Enzymatically synthesized polyaniline layer for extension of linear detection region of amperometric glucose biosensor

Abstract

In this article a new method for fabrication of enzymatic electrodes suitable for design of amperometric glucose biosensor and/or anode of biofuel cell powered by glucose is presented. Glucose oxidase (GOx) E.C. 1.1.3.4. from Penicillium vitale was immobilized on the carbon rod electrode by cross-linking it with glutaraldehyde (GOx-electrode). Catalytic activity of immobilized GOx was exploited for polymerisation of aniline by taking a high concentration of hydrogen peroxide produced during the catalytic action of immobilized GOx and locally lowered pH due to the formation of gluconic acid; it created optimal conditions for the polymerisation of aniline. The GOx layer was self-encapsulated within formed polyaniline (PANI) matrix (GOx/PANI-electrode). Properties of the GOx/PANI-electrode have been studied and results were compared with GOx-electrode. The results show that the upper detection limit of glucose using GOx-electrode was dramatically changed by the formation of PANI layer. An increase in the upper detection limit, optimal pH region for operation and stability of GOx based electrode modified by PANI was detected when comparing that of an unmodified GOx-electrode.

Introduction

Since the development of the first glucose biosensor (Clark and Lyons, 1962) vast numbers of research studies have been devoted for fabrication of enzyme electrodes reliable for generation of bioelectricity (Ramanavicius and Ramanaviciene, 2009) and/or measurement of glucose concentrations (Kakehi et al., 2007). Recently biosensors played an important role in the improvement of public health, food and beverage industry, environmental monitoring. This is all due to its rapid detection, high sensitivity, small size, and specificity. Among other biosensors, amperometric biosensors are prevalent because of their high selectivity and simple fabrication methods (Ramanavicius et al., 2004). Many advantages and new possibilities to detect biologically active compounds provide amperometric biosensors based on conducting polymers (Ramanavicius et al., 2005, Rahman et al., 2008, Ekanayake et al., 2008), because some of these polymers offer good biocompatibility in vivo (Ramanaviciene et al., 2007) and can be served as versatile immobilization matrix (Ramanaviciene and Ramanavicius, 2004). Proper immobilization of enzymes is very important procedure in biosensor design. Researchers constantly try to improve the sensitivity, stability, and the range of determinable concentrations and reproducibility of glucose biosensors by different techniques of immobilization of an enzyme (glucose oxidase (GOx) or glucose hydrogenase) on suitable electrodes with supportive materials (Kaimori et al., 2006). The immobilization of enzymes can be carried out using many different procedures such as physical adsorption, entrapment, chemical cross-linking and other methods, while retaining the biological recognition properties of enzymes. Characteristics of immobilized enzyme based electrodes depend on factors such as the immobilization method, thickness and stability of the layer or membrane formed during immobilization (Malhotra et al., 2005). Successfully immobilized enzymes have many operational advantages over free enzymes such as continuous operational mode, possible modulation of the catalytic properties and easier prevention of microbial growth, reduced cost of operation, reusability, enhanced stability and extended range of determinable concentrations.

Conducting polymers (CPs) are especially suitable for immobilization of various enzymes (Ramanavicius et al., 2006, Davis and Higson, 2007). The most widely used CPs for enzyme immobilization is polyaniline (PANI), polypyrrole and polythiophene. In comparison to other enzyme immobilization matrices, CPs have attracted attention due to their ability to bind oppositely charged complex entities in their oxidized conducting state and to release them in their neutral insulating state (Gaikwad et al., 2006). They provide stable and porous matrix for the immobilization and also facilitate the electron transfer process (Gaikwad et al., 2007). Out of many other conducting polymers, PANI is the well-known synthetic organic polymer firstly reported by Letheby (1862). PANI becomes very attractive due to its high conductivity (Kang et al., 2004), good environmental (MacDiarmid et al., 2001), thermal (Wang et al., 1995), electrochemical stability (Chiang and MacDiarmid, 1986), interesting electrochemical, electronic, optical and electro-optical properties (Heeger, 2001), and strong biomolecular interactions. Furthermore, due to its excellent conductivity and electroactivity, PANI can act as a redox mediator for enzyme-modified electrodes. It can accept electrons directly from the enzyme active site and transfer them to the electrode (Luo and Do, 2004, Shi et al., 2004, Luo et al., 2006, Michira et al., 2007). PANI is compatible to most enzymes and can be easily synthesized from aniline monomer in an aqueous solution. Because of the mentioned advantages PANI has found application as immobilization matrix in the design of conductometric (Ajay and Srivastava, 2007), potentiometric (Qaisar and Adeloju, 2009) and amperometric (Xu et al., 2009) biosensors. In the designing of amperometric biosensors PANI has been used as a matrix for covalent enzyme immobilization (Singh et al., 2006). Amperometric enzyme biosensors based on PANI nanoparticles have also been reported (Morrin et al., 2005). However, one of the simplest and the most widely used methods for immobilization of enzymes at the electrode surface is entrapment into PANI or other CPs films. Such entrapment provides a possibility to improve selectivity by preventing the active parts of the biosensors from disruptive materials and biocompatibility of biosensors prevents the enzyme from being leached out. At the same time it maintains the accessibility of the catalytic sites due to the permeability of the film to analytes (Ho et al., 2000). Moreover, after enzyme immobilization within conducting polymers due to increasing diffusion limitations such parameter as apparent Michaelis constant (K_M(apparent)) can be changed significantly thus causing an increase in the upper detection limit of the created biosensors and an extension of determinable concentration range (Persson et al., 1993, Baronas et al., 2003, Ramanavicius et al., 2008) or even reduction of influence of enzyme inactivation (Baronas et al., 2010). Similar diffusion-based effects of conducting (Gorton, 1995) and non-conducting (Somasundrum and Aoki, 2002) matrixes used in amperometric biosensors were reported.

Traditionally, PANI is synthesized via electrochemical and chemical methods (Huang et al., 1986). The electrochemical polymerisation usually yields a thin polymeric film at the electrode surface; while by chemical polymerisation the polymer is obtained as an amorphous suspension precipitated in bulk of the solution. During the electrochemical PANI polymerisation process negatively charged enzyme molecules could be easily entrapped into the positively charged backbone of the polymer (Skinner and Hall, 1997, Trojanowicz et al., 1995). The advantages of this method are: one-step, direct, easy and controlled localization of biologically active molecules in a defined area on the electrode surface, control of the film thickness and redox conductivity (Ahuja et al., 2007, Cosnier, 2003). However, the electrochemical PANI polymerisation has drawbacks as a necessity to use low-pH acidic aqueous solutions, high concentration of monomer and biomolecules (Cosnier, 2003). The PANI polymerisation drawbacks are: (i) extremely low-pH of polymerisation solution, (ii) application of toxic catalysts and/or oxidants, (iii) and formation of undesirable by-products. These limitations significantly reduce the applicability of PANI based systems in the design of amperometric biosensor.

An alternative approach for enzyme entrapment into electrochemical synthesised PANI films might be enzymatic PANI polymerisation (Cruz-Silva et al., 2004). This simple, one-step and environmentally friendly process does not require strong acidic media, it is free of oxidation by-products and uses catalysts derived from renewable resources (Xu et al., 2006). Studies of the enzymatic polymerisation of aniline using horseradish peroxidase (Nabid and Entezami, 2005), laccase (Karamyshev et al., 2003) or palm tree peroxidase (Sakharov et al., 2004) have been reported. Our previous studies demonstrated that glucose oxidase (GOx) dissolved in polymerisation bulk might be encapsulated within PANI by formation of colloid-particles (GOx/PANI-nanoparticles), which exhibits significantly different catalysed reaction kinetics if compared with the native enzyme (Kausaite et al., 2009). Therefore GOx/PANI-nanoparticles can be applied in the design of amperometric biosensors that allows the detection of glucose in a wider concentration interval when compared with biosensors based on native GOx immobilized in the similar way (Ramanavicius et al., 2005, Ramanavicius et al., 2008). These studies encouraged us to apply this GOx self-encapsulation into the PANI matrix method for increasing the glucose detection limits.

Thus the main aim of this study was to modify cross-linked GOx based graphite electrode by self-encapsulation of GOx within the formed PANI layer in order to show a possibility to increase the upper detection limit. Along with this an optimal pH region for operation and stability of created biosensor were investigated.