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Nouveaux biocapteurs et amelioration de senseurs existants grace au concept de "capteur intelligent"; application aux milieux aquatiques


The principal aims of this project are the:

- Development of sensitive and selective practical tools for aquatic media monitoring and management, with particular emphasis on methods used in waste water treatment plants.

- Use of existing or prospective measurement methods and integration in low-cost prototypes.

- Illustration and demonstration of the "smart sensor" concept:

. Development of a miniaturised micro-controller card

. Elaboration of signal treatment software performing the following functions:
decorrelation, parametric adjustment, extrapolation, compensation, outside world
action, use of other available information.

. Hardware/software integration.

Tests have been performed to compare different methods of measuring biological oxygen demand (BOD). Classical manometric BOD measurements have been improved by the use of differential pressure sensors. Data acquisition is automatic and classical BOD models can be fitted directly. Rapid BOD measurements using bioelectrodes or on bioreactors are being evaluated.

A selection of organic compound (eg tesazurin, resorufin, phenazine methosulphate, meldola blue, hexacyannoferrate) have been used to assess their potential as electrochemical mediators and/or artificial electron acceptors. To decide their future utilization, those compounds were fully characterized spectrophotometrically and electrochemically. Two categories of compounds can be defined: reversible and nonreversible systems. Irreversibility is an ideal property for quantitative measurement of dehydrogenasic activities of microorganisms. On the other hand, irreversibility is not desirable as part of chemically modified electrodes.

Measurements of electron transport system (ETS) activity have been carried out in the first instance with pure strain cultures of Pseudomonas aeruginosa, Alcaligenes, Zoogloea and a strain isolated from activated sludge. Assays of ETS activity being assessed are: activity measurements at different E0 representative of the main steps in respiratory chain; influence of dehydrogenases inhibitors on such measurements; correlation of those measurements with classical respirometric methods (oxygen consumption). Resazurin is being tested because of the reversibility of the redox system and also because both forms are soluble.

A flow injection analysis (FIA) system has been developed for the detection of pesticides in water. The detection is based on the inhibition of the cholinesterase. The inhibition of cholinesterase is the result of a stable covalent intermediate such as phosphoryl enzyme complexes which hydrolyse very slowly. For practical use, it is necessary to reactivate the inhibited enzyme. The reactivation of the enzyme after inhibition by increasing concentrations of paraoxon (PX) was studied using 2-PAM (2-pyridine aldoxime methiodide). The enzyme recovered 100% of its original activity after up to 80% inhibition.

The detection of very low concentration of organophosphorus insecticides in water required preconcentration or extraction and concentration using organic solvents. It is well known that the enzymes are capable of functioning in some organic solvents and various solvents were investigated. The hydrophobicity of the organic solvent plays a major role in determining stability and catalytic activity, the cholinesterase being inactivated in miscible organic solvents. The non polar organic solvent had little effect on the structure of the enzyme.

In order to apply the biosensor in the organic phase, the inhibition of the immobilized cholinesterase was measured after incubation for 30 minutes with an organic solvent pesticide solution. The determination of inhibited enzyme activity was performed in 0.05 M phosphate buffer pH 8 and nondenaturing solvents were tested. The percent of inhibition depends on the nature of the solvent. Compared to the reference inhibition rate achieved in buffer, the inhibitory capacity of paraoxon was good in all the solvents except butylacetate and ether.

For the isolation of pesticides from various types of waters, dichloromethane liquid liquid extraction (LLE), off line solid phase extraction (SPE) and supercritical fluid extraction (SFE) techniques have been used. A correlation has been carried out between the various analytical techniques and the enzymatic cholinesterase sensor, as applied to the trace determination of selected organophosphorus pesticides fenitrothion, parathion ethyl and fenamiphos stored in freeze dried water samples that contain also other pesticides. In this way the enzymatic biosensor can be validated and the problems encountered with its application to the determination of organophosphorus pesticides in water samples (eg matrix interferences, cross reactivity, influence of the extraction solvent used in LLE) is known. Correlations are being established between enzymatic response and gas chromatography with nitrogen phosphorus (GC-NPD) and liquid chromatography with diode array detection (LC-DAD). The whole methodology developed permits the determination of organophosphorus pesticides in spiked water samples at concentration levels varying from 0.03 to 6.9 ug/l.
The expected results are:

- new biosensors (pesticides, activity, fast BOD) and micro-sensors (CO2, O2, pH, NO3-, NH4+,
- an universal miniaturised micro-controller card
- prototypes of intelligent probes and tests (BOD; modified Redox sensor for nitrification-denitrification management).

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185,Av. de Longwy 185
6700 ARLON

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Participants (5)