Spectroelectrochemical Sensing Based on Multimode Selectivity

Sep 15, 1997 - A new type of spectroelectrochemical sensor that demonstrates three .... The selectivity of the third phase, the polyelectrolyte−SiO2...
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Anal. Chem. 1997, 69, 3679-3686

Spectroelectrochemical Sensing Based on Multimode Selectivity Simultaneously Achievable in a Single Device. 1. Demonstration of Concept with Ferricyanide Yining Shi, Andrew F. Slaterbeck, Carl J. Seliskar,* and William R. Heineman*

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

A new type of spectroelectrochemical sensor that demonstrates three modes of selectivity (electrochemistry, spectroscopy, and selective partitioning) is demonstrated. The sensor consists of an optically transparent electrode (OTE) coated with a selective film. Sensing is based on the change in the attenuation of light passing through the OTE that accompanies an electrochemical reaction of the analyte at the electrode surface. Thus, for an analyte to be detected, it must partition into the selective coating and be electrolyzed at the potential applied to the electrode, and either the analyte or its electrolysis product must absorb light at the wavelength chosen. Selectivity for the analyte relative to other solution components is obtained by choice of coating material, electrolysis potential, and wavelength for optical monitoring. The sensor concept is demonstrated with an OTE consisting of an indium-tin oxide coating on glass that has been overcoated with a sol-gel-derived charge-selective thin film. Attenuated total reflection (ATR) is used as the optical detection mode. The selective coating was an anionically charge-selective sol-gel-derived PDMDAAC-SiO2 composite film, where PDMDAAC ) poly(dimethyldiallylammonium chloride). Fe(CN)64- was used as a model analyte to demonstrate that the change in the transmittance of the ATR beam resulting from oxidation of Fe(CN)64- to Fe(CN)63- can be used to quantify an analyte. The unoptimized sensor exhibited the following characteristics: linear range, 8.0 × 10-6-5.0 × 10-5 M; sensitivity, 8.0 × 103∆A/M; and detection limit, 8.0 × 10-6 M. Spectroelectrochemistry has been employed for over three decades to investigate a wide variety of inorganic, organic, and biological redox systems.1-6 Its main application to these redox systems has been for the study of mechanisms of electrode reactions. Spectroelectrochemistry is generally accomplished with (1) Kuwana, T.; Darlington, R. K. Anal. Chem. 1964, 36, 2023. (2) Kuwana, T.; Winograd, N. Spectroelectrochemistry at Optically Transparent Electrodes. I. Electrodes under Semi-Infinite Diffusion Conditions. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1974; Vol. 7, pp 1-78. (3) Heineman, W. R.; Blount, H. N.; Hawkridge, F. M. Spectroelectrochemistry at Optically Transparent Electrodes. II. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; pp 1-113. (4) Kuwana, T.; Heineman, W. R. Acc. Chem. Res. 1976, 9, 241. (5) Heineman, W. R. Anal. Chem. 1978, 50, 390A. (6) Gale, R. J., Ed. Spectroelectrochemistry: Theory and Practice; Plenum Press: New York, 1988. S0003-2700(97)00322-3 CCC: $14.00

© 1997 American Chemical Society

optically transparent electrodes (OTEs) such as thin films of gold, platinum, and indium-tin oxide (ITO) deposited on glass, quartz, or plastic substrates and fine gold minigrids. Optical signals generated by electrochemical manipulation on these OTEs can be detected by either transmission or attenuated total reflection (ATR). In the first configuration, the interrogating beam of light is directed normal to the OTE surface and passes through the entire electrochemical cell. In the latter configuration, the interrogating light is coupled, at an appropriate angle, to and propagates within the OTE optical element by internal reflection. Attenuation of the light in the ATR configuration occurs within a wavelength or so adjacent to the OTE electrode surface through interaction between the evanescent field of the totally reflected light and light-absorbing species present in this region. The primary advantage of spectroelectrochemistry is the crosscorrelation of information attainable from simultaneous electrochemical and optical measurements. Thus, if such information correlation can be incorporated into the selectivity system of a sensor, i.e., the sensing is based on spectroelectrochemical responses in addition to other levels of selectivity, such as those which would be provided by a selective coating or membrane, then the overall selectivity of the sensor would be significantly enhanced. Thus far, only a few attempts have been made toward coupling electrochemistry to spectroscopy as the basis of chemical sensing. For example, although dual selectivity based on optical detection of electrochemically generated species7 and the electrochemical detection of photochemically generated species8 have been reported, the electrochemical potential merely functions to regenerate species for “resetting” purposes for reuse of these sensors. Also, neither of these devices has incorporated a selective coating or membrane as an additional level of selectivity in the sensor. Piraud and co-workers9,10 reported a planar waveguide sensor for chlorine. Their sensor consisted of an ion-exchanged channel waveguide coated with three different layers: a silica buffer layer, a thin conducting layer (ITO), and a chlorine-sensitive thin layer (7) Aizawa, M.; Tanaka, M.; Ikariyama, Y. A Fiber-Optic Electrode for Optoelectrochemical Biosensors. In Chemical Sensors and Microinstruments; ACS Symposium Series 403; American Chemical Society: Washington, DC, 1989, pp 129-138. (8) Cohen, C. B.; Weber, S. G. Anal. Chem. 1993, 65, 169. (9) Piraud, C.; Mwarania, E.; Wylangowski, G.; Wilkinson, J.; O’Dwyer, K.; Schiffrin, D. J. Anal. Chem. 1992, 64, 651-655. (10) Piraud, C.; Mwarania, E. K.; Yao, J.; O’Dwyer, K.; Schiffrin, D. J.; Wilkinson, J. S. J. Lightwave Technol. 1992, 10, 693-699.

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Figure 1. Diagram of the interfacial region of the new sensor. The optical phases for an ITO tin float glass-based structure are indicated. The selectivity of the third phase, the polyelectrolyte-SiO2 thin film, is also shown schematically. b, analyte; 2, excluded interferent; 9, included interferent.

of lutetium biphthalocyanine. The chemical mechanism of sensing chlorine was based on an oxidation-reduction reaction of chlorine with the sensing organic layer. Piraud et al. found sensitivity at the low parts-per-million level. Electrochemical modification of the lutetium biphthalocyanine layer was done only to regenerate (“reset”) the sensor after chlorine exposure. We report herein a new type of spectroelectrochemical sensor based on the incorporation of ATR spectroscopy at an OTE that is coated with a selective film. The sensor concept is illustrated by the schematic 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 the OTE surface, i.e., it is a chemically modified electrode. The evanescent wave at the reflection point penetrates the film so that electrochemical events within the film can be monitored optically. In its operation, a potential excitation signal is applied to the OTE to cause electrolysis of analyte that has partitioned into the film. This is illustrated in Figure 2 for potential step and cyclic potential scan excitation signals. The change in the light propagated by ATR due to disappearance of analyte by its electrolysis or appearance of an electrolysis product is monitored. Schematic optical response signals are shown below their respective potential excitation signals to illustrate these cases. The single potential step signal illustrates the case of optical monitoring of the species electrogenerated from the analyte by its oxidation. Transmittance decreases rapidly until the optical cell is “filled” with electrogenerated species, at which point it levels off. The cyclic potential scan illustrates a case in which the analyte itself is optically monitored and an increase in optical signal accompanies the conversion of analyte into a nonabsorbing species by reduction during the forward scan. Transmittance increases and decreases in concert with the cyclic scan for a reversible couple as it is cycled through its two redox forms and the optical cell is “emptied” and “refilled” by reversible electrolysis. Quantitation of the analyte is based on the magnitude of the change in transmittance (or calculated absorbance), which is proportional to concentration of analyte in the film, which, in turn, is proportional to its concentration in the sample as defined by the partition coefficient. Thus, the analytical signal is an optical change that occurs in response to an electrochemical event. The primary purpose of this initial research is to demonstrate that the change in the absorbance of a spectroelectrochemical sensor of this general type can be used to quantify an analyte. In 3680 Analytical Chemistry, Vol. 69, No. 18, September 15, 1997

Figure 2. Illustration of the applied electric potentials and expected transmittance changes. Column a depicts the case of an applied step potential; column b depicts a cycled triangular potential.

this paper, we demonstrate the detection of ferricyanide at a sensor consisting of ITO coated on glass that is overcoated with a solgel-derived anion-selective thin film. Fabrication, operation, and performance of the sensor are described and discussed. This sensor, though not yet optimized, clearly demonstrates the proposed method of quantitation.

Although not the subject of this paper, a significant aspect of this sensor is the three modes of selectivity that are simultaneously achieved in a single device. These three modes of selectivity are electrochemistry, spectroscopy, and selective partitioning. This concept, therefore, adds an additional component of selectivity compared with many of the existing chemical sensors and is very important for its practical application. Thus, for an analyte to be detected, it must partition into the selective coating and be electrolyzed at the potential applied to the electrode, and either the analyte or its electrolysis product must absorb light at the wavelength chosen. Selectivity for the analyte relative to other solution components is obtained by choice of coating material, electrolysis potential, and wavelength for optical monitoring. In a future paper, we will demonstrate the role of the three modes of selectivity in detection of analytes in the presence of direct interferents. EXPERIMENTAL SECTION Materials. The following chemicals were used: tetraethoxysilane (TEOS, Aldrich), poly(dimethyldiallylammonium chloride) (PDMDAAC, 20 wt % aqueous solution, Polysciences), potassium ferricyanide and potassium ferrocyanide (Aldrich), and potassium nitrate (Fisher Scientific). All reagents were used without further purification. Reagent solutions were all made by dissolving the appropriate amounts of chemicals into 0.1 M potassium nitrate solution (prepared with deionized water from a Barnstead water purification system). ITO tin float glass (11-50 Ω/in.2, 150 nm thick ITO layer over tin float glass) was purchased from Thin Film Devices. This ITO tin float glass was cut into 1 in. × 3 in. slides, scrubbed with Alconox, and rinsed thoroughly with deionized water and then 1-propanol prior to use. Then the alcohol-washed ITO glass slides were equilibrated with air before use. Preparation of Silica Sol-Gel and Polyelectrolyte Stock Solutions. Silica sol-gel solutions were prepared according to a previously reported protocol.11 Briefly, 4.0 mL of TEOS, 2.0 mL of deionized water, and 100 µL of 0.1 M HCl were combined in a sealed 30 mL vial and stirred at room temperature. After 3 h of stirring, a transparent and single-phase sol-gel solution resulted. Polyelectrolyte solutions were either used directly from the stock vial or made by a simple dilution with deionized water. Formation of Polyelectrolyte-SiO2 Composite Thin Films on ITO Glass Substrates. Incorporation of the polyelectrolyte, PDMDAAC, into silica sol-gel solutions was performed by mechanically blending the polyelectrolyte solution with the silica stock solution, i.e., the two solutions were blended under stirring for ∼5 min. The spin-coating solutions for making a PDMDAACSiO2 film were prepared using a solution ratio of PDMDAAC to SiO2 of 2:3 (v/v) and diluting the blend solution by 2:7 with deionized water. The polyelectrolyte-SiO2 films were then formed by spin-coating the blend solutions onto the ITO glass slides on a Headway spin-coater operated at 3000 rpm for 30 s. Both ends of the ITO side of the glass slides were masked with tape prior to spin-coating, leaving these sections of ITO surface uncoated for prism coupling. All PDMDAAC-SiO2 films on ITO glass were baked overnight in an oven at ∼50 °C. Thicknesses of the films were determined with a Hewlett-Packard 8453 diode array spectrophotometer using an interference fringe method.12 Instrumentation. The instrumental setup for performing ATR spectroelectrochemistry is standard and consists of the following. (11) Shi, Y.; Seliskar, C. J. Chem. Mater. 1997, 9, 821-829. (12) Goodman, A. M. Appl. Opt. 1978, 17, 2779.

Figure 3. Diagram of the ATR-based spectroelectrochemical sensor cell.

The light source was a xenon arc lamp (ILC Technology, Model 302UV). Light from the source was focused into a 1 mm optical fiber that was butt-coupled to a Spex Minimate or a Bausch and Lomb 0.25 m monochromator. Light of the monochromatorselected wavelength was coupled to a 600 µm silica step-index optical fiber (Fiberguide Superguide G, NA ) 0.22) with a microscope objective positioned directly against the exit slit of the monochromator. The light from this fiber was coupled into the ITO glass slide of the spectroelectrochemical cell with a Schott SF6 coupling prism (Karl Lambrecht, Chicago, IL). A highviscosity refractive index standard fluid (Cargille, n ) 1.517) was used to span the prism/ITO glass gap. The angle of the incident light into the prism and the alignment of the cell were adjusted to maximize the ATR throughput, as determined by measuring the intensity of the decoupled light. Light propagating through the ITO glass slide was decoupled by using another a SF6 coupling prism, and the emergent beam was focused into a silica fiber bundle (C Technology), which transported the light to a Spex 1870 0.5 m monochromator fitted with a phototube (Hamamatsu R955). The current from the phototube was detected by a gated integrator (Stanford Research Systems SR250) run continuously, digitized, and sent to a PC (IBM 80286). PC-logged data files were imported into commercial spreadsheet and graphics algorithms for data manipulation and plotting. For transmission spectroelectrochemistry at normal incidence, a Hewlett-Packard 8453 or 8452 diode array spectrophotometer was used to measure UV-visible absorption spectra. All electrochemistry was performed with a Bioanalytical Systems CV-27 potentiostat, and the resultant signals were plotted on a Houston Instruments Model 100 X-Y plotter. Spectroelectrochemical Cells. ATR spectroelectrochemical measurements were performed on the assembled plastic cell (1.0 cm × 4.5 cm × 2.5 cm) shown in Figure 3. Electrical contacts with the ITO glass slide were made with four copper clips attached to each exposed corner of the ITO glass. Transmission measurements at normal incidence to the ITO glass slides were done using a plastic cell (4 cm × 3 cm × 3 cm) which had UV-grade silica end windows. Electrical contact with the ITO glass slide in this cell was made with a single large metal clip. The reference electrodes for both spectroelectrochemical cells were BAS Ag/AgCl, and platinum wires were used as the auxiliary electrodes. Procedures for Spectroelectrochemistry. Aqueous 0.1 M KNO3 solution was employed as the supporting electrolyte in all experiments. All films coated on ITO glass were soaked in Analytical Chemistry, Vol. 69, No. 18, September 15, 1997

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deionized water overnight prior to any experiment to ensure sufficient equilibration with water. These water-equilibrated films were then stored in a capped, moisturized bottle containing a small amount of water for immediate or later use. For the first-time sensing measurement of Fe(CN)64- in pure sample solutions, the ATR cell assembled with a film-coated ITO glass slide was filled with ∼10 mL of pure 0.1 M KNO3 and cycled one or two times to ensure good electrical connections and electrochemical communication between the ITO glass slide and the electrolyte solution. This blank solution was then replaced with a Fe(CN)64--containing sample solution of about the same volume under opened-circuit condition. After 2-3 min, the system was switched to closed-circuit condition, for which the reduction potential was preset, and optical signal recording began. After being held for about 2-3 min, the potential was stepped to the oxidation potential with the optical signal recording continuing. The subsequent measurements were performed by simply replacing the used sample solution with a new one or by successive additions of a concentrated stock solution. RESULTS AND DISCUSSION General Description. The spectroelectrochemical sensor incorporates the elements of ATR spectroscopy, a charge-selective thin film, and an ITO OTE into one device. The general concept of the sensor is illustrated in Figure 1. The sensor consists of four distinct phases. Phase 1 consists of an underlying optical element that serves to define the channel or layer in which light propagates through the sensor by total internal reflection. This element can be as simple as a microscope slide or as sophisticated as a micromachined planar waveguide substrate. In general, phase 2 consists of the guided wave layer, in which total internal reflection of light is achieved. However, in the present case, we have used commercially available ITO-coated tin oxide float glass, where the first two phases are poorly defined due to the graded index of the tin oxide glass beneath the ITO coating. In the case of tin oxide float glass alone, it is known13 that the graded layer of SnO2 established by the float process can support one or more optical waveguide modes. We have intentionally arranged the prism coupling to the ITO-coated tin float glass to favor the base glass multiple reflection transmission of light rather than to excite the waveguide modes preferentially. Therefore, in our sensor configuration, like the multiple reflection spectroelectrochemical systems of Kuwana and coworkers,14 one has an optical guiding medium, essentially the tin float glass substrate of the ITO OTE (base glass 1 mm thick, n1 ) 1.53), as phase 1. Phase 2 is the ITO coating, which has been deposited on the tin-rich side of the glass substrate (0.150 µm thick, n2 ) 1.95). Beneath this coating lies a graded region of SnO2 established during the glass formation by the float process. Taken together with the ITO coating layer, our optical fringe measurements indicate an effective ITO/SnO2 layer thickness of about 400 nm. The third phase is the sol-gel-derived chargeselective film (0.75 µm thick, dry film, n3 ) 1.47) spin-coated on the ITO layer (Figure 1). The refractive index of the film is higher than that expected for silica (1.46) due to the addition of the polymer. Phases 1 and 2 are over-coated with phase 3, an analyteselective film. In the present example, we have used a new silica (13) Osterberg, H.; Smith, L. W. J. Opt. Soc. Am. 1964, 54, 1078. (14) Hansen, W. N.; Kuwana, T.; Osteryoung, R. A. Anal. Chem. 1966, 38, 18101821.

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composite material that consists of a polyelectrolyte-doped silica optical material made by the sol-gel technique.11 The refractive index of this selective film must be less than that of the underlying phase to achieve total internal reflection at this important interface. The final phase consists of the external medium surrounding the sensor, typically an aqueous solution of refractive index, n4, lower than that of the selective film. Our illustration uses ATR as the optical detection mode, although other optical modes are possible. In the ATR mode, the evansescent wave established at the electrode-selective film interface extends about one wavelength of light into the selective film phase and probes the selective coating. The sensor is capable of measuring a change in optical absorbance (complex refractive index) in the coating caused by electrolysis of the analyte at this surface. In this optical configuration, the thickness of the third phase is roughly the penetration depth of the evanescent field (i.e., dp), which decays exponentially into phase 3. Hence, contributions of optical attenuation due to interactions between the evanescent field and chromophore species that do not partition into phase 3 are negligible. Therefore, for an analyte to contribute substantially to the optical signal, it must partition into the selective coating. In order for it to contribute to a change in the optical signal resulting from electrolysis, the analyte must also reach the electrode surface and undergo electron transfer. It is important to note that signal acquisition for this type of sensor is both optical and electrochemical. This suggests the possibility of using signal modulation techniques which can enhance signal-to-noise characteristics for the improvement of detection limits, as has been previously demonstrated with spectroelectrochemistry.15 Also, further enhancement of selectivity of the sensor can be realized by signal correlation processing on the two sets of sensing response data (i.e., both optical and electrochemical responses). Chemical System. To demonstrate the basic functioning and selective sensing capability of the present sensors, we chose the ferri-/ferrocyanide couple, Fe(CN)63-/4-, as an example of an anionic analyte. This couple has a well-defined, reversible electro-

FeII(CN)64FeIII(CN)63+ e- a (absorbs at 420 nm) (transparent at 420 nm) (E° ) 0.25 V vs Ag/AgCl)

chemistry that is accompanied by distinct spectral changes in the visible wavelength region. In addition, this couple has well-studied partition properties into charge-selective materials,11 hence making it an ideal prototype analyte for our purposes. We have used a PDMDAAC-SiO2 film as an anion-exchange selective layer for partitioning of the Fe(CN)63-/4- couple. Solgel-derived PDMDAAC-SiO2 composite films have been shown to possess a series of properties suited to construction of both electrochemical16 and optochemical sensors,11 such as ionexchangeability, nanoscale porosity, high optical transparency, variable thickness (0.1-3.0 µm), and physicochemical stability. (15) Winograd, N.; Kuwana, T. Anal. Chem. 1971, 43, 252-259. (16) Petit-Dominguez, M. D.; Shen, H.; Heineman, W. R.; Seliskar, C. J. Anal. Chem. 1997, 69, 703-710.

Figure 4. (b) Series of cyclic voltammograms recorded with a PDMDDAC-SiO2 coated ITO OTE sensor during uptake of Fe(CN)64-. Voltammograms were recorded by cycling the sensor in 0.05 mM Fe(CN)64- (ν ) 20 mV/s, E/V vs Ag/AgCl). (a) Absorbance spectrum of the sensor at the positive potential limit of + 0.55 V. (c) Absorbance spectrum of the sensor at the negative potential limit of -0.30 V. The absorbance measurements were performed in a single-pass transmission geometry (normal incidence) after the sensor was immersed in 1.0 mM Fe(CN)64for 1 h.

Figure 5. Cyclic voltammograms for the same PDMDDAC-SiO2coated ITO OTE sensor described in Figure 4. For comparison, a cyclic voltammogram for a bare ITO electrode cycled in 0.05 mM Fe(CN)64- is shown as curve 1. Curve 2 represents the sensor of Figure 4 after 42 cycles in 0.05 mM Fe(CN)64-. Curve 3 shows the Fe(CN)64- -loaded sensor after cycling in neat 0.1 M KNO3 (supporting electrolyte). Cyclic voltammetry parameters were ν ) 10 mV/s, E/V vs Ag/AgCl.

Most importantly, it was found17 that this silica-based, polyelectrolyte-containing composite material can also form uniform and well-adhered thin films on ITO glass by the spin-coating method, thereby making it more useful in ITO OTE-based spectroelectrochemical sensing applications compared with, for example, SiO2 on gold surfaces. The reason for this affinity difference is unclear. However, enhanced adherence observed in the latter system might be attributed, in part, to the attractive molecular interactions originating from the reactive nature of the ITO material (a metal oxide semiconductor)18 and the high OH group content of the silica gel material.19 (17) Shi, Y.; Seliskar, C. J., unpublished results. (18) Moses, P. R.; Wier, L.; Murray, R. W. Anal. Chem. 1975, 12, 1882. (19) Brinker, C. J.; Scherer, G. W. Sol-Gel Science; Academic Press: San Diego, CA, 1990; p 370.

Cyclic Voltammetry and Transmission Spectroelectrochemistry of Fe(CN)63-/4- at the PDMDAAC-SiO2 ITO OTE. Electrochemical and spectral properties of Fe(CN)63-/4- at a solgel-derived PDMDAAC-SiO2 ITO OTE that are pertinent to the sensor performance were first investigated using cyclic voltammetry and transmission spectroelectrochemistry. Figure 4b shows repetitive scan cyclic voltammograms at a coated OTE that had been equilibrated with water beginning about 5 s after immersion into 5.0 × 10-5 M Fe(CN)64-. The increasing peak currents with each scan clearly show the time-dependent partitioning of Fe(CN)64into the PDMDAAC-SiO2 film from the aqueous solution by the anion-exchange process. The partitioned Fe(CN)64- permeates to the electrode surface, where it undergoes reversible electrochemical oxidation to Fe(CN)63-. As the first scan shows, a substantial amount of Fe(CN)64- has reached the electrode surface within about 30 s. After ∼42 cycles (60 min of cycling), the current peak heights became relatively constant, suggesting equilibration of Fe(CN)64- partitioned in the film with Fe(CN)64in the bulk solution. Figure 5 shows voltammograms taken at a bare ITO OTE, an ITO OTE coated with PDMDAAC-SiO2 equilibrated with Fe(CN)64-, and this same ITO OTE after transfer to supporting electrolyte and immersion in it for ∼10 min. A comparison of the three voltammograms reveals the following information. First, the formal redox potential of the film-confined Fe(CN)63-/4- couple shifted to a more negative potential (∆E°′ ) -100 mV), suggesting that the anion-exchange film stabilizes20 the species in the higher oxidation state, Fe(CN)63-. Second, a preconcentration factor of ∼50 was achieved at the coated OTE with the film equilibrated with Fe(CN)64- compared with the bare OTE. A 10% loss in signal occurred after exposing this film to a pure supporting electrolyte solution for ∼10 min. This gradual loss of the filmconfined redox species (as evidenced by the decrease in peak current) in pure electrolyte solution was retrievable when the OTE was re-exposed to the original Fe(CN)64--containing solution, (20) Shriver, D. F.; Atkins, P. W.; Langford, C. H. Inorganic Chemistry; W. H. Freeman: New York, 1990; p 255.

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which is indicative of some degree of dynamic equilibrium (on the voltammetric time scale) between Fe(CN)64- in the solution and Fe(CN)64- confined within the film. Thus, the PDMDAACSiO2 film functions as a reversible “accumulator” of anions and thereby, serves a useful preconcentration role for achieving lower detection limits. Third, both the anodic and cathodic peaks show a sharp, highly symmetrical shape with a relatively small ∆Ep (∼50 mV), an Efwhm (full width at half-maximum, 125 mV on average) close to 90.6 mV for a one-electron process, and a nearly linear dependence of the peak currents on rate of potential scan (r2 ) 0.992 for scan rates from 5 through 100 mV/s). This “intermediate” cyclic voltammetric behavior is more characteristic of a redox species confined in a thin layer adjacent to the electrode surface21 than for semi-infinite diffusion to the electrode surface. The concomitant spectral changes found during electrochemical cycling of Fe(CN)64- are shown in Figure 4a,c. The Fe(CN)64-, which is colorless and, thus, transparent in the visible region, shows a flat spectrum (Figure 4c). Application of a potential more positive (e.g., +0.55 V) than E°′ for ∼1 min causes the incorporated Fe(CN)64- to be electrooxidized to Fe(CN)63- (light yellow in color) as evidenced by the new spectrum with an absorption maximum at 420 nm (Figure 4a). When the potential was stepped more negative (e.g., -0.30 V) than E°′ for ∼30 s, the Fe(CN)63was electroreduced back to Fe(CN)64-. These spectroelectrochemical results show that the sol-gel polyelectrolyte-SiO2 films coated on ITO OTEs do have a series of properties suited to the fabrication of a charge-selective spectroelectrochemical sensor. Indeed, formation of uniform and robust thin films with appropriate polyelectrolyte content on ITO tin oxide float glass was found17 to be critical for success of the present work. Many other coatings were found to be unsuitable because of inadequate adhesion to the ITO OTE. Performance of the Sol-Gel-Derived PDMDAAC-SiO2 ITO OTE Sensor in the ATR Mode for Determination of Fe(CN)64- in Pure Samples. Although the above experiments show that the transmission configuration can be used to detect partitioned analyte spectroelectrochemically with a simple cell design and a relatively easy measurement technique, this configuration suffers from two drawbacks that might prevent its practical use as a sensor. First, since the light beam typically passes through the entire electrochemical cell including the sample solution, substantial light can be absorbed by sample components with spectra overlapping the analyte’s spectrum, even though these components may be excluded by the coating. Thus, benefits provided by the coating are compromised. By comparison, the ATR configuration only samples within the selective coating, unless the coating is so thin that the evanescent wave penetrates beyond it. (This is not the case in the sensor described here.) Thus, sample components must penetrate into the coating to contribute to the optical signal. Second, the transmission configuration usually has low sensitivity because the optical path length for monitoring spectroelectrochemical changes is defined by the diffusion layer for electrolysis, which is very short unless electrolysis times are long. Even though the optical path of the evanescent wave is very short in the ATR mode, sensitivity can be enhanced by orders of magnitude with multiple internal reflections. Both of these advantages were clearly demonstrated (21) Murray, R. W. Chemically Modified Electrodes. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 191368.

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in the sensing performance of the proposed ATR device, as described in following sections. Figure 6 is a compiled illustration of the sensing performance of the spectroelectrochemical sensor operating in the ATR mode for the detection of Fe(CN)64- in pure sample solutions. Although seven different concentrations of Fe(CN)64- were measured with one PDMDAAC-SiO2 ITO OTE sensor, only the results for two different concentrations (2.5 × 10-5 M, horizontal panel 1, and 2.5 × 10-3 M, horizontal panel 2) are shown. The three vertical columns of figure elements in Figure 6 correspond to (a) the transmittance change of the sensor induced by potential step modulation, (b) the transmittance change of the sensor induced by cyclic potential modulation, and (c) representative cyclic voltammograms responsible for giving rise to the transmittance changes shown in (b). Above panels 1 and 2, the corresponding electrical excitations and the timing of them are also shown by vertical dotted lines. As shown in Figure 6a, the colorless Fe(CN)64- anions did partition into the anion-exchange PDMDAAC-SiO2 film, where they were detected optically by a decrease in percent transmittance upon application of the potential step excitation signal shown at the top of the panel (from -0.30 to +0.55 V) to the sensor. In each of these experiments, the electrode potential was first held at -0.30 V to ensure that all the partitioned species were in the reduced, colorless form, giving a stable transmittance baseline. After a few minutes, the potential was stepped to +0.55 V, which electrooxidized the partitioned Fe(CN)64- to Fe(CN)63-. This electrochemically generated Fe(CN)63- then interacted with the evanescent field from the ITO OTE and caused an attenuation of the light (420 nm) propagating along the OTE optical element, as shown by the time-dependent step decrease in transmittance. Comparison of change in percent transmittance in panel 1 (2.5 × 10-5 M) and panel 2 (2.5 × 10-3 M) shows that the magnitude of the change in percent transmittance is proportional to the concentration of Fe(CN)64- in the sample. Close examination of Figure 6a indicates that the partitioning process is continuing during the course of these experiments, which take about 35 min from initial exposure of the OTE to Fe(CN)64- solution until the last cyclic voltammogram is recorded. Under the same conditions, electromodulation of the supporting electrolyte produced no detected change in transmittance. Calibration plots of absorbance change (calculated from the percent transmitance changes in Figure 6) illustrate the sensing performance of the spectroelectrochemical sensor for the determination of Fe(CN)64-. The large curve in Figure 7a shows the plot constructed from seven different samples that ranged in concentration from 8.0 × 10-6 to 5.0 × 10-3 M. The shape of the plot is determined by both the partition isotherm for Fe(CN)64into the coating and the depth of the evanescent wave penetration into the coating. The plot has a linear range at low concentrations (shown in the smaller inset figure) and deviates negatively at higher concentrations. This overall shape is characteristic of ionexchange partitioning, where the ion-exchanger approaches saturation at the higher concentrations. These results clearly demonstrate the first objective of this research: to demonstrate the quantitative relationship between sample concentration and the electrogenerated absorbance change. As mentioned above, one of the advantages exhibited by the ATR configuration over the transmission configuration is the sensitivity enhancement, which is clearly evident in this study.

Figure 6. A composite figure consisting of vertical columns and horizontal panels indicating spectroelectrochemical signals for the sensor. The horizontal panels depict results for the sensor exposed to two different concentrations of Fe(CN)64-. The concentrations are as follow: panel 1, 0.025 mM; panel 2, 2.5 mM. The electrical potentials applied to the sensor are illustrated above the panels and are appropriate for a potential step (column a) and a repeating triangular potential modulation (column b). Column a depicts the changes in transmittance of the sensor induced by a step potential from -0.30 to +0.55 V for the 0.025 mM (a1) and 2.5 mM (a2) Fe(CN)64- solution exposures. Column b depicts the changes in transmittance of the same sensor induced by cyclic potential modulation, between the limits of +0.55 and -0.30 V for the 0.025 mM (b1) and 2.5 mM (b2) solution exposures. Column c depicts the cyclic voltammograms recorded simultaneously with the transmittance changes shown in column b, with c1 and c2 corresponding to the sensor exposures to 0.025 and 2.5 mM Fe(CN)64- solutions, respectively. Cyclic voltammograms were recorded about three cycles after changing the oxidizing potential as shown in a1 and a2. Cyclic voltammetry and optical parameters were as follow: ν ) 10 mV/s, E/V vs Ag/AgCl; λ ) 420 nm.

Compared with the absorbance value at 420 nm obtained by the transmission configuration shown in Figure 4, an enhancement factor in sensitivity of ∼50 can be seen in the ATR measurements (Figure 7a) for the detection of Fe(CN)64- under similar conditions. The electrochemically modulated ATR optical detection of the colorless Fe(CN)64- down to 8.0 × 10-6 M in pure sample solution was demonstrated in this work. This is a reasonable detection limit, considering that its oxidized form, Fe(CN)63-, which provides the optical change, is a light yellow species with a molar absorptivity of only ∼1000 M-1 cm-1 in 0.1 M KNO3 and that the sensor is essentially unoptimized at this stage of development. Since the sensitivity of the sensor is determined by the magnitude of partitioning into the ITO coating, the magnitude of the electrogenerated optical change, the effective path length of the evanescent waves at the reflection points, and the noise of the system, improvements in any of these parameters will improve sensitivity. Thus, stronger chromophores and better preconcentrating films will enhance sensitivity. Also, sensitivity enhancement could be further increased, perhaps by as much as an order of magnitude, if the bulk ATR device were to be replaced by an

integrated optical waveguide-based ATR device, since the latter supports a much higher density of total internal reflections. Up to several thousand reflections per centimeter of guided channel are possible, resulting in substantially increased effective optical path length.22,23 OTEs based on optical waveguide structures have been reported.24,25 Detection limit can be further decreased by signal-averaging a repeating optical signal resulting from a repetitive potential pulse, as has been demonstrated for spectroelectrochemistry.15 Thus, further developments should lead to substantially improved detection limits. Figure 6b shows the changes in transmittance for the same system as described in Figure 6a when the ITO OTE was subjected to cyclic potential modulation. Since the potential applied was cycled between +0.55 and -0.30 V, the partitioned Fe(CN)64- was expected to cause a periodic change in transmit(22) Yang, L.; Saavedra, S. S.; Armstrong, N. R. Anal. Chem. 1996, 68, 1834. (23) Lee, J. E.; Saavedra, S. S. Anal. Chim. Acta 1994, 285, 265. (24) Itoh, K.; Fujishima, A. J. Phys. Chem. 1988, 92, 7043-7045. (25) Dunphy, D. R.; Mendes, S. B.; Saavedra, S. S.; Armstrong, N. R. Abstracts of Papers, 213th National Meeting of the American Chemical Society, San Francisco, CA, April 13-17, 1997; American Chemical Society: Washington, DC, 1997; ANYL 0234.

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sites of the sensing film with the partitioned analyte at higher sample concentration and longer exposure time. Thus, the two signals correlate well, as we would expect for a simple system such as this one. Thus, for this simple, one-component sample, there is no advantage in measuring the optical change over the current. However, as we will demonstrate in a future paper,26 this situation changes when samples with multiple components are used and the electrochemical signal for an inteference overlaps that for the analyte, which is often the case for real samples. CONCLUSIONS

Figure 7. Sensor calibration curves for both optical detection (a) and electrochemical detection (b). Curve a represents sensor absorbance for Fe(CN)63- generated by electrode step modulation vs the concentration of Fe(CN)64- added to the sensor cell. The concentration values of Fe(CN)64- in the plot are 8.0 µM, 25 µM, 50 µM, 0.25 mM, 0.50 mM, 2.5 mM, and 5.0 mM. The inset in (a) represents the linear portion of the calibration curve for the lower concentration range of 8.0-50 µM. Curve b shows a plot of peak current of the Fe(CN)64-/3- cycled between +0.55 and -0.30 V vs concentration of Fe(CN)64- added to the cell. The Fe(CN)64- concentration values are the same as those shown in (a). The inset in (b) represents the linear portion of the calibration curve for the concentration range from 8.0 µM to 0.25 mM.

tance of the ATR system that corresponded to the periodic formation and depletion of electrochemically generated Fe(CN)63-, which was the case as shown in Figure 6b. The transmittance changes are also proportional to the concentration of Fe(CN)64in the sample, as expected. Figure 6c shows representative cyclic voltammograms that were recorded while the optical measurements in Figure 6b were being made. The peak currents are proportional to the concentration of Fe(CN)64- in the sample. Although the concept of the sensor involves the measurement of the optical change for quantitation to enhance the selectivity of the sensor, we thought it interesting to compare, at this stage, the optical response with the current response. Figure 7b shows plots of the peak current (cathodic) from the cyclic voltammograms vs the Fe(CN)64concentration. Comparison of the plots in parts a and b of Figure 7a shows that both optical and current responses have good linearity over the lower concentration range, with the latter showing a wider linear concentration range. The nonlinearity observed at higher concentrations for both plots shows a similar trend, which is ascribed to saturation of the accessible exchange (26) Shi, Y.; Seliskar, C. J.; Heineman, W. R. Anal. Chem., submitted for review.

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These results demonstrate the ability to quantitate an electroactive analyte using an absorbance change accompanying its electrochemical oxidation/reduction at an OTE. Although this principle should also apply to spectroelectrochemistry at a bare OTE, modification of the OTE with an anion-exchanging film was used to improve the detection limit by preconcentration of the analyte in the optical path of the evanescent wave. A detection limit of 8.0 × 10-6 M was achieved for Fe(CN)64- with an unoptimized sensor. The detection limit should improve with more reflections, as would be achieved with a waveguide as the OTE and with signal averaging. The detection limit will also depend on the difference in molar absorptivities (∆) between the analyte and its electrogenerated product as well as the partitioning characteristics (i.e., partition isotherm) of the analyte into the electrode film. In the sensor reported here, the linear range is limited on the upper end by nonlinearity of the ionexchange process as the film approaches saturation. The linear range should be extended downward by procedures that improve the detection limit such as mentioned above. The ITO plates used in this report are a convenient way to demonstrate the technique; however, it is envisaged that practical sensors would be based on an integrated optical waveguide (IOW) that is easily mass produced. Although the sensor was demonstrated with UV-visible absorbance spectroscopy at an OTE, the concepts should also apply to other spectroscopic techniques, such as fluorescence, infrared, Raman, etc. It could also be used at nontransparent electrodes with techniques such as reflectance. The sensor was demonstrated with potential step and potential cycling excitation signals. It is not apparent that these are the best modes of operation to achieve the lowest detection limit, especially when signal averaging is applied. Although not demonstrated here, the selectivity of the sensor will be most important in practical applications. Thus, the effectiveness of the trimodal selectivity of electrochemistry, spectroscopy, and partitioning into the electrode film is the subject of a following paper26 and continuing investigations. ACKNOWLEDGMENT Financial support provided by the Department of Energy (Grant DE-FG07-96ER62311) is gratefully acknowledged.

Received for review March 25, 1997. Accepted July 1, 1997.X AC970322U X

Abstract published in Advance ACS Abstracts, August 15, 1997.