Mechanical manifestations of rare atomic jumps in dynamic force microscopy.

The resonance frequency and the excitation amplitude of a silicon cantilever have been measured as a function of distance to a cleaved KBr(001) surface with a low-temperature scanning force microscope (SFM) in ultrahigh vacuum. We identify two regimes of tip-sample distances. Above a site-dependent critical tip-sample distance reproducible data with low noise and no interaction-induced energy dissipation are measured. In this regime reproducible SFM images can be recorded. At closer tip-sample distances, above two distinct atomic sites, the frequency values jump between two limiting curves on a timescale of tens of milliseconds. Furthermore, additional energy dissipation occurs wherever jumps are observed. We attribute both phenomena to rarely occurring changes in the tip apex configuration which are affected by short-range interactions with the sample. Their respective magnitudes are related to each other. A specific candidate two-level system is also proposed.

Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA.

The availability of entire genome sequences has triggered the development of microarrays for clinical diagnostics that measure the expression levels of specific genes. Methods that involve labelling can achieve picomolar detection sensitivity, but they are costly, labour-intensive and time-consuming. Moreover, target amplification or biochemical labelling can influence the original signal. We have improved the biosensitivity of label-free cantilever-array sensors by orders of magnitude to detect mRNA biomarker candidates in total cellular RNA. Differential gene expression of the gene 1-8U, a potential marker for cancer progression or viral infections, has been observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. This qualifies the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression or therapy response. We foresee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

Energy level alignment at metal-octaethylporphyrin interfaces.

We studied the electronic structure of copper-octaethylporphyrin (CuEOP) adsorbed on three metal surfaces--Ag(001), Ag(111), and Cu(111)--by means of ultraviolet photoelectron spectroscopy (UPS). The adsorption-induced work function shifts saturate roughly beyond two monolayers. The saturation values are substrate dependent, negative, and range from -1.30 to -0.85 eV. This shift is larger than that for tetraphenylporphyrins. The two highest occupied molecular orbitals (HOMO and HOMO-1) of the organic are clearly resolved in the UPS spectra. The origin of the negative work function shift is discussed.

Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy.

Myomesin is the most prominent structural component of the sarcomeric M-Band that is expressed in mammalian heart and skeletal muscles. Like titin, this protein is an intracellular member of the Ig-fibronectin superfamily, which has a flexible filamentous structure and which is largely composed of two types of domain that are similar to immunoglobulin (Ig)-like and fibronectin type III (FNIII) domains. Several myomesin isoforms have been identified, and their expression patterns are highly regulated both spatially and temporally. Particularly, alternative splicing in the central part of the molecule gives rise to an isoform, EH (embryonic heart)-myomesin, containing a serine and proline-rich insertion with no well-defined secondary structure, the EH segment. EH-myomesin represents the major myomesin isoform at embryonic stages of mammalian heart and is rapidly down-regulated around birth, but it is re-expressed in the heart of patients suffering from dilated cardio-myopathy. Here, in order to facilitate a better understanding of the physiological, and possibly pathological, functions of myomesin proteins, we explore the mechanical stability, elasticity and force-driven structural changes of human myomesin's sub-molecular segments using single-molecule force spectroscopy and protein engineering. We find that human myomesin molecules are composed of modules (Ig and FNIII), that are designed to withstand force and we demonstrate that the human cardiac EH segment functions like an additional elastic stretch in the middle part of the EH-myomesin and behaves like a random coil. Consequently myomesin isoforms (proteins with or without the EH segment) have different elastic properties, the EH-myomesin being the more compliant one. These findings imply that the compliance of the M-band increases with the amount of EH-myomesin it contains. So, we provide the evidence that not only titin but also other sarcomeric proteins have complicated visco-elastic properties depending on the contractile parameters in different muscle types.

Octyl-decorated Fréchet-type dendrons: a general motif for visualisation of static and dynamic behaviour using scanning tunnelling microscopy?

A detailed STM study of monolayers of 3,5-bis[(3,5-bisoctyloxyphenyl)methyloxy]benzaldehyde and 3,5-bis[(3,5-bisoctyloxyphenyl)methyloxy]benzyl alcohol adsorbed on graphite is presented. Very highly resolved scanning tunnelling microscopy images are observed at room temperature in air allowing the analysis of the conformation of the adsorbed molecules. These long-chain alkyl-decorated Fréchet-type dendrons are a powerful assembly motif and initially form a pattern based on trimeric units, assembled into hexagonal host structures with a pseudo-unit cell of seven molecules, one of which remains highly mobile. Over time, the supramolecular ordering changes from a trimeric into a dimeric pattern. The chirality arising from the adsorption onto a surface of the dendrons is discussed.

Sublattice identification in scanning force microscopy on alkali halide surfaces.

We propose and apply to the KBr(001) surface a new procedure for species recognition in scanning force microscopy (SFM) of ionic crystal surfaces which show a high symmetry of the charge arrangement. The method is based on a comparison between atomistic simulations and site-specific frequency versus distance measurements. First, by taking the difference of force-distance curves extracted at a few judiciously chosen surface sites we eliminate site-independent long-range forces. The obtained short-range force differences are then compared with calculated ones assuming plausible tip apex models. This procedure allows for the first time identification of the tip apex polarity and of the positive and negative sublattices in SFM images of the (001) cleavage surface of an ionic crystal with the rock salt structure.

Quantitative measurement of short-range chemical bonding forces.

We report direct force measurements of the formation of a chemical bond. The experiments were performed using a low-temperature atomic force microscope, a silicon tip, and a silicon (111) 7x7 surface. The measured site-dependent attractive short-range force, which attains a maximum value of 2.1 nanonewtons, is in good agreement with first-principles calculations of an incipient covalent bond in an analogous model system. The resolution was sufficient to distinguish differences in the interaction potential between inequivalent adatoms, demonstrating the ability of atomic force microscopy to provide quantitative, atomic-scale information on surface chemical reactivity.

Low temperature scanning force microscopy of the Si(111)-(7x7) surface

A low temperature scanning force microscope (SFM) operating in a dynamic mode in ultrahigh vacuum was used to study the Si(111)- (7x7) surface at 7.2 K. Not only the twelve adatoms but also the six rest atoms of the unit cell are clearly resolved for the first time with SFM. In addition, the first measurements of the short range chemical bonding forces above specific atomic sites are presented. The data are in good agreement with first principles computations and indicate that the nearest atoms in the tip and sample relax significantly when the tip is within a few A of the surface.

Velocity dependence of atomic friction

Sliding friction between the tip of a friction force microscope and NaCl(100) was studied to deduce the velocity dependence of friction forces on the atomic scale. A logarithmic dependence of the mean friction force is revealed at low velocities. The experimental data are interpreted in terms of a modified Tomlinson model which is based on reaction rate theory.

Dependence of formation energies of tin nanoclusters on their size and shape

In a novel molecular beam experiment we have calorimetrically investigated the dependence of the formation energies of isolated Sn(N) clusters on their size. The experimentally determined size dependence of the formation energy for Sn(N) clusters consisting of between 95 and 975 atoms can be explained by the existence of two different types of cluster isomers: One class of isomers is characterized by formation energies proportional to N(-1/3), indicating compact spherical-like shapes. The other class has constant formation energies for the investigated size range, which is consistent with quasi-one-dimensional geometries.

Model energy landscapes and the force-induced dissociation of ligand-receptor bonds.

We discuss models for the force-induced dissociation of a ligand-receptor bond, occurring in the context of cell adhesion or single molecule unbinding force measurements. We consider a bond with a structured energy landscape which is modeled by a network of force dependent transition rates between intermediate states. The behavior of a model with only one intermediate state and a model describing a molecular zipper is studied. We calculate the bond lifetime as a function of an applied force and unbinding forces under an increasing applied load and determine the relationship between both quantities. The dissociation via an intermediate state can lead to distinct functional relations of the bond lifetime on force. One possibility is the occurrence of three force regimes where the lifetime of the bond is determined by different transitions within the energy landscape. This case can be related to recent experimental observations of the force-induced dissociation of single avidin-biotin bonds.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates.

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Translating biomolecular recognition into nanomechanics.

We report the specific transduction, via surface stress changes, of DNA hybridization and receptor-ligand binding into a direct nanomechanical response of microfabricated cantilevers. Cantilevers in an array were functionalized with a selection of biomolecules. The differential deflection of the cantilevers was found to provide a true molecular recognition signal despite large nonspecific responses of individual cantilevers. Hybridization of complementary oligonucleotides shows that a single base mismatch between two 12-mer oligonucleotides is clearly detectable. Similar experiments on protein A-immunoglobulin interactions demonstrate the wide-ranging applicability of nanomechanical transduction to detect biomolecular recognition.

Dynamic force spectroscopy of single DNA molecules.

To explore the analytic relevance of unbinding force measurements between complementary DNA strands with an atomic force microscope, we measured the forces to mechanically separate a single DNA duplex under physiological conditions by pulling at the opposite 5'-ends as a function of the loading rate (dynamic force spectroscopy). We investigated DNA duplexes with 10, 20, and 30 base pairs with loading rates in the range of 16-4,000 pN/s. Depending on the loading rate and sequence length, the unbinding forces of single duplexes varied from 20 to 50 pN. These unbinding forces are found to scale with the logarithm of the loading rate, which is interpreted in terms of a single energy barrier along the mechanical separation path. The parameters describing the energy landscape, i.e. , the distance of the energy barrier to the minimum energy along the separation path and the logarithm of the thermal dissociation rate, are found to be proportional to the number of base pairs of the DNA duplex. These single molecule results allow a quantitative comparison with data from thermodynamic ensemble measurements and a discussion of the analytic applications of unbinding force measurements for DNA.

Plasmon transmissivity and reflectivity of narrow grooves in a silver film.

Surface plasmon (SP) reflectivity and transmissivity of narrow grooves in silver films are studied. The SP source is the probe of a scanning near-field optical microscope. Locally detected leakage radiation from the SP provides detailed information on the paths of SP propagation, in particular the influence of perturbations. Global detection provides representative average data on the SP properties of a given metal film and its structures. A groove of 200 nm width, for instance, reflects/transmits about 15%/80% of 'blue-green' SP radiation at normal incidence.

Specific antigen/antibody interactions measured by force microscopy.

Molecular recognition between biotinylated bovine serum albumin and polyclonal, biotin-directed IG antibodies has been measured directly under various buffer conditions using an atomic force microscope (AFM). It was found that even highly structured molecules such as IgG antibodies preserve their specific affinity to their antigens when probed with an AFM in the force mode. We could measure the rupture force between individual antibody-antigen complexes. The potential and limitations of this new approach for the measurement of individual antigen/antibody interactions and some possible applications are discussed.

Binding strength between cell adhesion proteoglycans measured by atomic force microscopy.

Measurement of binding forces intrinsic to adhesion molecules is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. Atomic force microscopy was used to measure the binding strength between cell adhesion proteoglycans from a marine sponge. Under physiological conditions, the adhesive force between two cell adhesion molecules was found to be up to 400 piconewtons. Thus a single pair of molecules could hold the weight of 1600 cells. High intermolecular binding forces are likely to form the basis for the integrity of the multicellular sponge organism.