Anal. Chem. 2007, 79, 5328-5335
Electrochemical Behavior of L-Cysteine and Its Detection at Ordered Mesoporous Carbon-Modified Glassy Carbon Electrode Ming Zhou, Jie Ding, Li-ping Guo,* and Qing-kun Shang
Faculty of Chemistry, Northeast Normal University, Changchun, 130024, People’s Republic of China
In this paper, the electrochemical behavior of L-cysteine (CySH) was investigated thoroughly at an ordered mesoporous carbon-modified glassy carbon (OMC/GC) electrode. The voltammetric studies showed there were three anodic peaks for the electrooxidation of CySH in the pH range of 2.00-5.00; however, one peak disappeared above pH 5.00. This behavior has never been reported before. Through the studies of the effect of pH on the distribution fractions (δ) of the four chemical species of CySH, we conclude only CySH2+ (H3A+) and CyS- (HA-) are the electroactive substances and should be responsible for the electrooxidation of CySH. And for the first time, we successfully established the exact and systemic mechanisms based on the electroactive species to explain CySH oxidation at different pH values. On the other hand, a sensitive CySH sensor was developed based on an OMC/GC electrode, which shows a large determination range (18-2500 µmol L-1), a high sensitivity (23.6 µA mmol L-1), and a remarkably low detection limit (2.0 nmol L-1, which is the lowest value ever reported for direct CySH determination on the electrodes) at pH 2.00. At pH 7.00, the modified electrode can be still used to readily detect CySH in the range of the physiological levels. These make OMC/GC electrode a promising candidate for efficient electrochemical sensors for the detection of CySH. L-Cysteine (CySH) is an important amino acid owing to its crucial roles in biological systems. For example, it could be used as a prospective radiation protector and cancer indicator.1-4 Moreover, the couple L-cystine/L-cysteine is generally used as a model for the role of the disulfide bond and thiol group in proteins in a variety of biological media.1 Therefore, it is very important to investigate the electrochemical behavior and sensitive detection of CySH.5-12 Unfortunately, at ordinary electrodes (Pt, Au, graphite), the electrochemical behaviors of CySH are poor; no
* Corresponding author. Tel.: +86-431-85099762. Fax: +86-413-85099762. E-mail:
[email protected]. (1) Chen, H. L.; Li, M. S. Structure and Function of Biomacromolecules; Shanghai Science Press: Shanghai, 1999. (2) Kulys, J.; Drungiliene, A. Anal. Chim. Acta 1991, 243, 287-292. (3) Townshend, A. Encyclopedia of Analytical Science; Academic Press: London, 1995. (4) Filanovsky, B. Anal. Chim. Acta 1999, 394, 91-100. (5) Zen, J.; Kumar, A.; Chen, Y. Anal. Chem. 2001, 73, 1169-1175. (6) Spa˜taru, N.; Sarada, B. V.; Popa, E.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2001, 73, 514-519.
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electrochemical responses could be observed.13,14 It has been realized that the method to solve this problem may be to utilize new materials as electrodes; at such an electrode surface, the electrochemical responses could be simply obtained directly.5-23 Over the past decades, several carbon-based materials, including fullerene15 and boron-doped diamond, have been explored for the electrochemical oxidation and detection of CySH.6 Recently, due to their unique electronic, chemical, and mechanical properties, carbon nanotubes (CNTs) as a new class of carbon nanomaterials have been exploited for the electrochemical oxidation of CySH.7,22 The high electrocatalytic activity observed at CNTs is attributed to the presence of the oxygen-containing functional groups on the surface of CNTs and a large number of edge plane graphite sites within the walls and at the ends of CNTs.7,24,25 Besides the materials mentioned above, there has been significant interest in the development of one such novel carbon material, i.e., ordered mesoporous carbon (OMC). The OMC, which was first synthesized in 1999, is particularly promising (7) Gong, K.; Zhu, X.; Zhao, R.; Xiong, S.; Mao, L.; Chen, C. Anal. Chem. 2005, 77, 8158-8165. (8) Teixeiraa, M.; Dockalb, E.; Cavalheiroc, E. Sens. Actuators, B 2005, 106, 619-625. (9) Ralph, T. R.; Hitchman, M. L.; Millington, J. P.; Walsh, F. C. J. Electroanal. Chem. 1994, 375, 1-15. (10) Ralph, T. R.; Hitchman, M. L.; Millington, J. P.; Walsh, F. C. J. Electroanal. Chem. 1994, 375, 17-37. (11) Terashima, C.; Rao, T. N.; Sarada, B. V.; Kubota, Y.; Fujishima, A. Anal. Chem. 2003, 75, 1564-1572. (12) Fei, S.; Chen; J.; Yao, S.; Deng, G.; He, D.; Kuang, Y. Anal. Biochem. 2006, 339, 29-35. (13) Halbert, M. K.; Baldwin, R. P. Anal. Chem. 1985, 57, 591-595. (14) Wang, Z.; Pang, D. J. Electroanal. Chem. 1990, 283, 349-358. (15) Tan, W. T.; Bond, A. M.; Ngooi, S. W.; Lim, E. B.; Goh, J. K. Anal. Chim. Acta 2003, 491, 181-191. (16) Fujiwara, S.; Pessoa, C.; Gushikem, Y. Electrochim. Acta 2003, 48, 36253631. (17) Maree, S.; Nyokong, T. J. Electroanal. Chem. 2000, 492, 120-127. (18) Zhang, S.; Sun, W.; Zhang, W.; Qi, W. Anal. Chim. Acta 1999, 386, 21-30. (19) Zagal, J. H.; Aguirre, M. J.; Patodi, C. G. J. Electroanal. Chem. 1994, 374, 215-222. (20) Sugawara, K.; Hoshi, S.; Akatsuka, K.; Shimazu, K. J. Electroanal. Chem. 1996, 414, 253-256. (21) Reynaud, J. A.; Maltoy, B.; Canessan, P. J. Electroanal. Chem. 1980, 114, 195-211. (22) Zhao, Y. D.; Zhang, W. D.; Cheng, H.; Luo, Q. M. Sens. Actuators, B 2003, 92, 279-285. (23) Xu, J.; Wang, Y.; Xian, Y.; Jin, L.; Tanaka, K. Talanta 2003, 60, 11231130. (24) Moore, R. R.; Banks, C. E.; Compton, R. G. Anal. Chem. 2004, 76, 26772682. (25) Jia, N.; Wang, Z.; Yang, G.; Shen, H.; Zhu, L. Electrochem. Commun. 2007, 9, 233-238. 10.1021/ac0703707 CCC: $37.00
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because the SBA-15 template is inexpensive and easy to synthesize.26-28 Contrary to most porous silica-based materials (for example, SBA-15) that are electronic semiconductors, the mesoporous carbons are intrinsically conductors.26,27,29 And OMC also exhibits extremely high surface area and well-defined pore size as well as high thermal stability, flexible framework composition, and chemical inertness,25-30 which makes OMCs potential novel materials for investigating the electrochemical behavior of the substances. Despite such potential capability of OMC, there have only been a few studies on the electroanalytical applications for electrocatalysis, especially the electrocatalytic oxidation of biomolecules.25,31,32 In this paper, the electrochemical oxidation of CySH was investigated using a GC electrode modified with OMC, and the detailed and systematic investigation for the catalytic mechanisms of CySH oxidation at OMC/GC electrode were reported. An extraordinary sensitivity for CySH detection was obtained at the OMC/GC electrode. And CySH should be a good model for further electrochemical studies of other thiols. EXPERIMENTAL SECTION Reagents. L-Cysteine was purchased from Beijing Chemical Reagent Co. Nafion solution (5 wt % in 15-20% water/lower aliphatic alcohols) was obtained from Aldrich. All other chemicals not mentioned here were of analytical reagent grade and were used as received. Double-distilled water was used throughout. The 0.1 mol L-1 phosphate buffer solution (PBS), which was made from Na2HPO4, NaH2PO4, and H3PO4, was employed as a supporting electrolyte. Synthesis of OMC. SBA-15 mesoporous silicates were synthesized following a published hydrothermal procedure using amphiphilic poly(alkylene oxide)-type triblock copolymers in an aqueous medium.33 The SBA-15 silicate material was then used as a template to synthesize OMC using sucrose as the carbon source according to a reported carbonization procedure.30 Order mesoporous carbon (the carbonized SBA-15) was denoted as OMC in the literature. Electrode Preparation and Modification. GC electrodes (model CHI104, 3-mm diameter) were polished before each experiment with 1, 0.3- and 0.05-µm alumina powder, respectively, rinsed thoroughly with doubly distilled water between each polishing step, then washed successively with 1:1 nitric acid, acetone, and doubly distilled water in an ultrasonic bath, and dried in air. The OMC/GC electrodes were prepared by casting 5 µL of OMC suspension (1 mL of Nafion solution, 9 mL of doubledistilled water, and 5 mg of OMC) on the surface of a GC electrode (Nafion in the OMC suspension was used as a binder). Evaporation of water from such a solution yielded robust surface films that contained OMC. (26) Ryoo, R.; Joo, S. H.; Jun, S. J. Phys. Chem. B 1999, 103, 7743-7746. (27) Liang, C.; Dai, S. J. Am. Chem. Soc. 2006, 128, 5316-5317. (28) Kim, J. M.; Stucky, G. D. Chem. Commun. 2000, 13, 1159-1160. (29) Walcarius, A. C. R. Chim. 2005, 8, 693-712. (30) Jun, S.; Joo, S. H.; Ryoo, R.; Kruk, M.; Jaroniec, M.; Liu, Z.; Ohsuna, T.; Terasaki, O. J. Am. Chem. Soc. 2000, 122, 10712-10713. (31) Feng, J. J.; Xu, J. J.; Chen, H. Y. Biosens. Bioelectron. 2007, 22, 16181624. (32) Zhou, M.; Guo, L.; Lin, F.; Liu, H. Anal. Chim. Acta 2007, 587, 124-131. (33) Zhao, D.; Huo, Q.; Feng, J.; Chmelka, B. F.; Stucky, G. D. J. Am. Chem. Soc. 1998, 120, 6024-6036.
Apparatus and Measurements. Electrochemical experiments were performed with a CHI 830b electrochemical workstation (CH Instruments, Shanghai Chenhua Instrument Corp.) in a conventional three-electrode cell. The working electrode used was a GC electrode. A platinum electrode was applied as the auxiliary electrode, and an Ag/AgCl (in saturated KCl solution) electrode served as reference electrode. The sample solutions were purged with purified nitrogen for at least 15 min to remove oxygen prior to the beginning of a series of experiments. Small-angle X-ray diffraction (XRD) patterns were obtained on an X-ray Diffractor D4 (Brucker) operating at 40 kV and 20 mA and using Cu KR radiation (λ ) 0.154 06 nm). The transmission electron microscopy (TEM) image was recorded on a Hitachi H-600 operated at 75 keV. Nitrogen adsorption-desorption isotherms were measured on an ASAP 2020 (Micromeritics). The pore size distributions were calculated by the BJH method. Infrared spectrum of the sample was recorded with a Nicolet Magna 560 FT-IR spectrometer with a KBr plate. The controlled potential electrolysis was carried out using an OMC/GC electrode with the geometric area estimated to be 1 cm2. The applied potential was obtained using potentiostat (model DJS-292, Shanghai Rex Instruments Factory). This electrolysis was carried out in a single-compartment cell. A nitrogen stream and stirring were maintained in the solution during the electrolysis. The controlled potentials for peaks 1′ and 2′ were at +0.03 and +0.39 V in CySH pH 7.00 PBS, respectively. And the controlled potentials for peaks 1, 2, and 3 were at +0.32, +0.73, and +0.92 V in CySH pH 2.00 PBS, respectively. The initial concentration of the electrolysis solution was 5 µmol L-1 CySH; after chosen intervals (each 15 min), Five microliters of the electrolyzed solution was injected into the chromatographic system. In order to electrolyze the CySH solution completely, the products of the electrolyzed solution were not used for the MS measurements until the height of each peak in LC chromatograms was nearly unchanged within the consecutive injections. And in our system, the height of each peak in LC chromatograms was nearly unchanged after the CySH solution was electrolyzed for ∼120 min. RESULTS AND DISCUSSION Characterization of OMC. As shown in inset a of Figure 1A, the ordered arrangement of carbon nanorods can be observed by the well-resolved XRD peaks, which can be assigned to (100), (110) diffractions of hexagonal (p6 mm) structure. This demonstrates the carbon nanorods synthesized in this study are rigidly interconnected into a highly ordered hexagonal array by the carbon spacers, which are the inverse replica of SBA-15. Pore size distribution of the mesoporous carbon structure was analyzed by the nitrogen adsorption-desorption isotherms in Figure 1A. As shown, it exhibits capillary condensation steps on the nitrogen adsorption isotherm and consequently narrow mesopore size distributions. The result in inset b of Figure 1A indicates the carbon is mostly mesoporous with quite narrow pore size distribution centered at 4.5 nm. From the N2-adsorption experiments, the BET surface area is ∼1520 m2 g-1 and the total pore volume of carbon is ∼1.3 cm3 g-1. The oxygen-containing functional groups are presented on the surface of OMC, which were confirmed by Fourier transform infrared spectroscopy (FT-IR) in Figure 1B. The band around 1688 cm-1 is attributed to CdO stretch vibration, and the bands around Analytical Chemistry, Vol. 79, No. 14, July 15, 2007
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Figure 2. CVs obtained at the GC electrode (A) with 0 (curve a) and 5 mmol L-1 CySH (curve b), at the Nafion/GC electrode (B) with 0 (curve c) and 5 mmol L-1 CySH (curve d), and at the OMC/GC electrode (C) with 0, 5, 13, and 15 mmol L-1 CySH (curves e-h). Scan rate, 5 mV s-1. Electrolyte, 0.1 mol L-1 pH 7.00 PBS.
Figure 1. (A) Nitrogen adsorption-desorption isotherm for OMC. Inset a shows the powder XRD patterns of OMC, and inset b is the pore size distributions for OMC. (B) FT-IR spectra of OMC. (C) TEM image of OMC.
1550 cm-1 and 1515 cm-1 are ascribed to COO- stretch asymmetric vibration. The band around 1106 cm-1 is assignable to C-O stretch vibration, while the carboxylic acid O-H stretch vibration is responsible for the band around 3435 cm-1. Figure 1C shows the TEM image for OMC. With long-range ordering observed, this is in agreement with the result from the pore size distribution analysis, showing a main peak at 4-5 nm in the mesopore range. Cyclic Voltammetry of CySH at OMC/GC Electrode. Figure 2 shows the electrochemical oxidation of CySH at GC (A), Nafion/ GC (B), and OMC/GC (C) electrodes at pH 7.00. No obvious anodic peaks of CySH can be observed at GC and Nafion/GC electrodes. However, at the OMC/GC electrode, there are two anodic peaks at +0.02 (peak 1′) and +0.38 V (peak 2′), respectively. This means the oxidation of CySH at pH 7.00 undergoes two processes, and the OMC film clearly plays an important role in the observed electrocatalytic behavior. Mao et al. reported the oxidation of CySH at CNTs modified electrode at pH 7.0 undergoes two processes: at ∼0.0 V attributed to the oxygen-containing functional groups of CNTs, and at ∼+0.35 V ascribed to the edge5330 Analytical Chemistry, Vol. 79, No. 14, July 15, 2007
plane-like defective sites of CNTs,7 which are almost at the same potentials as those at the OMC/GC electrode of this work. This suggests the electrooxidation of CySH at OMC/GC and CNTsmodified electrodes at pH 7.0 may have the same processes and similar mechanisms. The quasi-reversible redox peak pairs (curve e) observed at OMC/GC electrode in Figure 2C can be also observed for OMC and CNTs-based materials,7,25 which indicates the pairs may originate from protonation/depronation of the oxygen-containing functional groups of OMC.25 So the oxygencontaining functional groups of OMC are thus mainly responsible for peak 1′. There are also significant edge-plane-like defective sites existing on the surface of OMC,25 which means peak 2′ at the OMC/GC electrode at pH 7.00 could be logically attributed to the edge-plane-like defective sites of OMC. We also investigate the electrochemical behavior of CySH at GC, Nafion/GC, and OMC/GC electrodes at pH 2.00. In Figure 3C, the addition of CySH in solution results in clear increases in the peak currents at peak 1 (+0.31 V), peak 2 (+0.72 V), and peak 3 (+0.91 V) at the OMC/GC electrode. To assess whether the preparation conditions (and not the OMC modifier itself) are responsible for the catalytic behavior, control experiments were conducted. For this purpose, the GC surface was coated with Nafion, which resulted in no obvious anodic peaks of CySH at the Nafion/GC electrode (Figure 3B). Therefore, the three welldefined anodic peaks observed in the presence of OMC clearly indicate the essential role of OMC in the observed electrocatalytic behavior. In Figure 3C, the quasi-reversible redox peak pairs (curve e) were observed at the OMC/GC electrode; therefore, peak 1 may also originate from the oxygen-containing functional groups of OMC.7,25 It should be noteworthy that the electrochemical behavior of the consecutive three steps for the oxidation of CySH at the OMC/GC electrode at pH 2.00 is dissimilar from that at the OMC/GC electrode at pH 7.00 (with two anodic peaks),
Figure 4. Effect of pH on the anodic peak currents of peaks I (A), II (B), and III (C) for 5 mmol L-1 CySH at the OMC/GC electrode by cyclic voltammetry. Scan rate, 5 mV s -1. Electrolyte, 0.1 mol L-1 PBS.
Figure 3. CVs obtained at the GC electrode (A) with 0 (curve a) and 5 mmol L-1 CySH (curve b), at the Nafion/GC electrode (B) with 0 (curve c) and 5 mmol L-1 CySH (curve d), and at the OMC/GC electrode (C) with 0, 5, 10, and 15 mmol L-1 CySH (curves e-h). Scan rate, 5 mV s-1. Electrolyte, 0.1 mol L-1 pH 2.00 PBS.
and it is also different from that at other electrodes reported before.5-23 Such phenomena not only indicate the essential role of OMC for the electrocatalysis of CySH but also suggest a high pH dependence of the electrochemical oxidation of CySH at the OMC/GC electrode. Effect of pH. Cyclic voltammograms (CVs) of 5 mmol L-1 CySH at different pH were recorded at an OMC/GC electrode (data not shown). In the pH range of 2.00-5.00, there are three anodic peaks for CySH oxidation at an OMC/GC electrode, but there are two when the pH is above ∼5.00. In order to illuminate the processes and the mechanisms for CySH oxidation at the OMC/GC electrode expediently, we name the first anodic peaks, which are mediated by the oxygen-containing functional groups of OMC in the pH range of 2.00-12.00 for CySH oxidation, as peaks I. Additionally, we name the second anodic peaks, which are attributed to the edge-plane-like defective sites, as peaks II and the third anodic peaks as peaks III. The effect of pH on the anodic peak currents and potentials of CySH at the OMC/GC electrode is shown in Figures 4 and 5. In Figure 4, for CySH oxidation with pH changing from 2.00 to 5.00, the currents of peaks I, II, and III all decreased and reached a minimum value at pH ∼5.00. When the solution pH was further increased, the currents of peaks I and II both increased accordingly but peaks III disappeared. Above pH 10.00, the peak currents of peaks I and II both decreased. From the results above, we can observe the effect of pH on the currents of the anodic peaks for CySH oxidation at the OMC/ GC electrode is more different and complicated than that at other electrodes reported before.5-23 This may have something to do with the ionization of CySH depending on pH in aqueous solutions, because CySH contains various functional groups of COOH, SH, and NH2 with pKa of 1.92, 8.37, and 10.70, respectively (Figure
Figure 5. Plot of solution pH against anodic peak potentials of peaks I, II, and III for 5 mmol L-1 CySH at the OMC/GC electrode in 0.1 mol L-1 PBS by cyclic voltammetry. Scan rate, 5 mV s -1.
6A).5,34 Figure 6B showed the distribution fraction (δ) of different chemical species of CySH (CySH2+ (H3A+), CySH (H2A), CyS(HA-), and CyS2- (A2-)) against pH.5,35 As shown, a decreasing tendency of the distribution fraction (δ) of CySH2+ (H3A+) was observed in the pH range of 2.00-5.00; accordingly, a decreasing trend of the anodic currents of peaks I, II, and III in the pH range of 2.00-5.00 was also recorded in Figure 4. With the solution pH increasing, the anodic currents of peaks I and II and the distribution fraction (δ) of CyS- (HA-) increased simultaneously. Above pH 10.00, the anodic currents of peaks I and II and the distribution fraction (δ) of CyS- (HA-) decreased at the same time. Therefore, it can be obviously observed that the changing trend of the distribution fractions (δ) of CySH2+ (H3A+) and CyS(HA-) with pH is well consistent with the changing tendency of the currents of peaks I, II, and III in the corresponding pH values. So we may rationally conclude that in the pH range of 2.00-12.00 only CySH2+ (H3A+) and CyS- (HA-) are electroactive substances for the four chemical species of CySH: in the pH range of 2.005.00 the electroactive substance is only CySH2+ (H3A+), and above pH 5.00 the electroactive substance is only CyS- (HA-). (34) Budavari, S., O’Neil, M. J., Smith, A., Heckelman, P. E., Eds.; The Merck Index, 11th ed.; Merck & Co. Inc.: Rahway, NJ, 1989. (35) Rubinson, K. A. Chemical Analysis; Little, Brown and Co.: Boston, 1987; p 34.
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Figure 6. (A) Various forms of CySH with respect to its pKa value. (B) Distribution fraction (δ) for various CySH species versus its pH.
In 2001, Fujishima et al. only noticed that at pH ∼9 (0.5 M KHCO3 solution), the electroactive substance is CyS- (HA-), but they did not propose whether CyS- (HA-) could be the electroactive substance at any other pH or which species are the electroactive substances at any other pH.6 Almost at the same time, Zen et al. suggested that when pH is
