Elsevier

Electrochimica Acta

Volume 55, Issue 22, 1 September 2010, Pages 6611-6616
Electrochimica Acta

Immobilization of laccase onto 1-aminopyrene functionalized carbon nanotubes and their electrocatalytic activity for oxygen reduction

https://doi.org/10.1016/j.electacta.2010.06.013Get rights and content

Abstract

Carbon nanotubes (CNTs) were non-covalent-functionalized with 1-aminopyrene (1-AP) and used for the first time to immobilize laccase (Lac) with the aid of glutaraldehyde (GA). The results of Fourier transform infrared (FTIR) spectra confirmed the successful modification of CNTs with 1-AP. The dispersibility of CNTs in aqueous solution was improved by the functionalization of 1-AP. The electrocatalytic properties of the Lac immobilized on the 1-AP functionalized CNTs (Lac/AP–CNTs) for oxygen reduction have been investigated by cyclic voltammetry in the presence of 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) in the Britton–Robinson (B–R) buffer solution (pH 3.0). Under the same experimental condition, the Lac/AP–CNTs catalyst shows higher electrocatalytic activity and better stability than the Lac immobilized on the pristine CNTs (Lac/CNTs). Additionally, effects of the mass ratio of 1-AP to CNTs in the AP–CNTs composites, the loading mass of the Lac/AP–CNTs catalyst and the pH value of the electrolyte on the electrocatalytic activity of the Lac/AP–CNTs/glassy carbon electrode for oxygen reduction were also optimized.

Introduction

Fuel cells are promising devices for converting chemical energy into electrical energy because of their high-energy conversion efficiency and environmental affinity. Oxygen, either pure or as a component of air, is the common fuel used in the cathode compartment of fuel cells because it is readily available and good oxidant, and its reduction product (water) is harmless. Electrochemical reduction of oxygen to water has been demonstrated with metallic catalysts [1], [2], [3], [4], [5], [6], [7] or biocatalysts [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. Biocatalysts such as microorganisms or enzymes with high selectivity and catalytic efficiency have been extensively investigated for the construction of biofuel cells. Among them, laccase (Lac) as a well known multicopper oxidase, which can reduce oxygen into water in a four-electron transfer step without intermediate formation of hydrogen peroxide, has been widely applied for the reduction of oxygen at the cathode of the enzyme-based biofuel cells [20], [21], [22], [23], [24], [25], [26], [27], [28], [29]. Recently, enzyme-based biofuel cells have attracted considerable research attention because of their unique advantages such as corrosion-free electrolyte, low cost, good biocompatibility and more moderate temperature [29].

In the case of enzyme-based biofuel cells, the major barriers to their practical application are short lifetime and low power density [30], both of which are related to enzyme immobilization. Therefore, immobilization of enzyme on the suitable electrochemical materials plays a crucial role in the construction of biofuel cells. Carbon nanotubes (CNTs), the “star” material of carbon, exhibit unique electronic properties, high chemical stability and good biocompatibility, making them as the promising candidates for enzyme immobilization in biofuel cells. It has been reported that Lac was immobilized on the CNT-modified electrode by physical adsorption and showed good electrocatalytic activity for oxygen reduction with ABTS as the mediator [31]. However, the electrochemical stability of the electrode should be improved because the adsorbed Lac would leak easily from the CNT-modified electrode.

Recently, non-covalent functionalization of CNTs, especially based on π-stacking interaction, has been paid increasing attention, which could not only enhance the dispersibility of CNTs but also maintain their attractive electronic and mechanical properties [32]. As reported previously, several kinds of organic compounds with π-conjugative structure, such as methylene blue [33] and 1-pyrenebutanoic acid succinimidyl ester [34], [35], could interact with CNTs through π-stacking interaction to introduce the functional groups on the CNTs uniformly and have been used to immobilize biomolecules (e.g., ferritin and horseradish peroxidase).

1-Aminopyrene (1-AP) is a bifunctional molecule with a pyrenyl group and an amino functional group. The pyrenyl group of 1-AP, being highly aromatic in nature, can interact strongly with the sidewalls of CNTs via π-stacking. Recently, 1-AP functionalized CNTs (AP–CNTs) were used successfully as the support to disperse PtRu and Pt nanoparticles for methanol electrooxidation with the assistance of the amino functional groups [36], [37]. It is well known that the amino functional group can be used to immobilize enzyme by a typical glutaraldehyde (GA) cross-linking reaction. By functionalization of CNTs with 1-AP, lots of amino functional groups can be introduced uniformly on the CNT surface and used to immobilize enzymes to construct biofuel cells. However, to our best knowledge, there are no studies on the immobilization of enzymes using 1-AP functionalized CNTs. In this paper, Lac was selected as the model due to its important applications in biosensors and biofuel cells, the non-covalent functionalization of CNTs with 1-AP and the immobilization of Lac on the AP–CNTs by GA cross-linking were investigated. Furthermore, the electrocatalytic properties of Lac immobilized on AP–CNTs (Lac/AP–CNTs) for oxygen reduction and the related influence factors have been evaluated and optimized.

Section snippets

Reagents and materials

Lac from Trametes versicolor (23.1 U/mg) and ABTS were purchased from Sigma–Aidrich. 1-AP was purchased from Alfa Aesar. CNTs (diameter, 20–40 nm) with multi-walls were purchased from Shenzhen Nanotech Port Co. Glutaraldehyde (GA) and other reagents were of analytical grade. Acetate buffer solution (pH 4.5) was 0.1 M HAc-NaAc aqueous solution. Britton–Robinson (B–R) buffer solutions with a series of pH values were prepared with equimolar amounts (0.04 M) of boric, acetic, and phosphoric acids,

Characterization of AP–CNTs

Fig. 1 shows the FTIR spectrum of AP–CNTs in the range of 400–4000 cm−1. For comparison, the FTIR spectrum of pure 1-AP is also present in Fig. 1. As shown in Fig. 1, the spectrum of AP–CNTs is nearly the same as that obtained from the pure 1-AP. The results confirm the successful functionalization of CNTs with 1-AP via the π–π interaction between the pyrenyl groups of 1-AP and the six-membered rings of the sidewalls of CNTs. Thus, the –NH2 group should be distributed uniformly on the surface of

Conclusions

1-AP functionalized CNTs were prepared and used for the first time to immobilize Lac in this paper. The electrochemical behavior of Lac at the Lac/AP–CNTs/GC electrode for oxygen reduction has been investigated by cyclic voltammetry. The functionalization of CNTs with 1-AP not only can improve the dispersibility of CNTs in aqueous solution, but also is helpful for the enzyme immobilization. Comparing with the Lac/CNTs/GC electrode, the Lac/AP–CNTs/GC electrode shows better electrochemical

Acknowledgements

This work was financially supported by NSFC (20975033, 20905024), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) and the National Basic Research Program of China (2009CB421601).

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