Spectroelectrochemical Sensing Based on ... - ACS Publications

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Anal. Chem. 2000, 72, 3461-3467

Spectroelectrochemical Sensing Based on Multimode Selectivity Simultaneously Achievable in a Single Device. 6. Sensing with a Mediator Jennifer M. DiVirgilio-Thomas, William R. Heineman,* and Carl J. Seliskar*

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

The use of a mediator to detect a nonabsorbing analyte during spectroelectrochemical modulation is demonstrated. The charge-selective composite film of NafionSiO2 was used to entrap the mediator, Ru(bipy)32+. The change in ∆A as detected by attenuated total reflection was then observed upon addition of the analyte, ascorbate. The effects of scan rate, concentration of mediator, film thickness, and analyte charge were studied to achieve optimal sensor conditions. The model sensor exhibited a linear range from 0.26 to 2.0 mM (R2 ) 0.998).

Recently, a new sensor concept that combines electrochemistry, spectroscopy, and selective partitioning in one device has been demonstrated.1-4 The sensor consists of an optically transparent electrode (OTE) coated with a thin perm-selective film. The OTE operates in the attenuated total reflection (ATR) spectroscopic mode for detection of species within the film. The output signal from the sensor is a change in absorbance (∆A) associated with electrolysis at the OTE. The sensor concept has been demonstrated by detection of ferrocyanide, Ru(bipy)32+, and Ru(CN)64- as analytes.1,2 In all of these examples, the analyte itself partitioned into the film and underwent electrochemical modulation, and one oxidation state was optically monitored as the sensor signal. The objective of this paper is to explore the possibility of using the spectroelectrochemical concept to detect an analyte that itself does not exhibit a measurable optical change associated with its electrolysis and, consequently, cannot be optically monitored directly. The approach being explored is to use a mediated electrode reaction in which the mediator undergoes spectroelectrochemical modulation. The analyte is detected indirectly by its effect on the optical signal from the modulated mediator. Mediators were introduced to aid in the electrochemistry of many biological redox species that exhibit slow electron transfer at an electrode due to a surrounding protein structure. Oxidation/ (1) Shi, Y.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 48194827. (2) Shi, Y.; Slaterbeck, A. F.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1997, 69, 3679-3686. (3) Slaterbeck, A. F.; Ridgway, T. H.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1998, 71, 1196-1203. (4) Gao, L.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 1999, 71, 40614068. 10.1021/ac991418m CCC: $19.00 Published on Web 06/29/2000

© 2000 American Chemical Society

reduction of the biological component (B) is, thus, indirect through the mediator (M) as shown below.5

In particular, mediators have been used in amperometric enzyme electrodes. In these types of electrodes, the mediators not only allowed for charge transfer but were also used to minimize interferences (due to low operating potential of a particular mediator) and to make measurements oxygen-independent.6 Several different types of mediators have been employed for these purposes. These include but are not limited to ferrocenes,7 potassium hexacyanoruthenate(II),8 [Ru(NH3)5py](PF6)2,8 tetrathiofulvalene,9 and Cu(II).10 This report demonstrates the spectroelectrochemical sensor concept with the mediator Ru(bipy)32+, which is trapped in the selective film, for the detection of ascorbate as a representative analyte. The primary purpose of this research is to demonstrate that the change in absorbance of a mediator in a spectroelectrochemical sensor can be used to determine the quantity of a nonabsorbing analyte. Factors such as scan rate, film thickness, mediator concentration in the film, and analyte charge are discussed in terms of their effect on sensor performance. EXPERIMENTAL SECTION Materials. The following chemicals were used: tetraethoxysilane (TEOS, Aldrich), Nafion (5% solution in lower aliphatic alcohols and water, Aldrich), tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate (Ru(bipy)32+, Aldrich), hydrochloric acid, potassium chloride, ascorbic acid, tris(hydroxymethyl)aminomethane (THAM), and citric acid monosodium salt. All reagents (5) Heineman, W. R. J. Chem. Educ. 1983, 60, 305. (6) Turner, A. P. F. Mediated Electrochemistry. In Advances in Biosensors; JAI Press Ltd.: Greenwich, CT, 1991; Vol. I. pp 125-169. (7) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill. H. A. O.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667-671. (8) Crumbliss, A. L.; Hill, H. A. O.; Page, D. J. J. Electoanal. Chem. 1986, 206, 327-331. (9) Wang, B.; Li, B.; Deng, Q.; Dong, S. Anal. Chem. 1998, 70, 3170-3174. (10) Haruyama, T.; Aizawa, M. Biosens. Bioelectron. 1998, 13, 1015-1022.

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were used without further purification. Ru(bipy)32+ solutions were made by dissolving the appropriate amounts in 0.2 M KCl solution. Ascorbate solutions were made by dissolving the appropriate amounts in 0.12 M KCl/0.04 M Tris HCl buffer (pH 7.0). Ascorbic acid solutions were made by dissolving the appropriate amount in 0.12 M KCl/0.04 M citric acid buffer (pH 3.6). ITO glass (1150 Ω/sq, 150 -nm-thick film on 1.1-mm glass, Thin Film Devices) was cut into 1-in. × 3-in. slides, scrubbed with Alconox, rinsed thoroughly with deionized water, dried, and then allowed to equilibrate with air prior to use. Preparation of Nafion-SiO2 Sensing Film. The selective film was prepared from a sol blended with a solution of Nafion in a ratio of 1:3 (v/v) as previously described.13,14 The sol was prepared from 4 mL of deionized water, 2 mL of TEOS, and 0.1 mL of 0.1 M HCl. The blend was mixed for 3 h until a singlephase sol resulted. The resulting blend of Nafion-SiO2 was spincoated onto ITO on a Headway spin-coater. Unless otherwise noted, slides were coated at a spin rate of 3000 rpm for 30 s. The ends of the ITO were masked with tape leaving areas uncoated for prism coupling. The coated slide was allowed to air-dry (at least overnight), submersed in supporting electrolyte (0.2 M KCl) overnight, and then submersed in Ru(bipy)32+ for mediator uptake (∼50 min). Instrumentation. The instrumental setup has been previously described.1-3 Briefly, a xenon arc lamp and monochromator (λ ) 450 nm, unless otherwise noted) served as the light source and was coupled to the sample cell using a 600-µm silica step-index optical fiber. The light from the fiber was coupled into the ITO slide using a microscope objective and a high-index coupling prism. The outcoming light was decoupled using another objective, prism, and silica fiber. Either a photomultiplier tube or a photodiode was used as a detector. Absorbance values were obtained by measuring the light intensity through the prismcoupled slide when the mediator was in the colorless form (Io) and the intensity when the mediator was in the colored form (I). Absorbance was defined as A ) log(Io/I). The electrochemistry was performed using a BAS potentiostat (CV-27) or a simple 2-A potentiostat built in our laboratory.3 The reference electrode was Ag/AgCl (3 M NaCl), and platinum was used as the auxiliary electrode. A syringe was used to make all of the sample injections into the cell. RESULTS AND DISCUSSION Chemical System. The model analyte/mediator system chosen for this study was ascorbic acid, in the form of ascorbate at pH 7.0, and Ru(bipy)32+/3+, respectively. Ascorbate was chosen because it is a good reducing agent with no optical changes in the visible range associated with its oxidation that could be used for its direct spectroelectrochemical detection. Ascorbate undergoes irreversible electrochemical oxidation to dehydroascorbic acid. Also, as the anionic conjugate base of neutral ascorbic acid, its charge can be controlled by pH, which provides a means of evaluating the effect of analyte charge. Ru(bipy)32+ was chosen (11) Heineman W. R. Introduction to Electroanalytical Chemistry. In Chemical Instrumentation: A Systematic Approach, 3rd ed.; Strobel, H. A., Heineman, W. R., John Wiley & Sons: New York; 1989; Chapter 27. (12) Goodman, A. M. Appl. Opt. 1978, 17, 2779-2787. (13) Shi, Y.; Seliskar, C. J. Chem. Mater. 1997, 9, 821-829. (14) Slaterbeck, A. F. Doctoral Dissertation, University of Cincinnati, 1998.

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Figure 1. Spectroelectrochemical modulation of Ru(bipy)32+ and its change in modulation upon addition of ascorbate (as designated by the arrow) at 450 nm for a Nafion-SiO2 thin film on ITO glass. Conditions: 0.1 M KCl, 0.04 M Tris HCl buffer (pH 7.0); Ru(bipy)32+ loading time, 50 min, 0.1 mM. (a) Cyclic voltammograms: The potential was scanned between 0.8 and 1.3 V at 5 mV/s. (1, 2) 0.0 mM ascorbate; (3, 4) 0.98 mM ascorbate. (b) Optical modulation: (1, 2) 0.0 mM ascorbate; (3, 4) 0.98 mM ascorbate.

as the mediator due to its reversible electrochemistry at a potential sufficiently positive to oxidize ascorbate (0.8-1.3 V), distinct spectral changes in the visible region ( ) 14 470 M-1cm-1 at λ ) 450 nm, Ru(II) complex), relative insensitivity to pH changes, suitable aqueous solubility, and negligible leaching after preconcentration into the film on the sensor surface. The spectroelectrochemical modulation of Ru(bipy)32+ incorporated into chemically selective films has been previously demonstrated.3,4 We chose the cation exchanger Nafion as the polymer for the film because it effectively traps the cationic mediator by electrostatic attraction. It was also chosen because of its suitable electrochemical properties and optical transparency.13 In addition, the selectivity of this coating has been demonstrated with Fe(CN)63- and Ru(bipy)32+.3,4 This will allow an additional parameter, analyte charge, to be evaluated. While this is probably not the most optimal mediator, it served as a good starting point for demonstration of the overall concept. Spectroelectrochemical Modulation of Mediator, Ru(bipy)32+. Mediation of ascorbate by Ru(bipy)32+/3+ is demonstrated by cyclic voltammetry and spectroelectrochemistry in Figure 1. Two cyclic voltammograms (1, 2) of Ru(bipy)32+ loaded into the film are shown in Figure 1a. The spectroelectrochemical modulation of Ru(bipy)32+/3+ preconcentrated into the selective film is shown as a function of time in Figure 1b. The first two optical modulations correspond to the two cyclic voltammograms (1, 2). The mediator ion exchanges into the film in the reduced, colored form Ru(bipy)32+. The oxidized, colorless form is then electrochemically generated by cycling from 0.8 to 1.3 V. This is seen by a decrease in absorbance in the absorbance versus time plot. The absorbance then increases upon electrochemical regeneration of the reduced form. The absorbance change due to modulation of the mediator (∆AM, where M designates mediator only) is proportional to the concentration of Ru(bipy)32+ loaded into the film.3,4 Note that the absorbance value does not reach zero for the oxidized form,

leaving a “trough” (minimum absorbance value greater than zero).4 This is due to the redox instability of the oxidized form, Ru(bipy)33+. This instability of the oxidized form of the mediator has been previously demonstrated in our laboratory in which the oxidized form is converted back to the reduced form without applying a potential to the electrode surface. Other factors (incomplete electrolysis, different potential sweep windows, potential scan rate) that have been previously investigated can also contribute to this trough.4 However, at the scan rate (5 mV/s) and potential sweep window used in the experiment shown in Figure 1, complete electrolysis of the mediator in the film is seen. The time for electrolysis of the analyte in the optical cell can be estimated using the Einstein equation (l ) (2Dt)1/2) and a few assumptions (the diffusion distance, l, is defined by the penetration of the evanescent wave (in this case, the thickness of the film since it is less than the penetration depth), and the diffusion coefficient is on the order of 1 × 10-11 cm2/s). In the case described, the calculated time for electrolysis is less than the time it takes to complete an electrochemical cycle; therefore, enough time has been allowed for all of the Ru(bipy)32+ to undergo electron transfer. Although this is only an approximation, it provides us with some confidence that all of the mediator is able to reach the electrode surface. Other evidence that provides us with confidence that complete electrolysis of the mediator in the film has occurred can be seen in the optical modulation peak shape as shown by the flattening of the peak as it reaches a minimum absorbance. Spectroelectrochemical Modulation with Ascorbate. Upon addition of ascorbate (cyclic voltamograms 3 and 4 in Figure 1a, peaks 3 and 4 in Figure 1b), a catalytic electrochemical reaction occurs which results in a change in both the electrochemical and optical responses. This change takes place by route of an electrode catalytic (EC) mechanism,11 in which Ru(bipy)32+ is being oxidized at the electrode surface to Ru(bipy)33+, which then chemically reacts with ascorbate to regenerate Ru(bipy)32+ and to form dehydroascorbic acid. This is seen in the cyclic voltammograms by a decrease in the cathodic peak and an increase in the anodic peak, which is characteristic of the EC mechanism. The decrease in cathodic peak is due to a large concentration of Ru(bipy)33+ being used up in the chemical reaction of converting ascorbate to its oxidized form. A concurrent change was seen in the optical response. The absorbance change shows a smaller modulation (∆AA, where A designates the addition of ascorbate to the modulation of Ru(bipy)32+) upon addition of ascorbate. This smaller optical modulation is due to ascorbate reducing some of the electrochemically generated Ru(bipy)33+ back to its reduced, colored form. The decrease in modulation amplitude (∆A ) ∆AM - ∆AA) is the basis for the spectroelectrochemical sensor. The electromodulation process irreversibly oxidizes ascorbate, which results in its depletion within the film (vide infra) and at the film/ solution interface. This causes a noticeable change in both the cyclic voltammogram and the optical modulation in the fourth scan, which was performed immediately after the third scan. This is noted by a larger optical modulation and a smaller anodic peak. This change is due to a smaller amount of ascorbate remaining within the film and at the film/solution interface to be converted to dehydroascorbic acid by Ru(bipy)33+ in the fourth voltammogram. Participation of the remaining ascorbate in solution is

Table 1. Effects of Mediator Concentration in the Film on ∆A with and without Ascorbate concn of mediatora (M)

concn of mediator (M) in the filmb

∆AMc

∆AAd

∆Ae

3.0 × 10-6 1.0 × 10-5 5.0 × 10-5 1.0 × 10-4 5.0 × 10-4

0.002 0.02 0.06 0.06 0.06

0.01 0.11 0.37 0.42 0.40

0.01 0.09 0.18 0.19 0.17

0.00 0.02 0.19 0.23 0.23

a Concentration of mediator used for preconcentration (t ) 50 min). Estimate of concentration of mediator in the film (as estimated by penetration depth and 10 reflections, and  ) 14 470 cm-1 M-1, where ∆A ) bc). c Where ∆AM is the change in absorbance of the mediator without ascorbate. d Where ∆AA is the change in absorbance of the mediator upon addition of ascorbate (1 × 10-3 M). e Where ∆A ) ∆AM - ∆AA. b

limited by its diffusion to the film interface. This behavior has also been noted in subsequent experiments in which the modulation was carried out further. To understand this system better, several parameters were evaluated to characterize conditions that affect sensor performance: scan rate, concentration of mediator in the film, thickness of the film, and analyte charge. Effect of Scan Rate. The absorbance versus time profiles (not shown) during modulation are affected by the scan rate. Results similar to those previously reported were found by varying the scan rate over a range of 2.5-20 mV/s.4 At faster scan rates, there is a larger trough under the optical modulations due to incomplete electrochemical cycling of the mediator. Slower scan rates reduce the presence of the trough and are more desirable. However, the trough is never eliminated in this system due to the redox instability of the mediator as previously mentioned. Upon the addition of ascorbate, a decrease in the peak amplitude of the mediator, Ru(bipy)32+, is seen at all of the scan rates studied. However, the change in peak amplitude is more significant at the slower scan rates associated with complete cycling of the optical cell. Therefore, if maximum ∆A is desired, slower scan rates need to be employed. For this reason, the experiments reported in the remainder of this paper were done at a scan rate of 5 mV/s, unless otherwise noted. Effect of Concentration of Mediator. The concentration of mediator in the film was controlled by varying the concentration of the mediator in the contact solution (preconcentration time, 50 min for a preequilibrated film). Under these conditions, the mediator loaded into the film should be homogeneously distributed across the film thickness. That is, the mediator is not concentrated at the outer edges of the film, creating a greater diffusion distance for the mediator to reach the electrode surface. The distribution of the mediator within the domains of the Nafion-SiO2 film is unknown at this time and will be investigated in further studies. It is probable that the Nafion-SiO2 film is not a homogeneous composite and will result in some segregation of the mediator throughout the film due to its different affinities for the Nafion and SiO2 domains. A summary of these experiments is given in Table 1. The concentration of the mediator in the contact solution used for preconcentration was varied from 3.0 × 10-6 to 5.0 × 10-4 M, yielding a concentration in the film varying from 0.002 to 0.06 M. At the highest concentrations in the films Analytical Chemistry, Vol. 72, No. 15, August 1, 2000

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studied in this experiment (0.06 M), ∼15% of the ion-exchange sites are occupied by the mediator.17 At low contact solution concentrations (3.0 × 10-6 and 1.0 × -5 10 M) only a small modulation (∆AM) in Ru(bipy)32+ is seen while at higher concentrations a larger modulation results. Upon addition of ascorbate at lower concentrations, the value for ∆AM ) ∆AA. That is, the colorless form of the mediator that was electrochemically generated was essentially immediately consumed by the reaction with ascorbate and this resulted in no change in absorbance (∆A) of the colored species. Higher concentrations of the mediator in the film resulted in a larger change in ∆AA upon addition of ascorbate. The range of a calibration curve is ultimately limited on the upper end by the concentration of the mediator in the film. It is apparent from these studies that the concentration of mediator in the film is a parameter that requires optimization. For these 1 mM ascorbate solutions, the highest concentration of mediator in the film (0.06 M) gave the greatest sensitivity of ∆A to ascorbate. Effect of Film Thickness. It has been previously shown that films of varying thickness can be achieved by varying the spincoating speed.13 The thickness of these films was determined using the optical interference fringe measurement.12 The thickness was calculated using

t)

Mabλaλb 2(λa - λb)(n12 - sin2θ0)1/2

(1)

where λa and λb are the wavelengths of the two fringe extremes (minimums or maximums), n1 is the refractive index of the bulk film, θo is the angle of incidence of the measuring beam (0°) with respect to the normal to the film surface, and Mab is the number of fringes between λa and λb. The refractive index used for the Nafion-SiO2 composite film was 1.46. Several assumptions have been made in order to compare the results obtained for soda lime glass to those obtained for ITO. First, the Nafion-SiO2 films were spin-coated onto soda lime glass slides under conditions similar to those prepared on ITO. By using soda lime glass slides in place of ITO, the observed fringe pattern is due solely to the Nafion-SiO2. In addition, although the wettability and chemical composition of these surfaces are different, they are assumed to not contribute significant differences to the measurement and provide adequate data for our use of the measurement. The effects of spin rate on film thickness are clearly shown in Figure 2. A large change in film thickness was seen between 500 and 1500 rpm, with a smaller change with increasing spin speed. It was therefore decided to compare a thick film (spin rate, 500 rpm; 480 nm) and a thin film (3000 rpm; 300 nm) in further studies. Figure 3 shows that film thickness has an effect on the optical modulation during incorporation of Ru(bipy)32+ into the film. (15) Wightman, R. M.; May, L. J.; Michael, A. C. Anal. Chem. 1988, 60, 769A79A. (16) Harrison, D. J.; Turner, R. F. B.; Baltes, H. P. Anal. Chem. 1988, 60, 20022007. (17) White, H. S.; Leddy, J.; Bard, A. J. J. Am. Chem. Soc. 1982, 104, 48114817.

3464 Analytical Chemistry, Vol. 72, No. 15, August 1, 2000

Figure 2. Variation in film thickness as a function of spin speed. Composite film, Nafion-SiO2 (1:3 v/v). Each point is an average of three slides with the error bar representing the standard deviation. Curve fit to exponential decay: y ) yo + ae-bx.

Figure 3. Optical modulation during incorporation of Ru(bipy)32+ into Nafion-SiO2 film (10 mV/s, 0.8-1.3 V (triangular wave)): (a) 130 µM Ru(bipy)32+ into a =300-nm thin film; (b) 130 µM Ru(bipy)32+ into a =480-nm thick film.

Nearly complete optical modulation of the Ru(bipy)32+/3+ couple occurred in the thin film. That is, the film was sufficiently thin for complete electrolysis to occur during a potential cycle. The small trough (absorbance minimum) below the modulation was due to the chemical instablity of the oxidized form, which was quickly converted back to the reduced state, biasing the colored species. In the thicker film, the trough was much more apparent. This is due to some Ru(bipy)32+ being farther from the electrode surface where the longer diffusional distance to reach the electrode for electrolysis results in a smaller fraction of the optically observable Ru(bipy)32+ being oxidized. Another apparent difference between the thick and thin film was the time in which equilibrium loading of the film was reached. Film equilibration happened in at least half of the time for the thin film (∼1 h compared to greater than 2 h for a thick film). Other factors that can affect the trough and preconcentration of the mediator have been discussed previously.4

Figure 5. Cyclic voltammetry for ascorbic acid (1.2 mM, 0.12 M KCl/0.04 M citric acid buffer (pH 3.6)) and ascorbate (1.2 mM, 0.12 M KCl/0.04 M Tris HCl buffer (pH 7.0)) in a Nafion-SiO2 film without any mediator on ITO glass. The potential was scanned between -0.3 and 1.3 V, at a scan rate of 50 mV/s.

Figure 4. Optical modulation for Ru(bipy)32+ at varied scan rates (50, 25, 5, and 1 mV/s). Scan rate changes marked by arrows. The potential was scanned from 0.8 to 1.3 V (triangular wave): (a) thin film =300 nm; (b) thick film =480 nm.

A scan rate study over the range of 50-1 mV/s was done on both of these films after preconcentration was completed, as depicted in Figure 4 by the absorbance versus time profiles. Again there were significant differences between the scans for the different film thicknesses. In the case of the thin film (Figure 4a), at 1 mV/s the optical modulation suggests substantial (78% electrolyzed) oxidation/reduction of the incorporated mediator. At faster scan rates, a larger trough appeared under the modulation. At these faster scan rates, response is limited by diffusion in the film, which has been previously reported.4 This phenomenon was more enhanced in a thicker film as shown in Figure 4b. In comparison, the percent electrolyzed at 1 mV/s is only 46% in the thicker film as evidenced by the substantially larger trough. This is due to a combination of the thickness of the film and the chemical instability of the mediator in the oxidized form. Therefore, to achieve a maximum ∆AM for this system, a thinner film is desired. A final comparison of films of different thickness was made by adding ascorbate to the solution. The change in ∆A was measured upon addition of ascorbate for both the thick (∆A ) 0.20) and thin (∆A ) 0.30) films. As anticipated, a larger change in ∆A was observed for the thinner film, which can be attributed to the more complete modulation of Ru(bipy)32+ in the thinner film. Because of their greater sensitivity to ascorbate, thinner films were then used for all subsequent studies. Effect of Charge of Analyte. Several experiments were done to evaluate the effect of analyte charge on sensor performance. The pH of the analyte solution was varied to give a comparison between ascorbic acid and ascorbate (pKa ) 4.2). At pH 7.0, the analyte (ascorbate) has a net negative charge whereas, at pH 3.6, ascorbic acid is neutral. One would expect the neutral analyte to partition into the film to a greater extent than the anionic analyte, which would be repelled by the anionic sulfonate groups in the

Nafion-SiO2 film. Nafion has been used as a protective or selective coating material on a number of different electrodes for a wide range of applications. Wightman et al. used a pure Nafion coating to repel ascorbate and attract positively charged neurotransmitters.15 Nafion membranes have also been used in glucose sensors to reduce interferences from ascorbate and urate.16 In both examples, a pure Nafion membrane was employed, which differs from our composite membrane of Nafion-SiO2. A pure Nafion membrane was not used in these experiments because it does not allow for adequate adhesion of the film onto the ITO surface. The partitioning of ascorbate and ascorbic acid into the film was studied using cyclic voltammetry. The two systems of varying pH were first studied using a Nafion-SiO2-coated ITO electrode without any mediator. Optical data are not shown since no change in absorbance was observed due to the lack of absorbance of ascorbic acid/ascorbate and their oxidation product in the visible region. The same electrode/film was used for this study in order to be able to quantify the effects of pH on the analyte. The presence of an electrochemical response, as shown by the cyclic voltammograms in Figure 5, showed that both forms of the analyte penetrated into the film. A difference in both the shape and magnitude of the anodic peak is seen between the two forms of the analyte. Ascorbic acid exhibited a significantly larger anodic peak, which is consistent with the neutral species, penetrating the film more efficiently than the negatively charged ascorbate. However, the negatively charged ascorbate was able to penetrate the film to some extent, even with the film composition containing Nafion. This could be due to the SiO2 microdomains that may exist in the Nafion-SiO2 film. These domains lacking the appropriate charge to repel the analyte from the film would allow some analyte to penetrate through the film to the electrode surface. A similar set of experiments was conducted after loading the film with the mediator. The corresponding cyclic voltammograms and optical modulation are shown in Figure 6. There was a slight difference in the cyclic voltammograms of Ru(bipy)32+ in buffer alone (1, and 3 compared with 4) at the two pH's studied. This slight change was also seen in the corresponding optical signal by a slightly smaller modulation obtained for the mediator at a pH 7.0, suggesting a small pH dependence. Upon addition of ascorbate (5) and ascorbic acid (2), the EC catalytic reaction occurred resulting in changes in both the electrochemical and optical modulation. A larger anodic peak current was seen in the cyclic voltammogram for ascorbic acid Analytical Chemistry, Vol. 72, No. 15, August 1, 2000

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Figure 8. Sensor calibration curve for optical detection of ascorbate: film thickness, =300 nm; scan rate, 5 mV/s; film composition Nafion-SiO2, 1:3 v/v. The concentration values of ascorbate in the plot are 0.26, 0.56, 0.87, 2.06, and 3.28 mM.

Figure 6. Cyclic voltammograms (a) and optical modulation (b) of Ru(bipy)32+ in buffer alone and in response to ascorbic acid and ascorbate: (1) Ru(bipy)32+ in 1.2 mM, 0.12 M KCl/0.04 M citric acid buffer (pH 3.6); (2) Ru(bipy)32+ upon addition of ascorbic acid in 1.2 mM, 0.12 M KCl/0.04 M citric acid buffer (pH 3.6); (3) Ru(bipy)32+ in 1.2 mM, 0.12 M KCl/0.04 M citric acid buffer (pH 3.6); (4) Ru(bipy)32+ in 1.2 mM, 0.12 M KCl/0.04 M Tris HCl buffer (pH 7.0); and (5) Ru(bipy)32+ upon addition of ascorbate in 1.2 mM, 0.12 M KCl/0.04 M Tris HCl buffer (pH 7.0). The potential was scanned between 0.8 and 1.3 V, at a scan rate of 5 mV/s.

Figure 7. Diagram of the sensor concept: (1) mediated detection of ascorbate using Ru(bipy)32+/3+; (2) direct oxidation of ascorbate at the electrode surface.

compared to that for ascorbate due to its greater partitioning into the film. Surprisingly, however, the optical modulation showed a larger change (1.2×) in absorbance for ascorbate. This contradiction can be explained by the varying degrees to which the analyte reacts with the mediator versus direct electrolysis at the electrode surface. Ascorbic acid penetrates the film to a greater extent than ascorbate, causing a substantial fraction of the ascorbic acid to undergo direct electron transfer at the electrode surface. This leaves less ascorbic acid to react with the mediator, resulting in a smaller effect on the optical modulation. This dual mechanism by which the analyte reacts with the mediator, and directly at the electrode surface, is depicted in Figure 7. The reaction between the analyte and mediator in this case occurs not only at the film/ solution interface but directly in the film. To achieve a larger ∆A, one would want to deny the analyte access to the electrode surface, by forcing it to react with a mediator. Quantification in Pure Samples. A representative calibration plot indicating the sensor performance for ascorbate is shown in Figure 8. Several other calibration curves were prepared on separate films/electrodes, and they differ by a small magnitude in ∆A. The differences in ∆A values may be attributed to differences in film composition, distribution of ion-exchange sites 3466 Analytical Chemistry, Vol. 72, No. 15, August 1, 2000

within the film, and differences in the electrodes. The calibration plot clearly demonstrates the quantification of ascorbate using this sensor design. The plot has a rather limited linear range at low concentrations (less than 2 × 10-3 M) and deviates negatively at higher concentrations. At higher concentrations, all of the Ru(bipy)23+ is being converted to Ru(bipy)22+ by ascorbate and cannot be elctrochemically regenerated sufficiently rapidly. This can be seen in the optical modulation by a greater trough (or decrease of ∆A) with increasing concentration of ascorbate. At a maximum amount of ascorbate (under these conditions, =3.2 × 10-3 M), no modulation of Ru(bipy)32+ can be seen. It may be possible to alter the range of linearity and response of the sensor by changing the mediator, its potential window, and the nature by which the analyte interacts with the mediator. Initial studies using methyl viologen (potential window -0.25 to -0.85 V) as a mediator altered the response of the sensor by decreasing the ∆A by a factor of 6 (∆A (Ru(bipy)32+) ) 0.30, ∆A (methyl viologen) ) 0.05). The mediator, Ru(bipy)32+, used in the present study suffers from autoreduction of electrogenerated Ru(bipy)33+. This causes a decrease in peak amplitude as noted by the trough in a previous section of this paper. By choosing a mediator without this problem, a larger peak amplitude should be achieved with subsequent lower detection limits. The detection limit will also depend on the difference in molar absorbtivities (∆) between the mediator and its electrogenerated product. CONCLUSIONS The concept of a spectroelectrochemical sensor with mediated detection for nonabsorbing analytes has been demonstrated. More specifically, we have demonstrated that both ascorbic acid and ascorbate can be detected using Ru(bipy)32+ as a mediator in this sensor design. Factors affecting sensor performance include scan rate, film thickness, concentration of the mediator in the film, and analyte charge. This sensor concept can be extended to many other analytes by appropriately choosing a mediator, potential window, and composite film. Furthermore this concept can be extended to biosensors, which will be discussed in a future paper. In this case, in order for an analyte to be detected it must first partition into the sensing film and undergo a reaction with the biosensing element, and either this product or a mediator must be electroactive at the applied potential of the working electrode and have a change in absorbance at the chosen wavelength of light.

Although the selectivity of the sensor has not been demonstrated in this paper, the current sensor design has three modes of selectivity: electrochemistry, spectroscopy, and the reaction of the mediator with the analyte of interest. A characteristic of this current sensor design is that it can suffer from interferences if the reaction with the mediator is not specific for a given

analyte. This will be explored further in a separate paper as well as other issues affecting the sensor’s selectivity. Received for review December 10, 1999. Accepted April 27, 2000. AC991418M

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