Published: March 10, 2011 r2011 American Chemical Society 5919 dx.doi.org/10.1021/jp1080065 | J. Phys. Chem. C 2011, 115, 5919–5929 ARTICLE pubs.acs.org/JPCC Direct Electron Transfer of Trametes hirsuta Laccase in a Dual-Layer Architecture of Poly(3,4-ethylenedioxythiophene) Films Xiaoju Wang, † Rose-Marie Latonen, ‡ Pia Sj€ oberg-Eerola, † Jan-Erik Eriksson, † Johan Bobacka, ‡ Harry Boer, § and Mikael Bergelin* ,† † Laboratory of Inorganic Chemistry, Process Chemistry Centre, Åbo Akademi University, Biskopsgatan 8, FIN-20500, Åbo/Turku, Finland ‡ Laboratory of Analytical Chemistry, Process Chemistry Centre, Åbo Akademi University, Bisopsgatan 8, FIN-20500, Åbo/Turku, Finland § VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland b S Supporting Information 1. INTRODUCTION The “blue” multicopper oxidases refer to a class of copper- containing oxidoreductases, which can catalyze the four-electron reduction of O 2 into water, coupled to one-electron oxidation of a variety of small organic (generally aromatic) or inorganic substrates. 1,2 The most commonly recognized multicopper oxi- dases include laccase, 3,4 bilirubin oxidase, 5 ceruloplasmin, 6 and ascorbate oxidase. 7,8 The catalytic sites of the multicopper oxidase consist of four copper ions, which can be classified in accordance with their spectroscopic characteristics as Type 1 (T1), Type 2 (T2), and Type 3 (T3) sites. A mononuclear T1 Cu is responsible for the blue color of the enzyme with a character- istic absorbance band at a wavelength around 610 nm in the UV-vis spectrum. T1 Cu is positioned near a wide, hydropho- bic, substrate-binding pocket, rich in π electron density, to which a range of substrates can bind and undergo rapid, one-electron oxidation to radical products that dissociate before further reaction. One T2 Cu and two T3 Cu form the trinuclear cluster site, where O 2 is bound between the two T3 copper nuclear and reduced into water. T1 Cu extracts one electron from electron donors (substrates) with a subsequent intramolecular electron transfer via a His-Cys-His bridge to the T2-T3 Cu cluster, where O 2 is reduced into water. 8,9 Laccase (EC 1.10.3.2) widely distributes in fungi, in higher plants, and also in some bacteria. 3,10,11 The enzymatic and physicochemical properties of laccase depend on the original source. Laccase has a relatively broad substrate spectrum and is a thermostable and environmentally friendly catalyst. 12 Its industrial importance is reflected by its diverse applications, from textile bleaching to pulping and paper making, 13 and from food applications 14 to bioremediation processes. 15 Recently, in development of bioelectronic devices, lac- case has been used as the “recognition” component in the fabrication of amperometric biosensors for detecting a large number of phenolic Received: August 24, 2010 Revised: February 18, 2011 ABSTRACT: Direct electron transfer (DET) type biocatalysis was accomplished for Trametes hirsuta laccase (ThL) on a glassy carbon (GC) electrode by immobilizing laccase into a well-designed dual- layer architecture of poly(3,4-ethylenedioxythiophene) (PEDOT). PEDOT films were subsequently deposited on a GC electrode via electropolymerization, with NO 3 - as the counterion for the first accommodation layer and poly(styrene-sulfonate) anions (PSS - ) for the second capping layer. The enzyme (ThL) was cast on top of the accommodation layer (PEDOT-NO 3 ), and then the capping layer (PEDOT-PSS) was electrodeposited to entrap ThL between the layers. This enzyme electrode is reported to be able to promote DET between ThL and the GC electrode and catalyze the reduction of O 2 into water. The influence of fabrication parameters on the enzyme electrode performance was investigated through chronoamperometric measurements. The investigated parameters included di fferent combinations of PEDOT films, ThL loading, and the thicknesses of both PEDOT layers. As a representative, one optimized dual-layer-architecture enzyme electrode of PEDOT-NO 3 (28 mC)/ThL (1.26 U)/ PEDOT-PSS (3.5 mC) performed fairly good reproducibility and operational stability. Its pH profile exhibited a bell-shape with an optimal pH in the range of 3.0-3.5. The influences of ionic strength and addition of a nonionic surfactant into the buffer solution on the enzyme electrode performance were also studied to obtain information about the DET mechanism of ThL in the dual-layer architecture. On the basis of the information obtained from different characterizations, π-π interaction between the PSS - ions and the hydrophobic substrate- binding pocket in the vicinity of the T1 Cu site was proposed to result in a favorable location of the conducting polymer chain close to the T1 Cu site and thus facilitate DET of ThL within this particular architecture. The applicability of this approach to various electrode materials is also underlined, which makes it a favorable approach to construct an O 2 -consuming cathode for biofuel cells.