Improving the Direct Electron Transfer in Monolithic Bioelectrodes Prepared by Immobilization of FDH Enzyme on Carbon-Coated Anodic Aluminum Oxide Films

Alberto Castro-Muñiz,Takashi Kyotani,Wataru Nakayama,Hiroshi Komiyama,Yasuto Hoshikawa,Tetsuji Itoh

doi:10.3389/fmats.2016.00007

Abstract

The present work reports the preparation of binderless carbon-coated porous films and the study of their performance as monolithic bioanodes. The films were prepared by coating anodic aluminum oxide (AAO) films with a thin layer of nitrogen-doped carbon by chemical vapor deposition. The films have cylindrical straight pores with controllable diameter and length. These monolithic films were used directly as bioelectrodes by loading the films with D-fructose dehydrogenase (FDH), an oxidoreductase enzyme that catalyzes the oxidation of D-fructose to 5-keto-D-fructose. The immobilization of the enzymes was carried out by physical adsorption in liquid phase and with an electrostatic attraction method. The latter method takes advantage of the fact that FDH is negatively charged during the catalytic oxidation of fructose. Thus the immobilization was performed under the application of a positive voltage to the CAAO film in a FDH-fructose solution in McIlvaine buffer (pH 5) at 25 oC. As a result, the FDH modified electrodes with the latter method show much better electrochemical response than that with the conventional physical adsorption method. Due to the singular porous structure of the monolithic films, which consists of an array of straight and parallel nanochannels, it is possible to rule out the effect of the diffusion of the D-fructose into the pores. Thus the improvement in the performance upon using the electrostatic attraction method can be ascribed not only to a higher uptake, but also to a more appropriate molecule orientation of the enzyme units on the surface of the electrodes.

Highlights

Electrodes modified with redox enzymes have been used as quick responsive biosensors with high accuracy and selectivity of a specific substrate and, more recently, as electrodes in enzymatic biofuel cells, i.e., a particular type of biofuel cells in which different oxidoreductase enzymes work as electrocatalysts
The pore diameter distributions of the films were determined with the Scanning electron microscopy (SEM) micrographs and thereby main pore sizes are found to be about 40 and 240 nm for the coated AAO (CAAO) and CAAO-P films, respectively
The CAAO film was loaded with fructose dehydrogenase (FDH) enzyme with the two different methods

Summary

Introduction

Electrodes modified with redox enzymes have been used as quick responsive biosensors with high accuracy and selectivity of a specific substrate and, more recently, as electrodes in enzymatic biofuel cells, i.e., a particular type of biofuel cells in which different oxidoreductase enzymes work as electrocatalysts. Because redox sites are deeply embedded into the framework structure of the enzymes, the electron transfer has to be helped by mediators, such as redox compounds or redox polymers, which act as relays of electrons between the enzymes and the electrodes. This mediated electron transfer (MET) type of electrode requires the use of complicated processes, e.g., in the case of the enzymatic biofuel cells, compartmentalized cells are needed to avoid the crossover of the mediators between the anode and the cathode compartments. The FDH-modified electrodes are, very convenient bioanodes for fructose detection in electrochemical biosensors and biofuel cells

Methods

Results

Conclusion