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Simultaneous voltammetric determination of dopamine and epinephrine in human body fluid samples using a glassy carbon electrode modified with nickel oxide nanoparticles and carbon nanotubes within a dihexadecylphosphate film† Luiz C. S. Figueiredo-Filho, Tiago A. Silva, Fernando C. Vicentini and Orlando Fatibello-Filho* A simple and highly selective electrochemical method was developed for the single or simultaneous determination of dopamine (DA) and epinephrine (EP) in human body fluids using a glassy carbon electrode
modified
with
nickel
oxide
nanoparticles
and
carbon
nanotubes
within
a
dihexadecylphosphate film using square-wave voltammetry (SWV) or differential-pulse voltammetry (DPV). Using DPV with the proposed electrode, a separation of ca. 360 mV between the peak reduction potentials of DA and EP present in binary mixtures was obtained. The analytical curves for the simultaneous determination of dopamine and epinephrine showed an excellent linear response, ranging from 7.0 108 to 4.8 106 and 3.0 107 to 9.5 106 mol L1 for DA and EP, respectively. The detection limits for the simultaneous determination of DA and EP were 5.0 108 mol L1 and 8.2
Received 30th January 2014 Accepted 28th February 2014
108 mol L1, respectively. The proposed method was successfully applied in the simultaneous
DOI: 10.1039/c4an00229f
determination of these analytes in human body fluid samples of cerebrospinal fluid, human serum and
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lung fluid.
1. Introduction Over the last decade, a considerable amount of research has focused on the preparation of novel nanoscale materials.1–3 Several different nanoparticles (NPs) have been the target of increasing interest in electrochemical studies, such as gold,4,5 silver,6,7 and platinum,8,9 as well as metal oxides such as ZnO,10 MnO2,11,12 NiO,13–15 and CuO.16,17 However, the literature contains a small number of studies that were carried out to investigate the electrochemistry of nickel oxide nanoparticles (NiONPs) and possible electroanalytical applications; most of them were used as biosensors.18–23 There are only three analytical methods using NiONP-modied electrodes described in the literature. NiONPs supported on a graphite24 electrode were used to determine aspirin levels, and a glassy carbon electrode and nickel–carbon nanotube-modied electrodes25,26 were applied in the voltammetric determination of amino acids. The combination of multilayer carbon nanotubes (MWCNTs) and NPs has been widely studied, as in most cases these
Department of Chemistry, Federal University of S˜ ao Carlos, Rod. Washington Lu´ıs km 235, P. O. Box 676, S˜ ao Carlos, 13560-970, SP, Brazil. E-mail: [email protected]; Fax: +55 16 33518350; Tel: +55 16 33518098 † Electronic supplementary 10.1039/c4an00229f
information
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(ESI)
available.
See
DOI:
materials produce a synergistic effect in terms of the electroanalytical response. Nevertheless, only a few electroanalytical methods using modied electrodes with MWCNTs and NiONPs have been proposed using electrodeposition of this metal oxide.15,21,26 Epinephrine (EP) and dopamine (DA) are important catecholamine neurotransmitters that play vital roles in the health of humans and other mammalians.27 Many diseases (neurological, psychiatric and cardiovascular) are related to changes in epinephrine (EP) concentrations. Thus, it is necessary to develop quantitative methods for this catecholamine for studies on physiological function and diagnosis in clinical medicine.28 Abnormal DA concentrations have also been associated with neurological disorders (Parkinson's disease and schizophrenia); thus, the determination of DA concentrations can be very important in diagnostic applications.29–31 Additionally, EP and DA are used in emergency health care in patients who show signal of hypertension, bronchial asthma, and myocardial infarction as well as during cardiac surgery,32 so the control of these compounds is of paramount importance. At present, a variety of methods are used to determine DA and EP simultaneously, including capillary electrophoresis,33 high performance liquid chromatography-mass spectrometry,34 ow injection analysis35 with spectrophotometric detection and electrochemical methods.36–41 Except for the electrochemical
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methods, the other methods are all difficult to use for rapid detection and cannot be miniaturized to determine these analytes in loco. The purpose of this work is to investigate the electrochemical behaviour of EP and DA in aqueous solutions at a glassy carbon electrode (GCE) modied with NiONP–MWCNT within a dihexadecylphosphate (DHP) lm (NiONP–MWCNT–DHP/GCE) by cyclic, square-wave and differential-pulse voltammetries. The aim was to develop a sensitive method to simultaneously determine both analytes in human body uid samples.
2.
Experimental
2.1. Reagents and solutions Dopamine (DA), epinephrine (EP), MWCNTs (95% purity, 20– 30 nm in diameter, 1–2 nm thick and 0.5–2 mm in length) and dihexadecylphosphate were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade. The solutions were prepared using ultrapure water (resistivity > 18.0 MU cm) obtained from a Millipore Milli-Q system (Billerica, USA). A stock solution of DA was prepared directly in ultrapure water and that of EP in 0.2 mol L1 sulfuric acid (better solubility in inorganic acid aqueous solutions). 2.2. Instrumentation The voltammetric measurements were performed using a threeelectrode system, including the modied electrode as the working electrode, a platinum plate as the counter electrode, and Ag/AgCl (3.0 mol L1 KCl) as the reference electrode. A potentiostat/galvanostat Autolab model PGSTAT12 (Ecochemie, the Netherlands) controlled by GPES 4.9 soware was used. The surface electrode morphology was characterized by a eld-emission gun scanning electron microscopy FEG/SEM mode (Supra 35-VP, Carl Zeiss, Germany). A histogram was constructed using the public domain ImageJ image processing soware. 2.3. MWCNTs functionalization and NiONP–MWCNT–DHP/ GCE preparation The carbon nanotubes were initially subjected to chemical pretreatment for 12 h using a mixture of concentrated sulfuric and nitric acids in a proportion of 3 : 1 v/v, respectively. Subsequently, the mixture was ltered, washed with deionized water until a pH of 6.5–7.0 was reached, and then dried at 120 C for 5 h.42,43 This pre-treatment was carried out to remove possible metal impurities, open the ends of the CNTs and add functional groups such as carboxylic groups onto the CNT surface.44 Next, the suspension of MWCNTs was prepared by dispersing 1.0 mg MWCNTs with 1.0 mg DHP in 1.0 mL of ultra-pure water, and this dispersion was placed under ultrasonic agitation for 40 min.45,46 Before the modication, the bare GCE (3.0 mm in diameter) was polished successively with alumina on a polishing cloth and then rinsed with ultra-pure water and ethyl alcohol. Next, 8 mL of the MWCNT–DHP dispersion was dropped onto the GCE surface and the solvent was evaporated at 25 C for 2 h. The
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MWCNT–DHP/GCE obtained was subjected to the NiONPs electrodeposition procedure. For this, in the rst step, the potential was cycled 40 times between 0 and 0.8 V at a scan rate of 100 mV s1 in an acetate buffer solution (pH 4) containing 0.002 mol L1 nickel nitrate.25 Finally, the potential was repetitively cycled (40 scans) from 0 to 0.6 V at a scan rate of 100 mV s1 in fresh 0.1 mol L1 NaOH solution for the electrodissolution and passivation of the NiONPs on the MWCNT–DHP/GCE.25 2.4. Analytical procedure Aer optimizing the experimental parameters for square-wave voltammetry (SWV) and differential-pulse voltammetry (DPV), analytical curves were constructed by adding small volumes of concentrated standard solutions of EP and DA. The detection limit was calculated to be three times the standard deviation for the blank solution (n ¼ 10) divided by the slope of the analytical curve. Samples of cerebrospinal uid, human serum and lung uid were prepared following the appropriate protocols.47–49 Next, an appropriate aliquot of EP and DA was added carefully to the samples (spiking) to obtain concentrations of 7.0 107 and 1.4 106 mol L1 of EP and 5.0 107 and 1.0 106 mol L1 of DA.
3.
Results and discussion
3.1. Electrodeposition of NiONPs on MWCNT–DHP/GCE and morphology characterization The preparation of the NiONP–MWCNT–DHP/GCE was performed/adapted following a procedure proposed in the literature,25 and it briey consisted of the electrodeposition of NiONPs. In the rst step, the potential was cycled between 0.0 V and 0.8 V in an acid solution containing nickel nitrate (0.002 mol L1) in order to electrodeposit metallic nickel. Next, the potential was cycled between 0.0 V and +0.6 V in an alkaline solution (0.1 mol L1 NaOH) for the electrodissolution and passivation of the NiONPs.25
Fig. 1 Cyclic voltammograms obtained for the NiONP–MWCNT– DHP/GCE in 0.1 mol L1 NaOH solution at different scan rates: (1) 10; (2) 20; (3) 30; (4) 40; (5) 50; (6) 60; (7) 70; (8) 80; (9) 90 and (10) 100 mV s1. Inset: log Ia vs. log v.
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The effective formation of the nickel oxide nanoparticles was conrmed by electrochemistry from the study of the electrochemical behaviour of the NiONP–MWCNT–DHP/GCE in 0.1 mol L1 NaOH solution. Fig. 1 shows the cyclic voltammograms obtained at different scan rates (v) for the NiONP– MWCNT–DHP/GCE in 0.1 mol L1 NaOH solution. As can be seen, a reversible process was veried, which was assigned to the equilibrium reaction (1):25 Ni(OH)2 + OH # NiO(OH) + H2O + e
(1)
Ni(OH)2 was generated during the electrodissolution and passivation step and subsequently was oxidized to NiO(OH) in the alkaline medium (anodic peak); this last species was reduced to Ni(OH)2 (cathodic peak) aer potential scanning inversion. Moreover, a plot of log Ia vs. log v revealed adsorptive behaviour, with a slope of 0.96. Fig. 2 presents the FEG-SEM images of (a) MWCNT–DHP/ GCE and (b) NiONP–MWCNT–DHP/GCE. A clear difference in terms of the surface is observed among these electrodes. It can be observed from Fig. 2(a) that a relatively dense lm (MWCNT– DHP) was formed on the GCE surface. In Fig. 2(b) an increase in the roughness of the MWCNTs surface can be observed indicating the incorporation of NiONPs. Also an EDX (energydispersive X-ray) analysis was performed to identify the elements present on the electrode surface and the results showed a high percentage of carbon (85.11%), oxygen (8.52%), nickel (1.14%), and phosphorus (3.89%), which was in agreement with the expectations considering the chemicals used in the lm preparation. Moreover, in Fig. S1 (ESI†) is shown a FEGSEM image of NiONPs electrodeposited on the GCE surface including a histogram of the NiONPs diameters with an average size of 40.8 nm.
3.2. Electrochemical behaviour of dopamine and epinephrine on the NiONP–MWCNT–DHP/GCE The glassy carbon electrode (GCE), glassy carbon electrode modied with MWCNT–DHP (MWCNT–DHP/GCE) and glassy carbon electrode modied with nickel oxide nanoparticles and MWCNT–DHP (NiONP–MWCNT–DHP/GCE) were used to investigate the electrochemical behaviour of EP and DA on the proposed electrodes via cyclic voltammetry (CV). Determinations were carried out on different samples via differential-pulse
Fig. 2 FEG-SEM images of the surface of: (a) MWCNT–DHP/GCE and (b) NiONP–MWCNT–DHP/GCE.
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Fig. 3 Cyclic voltammograms obtained for (a) 1.0 104 mol L1 DA and (b) 1.0 104 mol L1 EP in 0.2 mol L1 phosphate buffer solution (pH 7.0) using a GCE (black line), MWCNT–DHP/GCE (blue line) and NiONP–MWCNT–DHP/GCE (red line). (c) Overlap of the cyclic voltammograms for 1.0 104 mol L1 DA and 1.0 104 mol L1 EP in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP– MWCNT–DHP/GCE.
voltammetry (DPV) and square-wave voltammetry (SWV). The obtained voltammograms using GCE, MWCNT–DHP/GCE and NiONP–MWCNT–DHP/GCE are presented in Fig. 3(a) (DA), 3(b) (EP) and 3(c) (both analytes) using the proposed modied electrode. In the case of the GCE, small oxidation and reduction peaks were observed. With the incorporation of the MWCNT– DHP lm onto the GCE surface, the analytical signal was substantially increased and a decrease in the oxidation and reduction potentials was seen, a characteristic of the electrocatalytic effect. Likewise, the use of NiONPs combined with the MWCNT–DHP lm showed an increase in the oxidation current of 70% and in the reduction current of 45%; however, no additional electrocatalytic effects were seen when compared with the MWCNT–DHP lm. The increase of current could be explained by the interaction between the NiONPs and the –OH groups of catecholamines, leading to an increase of the analyte concentration on the electrode surface.50–52 However, the key problem of simultaneous sensing of catecholamines (Fig. 3(c)) by electrochemistry is the overlapping voltammetric signals. Also, as can be found in the literature, there is a large excess of coexisting substances in biological tissues such as ascorbic acid (AA) and uric acid (UA).29 One way to solve this problem is to focus on the reduction peak of EP and DA (Fig. 3(c)), as there is a difference between the reduction peaks of ca. 360 mV (EP ca. 192 mV and DA ca. 171 mV), which is a good value of peak separation for simultaneous determination. Aer the peak resolution studies, we turned to explore the other electrochemical characteristics. Scan rate studies where the voltammetric peak magnitude (Ip) was monitored as a function of scan rate (v) with a plot of peak height versus the square-root of the scan rate (Fig. 4(a) and (b)) revealed the
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Cyclic voltammograms obtained for (a) 1.0 104 mol L1 DA and (b) 1.0 104 mol L1 EP in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP–MWCNT–DHP/GCE at different scan rates (v): (1) 20; (2) 30; (3) 40; (4) 50; (5) 75; (6) 100; (7) 125; (8) 150; (9) 200 mV s1. Fig. 4
following trends: EP (cathodic peak), Ic (A) ¼ 3.60 105 A/(V s1)0.5 + 3.54 106 A (r ¼ 0.995); EP (anodic peak), Ia (A) ¼ 9.12 105 A/(V s1)0.5 2.97 106 A (r ¼ 0.998); DA (cathodic peak), Ic (A) ¼ 7.46 105 A/(V s1)0.5 + 1.04 105 A (r ¼ 0.996); DA (anodic peak), Ia (A) ¼ 1.10 104 A/(V s1)0.5 8.67 106 A (r ¼ 0.997). It was clear that, in both cases, a linear response was evident, indicating diffusional processes. Furthermore, as was expected for the case of the semi-innite diffusion model governed by the Randles–ˇ Sev´ cik equation, analysis of log Ip versus log v revealed gradients of 0.44 (EP cathodic peak), 0.54 (EP anodic peak), 0.53 (DA cathodic peak) and 0.48 (DA anodic peak), indicating the absence of thin-layer effects (e.g. the analytes were not trapped within the lm network (NiONP–MWCNT–DHP/GCE) of the GCE). 3.3. Simultaneous voltammetric determination of dopamine and epinephrine using the NiONP–MWCNT–DHP/GCE For EP and DA, an electroanalytical procedure was developed involving a systematic study and optimization of the experimental parameters (frequency, f; pulse amplitude, a; potential increment, DEs) that affect the SWV response (the value of the peak reduction current was registered at a concentration of 1.0 104 mol L1 for both analytes). The best results for the SWV parameters are shown in Table 1. Next, we determine the analytical curves. First, the analytical curve was constructed by varying the concentrations of EP from 9.8 107 mol L1 to 6.8 105 mol L1 while maintaining the DA concentration constant at 2.0 106 mol L1. Next, we varied the DA concentration from 7.0 108 mol L1 to 9.7 106 mol L1 and xed the concentration of EP (1.0
Table 1 Experimental parameters of the SWV and DPV techniques optimized for DA and EP simultaneous determination
Technique
Parameter
Range
Selected
SWV
Frequency, f (Hz) Amplitude, a (mV) Potential increment, DEs (mV) Scan rate potential, v (mV s1) Amplitude, a (mV) Pulse time, t (ms)
10–100 5–100 1–10 2.5–20 10–100 5–30
10 90 9 7.5 90 25
DPV
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105 mol L1). From the SW voltammograms (Fig. 5(a) and (b)), it can be seen that the EP and DA cathodic peak currents increased regularly with the concentration and the cathodic peak current for the analyte with xed concentration remained fairly constant (RSD values of 15.6% for DA (Fig. 5(a)) and 4.4% for EP (Fig. 5(b))), thus showing that they did not interfere with each other. Subsequently, an analytical curve was performed for both analytes (EP and DA) at the same time; the plot of the peak reduction current vs. EP concentration was linear in the concentration range from 7.0 107 mol L1 to 4.8 105 mol L1 and for DA it was linear in the concentration range from 7.0 108 mol L1 to 4.8 106 mol L1 (Fig. 5(c)). The corresponding equations were: EP: Ic (mA) ¼ 3.94 105 [EP] (mol L1) 1.91 (r ¼ 0.994) DA: Ic (mA) ¼ 3.83 106 [DA] (mol L1) 5.33 (r ¼ 0.991) The detection limits calculated using the relation 3 s/m, where s is the standard deviation of ten blank measurements and m is the slope of the analytical curve, were 4.8 107 mol L1 for EP and 3.8 108 mol L1 for DA. For DPV, the experiments were carried out in the same manner as for SWV. First, the parameters related to the technique were studied (scan rate potential, v; amplitude, a; pulse time, t); in Table 1, the optimum values selected can be seen. Next, we performed analytical curves, initially as in SWV, by varying the EP concentration (3.0 107 mol L1 to 9.7 106 mol L1) and maintaining the DA concentration (2.0 106 mol L1). Next, we carried out the opposite study, i.e. by varying the DA concentration (7.0 108 mol L1 to 6.8 106 mol L1) and maintaining the EP concentration (1.0 105 mol L1). Aer examining the DP voltammograms (Fig. 6(a) and (b)), a similar conclusion to the SWV case was obtained, i.e., the analytes did not interfere with each other, since the EP and DA cathodic peak currents increased regularly with the concentration and the cathodic peak current for the analyte with xed concentration remained fairly constant (RSD values of 3.4% for DA (Fig. 6(a)) and 2.4% for EP (Fig. 6(b))). Finally, the analytical curves were obtained by varying the concentration of both analytes at the same time; the plot of peak
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SW voltammograms obtained in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP–MWCNT–DHP/GCE for (a) various concentrations of EP: (1) 0.98; (2) 2.94; (3) 4.89; (4) 6.83; (5) 9.73; (6) 29.13; (7) 48.45 and (8) 67.70 mmol L1 at a fixed DA concentration (2.00 mmol L1), (b) various concentrations of DA: (1) 0.07; (2) 0.10; (3) 0.30; (4) 0.49; (5) 0.69; (6) 0.98; (7) 2.94; (8) 4.89; (9) 6.83 and (10) 9.73 mmol L1 at a fixed EP concentration (10 mmol L1) and (c) various concentrations of EP and DA: EP – (1) 0.70; (2) 0.98; (3) 2.93; (4) 4.87; (5) 6.80; (6) 9.66; (7) 28.85 and (8) 47.90 mmol L1; DA – (1) 0.07; (2) 0.10; (3) 0.30; (4) 0.49; (5) 0.68; (6) 0.96; (7) 2.87; (8) 4.77 mmol L1. Insets: analytical curves. Fig. 5
Fig. 6 DP voltammograms obtained in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP–MWCNT–DHP/GCE for (a) various concentrations of EP: (1) 0.30; (2) 0.49; (3) 0.69; (4) 0.98; (5) 2.94; (6) 4.89; (7) 6.83 and (8) 9.73 mmol L1 at fixed DA concentration (2.00 mmol L1), (b) various concentrations of DA: (1) 0.07; (2) 0.10; (3) 0.30; (4) 0.49; (5) 0.69; (6) 0.98; (7) 2.94; (8) 4.89; (9) 6.83 mmol L1 at fixed EP concentration (10 mmol L1) and (c) various concentrations of EP and DA: EP – (1) 0.30; (2) 0.49; (3) 0.68; (4) 0.97; (5) 2.89; (6) 4.80; (7) 6.69; (8) 9.50 mmol L1; DA – (1) 0.07; (2) 0.10; (3) 0.29; (4) 0.48; (5) 0.68; (6) 0.96; (7) 2.87; (8) 4.75 mmol L1. Inset: analytical curves.
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Table 2 Comparison between the SWV and DPV techniques for the simultaneous voltammetric determination of DA and EP
Technique Analyte
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DA
EP
Analytical parameter Sensitivity (mA L mol1) Detection limit (mol L1) Linear range (mol L1) Sensitivity (mA L mol1) Detection limit (mol L1) Linear range (mol L1)
SWV
DPV
3.8 106
1.9 106
3.8 108
5.0 108
7.0 108 to 4.8 106 3.9 105
7.0 108 to 4.8 106 9.0 105
4.8 107
8.2 108
7.0 107 to 4.8 105
3.0 107 to 9.5 106
current vs. EP concentration was linear in the concentration range from 3.0 107 mol L1 to 9.5 106 mol L1 and for DA it was linear in the concentration range from 7.0 108 mol L1 to 4.8 106 mol L1 (Fig. 6(c)). The analytical curve equations were: EP: Ic (mA) ¼ 9.03 105 [EP] (mol L1) 1.18 (r ¼ 0.993) DA: Ic (mA) ¼ 1.85 106 [DA] (mol L1) 0.55 (r ¼ 0.994) The detection limits calculated using the relation 3 s/m, where s is the standard deviation of ten blank measurements and m is the slope of the analytical curve, were 8.2 108 mol L1 for EP and 5.0 108 mol L1 for DA. Table 2 shows all the analytical parameters (sensitivity, detection limit and linear range) obtained using SWV and DPV for each analyte. Comparing the different analytical parameters,
we observed that for DA, the SWV and DPV techniques showed the same linear concentration range, although the SWV sensitivity was 2.1 times higher than the DPV sensitivity and the detection limits were very close (Detection limitDPV ¼ 1.3 Detection limitSWV). However, for EP detection, the DPV technique showed some important advantages in relation to the SWV technique: the DPV sensitivity was 2.3 times higher than the SWV sensitivity, the detection limit obtained for DPV was 6.0 times lower than that obtained with SWV and, although the linear concentration range veried for DPV was lower than that for SWV, in the case of the DPV technique, the procedure was capable of determining a low concentration of EP. The better performance of the DPV technique for EP determination can be related to the fact that this analyte presents higher irreversibility, and, the loss of sensitivity due to the irreversibility of the electrochemical reaction is lower for the differential pulse method than for other pulse techniques.53 From these observations, the DPV technique was chosen as the voltammetric technique to continue these studies. Table 3 presents the analytical parameters (detection limit and linear concentration range of the analytical curve) of electroanalytical methods using different electrodes and techniques that have been reported for the simultaneous determination of DA and EP. From this table, we can conclude that the concentration range is similar to those reported in the literature. Nevertheless, using the DPV technique with the NiONP– MWCNT–DHP/GCE, the detection limit was signicantly lower than the values published to date. 3.4. Repeatability studies and determination of dopamine and epinephrine in human body uids In order to determine the intra-day repeatability of the voltammetric procedure, various successive simultaneous measures (n ¼ 10) were performed using 0.2 mol L1 phosphate buffer solution (pH 7.0) containing DA and EP, both at 5.0 106 mol L1. Thus, the RSD of 3.2% and 1.2% was obtained for
Table 3 Comparison of the analytical parameters obtained for the simultaneous determination of DA and EP using the NiONP–MWCNT–DHP/ GCE with those from the literaturea
Electrode
Technique
Linear range (mol L1)
Detection limit (mol L1)
Reference
PLA/GCE
CV CV
Poly(caffeic acid)/GCE
CV
Poly(taurine)/GCE
DPV
a-CD/CNT/GCE
DPV
Poly(isonicotinic acid)/CPE
CV
NiONP–MWCNT–DHP/GCE
DPV
DA: 4.0 107 EP: 1.0 107 DA: 1.7 107 EP: 3.2 107 DA: 2.0 107 EP: 1.0 107 DA: 1.0 107 EP: 3.0 107 DA: 1.0 106 EP: 5.0 107 DA: 2.0 105 EP: 1.0 106 DA: 5.0 108 EP: 8.2 108
54
PAIUCPE
DA: 8.0 107 to 5.0 104 EP: 5.0 107 to 5.0 105 DA: 8.0 107 to 3.0 104 EP: 2.0 106 to 1.5 104 DA: 1.0 106 to 3.5 105 EP: 1.0 106 to 1.5 105 DA: 1.0 106 to 8.0 104 EP: 2.0 106 to 6.0 104 DA: 2.0 106 to 1.0 103 EP: 1.0 106 to 1.0 103 DA: 8.0 105 to 7.0 104 EP: 5.0 106 to 1.0 104 DA: 7.0 108 to 4.8 106 EP: 3.0 107 to 9.5 106
55 56 39 57 58 This work
a PLA – poly(L-arginine); GCE – glassy carbon electrode; CV – cyclic voltammetry; PAIUCPE – pre-anodized inlaying ultrathin carbon paste electrode; a-CD – a-cyclodextrin; CNT – carbon nanotubes; CPE – carbon paste electrode.
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Table 4 Simultaneous determination of DA and EP in human body fluid samples using the NiONP–MWCNT–DHP/GCE
Sample Cerebrospinal uid
Added (mmol L1) Level 1
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Level 2 Human serum
Level 1 Level 2
Lung uid
Level 1 Level 2
DA: 0.50 EP: 0.70 DA: 1.00 EP: 1.40 DA: 0.50 EP: 0.70 DA: 1.00 EP: 1.40 DA: 0.50 EP: 0.70 DA: 1.00 EP: 1.40
Found (mmol L1)
Recovery (%)
0.49 0.03 0.75 0.07 1.11 0.04 1.36 0.04 0.51 0.07 0.76 0.08 1.08 0.03 1.43 0.01 0.49 0.04 0.68 0.04 1.13 0.04 1.46 0.06
98.0 107.1 111.0 97.1 102.0 108.6 108.0 102.1 98.0 97.1 113.0 104.3
DA and EP. Also, the inter-day repeatability of the procedure was studied by carrying out measures on three different days (n ¼ 3) using, consequently, modied electrodes prepared on different days. In this study, DA and EP concentrations of 5.0 107 mol L1 and 7.0 106 mol L1 were used, providing an RSD of 4.8% and 7.0% for DA and EP, respectively. The results obtained in the repeatability studies show the good precision of the procedure, as well as the simplicity of preparing the modied electrodes, which allows obtaining reproducible measures using electrodes prepared at different times. Moreover, the electrode can be used for at least 40 determinations. Next, human body uid samples including cerebrospinal uid, human serum and lung uid spiked with two different levels of DA and EP were analyzed by the proposed method. Table 4 presents the results obtained for the analysis of these samples. As can be seen, excellent recovery values were obtained for both analytes in the three samples. From these results, one may conclude that the DPV procedure using the MWCNT–DHP/ GCE modied with electrodeposited NiONPs does not suffer interference from the synthetic sample matrix, thus presenting potential applicability in the simultaneous determination of DA and EP in real biological samples. Moreover, the NiONP– MWCNT–DHP/GCE presented good stability for repeated determinations (n ¼ 30) of biological samples, when the cathodic current response varied by only 7.6% and 8.9% for DA and EP, respectively.
4. Conclusions In this work, a NiONP–MWCNT–DHP/GCE was proposed to develop a novel DPV method for the single or simultaneous determination of DA and EP in human body uid samples. The proposed electrode presented good separation of the reduction peak potentials for DA and EP (about 360 mV) by DPV, enabling the simultaneous determination of these compounds. Very low detection limits were obtained in the simultaneous determination of these analytes: DA (5.0 108 mol L1) and EP (8.2 108 mol L1). Moreover, addition and recovery studies indicated that matrix effects did not present any signicant
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interference and the proposed method can be used for the simultaneous determination of DA and EP in real samples.
Acknowledgements The authors acknowledge nancial support from the following Brazilian funding agencies: CNPq, CAPES and FAPESP (Proc. 2010/20754-1 and 2013/16770-0).
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Analyst, 2014, 139, 2842–2849 | 2849
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Cite this: Analyst, 2014, 139, 2842
Simultaneous voltammetric determination of dopamine and epinephrine in human body fluid samples using a glassy carbon electrode modified with nickel oxide nanoparticles and carbon nanotubes within a dihexadecylphosphate film† Luiz C. S. Figueiredo-Filho, Tiago A. Silva, Fernando C. Vicentini and Orlando Fatibello-Filho* A simple and highly selective electrochemical method was developed for the single or simultaneous determination of dopamine (DA) and epinephrine (EP) in human body fluids using a glassy carbon electrode
modified
with
nickel
oxide
nanoparticles
and
carbon
nanotubes
within
a
dihexadecylphosphate film using square-wave voltammetry (SWV) or differential-pulse voltammetry (DPV). Using DPV with the proposed electrode, a separation of ca. 360 mV between the peak reduction potentials of DA and EP present in binary mixtures was obtained. The analytical curves for the simultaneous determination of dopamine and epinephrine showed an excellent linear response, ranging from 7.0 108 to 4.8 106 and 3.0 107 to 9.5 106 mol L1 for DA and EP, respectively. The detection limits for the simultaneous determination of DA and EP were 5.0 108 mol L1 and 8.2
Received 30th January 2014 Accepted 28th February 2014
108 mol L1, respectively. The proposed method was successfully applied in the simultaneous
DOI: 10.1039/c4an00229f
determination of these analytes in human body fluid samples of cerebrospinal fluid, human serum and
www.rsc.org/analyst
lung fluid.
1. Introduction Over the last decade, a considerable amount of research has focused on the preparation of novel nanoscale materials.1–3 Several different nanoparticles (NPs) have been the target of increasing interest in electrochemical studies, such as gold,4,5 silver,6,7 and platinum,8,9 as well as metal oxides such as ZnO,10 MnO2,11,12 NiO,13–15 and CuO.16,17 However, the literature contains a small number of studies that were carried out to investigate the electrochemistry of nickel oxide nanoparticles (NiONPs) and possible electroanalytical applications; most of them were used as biosensors.18–23 There are only three analytical methods using NiONP-modied electrodes described in the literature. NiONPs supported on a graphite24 electrode were used to determine aspirin levels, and a glassy carbon electrode and nickel–carbon nanotube-modied electrodes25,26 were applied in the voltammetric determination of amino acids. The combination of multilayer carbon nanotubes (MWCNTs) and NPs has been widely studied, as in most cases these
Department of Chemistry, Federal University of S˜ ao Carlos, Rod. Washington Lu´ıs km 235, P. O. Box 676, S˜ ao Carlos, 13560-970, SP, Brazil. E-mail: [email protected]; Fax: +55 16 33518350; Tel: +55 16 33518098 † Electronic supplementary 10.1039/c4an00229f
information
2842 | Analyst, 2014, 139, 2842–2849
(ESI)
available.
See
DOI:
materials produce a synergistic effect in terms of the electroanalytical response. Nevertheless, only a few electroanalytical methods using modied electrodes with MWCNTs and NiONPs have been proposed using electrodeposition of this metal oxide.15,21,26 Epinephrine (EP) and dopamine (DA) are important catecholamine neurotransmitters that play vital roles in the health of humans and other mammalians.27 Many diseases (neurological, psychiatric and cardiovascular) are related to changes in epinephrine (EP) concentrations. Thus, it is necessary to develop quantitative methods for this catecholamine for studies on physiological function and diagnosis in clinical medicine.28 Abnormal DA concentrations have also been associated with neurological disorders (Parkinson's disease and schizophrenia); thus, the determination of DA concentrations can be very important in diagnostic applications.29–31 Additionally, EP and DA are used in emergency health care in patients who show signal of hypertension, bronchial asthma, and myocardial infarction as well as during cardiac surgery,32 so the control of these compounds is of paramount importance. At present, a variety of methods are used to determine DA and EP simultaneously, including capillary electrophoresis,33 high performance liquid chromatography-mass spectrometry,34 ow injection analysis35 with spectrophotometric detection and electrochemical methods.36–41 Except for the electrochemical
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methods, the other methods are all difficult to use for rapid detection and cannot be miniaturized to determine these analytes in loco. The purpose of this work is to investigate the electrochemical behaviour of EP and DA in aqueous solutions at a glassy carbon electrode (GCE) modied with NiONP–MWCNT within a dihexadecylphosphate (DHP) lm (NiONP–MWCNT–DHP/GCE) by cyclic, square-wave and differential-pulse voltammetries. The aim was to develop a sensitive method to simultaneously determine both analytes in human body uid samples.
2.
Experimental
2.1. Reagents and solutions Dopamine (DA), epinephrine (EP), MWCNTs (95% purity, 20– 30 nm in diameter, 1–2 nm thick and 0.5–2 mm in length) and dihexadecylphosphate were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade. The solutions were prepared using ultrapure water (resistivity > 18.0 MU cm) obtained from a Millipore Milli-Q system (Billerica, USA). A stock solution of DA was prepared directly in ultrapure water and that of EP in 0.2 mol L1 sulfuric acid (better solubility in inorganic acid aqueous solutions). 2.2. Instrumentation The voltammetric measurements were performed using a threeelectrode system, including the modied electrode as the working electrode, a platinum plate as the counter electrode, and Ag/AgCl (3.0 mol L1 KCl) as the reference electrode. A potentiostat/galvanostat Autolab model PGSTAT12 (Ecochemie, the Netherlands) controlled by GPES 4.9 soware was used. The surface electrode morphology was characterized by a eld-emission gun scanning electron microscopy FEG/SEM mode (Supra 35-VP, Carl Zeiss, Germany). A histogram was constructed using the public domain ImageJ image processing soware. 2.3. MWCNTs functionalization and NiONP–MWCNT–DHP/ GCE preparation The carbon nanotubes were initially subjected to chemical pretreatment for 12 h using a mixture of concentrated sulfuric and nitric acids in a proportion of 3 : 1 v/v, respectively. Subsequently, the mixture was ltered, washed with deionized water until a pH of 6.5–7.0 was reached, and then dried at 120 C for 5 h.42,43 This pre-treatment was carried out to remove possible metal impurities, open the ends of the CNTs and add functional groups such as carboxylic groups onto the CNT surface.44 Next, the suspension of MWCNTs was prepared by dispersing 1.0 mg MWCNTs with 1.0 mg DHP in 1.0 mL of ultra-pure water, and this dispersion was placed under ultrasonic agitation for 40 min.45,46 Before the modication, the bare GCE (3.0 mm in diameter) was polished successively with alumina on a polishing cloth and then rinsed with ultra-pure water and ethyl alcohol. Next, 8 mL of the MWCNT–DHP dispersion was dropped onto the GCE surface and the solvent was evaporated at 25 C for 2 h. The
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MWCNT–DHP/GCE obtained was subjected to the NiONPs electrodeposition procedure. For this, in the rst step, the potential was cycled 40 times between 0 and 0.8 V at a scan rate of 100 mV s1 in an acetate buffer solution (pH 4) containing 0.002 mol L1 nickel nitrate.25 Finally, the potential was repetitively cycled (40 scans) from 0 to 0.6 V at a scan rate of 100 mV s1 in fresh 0.1 mol L1 NaOH solution for the electrodissolution and passivation of the NiONPs on the MWCNT–DHP/GCE.25 2.4. Analytical procedure Aer optimizing the experimental parameters for square-wave voltammetry (SWV) and differential-pulse voltammetry (DPV), analytical curves were constructed by adding small volumes of concentrated standard solutions of EP and DA. The detection limit was calculated to be three times the standard deviation for the blank solution (n ¼ 10) divided by the slope of the analytical curve. Samples of cerebrospinal uid, human serum and lung uid were prepared following the appropriate protocols.47–49 Next, an appropriate aliquot of EP and DA was added carefully to the samples (spiking) to obtain concentrations of 7.0 107 and 1.4 106 mol L1 of EP and 5.0 107 and 1.0 106 mol L1 of DA.
3.
Results and discussion
3.1. Electrodeposition of NiONPs on MWCNT–DHP/GCE and morphology characterization The preparation of the NiONP–MWCNT–DHP/GCE was performed/adapted following a procedure proposed in the literature,25 and it briey consisted of the electrodeposition of NiONPs. In the rst step, the potential was cycled between 0.0 V and 0.8 V in an acid solution containing nickel nitrate (0.002 mol L1) in order to electrodeposit metallic nickel. Next, the potential was cycled between 0.0 V and +0.6 V in an alkaline solution (0.1 mol L1 NaOH) for the electrodissolution and passivation of the NiONPs.25
Fig. 1 Cyclic voltammograms obtained for the NiONP–MWCNT– DHP/GCE in 0.1 mol L1 NaOH solution at different scan rates: (1) 10; (2) 20; (3) 30; (4) 40; (5) 50; (6) 60; (7) 70; (8) 80; (9) 90 and (10) 100 mV s1. Inset: log Ia vs. log v.
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The effective formation of the nickel oxide nanoparticles was conrmed by electrochemistry from the study of the electrochemical behaviour of the NiONP–MWCNT–DHP/GCE in 0.1 mol L1 NaOH solution. Fig. 1 shows the cyclic voltammograms obtained at different scan rates (v) for the NiONP– MWCNT–DHP/GCE in 0.1 mol L1 NaOH solution. As can be seen, a reversible process was veried, which was assigned to the equilibrium reaction (1):25 Ni(OH)2 + OH # NiO(OH) + H2O + e
(1)
Ni(OH)2 was generated during the electrodissolution and passivation step and subsequently was oxidized to NiO(OH) in the alkaline medium (anodic peak); this last species was reduced to Ni(OH)2 (cathodic peak) aer potential scanning inversion. Moreover, a plot of log Ia vs. log v revealed adsorptive behaviour, with a slope of 0.96. Fig. 2 presents the FEG-SEM images of (a) MWCNT–DHP/ GCE and (b) NiONP–MWCNT–DHP/GCE. A clear difference in terms of the surface is observed among these electrodes. It can be observed from Fig. 2(a) that a relatively dense lm (MWCNT– DHP) was formed on the GCE surface. In Fig. 2(b) an increase in the roughness of the MWCNTs surface can be observed indicating the incorporation of NiONPs. Also an EDX (energydispersive X-ray) analysis was performed to identify the elements present on the electrode surface and the results showed a high percentage of carbon (85.11%), oxygen (8.52%), nickel (1.14%), and phosphorus (3.89%), which was in agreement with the expectations considering the chemicals used in the lm preparation. Moreover, in Fig. S1 (ESI†) is shown a FEGSEM image of NiONPs electrodeposited on the GCE surface including a histogram of the NiONPs diameters with an average size of 40.8 nm.
3.2. Electrochemical behaviour of dopamine and epinephrine on the NiONP–MWCNT–DHP/GCE The glassy carbon electrode (GCE), glassy carbon electrode modied with MWCNT–DHP (MWCNT–DHP/GCE) and glassy carbon electrode modied with nickel oxide nanoparticles and MWCNT–DHP (NiONP–MWCNT–DHP/GCE) were used to investigate the electrochemical behaviour of EP and DA on the proposed electrodes via cyclic voltammetry (CV). Determinations were carried out on different samples via differential-pulse
Fig. 2 FEG-SEM images of the surface of: (a) MWCNT–DHP/GCE and (b) NiONP–MWCNT–DHP/GCE.
2844 | Analyst, 2014, 139, 2842–2849
Fig. 3 Cyclic voltammograms obtained for (a) 1.0 104 mol L1 DA and (b) 1.0 104 mol L1 EP in 0.2 mol L1 phosphate buffer solution (pH 7.0) using a GCE (black line), MWCNT–DHP/GCE (blue line) and NiONP–MWCNT–DHP/GCE (red line). (c) Overlap of the cyclic voltammograms for 1.0 104 mol L1 DA and 1.0 104 mol L1 EP in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP– MWCNT–DHP/GCE.
voltammetry (DPV) and square-wave voltammetry (SWV). The obtained voltammograms using GCE, MWCNT–DHP/GCE and NiONP–MWCNT–DHP/GCE are presented in Fig. 3(a) (DA), 3(b) (EP) and 3(c) (both analytes) using the proposed modied electrode. In the case of the GCE, small oxidation and reduction peaks were observed. With the incorporation of the MWCNT– DHP lm onto the GCE surface, the analytical signal was substantially increased and a decrease in the oxidation and reduction potentials was seen, a characteristic of the electrocatalytic effect. Likewise, the use of NiONPs combined with the MWCNT–DHP lm showed an increase in the oxidation current of 70% and in the reduction current of 45%; however, no additional electrocatalytic effects were seen when compared with the MWCNT–DHP lm. The increase of current could be explained by the interaction between the NiONPs and the –OH groups of catecholamines, leading to an increase of the analyte concentration on the electrode surface.50–52 However, the key problem of simultaneous sensing of catecholamines (Fig. 3(c)) by electrochemistry is the overlapping voltammetric signals. Also, as can be found in the literature, there is a large excess of coexisting substances in biological tissues such as ascorbic acid (AA) and uric acid (UA).29 One way to solve this problem is to focus on the reduction peak of EP and DA (Fig. 3(c)), as there is a difference between the reduction peaks of ca. 360 mV (EP ca. 192 mV and DA ca. 171 mV), which is a good value of peak separation for simultaneous determination. Aer the peak resolution studies, we turned to explore the other electrochemical characteristics. Scan rate studies where the voltammetric peak magnitude (Ip) was monitored as a function of scan rate (v) with a plot of peak height versus the square-root of the scan rate (Fig. 4(a) and (b)) revealed the
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Cyclic voltammograms obtained for (a) 1.0 104 mol L1 DA and (b) 1.0 104 mol L1 EP in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP–MWCNT–DHP/GCE at different scan rates (v): (1) 20; (2) 30; (3) 40; (4) 50; (5) 75; (6) 100; (7) 125; (8) 150; (9) 200 mV s1. Fig. 4
following trends: EP (cathodic peak), Ic (A) ¼ 3.60 105 A/(V s1)0.5 + 3.54 106 A (r ¼ 0.995); EP (anodic peak), Ia (A) ¼ 9.12 105 A/(V s1)0.5 2.97 106 A (r ¼ 0.998); DA (cathodic peak), Ic (A) ¼ 7.46 105 A/(V s1)0.5 + 1.04 105 A (r ¼ 0.996); DA (anodic peak), Ia (A) ¼ 1.10 104 A/(V s1)0.5 8.67 106 A (r ¼ 0.997). It was clear that, in both cases, a linear response was evident, indicating diffusional processes. Furthermore, as was expected for the case of the semi-innite diffusion model governed by the Randles–ˇ Sev´ cik equation, analysis of log Ip versus log v revealed gradients of 0.44 (EP cathodic peak), 0.54 (EP anodic peak), 0.53 (DA cathodic peak) and 0.48 (DA anodic peak), indicating the absence of thin-layer effects (e.g. the analytes were not trapped within the lm network (NiONP–MWCNT–DHP/GCE) of the GCE). 3.3. Simultaneous voltammetric determination of dopamine and epinephrine using the NiONP–MWCNT–DHP/GCE For EP and DA, an electroanalytical procedure was developed involving a systematic study and optimization of the experimental parameters (frequency, f; pulse amplitude, a; potential increment, DEs) that affect the SWV response (the value of the peak reduction current was registered at a concentration of 1.0 104 mol L1 for both analytes). The best results for the SWV parameters are shown in Table 1. Next, we determine the analytical curves. First, the analytical curve was constructed by varying the concentrations of EP from 9.8 107 mol L1 to 6.8 105 mol L1 while maintaining the DA concentration constant at 2.0 106 mol L1. Next, we varied the DA concentration from 7.0 108 mol L1 to 9.7 106 mol L1 and xed the concentration of EP (1.0
Table 1 Experimental parameters of the SWV and DPV techniques optimized for DA and EP simultaneous determination
Technique
Parameter
Range
Selected
SWV
Frequency, f (Hz) Amplitude, a (mV) Potential increment, DEs (mV) Scan rate potential, v (mV s1) Amplitude, a (mV) Pulse time, t (ms)
10–100 5–100 1–10 2.5–20 10–100 5–30
10 90 9 7.5 90 25
DPV
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105 mol L1). From the SW voltammograms (Fig. 5(a) and (b)), it can be seen that the EP and DA cathodic peak currents increased regularly with the concentration and the cathodic peak current for the analyte with xed concentration remained fairly constant (RSD values of 15.6% for DA (Fig. 5(a)) and 4.4% for EP (Fig. 5(b))), thus showing that they did not interfere with each other. Subsequently, an analytical curve was performed for both analytes (EP and DA) at the same time; the plot of the peak reduction current vs. EP concentration was linear in the concentration range from 7.0 107 mol L1 to 4.8 105 mol L1 and for DA it was linear in the concentration range from 7.0 108 mol L1 to 4.8 106 mol L1 (Fig. 5(c)). The corresponding equations were: EP: Ic (mA) ¼ 3.94 105 [EP] (mol L1) 1.91 (r ¼ 0.994) DA: Ic (mA) ¼ 3.83 106 [DA] (mol L1) 5.33 (r ¼ 0.991) The detection limits calculated using the relation 3 s/m, where s is the standard deviation of ten blank measurements and m is the slope of the analytical curve, were 4.8 107 mol L1 for EP and 3.8 108 mol L1 for DA. For DPV, the experiments were carried out in the same manner as for SWV. First, the parameters related to the technique were studied (scan rate potential, v; amplitude, a; pulse time, t); in Table 1, the optimum values selected can be seen. Next, we performed analytical curves, initially as in SWV, by varying the EP concentration (3.0 107 mol L1 to 9.7 106 mol L1) and maintaining the DA concentration (2.0 106 mol L1). Next, we carried out the opposite study, i.e. by varying the DA concentration (7.0 108 mol L1 to 6.8 106 mol L1) and maintaining the EP concentration (1.0 105 mol L1). Aer examining the DP voltammograms (Fig. 6(a) and (b)), a similar conclusion to the SWV case was obtained, i.e., the analytes did not interfere with each other, since the EP and DA cathodic peak currents increased regularly with the concentration and the cathodic peak current for the analyte with xed concentration remained fairly constant (RSD values of 3.4% for DA (Fig. 6(a)) and 2.4% for EP (Fig. 6(b))). Finally, the analytical curves were obtained by varying the concentration of both analytes at the same time; the plot of peak
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SW voltammograms obtained in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP–MWCNT–DHP/GCE for (a) various concentrations of EP: (1) 0.98; (2) 2.94; (3) 4.89; (4) 6.83; (5) 9.73; (6) 29.13; (7) 48.45 and (8) 67.70 mmol L1 at a fixed DA concentration (2.00 mmol L1), (b) various concentrations of DA: (1) 0.07; (2) 0.10; (3) 0.30; (4) 0.49; (5) 0.69; (6) 0.98; (7) 2.94; (8) 4.89; (9) 6.83 and (10) 9.73 mmol L1 at a fixed EP concentration (10 mmol L1) and (c) various concentrations of EP and DA: EP – (1) 0.70; (2) 0.98; (3) 2.93; (4) 4.87; (5) 6.80; (6) 9.66; (7) 28.85 and (8) 47.90 mmol L1; DA – (1) 0.07; (2) 0.10; (3) 0.30; (4) 0.49; (5) 0.68; (6) 0.96; (7) 2.87; (8) 4.77 mmol L1. Insets: analytical curves. Fig. 5
Fig. 6 DP voltammograms obtained in 0.2 mol L1 phosphate buffer solution (pH 7.0) using the NiONP–MWCNT–DHP/GCE for (a) various concentrations of EP: (1) 0.30; (2) 0.49; (3) 0.69; (4) 0.98; (5) 2.94; (6) 4.89; (7) 6.83 and (8) 9.73 mmol L1 at fixed DA concentration (2.00 mmol L1), (b) various concentrations of DA: (1) 0.07; (2) 0.10; (3) 0.30; (4) 0.49; (5) 0.69; (6) 0.98; (7) 2.94; (8) 4.89; (9) 6.83 mmol L1 at fixed EP concentration (10 mmol L1) and (c) various concentrations of EP and DA: EP – (1) 0.30; (2) 0.49; (3) 0.68; (4) 0.97; (5) 2.89; (6) 4.80; (7) 6.69; (8) 9.50 mmol L1; DA – (1) 0.07; (2) 0.10; (3) 0.29; (4) 0.48; (5) 0.68; (6) 0.96; (7) 2.87; (8) 4.75 mmol L1. Inset: analytical curves.
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Table 2 Comparison between the SWV and DPV techniques for the simultaneous voltammetric determination of DA and EP
Technique Analyte
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DA
EP
Analytical parameter Sensitivity (mA L mol1) Detection limit (mol L1) Linear range (mol L1) Sensitivity (mA L mol1) Detection limit (mol L1) Linear range (mol L1)
SWV
DPV
3.8 106
1.9 106
3.8 108
5.0 108
7.0 108 to 4.8 106 3.9 105
7.0 108 to 4.8 106 9.0 105
4.8 107
8.2 108
7.0 107 to 4.8 105
3.0 107 to 9.5 106
current vs. EP concentration was linear in the concentration range from 3.0 107 mol L1 to 9.5 106 mol L1 and for DA it was linear in the concentration range from 7.0 108 mol L1 to 4.8 106 mol L1 (Fig. 6(c)). The analytical curve equations were: EP: Ic (mA) ¼ 9.03 105 [EP] (mol L1) 1.18 (r ¼ 0.993) DA: Ic (mA) ¼ 1.85 106 [DA] (mol L1) 0.55 (r ¼ 0.994) The detection limits calculated using the relation 3 s/m, where s is the standard deviation of ten blank measurements and m is the slope of the analytical curve, were 8.2 108 mol L1 for EP and 5.0 108 mol L1 for DA. Table 2 shows all the analytical parameters (sensitivity, detection limit and linear range) obtained using SWV and DPV for each analyte. Comparing the different analytical parameters,
we observed that for DA, the SWV and DPV techniques showed the same linear concentration range, although the SWV sensitivity was 2.1 times higher than the DPV sensitivity and the detection limits were very close (Detection limitDPV ¼ 1.3 Detection limitSWV). However, for EP detection, the DPV technique showed some important advantages in relation to the SWV technique: the DPV sensitivity was 2.3 times higher than the SWV sensitivity, the detection limit obtained for DPV was 6.0 times lower than that obtained with SWV and, although the linear concentration range veried for DPV was lower than that for SWV, in the case of the DPV technique, the procedure was capable of determining a low concentration of EP. The better performance of the DPV technique for EP determination can be related to the fact that this analyte presents higher irreversibility, and, the loss of sensitivity due to the irreversibility of the electrochemical reaction is lower for the differential pulse method than for other pulse techniques.53 From these observations, the DPV technique was chosen as the voltammetric technique to continue these studies. Table 3 presents the analytical parameters (detection limit and linear concentration range of the analytical curve) of electroanalytical methods using different electrodes and techniques that have been reported for the simultaneous determination of DA and EP. From this table, we can conclude that the concentration range is similar to those reported in the literature. Nevertheless, using the DPV technique with the NiONP– MWCNT–DHP/GCE, the detection limit was signicantly lower than the values published to date. 3.4. Repeatability studies and determination of dopamine and epinephrine in human body uids In order to determine the intra-day repeatability of the voltammetric procedure, various successive simultaneous measures (n ¼ 10) were performed using 0.2 mol L1 phosphate buffer solution (pH 7.0) containing DA and EP, both at 5.0 106 mol L1. Thus, the RSD of 3.2% and 1.2% was obtained for
Table 3 Comparison of the analytical parameters obtained for the simultaneous determination of DA and EP using the NiONP–MWCNT–DHP/ GCE with those from the literaturea
Electrode
Technique
Linear range (mol L1)
Detection limit (mol L1)
Reference
PLA/GCE
CV CV
Poly(caffeic acid)/GCE
CV
Poly(taurine)/GCE
DPV
a-CD/CNT/GCE
DPV
Poly(isonicotinic acid)/CPE
CV
NiONP–MWCNT–DHP/GCE
DPV
DA: 4.0 107 EP: 1.0 107 DA: 1.7 107 EP: 3.2 107 DA: 2.0 107 EP: 1.0 107 DA: 1.0 107 EP: 3.0 107 DA: 1.0 106 EP: 5.0 107 DA: 2.0 105 EP: 1.0 106 DA: 5.0 108 EP: 8.2 108
54
PAIUCPE
DA: 8.0 107 to 5.0 104 EP: 5.0 107 to 5.0 105 DA: 8.0 107 to 3.0 104 EP: 2.0 106 to 1.5 104 DA: 1.0 106 to 3.5 105 EP: 1.0 106 to 1.5 105 DA: 1.0 106 to 8.0 104 EP: 2.0 106 to 6.0 104 DA: 2.0 106 to 1.0 103 EP: 1.0 106 to 1.0 103 DA: 8.0 105 to 7.0 104 EP: 5.0 106 to 1.0 104 DA: 7.0 108 to 4.8 106 EP: 3.0 107 to 9.5 106
55 56 39 57 58 This work
a PLA – poly(L-arginine); GCE – glassy carbon electrode; CV – cyclic voltammetry; PAIUCPE – pre-anodized inlaying ultrathin carbon paste electrode; a-CD – a-cyclodextrin; CNT – carbon nanotubes; CPE – carbon paste electrode.
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Table 4 Simultaneous determination of DA and EP in human body fluid samples using the NiONP–MWCNT–DHP/GCE
Sample Cerebrospinal uid
Added (mmol L1) Level 1
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Level 2 Human serum
Level 1 Level 2
Lung uid
Level 1 Level 2
DA: 0.50 EP: 0.70 DA: 1.00 EP: 1.40 DA: 0.50 EP: 0.70 DA: 1.00 EP: 1.40 DA: 0.50 EP: 0.70 DA: 1.00 EP: 1.40
Found (mmol L1)
Recovery (%)
0.49 0.03 0.75 0.07 1.11 0.04 1.36 0.04 0.51 0.07 0.76 0.08 1.08 0.03 1.43 0.01 0.49 0.04 0.68 0.04 1.13 0.04 1.46 0.06
98.0 107.1 111.0 97.1 102.0 108.6 108.0 102.1 98.0 97.1 113.0 104.3
DA and EP. Also, the inter-day repeatability of the procedure was studied by carrying out measures on three different days (n ¼ 3) using, consequently, modied electrodes prepared on different days. In this study, DA and EP concentrations of 5.0 107 mol L1 and 7.0 106 mol L1 were used, providing an RSD of 4.8% and 7.0% for DA and EP, respectively. The results obtained in the repeatability studies show the good precision of the procedure, as well as the simplicity of preparing the modied electrodes, which allows obtaining reproducible measures using electrodes prepared at different times. Moreover, the electrode can be used for at least 40 determinations. Next, human body uid samples including cerebrospinal uid, human serum and lung uid spiked with two different levels of DA and EP were analyzed by the proposed method. Table 4 presents the results obtained for the analysis of these samples. As can be seen, excellent recovery values were obtained for both analytes in the three samples. From these results, one may conclude that the DPV procedure using the MWCNT–DHP/ GCE modied with electrodeposited NiONPs does not suffer interference from the synthetic sample matrix, thus presenting potential applicability in the simultaneous determination of DA and EP in real biological samples. Moreover, the NiONP– MWCNT–DHP/GCE presented good stability for repeated determinations (n ¼ 30) of biological samples, when the cathodic current response varied by only 7.6% and 8.9% for DA and EP, respectively.
4. Conclusions In this work, a NiONP–MWCNT–DHP/GCE was proposed to develop a novel DPV method for the single or simultaneous determination of DA and EP in human body uid samples. The proposed electrode presented good separation of the reduction peak potentials for DA and EP (about 360 mV) by DPV, enabling the simultaneous determination of these compounds. Very low detection limits were obtained in the simultaneous determination of these analytes: DA (5.0 108 mol L1) and EP (8.2 108 mol L1). Moreover, addition and recovery studies indicated that matrix effects did not present any signicant
2848 | Analyst, 2014, 139, 2842–2849
interference and the proposed method can be used for the simultaneous determination of DA and EP in real samples.
Acknowledgements The authors acknowledge nancial support from the following Brazilian funding agencies: CNPq, CAPES and FAPESP (Proc. 2010/20754-1 and 2013/16770-0).
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