Détection des insecticides organophosphorés et

carbamates

Nerve cell communication: Synaptic membrane

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

Acetyl choline

AC receptor

AC esterase

Nerve agent

How nerve agents work

• Nerve agents block the breakdown of acetylcholine: muscle contracts continuously• This results in paralysis

STRATEGY

Hypersensitivity

Enzymes -genetically modified -synthetic

StabilityEncapsulation Sol-gel Affinity

Enzyme inhibition Screen printing

Enzyme inhibition Screen printing

STRATEGY

Amperometric Amperometric

BiosensorBiosensor

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

StabilityEncapsulation Sol-gel Affinity

STRATEGY

Entrapment of AChE in PVA-SbQ

Immobilisation of AChE in SOL-GEL

Ni

CH

CO

O

N

CH2 CO

O

O OH2

CH2

OC

OH2

O

GRAPHITE

Graphite-NTA-Ni

- (His)6AChE

HN

AChE

N

Ni

CH

CO

O

O

N

CH2 CO

O

O OH2

CH2

OC

GRAPHITE

Principle of Metal Affinity Chelate

Principle of Con A

G

R

A

P

H

I

TE

Sugar

Concanavalin A

AChE

AChE

Principle of Con A

G

R

A

P

H

I

TE

Sugar

Concanavalin A

AChE

AChE

Hypersensitivity

Enzymes -genetically modified -synthetic

STRATEGY

Electric eel

Bovine erythrocyte

Torpedo california

Horse serum

Drosophila melanogaster

CHOLINESTERASE

CHOLINESTERASE

Electric eel (98 % Biotools)

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 mole-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

Dm

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 0,03 42 2320

Detection of carbamate insecticides

SOURCE OF ENZYME

Drosophila melanogaster (Dm)

PESTICIDES

Pirimicarb

Carbaryl

Carbofuran

Detection principle

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

Amperometric determination of thiocholine, produced by enzymatic hydrolysis of acetylthiocholine substrate

+ R-COO- + H+H+ Cl-

(CH3)3N+-CH2-CH2-SH thiocholine

Cl-(CH3)3N+-CH2-CH2-SH thiocholine

(CH3)3N+-CH2-CH2-S-CO-CH3

Cl-

acetylthiocholineH2O

AChE

1. Measurement of the initial current, I0

Potentiostat

Buffer

Potentiostat

Buffer + Insecticide

2. Incubation time: 20 min

Potentiostat

Buffer

%Inhibition=(I0-I1)/I0 x 100

%I>10%

%I<10%

ILLUSTRATED PROCEDURE

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 30 17 12

B12(Y408A) 88 60 27 12

B41(I116V) 90 83 64 48 20 12

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 40 27 15

B41(I116V) 100 83 55 23 13

Carbaryl

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 82 67 37 11

B41(I116V) 95 65 48 23 11

Carbofuran

Real sample analysis

Standard solutions

of the insecticide

Tap waterFlat water

« Sémillante »

directly spiked directly spiked

B3 (E69W)

Pirimicarb 10-7 M30 8 38 3 30

B12 (Y408A)

Carbaryl 5x10-8 M40 8 37 7 42

B41(I116V)

Carbofuran 5x10-9 M48 5 42 8 36

OP-Prot SensorOP-Prot Sensor OP-Stick SensorOP-Stick Sensor

OP-Prot SensorOP-Prot Sensor

Solution to be tested

Control solution insecticide free

1

1

Shake and incubate 15 min.

2

2

Shake and incubate 10 min.

3

3

Shake and incubate 1 hour.

4

4

Shake and incubate

5 to 10 min.

Transfer 10ml to tube 1 yellow

Transfer to tube 2 Blue

Transfer to tube 3 red

Transfer to tube 4 green

Reference tube has to become yellow, meaning

there is no insecticide

Tested solution: If solution still uncolored, the test is positive. There are inhibiting substances

in the solution

ILLUSTRATED PROCEDURE

LIMIT OF DETECTIONCOMPOUND WATER (ppb or g/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 g/L)

Chlorpyriphos ethyl 0.1

Paraoxon 1

Parathion 1

Dichlorvos 1

Malaoxon 1

Malathion 1

Profenofos 10

Carbofuran 10

Carbaryl 100

Pirimicarb 1000

OR2

P OR1

S

HO

NO TOXIC

Parathion

Malathion

OR2

P OR1

O

HO

TOXIC

Paraoxon

Malaoxon

Oxidation

OP-Stick SensorOP-Stick Sensor

Insecticide OP-Protein Sensor

Amperometric

biosensor

Chlorpyriphos

Ethyl oxon

0.1 ppb 0.05 ppb

Carbofuran 100 ppb 40 ppb

LOD

Toulouse (FRANCE)