Sensors and Actuators B 188 (2013) 1101–1108 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical journal h om epage: www.elsevier.com/locat e/snb Tyrosinase biosensor based on a glassy carbon electrode modified with multi-walled carbon nanotubes and 1-butyl-3-methylimidazolium chloride within a dihexadecylphosphate film Fernando Campanhã Vicentini a , Bruno C. Janegitz a,b , Christopher M.A. Brett c , Orlando Fatibello-Filho a,d,∗ a Departamento de Química, Centro de Ciências Exatas e de Tecnologia, Universidade Federal de São Carlos, Caixa postal 676, CEP 13560-970 São Carlos, SP, Brazil b Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos, Universidade de São Paulo, CEP 13566-390 São Carlos, SP, Brazil c Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal d Instituto Nacional de Ciência e Tecnologia de Bioanalítica (INTC de Bioanalítica), Brazil a r t i c l e i n f o Article history: Received 19 May 2013 Received in revised form 2 July 2013 Accepted 29 July 2013 Available online xxx Keywords: 1-Butyl-3-methylimidazolium chloride Catechol determination Dihexadecylphosphate Ionic liquid Multi-walled carbon nanotubes Tyrosinase biosensor a b s t r a c t A glassy carbon electrode modified with functionalized multi-walled carbon nanotubes (MWCNT), 1-butyl-3-methylimidazolium chloride (ionic liquid = IL) and tyrosinase (Tyr) within a dihexadecylphos- phate (DHP) film for the development of a biosensor is proposed. MWCNT, IL and Tyr were efficiently immobilized in the film using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) as crosslinking agents, and which was characterized by cyclic voltammetry (CV) in the pres- ence of catechol. The IL-MWCNT nanocomposite showed good conductivity and biocompatibility with Tyr enzyme, since the biosensor presented biocatalytic activity toward the oxidation of catechol to o- quinone which was electrochemically reduced to catechol at a potential of 0.04 V. The analytical curve ranged from 4.9 × 10 -6 to 1.1 × 10 -3 mol L -1 with a detection limit of 5.8 × 10 -7 mol L -1 . The developed Tyr-IL-MWCNT-DHP/GCE biosensor showed a wide linear range, good reproducibility, sensitivity and stability and the biosensor was successfully applied to the determination of catechol in natural water samples, with satisfactory results compared with a spectrophotometric method, at the 95% confidence level. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Room temperature ionic liquids (RTIL) are defined as com- pounds composed entirely of ions, which are liquid at a temperature less than 100 ◦ C [1]. The use of ionic liquids in elec- troanalysis provides stability in the electrochemical responses and high ionic conductivity without the necessity of adding inert electrolyte. In recent years, RTILs have been used in the development of electrochemical sensors and biosensors, in most cases being incorporated in carbon paste [2] or carbon com- posite [3]. Nevertheless, there are very few biosensors using RTIL incorporated in film-modified electrodes. Film-modified elec- trodes are a good way to entrap proteins and enzymes, retaining their bioactivity whilst not interfering in the electrochemical response. ∗ Corresponding author. Tel.: +55 16 33518098; fax: +55 16 33518350. E-mail address: bello@ufscar.br (O. Fatibello-Filho). Carbon nanotubes (CNTs) present similarity in length scales with redox enzymes, they have high electronic conductivity and good mechanical properties, which can improve biosensing devices [4–7], such as tyrosinase biosensors. Ozoner et al. proposed a flow injection catechol biosensor based on tyrosinase entrapped in a CNT-modified polypyrrole biocomposite film on a glassy carbon electrode substrate [8]. Man and Yo prepared a biosen- sor containing tyrosinase, single-walled CNT and polypyrrole for amperometric detection of dopamine [9]. Tsai and Chiu developed an amperometric biosensor based on multi-walled CNT-Nafion- tyrosinase nanobiocomposites for the determination of phenolic compounds [10]. Dihexadecylphosphate (DHP), or dicetylphosphate, is a surfac- tant molecule with a polar head and two long hydrophobic tails [11]. It can produce stable films on electrode surfaces, probably via hydrogen bonds, and has been used in sensors [12] and biosensors [13]. Tyrosinase (Tyr) is a blue copper protein (with 2 copper atoms in the active center), which can be considered as a polyphenol oxidase (PPO). This important enzyme catalyzes two consecutive 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.07.109