Corrosion inhibition of pre-formed mussel adhesive protein (Mefp-1) film to magnesium alloy

Journal Pre-proof Corrosion inhibition of Pre-formed Mussel Adhesive Protein (Mefp-1) film to Magnesium alloy Rui-Qing Hou, Fan Zhang, Ping-Li Jiang, Shi-Gang Dong, Jin-Shan Pan, Chang-Jian Lin

PII:

S0010-938X(19)31081-9

DOI:

https://doi.org/10.1016/j.corsci.2019.108309

Reference:

CS 108309

To appear in:

Corrosion Science

Received Date:

25 May 2019

Revised Date:

12 August 2019

Accepted Date:

27 October 2019

Please cite this article as: Hou R-Qing, Zhang F, Jiang P-Li, Dong S-Gang, Pan J-Shan, Lin C-Jian, Corrosion inhibition of Pre-formed Mussel Adhesive Protein (Mefp-1) film to Magnesium alloy, Corrosion Science (2019), doi: https://doi.org/10.1016/j.corsci.2019.108309

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Corrosion inhibition of Pre-formed Mussel Adhesive Protein (Mefp-1) film to Magnesium alloy

Rui-Qing Hou a c *, Fan Zhang b, Ping-Li Jiang a c, Shi-Gang Dong d, Jin-Shan Pan , Chang-Jian Lin a *

a

State Key Laboratory for Physical Chemistry of Solid Surfaces, and College of Chemistry

ro of

b

and Chemical Engineering, Xiamen University, Xiamen 361005, China b

Div. of Surface and Corrosion Science, Dept. of Chemistry, KTH Royal Institute of

Technology, Drottning Kristinas väg. 51, SE-100 44 Stockholm, Sweden

Institute of Material Research, Helmholtz-Zentrum Geesthacht, Max-Planck Str. 1, 21502

-p

c

College of Energy, School of Energy Research, Xiamen University, Xiamen 361005, China

lP

d

re

Geesthacht, Germany

Corresponding authors: Ruiqing Hou, E-mail: [email protected] Phone: +49

na

(0)4152 871941, Max-Planck Str. 1, 21502 Geesthacht, Germany Changjian Lin E-mail: [email protected] Phone: +86 (0592) 2189354, College of Chemistry

ur

and Chemical Engineering, Xiamen University, 361005 Xiamen, China

Jo

Highlights: 

A protective film was formed on Mg alloy surface by the self-absorption of Mefp-1.



Mefp-1 films can effectively inhibit the localized corrosion of Mg alloy.



Mefp-1 film pre-formed at low pH imparts an increasing impedance resistance to Mg alloy.

Abstract 1 / 35

A range of ex situ and in situ analytical techniques were applied to gain insights into the formation and properties of the pre-formed Mefp-1 film on magnesium-1.0 wt.% calcium (Mg-1.0Ca) alloy. The results revealed that the Mefp-1 film pre-formed at pH 4.6 shows a netlike structure, whereas it is more packed at pH 8.5. In situ scanning micro-reference electrode technique results demonstrated the Mefp-1 films formed at both pH can effectively inhibit the localised corrosion of Mg-1.0Ca alloy. Moreover, the film pre-formed at pH 4.6 provides an

ro of

increasing corrosion inhibition to Mg-1.0Ca alloy during 7 days of exposure.

Keywords: Magnesium; Mussel adhesive protein; Scanning reference electrode technique;

-p

EIS

re

1. Introduction

Magnesium (Mg) alloys as biodegradable metallic materials, such as bone screws, plates and

lP

blood vessel stents, have been widely concerned due to their excellent biodegradability and remarkable biocompatibility [1-3]. It has been revealed that Mg-Ca alloy with 1.0 wt.% Ca

na

content (Mg-1.0Ca) shows promising mechanical properties and biocompatibility in vitro and in vivo [4-6]. However, the corrosion rate of this alloy is still too high to retard local gas

ur

accumulation and to match the bone healing process. Therefore, there is an urgent need to

Jo

discover a bio-safe and feasible method to modify the corrosion behaviour of Mg-1.0Ca alloy for its practical application. The use of protective surface films or coatings is one of the most convenient methods to control the corrosion of metallic material [7], such as plasma electrolytic oxidation (PEO) coating [8-10], chemical conversion coating [11-14], electroless plating [15] and organic coating/film [16-19]. Considering the demands of practical applications for the biosafety and suitable degradation rate of Mg alloy, a pre-formed protein adhesive film on Mg alloy surface 2 / 35

would be a good choice to modify the corrosion behaviour of Mg alloy and the biological response to Mg surface, because the protein adsorption on the implant surface is the first step when a material is implanted in the human body, which further induces the cell adhesion and surface biomineralization [20]. Compared with fibrin (one of the natural adhesive protein, non-toxic but poor adhesive properties) [21], the mussel adhesive proteins (MAPs), derived from the marine mussel Mytilus edulis, have drawn much attention due to its versatile, rapid and permanent adhesion and cohesion properties [22, 23]. Moreover, there is an increasing interest in the use of MAPs as biomimetic adhesives for medical as well as dental applications

ro of

[24-26]. Until now, six adhesive proteins have been isolated and named by Mytilus edulis foot protein-1 (Mefp-1) to Mefp-6 [27]. Mefp-1 (108 kDa, pI = 10.5) is the firstly purified and best characterized one [28]. Moreover, it has been studied as a non-toxic corrosion inhibitor on

-p

stainless steel and carton steel [29, 30]. Additionally, mussel aquaculture could be used to

re

treat eutrophication water, there is a surplus of mussels except for the demand of mussels as a food source. Therefore, on the basis of the unique adhesive and corrosion properties it

lP

presented and the environmental consideration, Mefp-1 will be a promising choice for an adhesive film preparation on Mg-1.0Ca alloy.

na

Mefp-1 is primarily composed of repeating deca-peptide and hexa-peptides, and contains a high percentage of hydroxylated amino acids: DOPA (dihydroxyphenyl alanine), diHYP

ur

(dihydroxy proline) and HYP (hydroxy proline) [27]. DOPA is recognized as a crucial functional role in the adhesive and cohesive properties of Mefp-1 [31, 32]. It enables Mefp-1

Jo

to interact with various substrates through H-bonding, coordination bonds (via catechol group), or oxidative cross-linking [32, 33], which enable the mussel to adsorb on a broad variety of different substrates, like glass, plastic and metal oxides [24, 34]. The pH of the solution has a dual role in the adhesion versatility of DOPA undermined by DOPA’s notorious susceptibility to oxidation [35]. Raising pH is favourable for DOPA selfoxidation cross-linking and the aggradation degree of Mefp-1 [36], which can enhance the 3 / 35

compactness of Mefp-1 film. However, the oxidation of DOPA may reduce the adhesion of Mefp-1 and the cross-linking of Mefp-1 further leads to the attenuation in the thickness of Mefp-1 film [37, 38]. Extensive research has been devoted to investigating the adhesion mechanism and anti-corrosion property of MAPs on different substrates and the development of new mussel-inspired adhesives [22, 39-41]. However, few studies have investigated the formation of Mefp-1 film on Mg alloy substrates by self-adsorption. The aim of this study was to investigate the anti-corrosion effect of the pre-formed Mefp-1

ro of

film on Mg-1.0Ca alloy and the influence of pH. Scanning electron microscopy (SEM), ex situ atomic force microscopy (AFM) and infrared spectrometer (IR) were used to obtain

detailed morphological and compositional information on the protein films pre-formed on

-p

Mg-1.0Ca alloy. In situ scanning micro-reference electrode technique (SRET) and

electrochemical impedance spectroscopy (EIS) measurements were performed on Mg-1.0Ca

re

alloy without and with the pre-formed protein films in 0.9 wt. % NaCl solution after different

na

2. Material and methods

lP

time intervals to investigate the anti-corrosion behaviour of the films.

2.1. Material and film preparation

ur

Extruded Mg-1.0Ca alloy (1.06 wt.% Ca, provided by Engineering College of Peking

Jo

University and produced as the literature [42]) cut into  12 mm × 4 mm disks was used as the substrate. The alloy specimens were embedded in epoxy resin with only one circular face ( 12 mm) exposed as the working surface. For the electrochemical measurements, the exposed specimen surface was successively wet ground up to 1500 grit with SiC abrasive paper, and then ultrasonically cleaned with ethanol. For the spectroscopic and SRET measurements, the specimens were further successively polished using 1.0 μm and 0.3 μm Al2O3 powders, and then ultrasonically cleaned with ethanol. 4 / 35

The hydrogen is prone to release from Mg alloy during the film preparation, which can seriously affect the adsorption of Mefp-1 on Mg alloy substrate, and further decrease the corrosion inhibition of this film to corrosion of the alloy. To obtain a relatively stable surface and ensure sufficient adsorption of the protein, the Mg alloy substrates were pre-conditioned in a 20 wt.% NaOH for one day to form a surface layer, which was called the stabilisation process in the following text. The stabilisation process was followed by rinsing with water and drying with a gentle stream of nitrogen gas prior to use.

ro of

The Mefp-1 (supplied by Biopolyer Products AB, Gothenburg, Sweden) used in this experiment was delivered in 1 wt.% citric acid solution at a concentration of 25 mg/mL. It was stored in the dark at 4 ◦C. The Mefp-1 solutions were prepared at a concentration of 1

-p

mg/mL in 1 wt.% critic acid solution at pH 4.6 or 8.5 prior to use. The pH was adjusted using a NaOH solution. 0.9 wt.% NaCl solution was prepared using NaCl and distilled water, which

re

was selected as the electrolyte for corrosion tests since it is a normal saline solution for evaluating the corrosion behaviour of biocompatible materials [43]. All the unspecified

were analytical grade.

lP

measurements were carried out at room temperature (25 ± 2 °C), and all the chemicals used

na

In this study, the Mefp-1 films were pre-formed by directly immersing the pre-stabilised substrates in 1 mg/mL Mefp-1 solutions for 1 hour. After the protein self-adsorption process,

ur

the sample surface was gently rinsed in deionized water and dried by a nitrogen jet.

Jo

2.2. Surface characterization after stabilisation X-ray photoelectron spectroscopy (XPS) measurements were performed by using electron spectrometer Quantum 2000 (VG) equipped with an Al Kα (1486.6 eV) source to obtain the dominate composition of the passive film on Mg-1.0Ca alloy. The base pressure of the analysis chamber was ca. 10-8 Pa. The analysed area was around 200 μm × 200 μm. Calibration for the spectra was conducted by adjusting the C 1s signal to 284.5 eV. For the 5 / 35

deconvolution of the spectra, background subtraction (linear) was performed, then the spectra were fitted with Gaussian function to find the peak positions. The reference for elementbinding energy used is the NIST Standard Reference Database 20, Version 3.5. 2.3. Surface characterization after the protein film formation The morphologies of the passive surface layer and the pre-formed protein films were analysed by utilizing a HITACHI S-4800 scanning electron microscopy (SEM) at an acceleration voltage of 5 kV and a CSPM-5500 AFM. The AFM probe used was a silicon tip with a

ro of

nominal spring constant of 40 N/m and a resonant frequency of 300 kHz. All images were taken in tapping mode with a scanning frequency of 1 Hz (the number of scanned rows per second). The surface roughness was analysed by the CSPM Imager Analysis software. Ra is

-p

the arithmetic average of the absolute values of the surface height deviations and it is calculated as: 1

re

𝑅a = 𝑁 ∑𝑁 𝑗=1 |𝑍𝑗 |

(Eq. 1)

lP

Rq, the root mean square of the Z-values of the surface, is calculated as: ∑(𝑍𝑗 )2 𝑁

(Eq. 2)

na

𝑅q = √

ur

Where Zj is the local Z value and N is the number of points. 2.4. Characterization of the protein films

Jo

IR analysis of the protein films on Mg-1.0Ca alloy was carried out with a Nicolet-8700 FTIR apparatus, which is equipped with a liquid nitrogen-cooled MCT-B detector. The spectra were obtained with Omnic E.S.P Software at a resolution of 2 cm-1 using 1024 scans on Mg-1.0Ca alloy. The resulting spectra were recorded in absorbance format. Here, the spectrum of stabilised Mg-1.0Ca alloy surface was used as the background. 2.5. In situ scanning micro-reference electrode technique (SRET) measurements 6 / 35

In situ SRET measurements were performed by using a home-built scanning tunnelling microscope (STM) assisted SRET system (CSPM5500-XMU-BY), which has been described in detail in previous publications [44, 45]. It has been proven to be an efficient method to monitor the localised corrosion of metals in solution [46]. A Pt/Ir scanning microprobe (< 1 μm) sealed by Apiezon wax except the tip was used to determine the surface potential difference with a micro-reference electrode. The distance between the scanning probe and the surface was set to be 50 μm with the aid of the STM mode. It took about 5 min to scan an area of 4 mm × 4 mm over the sample surface with a scanning frequency of 0.4 Hz (the number of

ro of

scanned rows per second). After 6 hours of immersion, the sample surface was cleaned with distilled water and then examined by optical microscopy (OM).

-p

2.6. Electrochemical measurements

An AUTOLAB instrument (ECO Chemie B.V., The Netherlands) was used for the continuous

re

electrochemical measurements of the samples with and without the protein films. All

lP

electrochemical measurements were conducted in a standard three electrodes cell. The sample, a saturated calomel electrode (SCE) and a platinum sheet were used as the working electrode, reference electrode and counter electrode, respectively. The EIS measurements were carried

na

out after 1 h, 4 h, 1 day, 3 days and 7 days of immersion in 0.9 wt.% NaCl solution at the open circuit potential (OCP) with the disturbing voltage amplitude of 10 mV in a frequency

ur

range of 100 kHz to 0.01 Hz (36 data points). The ZSimpWin software was used to analyse

Jo

the EIS data.

After 7 days of EIS measurements, potentiodynamic polarization was performed at a scanning rate of 10 mV/min in the range of -0.15 to 0.30 V vs. OCP. The corrosion current density (jcorr) was determined by the General Purpose Electrochemical System (GPES) of the Autolab CRP software according to the following equation: 𝐽 = 𝑗𝑐𝑜𝑟𝑟 {exp(2.3𝛥𝐸 ⁄𝑏𝑐 ) − exp(2.3𝛥𝐸 ⁄𝑏𝑎 )}

(Eq. 3)

7 / 35

J is the current density, ΔE is the applied potential, jcorr indicates the corrosion current density, ba and bc refer to the anodic and cathodic Tafel slopes, respectively. All the electrochemical measurements were carried out at room temperature with three replicates.

3. Results and discussion 3.1. Microstructure of Mg-1.0Ca alloy

ro of

The microstructure of Mg-1.0Ca alloy is presented in Fig. 1. SEM image (Fig. 1a) revealed the fine recrystallised grains and the uniform distribution of the bright phase along the grain boundary. According to our previous study and the reported literature [47, 48], the bright

phase is intermetallic Mg2Ca. Besides the dispersive and fine Mg2Ca along grain boundaries,

-p

some coarse Mg2Ca particles are also visible in the magnified image, as shown in Fig. 1b,

re

which results in the coexistence of -Mg + Mg2Ca region.

lP

3.2. Compositional characterization of the substrate

To characterize the compositional changes of Mg-1.0Ca surface after the stabilisation process,

na

XPS analysis was conducted for the samples. The deconvoluted spectra of Mg 2p and O 1s core-level for the samples are presented in Fig. 2. The Mg 2p spectrum (Fig. 2a) for the fresh

ur

sample can be resolved into two spectra with peaks at 51.84 and 50.93 eV, which are assigned to MgCO3 and MgO, respectively [49]. The presence of carbonates may be ascribed to the

Jo

exposure of the sample to the air. Whereas, the Mg 2p spectrum of the passivated sample (Fig. 2c) can be resolved into three peaks which are coordinated with MgCO3 (51.8 eV), MgO (50.9 eV) and Mg(OH)2 (49.92 eV) [49, 50]. Moreover, the O 1s binding energy spectrum of the fresh sample (Fig. 2b) also consist of two peaks at 531.66 eV and 533.07 eV, which are related to the lattice oxygen and carbonate oxygen, respectively [51]. The additional peak at 530.97 eV for the stabilised sample (Fig. 2d) is likely associated with the surface hydroxyl [52]. Based on the peak areas, the ratio of surface Mg was calculated to be 2747 (MgO) and 8 / 35

891 (MgCO3) for the fresh sample, and 283 (MgO), 151 (MgCO3) and 3350 (Mg(OH)2) for the stabilised sample. The results indicate that MgO is the major compound on the fresh Mg1.0Ca surface. Whereas, Mg ions dissolved from the matrix can react with hydroxyl in the electrolyte (NaOH solution), and form the Mg(OH)2 on the sample surface to inhibit the further dissolution of the Mg alloy to some extent. Another possibility is the transformation of MgO to Mg(OH)2 on the surface during the stabilisation. Therefore, on the stabilised alloy surface, Mg(OH)2 is the dominant component of the surface layer.

ro of

3.3. Morphology of the pre-formed Mefp-1 films The morphologies of pre-formed Mefp-1 films were investigated with SEM, as shown in Fig. 3. Before the Mefp-1 adsorption, scratches resulting from sample preparation process are still

-p

visible on the stabilised substrate surface (Fig. 3a), and the surface layer consist of densely packed particles. Whereas, after Mefp-1 films were pre-formed on the Mg alloy in the protein

re

solution at pH 4.6 (Figs. 3b and 3e) and pH 8.5 (Figs. 3c and 3f), no polishing scratches could

lP

be observed, indicating a high coverage of Mefp-1 in both low pH and high pH conditions. However, some cracks are visible on the protein films (Figs. 3b and 3c), which may be due to the dehydration of the protein films during the drying process. Some holes exist in the Mefp-1

na

film formed at low pH, which are the voids formed between Mefp-1 aggregates (Fig. 3b) due to the porous net structure of the protein film as revealed in Fig. 3e. In contrast, at high pH,

ur

the pre-formed Mefp-1 film consist of densely packed Mefp-1 aggregates as displayed in Fig.

Jo

3f, and the film structure is more uniform than that formed at low pH (Fig. 3b). The results demonstrate that Mefp-1 forms a more uniform and compact film on the Mg alloy substrate at high pH, which is in agreement with previous studies on the carbon steel [53]. In addition, some large aggregates also can be observed in both protein films (denoted by yellow arrows in Figs. 3b and 3c), which is most likely due to the high concentration of Mefp-1 in the film preparation solution and the increased pH near the alloy surface as a result of the dissolution of Mg alloy during the film preparation. 9 / 35

Fig. 4a shows the AFM topographic image of stabilised Mg-1.0Ca substrate, demonstrating that the substrate is covered by densely packed particles in an average size of 150 nm (according to the AFM sectional profiles in Fig. 4), which is in agreement with the result of SEM (Fig. 3d). Fig. 4b and 4c display the AFM images of the Mefp-1 films adsorbed on Mg1.0Ca alloy at pH 4.6 and 8.5, respectively. At pH 4.6 (Fig. 4b), the Mefp-1 film consist of particles in size of ca. 350 nm as shown in the sectional analysis. It seems to be inconsistent with the SEM result. The possible explanation is that the porous outmost layer of the protein film is too soft to be mapped by AFM due to the deformation effect of the AFM tip, as the

ro of

topographic image appears as a blur in Fig. 4b. In contrast, at pH 8.5 (Fig. 4c), the protein film appears to be closely packed with small Mefp-1 aggregates in size of ca. 150 nm, which is similar to the SEM result. The different morphologies of Mefp-1 films formed at different

-p

pH may result from the different pH near the alloy surface and the different dissolution

re

situations of the alloy in film preparation solutions [54, 55].

The statistic evaluation of the surface roughness of the images in Fig. 4 is provided in Table 1.

lP

The data revealed that the film preparation process increased the surface roughness, and the surface film prepared at low pH was rougher than that formed at high pH. The increase of the

na

surface roughness could be due to both the adsorption of the Mefp-1 and the corrosion of the substrate during the film preparation. At high pH, the formed protein film is more

ur

homogeneous than that formed at low pH [53, 56], and the corrosion of the substrate is more severe in lower pH electrolyte. Thus, it is reasonable that the roughness of the protein film

Jo

pre-formed at pH 8.5 is lower than that pre-formed at pH 4.6. However, although the surface topography has changed obviously after the protein adsorption, the direct evidence of the existence of the Mefp-1 on the substrate is still inadequate, which motivates the below studies. 3.3. IR analysis of the protein films on Mg-1.0Ca alloy Fig. 5 presents the IR spectra of the Mefp-1 films pre-formed at pH 4.6 and 8.5 on Mg alloy in the wavenumber range from 2000 cm-1 to 1000 cm-1. A comparison sample was prepared by 10 / 35

dripping 1 mg/mL Mefp-1 solution on the alloy surface and dried with nitrogen blowing within 30 seconds, which was marked as Mefp-1_Control in Fig. 5. The spectra in Fig. 5 exhibit several obvious Mefp-1 vibration bands: 1582 cm-1, 1399 cm-1, 1261 cm-1, 1153 cm-1 and 1083 cm-1, which can be assigned to vibrations of the protein backbone and of the DOPA side chain. The spectrum of the pre-formed Mefp-1 film at pH 4.6 was similar to that of the film pre-formed at pH 8.5. Based on the earlier IR measurements of Mefp-1 and Mefp-1-based films in the literature [38, 53, 57, 58], the strong and broad band at around 1582 cm-1 is associated with peptide bond vibrating of the backbone. The band at around 1399 cm-1 is

ro of

assigned to the aryl vibrating, while the band at 1261 cm-1 is attributed to C-O stretching of hydroxyl groups of DOPA. The band at around 1153 cm-1 is caused by the CH2 shaking. The IR peak at 1083 cm-1 is assigned to the combination of the C-O stretching of backbone and

-p

the vibration of the carbon backbone.

re

It should be noted that there is a strong band at around 1630 cm-1 on the control samples prepared both at pH 4.6 and 8.5. It has been confirmed that the amide bands and carbonyl

lP

bands appear in the 1532-1747 cm-1 region. Moreover, the shift of the peak is closely related to the secondary structure of the protein which is primarily caused by the H-bonding [57].

na

Therefore, the prominent bands at around 1630 cm-1 can be assigned to the β sheets of protein or the adsorbed water on the surface [38, 59], which also exists as a shoulder in the spectrum

ur

of Mefp-1 film pre-formed at pH 8.5 with another shoulder of the band at 1484 cm-1 (indicated by the arrow in Fig. 5b). The shoulder at 1484 cm-1 arises from the symmetrical coordination

Jo

of catechol of the DOPA resides to Mg hydroxide or oxides [30]. Whereas, these two shoulders were unrecognizable in the spectrum of Mefp-1 film prepared at pH 4.6. This may be due to the overlap of adjacent peak and the low adsorbed content of Mefp-1 on Mg alloy at this pH [58, 60]. 3.4. The localised corrosion measurements

11 / 35

To investigate the inhibition effect of the Mefp-1 films on the localised corrosion of Mg alloy, the potential distributions above the surface were followed by SRET measurements for the Mg alloy surface. In these 3D images presented in Figs. 6, 7 and 8, the Z scale was reverted and the Z-range was set to accommodate the potential difference in order to highlight the localised corrosion. Thus, since the detected potential is from the potential distribution in testing solution, the potential peak/valley (negative/positive potential) were associated with the activity of local cathode/anode on the sample surface, which is galvanically coupled with anodic/cathodic activity over the Mg alloy surface [47, 61, 62]. Therefore, the higher the

ro of

potential peak is, the stronger the intensity of localised corrosion would be.

Fig. 6 presents the surface potential difference of Mg-1.0Ca alloy without the protein film as a

-p

comparison. These negative potential peaks were indications of the local cathodic H2

evolution and the galvanic-coupled local anodic dissolution on Mg-1.0Ca alloy (as shown in

re

the inserted image in Fig. 9), which were continuously presented during the testing time and their intensities were enhanced as a function of exposure time. It is in agreement with our

studied in detail [47].

lP

previous report, in which the development of the localised corrosion for Mg-1.0Ca alloy was

na

In the case of the sample with the pre-formed Mefp-1 film at pH 4.6, the SRET potential images during 6 h of exposure are depicted in Fig. 7. During 10 min of initial immersion,

ur

many active corrosion spots with strong potential intensity were observed on the surface as

Jo

shown in Fig. 7a. The intensity of these spots gradually reduced within 4 h of exposure, indicating that the activity of localised corrosion of the alloy was attenuated with the exposure time. Moreover, the further significant decrease of the potential intensity was observed after 6 h of exposure, suggesting that the protein film pre-formed at pH 4.6 provides a pretty good resistance for the localised corrosion of Mg-1.0Ca alloy in 0.9 wt.% NaCl solution. The reasons for this may be related to the self-healing properties of Mefp-1 film [63] and/or the cooperative effect of Mefp-1 and corrosion products. 12 / 35

Fig. 8 displays the 3D potential images of the Mg alloy with a pre-formed protein film at pH 8.5 during 6 h of exposure. During the initial immersion time (Fig. 8a), many potential peaks distribute over the whole scanning area with high activity, indicating a strong intensity of localised corrosion. After exposure for half an hour (Fig. 8b), most of the negative potential peaks disappeared but several peaks still remained active on the surface. It indicates the effective inhibition of the Mefp-1 film to the localised corrosion of Mg-1.0Ca alloy, which gives a similar result to the pre-formed film at pH 4.6. Subsequent measurements showed a consistently declined intensity of active spots within 6 h of exposure. Therefore, the pre-

ro of

formed Mefp-1 film at pH 8.5 was also proved to afford effective protection for the localised corrosion of Mg-1.0Ca alloy.

-p

Furthermore, the sample surfaces before and after the immersion were determined by optical microscopy, as shown in Fig. 9. Before the immersion, a darkish phase could be visible in the

re

alloy, which has been proven to be the Mg2Ca phase [47]. After the adsorption of Mefp-1 on the alloy at different pH, a film could be observed on the surface despite some defects as

lP

indicated by the yellow arrows in Fig. 9c and 9d. The surface morphologies after the immersion exhibited a large difference between the sample with and without the Mefp-1 film.

na

The significant localised corrosion was examined for the control at the position indicated by the active corrosion spots in Fig. 9b, the centre of the active corrosion spots could be

ur

identified as corrosion pits (shown in the inserted SEM image), which is caused by the dissolution of Mg2Ca phase [47]. It agrees with the dissolution of Mg2Ca as a local anode in

Jo

the reported literature [48]. However, only the local cathodic potential signals were detected from SRET, which is caused by the resolution limits of SRET, the small size of Mg2Ca and the ratio of the large cathode-small anode during immersion, which has been discussed in our previous study [47]. In the cases of samples with the Mefp-1 film, although the localised corrosion still could be detected, the number of active spots decreased and the surface seemed

13 / 35

to be more homogenous, especially for the sample with Mefp-1 film pre-formed at pH 4.6. This is in accordance with the above SRET results. 3.5. Electrochemical tests The OCP curves of Mg-1.0Ca alloy with and without the protein films in 0.9 wt.% NaCl were recorded for 1800 s to disclose the variation of electrode potential with the immersion time, as shown in Fig. 10. The OCP of Mg-1.0Ca alloy without the protein film dramatically increased to around -1.74 mV within the initial 300 s of exposure, then the increasing speed slowed

ro of

down during the subsequent exposure, which is attributed to the formation of corrosion products on the surface during immersion. In contrast, the OCP of the alloy with the preformed Mefp-1 film decreased at the initial immersion, which is probably caused by the

-p

activation of the film surface and the dissolution of the substrate material [64]. It is in

agreement with the detected higher activity of localised corrosion during the initial immersion

re

time for samples with pre-formed protein films (Figs. 7 and 8). Subsequently, the OCP

lP

rapidly increased within 600 s of exposure for the sample with protein film pre-formed at pH 4.6, indicating the formation of corrosion products and their combination with proteins [65]. After 800 s of exposure, it reached a plateau at approximate -1.67 mV, suggesting the

na

establishment of a dynamic equilibrium between the substrate dissolution and the formation of products on the surface. A similar trend could be observed for the OCP curve of the alloy

ur

with the protein film pre-prepared at pH 8.5 during the initial immersion (about 500 s), but it

Jo

dropped to a same level as that of control sample in the following exposure. This decline at around 500 s of exposure most likely results from the partial breakdown or peeling off of the Mefp-1 film on the sample surface [66, 67]. Fig. 11 shows the consecutive EIS spectra of Mg-1.0Ca alloy with and without the Mefp-1 film after different immersion time. The EIS spectra mainly showed two semicircles in Nyquist plots and two time constants feature in Bode plots: a capacitive loop at the highfrequency region and another one at the middle-frequency area. For some spectra, a weak 14 / 35

inductive characteristic was visible, especially for the alloy without protein film, which may be related to the intermediate active species and/or the state of the surface film [68]. In general, the diameter of these two capacitive loops increased and the inductive characteristics disappeared for all the samples as the immersion time prolonged. This tendency was more significant for the alloy with Mefp-1 film pre-formed at pH 4.6, suggesting the stable evolution of surface film during the immersion. An equivalent circuit in Fig. 12 was used for the spectra fitting, which has been successfully

ro of

used for Mg alloy [69, 70]. The weak inductive component in some spectra was not taken into consideration to simplify the fitting process. Herein, Rs is the solution resistance between the reference electrode and working electrode, Rf and CPEf correspond to the resistance and the

-p

capacitance components of the surface layer, Rct and CPEdl represent the charge transfer

resistance and the capacitance components of the electrical double layer [71], respectively.

re

The surface layer mainly refers to the Mg oxide and hydroxide film for the control sample, and the protein/corrosion product film for the pre-filmed sample. The CPE, defined by Y0 and

lP

n, is normally used to replace the ideal capacitance due to the dispersion effects caused by the roughness and heterogeneity of the sample surface. Y0 is proportional to the capacitance of

na

pure capacitive electrodes, and n is the exponential factor, indicating the deviation degree of the capacitance from the ideal condition [72].

ur

The quantitative results of the EIS fitting are provided in Table. 2. The data presented the

Jo

mean values from three parallel measurements with the standard deviation, suggesting the reproducibility of the measurements. For the control samples, both Rf and Rct slightly increased with the immersion time. This increase of resistances is mainly ascribed to the formation and growth of the corrosion products on sample surface [73]. Whereas, in the case of the sample with the Mefp-1 film formed at pH 4.6, the resistance significantly increased over the immersion time, especially after 1 day of immersion, revealing the formation of a more resistant layer on the sample surface to prevent the Mg alloy dissolution. It demonstrates 15 / 35

that the Mefp-1 film pre-formed at pH 4.6 can enhance the resistance of surface layers to Mg dissolution during the exposure. However, samples with the Mefp-1 film pre-formed at pH 8.5 showed comparable resistance values to the control sample, which is likely ascribed to the state of the Mefp-1 film, such as, compactness, thickness, integrity of the film, adhesion strength of the protein layer, etc.. The EIS results obviously demonstrate that the Mefp-1 film pre-formed at pH 4.6 provides a better corrosion inhibition than that formed pH 8.5 for Mg1.0Ca alloy in the NaCl solution for a relatively long-term exposure, which is in good

ro of

consistence with the OCP results. The Mefp-1 film pre-formed at pH 4.6 showed relatively low inhibition for the corrosion of Mg alloy during the initial immersion. However, this inhibition effect significantly enhanced

-p

after 1 day of exposure. Moreover, at the initial stage of the exposure, samples with the Mefp1 film pre-formed show a higher Ydl value than the bare samples, and it decreases steadily

re

with the exposure time. According to the reported literature [72, 74], a higher Ydl value of sample indicates a more porous surface film and serious pitting corrosion of the alloy. Thus, it

lP

suggests the adsorbed porous protein film during the initial period of exposure. Nevertheless, this protein film becomes more and more compact with the immersion time, accompanied by

na

the formation of corrosion products on the surface, which could be one of the possible reason for the inhibited localised corrosion of the Mg alloy observed by SRET. Since the Mefp-1 film

ur

was prepared before exposure, this long-term enhancement of corrosion protection indicates the synergistic effect between the protein film and the corrosion products. Further studies

Jo

should be conducted to illustrate the details about this synergistic effect for Mg degradation. Fig. 13 presents polarization curves for the samples in 0.9 wt.% NaCl solution, obtained after 7 days of exposure in the absence and presence of the Mefp-1 film. Comparing with the control, the Mefp-1 film led to a slight shift of the corrosion potential of Mg-1.0Ca to the positive direction, and the slightly lower anodic polarization curves could be observed for Mg-1.0Ca with the Mefp-1 film, indicating the slower anodic dissolution of Mg caused by the 16 / 35

protein film. However, the insignificant difference for the film breakdown points was determined for the alloy with and without the Mefp-1 film, indicating the weak passivation of the Mefp-1 film for the alloy’s dissolution. It is determined by the nature of protein films, which is still permeable for ions and not enough robust. The fitted results (Table 3) showed a lower corrosion current density (jcorr) for the alloy with the Mefp-1 film pre-formed at pH 4.6, but a comparable jcorr for that with the film pre-formed at pH 8.5 to the control. This is consistent with the EIS results obtained after 7 days of exposure. Moreover, the corrosion

ro of

potentials of the sample with Mefp-1 film were more positive than that of the control sample. 3.6. The formation of Mefp-1 film and its corrosion inhibition

The formation of the Mefp-1 film on the alloy surface is mainly attributed to the strong

-p

adhesive ability of Mefp-1 which contains a high percentage of DOPA (dihydroxyphenyl

alanine) residue. DOPA plays a crucial functional role in the adhesive and cohesive properties

re

of Mefp-1 [32, 75-77]. Mefp-1 can adsorb on the metallic surface via the metal-catechol

lP

coordination bonds between DOPA and metal ion or oxides [33] and the hydrogen bonds between the catechol of DOPA and the surface [77]. Additionally, oxidation of DOPA leads to the formation of DOPA-quinone (benzoquinone). The high reactivity of DOPA-quinone

na

provides the opportunity for the cross-linking reactions of Mefp-1. On one hand, DOPAquinone can link with other chemical groups via the reaction of the aromatic ring with

ur

nucleophiles [78]. On the other hand, the auto-oxidation of DOPA-quinone with adjacent

Jo

DOPA can yield the free-radical-containing species, two of them can cross-link through the coupling of the aromatic rings. Thus, this can result in the formation of Mefp-1 film (polymerization of the proteins) on the surface [32, 79]. The thickness of the protein films was roughly evaluated < 500 nm (data are not shown), which is accordant with our previous reports on steel surface [80] and pure Mg surface [41]. It is clear that the adhesion and cohesion properties of the Mefp-1 film are largely determined by the oxidation rate and the auto-oxidation rate (the states of DOPA). Too fast oxidation rate 17 / 35

leads to inefficient cross-linking and interfacial adhesion failure due to the quick exhaustion of the DOPA [75]. Whereas, fast auto-oxidation rate can induce good molecule cross-linking and increase the cohesion property of the Mefp-1 film, but at the expense of adhesion property. Herein, pH is used to optimize the performance of Mefp-1 film, since pH has a large influence on the aggregation degree of Mefp-1 via DOPA-DOPA cross-linking [36]. As pH increases, the aggregation rate of Mefp-1 increases, which determines the adsorption amount of Mefp-1 on the surface. It agrees with the SEM observation that a porous net-like Mefp-1 film was formed at pH 4.6, while at pH 8.5 Mefp-1 film was composed of densely packed aggregates

ro of

(Fig. 3). However, at high pH, the affinity between the adsorption layer (aggregates-

aggregates) appears to be much smaller than the affinity of the first layer for the surface

(protein-surface) [36], combined with the different thickness of protein layer at different pH

-p

[53], which could be the reasons for the weak resistance of Mefp-1 film formed at pH 8.5 after

re

long-term immersion.

The prepared Mefp-1 films, irrespective of pH used, exhibit good inhibition to the localised

lP

corrosion of Mg-1.0Ca alloy (Figs. 6-8). The localised corrosion of Mg-1.0Ca alloy is different from other Mg alloys [69, 81] due to the more negative Volta potential of Mg2Ca

na

phase than the Mg matrix, indicating the dissolution of Mg2Ca as a local anode in the galvanic corrosion between Mg2Ca and Mg matrix. The pitting at the position of Mg2Ca after the

ur

immersion could be observed from the SEM image (inserted image in Fig. 9), which is in agreement with the reported results about the corrosion of Mg-Ca alloy [43, 82, 83].

Jo

Moreover, as we previously studied [47], the pitting expends inwards the bulk substrate as the immersion time prolonged. It should be noted that only negative local potentials were detected from the SRET measurement, indicating the local cathodic hydrogen evolution at these positions. This is mainly caused by the limitation of SRET resolution, the small Mg2Ca particle and the ratio of the large cathode-small anode, which has been discussed in [47]. Furthermore, the dissolution of Mg2Ca releases Mg2+ and Ca2+, which further affect the film 18 / 35

performance on Mg alloy [82]. These released ions can promote the complexation between Mefp-1 and ions/products, which is believed to enhance the anti-corrosion properties of the film during the immersion [30], thereby leading to the decreased localised corrosion of the alloy (Figs. 7 and 8) and the increased resistance to the corrosion of the sample (Fig. 11). A schematic illustration for the film formation and its corrosion inhibition mechanism is depicted in Fig. 14. It needs to be mentioned that, compared with the effect of proteins or organic molecules on Mg corrosion in testing solution [84, 85], the pre-formed Mefp-1 film in this study provides a higher corrosion inhibition to the corrosion of Mg alloy, and also gives

ro of

an opportunity to the further development of multifunctional Mg alloy surface, for example

-p

antibacterial surface [86].

re

4. Conclusions

In this study, the formation and corrosion protection of Mefp-1 films on Mg-1.0Ca alloy

lP

surface at pH 4.6 and 8.5 were investigated by SEM, AFM, IR, SRET and electrochemical measurements. The following conclusions can be drawn: Mefp-1 can adsorb and form a high

na

coverage film on the Mg alloy surface. The formed Mefp-1 film at higher pH (8.5) is composed of compact packed Mefp-1 aggregates, but a net-like protein structure is formed at

ur

a lower pH (4.6). This film can effectively inhibit the localised corrosion of Mg-1.0Ca alloy in the NaCl solution irrespective of solution pH used during film preparation. Furthermore,

Jo

the film formed at pH 4.6 provides increased corrosion resistance for Mg-1.0Ca alloy with the exposure time due to a synergistic effect between proteins and corrosion products in 0.9 % NaCl solution. Whereas, almost no enhancement of the corrosion resistance is determined for the film formed at pH 8.5 on Mg-1.0Ca alloy, possibly related to the state of this layer (integrity, thickness etc.) during immersion.

19 / 35

Acknowledgement: The authors are grateful for the financial supports from National Natural Science Foundation of China (51571169), State Key Project of Research and Development (2016YFC1100300) and the International Science & Technology Cooperation Program of China (2014DFG52350). The authors of KTH greatly acknowledge the financial supports from the Vinnova project of Sweden (No. 20132102073) and the Swedish Foundation for International Cooperation in Research and Higher Education (STINIT project for SwedishChina collaboration, CH2017-7255).

ro of

Funding: This work was supported by the National Natural Science Foundation of China [grant number: 51571169], State Key Project of Research and Development [grant number: 2016YFC1100300], the International Science & Technology Cooperation Program of China

-p

[grant number: 2014DFG52350], the Vinnova project of Sweden [grant number: No.

20132102073] and the Swedish Foundation for International Cooperation in Research and

re

Higher Education [grant number: CH2017-7255]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

lP

Authors’ contribution: Conceived and designed the study: RQH and CJL. Performed the experiments: RQH, and PLJ. Contributed reagents/materials/analysis tools: CJL and JSP.

na

Analysed and discussed the data: RQH, FZ, PLJ, JSP and CJL. Wrote the paper: RQH. Reviewed and edited the manuscript: FZ, PLJ, JSP and CJL. All authors read and approved

ur

the manuscript.

Jo

Declarations of interest: none. Data availability: The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. Reference: [1] D. Zhao, F. Witte, F. Lu, J. Wang, J. Li, L. Qin, Current status on clinical applications of magnesium-based orthopaedic implants: A review from clinical translational perspective, Biomaterials., 112 (2017) 287-302. https://doi.org/10.1016/j.biomaterials.2016.10.017. 20 / 35

Jo

ur

na

lP

re

-p

ro of

[2] R.B. Naqvi, Y.F. Joya, M.R.A. Karim, Next-Generation Biomaterials for Bone-Tissue Regeneration: Mg-Alloys on the Move, Key Eng. Mater., 778 (2018) 306-315. https://doi.org/10.4028/www.scientific.net/KEM.778.306. [3] Y. Liu, Y. Zheng, X.-H. Chen, J.-A. Yang, H. Pan, D. Chen, L. Wang, J. Zhang, D. Zhu, S. Wu, K.W.K. Yeung, R.-C. Zeng, Y. Han, S. Guan, Fundamental Theory of Biodegradable Metals— Definition, Criteria, and Design, Adv. Funct. Mater., 29 (2019) 1805402. https://doi.org/10.1002/adfm.201805402. [4] Z. Li, X. Gu, S. Lou, Y. Zheng, The development of binary Mg–Ca alloys for use as biodegradable materials within bone, Biomaterials., 29 (2008) 1329-1344. https://doi.org/10.1016/j.biomaterials.2007.12.021. [5] Y. Jeong, W. Kim, Enhancement of mechanical properties and corrosion resistance of Mg–Ca alloys through microstructural refinement by indirect extrusion, Corros. Sci., 82 (2014) 392-403. https://doi.org/10.1016/j.corsci.2014.01.041. [6] H. Bakhsheshi-Rad, E. Hamzah, M. Daroonparvar, R. Ebrahimi-Kahrizsangi, M. Medraj, In-vitro corrosion inhibition mechanism of fluorine-doped hydroxyapatite and brushite coated Mg-Ca alloys for biomedical applications, Ceram. Int., 40 (2014) 7971-7982. https://doi.org/10.1016/j.ceramint.2013.12.147. [7] M.-S. Song, R.-C. Zeng, Y.-F. Ding, R.W. Li, M. Easton, I. Cole, N. Birbilis, X.-B. Chen, Recent advances in biodegradation controls over Mg alloys for bone fracture management: A review, J. Mater. Sci. Technol., 35 (2018) 535-544. https://doi.org/10.1016/j.jmst.2018.10.008. [8] X. Lu, C. Blawert, D. Tolnai, T. Subroto, K.U. Kainer, T. Zhang, F. Wang, M.L. Zheludkevich, 3D reconstruction of plasma electrolytic oxidation coatings on Mg alloy via synchrotron radiation tomography, Corros. Sci., 139 (2018) 395-402. https://doi.org/10.1016/j.corsci.2018.05.019. [9] K. Dong, Y. Song, D. Shan, E.-H. Han, Corrosion behavior of a self-sealing pore micro-arc oxidation film on AM60 magnesium alloy, Corros. Sci., 100 (2015) 275-283. https://doi.org/10.1016/j.corsci.2015.08.004. [10] L.-Y. Cui, R.-C. Zeng, S.-K. Guan, W.-C. Qi, F. Zhang, S.-Q. Li, E.-H. Han, Degradation mechanism of micro-arc oxidation coatings on biodegradable Mg-Ca alloys: The influence of porosity, J. Alloy. Compd., 695 (2017) 2464-2476. https://doi.org/10.1016/j.jallcom.2016.11.146. [11] Z. Chunyan, L. Shangju, Y. Baoxing, L. Xiaopeng, C. Xiao-Bo, Z. Tao, W. Fuhui, Ratio of total acidity to pH value of coating bath: A new strategy towards phosphate conversion coatings with optimized corrosion resistance for magnesium alloys, Corros. Sci., 150 (2019) 279-295. https://doi.org/10.1016/j.corsci.2019.01.046. [12] G. Duan, L. Yang, S. Liao, C. Zhang, X. Lu, Y. Yang, B. Zhang, Y. Wei, T. Zhang, B. Yu, Designing for the chemical conversion coating with high corrosion resistance and low electrical contact resistance on AZ91D magnesium alloy, Corros. Sci., 135 (2018) 197-206. https://doi.org/10.1016/j.corsci.2018.02.051. [13] H.H. Elsentriecy, H. Luo, H.M. Meyer, L.L. Grado, J. Qu, Effects of pretreatment and process temperature of a conversion coating produced by an aprotic ammonium-phosphate ionic liquid on magnesium corrosion protection, Electrochim. Acta, 123 (2014) 58-65. https://doi.org/10.1016/j.electacta.2013.12.167. [14] R.-c. Zeng, H. Yan, F. Zhang, Y.-d. Huang, Z.-l. Wang, S.-q. Li, E.-h. Han, Corrosion resistance of cerium-doped zinc calcium phosphate chemical conversion coatings on AZ31 magnesium alloy, T. Nonferr. Metal Soc., 26 (2016) 472-483. https://doi.org/10.1016/S1003-6326(16)64102-X. [15] P. Zhou, W. Cai, Y. Yang, X. Li, T. Zhang, F. Wang, Effect of ultrasonic agitation during the activation process on the microstructure and corrosion resistance of electroless Ni-WP coatings on AZ91D magnesium alloy, Surf. Coat. Tech., 374 (2019) 103-115. https://doi.org/10.1016/j.surfcoat.2019.05.080. [16] A. Abdal-hay, N.A.M. Barakat, J.K. Lim, Hydroxyapatite-doped poly(lactic acid) porous film coating for enhanced bioactivity and corrosion behavior of AZ31 Mg alloy for orthopedic applications, Ceram. Int., 39 (2013) 183-195. https://doi.org/10.1016/j.ceramint.2012.06.008. [17] F. Singer, M. Schlesak, C. Mebert, S. Höhn, S. Virtanen, Corrosion properties of polydopamine coatings formed in one-step immersion process on magnesium, ACS Appl. Mater. Inter., 7 (2015) 26758-26766. https://doi.org/10.1021/acsami.5b08760. [18] L.C. Córdoba, C. Hélary, F. Montemor, T. Coradin, Bi-layered silane-TiO2/collagen coating to control biodegradation and biointegration of Mg alloys, Mat. Sci. Eng. C-Mater. Biol. Appl., 94 (2019) 126-138. https://doi.org/10.1016/j.msec.2018.09.032. 21 / 35

Jo

ur

na

lP

re

-p

ro of

[19] L.-Y. Li, L.-Y. Cui, R.-C. Zeng, S.-Q. Li, X.-B. Chen, Y. Zheng, M.B. Kannan, Advances in functionalized polymer coatings on biodegradable magnesium alloys-A review, Acta Biomater., (2018). https://doi.org/10.1016/j.actbio.2018.08.030. [20] K. Anselme, Osteoblast adhesion on biomaterials, Biomaterials., 21 (2000) 667-681. https://doi.org/10.1016/S0142-9612(99)00242-2. [21] A. Lauto, D. Mawad, L.J.R. Foster, Adhesive biomaterials for tissue reconstruction, J. Chem. Technol. Biot., 83 (2008) 464-472. https://doi.org/10.1002/jctb.1771. [22] P. Kord Forooshani, B.P. Lee, Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein, J. Polym. Sci. Pol. Chem, 55 (2017) 9-33. https://doi.org/10.1002/pola.28368. [23] L. Petrone, A. Kumar, C.N. Sutanto, N.J. Patil, S. Kannan, A. Palaniappan, S. Amini, B. Zappone, C. Verma, A. Miserez, Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins, Nat. Commun., 6 (2015) 8737-8748. https://doi.org/10.1038/ncomms9737. [24] H. Zhang, L.P. Bre, T. Zhao, Y. Zheng, B. Newland, W. Wang, Mussel-inspired hyperbranched poly(amino ester) polymer as strong wet tissue adhesive, Biomaterials., 35 (2014) 711-719. https://doi.org/10.1016/j.biomaterials.2013.10.017. [25] W.B. Tsai, W.T. Chen, H.W. Chien, W.H. Kuo, M.J. Wang, Poly(dopamine) coating to biodegradable polymers for bone tissue engineering, J. Biomater. Appl., 28 (2014) 837-848. https://doi.org/10.1177/0885328213483842. [26] H. Shen, Z. Qian, N. Zhao, J. Xu, Preparation and Application of Biomimetic Materials Inspired by Mussel Adhesive Proteins, in: G. Yang, L. Xiao, L. Lamboni (Eds.) Bioinspired Materials Science and Engineering, John Wiley & Sons, Inc., USA, 2018, pp. 103-118. [27] S. Haemers, M.C. van der Leeden, G. Frens, Coil dimensions of the mussel adhesive protein Mefp-1, Biomaterials., 26 (2005) 1231-1236. https://doi.org/10.1016/j.biomaterials.2004.04.032. [28] Q. Lin, D. Gourdon, C. Sun, N. Holten-Andersen, T.H. Anderson, J.H. Waite, J.N. Israelachvili, Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3, P. Natl. Acad. Sci. Usa., 104 (2007) 3782-3786. https://doi.org/10.1073/pnas.0607852104. [29] D. Hansen, S. Dexter, J. Waite, The inhibition of corrosion of S30403 stainless steel by a naturally occurring catecholic polymer, Corros. Sci., 37 (1995) 1423-1441. https://doi.org/10.1016/0010-938X(95)00050-T. [30] F. Zhang, M. Sababi, T. Brinck, D. Persson, J. Pan, P.M. Claesson, In situ investigations of Fe3+ induced complexation of adsorbed Mefp-1 protein film on iron substrate, J. Colloid Interf. Sci., 404 (2013) 62-71. https://doi.org/10.1016/j.jcis.2013.05.016. [31] L. Ma, H. Qin, C. Cheng, Y. Xia, C. He, C. Nie, L. Wang, C. Zhao, Mussel-inspired self-coating at macro-interface with improved biocompatibility and bioactivity via dopamine grafted heparin-like polymers and heparin, J. Mater. Chem. B, 2 (2014) 363-375. https://doi.org/10.1039/c3tb21388a. [32] Y. Chan Choi, J.S. Choi, Y.J. Jung, Y.W. Cho, Human gelatin tissue-adhesive hydrogels prepared by enzyme-mediated biosynthesis of DOPA and Fe3+ ion crosslinking, J. Mater. Chem. B, 2 (2014) 201-209. https://doi.org/10.1039/c3tb20696c. [33] B.J. Kim, S. Kim, D.X. Oh, A. Masic, H.J. Cha, D.S. Hwang, Mussel-inspired adhesive proteinbased electrospun nanofibers reinforced by Fe (III)–DOPA complexation, J. Mater. Chem. B, 3 (2015) 112-118. https://doi.org/10.1039/C4TB01496K. [34] Q. Lu, E. Danner, J.H. Waite, J.N. Israelachvili, H. Zeng, D.S. Hwang, Adhesion of mussel foot proteins to different substrate surfaces, J. R. Soc. Interface, 10 (2013) 20120759. https://doi.org/10.1098/rsif.2012.0759. [35] H. Lee, N.F. Scherer, P.B. Messersmith, Single-molecule mechanics of mussel adhesion, P. Natl. Acad. Sci. Usa., 103 (2006) 12999-13003. https://doi.org/10.1073/pnas.0605552103. [36] S. Haemers, M. Van der Leeden, E. Nijman, G. Frens, The degree of aggregation in solution controls the adsorbed amount of mussel adhesive proteins on a hydrophilic surface, Colloid Surface A, 190 (2001) 193-203. https://doi.org/10.1016/S0927-7757(01)00679-3. [37] J. Yu, W. Wei, M.S. Menyo, A. Masic, J.H. Waite, J.N. Israelachvili, Adhesion of mussel foot protein-3 to TiO2 surfaces: the effect of pH, Biomacromolecules, 14 (2013) 1072-1077. https://doi.org/10.1021/bm301908y. [38] W. Wei, J. Yu, C. Broomell, J.N. Israelachvili, J.H. Waite, Hydrophobic enhancement of Dopamediated adhesion in a mussel foot protein, J. Am. Chem. Soc., 135 (2013) 377-383. https://doi.org/10.1021/ja309590f. 22 / 35

Jo

ur

na

lP

re

-p

ro of

[39] S. Kim, D.S. Kim, S.M. Kang, Reversible Layer-by-Layer Deposition on Solid Substrates Inspired by Mussel Byssus Cuticle, Chem. Asian J., 9 (2014) 63-66. https://doi.org/10.1002/asia.201301291. [40] B.P. Lee, S. Konst, Novel hydrogel actuator inspired by reversible mussel adhesive protein chemistry, Adv. Mater., 26 (2014) 3415-3419. https://doi.org/10.1002/adma.201306137. [41] P.-L. Jiang, R.-Q. Hou, C.-D. Chen, L. Sun, S.-G. Dong, J.-S. Pan, C.-J. Lin, Controllable Degradation of Medical Magnesium by Electrodeposited Composite Films of Mussel Adhesive Protein (Mefp-1) and Chitosan, J. Colloid Interf. Sci., 478 (2016) 246-255. https://doi.org/10.1016/j.jcis.2016.06.001. [42] X.N. Gu, N. Li, W.R. Zhou, Y.F. Zheng, X. Zhao, Q.Z. Cai, L. Ruan, Corrosion resistance and surface biocompatibility of a microarc oxidation coating on a Mg–Ca alloy, Acta Biomater., 7 (2011) 1880-1889. https://doi.org/10.1016/j.actbio.2010.11.034. [43] J. Gonzalez, R.Q. Hou, E.P. Nidadavolu, R. Willumeit-Römer, F. Feyerabend, Magnesium degradation under physiological conditions–Best practice, Bioactive Materials, 3 (2018) 174-185. https://doi.org/10.1016/j.bioactmat.2018.01.003. [44] C.-Q. Ye, R.-G. Hu, Y. Li, C.-J. Lin, J.-S. Pan, Probing the vertical profiles of potential in a thin layer of solution closed to electrode surface during localized corrosion of stainless steel, Corros. Sci., 61 (2012) 242-245. https://doi.org/10.1016/j.corsci.2012.04.020. [45] H. Xu, Y. Liu, W. Chen, R.-G. Du, C.-J. Lin, Corrosion behavior of reinforcing steel in simulated concrete pore solutions: a scanning micro-reference electrode study, Electrochim. Acta, 54 (2009) 4067-4072. https://doi.org/10.1016/j.electacta.2009.02.046. [46] W. Wang, L. Xu, X. Li, Z. Lin, Y. Yang, E. An, Self-healing mechanisms of water triggered smart coating in seawater, J. Mater. Chem. A, 2 (2014) 1914-1921. https://doi.org/10.1039/c3ta13389c. [47] R.-Q. Hou, C.-Q. Ye, C.-D. Chen, S.-G. Dong, M.-Q. Lv, S. Zhang, J.-S. Pan, G.-L. Song, C.-J. Lin, Localized Corrosion of Binary Mg–Ca Alloy in 0.9 wt% Sodium Chloride Solution, Acta Metall. Sin.- Engl., 29 (2016) 46-57. https://doi.org/10.1007/s40195-015-0361-2. [48] R.-C. Zeng, W.-C. Qi, H.-Z. Cui, F. Zhang, S.-Q. Li, E.-H. Han, In vitro corrosion of as-extruded Mg–Ca alloys—the influence of Ca concentration, Corros. Sci., 96 (2015) 23-31. https://doi.org/10.1016/j.corsci.2015.03.018. [49] D. Aswal, K. Muthe, S. Tawde, S. Chodhury, N. Bagkar, A. Singh, S. Gupta, J. Yakhmi, XPS and AFM investigations of annealing induced surface modifications of MgO single crystals, J. Cryst. Growth, 236 (2002) 661-666. https://doi.org/10.1016/S0022-0248(02)00852-7. [50] S. Ardizzone, C. Bianchi, M. Fadoni, B. Vercelli, Magnesium salts and oxide: an XPS overview, Appl. Surf. Sci., 119 (1997) 253-259. https://doi.org/10.1016/S0169-4332(97)00180-3. [51] S.H. Tamboli, A. Jatratkar, J. Yadav, V. Puri, R. Puri, H. Cho, Ageing and vapor chopping effect on the properties of MgO thin films, J. Alloy. Compd., 588 (2014) 321-326. https://doi.org/10.1016/j.jallcom.2013.11.053. [52] M. Estrada, V.V. Costa, S. Beloshapkin, S. Fuentes, E. Stoyanov, E.V. Gusevskaya, A. Simakov, Aerobic oxidation of benzyl alcohol in methanol solutions over Au nanoparticles: Mg (OH) 2 vs MgO as the supportsupport, Appl. Catal. A-gen., 473 (2014) 96-103. https://doi.org/10.1016/j.apcata.2013.12.039. [53] F. Zhang, J. Pan, P.M. Claesson, T. Brinck, Electrochemical, atomic force microscopy and infrared reflection absorption spectroscopy studies of pre-formed mussel adhesive protein films on carbon steel for corrosion protection, Thin Solid Films, 520 (2012) 7136-7143. https://doi.org/10.1016/j.tsf.2012.07.115. [54] D.E. Fullenkamp, D.G. Barrett, D.R. Miller, J.W. Kurutz, P. Messersmith, pH-dependent crosslinking of catechols through oxidation via Fe 3+ and potential implications for mussel adhesion, RSC Adv., 4 (2014) 25127-25134. https://doi.org/10.1039/C4RA03178D. [55] M. Krogsgaard, M.A. Behrens, J.S. Pedersen, H. Birkedal, Self-healing mussel-inspired multipH-responsive hydrogels, Biomacromolecules, 14 (2013) 297-301. https://doi.org/10.1021/bm301844u. [56] F. Zhang, J. Pan, P.M. Claesson, Electrochemical and AFM studies of mussel adhesive protein (Mefp-1) as corrosion inhibitor for carbon steel, Electrochim. Acta, 56 (2011) 1636-1645. https://doi.org/10.1016/j.electacta.2010.10.033. [57] O. Zvarec, S. Purushotham, A. Masic, R.V. Ramanujan, A. Miserez, Catechol-functionalized chitosan/iron oxide nanoparticle composite inspired by mussel thread coating and squid beak interfacial chemistry, Langmuir., 29 (2013) 10899-10906. https://doi.org/10.1021/la401858s. 23 / 35

Jo

ur

na

lP

re

-p

ro of

[58] X. Cai, J. Yuan, S. Chen, P. Li, L. Li, J. Shen, Hemocompatibility improvement of poly(ethylene terephthalate) via self-polymerization of dopamine and covalent graft of zwitterions, Mat. Sci. Eng. CMater. Biol. Appl., 36 (2014) 42-48. https://doi.org/10.1016/j.msec.2013.11.038. [59] R.-Q. Hou, N. Scharnagl, F. Feyerabend, R. Willumeit-Römer, Exploring the effects of organic molecules on the degradation of magnesium under cell culture conditions, Corros. Sci., 132 (2018) 3545. https://doi.org/10.1016/j.corsci.2017.12.023. [60] J. Jiang, L. Zhu, L. Zhu, B. Zhu, Y. Xu, Surface characteristics of a self-polymerized dopamine coating deposited on hydrophobic polymer films, Langmuir., 27 (2011) 14180-14187. https://doi.org/10.1021/la202877k. [61] G. Williams, H. ap Llwyd Dafydd, R. Grace, The localised corrosion of Mg alloy AZ31 in chloride containing electrolyte studied by a scanning vibrating electrode technique, Electrochim. Acta, 109 (2013) 489-501. https://doi.org/10.1016/j.electacta.2013.07.134. [62] G. Williams, H. McMurray, R. Grace, Inhibition of magnesium localised corrosion in chloride containing electrolyte, Electrochim. Acta, 55 (2010) 7824-7833. https://doi.org/10.1016/j.electacta.2010.03.023. [63] C. Chen, F. Zhang, C. Lin, J. Pan, Corrosion protection and self-healing of a nanocomposite film of mussel adhesive protein and CeO2 nanoparticles on carbon steel, J. Electrochem. Soc., 163 (2016) C545-C552. https://doi.org/10.1149/2.0521609jes. [64] Y. Su, Y. Su, Y. Lu, J. Lian, G. Li, Composite microstructure and formation mechanism of calcium phosphate conversion coating on magnesium alloy, J. Electrochem. Soc., 163 (2016) G138G143. https://doi.org/10.1149/2.0801609jes. [65] J. Chen, Y. Song, D. Shan, E.-H. Han, In situ growth of Mg–Al hydrotalcite conversion film on AZ31 magnesium alloy, Corros. Sci., 53 (2011) 3281-3288. https://doi.org/10.1016/j.corsci.2011.06.003. [66] G. Williams, H.N. McMurray, Localized corrosion of magnesium in chloride-containing electrolyte studied by a scanning vibrating electrode technique, J. Electrochem. Soc., 155 (2008) C340-C349. https://doi.org/10.1149/1.2918900. [67] Y. Su, Y. Guo, Z. Huang, Z. Zhang, G. Li, J. Lian, L. Ren, Preparation and corrosion behaviors of calcium phosphate conversion coating on magnesium alloy, Surf. Coat. Tech., 307 (2016) 99-108. https://doi.org/10.1016/j.surfcoat.2016.08.065. [68] G.-L. Song, Z. Shi, Corrosion mechanism and evaluation of anodized magnesium alloys, Corros. Sci., 85 (2014) 126-140. https://doi.org/10.1016/j.corsci.2014.04.008. [69] P. Jiang, C. Blawert, R. Hou, N. Scharnagl, J. Bohlen, M.L. Zheludkevich, Microstructural influence on corrosion behavior of MgZnGe alloy in NaCl solution, J. Alloy. Compd., 783 (2019) 179192. https://doi.org/10.1016/j.jallcom.2018.12.296. [70] Z. Li, G.-L. Song, S. Song, Effect of bicarbonate on biodegradation behaviour of pure magnesium in a simulated body fluid, Electrochim. Acta, 115 (2014) 56-65. https://doi.org/10.1016/j.electacta.2013.10.131. [71] T.N. Vu, D. Veys-Renaux, E. Rocca, Potential bioactivity of coatings formed on AZ91D magnesium alloy by plasma electrolytic anodizing, J. Biomed. Mater. Res. B Appl. Biomater., 100 (2012) 1846-1853. https://doi.org/10.1002/jbm.b.32752. [72] C.-Q. Ye, R.-G. Hu, S.-G. Dong, X.-J. Zhang, R.-Q. Hou, R.-G. Du, C.-J. Lin, J.-S. Pan, EIS analysis on chloride-induced corrosion behavior of reinforcement steel in simulated carbonated concrete pore solutions, J. Electroanal. Chem., 688 (2013) 275-281. https://doi.org/10.1016/j.jelechem.2012.09.012. [73] R. Harrison, D. Maradze, S. Lyons, Y.F. Zheng, Y. Liu, Corrosion of magnesium and magnesium-calcium alloy in biologically-simulated environment, Prog. Nat. Sci.-Mater., 24 (2014) 539-546. https://doi.org/10.1016/j.pnsc.2014.08.010. [74] F. Zhang, T. Brinck, B.D. Brandner, P.M. Claesson, A. Dedinaite, J. Pan, In situ confocal Raman micro-spectroscopy and electrochemical studies of mussel adhesive protein and ceria composite film on carbon steel in salt solutions, Electrochim. Acta, 107 (2013) 276-291. https://doi.org/10.1016/j.electacta.2013.05.078. [75] J.H. Waite, Adhesion a la moule, Integr. Comp. Biol., 42 (2002) 1172-1180. https://doi.org/10.1093/icb/42.6.1172. [76] T.H. Anderson, J. Yu, A. Estrada, M.U. Hammer, J.H. Waite, J.N. Israelachvili, The contribution of DOPA to substrate–peptide adhesion and internal cohesion of mussel‐inspired synthetic peptide films, Adv. Funct. Mater., 20 (2010) 4196-4205. https://doi.org/10.1002/adfm.201000932. 24 / 35

Jo

ur

na

lP

re

-p

ro of

[77] W. Wei, L. Petrone, Y. Tan, H. Cai, J.N. Israelachvili, A. Miserez, J.H. Waite, An Underwater Surface‐Drying Peptide Inspired by a Mussel Adhesive Protein, Adv. Funct. Mater., 26 (2016) 34963507. https://doi.org/10.1002/adfm.201600210. [78] T.J. Deming, Mussel byssus and biomolecular materials, Curr. Opin. Chem. Biol., 3 (1999) 100105. https://doi.org/10.1016/S1367-5931(99)80018-0. [79] S. Haemers, G.J. Koper, G. Frens, Effect of oxidation rate on cross-linking of mussel adhesive proteins, Biomacromolecules, 4 (2003) 632-640. https://doi.org/10.1021/bm025707n. [80] F. Zhang, C. Chen, R. Hou, J. Li, Y. Cao, S. Dong, C. Lin, J. Pan, Investigation and application of mussel adhesive protein nanocomposite film-forming inhibitor for reinforced concrete engineering, Corros. Sci., 153 (2019) 333-340. https://doi.org/10.1016/j.corsci.2019.03.023. [81] S. Yu, R.-L. Jia, T. Zhang, F.-H. Wang, J. Hou, H.-X. Zhang, Effect of Different Scale Precipitates on Corrosion Behavior of Mg–10Gd–3Y–0.4 Zr Alloy, Acta Metall. Sin.- Engl., 32 (2019) 433-442. https://doi.org/10.1007/s40195-018-0792-7. [82] Z.-Y. Ding, L.-Y. Cui, X.-B. Chen, R.-C. Zeng, S.-K. Guan, S.-Q. Li, F. Zhang, Y.-H. Zou, Q.-Y. Liu, In vitro corrosion of micro-arc oxidation coating on Mg-1Li-1Ca alloy—The influence of intermetallic compound Mg2Ca, J. Alloy. Compd., 764 (2018) 250-260. https://doi.org/10.1016/j.jallcom.2018.06.073. [83] Z.-Y. Ding, L.-Y. Cui, R.-C. Zeng, Y.-B. Zhao, S.-K. Guan, D.-K. Xu, C.-G. Lin, Exfoliation corrosion of extruded Mg-Li-Ca alloy, J. Mater. Sci. Technol., 34 (2018) 1550-1557. https://doi.org/10.1016/j.jmst.2018.05.014. [84] Y. Wang, L.-Y. Cui, R.-C. Zeng, S.-Q. Li, Y.-H. Zou, E.-H. Han, In Vitro Degradation of Pure Magnesium―The Effects of Glucose and/or Amino Acid, Materials, 10 (2017) 725. https://doi.org/10.3390/ma10070725. [85] D. Mei, S.V. Lamaka, C. Feiler, M.L. Zheludkevich, The effect of small-molecule bio-relevant organic components at low concentration on the corrosion of commercially pure Mg and Mg-0.8 Ca alloy: An overall perspective, Corros. Sci., 153 (2019) 258-271. https://doi.org/10.1016/j.corsci.2019.03.039. [86] Y. Yang, Y. Zhang, R. Hu, Q. Huang, K. Wu, L. Zhang, P. Tang, C. Lin, Antibacterial and cytocompatible AgNPs constructed with the assistance of Mefp-1 for orthopaedic implants, RSC Adv., 7 (2017) 38434-38443. https://doi.org/10.1039/C7RA06449G.

25 / 35

ro of

Figure captions:

Jo

ur

na

lP

re

-p

Figure 1: The SEM microstructure of Mg-1.0Ca alloy (a) and the coarse Mg2Ca phase (b).

26 / 35

Figure 2: Mg 2p (a) and O 1s (b) XPS deconvoluted spectra for the Mg-1.0Ca alloy sample before

ro of

passivation, and Mg 2p (c) and O 1s (d) spectra of the sample after passivation.

-p

Figure 3: SEM images of Mg-1.0Ca surface without (a, d), and with theMefp-1 film prepared at pH 4.6

Jo

ur

na

lP

re

(b, e) and 8.5 (c, f).

Figure 4: AFM topographic images and corresponding section analysis of Mg-1.0Ca alloy substrates without Mefp-1 film (a), and with the Mefp-1 film formed at pH 4.6 (b) and pH 8.5 (c).

27 / 35

ro of -p re lP

Jo

ur

na

Figure 5: IR spectra of the Mefp-1 absorbed on Mg-1.0Ca alloy at pH 4.6 (a) and 8.5 (b).

28 / 35

Figure 6: Sequential SRET potential images (4 mm × 4 mm) on the surface of Mg-1.0Ca alloy without the Mefp-1 film but with an induced scratch in 0.9 % NaCl solution at OCP, obtained after 10 min (a), 1 h (b), 2 h (c), 3 h (d), 4 h (e) and 6 h (f) of exposure. The measurement solution was refreshed at 4 h

-p

ro of

of exposure.

re

Figure 7: Sequential SRET potential images (4 mm× 4 mm) on the surface of Mg-1.0Ca alloy with the Mefp-1 film pre-formed at pH 4.6 and an artificial scratch in 0.9% NaCl solution at OCP, obtained after 10 min (a), 1 h (b), 2 h (c), 3 h (d), 4 h (e) and 6 h (f) of exposure. The measurement solution was

Jo

ur

na

lP

replaced by a fresh 0.9 % NaCl solution at 4 h of exposure.

Figure 8: Sequential SRET potential images (4 mm× 4 mm) on the surface of Mg-1.0Ca alloy with the Mefp-1 film pre-formed at pH 8.5 and anartificial scratch in 0.9% NaCl solution at OCP, obtained 29 / 35

after 10 min (a), 1 h (b), 2 h (c), 3 h (d), 4 h (e) and 6 h (f) of exposure. The measurement solution was

Jo

ur

na

lP

re

-p

ro of

replaced by a fresh 0.9 % NaCl solution at 4 h of exposure.

Figure 9: Optical images of Mg-1.0Ca alloy with and without Mefp-1 film before and after 6 hours of immersion in 0.9% NaCl solution. The arrows indicates the active corrosion spots in the alloys as reflected by the SRET results in Figs. 6-8. (The inserted SEM image shows the morphology of a typical corrosion active spot)

30 / 35

ro of

Figure 10: OCP variations of Mg-1.0Ca alloy without (Control) and with the Mefp-1 films formed at

Jo

ur

na

lP

re

-p

pH = 4.6 or 8.5 during initial 1800 s exposure in 0.9% NaCl solution.

Figure 11: Nyquist and Bode plots of the Mg-1.0Ca alloy without the Mefp-1 film (a, b), with the Mefp-1 film formed at pH 4.6 (c, d), and with the Mefp-1 film formed at pH 8.5 (e, f), exposure in 0.9 wt. % NaCl solution up to 7 days. (The solid lines indicate the fitted curves according to the equivalent circuit in Fig. 12)

31 / 35

re

-p

ro of

Figure 12: Equivalent circuit used for EIS data fitting.

Figure 13: Tafel polarization curves of Mg-1.0Ca alloy without and with Mefp-1 film after 7 days

Jo

ur

na

lP

exposure in physiological saline solution.

Figure 14: Schematic illustration for the protein film formation and its inhibition for the corrosion of Mg-1.0Ca alloy.

32 / 35

Tables: Table 1: Surface roughness data (1024 pixels× 1024 pixels) of images in Fig. 4. Rq (nm)

Bare substrate

24.3

30.5

With pre-formed Mefp-1 film at pH 4.6

54.4

69.9

With pre-formed Mefp-1 film at pH 8.5

39.9

50.6

Jo

ur

na

lP

re

-p

ro of

Ra (nm)

33 / 35

Table 2: Data obtained from spectra fitting of the EIS results. Rs Time (Ω·cm2)

CPEf

Rf

Rct

CPEdl

(Ω·cm2)

Y(10-5 F·cm-2·sn-1)

n

(Ω·cm2)

Y(10-3 F·cm-2·sn-1)

n

Control 24 ± 3

1185 ± 178

1.88 ± 0.23

0.91

593 ± 163

1.75 ± 0.18

0.85

4h

26 ± 4

1389 ± 10

1.77 ± 0.14

0.91

676 ± 11

1.58 ± 0.02

0.83

1 day

32 ± 5

1600 ± 232

3.57 ± 0.67

0.88

669 ± 9

2.30 ± 0.35

0.90

3 day

31 ± 4

1594 ± 82

3.77 ± 1.15

0.85

981 ± 35

1.72 ± 0.01

0.82

7 day

36 ± 3

1780 ± 102

2.43 ± 0.12

0.88

0.89

ro of

1h

0.75

0.89

854 ± 159

2.55 ± 0.19

0.84

0.90

1022 ± 140

1.66 ± 0.12

0.80

0.89

1636 ± 211

0.95 ± 0.22

0.75

2.04 ± 0.16

0.86

2047 ± 116

0.55 ± 0.04

0.74

2.41 ± 0.14

0.90

480 ± 40

3.14 ± 0.21

0.94

2.67 ± 0.04

0.91

644 ± 55

3.37 ± 0.41

0.84

32 ± 9

1268 ± 259

2.48 ± 0.48

4h

34 ± 11

1716 ± 261

2.60 ± 0.36

1 day 52 ± 15

1993 ± 298

2.67 ± 0.46

3 day

29 ± 7

2568 ± 227

1.83 ± 0.41

7 day

41 ± 3

3975 ± 129

lP

re

1h

1.28 ± 0.24

657 ± 85

3.05 ± 0.60

0.81

-p

Mefp-1 film pre-formed at pH 4.6

1259 ± 6

Mefp-1 film pre-formed at pH 8.5

4h

23 ± 1

1 day

7 day

1270 ± 12

21 ± 0

1552 ± 23

5.46 ± 0.81

0.83

478 ± 75

5.48 ± 1.18

0.55

22 ± 1

1504 ± 32

2.65 ± 0.10

0.89

932 ± 10

2.07 ± 0.05

0.82

25 ± 1

1665 ± 6

2.08 ± 0.04

0.90

1088 ± 47

1.77 ± 0.11

0.84

Jo

3 day

991 ± 124

na

23 ± 1

ur

1h

34 / 35

Table 3: Data calculated from the polarization curves. Ecorr vs. SCE (V)

bc

ba

Rp (Ω cm2)

j (μA/cm2)

-1.70 -1.66 -1.65

-0.53 -0.30 -0.36

2.83 3.56 2.08

3559 6083 3298

55.49 18.64 42.48

Jo

ur

na

lP

re

-p

ro of

Control Mefp-1 pH 4.6 Mefp-1 pH 8.5

35 / 35