Elsevier

Biosensors and Bioelectronics

Volume 25, Issue 4, 15 December 2009, Pages 901-905
Biosensors and Bioelectronics

Glucose Oxidase–graphene–chitosan modified electrode for direct electrochemistry and glucose sensing

https://doi.org/10.1016/j.bios.2009.09.004Get rights and content

Abstract

Direct electrochemistry of a glucose oxidase (GOD)–graphene–chitosan nanocomposite was studied. The immobilized enzyme retains its bioactivity, exhibits a surface confined, reversible two-proton and two-electron transfer reaction, and has good stability, activity and a fast heterogeneous electron transfer rate with the rate constant (ks) of 2.83 s−1. A much higher enzyme loading (1.12 × 10−9 mol/cm2) is obtained as compared to the bare glass carbon surface. This GOD–graphene–chitosan nanocomposite film can be used for sensitive detection of glucose. The biosensor exhibits a wider linearity range from 0.08 mM to 12 mM glucose with a detection limit of 0.02 mM and much higher sensitivity (37.93 μA mM−1 cm−2) as compared with other nanostructured supports. The excellent performance of the biosensor is attributed to large surface-to-volume ratio and high conductivity of graphene, and good biocompatibility of chitosan, which enhances the enzyme absorption and promotes direct electron transfer between redox enzymes and the surface of electrodes.

Introduction

Electron transfer in biological systems is a very important phenomenon for the areas of biochemical and biophysical sciences. Direct electron transfer (DET) between redox enzymes and the surface of electrodes can be used to investigate the enzyme-catalyzed reactions in biological systems and to lay the electrochemical basis for the study of the structure of enzymes, kinetics and thermodynamics of redox transformations of enzyme molecules, and metabolic processes involving redox transformations (Gooding et al., 2003, Pulcu et al., 2007, Wang et al., 2002). Great effort has been made to develop new mediator-free (or reagentless) biosensors, enzymatic bioreactors, and biomedical devices based on DET by immobilizing enzymes on conducting substrates (Guiseppi-Elie et al., 2002, Nadzhafova et al., 2007). However, the redox center in biomolecules is usually embedded deeply into the large three-dimensional structure of enzyme molecules (Andreu et al., 2007, Zhao et al., 2008a). Controlling the interactions of enzymes with the substrate to optimize the electron transfer processes remains a challenge. Many methods and materials, including biopolymers (Zhao et al., 2008a, Zhou et al., 2008, Shan et al., 2007), nanostructures (Jia et al., 2002, Liu et al., 2007, Luo et al., 2006, Nadzhafova et al., 2007) and sol–gel matrices (Jia et al., 2002, Zhang et al., 2007), have been studied to immobilize enzymes and promote electron transfer of redox enzymes on the surface of electrodes.

Recently, a new class of large surface-to-volume ratio, high conductivity carbon material, graphene, has attracted increasing attention for optoelectronic devices (Wang et al., 2008), super-capacitors (Vivekchand et al., 2008), gas sensors (Ao et al., 2008, Leenaerts et al., 2008, Schedin et al., 2007), pH sensor (Ang et al., 2008), chemical sensor (Wang et al., 2009a, Wang et al., 2009b), biosensor (Shan et al., 2009) and nanocomposite (Li et al., 2008, Stankovich et al., 2006, Xu et al., 2008a, Xu et al., 2008b) applications. Graphene is made of monolayers of two-dimensional honeycomb graphite type carbon (Geim and Novoselov, 2007, Novoselov et al., 2004). This unique nanostructure material has high surface area, excellent electrical conductivity and electron mobility at room temperature, robust mechanical properties, and flexibility (Stankovich et al., 2006). The special 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 biomolecules (Stankovich et al., 2006). Graphene-based chemical sensors can also have a much higher sensitivity because of the low electronic noise from thermal effect (Ao et al., 2008, Peres et al., 2006). Furthermore, compared with CNTs, graphene can be obtained easily by chemical conversion of the inexpensive graphite (Xu et al., 2008a).

The successful dispersion of graphene has enabled the construction of various potentially useful graphene-based biosensors. Chemically functionalized graphene can be readily mixed with polymers in solution to form a stable dispersion and yield novel types of electrically conductive nanocomposites (Li and Kaner, 2008, Niyogi et al., 2006, Stankovich et al., 2006, Schniepp et al., 2006, Xu et al., 2008b). Graphene-based polymer nanocomposites display extraordinarily small electrical percolation threshold due to large conductivity and aspect ratio of the graphene sheets (Eda and Chhowalla, 2009, Liu et al., 2008a). Chitosan, a natural-biopolymer with unique structure features, possesses the primary amine at the C-2 position of the glucosamine residues and is soluble in aqueous acidic media at pH < 6.5. When dissolved and carried with the positive charge of –NH3+ groups, the chitosan can adhere to negatively charged surfaces or adsorb negatively charged materials. It is commonly used to disperse nanomaterials and immobilize enzymes for constructing biosensors due to its excellent capability for film formation, nontoxicity, biocompatibility, mechanical strength, and good water permeability. Chitosan can provide a good biocompatible microenvironment for proteins or enzyme (Kang et al., 2007, Yi et al., 2005, Zhang et al., 2004).

In this paper, the hybrid nanocomposite of graphene–chitosan was prepared and modified on the surface of glassy carbon electrode (GCE), and then GOD was absorbed on the nanocomposite film. The film was characterized with scanning electron microscopy and electrochemical methods. It was found that the nanocomposite film can provide a favorable microenvironment for GOD to realize DET. The GOD–Graphene–chitosan nanocomposite film can be used for glucose sensing and exhibit great sensitivity as compared with widely investigated carbon nanotubes-based ones. It opens up a new avenue for fabricating excellent electrochemical biosensors.

Section snippets

Reagents and apparatus

Phosphate buffer saline (PBS 0.05 M, pH 7.4) with 0.1 M KCl was used as the supporting electrolyte. Natural flake graphite, sized at 45 μm, was kindly provided by Asbury Carbons (Asbury, NJ). Sulfuric acid (95%), potassium chlorate (98%), hydrochloric acid (37%), GOD (EC 1.1.3.4, Type X-S, 40,300 U/g), D-glucose, and chitosan were purchased from Sigma–Aldrich. The stock GOD solution was prepared in the PBS buffer and stored at 4 °C. A stock solution of D-glucose (0.1 M) was prepared and allowed to

Dispersion of graphene by chitosan

When used as nanofiller into the chitosan matrix, similar to other polymers (Schniepp et al., 2006, Stankovich et al., 2006), graphene sheets may be performed for outstanding thermal, mechanical, and electrical properties. Here, a suspension containing graphene and chitosan was sonicated over 1 h. The graphene is well dispersed in the aqueous chitosan solution, forming a stable and dark suspension with only a small amount of graphene precipitated after 24 h. Fig. 1 is a SEM image of the

Conclusion

We have studied the electrochemical behavior of GOD at a graphene–chitosan modified electrode and demonstrated the direct electron transfer reaction of GOD at the modified electrode. The results indicate that the graphene can provide a favorable microenvironment for the enzyme and promote the direct electron transfer at the electrode surface. Chitosan also plays an important role in forming a well-dispersed graphene suspension and immobilizing the enzyme molecules. This graphene-based enzyme

Acknowledgments

This work was supported by a laboratory-directed research and development program (LDRD) at Pacific Northwest National Laboratory (PNNL). The work was performed at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy (DOE) and located at PNNL. PNNL is operated by Battelle for DOE under Contract DE-AC05-76RL01830. IAA acknowledges support from ARO/MURI under grant number W911NF-04-1-0170.

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