Materials Science and Engineering C 58 (2016) 53–59
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Construction of a sensitive and selective sensor for morphine using chitosan coated Fe3O4 magnetic nanoparticle as a modifier Sara Dehdashtian a, Mohammad Bagher Gholivand b,⁎, Mojtaba Shamsipur b, Samira kariminia b a b
Department of Chemistry, Behbahan Khatam Al-Anbia University of Technology, Behbahan, Iran Department of Analytical Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran
a r t i c l e
i n f o
Article history: Received 12 February 2015 Received in revised form 17 April 2015 Accepted 23 July 2015 Available online 28 July 2015 Keywords: Morphine Chitosan-coated magnetic nanoparticle Modified carbon paste electrode Differential pulse voltammetry Cyclic voltammetry Electrochemical impedance spectroscopy Chronoamperometry
a b s t r a c t A simple and sensitive sensor based on carbon paste electrode (CPE) modified by chitosan-coated magnetic nanoparticle (CMNP) was developed for the electrochemical determination of morphine (MO). The proposed sensor was characterized with scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The electrooxidation of MO was studied on modified carbon paste electrode using cyclic voltammetry, chronoamperometry and differential pulse voltammetry as diagnostic techniques. The oxidation peak potential of morphine on the CMNP/CPE appeared at 380 mV which was accompanied with smaller overpotential and increase in oxidation peak current compared to that obtained on the bare carbon paste electrode (CPE). Under optimum conditions the sensor provides two linear DPV responses in the range of 10–2000 nM and 2–720 μM for MO with a detection limit of 3 nM. The proposed sensor was successfully applied for monitoring of MO in serum and urine samples and satisfactory results were obtained. © 2015 Published by Elsevier B.V.
1. Introduction Morphine (MO) as a major component in opium is frequently used to relieve severe pain for patients, but when overdosed or abused, it is toxic and can cause disruption in the central nervous system. It is recommended by the World Health Organization (WHO) for the relief of moderate cancer-related pain [1]. Morphine is a precursor in the manufacture of a large number of opioids such as dihydromorphine, hydromorphone, nicomorphine, and heroin as well as codeine. Therefore, in order to prevent the toxicity, induced by overdosing or abusing, it is necessary to determine the concentrations of MO in patient's blood or urine using a sensitive method. Different analytical methods have been used for the determination of morphine in plasma, urine, and opium samples, such as gas chromatography (GC) [2], liquid chromatography (LC) [3], high performance liquid chromatography (HPLC) [4], ultraviolet (UV) spectroscopy [5], GC–mass spectroscopy (GC–MS) [6], fluorimetry [7], chemiluminescence [8], surface plasmon resonance (SPR) [9], radioimmunoassay (RIA) [7], and electrochemiluminescence (ECL) [10]. These methods
Abbreviations: MO, morphine; CPE, carbon paste electrode; CMNP, chitosan-coated magnetic nanoparticle; CV, cyclic voltammetry; DPV, differential pulse voltammetry; SEM, scanning electron microscopy; EIS, electrochemical impedance spectroscopy; LOD, limit of detection; LOQ, limit of quantitation; RSD, relative standard deviation. ⁎ Corresponding author. E-mail address: [email protected] (M.B. Gholivand).
http://dx.doi.org/10.1016/j.msec.2015.07.049 0928-4931/© 2015 Published by Elsevier B.V.
require advanced technical expertise and are expensive and timeconsuming. The electrochemical techniques as alternative methods have also received much interest due to their higher selectivity, lower cost and faster operation and thus, have become of growing importance in many fields including medicine and biotechnology, environmental monitoring, and different applications in industrial process control. Due to slow electron transfer reaction and electrode-fouling problem of most bare electrodes; they must be modified with suitable materials. Therefore, different modifiers such as cobalt hexacyanoferrate [11], Prussian blue-modified indium tin oxide (ITO) [12], molecularly imprinted polymer films [13], palladized aluminum [14], multiwalled carbon [15], MWNT-doped graphene oxide composite film [16], ZnO/ CNT nanocomposite [17], gold nanoparticles [18], ferrocene/gold nanoparticles [19], carbon nanotube/chitosan [20], Pt–Co alloy nanowire [21] and poly(3,4-ethylenedioxythiophene) [22] have been used for the morphine detection. Chitosan (CHT), as a natural polymer with abundant primary amino groups and hydroxyl groups, is non-toxic, readily decomposes in natural biological environments, and is compatible with living organism cells (both plant and animal) and displays a number of properties including hydrophilicity, gel-forming ability, doping feasibility, good mechanical stability, good permeability, cost-effectiveness, and availability of reactive functional groups for chemical modifications. CHT can effectively adsorb not only metal ions but also various organic compounds [23] and thus, can be widely used in construction of electrochemical sensors and biosensors [24–29]. To refine the electric conductivity of CHT, some
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materials such as metal nanoparticles, carbon nanotubes and redox mediators have been introduced to CHT films [30–35]. In the present study, metal oxide nanoparticles were used for improvement in the conductivity of CHT and enhancement in the sensitivity of the sensor. Furthermore, metal oxide nanoparticles can be helpful to obtain improved stability and sensitivity of a sensor, because they exhibit large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, strong adsorption ability and unique ability to promote fast electron transfer between electrode and target. Among them, Fe3O4 nanoparticles, with low toxicity are of interest due to its biocompatibility. Combination of CHT and Fe3O4 nanoparticles produces a nanocomposite that not only overcomes aggregation and rapid biodegradation of Fe3O4 nanoparticles by CHT [36–43] but dispersing of super paramagnetic Fe3O4 nanoparticles improves the optical and electrical properties of CHT for biosensor application [36,43]. Both metal oxide nanoparticles and chitosan can be used for removal of heavy metals [23] from aqueous environments and modification of electrode surface [24–29]. In this study, a simple and sensitive sensor based on carbon paste (CP) electrode modified by chitosan-coated magnetic nanoparticle was developed for the electrochemical determination of morphine in real samples. 2. Experimental
30 ml of chitosan solution. Then 30 ml of aqueous ammonia solution (25%) was very slowly added to the above solution for producing smaller sized nanoparticles. The resulting solution was stirred for an additional 30 min. The colloidal chitosan coated magnetic Fe3O4 nanoparticles were extensively washed with deionized water and separated by magnetic decantation for several times. Finally, the chitosan-coated MNPs were dried at 50 °C and stored at room temperature. 2.4. Characterization of chitosan coated magnetic nanoparticles (CMNPs) by Fourier transform infrared spectroscopy (FTIR) To confirm the chemical composition of synthesized nanoparticles, FTIR spectra were obtained. For the naked Fe3O4 (Fig. 1(a)), the peak at 579 cm−1 relates to Fe–O group in Fe3O4. However, from Fig. 1(b), it can be seen that the characteristic peak of Fe–O shifts to 581 cm−1 revealing the interaction between Fe3O4 and CHT. Compared with Fe3O4 nanoparticles, the spectrum of CHT–Fe3O4 has characteristic peak of CHT at 1070 cm−1 (C–N stretching vibrations) and 1636 cm−1 (N–H bending vibration). The results indicated that CHT was coated to the magnetic Fe3O4 nanoparticles successfully. Because the surface of iron oxide with negative charges has an affinity towards CHT, protonated CHT could coat the magnetite nanoparticles by the electrostatic interaction.
2.1. Apparatus and chemicals 2.5. Preparation of modified electrode Cyclic voltammetric (CV) and differential pulse voltammetric (DPV) experiments were performed using the μ-Autolab electrochemical system (Eco-Chemie, Utrecht, The Netherlands) equipped with GPES and FRA 4.9 software coupled with a conventional three-electrode cell. The working electrode was an unmodified or modified carbon paste electrode; the auxiliary and reference electrodes were a platinum wire and SCE respectively. A Metrohm 710 pH meter was used for pH measurements. The surface morphology of modified electrodes was characterized with a scanning electron microscope (SEM) (Philips XL 30). All experiments were carried out at ambient temperature (25 ± 1 °C). Morphine sulfate was used as received. Chitosan with low molecular weight and MO were of analytical grade from Sigma-Aldrich. Ferrous chloride tetrahydrate (FeCl2·4H2O) and ferric chloride hexahydrate (FeCl3·6H2O) were purchased from Sigma-Aldrich. The buffer solutions were prepared from orthophosphoric acid and its salts in the pH range of 2.0–10.0. All other reagents were of analytical grade and solutions were freshly prepared with double distilled water.
The unmodified and modified carbon pastes were prepared by thoroughly mixing analytical grade graphite and paraffin oil in the absence and presence of modifier and then each paste was mixed in a mortar for at least 10 min to become homogeneous. Each paste was packed into one end of a Teflon holder in which electrical contact was made with a copper rod that runs through the center of the electrode body. The surface of each electrode was polished using a butter paper to produce reproducible working surface and then was used for electrochemical studying of MO by voltammetric techniques. The unmodified carbon paste was prepared by mixing 70:30% of graphite and paraffin oil respectively. The modified carbon paste was prepared by mixing different percentages of graphite powder, paraffin oil, and modifier and the best results were obtained at 60:30:10% of graphite, paraffin oil and modifier. The modified electrode was activated in a 10 ml of 0.1 M phosphate buffer (pH 7) by potential scanning from − 0.25 to 1 V vs. SCE at a scan rate of 100 mV s−1, until a low and steady background was obtained.
2.2. Synthesis of Fe3O4 magnetic nanoparticles (MNPs) Magnetic iron oxide (Fe3O4) nanoparticles were synthesized by modified coprecipitation method [44]. Stoichiometric ratio of 1:2 ferrous chloride tetrahydrate (FeCl2·4H2O) and ferric chloride hexahydrate (FeCl3·6H2O) was dissolved in deionized water under nitrogen atmosphere with vigorous stirring at 70 °C. Chemical precipitation was achieved by dropwise adding NH4OH solution (25%) to reach pH 10 under vigorous stirring. The color of solution was turned from dark orange to the black immediately. After continuously stirring for 1 h, the black precipitates of Fe3O4 were magnetically decanted and washed several times with distilled water. The powder was then dried at 50 °C in an oven for 24 h. 2.3. Preparation of chitosan coated magnetic nanoparticles (CMNPs) Chitosan coated magnetic iron oxide nanoparticles were in situ synthesized by the co-precipitation of Fe (II) and Fe (III) salts in the presence of chitosan. Chitosan (0.125 g) was dissolved in 30 ml of 1% acetic acid and the pH was adjusted to 4.8 by 10 M NaOH. Under the nitrogen (N2) gas flow and by vigorously stirring at 50 °C, iron salts (1.34 g of FeCl2·4H2O and 3.40 g of FeCl3·6H2O) were dissolved in
Fig. 1. Fourier transform infrared spectra of (a) naked Fe3O4 and (b) chitosan coated Fe3O4.
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2.6. Analytical procedure 10 ml of 0.1 M phosphate buffer (pH 7) was introduced in a voltammetric cell and its DP voltammogram under optimum conditions (50 mV pulse amplitude, 40 ms pulse width and 60 m Vs−1 scan rate) in the potential range of 0.0 to 0.7 V was used for background correction. Then different volumes of sample solution were added to the above solution and their voltammograms were used for calibration plot. For recording the cyclic voltammograms, the potential was scanned from the 0 to 0.7 V using the scan rate of 100 m Vs−1. 2.7. Preparation of serum sample For the determination of MO in human serum sample, a serum sample of a healthy volunteer was stored frozen until assay. 2 ml of methanol was added to a 1.5 ml serum sample to remove serum protein. After vortexing of the serum sample for 2 min, the precipitated proteins were separated by centrifugation for 3 min at 6000 rpm. The clear supernatant layer was filtered and diluted to a definite volume using double distilled water. The urine sample used for measurement was centrifuged and diluted 10 times using double distilled water without any further pretreatment. For both real samples, known amounts of analyte were added to phosphate buffer solution (pH 7) containing deliberate amounts of real sample. 3. Results and discussion 3.1. Characterization of the modified electrode Electrochemical impedance spectroscopy (EIS) is an effective method for probing the features of surface modified electrodes. The Nyquist plot of impedance spectra includes a semicircle portion and a linear portion, with the former at higher frequencies corresponding to the electron transfer limited process and the latter at lower frequencies corresponding to the diffusion process. The electron transfer resistance (Rct) at the electrode surface is equal to the semicircle diameter, which can be used to describe the interface properties of the electrode. Fig. 2 presents the Nyquist diagrams of the bare CPE (curve a with Ret = 139.0 Ω) and CMNP–CPE (curve b with Ret = 77 Ω) in 5 mM [Fe(CN)6]3−/4 − and 0.1 M KCl. CMNP–CPE shows a small semicircle at the high frequency region when compared with the bare CPE, indicating lower electron transfer resistance. This can be attributed to the presented CMNP with good conductivity and large surface area in the modified electrode, which could effectively increase the rate electron transfer between
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electrode surface and [Fe(CN)6]3−/4− and decrease interface electron transfer resistance. The response of a modified electrode is related to its physical morphology. The SEM of bare and modified CPEs was shown in Fig. 3. Significant differences in the surface structure of these electrodes are observed. The surface of the bare CPE was predominated by isolated and irregularly shaped graphite flakes and separated layers were seen (Fig. 3A). The SEM image of MNP and CMNP–CPEs showed more uniform surface and no separated carbon layers could be observed in Fig. 3B and C. 3.2. Electrochemical behavior of MO at CMNP–CPE The electrochemical study of phosphate buffer solution (0.1 M, pH 7) in the absence (curve a) and presence (curve b) of 50 μM of MO at CMNP–CPE was performed using cyclic voltammetry. As can be seen from Fig. 4A in the presence of MO a well define anodic peak was observed at 0.38 V without any counterpart cathodic peak in the reversed scan indicating that the electrodic process is irreversible. As reported by Li et al., the observed oxidation peak could be ascribed to the oxidation of the phenolic group, which leads to the formation of pseudomorphine [1] (Fig. 5). The cyclic voltammetric response of MO at the CMNP–CPE was compared with those obtained at CP and MNP–CP electrodes and the results are presented in Fig. 4B. As it is shown, the oxidation of MO at the bare carbon paste electrode produces a week anodic peak at 0.44 V (curve a). At the surface of the CPE modified by MNP (MNP–CPE), the current associated with anodic peak of MO is increased in comparison with that obtained on CPE without any significant change in its potential. Modification of CPE by CMNP not only increases the peak current (almost three folds greater) but decreases the overvoltage accompanied by anodic peak of MO and shifts its peak potential towards less positive value. The absence of counterpart cathodic peak of the oxidation of MO in its cyclic voltammograms (CVs) at the surface of all tested electrodes reveals that the electrochemical reaction of MO is totally irreversible process. 3.3. The effect of pH Because of individual functional groups on the structure of MO, the effect of the pH on its oxidation behavior was studied. To find the optimum solution pH, the influence of the pH (between 3.0 and 10.0, using phosphate buffers) on peak current of a solution of 36 μM MO was studied (Fig. 6). The maximum peak current was obtained in pH 7 and thus this pH was selected for subsequent uses. Furthermore, our study showed that by increasing the pH the anodic peak potential of MO was shifted towards less positive values. The variations of anodic peak potential relative to pH of the solution were linear in the studied pH range (inset of Fig. 6). The linear regression equations for anodic peak are given by: Epa ðVÞ ¼ −0:0507pH þ 0:757
r2 ¼ 0:9948 :
The slope value of about −50 mV/pH was obtained which is nearly equal to the theoretical value of 59 mV suggesting the participation of equal number of protons and electrons in the oxidation of MO at the modified CPE. 3.4. The effect of the scan rate
Fig. 2. EIS for (a) CPE and (b) CMNP modified carbon paste electrode in 5 mM [Fe(CN)6]3−/4− with 0.1 M KCl.
To understand the reaction mechanism, the effect of scan rates on the peak currents of MO at the modified electrode was investigated. Therefore, the cyclic voltammograms of 50 μM MO at different scan rates including 50, 75, 100, 125 and 150 m Vs− 1 were investigated at the CMNP–CPE (Fig. 7). As shown in Fig. 7A, a linear relationship (Y = 2.5342v1/2–6.251 (Ip: μA, v: m Vs−1)) with a correlation
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Fig. 3. SEM images of (A) bare CPE, (B) MNP–CPE and CMNP–CPE.
coefficient of R = 0.9982 was obtained between the peak current and the square root of scan rate in the range of 50–150 m Vs− 1, which revealed that the oxidation of MO was rather a diffusion-controlled step than an adsorption controlled process. In the experiment, the relationship between the oxidation peak potentials and logarithm of scan rates can be described as follows: Epa = 0.0986 log v + 0.192, r = 0.9925. According to Laviron's theory [45], the slope was equal to 2.303RT/αnαF. Then the value of αnα was found to be 0.59. Thus, the rate-limiting step was obtained to be oneelectron transfer process when the transfer coefficient (α) was assumed to be 0.5. Since the equal numbers of electron and proton took part in the oxidation of MO, therefore, one electron and one proton transfer were involved in the electrode reaction process. These observations confirm the presented mechanism in Fig. 5.
3.5. Determination of electrochemical active surface area In order to measure the electrochemically active surface area of the bare and modified electrodes, the chronoamperogram of 0.1 mM potassium ferrocyanide as a redox probe was recorded. In chronoamperometric studies, the current, i, for the electrochemical reaction (at a mass-transfer-limited rate) of ferrocyanide which diffuses to an electrode surface is described by Cottrell equation [46]: i¼
1 nFAD =2 C
π
1= 1= 2 2
t
where A is the electrochemical active area, D (6.20 × 10−6 cm2 s−1) is the diffusion coefficient, C* (0.1 mM) is the bulk concentration of
Fig. 4. Cyclic voltammograms: (A) CMNP–CPE a) in the absence and b) in the presence of 50 μM MO; and (B) a) CPE, b) MNP–CPE, and c) CMNP–CPE in the presence of 50 μM MO. Measurement conditions: 0.1 M phosphate buffer (pH 7) and scan rate = 100 mV s−1.
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Fig. 5. Mechanism oxidation of MO.
ferrocyanide and the other parameters have their typical meanings. Under diffusion control, a plot of i vs. t−1/2 will be linear and from the slope, the value of A can be obtained. Chronoamperometry was also used for the determination of diffusion coefficient, D, of MO. The chronoamperograms were obtained at a fixed potential of 0.38 V over 40 s in phosphate buffer (pH 7.0) containing different concentrations of MO (Fig. 8A). According to Cottrell equation, D values were estimated from the slope of a plot of i vs. t−1/2 at different MO concentrations (Fig. 8B), the slopes of the resulting straight lines were then plotted vs. the MO concentration (Fig. 8C). An average value of D = 9.17 × 10−5 cm2 s−1 was obtained from the slopes.
3.6. Optimization of the amount of modifier in the electrode Primary experiments show that the amount of CMNP modifier in carbon paste electrode influences on the anodic peak current of MO. Therefore, five electrodes containing different percent of CMNP (0, 5, 10, 15 and 20%) were prepared and examined for detection of 50 μM MO under identical conditions (Fig. 9). As it is seen from the inset of Fig. 9, the peak current was increased up to 10% of the modifier (CMNP) and then decreased with further increasing in its amount that may be due to lower conductivity of the resulted paste. Therefore, the maximum sensitivity was obtained when the percent of the graphite powder, paraffin oil and CMNP was 60:30:10% (w/w) in the paste.
Fig. 6. Effect of pH on cyclic voltammograms of 0.1 M phosphate buffer solution containing 36 μM MO at 100 m Vs−1. Inset: variation of the peak potential with pH.
Fig. 7. Cyclic voltammograms of 0.1 M phosphate buffer (pH 7) containing 50 μM MO at different scan rates. The numbers of 1–5 correspond to 50, 75, 100, 125 and 150 mV s−1 respectively. Inset A: variation of the Ip with ʋ1/2. Inset B: variation of the Ep versus logʋ.
3.7. Analytical performance In order to increase the sensitivity of the proposed method, differential pulse voltammetry (DPV) was used for monitoring of MO. Therefore, the main important parameters such as pulse amplitude, pulse width and scan rate were optimized. During the study, each variable was changed while the other two were kept constant. The variables of interest were studied over the ranges 25–100 mV, 30–100 ms and 10–100 m Vs−1 for pulse amplitude, pulse width and scan rate respectively. The best sensitivity and well-shaped wave with relatively narrow
Fig. 8. A: Chronoamperograms of 0.1 M phosphate buffer (pH 7.0) containing various concentrations of MO: (1) 10, (2) 200, (3) 350, (4) 450, (5) 500 and (6) 550 μM. B: Plot of I vs. t−1/2 for different concentrations of MO. C: The plot of the slope of straight lines against the MO concentration.
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Fig. 11. DPV of 0.1 M phosphate buffer pH 7 containing 0.5 μM of MO in the absence (dashed line) and presence 0.5 μM DA (solid line).
Fig. 9. DPVs of 50 μM MO in 0.1 M phosphate buffer (pH 7) at the surface of the modified electrodes containing different CMNP% (a: 0%, b: 5%, c: 10%, d: 15% and e: 20%). Inset shows the plot of the peak current as a function of amount of CMNP (%).
peak width were obtained when the values of 50 mV, 40 ms and 60 m Vs− 1 were chosen for pulse amplitude, pulse width and scan rate, respectively. Optimal conditions were used for the determination of MO. The DPV signals obtained for tested different concentrations of MO as well as the plotted calibration curve are shown in Fig. 10. Two linear relationships are obtained over MO concentrations ranging from 0.01 to 2 (Fig. 10, inset A) and 2 to 720 μM respectively (Fig. 10, inset B). The linear regression equations are: IP ðμAÞ ¼ 1:4687 þ 27:276CMO ðμMÞ
r2 ¼ 0:9956
IP ðμAÞ ¼ 55:607 þ 0:1136CMO ðμMÞ r2 ¼ 0:998
A
B:
The limits of detection (LOD) and quantitation (LOQ) were calculated using the relation ks/m [47], where k = 3 for LOD and 10 for LOQ, s represents the standard deviation of the peak current of the blank (n = 10) and m represents the slope of the first calibration curve for MO. Both
LOD and LOQ values were found to be 3 and 10 nM respectively, in which these values indicated the sensitivity of the proposed method. Repeatability was examined by performing 10 replicate measurements for 20 μM MO. The recovery of analyzed target was calculated to be above 97.5%, and the relative standard deviation (RSD) was lower than 3.6%. This level of precision is suitable for the routine quality control analysis of the MO in biological fluids. Reproducibility of proposed electrode was investigated by using DPV. Three freshly packed electrodes were prepared on five consecutive days and the peak current values of a solution containing 10 μM of MO were measured for each electrode. The obtained results of five replicate measurements show a standard deviation less than ± 0.98 for DPV currents. This result indicates the acceptable reproducibility for the proposed electrode. The long term stability of the modified carbon paste electrode was also studied. The proposed electrode retained 97% of its initial activity after one month, demonstrating its stability.
3.8. Interference studies For possible analytical application of the method, the effects of some common interferences on the determination of 10 μM MO was examined. The tolerance limit was defined as the maximum concentration of the substances that caused an error of less than 5% in MO determination. The results showed that when a 1000-fold Na+, K+, Mg2+, Ca2+, Cl−, Li+, Al3 +, NH+ 4 , tryptophan, histidine, glycine, urea, dopamine and thiourea were present in the synthetic mixture, the average recovery in the determination of MO was 101.5%. For example, DP voltammograms of 0.5 μM MO and 0.5 μM dopamine (DA) mixtures were shown in Fig. 11. As can be seen in this figure, MO and DA yielded two welldefined oxidation peaks. From the results, the selectivity of the method was acceptable.
Table 1 Voltammetric determination of MO in real samples and recovery data obtained (n = 5). Sample
Added (μM)
Found (μM)
Recovery (%)
RSD (%)
Urine
5 10 15 20 25 10 20 30 40 50
4.8 10.6 14.3 20.8 24.9 10.3 20.5 29.1 38.5 52
96 106 95 104 99 103 102 97 96 104
2.3 3.7 2.6 3.4 2.8 3.8 3.9 2.4 3.7 2.5
Serum Fig. 10. DPVs 0.1 M phosphate buffer (pH 7) containing different concentrations of MO. Insets show the plots of the peak current as a function of MO concentration in the range of (A) 0.01–2 μM and (B) 2–720 μM.
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Table 2 Comparison of the performances of various electrodes for MO determination. Electrode
Method
Linear range (μM)
Detection limit (μM)
Real sample
Ref
Ordered mesoporous carbon modified glassy carbon electrode
0.1 to 20
0.01
Urine
[1]
0.4 to 200
0.004 7.83
Multi-walled carbon nanotube/chitosan composite modified glassy carbon electrode Gold nanoparticles–ferrocene modified carbon paste electrode ZnO/CNT nanocomposite room temperature ionic liquid modified carbon paste electrode Electrochemically reduced MWNT-doped graphene oxide (ER-MWNT-doped-GO) composite film modified glassy carbon electrode This work
DPV Chronoamperometry DPV Square wave voltammetry (SWV) Linear sweep voltammetry (LSV) DPV
23.5 to 2390 2 to 100 1 to 280 1 to 1800 0.1 to 700
Morphine sulfate tablet and urine Human serum
[18]
Pt–Co Alloy Nanowire Array-modified Electrode
cyclic voltammetry (CV) Differential pulse voltammetry (DPV) Chronoamperometry
0.24 0.4 0.0035 0.06
Human serum and urine
[20] [19] [17]
0.07–6.5
0.05
Urine Pharmaceutical (injection solution) and urine Human serum and urine
0.01–720
0.003
Human serum and urine
–
Gold Nanoparticles Modified Carbon Paste Electrode
3.9. Analytical application In order to demonstrate the application of the proposed method for voltammetric determination of MO in real sample, it was used for voltammetric determination of MO in serum and urine samples. After sample preparation as described in Section 2.7, the DPV method was applied to the MO determination by the standard addition method and the results were summarized in Table 1. The results in Table 1 show that the recoveries of the spiked samples are acceptable. Thus the modified electrode can be efficiently used for the determination of MO in real samples with different matrices. 4. Conclusions CMNP as a modifier was incorporated in carbon paste electrode for the determination of MO. It was more sensitive for a CMNP modified electrode to detect MO than a bare electrode. In comparison to other MO electrochemical sensors that have been reported, the proposed sensor has the lowest limit of detection (Table 2). The proposed method allows the determination of low levels of MO using DPV method. It was successfully applied for the determination of MO in serum and urine samples. This sensor has been shown to be promising for the determination of MO with many desirable properties including, simple fabrication procedure, high stability, good reproducibility and repeatability, high sensitivity and fast response time. Conflict of interest The authors declare that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Acknowledgments The authors gratefully acknowledge the financial support of this work by the Razi University Research. References [1] F. Li, J. Song, C. Shan, D. Gao, X. Xu, L. Niu, Biosens. Bioelectron. 25 (2010) 1408–1413. [2] H.-M. Lee, C.-W. Lee, J. Anal. Toxicol. 15 (1991) 182–187. [3] G. Chari, A. Gulati, R. Bhat, I.R. Tebbett, J. Chromatogr. B Biomed. Sci. Appl. 571 (1991) 263–270. [4] F. Tagliaro, D. Franchi, R. Dorizzi, M. Marigo, J. Chromatogr. B Biomed. Sci. Appl. 488 (1989) 215–228. [5] V.S.M.E. Soares, M.L. Bastos, J. Liq. Chromatogr. (1992) 1533.
[21]
[16]
[6] J. Guillot, M. Lefebvre, J. Weber, J. Anal. Toxicol. 21 (1997) 127–133. [7] R. Dams, T. Benijts, W. Lambert, A. De Leenheer, J. Chromatogr. B 773 (2002) 53–61. [8] S.W. Lewis, P.S. Francis, K.F. Lim, G.E. Jenkins, X.D. Wang, Analyst 125 (2000) 1869–1874. [9] G. Sakai, K. Ogata, T. Uda, N. Miura, N. Yamazoe, Sensors Actuators B Chem. 49 (1998) 5–12. [10] B. Qiu, X. Chen, H.L. Chen, G.N. Chen, Luminescence 22 (2007) 189–194. [11] F. Xu, M. Gao, L. Wang, T. Zhou, L. Jin, J. Jin, Talanta 58 (2002) 427–432. [12] K.-C. Ho, C.-Y. Chen, H.-C. Hsu, L.-C. Chen, S.-C. Shiesh, X.-Z. Lin, Biosens. Bioelectron. 20 (2004) 3–8. [13] C.-H. Weng, W.-M. Yeh, K.-C. Ho, G.-B. Lee, Sensors Actuators B Chem. 121 (2007) 576–582. [14] M. Pournaghi-Azar, A. Saadatirad, J. Electroanal. Chem. 624 (2008) 293–298. [15] A. Salimi, R. Hallaj, G.R. Khayatian, Electroanalysis 17 (2005) 873–879. [16] Y. Li, L. Zou, Y. Li, K. Li, B. Ye, Sensors Actuators B Chem. 201 (2014) 511–519. [17] E. Afsharmanesh, H. Karimi-Maleh, A. Pahlavan, J. Vahedi, J. Mol. Liq. 181 (2013) 8–13. [18] N.F. Atta, A. Galal, S.M. Azab, Int. J. Electrochem. Sci. 6 (2011). [19] N.F. Atta, A. Galal, A.A. Wassel, A.H. Ibrahim, Int. J. Electrochem. Sci. 7 (2012). [20] A. Babaei, M. Babazadeh, H. Momeni, Int. J. Electrochem. Sci. 6 (2011) 1382–1395. [21] M. Tao, F. Xu, Y. Li, Q. Xu, Y. Chang, Z. Wu, Y. Yang, Bull. Kor. Chem. Soc. 31 (2010) 1968–1972. [22] N.F. Atta, A. Galal, R.A. Ahmed, Electroanalysis 23 (2011) 737–746. [23] G. Lu, X. Yao, X. Wu, T. Zhan, Microchem. J. 69 (2001) 81–87. [24] M. Zhang, W. Gorski, Anal. Chem. 77 (2005) 3960–3965. [25] M. Zhang, A. Smith, W. Gorski, Anal. Chem. 76 (2004) 5045–5050. [26] X. Wei, J. Cruz, W. Gorski, Anal. Chem. 74 (2002) 5039–5046. [27] D.S. dos Santos, A. Riul, R.R. Malmegrim, F.J. Fonseca, O.N. Oliveira, L.H. Mattoso, Macromol. Biosci. 3 (2003) 591–595. [28] Y. Wang, J. Zhu, R. Zhu, Z. Zhu, Z. Lai, Z. Chen, Meas. Sci. Technol. 14 (2003) 831. [29] H.-S. Wang, Q.-X. Pan, G.-X. Wang, Sensors 5 (2005) 266–276. [30] M.G. Peter, J. Macromol. Sci., Part A: Pure Appl. Chem. 32 (1995) 629–640. [31] P. Norouzi, M. Pirali-Hamedani, M. Ganjali, F. Faridbod, Int. J. Electrochem. Sci. 5 (2010) 1434–1446. [32] H. Huang, N. Hu, Y. Zeng, G. Zhou, Anal. Biochem. 308 (2002) 141–151. [33] G. Wang, J.-J. Xu, L.-H. Ye, J.-J. Zhu, H.-Y. Chen, Bioelectrochemistry 57 (2002) 33–38. [34] J. Abdullah, M. Ahmad, N. Karuppiah, L.Y. Heng, H. Sidek, Sensors Actuators B Chem. 114 (2006) 604–609. [35] X. Tan, J. Zhang, S. Tan, D. Zhao, Z. Huang, Y. Mi, Z. Huang, Electroanalysis 21 (2009) 1514–1520. [36] A. Kaushik, R. Khan, P.R. Solanki, P. Pandey, J. Alam, S. Ahmad, B. Malhotra, Biosens. Bioelectron. 24 (2008) 676–683. [37] Y.-Y. Liang, L.-M. Zhang, W. Li, R.-F. Chen, Colloid Polym. Sci. 285 (2007) 1193–1199. [38] S.-F. Wang, Y.-M. Tan, Anal. Bioanal. Chem. 387 (2007) 703–708. [39] G. Zhao, J.-J. Xu, H.-Y. Chen, Electrochem. Commun. 8 (2006) 148–154. [40] M.S. Lin, H.J. Leu, Electroanalysis 17 (2005) 2068–2073. [41] J. Hrbac, V. Halouzka, R. Zboril, K. Papadopoulos, T. Triantis, Electroanalysis 19 (2007) 1850–1854. [42] H.L. Zhang, X.Z. Zou, G.S. Lai, D.Y. Han, F. Wang, Electroanalysis 19 (2007) 1869–1874. [43] A. Kaushik, P.R. Solanki, A.A. Ansari, S. Ahmad, B.D. Malhotra, Electrochem. Commun. 10 (2008) 1364–1368. [44] Z. Lei, X. Pang, N. Li, L. Lin, Y. Li, J. Mater. Process. Technol. 209 (2009) 3218–3225. [45] E. Laviron, J. Electroanal. Chem. Interfacial Electrochem. 52 (1974) 355–393. [46] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 2001. [47] The United States Pharmacopoeia, The National Formulary, USP 24, NF 19, USP Convention Inc., MD, 2000, p. 2151.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Construction of a sensitive and selective sensor for morphine using chitosan coated Fe3O4 magnetic nanoparticle as a modifier Sara Dehdashtian a, Mohammad Bagher Gholivand b,⁎, Mojtaba Shamsipur b, Samira kariminia b a b
Department of Chemistry, Behbahan Khatam Al-Anbia University of Technology, Behbahan, Iran Department of Analytical Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Iran
a r t i c l e
i n f o
Article history: Received 12 February 2015 Received in revised form 17 April 2015 Accepted 23 July 2015 Available online 28 July 2015 Keywords: Morphine Chitosan-coated magnetic nanoparticle Modified carbon paste electrode Differential pulse voltammetry Cyclic voltammetry Electrochemical impedance spectroscopy Chronoamperometry
a b s t r a c t A simple and sensitive sensor based on carbon paste electrode (CPE) modified by chitosan-coated magnetic nanoparticle (CMNP) was developed for the electrochemical determination of morphine (MO). The proposed sensor was characterized with scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The electrooxidation of MO was studied on modified carbon paste electrode using cyclic voltammetry, chronoamperometry and differential pulse voltammetry as diagnostic techniques. The oxidation peak potential of morphine on the CMNP/CPE appeared at 380 mV which was accompanied with smaller overpotential and increase in oxidation peak current compared to that obtained on the bare carbon paste electrode (CPE). Under optimum conditions the sensor provides two linear DPV responses in the range of 10–2000 nM and 2–720 μM for MO with a detection limit of 3 nM. The proposed sensor was successfully applied for monitoring of MO in serum and urine samples and satisfactory results were obtained. © 2015 Published by Elsevier B.V.
1. Introduction Morphine (MO) as a major component in opium is frequently used to relieve severe pain for patients, but when overdosed or abused, it is toxic and can cause disruption in the central nervous system. It is recommended by the World Health Organization (WHO) for the relief of moderate cancer-related pain [1]. Morphine is a precursor in the manufacture of a large number of opioids such as dihydromorphine, hydromorphone, nicomorphine, and heroin as well as codeine. Therefore, in order to prevent the toxicity, induced by overdosing or abusing, it is necessary to determine the concentrations of MO in patient's blood or urine using a sensitive method. Different analytical methods have been used for the determination of morphine in plasma, urine, and opium samples, such as gas chromatography (GC) [2], liquid chromatography (LC) [3], high performance liquid chromatography (HPLC) [4], ultraviolet (UV) spectroscopy [5], GC–mass spectroscopy (GC–MS) [6], fluorimetry [7], chemiluminescence [8], surface plasmon resonance (SPR) [9], radioimmunoassay (RIA) [7], and electrochemiluminescence (ECL) [10]. These methods
Abbreviations: MO, morphine; CPE, carbon paste electrode; CMNP, chitosan-coated magnetic nanoparticle; CV, cyclic voltammetry; DPV, differential pulse voltammetry; SEM, scanning electron microscopy; EIS, electrochemical impedance spectroscopy; LOD, limit of detection; LOQ, limit of quantitation; RSD, relative standard deviation. ⁎ Corresponding author. E-mail address: [email protected] (M.B. Gholivand).
http://dx.doi.org/10.1016/j.msec.2015.07.049 0928-4931/© 2015 Published by Elsevier B.V.
require advanced technical expertise and are expensive and timeconsuming. The electrochemical techniques as alternative methods have also received much interest due to their higher selectivity, lower cost and faster operation and thus, have become of growing importance in many fields including medicine and biotechnology, environmental monitoring, and different applications in industrial process control. Due to slow electron transfer reaction and electrode-fouling problem of most bare electrodes; they must be modified with suitable materials. Therefore, different modifiers such as cobalt hexacyanoferrate [11], Prussian blue-modified indium tin oxide (ITO) [12], molecularly imprinted polymer films [13], palladized aluminum [14], multiwalled carbon [15], MWNT-doped graphene oxide composite film [16], ZnO/ CNT nanocomposite [17], gold nanoparticles [18], ferrocene/gold nanoparticles [19], carbon nanotube/chitosan [20], Pt–Co alloy nanowire [21] and poly(3,4-ethylenedioxythiophene) [22] have been used for the morphine detection. Chitosan (CHT), as a natural polymer with abundant primary amino groups and hydroxyl groups, is non-toxic, readily decomposes in natural biological environments, and is compatible with living organism cells (both plant and animal) and displays a number of properties including hydrophilicity, gel-forming ability, doping feasibility, good mechanical stability, good permeability, cost-effectiveness, and availability of reactive functional groups for chemical modifications. CHT can effectively adsorb not only metal ions but also various organic compounds [23] and thus, can be widely used in construction of electrochemical sensors and biosensors [24–29]. To refine the electric conductivity of CHT, some
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materials such as metal nanoparticles, carbon nanotubes and redox mediators have been introduced to CHT films [30–35]. In the present study, metal oxide nanoparticles were used for improvement in the conductivity of CHT and enhancement in the sensitivity of the sensor. Furthermore, metal oxide nanoparticles can be helpful to obtain improved stability and sensitivity of a sensor, because they exhibit large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, strong adsorption ability and unique ability to promote fast electron transfer between electrode and target. Among them, Fe3O4 nanoparticles, with low toxicity are of interest due to its biocompatibility. Combination of CHT and Fe3O4 nanoparticles produces a nanocomposite that not only overcomes aggregation and rapid biodegradation of Fe3O4 nanoparticles by CHT [36–43] but dispersing of super paramagnetic Fe3O4 nanoparticles improves the optical and electrical properties of CHT for biosensor application [36,43]. Both metal oxide nanoparticles and chitosan can be used for removal of heavy metals [23] from aqueous environments and modification of electrode surface [24–29]. In this study, a simple and sensitive sensor based on carbon paste (CP) electrode modified by chitosan-coated magnetic nanoparticle was developed for the electrochemical determination of morphine in real samples. 2. Experimental
30 ml of chitosan solution. Then 30 ml of aqueous ammonia solution (25%) was very slowly added to the above solution for producing smaller sized nanoparticles. The resulting solution was stirred for an additional 30 min. The colloidal chitosan coated magnetic Fe3O4 nanoparticles were extensively washed with deionized water and separated by magnetic decantation for several times. Finally, the chitosan-coated MNPs were dried at 50 °C and stored at room temperature. 2.4. Characterization of chitosan coated magnetic nanoparticles (CMNPs) by Fourier transform infrared spectroscopy (FTIR) To confirm the chemical composition of synthesized nanoparticles, FTIR spectra were obtained. For the naked Fe3O4 (Fig. 1(a)), the peak at 579 cm−1 relates to Fe–O group in Fe3O4. However, from Fig. 1(b), it can be seen that the characteristic peak of Fe–O shifts to 581 cm−1 revealing the interaction between Fe3O4 and CHT. Compared with Fe3O4 nanoparticles, the spectrum of CHT–Fe3O4 has characteristic peak of CHT at 1070 cm−1 (C–N stretching vibrations) and 1636 cm−1 (N–H bending vibration). The results indicated that CHT was coated to the magnetic Fe3O4 nanoparticles successfully. Because the surface of iron oxide with negative charges has an affinity towards CHT, protonated CHT could coat the magnetite nanoparticles by the electrostatic interaction.
2.1. Apparatus and chemicals 2.5. Preparation of modified electrode Cyclic voltammetric (CV) and differential pulse voltammetric (DPV) experiments were performed using the μ-Autolab electrochemical system (Eco-Chemie, Utrecht, The Netherlands) equipped with GPES and FRA 4.9 software coupled with a conventional three-electrode cell. The working electrode was an unmodified or modified carbon paste electrode; the auxiliary and reference electrodes were a platinum wire and SCE respectively. A Metrohm 710 pH meter was used for pH measurements. The surface morphology of modified electrodes was characterized with a scanning electron microscope (SEM) (Philips XL 30). All experiments were carried out at ambient temperature (25 ± 1 °C). Morphine sulfate was used as received. Chitosan with low molecular weight and MO were of analytical grade from Sigma-Aldrich. Ferrous chloride tetrahydrate (FeCl2·4H2O) and ferric chloride hexahydrate (FeCl3·6H2O) were purchased from Sigma-Aldrich. The buffer solutions were prepared from orthophosphoric acid and its salts in the pH range of 2.0–10.0. All other reagents were of analytical grade and solutions were freshly prepared with double distilled water.
The unmodified and modified carbon pastes were prepared by thoroughly mixing analytical grade graphite and paraffin oil in the absence and presence of modifier and then each paste was mixed in a mortar for at least 10 min to become homogeneous. Each paste was packed into one end of a Teflon holder in which electrical contact was made with a copper rod that runs through the center of the electrode body. The surface of each electrode was polished using a butter paper to produce reproducible working surface and then was used for electrochemical studying of MO by voltammetric techniques. The unmodified carbon paste was prepared by mixing 70:30% of graphite and paraffin oil respectively. The modified carbon paste was prepared by mixing different percentages of graphite powder, paraffin oil, and modifier and the best results were obtained at 60:30:10% of graphite, paraffin oil and modifier. The modified electrode was activated in a 10 ml of 0.1 M phosphate buffer (pH 7) by potential scanning from − 0.25 to 1 V vs. SCE at a scan rate of 100 mV s−1, until a low and steady background was obtained.
2.2. Synthesis of Fe3O4 magnetic nanoparticles (MNPs) Magnetic iron oxide (Fe3O4) nanoparticles were synthesized by modified coprecipitation method [44]. Stoichiometric ratio of 1:2 ferrous chloride tetrahydrate (FeCl2·4H2O) and ferric chloride hexahydrate (FeCl3·6H2O) was dissolved in deionized water under nitrogen atmosphere with vigorous stirring at 70 °C. Chemical precipitation was achieved by dropwise adding NH4OH solution (25%) to reach pH 10 under vigorous stirring. The color of solution was turned from dark orange to the black immediately. After continuously stirring for 1 h, the black precipitates of Fe3O4 were magnetically decanted and washed several times with distilled water. The powder was then dried at 50 °C in an oven for 24 h. 2.3. Preparation of chitosan coated magnetic nanoparticles (CMNPs) Chitosan coated magnetic iron oxide nanoparticles were in situ synthesized by the co-precipitation of Fe (II) and Fe (III) salts in the presence of chitosan. Chitosan (0.125 g) was dissolved in 30 ml of 1% acetic acid and the pH was adjusted to 4.8 by 10 M NaOH. Under the nitrogen (N2) gas flow and by vigorously stirring at 50 °C, iron salts (1.34 g of FeCl2·4H2O and 3.40 g of FeCl3·6H2O) were dissolved in
Fig. 1. Fourier transform infrared spectra of (a) naked Fe3O4 and (b) chitosan coated Fe3O4.
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2.6. Analytical procedure 10 ml of 0.1 M phosphate buffer (pH 7) was introduced in a voltammetric cell and its DP voltammogram under optimum conditions (50 mV pulse amplitude, 40 ms pulse width and 60 m Vs−1 scan rate) in the potential range of 0.0 to 0.7 V was used for background correction. Then different volumes of sample solution were added to the above solution and their voltammograms were used for calibration plot. For recording the cyclic voltammograms, the potential was scanned from the 0 to 0.7 V using the scan rate of 100 m Vs−1. 2.7. Preparation of serum sample For the determination of MO in human serum sample, a serum sample of a healthy volunteer was stored frozen until assay. 2 ml of methanol was added to a 1.5 ml serum sample to remove serum protein. After vortexing of the serum sample for 2 min, the precipitated proteins were separated by centrifugation for 3 min at 6000 rpm. The clear supernatant layer was filtered and diluted to a definite volume using double distilled water. The urine sample used for measurement was centrifuged and diluted 10 times using double distilled water without any further pretreatment. For both real samples, known amounts of analyte were added to phosphate buffer solution (pH 7) containing deliberate amounts of real sample. 3. Results and discussion 3.1. Characterization of the modified electrode Electrochemical impedance spectroscopy (EIS) is an effective method for probing the features of surface modified electrodes. The Nyquist plot of impedance spectra includes a semicircle portion and a linear portion, with the former at higher frequencies corresponding to the electron transfer limited process and the latter at lower frequencies corresponding to the diffusion process. The electron transfer resistance (Rct) at the electrode surface is equal to the semicircle diameter, which can be used to describe the interface properties of the electrode. Fig. 2 presents the Nyquist diagrams of the bare CPE (curve a with Ret = 139.0 Ω) and CMNP–CPE (curve b with Ret = 77 Ω) in 5 mM [Fe(CN)6]3−/4 − and 0.1 M KCl. CMNP–CPE shows a small semicircle at the high frequency region when compared with the bare CPE, indicating lower electron transfer resistance. This can be attributed to the presented CMNP with good conductivity and large surface area in the modified electrode, which could effectively increase the rate electron transfer between
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electrode surface and [Fe(CN)6]3−/4− and decrease interface electron transfer resistance. The response of a modified electrode is related to its physical morphology. The SEM of bare and modified CPEs was shown in Fig. 3. Significant differences in the surface structure of these electrodes are observed. The surface of the bare CPE was predominated by isolated and irregularly shaped graphite flakes and separated layers were seen (Fig. 3A). The SEM image of MNP and CMNP–CPEs showed more uniform surface and no separated carbon layers could be observed in Fig. 3B and C. 3.2. Electrochemical behavior of MO at CMNP–CPE The electrochemical study of phosphate buffer solution (0.1 M, pH 7) in the absence (curve a) and presence (curve b) of 50 μM of MO at CMNP–CPE was performed using cyclic voltammetry. As can be seen from Fig. 4A in the presence of MO a well define anodic peak was observed at 0.38 V without any counterpart cathodic peak in the reversed scan indicating that the electrodic process is irreversible. As reported by Li et al., the observed oxidation peak could be ascribed to the oxidation of the phenolic group, which leads to the formation of pseudomorphine [1] (Fig. 5). The cyclic voltammetric response of MO at the CMNP–CPE was compared with those obtained at CP and MNP–CP electrodes and the results are presented in Fig. 4B. As it is shown, the oxidation of MO at the bare carbon paste electrode produces a week anodic peak at 0.44 V (curve a). At the surface of the CPE modified by MNP (MNP–CPE), the current associated with anodic peak of MO is increased in comparison with that obtained on CPE without any significant change in its potential. Modification of CPE by CMNP not only increases the peak current (almost three folds greater) but decreases the overvoltage accompanied by anodic peak of MO and shifts its peak potential towards less positive value. The absence of counterpart cathodic peak of the oxidation of MO in its cyclic voltammograms (CVs) at the surface of all tested electrodes reveals that the electrochemical reaction of MO is totally irreversible process. 3.3. The effect of pH Because of individual functional groups on the structure of MO, the effect of the pH on its oxidation behavior was studied. To find the optimum solution pH, the influence of the pH (between 3.0 and 10.0, using phosphate buffers) on peak current of a solution of 36 μM MO was studied (Fig. 6). The maximum peak current was obtained in pH 7 and thus this pH was selected for subsequent uses. Furthermore, our study showed that by increasing the pH the anodic peak potential of MO was shifted towards less positive values. The variations of anodic peak potential relative to pH of the solution were linear in the studied pH range (inset of Fig. 6). The linear regression equations for anodic peak are given by: Epa ðVÞ ¼ −0:0507pH þ 0:757
r2 ¼ 0:9948 :
The slope value of about −50 mV/pH was obtained which is nearly equal to the theoretical value of 59 mV suggesting the participation of equal number of protons and electrons in the oxidation of MO at the modified CPE. 3.4. The effect of the scan rate
Fig. 2. EIS for (a) CPE and (b) CMNP modified carbon paste electrode in 5 mM [Fe(CN)6]3−/4− with 0.1 M KCl.
To understand the reaction mechanism, the effect of scan rates on the peak currents of MO at the modified electrode was investigated. Therefore, the cyclic voltammograms of 50 μM MO at different scan rates including 50, 75, 100, 125 and 150 m Vs− 1 were investigated at the CMNP–CPE (Fig. 7). As shown in Fig. 7A, a linear relationship (Y = 2.5342v1/2–6.251 (Ip: μA, v: m Vs−1)) with a correlation
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Fig. 3. SEM images of (A) bare CPE, (B) MNP–CPE and CMNP–CPE.
coefficient of R = 0.9982 was obtained between the peak current and the square root of scan rate in the range of 50–150 m Vs− 1, which revealed that the oxidation of MO was rather a diffusion-controlled step than an adsorption controlled process. In the experiment, the relationship between the oxidation peak potentials and logarithm of scan rates can be described as follows: Epa = 0.0986 log v + 0.192, r = 0.9925. According to Laviron's theory [45], the slope was equal to 2.303RT/αnαF. Then the value of αnα was found to be 0.59. Thus, the rate-limiting step was obtained to be oneelectron transfer process when the transfer coefficient (α) was assumed to be 0.5. Since the equal numbers of electron and proton took part in the oxidation of MO, therefore, one electron and one proton transfer were involved in the electrode reaction process. These observations confirm the presented mechanism in Fig. 5.
3.5. Determination of electrochemical active surface area In order to measure the electrochemically active surface area of the bare and modified electrodes, the chronoamperogram of 0.1 mM potassium ferrocyanide as a redox probe was recorded. In chronoamperometric studies, the current, i, for the electrochemical reaction (at a mass-transfer-limited rate) of ferrocyanide which diffuses to an electrode surface is described by Cottrell equation [46]: i¼
1 nFAD =2 C
π
1= 1= 2 2
t
where A is the electrochemical active area, D (6.20 × 10−6 cm2 s−1) is the diffusion coefficient, C* (0.1 mM) is the bulk concentration of
Fig. 4. Cyclic voltammograms: (A) CMNP–CPE a) in the absence and b) in the presence of 50 μM MO; and (B) a) CPE, b) MNP–CPE, and c) CMNP–CPE in the presence of 50 μM MO. Measurement conditions: 0.1 M phosphate buffer (pH 7) and scan rate = 100 mV s−1.
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Fig. 5. Mechanism oxidation of MO.
ferrocyanide and the other parameters have their typical meanings. Under diffusion control, a plot of i vs. t−1/2 will be linear and from the slope, the value of A can be obtained. Chronoamperometry was also used for the determination of diffusion coefficient, D, of MO. The chronoamperograms were obtained at a fixed potential of 0.38 V over 40 s in phosphate buffer (pH 7.0) containing different concentrations of MO (Fig. 8A). According to Cottrell equation, D values were estimated from the slope of a plot of i vs. t−1/2 at different MO concentrations (Fig. 8B), the slopes of the resulting straight lines were then plotted vs. the MO concentration (Fig. 8C). An average value of D = 9.17 × 10−5 cm2 s−1 was obtained from the slopes.
3.6. Optimization of the amount of modifier in the electrode Primary experiments show that the amount of CMNP modifier in carbon paste electrode influences on the anodic peak current of MO. Therefore, five electrodes containing different percent of CMNP (0, 5, 10, 15 and 20%) were prepared and examined for detection of 50 μM MO under identical conditions (Fig. 9). As it is seen from the inset of Fig. 9, the peak current was increased up to 10% of the modifier (CMNP) and then decreased with further increasing in its amount that may be due to lower conductivity of the resulted paste. Therefore, the maximum sensitivity was obtained when the percent of the graphite powder, paraffin oil and CMNP was 60:30:10% (w/w) in the paste.
Fig. 6. Effect of pH on cyclic voltammograms of 0.1 M phosphate buffer solution containing 36 μM MO at 100 m Vs−1. Inset: variation of the peak potential with pH.
Fig. 7. Cyclic voltammograms of 0.1 M phosphate buffer (pH 7) containing 50 μM MO at different scan rates. The numbers of 1–5 correspond to 50, 75, 100, 125 and 150 mV s−1 respectively. Inset A: variation of the Ip with ʋ1/2. Inset B: variation of the Ep versus logʋ.
3.7. Analytical performance In order to increase the sensitivity of the proposed method, differential pulse voltammetry (DPV) was used for monitoring of MO. Therefore, the main important parameters such as pulse amplitude, pulse width and scan rate were optimized. During the study, each variable was changed while the other two were kept constant. The variables of interest were studied over the ranges 25–100 mV, 30–100 ms and 10–100 m Vs−1 for pulse amplitude, pulse width and scan rate respectively. The best sensitivity and well-shaped wave with relatively narrow
Fig. 8. A: Chronoamperograms of 0.1 M phosphate buffer (pH 7.0) containing various concentrations of MO: (1) 10, (2) 200, (3) 350, (4) 450, (5) 500 and (6) 550 μM. B: Plot of I vs. t−1/2 for different concentrations of MO. C: The plot of the slope of straight lines against the MO concentration.
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Fig. 11. DPV of 0.1 M phosphate buffer pH 7 containing 0.5 μM of MO in the absence (dashed line) and presence 0.5 μM DA (solid line).
Fig. 9. DPVs of 50 μM MO in 0.1 M phosphate buffer (pH 7) at the surface of the modified electrodes containing different CMNP% (a: 0%, b: 5%, c: 10%, d: 15% and e: 20%). Inset shows the plot of the peak current as a function of amount of CMNP (%).
peak width were obtained when the values of 50 mV, 40 ms and 60 m Vs− 1 were chosen for pulse amplitude, pulse width and scan rate, respectively. Optimal conditions were used for the determination of MO. The DPV signals obtained for tested different concentrations of MO as well as the plotted calibration curve are shown in Fig. 10. Two linear relationships are obtained over MO concentrations ranging from 0.01 to 2 (Fig. 10, inset A) and 2 to 720 μM respectively (Fig. 10, inset B). The linear regression equations are: IP ðμAÞ ¼ 1:4687 þ 27:276CMO ðμMÞ
r2 ¼ 0:9956
IP ðμAÞ ¼ 55:607 þ 0:1136CMO ðμMÞ r2 ¼ 0:998
A
B:
The limits of detection (LOD) and quantitation (LOQ) were calculated using the relation ks/m [47], where k = 3 for LOD and 10 for LOQ, s represents the standard deviation of the peak current of the blank (n = 10) and m represents the slope of the first calibration curve for MO. Both
LOD and LOQ values were found to be 3 and 10 nM respectively, in which these values indicated the sensitivity of the proposed method. Repeatability was examined by performing 10 replicate measurements for 20 μM MO. The recovery of analyzed target was calculated to be above 97.5%, and the relative standard deviation (RSD) was lower than 3.6%. This level of precision is suitable for the routine quality control analysis of the MO in biological fluids. Reproducibility of proposed electrode was investigated by using DPV. Three freshly packed electrodes were prepared on five consecutive days and the peak current values of a solution containing 10 μM of MO were measured for each electrode. The obtained results of five replicate measurements show a standard deviation less than ± 0.98 for DPV currents. This result indicates the acceptable reproducibility for the proposed electrode. The long term stability of the modified carbon paste electrode was also studied. The proposed electrode retained 97% of its initial activity after one month, demonstrating its stability.
3.8. Interference studies For possible analytical application of the method, the effects of some common interferences on the determination of 10 μM MO was examined. The tolerance limit was defined as the maximum concentration of the substances that caused an error of less than 5% in MO determination. The results showed that when a 1000-fold Na+, K+, Mg2+, Ca2+, Cl−, Li+, Al3 +, NH+ 4 , tryptophan, histidine, glycine, urea, dopamine and thiourea were present in the synthetic mixture, the average recovery in the determination of MO was 101.5%. For example, DP voltammograms of 0.5 μM MO and 0.5 μM dopamine (DA) mixtures were shown in Fig. 11. As can be seen in this figure, MO and DA yielded two welldefined oxidation peaks. From the results, the selectivity of the method was acceptable.
Table 1 Voltammetric determination of MO in real samples and recovery data obtained (n = 5). Sample
Added (μM)
Found (μM)
Recovery (%)
RSD (%)
Urine
5 10 15 20 25 10 20 30 40 50
4.8 10.6 14.3 20.8 24.9 10.3 20.5 29.1 38.5 52
96 106 95 104 99 103 102 97 96 104
2.3 3.7 2.6 3.4 2.8 3.8 3.9 2.4 3.7 2.5
Serum Fig. 10. DPVs 0.1 M phosphate buffer (pH 7) containing different concentrations of MO. Insets show the plots of the peak current as a function of MO concentration in the range of (A) 0.01–2 μM and (B) 2–720 μM.
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Table 2 Comparison of the performances of various electrodes for MO determination. Electrode
Method
Linear range (μM)
Detection limit (μM)
Real sample
Ref
Ordered mesoporous carbon modified glassy carbon electrode
0.1 to 20
0.01
Urine
[1]
0.4 to 200
0.004 7.83
Multi-walled carbon nanotube/chitosan composite modified glassy carbon electrode Gold nanoparticles–ferrocene modified carbon paste electrode ZnO/CNT nanocomposite room temperature ionic liquid modified carbon paste electrode Electrochemically reduced MWNT-doped graphene oxide (ER-MWNT-doped-GO) composite film modified glassy carbon electrode This work
DPV Chronoamperometry DPV Square wave voltammetry (SWV) Linear sweep voltammetry (LSV) DPV
23.5 to 2390 2 to 100 1 to 280 1 to 1800 0.1 to 700
Morphine sulfate tablet and urine Human serum
[18]
Pt–Co Alloy Nanowire Array-modified Electrode
cyclic voltammetry (CV) Differential pulse voltammetry (DPV) Chronoamperometry
0.24 0.4 0.0035 0.06
Human serum and urine
[20] [19] [17]
0.07–6.5
0.05
Urine Pharmaceutical (injection solution) and urine Human serum and urine
0.01–720
0.003
Human serum and urine
–
Gold Nanoparticles Modified Carbon Paste Electrode
3.9. Analytical application In order to demonstrate the application of the proposed method for voltammetric determination of MO in real sample, it was used for voltammetric determination of MO in serum and urine samples. After sample preparation as described in Section 2.7, the DPV method was applied to the MO determination by the standard addition method and the results were summarized in Table 1. The results in Table 1 show that the recoveries of the spiked samples are acceptable. Thus the modified electrode can be efficiently used for the determination of MO in real samples with different matrices. 4. Conclusions CMNP as a modifier was incorporated in carbon paste electrode for the determination of MO. It was more sensitive for a CMNP modified electrode to detect MO than a bare electrode. In comparison to other MO electrochemical sensors that have been reported, the proposed sensor has the lowest limit of detection (Table 2). The proposed method allows the determination of low levels of MO using DPV method. It was successfully applied for the determination of MO in serum and urine samples. This sensor has been shown to be promising for the determination of MO with many desirable properties including, simple fabrication procedure, high stability, good reproducibility and repeatability, high sensitivity and fast response time. Conflict of interest The authors declare that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Acknowledgments The authors gratefully acknowledge the financial support of this work by the Razi University Research. References [1] F. Li, J. Song, C. Shan, D. Gao, X. Xu, L. Niu, Biosens. Bioelectron. 25 (2010) 1408–1413. [2] H.-M. Lee, C.-W. Lee, J. Anal. Toxicol. 15 (1991) 182–187. [3] G. Chari, A. Gulati, R. Bhat, I.R. Tebbett, J. Chromatogr. B Biomed. Sci. Appl. 571 (1991) 263–270. [4] F. Tagliaro, D. Franchi, R. Dorizzi, M. Marigo, J. Chromatogr. B Biomed. Sci. Appl. 488 (1989) 215–228. [5] V.S.M.E. Soares, M.L. Bastos, J. Liq. Chromatogr. (1992) 1533.
[21]
[16]
[6] J. Guillot, M. Lefebvre, J. Weber, J. Anal. Toxicol. 21 (1997) 127–133. [7] R. Dams, T. Benijts, W. Lambert, A. De Leenheer, J. Chromatogr. B 773 (2002) 53–61. [8] S.W. Lewis, P.S. Francis, K.F. Lim, G.E. Jenkins, X.D. Wang, Analyst 125 (2000) 1869–1874. [9] G. Sakai, K. Ogata, T. Uda, N. Miura, N. Yamazoe, Sensors Actuators B Chem. 49 (1998) 5–12. [10] B. Qiu, X. Chen, H.L. Chen, G.N. Chen, Luminescence 22 (2007) 189–194. [11] F. Xu, M. Gao, L. Wang, T. Zhou, L. Jin, J. Jin, Talanta 58 (2002) 427–432. [12] K.-C. Ho, C.-Y. Chen, H.-C. Hsu, L.-C. Chen, S.-C. Shiesh, X.-Z. Lin, Biosens. Bioelectron. 20 (2004) 3–8. [13] C.-H. Weng, W.-M. Yeh, K.-C. Ho, G.-B. Lee, Sensors Actuators B Chem. 121 (2007) 576–582. [14] M. Pournaghi-Azar, A. Saadatirad, J. Electroanal. Chem. 624 (2008) 293–298. [15] A. Salimi, R. Hallaj, G.R. Khayatian, Electroanalysis 17 (2005) 873–879. [16] Y. Li, L. Zou, Y. Li, K. Li, B. Ye, Sensors Actuators B Chem. 201 (2014) 511–519. [17] E. Afsharmanesh, H. Karimi-Maleh, A. Pahlavan, J. Vahedi, J. Mol. Liq. 181 (2013) 8–13. [18] N.F. Atta, A. Galal, S.M. Azab, Int. J. Electrochem. Sci. 6 (2011). [19] N.F. Atta, A. Galal, A.A. Wassel, A.H. Ibrahim, Int. J. Electrochem. Sci. 7 (2012). [20] A. Babaei, M. Babazadeh, H. Momeni, Int. J. Electrochem. Sci. 6 (2011) 1382–1395. [21] M. Tao, F. Xu, Y. Li, Q. Xu, Y. Chang, Z. Wu, Y. Yang, Bull. Kor. Chem. Soc. 31 (2010) 1968–1972. [22] N.F. Atta, A. Galal, R.A. Ahmed, Electroanalysis 23 (2011) 737–746. [23] G. Lu, X. Yao, X. Wu, T. Zhan, Microchem. J. 69 (2001) 81–87. [24] M. Zhang, W. Gorski, Anal. Chem. 77 (2005) 3960–3965. [25] M. Zhang, A. Smith, W. Gorski, Anal. Chem. 76 (2004) 5045–5050. [26] X. Wei, J. Cruz, W. Gorski, Anal. Chem. 74 (2002) 5039–5046. [27] D.S. dos Santos, A. Riul, R.R. Malmegrim, F.J. Fonseca, O.N. Oliveira, L.H. Mattoso, Macromol. Biosci. 3 (2003) 591–595. [28] Y. Wang, J. Zhu, R. Zhu, Z. Zhu, Z. Lai, Z. Chen, Meas. Sci. Technol. 14 (2003) 831. [29] H.-S. Wang, Q.-X. Pan, G.-X. Wang, Sensors 5 (2005) 266–276. [30] M.G. Peter, J. Macromol. Sci., Part A: Pure Appl. Chem. 32 (1995) 629–640. [31] P. Norouzi, M. Pirali-Hamedani, M. Ganjali, F. Faridbod, Int. J. Electrochem. Sci. 5 (2010) 1434–1446. [32] H. Huang, N. Hu, Y. Zeng, G. Zhou, Anal. Biochem. 308 (2002) 141–151. [33] G. Wang, J.-J. Xu, L.-H. Ye, J.-J. Zhu, H.-Y. Chen, Bioelectrochemistry 57 (2002) 33–38. [34] J. Abdullah, M. Ahmad, N. Karuppiah, L.Y. Heng, H. Sidek, Sensors Actuators B Chem. 114 (2006) 604–609. [35] X. Tan, J. Zhang, S. Tan, D. Zhao, Z. Huang, Y. Mi, Z. Huang, Electroanalysis 21 (2009) 1514–1520. [36] A. Kaushik, R. Khan, P.R. Solanki, P. Pandey, J. Alam, S. Ahmad, B. Malhotra, Biosens. Bioelectron. 24 (2008) 676–683. [37] Y.-Y. Liang, L.-M. Zhang, W. Li, R.-F. Chen, Colloid Polym. Sci. 285 (2007) 1193–1199. [38] S.-F. Wang, Y.-M. Tan, Anal. Bioanal. Chem. 387 (2007) 703–708. [39] G. Zhao, J.-J. Xu, H.-Y. Chen, Electrochem. Commun. 8 (2006) 148–154. [40] M.S. Lin, H.J. Leu, Electroanalysis 17 (2005) 2068–2073. [41] J. Hrbac, V. Halouzka, R. Zboril, K. Papadopoulos, T. Triantis, Electroanalysis 19 (2007) 1850–1854. [42] H.L. Zhang, X.Z. Zou, G.S. Lai, D.Y. Han, F. Wang, Electroanalysis 19 (2007) 1869–1874. [43] A. Kaushik, P.R. Solanki, A.A. Ansari, S. Ahmad, B.D. Malhotra, Electrochem. Commun. 10 (2008) 1364–1368. [44] Z. Lei, X. Pang, N. Li, L. Lin, Y. Li, J. Mater. Process. Technol. 209 (2009) 3218–3225. [45] E. Laviron, J. Electroanal. Chem. Interfacial Electrochem. 52 (1974) 355–393. [46] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 2001. [47] The United States Pharmacopoeia, The National Formulary, USP 24, NF 19, USP Convention Inc., MD, 2000, p. 2151.
