Investigation of Ig.G Adsorption and the Effect on Electrochemical

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Langmuir 2005, 21, 316-322

Investigation of Ig.G Adsorption and the Effect on Electrochemical Responses at Titanium Dioxide Electrode Simon E. Moulton,† Joseph N. Barisci,† Andrew Bath,‡ Rita Stella,‡ and Gordon G. Wallace*,† ARC Centre for Nanostructured Electromaterials, Intelligent Polymer Research Institute, University of Wollongong, NSW 2522, Australia, and Polartechnics Ltd., Level 1, 140 William Street, Sydney, NSW 2011, Australia Received May 24, 2004. In Final Form: October 12, 2004 The adsorption of Immunoglobulin G on a titanium dioxide (TiO2) electrode surface was investigated using 125I radiolabeling and electrochemical impedance spectroscopy (EIS). 125I radiolabeling was used to determine the extent of protein adsorption, while EIS was used to ascertain the effect of the adsorbed protein layer on the electrode double layer capacitance and electron transfer between the TiO2 electrode and the electrolyte. The adsorbed amounts of Ig.G agreed well with previous results and showed approximately monolayer coverage. The amount of adsorbed protein increased when a positive potential was applied to the electrode, while the application of a negative potential resulted in a decrease. Exposure to solutions of Ig.G resulted in a decrease of the double layer capacitance (C) and an increase in the charge-transfer resistance (R2) at the electrode solution interface. As more Ig.G adsorbed onto the electrode surface, the extent of C and R2 variation increased. These capacitance and charge-transfer resistance variations were attributed to the formation of a proteinaceous layer on the electrode surface during exposure.

1. Introduction Adsorption of proteins on surfaces is important in a number of fields, including biology, medicine, biotechnology, and food processing.1 The phenomenon of protein adsorption has attracted the attention of scientists from several fields who have used a diverse array of techniques to study the processes involved. Techniques such as iodine 125 (125I) radiolabeling,2,3 cyclic voltammetry,4,5 scanning tunneling microscopy,6 ellipsometry,7 and more recently electrochemical impedance spectroscopy8,9 have been employed to investigate the adsorption of proteins on various surfaces. Protein interaction with passive films such as titanium dioxide (TiO2) is of major concern in the medical industry as this may have bearing on film breakdown and ultimately implant failure.8 The interaction of TiO2 with proteins in solution forms the basis of many studies into the biocompatibility of this material.3-11 This oxide layer plays a vital role in the biocompatibility of surgical implants because this oxide layer acts as a passive layer protecting the implant from corrosion.8 * Corresponding author. Tel.: +61 (0)2 4221 3127. Fax: +61 (0)2 42213114. E-mail: [email protected]. † University of Wollongong. ‡ Polartechnics Ltd. (1) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233. (2) Duncan, M.; Gilbert, M.; Lee, J.; Warchol, M. J. Colloid Interface Sci. 1994, 165, 341. (3) Liu, F.; Zhou, M.; Zhang, F. Appl. Radiat. Isot. 1998, 49, 67. (4) Guo, B.; Anzai, J.; Osa, T. Chem. Pharm. Bull. 1996, 44, 800. (5) Cabilio, N. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16, 8480. (6) Davis, J. J.; Halliwell, C. M.; O’Hill, H. A.; Canters, G. W.; van Amsterdam, M. C.; Verbeet, M. P. New J. Chem. 1998, 1119. (7) Elwing, H. Biomaterials 1998, 19, 397. (8) Jackson, D. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16, 5449. (9) Phillips, R. K. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2001, 17, 2471. (10) Serro, A. P.; Fernandes, A. C.; Saramago, B. J.; Norde, W. J. Biomed. Mater. Res. 2000, 47, 376. (11) Serro, A. P.; Fernandes, A. C.; Saramago, B.; Lima, J.; Barbosa, M. A. Biomaterials 1997, 18, 963.

Topoglidis et al.12 studied the factors that affect protein adsorption on nanostructured TiO2 films and found immobilized protein to be remarkably stable, which they attributed to secondary binding processes occurring as immobilization time increased. Giacomelli et al.,13 using ellipsometry to study the adsorption of bovine serum albumin (BSA) on Ti/TiO2 electrodes, found that adsorption occurs in a two-step process. The first step involves rapid attachment and is governed by electrostatic interactions. The second step is much slower and involves rearrangement of the adsorbed protein molecules to find their more energetically favored orientation with respect to the TiO2 surface. While there are many papers published regarding protein adsorption and electrochemical effects at metal electrodes,14-18 there is very little information regarding the effects of protein adsorption on semiconductor electrochemistry. A recent paper by Oliva et al.19 has gone some way to filling this void and provides a valuable insight into the semiconductor-protein interface. The work presented in this paper arises from the development of TruScreen by Polartechnics Ltd. (Sydney, Australia). TruScreen is an in-vivo optoelectronic medical device used in the detection of cervical cancer and precancer. TruScreen uses a combination of optical and electrical measurements to classify cervical tissue. TruScreen employs the use of a disposable single use sensor (SUS) that fits over the handle section of the device (12) Topoglidis, E.; Campbell, C. J.; Cass, A. E. G.; Durrant, J. R. Langmuir 2001, 17, 7899. (13) Giacomelli, C. E.; Esplandiu, M. J.; Ortiz, P. I.; Avena, M. J.; De Pauli, C. P. J. Colloid Interface Sci. 1999, 218, 404. (14) Sanchez, J.; Augstynski, J. J. Electrochem. Soc. 1979, 103, 423. (15) Caprani, A.; Lacour, F. J. Electroanal. Chem. 1991, 320, 241. (16) Omanovic, S.; Roscoe, S. G. J. Colloid Interface Sci. 2000, 227, 452. (17) Bernabeu, P.; Tamisier, L.; De Cesare, A.; Caprani, A. Electrochim. Acta 1988, 33, 1129. (18) Omanovic, S.; Metikos-Hukovic, M. Thin Solid Films 1995, 266, 31. (19) Oliva, F. Y.; Avalle, L. B.; Macagno, V. A.; De Pauli, C. P. Biophys. Chem. 2001, 91, 141.

10.1021/la0487242 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/07/2004

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Table 1. Capacitance Values (µF cm-2) Measured for a TiO2 Electrode (Initial) before and (Final) after Exposure to PBS and 1.0 mg mL-1 Ig.G Prepared in PBS for 30 mina -400 mV

OCP

400 mV

exposure to

initial

final

∆Capb (%)

initial

final

∆Capb (%)

initial

final

∆Capb (%)

PBS Ig.G total ∆Capc

4.6 4.1

6.3 5.7

36.9 39.0 3.9((0.3)%

3.7 2.2

4.4 2.4

18.9 9.09 -9.8((0.5)%

3.3 3.0

4.0 3.2

21.2 6.6 -14.6((1.2)%

a Impedance measured in PBS at OCP. The potentials shown indicate the potential applied during exposure. b ∆Cap(%) ) (FinalCap - InitialCap/InitialCap) × 100%. c Total ∆Cap(%) ) (∆CapIg.G - ∆CapPBS) × 100%.

to eliminate cross contamination between patients. The SUS incorporates at its distal tip a symmetrical arrangement of three kidney-shaped TiO2 electrodes. This SUS was used for the electrochemical studies in this paper. From the electrical point of view, TruScreen monitors the electrical decay of the tissue following the application of low energy electrical pulses. TruScreen applies a potential pulse of 800 mV across a two electrode system (one electrode as the working and the other two electrodes as the reference/auxiliary electrode) during the cervical screening process; therefore, the potentials used in this work were chosen to best represent the voltage applied to each individual electrode during each pulse. For this reason, it is crucial to characterize any separate variations measured at the electrode tip due to protein interaction from changes in the structure of cervical tissue. Our previous publications have investigated the extent of protein adsorption at metal electrodes and the subsequent effect this adsorption has on charge-transfer properties at the electrode/protein interface.20,21 This paper, and those previously published,20,21 constitutes part of a detailed study into the effects of protein adsorption on the electrochemical behavior of metal electrodes. The main focus of the study involves the polyclonal antibody Ig.G prepared in a phosphate buffer. This protein was chosen, as it is representative of the type of protein found in cervical mucus22-24 which lines the cervix. The present study focuses on investigating the adsorption trends of Ig.G and the subsequent electrochemical variation at the electrode/solution interface at a TiO2 electrode using 125I radiolabeling and electrochemical impedance spectroscopy (EIS), respectively. Electrical equivalent circuit modeling of the impedance data was also performed. 2. Experimental Section 2.1. Reagents and Material. All chemicals and reagents were used as received unless otherwise stated. Human polyclonal Immunoglobulin G (Ig.G) was obtained from Sigma Aldrich and was reagent grade isolated from pooled normal serum. Sodium chloride, sodium nitrate, potassium ferricyanide, disodium orthophosphate, and sodium dihydrogen orthophosphate were all obtained from Sigma Aldrich. All solutions were prepared in Milli-Q water. The buffer used was phosphate buffer saline solution (PBS), which was prepared by combining 0.2 M Na2HPO4 and 0.2 M NaH2PO4. Enough NaCl was added to produce a final saline concentration of 0.15 M. This buffer was prepared at pH 7.0. The 0.2 M PBS solution was diluted by a factor of 50 to prepare the 4.0 × 10-3 M PBS solution used for the potential of (20) Moulton, S. E.; Barisci, J. N.; Bath, A.; Stella, R.; Wallace, G. G. J. Colloid Interface Sci. 2003, 261, 312-319. (21) Moulton, S. E.; Barisci, J. N.; McQuillan, A. J.; Wallace, G. G. Colloids Surf., A 2002, 220, 159. (22) Strous, G. J.; Dekker, J. Crit. Rev. Biochem. Mol. Biol. 1992, 27, 57-92. (23) Saltzman, W. M.; Radomsky, M. L.; Whaley, K. J.; Cone, R. A. Biophys. J. 1994, 66, 508-515. (24) Bansil, R.; Stanley, E.; LaMont, J. T. Annu. Rev. Physiol. 1995, 57, 635-657.

zero charge measurements. The 0.2 M PBS was used to prepare the electroactive PBS containing 0.1 M NaNO3 and 0.01 M K3Fe(CN)6. The 125I isotope was obtained from Amrad/Biotech. The IODO Beads and PD-10 Sefadex Gel Filtration columns used in the labeling process were purchased from Lab Supply and Amersham, respectively. The TiO2 electrode used in the 125I adsorption study was fabricated by electrochemically oxidizing a Ti coated polycarbonate sheet in 1.0 M H2SO4. The TiO2 electrodes used in the EIS study were obtained from Polartechnics Pty. Ltd. For both the adsorption and the EIS experiments, the TiO2 samples had an area of 0.50 cm2. No pretreatment of the TiO2 samples was performed prior to Ig.G adsorption. 2.2. Radiolabeling. All radiolabeling experiments were performed in a controlled “hot” laboratory. The radiolabeled samples were counted using a Whitman-3 γ-counter. Ig.G was prepared into 0.1 mg mL-1 solution using 0.2 M PBS (pH 7.0) (hereafter referred to as PBS) with aliquots being freeze-dried and stored at -2.0 °C. The protein was labeled using the protocol outlined in the Amrad/Pharmacia Technical Notes. Solutions containing a ratio of 50:2 (unlabeled:labeled) protein were prepared using the unlabeled and labeled protein prepared in PBS. These solutions were used in the adsorption experiments. Labeled solutions with varying Ig.G concentration were used for the adsorption isotherm experiments. When adsorption occurred at constant applied potential, the samples were exposed to the protein solution for various lengths of time. For the adsorption isotherm experiments, the samples were immersed in labeled Ig.G, at various concentrations, for 60 min. After adsorption, the electrodes were removed from the cell, rinsed in PBS then Milli-Q water, and allowed to dry at room temperature. The radioactivity of the electrode was then counted for 10 min using the γ-counter. The adsorbed amount was calculated from the counts per minute and the labeled protein specific activity. This adsorption/counting procedure was used for all radiolabeling experiments. 2.3. Electrochemical Impedance Spectroscopy. All electrochemical measurements were performed at room temperature, in a three-electrode cell comprised of a SUS TiO2 (electrochemically generated on a Ti electrode) working electrode (0.50 cm2), platinum mesh auxiliary (95.0 cm2) electrode, and Ag/AgCl (3 M NaCl) reference electrode. Cyclic voltammograms presented in this paper were recorded using a Princeton Applied Research (PAR) 263 potentiostat at a scan rate of 100 mV s-1. All impedance measurements were recorded between 50 mHz and 100 kHz with an AC amplitude of 10 mV, using a Princeton Applied Research BES Impedance system. All electrochemical measurements were performed in oxygenated PBS (unless otherwise stated). The reason for not degassing the PBS solution was to ensure that the solution conditions during electrochemical measurements were similar to the environment in which TruScreen operates. All EIS measurements were performed at OCP in PBS unless otherwise stated. Because the TiO2 electrode used in the EIS study was a single use electrode, it was not possible to measure impedance after exposure to PBS and Ig.G on the same electrode. Therefore, for each electrode, an “initial” impedance measurement was made before exposure and a “final” one was made after exposure. The changes in capacitance (initial capacitance - final capacitance), due to exposure to Ig.G and the interaction of phosphate from the PBS, were converted to percentages (see Table 1) and were compared for separate electrodes for PBS and

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Figure 2. Adsorption isotherms for Ig.G measured for TiO2. Adsorption occurred from an Ig.G solution of pH ) 7.0 at OCP. The error bars show a measurement error of approximately 8.3%.

Figure 1. (a) Cyclic voltammogram showing the oxidation of Ti in 1.0 M H2SO4 to form TiO2. Scan rate was 100 mV s-1 with the arrows showing the direction of the scan. AFM images of (b) Ti electrode and (c) TiO2 electrode after electrochemical oxidation of Ti electrode. Ig.G interaction. In previous studies,25 exposure to protein solutions for 30 min was sufficient to reach electrode surface saturation. The impedance data recorded in PBS were modeled using Zview version 2.2 (Scribner - USA) software. The impedance spectra were fitted to the electrical equivalent circuit using initial starting resistance and capacitance values calculated from the impedance data. For each set of experimental conditions, all spectra were modeled using the same circuit.

3. Results and Discussion 3.1. Conversion of Ti to TiO2. The CV for the formation of the TiO2 film is presented in Figure 1a. The large oxidation current is associated with the formation of the oxide layer. The formation of the oxide layer was confirmed using AFM. Upon oxidation of Ti in 1.0 M H2SO4, an increase in surface roughness was observed resulting in a total surface area increase of 19.2%, as compared to a flat Ti surface (Figure 1b and c, respectively). The nature of the oxide layer varies depending on the final oxidizing (25) Moulton, S. E.; Barisci, J. N.; McQuillan, A. J.; Wallace, G. G. Colloids Surf., A 2003, 220, 159-167.

potential,26 and the TiO2 film forms as a polycrystalline layer consisting of a mixture of rutile and anatase. 3.2. Ig.G Adsorption. Radiolabeling was used to investigate the adsorption of Ig.G on the TiO2 electrode from solutions at pH 7.0 and with different potentials applied to the electrode. The protein concentration used in this work was 0.10 mg mL-1 because surface saturation occurs at this concentration (Figure 2). All adsorbed amounts are reported in mass (mg) per actual surface area (m2 - geometric surface area plus 19.2%). At each Ig.G concentration, the TiO2 electrode was exposed to the solution for 60 min. A well-defined maximum adsorption plateau is observed at concentrations greater than 0.10 mg mL-1. There appears to be two plateau regions in the isotherm, one at low concentrations and the other at high concentrations. The presence of a step or “kink” has been reported elsewhere for globular protein isotherms.27 The presence of the step indicates that protein binding to the surface is bimodal. To explain the appearance of this kink, Koutsouko et al.28 proposed that orientation of the adsorbed protein varies with surface coverage, that is, a side-on to end-on transition. Evidence for such an orientation transition has come from surface force measurements of albumin on various hydrophilic surfaces.27-29 When a potential is applied to the TiO2 electrode during exposure, the isotherms exhibited a much less pronounced two-plateau region. The adsorption profiles for Ig.G with different applied potentials are shown in Figure 3. It was observed that the application of a potential to the electrode has varying effects on the adsorption of Ig.G. The exact pI of the polyclonal Ig.G antibody used for this work is unknown, but it is considered to be between pH 5.0 and 8.0.30 Protein adsorption was greatest when the electrode potential was 400 mV; it decreased at OCP (-100 mV) and further decreased at -400 mV. This trend in the amount of protein adsorbed was observed for adsorption of HSA and Ig.G at a gold electrode.20 Increased adsorption of Ig.G at positive potentials was also observed by Guo et al.4 Adsorption appeared to still be occurring after 60 min; however, the rate of adsorption had reached a pseudo plateau value. The amount of Ig.G adsorbed after 60 min was taken to be the saturation point. (26) Finklea, H. O., Ed. Studies in Physical and Theoretical Chemistry - Semiconductor Electrodes; Elsevier Science: New York, 1988; Vol. 55. (27) Haynes, C. A.; Norde, W. Colloids Surf., B 1994, 2, 517. (28) Koutsoukos, P.; Mumme-Young, C.; Norde, W.; Lyklema, J. Colloids Surf. 1982, 5, 93. (29) Blomberg, E.; Claesson, P.; Golander, C. J. Dispersion Sci. Technol. 1991, 12, 176. (30) Hidalgo-Alvarez, R.; Galisteo-Gonzalez, F. Heterog. Chem. Rev. 1995, 2, 249.

Investigation of Ig.G Adsorption

Figure 3. Adsorption profiles for TiO2 samples in 0.1 mg mL-1 Ig.G at pH ) 7.0. Applied potential during adsorption: (O) 400 mV, (0) OCP, and (4) -400 mV. The error bars show a measurement error of approximately 3.2% (400 mV), 2.1% (OCP), and 3.7% (-400 mV).

The overall adsorption behavior observed in Figure 3 may be considered to be the result of a number of processes: redistribution of charged groups, van der Waals interactions between the protein and the sorbent, and structural rearrangement in the adsorbing protein molecule.31 Roscoe et al.5,8,32 and others,33,34 using thermodynamic and FTIR studies, respectively, have shown this last point to be significant for “soft proteins” such as Ig.G.35 Immunoglobulin G usually adsorbs to adopt a Y shape with dimensions of 4.5 × 4.5 × 23.5 nm.36 Adsorption of proteins has been considered to be either horizontal or vertical, and many papers provide calculated data for both orientations of adsorbed proteins at complete surface coverage.1,4,37 Using the dimensions of the Ig.G molecule, the adsorbed amount at complete electrode surface coverage for vertical and horizontally oriented adsorption is 12.02 and 3.30 mg m-2. Based on the calculated adsorbed amounts for each adsorption orientation, the adsorbed amount at OCP (3.05 mg m-2), 400 mV (3.58 mg m-2), and -400 mV (2.32 mg m-2) in Figure 3 corresponds to approximately a monolayer of horizontally adsorbed Ig.G molecules. However, these adsorbed amounts do not correspond to the vertical orientation proposed by Malmsten36 based on ellipsometry studies. 3.3. Electrochemical Impedance Spectroscopy Studies. 3.3.1. Electrode Characterization. Initially, electrode processes at the TiO2 electrode in PBS were characterized by cyclic voltammetry (CV) (Figure 4). Between 0.0 and 700 mV, no Faradaic (electron-transfer) processes where noted with only double layer charging observed. At potentials below 0.0 mV, a reduction response is observed associated with the reduction of dissolved oxygen, hydrogen adsorption, and Ti(IV) reduction (Figure 4). Upon degassing the PBS with nitrogen gas, the reduction current decreases associated with the partial removal of dissolved oxygen. The electrode open circuit potential (OCP) was measured in PBS using the EIS system by monitoring the potential between the working and reference electrode until a steady (31) Norde, W. L., Ed. Polymer Science and Technology; Plenum Press: New York, 1980; Vol. 12B. (32) Hanrahan, K.; Macdonald, S. M.; Roscoe, S. G. Electrochim. Acta 1996, 41, 2469. (33) Buijs, J.; Norde, W.; Lichtenbelt, J. W. Langmuir 1996, 12, 1605. (34) Servagent-Noinville, S.; Revault, M.; Quiquampoix, H.; Baron, M. H. J. Colloid Interface Sci. 2000, 221, 273. (35) Blomberg, E., Claesson, P. M., Eds. Proteins at Interfaces; 1993; Vol. 2. (36) Malmsten, M. Colloids Surf., B 1995, 3, 297. (37) Lassen, B.; Malmsten, M. J. Colloid Interface Sci. 1996, 180, 339.

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Figure 4. TiO2 electrode cyclic voltammogram recorded in 0.2 M PBS (pH 7.0), before and after deoxygenation with N2 gas at a scan rate of 100 mV s-1. The arrows show the direction of the scan.

Figure 5. Capacitance versus potential plot for the TiO2 electrode. Capacitance values were obtained from the diameter of the circle fit applied to each impedance complex capacitance plot recorder at each potential. Impedance was measured at each potential in a solution of 4.0 × 10-3 M PBS at pH 7.0. The error bars show a measurement error of approximately 7.4%.

value was obtained. Steady state was achieved after approximately 180 min with a final OCP of -100 mV. This value was subsequently used for all TiO2 impedance measurements performed at OCP. To determine the potential of zero charge for the TiO2 electrode, a plot of capacitance as a function of potential was obtained in 4 × 10-3 M PBS (Figure 5). Capacitance values were obtained from impedance spectra recorded between 50 mHz and 100 kHz in PBS. The capacitance reached a minimum between -200 and 0 mV. The point of zero charge (pzc) was estimated to be -100 mV. This value agrees reasonably well with the value obtained by Oliva et al.19 for a nanocrystalline Ti/TiO2 electrode in 0.1 M NaCl. For potentials more positive than -100 mV, the electrode surface carries a positive charge, and for potentials more negative than -100 mV, the electrode charge is negative. 3.3.2. Non-Faradaic - Double Layer Effects. The shape of the impedance spectra is dependent on the potential applied to the electrode during the impedance measurement (Figure 6). The impedance spectra show three distinct frequency regions. In the high-frequency region, the impedance modulus (log |Z|) is independent of frequency with the phase angle value at or near 0° as represented in the phase angle Bode plot. This behavior is attributed to the resistance of the phosphate solution between the working and the reference electrode (ohmic resistance - R1).38 In the medium-frequency region, a linear relationship can be observed between the impedance (38) Omanovic, S.; Roscoe, S. G. Langmuir 1999, 15, 8315.

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Figure 6. log |Z| (a) and phase angle (b) plots of TiO2 electrode recorded in 0.2 M PBS (pH 7.0). The potentials applied during the impedance measurement are shown. The frequency range was 50 mHz to 50 kHz.

modulus and log frequency. This frequency region corresponds to the capacitive behavior of the electrode/ electrolyte interface.38 A third region appears in the lowfrequency region (50 Hz to 50 mHz) for impedance measured at -400 mV, where the phase angle reaches a maximum, after which time it starts to decrease. Variation in this region may be attributed to the application of the reducing potential of -400 mV generating Faradaic processes at the electrode/PBS interface. It has been shown21 that Ig.G and phosphate spontaneously adsorb on to a TiO2 surface at pH 7.0. The impedance spectra of a TiO2 electrode were recorded before and after exposure to a solution of PBS and a solution of 1.0 mg mL-1 Ig.G prepared in PBS. It was necessary to increase the Ig.G concentration by an order of magnitude (0.1 mg mL-1 for 125I adsorption study to 1.0 mg mL-1 for EIS study) to observe measurable capacitive changes at the TiO2 electrode. While adsorption was performed at three DC potentials (-400 mV, OCP, and 400 mV), all impedance measurements were performed at OCP (-100 mV) to minimize Faradaic processes influencing the impedance data. The capacitance was measured from the fit applied to the recorded capacitance plot. At all potentials, the capacitance increased after exposure to solutions of PBS and 1.0 mg mL-1 Ig.G prepared in PBS, with the ∆ values indicating the increase with respect to the initial capacitance value (Table 1). This increase in electrode capacitance observed in Table 1 (∆Cap values) agrees with results published elsewhere,26 where an increase in capacitance occurs when the electrode is biased from the Vfb (flatband potential) at a given pH. Oliva et al.19 also found an increase in electrode capacitance of ∼5% after protein adsorption near the flat band

Figure 7. Nyquist plot for a TiO2 electrode recorded in PBS at OCP, before (initial) and after (final) exposure to Ig.G at OCP (a), -400 mV (b), and 400 mV (c) for 30 min. The symbol ([) represents the experimental data, while (- - - -) is the simulated fit using the EEC shown. Zim ) imaginary impedance, ZR ) real impedance.

potential of Ti/TiO2 nanocrystalline electrode (-0.6 V). Finklea26 also points out that it is possible to dope TiO2 electrochemically at room temperature by biasing the electrode negative of the flatband potential and the increased doping accounts for this capacitance change. The extent of capacitance increase after Ig.G adsorption at OCP and 400 mV was less than after exposure to the PBS solution. When the ∆ value due to PBS interaction is subtracted from the ∆ value due to Ig.G interaction, the

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Table 2. Charge-Transfer Resistance Values Measured for a TiO2 Electrode (Initial) before and (Final) after Exposure to PBS and 1.0 mg mL-1 Ig.G Prepared in PBS for 30 mina -400 mV

OCP

400 mV

exposure to

initial

final

∆R2b (%)

initial

final

∆R2b (%)

initial

final

∆R2b (%)

PBS Ig.G total ∆R2c

0.021 0.120

0.015 0.079

-40 -34.2 5.8((0.9)%

1.045 4.158

0.538 2.828

-48.5 -31.9 16.6((1.3)%

8.544 11.14

4.776 9.70

-44.1 -12.9 31.2((2.1)%

a Impedance was measured in electroactive PBS at OCP. The potentials shown indicate the potential applied during exposure. b ∆R (%) 2 ) (FinalR2 - InitialR2/InitialR2) × 100%. c Total ∆R2(%) ) (∆R2Ig.G - ∆R2PBS) × 100%.

net effect due to exposure to a solution of Ig.G is a decrease in the TiO2 electrode capacitance. This net decrease agrees with Wang et al.39 and Contu et al.,40 who observed a slight decrease in capacitance when Ti electrodes were immersed in a solution of simulated body fluid and fetal bovine serum, respectively. The extent to which the capacitance decreased was 9.8% and 14.6% for the impedance measured after adsorption at an electrode potential of OCP and 400 mV, respectively. The decrease in capacitance has been attributed to the adsorption of a continuous proteinaceous layer.40 The extent of capacitance decrease agrees with the amount of adsorbed Ig.G stated in section 3.1, the greater the adsorbed amount, the greater the capacitance decrease. In addition, a decrease in electrode capacitance could be envisaged because the dielectric constant of the organic species in the proteinaceous layer present in the Helmholtz double layer is lower than that of water.17 Adsorption occurring at an electrode potential of -400 mV resulted in a slight increase in electrode capacitance (3.9%). This increase in capacitance may be the result of electrode processes occurring during exposure, because at -400 mV Faradaic processes are occurring. EEC modeling was applied to the impedance data obtained from measurements performed in PBS (section 3.3.2). The circuit shown in the inset of Figure 7a produced a good fit to the experimental data obtained. The correlation between experimental data and simulated fit was observed for initial and final impedance data (PBS and Ig.G) recorded after exposure at all potentials investigated. The circuit was comprised of a resistor (R1 - ohmic resistance) in series with a constant phase element (CPE) in parallel with a resistor (R2 - charge-transfer resistance). In place of pure capacitance, a CPE was introduced in the modeling procedure representing the electrical double layer of the electrode/electrolyte interface. The use of CPE in the model shown in Figure 7 is due to a distribution of

relaxation times as a result of inhomogeneities present at the microscopic level at the electrode/electrolyte interface.38 This may result from the contributions from static disorder due to porosity41 as well as the nonideal capacitive response of the interface.40-42 3.3.3. Faradaic - Electron-Transfer Effects. The application of OCP to the electrode in PBS does not induce any Faradaic response (Figure 4). When the electroactive moiety (potassium ferricyanide - 0.01 M K3Fe(CN)6) is added to the PBS solution, the application of OCP (-100 mV) to the TiO2 electrode causes the reduction of ferricyanide to occur (Figure 8). All of the initial CVs recorded prior to exposure to the protein solutions showed no or only a very slight redox couple associated with the oxidation and reduction of the ferricyanide ions. However, after exposure, the current density associated to the redox couple was much more pronounced (Figure 8). This result suggests that during the adsorption process the electrode oxide surface may be modified (activated) in some way. By performing impedance measurement of the TiO2 electrode in the electroactive PBS, before and after exposure to PBS and Ig.G prepared in PBS, it is possible to monitor the change in the Faradaic charge transfer. Impedance spectra of the TiO2 electrode were recorded in electroactive PBS at OCP (-100 mV) prior to exposure to either PBS or Ig.G prepared in PBS (initial). The TiO2 electrode was then exposed to either PBS or Ig.G prepared in PBS at OCP, -400 mV, and 400 mV for 30 min. After exposure, the TiO2 electrode was placed back into the electroactive PBS solution, and the impedance spectra were recorded at OCP (final). By comparing the Nyquist plot before and after exposure of the TiO2 electrode to solutions of PBS and Ig.G prepared in PBS, it is possible to observe the changes in the electrode charge-transfer resistance (R2) brought about by adsorption. As adsorption occurred at the electrode surface, the semicircle feature became smaller, indicating a reduction in R2 (Figure 9). This reduction in R2 after adsorption is the opposite of what was observed previously for a gold electrode.20 This could be due to modification of the TiO2 surface during protein adsorption. The surface modification appears to activate the surface of TiO2. The decrease in R2 after exposure to Ig.G was less than that after exposure to a solution of PBS (Figure 9). This agrees with what was observed for the gold electrode where an increase in R2 was observed after exposure to a solution of Ig.G but not PBS.20 If the activation of the TiO2 electrode were the same after exposure to both PBS and Ig.G, then the percentage change in R2 (∆R2%) would be similar. However, if during exposure, adsorption of Ig.G causes an increase in R2, then the ∆R2% would be less than that of PBS. The extent of ∆R2% upon exposure to PBS and Ig.G is shown in Table 2. From Table 2 and Figure 9, it is clear that the overall effect of Ig.G adsorption at the TiO2 electrode is an increase

(39) Wang, C. X.; Wang, M.; Zhou, X. Langmuir 2002, 18, 7641. (40) Contu, F.; Elsener, B.; Bohni, H. J. Biomed. Mater. Res. 2002, 62, 412-421.

(41) Kramer, M.; Tomkiewicz, M. J. Electrochem. Soc. 1984, 131, 1283. (42) Mulder, W. H.; Sluyters, J. H. Electrochim. Acta 1988, 33, 303.

Figure 8. A TiO2 CV recorded in electroactive PBS (initial) before and (final) after exposure to a solution containing 1.0 mg mL-1 Ig.G (pH 7.0) prepared in PBS. The scan rate used was 100 mV s-1. The arrows show the direction of the scan.

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effective current density and the over voltage. From the results presented in Figure 3 and Table 2, it appears that as more protein adsorbs at the electrode surface the greater the increase in R2. EEC modeling was also applied to the impedance data obtained from measurements performed in electroactive PBS. The circuit shown in the inset of Figure 9a produced a good fit to the experimental data obtained, except at low frequencies. The poor fit observed at low frequencies may be attributed to the movement of ions, due to the Faradaic process, influencing the recorded impedance measurement in this region. The correlation between experimental data and simulated fit was observed for initial and final impedance data (PBS and Ig.G) recorded after exposure at all potentials investigated. The circuit used is the same as that used to model the impedance data obtained in section 3.3.2, with the electronic components of the circuit being the same as those listed for the circuit shown in Figure 7a. The variation in R2 of the circuit for the simulated fit agrees with that found experimentally. That is, adsorption of Ig.G at the TiO2 electrode caused an overall increase in R2. 4. Conclusions

Figure 9. Nyquist plot of impedance spectra recorded for a TiO2 electrode in 0.2 M PBS containing 0.01 M K3Fe(CN)6 and 0.1 M NaNO3 at OCP (pH 7.0). The spectra were recorded (initial) before and (final) after exposure at OCP to (a) a solution of 0.2 M PBS and (b) a solution containing 1.0 mg mL-1 Ig.G (pH 7.0) prepared in PBS. The symbol ([) represents the experimental data, while (- - - -) is the simulated fit using the EEC shown. Zim ) imaginary impedance, ZR ) real impedance.

in R2. This result agrees with our previous results for a gold electrode.20 The increase in R2 is due to the electrode surface becoming coated with a continuous protein layer. This continuous layer acts as a barrier, preventing the ferricyanide ions from approaching the electrode surface. This view is supported by the work performed by Anzani et al.43 and Willner et al.,44 where they noted that coverage of the electrode by a foreign substance decreases the

Adsorption of Ig.G at a TiO2 electrode occurs spontaneously at pH 7.0. The amount of adsorbed protein increases upon application of a potential positive of the OCP and decreased upon application of a potential negative of OCP. Using electrochemical impedance spectroscopy, it was possible to study the effects of protein interaction on the double layer capacitance and electron-transfer processes at the electrode/electrolyte interface. Under all experimental conditions, the interaction of protein with the electrode surface resulted in a decrease in double layer capacitance and an increase in charge-transfer resistance, except at -400 mV. These changes are attributed to the formation of a continuous protein layer at the electrode surface, and the extent of these changes varied with the amount of adsorbed Ig.G. The greater was the adsorbed amount, the greater were the changes. The experimental impedance data measured in PBS and electroactive PBS at OCP before and after exposure to PBS and Ig.G were successfully modeled using an electrical equivalent circuit. Acknowledgment. G.G.W. acknowledges the continued support of the Australian Research Council (ARC). S.E.M. was supported by an ARC Linkage Scholarship with Polartechnics Ltd. LA0487242 (43) Anzani, J.; Guo, B.; Osa, T. Chem. Pharm. Bull. 1994, 42, 2391. (44) Willner, I.; Rubin, S.; Cohen, Y. J. Am. Chem. Soc. 1993, 115, 4937.