Biosensors and Bioelectronics 25 (2010) 1474–1480 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Micro-biofuel cell powered by glucose/O 2 based on electro-deposition of enzyme, conducting polymer and redox mediators: Preparation, characterization and performance in human serum Malika Ammam ∗ , Jan Fransaer Department of Metallurgy and Materials Engineering (MTM), KU Leuven, Kasteelpark Arenberg 44, B-3001, Heverlee, Belgium article info Article history: Received 14 July 2009 Received in revised form 12 October 2009 Accepted 2 November 2009 Available online 18 November 2009 Keywords: Biofuel cells Laccase Glucose oxidase Redox mediators Electro-deposition abstract In this study we report a new simple process to manufacture a biofuel cell consisting of a glucose oxidase (GOx) based anode and a laccase (LAc) based cathode. The process is based on the electro-deposition of the enzymes, conducting polymer and redox mediators from ultrapure water at a potential of 4 V vs. AgCl/Ag. Contrary to the conventional electro-deposition from high ionic strength (buffer solution) at low applied potential (1 V vs. AgCl/Ag) where only thin films could be deposited, leading to BFC with moderate power, the electro-deposition from ultrapure water at 4 V allows the growth of thick films leading to BFC with high power output. It was observed that the combination of polypyrrole (PPy), with ferrocenium hexafluorophosphate (FHFP) and pyrroloquinoline quinone (PQQ) to be appropriate for the electron transfer at the GOx bioanode, while the combination of polypyrrole with bis-(bipyridine)-(5- amino-phenanthroline) ruthenium bis (hexafluorophosphate)(RuPy) and 4,4-sulfonyldiphenol (SDP) to be effective for the electron transfer at the LAc biocathode. The working biofuel cell was studied at 37 ◦ C in phosphate buffer solution at pH 7.4 containing 10 mM glucose and in human serum. Under these conditions, the maximum power density reached 3.1 W mm -2 at a cell voltage of 0.28 V in buffer solution and 1.6 W mm -2 at a cell voltage of 0.21 V in human serum. This study offers a new route to the development of enzymatic BFCs with high performance and provides information on enzymatic BFCs as in vivo power sources. © 2009 Elsevier B.V. All rights reserved. 1. Introduction An enzyme fuel cell is an electrochemical device that converts chemical energy contained in e.g. glucose into electrical energy. Considerable attention has recently been paid to enzymatic biofuel cells (BFCs) because they are recognized as an energy conversion technology that possess interesting properties such as ambient working temperature, neutral working pH, and most importantly the possibility to be used for in vivo implantation in animals or humans as a power source for micro-pumps and pacemakers (Barton et al., 2004; Kim et al., 2006; Bullen et al., 2006; Ikeda and Kano, 2003). Several new electrochemical concepts to enzymatic biofuel cells have been demonstrated in recent years (Soukharev et al., 2004; Palmore et al., 1998; Palmore and Kim, 1999; Lim and Palmore, 2007; Moore et al., 2005; Topcagic and Minteer, 2006; Ramanaviciusa et al., 2005; Sato et al., 2005; Ivnitski et al., 2006; Barriere et al., 2004; Liu et al., 2005). For glucose/O 2 biofuel cells, glucose is electro-oxidized at the anode to -gluconolactone by ∗ Corresponding author. Tel.: +32 16 321260; fax: +32 16 321991. E-mail address: Malika.Ammam@mtm.kuleuven.be (M. Ammam). glucose oxidase (GOx), and oxygen is reduced to water at the cathode by enzymes such as laccase or bilirubin oxidase. How- ever, enzymes have a complex 3D structure comprised of proteins. The electron transferring unit of the enzyme, namely the apoen- zyme, is deeply buried inside its complex structure. Hence, efficient electrical communication between the electrode and the enzyme biocatalyst is difficult. Small redox molecules that act as media- tors and conducting polymers are useful for the electron transfer between the redox center of the enzyme and the electrode. On the other hand, the supply of oxygen and glucose mixture directly in a non-compartmentalized cell leads to its reduction at the biocath- ode but likewise at the bioanode, which decreases the power output of the biofuel cell (Mano et al., 2003; Kim et al., 2003). There are several approaches to limit the decrease in the power output due to the parasitic reactions. One approach was the use of reconstructed GOx that is less sensitive to the presence of oxygen (Katz et al., 1999). Reconstituted GOx with the FAD active center removed and connected to a layer of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) was used as anode. The cathode was constructed from cytochrome c oxidase adsorbed on a monolayer of cytochrome c as redox mediator. The biofuel cell had a power out- put of 0.05 W mm -2 at pH 7 and 25 ◦ C. A second approach was the 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.11.001