- Email: [email protected]
Simultaneous determination of epinephrine and ascorbic acid at the electrochemical sensor of triazole SAM modified gold electrode
Sensors and Actuators B 113 (2006) 156–161
Simultaneous determination of epinephrine and ascorbic acid at the electrochemical sensor of triazole SAM modified gold electrode Yi-Xin Sun, Sheng-Fu Wang ∗ , Xiu-Hua Zhang, Yin-Fang Huang Faculty of Chemistry and Material Science, Hubei University, Wuhan 430062, PR China Received 11 October 2004; received in revised form 15 February 2005; accepted 17 February 2005 Available online 22 March 2005
Abstract The electrochemical sensor of triazole (TA) self-assembled monolayer (SAM) modified gold electrode (TA SAM/Au) was fabricated. The electrochemical behaviors of epinephrine (EP) at TA SAM/Au have been studied. The TA SAM/Au shows an excellent electrocatalytic activity for the oxidation of EP and accelerates electron transfer rate. The diffusion coefficient is 1.135 × 10−6 cm2 s−1 . Under the optimum experiment conditions (i.e. 0.1 mol L−1 , pH 4.4, sodium borate buffer, accumulation time: 180 s, accumulation potential: 0.6 V, scan rate: 0.1 Vs−1 ), the cathodic peak current of EP versus its concentration has a good linear relation in the ranges of 1.0 × 10−7 to 1.0 × 10−5 mol L−1 and 1.0 × 10−5 to 6.0 × 10−4 mol L−1 by square wave adsorptive stripping voltammetry (SWASV), with the correlation coefficient of 0.9985 and 0.9996, respectively. Detection limit is down to 1.0 × 10−8 mol L−1 . The TA SAM/Au can be used for the determination of EP in practical injection. Meantime, the oxidative peak potentials of EP and ascorbic acid (AA) are well separated about 200 ± 10 mV at TA SAM/Au, the oxidation peak current increases approximately linearly with increasing concentration of both EP and AA in the concentration range of 2.0 × 10−5 to 1.6 × 10−4 mol L−1 . It can be used for simultaneous determination of EP and AA. © 2005 Elsevier B.V. All rights reserved. Keywords: Triazole; SAM; Epinephrine; Ascorbic acid; Simultaneous determination
1. Introduction Catecholamines, such as dopamine and epinephrine, are kinds of important compounds for the message transfer in the mammalian central nervous system, which exist as an organic cation in the nervous tissue and biological body fluid [1]. Many diseases are related to changes of their concentration. Quantitative determinations of them are significant method for developing nerve physiology, making diagnosis and controlling medicine [2]. The methods for epinephrine (EP) analysis are based on the native fluorescence of original amines. The trihidroxiindol method requires prior separation in a weak cationic resin for subsequent determination by ∗
Corresponding author. Tel.: +86 27 50865498; fax: +86 27 88663043. E-mail addresses: [email protected] (Y.-X. Sun), [email protected] (S.-F. Wang). 0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.02.042
fluorescence. Present methods focus on electrochemical detection [3]. The electron transfer rate of EP at bare electrode is slow, because it is often adsorbed on the surface of bare electrode for subsequent passivation. Recently, an enormous amount of research has been devoted to the development of new modified electrode for monitoring EP [4–6]. EP and ascorbic acid (AA) always exist together in biological environment, at most solid electrodes; AA is oxidized at potentials close to that of the EP, resulting in an overlapping voltammetric response. Therefore, a lot of chemically modified electrodes have been developed to eliminate the interference of AA to EP determination [7] or separate the electrochemical response of EP and AA [4,8]. Self-assembled monolayer (SAM) provides a means for controlling the chemical nature of the electrode solution interface. The field of SAM modified electrode has witnessed tremendous growth in electrochemistry over the past 15 years.
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
Many self-assembly systems have been invested, but monolayers of thiolates on gold electrode are probably the most studied SAM to date [9–11]. The use of SAM modified electrodes to improve the selectivity and sensitivity of gold electrodes has been reported [12]. Usually, long-chain thiols can form stable, well-ordered SAM; however, their transfer rate of electron was much slower. In comparison with long-chain thiols, short-chain thiols lead to less order structures but their transfer rate was much quicker [13]. In recent years, our group has studied many kinds of short chain mercaptocompounds as SAM [14–17]. In this paper, TA (3-amino-5-mercapto-1,2,4-triazole) was used as the assembling molecule and the triazole selfassembled monolayer modified gold electrode (TA SAM/Au) was prepared for the determination of EP injection and simultaneous determination of EP and AA. The scheme of TA is shown as follows:
2. Experimental 2.1. Apparatus Electrochemical measurements were carried out on CHI660 (USA). A three-electrode system is used in the measurements, with a bare gold electrode (d =2 mm) or TA SAM/Au as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and Pt wire as the counter electrode. All potentials given are referred to the SCE. The pH values were measured with a pHS-3C pH meter (Shanghai, China). 2.2. Reagents
157
soaked in methanol for 2 h to remove the physically adsorbed TA, and then a TA SAM/Au was obtained.
3. Results and discussion 3.1. Cyclic voltammetric behaviors of EP at TA SAM/Au There are no redox peaks in cyclic voltammograms of the bare electrode and TA SAM/Au (Fig. 1a) in the potential range of 0.8 V to −0.4 V in 0.1 mol L−1 pH 4.4 BR buffer. Compared with bare gold electrode, its background current dropped greatly. It is probable that the bare electrode was modified efficiently by TA SAM, and the AZ SAM blocks access of solvent molecules and electrolyte ions to the gold surface. As can be seen, EP exhibits a weak electrochemical response at a bare gold electrode in the given potential range (Fig. 1b). The peak current of EP at the bare gold electrode is small and is not suitable for the voltammetric determination of EP. But the cyclic voltammogram of EP at TA SAM/Au (Fig. 1c) shows two pairs of redox peaks: the first pair of redox peak potential is 0.239 V (cathodic peak potential, Epc1 ) and 0.312 V (anodic peak potential, Epa1 ), and the second pair of redox peak potential is −0.142 V (Epc2 ) and −0.078 V (Epa2 ), and the formal potentials (E0 ) of EP are 0.276 V and −0.110 V, respectively. 3.2. Relationship between pH values peak potentials An increase of pH leads to a negative shift in potential for both the second reduction and oxidation peak. Fig. 2 shows the relationship between the second redox peak potential of EP and pH, the equations were as following: Epc2 (V) = 0.2104–0.06061 pH, R = 0.9988; Epa2 (V) = 0.1675–0.05876 pH, R = 0.9989; E20 (V) = 0.1890– 0.0597 pH, R = 0.9989.
TA and EP were purchased from Sigma Chemical Co. (USA), the injection is an aqueous solution of EP with a concentration of EP of 1 mg mL−1 (Pharmacal Factory of Jin Tan, Jiangshu), a piece of AA tablets is 0.1 g (Hospital of Hubei Province). Other reagents were of analytical grade. All solutions were prepared with twice-distilled water. The experimental results are obtained at room temperature. 2.3. Preparation of TA SAM/Au The bare gold electrode was polished to a mirror-like surface with 0.5 m, 0.05 m ␣-Al2 O3 , and then rinsed ultrasonically with water and absolute ethanol and sonicated in twice-distilled water. This electrode was voltammetrically cycled and characterized in 1.0 mol L−1 H2 SO4 until a stable cyclic voltammogram was obtained. The cleaned gold electrode was immersed in TA methanol solution for 12 h at room temperature, and then washed thoroughly with methanol and
Fig. 1. Cyclic voltammograms: 1.0 × 10−4 mol L−1 EP in 0.1 mol/L pH 4.4 BR buffer solution, at a bare electrode (b) and TA SAM/Au (c); absence of EP in solution at TA SAM/Au (a), scan rate: 0.1 V/s.
158
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
Fig. 2. The relationship between Epc2 and pH (inset: SWASV of the Epc2 of 1.0 × 10−5 mol L−1 EP at TA SAM/Au at different pH: (a–g) 3.0, 4.0, 4.4, 5.0, 6.2, 7.0, 8.0).
Fig. 3. Plot of ipc and Eacc in EP concentration 1.0 × 10−5 mol L−1 (tacc : 180 s, pH 4.4).
3.3. Selection of the optimum experimental conditions All these slope values of linear relationship of Ep versus pH were approximately close to the theoretical value of −57.6 mV pH−1 at 18 ◦ C for a reversible proton-coupled single electron transfer [18,19]. Because the number of H+ is 2 and so the number of electron transfer is also 2. The results are agreed with CV above. A coulometeric study above showed that the number of the electrons involved in the process, n, was 2. Thus, the proton numbers intervening in the redox process could also be calculated and found to be approximately 2 from the slope of above equations. Therefore, the proposed redox mechanism for EP can be written as follows, this is in agreement with the previous report [20–23]:
The redox peaks Ep1 and Ep2 of EP (Fig. 1) are consistent with combination of the electron transfer processes of Eqs. (1) and (3), respectively. In pH 4.4 BR buffer solution, we found that the amplitude of the second wave for EP is enhanced obviously through deposition on TA/Au SAM surface. This indicates that the oxidation process at the second wave must be preceded by adsorption of the o-quinone at the electrode surface at this case [2]. So, we chose the second redox couple as our research emphases in subsequent experiment.
3.3.1. The optimum accumulation potential We analyzed the accumulation potential between 0 and 0.9 V by SWASV (Fig. 3). A solution of EP (1.0 × 10−5 mol L−1 ) is used in 0.1 mol L−1 BR buffer solution at pH 4.4, varying the accumulation potential, with a 180 s accumulation time. The peak current value reaches the highest at 0.6 V; therefore, it is the optimum accumulation potential. The effect of accumulation time had a great influence on the peak current value (Fig. 4). To accomplish this, we varied the accumulation time between 0 and 600 s for 1.0 × 10−5 mol L−1 EP in the measurement cell in a BR buffer at pH 4.4, in all cases without shaking. Measurements were carried out applying an accumulation potential of 0.6 V. As can be seen, the peak current increases as the tacc increases and reaches a maximum around 180 s. In light of these results, we chose 180 s as the accumulation time for subsequent analyses. The SWV parameters that were investigated are the frequency, the pulse amplitude and the pulse increment. These parameters are interrelated and have a combined effect on
Fig. 4. Plot of ipc and tacc in EP concentration 1.0 × 10−5 mol L−1 (Eacc : 0.6 V, pH 4.4).
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
159
Table 1 Recoveries of EP in injection
Fig. 5. SWSVs of EP at TA SAM/Au at different EP concentrations (bottom to top) 0.1, 0.4, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 × 10−6 mol L−1 , linear relationship between ipc and cEP (inset).
the current response. The experiment showed the frequency of 30 Hz, the pulse amplitude of 25 mV and the pulse increment of 4 mV were the optimum. 3.4. Analytical application 3.4.1. The linear relation range and the relative standard deviation There are two good linear ranges as follows (Figs. 5 and 6), which the regression equations are ipc = 6.954 × 10−7 + 0.083c (ipc : A, c: mol L−1 , 1.0 × 10−7 to 1.0 × 10−5 mol L−1 ) and ipc = 1.519 × 10−6 + 0.012c (ipc : A, c: mol L−1 , 1.0 × 10−5 to 6.0 × 10−4 mol L−1 ), their correlation coefficients are 0.9985 and 0.9996, respectively. A low detection limit (three times the signal blank/slope) of 1.0 × 10−8 mol L−1 is obtained. The relative standard deviation (R.S.D.) of 1.5% for 1.0 × 10−6 mol L−1 EP (n = 9) showed excellent stability and reproducibility. It is fit for quantitative determination of EP.
Fig. 6. SWSVs of EP at TA SAM/Au at different EP concentrations (bottom to top): 0.1, 0.3, 0.6, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 × 10−4 mol L−1 , linear relationship between ipc and cEP (inset).
Samples (c/10−5 mol L−1 )
Added (c/10−5 mol L−1 )
Found (c/10−5 mol L−1 )
Recoveries (%)
1.09 1.09 1.09 1.09 1.09
0.90 1.50 3.50 4.50 5.50
1.92 2.47 4.60 5.49 6.41
92.2 92.0 100.3 97.8 96.7
3.4.2. Determination of EP Take EP injection quantitatively. The average result of EP in injection of epinephrine hydrochloride by TA SAM/Au is 0.98 ± 0.05 mg mL−1 , which is quite corresponding to reference value (1 mg mL−1 ) and was given by injection specification. Table 1 show the result of the recoveries test. Recoveries of 92.0% and 100.3% of EP from the injection EP samples were obtained using the modified electrode. In this study, 18, 30, 70, 90, 110 L of 2.5 × 10−3 mol L−1 EP injection were added to 5.00 mL diluted (500-fold) EP injection, respectively. The good agreement with the method is a promising feature for the applicability of the modified electrode for direct determination of EP in real sample. 3.4.3. Simultaneous determination of EP and AA EP and AA always exist together in biological environment. Simultaneous determination of EP and AA is difficult at a bare gold electrode and other solid electrode. But the peak potentials can be distinguished at TA SAM/Au. According to the remarkable effect of pH on the peak potential, there was a good separation of peak potentials in pH 2.0 BR solution, so we chose pH 2.0 in the simultaneous determination of EP and AA. Fig. 7 shows semi-derivative linear sweep voltammetry (SLSV) curves at different concentrations of AA in the presence of EP. The peak current and potential of EP were almost unchanged during the increasing concentration of AA, and the peak current of AA increased linearly with increasing AA concentration (9.900 × 10−6 to 2.153 × 10−4 mol L−1 ) with a correlation coefficient of 0.9989. The detection limit (3σ) for AA in the presence of 100-fold excess of EP was found to be 1.0 × 10−6 mol L−1 . Fig. 8 shows the SLSV curves of EP and AA in simultaneously changing the concentration. The oxidation peak current increases approximately linearly with increasing concentration of both EP and AA in the concentration range of 2.0 × 10−5 to 1.6 × 10−4 mol L−1 .The linear regression equations were: ipa =−3.234 × 10−7 − 0.0070c, R = 0.9965 (EP); ipa = −1.459 × 10−7 − 0.0066c, R = 0.9946 (AA). The method can be used for simultaneous determination of EP and AA. EP and AA in simulated samples containing 1 × 10−3 mol L−1 EP and 1 × 10−3 mol L−1 AA were determined by the calibration curve method with the TA/Au SAM modified electrode. In this study, 10 L, 20 L,
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
160
Table 2 Recoveries of AA and EP in simulated samples Samples (c/10−5 mol L−1 )
Added (c/10−5 mol L−1 )
Found (c/10−5 mol L−1 )
Recoveries (%)
cAA
cEP
cAA
cEP
cAA
cEP
AA
EP
4.00 4.00 4.00 4.00 4.00
4.00 4.00 4.00 4.00 4.00
0.199 0.398 0.597 0.796 0.995
0.54 1.08 1.62 2.16 2.70
4.210 4.398 4.602 4.753 4.970
4.551 5.110 5.605 2.158 6.668
106 99.5 100.8 94.6 97.5
102 103 101.3 99.9 98.8
Fig. 7. SLSV curves of different AA concentration in the presence of EP (1.0 × 10−4 mol L−1 ) at TA SAM/Au. The concentrations of AA (bottom to top): 0.990, 1.996, 2.996, 3.984, 5.964, 7.937, 9.901, 11.86, 13.81, 15.75, 17.68, 19.61, 21.53 × 10−5 mol L−1 , pH 2.0: (a), quite time: 90 s, plot of ipa vs. cAA (b).
Fig. 8. SLSV curves of EP and AA at TA SAM/Au. Concentrations of both were changed simultaneously. The concentrations of AA (bottom to top): 1.99, 3.97, 6.93, 7.87, 9.80, 11.72, 13.62, 15.5 × 10−5 mol L−1 . The concentration of EP (bottom to top): 1.99, 3.97, 5.92, 7.87, 9.80, 11.72, 13.62, 15.5 × 10−5 mol L−1 .
30 L, 40 L, 50 L of 1.0 × 10−3 mol L−1 AA and 10 L, 20 L, 30 L, 40 L, 50 L of 2.7 × 10−3 mol L−1 EP were added to 5.00 mL containing 1.0 × 10−5 mol L−1 AA and 1.0 × 10−5 mol L−1 EP solution, respectively. The result is listed in Table 2.
cessfully at TA SAM/Au by LSV at pH 2.0 BR, it is possible that TA SAM/Au can be used for simultaneous determination of EP and AA.
Acknowledgements 4. Conclusions TA SAM/Au was fabricated and several methods are used to characterize the modified monolayer. CV results show that TA can act as a promoter to accelerate the electrochemical reaction of EP. There are good linear relations between ipc and cEP in the ranges of 1 × 10−7 to 1 × 10−5 mol L−1 and 1 × 10−5 to 6 × 10−4 mol L−1 , so TA SAM/Au is fit for the quantitative determination of EP in EP injection. In the meantime, the peak potentials of AA and EP can be separated suc-
This work was supported by Natural Science Foundation of Hubei province (No. 2002AB047) and Natural Science Foundation of Hubei province Education Department (No. 2002A00018).
References [1] Q.M. Xue, Physiological and Pathological Chemistry of Nervous System, Science Press, Beijing, 1978, p. 102.
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161 [2] K. Pihel, T.J. Schroeder, R.M. Wightman, Rapid and selective cyclic voltammetric measurements of epinephrine and norepinephrine as a method to measure secretion from single bovine adrenal medullary cells, Anal. Chem. 66 (1994) 4532–4537. [3] M. Ponti´e, C. Gobin, T. Pauport´e, F. Bedioui, J. Devynck, Electrochemical nitric oxide microsensors: sensitivity and selectivity characterization, Anal. Chim. Acta 411 (1–2) (2000) 175–185. [4] H.-M. Zhang, X.-L. Zhou, R.-T. Hui, N.-Q. Li, D.-P. Liu, Studies of the electrochemical behavior of epinephrine at a homocysteine self-assembled electrode, Talanta 56 (2002) 1081–1088. [5] S.H. Kim, J.W. Lee, I.-H. Yeo, Spectroelectrochemical and electrochemical behavior of epinephrine at a gold electrode, Electrochim. Acta 45 (2000) 2889–2895. [6] H.-M. Zhang, X.-B. Wang, L.-J. Wan, Y.-Q. Liu, C.-L. Bai, Electrochemical behavior of multi-wall carbon nanotubes and electrocatalysis of toluene-filled nanotube film on gold electrode, Electrochim. Acta 49 (2004) 715–719. [7] J-A. Ni, H-X. Ju, H-Y. Chen, D´onal Leech, Amperometric determination of epinephrine with an osmium complex and Nafion doublelayer membrane modified electrode, Anal. Chim. Acta 378 (1999) 151–157. [8] J. Wang, P. Tuzhi, T. Golden, Amperometric detection of cationic neurotransmitters at nafion-coated glassy carbon electrodes in flow streams, Anal. Chim. Acta 194 (1987) 129–138. [9] S. Arnold, Z.-Q. Feng, T. Kakiuchi, W. Knoll, K. Niki, Investigation of the electrode reaction of cytochrome c through mixed selfassembled monolayers of alkanethiols on gold(III) surfaces, J. Electroanal. Chem. 438 (1997) 91–97. [10] W. Pan, C.J. Durning, N.J. Turro, Kinetics of alkanethiol adsorption on gold, Langmuir 12 (18) (1996) 4469–4473. [11] T. Liu, M.X. Li, Q.Y. Li, Electroanalysis of dopamine at a gold electrode modified with N-acetylcysteine self-assembled monolayer, Talanta 63 (2004) 1053–1059. [12] C.R. Raj, T. Ohsaka, Voltammetric detection of uric acid in the presence of ascorbic acid at a gold electrode modified with a selfassembled monolayer of heteroaromatic thiol, J. Electroanal. Chem. 540 (2003) 69–77. [13] I. Turyan, D. Mandler, Self-assembled monolayers in electroanalytical chemistry: Application of omega-mercaptocarboxylic acid monolayers for electrochemical determination of ultralow levels of cadmium(II), Anal. Chem. 66 (1) (1994) 58. [14] S.-F. Wang, D. Du, Q.-C. Zou, Electrochemical behavior of epinephrine at l-cysteine self-assembled monolayers modified gold electrode, Talanta 57 (2002) 687–692. [15] S.-F. Wang, D. Du, Preparation and electrochemical properties of Keggin-type phosphomolybdic anions in electrostaticly linked lcysteine self-assembled monolayers, Sens. Actuators B: Chem. 94 (3) (2003) 282–289.
161
[16] X.-H. Zhang, S.-F. Wang, Differential pulse adsorptive stripping voltammetry determination of epinephrine with -mercaptoethanol self-assembled monolayers modified electrode, Anal. Lett. 35 (6) (2002) 995–1006. [17] X.-H. Zhang, S.-F. Wang, Determination of ethamsylate in the presence of catecholamines using 4-amino-2-mercaptopyrimidine selfassembled momolayer gold electrode, Sens. Actuators B 104 (2005) 29–34. [18] A.M. Bond, Modern Polarographic Methods In Analytical Chemistry, Marcel Dekker, New York, 1980, p. 29. [19] L. Meites, Polarographic Techniques, second ed., Wiley, New York, 1965, p. 282. [20] I. Hern´andez, F. S´anchez, L. Pat´on, Hern´andez cyclic voltammetry determination of epinephrine with a carbon fiber ultramicroelectrode, Talanta 46 (1998) 985–991. [21] X.-L. Wen, Y.-H. Jia, Z.-L. Liu, Micellar effects on the electrochemistry of dopamine and its selective detection in the presence of ascorbic acid, Talanta 50 (1999) 1027–1033. [22] J. Wang, A. Walcarius, Zeolite-modified carbon paste electrode for selective monitoring of dopamine, J. Electroanal. Chem. 407 (1996) 183–187. [23] S.-M. Chen, K.-T. Peng, The electrochemical properties of dopamine, epinephrine, norepinephrine, and their electrocatalytic reactions on cobalt (II) hexacyanoferrate films, J. Electroanal. Chem. 547 (2003) 179–189.
Biographies Yi-Xin Sun received her BSc in analytical chemistry in 1995 from Hubei University, and will obtain her MSc in analytical chemistry in 2005 from Hubei University. Sheng-Fu Wang received his BSc in analytical chemistry in 1987 from Hubei University and MSc in analytical chemistry in 1992 from Wuhan University, and will obtain his PhD in analytical chemistry in 2005 from Wuhan University. He has been a professor at Hubei University since 1996. His current interests include the development of new chemical sensors and biosensors. Yin-Fang Huang received her BSc in analytical chemistry from Hubei University in 2004. Xiu-Hua Zhang received his BSc and MSc in analytical chemistry from Hubei University in 1991 and 2003, respectively. He has been an Assistant Professor at Hubei University since 2004. His current interests include the development of new chemical sensors and nanoelectrochemistry.
Simultaneous determination of epinephrine and ascorbic acid at the electrochemical sensor of triazole SAM modified gold electrode Yi-Xin Sun, Sheng-Fu Wang ∗ , Xiu-Hua Zhang, Yin-Fang Huang Faculty of Chemistry and Material Science, Hubei University, Wuhan 430062, PR China Received 11 October 2004; received in revised form 15 February 2005; accepted 17 February 2005 Available online 22 March 2005
Abstract The electrochemical sensor of triazole (TA) self-assembled monolayer (SAM) modified gold electrode (TA SAM/Au) was fabricated. The electrochemical behaviors of epinephrine (EP) at TA SAM/Au have been studied. The TA SAM/Au shows an excellent electrocatalytic activity for the oxidation of EP and accelerates electron transfer rate. The diffusion coefficient is 1.135 × 10−6 cm2 s−1 . Under the optimum experiment conditions (i.e. 0.1 mol L−1 , pH 4.4, sodium borate buffer, accumulation time: 180 s, accumulation potential: 0.6 V, scan rate: 0.1 Vs−1 ), the cathodic peak current of EP versus its concentration has a good linear relation in the ranges of 1.0 × 10−7 to 1.0 × 10−5 mol L−1 and 1.0 × 10−5 to 6.0 × 10−4 mol L−1 by square wave adsorptive stripping voltammetry (SWASV), with the correlation coefficient of 0.9985 and 0.9996, respectively. Detection limit is down to 1.0 × 10−8 mol L−1 . The TA SAM/Au can be used for the determination of EP in practical injection. Meantime, the oxidative peak potentials of EP and ascorbic acid (AA) are well separated about 200 ± 10 mV at TA SAM/Au, the oxidation peak current increases approximately linearly with increasing concentration of both EP and AA in the concentration range of 2.0 × 10−5 to 1.6 × 10−4 mol L−1 . It can be used for simultaneous determination of EP and AA. © 2005 Elsevier B.V. All rights reserved. Keywords: Triazole; SAM; Epinephrine; Ascorbic acid; Simultaneous determination
1. Introduction Catecholamines, such as dopamine and epinephrine, are kinds of important compounds for the message transfer in the mammalian central nervous system, which exist as an organic cation in the nervous tissue and biological body fluid [1]. Many diseases are related to changes of their concentration. Quantitative determinations of them are significant method for developing nerve physiology, making diagnosis and controlling medicine [2]. The methods for epinephrine (EP) analysis are based on the native fluorescence of original amines. The trihidroxiindol method requires prior separation in a weak cationic resin for subsequent determination by ∗
Corresponding author. Tel.: +86 27 50865498; fax: +86 27 88663043. E-mail addresses: [email protected] (Y.-X. Sun), [email protected] (S.-F. Wang). 0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2005.02.042
fluorescence. Present methods focus on electrochemical detection [3]. The electron transfer rate of EP at bare electrode is slow, because it is often adsorbed on the surface of bare electrode for subsequent passivation. Recently, an enormous amount of research has been devoted to the development of new modified electrode for monitoring EP [4–6]. EP and ascorbic acid (AA) always exist together in biological environment, at most solid electrodes; AA is oxidized at potentials close to that of the EP, resulting in an overlapping voltammetric response. Therefore, a lot of chemically modified electrodes have been developed to eliminate the interference of AA to EP determination [7] or separate the electrochemical response of EP and AA [4,8]. Self-assembled monolayer (SAM) provides a means for controlling the chemical nature of the electrode solution interface. The field of SAM modified electrode has witnessed tremendous growth in electrochemistry over the past 15 years.
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
Many self-assembly systems have been invested, but monolayers of thiolates on gold electrode are probably the most studied SAM to date [9–11]. The use of SAM modified electrodes to improve the selectivity and sensitivity of gold electrodes has been reported [12]. Usually, long-chain thiols can form stable, well-ordered SAM; however, their transfer rate of electron was much slower. In comparison with long-chain thiols, short-chain thiols lead to less order structures but their transfer rate was much quicker [13]. In recent years, our group has studied many kinds of short chain mercaptocompounds as SAM [14–17]. In this paper, TA (3-amino-5-mercapto-1,2,4-triazole) was used as the assembling molecule and the triazole selfassembled monolayer modified gold electrode (TA SAM/Au) was prepared for the determination of EP injection and simultaneous determination of EP and AA. The scheme of TA is shown as follows:
2. Experimental 2.1. Apparatus Electrochemical measurements were carried out on CHI660 (USA). A three-electrode system is used in the measurements, with a bare gold electrode (d =2 mm) or TA SAM/Au as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and Pt wire as the counter electrode. All potentials given are referred to the SCE. The pH values were measured with a pHS-3C pH meter (Shanghai, China). 2.2. Reagents
157
soaked in methanol for 2 h to remove the physically adsorbed TA, and then a TA SAM/Au was obtained.
3. Results and discussion 3.1. Cyclic voltammetric behaviors of EP at TA SAM/Au There are no redox peaks in cyclic voltammograms of the bare electrode and TA SAM/Au (Fig. 1a) in the potential range of 0.8 V to −0.4 V in 0.1 mol L−1 pH 4.4 BR buffer. Compared with bare gold electrode, its background current dropped greatly. It is probable that the bare electrode was modified efficiently by TA SAM, and the AZ SAM blocks access of solvent molecules and electrolyte ions to the gold surface. As can be seen, EP exhibits a weak electrochemical response at a bare gold electrode in the given potential range (Fig. 1b). The peak current of EP at the bare gold electrode is small and is not suitable for the voltammetric determination of EP. But the cyclic voltammogram of EP at TA SAM/Au (Fig. 1c) shows two pairs of redox peaks: the first pair of redox peak potential is 0.239 V (cathodic peak potential, Epc1 ) and 0.312 V (anodic peak potential, Epa1 ), and the second pair of redox peak potential is −0.142 V (Epc2 ) and −0.078 V (Epa2 ), and the formal potentials (E0 ) of EP are 0.276 V and −0.110 V, respectively. 3.2. Relationship between pH values peak potentials An increase of pH leads to a negative shift in potential for both the second reduction and oxidation peak. Fig. 2 shows the relationship between the second redox peak potential of EP and pH, the equations were as following: Epc2 (V) = 0.2104–0.06061 pH, R = 0.9988; Epa2 (V) = 0.1675–0.05876 pH, R = 0.9989; E20 (V) = 0.1890– 0.0597 pH, R = 0.9989.
TA and EP were purchased from Sigma Chemical Co. (USA), the injection is an aqueous solution of EP with a concentration of EP of 1 mg mL−1 (Pharmacal Factory of Jin Tan, Jiangshu), a piece of AA tablets is 0.1 g (Hospital of Hubei Province). Other reagents were of analytical grade. All solutions were prepared with twice-distilled water. The experimental results are obtained at room temperature. 2.3. Preparation of TA SAM/Au The bare gold electrode was polished to a mirror-like surface with 0.5 m, 0.05 m ␣-Al2 O3 , and then rinsed ultrasonically with water and absolute ethanol and sonicated in twice-distilled water. This electrode was voltammetrically cycled and characterized in 1.0 mol L−1 H2 SO4 until a stable cyclic voltammogram was obtained. The cleaned gold electrode was immersed in TA methanol solution for 12 h at room temperature, and then washed thoroughly with methanol and
Fig. 1. Cyclic voltammograms: 1.0 × 10−4 mol L−1 EP in 0.1 mol/L pH 4.4 BR buffer solution, at a bare electrode (b) and TA SAM/Au (c); absence of EP in solution at TA SAM/Au (a), scan rate: 0.1 V/s.
158
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
Fig. 2. The relationship between Epc2 and pH (inset: SWASV of the Epc2 of 1.0 × 10−5 mol L−1 EP at TA SAM/Au at different pH: (a–g) 3.0, 4.0, 4.4, 5.0, 6.2, 7.0, 8.0).
Fig. 3. Plot of ipc and Eacc in EP concentration 1.0 × 10−5 mol L−1 (tacc : 180 s, pH 4.4).
3.3. Selection of the optimum experimental conditions All these slope values of linear relationship of Ep versus pH were approximately close to the theoretical value of −57.6 mV pH−1 at 18 ◦ C for a reversible proton-coupled single electron transfer [18,19]. Because the number of H+ is 2 and so the number of electron transfer is also 2. The results are agreed with CV above. A coulometeric study above showed that the number of the electrons involved in the process, n, was 2. Thus, the proton numbers intervening in the redox process could also be calculated and found to be approximately 2 from the slope of above equations. Therefore, the proposed redox mechanism for EP can be written as follows, this is in agreement with the previous report [20–23]:
The redox peaks Ep1 and Ep2 of EP (Fig. 1) are consistent with combination of the electron transfer processes of Eqs. (1) and (3), respectively. In pH 4.4 BR buffer solution, we found that the amplitude of the second wave for EP is enhanced obviously through deposition on TA/Au SAM surface. This indicates that the oxidation process at the second wave must be preceded by adsorption of the o-quinone at the electrode surface at this case [2]. So, we chose the second redox couple as our research emphases in subsequent experiment.
3.3.1. The optimum accumulation potential We analyzed the accumulation potential between 0 and 0.9 V by SWASV (Fig. 3). A solution of EP (1.0 × 10−5 mol L−1 ) is used in 0.1 mol L−1 BR buffer solution at pH 4.4, varying the accumulation potential, with a 180 s accumulation time. The peak current value reaches the highest at 0.6 V; therefore, it is the optimum accumulation potential. The effect of accumulation time had a great influence on the peak current value (Fig. 4). To accomplish this, we varied the accumulation time between 0 and 600 s for 1.0 × 10−5 mol L−1 EP in the measurement cell in a BR buffer at pH 4.4, in all cases without shaking. Measurements were carried out applying an accumulation potential of 0.6 V. As can be seen, the peak current increases as the tacc increases and reaches a maximum around 180 s. In light of these results, we chose 180 s as the accumulation time for subsequent analyses. The SWV parameters that were investigated are the frequency, the pulse amplitude and the pulse increment. These parameters are interrelated and have a combined effect on
Fig. 4. Plot of ipc and tacc in EP concentration 1.0 × 10−5 mol L−1 (Eacc : 0.6 V, pH 4.4).
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
159
Table 1 Recoveries of EP in injection
Fig. 5. SWSVs of EP at TA SAM/Au at different EP concentrations (bottom to top) 0.1, 0.4, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 × 10−6 mol L−1 , linear relationship between ipc and cEP (inset).
the current response. The experiment showed the frequency of 30 Hz, the pulse amplitude of 25 mV and the pulse increment of 4 mV were the optimum. 3.4. Analytical application 3.4.1. The linear relation range and the relative standard deviation There are two good linear ranges as follows (Figs. 5 and 6), which the regression equations are ipc = 6.954 × 10−7 + 0.083c (ipc : A, c: mol L−1 , 1.0 × 10−7 to 1.0 × 10−5 mol L−1 ) and ipc = 1.519 × 10−6 + 0.012c (ipc : A, c: mol L−1 , 1.0 × 10−5 to 6.0 × 10−4 mol L−1 ), their correlation coefficients are 0.9985 and 0.9996, respectively. A low detection limit (three times the signal blank/slope) of 1.0 × 10−8 mol L−1 is obtained. The relative standard deviation (R.S.D.) of 1.5% for 1.0 × 10−6 mol L−1 EP (n = 9) showed excellent stability and reproducibility. It is fit for quantitative determination of EP.
Fig. 6. SWSVs of EP at TA SAM/Au at different EP concentrations (bottom to top): 0.1, 0.3, 0.6, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 × 10−4 mol L−1 , linear relationship between ipc and cEP (inset).
Samples (c/10−5 mol L−1 )
Added (c/10−5 mol L−1 )
Found (c/10−5 mol L−1 )
Recoveries (%)
1.09 1.09 1.09 1.09 1.09
0.90 1.50 3.50 4.50 5.50
1.92 2.47 4.60 5.49 6.41
92.2 92.0 100.3 97.8 96.7
3.4.2. Determination of EP Take EP injection quantitatively. The average result of EP in injection of epinephrine hydrochloride by TA SAM/Au is 0.98 ± 0.05 mg mL−1 , which is quite corresponding to reference value (1 mg mL−1 ) and was given by injection specification. Table 1 show the result of the recoveries test. Recoveries of 92.0% and 100.3% of EP from the injection EP samples were obtained using the modified electrode. In this study, 18, 30, 70, 90, 110 L of 2.5 × 10−3 mol L−1 EP injection were added to 5.00 mL diluted (500-fold) EP injection, respectively. The good agreement with the method is a promising feature for the applicability of the modified electrode for direct determination of EP in real sample. 3.4.3. Simultaneous determination of EP and AA EP and AA always exist together in biological environment. Simultaneous determination of EP and AA is difficult at a bare gold electrode and other solid electrode. But the peak potentials can be distinguished at TA SAM/Au. According to the remarkable effect of pH on the peak potential, there was a good separation of peak potentials in pH 2.0 BR solution, so we chose pH 2.0 in the simultaneous determination of EP and AA. Fig. 7 shows semi-derivative linear sweep voltammetry (SLSV) curves at different concentrations of AA in the presence of EP. The peak current and potential of EP were almost unchanged during the increasing concentration of AA, and the peak current of AA increased linearly with increasing AA concentration (9.900 × 10−6 to 2.153 × 10−4 mol L−1 ) with a correlation coefficient of 0.9989. The detection limit (3σ) for AA in the presence of 100-fold excess of EP was found to be 1.0 × 10−6 mol L−1 . Fig. 8 shows the SLSV curves of EP and AA in simultaneously changing the concentration. The oxidation peak current increases approximately linearly with increasing concentration of both EP and AA in the concentration range of 2.0 × 10−5 to 1.6 × 10−4 mol L−1 .The linear regression equations were: ipa =−3.234 × 10−7 − 0.0070c, R = 0.9965 (EP); ipa = −1.459 × 10−7 − 0.0066c, R = 0.9946 (AA). The method can be used for simultaneous determination of EP and AA. EP and AA in simulated samples containing 1 × 10−3 mol L−1 EP and 1 × 10−3 mol L−1 AA were determined by the calibration curve method with the TA/Au SAM modified electrode. In this study, 10 L, 20 L,
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161
160
Table 2 Recoveries of AA and EP in simulated samples Samples (c/10−5 mol L−1 )
Added (c/10−5 mol L−1 )
Found (c/10−5 mol L−1 )
Recoveries (%)
cAA
cEP
cAA
cEP
cAA
cEP
AA
EP
4.00 4.00 4.00 4.00 4.00
4.00 4.00 4.00 4.00 4.00
0.199 0.398 0.597 0.796 0.995
0.54 1.08 1.62 2.16 2.70
4.210 4.398 4.602 4.753 4.970
4.551 5.110 5.605 2.158 6.668
106 99.5 100.8 94.6 97.5
102 103 101.3 99.9 98.8
Fig. 7. SLSV curves of different AA concentration in the presence of EP (1.0 × 10−4 mol L−1 ) at TA SAM/Au. The concentrations of AA (bottom to top): 0.990, 1.996, 2.996, 3.984, 5.964, 7.937, 9.901, 11.86, 13.81, 15.75, 17.68, 19.61, 21.53 × 10−5 mol L−1 , pH 2.0: (a), quite time: 90 s, plot of ipa vs. cAA (b).
Fig. 8. SLSV curves of EP and AA at TA SAM/Au. Concentrations of both were changed simultaneously. The concentrations of AA (bottom to top): 1.99, 3.97, 6.93, 7.87, 9.80, 11.72, 13.62, 15.5 × 10−5 mol L−1 . The concentration of EP (bottom to top): 1.99, 3.97, 5.92, 7.87, 9.80, 11.72, 13.62, 15.5 × 10−5 mol L−1 .
30 L, 40 L, 50 L of 1.0 × 10−3 mol L−1 AA and 10 L, 20 L, 30 L, 40 L, 50 L of 2.7 × 10−3 mol L−1 EP were added to 5.00 mL containing 1.0 × 10−5 mol L−1 AA and 1.0 × 10−5 mol L−1 EP solution, respectively. The result is listed in Table 2.
cessfully at TA SAM/Au by LSV at pH 2.0 BR, it is possible that TA SAM/Au can be used for simultaneous determination of EP and AA.
Acknowledgements 4. Conclusions TA SAM/Au was fabricated and several methods are used to characterize the modified monolayer. CV results show that TA can act as a promoter to accelerate the electrochemical reaction of EP. There are good linear relations between ipc and cEP in the ranges of 1 × 10−7 to 1 × 10−5 mol L−1 and 1 × 10−5 to 6 × 10−4 mol L−1 , so TA SAM/Au is fit for the quantitative determination of EP in EP injection. In the meantime, the peak potentials of AA and EP can be separated suc-
This work was supported by Natural Science Foundation of Hubei province (No. 2002AB047) and Natural Science Foundation of Hubei province Education Department (No. 2002A00018).
References [1] Q.M. Xue, Physiological and Pathological Chemistry of Nervous System, Science Press, Beijing, 1978, p. 102.
Y.-X. Sun et al. / Sensors and Actuators B 113 (2006) 156–161 [2] K. Pihel, T.J. Schroeder, R.M. Wightman, Rapid and selective cyclic voltammetric measurements of epinephrine and norepinephrine as a method to measure secretion from single bovine adrenal medullary cells, Anal. Chem. 66 (1994) 4532–4537. [3] M. Ponti´e, C. Gobin, T. Pauport´e, F. Bedioui, J. Devynck, Electrochemical nitric oxide microsensors: sensitivity and selectivity characterization, Anal. Chim. Acta 411 (1–2) (2000) 175–185. [4] H.-M. Zhang, X.-L. Zhou, R.-T. Hui, N.-Q. Li, D.-P. Liu, Studies of the electrochemical behavior of epinephrine at a homocysteine self-assembled electrode, Talanta 56 (2002) 1081–1088. [5] S.H. Kim, J.W. Lee, I.-H. Yeo, Spectroelectrochemical and electrochemical behavior of epinephrine at a gold electrode, Electrochim. Acta 45 (2000) 2889–2895. [6] H.-M. Zhang, X.-B. Wang, L.-J. Wan, Y.-Q. Liu, C.-L. Bai, Electrochemical behavior of multi-wall carbon nanotubes and electrocatalysis of toluene-filled nanotube film on gold electrode, Electrochim. Acta 49 (2004) 715–719. [7] J-A. Ni, H-X. Ju, H-Y. Chen, D´onal Leech, Amperometric determination of epinephrine with an osmium complex and Nafion doublelayer membrane modified electrode, Anal. Chim. Acta 378 (1999) 151–157. [8] J. Wang, P. Tuzhi, T. Golden, Amperometric detection of cationic neurotransmitters at nafion-coated glassy carbon electrodes in flow streams, Anal. Chim. Acta 194 (1987) 129–138. [9] S. Arnold, Z.-Q. Feng, T. Kakiuchi, W. Knoll, K. Niki, Investigation of the electrode reaction of cytochrome c through mixed selfassembled monolayers of alkanethiols on gold(III) surfaces, J. Electroanal. Chem. 438 (1997) 91–97. [10] W. Pan, C.J. Durning, N.J. Turro, Kinetics of alkanethiol adsorption on gold, Langmuir 12 (18) (1996) 4469–4473. [11] T. Liu, M.X. Li, Q.Y. Li, Electroanalysis of dopamine at a gold electrode modified with N-acetylcysteine self-assembled monolayer, Talanta 63 (2004) 1053–1059. [12] C.R. Raj, T. Ohsaka, Voltammetric detection of uric acid in the presence of ascorbic acid at a gold electrode modified with a selfassembled monolayer of heteroaromatic thiol, J. Electroanal. Chem. 540 (2003) 69–77. [13] I. Turyan, D. Mandler, Self-assembled monolayers in electroanalytical chemistry: Application of omega-mercaptocarboxylic acid monolayers for electrochemical determination of ultralow levels of cadmium(II), Anal. Chem. 66 (1) (1994) 58. [14] S.-F. Wang, D. Du, Q.-C. Zou, Electrochemical behavior of epinephrine at l-cysteine self-assembled monolayers modified gold electrode, Talanta 57 (2002) 687–692. [15] S.-F. Wang, D. Du, Preparation and electrochemical properties of Keggin-type phosphomolybdic anions in electrostaticly linked lcysteine self-assembled monolayers, Sens. Actuators B: Chem. 94 (3) (2003) 282–289.
161
[16] X.-H. Zhang, S.-F. Wang, Differential pulse adsorptive stripping voltammetry determination of epinephrine with -mercaptoethanol self-assembled monolayers modified electrode, Anal. Lett. 35 (6) (2002) 995–1006. [17] X.-H. Zhang, S.-F. Wang, Determination of ethamsylate in the presence of catecholamines using 4-amino-2-mercaptopyrimidine selfassembled momolayer gold electrode, Sens. Actuators B 104 (2005) 29–34. [18] A.M. Bond, Modern Polarographic Methods In Analytical Chemistry, Marcel Dekker, New York, 1980, p. 29. [19] L. Meites, Polarographic Techniques, second ed., Wiley, New York, 1965, p. 282. [20] I. Hern´andez, F. S´anchez, L. Pat´on, Hern´andez cyclic voltammetry determination of epinephrine with a carbon fiber ultramicroelectrode, Talanta 46 (1998) 985–991. [21] X.-L. Wen, Y.-H. Jia, Z.-L. Liu, Micellar effects on the electrochemistry of dopamine and its selective detection in the presence of ascorbic acid, Talanta 50 (1999) 1027–1033. [22] J. Wang, A. Walcarius, Zeolite-modified carbon paste electrode for selective monitoring of dopamine, J. Electroanal. Chem. 407 (1996) 183–187. [23] S.-M. Chen, K.-T. Peng, The electrochemical properties of dopamine, epinephrine, norepinephrine, and their electrocatalytic reactions on cobalt (II) hexacyanoferrate films, J. Electroanal. Chem. 547 (2003) 179–189.
Biographies Yi-Xin Sun received her BSc in analytical chemistry in 1995 from Hubei University, and will obtain her MSc in analytical chemistry in 2005 from Hubei University. Sheng-Fu Wang received his BSc in analytical chemistry in 1987 from Hubei University and MSc in analytical chemistry in 1992 from Wuhan University, and will obtain his PhD in analytical chemistry in 2005 from Wuhan University. He has been a professor at Hubei University since 1996. His current interests include the development of new chemical sensors and biosensors. Yin-Fang Huang received her BSc in analytical chemistry from Hubei University in 2004. Xiu-Hua Zhang received his BSc and MSc in analytical chemistry from Hubei University in 1991 and 2003, respectively. He has been an Assistant Professor at Hubei University since 2004. His current interests include the development of new chemical sensors and nanoelectrochemistry.