Particle sorting by a structured microfluidic ratchet device with tunable selectivity: Theory and Experiment

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

Probing single biomolecules with atomic force microscopy

Fritz J, Anselmetti D, Jarchow J, Fernandez-Busquets X. Probing single biomolecules with atomic force microscopy. Journal of structural biology. 1997;119(2):165-171.During the last years, atomic force microscopy (AFM) has developed from a microscopy tool for solid state surface science towards a method employed in many scientific disciplines such as biology to investigate individual molecules on a nanometer scale. This article describes the current status of the imaging possibilities of AFM on RNA, IgG and gold-labelled cell adhesion molecules, as well as of measurements of intermolecular binding forces between biomolecules in order to investigate their molecular structure, function and elasticity

Single molecule DNA biophysics with atomic force microscopy

Anselmetti D, Fritz J, Smith B, Fernandez-Busquets X. Single molecule DNA biophysics with atomic force microscopy. Single molecules. 2000;1(1):53-58.Structural and functional properties of double stranded deoxyribonucleic acid (dsDNA) are investigated by atomic force microscopy (AFM) on a single molecule level. Here, we characterize different linear and circular DNA systems in terms of their geometry and topology, and visualize enzyme binding of restriction endonuclease Hae III to DNA. Manipulation of single DNA molecules is demonstrated by dissecting individual DNA strands. Furthermore, the elastic response of single DNA molecules to an externally applied force is investigated by AFM force spectroscopy experiments. This gives information about structural properties of the DNA double helix. Specifically, transition from B-form to S-form DNA and a melting transition from double stranded to single stranded DNA is observed. This allows monitoring of specific interaction and binding of small intercalator molecules such as ethidium bromide (EtBr) to DNA by means of a mechanical, non-fluorescent detection scheme

Chiral particle separation by a non-chiral micro-lattice

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: Micro-particles 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 relatively to the fluid flow

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

Bartels FW, Baumgarth B, Anselmetti D, Ros R, Becker A. 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. Journal of structural biology. 2003;143(2):145-152.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 165pN 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) × 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

Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA

Baumgarth B, Bartels FW, Anselmetti D, Becker A, Ros R. Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA. Microbiology. 2005;151(1):259-268.The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His)6 fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His)6 to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction

An Oil-Based Lubrication System Based on Nanoparticular TiO2 with Superior Friction and Wear Properties

Bogunovic L, Zünkeler S, Tönsing K, Anselmetti D. An Oil-Based Lubrication System Based on Nanoparticular TiO2 with Superior Friction and Wear Properties. Tribology Letters. 2015;29(2): 59.We evaluated the performance of five different commercially available nanoparticle classes as additives for an oil-based lubrication system. While the silicon dioxide particles Aerosil® 300, RY300, and R972V tended to increase wear and friction in our 100Cr6 versus cast iron disc–disc contact, Aeroxide® P 25 and especially T 805 TiO2 nanoparticles showed superior anti-wear and anti-friction properties. The underlying tribological mechanism was investigated with optical microscopy, helium ion microscopy, and X-ray photoelectron spectroscopy. Subsequently, we formulated a stable lubrication system based on the best performing T 805 particles. Here, the base oil is a highly purified paraffin oil which was supplemented with 1 wt% T 805 TiO2 particles, 1 wt% Estisol® 242 or 1 wt% oleic acid, 0.15 wt% oleylamine, and 0.15 wt% Pluronic® RPE 2520. Superior lubrication and anti-wear properties of this formulation were demonstrated in 4-h test runs with a normal force of F N = 2.5 kN and a sliding velocity of 0.15 m/s in our disc–disc contact. Wear was significantly reduced along with a nearly 12-fold reduction in the friction coefficient, compared to the base oil (μftobase=0.155vs.μftoT805≈0.01). Using 100Cr6 disc–ball contacts, we additionally analyzed the properties of our lubrication system in the border friction regime under higher loads (F N = 0.5 kN) in 2-h runs. In particular, on the discs with lower engagement ratio, chemo-tribological protective layers were built, which protected the parts very well against wear

Two-State Migration of DNA in a structured Microchannel

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 of Lambda- and 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 phase transition between two different migration states, a slow one and a fast one, which can also be observed experimentally under appropriate conditions.Comment: Revised edition corresponding to the comments by the referees, submitted to Physical Review

Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin

Harder A, Dieding M, Walhorn V, et al. Apertureless scanning near-field optical microscopy of sparsely labeled tobacco mosaic viruses and the intermediate filament desmin. Beilstein Journal of Nanotechnology. 2013;4:510-516.Both fluorescence imaging and atomic force microscopy (AFM) are highly versatile and extensively used in applications ranging from nanotechnology to life sciences. In fluorescence microscopy luminescent dyes serve as position markers. Moreover, they can be used as active reporters of their local vicinity. The dipolar coupling of the tip with the incident light and the fluorophore give rise to a local field and fluorescence enhancement. AFM topographic imaging allows for resolutions down to the atomic scale. It can be operated in vacuum, under ambient conditions and in liquids. This makes it ideal for the investigation of a wide range of different samples. Furthermore an illuminated AFM cantilever tip apex exposes strongly confined non-propagating electromagnetic fields that can serve as a coupling agent for single dye molecules. Thus, combining both techniques by means of apertureless scanning near-field optical microscopy (aSNOM) enables concurrent high resolution topography and fluorescence imaging. Commonly, among the various (apertureless) SNOM approaches metallic or metallized probes are used. Here, we report on our custom-built aSNOM setup, which uses commercially available monolithic silicon AFM cantilevers. The field enhancement confined to the tip apex facilitates an optical resolution down to 20 nm. Furthermore, the use of standard mass-produced AFM cantilevers spares elaborate probe production or modification processes. We investigated tobacco mosaic viruses and the intermediate filament protein desmin. Both are mixed complexes of building blocks, which are fluorescently labeled to a low degree. The simultaneous recording of topography and fluorescence data allows for the exact localization of distinct building blocks within the superordinate structures

Na,K-ATPase on a waveguide sensor : supramolecular assembly and side directed binding studies by surface-confined fluorescence

Grell E, Pawlak M, Anselmetti D, Schick E, Lewitzki E, Ehrat M. Na,K-ATPase on a waveguide sensor : supramolecular assembly and side directed binding studies by surface-confined fluorescence. In: Taniguchi K, Kaya S, eds. Na/K-ATPase and related ATPases: proceedings of the 9th International Conference on the Na/K-ATPase and Related ATPases. Excerpta Medica international congress series. Vol 1207. Amsterdam: Elsevier; 2000: 437-440.The functional assembly of FITC-Na,K-ATPase membrane fragments on a surface-modified Ta2O5 waveguide allows to investigate the directed binding of ligands by surface-confined fluorescence studies. The results allow to draw conclusions about the sidedness of interactions. The fluorescence intensity decrease observed upon the selective binding of K+ is attributed to its coordination to a site accessible from the former intracellular membrane side