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Activated Nickel Platform for Electrochemical Sensing of Phosphate Wan-Ling Cheng, Jun-Wei Sue, Wei-Chung Chen, Jen-Lin Chang, and Jyh-Myng Zen* Department of Chemistry, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan We report here a highly selective enzymeless approach for the determination of phosphate (PO43-) by flow injection analysis (FIA). In this system, the activation of barrel plated nickel electrode (Ni-BPE) in alkaline media to form a Ni(OH)2/NiO(OH) film was found to trigger the adsorption of phosphate at the electrode surface. Based on the suppressed current of the electrocatalytic oxidation of glucose at the activated NiBPE in 0.1 M NaOH solution caused by adsorption of phosphate, we develop an FIA detection scheme for the determination of phosphate. Under the optimized conditions of flow rate )300 µL/min and detection potential )0.55 V vs Ag/AgCl with 25 µM glucose in 0.1 M NaOH as carrier solution, the calibration curve showed a linear range up to 1 mM. Possible interferences from the coexisting ions were also investigated. The results demonstrated that sensor could be used for the determination of phosphate in the presence of nitrate, chloride, sulfate, acetate, oxalate, carbonate, and some anionic species of toxicological and environmental interest, such as chlorate, chromate, and arsenate ions. The electrode can be effectively regenerated without extra treatment under the hydrodynamic condition. For eight continuous injections of 40 µM PO43-, a relative standard deviation of 0.28% was obtained, indicating good reproducibility of the proposed method. The detection limit (S/N ) 3) was calculated as 0.3 µM. Quantitative analysis of phosphate anions, both inorganic and organic, is important in biological diagnosis, environmental monitoring, and biomedical research. The analytical range of phosphorus is from 0.2 to 10 mg/L in natural and waste waters and from 0.2 to 50 mg/kg in soil. A maximum permissible concentration of phosphate in river water is 0.32 µM and ranges from 0.0143 to 0.143 mM in wastewater. As for as a diagnostic fluid, the concentration of phosphate ion in human saliva are variable, ranging from 5 to 14 mM. It is in the range of 0.81 to 1.45 mM PO43- in adult human serum. Various detection strategies for phosphate have been developed and include phosphate ion selective electrodes, chromatography, and spectroscopy and the development of sensors exploiting enzymatic reactions.1-14 Recently, great efforts have also been devoted to * To whom correspondence should be addressed. Fax: +886 4 22854007. E-mail: [email protected]. (1) Engblom, S. O. Biosens. Bioelectron. 1998, 13, 981–994. 10.1021/ac9025253  2010 American Chemical Society Published on Web 12/29/2009

design new sensors for recognition of phosphate ions in water at biological pH values using various receptors to bind dihydrogen phosphate ions.15-19 We report here a novel method using a barrel plated nickel electrode (Ni-BPE) for flow injection analysis (FIA) of phosphate (PO43-). Note that our group has successfully applied the engineering process of barrel platting technology for mass production of disposable-type electrodes for various analytical applications.20-25 Especially, the barrel platting technology offers a relatively straightforward means to manufacture sensor strips on a mass scale and represents a useful platform for the development of a highly precise glucose sensor.26-29 (2) Quintana, J. B.; Rodil, R.; Reemtsma, T. Anal. Chem. 2006, 78, 1644–1650. (3) Zyryanov, G. V.; Palacios, M. A.; Anzenbacher, P. Angew. Chem., Int. Ed. 2007, 119, 7995–7998. (4) Zhang, J. Z.; Chi, J. Environ. Sci. Technol. 2002, 36, 1048–1053. (5) Villalba, M. M.; McKeeganb, K. J.; Vaughanb, D. H.; Cardosic, M. F.; Davis, J. J. Mol. Catal. B 2009, 59, 1–8. (6) de Marco, R.; Clarke, G.; Pejcic, B. Electroanalysis 2007, 19, 1987–2001. (7) Parra, A.; Ramon, M.; Alonso, J.; Lemos, S. G.; Vieira, E. C.; Nogueira, A. R. A. J. Agric. Food Chem. 2005, 53, 7644–7648. (8) Ganjali, M. R.; Norouzi, P.; Ghomi, M.; Salavati-Niasari, M. Anal. Chim. Acta 2006, 567, 196–201. (9) Quintana, J. C.; Idrissi, L.; Palleschi, G.; Albertano, P.; Amine, A.; Raiz, M.; Moscone, D. Talanta 2004, 63, 567–574. (10) Akyilmaz, E.; Yorganci, E. Electrochim. Acta 2007, 52, 7972–7977. (11) Kwan, R. C. H.; Leung, H. F.; Hon, P. Y. T.; Bradford, J. P.; Renneberg, R. Appl. Microbiol. Biotechnol. 2005, 66, 377–383. (12) Mak, W. C.; Chan, C. Y.; Barford, J.; Renneberg, R. Biosens. Bioelectron. 2003, 19, 233–237. (13) Preechaworapun, A.; Dai, Z.; Xiang, Y.; Chailapakul, O.; Wang, J. Talanta 2008, 76, 424–431. (14) Mousty, C.; Cosnier, S.; Shan, D.; Mu, S. L. Anal. Chim. Acta 2001, 443, 1–8. (15) Nishizawa, S.; Kato, Y.; Teramae, N. J. Am. Chem. Soc. 1999, 121, 9463– 9464. (16) Lu, H.; Xu, W.; Zhang, D.; Zhu, D. Chem. Commun. 2005, 4777–4779. (17) Han, M. S.; Kim, D. H. Angew. Chem., Int. Ed. 2002, 41, 3809–3811. (18) O’Toole, M.; Lau, K. T.; Shepherd, R.; Slater, C.; Diamond, D. Anal. Chim. Acta 2007, 596, 290–294. (19) Worsfold, P. J.; Gimbert, L. J.; Mankasingh, U.; Omaka, O. N.; Hanrahan, G.; Gardolinski, P. C. F. C.; Haygarth, P. M.; Turner, B. L.; Keith-Roach, M. J.; McKelvie, I. D. Talanta 2005, 66, 273–293. (20) Sue, J.-W.; Senthil Kumar, A.; Chung, H.-H.; Zen, J.-M. Electroanalysis 2005, 17, 1245–1250. (21) Su, J.-W.; Hung, C.-J.; Chen, W.-C.; Zen, J.-M. Electroanalysis 2008, 20, 1647–1654. (22) Shih, Y.; Wu, K.-L.; Sue, J.-W.; Kumar, S. A.; Zen, J.-M. J. Pharm. Biomed. Anal. 2008, 48, 1446–1450. (23) Sue, J.-W.; Tai, C.-Y.; Cheng, W.-L.; Zen, J.-M. Electrochem. Commun. 2008, 10, 277–282. (24) Sue, J.-W.; Ku, H.-H.; Chung, H.-H.; Zen, J.-M. Electrochem. Commun. 2008, 10, 987–990. (25) Tai, C.-Y.; Chang, J.-L.; Zen, J.-M. Chem. Commun. 2009, 6083–6085. (26) Wu, M.-H.; Fang, M.-Y.; Jen, L.-N.; Hsiao, H.-C.; Muller, A.; Hsu, C.-T. Clin. Chem. 2008, 54, 1689–1695. (27) Hsu, C.-T.; Hsiao, H.-C.; Lee, M.-S.; Chang, S.-F.; Lee, T.-C.; Tsai, Y.-S.; Zen, J.-M. Clin. Chim. Acta 2009, 402, 119–123.

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Previously, extensive studies have been done by soil scientists regarding phosphate reactions with iron and aluminum oxides and hydroxides.30-33 It is reported that a phosphonic monolayer format on many substrates, which are dominated by atoms with an empty valence shell and have a strong affinity for oxide surfaces, such as ZrO2, TiO2, and Al2O3 by condensation reactions of the acid, function with metal hydroxyl species to form bound phosphonate salts.34-39 Adsorption of phosphate on indium tin oxide (ITO) as well as on a cobalt electrode surface have also been investigated.40-43 An interesting sensor was recently reported for the determination of phosphate based on the suppressed current of an [Fe(CN)6]3- redox probe caused by ionic adsorption of phosphate on a Zr(IV)-immobilized gold-mercaptopropionic acid self-assembled monolayer electrode.44 The strategy inspires us to design a relatively stable and simple detection scheme for the determination of phosphate in the present work. In our system, the activation of Ni-BPE in alkaline media to form a Ni(OH)2/NiO(OH) film was found to trigger the adsorption of phosphate (PO43-) at the electrode surface. Meanwhile, the electrocatalytic oxidation of glucose at the activated Ni-BPE in 0.1 M NaOH solution was used as a redox probe similar to the role of [Fe(CN)6]3- in the Zr system mentioned earlier.44 Similarly, the suppressed current of electrocatalytic oxidation of glucose caused by adsorption of phosphate on the activated Ni-BPE can then be used for the detection of PO43-. Interestingly, the activated Ni-BPE was found highly selective in the adsorption of PO43- under alkaline condition. This is indeed a major advantage of the proposed sensor compared to enzyme-based biosensors, which are not suited for continuous operation and repetitive use.5 It is because these enzyme-based biosensors need an appropriate medium for optimum activity and stability of all the involved sensing elements, and the sensor performance can be largely influenced by variations of buffer, electrolyte, or immobilization strategy. When the FIA current response as the detection signal was measured, such a sensing device was demonstrated to detect low levels of PO43- with good selectivity. Overall, important (28) Hsu, C.-T.; Hsiao, H.-C.; Fang, M.-S.; Zen, J.-M. Biosen. Bioelectron. 2009, 25, 383–387. (29) Hsu, C.-T.; Chung, H.-H.; Tsai, D.-M.; Fang, M.-Y.; Hsiao, H.-C.; Zen, J.-M. Anal. Chem. 2009, 81, 515–518. (30) Daou, T. J.; Begin-Colin, S.; Grene`che, J. M.; Thomas, F.; Derory, A.; Bernhardt, P.; Legare´, P.; Pourroy, G. Chem. Mater. 2007, 19, 4494–4504. (31) Nooney, M. G.; Campbell, A.; Murrell, T. S.; Lin, X.-F.; Hossner, L. R.; Chusuei, C. C.; Goodman, D. W. Langmuir 1998, 14, 2750–2755. (32) Hongahao, Z.; Stanforth, R. Environ. Sci. Technol. 2001, 35, 4753–4757. (33) Ler, A.; Stanforth, R. Environ. Sci. Technol. 2003, 37, 2694–2700. (34) Pellerite, M. J.; Dunbar, T. D.; Boardman, L. D.; Wood, E. J. J. Phys. Chem. B 2003, 107, 11726–11736. (35) Pawsey, S.; Yach, K.; Reven, L. Langmuir 2002, 18, 5205–5212. (36) Baker, M. V.; Jennings, G. K.; Laibinis, P. E. Langmuir 2000, 16, 3288– 3293. (37) Yim, C. T.; Pawsey, S.; Morin, F. G.; Reven, L. J. Phys. Chem. B 2002, 106, 1728–1733. (38) Van Alsten, J. G. Langmuir 1999, 15, 7605–7614. (39) Harvey, O. R.; Rhue, R. D. J. Colloid Interface Sci. 2008, 322, 384–393. (40) Gardner, T. J.; Frisbie, C. D.; Wrighton, M. S. J. Am. Chem. Soc. 1995, 117, 6927–6933. (41) Kato, D.; Xu, G.; Iwasaki, Y.; Hirata, Y.; Kurita, R.; Niwa, O. Langmuir 2007, 23, 8400–8405. (42) Meruva, R. K.; Meyerhoff, M. E. Anal. Chem. 1996, 68, 2022. (43) Shimizu, Y.; Yamashita, T.; Takase, S. Jpn. J. Appl. Phys. 2000, 39, L384– L386. (44) Shervedani, R. K.; Pourbeyram, S. Biosens. Bioelectron. 2009, 24, 2199– 2204.

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practical features of FIA include simplicity, lower cost, and better analytical characteristics. Besides, the instrumentation is lightweight, compact, easily taken into the field, and readily automated. The proposed scheme cannot only increase the sensitivity but also is suitable for continuous operation and repetitive use. EXPERIMENTAL SECTION Chemicals and Reagents. Sodium hydroxide and glucose were used as received from Sigma. All other compounds used in this work were ACS-certified reagents and used without any further purification. Aqueous solutions and real samples were prepared using double distilled deionized water. Unless otherwise stated, the base electrolyte used in the study was made of 0.1 M NaOH solution. Apparatus and Electrochemical Detector Setup. Cyclic voltammetric and chronoamperometric experiments were carried out using a CHI 900 electrochemical workstation (CH Instruments, U.S.A.). The Ni-BPE three-electrode system is the same as reported in our previous study.26 Typical pictures of the Ni-BPE working system used in this work is illustrated in Supporting Information Figure S-1. It consists of a Ni-BPE working electrode, a stainless tube counter electrode (outlet), and an Ag/AgCl reference electrode. The Ni-BPE (1.25 mm diameter, 31 mm length), obtained from Zensor R&D (Taichung, Taiwan) with an average weight of 392.4 ± 0.6 mg (n ) 10) was fabricated by barrel plating nickel thin film onto a copper rod, and the metallic thin film was measured as 50 ± 2 µm after the barrel plating procedure. Similar to our previous studies, the Ni-BPE surface was activated by cycling from +0.6 to -0.2 V in 0.1 M NaOH solution, and the activated Ni-BPE was equilibrated in 0.1 M NaOH of carrier solution at +0.55 V vs Ag/AgCl until the current became constant.21-24 It usually took a few minutes. All the experiments were performed at room temperature (25 °C). RESULTS AND DISCUSSION Physical Characterization. The electrochemical behavior of the proposed system was first characterized. Figure 1 shows the observed cyclic voltammograms in the presence/absence of respective analytes at the activated Ni-BPE. The characteristic oxidation peak at +0.47 V and reduction wave at +0.38 V are originated from multilayer formation and reduction of nickel oxide (i.e., NiO + OH- f NiO(OH) + e- or Ni(OH)2 + OH- f NiO(OH) + H2O + e-) during activation.35-39 The activation was carried out by cycling from +0.6 to -0.2 V for 50 cycles at a scan rate of 100 mV/s. Upon glucose addition, there is an increase in the anodic peak current together with a slight decrease in the cathodic peak current. Note that the electrocatalytic function toward the oxidation of glucose can be observed only after the Ni-BPE is activated. Most importantly, an intriguing electrochemical behavior of PO43- at the activated Ni-BPE in the presence of glucose in alkaline media was indeed observed with a suppressed anodic peak after the addition of 3 mM PO43-. A systematic decrease in current signal of glucose was further observed upon the continuous addition of PO43-. To confirm that the suppressed current was related to the adsorption of PO43- at the activated Ni-BPE, electron spectroscopy of chemical analysis (ESCA) was employed to examine the change on the surface of Ni-BPE. As also can be seen in

Figure 1. Typical cyclic voltammetric responses for the activated Ni-BPE in (a) 0.1 M NaOH and (b) 0.1 M NaOH in the presence of 1 mM glucose and (c) 0.1 M NaOH in the presence of 1 mM glucose and 3 mM PO43- at a scan rate of 50 mV/s. Corresponding ESCA of the activated Ni-BPE surface in the presence of 1 mM glucose before/ after the adsorption of 3 mM PO43-.

Figure 1, only in the presence of PO43-, a small peak with a binding energy of 133.4 eV indicating the existence of P(2p) on the surface can be observed. In other words, there is definitely an adsorption of PO43- at the activated Ni-BPE in the presence of glucose. To further clarify that the adsorption of PO43- was not a mixed effect with glucose, surface characterization of the activated Ni-BPE in the absence of glucose was then carried out by atomic force microscopy (AFM). Figure 2 compares the AFM images of the activated Ni-BPE surface before/after immersing in 10 mM PO43- for ∼20 min. Again, a change of the appearance at 2.5 × 2.5 µm2 area with a small amount of lump (∼20 nm height) was observed, presumably due to the adsorption of PO43- at the activated Ni-BPE. The change was clearly differentiated by the increase in surface contour level, as shown in Figure 2c, after the adsorption of PO43- on the surface. Note that this result was in accordance with the ESCA result that revealed a small ratio of P/O atom counts (1.74%) (Supporting Information Figure S-2). Analytical Performance. Subsequently, this characteristic of PO43- at the activated Ni-BPE was employed in designing a detection scheme for PO43- by FIA. By taking 0.1 M NaOH as the mobile phase, the suppressed current of electrocatalytic oxidation of glucose caused by adsorption of phosphate on the activated Ni-BPE was utilized to electroanalysis of phosphate based on the detection scheme as illustrated in Figure 3A. Figure 3B shows typical amperometric responses under conditions of Hf ) 300 µL/min and Eapp ) +0.55 V vs Ag/AgCl for the oxidation signal from 0.1 M NaOH carrier solution (a), the electrocatalytic current after adding 25 µM glucose (b), and the suppressed current with injection of 500 µM PO43- (c), respectively. More importantly, the detection potential of Eapp ) +0.55 V is quite consistent with the observed cyclic

Figure 2. AFM images of the activated Ni-BPE before (A) and after (B) immersion in 0.1 M NaOH in the presence of 10 mM PO43- at room temperature for 20 min. (C) The surface outlines of Ni-BPE before (dashed line) and after (solid line) PO43- adsorption.

voltammogram, and a systematic decrease in current signal of glucose was observed upon the increase in concentration of PO43-. Under the optimal conditions of Hf ) 300 µL/min and Eapp ) +0.55 V vs Ag/AgCl (Supporting Information Figure S-3), Figure 4A shows the obtained calibration plot with the optimized glucose concentration of 25 µM in carrier solution (Supporting Information Figures S-4 and S-5). A good linearity in the window of 40 µM to 1 mM with sensitivity and regression coefficient of 4.29 µA/µM and 0.997, respectively, was observed. It is interesting that the electrode can be effectively regenerated without extra treatment under the hydrodynamic condition. The procedure of regeneration was accomplished by applying +0.55 V (vs Ag/AgCl) for 60 s under continuous hydrodynamic flow of 0.1 M NaOH solution in the presence of 25 µM glucose. A simple regeneration procedure enhances the potential applicability of the sensor. Continuous hydrodynamic flow of carrier solution at regular PO43- injections was performed to verify the reproducibility of the present approach. For eight continuous injections of 40 µM PO43-, a relative standard deviation of 0.28% was obtained, indicating good reproducibility of the proposed Analytical Chemistry, Vol. 82, No. 3, February 1, 2010

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Figure 3. (A) Detection scheme of the proposed system. (B) FIA responses of the activated Ni-BPE in 0.1 M NaOH (a), in 0.1 M NaOH with 25 µM glucose (b), and sequential injection of 500 µM PO43- in 0.1 M NaOH with 25 µM glucose as carrier solution (c) at Eapp ) +0.55 V vs Ag/AgCl.

Figure 4. (A) Typical calibration curve of PO43- at Eapp ) +0.55 V (vs Ag/AgCl) and Hf ) 300 µL/min in 25 µM glucose with 0.1 M NaOH as carrier solution. (B) FIA responses of 40 µM PO43- for eight continuous injections.

method (Figure 4B). The detection limit (S/N ) 3) was calculated as 0.3 µM. Overall, the stability of the activated Ni-BPE in the carrier stream is sufficiently good to allow a correct performance of the amperometric sensor under hydrodynamic conditions. The most important aspect of this work is that we apply a simple detection scheme to achieve good selectivity. The effect of some coexisting anions on the response of phosphate was investigated by FIA. Figure 5 shows the interference effect of nitrate (NO3-), chloride (Cl-), sulfate (SO42-), acetate (CH3COO-), oxalate (C2O42-), carbonate (CO32-), chlorate 1160

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Figure 5. FIA responses of 500 µM PO43- and (a) NO3-, (b) SO42-, (c) C2O42-, (d) Cl-, (e) CH3COO-, (f) CO32-, and (g) all anions (including 5 µM ClO4-, CrO42-, and HAsO42-) wherein the interfering anion and phosphate are present simultaneously. FIA conditions: Eapp ) +0.55 V vs Ag/AgCl and Hf ) 300 µL/min, respectively, in 25 µM glucose with 0.1 M NaOH as carrier solution.

(ClO4-), chromate (CrO42-), and arsenate (HAsO42-) ions in comparison with the results of 500 µM PO43-. As can be seen, in contrast to the suppressed current observed in the presence of phosphate ion, no such an effect was observed in the presence of these anions at the proposed system. Note that such freedom can indeed extend to conditions wherein the interfering anion and phosphate are present simultaneously. These results clearly demonstrate the high selectivity toward

PO43-, which is important whether investigating environmental or biological samples. CONCLUSION The proposed system is a new electrochemical sensor that can detect PO43- with good sensitivity and selectivity. The ESCA analysis and AFM images confirm the selective adsorption of phosphate on the Ni(OH)2/NiO(OH) film in alkaline media. Upon the addition of PO43-, the suppressed current signal of glucose was observed due to the inhibitory action at the activated Ni-BPE. These results are significant and noteworthy for a variety of reasons. First, in this report, we have utilized the barrel plating technique for the fabrication of sensor. Barrel plated electrodes have attracted analytical chemists due to their easy and simple preparation procedures with the important advantages of low cost (thus disposable) and flexible design. A second important aspect of this work is that we apply a simple detection scheme to achieve good sensitivity and selectivity. A final significant aspect of this work is the good catalytic

response of the sensors without the use of expensive chemicals in the system (e.g., glucose and NaOH). The development of a selective phosphate sensing system by FIA offers important practical features not only in analytical characteristics but also in the instrumentation into the field and can readily be automated. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Science Council of Taiwan. This work is supported in part by the Ministry of Education, Taiwan, under the ATU plan. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 14, 2009.

November

5,

2009.

Accepted

AC9025253

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