Biomedical subjects

P Skládal

Publications and source records attributed to P Skládal.

10 recordsLinked to original sources

PubMed2005

Sensitive detection of organophosphates in river water by means of a piezoelectric biosensor.

A highly sensitive piezoelectric biosensor has been developed for detection of cholinesterase inhibitors. The inhibitor benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on a monolayer of 11-mercaptomonoundecanoic acid (MUA) self-assembled on the gold surface of the sensor. The binding of high-molecular-weight cholinesterase to the immobilized cocaine derivative was monitored with a mass sensitive piezoelectric quartz crystal (quartz crystal nanobalance; QCN). In the presence of an inhibiting substance in the sample, the binding of cholinesterase to the immobilized inhibitor was reduced. The decrease of the rate of mass change was proportional to the concentration of free inhibitor in the sample. This way the affinity sensor followed anti-cholinesterase toxicity and the enzyme activity of ChE was not addressed. A assay for detection of organophosphates (OP) was optimized. Regeneration of the sensor surface was achieved with 1 mol L(-1) formic acid, which enabled 40 measurements with one sensor. All assays were carried out in a flow-through arrangement. The total measurement time (binding+regeneration) was 25 min and the detection limit for different OP (paraoxon, diisopropylfluorophosphate, chlorpyriphos, and chlorfenvinphos) was down to 10(-10) mol L(-1) (0.02 microg L(-1)). This sensor was used for determination of organophosphate (diisopropylfluorophosphate) levels in river water samples.

Biosensing Techniques↗

PubMed2003

Screen-printed multienzyme arrays for use in amperometric batch and flow systems.

Screen-printing technology for electrode fabrication enables construction of amperometric devices suitable for combination of several enzyme electrodes. To develop a biosensor array for characterisation of wastewaters, tyrosinase and horseradish peroxidase (HRP) or cholinesterase-modified electrodes were combined on the same array. The behaviour of the tyrosinase-modified electrode in the presence of hydrogen peroxide (required co-substrate for the HRP-modified electrode) and acetylthiocholine chloride (required co-substrate for cholinesterase) was studied. Performance of bi-enzyme biosensor arrays in the batch mode and in the flow-injection system are discussed.

Acetylcholinesterase↗

PubMed2003

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus.

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.

Amino Acid Sequence↗

PubMed2001

Investigation of highly sensitive piezoelectric immunosensors for 2,4-dichlorophenoxyacetic acid.

The improved highly sensitive piezoelectric immunosensor has been developed and evaluated using a model interaction of antibody with the model hapten-herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). For immobilization of 2,4-D, the self-assembled layers of cystamine, 4-aminothiophenol or 3,3'-dithio-bis(propionic acid N-hydroxysuccinimide ester) were formed on smooth and rough crystals coated with gold or silver electrodes. The immunochemical interactions performed well in all cases, the aminothiophenol on gold was chosen as the optimum with regard to regeneration of immunosensing surfaces. The kinetics of interaction of surface-bound 2,4-D with free antibody provided significantly higher kinetic parameters (kinetic association rate constant) when using optically smooth crystals compared to common rough crystal. Therefore, the smooth crystal should be preferred for future kinetic studies. The competitive assay of the herbicide 2,4-D achieved a limit of detection of 10 ng/l using the monoclonal anti-2,4-D antibody F6C10. Finally, a direct assay format has been evaluated using a thicker layer of glutaraldehyde-crosslinked antibody on the sensing surface. The direct binding of a small herbicide molecule was followed in real time. The detected concentration of 2,4-D (5 microg/l) was low enough for future direct monitoring of this herbicide in water.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1999

Effect of methanol on the interaction of monoclonal antibody with free and immobilized atrazine studied using the resonant mirror-based biosensor.

The interaction of monoclonal anti-atrazine antibody D6F3 with free and immobilized atrazine was studied using the resonant mirror-based optical biosensor system IAsys. The binding of antibody to atrazine immobilized on silanized surface through albumin spacer was studied in the presence of methanol. The highest affinity was observed in 10% methanol, the kinetic equilibrium association constant KA was 1.16 x 10(9) mol-1 l compared to 4.3 x 10(8) mol-1 l determined in water. The surface binding capacity was 2-fold lower in the presence of methanol compared to aqueous buffer solution. The kinetic rate constants were significantly improved with low contents of methanol; the fastest association and slowest dissociation occurred at 5 and 10% methanol, respectively. The formation of immunocomplexes was observed even in the presence of 50% methanol. To avoid possible disturbing effects resulting from the immobilization of atrazine, the interaction of free atrazine and antibody was studied using the competitive procedure. The determined values of KA were 9.35, 0.73 and 410 x 10(6) mol-1 l for interactions carried out in 10, 30 and 50% methanol, respectively. For practical determination of atrazine using this antibody, the content of 10% methanol in the assay mixture seems to be the best choice. Thirty percent methanol resulted in the lowest affinity; 50% methanol provides the highest affinity, but much smaller signal is measured. The affinity biosensor system IAsys appeared to be a suitable, rapid and convenient tool for studies of binding interactions in the presence of organic solvents.

Antibodies, Monoclonal↗

PubMed1997

Construction and characterization of the direct piezoelectric immunosensor for atrazine operating in solution.

The direct immunosensor for determination of the herbicide atrazine was studied. The gold electrodes of the piezoelectric quartz crystal were silanized and activated using glutaraldehyde. The bioaffinity ligand atrazine was linked through albumin as a spacer molecule. The modified piezoelectric crystal was placed in a flow cell and all measurements were performed directly in flowing solution. The interaction of the anti atrazine monoclonal antibody (MAb, clone D6F3) with the immobilized atrazine was characterized using both crude ascitic fluid and Protein A-purified MAb preparates. The association and dissociation rate constants were determined, ka = 1.21 x 10(5) M-1S-1 and kd = 4.0 x 10(-4)S-1. The competitive determination of free atrazine was studied using different dilutions (100x, 250x and 1000x) of the ascitic fluid containing MAb. MAb was preincubated with atrazine (concentrations 0-1 microgram/l) for 15 min and the mixture was then introduced to the flow cell. As a signal, either the rate of frequency decrease or the relative change of frequency after the fixed binding period (10 min) was evaluated. As expected, the higher dilutions of MAb provided improved sensitivity for the analyte. For the 1000x diluted ascitic fluid, 0.1 and 1 microgram/l atrazine caused 5 and 30% decreases of the relative binding of MAb, respectively. Repeated use of the crystals was achieved using a 5 min flow of 100 mM NaOH for regeneration. The results obtained seem to be promising for determination of atrazine in drinking water using direct piezoelectric immunosensors.

Antibodies, Monoclonal↗

PubMed1994

Characterization of monoclonal antibodies to 2,4-dichlorophenoxyacetic acid using a piezoelectric quartz crystal microbalance in solution.

Piezoelectric quartz crystals (resonance frequency 10 MHz) were used for an investigation of the immunochemical reaction between 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide and several monoclonal antibodies prepared against 2,4-D. The herbicide was immobilized on the gold electrodes of the crystals silanized with 3-aminopropyltriethoxysilane. The activated carboxylic group of 2,4-D was linked either directly to the silanized surface or through hexamethylenediamine or albumin as a macromolecule. The interaction of the immobilized antigen with monoclonal antibody in solution was followed as a change in the resonant frequency of the crystals. The best results were achieved using 2,4-D attached to albumin. The affinity binding of five monoclonal antibodies was characterized by association (ka) and dissociation (kd) kinetic rate constants and by equilibrium association constants (KA) which were obtained from the experimental frequency vs. time curves.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1992

Amperometric glucose biosensor with extended concentration range utilizing complexation effect of borate.

Borate buffer strongly decreases amperometric response of a glucose oxidase linked pO2 or H2O2 sensing electrode, extending substantially its linear calibration range. With increasing pH and concentration of the buffer the upper limit for glucose can be varied between 1 and 30 mmol l-1 glucose. The effect of borate ion is explained by the rapid complexation of glucose decreasing the equilibrium concentration of free beta-anomer, the specific substrate of glucose oxidase. The high loading of cross-linked enzyme inside the sensor membrane is necessary for the measurement to ensure an almost constant response factor (delta i per 1 mmol l-1) between pH 5 and 10. Analysis in stirred solution and in a flow-through system has been employed for the measurement of elevated glucose levels in heparinized human blood or plasma samples.

Biosensing Techniques↗

PubMed1990

Formation of a potent respiratory inhibitor at nitrite reduction by nitrite reductase isolated from the bacterium Paracoccus denitrificans.

A new method of dissimilatory nitrite reductase (cytochrome cd1) isolation from the periplasmic fraction of anaerobically grown cells of the bacterium Paracoccus denitrificans was developed, using ionex and gel permeation chromatography with FPLC system (Pharmacia, Sweden). In experiments with isolated enzyme it was shown that through a nitrite reduction, catalysed by this enzyme, a substance (presumably nitric oxide) was formed which at submicromolar concentrations inhibited terminal cytochrome oxidase of the respiratory chain of the same bacterium. These results help to explain formerly observed sensitivity of bacterial oxidase activity to NO2- and the mechanism of switching the electron flow from O2 to nitrogen terminal acceptors.

Chromatography, High Pressure Liquid↗

PubMed1990

Hydroxylamine as an inhibitor and terminal acceptor in the respiratory chain of the bacterium Paracoccus denitrificans.

Three sites of inhibitory action of hydroxylamine were identified in the respiratory chain of anaerobically grown bacterium Paracoccus denitrificans. Terminal oxidases were blocked at concentrations of 10(-4) to 10(-3) mol.l-1, and the inhibitor competed with artificial donor of electrons N, N, N', N'-tetramethyl-l, 4-phenylenediamine. In the anaerobic part of the respiratory chain inhibition of nitrite reductase and apparently also nitric oxide reductase occurred, resulting in the increased accumulation of nitric oxide during denitrification. These effects together with the inhibition of terminal oxidases by nitric oxide are probably realized through switching the electron flow from oxygen to nitrogen terminal acceptors in the presence of hydroxylamine. By means of difference spectroscopy, the respiratory inhibitor mucidin and a cytochrome c-deficient mutant of Paracoccus denitrificans, hydroxylamine could be shown to serve also as a terminal acceptor of the cytochrome c region. Reduction of hydroxylamine to ammonia was at the same time accompanied by the formation of transmembrane electrical gradient. Hydroxylamine reductase was purified 123-fold from the periplasmatic cell fraction by FPLC; the product obtained showed the features of respiratory nitrite reductase of the cytochrome cd1 type.

Anaerobiosis↗