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 How nerve/muscle junctions work Acetylcholine is released by the nerve cell Binds to protein receptor in muscle cell membrane to trigger contraction

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

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

STRATEGY Enzyme inhibition Screen printing

Amperometric Biosensor

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

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

STRATEGY Stability Encapsulation Sol-gel Affinity

Entrapment of AChE in PVA-SbQ

Immobilisation of AChE in SOL-GEL

Principle of Metal Affinity Chelate 2 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

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

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

STRATEGY Hypersensitivity Enzymes -genetically modified -synthetic

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

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

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

Acetylcholinesterase structure

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

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 63 300 000 Carbaryl 0,07 3 40 Omethoate 42 2320

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

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

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%

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

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

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

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

OP-Prot Sensor OP-Stick Sensor

OP-Prot Sensor

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

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

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

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

OP-Stick Sensor

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

Toulouse (FRANCE)