Functionalized carbon nanotube-bienzyme biocomposite for amperometric sensing

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Functionalized carbon nanotube-bienzyme biocomposite for amperometric sensing D.R. Shobha Jeykumari, S. Sriman Narayanan* Department of Analytical Chemistry, School of Chemical Sciences, University of Madras, Guindy Campus, Chennai 600 025, India

A R T I C L E I N F O

A B S T R A C T

Article history:

An approach to design a biocomposite bienzyme biosensor with the aim of evaluating its

Received 4 June 2008

suitability as an amperometric sensor using functionalized multiwalled carbon nanotubes

Accepted 23 November 2008

(MWCNTs) is presented. The biosensor is based on a bienzyme-channelling configuration,

Available online 10 December 2008

employing the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP), which were immobilized with toluidine blue (TB) functionalized MWCNTs. The proposed method demonstrates an easy electron transfer between the immobilized enzymes and the electrode via functionalized MWCNTs in a Nafion matrix. Co-immobilization of GOx and HRP was employed to establish the feasibility of fabricating highly effective bienzymebased biosensors for low-level glucose determination. Bienzyme immobilized TB functionalized MWCNTs were attached to a glassy carbon electrode, and the electrochemical behavior of the sensor was studied using electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry. The excellent electrocatalytic activity of the biocomposite film resulted in the detection of glucose under reduced over potential with a wider range of determination from 1.5 · 108 M to 1.8 · 103 M and with a detection limit of 3 · 109 M. The sensor showed a short response time (within 2 s), good stability and anti-interferant ability. The proposed biosensor exhibits good analytical performance in terms of repeatability, reproducibility and shelf-life stability.  2008 Elsevier Ltd. All rights reserved.

1.

Introduction

Biosensors combine the exquisite selectivity of biology with the processing power of modern microelectronics and optoelectronics to offer new powerful analytical tools with major applications in medicine, environmental diagnostics, and the food and processing industries. The rapid progress in nanoscience and nanotechnology introduced a fast growth in the field of electrochemical biosensors during the past years [1–4]. Nano-dimensional materials, e.g. nanoparticles, nanotubes, nanofibres and nanorods, have been universally employed for the construction of electrochemical sensing and biosensing devices with favorable analytical performances. As promising building blocks for biosensing plat-

forms with high complexity and controlled structure, carbon nanotubes (CNTs) have gained special attention owing to their unique chemical, electronic and mechanical properties [5–7]. CNT can be described as a sheet of carbon atoms rolled up into a tube with a diameter of around tens of nanometers. Since their rediscovery in 1991 [8] numerous attempts have been made all over the world to develop technologies that can yield highly purified single- or multiwalled carbon nanotubes. Multiwalled carbon nanotubes (MWCNTs) are made up of several concentric cylinders of graphite sheets, with about ˚ spacing between the layers. MWCNTs usually have 3.4 A 2–100 nm diameter (typically 2–10 nm in internal diameter) compared to the single layer cylindrical graphite sheet of

* Corresponding author: Fax: +91 44 2235 2494. E-mail addresses: [email protected], [email protected] (S.S. Narayanan). 0008-6223/$ - see front matter  2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2008.11.050

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single-walled carbon nanotubes (SWCNTs) that is usually about 0.2–2 nm in diameter. The length of the CNTs can vary from micrometers to centimeters with a very high aspect ratio (length/diameter). The nanotubes usually have a high surface area to weight ratio of 300 m2/g, and most of this surface area is accessible to both electrochemistry and immobilization of biomolecules [9]. CNTs have been extensively studied for their properties and applications [10,11]. Unique electrical properties together with significant surface enlargement make them an important component in sensing applications [12,13]. CNTs have been extensively used in the design of electrochemical sensors and biosensors. Their use in these devices is based on the fact that CNTs can play dual roles. They can be used as immobilization platform for biomolecules, while at the same time they can relay the electrochemical signal acting as transducers. Kong et al. [14] were the first to build a CNT-based chemical sensor for detecting NH2 and NH3 gas. Chen et al. [15] immobilized proteins on the sidewall of CNTs through a linking molecule. Besteman et al. [16], Lin et al. [17] and Wang and Musameh [3] demonstrated the use of CNTs as biological sensors for detecting glucose. (see Table 1) The use of CNTs in biosensors is limited by the fact that their closed shell does not allow for a high degree of functionalization [18]. This is because adsorption or covalent immobilization can be achieved only at the opened end of the functionalized nanotubes. Immobilization of biomolecules on the sidewalls of CNTs primarily requires the oxidation [19] and chemical modification [20] of the nanotube structure. Formation of complexes between CNTs and different types of polymers or organic functionalization of CNTs drastically increased the solubility of CNTs. Depending on the type of bonding (covalent or noncovalent) and nature of moiety attached to or interacting with the tubes, solubility can be modulated in different solvents. As electrode materials, CNTs can be used for promoting electron-transfer between the electroactive species and the electrode and provide a novel platform for fabricating chemical sensor or biosensors. Recent studies have shown the excellent electrocatalytic activity and antifouling properties of CNTs to improve the electrochemical reactions of nicotinamide adenine dinucleotide and hydrogen peroxide at lower working potentials. [21–23]. The unique properties of the CNTs make them very promising in electro-

Table 1 – Effect of interferants on analyte (0.1 mM glucose) determination. Interferant compounds L-Cysteine L-Tyrosine L-Leusine L-Tryptophan L-Histidine L-Aspartic

acid acid Ascorbic acid Uric acid Acetaminophen

L-Glutamic

Molar ratio of anlayte: interferant compounds 1:2 1:2 1:2 1:2 1:2 1:2 1:2 1:5 1.2 1:2

Relative error (%) 1.9 1.7 1.3 1.6 1.5 1.4 1.8 1.2 1.0 1.1

chemical applications, especially in electrochemical biosensors [24]. Composite materials based on CNTs with various polymers especially Nafion (Nf) have been reported. Because of the unique ion exchange, discriminative and biocompatible properties, Nf films containing various electrocatalytic materials [25–27] have been extensively employed for the modification of the electrode surfaces and applied for the amperometric sensing in electrochemical sensors and biosensors. On the other hand, Nf as a perfluorosulfonated cation exchange polymer, is a perm-selective polymer known for its ability to incorporate positively charged ions and reject anionic species [28,29]. This cation inclusive characteristic of Nf, with some other properties such as exceptional chemical and thermal stability, makes it a preferable candidate for successful incorporation of various cationic electrocatalysts (for example, metal complexes and organic dyes) into the Nf films at the surface of modified electrodes [30,31]. It can be predicted that due to the presence of the anionic sites in the structure of Nf polymer, some analyte species with negative charge are repelled from electrode surface. Therefore, the resolution of the adjacent overlapping voltammetric peaks can be achieved [32]. Presently, we are interested in the study of the possible cooperation of two enzymes with functionalized MWCNTs to improve the performances, in particular, the selectivity of glucose oxidase (GOx) based biosensors. Horseradish peroxidase (HRP) modified electrodes coupled with various oxidases have already been proposed for biosensor to detect glucose, lactate, alcohol, choline ester and others [33–37]. In these systems, hydrogen peroxide produced by the GOx is subsequently reduced by the HRP. HRP is then reduced either through mediator or directly at the electrode at low applied potentials. Until now, most of the studies on bienzyme-modified electrodes have been performed on systems where enzymes were entrapped within polymer layers [38–40]. Direct electron transfer between conventional electrode and immobilized HRP is a slow process and mediators have been used to improve the rate of the electron transfer at low potentials. To overcome the problems arising from the presence of the free mediators in solution, sensors were designed in which the electrical communication between the active centers of the HRP and the electrode is accomplished via an oxidase–peroxidase sequence and a network of donor–acceptor relays immobilized on electrode surfaces. Organic dyes like neutral red, toluidine blue (TB), thionine, methylene blue have been used as mediators for biosensor applications [41–44]. In this paper, we report a method for the development of an amperometric biosensor to interference-free determination of glucose using bienzyme-based biosensor constructed with TB functionalized CNTs. The basic transducer employs TB, a phenothiazine dye functionalized with MWCNTs as an electrocatalyst, and HRP as a bio-electrocatalyst. The bifunctional biosensor is used as an amperometric transducer in designing a glucose biosensor with GOx as enzyme for H2O2 generation. Nf is used to disperse the bienzyme immobilized TB functionalized MWCNTs composite as a thin film is made on the glassy carbon (GC) electrode surface. By a combination of MWCNTs, TB, GOx, HRP and Nf, an electroanalytical biocomposite electrode was produced by simple solvent casting.

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TB immobilized MWCNTs acts as an efficient conduit for electron transfer, while GOx and HRP act as effective biological catalysts and Nf as the polymeric binder. A wider linear range, a lower detection limit and fast response of the MWCNT–TB– GOx–HRP–Nf modified GCE imply that the proposed method provides an excellent platform for sensitive electrochemical sensing and biosensing.

2.

Experimental

2.1.

Reagents and materials

The MWCNT sample used in this work has been prepared by the catalytic decomposition of acetylene over Ni/Cr hydrotalcite – type anionic clay catalyst and the purity is more than 95% [45]. GOx (E.C.1.1.3.4, activity 250 EU.mg1, from Aspergillus niger) and HRP (E.C.1.11.1.7, activity 90 EU.mg1) were from Sigma Chemical Co. (St. Louis, MO), and were used without further purification. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and N-hydroxy sulfo succinimide (sulfo-NHS) and TB were from Himedia. Nafion was also obtained from Aldrich as 5 wt% solution and used as 0.5% solution after dilution to using 0.1 M phosphate buffer (PB) solution (pH 7.4). All other chemicals were of analytical grade and were used as received, unless otherwise noted. The aqueous solutions were prepared with doubly distilled water. PB solutions (0.1 M) with various pHs were prepared by mixing standard solutions of K2HPO4 and KH2PO4 and adjusting the pH with 0.1 M H3PO4 or NaOH. Glucose stock solution was allowed to mutarotate at room temperature overnight before use.

2.2.

Apparatus and measurements

The morphology and microscopic structure of MWCNT and MWCNT–TB–GOx–HRP–Nf biocomposite were characterized by Transmission electron microscopy (TEM, JEOL-JEM200 FX II) and scanning electron microscopy (SEM, JEOL JSM 6360, Japan) respectively; Fourier transform infrared (FTIR) spectra were recorded with a Shimadzu FT-IR 8300 spectrophotometer. The electrochemical measurements were performed at room temperature (27 C except for experiments on temperature effects) in a conventional one compartment cell with a three-electrode configuration using a CHI 660B electrochemical workstation (CH Instruments, USA) linked to a personal computer for data acquisition and potential control. The working electrodes were MWCNT–TB–GOx– HRP–Nf biocomposite bienzyme electrode, MWCNT–GOx–Nf, MWCNT–HRP–Nf modified and unmodified GC electrodes, the auxiliary electrode was a Pt wire and the saturated calomel electrode (SCE) served as the reference electrode. All potentials were measured and quoted versus SCE. Electrochemical impedance spectra measurements were performed in a solution containing 5.0 · 103 M K3[Fe(CN)6]/ K4[Fe(CN)6] (v/v = 1:1), and 0.1 M PB solution (pH 7.4). Measurements have been recorded where the bias potential equals the formal potential of the redox probe and it was set at 0.26 V, and 5 mV amplitude voltage was applied in the frequency range of 0.1 Hz–100 kHz, and then was plotted in the form of complex plane diagrams (Nyquist plots).

2.3.

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Functionalization of multiwalled carbon nanotubes

The CNT functionalization was carried out according to our earlier reports [46,47]. MWCNTs were purified by controlled thermal oxidation in air followed by sonication in conc. HCl. The treatment with conc. HCl is to eliminate the residual metal catalyst from the nanotubes. Moreover, the acid treatment modifies the surface of MWCNTs to some extent. The CNTs were then refluxed in 12.8 M nitric acid for 12 h, washed extensively with water and then dried at 80 C overnight. MWCNTs thus obtained were water-soluble, since they were disconnected and functionalized with carboxylic acid groups during the oxidation process. The redox mediator TB was attached to the acid groups of the MWCNTs using water-soluble coupling agents namely EDAC and sulfo-NHS. The reaction was carried out by immersing the activated CNTs in a freshly prepared aqueous solution of EDAC (10 mg/ml). With stirring sulfo-NHS (300 mg) was then added to the solution [48]. The reaction was allowed to occur at room temperature for 2 h and then the CNTs were washed thoroughly with water, filtered and dried. The enzyme mixture was prepared by dissolving 4 mg of GOx and 3 mg of HRP were in 0.3 ml of PB solution (pH 7.0). Enzyme immobilization with TB-functionalized CNTs (1 mg) was achieved by mixing thoroughly with 20ll of the enzyme mixture. Due to the ability of CNTs to bind biomolecules, the two enzymes became electrostatically and hydrophobically adsorbed onto the surface of TB functionalized CNTs during mixing. The enzyme-CNTs mixture was then dispersed in 1 ml of 0.5 wt% Nf solution with the aid of ultrasonic agitation for 5 min to form a homogeneous biocomposite colloidal solution. For comparison, a bienzyme electrode with mere MWCNTs (without TB) was also prepared.

2.4.

Electrode preparation and modification

The GC electrode used in experiments was a 3 mm glassy carbon disk insulated in a 7 mm diameter Teflon rod. Before modification, the GC electrode was polished with chamois leather, and then ultrasonically cleaned in distilled ethanol and water. The bienzyme biocomposite electrodes were prepared by casting 10 lL of MWCNT–TB–GOx–HRP–Nf or MWCNT–GOx– HRP–Nf biocomposite colloidal solutions on the surface of GC electrode followed by air drying for about 2–3 h, rinsed by water several times before use. The GC electrodes coated with biocomposite films were used as the working electrodes. When not in use all the modified electrodes were stored at 4 C.

3.

Results and discussion

The CNT consists of seamlessly rolled-up graphene sheets of carbon with a highly hydrophobic surface and hence are insoluble in most solvents, especially in water. The successful production of CNT-based sensors requires their functionalization and surface immobilization. Functionalization also improves the solubility of the nanotubes. By surface modification, new properties can be incorporated to CNTs and the resulting new materials will have wider applications [19,20].

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3.1. Physical characterizations biocomposite film

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and

TEM and SEM were used for examining the morphology of the MWCNTs and biocomposite film. Fig. 1A shows the TEM image of the purified MWCNTs, They were randomly oriented and entangled network structure showing a very clean surface and almost no impurities were observed for all of the nanotubes. Dispersion of the MWCNTs in the Nf matrix is an important consideration during composite preparation. SEM image (Fig. 1B) of the MWCNT–TB–GOx–HRP–Nf biocomposite on the electrode surface was recorded. A random distribution of MWCNTs was observed and this showed that the MWCNTs were homogeneously dispersed into the Nf matrix. In this matrix, MWCNTs will act as a high conducting nanowire connecting biocomposite film domains throughout the MWCNT– Nf to the electrode surface. In the FTIR spectra (Fig. 1C), the peaks at 1729 and 3408 cm1 are due to the presence of the C@O and O–H stretching vibrations of the carboxylic groups of MWCNTs [48]. For the TB immobilized MWCNTs the C@O stretching frequencies of the amide bond formed by the functionalization reaction appeared at 1640 cm1. The bands at 2922 and 2852 cm1 can be clearly assigned to the C–H stretching vibrations of the attached mediator [47]. The successful attachment of GOx and HRP to MWCNT is indicated by the appearance of IR absorption bands of the amide I and amide II at 1673, 1661, 1531 and 1542 cm1. A photograph of vials containing of 2 mg L1 pristine MWCNTs and oxidized MWCNTs in aqueous solutions taken 24 h after sonication is shown in Fig. 1D. When pristine MWCNTs were dissolved in PB solution, the resulting solution

was not homogeneous, but a homogeneous and well-dispersed solution was observed for oxidized MWCNTs. This is attributed to the presence of carboxyl groups, which lead to a reduction of van der Waals interactions between the MWCNTs. It will facilitate the separation of MWCNT bundles into individual nanotubes. The attachment of carboxyl groups assists the solubilization of MWCNTs in aqueous solution. The third vial shows the incorporation of the mediator toluidine blue and the enzymes GOx and HRP over the surface of the nanotubes. The resulting MWCNT–TB–GOx–HRP biocomposite has a high function density and is soluble in aqueous solution.

3.2. Electrochemical characterization of the modified electrodes 3.2.1.

Electrochemical impedance spectroscopic measurements

The electrochemical impedance spectroscopy (EIS) can give information on the impedance changes of the electrode surface during the modification process [49]. In an EI spectra, the semicircle part at higher frequencies correspond to the electron transfer limited process or the electron transfer resistance (Ret). This resistance controls the electron transfer kinetics of the redox probe at the electrode interface. The linear segment at lower frequencies shows a controlled diffusion process. Fig. 2 compares the Nyquist plots of the impedance spectroscopy for different biocomposite modified electrodes. Here, Z 0 and Z 0 0 are the real variable and the negative value of the imaginary variable of impedance, respectively. In Fig. 2, curve (a) shows the EI spectra of the bare GC electrode. It exhibits an almost straight line that is characteristics of electron transfer limited by diffusion. The plot for oxidized MWCNTs–GCE showed the greatest semicircle with a higher

Fig. 1 – (A) TEM micrograph of the purified MWCNTs, (B) SEM image of MWCNT–TB–GOx–HRP–Nf biocomposite, (C) FT-IR spectra of TB functionalized MWCNTs immobilized with GOx and HRP and (D) photographs of vials containing (1) the pristine MWCNTs, (2) a stable suspension of purified MWCNTs and (3) MWCNTs–TB–GOx–HRP biocomposite in PB solution.

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Fig. 2 – Nyquist diagram (Z 0 vs Z 0 0 ) for the Electrochemical impedance measurements in the presence of 5 mM ½FeðCNÞ6 3=4 for: (a) bare GC electrode, (b) MWCNT–Nf modified (c) MWCNT–TB–Nf modified (d) MWCNT–TB–GOx– Nf modified (e) MWCNT–TB–HRP-Nf modified and (f) MWCNT–TB–GOx–HRP–Nf modified GC electrodes in 0.1 M PB solutions (pH 7.4). Electrode potential:0.26 V vs SCE. Inset: Randles equivalent circuit. electron transfer resistance (Ret = 621 X). This is due to the electrostatic repulsion between the probe molecule, FeðCNÞ63=4 and negatively charged CNT surface, as oxidized CNTs have carboxylic acid groups (curve b). Upon covalent attachment of TB with MWCNTs (curve c), the electron transfer resistance decreased dramatically to 143 X, due to the decrease in the negative charges on the surface of CNTs, making it easier for the electron transfer to take place. Further immobilization of monoenzymes, GOx and HRP (curve d and e) separately over the surface of the nanotubes, generated a hydrophobic insulating layer on the electrode and introduced a barrier to the electron transfer. As a result the electron transfer resistance increased to 262 and 310 X for GOx and HRP, respectively. Upon immobilization of both the enzymes over the TB functionalized MWCNTs, the electron transfer resistance further increased to 442 X (curve f). This is due to the attachment of both enzymes, which increases the extent of insulation of the conductive support by the hydrophobic protein layers. As a result, the interfacial electron transfer resistance gradually increased in their values upon the immobilization of the two biocatalysts. These data showed that the MWCNT–TB–GOx–HRP–Nf biocomposite was successfully attached to the electrode surface and formed a tunable kinetic barrier.

3.2.2.

Voltammetric characterization

The homogeneity of the MWCNT-TB–GOx–HRP–Nf biocomposite is the key factor in electroanalytical applications. We used cyclic voltammetric (CV) method to investigate the electrochemical characteristics of the enzyme-modified electrodes. The cyclic voltammograms of various modified electrodes were obtained (Fig. 3) in the potential range of 0 to 0.7 V in 0.1 M PB solution (pH 7.4), except for the MWCNT–TB–GOx–HRP–Nf modified bienzyme electrode which was obtained in the range of 0–0.6 V. No redox peak was observed for the bare GC electrode and MWCNT-Nf modified GC electrode. Compared with bare GC

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electrode, the background current for MWCNT–Nf electrode is higher, which indicates that the effective electrode surface area is significantly larger for MWCNTs incorporated GCE (not shown here). Fig. 3A shows the typical CV for MWCNT–GOx– Nf modified electrode. A pair of well-defined and nearly symmetric redox peaks is observed with the formal potential of 0.47 V vs. SCE. The experiments indicate that the redox peaks were derived from GOx, which is similar to the results reported [50,51]. Fig. 3B shows the CV for MWCNT–HRP–Nf modified GC electrode, in which a pair of redox peaks were observed with the formal potential at 0.29 V for the HRP(Fe(III)/HRP(Fe(II)) redox couple transformation which could be ascribed to the direct electron transfer between the HRP and the underlying electrode. Fig. 3C shows the CV for MWCNT–GOx–HRP–Nf modified electrode, which showed two pairs of redox peaks with formal potentials at 0.36 and 0.51 V, which may be due to the presence of HRP and GOx adsorbed over the surface of the nanotubes. However MWCNT–TB–GOx–HRP–Nf modified biocomposite electrode displayed a CV in a different form from other three electrodes (Fig. 3D), which exhibited two well defined reversible redox waves at very low potentials with formal potentials at 0.26 and 0.43 V, with higher cathodic and anodic peak currents, which should be attributed to the presence of TB in the biocomposite. The voltammogram of the MWCNTs provide two pieces of information. Firstly, it is clear that they have a metallic character in the range of potentials applied, since there is no apparent oxidation or reduction peaks. Based on this, nanotubes can donate and accept electrons in a wide range of potentials, and could, therefore be used as transducer in biosensor systems. If the enzymes can be trapped in the inner cavities of the nanotubes, then transduction and mediation may be achieved at the same time. The relative activity of the enzymes is improved after mixing with MWCNTs. The carboxyl groups, which are present on the CNTs, could combine with amino group of enzymes during the mixing of MWCNTs with enzyme solution. The combination could form a stable MWCNT–GOx-HRP complex. Secondly, the CNTs have excellent electrocatalytic ability, and the CNTs attached to the enzyme could improve the electron transfer between the active redox center of the enzyme and the TB immobilized CNTs. This effect could accelerate the regeneration of GOx and increase the relative activity of the enzymes finally. It is thought to be the main reason for the higher response of MWCNTs modified glucose biosensor than sensors made with other electrodes. The dependence of the peak current towards the scan rate has also been studied for the MWCNT–TB–GOx–HRP–Nf modified bienzyme electrode. A good linearity in the plot of peak current versus scan rate in the range from 0.1 to 2.0 V s1 was observed as expected for surface confined redox process. In addition, with increasing scan rates, the peak separation begins to increase, indicating the limitation arising from charge transfer kinetics. Based on Laviron’s theory [52], the electron transfer rate constant (Ks) and charge transfer coefficient (a) can be determined by measuring the variation of peak potential with scan rate. The values of peak potentials were proportional to the logarithm of the scan rate for scan rates higher than 2.0 V s1. The calculated values for (Ks)

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Fig. 3 – Cyclic voltammograms of: (A) MWCNT–GOx–Nf, (B) MWCNT–HRP–Nf, (C) MWCNT–GOx–HRP–Nf (D) MWCNT–TB–GOx– HRP–Nf modified GC electrode in 0.1 M PB (pH 7.4) at a scan rate of 20 mV s1.

and (a) were about 7.75 s1 and 0.56, respectively. The surface concentration (C) of the biocatalyst/mediator was calculated to be 9.76 · 1010 M cm2 using Laviron’s equation [53].

3.2.3. Analytical biosensor

utility

of

the

biocomposite

bienzyme

The advantage of the biocomposite sensor is illustrated in connection with the quantification of glucose. The working mechanism of the GOx–HRP bienzyme electrodes studied in this work is depicted in Fig. 4. The substrate glucose reacts with the GOx enzyme, in the presence of the natural co-substrate O2, to produce H2O2. The hydrogen peroxide then serves as substrate for HRP, which is converted to oxidized form is in turn reduced by the redox mediator TB immobilized over MWCNTs. Depending on the applied potential and the spatial distribution of the enzymes on the electrode surface, a direct electron transfer to/from H2O2 is also likely to occur. In the absence of glucose, both GOx and HRP electrodes yield a voltammogram, which exhibits two redox peaks corresponding to the redox behavior of both the enzymes for the MWCNT–GOx–HRP–Nf modified biocomposite electrode. Upon addition of glucose, the bienzyme-based biosensor (MWCNT-TB–GOx–HRP–Nf) triggers bioelectrocatalytic reaction, which dramatically changes the cyclic voltammogram with a sharp increase in the cathodic current and a concomitant decrease in the anodic current (Fig. 5A). The increase in cathodic current is due to the cascade of reactions that are taking place at the electrode. As the charging current contribution to the background current is the limiting factor in the analytical determination of any electroactive species, differential pulse voltammetry (DPV) experiments were carried out. When the DPV was per-

formed instead of CV, an obvious improvement in the sensitivity was observed (Fig. 5B), which was quite important in the detection of low levels of glucose. The linear range with DPV was from 1.5 · 108 M to 1.8 · 103 M. Hydrodynamic voltammetric (HDV) experiments were then carried out for determining the optimum potential necessary for the chronoamperometric determination of glucose. Fig. 6A compares the HDV of 0.1 lM glucose on (a) bare GC electrode (b) MWCNT–GOx–HRP–Nf modified and (c) MWCNT–TB–GOx–HRP–Nf modified GC electrode. The currents were measured at constant interval of potentials after the addition of glucose to a stirred electrolyte solution at a pH of 7.4. As expected no response was observed at the bare GC electrode in the entire potential range studied. For the MWCNT–GOx–HRP–Nf modified electrode the reduction

Fig. 4 – Illustration of the reactions occurring at the GC electrode modified with MWCNT–TB–GOx–HRP–Nf biocomposite.

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Fig. 5 – (A) Cyclic voltammograms of MWCNT–TB–GOx–HRP–Nf biocomposite biosensor (a) in the absence and (b) in the presence of 1.0 lM glucose; (B) Differential pulse voltammograms of biocomposite biosensor (a) in the absence and (b–k) successive addition of 0.1 lM glucose.

20

c

Current /µA

A 15

b

10

5

0

a 0

-0.2

-0.4

-0.6

-0.8

Potential / V

B

I / µA

100 50 0 0

10

20

30

Conc. mM

sharp increase around0.25 V and leveled off above 0.3 V. Such a potential dependence profile is in agreement with the cyclic voltammogram results shown in Fig. 4A. All subsequent experiments with MWCNT–TB–GOx–HRP–Nf modified GC were carried out at 0.3 V. The catalytic action of the mediator and bienzyme functionalized CNTs facilitate the sensitive fixed-potential amperometric determination of glucose. Fig. 6B shows the chronoamperometric response of the biosensor to successive increments (0.1 lM) of glucose to the PB solutions under stirring condition at an applied potential of 0.3 V. The time required to reach 95% of the maximum steady-state current was 2 s. The response was also linear in the range from 1.5 · 108 M to 1.8 · 103 M. The detection limit was 3 · 109 M for a signal to noise ratio of 3. In the linear range, the sensor has a high sensitivity of 8.3 lAm M1 cm2. The apparapp ent Michaelis–Menten constant K M was calculated using Michaelis– Menten equation.   app i i ¼ imax  KM C Where i is the steady-state catalytic current, imax the maximum current measured under saturated substrate conapp ditions, C referred to the glucose concentration and K M stands for the apparent Michaelis–Menten constant of the app system. K M in this work, was evaluated as 0.89 mM and is in agreement with the earlier reports [54]. The results show that the biosensor possesses higher biological affinity to glucose.

Fig. 6 – (A) Hydrodynamic voltammograms for 0.1 lM glucose in 0.1 M phosphate buffer solution (pH 7.4) at (a) unmodified (b) MWCNT–GOx–HRP–Nf modified and (c) MWCNT–TB–GOx–HRP–Nf modified GC electrodes. (B) Amperometric response of MWCNT–TB–GOx–HRP–Nf biocomposite electrode (a) in the absence and (b–k) successive addition of 0.1 lM glucose in 0.1 M PB (pH 7.4); Inset shows the linear calibration plot.

started at 0.2 V, then the current increased slowly until 0.4 V. In contrast the voltammetric response of the MWCNT–TB–GOx–HRP–Nf modified GC electrode showed a

3.2.4.

Factors affecting the biocomposite electrode

The effect of pH on the response of the biocomposite electrode was also evaluated. The increase in catalytic current to the addition of 0.1 mM glucose was measured in the pH range from 4 to 9. Studies under highly acidic (below pH 4) and basic conditions (above pH 9) were avoided due to the instability of the enzyme GOx in these pHs. No appreciable variation of response towards the quantification of glucose was observed when the pH was varied from 4 to 9. However a maximum response was observed in the pH range between 6 and 9. Thus a working pH of 7.4 was chosen for the determination of glucose considering the physiological pH condition.

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The effect of temperature has been reported to be very important on the functioning of the biosensors. When the temperature was varied from 15 to 70 C in the presence of 0.1 mM glucose, the current response increased with increasing temperature, reaching a maximum value at 60 C. It was evident that the immobilized bienzymes had good thermal stability upto 60 C because of the unchanging microenvironment. At high temperatures, the response was found to decrease and this may be due to the denaturation of the enzymes [55]. However, for practical convenience, all experiments were carried out at room temperature.

3.2.5.

Effect of interference on glucose monitoring

The selectivity of the proposed sensor was examined by studying the effects of foreign species on the determination of 0.1 mM glucose (1). The high selectivity of the biosensor is attributed to the low working potential and the biosensor construction. The bienzyme electrode had a relative standard deviation of 3% for five repeated analyses of 0.1 mM glucose, indicating a good precision for analysis. Using oxidase/peroxidase bienzyme systems the detection principle switches from an electrochemical oxidation to a reduction process that takes place at much lower potentials, and therefore, the selectivity of the device is improved considerably.

3.2.6.

Reproducibility and stability of the enzyme sensor

The reproducibility and stability are the two important parameters for the evaluation of the performance of the sensor. The reproducibility of the biosensors was examined at 1 mM glucose solution. The relative standard deviation was 1.9% (n = 8). The electrode-to-electrode reproducibility was also examined between 10 different electrodes in the above solutions, and the relative standard deviation was calculated to be 2.5%. Good reproducibility may be explained by the fact that the functionalized CNTs and the enzymes were consistent and the GOx/HRP molecules are firmly attached on the surface of the functionalized CNTs. In order to demonstrate long-term stability, the response to glucose was measured every day for 5 months. All electrodes, when not in use, were stored under dry conditions and were stable for at least 4 months. The current response of the sensor to glucose remained almost constant during the first 90 days. This was confirmed by electrochemical impedance measurement in which similar Ret was obtained for the enzyme electrode in three months of operation, suggesting the same amount of insulating material on the electrode surface till 3 months. After 4 months 95% of the original response remained. The decrease in current response may be due to the denaturation of the enzymes in long-time keeping. Good long-term stability of the biosensor can be attributed to the excellent biocompatibility of the MWCNTs for preserving the activity of GOx, HRP and to the strong covalent interaction between CNTs, mediator and the enzymes.

4.

Conclusions

The results demonstrate the possibility for co-immobilization of GOx and HRP over TB immobilized MWCNTs modified elec-

trode to conveniently construct a new bienzyme glucose biosensor. The MWCNT–TB–GOx–HRP–Nf film has a highly permeable structure. The functionalized CNTs markedly influenced the interface property of the modified electrode and played an important role in the biosensor response. The proposed method where a mediator transfers electrons between the enzyme and electrode reduced the problem of interferences by the other electroactive species. A further advantage is the use of multiple enzymes, which can be combined to create a new biosensor, to enhance sensor selectivity, and to chemically amplify the sensor response. This strategy prolongs the operational lifetime of the sensor. Thus the sensor showed high performance characteristics with a broad detection range, a short measuring time, a good stability and an interference-free determination. The proposed method can be extended to other enzymes and bioactive macromolecules to provide promising platforms for biosensor and bioelectronics applications.

Acknowledgements The financial supports from the Department of Science and Technology (DST), New Delhi, India, under Nanomaterials Science and Technology Initiative programme and Council of Scientific and Industrial Research (CSIR), New Delhi, India, for the award of SRF are gratefully acknowledged.

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