Fluorescence Spectroelectrochemical Sensor for 1-Hydroxypyrene

Anal. Chem. 2010, 82, 9743–9748

Fluorescence Spectroelectrochemical Sensor for 1-Hydroxypyrene Tatyana S. Pinyayev, Carl J. Seliskar, and William R. Heineman* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, United States A spectroelectrochemical sensor was demonstrated for an organic compound whose oxidation proceeds through an electron transfer-chemical reaction-electron transfer (ECE) mechanism to generate new chemical species that are used for detection by fluorescence. The polycyclic aromatic hydrocarbon 1-hydroxypyrene (1-PyOH) served as a representative model analyte. The spectroelectrochemical properties of 1-PyOH in solution were explored with an optically transparent thin layer electrode. Electrochemical oxidation of 1-PyOH under acidic conditions proceeds via the ECE mechanism to a diquinonepyrene, which shows reversible electrochemistry and fluoresces at 425 nm in its reduced form, dihydroxypyrene. The sensor consisted of a tin-doped indium optically transparent electrode coated with a Nafion thin-film (20 nm) that rapidly preconcentrated the analyte at the sensor surface. Fluorescence in the film was excited by the evanescent wave from attenuated total reflection spectroscopy. Electrochemical modulation of dihydroxypyrene fluorescence at 425 nm in the 500 to -200 mV (vs Ag/AgCl) potential range was used for indirect detection of 1-PyOH. The spectroelectrochemical sensor calibration curve had a range of 5 × 10-9 to 1 × 10-6 M with a calculated detection limit of 1 × 10-9 M. Spectroelectrochemistry offers a strategy for developing sensors with improved selectivity by electrochemically changing the analyte’s optical response to distinguish it from interferences that have overlapping optical spectra.1 We have demonstrated this concept mainly with charged metal complexes such as Fe(CN)64and Ru(bpy)62+ using both absorbance and fluorescence detection.2,3 One organic compound, ascorbate, was detected indirectly using Ru(bpy)62+ as a mediator.4 Here we expand the sensor concept to the detection of other organic compounds using the polycyclic aromatic hydrocarbon (PAH) 1-hydroxypyrene (1-PyOH) as a model compound. 1-PyOH is a particularly interesting example, because its oxidation proceeds through an electron transfer-chemical reaction-electron transfer (ECE) mechanism generating new chemical species, * To whom correspondence should be addressed. E-mail: [email protected]. (1) Andria, S. E.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2009, 81, 9599. (2) Kaval, N.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2003, 75, 6334. (3) Maizels, M.; Stegemiller, M. L.; Ross, S.; Slaterbeck, A.; Shi, Y.; Ridgway, T. H.; Heineman, W. H.; Seliskar, C. J.; Bryan, S. A. In Nuclear Site Remediation; American Chemical Society: Washington, DC, 2000; p 364. (4) DiVirgilio-Thomas, J. M.; Heineman, W. R.; Seliskar, C. J. Anal. Chem. 2000, 72, 3461. 10.1021/ac101883a  2010 American Chemical Society Published on Web 11/05/2010

which makes developing the sensor more complicated than for species with simple, reversible redox reactions. PAHs are ubiquitous environmental contaminants that come from a variety of industrial and household sources.5 1-PyOH was chosen for this study because it is widely used as a biomarker for human exposure to PAHs.6 1-PyOH is also an intermediate metabolite of pyrene in other eukaryotes7,8 and has been identified as one of the initial microbial pyrene oxidation products.9,10 1-PyOH and its glucuronide and sulfate conjugates are excreted by organisms in relative concentrations largely dependent on genetically determined enzymatic patterns for metabolic PAH conversion.11 The most widely used methods for 1-PyOH determination are liquid12 and gas13 chromatography that involve lengthy enzymatic hydrolysis to release the analyte from metabolic conjugates. In order to minimize the analysis time, several groups have developed methods for direct quantification of 1-PyOglucuronide using immunoaffinity liquid chromatography14 and HPLC-MS,15 which allow the sample pretreatment time to be shortened by several hours. Despite the effectiveness of chromatographic methods, they fail to satisfy the increasing demand for portable environmental detection instruments, particularly for groundwater monitoring and hazardous waste site assessment. Rapidly developing sensor technologies, on the other hand, offer on-site analytical measurements with shorter analysis time and improved cost effectiveness for environmental monitoring and remediation.16-18 (5) Harvey, R. G. Polycyclic Aromatic Hydrocarbons Chemistry and Carcinogenicity; Cambridge University Press: Cambridge, 1991. (6) Jongeneelen, F. J. Ann. Occup. Hyg. 2001, 45, 3. (7) Giessing, A.; Mayer, L.; Forbes, T. Environ. Toxicol. Chem. 2003, 1107. (8) Stroomberg, G. J.; Gestel, F.; Hattum, C. A.; Velthorst, B.; Straalen, N. H. Environ. Toxicol. Chem. 2003, 22, 224. (9) Chaudhry, G. R. In Biological Degradation and Bioremediation of Toxic Chemicals; Dioscorides Press: Portland, OR, 1994; p 92. (10) Lambert, M.; Kremer, S.; Sterner, O.; Anke, H. Appl. Environ. Microbiol. 1994, 3597. (11) Singh, R.; Tucek, M.; Maxa, K.; Jana, T.; Weyand, E. H. Carcinogenesis 1995, 16, 2909. (12) Jongeneelen, F. J.; Anzion, R. B.; Henderson, P. T. J. Chromatogr. 1987, 413, 227. (13) Smith, C. J.; Huang, W.; Walcott, C. J.; Turner, W.; Grainger, J.; Patterson, D. G. Anal. Bioanal. Chem. 2002, 372, 216. (14) Lai, C. H.; Liou, S. H.; Shih, T. S.; Tsai, P. J.; Chen, H. L.; Buckley, T. J.; Strickland, P. T.; Jaakkola, J. J. Arch. Environ. Health 2004, 59, 61. (15) Kakimoto, K.; Oriba, A. T.; Ohno, T.; Ueno, M.; Kameda, T.; Tang, N.; Hayakawa, K. J. Chromatogr. B 2008, 867, 259. (16) Alarie, J. P.; Vo-Dinh, T.; Miller, G.; Ericson, M. N.; Maddox, S. R.; Watts, W. Rev. Sci. Instrum. 1993, 64, 2541. (17) Campiglia, A.; Vo-Dinh, T. Talanta 1996, 43, 1805. (18) Vo-Dinh, T.; Tromberg, B.; Griffin, G.; Ambrose, K.; Sepaniak, M.; Gardenhire, E. M. Appl. Spectrosc. 1987, 41, 735.

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The spectroelectrochemical sensor19 takes advantage of three modes to provide the selectivity needed for accurate analyte determination in complex and harsh sample environments, such as nuclear waste.20 In order to be detected by a spectroelectrochemical sensor, the analyte must partition into a thin film, be electroactive, and absorb or emit light in its reduced or oxidized form. To date, we have studied both anion- and cation-exchange films for selective analyte preconcentration and have also demonstrated that a 1 × 10-10 M detection limit can be achieved using fluorescence as the mode of optical detection.2 In order to develop a spectroelectrochemical sensor for 1-PyOH, the analyte must fulfill all three requirements mentioned above. It is known that 1-PyOH both absorbs UV light and fluoresces21 and undergoes oxidation followed by chemical reactions to produce various electrochemically active pyrene quinones and dimers.22 The oxidation of 1-PyOH is reported to proceed via two consecutive one-electron oxidations. The first one-electron oxidation yields a neutral radical, which is liable to undergo dimerization to produce an electrochemically reversible pyrene quinone dimer.23 An alternative route for the neutral pyrenolate radical is to undergo the second one-electron oxidation to produce a pyrenolate cation that is susceptible to a nucleophilic attack by water to yield electrochemically reversible dihydroxypyrenes.22 Here we report the proof of concept of a spectroelectrochemical sensor for indirect detection of 1-PyOH that is based on the fluorescence of the product of the ECE mechanism for electrochemical oxidation of 1-PyOH. EXPERIMENTAL SECTION Reagents and Solutions. 1-Hydroxypyrene was purchased from Toronto Research Chemicals (98% pure) and used as received. Ethanolic 1-PyOH stock solution (1 × 10-2 M) was prepared by dissolving 1-PyOH in HPLC-UV-grade ethanol purchased from Pharmco (Brookfield, CT) and was kept in the dark until used. Sodium chloride (Fisher, Pittsburgh, PA) was used to prepare 1.0 M supporting electrolyte stock solution and then used in appropriate amounts to prepare supporting electrolyte solutions of different concentrations. Acidic buffer solution was prepared by dissolving an appropriate amount of potassium dihydrogen phosphate (Fisher) in deionized water (Barnstead water purification system) and titrated with concentrated phosphoric acid (Fisher) to pH 3.5. Working acidic 1-PyOH solutions contained 30% ethanol (v:v). Nafion (5% solution in lower aliphatic alcohols and water, obtained from Sigma-Aldrich, Milwaukee, WI) was diluted to 2% with aqueous isopropanol solution (20% water v:v) and used to prepare films as described previously.24 Equipment. Indium tin oxide (ITO) coated glass slides (Corning 1737F and 7059, 11-50 Ω/sq, 130 nm thick film on 1.1 (19) Yining Shi, S. A. F.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 3679. (20) Stegemiller, M. L.; Heineman, W. R.; Seliskar, C. J.; Ridgway, T. H.; Bryan, S. A.; Hubler, T.; Sell, R. L. Environ. Sci. Technol. 2003, 37, 123. (21) Milosavljevic, B. H.; Thomas, J. K. Photochem. Photobiol. Sci. 2002, 1, 100. (22) Honeychurch, K. C.; Hart, J. P.; Kirsch, N. Electrochim. Acta 2004, 49, 1141. (23) Kirsch, N.; Hart, J. P.; Bird, D. J.; Luxton, R. W.; McCalley, D. V. Analyst 2001, 126, 1936. (24) Andria, S. E.; Richardson, J. N.; Kaval, N.; Zudans, I.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2004, 76, 3139.

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mm glass, Thin Film Devices, Anaheim, CA) with dimensions of 10 mm × 40 mm were used as optically transparent electrodes. Nafion-coated ITO was prepared by spin-coating bare ITO with diluted Nafion solution to obtain 200- and 20-nm thick films as described previously.24 For sensor calibration, thin 20 nm films were used, whereas thicker 200 nm films were used for the basic development experiments. An optically transparent thin layer electrode (OTTLE) and a unique cell holder fabricated by rapid prototyping were used for the fluorescence thin-layer spectroelectrochemical measurements.25 A Ag/AgCl (3 M KCl) reference electrode and platinum mesh or platinum wire auxiliary electrode were used. Approximately 35 µL of 1 × 10-4 M 1-PyOH sample was introduced into the OTTLE cell by capillary action. About 1 mL of 0.1 M NaCl supporting electrolyte solution was then placed on the bottom of the cell holder in order to maintain electrical contact between the electrodes. The cell holder was then placed in a Cary Eclipse fluorometer, the quartz slide facing the incident light beam at 45° for fluorescence measurements. Electrochemistry was done with either a BAS 100-B or Epsilon electrochemical workstation (Bioanalytical Systems, West Lafayette, IN). Attenuated total reflectance (ATR) experiments were carried out using the spectroelectrochemical flow cell previously described.2 The flow cell was modified to increase the sample volume from ∼4 µL to ∼1 mL for practical considerations. Light from a 385 nm LED (Torrance, CA) was directed through a multimode optical fiber (Romack, 400 µm core step index, NA ) 0.22) to a collimating objective (Newport, 10×, NA ) 0.25). The collimated light was then passed in and out of the ATR cell by way of two Schott SF6 coupling prisms (Karl Lambrecht, Chicago, IL), attached to the ITO electrode slide by a mounting compound (Cargille Meltmount, n ) 1.59). The angle of the incident light into the prism and the alignment of the cell were adjusted using attenuated absorbance light. ATR light was focused by an objective lens (Newport, 20×, NA ) 0.40) 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. The fluorescence signal was collected by a fiberoptic bundle butted up against a single fluorescence spot. Data collection was achieved using Acton Research NCL monochromator controller electronics and Spectrasense software. A potentiostat (Bioanalytical Systems CV-27) was used to perform the electrochemical modulations. The solutions were introduced into the cell at 0.5 mL/min flow rate by a Sage Instruments syringe pump (model 341 B). Nafion film thickness was measured by elipsometry using a J.A. Woollam, Inc. variable angle spectroscopic ellipsometer (vertical configuration). RESULTS AND DISCUSSION Semi-Infinite Diffusion Electrochemistry. As the first step in developing the spectroelectrochemical sensor, the electrochemistry of 1-PyOH was evaluated by cyclic voltammetry (CV) under semi-infinite diffusion conditions using different working electrodes made from three materials widely used for optically transparent electrodes: platinum, gold, and ITO. 1-PyOH acidity (25) Wilson, R. A.; Pinyayev, T. S.; Membrano, N.; Heineman, W. R. Electroanalysis 2010, 22, 2162.

Figure 1. Emission spectra obtained (A) during controlled potential electrolysis at +900 mV of 1 × 10-5 M 1-PyOH in 1 M NaCl, 0.5 M (pH 3.5) phosphate buffer with 30% EtOH (v/v) and (B) during subsequent electrolysis at -100 mV in an OTTLE cell. Controlled potential electrolyses were carried out for 3 min each. λexc ) 280 nm. (C) Cyclic voltammogram obtained in the OTTLE cell of 0.1 mM 1-PyOH at 2 mV/s.

depends on whether the analyte is in the ground state (pKa ) 9.0) or the excited state (pKa* ) 4.5).21 In order to ensure that the sample solutions contain over 90% of 1-PyOH (even when excited by light), we used acidic solutions (pH 3.5) in this study. The most well-defined cyclic voltammograms were obtained on gold and ITO electrodes (results not shown). Since ITO has been used successfully with previous spectroelectrochemical sensors, we chose it for the sensor platform. Thin-Layer Spectroelectrochemistry. Figure 1C shows a thin-layer cyclic voltammogram obtained for 1-PyOH using an OTTLE with ITO as the transparent electrode.26 The scan was initiated at the open-circuit potential in the positive direction. As expected on the basis of the information available in the literature, 1-PyOH showed a distinct anodic peak due to oxidation. The subsequent negative and positive scans show no corresponding (26) Heineman, W. R.; Meckstroth, M. L.; Norris, B. J.; Su, C.-H. J. Electroanal. Chem. 1979, 104, 577.

reduction peak, but rather the appearance of a new, reversible redox couple centered at about 200 mV. This behavior is consistent with the reported ECE mechanism of 1-PyOH and other PAHs. HPLC-MS has shown that the major product of 1-PyOH oxidation is a diquinonepyrene,22 the product of nucleophilic attack of the electrochemically generated intermediate cation by water. Thus, the new redox couple measured here is associated with the reversible electrochemistry of diquinonepyrene, which can be reduced to dihydroxypyrene. On the basis of the CV results, we concluded that spectroelectrochemical detection of 1-PyOH can be based on an optical change associated with its primary oxidation, or the diquinonepyrene product of the ECE mechanism (redox couple 1a/1b in Figure 2), assuming that these electrode processes give an appropriate spectral change. Fluorescence changes associated with the observed electrode reactions were studied using the OTTLE.26 The fluorescence emission spectrum of 1-PyOH obtained in the OTTLE before electrolysis (Figure 1A) shows three emission peaks at 385, 405, and 430 nm. Oxidation at +900 mV causes a decrease in fluorescence associated with oxidation of the alcohol group to presumably a ketone. Stepping the potential to -100 mV for 3 min reduces the oxidation product and results in a fluorescence spectrum similar to 1-PyOH, but shifted to longer wavelengths by about 20 nm (Figure 1B). The wavelength shift is apparently caused by the additional alcohol group resulting from the ECE mechanism (Figure 2). We note that the fluorescence spectrum of the new compound shows a strong resemblance to that of 1,6dihydroxypyrene previously reported by Mazura et al.,27 which also shows two major emission bands at 405 and 425 nm. Most importantly, the fluorescence of the new compound can be turned “on” and “off” by cycling the potential between +400 and -100 mV and thereby cycling it between the nonfluorescing diquinonepyrene and the fluorescing dihyroxypyrene. However, continuous cycling causes the fluorescence intensity of this new compound to gradually decreases (about 10-15% of the intensity is lost after about 20 min of cycling), which may indicate some photobleaching or loss of the compound by a slow side reaction. Results of this experiment also point to the formation via the ECE mechanism of one dominant redox product during 1-PyOH oxidation. In order to further characterize the species produced by the ECE mechanism of 1-PyOH electrochemical oxidation, a spectroelectrochemical Nernst plot for the product was obtained using the OTTLE. The 1-PyOH was first oxidized by holding the potential at +800 mV in order to produce the dihydroxypyrene. After 3 min of oxidation, all of the analyte was converted to its oxidized form and its fluorescence was measured. Then, the electrode potential was stepped at random intervals from positive to negative for reduction to dihydroxypyrene (Figure 3A). As the electrode potential reached +400 mV, which is negative enough to reduce some of the analyte, a slight increase in fluorescence in the 380-480 nm optical window was observed. Fluorescence intensity continued increasing until the electrode potential reached -100 mV, where all of the analyte was in its reduced form. The procedure was then repeated by stepping from negative to positive for oxidation of the dihydroxypyrene. The thin layer spectroelec(27) Mazura, M.; Blanchard, G. J. Bioelectrochemistry 2005, 66, 89.


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Figure 2. Assumed oxidation mechanism of 1-PyOH in acidic solution.

Figure 3. Spectroelectrochemistry in OTTLE obtained for reversible redox reaction of the diquinone-dihydroxypyrene formed by oxidation of 1 × 10-5 M 1-PyOH in 1 M NaCl, 0.5 M (pH 3.5) phosphate buffer with 30% EtOH (v/v) by holding the electrode potential at +800 mV for 3 min. (A) Increase in fluorescence intensity during diquinonepyrene reduction as electrode potential is stepped from positive to negative. Each potential was held for 30 s before fluorescence measurements were taken to allow equilibrium to be established between the redox species and the electrode potential. (B) Nernst plot constructed for the process in A at 425 nm.

trochemical Nernst plot,28 where the reaction quotient [Red]/ [Ox] was obtained from the ratio of fluorescence intensities at 425 nm corresponding to each applied potential, is shown in Figure 9746


3B (y ) -30.96x + 151.6, R2 ) 0.9897). The slopes of the Nernst plots for both reduction and oxidation processes were 30.1 and 32.3 mV, respectively, indicating a two-electron redox process for the product of 1-PyOH electrochemical oxidation in acidic media. Also obtained from the plot, the intercept of the line gives a formal reduction potential of +152 mV. This value is consistent with the results of Kirsch et al.,23 who reported a new electrochemically reversible couple produced by the ECE mechanism for 1-PyOH with a pH-dependent standard redox potential of ∼150 mV (vs Ag/AgCl) at pH 3.5. Signal Enhancement by Modification of the Electrode with Nafion. In our previous work, we have used ion-exchange films to preconcentrate charged analytes and partially exclude interferences.24,29 This ion exchange strategy does not apply to 1-PyOH in acidic samples, where it is neutral. However, we found that the cation-exchanger sulfonated tetrafluoroethylene copolymer Nafion30 preconcentrated 1-PyOH, presumably due to the hydrophobic pockets that comprise a significant part of this polymer’s morphology. Cyclic voltammograms of 1-PyOH in acidic samples at the Nafion-modified ITO electrode show a significant increase in current with repetitive scanning compared to the bare electrode (Figure 4). Interestingly, the peaks at ca. 0.2 V associated with the chemical product of the ECE mechanism, dihydroxypyrene, are significantly larger compared to the bare electrode. As more 1-pyOH partitions into the film during the cycling, it is converted into dihydroxypyrene by the ECE mechanism. The sharpness of the waves is consistent with the dihydroxypyrene being constrained within a thin polymer film (i.e., restricted diffusion or thin-layer behavior). It is known that Nafion is a good catalyst for chemical oxidation reactions,31,32 and it appears that 1-PyOH is oxidized during its uptake in Nafion film, probably by molecular oxygen. The spectroelectrochemical fluorescence properties of 1-PyOH loaded into a thin Nafion film were similar to those exhibited in the OTTLE. The fluorescence of 1-PyOH preconcentrated into a 200 nm Nafion film for 15-min and then oxidized by holding the electrode potential at +800 mV for 3 min to produce the (28) Kissinger, P. T.; Heineman, W. R., Eds. Laboratory Techniques in Electroanalytical Chemistry, 2nd ed.; CRC Press, Boca Raton, FL, 1996. (29) Conklin, S. D.; Heineman, W. R.; Seliskar, C. J. Electroanalysis 2005, 17, 1433. (30) Mauritz, K. A.; Moore, R. B. Chem. Rev. 2004, 104, 4535. (31) Jain, S. L.; Sain, B. Appl. Catal., A 2006, 301, 259. (32) Venkatesan, S.; Kumar, A. S.; Zen, J. M. Tetrahedron Lett. 2008, 49, 4339.

Figure 4. Cyclic voltammogram of 1 × 10-5 M 1-PyOH in 0.1 M (pH 3.5) buffer at Nafion-modified ITO and at bare ITO. Scan rate 10 mV/s.

Figure 6. Calibration curve constructed for indirect detection of 1-PyOH from acidic sample by fluorescence of electrochemically generated dihydroxypyrene at an ITO electrode modified with 20 nm Nafion film. Fluorescence of dihydroxypyrene was excited at 385 nm. Plotted here is the magnitude of dihydroxypyrene fluorescence (λem ) 425 nm) modulation (change in fluorescence) during cyclic voltammetry vs 1-PyOH sample concentration.

Figure 5. Fluorescence modulation of dihydroxypyrene on Nafionmodified (A) and bare (B) electrodes in a flow cell. Diquinonepyrene (oxidized form of dihydroxypyrene) was generated during 15-min uptake of 1 × 10-5 M 1-PyOH into 200 nm Nafion film. After uptake, the electrode potential was held at +800 mV for complete 1-PyOH oxidation to produce diquinonepyrene. Generated diquinonepyrene was electrochemically reduced during cyclic voltammetry in the +500 to -200 mV potential window, which generated fluorescent dihydroxypyrene. Fluorescence of dihydroxypyrene was excited at 380 nm. Modulation of dihydroxypyrene fluorescence emission at 425 nm was monitored during cyclic voltammetry.

diquinonepyrene by the ECE mechanism was found to reversibly modulate when the potential was scanned between 500 and -200 mV. The fluorescence at 425 nm increased when the dihydroxypyrene was formed and decreased when the diquinonepyrene was formed (Figure 5, curve A). In order to show the preconcentration effect, we also ran the same experiment using bare ITO. Figure 5, curve B shows slight fluorescence modulation of the dihydroxypyrene, some of which was probably adsorbed on the electrode surface. Similar to that observed during previous studies with Ru(bpy)33+, some loss of fluorescence intensity occurs with each consecutive electrode potential scan. This decrease is attributed to photobleaching or loss of the compound by a slow side reaction. Importantly, this experiment demonstrated that, upon uptake by Nafion, 1-PyOH can be indirectly detected by measuring the fluorescence change during electrochemical

cycling of the 1-PyOH oxidation product formed by the ECE mechanism, dihydroxypyrene/diquinonepyrene. Calibration Curve. The sensor’s quantitative response to fluorescence modulation of electrochemically generated dihydroxypyrene for indirect detection of 1-PyOH was evaluated with a series of standard 1-PyOH solutions. An ITO electrode was modified with a 20 nm Nafion film for rapid analyte preconcentration. For each data point, a 1-PyOH sample with a given concentration was pumped through a flow cell at 0.3 mL/s for 20 min for analyte uptake. Then, the electrode potential was held at +800 mV for 3 min to ensure complete 1-PyOH oxidation. The film’s contents were then subjected to cyclic voltammetry in the +500 to -200 mV potential window, which generated fluorescent dihydroxypyrene at the negative electrode potential and nonfluorescent diquinonepyrene at the positive end analogous to that shown in Figure 5. The calibration curve in Figure 6 covering a wide concentration range has the usual sigmoid-like shape associated with fluorescence. The curve has a linear range [y ) (0.672 ± 0.07)x + (8.43 ± 0.6)] covering about about 2 orders of magnitude, as shown in the inset figure. For the calibration curve, the magnitude of dihydroxypyrene fluorescence modulation (λex ) 385 nm, λem ) 425 nm) during cyclic voltammetry vs 1-PyOH concentration in a sample solution was plotted. An average of the first three modulations was used for each data point. The measurement was then repeated two more times, each with a different sensor, and the RSD was calculated on the basis of the triplicate data. Although the magnitude of the modulation signal decreases with each cycle, averaging the first three cycles resulted in a good calibration curve. Since a new sensor was used for each measurement, the uncertainty for each point on the plot is primarily due to the irreproducibility in the different sensors. The calibration curve shown in Figure 6 was constructed for sensors with a 20-nm Nafion film coating. The film spin-coating procedure yields a film thickness of 20 ± 2 nm, which results in high (up to 20%) RSDs for replicate Analytical Chemistry, Vol. 82, No. 23, December 1, 2010

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measurements. Optimization of the film-coating procedure (such as spin-coating a large ITO-glass substrate and then cutting it into smaller electrodes) should address this issue. As seen in Figure 6, the linear range of the calibration plot extends from about 5 × 10-9 to 1 × 10-6 M 1-PyOH. The detection limit, calculated using fluorescence modulation of the blank solution plus three standard deviations, was 1 × 10-9 M. These results are somewhat higher than those obtained earlier for detection of Ru(bpy)32+2. However, spectroelectrochemical detection of Ru(bpy)32+ ion relies directly on decrease of fluorescence during electrochemical conversion of fluorescent Ru(II) to nonfluorescent Ru(III). In the case of 1-PyOH detection, the analyte must first be converted into a nonfluorescent compound (pyrene quinone), which is then electrochemically reduced to fluorescent dihydroxypyrene for detection. CONCLUSIONS The proof of principle of a spectroelectrochemical sensor that detects an analyte indirectly via another compound formed by an ECE mechanism has been demonstrated with 1-PyOH, one of the biomarkers used for evaluation of human PAH exposure. The sensor relies on detection of fluorescent dihydroxypyrene, which is generated via an ECE mechanism. Fluorescent dihydroxypyrene can be electrochemically reduced to nonfluorescent diquinonepyrene, and the magnitude of this fluorescence modulation is proportional to the 1-PyOH concentration in the sample solution. Nafion, a commonly used cation exchanger, was found to preconcentrate neutral 1-PyOH. Importantly, fluorescence of dihy(33) Levin, J. O. Sci. Total Environ. 1995, 163, 165.

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droxypyrene formed in the film was not quenched by Nafion. Analtye preconcentration and the sensitivity of fluorescence detection enabled the sensor to have a wide linear operating range, from 5 × 10-9 to 1 × 10-6 M, and a low limit of detection, 1 × 10-9 M. These parameters compare favorably with those obtained by a fluorescence-based sensor for Ru(bpy)2+ that also used Nafion as the preconcentrating film. According to Levin,33 average background 1-PyOH concentrations found in urine vary from 1.3 × 10-9 up to 2.75 × 10-4 M and are greatly influenced by factors such as smoking, diet, and industrial pollution. The limit of detection for the spectroelectrochemical sensor is 1 nM, which is adequate for 1-PyOH detection for many of the categories listed in this reference. This performance is adequate for some practical applications, such as detection of 1-PyOH in urine for measuring bioexposure to PAHs and some environmental measurements. The limit of detection is dependent on the film material and therefore could be improved by finding a film that preconcentrates better without quenching fluorescence. ACKNOWLEDGMENT The authors gratefully acknowledge Robert Wilson for stimulating discussions. This research was supported in part by the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG02-07ER64353. Received for review July 14, 2010. Accepted October 17, 2010. AC101883A

Fluorescence Spectroelectrochemical Sensor for 1-Hydroxypyrene

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