Biomedical subjects

W Schuhmann

Publications and source records attributed to W Schuhmann.

18 recordsLinked to original sources

PubMed2006

Electrochemically deposited Pd islands on an organic surface: the presence of Coulomb blockade in STM I(V) curves at room temperature.

Palladium islands with a thickness of a few monolayers were deposited on top of a self-assembled monolayer (SAM) fabricated from 4-mercaptopyridine. In the I(V) curves obtained using the scanning tunneling microscope (STM) clearly the signature of Coulomb blockade is observed, explicitly demonstrating that these islands are coupled to the underlying gold substrate only via a tunneling barrier; this spectroscopic feature also allows to distinguish the palladium islands from similar morphological features present on the gold substrate prior to palladium deposition.

Adsorption↗

PubMed2002

Pulse-mode scanning ion conductance microscopy--a method to investigate cultured hippocampal cells.

Scanning ion conductance microscopy (SICM) takes advantage of the increase in the resistance which occurs if a glass microelectrode is closely approached to a poorly conducting membrane (Science 243 (1989) 641) and has been shown to be a promising technique to study membranes of living cells (Biophys J 73 (1997a) 653; J Microsc 188 (1997b) 17). Based on a newly designed set-up on top of an inverted light microscope in combination with a speed optimized low noise intracellular amplifier, a novel mode for control of the distance between the probe and surface has been developed. By application of current pulses, the change in the resistance is monitored independently from electrode drift and parasitic DC currents. We demonstrate the applicability by showing first high-resolution images of neural cells produced with the pulse-mode operated SICM.

Animals↗

PubMed2001

A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser.

Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.

Biosensing Techniques↗

PubMed2001

Determination of diffusion coefficients of electroactive species in time-of-flight experiments using a microdispenser and microelectrodes.

Two novel methods for the determination of diffusion coefficients of redox species combining the special properties of microdispensing devices and microelectrodes are presented. Both are based on the local application of tiny volumes of the redox-active species by means of a dispenser nozzle at a defined distance from the surface of a microelectrode. The microelectrode, which is inserted through the bottom into an electrochemical cell, is held at a constant potential sufficient to oxidize or reduce the electro-active species under diffusional control. The dispenser, which is filled with the electro-active species, can be positioned by means of micrometer screws over the microelectrode. After dispensing a defined number of droplets near the microelectrode surface, the current through the microelectrode is recorded, usually yielding a peak-shaped curve having a defined time delay between the shooting of the droplets and the maximum current. The time that is necessary to attain maximum current, together with the known distance between two dispensing points, can be used to determine the diffusion coefficient of the electroactive species without knowledge of any system parameters, such as concentration of the redox species, diameter of the electroactive surface or number of transferred electrons. A similar method for the determination of diffusion coefficient of redox species involves a second redox species for calibration purposes. A mixture of both species is shot close to the microelectrode surface. Due to the different formal potentials of the redox species that are used, they can be distinguished in sequential experiments by variation of the potentials that are applied to the microelectrode, and it is thus possible to determine the individual transit times of the redox species independently. The difference in the transit times, together with the known diffusion coefficient of one of the redox species, can be used to calculate the unknown diffusion coefficient of the second one.

Journal Article↗

PubMed2001

A miniaturised electrochemical affinity assay based on a wall-free sample droplet and micro-dispensing of the redox-labelled binding partner.

An affinity-assay was developed that is based on the modulation of the diffusion coefficient of a redox-labelled hapten upon complementary recognition of the analyte leading to an increase of molecular weight and hence to a decrease of the diffusion coefficient. The slower diffusion is monitored by means of cyclic voltammetry. In order to demonstrate the feasibility of this assay format, recognition of biotin by streptavidin has been chosen as a model system. Labelling of biotin was achieved by covalent binding of a ferrocene derivative to the biotin unit. To reduce the consumption of expensive compounds and to allow automatisation of the assay a novel miniaturised set-up was developed based on a wall-free sample droplet which forms the electrochemical cell with typical volumes of up to 10 microl. This droplet is dispensed by means of a step-motor driven syringe pump through a specially designed electrode holder spanning the gap between a micro-working electrode and a macroscopic counter electrode. By means of a piezo-driven micro-dispenser a predefined number of nano-droplets (100 pl volume each) containing the redox-labelled hapten are shot into the sample droplet. By this, any physical contact and hence any cross-contamination between the sample and the reagent solution could be avoided. Signal amplification can be achieved by redox recycling between the micro-electrode and the perpendicular positioned macroscopic counter electrode.

Biosensing Techniques↗

PubMed2001

Picodroplet-deposition of enzymes on functionalized self-assembled monolayers as a basis for miniaturized multi-sensor structures.

We are reporting on a novel approach for structured immobilisation of enzymes on gold surfaces modified with monolayers of functionalised alkylthiols. The formation of enzyme spots is achieved by shooting very small volumes of an appropriate enzyme solution (down to 100 pl) onto a thiol-monolayer modified gold surface using a micro-dispenser. Formation of enzyme patterns is obtained by moving the micro-dispenser relative to the modified gold surface using a micro-positioning device. Enzyme spots with typical lateral dimensions of 100 microm are obtained, but also, more complex structures, e.g. lines or meander structures, can be achieved by multiple droplets dispensed during the concomitant movement of the micro-dispenser. The first enzyme layer on top of the functionalised thiol-monolayer is subsequently covalently immobilised using either carbodiimide activation of carboxilic headgroups at the enzyme or via already introduced activated ester functions at the monolayer. Immobilised enzyme activities of glucose oxidase and lactate oxidase patterns have been characterised by means of scanning electrochemical microscopy. The product of the enzyme-catalysed reaction, H(2)O(2), is detected with an micro-electrode in the presence of either or both substrates, glucose and lactate, leading to a visualisation of the corresponding enzyme pattern and the lateral enzymatic activity.

Biosensing Techniques↗

PubMed2001

Redox modification of proteins using sequential-parallel electrochemistry in microtiter plates.

Redox modification of proteins has frequently been used to improve the electron-transfer properties in amperometric biosensors. One approach is the coordinative labelling of histidine residues with metal complexes like [Ru(bpy)2Cl2] and [Ru(bpy)2CO3]. Although the reaction depends on a variety of parameters no detailed optimisation of these modification procedures has been done, most probably due to the complexity of the parameter matrix and the expected differences for any individual protein. A multi electrode sequential analyser (MESA) system has been developed which allows one to follow in a sequential-parallel scheme a number of modification reactions by performing electrochemical measurements such as cyclic voltammetry or differential pulse voltammetry in individual wells of a conventional microtiter plate. Using this system, the ligand exchange reaction leading to the binding of the Ru-complex to histidine residues could be investigated with imidazole as a model compound. Furthermore, the selective labelling of soluble PQQ (pyrrolochinolinquinone)-dependent glucose dehydrogenase (sGDH) and glucose oxidase (GOx) with Ru complexes could be optimised and the electrochemical and biological properties of the obtained 'electroenzymes' were examined.

Biomarkers↗

PubMed2000

Facilitated tip-positioning and applications of non-electrode tips in scanning electrochemical microscopy using a shear force based constant-distance mode.

In scanning electrochemical microscopy (SECM) a microelectrode is usually scanned over a sample without following topographic changes (constant-height mode). Therefore, deconvolution of effects from distance variations arising from non-flat sample surface and electrochemical surface properties is in general not possible. Using a shear force-based constant distance mode, information about the morphology of a sample and its localized electrochemical activity can be obtained simultaneously. The setup of the SECM with integrated constant-distance mode and its application to non-flat or tilted surfaces, as well as samples with three-dimensional surface structures are presented and discussed. The facilitated use of non-amperometric tips in SECM like enzyme-filled glass capillaries is demonstrated.

Electrochemistry↗

PubMed2000

Anisotropic orientation of horseradish peroxidase by reconstitution on a thiol-modified gold electrode.

Horseradish peroxidase (HRP) was reconstituted on the surface of a gold electrode that was modified first with a hemin-carbon-chain-thiol derivative followed by addition of the apo protein to the contacting solution. To facilitate the reconstitution of the holo enzyme, the hemin needs to be immobilised on a carbon-chain spacer arm. To achieve this, an immobilisation protocol was developed that is based on the initial formation of a mixed self-assembled monolayer on the gold surface consisting of 3-carboxypropyl disulphide and an activated disulphide (3,3'-dithiodipropionic acid di-(N-succinimidyl ester)) followed by binding of a diaminoalkane to the activated disulphide. The hemin was then coupled to the second amino group of the diaminoalkane by means of a carbodiimide coupling reagent. Finally, the enzyme was reconstituted on the hemin-modified surface by immersion of the electrode in a solution containing apo-HRP. The advantage of this method is that the length of the spacer arm can be changed easily, because diaminoalkanes of different chain lengths are available. The electrochemistry of the hemin and the reconstituted HRP electrodes was studied by means of cyclic voltammetry and differential-pulse voltammetry. The catalytic ability for reduction of hydrogen peroxide was investigated for both direct and mediated electrochemistry with a soluble electron donor (ortho-phenylenediamine).

Electrochemistry↗

PubMed2000

Electron-transfer pathways between redox enzymes and electrode surfaces: reagentless biosensors based on thiol-monolayer-bound and polypyrrole-entrapped enzymes.

Based on previous results which showed that quinohemo-protein alcohol dehydrogenase (QH-ADH) entrapped within polypyrrole is able to directly transfer electrons via the conducting polymer to the electrode surface, the electron-transfer properties of this multi-cofactor enzyme adsorbed and covalently-bound to self-assembled thiol monolayers and bare electrode surfaces has been investigated more closely. While the dissolved enzyme is able to transfer electrons to the electrode via heme c as well as via the more deeply buried PQQ (fast adsorption-chemical reaction-desorption mechanism), an orientation of adsorbed QH-ADH on hydrophobic electrode surfaces, as well as of adsorbed and covalently bound QH-ADH on negatively-charged thiol monolayers could be observed. In these cases the heme c units are pointing towards the electrode surfaces resulting in an optimised direct ET rate.

Alcohol Dehydrogenase↗

PubMed1999

Polypyrrole-entrapped quinohemoprotein alcohol dehydrogenase. Evidence for direct electron transfer via conducting-polymer chains.

It is reported for the first time that direct electron-transfer processes between a polypyrrole (PPY) entrapped quinohemoprotein alcohol dehydrogenase from Gluconobacter sp. 33 (QH-ADH) and a platinum electrode take place via the conducting-polymer network. The cooperative action of the enzyme-integrated prosthetic groups--pyrroloquinoline-quinone and hemes--is assumed to allow this electron-transfer pathway from the enzyme's active site to the conducting-polymer backbone. A hypothetical model of the electron transfer is proposed which is supported by the influence of various parameters, such as, e.g., ionic strength and nature of the buffer salts. This unusual electron-transfer pathway leads to an accentuated increase of the K M app value (102 mM) and hence to a significantly increased linear detection range of an ethanol sensor based on this enzyme.

Acetobacteraceae↗

PubMed1997

Pulse technique for the electrochemical deposition of polymer films on electrode surfaces.

Often, electrochemically-induced deposition of conducting polymer films on electrode surfaces fails using potentiostatic, galvanostatic or multisweep deposition procedures if bulky substituents at the monomer, nucleophilic attack at intermediate radical cations, hindered diffusional mass transport of the monomer to the electrode surface or the copolymerization of monomers with different oxidation potentials prevent fast chain propagation. A pulse profile for the electrochemical deposition of conducting polymer films has been developed based on the rationalization of the limiting steps and the concentration profiles in front of the electrode surface. The pulse deposition method could be advantageously applied for the localized deposition of conducting polymers using scanning electrochemical microscopy, for the copolymerization of pyrrole/[Os(2,2'-bipyridine)2(3-¿pyrrole-1-ylmethyl¿pyridine)C1]+ and for the entrapment of enzymes within the growing ramified network of the polymer.

Biosensing Techniques↗

PubMed1996

Poly(methylene blue)-modified thick-film gold electrodes for the electrocatalytic oxidation of NADH and their application in glucose biosensors.

Electropolymerization of the phenothiazine derivative methylene blue (MB) on screen-printed, thick-film gold electrodes leads to electrocatalytically active and conducting layers of poly(methylene blue) (PMB) in intimate and stable contact with the electrode surface. The catalytic properties of the PMB films allow anodic oxidation of NADH at potentials as low as +200 mV vs. the saturated calomel electrode (SCE) reducing interferences from cooxidizable species as well as minimizing electrode fouling by enabling a simultaneous two-electron transfer mechanism. Dehydrogenase-based biosensors employing PMB-modified thick-film electrodes are obtained either by entrapment of the enzyme into the PMB layer itself or by laminating an enzyme membrane made of an aqueous poly(vinylacetate) dispersion over the PMB-modified electrode. Both methods are used to fabricate glucose biosensors which can be operated at low overpotentials, i.e. +200 mV vs. SCE.

Biosensing Techniques↗

PubMed1993

An automatic dehydrogenase-based flow-injection system: application for the continuous determination of glucose and lactate in mammalian cell-cultures.

A concept for the development of an automatic flow-injection analyzer with integrated dehydrogenase columns and its application in the control of industrial processes is presented. The system is based upon a kernel consisting of a nested-loop injection unit, pumps for the filling of the injection loops and the transport of buffer and values for switching on the one hand between sample and standard solutions and on the other hand between different enzyme columns. A Microsoft Windows 3.x application 'WIN-FIA' controls interactively the whole system and can be easily adapted to a specific solution of an analytical problem. As an example, the flow-injection system was used for the continuous determination of glucose and lactate, using glucose dehydrogenase (GDH) and lactate dehydrogenase (LDH) as indicator enzymes, in a mammalian cell-culture fermentation process. The resulting concentration values are in good agreement with those obtained by discontinuously taken standard spectrophotometric enzyme assays.

Animals↗

PubMed1993

Non-leaking amperometric biosensors based on high-molecular ferrocene derivatives.

Poly(ethylene glycol)-bound ferrocene derivatives, synthesized after activation of ferrocene carboxylic acid with 1,1'-bis carbonyl diimidazol, are able to transfer electrons from the active site of reduced glucose oxidase to electrode surfaces. This opens a route to non-leaking mediated enzyme electrodes for the determination of glucose.

Biosensing Techniques↗

PubMed1992

Stabilization of NAD(+)-dependent dehydrogenases and diaphorase by bilayer encagement.

A feasibility study aimed at stabilization of L-lactate-dehydrogenase, L-malate-dehydrogenase, alcohol-dehydrogenase and diaphorase by the recently described method of enzyme 'encagement' was conducted. This method involves derivatizing the enzymes with polyglutaraldehyde, followed by secondary crosslinking with amino derivatives of polyacrylamide. Encagement conditions were optimized for each of the four enzymes, so as to achieve the highest thermal stability combined with highest catalytic activity. Depending on the encagement conditions, residual activities were in the range of 18% to 96% with higher values in the presence of cofactors. Increases in thermal stabilization of up to 26-fold were obtained. For high retention of enzymic activity and stability, the most significant factor was the concentration of polyglutaraldehyde; the crosslinking polymers had only a negligible effect. Furthermore, the significant enhancement in thermal stability could be attained without perturbing the kinetic parameters: Km values for NADH and pH optima remained unaltered for the stabilized enzymes.

Acrylic Resins↗

PubMedDate not supplied

Electron-transfer mechanisms in amperometric biosensors.

The function of amperometric biosensors is related to electron-transfer processes between the active site of an (immobilized) enzyme and an electrode surface which is poised to an appropriate working potential. Problems and specific features of architectures for amperometric biosensors using different electron-transfer pathways such as mediated electron transfer, electron-hopping in redox polymers, electron transfer using mediator-modified enzymes and carbon-paste electrodes, direct electron transfer by means of self-assembled monolayers or via conducting-polymer chains are discussed.

Animals↗

PubMedDate not supplied

[Amperometric biosensor for ethanol analysis in wines and grape must during wine fermentation].

The amperometric biosensor for ethanol determination based on alcohol oxidase immobilised by the method of electrochemical polymerization has been developed. The industrial screen-printed platinum electrodes were used as transducers for creation of amperometric alcohol biosensor. Optimal conditions for electrochemical deposition of an active membrane with alcohol oxidase has been determined. Biosensors are characterised by good reproducibility and operational stability with minimal detection limit of ethanol 8 x 10(-5) M. The good correlation of results for ethanol detection in wine and during wine fermentation by using the developed amperometric biosensor with the data obtained by the standard methods was shown (r = 0.995).

Biosensing Techniques↗