Colloids and Surfaces B: Biointerfaces 116 (2014) 674–680
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Electropolymerization of curcumin on glassy carbon electrode and its electrocatalytic application for the voltammetric determination of epinephrine and p-acetoaminophenol Balamurugan Devadas, Muniyandi Rajkumar, Shen-Ming Chen ∗ Electroanalysis and Bio Electrochemistry Lab, Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan, ROC
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Article history: Received 5 August 2013 Received in revised form 31 October 2013 Accepted 2 November 2013 Available online 15 November 2013 Keywords: Poly curcumin Epinephrine p-Acetoaminophenol and real sample
a b s t r a c t Here in, we report the simultaneous voltammetric determination of epinephrine (EP) and pacetoaminophenol (AP) on a poly curcumin (1,7 Bis ((4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5 dione), poly CM) modified glassy carbon electrode (GCE) for the first time. The CM was polymerized on to the GCE surface by simple electro polymerization process. A low peak to peak (Ep ) separation of 60 mV was observed, indicating fast electron transfer between poly CM and the electrode surface. The electrochemical measurements and surface morphology of the as prepared poly CM film modified electrode were studied using cyclic voltammetry (CV), and field emission scanning electron microscopy (SEM), respectively. Moreover, poly CM modified GCE exhibits enhanced electro catalytic activity toward EP and AP in the linear range of 4.97–230.76 M and 0.99–230.76 M and with very low detection limit (LOD) of 0.05 M and 0.1 M. The sensitivity is 0.621 and 0.303 A M−1 cm2 for EP and AP, respectively. The practical feasibility of the proposed poly CM/GCE was evaluated in adrenaline injection (1 mg mL−1 ) solutions and paracetamol tablets for the simultaneous determination of EP and AP. We found maximum recovery of 99.2% for adrenaline injection and 97.4% for paracetamol tablets. Finally the modified electrode exhibit excellent repeatability, reproducibility and stability for the selective and simultaneous determination of EP and AP. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Curcumin is a bioactive polyphenol component present in the rhizomes of Curcumin longa, which is also known as diferuloylmethane (C21 H20 O6 ). Curcumin has become an intense topic of research due to its interesting biological properties and pharmacological applications such as anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects [1–4]. The powerful curcumin antioxidant activity, working especially when diverse free radical are produced as a result of physiological process is essentially an electrochemical property, so it has to be investigated from an electrochemical viewpoint in order to characterize its redox behavior and its electrocatalytic role. As a result, investigating the redox process and the catalytic activity of curcumin are of biological importance. Hitherto, there were only few reports on curcumin modified electrodes for various types of electrochemical sensors. So far several reports for the metal incorporated curcumin modified electrodes such as, Ni–curcumin modified glassy carbon electrodes for electrocatalytic oxidation on simple alcohols [5,6],
∗ Corresponding author. Tel.: +886 2270 17147; fax: +886 2270 25238. E-mail address: [email protected] (S.-M. Chen). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.002
glucose [7] and some amino acids [8], respectively, based on the redox couple Ni (III)/Ni (II) of Ni (II)–curcumin complex as the catalytic center. To the best of our knowledge, there was no report about the direct electrocatalytic activity of curcumin. Glassy carbon electrode (GCE) has been made by special type of carbon which was fabricated by pyrolysis of polymer resin [9]. Moreover most of the films were fabricated on GCE and it avenues to the field of electrocatalysis and biosensor applications. Surface treatment of a solid electrode has been used extensively to improve the electrochemical performance of the electrode. Especially, electrochemical pretreatment has been used for cleaning and activating the surface of electrode [10–12]. The surface of the metal and carbon electrodes can be oxidized and various kinds of oxygenous groups, such as phenolic, quinoidal, and carboxyl functionalities, added on the surfaces. The existences of these active groups have been proved by such methods as cyclic voltammetry (CV), differential pulse polarography, scanning probe microscope, scanning electron microscope and X-ray photoelectron spectrum [13–15], respectively. On the other hand, epinephrine (EP) belongs to the family of catecholamine neurotransmitters, which is widely distributed in the mammalian central nervous system for message transfer process [16]. It is as an important chemical mediator for conveying nerve
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impulse in the mammalian central nervous systems. Furthermore, EP can be used to treat cardiac arrest and bronchodilator for asthma patients [17]. The normal EP concentrations for adults are less than 10 ng/l, hence, abnormal concentration of EP in human body causes to symptoms of several diseases [18]. Hence, a quantitative determination of EP concentration is quite helpful for developing research in disease diagnosis, pharmacological research and life science. Electrochemical methods have been developed to determine epinephrine due to its electroactive nature [19]. In addition, acetoaminophenol (AP) or paracetamol has been widely used as an analgesic drug, relief of moderate pain for headache, backache and for reduction of fevers, respectively [20,21]. So far many analytical techniques have been employed for the detection of acetoaminophenol such as spectrophotometric [22,22], HPLC [23], Flow injection analysis [24] and electrophoresis [25], respectively. Relative to the above methods, we need a simple, highly sensitive and low cost instrument for the detection of acetoaminophenol. Electrochemical methods are found to be convenient, reliable, cheapest and important one for the acetoaminophenol detection [26–28]. However, the oxidation peaks of EP and AP are nearly at the same potentials at the bare electrodes. This results to overlap and fouling during the determination of these compounds. Therefore, fabrication of economically viable electrochemical sensor with simultaneous determination of EP and AP with higher selectivity, stability and sensitivity is still one of the challenging tasks for the electrochemists. In this paper, we report a highly stable poly curcumin film on the activated GCE for the simultaneous determination of EP and AP. To the best of our knowledge this is the first report to use poly curcumin modified electrode as electrochemical sensor for simultaneous determination of EP and AP. The poly curcumin modified film also offers several distinct advantages including extraordinary stability, high electron transfer rate constant and low detection limit for simultaneous and selective detection for EP and AP. Linear pulse voltammetry (LSV) has been used to evaluate the analytical performance of the sensor in quantification of EP in the presence of AP. Finally, to evaluate the utility of the poly curcumin modified electrode for analytical applications, it also used for the simultaneous voltammetric determination of EP and AP in real samples. 2. Experimental 2.1. Materials and methods Curcumin (CM) from curcuma longa (Turmeric) was purchased from Sigma Aldrich and the required concentration was prepared by using pure ethanol (99%). (−)-Epinephrine, and pacetoaminophenol were obtained from Sigma Aldrich. Sodium bicarbonate (NaHCO3 ) was purchased from Wako Chemicals. All electrochemical studies were performed in phosphate buffer (pH 8) using Na2 HPO4 and NaH2 PO4 . The pH was adjusted by using NaOH. All the aqueous solutions were prepared using doubly distilled (DD) water. Pre-purified N2 gas was purged through the experimental solutions for 15 min prior to electrochemical measurements. All the electrochemical studies were performed in a CHI 1205A work station. A conventional three electrode system was used for electrochemical measurements. Glassy carbon electrode (GCE) with a working area of 0.079 cm2 , Ag|AgCl in saturated KCl and Pt wire were used as working, reference and counter electrodes, respectively. The surface morphological studies were carried out using Scanning electron microscopy (Hitachi S-3000H, Japan). 2.2. Preparation of poly CM modified GCE GCE surface was polished with 0.05 m of alumina slurry using a Buhler polishing kit. The electrode surface was washed several
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times in deionized water and then ultrasonicated for 2 min to remove the adsorbed alumina particles. Then the GCE surface was activated by performing 20 consecutive potential scanning cycles in 0.1 M NaHCO3 solution in the potential range of −1.0 to 0.55 V at the scan rate of 100 mV s−1 . The activated GCE was washed again with deionized water and transferred into an electrochemical cell containing 50 M CM in fresh PBS (pH 8) solution. Then 16 consecutive cycles were performed for the electropolymerization process in the potential range of 0.15–0.55 V at the scan rate of 100 mV s−1 . Finally the poly CM modified GCE was dried in open air and used for further electrocatalytic studies. 3. Result and discussion 3.1. Electropolymerization of CM on activated GCE and its electrochemical behavior During electro polymerization of curcumin on bare GCE, no significant peaks were observed, because of its smooth surface and due to the absence of active sites (Fig. 1A bare GCE). However, CM can polymerize on electrochemically activated GCE, mainly due to its high surface roughness and increase in the active sites which lead to enhance in the electron transfer process. Fig. 1A shows the electropolymerization of CM on the activated GCE and Bare GCE. In the first cycle of activated GCE, an anodic peak (a) appears at 0.4 V, which indicates the oxidation of CM leading to the formation of an o-quinone derivative. Upon increasing the number of cycles, the anodic current density significantly decreased while the corresponding anodic peak (a) disappears completely by the end of 16 cycles. In the second cycle, an anodic peak (b) and a cathodic peak (c) appears at 0.14 V and 0.12 V, respectively that are readily assigned to the quinone/hydroquinone redox couple involving a two electron coupled two proton transfer process [29]. The anodic and cathodic current density increases with increases in the number of cycles, revealing the polymerization of CM on the activated GCE surface. The polymerization process occurred through the nucleophilic reaction of 3,5 dione group and o-quinone group with the functional groups on the activated GCE resulting in the bond formation. The two electron two proton process of poly CM cyclization step was favored by increasing pH [30]. Moreover the pH of the electrolyte solution (pH 8) used for electrocatalytic purpose, which is almost same with pH of the NaHCO3 solution (pH 8.2). The cyclic voltammogram response of the activated GCE and poly CM modified GCE in PBS (pH 8) solution is showed in Fig. 1B. In Fig. 1B the activated GCE (peak b) does not exhibit any redox peak in PBS (pH 8) when compared to poly CM modified GCE (peak a). Fig. 1C shows the CVs of activated GCE/CM and bare GCE/CM in PBS containing 100 M of EP and AP. The activated GCE/CM shows well defined obvious electro catalytic peaks for the simultaneous detection of EP and AP. At the same time the bare GCE/CM electrode shows null response. These results reveals that polymerization process occurred on the activated GCE shows well clear redox peaks with peak to peak separation (Ep ) of 60 mV toward detection of EP and AP. The possible mechanism of electro polymerization reaction (Scheme 1) was reported earlier, shown in below scheme [29]. 3.2. EIS, surface morphology and scan rate studies EIS study has been employed to analyze the detailed electrochemical activities of modified electrode with individual or mixed components. Here the complex impedance can be presented as a sum of the real, Z (ω), and imaginary Z (ω), components that originate mainly from the resistance and capacitance of the cell. From the shape of an impedance spectrum, the electron-transfer
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Fig. 1. (A) Comparison CVs of electro polymerization of CM on activated GCE and bare GCE in a 0.1 M Phosphate buffer solution (pH 8) at the scan rate 100 mV s−1 (16 cycles). (B) CVs obtained at (a) CM/GCE, (b) activated GCE in phosphate buffer solution (pH 8). Scan rate 100 mV s−1 . (C) CVs of activated GCE/CM and bare GCE/CM in PBS (pH 8) containing 100 M of EP and AP (scan rate 50 mV s−1 ). (D) EIS spectra of activated GCE (red color) and act. GCE/CM (black color). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
kinetics and diffusion characteristics can be extracted. The respective semicircle parameters correspond to the electron transfer resistance (Ret) and the double layer capacity (Cdl) nature of the modified electrode. Fig. 1D shows the Faradaic impedance spectra, presented as Nyquist plots (Z vs Z ) for the Poly CM modified activated GCE and activated GCE, respectively. The activated GCE electrode exhibits an almost straight line (Red) with a very small enlarged semi-circle arc which represents the characteristics of diffusion limited electron-transfer process on the electrode surface. At the same time, the Poly CM film shows like a enlarged semi-circle arc with an interfacial resistance due to the electrostatic repulsion between the charged surface and probe molecule Fe(CN)6 3−/4− . This enlarged semi-circle arc clearly indicates the capacitance behavior of the poly CM activated GCE. Thus, the
electron transfer process will become as a slow process on the poly CM activated GCE. Finally, based on these illustrations, the slow electron transfer kinetics nature of poly CM activated GCE film has been authenticated. SEM image of the poly CM modified GCE reveals that smooth, rod-like structures were formed through the electro-deposition process (Fig. 2A). The surface morphology of the activated GCE is shown in inset Fig. 2A, relative to bare electrode, being highly rough and porous. The polymerization process occurs on activated GCE surface more efficiently. The effect of scan rate at the poly CM/GCE in phosphate buffer solution (pH 8) is shown in Fig. 2B. The anodic and cathodic peak currents observed at the CM/GCE increased with increases in the scan rates from 100 to 1000 mV s−1 , revealing a diffusion controlled process.
Scheme 1. Electropolymerization of curcumin on glassy carbon electrode.
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Fig. 2. (A) SEM image of poly CM on activated GCE. Inset image of activated GCE. (B) CVs of CM/GCE in 0.1 M phosphate buffer solution at different scan rates 100 mV s−1 to 1000 mV s−1 (from inner to outer). Inset: linear dependence of Ipa and Ipc on scan rates.
Scheme 2. Schematic representation of electro polymerization process of curcumin and its electro catalytic activity.
pH 8 (inset of Fig. 3); therefore we utilize pH 8 for all our electro catalytic experiments.
Fig. 3. CVs obtained at poly CM modified GCE in various buffer solutions (pH 4–10). Scan rate 100 mV s−1 . Inset plot of pH vs E0 .
3.3. Different pH study Fig. 3 shows the effect of pH on the poly CM/GCE electrode in various buffer solutions (pH 4–11). Well defined peaks were observed for the wide pH range, and the redox couples are reproducible when the modified GCE was transferred from one pH solution to the other. With increase in pH of the solutions, both these redox peaks shifted toward the negative potential. On each occasion, before transferring the modified GCE to another buffer solution the film was washed several times with doubly distilled water. However, there was no considerable decrease in the peak currents, which validates the good stability of the poly CM film. We have presented the influence of pH vs E0 (peak potential) values in Fig. 3 inset. It is apparent that values of the peaks exhibit a linear dependence on pH. The slope value is 61 mV/pH, which is close to the slope value of 59 mV/pH for equal number of proton and electron transfer processes. Thus the poly CM modified film involves an equal number of proton and electron transfer process. Moreover, the poly CM/GCE shows well-defined, stable and enhanced redox peaks in
3.4. Individual determination of epinephrine and p-acetoaminophenol Before evaluating the simultaneous determination of EP and AP, it was also necessary to determine the EP and AP individually. Fig. 4A depicts the linear sweep voltammogram (LSV) response of different concentration of EP on poly CM modified GCE. Initially 5 M of EP added to electrochemical cell containing 10 ml of PBS (pH 8), then the CM modified GCE placed in the electrochemical cell and recorded in LSV. The peak potential occurs at 168 mV due to EP oxidation. Further increasing the concentration of EP from 5 to 300 M the peak current also increases linearly with increase in concentration. The inset of Fig. 4A shows the linear calibration plot of EP concentration vs current. The linear regression can be written as Ipa (A) = 0.0491C (M) + 0.4939, R2 = 0.9855. Hence the poly CM modified GCE have ability to determine the EP without any corelated substance. Likewise the individual determination of AP have also been investigated using LSV. The oxidation peak of AP appears at 384 mV (Fig. 4B). When the concentration of AP increase (1–230 M) the peak current also increases linearly Moreover the linear calibration plot validates the Concentration of AP vs Ipa was linear. The linear regression equation for AP from linear calibration plot (inset) can be expressed as Ipa (A) = 0.0248C (M) + 0.7341, R2 = 0.98. Scheme 2 could explain the fabrication and electro
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Fig. 4. (A and B) LSV of poly CM modified GCE in 0.1 M PBS containing different concentration of EP and AP (from 5 to 300 M & from 1 to 300 M). Scan rate 50 mV s−1 . Inset linear plot of Ipa vs concentration.
Table 1 Experimetnal performance of polyCM modified GCE for detection of EP and AP. Analyte EPa APb a b
Linear range (M) 4.9–230.7 0.99–230.7
Detection limit (M)
Sensitivity (A M−1 cm2 )
0.054 0.11
0.621 0.303
EP, epinephrine. AP, p-acetaminophen.
catalytic properties of CM polymerization film toward the selective oxidation of EP and AP. Table 1 indicates the performances of individual analyte. 3.5. Selective determination of epinephrine and p-acetoaminophenol The selective determination of EP and AP has been employed using LSV technique. For the selective determination of AP, 10 M of AP added to electrochemical cell containing 10 ml PBS with constant concentration of 0.02 mM EP shown in Fig. 5A. The oxidation peak current at 384 mV increased linearly with increases in AP
concentrations ranging from 10 to 100 M. Interestingly there is no increse in EP oxidation current. Similarly the experiment was again carried out with PBS containing constant concentration of AP (0.1 mM). The oxidation peak current at 168 mV increased linearly with increase in EP concentration ranging from 30 to 100 M (Fig. 5B). Here the oxidation peak current of AP does not increases, but there is slight change in the background current which may be due to increasing the oxidation peak current of EP. Hence from these results we conclude that the poly CM modified GCE promising choice for selective determination of EP and AP. 3.6. Simultaneous determination of epinephrine and p-acetoaminophenol The simultaneous determination of EP and AP has been recorded using poly CM modified GCE by LSV technique. Equal concentration of EP and AP were added to the electrochemical cell containing PBS (pH 8) and LSVs were recorded (Fig. 6). The LSV results shows well defined separate peaks at 168 mV (EP) and 384 mV (AP) consistent to the oxidation of EP and AP, respectively. Furthermore, the peak to peak separation (Ep ) of EP and AP at CM modified GCE is 216 mV,
Fig. 5. (A) LSV obtained at poly CM/GCE in PBS containing 0.02 mM of EP, with addition of different concentration of AP (10–100 M). Scan rate 50 mV s−1 . (B) LSV obtained at CM/GCE in PBS containing 0.1 mM of AP, with addition of different concentration of EP (30–100 M). Scan rate 50 mV s−1 .
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Table 2 Detection of EP and AP using injection and tablet samples at polyCM modified GCE. ADa injection sample
Added (M)
Found (M)
Recovery %
PAb tablet sample
Added (M)
Found (M)
Recovery %
1 2
5 10
4.9 9.92
98 99.2
1 2
30 40
28.8 38.9
96.2 97.4
a b
AD, adrenaline injection. PA, paracetamol tablets.
stability of the proposed film has been examined by storing the CM modified electrode in PBS (pH 8) at 4 ◦ C and its response toward to EP and AP were monitored everyday using CV. The modified electrode retains 95% of its initial response even after one week, revealing its good stability. This confirms that the proposed film was electrochemically active and stable. 4. Conclusion
Fig. 6. LSV obtained at poly CM modified GCE in 0.1 M PBS containing equal amount mixture of each addition of 10 M EP and AP. (a–h) 30–100 M.
showing that their oxidation peaks are well separated. Further the oxidation peak currents of EP and AP increases linearly in conjunction with increasing concentrations. These results clearly indicates that the proposed poly CM modified GCE possess the promising choice for the simultaneous as well as individual determination of EP and AP, respectively. 3.7. Real sample analysis The practical feasibility of the polyCM modified GCE has been recorded with injection sample and paracetamol tablets, respectively. The adrenalin injection solution (1 mg ml−1 ) and Paracetamol tablets were purchased from nearby hospital and utilized for the real sample analysis. Table 2 shows the real sample analysis results for the selective detection of EP and AP in injection and tablet mixtures. The adrenaline injection and paracetamol tablets were diluted to required concentration with PBS (pH 8) solution and used for further analysis on poly CM modified GCE by LSV method. The added real samples shows good recoveries. The maximum recovery was specified in Table 2. The recoveries obtained here validates the pertinent nature of the poly CM modified GCE for the sensor applications, respectively. 3.8. Repeatability, reproducibility and stability Repeatability, reproducibility and stability studies were evaluated using CV technique. Four different poly CM modified GCEs were prepared and their responses toward the constant concentration of EP (0.1 mM) and AP (0.1 mM) in PBS (pH 8) were tested. The relative standard deviation (R.S.D) for the determination of EP and AP at four CM/GCEs is 4.1%, indicating good repeatability. Similarly, the RSD values for four repetitive measurements of EP and AP at a CM/GCE is 2.7%, indicating good reproducibility. In addition the
In conclusion, we have successfully fabricated poly CM on GCE by simple electro deposition process. This type of film has been found as electrochemically active and stable in pH 8 PBS. The poly CM modified GCE possess the high electro active surface area and excellent electro catalytic activity toward the selective and simultaneous detection of EP and AP at less positive potential of 0.16 V and 0.38 V. The proposed poly CM modified GCE remarkably suppressed the interference effect and showed well defined oxidation peaks for the selective detection of EP and AP. In addition, the high surface area of the poly CM film modified GCE electrode well suited for the simultaneous determination of these compounds. Finally, the poly CM modified GCE applied for the detection of EP and AP in injection sample and tablet mixtures. Thus, the poly CM film modified GCE electrode showed higher stability, reproducilibity, exhibits promising electrocatalytic activityand rapid response toward selective and simutaneous determination of EP and AP in both lab and real sample analysis. Acknowledgements The authors are gratefully acknowledged to the National Science Council and the Ministry of Education of Taiwan (Republic of China). The heart fully thank to Dr. Arunprakashperiasamy for his valuable suggestion throughout this work. References [1] R.A. Sharma, A.J. Gescher, W.P. Steward, Curcumin: the story so far, Eur. J. Cancer 41 (2005) 1955–1968. [2] P. Anand, S.G. Thomas, A.B. Kunnumakkara, C. Sundaram, K.B. Harikumar, B. Sung, S.T. Tharakan, K. Misra, I.K. Priyadarsini, K.N. Rajasekharan, B.B. Aggarwal, Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature, Biochem. Pharmacol. 76 (2008) 1590–1611. [3] R.K. Maheshwari, A.K. Singh, J. Gaddipati, R.C. Srimal, Multiple biological activities of curcumin: a short review, Life Sci. 78 (2006) 2081–2087. [4] I. Chattopadhyay, K. Biswas, U. Bandyopadhyay, R.K. Banerjee, Turmeric and Curcumin Biological actions and medicinal applications, Curr. Sci. 87 (2004) 44–53. [5] A. Ciszewski, Catalytic oxidation of methanol on a glassy carbon electrode electrochemically modified by a conductive Ni (II) curcumin film, Electroanalysis 7 (1995) 1132–1135. [6] A. Ciszewski, G. Milczarek, B. Lewandowska, K. Krutowski, Electrocatalytic properties of electropolymerized Ni(II) curcumin complex, Electroanalysis 15 (2003) 518–523. [7] M.Y. Elahi, H. Heli, S.Z. Bathaie, M.F. Mousavi, Electrocatalytic oxidation of glucose at a Ni-curcumin modified glassy carbon electrode, J. Solid State Electrochem. 11 (2007) 273–282. [8] S. Majdi, A. Jabbari, H. Heli, A.A. Moosavi-Movahedi, Electrocatalytic oxidation of some amino acids on a nickel–curcumin complex modified glassy carbon electrode, Electrochim. Acta 52 (2007) 4622–4629. [9] G.M. Jenkins, K. Kawamura, Polymeric Carbons, Carbon Fiber, Glass and Char, Cambridge University Press, Cambridge, England, 1976. [10] R.J. Taylor, A.A. Humffray, Electrochemical studies on glassy carbon electrodes: I. Electron transfer kinetics, J. Electroanal. Chem. 42 (1973) 347–354.
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Electropolymerization of curcumin on glassy carbon electrode and its electrocatalytic application for the voltammetric determination of epinephrine and p-acetoaminophenol Balamurugan Devadas, Muniyandi Rajkumar, Shen-Ming Chen ∗ Electroanalysis and Bio Electrochemistry Lab, Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 31 October 2013 Accepted 2 November 2013 Available online 15 November 2013 Keywords: Poly curcumin Epinephrine p-Acetoaminophenol and real sample
a b s t r a c t Here in, we report the simultaneous voltammetric determination of epinephrine (EP) and pacetoaminophenol (AP) on a poly curcumin (1,7 Bis ((4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5 dione), poly CM) modified glassy carbon electrode (GCE) for the first time. The CM was polymerized on to the GCE surface by simple electro polymerization process. A low peak to peak (Ep ) separation of 60 mV was observed, indicating fast electron transfer between poly CM and the electrode surface. The electrochemical measurements and surface morphology of the as prepared poly CM film modified electrode were studied using cyclic voltammetry (CV), and field emission scanning electron microscopy (SEM), respectively. Moreover, poly CM modified GCE exhibits enhanced electro catalytic activity toward EP and AP in the linear range of 4.97–230.76 M and 0.99–230.76 M and with very low detection limit (LOD) of 0.05 M and 0.1 M. The sensitivity is 0.621 and 0.303 A M−1 cm2 for EP and AP, respectively. The practical feasibility of the proposed poly CM/GCE was evaluated in adrenaline injection (1 mg mL−1 ) solutions and paracetamol tablets for the simultaneous determination of EP and AP. We found maximum recovery of 99.2% for adrenaline injection and 97.4% for paracetamol tablets. Finally the modified electrode exhibit excellent repeatability, reproducibility and stability for the selective and simultaneous determination of EP and AP. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Curcumin is a bioactive polyphenol component present in the rhizomes of Curcumin longa, which is also known as diferuloylmethane (C21 H20 O6 ). Curcumin has become an intense topic of research due to its interesting biological properties and pharmacological applications such as anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects [1–4]. The powerful curcumin antioxidant activity, working especially when diverse free radical are produced as a result of physiological process is essentially an electrochemical property, so it has to be investigated from an electrochemical viewpoint in order to characterize its redox behavior and its electrocatalytic role. As a result, investigating the redox process and the catalytic activity of curcumin are of biological importance. Hitherto, there were only few reports on curcumin modified electrodes for various types of electrochemical sensors. So far several reports for the metal incorporated curcumin modified electrodes such as, Ni–curcumin modified glassy carbon electrodes for electrocatalytic oxidation on simple alcohols [5,6],
∗ Corresponding author. Tel.: +886 2270 17147; fax: +886 2270 25238. E-mail address: [email protected] (S.-M. Chen). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.002
glucose [7] and some amino acids [8], respectively, based on the redox couple Ni (III)/Ni (II) of Ni (II)–curcumin complex as the catalytic center. To the best of our knowledge, there was no report about the direct electrocatalytic activity of curcumin. Glassy carbon electrode (GCE) has been made by special type of carbon which was fabricated by pyrolysis of polymer resin [9]. Moreover most of the films were fabricated on GCE and it avenues to the field of electrocatalysis and biosensor applications. Surface treatment of a solid electrode has been used extensively to improve the electrochemical performance of the electrode. Especially, electrochemical pretreatment has been used for cleaning and activating the surface of electrode [10–12]. The surface of the metal and carbon electrodes can be oxidized and various kinds of oxygenous groups, such as phenolic, quinoidal, and carboxyl functionalities, added on the surfaces. The existences of these active groups have been proved by such methods as cyclic voltammetry (CV), differential pulse polarography, scanning probe microscope, scanning electron microscope and X-ray photoelectron spectrum [13–15], respectively. On the other hand, epinephrine (EP) belongs to the family of catecholamine neurotransmitters, which is widely distributed in the mammalian central nervous system for message transfer process [16]. It is as an important chemical mediator for conveying nerve
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impulse in the mammalian central nervous systems. Furthermore, EP can be used to treat cardiac arrest and bronchodilator for asthma patients [17]. The normal EP concentrations for adults are less than 10 ng/l, hence, abnormal concentration of EP in human body causes to symptoms of several diseases [18]. Hence, a quantitative determination of EP concentration is quite helpful for developing research in disease diagnosis, pharmacological research and life science. Electrochemical methods have been developed to determine epinephrine due to its electroactive nature [19]. In addition, acetoaminophenol (AP) or paracetamol has been widely used as an analgesic drug, relief of moderate pain for headache, backache and for reduction of fevers, respectively [20,21]. So far many analytical techniques have been employed for the detection of acetoaminophenol such as spectrophotometric [22,22], HPLC [23], Flow injection analysis [24] and electrophoresis [25], respectively. Relative to the above methods, we need a simple, highly sensitive and low cost instrument for the detection of acetoaminophenol. Electrochemical methods are found to be convenient, reliable, cheapest and important one for the acetoaminophenol detection [26–28]. However, the oxidation peaks of EP and AP are nearly at the same potentials at the bare electrodes. This results to overlap and fouling during the determination of these compounds. Therefore, fabrication of economically viable electrochemical sensor with simultaneous determination of EP and AP with higher selectivity, stability and sensitivity is still one of the challenging tasks for the electrochemists. In this paper, we report a highly stable poly curcumin film on the activated GCE for the simultaneous determination of EP and AP. To the best of our knowledge this is the first report to use poly curcumin modified electrode as electrochemical sensor for simultaneous determination of EP and AP. The poly curcumin modified film also offers several distinct advantages including extraordinary stability, high electron transfer rate constant and low detection limit for simultaneous and selective detection for EP and AP. Linear pulse voltammetry (LSV) has been used to evaluate the analytical performance of the sensor in quantification of EP in the presence of AP. Finally, to evaluate the utility of the poly curcumin modified electrode for analytical applications, it also used for the simultaneous voltammetric determination of EP and AP in real samples. 2. Experimental 2.1. Materials and methods Curcumin (CM) from curcuma longa (Turmeric) was purchased from Sigma Aldrich and the required concentration was prepared by using pure ethanol (99%). (−)-Epinephrine, and pacetoaminophenol were obtained from Sigma Aldrich. Sodium bicarbonate (NaHCO3 ) was purchased from Wako Chemicals. All electrochemical studies were performed in phosphate buffer (pH 8) using Na2 HPO4 and NaH2 PO4 . The pH was adjusted by using NaOH. All the aqueous solutions were prepared using doubly distilled (DD) water. Pre-purified N2 gas was purged through the experimental solutions for 15 min prior to electrochemical measurements. All the electrochemical studies were performed in a CHI 1205A work station. A conventional three electrode system was used for electrochemical measurements. Glassy carbon electrode (GCE) with a working area of 0.079 cm2 , Ag|AgCl in saturated KCl and Pt wire were used as working, reference and counter electrodes, respectively. The surface morphological studies were carried out using Scanning electron microscopy (Hitachi S-3000H, Japan). 2.2. Preparation of poly CM modified GCE GCE surface was polished with 0.05 m of alumina slurry using a Buhler polishing kit. The electrode surface was washed several
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times in deionized water and then ultrasonicated for 2 min to remove the adsorbed alumina particles. Then the GCE surface was activated by performing 20 consecutive potential scanning cycles in 0.1 M NaHCO3 solution in the potential range of −1.0 to 0.55 V at the scan rate of 100 mV s−1 . The activated GCE was washed again with deionized water and transferred into an electrochemical cell containing 50 M CM in fresh PBS (pH 8) solution. Then 16 consecutive cycles were performed for the electropolymerization process in the potential range of 0.15–0.55 V at the scan rate of 100 mV s−1 . Finally the poly CM modified GCE was dried in open air and used for further electrocatalytic studies. 3. Result and discussion 3.1. Electropolymerization of CM on activated GCE and its electrochemical behavior During electro polymerization of curcumin on bare GCE, no significant peaks were observed, because of its smooth surface and due to the absence of active sites (Fig. 1A bare GCE). However, CM can polymerize on electrochemically activated GCE, mainly due to its high surface roughness and increase in the active sites which lead to enhance in the electron transfer process. Fig. 1A shows the electropolymerization of CM on the activated GCE and Bare GCE. In the first cycle of activated GCE, an anodic peak (a) appears at 0.4 V, which indicates the oxidation of CM leading to the formation of an o-quinone derivative. Upon increasing the number of cycles, the anodic current density significantly decreased while the corresponding anodic peak (a) disappears completely by the end of 16 cycles. In the second cycle, an anodic peak (b) and a cathodic peak (c) appears at 0.14 V and 0.12 V, respectively that are readily assigned to the quinone/hydroquinone redox couple involving a two electron coupled two proton transfer process [29]. The anodic and cathodic current density increases with increases in the number of cycles, revealing the polymerization of CM on the activated GCE surface. The polymerization process occurred through the nucleophilic reaction of 3,5 dione group and o-quinone group with the functional groups on the activated GCE resulting in the bond formation. The two electron two proton process of poly CM cyclization step was favored by increasing pH [30]. Moreover the pH of the electrolyte solution (pH 8) used for electrocatalytic purpose, which is almost same with pH of the NaHCO3 solution (pH 8.2). The cyclic voltammogram response of the activated GCE and poly CM modified GCE in PBS (pH 8) solution is showed in Fig. 1B. In Fig. 1B the activated GCE (peak b) does not exhibit any redox peak in PBS (pH 8) when compared to poly CM modified GCE (peak a). Fig. 1C shows the CVs of activated GCE/CM and bare GCE/CM in PBS containing 100 M of EP and AP. The activated GCE/CM shows well defined obvious electro catalytic peaks for the simultaneous detection of EP and AP. At the same time the bare GCE/CM electrode shows null response. These results reveals that polymerization process occurred on the activated GCE shows well clear redox peaks with peak to peak separation (Ep ) of 60 mV toward detection of EP and AP. The possible mechanism of electro polymerization reaction (Scheme 1) was reported earlier, shown in below scheme [29]. 3.2. EIS, surface morphology and scan rate studies EIS study has been employed to analyze the detailed electrochemical activities of modified electrode with individual or mixed components. Here the complex impedance can be presented as a sum of the real, Z (ω), and imaginary Z (ω), components that originate mainly from the resistance and capacitance of the cell. From the shape of an impedance spectrum, the electron-transfer
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Fig. 1. (A) Comparison CVs of electro polymerization of CM on activated GCE and bare GCE in a 0.1 M Phosphate buffer solution (pH 8) at the scan rate 100 mV s−1 (16 cycles). (B) CVs obtained at (a) CM/GCE, (b) activated GCE in phosphate buffer solution (pH 8). Scan rate 100 mV s−1 . (C) CVs of activated GCE/CM and bare GCE/CM in PBS (pH 8) containing 100 M of EP and AP (scan rate 50 mV s−1 ). (D) EIS spectra of activated GCE (red color) and act. GCE/CM (black color). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
kinetics and diffusion characteristics can be extracted. The respective semicircle parameters correspond to the electron transfer resistance (Ret) and the double layer capacity (Cdl) nature of the modified electrode. Fig. 1D shows the Faradaic impedance spectra, presented as Nyquist plots (Z vs Z ) for the Poly CM modified activated GCE and activated GCE, respectively. The activated GCE electrode exhibits an almost straight line (Red) with a very small enlarged semi-circle arc which represents the characteristics of diffusion limited electron-transfer process on the electrode surface. At the same time, the Poly CM film shows like a enlarged semi-circle arc with an interfacial resistance due to the electrostatic repulsion between the charged surface and probe molecule Fe(CN)6 3−/4− . This enlarged semi-circle arc clearly indicates the capacitance behavior of the poly CM activated GCE. Thus, the
electron transfer process will become as a slow process on the poly CM activated GCE. Finally, based on these illustrations, the slow electron transfer kinetics nature of poly CM activated GCE film has been authenticated. SEM image of the poly CM modified GCE reveals that smooth, rod-like structures were formed through the electro-deposition process (Fig. 2A). The surface morphology of the activated GCE is shown in inset Fig. 2A, relative to bare electrode, being highly rough and porous. The polymerization process occurs on activated GCE surface more efficiently. The effect of scan rate at the poly CM/GCE in phosphate buffer solution (pH 8) is shown in Fig. 2B. The anodic and cathodic peak currents observed at the CM/GCE increased with increases in the scan rates from 100 to 1000 mV s−1 , revealing a diffusion controlled process.
Scheme 1. Electropolymerization of curcumin on glassy carbon electrode.
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Fig. 2. (A) SEM image of poly CM on activated GCE. Inset image of activated GCE. (B) CVs of CM/GCE in 0.1 M phosphate buffer solution at different scan rates 100 mV s−1 to 1000 mV s−1 (from inner to outer). Inset: linear dependence of Ipa and Ipc on scan rates.
Scheme 2. Schematic representation of electro polymerization process of curcumin and its electro catalytic activity.
pH 8 (inset of Fig. 3); therefore we utilize pH 8 for all our electro catalytic experiments.
Fig. 3. CVs obtained at poly CM modified GCE in various buffer solutions (pH 4–10). Scan rate 100 mV s−1 . Inset plot of pH vs E0 .
3.3. Different pH study Fig. 3 shows the effect of pH on the poly CM/GCE electrode in various buffer solutions (pH 4–11). Well defined peaks were observed for the wide pH range, and the redox couples are reproducible when the modified GCE was transferred from one pH solution to the other. With increase in pH of the solutions, both these redox peaks shifted toward the negative potential. On each occasion, before transferring the modified GCE to another buffer solution the film was washed several times with doubly distilled water. However, there was no considerable decrease in the peak currents, which validates the good stability of the poly CM film. We have presented the influence of pH vs E0 (peak potential) values in Fig. 3 inset. It is apparent that values of the peaks exhibit a linear dependence on pH. The slope value is 61 mV/pH, which is close to the slope value of 59 mV/pH for equal number of proton and electron transfer processes. Thus the poly CM modified film involves an equal number of proton and electron transfer process. Moreover, the poly CM/GCE shows well-defined, stable and enhanced redox peaks in
3.4. Individual determination of epinephrine and p-acetoaminophenol Before evaluating the simultaneous determination of EP and AP, it was also necessary to determine the EP and AP individually. Fig. 4A depicts the linear sweep voltammogram (LSV) response of different concentration of EP on poly CM modified GCE. Initially 5 M of EP added to electrochemical cell containing 10 ml of PBS (pH 8), then the CM modified GCE placed in the electrochemical cell and recorded in LSV. The peak potential occurs at 168 mV due to EP oxidation. Further increasing the concentration of EP from 5 to 300 M the peak current also increases linearly with increase in concentration. The inset of Fig. 4A shows the linear calibration plot of EP concentration vs current. The linear regression can be written as Ipa (A) = 0.0491C (M) + 0.4939, R2 = 0.9855. Hence the poly CM modified GCE have ability to determine the EP without any corelated substance. Likewise the individual determination of AP have also been investigated using LSV. The oxidation peak of AP appears at 384 mV (Fig. 4B). When the concentration of AP increase (1–230 M) the peak current also increases linearly Moreover the linear calibration plot validates the Concentration of AP vs Ipa was linear. The linear regression equation for AP from linear calibration plot (inset) can be expressed as Ipa (A) = 0.0248C (M) + 0.7341, R2 = 0.98. Scheme 2 could explain the fabrication and electro
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Fig. 4. (A and B) LSV of poly CM modified GCE in 0.1 M PBS containing different concentration of EP and AP (from 5 to 300 M & from 1 to 300 M). Scan rate 50 mV s−1 . Inset linear plot of Ipa vs concentration.
Table 1 Experimetnal performance of polyCM modified GCE for detection of EP and AP. Analyte EPa APb a b
Linear range (M) 4.9–230.7 0.99–230.7
Detection limit (M)
Sensitivity (A M−1 cm2 )
0.054 0.11
0.621 0.303
EP, epinephrine. AP, p-acetaminophen.
catalytic properties of CM polymerization film toward the selective oxidation of EP and AP. Table 1 indicates the performances of individual analyte. 3.5. Selective determination of epinephrine and p-acetoaminophenol The selective determination of EP and AP has been employed using LSV technique. For the selective determination of AP, 10 M of AP added to electrochemical cell containing 10 ml PBS with constant concentration of 0.02 mM EP shown in Fig. 5A. The oxidation peak current at 384 mV increased linearly with increases in AP
concentrations ranging from 10 to 100 M. Interestingly there is no increse in EP oxidation current. Similarly the experiment was again carried out with PBS containing constant concentration of AP (0.1 mM). The oxidation peak current at 168 mV increased linearly with increase in EP concentration ranging from 30 to 100 M (Fig. 5B). Here the oxidation peak current of AP does not increases, but there is slight change in the background current which may be due to increasing the oxidation peak current of EP. Hence from these results we conclude that the poly CM modified GCE promising choice for selective determination of EP and AP. 3.6. Simultaneous determination of epinephrine and p-acetoaminophenol The simultaneous determination of EP and AP has been recorded using poly CM modified GCE by LSV technique. Equal concentration of EP and AP were added to the electrochemical cell containing PBS (pH 8) and LSVs were recorded (Fig. 6). The LSV results shows well defined separate peaks at 168 mV (EP) and 384 mV (AP) consistent to the oxidation of EP and AP, respectively. Furthermore, the peak to peak separation (Ep ) of EP and AP at CM modified GCE is 216 mV,
Fig. 5. (A) LSV obtained at poly CM/GCE in PBS containing 0.02 mM of EP, with addition of different concentration of AP (10–100 M). Scan rate 50 mV s−1 . (B) LSV obtained at CM/GCE in PBS containing 0.1 mM of AP, with addition of different concentration of EP (30–100 M). Scan rate 50 mV s−1 .
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Table 2 Detection of EP and AP using injection and tablet samples at polyCM modified GCE. ADa injection sample
Added (M)
Found (M)
Recovery %
PAb tablet sample
Added (M)
Found (M)
Recovery %
1 2
5 10
4.9 9.92
98 99.2
1 2
30 40
28.8 38.9
96.2 97.4
a b
AD, adrenaline injection. PA, paracetamol tablets.
stability of the proposed film has been examined by storing the CM modified electrode in PBS (pH 8) at 4 ◦ C and its response toward to EP and AP were monitored everyday using CV. The modified electrode retains 95% of its initial response even after one week, revealing its good stability. This confirms that the proposed film was electrochemically active and stable. 4. Conclusion
Fig. 6. LSV obtained at poly CM modified GCE in 0.1 M PBS containing equal amount mixture of each addition of 10 M EP and AP. (a–h) 30–100 M.
showing that their oxidation peaks are well separated. Further the oxidation peak currents of EP and AP increases linearly in conjunction with increasing concentrations. These results clearly indicates that the proposed poly CM modified GCE possess the promising choice for the simultaneous as well as individual determination of EP and AP, respectively. 3.7. Real sample analysis The practical feasibility of the polyCM modified GCE has been recorded with injection sample and paracetamol tablets, respectively. The adrenalin injection solution (1 mg ml−1 ) and Paracetamol tablets were purchased from nearby hospital and utilized for the real sample analysis. Table 2 shows the real sample analysis results for the selective detection of EP and AP in injection and tablet mixtures. The adrenaline injection and paracetamol tablets were diluted to required concentration with PBS (pH 8) solution and used for further analysis on poly CM modified GCE by LSV method. The added real samples shows good recoveries. The maximum recovery was specified in Table 2. The recoveries obtained here validates the pertinent nature of the poly CM modified GCE for the sensor applications, respectively. 3.8. Repeatability, reproducibility and stability Repeatability, reproducibility and stability studies were evaluated using CV technique. Four different poly CM modified GCEs were prepared and their responses toward the constant concentration of EP (0.1 mM) and AP (0.1 mM) in PBS (pH 8) were tested. The relative standard deviation (R.S.D) for the determination of EP and AP at four CM/GCEs is 4.1%, indicating good repeatability. Similarly, the RSD values for four repetitive measurements of EP and AP at a CM/GCE is 2.7%, indicating good reproducibility. In addition the
In conclusion, we have successfully fabricated poly CM on GCE by simple electro deposition process. This type of film has been found as electrochemically active and stable in pH 8 PBS. The poly CM modified GCE possess the high electro active surface area and excellent electro catalytic activity toward the selective and simultaneous detection of EP and AP at less positive potential of 0.16 V and 0.38 V. The proposed poly CM modified GCE remarkably suppressed the interference effect and showed well defined oxidation peaks for the selective detection of EP and AP. In addition, the high surface area of the poly CM film modified GCE electrode well suited for the simultaneous determination of these compounds. Finally, the poly CM modified GCE applied for the detection of EP and AP in injection sample and tablet mixtures. Thus, the poly CM film modified GCE electrode showed higher stability, reproducilibity, exhibits promising electrocatalytic activityand rapid response toward selective and simutaneous determination of EP and AP in both lab and real sample analysis. Acknowledgements The authors are gratefully acknowledged to the National Science Council and the Ministry of Education of Taiwan (Republic of China). The heart fully thank to Dr. Arunprakashperiasamy for his valuable suggestion throughout this work. References [1] R.A. Sharma, A.J. Gescher, W.P. Steward, Curcumin: the story so far, Eur. J. Cancer 41 (2005) 1955–1968. [2] P. Anand, S.G. Thomas, A.B. Kunnumakkara, C. Sundaram, K.B. Harikumar, B. Sung, S.T. Tharakan, K. Misra, I.K. Priyadarsini, K.N. Rajasekharan, B.B. Aggarwal, Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature, Biochem. Pharmacol. 76 (2008) 1590–1611. [3] R.K. Maheshwari, A.K. Singh, J. Gaddipati, R.C. Srimal, Multiple biological activities of curcumin: a short review, Life Sci. 78 (2006) 2081–2087. [4] I. Chattopadhyay, K. Biswas, U. Bandyopadhyay, R.K. Banerjee, Turmeric and Curcumin Biological actions and medicinal applications, Curr. Sci. 87 (2004) 44–53. [5] A. Ciszewski, Catalytic oxidation of methanol on a glassy carbon electrode electrochemically modified by a conductive Ni (II) curcumin film, Electroanalysis 7 (1995) 1132–1135. [6] A. Ciszewski, G. Milczarek, B. Lewandowska, K. Krutowski, Electrocatalytic properties of electropolymerized Ni(II) curcumin complex, Electroanalysis 15 (2003) 518–523. [7] M.Y. Elahi, H. Heli, S.Z. Bathaie, M.F. Mousavi, Electrocatalytic oxidation of glucose at a Ni-curcumin modified glassy carbon electrode, J. Solid State Electrochem. 11 (2007) 273–282. [8] S. Majdi, A. Jabbari, H. Heli, A.A. Moosavi-Movahedi, Electrocatalytic oxidation of some amino acids on a nickel–curcumin complex modified glassy carbon electrode, Electrochim. Acta 52 (2007) 4622–4629. [9] G.M. Jenkins, K. Kawamura, Polymeric Carbons, Carbon Fiber, Glass and Char, Cambridge University Press, Cambridge, England, 1976. [10] R.J. Taylor, A.A. Humffray, Electrochemical studies on glassy carbon electrodes: I. Electron transfer kinetics, J. Electroanal. Chem. 42 (1973) 347–354.
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