Biomedical subjects

Alexandra Ros

Publications and source records attributed to Alexandra Ros.

9 recordsLinked to original sources

PubMed2006

Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices.

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 (2005) 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.

Animals↗

PubMed2006

Bioanalysis in structured microfluidic systems.

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 mum 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.

Animals↗

PubMed2005

Brownian motion: absolute negative particle mobility.

Noise effects in technological applications, far from being a nuisance, can be exploited with advantage - for example, unavoidable thermal fluctuations have found application in the transport and sorting of colloidal particles and biomolecules. Here we use a microfluidic system to demonstrate a paradoxical migration mechanism in which particles always move in a direction opposite to the net acting force ('absolute negative mobility') as a result of an interplay between thermal noise, a periodic and symmetric microstructure, and a biased alternating-current electric field. This counterintuitive phenomenon could be used for bioanalytical purposes, for example in the separation and fractionation of colloids, biological molecules and cells.

Journal Article↗

PubMed2005

Poly(oxyethylene) based surface coatings for poly(dimethylsiloxane) microchannels.

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.

Adsorption↗

PubMed2005

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 bistability between two different migration states, a slow one and a fast one. The latter can also be observed experimentally under appropriate conditions.

Computer Simulation↗

PubMed2005

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology.

Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.

Animals↗

PubMed2004

Mechanisms of DNA separation in entropic trap arrays: a Brownian dynamics simulation.

Using Brownian dynamics simulations, we study the migration of long charged chains in an electrophoretic microchannel device consisting of an array of microscopic entropic traps with alternating deep regions and narrow constrictions. Such a device has been designed and fabricated recently by Han and Craighead [Science 288 (2000) 1026] for the separation of DNA molecules. Our simulation reproduces the experimental observation that the mobility increases with the length of the DNA. A detailed data analysis allows to identify the reasons for this behavior. Two distinct mechanisms contribute to slowing down shorter chains. One has been described earlier by Han and Craighead [Science 288 (2000) 1026]: the chains are delayed at the entrance of the constriction and escape with a rate that increases with chain length. The other, actually dominating mechanism is here reported for the first time: some chains diffuse out of their main path into the corners of the box, where they remain trapped for a long time. The probability that this happens increases with the diffusion constant, i.e., the inverse chain length.

Computer Simulation↗

PubMed2003

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.

Amino Acid Motifs↗

PubMed2002

Protein purification by Off-Gel electrophoresis.

A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.

Bacterial Proteins↗