Spectroelectrochemical Sensing Based on Multimode Selectivity

Oct 9, 2003 - trode (OTE) coated with a selective film to enhance detection limits by preconcentrating the analyte at the OTE surface. Nafion was used...
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Anal. Chem. 2003, 75, 6334-6340

Spectroelectrochemical Sensing Based on Multimode Selectivity Simultaneously Achievable in a Single Device. 16. Sensing by Fluorescence Necati Kaval, Carl J. Seliskar,* and William R. Heineman*

Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, Ohio 45221-0172

A fluorescence spectroelectrochemical sensor capable of detecting very low concentrations of metal complexes is described. The sensor is based on a novel spectroelectrochemical sensor that incorporates multiple internal reflection spectroscopy at an optically transparent electrode (OTE) coated with a selective film to enhance detection limits by preconcentrating the analyte at the OTE surface. Nafion was used as the selective cation exchange film for detecting Ru(bpy)32+, the model analyte, which fluoresces at 605 nm when excited with a 441.6-nm HeCd laser. The unoptimized linear dynamic range of the sensor for Ru(bpy)32+ is between 1 × 10-11 and 1 × 10-7 M with a calculated 2 × 10-13 M detection limit. The sensor employs extremely thin films (∼12 nm) without significantly sacrificing its sensitivity. The sensor response is demonstrated with varying film thicknesses. A state-ofthe-art flow cell design allows variable cell volumes as low as ∼4 µL. Fluorescence of the sample can be controlled by electromodulation between 0.7 and 1.3 V. Sensor operation is not reversible for the chosen model film (Nafion) and sample (Ru(bpy)32+) but it can be regenerated with ethanol for multiple uses. For the last several years, we have been developing a functional spectroelectrochemical sensor that combines electrochemistry, optical spectroscopy, and selective partitioning into a single device.1-12 The sensor is based on a novel device that incorporates multiple internal reflection (MIR) spectroscopy in an optically * Corresponding authors. E-mail: [email protected]; carl.seliskar@ uc.edu. (1) Shi, Y.; Slaterbeck, A. F.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 3679. (2) Shi, Y.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 4819. (3) Slaterbeck, A. F.; Ridgway, T. H.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1999, 71, 1196. (4) Slaterbeck, A. F.; Stegemiller, M. L.; Seliskar, C. J.; Ridgway, T. H.; Heineman, W. R. Anal. Chem. 2000, 72, 5567. (5) DiVirgilio-Thomas, J. M.; Heineman, W. R.; Seliskar, C. J. Anal. Chem. 2000, 72, 3461. (6) Gao, L.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1999, 71, 4061. (7) Gao, L.; Seliskar, C. J.; Heineman, W. R. Electroanalysis 2001, 13, 613619. (8) Maizels, M.; Seliskar, C. J.; Heineman, W. R. Electroanalysis 2000, 12, 1356. (9) Maizels, M.; Seliskar, C. J.; Heineman, W. R.; Bryan, S. A. Electroanalysis 2002, 14, 1345-1352. (10) Richardson, J. N.; Dyer, A. L.; Stegemiller, M. L.; Zudans, I.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2002, 74, 3330. (11) 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.

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transparent electrode (OTE) coated with a selective film.1 The sensor concept is illustrated by the diagram of the interfacial region shown in Figure 1. The OTE is coated by a thin film that serves to enhance detection limit by preconcentrating the analyte. At each reflection point, the evanescent field penetrates the film, and as a result, the reduced or oxidized form of the analyte can be monitored optically. In its operation, an electrical potential is applied to the OTE to cause electrolysis of analyte that has partitioned into the film. Quantification of the analyte is based on the magnitude of the change in the optical signal, which is proportional to concentration of analyte in the film, as defined by the partition coefficient and, in turn, is proportional to its concentration in the sample. All of our work thus far has used absorption spectroscopy (“sensor absorbance”1-12) as the mode of optical detection. Because detection is spectroscopic, rather than electrochemical, sensitivity is determined in part by the two parameters of effective path length13,14 (b) and the molar extinction coefficient () of the light-absorbing species. In the case of MIR, the effective path length is determined14 by the penetration depth of the evanescent field into the selective film and the number of reflections of light. A third parameter of importance is the partition coefficient of the analyte from the solution into the selective film, which determines the concentration enhancement in the optical path length that the film provides. Because electrochemistry is used only for modulation of the optical signal, the usual Faraday’s law parameters are not a factor in determining sensitivity. Improvements in the sensitivity generally translate into a lower limit of detection. We have reported detection limits in the 10-6-10-5 M range for the analytes investigated so far using sensor absorbance techniques.9 These analytes all had ∆ values of ∼1000 L mol-1 cm-1. These detection limits are quite modest by today’s standards and restrict the applicability of the sensor to samples with relatively high concentrations of analyte. We have been investigating various ways of lowering the detection limits of our spectroelectrochemical sensor and have found that one of the most promising of these is by using fluorescence spectroscopy. The excellent detection limits of fluorescence spectroscopy are well known, and fluorescence is often the method of choice when (12) Heineman, W. R.; Seliskar, C. J.; Richardson, J. N. Aust. J. Chem 2003, 56, 93-102. (13) Mendes, S. B.; Saavedra, S. S. Appl. Opt. 2000, 39, 612-621. (14) Fornel, d. F. Evanescent Waves From Newtonian Optics to Atomic Optics; Springer: Berlin, 2001. 10.1021/ac0347664 CCC: $25.00

© 2003 American Chemical Society Published on Web 10/09/2003

Figure 1. Illustration of the interfacial region of the fluorescence spectroelectrochemical sensor. Coupling of the laser excitation is achieved by using a high-index prism on the backside of the ITO glass slide that has been coated on the ITO side with a chemically selective film. Light traveling through the OTE by MIR generates an evanescent field that penetrates the selective film at each reflection point. In turn, this evanescent field can pump the analyte, producing fluorescence. The selective film is exposed to solution containing analyte and, in turn, preconcentrates it. A fiber-optic bundle gathers fluorescence generated by laser excitation of the analyte entrapped in the selective film.

achieving a low detection limit is important.15,16 The literature is rich in reports of combining electrochemistry and fluorometry to study the more fundamental aspects of electrochemically initiated chemical reactions.17-22 However, there are relatively few reports of combined fluorescence spectroscopy and electrochemistry for chemical sensing. The work of Weber and co-workers23,24 established that by using a gold-coated fiber optic one could make a small probe that could sensitively detect analytes by manipulating their redox states. They also demonstrated the extension of this type of sensing to biosensing. Calvo and co-workers20,25 demonstrated the use of fluorometry at the surfaces of fluor-hydrogelmodified electrodes. Importantly, this group demonstrated electromodulation of the fluorescence excited externally at the electrode surface. Most recently, Pantano and co-workers reported an imaging-fiber optic electrode chemical sensor.26 This device incorporated a Nafion film within which a fluorescent dye was trapped and functioned by controlling the redox state of the dye at a gold electrode surface. However, no analytical figures of merit were reported. In summary, although there have been several important reports pertaining to certain narrowly defined aspects of spectroelectrochemical sensing using fluorescence, no one has combined the three modes of selectivity together in a single device (15) Ingle, J. D.; Crouch, S. R. Spectrochemical Analysis; Prentice Hall: Englewood Cliffs, NJ, 1988. (16) Pizes, M.; Bartos, J. Colorimetric and Fluorometric Analysis of Organic Compounds and Drugs; Marcel Dekker: New York, 1974. (17) Jones, T. T.; Faulkner, L. R. J. Electroanal. Chem. 1984, 179, 53-64. (18) Wilson, R.; Schiffrin, D. J. Analyst 1995, 120, 175-178. (19) Rubim, J. C.; Gutz, I. G. R.; Sala, O. J. Electroanal. Chem. 1985, 190, 5563. (20) Bonanzola, C.; Brust, M.; Calvo, E. J. J. Electroanal. Chem. 1996, 407, 203207. (21) Buttry, D. A.; Anson, F. C. J. Am. Chem. Soc. 1982, 104, 4824-4829. (22) Simone, M. J.; Heineman, W. R.; Kreishman, G. P. Anal. Chem. 1982, 54, 2382-2384. (23) Cohen, C. B.; Weber, S. G. Anal. Chem. 1993, 65, 169-175. (24) Kuhn, L. S.; Weber, A.; Weber, S. G. Anal. Chem. 1990, 62, 1631-1636. (25) Battaglini, F.; Bonanzola, C.; Calvo, E. J. J. Electroanal. Chem. 1991, 309, 347-353. (26) Khan, S. S.; Jin, E. S.; Sojic, N.; Pantano, P. Anal. Chim. Acta 2000, 404, 213-221.

Figure 2. Diagram of the complete sensor system. λex and λf denote excitation wavelength and fluorescence wavelength, respectively.

as we propose to achieve both high selectivity and low detection limits. Here we introduce a fluorescence-based spectroelectrochemical sensor that extends the applicability of our film-based spectroelectrochemical sensor to significantly lower limits of detection. In addition, this mode of operation of the sensor offers a faster response time and a much smaller sample volume than our previously reported designs. EXPERIMENTAL SECTION Chemicals and Materials. The following chemicals were used: Nafion (5% solution in lower aliphatic alcohols and water, Aldrich), tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate ([Ru(bpy)3Cl2‚6H2O], Aldrich), potassium nitrate (Fisher Scientific). All reagents were used without further purification. Ru(bpy)32+ solutions were prepared by dissolving the appropriate amounts in 0.1 M KNO3 solution (prepared with deionized water from a Barnstead water purification system). Indium tin oxide (ITO)-coated glass slides (Corning 1737F and 7059, 11-50 Ω/square, 130-nm-thick film on 1.1-mm glass, Thin Film Devices) with dimensions of 10 mm × 40 mm were used as OTEs. The ITO slides were scrubbed with Alconox and rinsed thoroughly with deionized water. Finally, the cleaned slides were rinsed with methanol and dried in air before use. Instrumental Arrangement and Spectroelectrochemical Sensor. The instrumental arrangement is shown in Figure 2. The system consists of a laser (441.6-nm HeCd model 1K4153R-C, Kimmon Electric Co.) and light control modules (shutter, attenuator, and focusing optics), a spectroelectrochemical flow cell, a monochromator (0.3-m focal length, triple grating turret) and photon-counting phototube (Acton Research Corp.), a computer, and data acquisition and control electronics (NCL and SpectraSense software, Acton), a potentiostat (CV-27 potentiostat, Bioanalytical Systems), and a syringe pump (model 341B, Sage Instruments). Light from the laser was focused onto the polished end of a 400-µm silica step-index optical fiber (NA ) 0.22, RoMack, Inc.) with a microscope objective (10×, NA ) 0.25, Newport). A homemade shutter and a variable neutral density filter (Newport) were used to control the laser intensity and the exposure time in Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

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Figure 3. Detailed illustration of the spectroelectrochemical sensor for fluorescence-based measurements.

the sensor. Laser power was attenuated to 0.1 mW at the launching prism face, and the sample was exposed to laser light only during data acquisition to minimize photodegradation. The light was coupled into the ITO glass slide of the spectroelectrochemical cell using another microscope objective (10×, NA ) 0.25, Newport) and a coupling prism (Schott SF6, Karl Lambrecht, Chicago, IL). The angle of the incident light into the prism and the alignment of the cell were adjusted to maximize the MIR throughput by monitoring the intensity of the decoupled light from the other end of the slide or by monitoring the fluorescence intensity. Electromodulation was done using the potentiostat, and the sample solutions were delivered to the cell with the syringe pump at a 0.1 mL/min rate using a 5-mL syringe. For fluorescence data acquisition, the end of a fiber-optic bundle was butted up against a single spot (see Figure 1). All experiments were carried out at room temperature. All data were acquired digitally, and subsequent data analysis and manipulation were accomplished using commercial spreadsheet and graphics algorithms. Fluorescence-based spectroelectrochemical measurements were acquired using a thin-film flow cell (fashioned from a BAS electrochemical flow cell, model CC-44, Bioanalytical Systems). The sensor itself (Figure 3) is composed of several components: (1) A stainless steel body (auxiliary electrode) with a highly polished surface accommodated liquid inlet and outlet. (2) A 51-µm-thick Teflon spacer (BAS) with an oval opening at the center was used to create a liquid well with a ∼4-µL volume. (3) A 40 mm × 10 mm × 1.1 mm ITO glass slide was used as an OTE. Electrical contact to the ITO slide was provided by linear spring finger arrays (Laird Technologies), mounted on each side of the stainless steel body using Delrin insulators. (4) A Schott SF6 coupling prism was mounted on one end of the ITO slide by using high-viscosity refractive index standard fluid (Cargille, n ) 1.59) to span the prism/ITO slide gap. (5) Two Delrin pieces, one with an oval hole (0.25 in. × 0.75 in.) and the other with a circular hole (i.d. ) 0.5 in.), were used to fasten the ITO slide onto the flow cell and to hold the prism in place. The tip of the fiber optic was butted against the backside of the MIR optic and fluorescence from only one fluorescent spot was gathered (see also Figure 1). 6336

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For fluid introduction, the cell was plumbed with Teflon tubing (1/16-in. o.d. and 0.030-in. i.d.), and the cell can operate either under flow or nonflow conditions. Volume of the flow cell can be varied by using Teflon spacers with different thicknesses or by varying the well area. For smaller sample volumes, the dead volume in the tubing can be decreased by using 1/16-in. tubing with a narrower internal diameter. All electrochemical modulations were performed against an Ag/AgCl, 3 M NaCl reference electrode (BAS). Preparation of Nafion Films. Nafion films were prepared using a spin coater (model 1-PM101DT-R485, Headway Research, Inc.). Film thicknesses were determined using a variable-angle spectroscopic ellipsometer (VASE) and associated software (Woollam Co., Inc.). A 5% stock Nafion solution was diluted to the desired concentration using 4:1 2-propanol/water mixture.27 A 100µL aliquot of diluted solution was pipetted onto an ITO-coated slide and spun at 6000 rpm for 30 s. In this manner, film thicknesses of about 10-50 nm were routinely obtained by varying the concentration of Nafion solution between 0.5 and 2.0% (w/v). The thicknesses of spin-coated Nafion films routinely obtained were 12, 24, and 50 nm, by using 0.5, 1.0, and 2.0% Nafion solutions, respectively. Before spin coating, a 1-cm portion of each end of the slide was masked with tape, leaving a 2 cm × 1 cm area in the center for film coating. The two areas of the ITO slide not covered by Nafion film were used for electrical contact. RESULTS AND DISCUSSION To demonstrate the use of fluorescence in spectroelectrochemical sensing, we have chosen a model system that highlighted all the aspects of this technique: chemical uptake of an analyte, reversible electrochemical oxidation/reduction of the analyte, and modulation of the fluorescence of one of the redox forms of the analyte. That model system is the Ru(bpy)32+/Nafion system that we and others have used previously. This model system satisfies several important detailed requirements. First, it is well known that Ru(bpy)32+ is incorporated into Nafion by ion exchange.28,29 For example, Nafion has been extensively used with Ru(bpy)32+ in modified electrodes.30 We have experience with Nafion films and its silica composites having, for example, even studied the film dynamics of Nafion while it takes up Ru(bpy)32+.31 Nafion films of a wide variety of thicknesses can be easily formed on glass or ITO-coated glass by spin coating. Because of their thermal and chemical resistance, mechanical strength, and insolubility in water, Nafion films remain intact on a compatible surface for a very long time without degradation. Second, the electrochemistry of this analyte at ITO is well behaved and this allows us to use the ITO OTE (potential window in 0.1 M KNO3, -1.0 to +1.4 V vs Ag/AgCl)32 for electrochemical modulation of the optical signal. Third, both the oxidized and reduced forms of Ru(bpy)32+ are sufficiently stable chemically and photochemically to demonstrate the technique. Fourth, the reduced form, i.e., (27) Shi, M.; Anson, F. C. J. Electroanal. Chem. 1997, 425, 117-123. (28) Szentirmay, M. N.; Martin, C. R. Anal. Chem. 1984, 56, 1898-1902. (29) Khramov, A. N.; Colinson, M. M. Anal. Chem. 2000, 72, 2943-2948. (30) Downey, T. M.; Nieman, T. A. Anal. Chem. 1992, 64, 261-268. (31) Zudans, I.; Heineman, W. R.; Seliskar, C. J. Manuscript in preparation. (32) Zudans, I.; Paddock, J. R.; Kuramitz, H.; Maghasi, A. T.; Wansapura, C. M.; Conklin, S. D.; Kaval, N.; Shtoyko, T.; Monk, D. J.; Bryan, S. A.; Hubler, T. L.; Richardson, J. N.; Seliskar, C. J.; Heineman, W. R. J. Electroanal. Chem. Manuscript in press.

Ru(bpy)32+, absorbs with a moderately high molar absorptivity (14 600 M-1 cm-1 at 452 nm)33 and fluoresces with a moderate quantum yield. (We use the term fluorescence in this paper to describe the emission of Ru(bipy)32+. We note that the emissions of ruthenium complexes are more correctly called phosphorescence (i.e., emission from nominally triplet spin states). Nonetheless, it is common in the literature to call this emission fluorescence.) We have reported the electrochemistry and absorption spectroscopy of Ru(bpy)32+ in Nafion/SiO2 and other film-coated sensors.1,4-6,34,35 The oxidized form, Ru(bpy)33+, does not fluoresce under conditions where the reduced form is excited at 441.6 nm, which permits electrochemical modulation of the fluorescence signal. Demonstration of Fluorescence Detection. The HeCd laser line at 441.6 nm efficiently excites the fluorescence21,36-40 of Ru(bpy)32+ generating discrete bright red spots in Nafion on a coated OTE. For simplicity, the fluorescence was measured by collecting light from a single spot on the MIR optic (see also Figure 1). Alternatively, one could collect the fluorescence from several spots and we are currently devising collection optics for this approach. Ru(bpy)32+ preconcentrated in a 24-nm-thick Nafion film fluoresces red-orange with a peak maximum at ∼605 nm. In a typical experiment, three fluorescent spots each with ∼4-mm diameter appear in the flow cell optical window. The brightness of the spots increases with increasing Ru(bpy)32+ concentration. The spots are intense enough to be seen by the naked eye at solution concentrations higher than 1 × 10-7 M. Sensor response (as log of photon counts) to Ru(bpy)32+ is shown in Figure 4 as a function of the log of the sample concentration. Sample solutions ranging from 1 × 10-12 to 1 × 10-5 M were introduced into the sensor in random order by pumping into the flow cell at 0.1 mL/min flow rate for 45 min. Fluorescence spectra were recorded every 5 min. The log of the fluorescence intensity after 45 min of preconcentration was plotted against the log of solution concentration to obtain the calibration curve shown in Figure 4. The Nafion film was regenerated by flushing with ethanol after each data collection period. This regeneration process is discussed in more detail below. The inset in Figure 4 shows that the linear region of the curve extends from 1 × 10-11 to 1 × 10-7 M. The slope of the curve at concentrations higher than 1 × 10-7 M and lower than 1 × 10-11 M changes and becomes smaller. Sensor response time (the time to reach equilibrium) is strongly dependent on sample concentration. At solution concentrations higher than 1 × 10-7 M, the fluorescence signal levels off very quickly. It takes ∼10 min in 1 × 10-6 M solution (Figure 5A) and less than 5 min in 1 × 10-5 M solution (not shown). The time increases dramatically as the Ru(bpy)32+ concentration in solution decreases. It takes many hours (or even days at extremely low concentrations) to reach (33) Kalyanasundaram, K. Coord. Chem. Rev. 1982, 46, 159-244. (34) Dasenbrock, C. O.; Ridgway, T. H.; Seliskar, C. J.; Heineman, W. R. Electrochim. Acta 1998, 43, 3497-3502. (35) Gao, L.; Seliskar, C. J. Chem. Mater. 1998, 10, 2481-2489. (36) Matsui, K.; Momose, F. Chem. Mater. 1997, 9, 2588-2591. (37) Colon, J. L.; Martin, C. R. Langmuir 1993, 9, 1066-1070. (38) Castellano, F. N.; Heimer, T. A.; Tandhasetti, M. T.; Meyer, G. J. Chem. Mater. 1994, 6, 1041-1048. (39) Xu, W.; McDonough, R. C.; Langsdorf, B.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1994, 66, 4133-4141. (40) Demas, J. N.; DeGraff, B. A. Anal. Chem. 1991, 63, 829A-837A.

Figure 4. Calibration curve for the fluorescence detection of Ru(bpy)32+ in 0.1 M KNO3 solution. A 24-nm-thick Nafion film on the sensor was exposed to varying concentrations of Ru(bpy)32+ for 45 min. Concentrations vary from 1 × 10-12 to 1 × 10-5 M. Integration time in this study was 100 ms. The inset shows the linear range extending from 1 × 10-11 to 1 × 10-7 M; λex) 441.6 nm and λem max ) 605 nm.

Figure 5. Preconcentration kinetics of 1 × 10-6 (A) and 1 × 10-12 M (B) Ru(bpy)32+ solutions into 24-nm-thick Nafion film.

equilibrium. On the other hand, a measurable fluorescence signal can be easily obtained in about 15-20 min at very low concentrations (e.g., 1 × 10-12 M). The initial increase in the fluorescence Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

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intensity with time is quite linear as shown in Figure 5B for 1 × 10-12 M Ru(bpy)32+ solution. As the solution concentration increases, the slope of the fluorescence intensity versus time curve increases. Because of the long preconcentration periods to reach equilibrium at low solution concentrations, it was not practical to construct a calibration curve at equilibrium (or at steady state). Instead we chose to construct the calibration curve using the fluorescence intensity of each concentration at a 45-min preconcentration period. Alternatively, one could construct a calibration curve using the initial slope of the photon counts versus time plot. As long as the flow rate is kept constant, the amount of the Ru(bpy)32+ accumulated in the film should be proportional to the Ru(bpy)32+ concentration in the solution. It is obvious from Figure 1 that the fiber bundle collected only a small portion of the fluorescence from a single 4-mm-wide source. Nonetheless, this simple optical arrangement can easily result in the detection of solution concentrations at the picomolar level. This, in turn, suggests that lower detection limits might be reached by using more efficient light collection optics. The detection limit of the sensor in the current form and configuration was calculated using the calibration curve equation shown in the inset of Figure 4. In this calculation, we assumed that the linearity of the calibration curve extended to lower concentrations. Using the slope of the calibration curve and the standard deviation of the baseline signal at 605 nm, detection limit was determined with the following equation,15

DL ) ksbk/m where DL is photon counts at the detection limit, k is a confidence factor often chosen to be 2 or 3, sbk is the standard deviation of blank (bk) measurements, and m is the slope of the calibration curve. For the confidence factor, we used k ) 3. The standard deviation of the blank (sbk) was determined experimentally as 11 photon counts. Using these parameters, the detection limit of the sensor for Ru(bpy)32+ was calculated to be 2 × 10-13 M after a 45-min preconcentration period. This limit of detection based on fluorescence change from one fluorescing Ru(bpy)32+ spot is ∼7 orders of magnitude lower than we have reported with sensor absorbance using similar sensors. Furthermore, this detection limit is for unoptimized conditions, suggesting that we can achieve even lower detection limits. Selectivity of the sensor was not a focus of this paper; rather we have elected to focus on the extension of our absorbancebased spectroelectrochemical sensor to fluorescence. For this reason, all sample solutions were prepared using pure Ru(bpy)32+ in 0.1 M aqueous KNO3 solution. The trimodal selectivity of our sensor technology has already been discussed in detail elsewhere.2,4-11,41 Film Thickness and Sensor Response. The film thicknesses we have employed in our absorption-based sensors have typically ranged from a few hundred nanometers to 1 µm.5,6,11 We have found that it is possible and even preferable to use much thinner films in the fluorescence-based sensor. Figure 6 shows that a film thickness as low as 12 nm is useable when the fluorescence-based sensor in the thin-layer flow cell is used. As expected, film (41) Ross, S. E.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2000, 72, 55495555.

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Figure 6. Change in the sensor fluorescence intensity as a function of time beginning at initial exposure to a solution containing 1 × 10-6 M Ru(bipy)32+ under flow conditions (0.1 mL/min) for three Nafion film thicknesses. The inset shows the relationship between the film thickness and response time for these three films.

thickness affected the response time of the sensor and this is illustrated in Figure 6. A 1 × 10-6 M Ru(bpy)32+ solution in 0.1 M aqueous KNO3 was introduced into the sensor at a 0.1 mL/min flow rate. For each film thickness shown, 90% of the maximum fluorescence was recorded as the response time for comparison. As the figure shows, decrease in the film thickness resulted in a decrease in preconcentration time (or the response time) of the sensor. For the 12-nm-thick film, it took ∼3 min for the fluorescence signal to level off. In comparison, the response times of the sensor are 6 and 16 min for the 24- and 50-nm thicknesses, respectively. The inset in the Figure 6 shows that sensor response time increases linearly with increasing film thickness over this narrow range of thickness variation. Furthermore, from Figure 6, response time approximately doubled as the film thickness was doubled. In our ongoing studies, we will continue investigating the interplay of film thickness, sensitivity, limit of detection, and response time. Electrochemical Modulation of Fluorescence. In general, the spectroelectrochemical sensor analytical signal is based on the change in the optical signal on electrochemical modulation. Electromodulation provides selectivity against nonelectroactive interfering species or against the species that are not electromodulated at the chosen potential window. Before electrochemical modulation, the film was equilibrated with a 0.1 M solution of KNO3 overnight. Here, we used 12-nm-thick Nafion films due to reasons described below. The cyclic voltammogram shown in Figure 7A corresponds to Ru(bpy)32+ preconcentrated from 1 × 10-7 M solution in a 12-nm Nafion film on ITO. Figure 7B demonstrates the electrochemical modulation of the same film while it was preconcentrated from a 1 × 10-7 M solution of Ru(bpy)32+. Potential modulation was accomplished using square waves with 50-s periods by stepping the potential from 0.7 V, at which Ru(bpy)32+ preconcentrated in the film emits red light, to 1.3 V at which fluorescence emission ceases because Ru(bpy)32+ is oxidized to nonfluorescent Ru(bpy)33+. The amplitude of the fluorescence modulation can also be employed to determine analyte concentration, yielding calibration curves analogous to that

Figure 8. Regeneration of a Nafion film (24 nm thick) preconcentrated in 1 × 10-6 M Ru(bpy)32+ by using 95% ethanol.

Figure 7. (A) Cyclic voltammogram of Ru(bipy)32+ preconcentrated from a 1 × 10-7 M solution into a 12-nm-thick Nafion film. Potential was scanned at a 10 mV/min rate (vs Ag/AgCl ref). (B) Electromodulation of the same film by stepping the potential between 0.7 and 1.3 V at 50-s periods while preconcentrating it in a 1 × 10-7 M Ru(bipy)32+ solution.

in Figure 4. We constructed a calibration curve (not shown) using the Ru(bpy)32+ solutions ranging between 1 × 10-11 and 1 × 10-5 M and noticed that the linear range extended from 1 × 10-10 to 1 × 10-7, which is 1 order of magnitude smaller than what we obtained with the unmodulated curve shown in Figure 4. The calibration curve equation for the linear range, y ) 0.46x + 6.85, was similar to that we obtained using the unmodulated intensity. We did not obtain measurable modulations at concentrations equal to and lower than 1 × 10-12 M with the current instrumental configuration. We also noticed that the film thickness significantly affects the magnitude of modulation at low concentrations. When we used 24-nm-thick film, the lowest Ru(bpy)32+ concentration that we observed electrochemical modulation was 1 × 10-9 M after 35 min of preconcentration period. We are still investigating the effect of film thickness on electromodulation. As seen in Figure 7B, in concert with the potential modulation, the fluorescence intensity increases and decreases as Ru(bpy)32+ is cycled between the two redox forms. Comparing the several step cycles shown, it can be seen that there is an overall gradual decrease in the peak level of fluorescence as the number of such step cycles increases. This overall decrease might be attributed to several things, which include photodegradation and leaching of one of the redox forms of the analyte. According to Rubinstein

and Bard, it is very likely that the excited form of complex (Ru(bpy)32+*) can be quenched by its oxidized form (Ru(bpy)33+) via electron-transfer reaction.42 This effect is decreased if the potential is scanned or stepped more rapidly between oxidative and reductive potentials.21 Even though Ru(bpy)32+ binds very strongly to Nafion, Ru(bpy)33+ is not held as strongly as its 2+ form.43 This suggests that some of the 3+ form may leach out of the film at each cycle. Our speculation is that photodegradation of the analyte by laser excitation during the electrochemical stepping process was responsible for much of the signal decrease. However, we note that lowering the laser power did not stop the gradual decrease in fluorescence peak intensity. Since it was beyond the scope of this paper, we did not investigate the specific sources of this decrease. Regeneration of Nafion Films. Ru(bpy)32+ binds strongly to Nafion, and the interaction is not easily reversed under most conditions. For example, we were not able to completely remove Ru(bpy)32+ by flushing the flow cell with copious amounts of electrolyte solution. An ideal sensor, on the other hand, should be reversible or at least be able to be regenerated, for continuous monitoring or multiple uses. We discovered that we can regenerate the Nafion film by extracting Ru(bpy)32+ with 95% ethanol. The procedure entails simply flushing the flow cell with ethanol at a 0.1 mL/min flow rate until the film is completely purged of Ru(bpy)32+. Figure 8 shows that the fluorescence of a 24-nm-thick Nafion film preconcentrated in the 1 × 10-6 M Ru(bpy)32+ solution returns to the baseline level after flushing the film with ethanol. Surprisingly, regeneration occurs in less than 1 min under flow conditions. To show that disappearance of fluorescence is not due to quenching caused by ethanol, we reintroduced 0.1 M KNO3 solution (in which the analyte solution was also prepared) to replace ethanol. This did not produce any new fluorescence, thus showing that Ru(bpy)32+ was completely removed from the film by ethanol. When a fresh Ru(bpy)32+ solution was then reintroduced, the fluorescence returned to its initial level. Such regeneration and reuse cycles are reproducible as shown in Figure 8. Even after repeating this process many times, the Nafion film was not (42) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 5007-5013. (43) Martin, C. R.; Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1982, 104, 48174824.

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dissolved or otherwise stripped off the OTE surface. Others30 have also tried regenerating Nafion film on electrodes by using alcohol (methanol) but experienced solubility and stability problems when aqueous methanol higher than 30% was used. This might be due in part to film thickness because the films employed were much thicker (10 µm) than the films used by us (24 nm). Indeed, it is our experience that thinner films adhere to a surface much more tightly than thicker ones. CONCLUSIONS The conclusions of this work are as follows: (1) the new small volume sensor allows direct detection for a model fluorescent analyte at least at the 1 × 10-12 M level; (2) from photon-counting statistics, it is clear that the limit of detection for the current version of the sensor could be as low as 2 × 10-13 M, with improvements in the collection and excitation optics of the cell possibly yielding even lower detection limits; (3) the sensing volume has been dramatically reduced compared to our earlier cell for absorbance measurements and now is only ∼4 µL; (4) film thickness can be as small as 12 nm, which results in faster response time; (5) Nafion films loaded with Ru(bpy)32+ can be regenerated with ethanol for multiple uses; (6) electrochemical modulation has been demonstrated and this adds the third mode of selectivity.

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We are currently working on extending the fluorescence spectroelectrochemical sensor to different films and analytes while optimizing it for better performance. The sensor flow cell can be easily modified by dividing the transparent electrode surface into smaller sections and coating it with different films for multisensing applications. Its high sensitivity, fast response time, reusability, and very small sensing volume make it promising for a variety of applications including clinical monitoring, biosensor applications, and environmental monitoring. Due to the improvements in laser and LED technologies in the past decade, our sensor technology may be easily miniaturized and even mass-produced with reasonably low cost. ACKNOWLEDGMENT This work was supported by a grant awarded by the Environmental Management Sciences Program of the U.S. Department of Energy, Office of Environmental Management (DE-FG0799ER62311-70010). A Woollam spectroscopic ellipsometer (VASE) was purchased using a grant from the Hayes Fund of the State of Ohio. Received for review July 8, 2003. Accepted August 29, 2003. AC0347664