Please cite this article in press as: F.N. Crespilho, et al., Talanta (2008), doi:10.1016/j.talanta.2008.04.054 ARTICLE IN PRESS G Model TAL-9787; No. of Pages 7 Talanta xxx (2008) xxx–xxx Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Enzyme immobilisation on electroactive nanostructured membranes (ENM): Optimised architectures for biosensing Frank N. Crespilho a , M. Emilia Ghica b , Carla Gouveia-Caridade b , Osvaldo N. Oliveira Jr. a , Christopher M.A. Brett b,∗ a Instituto de F´ ısica de S˜ ao Carlos, Universidade de S˜ ao Paulo, 13560-970 S˜ ao Carlos-SP, Brazil b Departamento de Qu´ ımica, Faculdade de Ciˆ encias e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal article info Article history: Received 9 January 2008 Received in revised form 17 April 2008 Accepted 23 April 2008 Available online xxx Keywords: Layer-by-layer Glucose oxidase Gold nanoparticles PAMAM dendrimer Cobalt hexacyanoferrate redox mediator abstract Electroactive nanostructured membranes have been produced by the layer-by-layer (LbL) technique, and used to make electrochemical enzyme biosensors for glucose by modification with cobalt hexacyano- ferrate redox mediator and immobilisation of glucose oxidase enzyme. Indium tin oxide (ITO) glass electrodes were modified with up to three bilayers of polyamidoamine (PAMAM) dendrimers contain- ing gold nanoparticles and poly(vinylsulfonate) (PVS). The gold nanoparticles were covered with cobalt hexacyanoferrate that functioned as a redox mediator, allowing the modified electrode to be used to detect H 2 O 2 , the product of the oxidase enzymatic reaction, at 0.0 V vs. SCE. Enzyme was then immobilised by cross-linking with glutaraldehyde. Several parameters for optimisation of the glucose biosensor were investigated, including the number of deposited bilayers, the enzyme immobilisation protocol and the concentrations of immobilised enzyme and of the protein that was crosslinked with PAMAM. The latter was used to provide glucose oxidase with a friendly environment, in order to preserve its bioactivity. The optimised biosensor, with three bilayers, has high sensitivity and operational stability, with a detection limit of 6.1 M and an apparent Michaelis–Menten constant of 0.20 mM. It showed good selectivity against interferents and is suitable for glucose measurements in natural samples. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Monolayers prepared by direct adsorption of dendrimers onto solid substrates have been proven to be highly stable and amenable to functionalisation without loss of dendrimers into solution [1]. The large surface area of dendrimers allows various functional units to be immobilised, which is important in applications such as sen- sors. Biosensors based on enzymes have been built which exploit the properties of dendrimers. Dendrimer-encapsulated platinum nanoparticles have been used for layer-by-layer (LbL) glucose biosensor development on platinum electrodes [2] and on carbon nanotubes [3], and for a glu- tamate biosensor on carbon nanotubes [4], without redox mediator. Yoon and Kim [5] prepared layer-by-layer films with polyami- doamine (PAMAM) dendrimer alternated with periodate oxidized glucose oxidase, on a gold substrate, and detected glucose using ferrocenemethanol in solution as diffusional electron-transfer mediator. A similar method, with the same redox mediator, was used by Yang et al. [6] but with gold nanoparticles in combina- ∗ Corresponding author. Tel.: +351 239835295; fax: +351 239835295. E-mail address: brett@ci.uc.pt (C.M.A. Brett). tion with the covalently attached cross-linker cysteamine instead of PAMAM. The main disadvantage of these methods is the addition of a mediator to the reaction medium. A new method for enzymatic immobilisation was recently demonstrated by us, which uses a combination of LbL self-assembly, redox mediator electrodeposition and cross-linking [7]. It was shown how one can benefit from combining the biocatalytic properties of the enzyme with the conductivity and electrocat- alytic properties of gold nanoparticles. The strategy used consists in applying a new concept in nanoarchitecture: electroactive nanostructured membranes (ENM) with ITO-PVS/PAMAM-Au LbL electrodes [8], where indium tin oxide (ITO) is the electrode substrate and PVS is poly(vinylsulfonate), which alternates with PAMAM-Au (PAMAM containing gold nanoparticles) in the bilayer structure. A redox mediator (Me) is then electrodeposited around the Au nanoparticles to form an ITO-PVS/PAMAM-Au@Me system. The redox mediators used were Co, Fe, Ni and Cu hexacyanoferrates. These hexacyanoferrate modified electrodes showed electrocat- alytic activity towards hydrogen peroxide, demonstrating that this new approach can be used in biosensors and nanodevices, where a redox mediator is required. The LbL assembly of oppositely charged species is a simple and powerful method to provide a suitable nanoenvironment to 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.04.054