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
How nerve/muscle junctions work
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 agents block the breakdown of acetylcholine: muscle contracts continuously
This results in paralysis

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

6. STRATEGY
Enzyme inhibition
Screen printing

7. Amperometric
Biosensor

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

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

10. STRATEGY
Stability
Encapsulation Sol-gel Affinity

11. Entrapment of AChE in PVA-SbQ

12. Immobilisation of AChE in SOL-GEL

13. Principle of Metal Affinity Chelate2 CO OC HN
AChE N Ni C H O 2 CO OC G R A P I T E O G R A P H I T E
- (His)6
AChE Ni
Graphite-NTA-Ni

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

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

16. STRATEGY
Hypersensitivity
Enzymes genetically modified synthetic

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

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

19. Drosophila melanogaster
Insecticide
Electric eel
Drosophila melanogaster
Factor
Paraoxon
0,29
1,55 5
Azinphos
0,36
33,00 92
Chlorpyrifos oxon
2,85
1135,00
398
Dichlorvos
0,03
1,90 63
Carbaryl
0,07
0,20 3
Omethoate
0,001
0,042 42
Values of Ki in mmole-1.L-1

20. Acetylcholinesterase structure

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

22. Insecticide
Electric
eel
Wild Dm
Modified
Paraoxon
0,29 5 66
Azinphos
0,36 92
1922
Chlorpyrifos oxon
2,85
398
568
Dichlorvos
0,03 63
Carbaryl
0,07 3 40
Omethoate 42
2320

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

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

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

26. Pirimicarb 1x10-6 8x10-7 5x10-7 3x10-7 1x10-7 7x10-8 4x10-8 2x10-8
AChE-wt 87 68 51 30 13
B3(E69W)
100 69 17 12
B12(Y408A) 88 60 27
B41(I116V) 90 83 64 48 20

27. Carbaryl 8x10-7 5x10-7 3x10-7 1x10-7 7x10-8 5x10-8 3x10-8 1x10-8
AChE-wt 73 59 18 10
B3(E69W) 87 75 47 40 13
B12(Y408A) 98 64 27 15
B41(I116V)
100 83 55 23

28. Useless due to fast reactivation
Carbofuran
5x10-7
1x10-7
5x10-8
1x10-8
5x10-9
1x10-9
8x10-10
AChE-wt
Useless due to fast reactivation
B3(E69W) 82 74 39 10
B12(Y408A) 96 67 37 11
B41(I116V) 95 65 48 23

29. Standard solutions of the insecticide
Real sample analysis
Standard solutions of the insecticide
Tap water
Flat water
« Sémillante »
directly
spiked
B3 (E69W)
Pirimicarb 10-7 M 30 8 38 3
B12 (Y408A)
Carbaryl 5x10-8 M 40 37 7 42
B41(I116V)
Carbofuran 5x10-9 M 48 5 36

30. OP-Prot Sensor
OP-Stick Sensor

31. OP-Prot Sensor

32. 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
15 min.
Shake and incubate
10 min.
Shake and incubate
1 hour.
Shake and incubate
5 to 10 min.
Reference tube has to become yellow, meaning there is no insecticide

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
Carbofuran
Carbaryl
Pirimicarb

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

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

36. OP-Stick Sensor

37. LOD Insecticide OP-Protein Sensor Amperometric biosensor Chlorpyriphos
Ethyl oxon
0.1 ppb
0.05 ppb
Carbofuran
100 ppb
40 ppb

38. Toulouse (FRANCE)