Biomedical subjects

W Knoll

Publications and source records attributed to W Knoll.

At least 37 records · Page 2Linked to original sources

PubMed1999

Specific bio-recognition reactions observed with an integrated Mach-Zehnder interferometer.

The combination of an integrated Mach-Zehnder-interferometer (iMZI) at the bottom of a fluidic microchannel system with supramolecular interfacial binding layers optimized for biosensing purposes is described. The model system used is based on the highly specific interaction of streptavidin to its 'ligand' biotin: a single monolayer of a correspondingly derivatized silane-compound is formed by a self-assembly procedure on top of the channel rib guiding the light through the channels. Injection of a streptavidin solution which leads to the formation of a protein monolayer of d = 2.8 nm in effective thickness results in a phase shift of the sample light relative to the reference channel of delta phi = 6 pi, in good agreement with the theoretical sensitivity of delta phi/delta df = 2.9 pi/nm for a protein layer (n = 1.45) calculated for the device.

Biosensing Techniques↗

PubMed1999

Bioassay development: the implications of cardiac myocyte motility in vitro.

Cardiac myocytes cultured over microfabricated extracellular recording devices can be used to assay bioactive compounds. However, electrophysiological signals recorded from these devices vary in amplitude with time. Theoretically, changes in signal amplitude arise from myocytes being moved over recording sites by cocultured fibroblasts. To test this, neonatal rat cardiac myocytes were cultured at high densities and low densities on fibronectin-coated glass. After 36.5 h, myocytes were identified by their rhythmic contractions and then time-lapse-recorded for 3.5 h. Length, width, and angle of orientation was then determined every 30 min for five cells in low density and five cells in high-density culture. Low-density cells had mean lengths of 65.3 microm and widths of 35.1 microm, whereas cells in high-density culture had greater mean lengths of 74.2 microm and lower mean widths of 24.3 microm. Length, width, and angle of orientation of cells in low- and high-density culture changed by 4.1%, 11.8%, and 2.7 degrees, and 6.4%, 10%, and 4.6 degrees, respectively, every half hour. We found no evidence of myocyte-fibroblast interactions influencing cell position or shape in low density, but in high density, we found evidence that fibroblast-myocyte interactions could transiently influence cell shape. We conclude that fibroblast-independent changes in cell shape are largely responsible for the changes in signal amplitude recorded from cardiac myocytes cultured on microfabricated extracellular recording devices. However, there is some evidence that myocyte-fibroblast interactions may augment this process in high-density culture. The implications of these findings for bioassay development are discussed.

Animals↗

PubMed1999

Novel neuronal effects of midkine on embryonic cerebellar neurons examined using a defined culture system.

1. Midkine (MK) is known to be a member of a family of heparin-binding neurotrophic factors. We used a chemically defined culture system to examine neuronal activities of MK on embryonic rat cerebellar cells. 2. In the culture system, a substrate surface was chemically modified either with amine or with laminin peptide to homogenize substrate conditions for culturing neurons. 3. At the optimal concentration (2.5 ng/ml), MK moderately promoted survivability (1.3-fold) and accelerated neurite outgrowth (1.4-fold) of cerebellar cells, putatively granule neurons, grown on an amine-modified surface. 4. Higher dosages (10 ng/ml or more) of MK, however, caused cellular fragmentation and detachment. Such degenerative effects were diminished by increasing the surface adhesiveness using laminin peptide, suggesting that the cellular degeneration might be caused by changes in the adhesive property of the neuron. 5. Using this culture system, we have found that MK has a novel modulatory activity of neuronal adhesiveness on the cultured cerebellar granule cells. Together with the expression pattern of MK, our study supports the idea that MK may be involved in the developmental events of the cerebellum.

Animals↗

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↗

PubMed1999

Tailoring of surfaces with ultrathin polymer films for survival and growth of neurons in culture.

We have studied the adhesion, growth, and survival of dissociated cells of rat cerebellum onto glass surfaces derivatized with covalently bound ultrathin polymer layers. The surface coatings were prepared by growing polymer chains directly at the surfaces of solid substrates by using self-assembled monolayers of radical chain initiators. Methacrylate and acrylamide polymers with different polarities were covalently attached to the surfaces and the adhesion behaviour of the neuronal cells to these surfaces was studied. Best adhesion and neurite outgrowth properties were found using a positively charged polymer (poly(methacryloyl oxypropyl (trimethyl ammonium) bromide)). This 'grafting from' method for the generation of surface modifications can be used for tailoring the composition of the surface of the substrates. Our experiments demonstrate the potential of these layers for the design of polymeric surface coatings for long term stability of neuronal culture.

Animals↗

PubMed1998

Incorporation of the acetylcholine receptor dimer from Torpedo californica in a peptide supported lipid membrane investigated by surface plasmon and fluorescence spectroscopy.

The dimer species (M(r) 580,000) of the nicotinic acetylcholine receptor, isolated from the electric organ of Torpedo californica, was incorporated into a thiopeptide supported lipid bilayer. The incorporation was achieved by fusion of liposomes with reconstituted receptor onto a gold-supported thiopeptide lipid monolayer. Surface plasmon resonance spectroscopy (SPS) was used to monitor in real time the fusion process as well as the specific binding of the antagonist alpha-bungarotoxin. A recently developed extension of SPS offering enhanced sensitivity and specificity, surface plasmon fluorescence spectroscopy (SPFS), was then used to monitor subsequent binding of the monoclonal WF6 and polyclonal antibody, respectively. The latter was fluorescence labeled with Cy5. The different binding assays indicate the successful incorporation of the receptor in the lipid bilayer.

Animals↗

PubMed1998

Molecular gradient along the axon pathway is not required for directional axon growth.

We show that axon guidance of embryonic hippocampal neurons is promoted by pathways of a decapeptide (RDIAEIIKDI) derived from a neurite outgrowth domain of the gamma1 chain of laminin-1. This guidance is directly dependent on: (1) a concentration difference of the decapeptide between the peptide pathway and its surrounding areas, and (2) the optimal surface geometry of the decapeptide pathway. These results indicate that axon guidance of central neurons may proceed along a preferred substratum pathway without a concentration gradient of the guiding molecule along this pathway, or without a repulsive molecule next to the axon pathway.

Animals↗

PubMed1998

Translocation of alkali metal cations by lipophilic cyclodextrin derivatives through black lipid membranes.

Lipophilic cyclodextrin (CD) derivatives, synthetic ionophores, were prepared to transport alkali metal cations across a black lipid membrane (BLM). The purpose of this study is to develop a new class of an artificial transportation system of alkali metal cations via bilayer lipid membranes, by using CD derivatives as a cation carrier. A lipophilic CD derivative incorporated into a BLM forms a complex with an alkali metal cation at one surface of the membrane. This charged complex migrates to the opposite side of the membrane and then releases the cation into the subphase. CD derivatives have various types of acyl groups as a complexing site and formed a 1:1 complex with the alkali metal cation. The complex formation was interpreted by an induced-fit mechanism. It is found that the ability of CD derivative for forming a complex and/or transporting cations across the BLM depends on the bulkiness of acyl groups. The conductivities of heptakis (2,6-di-O-propyl-3-O-propionyl)-beta-CD were higher than those of valinomycin regardless of sizes of cations. The order of the conductivity in all derivatives is Li+ < Na+ < K+ approximately = Rb+ approximately = Cs+, regardless of the types of acyl groups in the derivatives. The effects of alkali metal cation concentration in the aqueous phase and CD concentration in the membrane on the translocation are also discussed.

Cations↗

PubMed1998

A de novo translocation 46,X,t(X;15) causing haemophilia B in a girl: a case report.

Haemophilia B is an X-linked recessive bleeding disorder caused by mutations in the factor IX gene with an incidence of 1:25000-30000. Usually female carriers are clinically normal, and severe phenotypic expression of the disease in females is extremely rare. In this report we describe a girl with a clinically severe course of haemophilia B who had no signs of Turner syndrome or any other dysmorphic features. Cytogenetic and molecular studies in the patient and her parents showed a de novo translocation 46,X,t(X;15)(q27.1;p11.2) in the patient, indicating a possible break near the factor IX gene. The structurally normal X chromosome was late replicating and inactivated in all metaphases as shown by high-resolution R-banding. By fluorescence in situ hybridization (FISH) with YAC and cosmid probes we could further characterize the breakpoint region on the X chromosome and the involvement of the factor IX gene.

Adult↗

PubMed1998

Interfaces and thin films as seen by bound electromagnetic waves.

This contribution summarizes the use of plasmon surface polaritons and guided optical waves for the characterization of interfaces and thin organic films. After a short introduction to the theoretical background of evanescent wave optics, examples are given that show how this interfacial "light" can be employed to monitor thin coatings at a solid/air or solid/liquid interface. Examples are given for a very sensitive thickness determination of samples ranging from self-assembled monolayers, to multilayer assemblies prepared by the Langmuir/Blodgett/Kuhn technique or by the alternate polyelectrolyte deposition. These are complemented by the demonstration of the potential of the technique to also monitor time-dependent processes in a kinetic mode. Here, we put an emphasis on the combination set-up of surface plasmon optics with electrochemical techniques, allowing for the on-line characterization of various surface functionalization strategies, e.g. for (bio-) sensor purposes.

Journal Article↗

PubMed1997

Electrophysiological development of embryonic hippocampal neurons from the rat grown on synthetic thin films.

We have studied the electrophysiological properties of hippocampal neurons grown on surfaces of organic thin films formed on glass or silicon substrates and on microelectronic device surfaces in culture. Hippocampal neurons were dissociated from embryonic rats and plated on substrates chemically modified with laminin peptide in a chemically defined medium. The electrophysiological properties of the neurons were studied using patch-clamp amplifier technique. We observed that the neurons grown on these substrates develop resting membrane potentials more negative than -33 mV after 3 days in culture and are able to produce action potentials. More interestingly we found that the neurons when grown on the microelectronic surfaces develop similar electrophysiological characteristics as those on the glass surfaces. Passive electrical properties (Cm = 27 +/- 5 pF, Rm > or = 1 G omega) of the neurons studied by impedance spectroscopy did not change considerably during the first week in culture.

Action Potentials↗

PubMed1997

Reduced protein adsorption on plastics via direct plasma deposition of triethylene glycol monoallyl ether.

The direct plasma-induced deposition of tri(ethylene glycol) monoallyl ether is reported. RF plasma polymerization of this monomer was carried out under both continuous wave (CW) and pulsed plasma operation. The major focus of this work was optimization of the degree of retention of the C-O-C bonds of the starting monomer during the deposition process. This successfully was accomplished using low RF power during the CW runs and low RF duty cycles during the pulsed plasma experiments. Spectroscopic analysis of the plasma films revealed a strong dependence of film composition on the RF power and duty cycles employed. In particular, an unusually high level of film chemistry compositional control was demonstrated for the pulsed plasma studies, with film composition varying in a steady, progressive fashion with sequential changes in the ratios of plasma on to plasma off times. This film chemistry controllability is demonstrated despite the relatively low volatility of the starting monomer. The utility of this plasma deposition approach in introducing polyethylene oxide (PEO) structures on solid substrates was evaluated via protein adsorption studies. Radiolabeled bovine albumin adsorption was studied on plasma-modified poly(ethylene teraphthalate) (PET) substrates. Dramatic reductions in both initial adsorption and retention of this protein were observed on PET samples having maximal PEO content relative to its adsorption on untreated PET surfaces. Good stability and adhesion of the plasma films to the underlying PET substrates were observed, as evidenced from prolonged immersion of plasma-treated surfaces in aqueous solution. Overall, the results obtained from the present work provide additional support for the utility of one-step plasma process to reduce biological fouling of surfaces via deposition of PEO surface units.

Animals↗

PubMed1997

Supramolecular architectures for the functionalization of solid surfaces.

Surface plasmon optical techniques are described as sensitive tools that allow for the on-line characterization of supramolecular biofunctional architectures at solid/solution interfaces. After a short introduction into the fundamentals of surface plasmon optics the observation of the build up of a functional bio-interface by the self-assembly process of long chain thiolates at an Au surface is described. Criteria are developed for tailoring the SAM architectures optimized for maximum protein binding from solution by specific bio-recognition reactions. SPM is employed to image the selective binding of streptavidin to a functionalized SAM laterally patterned by UV-photolithographic techniques.

Bacterial Proteins↗

PubMed1997

Electropolymerization of a phenol-modified peptide for use in receptor-ligand interactions studied by surface plasmon resonance.

The combination of surface plasmon resonance (SPR) with an electrochemical method for surface modification is presented. The SLP1 sequence of the sodium channel protein of rat cardiac muscle cells was N-terminally modified with an electropolymerizable group and immobilized on a gold-coated glass slide by oxidative polymerization. The resulting peptide-functionalized substrate was incubated with a polyclonal-specific anti-SLP1 serum. Growth of the peptide layer and the immunological reaction between ligand and receptor were detected on-line by SPR. The applicability of this approach for the rapid and selective analysis of receptor-ligand interactions is demonstrated.

Amino Acid Sequence↗

PubMed1997

Field-effect transistor array for monitoring electrical activity from mammalian neurons in culture.

A field-effect transistor (FET) array has been fabricated and used for recording of electrical signals from neural cells. The array consists of p-channel FETs with non-metalized gates. The size of the gates of the 16 FETs are from 28 x 12 microns2 down to 10 x 4 microns2 and are arranged in a 4 x 4 matrix on 200 microns centers. For the device fabrication process we have especially focused on high sensitivity, good long-term stability in physiological conditions, and sufficient reduced signal-to-noise ratio. Special care was taken on the encapsulation technique of the device to allow surface modification based on the self-assembly technique. It can be shown that the microelectronic device surface can be modified with a synthetic peptide linked to the surface. Tailoring of the surface composition using this method allows hippocampal neurons to adhere and grow for days. More importantly, these cells develop typical electrical characteristics when cultured on this artificial surface. Using this approach neuron-FET couplings were recorded.

Animals↗

PubMed1996

Chemically modifying glass surfaces to study substratum-guided neurite outgrowth in culture.

We describe here a modification procedure for chemically fabricating neuron adhesive substrates to study the substratum-guided neurite outgrowth in culture. These substrates were fabricated by chemically attaching a synthetic peptide derived from a neurite-out-growth-promoting domain of the B2 chain of laminin. The attachment was carried out by coupling the peptide to an amine-derived glass surface using a heterobifunctional crosslinker. Hippocampal neurons were dissociated from embryonic rats and placed on the substrate at low-density in a chemically defined medium to examine the direct effect of the modified surface on their outgrowth. We observed that the neurons developed a morphology typical to that of hippocampal neurons having multiple short and single long processes within 24 h in culture. The chemical modification procedure was then combined with a UV-photo-masking technique to fabricate patterns of peptide surface on glass substrates. By culturing the hippocampal neurons on substates having alternate stripes of peptide surface and non-adhesive surface, we demonstrated substratum-controlled changes in the neuronal morphology. The modification procedure presented here can be easily achieved in the standard culture facility and should be useful in fabricating an in vitro tool for studying substratum-guided neurite outgrowth.

Animals↗

PubMed1996

Neural cell pattern formation on glass and oxidized silicon surfaces modified with poly(N-isopropylacrylamide).

Control over the adsorption of proteins and over the adsorption and spatial orientation of mammalian cells onto surfaces has been achieved by modification of glass and other silicon oxide substrates with poly(N-isopropylacrylamide) (PNIPAM). The functionalization of the substrates was achieved either by a polymer-analogous reaction of aminosilanes with reactive N-(isopropylacrylamide) (NIPAM)-copolymers and by copolymerization of NIPAM with surface-bound methacrylsilane. The obtained coatings were characterized by FT-1R, ellipsometry, and surface plasmon resonance measurements. The adsorption of two proteins-fibrinogen and ribonuclease A-on these surfaces was studied in situ by real time surface plasmon resonance measurements. The PNIPAM-grafted surfaces prepared by either chemical procedure inhibited the adsorption of both proteins. More importantly they prevented the adhesion of neuroblastomaXglioma hybrid cells cultured either in serum-free medium or in a medium containing serum proteins. Deep-UV irradiation was used to perform ablation processes and to create patterns permitting the examination of spatially controlled adhesion and growth of cells. This study showed that patterned ultrathin polymer films on glass are suitable substrates for controlling the interactions of cells with surfaces and are capable of directing the attachment and spreading of cells.

Acrylamides↗

PubMed1994

Electron microscopic investigations on free-standing mixed lipid Langmuir-Blodgett-Kuhn monolayers: phase separation and aging process.

Lipid monolayers were prepared by the Langmuir-Blodgett-Kuhn technique (LBK) as free-standing films spanning a diameter of up to 1 micron. These films were investigated by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The free-standing monolayer is shown to be in a transient state in which an aging process is proceeding: after storage for two weeks in air at room temperature the films tear off the edges of the perforated supporting film. Ca2+ ions induced lateral phase separation in these films prepared from a 50:50 mixture of lecithin/glycerol could be visualized by means of ESI, i.e., by comparing micrographs below and above the Ca absorption edge in the EEL spectrum. The domain sizes of the demixed phases were determined to vary between 30 and 60 nm. In addition it was shown that the counter ion of the negatively charged glycerol in these films is Ca2+ and not Na+.

Calcium↗