Amperometric sensor based on carbon nanotubes and electropolymerized vanillic acid for simultaneous determination of ascorbic acid, dopamine, and uric acid L. V. da Silva, F. A. S. Silva, L. T. Kubota, C. B. Lopes, P. R. Lima, E. O. Costa, W. Pinho Júnior & M. O. F. Goulart Journal of Solid State Electrochemistry Current Research and Development in Science and Technology ISSN 1432-8488 J Solid State Electrochem DOI 10.1007/s10008-016-3129-3
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Author's personal copy J Solid State Electrochem DOI 10.1007/s10008-016-3129-3
SHORT COMMUNICATION
Amperometric sensor based on carbon nanotubes and electropolymerized vanillic acid for simultaneous determination of ascorbic acid, dopamine, and uric acid L. V. da Silva 1,2,6 & F. A. S. Silva 4,6 & L. T. Kubota 4,6 & C. B. Lopes 3 & P. R. Lima 3,6 & E. O. Costa 1,6 & W. Pinho Júnior 1,6 & M. O. F. Goulart 1,6
Received: 13 October 2015 / Revised: 13 January 2016 / Accepted: 17 January 2016 # Springer-Verlag Berlin Heidelberg 2016
Abstract This paper describes the development of a simple and efficient nanostructured platform based on multi-walled carbon nanotubes (MWCNT) functionalized with an in situ generated vanillic acid (VA) polymer. It was used as an analytical sensor for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The electropolymerization process of VA, performed on MWCNT-modified glassy carbon electrode, produces three redox systems based on quinone/hydroquinone functionality, as observed by cyclic voltammetry. The amperometric sensor has as figures of merit for the simultaneous determination of AA, DA, and UA the following values: for AA, a linear range of 5–120 μM and detection limit of 3.5 μM; for DA, a linear range of 5–120 μM and detection limit of 4.5 μM; and for UA, a linear range of 5–120 μM and a detection limit of
* M. O. F. Goulart
[email protected]
1
Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Campus A. C. Simões, Avenida Lourival Melo Mota, s/n, Tabuleiro dos Martins, Maceió, AL 57072-970, Brazil
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Instituto Federal de Educação, Ciência e Tecnologia de Alagoas, IFAL- Campus Palmeira dos Índios, Palmeira dos Índios, AL 57608-180, Brazil
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Instituto Federal de Educação, Ciência e Tecnologia de Alagoas, IFAL, Maceió, AL 57020-600, Brazil
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Instituto Federal de Educação, Ciência e Tecnologia de Goiás, IFGcampus Uruaçu, Uruaçu, GO 76400000, Brazil
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Instituto de Química, UNICAMP, C. Postal 6154, Campinas, SP 13084-971, Brazil
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Instituto Nacional de Ciência e Tecnologia de Bioanalítica, UNICAMP, C. Postal 6154, Campinas, SP 13084-971, Brazil
1.5 μM. From the obtained performance, the development of the platform based on MWCNT/poly-VA is justified for the simultaneous determination of AA, DA, and UA. Keywords Vanillic acid . Electroanalysis . Ascorbic acid . Dopamine . Uric acid . Chemically modified electrodes . Quinone/hydroquinone system
Introduction Vanillic acid (4-hydroxy-3-methoxybenzoic acid) (VA), a biologically active phenolic acid, behaves as an antioxidant and was shown to reduce lipid peroxidation products (thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes) and significantly restored enzymatic antioxidants (superoxide dismutase, catalase, and glutathione peroxidase) and non-enzymatic antioxidants (vitamin C, vitamin E, and reduced glutathione) in the plasma [1]. As in nature, phenol polymerization can occur [1]. The investigation of the electrochemistry of VA for a better understanding of its oxidation mechanism and possible electroanalytical application is relevant, specially, for the determination of molecules, which have biological relevance, such as ascorbic acid (AA), uric acid (UA), and dopamine (DA). To the best of our knowledge, this is the first report on the electrochemical polymerization of vanillic acid on MWCNT/glassy carbon electrode (GCE) and its application as an electroanalytical redox mediator. Due to the biological relevance of AA, UA, and DA, it is necessary to develop tools for their analyses that present low cost, portability, high sensitivity, low detection limits, selectivity, and response in a short period of time [2–4]. Electroanalysis has several desirable features for use in routine. The efficiency of electroanalysis can be increased by the use of electrochemically generated redox agents. Various
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sensors based on multi-walled carbon nanotubes (MWCNT) and other materials [5–13], as well as chemically modified electrodes (CME), based on the electropolymerization of phenolic compounds [14–16], were used for simultaneous determination of such biomolecules, along with serotonin and others. Polymers are interesting materials for the development of analytical devices, working as a support matrix for the immobilization of biomolecules and allowing electrocatalysis. This article describes, briefly, and for the first time, the preparation, characterization by cyclic voltammetry, and application of a platform based on GCE (glassy carbon electrode), modified with MWCNTs and by electropolymerization of VA, for simultaneous quantification of AA, DA, and UA, in several matrixes.
Experimental
electrode was estimated using 1 mM Fe[(CN)6] 3−/4− in 0.1 M KCl, employing the Randles-Sevcik equation, as reported [17]. The analytical curves were obtained with the modified electrode (GCE/MWCNT/poly-VA) by pulsed potential amperometric technique, applying different potentials for the oxidation of AA, DA, and UA in 0.1 M PBS (pH 7.0). Real sample analysis and interferent tests The proposed modified electrode was used to investigate real samples, by a direct analysis of AA and UA in human urine, obtained from volunteers. At first, a human urine sample was diluted 25 times with a 0.1-μM phosphate buffer (pH 7.0). Then the urine samples were spiked with 100 μM, each, of AA and UA. In the interferent test, all analytes were at the same concentration (100 μM), as AA and UA. The current change was measured by chronoamperometry.
Chemicals and solutions
Results and discussion All chemicals were of analytical grade and used as received, without further purification. Multi-walled carbon nanotubes (MWCNT), vanillic acid (VA), ascorbic acid (AA), dopamine (DA), uric acid (UA), serotonin (SER), N-acetyl-L-cysteine (NAC), homocysteine (HCys), glutathione (GSH) and cysteine (Cys), KCl, FeCl3, ZnCl2, CaCl2, and glucose were acquired from Sigma-Aldrich, St. Louis, USA. All the solutions were freshly prepared prior to each experiment. Construction of the sensors and electrochemical measurements GCE (0.07 cm2) was manually polished with alumina powder (Al2O3, 1 μm) and rinsed with Milli-Q water. After cleaning the electrode, a suspension was prepared by sonication, mixing 1.0 mg of MWCNT and 1 mL of DMF. Five microliters of this suspension was, then, deposited directly on the GCE surface and dried at 80 °C, to form a modified surface (GCE/MWNCT). The electroactive system was electrogenerated in situ from VA oxidation, after cycling (20 scans) in the potential range of −0.5 up to +1.0 V vs. Ag/AgCl (KCl, sat.) at 50 mV s−1 in a solution of VA (0.8 mM) in 0.1 M PBS (pH 5.5). Prior to all the electropolymerization experiments, the PBS was bubbled with nitrogen for 10 min. The voltammetric measurements were performed with an Echo Chemie Autolab PGSTAT-30 potentiostat (Utrecht, The Netherlands). All the measurements were performed using an electrochemical cell with three electrodes, with an Ag/AgCl (KCl, sat) electrode as reference, a Pt wire as auxiliary, and an unmodified or chemically modified glassy carbon, in the latter case, with MWCNT and poly-VA (GCE/ MWCNT/poly-VA) as working electrodes. The actual surface area (0.147 cm2) of the GCE/MWCNT/poly-VA modified
Polymerization process of VA on MWCNT/GCE-modified electrode In this study, the electroactive species were electrogenerated, in situ, from VA by an electropolymerization process on the GCE/MWCNT, after potential cycling in the range of +0.5 to +1.0 V vs. Ag/AgCl in 0.1 M PBS (pH 5.5) with a scan rate of 0.05 V s−1. The VA electropolymerization process was optimized and the best experimental conditions were: number of successive cycles (20 scans) applied to the electrode, VA concentration (0.8 mM), and pH value of the solution 5.5. Initially, the cyclic voltammogram recorded with a GCE/ MWCNT-modified electrode using dissolved VA (PBS, pH 5.5) displayed an irreversible oxidation peak at around + 0.65 V vs. Ag/AgCl (Fig. 1-IVa), which corresponds to the beginning of the electropolymerization process [18] and is probably related to the formation of cation radicals. The presence of MWCNT precludes the passivation of the electrode that is commonly observed on GCE, when a phenolic species is present [19]. This group is responsible for the main oxidation peak, generating intermediate reaction products that polymerize and produce a gradual fouling of the electrode surface. Thus, as Fig. 1 indicates, peak IVa decreases and three new pair of peaks appear and increase (Ia/Ic, IIa/IIc, IIIa/IIIc) with successive scanning, indicating that the surface of the working electrode is gradually covered with an electroactive film. The modified GCE/MWCNT/poly-VA, obtained by the electropolymerization procedure described above, was thoroughly washed with distilled water and immersed in a fresh PBS (pH 7.0) for detection of analytes. Electrochemical oxidation of VA in MWCNT leads to the formation of some reactive functional groups, which can react within themselves
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AA, UA, and DA were very close. It is practically impossible to analyze the contribution current of each compound in the mixture principally when unmodified GCE (Fig. 2a) and GCE/poly-VA (Fig. 2b) were used. In the last platform, only one peak for oxidation of DA (positively charged at this pH value) is observed, possibly because the film is negatively charged (Fig. 2b), hampering the approach of the negatively charged AA and UA. This result shows a synergistic effect between the MWCNT and the modified VA polymer film-surface toward AA catalytic oxidation and for the simultaneous determination of AA, UA, and DA.
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E (V) vs. Ag/AgCl Fig. 1 Successive cyclic voltammograms of VA, on GCE/MWCNT, leading to its electropolymerization. Experimental conditions: VA 0.8 mM, in PBS 0.1 M, pH 5.5, and ν = 0.05 V s−1, 20 cycles
and with nucleophiles present in the solution [20]. Due to the polymeric nature of the obtained material, it was difficult to determine the chemical structure, as earlier observed [21]. However, the presence of the quasi-reversible redox couples points to the presence of quinone/hydroquinone systems [20], which would work as mediators. The polymerization can occur through Michael addition or other mechanisms, on the available ring positions, mainly para or ortho to the generated carbonyls, hampering lixiviation to the solution [22]. The platform is a very stable one. The exact mechanism of the polymerization and the participation of the MWCNT are being investigated and will be reported elsewhere. Electrochemical oxidation of AA, DA, and UA The electrocatalytic activity of the GCE/MWCNT/poly-VA was evaluated toward the oxidation of AA, UA, and DA. In Fig. 2d, the CV represents GCE/MWCNT/poly-VA. When a solution of AA is added, the oxidation peak (Epa(AA) = 0.0 V vs. Ag/AgCl) significantly increased with the concomitant disappearance of the reduction peak. However, upon the addition of UA and DA solutions, a catalytic effect is not observed and UA oxidation occurs at Epa(UA) = 0.35 V and Epa(DA) = 0.22 V vs. Ag/AgCl (Fig. 2d). To confirm the catalytic activity of the modified electrode toward AA oxidation and the possibility of a simultaneous quantitative analysis, AA, UA, and DA, in solution, were added, all together. The analysis of Fig. 2d allows us to suggest the catalytic nature toward AA oxidation with no such effect on UA and DA oxidations. Furthermore, the difference between the peak potentials for AA and UA is close to 300 mV and the one for AA and DA of 225 mV vs. Ag/AgCl, large enough to allow their selective and simultaneous determination, in their ternary mixture. Figure 2a, c displays control experiments, showing first, GCE/MWCNT, where the oxidation potential peaks of
Amperometry under stirred conditions has a much higher current sensitivity than cyclic voltammetry. Thus, for this purpose, the pulsed potential amperometric detection was used. The method is based on the application of multistep potentialtime waveforms to manage the sequential analytical process as earlier reported [17]. For AA and UA oxidations, it was carried out simultaneously at E1 = 0.100 V, E2 = 0.225 V, and E3 = 0.325 V vs. Ag/AgCl, respectively (at E1, E2, and E3, there is oxidation of AA, DA, and UA, respectively), with a pulse time of 100 ms in each potential. However, at Eapp. = 0.325 V, oxidation of the ternary mixture (AA, DA, and UA) occurs. For that reason, to obtain the net amperogram or UA analytical curve, without the contribution of the oxidation current of AA and DA, it is necessary to subtract the currents of the amperograms, at different concentrations in 0.1 M phosphate buffer at pH 7.0, after optimizing the experimental parameters. Detection limits calculated for AA, DA, and UA were, respectively, 3.5, 4.5, and 1.5 μM, which was determined using 3 σ/b, where σ is the standard deviation of the mean value for 10 amperograms and b is the slope of the calibration curve, determined in accordance with the recommendations of the IUPAC. The relations between currents and concentrations for electro-oxidation of AA, DA, and UA can be expressed, according to the following equations: ΔIðμAÞ ¼ 5:39 ð0:07Þ þ 0:0625 ð0:007Þ½AAðμMÞ⋅R ¼ 0:998
ð1Þ
ΔIðμAÞ ¼ 0:81 ð0:2Þ þ 0:048 ð0:004Þ½DAðμMÞ⋅R ¼ 0:998
ð2Þ
ΔIðμAÞ ¼ 3:58 ð0:13Þ þ 0:080 ð0:007Þ½UAðμMÞ⋅R ¼ 0:996
ð3Þ
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Conclusions
The interference from serotonin (SER) was investigated by comparing oxidation potentials of AA, DA, and UA with the oxidation potential of SER. It can be observed that the separation of the oxidation peak potential is large enough for the detection and quantitation of analytes of interest in this study, except for UA. The following compounds were also analyzed and did not show any interference toward the main analytes: KCl, FeCl3, ZnCl2, CaCl2, and glucose (all with c = 100 μM). The developed platform was applied for the determination of UA in urine samples, in triplicate, using the standard addition method. To fit into the linear range of UA determination, the urine samples were diluted 25 times. This dilution can actually help in reducing the matrix effects of real samples. To check the correctness of the results, the samples were spiked with standard solutions of UA and then the total amounts were detected. The recovery values of the spiked samples were determined to be 100.1 %. The chemicals used are inexpensive, commercially available, in addition to the applied amperometric technique, which shows high sensitivity and shorter period of analysis, as compared with voltammetric techniques. Compared to sensors based on polymers already reported [14–16], it has better or similar values.
This work demonstrated that a GCE/MWCNT modified by electropolymerization of VA has been shown to be an alternative for the detection of AA at low potentials and simultaneous determination of AA, DA, and UA at neutral pH values. This nanostructured platform is a selective and stable system. The proposed sensor is easy to prepare, inexpensive, and has high sensitivity and short analysis time.
Acknowledgments The authors gratefully acknowledge the financial support of CNPq (grants 458114/2014-6, 407963/2013-8, 484044/20117), INCT-Bioanalítica, CAPES, FAPEAL (PRONEX–2009-0-006), UFAL, and IFAL.
References 1. 2.
3.
Podeshvo IV, Mikhailov GM, Fedorova GN, et al. (2002) Polym Mater Derived Vanillic Acid 75:777–780 Dey RS, Gupta S, Paira R, et al. (2012) Flow injection amperometric sensing of uric acid and ascorbic acid using the self-assembly of heterocyclic thiol on Au electrode. J Solid State Electrochem 16: 173–178 Salimi A, Mamkhezri H, Hallaj R (2006) Simultaneous determination of ascorbic acid, uric acid and neurotransmitters with a carbon ceramic electrode prepared by sol-gel technique. Talanta 70:823– 832
Author's personal copy J Solid State Electrochem 4.
5.
6.
7.
8.
9. 10.
11.
12.
Zhao Y, Gao Y, Zhan D, et al. (2005) Selective detection of dopamine in the presence of ascorbic acid and uric acid by a carbon nanotubes-ionic liquid gel modified electrode. Talanta 66:51–57 Bi H, Li Y, Liu S, et al. (2012) Carbon-nanotube-modified glassy carbon electrode for simultaneous determination of dopamine, ascorbic acid and uric acid: the effect of functional groups. Sensors Actuators B Chem 171-172:1132–1140 Zhou Y, He M, Huang C, et al. (2012) A novel and simple biosensor based on poly(indoleacetic acid) film and its application for simultaneous electrochemical determination of dopamine and epinephrine in the presence of ascorbic acid. J Solid State Electrochem 16: 2203–2210 Shahrokhian S, Zare-Mehrjardi H, Khajehsharifi H (2009) Modification of carbon paste with Congo red supported on multiwalled carbon nanotube for voltammetric determination of uric acid in the presence of ascorbic acid. J Solid State Electrochem 13: 1567–1575 Yang SL, Li G, Yang R, et al. (2011) Simultaneous voltammetric detection of dopamine and uric acid in the presence of high concentration of ascorbic acid using multi-walled carbon nanotubes with methylene blue composite film-modified electrode. J Solid State Electrochem 15:1909–1918 Tsierkezos NG, Ritter U (2012) Oxidation of dopamine on multiwalled carbon nanotubes. J Solid State Electrochem 16:2217–2226 Li NB, Ren W, Luo HQ (2007) Simultaneous voltammetric measurement of ascorbic acid and dopamine on poly(caffeic acid)-modified glassy carbon electrode. J Solid State Electrochem 12:693– 699 Parvin MH (2015) Simultaneous determination of ascorbic acid, dopamine and uric acid, at a graphene paste electrode modified with functionalized graphene sheets. Electroanalysis 27:1394–1402 Temoçin Z (2013) Modification of glassy carbon electrode in basic medium by electrochemical treatment for simultaneous determination of dopamine, ascorbic acid and uric acid. Sensors Actuators, B Chem 176:796–802
13.
Xie Y-L, Yuan J, Ye H-L, et al. (2015) Facile ultrasonic synthesis of graphene/SnO2 nanocomposite and its application to the simultaneous electrochemical determination of dopamine, ascorbic acid, and uric acid. J Electroanal Chem 749:26–30 14. Wang Y, Bi C (2013) Simultaneous electrochemical determination of ascorbic acid, dopamine and uric acid using poly (tyrosine)/functionalized multi-walled carbon nanotubes composite film modified electrode. J Mol Liq 177:26–31 15. Zhang R, Di JG, Chen D, Hu XY (2009) Simultaneous electrochemical determination of dopamine, ascorbic acid and uric acid using poly(acid chrome blue K) modified glassy carbon electrode. Sensors Actuators, B Chem 138:174–181 16. Zhang L, Shi Z, Lang Q (2011) Fabrication of poly(orthanilic acid)multiwalled carbon nanotubes composite film-modified glassy carbon electrode and its use for the simultaneous determination of uric acid and dopamine in the presence of ascorbic acid. J Solid State Electrochem 15:801–809 17. Silva FDADS, Lopes CB, Kubota LT, et al. (2012) Polyxanthurenic acid modified electrodes: an amperometric sensor for the simultaneous determination of ascorbic and uric acids. Sensors Actuators B Chem 168:289–296 18. Ferreira LF, Souza LM, Franco DL, et al. (2011) Formation of novel polymeric films derived from 4-hydroxybenzoic acid. Mater Chem Phys 129:46–52 19. Ferreira M, Varela H, Torresi RM, Tremiliosi-Filho G (2006) Electrode passivation caused by polymerization of different phenolic compounds. Electrochim Acta 52:434–442 20. Chambers JQ (1988) Electrochemistry of Quinones. In: Quinonoid Compd. pp 719–757 21. Hapiot P, Pinson J, Neta P, Al E (1994) Mechanism of oxidative coupling of coniferyl alcohol. Phytochemistry 36:1013–1020 22. Rodrigues LP, Ferreira DC, Sonoda MT, Madurro AGB, Abrahão O Jr, Madurro JM (2014) Electropolymerization mechanisms of hydroxyphenylacetic acid isomers. J Mol Struct 1072:298–306
