Enzyme immobilisation on electroactive nanostructured membranes (ENM): Optimised architectures for biosensing

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 hexacyanoferrate 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 containing 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 mu 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. (C) 2008 Elsevier B.V. All rights reserved.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

FAPESP

CAPES

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

CNPq

IMMP/MCT (Brazil)[HPRN-CT-2002-00186]

Ministério da Ciência, Tecnologia e Inovação do Brasil (MCTI)

Fundcado para a Ciencia e Tecnologia (FCT) Portugal

Fundcado para a Ciencia e Tecnologia (FCT) Portugal[PTDC/QUI/65255/2006]

Fundcado para a Ciencia e Tecnologia (FCT) Portugal[POCI 2010]

Fundcado para a Ciencia e Tecnologia (FCT) Portugal

European Community

European Community Fund FEDER[SFRH/BD/14014/2003]

European Community Fund FEDER[SFRH/BD/18659/2004]

European Community

ICEMS (Research Unit 103)

ICEMS (Research Unit 103)