Dario Anselmetti

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.

Forces in scanning probe methods

Preface. Introduction to Scanning Probe Methods. Instrumentation. Theory. Metallic Adhesion. Photons. Friction. Nano and Micromechanics. Magnetic Storage and Magnetic Forces. Applications. AFM in Liquids. Organics and Biology. Author Index. Subject Index.

Soft, entirely photoplastic probes for scanning force microscopy

A new probe made entirely of plastic material has been developed for scanning probe microscopy. Using a polymer for the cantilever facilitates the realization of mechanical properties that are difficult to achieve with classical silicon technology. The new cantilever and tip presented here are made of an epoxy-based photoplastic. The fabrication process is a simple batch process in which the integrated tip and the lever are defined in one photolithography step. The simplicity of the fabrication step, the use of a polymer as material, and the ability to reuse the silicon mold lead to a soft low-cost probe for scanning force microscopy. Imaging soft condensed matter with photoplastic levers, which uses laser beam deflection sensing, exhibits a resolution that compares well with that of commercially available silicon cantilevers.

Screw dislocation mediated growth of sputtered and laser-ablated YBa2Cu3O7−δ films

By imaging the as-grown surfaces of sputtered and laserablated YBa2Cu3O7−δ films with scanning tunneling microscopy (STM), we have directly observed spiral-shaped growth terraces which emanate from screw dislocations. The density of screw dislocations was observed to decrease with increasing growth temperature and substrate misorientation. The surface structures observed by STM together with cross-sectional transmission electron microscope (TEM) images provide insights into the mechanisms of crystal growth operative during the formation of YBa2Cu3O7−δ films grown using these two widespread techniques.

De novo desmin mutation N116S is associated with arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease, frequently accompanied by sudden cardiac death and terminal heart failure. Genotyping of ARVC patients might be used for palliative treatment of the affected family. We genotyped a cohort of 22 ARVC patients referred to molecular genetic screening in our heart center for mutations in the desmosomal candidate genes JUP, DSG2, DSC2, DSP and PKP2 known to be associated with ARVC. In 43% of the cohort, we found disease-associated sequence variants. In addition, we screened for desmin mutations and found a novel desmin-mutation p.N116S in a patient with ARVC and terminal heart failure, which is located in segment 1A of the desmin rod domain. The mutation leads to the aggresome formation in cardiac and skeletal muscle without signs of an overt clinical myopathy. Cardiac aggresomes appear to be prominent, especially in the right ventricle of the heart. Viscosimetry and atomic force microscopy of the desmin wild-type and N116S mutant isolated from recombinant Escherichia coli revealed severe impairment of the filament formation, which was supported by transfections in SW13 cells. Thus, the gene coding for desmin appears to be a novel ARVC gene, which should be included in molecular genetic screening of ARVC patients.

Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti - a combined molecular biology and force spectroscopy investigation

Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28 bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165 pN in a logarithmic dependence from the loading rates of 70-79000 pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of k(off)=(1.2+/-1.0) x 10(-3)s(-1) were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism.

Electrodeless dielectrophoresis for bioanalysis: Theory, devices and applications

Dielectrophoresis is a non‐destructive, label‐free method to manipulate and separate (bio‐) particles and macromolecules. The mechanism is based on the movement of polarizable objects in an inhomogeneous electric field. Here, microfluidic devices are reviewed that generate those inhomogeneous electric fields with insulating posts or constrictions, an approach called electrodeless or insulator‐based dielectrophoresis. Possible advantages compared to electrode‐based designs are a less complex, monolithic fabrication process with low‐cost polymeric substrates and no metal surface deterioration within the area of sample analysis. The electrodeless design has led to novel devices, implementing the functionality directly into the channel geometry and covering many areas of bioanalysis, like manipulation and separation of particles, cells, DNA, and proteins.

CORRELATION BETWEEN JC AND SCREW DISLOCATION DENSITY IN SPUTTERED YBA2CU3O7-DELTA FILMS

Electric transport properties of sputtered YBa2Cu3O7−δ films were studied as a function of screw dislocation density, ranging from 5·107 cm−2 to 1.3·109 cm−2 as determined at the film surface. A correlation was found between the number of screw dislocations and the critical current density (Jc). Films with higher screw dislocation densities have higher critical current densities and a slower drop ofJc as a function of applied magnetic fieldH.

Identification of Binding Mechanisms in Single Molecule–DNA Complexes

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.