Malaoxon Analysis Essay

1. Introduction

Olive cultivation is widespread throughout the Mediterranean region and is important for the rural economy, local heritage and environment [1]. To ensure crop protection, the use of pesticides is often required for blocking attacks of pests and diseases, as well as presence of weeds. The olive fruit fly Bactrocera oleae is the most serious pest of olives in the Mediterranean countries, causing economic losses reaching up to 15% of olive production [2]. Insecticide treatments are applied every year to control the fly population, mainly based on pesticides belonging to the organophosphates class. These chemicals can persist to the harvest stage and are likely to contaminate olive oil. Therefore, both European Union and the Codex Alimentarius Commission of the Food and Agriculture Organization of the United Nations (FAO) have established maximum pesticide residues limits (MRLs) for olives and olive oil [3,4].

Conventional methods of detection of organophosphate pesticides rely on an analysis by gas chromatography with specific detection. Although these techniques are very powerful and can detect very low concentrations, they are still very expensive and require highly skilled personnel, expensive purification steps and specialized major equipment [5]. In the last decades, new technologies based on biological detection systems have emerged. Among these techniques, biosensors have been shown to be very promising due to their simplicity and cost effectiveness compared to conventional techniques. Biosensors based on the inhibition of acetylcholinesterase (AChE) have been intensively studied in the aim of detecting organophosphorus insecticides [6]. Cholinesterases are important enzymes present in vertebrates and insects, which hydrolyze the neurotransmitter acetylcholine in the nervous system [7]. Organophosphorus pesticides are esters, amides or thiol derivatives of phosphoric acid esters. They primarily exist in the thionate form which is stable, but not very active. Activation occurs during metabolic oxidation into the biologically active oxon form, which is much less stable [8]. These insecticides act by phosphorylation of the serine located in the catalytic site of AChE, they can be considered as pseudo-substrates [9]. As this phosphorylation is very difficult to reverse, organophosphates are considered as irreversible inhibitors. This irreversibility is probably the main problem related to AChE-based biosensors, because of the difficulty in performing multiple assays using the same sensor [6]. Several methods have been investigated to overcome this problem, including mainly reactivation using oximes [10] and original immobilisation techniques.

Among these immobilisation methods, magnetic particles have recently gained a great attention due to their potential for providing control of electrochemical processes [11] and creating magneto-switchable devices [12,13]. Immobilization of enzymes, antibodies, oligonucleotides, and other biologically active compounds onto magnetic nanoparticles platforms is a key element in using these structures for biosensing purposes. Fabricating biofunctionalized magnetic materials containing a high amount of the biological element with high activity and stability is essential for the design of robust sensors that take advantage of the magnetic capabilities. The different routes for the fabrication of biofunctionalized magnetic nanoparticles include traditional methods such as covalent binding, adsorption, specific affinity interactions, and entrapment in porous surface layers [14]. Immobilisation of acetylcholinesterase on magnetic microbeads was already described in the literature, based on nickel-histidine affinity [6,15]. In this work we propose an immobilisation method that can be applied to the native acetylcholinesterase from electric eel, based on covalent coupling on magnetic microbeads. This method allows designing cheaper biosensors allowing the detection of insecticides in olive oil (Figure 1). The modified beads have been used either in bioassay or in biosensor configurations, based respectively on spectrophotometric or amperometric detection methods.

2. Experimental Section

2.1. Chemicals and Stock Solutions

AChE (EC from electric eel (EE) (Type V-S, 1,000 U/mg) was purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Acetylthiocholine chloride (ATChCl), acetylthiocholine iodide (ATChI) and 5,5-dithiobis(2-nitrobenzoic acid) (DTNB-Ellman's reagent) were provided by Sigma. In order to minimize hydrolysis, ATChCl and ATChI solutions were prepared daily in 0.9% NaCl (Sigma-Aldrich) solution. Stock solutions of enzymes and DTNB were prepared in 0.1 M phosphate buffer (Na2HPO4/KH2PO4, Sigma-Aldrich) at pH 7. The organophosphorus insecticides malaoxon, omethoate and methidathion were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Pesticide stock solutions (concentration 10−3 M) were prepared in acetonitrile (Sigma) and stored at 4 °C, working pesticide solutions were prepared daily in distilled water by dilution of the stock solution. The oxidation of methidathion was achieved using N-bromosuccinimide provided by Sigma-Aldrich. The glutaraldehyde used for activation of magnetic beads was also purchased from Sigma-Aldrich. Carbon (Electrodag 423SS) and silver/silver chloride (Electrodag 418SS) inks were obtained from Acheson (Plymouth, UK). Cobalt phtalocyanine-modified carbon paste was purchased from Gwent Electronic Materials, Ltd. (Gwent, UK). Poly(vinyl)chloride (PVC) sheets (200 mm × 100 mm × 0.5 mm), supplied by SKK (Denzlingen, Germany), were used as support for the screen-printed electrodes. A glycerophthalic paint (Astral, France) was used as insulating layer.

2.2. Apparatus

Spectrophotometric measurements were performed using a Hewlett Packard diode array 8451A spectrophotometer. Colorimetric measurements on PS-microtiter plates, U form (Greiner, Germany) were performed with a Labsystems Multiskan EX microtiter plate reader (Thermo Life Sciences, France). Amperometric measurements were carried out with a 641VA potentiostat (Metrohm, Switzerland), connected to a BD40 (Kipp & Zonen, The Netherlands) flatbed recorder.

Screen-printed electrodes were produced using a semi-automatic DEK248 printing machine according to a procedure previously described [15], but in a three-electrode configuration. The working electrode was a 4 mm-diameter disk, the auxiliary electrode was a 16 mm × 1.5 mm curved line and the Ag/AgCl pseudo-reference electrode was a 5 mm × 1.5 mm straight track. For experiments with magnetic beads, a small 4 mm-diameter magnet was placed on the backside of the working electrode to magnetically attach the enzyme-functionalised beads to the electrode surface.

2.3. Determination of Acetylcholinesterase Activity

The activity of AChE was measured spectrophotometrically by monitoring at 412 nm the appearance of thionitrobenzoate resulting from the reaction of DTNB with thiocholine, the product of the enzymatic hydrolysis of acetylthiocholine substrate, according to the procedure described by Ellman et al. [16]. This method is based on the use of a synthetic substrate: acetylthiocholine, whose hydrolysis liberates thiocholine and acetic acid according to the reaction:

The thiocholine reacts with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) yielding a yellow complex absorbing at 412 nm (ε = 1.36 × 104 M−1cm−1).

2.4. Determination of the Inhibition Constant ki

The mechanism of inhibition of AChE by organophosphate compounds is well-known [17]. The inhibitor phosphorylates a serine located in the active site and the inhibition can be considered as irreversible in the first 30 min [18]:

With E = enzyme, PX = organophosphate and X = leaving group. This scheme can be simplified with the bimolecular constant ki = k2/Kd:

To follow the inhibition, the enzyme was incubated with the pesticide during different periods of time, at 30 °C in 0.1 M phosphate buffer, pH 7. The change in remaining free enzyme concentration [E]/[E0] with time was estimated by sampling aliquots at various times and recording the remaining activity in the presence of 1mM acetylthiocholine [19].

The experimental procedure was as follows: 300 μL of 2.5 × 10−3 M DTNB and 100 μL of 0.01 M ATChI were added to 500 μL of 0.1 M phosphate buffer at pH 7; then 100 μL of the enzyme-inhibitor solution were taken at fixed time intervals and added to the cell. The incubation times used to study EE-AChE inhibition were 0, 1, 3, 5, 7, 10, 15 and 20 min.

The residual activity of AChE was calculated by comparing the slope of obtained kinetics before and after inhibition. The graphs obtained by plotting log of residual activity vs. incubation time for each inhibitor showed a linear representation. The apparent reaction rate kobs (min−1) were obtained by measuring the slope of this straight line. Plotting 1/kobsvs. 1/[I] allowed calculating the inhibition constant ki, which corresponds to the reciprocal value of the obtained slope.

2.5. Immobilisation on Magnetic Nanoparticles by Covalent Coupling

Nickel magnetic beads with a diameter of 200 nm were activated according to the following steps [20]:

  • Oxidation of the beads: 60 mg of magnetic beads were stirred for 4 h in 1 mL of 0.5 M sulfuric acid, and then washed twice with distilled water.

  • Functionalization with an amine group: 70 μL of 3-aminopropyltriethoxysilane were added to the beads previously poured in 100 mL of ethanol and ultrasonicated during 5 min, the suspension was kept under mechanical stirring overnight and finally washed three times with ethanol and twice with distilled water.

  • Covalent coupling with glutaraldehyde: 30 μL of aminated beads were washed twice with 1 mL of 0.1 M pH 7 buffer. 820 μL of buffer, 100 μL of electric eel AChE (4.41 UI/mL) and 80 μL of a 25% glutaraldehyde solution were added to the beads and stirred during 30 min at room temperature.

  • 1 μL of the obtained enzyme-linked beads suspension was placed either on the surface of the working electrode, beforehand fitted with a 4 mm-diameter magnet (amperometric detection), or in each well of the microplate (colorimetric detection).

2.6. Measurements

2.6.1. Amperometric Measurements

The electrode was vertically immersed in a thermostated cell (30 °C) containing 10 mL phosphate buffer pH 7 under constant magnetic stirring (417 rpm). The applied potential was 100 mV vs. Ag/AgCl reference electrode, using cobalt phtalocyanine as mediator. The current intensity was recorded and, after current stabilisation, 1 mM ATCh (final concentration) was added in the cell. The measured signal corresponded to the difference of current intensity between the baseline and the plateau. The cell was washed with distilled water between measurements.

The pesticide detection was made in a three-step procedure as follows: first, the initial response of the electrode to 1 mM ATCh was recorded three times, then the electrode was incubated in a solution containing a known concentration of insecticide, and finally the residual response of the electrode was recorded again. Electrodes were thoroughly washed with distilled water between each measurement. The percentage of the inhibition was correlated with the insecticide concentration, the inhibition rate was calculated according to the following formula: I (%) = [(I0 − I)/I0]100, I and I0 being respectively the current after the and before inhibition.

2.6.2. Colorimetric Measurements

Two hundred μL of phosphate buffer pH 8 were added in each well containing AChE-modified magnetic beads suspension in order to equilibrate the enzyme. After removal of the liquid using the Adem-Mag96 [21] 200 μL of phosphate buffer containing 2 mM ACTh-I and 6% DTNB were added and the microplate was incubated for 30 min under constant orbital stirring (300 rpm). The absorbance was then measured at 405 nm using 100 μL of the solution taken from each well. Inhibition experiments were performed by incubating the magnetic beads (1 μL) with 100 μL of different concentrations of pesticide during 10 min. The measurement procedure was the same as described above.

2.8. Oxidation of Methidathion

In this study we have focused on the detection of oxidized forms of each pesticide, which are less stable but more toxic than the normal forms. The oxidized forms of dimethoate and malathion, respectively called omethoate and malaoxon are commercially available, but in the case of methidathion an oxidation step must be carried out using N-bromosuccinimide (NBS). The efficiency of this oxidation step was controlled using reverse-phase HPLC, it was shown that 3 × 10−5 M NBS was sufficient for the total oxidation of a 10−5 M methidathion solution. The effect of NBS on AChE was investigated to ensure that the enzyme is not affected by the oxidizing agent, it was shown that in assays conditions NBS did not inhibit AChE.

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