Copper–zinc alloy nanoparticle based enzyme-free superoxide radical sensing on a screen-printed electrode Burak Derkus, Emel Emregul n , Kaan C. Emregul Ankara University, Science Faculty, Department of Chemistry, Tandoğan, Ankara 06100, Turkey article info Article history: Received 11 August 2014 Received in revised form 29 October 2014 Accepted 2 November 2014 Available online 18 November 2014 Keywords: Superoxide dismutase Enzyme-free biosensor Screen-printed electrode CuZn nanoparticles Nanobiosenosors abstract In this paper, amperometric enzyme-free sensors using superoxide dismutase (SOD) enzyme as a catalyst for the dismutation reaction of superoxides into oxygen and hydrogen peroxide, enabling superoxide radical detection have been described. For this purpose, the surfaces of screen-printed platinum electrodes have been modified with gelatin composites of CuO, ZnO and CuZn nanoparticles with the expectation of an increase in catalytic effect toward the dismutation reaction. SOD containing electrodes were also prepared for comparative studies in which glutaraldehyde was used as a cross- linker for the immobilization of SOD to the nanocomposite materials. Electrochemical measurements were carried out using a screen-printed electrochemical system that included potassiumferrocyanide (K 4 [Fe(CN) 6 ]) and potassiumferricyanide (K 3 [Fe(CN) 6 ]) as the redox probes. The results revealed that the enzyme-free detection method using CuZn nanoparticles can determine superoxide radicals with high performance compared to other detection methods prepared with different nanoparticles by mimicking the active region of superoxide dismutase enzyme. The anodic (ks a ) and cathodic (ks c ) electron transfer rate constants and the anodic (α a ) and cathodic (α c ) transfer coefficients were evaluated and found to be ks a ¼6.31 s 1 and α a ¼0.81, ks c ¼1.48 s 1 and α c ¼0.19 for the gelatin–CuZn–SOD electrode; ks a ¼6.15 s 1 and α a ¼0.79, ks c ¼1,63 s 1 and α c ¼0.21 for the enzyme-free gelatin–CuZn electrode. The enzyme-free electrode showed nearly 80% amperometric performance with respect to the enzyme containing electrode indicating the superior functionality of enzyme-free electrode for the detection of superoxide radicals. & 2014 Elsevier B.V. All rights reserved. 1. Introduction Due to its overwhelming reaction rate owing to a radical mechanism and high specificity [1], the superoxide dismutase (SOD) enzyme offers a great potential for sensitive quantification of superoxide radicals in various biological samples especially in cancerous tissues using biosensing technology. Superoxide radicals (O 2 d  ) are known to damage some biological molecules [2–4] and signal pathways, and also play important roles in heart disease [5], cancer [6] and neuronal dejeneration [7]. Hence, the study of super- oxide radicals has attracted considerable attention in recent years. SODs are metalloenzymes which have iron (Fe), manganese (Mn), nickel (Ni), or copper–zinc (CuZn) ions in their active region that catalyze the dismutation of superoxide to oxygen and hydrogen peroxide [8]. Copper–zinc superoxide dismutase is present in the cytosol, nucleus, peroxisomes, and mitochondrial intermembrane space of human cells, acting as an antioxidant enzyme by lowering the steady-state concentration of superoxide. The human enzyme is a 32-kDa homodimer, with a copper- and zinc-binding site each per 153-amino acid subunits [9–11]. The copper site is the heart of the enzymatic active site where SOD1 protein catalyzes the dispro- portionation of superoxide to give dioxygen and hydrogen per- oxide. This catalysis is a two-step process: one molecule of superoxide first reduces the cupric ion to form dioxygen and then a second molecule of O 2 d  reoxidizes the cuprous ion to form hydrogen peroxide. Detailed information about the catalyzer effect of CuZn can be found in literature [12]. The dismutation of superoxide proceeds via a two-step reaction if the enzyme includes CuZn [13]: O 2  þ Cu(II)ZnSOD-O 2 þ Cu(I)ZnSOD O 2  þ Cu(I)ZnSOD þ 2H þ -H 2 O 2 þ Cu(II)ZnSOD The overall reaction is O 2  þ 2H þ -H 2 O 2 þ O 2 This reaction is often used to determine the superoxide anion. In order to understand the role of O 2 d  in pathology and physiology and the relationship between O 2 d  and environmental stresses, Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta http://dx.doi.org/10.1016/j.talanta.2014.11.003 0039-9140/& 2014 Elsevier B.V. All rights reserved. n Corresponding author. Tel.: þ90 312 212 60 40. E-mail address: eemregul@yahoo.com (E. Emregul). Talanta 134 (2015) 206–214