Application of Cysteine Monolayers for Electrochemical Determination

Anal. Chem. 1999, 71, 1549-1552

Application of Cysteine Monolayers for Electrochemical Determination of Sub-ppb Copper(II) An-Chiang Liu, Der-chang Chen, Chun-Cheng Lin, Hsien-Hsin Chou, and Chun-hsien Chen*

Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan 80424, R.O.C.

Electrodes with the functionality of amino acids can be easily prepared by taking advantage of strong sulfur-gold interactions and immersing gold electrodes into cysteine solutions. Such electrodes exhibit excellent sensitivity and selectivity toward copper(II) determination, because the bidentate chelation of amino acids toward copper(II) is effective and far superior than that toward any other metal ions. In this study, we demonstrate that at a preconcentration time of 5 min the cysteine-modified electrode has a dynamic range of 2 orders of magnitude (from 5.0 to 0.050 µg/L) and the detection limit is as low as 25 ng/L (0.39 nM). This method is applied to the determination of copper(II) in two reference materials, SLEW-2 (river water, 1.62 µg/L) and CASS-3 (seawater, 0.517 µg/L). The results agree satisfactorily with the certified values. Development of chemosensors for the detection and measurement of copper(II) is an active research field due to the environmental and biological importance of this ion.1,2 To improve the sensitivity and selectivity of these sensors, most reported methods utilize multidentate ligands to form complexes with copper(II). Concentrations of copper(II) are then determined by either the attenuation of fluorescence3-8 of the chromophore attached to the ligands or by the electrochemical response9-15 of * Corresponding author: (e-mail) [email protected]; (fax) +886-75253909. (1) Bond, R. G., Straub, C. P., Eds. CRC Handbook of Environmental Control; CRC Press: Cleveland, OH, 1973; Vol. 3. (2) Wang, J. Stripping Analysis: Principles, Instrumentation, and Applications; VCH: Deerfield Beach, FL, 1985. (3) Torrado, A.; Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1998, 120, 609610. (4) Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119, 73867387. (5) Corradini, R.; Dossena, A.; Galaverna, G.; Marchelli, R.; Panagia, A.; Sartor, G. J. Org. Chem. 1997, 62, 6283-6289. (6) De Santis, G.; Fabbrizzi, L.; Licchelli, M.; Mangano, C.; Sacchi, D.; Sardone, N. Inorg. Chim. Acta 1997, 257, 69-76. (7) Thompson, R. B.; Ge, Z. F.; Patchan, M.; Huang, C.-C.; Fierke, C. A. Biosens. Bioelectron. 1996, 11, 557-564. (8) Sasaki, D. Y.; Shnek, D. R.; Pack, D. W.; Arnold, F. H. Angew. Chem., Int. Ed. Engl. 1995, 34, 905-907. (9) Zen, J.-M.; Lee, M.-L.; Huang, S.-Y.; Hsu, F.-S.; Chi, N.-Y.; Chung, M.-J. J. Chin. Chem. Soc. 1998, 45, 39-46. (10) Riso, R. D.; Le Corre, P.; Chaumery, C. J. Anal. Chim. Acta 1997, 351, 83-89. (11) Stora, T.; Hovius, R.; Dienes, Z.; Pachoud, M.; Vogel, H. Langmuir 1997, 13, 5211-5214. (12) Wang, J.; Sucman, E.; Tian, B. Anal. Chim. Acta 1994, 286, 189-195. 10.1021/ac980956g CCC: $18.00 Published on Web 03/04/1999

© 1999 American Chemical Society

copper(II). Monitoring of transition metal ions in complicated matrixes by fluorescent chemosensors is still problematic due to unsatisfactory differentiation between the complexation affinity of the ligand for the target ion and the complexation affinity of the ligand for interference.16 Electrochemical methods, in general, are more sensitive and selective than fluorescence methods. Conventional stripping analysis has the advantages of low detection limit, ease in operation, low cost, and multielement and speciation capabilities, but it suffers interference from relatively complex matrixes containing substances such as surfactants.2 Turyan and Mandler17,18 demonstrated that problems of surfactant adsorption can be circumvented by using electrodes modified by selfassembled monolayers (SAMs) of thiols or sulfur-containing molecules chemisorbed onto gold surfaces.19,20 For the determination of ultralow levels of analytes, electrodes modified by monolayers are expected to provide the best sensitivity in a relatively short time frame, and SAMs appear to be the most straightforward method to prepare monolayer-modified electrodes.21,22 Because sulfur-gold interactions are strong, the monolayers can be assembled in a short time and are reasonably stable in aqueous solutions. Unique and desired properties of an electrode surface can thus be fabricated by chemisorption of appropriately functionalized thiols, conceptually resonating with the well-practiced chemically modified electrodes.23,24 However, there are only a few reports of SAMs applied in quantitative analysis of real samples17,18 because the termini of SAM molecules are often composed of only one type of functional group and the chelation specificity is insufficient. A way to improve the selectivity is to use multidentate13,15,25 or multifunctional11 terminal groups (13) Steinberg, S.; Tor, Y.; Sabatani, E.; Rubinstein, I. J. Am. Chem. Soc. 1991, 113, 5176-5182. (14) Cha, S. K.; Abruna, H. D. Anal. Chem. 1990, 62, 274-278. (15) Rubinstein, I.; Steinberg, S.; Tor, Y.; Shanzer, A.; Sagiv, J. Nature 1988, 332, 426-429. (16) Yoon, J.; Ohler, N. E.; Vance, D. H.; Aumiller, W. D.; Czarnik, A. W. Tetrahedron Lett. 1997, 38, 3845-3848. (17) Turyan, I.; Mandler, D. Anal. Chem. 1994, 66, 58-63. (18) Turyan, I.; Mandler, D. Anal. Chem. 1997, 69, 894-897. (19) Ulman, A. An Introduction to Ultrathin Organic Films: From LangmuirBlodgett to Self-Assembly; Academic Press: Boston, 1991. (20) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (21) Mandler, D.; Turyan, I. Electroanalysis 1996, 8, 207-213. (22) Zhong, C.-J.; Porter, M. D. Anal. Chem. 1995, 67, 709A-715A. (23) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 191-368. (24) Molecular Design of Electrode Surfaces; Murray, R. W., Ed.; Wiley: New York, 1992. (25) Steinberg, S.; Rubinstein, I. Langmuir 1992, 8, 1183-1187.

Analytical Chemistry, Vol. 71, No. 8, April 15, 1999 1549

so that the chelation discrimination between analytes and the interfering substances is enhanced. Such attempts involve intensive synthetic efforts and cannot as yet be successfully applied to real sample determination. We introduce here a cysteine-modified electrode as a highly sensitive and selective copper(II) sensor, in which a stable fivemembered ring is formed by the copper(II), the R-amino, and the R-carboxy groups.26 It has been documented that the complexation constant of the amino acid moiety (R-alanine) with copper(II) is 4 orders of magnitude larger than with any other metal ions,27 indicating that surfaces containing such functionality will be unique for copper(II) determination. We employ cysteine (HSCH2CH(NH2)COOH) to modify gold surfaces because XPS28 and FTIR29 spectroscopic studies have shown that cysteine binds onto the gold surface through the sulfur atom and the surface is thus functionalized with amino acids.30 By utilizing such electrodes, results validated with standard reference materials of seawater and river water containing copper(II) at sub-ppb levels are presented.

amplitude, step potential, frequency, and potential range were 60 mV, 5 mV, 60 Hz, and 0.4 to -0.15 V vs EAgAg/Cl, respectively. Reagents and Apparatus. Solutions were prepared with purified water (18 MΩ‚cm, Millipore-Q, Millipore Inc., MD). All glassware were soaked in 6 N HNO3 and carefully cleaned before use to avoid contamination. All chemicals were ACS-certified reagent grade from Aldrich, Sigma, or Merck. Two reference materials, SLEW-2 (river water) and CASS-3 (seawater), were obtained from the National Research Council Canada. Real samples (local tap water) were sonicated (Branson, Danbury, CT) and filtered through a syringe filter with pore size of 0.45 µm (Lida Manufacturing Co., Kenosha, WI). In the case of determination of copper(II) concentration in humic acid matrix, a UV digester (Metrohm, model 705, Herisau, Switzerland) was employed to decompose the multifunctional composition of humic acid. Electrochemical measurements were carried out on a PAR VersaStat potentiostat (EG&G Instruments Corp., NJ). The threeelectrode system consisted of a gold-film working electrode, an Ag/AgCl reference electrode, and a Pt-foil counter electrode.

EXPERIMENTAL SECTION Methods. Working electrodes were prepared by thermally evaporating (1 × 10-8 Torr, KV-301, KEY High Vacuum Co., Nesconset, NY) ∼150-nm-thick Au films onto piranha-cleaned glass slides. (Piranha solution is a 1:4 (v/v) mixture of 30% H2O2 and concentrated H2SO4. Warning: This solution reacts violently with organic materials and should be handled with great care.) A chromium underlayer (5 nm thick) was precoated to improve the adhesion of the gold film. The gold-coated slides were cut into 2.54 × 0.8 cm pieces to which electric contacts were cemented with conductive silver epoxy (Epoxy Technology, Billerica, MA). After the silver epoxy was oven-dried, the contacts were covered and the area of the working electrode was defined with a layer of nonconductive epoxy (Epoxi-Patch, Dexter, Seabrook, NH). Modification of the gold electrodes was performed by immersion of the electrodes into 10-mL solutions of 8 mM L-cysteine in 0.2 M phosphate (pH 5) for more than 30 min. Upon removal from the cysteine solution, the electrodes were then copiously rinsed with water and blown dry with nitrogen. Preconcentration of copper(II) was accomplished by immersing a modified electrode into 10 mL of a stirred copper(II)-containing phosphate solution (pH ) 5) for 5 min without potential control. The electrode was then removed and rinsed with a copper-free phosphate solution. Osteryoung square wave voltammetry experiments (OSWV) were conducted in a 10-mL copper-free phosphate solution (pH 5). Deaeration was not necessary because dissolved oxygen did not interfere with the voltammetry. Regeneration of a copper-free electrode was achieved by holding the working electrode at 0.5 V vs EAg/AgCl for 20 s in 0.1 M HClO4. If regeneration of the cysteine monolayer became necessary, soaking the electrode for 5 min in the cysteine solution was satisfactory. For the OSWV employed in this study, the optimized parameters of pulse

RESULTS AND DISCUSSION Optimization. Optimization of the sensor’s response relies on four system variables. These variables include surface concentration of the monolayer, pH of the preconcentration solutions, preconcentration time in the copper(II) solution, and parameters of Osteryoung square wave voltammetry. The surface concentration of cysteine affects the electrode performance in terms of sensitivity and reproducibility, both of which can be optimized at the saturated coverage of cysteine. The surface coverage of cysteine can be estimated from the method developed by Porter et al.31 The cyclic voltammogram of the modified electrode in a deaerated KOH solution (0.5 M) from 0.0 to -1.4 V (vs EAg/AgCl) exhibits two reduction peaks32 corresponding to the desorption of thiolates from a gold surface. This desorption is assumed to be a one-electron reduction process and is directly related to the surface coverage. Figure 1A is the adsorption isotherm plotted as the desorption charge ratio (Q(t)/ Qsaturation) against immersion time of the electrode in a 10-mL solution of 0.2 M phosphate (pH 5) containing 8 mM L-cysteine. Saturation of cysteine is reached at ∼10 min. To ensure saturation, an immersion time of 30 min is chosen for a fresh gold electrode. If regeneration becomes necessary, 5 min of immersion time appears sufficient to restore the current response to a satisfactory level. The pH of copper(II) solutions affects protonation of the amino acid moiety, its ligation strength toward copper(II), and consequently the sensitivity of the electrodes. The dependence of pH effects on the OSWV response is shown in Figure 1B. The best performance appears at pH 5, the isoelectric point of L-cysteine,33 indicating that the zwitterionic (HSCH2CH(NH3+)CO2-) form is predominant. This result is verified by spectroscopic determinations of cysteine monolayer orientation. Ihs et al. studied the structure of cysteine adsorption on gold from solutions at pH 11.7, 5.7, and 1.5, by utilizing XPS28 and infrared reflection-absorption

(26) Gilon, C.; Grushka, E.; Leshem, R. J. Chromatogr. 1981, 203, 365-375. (27) Inczedy, J. Analytical Applications of Complex Eequilibria; Halsted Press: New York, 1976; p 330. (28) Uvdal, K.; Bodo, P.; Liedberg, B. J. Colloid Interface Sci. 1992, 149, 162173. (29) Ihs, A.; Liedberg, B. J. Colloid Interface Sci. 1991, 144, 282-292. (30) Schlereth, D. D.; Katz, E.; Schmidt, H. L. Electroanalysis 1995, 7, 46-54.

1550 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

(31) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335-359. (32) Tudos, A. J.; Johnson, D. C. Anal. Chem. 1995, 67, 557-560. (33) Ralph, T. R.; Hitchman, M. L.; Millington, J. P.; Walsh, F. C. J. Electroanal. Chem. 1994, 375, 1-15.

Figure 1. Effect of (A) immersion time, (B) pH, and (C) preconcentration time on the OSWV current of a phosphate solution containing 1.0 µg/L copper(II) by using the cysteine-modified electrodes in 0.2 M phosphate solution.

spectroscopy (IRRAS).29 XPS spectra show that cysteine binds to the surface through the sulfur atom at all three pHs.28 At high pH, the NH2 modes above 3200 cm-1 were absent in IRRAS spectra. Strong interaction between the amino nitrogen and the gold surface was proposed because such a structure allows the transition dipole moments of the NH2 modes to align parallel to the surface and therefore to be invisible in the spectrum. Such nitrogen-gold interaction will weaken the ligation of an amino group toward copper(II). At pH lower than the isoelectric point, the chelating ability decreases because the carboxylate is protonated. A solution of pH 5 is therefore used for the subsequent experiments. At the measurement pH of 5, the predominant inorganic form of copper will be copper(II).34 Organic ligand may, however, bind copper(II) in nonlabile forms that may not exchange with cysteine in the time scale of the experiments. Shown in Figure 1C is the effect of preconcentration time on the OSWV response obtained from a stirred, 15-mL phosphate solution containing 1.0 µg/L copper(II). The response does not level off within 20 min. The preconcentration time was set to 5 min for two reasons. First, we wish to keep the total time for one analysis within 15 min. Second, by employing a preconcentration time shorter than that of the saturation level, the dynamic range can be improved to 2 orders of magnitude (vide infra), despite a sacrifice in sensitivity. For comparison, the linear range reported from the only two publications17,18 of SAM-modified electrodes applied in real samples is 1 order of magnitude. The SW parameters are interrelated. After careful examination, we find that the optimized instrument settings of frequency, pulse height, pulse increment, and potential range are 60 Hz, 60 mV, 5 mV, and from 0.4 to -0.15 V, respectively. Deaeration is not necessary because oxygen reduction is suppressed by the cysteine monolayer. Under optimum conditions, the square wave voltammogram after preconcentration of copper(II) shows a cathodic wave with the peak potential at 0.12 V vs EAg/AgCl. Calibration and Interference Studies. Following the preconcentration and rinsing protocol, control experiments with the (34) Pourbaix, M. Atlas of Electrochemical Equilibria in Aqueous Solutions, 2nd ed.; NACE: Houston, 1974.

bare gold surfaces show no copper(II) signal in a preconcentration solution of 50.0 ppm copper(II). The reproducibility of different films is examined by utilizing an O-ring and a Teflon cell to confine the area of the working electrodes. The RSD of 1.0 µg/L copper(II) signals is 4.8% (n ) 5). The dynamic range of the calibration curve extends from 0.050 to 5.0 µg/L. The calibration curve shows a very linear behavior with slope of 7.88 µA‚L/µg, intercept of 0.39 µA, and correlation coefficient of 0.9996. The detection limit, equal to the three times the standard deviation of the background, is 0.025 µg/L (0.39 nM). Consecutive measurements of 10 1.0 µg/L copper(II) samples without regeneration of cysteine films result in an RSD of 3.9%. In the interference study of the cysteine-modified electrodes against copper(II) determination, the possible interference are chosen on the basis of their complexation affinities with amino acids,27 their abundance in biological and environmental samples, or their use as electrolytes commonly employed in electrochemical studies. The concentrations of the interfering substances examined are as large as 1 g/L or their saturation concentrations. The determination of 1.0 µg/L copper(II) is not affected at all by the presence of CH3COO-, CO32-, Cl-, ClO4-, NO3-, SCN-, NH4+, Cd2+, Co3+, Cr3+, and Fe3+ (1.0 g/L each), Zn2+ (0.10 g/L), Ag+, Bi3+, Pb2+, and Sb6+ (10 mg/L). Organic species, potential interference of conventional stripping analysis, are also examined. Dimethylglyoxime (DMG), octanoic acid, and Triton X-100 (1.0 g/L each) also show no interfering effect. The high selectivity is a result of the fact that the ligation affinity of amino acid moiety toward copper(II) is far superior than other ions. For example, the formation constant of copper(II) with R-alanine, which has the same amino acid moiety as cysteine, is 15.37,27 more than 4 orders of magnitude larger than the log of the second largest formation constant, that of nickel(II), 10.66. In fact, of all the substances examined in this study, nickel(II) is the only species that affects the sensitivity of copper(II) determination. Nickel(II) interference is not pronounced until the concentration is larger than 5.0 mg/L, a 5000-fold excess of the copper(II) concentration (Figure 2). The percentages of SW current decrease of 1.0 µg/L copper(II) due to the interference Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

1551

Table 1. Measurements of Copper(II) on Two Certified Reference Samples and Local Tap Water

sample

measd concna (µg/L)

coeff of variation (%)

certified values (µg/L)

SLEW-2 (river water) CASS-3 (seawater) local tap water

1.58 ( 0.05 0.493 ( 0.027 88.71 ( 0.91

3.2 5.5 1.0

1.62 ( 0.11 0.517 ( 0.062 b

a Number of measurements, 6. b The recovery rate of spiking 1.0 µg/L copper(II) into 100-fold diluted samples is 98.0%.

Figure 2. Effect of DMG on the removal of nickel(II) interference. The samples contain 1.0 µg/L copper(II), 1.0 µg/L copper(II) + 10.0 mg/L nickel(II), and 1.0 µg/L copper(II) + 10.0 mg/L nickel(II) + 50 mg/L DMG, respectively, for the circles and solid and dashed curves.

of nickel(II) at 5.0 and 10.0 mg/L are 8.9 and 16.1, respectively. The nickel(II) interference can be completely suppressed with the addition of DMG, which does not interfere with copper(II) signals. In Figure 2, the open circles are results obtained from 1.0 µg/L copper(II) solutions. When the solutions contain 10.0 mg/L nickel(II), the SW signal of copper(II) decreases significantly and is shown as the solid curve. With the addition of 50 mg/L DMG, the SW voltammogram of open circles is restored, as shown by the dashed line, because DMG complexes strongly with nickel(II). Overall, nickel(II) always interferes significantly with the detection of copper(II) when a chelation methodology is employed.3,9,11 Figure 2, however, demonstrates that with the addition of DMG the cysteine-modified electrodes are free of nickel(II) interference. Analytical Application. The analytical application of the cysteine-modified electrode is demonstrated in the determination of copper(II) on two certified reference materials of river water (SLEW-2) and seawater (CASS-3). Summarized in Table 1 are the measured values obtained from six analyses. The values are within the certified limits, indicating good accuracy in such complex matrixes and at ultralow concentration levels. The coefficients of variation are 3.2% and 5.5%, better precision than those of certified values. Also examined is local tap water (Kaohsiung, Taiwan). Diluting the sample 100-fold is necessary so that the concentration (35) GardeaTorresdey, J. L.; Tang, L.; Salvador, J. M. J. Hazard. Mater. 1996, 48, 191-206. (36) Lu, X. Q.; Yang, X. H.; Chen, Z. L. Electroanalysis 1997, 9, 1278-1282. (37) Lu, X. Q.; Johnson, W. D. Sci. Total Environ. 1997, 203, 199-207.

1552 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

of copper falls in the linear range of the calibration curve. By doing so, any matrix problems become less significant. The result is listed in Table 1. The samples are spiked with 1.0 µg/L copper(II) to ascertain the correctness of the results. The recovery rate of the spiked samples is 98.0%, reassuring the validity of this method in the application of real samples. Environmental samples frequently contain humic acid, an organic degradation product, and therefore the impact of humic acid on copper(II) analyses is important. Determination of copper(II) in humic acid by utilizing cysteine-modified electrodes represents an unusual case because humic acid is a complicated substance of variable composition, containing multifunctional groups which chelate strongly with metal ions, especially copper(II).14,35-37 We found that experiments with added humic acid showed that 26% depression of a 1.0 µg/L copper(II) signal was obtained with a 10.0 mg/L humic acid solution. The humic acid concentration is higher than might normally be expected in natural waters, which are typically 1-2 mg/L dissolved organic carbon. This would result in some 5% depression in signal. Where this is likely to be a problem, UV irradiation satisfactorily decomposes the humic complexes and releases copper(II). The results show that the cysteine-modified electrodes can accurately measure trace amount of copper(II) in complicated matrixes such as humic acid. ACKNOWLEDGMENT This work was supported by the National Science Council, R.O.C. C.h.C. gratefully acknowledges Chemistry Department of National Sun Yat-Sen University for generous research support. The authors appreciate the kind help of Dr. R. Pyati for critical reading and revising of the manuscript. We also thank reviewers for their helpful comments and suggestions. Received for review August 25, 1998. Accepted February 2, 1999. AC980956G