A simple electrochemical approach to fabricate a glucose biosensor based on graphene–glucose oxidase biocomposite Binesh Unnikrishnan, Selvakumar Palanisamy, Shen-Ming Chen n Electroanalysis and Bioelectrochemistry Laboratory, Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan, ROC article info Article history: Received 17 March 2012 Received in revised form 19 June 2012 Accepted 21 June 2012 Available online 1 July 2012 Keywords: Glucose oxidase Immobilization Reduced graphene oxide Biosensor Amperometry Electrochemical reduction abstract We report a simple electrochemical approach for the immobilization of glucose oxidase (GOx) on reduced graphene oxide (RGO). The immobilization of GOx was achieved in a single step without any cross linking agents or modifiers. A simple solution phase approach was used to prepare exfoliated graphene oxide (GO), followed by electrochemical reduction to get RGO–GOx biocomposite. The direct electrochemistry of GOx was revealed at the RGO–GOx modified glassy carbon electrode (GCE). The electrocatalytic and electroanalytical applications of the proposed film were studied by cyclic voltammetry (CV) and amperometry. It is notable that the glucose determination has been achieved in mediator-free conditions. RGO–GOx film showed very good stability, reproducibility and high selectivity. The developed biosensor exhibits excellent catalytic activity towards glucose over a wide linear range of 0.1–27 mM with a sensitivity of 1.85 mA mM 1 cm 2 . The facile and easy electro- chemical approach used for the preparation of RGO–GOx may open up new horizons in the production of cost-effective biosensors and biofuel cells. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Immobilizing enzymes efficiently on electrode surface is one of the most challenging tasks in a biosensor fabrication. Among the various enzymes, glucose oxidase (GOx) has received consider- able importance in real-time glucose monitoring and biofuel cells related applications, due to its high selectivity to glucose. The two major limitations in immobilizing GOx on solid electrodes are: the poor electrical communication between the active site of the enzyme and the electrode, and enzyme leaching (Jody et al., 2007; Yang et al., 2003). To date, several methods have been employed to effectively immobilize GOx on the electrode surface. Entrap- ping or covalently immobilizing GOx in stable matrices including nano and mesostructured metal oxides (Cao et al., 2008; Fang et al., 2011), metal nanoparticles (Bharathi and Nogami, 2001; Baby et al., 2010), conducting polymers (Ekiz et al., 2010; Foulds et al., 1986; Alwarappan et al., 2010a), mesostructured silica (Blin et al., 2005), sol–gel matrix (Jia et al., 2007; Chen et al., 1998), carbon nanotubes (Lin et al., 2004; Periasamy et al., 2011), graphene (Shan et al., 2009; Kang et al., 2009; Zhou et al., 2010), etc., leads to the enhanced electron transfer and improved enzyme stability (Alwarappan et al., 2009, 2010b). Recently, Fu et al. (2011) demon- strated the use of metal-organic coordination polymers as a suitable immobilization matrix for GOx. Besides, GOx immobilized on conducting polymer/metal oxide composite (Xian et al., 2010) also exhibits enhanced direct electron transfer. All these findings reveal that choosing an immobilization matrix with good electrical con- ductivity, stability, and antifouling property is mandatory for biosensor applications. Graphene is an inexpensive material with good mechanical, electrical and thermal properties (Novoselov et al., 2004; Geim and Novoselov, 2007) and it has perceived considerable attention recently. It has large surface area to volume ratio and good biocompatibility. Owing to the aforementioned properties, gra- phene is a potential matrix for electrochemical biosensors (Kuila et al., 2011). However, graphene sheets tend to form graphite by restacking (Li et al., 2008) due to the van der Waals force of attraction. In addition, dispersion of graphene in aqueous media is difficult due to its hydrophobic nature. So far, versatile strategies have been employed by several research groups to produce large yield and high-electronic quality graphene (Gengler et al., 2010; Shen et al., 2009). Though the chemical exfoliation method is much preferred for the large scale production of graphene sheets (Song et al., 2012), as-produced graphene sheets are not good enough for nanoelectronics applications (Guo et al., 2009). As an alternative, the electrochemical reduction method is more green (without using any toxic solvents), cost-effective and it is more suitable for preparing less defective graphene sheets. Till date, numerous strategies have been successfully employed for immobilizing GOx efficiently on graphene (Wu et al., 2010), Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.06.045 n Corresponding author. Tel.: þ886 2270 17147; fax: þ886 2270 25238. E-mail address: smchen78@ms15.hinet.net (S.-M. Chen). Biosensors and Bioelectronics 39 (2013) 70–75