Electrochemical Quartz Crystal Microbalance and X-Ray Photoelectron Spectroscopy study of cathodic reactions in Bovine Serum Albumin containing solutions on a Physical Vapour Deposition-CoCrMo biomedical alloy

Electrochemical Quartz Crystal Microbalance and X-Ray Photoelectron Spectroscopy study of cathodic reactions in Bovine Serum Albumin containing solutions on a Physical Vapour Deposition-CoCrMo biomedical alloy

Electrochimica Acta 180 (2015) 96–103 Contents lists available at ScienceDirect Electrochimica Acta journal homepage: www.elsevier.com/locate/electa...

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Electrochimica Acta 180 (2015) 96–103

Contents lists available at ScienceDirect

Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta

Electrochemical Quartz Crystal Microbalance and X-Ray Photoelectron Spectroscopy study of cathodic reactions in Bovine Serum Albumin containing solutions on a Physical Vapour Deposition-CoCrMo biomedical alloy A. Igual Muñoza,* , S. Mischlerb a b

Departamento de Ingeniería Química y Nuclear, E.T.S.I. Industriales, Universidad Politécnica de Valencia, E-46071 Valencia, España Ecole Polytechnique Fédérale de Lausanne EPFL, Tribology and Interface Chemistry Group, Station 12, CH-1015 Lausanne, Switzerland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 June 2015 Received in revised form 4 August 2015 Accepted 4 August 2015 Available online 14 August 2015

Protein adsorption on biomedical CoCrMo alloys plays a crucial role in biocompatibility, corrosion and wear properties of implants. So far, protein adsorption was studied only on passive CoCrMo alloys above the open circuit potential. In this work the adsorption of Bovine Serum Albumin (BSA) under cathodic conditions was investigated using a combination of Electrochemical Quartz Cristal Microbalance (EQCM) and X-Ray Photoelectron Spectroscopy (XPS) surface analysis. Results show that cathodic polarization yields larger BSA adsorption than what reported at passive potentials. The involved adsorption mechanism is related to the electrochemical controlled reduction of BSA. ã 2015 Elsevier Ltd. All rights reserved.

Keywords: Electrochemical Quartz Crystal Microbalance X-Ray Photoelectron Surface Bovine Serum Albumin adsorption CoCrMo alloy passivity

1. Introduction Albumin is the most abundant protein found in the body fluids. It has a molecular mass Mr of 66300 Da and dimensions of 15  3.8  3.8 nm3. The typical concentration of Albumin is 37.6– 54.9 mg mL1 and 6–10 in the plasma (serum) and in the synovial fluid respectively [1]. Adsorption of Albumin on implant surfaces plays a key role in determining surface functionalities such as, biocompatibility, corrosion and tribology [2]. Indeed protein adsorption is considered the first stage enabling the adhesion of cells on the biomaterial surface and thus relevant clinical phenomena such as osseointegration of orthopaedic implants [3]. Thanks to their unique combination of favourable corrosion and tribological properties, CoCrMo alloys are commonly used for the fabrication of orthopaedic implants for hip or knee joint replacement. Albumin influences the corrosion process of CoCrMo by enhancing passive [4,5] and transpassive dissolution [6] as well as modifying surface chemistry [5,7]. Wear of these alloys is also highly dependent on the presence of Albumin [8] in the electrolyte, which can affect the corrosion response to friction as well as the

* Corresponding author. Tel.: +34 963879632; fax.: +34963877639. http://dx.doi.org/10.1016/j.electacta.2015.08.017 0013-4686/ ã 2015 Elsevier Ltd. All rights reserved.

mechanical mechanisms leading to wear. Proteins from synovial fluids were found to generate protective carbonaceous tribochemical films on CoCrMo hip joints in-vivo and in-vitro [9]. The material properties play an important role on the adsorption process: electrode potential, wettability, polar or ionic interaction, chemical structure and topography of the surface among others affect the nature and the amount of the adsorbed protein. Generally, hydrophobic surfaces are considered to be more protein-adsorbed than hydrophilic surfaces because of the strong hydrophobic interactions occurring at these surfaces, contrary to the repulsive solvation forces arising from strongly bound water at the hydrophilic surfaces [10,11]. The solution pH has also an important role in the protein adsorption since the overall charge of the protein changes depending on the acidity or alkalinity of the electrolyte. In acidic media the protein has positive charge as consequence protonation of the amino group (-NH2), which becomes –NH3+. In alkaline solutions the carboxyl group (-COOH) loses a proton and becomes –COO thus negatively charging the protein. The electrical charge of the adsorbing surface can also affect adsorption as it determines electrostatic forces between proteins and surface. Such forces were for example found to govern adsorption of BSA onto functionalized hydroxyapatite particles [12]. Therefore the electrochemical conditions are supposed to

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influence protein adsorption. Indeed, electrode potential dependent Albumin adsorption was reported by Ithurbide et al. on chromium [13] and Valero Vidal et al. on CoCrMo alloys [4]. The latter authors showed that at low passive potentials BSA (Bovine Serum Albumin) only adsorbs on a small fraction of the CoCrMo surface, while it forms a nearly complete monolayer at higher passive potentials. Under certain circumstances the implant material can loose its passivity and experience lower electrode potential. This is for example the case of artificial joint surface where passive film can be mechanically abraded in the rubbed zone. Thus galvanic coupling with the surrounding area can yield very low potentials close to the reversible potential of the metal [14–16]. It would be therefore interesting to assess the adsorption of BSA on CoCrMo alloys under cathodic conditions. The aim of this work is to explore the adsorption processes occurring at potentials well below the Open Circuit Potential (OCP) of CoCrMo alloys. For this potentiodynamic and potentiostatic Electrochemical Quartz Microbalance (EQCM) experiments were conducted in 0.9% NaCl with and without BSA on CoCrMo PVD alloys. Bulk alloys were used to investigate larger potential domains. Surface analysis by X-Ray Photoelectron Spectroscopy (XPS) was used to characterize surface composition after polarization. A similar protocol was previously used for investigating adsorption of protein at passive potentials [17]. 2. Experimental 2.1. Sample preparation and electrolytes Low carbon CoCrMo biomedical alloy was used as bulk material. Samples were cut by spark erosion from hip joint simulator heads made out of low carbon ASTM F 1537-00CoCrMo alloy (heads supplied by PlusOrthopedics, Aarau). A surface area of 0.28 cm2 was exposed to the test solution during experiments. Prior to test sample surfaces were polished until surface like mirror finish using 1 mm diamond paste. Electrochemical quartz crystal microbalance measurements were carried out on CoCrMo layers deposited from Stellite 21 targets on quartz crystals (AT-cut 10 MHz) by Physical Vapour Deposition (PVD). Thus, working electrodes of 23 mm2 surface area were covered by a CoCrMo coating of 1.5 mm thickness. The deposition procedure of CoCrMo layer on quartz crystal and crystal characterization has been described in detail elsewhere [17]. A double walled three-electrode cell was used for all the electrochemical measurements. An Ag/AgCl reference electrode (0.195 V versus SHE) and a gold wire counter electrode were used. More details on the experimental set-up can be found in previous work [4,17]. The experimental tests were carried out using two different electrolytes: (i) 0.14 M NaCl solution and (ii) 0.14 M NaCl solution with 30 g L1 of BSA (NaCl30BSA). Both solutions had a pH of 7.4. All tests were carried out at 37  C under stagnant conditions and in direct contact with air in order to assure oxygen presence in the electrolyte.

AgCl, a value close to the actual OCP, and moved into the cathodic direction down to 1 VAg/AgCl at a scan rate of 1 mV s1. Lower potentials could not be reached since below 1.2 VAg/AgCl the PVD coating started delaminating locally. Potentiostatic tests were carried out by applying a cathodic potential of 1 VAg/AgCl for 300 seconds and registering the response in current. In the EQCM tests the total frequency was measured simultaneously to the current and it was corrected for viscous loading to obtain the frequency variation due to the mass loading contribution only. The Sauerbrey equation (Eq. (1)) [18] was applied to convert this frequency shift Dfm into mass change on the working electrode (massEQCM). Correction constant for viscous loading (K) is 8.6 Hz V1 and Sauerbrey constant (Cf) is 0.21 Hz cm2 ng1 as calibrated using copper deposition [17].

Df m ¼ C f massEQCM

ð1Þ

In order to check wider ranges beyond the PVD coating delamination, polarization curves of the bulk CoCrMo alloy were measured with the same procedure and scan rate but reaching potentials below 1 V. 2.3. X-ray Photoelectron Spectroscopy After the electrochemical experiments the samples were removed from the cell while maintaining the applied potential. They were subsequently rinsed with water, blow-dried with argon and stored in a desiccator for at least one week before being transferred to the ultra-high vacuum (UHV) chamber of the XPS. The XPS measurements were performed using a Kratos AXIS ultrahigh resolution spectrometer, using a monochromatic Al Ka X-ray source and a take-off angle of 0 with respect to the surface normal. No sputtering was performed prior to analysis. The energy scale was calibrated by fixing the adventitious carbon peak at 285 eV. The fitting of the XPS-spectra was performed using the commercial software package CasaXPS and the peaks listed in Table I. The evaluation procedure included an iterated Shirley procedure for background subtraction. The metal peak positions were allowed to slightly float to compensate for possible charging

Table I XPS peaks considered for fitting and quantificatio n. Element

Peak (sensitivity factor)

Oxidation state

Binding Energy

Cr

2p2/3 (1.583)

Cr

573.8  0.4 +3

Co

2p2/3 (2.362)

Mo

3d5/2 (2.005)

2.2. Electrochemical measurements on CoCrMo EQCM experiments were carried out by an oscillatory circuit Maxtek PLO 10i coupled with a Fluke PM6680B frequency counter and to a HP 34401 voltmeter. The whole electrochemical system was controlled by a Schlumberger Solartron 1286 potentiostat and a National Instruments Labview interface. Two different electrochemical tests were conducted on the PVD-CoCrMo quartz crystal using the described EQCM set-up: cathodic potentiodynamic curves and potentiostatic tests. Initially, open circuit potential (OCP) was measured for 30 min. The scans were started at 0.3 VAg/

97

O

C

N

1s (0.780)

1s (0.278)

1s (0.477)

Cr oxide Cr+3 hydroxide Cr+6 Co

+2.2 +3.4 +4.9 778  0.6

Co oxidized Mo

+2.5 227.5  0.3

Mo+4 oxide Mo+4 hydroxide Mo+6 O2

+1.5 +3.4 +4.9 530.2  0.7

OH H2O C-C

+1.6 +3.0 285  0.5

C-OH C=O Organic compounds

+1.6 3.4 400  1

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due to the passive film. For quantification, relative sensitivity factors given by the XPS instrument supplier were used.

a)

3. Results 3.1. Electrochemical response of the bulk material Fig. 1 shows the potentiodynamic curves of the bulk CoCrMo alloy in NaCl and NaCl30BSA solutions measured by starting the scan from the corresponding OCP potentials first towards the cathodic direction and subsequently by reversing the scan towards the anodic direction. OCP and cathodic current values of the bulk alloy are higher in the protein free solution. In the NaCl solution, the cathodic current density linearly increases from the OCP down to 0.4 VAg/AgCl where a current plateau extending to 1 VAg/AgCl is reached. In this plateau, mass transport of the oxidant (oxygen) to the metallic surface is expected to limit the current. In the BSA containing solution, cathodic current values are lower in the potential range from 0.4 to 1 VAg/AgCl. In this case, no current plateau can be observed. Below 1 VAg/AgCl, when water reduction starts, current density in the BSA-containing solution is larger compared to the NaCl solution. In the reverse scan, from 1 VAg/AgCl towards anodic applied potentials, three different potential domains can be distinguished. Below 1 VAg/AgCl and up to the corrosion potential the cathodic domain is defined by the reduction of water and of the dissolved oxygen. The cathodic-anodic transition occurs at the corrosion potential, which is shifted towards lower values in presence of BSA. Finally the passive domain is extended from the corrosion potential to 0.5 VAg/AgCl, just before the transpassive dissolution takes place. Passive current density within the passive domain is lower in the NaCl solution.

b)

3.2. EQCM cathodic potentiodynamic polarization Fig. 2 shows the cathodic potentiodynamic curves of the PVDCoCrMo alloy carried out from 0.3 VAg/AgCl to 1 VAg/AgCl in both studied solutions. The graphs represent the current and the mass evolution with the applied potential. Mass variation measured by the EQCM (Dm) was determined following the procedure described in the experimental section applying the Sauerbrey

Fig. 1. Potentiodynamic curves (OCP ! 1 VAg/AgCl ! 0.5 VAg/AgCl) of the bulk CoCrMo alloy in NaCl (black) and NaCl30BSA (grey) solutions.

Fig. 2. Mass and current density evolution with the applied potential in a) NaCl and b) NaCl30BSA solution.. Two different tests for each condition are shown.

equation (Eq. (1)) [18]. No significance differences in viscous load were observed in all tests. In the NaCl solution, the current steadily decreases from 0.45 VAg/AgCl to 1 VAg/AgCl. Interestingly no plateau is observed here suggesting that oxygen reduction is under kinetic control in the PVD-CoCrMo while mass transport limited in the bulk alloy. This difference can be due to the presence of defects such as impurities or second phases present in the bulk alloy but not in the PVD-alloy that may catalyse oxygen reduction reaction. Another possible explanation is that the bulk sample was polished prior to the experiments, which resulted in the typical air-formed native passive film on the surface. The oxide layer formed on the PVD sample was formed under different conditions leading to different composition or thickness and a more kinetics blocking nature. The mass does not exhibit significant variations with potential until values between 0.8 and 0.9 VAg/AgCl are reached. Below 0.8 VAg/AgCl the mass starts slightly increasing gaining approximately 50 ng/cm2 at the end of the potentiodynamic test. In the NaCl30BSA electrolyte, the current remains very low for potentials comprised between 0.3 to 0.8 VAg/AgCl. At more cathodic potentials the current increases rapidly with potential.

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During the low current potential range mass oscillates around 100 ng/cm2 until the potential of around 0.9 VAg/AgCl is reached, then the mass steadily increases. The mass gain between 0.8 and 1 VAg/AgCl in the case of NaCl30BSA solution is around 150 ng/cm2. 3.3. EQCM potentiostatic tests Fig. 3 shows the current and mass evolution with time at the applied potential of 1 VAg/AgCl. In both solutions the cathodic current remains constant during all the experiments. The current is larger in NaCl solution (80 mA/cm2) than in NaCl30BSA (60 mA/ cm2). The mass shows in average a linear increase with time, which is much more marked in the BSA solution. The final mass increase is of 950 ng/cm2 and 200 ng/cm2 in the NaCl30BSA and NaCl, respectively. This difference is most likely related to protein adsorption.

a)

b)

Fig. 3. a) Current density and b) mass evolution with time at an applied potential of 1 V in NaCl and NaCl30BSA solutions. Two different tests for each condition are shown.

Fig. 4. XPS spectra and corresponding fitted peaks measured on the CoCrMo alloy after the potentiostatic test at 1 VAg/AgCl in NaCl30BSA at 37  C and pH 7.4 for (a) cobalt (b) chromium and (c) molybdenum.

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Table II Cationic fraction in the oxide film after the EQCM tests at 1 VAg/AgCl in NaCl and NaCl30BSA. Cationic fraction Solution

Co

Cr

Mo

NaCl

14.2 22.2 11.5 11.4

79.3 71.8 79.4 80.6

6.5 6.0 9.1 8.0

NaCl30BSA

Fig. 6. XPS spectra of nitrogen on the CoCrMo alloy after the potentiostatic test at 1 VAg/AgCl in NaCl30BSA at 37  C and pH 7.4.

indicates that an oxide film is present on the electrode surface at 1 VAg/AgCl. This film can be generated by passivation during OCP stabilization and/or oxidation during transfer to the XPS chamber. The cationic fraction in the oxide film was calculated from the deconvoluted metallic peaks by considering the area sensitivity factors given by the XPS instrument manufacturer (Table II). In this, results from two independent tests under the same experimental conditions are shown. The data from Table II indicate that BSA reduces the amount of Co in the oxide film while increasing the concentration of Mo. Table II exhibits higher fractions of Co and Mo compared to results obtained in similar experiments but under passive potential [4]. All samples showed adventitious carbon contamination as indicated by the C1s peak situated at 285 eV (Fig. 5(a)), which corresponds to the C-H bond. Smaller C-OH and C¼O contributions were found in all conditions although their intensity is significantly larger in presence of BSA. The nitrogen peak, Fig. 6, was only observed in the NaCl30BSA solution. Together with the C-H, C-OH and C¼0 states, nitrogen is a constitutive element of BSA. Its presence is therefore indicative of BSA adsorption. The oxygen peak shows the presence of O2, OH and H2O, Fig. 7, on the electrodes tested in both solutions. The respective intensities are affected by the presence of BSA. Table III lists the overall atomic concentrations of all detected elements. The samples tested in NaCl30BSA show larger concentration of the elements composing BSA (O, C, N) confirming protein adsorption. The C/N ratio calculated from Table III for the NaCl30BSA solution is 3.8, which corresponds well to the theoretical one (3.7). Fig. 5. XPS spectra and corresponding fitted peaks measured of carbon on the CoCrMo alloy after the potentiostatic test at 1 VAg/AgCl (a) in NaCl and (b) in NaCl30BSA at 37  C and pH 7.4.

4. Discussion 4.1. Theoretical considerations on reduction reactions

3.4. XPS Results Examples of the fitting of cobalt (Co2p), chromium (Cr2p) and molybdenum (Mo3d) peaks of the CoCrMo in the NaCl30BSA solution after the potentiostatic test at 1 VAg/AgCl are shown in Fig. 4. In NaCl these element spectra are very similar and for the shake of clarity they are not shown here. All peaks exhibit a significant contribution from an oxidized state of each metal. This

Cathodic polarization curves of the bulk CoCrMo show that BSA reduces the reduction reaction kinetics at potentials from OCP to 1 VAg/AgCl, (Fig. 1). The possible reactions that may occur at the considered potential of 1 VAg/AgCl are the oxygen, water and proton reduction as well as BSA reduction [19]. The former three reactions are relatively well understood [20]. The proton reduction can thermodynamically occur for potentials below 0.66 VAg/AgCl for the present pH [21]. However, because of

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Table III Overall mass concentration calculated by the convolution of the XPS peaks of the different elements at different applied potentials of the CoCrMo alloy in NaCl and NaCl30BSA at 37  C. Results from two independent tests at each experimental condition are shown. Overall Atomic Concentration (% at.) Solution

Co

Cr

Mo

O

C

N

NaCl

22 23.9 8.8 12.3

20.2 18.5 10.7 13.7

5.2 5.0 3.8 4.3

37.9 36.8 42.4 41.7

14.3 15.8 26.9 22.4

– – 7.3 5.5

NaCl30BSA

Fig. 8 summarizes the potential range at which those electrochemical reactions may occur. Adsorption of species such as OH, H, BSA and Cl constitute further non-electrochemical reactions that can take place on the metallic surface. 4.2. Effect of BSA on cathodic current response

Fig. 7. XPS spectra and corresponding fitted peaks measured of oxygen on the CoCrMo alloy after the potentiostatic test at 1 VAg/AgCl (a) in NaCl and (b) in NaCl30BSA at 37  C and pH 7.4.

the low concentration of protons at pH 7, its contribution to the overall current is expected to be below 0.1 mA/cm2 [20], therefore it will be neglected in the following discussion. The water reaction reduction is cathodically inhibited for potentials higher than 0.75 VAg/AgCl. Oxygen may undergo reduction at potentials below 0.6 VAg/AgCl. However its reduction is kinetically inhibited above approximately 0.3 VAg/AgCl. Note that kinetics oxygen reduction inhibition can extend to lower potentials in case of passive surfaces [20]. Below 0.6 VAg/AgCl the maximum reduction rate is limited by mass transport from the solution towards the electrode surface. BSA is a globular protein made up of 584 amino acid residues and contains 17 disulfide bonds. Since BSA is a large complex molecule the mechanism of its reduction is little understood. Previous studies by Stankovich et al. [19] have suggested that when a potential below 0.6 VAg/AgCl is applied for times shorter than 1 hour, 3 or 4 of the 17 disulfide bonds in BSA are reduced [19]. After reduction, the BSA molecules remain adsorbed on to the electrode surface. For longer polarization times, the number of reduced disulfide bonds can increase up to 8 or 9 yielding insoluble products.

In order to analyze the BSA effect on the cathodic reactions, the difference between the cathodic polarisation curve in NaCl and in NaCl30BSA (as determined by averaging the results from the two experiments in each solution for the PVD coating and the bulk alloy) was calculated and plotted in Fig. 9. This difference represents the deficit in cathodic kinetics induced by BSA. For comparison reasons, the cathodic polarization curve for oxygen reduction measured on a Pt rotating disk (1000 rpm) in 3% NaCl taken from literature [20] was added. The latter shows the oxygen limiting current plateau in the potential range 0.7 to 1 VAg/AgCl that corresponds to the maximum effect of BSA on the cathodic kinetics as observed in the present experiments. This clearly indicates that BSA significantly inhibits the oxygen reduction. Fig. 9 also shows that BSA accelerates the water reduction kinetics (as discussed later, the possible contribution of BSA reduction is negligible) as the deficit becomes negative at potentials below 1 VAg/AgCl. This behaviour is rather surprising since XPS shows BSA adsorption on the electrode surface (Table III) that is expected to limit surface reactivity. Two factors may justify the difference in electrochemical kinetics induced by BSA: the adsorbed BSA layer and the different surface composition as evidenced by XPS, Table III. The adsorbed layer could limit the accessibility of the reactants (water and oxygen) to the metallic surface. This phenomenon could explain the inhibition of the oxygen reaction but not the enhancement of water reduction. The hypothesis that BSA selectively blocks oxygen transport but not water transport seems conceptually difficult to accept since oxygen is dissolved in water. Therefore other mechanisms should be considered. Both oxygen and water reduction are in the present situation under kinetic control. Therefore their kinetics is highly dependent on surface state of the electrode that, as XPS results show, is significantly modified by the presence of BSA (Tables II and III).

Fig. 8. Range of potentials at which the different reduction reactions occur. In the BSA reduction “R” represents the rest of the molecule.

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Fig. 9. Plot of the deficit in cathodic current caused by the presence of BSA on bulk (red) and PVD (blue) CoCrMo alloys as a function of potential. For comparison the cathodic polarization curve (current density in arbitrary units) for oxygen reduction on Pt in 3% NaCl (grey) [20] is also plotted. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4.3. Mass balance

desorption of a whole monolayer of water molecules would lead to a negligible mass variation on the present electrode of 2 106 ng. In order to appraise the mechanisms responsible for mass changes, the Dm was plotted versus the consumed charge in Fig. 10. The good linear correlation between mass and charge observed in both solutions (Fig. 10) is consistent with a faradaic nature of the involved mass change mechanisms. Based on this hypothesis and neglecting adsorption of non-reactive species, analysis of Fig. 10 leads to interesting insights into prevailing mechanisms. From the slope of the lines represented in Fig. 10Mr/n values of 0.9 and 6 in NaCl and NaCl30BSA respectively were obtained. In the NaCl solution the Mr/n value is consistent with a mechanism involving water reduction and subsequent adsorption of hydrogen into the metal. The presence of the anodic peak at 0.7 VAg/AgCl in the NaCl30BSA solution, Fig. 1, could be then correlated to the oxidation of the hydrogen adsorbed during the cathodic scan, which is

The XPS results clearly show the presence of BSA on the electrode surface (Fig. 6). Assuming the formation of a uniform BSA surface layer covering the passive metal surface the thickness of that layer can be calculated from the ratio between the N1s and Cr(III)hydroxide signal intensities (3100 and 2600CPS, respectively) as previously reported [4,13]. For this, the parameters reported by Valero Vidal et al. [4] were used. The thickness of the hydroxide layer (which constitutes the outer part of the passive film [4]) was assumed to be 1 nm. This yields BSA film thickness of approximately 3 to 4 nm, corresponding to a BSA monolayer with a mass density of 200 ng/cm2. The latter value is much smaller than the total mass change (950 ng/cm2 i.e. 5 monolayers) measured in-situ by EQCM during polarization on the same electrodes (Fig. 3). This difference indicates that a significant part of the BSA adsorbed during polarization is removed by the cleaning procedure of the electrode carried out prior to XPS analysis. Several mechanisms beside BSA adsorption can in principle contribute to mass change during polarization. The imposed cathodic conditions may lead to the reduction of the passive film generated during electrode immersion. However, passive film on CoCrMo alloys is mainly composed by chromium oxide or hydroxide that can not be reduced cathodically [22]. Ions (chloride, OH-), hydrogen and water can adsorb on the electrode surface. Further, water adsorbed at OCP can desorb during cathodic polarization. These effects are thought to be marginal as the adsorption or

Fig. 10. Representation of the Dm versus the charge in order to corroborate the Faraday’s law under cathodic conditions (91 V) forPVD- CoCrMo at 37  C.

So BSA seems to provide a catalytic surface for water reduction while kinetically inhibiting oxygen reduction. According to Fig. 8, BSA undergoes reduction at cathodic potentials (below 0.6 VAg/AgCl). In order to assess the contribution of BSA reduction to the overall cathodic current the former can be calculated using the EQCM results (Fig. 3b) by assuming that adsorption of reduced BSA only contributes to mass change. In the NaCl30BSA solution at 1 VAg/AgCl the rate of mass increase in the BSA-containing solution is 3 ng/s cm2 (obtained from the slope Dm vs t in Fig. 3b). By assuming an oxidation valence, n, equal to 8 (Section 4.1) and Mr of BSA 66300 Da, this rate corresponds to a BSA reduction current of 35 nA/cm2. This value is much smaller than the measured cathodic current density (60 mA/cm2,). Thus protein reduction little contributes to the overall cathodic current.

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expected to occur at the equilibrium potential of 0.66 VAg/AgCl. In case of the BSA-free solution no peak can be observed due to the overwhelming contribution of oxygen reduction. In the NaCl30BSA solution the Mr/n value can not be explained by a direct reduction of the BSA according to the mechanism proposed by Stankovich et al. [19]. Indeed the proposed range of reduction valences for BSA (8 to 34) would yield Mr/n ratios ranging from 8287 to 1950 g/equivalent (average 5000 g/equivalent). However in our experiments water reduction dominates the cathodic current at 1 VAg/AgCl applied potential. According to the previous estimation of the partial currents for BSA and water reduction, the fraction of charge due to BSA reduction is approximately 0.1%. Therefore, the expected Mr/n ratio should correspond to the sum of the Mr/n ratios for water and BSA reduction (1 and 5000 g/equivalent, respectively) multiplied by the respective charge fraction (0.999 and 0.001, respectively). This yields to Mr/n value of approximately 6 g/equivalents, very close to the experimental one (5.2–5.7 g/equivalents, Fig. 10). These considerations are in good agreement with the reaction model proposed by Stankovich et al. [19] indicating that BSA reduction acts as precursor for protein adsorption. Note that BSA reduction under cathodic potentials in aqueous solutions could in principle involve preliminary hydrogen generation by water reduction (Reaction (1)) followed by bonding of the former to the disulfides groups of BSA (Reaction (2)) according to the following reactions: H2O + 2e ! 2HADS + OH

(1)

RSSR + 2HADS ! RSHHSRADS

(2)

This mechanism is consistent with the significant mass increase observed in Fig. 2b only when reaching the onset potential for water reduction. 5. Conclusions This study on the cathodic reactions on bulk and PVD-CoCrMo alloys in a NaCl and NaCl30BSA solutions by EQCM and XPS has lead to the following conclusions: - Under applied potential of 1 VAg/AgCl, BSA forms thick adsorption films, equivalent to five monolayers, and modifies the passive film composition of CoCrMo alloys. - BSA inhibits oxygen reduction and promotes water reduction. These effects are attributed to the observed chemical modifications of the alloy surface induced by BSA that influences the catalytic properties of the metallic surfaces. - The adsorption of BSA under cathodic conditions is consistent with an electrochemical process involving hydrogen generation by water reduction followed by the hydrogenation of disulfide bonds in BSA molecule. - In NaCl solution water reduction results in hydrogen intake into the CoCrMo alloy.

Acknowledgements Authors would like to thank you the Spanish Government “Ministry of Education, Culture and Sport” for the financial support

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