Enzyme and Microbial Technology 47 (2010) 153–158 Contents lists available at ScienceDirect Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt Biosensor based on pequi polyphenol oxidase immobilized on chitosan crosslinked with cyanuric chloride for thiodicarb determination Fábio de Lima a , Bruno G. Lucca a , Antônio M.J. Barbosa a , Valdir S. Ferreira a , Sally K. Moccelini b , Ana C. Franzoi b,∗ , Iolanda C. Vieira b a Departamento de Química, Universidade Federal do Mato Grosso do Sul, 79070-900, Campo Grande, MS, Brazil b Departamento de Química, Laboratório de Biossensores, Universidade Federal de Santa Catarina, 88040-970, Florianópolis, SC, Brazil article info Article history: Received 2 March 2010 Received in revised form 13 April 2010 Accepted 15 May 2010 Keywords: Biosensor Thiodicarb Polyphenol oxidase Pequi fruit abstract A novel pequi fruit (Caryocar brasiliense Camb.) homogenate source of polyphenol oxidase was obtained and immobilized in chitosan crosslinked with cyanuric chloride (CHcych-PPO). A biosensor was devel- oped and used for the square-wave voltammetric determination of thiodicarb. Several parameters were investigated to evaluate the performance of this biosensor in the presence of hydroquinone and thiodi- carb. The best response was obtained using 70:20:10% (w/w/w) of graphite powder:Nujol:CHcych-PPO, 250 U mL -1 of PPO and phosphate buffer solution (0.1 M; pH 7.0) with frequency, pulse amplitude and scan increment of 10 Hz, 150 mV, and 15 mV, respectively. Under optimized operational conditions the thiodicarb concentration was linear in the range of 3.75 × 10 -7 to 2.23 × 10 -6 M with a detection limit of 1.58 × 10 -7 M. The biosensor was applied in the determination of thiodicarb in fresh fruit and vegetable samples and the results compared with those obtained using high-performance liquid chromatography. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Polyphenol oxidase (PPO) has received continuous attention from food chemists and processors since its discovery over 90 years ago. This is because it is involved in the enzymatic browning of many edible plant products, especially fruits and vegetables. The positive attributes of high catalytic activity and the ability to uti- lize different phenolic compounds as a substrate have led to a wide interest in its use for the construction of new biosensors. The level of PPO in plants is dependent on the species, cultivar, maturity and age. It is located in chloroplasts, mitochondria, microsomes, peroxisomes and cellular plasma [1–4]. PPO belongs to the group of oxidoreductases, and is a copper-containing enzyme capable of catalyzing the oxidation of monophenols and diphenols to the corresponding o-quinones [1–4]. The development of biosensors using plant homogenates as alternative biological materials to replace isolated enzymes has received considerable attention and has been successfully used by our group [5–9]. This class of materials maintains the enzyme of interest in its natural environment with higher stability and enzyme activity [1–4]. A biosensor can be defined as an analytical device that com- bines a biological material (e.g. enzymes, microorganisms, tissues) ∗ Corresponding author. Tel.: +55 48 3721 9852. E-mail address: aninhafranzoi@yahoo.com.br (A.C. Franzoi). with an appropriate transducer (e.g. electrochemical, piezoelectric) capable of giving selective and/or quantitative analytical informa- tion. Electrochemical biosensors combine the analytical power of known techniques with the specificity of biological recognition processes. The aim is to biologically produce an electrical signal that relates to the concentration of the analyte. Enzymes may be immobilized by a variety of supports and methods, which may be classified as physical, where there are weak interactions between the support and the enzyme, and chemical, where covalent bonds are formed with the enzyme. A key factor in the construction of a biosensor is the need to achieve adequate and effective enzyme immobilization [10,11]. The PPO enzyme has been obtained from different plant tissues and immobilized in a variety of supports (e.g. chitosan, chitin) and these PPO biosensors employing alter- native biological materials have been extensive used due to their high stability and catalytic activity [9,12–14]. Chitosan (CH) is a natural polysaccharide consisting of glu- cosamine and N-acetylglucosamine. It can be derived through the partial deacetylation of chitin, the major compound of the exoskeletons of crustaceans. The molecular unit of CH has one amino group and two hydroxyl groups that are potentially capa- ble of being crosslinked with different substances. It is one of the most promising polymers and has considerable potential due to its biocompatibility, biodegradability, and low toxicity, along with a variety of biological activities including antimicrobial activity [15–17]. This biopolymer can be employed in the immobiliza- tion of the enzyme for the construction of biosensors for use in 0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2010.05.006