Enzyme and Microbial Technology 39 (2006) 131–140 In situ measurement of activity and mass transfer effects in enzyme immobilized electrodes Wayne Johnston 1 , Nathan Maynard, Bor Yann Liaw ∗∗ , Michael J. Cooney ∗ Hawaii Natural Energy Institute, University of Hawaii – Manoa, 1680 East West Road, Post 109, Honolulu, HI 96822, USA Received 20 July 2005; received in revised form 8 October 2005; accepted 10 October 2005 Abstract Enzyme catalyzed biofuel cells have been proposed as an alternative to transition metal catalysts for power generation as they oxidize alcohols at relatively low overpotential without the production of detrimental carbon monoxide, and are capable of operation at lower temperatures [Palmore GT, Bertschy H, Bergens SH, Whitesides GM. A methanol/dioxygen biofuel cell that uses NAD + -dependent dehydrogenase as catalysts: application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at low overpotentials. J Electroanal Chem 1998;443:155–61]. Additionally, the immobilization procedure prevents internal leakage or cross-contamination of electron mediators. However, full realization of the membrane-less biofuel cell as a power source requires a three-dimensional boundary structure which balances the overall effective surface area against porosity, thus ensuring the maximum number of catalyst sites are available without suffering the blockage of fuel transport, which occurs if the pore size is too small. In this work, a simple and in situ method using a simplified diffusion model is presented to estimate the total activity immobilized enzyme in the absence of mass transfer effects. The method, which also calculates a combined mass transfer parameter including an effective diffusion coefficient, models the reactant concentration at the enzyme surface using bulk concentrations which then can be measured in situ by spectrophotometric detection or ex situ by HPLC analysis. The method was then applied to evaluate two methods of alcohol dehydrogenase electrode fabrication: direct adsorption to carbon felt and entrapment within the conductive polymer polypyrrole. Results showed that direct adsorption provided 26 times the activity versus the method of direct entrapment and better mass transfer characteristics. Correlation of these results to scanning electron micrographs suggested that the polypyrrole entrapment method lacked the expected diffusive pathways and most likely expelled enzyme during growth of the film. © 2005 Elsevier Inc. All rights reserved. Keywords: Enzyme; Alcohol dehydrogenase; Electrode; Mass transfer; Activity; Immobilization; Polypyrrole 1. Introduction Fuel cells generate power through the electrochemical oxi- dation of fuels. As the resulting electrical energy is gener- ated without the limitation of the thermal–mechanical Carnot Cycle, enzymatic fuel cells are desirable from an environmen- tal point of view. Recently, there has been increasing interest in direct alcohol fuel cells (DAFCs) using platinum based cat- alysts [2–6]. Both methanol and ethanol have been used as reactants [3,4] although ethanol is preferred because it lacks toxicity relative to methanol and is readily produced from ∗ Corresponding author. Tel.: +1 808 956 7337; fax: +1 808 956 2336. ∗∗ Corresponding author. Tel.: +1 808 956 2339; fax: +1 808 956 2336. E-mail addresses: bliaw@hawaii.edu (B.Y. Liaw), mcooney@hawaii.edu (M.J. Cooney). 1 Current address: School of Biomedical Sciences, University of Queensland, Brisbane, Australia. renewable resources [3–9]. Direct oxidation of ethanol on plat- inum, however, is hindered by fouling of the electrode surface by reactive intermediates containing CO groups. In response, researchers have explored the use of bi- and tri-metal cata- lysts to further oxidize the intermediates at potentials more negative than those used to oxidize ethanol [3,5–9]. Despite improvements such as the use of ordered intermetallic phases [10], fouling of the electrode remains problematic because the extra C–C bond increases the number of detrimental chem- ical intermediates produced [5,9,10]. Enzyme catalyzed bio- fuel cells have been proposed as an alternative to transition metal catalysts for power generation as they oxidize alco- hols at relatively low overpotential without producing carbon monoxide, and are capable of operation at lower temperatures [1]. Work on enzyme electrodes has generally focused on reac- tant specificity [11–40]. A critical aspect of the fabrication of such electrodes is the electrical wiring of the enzyme’s buried 0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.10.008