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Multi-walled carbon nanotube modified carbon paste electrode as a sensor for the amperometric detection of l -tryptophan in biological samples
Journal of Colloid and Interface Science 402 (2013) 223–229
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Multi-walled carbon nanotube modified carbon paste electrode as a sensor for the amperometric detection of L-tryptophan in biological samples Tony Thomas a, Ronald J. Mascarenhas a,⇑, Ozma J. D’Souza a, Praveen Martis b, Joseph Dalhalle b, B.E. Kumara Swamy c a b c
Electrochemistry Research Group, Department of Chemistry, St. Joseph’s College, Lalbagh Road, Bangalore 560 027, Karnataka, India Laboratoire de Chimie et d’Electrochimie des Surface, Faculteı˘s Universitaires Notre-Dame de la Paix, 61 Rue de Bruxelles, B-5000 Namur, Belgium Department of Post Graduate Studies and Research in Industrial Chemistry, Jnana Sahyadri, Kuvempu University, Shankaraghatta, Shimoga 577 451, Karnataka, India
a r t i c l e
i n f o
Article history: Received 28 January 2013 Accepted 28 March 2013 Available online 11 April 2013 Keywords: Tryptophan Modified carbon paste electrode Multi-walled carbon nanotubes Casting Amperometry
a b s t r a c t An electrochemical sensor for the amperometric determination of L-tryptophan (Trp) was fabricated by modifying the carbon paste electrode (CPE) with multi-walled carbon nanotubes (MWCNTs) using drop cast method. 4.0 lL of the dispersion containing 2.0 mg of MWCNTs in 1.0 mL of ethanol was drop cast onto the electrode surface and dried in hot air oven to form a stable layer of MWCNTs. The electro-catalytic activity of the modified electrode towards the oxidation of Trp was thoroughly investigated. The modification with MWCNTs has greatly improved the current sensitivity of CPE for the oxidation of Trp. A very minimal amount of the modifier was required to achieve such a high sensitivity. The field emission scanning electron microscopy (FESEM) images revealed a uniform coverage of the surface of CPE by MWCNTs. Nyquist plots revealed the least charge transfer resistance for the modified electrode. The analytical performance of the modified electrode was examined using amperometry under hydro-dynamic conditions. The two linear dynamic ranges observed for Trp were 0.6–9.0 lM and 10.0–100.0 lM. The amperometric determination of Trp did not suffer any interference from other biomolecules. The detection limit of Trp at modified electrode was (3.30 ± 0.37) 108 M (S/N = 3). The analytical applications of the modified electrode were demonstrated by estimating Trp in the spiked milk and biological fluid such as blood serum. The modified electrode showed good reproducibility, long-term stability and anti-fouling effects. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Tryptophan (Trp) is one of the most important essential amino acids having biochemical, nutritional and clinical significance in humans and herbivores [1]. Trp is commonly synthesized in plants and microorganisms from shikimic acid [2]. Trp is abundantly present in oats, milk, chocolates, bananas, yogurt, dried dates, etc. as component of dietary protein while it is scarcely present in vegetables. Hence, it has been supplied through food products to avoid its deficiency [3–5]. Trp is one of the basic constituents of protein and a requisite in human nutrition to establish and maintain a positive nitrogen balance [6]. Trp acts as precursor for serotonin, melatonin and niacin [7]. Improper metabolism of Trp accumulates toxic products in brain which causes hallucinations, delusions and schizophrenia [8]. An overdose of Trp creates drowsiness, nausea, dizziness and loss of appetite [9]. The level of Trp in blood closely relates to serotonin and melatonin level in the brain
⇑ Corresponding author. Fax: +91 8022245831. E-mail address: [email protected] (R.J. Mascarenhas). 0021-9797/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2013.03.059
while the same in plasma relates to hepatic disease [10]. Hence, Trp is very essential for people with anxiety, sleep deprivation and the need for mood enhancement. It is also used as a sleep aid, nutraceutical and antidepressant [11]. Therefore, developing a simple, fast, inexpensive and accurate method for the determination of Trp in food products, pharmaceuticals and biological fluids is necessary and is likely to have great significance in life science research and drug analysis. Several methods that have been used for the determination of Trp in different samples include chromatography, chemiluminescence, spectrophotometry, fluorimetry, flow injection analysis and electrophoresis [12,13]. Nevertheless, these methods are complex, time-consuming, expensive and often suffer from selectivity or specificity and pretreatment or require derivatization prior to its determination [14]. Trp being an electroactive compound, electroanalytical techniques provide an alternate way to analyze Trp with certain advantages such as quick response, high sensitivity, high selectivity, inexpensiveness, amenability to miniaturization, low power consumption and wide linear dynamic range [15]. However, the electrochemical detection of Trp faces some problems. At traditional working electrodes, Trp follows a sluggish kinetics and
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has very high oxidation overpotential [16,17]. The other electroactive biomolecules which coexist with Trp in biological matrices interfere with the determination of Trp due to their similar oxidation peak potentials. These problems are solved by modifying the electrodes with suitable materials using various modification methods [18]. Many materials have been used to modify the traditional working electrodes for the determination of Trp [19]. The carbon paste electrode (CPE) has been used in the development of electrochemical sensors for various biologically significant molecules because of its simple method of preparation, renewable surface, low background current, applicability of various modification procedures and above all, its biocompatibility [20–22]. Carbon nanotubes (CNTs) are most promising materials to modify the electrodes because of their unique ability to promote electron transfer kinetics [23]. CNTs are of ultra-light weight with excellent electronic conductivity, high surface area and have thermal and chemical stability [24]. Basically, there are two types of CNTs – single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). SWCNTs contain single graphene roll while MWCNTs contain many such concentric graphene rolls [25]. Different types of CNT modified electrodes are reported in literature to study biologically important compounds [26,27]. They are CNT-polymer nanocomposite electrodes, CNTpaste electrodes, CNT/ sol–gel nanocomposite electrodes and layer-by-layer assembly of CNT film electrode. MWCNTs modified electrodes have also been used for the determination of Trp [28]. Casting is a common route to modify the electrodes. To date, extensively used surfaces for casting are glassy carbon, gold and graphite electrodes, but the surface of carbon paste is not commonly used for this kind of modification. Unlike the other methods of modification, in casting, the modifier covers the entire surface of the electrode and is completely exposed to analyte solution. Hence, it can improve the analytical performance of the electrode in terms of selectivity or specificity towards a particular analyte. A minimum amount of the modifier is used in this kind of modification. Casting of MWCNTs films at CPE have not yet been reported for the determination of Trp. In continuation of our efforts to use CPE and modified CPE for different applications [29–33], our objective in the present work was to develop a simple amperometric sensor for the determination of Trp which is free from all interfering molecules. Even though there are several reports for the fabrication of a sensor for the determination of Trp, the focus happens to be on complicated procedures of modifications. Hence, a sensor with a simple method of preparation is much desired. In this study, our aim was to use a simple and convenient method of modification i.e. casting the MWCNTs on a carbon paste as the underlying surface in order to fabricate a highly sensitive sensor with better lower detection limits and free from interferences from other bio-molecules which coexist with Trp. Electrochemical investigation of Trp at this modified electrode reveals its electro-catalytic activity towards Trp. Analytical applications of the modified electrode were demonstrated by estimating the Trp content in milk and biological sample such as the blood serum.
2. Experimental 2.1. Reagents L-Tryptophan, L-Tyrosine, L-Cysteine, Uric acid, Folic acid (SRL), Dopamine hydrochloride, Epinephrine hydrochloride (Aldrich), Acetaminophen (Micro Labs Ltd.), Ascorbic acid, KH2PO4, H3PO4, NaOH pellets and HClO4 (Merck) were of analytical grade and used as received. All aqueous solutions were prepared with ultra pure water (>18.2 MX cm) from Milli-Q Plus system (Millipore).
Stock solutions of EP, DA were prepared in 0.1 M HClO4, UA, L-Cysteine, Folic acid and L-Tyrosine in 0.1 M NaOH and L-Tryptophan, Acetaminophen and Ascorbic acid in water. Phosphate buffer solutions were prepared from KH2PO4 and NaOH and pH was adjusted using H3PO4 or NaOH. The graphite powder was obtained from Graphite India Ltd.. The thin MWCNTs obtained from Nanocyl SA (Belgium) were synthesized by decomposition of ethylene using the combustion chemical vapor deposition method. The MWCNTs have an average diameter of 10.0 nm and length of several (0.1–10.0) lm. 2.2. Apparatus All electrochemical experiments were performed using ChemiLink model EA-201 Electro Analyzer. A conventional three electrode system was used for all electrochemical experiments, which comprise a bare or modified CPE as working electrode, a platinum wire as auxiliary electrode and all potentials were measured and applied using saturated calomel electrode (SCE) as a reference electrode. The tip of Lugin capillary was set approximately at a distance of 1 mm from the surface of the working electrode – bare and modified CPE in order to minimize the error due to IR drop in the electrolyte. The cyclic voltammetric (CV) studies were performed in quiescent solution and the amperometric experiments were carried out under the hydrodynamic conditions. The electrochemical experiments and voltammetric curves were recorded at room temperature (300.0 K). The surface morphology of the electrodes was studied using field-emission scanning electron microscopy (FESEM) using Quanta 200, FEI, Germany; SUPRA 40 VP, Gemini, Zeiss, Germany. Electrochemical impedance spectroscopy (EIS) was performed using VersaSTAT 3. A digital pH/ mV meter (ELICO LI 614) was employed to measure the pH of the prepared buffer solutions. 2.3. Generation of oxygen functionalities on MWCNTs The oxygen functionalities on the surface of MWCNT are known to improve their electrochemical properties. Hence, the same were generated by treating them with a mixture of concentrated H2SO4 and HNO3 (molar ratio 3:1). In a typical experiment 75.0 mL of conc. H2SO4 (97%) and 25.0 mL of conc. HNO3 (65%) were mixed and added to 1.0 g of MWCNTs in a round-bottomed flask and heated under constant agitation at 50 °C for 8.0 h. It was allowed to cool down to room temperature after which an equal quantity of deionised water was added. It was filtered and the residue was washed several times with deionised water until neutral pH was attained. The residue was then filtered and freeze-dried [34]. 2.4. Preparation of bare and MWCNTs modified carbon paste electrodes After optimization of the ratio of graphite powder to binder, the CPE was prepared by thoroughly hand-mixing the graphite powder and silicone oil in the ratio 70:30 (w/w) in an agate mortar using a pestle to obtain a homogeneous paste. A portion of the resulting homogeneous paste was packed into the cave of the Teflon tube. A copper wire fixed to a graphite rod and inserted into the Teflon tube served to establish electrical contact with the external circuit. 2.0 mg of MWCNTs were dispersed in 1.0 mL of ethanol under ultrasonication to prepare MWCNTs modified carbon paste electrode. 4.0 lL of the above dispersion was drop cast onto the electrode surface and dried in hot air oven to form a stable layer of MWCNTs. The prepared electrode is designated as MCPE/MWCNTs. The surface morphology of CPE and MCPE/MWCNTs was analyzed by recording the FESEM images as shown in Fig. 1a and b.
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3.2. Cyclic voltammetric behaviour of tryptophan at different electrodes
Fig. 1. FESEM images of (a) CPE and (b) MCPE/MWCNTs.
The FESEM images recorded at higher magnification indicate a complete coverage of CPE surface by MWCNTs. The non uniform surface was possibly due to the flakes of graphite in Fig. 1a which was shown to be responsible for the rough irregular surface of CPE and this gets uniformly covered with MWCNTs after the drop cast as shown in Fig. 1b. Formation of MWCNTs layer makes the surface of MCPE/MWCNTs smooth.
3. Results and discussion 3.1. Characterization by electrochemical impedance spectroscopy The interface properties of CPE and MCPE/MWCNTs were compared by recording EIS. The Nyquist plots for CPE and MCPE/ MWCNTs are shown in Fig. 2. EIS data of the electrodes were obtained in ac frequency range of 0.1 Hz to 100 kHz. All experiments were carried out in presence of 5.0 104 M Trp in 0.1 M phosphate buffer of pH 7.0 with an applied potential corresponding to anodic peak potential (Epa) of Trp. The charge transfer resistance (Rct) values can be directly correlated with electron transfer kinetics of Trp at electrode/electrolyte interface. Rct values of Trp at CPE and MCPE/MWCNTs were obtained by fitting the impedance data to a suitable equivalent circuit. The equivalent circuit is shown in the inset of Fig. 2. The Rct values of 5.0 104 M Trp at CPE and MCPE/MWCNTs are 1.61 102 X and 1.13 102 X respectively. It is clear from the Rct values that presence of MWCNTs at CPE facilitates the electron transfer kinetics. Hence, MCPE/MWCNTs were employed for the development of electrochemical sensor for Trp.
Fig. 3 shows the cyclic voltammograms of 5.0 104 M Trp recorded at CPE and MCPE/MWCNTs in 0.1 M phosphate buffer solution of pH 7.0. Trp shows an irreversible electrochemical behaviour at both the electrodes as represented by the equation in Scheme 1. Epa of Trp at CPE and MCPE/MWCNTs were observed at 721.7 ± 6.4 and 641.3 ± 8.7 mV respectively. The anodic peak current of Trp (Ipa) at CPE and MCPE/MWCNTs were measured to be 15.6 ± 1.0 and 75.3 ± 2.4 lA. It is obvious from electrochemical parameters that oxidation of Trp happens more easily at MCPE/ MWCNTs with an enhancement in peak current. Five times increase in current sensitivity was observed for Trp oxidation at MCPE/MWCNTs. Hence, it can be concluded that MCPE/MWCNTs can electrochemically catalyse the oxidation of Trp because of the presence of MWCNTs at the surface of CPE. MWCNTs carry edge plane sites/defects at its open ends and along the tube axis. The edge plane sites act as reactive centres for different biomolecules. These edge plane and the defect sites facilitate the electron transfer kinetics of Trp at MCPE/MWCNTs. This in turn results in improved current sensitivity and low oxidation over potential [35,36]. Also, the increase in surface area upon modification contributes to enhanced current sensitivity. The effect of potential scan rate on the electrochemical oxidation of Trp was investigated at MCPE/MWCNTs using the cyclic voltammetry technique. It was observed that Ipa varies linearly with scan rate in the range 10–150 mV s1 as shown in Fig. 4a. This suggests that electrochemical oxidation of Trp at this electrode is adsorption controlled [37]. The linear regression equation for above range is Ipa (lA) = 29.83–0.8376m (mV s1) with R2 = 0.9895. At CPE, Ipa was observed to vary linearly with square root of scan rate in the scan rate range 10–150 mV s1 as shown in Fig. 4b. The linear regression equation for the same was found to be pffiffiffi Ipa (lA) = 2.06–3.17 m (mV s1)1/2 with R2 = 0.9986. Hence, it was evident that the electrochemical oxidation of Trp was diffusion controlled at CPE. As the concentration of Trp was increased from 1.0 104 M to 4.0 103 M, the Ipa increased linearly with
Fig. 3. Cyclic voltammograms of 5.0 104 M Trp in 0.1 M phosphate buffer of pH 7.0 at CPE (dotted line) and MCPE/MWCNTs (solid line). Scan rate: 50 mV s1.
Fig. 2. Nyquist plots of 5.0 104 M Trp in 0.1 M phosphate buffer of pH 7.0 using different electrodes at the oxidation peak potential of Trp. Inset shows the equivalent circuit used for analysis.
Scheme 1. Electrochemical oxidation reaction of Trp at MCPE/MWCNTs.
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Fig. 4. (a) Plot of variation of Ipa with scan rate for 5.0 104 M Trp at MCPE/ MWCNTs and (b) plot of variation of Ipa with square root of scan rate for 5.0 104 M Trp at CPE in 0.1 M phosphate buffer of pH 7.0. Fig. 6. Plot of Ipa of 1.0 103 M Trp with different quantities of MWCNTs.
a coefficient of determination, R2 = 0.9831 at MCPE/MWCNTs as shown in Fig. 5. The corresponding linear regression equation for Fig. 5 is Ipa (lA) = 40.02–64.19 C (mM). As expected for an irreversible charge transfer, Epa shifts in positive direction with both, the increase in concentration as well as the scan rate.
3.3. Effect of amount of MWCNTs on tryptophan oxidation The amount of MWCNTs used to modify the electrode plays an important role in determining the performance of the sensor. Here, different amount of MWCNTs (1.0–5.0 mg) were dispersed in ethanol keeping the volume of the suspension a constant at 4.0 lL; the dispersion was cast onto the surface of the CPE. The experiments were performed and the corresponding Ipa were recorded at MCPE/MWCNTs. Fig. 6 depicts the variation of Ipa of 1.0 103 M Trp at MCPE/MWCNTs at different amounts of MWCNTs. Maximum current sensitivity was achieved when the electrode was modified with 2.0 mg mL1 suspension. Consequently, thickness of MWCNTs film at modified electrode determines the current sensitivity of electrode. When we used the suspension above 2.0 mg mL1, the thickness of modifier film at CPE surface increased and the electron transfer kinetics became sluggish. Moreover, the back-ground current increased with higher amount of the modifier. These factors led to decreased sensitivity of the electrode. When the suspension was less than 2.0 mg mL1, decrease in current was observed possibly due to an inadequate surface coverage.
Fig. 5. Plot of variation of Ipa with concentration of Trp at MCPE/MWCNTs in phosphate buffer of pH 7.0. Scan rate 50 mV s1.
3.4. Effect of pH The influence of pH on the electrochemical parameters of Trp was investigated by recording the cyclic voltammograms of 1.0 103 M Trp in 0.1 M phosphate buffer solution of different pH ranging from 3.0 to 8.0. As displayed in Fig. 7a maximum Ipa was observed at pH 3.0. The isoelectric point of Trp is 5.86 [38]. Trp exists predominantly as a cation in the pH range 3.0–5.0 due to the protonation of the amino group. The modification with MWCNTs provides a negatively charged surface to CPE. The electrostatic attraction of Trp cation towards MCPE/MWCNTs results in maximum Ipa at pH 3.0. The Ipa decreased till pH 5.0 and again gradually attained a maximum value at pH 7.0. At pH range 5.0– 7.0, Trp behaves as zwitterion and p–p interaction between Trp and MWCNTs overweighs the electrostatic interaction [39]. Hence, the increase in Ipa at pH 7.0 is attributed to the above factor. Trp acts as an anion at pH 8.0 due to deprotonated –COO group and it gets repelled electrostatically from the surface of MCPE/
Fig. 7. Plot of variation of (a) Ipa and (b) Epa of 5.0 104 M Trp in 0.1 M phosphate buffer solutions of different pH at MCPE/MWCNTs. Scan rate 50 mV s1.
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MWCNTs. This accounts for a steep decrease in current observed at pH 8.0. Moreover, it was observed that Epa of Trp at MCPE/MWCNTs shifted towards more negative potential with an increase in pH. This phenomenon indicates the involvement of protons in the electrochemical oxidation of Trp. The linear regression equation can be expressed as; Epa (mV) = 1052.23–54.17 pH with a correlation coefficient of 0.9933. The slope of Fig. 7b is 54.17 mV pH1 which is very close to Nernstian value of 59.1 mV pH1. This suggests that equal number of protons and electrons are transferred in the electrochemical reaction of Trp at MCPE/MWCNTs. This is in accordance with the mechanism of oxidation of Trp as reported elsewhere [40,41].
3.5. Analytical characterization of MCPE/MWCNTs using amperometry Amperometry under hydrodynamic conditions provides a facile electrochemical method to detect Trp at very low concentrations. The amperometric responses of various concentrations of Trp at MCPE/MWCNTs are shown in Fig. 8. Aliquots of Trp were added to the buffer solution under stirred condition and MCPE/MWCNTs gave quick responses. The responses attained stability in 6.0 s. A calibration plot of Ipa versus concentrations of Trp was shown in Fig. 9. The amperometric current response was linear with the concentrations of Trp in the range 0.6–9.0 lM and 10.0– 100.0 lM. The corresponding linear regression equations were Ipa (lA) = 0.0344–0.1065 C (lM) and Ipa (lA) = 1.913–0.109 C (lM) with R2 = 0.9921 and 0.9914 respectively. The detection limit of Trp at MCPE/MWCNTs is (3.30 ± 0.37) 108 M. The quantification limit for Trp at MCPE/MWCNTs is (4.20 ± 0.58) 107 M. Analytical performance of MCPE/MWCNTs has been compared with other reported electrodes and the results are shown in
Fig. 8. Amperometric current response of different concentrations of Trp at MCPE/ MWCNTs in 0.1 M phosphate buffer solution of pH 7.0 under hydrodynamic conditions. Applied potential: 645.0 mV.
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Table 1. Binuclear manganese (II) complex modified CPE requires 60.0 s accumulation time prior to the determination of Trp and moreover, the energy required to oxidize Trp at this electrode was more as compared to MCPE/MWCNTs [42]. The preparation of carbon nanofibers (CNFs) needs sophistication and apart from that none of the analytical applications of CNF–CPE have been reported [43]. Acidic medium was required for the electrocatalysis of CILE towards Trp [44]. An accumulation time of 1100.0 s and 180.0 s was required for the determination of Trp at CPE/ SiO2[45] and ERGO/GCE [46] respectively. PAA/GCE suffers serious interference from tyrosine (Tyr) [47]. This setback was not observed at MCPE/MWCNTs. Trp oxidation at CoSal–CNTPE required a very high oxidation over potential [48]. Ag@C core–shell nanocomposites preparation was tedious and it required an electrochemical pre-treatment of glassy carbon electrode prior to the preparation of Ag@C/GCE [49]. The oxidation overpotential at expensive BDD NWs [50] was very high and it required a highly basic medium for the electrocatalytic oxidation of Trp. A two step procedure was involved in the preparation of GNP/CILE [51]. The MCPE/MWCNTs has certain advantages as compared to glassy carbon (GC) electrode modified with MWCNTs; GC/MWCNT [28] which is cited earlier in section 1 under introduction. The GC/ MWCNT requires an accumulation time before each measurement while MCPE/MWCNTs has an advantage of easily renewable surface and accumulation time is not required before any measurement. The electrochemical oxidation of Trp was observed at 978.0 mV by GC/MWCNT while the same by MCPE/MWCNTs were observed at 641.3 ± 8.7 mV. Hence, Trp requires less oxidation over potential at MCPE/MWCNTs as compared to GC/MWCNT. The trace level detection of Trp at GC/MWCNT was achieved in a solution of pH 3.5. The detection limit of Trp at GC/MWCNT was 2.7 108 M and the linear dynamic range reported was 2.5 107–1 104 M. The detection limit and the linear dynamic range achieved at MCPE/MWCNTs were comparable with that achieved at GC/MWCNT apart from having an additional advantage of achieving it at physiological pH. The interference studies using GC/MWCNT reveal that an accurate determination of Trp was possible in presence of not more than fivefold excess of Tyrosine (Tyr) while even in the presence of 10-fold excess of Tyr the quantification of Trp could be performed at MCPE/ MWCNTs. The applicability of GC/MWCNT has been demonstrated for the determination of Trp in pharmaceuticals while MCPE/ MWCNTs was applicable for the determination of Trp content both in pharmaceuticals as well as in biological samples. Comparing with most of the aforementioned modified electrodes, a much better current sensitivity and wide linear dynamic range is achieved at MCPE/MWCNTs without involving any complicated and time-consuming methods of preparation. The oxidation over potential of Trp was considerably reduced at MCPE/MWCNTs under physiological conditions and so we can easily extend its applications to biological fields. These facts make MCPE/MWCNTs a potential electrochemical sensor for the determination of Trp for various applications. 3.6. Interference studies
Fig. 9. Calibration plot of Ipa versus concentration of Trp at MCPE/MWCNTs.
Trp is found to coexist with many other molecules present in real matrices such as food items and biological samples. Hence, we have examined the amperometric response of Trp in presence of 1.0 105 M L-Tyrosine (Tyr), L-Cysteine (Cys), Uric acid (UA), Folic acid (FA), Dopamine (DA), Epinephrine (EP), Acetaminophen (AAP) and Ascorbic acid (AA). Amperometric response of Trp remains intact in presence of these interfering molecules as shown in Fig. 10. In the presence of 100 fold excess of interfering molecules, amperometric responses of Trp were completely diminished, possibly due to the fouling of the electrode surface by
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Table 1 Comparison of MCPE/MWCNTs with other working electrodes. Electrode CPE/binuclear manganese(II) complex CNF–CPE CILE CPE/SiO2 ERGO/GCE PAA/GCE CoSal–CNTPE Ag@C/GCE BDD NWs GNP/CILE MCPE/MWCNTs
pH 4.1 7.0 2.8 2.0 6.5 7.4 4.0 2.0 11 7.0 7.0
Linear dynamic range (M) 7
6
Detection limit (M) 6
5
1.0 10 –1.0 10 and 1.0 10 –8.0 10 1.0 107–1.19 104 8.0 106–1.0 103 1.0 107–5.0 106 and 5.0 106–5.0 105 2.0 107–4.0 105 1.0 106–5.0 104 5.0 107–5.0 105 1.0 107–1.0 104 5.0 107–5.0 105 5.0 106–9.0 104 6.0 107–9.0 106 and 1.0 105–1.0 104
8
8.0 10 1.0 107 4.8 106 3.6 106 1.0 107 8.1 107 1.0 107 4.0 108 5.0 107 4.0 106 (3.30 ± 0.37) 108
Technique used
Refs.
LSV Amperometry CV LSV LSV DPV DPV LSV DPV SWV Amperometry
[42] [43] [44] [45] [46] [47] [48] [49] [50] [51] Present work
0.1 M phosphate buffer solution of pH 7.0 without subjecting it to any further pretreatment. The amperometric response of Trp was recorded at MCPE/MWCNTs and displayed in Table 2. The good quantitative recoveries and precision of the results observed suggests that the present method is reliable and suitable for the determination of Trp in food and biological samples. Hence, MCPE/ MWCNTs can be successfully employed for the determination of Trp in real samples. 3.8. Reproducibility and stability of the electrode
Fig. 10. Amperometric current response of different concentrations of Trp at MCPE/ MWCNTs in presence of 1.0 105 M L-Tyrosine, L-Cysteine, Uric acid, Folic acid, Dopamine, Epinephrine, Acetaminophen and Ascorbic acid in 0.1 M phosphate buffer solution of pH 7.0 under hydrodynamic conditions. Applied potential: 645.0 mV.
oxidized products of interfering molecules. Fig. 11 shows calibration plot of Trp in presence of interfering molecules. Two linear segments were observed over 0.6–9.0 lM and 10.0–100.0 lM. The linear regression equations for these ranges were Ipa (lA) = 0.0941–0.0967 C (lM) and Ipa (lA) = 1.357–0.094 C (lM) with R2 = 0.9958 and 0.9982 respectively. 3.7. Analytical applications of MCPE/MWCNTs The practical utility of MCPE/MWCNTs was evaluated by employing it for the determination of Trp in the blood serum and cow’s milk. The content of Trp was estimated by standard addition method. Before usage, the milk was filtered through a normal filter paper. The blood serum and the milk were diluted five times with
Fig. 11. Calibration plot of Ipa versus concentration of Trp at MCPE/MWCNTs in presence of 1.0 105 M L-Tyrosine, L-Cysteine, Uric acid, Folic acid, Dopamine, Epinephrine, Acetaminophen and Ascorbic acid in 0.1 M phosphate buffer solution of pH 7.0 under hydrodynamic conditions.
Since the stability of the electrode is an important parameter to decide the fate of a sensor; it was investigated using amperometric technique. The amperometric response of 1.0 104 M Trp at MCPE/MWCNTs was studied by keeping the oxidation potential a constant at 645.0 mV as shown in Fig. 12. A constant amperometric response was achieved for half an hour indicating the antifouling effects and stability of MCPE/MWCNTs towards Trp oxidation. The oxidation products of Trp are not known to adsorb at the electrode surface and had no adverse effect on the analytical performance of MCPE/MWCNTs. The long-term stability of electrode was analyzed by storing it in a dry place for a week. The amperometric response of 5.0 106 M Trp at MCPE/MWCNTs retains 94.6% of its initial activity. Table 2 Results of real sample analysis at MCPE/MWCNTs. Real samples
Trp added (lM)
Trp found (lM)
Recovery (%)
Blood serum
1.0 2.0 3.0
0.95 ± 0.0012 1.91 ± 0.0016 2.93 ± 0.0009
95.0 95.5 97.7
Milk
2.0 4.0 6.0
1.93 ± 0.0014 3.89 ± 0.0018 5.91 ± 0.0011
96.5 97.3 98.5
Fig. 12. Amperometric response of 1.0 104 M Trp at MCPE/MWCNTs for 30.0 min in 0.1 M phosphate buffer of pH 7.0. Applied potential: 645.0 mV.
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MCPE/MWCNTs were prepared separately for five times following the same procedure and the amperometric response of 5.0 106 M Trp was recorded. All electrodes exhibited similar response with a relative standard deviation (RSD) was 3.6%. Therefore, MCPE/MWCNTs possess good reproducibility.
[9] [10] [11] [12]
4. Conclusions
[15] [16] [17] [18]
In this work, we have described a simple and rapid amperometric method for the quantification of Trp under physiological condition by modifying the CPE with MWCNTs. CPE provides a good base for drop casting. Decrease in oxidation overpotential and enhancement in current proved the electrocatalytic activity of MCPE/ MWCNTs as a sensor. The fabricated electrochemical sensor has an excellent stability, reproducibility and anti fouling effects. A very minimum amount of MWCNTs was used for the modification procedure makes the sensor totally inexpensive. Moreover, by this simple method of fabrication a much lower detection limit was achieved without involving any pre-treatment or activation steps. The analytical applicability of the modified electrode has been evaluated by successfully employing it for the determination of Trp in the blood serum and milk. The low cost, simple method of preparation, high efficiency and quick response time makes employing this electrode convenient for routine analysis of Trp in different real samples. Acknowledgments Authors Tony, Swamy and Ronald gratefully acknowledge the financial support rendered by the University Grants Commission, New Delhi of India under the Major Research Project No. UGC. F. No. 38-232/2009 (SR) to carry out the present research work. Authors gratefully acknowledge St. John’s Medical College, Bangalore for providing serum for biological sample analysis. Authors thank Dr Suresh of SSMRV College, Bangalore for providing EIS facility. References [1] H. Wang, H. Cui, A. Zhang, R. Liu, Anal. Commun. 33 (1996) 275–277. [2] A.A. Ensafi, H.K. Maleh, S. Mallakpour, Electroanalysis 24 (2012) 666–675. [3] M. Kia, A. Islamnezhad, S. Shariati, P. Biparva, Korean J. Chem. Eng. 28 (2011) 2064–2068. [4] C. Li, Y. Ya, G. Zhan, Colloids Surf., B 76 (2010) 340–345. [5] J.-B. Raoof, R. Ojani, M. Baghayeri, Sens. Actuat., B 143 (2009) 261–269. [6] W. Li, C. Li, Y. Kuang, P. Deng, S. Zhang, J. Xu, Microchim. Acta 176 (2012) 455– 461. [7] F. Mirrahimi, M.A. Taher, H. Beitollahi, R. Hosseinzadeh, Appl. Organomet. Chem. 26 (2012) 194–198. [8] R.N. Goyal, S. Bishnoi, H. Chasta, M.A. Aziz, M. Oyama, Talanta 85 (2011) 2626– 2631.
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Multi-walled carbon nanotube modified carbon paste electrode as a sensor for the amperometric detection of L-tryptophan in biological samples Tony Thomas a, Ronald J. Mascarenhas a,⇑, Ozma J. D’Souza a, Praveen Martis b, Joseph Dalhalle b, B.E. Kumara Swamy c a b c
Electrochemistry Research Group, Department of Chemistry, St. Joseph’s College, Lalbagh Road, Bangalore 560 027, Karnataka, India Laboratoire de Chimie et d’Electrochimie des Surface, Faculteı˘s Universitaires Notre-Dame de la Paix, 61 Rue de Bruxelles, B-5000 Namur, Belgium Department of Post Graduate Studies and Research in Industrial Chemistry, Jnana Sahyadri, Kuvempu University, Shankaraghatta, Shimoga 577 451, Karnataka, India
a r t i c l e
i n f o
Article history: Received 28 January 2013 Accepted 28 March 2013 Available online 11 April 2013 Keywords: Tryptophan Modified carbon paste electrode Multi-walled carbon nanotubes Casting Amperometry
a b s t r a c t An electrochemical sensor for the amperometric determination of L-tryptophan (Trp) was fabricated by modifying the carbon paste electrode (CPE) with multi-walled carbon nanotubes (MWCNTs) using drop cast method. 4.0 lL of the dispersion containing 2.0 mg of MWCNTs in 1.0 mL of ethanol was drop cast onto the electrode surface and dried in hot air oven to form a stable layer of MWCNTs. The electro-catalytic activity of the modified electrode towards the oxidation of Trp was thoroughly investigated. The modification with MWCNTs has greatly improved the current sensitivity of CPE for the oxidation of Trp. A very minimal amount of the modifier was required to achieve such a high sensitivity. The field emission scanning electron microscopy (FESEM) images revealed a uniform coverage of the surface of CPE by MWCNTs. Nyquist plots revealed the least charge transfer resistance for the modified electrode. The analytical performance of the modified electrode was examined using amperometry under hydro-dynamic conditions. The two linear dynamic ranges observed for Trp were 0.6–9.0 lM and 10.0–100.0 lM. The amperometric determination of Trp did not suffer any interference from other biomolecules. The detection limit of Trp at modified electrode was (3.30 ± 0.37) 108 M (S/N = 3). The analytical applications of the modified electrode were demonstrated by estimating Trp in the spiked milk and biological fluid such as blood serum. The modified electrode showed good reproducibility, long-term stability and anti-fouling effects. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Tryptophan (Trp) is one of the most important essential amino acids having biochemical, nutritional and clinical significance in humans and herbivores [1]. Trp is commonly synthesized in plants and microorganisms from shikimic acid [2]. Trp is abundantly present in oats, milk, chocolates, bananas, yogurt, dried dates, etc. as component of dietary protein while it is scarcely present in vegetables. Hence, it has been supplied through food products to avoid its deficiency [3–5]. Trp is one of the basic constituents of protein and a requisite in human nutrition to establish and maintain a positive nitrogen balance [6]. Trp acts as precursor for serotonin, melatonin and niacin [7]. Improper metabolism of Trp accumulates toxic products in brain which causes hallucinations, delusions and schizophrenia [8]. An overdose of Trp creates drowsiness, nausea, dizziness and loss of appetite [9]. The level of Trp in blood closely relates to serotonin and melatonin level in the brain
⇑ Corresponding author. Fax: +91 8022245831. E-mail address: [email protected] (R.J. Mascarenhas). 0021-9797/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2013.03.059
while the same in plasma relates to hepatic disease [10]. Hence, Trp is very essential for people with anxiety, sleep deprivation and the need for mood enhancement. It is also used as a sleep aid, nutraceutical and antidepressant [11]. Therefore, developing a simple, fast, inexpensive and accurate method for the determination of Trp in food products, pharmaceuticals and biological fluids is necessary and is likely to have great significance in life science research and drug analysis. Several methods that have been used for the determination of Trp in different samples include chromatography, chemiluminescence, spectrophotometry, fluorimetry, flow injection analysis and electrophoresis [12,13]. Nevertheless, these methods are complex, time-consuming, expensive and often suffer from selectivity or specificity and pretreatment or require derivatization prior to its determination [14]. Trp being an electroactive compound, electroanalytical techniques provide an alternate way to analyze Trp with certain advantages such as quick response, high sensitivity, high selectivity, inexpensiveness, amenability to miniaturization, low power consumption and wide linear dynamic range [15]. However, the electrochemical detection of Trp faces some problems. At traditional working electrodes, Trp follows a sluggish kinetics and
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has very high oxidation overpotential [16,17]. The other electroactive biomolecules which coexist with Trp in biological matrices interfere with the determination of Trp due to their similar oxidation peak potentials. These problems are solved by modifying the electrodes with suitable materials using various modification methods [18]. Many materials have been used to modify the traditional working electrodes for the determination of Trp [19]. The carbon paste electrode (CPE) has been used in the development of electrochemical sensors for various biologically significant molecules because of its simple method of preparation, renewable surface, low background current, applicability of various modification procedures and above all, its biocompatibility [20–22]. Carbon nanotubes (CNTs) are most promising materials to modify the electrodes because of their unique ability to promote electron transfer kinetics [23]. CNTs are of ultra-light weight with excellent electronic conductivity, high surface area and have thermal and chemical stability [24]. Basically, there are two types of CNTs – single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). SWCNTs contain single graphene roll while MWCNTs contain many such concentric graphene rolls [25]. Different types of CNT modified electrodes are reported in literature to study biologically important compounds [26,27]. They are CNT-polymer nanocomposite electrodes, CNTpaste electrodes, CNT/ sol–gel nanocomposite electrodes and layer-by-layer assembly of CNT film electrode. MWCNTs modified electrodes have also been used for the determination of Trp [28]. Casting is a common route to modify the electrodes. To date, extensively used surfaces for casting are glassy carbon, gold and graphite electrodes, but the surface of carbon paste is not commonly used for this kind of modification. Unlike the other methods of modification, in casting, the modifier covers the entire surface of the electrode and is completely exposed to analyte solution. Hence, it can improve the analytical performance of the electrode in terms of selectivity or specificity towards a particular analyte. A minimum amount of the modifier is used in this kind of modification. Casting of MWCNTs films at CPE have not yet been reported for the determination of Trp. In continuation of our efforts to use CPE and modified CPE for different applications [29–33], our objective in the present work was to develop a simple amperometric sensor for the determination of Trp which is free from all interfering molecules. Even though there are several reports for the fabrication of a sensor for the determination of Trp, the focus happens to be on complicated procedures of modifications. Hence, a sensor with a simple method of preparation is much desired. In this study, our aim was to use a simple and convenient method of modification i.e. casting the MWCNTs on a carbon paste as the underlying surface in order to fabricate a highly sensitive sensor with better lower detection limits and free from interferences from other bio-molecules which coexist with Trp. Electrochemical investigation of Trp at this modified electrode reveals its electro-catalytic activity towards Trp. Analytical applications of the modified electrode were demonstrated by estimating the Trp content in milk and biological sample such as the blood serum.
2. Experimental 2.1. Reagents L-Tryptophan, L-Tyrosine, L-Cysteine, Uric acid, Folic acid (SRL), Dopamine hydrochloride, Epinephrine hydrochloride (Aldrich), Acetaminophen (Micro Labs Ltd.), Ascorbic acid, KH2PO4, H3PO4, NaOH pellets and HClO4 (Merck) were of analytical grade and used as received. All aqueous solutions were prepared with ultra pure water (>18.2 MX cm) from Milli-Q Plus system (Millipore).
Stock solutions of EP, DA were prepared in 0.1 M HClO4, UA, L-Cysteine, Folic acid and L-Tyrosine in 0.1 M NaOH and L-Tryptophan, Acetaminophen and Ascorbic acid in water. Phosphate buffer solutions were prepared from KH2PO4 and NaOH and pH was adjusted using H3PO4 or NaOH. The graphite powder was obtained from Graphite India Ltd.. The thin MWCNTs obtained from Nanocyl SA (Belgium) were synthesized by decomposition of ethylene using the combustion chemical vapor deposition method. The MWCNTs have an average diameter of 10.0 nm and length of several (0.1–10.0) lm. 2.2. Apparatus All electrochemical experiments were performed using ChemiLink model EA-201 Electro Analyzer. A conventional three electrode system was used for all electrochemical experiments, which comprise a bare or modified CPE as working electrode, a platinum wire as auxiliary electrode and all potentials were measured and applied using saturated calomel electrode (SCE) as a reference electrode. The tip of Lugin capillary was set approximately at a distance of 1 mm from the surface of the working electrode – bare and modified CPE in order to minimize the error due to IR drop in the electrolyte. The cyclic voltammetric (CV) studies were performed in quiescent solution and the amperometric experiments were carried out under the hydrodynamic conditions. The electrochemical experiments and voltammetric curves were recorded at room temperature (300.0 K). The surface morphology of the electrodes was studied using field-emission scanning electron microscopy (FESEM) using Quanta 200, FEI, Germany; SUPRA 40 VP, Gemini, Zeiss, Germany. Electrochemical impedance spectroscopy (EIS) was performed using VersaSTAT 3. A digital pH/ mV meter (ELICO LI 614) was employed to measure the pH of the prepared buffer solutions. 2.3. Generation of oxygen functionalities on MWCNTs The oxygen functionalities on the surface of MWCNT are known to improve their electrochemical properties. Hence, the same were generated by treating them with a mixture of concentrated H2SO4 and HNO3 (molar ratio 3:1). In a typical experiment 75.0 mL of conc. H2SO4 (97%) and 25.0 mL of conc. HNO3 (65%) were mixed and added to 1.0 g of MWCNTs in a round-bottomed flask and heated under constant agitation at 50 °C for 8.0 h. It was allowed to cool down to room temperature after which an equal quantity of deionised water was added. It was filtered and the residue was washed several times with deionised water until neutral pH was attained. The residue was then filtered and freeze-dried [34]. 2.4. Preparation of bare and MWCNTs modified carbon paste electrodes After optimization of the ratio of graphite powder to binder, the CPE was prepared by thoroughly hand-mixing the graphite powder and silicone oil in the ratio 70:30 (w/w) in an agate mortar using a pestle to obtain a homogeneous paste. A portion of the resulting homogeneous paste was packed into the cave of the Teflon tube. A copper wire fixed to a graphite rod and inserted into the Teflon tube served to establish electrical contact with the external circuit. 2.0 mg of MWCNTs were dispersed in 1.0 mL of ethanol under ultrasonication to prepare MWCNTs modified carbon paste electrode. 4.0 lL of the above dispersion was drop cast onto the electrode surface and dried in hot air oven to form a stable layer of MWCNTs. The prepared electrode is designated as MCPE/MWCNTs. The surface morphology of CPE and MCPE/MWCNTs was analyzed by recording the FESEM images as shown in Fig. 1a and b.
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3.2. Cyclic voltammetric behaviour of tryptophan at different electrodes
Fig. 1. FESEM images of (a) CPE and (b) MCPE/MWCNTs.
The FESEM images recorded at higher magnification indicate a complete coverage of CPE surface by MWCNTs. The non uniform surface was possibly due to the flakes of graphite in Fig. 1a which was shown to be responsible for the rough irregular surface of CPE and this gets uniformly covered with MWCNTs after the drop cast as shown in Fig. 1b. Formation of MWCNTs layer makes the surface of MCPE/MWCNTs smooth.
3. Results and discussion 3.1. Characterization by electrochemical impedance spectroscopy The interface properties of CPE and MCPE/MWCNTs were compared by recording EIS. The Nyquist plots for CPE and MCPE/ MWCNTs are shown in Fig. 2. EIS data of the electrodes were obtained in ac frequency range of 0.1 Hz to 100 kHz. All experiments were carried out in presence of 5.0 104 M Trp in 0.1 M phosphate buffer of pH 7.0 with an applied potential corresponding to anodic peak potential (Epa) of Trp. The charge transfer resistance (Rct) values can be directly correlated with electron transfer kinetics of Trp at electrode/electrolyte interface. Rct values of Trp at CPE and MCPE/MWCNTs were obtained by fitting the impedance data to a suitable equivalent circuit. The equivalent circuit is shown in the inset of Fig. 2. The Rct values of 5.0 104 M Trp at CPE and MCPE/MWCNTs are 1.61 102 X and 1.13 102 X respectively. It is clear from the Rct values that presence of MWCNTs at CPE facilitates the electron transfer kinetics. Hence, MCPE/MWCNTs were employed for the development of electrochemical sensor for Trp.
Fig. 3 shows the cyclic voltammograms of 5.0 104 M Trp recorded at CPE and MCPE/MWCNTs in 0.1 M phosphate buffer solution of pH 7.0. Trp shows an irreversible electrochemical behaviour at both the electrodes as represented by the equation in Scheme 1. Epa of Trp at CPE and MCPE/MWCNTs were observed at 721.7 ± 6.4 and 641.3 ± 8.7 mV respectively. The anodic peak current of Trp (Ipa) at CPE and MCPE/MWCNTs were measured to be 15.6 ± 1.0 and 75.3 ± 2.4 lA. It is obvious from electrochemical parameters that oxidation of Trp happens more easily at MCPE/ MWCNTs with an enhancement in peak current. Five times increase in current sensitivity was observed for Trp oxidation at MCPE/MWCNTs. Hence, it can be concluded that MCPE/MWCNTs can electrochemically catalyse the oxidation of Trp because of the presence of MWCNTs at the surface of CPE. MWCNTs carry edge plane sites/defects at its open ends and along the tube axis. The edge plane sites act as reactive centres for different biomolecules. These edge plane and the defect sites facilitate the electron transfer kinetics of Trp at MCPE/MWCNTs. This in turn results in improved current sensitivity and low oxidation over potential [35,36]. Also, the increase in surface area upon modification contributes to enhanced current sensitivity. The effect of potential scan rate on the electrochemical oxidation of Trp was investigated at MCPE/MWCNTs using the cyclic voltammetry technique. It was observed that Ipa varies linearly with scan rate in the range 10–150 mV s1 as shown in Fig. 4a. This suggests that electrochemical oxidation of Trp at this electrode is adsorption controlled [37]. The linear regression equation for above range is Ipa (lA) = 29.83–0.8376m (mV s1) with R2 = 0.9895. At CPE, Ipa was observed to vary linearly with square root of scan rate in the scan rate range 10–150 mV s1 as shown in Fig. 4b. The linear regression equation for the same was found to be pffiffiffi Ipa (lA) = 2.06–3.17 m (mV s1)1/2 with R2 = 0.9986. Hence, it was evident that the electrochemical oxidation of Trp was diffusion controlled at CPE. As the concentration of Trp was increased from 1.0 104 M to 4.0 103 M, the Ipa increased linearly with
Fig. 3. Cyclic voltammograms of 5.0 104 M Trp in 0.1 M phosphate buffer of pH 7.0 at CPE (dotted line) and MCPE/MWCNTs (solid line). Scan rate: 50 mV s1.
Fig. 2. Nyquist plots of 5.0 104 M Trp in 0.1 M phosphate buffer of pH 7.0 using different electrodes at the oxidation peak potential of Trp. Inset shows the equivalent circuit used for analysis.
Scheme 1. Electrochemical oxidation reaction of Trp at MCPE/MWCNTs.
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Fig. 4. (a) Plot of variation of Ipa with scan rate for 5.0 104 M Trp at MCPE/ MWCNTs and (b) plot of variation of Ipa with square root of scan rate for 5.0 104 M Trp at CPE in 0.1 M phosphate buffer of pH 7.0. Fig. 6. Plot of Ipa of 1.0 103 M Trp with different quantities of MWCNTs.
a coefficient of determination, R2 = 0.9831 at MCPE/MWCNTs as shown in Fig. 5. The corresponding linear regression equation for Fig. 5 is Ipa (lA) = 40.02–64.19 C (mM). As expected for an irreversible charge transfer, Epa shifts in positive direction with both, the increase in concentration as well as the scan rate.
3.3. Effect of amount of MWCNTs on tryptophan oxidation The amount of MWCNTs used to modify the electrode plays an important role in determining the performance of the sensor. Here, different amount of MWCNTs (1.0–5.0 mg) were dispersed in ethanol keeping the volume of the suspension a constant at 4.0 lL; the dispersion was cast onto the surface of the CPE. The experiments were performed and the corresponding Ipa were recorded at MCPE/MWCNTs. Fig. 6 depicts the variation of Ipa of 1.0 103 M Trp at MCPE/MWCNTs at different amounts of MWCNTs. Maximum current sensitivity was achieved when the electrode was modified with 2.0 mg mL1 suspension. Consequently, thickness of MWCNTs film at modified electrode determines the current sensitivity of electrode. When we used the suspension above 2.0 mg mL1, the thickness of modifier film at CPE surface increased and the electron transfer kinetics became sluggish. Moreover, the back-ground current increased with higher amount of the modifier. These factors led to decreased sensitivity of the electrode. When the suspension was less than 2.0 mg mL1, decrease in current was observed possibly due to an inadequate surface coverage.
Fig. 5. Plot of variation of Ipa with concentration of Trp at MCPE/MWCNTs in phosphate buffer of pH 7.0. Scan rate 50 mV s1.
3.4. Effect of pH The influence of pH on the electrochemical parameters of Trp was investigated by recording the cyclic voltammograms of 1.0 103 M Trp in 0.1 M phosphate buffer solution of different pH ranging from 3.0 to 8.0. As displayed in Fig. 7a maximum Ipa was observed at pH 3.0. The isoelectric point of Trp is 5.86 [38]. Trp exists predominantly as a cation in the pH range 3.0–5.0 due to the protonation of the amino group. The modification with MWCNTs provides a negatively charged surface to CPE. The electrostatic attraction of Trp cation towards MCPE/MWCNTs results in maximum Ipa at pH 3.0. The Ipa decreased till pH 5.0 and again gradually attained a maximum value at pH 7.0. At pH range 5.0– 7.0, Trp behaves as zwitterion and p–p interaction between Trp and MWCNTs overweighs the electrostatic interaction [39]. Hence, the increase in Ipa at pH 7.0 is attributed to the above factor. Trp acts as an anion at pH 8.0 due to deprotonated –COO group and it gets repelled electrostatically from the surface of MCPE/
Fig. 7. Plot of variation of (a) Ipa and (b) Epa of 5.0 104 M Trp in 0.1 M phosphate buffer solutions of different pH at MCPE/MWCNTs. Scan rate 50 mV s1.
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MWCNTs. This accounts for a steep decrease in current observed at pH 8.0. Moreover, it was observed that Epa of Trp at MCPE/MWCNTs shifted towards more negative potential with an increase in pH. This phenomenon indicates the involvement of protons in the electrochemical oxidation of Trp. The linear regression equation can be expressed as; Epa (mV) = 1052.23–54.17 pH with a correlation coefficient of 0.9933. The slope of Fig. 7b is 54.17 mV pH1 which is very close to Nernstian value of 59.1 mV pH1. This suggests that equal number of protons and electrons are transferred in the electrochemical reaction of Trp at MCPE/MWCNTs. This is in accordance with the mechanism of oxidation of Trp as reported elsewhere [40,41].
3.5. Analytical characterization of MCPE/MWCNTs using amperometry Amperometry under hydrodynamic conditions provides a facile electrochemical method to detect Trp at very low concentrations. The amperometric responses of various concentrations of Trp at MCPE/MWCNTs are shown in Fig. 8. Aliquots of Trp were added to the buffer solution under stirred condition and MCPE/MWCNTs gave quick responses. The responses attained stability in 6.0 s. A calibration plot of Ipa versus concentrations of Trp was shown in Fig. 9. The amperometric current response was linear with the concentrations of Trp in the range 0.6–9.0 lM and 10.0– 100.0 lM. The corresponding linear regression equations were Ipa (lA) = 0.0344–0.1065 C (lM) and Ipa (lA) = 1.913–0.109 C (lM) with R2 = 0.9921 and 0.9914 respectively. The detection limit of Trp at MCPE/MWCNTs is (3.30 ± 0.37) 108 M. The quantification limit for Trp at MCPE/MWCNTs is (4.20 ± 0.58) 107 M. Analytical performance of MCPE/MWCNTs has been compared with other reported electrodes and the results are shown in
Fig. 8. Amperometric current response of different concentrations of Trp at MCPE/ MWCNTs in 0.1 M phosphate buffer solution of pH 7.0 under hydrodynamic conditions. Applied potential: 645.0 mV.
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Table 1. Binuclear manganese (II) complex modified CPE requires 60.0 s accumulation time prior to the determination of Trp and moreover, the energy required to oxidize Trp at this electrode was more as compared to MCPE/MWCNTs [42]. The preparation of carbon nanofibers (CNFs) needs sophistication and apart from that none of the analytical applications of CNF–CPE have been reported [43]. Acidic medium was required for the electrocatalysis of CILE towards Trp [44]. An accumulation time of 1100.0 s and 180.0 s was required for the determination of Trp at CPE/ SiO2[45] and ERGO/GCE [46] respectively. PAA/GCE suffers serious interference from tyrosine (Tyr) [47]. This setback was not observed at MCPE/MWCNTs. Trp oxidation at CoSal–CNTPE required a very high oxidation over potential [48]. Ag@C core–shell nanocomposites preparation was tedious and it required an electrochemical pre-treatment of glassy carbon electrode prior to the preparation of Ag@C/GCE [49]. The oxidation overpotential at expensive BDD NWs [50] was very high and it required a highly basic medium for the electrocatalytic oxidation of Trp. A two step procedure was involved in the preparation of GNP/CILE [51]. The MCPE/MWCNTs has certain advantages as compared to glassy carbon (GC) electrode modified with MWCNTs; GC/MWCNT [28] which is cited earlier in section 1 under introduction. The GC/ MWCNT requires an accumulation time before each measurement while MCPE/MWCNTs has an advantage of easily renewable surface and accumulation time is not required before any measurement. The electrochemical oxidation of Trp was observed at 978.0 mV by GC/MWCNT while the same by MCPE/MWCNTs were observed at 641.3 ± 8.7 mV. Hence, Trp requires less oxidation over potential at MCPE/MWCNTs as compared to GC/MWCNT. The trace level detection of Trp at GC/MWCNT was achieved in a solution of pH 3.5. The detection limit of Trp at GC/MWCNT was 2.7 108 M and the linear dynamic range reported was 2.5 107–1 104 M. The detection limit and the linear dynamic range achieved at MCPE/MWCNTs were comparable with that achieved at GC/MWCNT apart from having an additional advantage of achieving it at physiological pH. The interference studies using GC/MWCNT reveal that an accurate determination of Trp was possible in presence of not more than fivefold excess of Tyrosine (Tyr) while even in the presence of 10-fold excess of Tyr the quantification of Trp could be performed at MCPE/ MWCNTs. The applicability of GC/MWCNT has been demonstrated for the determination of Trp in pharmaceuticals while MCPE/ MWCNTs was applicable for the determination of Trp content both in pharmaceuticals as well as in biological samples. Comparing with most of the aforementioned modified electrodes, a much better current sensitivity and wide linear dynamic range is achieved at MCPE/MWCNTs without involving any complicated and time-consuming methods of preparation. The oxidation over potential of Trp was considerably reduced at MCPE/MWCNTs under physiological conditions and so we can easily extend its applications to biological fields. These facts make MCPE/MWCNTs a potential electrochemical sensor for the determination of Trp for various applications. 3.6. Interference studies
Fig. 9. Calibration plot of Ipa versus concentration of Trp at MCPE/MWCNTs.
Trp is found to coexist with many other molecules present in real matrices such as food items and biological samples. Hence, we have examined the amperometric response of Trp in presence of 1.0 105 M L-Tyrosine (Tyr), L-Cysteine (Cys), Uric acid (UA), Folic acid (FA), Dopamine (DA), Epinephrine (EP), Acetaminophen (AAP) and Ascorbic acid (AA). Amperometric response of Trp remains intact in presence of these interfering molecules as shown in Fig. 10. In the presence of 100 fold excess of interfering molecules, amperometric responses of Trp were completely diminished, possibly due to the fouling of the electrode surface by
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Table 1 Comparison of MCPE/MWCNTs with other working electrodes. Electrode CPE/binuclear manganese(II) complex CNF–CPE CILE CPE/SiO2 ERGO/GCE PAA/GCE CoSal–CNTPE Ag@C/GCE BDD NWs GNP/CILE MCPE/MWCNTs
pH 4.1 7.0 2.8 2.0 6.5 7.4 4.0 2.0 11 7.0 7.0
Linear dynamic range (M) 7
6
Detection limit (M) 6
5
1.0 10 –1.0 10 and 1.0 10 –8.0 10 1.0 107–1.19 104 8.0 106–1.0 103 1.0 107–5.0 106 and 5.0 106–5.0 105 2.0 107–4.0 105 1.0 106–5.0 104 5.0 107–5.0 105 1.0 107–1.0 104 5.0 107–5.0 105 5.0 106–9.0 104 6.0 107–9.0 106 and 1.0 105–1.0 104
8
8.0 10 1.0 107 4.8 106 3.6 106 1.0 107 8.1 107 1.0 107 4.0 108 5.0 107 4.0 106 (3.30 ± 0.37) 108
Technique used
Refs.
LSV Amperometry CV LSV LSV DPV DPV LSV DPV SWV Amperometry
[42] [43] [44] [45] [46] [47] [48] [49] [50] [51] Present work
0.1 M phosphate buffer solution of pH 7.0 without subjecting it to any further pretreatment. The amperometric response of Trp was recorded at MCPE/MWCNTs and displayed in Table 2. The good quantitative recoveries and precision of the results observed suggests that the present method is reliable and suitable for the determination of Trp in food and biological samples. Hence, MCPE/ MWCNTs can be successfully employed for the determination of Trp in real samples. 3.8. Reproducibility and stability of the electrode
Fig. 10. Amperometric current response of different concentrations of Trp at MCPE/ MWCNTs in presence of 1.0 105 M L-Tyrosine, L-Cysteine, Uric acid, Folic acid, Dopamine, Epinephrine, Acetaminophen and Ascorbic acid in 0.1 M phosphate buffer solution of pH 7.0 under hydrodynamic conditions. Applied potential: 645.0 mV.
oxidized products of interfering molecules. Fig. 11 shows calibration plot of Trp in presence of interfering molecules. Two linear segments were observed over 0.6–9.0 lM and 10.0–100.0 lM. The linear regression equations for these ranges were Ipa (lA) = 0.0941–0.0967 C (lM) and Ipa (lA) = 1.357–0.094 C (lM) with R2 = 0.9958 and 0.9982 respectively. 3.7. Analytical applications of MCPE/MWCNTs The practical utility of MCPE/MWCNTs was evaluated by employing it for the determination of Trp in the blood serum and cow’s milk. The content of Trp was estimated by standard addition method. Before usage, the milk was filtered through a normal filter paper. The blood serum and the milk were diluted five times with
Fig. 11. Calibration plot of Ipa versus concentration of Trp at MCPE/MWCNTs in presence of 1.0 105 M L-Tyrosine, L-Cysteine, Uric acid, Folic acid, Dopamine, Epinephrine, Acetaminophen and Ascorbic acid in 0.1 M phosphate buffer solution of pH 7.0 under hydrodynamic conditions.
Since the stability of the electrode is an important parameter to decide the fate of a sensor; it was investigated using amperometric technique. The amperometric response of 1.0 104 M Trp at MCPE/MWCNTs was studied by keeping the oxidation potential a constant at 645.0 mV as shown in Fig. 12. A constant amperometric response was achieved for half an hour indicating the antifouling effects and stability of MCPE/MWCNTs towards Trp oxidation. The oxidation products of Trp are not known to adsorb at the electrode surface and had no adverse effect on the analytical performance of MCPE/MWCNTs. The long-term stability of electrode was analyzed by storing it in a dry place for a week. The amperometric response of 5.0 106 M Trp at MCPE/MWCNTs retains 94.6% of its initial activity. Table 2 Results of real sample analysis at MCPE/MWCNTs. Real samples
Trp added (lM)
Trp found (lM)
Recovery (%)
Blood serum
1.0 2.0 3.0
0.95 ± 0.0012 1.91 ± 0.0016 2.93 ± 0.0009
95.0 95.5 97.7
Milk
2.0 4.0 6.0
1.93 ± 0.0014 3.89 ± 0.0018 5.91 ± 0.0011
96.5 97.3 98.5
Fig. 12. Amperometric response of 1.0 104 M Trp at MCPE/MWCNTs for 30.0 min in 0.1 M phosphate buffer of pH 7.0. Applied potential: 645.0 mV.
T. Thomas et al. / Journal of Colloid and Interface Science 402 (2013) 223–229
MCPE/MWCNTs were prepared separately for five times following the same procedure and the amperometric response of 5.0 106 M Trp was recorded. All electrodes exhibited similar response with a relative standard deviation (RSD) was 3.6%. Therefore, MCPE/MWCNTs possess good reproducibility.
[9] [10] [11] [12]
4. Conclusions
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In this work, we have described a simple and rapid amperometric method for the quantification of Trp under physiological condition by modifying the CPE with MWCNTs. CPE provides a good base for drop casting. Decrease in oxidation overpotential and enhancement in current proved the electrocatalytic activity of MCPE/ MWCNTs as a sensor. The fabricated electrochemical sensor has an excellent stability, reproducibility and anti fouling effects. A very minimum amount of MWCNTs was used for the modification procedure makes the sensor totally inexpensive. Moreover, by this simple method of fabrication a much lower detection limit was achieved without involving any pre-treatment or activation steps. The analytical applicability of the modified electrode has been evaluated by successfully employing it for the determination of Trp in the blood serum and milk. The low cost, simple method of preparation, high efficiency and quick response time makes employing this electrode convenient for routine analysis of Trp in different real samples. Acknowledgments Authors Tony, Swamy and Ronald gratefully acknowledge the financial support rendered by the University Grants Commission, New Delhi of India under the Major Research Project No. UGC. F. No. 38-232/2009 (SR) to carry out the present research work. Authors gratefully acknowledge St. John’s Medical College, Bangalore for providing serum for biological sample analysis. Authors thank Dr Suresh of SSMRV College, Bangalore for providing EIS facility. References [1] H. Wang, H. Cui, A. Zhang, R. Liu, Anal. Commun. 33 (1996) 275–277. [2] A.A. Ensafi, H.K. Maleh, S. Mallakpour, Electroanalysis 24 (2012) 666–675. [3] M. Kia, A. Islamnezhad, S. Shariati, P. Biparva, Korean J. Chem. Eng. 28 (2011) 2064–2068. [4] C. Li, Y. Ya, G. Zhan, Colloids Surf., B 76 (2010) 340–345. [5] J.-B. Raoof, R. Ojani, M. Baghayeri, Sens. Actuat., B 143 (2009) 261–269. [6] W. Li, C. Li, Y. Kuang, P. Deng, S. Zhang, J. Xu, Microchim. Acta 176 (2012) 455– 461. [7] F. Mirrahimi, M.A. Taher, H. Beitollahi, R. Hosseinzadeh, Appl. Organomet. Chem. 26 (2012) 194–198. [8] R.N. Goyal, S. Bishnoi, H. Chasta, M.A. Aziz, M. Oyama, Talanta 85 (2011) 2626– 2631.
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