Analytica Chimica Acta 587 (2007) 22–32 Organophosphorus and carbamate pesticide analysis using an inhibition tyrosinase organic phase enzyme sensor; comparison by butyrylcholinesterase + choline oxidase opee and application to natural waters L. Campanella, D. Lelo, E. Martini, M. Tomassetti ∗ Department of Chemistry, University of Rome “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy Received 7 November 2006; received in revised form 29 December 2006; accepted 10 January 2007 Available online 19 January 2007 Abstract Recent research performed in our laboratory (using a butyrylcholinesterase + choline oxidase enzyme electrode) suggested the validity of the biosensor approach using enzyme inhibition OPEEs (i.e. enzyme electrodes working in organic phase) in the case of organophosphorus and carbamate pesticides, which are poorly soluble in aqueous solutions. Since these pesticides are generally much more soluble in chloroform than in water, the present research aimed at analysing this class of pesticides using a tyrosinase inhibition OPEE operating in water-saturated chloroform medium. The tyrosinase biosensor was assembled using an oxygen amperometric transducer coupled to the tyrosinase enzyme, immobilized in kappa-carrageenan gel. Lastly a detailed comparison between the inhibition monoenzymatic tyrosinase and inhibition bienzymatic (butyrylcholinesterase + choline oxidase) OPEEs was performed and discussed in this work. © 2007 Elsevier B.V. All rights reserved. Keywords: Biosensor pesticide analysis; Carbamate; Organophosphorus; Tyrosinase inhibition 1. Introduction It is not necessary to recall the importance and the need for analysing pesticides in many real samples. In recent years a series of researches were performed on the development of inhibition biosensors for pesticide determination (mainly organophosphorus and carbamates) [1–6]. Practically, all these biosensors exploited the inhibition of the activity of the acetyl- cholinesterase (or butyrylcholinesterase) enzyme [3–10] and their functioning was based on one or two enzymatic reactions in series: butyrylcholine butyrylcholinesterase -→ choline + butyric acid choline + 2O 2 choline oxidase -→ betaine + 2H 2 O 2 All these biosensors usually operated in aqueous environ- ment. The principal applications difficulties encountered were ∗ Corresponding author. Tel.: +39 06 4991 3722. E-mail address: mauro.tomassetti@uniroma1.it (M. Tomassetti). due precisely to the low solubility in aqueous solution of several pesticides to be determined as well as of the real substrates in which they were contained. The recent development of organic phase enzyme electrodes (OPEEs) [11–14] has opened up new horizons in this regard, the possibility of developing inhibition biosensors operating in organic solvents for pesticide determi- nation. Our group has acquired considerable experience in the development of OPEEs [11,15,16]. In recent years it has devel- oped also inhibition OPEEs based on the bienzymatic system butyrylcholinesterase + choline oxidase for the determination of organophosphorus or carbamate pesticides [15,16]. Recently published work showed that inhibition of the tyrosinase enzyme can be conveniently exploited to determine triazinic pesticides [17,18]. Phenol + O 2 tyrosinase -→ o-quinone + H 2 O In view of the fact that we have recently developed excel- lent tyrosinase OPEEs, and since the solubility of pesticides is generally higher in organic solvents than in aqueous sol- vents, we recently decided to study the possibility of using a 0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.01.023