Detection of pesticides using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic system

Abstract

A new highly sensitive amperometric method for the detection of organophosphorus compounds has been developed. The method is based on a ferophthalocyanine chemically modified carbon paste electrode coupled with acetylcholinesterase and choline oxidase co-immobilized onto the surface of a dialysis membrane. The activity of cholinesterase is non-competitively inhibited in the presence of pesticides. The highest sensitivity to inhibitors was found for a membrane containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetylcholine as substrate; choline produced by hydrolysis in the enzymatic layer was oxidized by choline-oxidase and subsequently H₂O₂ produced was electrochemically detected at +0.35 V vs. Ag/AgCl. The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, up to 10⁻¹⁰ M of paraoxon and carbofuran can be detected.

Introduction

Pesticides (herbicides, fungicides, insecticides) are widely used throughout the world, and millions of tons are used each year in agriculture, medicine, industry and related activities (Sherma, 1987). Because similar compounds have been produced as possible nerve poisons a further area of application is in the military (Sklâdal, 1991). Many of them are highly toxic, however, and their accumulation in living organisms can be cause of serious diseases. Even if they present a low persistence, their high acute toxicity creates a need for fast responding detection systems in order to protect human health during manufacturing and application processes and subsequently sensitive systems for reliable control of food products and environment pollution (Sklâdal and Mascini, 1992).

The mode of action of these pesticides is based on irreversible inhibition (non-competitive) of acetylcholinesterase (Eto, 1974) and the same principle is utilized for analysis. Free (Guilbault et al., 1970) or immobilized (Leon-Gonzales and Townshend, 1990) enzyme can be used. According to the method selected for measurement of enzyme activity, a variety of spectrophotometric (Wolfbeis and Koller, 1989, Leon-Gonzales and Townshend, 1990), fluorimetric (Guilbault and Kramer, 1965), piezoelectric (Guilbault and Ngwainbi, 1989), potentiometric (Goodson and Jacobs, 1976, Tran-Minh et al., 1990) and voltammetric (Medyantseva et al., 1990) methods were developed, but recently attention has been directed especially to amperometric sensors.

With acetyl- or butyryl-choline as the substrate, bienzyme electrodes coupling a cholinesterase (acetyl- or butyryl-cholinesterase) with choline oxidase (ChOx) have been reported, with measurement based on the detection of oxygen (Campanella et al., 1991, Vrbova et al., 1993) or hydrogen peroxide (Yao, 1983, Mascini and Moscone, 1986, Bernabei et al., 1991, Wollenberger et al., 1991, Palleschi et al., 1992), this latter being more sensitive.

The design of biosensor and optimization of its performances towards pesticides can be simplified using mono-enzymatic systems based on the hydrolysis of acetyl- or butyryl-thiocholine by a selected cholinesterase. In this case, detection is based on the oxidation of thiocholine produced at a platinum electrode. Using such an approach, the applied potential is lower than that used for the previously described oxidation of hydrogen peroxide (Marty et al., 1995).

The platinum anode is not convenient for detection of thiol compounds since a large overvoltage is necessary (Halbert and Baldwin, 1985). Similar problems in detection of other sulphydryl compounds were overcome by using a variety of carbon-based electrodes chemically modified with cobaltphthalocyanine (Qi et al., 1991, Ciucu and Baldwin, 1992). By using different electronic mediators such as cobaltphthalocyanine (Wring et al., 1989, Wring et al., 1991, Sklâdal, 1991, Sklâdal and Mascini, 1992), tetracyano p-quinodimethane, tetrathiafulvalene and 1,1′-dimethyl-ferrocene (Kulys and Drungiliene, 1991, Kulys and D'Costa, 1991, Kulys and Schimd, 1991) it was possible to decrease the applied potential. The interference due to oxidizable substances possibly present in real samples was dramatically decreased in this way. Some studies have been successfully validated using chromatographic methods (Barceló et al., 1995, Marty et al., 1995).

In our work, we chose to examine acetylcholine/choline system at carbon paste ferrophthalocyanine (FePC) chemically modified electrodes by using acetylcholinesterase (AChE)/ChOx bienzymatic system, and subsequently to evaluate the suitability of these electrodes for pesticides detection, which to our knowledge is the first report. The FePC chemically modified carbon paste electrodes exhibited an electrocatalytic response for the reduction of peroxides (Qi and Baldwin, 1993).

We developed a sensitive and effective bienzymatic sensor for pesticide detection based on the following equations:

$$\text{H}{}_3\text{C}\text{}\text{COO}\text{(}\text{CH}{}_2\text{)}{}_2\text{N}{}^{\mathrm{+}}\text{(}\text{CH}{}_3\text{)}{}_3\text{Acetylcholine}\text{(}\text{AChO}\text{)}\text{+}\text{H}{}_2\text{O}\text{→}\text{AChE}\text{OH}\text{(}\text{CH}{}_2\text{)}{}_2\text{}\text{N}{}^{\mathrm{+}}\text{(}\text{CH}{}_3\text{)}{}_3\text{Choline}\text{(}\text{ChO}\text{)}\text{+}\text{CH}{}_3\text{}\text{COOH}$$

$$\text{(}\text{CH}{}_3\text{)}{}_3\text{N}{}^{\mathrm{+}}\text{(}\text{CH}{}_2\text{)}{}_2\text{}\text{OH}\text{+2}\text{O}{}_2\text{+}\text{H}{}_2\text{O}\text{→}\text{ChOx}\text{(}\text{CH}{}_3\text{)}{}_3\text{N}\text{}\text{CH}{}_2\text{}\text{COOH}\text{+2}\text{H}{}_2\text{O}{}_2\text{Betaine}$$

$$\text{H}{}_2\text{O}{}_2\text{→}\text{350}\text{mV}\text{vs}\text{.}\text{Ag}\text{/}\text{AgCl}\text{Cathodic}\text{reduction}\text{peroxide}\text{reduction}\text{product}$$

A diminished sensitivity was obtained for acetylcholine with an increased sensitivity towards inhibitors such as pesticides when a kinetically controlled bienzyme sensor with a low activity of acetylcholinesterase was used. Inhibition of AChE, proportional to pesticide concentration, was measured as a decrease of the formation rate of hydrogen peroxide, which was correlated with the corresponding decrease in the cathodic current generated by the biosensor. In this paper the results obtained with co-immobilized enzymes are presented. By this approach it was possible to detect down to 10⁻¹⁰ M paraoxon and carbofuran.