Tunable Adsorption and Film Formation of Mussel Adhesive Protein by

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Tunable adsorption and film formation of mussel adhesive protein by potential control Fan Zhang, Guoxin Xie, and Jinshan Pan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04125 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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Tunable adsorption and film formation of mussel adhesive protein by potential control Fan Zhanga*, Guoxin Xiea,b*, Jinshan Pana

2 3

a) Division of Surface and Corrosion Science, Department of Chemistry, School of

4

Chemical Science and Engineering, KTH Royal Institute of Technology, Drottning

5

Kristinas väg 51, SE-100 44 Sweden

6

b) State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, China

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*) Corresponding author

8

Abstract

9

Mussel adhesive proteins are of great interest in many applications due to their outstanding

10

adhesive property and film-forming ability. Understanding and controlling the film formation

11

and its performance is crucial for the effective use of such proteins. In this study, we focus on

12

the potential controlled film formation and compaction of one mussel adhesive protein, Mefp-

13

1. Adsorption and film forming behavior of Mefp-1 on platinum (Pt) substrate under applied

14

potentials were investigated by cyclic voltammetry, potential controlled electrochemical

15

impedance spectroscopy (EIS) and quartz crystal microbalance with dissipation monitoring

16

(QCM-D). Moreover, micro-friction measurements were performed to evaluate mechanical

17

properties of the Mefp-1 films formed at selected potentials. The results lead to the

18

conclusions that, Mefp-1 adsorbs on the Pt substrate through both electrostatic and non-

19

electrostatic interactions, and shows effective blocking effect for the electroactive sites on the

20

substrate. The properties of the adsorbed Mefp-1 film vary with applied potential, and the

21

compactness of the adsorbed Mefp-1 film can be reversibly tuned by applied potential.

22

Keyword: mussel adhesive protein, EIS, QCM-D, In-situ friction, potential control

1

ACS Paragon Plus Environment

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Introduction

2

Marine mussels are capable of anchoring themselves to a wide range of surfaces under

3

seawater. Adhesion of mussels is rapid, versatile and permanent, and the cohesion is able to

4

maintain the adhesive strength in various environments. Mussel adhesive proteins (MAP),

5

derived from marine mussel Mytilus edulis, have attracted considerable attention in recent

6

years for their universal adhesive, film-forming and corrosion inhibiting properties 1-9. Mefp-

7

1, the first polyphenolic protein derived and identified from the M. Edulis foot 1, 10, is a large

8

and basic hydrophilic protein, having a molecular weight of about 108 kDa and a high

9

isoelectric point about 10

5, 11

. Mefp-1 consists of a large number (75-80) of repetitive

10

decapeptide units 12, and the structure is found to be open and extended in the solution with

11

minimized secondary structure 13.

12

It

is

generally

accepted

that

a

high

level

10~15

mol%

of

as

L-3,4-

13

dihydroxyphenylalanine (DOPA) in Mefp-1 is responsible for adhesion, film forming and

14

cross-linking properties of the protein. DOPA group can form hydrogen bonds 3, 14 and strong

15

covalent bonds 15, 16 with surfaces, and complexes with metal ions 17-19 and metal oxides 20, 21.

16

DOPA also enables Mefp-1 molecules to cross-link to each other by oxidative conversion to

17

DOPA-quinone

18

which then couple and re-oxidize to form coupled diphenols

19

undergo auto-oxidation above a certain pH, forming aggregates in bulk solution. However,

20

the adhesive properties of oxidized Mefp-1 due to increasing pH of the solution cannot be

21

recovered after the pH of the solution is lowered again 25. The pre-adsorbed Mefp-1 film can

22

be chemically oxidized, leading to cross-linking of the DOPA and thus compaction of the

23

film. However, chemically induced oxidation of Mefp-1 may result in interfacial failure, and

24

chemically induced reduction of oxidized protein such as thiol-quinone is restrictive

25

Moreover, pH and chemical control of Mefp-1 redox reaction are not feasible for practical

22, 23

, and the DOPA-quinone reverse dismutation to aryloxy free radicals,

2


24

. Mefp-1 molecules can

26, 27

.

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industrial applications. Therefore, it is of great importance to develop a smart approach to

2

enhance the adherence of Mefp-1 and control the redox state of the adsorbed protein film on a

3

metallic substrate, which has great implications for anticorrosion coating engineering and

4

antifouling strategies.

5

Due to its high isoelectric point, Mefp-1 is a positively charged protein in an acidic solution.

6

It is known that electrostatic force between the positively charged Mefp-1 and negatively

7

charged surface can also contribute to the protein adsorption

8

determination of the potential of zero total charge (PZTC) of Pt electrode is complicated and

9

out of the scope of this work. However, the PZTC is a matter of charge distribution in the

10

voltammogram and can be correlated with the voltammetric shape of the electrodes, therefore,

11

the PZTC value of an electrode can be accurately estimated from the electrode having the

12

same voltammetric profile shape and a known PZTC, without having to perform the

13

experiment 28. As reported in the literature, the Pt surface exhibits a non-monotonic charging

14

behavior, i.e. negatively charged in a low potential region, positively charged in an

15

intermediate potential region, and negatively charged in a high potential region due to surface

16

oxide dipoles

17

molecules can be electrochemically oxidized/reduced, and the redox reaction is reversible 32.

18

In this study, by applying electrochemical potentials to the substrate, we explore the

19

possibility to control the adsorption amount of Mefp-1 and modify the properties of the

20

adsorbed protein film. To elucidate the role of applied potential, we utilized a platinum (Pt)

21

sheet as the substrate, which allows us to avoid any influence of anodic dissolution of metal

22

ions from more active metals. Electrochemical properties of the substrate and the adsorbed

23

Mefp-1 film were investigated by means of cyclic voltammetry (CV) and potential controlled

24

electrochemical impedance spectroscopy (EIS). Quartz crystal microbalances with dissipation

25

(QCM-D) and in-situ micro-friction analysis under selected applied potentials were employed

4

. The experimental

29-31

. Moreover, the previous study has demonstrated that the dopamine

3


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to reveal kinetics of the protein adsorption, film compaction and decompaction. The

2

reversibility of the properties of the adsorbed Mefp-1 film by the potential control was

3

investigated, showing the applicability of Mefp-1 as a smart surface film with tunable

4

properties.

5

2. Experimental

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2.1. Protein and solution

7

Mussel adhesive protein, Mefp-1, with a purity of 92 wt. % (the rest is mainly

8

aggregation/degradation products of Mefp-1) was supplied by Biopolymer Products AB

9

(Gothenburg, Sweden). The details of the extraction and purification processes of Mefp-

10

1 were described elsewhere 33. The Mefp-1 is highly positively charged in acidic solutions 34.

11

The Mefp-1 in a concentration of 25 mg/ml was delivered in 1 wt. % citric acid buffer

12

solution, and stored in the dark at +4 ˚C before use. The 1 wt. % citric acid solution was used

13

as the buffer solution, and the pH was adjusted to 4.6 by adding small amount of 5.7 M

14

sodium hydroxide (Sigma Aldrich, 98% pure). Just 3 minutes prior to measurement, Mefp-1

15

was added to the buffer solution to give a concentration of 0.1 mg/ml. Aqueous solutions

16

were made using pure water with a resistivity of 18 MΩ cm as prepared by a Milli-Q system.

17

2.2 Substrate

18

Pure Pt (99.99%) sheet samples were used for CV, EIS and friction measurements. The

19

substrate for the QCM-D measurements was a 100 nm thick Pt film coated on quartz crystals

20

(Q-Sense, Gothenburg, Sweden), having a fundamental frequency of 4.95 MHz. For the CV

21

measurements, the Pt sheet samples were cleaned in 0.2 M H2SO4 solution by 20 cycles of

22

CV in the measured range, and then rinsed with water. For other measurements, Pt samples

4


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were ultrasonically cleaned with acetone, ethanol, rinsed with water and dried with nitrogen

2

gas.

3

2.3 Electrochemical measurements

4

All the CV and EIS data were obtained by using a Multi Autolab instrument (Metrohm

5

Autolab B.V. Netherlands), and a three-electrode electrochemical cell with a saturated

6

Ag/AgCl in Luggin capillary as the reference electrode, a Pt mesh as the counter electrode,

7

and a Pt sheet sample as working electrode having an exposed area of 1 cm2. The CV was

8

performed by applying a potential sweep at a scan rate of 40 mV/s in the range of -0.4 to 0.8

9

V, and 10 scanned cycles were recorded. The EIS measurements were performed at the OCP

10

and at applied potentials in the buffer and Mefp-1 solutions. The EIS spectra were recorded

11

15 minutes after stepping potential to each applied potential (steady-state condition), with the

12

perturbation amplitude of 10 mV and in the frequency range from 104 Hz to 10-2 Hz.

13

2.4 QCM-D

14

A QCM-D instrument, highly sensitive balance on the basis of measuring the resonance

15

frequency of a quartz crystal oscillator, was used to measure the surface sensed mass and

16

evaluate visco-elastic properties of the adsorbed layer as a function of applied potential. The

17

instrument used was a Q-sense E4 microbalance (Q-sense, Gothenburg). Adsorption (or

18

desorption) of material to the Pt-coated crystal surface will give rise to a frequency change,

19

from which the sensed mass can be calculated according to the Sauerbrey equation (assuming

20

a rigid adsorbed layer) given below. The method is described in detail by Höök et al. 35

∆ =

∆ ×

5


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1

where ∆f is the measured frequency change due to adsorption (or desorption), C is the mass

2

sensitivity constant of the quartz crystal (0.177 mg·m-2·Hz-1 for a resonance frequency of 5

3

MHz), and n is the overtone number (n = 1, 3, 5…). This formula was, due to a low ratio of

4

the dissipation change to the frequency change, used for evaluating the experimental data.

5

QCM-D also gives information about shear viscoelastic properties of the film by measuring

6

the energy dissipation (D). This parameter is obtained from the rate of decay of the crystal

7

oscillation when the voltage is switched off. For a soft film, the decay time is small and the

8

dissipation value is high, whereas for a rigid film the dissipation value is relatively low.

9

2.6 In-situ friction measurements

10

In-situ friction measurements were performed on a Pt plate in the buffer and Mefp-1 solutions.

11

A micro-tribometer with a linear reciprocating ball-on-disk configuration was employed, as

12

schematically shown in Fig. 1. The tribopairs consist of a polished Pt sheet, and a ZrO2 ball

13

with a diameter of 4 mm and a surface roughness (Rq) of ca. 15 nm. During the in-situ friction

14

measurement, a Pt plate sample was the working electrode, and a Pt wire and a micro-

15

reference electrode (Ag/AgCl) were used as the counter and the reference electrode,

16

respectively. The normal load of the ZrO2 ball onto the Pt surface was 6 N, and the

17

corresponding maximum contact pressure was 1.42 GPa. The reciprocating amplitude in

18

sliding was 2 mm, and the average sliding speed was 1 mm/s. The friction measurements

19

were performed in the buffer and Mefp-1 solutions, starting at OCP and then at different

20

applied potentials.

6


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Load motor Friction force sensor RE

CE Solution WE Seal ring Sample

1 2

Tribopair

Figure 1. The schematic of the micro-tribometer under applied potential.

3

4

3. Results and discussion

5

3.1 CV curves

6

CV measurements were performed on a Pt sheet in the buffer and Mefp-1 solutions. The

7

potential sweep (40 mV/s) was started from 0 V in the cathodic direction to -0.4 V, then in

8

the anodic direction to 0.8 V, and reversed back to 0 V. It has been reported that citric acid

9

was not electrochemically active in the potential range studied 36. Fig. 2 shows the stable CV

10

curves obtained (the 4th cycle), where a negative current shift appeared especially at lower

11

and middle range of potential. It is mainly originated from the oxygen reduction reaction

12

(ORR), due to the existence of O2 in the solutions (not deaerated). In this case, the possible

13

ORR pathway is the so-called “2-electron” pathway 37, 38:

14

+ 2 + 2 ⇆

(1)

15

+ 2 + 2 ⇆

(2)

7


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1

The CV curves can be divided into three regions according to the different processes taking

2

place on the sample surface, i.e., region I: underpotential deposited hydrogen (UPD H)

3

adsorption-desorption; region II: double layer charging and discharging; and region III:

4

oxidation/reduction of Pt and possibly also the adsorbed compounds 39, 40.

5

In region I, two peaks appear in the anodic sweep (Ha1 and Ha2) of the CV for the buffer

6

solution (curve a, Fig. 2). Anodic peaks Ha1 and Ha2 are associated with hydrogen

7

desorption at sites of Pt (110) and Pt (100) crystal planes, respectively

8

previously reported for the polycrystalline Pt sample, the PZTC value should lie in the

9

potential region in which the adsorption state linked to (100) linear domains develops

41

. It has been

28

.

10

Therefore the PZTC value of our Pt sample in the studied solutions should be about -0.2 V,

11

and the negatively charged surface may promote the electrostatic adsorption of positively

12

charged Mefp-1. As shown in Fig. 2, the presence of Mefp-1 in the solution leads to a

13

decreased height of Ha1 and Ha2 peaks (curve b, Fig. 2), suggesting that Mefp-1 adsorbs on

14

the Pt surface and occupies some of the hydrogen adsorption sites. Moreover, in the Mefp-1

15

solution, cathodic peaks Hc1 and Hc2 are lower in magnitude and appear at a bit to more

16

negative potentials, also indicating that adsorbed Mefp-1 modifies the energy of the hydrogen

17

adsorption/desorption.

18

In region II, the potential is more positive than the PZTC, therefore the Pt surface is

19

positively charged. Despite the existence of an electrostatic repulsion between the sample and

20

Mefp-1 molecules, the difference of curves a and b (Fig. 2) suggests the present of the protein

21

on the Pt surface. Moreover, the width of the double-layer-charging zone is larger in the

22

Mefp-1 solution, indicating that the adsorbed protein is subjected to an electrochemical

23

oxidation/reduction in this region 42.

8


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Pt oxidation and reduction processes occur in region III. Oxidation of Pt in acidic solutions

2

involves following two steps: discharge of H2O forming chemisorbed oxygen (Eq. 3), and

3

interfacial place-exchange forming quasi-3D lattice comprising Pt2+ and O2- (Eq. 4) 43-45.

4

Pt + H2O "#############$ Pt % − O% '( + 2H +2e

5

Pt % − O% − O '( "###################$ Pt

6

It has been reported that O2 has a negligible effect on the Pt oxide formation, however, the

7

place-exchange process occurs at lower potentials in the presence of O2 46. Therefore, the DPtO

8

peak appearing upon the cathodic sweep in Fig. 2 is associated with the reduction of quasi-3D

9

lattice PtO. The sample surface is negatively charged in this potential region due to surface

10

oxide dipoles 29. The magnitude of that peak is lower in Mefp-1 solution, indicating that less

11

PtO is formed, because the adsorbed Mefp-1 partially blocks active sites of Pt oxidation.

12

Upon the anodic sweep, the presence of Mefp-1 leads to a slight increase of current density

13

before 0.56 V (curve b, Fig. 2), which is probably due to oxidation of the adsorbed DOPA

14

content as reported in literature 32, 47-49. On the other hand, at anodic potentials of 0.56 V and

15

higher, the current density is lower for the sample in Mefp-1 solution than that in the buffer

16

solution, suggesting some blocking effect of the adsorbed Mefp-1 as discussed above.

!

*+ ,- .( !

9


(3)

(4)

0.10

I

0.05

Ha2

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II

III

2

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Current density (mA/cm )

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Ha1

0.00 -0.05 -0.10

Hc1

DPtO

Hc2

a b

-0.15 -0.20

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

Potential E (V) vs. Ag/AgCl

1 2

Figure 2. CV curves of Pt in the buffer (a) and Mefp-1 (b) solutions. Scan rate: 40 mV/s, the

3

starting points are marked with arrows.

4

5

3.2 Potential controlled EIS measurements

6

EIS measurements were performed at different applied dc potentials selected from the three

7

regions in the CV profile (Fig. 2). EIS spectra were recorded at the controlled potentials and

8

after the metal/solution interface had reached a steady-state condition.

9

The spectra obtained at the applied potentials are plotted in Nyquist and Bode formats, as

10

shown in Fig. 3. All the spectra show only one time constant feature. The data can be fitted

11

satisfactorily with the simplest equivalent circuit describing the metal-electrolyte interface,

12

which consists of a charge transfer resistance (Rct) and an interfacial capacitance (C) in

13

parallel, and a solution resistance Rs connected in series. As a common practice, a constant

14

phase element (CPE) is used for spectra fitting instead of capacitance, since the capacitive 10


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50-53

1

response of the interface is not ideal

. The fitted data of Rct and CPE are plotted as a

2

function of potential in Fig. 4, where the shapes of the curves in the buffer and Mefp-1

3

solutions resemble each other, but the values are slightly different. Apparently, the EIS

4

results indicate that hydrogen adsorption/desorption as well as Pt oxidation/reduction are also

5

the dominant reactions in the Mefp-1 solution. On the other hand, the different values for the

6

two solutions imply a certain influence of the adsorbed Mefp-1 on the properties of the

7

surface/solution interface.

8

As shown in Fig. 4, Rct increases and the capacitance decreases as potential increasing from -

9

0.4 V to -0.25 V for both cases, which is due to the decreasing of the over-potential and a

10

decreased coverage of adsorbed hydrogen on the Pt surface, respectively. The measured

11

capacitance here is the sum of the double-layer capacitance and the pseudo-capacitance of

12

hydrogen adsorption (adsorption capacitance) 54. In the applied potential range from -0.1 V to

13

0.4 V, the capacitance value is minimal and nearly constant due to the presence of the double-

14

layer region 55. Furthermore, Rct and capacitance increase with potential in the range of 0.4 V

15

– 0.65 V, corresponding to the oxidation of Pt, forming a less conducting PtO film.

16

The difference between the results of the buffer and Mefp-1 solutions relates to the adsorption

17

of the protein on the Pt surface at different potentials. It has been reported that in the

18

hydrogen adsorption region, the Rct is proportional to the fraction of blocked sites by

19

adsorbed compounds

20

some of the active sites for hydrogen adsorption. Therefore, the higher Rct in the Mefp-1

21

solution suggests more blocked sites due to the adsorption of the positively charged Mefp-1

22

molecules on the negatively charged Pt surface. Moreover, Mefp-1 has a higher relative

23

permittivity ɛr than that of H2 because the non-polar character of the H2. Therefore, according

24

to the following equation:

54

. As discussed in section 3.1, the adsorbed Mefp-1 molecules block

11


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/0 /1 2

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1

=

2

the adsorption of Mefp-1 leads to a higher value of the corresponding pseudo-capacitance in

3

the potential range from -0.4 V to -0.1 V as seen in Fig. 4b. It is worth mentioning that the Rct

4

is higher in the Mefp-1 solution even in the double-layer region where the Pt is positively

5

charged. This proved that the adsorption of Mefp-1 is not electrostatically irreversible.

6

The results in the oxidation region also confirmed the existence of the Mefp-1, however, the

7

EIS technique is not sensitive to the Mefp-1 oxidation process. Other techniques should be

8

employed as discussed in following sections.

(5)

3

9

10 11

Figure 3. EIS spectra of Pt in (a) buffer solution and (b) Mefp-1 solution at different applied

12

potentials.

13

12


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2 3

Figure 4. Fitted values of Rct (a) and magnitude of CPE (b) from the EIS data obtained in the

4

buffer and Mefp-1 solutions, plotted against applied potential. Error bars show standard

5

deviations of triplicated measurements.

6

7

3.3 Potential controlled QCM-D measurements

8

The sensed mass and viscoelastic properties of the adsorbed surface layer as a function of

9

applied potential were investigated by potential controlled QCM-D measurements, using a Pt13


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1

coated QCM crystal immersed in Mefp-1 solution. Before the experiment, a baseline of the

2

buffer solution was established. The measurements were repeated twice and the results

3

showed quite a good reproducibility. The applied potential values were selected based on the

4

different regions in the CV profile (Fig. 2).

5

Fig. 5 shows that the sensed mass and dissipation are influenced by the applied potential. At -

6

0.4 V the sensed mass continually increases, while the dissipation decreases with time. The

7

increase of the sensed mass is mainly a result of enhanced adsorption of both the Mefp-1 and

8

UPD hydrogen due to a negatively charged Pt surface at this potential 39. The decrease of the

9

dissipation most likely related to a compaction process of the preformed Mefp-1 film at OCP

10

due to the electrostatic force between the negatively charged substrate and the positively

11

charged Mefp-1 molecules. After stepping the potential to -0.1 V, the sensed mass drops

12

immediately at the beginning and then remains to be stable over time. The sudden drop of the

13

sensed mass and a slight decrease of dissipation are mainly due to some desorption of the

14

UPD hydrogen. The stable level of sensed mass suggests that the Mefp-1 adsorbed at -0.4 V

15

remains on the positively charged Pt surface at -0.1 V. Meanwhile, the relatively stable

16

dissipation value indicates that there is no significant change in the structure of the pre-

17

adsorbed Mefp-1 film. At 0.2 V (positively charged surface), the sensed mass and the

18

dissipation exhibit a similar trend, i.e., a gradual increase followed by a stabilization with

19

time. The initial increases in the sensed mass and the dissipation is most likely the

20

consequence of the swelling of the Mefp-1 film due to the electrostatic repulsion between the

21

substrate and lysine group of the Mefp-1. After stepping the potential to 0.5 V, the sensed

22

mass and the dissipation rises initially, and then decreases gradually over time. The initial

23

increase is likely due to oxygen plating on the Pt surface. Whereas the subsequent decrease of

24

the sensed mass and dissipation suggests an electrochemical oxidation process of the Mefp-1

25

film. It has been reported that oxidation of Mefp-1 normally followed by the cross-linking - a 14


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1

compaction process

, therefore squeezing out of water is probably responsible for the

2

decrease of the sensed mass. At 0.8 V, a noticeable increase of the sensed mass occurs, which

3

is due to the oxidation of Pt to PtO (Eqs. 1 and 2). Further oxidation of the Mefp-1 film may

4

also occur at this potential, but is hidden by the Pt oxide formation in the sensed mass data.

5

However, the slight decrease of the dissipation may be regarded as evidence for the oxidation

6

of the Mefp-1 film. The oxidation of Pt should result in a higher dissipation, because the

7

quasi-3D lattice PtO film is less compact than the Pt film.

8

Although the adsorption of Mefp-1 is irreversible upon the substrate charge as demonstrated

9

in electrochemical results, the QCM-D data imply the film structure can be varied.

10

11 12

Figure 5. Sensed mass (a) and dissipation (b) obtained from the potential controlled QCM-D

13

measurement on a Pt-coated QCM crystal, in 0.1 mg/ml Mefp-1 solution at pH 4.6. 15


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1

3.4 Potential controlled friction measurement

2

The friction behaviour is highly sensitive to surface properties. Therefore, potential

3

controlled in-situ friction measurements were performed on a Pt sheet in the buffer and Mefp-

4

1 solutions, to obtained complementary information about the structure and mechanical

5

property of the adsorbed Mefp-1 layers. The coefficient of friction (COF) and the

6

corresponding electrical current obtained at applied potentials are plotted against time in Figs.

7

6a and 6b. To interpret these data, it is necessary to combine the discussions with the CV and

8

QCM-D results.

9

At OCP, the measured COF in both solutions experiences a running-in process where it

10

decreases gradually over time. The steady-state value of the COF after 200 s in the Mefp-1

11

solution is significantly larger than that in the buffer solution, implying that the adhesive

12

property of the adsorbed Mefp-1 film on the Pt surface leads to greater resistance to

13

movement. Moreover, the electrical current at OCP is slightly negative (-5 µA/cm2) in the

14

Mefp-1 solution during the running-in process (Fig. 6b). This phenomenon originates from

15

the dynamic process of the natural adsorption of the Mefp-1 molecules onto the rubbing track

16

of the specimen surface, where the adsorbed molecules could be removed by friction.

17

In the buffer solution, the measured COF and the electrical current are relatively stable at -0.4

18

and 0.2 V, due to the potential-inactive feature of the citric acid. However, at 0.8 V, the COF

19

in the buffer solution decreases first and increases later over time. The decrease of the COF

20

might be related to the formation of the boundary lubrication layer due to the adsorption of

21

abundant citrate anions 56, 57. Because sufficient positive charges would build up on the sheet

22

surface before the oxidation of Pt. Nevertheless, the increase of the COF is associated with

23

the oxidation of the Pt surface under the anodic potentials, as mentioned in the discussion of

24

the CV results, and this is confirmed by the gradual decrease of the electric current in Fig. 6b. 16


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1

In contrast, the COF is more sensitive to the applied potential in the Mefp-1 solution,

2

suggesting a potential active character of the protein. At -0.4V, as seen in Fig. 6a, the COF

3

exhibits fluctuations, and the electrical current shows a transient decay. For interpreting this

4

result, it is necessary to recall the QCM-D result that there is a co-adsorption process of the

5

Mefp-1 and hydrogen molecules in this potential range. Different from the QCM experiments,

6

here the tribopairs are in relative motion besides the co-adsorption process. The mechanical

7

movement of the tribopair displaces the adsorbed hydrogen atoms more easily, resulting in

8

the re-adsorption of hydrogen molecules. This is confirmed by the transient decay of the

9

electrical current. These complex processes of co-adsorption of Mefp-1 and hydrogen

10

molecules, as well as the mechanical displacement and re-adsorption of hydrogen molecules,

11

give rise to the fluctuations of the COF. When raising the potential to 0.2V, the COF in Mefp-

12

1 solution increases suddenly and then decreases gradually to a stable value, which is still

13

higher than that at -0.4 V. This phenomenon is consistent with the QCM-D result, where the

14

increase of the COF implies a less compact structure of the Mefp-1 film. Specifically, the

15

swelling of adsorbed Mefp-1 film with more penetrating water molecules occurs due to the

16

repulsive interaction with the positively charged substrate. This inference is confirmed by the

17

QCM result where both the sensed mass and the dissipation increase after the potential is

18

stepped to 0.2 V. In this case, the Mefp-1 film becomes more viscous, leading to the increase

19

of the COF. Moreover, the following decrease of the COF might result from oxidation of

20

Mefp-1 and squeezing of water molecules of the Mefp-1 molecules, as demonstrated by the

21

QCM result that both the sensed mass and the dissipation increase in the later period

22

(4800~5100 s in Fig. 5). At the final step, the potential is increased to 0.8V, the COF in the

23

Mefp-1 solution exhibits another sudden drop, and then increases slightly over time. The

24

sudden drop of the COF may result from the oxidation of Mefp-1. Nevertheless, during the

25

initial stage of 0.8 V, the COF in the Mefp-1 solution is higher than that in the buffer solution. 17


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

This fact confirms the existence of oxidized Mefp-1 on the surface, and desorption of Mefp-1

2

is not the dominant process during the initial stage. The later increase of the COF may be

3

related to the oxidation of the Pt surface. The slower increase rate implies a slower Pt

4

oxidation process in the Mefp-1 solution than that in the buffer solution, which is most likely

5

due to the blocking of the active sites for Pt oxidation by the adsorbed protein molecules.

6

In order to test the reversibility of adsorption/desorption and oxidation/reduction processes of

7

the Mefp-1 film under applied potential, in-situ friction measurements were performed in the

8

Mefp-1 solution with switching potentials between -0.4 V and 0.8 V, as well as between -0.4

9

V and 0.2 V. The potentials were selected from different regions according to the CV curve

10

shown in Fig. 2. The results in Fig. 7 show that the COF varies periodically in response to the

11

potential change, with good reversibility in both cases. However, a general and slight increase

12

of the COF over time is also evident in Fig. 7b, which is likely due to the adsorption of Mefp-

13

1 molecules on the tribopair (ZrO2 ball) surface, making a contribution to the adhesive

14

bridging with the specimen surface. Overall, these results indicate that the potential induced

15

adsorption, compaction and oxidation processes of Mefp-1 on Pt surface are reversible.

18


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1

2 3

Figure 6. (a) Variations of COF over time at different potentials in the buffer solution without

4

and with 0.1 mg/mL MAP. (b) Corresponding variations of the electrical currents over time.

19


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1

2 3

Figure 7 Variations of COF over time at different switching potentials in Mefp-1 solution: (a)

4

switch potentials between -0.4 V and 0.8 V; (b) switch potentials between -0.4 V and 0.2 V.

5 6

The Mefp-1 film can be formed naturally by immersing substrates to the protein

7

containing solutions, however, the adsorbed amount can be limited by the surface charge of

8

the substrate, and the adsorbed protein film is not compact enough without a further

9

compaction treatment 7. Mefp-1 films compaction through pH adjustment

58

, iron ions

10

complexation 59, chemically induced oxidization 6, as well as addition of ceria nanoparticles

11

60

12

an anticorrosion inhibitor. In this paper, we present an alternative Mefp-1 film formation and

and phosphate 61, have been previously reported showing great potential of using Mefp-1 as

20


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1

compaction controlling approach – applying electrochemical potential to the metallic

2

substrate. The film forming and compacting processes are relatively simple and fast as

3

compared with layer-by-layer composite film assembling for instance, which is of great

4

importance for industrial applications. Furthermore, this approach does not involve addition

5

of more chemical compounds showing financial and environmental advantages as compared

6

to previously reported methods.

7

8

4. Conclusions

9

In this study, we have demonstrated the feasibility of potential-tuned adsorption of Mefp-1, as

10

well as compaction and decompaction of the pre-adsorbed Mefp-1 film, by using CV and

11

potential controlled EIS, QCM-D and in-situ friction measurements. The results give insights

12

into the mechanisms of the potential induced adsorption of Mefp-1, and the structural changes

13

of the pre-adsorbed Mefp-1 film. The combined data provide a consistent picture and lead to

14

the following conclusions:

15

Mefp-1 adsorbs on Pt substrate at the studied potential range. In the UPD hydrogen

16

adsorption region, an increased potential enhances Mefp-1 adsorption leading to higher

17

protein coverage of the Pt surface. In the electrochemical double layer region, an increased

18

potential results in decompaction of the pre-adsorbed Mefp-1 film, forming a thicker layer

19

with higher water content. In the Pt oxidation region, an increased potential leads to oxidation

20

of pre-adsorbed Mefp-1 film resulting in a film compaction. Moreover, the adsorbed protein

21

molecules

22

oxidation/reduction. The potential-induced adsorption, compaction and oxidation processes

23

of Mefp-1 on Pt surface are reversible.

can

block

active

sites

for

hydrogen

adsorption/desorption

21


and

Pt

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1

2

5. Acknowledgement

3

Guoxin Xie acknowledges the financial support from the National Natural Science

4

Foundation of China (Grant No. 51475256), the Ragnar Holm postdoc fellowship at KTH,

5

Sweden, and the Foundation for the Author of National Excellent Doctoral Dissertation of

6

China (FANEDD No. 201429). Biopolymer Products AB, Sweden, is acknowledged for the

7

supply of the protein.

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

6. References 1. Waite, J. H. Evidence for a repeating 3,4-dihydroxyphenylalanine- and hydroxyprolinecontaining decapeptide in the adhesive protein of the mussel, Mytilus edulis L. J. Biol. Chem. 1983, 258 (5), 2911-2915. 2. Bell, E.; Gosline, J. Mechanical design of mussel byssus: material yield enhances attachment strength. J. Exp. Biol. 1996, 199 (4), 1005-1017. 3. Lu, Q.; Danner, E.; Waite, J. H.; Israelachvili, J. N.; Zeng, H.; Hwang, D. S. Adhesion of mussel foot proteins to different substrate surfaces. J. R. Soc. Interface 2013, 10 (79). 4. Krivosheeva, O.; Dėdinaitė, A.; Claesson, P. M. Adsorption of Mefp-1: Influence of pH on adsorption kinetics and adsorbed amount. J. Colloid Interface Sci. 2012, 379 (1), 107-113. 5. Holten-Andersen, N.; Zhao, H.; Waite, J. H. Stiff Coatings on Compliant Biofibers: The Cuticle of My?lus californianus Byssal Threads†,‡. Biochemistry 2009, 48 (12), 2752-2759. 6. Zhang, F.; Pan, J.; Claesson, P. M.; Brinck, T. 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 2012. 7. Zhang, F.; Pan, J.; Claesson, P. M. Electrochemical and AFM studies of mussel adhesive protein ( Mefp-1) as corrosion inhibitor for carbon steel. Electrochim. Acta 2011, 56 (3), 1636-1645. 8. Hansen, D. C.; Dexter, S. C.; Waite, J. H. The inhibition of corrosion of S30403 stainless steel by a naturally occurring catecholic polymer. Corros. Sci. 1995, 37 (9), 1423-1441. 9. Hansen, D. C.; Luther III, G. W.; Waite, J. H. The Adsorption of the Adhesive Protein of the Blue Mussel Mytilus edulis L onto Type 304L Stainless Steel. J. Colloid Interface Sci. 1994, 168 (1), 206-216. 10. Waite, J. H.; Housley, T. J.; Tanzer, M. L. Peptide repeats in a mussel glue protein: theme and variations. Biochemistry 1985, 24 (19), 5010-5014. 11. Filpula, D. R.; Lee, S.-M.; Link, R. P.; Strausberg, S. L.; Strausberg, R. L. Structural and Functional Repetition in a Marine Mussel Adhesive Protein. Biotechnol. Progr. 1990, 6 (3), 171-177. 12. Waite, J. H. Adhesion à la Moule. Integr. Comp. Biol. 2002, 42 (6), 1172-1180. 13. Williams, T.; Marumo, K.; Waite, J. H.; Henkens, R. W. Mussel glue protein has an open conformation. Arch. Biochem. Biophys. 1989, 269 (2), 415-422. 22


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56. Zhu, S.; Yue, J.; Qin, X.; Wei, Z.; Liang, Z.; Adzic, R. R.; Brankovic, S. R.; Du, Z.; Shao, M. The Role of Citric Acid in Perfecting Platinum Monolayer on Palladium Nanoparticles during the Surface Limited Redox Replacement Reaction. J. Electrochem. Soc. 2016, 163 (12), D3040-D3046. 57. Attard, G. A.; Ye, J.-Y.; Jenkins, P.; Vidal-Iglesias, F. J.; Herrero, E.; Sun, S.-G. Citrate adsorption on Pt {hkl} electrodes and its role in the formation of shaped Pt nanoparticles. J. Electroanal. Chem. 2013, 688, 249-256. 58. Zhang, F. The Mussel Adhesive Protein (Mefp-1): A GREEN Corrosion Inhibitor. 2013, 24-28. 59. Zhang, F.; Sababi, M.; Brinck, T.; Persson, D.; Pan, J.; Claesson, P. M. In situ investigations of Fe3+ induced complexation of adsorbed Mefp-1 protein film on iron substrate. J. Colloid Interface Sci. 2013, 404 (0), 62-71. 60. Sababi, M.; Zhang, F.; Krivosheeva, O.; Forslund, M.; Pan, J.; Claesson, P. M.; Dedinaite, A. Thin Composite Films of Mussel Adhesive Proteins and Ceria Nanoparticles on Carbon Steel for Corrosion Protection. J. Electrochem. Soc. 2012, 159 (8), C364-C371. 61. Zhang, F.; Brinck, T.; Brandner, B. D.; Claesson, P. M.; Dedinaite, A.; Pan, J. 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 2013, 107, 276-291.

17

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Table of content 315x249mm (144 x 144 DPI)


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Tunable Adsorption and Film Formation of Mussel Adhesive Protein by

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