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Letters to Analytical Chemistry Nanoarray Membrane Sensor Based on a Multilayer Design For Sensing of Water Pollutants Lin Zhuo, Yan Huang, Ming Soon Cheng, Hian Kee Lee, and Chee-Seng Toh* Department of Chemistry, Faculty of Science, 3 Science Drive 3, National University of Singapore, Singapore 117543 A ubiquitous electrochemical sensor which can detect pollutants in nonconducting aqueous solutions is prepared using a triple layer design, comprising a polyelectrolyte entrapped within micrometer-length nanochannels and sandwiched between two nanometer-thick electrode layers. Replacement of the polyelectrolyte with an enzyme-polyelectrolyte mixture within the nanochannels confers excellent biosensing characteristics. Its superior analytical performance of quantitating copper ions and formaldehyde at trace levels without additional sample treatment steps is demonstrated in freshwater samples derived from a local reservoir. Water pollution is one of the most challenging issues in recent times where one-fifth of world’s population has no access to clean water giving rise to million of deaths yearly, besides increasing ecological hazards and health impacts. Thus there is an urgent need to develop sensors for rapid, accurate, selective, and sensitive detection of a wide range of pollutants in waters with early warning capability and of low cost. Electrochemical methods which are of low cost and portable have reported superior performances for the detection of a wide range of pollutants.1-3 However, applications in freshwater analyses remain limited because a moderate level of electrolyte concentration is required for electrochemical methods. An electrochemical sensor that can operate in negligible micromolar ionic strength conditions is expected to be extremely useful and versatile with potential enhanced performance when miniaturized to the microscale such as for integration in microand nanofluidics systems.4 Multilayer electrochemical sensors utilizing an electrodeceramic-electrode design can operate in a nonconductive environment such as air.5 Because the sensor response relies on the mass transfer of specific analytes or their redox products through the ceramic matrix, a general electrochemical sensor that operates * To whom correspondence should be addressed. Phone: +65-65163887. Fax: +65-67791691. E-mail:
[email protected]. (1) Daniele, S.; Bragato, C.; Baldo, M. A.; Wang, J.; Lu, J. M. Analyst 2000, 125, 731–735. (2) Trouwborst, R. E.; Clement, B. G.; Tebo, B. M.; Glazer, B. T.; Luther, G. W. Science 2006, 313, 1955–1957. (3) Brendel, P. J.; Luther, G. W. Environ. Sci. Technol. 1995, 29, 751–761. (4) Bianchi, F.; Chevolot, Y.; Mathieu, H. J.; Girault, H. H. Anal. Chem. 2001, 73, 3845–3853. (5) Knake, R.; Jacquinot, P.; Hodgson, A. W. E.; Hauser, P. C. Anal. Chim. Acta 2005, 549, 1–9. 10.1021/ac100776p 2010 American Chemical Society Published on Web 05/12/2010
Scheme 1. Triple Layer Design of the NENE Sensor and the Scanning Electron Micrograph Showing Nafion Deposits within the 200 nm Wide Membrane Nanochannelsa
a Enlarged drawing shows the Nafion polyelectrolyte structure and associated mobile cations within a nanochannel.
in nonconductive environments remains a challenge. Conversely, a triple layer electrochemical sensor using a general ion-conducting polyelectrolyte layer with mobile aqueous ions would be expected to partly overcome obstacles of ubiquitous use, since ionic conduction of current can occur between the two electrodes even in low-conductivity bulk solutions such as nonconductive ultrapure water or freshwater samples. This follows the design of molecular devices which utilize general electronic-conducting materials between two electrode layers and find widespread useful electronic applications.6 Herein, our approach entraps the perfluorinated ionmer Nafion within the nanochannels of a 60 µm thick nanoporous alumina membrane, which connects two 50 nm thick porous electrode layers coated on both sides of the membrane (Scheme 1). Overall, the new construct operates like a conventional two-electrode electrochemical sensor but uses a multilayer pseudo solid-state design. Nanostructuring of sensors have tremendously improved their performances because of increased signal-to-noise ratio and sensitivity owing to fast mass transport of analytes.7,8 The nanoporous membrane electrodes with ∼15-150 nm pore sizes (6) McCreery, R. L.; Bergren, A. J. Adv. Mater. 2009, 21, 4303–4322. (7) Godino, N.; Borrise, X.; Munoz, F. X.; Del Campo, F. J.; Compton, R. G. J. Phys. Chem. C 2009, 113, 11119–11125.
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(after sputter-coating) used in this work showed peak-shaped and plateau-shaped voltammograms, respectively, which can be explained by changes in diffusion profiles.7,8 To further improve sensing performance, the sputtering condition was optimized to give coatings which comprise nanoparticles with ∼20-50 nm diameters to significantly increase electrode surface area. As such, the sensor current responses are in range of tens of microamps to milliamps, exceedingly larger than those of single microelectrodes (typically picoamps to nanoamps). Thus the sensor is compatible with low-cost potentiostats and potentially useful in rugged field measurement conditions. Herein, we integrate a nanoarray with a multilayer design to construct a powerful and novel sensor with broad-based electrochemical sensing capability in nonconductive ultrapure water. Trace detection of two major pollutants, formaldehyde and copper ions, are demonstrated by using amperometric biosensing and anodic stripping voltammetry methods. EXPERIMENTAL SECTION The NENE (nanoarray electrode-Nafion-electrode) sensor was fabricated by sputter-coating of ∼50 nm platinum metal layers on both sides of a nanoporous alumina membrane (Whatman) with a nominal pore size of 20-200 nm, as previously reported.9 A volume of 15 µL of Nafion solution was applied onto one side of the porous metal-coated membrane and allowed to penetrate into the membrane nanochannels. Subsequent potential cycling of the device was carried out in 1 mM ferrocenemethanol, 10× PBS buffer (1.37 M NaCl, 27 mM KCl, 0.1 M phosphate buffer, pH 6.86) for 20 cycles from -0.6 to 0.6 V at 50 mV s-1. This necessary step improved the electroactivity of the NENE sensor in 18 MΩ cm water significantly, owing to exchange of the polyelectrolyte protons with Na+ and K+ cations10 with an increase in conductivity from 2 to 4 mS cm-1 (conductivity meter Philips PW9526). The NENE biosensor was fabricated by applying 10 µL of 15 U mL-1 alcohol oxidase enzyme solution (Hansenula sp., Sigma-Aldrich) onto one side of the NENE sensor surface, so the enzyme solution infiltrated into the Nafion-filled nanochannels and then left to dry over 10 min. See the Supporting Information for reagents, equipment, and other procedures. RESULTS AND DISCUSSION The Nafion-impregnated NENE sensor shows a large signal response toward redox probe ferrocenemethanol in 18 MΩ cm water in contrast to a control sensor without Nafion (Figure 1A). This remarkably high electroactivity of the NENE sensor in ultrapure water is comparable to similar sensors with or without Nafion in conductive 10× PBS buffer (Figure 1A). Further evaluation of the NENE sensor in ultrapure water using voltammetry and electrochemical impedance techniques revealed similar behaviors to conventional electrodes placed in conductive electrolyte solutions (Figures SI1 and SI2 in the (8) Dai, X.; Wildgoose, G. G.; Salter, C.; Crossley, A.; Compton, R. G. Anal. Chem. 2006, 78, 6102–6108. (9) Cheow, P. S.; Zhi, E.; Ting, C.; Tan, M. Q.; Toh, C. S. Electrochim. Acta 2008, 53, 4669–4673. (10) Yeager, H. L.; Eisenberg, A. In Perfluorinated Ionomer Membranes; Yeager, H. L., Eisenberg, A., Eds.; American Chemical Society: Washington, DC, 1982; p 3.
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Figure 1. (A) Cyclic voltammograms of NENE sensors in 10× PBS, pH 6.86 (- - -), ultrapure water (- · -), and controls without Nafion in 10× PBS, pH 6.86 (---), and ultrapure water (s). Conditions: 1 mM ferrocenemethanol; scan rate ) 50 mV s-1; electrode pore size ∼15 nm. (B) Chronoamperometric response of NENE sensor during successive additions of H2O2 in ultrapure water. Potential applied between electrodes ) 0.6 V, with solution stirring. Inset: linear calibration plot of NENE sensor for H2O2 detection.
Supporting Information). Its electrochemical behavior can be explained using an equivalent circuit model of a simple two electrode system (Figure SI3 and Table 1 in the Supporting Information). On the basis of the significantly high signal response of the NENE sensor toward a redox probe in ultrapure water which cannot be achieved using conventional electrodes, it is worthwhile to investigate if the NENE sensor can detect water pollutants. Hydrogen peroxide is the product of several hundreds of oxidoreductase enzymes used widely in many biosensor-based analytical applications and are also useful stress indicators of living cells.11,12 Figure 1B shows the chronoamperometric response of the sensor toward successive additions of hydrogen peroxide in ultrapure water with a linear range from 3 to 50 µM (R2 ) 0.998) and detection limit of 1.2 µM. Because of the high sensitivity response of the NENE sensor toward hydrogen peroxide, we incorporated alcohol oxidase enzyme to fabricate a NENE biosensor to detect formaldehyde, one of the most important commercial chemicals, according to the following reaction: HCHO + H2O + O2 f HCOOH + H2O2
(1)
Formaldehyde is toxic, mutagenic, and known to be released by ozonation and chlorination of some natural organic matter (11) Amatore, C.; Arbault, S.; Koh, A. C. W. Anal. Chem. 2010, 82, 1411–1419. (12) Ferreira, D. C. M.; Tapsoba, I.; Arbault, S.; Bouret, Y.; Moreira, M. S. A.; Pinto, A. V.; Goulart, M. O. F.; Amatore, C. ChemBioChem 2009, 10, 528– 538.
Figure 2. Chronoamperometric response of a NENE biosensor during successive additions of formaldehyde in ultrapure water. Potential applied between electrodes ) 0.3 V with solution stirring; electrode pore size ∼15 nm. Inset: linear calibration plot for formaldehyde detection.
during recycling of wastewater13 and thus necessitates the development of simple, sensitive, selective, and rapid formaldehyde detection methods in potable water sources. Figure 2 shows the chronoamperometric response of the NENE biosensor upon successive additions of formaldehyde in nonconducting ultrapure water held at a constant potential difference of 0.3 V between two sides of the membrane. The response toward formaldehyde gave a rapid response time of 3-30 s and an extremely wide linear range from 1 µM to 16 mM (R2 ) 0.99) with an excellent detection limit of 0.5 µM (∼15 ppb). These figures of merit obtained in nonconducting water outperforms solid-state gas sensor14 and is comparable to the best performing formaldehyde biosensors operating in aqueous electrolyte media.15 Nonlinear curve fitting of the calibration curve gave apparent Michaelis Km(formaldehyde) value of 246 (±9) mM, similar to the reported value (300 mM) of free enzyme and thus indicates an enzyme compatible environment within the Nafion-filled nanochannels. At low analyte concentrations, the correlation between current signal and formaldehyde concentration is somewhat linear (R2 ) 0.99) and could be used for analytical measurement purposes. Nonhomogeneous coating of enzyme within the Nafion infiltrated nanochannels as well as remnant surface adsorbed enzyme on the electrode layer surface likely contribute toward a small rise in currents during the chronoamperometric plateau current responses in Figure 2. To evaluate the general utility of the NENE sensor in metal ion sensing, one of the most common trace heavy metals was chosen as our target analyte, the copper ion. Copper is present in amounts varying from micrograms per liter to low milligrams per liter in most domestic water supplies due to the prevailing use of copper piping in household plumbing.13 These levels in aquatic environment can cause acute deleterious effects on aquatic life, including oxidative stress, lipid peroxidation, and DNA damage. Thus, there is considerable interest in accurate deter-
mination of copper concentrations in aquatic environment. Herein, we applied -1.5 V reduction potential, followed by a differential pulse voltammetric sweep from -1.5 to 1.0 V. Figure 3A shows the anodic stripping curve of an ultrapure water solution containing 40 µM Cu2+ in the presence of 60 µM Pb2+ and indicates excellent resolution of the two metal ions. The calibration plot obtained under optimal deposition conditions gave two linear ranges, from 1.3-35.2 µM (R2 ) 0.998) and 35.2-98.0 µM (R2 ) 0.992), respectively (Figure 3B). This is likely due to the partial removal of Cu2+ through an ion-exchange process in the Nafion layer of the sensor causing the gentle slope response, followed by a steeper sensitive slope at high Cu2+ concentrations when the limited amount of Nafion entrapped in the nanochannels becomes saturated with Cu2+ counterions. The detection limit of 0.4 µM outperforms some high-resolution methods in aqueous electrolyte solutions.16-18 These remarkable results achieved in ultrapure water conditions have not been previously reported. To demonstrate useful applications of the NENE sensor for the analyses of formaldehyde and Cu2+ in freshwater samples, separate analyses were performed on untreated reservoir water samples collected in clean sterile containers and subsequently spiked with 1.0 µM formaldehyde or 4.0 µM Cu2+. Cu2+ was determined using the standard addition method, while form-
(13) WHO. In Guidelines for Drinking-Water Quality, 3rd ed.; World Health Organization: Geneva, Switzerland, 2004; Vol. 1, p 335. (14) Gou, X. L.; Wang, G. X.; Kong, X. Y.; Wexler, D.; Horvat, J.; Yang, J.; Park, J. Chem.sEur. J. 2008, 14, 5996–6002. (15) Ben Ali, M.; Korpan, Y.; Gonchar, M.; El’skaya, A.; Maaref, M. A.; JaffrezicRenault, N.; Martelet, C. Biosens. Bioelectron. 2006, 22, 575–581.
(16) Orozco, J.; Fernandez-Sanchez, C.; Jimenez-Jorquera, C. Environ. Sci. Technol. 2008, 42, 4877–4882. (17) Wang, S. P.; Forzani, E. S.; Tao, N. J. Anal. Chem. 2007, 79, 4427–4432. (18) Loe-Mie, F.; Marchand, G.; Berthier, J.; Sarrut, N.; Pucheault, M.; BlanchardDesce, M.; Vinet, F.; Vaultier, M. Angew. Chem., Int. Ed. 2010, 49, 422– 425.
Figure 3. (A) Differential pulse anodic stripping voltammetric response of a NENE sensor with an electrode pore size ∼150 nm in ultrapure water containing 40 µM Cu2+ and 60 µM Pb2+ after 15 s deposition time at -1.5 V. (B) Calibration plot of anodic differential peak current response of the sensor versus Cu2+ concentration that shows two linear ranges.
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aldehyde was analyzed by comparing the biosensor amperometric signal responses to the standard calibration curve obtained under ultrapure water conditions. Real sample analyses of formaldehyde and Cu2+ in the reservoir water samples gave excellent correlation of spiked concentrations and experimentally determined values at 90% and 80% confidence levels, respectively. In conclusion, we describe the first report of a Nafionimpregnated nanoarray sensor using a multilayer design which demonstrates its outstanding general electrochemical sensing utility in ultrapure water. Incorporation of alcohol oxidase enzyme within the sensor achieves sensitive detection of formaldehyde and demonstrates specific detection of a wide range of analytes that can be created using a biosensing approach. Its superior analytical performance of quantitating copper ions and formaldehyde at trace levels without additional sample treatment steps is demonstrated in freshwater samples derived from a local reservoir. Furthermore, the high current outputs in range of microamps to milliamps even at low analyte concentrations suggest potentially useful field applications using low-cost potentiostats and setups,
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unlike measurements relying on single microelectrodes. Because of the potential ease of miniaturizing this simple two-electrode multilayer design, microsized NENE sensors could be developed for powerful hyphenated methods in new environmentally sustainable monitoring applications, such as in situ and on-site analyses of water derived from freshwater sources. ACKNOWLEDGMENT L.Z. and Y.H. contributed equally to this work. We gratefully acknowledge financial support from URC MOE Tier 1 Research Fund (Grant R143-000-382-112), NUS, and the Environmental & Water Industry (EWI) for graduate research scholarships. 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 March 26, 2010. Accepted May 7, 2010. AC100776P
