Sensors and Actuators B 142 (2009) 308–315 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb Application of functionalised carbon nanotubes immobilised into chitosan films in amperometric enzyme biosensors Mariana Emilia Ghica a , Rasa Pauliukaite a , Orlando Fatibello-Filho a,b , Christopher M.A. Brett a,∗ a Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal b Departamento de Química, Universidade Federal de São Carlos, C.P. 676, 13560-970 São Carlos-SP, Brazil article info Article history: Received 4 June 2009 Received in revised form 5 August 2009 Accepted 6 August 2009 Available online 20 August 2009 Keywords: Graphite-epoxy resin composite Functionalised carbon nanotubes Chitosan Glucose Crosslinking Hexaammineruthenium (III) chloride abstract A new approach for building a bio-conductive interface for enzyme immobilisation is described. This strat- egy permits very simple preparation of the enzyme biosensor and also reveals direct electron transfer features. A graphite-epoxy resin composite (GrEC) electrode modified with functionalised multi-wall car- bon nanotubes (MWCNTs) immobilised by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide together with N-hydroxysuccinimide (EDC–NHS) in a chitosan (Chit) matrix was prepared and characterised by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of hexaam- mineruthenium (III) chloride. It was then used as a base for glucose oxidase (GOx) immobilisation by the simple method of crosslinking with glutaraldehyde (GA) with bovine serum albumin (BSA) as carrier protein. The resulting mediator-free biosensor was applied to the determination of glucose in amper- ometric mode at different applied potentials and the mechanism of reaction was also investigated by cyclic voltammetry, with and without dissolved oxygen in solution. Analytical parameters, as well as reproducibility, repeatability and stability were determined. Interferences were assessed using different compounds usually present in natural samples, such as wines, juices or blood, in order to evaluate the selectivity of the developed biosensor. The novel combination of carbon nanotubes immobilised with chi- tosan crosslinked with EDC–NHS and glucose oxidase immobilised by crosslinking with glutaraldehyde offers an excellent, easy to make biosensor for glucose determination without interferences. © 2009 Elsevier B.V. All rights reserved. 1. Introduction A new era in the field of nanotechnology has begun with the discovery of carbon nanotubes (CNTs) by Iijima in 1991 [1]. Con- sisting in single-wall carbon nanotubes (SWCNTs)—a single sheet of graphene rolled seamlessly in a cylinder of 1–2 nm diameter and multi-wall carbon nanotubes (MWCNTs)—several concentric tubes of graphene inside one other with diameters typically rang- ing from 2 to 100 nm, separated by a distance of 0.3–0.4 nm [2–9], this class of nanomaterials has attracted enormous interest. Due to their unique physical and chemical properties, CNTs have been extensively researched for electrocatalytic and sensing applications including fabrication of electrochemical sensors and biosensors [2–9]. CNTs promote electron transfer reactions of many compounds and their use as electrode modifiers leads to a decrease of the overpotential, a decrease of the electrode response time and/or an increase of the reaction rate of various electroactive substrates [2–10], in comparison with conventional carbon electrodes [3,11]. ∗ Corresponding author. Tel.: +351 239835295; fax: +351 239835295. E-mail address: brett@ci.uc.pt (C.M.A. Brett). The electroactivity of CNTs is ascribed to the presence of reactive groups on its surface and/or defect-areas of the nanotubes [4,7,9], and the advantages of using CNTs for electrode surface modifica- tion in the development of new designs of electrochemical sensors and biosensors have been recently highlighted by many authors [2–16]. Nevertheless, the low solubility of CNTs in most solvents is the major challenge to their use as modifiers in the fabrica- tion of chemical sensors and/or biosensors. The strategies most employed to disperse CNTs are end and sidewall functionalisa- tion [4,17,18], use of surfactants with sonication [19], and polymer wrapping [20]. In an attempt to develop more sensitive biosensors, different enzymes have been immobilised together with carbon nanotubes [9,21]. The immobilisation of enzymes is a key step in the fabrication of biosensors and the biocompatibility of the matrix, its easy preparation and/or its stability is of extreme importance. Chitosan (Chit), a linear -1,4-linked polysaccharide (similar to cel- lulose) that is obtained by the partial deacetylation of chitin, a major component of the shells of crustaceans such as crab, shrimp, and crawfish fulfils these requirements. Chitosan (Chit) possesses dis- tinct chemical and biological properties [22], because chitosan has reactive amino and hydroxyl groups in its linear polyglucosamine high molar mass chains which are amenable to chemical modifica- tion [22–26]. In addition, Chit is biocompatible, biodegradable, is a 0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2009.08.012