Détection des insecticides organophosphorés et carbamates
description
Transcript of Détection des insecticides organophosphorés et carbamates
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
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
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
Electric eel
Bovine erythrocyte
Torpedo california
Horse serum
Drosophila melanogaster
CHOLINESTERASE
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
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
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
Insecticide OP-Protein Sensor
Amperometric
biosensor
Chlorpyriphos
Ethyl oxon
0.1 ppb 0.05 ppb
Carbofuran 100 ppb 40 ppb
LOD