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.     

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.     

Following aptamer-ricin specific binding by single molecule recognition and force spectroscopy measurements

Single molecule recognition imaging and dynamic force spectroscopy (DFS) analysis showed strong binding affinity between an aptamer and ricin, which was comparable with antibody-ricin interaction. Molecular simulation showed a ricin binding conformation with aptamers and gave different ricin conformations immobilizing on substrates that were consistent with AFM images.     

Covalent attachment of proteins to calcite surface for the single molecule experiments

Two protocols of covalent attachment of proteins onto the calcite surface, viz. one using the metallochelat and second using the aminohexil, are elaborated. Single molecule force spectroscopy method has been used to test their efficiency and practical applicability. Experiments were performed measuring the specific interaction force between bovine serum albumin (BSA) fixed onto the freshly cleaved calcite single crystal surface (procedure under the study here) and its polyclonal antibody (Ab-BSA) immobilized onto an AFM tip using standard and well studied procedure. We found the conditions, when up to 3–3.5% of tip-sample approaches lead to the formation of a single specific bond.     

Single molecule force spectroscopy using polyproteins

In recent years single molecule force spectroscopy has emerged as a powerful new tool to explore the mechanical stability and folding pathways of individual proteins. This technique is used to apply a stretching force between two points of a protein, unfolding the protein to an extended state. By measuring the unfolding and folding trajectories of individual proteins, insight can be gained into the physical mechanisms of protein folding. In this tutorial review we introduce the reader to single molecule force spectroscopy using the atomic force microscope (AFM), and explain the two main modes of operation of the AFM for force spectroscopy: force-extension and force-clamp. We introduce the approach of using polyproteins to obtain a clear mechanical fingerprint for monitoring the response of proteins to an applied mechanical force. In addition, we provide an informative and representative review of recent research on proteins using single molecule force spectroscopy. We focus on areas which have made a significant contribution to the single molecule protein folding field and highlight emerging areas of research which have wider implications for the general scientific community.     

Direct detection of the formation of V-amylose helix by single molecule force spectroscopy

An important polysaccharide, amylose crystallizes as a regular single left-handed helix from a propanol, butanol, or iodine solution. However, its solution structure remains elusive because amylose does not form molecular solutions in these solvents, and standard spectroscopic techniques cannot be exploited to determine its structure. Using AFM, we forced individual amylose chains adsorbed to a surface to enter these poor solvents and carried out stretch-release measurements on them in solution. In this manner, we directly captured the formation of individual amylose helices induced by butanol and iodine. With an accuracy approaching that of X-ray diffraction on amylose crystals, we determined that the pitch of the helix in solution is 1.3 angstroms/ring. We also directly measured the force driving the formation of the helix in solution to be 50 pN. SMD simulations in explicit butanol reproduced the AFM-measured force-extension curves and revealed that the long plateau feature is caused by the rupture of O(2)n-O(6)(n+6) and O(3)n-O(6)(n+6) hydrogen bonds and by the unwinding of the helix. We also found that amylose helices formed in iodine solution are more compliant and hysteretic as compared to helices in butanol, which extend/relax reversibly. In iodine solution, the formation of the helix is inhibited by force and limited by the slow kinetics of the amylose-iodine complex. By forcing individual molecules into poor solvents and performing force spectroscopy measurements in solution, our AFM approach uniquely supplements X-ray diffraction and NMR methods for investigating solution conformations of insoluble biopolymers.     

Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.     

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.     

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.     

Functionalized self-assembled monolayers on ultraflat gold as platforms for single molecule force spectroscopy and imaging

Single molecule force spectroscopy is a valuable tool for studying unfolding and nanomechanical properties of proteins. The common practice is to stretch proteins from a surface that was dosed to give a reasonable hit rate and to analyze the curves that exhibit the expected characteristics of a single polymer. Whether the surface-bound proteins are indeed single and isolated remains unclear, and the undesirable protein/surface interactions that obscure informative features of the force curves are implicitly assumed to be absent. In this study, mixed self-assembled monolayers (SAMs) consisting of N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG) terminated thiols on an ultraflat gold surface were used to covalently immobilize proteins via lysine residues. By the optimization of attachment sites via lysine-NHS linkages amidst a protein-resistant layer of the OEG SAM, it was possible to isolate single proteins for study in a controlled fashion. The single protein distribution on the surface is clearly demonstrated by atomic force microscopy (AFM) imaging. The OEG also significantly reduces nonspecific tip-surface interactions between the cantilever and surface. Stretching covalently attached single proteins produces high-quality and reproducible force-extension curves. This experimental strategy is an attractive platform with which to study protein structure, interactions, and nanomechanical properties of single proteins.     

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．     

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).     

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.     

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.     

Oriented purple membrane monolayers covalently attached to gold by multiple thiole linkages analyzed by single molecule force spectroscopy

Highly oriented monolayers of bacteriorhodopsin (BR) in purple membrane (PM) form are obtained by the reaction of BR-Q3C, where a cysteine was introduced into the N-terminal region, with a gold surface. Single molecule force spectroscopy was used to show that about 50% of the BRs are covalently bound to the surface. The linkage between the cysteine and the gold causes an additional characteristic peak in the force-distance curves to appear. Because several thousand cysteine-gold bonds exist between each PM patch and the surface, the PM is irreversibly bound. Such oriented PM monolayers may serve as an interface between metal surfaces and biomaterials, which may be linked to the PM surface chemically. Photoelectric applications of BR will benefit from the high degree of orientation obtained by this method.     

High-throughput single molecule screening of DNA and proteins

We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification.     

Single molecule force spectroscopy on poly(vinyl alcohol) by atomic force microscopy

The elastic properties of poly(vinyl alcohol) (PVA) were investigated on the nanoscale using the new technique of single molecule force spectroscopy by atomic force microscopy (AFM). It was found that the elastic properties of PVA molecules scale linearly with their contour lengths. This finding corroborates that the deformation of individual PVA chains is measured. The force spectra of PVA show a kink at around 200 pN and cannot be fitted by an extended Langevin function. The deviation of the elastic behavior of PVA from a freely jointed chain model may indicate the presence of a suprastructure of PVA in NaCl solution.     

Flexibility of phenylene oligomers revealed by single molecule spectroscopy

The rigidity of a p-phenylene oligomer (p-terphenyl) has been investigated by single molecule confocal fluorescence microscopy. Two different rylene diimide dyes attached to the terminal positions of the oligomer allowed for wavelength selective excitation of the two chromophores. In combination with polarization modulation the spatial orientation of the transition dipoles of both end groups could be determined independently. We have analyzed 597 single molecules in two different polymer hosts, polymethylmethacrylate and Zeonex. On average we find a 22 degrees deviation from the linear gas phase geometry (T = 0 K), indicating a rather high flexibility of the p-phenylene oligomer independent of the matrix. To substantiate our experimental results, we have performed quantum chemical calculations at the density functional theory level for the molecular geometry and the electronic excitations. Our findings are in agreement with former experiments on the persistence length of poly(p-phenylenes).     

Super- and sub-Poissonian photon statistics for single molecule spectroscopy

We investigate the distribution of the number of photons emitted by a single molecule undergoing a spectral diffusion process and interacting with a continuous wave laser field. The spectral diffusion is modeled based on a stochastic approach, in the spirit of the Anderson-Kubo line shape theory. Using a generating function formalism we solve the generalized optical Bloch equations and obtain an exact analytical formula for the line shape and Mandel's Q parameter. The line shape exhibits well-known behaviors, including motional narrowing when the stochastic modulation is fast and power broadening. The Mandel parameter, describing the line shape fluctuations, exhibits a transition from a quantum sub-Poissonian behavior in the fast modulation limit to a classical super-Poissonian behavior found in the slow modulation limit. Our result is applicable for weak and strong laser fields, namely, for arbitrary Rabi frequency. We show how to choose the Rabi frequency in such a way so that the quantum sub-Poissonian nature of the emission process becomes strongest. A lower bound on Q is found and simple limiting behaviors are investigated. A nontrivial behavior is obtained in the intermediate modulation limit, when the time scales for spectral diffusion and the lifetime of the excited state become similar. A comparison is made between our results and previous ones derived, based on the semiclassical generalized Wiener-Khintchine formula.