Publications (91)335.52 Total impact
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Dataset: PicoTweezers Brochure
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Article: Analysis of DNA interactions using single-molecule force spectroscopy.
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ABSTRACT: 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 single-molecule 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.Amino Acids 03/2013; · 3.25 Impact Factor -
Article: Fast and continuous-flow separation of DNA-complexes and topological DNA variants in microfluidic chip format.
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ABSTRACT: The efficient detection, separation and purification of topological and (protein-)complexed DNA variants is mandatory for many state-of-the-art molecular medicine technologies, like medical diagnostics, gene- and cancer-therapy as well as plasmid vaccination. Here, we present the proof-of-concept of a novel micro-nanofluidic device for a fast and efficient, continuous-flow, and virtually label-free detection/purification protocol that goes beyond the standard methods of electrophoretic mobility shift assays, capillary electrophoresis and affinity chromatography. Based on dielectrophoretic trapping, analyte mixtures of small linear DNA-fragments (2.868 kbp and 6.0 kbp), topological DNA variants like plasmids (6.766 kbp) and minicircle-DNA (2.257 kbp), or cytostatic- and protein-DNA complexes were separated in the vicinity of a channel-spanning bowed ridge (creating a nanoslit). One analyte is continuously deflected due to dielectrophoretic trapping at the ridge whereas other species pass the nanoslit unhindered, resulting in two molecule specific pathways with baseline separated resolution. This offers one-step real-time separation of low analyte volumes on a one-minute timescale at low-costs. The underlying dielectrophoretic mechanism was quantified by determining the electrical polarizabilities of the molecules. Additionally, we compared the continuous-flow detection of DNA-complexes with well-established electrophoretic mobility shift assays. Future analytical and preparative applications, such as for plasmid pharmaceuticals as well as continuous sample harvesting in parallel microchip format, are discussed.The Analyst 11/2012; · 4.23 Impact Factor -
Article: Functional characterization of desmin mutant p.P419S.
European journal of human genetics: EJHG 10/2012; · 3.56 Impact Factor -
Article: Video-based and interference-free axial force detection and analysis for optical tweezers.
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ABSTRACT: For measuring the minute forces exerted on single molecules during controlled translocation through nanopores with sub-piconewton precision, we have developed a video-based axial force detection and analysis system for optical tweezers. Since our detection system is equipped with a standard and versatile CCD video camera with a limited bandwidth offering operation at moderate light illumination with minimal sample heating, we integrated Allan variance analysis for trap stiffness calibration. Upon manipulating a microbead in the vicinity of a weakly reflecting surface with simultaneous axial force detection, interference effects have to be considered and minimized. We measured and analyzed the backscattering light properties of polystyrene and silica microbeads with different diameters and propose distinct and optimized experimental configurations (microbead material and diameter) for minimal light backscattering and virtually interference-free microbead position detection. As a proof of principle, we investigated the nanopore threading forces of a single dsDNA strand attached to a microbead with an overall force resolution of ±0.5 pN at a sample rate of 123 Hz.The Review of scientific instruments 10/2012; 83(10):103704. · 1.52 Impact Factor -
Article: Chiral Particle Separation by a Nonchiral Microlattice
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ABSTRACT: We conceived a model experiment for a continuous separation strategy of chiral molecules (enantiomers) without the need of any chiral selector structure or derivatization agents: Microparticles that only differ by their chirality are shown to migrate along different directions when driven by a steady fluid flow through a square lattice of cylindrical posts. In accordance with our numerical predictions, the transport directions of the enantiomers depend very sensitively on the orientation of the lattice relative to the fluid flow.Physical Review Letters 09/2012; 109(10):100603. · 7.37 Impact Factor -
Article: Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.
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ABSTRACT: Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.Journal of Biological Chemistry 03/2012; 287(19):16047-57. · 4.77 Impact Factor -
Article: Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis.
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ABSTRACT: In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensemble-averaging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N = 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N = 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFP-labeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein γ-PKC and envisions a further feasible identification of more than one single protein in the future.Biomicrofluidics 03/2012; 6(1):14104-1410410. · 3.37 Impact Factor -
Article: Particle sorting by a structured microfluidic ratchet device with tunable selectivity: Theory and Experiment
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ABSTRACT: We theoretically predict and experimentally demonstrate that several different particle species can be separated from each other by means of a ratchet device, consisting of periodically arranged triangular (ratchet) shaped obstacles. We propose an explicit algorithm for suitably tailoring the externally applied, time-dependent voltage protocol so that one or several, arbitrarily selected particle species are forced to migrate oppositely to all the remaining species. As an example we present numerical simulations for a mixture of five species, labelled according to their increasing size, so that species 2 and 4 simultaneously move in one direction and species 1, 3, and 5 in the other. The selection of species to be separated from the others can be changed at any time by simply adapting the voltage protocol. This general theoretical concept to utilize one device for many different sorting tasks is experimentally confirmed for a mixture of three colloidal particle species.Soft Matter 03/2012; 8:3900. · 4.39 Impact Factor -
Article: Nanopores: Tiny holes with great promise.
Nature Nanotechnology 01/2012; 7(2):81-2. · 27.27 Impact Factor -
Article: Continuous and reversible mixing or demixing of nanoparticles by dielectrophoresis.
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ABSTRACT: Mixing and demixing (separation) are essential tasks in microfluidic devices, which seem to be contrary in nature. Accordingly, completely different strategies and devices are usually employed for their realization. We here present a microfluidic device which is capable of performing both these tasks as it can be operated in either mixing or demixing mode. The mixing and demixing processes are reversible and are accomplished by continuous operation of the device. An asymmetric S-shaped ridge extends over the full width of a microfluidic channel (200 μm) creating a constriction of 620 nm in height with an aspect ratio of 1 : 500. Appropriate AC and DC voltages generate electrodeless dielectrophoresis at the constriction as well as (linear) electrokinetic driving forces along the channel. These de/mixing parameters can be adapted in real time in such a way that continuous separation and mixing efficiencies of 85-100% can be achieved. As a proof of concept we demonstrate continuous mixing and demixing of polystyrene nanoparticles (20 and 100 nm). The experimental results are complemented by numerical simulations illustrating the particles' motion under the influence of the electrokinetic effects and thermal noise (diffusion). The monolithic one-step fabrication process by soft lithography (with PDMS in our case) will make integration and combination of several mixing and demixing functions into a more complex lab-on-a-chip device possible.Lab on a Chip 12/2011; 12(3):485-94. · 5.67 Impact Factor -
Article: Functional characterization of a supramolecular affinity switch at the single molecule level.
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ABSTRACT: Surface-immobilized and switchable resorcin[4]arene receptor molecules were quantitatively investigated with atomic force microscopy (AFM) and AFM-single molecule force spectroscopy (AFM-SMFS). The upper rim of the supramolecular receptor cavities was modified with two photodimerizable anthracene moieties. The molecular constitution can be externally switched and controlled by exposure to ultraviolet (UV) light and heat. The topography as well as the complexation affinity against small ammonium guest ions of the two isomers were investigated at the single molecule level. Our results demonstrate the feasibility to externally control the supramolecular receptor's affinity and simultaneously quantify and associate these binding properties with the structural change of the resorcin[4]arenes structure on the basis of the measured molecule corrugation height.Nanoscale 11/2011; 3(11):4859-65. · 5.91 Impact Factor -
Article: Electrodeless dielectrophoresis for bioanalysis: Theory, devices and applications.
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ABSTRACT: 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.Electrophoresis 09/2011; 32(17):2253-73. · 3.30 Impact Factor -
Article: Supramolecular Capsules Derived from Resorcin[4]arenes by H‐Bonding and Metal Coordination: Synthesis, Characterization, and Single‐Molecule Force Spectroscopy
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ABSTRACT: Self-assembly by H-bonding and by metal-coordination of functionalized calix[4]arenes and cavitands to large supramolecular capsules is described. In addition, a new method of analyzing supramolecular recognition processes at the single molecule level is discussed. By measuring interaction forces in a hydrogen-bonded assembly using single-molecule force spectroscopy (SMFS), the dynamics of the self-assembly process can be evaluated. In the future, consequent application of this new technique will influence supramolecular design principles and the use of non-covalent interactions as construction elements in the field of nanotechnology.Israel Journal of Chemistry. 08/2011; 51(7):725 - 742. -
Article: Nanopore translocation dynamics of a single DNA-bound protein.
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ABSTRACT: We study the translocation dynamics of a single protein molecule attached to a double-stranded DNA that is threaded through a solid-state nanopore by optical tweezers and an electric field (nanopore force spectroscopy). We find distinct asymmetric and retarded force signals that depend on the protein charge, the DNA elasticity and its counterionic screening in the buffer. A theoretical model where an isolated charge on an elastic, polyelectrolyte strand is experiencing an anharmonic nanopore potential was developed. Its results compare very well with the measured force curves and explain the experimental findings that the force depends linearly on the applied electric field and exhibits a small hysteresis during back and forth translocation cycles. Moreover, the translocation dynamics reflects the stochastic nature of the thermally activated hopping between two adjacent states in the nanopore that can be adequately described by Kramers rate theory.Nano Letters 06/2011; 11(7):2978-82. · 13.20 Impact Factor -
Article: Continuous-flow separation of nanoparticles by electrostatic sieving at a micro-nanofluidic interface.
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ABSTRACT: Continuous-flow separation of nanoparticles (NPs) (15 and 39 nm) is demonstrated based on electrostatic sieving at a micro-nanofluidic interface. The interface is realized in a poly(dimethylsiloxane) device with a nanoslit of 525 nm laterally spanning the microfluidic channel (aspect ratio of 540:1). Within this nanoslit, the Debye layers overlap and generate an electrostatic sieve. This was exploited to selectively deflect and sort NPs with a sorting purity of up to 97%. Because of the continuous-flow operation, the sample is continuously fed into the device, immediately separated, and the parameters can be adapted in real time. For bioanalytical purposes, we also demonstrate the deflection of proteins (longest axis 6.8 nm). The continuous operation mode and the general applicability of this separation concept make this method a valuable addition to the current Lab-on-a-Chip devices for continuous sorting of NPs and macromolecules.Journal of Separation Science 03/2011; 34(10):1180-3. · 2.73 Impact Factor -
Article: Photolithographic fabrication of arbitrarily shaped SU-8 microparticles without sacrificial release layers
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ABSTRACT: We report on an efficient high throughput method for the photolithographic fabrication ofJournal of Micromechanics and Microengineering 01/2011; 21:027003. · 2.11 Impact Factor -
Conference Proceeding: Towards Selectorfree Separation of Chiral Molecules: Enantioselective Separation of Microparticles in a Microfluidic Device
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ABSTRACT: We developed two model experiments for a continuous separation strategy for chiral molecules without the need for any chemical selection- or derivatization agents. By breaking all relevant spatial symmetries with either a structured sidewall or a tilted array of posts in a microfluidic channel, chiral microparticles are forced to migrate into different directions. In our experiments, separation efficiencies of 85% and up to 100%, respectively, could be demonstrated by introducing asymmetric flow environments in microchip systems.Micro total analysis systems (µTAS), Seattle, Washington, USA; 01/2011 -
Article: Distance dependence of near-field fluorescence enhancement and quenching of single quantum dots.
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ABSTRACT: 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.Beilstein Journal of Nanotechnology 01/2011; 2:645-52. · 0.79 Impact Factor -
Article: De novo desmin-mutation N116S is associated with arrhythmogenic right ventricular cardiomyopathy.
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ABSTRACT: 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.Human Molecular Genetics 12/2010; 19(23):4595-607. · 7.64 Impact Factor
Top co-authors
Top Journals
Institutions
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2003–2013
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Universität Bielefeld
- • Organische Chemie
- • Faculty of Physics
- • Experimental Physics
Bielefeld, North Rhine-Westphalia, Germany
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2012
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Ruhr-Universität Bochum
Bochum, North Rhine-Westphalia, Germany
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2009
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Institute for Bioengineering of Catalonia (IBEC)
Barcelona, Catalonia, Spain -
El Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)
Zaragoza, Aragon, Spain
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2006
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University of Barcelona
Barcelona, Catalonia, Spain
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