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Voltammetric determination of tryptophan in the presence of uric acid and dopamine using carbon paste electrode modified with multi-walled carbon nanotubes
⁎Corresponding author. Tel.: +98 3615912395; fax: +98 3615552930. s.m.ghoreishi@kashanu.ac.ir (Sayed Mehdi Ghoreishi)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A carbon paste electrode modified with multi-walled carbon nanotubes (CPE-MWCNTs) was prepared for determination of tryptophan (Trp) in the presence of uric acid (UA) and dopamine (DA). Cyclic voltammetry, chronoamperometry and differential pulse voltammetry (DPV) techniques were used to investigate the modified electrode for the electrocatalytic oxidation of Trp, UA and DA in aqueous solutions. The separation of the oxidation peak potentials for Trp-UA and UA-DA by CPE-MWCNTs was 386 and 144 mV, respectively. The calibration curves obtained for Trp, UA and DA were in the range of 0.60–100.00, 0.40–100.00 and 2.00–170.00 μM, respectively. The detection limits (S/N = 3) were 6.50 × 10−8, 2.70 × 10−7 and 3.60 × 10−7 M for Trp, UA and DA, respectively. The diffusion coefficient for the oxidation of Trp at the surface of modified electrode was calculated as 1.00 × 10−5 cm2 s−1 by chronoamperometry. The chemically modified electrode was successfully used for the quantification of Trp in the presence of UA and DA in serum samples.
Keywords
Tryptophan
Uric acid
Dopamine
Multi-walled carbon nanotubes
Modified carbon paste electrode
Voltammetry
1 Introduction
Tryptophan (Trp) is an essential amino acid for humans and also is a precursor of serotonin (a neurotransmitter), melatonin (a neurohormone), and niacin (Kochen and Steinhart, 1994). It is sometimes added to dietary and feed products as a food fortifier and to pharmaceutical formulations in order to correct possible dietary deficiencies (Fitznar et al., 1999).
Uric acid (UA) is another important compound in our body that abnormal levels are symptoms of several diseases such as hyperuricaemia, gout, and Lesch–Nyhan disease.
Dopamine (DA) is an important monoamine neurotransmitter in central nervous system of mammalians (Demier et al., 1999). Low levels of DA are related to neurological disorders such as parkinson’s disease, schizophrenia and to HIV infection (Wightman et al., 1988). Therefore it is essential to develop simple and rapid methods for determination of these biological molecules in routine analysis.
Methods for the determination of Trp are mainly based on HPLC (Fitznar et al., 1999; Hanaoka et al., 2000) and spectrophotometric procedures (Evgen’ev and Evgen’ev, 2000). Most of the spectrophotometric methods involve laborious and slow procedures with the modification of Trp by numerous reagents. Chromatographic separation is often complex and time-consuming. Electroanalytical methods, with respect to their sensitivity, accuracy and simplicity, have been more of interest in recent years for Trp analysis. The development of voltammetric sensors for determination of Trp, UA and DA in human fluid, food processing, pharmaceuticals, and clinical analysis has received considerable interest in recent years (Behpour et al., 2011a,b; Saurina et al., 2000; Fiorucci and Cavalheiro, 2002; Shahrokhian and Fotouhi, 2007; Wu et al., 2004). However, the voltammetric response of Trp is not satisfactory due to its slow heterogeneous electron transfer rate at electrode surfaces. Therefore applications of modified electrodes to increase the electron transfer rate and also the sensitivity were necessary.
Carbon paste electrode (CPE) is a special kind of heterogeneous carbon electrode consisting of mixture prepared from carbon powder (as graphite, glassy carbon and others carbonaceous materials) and a suitable water-immiscible or non-conducting binder (Kalcher, 1990). The use of carbon paste as an electrode was initially reported in 1958 by Adams (1958). In later researches a wide variety of modifiers including enzymes (Kozan et al., 2007; Cubukcu et al., 2007), polymers (nafion, chitosan, etc.) (Shahrokhian and Ghalkhani, 2006; Mandong et al., 2007) and nanomaterials (Ghoreishi et al., 2012a,b; Mashhadizadeh and Akbarian, 2009; Behpour et al., 2010; Hočevar and Ogorevc, 2007) have been used with these versatile electrodes. CPEs are widely applicable in both electrochemical studies and electroanalysis because of their advantages such as very low background current (compared to solid graphite or noble metal electrodes), facility to prepare, low cost, large potential window, simple surface renewal process and easiness of miniaturization (Ghoreishi et al., 2012a,b, 2013a,b). Besides the properties and characteristics listed before, the feasibility of incorporation of different substances during the paste preparation (which resulting in the so-called modified carbon paste electrode), allows the fabrication of electrodes with desired composition, and hence, with predetermined properties (Tashkhourian et al., 2009).
Since the discovery of carbon nanotubes (CNTs) in 1991 (Iijima, 1991), numerous investigations were focused on the studies of their properties and applications (Khoobi et al., 2013; Ghalkhani et al., 2009; Ghoreishi et al., 2013a,b; Tuzen and Soylak, 2007; Tuzen et al., 2008a,b; Duran et al., 2009). CNTs possess several unique properties such as good electrical conductivity, high chemical stability and extremely high mechanical strength because of the special tube structure (Gregory et al., 2006). In addition, the subtle electronic behavior of CNTs reveals that they have the ability to promote electron-transfer reaction and have a high electrocatalytic effect when used as electrode materials (Pai et al., 2006). All these fascinating properties make CNTs a suitable candidate for the modification of electrodes (Huang et al., 2008).
This work describes a novel strategy based on the simultaneous determination of Trp, UA and DA at the surface of a carbon paste electrode modified with multi-walled carbon nanotubes (CPE-MWCNTs). The ability of the modified electrode to recognize Trp, UA and DA is enhanced with the faster electron transfer process inherent with the CNT electrode interface. Also the analytical performance of this modified electrode for determinations of Trp in the presence of UA and DA in human serum was evaluated by differential pulse voltammetry (DPV).
2 Experimental
2.1 Chemicals and reagents
Trp, UA and DA and other chemicals with analytical grade were purchased from Merck. MWCNTs with purity of 95% (40–60 nm in diameter) were obtained from the Chinese Academy of Sciences. Phosphate buffer (PB) solution (0.10 M) was prepared by dissolving 1.67 mL concentrated ortho phosphoric acid in water and diluting to 0.25 L in a volumetric flask. The buffer solutions were prepared by the addition of 0.50 M sodium hydroxide to the phosphoric acid solution to reach appropriate pH values. All reagents were of analytical grade and used as received. All solutions were prepared with deionized water.
2.2 Electrodes and instrumentation
All the electrochemical measurements were carried out with a M273A Electrochemical Workstation (EG&G Corporation, America). A conventional three electrode system was employed, consisting of a MWCNT modified CPE as a working electrode, a silver/silver chloride (Ag/AgCl/KClsat) (Metrohm, Switzerland) and a Pt wire counter electrode (Azar electrode, Iran). Solution pH values were determined using a 691 pH meter (Metrohm, Switzerland) combined with glass electrode (Metrohm, Switzerland). Deionized water was formed with an ultrapure water system (smart 2 pure, TKA, Germany). MWCNTs were dispersed with an ultrasonic bath (SONOREX DIGITAL, 10P, BANDELIN).
2.3 Fabrication of CPE-MWCNTs
For preparation of CPE-MWCNTs, 6.00 mg MWCNTs were dispersed into 5.00 mL of deionized water, and then sonicated for about 45 min to give a stable and homogeneous MWCNT suspension. This suspension was mixed with 0.50 g graphite powder. After evaporation of the solvent in room temperature, a portion of it was mixed with nujol in a mortar and pestle. A portion of the mixture was packed into the end of a polyethylene tube. Electrical contact was made by forcing a copper pin down into the tube and into the back of the composite. For the preparation of unmodified CPE we carried out the same preparation of CPE-MWCNTs except the addition step of MWCNTs.
3 Results and discussion
3.1 Electrochemical studies of Trp at CPE-MWCNTs
The cyclic voltammograms of Trp at a CPE (curve a) and CPE-MWCNTs (curve b) in phosphate buffer (pH 3.0) are shown in Fig. 1. It can be seen the oxidation peak at the bare CPE is broad due to slow electron transfer, but at the surface of the CPE-MWCNTs the peak is sharper and the peak current is also increased. On the other hand, at CPE the peak potential is at 0.99 V along with a peak current of 0.65 μA, whereas an oxidation peak at 0.98 V with a peak current of 2.20 μA is observed at the CPE-MWCNTs. Also no reduction peak is observed in Fig. 1 that indicates the electrochemical reaction of Trp is a totally irreversible process.
3.2 Effect of pH on the oxidation of Trp
The pH of the supporting electrolyte has a significant influence on the electro-oxidation of Trp on the modified electrode. The electrooxidation of Trp was studied over the pH range of 2.0–8.0 in a 0.10 M phosphate buffer. The potential of the oxidation peak shifted to less positive potentials with increasing of pH. The peak current in pH = 3.0 was maximum, therefore, pH = 3.0 was selected as the optimum pH. The relationship of the peak potential versus pH can be expressed by the following equation:
The slope 52 mV/pH is close to the theoretical 59.1 mV/pH slope which showed that the overall process was proton dependent and the electron transfer was accompanied by the transfer of an equal number of protons. The probable catalytic processes of DA may be expressed as in Scheme 1 (Pournaghi-Azar et al., 2010).
3.3 Effect of potential scan rate
The effect of the potential scan rate on the peak current of every three compounds was investigated in the buffer solution containing 100.00 μM of Trp, 40.00 μM of UA (pH = 3.0) and 100.00 μM of DA (pH = 4.0). The cyclic voltammetric results indicated that the anodic peak current of Trp, UA and DA has a linear relationship with the square root of the potential sweep rate over a wide range of 20.0–300.0 mV s−1 with correlation coefficient values (R2) greater than 0.99. On the other hand, for the three compounds the peak potential shifted in a positive direction with increasing of the scan rate. Such behaviors revealed that the anodic oxidations of these species on the surface of CPE-MWCNTs are diffusion controlled.
3.4 Chronoamperometric studies
The catalytic oxidation of Trp at the surface of CPE-MWCNTs was also studied by chronoamperometry. Chronoamperometric measurements of different concentrations of Trp at CPE-MWCNTs were implemented by setting the working electrode potential at 1200.0 mV. Under diffusion limited transport (mass transport), a plot of I versus t−1/2 (s−1/2) will be linear, and the value of D can be extracted from the slope. Fig. 2(B) and (C) shows the fitted experimental plots for different concentrations of Trp. The slopes of the resulting straight lines were plotted versus the Trp concentration, and the mean value of D was found to be 1.0 × 10−5cm2 s−1.
3.5 Determination of Trp, UA and DA
Since differential pulse voltammetry has a much higher current sensitivity and better resolution than cyclic voltammetry, it was used in determination of Trp, UA and DA concentration at the CPE-MWCNT modified electrode and estimating the lower detection limit. The oxidation peak currents of Trp, UA and DA were measured in 0.10 M phosphate buffer solution, and plotted against the bulk concentration of Trp, UA and DA and are shown in Figs. 3–5, respectively. The dependence of peak currents on the concentration of Trp, UA and DA is linear relationship in the ranges of 0.60–100.00, 0.40–100.00 and 2.00–170.00 μM, respectively. The linear regression equations are as follows:
The detection limits (S/N = 3) are 6.50 × 10−8 M, 2.70 × 10−7 M and 3.60 × 10−7 M for Trp, UA and DA, respectively.
3.6 Determination of Trp in the presence of UA and DA
It is well known that the electrochemical detection of UA in the presence of high levels of DA on untreated carbon paste electrodes or on ordinary electrodes severely struggles due to the catalytic oxidation of UA by DA. The ability of the modified electrode to promote the voltammetric resolution of DA, UA and Trp was investigated. Fig. 6 shows the DPV responses of a mixture containing 10.00 μM Trp, 10.00 μM DA and 30.00 μM UA in 0.10 M buffer solution (pH 3.0) at a bare CPE and CPE-MWCNTs. Fig. 6(a) shows the voltammogram of the mixture at the bare CPE. According this Fig. a rather broad oxidation peak with indistinguishable peak potentials was obtained for UA and DA at the bare CPE. Thus it is impossible to determine the individual concentrations of these two compounds from the merged voltammetric peak. But as shown in Fig. 6(b), modification of CPE surface with multi walled carbon nanotubes resolved the merged voltammetric peak into three well defined voltammetric peaks at potentials around 0.39, 0.53 and 0.92 V for DA, UA and Trp, respectively. On the other hand, at the surface of CPE-MWCNTs, the peak currents of Trp, DA and UA increased therefore, the sensitivity is also improved with comparison to bare CPE. The ability of the modified electrode in determination of Trp in the presence of DA and UA was investigated in three steps. First, it was investigated by increasing of Trp concentration (from 0.20 to 6.00 μM) in the presence of constant concentration of DA (30.00 μM) and UA (20.00 μM) (Fig. 7). No obvious changes in the DA and UA oxidation currents were observed while varying the concentration of Trp, and the peak current of Trp increased linearly with increasing Trp concentration with a correlation coefficient of 0.9919. In the second step, in mixtures of Trp, DA and UA, the concentration of Trp and DA was kept at 80.00 and 100.00 μM, respectively, while the concentration of UA was changed between 20.00 and 120.00 μM. It was observed the peak current of UA increases proportionally to its concentration with a correlation coefficient of 0.990 without influencing the peak corresponding to Tyr and DA concentration. This result is indicating that the responses of Trp, UA and DA at the surface of CPE-MWCNTs are relatively independent. Finally in the third step, we carefully examined the oxidation currents of Trp (120.00 μM) and UA (30.00 μM) with increasing of DA concentration at the CPE-MWCNTs. No obvious changes in the Trp and UA oxidation currents were observed while varying the concentration of DA, and the peak current of DA increased linearly with increasing DA concentration (20.00–60.00 μM) with a correlation coefficient of 0.992. It is interesting to note that the sensitivities of the modified electrode toward Trp, UA and DA in the absence and presence of each other are virtually the same, which indicates that the oxidation processes of Trp, DA and UA at the CPE-MWCNTs are independent. This indicates that simultaneous or independent measurements of the three analytes are possible without any interference.
3.7 Interference studies
To evaluate the selectivity of the CPE-MWCNT modified electrode, the interference effects were investigated by detecting the response of the modified electrode to 20.00 μM Trp, 20.00 μM UA and 20.00 μM DA in the presence of cysteine, leucine, methionine, arginine, phenylalanine, glycine, alanine, proline each in concentration 50 times of that of three analytes, 200 times of K+, Na+, Cl− and Br−. The results showed that the aforementioned species did not cause observable interference at the concentrations given.
3.8 Determination of Trp in the presence of UA and DA in human blood serum
In order to verify the reliability of the method for simultaneous determination of the considered compounds in clinical samples, the prepared modified electrode was also applied for the analysis in human blood serum samples. The serum sample was centrifuged and then after filtering, diluted with 0.10 M phosphate buffer solution of pH 4.0 without any further treatment. The diluted serum sample was spiked with different amounts of Trp, UA and DA. In these measurements the concentration of one species changed in the presence of a constant concentration of two other compounds. Using the DPV method, the oxidation peak currents were linearly proportional to Trp, UA and DA concentration with very good correlation coefficients (Fig. 8). The results indicated very good recoveries for the determinations of these species in clinical samples, in the range of 97.50–102.00% (Table 1). Therefore, it is possible to determine Trp, UA and DA by using the CPE-MWCNTs in complex matrix samples.
| Sample No. | Added (μM) | Found (μM) | Recovery (%) |
|---|---|---|---|
| Trp | |||
| 1 | 0.00 | 0.80 | – |
| 2 | 5.00 | 5.70 | 98.00 |
| 3 | 10.00 | 10.60 | 97.90 |
| UA | |||
| 1 | 0.00 | 8.00 | – |
| 2 | 10.00 | 18.20 | 102.00 |
| 3 | 20.00 | 27.50 | 97.50 |
| DA | |||
| 1 | 0.00 | 2.10 | – |
| 2 | 10.00 | 12.60 | 102.00 |
| 3 | 20.00 | 22.10 | 98.50 |
4 Conclusions
This study has indicated that CPE-MWCNT electrode exhibits electrocatalytic activity to Trp oxidation. The electrochemical behavior of the modified electrode is strongly dependent on the pH of solution. Trp, DA and UA coexist in a homogeneous solution and could be simultaneously detected by the modified electrode, and the separation of the oxidation peak potentials for UA-DA and Trp-UA is about 144 and 386 mV, respectively. Therefore, simultaneous or independent measurements of the three analytes are possible without any interference. The proposed method can be applied to the determination of Trp, UA and DA in real samples with satisfactory results.
Acknowledgment
The authors are grateful to the University of Kashan for supporting this work by Grant No. 211037-2.
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