Direct electron transfer of horseradish peroxidase and its biosensor based on chitosan and room temperature ionic liquid

https://doi.org/10.1016/j.elecom.2006.03.026Get rights and content

Abstract

A reagentless horseradish peroxidase (HRP) biosensor, which is based on the direct electron transfer between the enzyme and the electrode, has been shown by direct electrochemistry of HRP and direct bioelectrocatalysis towards H2O2. Composite material based on biocompatible chitosan (Chi) and room temperature ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate (BMIM · BF4) was used to construct the HRP biosensor. A pair of stable and well-defined quasi-reversible redox peaks of HRP for the HRP(Fe(III))/HRP(Fe(II)) redox couple with a formal potential of about −0.34 V (vs. Ag/AgCl) in a pH 7.0 phosphate buffer solution (PBS) were observed at the Chi-BMIM · BF4-HRP composite film modified glassy carbon (GC) electrode. The biosensor exhibited good sensitivity and reproducibility, wide linear range, low detection limit and excellent long-term stability. The Chi-BMIM · BF4-HRP film was also characterized by UV–visible spectroscopy, indicating that HRP in the composite film could retain its native structure. Both biocompatibility of chitosan and inherent conductivity of BMIM · BF4 enable the composite material to become an excellent biosensing platform for realizing direct electrochemistry and electrocatalysis of HRP along with good stability.

Introduction

Direct electrochemistry of metalloenzymes is of immense interest both for fundamental studies of electron transfer in proteins and for the development of highly selective bioelectrocatalyst and biosensors [1]. Horseradish peroxidase (HRP), which can catalyze the oxidation of a variety of substrates by hydrogen peroxide or related compounds, is one of the most commonly used metalloenzymes for the construction of electrochemical biosensors [2]. The protein contains heme as active site; in the resting state, the heme-iron oxidation state is Fe(III). The basic catalytic mechanism of HRP is carried out through the rapid reaction with hydrogen peroxide to give a two-equivalent oxidized form, called compound I (an oxidized form of HRP and consists of oxyferryl iron (Fe4+=O) and a porphyrin π-cation radical); the rapid reaction of compound I with the substrate then regenerates the Fe(III) ground state form via an intermediate called compound II (contains an additional proton compared to the native form and compound I) [3], [4]. Although HRP can directly catalyze the electrochemical reduction of H2O2, direct electrochemistry of HRP itself is rarely observed, possibly due to their deeply located redox centers [5], [6]. Since, for most redox enzymes, direct electron transfer is a very inefficient process, mediated electrochemical biosensors are typically used [7], [8], [9], [10]. Because of the well-known limitation (known as “oxygen concentration dependence”) of the first-generation biosensor (based on natural mediator, i.e., oxygen), the second-generation biosensors (based on artificial redox-mediators, e.g., ferrocene and ferricyanide) have been introduced. However, the problems such as potential leaching and toxicity of mediators restrict the further development of the second-generation biosensor [11]. The third-generation biosensor, which is based on the direct electron transfer between the enzyme and the electrode, is certainly desirable for avoiding the limitations and complications of mediated biosensor.

Room temperature ionic liquids (RTILs) are compounds consisting entirely of ions that exist in liquid state around room temperature [12]. As novel attractive solvents, they possess unique properties such as negligible vapor pressure, wide potential windows, high thermal stability and viscosity, good conductivity, and solubility [13]. Several groups have reported increased thermal stability and activity of enzymes (HRP and papain) in aqueous mixture of BMIM · BF4 ionic liquids as compared with conventional organic solvents or aqueous buffer solution [14], [15]. Another interesting application is to incorporate ionic liquids into conventional matrices, such as cellulose [16], carbon materials [17], [18] and sol–gel-based silica matrices [19]. The combination of RTIL with conventional matrices can created unique materials that might open up new opportunities for the development of biosensors, biocatalysis, and bioelectronics. However, the combinations of RTILs with these conventional matrices are either a complicated and time-consuming process or lack of good biocompatibility, which limit their widely applications.

In the present work, we constructed a mediator-free biosensor by simply entrapping HRP into a novel chitosan/BMIM · BF4 composite. Chitosan (pKa = 6.3) is a linear hydrophilic polysaccharide composed of β(1  4) linked glucosamine units together with some proportion of N-acetylglucosamine units. It is a biocompatible, biodegradable, and nontoxic natural biopolymer that exhibits excellent film-forming ability [20]. BMIM · BF4 is a hydrophilic RTIL that has been widely used in biocatalysis [21]. Due to strong charge–charge interaction associated with Van der Waals forces between solvent molecules, BMIM · BF4 has an inherent high viscosity similar to ethylene glycol (19.6 cP at 25 °C vs. 16 cP for ethylene glycol at 25 °C) [21]. BMIM · BF4 can mix well with chitosan aqueous solution to form a homogenous solution, which can be readily immobilized onto the GC electrode surface and form a film after it is being dried. The resulting polymer/RTIL composite film can provide a favorable microenvironment for fabricating the third-generation HRP biosensor, in which BMIM · BF4 (0.17 S m−1 at 25 °C) can facilitate the electron transfer due to its intrinsic good conductivity. Both biocompatibility of chitosan and inherent conductivity of BMIM · BF4 enable the composite material to become an excellent biosensing platform for realizing direct electrochemistry and electrocatalysis of HRP along with good stability.

Section snippets

Materials

Chitosan (from Crab Shells, minimum 85% deacetylated) and HRP (285 U g−1) were purchased from Sigma. BMIM · BF4 (>98%, from Solvent Innovation, Cologne, Germany) was purified according to Ref. [22]. 6 mg ml−1 chitosan aqueous solution (pH 5.0) was prepared mainly according to the previously reported procedure [23]. Briefly, chitosan was dissolved in 0.05 M HCl aqueous solution, and the pH of the chitosan solution was adjusted to 5.0 using a 1.0 M NaOH solution. Then, the chitosan solution was filtered

Characterization by UV–vis absorption spectra

HRP entraped in the composite film was firstly investigated by spectroscopic analysis. UV–vis spectroscopy is an effective means to probe into the characteristic structure of proteins [4]. The position of Soret absorption band of heme may provide information about possible denaturation of heme proteins [24]. As can been seen from Fig. 1, HRP entrapped in the composite film (curve b in Fig. 1) has a characteristic Soret absorption band at 403 nm, same as that of native HRP in pH 7.0, PBS (curve a

Conclusions

An excellent HRP biosensor, which is based on the direct electron transfer between the enzyme and the electrode, has been shown by direct electrochemistry of HRP and direct bioelectrocatalysis towards H2O2. The prepared biosensor exhibited good sensitivity and reproducibility, wide linear range, low detection limit, and good long-term stability. The simple method by entrapping enzyme into biocompatible chitosan and RTIL (BMIM · BF4) composite, provided an effective means for realizing direct

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 20125513, No. 20575032), Specialized Research Fund for the Doctoral Program of Higher Education (No. 20050003035).

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