Dtection des insecticides organophosphors et carbamates

Nerve cell communication: Synaptic membrane

Acetyl choline

AC receptor

AC esterase

Nerve agentHow nerve agents work Nerve agents block the breakdown of acetylcholine: muscle contracts continuously This results in paralysis

STRATEGY

STRATEGY

Amperometric Biosensor

Screen printed method

LOW COSTPORTABLESINGLE USEHIGHLY REPRODUCIBLESAMPLE HANDLINGMASS PRODUCTIONNO SKILLED PERSONNALFAST DETECTION

STRATEGY

Entrapment of AChE in PVA-SbQ

Immobilisation of AChE in SOL-GEL

NiGraphite-NTA-NiPrinciple of Metal Affinity Chelate

Principle of Con A

Principle of Con A

STRATEGY

Electric eel Bovine erythrocyte Torpedo california Horse serum Drosophila melanogaster CHOLINESTERASE

CHOLINESTERASE

Electric eel (98 % Biotools)Drosophila melanogaster

Values of Ki in mmole-1.L-1

InsecticideElectric eelDrosophila melanogasterFactorParaoxon0,291,555Azinphos0,3633,0092Chlorpyrifos oxon2,851135,00398Dichlorvos0,031,9063Carbaryl0,070,203Omethoate0,0010,04242

Acetylcholinesterase structure

Engineered cholinesterase

InsecticideElectriceelWild DmModifiedDmParaoxon0,29566Azinphos0,36921922Chlorpyrifos oxon2,85398568Dichlorvos0,0363300 000Carbaryl0,07340Omethoate0,03422320

Detection of carbamate insecticidesSOURCE OF ENZYME

Drosophila melanogaster (Dm)PESTICIDES

PirimicarbCarbarylCarbofuran

Detection principle

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

Pirimicarb

1x10-68x10-75x10-73x10-71x10-77x10-84x10-82x10-8AChE-wt8768513013B3(E69W)10069301712B12(Y408A)88602712B41(I116V)908364482012

Graph2

870.0000010.00000190

680.00000080.000000883

511008864

30696048

13302720

0.00000004171212

0.00000002120.00000002

B1-wt

B3

B12

B41

M

Inh (%)

Feuil1

pirimicarb1.00E-068.00E-075.00E-073.00E-071.00E-077.00E-084.00E-082.00E-08

B1-wt87685130130.208

B3100693017120.85

B12886027120.186

B419083644820120.154

pirimicarb1.00E-068.00E-075.00E-073.00E-071.00E-074.00E-082.00E-08

B1-wt8768513013

B310069301712

B1288602712

1.00E-068.00E-075.00E-073.00E-071.00E-077.00E-08

B41908364482012

Feuil1

0000

0000

0000

0000

0000

0000

000

B1-wt

B3

B12

B41

M

Inh (%)

Feuil2

Feuil3

Carbaryl

8x10-75x10-73x10-71x10-77x10-85x10-83x10-81x10-8AChE-wt73591810B3(E69W)8775474013B12(Y408A)9864402715B41(I116V)10083552313

Graph2

0.00000080.000000898100

73876483

59754055

18472723

10401513

0.0000000513

B1-wt

B3

B12

B41

(M)

Inh (%)

Feuil1

carbaryl8.00E-075.00E-073.00E-071.00E-077.00E-085.00E-08

B1-wt73591810

B4110083552313

B38775474013

carbaryl5.00E-071.00E-075.00E-083.00E-081.00E-08

B129864402715

Feuil1

0000

0000

0000

0000

0000

00

B1-wt

B3

B12

B41

Carbaryl (M)

Inh (%)

Feuil2

Feuil3

Carbofuran

5x10-71x10-75x10-81x10-85x10-91x10-98x10-10AChE-wtUseless due to fast reactivationB3(E69W)82743910B12(Y408A)9682673711B41(I116V)9565482311

Graph3

95

748265

396748

103723

0.0000000051111

0.0000000010.000000001

0.00000000080.0000000008

B3

B12

B41

M

Inh (%)

Feuil1

carbofuran1.00E-075.00E-081.00E-085.00E-091.00E-098.00E-10

B3743910

B1282673711

1.00E-071.00E-085.00E-091.00E-098.00E-10

B419565482311

Feuil1

000

000

000

000

000

00

00

B3

B12

B41

M

Inh (%)

Feuil2

Feuil3

Real sample analysis

Standard solutions of the insecticideTap waterFlat water SmillantedirectlyspikeddirectlyspikedB3 (E69W)Pirimicarb 10-7 M30838330B12 (Y408A)Carbaryl 5x10-8 M40837742B41(I116V)Carbofuran 5x10-9 M48542836

OP-Prot Sensor

Solution to be testedControl solution insecticide free

1

1Shake and incubate 15 min.

2

2Shake and incubate 10 min.

3

3Shake and incubate 1 hour.

4

4Shake and incubate 5 to 10 min.Transfer 10ml to tube 1 yellowTransfer to tube 2 BlueTransfer to tube 3 redTransfer to tube 4 green

Reference tube has to become yellow, meaning there is no insecticideTested solution: If solution still uncolored, the test is positive. There are inhibiting substances in the solutionILLUSTRATED PROCEDURE

LIMIT OF DETECTIONCOMPOUNDWATER (ppb or mg/L)Chlorpyriphos ethyl0.1Paraoxon1Parathion1Dichlorvos1Malaoxon1Malathion1Profenofos10Carbofuran10Carbaryl100Pirimicarb1000

LIMIT OF DETECTIONCOMPOUNDWATER (ppb or mg/L)Chlorpyriphos ethyl0.1Paraoxon1Parathion1Dichlorvos1Malaoxon1Malathion1Profenofos10Carbofuran10Carbaryl100Pirimicarb1000

OP-Stick Sensor

LOD

InsecticideOP-Protein SensorAmperometricbiosensorChlorpyriphosEthyl oxon0.1 ppb0.05 ppbCarbofuran100 ppb40 ppb

Toulouse (FRANCE)