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Détection des insecticides organophosphorés et carbamates

Détection des insecticides organophosphorés et carbamates. Nerve cell communication: Synaptic membrane. How nerve/muscle junctions work. Acetyl Choline (AC) Hydrolysed AC AC receptor AC esterase. Acetylcholine is released by the nerve cell

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Détection des insecticides organophosphorés et carbamates

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  1. Détection des insecticides organophosphorés et carbamates

  2. Nerve cell communication: Synaptic membrane

  3. How nerve/muscle junctions work Acetyl Choline (AC) Hydrolysed AC AC receptor AC esterase • Acetylcholine is released by the nerve cell • Binds to protein receptor in muscle cell membrane to trigger contraction

  4. How nerve agents work Acetyl choline AC receptor AC esterase Nerve agent • Nerve agents block the breakdown of acetylcholine: muscle contracts continuously • This results in paralysis

  5. Enzyme inhibition Screen printing Encapsulation Sol-gel Affinity Stability Enzymes -genetically modified -synthetic Hypersensitivity STRATEGY

  6. Enzyme inhibition Screen printing STRATEGY

  7. Amperometric Biosensor

  8. Silver conducting tracks Graphite pads Graphite working electrode Ag/AgCl Reference electrode Insulating layer Screen printed method

  9. LOW COST PORTABLE SINGLE USE HIGHLY REPRODUCIBLE SAMPLE HANDLING MASS PRODUCTION NO SKILLED PERSONNAL FAST DETECTION

  10. Encapsulation Sol-gel Affinity Stability STRATEGY

  11. Entrapment of AChE in PVA-SbQ

  12. Immobilisation of AChE in SOL-GEL

  13. C O C O O O G G R R C H CO C H CO 2 2 AChE A A C H - (His)6 C H P P O O H H O N O N I I C H T E T E C H 2 Ni 2 OC OC O H O O H O 2 2 O H N 2 HN AChE Principle of Metal Affinity Chelate Ni Graphite-NTA-Ni

  14. G R A AChE AChE P H I T E Sugar Concanavalin A Principle of Con A

  15. G R A AChE AChE P H I T E Sugar Concanavalin A Principle of Con A

  16. Enzymes -genetically modified -synthetic Hypersensitivity STRATEGY

  17. CHOLINESTERASE • Electric eel • Bovine erythrocyte • Torpedo california • Horse serum • Drosophila melanogaster

  18. CHOLINESTERASE • Electric eel (98 % Biotools) • Drosophila melanogaster

  19. Values of Ki in mmole-1.L-1

  20. Acetylcholinesterase structure

  21. Engineered cholinesterase Modification of the active site • Residue replacement • Insertion • Deletion • Combination of mutations Obtention of sensitive mutants

  22. Detection of carbamate insecticides PESTICIDES Pirimicarb Carbaryl Carbofuran SOURCE OF ENZYME • Drosophila melanogaster (Dm)

  23. Amperometric determination of thiocholine, produced by enzymatic hydrolysis of acetylthiocholine substrate Cl- (CH3)3N+-CH2-CH2-SH thiocholine H+ + R-COO- + Cl- H2O (CH3)3N+-CH2-CH2-S-CO-CH3 100 mV vs Ag/AgCl acetylthiocholine TCNQ(ox) Thiocholine(ox) + 2H+ Thiocholine(red) 2e- TCNQ(red) SPE AChE TCNQ mediator decreases the working potential from 410 mV to 100 mV vs. Ag/AgCl Detection principle

  24. Potentiostat Potentiostat Potentiostat Buffer + Insecticide Buffer Buffer ILLUSTRATEDPROCEDURE 1. Measurement of the initial current, I0 2. Incubation time: 20 min %I>10% %Inhibition=(I0-I1)/I0 x 100 %I<10%

  25. Pirimicarb

  26. Carbaryl

  27. Carbofuran

  28. Real sample analysis

  29. OP-Prot Sensor OP-Stick Sensor

  30. OP-Prot Sensor

  31. ILLUSTRATED PROCEDURE Tested solution: If solution still uncolored, the test is positive. There are inhibiting substances in the solution Transfer 10ml to tube 1 yellow Transfer to tube 2 Blue Transfer to tube 3 red Transfer to tube 4 green Solution to be tested 1 2 3 4 Control solution insecticide free 1 2 3 4 Shake and incubate 10 min. Shake and incubate 15 min. Shake and incubate 1 hour. Shake and incubate 5 to 10 min. Reference tube has to become yellow, meaning there is no insecticide

  32. LIMIT OF DETECTION COMPOUND WATER (ppb or mg/L) Chlorpyriphos ethyl 0.1 Paraoxon 1 Parathion 1 Dichlorvos 1 Malaoxon 1 Malathion 1 Profenofos 10 Carbofuran 10 Carbaryl 100 Pirimicarb 1000

  33. LIMIT OF DETECTION COMPOUND WATER (ppb or mg/L) Chlorpyriphos ethyl 0.1 Paraoxon 1 Parathion 1 Dichlorvos 1 Malaoxon 1 Malathion 1 Profenofos 10 Carbofuran 10 Carbaryl 100 Pirimicarb 1000

  34. Oxidation O S HO P OR1 HO P OR1 OR2 OR2 NO TOXIC Parathion Malathion TOXIC Paraoxon Malaoxon

  35. OP-Stick Sensor

  36. LOD

  37. Toulouse(FRANCE)

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