Pinpointing Mechanochemical Bond Rupture by Embedding the Mechanophore into a Macrocycle

Mechanophores contain a mechanically labile bond that can be broken by an external mechanical force. Quantitative measurement and control of the applied force is possible through atomic force microscopy (AFM). A macrocycle was synthesized that contains both the mechanophore and an aliphatic chain that acts as a “safety line” upon bond breaking. This ring‐opening mechanophore unit is linked to poly(ethylene glycol) spacers, which allow investigation by single molecule force spectroscopy. The length increase upon rupture of the mechanophore was measured and compared with quantum chemical calculations.     

Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces

PEGylated Nb2O5 surfaces were obtained by the adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers, allowing control of the PEG surface density, as well as the surface charge. PEG (MW 2 kDa) surface densities between 0 and 0.5 nm(-2) were obtained by changing the PEG to lysine-mer ratio in the PLL-g-PEG polymer, resulting in net positive, negative and neutral surfaces. Colloid probe atomic force microscopy (AFM) was used to characterize the interfacial forces associated with the different surfaces. The AFM force analysis revealed interplay between electrical double layer and steric interactions, thus providing information on the surface charge and on the PEG layer thickness as a function of copolymer architecture. Adsorption of the model proteins lysozyme, alpha-lactalbumin, and myoglobin onto the various PEGylated surfaces was performed to investigate the effect of protein charge. In addition, adsorption experiments were performed over a range of ionic strengths, to study the role of electrostatic forces between surface charges and proteins acting through the PEG layer. The adsorbed mass of protein, measured by optical waveguide lightmode spectroscopy (OWLS), was shown to depend on a combination of surface charge, protein charge, PEG thickness, and grafting density. At high grafting density and high ionic strength, the steric barrier properties of PEG determine the net interfacial force. At low ionic strength, however, the electrical double layer thickness exceeds the thickness of the PEG layer, and surface charges "shining through" the PEG layer contribute to protein interactions with PLL-g-PEG coated surfaces. The combination of AFM surface force measurements and protein adsorption experiments provides insights into the interfacial forces associated with various PEGylated surfaces and the mechanisms of protein resistance.     

Single-molecule force spectroscopy measurements of "hydrophobic bond" between tethered hexadecane molecules

The hydrophobic effect is important for many biological and technological processes. Despite progress in theory, experimental data clarifying water structure and the interaction between hydrophobic solutes at the nanometer scale are scarce due to the very low solubility of hydrophobic species. This article describes an AFM single molecule force spectroscopy method to probe the interaction between molecules with low solubility and reports measurements of the strength and the length scale of the "hydrophobic bond" between hexadecane molecules. Hexadecane molecules are tethered by flexible poly(ethylene glycol) linkers to AFM probes and substrates, removing the aggregation state uncertainty of solution-based approaches as well as spurious surface effects. A shorter hydrophilic polymer layer is added to increase the accessibility of hydrophobic molecules for the force spectroscopy measurements. Statistical analysis of the rupture forces yields a barrier width of 0.24 nm, and a dissociation rate of 1.1 s(-1). The results of single molecule measurements are related to the theoretical predictions of the free energy of cavitation in water and to the empirical model of micellization of nonionic surfactants. It is estimated that approximately one-quarter of each molecule's surface is hydrated during forced dissociation, consistent with an extended (nonglobular) conformation of the hexadecane molecules in the dimer.     

Evidence of Splitting 1,2,3‐Triazole into an Alkyne and Azide by Low Mechanical Force in the Presence of Other Covalent Bonds

The cycloaddition reaction of an alkyne and azide to form a 1,2,3‐triazole is widely used in many areas. However, the stability of the triazole moiety under mechanical stress is unclear. To see if a triazole could be selectively split into an alkyne and azide in the presence of other typical covalent bonds, a mica surface functionalized with a molecule containing a triazole moiety in the middle and an activated ester at the end was prepared. An atomic force microscope (AFM) tip with amino groups on its surface was ramped over the mica surface at predefined locations, which could temporarily link the tip to the surface through amide bond formation. During retraction, the triazole or another bond in the linkage broke, and a force was recorded. The forces varied widely at different ramps from close to 0 pN to 860 pN due to nonspecific adhesions and to the inherent inconsistency of single bond rupture. If some of the forces were from triazole cycloreversion, there would be alkynes at the predefined ramping locations. The surface was reacted with an azide carboxylic acid followed by labeling with amino Au nanoparticles (AuNPs). AFM imaging revealed AuNPs at the predicted locations, which provided evidence that under certain conditions triazole could be split selectively in the presence of other bonds at forces below 860 pN.     

Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.     

Direct measurements of the interaction between pyrene and graphite in aqueous media by single molecule force spectroscopy: understanding the pi-pi interactions

Pyrene derivatives can absorb onto the surface of carbon nanotubes and graphite particles through pi-pi interactions to functionalize these inorganic building blocks with organic surface moieties. Using single molecule force spectroscopy, we have demonstrated the first direct measurement of the interaction between pyrene and a graphite surface. In particular, we have connected a pyrene molecule onto an AFM tip via a flexible poly(ethylene glycol) (PEG) chain to ensure the formation of a molecular bridge. The pi-pi interaction between pyrene and graphite is thus indicated to be approximately 55 pN with no hysteresis between the desorption and adhesion forces.     

Reduced hydrophobic interaction of polystyrene surfaces by spontaneous segregation of block copolymers with oligo (ethylene glycol) methyl ether methacrylate blocks: force measurements in water using atomic force microscope with hydrophobic probes

Reduction of hydrophobic interaction in water is important in biological interfaces. In our previous work, we have found that poly(styrene- b-triethylene glycol methyl ether methacrylate) (PS-PME3MA) segregates the PME3MA block to the surface in hydrophobic environment, such as in air or in a vacuum, and shows remarkable resistance against adsorption or adhesion of proteins, platelets, and cells in water. In this paper, we report that atomic force microscopy (AFM) with hydrophobic probes can directly monitor the reduced hydrophobic interaction of the PS surfaces modified by poly(styrene- b-origoethylene glycol methyl ether methacrylate) (PS-PME NMA), where N is the number of ethylene glycol units. The pull-off forces between the hydrophobic probes that are coated with octyltrichlorosilane (OLTS) and the PS-PME NMA modified polystyrene (PS) surfaces in water were measured. The absolute spring constants and tip-curvatures of the AFM cantilevers were measured to compute the work of adhesion by the Johnson, Kendall, and Roberts (JKR) theory, which relates the pull-off force at which the separation occurs between a hemisphere and a plane to the work of adhesion. The hydrophobic interactions between the hydrophobic tip and polymer surfaces in water were greatly reduced with the segregated PME NMA blocks. The hydrophobic interactions decrease with increasing N of the series of PS-PME NMA and show a correlation with the amount of protein adsorbed.     

Viscoelastic properties of single poly(ethylene glycol) molecules.

The viscoelastic properties of single poly(ethylene glycol) (PEG) molecules were measured by analysis of thermally and magnetically driven oscillations of an atomic force microscope (AFM) cantilever/molecule system. The molecular and monomer stiffness and friction of the PEG polymer were derived using a simple harmonic oscillator (SHO) model. Excellent agreement between the values of these two parameters obtained by the two approaches indicates the validity of the SHO model under the experimental regimes and the excellent reproducibility of the techniques. A sharp minimum in the monomeric friction is seen at around 180 pN applied force which we propose is due to a force induced change in the shape of the energy landscape describing the conformational transition of PEG from a helical to a planar state, which in turn affects the timescale of the transition and therefore modifies the measured internal friction. A knowledge of the viscoelastic response of PEG monomers is particularly important since PEG is widely used as a linker molecule for tethering groups of interest to the AFM tip in force spectroscopy experiments, and we show here that care must be exercised because of the force-dependent viscoelastic properties of these linkers.     

Comparison of antibody--antigen interactions on collagen measured by conventional immunological techniques and atomic force microscopy

We have developed a means of using atomic force microscopy (AFM) to repeatedly localize a small area of interest (4 x 4 microm(2)) within a 0.5-cm(2) area on a heterogeneous sample, to obtain and localize high-resolution images and force measurements on nonideal samples (i.e., samples that better reflect actual biological systems, not prepared on atomically flat surfaces). We demonstrate the repeated localization and measurement of unbinding forces associated with antibody--antigen (ab--ag) interactions, by applying AFM in air and in liquid to visualize and measure polyclonal ab--ag interactions, using chicken collagen as a model system. We demonstrate that molecular interactions, in the form of ab--ag complexes, can be visualized by AFM when secondary antibodies are conjugated to 20-nm colloidal gold particles. We then compare those results with established immunological techniques, to demonstrate broader application of AFM technology to other systems. Data from AFM studies are compared with results obtained using immunological methods traditionally employed to investigate ab--ag interactions, including enzyme-linked immunosorbent assay, immunoblotting, and in situ immunofluorescence. Finally, using functionalized AFM tips with a flexible tether [poly(ethylene glycol) 800] to which a derivatized antibody was attached, we analyzed force curve data to measure the unbinding force of collagen antibody from its antigen, obtaining a value of approximately 90 +/- 40 pN with a MatLab code written to automate the analyses of force curves obtained in force--volume mode. The methodology we developed for embedded collagen sections can be readily applied to the investigation of other receptor--ligand interactions.     

Correction of systematic errors in single-molecule force spectroscopy with polymeric tethers by atomic force microscopy

Single-molecule force spectroscopy has become a valuable tool for the investigation of intermolecular energy landscapes for a wide range of molecular associations. Atomic force microscopy (AFM) is often used as an experimental technique in these measurements, and the Bell-Evans model is commonly used in the statistical analysis of rupture forces. Most applications of the Bell-Evans model consider a constant loading rate of force applied to the intermolecular bond. The data analysis is often inconsistent because either the probe velocity or the apparent loading rate is being used as an independent parameter. These approaches provide different results when used in AFM-based experiments. Significant variations in results arise from the relative stiffness of the AFM force sensor in comparison with the stiffness of polymeric tethers that link the molecules under study to the solid surfaces. An analytical model presented here accounts for the systematic errors in force-spectroscopy parameters arising from the nonlinear loading induced by polymer tethers. The presented analytical model is based on the Bell-Evans model of the kinetics of forced dissociation and on the asymptotic models of tether stretching. The two most common data reduction procedures are analyzed, and analytical expressions for the systematic errors are provided. The model shows that the barrier width is underestimated and that the dissociation rate is significantly overestimated when force-spectroscopy data are analyzed without taking into account the elasticity of the polymeric tether. Systematic error estimates for asymptotic freely jointed chain and wormlike chain polymer models are given for comparison. The analytical model based on the asymptotic freely jointed chain stretching is employed to analyze and correct the results of the double-tether force-spectroscopy experiments of disjoining "hydrophobic bonds" between individual hexadecane molecules that are covalently tethered via poly(ethylene glycol) linkers of different lengths to the substrates and to the AFM probes. Application of the correction algorithm decreases the spread of the data from the mean value, which is particularly important for measurements of the dissociation rate, and increases the barrier width to 0.43 nm, which might be indicative of the theoretically predicted hydrophobic dewetting.     

Thermal [2 + 2] cycloreversion of a cyclobutane moiety via a biradical reaction

The nature of the Woodward-Hoffmann-forbidden, thermal activated cycloreversion mechanism of cyclobutane has long been the subject of speculation and intense research. We were now able to prove the theoretically postulated biradicalic mechanism directly from radical scavenging reactions and electron paramagnetic resonance (EPR) experiments on [2 + 2] heterodimers of 5-fluoro-1-heptanoyluracil and 7-methoxy-1,1-dimethylnaphthalenon. The dimers show both the "allowed" photochemically as well as the "forbidden" thermally triggered [2 + 2] cycloreversion of the cyclobutane ring. The quantum efficiency of the photochemical cleavage is about 1%. The thermal cycloreversion reaction is independent from solvent and occurs at low activation energies of about 13 kcal/mol, even in the solid state. The radical scavenger and EPR results are further supported by the finding, that the reaction products are solely the educts for the anti-head-to-tail heterodimer. But for the syn-head-to-head heterodimer two additional products are observed, which require a sufficiently stable biradical intermediate to facilitate the required intramolecular rearrangements. Because of the surprisingly high lifetime of the radical species of these heterodimers it was possible to prove the long-discussed biradical mechanism experimentally.     

Interaction between dendrons directly studied by single-molecule force spectroscopy

In this article, we have investigated the interaction between two poly(benzyl ether) dendrons directly by single-molecule force spectroscopy. For this purpose, one dendron was immobilized on an AFM tip through a poly(ethylene glycol) (PEG) spacer, and the other dendron was anchored on a gold substrate as a self-assembled monolayer. Two dendrons approached and then interacted with each other when the AFM tip and the substrate moved close together. The rupture force between dendrons was measured while the AFM tip and the substrate separated. PEG as a flexible spacer can function as a length window for recognizing the force signals and avoiding the disturbance of the interaction between the AFM tip and the substrate. The interaction between two first-generation dendrons is measured to be about 224 pN at a force loading rate of 40 nN/s. The interaction between second- and first-generation dendrons rises to 315 pN at the same loading rate. Such interactions depend on the force loading rate in the range of several to hundreds of nanonewtons per second, indicating that the rupture between dendrons is a dynamic process. The study of the interaction between surface-bound dendrons of different generations provides a model system for understanding the surface adhesion of molecules with multiple branches. In addition, this multiple-branch molecule may be used to mimic the sticky feet of geckos as a man-made adhesive.     

疏水链段对两亲性三嵌段共聚物在水中聚集行为的影响

以结构明确的两端为短的聚苯乙烯(PS)或聚甲基丙烯酸甲酯(PMMA)链段,中间为长的聚乙二醇(PEG)链段的PS-b-PEG-b-PS和PMMA-b-PEG-b-PMMA两亲性三嵌段共聚物为对象,研究了PS和PMMA链段对其在水中形成胶束和凝胶的影响.两种三嵌段共聚物在水中形成以PS或PMMA链段为核、PEG链段为壳的球形胶束,流体力学半径Rh,app为15.3~24.3 nm,并随PEG链段长度增长而增大.临界胶束浓度CMC均小于0.01 mg/mL,随着PS和PMMA链段长度的增加而减小.PS-b-PEG-b-PS浓度高于4.5 wt%可形成较强的疏水缔合的物理凝胶,平衡模量Ge可达到103Pa;PMMA-b-PEG-b-PMMA浓度高于7.5 wt%可以形成弱的凝胶,Ge<10 Pa.凝胶的储存模量G′和损耗模量G″均随着PS或PMMA链段的增长而增大.     

Simple test system for single molecule recognition force microscopy

We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG₈₀₀ diamine was glutarylated, the mono-adduct NH₂-PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy.     

A molecular shuttle for driving a multilevel fluorescence switch

A [2]rotaxane-based molecular shuttle comprised a macrocycle mechanically interlocked to a chemical "dumbbell" has been prepared in high yields by a thermodynamically controlled, template-induced clipping procedure. This molecular shuttle has two different recognition sites, namely, -NH2 +- and amide, separated by a phenyl unit. The macrocycle exhibits high selectivity for the -NH2+- recognition sites in the protonated form through noncovalent interactions, which include 1) N+-H...O hydrogen bonds; 2) C-H...O interactions between the CH2NH2+CH2 protons on the thread and the oligo(ethylene glycol) unit in the macrocycle; 3) pi...pi stacking interaction between macrocycle and aromatic unit. Upon deprotonation of the [2]rotaxane the macrocycle glides to the amide recognition site due to the hydrogen bonds between the -CONH- group and the oligo(ethylene glycol) unit in the macrocycle. The deprotonation process requires about 10 equivalents of base (iPr2NEt) in polar acetone, while the amount of base is only 1.2 equivalents in apolar tetrachloroethane. Upon addition of Li+, the conformation of the [2]rotaxane was altered as a result of the collective interactions of 1) hydrogen bonds between pyridine nitrogen and amide hydrogen atoms; 2) coordination between the oligo(ethylene glycol) unit, amide oxygen atom and Li+ cation. Then, when Zn2+ ions are added, the macrocycle returns to the deprotonated -NH- recognition site owing to coordination of the macrocycle and -NH- from the axle with the Zn2+ ion. All the above-mentioned movement processes are reversible through the alternate addition of TFA/iPr2NEt, Li/[12]-crown-4 and Zn2+/ethylenediaminetetraacetate (EDTA), by virtue of hydrogen bonding and metal-ion complexation. Significantly, the three independent movement processes are all accompanied by fluorescent responses: 1) complete repression in the protonated form; 2) low-level expression in the deprotonated form; 3) medium-level expression following addition of Li+; 4) high-level expression on complexation with Zn2+.     

Direct force measurement of the stability of poly(ethylene glycol)-polyethylenimine graft films

The stability and passivity of poly(ethylene glycol)-polyethylenimine (PEG-PEI) graft films are important for their use as antifouling coatings in a variety of biotechnology applications. We have used AFM colloidal-probe force measurements combined with optical reflectometry to characterize the surface properties and stability of PEI and dense PEG-PEI graft films on silica. Initial contact between bare silica probes and PEI-modified surfaces yields force curves that exhibit a long-range electrostatic repulsion and short-range attraction between the surfaces, indicating spontaneous desorption of PEI in the aqueous medium. Further transfer of PEI molecules to the probe occurs with subsequent application of forces between FR = 300 and 500 microN/m. The presence of PEG reduces the adhesive properties of the PEI surface and prevents transfer of PEI molecules to the probe with continuous contact, though an initial desorption of PEI still occurs. Glutaraldehyde crosslinking of the graft films prevents both the initial desorption and subsequent transfer of the PEI, resulting in sustained attractive interaction forces of electrostatic origin between the negatively charged probe and the positively charged copolymer graft films.     

Colloid probe AFM investigation of interactions between fibrinogen and PEG-like plasma polymer surfaces

Interaction forces between surfaces designed to be protein resistant and fibrinogen (Fg) were investigated in phosphate-buffered saline with colloid probe atomic force microscopy. The surfaces of the silica probes were coated with a layer of fibrinogen molecules by adsorption from the buffer. The technique of low-power, pulsed AC plasma polymerization was used to make poly(ethylene glycol) (PEG)-like coatings on poly(ethylene teraphthalate) by using diethylene glycol vinyl ether as the monomer gas. The degree of PEG-like nature of the films was controlled by use of a different effective plasma power in the chamber for each coating, ranging from 0.6 to 3.6 W. This produced a series of thin films with a different number of ether carbons, as assessed by X-ray photoelectron spectroscopy. The interaction force measurements are discussed in relation to trends observed in the reduction of fibrinogen adsorption, as determined quantitatively by (125)I radio-labeling. The plasma polymer coatings with the greatest protein-repelling properties were the most PEG-like in nature and showed the strongest repulsion in interaction force measurements with the fibrinogen-coated probe. Once forced into contact, all the surfaces showed increased adhesion with the protein layer on the probe, and the strength and extension length of adhesion was dependent on both the applied load and the plasma polymer surface chemistry. When the medium was changed from buffer to water, the adhesion after contact was eliminated and only appeared at much higher loads. This indicates that the structure of the fibrinogen molecules on the probe is changed from an extended conformation in buffer to a flat conformation in water, with the former state allowing for stronger interaction with the polymer chains on the surface. These experiments underline the utility of aqueous surface force measurements toward understanding protein-surface interactions, and developing nonfouling surfaces that confer a steric barrier against protein adsorption.     

Stretching single molecules along unbinding and unfolding pathways with the scanning force microscope

Individual molecules can be stretched with a scanning force microscope and the forces required to rupture bonds and to mechanically drive their structures towards new conformations and states can be measured. The tailoring of the experiments, the possibility of carrying them out in quasi-equilibrium conditions, the relationships between single molecule force measurements, and macroscopic kinetics or thermodynamic data are discussed. Mechanochemical experiments are expanding chemistry into new realms between biology and material science.     

Dip-pen patterning and surface assembly of peptide amphiphiles

This paper presents results on controlling the surface morphology of evaporation-driven self-assembly of peptide amphiphile (PA) nanofibers by dip-pen nanolithography. These PA nanofibers, which measure only a few nanometers in diameter, can be oriented perpendicularly to the receding edge of a solution. Dragging a meniscus of PA ink with an atomic force microscope (AFM) tip creates reproducibly aligned arrays of isolated and close-packed PA nanofiber patterns on silicon substrates, utilizing surface coating of poly(ethylene glycol) to suppress the self-assembly of nanofibers on AFM tips. We also demonstrate the ability to construct double-layer patterns of differing nanofiber orientations at the same position. This result could be important in producing a complex, multilayer pattern of these peptide-based supramolecular nanostructures.     

Unbinding of the streptavidin-biotin complex by atomic force microscopy: a hybrid simulation study

A hybrid molecular simulation technique, which combines molecular dynamics and continuum mechanics, was used to study the single-molecule unbinding force of a streptavidin-biotin complex. The hybrid method enables atomistic simulations of unbinding events at the millisecond time scale of atomic force microscopy (AFM) experiments. The logarithmic relationship between the unbinding force of the streptavidin-biotin complex and the loading rate (the product of cantilever spring constant and pulling velocity) in AFM experiments was confirmed by hybrid simulations. The unbinding forces, cantilever and tip positions, locations of energy barriers, and unbinding pathway were analyzed. Hybrid simulation results from this work not only interpret unbinding AFM experiments but also provide detailed molecular information not available in AFM experiments.