Biosensors and Bioelectronics 26 (2010) 1432–1436 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Peroxygenase based sensor for aromatic compounds Lei Peng a , Ulla Wollenberger a , Matthias Kinne b , Martin Hofrichter b , René Ullrich b , Katrin Scheibner c , Anna Fischer d , Frieder W. Scheller a,e,∗ a Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Golm, Germany b Unit of Environmental Biotechnology, International Graduate School of Zittau, 0276 Zittau, Germany c Department of Biotechnology, Lausitz University of Applied Sciences, 01968 Senftenberg, Germany d Institute of Chemistry, Technical University Berlin, 10967 Berlin, Germany e Fraunhofer Institute for Biomedical Engineering IBMT, D-14476 Potsdam, Germany article info Article history: Received 12 April 2010 Received in revised form 6 July 2010 Accepted 7 July 2010 Available online 29 July 2010 Keywords: Peroxygenase Direct electron transfer Nanoparticles Naphthalene biosensor Bioelectrocatalysis abstract We report on the redox behaviour of the peroxygenase from Agrocybe aegerita (AaeAPO) which has been electrostatically immobilized in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode. AaeAPO contains a covalently bound heme-thiolate as the redox active group that exchanges directly electrons with the electrode via the gold nanoparticles. The formal potential E ◦′ of AaeAPO in the gold nanoparticles-chitosan film was estimated to be -(286 ± 9) mV at pH 7.0. The heterogeneous electron transfer rate constant (k s ) increases from 3.7 in the scan rate range from 0.2 to 3.0 V s -1 and level off at 63.7 s -1 . Furthermore, the peroxide-dependent hydroxylation of aromatic compounds was applied to develop a sensor for naphthalene and nitrophenol. The amperometric measurements of naphthalene are based on the indication of H 2 O 2 consumption. For the chitosan-embedded gold nanoparticle system, the linear range extends from 4 to 40 M naphthalene with a detection limit of 4.0 M (S/N = 3) and repeatability of 5.7% for 40 M naphthalene. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Electrochemical enzyme assays and biosensors are attractive tools for the study of drug biotransformation since the majority of hydrophobic xenobiotics are metabolized in vivo by enzymes of the cytochrome P-450 family (P450s). Such biosensors pro- vide quantitative information and contribute to the elucidation of the oxidative pathway of the investigated substances. In this respect reactive intermediates may be enzymatically generated and detected at the redox electrode. At the beginning liver P450s have been applied as the recognition elements in substrate detecting biosensors (Renneberg et al., 1978; Bistolas et al., 2005). By engi- neering the P450 biocatalysis, the specificity was improved (Rabe et al., 2008). As an alternative to these concepts, horseradish per- oxidase (HRP) has been used in amperometric biosensors for the detection of different drugs, e.g. closapin and paracetamol, despite its radical reaction mechanism (Yu et al., 2006, 2007). Recently we presented a biosensor based on the aromatic peroxygenase from ∗ Corresponding author at: Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Golm, Germany. Tel.: +49 331 977 5121; fax: +49 331 977 5050. E-mail address: fschell@uni-potsdam.de (F.W. Scheller). the agaric fungus Agrocybe aegerita (AaeAPO) for the indication of p-nitrophenol (Peng et al., 2010). Functional combinations of conducting nanostructures, e.g. metallic nanoparticles, carbon nanotubes or nanoporous semicon- ductor films with redox enzymes, have a high potential as building blocks for bioelectronic devices such as biomolecular sensors or biofuel cells and microreactors (Willner and Katz, 2005). These con- ducting nanostructures allow binding of redox proteins to exploit their catalytic properties and can be immobilized on solid sup- ports, e.g. on electrodes or optically transparent sensor interfaces. The binding of the proteins to the conducting nanostructures is accomplished by methods for protein immobilization developed for macroscale interfaces, including chemisorption via thiol groups, electrostatic adsorption to modifiers, or coupling by bifunctional reagents to surface functionalities. For electrostatic adsorption, chi- tosan, a deacetylated derivative of chitin has been widely used in the development of biosensors due to its polycationic, biocompat- ible and film-forming properties (Yi et al., 2005). The hydrophilic chitosan is also compatible with conductive nanostructures such as gold nanoparticles and can be used as surface functionalization agent but also as direct reducing agent for the one-step synthesis of biocompatible gold nanoparticles (Au-NPs), as used in this work. In this paper we report for the first time on the direct electron transfer and on the enzymatic activity of AaeAPO immobilized in a 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.07.075