Papers by Dario Anselmetti
Beilstein Journal of Nanotechnology, Sep 29, 2011
In fluorescence microscopy and spectroscopy, energy transfer processes between single fluorophore... more In fluorescence microscopy and spectroscopy, energy transfer processes between single fluorophores and fluorophore quencher pairs play an important role in the investigation of molecular distances or orientations. At distances larger than about 3 nm these effects originate predominantly from dipolar coupling. As these experiments are commonly performed in homogenous media, effects at the interface boundaries can be neglected. Nevertheless, the combination of such assays with single-molecule manipulation techniques such as atomic force microscopy (AFM) requires a detailed understanding of the influence of interfaces on dipolar coupling effects. In the presented work we used a combined total internal reflection fluorescence microscopy (TIRFM)-AFM setup to elucidate this issue. We measured the fluorescence emission emanating from single quantum dots as a function of distance from the apex of a gold-coated cantilever tip. As well as fluorescence quenching at close proximity to the tip, we found a nonlinear and nonmonotonic distance dependence of the fluorescence emission. To confirm and interpret our findings we performed calculations on the basis of a simplified multiple multipole (MMP) approach, which successfully supports our experimental data. Moreover, we revealed and quantified the influence of interfering processes such as field enhancement confined at interface boundaries, mirror dipoles and (resonant) dipolar coupling.
Physical Chemistry Chemical Physics, 2010
The specific interaction of a supramolecular binding motif was quantitatively evaluated by dynami... more The specific interaction of a supramolecular binding motif was quantitatively evaluated by dynamic single-molecule force spectroscopy (SMFS) using an atomic force microscope (AFM). The supramolecular capsule forms by two different cavitands stitched together by four hydrogen bonds between carboxylic acid and pyridyl groups. The tetra(carboxyl)cavitand is monofunctionalized at the lower rim with a flexible poly(ethylene glycol) linker and attached to the AFM sensor tip. Single-molecule association experiments are accomplished using a diluted self-assembled monolayer (SAM) of the tetra(pyridyl)cavitand on a gold substrate. The measured single-molecule dissociation forces of the heterodimeric capsule represent the mechanical stability of the supramolecular system and allow a quantitative evaluation of the interaction according to the Bell-Evans model yielding dissociation rate constant k off = (0.14 AE 0.14) s À1 , reaction length x b = (0.56 AE 0.076) nm and an estimated value of DG 0 = À27 kJ mol À1 .
Biophysical Journal, Apr 1, 2015
In biological adhesion, the biophysical mechanism of specific biomolecular interaction can be div... more In biological adhesion, the biophysical mechanism of specific biomolecular interaction can be divided in slip and catch bonds, respectively. Conceptually, slip bonds exhibit a reduced bond lifetime under increased external force and catch bonds, in contrast, exhibit an increased lifetime (for a certain force interval). Since 2003, a handful of biological systems have been identified to display catch bond properties. Upon investigating the specific interaction between the unique hydrophilic domain (HD) of the human cell-surface sulfatase Sulf1 against its physiological glycosaminoglycan (GAG) target heparan sulfate (HS) by single molecule force spectroscopy (SMFS), we found clear evidence of catch bond behavior in this system. The HD, ~320 amino acids long with dominant positive charge, and its interaction with sulfated GAG-polymers were quantitatively investigated using atomic force microscopy (AFM) based force clamp spectroscopy (FCS) and dynamic force spectroscopy (DFS). In FCS experiments, we found that the catch bond character of HD against GAGs could be attributed to the GAG 6-O-sulfation site whereas only slip bond interaction can be observed in a GAG system where this site is explicitly lacking. We interpreted the binding data within the theoretical framework of a two state two path model, where two slip bonds are coupled forming a double-well interaction potential with an energy difference of DE z 9 k B T and a compliance length of Dx z 3.2 nm. Additional DFS experiments support this assumption and allow identification of these two coupled slip-bond states that behave consistently within the Kramers-Bell-Evans model of force-mediated dissociation.
Hexagonal and nonhexagonal superlattice structures on stage-1 alkali metal graphite intercalation compounds studied by scanning tunnelling microscopy
Synthetic Metals, Sep 1, 1990
ABSTRACT
Amino Acids, Mar 7, 2013
Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the ... more Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based singlemolecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide-and protein-DNA interactions are given.
Physical review, May 15, 1989
Scanning tunneling microscopy (STM) has been used to study the surface of C6Li as a stage-1 donor... more Scanning tunneling microscopy (STM) has been used to study the surface of C6Li as a stage-1 donor graphite intercalation compound from a submicrometer down to the atomic scale. Ordered superlattices commensurate as well as incommensurate with the graphite lattice have been observed. The measured STM corrugation at small bias voltage (&200 mV) has been found to be similar to graphite whereas at larger bias voltage a strong decrease of the corrugation was observed. This ex- perimental result is compared with recent theoretical predictions. Graphite intercalation compounds (GIC's)' have been the object of numerous experimental and theoretical in- vestigations in recent years due to their interesting elec- tronic, optical, and transport properties. Their microscopic bulk structure has been studied in detail by high- resolution transmission electron microscopy. On the other hand, only a little is known about the surface struc- ture of GIC's on a submicrometer scale. Pioneering work in this field has been reported by Levi-Setti et aI. ' They developed a high-resolution scanning-ion microprobe
Ultramicroscopy, Sep 1, 2010
We systematically studied the origin of surface patterns observed on single Sinorhizobium melilot... more We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.
Biological materials studied with dynamic force microscopy
Journal of vacuum science & technology, May 1, 1994
Biological materials such as hexagonal packed intermediate (HPI) layers, DNA, tobacco mosaic viru... more Biological materials such as hexagonal packed intermediate (HPI) layers, DNA, tobacco mosaic virus and collagen deposited on various substrates with noncontact dynamic force microscopy under ambient conditions were investigated. This method is highly suited for the investigation of soft organic matter where a minimized interaction between tip and sample is needed for nondestructive and reliable operation. Hence, additional anchoring of the biological specimens was no longer found to be crucial. The vertical and lateral resolution limits of this gentle method were determined to be <0.1 nm and 1–2 nm, respectively, allowing very stable and high resolution results on all investigated systems. By taking approach curves and monitoring the dynamic properties of the cantilever (resonance frequency and Q value) during the experiment, the interaction mechanism between tip and sample was found to be dominated by attractive van der Waals interaction and capillary forces. Furthermore, initial results from a HPI layer imaged with noncontact dynamic force microscopy in a water environment are presented.
Nanotechnology, Apr 1, 1994
We have applied the non-contact dynamic force microscopy method to investigate soft biological ma... more We have applied the non-contact dynamic force microscopy method to investigate soft biological materials such as hexagonally-packed intermediate layers, DNA, and tobacco mosaic virus under ambient conditions. This method, where a stiff cantilever is oscillated close to its resonance frequency with an amplitude of 0.3-1.5 nm above the sample, allows highly reliable investigation of soft organic matter with minimized normal and lateral forces between tip and sample. The vertical and lateral resolution are determined to be c 1 A and 1-2 nm, respectively, comparing favorably to established results from repulsive-mode scanning force microscopy experiments on adsorbate covered surfaces in liquids. The interaction forces are found to be attractive, dominated by damping mechanisms and attractive force gradients of capillary and van der Waals interactions.
Zeitschrift für Physik, Jun 1, 1992
Electric transport properties of sputtered YBa2Cu307_ ~ films were studied as a function of screw... more Electric transport properties of sputtered YBa2Cu307_ ~ 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 (J~). Films with higher screw dislocation densities have higher critical current densities and a slower drop of J~ as a function of applied magnetic field H.
In this paper, we report on the performance of electrophoretical separation and laser-induced flu... more In this paper, we report on the performance of electrophoretical separation and laser-induced fluorescence (LIF) detection of dyes and fluorescently labeled biomolecules in poly(dimethylsiloxane) (PDMS) microdevices. The dyes fluorescein and fluorescein isothiocyanate (FITC) have been separated effectively in nM concentrations. Fluorescein injections gave linear concentration response in the range from 4 to 100 pM. As ultimate detection sensitivity, 100 fM injected fluorescein was obtained. Further, 100 fM injected fluorescein could be detected. This is to our knowledge the smallest electrokinetically injected dye concentration detected on a microchip. Injection studies of fluorescently labeled avidin revealed a theoretical detection limit of 25 nM for laser-induced fluorescence detection in good agreement with separations in glass chips. Furthermore, the injection of several and even one single DNA molecule using a PDMS cross injector has been demonstrated as well as free solution separation ofand T2-DNA (60 pM each) in periodically structured channels.
Single cell analytics is a key method in the framework of proteom research allowing analyses, whi... more Single cell analytics is a key method in the framework of proteom research allowing analyses, which are not subjected to ensemble-averaging, cell-cycle or heterogeneous cell-population effects. Our previous studies on single cell analysis in poly(dimethylsiloxane) microfluidic devices with native label-free laser induced fluorescence detection [W. Hellmich, C. Pelargus, K. Leffhalm, A. Ros, D. Anselmetti, Electrophoresis 26 ( ) 3689] were extended in order to improve separation efficiency and detection sensitivity. Here, we particularly focus on the influence of poly(oxyethylene) based coatings on the separation performance. In addition, the influence on background fluorescence is studied by the variation of the incident laser power as well as the adaptation of the confocal volume to the microfluidic channel dimensions. Last but not least, the use of carbon black particles further enhanced the detection limit to 25 nM, thereby reaching the relevant concentration ranges necessary for the label-free detection of low abundant proteins in single cells. On the basis of these results, we demonstrate the first electropherogram from an individual Spodoptera frugiperda (Sf9) cell with native label-free UV-LIF detection in a microfluidic chip.
Sorting in Structured Microfluidic Devices
Control of surface properties in microfluidic systems is an indispensable prerequisite for succes... more Control of surface properties in microfluidic systems is an indispensable prerequisite for successful bioanalytical applications. Poly(dimethylsiloxane) (PDMS) microfluidic devices are hampered from unwanted adsorption of biomolecules and lack of methods to control electroosmotic flow (EOF). In this paper, we propose different strategies to coat PDMS surfaces with poly(oxyethylene) (POE) molecules of varying chain lengths. The native PDMS surface is pretreated by exposure to UV irradiation or to an oxygen plasma, and the covalent linkage of POE-silanes as well as physical adsorption of a triblock-copolymer (F108) are studied. Contact angle measurements and atomic force microscopy (AFM) imaging revealed homogeneous attachment of POE-silanes and F108 to the PDMS surfaces. In the case of F108, different adsorption mechanisms to hydrophilic and hydrophobic PDMS are discussed. Determination of the electroosmotic mobilities of these coatings in PDMS microchannels prove their use for electrokinetic applications in which EOF reduction is inevitable and protein adsorption has to be suppressed.
Microfluidic and lab-on-a-chip devices have attracted widespread interest in separation sciences ... more Microfluidic and lab-on-a-chip devices have attracted widespread interest in separation sciences and bioanalysis. Recent designs in microfluidic devices extend common separation concepts by exploiting new phenomena for molecular dynamics on a length scale of 10 mm and below, giving rise to novel manipulation tools and nonintuitive phenomena for microseparations. Here, we focus on three very recent developments for bioseparations based on tailored microfluidic systems: Single cell navigation, trapping and steering with subsequent on-chip lysis, protein separation and LIF detection (Section 3.1), then we report dielectrophoretic trapping and separation of large DNA fragments in structured microfluidic devices (Section 3.2). Finally, a paradoxial migration phenomenon based on thermal fluctuations, periodically arranged microchannels and a biased alternating current electric field is presented in Section 3.3.
DNA migration in topologically structured microchannels with periodic cavities is investigated ex... more DNA migration in topologically structured microchannels with periodic cavities is investigated experimentally and with Brownian dynamics simulations of a simple bead-spring model. The results are in very good agreement with one another. In particular, the experimentally observed migration order ofand T2-DNA molecules is reproduced by the simulations. The simulation data indicate that the mobility may depend on the chain length in a nonmonotonic way at high electric fields. This is found to be the signature of a nonequilibrium bistability between two different migration states, a slow one and a fast one. The latter can also be observed experimentally under appropriate conditions.
Acceleration of Absolute Negative Mobility: Journal of Separation Science
We demonstrate the proof-of-principle of a new separation concept for micrometer-sized particles ... more We demonstrate the proof-of-principle of a new separation concept for micrometer-sized particles in a structured microfluidic device. Under the action of externally applied, periodic voltage-pulses two different species of like-charged polystyrene beads are observed to simultaneously migrate into opposite directions. Based on a theoretical model of the particle motion in the microdevice that shows good agreement with the experimental measurements, the underlying separation mechanism is identified and explained. Potential biophysical applications, such as cell sorting, are briefly addressed.
In order to investigate the individual and inhomogenous cellular response, e.g. to external stimu... more In order to investigate the individual and inhomogenous cellular response, e.g. to external stimuli, single cell analysis is mandatory and may provide new cognitions in proteomics as well as in other fields of systems biology in the future. Here, we report on novel chip architectures for single cell analysis based on full body quartz glass microfluidic chips (QG chips) that extend our previous studies in polydimethylsiloxane (PDMS) chips, and enhance the detection sensitivity of native UV laser-induced fluorescence (UV-LIF) detection. Detection of a 10 nM tryptophan solution with an S/N ratio of 11.9, which gives a theoretical limit of detection of 2.5 nM (with S/N = 3), was possible. With these optimizations the three proteins ␣chymotrypsinogen A, ovalbumin and catalase each at a concentration of 100 g/mL (equal to 4 M, 0.4 M and 2.2 M) were injected electrokinetically and could be separated with nearly baseline resolution. Furthermore, fluorescence spectra (excitation wavelength, ex = 266 nm) clearly demonstrate the favourable properties like the very high UV transparency and the nearly vanishing background fluorescence of the QG chips as compared to PDMS chips and to PDMS quartz window (PQW) chips. Finally we exploit the improved sensitivity for single cell electropherograms of Spodoptera frugiperda (Sf9) insect cells.
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Papers by Dario Anselmetti