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Novel choline and acetylcholine modified glassy carbon electrodes for simultaneous determination of dopamine, serotonin and ascorbic acid
Journal of
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 569 (2004) 135–142 www.elsevier.com/locate/jelechem
Novel choline and acetylcholine modified glassy carbon electrodes for simultaneous determination of dopamine, serotonin and ascorbic acid Guan-Ping Jin, Xiang-Qin Lin *, Jing-ming Gong Department of Chemistry, University of Science and Technology of China, 96 Jinzhai, Hefei 230026, China Received 24 November 2003; received in revised form 18 February 2004; accepted 29 February 2004 Available online 6 May 2004
Abstract Choline (Ch) and acetylcholine (ACh) modified glassy carbon electrodes have been prepared and characterized by X-ray photoelectron spectroscopy (XPS), UV–visible spectroelectrochemistry (UV–Vis), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The modification of ACh was that the Ch was covalently bounded on the electrode surface accompanied by acetate anions embedded and adsorbed by electrostatics in the Ch modified layer, forming a novel choline/acetate interdigitate assembly. This provided an excellent example of a mixed two-component modification from a native acetylcholine molecule on a carbon electrode. These two modified electrodes were applied to the electrocatalytic oxidation of dopamine (DA), serotonin (5-HT) and ascorbic acid (AA), and resolved the overlapping of the anodic peaks of DA, 5-HT and AA into three well-defined voltammetric peaks in CV or different pulse voltammetry (DPV). This can be used for simultaneous determination of these species in a mixture. The stability of the acetate/choline/glass carbon electrode (ACh/GCE) sensing system was good, with up to at least 30 days of continual operation. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Choline; Acetylcholine; Modification; Separation
1. Introduction Dopamine (DA) is an important neuron-transmitter compound widely distributed in the brain for message transfer in the mammalian central nervous system. Low levels of DA have been found in patients with Parkinson’s disease [1]. Serotonin (5-HT) plays a crucial role in the emotional system together with other monoamine transmitters. Because DA and 5-HT are readily oxidized, hence, electrochemical methods have been explored for their analyses [1–7]. However, a major problem encountered in these determinations is the interference from ascorbic acids (AA) [8], which can be oxidized at potentials close to those of DA and 5-HT (while at pH >6, DA, AA and 5-HT overlap, at pH <5, *
Corresponding author. Tel.: +8005513606646; fax: +8005513601 592. E-mail address: [email protected] (X.-Q. Lin). 0022-0728/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2004.02.022
AA and DA overlap, and 5-HT is a single peak) at GCE electrodes. The ability to determine DA, 5-HT and AA selectively in a mixture is significant. Various methods, mainly based on the chemical modification of traditional electrode materials, have been developed to resolve the problem [8–13]. In particular polymer modified electrodes appear to have distinct advantages for their high catalytic function, good stability and broad potential window [14–21]. ACh has long been recognized as a crucial neurotransmitter in mammals. The distribution of neurons that contains ACh in the rat brain has been documented [22]. Ch is a precursor of ACh synthesis. Recently, there has been considerable interest in developing chemically modified electrodes for electroanalysis of ACh and Ch [23–26]. Although choline has an HO–, end group, which could be covalently bound to the edge plane sites of the carbon surface through the oxygen atom, similarly to alkanols [27–31], to the best of our knowledge,
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no surface modification of Ch or ACh has been studied for the fabrication of biosensors. Only the HO(CH2 )5 OH carbon fiber modified electrode has been reported for selective determination of DA in coexistence with AA [31]. The significance of Ch and ACh modification might provide a modified monolayer containing positively charged –Nþ (CH3 )3 polar head groups, which may play an important role in bio-active interactions. Evaluation of the electrostatic interaction between this positively charged modified layer and the cation and anions is of interest in designing biosensors. On the other hand, neurotransmitter molecule modified electrodes used for neurotransmitter determination should be an interesting way to evaluate the interactions between different kinds of neurotransmitter molecules. Actually, we have found interesting catalytic activities of Ch and ACh modified electrodes for DA, 5-HT and AA determinations. This report describes the preparation, characterization and mechanisms of these modified electrodes. It was found that the Ch and ACh modified electrodes not only exhibited strong catalytic ability toward the oxidation of DA, 5-HT and AA, but resolved their voltammetric responses into three well-defined voltammetric peaks, which can be used to determine DA, 5-HT and AA, simultaneously.
2. Experimental 2.1. Apparatus Electrochemical experiments such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed with a model CHI832 electrochemical analyzer (Cheng-Hua, Shanghai, China). Electrochemical impedance spectroscopy (EIS) was carried out with a CHI 660A workstation (Cheng-Hua, Shanghai, China). A conventional three-electrode system was used, which consisted of a working electrode, a twisted platinum wire counter electrode and a saturated calomel reference electrode (SCE). All potentials given in this paper are versus SCE. All experiments were performed at room temperature. The electrochemical solutions were thoroughly deoxygenated by N2 before sampling and an N2 atmosphere was maintained throughout the experiments. A home-made versatile long path-length thin-layer electrochemical cell was used for in situ spectroelectrochemical measurements as described previously [32]. Glassy carbon disk electrodes (GCE) with a geometric area of 0.125 cm2 were obtained from Tianjin-Lan-Like High Chemical Electronic Technology Company (Tianjin China). UV–Vis absorption spectra were measured with a UV-2401PC spectrophotometer (Shimadzu, Japan).
XPS was carried out using an ESCALAB MK2 spectrometer (VG Corporation, UK) with Mak-Alpha X-ray radiation as the excitation source. 2.2. Chemicals and solutions Choline (Ch) and acetylcholine (ACh) were purchased from the Chemical Reagent Factory of Beijing (Beijing China). Dopamine hydrochloride (DA) and serotonin (5-HT) were obtained from Sigma (USA). Ascorbic acid (AA) was obtained from Chemical Reagent Company of Shanghai (Shanghai China). All other reagents used were of analytical grade. Solutions of ACh and Ch were prepared in 0.01 M LiClO4: Solutions of DA, 5-HT and AA were prepared in water, prior to use. Acetonitrile (ACN) was of analytical quality and was dried over molecular before use. Phosphate-buffered saline (PBS; sieves (3 A) 0.1 M) solutions of different pH were prepared by mixing four stock solutions of 0.1 M H3 PO4 , KH2 PO4 , K2 HPO4 and K3 PO4 . Twice distilled water was used. High purity nitrogen was used for deaeration. All experiments were carried out at ambient temperature. 2.3. Preparation of modified electrode The GCE was prepared by polishing to a mirror-like finish with fine wet emery paper (grain size 4000). After sonicating in water for 15 min, it was resurfaced using 1.0 lM alumina slurry. After cleaning, the electrode was electrochemically pre-treated by cyclic scanning in 1.0 103 M ACh or Ch solution containing 0.01 M LiClO4 in the potential range of )1.7 to 1.8 V at 20 mV s1 for six scans. The electrode was then rinsed with ethanol and sonicated for 15 min in water to remove any physisorbed unreacted materials from the surface. The acetylcholine and choline modified electrodes were designated as ACh/GCE and Ch/GCE, respectively. They were stored in 0.1 M PBS (pH 7.0) and twice distilled water at 4 °C, respectively, and were ready for use. 2.4. Spectroelectrochemistry A 1 ml volume of ACh solution was injected into the cell through its reference electrode chamber and thinlayer compartment [32]. In this way, any air bubbles in the thin-layer compartment could be removed. A similar cell was set in the reference beam of the spectrophotometer for background subtraction during in situ measurement of UV–Vis absorption spectra. Each spectrum was measured after the thin-layer solution in the sample cell was electrolyzed at a steady potential for 2 min. 2.5. X-ray photoelectron spectroscopy (XPS) Surface analysis of the electrode was performed with an ESCA lab MK2 (VG, UK) with a Mo Ka radiation
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source at 50 and 0.05 eV per step. The elemental nitrogen-to-carbon ratio (N/C) was used to assess the extent of modifier coverage calculated by dividing the total number of counts under the N (1s) band by that under the C (1s) band.
137
(a)
2.6. Electrochemical impedance spectroscopy
50 µA
Electrochemical impedance spectroscopy (EIS) was carried out via the workstation. The glassy carbon electrodes before and after modification with ACh or Ch 4=3 were characterized in 0.1 M Fe(CN)6 + 0.1 M PBS (PH 7.0) via EIS methods. EIS data were measured at 100 kHz to 0.05 Hz and at an electrode-potential of 0.215 V, the formal potential of the ferrocyanide/ferricyanide couple.
(b)
25 µA
(c)
3. Results and discussion 3.1. Electrochemical modification of Ch and ACh on GCE The ACh modification was conducted under cyclic voltammetric (CV) conditions as shown in Fig. 1(a). Approximately, six CV scans were required to obtain a steady-state response, at which a single, broad and irreversible oxidation peak appeared at about 1.38 V (vs. SCE). The same result can be observed at Ch following the same method. The modified electrodes formed were denoted as Ch/GCE and ACh/GCE, respectively. To understand the mechanism of the ACh modification, a thin-layer spectroelectrochemical technique was used for characterization of the intermediates generated during the six CV scans. Before the CV scan, ACh showed a sharp absorption peak at 195 nm. However, this peak increased and shifted to 197 nm during the CV scans, and an absorption shoulder in the range of about 197–235 nm arose with increasing scan number. Ch showed a sharp absorption peak at about 197 nm, which did not change during the CV scans. Acetate showed a broad absorption plateau at about 197–235 nm. The comparison of the in situ spectrum of ACh with the spectrum of Ch and acetate shows that the ACh was hydrolyzed to Ch and acetate species in the thin-layer solution. The ease of hydrolysis of ACh is certainly well known [33]. The question is whether acetate, the hydrolysis product, was incorporated into the modified layer or not. This will be discussed in a later section. XPS was used for characterization of the electrode surfaces and is shown in Fig. 2. It is found that in the N (1s) region, the ratio of the surface nitrogen to carbon (N/C) increased to 167% and 175% for the prepared Ch/ GCE and ACh/GCE at 399.95 eV in comparison to that for the basal bare GCE at 400.20 eV. It is clear that the nitrogen containing residues have been immobilized on the GCE surfaces for the Ch/GCE and ACh/GCE.
10 µA -1.8 -1.2 -0.6 0.0 0.6 E / V vs SCE
1.2
1.8
Fig. 1. CVs of a GCE in 10 mM ACh + 10 mM LiCLO4 (a); CVs of ACh/GCE (b) and Ch/GCE (c) in ACN + 0.1 M NBu4 BF4 .
399.95 200 cps a
b 400.20 c 390
395 400 405 Binding Energy / eV
410
Fig. 2. XPS of the N(1) region of ACh/GCE (a), Ch/GCE (b) and GCE (c).
Table 1 summarizes the results of XPS data. It can also be seen that the content of oxygen (1s, O) increased by 11.1% and 6.8% for the Ch/GCE and ACh/GCE, respectively, in comparison with the bare GCE. This is
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Table 1 The XPS characterization of N (1s), C (1s) and O (1s) Peak-Id (X)
C1s, COO– O1s, 1s N1s
Peak position (eV)
X/C total, 1s (%)
Ch/GCE
ACh/GCE
GCE
Ch/GCE
ACh/GCE
GCE
288.35 531.80 399.95
288.57 532.65 399.95
288.31 532.85 400.20
5.15 21.3 2.0
7.5 18.1 2.1
2.2 11.3 1.2
characteristic of the formation of C–O–C bonding on the electrode. The carbon content (1s, COO–) increased by 3.0% and 5.2% for the Ch/GCE and ACh/GCE, respectively. The increase of carbon and oxygen contents is attributed to the surface anodization process during the CV scans, similarly to the case for electrochemical pretreatment of GCE [34–39], which was the preparative step for Ch or ACh assembly. The carbon content (1s, COO ) at the ACh/GCE was apparently greater than that at the Ch/GCE, which may reflect the immobilization of the acetate residues from the hydrolysis of ACh. The chemistry of Ch modification could be related to the covalent bonding of Ch with the anodized carbon surface [22–26,33,40]. A mechanism for the Ch and ACh modification is proposed as shown in Scheme 1. It shows that the surface of the GCE is anodized first, generating cation radicals (reaction 1); the surface cation radicals then react with Ch by nucleophilic attack forming ether bound –O– linkages for the Ch assembly (reaction 2). Similarly, the surface cation radicals can also react with ACh forming –O– bound linkages; for Ch covalent assembly, however, the released acetate residues could be embedded in the assembled Ch layer (reaction 3). It is probable that the acetate embedding is not only due to the electrostatic interaction with –Nþ (CH3 )3 head groups but also due to the hydrophobic effect of the hydrocarbon chains. To verify the existence of the acetate residues, CVs of the prepared Ch/GCE and ACh/GCE were conducted in ACN solution containing only ACN + 0.1 M NBu4 BF4 , after the electrodes were carefully rinsed by ultrasonicating in water. As seen in Fig. 1(b), an irreversible oxidation peak appeared at 1.32 V; however, for a continuous CV scan of 60 min, the peak height was reduced by only 12%. This is attributed to the one-electron oxidation of the assembled Ch groups to the super-oxide state. Certainly, the Ch/GCE also showed this oxidation
Scheme 1.
peak, as shown in Fig. 1(c). Based on the anodic peak area, the surface concentration of Ch at the Ch/GCE was calculated as 4.8 1010 mol cm2 . A pair of reversible redox peaks appeared at Em of 0.82 V with DEp of 260 V at the ACh/GCE. This is attributed to the redox reaction of carboxylic functionalities embedded in the surface layer of the ACh/GCE by a four-electron process [21]. Accordingly, the Ch/GCE had, as expected, no such redox waves in this region (Fig. 1(c)). Only a small and irreversible reduction peak appeared at about 0.90 V with no reoxidation peak, which is attributed to the reduction of carboxy sites generated during the anodization of the GCE [41]. It is interesting that the positively charged Ch/GCE may require counter anions for charge balancing. Certainly, after immersing the Ch/GCE in an acetate solution for 6 h, a pair of small redox peaks for carboxylic functionalities appeared at about )0.90 and )0.65 V. For a modified electrode prepared in a Ch + acetate (1:1) mixed solution, the electrode obtained also showed that a pair of reversible redox peaks of carboxylic functionalities at these potentials; however, calculation showed the ratio of surface Ch and acetate was 17:1, indicating the existence of a small amount of acetate in the layer. It is probable that the Ch layer was balanced by a large number of anions from the supporting electrolyte instead of the acetate. 0 The surface concentrations of Ch (ICH ) and acetate 0 (Iacetate ) for the ACh/GCE were calculated as 3.7 1010 and 4.1 1010 mol cm2 based on the data of Fig. 1(b), showing a monolayer modification. The approximate 1:1 ratio of Ch and acetate is reasonable for the charge balancing of the modified monolayer. The differences between ACh/GCE, Ch/GCE and bare GCE were first investigated in aqueous solution for the Fe(CN)3 6 redox reaction at various pHs, and the results are summarized in Fig. 3. At all three electrodes, the anodic peak current of Fe(CN)3 decreased with 6 increase of pH. This is due to the fact that the increase of the hydroxyl anionic concentration on the modified layer may prevent the anionic ferrocyanide anions from approaching. However, the current – pH curve was linear for a GCE, and a clear inflection appeared for the ACh/GCE. From the inflection point, 3.2 of the pKa value for the ACh modified layer could be estimated [42–46]. This is somewhat smaller than the value expected for acetic acid in solution (pKa 4.76). The reason
G.-P. Jin et al. / Journal of Electroanalytical Chemistry 569 (2004) 135–142
Cm
-3
25
139
Cdl
R sol
Zima / KΩ
I / µA
20 15 10
Rm
-2
Rct
Zw
2 1
-1
3
5 0
0 0
2
4
6
8
10
0
1
2
pH Fig. 3. The pH dependence of the cathodic peak current of 0.6 mM Fe(CN)3 6 at a bare GCE (N), Ch/GCE (j) and ACh/GCE ðÞ.
may be that the attraction from the Ch residues with positive charges enables the embedded acetate to lose Hþ more easily. For the case of Ch/GCE, a similar inflection of the current – pH curve can be observed, but with significantly scattered data points. The static interaction of the quaternary ammonium –Nþ (CH3 )3 head groups of Ch residues should attract some kinds of anions from the solution for temporary charge balancing; phosphate and even the ferrocyanide anions may thus be adopted into the surface layer, leading to complicated situations for analysis. The apparent difference between ACh/GCE and Ch/ GCE supports the reaction mechanism of the ACh modification shown in Scheme 1. The ACh could also be decomposed to Ch and acetate first, and then the intermediates could react with the anodized carbon surface forming ACh/GCE. However, the 1:1 stoichiometry of Ch and acetate may imply that a self-assembly process may be involved in the reactions. The alternative assembly of Ch and acetate on GCE gave a neutralized surface monolayer, which may be the major advantage over the Ch/GCE. Electrochemical impedance spectroscopy (EIS) can be used to evaluate the surface layer using the redox 3=4 probe Fe(CN)6 [42]. The EIS data for the GCE, ACh/GCE and Ch/GCE are shown in Fig. 4. The same equivalent circuit was used for ACh/GCE and Ch/GCE analysis as is shown in Fig. 4 (the inset), which com-
3
Zreal / KΩ Fig. 4. EIS at Ch/GCE (1), bare GCE (2) and ACh/GCE (3) in a 0.1 mM K3 [Fe(CN)6 ] + K4 [Fe(CN)6 ] + 0.1 M pH 7.0 PBS at 0.215 V vs. SCE. Data were measured from 100 kHz to 0.05 Hz.
prises the solution resistance (Rsol ), the film resistance (Rm ), the film capacitor (Cm ), the charge-transfer resistance (Rct ), the Warburg resistance (ZW ), and the double layer capacitance (Cdl ). Comparison of curves 2 and 3 4=3 shows that the electron transfer of Fe(CN)6 became difficult (larger semicircle) due to the ACh modification, indicating a significant blocking effect of the ACh layer. Interestingly, a dramatic decrease of the Rct (smaller semicircle) resulted due to the Ch modification (curve 1), suggesting that a significant acceleration of the 4=3 Fe(CN)6 redox reaction occurred due to the presence of Ch assembly. Table 2 summarizes the results of EIS data. It can be seen that the fitted Cdl value for the ACh/GCE or Ch/ GCE is larger than that for GCE. This is mainly due to the surface anodizing treatment, which generates polar functional groups at the surface and is accompanied by ion incorporation in the inner or outer Helemoltz plane as a result of the increase of the double layer capacitance [38]. 3.2. Electrochemical behavior of DA, 5-HT and AA Since the ACh/GCE presented a flat background from )0.8 to 0.8 V in 0.1 M PBS (pH 4.0), a wide potential window was available for investigation of the voltammetric behavior of DA, 5-HT and AA.
Table 2 3=4 EIS data of GCE, ACh/GCE and Ch/GCE in Fe(CN)6 Electrode
Rct (X)
Cdl (lF)
Sa
Rs (X)
Cm (lF)
Rm (X)
GCE Ch/GCE ACh/GCE
876.44 192.49 1967.5
1.74 11.12 17.66
708.83 842.26 1688.7
62.50 77.45 40.02
–
– 179.17 213.44
a
Relative Warburg resistance (ZW ).
1.73 30.42
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The CV curves of DA, 5-HT and AA at a bare GCE and the ACh/GCE are shown in Fig. 5. Fig. 5(c) shows a broad oxidation peak of AA at about 0.36 V at bare GCE (1) and this peak shifts to about 0.17 V with a slightly higher peak current at the ACh/GCE (2). Fig. 5(b) shows a drawn out peak for DA oxidation at about 0.39 V at bare GCE (1); however, it shifted to 0.34 V with an obviously increased peak current at the ACh/ GCE (2). Fig. 5(c) shows a well-defined oxidation peak of 5-HT at a potential of about 0.53 V at a bare GCE (1); however, the oxidation peak shifted to about 0.49 V, while a similar peak current was observed at the ACh/ GCE (2). The ACh/GCE showed an obvious catalytic effect toward all of these species; the reduction in overpotentials for these oxidations was 190 mV for AA, 50 mV for DA and 40 mV for 5-HT. Linear scan voltammetry (LSV) at the ACh/GCE showed that the peak currents of DA and 5-HT were linearly proportional to the scan rate in the range of 10– 120 and 10–200 mV s1 in pH 4.0, respectively. This indicated that DA and 5-HT were accumulated in the
(a)
AA
2 1
2 µA
(b)
DA 2 1 4 µA
(c)
5-H T 2 1
4 µA 0.0
0.1
0.2 0.3 0.4 E / V (vs. SCE)
0.5
0.6
Fig. 5. CVs of 0.1 mM DA, AA and 5-HT at GCE (1) and ACh/GCE (2) in 0.1 M pH 4.0 PBS. Scan rate 50 mV/s.
surface layer for the oxidations. The LSV current for AA oxidation at the ACh/GCE was linearly proportional to the scan rate in the range of 10–50 mV s1 ; however, it was linearly proportional to the square root of scan rate in the range of 60–200 mV s 1 . This indicates that the electrode reaction of AA was controlled mainly by a diffusion process. Solution switching experiments also demonstrated that a memory effect existed for DA and 5-HT at the ACh/GCE. The memory effect can be eliminated and the clean electrode surface reset by electrolysis at 0.6 V for more then 1 min in blank solution. The effects of pH on the electrode single and oxidation potentials were investigated using CV. The results showed that the slope of the peak potential for AA between pH 2.0 and 4.0 is about 35 3 mV per pH, indicating that a 2e /Hþ reaction was involved in the oxidation process. At higher pH values (4.0–8.0) the slope decreased to about 52 3 mV, suggesting a 2e /2Hþ transfer process. Thus, the electrode reaction can be classified as an electrochemical reaction followed by a chemical reaction process, as reported previously [19–21]. The peak potential of DA and 5HT oxidation varied linearly with pH and shifted to more negative potentials with a slope of )56 2 and )58 2 mV per pH unit, respectively. These are very close to the theoretical value of )59 mV per pH unit for a two electron two-proton process. For comparison, the changes in AA, DA and 5-HT peak currents with pH were also investigated; the peak current of AA in acidic solution was higher than that in basic solution and reached a maximum at about pH 4.0. The peak currents of DA and 5-HT gradually increase with increasing pH, and reached maximal values at about pH 7.0. This suggested good electrode stability and selectivity. Therefore, the optimum solution pH selected was pH 4.0. As shown in Fig. 6(a), curve 1, the CV of a sample solution containing DA, 5-HT and AA shows two broad and overlapped anodic peaks at (0.30 and 0.49 V) at GCE. So the peak potentials for DA, 5-HT and AA are indistinguishable at a bare GCE. Therefore, it is impossible to deduce any information from the broad and overlapped voltammetric peak. But at the ACh/GCE, the overlapped voltammetric peak is resolved into three well-defined CV peaks (Fig. 6(a), curve 2) at about 0.33, 0.46 and 0.17 V or DPV peaks (Fig. 6(b)) at about 0.31, 0.43 and 0.16 V, corresponding to the oxidation of DA, 5-HT and AA, respectively. When the concentration of AA is 200 and forty times that of DA, simultaneous detection can still be obtained. Ch/GCE also had a similar catalytic effect toward DA, AA and 5-HT at pH <5. However, the DPV selectivity for DA, AA and 5-HT decreased significantly at pH 7. ACh/GCE could be used satisfactorily at pH 7.0;
G.-P. Jin et al. / Journal of Electroanalytical Chemistry 569 (2004) 135–142
(a)
5 -H T DA AA
4. Conclusions 2
1
4 µA (b)
AA
DA 5 -H T
4 µA
0.0
0.1
0.2
0.3
0.4
0.5
141
0.6
E /V vs. SCE Fig. 6. (a) CVs in a solution of 0.1 M pH 4.0 PBS containing 0.1 mM DA + 0.1 mM AA + 0.1 mM 5-HT and ACh/GCE (2) at 50 mV/s and (b) DPVs at Ach/GCE in solutions containing 0.7, 0.9, 3, 5, 7, 9, 15, 35, 50 lM DA; 1, 3, 5, 7, 9, 15, 30, 50, 90 lM 5-HT; 3, 5, 7, 9, 30, 50, 70, 90, 100 lM AA, respectively.
the DPV peaks for DA, AA and 5-HT were moved to 0.034, 0.143 and 0.312 V, respectively. Under the optimum conditions, using the DPV mode, the catalytic peak current was linearly related to DA, 5HT and AA concentration in the range 7 107 – 5 106 , 1 106 – 3 105 and 7 106 – 9 105 M, with correlation coefficients of 0.995, 0.997 and 0.944, respectively. The practical detection limit was 3 107 , 5 107 and 9 107 M, respectively.
This study provides the possibility to create chemically modified electrodes by electrooxidations forming choline and acetylcholine modified glassy carbon electrodes, Ch/GCE and ACh/GCE. It provides excellent examples of mixed two-component modification from native molecular acetylcholine on a carbon electrode. The preliminary application showed that the ACh/GCE not only improved the electrochemical catalytic oxidation of DA, 5-HT and AA, but also resolved the overlapping anodic DA, 5-HT and AA peaks into three welldefined peaks; thus the DA, 5-HT and AA content of a mixture can be determined simultaneously. These modified electrodes had high stability, selectivity and reliability. Together with low cost and ease of preparation, these modified electrodes could be used for in vivo and in vitro detections. The Ch/GCE and ACh/GCE have positively charged quaternary ammonium [–Nþ (CH3 )3 ] functional groups on their surface modified layer. Both of these electrodes displayed strong catalytic effects toward positively charged DA and 5-HT in pH P 7 solutions, which is obviously different from the electrostatic adsorption characteristic at the modified electrodes with a negatively charged surface modified layer. Accordingly, it is probable that the [–Nþ (CH3 )3 ] functional groups play an activation role toward neurotransmitters such as DA and 5-HT. A further study is in progress.
Acknowledgements The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (No. 20075025).
3.3. Electrode stability References To maintain the reproducibility of the ACh/GCE and eliminate adsorption, the modified electrode was cleaned with voltammetric cycles in PBS solution after measurements, and was stored in PBS (pH 7.0). The ACh/ GCE showed high stability. For example, over the first 2 days the signal showed a 2% decrease, over 5 days the current response decreased by about 7% of its initial response and in the following month the decrease was 17%. On the other hand, Ch/GCE was stored in PBS (7.0) and showed a low stability; over the first day the signal showed a 37% decrease. If Ch/GCE was stored in twice distilled water it showed good stability; over the first 2 days the signal showed a 5% decrease, over 5 days the current response decreased by about 9% of its initial response and over the following month it decreased by 19%.
[1] M.A. Dayton, J.C. Brown, K.J. Stutts, R.M. Wightman, Anal. Chem. 52 (1980) 946. [2] T.-F. Kang, G.-L. Shen, R.-Q. Yu, Anal. Chim. Acta 356 (1997) 245. [3] E.L. Ciolkowski, K.M. Maness, S. Cahill, R.M. Wightman, D.H. Evans, B. Fosset, C. Amatore, Anal. Chem. 66 (1994) 3611. [4] M.B. Gelbert, D.J. Curran, Anal. Chem. 58 (1986) 1028. [5] F. Malem, D. Mandler, Anal. Chem. 65 (1993) 37. [6] J.A. Stamford, J.B.J. Justice, Anal. Chem. 69 (1996) 359. [7] J. Oni, T. Nyokong, Anal. Chim. Acta 434 (2001) 9. [8] R.N. Adams, Anal. Chem. 48 (1976) 1126. [9] K. Miyazaki, G. Matsumoto, M. Yamada, S. Yasu, H. Kaneko, Electrochim. Acta 44 (1999) 3809. [10] E.S. Forzani, G.A. Rivas, V.M. Solis, J. Electroanal. Chem. 435 (1997) 77. [11] R.L. McCreery, C.B. Baker, R.N. Adams, Neuromethods Humana 27 (1995) 1. [12] J.M. Zen, C.T. Hsu, Talanta 46 (1998) 1363.
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[13] F. Gonon, C.B. Baker, R.N. Adams, Neuromethods Humana 27 (1995) 153. [14] E.W. Kristensen, W.G. Kuhr, R.M. Wightman, Anal. Chem. 59 (1989) 1752. [15] R.L. McCreery, in: A.J. Bard (Ed.), Electroanlytical Chemistry, vol. 17, Marcel Dekker, New York, 1991, p. 221. [16] J. Wang, M.S. Lin, Electroanalysis 10 (1980) 861. [17] L.A. Coury, E.W. Huber, E.M. Birth, W.R. Heineman, J. Electrochem. Soc. 136 (1989) 2603. [18] A. Yu, H. Chen, Anal. Chim. Acta 344 (1997) 181. [19] L. Zhang, Y.G. Sun, X.Q. Lin, Analyst 126 (2001) 1760. [20] L. Zhang, X.Q. Lin, Analyst 126 (2001) 367. [21] L. Zhang, X.Q. Lin, Fresenius, J. Anal. Chem. 370 (2001) 956. [22] F.M. Zhou, Y. Liang, J.A. Dani, Nature 4 (2001) 224. [23] P.C. Pandet, S. Upadhyay, H.C. Pathak, C.M.D. Pandey, I. Tiwri, Sensors and Actuators B 62 (2001) 109. [24] P.C. Pandey, C. Tran-Minh, F. Lantreibecq, Appl. Biochem. Biotechnol. 31 (1991) 145. [25] M. Mascini, D. Moscone, Anal. Chim. Acta 179 (1986) 439. [26] J. Kehr, P. Dechent, T. Kato, S.O. Ogren, J. Neurosci. Methods 83 (1998) 143. [27] H. Maeda, Y. Yamauchi, M. Hosoe, T.X. Li, E. Yamaguchi, M. Kasamatsu, H. Ohmori, Chem. Pharm. Bull. 42 (1994) 1870. [28] H. Maeda, T.X. Li, M. Hosoe, M. Itami, Y. Yamauch, H. Ohmori, Chem. Pharm. Bull. 10 (1994) 963. [29] B. Guo, T. Anzai, T. OsA, Chem. Pharm. Bull. 44 (1996) 860. [30] H. Maeda, M. Itami, Y. Yamauchi, H. Ohmori, Chem. Pharm. Bull. 44 (1996) 2294.
[31] H. Maeda, M. Hosoe, T.X. Li, M. Itami, H. Ohmori, Chem. Pharm. Bull. 44 (1996) 559. [32] H. Cui, L. Wu, J. Chen, X.Q. Lin, J. Electroanal. Chem. 504 (2001) 195. [33] G. Zou, Basic Nerve Pharmacology, Science Book Concern, Beijing, 1999, p. 153. [34] R.M. Wightman, M.R. Deakin, P.M. Kovach, W.G. Kuhr, K.J. Stutts, J. Electrochem. Soc. 131 (1984) 1578. [35] L. Falat, H.Y. Cheng, Anal. Chem. 54 (1982) 2108. [36] R.C. Engstrom, Anal. Chem. 56 (1984) 136. [37] M.S. Wrighton, Science 231 (1986) 32. [38] T. Nagaokaand, T. Yoshino, Anal. Chem. 58 (1998) 1037. [39] P.L. Runnels, J.D. Joseph, M.J. Logman, R.M. Wightman, Anal. Chem. 71 (1999) 2782. [40] R.L. McCreery, in: A.J. Bard (Ed.), Electroanalytical Chemistry, Marcel Dekker, New York, 1991, p. 17221. [41] P. Bianco, J. Haladjian, J. Electroanal. Chem. 151 (1990) 163. [42] C. Saby, B. Ortiz, G.Y. Champagne, D. Belanger, Langmuir 13 (1997) 6805. [43] B. Ortiz, C. Saby, G.Y. Champagne, D. Belanger, J. Electroanal. Chem. 455 (1998) 75. [44] H.M. Wu, R. Oliver, R.N. Jattrezic, P. Clechet, A. Nyamsi, C. Martelt, Electrochim. Acta 39 (1994) 331. [45] P.T. Kissinger, W.R. Heineman, Laboratory Techniques in Electroanalytical Chemistry, Marcel Dekker, New York, 1996 (Chapter 3). [46] A. Ciszewski, G. Milezarek, Anal. Chem. 71 (1999) 1055.
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 569 (2004) 135–142 www.elsevier.com/locate/jelechem
Novel choline and acetylcholine modified glassy carbon electrodes for simultaneous determination of dopamine, serotonin and ascorbic acid Guan-Ping Jin, Xiang-Qin Lin *, Jing-ming Gong Department of Chemistry, University of Science and Technology of China, 96 Jinzhai, Hefei 230026, China Received 24 November 2003; received in revised form 18 February 2004; accepted 29 February 2004 Available online 6 May 2004
Abstract Choline (Ch) and acetylcholine (ACh) modified glassy carbon electrodes have been prepared and characterized by X-ray photoelectron spectroscopy (XPS), UV–visible spectroelectrochemistry (UV–Vis), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The modification of ACh was that the Ch was covalently bounded on the electrode surface accompanied by acetate anions embedded and adsorbed by electrostatics in the Ch modified layer, forming a novel choline/acetate interdigitate assembly. This provided an excellent example of a mixed two-component modification from a native acetylcholine molecule on a carbon electrode. These two modified electrodes were applied to the electrocatalytic oxidation of dopamine (DA), serotonin (5-HT) and ascorbic acid (AA), and resolved the overlapping of the anodic peaks of DA, 5-HT and AA into three well-defined voltammetric peaks in CV or different pulse voltammetry (DPV). This can be used for simultaneous determination of these species in a mixture. The stability of the acetate/choline/glass carbon electrode (ACh/GCE) sensing system was good, with up to at least 30 days of continual operation. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Choline; Acetylcholine; Modification; Separation
1. Introduction Dopamine (DA) is an important neuron-transmitter compound widely distributed in the brain for message transfer in the mammalian central nervous system. Low levels of DA have been found in patients with Parkinson’s disease [1]. Serotonin (5-HT) plays a crucial role in the emotional system together with other monoamine transmitters. Because DA and 5-HT are readily oxidized, hence, electrochemical methods have been explored for their analyses [1–7]. However, a major problem encountered in these determinations is the interference from ascorbic acids (AA) [8], which can be oxidized at potentials close to those of DA and 5-HT (while at pH >6, DA, AA and 5-HT overlap, at pH <5, *
Corresponding author. Tel.: +8005513606646; fax: +8005513601 592. E-mail address: [email protected] (X.-Q. Lin). 0022-0728/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2004.02.022
AA and DA overlap, and 5-HT is a single peak) at GCE electrodes. The ability to determine DA, 5-HT and AA selectively in a mixture is significant. Various methods, mainly based on the chemical modification of traditional electrode materials, have been developed to resolve the problem [8–13]. In particular polymer modified electrodes appear to have distinct advantages for their high catalytic function, good stability and broad potential window [14–21]. ACh has long been recognized as a crucial neurotransmitter in mammals. The distribution of neurons that contains ACh in the rat brain has been documented [22]. Ch is a precursor of ACh synthesis. Recently, there has been considerable interest in developing chemically modified electrodes for electroanalysis of ACh and Ch [23–26]. Although choline has an HO–, end group, which could be covalently bound to the edge plane sites of the carbon surface through the oxygen atom, similarly to alkanols [27–31], to the best of our knowledge,
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no surface modification of Ch or ACh has been studied for the fabrication of biosensors. Only the HO(CH2 )5 OH carbon fiber modified electrode has been reported for selective determination of DA in coexistence with AA [31]. The significance of Ch and ACh modification might provide a modified monolayer containing positively charged –Nþ (CH3 )3 polar head groups, which may play an important role in bio-active interactions. Evaluation of the electrostatic interaction between this positively charged modified layer and the cation and anions is of interest in designing biosensors. On the other hand, neurotransmitter molecule modified electrodes used for neurotransmitter determination should be an interesting way to evaluate the interactions between different kinds of neurotransmitter molecules. Actually, we have found interesting catalytic activities of Ch and ACh modified electrodes for DA, 5-HT and AA determinations. This report describes the preparation, characterization and mechanisms of these modified electrodes. It was found that the Ch and ACh modified electrodes not only exhibited strong catalytic ability toward the oxidation of DA, 5-HT and AA, but resolved their voltammetric responses into three well-defined voltammetric peaks, which can be used to determine DA, 5-HT and AA, simultaneously.
2. Experimental 2.1. Apparatus Electrochemical experiments such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed with a model CHI832 electrochemical analyzer (Cheng-Hua, Shanghai, China). Electrochemical impedance spectroscopy (EIS) was carried out with a CHI 660A workstation (Cheng-Hua, Shanghai, China). A conventional three-electrode system was used, which consisted of a working electrode, a twisted platinum wire counter electrode and a saturated calomel reference electrode (SCE). All potentials given in this paper are versus SCE. All experiments were performed at room temperature. The electrochemical solutions were thoroughly deoxygenated by N2 before sampling and an N2 atmosphere was maintained throughout the experiments. A home-made versatile long path-length thin-layer electrochemical cell was used for in situ spectroelectrochemical measurements as described previously [32]. Glassy carbon disk electrodes (GCE) with a geometric area of 0.125 cm2 were obtained from Tianjin-Lan-Like High Chemical Electronic Technology Company (Tianjin China). UV–Vis absorption spectra were measured with a UV-2401PC spectrophotometer (Shimadzu, Japan).
XPS was carried out using an ESCALAB MK2 spectrometer (VG Corporation, UK) with Mak-Alpha X-ray radiation as the excitation source. 2.2. Chemicals and solutions Choline (Ch) and acetylcholine (ACh) were purchased from the Chemical Reagent Factory of Beijing (Beijing China). Dopamine hydrochloride (DA) and serotonin (5-HT) were obtained from Sigma (USA). Ascorbic acid (AA) was obtained from Chemical Reagent Company of Shanghai (Shanghai China). All other reagents used were of analytical grade. Solutions of ACh and Ch were prepared in 0.01 M LiClO4: Solutions of DA, 5-HT and AA were prepared in water, prior to use. Acetonitrile (ACN) was of analytical quality and was dried over molecular before use. Phosphate-buffered saline (PBS; sieves (3 A) 0.1 M) solutions of different pH were prepared by mixing four stock solutions of 0.1 M H3 PO4 , KH2 PO4 , K2 HPO4 and K3 PO4 . Twice distilled water was used. High purity nitrogen was used for deaeration. All experiments were carried out at ambient temperature. 2.3. Preparation of modified electrode The GCE was prepared by polishing to a mirror-like finish with fine wet emery paper (grain size 4000). After sonicating in water for 15 min, it was resurfaced using 1.0 lM alumina slurry. After cleaning, the electrode was electrochemically pre-treated by cyclic scanning in 1.0 103 M ACh or Ch solution containing 0.01 M LiClO4 in the potential range of )1.7 to 1.8 V at 20 mV s1 for six scans. The electrode was then rinsed with ethanol and sonicated for 15 min in water to remove any physisorbed unreacted materials from the surface. The acetylcholine and choline modified electrodes were designated as ACh/GCE and Ch/GCE, respectively. They were stored in 0.1 M PBS (pH 7.0) and twice distilled water at 4 °C, respectively, and were ready for use. 2.4. Spectroelectrochemistry A 1 ml volume of ACh solution was injected into the cell through its reference electrode chamber and thinlayer compartment [32]. In this way, any air bubbles in the thin-layer compartment could be removed. A similar cell was set in the reference beam of the spectrophotometer for background subtraction during in situ measurement of UV–Vis absorption spectra. Each spectrum was measured after the thin-layer solution in the sample cell was electrolyzed at a steady potential for 2 min. 2.5. X-ray photoelectron spectroscopy (XPS) Surface analysis of the electrode was performed with an ESCA lab MK2 (VG, UK) with a Mo Ka radiation
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source at 50 and 0.05 eV per step. The elemental nitrogen-to-carbon ratio (N/C) was used to assess the extent of modifier coverage calculated by dividing the total number of counts under the N (1s) band by that under the C (1s) band.
137
(a)
2.6. Electrochemical impedance spectroscopy
50 µA
Electrochemical impedance spectroscopy (EIS) was carried out via the workstation. The glassy carbon electrodes before and after modification with ACh or Ch 4=3 were characterized in 0.1 M Fe(CN)6 + 0.1 M PBS (PH 7.0) via EIS methods. EIS data were measured at 100 kHz to 0.05 Hz and at an electrode-potential of 0.215 V, the formal potential of the ferrocyanide/ferricyanide couple.
(b)
25 µA
(c)
3. Results and discussion 3.1. Electrochemical modification of Ch and ACh on GCE The ACh modification was conducted under cyclic voltammetric (CV) conditions as shown in Fig. 1(a). Approximately, six CV scans were required to obtain a steady-state response, at which a single, broad and irreversible oxidation peak appeared at about 1.38 V (vs. SCE). The same result can be observed at Ch following the same method. The modified electrodes formed were denoted as Ch/GCE and ACh/GCE, respectively. To understand the mechanism of the ACh modification, a thin-layer spectroelectrochemical technique was used for characterization of the intermediates generated during the six CV scans. Before the CV scan, ACh showed a sharp absorption peak at 195 nm. However, this peak increased and shifted to 197 nm during the CV scans, and an absorption shoulder in the range of about 197–235 nm arose with increasing scan number. Ch showed a sharp absorption peak at about 197 nm, which did not change during the CV scans. Acetate showed a broad absorption plateau at about 197–235 nm. The comparison of the in situ spectrum of ACh with the spectrum of Ch and acetate shows that the ACh was hydrolyzed to Ch and acetate species in the thin-layer solution. The ease of hydrolysis of ACh is certainly well known [33]. The question is whether acetate, the hydrolysis product, was incorporated into the modified layer or not. This will be discussed in a later section. XPS was used for characterization of the electrode surfaces and is shown in Fig. 2. It is found that in the N (1s) region, the ratio of the surface nitrogen to carbon (N/C) increased to 167% and 175% for the prepared Ch/ GCE and ACh/GCE at 399.95 eV in comparison to that for the basal bare GCE at 400.20 eV. It is clear that the nitrogen containing residues have been immobilized on the GCE surfaces for the Ch/GCE and ACh/GCE.
10 µA -1.8 -1.2 -0.6 0.0 0.6 E / V vs SCE
1.2
1.8
Fig. 1. CVs of a GCE in 10 mM ACh + 10 mM LiCLO4 (a); CVs of ACh/GCE (b) and Ch/GCE (c) in ACN + 0.1 M NBu4 BF4 .
399.95 200 cps a
b 400.20 c 390
395 400 405 Binding Energy / eV
410
Fig. 2. XPS of the N(1) region of ACh/GCE (a), Ch/GCE (b) and GCE (c).
Table 1 summarizes the results of XPS data. It can also be seen that the content of oxygen (1s, O) increased by 11.1% and 6.8% for the Ch/GCE and ACh/GCE, respectively, in comparison with the bare GCE. This is
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Table 1 The XPS characterization of N (1s), C (1s) and O (1s) Peak-Id (X)
C1s, COO– O1s, 1s N1s
Peak position (eV)
X/C total, 1s (%)
Ch/GCE
ACh/GCE
GCE
Ch/GCE
ACh/GCE
GCE
288.35 531.80 399.95
288.57 532.65 399.95
288.31 532.85 400.20
5.15 21.3 2.0
7.5 18.1 2.1
2.2 11.3 1.2
characteristic of the formation of C–O–C bonding on the electrode. The carbon content (1s, COO–) increased by 3.0% and 5.2% for the Ch/GCE and ACh/GCE, respectively. The increase of carbon and oxygen contents is attributed to the surface anodization process during the CV scans, similarly to the case for electrochemical pretreatment of GCE [34–39], which was the preparative step for Ch or ACh assembly. The carbon content (1s, COO ) at the ACh/GCE was apparently greater than that at the Ch/GCE, which may reflect the immobilization of the acetate residues from the hydrolysis of ACh. The chemistry of Ch modification could be related to the covalent bonding of Ch with the anodized carbon surface [22–26,33,40]. A mechanism for the Ch and ACh modification is proposed as shown in Scheme 1. It shows that the surface of the GCE is anodized first, generating cation radicals (reaction 1); the surface cation radicals then react with Ch by nucleophilic attack forming ether bound –O– linkages for the Ch assembly (reaction 2). Similarly, the surface cation radicals can also react with ACh forming –O– bound linkages; for Ch covalent assembly, however, the released acetate residues could be embedded in the assembled Ch layer (reaction 3). It is probable that the acetate embedding is not only due to the electrostatic interaction with –Nþ (CH3 )3 head groups but also due to the hydrophobic effect of the hydrocarbon chains. To verify the existence of the acetate residues, CVs of the prepared Ch/GCE and ACh/GCE were conducted in ACN solution containing only ACN + 0.1 M NBu4 BF4 , after the electrodes were carefully rinsed by ultrasonicating in water. As seen in Fig. 1(b), an irreversible oxidation peak appeared at 1.32 V; however, for a continuous CV scan of 60 min, the peak height was reduced by only 12%. This is attributed to the one-electron oxidation of the assembled Ch groups to the super-oxide state. Certainly, the Ch/GCE also showed this oxidation
Scheme 1.
peak, as shown in Fig. 1(c). Based on the anodic peak area, the surface concentration of Ch at the Ch/GCE was calculated as 4.8 1010 mol cm2 . A pair of reversible redox peaks appeared at Em of 0.82 V with DEp of 260 V at the ACh/GCE. This is attributed to the redox reaction of carboxylic functionalities embedded in the surface layer of the ACh/GCE by a four-electron process [21]. Accordingly, the Ch/GCE had, as expected, no such redox waves in this region (Fig. 1(c)). Only a small and irreversible reduction peak appeared at about 0.90 V with no reoxidation peak, which is attributed to the reduction of carboxy sites generated during the anodization of the GCE [41]. It is interesting that the positively charged Ch/GCE may require counter anions for charge balancing. Certainly, after immersing the Ch/GCE in an acetate solution for 6 h, a pair of small redox peaks for carboxylic functionalities appeared at about )0.90 and )0.65 V. For a modified electrode prepared in a Ch + acetate (1:1) mixed solution, the electrode obtained also showed that a pair of reversible redox peaks of carboxylic functionalities at these potentials; however, calculation showed the ratio of surface Ch and acetate was 17:1, indicating the existence of a small amount of acetate in the layer. It is probable that the Ch layer was balanced by a large number of anions from the supporting electrolyte instead of the acetate. 0 The surface concentrations of Ch (ICH ) and acetate 0 (Iacetate ) for the ACh/GCE were calculated as 3.7 1010 and 4.1 1010 mol cm2 based on the data of Fig. 1(b), showing a monolayer modification. The approximate 1:1 ratio of Ch and acetate is reasonable for the charge balancing of the modified monolayer. The differences between ACh/GCE, Ch/GCE and bare GCE were first investigated in aqueous solution for the Fe(CN)3 6 redox reaction at various pHs, and the results are summarized in Fig. 3. At all three electrodes, the anodic peak current of Fe(CN)3 decreased with 6 increase of pH. This is due to the fact that the increase of the hydroxyl anionic concentration on the modified layer may prevent the anionic ferrocyanide anions from approaching. However, the current – pH curve was linear for a GCE, and a clear inflection appeared for the ACh/GCE. From the inflection point, 3.2 of the pKa value for the ACh modified layer could be estimated [42–46]. This is somewhat smaller than the value expected for acetic acid in solution (pKa 4.76). The reason
G.-P. Jin et al. / Journal of Electroanalytical Chemistry 569 (2004) 135–142
Cm
-3
25
139
Cdl
R sol
Zima / KΩ
I / µA
20 15 10
Rm
-2
Rct
Zw
2 1
-1
3
5 0
0 0
2
4
6
8
10
0
1
2
pH Fig. 3. The pH dependence of the cathodic peak current of 0.6 mM Fe(CN)3 6 at a bare GCE (N), Ch/GCE (j) and ACh/GCE ðÞ.
may be that the attraction from the Ch residues with positive charges enables the embedded acetate to lose Hþ more easily. For the case of Ch/GCE, a similar inflection of the current – pH curve can be observed, but with significantly scattered data points. The static interaction of the quaternary ammonium –Nþ (CH3 )3 head groups of Ch residues should attract some kinds of anions from the solution for temporary charge balancing; phosphate and even the ferrocyanide anions may thus be adopted into the surface layer, leading to complicated situations for analysis. The apparent difference between ACh/GCE and Ch/ GCE supports the reaction mechanism of the ACh modification shown in Scheme 1. The ACh could also be decomposed to Ch and acetate first, and then the intermediates could react with the anodized carbon surface forming ACh/GCE. However, the 1:1 stoichiometry of Ch and acetate may imply that a self-assembly process may be involved in the reactions. The alternative assembly of Ch and acetate on GCE gave a neutralized surface monolayer, which may be the major advantage over the Ch/GCE. Electrochemical impedance spectroscopy (EIS) can be used to evaluate the surface layer using the redox 3=4 probe Fe(CN)6 [42]. The EIS data for the GCE, ACh/GCE and Ch/GCE are shown in Fig. 4. The same equivalent circuit was used for ACh/GCE and Ch/GCE analysis as is shown in Fig. 4 (the inset), which com-
3
Zreal / KΩ Fig. 4. EIS at Ch/GCE (1), bare GCE (2) and ACh/GCE (3) in a 0.1 mM K3 [Fe(CN)6 ] + K4 [Fe(CN)6 ] + 0.1 M pH 7.0 PBS at 0.215 V vs. SCE. Data were measured from 100 kHz to 0.05 Hz.
prises the solution resistance (Rsol ), the film resistance (Rm ), the film capacitor (Cm ), the charge-transfer resistance (Rct ), the Warburg resistance (ZW ), and the double layer capacitance (Cdl ). Comparison of curves 2 and 3 4=3 shows that the electron transfer of Fe(CN)6 became difficult (larger semicircle) due to the ACh modification, indicating a significant blocking effect of the ACh layer. Interestingly, a dramatic decrease of the Rct (smaller semicircle) resulted due to the Ch modification (curve 1), suggesting that a significant acceleration of the 4=3 Fe(CN)6 redox reaction occurred due to the presence of Ch assembly. Table 2 summarizes the results of EIS data. It can be seen that the fitted Cdl value for the ACh/GCE or Ch/ GCE is larger than that for GCE. This is mainly due to the surface anodizing treatment, which generates polar functional groups at the surface and is accompanied by ion incorporation in the inner or outer Helemoltz plane as a result of the increase of the double layer capacitance [38]. 3.2. Electrochemical behavior of DA, 5-HT and AA Since the ACh/GCE presented a flat background from )0.8 to 0.8 V in 0.1 M PBS (pH 4.0), a wide potential window was available for investigation of the voltammetric behavior of DA, 5-HT and AA.
Table 2 3=4 EIS data of GCE, ACh/GCE and Ch/GCE in Fe(CN)6 Electrode
Rct (X)
Cdl (lF)
Sa
Rs (X)
Cm (lF)
Rm (X)
GCE Ch/GCE ACh/GCE
876.44 192.49 1967.5
1.74 11.12 17.66
708.83 842.26 1688.7
62.50 77.45 40.02
–
– 179.17 213.44
a
Relative Warburg resistance (ZW ).
1.73 30.42
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The CV curves of DA, 5-HT and AA at a bare GCE and the ACh/GCE are shown in Fig. 5. Fig. 5(c) shows a broad oxidation peak of AA at about 0.36 V at bare GCE (1) and this peak shifts to about 0.17 V with a slightly higher peak current at the ACh/GCE (2). Fig. 5(b) shows a drawn out peak for DA oxidation at about 0.39 V at bare GCE (1); however, it shifted to 0.34 V with an obviously increased peak current at the ACh/ GCE (2). Fig. 5(c) shows a well-defined oxidation peak of 5-HT at a potential of about 0.53 V at a bare GCE (1); however, the oxidation peak shifted to about 0.49 V, while a similar peak current was observed at the ACh/ GCE (2). The ACh/GCE showed an obvious catalytic effect toward all of these species; the reduction in overpotentials for these oxidations was 190 mV for AA, 50 mV for DA and 40 mV for 5-HT. Linear scan voltammetry (LSV) at the ACh/GCE showed that the peak currents of DA and 5-HT were linearly proportional to the scan rate in the range of 10– 120 and 10–200 mV s1 in pH 4.0, respectively. This indicated that DA and 5-HT were accumulated in the
(a)
AA
2 1
2 µA
(b)
DA 2 1 4 µA
(c)
5-H T 2 1
4 µA 0.0
0.1
0.2 0.3 0.4 E / V (vs. SCE)
0.5
0.6
Fig. 5. CVs of 0.1 mM DA, AA and 5-HT at GCE (1) and ACh/GCE (2) in 0.1 M pH 4.0 PBS. Scan rate 50 mV/s.
surface layer for the oxidations. The LSV current for AA oxidation at the ACh/GCE was linearly proportional to the scan rate in the range of 10–50 mV s1 ; however, it was linearly proportional to the square root of scan rate in the range of 60–200 mV s 1 . This indicates that the electrode reaction of AA was controlled mainly by a diffusion process. Solution switching experiments also demonstrated that a memory effect existed for DA and 5-HT at the ACh/GCE. The memory effect can be eliminated and the clean electrode surface reset by electrolysis at 0.6 V for more then 1 min in blank solution. The effects of pH on the electrode single and oxidation potentials were investigated using CV. The results showed that the slope of the peak potential for AA between pH 2.0 and 4.0 is about 35 3 mV per pH, indicating that a 2e /Hþ reaction was involved in the oxidation process. At higher pH values (4.0–8.0) the slope decreased to about 52 3 mV, suggesting a 2e /2Hþ transfer process. Thus, the electrode reaction can be classified as an electrochemical reaction followed by a chemical reaction process, as reported previously [19–21]. The peak potential of DA and 5HT oxidation varied linearly with pH and shifted to more negative potentials with a slope of )56 2 and )58 2 mV per pH unit, respectively. These are very close to the theoretical value of )59 mV per pH unit for a two electron two-proton process. For comparison, the changes in AA, DA and 5-HT peak currents with pH were also investigated; the peak current of AA in acidic solution was higher than that in basic solution and reached a maximum at about pH 4.0. The peak currents of DA and 5-HT gradually increase with increasing pH, and reached maximal values at about pH 7.0. This suggested good electrode stability and selectivity. Therefore, the optimum solution pH selected was pH 4.0. As shown in Fig. 6(a), curve 1, the CV of a sample solution containing DA, 5-HT and AA shows two broad and overlapped anodic peaks at (0.30 and 0.49 V) at GCE. So the peak potentials for DA, 5-HT and AA are indistinguishable at a bare GCE. Therefore, it is impossible to deduce any information from the broad and overlapped voltammetric peak. But at the ACh/GCE, the overlapped voltammetric peak is resolved into three well-defined CV peaks (Fig. 6(a), curve 2) at about 0.33, 0.46 and 0.17 V or DPV peaks (Fig. 6(b)) at about 0.31, 0.43 and 0.16 V, corresponding to the oxidation of DA, 5-HT and AA, respectively. When the concentration of AA is 200 and forty times that of DA, simultaneous detection can still be obtained. Ch/GCE also had a similar catalytic effect toward DA, AA and 5-HT at pH <5. However, the DPV selectivity for DA, AA and 5-HT decreased significantly at pH 7. ACh/GCE could be used satisfactorily at pH 7.0;
G.-P. Jin et al. / Journal of Electroanalytical Chemistry 569 (2004) 135–142
(a)
5 -H T DA AA
4. Conclusions 2
1
4 µA (b)
AA
DA 5 -H T
4 µA
0.0
0.1
0.2
0.3
0.4
0.5
141
0.6
E /V vs. SCE Fig. 6. (a) CVs in a solution of 0.1 M pH 4.0 PBS containing 0.1 mM DA + 0.1 mM AA + 0.1 mM 5-HT and ACh/GCE (2) at 50 mV/s and (b) DPVs at Ach/GCE in solutions containing 0.7, 0.9, 3, 5, 7, 9, 15, 35, 50 lM DA; 1, 3, 5, 7, 9, 15, 30, 50, 90 lM 5-HT; 3, 5, 7, 9, 30, 50, 70, 90, 100 lM AA, respectively.
the DPV peaks for DA, AA and 5-HT were moved to 0.034, 0.143 and 0.312 V, respectively. Under the optimum conditions, using the DPV mode, the catalytic peak current was linearly related to DA, 5HT and AA concentration in the range 7 107 – 5 106 , 1 106 – 3 105 and 7 106 – 9 105 M, with correlation coefficients of 0.995, 0.997 and 0.944, respectively. The practical detection limit was 3 107 , 5 107 and 9 107 M, respectively.
This study provides the possibility to create chemically modified electrodes by electrooxidations forming choline and acetylcholine modified glassy carbon electrodes, Ch/GCE and ACh/GCE. It provides excellent examples of mixed two-component modification from native molecular acetylcholine on a carbon electrode. The preliminary application showed that the ACh/GCE not only improved the electrochemical catalytic oxidation of DA, 5-HT and AA, but also resolved the overlapping anodic DA, 5-HT and AA peaks into three welldefined peaks; thus the DA, 5-HT and AA content of a mixture can be determined simultaneously. These modified electrodes had high stability, selectivity and reliability. Together with low cost and ease of preparation, these modified electrodes could be used for in vivo and in vitro detections. The Ch/GCE and ACh/GCE have positively charged quaternary ammonium [–Nþ (CH3 )3 ] functional groups on their surface modified layer. Both of these electrodes displayed strong catalytic effects toward positively charged DA and 5-HT in pH P 7 solutions, which is obviously different from the electrostatic adsorption characteristic at the modified electrodes with a negatively charged surface modified layer. Accordingly, it is probable that the [–Nþ (CH3 )3 ] functional groups play an activation role toward neurotransmitters such as DA and 5-HT. A further study is in progress.
Acknowledgements The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (No. 20075025).
3.3. Electrode stability References To maintain the reproducibility of the ACh/GCE and eliminate adsorption, the modified electrode was cleaned with voltammetric cycles in PBS solution after measurements, and was stored in PBS (pH 7.0). The ACh/ GCE showed high stability. For example, over the first 2 days the signal showed a 2% decrease, over 5 days the current response decreased by about 7% of its initial response and in the following month the decrease was 17%. On the other hand, Ch/GCE was stored in PBS (7.0) and showed a low stability; over the first day the signal showed a 37% decrease. If Ch/GCE was stored in twice distilled water it showed good stability; over the first 2 days the signal showed a 5% decrease, over 5 days the current response decreased by about 9% of its initial response and over the following month it decreased by 19%.
[1] M.A. Dayton, J.C. Brown, K.J. Stutts, R.M. Wightman, Anal. Chem. 52 (1980) 946. [2] T.-F. Kang, G.-L. Shen, R.-Q. Yu, Anal. Chim. Acta 356 (1997) 245. [3] E.L. Ciolkowski, K.M. Maness, S. Cahill, R.M. Wightman, D.H. Evans, B. Fosset, C. Amatore, Anal. Chem. 66 (1994) 3611. [4] M.B. Gelbert, D.J. Curran, Anal. Chem. 58 (1986) 1028. [5] F. Malem, D. Mandler, Anal. Chem. 65 (1993) 37. [6] J.A. Stamford, J.B.J. Justice, Anal. Chem. 69 (1996) 359. [7] J. Oni, T. Nyokong, Anal. Chim. Acta 434 (2001) 9. [8] R.N. Adams, Anal. Chem. 48 (1976) 1126. [9] K. Miyazaki, G. Matsumoto, M. Yamada, S. Yasu, H. Kaneko, Electrochim. Acta 44 (1999) 3809. [10] E.S. Forzani, G.A. Rivas, V.M. Solis, J. Electroanal. Chem. 435 (1997) 77. [11] R.L. McCreery, C.B. Baker, R.N. Adams, Neuromethods Humana 27 (1995) 1. [12] J.M. Zen, C.T. Hsu, Talanta 46 (1998) 1363.
142
G.-P. Jin et al. / Journal of Electroanalytical Chemistry 569 (2004) 135–142
[13] F. Gonon, C.B. Baker, R.N. Adams, Neuromethods Humana 27 (1995) 153. [14] E.W. Kristensen, W.G. Kuhr, R.M. Wightman, Anal. Chem. 59 (1989) 1752. [15] R.L. McCreery, in: A.J. Bard (Ed.), Electroanlytical Chemistry, vol. 17, Marcel Dekker, New York, 1991, p. 221. [16] J. Wang, M.S. Lin, Electroanalysis 10 (1980) 861. [17] L.A. Coury, E.W. Huber, E.M. Birth, W.R. Heineman, J. Electrochem. Soc. 136 (1989) 2603. [18] A. Yu, H. Chen, Anal. Chim. Acta 344 (1997) 181. [19] L. Zhang, Y.G. Sun, X.Q. Lin, Analyst 126 (2001) 1760. [20] L. Zhang, X.Q. Lin, Analyst 126 (2001) 367. [21] L. Zhang, X.Q. Lin, Fresenius, J. Anal. Chem. 370 (2001) 956. [22] F.M. Zhou, Y. Liang, J.A. Dani, Nature 4 (2001) 224. [23] P.C. Pandet, S. Upadhyay, H.C. Pathak, C.M.D. Pandey, I. Tiwri, Sensors and Actuators B 62 (2001) 109. [24] P.C. Pandey, C. Tran-Minh, F. Lantreibecq, Appl. Biochem. Biotechnol. 31 (1991) 145. [25] M. Mascini, D. Moscone, Anal. Chim. Acta 179 (1986) 439. [26] J. Kehr, P. Dechent, T. Kato, S.O. Ogren, J. Neurosci. Methods 83 (1998) 143. [27] H. Maeda, Y. Yamauchi, M. Hosoe, T.X. Li, E. Yamaguchi, M. Kasamatsu, H. Ohmori, Chem. Pharm. Bull. 42 (1994) 1870. [28] H. Maeda, T.X. Li, M. Hosoe, M. Itami, Y. Yamauch, H. Ohmori, Chem. Pharm. Bull. 10 (1994) 963. [29] B. Guo, T. Anzai, T. OsA, Chem. Pharm. Bull. 44 (1996) 860. [30] H. Maeda, M. Itami, Y. Yamauchi, H. Ohmori, Chem. Pharm. Bull. 44 (1996) 2294.
[31] H. Maeda, M. Hosoe, T.X. Li, M. Itami, H. Ohmori, Chem. Pharm. Bull. 44 (1996) 559. [32] H. Cui, L. Wu, J. Chen, X.Q. Lin, J. Electroanal. Chem. 504 (2001) 195. [33] G. Zou, Basic Nerve Pharmacology, Science Book Concern, Beijing, 1999, p. 153. [34] R.M. Wightman, M.R. Deakin, P.M. Kovach, W.G. Kuhr, K.J. Stutts, J. Electrochem. Soc. 131 (1984) 1578. [35] L. Falat, H.Y. Cheng, Anal. Chem. 54 (1982) 2108. [36] R.C. Engstrom, Anal. Chem. 56 (1984) 136. [37] M.S. Wrighton, Science 231 (1986) 32. [38] T. Nagaokaand, T. Yoshino, Anal. Chem. 58 (1998) 1037. [39] P.L. Runnels, J.D. Joseph, M.J. Logman, R.M. Wightman, Anal. Chem. 71 (1999) 2782. [40] R.L. McCreery, in: A.J. Bard (Ed.), Electroanalytical Chemistry, Marcel Dekker, New York, 1991, p. 17221. [41] P. Bianco, J. Haladjian, J. Electroanal. Chem. 151 (1990) 163. [42] C. Saby, B. Ortiz, G.Y. Champagne, D. Belanger, Langmuir 13 (1997) 6805. [43] B. Ortiz, C. Saby, G.Y. Champagne, D. Belanger, J. Electroanal. Chem. 455 (1998) 75. [44] H.M. Wu, R. Oliver, R.N. Jattrezic, P. Clechet, A. Nyamsi, C. Martelt, Electrochim. Acta 39 (1994) 331. [45] P.T. Kissinger, W.R. Heineman, Laboratory Techniques in Electroanalytical Chemistry, Marcel Dekker, New York, 1996 (Chapter 3). [46] A. Ciszewski, G. Milezarek, Anal. Chem. 71 (1999) 1055.