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ABSTRACT: Platelets are essential in hemostasis. Upon activation they undergo a shape-change accompanied with receptor presentation. Atomic Force Microscopy (AFM) imaging and Single Molecule Force Spectrocopy (SMFS) were used as powerful tools for exploring morphological changes as well as receptor activities of platelets. Imaging time series was accomplished with and without fixation steps at the single platelet level. Hereby the response of mechanical stimulation of the platelet by the AFM cantilever tip was directly observed. We demonstrate that living and fixed platelets develop filopodia after a short activation time followed by their disappearance including cellular bleb formation. Thereafter a second filopodia formation (filopodia extrusion) was observed; those filopodia subsequently disappeared again, and finally platelets detached from the support due to cell death. We determined the influence of mechanical stress on the chronology of morphological changes of platelets and demonstrated shear force induced filopodia formation. Through recordings over several hours, topographical AFM images over the full platelet lifetime - from early activation up to apoptosis - are presented. SMFS measurements on living platelets allowed determining the activation state of the most prominent membrane receptor integrin αIIbβ3 at all different phases of activation. αIIbβ3 was fully activated, independent of the morphological state.
Methods 04/2013; · 4.01 Impact Factor
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ABSTRACT: Atomic scale x-ray photon correlation spectroscopy (aXPCS) was used to study atomic diffusion in the Ni(97)Pt(3) solid solution with both a single crystal and a polycrystalline sample. Different jump diffusion models are discussed using experimental results and Monte Carlo simulations. The sensitivity of aXPCS experiments to short-range order (in this case governed by a strong Pt-Pt repulsive force) is demonstrated. The activation energy of 2.93(10) eV as well as diffusivities in the range of 10(-23) m(2) s(-1) at 830 K agree very well with the results of tracer diffusion studies at much higher temperatures.
Journal of Physics Condensed Matter 01/2013; 25(6):065401. · 2.55 Impact Factor
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ABSTRACT: Nucleic acids (NAs), including antisense oligonucleotides, small interfering RNA (siRNA), aptamers and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), that were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases . The phage protein/siRNA complexes can make a new type of highly selective, stable, active and physiologically acceptable cancer nanomedicines.
Molecular Pharmaceutics 12/2012; · 4.78 Impact Factor
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ABSTRACT: In this study, we demonstrate the increased performance in speed and sensitivity achieved by the use of small AFM cantilevers on a standard AFM system. For this, small rectangular silicon oxynitride cantilevers were utilized to arrive at faster atomic force microscopy (AFM) imaging times and more sensitive molecular recognition force spectroscopy (MRFS) experiments. The cantilevers we used had lengths between 13 and 46 μm, a width of about 11 μm, and a thickness between 150 and 600 nm. They were coated with chromium and gold on the backside for a better laser reflection. We characterized these small cantilevers through their frequency spectrum and with electron microscopy. Due to their small size and high resonance frequency we were able to increase the imaging speed by a factor of 10 without any loss in resolution for images from several μm scansize down to the nanometer scale. This was shown on bacterial surface layers (s-layer) with tapping mode under aqueous, near physiological conditions and on nuclear membranes in contact mode in ambient environment. In addition, we showed that single molecular forces can be measured with an up to 5 times higher force sensitivity in comparison to conventional cantilevers with similar spring constants.
Micron 06/2012; 43(12):1399-407. · 1.53 Impact Factor
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ABSTRACT: Recognizing pDNA supercoils: Differently supercoiled species of the same plasmid DNA can be separated by topology-selective chromatography. Two-dimensional HPLC proved that the supercoiling changes during fermentation. Thus, a new quality criterium might help to optimize the effectivity of future genetic drugs and vaccines.
Angewandte Chemie International Edition 11/2011; 51(1):267-70. · 13.45 Impact Factor
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ABSTRACT: Single-molecule characterization is essential for ascertaining the structural and functional properties of bottom-up DNA nanostructures. Here we enlist three atomic force microscopy (AFM) techniques to examine tetrahedron-shaped DNA nanostructures that are functionally enhanced with small chemical tags. In line with their application for biomolecule immobilization in biosensing and biophysics, the tetrahedra feature three disulfide-modified vertices to achieve directed attachment to gold surfaces. The remaining corner carries a single bioligand that can capture and present individual cargo biomolecules at defined lateral nanoscale spacing. High-resolution AFM topographic imaging confirmed the directional surface attachment as well as the highly effective binding of individual receptor molecules to the exposed bioligands. Insight into the binding behavior at the single-molecule level was gained using molecular recognition force spectroscopy using an AFM cantilever tip with a tethered molecular receptor. Finally, simultaneous topographic and recognition imaging demonstrated the specific receptor-ligand interactions on individual tetrahedra. In summary, AFM characterization verified that the rationally designed DNA nanostructures feature characteristics to serve as valuable immobilization agents in biosensing, biophysics, and cell biology.
ACS Nano 08/2011; 5(9):7048-54. · 10.77 Impact Factor
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ABSTRACT: Herein, we report on the in vitro change of DNA conformation of plasmids bound to a 3-aminopropyl-modified mica surface and monitoring the events by atomic force microscopy (AFM) imaging under near physiological conditions. In our study, we used an intercalating drug, chloroquine, which is known to decrease the twist of the double helix and thus altered the conformation of the whole DNA. During our experiments, a chloroquine solution was added while imaging a few highly condensed plasmid nanoparticles in solution. AFM images recorded after the drug addition clearly show a time-resolved relaxation of these bionanoparticles into a mixture of loose DNA strands.
Analytical and Bioanalytical Chemistry 07/2011; 402(1):373-80. · 3.78 Impact Factor
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ABSTRACT: Incoherent quasi-elastic scattering of neutrons or gamma rays determines the jump vectors of diffusing single atoms. Coherent quasi-elastic scattering beyond that contains additional information on the interaction between atoms. Here we firstly derive the influence of weak interactions (i.e. the high-temperature limit) on the correlation times taking an analytical approach via transition state theory, different from and, in our opinion, easier to follow than the lattice dynamics approach by Sinha and Ross. As de Gennes had already argued earlier, short-range order increases the correlation time (or equivalently narrows the quasi-elastic line) proportional to the diffuse intensity at the corresponding wavevector transfer. Secondly we show by way of Monte Carlo simulations that for strong interactions the deviations from the analytical theory bear additional information about the jump processes. We compare our theory with the results of a recent XPCS experiment.
Journal of Physics Condensed Matter 06/2011; 23(25):254206. · 2.55 Impact Factor
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ABSTRACT: Knowledge of atomic diffusion is a fundamental issue in synthesis and stability of materials. Direct studies of the elementary diffusion event, that is, how the individual atoms 'jump', are scarce, as the available techniques are limited to selected systems. Here we show how by monitoring the spatial and temporal variations of the scattered coherent X-ray intensity the diffusion of single atoms can be studied. This is demonstrated for the intermetallic alloy Cu(90)Au(10). By measuring along several directions in reciprocal space, we can elucidate the dynamical behaviour of single atoms as a function of their neighbourhood. This method, usually referred to as X-ray photon correlation spectroscopy (XPCS), does not rely on specific atomic species or isotopes and can thus be applied to almost any system. Thus, given the advent of the next-generation X-ray sources, XPCS has the potential to become the main method for quantitatively understanding diffusion on the atomic scale.
Nature Material 08/2009; 8(9):717-20. · 32.84 Impact Factor
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Jilin Tang,
Andreas Ebner,
Bernhard Kraxberger, Michael Leitner,
Alba Hykollari,
Christian Kepplinger,
Christian Grunwald,
Hermann J Gruber,
Robert Tampé,
Uwe B Sleytr,
Nicola Ilk,
Peter Hinterdorfer
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ABSTRACT: Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His(6)-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His(6)-tags. The accessibility of the His(6)-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His(6)-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris-NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His(6)-tags in the presence of nickel ions. Thus the His(6)-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris-NTA derivatives.
Journal of Structural Biology 03/2009; 168(1):217-22. · 3.41 Impact Factor
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Jilin Tang,
Andreas Ebner,
Helga Badelt-Lichtblau,
Christine Völlenkle,
Christian Rankl,
Bernhard Kraxberger, Michael Leitner,
Linda Wildling,
Hermann J Gruber,
Uwe B Sleytr,
Nicola Ilk,
Peter Hinterdorfer
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ABSTRACT: Functional nanoarrays were fabricated using the chimeric bacterial cell surface layer (S-layer) protein rSbpA fused with the affinity tag Strep-tagII and characterized using various atomic force microscopy (AFM) techniques in aqueous environment. The accessibility of Strep-tagII was verified by single-molecule force spectroscopy studies employing Strep-Tactin as specific ligand. Simultaneous topography and recognition imaging (TREC) of the nanoarray yielded high resolution maps of the Strep-tagll binding sites with a positional accuracy of 1.5 nm. The nanoarrays were used as template for constructing highly ordered molecular binding blocks.
Nano Letters 01/2009; 8(12):4312-9. · 13.20 Impact Factor
