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Determination of tryptophan using electrode modified with poly(9-aminoacridine) functionalized multi-walled carbon nanotubes
Electrochimica Acta 57 (2011) 290–296
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Determination of tryptophan using electrode modified with poly(9-aminoacridine) functionalized multi-walled carbon nanotubes Sevgi Güney ∗ , Gülcemal Yıldız Department of Chemistry, Faculty of Science and Letters, Istanbul Technical University, Maslak, Istanbul 34469, Turkey
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Article history: Received 29 November 2010 Received in revised form 31 May 2011 Accepted 2 June 2011 Available online 13 June 2011 Keywords: Carbon nanotubes 9-Aminoacridine Tryptophan Tyrosine Electrochemical sensor
a b s t r a c t Two new kinds of electrochemical sensors were prepared using glassy carbon electrodes modified with poly(9-aminoacridine) (PAA) and PAA-functionalized multi-walled carbon nanotubes (PAA-MWCNTs) for the determination of tryptophan (Trp) in the presence of tyrosine (Tyr). The electrocatalytic response of the modified electrodes towards electrooxidation of Trp and Tyr was investigated and compared by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The surface of the modified electrodes was characterized using CV, scanning electron microscopy, scanning-tunneling microscopy and scanningtunneling spectroscopy. The anodic peak current of the electrooxidation of Trp on PAA-MWCNTs electrode was 5.5-fold increased comparing to PAA modified electrode. The anodic peak potential of Trp is shifted about 0.12 V to more negative value indicated that PAA-MWCNTs electrode has better electrocatalytic activity for electrooxidation of Trp. Furthermore, the anodic peak current which was attributed to the electrooxidation of Trp did almost not change with the addition of Tyr in buffer solution at pH 3.5. Besides this, at pH 7.4, the anodic peak current obtained at 0.6 V increased as the concentration of Tyr was increased. The results demonstrate that PAA-MWCNTs electrode can be used for the simultaneous determination of Trp and Tyr by adjusting the pH of the solution. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Determination of amino acids has been attracting more attention since they are the basic units of enzymes and proteins. A lot of research papers related to the electroanalytical determination of amino acids by chemically modified electrodes (CMEs) have been published in the last few decades [1,2]. Trp is a vital constituent of proteins and indispensable in human nutrition for establishing and maintaining a positive nitrogen balance and is also a precursor of the neurotransmitter serotonin. Tyr is the precursor of dopamine, thyroxin, dopa and epinephrine hormone or neurotransmitters in mammalian central nervous systems. The concentration of Tyr in culture medium is very important. While a high concentration of Tyr results increase in sister chromatid exchange, the absence of Tyr could cause albinism and alkaptonuria. Therefore, the determination of Tyr is a vital issue. There are several techniques for determination of Trp and Tyr including electroanalytical methods [3–6]. However, the voltammetric response of electroanalytical methods is not sufficient
∗ Corresponding author. Tel.: +90 212 285 32 46; fax: +90 212 285 63 86. E-mail address: [email protected] (S. Güney). 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.06.003
because of slow heterogeneous electron transfer at the electrode surface. Therefore, CMEs have been designed to improve the electron transfer characteristics and the sensitivity. CMEs have been constructed by immobilization of different organic and inorganic modifiers on the surface of electrode. CMEs were developed for the improvement of sensitivity, stability and selectivity in analytical applications to use as amperometric, voltammetric and potentiometric sensors. CMEs provide higher rates for the electrode reaction and show higher selectivity and sensitivity for determination of various compounds compared with ordinary electrodes [7,8]. One of the common methods for designing of CMEs involves applying of polymer films to the surface of the solid electrodes. This method offers several advantages, such as a chemical and physical stability, a good conduction, simplicity of preparation and reusability. The modification of electrode with the polymer offers superior properties for the determination of bio-molecules since the polymers are chemically stable in electrochemical deposition and also strongly and homogeneously adherent to electrode surface as a film [9,10]. Carbon nanotubes (CNTs) have been attractive electrode materials because of their excellent mechanical strength and electrical conductivity as well as being comparatively chemically inert in most electrolyte solutions. Usage of CNTs as the modification
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material on glassy carbon (GC) electrode supply better surface property for immobilizing of chemical and biological active species on the sensing electrode and also improved sensitivity due to great chemical and electrical properties of CNTs [11]. In addition, GC electrode (GCE) modified with CNTs exhibits electrochemical behavior with antifouling of the electrode. The other promising molecule as a modification agent, acridine, is structurally similar to anthracene but contains a nitrogen atom in its central ring. The usage of 9-aminoacridine (9-AA) and multiwalled carbon nanotubes (MWCNTs) together as a modifier for the fabrication of biosensor has never been reported until our current work. In this study, we applied 9-AA as a modifier to fabricate a PAA modified GCE (PAA/GCE) by electropolymerization. The surface of the modified electrode was characterized using CV method, scanning electron microscopy (SEM), scanning-tunneling microscopy (STM) and scanning-tunneling spectroscopy (STS). The electrocatalytic activities of the modified and unmodified electrodes towards aminoacid detection were studied and compared. Furthermore, we fabricated PAA-functionalized multi-walled carbon nanotubes modified GCE (PAA-MWCNTs/GCE) by drop deposition of homogeneous solution of 9-AA and MWCNTs on to the electrode surface followed by electrocycling in phosphate buffer solution at pH 6.0. The peak current obviously increased and the peak potential shifted to negative direction by using PAA-MWCNTs modified GCE compared with the bare, PAA only and MWCNTs only modified GCEs. The experimental results revealed that Trp and Tyr can be determined simultaneously by adjusting the pH of the solution.
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2.3. Preparation of PAA modified electrode Prior to modification, GCE surface was polished with 1, 0.3 and 0.05 m alumina slurry using a polishing cloth to produce a mirrorlike surface, respectively and then rinsed distilled water, sonicated 3 min into water and ethanol, and dried in air. For the surface characterization, prior to the modification, ITO coated glass sheet was sonicated in acetone, ethanol and distilled water, dried with a stream of nitrogen. Next, the working electrode was immersed into 0.1 M PBS at pH 6.0 and electroactivation was performed by applying 20 voltammetric cycles between −1.0 and 2.0 V versus Ag/AgCl at 100 mV s−1 . Finally, the solution of 9-AA with 5.0 × 10−5 M was added to the buffer solution and the electrode was modified by applying 20 voltammetric cycles between −1.2 and 2.5 V versus Ag/AgCl at 100 mV s−1 . When the electropolymerization was completed, the electrode was rinsed with distilled water to remove soluble species from the surface of the film. The modified electrode was stored in PBS at pH 6.0 at room temperature when not in use. The modification of ITO electrode was performed by the same procedure.
2.4. Preparation of MWCNTs-modified electrode 0.1 mg of MWCNTs was dispersed in DMF with the ultrasonic stirring to form a black solution. The 20 L of this solution was dropped on the polished GCE surface and spun at 3500 rpm for 120 s. DMF was evaporated in the air to form a MWCNTs modified electrode.
2. Experimental 2.1. Apparatus 2.5. Preparation of PAA-MWCNTs modified electrode Voltammetric measurements were performed with an Autolab PGSTAT 30 (Eco Chemie) potentiostat. All voltammetric experiments were conducted in a general-purpose cell stand (BAS C-3), having classical three-electrode cell with a platinum wire as a counter electrode (BAS MW 1032) and an Ag/AgCl/3 M KCl as a reference (BAS MF 2052) electrode. The working electrode was a GCE with 3 mm diameter (BAS MF-2012). For the microscopic measurements, an indium tin oxide (ITO) coated glass sheet (1.0 cm2 ) was used as the working electrode. All solutions were purged for 10 min with pure nitrogen and an inert atmosphere was maintained during the experiments. The pH values of the solutions were recorded using a pH-meter (WTW Inolab pH 720). An ultrasonic bath was used for preparing the solutions (Bandelin Sonorex RK 100H). SEM measurements were performed with a Nanoeye SNE-3000M MiniSEM. STM and STS measurements were performed with a Nanosurf Easyscan 2 Controller NanoBEEs.
The aqueous solution of 9-AA.HCl.H2 O was prepared and titrated with ammonium carbonate to obtained the neutral form of 9-AA. The precipitated product in aqueous solution was filtered and washed with distilled water to eliminate the ammonium chloride from the precipitation and dried in vacuum oven at 40 ◦ C. The homogeneous solutions of 9-AA and MWCNTs in DMF were prepared by dissolving of 0.2 mg each of them in different glass tubes. After that, two solutions were mixed. The 20 L aliquot of this solution was dropped on the polished GCE surface and spun at 3500 rpm for 120 s. ITO electrode was also modified with the same procedure for microscopic measurements. Then, the electrode was dipped into the electrochemical cell containing PBS at pH 6.0 and the electrochemical modification conditions which were discussed in Section 2.3 were applied.
2.2. Reagents and solutions
2.6. Electrochemical measurements
Trp, Tyr and 9-aminoacridine hydrochloride monohydrate (9AA.HCl.H2 O) 98% were obtained from Sigma–Aldrich. The stock solution of 9-AA with 1 × 10−3 M was prepared with ultra pure water and stored in a refrigerator at 4 ◦ C. Multi-walled carbon nanotubes (MWCNTs) was purchased from ARRY International group Limited (Germany). The stock solution of MWCNTs with 0.2 mg/ml was prepared in DMF (dimethylformamide). All reagents used were of analytical grade. The phosphate buffer solution (PBS) was prepared by 0.1 M KH2 PO4 –K2 HPO4 , and adjusting pH with 0.1 M H3 PO4 and 0.1 M KOH solutions. All solutions were prepared using ultra pure water from a Millipore Milli-Q system (system resistivity equal to 18 M cm).
Electrochemical behavior of PAA and PAA-MWCNTs modified electrodes was investigated by CV in the potential range between −0.4 and +0.4 V versus Ag/AgCl. The electrocatalytic properties of Trp at PAA/GCE and PAA-MWCNTs/GCE were investigated by using CV. Determination of Trp was carried out by DPV obtained by scanning the potential in the range from 0.1 to +1.2 V at differential pulse step potential of 5 mV and modulation amplitude of 50 mV. After the background current declined to a steady value, Trp solution were added, and the currents produced as a result of electrocatalytic oxidation of Trp was recorded. Trp quantities were gained by measuring the heights of the oxidative peaks. 0.1 M PBS was used as the supporting electrolyte for the most of the experiments.
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Fig. 1. Electropolymerization of 5 × 10−5 M 9-AA at GCE in 0.1 M PBS at pH 6.0, v = 100 mV s−1 . Inset: CVs of supporting electrolyte at (a) bare GCE and (b) PAA/GCE.
3. Results and discussion 3.1. Electrochemical characterization of PAA/GCE PAA/GCE was prepared by electropolymerization of 9-AA at GCE as described before [12]. Fig. 1 shows successive CVs displaying the polymer growth with cycling expressed by the increase in the anodic and cathodic peaks of PAA. Since the polymerization reaction would not occur lower than 1.5 V and higher than −0.8, the potential scan window was selected as from −1.2 to 2.5 V. During the electropolymerization process, an anodic peak (Pa1 ) at 1.7 V corresponding to the electrooxidation of –NH2 group of 9-AA monomers was observed. Consequently, oxidized species result one cathodic peak (Pc = −0.5 V) and one new anodic peak (Pa2 = +0.5 V) with continuous scans, and the peak currents also increased substantively. Anodic and cathodic peaks remained constant after 10 cycles. The broader peaks were observed during the ongoing scanning, indicating the permanent growth of the polymer film. These results revealed that PAA films were formed on the surface of GCE by electropolymerization. A blue polymer film with uniformly and tightly bounded to GCE surface was obtained. PAA film was rinsed with doubly distilled water to remove the physically adsorbed 9-AA and stored in PBS at pH 6.0. The electrochemical behavior of PAA/GCE was in good agreement with previous results referring to the electrochemical responses of azo compounds at solid electrode [13,14]. The CVs of PBS (0.1 M) at pH 6.0 in the range of −0.4 to 0.4 V at the surfaces of bare and PAA modified GC electrodes are shown in Fig. 1. As seen from Fig. 1, PAA modified electrode has chemically reversible redox couple (Pa and Pc ) where potentials are 0.14 and −0.08 V, respectively. Meanwhile there is no peak on bare GCE (curve a). This finding proves that GCE was modified with PAA.
Fig. 2. CVs of 1 × 10−4 M Trp at bare GCE (a) and PAA/GCE (b). Inset: DPVs of 1 × 10−4 M Trp at bare GCE (a) and PAA/GCE (b) in 0.1 M PBS at pH 7.4, v = 50 mV s−1 .
This indicates that PAA modified GCE has better electrocatalytic effect than bare GCE for the electrooxidation of Trp (Fig. 2). The effect of the cycle number, which was applied during PAA growth, was investigated with the modified electrode response to electrooxidation of Trp. The anodic peak current of Trp increased with the number up to 20 voltammetric cycles, and then it decreased (data was not shown). This behavior was attributed to the decrease of PAA conductivity with increasing the thickness of the film. Taking into account the results obtained 20 voltammetric cycles were selected for the growth of PAA. The effect of 9-AA monomer concentration was also evaluated (data was not shown). The peak currents of electrooxidation of Trp increased with 9-AA concentration up to 5 × 10−5 M, and then decreased. This behavior is related to higher yield of electropolymerization reaction and hence better electrode covering occurs as the monomer concentration increases. However, a large thickness of the polymeric film can reduce the overall conductivity of the electrode. 3.3. Effect of pH We also investigated the effect of pH on the anodic peak potential (Epa ) of Trp at PAA modified GCE. It was observed that PAA modified GCE showed the stable electrocatalytic activity in broad range of pH values. DPV measurements of Trp at different pHs, ranging from 4.0 to 9.0 in a buffer solution indicated that the best peak height and the peak shape could be obtained at pH 7.4 (Fig. 3). As
3.2. Electrocatalytic oxidation of Trp at PAA/GCE The parameters involved in the manufacturing of the polymer modified electrode were optimized by testing the electrochemical response of 1.0 × 10−4 M Trp solutions. Fig. 2 shows CVs and DPVs recorded at bare and PAA modified GCE in 0.1 M PBS at pH 7.4 containing 1.0 × 10−4 M Trp. As expected, a small irreversible electrooxidation peak at 0.93 V was observed with bare GCE, whereas a well-defined oxidation peak at 0.78 V was obtained with PAA modified GCE (Fig. 2). The anodic peak of Trp at PAA modified GCE increased and oxidative peak potentials negatively shifted nearly to 11 mV compared with bare electrode.
Fig. 3. Effect of pH on DPV peak current (a) and peak potential (b) of 1 × 10−4 M Trp at PAA/GCE in 0.1 M PBS at pH 7.4.
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Table 1 Determination of Trp in pharmaceutical sample. Added L-Trp (mol dm−3 )
Found L-Trp ± SD (mol dm−3 )
Found L-Trp ± SD (g dm−3 )
Recovery (%)
RSD (%)
0.00 0.99 1.98 3.96
0.746 ± 0.099 1.66 ± 0.21 2.59 ± 0.29 4.52 ± 0.48
0.152 ± 0.020 0.338 ± 0.042 0.528 ± 0.059 0.922 ± 0.097
– 92.32 93.13 95.30
13.27 12.65 11.19 10.61
3.6. Interference effect
Fig. 4. DPVs of Trp with different concentrations: (a) 10−6 M, (b) 10−5 M and (c) 10−4 M at PAA/GCE in 0.1 M PBS at pH 7.4. Inset: Calibration curve depending on log Ip versus log cTrp based on DPV measurements.
seen from Fig. 3, the anodic peak of Trp shifted to negative potential with the increasing of pH since the electrooxidation process of Trp facilitated at higher pH values. In Fig. 3, a plot of DPV peak potential versus pH was linear with a linear regression equation, Epa (V) = 1.026 − 0.052 pH (r2 = 0.999). The slope of Epa versus pH was −52 mV/pH, indicating that the number of protons involved equal to the number of electrons transferred in the electrochemical reaction [11,12].
3.4. Effect of scan rate The influence of scan rate on peak potential and peak current (Ip ) of Trp at PAA/GCE was investigated by CV. The oxidative peak current of Trp increased linearly with the scan rate in the range from 0.01 to 0.5 V s−1 . The linear regression equation was expressed as Ip (A) = 0.0104v (mV s−1 ) + 0.432 with a correlation coefficient of r2 = 0.9981 revealing that the oxidation of Trp was an adsorptioncontrolled step (plot was not shown). Change in the peak potential with the logarithm of the scan rate (log v) in the range of 0.01–0.5 V s−1 for the electrooxidation of Trp was expressed as Epa = 0.06388 log v + 0.55845 with r2 = 0.9959. According to Laviron’s theory [15], the slope is equal to 2.303RT/˛n˛ F. Using linear regression equation above, the value of ˛n˛ was calculated as 0.935. As for a totally irreversible electrode reaction process, ˛ was assumed to be 0.5. On the basis of the above discussion, the n˛ was calculated to be 1.87. This result shows that transfer of two electrons and two protons takes place in the process of the electrode reaction since the equal numbers of electron and proton involved in the electrooxidation of Trp.
The possible interferences of different amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, aspartic acid, glutamic acid, lysine, arginine, serine, threonine, hydroxy pro, methionine, cysteine, proline) were investigated for the determination of 1.0 × 10−5 M Trp at PAA/GCE under optimized experimental conditions described above. The results showed that these amino acids, except Tyr, did not exhibit significant interference effect on the determination of Trp. On the other hand, the effect of Tyr might be neglected since the electrooxidation peak potential of Tyr was far away and the peak current was much smaller than that of Trp. 3.7. Determination of Trp in pharmaceutical sample The proposed method was applied to detect the concentration of Trp in amino acid injection. The content of Trp was determined with the standard addition method with DPV. The analytical procedure was the same as described in Section 3.1. The results were shown in Table 1. The application of PAA modified GCE for amino acid injection suggested that a practical method had been successfully developed for the determination of Trp. 3.8. Electrochemical characterization of PAA-MWCNTs/GCE and electrooxidation of Trp at the modified electrode PAA-MWCNTs/GCE was prepared as described in Section 2.4 and electrochemical characterization of the electrode was carried out in 0.1 M PBS at pH 6.0 by CV. The cyclic voltammetric behavior of MWCNTs/GCE was also investigated for comparison. Both of the modified electrodes show significant diffencence in background currents (Fig. 5). The background current of PAA-MWCNTs/GCE is larger than that of MWCNTs/GCE since the incorporation of the
3.5. Analytical application DPVs of Trp at PAA/GCE at various concentrations were shown in Fig. 4. As seen from Fig. 4, DPV peak currents increased linearly with Trp concentration. The voltammetric calibration curve of Trp had a linear range of 1 × 10−6 –5 × 10−4 M with a detection limit of 8.1 × 10−7 M Trp (S/N = 3) (Fig. 4). The slope of the calibration curve was 0.51 log cTrp (mol dm−3 ) and the correlation coefficient (r2 ) was 0.9988. The voltammetric detection with PAA modified GCE was very stable and R.S.D. (95% confidence interval) was 3.5% for the slope variation based on 10 measurements during 2 months.
Fig. 5. DPVs of 5 × 10−5 M Trp at MWCNTs/GCE (a) and PAA-MWCNTs/GCE (b) in 0.1 M PBS at pH 7.4. Inset: CVs of 5 × 10−5 M Trp at MWCNTs/GCE (a) and PAAMWCNTs/GCE (b) in 0.1 M PBS at pH 6.0, v = 100 mV s−1 .
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Table 2 Peak currents and peak potentials of DPVs with the different modified electrodes for the electrochemical oxidation of 5 × 10−5 M Trp. Electrode
pH
Ip (A)
Ep (V)
Bare GC PAA/GC MWCNT/GC MWCNT-PAA/GC
7.4 7.4 3.5 3.5
0.93 3.00 4.40 16.5
0.87 0.70 0.78 0.58
acridine molecule in nanotube matrix causing an increase in the surface area of the electrode. The electrooxidation of Trp at MWCNTs/GC and PAAMWCNTs/GC electrodes were investigated and the differential pulse voltammograms were recorded in 0.1 M PBS at pH 7.4 containing 5 × 10−5 M Trp (Fig. 5). When compared with MWCNTs/GCE, the anodic peak current of Trp at PAA-MWCNTs/GCE increased 3.5-fold and oxidative peak potential negatively shifted to 0.2 V, which indicates that PAA-MWCNTs/GCE had better electrocatalytic effect for the electrooxidation of Trp (Fig. 5). The electrocatalytic activities of different modified electrodes towards the electrochemical oxidation of Trp were shown in Table 2. As seen from Table 2, the current measured for electrooxidation of Trp using PAA-MWCNTs modified GCE was higher than those of the PAA/GCE and MWCNTs/GCE. This shows more excellent electrocatalytic activity of PAA-MWCNTs/GCE electrode towards to the electrooxidation of Trp. This is due to the larger active surface area of PAA-MWCNTs/GCE contributing to the increase in electrooxidation current of Trp and a synergic effect with the hybrid modifier material. These results agree with that published for poly (aniline)-CNTs- GCE [16], and can be associated with the fact that the conducting polymer can immobilize and adhere to MWCNTs. Meanwhile, MWCNTs can interact with the polymer and some of the polymer-MWCNTs aggregates formed can reduce the ion intercalation distance, thus facilitating the charge transfer and improving the conductivity of the polymeric film [16,17]. It is difficult to determine Trp and Tyr simultaneously by direct voltammetry at PAA-MWCNTs modified GCE since oxidative peaks of these amino acids could not be completely separated. For this reason, the effect of pH of the buffer solution was investigated to distinguish the oxidative peaks. PAA-MWCNTs/GCE was dipped into the PBS (0.1 M) at pH 3.5 containing 5 × 10−5 M Trp, and then CV and DPV were performed, respectively. The solution of Tyr with increasing concentrations was added into the same solution, and then CV and DPV were applied. DPVs of Trp at PAA-MWCNTs modified GCE in a solution containing different concentration of Tyr were obtained (Fig. 6). As seen from Fig. 6, the oxidation peak current at about 0.56 V which was attributed to the electrooxidation of
Fig. 6. DPVs of 5 × 10−5 M Trp at PAA-MWCNTs/GCE containing (a) 0 M, (b) 5 × 10−5 M and (c) 1 × 10−4 M Tyr in 0.1 M H3 PO4 buffer at pH 3.5. Inset: the same experiment was carried out in 0.1 M PBS at pH 7.4.
Trp did almost not change with the addition of Tyr into the solution. This result also shows that there was no interference effect caused by Tyr on electrooxidation of Trp. When the same experiment was carried out in PBS at pH 3.5, the oxidation peak current observed at 0.6 V increased with the increasing of Tyr concentrations (Fig. 6). This indicates that the oxidation peak located at 0.6 V is due to the electrooxidation of Tyr. So, the results revealed that Trp and Tyr can be determined simultaneously by adjusting pH of the solution. 3.9. Surface characterization of PAA and PAA-MWCNTs modified ITO electrodes The surface of the modified electrodes was also characterized using SEM, STM and STS. SEM images of PAA modified ITO (PAA/ITO) and PAA-MWCNTs modified ITO electrodes (PAA-MWCNTs/ITO) are shown in Fig. 7. PAA/ITO electrode (Fig. 7B) has a fine structure and forms aggregates of microparticles compared with the bare ITO (Fig. 7A). SEM image of PAA/ITO electrode has been verified that the thin PAA film almost uniformly covered the electrode surface. On the other hand, PAA particles have been seen on MWCNTs (Fig. 7C). STM images of bare and PAA modified ITO electrodes were obtained (Fig. 8). Highly ordered prolytic graphite (HOPG) was used as a reference substance in microscopic measurements (Fig. 8A). STM image of bare ITO surface shown in Fig. 8B is obviously seen. Whereas the image of PAA (Fig. 8C) could not fully observe since the tunneling currents was not stable. The reason for that the gaps
Fig. 7. SEM images of (A) ITO, (B) PAA/ITO and (C) PAA-MWCNTs/ITO.
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Fig. 8. STM images of (A) HOPG, (B) ITO and (C) PAA/ITO.
were obtained (Fig. 9). As shown in Fig. 9, LUMO of PAA is above +0.8 eV, and LUMO is below −1.7 eV. The band gap of HOMO-LUMO is over 2.5 eV. Fig. 10 also shows the tri-dimensional STM images of bare and PAA modified ITO electrodes. In addition, STS value of PAA/ITO electrode is bigger than the bare ITO when STS data of the PAA modified and bare ITO electrodes are compared. The conductivity of the PAA modified electrode increases at higher than 0.7 eV since there is an electronic level formation on PAA layer. Furthermore, small hills indicating PAA layers were observed around −0.55 and −0.9 V on the modified electrode. On the other hand, these hills were not observed on the bare ITO. 4. Conclusions
Fig. 9. STS graphs of (a) HOPG, (b) ITO and (c) PAA/ITO.
of ITO hills were full of PAA causing to reduce in conductivity. Therefore, the tip of STM loses the surface and describes the discontinuous structure which has been seen in Fig. 8C. Probably, PAA takes place as a less and thinner layer on ITO hills of which STS data
A selective and sensitive electrochemical technique by using modified electrode has been developed for Trp detection in the presence of large quantities of other amino acids. The mechanism of electrochemical behavior of Trp on the surface of modified electrode was analyzed by investigating the effect of sweep rate and pH on the responses of CV method. The kinetics of the electrooxidation of Trp has considerably enhanced due to the lowering of anodic overpotential through a catalytic fashion. Electrochemical and microscopic characterizations of the modified electrodes were also examined. The modified electrode showed highly sensitive
Fig. 10. Three-dimensional STM images of (A) ITO and (B) PAA/ITO.
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and selective voltammetric responses for electrooxidation of Trp. It has been shown that PAA-modified electrode is suitable for simultaneous voltammetric detection of Trp in the presence of other potentially interfering species. However, PAA modified electrode was inadequate for the simultaneous determination of Trp and Tyr. For this purpose, a new electrochemical modification method based on the application of PAA-MWCNTs modified electrode was developed. The proposed technique has been used for the simultaneous determination of Trp and Tyr by adjusting the pH of the solution. On the other hand, the presence of MWCNTs improves the mechanical compactness and electrical conductivity of PAA modified GCE.
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Acknowledgements We thank the Research Funds of ITU for financial support (BAP: Project No: 32646) and Dr O˘guzhan Gürlü of ITU for taking the STM images and for stimulating discussion.
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Determination of tryptophan using electrode modified with poly(9-aminoacridine) functionalized multi-walled carbon nanotubes Sevgi Güney ∗ , Gülcemal Yıldız Department of Chemistry, Faculty of Science and Letters, Istanbul Technical University, Maslak, Istanbul 34469, Turkey
a r t i c l e
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Article history: Received 29 November 2010 Received in revised form 31 May 2011 Accepted 2 June 2011 Available online 13 June 2011 Keywords: Carbon nanotubes 9-Aminoacridine Tryptophan Tyrosine Electrochemical sensor
a b s t r a c t Two new kinds of electrochemical sensors were prepared using glassy carbon electrodes modified with poly(9-aminoacridine) (PAA) and PAA-functionalized multi-walled carbon nanotubes (PAA-MWCNTs) for the determination of tryptophan (Trp) in the presence of tyrosine (Tyr). The electrocatalytic response of the modified electrodes towards electrooxidation of Trp and Tyr was investigated and compared by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The surface of the modified electrodes was characterized using CV, scanning electron microscopy, scanning-tunneling microscopy and scanningtunneling spectroscopy. The anodic peak current of the electrooxidation of Trp on PAA-MWCNTs electrode was 5.5-fold increased comparing to PAA modified electrode. The anodic peak potential of Trp is shifted about 0.12 V to more negative value indicated that PAA-MWCNTs electrode has better electrocatalytic activity for electrooxidation of Trp. Furthermore, the anodic peak current which was attributed to the electrooxidation of Trp did almost not change with the addition of Tyr in buffer solution at pH 3.5. Besides this, at pH 7.4, the anodic peak current obtained at 0.6 V increased as the concentration of Tyr was increased. The results demonstrate that PAA-MWCNTs electrode can be used for the simultaneous determination of Trp and Tyr by adjusting the pH of the solution. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Determination of amino acids has been attracting more attention since they are the basic units of enzymes and proteins. A lot of research papers related to the electroanalytical determination of amino acids by chemically modified electrodes (CMEs) have been published in the last few decades [1,2]. Trp is a vital constituent of proteins and indispensable in human nutrition for establishing and maintaining a positive nitrogen balance and is also a precursor of the neurotransmitter serotonin. Tyr is the precursor of dopamine, thyroxin, dopa and epinephrine hormone or neurotransmitters in mammalian central nervous systems. The concentration of Tyr in culture medium is very important. While a high concentration of Tyr results increase in sister chromatid exchange, the absence of Tyr could cause albinism and alkaptonuria. Therefore, the determination of Tyr is a vital issue. There are several techniques for determination of Trp and Tyr including electroanalytical methods [3–6]. However, the voltammetric response of electroanalytical methods is not sufficient
∗ Corresponding author. Tel.: +90 212 285 32 46; fax: +90 212 285 63 86. E-mail address: [email protected] (S. Güney). 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.06.003
because of slow heterogeneous electron transfer at the electrode surface. Therefore, CMEs have been designed to improve the electron transfer characteristics and the sensitivity. CMEs have been constructed by immobilization of different organic and inorganic modifiers on the surface of electrode. CMEs were developed for the improvement of sensitivity, stability and selectivity in analytical applications to use as amperometric, voltammetric and potentiometric sensors. CMEs provide higher rates for the electrode reaction and show higher selectivity and sensitivity for determination of various compounds compared with ordinary electrodes [7,8]. One of the common methods for designing of CMEs involves applying of polymer films to the surface of the solid electrodes. This method offers several advantages, such as a chemical and physical stability, a good conduction, simplicity of preparation and reusability. The modification of electrode with the polymer offers superior properties for the determination of bio-molecules since the polymers are chemically stable in electrochemical deposition and also strongly and homogeneously adherent to electrode surface as a film [9,10]. Carbon nanotubes (CNTs) have been attractive electrode materials because of their excellent mechanical strength and electrical conductivity as well as being comparatively chemically inert in most electrolyte solutions. Usage of CNTs as the modification
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material on glassy carbon (GC) electrode supply better surface property for immobilizing of chemical and biological active species on the sensing electrode and also improved sensitivity due to great chemical and electrical properties of CNTs [11]. In addition, GC electrode (GCE) modified with CNTs exhibits electrochemical behavior with antifouling of the electrode. The other promising molecule as a modification agent, acridine, is structurally similar to anthracene but contains a nitrogen atom in its central ring. The usage of 9-aminoacridine (9-AA) and multiwalled carbon nanotubes (MWCNTs) together as a modifier for the fabrication of biosensor has never been reported until our current work. In this study, we applied 9-AA as a modifier to fabricate a PAA modified GCE (PAA/GCE) by electropolymerization. The surface of the modified electrode was characterized using CV method, scanning electron microscopy (SEM), scanning-tunneling microscopy (STM) and scanning-tunneling spectroscopy (STS). The electrocatalytic activities of the modified and unmodified electrodes towards aminoacid detection were studied and compared. Furthermore, we fabricated PAA-functionalized multi-walled carbon nanotubes modified GCE (PAA-MWCNTs/GCE) by drop deposition of homogeneous solution of 9-AA and MWCNTs on to the electrode surface followed by electrocycling in phosphate buffer solution at pH 6.0. The peak current obviously increased and the peak potential shifted to negative direction by using PAA-MWCNTs modified GCE compared with the bare, PAA only and MWCNTs only modified GCEs. The experimental results revealed that Trp and Tyr can be determined simultaneously by adjusting the pH of the solution.
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2.3. Preparation of PAA modified electrode Prior to modification, GCE surface was polished with 1, 0.3 and 0.05 m alumina slurry using a polishing cloth to produce a mirrorlike surface, respectively and then rinsed distilled water, sonicated 3 min into water and ethanol, and dried in air. For the surface characterization, prior to the modification, ITO coated glass sheet was sonicated in acetone, ethanol and distilled water, dried with a stream of nitrogen. Next, the working electrode was immersed into 0.1 M PBS at pH 6.0 and electroactivation was performed by applying 20 voltammetric cycles between −1.0 and 2.0 V versus Ag/AgCl at 100 mV s−1 . Finally, the solution of 9-AA with 5.0 × 10−5 M was added to the buffer solution and the electrode was modified by applying 20 voltammetric cycles between −1.2 and 2.5 V versus Ag/AgCl at 100 mV s−1 . When the electropolymerization was completed, the electrode was rinsed with distilled water to remove soluble species from the surface of the film. The modified electrode was stored in PBS at pH 6.0 at room temperature when not in use. The modification of ITO electrode was performed by the same procedure.
2.4. Preparation of MWCNTs-modified electrode 0.1 mg of MWCNTs was dispersed in DMF with the ultrasonic stirring to form a black solution. The 20 L of this solution was dropped on the polished GCE surface and spun at 3500 rpm for 120 s. DMF was evaporated in the air to form a MWCNTs modified electrode.
2. Experimental 2.1. Apparatus 2.5. Preparation of PAA-MWCNTs modified electrode Voltammetric measurements were performed with an Autolab PGSTAT 30 (Eco Chemie) potentiostat. All voltammetric experiments were conducted in a general-purpose cell stand (BAS C-3), having classical three-electrode cell with a platinum wire as a counter electrode (BAS MW 1032) and an Ag/AgCl/3 M KCl as a reference (BAS MF 2052) electrode. The working electrode was a GCE with 3 mm diameter (BAS MF-2012). For the microscopic measurements, an indium tin oxide (ITO) coated glass sheet (1.0 cm2 ) was used as the working electrode. All solutions were purged for 10 min with pure nitrogen and an inert atmosphere was maintained during the experiments. The pH values of the solutions were recorded using a pH-meter (WTW Inolab pH 720). An ultrasonic bath was used for preparing the solutions (Bandelin Sonorex RK 100H). SEM measurements were performed with a Nanoeye SNE-3000M MiniSEM. STM and STS measurements were performed with a Nanosurf Easyscan 2 Controller NanoBEEs.
The aqueous solution of 9-AA.HCl.H2 O was prepared and titrated with ammonium carbonate to obtained the neutral form of 9-AA. The precipitated product in aqueous solution was filtered and washed with distilled water to eliminate the ammonium chloride from the precipitation and dried in vacuum oven at 40 ◦ C. The homogeneous solutions of 9-AA and MWCNTs in DMF were prepared by dissolving of 0.2 mg each of them in different glass tubes. After that, two solutions were mixed. The 20 L aliquot of this solution was dropped on the polished GCE surface and spun at 3500 rpm for 120 s. ITO electrode was also modified with the same procedure for microscopic measurements. Then, the electrode was dipped into the electrochemical cell containing PBS at pH 6.0 and the electrochemical modification conditions which were discussed in Section 2.3 were applied.
2.2. Reagents and solutions
2.6. Electrochemical measurements
Trp, Tyr and 9-aminoacridine hydrochloride monohydrate (9AA.HCl.H2 O) 98% were obtained from Sigma–Aldrich. The stock solution of 9-AA with 1 × 10−3 M was prepared with ultra pure water and stored in a refrigerator at 4 ◦ C. Multi-walled carbon nanotubes (MWCNTs) was purchased from ARRY International group Limited (Germany). The stock solution of MWCNTs with 0.2 mg/ml was prepared in DMF (dimethylformamide). All reagents used were of analytical grade. The phosphate buffer solution (PBS) was prepared by 0.1 M KH2 PO4 –K2 HPO4 , and adjusting pH with 0.1 M H3 PO4 and 0.1 M KOH solutions. All solutions were prepared using ultra pure water from a Millipore Milli-Q system (system resistivity equal to 18 M cm).
Electrochemical behavior of PAA and PAA-MWCNTs modified electrodes was investigated by CV in the potential range between −0.4 and +0.4 V versus Ag/AgCl. The electrocatalytic properties of Trp at PAA/GCE and PAA-MWCNTs/GCE were investigated by using CV. Determination of Trp was carried out by DPV obtained by scanning the potential in the range from 0.1 to +1.2 V at differential pulse step potential of 5 mV and modulation amplitude of 50 mV. After the background current declined to a steady value, Trp solution were added, and the currents produced as a result of electrocatalytic oxidation of Trp was recorded. Trp quantities were gained by measuring the heights of the oxidative peaks. 0.1 M PBS was used as the supporting electrolyte for the most of the experiments.
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Fig. 1. Electropolymerization of 5 × 10−5 M 9-AA at GCE in 0.1 M PBS at pH 6.0, v = 100 mV s−1 . Inset: CVs of supporting electrolyte at (a) bare GCE and (b) PAA/GCE.
3. Results and discussion 3.1. Electrochemical characterization of PAA/GCE PAA/GCE was prepared by electropolymerization of 9-AA at GCE as described before [12]. Fig. 1 shows successive CVs displaying the polymer growth with cycling expressed by the increase in the anodic and cathodic peaks of PAA. Since the polymerization reaction would not occur lower than 1.5 V and higher than −0.8, the potential scan window was selected as from −1.2 to 2.5 V. During the electropolymerization process, an anodic peak (Pa1 ) at 1.7 V corresponding to the electrooxidation of –NH2 group of 9-AA monomers was observed. Consequently, oxidized species result one cathodic peak (Pc = −0.5 V) and one new anodic peak (Pa2 = +0.5 V) with continuous scans, and the peak currents also increased substantively. Anodic and cathodic peaks remained constant after 10 cycles. The broader peaks were observed during the ongoing scanning, indicating the permanent growth of the polymer film. These results revealed that PAA films were formed on the surface of GCE by electropolymerization. A blue polymer film with uniformly and tightly bounded to GCE surface was obtained. PAA film was rinsed with doubly distilled water to remove the physically adsorbed 9-AA and stored in PBS at pH 6.0. The electrochemical behavior of PAA/GCE was in good agreement with previous results referring to the electrochemical responses of azo compounds at solid electrode [13,14]. The CVs of PBS (0.1 M) at pH 6.0 in the range of −0.4 to 0.4 V at the surfaces of bare and PAA modified GC electrodes are shown in Fig. 1. As seen from Fig. 1, PAA modified electrode has chemically reversible redox couple (Pa and Pc ) where potentials are 0.14 and −0.08 V, respectively. Meanwhile there is no peak on bare GCE (curve a). This finding proves that GCE was modified with PAA.
Fig. 2. CVs of 1 × 10−4 M Trp at bare GCE (a) and PAA/GCE (b). Inset: DPVs of 1 × 10−4 M Trp at bare GCE (a) and PAA/GCE (b) in 0.1 M PBS at pH 7.4, v = 50 mV s−1 .
This indicates that PAA modified GCE has better electrocatalytic effect than bare GCE for the electrooxidation of Trp (Fig. 2). The effect of the cycle number, which was applied during PAA growth, was investigated with the modified electrode response to electrooxidation of Trp. The anodic peak current of Trp increased with the number up to 20 voltammetric cycles, and then it decreased (data was not shown). This behavior was attributed to the decrease of PAA conductivity with increasing the thickness of the film. Taking into account the results obtained 20 voltammetric cycles were selected for the growth of PAA. The effect of 9-AA monomer concentration was also evaluated (data was not shown). The peak currents of electrooxidation of Trp increased with 9-AA concentration up to 5 × 10−5 M, and then decreased. This behavior is related to higher yield of electropolymerization reaction and hence better electrode covering occurs as the monomer concentration increases. However, a large thickness of the polymeric film can reduce the overall conductivity of the electrode. 3.3. Effect of pH We also investigated the effect of pH on the anodic peak potential (Epa ) of Trp at PAA modified GCE. It was observed that PAA modified GCE showed the stable electrocatalytic activity in broad range of pH values. DPV measurements of Trp at different pHs, ranging from 4.0 to 9.0 in a buffer solution indicated that the best peak height and the peak shape could be obtained at pH 7.4 (Fig. 3). As
3.2. Electrocatalytic oxidation of Trp at PAA/GCE The parameters involved in the manufacturing of the polymer modified electrode were optimized by testing the electrochemical response of 1.0 × 10−4 M Trp solutions. Fig. 2 shows CVs and DPVs recorded at bare and PAA modified GCE in 0.1 M PBS at pH 7.4 containing 1.0 × 10−4 M Trp. As expected, a small irreversible electrooxidation peak at 0.93 V was observed with bare GCE, whereas a well-defined oxidation peak at 0.78 V was obtained with PAA modified GCE (Fig. 2). The anodic peak of Trp at PAA modified GCE increased and oxidative peak potentials negatively shifted nearly to 11 mV compared with bare electrode.
Fig. 3. Effect of pH on DPV peak current (a) and peak potential (b) of 1 × 10−4 M Trp at PAA/GCE in 0.1 M PBS at pH 7.4.
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Table 1 Determination of Trp in pharmaceutical sample. Added L-Trp (mol dm−3 )
Found L-Trp ± SD (mol dm−3 )
Found L-Trp ± SD (g dm−3 )
Recovery (%)
RSD (%)
0.00 0.99 1.98 3.96
0.746 ± 0.099 1.66 ± 0.21 2.59 ± 0.29 4.52 ± 0.48
0.152 ± 0.020 0.338 ± 0.042 0.528 ± 0.059 0.922 ± 0.097
– 92.32 93.13 95.30
13.27 12.65 11.19 10.61
3.6. Interference effect
Fig. 4. DPVs of Trp with different concentrations: (a) 10−6 M, (b) 10−5 M and (c) 10−4 M at PAA/GCE in 0.1 M PBS at pH 7.4. Inset: Calibration curve depending on log Ip versus log cTrp based on DPV measurements.
seen from Fig. 3, the anodic peak of Trp shifted to negative potential with the increasing of pH since the electrooxidation process of Trp facilitated at higher pH values. In Fig. 3, a plot of DPV peak potential versus pH was linear with a linear regression equation, Epa (V) = 1.026 − 0.052 pH (r2 = 0.999). The slope of Epa versus pH was −52 mV/pH, indicating that the number of protons involved equal to the number of electrons transferred in the electrochemical reaction [11,12].
3.4. Effect of scan rate The influence of scan rate on peak potential and peak current (Ip ) of Trp at PAA/GCE was investigated by CV. The oxidative peak current of Trp increased linearly with the scan rate in the range from 0.01 to 0.5 V s−1 . The linear regression equation was expressed as Ip (A) = 0.0104v (mV s−1 ) + 0.432 with a correlation coefficient of r2 = 0.9981 revealing that the oxidation of Trp was an adsorptioncontrolled step (plot was not shown). Change in the peak potential with the logarithm of the scan rate (log v) in the range of 0.01–0.5 V s−1 for the electrooxidation of Trp was expressed as Epa = 0.06388 log v + 0.55845 with r2 = 0.9959. According to Laviron’s theory [15], the slope is equal to 2.303RT/˛n˛ F. Using linear regression equation above, the value of ˛n˛ was calculated as 0.935. As for a totally irreversible electrode reaction process, ˛ was assumed to be 0.5. On the basis of the above discussion, the n˛ was calculated to be 1.87. This result shows that transfer of two electrons and two protons takes place in the process of the electrode reaction since the equal numbers of electron and proton involved in the electrooxidation of Trp.
The possible interferences of different amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, aspartic acid, glutamic acid, lysine, arginine, serine, threonine, hydroxy pro, methionine, cysteine, proline) were investigated for the determination of 1.0 × 10−5 M Trp at PAA/GCE under optimized experimental conditions described above. The results showed that these amino acids, except Tyr, did not exhibit significant interference effect on the determination of Trp. On the other hand, the effect of Tyr might be neglected since the electrooxidation peak potential of Tyr was far away and the peak current was much smaller than that of Trp. 3.7. Determination of Trp in pharmaceutical sample The proposed method was applied to detect the concentration of Trp in amino acid injection. The content of Trp was determined with the standard addition method with DPV. The analytical procedure was the same as described in Section 3.1. The results were shown in Table 1. The application of PAA modified GCE for amino acid injection suggested that a practical method had been successfully developed for the determination of Trp. 3.8. Electrochemical characterization of PAA-MWCNTs/GCE and electrooxidation of Trp at the modified electrode PAA-MWCNTs/GCE was prepared as described in Section 2.4 and electrochemical characterization of the electrode was carried out in 0.1 M PBS at pH 6.0 by CV. The cyclic voltammetric behavior of MWCNTs/GCE was also investigated for comparison. Both of the modified electrodes show significant diffencence in background currents (Fig. 5). The background current of PAA-MWCNTs/GCE is larger than that of MWCNTs/GCE since the incorporation of the
3.5. Analytical application DPVs of Trp at PAA/GCE at various concentrations were shown in Fig. 4. As seen from Fig. 4, DPV peak currents increased linearly with Trp concentration. The voltammetric calibration curve of Trp had a linear range of 1 × 10−6 –5 × 10−4 M with a detection limit of 8.1 × 10−7 M Trp (S/N = 3) (Fig. 4). The slope of the calibration curve was 0.51 log cTrp (mol dm−3 ) and the correlation coefficient (r2 ) was 0.9988. The voltammetric detection with PAA modified GCE was very stable and R.S.D. (95% confidence interval) was 3.5% for the slope variation based on 10 measurements during 2 months.
Fig. 5. DPVs of 5 × 10−5 M Trp at MWCNTs/GCE (a) and PAA-MWCNTs/GCE (b) in 0.1 M PBS at pH 7.4. Inset: CVs of 5 × 10−5 M Trp at MWCNTs/GCE (a) and PAAMWCNTs/GCE (b) in 0.1 M PBS at pH 6.0, v = 100 mV s−1 .
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Table 2 Peak currents and peak potentials of DPVs with the different modified electrodes for the electrochemical oxidation of 5 × 10−5 M Trp. Electrode
pH
Ip (A)
Ep (V)
Bare GC PAA/GC MWCNT/GC MWCNT-PAA/GC
7.4 7.4 3.5 3.5
0.93 3.00 4.40 16.5
0.87 0.70 0.78 0.58
acridine molecule in nanotube matrix causing an increase in the surface area of the electrode. The electrooxidation of Trp at MWCNTs/GC and PAAMWCNTs/GC electrodes were investigated and the differential pulse voltammograms were recorded in 0.1 M PBS at pH 7.4 containing 5 × 10−5 M Trp (Fig. 5). When compared with MWCNTs/GCE, the anodic peak current of Trp at PAA-MWCNTs/GCE increased 3.5-fold and oxidative peak potential negatively shifted to 0.2 V, which indicates that PAA-MWCNTs/GCE had better electrocatalytic effect for the electrooxidation of Trp (Fig. 5). The electrocatalytic activities of different modified electrodes towards the electrochemical oxidation of Trp were shown in Table 2. As seen from Table 2, the current measured for electrooxidation of Trp using PAA-MWCNTs modified GCE was higher than those of the PAA/GCE and MWCNTs/GCE. This shows more excellent electrocatalytic activity of PAA-MWCNTs/GCE electrode towards to the electrooxidation of Trp. This is due to the larger active surface area of PAA-MWCNTs/GCE contributing to the increase in electrooxidation current of Trp and a synergic effect with the hybrid modifier material. These results agree with that published for poly (aniline)-CNTs- GCE [16], and can be associated with the fact that the conducting polymer can immobilize and adhere to MWCNTs. Meanwhile, MWCNTs can interact with the polymer and some of the polymer-MWCNTs aggregates formed can reduce the ion intercalation distance, thus facilitating the charge transfer and improving the conductivity of the polymeric film [16,17]. It is difficult to determine Trp and Tyr simultaneously by direct voltammetry at PAA-MWCNTs modified GCE since oxidative peaks of these amino acids could not be completely separated. For this reason, the effect of pH of the buffer solution was investigated to distinguish the oxidative peaks. PAA-MWCNTs/GCE was dipped into the PBS (0.1 M) at pH 3.5 containing 5 × 10−5 M Trp, and then CV and DPV were performed, respectively. The solution of Tyr with increasing concentrations was added into the same solution, and then CV and DPV were applied. DPVs of Trp at PAA-MWCNTs modified GCE in a solution containing different concentration of Tyr were obtained (Fig. 6). As seen from Fig. 6, the oxidation peak current at about 0.56 V which was attributed to the electrooxidation of
Fig. 6. DPVs of 5 × 10−5 M Trp at PAA-MWCNTs/GCE containing (a) 0 M, (b) 5 × 10−5 M and (c) 1 × 10−4 M Tyr in 0.1 M H3 PO4 buffer at pH 3.5. Inset: the same experiment was carried out in 0.1 M PBS at pH 7.4.
Trp did almost not change with the addition of Tyr into the solution. This result also shows that there was no interference effect caused by Tyr on electrooxidation of Trp. When the same experiment was carried out in PBS at pH 3.5, the oxidation peak current observed at 0.6 V increased with the increasing of Tyr concentrations (Fig. 6). This indicates that the oxidation peak located at 0.6 V is due to the electrooxidation of Tyr. So, the results revealed that Trp and Tyr can be determined simultaneously by adjusting pH of the solution. 3.9. Surface characterization of PAA and PAA-MWCNTs modified ITO electrodes The surface of the modified electrodes was also characterized using SEM, STM and STS. SEM images of PAA modified ITO (PAA/ITO) and PAA-MWCNTs modified ITO electrodes (PAA-MWCNTs/ITO) are shown in Fig. 7. PAA/ITO electrode (Fig. 7B) has a fine structure and forms aggregates of microparticles compared with the bare ITO (Fig. 7A). SEM image of PAA/ITO electrode has been verified that the thin PAA film almost uniformly covered the electrode surface. On the other hand, PAA particles have been seen on MWCNTs (Fig. 7C). STM images of bare and PAA modified ITO electrodes were obtained (Fig. 8). Highly ordered prolytic graphite (HOPG) was used as a reference substance in microscopic measurements (Fig. 8A). STM image of bare ITO surface shown in Fig. 8B is obviously seen. Whereas the image of PAA (Fig. 8C) could not fully observe since the tunneling currents was not stable. The reason for that the gaps
Fig. 7. SEM images of (A) ITO, (B) PAA/ITO and (C) PAA-MWCNTs/ITO.
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Fig. 8. STM images of (A) HOPG, (B) ITO and (C) PAA/ITO.
were obtained (Fig. 9). As shown in Fig. 9, LUMO of PAA is above +0.8 eV, and LUMO is below −1.7 eV. The band gap of HOMO-LUMO is over 2.5 eV. Fig. 10 also shows the tri-dimensional STM images of bare and PAA modified ITO electrodes. In addition, STS value of PAA/ITO electrode is bigger than the bare ITO when STS data of the PAA modified and bare ITO electrodes are compared. The conductivity of the PAA modified electrode increases at higher than 0.7 eV since there is an electronic level formation on PAA layer. Furthermore, small hills indicating PAA layers were observed around −0.55 and −0.9 V on the modified electrode. On the other hand, these hills were not observed on the bare ITO. 4. Conclusions
Fig. 9. STS graphs of (a) HOPG, (b) ITO and (c) PAA/ITO.
of ITO hills were full of PAA causing to reduce in conductivity. Therefore, the tip of STM loses the surface and describes the discontinuous structure which has been seen in Fig. 8C. Probably, PAA takes place as a less and thinner layer on ITO hills of which STS data
A selective and sensitive electrochemical technique by using modified electrode has been developed for Trp detection in the presence of large quantities of other amino acids. The mechanism of electrochemical behavior of Trp on the surface of modified electrode was analyzed by investigating the effect of sweep rate and pH on the responses of CV method. The kinetics of the electrooxidation of Trp has considerably enhanced due to the lowering of anodic overpotential through a catalytic fashion. Electrochemical and microscopic characterizations of the modified electrodes were also examined. The modified electrode showed highly sensitive
Fig. 10. Three-dimensional STM images of (A) ITO and (B) PAA/ITO.
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and selective voltammetric responses for electrooxidation of Trp. It has been shown that PAA-modified electrode is suitable for simultaneous voltammetric detection of Trp in the presence of other potentially interfering species. However, PAA modified electrode was inadequate for the simultaneous determination of Trp and Tyr. For this purpose, a new electrochemical modification method based on the application of PAA-MWCNTs modified electrode was developed. The proposed technique has been used for the simultaneous determination of Trp and Tyr by adjusting the pH of the solution. On the other hand, the presence of MWCNTs improves the mechanical compactness and electrical conductivity of PAA modified GCE.
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Acknowledgements We thank the Research Funds of ITU for financial support (BAP: Project No: 32646) and Dr O˘guzhan Gürlü of ITU for taking the STM images and for stimulating discussion.
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