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Anal. Chem. 2010, 82, 1720–1726

Simultaneous Detection of Two Analytes Using a Spectroelectrochemical Sensor Sara E. Andria, Carl J. Seliskar, and William R. Heineman* Department of Chemistry, University of Cincinnati, 301 Clifton Court, Cincinnati, Ohio 45221-0172 Spectroelectrochemical sensors developed in our group achieve three modes of selectivity by combining electrochemistry, spectroscopy, and a chemically selective membrane in a single device. Analyte detection is based upon a change in the optical response due to the conversion of the analyte between two oxidation states that results from the cycling or stepping of the applied potential. We have demonstrated a novel approach to simultaneously detect two metals by combining optical stripping voltammetry for one metal (Pb2+) and the in situ ligand complexation in a film for the other metal (Fe2+). Using an indium tin oxide (ITO) sensor platform with a 50 nm Nafion film to preconcentrate the analytes, equimolar mixtures of Pb2+ and Fe2+ in 0.1 M sodium acetate buffer (pH 5) were detected. Pb2+ was detected by optical stripping voltammetry, in which lead was deposited as metal on the ITO and detected by the optical change as it was removed by stripping. The ferrous ion was detected by the in situ ligand complexation method in which Fe2+ was complexed with 2,2′-bipyridyl in the Nafion in the film to form an intense red complex that was detected by absorbance at 520 nm. Detection limits of 300 and 400 nM were obtained for Pb2+ and Fe2+, respectively. The presence of the film had no effect on the optical signal that results from the deposition and stripping of the Pb2+. In addition, competition between the Pb2+ and Fe2+ for sites in the film and for the organic ligand was investigated. Spectroelectrochemical techniques have demonstrated the capability of detecting various analytes in aqueous solutions. Their potential utility was examined early on by the use of a grazing angle optical configuration.1,2 In one study, the absorbance of electrochemically generated metal-hydroxo complexes was monitored and used to quantitate the free metal ions of interest.3 The same optical configuration also was used to demonstrate the detection of electroactive organic compounds.4 Furthermore, in another study, gold coated piezoelectric quartz crystal was used as the working electrode in order to study the electrochemical processes.5 * To whom correspondence should be addressed. (1) Tyson, J. F. Talanta 1985, 33, 51–54. (2) Xie, Y.; Dong, S. J. Electroanal. Chem. 1990, 294, 21–32. (3) Tyson, J. F.; West, T. S. Talanta 1978, 26. (4) Tyson, J. F.; West, T. S. Talanta 1979, 27, 335–342. (5) Xie, Q.; Shen, D.; Nie, L.; Yao, S. Electrochimica Acta 1993, 38, 2277– 2280.

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The spectroelectrochemical sensors developed in our group are unique in that three modes of selectivity are achieved by combining electrochemistry, spectroscopy, and selective partitioning into a single device.6-13 These modes are integrated at an optically transparent electrode (OTE), which simultaneously serves as the working electrode, optical waveguide, and the support for the selective film. In order for an analyte to be detected, (1) it must partition into the film, (2) it must be electrochemically active in the selected potential window, and (3) either the analyte or its electrolysis product must absorb or emit light at the selected wavelength. Analyte detection is based upon a change in optical response due to the conversion of the analyte between two oxidation states that results from the cycling of the applied potential. In essence, electrochemistry is used to select a particular optical signal to monitor. Typically, free metal ions alone do not possess the desired optical and/or electrochemical properties needed for this type of detection. Our group has explored several strategies to render these analytes detectable by the spectroelectrochemical sensor. In one example, the detection of weakly absorbing Fe2+ was achieved by loading the organic ligand 2,2′-bipyridyl into the preconcentrating film prior to analysis.9,14,15 The free metal ion partitions into the film where it binds with the ligand in situ to form Fe(bpy)32+, which has a molar absorptivity 1000-fold greater than Fe2+ alone. In addition, Fe(bpy)32+ has reversible electrochemistry and is colorless in its oxidized state, qualities that are ideal for this type of detection. In another example, the spectroelectrochemical sensor in conjunction with anodic stripping voltammetry (ASV) was used for the detection of analytes that do not meet the three requirements described in (6) Shi, Y.; Slaterbeck, A. F.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 3679–3686. (7) Shi, Y.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 4819– 4827. (8) DiVirgilio-Thomas, J. M.; Heineman, W. R.; Seliskar, C. J. Anal. Chem. 2000, 72, 3461–3467. (9) Richardson, J. N.; Dyer, A. L.; Stegemiller, M. L.; Zudans, I.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2002, 74, 3330–3335. (10) Stegemiller, M. L.; Heineman, W. R.; Seliskar, C. J.; Bryan, S. A.; Hubler, T.; Sell, R. L. Environ. Sci. Technol. 2003, 37, 123–130. (11) Kaval, N.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2003, 75, 6334– 6340. (12) Andria, S. E.; Richardson, J. N.; Kaval, N.; Zudans, I.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2004, 76, 3139–3144. (13) Monk, D. J.; Ridgway, T. H.; Heineman, W. R.; Seliskar, C. J. Electroanalysis 2003, 15, 1198–1203. (14) Wansapura, C. M.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2007, 79, 5594–5600. (15) Pantelic, N.; Wansapura, C. M.; Heineman, W. R.; Seliskar, C. J. J. Phys. Chem. B 2005, 109, 13971–13979. 10.1021/ac902243u  2010 American Chemical Society Published on Web 02/09/2010

the previous paragraph.16-18 For this approach, a selective film is not necessary for detection. Instead, the nonabsorbing, free metal ion is deposited as absorbing metal atoms directly on the electrode surface and then are subsequently stripped, which results in the desired change in optical response. This approach has been demonstrated using Pb2+, Hg2+, Cd2+, and Cu2+. As a next step, we have explored the possibility of detecting multiple analytes using the spectroelectrochemical sensor. Detecting multiple analytes simultaneously offers simplicity as fewer steps and sensors are required for the intended application. However, the challenge in developing such a method is (1) finding a set of conditions under which all target analytes can be detected and (2) producing distinguishable signals for each analyte. Very few examples of multiple analyte detection using spectroelectrochemistry have been reported. One such sensor was demonstrated for the simultaneous detection of Fe2+ and H+ by immobilizing Ferrozine, an iron indicator, and HPTS (8-hydroxyl-1,3,6pyrenesulfonic acid, trisodium salt), a pH indicator, in adjacent sections of the film.19 In another study, iron and copper were deposited onto a bare glassy carbon working electrode as a preconcentration step and then allowed to complex with 2,2′bipyridine after the stripping step, which produced an optical response that was monitored using the grazing angle optical configuration.20 Here, we report a novel method for the simultaneous detection of two analytes by combining the spectroelectrochemical sensing methods that employ in situ ligand complexation and ASV into a single experiment. This two step approach recently was reported in conjunction with a fiber optic spectroelectrochemical sensor. The detection of aqueous copper was previously demonstrated using ASV as a preconcentration step and complexation of the copper with a ligand as the detection step.21 However, we have employed these two steps as separate methods of detection for two separate analytes. Potential complications of this method, such as ASV at a film-coated electrode and competition for the complexing ligand and ion-exchange sites in the film, were also investigated. EXPERIMENTAL SECTION Chemicals and Materials. The following reagents were used without further purification: lead nitrate (Fisher), ferrous ammonium sulfate (Fe(NH4)2(SO4)2 · 6H2O, Mallinckrodt), 2,2′bipyridyl (bpy, Aldrich), sodium acetate (Fisher), acetic acid (Fisher), and Nafion (5% solution in lower aliphatic alcohols and water, Aldrich). All solutions were prepared using deionized water (D3798 Nanopure water purification system, Barnstead, Boston, MA) in a 0.1 M sodium acetate buffer, pH 5. Indium tin oxide (ITO) coated glass slides (Corning 1737F glass, 11-50 Ω/sq, 135 nm thick film on 1.1 mm glass, Thin Film Devices) with the dimensions 10 mm × 40 mm were used as optically transparent electrodes (OTEs). (16) Shtoyko, T.; Maghasi, A. T.; Richardson, J. N.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2003, 75, 4585–4590. (17) Maghasi, A. T.; Conklin, S.; Shtoyko, T.; Piruska, A.; Richardson, J. N.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2004, 76, 1458–1465. (18) Shtoyko, T.; Conklin, S.; Maghasi, A. T.; Richardson, J. N.; Piruska, A.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2004, 76, 1466–1473. (19) Shi, Y.; Seliskar, C. J.; Heineman, W. R. Talanta 1998, 47, 1071–1076. (20) Xie, Q. J.; Nie, L. H.; Yao, S. Z. Anal. Sci. 1997, 13, 453–456. (21) Flowers, P. A.; Arnett, K. A. Spectrosc. Lett. 2007, 40, 501–511.

Preparation of Nafion Films on ITO Slides. The ITOs were first scrubbed with Neutrad glass cleaner (Decon Laboratories, Inc.) and rinsed thoroughly with water. The cleaned slides were rinsed with ethanol and then deionized water and allowed to dry prior to use. The Nafion films were prepared using a Model 1-PM1-1DT-R485 spin coater from Headway Research, Inc. The film thickness used throughout the study was 50 nm. To achieve this thickness, the 5% Nafion solution was diluted to 2% using 2-propanol. A 100 µL aliquot of the 2% Nafion solution was transferred onto the slide and spun at 6000 rpm for 30 s. For spectroelectrochemical experiments, a 1 cm portion of each end of the ITO was masked with tape before spin-coating. For electrochemical experiments, only one end of the ITO was masked with tape before spin-coating, allowing an area of 1.9 cm2 to be covered with film. These areas free of film were then used for electrical contacts. Instrumentation. Normal incidence transmission measurements were made using a UV-vis spectrophotometer (Cary-50). All electrochemical measurements were acquired using a potentiostat (Epsilon, BAS). Some of the electrochemical measurements were done under quiescent conditions. For these measurements, the cell configuration included the bare or Nafion-modifed ITO working electrode, a Ag/AgCl reference electrode (BAS), and a Pt mesh auxiliary electrode, which were assembled in a vial (V ) 14.8 mL). Spectroelectrochemical Measurements. A detailed description of the experimental setup has been described in a previous publication.11 Briefly, incident radiation was directed through a multimode optical fiber (Romack, 400 mm core step index, NA ) 0.22) to a collimating objective (Newport, M-10×, NA ) 0.25). A xenon arc lamp (model ILC302UV, ILC Technology, Inc.) was used as the light source. The collimated light was then passed in and out of the ATR cell by way of two Schott SF6 coupling prisms (Karl Lambrecht). The prisms were attached to the rear of the slide by a high refractive index mounting compound (Cargille Meltmount, n ) 1.704). The attenuated light was then focused by a microscope objective lens (Newport, 5×, NA ) 0.12) into another optical fiber, which directed the light into a monochromator (SpectraPro 300i, Acton Research Corp., 0.3 m focal length) outfitted with a photon counting PMT. Data collection was achieved using Acton Research NCL monochromator controller electronics and Spectrasense software. The flow cell used for the ATR measurements was slightly different from that previously described. The body of the flow cell was made of black Delrin and had a volume of ∼0.4 mL. A platinum wire was affixed inside the cell, which served as the auxiliary electrode. In addition, a miniature Ag/AgCl reference electrode (3 M KCl, Cypress) was used. The solutions were pumped through the sensor cell at 0.2 mL/min using a syringe pump (model NE-1000, New Era Pump Systems, Inc.). Stripping and cyclic voltammograms were acquired simultaneously with the optical measurements. All data analysis and manipulations were carried out using commercial spreadsheet and graphics algorithms. RESULTS AND DISCUSSION Sensor Concept. The novel sensing method presented in this work allows for the detection of two analytes in a single analysis. To achieve this, two previously established approaches to spectroelectrochemical sensing have been combined into a single Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

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Figure 1. Illustration of sensor concept. The sensing platform consists of an ITO-OTE coated with a 50 nm thick Nafion film, which contacts the sample solution. (A) The solution of Fe2+ and Pb2+ is exposed to the sensor as the applied potential is held at -900 mV. Fe2+ complexes with the bpy preloaded in the film and Pb2+ diffuses through the film to the ITO surface where it is deposited as Pb0. (B) The potential is scanned from -900 to 0 mV at 50 mV/s, resulting in the oxidation of the deposited Pb0 to Pb2+. (C) The Fe(bpy)32+ is cycled between its colored and colorless form by scanning the potential between 500 and 1300 mV at 5 mV/s. (D) Absorbance versus time plot showing the optical responses that result from the analysis of the mixture.

experiment: (1) in situ complexation of a free metal ion with an organic ligand to form an “ideal” complex, and (2) spectroelectrochemical sensing at a bare OTE in conjunction with ASV. The analytes chosen to demonstrate the sensor concept are Fe2+ and Pb2+ because both have been used in previous studies characterizing the spectroelectrochemical sensor, and their behavior is well-known.9,14,16,17 Figure 1 is a schematic that illustrates the concept on which this sensing method is based. The sensing platform is an OTE, which consists of a glass substrate coated with ITO. A 50 nm thick Nafion film was deposited onto the OTE surface by spin-coating and was preloaded with 2,2′-bipyridyl (bpy) by soaking in the ligand solution overnight. The optical response of the analytes in the film was measured using attenuated total reflectance (ATR) spectroscopy. Figure 1A shows the first step in analyte detection. The analyte mixture was exposed to the sensor while simultaneously applying a potential of -900 mV. Through ion exchange, the metal cations partitioned into the film. Because the bpy was loaded into the film prior to analysis, the Fe2+ complexed with the ligand in the film to form Fe(bpy)32+. Additionally, the Pb2+ diffused through the film toward the electrode surface where it was reduced and deposited onto the electrode surface as Pb0. 1722

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Both of these processes (i.e., formation of Fe(bpy)32+ and deposition of Pb0) absorbed the incident light. The Fe(bpy)32+ absorbs strongly at 520 nm (εmol ) 7700 M-1 cm-1).22 The wavelength of maximum absorbance for Pb0 is 750 nm;16 however, the deposited metal absorbs significantly in the rest of the visible region as well. For this reason, we chose to monitor the sensor absorbance response for both analytes at 520 nm. The optical response that results from the first step of analyte detection was the steady increase in the absorbance marked as A in Figure 1D. Step two of analyte detection involved quantification of the Pb2+ in the analyte mixture and is illustrated in Figure 1B. After allowing 30 min for the deposition of Pb2+, the applied potential was scanned from -900 mV to 0 mV at 50 mV/s. This caused the oxidation of Pb0 to Pb2+ (the “stripping” step) and resulted in an abrupt decrease in the absorbance marked as B in Figure 1D. The change in absorbance (∆A) in this step was the analytical signal used to determine the concentration of the Pb2+ in the sample. (22) Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis, 5th ed.; Saunders: Philadelphia, 1998.

The final step in analyte detection involved quantification of the Fe2+. The absorbance response for this 10 min time period remained constant, indicating that the Fe(bpy)32+ had equilibrated in the Nafion film. The sensor sat at open circuit for 10 min while the analyte mixture continued to flow through the cell to allow for adequate separation between the Pb2+ signal and the ensuing Fe2+ signal. The potential was then cycled between 500 and 1300 mV at 5 mV/s for 10 segments. The first segment (500 to 1300 mV) oxidized Fe(bpy)32+ to Fe(bpy)33+, which does not absorb in the visible region. The absorbance in turn decreased due to the conversion of the complex to its colorless form. The second segment (1300 to 500 mV) reduced Fe(bpy)33+ back to Fe(bpy)32+ (the colored form) and returned the absorbance close to its original magnitude. The iron complex was continually cycled between its two oxidation states (Figure 1C) for the remainder of the segments. The resulting optical response was a modulation of the absorbance signal, which is marked as C in Figure 1D. The average ∆A of the absorbance modulation was the signal used to determine the concentration of the Fe2+ in the analyte mixture. Sensor Response of Pb2+ at a Nafion-Modified ITO. One concern in combining these two sensing methods in a single device was the effect, if any, the presence of the film would have on the deposition and stripping of the Pb2+. This was investigated by comparing the spectroelectrochemical responses of the deposition and stripping of Pb2+ at a bare ITO to that at a Nafion-modified ITO. The procedure for acquiring the spectroelectrochemical responses of the deposition/stripping of Pb2+ at both the bare and Nafion-modified electrodes was essentially the same: The optical response of only the 0.1 M sodium acetate (NaAc) supporting electrolyte was monitored for 250 s to obtain a baseline. The Pb2+ solution was introduced into the flow cell and allowed to deposit for 20 min before being stripped. New Pb2+ solution was continuously pumped through the flow cell during the entire experiment. For the bare ITO experiments, the same electrode was used for all trials. Between each trial, the NaAc was pumped through the flow cell for 10 min to rinse the Pb2+ solution from the cell. For the Nafion-modified ITO experiments, a new film was used for each trial. The NaAc was pumped for 10 min prior to the beginning of the experiment. Three trials were completed for each study. Figure 2 shows the comparison of the deposition/stripping of Pb2+ at the bare (A) and Nafion-modified (B) electrodes. The sensor response seen with the Nafion-modified ITO was similar to that of the bare ITO, namely, a steady increase in the absorbance due to the deposition of the Pb0 onto the electrode surface and a subsequent drop in absorbance associated with stripping the Pb0 from the electrode surface. The ∆A values for each study were calculated and averaged. Although slightly more variability was observed with the Nafion-modified electrodes, the average ∆A values for the Pb2+ at both the bare and Nafion-modified electrodes were found to be 0.04. For comparison, the electrochemical responses of the stripping step were acquired simultaneously with the absorbance measurements. One representative voltammogram each for bare and Nafion-coated electrodes is displayed in Figure 2C. Even though the voltammograms do not overlay each other, the ip values are essentially the same when measured from their respective

Figure 2. Absorbance response of the deposition/stripping of Pb2+ at a bare (A) and Nafion-modified (B) ITO. The applied potential was held at -900 mV for 20 min, and then scanned from -900 to 0 mV at 50 mV/s. (C) One stripping voltammogram from each study that was acquired simultaneously with the optical measurements: bare ITO (---), Nafion-modified ITO (s).

baselines. These results indicate that the presence of the Nafion film on the electrode surface does not significantly interfere with the deposition/stripping of Pb2+. Competition for the 2,2′-Bipyridine Ligand. Both Fe2+ and 2+ Pb are capable of forming complexes with the bpy ligand. However, the equilibrium constant (reported as log K) for the formation of Fe(bpy)32+ is 17.2 (±0.2), which is significantly larger compared to the value of 2.9 for the formation of Pb(bpy)2+.23 This difference allows for the assumption that the Pb2+ ions in the film would not compete with the Fe2+ for the bpy ligand. This aspect was confirmed by comparing bulk solution absorbance spectra and electrochemical responses of the Fe(bpy)32+ when the Pb2+ concentration was varied. Absorbance spectra were acquired for solutions consisting of 0.1 mM Fe2+, 0.3 mM bpy, and Pb2+ concentrations ranging from 1 µM to 1 mM. The absorbance spectrum for the control, 0.1 mM Fe(bpy)32+, was also obtained. As expected, no difference was observed between the absorbance spectra of (23) Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenun Press: New York, 1974.

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Figure 3. Stripping voltammograms of Pb2+ (A) and cyclic voltammograms for Fe(bpy)32+ (B) recorded for the Nafion-modified ITO loaded with 10 µM Pb2+ on day 1 (---) and the 10 µM Fe(bpy)32+/ Pb2+ mixture on day 2 (s). Stripping voltammograms of Pb2+ (C) and cyclic voltammograms for Fe(bpy)32+ (D) recorded for the Nafion-modified ITO loaded with 10 µM Fe(bpy)32+ on day 1 (---) and the 10 µM Fe(bpy)32+/ Pb2+ mixture on day 2 (s). (A, C): The applied potential was held at -900 mV for 30 min before scanning from -900 to 0 mV at 50 mV/s. (B, D): The potential was scanned between 500 and 1300 mV at 5 mV/s. Each 50 nm thick Nafion film was loaded with the individual analyte or mixture by soaking overnight in the appropriate solution. The same film was used for both days. Measurements were made in the soaking solution under quiescent conditions.

the control and the Fe2+/Pb2+/bpy mixtures (data not shown).24 Cyclic voltammograms of the same mixtures also were acquired at a bare ITO (data not shown).24 The voltammograms observed for the mixtures having a Pb2+ concentration of less than 10 µM were essentially the same and were consistent with the control. The voltammograms for the mixtures having a concentration of 10 µM Pb2+ or greater were significantly larger than the Fe(bpy)32+ control and were severely distorted. This response was most likely due to an oxidative process involving the Pb2+ and not due to an interaction between the Pb2+ and the bpy. Pb2+ can oxidize to form PbO2, which would coat the ITO and thereby affect the voltammograms for Fe(bpy)32+. In summary, it is reasonable to conclude that Pb2+ and Fe2+ do not compete for the ligand in the Nafion film during the preconcentration step, especially since the Pb2+ is being continuously removed from the film by deposition on the underlying electrode. Competition for Ion-Exchange Sites. Although different cations in solution are able to exchange with the protons of the Nafion film, the Nafion’s selectivity for one cation over the others is a concern in that the presence of both Pb2+ and Fe(bpy)32+ in the film could affect preconcentration. Ion exchange selectivity (24) Andria, S. E. Ph.D. Dissertation, University of Cincinnati, Cincinnati, OH, July 2009.

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coefficients can be measured; however, they are typically reported for a species in the presence of only one competing ion.25-27 The mixtures in this study contain three competing cations (Fe2+, Pb2+, and Na+), making the determination of a selectivity coefficient more complicated. Instead of quantifying the selectivity of the film for each of the analytes, we compared the electrochemical responses of only one analyte in the film to that of the mixture in the film under steady-state conditions. This approach is more of an estimation of the ion-exchange competition, yet it still provides useful information regarding the practicality of this method. The general procedure was as follows: ITO electrodes coated with a 50 nm thick Nafion film were allowed to soak overnight in solutions containing either 10 µM Pb2+ or 10 µM Fe(bpy)32+ prepared in 0.1 M NaAc. On day 1, each Nafion-modified ITO loaded with only one analyte was assembled in a vial containing the same soaking solution. The electrochemical excitation potential sequence previously described for analyte detection was then implemented: The Pb2+ was allowed to deposit for 30 min, and then, it was stripped. The cell was held at open circuit for 10 min, after which the Fe(bpy)32+ was cycled between its two oxidation states. Voltammograms for both the Pb2+ stripping step and (25) Szentirmay, M. N.; Martin, C. R. Anal. Chem. 1984, 56, 1898–1902. (26) Ugo, P.; Moretto, L. M. Electroanalysis 1995, 7, 1105–1113. (27) Wang, J. Talanta 1994, 41, 857–863.

Fe(bpy)32+ electrolysis were acquired. The same Nafionmodified ITOs were then soaked overnight in the same concentration of the mixture (Pb2+ and Fe(bpy)32+). On day 2, the Nafion-modified ITOs were assembled in vials containing the same mixture soaking solution; the electrochemical excitation sequence was again performed. The effect of excess Fe(bpy)32+ on the determination of Pb2+ is shown in Figure 3A,B, which shows the results of the film that was soaked in Pb2+ on day 1 (dotted line) and then soaked in the mixture on day 2 (solid line). In Figure 3A, the voltammograms shown are the result of the oxidation of Pb (deposited onto the ITO) to Pb2+. If the Nafion were more selective for the Fe(bpy)32+, then it would be reasonable to expect a decrease in the peak current for the Pb oxidation after being exposed to the mixture overnight. However, this was not the case; the peak current for the stripping step actually increased by ∼150 µA when measured on day 2. Furthermore, the anodic peak shifted 27 mV more positive. Although only the Pb2+ electrochemical response was really of interest for this film, the cyclic voltammogram illustrating the electrolysis of Fe(bpy)32+ in the film is shown in Figure 3B. The small peaks observed for the voltammogram acquired on day 1 are due to oxidation of the Pb2+, which is consistent with the results from the bpy-competition study. The large current response corresponding to the electrolysis of Fe(bpy)32+ is only observed on day 2 after the film was exposed to the mixture. Figure 3C,D shows the results of the film that was soaked in Fe(bpy)32+ on day 1 (dotted line) and soaked in the mixture on day 2 (solid line). For this film, the response of the Fe(bpy)32+ is of more interest and is shown in Figure 3D. The cyclic voltammogram acquired on day 1 is nearly identical to that acquired on day 2. It is apparent from the voltammogram shown in Figure 3C that Pb2+ has loaded into the film. However, the partitioning of the Pb2+ has not caused any leaching of the Fe(bpy)32+. Dynamic Range and Detection Limits. The dynamic range of the sensor was determined by generating two calibration curves, one for each analyte. Solutions containing equal concentrations of Pb2+ and Fe2+ were analyzed using 50 nm thick Nafion films. The films were preloaded with bpy by soaking overnight in a solution of 5 mM bpy prepared in 0.1 M NaAc. The procedure used for each trial is as follows: The Nafion-modified ITO was assembled in the flow cell and a solution of 0.1 M NaAc was pumped for 3 min before beginning data acquisition. The optical response of the NaAc was monitored for 250 s to obtain a baseline. The analyte mixture was then introduced into the flow cell, and at the same time, the potential was applied for the Pb2+ deposition step. After 30 min, the potential was scanned positively to strip the deposited Pb. The cell remained at open circuit for 10 min before the potential was then cycled to modulate the Fe(bpy)32+. New analyte solution was continually pumped through the flow cell at 0.2 mL/min throughout the entire experiment. A new Nafion-modified ITO was used for each trial. The calibration curve compiled for Pb2+ is shown in Figure 4A. A linear relationship between ∆A and concentration was observed. Each error bar represents one standard deviation on either side of the mean of three trials. The detection limit for Pb2+

Figure 4. Calibration curves for the simultaneous detection of Pb2+ (A) and Fe2+ (B) using a 50 nm Nafion film.

was calculated to be 300 nM, which was determined using the slope of the curve and three times the standard deviation of the blank. In a previous, similar study, a spectroelectrochemical sensor using a bare ITO for the detection of Pb2+ exhibited a detection limit of 50 nM.16 However, it is important to note that in this previous report, a larger ITO slide was used (1 × 3 in.), and therefore, a longer path length resulted due to the ability to achieve more reflections. In addition, the λmax of Pb0 (750 nm) was used for the detection wavelength. In the present study, 520 nm was used as the detection wavelength, where ε is approximately 2 times less. The longer path length and larger ε are the main contributing factors for the lower detection limit achieved in the previous study. Figure 4B shows the calibration curve generated for Fe2+, which is nonlinear. The data point on the y-axis is the response observed from the blank. This response is attributed to iron impurities in the sodium acetate. The detection limit for Fe2+ was estimated using three times the standard deviation of the blank when the potential was not cycled and the slope of the first three points of the calibration curve (not including the data point from the blank); this value was determined to be ∼400 nM. In a previous, similar study, a spectroelectrochemical sensor for the detection of Fe2+ using Nafion films Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

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preloaded with bpy exhibited a detection limit of 600 nM.14 For the previous study, larger ITO and a much thicker film (∼280 nm) were used. Theoretically, for a purely optical sensor, a larger signal should result when a thicker film is used since it can accommodate more analyte. However, detection with the spectroelectrochemical sensor requires a change in the optical signal that results from the electrochemical step. The analyte must diffuse to the OTE surface for the oxidation or reduction that causes the optical change, which can take longer in thicker films and slows the response time. When comparing the %RSD (relative standard deviation) for each concentration of the calibration curves, repeatability for the detection of Pb2+ was comparable to that of the detection of Fe2+. Variability observed for the Fe2+ is most likely attributed to the film-to-film irreproducibility that contributes to the error using a different film for every experiment. Detection of Fe2+ relies on the complexation with the ligand, and the amount of ligand available depends on the volume of the film. The irreproducibility in casting films most likely has little effect on the Pb2+ deposition and stripping. However, the variability observed for the Pb2+ detection could be attributed to the use of a different ITO electrode for every experiment. CONCLUSION Proof of principle for the simultaneous detection of two metal ions by spectroelectrochemistry has been demonstrated. A unique feature is the use of two quite different strategies for each metal ion: stripping voltammetry to preconcentrate and provide the optical signal to detect one analyte and preconcentration in a film with in situ ligand exchange to provide the optical signal for detection for the other analyte. The film needed for the latter mode of detection only minimally interferes with the stripping voltam(28) Heineman, W. R.; Kuwana, T. Anal. Chem. 1972, 44, 1972–1978.

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metry mode, which does not need a film. This makes simultaneous detection of both cations at the same electrode feasible. The model analytes chosen for this demonstration have potential regions for detection that are well separated, which enables the optical changes associated with the two analytes to be easily distinguished, as shown in Figure 1. This strategy should work for other heavy metals such as Cd2+, Cu2+, and Zn2+ that can be electrochemically deposited on ITO and give an optical change when stripped. Likewise, the in situ ligand exchange should be effective with other metal ions that form strongly absorbing metal complexes. It is interesting to note that the limits of detection for the two analytes were in the same ball park, 300 and 400 nM, even though the modes of preconcentration and optical detection were different. The sensitivity of optical detection is directly dependent on molar absorptivity, ε. In this case, ε for Fe(bpy)32+ is 7700 M-1 cm-1 at 520 nm, and the effective ε for metallic Pb (dissolved in mercury) is 7700 M-1 cm-1 at 589 nm.28 Thus, all other things equal, a similar limit of detection for both analytes is not unexpected. Very importantly, the presence of the Nafion film on the OTE did not significantly interfere with the deposition and stripping of the Pb2+. Furthermore, under the conditions studied, there was no indication that a competition existed between the Fe2+ and Pb2+ for the bpy ligand or the ion-exchange sites in the film. ACKNOWLEDGMENT The authors gratefully acknowledge support for the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG0207ER64353. Received for review October 5, 2009. Accepted January 17, 2010. AC902243U