Electrochemical Biosensors for photosynthetic herbicides

During the last several decades herbicide application in agriculture has enormously increased, resulting in their mass production and herbicide pollution of soil and water. Because herbicides can be highly toxic to human and animal health, their indiscriminate use has serious environmental implications. Understandably, therefore, dinoseb was prohibited in the USA and in most other countries for its high toxicity (US Federal Register, 1986) while atrazine, a possible human carcinogen, was also banned (US EPA, 1988). The European Union introduced the European Drinking Water Act of 1980, which does not allow concentrations of pesticides in drinking water to exceed 0.1 mg/L of an individual pesticide or 0.5 mg/L of total pesticides. To monitor such low residual herbicide levels a need has arisen for developing sensitive and reliable detection methods. Herbicides inhibiting photosynthesis via targeting Photosystem II (PSII) function still represent the basic means of weed control. This group consists of several classes of chemicals such as triazines (e.g. atrazine, simazine, cyanazine), phenylureas (linuron, diuron) or phenols (e.g. ioxynil, bromoxynil) (Draber et al 1991). Triazine herbicides are used every year in large quantities, for example, in the USA about 35 × 10^6 kg of atrazine, 9 × 10^6 kg of cyanazine and 3 × 10^6 kg of simazine have been applied every year (U.S. EPA). This practice frequently leads to soil contamination and subsequent pollution of surface and ground water. Triazines are relatively persistent in water and represent the most frequently detected pesticides in ground water.

Currently, three methods are generally employed to determine most of the herbicides, HPLC, GC-MS and ELISA. HPLC and GC-MS represent reliable and routine methods but their disadvantages are that they require expensive equipment, organic solvents and purification of samples prior to assay, thus, limiting the number of samples that can be analyzed (Pacakova et al., 1996). A recently developed, immunochemical method (ELISA) has high sensitivity (2 x 10-10 M for diuron; 1 x 10-10 M for atrazine) (Schneider and Hammock, 1992; Giersch, 1993; Schneider et al., 1994) but involves preparation of monoclonal antibodies, which is difficult, time consuming and expensive. Furthermore, the antibodies generated are specific either to one compound or a few of its structural analogues.

About one half of the herbicides used at present in agriculture inhibit the light reactions in photosynthesis, mostly by targeting the Photosystem II (PS II) complex (Draber et al., 1991). PS II is a pigment-protein membrane complex made up of the reaction center D1/D2 heterodimer carrying the main functional groups of PS II, the chlorophyll(Chl)-proteins CP47 and CP43 acting as inner antennae, α and β subunits of cytochrome b559 and the oxygen-evolving complex (Mattoo et al., 1989). The D1 protein is the main target of herbicides that inhibit photosynthesis. Based on the chemical structure and binding properties the PS II herbicides fall into two main groups: phenylureas and triazines, and phenols (Trebst and Draber, 1979). Although both classes replace the QB acceptor on the D1 protein (Pfister et al., 1981; Mattoo et al., 1981), they interact with different amino acid residues on D1 (Draber et al., 1991). The high binding affinity of these herbicides to D1 offers a unique possibility to use PSII complex for herbicide detection. Interestingly, in the 1950's, selection for the most effective herbicides took advantage of the fact that herbicides could inhibit Hill reaction in isolated chloroplasts (Wessels and Van der Veen, 1956; Good, 1961).

Based on a similar strategy, isolated chloroplasts and thylakoids have been used, conversely, to detect herbicides, by testing inhibition of the Hill reaction (Loranger and Carpentier, 1994; Rouillon et al., 1994), inhibition of DCPIP photoreduction (Brewster and Lightfield, 1993; Brewster et al., 1995), or change in Chl fluorescence (Conrad et al., 1993; Merz et al., 1996). These observations have initiated interest in developing biological sensors to detect low levels of herbicides in water and soil using PS II. So far, the practical use of herbicide biosensors based on isolated PS II preparations has been limited by their instability, particularly upon illumination.

Because a large number of herbicides inhibit Photosystem II activity (Moreland, 1992) has resulted in its use as an analytical tool (bioassay) for designing (Wessels and Van der Veen, 1956; Good, 1961) or detection of herbicides (Conrad et al., 1993; Brewster and Lightfield, 1993; Loranger and Carpentier, 1994; Rouillon et al., 1995; Merz et al., 1996; Soukupová et al., 1999; Giardi et al., 2000). We have constructed a biosensor by immobilization of PSII complex from thermophilic cyanobacterium Synechococcus elongatus on a Clark oxygen electrode. The system exhibited a good stability at laboratory temperature as well as high sensitivity to herbicides (Koblížek et al., 1998).

However, the use of Clark electrode as the transductor prevents a simple and potentially mass production of this system. For this reason screen-printing was chosen as suitable technology offering batch production of electrochemical biosensors with high reproducibility at low price.

The electrochemical sensors are made by sequential, multi-layer, deposition of metal conductors (Pt, Au, Ag, Pd, Rh, Ru, Ti), dielectric insulators (Al or Zr oxide powders) and polymeric pastes on inorganic (Al2O3 ceramic) or polymeric (PVC) substrates. The layout drawn on the open mesh of a screen is transferred onto a substrate, providing two-dimensional microcircuits and electrodes (Karlberg and Pacey, 1989; Scheller and Schubert, 1992; Prudenziati, 1994). A herbicide biosensor based on isolated PSII particles immobilized to a graphite-Ag/AgCl screen-printed electrode exhibits selective sensitivity to phenylurea and triazine herbicides, whereas phenyl herbicides are not registered. The use of screen-printed electrodes allows a cheap, large scale, production of these devices.

1. A Biosensor for the Detection of Triazine and Phenylurea Herbicides Designed Using Photosystem II Coupled to a Screen-Printed Electrode - M Koblížek, J Malý, J Masojídek, J Komenda, T Kučera, MT Giardi, AK Mattoo and R.Pilloton - Biotechnology and bioengineering (2002) 78 (1), 110-116
2. A Sensitive Photosystem II-Based Biosensor for Detection of a Class of Herbicides - M Koblizek, J Masojidek, J Komenda, T Kucera, R Pilloton, AK Mattoo, MT Giardi Biotechnology and Bioengineering (1998) 60 (6), 664-669

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