Materials Science and Engineering C 40 (2014) 49–54
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Determination of serotonin on platinum electrode modified with carbon nanotubes/polypyrrole/silver nanoparticles nanohybrid Ivana Cesarino ⁎, Heloisa V. Galesco, Sergio A.S. Machado Instituto de Química de São Carlos, Universidade de São Paulo, C.P. 780, 13560-970, São Carlos, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 5 November 2013 Received in revised form 17 February 2014 Accepted 17 March 2014 Available online 25 March 2014 Keywords: Silver nanoparticles Carbon nanotubes Polypyrrole Nanohybrid Serotonin
a b s t r a c t A new sensor has been developed by a simple electrodeposition of multi-walled carbon nanotubes (MWCNT), polypyrrole (PPy) and colloidal silver nanoparticles on the platinum (Pt) electrode surface. The Pt/MWCNT/ PPy/AgNPs electrode was applied to the detection of serotonin in plasmatic serum samples using differential pulse voltammetry (DPV). The synergistic effect of MWCNT/PPy/AgNPs nanohybrid formed yielded a LOD of 0.15 μmol L−1 (26.4 μg L−1). Reproducibility and repeatability values of 2.2% and 1.7%, respectively, were obtained compared to the conventional procedure. The proposed electrode can be an effective material to be used in biological analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction There is considerable interest in developing electrochemical techniques for determination of neurotransmitters, given the vast number of publications in this regard. Serotonin (5-hydroxytryptamine) is a monoamine neurotransmitter synthesised in serotonergic neurons in the central nervous system and plays a crucial role in the emotional system together with other monoamine transmitters such as regulation of mood, sleep, emesis (vomiting), sexuality and appetite. Low levels of serotonin have been associated with several disorders, notably depression, migraine, bipolar disorder and anxiety [1,2]. Recently, the development of versatile materials to modify electrode surface has been the target of numerous research in biological and environmental analysis using electrochemical methods. In most cases, this modification increases the sensitivity, selectivity, and reproducibility compared to conventional electrodes. One of the most widely used materials as a surface modifier is the nanostructured carbon, especially carbon nanotubes (CNTs). The use of CNTs in electrochemical sensors is due to their unique properties, such as high chemical stability, good electrical conductivity, high surface–volume ratio, and high adsorption capacity [3–5]. Moreover, similar to the electrically conducting polymers [6,7], the CNTs present functional groups anchored onto them, such as hydroxyl (\OH), carboxyl (\COOH), and carbonyl (\C_O), making these materials an excellent support that can be modified with several species, including metallic nanoparticles (NPs) [8–10]. ⁎ Corresponding author. Tel.: +55 16 3373 9924. E-mail address: [email protected] (I. Cesarino).
http://dx.doi.org/10.1016/j.msec.2014.03.030 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Silver nanoparticles (AgNPs) find use in many fields, and the major applications include their use as catalysts, as optical sensors, in textile engineering, in electronics, and in the medical field as a bactericidal and as a therapeutic agent [11]. When AgNPs were supported on CNTs, the CNT–AgNPs exhibited good electrocatalytic activity, remarkable antibacterial activity, and excellent surface-enhanced Raman scattering (SERS), as well as high chemical stability, excellent absorption capacity, and high selectivity [12,13]. One of the polymers most studied and applied in the development of sensors and biosensors is the polypyrrole (PPy) [14]. The use of PPy in electroanalysis is due to its suitable characteristics, such as stability at ambient conditions, high conductivity, efficient polymerisation at neutral pH, thickness controllability and a good reversibility between conducting and insulating states. In this sense, Harley et al. [15] fabricated a sensor by doping PPy with a sulfonated β-cyclodextrin to determine dopamine levels. Lu et al. [16] used a gold (Au) electrode modified with PPy/CNT/Au nanoparticles as a sensor to detect epinephrine (EP) sensitively when ascorbic acids and uric acids also exist. Another study [17] also determined EP using an overoxidised PPy/CNT composite on a glass carbon (GC) electrode. Considering that described above, an electrochemical sensor for selective determination of serotonin using CNTs/PPy/AgNPs nanohybrid is proposed. The sensor was prepared by a simple electrodeposition of a certain ratio of multi-walled carbon nanotubes (MWCNT) and PPy on the platinum (Pt) electrode surface, followed by the electrodeposition of colloidal AgNPs. The nanocomposite formed showed excellent properties through the synergistic effects of the component materials.
50
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
2. Experimental 2.1. Apparatus and procedures Cyclic voltammetry (CV), chronoamperometry (CA), and differential pulse voltammetry (DPV) experiments were performed using a model PGSTAT 30 Autolab electrochemical system (Eco Chemie, Utrecht, the Netherlands) equipped with GPES software (Eco Chemie Utrecht, the Netherlands). The cell was assembled with a conventional threeelectrode electrochemical system: Pt modified with MWCNT/PPy/ AgNPs nanohybrid as a working electrode, an Ag/AgCl/KCl (3.0 mol L− 1) electrode as the reference electrode and a Pt plate as the auxiliary electrode. All experiments were carried out at a 25 °C. DPV measurements were obtained over a relevant potential range, with a scan rate of 10 mV s−1, pulse amplitude of 100 mV, and a step potential of 2 mV in a 0.2 mol L −1 phosphate buffer solution (PBS) pH 8.0, containing 50.0 μmol L−1 of serotonin standards. The morphologies of the MWCNT/PPy and MWCNT/PPy/AgNPs nanohybrid were examined using a field-emission gun scanning electron microscope (FEG-SEM), and the images were recorded using a model FEI Inspect F50 microscope (FEI Company, Hillsboro, USA). 2.2. Chemicals and solutions All reagents used in this study were of analytical grade and were used without further purification. Pyrrole, MWCNT (90% purity), silver nitrate and serotonin hydrochloride were purchased from SigmaAldrich (Germany). All solutions were prepared with Nanopure water from a Barnsted Nanopure System (Thermo Scientific, USA).1.0 g of MWCNT was mixed with 500 mL of a 1:3 mixture of HNO3/H2SO4 for 12 h, in order to promote its functionalisation. They were then filtered through a 0.45 μm Millipore Nylon filter membrane. The resulting MWCNT were continuously washed using distilled water until the pH of the filtrate was neutral, at which point the MWCNT were dried overnight in a vacuum oven at 120 °C. 2.3. Synthesis of colloidal silver nanoparticles (AgNPs) The AgNPs were prepared from dropwise addition of 10 mL of a 1.0 mmol L −1 AgNO3 solution in 30 mL of a 2.0 mmol L −1 NaBH4 solution. The addition of silver nitrate was performed slowly (1 drop per second), under magnetic stirring and ice bath. Initially the solution is slightly yellow, but becomes bright yellow when the whole solution of AgNO3 is added. The colloidal AgNPs is stored under refrigeration. 2.4. Preparation of the Pt/MWCNT/PPy/AgNPs electrode Prior to modification, the Pt electrode surface was polished with 0.5 μm alumina slurries, rinsed thoroughly with double-distilled water, sonicated for 5 min in ethanol and 5 min in water, and then dried in air. 25.0 mL of 0.2 mol L−1 NaCl solution containing 443 μL of pyrrole and 5.0 mg of MWCNT was sonicated for 10 min with 70% amplitude. The Pt electrode was immersed in this solution and electrocodeposition of PPy/MWCNT was performed using 10 scans of CV in the potential range of − 0.2 and + 0.8 V (vs. Ag/AgCl) with a scan rate of 50 mV s −1. The Pt electrode containing the MWCNT/PPy film was removed from the solution and the electrodeposition of AgNPs is performed using CA at a fixed potential of − 1.0 V for 420 s in 12.0 mL of a 0.1 mol L −1 KCl solution containing 100.0 μmol L −1 of colloidal AgNPs. After this step, the proposed Pt/MWCNT/PPy/AgNPs electrode is washed with distilled water and air dried. 2.5. Preparation of the plasmatic serum and analysis of serotonin Samples similar to plasmatic serum were Krebs–Ringer solution and PBS. The artificial body fluid was prepared similarly as described by
Brett et al. [18]. The solution composition per litre of solution was: 6.98 g NaCl, 0.36 g KCl, 0.28 g CaCl2, 0.15 g MgSO4, 210 mL PBS, and pH 8.0. An aliquot of 10 mL of the plasmatic serum was spiked with 2.0 μmol L −1 of serotonin. The sample analyses were performed using the standard addition method and DPV experiments were carried out directly on these samples without pre-treatment procedures. 3. Results and discussion 3.1. Morphological characterisation of the nanohybrid The morphologies of the PPy, MWCNT/PPy, and the MWCNT/PPy/ AgNPs nanohybrid were examined by FEG-SEM analysis. Fig. 1 displays typical images of the PPy, MWCNT/PPy and MWCNT/PPy/AgNPs films assembled onto Pt electrodes. Fig. 1A characterises the electropolymerised PPy structure as compact layers of a globular shape, where each globule has an average size of 400 nm. Fig. 1B indicates that MWCNT structures were totally covered by a compact layer of PPy in a core–shell structure [14,19]. The average diameter of the MWCNT covered by a PPy layer was calculated at 750 nm, which was much larger than that of the uncovered MWCNT (80 nm). The MWCNT/PPy/AgNPs nanohybrid displayed in Fig. 1C shows that the MWCNT/PPy was modified with the colloidal AgNPs. The AgNPs were preferentially supported in the sidewalls of the MWCNT/PPy and had sizes varying from 55 to 85 nm. 3.2. Electrocatalytic oxidation of serotonin on the Pt/MWCNT/PPy/AgNPs electrode The effect of scan rate on the electrocatalytic oxidation of serotonin at the Pt/MWCNT/PPy/AgNPs electrode was carried out in 0.2 mol L −1 PBS pH 7.0 containing 100.0 μmol L −1 of serotonin by cyclic voltammetry experiments, using several scan rates in a range varying from 10 to 100 mV s− 1, and the results obtained are presented in Fig. 2. In the voltammetric response using a scan rate of the 50 mV s−1 (open circle in Fig. 2), it can be seen a well-defined oxidation peak at a potential value of +0.42 V vs. Ag/AgCl/KCl (3.0 mol L−1). This peak is attributed to an irreversible oxidation of the hydroxyl group present in the aromatic ring of the serotonin forming ketone species. As can be observed in the inset of Fig. 2, there is a linear relationship between the anodic current peak and the square root of scan rate (v1/2) in the range between 10 and 100 mV s −1, which can be described by the equation Ipa = 3.99 + 2.61 v (Ipa in μA and scan rate in mV s −1, r = 0.993), which reflects the control of the electrochemical process by adsorbed species. 3.3. Parameters for the optimisation of the serotonin response on the Pt/MWCNT/PPy/AgNPs electrode To maximise the DPV analytical signal, the effects of experimental parameters (electrode composition, pulse amplitude, potential step height, scan rate, pre-treatment potential and time for cleaning the electrode surface) were studied using Pt/MWCNT/PPy/AgNPs electrode in a 0.2 mol L −1 PBS pH 7.0 containing 50.0 μmol L −1 of serotonin. The influence of the composition of the electrode material was evaluated first. Accordingly, the colloidal AgNPs concentration on the nanohybrid electrode was optimised in the range of 37.5 to 125.0 μmol L −1 in the potential interval of 0.0 to +0.6 V vs. Ag/AgCl/KCl (3.0 mol L−1) as shown in Fig. 3. An increase by a factor of 2.3 was observed in the anodic peak current when the amount of colloidal AgNPs was changed from 37.5 to 100.0 μmol L −1, reaching the maximum current at 100.0 μmol L −1. When 125.0 μmol L −1 of colloidal AgNPs was used in the nanohybrid electrode preparation the anodic peak current decreased significantly. This occurs due to the loss of the AgNPs nanostructuration. This fact is attributed to the disordered formation of Ag clusters, leading to an increase in particle size. Based on these results, 100.0 μmol L −1 of colloidal AgNPs was used for the nanohybrid preparation.
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
51
Fig. 2. Cyclic voltammograms for Pt/MWCNT/PPy/AgNPs electrode in 0.2 mol L −1 PBS pH 7.0 containing 100 μmol L−1 of serotonin at the following scan rates: 10, 20, 30, 40, 50, 75 and 100 mV s−1. Inset: linear relationship between the anodic peak current and v½.
The following pre-treatment potential values: 0.0, − 0.1, − 0.2, −0.3, −0.4 and − 0.5 V were investigated, and the results are shown in Fig. 4. Complete recovery of the original response was obtained between −0.3 and −0.2 V. At more positive cleaning potentials the peak current, Ipa, obtained during the following experiment begins to decrease, reaching 65% at 0.0 V pre-treatment cleaning potential. Thus, − 0.2 V was chosen as the pre-treatment cleaning potential in further studies. The influence of the pre-treatment time was also evaluated, varying it from 20 to 90 s, at which the complete recovery of the analytical signal was obtained in 45 s. Hence 45 s was chosen as time of pre-treatment for cleaning the electrode surface. The influence of DPV voltammetry parameters was then investigated. First, the amplitude was varied from 10 to 100 mV, while holding the step potential constant at 2 mV and the scan rate at 10 mV s −1. It did not observe any significant increase in peak widths for serotonin oxidation, even at amplitudes greater than 50 mV. Since the peak currents were proportional to the increase in amplitude, 100 mV was chosen as the optimised DPV amplitude. The next step was keeping the amplitude constant at 100 mV and the scan rate in 10 mV s −1, and studying the effect of step potential increments within the range of 1 to 10 mV. For step
Fig. 1. FEG-SEM micrographs for (A) Pt/PPy, (B) Pt/MWCNT/PPy and (C) Pt/MWCNT/PPy/ AgNPs.
In general, the electrochemical detection of serotonin is accompanied by adsorption of the serotonin or its oxidation products. Thus, there is a poisoning of the electrode surface and consequent decrease of the analytical signal. Therefore, the next study performed was the pre-treatment potential and time for cleaning the electrode surface.
Fig. 3. Effect of the colloidal AgNPs concentration on the nanohybrid electrode. DPV voltammograms collected in 0.2 mol L−1 PBS pH 7.0 containing 50.0 μmol L−1 of serotonin. Inset: Dependence of the serotonin oxidation peak current and colloidal AgNPs concentration.
52
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
with the current observed for the other electrodes. Serotonin oxidation on the proposed electrode showed an increase by a factor of 1.8 in the current peak when compared to the electrode that was prepared with the AgNPs support directly on MWCNT, namely Pt/MWCNT–AgNPs/ PPy electrode (curve c). This occurs because the colloidal AgNPs are more exposed on the surface electrode and its synergistic effect with the MWCNT and PPy is increased. Regarding the Pt/MWCNT/PPy electrode (curve b), an increase by a factor of 2.4 was observed for serotonin oxidation. As expected, when the Pt/MWCNT/PPy/AgNPs electrode was compared with the Pt/PPy electrode (curve a) an even higher increase was observed: by a factor of 10.0 for serotonin oxidation. The increase in current values reflects the increase of the electroactive surface area by the MWCNT/PPy/AgNPs nanohybrid formed. Such properties make the Pt/MWCNT/PPy/AgNPs electrode a promising setup for serotonin detection. 3.5. Effect of pH on serotonin oxidation process Fig. 4. Effect of pre-treatment potential on the serotonin oxidation peak current using DPV in 0.2 mol L−1 PBS pH 7.0 containing 50.0 μmol L −1 of serotonin.
potential greater than 2 mV, deformation of the voltammetric profiles was observed and the anodic peak currents for serotonin decreased in height. Hence, a 2 mV step potential increment was chosen. The effect of scan rate was studied in the range of 2.5 to 15 mV s −1, with the step potential constant at 2 mV and the amplitude at 100 mV. The scan rate of 10 mV s −1 was chosen because it presented more anodic peak current. Higher scan rates caused loss in peak definition and lower anodic peak current. 3.4. Comparison of the voltammetric behaviour of serotonin on modified electrodes The DPV experiments were performed in the potential range of 0.0 to + 0.6 V vs. Ag/AgCl/KCl (3.0 mol L−1) in 0.2 mol L −1 PBS at pH 7.0 containing 50.0 μmol L −1 of serotonin for the following electrodes: Pt/PPy (curve a), Pt/MWCNT/PPy (curve b), Pt/MWCNT–AgNPs/PPy (curve c), and Pt/MWCNT/PPy/AgNPs (curve d), as shown in Fig. 5. The electrode used to obtain the curve c was prepared with the AgNPs supported directly on MWCNT as previously described [10]. The voltammetric profiles had peaks at almost identical potentials of approximately +0.4 V vs. Ag/AgCl/KCl (3.0 mol L−1) for all electrodes studied. However, the Pt/MWCNT/PPy/AgNPs electrode (curve d) displayed higher anodic current intensities for serotonin in comparison
Fig. 5. DPV voltammograms obtained under the optimised parameters in 0.2 mol L−1 PBS pH 7.0 containing 50.0 μmol L−1 of serotonin for the following electrodes: (a) Pt/PPy, (b) Pt/MWCNT/PPy, (c) Pt/MWCNT-AgNPs/PPy and, (d) Pt/MWCNT/PPy/AgNPs.
Aiming to confirm the mechanism of serotonin oxidation on the Pt/MWCNT/PPy/AgNPs electrode, the dependence on the electrochemical oxidation of serotonin on pH was studied by DPV experiments at pHs ranging from 4.0 to 10.0 in 0.2 mol L−1 PBS containing 25.0 μmol L−1 of serotonin. The results are shown in Fig. 6, which is a plot of the DPV peak current (Ipa) and peak potential (Epa) as a function of pH. The variation of Epa with pH can provide valuable information about the serotonin oxidation process. For serotonin, reducing the hydrogen ionic concentration of the electrolyte causes a shift in peak potential towards more negative values, as illustrated Fig. 5. This is a consequence of deprotonation in the oxidation process that is facilitated at higher pHs. The Epa vs. pH showed a linear relationship, with a slope of 61.7 mV per pH unit. Therefore, an electrochemical process involving the same number of protons and electrons during the electrooxidation of serotonin can be proposed, which is in agreement with previous reports [20–22]. The plot of Ipa vs. pH, as shown in Fig. 6, demonstrates that the peak current has a maximum value at pH 8.0 decreasing for lower and higher pH values. Thus, a 0.2 mol L−1 PBS at pH 8.0 was used in the further studies. 3.6. Analytical characteristics DPV experiments were carried out in triplicate using the optimised experimental parameters to obtain an analytical curve for the determination of serotonin with the Pt/MWCNT/PPy/AgNPs electrode. The analytical response shown in Fig. 7 has a linear response in the
Fig. 6. Effect of pH on the peak potential (■) and peak current (●) for serotonin oxidation on the Pt/MWCNT/PPy/AgNPs electrode using 0.2 mol L−1 PBS containing 25.0 μmol L−1 of serotonin.
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
53
process of the serotonin on the Pt/MWCNT/PPy/AgNPs surface was evaluated. The experiments were carried out using DPV with optimised parameters in solutions with 0.2 mol L −1 of PBS (pH 8.0), concentrations of AA and UA fixed at 15.0 μmol L −1, and sequential additions of 10.0, 15.0, and 25.0 μmol L −1 of serotonin. Fig. 8 shows some of the results. The peak at +0.22 V is due to the AA oxidation process; however no signal associated with UA could be detected in these experimental conditions using the proposed electrode. It can be noted that, even at equal concentrations of AA, UA, and serotonin, it was not observed an overlap process of the serotonin oxidation peak and the oxidation peaks of the interfering substances studied. Also, the interfering substances studied did not shift the serotonin oxidation peak, indicating that the analytical signal did not suffer interference of the AA and UA. Accordingly, the Pt/MWCNT/PPy/AgNPs electrode is adequate to be used for selective determination of serotonin in the presence of AA and UA. 3.8. Analysis of serotonin in plasmatic serum sample Fig. 7. DPV voltammograms for Pt/MWCNT/PPy/AgNPs electrode, with the optimised parameters. The serotonin concentrations in μmol L−1 are: (a) 0.50, (b) 1.00, (c) 1.50, (d) 2.00, (e) 2.50, (f) 3.00, (g) 4.00, and (h) 5.00. Inset: linear dependence of the peak current with serotonin concentrations.
range from 0.50 to 5.0 μmol L −1, in accordance with the following equation: Ipa ðμAÞ ¼ 0:16 ðμAÞ −1 −1 ½serotonin μmol L þ 0:43 μA=μmol L
ð1Þ
with a correlation coefficient of 0.998 (n = 8). The LOD obtained was 0.15 μmol L − 1 (26.4 μg L − 1), being determined using a 3σ/slope ratio, where σ is the standard deviation of the mean value for 10 voltammograms of the blank. Comparing the results at the Pt/MWCNT/PPy/AgNP electrode with other electrochemical methods for serotonin detection, higher detection limits were observed at carbon electrodes: 500 nM for polycrystalline boron-doped and 2 μM for glassy carbon [23]. Rand et al. [24] also found high detection limit of 250 μM using a biosensor based on a carbon nanofiber. Lower detection limits of 0.003 μmol L −1, 0.023 μmol L −1 and 0.2 nmol L −1 were calculated using a Nafion/Ni(OH)2 nanoparticle– MWCNT modified glassy carbon electrode [25], a carbon ionic liquid electrode modified with Co(OH)2 nanoparticles and MWCNT [20] and a glass/ PDMS hybrid microfluidic device integrating vertically aligned SWCNTs [26], respectively. The advantages of the Pt/MWCNT/PPy/AgNPs electrode are that this sensor has a simple preparation and it exhibits long term stability, since stored in 0.2 mol L −1 PBS pH 8.0 at 4 °C. The reproducibility of the Pt/MWCNT/PPy/AgNPs electrode was measured from seven experiments, in which each experiment consisted of five sequential DPV voltammograms. These experiments were performed on different days. Prior to each experiment, the electrode surfaces were rinsed thoroughly with double-distilled water. Thus, the DPV voltammograms were performed in 0.2 mol L −1 PBS at pH 8.0 containing 50.0 μmol L −1 of serotonin. The RSD was calculated as 2.2%. In addition, intra-assay precision tests were performed from ten DPV voltammograms of that same solution. The RSD was found to be 1.7%.
The developed sensor was used for the quantification of serotonin in plasmatic serum, prepared as described in the Experimental section. The corresponding DPV voltammograms obtained for the analysis of the plasmatic serum sample are shown in Fig. 9, along with the respective standard addition plots. Serotonin determinations were performed in triplicate, without any treatment procedure, using the standard addition method. The results obtained (mean ± SD) for three determinations were: 1.99 ± 0.05 μmol L −1 for serotonin. Recoveries between 97.9% and 103.1% of serotonin from plasmatic serum samples (n = 3) were obtained for samples spiked with 1.0, 1.5, and 2.0 μmol L −1 of serotonin. There were no significant differences between the found and added concentrations of serotonin, indicating that the Pt/MWCNT/PPy/AgNP electrode can be successfully used for the determination of serotonin in plasmatic serum samples under the optimised conditions and using the standard addition approach. 4. Conclusions A newly modified Pt electrode was developed using MWCNT/PPy/ AgNPs nanohybrid, which can be used for the determination of serotonin in plasmatic serum samples. The MWCNT/PPy/AgNPs nanohybrid was successfully characterised by FEG-SEM microscopy, which indicated that MWCNT structures were totally covered by a compact layer of PPy and the colloidal AgNPs were
3.7. Selective determination of serotonin in the presence of interfering The electrochemical determination of serotonin in biological fluids is inhibited by the interference of ascorbic acid (AA) and uric acid (UA) also present in such matrices in high concentrations. Advanced chemical sensors for such determination should be able to reduce such interferences without diminishing the serotonin signal. In this sense, the intensity of the interference of these species in the electrooxidation
Fig. 8. DPV experiments for Pt/MWCNT/PPy/AgNPs electrode using the optimised parameters in 0.2 mol L−1 PBS pH 8.0 with concentrations of ascorbic acid (AA) and uric acid (UA) fixed at 15.0 μmol L −1, and sequential additions of serotonin standards in the following concentrations: 10.0 (open circle), 15.0 (close circle), and 25.0 (solid line) μmol L−1.
54
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
Fig. 9. DPV responses obtained on a Pt/MWCNT/PPy/AgNPs electrode for the determination of serotonin in plasmatic serum: S) sample; 1) sample plus 1.0 μmol L−1 serotonin; 2) sample plus 1.5 μmol L−1 serotonin; 3) sample plus 2.0 μmol L−1 serotonin. Inset: linear dependence of the peak current with serotonin concentrations.
supported onto MWCNT/PPy. Furthermore, the synergistic effect of MWCNT/PPy/AgNPs nanohybrid formed yielded lower LOD and improved the reproducibility, repeatability, and the sensitivity of the Pt electrode in the analysis of serotonin. Finally, the Pt/MWCNT/PPy/AgNPs electrode can be an effective material for serotonin electrochemical determination and is an alternative material to be used in biological analysis. Acknowledgments We are grateful for the financial support from CAPES, CNPq (grant 471467/2012-0) and FAPESP (2012/19633-0). References [1] J.M. Zen, I.L. Chen, Y. Shih, Voltammetric determination of serotonin in human blood using a chemically modified electrode, Anal. Chim. Acta. 369 (1998) 103–108. [2] Z.H. Wang, Q.L. Liang, Y.M. Wang, G.A. Luo, Carbon nanotube-intercalated graphite electrodes for simultaneous determination of dopamine and serotonin in the presence of ascorbic acid, J. Electroanal. Chem. 540 (2003) 129–134. [3] F.C. Moraes, T.A. Silva, I. Cesarino, M.R.V. Lanza, S.A.S. Machado, Antibiotic detection in urine using electrochemical sensors based on vertically aligned carbon nanotubes, Electroanalysis 25 (2013) 2092–2099. [4] I. Cesarino, F.C. Moraes, S.A.S. Machado, J. Passaretti-Filho, A.A. Cardoso, A new indirect electrochemical method for determination of ozone in water using multiwalled carbon nanotubes, Electroanalysis 23 (2011) 1512–1517. [5] A. Merkoci, M. Pumera, X. Llopis, B. Pérez, M. Del Valle, S. Alegret, New materials for electrochemical sensing VI: Carbon nanotubes, TrAC, Trends Anal. Chem. 24 (2005) 826–838. [6] X.G. Li, H. Feng, M.R. Huang, G.L. Gu, M.G. Moloney, Ultrasensitive Pb(II) potentiometric sensor based on copolyaniline nanoparticles in a plasticizer-free membrane with a long lifetime, Anal. Chem. 84 (2012) 134–140.
[7] M.R. Huang, Y.B. Ding, X.G. Li, Lead-ion potentiometric sensor based on electrically conducting microparticles of sulfonic phenylenediamine copolymer, Analyst 138 (2013) 3820–3829. [8] F.C. Moraes, I. Cesarino, V. Cesarino, L.H. Mascaro, S.A.S. Machado, Carbon nanotubes modified with antimony nanoparticles: A novel material for electrochemical sensing, Electrochim. Acta 85 (2012) 560–565. [9] I. Cesarino, V. Cesarino, M.R.V. Lanza, Carbon nanotubes modified with antimony nanoparticles in a paraffin composite electrode: Simultaneous determination of sulfamethoxazole and trimethoprim, Sensors Actuators B 188 (2013) 1293–1299. [10] I. Cesarino, V. Cesarino, F.C. Moraes, T.C.R. Ferreira, M.R.V. Lanza, L.H. Mascaro, S. A.S. Machado, Electrochemical degradation of benzene in natural water using silver nanoparticle-decorated carbon nanotubes, Mater. Chem. Phys. 141 (2013) 304–309. [11] S. Prabhu, E.K. Poulose, Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects, Int. Nano Lett. 32 (2012) 1–10. [12] A. Balamurugan, S.M. Chen, Silver nanograins incorporated PEDOT modified electrode for electrocatalytic sensing of hydrogen peroxide, Electroanalysis 21 (2009) 1419–1423. [13] Y.C. Tsai, P.C. Hsu, Y.W. Lin, T.M. Wu, Silver nanoparticles in multiwalled carbon nanotube-Nafion for surface-enhanced Raman scattering chemical sensor, Sensors Actuators B 138 (2009) 5–8. [14] I. Cesarino, H.V. Galesco, F.C. Moraes, M.R.V. Lanza, S.A.S. Machado, Biosensor based on electrocodeposition of carbon nanotubes/polypyrrole/laccase for neurotransmitter detection, Electroanalysis 25 (2013) 394–400. [15] C.C. Harley, A.D. Rooney, C.B. Breslin, The selective detection of dopamine at a polypyrrole film doped with sulfonated-cyclodextrins, Sensors Actuators B 150 (2010) 498–504. [16] X. Lu, Y. Li, J. Du, X. Zhou, Z. Xue, X. Liu, Z. Wang, A novel nanocomposites sensor for epinephrine detection in the presence of uric acids and ascorbic acids, Electrochim. Acta 56 (2011) 7261–7266. [17] S. Shahrokhian, R.S. Saberi, Electrochemical preparation of over-oxidized polypyrrole/ multi-walled carbon nanotube composite on glassy carbon electrode and its application in epinephrine determination, Electrochim. Acta 57 (2011) 132–138. [18] C.M.A. Brett, I. Muresan, The influence of artificial body fluids on metallic corrosion, Key Eng. Mater. 230–232 (2002) 459–462. [19] I. Cesarino, F.C. Moraes, S.A.S. Machado, A biosensor based on polyaniline-carbon nanotube core-shell for electrochemical detection of pesticides, Electroanalysis 23 (2011) 2586–2593. [20] A. Babaei, A.R. Taheri, M. Aminikhah, Nanomolar simultaneous determination of levodopa and serotonin at a novel carbon ionic liquid electrode modified with Co(OH)2 nanoparticles and multi-walled carbon nanotubes, Electrochim. Acta 90 (2013) 317–325. [21] Y. Li, X. Huang, Y. Chen, L. Wang, X. Lin, Simultaneous determination of dopamine and serotonin by use of covalent modification of 5-hydroxytryptophan on glassy carbon electrode, Microchim. Acta 164 (2009) 107–112. [22] R.T. Kachoosangi, R.G. Compton, A simple electroanalytical methodology for the simultaneous determination of dopamine, serotonin and ascorbic acid using an unmodified edge plane pyrolytic graphite electrode, Anal. Bioanal. Chem. 387 (2007) 2793–2800. [23] A.G. Guell, K.E. Meadows, P.R. Unwin, J.V. Macpherson, Trace voltammetric detection of serotonin at carbon electrodes: comparison of glassy carbon, boron doped diamond and carbon nanotube network electrodes, Phys. Chem. Chem. Phys. 12 (2010) 10108–10114. [24] E. Rand, A. Periyakaruppan, Z. Tanaka, D.A. Zhang, M.P. Marsh, R.J. Andrews, K.H. Lee, B. Chen, M. Meyyappan, J.E. Koehne, A carbon nanofiber based biosensor for simultaneous detection of dopamine and serotonin in the presence of ascorbic acid, Biosens. Bioelectron. 42 (2013) 434–438. [25] A. Babaei, A.R. Taheri, Nafion/Ni(OH)2 nanoparticles-carbon nanotube composite modified glassy carbon electrode as a sensor for simultaneous determination of dopamine and serotonin in the presence of ascorbic acid, Sensors Actuators B 176 (2013) 543–551. [26] F.C. Moraes, R.S. Lima, T.P. Segato, I. Cesarino, J.L.M. Cetino, S.A.S. Machado, F. Gomez, E. Carrilho, Glass/PDMS hybrid microfluidic device integrating vertically aligned SWCNTs to ultrasensitive electrochemical determinations, Lab Chip 12 (2012) 1959–1962.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Determination of serotonin on platinum electrode modified with carbon nanotubes/polypyrrole/silver nanoparticles nanohybrid Ivana Cesarino ⁎, Heloisa V. Galesco, Sergio A.S. Machado Instituto de Química de São Carlos, Universidade de São Paulo, C.P. 780, 13560-970, São Carlos, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 5 November 2013 Received in revised form 17 February 2014 Accepted 17 March 2014 Available online 25 March 2014 Keywords: Silver nanoparticles Carbon nanotubes Polypyrrole Nanohybrid Serotonin
a b s t r a c t A new sensor has been developed by a simple electrodeposition of multi-walled carbon nanotubes (MWCNT), polypyrrole (PPy) and colloidal silver nanoparticles on the platinum (Pt) electrode surface. The Pt/MWCNT/ PPy/AgNPs electrode was applied to the detection of serotonin in plasmatic serum samples using differential pulse voltammetry (DPV). The synergistic effect of MWCNT/PPy/AgNPs nanohybrid formed yielded a LOD of 0.15 μmol L−1 (26.4 μg L−1). Reproducibility and repeatability values of 2.2% and 1.7%, respectively, were obtained compared to the conventional procedure. The proposed electrode can be an effective material to be used in biological analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction There is considerable interest in developing electrochemical techniques for determination of neurotransmitters, given the vast number of publications in this regard. Serotonin (5-hydroxytryptamine) is a monoamine neurotransmitter synthesised in serotonergic neurons in the central nervous system and plays a crucial role in the emotional system together with other monoamine transmitters such as regulation of mood, sleep, emesis (vomiting), sexuality and appetite. Low levels of serotonin have been associated with several disorders, notably depression, migraine, bipolar disorder and anxiety [1,2]. Recently, the development of versatile materials to modify electrode surface has been the target of numerous research in biological and environmental analysis using electrochemical methods. In most cases, this modification increases the sensitivity, selectivity, and reproducibility compared to conventional electrodes. One of the most widely used materials as a surface modifier is the nanostructured carbon, especially carbon nanotubes (CNTs). The use of CNTs in electrochemical sensors is due to their unique properties, such as high chemical stability, good electrical conductivity, high surface–volume ratio, and high adsorption capacity [3–5]. Moreover, similar to the electrically conducting polymers [6,7], the CNTs present functional groups anchored onto them, such as hydroxyl (\OH), carboxyl (\COOH), and carbonyl (\C_O), making these materials an excellent support that can be modified with several species, including metallic nanoparticles (NPs) [8–10]. ⁎ Corresponding author. Tel.: +55 16 3373 9924. E-mail address: [email protected] (I. Cesarino).
http://dx.doi.org/10.1016/j.msec.2014.03.030 0928-4931/© 2014 Elsevier B.V. All rights reserved.
Silver nanoparticles (AgNPs) find use in many fields, and the major applications include their use as catalysts, as optical sensors, in textile engineering, in electronics, and in the medical field as a bactericidal and as a therapeutic agent [11]. When AgNPs were supported on CNTs, the CNT–AgNPs exhibited good electrocatalytic activity, remarkable antibacterial activity, and excellent surface-enhanced Raman scattering (SERS), as well as high chemical stability, excellent absorption capacity, and high selectivity [12,13]. One of the polymers most studied and applied in the development of sensors and biosensors is the polypyrrole (PPy) [14]. The use of PPy in electroanalysis is due to its suitable characteristics, such as stability at ambient conditions, high conductivity, efficient polymerisation at neutral pH, thickness controllability and a good reversibility between conducting and insulating states. In this sense, Harley et al. [15] fabricated a sensor by doping PPy with a sulfonated β-cyclodextrin to determine dopamine levels. Lu et al. [16] used a gold (Au) electrode modified with PPy/CNT/Au nanoparticles as a sensor to detect epinephrine (EP) sensitively when ascorbic acids and uric acids also exist. Another study [17] also determined EP using an overoxidised PPy/CNT composite on a glass carbon (GC) electrode. Considering that described above, an electrochemical sensor for selective determination of serotonin using CNTs/PPy/AgNPs nanohybrid is proposed. The sensor was prepared by a simple electrodeposition of a certain ratio of multi-walled carbon nanotubes (MWCNT) and PPy on the platinum (Pt) electrode surface, followed by the electrodeposition of colloidal AgNPs. The nanocomposite formed showed excellent properties through the synergistic effects of the component materials.
50
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
2. Experimental 2.1. Apparatus and procedures Cyclic voltammetry (CV), chronoamperometry (CA), and differential pulse voltammetry (DPV) experiments were performed using a model PGSTAT 30 Autolab electrochemical system (Eco Chemie, Utrecht, the Netherlands) equipped with GPES software (Eco Chemie Utrecht, the Netherlands). The cell was assembled with a conventional threeelectrode electrochemical system: Pt modified with MWCNT/PPy/ AgNPs nanohybrid as a working electrode, an Ag/AgCl/KCl (3.0 mol L− 1) electrode as the reference electrode and a Pt plate as the auxiliary electrode. All experiments were carried out at a 25 °C. DPV measurements were obtained over a relevant potential range, with a scan rate of 10 mV s−1, pulse amplitude of 100 mV, and a step potential of 2 mV in a 0.2 mol L −1 phosphate buffer solution (PBS) pH 8.0, containing 50.0 μmol L−1 of serotonin standards. The morphologies of the MWCNT/PPy and MWCNT/PPy/AgNPs nanohybrid were examined using a field-emission gun scanning electron microscope (FEG-SEM), and the images were recorded using a model FEI Inspect F50 microscope (FEI Company, Hillsboro, USA). 2.2. Chemicals and solutions All reagents used in this study were of analytical grade and were used without further purification. Pyrrole, MWCNT (90% purity), silver nitrate and serotonin hydrochloride were purchased from SigmaAldrich (Germany). All solutions were prepared with Nanopure water from a Barnsted Nanopure System (Thermo Scientific, USA).1.0 g of MWCNT was mixed with 500 mL of a 1:3 mixture of HNO3/H2SO4 for 12 h, in order to promote its functionalisation. They were then filtered through a 0.45 μm Millipore Nylon filter membrane. The resulting MWCNT were continuously washed using distilled water until the pH of the filtrate was neutral, at which point the MWCNT were dried overnight in a vacuum oven at 120 °C. 2.3. Synthesis of colloidal silver nanoparticles (AgNPs) The AgNPs were prepared from dropwise addition of 10 mL of a 1.0 mmol L −1 AgNO3 solution in 30 mL of a 2.0 mmol L −1 NaBH4 solution. The addition of silver nitrate was performed slowly (1 drop per second), under magnetic stirring and ice bath. Initially the solution is slightly yellow, but becomes bright yellow when the whole solution of AgNO3 is added. The colloidal AgNPs is stored under refrigeration. 2.4. Preparation of the Pt/MWCNT/PPy/AgNPs electrode Prior to modification, the Pt electrode surface was polished with 0.5 μm alumina slurries, rinsed thoroughly with double-distilled water, sonicated for 5 min in ethanol and 5 min in water, and then dried in air. 25.0 mL of 0.2 mol L−1 NaCl solution containing 443 μL of pyrrole and 5.0 mg of MWCNT was sonicated for 10 min with 70% amplitude. The Pt electrode was immersed in this solution and electrocodeposition of PPy/MWCNT was performed using 10 scans of CV in the potential range of − 0.2 and + 0.8 V (vs. Ag/AgCl) with a scan rate of 50 mV s −1. The Pt electrode containing the MWCNT/PPy film was removed from the solution and the electrodeposition of AgNPs is performed using CA at a fixed potential of − 1.0 V for 420 s in 12.0 mL of a 0.1 mol L −1 KCl solution containing 100.0 μmol L −1 of colloidal AgNPs. After this step, the proposed Pt/MWCNT/PPy/AgNPs electrode is washed with distilled water and air dried. 2.5. Preparation of the plasmatic serum and analysis of serotonin Samples similar to plasmatic serum were Krebs–Ringer solution and PBS. The artificial body fluid was prepared similarly as described by
Brett et al. [18]. The solution composition per litre of solution was: 6.98 g NaCl, 0.36 g KCl, 0.28 g CaCl2, 0.15 g MgSO4, 210 mL PBS, and pH 8.0. An aliquot of 10 mL of the plasmatic serum was spiked with 2.0 μmol L −1 of serotonin. The sample analyses were performed using the standard addition method and DPV experiments were carried out directly on these samples without pre-treatment procedures. 3. Results and discussion 3.1. Morphological characterisation of the nanohybrid The morphologies of the PPy, MWCNT/PPy, and the MWCNT/PPy/ AgNPs nanohybrid were examined by FEG-SEM analysis. Fig. 1 displays typical images of the PPy, MWCNT/PPy and MWCNT/PPy/AgNPs films assembled onto Pt electrodes. Fig. 1A characterises the electropolymerised PPy structure as compact layers of a globular shape, where each globule has an average size of 400 nm. Fig. 1B indicates that MWCNT structures were totally covered by a compact layer of PPy in a core–shell structure [14,19]. The average diameter of the MWCNT covered by a PPy layer was calculated at 750 nm, which was much larger than that of the uncovered MWCNT (80 nm). The MWCNT/PPy/AgNPs nanohybrid displayed in Fig. 1C shows that the MWCNT/PPy was modified with the colloidal AgNPs. The AgNPs were preferentially supported in the sidewalls of the MWCNT/PPy and had sizes varying from 55 to 85 nm. 3.2. Electrocatalytic oxidation of serotonin on the Pt/MWCNT/PPy/AgNPs electrode The effect of scan rate on the electrocatalytic oxidation of serotonin at the Pt/MWCNT/PPy/AgNPs electrode was carried out in 0.2 mol L −1 PBS pH 7.0 containing 100.0 μmol L −1 of serotonin by cyclic voltammetry experiments, using several scan rates in a range varying from 10 to 100 mV s− 1, and the results obtained are presented in Fig. 2. In the voltammetric response using a scan rate of the 50 mV s−1 (open circle in Fig. 2), it can be seen a well-defined oxidation peak at a potential value of +0.42 V vs. Ag/AgCl/KCl (3.0 mol L−1). This peak is attributed to an irreversible oxidation of the hydroxyl group present in the aromatic ring of the serotonin forming ketone species. As can be observed in the inset of Fig. 2, there is a linear relationship between the anodic current peak and the square root of scan rate (v1/2) in the range between 10 and 100 mV s −1, which can be described by the equation Ipa = 3.99 + 2.61 v (Ipa in μA and scan rate in mV s −1, r = 0.993), which reflects the control of the electrochemical process by adsorbed species. 3.3. Parameters for the optimisation of the serotonin response on the Pt/MWCNT/PPy/AgNPs electrode To maximise the DPV analytical signal, the effects of experimental parameters (electrode composition, pulse amplitude, potential step height, scan rate, pre-treatment potential and time for cleaning the electrode surface) were studied using Pt/MWCNT/PPy/AgNPs electrode in a 0.2 mol L −1 PBS pH 7.0 containing 50.0 μmol L −1 of serotonin. The influence of the composition of the electrode material was evaluated first. Accordingly, the colloidal AgNPs concentration on the nanohybrid electrode was optimised in the range of 37.5 to 125.0 μmol L −1 in the potential interval of 0.0 to +0.6 V vs. Ag/AgCl/KCl (3.0 mol L−1) as shown in Fig. 3. An increase by a factor of 2.3 was observed in the anodic peak current when the amount of colloidal AgNPs was changed from 37.5 to 100.0 μmol L −1, reaching the maximum current at 100.0 μmol L −1. When 125.0 μmol L −1 of colloidal AgNPs was used in the nanohybrid electrode preparation the anodic peak current decreased significantly. This occurs due to the loss of the AgNPs nanostructuration. This fact is attributed to the disordered formation of Ag clusters, leading to an increase in particle size. Based on these results, 100.0 μmol L −1 of colloidal AgNPs was used for the nanohybrid preparation.
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
51
Fig. 2. Cyclic voltammograms for Pt/MWCNT/PPy/AgNPs electrode in 0.2 mol L −1 PBS pH 7.0 containing 100 μmol L−1 of serotonin at the following scan rates: 10, 20, 30, 40, 50, 75 and 100 mV s−1. Inset: linear relationship between the anodic peak current and v½.
The following pre-treatment potential values: 0.0, − 0.1, − 0.2, −0.3, −0.4 and − 0.5 V were investigated, and the results are shown in Fig. 4. Complete recovery of the original response was obtained between −0.3 and −0.2 V. At more positive cleaning potentials the peak current, Ipa, obtained during the following experiment begins to decrease, reaching 65% at 0.0 V pre-treatment cleaning potential. Thus, − 0.2 V was chosen as the pre-treatment cleaning potential in further studies. The influence of the pre-treatment time was also evaluated, varying it from 20 to 90 s, at which the complete recovery of the analytical signal was obtained in 45 s. Hence 45 s was chosen as time of pre-treatment for cleaning the electrode surface. The influence of DPV voltammetry parameters was then investigated. First, the amplitude was varied from 10 to 100 mV, while holding the step potential constant at 2 mV and the scan rate at 10 mV s −1. It did not observe any significant increase in peak widths for serotonin oxidation, even at amplitudes greater than 50 mV. Since the peak currents were proportional to the increase in amplitude, 100 mV was chosen as the optimised DPV amplitude. The next step was keeping the amplitude constant at 100 mV and the scan rate in 10 mV s −1, and studying the effect of step potential increments within the range of 1 to 10 mV. For step
Fig. 1. FEG-SEM micrographs for (A) Pt/PPy, (B) Pt/MWCNT/PPy and (C) Pt/MWCNT/PPy/ AgNPs.
In general, the electrochemical detection of serotonin is accompanied by adsorption of the serotonin or its oxidation products. Thus, there is a poisoning of the electrode surface and consequent decrease of the analytical signal. Therefore, the next study performed was the pre-treatment potential and time for cleaning the electrode surface.
Fig. 3. Effect of the colloidal AgNPs concentration on the nanohybrid electrode. DPV voltammograms collected in 0.2 mol L−1 PBS pH 7.0 containing 50.0 μmol L−1 of serotonin. Inset: Dependence of the serotonin oxidation peak current and colloidal AgNPs concentration.
52
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
with the current observed for the other electrodes. Serotonin oxidation on the proposed electrode showed an increase by a factor of 1.8 in the current peak when compared to the electrode that was prepared with the AgNPs support directly on MWCNT, namely Pt/MWCNT–AgNPs/ PPy electrode (curve c). This occurs because the colloidal AgNPs are more exposed on the surface electrode and its synergistic effect with the MWCNT and PPy is increased. Regarding the Pt/MWCNT/PPy electrode (curve b), an increase by a factor of 2.4 was observed for serotonin oxidation. As expected, when the Pt/MWCNT/PPy/AgNPs electrode was compared with the Pt/PPy electrode (curve a) an even higher increase was observed: by a factor of 10.0 for serotonin oxidation. The increase in current values reflects the increase of the electroactive surface area by the MWCNT/PPy/AgNPs nanohybrid formed. Such properties make the Pt/MWCNT/PPy/AgNPs electrode a promising setup for serotonin detection. 3.5. Effect of pH on serotonin oxidation process Fig. 4. Effect of pre-treatment potential on the serotonin oxidation peak current using DPV in 0.2 mol L−1 PBS pH 7.0 containing 50.0 μmol L −1 of serotonin.
potential greater than 2 mV, deformation of the voltammetric profiles was observed and the anodic peak currents for serotonin decreased in height. Hence, a 2 mV step potential increment was chosen. The effect of scan rate was studied in the range of 2.5 to 15 mV s −1, with the step potential constant at 2 mV and the amplitude at 100 mV. The scan rate of 10 mV s −1 was chosen because it presented more anodic peak current. Higher scan rates caused loss in peak definition and lower anodic peak current. 3.4. Comparison of the voltammetric behaviour of serotonin on modified electrodes The DPV experiments were performed in the potential range of 0.0 to + 0.6 V vs. Ag/AgCl/KCl (3.0 mol L−1) in 0.2 mol L −1 PBS at pH 7.0 containing 50.0 μmol L −1 of serotonin for the following electrodes: Pt/PPy (curve a), Pt/MWCNT/PPy (curve b), Pt/MWCNT–AgNPs/PPy (curve c), and Pt/MWCNT/PPy/AgNPs (curve d), as shown in Fig. 5. The electrode used to obtain the curve c was prepared with the AgNPs supported directly on MWCNT as previously described [10]. The voltammetric profiles had peaks at almost identical potentials of approximately +0.4 V vs. Ag/AgCl/KCl (3.0 mol L−1) for all electrodes studied. However, the Pt/MWCNT/PPy/AgNPs electrode (curve d) displayed higher anodic current intensities for serotonin in comparison
Fig. 5. DPV voltammograms obtained under the optimised parameters in 0.2 mol L−1 PBS pH 7.0 containing 50.0 μmol L−1 of serotonin for the following electrodes: (a) Pt/PPy, (b) Pt/MWCNT/PPy, (c) Pt/MWCNT-AgNPs/PPy and, (d) Pt/MWCNT/PPy/AgNPs.
Aiming to confirm the mechanism of serotonin oxidation on the Pt/MWCNT/PPy/AgNPs electrode, the dependence on the electrochemical oxidation of serotonin on pH was studied by DPV experiments at pHs ranging from 4.0 to 10.0 in 0.2 mol L−1 PBS containing 25.0 μmol L−1 of serotonin. The results are shown in Fig. 6, which is a plot of the DPV peak current (Ipa) and peak potential (Epa) as a function of pH. The variation of Epa with pH can provide valuable information about the serotonin oxidation process. For serotonin, reducing the hydrogen ionic concentration of the electrolyte causes a shift in peak potential towards more negative values, as illustrated Fig. 5. This is a consequence of deprotonation in the oxidation process that is facilitated at higher pHs. The Epa vs. pH showed a linear relationship, with a slope of 61.7 mV per pH unit. Therefore, an electrochemical process involving the same number of protons and electrons during the electrooxidation of serotonin can be proposed, which is in agreement with previous reports [20–22]. The plot of Ipa vs. pH, as shown in Fig. 6, demonstrates that the peak current has a maximum value at pH 8.0 decreasing for lower and higher pH values. Thus, a 0.2 mol L−1 PBS at pH 8.0 was used in the further studies. 3.6. Analytical characteristics DPV experiments were carried out in triplicate using the optimised experimental parameters to obtain an analytical curve for the determination of serotonin with the Pt/MWCNT/PPy/AgNPs electrode. The analytical response shown in Fig. 7 has a linear response in the
Fig. 6. Effect of pH on the peak potential (■) and peak current (●) for serotonin oxidation on the Pt/MWCNT/PPy/AgNPs electrode using 0.2 mol L−1 PBS containing 25.0 μmol L−1 of serotonin.
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
53
process of the serotonin on the Pt/MWCNT/PPy/AgNPs surface was evaluated. The experiments were carried out using DPV with optimised parameters in solutions with 0.2 mol L −1 of PBS (pH 8.0), concentrations of AA and UA fixed at 15.0 μmol L −1, and sequential additions of 10.0, 15.0, and 25.0 μmol L −1 of serotonin. Fig. 8 shows some of the results. The peak at +0.22 V is due to the AA oxidation process; however no signal associated with UA could be detected in these experimental conditions using the proposed electrode. It can be noted that, even at equal concentrations of AA, UA, and serotonin, it was not observed an overlap process of the serotonin oxidation peak and the oxidation peaks of the interfering substances studied. Also, the interfering substances studied did not shift the serotonin oxidation peak, indicating that the analytical signal did not suffer interference of the AA and UA. Accordingly, the Pt/MWCNT/PPy/AgNPs electrode is adequate to be used for selective determination of serotonin in the presence of AA and UA. 3.8. Analysis of serotonin in plasmatic serum sample Fig. 7. DPV voltammograms for Pt/MWCNT/PPy/AgNPs electrode, with the optimised parameters. The serotonin concentrations in μmol L−1 are: (a) 0.50, (b) 1.00, (c) 1.50, (d) 2.00, (e) 2.50, (f) 3.00, (g) 4.00, and (h) 5.00. Inset: linear dependence of the peak current with serotonin concentrations.
range from 0.50 to 5.0 μmol L −1, in accordance with the following equation: Ipa ðμAÞ ¼ 0:16 ðμAÞ −1 −1 ½serotonin μmol L þ 0:43 μA=μmol L
ð1Þ
with a correlation coefficient of 0.998 (n = 8). The LOD obtained was 0.15 μmol L − 1 (26.4 μg L − 1), being determined using a 3σ/slope ratio, where σ is the standard deviation of the mean value for 10 voltammograms of the blank. Comparing the results at the Pt/MWCNT/PPy/AgNP electrode with other electrochemical methods for serotonin detection, higher detection limits were observed at carbon electrodes: 500 nM for polycrystalline boron-doped and 2 μM for glassy carbon [23]. Rand et al. [24] also found high detection limit of 250 μM using a biosensor based on a carbon nanofiber. Lower detection limits of 0.003 μmol L −1, 0.023 μmol L −1 and 0.2 nmol L −1 were calculated using a Nafion/Ni(OH)2 nanoparticle– MWCNT modified glassy carbon electrode [25], a carbon ionic liquid electrode modified with Co(OH)2 nanoparticles and MWCNT [20] and a glass/ PDMS hybrid microfluidic device integrating vertically aligned SWCNTs [26], respectively. The advantages of the Pt/MWCNT/PPy/AgNPs electrode are that this sensor has a simple preparation and it exhibits long term stability, since stored in 0.2 mol L −1 PBS pH 8.0 at 4 °C. The reproducibility of the Pt/MWCNT/PPy/AgNPs electrode was measured from seven experiments, in which each experiment consisted of five sequential DPV voltammograms. These experiments were performed on different days. Prior to each experiment, the electrode surfaces were rinsed thoroughly with double-distilled water. Thus, the DPV voltammograms were performed in 0.2 mol L −1 PBS at pH 8.0 containing 50.0 μmol L −1 of serotonin. The RSD was calculated as 2.2%. In addition, intra-assay precision tests were performed from ten DPV voltammograms of that same solution. The RSD was found to be 1.7%.
The developed sensor was used for the quantification of serotonin in plasmatic serum, prepared as described in the Experimental section. The corresponding DPV voltammograms obtained for the analysis of the plasmatic serum sample are shown in Fig. 9, along with the respective standard addition plots. Serotonin determinations were performed in triplicate, without any treatment procedure, using the standard addition method. The results obtained (mean ± SD) for three determinations were: 1.99 ± 0.05 μmol L −1 for serotonin. Recoveries between 97.9% and 103.1% of serotonin from plasmatic serum samples (n = 3) were obtained for samples spiked with 1.0, 1.5, and 2.0 μmol L −1 of serotonin. There were no significant differences between the found and added concentrations of serotonin, indicating that the Pt/MWCNT/PPy/AgNP electrode can be successfully used for the determination of serotonin in plasmatic serum samples under the optimised conditions and using the standard addition approach. 4. Conclusions A newly modified Pt electrode was developed using MWCNT/PPy/ AgNPs nanohybrid, which can be used for the determination of serotonin in plasmatic serum samples. The MWCNT/PPy/AgNPs nanohybrid was successfully characterised by FEG-SEM microscopy, which indicated that MWCNT structures were totally covered by a compact layer of PPy and the colloidal AgNPs were
3.7. Selective determination of serotonin in the presence of interfering The electrochemical determination of serotonin in biological fluids is inhibited by the interference of ascorbic acid (AA) and uric acid (UA) also present in such matrices in high concentrations. Advanced chemical sensors for such determination should be able to reduce such interferences without diminishing the serotonin signal. In this sense, the intensity of the interference of these species in the electrooxidation
Fig. 8. DPV experiments for Pt/MWCNT/PPy/AgNPs electrode using the optimised parameters in 0.2 mol L−1 PBS pH 8.0 with concentrations of ascorbic acid (AA) and uric acid (UA) fixed at 15.0 μmol L −1, and sequential additions of serotonin standards in the following concentrations: 10.0 (open circle), 15.0 (close circle), and 25.0 (solid line) μmol L−1.
54
I. Cesarino et al. / Materials Science and Engineering C 40 (2014) 49–54
Fig. 9. DPV responses obtained on a Pt/MWCNT/PPy/AgNPs electrode for the determination of serotonin in plasmatic serum: S) sample; 1) sample plus 1.0 μmol L−1 serotonin; 2) sample plus 1.5 μmol L−1 serotonin; 3) sample plus 2.0 μmol L−1 serotonin. Inset: linear dependence of the peak current with serotonin concentrations.
supported onto MWCNT/PPy. Furthermore, the synergistic effect of MWCNT/PPy/AgNPs nanohybrid formed yielded lower LOD and improved the reproducibility, repeatability, and the sensitivity of the Pt electrode in the analysis of serotonin. Finally, the Pt/MWCNT/PPy/AgNPs electrode can be an effective material for serotonin electrochemical determination and is an alternative material to be used in biological analysis. Acknowledgments We are grateful for the financial support from CAPES, CNPq (grant 471467/2012-0) and FAPESP (2012/19633-0). References [1] J.M. Zen, I.L. Chen, Y. Shih, Voltammetric determination of serotonin in human blood using a chemically modified electrode, Anal. Chim. Acta. 369 (1998) 103–108. [2] Z.H. Wang, Q.L. Liang, Y.M. Wang, G.A. Luo, Carbon nanotube-intercalated graphite electrodes for simultaneous determination of dopamine and serotonin in the presence of ascorbic acid, J. Electroanal. Chem. 540 (2003) 129–134. [3] F.C. Moraes, T.A. Silva, I. Cesarino, M.R.V. Lanza, S.A.S. Machado, Antibiotic detection in urine using electrochemical sensors based on vertically aligned carbon nanotubes, Electroanalysis 25 (2013) 2092–2099. [4] I. Cesarino, F.C. Moraes, S.A.S. Machado, J. Passaretti-Filho, A.A. Cardoso, A new indirect electrochemical method for determination of ozone in water using multiwalled carbon nanotubes, Electroanalysis 23 (2011) 1512–1517. [5] A. Merkoci, M. Pumera, X. Llopis, B. Pérez, M. Del Valle, S. Alegret, New materials for electrochemical sensing VI: Carbon nanotubes, TrAC, Trends Anal. Chem. 24 (2005) 826–838. [6] X.G. Li, H. Feng, M.R. Huang, G.L. Gu, M.G. Moloney, Ultrasensitive Pb(II) potentiometric sensor based on copolyaniline nanoparticles in a plasticizer-free membrane with a long lifetime, Anal. Chem. 84 (2012) 134–140.
[7] M.R. Huang, Y.B. Ding, X.G. Li, Lead-ion potentiometric sensor based on electrically conducting microparticles of sulfonic phenylenediamine copolymer, Analyst 138 (2013) 3820–3829. [8] F.C. Moraes, I. Cesarino, V. Cesarino, L.H. Mascaro, S.A.S. Machado, Carbon nanotubes modified with antimony nanoparticles: A novel material for electrochemical sensing, Electrochim. Acta 85 (2012) 560–565. [9] I. Cesarino, V. Cesarino, M.R.V. Lanza, Carbon nanotubes modified with antimony nanoparticles in a paraffin composite electrode: Simultaneous determination of sulfamethoxazole and trimethoprim, Sensors Actuators B 188 (2013) 1293–1299. [10] I. Cesarino, V. Cesarino, F.C. Moraes, T.C.R. Ferreira, M.R.V. Lanza, L.H. Mascaro, S. A.S. Machado, Electrochemical degradation of benzene in natural water using silver nanoparticle-decorated carbon nanotubes, Mater. Chem. Phys. 141 (2013) 304–309. [11] S. Prabhu, E.K. Poulose, Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects, Int. Nano Lett. 32 (2012) 1–10. [12] A. Balamurugan, S.M. Chen, Silver nanograins incorporated PEDOT modified electrode for electrocatalytic sensing of hydrogen peroxide, Electroanalysis 21 (2009) 1419–1423. [13] Y.C. Tsai, P.C. Hsu, Y.W. Lin, T.M. Wu, Silver nanoparticles in multiwalled carbon nanotube-Nafion for surface-enhanced Raman scattering chemical sensor, Sensors Actuators B 138 (2009) 5–8. [14] I. Cesarino, H.V. Galesco, F.C. Moraes, M.R.V. Lanza, S.A.S. Machado, Biosensor based on electrocodeposition of carbon nanotubes/polypyrrole/laccase for neurotransmitter detection, Electroanalysis 25 (2013) 394–400. [15] C.C. Harley, A.D. Rooney, C.B. Breslin, The selective detection of dopamine at a polypyrrole film doped with sulfonated-cyclodextrins, Sensors Actuators B 150 (2010) 498–504. [16] X. Lu, Y. Li, J. Du, X. Zhou, Z. Xue, X. Liu, Z. Wang, A novel nanocomposites sensor for epinephrine detection in the presence of uric acids and ascorbic acids, Electrochim. Acta 56 (2011) 7261–7266. [17] S. Shahrokhian, R.S. Saberi, Electrochemical preparation of over-oxidized polypyrrole/ multi-walled carbon nanotube composite on glassy carbon electrode and its application in epinephrine determination, Electrochim. Acta 57 (2011) 132–138. [18] C.M.A. Brett, I. Muresan, The influence of artificial body fluids on metallic corrosion, Key Eng. Mater. 230–232 (2002) 459–462. [19] I. Cesarino, F.C. Moraes, S.A.S. Machado, A biosensor based on polyaniline-carbon nanotube core-shell for electrochemical detection of pesticides, Electroanalysis 23 (2011) 2586–2593. [20] A. Babaei, A.R. Taheri, M. Aminikhah, Nanomolar simultaneous determination of levodopa and serotonin at a novel carbon ionic liquid electrode modified with Co(OH)2 nanoparticles and multi-walled carbon nanotubes, Electrochim. Acta 90 (2013) 317–325. [21] Y. Li, X. Huang, Y. Chen, L. Wang, X. Lin, Simultaneous determination of dopamine and serotonin by use of covalent modification of 5-hydroxytryptophan on glassy carbon electrode, Microchim. Acta 164 (2009) 107–112. [22] R.T. Kachoosangi, R.G. Compton, A simple electroanalytical methodology for the simultaneous determination of dopamine, serotonin and ascorbic acid using an unmodified edge plane pyrolytic graphite electrode, Anal. Bioanal. Chem. 387 (2007) 2793–2800. [23] A.G. Guell, K.E. Meadows, P.R. Unwin, J.V. Macpherson, Trace voltammetric detection of serotonin at carbon electrodes: comparison of glassy carbon, boron doped diamond and carbon nanotube network electrodes, Phys. Chem. Chem. Phys. 12 (2010) 10108–10114. [24] E. Rand, A. Periyakaruppan, Z. Tanaka, D.A. Zhang, M.P. Marsh, R.J. Andrews, K.H. Lee, B. Chen, M. Meyyappan, J.E. Koehne, A carbon nanofiber based biosensor for simultaneous detection of dopamine and serotonin in the presence of ascorbic acid, Biosens. Bioelectron. 42 (2013) 434–438. [25] A. Babaei, A.R. Taheri, Nafion/Ni(OH)2 nanoparticles-carbon nanotube composite modified glassy carbon electrode as a sensor for simultaneous determination of dopamine and serotonin in the presence of ascorbic acid, Sensors Actuators B 176 (2013) 543–551. [26] F.C. Moraes, R.S. Lima, T.P. Segato, I. Cesarino, J.L.M. Cetino, S.A.S. Machado, F. Gomez, E. Carrilho, Glass/PDMS hybrid microfluidic device integrating vertically aligned SWCNTs to ultrasensitive electrochemical determinations, Lab Chip 12 (2012) 1959–1962.
