Flow Injection Analysis System for Screening Organophosphorus Pesticides by their Inhibitory Effect on the Enzyme Acethylcholinesterase

A flow-injection spectrophotometric procedure was developed for screening organophosphorus pesticides. The method is based on the inhibition of acetylcholinesterase immobilized on controlled porous glass beads with acetylcholine chloride as the substrate. Methyl parathion, chlorpyrifos, malathion and dichlorvos have been tested. The analytical peak height for a given acetylcholine chloride concentration correlates linearly with the logarithmic concentration of the pesticides between 1.0 × 10^-3 mol L^-1 and 1.0 × 10^-6 mol L-1. If bromine water is added to the pesticide solution, a dramatic increase is observed in the analytical signal, and correlations with the logarithm of the concentrations are observed from 1.0 × 10^-3 to 1.0 × 10-10 mol L^-1.

pesticides; organophosphorus; acetylcholinesterase; biosensor

Authorship

Marcos P. Silva

Institute of Chemistry, University of Campinas (UNICAMP), PO Box 6154, 13083-970 Campinas-SP, BrazilUniversity of Campinas (UNICAMP)BrazilCampinas, SP, BrazilInstitute of Chemistry, University of Campinas (UNICAMP), PO Box 6154, 13083-970 Campinas-SP, Brazil

Matthieu Tubino ^**e-mail: tubino@iqm.unicamp.br

Tereza C. R. Elsholz

Department of Chemistry, University of Minas Gerais State (UEMG), 38302-192 Ituiutaba-MG, BrazilUniversity of Minas Gerais State (UEMG)BrazilItuiutaba, MG, BrazilDepartment of Chemistry, University of Minas Gerais State (UEMG), 38302-192 Ituiutaba-MG, Brazil

Olaf Elsholz

Hochschule für Angewandte Wissenschaften Hamburg, Life Sciences, Ulmenliet 20, 21033 Hamburg, GermanyHochschule für Angewandte Wissenschaften HamburgGermanyHamburg, GermanyHochschule für Angewandte Wissenschaften Hamburg, Life Sciences, Ulmenliet 20, 21033 Hamburg, Germany

Sabir Khan

Marta M. D. C. Vila

Pharmacy Department, University of Sorocaba, 18023-000 Sorocaba-SP, BrazilUniversity of SorocabaBrazilSorocaba, SP, BrazilPharmacy Department, University of Sorocaba, 18023-000 Sorocaba-SP, Brazil

*e-mail: tubino@iqm.unicamp.br

SCIMAGO INSTITUTIONS RANKINGS

Pharmacy Department, University of Sorocaba, 18023-000 Sorocaba-SP, BrazilUniversity of SorocabaBrazilSorocaba, SP, BrazilPharmacy Department, University of Sorocaba, 18023-000 Sorocaba-SP, Brazil

Figures | Tables

Figures (3)
Tables (3)

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Scheme 1

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Figure 1
Scheme for the flow injection system used: F₁ = phosphate buffer 0.1 mol L^-1 pH 7.0; F₂ = sulfuric acid 0.5 mol L^-1; F₃ = bromocresol purple solution 5.0 × 10^-5 mol L-1 pH 7.00. F₁ = F₂ = F₃ = 1.0 mL min^-1; P = peristaltic pump; ER = enzymatic reactor; V = sampling valve; PC = permeation cell; S = sample introduction where S is as desired: acetylcholine chloride = 5.0 × 10^-3 mol L^-1, TMB-4 = 5.0 × 10^-3, standard or sample solutions containing organophosphorus pesticides; W= waste; SP = spectrophotometer, λ = 590 nm; R = recorder.

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Figure 2
Example of the analytical signal. The group of peaks labeled 1 is related to signal 1 of the acetic acid produced from 5.0 × 10-5 mol L^-1 acetylcholine prior to the inhibition of the enzyme by the pesticide. Signal 2 corresponds to the introduction of the pesticide solution in the FIA system. Group 3 is related to the signal after the inhibition of the enzyme by the pesticide. Peak 4 is related to the introduction of the TMB‑4 solution into the FIA system. Group 5 is related to the signals for the acetylcholine after the regeneration of the enzyme.

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Table 1
The studied pesticides and their molecular formulas²⁸28 The Merck Index, an Encyclopedia of Chemicals, Drugs, and Biologicals, 14th ed.; Merck & Co. Inc.: Whitehouse Station, NJ, USA, 2006.

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Table 2
Optimized parameters of the FIA system

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Table 3
Inhibition of the enzyme acetylcholinesterase as functions of the concentration of the pesticides methyl parathion, chlorpyrifos, malathion and dichlorvos. Comparison of the inhibition power of the pesticides with respect to dichlorvos arbitrarily taken as reference. Determination of these pesticides in fresh orange juice purposely contaminated

Scheme 1

Figure 1 Scheme for the flow injection system used: F₁ = phosphate buffer 0.1 mol L^-1 pH 7.0; F₂ = sulfuric acid 0.5 mol L^-1; F₃ = bromocresol purple solution 5.0 × 10^-5 mol L-1 pH 7.00. F₁ = F₂ = F₃ = 1.0 mL min^-1; P = peristaltic pump; ER = enzymatic reactor; V = sampling valve; PC = permeation cell; S = sample introduction where S is as desired: acetylcholine chloride = 5.0 × 10^-3 mol L^-1, TMB-4 = 5.0 × 10^-3, standard or sample solutions containing organophosphorus pesticides; W= waste; SP = spectrophotometer, λ = 590 nm; R = recorder.

Figure 2 Example of the analytical signal. The group of peaks labeled 1 is related to signal 1 of the acetic acid produced from 5.0 × 10-5 mol L^-1 acetylcholine prior to the inhibition of the enzyme by the pesticide. Signal 2 corresponds to the introduction of the pesticide solution in the FIA system. Group 3 is related to the signal after the inhibition of the enzyme by the pesticide. Peak 4 is related to the introduction of the TMB‑4 solution into the FIA system. Group 5 is related to the signals for the acetylcholine after the regeneration of the enzyme.

Table 1 The studied pesticides and their molecular formulas²⁸28 The Merck Index, an Encyclopedia of Chemicals, Drugs, and Biologicals, 14th ed.; Merck & Co. Inc.: Whitehouse Station, NJ, USA, 2006.

Table 2 Optimized parameters of the FIA system

Parameters of the FIA system
Wavelength / nm	590
Temperature / °C	25.0
Acetylcholine / (mol L^-1)	5 × 10^-3
Injection loop / μL	150
Flow rate / (mL min^-1)	1.0
H₂SO₄ / (mol L^-1)	0.5
Sorensen buffer / pH (carrier)	7.5
TMB-4 / (mol L^-1)	5 × 10^-5
Sampling rate / h	12

Table 3 Inhibition of the enzyme acetylcholinesterase as functions of the concentration of the pesticides methyl parathion, chlorpyrifos, malathion and dichlorvos. Comparison of the inhibition power of the pesticides with respect to dichlorvos arbitrarily taken as reference. Determination of these pesticides in fresh orange juice purposely contaminated

Conc. / (mol L^-1)	Pesticide methyl malathion										Pesticide chlorpyrifos
	Not oxidized			Oxidized with bromine water							Not oxidized			Oxidized with bromine water
	p^a a p = percentage of inhibition;	± sd^b b sd = standard deviation;	p	± sd	dp^c c dp: inhibition of the pesticide proportional to dichlorvos 1.0 × 10 3 mol L 1 inhibition;	± sd	oj^d d oj: pesticide spiked in fresh orange juice;	± sd	ojdp^e e ojdp: inhibition of the pesticide dissolved in orange juice proportional to dichlorvos 1.0 × 10-3 mol L-1 inhibition.	± sd	p	± sd	p	± sd	dp	± sd	oj	± sd	ojdp	± sd
1.0 × 10^-10	0.0	-	0.0	-	0.00	-	-	-	-	-	0.0	-	0.0	-	0.00	-	-	-	-	-
1.0 × 10^-9	0.0	-	2.3	0.7	0.03	0.01	-	-	-	-	0.0	-	0.0	-	0.00	-	-	-	-	-
1.0 × 10^-8	0.0	-	15.0	2.2	0.17	0.02	13.5	1.1	0.16	0.01	0.0	-	10.2	0.8	0.12	0.01	8.3	1.0	0.10	0.01
1.0 × 10^-7	0.0	-	26.2	1.4	0.31	0.02	-	-	-	-	0.0	-	27.7	1.7	0.32	0.02	-	-	-	-
1.0 × 10^-6	0.0	-	50.0	1.0	0.58	0.02	47.4	1.1	0.55	0.02	0.0	-	45.4	1.3	0.53	0.02	42.5	1.4	0.49	0.02
1.0 × 10^-5	7.8	1.0	71.0	0.8	0.83	0.02	-	-	-	-	0.0	-	64.8	1.3	0.75	0.02	-	-	-	-
1.0 × 10^-4	19.0	1.0	89.1	1.6	1.04	0.03	-	-	-	-	8.3	0.5	83.6	0.8	0.97	0.02	-	-	-	-
1.0 × 10^-3	31.6	1.5	97.3	0.8	1.13	0.02	-	-	-	-	20.3	0.8	95.0	0.8	1.11	0.02	-	-	-	-
Conc. / (mol L^-1)	Pesticide malathion										Pesticide dichlorvos
	Not oxidized			Oxidized with bromine water							Not oxidized			Oxidized with bromine water
	p	± sd	p	± sd	dp	± sd	oj	± sd	ojdp^e e ojdp: inhibition of the pesticide dissolved in orange juice proportional to dichlorvos 1.0 × 10-3 mol L-1 inhibition.	± sd	p	± sd	p	± sd	dp	± sd	oj	± sd	ojdp	± sd
1.0 × 10^-10	0.0	-	0.0	-	0.00	-	-	-	-	-	0.0	-	6.1	1.1	0.07	0.01	-	-	-	-
1.0 × 10^-9	0.0	-	0.0	-	0.00	-	-	-	-	-	0.0	-	8.8	1.3	0.10	0.01	7.2	1.1	0.08	0.01
1.0 × 10^-8	0.0	-	0.0	-	0.00	-	-	-	-	-	0.0	-	17.9	0.8	0.21	0.01	-	-	-	-
1.0 × 10^-7	0.0	-	3.2	1.0	0.04	0.01	-	-	-	-	0.0	-	25.6	1.2	0.30	0.01	-	-	-	-
1.0 × 10^-6	0.0	-	8.4	2.0	0.10	0.02	7.2	1.5	0.08	0.01	0.0	-	38.0	1.3	0.44	0.02	52.1	1.3	0.61	0.02
1.0 × 10^-5	0.0	-	16.3	1.6	0.19	0.02	-	-	-	-	4.2	0.8	53.5	1.3	0.62	0.02	-	-	-	-
1.0 × 10^-4	0.0	-	52.8	2.5	0.61	0.03	49.9	2.0	0.58	0.02	14.8	0.8	73.2	2.4	0.85	0.03	-	-	-	-
1.0 × 10^-3	4.3	0.6	75.0	2.2	0.87	0.03	-	-	-	-	24.6	0.8	86.0	0.8	1.00	0.01	-	-	-	-

^a
p = percentage of inhibition;
^b
sd = standard deviation;
^c
dp: inhibition of the pesticide proportional to dichlorvos 1.0 × 10 ³ mol L ¹ inhibition;
^d
oj: pesticide spiked in fresh orange juice;
^e
ojdp: inhibition of the pesticide dissolved in orange juice proportional to dichlorvos 1.0 × 10^-3 mol L^-1 inhibition.

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Flow Injection Analysis System for Screening Organophosphorus Pesticides by their Inhibitory Effect on the Enzyme Acethylcholinesterase

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[1] *e-mail: tubino@iqm.unicamp.br