Direct Voltammetric Specific Recognition of Dopamine Using Al III -DA Complexes at the Hanging Mercury Drop Electrode

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1 2007 The Japan Society for Analytical Chemistry 1325 Direct Voltammetric Specific Recognition of Dopamine Using Al III -DA Complexes at the Hanging Mercury Drop Electrode Fuping ZHANG, Min ZHANG, Jiongjia CHENG, Li YANG, Ming JI, and Shuping BI School of Chemistry & Chemical Engineering, MOE Key Laboratory for Life Science & State Key Laboratory of Coordination Chemistry of China, Nanjing University, Nanjing , China In this paper, we firstly report the direct voltammetric recognition and determination of dopamine (DA) by using Al III -DA complexes at the hanging mercury drop electrode (HMDE). A new sensitive cathodic peak of Al III -DA can be detected at 900 mv (vs. SCE) in 0.1 M NH 4Cl NH 3 H 2O 0.1 M KCl buffer solution at ph 8.5. This unique 900 mv cathodic peak arises from the specific interaction between Al III and DA on the HMDE, whereas other substances with similar structures, such as L-dopa, epinephrine (EP), norepinephrine (NE), catechols, caffeic acid (CA), trihydric phenols and tiron, do not yield any new peak on the voltammograms in the potential range from 100 to 1200 mv when Al III is added. The distinct voltammetric characteristic of the recognition of DA can effectively inhibit the interferences of both ascorbic acid and uric acid in the DA determination by the direct electrochemistry, which is a major difficulty when a solid electrode is used. The proposed method can be anticipated as an effective means for the recognition of DA in the elucidation of the mechanisms of Parkinson s disease (PD) and Alzheimer s disease (AD) in the presence of Al III. (Received January 9, 2007; Accepted May 18, 2007; Published November 10, 2007) Introduction It is well known that dopamin (DA), one of the neurotransmitters in the brain, plays an important role in regulating the body movements and mental activity. Psychomotor stimulants and neuroleptics exert multiple effects on dopaminergic signaling and produce the DA-related behaviors of motor activation and catalepsy, respectively. 1 On the other hand, some types of neurodementia such as Pakinson s disease (PD), Alzheimer s disease (AD), and dialysis encephalopathy (DE) are believed to relate to an abnormally high level of aluminum (Al III ) in the brain. 2 Therefore, the study of DA in the nerve center system and its interaction with Al III are of great significance to the biomedicine for the treatment of neuropathic disease and exploitation of the relevant drugs. DA can be analyzed electrochemically due to its redox reaction at the electrode surface. The electrochemical behaviors of DA on various kinds of electrodes, including gold electrode, carbon fiber microelectrodes, glassy carbon electrodes, carbon paste electrode and hanging mercury drop electrode (HMDE), have been investigated extensively in the last two decades. 3 9 However, a series of biomolecules possessing similar structures and characters such as epinephrine (EP) and norepinephrine (NE), coexist with DA in biosystems, making their determination and recognition quite difficult. In addition, a high concentration of ascorbic acid in a biosystem greatly disturbs the anodic responses of DA. A common problem of electrochemical analysis at unmodified electrodes is the lack of selectivity due to the presence of interfering compounds that overlap with the signal of the compound of interest. 10 Consequently, some modified electrodes were developed To whom correspondence should be addressed. bisp@nju.edu.cn These modified electrodes differentiate well between DA and ascorbic acid, and improve the sensitivity of DA detection. However, until now, DA can not be partitioned among the coexisting biomolecules with similar structures such as EP, NE, catechols, and L-dopa through the means mentioned above and so the related target of recognition still cannot be achieved. Therefore, establishing a novel electrochemical method to directly recognize and determine DA will be welcomed. In recent years, there are some reports with regard to the interaction between some catecholamines and other metal ions such as Fe III, Ge IV, Bi III, Pb II, Co II, and Cd II at the HMDE, but the investigation of Al III -DA at the HMDE has never been reported before. In this paper, the electrochemical behaviors of Al III -DA at the HMDE have been investigated systemically. A new sensitive linear scan voltammetry (LSV) cathodic peak of Al III -DA complex was detected at 900 mv in 0.1 M NH 4Cl NH 3 H 2O 0.1 M KCl buffer solution at ph 8.5, while other catecholamine neurotransmitters were not able to produce the corresponding electrochemical cathodic peaks under the same experimental conditions. This Al III -DA complex peak can be effectively used for the recognition of DA. Experimental Apparatus and reagents A three-electrode system was used, which consisted of a HMDE, a platinum foil counter electrode, and a saturated calomel reference electrode. All electrochemical experiments were performed with a BAS-100 B electrochemical system (BAS Inc. IN, USA). The LSV rate was 100 mv/s. PH measurements were carried out with a PHSJ-4A digital ion meter (Shanghai Instrument Company, China). The solutions were stirred with a 79-1 magnetic stirrer (Guohua Instrument Factory, China) and the experiments were carried out in the

2 1326 ANALYTICAL SCIENCES NOVEMBER 2007, VOL. 23 Fig. 1 LSV cathodic responses of DA with and without adding Al III, v = 100 mv/s. (a) ph 8.5, 0.1 M NH 4Cl NH 3 H 2O 0.1 M KCl, (b) a M DA, (c) b M Al III. ambient of nitrogen at room temperature. Most chemicals were obtained from Shanghai Chemical Reagent Factory, Beijing Chemical Factory or Nanjing Chemical Reagent Factory and were used as received unless otherwise noted. Most chemicals were of analytical reagent grade. A stock solution of M DA (99%, Acros Organics Inc., Swiss) was prepared freshly every day. L-Dopa, epinephrine and norepinephrine were obtained from Sigma (St. Louis, MO). Caffeic acid (CA), gallic acid (GA), pyrogallic acid (PA), tiron and catechol were obtained from Aldrich. Al III stock solution (0.01 M) was prepared by dissolving super-purity Al powder (99.99%) in 1:10 HCl solution. It was diluted to the proper volume when used. To prevent hydrolysis of the metal ion, we prepared the Al III stock solutions at ph < 2. The 0.1 M NH 3 H 2O NH 4Cl 0.1 M KCl buffer solution was prepared by mixing a certain amount of NH 4Cl or KCl with an appropriate volume of NH 3 H 2O at ph All dilute solutions were prepared by diluting this solution with doubly-distilled water. Necessary polyethylene vessels were used. All pieces of labwares were soaked in 10% HNO 3 for at least 24 h, before being washed carefully with water and doubly-distilled water in turn. Procedures In analytical tests, 25 ml of buffer solution was transferred into the electrolytic cell and then the current response was recorded after applying LSV mode. The M DA testing solution was prepared by mixing an appropriate amount of solid DA with the buffer solution. The other DA solution was prepared by mixing the buffer solution with the stock solution of M DA properly. The corresponding LSV cathodic current response of the M DA testing solution was recorded after a 2-min stirring and 1-min resting to restore the calmness. After adding a certain amount of Al III, we followed the same stirring and resting procedure again. Then the related cathodic current response of Al III -DA complex was recorded again after applying LSV mode. Under the same experimental conditions, the experiments for L-dopa, EP, NE, catechol, CA, PA, GA, tiron, ascorbic acid and uric acid were carried out. Results and Discussion LSV cathodic responses of Al III -DA complex at HMDE Figure 1 shows the LSV cathodic responses of DA at HMDE Fig. 2 The optimized experimental conditions and the linear response of I p vs. C DA, v = 100 mv/s. (A) Effect of ph, (B) effect of Al III concentrations, (C) effect of the concentrations of buffer solutions. Other conditions are the same as Fig. 1. in both the absence and presence of Al III. In the supporting electrolyte (Fig. 1a), there is no current peak appearing in the potential range of 100 to 1200 mv. After the addition of DA (Fig. 1b), the LSV cathodic peak of DA appears at 300 mv. It is ascribed to the reduction of complex of Hg II and DA according to the literature. 8,9 After addition of Al III (Fig. 1c), a new sharp peak appears at 900 mv; that the height of the peak is proportional to the DA concentration was observed in further study. This peak can be used to recognize and detect DA. Recognition of DA by Al III -DA electrochemical response The experimental conditions for the recognition of DA were optimized. As shown in Fig. 2, the optimum experimental conditions are ph 8.5, M Al III, and 0.1 M NH 4Cl NH 3 H 2O 0.1 M KCl buffer solution. A linear relationship between the cathodic peak current of Al III -DA was established; the linear regression equation is: I pc (μa) = C DA (M) ( M, R = ). The relative standard deviation for M DA is 2.1% (n = 16) and the detection limit is M. The prominent characteristic of the electrochemical response of Al III -DA at HMDE is that it can efficiently inhibit the

3 1327 Fig. 4 CV voltammograms of the DA in the absence and presence of Al III (v = 100 mv/s). Cathodic process: (a) ph 8.5, 0.1 M NH 4Cl NH 3 H 2O 0.1M KCl, M DA, (b) a M Al III, (c) a M Al III, (d) a M Al III. Voltammograms from (a ) to (d ) are the related anodic processes. Fig. 3 LSV cathodic responses of ascorbic acid (A) and uric acid (B) in the absence and presence of Al III. A: (a) ph 8.5, 0.1 M NH 4Cl NH 3 H 2O 0.1 M KCl, (b) a M ascorbic acid, (c) b M Al III, (d) c M DA. B: (a) ph 8.5, 0.1 M NH 4Cl NH 3 H 2O 0.1 M KCl, (b) a M uric acid, (c) b M Al III, (d) c M DA. interferences of both ascorbic acid (AA) and uric acid (UA) which are the most two important substances to interfere with the detection of DA. This Al III -DA peak is quite sensitive to recognize DA. Figure 3 shows the electrochemical behaviors of both AA and UA at HMDE with and without both DA and Al III. No cathodic peak of AA at HMDE could be observed either with or without Al III (Figs. 3A, b and c). But after the addition of DA, the Al III -DA cathodic peak appears at about 960 mv (Fig. 3A, d). Although two cathodic peaks of UA at HMDE were observed at 500 and 700 mv, respectively (Figs. 3B, b and c), there are no new cathodic peaks that appear after Al III is added. Nor are any obvious changes of the peak height observed. However, with the addition of DA, the Al III -DA cathodic peak appears also at about 960 mv (Fig. 3B, d). At the same time, the cathodic peaks of uric acid disappear. It is quite possible that the complexes of both Al III -AA and Al III -UA are not adsorbed at HMDE even if the complexes are formed. This method can easily partition DA from the mixtures of both DA-AA and DA-UA. In order to get further evidence for this unique recognition mechanism of Al III -DA, we chose some chemicals with similar structures to DA as the objects for LSV investigations in the same potential window. The experimental results indicate that L-dopa, EP, NE, catechol, CA, PA, GA and tiron all yield the cathodic peaks at about 300 mv in the absence of Al III. But, no cathodic peak of catechol was observed. In the presence of Al III, all of the cathodic peak currents at 300 mv ascribed to L- dopa, E, NE, CA, PA, GA and tiron decreased with the increase of the Al III concentration (Figures not shown), but no new cathodic peak were observed within mv. Furthermore, some possible interfering metal ions were also inspected. The cathodic peak potentials for the complexes of DA with Zn II, Fe II, Pb II and Ca II were found at about 1200, 600, 650 and 500 mv, respectively (figures not shown). However, these peaks are not sensitive enough to DA and cannot be applied to recognize DA. Only the complex of Al III - DA can yield a unique and sensitive cathodic peak at about 900 mv which can be utilized to recognize DA effectively. Exploration of the electrode process and reaction mechanism of Al III -DA complex at HMDE We propose that both cathodic and anodic waves of Al III -DA possess adsorptive behaviors, which might be a quasi-reversible catalytic pre-wave on a mercury electrode. 25 This proposal for the electrode process is based on the following experimental results. (a) The cyclic voltammogram of DA in the presence of M Al III (Fig. 4) shows a pair of symmetric cathodic (i pc) and anodic (i pa) current peaks. The cathodic and anodic peak potentials are at 900 and 830 mv, respectively. This is in agreement with an one-electron quasi-reversible electrode process. (b) Both the i pc v and the i pa v show a linear relationship. I pc (μa) = v (mv/s), R = and I pa (μa) = v (mv/s), R = (0.1 mol/l NH 3 H 2O NH 4Cl 0.1 mol/l KCl, ph 8.5, mol/l Al III, mol/l DA, v = mv/s). This is the typical adsorptive behavior of the complex at HMDE. 25,26 (c) H + is not involved in the electrode reaction because the peak potentials do not change with ph values from 8.3 to 9.5 (figures not shown). This is quite different from the electrode reaction of Al(III)-norepinephrine. 27 (d) When the concentrations of both the animal glue and Triton X-100 reach 0.1%, both the cathodic and anodic peaks disappear. (e) After the solution is electrolyzed with controlling the constant potential for a long time, the cathodic and anodic peaks disappear and there are no small bubbles appearing on the electrode surface in the electrolytic process. These facts indicate that the cathodic peak cannot be a catalytic hydrogen wave. 26,28 If we use the difference of voltage (70 mv) between cathodic and anodic peaks, the molar ratio of 1:1 for Al III -DA can also be obtained, because this voltage difference is close to that of the theoretic 59 mv for one electron transfer redox reaction. Only the molar ratio of 1:1 Al III -DA complex would show an apparent one electron transfer redox reaction. Thus it is very possible that in alkaline buffer solution the amino group NH 2 and two phenolics O in DA molecule participate in the binding to Al III

4 1328 ANALYTICAL SCIENCES NOVEMBER 2007, VOL. 23 Fig. 5 Proposed chemical structure of Al III -DA complex on the HMDE surface. complex at HMDE can be directly observed on the LSV voltammogram. This success has practical meaning in pathologic research of neurodegenerative disorders in the future. This method holds great promise for its potential application in situ by coating Hg on microelectrodes with capillary electrophoresis and dialysis techniques. Combined with other modern analytical techniques, it might be anticipated to play a unique role in direct detecting free radicals generated in biooxidation of DA for the studies of the mechanism of AD relating to the Al toxicity. 29,39 Acknowledgements This project was supported by the National Natural Science Foundation of China (No ), MOE PhD Program Research Funding ( ), Analytical Measurement Grants of Nanjing University and a Visiting Fellowship from the Berkeley Lawrence National Laboratory of the USA. References Fig. 6 Chemical structures of various substances. a, Dopamine (DA); b, L-dopa; c, epinephrine (E); d, norepinephrine (NE); e, catechol; f, caffeic acid (CA); g, tiron; h, pyrogallic acid (PA); i, gallic acid (GA). and form 2:2 complexes (Fig. 5). Therefore, there are no cathodic and anodic peaks that appear in acidic NaAc-HAc and neutral NH 4Ac buffer solutions in the presence of DA and Al III. Because in acidic condition, an NH 2 group will combine with a H + to form NH 3+ which is hard to unite to Al III due to the electrostatic repulsive effect. 29 Consequently, this specific cathodic response of the Al III -DA complex at HMDE only exists in an alkaline buffer solution. This proves again that NH 2 probably participates in binding to Al III in alkaline condition. Why do other analogs (Fig. 6) not bring the similar pair of cathodic and anodic peaks under the same conditions? It is possible that they do not form the analogical complexes like Al III -DA at HMDE. L-Dopa will form a molecular inner-salt owing to the existing of NH 2 and COO groups. The CH 3 group of epinephrine will affect binding in virtue of steric hindrance. Both NH 2 and OH of norepinephrine are prone to form an intermolecular hydrogen bond with a five-member ring. Due to the double-bond and no NH 2 group, as well as to a plane configuration, caffeic acid will not complex similarly to that of Al III -DA. The others will not form the complexes apparently because of the lacking of a NH 2 group. Conclusions In summary, a novel sensitive LSV cathodic electrochemical response of Al III -DA has been discovered and applied for the recognizing DA from a series of chemicals with similar structures. Unlike the cases at solid electrodes, the Al III -DA 1. P. A. Garris, E. A. Budygin, P. E. M. Phillips, B. J. Venton, D. L. Robinson, B. P. Bergstrom, G. V. Rebec, and R. M. Wightman, Neuroscience, 2003, 118, D. R. Williams, J. Inorg. Biochem., 2000, 79, P. L. Runnels, J. D. Joseph, M. J. Logman, and R. M. Wightman, Anal. Chem., 1999, 71, A. R. Bernardo, J. F. Stoddart, and A. E. Kaifer, J. Am. Chem. Soc., 1992, 114, K. Pihel, T. J. Schroeder, and R. M. Wightman, Anal. Chem., 1994, 66, C. D. Allred and R. L. McCreery, Anal. Chem., 1992, 64, T. G. Strein and A. G. Ewing, Anal. Chem., 1991, 63, R. Kalvoda, Anal. Chim. Acta, 1984, 162, R. Kalvoda, J. Electroanal. Chem., 1986, 214, A. Dalmia, C. C. Liu, and R. F. Savinell, J. Electroanal. Chem., 1997, 430, J. W. Mo and B. Ogorevc, Anal. Chem., 2001, 73, S. H. DuVall and R. L. McCreery, Anal. Chem., 1999, 71, J. M. Zen, G. Ilangovan, and J. J. Jou, Anal. Chem., 1999, 71, A. Ciszewski and G. Milczarek, Anal. Chem., 1999, 71, J. M. Zen and P. J. Chen, Anal. Chem., 1997, 69, R. S. Deinhammer, M. Ho, J. W. Anderegg, and M. D. Porter, Langmiur, 1994, 10, P. D. Lyne and R. D. O Neill, Anal. Chem., 1990, 62, M. L. McGlashen, K. L. Davis, and M. D. Morris, Anal. Chem., 1990, 62, A. J. Tudos, W. J. J. Ozinga, H. Poppe, and W. T. Kok, Anal. Chem., 1990, 62, L. M. Jesus, A. G. Josefa, and M. L. Juan, Talanta, 2001, 53, H. Obata and C. M. G. van den Berg, Anal. Chem., 2001, 73, J. Limson and T. Nyokong, Anal. Chim. Acta, 1997, 344, K. Hasebe, S. Hikima, T. Kakizaki, T. Iwashimizu, and K. Aoki, Analyst, 1990, 115, L. M. Jesus, A. G. Josefa, and M. L. Juan, Electroanalysis, 1999, 11, 656.

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