Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein, we use the combined techniques of atomic force microscopy （AFM） imaging and force spectroscopy to pull a single polyethylene oxide （PEO） chain out of its spiral crystal in amyl acetate. From these data, the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area, leading to the decrease in the apparent lamellar height. While in the area of screw dislocation, the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level.

Detection of weak non-covalent cation-π interactions in NGAL by single-molecule force spectroscopy

Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene.It plays an essential role in many biological systems as a vital driving force for protein folding,stability,and receptor-ligand interaction/recognition.To date,the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional(3D)protein structures and corresponding computational calculations.However,their experimental verification and quantification remain sparse at the molecular level,mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins.Here,we use atomic force microscopy-based single-molecule force spectroscopy(AFM-SMFS)to measure the stability of protein neutrophil gelatinase-associated lipocalin(also known as NGAL,siderocalin,lipocalin 2)that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols.Based on a site-specific cysteine engineering and anchoring method,we first characterized the stability and unfolding pathways of apo-NGAL.Then,the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized.AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites,F122 and F133.Here,NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect.Thus,our work demonstrates high-precision detection of the weak cation-π interaction in NGAL.

Combination of Click Chemistry and Enzymatic Ligation for Stable and Efficient Protein Immobilization for Single-Molecule Force Spectroscopy

Protein immobilization is an essential method for both basic and applied research for protein,and covalent,site-specific attachment is the most desirable strategy.Classic methods typically rely on a heterobifunctional cross-linker,such as N-hydroxysuccinimide(NHS)-linker-maleimide,or a similar two-step process.It utilizes the amino-reactive NHS and the thiolreactive maleimide to conjugate protein to the solid support.However,NHS as a chemical is susceptible to hydrolysis during storage and handling,and maleimide reacts nonspecifically with all cysteines available in the protein,leading to an inconsistent result.To solve these problems,we have developed a method by combining a strain-promoted azide–alkyne cycloaddition(SPAAC)click reaction and an OaAEP1(C247A)-based enzymatic ligation.The method was demonstrated by the successful immobilization of enhanced green fluorescent protein(eGFP),which was visualized by fluorescent imaging.Moreover,the correct folding and stability of the immobilized protein were verified by atomic force microscopy-based single-molecule force spectroscopy(AFM-SMFS)measurement with a high success rate(89%).Finally,the strength of the 1,2,3-triazole linkage from the azide-dibenzocyclooctyne(DBCO)-based SPAAC reaction was quantified with an ultrahigh rupture force>1.7 nN.Thus,this stable,efficient,and site-specific immobilization method can be used for many challenging systems,especially SMFS studies.

Anisotropic robustness of talc particles after surface modifications probed by atomic force microscopy force spectroscopy

As a versatile mineral,the crystalline hydrated magnesium silicate talcum,or talc,has been widely used in numerous industries from pharmaceutical formulations to composite material designs.Its efficient application as filler/additives incorporates the improvement in concomitant properties within materials,e.g.,strength,which involves interactions between talc particles and aqueous/nonaqueous matrices.Successful property enhancement imposes ideal mixing and homogenous adhesion within a talc particle,but they are limited by the coexistence of face and edge surfaces of talc,which exhibit different level of hydrophobicity.Here,using atomic force microscopy force spectroscopy,we showed that although hydrophilic talc particles obtained from acid treatment or aminosilanization better adhered with materials representing a matrix,the anisotropic characters of the two surface types persisted.Conversely,the degree of talc’s surface anisotropy reduced with the surface hydrophobization by aliphatic methylsilanization,but followed by the decrease in adhesion.With ten-fold difference in Hamaker constants of the probe/talc surface interacting pairs,we showed that the adhesions resulted from van der Waals interactions that suggested the influence of surface polarity.The insight from this work would provide grounds for strategies to modulate talc’s adhesion,hydrophobicity and surface uniformity.

Single-molecule-force spectroscopy study of the mechanism of interactions between TSP-1 and CD47

The 4N1K peptide,which is derived from the C-terminal domain of thrombospondin-1(TSP-1),is usually used as a functional mimic peptide for TSP-1.Knowledge about the interaction force of 4N1K/CD47 is important in explaining how TSP-1 affects the biological effect of CD47.Here we used a single-molecule force spectroscopy(SMFS)technique to explore the interaction of 4N1K/CD47 on both normal and oxidative human red blood cells(h RBCs)at single-molecule level.There was no interaction force between 4N1K and CD47 on normal h RBCs;however,we did find 4N1K-bound CD47 on oxidative h RBCs.We also detected interaction forces for 4N1K/CD47ex(extracellular domain of human CD47),and 4N1K/oxidative CD47ex.The interaction forces of 4N1K/CD47ex were almost consistent with those of 4N1K/oxidative CD47ex at the same loading rate.These results suggest that the conformational change of CD47 is critical for 4N1K-CD47 interaction on oxidative h RBCs.

Revealing the Flexibility of Inorganic Sub-Nanowires by Single-Molecule Force Spectroscopy

Sub-nanowires(SNWs),∼1 nm in thickness,possess an inorganic skeleton but display characteristics akin to those of carbon–carbon backbone polymers,such as flexibility,adhesion,gelation,self-assembling behaviors,and shear-thinning properties.Despite this,the underlying mechanism for these polymer-like properties remains unexplored.This study investigates the origin of SNWs’unique behavior from three distinct perspectives.Utilizing single-molecule force spectroscopy,we quantitatively measure the persistence lengths of SNWs,which provide a measure of flexibility.In addition,we evaluate the macroscopic mechanical properties of SNW materials,including the strength of electrospun fibers and gelation in solutions.Finally,we apply molecular dynamics to simulate the behaviors of SNWs under elongating and rotating conditions.The three perspectives mentioned above in the study collectively provide evidence for the structure-activity relationship of nanomaterials:a freely rotated backbone results in a flexible SNW,which is inclined to bend and entangle to form gels in solutions.Conversely,a stiff backbone leads to a rigid SNW,which induces strong fibers.

A Rising Force for the World-Wide Development of Laser-Induced Breakdown Spectroscopy

Laser-induced breakdown spectroscopy （LIBS） has attracted many academic and industrial interests world-wide due to its unique advantages, such as little or no sample preparation requirement, in-situ/online and multi-elemental analysis, and remote sensing etc., and it has been regarded as a ＂future super star＂ for chemical analysis for many years . In China,

Effect of Electropolishing on Electrochemical Behaviours of Titanium Alloy Ti-10V-2Fe-3Al

The electrochemical behaviours of titanium alloy Ti-10V-2Fe-3Al after electropolishing in a self-developed electrolyte in comparison with conventional grinding were studied by electrochemical impedance spectroscopy （EIS）.Optical microscopy （OM）,scanning electron microscopy （SEM）,atomic force microscopy （AFM） and X-ray photoelectron spectroscopy （XPS） were used to evaluate the surface characteristics of the alloy.It was found from the EIS experiments that the polarization resistance （Rp） was increased,the double layer capacitance （Qc） was decreased and the electrochemical impedance of the alloy was enhanced by electropolishing.The electropolished surface was flat,smooth and bright and its roughness was 3.310 nm.To underline the advantage of electropolishing process against grinding to provide the anodic oxidation process with a higher quality substrate,the ground and electropolished titanium alloys were anodized in parallel under the same conditions.The corrosion behaviours of the two kinds of anodized titanium alloys were compared.It was revealed that electropolishing generated a high quality substrate and improved the corrosion resistance of anodic oxide film formed on titanium alloy Ti-10V-2Fe-3Al.Furthermore,the mechanism of electropolishing improving the corrosion resistance of the anodic oxide film was proposed.

Review on near-field detection technology in the biomedical field

We review the recent biomedical detection developments of scanning near-field optical microscopy(SNOM),focusing on scattering-type SNOM,atomic force microscope-based infrared spectroscopy,peak force infrared microscopy,and photo-induced force microscopy,which have the advantages of label-free,noninvasive,and specific spectral recognition.Considering the high water content of biological samples and the strong absorption of water by infrared waves,we divide the relevant research on these techniques into two categories:one based on a nonliquid environment and the other based on a liquid environment.In the nonliquid environment,the chemical composition and structural information of biomedical samples can be obtained with nanometer resolution.In the liquid environment,these techniques can be used to monitor the dynamic chemical reaction process and track the process of chemical composition and structural change of single molecules,which is conducive to exploring the development mechanism of physiological processes.We elaborate their experimental challenges,technical means,and actual cases for three microbiomedical samples(including biomacromolecules,cells,and tissues).We also discuss the prospects and challenges for their development.Our work lays a foundation for the rational design and efficient use of near-field optical microscopy to explore the characteristics of microscopic biology.

Microstructure and Mechanical Property of Magnetron Sputtering Deposited DLC Film

A diamond-like carbon（DLC） film was deposited on YT14 substrate using magnetron sputtering（MS）. The surface morphologies, roughness and bonding spectra of obtained film were characterized using scanning electron microscopy（SEM）, atomic force microscopy（AFM）, and X-ray photoelectron spectroscopy（XPS）, respectively, and its mechanical property and bonding strength were measured using a nanoindentation and scratch tester, respectively. The results show that the C-enriched DLC film exhibits a denser microstructure and smoother surface with lower surface roughness of 21.8 nm. The ratio of C sp2 at 284.4 e V that corresponds to the diamond（111） and the C sp3 at 285.3 e V that corresponds to the diamond（220） plane for the as-received film is 0.36： 0.64, showing that the C sp3 has the high content. The hardness and Young＇s modulus of DLC film by nanoindentation are 8.534 41 and 142.158 1 GPa, respectively, and the corresponding bonding strength is 74.55 N by scratch test.

The effect of cigarette smoke extract on thrombomodulinthrombin binding: an atomic force microscopy study

Cigarette smoking is a well-known risk factor for cardiovascular disease. Smoking can cause vascular endothelial dysfunction and consequently trigger haemostatic activation and thrombosis. However, the mechanism of how smoking promotes thrombosis is not fully understood. Thrombosis is associated with the imbalance of the coagulant system due to endothelial dysfunction. As a vital anticoagulation cofactor, thrombomodulin (TM) located on the endothelial cell surface is able to regulate intravascular coagulation by binding to thrombin, and the binding results in thrombosis inhibition. This work focused on the effects of cigarette smoke extract (CSE) on TM-thrombin binding by atomic force microscopy (AFM) based single-molecule force spectroscopy. The results from both in vitro and live-cell experiments indicated that CSE could notably reduce the binding probability of TM and thrombin. This study provided a new approach and new evidence for studying the mechanism of thrombosis triggered by cigarette smoking.

Detecting CD20-Rituximab specific interactions on lymphoma cells using atomic force microscopy

Elucidating the underlying mechanisms of cell physiology is currently an important research topic in life sciences. Atomic force microscopy methods can be used to investigate these molecular mechanisms. In this study, single-molecule force spectroscopy was used to explore the specific recognition between the CD20 antigen and anti-CD20 antibody Rituximab on B lymphoma cells under near-physiological conditions. The CD20-Rituximab specific binding force was measured through tip functionalization. Distribution of CD20 on the B lymphoma cells was visualized three-dimensionally. In addition, the relationship between the intramolecular force and the molecular extension of the CD20-Rituximab complex was analyzed under an external force. These results facilitate further investigation of the mechanism of Rituximab’s anti-cancer effect.

Re-evaluation of the Widely Applied Force-Frequency Relation for Frequency-Modulation AFM Under Solution

Frequency-modulation atomic force microscopy(FM-AFM) is a highly versatile tool for surface science.Besides imaging surfaces, FM-AFM is capable of measuring interactions between the AFM probe and the surface with high sensitivity, which can provide chemical information at sub-nanometer resolution. This is achieved by deconvoluting the frequency shift, which is directly measured in experiments, into the force between the probe and sample. At present, the widely used method to perform this deconvolution has been shown to be accurate under high quality(high-Q) factor vacuum conditions. However, under low quality(low-Q) factor conditions, such as in solution, it is not clear if this method is valid. A previous study apparently verified this relation for experiments in solution by comparing the force calculated by this equation with that obtained in separate experiments using the surface force apparatus(SFA). Here we show that, in solution, a more direct comparison of the force calculated by this relation with that directly measured by the cantilever deflection in AFM reveals significant differences,both qualitative and quantitative. However, we also find that there are complications that hinder this comparison.Namely, while contact with the surface is clear in the direct measurements(including the SFA data), it is less certain in the FM-AFM case. Hence, it is not clear if the two methods are measuring the same tip-sample distance regimes. Thus, our results suggest that a more thorough verification of this relation is required, as application of this formulation for experiments in solution may not be valid.

FluidFM for single-cell biophysics

Fluidic force microscopy(FluidFM),which combines atomic force microscopy(AFM)with microchanneled cantilevers connected to a pressure controller,Is a technique allowing the realization of force-sensitive nanopipette under aqueous conditions.FluidFM has unique advantages in simultaneous three-dimensional manipulations and mechanical measurements of biological specimens at the micro-/nanoscale.Over the past decade,FluidFM has shown its potential in biophysical assays particularly in the investigations at single-cell level,offering novel possibilities for discovering the underlying mechanisms guiding life activities.Here,we review the utilization of FluidFM to address biomechanical and biophysical issues in the life sciences.Firstly,the fundamentals of FluidFM are represented.Subsequently,the applications of FluidFM for biophysics at single-cell level are surveyed from several facets,including single-cell manipulations,single-cell force spectroscopy,and single-cell electrophysiology.Finally,the challenges and perspectives for future progressions are provided.

Exploring the Nanomechanical Properties of a Coordination-bond Based Supramolecular Polymer

Due to their dynamic nature and strength tunability,metallo-supramolecular polymers have been introduced into various materials.The mechanical strength of the metallo-supramolecular polymers in the system directly influences the mechanical properties(e.g.,the toughness)of the materials.Therefore,it is necessary to explore the mechanical behavior of the metallo-supramolecular polymers.Herein,we present a single-molecule method to systematically explore the chain structure and mechanical properties of metallo-supramolecular polymer by using a loop protected architecture.Notably,we found that the mechanical stability of the individual chain,which is determined by the strength of terpyridine-Fe^(2+) -terpyridine(tpy-Fe^(2+)-tpy)bonds,was about 0.6–1.0 nN,depending on the pulling speed.This value is around three times higher than those measured using old methods.In addition,the unique loop protected structure further reduces the interference of non-specific polymer-AFM tip(or polymer-substrate)interactions on the quantification of the actual strength and kinetic parameter of noncovalent interactions in supramolecular polymers.Furthermore,the single chain flexibility of the metallo-supramolecular polymer was investigated and found to be comparable to the corresponding covalent analogues.Our findings provide a new way to explore the force response of supramolecular polymers composed of metal-ligand interactions and will be useful for the design of metallo-supramolecular polymer-based functional materials with tailored mechanical properties.

Mechanochemistry of an Interlocked Poly[2]catenane:From Single Molecule to Bulk Gel

Mechanically interlocked molecules(MIMs)are prototypical molecular machines with parts that enable controlled,large-amplitude movement with one component positioned relative to another.Incorporating MIMs into polymeric matrices is promising for the designing of functional materials with unprecedented properties.However,the central issue is the challenges involved with establishing the mechanistic linkage between the single-molecule and the bulk material.Herein,we explore the mechanochemical properties and energetic details of a linear poly[2]catenane with strong intercomponent hydrogen bonding(IHB)revealed by single-molecule force spectroscopy.Our results showed that the individual linear poly[2]catenane chain exhibited typical sawtooth pattern,corresponding to the reversible unlocking and relocking transitions under external force or upon stimulations to dissociate or re-form the strong IHB.Furthermore,when a poly[2]catenane-based polymer gel was prepared using a thiol-ene click reaction between thiol-ended poly[2]catenane and a low-molecule-weight cross-linker,the resultant gel showed excellent mechanical adaptability and dynamic properties,which correlated well with the molecular-level observations.The unique poly[2]catenane structure also contributed to the gel formation with an extraordinary IHB-mediated swelling behavior and shape memory property.Thus our present results demonstrate the functioning of bulk material in a linear tandem manner from the behavior of a single molecule,a finding which should be applicable to other systems with versatile properties and promising applications.

Harvesting Mechanical Work From Folding-Based Protein Engines: From Single-Molecule Mechanochemical Cycles to Macroscopic Devices

Mechanochemical coupling cycles underlie the work-generation mechanisms of biological systems and are realized by highly regulated conformational changes of the protein machineries.However,it has been challenging to utilize protein conformational changes to do mechanical work at the macroscopic level in biomaterials,and it remains elusive to con-struct macroscopic mechanochemical devices based on molecular-level mechanochemical coupling sys-tems.Here,the authors demonstrate that protein folding can be utilized to realize protein’s mechano-chemical cycles at both single-molecule and macro-scopic levels.

Single-molecule observation of mechanical isomerization of spirothiopyran and subsequent Click addition

Spirothiopyran(STP)is particularly attractive when used as a mechanophore to endow polymers with both damage-signaling and.self-reinforcing capacity.It is,however,not clear the actual force required to induce the cycloreversion of STP into ring-opened thiomerocyanine(TMC),which reacts spontaneously with activated C=C bonds.Here,we used atomic force microscopy(AFM)-based single molecule force spectroscopy(SMFS)to study the mechanochemistry of STP mechanophore.It is found that the ring-opening of STP at room temperature requires forces of-200-400 pN,depending on the pulling speed.In addition,the reversibility of STP to TMC isomerization is demonstrated.Finally,mechanochemically induced intermolecular Click addition is achieved in single'molecule level by pulling STP in the presence of maleimide.

Stretching Elasticity and Flexibility of Single Polyformaldehyde Chain

In this work,the single-chain elasticity of polyformaldehyde(POM)is studied,for the first time,by employing atomic force microscopy(AFM)-based single molecule force spectroscopy(SMFS).We find that the single-chain elasticity of POM in a nonpolar organic solvent(nonane)can be described well by a theoretical model(QM-FRC model),when the rotating unit length is 0.144 nm(C―O bond length).After comparison,POM is more flexible than polystyrene(a typical polymer with C―C backbone)at the single-chain level,which is reasonable since the C―O bond has a lower rotation barrier than C―C bond.This result indicates that the flexibility of a polymer chain can be tuned by the C―O bond proportion in backbone,which casts new light on the rational design of new synthetic polymers in the future.