Electrochemical Sensor for Organophosphate Pesticides and Nerve

An electrochemical sensor for detection of organophosphate (OP) pesticides and nerve agents using zirconia (ZrO2) nanoparticles as selective sorbents ...
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Anal. Chem. 2005, 77, 5894-5901

Electrochemical Sensor for Organophosphate Pesticides and Nerve Agents Using Zirconia Nanoparticles as Selective Sorbents Guodong Liu and Yuehe Lin*

Pacific Northwest National Laboratory, Richland, Washington 99352

An electrochemical sensor for detection of organophosphate (OP) pesticides and nerve agents using zirconia (ZrO2) nanoparticles as selective sorbents is presented. Zirconia nanoparticles were electrodynamically deposited onto the polycrystalline gold electrode by cyclic voltammetry. Because of the strong affinity of zirconia for the phosphoric group, nitroaromatic OPs strongly bind to the ZrO2 nanoparticle surface. The electrochemical characterization and anodic stripping voltammetric performance of bound OPs were evaluated using cyclic voltammetric and square-wave voltammetric (SWV) analysis. SWV was used to monitor the amount of bound OPs and provide simple, fast, and facile quantitative methods for nitroaromatic OP compounds. The sensor surface can be regenerated by successively running SWV scanning. Operational parameters, including the amount of nanoparticles, adsorption time, and pH of the reaction medium have been optimized. The stripping voltammetric response is highly linear over the 5-100 ng/mL (ppb) methyl parathion range examined (2-min adsorption), with a detection limit of 3 ng/mL and good precision (RSD ) 5.3%, n ) 10). The detection limit was improved to 1 ng/mL by using 10-min adsorption time. The promising stripping voltammetric performances open new opportunities for fast, simple, and sensitive analysis of OPs in environmental and biological samples. These findings can lead to a widespread use of electrochemical sensors to detect OP contaminates. Organophosphates (OPs) are known to be highly neurotoxic; they disrupt the cholinesterase enzyme that regulates acetylcholine,1-5 a neurotransmitter needed for proper nervous system function. Because of their high neurotoxicity, the OPs are widely used as pesticides and as nerve agents as part of chemical and biological warfare agents. OP residuals in crop, livestock, and * Corresponding author. Tel.: 01-509-376-0529. Fax: 01-509 376-5106. E-mail: [email protected]. (1) Rosenberry, T. L. Advances in enzymology and related areas of molecular biology; John Wiley & Sons: New York, 1975. (2) Zhang, S.; Zhao, H.; John, R. Biosens. Bioelectron. 2001, 16, 1119-1126. (3) Fennouh, S.; Casimiri, V.; Burstein, C. Biosens. Bioelectron. 1997, 12, 97104. (4) Cremisini, C.; Disario, S.; Mela, J.; Pilloton, R.; Palleschi, G. Anal. Chim. Acta 1995, 311, 273-280. (5) Guerrieri, A.; Monaci, L.; Quinto, M.; Palmisano, F. Analyst 2002, 127, 5-7.

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poultry products are clearly dangerous to human health. The related clinical signs include negative effects on the visual system, sensory function, cognitive function, and nervous system. Specifically, exposure to OPs has been shown to cause headache, dizziness, profuse sweating, blurred vision, nausea, vomiting, reduced heart beat, diarrhea, loss of coordination, slow and weak breathing, fever, coma, and death.6 Infants and children may be especially sensitive to health risks posed by pesticides: an estimated 74 000 children were involved in common household pesticide-related poisonings or exposures in the United States in 1994.7 Because of the high toxicity of OPs, the rapid detection of these toxic agents in the environment, public places, or workplaces and the monitoring of individual exposures to chemical warfare agents have become increasingly important for homeland security and health protection.8-11 Early detection of OPs may give an indication of terrorist activity, allowing proper procedures to be followed to mitigate dangers. It is still an extremely difficult challenge to detect low concentrations of OPs accurately in environmental samples. Soil and water samples are very likely to contain OPs because of heavy urban and rural use of these compounds. Military and terrorist activities may result in air, water, and soil contamination with different chemical warfare agents. Analysis of OPs in environmental and biological samples is routinely carried out using analytical techniques, such as gas or liquid chromatography and mass spectrometry.12 Such analysis is generally performed at centralized laboratories, requiring extensive labor and analytical resources, and often results in a lengthy turnaround time. These analysis methods have a number of disadvantages that limit their applications primarily to laboratory settings and prohibit their use for rapid analyses under field conditions. Biological methods, such as immunoassay, have also been reported.13 Long analysis time and extensive sample handling with multiple washing steps limit the applications. In recent years, OP pesticide kits have become commercially available that offer advantages, including portability, rapid turnaround time, and costeffectiveness.14 Drawbacks of these test kits include the compli(6) http://www.beyondpesticides.org/. (7) http://www.epa.gov/pesticides/. (8) Wang, J. Anal. Chim. Acta 2003, 507, 3-10. (9) Sadik, O. A.; Land, W. H.; Wang, J. Electroanalysis 2003, 15, 1149-1159. (10) Lin, Y.; Lu, F.; Wang, J. Electroanalysis 2004, 16, 145-149. (11) Wang, J.; Pumera, M.; Collins, G.; Mulchandani, A.; Lin, Y.; Olsen, K. Anal. Chem. 2002, 74, 1187-1191. (12) Sherma, J. Anal. Chem. 1993, 65, 40R-54R. (13) Miller J. K.; Lenz D. J. Appl. Toxicol. 2001, 21, S23-S26. 10.1021/ac050791t CCC: $30.25

© 2005 American Chemical Society Published on Web 08/09/2005

Figure 1. Structure of nitroaromatic OP compounds. (A) Paraoxon; (B) fenitrothion; (C) methyl parathion.

cated handling procedure and often a lack of sensitivity (ppm level) and precision. Moreover, in most cases, these tests are qualitative or semiquantitative and show false positive and negative results. To meet the requirements of rapid warning and field deployment, more-compact low-cost instruments, coupled to smaller sensing probes, are highly desirable for facilitating the task of on-site monitoring of OP compounds. Various inhibition and noninhibition biosensor systems, based on the immobilization of acetylcholinesterase or OP hydrolase onto various electrochemical or optical transducers, have been proposed for field screening of OP neurotoxins.15-19 Specific antibodies against OP pesticides have been recently developed for enzyme-linked immunoassay and immunosensors.20,21 Although acetylcholinesterase is commercially available, OP hydrolase and antibodies against OPs are still only produced in laboratories, which limits wide applications of biosensors. To avoid the use of enzymes and antibodies, molecular imprint technologies with high selectivity toward specific OP species have been developed and applied to the detection of pesticides in environmental samples.22,23 Nitroaromatic OPs, such as paraoxon, methyl parathion, and fenitrothion (Figure 1), exhibit good redox activities at the electrode surface.11 Electrochemical detection of nitroaromatic OPs showed great promise when it was coupled with different separation technologies, such as highperformance liquid chromatography24 or capillary electrophoresis.25 Surprisingly, little attention has been given to direct electrochemical sensing of nitroaromatic OP compounds, despite (14) The EnviroLogix Cholinesterase Screening Test (EP 014). EnviroLogix Inc., www.envirologix.com. (15) La Rosa, C.; Pariente, F.; Hernandez, L.; Lorenzo, E. Anal. Chim. Acta 1994, 295, 273-282. (16) Mulchandani, A.; Kaneva, I.; Chen, W. Anal. Chem. 1998, 70, 5042-5046. (17) Wang, J.; Mulchandani, A.; Chen, L.; Mulchandani, P.; Chen, W. Anal. Chem. 1999, 71, 2246-2249. (18) Mulchandani, A.; Mulchandani, P.; Kaneva, I.; Chen, W. Anal. Chem. 1998, 70, 4140-4145. (19) Wang, J.; Mulchandani, A.; Chen, L.; Mulchandani, P.; Chen, W. Electroanalysis 1999, 11, 866-869. (20) Hu, S.; Xie, J.; Xu, Q.; Rong, K.; Shen, G.; Yu, R. Talanta 2003, 61, 769777. (21) Marty, I.-L.; Leca, B.; Noguer, T. Analusis Mag. 1998, 26, M144-M149. (22) Turiel, E.; Matin-Esteban, A.; Femandez, P.; Perez-Conde, C.; Camara, C. Anal. Chem. 2001, 73, 5133-5141. (23) Muldoon, M. T.; Stanker, L. H. Anal. Chem. 1997, 69, 803-808. (24) Martinez, R. C.; Gonzalo, E. R.; Garcı´a, F. G.; Me´ndez, J. H. J. Chromatogr. 1993, 644, 49-58. (25) Wang, J.; Chatrathi, M.; Mulchandani, A.; Chen, W. Anal. Chem. 2001, 73, 1804-1808.

their inherent redox activity and the compact nature of electrochemical instruments. Zirconia (ZrO2) is an inorganic oxide with thermal stability, chemical inertness, and lack of toxicity.26-29 Researchers have demonstrated that zirconia has a strong affinity for the phosphoric group. This has been used to prepare multiple films by selfassembly,27-29 or a DNA probe30,31 was attached with the phosphate group at the 5′ end to develop a DNA biosensor. Zirconia films or microcrystals were prepared by electrodeposition of ZrOCl2 at bare or functionalized gold surfaces.32-34 In this paper, we describe the electrochemical sensing nitroaromatic OPs based on a gold electrode modified with zirconia nanoparticles (Figure 2). The new ZrO2 nanoparticle-based electrochemical sensing protocol involves electrodynamically depositing ZrO2 nanoparticles onto a gold electrode surface (A), followed by OP adsorption (B), and electrochemical stripping detection of adsorbed electroactive OPs (C). The electrochemical characterization and anodic stripping voltammetric performance of bound nitroaromatic OP compounds were evaluated using cyclic voltammetric and square-wave voltammetric (SWV) analysis. The promising stripping voltammetric performances open new opportunities for fast, simple, and sensitive analysis of OPs. A disposable screen-printed gold electrode and portable electrochemical instrument would benefit the field monitoring of OPs. EXPERIMENTAL SECTION Reagents. Paraoxon, methyl parathion, and fenitrothion were purchased from Sigma-Aldrich, and their 10 000 mg/L stock solutions were prepared in acetonitrile. Stock solutions of 5 mg/L trinitrotoluene (TNT) were prepared from a 1000 mg/L standard solution of TNT in acetonitrile (Cerilliant, Austin, TX) in 0.1 M potassium chloride, which was used as the supporting electrolyte and also served as the adsorption medium during the adsorption experiments. Zirconium oxychloride (ZrOCl2), nitrobenzene, and p-nitrophenol were obtained from Sigma-Aldrich (St. Louis, MO) and used without further purification. Other reagents were commercially available and were of analytical reagent grade. Solutions were prepared with ultrapure water from a Millipore Milli-Q water purification system (Billerica, MA). Instruments. Cyclic voltammetric and SWV measurements were performed using an electrochemical analyzer CHI 660 (CH Instruments, Austin, TX) connected to a personal computer. A three-electrode configuration was employed, consisting of a zirconia nanoparticle-modified gold electrode (3-mm diameter) serving as a working electrode, while Ag/AgCl/3 M KCl and platinum wire served as the reference and counter electrodes, respectively. Electrochemical experiments were carried out in a (26) Thomas Buscher, C.; McBranch, D.; Li, D. J. Am. Chem. Soc. 1996, 118, 2950-2953. (27) Fang, M.; Kaschak, D. M.; Sutorik, A. C.; Mallouk T. E. J. Am. Chem. Soc. 1997, 119, 12184-12191. (28) Lee, H.; Kepley, L. J.; Hong, H.; Mallouk T. E. J. Am. Chem. Soc. 1988, 110, 618-620. (29) Hong, H.; Sackett, D. D.; Mallouk, T. E. Chem. Mater. 1991, 3, 521-527. (30) Zhu, N.; Zhang A.; Wang, Q.; He, P.; Fang, Y. Anal. Chim. Acta 2004, 510, 163-168. (31) Liu, S.; Xu, J.; Chen, H. Bioelectrochemistry 2002, 57, 149-154. (32) Yu, H.; Rowe, A.; Waugh, D. M. Anal. Chem. 2002, 74, 5742-5747. (33) Aslam, M.; Pethkar, S.; Bandyopadhyay, K.; Mulla, I. S.; Sainkar, S. R.; Mandale, A. B.; Vijayamohanan, K., J. Mater. Chem. 2000, 10, 1737-1743. (34) Bandyopadhyay, K.; Vijayamohanan, K. Langmuir 1998, 14, 6924-6929.

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Figure 2. Scheme of electrochemical sensing nitroaromatic OP compounds. (A) Electrodeposition ZrO2 nanoparticle to gold electrode surface; (B) nitroaromatic OP compounds adsorb to ZrO2 nanoparticle surface; (C) electrochemical stripping detection of nitroaromatic OP compounds; X ) O or S and R ) nitroaromatic OP group.

2-mL voltammetric cell at room temperature (25 °C). All potentials are referred to the Ag/AgCl reference electrode (CH Instruments). Scanning electron microscopy (SEM) was carried out using a JEOL JSM-5900 LV machine. All samples were imaged under vacuum conditions using secondary electron imaging. The typical accelerating voltage of the electron beam used was 10 kV. All samples were grounded with a piece of copper tape to curtail specimen charging. Preparation of Gold Electrode Modified with Zirconia Nanoparticles. A gold electrode (3-mm diameter) from CH instruments was polished carefully to a mirrorlike surface with 0.3- and 0.05-µm alumina slurry and sequentially sonicated for 2 min in 6 M nitric acid, acetone, and water. Before the experiment, the bare gold electrode was cyclic-potential scanned within the potential range 0.5-1.5 V in freshly prepared 0.2 M H2SO4 until a voltammogram characteristic of the clean polycrystalline gold was established. Then it was washed with distilled water and dried by nitrogen. Zirconia nanoparticles were deposited onto bare gold electrodes in an aqueous electrolyte of 5.0 mM ZrOCl2 and 0.1 M KCl by cycling the potential between -1.1 and + 0.7 V (versus Ag/AgCl) at a scan rate of 20 mV/s for 10 consecutive scans.32 The gold electrodes modified with zirconia nanoparticles (ZrO2/ Au) were rinsed with water and dried with N2 for further experiments. Electrochemical Stripping Detection. A ZrO2/Au electrode was dipped into a stirring 0.1 M KCl solution containing the desired concentration of OP pesticides for 2 min, washed with distilled water carefully, and transferred to a 2-mL electrochemical cell containing 0.1 M KCl solution. Before electrochemical measurements, the electrolyte solution was purged with nitrogen for 5 min. SWV measurements were performed from -0.4 to +0.3 V with a step potential of 4 mV, an amplitude of 20 mV, and a frequency of 25 Hz (unless otherwise stated). Baseline correction of the resulting voltammogram was performed using the “linear baseline correction” mode of the CHI 660 (CH Instruments, Austin, TX) software. Cyclic voltammetric measurements were performed under batch conditions. The cyclic voltammogram was recorded between -0.8 and +0.5 V at a scan rate of 100 mV/s. All measurements were performed at room temperature. 5896 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

Regeneration of Electrode Surface. After the electrochemical stripping measurement, multiple successive SWV scanning was used to remove the bound OPs until the anodic stripping peak disappeared. The electrode was washed with distilled water for the next measurement. Safety Considerations. OP pesticides are highly toxic and should be handled in a fumehood. Skin and eye contact and accidental inhalation or ingestion should be avoided. RESULTS AND DISCUSSION In the current study, the ZrO2 nanoparticles were electrodynamically deposited onto a cleaned gold electrode surface in an aqueous electrolyte of 5.0 mM ZrOCl2 and 0.5 M KCl by cycling the potential scanning between -1.1 and +0.7 V (versus Ag/AgCl) for 10 consecutive scans at a scan rate of 20 mV/s (unless otherwise stated). Figure 3A shows a representative cyclic voltammogram of the formation processes of ZrO2 nanoparticles on the cleaned gold electrode surface (curve a, red line). A normal electropolymerization growth, with increasing current upon repetitive scanning, is observed during the electrodeposition processes. The steep rise in the cathodic and anodic current at the potential range of -0.6 to -1.1 V corresponds to the complex redox behavior of ZrOCl2 on gold.32 Such redox behavior was not observed in the absence of ZrOCl2 (curve b, blue line). Note that the increasing cathodic and anodic current is different from the results reported by Yu et al. The cathodic current decreased with a thiol self-assembled monolayer modified gold electrode.32 The observed difference may come from electrode material or a different electrodeposition mechanism of zirconia. Different cycling potential ranges (between -1.1 V to varying high potential from 0.7 to 1.2 V) were used to prepare ZrO2 nanoparticles on the gold electrode surface. Experimental results showed there was no significant difference observed including the shape, density of formed ZrO2 nanoparticle, and stripping voltammetric characteristics of bound OPs. A cycling potential range between -1.1 V and +0.7 V was used to prepare the ZrO2/Au electrode. A SEM image (Figure 3B) confirms the distinct ZrO2 nanoparticle formation on the gold electrode surface. The ZrO2 nanoparticles formed by 10 consecutive potential cycling possess an average size of 50-

attributed to a two-electron-transfer process (reactions 2 and 3), as shown below:

Figure 3. (A) Cyclic voltammograms of gold electrode (curve a, red line) during electrodeposition process in 5.0 mM ZrOCl2 and 0.1 M KCl aqueous solution at a scan rate of 20 mV/s. Potential cycles, 10; curve b (blue line) is the cyclic voltammograms of gold electrode in 0.1 M KCl aqueous solution in the absence of ZrOCl2 under the same conditions. (B) Typical SEM image of zirconia nanoparticles formed with 10 consecutive potential cycling on a gold electrode.

150 nm with a small interparticle distance that is much smaller than the ZrO2 microcrystalline (7-15 µm), which has a 17-µm interparticle distance, formed on the dithiol functionalized gold surface or bare vacuum-deposited 2000-Å gold surface.34 The formed nanosize ZrO2 particles in our experiments may benefit from the polycrystalline gold seeds that were formed during the electrochemical cleaning step in the 0.2 M H2SO4 solution. The amount of zirconia nanoparticle on the gold electrode surface increases with the increase in the number of potential cycles. An average of 300 zirconia nanoparticles/µm2 was observed on the prepared ZrO2/Au electrode surface (based on counting at six different locations) after 10 potential cycles. Zirconia has a strong affinity to the phosphoric group and provides a facile method to attach OPs to an electrode surface. Nanosize ZrO2 particles offer a large electrode surface area and increase the interacting opportunities of OPs. Figure 4 shows the cyclic voltammograms of methyl parathion/ZrO2/Au electrode (a) and ZrO2/Au (b) in a 0.1 M potassium chloride solution. A pair of rather well-defined redox peaks (Epa, 0.093 V and Epc, 0.037) and an irreversible reduction peak (Epc, -0.61 V) were observed with a methyl parathion/ZrO2/Au electrode in the potential range of -0.8 to +0.5 V (Figure 4 a). The irreversible reduction peak corresponds to the reduction of the nitro group to the hydroxylamine group (reaction 1), and the reversible redox peaks are

These profiles are consistent with those described elsewhere for nitroaromatic OP pesticides and nitrophenyl derivates.25,35-37 A control experiment (Figure 4b) was performed under the same conditions with the ZrO2/Au electrode in the absence of methyl parathion; no redox peak appeared at the selected potential range (the standard reduction potential of ZrO2 is -1.544 V). SWV analysis has a higher sensitivity than other electrochemical technologies, such as cyclic voltammetry and differential pulse voltammetry. The inset of Figure 4 shows corresponding SWV voltammograms of methyl parathion /ZrO2/Au electrode (a) and ZrO2/Au electrode (b) in 0.1 M KCl. There is no anodic stripping peak observed at the ZrO2/Au electrode (inset, curve b). The methyl parathion/ZrO2/Au electrode exhibits a very sharp and well-defined stripping peak at the potential range from -0.8 V to +0.4 V (inset, curve a). The peak potential of the oxidation peak (0.06 V) shifts 20 mV to a negative potential direction compared with that in the cyclic voltammogram. The attracting voltammetric characteristics of adsorbed methyl parathion on the ZrO2/Au show that ZrO2 nanoparticles have a strong affinity to the OP compound, which possesses a phosphate group. To confirm that the affinity occurred between ZrO2 and methyl parathion instead of the nonspecific adsorption between the exposed gold surface and methyl parathion, Figure 5 shows a comparison of the SWV signals of a cleaned bare gold electrode (a) and a ZrO2/Au electrode (b) after incubating 2 min in 0.1 M KCl containing 200 ng/mL methyl parathion. A substantially smaller signal (35 times less compared to the ZrO2/Au electrode) is observed for a bare electrode. Such a big difference in SWV signals is attributed to the specific adsorbing between ZrO2 (35) Lin, Y.; Zhang, R. Electroanalysis 1994, 6, 1126-1131. (36) Roston, D. A.; Kissinger, P. T. Anal. Chem. 1982, 54, 429-434. (37) Kastening, B., Zuman, P., Meites, L., Kolthoff, I. M, Eds. Progress in Polarography; Wiley-Interscience: New York, 1972; Vol. 3, p 259.

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Figure 4. Cyclic voltammograms of methyl parathion/ZrO2/Au (a) and ZrO2/Au electrode (b) in 0.1 M KCl solution (pH 7.0). Potential scanning rate, 100 mV/s. Methyl parathion/ZrO2/Au electrode was prepared by dipping the ZrO2/Au electrode in stirring 0.1 M of KCl solution containing 800 ng/mL methyl parathion for 2 min and carefully washing with distilled water before electrochemical measurement. Inset is corresponding stripping voltammograms. SWV conditions: scanning potential range, -0.8 to +0.4 V; frequency, 25 Hz; increasing potential, 4 mV.

Figure 5. Stripping voltammograms of bare gold electrode (a) and ZrO2/Au electrode (b) after 2-min adsorption in stirring 0.1 M of KCl solution containing 200 ng/mL methyl parathion. Potential scanning potential. -0.4 to +0.3 V; other conditions, same as Figure 4.

nanoparticles and methyl parathion. Also to be noted is that the peak potentials for the stripping voltammograms shift to positive potential direction with the increase of concentration of methyl parathion on the ZrO2/Au electrode surface. The peak potential for the stripping voltammogram (Figure 5, 200 ng/mL methyl parathion) is shifted around 100 mV from that observed in Figure 4 (800 ng/mL methyl parathion). Similar behavior was observed in the stripping voltammograms of different concentrations of methyl parathion (see Figure 9). Another two electroactive OPs, paraoxon and fenitrothion, which possess a structure similar to that of methyl parathion 5898 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

(Figure 1), exhibit similar voltammetric characteristics after they are adsorbed on the ZrO2/Au electrode surface. The mixture of three identical concentrations of OP compounds shows a big stripping response, which almost equals the sum of the individual OPs. To confirm that the affinity occurred between ZrO2 and the phosphate group instead of the nitro group, trinitrotoluene was used to perform the comparison experiment. A negligible signal was obtained even though the concentration of TNT (1 µg/mL) is 5 times more than methyl parathion (not shown). One of the most important issues in the development of a chemical biosensor is the regeneration of the sensor surface. Electrochemical stripping analysis includes built-in preconcentration and stripping steps. The target analyte is normally accumulated on the working electrode by applying a constant potential followed with a stripping step, which can also be regarded as a cleaning step to remove the target from the electrode surface. In our experiments, the adsorption process of methyl parathion corresponds to the stripping step to obtain the electrochemical stripping signal of the analyte, which automatically removes the adsorbed OPs. Figure 6 presents a typical successive SWV voltammogram of a methyl parathion/ ZrO2/Au electrode. It was found that the stripping peak currents decreased rapidly with the increase of scanning times, and the anodic stripping peak disappeared completely after multiple scanning, indicating that the methyl parathion-ZrO2 complex is dissociated. The electrode was washed carefully with distilled water and measured again in fresh 0.1 M KCl solution; no stripping peak was obtained. Also to be noted is that the SWV scanning times depended on the concentration of adsorbed OPs. More scanning times are necessary for a higher amount of bound OPs.

Figure 6. Stripping voltammograms (without baseline correction) of the regeneration process of methyl parathion/ZrO2/Au electrode; other conditions, same as Figure 5.

The attracting stripping voltammetric characteristics of methyl parathion on the ZrO2/Au provide a facile electrochemical quantitative method for analyzing OPs. Parameters of the assay procedure would affect the stripping response of OPs. The amount of ZrO2 nanoparticles influences the amount of OPs bound to the surface of the ZrO2/Au electrode. The cycles of cyclic-potential scanning were used to control the amount of ZrO2 nanoparticle on the gold electrode surface. Figure 7A shows the effect of the cycles of cyclic-potential scanning on the adsorption of methyl parathion. The stripping current of methyl parathion rises with the cycles at first up to 10 cycles and then decreases. The increase of the stripping current indicates that the amount of adsorbed methyl parathion is increasing with the increase of the amount of zirconia nanoparticle on the electrode surface. The decrease of the stripping current can be understood by considering the continual buildup of zirconia nanoparticles, which consequently cause aggregation of zirconia nanoparticles and generate higher resistance for the electrochemical stripping processes, leading to a change of sensing characteristics of the electrode. The change of electrochemical sensing characteristics of ZrO2/Au was investigated by cyclic voltammetry of a 5 mM Fe(CN)63-/0.1 M KCl. Figure 7B presents cyclic voltammograms of a bare Au electrode, and different amounts of ZrO2 nanoparticles modified the Au electrodes. We can see the redox peak currents of Fe(CN)63decrease with the increase of potential cycle times (from top to bottom, 0, 2, 4, 6, 8, 10, 15, and 20 cycles). Although more potential cycles increase the amount of ZrO2 nanoparticles on the electrode surface, aggregations of nanoparticles increase the electrontransfer distance, which leads to the decrease of redox peak current of Fe (CN)63- and decreases the sensitivity of the electrode. So 10 potential cycles were used to prepare the ZrO2 nanoparticle modified gold electrode. The effect of adsorption time on the stripping peak current was investigated (Figure 8A). The peak currents increase rapidly

Figure 7. (A) Effect of the amount of zirconia nanoparticle on methyl parathion adsorption. ZrO2/Au electrodes were prepared by different potential scanning cycles (2, 4, 6, 8, 10, 15, 20). The adsorption experiments were performed for 2 min in 0.1 M KCl solution containing 200 ng/mL methyl parathion. Stripping detection conditions, same as Figure 5. (B) Cyclic voltammograms of corresponding electrodes in 5 mM Fe(CN)63-/0.1 M KCl solution (from top to bottom, 0, 2, 4, 6, 8, 10, 15, 20 cycles), potential scanning rate. 100 mV/s.

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Figure 8. Effect of adsorption time (A) and the pH of adsorption medium (B) on the adsorption of methyl parathion. The concentration of methyl parathion in adsorption medium was 200 ng/mL. The adsorption experiments of (A) were performed in a pH 7.0 of 0.1 M KCl. Electrochemical stripping detection conditions, same as Figure 5.

with the accumulation time at first and then more slowly from 2 min. The resulting current versus time plot displays a curvature consistent with adsorption processes. No such surface adsorption is indicated in analogous measurements at the cleaned bare gold

electrode surface (not shown). We also observed the adsorption of methyl parathion under constant potential conditions (not shown); there is no significant increase of the stripping peak current. Two minutes of adsorption time under open-circuit conditions was thus employed. An additional parameter that affected the adsorption of methyl parathion was the pH of the adsorption medium. The pH of the adsorption solution (0.1 M KCl) was adjusted with 1.0 M NaOH or HCl solution and varied from 3.0 to 9.0. Figure 8B presents the pH effect of the adsorption solution on the adsorption of methyl parathion. We can see that the stripping signal increases with an increase of pH up to 7.0, and then it decreases at higher pH. It indicates that ZrO2 has the maximum adsorption to methyl parathion in a neutral environment. The loss of signal at acidic or basic environment may be attributed to the effect of H+ or OHon adsorption. The mechanism of the pH effect is under investigation in our laboratory. So a pH 7.0 of 0.1 M KCl solution was used as the adsorption medium in most experiments. Analytical Performance. Figure 9 displays the SWV response of the ZrO2/Au electrode incubated in increasing concentrations of methyl parathion solution under optimum experimental conditions. Well-defined peaks, proportional to the concentration of the corresponding methyl parathion, were observed. A linear relationship between the stripping current and the methyl parathion concentration was obtained covering the concentration range from 5 to 100 ng/mL, the linear regression equation being I (nA) ) 1.0696C + 5.4453, with a correlation coefficient of 0.9939. A wide linear range will be realized by increasing the amount of zirconia nanoparticle on the gold electrode surface. A detection limit of 3 ng/mL (based on signal-to-noise ratio equal to 3) was obtained under the optimum experimental conditions. The detection limit was improved significantly by increasing the accumulation time. A detection limit of 1 ng/mL was estimated on the basis of a s/n ) 3 characteristic of the 3 ng/mL data points in connection with a 600-s incubating time. The detection limit obtained is

Figure 9. Stripping voltammograms of increasing methyl parathion concentration, from bottom to top, 5, 10, 20, 40, 60, 80, 100, and 200 ng/mL, respectively. The inset shows the calibration curve. Electrochemical stripping detection conditions, same as Figure 5. 5900

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and electroactive species, whose oxidation potentials are more than 0.3 V. An electrochemical stripping analysis used in conjunction with a zirconia nanoparticle modified gold electrode thus holds great promise for direct analysis of relevant water samples without any prior separation or pretreatment.

Figure 10. Electrochemical stripping signals of methyl parathion/ ZrO2/Au electrodes. Adsorption experiments were performed with pH 7 0.1 M KCl containing 100 ng/mL methyl parathion in the absence and presence of 100 ng/mL p-nitrophenol, 100 ng/mL nitrobenzene, 100 ng/mL TNT, 0.1 M PO43-, 0.1 M SO42-, and 0.1 M NO3-, respectively. Electrochemical stripping detection conditions, same as Figure 5.

comparable with that reported so far with an enzyme-based biosensor.18 A series of 10 repetitive measurements of a solution containing 20 ng/mL yielded reproducible peak currents with relative standard deviations of 5.3. Interferences arising from the other electroactive nitrophenyl derivatives and oxygen-containing inorganic ions (PO43-, SO42-, NO3-) that are expected to coexist in solution were used to evaluate the selectivity of the ZrO2/Au electrode to nitroaromatic OPs. Separate adsorbing experiments were performed with 100 ng/mL methyl parathion in 0.1 M KCl solution in the absence and presence of 100 ng/mL of p-nitrophenol, 100 ng/mL nitrobenzene, 100 ng/mL TNT, 0.1 M of PO43-, 0.1 M of SO42-, and 0.1 M of NO3-. Figure 10 shows the electrochemical stripping signals of methyl parathion at different experimental conditions. One can see that electroactive nitrophenyl derivatives and oxygen-containing inorganic ions do not interfere with the adsorption of methyl parathion, and the stripping peak current varies slightly. Also note that it was reported in the literature that zirconia has a good affinity to PO43-,33, 34 but in this case, it does not interfere with the adsorption of methyl parathion. The reason may be that the adsorption capability of methyl parathion to zirconia is much stronger than PO43-. Further experiments are being conducted in our laboratory. The stripping peak potential of OPs is ∼0 mV, which also avoids the interferences from other phenol compounds

CONCLUSION We have demonstrated a sensitive electrochemical sensing protocol for nitroaromatic OPs based on the use of zirconia nanoparticles as selective sorbents. The strong affinity of zirconia nanoparticles for the phosphoric group and the promising SWV characteristics of nitroaromatic OPs provide a facile quantitative method for a group of electroactive OPs. Other electroactive nitrophenyl derivatives do not interfere with the adsorption of OPs. An anodic stripping analysis with a very low stripping peak potential avoids the interferences from other electroactive species. The results obtained from this work imply that the combination of a disposable screen-printed gold electrode with a portable electrochemical instrument would benefit the field monitoring of OPs. Current methods are limited to the detection of a group of nitroaromatic OPs. Nonelectroactive OPs can be monitored by combining zirconia nanoparticles (selective absorbents) with enzyme or metal nanoparticle-labeled antibodies against OPs (recognition elements) and electrochemically measuring the enzymatic product or dissolved metal ions. The proposed electrochemical sensing technology is thus expected to open new opportunities for detecting OP pesticides and nerve agents in the environment, public places, or workplaces and for monitoring the exposures of individuals to chemical warfare agents. ACKNOWLEDGMENT The work is supported by a laboratory-directed research and development program at Pacific Northwest National Laboratory (PNNL). The research described in this paper was performed at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy’s (DOE’s) Office of Biological and Environmental Research and located at PNNL. PNNL is operated by Battelle for DOE under Contract DE-AC05-76RL01830.

Received for review May 6, 2005. Accepted July 12, 2005. AC050791T

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

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