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Biosensors for Organophosphorus Compounds

#1055


Development of Biosensor Methods for the Analysis of Organophosphorus Compoumds Induced Acute and Delayed Neurotoxicity

Tech Area / Field

  • ENV-MIN/Monitoring and Instrumentation/Environment

Status
8 Project completed

Registration date
01.08.1997

Completion date
22.06.1999

Senior Project Manager
Kondratenkov Yu B

Leading Institute
Research Center of Molecular Diagnostics and Theraphy, Russia, Moscow

Collaborators

  • Lawrence Livermore National Laboratory, USA, CA, Livermore\nUS Environmental Protection Agency / National Health and Environmental Effects Research Laboratory, USA, NC, Research Triangle Park\nBattelle Memorial Institute, USA, VA, Arlington\nPacific Northwest National Laboratory, USA, WA, Richland\nUniversity of Michigan / School of Public Health, USA, MI, Ann Arbor\nChemical Industry institute of Toxicology, USA, NC, Research Triangle Park\nUS Army Medical Research Institute of Chemical Defence, USA, MD, Aberdeen Proving Ground

Project summary

Objective

Development of highly-sensitive biosensor's methods for biomedical and ecological monitoring organophosphorus compounds (OPhC) induced acute and delayed neurotoxicity at the people and animals and also realization of direct detection of these compounds.

Technical approach

A number biosensors for analysis of OPhC and their enzymatic targets (butyrylcholinesterase and neurotoxic esterase) will be developed in the project.


1) A potentiometric biosensor for butyrylcholinesterase inhibitor (OPhC induced acute neurotoxicity) analysis based on mediatorless bioelectrocatalysis. The method includes coimmobilization of three enzymes (butyrylcholinesterase, choline oxidase and peroxidase) on composite carbon electrode. Catalytic hydrolysis of butyrylcholine with subsequent oxidation of choline result in the formation of hydrogen peroxide leads to a shift in electrode potential.
2) A potentiometric biosensor for butyrylcholinesterase detection based on application of bienzyme electrode with choline oxidase and peroxidase.
3) An amperometric biosensor for assay of neurotoxic esterase inhibitor (OPhC induced delayed neurotoxicity) analysis and neurotoxic esterase activity in biological samples. The principle of neurotoxic esterase monitoring consists of the combination of the enzymatic hydrolysis of phenyl valerate with amperometric detection of phenol by tyrosinase (laccase) immobilized on Clark-type O2-electrode. Tyrosinase and laccase are copper enzymes which catalyze the oxidation of phenols by dissolved oxygen.
4) The method for direct detection of OPhC using bienzyme conjugate butyrylcholinesterase-peroxidase. According to the method the OPhC binds to the bienzyme conjugate containing butyrylchohnesterase as a receptor part and horseradish peroxidase as a signal amplification part. The separation of the OPhC-bound and the OPhC-free bienzyme conjugate is going onto an affinity support with OPhC derivative. OPhC-free bienzyme conjugate practically irreversible binds to the affinity support due to interaction of the butyrylcholinesterase from conjugate with the support. Only, OPhC-bound bienzyme conjugate can be detected in the solution by the measure of horseradish peroxidase activity. The peroxidase activity is direct proportional to the analyte (OPhC) concentration.

The project is based on the following advanced technological and methodological approaches: a) chemical and physical immobilization of low molecular weight compounds and enzymes; chemical modification of protein molecules; b) original methods of synthesis of analogs OPhC and modes of their stabilization; c) a process engineering of manufacture of the biosensor's elements and electrodes; d) modern affinity perfusion chromatography; e) scanning probe microscopy; f) electrochemistry of protein molecules and mediatorless direct electron transfer effects.

During of the project the following tasks will be solved;


1. Development of the modes of enzymes immobilization (peroxidase, choline oxidase, butyrylcholinesterase, tyrosinase, laccase) on surface from carbon materials and other support. Study of structural, functional and kinetic properties of immobilized enzymes, and also their analytical possibilities at an electrochemical mode of detection.
2. Isolation and purification of neurotoxic esterase from various biological tissues (hens brain, rats brain and bovine adrenal medulla). Study of a kinetics and molecular mechanism the enzyme inhibition.
3. Synthesis of conjugate of cholinesterase with peroxidase.
4. Synthesis of various OPhC derivatives and their immobilization on a solid substrate with the purpose of creation of the affinity support.
5. Development of interfaces and softwares for the electrochemical analysis of OPhC and their enzymatic targets.

Expected outcomes

During the project the biosensor's methods for analysis of OPhC induced acute and delayed neurotoxicity at the people and animals and monitoring their enzymatic target will be developed. The developed complex of biosensor's methods, not having of analogs in world practice, can be used in biomedical (toxicological) practice, and also for environmental monitoring on industrial, agricultural and military plants.

Potential role of foreign collaborators

Joint development, approbation and application of biosensor's methods for the analysis of OPhC. Possible application of developed methods as tools of ecological and biomedical international monitoring at the destruction of the chemical weapon.

Scientific and technical background

The authors of the project have activities in the biosensor field last 17 years. The area of interest is bioelectrocatalytical, electrochemical, and fibre-optic biosensors for the analysis of superecotoxicant, drugs, typical metabolites and ecological monitoring.

Last years the main works have been done in the frames of the programs:


- Russian state scientific and technical program "New methods of bioengineering",
- international Russian-German project "Biosensors",
- research project of the President's Committee for Conventional Problems of Chemical and Biological Weapons of Russian Federation,
- research project of the German Federal Ministry for Research and Technology.

Laboratory prototype of the bioelectrocatalytical biosensor for the detection of metabolites and cholinesterase inhibitors was demonstrated in the international exhibition "Advances in Russian Biotechnology" (Berlin, 1996).

Last 5 years the main principles for the creation of the biosensor system for direct detection of cholinesterase inhibitors based on bienzyme conjugate and substrate amplification systems have been developed. Biochemical background for the creation of the biosensors for the detection of neurotoxic esterase inhibitors and neurotixic esterase activity has been developed also. The results of these investigations are presented in the 35 publications (see main part) and one patent of Russia.


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