Biomedical subjects

A Offenhäusser

Publications and source records attributed to A Offenhäusser.

At least 19 recordsLinked to original sources

PubMed2006

Label-free detection of single nucleotide polymorphisms utilizing the differential transfer function of field-effect transistors.

We present a label-free method for the detection of DNA hybridization, which is monitored by non-metallized silicon field-effect transistors (FET) in a microarray approach. The described method enables a fast and fully electronic readout of ex situ binding assays. The label-free detection utilizing the field-effect is based on the intrinsic charge of the DNA molecules and/or on changes of the solid-liquid interface impedance, when biomolecules bind to the sensor surface. With our sensor system, usually a time-resolved, dc readout is used. In general, this FET signal suffers from sensor drift, temperature drift, changes in electrolyte composition or pH value, influence of the reference electrode, etc. In this article, we present a differential ac readout concept for FET microarrays, which enables a stable operation of the sensor against many of these side-parameters, reliable readout and a possibility for a quick screening of large sensor arrays. We present the detection of point mutations in short DNA samples with this method in an ex situ binding assay.

Biosensing Techniques↗

PubMed2006

Field-effect sensors with charged macromolecules: characterisation by capacitance-voltage, constant-capacitance, impedance spectroscopy and atomic-force microscopy methods.

Field-effect-based capacitive electrolyte-insulator-semiconductor (EIS) sensors have been utilised for the deoxyribonucleic acid (DNA) immobilisation and hybridisation detection as well as for monitoring the layer-by-layer adsorption of polyelectrolytes (anionic poly(sodium 4-styrene sulfonate) (PSS) and cationic poly(allylamine hydrochloride) (PAH)). The EIS sensors with charged macromolecules have been systematically characterised by capacitance-voltage, constant-capacitance, impedance spectroscopy and atomic-force microscopy methods. The effect of the number and polarity of the polyelectrolyte layers on the shift of the capacitance-voltage curves has been investigated. Alternating potential shifts of about 30-90 mV have been observed after the adsorption of each polyanion and polycation layer, respectively. The DNA immobilisation and hybridisation signals were 35-55 and 24-33 mV, respectively. The possible mechanisms for the sensor responses are discussed.

DNA↗

PubMed2006

Influence of the first amplifier stage in MEA systems on extracellular signal shapes.

Recording of extracellular signals with planar metal microelectrodes (ME) has already been presented more than 30 years ago. To date, microelectrode array (MEA) systems are able to measure extracellular signals at about 64 sites, simultaneously. This enables monitoring of electrical activity of many cells in a large area. The extracellular recording technique has become a widely used method for neurological, toxicological or pharmacological studies. It already proved its potential to supplement the classical methods in electrophysiology. The interpretation of the recorded signal shapes in order to extract electrophysiological meaningful data--however--is still under discussion. In this article, we analyse the preamplifier circuit for extracellular recording of cardiac myocyte signals. We use a circuit model for the cell-electrode contact including the first amplification stage. In test experiments, we observe different signal shapes, when different shunt resistors are introduced at the input of the preamplifier. According to the frequency spectra of the recordings, we evaluate the transfer function between the source signal and the readout signal. As a result of our studies, an optimum readout electronics for originally, preserved extracellular signal shapes is proposed. Our amplifier design will be most valuable, if the use of small microelectrodes with high input impedances for in vitro as well as for in vivo experiments is desired.

Action Potentials↗

PubMed2005

Independence of synaptic specificity from neuritic guidance.

Neuronal circuits are interconnected with a high degree of specificity. While axonal guidance has been demonstrated to be crucial for the choice of the correct target region, its role in specificity at the level of individual cells remains unclear. Specificity of synapse formation may either result from precise guidance of axonal outgrowth onto the target or depend on a molecular "match" between pre- and postsynapse. To distinguish between these possibilities, an in vitro system was used in which neuritic outgrowth of rat cortical neurons is accurately guided along the narrow pathways of a surface micropattern. The micropattern consisted of a blend of extracellular matrix molecules applied to a cell repellent background of polystyrene by microcontact printing. The system reproduces guidance by attractant and repellent surface cues while no other signals that may influence synapse formation, like gradients of trophic factors or accumulations of signaling molecules, are provided. While the number of contact points between neighboring cells was strongly reduced on patterned substrates due to the geometrical restrictions, frequency of synapse formation was not different from homogeneous cultures. Thus it was unaffected by stringent guidance onto the target cell or by the number of cell-cell contacts. Moreover, a statistically significant enrichment of reciprocal contacts between mixed pairs of excitatory and inhibitory neurons over probabilistic predictions was found, which has similarly been shown by others in dissociated neuronal cultures. Our results indicate that precise axonal guidance is insufficient for target-specific synapse formation and suggest that instead recognition between individual cells is required.

Animals↗

PubMed2004

Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device.

The labelfree detection of nucleic acid sequences is one of the modern attempts to develop quick, cheap and miniaturised hand-held devices for the future genetic testing in biotechnology and medical diagnostics. We present an approach to detect the hybridisation of DNA sequences using electrolyte-oxide-semiconductor field-effect transistors (EOSFETs) with micrometer dimensions. These semiconductor devices are sensitive to electrical charge variations that occur at the surface/electrolyte interface, i.e. upon hybridisation of oligonucleotides with complementary single-stranded (ss) oligonucleotides, which are immobilised on the oxide surface of the transistor gate. This method allows direct, time-resolved and in situ detection of specific nucleic acid binding events without any labelling. We focus on the detection mechanism of our sensors by using oppositely charged polyelectrolytes (PAH and PSS) subsequently attached to the transistor structures. Our results indicate that the sensor output is charge sensitive and distance dependent from the gate surface, which pinpoints the need for very defined surface chemistry at the device surface. The hybridisation of natural 19 base-pair sequences has been successfully detected with the sensors. In combination with nano-transistors a PCR free detection system might be feasible in future.

Biosensing Techniques↗

PubMed2004

Analysis of electrotonic coupling in patterned neuronal networks.

Microcontact printing of laminin is known as an efficient approach for guiding neuronal cell migration and neurite outgrowth on artificial surfaces. In the present study, ultrathin (approximately 250 microm) brain stem slices of Sprague-Dawley rats (E15-E18) were cultured on laminin-patterned substrates, such that neuronal cells migrating out of the slices formed grid-shaped neuronal networks along the geometry defined by the pattern. The interconnections between neighbouring pairs of neurons within these artificial networks were assessed electrophysiologically by double patch-clamp recordings and optically by microinjection of fluorescent dyes. Both functional and electrotonic synapses were detected. Based on the recorded data and simulations in PSpice, an electrical model for electrotonically coupled cells was derived. In this model the neuritic pathway is described as a cylindric cable, and gap junctions are represented by an ohmic resistor. Applying this model in the data analysis, the average inner radius of neurites could be determined to be approximately 0.1 microm. In addition, evidence was found for a correlation between the path-width of the applied pattern and the diameter of neurites growing along these paths.

Journal Article↗

PubMed2003

Fusion of small unilamellar vesicles onto laterally mixed self-assembled monolayers of thiolipopeptides.

Monolayers of the thiolipopeptide NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-(Myr)Lys(Myr)-OH (III) were formed on gold surfaces by self-assembly, mixed with a lateral spacer of the same peptide composition, NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-Lys-OH (I). Different mixing ratios were employed ranging from 0.1 to 1, corresponding to 10-100% thiolipopeptide. These self-assembled monolayers (SAMs) were then exposed to a suspension of liposomes with the aim of forming lipid bilayers as a function of the mixing ratio. A clear optimum with respect to homogeneity and electrical properties of the membranes was obtained in the middle region (0.5) of mixing ratio, as revealed by surface plasmon resonance spectroscopy, impedance spectroscopy, and fluorescence microscopy. The combination of these methods was shown to be a powerful tool, although a true lipid bilayer was not obtained. Instead, vesicle adsorption was shown to be the predominant process, and FRAP (fluorescence recovery after photobleaching) measurements showed that the films were not fluid on the micrometer length scale.

Lipoproteins↗

PubMed2003

Backside contacted field effect transistor array for extracellular signal recording.

A new approach to the design of field-effect transistor (FET) sensors and the use of these FETs in detecting extracellular electrophysiological recordings is reported. Backside contacts were engineered by deep reactive ion etching and a gas phase boron doping process of the holes using planar diffusion sources. The metal contacts were designed to fit on top of the bonding pads of a standard industrial 22-pin DIL (dual inline) chip carrier. To minimise contact resistance, the metal backside contacts of the chips were electroless plated with gold. The chips were mounted on top of the bonding pads using a standard flip-chip process and a fineplacer unit previously described. Rat embryonic myocytes were cultured on these new devices (effective growth area 6 x 6 mm(2)) in order to confirm their validity in electrophysiological recording.

Action Potentials↗

PubMed2002

Electrophysiological recordings of patterned rat brain stem slice neurons.

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information in the past. However, although the culture of brain slices has many advantages over dissociated neuronal cultures, its feasibility on patterned substrates has not been demonstrated to date. In the present study, neuronal outgrowth from brain stem slices onto homogeneous control substrates, and onto laminin structures of grid- and line-shape was achieved. Cultures were evaluated by means of phase contrast microscopy, antibody staining, and patch-clamp measurements. Only patterns with line sizes of more than 4 microm yielded satisfactory neuronal outgrowth. The size of the nodes in the pattern influenced the nodal compliance of the spreading cells and the amount of unstructured overgrowth. Best grid patterns were 4 microm lines and 10 microm nodes, best line patterns were 4 microm lines and 20 microm nodes. On patterned substrates, average sodium and potassium currents were reduced by approximately 50% compared to controls, whereas area-normalized ion-currents were in the same order of magnitude. This indicates that as a consequence of the pattern-enforced geometrical confinement, neurons tend to have a smaller surface. In addition, neurons on patterned substrates were rapidly covered with glial overgrowth. This was shown by antibody staining.

Animals↗

PubMed2002

Proton transport through a peptide-tethered bilayer lipid membrane by the H(+)-ATP synthase from chloroplasts measured by impedance spectroscopy.

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.

Chloroplasts↗

PubMed2001

Modulation of the growth and guidance of rat brain stem neurons using patterned extracellular matrix proteins.

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information regarding the physiological characteristics of neuronal cells behaviour in vitro. However, neuronal patterning using long term embryonic brain slice cultures has not been comprehensively demonstrated to-date. Structuring was performed by micro contact printing of laminin. The slice cultures were evaluated by means of phase contrast microscopy at 3-22 days in culture. We were able to consistently achieve outgrowth of neurons, neurites and filopodia from brain stem slices cultured on ECM proteins structures of grid- and line-shapes. We believe that brain slice cultures on patterned substrates is a favourable approach to study functional synapses in vitro under defined conditions. The use of appropriate structures and the subsequent cell patterning may help to gain further understanding of axonal, dendritic and synaptic signal transductions and processes.

Animals↗

PubMed2001

Spot compliant neuronal networks by structure optimized micro-contact printing.

Neuronal cell growth in vitro can be controlled with micropatterned structures of extracellular matrix proteins such as laminin. This technique is a powerful tool for studying neuronal cell function in order to increase experimental reproducibility and to specifically design innovative experimental setups. In this paper the correlation between the structural dimensions of the ECM pattern and the shape of the resulting cellular network is analyzed. The aim of the present study was to position neuronal cell bodies as precisely as possible and to induce directed cell differentiation. PCC7-MzN cells were cultured on laminin patterns. The line width, node size and gap size in-between cell adhesion sites was varied systematically. Micrographs of the samples were taken and statistically analyzed using Student's t-test and linear correlation methods. Precise cell positioning has successfully been performed and evidence for controlled neuronal polarization has been found. With a structure geometry of 4 microm line width, 20 microm node size and 10 microm gap size a nodal compliance of 86% (+/- 10%) has been achieved.

Animals↗

PubMed2001

Cell-transistor hybrid systems and their potential applications.

Electrogenic cells fire spontaneous or triggered action potentials (transient changes of their membrane potentials) and can be electronically coupled to external electrodes (arrays). Signals from rat heart-muscle cells were recorded by a field-effect transistor and the results described on the basis of an equivalent circuit. This technique has potential applications in drug screening, such as measuring the dose-response curve of isoproterenol, a beta-adrenergic agonist with a positive chronotropic effect.

Action Potentials↗

PubMed2001

Cardiomyocyte-transistor-hybrids for sensor application.

An extracellular recording system has been designed for the detection of electrical cell signals using p-channel or n-channel field-effect transistor (FET) arrays with non-metallized gates. Signals from rat heart muscle cell were recorded by these devices and the results described on the basis of an equivalent circuit. This technique is sensitive enough to detect minute changes of the extracellular membrane voltage and has potential applications in drug screening. We show that known cardiac stimulants (isoproterenol, norepinephrine) and relaxants (verapamil, carbamylcholine) have characteristic effects on the heart cells in terms of the changes of beat frequencies in the absence or presence of corresponding agents.

Action Potentials↗

PubMed2001

Aligned microcontact printing of biomolecules on microelectronic device surfaces.

Microcontact printing (muCP) of extracellular matrix proteins is a fascinating approach to control cell positioning and outgrowth, which is essential in the development of applications ranging from cellular biosensors to tissue engineering. Microelectronic devices can be used to detect the activity from a large number of recording sites over the long term. However, signals from cells can only be recorded at small sensitive spots. In this paper, we present an innovative setup to perform aligned muCP of extracellular matrix proteins on microelectronic devices in order to guide the growth of electrogenic cells specifically to these sensitive spots. Our system is based on the combination of a fine-placer with redesigned micro stamps having a rigid glass cylinder as backbone for attachment in the alignment tool. Alignment is performed moving the device with an optical table under microscopic control of the superimposed images from stamp and device surface. After successful alignment, the stamp is brought into contact with the device surface by means of a high-precision lever. With our setup, we were able to pattern up to 40 devices per hour. A lateral alignment accuracy of < 2microm has been achieved. Aligned neuronal growth on patterned devices was demonstrated with dissociated hippocampal neurons.

Animals↗

PubMed2000

Ordered networks of rat hippocampal neurons attached to silicon oxide surfaces.

The control of neuronal cell position and outgrowth is of fundamental interest in the development of applications ranging from cellular biosensors to tissue engineering. We have produced rectangular networks of functional rat hippocampal neurons on silicon oxide surfaces. Attachment and network formation of neurons was guided by a geometrical grid pattern of the adhesion peptide PA22-2 which matches in sequence a part of the A-chain of laminin. PA22-2 was applied by contact printing onto the functionalised silicon oxide surface and was immobilised by hetero-bifunctional cross-linking with sulfo-GMBS. Geometric pattern matching was achieved by microcontact printing using a polydimethylsiloxane (PDMS) stamp. In this way the produced grid pattern ranged from 3 to 20 microm in line width and from 50 to 100 microm in line distances. As shown by atomic force microscopy (AFM), line widths and line distances of the peptide pattern differ less than 0.5 microm from the used PDMS stamp. The height of the layer of immobilised PA22-2 was approximately 3.5 nm implying the layer to be monomolecular. Immobilised PA22-2 was capable of binding anti-PA22-2 antibodies indicating that the function of the peptide was not compromised by immobilisation. Rat hippocampal neurons, cultured at low density in serum-free medium, were applied to the growth matrix of PA22-2-coated substrates and, within 1-3 h of culture, formed a network-like pattern that more or less matched the printed grid. Reliability and reproducibility of neuronal network formation depended on the geometry, line width and node diameter of the grid pattern. The immobilised neurons showed resting membrane potentials comparable with controls and, already after 1 day of culture, were capable of eliciting action potentials. The suitability of the immobilised neurons for the study of man-made neural networks and for multi-site recordings from a functional neuronal network is discussed.

Animals↗

PubMed2000

Functional tethered lipid bilayers.

Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support. Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support. An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite. And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates. All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques. For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer. Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step. Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e. the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively. The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes. The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies. The results obtained for reconstituted H(+)-ATPase from chloroplasts and E. coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.

Biopolymers↗

PubMed1999

Electrical recordings from rat cardiac muscle cells using field-effect transistors.

Extracellular electrophysiological recordings were made from cardiac cells cultured for up to seven days over microfabricated arrays of field-effect transistors. The recorded signals can be separated mainly into two types of cell transistor couplings: one that can be explained entirely by purely passive circuitry elements, and a second where voltage-gated ion channels contribute greatly to the measured extracellular signal.

Action Potentials↗