基于原子力显微镜的高分子单分子力学研究

原子力显微镜(AFM)从根本上改变了人们对单个原子和分子的作用和认识方式。单分子力谱是基于原子力显微镜力的测量方法。概速了近年来利用基于原子力显微镜的单分子力谱研究单个高分子分子内及分子闻作用力的进展。     

Supramolecular research by single molecule force spectroscopy

Single molecule force spectroscopy (SMFS) is a new kind of technique based on atomic force microscope, which allows detecting force as low as pico-newtons directly. Herein based on our recent work, we want to demonstrate the investigation of supramolecular structures and interactions in polymer systems by SMFS, such as desorption force between polymer and substrate, identifiability of polymer micelle and its interaction with surfactant, splitting force of ion-induced helical structure in polysaccharide. It shows well that SMFS is a powerful tool in the study of supramolecular science.     

单分子水平高分子相互作用的AFM成像与单分子力谱研究

结合作者近期的研究工作,重点介绍了如何把原子力显微镜(AFM)成像及单分子力谱结合(包括原位结合或者离位结合)起来,研究高分子之间的相互作用.本文涉及生物高分子(主要是核酸-蛋白质体系)以及合成高分子体系(如聚氧乙烯,PEO)的相关研究工作.对于生物高分子体系,主要以长链核酸(如双螺旋DNA及RNA)为探针,首先利用A...     

Unexpected temperature-dependent single chain mechanics of poly(N-isopropyl-acrylamide) in water

Poly(N-isopropyl-acrylamide) (PNIPAM) is a paradigm thermally sensitive polymer, which has a lower critical solution temperature (LCST) of ~32 °C in water. Herein by AFM-based single molecule force spectroscopy (SMFS), we measured the single chain elasticity of PNIPAM across the LCST in water. Below LCST, the force curves obtained at different temperatures have no remarkable difference; while above LCST, an unexpected temperature dependent elasticity is observed, mainly in the middle force regime. We found that 35 °C is a turning point of the variation: from 31 to 35 °C, the middle parts of the force curves drop gradually, whereas from 35 to 40 °C, the middle parts rise gradually. A possible mechanism for the unexpected temperature dependent mechanics is proposed. The single chain contraction against external force upon heating from 35 to 40 °C may cast new light on the design of molecular devices that convert thermal energy to mechanical work.     

A facile and environment-friendly method for fabrication of polymer brush

A novel environment-friendly system is proposed tofabricate polymer brush, which has the advantages including non-toxic and inexpensive initiator(eosin Y), visible-light exposure(λ= 515 nm), water medium and ambient environment. The experimental results from UV-Vis spectroscopy, AFM-based single molecule force spectroscopy(SMFS) and other measurements indicate thata polymer brush with a living nature is fabricated via free radical polymerization. This polymer brush may find applications incoatings, bio-interfaces and so forth.     

Stretching single polysaccharides and proteins using atomic force microscopy

The past years have witnessed remarkable advances in our use of atomic force microscopy (AFM) for stretching single biomolecules, thereby contributing to answering many outstanding questions in biophysics and chemical biology. In these single-molecule force spectroscopy (SMFS) experiments, the AFM tip is continuously approached to and retracted from the biological sample, while monitoring the interaction force. The obtained force-extension curves provide key insight into the molecular elasticity and localization of single molecules, either on isolated systems or on cellular surfaces. In this tutorial review, we describe the principle of such SMFS experiments, and we survey remarkable breakthroughs made in manipulating single polysaccharides and proteins, including understanding the conformational properties of sugars and controlling them by force, measuring the molecular elasticity of mechanical proteins, unfolding and refolding individual proteins, probing protein-ligand interactions, and tuning enzymatic reactions by force. In addition, we show how SMFS with AFM tips bearing specific bioligands has enabled researchers to stretch and localize single molecules on live cells, in relation with cellular functions.     

聚合物单分子力谱的研究进展

在单分子水平研究聚合物体系的分子内及分子间相互作用, 对于揭示其结构-性能的关系, 进而实现对相应功能的调控极为重要. 基于原子力显微镜技术(AFM)的单分子力谱, 由于其操作简单且适用面广, 在单分子研究领域得到了广泛的应用. 本文概括了该技术在生物高分子及合成高分子体系的研究进展. 对于生物高分子体系, 主要介绍了核酸(DNA/RNA)、 蛋白质和多糖(淀粉)的单分子力谱研究及利用各自力学指纹谱对其它分子间的相互作用的研究. 对于合成高分子体系, 主要介绍了聚合物的一级结构与单链弹性的关系及溶剂和聚集态结构等对高分子单链力学性质的影响规律.     

Grafting of single, stimuli-responsive poly(ferrocenylsilane) polymer chains to gold surfaces

Redox-responsive poly(ferrocenylsilane) (PFS) polymer molecules were attached individually to gold surfaces for force spectroscopy experiments on the single molecule level. By grafting ethylenesulfide-functionalized PFS into the defects of preformed self-assembled monolayers (SAMs) of different omega-mercaptoalkanols on Au(111), the surface coverage of PFS macromolecules could be conveniently controlled. Atomic force microscopy (AFM), contact angle, as well as cyclic and differential pulse voltammetry measurements were carried out to characterize the morphology, wettability, and surface coverage of the grafted layers. The values of the PFS surface coverage were found to depend on the chain length of the omega-mercaptoalkanol molecules and on the concentration of the PFS solution but not on the insertion time or on the molar mass of PFS. The equilibrium surface coverages were successfully described by Langmuir adsorption isotherms. For low-surface coverage values (< 6.2 x 10(-4) chain/nm2), achieved by PFS insertion from very dilute solutions (8 x 10(-6) M) into long-chain SAMs, AFM and differential pulse voltammetry showed that surfaces exposing isolated individual polymer chains were obtained. The isolated PFS macromolecules were subjected to in situ AFM-based single molecule force spectroscopy (SMFS) measurements. The single chain elasticity of PFS in isopropanol (and ethanol) was fitted with the modified freely jointed chain (m-FJC) model. This procedure yielded a Kuhn segment length of 0.33 +/- 0.05 nm and a segment elasticity of 32 +/- 5 nN/nm.     

Perfluorophenyl azide immobilization chemistry for single molecule force spectroscopy of the concanavalin A/mannose interaction

The versatility of perfluorophenyl azide (PFPA) derivatives makes them useful for attaching a wide variety of biomolecules and polymers to surfaces. Herein, a single molecule force spectroscopy (SMFS) study of the concanavalin A/mannose interaction was carried out using PFPA immobilization chemistry. SMFS of the concanavalin A/mannose interaction yielded an average unbinding force of 70-80 pN for loading rates between 8000 and 40,000 pN/s for mannose surfaces on aminated glass, and an unbinding force of 57 ± 20 pN at 6960 pN/s for mannose surfaces on gold-coated glass. Dynamic force spectroscopy was used to determine the dissociation rate constant, k(off), for this interaction to be 0.16 s(-1).     

Interrogation of single synthetic polymer chains and polysaccharides by AFM-based force spectroscopy.

This contribution reviews selected mechanical experiments on individual flexible macromolecules using single-molecule force spectroscopy (SMFS) based on atomic force microscopy. Focus is placed on the analysis of elasticity and conformational changes in single polymer chains upon variation of the external environment, as well as on conformational changes induced by the mechanical stress applied to individual macromolecular chains. Various experimental strategies regarding single-molecule manipulation and SMFS testing are discussed, as is theoretical analysis through single-chain elasticity models derived from statistical mechanics. Moreover, a complete record, reported to date, of the parameters obtained when applying the models to fit experimental results on synthetic polymers and polysaccharides is presented.     

Rupture force of single supramolecular bonds in associative polymers by AFM at fixed loading rates

Atomic-force-microscopy-based single-molecule force spectroscopy (AFM-SMFS) was used to study the bond strength of self-complementary hydrogen-bonded complexes based on the 2-ureido-4[1H]-pyrimidinone (UPy) quadruple H-bond motif in hexadecane (HD). The unbinding force corresponding to single UPy-UPy dimers was investigated at a fixed piezo retraction rate in the nonequilibrium loading rate regime. The rupture force of bridging supramolecular polymer chains formed between UPy-functionalized substrates and AFM tips in the presence of a bis-UPy derivative was found to decrease with increasing rupture length. The rupture length was identified as the chain length of single, associating polymers, which allowed us to determine the number of supramolecular bonds (N) at rupture. The rupture force observed as a function of N was in quantitative agreement with the theory on uncooperative bond rupture for supramolecular linkages switched in a series. Hence, the value of the dimer equilibrium constant Keq=(1.3+/-0.5) x 10(9) M(-1), which is in good agreement with previously estimated values, was obtained by SMFS of supramolecular polymers at a single loading rate.     

Single-molecule force spectroscopy of selenium-containing amphiphilic block copolymer: toward disassembling the polymer micelles

Selenium-containing polymers are a new type of responsive polymer material. Here, a selenium-containing amphiphilic block copolymer (PEG-PUSe-PEG) has been investigated using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). The deviation between force-extension curves of PEG-PUSe-PEG in water and in DMSO is found to be related to the disassembly of the micellar structures in water. SMFS experiments on PEG-PUSeox-PEG suggest that the change from selenide to oxidized selenone contributes significantly to the change in amphiphilicity, without obviously influencing the single-chain elasticity.     

AFM单分子力谱在真实材料及生物体系中的应用——挑战与机遇并存

基于原子力显微镜技术（AFM）的单分子力谱是研究分子间分子内相互作用的有效手段．为了简化样品体系及数据的解析,真实的生物或材料体系通常被简化,其中的目标分子被提取并桥连于AFM的针尖与固体基片之间进行研究,这是认识真实体系的有效途径．随着技术的不断进步（包括样品固定方法的改进）,使得直接研究真实生物及材料体系中的各种弱相互作用成为可能,此种条件下获得的信息对相关生命过程的调控及高性能材料的设计更具指导意义．本文概述了近几年基于AFM力谱技术在活体细胞以及高分子材料领域的研究进展,分析了存在的主要问题,并对相关领域的未来进行了展望．     

Single molecule atomic force microscopy and force spectroscopy of chitosan

Atomic force microscopy (AFM) and AFM-based force spectroscopy was used to study the desorption of individual chitosan polymer chains from substrates with varying chemical composition. AFM images of chitosan adsorbed onto a flat mica substrate show elongated single strands or aggregated bundles. The aggregated state of the polymer is consistent with the high level of flexibility and mobility expected for a highly positively charged polymer strand. Conversely, the visualization of elongated strands indicated the presence of stabilizing interactions with the substrate. Surfaces with varying chemical composition (glass, self-assembled monolayer of mercaptoundecanoic acid/decanethiol and polytetrafluoroethylene (PTFE)) were probed with chitosan modified AFM tips and the corresponding desorption energies, calculated from plateau-like features, were attributed to the desorption of individual polymer strands. Desorption energies of 2.0±0.3×10(-20)J, 1.8±0.3×10(-20)J and 3.5±0.3×10(-20)J were obtained for glass, SAM of mercaptoundecanoic/dodecanethiol and PTFE, respectively. These single molecule level results can be used as a basis for investigating chitosan and chitosan-based materials for biomaterial applications.     

Single molecule force spectroscopy of asphaltene aggregates

Asphaltene aggregation and deposition cause severe problems in nearly all phases of petroleum processing. To resolve those problems, understanding the aggregation mechanisms is a prerequisite and has attracted the interest of a great number of investigators. However, to date, the nature and extent of asphaltene aggregation remain widely debated. In the present study, we attempt to investigate asphaltene aggregation from a completely new perspective. The technique of single molecule force spectroscopy (SMFS) was used to investigate the response of single asphaltene aggregates under an external pulling force. Force curves representing the stretching of single asphaltene aggregates were obtained in simple electrolyte solutions (KCl and calcium) and organic solvents (toluene and heptane). These force curves were well-fitted by the modified worm-like chain model, indicating that those asphaltene aggregates acted like long-chain polymers under pulling by an external force. It was found that lower solution pH values and the presence of divalent cations resulted in a lower bending rigidity of the formed aggregates. The information retrieved from the force curves suggests that asphaltene molecules with a structure featuring small aromatic clusters connected by aliphatic chains do exist and that asphaltene aggregation could occur through a linear polymerization mechanism. The current study extends the application scope of SMFS.     

Intercalation interactions between dsDNA and acridine studied by single molecule force spectroscopy

In this letter, we report on the direct measurement of the intercalation interactions between acridine and double-stranded DNA (dsDNA) using single molecule force spectroscopy. The interaction between acridine and dsDNA is broken by force of 36 pN at a loading rate of 5.0 nN/s. The most probable rupture force between acridine and dsDNA is dependent on the loading rate, indicating that the binding of acridine and dsDNA is a dynamic process. The combination of SMFS experimental data with the theoretical model clearly suggests the presence of two energy barriers along with an unbinding trajectory of acridine-dsDNA.     

EMSA and single-molecule force spectroscopy study of interactions between Bacillus subtilis single-stranded DNA-binding protein and single-stranded DNA

In this article, interactions between Bacillus subtilis single-stranded DNA binding proteins (BsSSB) and single-stranded DNA (ssDNA) were systematically studied. The effect of different molar ratios between BsSSB and ssDNA on their binding modes was first investigated by electrophoretic mobility shift assays (EMSAs). It is found that a high molar ratio of BsSSB to ssDNA can produce BsSSB-ssDNA complexes formed in the mode of two proteins binding one 65-nt (nucleotide) ssDNA whereas a low molar ratio facilitates the formation of BsSSB-ssDNA complexes in the mode of one protein binding one 65-nt ssDNA. Furthermore, two binding modes are in dynamic equilibrium. The unbinding force of BsSSB-ssDNA complexes was measured quantitatively in solutions with different salt concentrations by using AFM-based single-molecule force spectroscopy (SMFS). Our results show that the unbinding force is about 10 pN higher at high salt concentration (0.5 M NaCl) than at low salt concentration (0.1 M NaCl) and the lifetime of BsSSB-ssDNA complexes at high salt concentration is twice as long as that at low salt concentration. These results indicate that more tightly packed BsSSB-ssDNA complexes can form at high salt (0.5 M NaCl) concentration. In addition, the results of EMSA show that ssDNA, which is bound to BsSSB, can dissociate from BsSSB in the presence of the cDNA strand, indicating the dynamic nature of BsSSB-ssDNA interactions.     

Hydration and conformational mechanics of single, end-tethered elastin-like polypeptides

We investigated the effect of temperature, ionic strength, solvent polarity, and type of guest residue on the force-extension behavior of single, end-tethered elastin-like polypeptides (ELPs), using single molecule force spectroscopy (SMFS). ELPs are stimulus-responsive polypeptides that contain repeats of the five amino acids Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. We fitted the force-extension data with a freely jointed chain (FJC) model which allowed us to resolve small differences in the effective Kuhn segment length distributions that largely arise from differences in the hydrophobic hydration behavior of ELP. Our results agree qualitatively with predictions from recent molecular dynamics simulations and demonstrate that hydrophobic hydration modulates the molecular elasticity for ELPs. Furthermore, our results show that SMFS, when combined with our approach for data analysis, can be used to study the subtleties of polypeptide-water interactions and thus provides a basis for the study of hydrophobic hydration in intrinsically unstructured biomacromolecules.     

Single-molecule force spectroscopy of bimolecular reactions: system homology in the mechanical activation of ligand substitution reactions

The mechanochemistry of the bimolecular nucleophilic substitution of DMSO for substituted pyridines at a square-planar pincer Pd(II) center was investigated using single-molecule force spectroscopy (SMFS). The SMFS data are interpreted in terms of the Bell-Evans model, which gives thermal off-rates for two reactions that agree well with previous, stress-free measurements. The characteristic force dependency of the rupture rate, fbeta, is effectively constant for the two reactions examined (22 +/- 2 and 24 +/- 2 pN), and the system homology in the mechanical response is consistent with expected similarities in the reaction potential energy surfaces.     

Feeling Inter‐ or Intramolecular Interactions with the Polymer Chain as Probe: Recent Progress in SMFS Studies on Macromolecular Interactions

Single‐molecule force spectroscopy (SMFS) opens new avenues for elucidating the structures and functions of large coiled molecules such as synthetic and biopolymers at the single‐molecule level. In addition, some of the features in the force–extension curves (i.e. force spectra) are closely related to primary/secondary structures of the molecules being stretched. For example, the long force plateau in the DNA stretching curve is related to the double‐helix structure. These features can be regarded as the force fingerprints of individual macromolecules. These force fingerprints can therefore be used as indicators/criteria of single‐molecule manipulation during the measurement of some unknown intra‐ or intermolecular interactions. By comparing the force spectra of a single polymer chain before and after interaction with other molecules, the mode/strength of such molecular interactions can be derived. This Review focuses on recent advances in AFM‐based SMFS studies on molecular interactions in both synthetic and biopolymer systems using a single macromolecular chain as probe, including interactions between nucleic acids and proteins, mechanochemistry of covalent bonds, conformation‐regulated enzymatic reactions, adsorption and desorption of biopolymers on a flat surface or from the nanopore of a carbon nanotube, and polymer interactions in the condensed state.