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Electrochemical Studies of the Adsorption Behavior of Serum Proteins on Titanium Douglas R. Jackson, Sasha Omanovic, and Sharon G. Roscoe* Department of Chemistry, Acadia University, Wolfville, Nova Scotia, B0P 1X0 Canada Received November 16, 1999. In Final Form: March 7, 2000 Cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were used to examine the adsorption behavior of bovine serum albumin (BSA) and bovine fibrinogen on titanium in phosphate buffer pH 7.4, over the temperature range 295-343 K. It was shown that the surface charge density is directly proportional to the amount of the adsorbed protein (surface concentration), thus indicating that the adsorption is accompanied by the transfer of charge, i.e. chemisorption. On the other hand, the resulting adsorption pseudocapacitance obtained under the potentiostatic conditions not only depends on the protein surface concentration but also is a very complex function of parameters that are, in turn, dependent on structural, physical, and chemical properties of the proteins. Both techniques were shown to be very sensitive to the conformational behavior of the proteins. The adsorption of BSA onto a Ti surface resulted in a bimodal isotherm at all the temperatures studied, while the adsorption of fibrinogen resulted in a single saturation plateau. The adsorption process was modeled with a Langmuir adsorption isotherm. It was found that fibrinogen exhibits more than twice the affinity for adsorption onto a Ti surface compared to BSA. At lower surface coverage, adsorption appears to be mainly surface binding rate limited. The calculated standard Gibbs energies of adsorption also suggested a very strong adsorption of both proteins through a chemisorption process. The adsorption process for both proteins was found to be endothermic, resulting from the excess energetics required for the disruption of intramolecular interactions relative to those involved in the formation of protein-metal interactions, i.e. chemisorption at the electrode surface. In addition, adsorption of BSA onto a Ti surface at low concentrations was shown to be an entropically controlled process, also suggesting structural unfolding of the protein occurs at the electrode surface.
Introduction In recent years, the corrosion and wear of titanium implants has become a concern.1-5 Most metallic materials used in implants rely on passive oxide films on their surface to develop good corrosion resistance.3,6 Conditions that adversely affect the stability of these passive films and reduce its ability to control undesirable metal dissolution are the main concerns in corrosion. As a result, protein interactions with the passive film on the metal surfaces are therefore of considerable interest in medicine as they may have a bearing on film breakdown, metal ion release, and film repair processes.3 When titanium is released into the surrounding tissue in the form of metal ions either by passive diffusion or by protein adsorption and subsequent desorption of formed metal-protein complexes, the reaction of the tissue varies from the discoloration of the tissue to more severe reactions occurring as the implant continues to corrode. The pocket formed between the implant and the receded tissue provides a suitable microenvironment for bacterial and viral infections of the adsorbed protein biofilm on the implant. These infections can result in increased corrosion caused by bacterial metabolic products, localized temperature increases, and pH changes. Since implants are often surrounded by blood-rich tissue, serum proteins have been identified as being the main * To whom correspondence should be addressed. (1) Rosengren, A.; Johansson, B. R.; Danielsen, N.; Thomsen, P.; Ericson, L. E. Biomaterials 1996, 17, 1779. (2) Williams, R. L.; Brown, S. A.; Merritt, K. Biomaterials 1988, 9, 181. (3) Okazaki, Y.; Tateishi, T.; Ito, Y. Mater. Trans. 1997, 38, 78. (4) Kanagaraja, S.; Lundstrom, I.; Nygren, H.; Tengvall, P. Biomaterials 1996, 17, 2225. (5) Kornu, R.; Maloney, W. J.; Kelly, M. A.; Smith, R. L. J. Orthopaedic Res. 1996, 14, 871. (6) McAleer, J. F.; Peter, L. M. J. Electrochem. Soc. 1982, 129, 1252.
contributors to biofilm formation.7 Two serum proteins of particular interest are albumin and fibrinogen. There has been an extensive amount of literature on studies of the adsorption of serum albumin and fibrinogen on glass8-16 and various polymers.8-13,17-20 These polymers are widely used for artificial heart valves, dialysis machine membranes and tubing, and joint implants. Most of the reported work relies heavily on trace labeling methods with 131I and 125I, but techniques such as infrared internal reflection spectroscopy (IIRS)17 and differential scanning calorimetry (DSC)14 have also been used in these measurements. A number of studies have been made on the interaction of proteins with metal surfaces to determine the molecular conformation or orientation of the adsorbed molecules. (7) Ratner, B. D. In Biomaterials science: An Introduction to Materials in Medicine; Ratner, B. D., Hoffman, A. S., Schoen, F. J., Lemons, J. E., Eds.; Academic Press: Toronto, 1996; pp 1-50. (8) Brash, J. L.; Davidson, V. J. Thromb. Res. 1976, 9, 249. (9) Chan, B. M. C.; Brash, J. L. J. Colloid Interface Sci. 1981, 82, 217. (10) Schmitt, A.; Varoqui, R.; Uniyal, S.; Brash, J. L.; Pusineri, C. J. Colloid Interface Sci. 1983, 92, 25. (11) Brash, J. L.; ten Hove, P. Thromb Haemostas 1984, 51, 326. (12) Wojciechowski, P.; ten Hove, P.; Brash, J. L. J. Colloid Interface Sci. 1986, 111, 455. (13) Brash, J. L.; ten Hove, P. J. Biomed. Mater. Res. 1989, 23, 157. (14) De Baillou, N.; Dejardin, P.; Schmitt, A.; Brash, J. L. J. Colloid Interface Sci. 1984, 100, 167. (15) Brash, J. L.; Uniyal, S.; Chan, B. M. C.; Yu, A. ACS Symp. Ser. 1984, 256, 45. (16) Wojciechowski, P.; Brash, J. L. J. Biomater. Sci. Polym. Ed. 1991, 2, 203. (17) Lyman, D. J.; Brash, J. L.; Chaikin, S. W.; Klein, K. G.; Carini, M. Trans. Am. Soc. Artif. Int. Organs 1968, 14, 250. (18) Brash, J. L.; Uniyal, S. Trans. Am. Soc. Artif. Int. Organs 1976, 22, 253. (19) Brash, J. L.; Uniyal, S. J. Polym. Sci., Polym. Symp. 1979, 66, 377. (20) Santerre, J. P.; ten Hove, P.; VanderKamp, N. H.; Brash, J. L. J. Biomed. Mater. Res. 1992, 26, 39.
10.1021/la991497x CCC: $19.00 © 2000 American Chemical Society Published on Web 05/05/2000
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Recent studies in our laboratory have been made on globular proteins such as β-lactoglobulin,21-23 R-lactalbumin,24 κ-casein,25 ribonuclease,22,23 lysozyme,22,23 BSA,24 insulin,26 cytochrome c,27 myoglobin,27 and hemoglobin27 at a platinum electrode using electrochemical techniques. The surface charge density resulting from protein adsorption was found to be sensitive to the conformational behavior of the proteins. The carboxylate groups were determined to play a major role as the surface-active functional group of the proteins at anodic potentials, and a mechanism has been proposed. A number of studies have evaluated the effect of proteins on the corrosion of materials of interest to medicine.1-5,28-36 Typically, the techniques that have been used include electrochemical impedance spectroscopy (EIS),28 cyclic linear polarization (“pitting” polarization),29,30 Tafel polarization,2 solution depletion methods,31 X-ray photoelectron spectroscopy (XPS),32,33 scanning electron microscopy (SEM),32,33 atomic force microscopy (AFM),33 and atomic absorption spectrophotometry (AAS).34 It was established that generally proteins interact and alter the corrosion behavior of the surface in two ways: adsorption and chelation. Omanovic and Roscoe,28 using electrochemical impedance spectroscopy, found that adsorption of BSA onto an austenitic stainless steel surface resulted in an increased corrosion rate up to 55%. Clarke and Williams,34 using atomic absorption spectrophotometry, showed the effects of serum proteins (BSA and fibrinogen) on metallic corrosion. They found that protein adsorption greatly enhanced the corrosion of the first-row transition metals due to their ability to form complexes. Proteincatalyzed dissolution of these metals was also suggested. Williams, Brown, and Merritt2 found that 316L stainless steel and commercially pure titanium exhibited greater corrosion in the presence of serum proteins. Bernabeu and Caprani35 examined the adsorption behavior of BSA and fibrinogen as a function of electrode surface charge on platinum and carbon rotating disk electrodes and found that the rate of adsorption and the area of the adsorbed protein in close contact with the electrode surface increase with the increasing negative charge of the surface. In the present paper, we report on the adsorption behavior of BSA and fibrinogen onto commercially pure titanium, (cp)Ti, in phosphate buffer pH 7.4, under potentiodynamic and potentiostatic conditions. The ad(21) Roscoe, S. G.; Fuller, K. L.; Robitaille, G. J. Colloid Interface Sci. 1993, 160, 245. (22) Roscoe, S. G.; Fuller, K. L. J. Colloid Interface Sci. 1992, 152, 429. (23) Roscoe, S. G. In Modern Aspects of Electrochemistry; Bockris, J. O’M., Conway, B. E., White, R. E., Eds.; Plenum Press: New York, 1996; Vol. 29, p 319. (24) Rouhana, R.; Budge, S. M.; MacDonald, S. M.; Roscoe, S. G. Food Res. Int. 1997, 30, 303. (25) Roscoe, S. G.; Fuller, K. L. Food Res. Int. 1993, 26, 343. (26) MacDonald, S. M.; Roscoe, S. G. J. Colloid Interface Sci. 1996, 184, 449. (27) Hanrahan, K. L.; MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1996, 41, 2469. (28) Omanovic, S.; Roscoe, S. G. Langmuir 1999, 15, 8315. (29) Tomas, H.; Freire, A. P.; Abrantes, L. M. J. Mater. Sci.: Mater. Med. 1994, 5, 446. (30) Hansen, D. C.; Dexter, S. C.; Waite, J. H. Corros. Sci. 1995, 37, 1423. (31) Hansen, D. C.; Luther III, G. W.; Waite, J. H. J. Colloid Interface Sci. 1994, 168, 206. (32) Serro, A. P.; Fernandes, A. C.; Saramago, B.; Lima, J.; Barbosa, M. A. Biomaterials 1997, 18, 963. (33) Nanci, A.; Wuest, J. D.; Peru, L.; Brunet, P.; Sharma, V.; Zalzal, S.; McKee, M. D. J. Biomed. Mater. Res. 1998, 40, 324. (34) Clarke, G. C. F.; Williams, D. F. J. Biomed. Mater. Res. 1982, 16, 125. (35) Bernabeu, P.; Caprani, A. Biomaterials 1990, 11, 258. (36) Pan, J.; Thierry, D.; Leygraf, C. J. Biomed. Mater. Res. 1994, 28, 113.
Jackson et al. Table 1. Chemical Composition of Commercially Pure Titanium (Maximum Amount by % Mass)1 Ti
C
H
Fe
N
O
bulk
0.10
0.0125-0.015
0.5
0.05
0.4
sorption of the proteins to the surface directly from an aqueous solution is discussed on the basis of cyclic voltammetry and electrochemical impedance spectroscopy measurements. Saturated surface coverages and thermodynamic adsorption values are presented. Experimental Section Reagents and Solutions. Solutions of bovine fibrinogen (Sigma Chemical Co., F-8630) and bovine serum albumin (Sigma Chemical Co., A-6793) were prepared by dissolving reagents in 0.05 M phosphate buffer (pH 7.4). The buffer was made by dissolving monobasic, anhydrous KH2PO4 (Sigma Chemical Co.) in conductivity water (Nanopure, resistivity ) 18.2 MΩ‚cm) and adding 0.10 M sodium hydroxide (made from concentrated volumetric solution, ACP Chemical Inc.) to adjust the pH of the solution. Electrochemical Cell and Electrodes. A single compartment, glass electrochemical cell was used for all measurements. The working electrode was commercially pure titanium rod, (cp)Ti (99.7%, 6.4 mm diameter, alpha structure, Johnson, Matthey and Mallory). The rod was sealed in Teflon TFE tubing (ColeParmer Instrument Co.) with Torr Seal (Varian Vacuum Products) to produce a two-dimensional surface. The real surface area of the electrode was calculated on the basis of capacitance measurements performed with the electrode polished using 1500 grit sandpaper and 1 µm diamond paste, for which a roughness factor was found to be 2. The chemical composition of (cp)Ti used in this research is given in Table 1. The counter electrode was a large-area platinum electrode (mesh) of high purity (99.99%, Johnson-Matthey), which was degreased by refluxing in acetone, sealed in soft glass, electrochemically cleaned by potential cycling in 0.5 M sulfuric acid, and stored in 98% sulfuric acid. Before each use, the electrode was washed with mixed acid (1:1 concentrated H2SO4 to concentrated HNO3) and then rinsed with Nanopure water. A saturated calomel reference electrode (Corning Scientific Instruments, Cat. No. 476002) was used as the reference electrode and stored in a saturated potassium chloride solution. All potentials in this paper are referred to the SCE. Electrochemical Equipment. Electrochemical data were obtained using a VoltaLab 40: Dynamic EIS and Voltammetry Electrochemical Laboratory (Radiometer). All measurements were recorded and graphically displayed using Voltamaster 4 (Radiometer) computer software. Impedance data were modeled using Boukamp’s Equivalent Circuit 3.97 software. Experimental Methodology. Prior to each experiment, the working electrode was wet polished with 1500 grit emery paper, thoroughly rinsed with Nanopure water, degreased with acetone, and again rinsed with Nanopure water. This removed the thick oxide layer previously formed and produced a reproducible surface. All measurements were carried out in an oxygen-free solution, which was achieved by continuous purging of the cell with argon (Praxair Products Inc.). This bubbling also provided a well-mixed bulk solution. The protein solution was prepared in a separate container and allowed to equilibrate for at least 1 h in the constant-temperature bath, fitted with a Julabo P temperature regulator, at the same temperature as the electrochemical cell. To characterize the adsorption behavior of BSA and fibrinogen by cyclic voltammetry, it was first necessary to determine the response of the Ti electrode in a protein-free solution (phosphate buffer, pH 7.4) at each temperature. A large potential region was initially examined, -1.5 to 5.0 V, to find a region where the current response of processes occurring at the electrode surface was reproducible with continuous cycling. Titanium forms a thick oxide layer, and its formation is evident in the anodic region. A gradual decrease in the charge was recorded in each subsequent cycle, due to the passivation of the electrode surface as a result of the development and thickening of the oxide layer. However, a stable and reproducible surface
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Figure 1. Cyclic voltammograms of a (cp)Ti electrode recorded in phosphate buffer pH 7.4 (-) without and (- - -) with addition of 0.42 g L-1 of fibrinogen at 310 K. Scan rate: v ) 500 mV s-1. Inset: time dependence of the open-circuit potential. was found in the potential region from -1.3 to -0.5 V, even after prolonged cyclization of the electrode. Before each particular protein addition, 200 cycles with the scan rate of 500 mV s-1 were made. Cyclic voltammograms for the stepwise additions of BSA and fibrinogen were then obtained. Electrochemical impedance spectroscopy data were recorded immediately following a cyclic voltammetry measurement. To ensure complete characterization of the solution/surface interface and surface processes, EIS measurements were made over seven frequency decades, from 50 kHz to 30 mHz. Before each measurement, the electrode was stabilized at -0.94 mV for 10 min. Following each EIS measurement, before the sequential aliquot of protein was added, a cyclic voltammogram was recorded and compared with that taken before the EIS measurement to ensure the stability of the electrode surface during the EIS measurements. This procedure ensured high reproducibility of the surface investigated.
Results and Discussion Cyclic Voltammetry. Figure 1 shows a cyclic voltammogram (solid line) of a Ti electrode recorded in a potential region between the hydrogen evolution region (-1.3 V) and an oxide region (-0.5 V), i.e., around a measured open-circuit-potential (EOCP ) -0.7 V, inset in Figure 1). Our experimental value of the OCP agreed well with the value of -0.65 V obtained in a deaerated phosphate-buffered saline solution.36 This region was found to be highly reproducible following the pretreatment described in the Experimental Section of the paper, even after prolonged cyclization of the electrode. It is wellknown that Ti forms passive oxide films on its surface, even with a brief contact with air, which cannot be completely removed with the mechanical polishing of the surface37 used in this paper. However, avoiding the polarization of the electrode to higher potentials (above -0.5 V), a Ti electrode covered with the thin oxide film formed in air and then stabilized during continuous cycling between -1.3 and -0.5 V could be regarded as an “inert and electron-transparent” electrode surface, and the potential drop across the oxide film can be neglected. The cyclic voltammogram in Figure 1 (solid line) shows a very broad current peak in the anodic scan, with a maximum around -0.95 V. This peak is attributed to the oxidation (desorption) of adsorbed hydrogen:37,38
H+ + e- a HADS
(1)
(37) Bonilla, S. H.; Zinola, C. F. Electrochim. Acta 1998, 43, 423. (38) Clark, T.; Johnson, D. C. Electroanalysis 1997, 9, 273.
Figure 2. Surface charge density of (4) fibrinogen and (O) BSA in phosphate buffer pH 7.4 at 310 K on (cp)Ti. Inset: semilogarithmic plot showing the dependence of the surface concentration on the protein concentration in the bulk solution.
In the returning cathodic scan, no current peak was observed. Instead, the cathodic current increases, and a small shoulder, which could be attributed to the adsorption of hydrogen, is apparent below -1.0 V. It was noticed that when the electrode was polarized to more negative potentials (below -1.3 V), this shoulder extended further, and finally, a sharp increase in the cathodic current, due to hydrogen evolution, was recorded. The addition of fibrinogen to the phosphate buffer solution resulted in a change in a voltammetric response (Figure 1, dashed line). However, although the shape (profile) of the cyclic voltammogram remained the same, the resulting current, i.e. charge, increased considerably. This is very strong evidence of the interaction of the protein molecules with the Ti surface. The same was observed after addition of BSA to the phosphate buffer solution. Oxidation of the proteins could be excluded as a reason for the observed increase of the charge, since the measurements were done in a low cathodic potential region. Therefore, some other electron-transfer-related process should be taken into account. As previously stated, the surface charge recorded in a protein-free solution (Figure 1, solid line) is due to the adsorption/desorption of hydrogen (eq 1). Since the shape of the voltammogram remained essentially the same after addition of the protein, the observed increase in charge could be due to “external donation” of hydrogen atoms, as a result of the protein adsorption. At pH 7.4, the amino groups in fibrinogen and BSA molecules carry a positive charge, due to the protonation with a hydrogen atom of the basic amino acids, i.e. histidine, lysine, and arginine. Therefore, we believe that the “external donation” of hydrogen, after addition of protein (Figure 1), occurs via the protonated amino groups. In addition, a Ti surface has a net negative charge in the investigated potential region, which facilitates the involvement of these positively charged amino groups of the proteins as anchoring sites in the contact region between the proteins and the Ti surface. Figure 2 shows the dependence of the charge density obtained from cyclic voltammograms, QADS, against the protein concentration in the bulk solution. Raw charge density values were corrected for the value obtained in a protein-free solution, thus giving only charge density resulting from protein adsorption. As an example, the effect of adsorption of fibrinogen (triangles) and BSA (circles) onto a Ti surface at 310 K is presented. In this figure, the solid lines do not represent any attempt to
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Table 2. Data for Fibrinogen and BSA17,46-48
protein
molar mass, g mol-1
no. of basic amino acids
diameter, nm
projected area end-on, nm2/surface monolayer concn, mg m-2
length, nm
projected area side-on, nm2/surface monolayer concn, mg m-2
fibrinogen BSA
267 706 69 000
347 100
6.5 4.0
42/11.1 17.0/6.7
47.5 11.5
(130-300)/(3.6-1.6) 46/2.5
model the data but are shown to aid visual presentation. A comparison of the fibrinogen curve presented in Figure 2 with those reported elsewhere (nonelectrochemical techniques)39-42 shows quite similar trends in behavior; with increase of the fibrinogen concentration in the bulk solution, the surface charge density rapidly increased and finally reached a plateau level at ca. 54 µC cm-2 with a bulk concentration of approximately 0.1 g L-1. This “threshold” concentration agrees well with the results reported by Cornelius et al.39 and LeDuc et al.40 Using surface charge density values, QADS, it is possible to calculate the protein surface concentration, Γ in mg m-2:21-27
Γ)
QADSMr nF
(2)
where QADS is the surface charge density in C cm-2, Mr is the molar mass of the protein in g mol-1, n is the number of electrons transferred (in this case, one electron for each protonated amino group in the protein, Table 2, eq 1), and F is the Faraday constant in C mol-1. Using data presented in Table 2, the saturated surface concentration for fibrinogen on Ti at 310 K (Figure 2) was calculated to be 4.3 ( 0.1 mg m-2. This surface concentration is slightly larger than a theoretical close-packed monolayer concentration of fibrinogen with a “side-on” orientation of adsorbed molecules to the surface (Table 2). However, our value is in accordance with values reported in the literature, obtained using nonelectrochemical techniques. Brash’s research group9,12-15,39,40,43,44 reported surface monolayer concentration of fibrinogen ranging from 2 to 8 mg m-2, depending on the surface investigated. Malmsten et al.,41,42 using in-situ ellipsometry and TIRF (total internal reflection fluorescence spectroscopy), investigated adsorption of fibrinogen at methylated silica surfaces, and they obtained maximum surface concentration around 5 mg m-2 and the layer thickness of 28 nm. They concluded that fibrinogen adsorbed in several different orientations. Liu et al.45 studied adsorption of fibrinogen onto a titanium oxide surface using radiotracing with 125I, and they reported surface concentration around 4.7 mg m-2. Contrary to the fibrinogen curve, which shows one plateau level (Figure 2), the BSA curve shows a trend of a bimodal isotherm; i.e., after an initial sharp increase, the curve levels out around 26 µC cm-2 at a concentration of approximately 0.04 g L-1 and then begins to rise again to a final plateau level of ca. 38 µC cm-2, at a concentration around 0.23 g L-1. Stepped isotherms for adsorption of albumin onto solid surfaces have been reported in the (39) Cornelius, R. M.; Wojciechowski, P. W.; Brash, J. L. J. Colloid Interface Sci. 1992, 150, 121. (40) Leduc, C.; ten Hove, P.; Park, S.; Vroman, L.; Brash, J. L.; Leonard, E. F. J. Biomater. Sci. Polym. Ed. 1995, 7, 531. (41) Malmsten, M. J. Colloid Interface Sci. 1994, 166, 333. (42) Malmsten, M.; Lassen, B. J. Colloid Interface Sci. 1994, 166, 490. (43) Santerre, J. P.; ten Hove, P.; Brash, J. L. J. Biomed. Mater. Res. 1992, 26, 1003. (44) Brash, J. L.; ten Hove, P. J. Biomater. Sci. Polym. Ed. 1993, 4, 591. (45) Liu, F.; Zhou, M.; Zhang, F. Appl. Radiat. Isot. 1998, 49, 67.
literature.49,50 The existence of the stepped BSA isotherm (Figure 2) can be explained by adapting Fair and Jamieson’s49 approach; at low concentrations, i.e. below ca. 0.12 g L-1 (Figure 2, first plateau), adsorption proceeds by a random uncorrelated mode (which likely involves conformational changes as discussed later), resulting in a completely disorganized structure containing approxi/Q2.plateau ) 0.67). mately 70% occupied area (Q1.plateau ADS ADS -1 Above 0.12 g L , the surface free energy of the protein layer favors a transition from the less dense random layer to a more dense, partly ordered structure, whose degree of order is kinetically limited by the configurational relaxation time, τs, at the surface. However, the collision frequency νc of the protein molecules with the surface is still too low to maintain growth of the more “ordered” structure. At ca. 0.23 g L-1 (Figure 2, beginning of the second plateau) the collision frequency is sufficiently large to overcome the kinetic limitation, and therefore, a transition to a cooperative adsorption mode occurs in which a close-packed more “ordered” surface phase of the protein is formed. Thus, the “critical bulk concentration” (c* ) 0.23 g L-1) may be regarded as a concentration at which the average time between consecutive collision of the protein molecules at a growth site on the surface becomes comparable to the configurational relaxation time at the surface τs. A collision frequency of a BSA molecule with the stainless steel surface was calculated using the Smoluchowski equation:49
νc ) 2πDc*dNA
(3)
where D ) 8.83 × 10-7 cm2 s-1 is the diffusion coefficient of the BSA molecule at 310 K,51 d ) 7.2 nm is the “diameter” of BSA which is equivalent to twice the Stokes radius,49 NA ) 6.023 × 1023 mol-1 is the Avogadro number, and c* ) 0.23 g L-1 (3.3 µM) is the critical bulk supersaturation concentration. For the BSA curve shown in Figure 2, a collision frequency was calculated to be 8 × 103 molecules s-1. Then, using the relation νcτs ) 1, the mean lifetime of the protein molecule at the surface before immobilization, i.e. the configurational relaxation time, was calculated to be τs ) 1.3 × 10-4 s. Using eq 2 and data from Table 2, the BSA surface concentration on Ti at 310 K was calculated to be 1.87 ( 0.04 mg m-2 at the first plateau level and 2.76 ( 0.01 mg m-2 at the second one. Although the latter value is close to a monolayer concentration of native BSA with a “sideon” orientation of adsorbed molecules to the surface (Table 2), our work suggests that denaturation occurs at the (46) Doolittle, R. F. In Haemostasis and Thrombosis; Churchill Livinstone: New York, 1987; Vol. 2, pp 192-215. (47) Doolittle, R. F. In Annual Review of Biochemistry; Richardson, C. C., Boyer, P. D., Meister, A., Eds.; Annual Reviews Inc.: Paolo Alto, CA, 1984; Vol. 53, pp 195-229. (48) Sequence Retrival System Network, online protein database, http://srs.ebi.ac.uk. (49) Fair, B. D.; Jamieson, A. M. J. Colloid Interface Sci. 1980, 77, 525. (50) Wojciechowski, P. W.; Brash, J. L. Colloids Surf. B: Biointerfaces 1993, 1, 107. (51) Gaigalas, A. K.; Hubbard, J. B.; McCurley, M.; Woo, S. J. Phys. Chem. 1992, 96, 2355.
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Table 3. Saturated Surface Concentrations, Affinity Constants, and Standard Gibbs Energies for BSA and Fibrinogen at Ti, Obtained from Cyclic Voltammetry Measurements at Various Temperatures BSA T/K
Γmax/mg m-2
BADS × 10-7/ dm3 mol-1
295 299 310 316 323 333 343
2.52 ( 0.01 2.76 ( 0.01 2.85 ( 0.01 6.50 ( 0.06 4.9 ( 0.1 10.2 ( 0.2
0.47 ( 0.02 1.08 ( 0.05 4.8 ( 0.2 4.8 ( 0.2 4.8 ( 0.2 45 ( 2
fibrinogen ∆G°ADS/ kJ mol-1 -48.2 ( 0.1 -52.1 ( 0.1 -57.0 ( 0.1 -58.3 ( 0.2 -60.1 ( 0.2 -68.3 ( 0.2
surface, and this will be discussed in more detail later. Fair et al.,49 using a solution-depletion technique, reported a value of 1.4 mg m-2 for the first plateau level and 2.6 mg m-2 for the second one on a polysterene surface at 295 K. Wojciechowski et al.50 also noted bimodal isothermic behavior of albumin on various surfaces, but they obtained lower surface concentration values, ranging from 0.24 to 1.24 mg m-2. On the other hand, Liu et al.,45 using 125I labeling, reported values between 6.4 and 7.2 mg m-2 on titanium oxide, depending on the substrate surface formation. However, Sheardown et al.52 showed that extreme caution should be used when interpreting data on the adsorption of 125I-labeled proteins to metal surfaces, since artifacts can arise due to metal-iodide ion interactions. Using a “standard” 125I-labeling procedure, they obtained a surface concentration of albumin around 4.2 mg m-2 on titanium, but with the “SDS treatment” (sodium dodecyl sulfate), the measured surface concentration was around 1.2 mg m-2. However, they suspected that the latter one was rather low, due to the incomplete elution with SDS. Enckevort et al.53 reported a BSA saturated surface concentration on stainless steel to be 2.2 mg m-2. Cyclic voltammetry was also used to determine the surface concentrations of BSA on platinum,24 and a value of 2.7 mg m-2 was reported. The inset in Figure 2 shows a semilogarithmic dependence of the surface concentration of fibrinogen and BSA on the equilibrium concentration of proteins in the bulk solution. Using this presentation, from the slope of the curves it is possible to calculate the “affinity” of the proteins toward adsorption onto a solid surface.50 (We put “affinity” between the quotation marks because the real affinity, BADS, will be calculated later in the paper; see Table 3.) Calculated values are 2.00 mg m-2 decade-1 for fibrinogen and 0.88 mg m-2 decade-1 for BSA. This shows that fibrinogen exhibits more than twice the affinity for adsorption onto a Ti surface, compared to the case of BSA. Wojciechowski et al.50 reported similar observation in their study of fibrinogen and albumin adsorption onto chemically functionalized silica substrates.50 Although we did not investigate a competitive adsorption behavior of fibrinogen and BSA from the mixture, the present results indicate that the “Vroman” effect12,16,44,50 would probably appear if fibrinogen and BSA were adsorbed from the mixture; i.e., the abundant protein, with a smaller affinity, would adsorb first (BSA) but would be later replaced by a protein with a higher affinity (fibrinogen). Lelah et al.54 showed that, in adsorption from solution, fibrinogen can compete favorably for surface sites even in the presence of a significantly higher albumin concentration. Brash et (52) Sheardown, H.; Cornelius, R. M.; Brash, J. L. Colloids Surf. B: Biointerfaces 1997, 10, 29. (53) Van Enckevort, H. J.; Dass, D. V.; G.Langdon, A. J. Colloid Interface Sci. 1984, 98, 138. (54) Lelah, M. D.; Cooper, S. L. In Polyurethanes in Medicine; CRC Press: Boca Raton, FL, 1986; p 125.
Γmax/mg m-2
BADS × 10-7/ dm3 mol-1
∆G°ADS/ kJ mol-1
1.90 ( 0.09
0.021 ( 0.001
-39.9 ( 0.1
4.2 ( 0.1 5.2 ( 0.8
2.4 ( 0.1 86 ( 4
-54.2 ( 0.1 -64.6 ( 0.2
al.8,19 also reported preferential adsorption of fibrinogen over albumin. The affinity values calculated from the inset of Figure 2 also show that, at lower surface coverage, adsorption appears to be mainly surface binding rate limited (rather than diffusion rate limited) because the ratio between the affinity values for fibrinogen and BSA on titanium is 2.27 (the same is obtained if the real affinity values from Table 3 are used), while the ratio between corresponding diffusion coefficients is 0.23 (Dfibrinogen ) 2 × 10-7 cm2 s-1).43 According to the literature,31,40,55,56 adsorption of fibrinogen and BSA has been described by the Langmuir isotherm:
Γ)
BADSΓmaxc 1 + BADSc
(4)
in which c (mol cm-3) is the equilibrium concentration of the adsorbate in the bulk solution, Γ (mol cm-2) is the amount of protein adsorbed, i.e. surface concentration, Γmax (mol cm-2) is the maximum value of Γ, and the parameter BADS (cm3 mol-1) reflects the affinity of the adsorbate molecules toward adsorption sites. Equation 4 can be rearranged to give
1 c c ) + Γ BADSΓmax Γmax
(5)
A plot of c/Γ versus concentration c should yield a straight line with parameters Γmax and BADS derived from the slope and intercept, respectively. As an example, adsorption of fibrinogen at 310 K is presented in Figure 3, and indeed, the c/Γ vs c dependence is linear, with a correlation coefficient r2 ) 0.9995. Calculated maximum surface coverage Γmax was found to be (1.66 ( 0.06) × 10-8 mol m-2, or 4.4 ( 0.2 mg m-2, which is quite close to the experimentally obtained value (4.3 ( 0.1 mg m-2). The intercept yielded BADS ) (2.4 ( 0.1) × 107 dm3 mol-1. Using these values, the isotherm for the adsorption of fibrinogen onto the Ti surface at 310 K was calculated and plotted according to eq 4 (inset in Figure 3). Such good agreement between experimental and simulated values confirmed the applicability of the Langmuir isotherm in the description of adsorption of fibrinogen onto Ti surface. The parameter BADS, which reflects the affinity of the adsorbate molecules toward adsorption sites at a constant (55) Fukuzaki, S.; Urano, H.; Nagata, K. J. Ferment. Bioeng. 1995, 80, 6. (56) Klinger, A.; Steinberg, D.; Kohavi, D.; Sela, M. N. J. Biomed. Mater. Res. 1997, 36, 387.
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Figure 3. Adsorption isotherm of fibrinogen adsorbed onto (cp)Ti in phosphate buffer pH 7.4 at 310 K. Symbols are experimental values, and solid line is the best fit. Inset: experimental data (symbols) fitted using the Langmuir isotherm equation and calculated parameters (Γmax and BADS).
temperature, could be presented as57
BADS )
1 csolvent
(
exp
)
-∆G°ADS RT
(6)
where R (J mol-1 K-1) is the gas constant, T (K) the temperature, ∆G°ADS (J mol-1) the standard Gibbs energy of adsorption, and csolvent the molar concentration of a solvent, which is for this case water (cH2O ) 55.5 mol dm-3). Using this equation, the standard Gibbs energy of adsorption of fibrinogen onto the Ti surface in phosphate buffer solution at 310 K was calculated to be -54.2 ( 0.2 kJ mol-1. Such a high value indicates strong adsorption of fibrinogen onto the Ti surface via chemisorption. This confirmed our previous assumption that the protein molecules are chemically bound to the Ti surface through “hydrogen donation” from amino groups. From the fibrinogen adsorption curves presented elsewhere (obtained using radiolabeling with 125I),40,43 we calculated the free energy of adsorption of fibrinogen on hydrophilic glass and various polyurethane surfaces using the Langmuir isotherm and obtained values -52 kJ mol-1 and -52 to -55 kJ mol-1, respectively, which agree very well with our present results. Temperature Dependence of Protein Adsorption. Figure 4 shows curves for adsorption of BSA on Ti at different temperatures. The common feature is that all the curves show a double plateau. However, a distinct difference in surface concentration can be observed in a temperature range below and above 316 K. Below this “threshold” temperature, all the curves are almost overlayed, and above 316 K, the surface concentration suddenly increases. This increase in surface concentration of BSA above 316 K could be related to the reversible partial unfolding that occurs between 315 and 323 K in the bulk solution.24,58-60 This change is believed to be similar to the transition from the N-form to F-form observed at increased pH.58 However, at temperatures above 333 K, thermal denaturation and unfolding of BSA begins,24,51,55 (57) Gomma, G. K.; Wahdan, M. H. Mater. Chem. Phys. 1994, 39, 142. (58) Carter, D. C.; Ho, J. X. In Advances in Protein Chemistry; Schumaker, V. N., Ed.; Academic Press: Toronto, 1994; Vol. 45, pp 153-203. (59) Lin, V. J. C.; Koenig, J. L. Biopolymers 1976, 15, 203. (60) Kiss, E. Colloids Surf. A: Physicochem. Eng. Aspects 1993, 76, 135.
Figure 4. Surface concentration of BSA on (cp)Ti in phosphate buffer pH 7.4 at (b) 299, (O) 310, (1) 316, (3) 323, (9) 333, and (0) 343 K.
which resulted in sharp increase of the surface concentration of BSA to 10.3 mg m-2 (Table 3). This sudden increase in surface concentration is in agreement with results reported for BSA on platinum24 and stainless steel.55 The constant BADS also increased by a factor of 10, indicating high increase in the affinity of BSA toward adsorption sites at the Ti surface. This increase in adsorption likely results from a higher affinity of the denatured structure of the BSA molecule in the bulk solution for the surface, possibly resulting in multilayer adsorption. This will be discussed in more detail later. Fukuzaki et al.55 also noticed increased adsorption of BSA above 333 K. The maximum surface concentration of fibrinogen on Ti was found to increase linearly between 295 and 316 K, with the rate of 0.16 mg m-2K-1 (r2 ) 0.9996). At the same time, the affinity constant increased 2 orders of magnitude between 295 and 310 K (Table 3) and then reached a value as high as 86 × 107 dm3 mol-1, thus indicating very high affinity and strong adsorption of fibrinogen toward the Ti surface. At 323 K a fibrinogen adsorption isotherm could not be plotted due to the huge scattering of the data. At this temperature the agglomeration of long filaments of fibrinogen molecules in the bulk solution was clearly observed. Therefore, it appears that protein-protein interactions are apparently greater at 323 K than proteinmetal surface interactions. A similar temperature effect was also observed by De Baillou et al.14 when studying the thickness of fibrinogen adsorption layers by flow rate differences through a system of capillaries constructed of synthetic polymers used for implants. Table 3 also shows the temperature dependence of the standard Gibbs energy of adsorption of BSA and fibrinogen onto the Ti surface. To calculate ∆G°ADS values for BSA, only the lower concentration region was used (first plateau). Figure 5 shows the linear dependence of ∆G°ADS versus temperature for BSA. From the relationship ∆G°ADS ) ∆H°ADS - T∆S°ADS, the slope yielded the value of standard entropy ∆S°ADS ) 425 ( 1 J mol-1 K-1, and the standard enthalpy of adsorption was calculated to be ∆H°ADS ) 79 ( 2 kJ mol-1. Such a large positive value of standard enthalpy indicates that the adsorption of BSA on (cp)Ti is an endothermic process under the present experimental conditions. The same can be concluded from the affinity constant values presented in Table 3, since these also increase with increasing temperature, indicating an endothermic process. Our results suggest that some conformational changes of BSA are occurring during the adsorption process; i.e., the overall energetics in the
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Figure 5. Dependence of the standard Gibbs energy of adsorption on the temperature for BSA adsorbed onto (cp)Ti in phosphate buffer pH 7.4.
breaking of intramolecular interactions is greater than that released in the formation of protein-metal interactions during the adsorption process. This would result if conformational changes of the protein occur on the surface relative to the protein structure in the bulk solution. In addition, from the calculated thermodynamic values it is apparent that changes in entropy govern the standard Gibbs energy values for the adsorption of BSA onto a Ti surface. The T∆S°ADS product ranges from 127 to 146 kJ mol-1, depending on the temperature, which is almost twice the standard enthalpy value. This is also in accordance with the explanation of the appearance of the first BSA adsorption plateau (Figure 2); i.e., at low concentrations BSA adsorption proceeds by a random uncorrelated mode resulting in a completely disorganized structure.49 Conformational changes of the protein would also contribute to a large positive entropy. Standard enthalpy and entropy values for fibrinogen could not be calculated due to the lack of experimental data as a result of the formation of fibrils at temperatures below 295 K and above 323 K. However, from the affinity constant values (Table 3) it appears that the adsorption of fibrinogen onto a Ti surface is an endothermic process. Electrochemical Impedance Spectroscopy (EIS). The electrochemical impedance spectroscopy (EIS) technique was also applied to investigate the electrode/ electrolyte interface and processes that occur on the surfaces in the presence of protein molecules. To ensure complete characterization of the interface and surface processes, EIS measurements were made over seven frequency decades, from 50 kHz to 30 mHz. Figure 6 shows an example of an EIS spectra recorded on a Ti electrode in a protein-free solution (circles) and after addition of fibrinogen (triangles). The spectra presented show two distinctive segments. In the high-frequency region, the absolute impedance curve is almost independent of the frequency, with the phase angle value approaching 0°. This is a typical response for a resistive behavior and corresponds to the resistance of the phosphate solution between the working and the reference electrode. In the lower medium-frequency region, a linear relationship can be observed between the absolute impedance and the frequency with a slope of -0.85 and phase angle approximately -75°. This frequency region corresponds to a purely capacitive behavior of the electrode/electrolyte interface. In the low-frequency region, the resistive behavior of the electrode is not visible; i.e., the region where the absolute impedance is independent of the frequency (dc limit) is not attained. In addition, it is also
Figure 6. Bode plot of a (cp)Ti electrode in phosphate buffer pH 7.4, recorded at -0.94 V and 310 K (O) in a protein-free solution and (4) in a fibrinogen-containing solution (0.42 g L-1). Symbols are experimentally measured values, and lines are simulated values. Inset: equivalent electrical circuit used to model the EIS data.
interesting to note that the capacitive frequency region commences at rather lower frequencies and extends deep into the low-frequency region (Figure 6). This denotes that the capacitive behavior of the electrode dominates and that the time constant for the process of interest (eq 1) is relatively high. Therefore, a nonlinear least-square-fit analysis (NLLS)61 was employed in a modeling procedure, using the electrical equivalent circuit (EEC) comprised of only one RC time constant. The fitting procedure showed that a better agreement between theoretical and experimental data was obtained if a frequency-dependent constant-phase element (CPE) was introduced instead of a pure capacitance (C). Generally, usage of a CPE is required due to a distribution of the relaxation times as a result of inhomogeneities present on the microscopic level under the oxide phase and at the oxide/electrolyte interface.62 This was also in agreement with the observed deviation in the slope of the impedance curve from the value of -1, as well as the deviation of the phase angle from the value of -90°. The impedance transfer function for the one-time constant circuit used in the fitting procedure (inset in Figure 6) could be written as
Z ) Rel +
Rp 1 + Q(jω)nRp
(7)
Electronic elements in Figure 6 and in eq 7 have the following meanings: Rel is the ohmic resistance between the working and the reference electrode, Rp is the polarization resistance, and CPE (or Q in eq 7) is the capacitance represented by the constant-phase element and its exponent n. Since its value in a protein-free solution was found to be relatively high (300 × 10-6 Ω-1sn cm-2; note when n ) 1, “Ω-1 sn” becomes “F cm-2”), it cannot be attributed either to a double-layer capacitance or to a capacitance of a surface oxide film. The potential region where the measurements were made corresponds to a (61) Boukamp, B. A. In Equivalent Circuit Users Manual, Report CT88/265/128, University of Twente, Department of Chemical Technology, The Netherlands, 1989. (62) Omanovic, S.; Metikos-Hukovic, M. Thin Solid Films 1995, 266, 31.
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Figure 7. Adsorption pseudocapacitance of (4) fibrinogen and (O) BSA at 310 K on (cp)Ti in phosphate buffer pH 7.4. Inset: dependence of the adsorption pseudocapacitance on the adsorption charge density of protein obtained from cyclic voltammetry measurements.
hydrogen adsorption/desorption region, and therefore, this pseudocapacitance can be related to the process described by eq 1. Conway et al.63 obtained hydrogen pseudocapacitance values on single-crystal Pt electrodes ranging from ca. 100 µF cm-2 to even 4.5 mF cm-2, depending on applied potential. High values have also been obtained on various h.e.r. electrocatalysts (Ni-Alx, Ni-Si2, Ni-MoCd).64 Paseka65 also recorded high hydrogen pseudocapance values on amorphous Ni-Sx electrodes, ranging from ca. 80 to 1300 µF cm-2, depending on the potential applied and the surface investigated. Using the EEC from Figure 6, a very good agreement between the experimental (symbols) and simulated (lines) values was obtained in the modeling procedure (Figure 6). In all the measurements, the fitting procedure resulted in a value of the exponent n around 0.9, which justified the use of a CPE instead of a pure capacitive element. However, due to the absence of the dc limit in the spectra at low frequencies, i.e. almost pure capacitive behavior of the electrode, the accuracy in determining the polarization resistance values was rather poor, and calculated values were not used for analysis and discussion of the results in the paper. Therefore, only capacitance values are used in further analysis. After the protein was added to the solution, the recorded EIS spectrum (triangles, Figure 6) showed features similar to those observed in a protein-free solution (circles). However, the adsorption of the protein onto the surface changed the impedance response of the electrode, which is apparent from the lower impedance values obtained (Z curve) and from the shift of the phase angle curve toward lower frequencies. The modeling procedure was performed for the spectra recorded in a wide concentration range. The calculated pseudocapacitance, CADS, resulting from the adsorption of fibrinogen and BSA onto a Ti surface, is plotted in Figure 7 against the protein concentration in the bulk solution, c. The pseudocapacitance CADS presented in Figure 7 is directly related to effects produced by protein adsorption, since it represents the difference between the capacitance measured in a protein solution and in a protein-free (phosphate buffer) solution. Again, the shape of the curves is quite similar to those obtained from the cyclic voltammetry measurements (Figure 2). (63) Conway, B. E. Electrochim. Acta 1998, 44, 1108. (64) Sˇ impraga, R. P.; Conway, B. E. Electrochim. Acta 1998, 43, 3045. (65) Paseka, I. Electrochim. Acta 1993, 16, 2449.
Jackson et al.
Figure 8. Normalized adsorption charge (filled symbols) and pseudocapacitance (hollow symbols) curves for fibrinogen and BSA in phosphate buffer pH 7.4 at 310 K on (cp)Ti.
The adsorption of fibrinogen resulted in a single saturation plateau, while the adsorption of BSA again resulted in a double plateau. However, the difference between the fibrinogen and BSA plateaus is somewhat smaller, compared to that in Figure 2. To directly compare the data obtained with the two different electrochemical techniques, the adsorption charge and the pseudocapacitance curves presented in Figures 2 and 7 were normalized to their initial values obtained in a protein-free solution according to
ηQ ) 1 -
Q0 C0 or ηC ) 1 Qi Ci
(8)
where Qi (C cm-2) and Ci (F cm-2) are the charge and pseudocapacitance at a particular protein concentration (i), respectively, and Q0 and C0 are the charge and pseudocapacitance obtained in a protein-free solution, respectively. Figure 8 shows that the difference between the normalized charge and pseudocapacitance points is much smaller with BSA than with fibrinogen. The fibrinogen normalized pseudocapacitance curve is situated below the charge curve in the whole concentration region. Further, the slopes of the lines presented in the inset of Figure 7 (CADS vs QADS) are not equal, indicating that although a linear relation is obtained, the direct relationship for the two proteins between the capacitance and the charge (∆C ) ∆Q/E)E)const cannot be established, although both sets of measurements were made at the same potential (-0.94 V) and with the same ac amplitude ((10 mV). The origin of differences observed in Figure 8 and in the inset of Figure 7 results from a number of factors. The adsorption pseudocapacitance, CADS, obtained from the EIS measurements (Figure 7) is not just dependent on the amount of adsorbed protein, i.e. “donated hydrogen”, but also is a very complex function of other parameters related to the electrode/electrolyte interface, which are, in turn, dependent on structural, physical, and chemical properties of proteins (thickness and structure of a protein layer and an electrode/protein/electrolyte interface, surface charge distribution, etc.). The rate of protein unfolding on the surface may be a large contributory factor. It is wellknown that BSA is a globular protein of a relatively small size (it is the smallest serum protein), while fibrinogen is a fibrous protein and is approximately 11 times larger
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Table 4. Adsorption Capacitance Plateau Values for BSA and Fibrinogen at Ti, Obtained from EIS Measurements at Various Temperatures CADS,max/Ω-1 sn cm-2 T/K
BSA
295 299 310 316 323 333 343
136 ( 2 135 ( 2 148 ( 1 190 ( 6 102 ( 7 108 ( 7
fibrinogen 90 ( 3 142 ( 3 157 ( 2
than BSA. This difference in size and structure may account for the large discrepancy between the charge and capacitance of the fibrinogen measurements. The plateau pseudocapacitance values obtained at various temperatures are presented in Table 4. Again, there is a sharp increase in the value above 316 K for BSA due to the reversible partial unfolding that occurs between 315 and 323 K in the bulk solution.24,58-60 At 333 K the pseudocapacitance dropped abruptly, and at 343 K it again increased, thus showing a trend similar to that observed for the surface concentration values presented in Table 3. Similarly, the fibrinogen pseudocapacitance values (Table 4) increase with increase in temperature, which is also comparable to the trend shown by the surface concentration values presented in Table 3. However, at temperatures above 316 K, fibrinogen agglomerated to form long filaments which were visible in solution, and therefore measurements were not made at these temperatures. The results obtained by cyclic voltammetry and electrochemical impedance spectroscopy have shown very good agreement with results obtained from accepted nonelectrochemical methods used for the evaluation of protein adsorption on biomaterials. This indicates that these electrochemical techniques are a valuable tool in the examination of protein adsorption onto metallic materials. Conclusions Studies of the adsorption behavior of fibrinogen and BSA at the commercially pure titanium surface were made over the temperature range 295-343 K, between the hydrogen and irreversible oxide formation potential regions (around the open circuit potential), using potentiodynamic dc (cyclic voltammetry) and potentiostatic ac (electrochemical impedance spectroscopy) electrochemical techniques. It was shown that the surface charge density, resulting from protein adsorption, is directly proportional to the
amount of adsorbed protein (surface concentration), indicating that adsorption is accompanied by the transfer of charge, i.e. chemisorption through the basic amino groups on the protein at these potentials. On the other hand, the adsorption pseudocapacitance values, obtained under the potentiostatic conditions, depend not only on the protein surface concentration, but it is also a very complex function of other parameters related to the electrode/electrolyte interface, which are, in turn, dependent on structural, physical, and chemical properties of proteins. Under the present experimental conditions, the adsorption of BSA onto a Ti surface showed a bimodal isothermic behavior at all the temperatures studied, while the adsorption of fibrinogen resulted in a single surface concentration saturation plateau. The adsorption process was described with a Langmuir adsorption isotherm. It was found that fibrinogen exhibits more than twice the affinity for adsorption onto a Ti surface compared to the case of BSA. At lower surface coverage, adsorption appears to be mainly surface binding rate limited. The calculated standard Gibbs energies of adsorption suggest a very strong adsorption of both proteins through chemisorption. The adsorption process for both proteins is endothermic, presumably resulting from the excess energetics required for the disruption of intramolecular interactions relative to those involved in the formation of protein-metal interactions. The initial adsorption of BSA onto a Ti surface was found to be an entropically governed process, also suggesting structural unfolding of the protein at the electrode surface. Therefore, the evidence from the present work suggests that denaturation occurs on the surface and that the disruption of intramolecular interactions during the adsorption process governs the energetics of the process. In the past, the examination of protein adsorption on biomaterials has relied primarily on nonelectrochemical methods. However, the present study shows that the electrochemical techniques (cyclic voltammetry and electrochemical impedance spectroscopy) can be successfully used to study the interfacial behavior of proteins on metallic biomaterials. Both techniques were shown to be very sensitive to the conformational behavior of the proteins. Acknowledgment. We gratefully acknowledge the Natural Science and Engineering Research Council of Canada for support of this research. LA991497X
