PubMed Health⌕ Search

Biomedical subjects

E Csöregi

Publications and source records attributed to E Csöregi.

At least 19 recordsLinked to original sources
PubMed2005

Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline.

This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H2O2 (detection limit at 0.5 microM). This electrode was also modified through the co-immobilisation of acetylcholinesterase (AChE) and/or choline oxidase (ChOx) in a bovine serum albumin (BSA) membrane for the development of a sensor for in vivo measurements of acetylcholine and choline. Amperometric measurements were performed using a conventional three-electrode system forming part of a flow-injection set-up at an applied potential of 800-1100 mV relative to an Ag/AgCl reference electrode. The optimised biosensor obtained was reproducible and stable, and exhibited a detection limit of 1 microM for both acetylcholine and choline. However, due to the high operating potential used, the biosensor was prone to substantial interference from other electroactive compounds, such as ascorbic acid. Therefore, in a further step, a mediated electron transfer approach was used that incorporated horseradish peroxidase into an osmium-based redox hydrogel layered onto the active surface of the electrode. Afterwards, a Nafion layer and a coating containing AChE and/or ChOx co-immobilised in a BSA membrane were successively deposited. This procedure further increased the selectivity of the biosensor, when operated in the same flow-injection system but at an applied potential of -50 mV relative to an Ag/AgCl reference electrode. The sensor exhibited good selectivity and a high sensitivity over a concentration range (0.3-100 microM) suitable for the measurement of choline and acetylcholine in vivo.

Acetylcholine↗
PubMed2004

Adaptive responses to static conditions in nutrient-rich cultures of luminous Ralstonia eutropha.

The lux-gene fused Ralstonia eutropha, when adapting to static conditions, causes stratification of air-exposed and nutrient-rich cultures at above 0.15 mg biomass ml(-1). The O2 respiring biofilm (luminous neuston) phase, along with the dark sub-neustonic suspension phase, develops within 5-60 min. The instability of the biphasic static culture was identified as a reason for occasionally observable oscillatory bioluminescence.

Adaptation, Physiological↗
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

Monitoring of alpha-ketoglutarate in a fermentation process using expanded bed enzyme reactors.

A bienzyme flow injection system is presented for the monitoring of alpha-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, alpha-ketoglutarate was converted to L-glutamate by GDH in the presence of ammonia and NADH. In the following reactor, L-glutamate was converted by GlOx to alpha-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of alpha-ketoglutarate in the coupled packed bed reactors was 1 microM (defined as 3 S/N), the linear range 0-100 microM, and the sensitivity 0.80 nA/microM (R(2) 0.99). In the coupled expanded bed reactors, the detection limit of alpha-ketoglutarate was 7 microM (defined as 3 S/N), the linear range and the sensitivity being 0-500 microM and 0.11 nA/microM (R(2) 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.

Amino Acid Oxidoreductases↗
PubMed2000

Electrooxidation mechanism of biogenic amines at amine oxidase modified graphite electrode.

Amine oxidase (AO, EC. 1.4.3.6) was previously shown to be a very efficient biological recognition element of amperometric biosensors for monitoring biogenic amines. The enzyme was effectively working in both mono- and bienzyme electrode designs, based on either a direct or a mediated electron-transfer pathway. This work focuses on the elucidation of the electron-transfer mechanism of the monoenzymatic unmediated AO-modified biosensor. The observed unmediated catalytic currents were assumed to be caused by (i) a direct electron-transfer process, (ii) the electrooxidation of the formed product, or (iii) their combination. Experiments supporting these assumptions are discussed in detail.

Amine Oxidase (Copper-Containing)↗
PubMed2000

Bacterial metal-resistance proteins and their use in biosensors for the detection of bioavailable heavy metals.

We have expressed and purified metal-resistance and metal regulatory proteins from the bacterial determinants of resistance to heavy metals and utilised these in the development of biosensors for heavy metals. Both the metallothionein from the cyanobacterium Synechococcus PCC 7942 and the MerR regulatory protein from transposon Tn501 allow the detection of non-specific metal binding down to 10(-15) M concentrations of Hg(II), Cu(II), Zn(II) and Cd(II) in pure solution. Differential effects of the metals can be detected at both low and high concentrations, and the shape of the capacitance curves may reflect biologically relevant responses of the proteins to metals. Further work is required to establish the relationship between the detected binding of metal and the biological response of the protein, or to provide biosensors of use in the natural environment.

Bacterial Proteins↗
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↗
PubMed1999

Redox hydrogel based bienzyme electrode for L-glutamate monitoring.

Amperometric bienzyme electrodes based on coupled L-glutamate oxidase (GlOx) and horseradish peroxidase (HRP) were constructed for the direct monitoring of L-glutamate in a flow injection (FI)-system. The bienzyme electrodes were constructed by coating solid graphite rods with a premixed solution containing GlOx and HRP crosslinked with a redox polymer formed of poly(1-vinylimidazole) complexed with (osmium (4-4'-dimethylbpy)2 Cl)II/III. Poly(ethylene glycol) diglycidyl ether (PEGDGE) was used as the crosslinker and the modified electrodes were inserted as the working electrode in a conventional three electrode flow through amperometric cell operated at -0.05 V versus Ag¿AgCl (0.1 M KCl). The bienzyme electrode was optimized with regard to wire composition, Os-loading of the wires, enzyme ratios, coating procedure, flow rate, effect of poly(ethyleneimine) addition, etc. The optimized electrodes were characterized by a sensitivity of 88.36 +/- 0.14 microA mM(-1) cm(-2), a detection limit of 0.3 microM (calculated as three times the signal-to-noise ratio), a response time of less than 10 s and responded linearly between 0.3 and 250 microM (linear regression coefficient = 0.999) with an operational stability of only 3% sensitivity loss during 8 h of continuous FI operation at a sample throughput of 30 injections h(-1).

Amino Acid Oxidoreductases↗
PubMed1998

Detection of heavy metal ions at femtomolar levels using protein-based biosensors.

Sensors based on proteins (GST-SmtA and MerR) with distinct binding sites for heavy metal ions were developed and characterized. A capacitive signal transducer was used to measure the conformational change following binding. The proteins were overexpressed in Escherichia coli, purified, and immobilized in different ways to a self-assembled thiol layer on a gold electrode placed as the working electrode in a potentiostatic arrangement in a flow analysis system. The selectivity and the sensitivity of the two protein-based biosensors were measured and compared for copper, cadmium, mercury, and zinc ions. The GST-SmtA electrodes displayed a broader selectivity (sensing all four heavy metal ions) compared with the MerR-based ones, which showed an accentuated selectivity for mercury ions. Metal ions could be detected with both electrode types down to femtomolar concentration. The upper measuring limits, presumably due to near saturation of the proteins' binding sites, were around 10(-10) M. Control electrodes similarly constructed but based on bovine serum albumin or urease did not yield any signals. The electrodes could be regenerated with EDTA and used for more than 2 weeks with about 40% reduction in sensitivity.

Amino Acid Sequence↗
PubMed1995

Design and optimization of a selective subcutaneously implantable glucose electrode based on "wired" glucose oxidase.

An implantable 0.29 mm o.d. flexible wire electrode was designed for subcutaneous monitoring of glucose. The electrode was formed by sequentially depositing in a 0.09 mm deep shielded recess at the tip of a polyimide-insulated 0.25 mm gold wire a "wired" glucose oxidase (GOX) sensing layer, a mass transport limiting layer, and a nonfouling biocompatible layer. The glucose sensing layer was formed by cross-linking (poly[(1-vinylimidazolyl)osmium(4,4'-dimethylbipyridine)2Cl] )+/2+(PVI13-dme - Os) and GOX with poly(ethylene glycol) diglycidyl ether (PEG). The glucose mass transport restricting layer consisted of a poly(ester sulfonic acid) film (Eastman AQ 29D) and a copolymer of polyaziridine and poly(vinyl pyridine) partially quaternized with methylene carboxylate. The outer biocompatible layer was formed by photo-cross-linking tetraacrylated poly(ethylene oxide). The three layers contained no leachable components and had a total mass less than 2.2 micrograms (approximately 50 ng of Os). When poised at +200 mV vs SCE and operated at 37 degrees C, the 5 x 10(-4) cm2 electrode had in vitro a sensitivity of 1-2.5 nA mM-1. The current increased with the glucose concentration up to 60 mM, and the 10-90% response time was approximately 1 min when the glucose concentration was abruptly raised from 5 to 10 mM. The sensitivity decreased by less than 4% over a test period of 1 week, during which the electrode was operated continuously in a 10 mM glucose physiological buffer solution at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Design, characterization, and one-point in vivo calibration of a subcutaneously implanted glucose electrode.

A 0.29-mm-diameter flexible electrode designed for subcutaneous in vivo amperometric monitoring of glucose is described. The electrode was designed to allow "one-point" in vivo calibration, i.e., to have zero output current at zero glucose concentration, even in the presence of other electroreactive species of serum or blood. A valid zero point, along with a measurement of the glucose concentration in a withdrawn sample of blood at which the current is known, defined the sensitivity in the linear response range. The electrode was four-layered, with the layers serially deposited within a 0.125-mm recess upon the tip of a polyimide-insulated 0.25-mm gold wire. The recessed structure reduced the sensitivity to movement and allowed, through control of the depth of the recess, control of the transport of glucose and thus the range of linearity. The recess contained the four polymeric layers, with a total mass less than 5 micrograms and no leachable components. The bottom glucose concentration-to-current transducing layer consisted of the enzyme "wiring" redox polymer poly[(vinylimidazole)Os(bipyridine)2Cl]+ , complexed with recombinant glucose oxidase and cross-linked with poly(ethylene glycol) diglycidyl ether, to form an electron-conducting hydrogel. The layer was overcoated with an electrically insulating layer of polyaziridine-cross-linked poly(allylamine), on which an immobilized interference-eliminating horseradish peroxidase based film was deposited. An outer biocompatible layer was formed by photo-cross-linking derivatized poly(ethylene oxide). The current output of a typical electrode at 10 mM glucose and at 37 degrees C was 35 nA, the apparent Km was 20 mM, and the 10-90% response time was approximately 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A reagentless amperometric biosensor for alcohol detection in column liquid chromatography based on co-immobilized peroxidase and alcohol oxidase in carbon paste.

A reagentless carbon paste electrode chemically modified with covalently bound alcohol oxidase and horse-radish peroxidase was examined as a selective sensor in flow injection and column liquid chromatography. A combination of carbodiimide, glutaraldehyde, and polyethyleneimine was used for immobilizing the enzymes in the paste. The surface of the electrodes was protected by first forming a layer of electropolymerized ortho-phenylenediamine followed by deposition of a cation exchange membrane (Eastman AQ 29D). The electrodes were used for detection of hydrogen peroxide, methanol, ethanol, propanol, isopropanol, and butanol. Preliminary investigations of the use of this sensor for bioprocess control are reported.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Preliminary investigations of an amperometric oligosaccharide dehydrogenase-based electrode for the detection of glucose and some other low molecular weight saccharides.

Biosensors for the determination of sugars were constructed using oligosaccharide dehydrogenase (ODH) and soluble phenazine methosulfate (PMS) or an osmium-based three-dimensional redox hydrogel. In the latter case the enzyme and poly(1-vinylimidazole) complexed with osmium (4,4'-dimethylbpy)2Cl were cross-linked with poly(ethylene glycol) diglycidyl ether. Both electrode configurations showed similar sensitivities for glucose in the range between 8 and 21 muAmM-1 cm-2. The responses for 10 mono and oligosaccharides were studied. There was no response for fructose. In the concentration range 0.1-20 mM the relative sensitivities were determined for arabinose (96%), xylose (3%), mannose (50%), galactose (11%), glucose (100%), maltose (24%), lactose (12%), cellobiose (34%) and maltotriose (10%).

Biosensing Techniques↗