Copper(II) Nanosensor Based on a Gold Cysteamine Self-Assembled

The sensor is based on gold cysteamine self-assembled monolayer functionalized with salicylaldehyde by means of Schiff's base formation. Cyclic voltam...
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Anal. Chem. 2006, 78, 4957-4963

Copper(II) Nanosensor Based on a Gold Cysteamine Self-Assembled Monolayer Functionalized with Salicylaldehyde Reza Karimi Shervedani* and Seyed Ahmad Mozaffari

Department of Chemistry, University of Isfahan, Isfahan 81746-73441, I.R. Iran

Fabrication and electrochemical characterization of a novel nanosensor for determination of Cu2+ in subnanomolar concentrations is described. The sensor is based on gold cysteamine self-assembled monolayer functionalized with salicylaldehyde by means of Schiff’s base formation. Cyclic voltammetry, Electrochemical impedance spectroscopy (EIS), and electrochemical quartz crystal microbalance were used to probe the fabrication and characterization of the modified electrode. The sensor was used for quantitative determination of Cu2+ by the EIS in the presence of parabenzoquinone in comparison with stripping Osteryoung square wave voltammetry (OSWV). The attractive ability of the sensor to efficiently preconcentrate trace amounts of Cu2+ allowed a simple and reproducible method for copper determination. A wide range linear calibration curve was observed, 5.0 × 10-11-5.0 × 10-6 and 5.0 × 10-10-5.0 × 10-6 M Cu2+, by using the EIS and OSWV, respectively. Moreover, the sensor presented excellent stability with lower than 10% change in the response, as tested for more than three months daily experiments, and a high repeatability with relative standard deviations of 6.1 and 4.6% obtained for a series of eight successive measurements in 5.0 × 10-7 M Cu2+ solution, by the EIS and OSWV, respectively. Monomolecular-level modification of the electrode surface through the self-assembly approach is, of late, gaining importance in view of its many functional applications in different areas of science and technology such as the following: modification of the surface electronic properties, charge-transfer kinetic studies at the controlled distances, and recognition of biological and inorganic species in electroanalysis. Application of chemisorbed organosulfur self-assembled monolayer (SAM) films on gold surface has received much attention in recent years. The benefits of SAM films for sensor applications include chemical specificity, rapid response, high sensitivity, and possibility for in situ immobilization of biological recognition agents (e.g., enzymes) or functionalization of the film terminals by chemical reagents.1-4 * Corresponding author. Tel.: +98-311-7932715. E-mail: [email protected]. (1) Mandler, D.; Turyan, I. Electroanalysis 1996, 8, 207-213. (2) (a) Wink, Th.; van Zuilen, S. J.; Bult, A.; van Bennekom, W. P. Analyst 1997, 122, 43R-50R. (b) Mirsky, V. M. Trends Anal. Chem. 2002, 21, 439-450. (3) Postlethwaite, T. A.; Hutchison, J. E.; Hathcock, K. W.; Murray, R. W. Langmuir 1995, 11, 4109-4116. 10.1021/ac052292y CCC: $33.50 Published on Web 06/17/2006

© 2006 American Chemical Society

Exploiting the molecular-level control over the fabrication of a sensing interface afforded by SAMs for metal ions was first demonstrated by Rubinstein et al., where a modified gold electrode with mixed SAMs, which could detect a low level of Cu2+ (∼10-7 M) with minimal interference from Fe2+, was introduced.5-7 Then, Turyan and Mandler developed the ω-mercaptocarboxylic acid SAMs on mercury and gold for determination of Cd2+, which resulted in a detection limit of 4.0 × 10-12 M Cd2+ with a relatively high selectivity.8 They also reported a highly selective sensor based on gold 4-(alkylthio)pyridine SAM with a detection limit lower than 1 ppt, which was intended to determine Cr6+ in the presence of Cr3+.9 Mercaptopropionic acid10 and 3,3′-thiodipropionic acid11 SAM-modified electrodes were investigated for Cu2+ and Pb2+, and Cu2+ and Ag+ ions, respectively. Liu et al.,12 and Yang et al.13 used the gold cysteine SAM-modified electrode and determined Cu2+ ions in solution with a detection limits of 3.9 × 10-10 and 3.0 × 10-9 M, respectively. The gold 3-mercaptopropionic acid14 and mercaptoacetic acid SAMs15 have been used as highly sensitive voltammetric sensor for Cu2+ with the linear range from 1.0 × 10-12 to 1.0 × 10-9 and 8.0 × 10-7 to 1.0 × 10-5 M Cu2+, respectively. Several reports demonstrate application of other SAM-modified surfaces in chemical analysis. For example, 2-mercaptobenzimidazole for Hg2+,16 calix[4]arene disulfide diquinone for Ba2+,17 functionalized cysteamine monolayers with calix[6]arene18 and disulfide derivative of calix[4]arene19 for uranyl ions, carbam(4) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103-1169. (5) Rubinstein, I.; Steinberg, S.; Tor, Y.; Shanzer, A.; Sagiv, J. Nature 1988, 332, 426-429. (6) Steinberg, S.; Tor, Y.; Sabatani, E.; Rubinstein, I. J. Am. Chem. Soc. 1991, 113, 5176-5182. (7) Steinberg, S.; Rubinstein, I. Langmuir 1992, 8, 1183-1187. (8) Turyan, I.; Mandler, D. Anal. Chem. 1994, 66, 58-63. (9) Turyan, I.; Mandler, D. Anal. Chem. 1997, 69, 894-897. (10) Shen, H.; Mark, J. E.; Seliskar, C. J.; Mark, H. B., Jr.; Heineman, W. R. J. Solid State Electrochem. 1997, 1, 241-247. (11) Nagaoka, T.; Chen, Z. D.; Okuno, H.; Nakayama, M.; Ogura, K. Anal. Sci. 1999, 15, 857-862. (12) Liu, A. C.; Chen, D. C.; Lin, C. C.; Chou, H. H.; Chen, C. H. Anal. Chem. 1999, 71, 1549-1552. (13) Yang, W.; Gooding, J. J.; Hibbert, D. B. J. Electroanal. Chem. 2001, 516, 10-16. (14) Freire, R. S.; Kubota, L. T. Electrochim. Acta 2004, 49, 3795-3800. (15) Xia, J.; Wei, W.; Hu, Y.; Tao, H.; Wu, L. Anal. Sci. 2004, 20, 1037-1041. (16) Berchmans, S.; Arivukkodi, S.; Yegnarman, V. Electrochem. Commun. 2000, 2, 226-229. (17) Chung, T. D.; Park, J.; Kim, J.; Lim, H.; Choi, M.-J.; Kim, J. R.; Chang, S.-K.; Kim, H. Anal. Chem. 2001, 73, 3975-3980.

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oylphosphonic acid for simultaneous detection of Cd2+, Cu2+, and Pb2+,20 urease for Cd2+,21 and bisthiourea receptor for inorganic phosphate22 have been investigated. A great deal has been devoted to the utilization of functional SAMs for preparation of electrochemical biosensors.23-25 Most of the aforementioned studies have been based on amperometric transducers. However, it is reasonable to investigate other transducers such as electrochemical impedance sprectroscopy (EIS). The EIS is a powerful, informative, and nondestructive method, which can be used to study the interfacial events and trace the blocking behaviors (as complexation and precipitation) or diffusion effects at modified electrodes and serve as a transducer.26-29 For example, gold SAM-modified electrodes functionalized with crown ethers for electrochemically inactive ions such as Na+, K+, and Li+ 30 modified electrodes by poly(crown ether ferrocene) for recognition of Ca2+ and Ba2+ 31 and thiacalix[4]arene thin films for Cu2+ 32 have been investigated using the EIS. The common approaches for introducing selective functions into the electrode surface via thiol SAM coatings involve the following: (i) ex situ functionalization of thiols and then adsorption onto the surface. A number of drawbacks are inherent in this method.33 For example, preparation of functionalized thiols requires multistep synthesis and usually includes different reagents, which can contaminate the synthesized ligands and analytical chemists often try to avoid. Furthermore, assembling the macromolecules of these types onto the surface produces nondense SAMs exposing a large number of uncoated sites, which are adverse and can be occupied by underpotential deposition of metal ions or fouled by biological species. These effects can reduce the selectivity and sensitivity of the sensor. (ii) In situ functionalization by using a simple SAM (e.g., cysteamine) as a platform to immobilize biological species for biosensor applications or to anchor desired chemical functions to the surface. Although some interesting advantages have been cited for this procedure,4 still some problems as contamination of the modified electrode by intermediate reagents may persist. This can be overcome by functionalization of the SAM-modified electrode through Schiff’s base formation as a reagentless method. (18) Lee, M.; Hur, Y.; Kim, J.; Choi, H.; Koh, K. Mol. Cryst. Liq. Cryst. 2002, 377, 237-240. (19) Evans, C. J.; Nicholson, G. P. Sens. Actuators, B 2005, 105, 204-207. (20) Yantasee, W.; Lin, Y.; Fryxell, G. E.; Busche, B. J. Anal. Chim. Acta 2004, 502, 207-212. (21) May, L. M.; Russell, D. A. Anal. Chim. Acta 2003, 500, 119-125. (22) Aoki, H.; Hasegawa, K.; Tohda, K.; Umezawa, Y. Biosens. Bioelectron. 2003, 18, 261-267. (23) Willner, I.; Riklin, A. Anal. Chem. 1994, 66, 1535-1539. (24) Creager, S. E.; Olsen, K. G. Anal. Chim. Acta 1995, 307, 277-289. (25) Jung, S. K.; Wilson, G. S. Anal. Chem. 1996, 68, 591-596. (26) (a) Macdonalds, J. R. Impedance Spectroscopy, 1st ed.; Wiley: New York, 1987. (b) Janek, R. P.; Fawcett, W. R.; Ulman, A. J. Phys. Chem. B 1997, 101, 8550-8558. (27) Flink, S.; Boukamp, B. A.; van den Berg, A.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Am. Chem. Soc. 1998, 120, 4652-4657. (28) Alfonta, L.; Katz, E.; Willner, I. Anal. Chem. 2000, 72, 927-935. (29) Shervedani, R. K.; Mozaffari, S. A. Surf. Coat. Technol. 2005, 198, 123128. (30) Flink, S.; Schonherr, H.; Vancso, G. J.; Geurts, F. A. J.; van Leerdan, K. G. C.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Chem. Soc., Perkin Trans. 2 2000, 2141-2146. (31) Ion, A. C.; Moutet, J.-C.; Pailleret, A.; Popescu, A.; Saint-Aman, E.; Siebert, E.; Ungureanu, E. M. J. Electroanal. Chem. 1999, 464, 24-30. (32) Ben Ali, M.; Bureau, C.; Martelet, C.; Jafferzic-Renault, N.; Lamartine, R.; Ben Ouada, H. Mater. Sci. Eng. C7 2000, 83-89. (33) Markovich, I.; Mandler, D. Analyst 2001, 126, 1850-1856.

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The Schiff’s bases of salicylaldehyde (SAL) and its derivatives have been often used as complexing agents,34-36 especially for copper.37 The copper SAL complexes have been used for fabrication of bulky modified selective electrodes,35 modification of the gold SAM electrodes,36 and catalysts of quinones/hydroquinones redox reaction.38 Despite these interesting behaviors, no quantitative data have been reported on preparation, electrochemical characterization, and application of the gold-cysteamine-salicylaldehyde self-assembled monolayer (Au-CA-SAL SAM) toward Cu2+. In this paper, in situ functionalization of gold-cysteamine (AuCA) SAM by simple Schiff’s base formation between aldehyde group of SAL and amine group of Au-CA is reported. The response of the prepared sensor (Au-CA-SAL SAM) is tested for common metallic ions and finally demonstrated for preconcentration and determination of Cu2+. Stepwise assembly and functionalization of the layered electrode are probed by cyclic voltammetry (CV), the EIS, and electrochemical quartz crystal microbalance (EQCM). Quantitative response of the sensor toward Cu2+ was studied by stripping Osteryoung square wave voltammetry (OSWV) and compared with that obtained by the EIS transducer based on electrocatalytic effect of Cu2+ for redox reaction of p-benzoquinone (PBQ). The data are presented and discussed, from which a quantitative analytical method developed for Cu2+ is presented. The Au-CA-SAL SAM-modified electrode was electrochemically stable during three months of daily checking experiments. The major advantages of the present work are (i) development of a faradaic impedimetric sensor for copper determination based on its electrocatalytic effect for PBQ redox reaction, (ii) expansion the linear range of the calibration curve with a relatively lower detection limit, and (iii) simplicity of the sensor fabrication. EXPERIMENTAL SECTION Reagents. Solutions were prepared with purified water (18 MΩ cm, Millipore-MilliQ, Millipore Inc.). The glassware were soaked in 6 M HNO3 and carefully cleaned before use to avoid contamination. All chemicals were of analytical grade and used as received, except PBQ, which was further purified by recrystallization from its hot n-hexane solution. Phosphate buffer solution (PBS) contained 0.05 M K2HPO4/KH2PO4 in 0.05 M NaClO4, and the pH was adjusted with NaOH or HNO3 solutions. The PBS was electrochemically polished before use. Dilute solutions of Cu2+ were prepared immediately before use from a stock solution (1.0 × 10-2 M). Methods. Before chemical modification, the electrode surface was cleaned first physically by polishing with alumina slurries (Buehler, starting from 0.3 µm down to 0.05 µm), rinsing with ethanol and deionized water, and sonicating in water/chloroform/ water, respectively, for 2 min, and then electrochemically by (34) Paschke, R.; Liebsch, S.; Tschierske, C.; Oakley, M. A.; Sinn, E. Inorg. Chem. 2003, 42, 8230-8240. (35) Alizadeh, N.; Ershad, S.; Naeimi, H.; Sharghi, H.; Shamsipur, M. Fresenius J. Anal. Chem. 1999, 365, 511-515. (36) Tsutsumi, H.; Hatagishi, T. Electrochim. Acta 1994, 39, 553-555. (37) Nagel, J.; Oertel, U.; Friedel, P.; Komber, H.; Mobius, D. Langmuir 1997, 13, 4693-4698. (38) (a) Mandal, S.; Kazmi, N. H.; Sayre, L. M. Arch. Biochem. Biophys. 2005, 435, 21-31. (b) Tuken, T.; Arslan, G.; Yazici, B.; Erbil, M. Prog. Org. Coat. 2004, 49, 153-159.

Scheme 1. Proposed Mechanism for Self-Assembly Process and Copper Interaction with the Modified Electrode

cycling the electrode potential between 0.0 to +1.5 V versus Ag/ AgCl in 0.5 M H2SO4 until a reproducible voltammogram was obtained. A roughness factor of 1.8 ( 0.2 was calculated from the ratio of the real to geometric surface area. The real surface area was calculated by integration of a cathodic peak during the redox of gold assuming 482 µC cm-2 charge for reduction of one monolayer of AuO on Au(111).39 Modification of gold electrodes was performed in a two-step method: (i) immediately after cleaning, the bare gold was placed into 18 mM CA aqueous solution at room temperature in darkness for 4 h to form Au-CA-modified electrode, removed from the CA solution, rinsed copiously with water to eliminate physically adsorbed CA, and blown dry with nitrogen gas stream, (ii) the Au-CA-modified electrode was dipped into a saturated salicylaldehyde ethanolic solution in 40 °C for 1 h to form Au-CASAL SAM by means of Schiff’s base formation (Scheme 1). Preconcentration of Cu2+ was carried out by dipping the AuCA-SAL SAM-modified electrode into 5 mL of stirred solution containing supporting electrolyte, with the desired concentration of Cu2+ with a known pH, under open-circuit potential for a set time to form Au-CA-SAL-Cu2+. The electrode was then removed and rinsed with a copper-free PBS and used immediately for electrochemical measurements. Electrochemical Measurements. The CV, OSWV, EIS, and EQCM measurements were performed using an Autolab 30 potentiostat/galvanostat equipped with a frequency response analyzer, interfaced with a PCIII 800 MHz, and controlled by GPES and FRA 4.9 softwares (Eco Chemie, Utrecht, The Netherlands). The electrochemical measurements were performed in a conventional three-electrode glass cell including a gold disk (0.0962 cm2, Azar Electrode Co., Urmia, Iran) as working electrode, a large surface area Pt plate (99.99%, 5 cm2) as auxiliary electrode, and a Ag/AgCl electrode as reference. All reported potentials are referenced to the Ag/AgCl electrode. The cell was placed in a grounded Faraday cage to eliminate environmental stray effects. (39) (a) Hoare, J. P. J. Electrochem. Soc. 1984, 131, 1808-1815. (b) Oesch, U.; Janata, J. Electrochim. Acta 1983, 28, 1237-1246.

Table 1. Graphite Furnace Atomizer Program step

temp/οC

ramp time/s

hold time/s

argon flow rate/mL min-1

drying pyrolysis atomization cleaning

130 1200 2000 2540

1 10 0 1

30 20 5 3

250 250 0 250

The OSWV experiments were conducted in a 10-mL copperfree pre-electrolyzed PBS (pH 5.5) under optimized conditions. Electrochemical blank tests were always carried out by immersing the modified electrode in the preconcentration solution before adding Cu2+ ions. The regeneration procedure involving Cu2+ elimination from the electrode surface was performed by holding the electrode potential at +0.5 V in a stirred 0.1 M EDTA solution (pH 3.0) for ∼100 s. The EIS measurements were performed in the presence of 5 mM PBQ as a redox probe. A 5-mV ac amplitude potential superimposed on the formal potential of the redox probe (E0′ ) -0.003 V) was applied and a wide range of frequencies from 10 kHz to 10 mHz was scanned. The EIS data analysis was performed using ZView version 2.3f software and CNLS approximation method. A CPE model was enough to explain the whole frequency range of data from which kinetic and analytical information was extracted.40 An EQCM analyzer (PM-700 Plating Monitor, Maxtek Inc.) linked to a personal computer through MPS-550 probe equipped with a quartz crystal (AT-cut, 5 MHz) sandwiched between two polished Au electrodes (1.370 cm2, model SC-501-1) was employed for microgravimetric monitoring of stepwise self-assembly and layer addition. The EQCM measurements were performed under open-circuit potential. Graphite Furnace Atomic Absorption Spectrometry. A Perkin-Elmer Zeeman atomic absorption spectrometer equipped with a heated graphite atomizer (HGA-600) furnace together with an autosampler (AS-800) was used for validation of the method. Instrumental parameters were adjusted according to the manufacturer’s recommendations. Typical operating conditions are presented in Table 1. RESULTS AND DISCUSSION Characterization. Cyclic Voltammetry. Figure 1 shows the voltammograms obtained in copper-free PBS (pH 7.0) on Au (curve a), Au-CA (curve b), Au-CA-SAL (curve c), and AuCA-SAL-Cu2+ SAM (curve d) in the absence of PBQ. The voltammograms (a-c) suggest that the layers are electrochemically inactive in the range of +0.6 to -0.6 V. The small current peak found around +0.2 V is attributed to the redox reaction of adsorbed Cu2+ on the topside of self-assembled bilayers (curve d). Figure 2A shows the cyclic voltammograms of the layers mentioned in Figure 1 in the presence of PBQ. The reversible electrochemical behavior and large current density of the redox waves around -100 mV indicate that PBQ can easily access the (40) (a) Jurczakowski, R.; Hitz, C.; Lasia, A. J. Electroanal. Chem. 2004, 572, 355-366. (b) Jurczakowski, R.; Hitz, C.; Lasia, A. J. Electroanal. Chem. 2005, 582, 85-96. (c) Lasia, A.; Rami, A. J. Electroanal. Chem. 1990, 294, 123141. (d) Boukamp, B. A. Solid State Ionics 1986, 18, 19, 136-140. (e) Boukamp, B. A. Solid State Ionics 1986, 20, 31-44.

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Figure 1. Voltammograms obtained in copper-free PBS (pH 7.0) and the absence of PBQ on Au (curve a), Au-CA (curve b), AuCA-SAL (curve c), and Au-CA-SAL modified electrode after 5-min preconcentration in 5.0 × 10-5 M Cu2+, Au-CA-SAL-Cu2+ (curve d) at 18 °C and in argon atmosphere; scan rate, 100 mV s-1.

surface of the bare Au electrode (curve a). While, addition of cysteamine monolayer onto the electrode does not show a considerable effect in the peak characteristics (curve b), the heterogeneous charge transfer between the gold electrode and PBQ was strictly influenced by the presence of SAL on Au-CA SAM (curve c), and indicates that PBQ can hardly access the electrode surface. These results are consistent with the fact that further layer-by-layer assembly on the electrode retards the interfacial charge-transfer kinetics of PBQ. However, a 5-min preconcentration of the Au-CA-SAL electrode in 5.0 × 10-7 M Cu2+ (curve d) improves the reduction process (the cathodic peak is shifted to positive direction) and shows the electrocatalytic activity of Au-CA-SAL-Cu2+-modified electrode for redox reaction of PBQ. Electrochemical Impedance Spectroscopy. The EIS measurements were used to trace the events during the formation of the Au-CA-SAL electrode. Figure 2B shows the complex plane (Nyquist) plots (Zim vs Zre) of the layers mentioned in Figure 2A in the presence of PBQ. The EIS data obtained on the modified electrode in different steps are well described by a constant phase element (CPE) model (Figure 3).40 The bare Au electrode exhibits an almost straight line (curve a and inset) that is characteristic of a mass diffusional limiting charge-transfer process. Assembly of the CA monolayer on the Au electrode generates a layer that introduces a small barrier to the interfacial charge transfer. This is reflected by the appearance of the semicircle part on the spectrum (curve b), corresponding to a charge-transfer resistance (Rct) of 5268 Ω. The formation of a Schiff base between the CA monolayer and SAL results in an increase in the charge-transfer resistance, Rct ) 20 380 Ω (curve c). A 5-min preconcentration of the Au-CA-SAL electrode in 5.0 × 10-7 M Cu2+ (curve d) shows an attenuation in the redox probe charge-transfer resistance, Rct ) 12 190 Ω. This effect is used in this work to develop a faradaic impedimetric method for determination of Cu2+. Curve d implies that complexation of Cu2+ ions by the modified electrode increases the kinetics of the redox probe and is consistent with findings of Figure 2A. 4960 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

Figure 2. (A). Voltammograms obtained in copper-free PBS (pH 7.0) and the presence of 5 mM PBQ on Au (curve a), Au-CA (curve b), Au-CA-SAL (curve c), and Au-CA-SAL modified electrode after 5-min preconcentration in 5.0 × 10-7 M Cu2+, Au-CA-SAL-Cu2+ (curve d) at 18 °C and in argon atmosphere; scan rate, 100 mV s-1. (B) Complex plane plots (Zim vs Zre) obtained for the faradaic impedance measurements in the same conditions as (A). The inset shows curves a and b with a higher resolution scale. Symbols indicate experimental and solid lines approximated data.

Figure 3. Equivalent circuit (CPE model) used for impedance data approximation: Rs, Rct, CPE, and Zw represent the solution resistance, charge-transfer resistance, constant-phase element, and Warburg impedance, respectively.

Electrochemical Quartz Crystal Microbalance. The EQCM as a powerful tool of response in situ to the electrode mass change was also used to assay the formation (Figure 4A) and functionalization (Figure 4B) of the cysteamine SAM.41 In both cases, the frequency, f, decreased quickly for the first ∼30 min of immersion (41) (a) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1355-1379. (b) Schneider, T. W.; Buttry, D. A. J. Am. Chem. Soc. 1993, 115, 12391-12397. (c) Jerkiewicz, G.; Vatankhah, G.; Zolfaghari, A.; Lessard, J. Electrochem. Commun. 1999, 1, 419-424. (42) Manzoori, J. L.; Bavili-Tabrizi, A. Microchem. J. 2002, 72, 1-7.

Figure 5. Response of Au-CA-SAL SAM-modified electrode as a function of pH (a) preconcentration and (b) measurement solutions. Preconcentration conditions: 1.0 × 10-6 M Cu2+, 0.1 M KNO3 (pH 7.0), time (5 min) at the open-circuit potential. Measurement conditions, 0.1 M copper-free PBS (pH 5.5); scan rate, 100 mV s-1.

Figure 4. Time-dependent frequency changes of an EQCM for formation of (A) Au-CA and (B) Au-CA-SAL SAM. The insets show higher resolution scale of the first 2400 s.

and then became almost constant after 40 min. This behavior implies that the formation of Au-CA SAM and its functionalization by SAL can be finished in about 30 and 40 min, respectively. The frequency shift, ∆f, measured for the formation of Au-CA and Au-CA-SAL was about 4.66 and 5.22 Hz. These values were further translated and 1.46 × 10-9 and 1.20 × 10-9 mol cm-2 were found for the surface coverages of CA and SAL, using the Sauerbrey equation, ∆f ) -2f02[∆m/A(µqFq)1/2], in which, f0 is the frequency of the quartz crystal prior to a mass change (Hz), ∆f is the measured frequency shift, ∆m is the mass change (gram), A is the piezoelectrically active area (cm-2), Fq is the density of quartz (2.648 g cm-3), and µq is the shear modulus (2.947 × 1011 dyn cm-2 for AT-cut quartz). Optimization of the Conditions and Analytical Application. Time. The dependence of the cathodic stripping peak current on the preconcentration time at the modified gold electrodes was studied. The modified electrodes were immersed in a 5.0 × 10-6 M Cu2+ stirred solution for different periods of time. The peak current was increased as a function of preconcentration time, and the asymptotic value was reached within 5 min (data not shown). pH. To achieve high sensitivity and selectivity, the pH effect was investigated and the best pH conditions for preconcentration and determination steps were achieved around pH 7.0 and 5.5, respectively (Figure 5). Such a profile indicates that, for pH values

Figure 6. Response of Au-CA-SAL SAM-modified electrode for different types of electrolyte solutions. Preconcentration conditions: 1.0 × 10-6 M Cu2+, 0.1 M electrolyte (pH 7.0), time (5 min) at the open-circuit potential. Measurement conditions, 0.1 M copper-free electrolyte (pH 5.5); scan rate, 100 mV s-1.

lower and higher than 7.0 in the preconcentration step, the accumulated Cu2+ on the functionalized monolayer decreases, due to H+ competition for functional groups of the surface, and OHfor Cu2+ in solution, respectively (Figure 5, curve a). In the stripping step, for pH values higher than 5.5, the interaction between Cu2+ and the functionalized monolayer becomes stronger, hindering the charge-transfer kinetics between Cu2+ and the metallic electrode base, and at lower pH, some part of the Cu2+ ions will leave the surface before starting the stripping step (Figure 5, curve b). Type of Electrolyte. To investigate the influence of the solution matrix on the extraction and determination steps of Cu2+ ions, the PBS, NaOAc, KHP, NaClO4, KCl, and KNO3 electrolytes were studied. Figure 6 shows the relative response of the AuCA-SAL SAM-modified electrode in different electrolyte solutions. A maximum response was observed when 0.1 M KNO3 and 0.1 M PBS were used as electrolytes in preconcentration and determination steps, respectively. Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

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Figure 7. OSWVs obtained on Au-CA-SAL SAM-modified electrode as a function of Cu2+ ion concentrations: (1) 0, (2) 5.0 × 10-10, (3) 5.0 × 10-9, (4) 5.0 × 10-8, (5) 5.0 × 10-7, and (6) 5.0 × 10-6 M in 0.1 M KNO3 solution, pH 7.0, and 5-min preconcentration time. The inset shows calibration curve. Stripping conditions: 0.1 M PBS pH 5.5; frequency, pulse height, and pulse increment are 50 Hz, 100 mV, and 6 mV, respectively.

Instrument Parameters. The OSWV parameters are interrelated. After careful examination, we find that the optimized instrument settings of frequency, pulse height, and pulse increment are 50 Hz, 100 mV, and 6 mV, respectively. Under optimum conditions, the square wave voltammogram shows a cathodic wave around 0.1 V for preadsorbed Cu2+ on Au-CA-SAL SAM electrode. Calibration and Detection Limit. The calibration curve (Figure 7) was obtained under optimized conditions by systematically increasing the concentration of Cu2+ from 5.0 × 10-10 to 5.0 × 10-6 M in preconcentration solution and monitoring the response of Au-CA-SAL-Cu2+ electrode in determination solution by OSWV [-log(i/µA) ) 3.484((0.062) + 0.244((0.083) pCu, r2 ) 0.997]. The detection limit of this electrode, calculated from the standard deviation of the background (signal equals 3δ of the background noise), is 7.5 × 10-11 M Cu2+, with a relative standard deviation of 4.6% (n ) 8) for 5.0 × 10-7 M Cu2+. The EIS measurements were also performed to determine the Cu2+ ion concentration using the electrocatalytic effect of copper on the redox reaction of PBQ (Figure 2B). The preconcentration and determination conditions for EIS were the same as those optimized for OSWV measurements. The complex plane plots were recorded on Au-CA-SAL-Cu2+ as a function of Cu2+ ion concentration at -0.003 V in a copper-free PBQ solution (Figure 8). The data were approximated by using the CPE model and parameters such as Rct were evaluated. The magnitude of Rct decreases as a function of copper concentration, which indicates the electrocatalytic effect of complexed Cu2+ for the PBQ redox reaction. The calibration curve (from 5.0 × 10-11 to 5.0 × 10-6 M) was constructed from variation of differential charge-transfer resistance, ∆Rct versus Cu2+ ion concentration (Figure 8 inset). A wider dynamic range and a lower detection limit were obtained [∆Rct(kΩ) ) 17.667((0.585) - 1.512((0.128) pCu, r2 ) 0.991] relative to those obtained by OSWV. The remarkable detection limit of this electrode was found as 8.3 × 10-12 M Cu2+ with a relative standard deviation of 6.1% (n ) 8) for 5.0 × 10-7 M Cu2+. 4962 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

Figure 8. Complex plane plots (Zim vs Zre) obtained on Au-CASAL SAM-modified electrode after 5-min preconcentration in 0.1 M KNO3 (pH 7.0) containing (a) 0, (b) 5.0 × 10-11, (c) 5.0 × 10-10, (d) 5.0 × 10-9, (e) 5.0 × 10-8, (f) 5.0 × 10-7, and (g) 5.0 × 10-6 M Cu2+. The inset shows variation of the differential charge-transfer resistance (∆Rct) as a function of Cu2+ concentration. Symbols indicate experimental and solid lines approximated data. Measurement solution: 5 mM PBQ in 0.1 M copper-free PBS (pH 5.5). Table 2. Mean Values (µg L-1) and Standard Deviation (n ) 3) for Determination of Cu2+

sample synthetic soln 0.10 ppb synthetic soln 30.00 ppb blood serumb,d blood serumc,d

SWV on Au-CA-SAL electrode

EIS on Au-CA-SAL electrode

0.09 ( 0.01

0.08 ( 0.01

31.40 ( 1.51

26.62 ( 2.75

29.20 ( 1.50

190.60 ( 12.60 473.05 ( 15.20

11.75 ( 1.50 426.50 ( 32.00

611.25 ( 26.00 540.50 ( 18.00

GFAAS nda

a nd, not detected. b Without pretreatment. c Pretreated as described in ref 42. d Standard addition method.

These results show that the Au-CA-SAL SAM-modified electrode is an appropriate impedimetric sensor for detection of Cu2+ at low levels. The stability of the Au-CA-SAL sensor was tested by recording its electroanalytical response for a relatively long period of time. The sensor was stable for more than three months of daily testing with relative standard deviation less than 10%. To verify our results, several samples were analyzed simultaneously by graphite furnace atomic absorption spectroscopy (GFAAS) as well as by the sensor fabricated in this work. The results obtained by OSWV and EIS for recovery tests and real sample analysis of Cu2+ in comparison with those obtained by GFAAS are shown in Table 2. These results clearly imply the potential application of this electrode for the analysis of low levels of Cu2+. However, the matrix of the real samples, like blood serum, is complicated and the most of the differences observed between the results obtained by GFAAS and those obtained by OSWV and EIS for nondigested blood serum are probably due to the fact that Cu2+ ions are tightly bound to the celators such as proteinaceous matter in the blood serum sample, which prevents effective accumulation of copper on the Au-CA-SAL SAM electrode. Still, surface fouling effects from the matrix of nondigested sample cannot be completely negligible.

The effect of some cations on the response of Au-CA-SAL SAM to the Cu2+ ions was investigated. The interfering effect is defined as the concentration of interfering species that can change the electrode response toward the analyte by more than 3δ, where δ is standard deviation of the replicate analyte measured signal. The results showed that the peak current of 5.0 × 10-8 M Cu2+ was not affected by 60-fold Zn2+, Co2+, and Fe3+, 40-fold Pb2+, Cd2+, and Ni2+, 20-fold Ag+, and 10-fold Fe2+ for OSWV; and also 60fold Zn2+, Ni2+, Co2+, and Cd2+, 40-fold Pb2+ and Fe3+, 20-fold Ag+, and 10-fold Fe2+ for the EIS measurements. However, when 0.01 M S2O32- was added to the preconcentration solution, the interference of 200-fold excess of Ag+ was eliminated. In conclusion, a novel sensor was developed based on goldcysteamine self-assembled monolayers functionalized with salicylaldehyde for determination of Cu2+. The ability of the sensor

for analysis of Cu2+ was demonstrated by voltammetric and impedimetric methods. Better detection limit (8.3 × 10-12 M Cu2+) and wider linear range (5.0 × 10-11-5.0 × 10-6 M Cu2+) was obtained by the EIS based on the electrocatalytic effect of Cu2+ for the PBQ redox reaction. In addition, the sensor enjoys some other advantages as simple fabrication, easy regeneration, and high stability for long working time. ACKNOWLEDGMENT The authors acknowledge the University of Isfahan providing research facilities. Received for review December 29, 2005. Accepted May 14, 2006. AC052292Y

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