Biosensors & Bioelectronics 15 (2000) 499 – 502 Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring K. Rekha, M.D. Gouda, M.S. Thakur, N.G. Karanth * Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute, Mysore, 570013, India Received 2 November 1999; received in revised form 30 November 1999; accepted 20 June 2000 Abstract An amperometric principle based biosensor containing tissues of cucumber, rich in ascorbic acid oxidase, was used for the detection of organophosphorous (OP) pesticide ethyl paraoxon, which inhibits the activity of ascorbic acid oxidase. The optimal concentration of ascorbic acid used as substrate was found to be 5.67 mM. The biosensor response was found to reach steady state within 2 min. A measurable inhibition ( 10%) was obtained with 10 min incubation of the enzyme electrode with different concentrations of the pesticide. There was a linear relationship between the percentage of inhibition of the enzyme substrate reaction and the pesticide (ethyl paraoxon) concentration in the range 1 – 10 ppm with a regression value 0.9942. © 2000 Elsevier Science S.A. All rights reserved. Keywords: Ascorbate oxidase; Organophosphorous pesticide; Amperometry; Biosensor www.elsevier.com/locate/bios 1. Introduction The application of organochlorine pesticides in agri- culture has been discontinued in many countries and other classes of pesticides such as organophosphates (OP) and carbamates have become popular. As a result of increased use of OP pesticides in agriculture in the last few decades, ground water, raw food materials and processed foods are becoming contaminated with pesti- cide residues. OP pesticides inhibit the enzyme acetyl- cholinesterase, which is essential in insect and animal systems for nerve impulse transmission, by forming stable covalent intermediates (Kumaran and Tran- Minh, 1992). The solubility of OP compounds in water is more than that of organochlorides, thus giving a higher environmental mobility and therefore causing severe threat to aquatic life (Palleschi et al., 1992). The most widely used methods for determination of OP pesticides are high performance liquid chromatography (HPLC) and gas chromatography (GC). Other methods include ELISA and calorimetric techniques. These con- ventional methods have several disadvantages as they are laborious, time consuming and need expensive in- struments and sample pretreatment. Biosensors present an efficient alternative technique which overcomes many of these disadvantages, including minimum sam- ple pretreatment and the fact that the colour of the sample does not interfere with measurements. There are reports on both single as well as multien- zyme based systems for OP pesticide detection. Single enzyme based biosensors use either acetyl cholinesterase (AChE) or butyryl cholinesterase (BuChE) as the bio- logical component and thiocholine production is moni- tored amperometrically (Hartley and Hart, 1994) or acid production is monitored potentiometrically (Imato and Ishibashi, 1995). Multienzyme based biosensors use cholinesterase in conjunction with choline oxidase and measure hydrogen peroxide production (Wollenberger et al., 1991) or oxygen consumption (Fennouh et al., 1997). There are also reports of using acid phosphatase (Gouda et al., 1997) and alkaline phosphatase (Ayyan- gari et al., 1995) for OP pesticide determination. Multienzyme based biosensors for OP pesticide de- tection reported in the literature use cholinesterase (ChE) in association with choline oxidase (ChO) (Wol- * Corresponding author. Tel.: +91-821-517233; fax: +91-821- 515792. E-mail address: ferm@cscftri.ren.nic.in (N.G. Karanth). 0956-5663/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved. PII: S0956-5663(00)00077-4