Direct electrochemistry of glucose oxidase assembled on graphene and application to glucose detection

Abstract

The direct electrochemistry of glucose oxidase (GOx) integrated with graphene was investigated. The voltammetric results indicated that GOx assembled on graphene retained its native structure and bioactivity, exhibited a surface-confined process, and underwent effective direct electron transfer (DET) reaction with an apparent rate constant (k_s) of 2.68 s⁻¹. This work also developed a novel approach for glucose detection based on the electrocatalytic reduction of oxygen at the GOx–graphene/GC electrode. The assembled GOx could electrocatalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction current decreased, which could be used for glucose detection with a high sensitivity (ca. 110 ± 3 μA mM⁻¹ cm⁻²), a wide linear range (0.1–10 mM), and a low detection limit (10 ± 2 μM). The developed approach can efficiently exclude the interference of commonly coexisting electroactive species due to the use of a low detection potential (−470 mV, versus SCE). Therefore, this study has not only successfully achieved DET reaction of GOx assembled on graphene, but also established a novel approach for glucose detection and provided a general route for fabricating graphene-based biosensing platform via assembling enzymes/proteins on graphene surface.

Introduction

Carbon materials have been widely used in both analytical and industrial electrochemistry because of their chemical inertness, their low residual current, their excellent conductivity, their wide potential window, and their electrocatalytic activity to a variety of redox reactions. Several forms of carbon materials such as graphite [1], [2], highly ordered mesoporous carbon [3], carbon nanofiber [4], carbon nanotube [5], [6], [7], [8], etc. have been studied to immobilize redox enzymes and used for developing enzyme-based electrochemical devices. Recently, a new form of large surface-to-volume ratio carbon material, graphene, which is a flat monolayer of carbon atoms tightly packed into a two-dimensional (2D) honeycomb lattice [9], has received a considerable attention. One of the factors makes graphene so attractive is its low energy dynamics of electrons with atomic thickness [10]. It is a semiconductor with zero band gap and high carrier mobility and concentration and shows nearly ballistic transport at room temperature [11], [12]. These unusually electronic properties make graphene one of the most promising candidate materials for future nanoelectronic applications, including graphene-based field effect transistors [13], gas sensors [14], nanoelectromechanical switch [15], supercapacitors [16], lithium secondary batteries [17], and so forth. Of particular interest for us is to explore its application in the field of electrochemical research [18], [19]. To fully exploit the electrochemical properties of graphene, it is important to understand the electrochemical characteristics of the graphene surface, including adsorption, the electron transfer (ET) kinetics of the redox system, and electrocatalysis. Several groups have demonstrated that graphene sheets show fast ET kinetics and excellent electrocatalytic characteristics compared with graphite and glassy carbon (GC) [18], [20], [21], [22], [23]. More recently, it has been found that graphene oxide can facilitate direct electron transfer (DET) of metalloproteins including cytochrome c, myoglobin, and horseradish peroxidase [24], and an ionic liquid, Nafion or chitosan modified graphene can support the DET of glucose oxidase (GOx) [25], [26], [27]. However, the reported procedures of immobilizing GOx were relatively complicated.

The unique properties of graphene may provide insight to fabricate novel biosensors for virtual applications. The high surface area is helpful in increasing the surface loading of the target enzyme molecules on the surface. The excellent conductivity and small band gap are favorable for conducting electrons from the biomolecule. Here, we describe the fabrication and characterization of a novel hybrid (GOx–graphene) in which glucose oxidase (GOx) is directly assembled onto the unmodified graphene surface. GOx is chosen as a model for its stability, its ability to be immobilized on the electrode surface, and its electrical communication with the electrode surface [28], [29]. The results demonstrated that GOx immobilized on the surface of graphene could undergo the effective DET reaction and the hybrid exhibited good electrocatalytic activity toward the reduction of oxygen. Moreover, the concentration of glucose can be determined quantitatively based on the reduction of O₂ catalyzed by the GOx–graphene hybrid. In comparison with the previous report [27], although DET characteristics of GOx obtained in presented work are practically the same as that obtained by Kang et al. [27] at the GOx–graphene–chitosan/GC electrode, this work simplifies the electrode fabrication procedures significantly by directly dispersing graphene into water. However, Kang et al. dispersed graphene in chitosan solution, therefore complicating the electrode fabrication procedures. More importantly, the analytical performances of the GOx–graphene/GC electrode are much better that those of GOx–graphene–chitosan/GC electrode regarding the sensitivity (110 ± 3 μA mM⁻¹ cm⁻² versus 37.93 μA mM⁻¹ cm⁻²) and the detection limit (10 ± 2 μM versus 20 μM). Therefore, this study expands the scope of graphene applications to the field of bioelectroanalytical chemistry, which may open up a new challenge and approach to explore the electrochemical features of graphene or its hybrid materials for the potential utilizations. Our goal is not only to design a novel biosensing platform but also to present a new approach for preparation of a graphene-based hybrid, which has potential utility to bioelectroanalytical chemistry.