Human sulfite oxidase electrochemistry on gold nanoparticles modified electrode Stefano Frasca a, ⁎, Oscar Rojas b , Johannes Salewski c , Bettina Neumann a , Konstanze Stiba a , Inez M. Weidinger c , Brigitte Tiersch b , Silke Leimkühler a , Joachim Koetz b , Ulla Wollenberger a, ⁎ a Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht-Str.24-25, Haus 25 14476 Golm, Germany b Institut für Chemie, Universität Potsdam, Karl-Liebknecht-Str.24-25, Haus 25 14476 Golm, Germany c Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany abstract article info Article history: Received 29 July 2011 Received in revised form 3 November 2011 Accepted 28 November 2011 Available online 8 December 2011 Keywords: Direct electron transfer Gold nanoparticle Human sulfite oxidase Ionic liquid Sulfite biosensor The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron trans- fer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl 4 with branched poly(ethylenei- mine) in an ionic liquids resulting particles with a diameter less than 10 nm. These nanoparticles were co- valently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range be- tween 0.5 and 5.4 μM with a high sensitivity (1.85 nA μM -1 ). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Over the last decades, electron exchange of redox enzymes with electrodes has gained interest for technological applications like in biosensors and other bioelectronic devices and biosynthesis [1–11]. Therefore, an efficient communication between protein and electrode continues being a challenge and the subject of extensive research. Several approaches have been developed including the addition of free and immobilized electron mediators [7–14], the modification of the enzyme [15–17], and the immobilization of enzymes on electro- active polymers [18–20]. The incorporation of gold nanoparticles (AuNPs) to assemble elec- trochemically active enzymes has been suggested [21–22]. For example, AuNPs have been successfully employed in a biocompatible matrix (chitosan) for the development of biosensors [23–26]. Based on the polyelectrolyte characteristics of this biopolymer, different enzymes were immobilized onto AuNPs by electrostatic interactions. Other approaches for the construction of biosensors have been reported using proteins adsorbed gold nanoparticles coated with polyelectro- lytes like polyamidoamine and polypropyleneimine [27–28]. Alterna- tively, Willner and co-workers have reported the reconstruction of apo-glucose oxidase on 1.4 nm AuNPs functionalized with the cofactor flavin adenine dinucleotide into a conductive film to yield a highly efficient electrical contact with the electrode support [29–30]. Similar effect of AuNPs was observed by Jensen and Ulstrup who described an electrostatically conjugated system of cytochrome c and 3–4 nm size AuNPs coated with thiol ligands [31]. Animal sulfite oxidizing enzymes are molybdo- and heme- containing redox enzymes [32–33]. The enzyme catalyzes the conver- sion of sulfite to sulfate, the terminal reaction in the oxidative degra- dation of cysteine and methionine. The catalytic activity of eukaryotic sulfite oxidase (SO) is attributed to the functionality of distinct do- mains. The enzyme contains a N-terminal heme domain (HD) with a non-covalently bound heme b5 cofactor connected by a flexible loops to large central domain, containing the Mo atom (molybdenum domain, MD) and a C-terminal dimerization domain [34–36]. Sulfite is oxidized to sulfate at the Moco center, and the reducing equivalents are transferred via intramolecular electron transfer (IET) to the heme b5, where the terminal electron carrier cytochrome c is reduced. It is generally accepted that conformational rearrangement between the MD and the HD occur before IET to reach a more effective orientation. Once the IET has taken place the HD moves away from the MD to interact with the positively charged cytochrome c [37]. This conformational change is thought to be induced by a cyto- chrome c binding event and can be largely affected by the ionic strength, viscosity and pH and the components of the solution [38–41]. Bioelectrochemistry 87 (2012) 33–41 ⁎ Corresponding authors. Tel.: + 49 3319775122; fax: + 49 3319775051. E-mail address: uwollen@uni-potsdam.de (U. Wollenberger). 1567-5394/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2011.11.012 Contents lists available at SciVerse ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem