Amperometric biosensing of glutamate using carbon nanotube based electrode

Electrochemistry Communications 9 (2007) 1323–1330 www.elsevier.com/locate/elecom

Amperometric biosensing of glutamate using carbon nanotube based electrode Sudip Chakraborty, C. Retna Raj

*

Department of Chemistry, Indian Institute of Technology, Kharagpur 721 302, India Received 8 November 2006; received in revised form 20 January 2007; accepted 22 January 2007 Available online 26 January 2007

Abstract Amperometric biosensing of glutamate using nanobiocomposite derived from multiwall carbon nanotube (CNT), biopolymer chitosan (CHIT), redox mediator meldola’s blue (MDB) and glutamate dehydrogenase (GlDH) is described. The CNT composite electrode shows a reversible voltammetric response for the redox reaction of MDB at 0.15 V; the composite electrode efficiently mediates the oxidation of NADH at 0.07 V, which is 630 mV less positive than that on an unmodified glassy carbon (GC) electrode. The CNTs in the composite electrode facilitates the mediated electron transfer for the oxidation of NADH. The CNT composite electrode is highly sensitive (5.9 ± 1.52 nA/lM) towards NADH and it could detect as low as 0.5 lM of NADH in neutral pH. The CNT composite electrode is highly stable and does not undergo deactivation by the oxidation products. The electrode does not suffer from the interference due to other anionic electroactive compounds such as ascorbate (AA) and urate (UA). Separate voltammetric peaks have been observed for NADH, AA and UA, allowing the individual or simultaneous determination of these bioanalytes. The glutamate biosensor was developed by combining the electrocatalytic activity of the composite film and GlDH. The enzymatically generated NADH was electrocatalytically detected using the biocomposite electrode. Glutamate has been successfully detected at 0.1 V without any interference. The biosensor is highly sensitive, stable and shows linear response. The sensitivity and the limit of detection of the biosensor was 0.71 ± 0.08 nA/lM and 2 lM, respectively.  2007 Elsevier B.V. All rights reserved. Keywords: Carbon nanotube; Composite electrode; Biosensor; Glutamate dehydrogenase; Amperometry

1. Introduction Glutamate is the major excitatory neurotransmitter in the central nervous system and it is a useful marker for the diagnosis of myocardial and heptical disease [1,2]. Neuronal pathways in the brain that use glutamate are implicated to great extent in many neurological disorders [3,4]. Precise monitoring of glutamate level in the extracellular space of brain tissue is essential to understand the role of glutamate in these disorders. The concentration of glutamate in extracellular space is in the range between 4 and 350 lM [5,6]. Various analytical methods have been reported for the sensing of glutamate; the

optical and electrochemical methods have been widely used [7–14]. The electrochemical methods have been used for in vivo measurement in several occasions [15,16]. However, the selective electrochemical detection of glutamate is a challenging task because the easily oxidizable electroactive interferents such as ascorbate (AA) and urate (UA) often interfere the amperometric measurement of glutamate [9,14]. The electrochemical methods are based on the use of enzymes glutamate oxidase (GlOD) and glutamate dehydrogenase (GlDH) [7–14]. The electrochemical glutamate biosensors based on GlOD involve the monitoring of enzymatically GIOD

glutamate þ O2 þ H2 O ! 2-oxoglutarate þ NH3 þ H2 O2 *

Corresponding author. Fax: +91 3222 282522. E-mail address: [email protected] (C. Retna Raj).

1388-2481/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2007.01.039

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generated H2O2 (Eq. (1)) either by its direct oxidation or by using another enzyme horseradish peroxidase. The detection of H2O2 by its oxidation requires high overpotential and it often invites interference. The artificial peroxidases and osmium containing hydrogel have been used for development of glutamate sensors [9,13]. The electrochemical biosensors based on GlDH involves the electrochemistry of NAD+/NADH redox couple (Eq. (2)) [17–19]. The biosensing of glutamate using GlDH requires highly sensitive NADH transducer because the transduced signal of the biosensor is based on the detection GIDH

glutamate þ NADþ þ H2 O ! 2-oxoglutarate þ NADH þ þ NHþ ð2Þ 4 þH of enzymatically generated NADH. The selective detection of glutamate in the presence of other interfering compounds by the dehydrogenase-based biosensor is successful only if NADH can be detected at less negative potential (<0 V). The direct oxidation of NADH at unmodified electrode requires large overpotential (>1 V) owing to the slow electron transfer kinetics [20]. The unmodified electrodes very often suffer from fouling by the adsorption of oxidation products. Various methodologies have been developed to facilitate the electron transfer kinetics for the oxidation of NADH [21]. A conventional way to decrease the high overpotential and avoid the surface fouling is to use redox mediators that can promote the electron transfer kinetics. In this way, much effort has been devoted to identify/develop new materials which can effectively overcome the kinetic barriers for the electrochemical regeneration of enzymatically active NAD+ [21–25]. In the recent years, carbon nanotubes (CNTs) have emerged as a useful material in the field of electroanalytical chemistry due to their unique structural and electronic properties. The high electrical conductivity and large length-to-diameter ratio makes them an excellent candidate in the development of electrochemical sensors. It has been demonstrated that CNTs can impart strong electrocatalytic effect and promote the electrochemical properties of bioanalytes and redox enzymes [26–29]. Wang and co-workers have first explored the electroanalytical application of CNTs for the oxidation of biomolecules [29]. Significant decrease in the overpotential for the oxidation of biomolecules such as AA, NADH, etc. has been observed at CNT modified electrodes [28–31]. Recently, Compton and coworkers questioned the electrocatalytic properties of CNTs and they have shown that CNTs are no more electrocatalytically active than the edge planes of pyrolytic graphite [32]. It has been further demonstrated that both CNTs and the edge plane pyrolytic graphite have similar electrocatalytic activity [33]. Although, the CNTs based electrodes are known to decrease the overpotential for the oxidation of NADH [30,34], the extent of decrease is not sufficient enough for the selective detection of NADH. The oxidation of NADH at more positive potential (>0.1 V) on any electrode often suffers from the interference due to eas-

ily oxidizable bioanalytes such as AA and UA [35]. Therefore, it is essential to develop an electrode, which can catalyze the oxidation of NADH at less negative potential. The conventional approach to decrease overpotential is the use of redox mediators, which can efficiently mediates the oxidation process. It is well known that quinones and diimines are the excellent candidates for the oxidation of NADH. The diimines effectively catalyzes the oxidation of NADH because they can engage ‘‘two-electron’’ and ‘‘one-plus-one-electron’’ redox process [36,37]. The extended aromatic molecules with diimine function such as meldola’s blue can efficiently mediate the oxidation of NADH [37]. It has been shown already that redox dyes can be conveniently adsorbed on the walls of CNTs [38,39]. The adsorbed redox molecules displayed stable and well defined voltammetric response [38,39]. Because CNTs have excellent catalytic properties, the integration of redox mediator having diimine functionality with CNTs would be promising for the selective detection of NADH at low potential. In the present investigation, we describe the electrocatalytic detection of NADH using the integrated assembly of CNT, chitosan (CHIT) and a redox mediator, medola’s blue (MDB) and the development of an amperometric biosensor for glutamate. 2. Experimental 2.1. Materials Multiwalled CNTs (Cat. No. 636487, P95% purity), CHIT, MDB, NADH and GlDH and glutamate were purchased from Sigma-Aldrich. All other chemicals used in this investigation were of analytical grade. All solutions were prepared using Millipore water. 2.2. Instrumentation Electrochemical measurements were performed using two-compartment three-electrode cell with a glassy carbon (GC) working electrode, a Pt wire auxiliary electrode and Ag/AgCl (3 M NaCl) reference electrode. Cyclic voltammograms and amperometric curves were recorded using a computer controlled CHI643B electrochemical analyzer attached to a pico amp booster-Faraday cage. 2.3. Preparation of CNT composite electrode The GC electrodes (0.07 cm2) were used as substrate for making CNT–CHIT–MDB composite film. Before modification, the GC electrodes were polished well with fine emery paper and alumina (0.05 lm) slurry and then sonicated in Millipore water for 10–15 min. Finally the electrode was thoroughly rinsed with Millipore water and used for modification. The CNTs were purified according to the literature procedure [32]. A 0.6 mg of purified CNT was dispersed in 300 lL of CHIT (0.05% solution in 0.05 M HCl) and 5 lL of 5 mM ethanolic solution of

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MDB was added. The mixture was stirred in a magnetic stirrer vigorously for 30 min to obtain a homogeneous suspension. An aliquot of 5 lL of the suspension was uniformly coated on the clean GC electrode and allowed to dry at room temperature for 30 min. For control experiments, the GC electrode was modified with CHIT–MDB mixture and dried at room temperature. Hereafter, the electrodes modified with CHIT–MDB and CNT–CHIT–MDB composite film will be referred as GC/CHIT–MDB and CNT composite electrodes, respectively. Amperometric traces for the detection of NADH have been recorded in a stirred phosphate buffer solution (PBS) of pH 7.2 (0.1 M) at a potential of 0.14 V. All the experiments were carried out in argon atmosphere.

6

The following optimized procedure was used for the preparation of glutamate biosensor: First the CNT composite electrode was prepared as described earlier and an aliquot of 10 lL of GlDH (2.7 U/lL) in 5 mM PBS was caste on the CNT composite electrode and dried at 4 C for 30–40 min. This biocomposite electrode was stored in PBS before subjecting to amperometric experiments. The amperometric measurements of glutamate were performed in 0.1 M PBS (pH 7.2) containing 4 mM of NAD+ using the enzyme-immobilized electrode. The electrode was polarized at 0.1 V and glutamate was injected at regular intervals. All the experiments have been repeated at least for three times and reproducible results were obtained. 3. Results and discussion 3.1. Electrochemical behavior The CNT composite electrode shows a well-defined reversible voltammogram corresponding to the redox reaction of MDB (Fig. 1a). The peak-to-peak separation (DEp) is typically small (30 mV), although not zero, as expected for a reversible ideal case; the ratio of anodic and cathodic peak current ðI ap =I cp Þ is close to unity. On the other hand, the GC/CHIT–MDB electrode does not show such reversible voltammetric response for MDB (Fig. 1, inset). As shown, the anodic and cathodic peaks are rather broad and the magnitude of the peak current is significantly lower than that observed on the CNT composite electrode. Furthermore, the DEp value at the GC/CHIT–MDB electrode is relatively large, suggesting a sluggish electron transfer kinetics. The anodic and cathodic peak current gradually decreases during the subsequent sweeps, possibly due to the leaching of MDB from the electrode surface. The well-defined voltammogram obtained for the CNT composite electrode is due to the presence of CNTs in the composite film; CNTs on the electrode surface provide an ideal environment for the redox reaction of MDB. The increased reversibility indicates that the CNTs facilitate the electron transfer for the redox reaction. The formal potential

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E/ V Vs Ag/AgCl Fig. 1. Cyclic voltammograms obtained for (a) CNT composite and (b) CNT (without MDB) modified electrodes in 0.1 M PBS of pH 7.2. Scan rate 100 mV/s. Inset shows the voltammogram obtained for GC/CHIT– MDB electrode.

ðEap þ Ecp =2Þ of MDB on the CNT composite electrode was found to be 0.14 V, which is much less negative than that of the GC/CHIT–MDB electrode. The peak current obtained for the CNT composite electrode linearly increases with sweep rate, showing that the voltammetric response corresponds to a surface confined redox species. The voltammogram at the CNT composite electrode is very stable and the peak current and peak potential does not change upon repeated sweeps, implying that MDB is strongly adsorbed on the walls of CNTs. The surface coverage (C) of MDB on the CNT composite electrode was calculated from the area under the anodic peak and it was 6.23 · 1010 mol/cm2. 3.2. Electrocatalytic oxidation of NADH The main objective of the present investigation is to develop NAD+ dependent dehydrogenase amperometric biosensor for the sensing of glutamate. Since the function of dehydrogenase biosensors is based on the detection of enzymatically generated NADH, first we have investigated the electrocatalytic property of CNT composite electrode towards oxidation of NADH. Fig. 2 shows the voltammograms obtained for the oxidation of NADH at CNT composite electrode. The oxidation of NADH occurs at 0.07 V, which is very close to the anodic peak potential of MDB. The cathodic peak for MDB completely disappears in the presence of NADH, indicating the strong electrocatalytic effect of the electrode due to mediated oxidation. The onset potential for the oxidation of NADH is much less negative (0.3 V) than the oxidation peak actually appears. At lower concentration (0.05 mM), the oxidation of NADH occurs at the formal potential of MDB (data not shown) and the peak potential shifts to more positive side with the concentration of NADH. The

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Y=24.34 + 5.97X Fig. 2. Cyclic voltammograms illustrating the electrocatalytic activity of CNT composite electrode towards oxidation of NADH in 0.1 M PBS (pH 7.2). [NADH]: (a) 0 and (b) 5 mM. Scan rate 10 mV/s.

oxidation of NADH on the electrode modified only with CNT occurs at P0.3 V (data not shown) and this is in agreement with literature reports [30]. In the case of unmodified GC electrode, the oxidation occurs at much positive potential (>0.6 V) and the voltammogram is not stable, possibly due to the fouling of electrode surface. It has been reported earlier that the oxidation of NADH on single and multiwall CNTs occurs at more positive potential [28–30,40]. For instance, Wang’s group has observed the oxidation of NADH at >0.2 V on CNT modified electrodes [29,30]. Chen and co-workers have modified the GC electrodes with ordered CNTs and they observed the oxidation peak at 0 V (SCE) in pH 6.8 [40]. The single wall CNT paste electrode catalyzes the oxidation of NADH at 0.54 V [28]. Interestingly, the oxidation at our CNT composite electrode occurs at 0.07 V, which is much less positive than those reported in the literature. Six hundred and seventy millivolts decrease in the overpotential with respect to the unmodified GC electrode has been observed; this is significantly higher than the other CNT based electrodes. The analytical performance of the electrode was tested by recording the amperometric response of the electrode. Fig. 3 shows the amperometric trace obtained for the oxidation of NADH on the CNT composite electrode. The electrode was polarized at 0.14 V in a stirred PBS of pH 7.2 and NADH was injected at regular interval. Gradual increase in the current after each addition was observed. The current flowing through the electrode was stable and the response time of the electrode was 4 s. No response was obtained with the electrode modified with CNT in the absence of MDB, confirming that the amperometric current obtained is due to the mediated oxidation of NADH by MDB on the electrode surface. The amperometric response was very stable upon repeated injection, demonstrating that the electrode does not undergo fouling by the oxidation products. The electrode linearly responds to

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[NADH], M Fig. 3. Amperometric i–t curve (A) for the oxidation of NADH at CNT composite electrode and the corresponding calibration plot (B). The electrode was polarized at 0.14 V in a stirred supporting electrolyte of 0.1 M PBS (pH 7.2). Each addition increased the concentration of NADH by 10 lM.

NADH upto 80 lM and it can detect as low as 0.5 lM NADH in neutral pH. The sensitivity of the electrode was determined to be 5.9 ± 1.52 nA/lM. The limit of detection (LOD) obtained at our CNT composite electrode is comparable with those reported in the literature. The LOD of the existing CNT based electrodes ranges from 0.1 to 6 lM [28,33,40–42]. It should be mentioned here that the easily oxidizable electroactive analytes such as AA and UA strongly interfere the measurement of NADH on these electrodes. However, our CNT composite electrode can be operated at low potential without any interference (vide infra) and it can detect NADH at sub micromolar level. The excellent analytical performance of the electrode can be explained by considering the improved electronic and ionic transport capacity and efficient mediation of the composite electrode. The surface fouling of electrode by the oxidation product is a serious concern in the development of transducers for the detection of NADH. The electrode surface under-

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goes deactivation by the adsorption of NAD+ during the oxidation process and hence stable electrochemical signal cannot be obtained. We have examined the stability by using the same electrode for repeated measurements. The voltammetric measurement with CNT composite electrode was carried out in PBS containing 0.2 mM of NADH, and the peak height and peak position was measured at an interval of 20 min for 1 h. Note that the electrode was kept inside the electrochemical cell containing supporting electrolyte solution and NADH throughout the experiment. As shown in Fig. 4A, 1.26% decrease in the current and 15 mV positive shift in the peak potential was noticed after 1 h, confirming that the electrode is highly stable against the surface fouling. To further ascertain the operational stability of the electrode during the amperometric measurement, the electrode was polarized at the working potential of 0.14 V for long time; no appreciable change in the initial current was observed (Fig. 4B), indicating that the electrode can be used for continuous measurement of NADH. As the CNT composite electrode facilitates the electron transfer for the oxidation of NADH, the generation of intermediates that are responsible for fouling of electrode surface is avoided. Selective detection of NADH is one of the main challenges in the development of NADH sensor. The interference due to other electroactive compounds like AA and UA is a serious problem in the electrochemical sensing of NADH. The oxidation of NADH and AA involve two electrons and AA interferes in the measurement of NADH at the unmodified electrodes. Very often the redox mediator modified electrodes, which shows the excellent electrocatalytic activity towards oxidation of NADH failed to overcome the interference due to AA. Despite the fact that the mediator modified electrodes decrease the overpotential

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for the oxidation of NADH to large extent, the selective oxidation of NADH in the presence of AA is still a challenging task for these electrodes, as the electrocatalyst can also catalyze the oxidation of AA [35]. To avoid the interference due to AA, two different approaches have been used in the past: (i) coating the electrode surface with anionic polymers [22] and (ii) immobilization of ascorbate oxidase, an enzyme capable of selectively oxidizing AA in the presence of O2 [35]. In the former approach, the anionic polymers would prevent the approach of AA, as AA is negatively charged in neutral pH. However, this anionic polymer will hinder the approach of NADH as it is also negatively charged at the neutral pH and therefore, this approach would not overcome the problem. In the present investigation, the analytical performance of the CNT composite electrode towards NADH in the presence of the common interferents AA and UA has been tested. The electrode was polarized at 0.14 V and aliquots of NADH and the interferents (AA and UA) have been injected into the supporting electrolyte solution. Interestingly, as shown in Fig. 5A there is no change in amperometric signal obtained for the oxidation of NADH upon the injection of AA and UA, showing that the CNT composite electrode does not suffer from the interference due to AA and UA. It is interesting to note that the CNT composite electrode can successfully be used for the simultaneous detection of NADH, AA and UA, as it shows individual voltammetric peaks for these analytes at 0.15, 0.05 and 0.25, respectively (Fig. 5B). Because the separation between the voltammetric peaks is large enough, the selective or the simultaneous sensing of these analytes is feasible with this electrode. It is worth to compare the electrochemical and electrocatalytic behavior of MDB on CNT composite electrode

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E/ V Vs Ag/AgCl Fig. 4. (A) Voltammetric response of CNT composite electrode before (a) and after (b) leaving in 0.1 M PBS containing 2 mM of NADH for 1 h. Scan rate 25 mV/s. (B) Operational stability of CNT composite electrode in 0.1 M PBS of pH 7.2. The electrode was polarized at 0.14 V and 0.1 mM NADH was injected into the supporting electrolyte.

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E/V vs Ag/AgCl Fig. 5. (A) Amperometric i–t curve illustrating the interference free sensing of NADH at CNT composite electrode in 0.1 M PBS of pH 7.2. NADH, AA, and UA (0.1 mM each) were injected at regular interval as indicated. (B) Linear sweep voltammogram demonstrating the simultaneous sensing of NADH, AA and UA (0.5 mM each) using the CNT composite electrode. Scan rate 25 mV/s.

with other electrodes based on carbon composite, calcium, titanium and zirconium phosphates, niobium oxide, etc. [43–45]. Sampath and Lev investigated the electrochemical behavior of MDB using surface renewable sol–gel carbon composite electrode. Sluggish voltammetric response was obtained for MDB at this electrode [43]. The calcium, titanium and zirconium phosphates based electrodes show the redox peak for MDB at more positive potentials [44,45] than that is observed in the present CNT composite electrode; the DEp value at these electrodes is relatively larger [44,45]. Furthermore, the electrocatalytic oxidation of NADH on these electrodes occurs at more positive potential than that on the present CNT composite electrode. The LOD achieved on this electrode is significantly higher than the other existing MDB based electrodes. These results show that the CNTs on the composite electrode plays major role in facilitating the electron transfer reaction.

sured quantitatively from the transduced signal of the biosensor. The electrode linearly responds to glutamate at lower concentration and attained saturation level at higher concentration as expected for Michaelis–Menten type enzyme kinetics. The apparent Michaelis–Menten constant ðK app M Þ was calculated from the double reciprocal plot and was 158 lM. The sensitivity and detection limit was 0.71 ± 0.08 nA/lM and 2 lM, respectively. The amperometric response of the biosensor depends on the concentration of the cofactor NAD+. The effect of cofactor concentration on the amperometric response was investigated with constant amount of enzyme on the electrode surface and glutamate in solution (25 lM). The amperometric response of the biosensor increases with the concentration of NAD+ and maximum response was obtained at 4 mM of NAD+. Further increase in the concentration of NAD+ results in a decrease in the amperometric response preferably due to the inhibitory effect.

3.3. Amperometric glutamate biosensor Since the CNT composite electrode is highly sensitive and selective towards NADH, it can be successfully used for the development of dehydrogenase-based biosensor. The glutamate biosensor was developed using the CNT composite electrode and GlDH, as described earlier (Scheme 1). Fig. 6 displays the amperometric response of the biosensor towards glutamate; fast and stable response was obtained upon every injection. Such response was not obtained in the absence of either the cofactor NAD+ or glutamate, showing that the amperometric response was due to the enzymatically generated NADH (Eq. (2)). The CNT composite electrode could electrocatalytically detect NADH at low potential. As the concentration of enzymatically generated NADH is a measure of glutamate injected into the test solution, glutamate has been mea-

Scheme 1. Schematic representation of the CNT based glutamate biosensor.

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[Glutamate], M Fig. 6. (A) Amperometric i–t curve for the sensing of glutamate. The electrode was polarized at 0.1 V and aliquots of glutamate were injected at regular intervals into 0.1 M PBS containing NAD+ (4 mM). Each addition increased the concentration of glutamate by 25 lM. (B) Corresponding calibration plot.

4. Conclusion The CNT composite electrode prepared from the biopolymer CHIT, CNT and redox mediator has been successfully used for the sensing of NADH and for the development of glutamate biosensor. The redox mediator on the CNT composite electrode shows fast electron transfer due to the ideal environment of CNT. The composite electrode is highly sensitive towards NADH due to the improved charge transport in the film and the mediation by the redox mediator. The common interfering agents AA and UA do not interfere the amperometric measurement of NADH and glutamate. The simultaneous detection of three different negatively charged bioanalytes at the CNT composite electrode has been demonstrated for the first time. The amperometric glutamate biosensor has been developed using CNT composite electrode and GlDH and it could successfully detect glutamate in the physiological level. Acknowledgements This work was supported by grants from Department of Science and Technology (DST) and Council of Scientific and Industrial Research (CSIR), New Delhi. S.C. acknowledges UGC, New Delhi for fellowship. References [1] [2] [3] [4] [5]

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