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Biomedical subjects

J Rishpon

Publications and source records attributed to J Rishpon.

At least 19 recordsLinked to original sources
PubMed2003

Electrochemical detection of protein-protein interactions using a yeast two hybrid: 17-beta-estradiol as a model.

In this work we present a modified yeast two-hybrid bioassay for the highly sensitive detection of protein-protein interactions, based on the electrochemical monitoring of beta-D-galactosidase reporter gene activity, using p-aminophenyl-beta-D-galactopyranoside (PAPG) as a synthetic substrate. In a model system, the sensitive detection of 17-beta-estradiol was achieved at concentrations as low as 10(-11)M (approx 2 pg/ml) by monitoring 17-beta-estradiol receptor dimerization after exposure to 17-beta-estradiol. The sensitivity of this system was higher than that of standard optical methods by three orders of magnitude.

Dimerization↗
PubMed2003

Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria.

Here, we describe a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria. The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometic measurement of enzymatic activity. As a model system, we used virulent phage typing and cell-marker enzyme activity (beta-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655). Filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 h. In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage.

Bacteria↗
PubMed2000

Immobilized parathion hydrolase: an amperometric sensor for parathion.

An amperometric enzyme biosensor for the direct measurement of parathion was developed. The biosensor is based on parathion hydrolase from Pseudomonas sp. isolated from contaminated soil. The enzyme, which was immobilized on a carbon electrode, catalyzes the hydrolysis of parathion to form p-nitrophenol, which was detected by its anodic oxidation. The enzymatic and electrochemical reactions were examined and optimized. Screen-printed electrodes and a microflow injection system provide the means to significantly reduce the volume of the detected samples. Pulsed techniques further increased the sensitivity of the measurement. The current signal was linearly related to the parathion concentration, and the detection limit was less than 1 ng/mL. The biosensor is rapid as well and can be used outdoors and indoors by a nonqualified person.

Aryldialkylphosphatase↗
PubMed2000

A micro flow injection electrochemical biosensor for organophosphorus pesticides.

We describe a disposable, amperometric micro flow injection electrochemical biosensor that can be applied to the identification and quantification of highly toxic organophosphorus (OP) compounds in the environment, on the spot and in a short time. The system traces very small quantities of OP by monitoring the enzymatic reaction of acetylcholine esterase (AChE) and its inhibition. The sensor is sensitive, rapid, small, inexpensive, disposable and can be operated by non-professional technicians. The electrochemical cell consists of screen-printed electrodes covered with an enzymatic membrane and placed in a home-made flow cell. The electrodes are connected to a computer-controlled potentiostat. We quantitatively detected the OP compound, dimethyl 2,2-dichlorovinyl phosphate (DDVP), by monitoring the OP induced decrease in enzymatic degradation of the substrate, acetylthiocholine chloride (ATCh), to thiocholine and acetic acid. Thiocholine reacts with hexacyanoferrate ion in the working solution and the reduction of [Fe(CN)6](-3) to [Fe(CN)6](-4) and its subsequent reoxidization by the electrode generates very sharp, rapid and reproducible electric signals. The ability to detect low quantities is extremely important when dealing with hazardous environmental pollutants.

Acetylcholinesterase↗
PubMed2000

Online and in situ monitoring of environmental pollutants: electrochemical biosensing of cadmium.

Online sensitive monitoring of gene expression is essential for understanding microbial life and microbial communities, especially under stress-inducing conditions, such as the presence of environmental pollutants. We describe here a novel use of promoter-based electrochemical biosensing for online and in situ monitoring of gene expression in response to pollutants. As a model system, we used a cadmium-responsive promoter from Escherichia coil fused to a promoterless lacZ gene, which was monitored using an electrochemical assay of beta-galactosidase activity. This whole-cell biosensor could detect, within minutes, nanomolar concentrations of cadmium in water, sea water and soil samples, and it can be used for continuous online and in situ monitoring.

Adenosine Triphosphatases↗
PubMed2000

A three-cascaded-enzymes biosensor to determine lactose concentration in raw milk.

The increasing demand for on-line measurement of milk composition directs science and industry to search for practical solutions, and biosensors may be a possibility. The specific objective of this work was to develop an electrochemical biosensor to determine lactose concentration in fresh raw milk. The sensor is based on serial reactions of three enzymes--beta-galactosidase, glucose oxidase, and horseradish peroxidase--immobilized on a glassy carbon electrode. The sequential enzymatic reactions increase the selectivity and sensitivity of the sensor. The sensor requires dilution of the raw milk and the addition of 5-aminosalicylic acid. Lactose concentrations in raw milk measured by the sensor were in good agreement with those measured by a reference laboratory using infrared technology. The results were obtained in milk samples that varied in fat and protein composition. From the results, we conclude that an electrochemical biosensor for determination of lactose concentration in fresh raw milk can be developed, and that the biosensor presented in this study maintained the qualities required for further development into an online sensor in the milking parlor.

Animals↗
PubMed1998

Interactions of calmodulin with metal ions and with its target proteins revealed by conformation-sensitive monoclonal antibodies.

Two monoclonal antibodies (mAbs) raised against bovine calmodulin (CaM), CAM1 and CAM4, enable one to monitor conformational changes that occur in the molecule. The interaction of CAM1 with CaM depends on the Ca2+ occupancy of its Ca(2+)-binding sites. CAM4, in contrast, interacts with CaM in a Ca(2+)-independent manner, interacting with both holoCaM and EGTA-treated CaM to a similar extent. Their interaction with various CaMs, CaM tryptic fragments and chemically modified CaM, as well as molecular graphics, led to identification of the CAM1 and CAM4 epitopes on the C- and N-terminal lobes of CAM respectively. The two mAbs were used as macromolecular probes to detect conformational changes occurring in the CaM molecule upon binding of metal ions and target proteins and peptides. MAb CAM1 successfully detected changes associated with Al3+ binding even in the presence of Ca2+, indicating that Al3+ and Ca2+ ions may bind to the protein simultaneously, leading to a new conformation of the molecule. MAbs CAM1 and CAM4 were used to follow the interactions of CaM with its target peptides and proteins. Complexes with melittin, mastoparan, calcineurin and phosphodiesterase showed different immunological properties on an immuno-enzyme electrode, indicating unique structural properties for each complex.

Aluminum↗
PubMed1997

An amperometric enzyme-channeling immunosensor.

This paper presents a new disposable amperometric, enzyme-channeling immunosensor for a quantitative, rapid, separation-free enzyme immunoassay (EIA) that can be used in clinical diagnostics, as well as in biomedical, biochemical, and environmental research. The sensor consists of a disposable, polymer-modified, carbon electrode on which enzyme 1 is coimmobilized with a specific antibody that binds the corresponding antigen in a test solution. The solution also contains a conjugate of enzyme 2. An immunological reaction brings the two enzymes into close proximity at the electrode surface, and the signal is amplified through enzyme channeling. The localization of both enzymes on the electrode surface limits the enzymatic reactions to the polymer/membrane/electrode interface. The sensor overcomes the problem of discriminating between the signal that is produced by the immuno-bound enzyme label on the electrode surface and the background level of signal that emerges from the bulk solution. Combining enzyme-channeling reactions, optimizing hydrodynamic conditions, and electrochemically regenerating mediators within the membrane layer of the antibody electrode significantly increased the signal-to-noise ratio of the sensor. The amperometric enzyme-channeling immunosensor enabled the performance of separation-free EIAs without washing steps, resulting in a relatively short assay time of 5-30 min for the complete immunoassay, compared with at least 1-3 h for ELISA methods. Model systems using peroxidase-antibody, biotin-avidin, viral antigens (CD4-gp120), and bacteria (Staphylococcus aureus) were investigated. S. aureus cells were detected in pure culture at concentrations as low as 1000 cells/ml.

Biosensing Techniques↗
PubMed1996

A one-step, separation-free amperometric enzyme immunosensor.

A new one-step, separation-free, amperometric enzyme immunosensor is described. The sensor consists of an antibody electrode that is low cost, disposable, and operates without washing or separation steps. The immunosensor combines the following signal-amplification systems: enzyme-channeling immunoassay; accumulation of the redox mediators (I2/I-); cyclic regeneration of an enzyme (peroxidase) substrate at the (polyethylenimine) polymer/electrode interface; and control of the hydrodynamic conditions at the interface of the antibody electrode. The immunological reactions were monitored electrochemically in situ, and the binding curves were directly visualized on a computer screen. The complete immunoassay can be performed in 5-20 min depending on the complexity of the immunological reactions. Model systems using rabbit IgG and human luteinizing hormone (hLH) in a 'sandwich' immunoassay revealed that the immunosensor can detect concentrations of hLH in human serum as low as 1 ng ml-1.

Antibodies↗
PubMed1993

Novel approaches for the use of mediators in enzyme electrodes.

This work describes the preparation of glucose electrodes consisting of an enzyme (glucose oxidase) and a mediator embedded in a colloidal graphite emulsion matrix. These components are homogeneously mixed in an organic medium that evaporates rapidly while the enzyme activity remains intact. The appropriate conditions for preparation and measurement, as well as electrochemical characterization of these electrodes, are discussed. The problem of mediator leaching in electrodes using vinyl ferrocene was overcome by the use of a special membrane that prevented leaching of positive ferricinium ions by electrostatic repulsion under the appropriate measurement conditions, including minimal exposure of the electrode to anodic potentials, thus increasing the long-term stability. The resulting electrodes are easily prepared, have fast and high response independent of oxygen tension and can be stored in dry form. The linear range can be extended by the introduction of a dialysis membrane and can thus be adjusted to the measurement of glucose concentration in the range of medical interest.

Biosensing Techniques↗
PubMed1992

A rapid and sensitive heterogeneous immunoelectrochemical assay using disposable electrodes.

In this novel enzyme-tagged immuoelectrochemical assay, disposable carbon felt discs serve both as electrodes and as the heterogeneous solid phase. Antibodies are immobilized on the carbon felt via a diaminoalkane-biotin-avidin-biotin bridge. Alkaline phosphatase is used as a label. Bound antibodies are monitored by following the electro-oxidation of aminophenol, produced enzymatically from p-amino-phenyl phosphate by the immobilized alkaline phosphatase at the electrode surface. A model system designed for determination of mouse IgG concentration yielded a calibration curve ranging from 10 pg/ml to 100 micrograms/ml. This assay can be performed rapidly and a single determination completed within 20 minutes. The system is useful also for rapid quantitation of a small number (approximately 80 organisms per ml) of bacteria.

Animals↗
PubMed1991

Investigation of polypyrrole/glucose oxidase electrodes by ellipsometric, microgravimetric and electrochemical measurements.

We describe the simultaneous application of two in-situ techniques for the study of the electrochemical growth of a conducting polymer film (polypyrrole) in the presence of an enzyme (glucose oxidase). The combination of optical (ellipsometric) and microgravimetric (QCMB) measurements employed in this study provides information on fundamental properties of the enzyme-containing film, including film thickness, mass and density. Our results show that incorporation of the enzyme results in changes in the apparent optical properties and in the apparent density of the electrochemically grown film which suggest mutual stabilization of the polypyrrole and the enzyme in the composite layer.

Biosensing Techniques↗
PubMed1989

The development of an immunosensor for the electrochemical determination of the isoenzyme LDH5.

In this work we describe an immunoelectrochemical technique for the determination of LDH isoenzymes. The method is based on the covalent binding of monoclonal antibody against the isoenzyme LDH5 to a glassy carbon electrode, and the exploitation of the electrode surface as the solid phase for enzyme immunoassay. The resulting enzyme electrode was studied by a computerized electrochemical system. This combination formed the basis of a highly sensitive fast and convenient technique for the determination of the LDH isoenzymes in sera.

Animals↗
PubMed1982

Interaction between ATP, metal ions, glycine, and several minerals.

The adsorption of ATP and ADP on montmorillonite, kaolinite, and A1(OH)3 was studied as a function of pH and, for montmorillonite and kaolinite, as a function of the ionic composition of the system. The three minerals exhibit different adsorption characteristics. Mg2+- and Zn2+-montmorillonite adsorb ATP and ADP more than Na+-montmorillonite, presumably because of complex formation. In kaolinite, the effect of these divalent cations is small. Pure ATP decomposes upon heating, and the rate of the decomposition is accelerated by the presence of glycine. Drying and heating glycine to 70 degrees C under vacuum in the presence of ATP results in abiotic peptide formation with yields up to 0.25%. This peptide formation also occurs when kaolinite or montmorillonite is added to the system. The presence of kaolinite, Mg2+- or Zn2+-kaolinite, or Mg2+-montmorillonite results in a reduction in the rate of the ATP decomposition in the abiotic peptide synthesizing system. These results suggest that one role for clays and metal ions in chemical evolution may have been the stabilization of nucleotides during prebiotic peptide synthesis.

Adenosine Diphosphate↗