Simultaneous Quartz Crystal Microbalance Impedance and

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Anal. Chem. 2002, 74, 3304-3311

Simultaneous Quartz Crystal Microbalance Impedance and Electrochemical Impedance Measurements. Investigation into the Degradation of Thin Polymer Films Andrea Sabot and Steffi Krause*

Department of Chemistry, The University of Sheffield, Sheffield S3 7HF, U.K.

The reproducible degradation of thin polymer films in the presence of an analyte or a reaction product of an analyte has potential applications in the development of highly sensitive, disposable biosensors. In this study, a novel combination of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS) has been developed to monitor the degradation of thin polymer films. Unlike a conventional QCM, the instrument described here allows rapid in situ measurement of quartz crystal impedance spectra. Simultaneously, classical electrochemical impedance spectra are measured in situ, affording the polymer film capacitance and bulk resistance. The combination of QCM impedance and classical EIS provides a wealth of information about the process of degradation of thin polymer films such as mass variation, swelling, delamination, viscoelasticity, and pore formation. Three different systems have been analyzed with this experimental setup; in two of the systems, polymer degradation was promoted by hydrolytic enzymes, and in the third one by a pH change. The results obtained show that the degradation of these three systems follows very different mechanisms. It is also underlined how the complementary information obtained by the two techniques allows a detailed description of the dissolution process. The reproducible degradation of thin polymer films in the presence of an analyte or a reaction product of an analyte can be utilized to produce highly sensitive, disposable biosensors.1-3 A sensor based on the measurement of capacitance changes produced during enzyme-catalyzed dissolution of polymer coatings on electrodes was described by McNeil et al.1 Thin films of a pHsensitive polymer, a copolymer of methyl methacrylate and methacrylic acid, were deposited onto gold-coated electrodes. A localized increase in pH, caused by the enzymatic action of urease * Corresponding author: (fax) +44 (0) 114 2738673; (e-mail) s.krause@ sheffield.ac.uk. (1) McNeil, C. J.; Athey, D.; Ball, M.; Ho, W. O.; Krause, S.; Armstrong, R. D.; Wright, J. D.; Rawson, K. Anal. Chem. 1995, 67, 3928-3935. (2) Sumner, C.; Sabot, A.; Turner, K.; Krause, S. Anal. Chem. 2000, 72, 52255232. (3) Saum, A. G. E.; Cumming, R. H.; Rowell, F. J. Biosens. Bioelectron. 1998, 13, 511-518.

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on urea, triggered the dissolution of the polymer films. Degradation of the films was accompanied by an increase in capacitance of up to 4 orders of magnitude. The method was developed into a fast and simple disposable sensor for urea in serum and whole blood.1 More recently, it has been shown that this type of transducer has great potential for the detection of enzyme concentrations. This new sensor format uses biodegradable polymers, which are directly degraded by an enzyme rather than by the product of an enzymatic reaction. Surface plasmon resonance (SPR) has been used as an alternative technique to monitor the enzymatic degradation of polymer films.2 This technique has been used successfully for the detection of R-chymotrypsin using thin films of poly(ester amide) as the biodegradable material and for measuring Dextranase concentrations using a dextran hydrogel to form the biodegradable films. R-Chymotrypsin and Dextranase concentrations down to 2 × 10-11 M could be detected in less than 20 min. In this paper, we report the design and application of a novel in situ combination of quartz crystal impedance measurements and electrochemical impedance spectroscopy (EIS) to monitor the degradation of thin polymer films. Previously, quartz crystal microbalance (QCM) impedance and EIS were used independently to study a variety of polymer film properties including the growth and degradation of polymer films. QCM Impedance. The change of the QCM resonant frequency is often used to monitor mass variations of thin films by employing the linear relation given by the Sauerbrey equation.4 However, the linear relationship between mass variation and frequency shift holds true only in the case of ideal mass layers, i.e., in the case of rigid coatings or very thin films where the phase shift of the acoustic wave across the film is ,π/2. In more general situations, such as nonrigid or viscoelastic films, QCM impedance measurements around the resonance frequency of the quartz crystal have been successfully employed. For example, QCM impedance measurements have been used to show the variation of viscoelastic properties during the electrodeposition of conducting polymer films5,6 or during the exchange of ions and solvent between hydrogel films and the electrolyte solution.7 In the presence of a liquid phase, not only the viscoelasticity of the film but also parameters such as solvent density, viscosity, (4) Sauerbrey, G. Z. Phys. 1959, 155, 206-222. 10.1021/ac0200724 CCC: $22.00

© 2002 American Chemical Society Published on Web 05/29/2002

Figure 1. Butterworth-Van Dyke equivalent circuit model for a QCM resonator with a surface loading. The surface loading arises from the presence of a coating and from the contact with a liquid. The surface loading is resolved into motional inductance L2 and resistance R2.

wetting properties of the coating, and its surface roughness affect the QCM resonance mode.8 Then, a more detailed model than the Sauerbrey equation must be considered to correctly describe the interaction of the quartz resonator with the surface loading (film + liquid). The Butterworth-Van Dyke (BVD) equivalent circuit (Figure 1) has been used to describe the near-resonant electrical characteristic of the quartz resonator coated by a viscoelastic film.8 In the BVD circuit, elements C0*, C1, L1, and R1 refer to the unperturbed quartz resonator, while elements L2 and R2 represent surface loading perturbation (film + liquid). The circuit consists of a “static” and a “motional” arm in parallel. The static arm consists of a capacitance, C0*, while the motional arm, containing C1, L1, and R1, describes the electromechanical coupling of piezoelectric quartz responsible for the quartz oscillations. The capacitive component, C1, represents the quartz mechanical elasticity, L1 represents the inertial mass, and R1 represents the energy dissipation due to internal friction and damping from the crystal mounting. A surface loading modifies the electrical impedance of the unperturbed quartz resonator. In the BVD circuit, this change is described by an additional inductance L2, which represents the inertial mass of the film and liquid coupled in the oscillations, and by a resistance R2, which represents the energy dissipated by the viscoelastic and damping effects of the film and liquid overlayer. It should be noted that we were only interested in relative changes in the polymer film, with constant viscoelastic properties of the liquid and the quartz. Electrochemical Impedance Spectroscopy. Electrochemical impedance spectroscopy has long been used as a tool for investigating bulk and interfacial properties of polymer coated electrodes.9 The degradation of Eudragit S-100, a copolymer of methyl methacrylate and methacrylic acid, was studied in context with the biosensor principle mentioned above.1,10 This is an enteric polymer, also used for drug delivery, which is stable at low pH (5) Glidle, A.; Hillman, A. R.; Bruckenstein, S. J. Electroanal. Chem. 1991, 318, 411-420. (6) Bandey, H. L.; Hillman, A. R.; Brown, M. J.; Martin, S. J. Faraday Discuss. 1997, 107, 105-121. (7) Etchenique, R. A.; Calvo, E. J. Anal. Chem. 1997, 69, 4833-4841. (8) Ballantine, D. S.; White, R. M.; Martin, S. J.; Ricco, A. J.; Zellers, E. T.; Frye, G. C.; Wohltjen H. Acoustic Wave Sensors: Theory, Design and PhysicoChemical Applications; Academic Press: San Diego, 1997. (9) MacDonald, J. R. Impedance Spectroscopy; Wiley: New York, 1987. (10) Krause, S.; McNeil, C. J.; Armstrong R. D.; Ho, W. o. J. Appl. Electrochem. 1997, 27, 291-298.

Figure 2. Equivalent circuits for modeling an electrode coated with a porous film. CPEg and CPEdl are the geometric and double-layer capacitance described by constant-phase elements (CPE). Rb and Rel are the coating bulk resistance and electrolyte resistance. Circuit a is used when CPEdl is negligible; i.e., the metal electrode is not wetted by the electrolyte solution. Circuit b is more general because it also contains the electrolyte resistance Rel and the double-layer capacitance CPEdl (wet electrode). Circuit c is used for the bare metal electrode in solution.

and dissolves above pH 7.10 In this early work, it was shown that Eudragit S-100 films dissolved by initially forming pores through which the electrolyte penetrated and then spread along the electrode surface. The impedance spectra were described in terms of equivalent circuits. An unperturbed Eudragit film could be modeled with a pure geometric capacitance or a constant phase element (CPEg), a nonideal capacitance the impedance of which is defined as ZCPE ) -1/(iωQ)n. Pores in the coating filled with electrolyte caused an apparent conductivity of the polymer, which could be described by a bulk resistance Rb in parallel with the geometric capacitance (Figure 2a). Once the electrolyte penetrated the pores and got into contact with the underlying electrode, an electrochemical double layer appeared at the metal-solution interface, which was described by an additional constant phase element (CPEdl) in the equivalent circuit (Figure 2b). The highfrequency part of the impedance spectrum of a partially degraded film was dominated by the electrolyte resistance Rel (equivalent circuits in Figure 2b and c). Further degradation resulted in partial coverage of the electrode with a thin polymer film, making it impossible to distinguish between geometrical capacitance and double-layer capacitance. Hence, circuit 2c was used for fitting spectra during the final stages of degradation. A common problem with impedance spectra measured using a frequency response analyzer is that it is difficult to measure in real time because a full spectrum acquisition requires minutes, making this technique unsuitable for monitoring processes of similar time scale. Krause et al.10 overcame this problem by halting the polymer breakdown at different times and then measuring EIS ex situ. However, this approach has the disadvantage that the quenching step might interfere with the process of degradation. One way to reduce the total measurement time of impedance measurements is to employ so-called time domain or fast Fourier transform (FFT) techniques.11 We recently introduced fast impedance measurements based on a network analyzer to follow the degradation of thin films for the biosensor format described above.2 The first biodegradable polymer under study was a poly(ester amide) based on bis(L-phenylalanine)R,ω-alkylene diesters, which is degraded in the presence of the hydrolytic enzyme R-chymotrypsin.12 The poly(ester amide) was an insulating polymer, which was shown to degrade layer by layer. The second (11) Popkirov, G. S. Electrochim. Acta 1996, 42, 1023-1027.

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Figure 3. Experimental setup for in situ QCM/EIS measurements. The working electrode, coated QCM side, is common for both QCM and EIS, while the counter electrode is different. A computer-controlled rf relay switches continuously between the two counter electrodes; the Network Analyzer provides the appropriate electrical stimulus to the different electrodes and measures the electrical response returning from the cell.

system was a dextran hydrogel obtained by cross-linking dextran molecules with hexamethylene diisocyanate. Since hydrogels are strongly hydrated, this was an example of a material with poor insulating properties. In this work, the performance of the novel in situ combination of quartz crystal impedance measurements and electrochemical impedance spectroscopy was assessed using three model systems. Due to their different degradation mechanisms, the polymer systems described above, Eudragit S-100 degraded due to an increase in pH, poly(ester amide) degraded by R-chymotrypsin, and dextran hydrogel degraded by Dextranase, were particularly suitable for this investigation. EXPERIMENTAL SECTION Materials and Sample Preparation. The synthesis of the biodegradable polymer, poly(ester amide) MW 6400, was described previously.12 R-Chymotrypsin, type II, MW 25 000, from bovine pancreas, with an activity of 60 units mg-1 was purchased from Aldrich (Dorset, U.K.). Dextranase from Paecilomyces lilacinus, activity 60.1 units mg-1, was purchased from Fluka (Dorset, U.K.). Dextran hydrogel was obtained by cross-linking dextran (molecular weight 70 000; Aldrich) with hexamethylene diisocyanate (Fluka), adapting a procedure described in the literature.13 Since the hydrogel is not soluble and therefore not suitable for spin coating, the cross-linking was carried out directly onto the QCM substrates (see below). Eudragit S-100 polymer was obtained from Dumas Ltd. (Kent, U.K.). Phosphate buffers pH 5.1 and pH 7.3 containing 140 mM NaCl and 10 mM KH2PO4 were prepared. Water was purified through a Milli-Q ion exchange system (Millipore) and used to prepare all the solutions. Organic solvents such as acetone, chloroform, and DMSO were purchased from BDH (Dorset, U.K.). Polished, gold-coated QCM crystals (10 MHz) were purchased from Elchema. Prior to film deposition, the quartz crystals were cleaned by boiling in piranha solution (12) Arabuli, N.; Tsitlanadze, G.; Edilashvili, L.; Kharadze, D.; Goguadze, T.; Beridze, V.; Gomurashvili, Z. and Katsarava, R. Macromol. Chem. Phys. 1994, 195, 2279-2289. (13) Brondsted, H.; Hovgaard, L.; Simonsen, L. Eur. J. Pharm. Biopharm. 1995, 41, 341-345.

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(7:3 v/v concentrated H2SO4 and 20% H2O2) for 15 min, rinsed thoroughly, first with deionized water, then with spectrophotometric-grade ethanol (Aldrich), and finally dried with a stream of nitrogen. Film Preparation. Films of poly(ester amide), ∼65 nm thick, were produced by spin-coating a solution of the polymer, 10 mg mL-1 in chloroform, at a speed of 3000 rpm for 40 s onto the freshly cleaned QCM crystals. The solvent was allowed to evaporate at room temperature overnight. Dextran (2.5 g) was dissolved in 14 mL of DMSO. Some 0.03 mL (i.e., 6% mol per dextran unit) of hexamethylene diisocyanate was added under continuous stirring. The resulting solution was immediately applied to a QCM substrate and spun at a speed of 3500 rpm for 40 s. Prior to the dextran deposition, the QCM crystals were treated with a 5 mM solution of 11-mercapto-1undecanol (Aldrich) in Spec-grade ethanol overnight. This treatment improved the substrate wettability, and more uniform films could be obtained. Substrates were then left on a hot plate at 70 °C overnight for the cross-linking reaction to proceed in a DMSOsaturated atmosphere. Then, the films were washed with distilled water for 1 h and dried on a hot plate (100 °C, 10 min). Films of Eudragit S-100 were spin-coated onto quartz crystals at a spinning speed of 4000 rpm for 40 s from a 7.5% w/w solution of polymer in acetone. The films were dried under vacuum overnight at room temperature and cured at 100 ˚C for 12 h (this treatment greatly improved the reproducibility of dissolution). The thickness of each films was calculated using the Sauerbrey equation from the shift of the QCM resonant frequency for the dry film and assuming the density dfilm ) 1 g cm-3. Experimental Setup for QCM Impedance and EIS Measurements. High-frequency (∼10 MHz) QCM impedance and low-frequency (5 Hz-100 kHz) electrochemical impedance measurements were carried out using a Hewlett-Packard HP 8751A (5 Hz-500 MHz) Network Analyzer in reflectance mode. This was made possible by the dual independent channel capability of this instrument for spectra acquisition. A computer-controlled rf switch was built in-house to switch between electrodes (see Figure 3) so that alternating QCM impedance and electrochemical impedance measurements could be made. Electrochemical impedance

measurements were performed at zero potential in a two-electrode arrangement. The polymer-coated QCM gold electrode (the working electrode) was common for both QCM impedance and electrochemical impedance, while two different counter electrodes were used. During QCM impedance measurements, the Network Analyzer was connected to the uncoated QCM gold electrode. Instead, a platinum foil placed opposite and at a distance of ∼5 mm from the working electrode was used as a counter electrode to measure the electrochemical impedance. The counter electrode was made large enough to avoid current limitations. The accuracy of the measured electrochemical impedance was verified using a threeelectrode arrangement connected to a frequency response analyzer (FRA2, Autolab) with potentiostat (PGSTAT 10, Autolab). The electrodes were connected via a 50 Ω coaxial cable and a HP 87512A transmission/reflection unit. The Network Analyzer was connected to a PC through a GPIB board (National Instruments), and the impedance data acquisition was computer controlled by a program developed in-house using LabView 5.0 (National Instruments). For QCM measurements, one full impedance spectrum (201 points, ac stimulus 160 mV, acquisition time 1 s) was recorded over a range of 10 kHz centered at the QCM resonant frequency (∼10 MHz) every 30 s. For electrochemical impedance measurements, an ac stimulus of 20 mV was applied and impedance was recorded in the range between 5 Hz and 100 kHz, one full spectrum (25 points, acquisition time 6 s) every 30 s. Degradation Experiments. QCM crystals, one side coated with the biodegradable polymer, were integrated into a customdesigned cell (shown in Figure 3) with the polymer-coated side in contact with the solution while the other side was kept dry. Sealing was ensured by a silicone rubber O-ring and a spring pressing from the back of the crystal. Damping of the QCM signal was reduced to a minimum by making the O-ring slightly bigger than the gold electrode. The cell was filled with phosphate buffer, and the system was allowed to equilibrate for at least 20 min under magnetic stirring (600 rpm). For the degradation of the poly(ester amide) and the dextran hydrogel films, 0.98 mL of phosphate buffer at pH 7.3 were used. Then, 20 µL of a concentrated solution of enzyme (3 units mL-1) was added using a micropipet (Gilson): R-chymotrypsin (final concentration 0.06 unit mL-1) for the poly(ester amide) and Dextranase (final concentration 0.06 unit mL-1) for the dextran hydrogel. In the case of Eudragit S-100 films, the cell was filled with 2.00 mL of phosphate buffer at pH 5.1 and left to equilibrate. Then, a measured amount of 1 M NaOH (13.5 µL) was added to raise the pH to 7.0. A magnetic stirring bar ensured continuous mixing of the solution throughout the degradation. Degradation of the polymer films was monitored by recording the impedance spectrum at regular time intervals (every 30 s). Enzyme solutions were freshly prepared, using the same phosphate buffer before each experiment in order to avoid loss of enzymatic activity. Experiments were conducted at room temperature.

Data Analysis. The degradation was monitored using the shift of the QCM resonant frequency. After the experiments, the QCM admittance (Y ) 1/Z) rather than the impedance Z was fitted with the BVD equivalent circuit. The impedance of the “motional” arm, Zm, of the BVD circuit, including the surface loading, is given by eq 1 and the total

Zm ) (R1 + R2) + iω(L1 + L2) + 1/iωC1

(1)

ZBVD ) (iωC0* + 1/Zm)-1

(2)

impedance, ZBDV, is shown in eq 2, where i ) -11/2 and ω is the angular frequency (ω ) 2πf, where f is frequency). Subsequent fitting of the experimental impedance data yields the parameters for the BVD equivalent circuit R ) R1 + R2, L ) L1 + L2, C1, and C0*. The value of the quartz compliance C1 was determined in a four-parameter fit and then held constant during fitting of the rest of the data. A highly efficient nonlinear least-squares fitting program was written using LabView 5.0 for this purpose. The variations of the electroacoustic impedance components ∆XL ) ω∆L and ∆R were determined with respect to the system before degradation (with f ) 10 MHz). Changes in the mass of the polymer are directly related to ∆XL while changes in the viscoelastic properties of the film only affect ∆R. The electrochemical impedance spectra were fitted using the equivalent circuits in Figure 2 yielding the circuit parameters. All three models (Figure 2) were tested, and the choice of the appropriate equivalent circuit to be used was made case by case on the basis of the best fit using the least number of parameters. Due to the variability of the fitting function and the high number of spectra to process, a nonlinear least-squares fitting program for EIS was written using MathLab 4.0. Microscope Analysis. Images of Eudragit S-100 films were taken before degradation and after they have been partially dissolved using an optical microscope equipped with a digital camera. The films were spin-coated onto glass slides cleaned with piranha solution as described above. These were immersed into buffer at pH 7.0 for the required period of time, rinsed with diluted HCl (pH 3) to halt the degradation and with deionized water, and finally dried with a stream of nitrogen. RESULTS AND DISCUSSION Poly(ester amide) Films. Spin-coated films of poly(ester amide), ∼65 nm thick, were degraded by the hydrolytic action of the enzyme R-chymotrypsin. The QCM admittance spectra (a) and the electrochemical impedance spectra (b) before, during, and after degradation are shown in Figure 4. For clarity of presentation, only the magnitude is reported even though both magnitude and phase were recorded and used for the subsequent analysis. The QCM admittance spectra contain a maximum and a minimum, which are called series, ωs, and parallel, ωp, resonant frequencies, respectively. An added mass to the QCM surface increases L2, which, according to eqs 1 and 2, shifts both ωs and ωp toward lower frequencies. Furthermore, if the mass layer is nonrigid (R2 > 0), there is an increase in the damping of the QCM oscillations resulting in a decrease of the maximum admittance at ωs. As the poly(ester amide) film degraded, the QCM spectrum Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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Figure 4. Poly(ester amide) QCM admittance spectra (top) and EIS spectra (bottom) while the film is being degraded. ωs and ωp are the QCM series and parallel resonant frequency, respectively. One spectrum every 3 min is displayed.

shifted toward higher frequencies (Figure 4a) with little increase in the maximum admittance, indicating that the film was initially fairly rigid and remained rigid throughout the degradation. The QCM spectra in Figure 4a were fitted with the BVD circuit (Figure 1) affording the parameters ∆XL and ∆R; these were plotted against time and are shown in Figure 5a. The reactive inductance ∆XL is proportional to mass changes within the film while ∆R, the variation of the acoustic energy dissipated, is affected by changes in both the film thickness and viscoelasticity. ∆R is positive if the film becomes more viscoelastic, or at constant viscoelasticity, if the film thickness increases. It should be noted that the morphology of the polymer-liquid interface also affects ∆R. Figure 5a shows that the mass of the polymer was constant in buffer and began to decrease immediately after the enzyme was added. The rate of mass loss, according to ∆XL, was constant for most of the degradation, slowing down asymptotically at the end. After a few minutes required for equilibrating the dry film with the buffer, R was constant, confirming the stability of the film in buffer solution. During degradation, R decreased only very little, meaning that the initial film dissipated as much energy as the bare QCM or, in other words, that the film was rigid. These data exclude processes such as swelling or formation of pores wider than the hydrodynamic decay length (∼180 nm), where XL and R would increase. Formation of pores smaller than 180 nm is incompatible with the steady decay of XL. 3308 Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

Figure 5. Variation of the impedance parameters during the degradation of a poly(ester amide) film, parameters obtained by fitting the spectra in Figure 4. One point corresponds to a time interval of 1 min. Top: QCM impedance parameters obtained from the BDV circuit (Figure 1). Bottom: EIS parameters from fitting with the equivalent circuit (c) in Figure 2.

Electrochemical impedance spectra, shown as a Bode plot in Figure 4b, have a slope of about -1 at low frequencies, which becomes zero at higher frequencies. This indicates capacitive behavior at low frequencies (the geometric capacitance of the insulating polymer layer) and a resistive behavior at higher frequency (the solution resistance due to the electrolyte). During the course of degradation, the impedance at high frequency remained constant while the impedance at low frequency decreased, indicating a thinning of the capacitive dielectric layer. Electrochemical impedance data from Figure 4b were also fitted, and the parameters obtained are shown in Figure 5b. The fit was based on the equivalent circuit shown in Figure 2c because this was the simplest and most effective circuit that could describe the electrochemical impedance spectrum, before, during, and after degradation. In this case, the CPEdl (Figure 2c) describes the geometric capacitance of the film, which eventually becomes a double-layer capacitance of the metal-electrolyte interface once the film is degraded. A purely capacitive behavior implies that the film was completely insulating and that it did not take up electrolyte. The geometric capacitance of the film is approximated with the parameter Q in the constant-phase element; this approximation is reasonable because the exponent n is close to 1. As shown from Figure 5b, Q increased considerably, from 0.012 to 0.85 µF, as a direct consequence of the thinning of the polymer layer. The parameter n also increased, indicating that the polymer layer was becoming smoother during degradation. Interestingly, Q increased with some delay with respect to the mass decrease (∆XL). This observation, plus the fact that n increased immediately

Figure 7. Change of the QCM impedance parameters ∆XL and ∆R before, during, and after the degradation of a hydrogel film. Data obtained by BDV fit of the spectra in Figure 6. One point corresponds to a 1-min time interval.

Figure 6. Dextran hydrogel degradation: QCM admittance spectra (top) and EIS spectra (bottom), one spectrum every 2 min.

after adding the enzyme, leads to the conclusion that initially the degradation occurred at rough features or edges present on the polymer surface which are more readily attacked by the enzyme. However, this part of the polymer layer contributes little to the overall film capacitance Q. Dextran Hydrogel Films. Dextran hydrogel films with thickness of 30 nm were degraded by the enzyme Dextranase. During degradation, the QCM admittance was shifting to higher frequencies. At the same time, the maximum admittance increased in magnitude, meaning that the energy dissipated in the film was decreasing, showing a clear viscoelastic behavior. From Figure 7, it is evident that, unlike the case of poly(ester amide), both XL and R decreased after the Dextranase was added, confirming the nonrigidity of the hydrogel layer. The decrease in R suggests that there is no further softening of the polymer following the addition of enzyme, as this would be accompanied by an increase in R. However, it should be pointed out that the interpretation of ∆R in terms of viscoelastic changes is complicated due to the fact that the mass of the film is not constant. Figure 6b shows that there is no difference in the electrochemical impedance spectrum before and after the degradation. This observation implies that the hydrogel takes up sufficient electrolyte to make its impedance similar to or smaller than the impedance of the surrounding electrolyte solution. Consequently, EIS could not be used to study the degradation for this particular system. Eudragit S-100 Films. To study the mechanism of degradation of Eudragit S-100 films the process had to be slowed

Figure 8. Degradation of Eudragit S-100. Top: QCM admittance spectra, the arrows indicate the shift of the maximum admittance (ωs). Bottom: EIS spectra. One spectrum every minute is displayed.

sufficiently. Therefore, fairly thick films (0.76 µm) of the polymer were produced. The mechanism of dissolution of Eudragit S-100 was clearly different from the previous two systems as can be seen from Figure 8. Immediately after the pH was raised from 5.1 to 7.0, both the maximum admittance and the resonant frequency ωs decreased (Figure 8a). Then, ωs increased with a steady rate until it reached the value of the bare quartz crystal. The electrochemical impedance spectrum also changed substantially while the Eudragit films were degrading (Figure 8b). Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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It was not possible to fit the electrochemical impedance data using only one equivalent circuit throughout the whole dissolution process. Basically, three different stages of the polymer-coated electrodes were characterized using EIS: the film before degradation and initial stage of degradation (1), intermediate stages of degradation (2), and final stage of degradation including the bare gold electrode (3). Vertical dashed lines separate these three stages in Figure 9. The impedance spectra of the initial Eudragit S-100 film, in contact with buffer at pH 5.1, showed capacitive behavior over a large frequency region (Figure 8b, “before”), these spectra could be properly described by the circuit shown in Figure 2a: a geometric capacitance, CPEg, in parallel with a residual bulk resistance, Rb. The presence of a bulk resistance Rb indicated some porosity already present in the initial film. Equivalent circuit 2b described very well the complex electrochemical impedance spectrum obtained during the intermediate stage of dissolution. The decrease in Rb (Figure 9b) indicated an increasing porosity. At stage 1, there was no double-layer capacitance, CPEdl, showing that the electrolyte was not wetting the electrode. At stage 2, a CPEdl contribution became apparent at low frequencies (Figures 8 and 9c), indicating that the electrolyte had reached the gold electrode. The relative porosity P and the fraction of wetted metal W can be calculated in the following way:10

Figure 9. Impedance parameters for the degradation of the Eudragit S-100 polymer film obtained by fitting the spectra in Figure 8. One point every 30 s. (a) QCM impedance parameters ∆XL and ∆R. (b) EIS parameters Rel and Rb obtained for three different stages of the degradation: before degradation and initial stage of degradation (1), intermediate stages of degradation (2), and final stage of degradation including the bare gold electrode (3). The three equivalent circuits in Figure 2 where used to fit EIS data at these different stages. (c) Same as (b) but the parameters CPEg and CPEdl are shown.

The electroacoustic parameters (∆XL and ∆R) and the electrochemical parameters (Rel, Rb, CPEg, CPEdl) obtained by fitting are shown in Figure 9. After an initial increase in ∆XL and therefore the mass, the rate of dissolution was constant as indicated by the steady decrease of ∆XL. We deduced that the polymer film was initially rigid because ∆R was negligible at the end. However, during the dissolution of the film, ∆R increased to a constant value of roughly 50 Ω. We believe that this increase of ∆R and the initial increase in ∆XL were mainly due to the formation of pores in the film and trapping of electrolyte in these pores (see below), which increased the acoustic damping at the film-liquid interface and the mass of the film. 3310

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P ) Rbt/Rb

(3)

W ) Cdl/Cdl0

(4)

where Rbt is the resistance of a layer of electrolyte having the same area and thickness as the polymer coating, while Cdl and Cdl0 are the double-layer capacitance of the coated and bare electrode, respectively. We have estimated, by approximating C ) Q, that W was ∼5 × 103 times higher than P in the middle of stage 2; i.e., after penetrating the pores, the electrolyte spread as a thin film between metal and coating, increasing further the fraction of wetted metal area. Simultaneously, the exponent n of CPEg (not shown here) decreased from an initial value of 0.83 to a minimum of 0.56 in the middle of stage 2 (at t ) 115 min, Figure 9), confirming the inhomogeneous structure of the partially broken layer. After this point, n increased again. It can be seen from Figure 9c that at t ) 118 min CPEg and CPEdl became very similar in value. Hence, the error in the simultaneous determination of CPEg and CPEdl by fitting was very high. For this reason, circuit 2c was used to fit the impedance spectra obtained during the last part of the dissolution producing an average capacitance value, which increased until the capacitance of a bare electrode was obtained. Microscope images taken ex situ at different times clearly confirmed formation of pores in the polymer layer and the spreading of the electrolyte between the polymer and the metal (Figure 10). CONCLUSIONS A novel experimental setup, which combines QCM impedance spectroscopy and EIS, has been developed to study properties of thin polymer films. One full EI spectrum can be recorded in ∼6

Figure 10. Microscope images of Eudragit S-100 polymer films before degradation (a) and partially broken down after soaking for 2 min in buffer at pH 7 (b).

s; one QCM admittance spectrum takes less than 1 s to acquire, allowing the study of the degradation process in real time and in situ. The simplicity of the setup derives from the fact that the same instrument, a Network Analyzer, is used for both types of measurements. QCM impedance data were analyzed with the Butterworth-Van Dyke equivalent circuit affording the electroacoustic reactive inductance XL and resistance R, while electrochemical impedance data were analyzed using different equivalent circuits. By combining QCM impedance and EIS, the degradation mechanism of different polymers could be studied in great detail. The information obtained using both techniques is truly complimentary. For example, it was shown by EIS that the poly(ester amide) degrades layer by layer since there was a steady increase in the geometric capacitance throughout the degradation process. However, only by comparing the EIS data with the results of the QCM impedance measurement could it unambiguously be concluded that there is a true mass loss from the surface and not just a swelling of the upper fraction of the polymer film since the latter would have resulted in a significant increase of the electroacoustic parameters R (increased viscoelasticity) and XL (increased mass).

Changes in the exponent n of a constant-phase element (see Figure 5) are usually difficult to interpret as nonideality in the geometric capacitance of a polymer film can be caused by different factors such as inhomogeneity in the material, pores, or surface roughness. The observation that the increase in n at constant geometric capacitance was accompanied by mass loss from the surface of the poly(ester amide) film permitted the conclusion that the exponent n increased due to a decrease of the surface roughness of the film. The negligible reduction in the electroacoustic parameter R can be explained with the fact that the rough features on the polymer surface have dimensions smaller than the hydrodynamic decay length. In case of the dextran hydrogel, which was found to take up electrolyte and to show viscoelastic behavior, the degradation could only be detected by QCM impedance measurements. No change in electrochemical impedance was observed during degradation. Eudragit S-100 films were shown to be rigid and did not swell but formed pores, and the electrolyte spread underneath the film on the surface of the electrode after penetrating the polymer. This complex degradation mechanism could not have been resolved by means of a single technique. Difficulty in interpreting the small increase in the electroacoustic parameter ∆R after the onset of degradation arises from the fact that this parameter can depend on many factors such as viscoelastic properties and pore formation. The decrease in the bulk resistance and the presence of a doublelayer capacitance obtained from EIS could also have been interpreted as electrolyte uptake by the polymer layer. However, from the additional information obtained from QCM impedance measurements about the loss of mass and the constant electroacoustic parameter R throughout the degradation, it could be concluded that electrolyte uptake was less likely than the formation of pores and the delamination of the polymer layer. It should be noted that the process of delamination was only partial because the film and solvent remained rigidly coupled to the surface (constant R). Decoupling of the film from the surface, i.e., sudden decrease of XL and R, was actually observed when the film had not been annealed before degradation (data not shown here). The three example systems studied in this work have shown that, by combining QCM and EIS, polymers with substantially different properties can be characterized and ambiguities in the data interpretation can be resolved. ACKNOWLEDGMENT The authors are grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) for funding this project.

Received for review February 1, 2002. Accepted April 15, 2002. AC0200724

Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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