Simultaneous Electrochemical Determination of Glutathione and

Aug 14, 2009 - Ying Wang , Jin Lu , Longhua Tang , Haixin Chang and Jinghong Li. Analytical Chemistry 2009 81 (23), 9710-9715. Abstract | Full Text HT...
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Anal. Chem. 2009, 81, 7538–7543

Simultaneous Electrochemical Determination of Glutathione and Glutathione Disulfide at a Nanoscale Copper Hydroxide Composite Carbon Ionic Liquid Electrode Afsaneh Safavi,* Norouz Maleki, Elaheh Farjami, and Farzaneh Aghakhani Mahyari Department of Chemistry, College of Sciences, and Nanotechnology Research Institute, Shiraz University, Shiraz, Iran Direct simultaneous electrochemical determination of glutathione (GSH) and glutathione disulfide (GSSG) has been presented using a nanoscale copper hydroxide carbon ionic liquid composite electrode. To the best of our knowledge, this is the first report on the simultaneous determination of these two biologically important compounds based on their direct electrochemical oxidation. Incorporation of copper(II) hydroxide nanostructures in the composite electrode results in complexation of Cu(II) with the thiol group of GSH and leads to a significant decrease in GSH oxidation overpotential, while an anodic peak corresponding to the direct oxidation of GSSG as the product of GSH oxidation is observed at higher overvoltages. Low detection limits of 30 nM for GSH and 15 nM for GSSG were achieved based on a signal-to-noise ratio of 3. The proposed method is free from interference of cysteine, homocystein, ascorbic acid (AA), and uric acid (UA). No electrode surface fouling was observed during successive scans. Stability, high sensitivity, and low detection limits made the proposed electrode applicable for the analysis of biological fluids. Glutathione (GSH) and glutathione disulfide (GSSG) play an important role in the metabolism of living cells. Reduced glutathione is the main nonprotein thiol which is found at high concentrations in many living cells.1,2 Under oxidative stress, glutathione in reduced form (GSH), can be converted to the oxidized form, GSSG, and rapidly reverts back to GSH by the action of the enzyme glutathione reductase. The redox status cell depends on the ratio of GSH to GSSG as a sensitive indicator of the oxidative stress which is a critical determinant in cells.3 Therefore, sensitive detection methods for the analysis of these compounds are highly demanded. Some analytical methods using liquid chromatography (LC) combined with different detection techniques have been developed for the analysis of GSH and GSSG. Since GSH and GSSG do not * To whom correspondence should be addressed. E-mail: safavi@ chem.susc.ac.ir. Tel: +98-711-6137351. Fax: +98-711-2286008. (1) Pastore, A.; Federici, G.; Bertini, E.; Piemonte, F. Clin. Chim. Acta 2003, 333, 19–39. (2) Mate´s, J. M.; Pe´rez-Go´mez, C.; Blanca, M. Clin. Chim. Acta 2000, 296, 1–15. (3) Morel, Y.; Barouki, R. Biochem. J. 1999, 342, 481–496.

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have spectroscopically active groups, detection systems based on fluorimetric4,5 or other photometric methods6,7 need derivatization, which is time-consuming, and it is usually difficult to avoid oxidation of GSH. Simplicity, rapidity, high sensitivity, and low cost are the main advantages of electrochemical techniques for the analysis of biological compounds. Several electrochemical detection methods, mostly based on either the reduction of GSSG on a hanging mercury drop electrode8 or oxidation of both GSH and GSSG on unmodified9,10 or chemically modified electrodes,11-13 have been reported. However, the main problem with these methods is the high overpotential for oxidation of these compounds on common carbon based electrodes.14 Moreover, surface oxides formed on metal electrodes, such as platinum and gold, mediated the anodic oxidation of disulfides in the same potential range15,16 and, thus, complicate their analytical applications. The detection of thiol compounds (not disulfides) using inorganic electrocatalyst modified electrodes12 suffers from a gradual decrease in response with time. A recent report explains the use of a functionalized carbon nanotube modified electrode coupled with HPLC as a detector for the determination of GSH and GSSG.11 However, this kind of electrode modification was more effective in increasing the current of GSSG and did not overcome the high overpotential of GSSG oxidation.11 As mentioned previously, common carbon electrodes such as glassy carbon show no signal corresponding to GSSG oxidation, (4) Wang, H.; Liang, S. C.; Zhang, Z. M.; Zhang, H. S. Anal. Chim. Acta 2004, 512, 281–287. (5) Kamencic, H.; Lyon, A.; Paterson, P. G.; Juurlink, B. H. J. Anal. Biochem. 2000, 286, 35–37. (6) Katrusiak, A. E.; Paterson, P. G.; Kamencic, H.; Shoker, A.; Lyon, A. W. J.Chromatogr., B 2001, 758, 207–212. (7) Muscari, C.; Pappagallo, M.; Ferrari, D.; Giordano, E.; Capanni, C.; Caldarera, C. M.; Guarnieri, C. J. Chromatogr. B 1998, 707, 301–307. (8) Ba˘nica˘, F. G.; Fogg, A. G.; Moreira, J. C. Talanta 1995, 42, 227–235. (9) Smith, N. C.; Dunnett, M.; Mills, P. C. J. Chromatogr., B 1995, 673, 35– 41. (10) Lakritz, J.; Plopper, C. G.; Buckpitt, A. R. Anal. Biochem. 1997, 247, 63– 68. (11) Zhang, W.; Wan, F.; Zhu, W.; Xu, H.; Ye, X.; Cheng, R.; Jin, L. T. J. Chromatogr., B 2005, 818, 227–232. (12) Nalini, B.; Narayanan, S. S. Electroanalysis 1998, 10, 779–783. (13) Inoue, T.; Kirchhoff, J. R. Anal. Chem. 2000, 72, 5755–5760. (14) Tang, H.; Chen, J.; Nie, L.; Yao, S.; Kuang, Y. Electrochim. Acta 2006, 51, 3046–3051. (15) Vandeberg, P.; Johnson, D. C. Anal. Chem. 1993, 65, 2713–2718. (16) Hoekstra, J. C.; Johnson, D. C. Anal. Chem. 1998, 70, 83–88. 10.1021/ac900501j CCC: $40.75  2009 American Chemical Society Published on Web 08/14/2009

and the signal for GSH is observed at a relatively high overpotential of ∼1.0 V vs SCE.17 To the best of our knowledge, there is no report on simultaneous direct electrochemical oxidation of GSH and GSSG. Therefore, the design of suitable electrode materials that can simultaneously detect these important biocompounds is highly demanding, particularly for the analysis of biological samples. High performance carbon ionic liquid electrode (CILE) has been proposed in our research group with many good features, particularly provision of high rates of electron transfer.18-25 On the other hand, in recent years, fast growth in the design of electrochemical sensors based on nanomaterials has been observed due to their special physical and chemical properties such as increasing the surface area, mass transport, and catalysis.26 In a previous report, we introduced a composite CILE based on nanoscale nickel hydroxide as an extraordinary stable nanosensor for highly sensitive nonenzymatic detection of glucose.27 The presence of a thiol moiety in the GSH molecule gives it the ability to interact directly with Cu2+ ions. There is evidence which implicates the interaction between GSH and Cu2+ ions in the promotion of an antioxidant effect action of GSH.28,29 In this paper, a simple and facile method has been used for the synthesis of nanosized copper hydroxide powder. Then, a composite electrode was fabricated based on mixing nanoscale copper hydroxide with graphite powder and ionic liquid (octylpyridinum hexafluorophosphate, OPy+PF6-). The mentioned composite electrode largely decreased the overpotential of the oxidation of GSH compared to bare CILE. Moreover, the product of GSH oxidation, GSSG, can be oxidized in the available potential window. The electrode surface could be easily and reproducibly renewed by slight polishing on a smooth paper. Due to its excellent resistance to surface fouling and its renewable surface, the electrode is suggested for direct analysis of GSH and GSSG in blood serum samples. EXPERIMENTAL SECTION Materials. 1-Iodooctane, pyridine, the reduced form of glutathione, and glutathione disulfide were obtained from Merck. Ammonium hexafluorophosphate, copper sulfate hexahydrate, and graphite powder (mesh size +1.0 V vs Ag/AgCl), oxygencontaining functional groups, such as carbonyl or hydroxyl groups, are formed on the facets of the electrode surface which form a negative dipolar field, which electrostatically attracts the positively charged GSSG molecule.37 (35) Johnson, A. M.; Holcombe, J. A. Anal. Chem. 2005, 77, 30–35. (36) Miller, T. C.; Kwak, E.-S.; Howard, M. E.; Vanden Bout, D. A.; Holcombe, J. A. Anal. Chem. 2001, 73, 4087–4095. (37) Chiku, M.; Nakamura, J.; Fujishima, A.; Einaga, Y. Anal. Chem. 2008, 80, 5783–5787. (38) Zhou, M.; Ding, J.; Guo, L.; Shang, Q. Anal. Chem. 2007, 79, 5328–5335.

Figure 4. Cyclic voltammograms of 1 mM GSH obtained in 0.1 M phosphate buffer solution at: (a) Cu(OH)2 bulk scale modified CILE and (b) nano Cu(OH)2 modified CILE, 1% in both cases.

Figure 5. Nyquist plots for 1 mM GSH 0.1 M phosphate buffer solution (pH 7.0) at (a) bare CILE and (b) nano Cu(OH)2 modified CILE. 0.1-105 Hz frequency ranges.

A higher sensitivity was observed at nanoscale Cu(OH)2 platelet-like modified CILE mainly due to its higher active surface area compared to bulk scale Cu(OH)2 modified CILE, with the same percentage (Figure 4). Different ratios of the copper(II) hydroxide nanoscale, 0.5-20% (wt %), were incorporated to the carbon ionic liquid electrode, and the optimum response was observed by incorporation of 1% (wt %) of nano Cu(OH)2 to the CILE. Increasing the amount of nanoscale Cu(OH)2 caused an increase in background current (not shown). The results for GSH oxidation in neutral and acidic media show that the peak potential shifts linearly toward more positive potentials. However, in alkaline solutions, the process is not pH dependent. By plotting Ep vs pH, a pKa value of 9.12 was obtained which agrees well with the pKa value of the dissociation of the proton from the thiol group in GSH (pKSH ) 9.65). In this work, pH 7.0 was selected as the working pH, because of its physiological significance. Electrochemical impedance spectroscopy (EIS) is a suitable technique for investigation of the electrode surface dependent charge transfer process. In our work, EIS results obtained from a Nyquist plot show a large decrease in charge transfer resistance for 1 mM GSH phosphate buffer solution (pH 7.0) at nano Cu(OH)2 modified CILE (Figure 5b) compared to bare CILE (Figure 5a) indicating a significant increase in the rate of oxidation of GSH due to the complexation between its thiol group and

Figure 6. Cyclic voltammograms for the oxidation of 1 mM GSH obtained in 0.1 M phosphate buffer solution at (a) nano Cu(OH)2 modified CILE and (b) nano Cu(OH)2 modified CPE.

copper(II) ions present on the modified electrode. The advantages of the presence of ionic liquid in carbon paste electrodes have been discussed in our previous report.19 In order to elucidate the role of ionic liquid as the binder in the proposed composite electrode, Nujol was used as the binder instead of ionic liquid. A carbon paste electrode modified with nano Cu(OH)2 shows no signal corresponding to the oxidation of 1 mM GSH and disulfide as the product of thiol oxidation (Figure 6). This observation signifies the fact that the nanoparticle microenvironment is greatly influenced by the support on which it is deposited.23,39 No surface fouling was observed on nano Cu(OH)2 modified CILE, due to the existence of adsorbed reaction products during GSH and GSSG oxidation (Figure 7A,B). Therefore, the oxidation peak current and potential at the modified composite electrode were essentially unchanged after repetitive scans. A chronocoulometric study was carried out by applying a potential step in the presence and absence of low concentrations of GSSG (100 µM) to investigate the adsorption process at the proposed modified electrode. The difference between the intercepts of Qtotal and Qdl (blank) vs t1/2 plots was approximately zero (Figure S-2 in the Supporting Information), indicating the absence of adsorption of reactant within the potential range of GSSG oxidation. The obtained results show that the nano Cu(OH)2 modified CILE is free from adsorption of GSSG. At the copper hydroxide modified CILE, no cathodic peak due to the oxidation products of GSSG was observed on the reverse scan within the potential range of +1.4 to -0.2 V, which indicates that the byproduct of GSSG oxidation is not electroreducible in the investigated potential range on the modified electrode. The advantage of the proposed composite electrode is its ability in simultaneous electrochemical detection of GSH and GSSG with good sensitivity and without the need for separation of the two compounds prior to electrochemical measurements. The analytical performance of the proposed electrode was examined using square wave voltammetry. The slopes of calibration curves for GSH and GSSG were calculated by a linear regression method in the two linear concentration ranges of 1-50 µM and 0.1-1.8 mM for GSH and 0.4-120 µM for GSSG using their relevant oxidation (39) Welch, C. M.; Compton, R. G. Anal. Bioanal. Chem. 2006, 384, 601–619.

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Table 1. Results of Recovery Studies of the Analysis of GSH and GSSG in Human Plasmaa compound added

added/µM

found/µM

GSH

2 5 2 5

3.10 ± 0.11 5.21 ± 0.20 7.92 ± 0.53 0.20 ± 0.04 2.15 ± 0.32 5.12 ± 0.40

GSSG

a

Figure 7. Consecutive cyclic voltammograms of (A) 1 mM GSH and (B) 0.25 mM GSSG obtained in 0.1 M phosphate buffer solution at nano Cu(OH)2 modified CILE, first and fifth cycles.

peaks. The detection limits of 30 nM for GSH and 15 nM for GSSG were achieved based on a signal-to-noise ratio of 3. The relative standard deviation (RSD) values were evaluated by replicate measurements (n ) 5) as 3.8 and 4.4% for GSH and GSSG, respectively. It is interesting to note that the calibration curve obtained for GSSG alone resembles that obtained for GSSG produced via GSH oxidation. The proposed composite electrode was stored in air at ambient conditions, and its sensitivity toward 0.5 mM GSH was checked every week. The response was 93% of its initial value after 70 days which shows long-term stability and very good sensitivity for the analysis of real samples. The responses of four similar electrodes toward 0.5 mM GSH were measured, and a RSD of 3.1% was obtained confirming high reproducibility of the fabrication method. Cysteine is the main biological compound existing in living cells which can interfere with glutathione detection owing to the structural similarity between this compound and the GSH molecule, which contains a cysteinyl residue. In the present work, complexation between copper(II) ions and cysteine gives an anodic peak for oxidation of cysteine at a less positive potential (0.03 V vs Ag/AgCl) compared to the corresponding anodic peak for GSH oxidation, (∼0.12 V vs Ag/AgCl). Therefore, there is a significant difference between their peak potentials, and it is quite possible to detect GSH oxidation responses with minimal interfrerence from cysteine. Only a 7% decrease in GSH oxidation current (0.1 mM) was observed in the presence of five times molar excess of cysteine. 7542

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recovery/% 100.5 96.4 97.5 98.4

Triplicate analysis was performed for each analysis. -: not added.

Homocysteine is an important amino acid which exists in either its reduced or oxidized form and plays important roles within physiological matrixes. At physiological pH values, homocysteine is readily oxidized and less than 1% will be present as its thiolate anion.40 The response of the proposed electrode was studied in the presence of 1 mM homocysteine in phosphate buffer solution (pH 7.0). Two peaks were observed at high anodic potentials (+0.9 and +1.3 V vs Ag/AgCl) on the nano Cu(OH)2 modified CILE while no peak was observed in the potential range of -0.2 to +0.8 V, where the peak due to GSH appeared (Figure S-3 in the Supporting Information). Thus, the presence of 1 mM homocysteine in the PBS solution containing 1 mM GSH does not have an appreciable effect on direct oxidation of GSH (Figure S-4 in the Supporting Information). It should be noted that the homocysteine concentration (1 mM) reported in Figure S-4 in the Supporting Information is much higher than the level of homocysteine in blood serums (typically 5-15 µM).40 In the presence of physiological concentrations of homocysteine, no effect was observed on the glutathione response. The response of the proposed composite electrode toward GSH was also evaluated in the presence of some common oxidizable species found in biological fluids such as glucose, ascorbic acid, and uric acid. No interfereing effect was observed from the presence of 1 mM of either glucose (Figure S-5 in the Supporting Information) or ascorbic acid (Figure S-6 in the Supporting Information), while the oxidation peak current corresponding to 1 mM GSH was decreased to 81% of its initial value in the presence of 1 mM uric acid (Figure S-7 in the Supporting Information). The obtained results show the satisfactory selectivity of the proposed composite electrode even at higher overpotentials. The applicability of the proposed electrode was further evaluated by the simultaneous determination of GSH and GSSG in human serum plasma after the protein precipitation process. Here, 0.5 mL of blood serum sample was added to 10 mL of phosphate buffer (pH 7.0), and analysis of the unknown sample was performed by the standard addition method. The GSH and GSSG concentrations (Table 1) were in agreement with the range of GSH and GSSG concentrations in human plasma.41 Recovery experiments were also carried out by adding known amounts of GSH and GSSG to the human plasma sample. Very satisfactory results were obtained (Table 1). CONCLUSION The unique behavior of nano Cu(OH)2 modified CILE has allowed simultaneous electrochemical determination of GSH (40) Nekrassova, O.; Lawrence, N. S.; Compton, R. G. Talanta 2003, 60, 1085– 1095. (41) Camera, E.; Picardo, M. J. Chromatogr., B 2002, 781, 181–206.

and GSSG with a significant difference between the oxidation responses (∼1 V vs Ag/AgCl) of the two compounds. Thus, there is no need for prior separation of these two substances using expensive analytical techniques. The proposed method is free from interference of important common oxidizable species found in biological fluids. Surface fouling due to the adsorption of byproducts of the oxidation process did not occur on the proposed nanocomposite electrode during successive scans for direct oxidation of GSH and GSSG. High stability, sensitivity, and reproducibility as well as low detection limits for GSH and GSSG, ease of preparation, low cost, and surface renewal made the proposed nanocomposite electrode ideal for simultaneous detection of GSH and GSSG in biological fluids. ACKNOWLEDGMENT The authors wish to express their gratitude to the Shiraz University Nanotechnology Research Institute, Shiraz University

Research Council, and the Third World Academy of Sciences, Iran Chapter (TWASIC), for the support of this work. SUPPORTING INFORMATION AVAILABLE Absorption spectral changes of adding GSH to nanoscale copper hydroxide with increasing time. Linear chronocoulometric plots at Cu(OH)2 nano platelet-like modified CILE. Cyclic voltammogram of homocysteine. Cyclic voltammograms of GSH in the presence of homocysteine, glucose, ascorbic acid, and uric acid. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 8, 2009. Accepted August 1, 2009. AC900501J

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