BSA adsorption on a plasma-deposited silver nanocomposite film controls silver release: A QCM and XPS-based modelling

    BSA adsorption on a plasma-deposited silver nanocomposite film controls silver release: A QCM and XPS-based modelling Chun Wang, Sandrine Zanna, Isabelle Frateur, Bernard Despax, Patrice Raynaud, Muriel Mercier-Bonin, Philippe Marcus PII: DOI: Reference:

S0257-8972(16)30666-1 doi: 10.1016/j.surfcoat.2016.07.063 SCT 21393

To appear in:

Surface & Coatings Technology

Received date: Revised date: Accepted date:

7 April 2016 11 July 2016 21 July 2016

Please cite this article as: Chun Wang, Sandrine Zanna, Isabelle Frateur, Bernard Despax, Patrice Raynaud, Muriel Mercier-Bonin, Philippe Marcus, BSA adsorption on a plasma-deposited silver nanocomposite film controls silver release: A QCM and XPS-based modelling, Surface & Coatings Technology (2016), doi: 10.1016/j.surfcoat.2016.07.063

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT BSA adsorption on a plasma-deposited silver nanocomposite film controls

T

silver release: a QCM and XPS-based modelling

IP

Chun Wang1, Sandrine Zanna1,*, Isabelle Frateur1,2,3, Bernard Despax4, Patrice Raynaud4,

SC R

Muriel Mercier-Bonin5, Philippe Marcus1

1

NU

PSL Research University, Chimie ParisTech-CNRS, Institut de Recherche de Chimie Paris,

2

MA

Research Group Physical Chemistry of Surfaces, 75005, Paris, France

CNRS, UMR 8235, Laboratoire Interfaces et Systèmes Electrochimiques, F-75005, Paris,

Sorbonne Universités, UPMC Univ Paris 06, UMR 8235, LISE, F-75005, Paris, France

TE

3

D

France

4

CE P

France

LAPLACE, Université de Toulouse, CNRS, INPT, UPS, 118 Route de Narbonne , 31062

Toulouse cedex 9 - France

LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, F-31077

AC

5

Toulouse, France

Keywords: silver; nanocomposite film; protein; QCM; XPS

*

Corresponding author: [email protected], tel:+33 1 44 27 80 17, PSL Research

University, Chimie ParisTech-CNRS, Institut de Recherche de Chimie Paris, Research Group Physical Chemistry of Surfaces, 75005, Paris, France

1

ACCEPTED MANUSCRIPT Abstract The adsorption of a model protein, the bovine serum albumin (BSA), on plasma-deposited

T

silver nanocomposite thin films was investigated in situ by Quartz Crystal Microbalance

IP

(QCM) and ex situ by X-ray Photoelectron Spectroscopy (XPS). For comparison, BSA

SC R

adsorption was also studied on pure silver sample and on the polymeric matrix without silver nanoparticles. Both techniques showed that BSA adsorption systematically occurred,

NU

regardless of the chemical composition of the solid surface. BSA adsorption was found to be a fast and irreversible process. For the adsorbed BSA layer characterization, a general island

MA

model was considered. The height of the protein islands (h) and their surface coverage () were estimated from combined QCM and XPS data. On the polymeric matrix, the surface

D

coverage was low whereas on pure silver sample and on the nanocomposite film, it was

TE

significantly increased. From QCM measurements, mass loss at a constant rate, ascribed to

CE P

the release and dissolution of Ag particles from the nanocomposite film into the surrounding solution, was observed before and after BSA adsorption, with two different associated rates. The decrease of the silver release rate after BSA adsorption is explained by silver particles

AC

coverage by protein islands. BSA molecules adsorbed on silver nanoparticles have a "blocking" effect, decreased the rate of silver nanoparticle release. However, silver dissolution as

Ag+

ions

may

still

occur.

2

ACCEPTED MANUSCRIPT Introduction Microbial adhesion followed by biofilm formation on the surfaces of medical devices, food processing equipments, heat exchangers and ship hulls has been recognized as a widespread

T

phenomenon. Its prevention has a major impact in limiting medical- and food-related

IP

problems. An effective and desired approach to reduce microbial adhesion is to modify the

SC R

solid surface and make it less adhesive towards micro-organisms and/or anti-microbial using a physical, chemical treatment or natural bactericidal substances [1-3]. Materials containing

NU

silver, which is a well-known antiseptic, have been considered as good candidates for reducing microbial adhesion, and have been widely used in the biomedical industry for

MA

catheters, dental materials, medical devices and implants. Collinge et al. reported that silvercoated pins resulted in decreasing infection and motion at pin sites [4]. Bosetti et al. showed

D

that silver-coated external fixation devices exhibited good biocompatibility properties and

TE

inhibited the initial stages of bacterial colonisation [5]. Silver causes a dramatic decrease in

CE P

biofilm activity; e.g. silver-coated or silver-impregnated materials have a particular potential in preventing catheter-associated urinary tract infections [6]. Another relevant strategy consists in modifying the material surface by silver-containing nanocomposite coatings using

AC

plasma deposition [7-10]. Saulou et al. [11] and, more recently, Allion-Maurer et al. [12] demonstrated antimicrobial properties of silver nanocomposite films against adhering-cells, including the yeast Saccharomyces cerevisiae and the bacteria Escherichia coli and Staphylococcus aureus, respectively. The initial stage involved in microbial adhesion to solid surfaces is the adsorption of proteins present in the surrounding medium. Interactions between micro-organisms and surfaces are then mediated by these pre-adsorbed proteins. Bovine serum albumin (BSA) is commonly used as a model protein for studying protein–surface interactions, and the quartz crystal microbalance (QCM) technique is now recognised as a powerful tool for in situ studies of protein adsorption [13-17].

3

ACCEPTED MANUSCRIPT In this study, silver nanocomposite thin films were produced on chromium (Cr) quartz crystals by using

the

combination

of

silver

sputtering

and

plasma

polymerization

of

hexamethyldisiloxane (HMDSO), based on previous works [10,11]. Polymeric matrix thin

T

films (i.e. with no embedded silver) were also deposited by plasma on Cr quartz crystals.

IP

Such crystals have been chosen since Cr is a major constituent of stainless steels which are

SC R

often used in protein-containing environments, including marine, food, and biomedical applications. The adsorption of BSA on the deposited films and, for comparison, on

NU

commercial pure Ag-coated quartz crystals was investigated in situ and in real time using a QCM combined to a switch-flow cell. The chemical characterization of the deposited films

MA

was carried out ex situ by X-ray Photoelectron Spectroscopy (XPS), before and after BSA

AC

CE P

TE

D

adsorption

4

ACCEPTED MANUSCRIPT Experimental

T

Quartz crystals

IP

Polished AT-cut, 5 MHz, 1-inch diameter commercial quartz crystals (Maxtek-Inficon), with a

SC R

nominal thickness of 333 m, were sputter-coated on both faces with a 550-nm Cr layer or a 360-nm Ag layer. The Cr deposit was amorphous with 99.99% purity. In the case of massive

MA

structure was observed by AFM [18].

NU

Ag quartz crystals, the Ag layer was deposited on a 70-nm Ti sublayer, and a granular

D

Protein and solutions

TE

Bovine serum albumin (BSA, Fraction V) with a purity of about 99% was purchased from

CE P

Sigma-Aldrich. Two solutions were prepared in a 0.15 M NaCl supporting electrolyte using high purity Milli-Q grade water (COT< 5ppb): one without protein and the other one with 20 mg. L-1 of BSA. Both solutions were deaerated by nitrogen bubbling for at least 1 h before the

AC

beginning of the QCM experiment, and were thermostated at 30°C.

Plasma deposition

The deposition process relies on a dual strategy, associating silver sputtering and simultaneous Plasma Enhanced Chemical Vapour Deposition (PE-CVD) in an argon-HMDSO plasma, using an asymmetrical radiofrequency (RF, 13.56 MHz) discharge [10]. The plasma process conditions were identical to those used by Saulou et al. [11] for stainless steel plasma-coating. In brief, a RF power of 100 W was applied through an impedance-matching network.

5

ACCEPTED MANUSCRIPT The RF powered electrode was a silver disc, capacitively coupled to a RF generator. The Cr quartz crystals were placed on the sample holder and mounted on the ground electrode. The

T

system was pumped down and gased out to a limit pressure of 1.33 10-4 Pa. The chamber was

IP

then filled with pure argon until reaching a pressure of 5.32 Pa. The mixture of argon gas

SC R

(alphagaz 2, 99.9999% purity, Air Liquide, France) and HMDSO (Sigma-Aldrich, NMR grade ≥ 99.5%) was obtained into the chamber by pulsing HMDSO injection in argon, which

NU

allowed silver sputtering and plasma polymerization to be balanced. The duration of injection over a period of 5 s was set to 3 s for polymeric matrix films and 1.6 s for silver

MA

nanocomposite ones. The injection of HMDSO was not continuous, so the pressure oscillated during the 5-s period (1.6-s injection (TON) and 3.4-s no injection (TOFF)). Complementary to

D

the control of the discharge parameters (power, pressure, gas flow rates, and duration of

TE

injection), the plasma phase composition was followed by in-line Optical Emission

CE P

Spectroscopy (OES, acquisition range: 480-550 nm). The Ag/Ar ratio, which has been demonstrated by our group to offer a reliable in-line monitoring of the silver content in the plasma phase [19] was fixed to a mean value of 0.19. The OES monitoring thus enabled to

AC

control the silver flux from the Ag target to the sample during the 5-s cycle and, consequently, to obtain an indirect image of the expected metal amount in the nanocomposite film being synthesized.

For all experiments, the duration for plasma deposition was set to 10 min, with a deposition rate of about 17 nm/min. Under the same experimental conditions, Saulou et al. [11] clearly demonstrated by electron microscopy the nanocomposite structure of the deposited films, with the inclusion of metallic, spherical and nano-sized (3 to 15 nm) silver nanoparticles within the polymeric matrix. Investigation of the film physico-chemical properties was also conducted by X-ray photoelectron spectroscopy and transmission FTIR spectroscopy [11]. In particular, plasma-mediated coatings were composed of C, O, Si and Ag, the latter being predominantly

6

ACCEPTED MANUSCRIPT under its metallic form. The presence of Si–H, Si–O–Si, Si–(CH)n–Si and C–H groups was established.

IP

T

Quartz crystal microbalance (QCM) measurements

SC R

The switch-flow cell was a 3-electrode and a 2-compartment cell that allowed QCM experiments to be performed in deaerated solution. Details on the flow cell set-up have been

NU

reported elsewhere [14]. To perform gravimetric measurements under electrochemical control, the flow cell was connected to a microbalance control unit (RQCM, provided by Maxtek-

MA

Inficon) and to a potentiostat, both devices being computer controlled. This system allows for real-time and simultaneous recording of the quartz crystal frequency and resistance. In this

D

work, the resistance signal was not taken into account. Before the QCM experiments, the

TE

quartz crystals were cleaned with high purity Milli-Q grade water and dried with nitrogen. All

CE P

experiments were performed at the open circuit potential (OCP) as follows: (1) the microbalance signals were recorded under BSA-free solution flow until stabilization (step I), then (2) the BSA-containing solution was switched to the cell and the signals were recorded

AC

for at least 60 min (protein adsorption study; step II) and, finally, (3) once the adsorption plateau was reached, the BSA-free solution was switched again to the cell and the signals were recorded for at least 60 min (protein desorption study; step III). Table II presents the three steps monitoring. The total frequency variation (f) was converted to mass variation per unit area (m) by application of the Sauerbrey equation [20], using the theoretical sensitivity coefficient (C) equal to 0.0566 Hz.cm2.ng-1:

f   C  m

(1)



7

ACCEPTED MANUSCRIPT

T

X-Ray Photoelectron Spectroscopy (XPS) analyses

IP

The chemical state of the plasma-deposited coatings was determined by X-ray Photoelectron

SC R

Spectroscopy (XPS) using a Thermo Electron ESCALAB 250 spectrometer, with a monochromatised Al Kα radiation (1486.6 eV). The analyser pass energy was 100 eV for

NU

survey spectra and 20 eV for high resolution spectra. The spectrometer was calibrated using Au 4f7/2 at 84.1 eV. The following core levels were analysed: Ag 3d, Si 2p, O 1s, C 1s, N 1s.

MA

Samples were analysed before and after BSA adsorption/desorption followed by QCM. At the end of each QCM measurement, the cell was dismounted, the sample was then dipped tenfold

D

in high purity Milli-Q grade water to remove BSA molecules loosely bound to the surface,

TE

and finally dried with nitrogen before introduction in the fast-entry lock chamber of the XPS

CE P

spectrometer. Peak parameters used to decompose the C1s spectra were described in a previously published paper [21]. All spectra were referred to the C 1s peak for carbon involved in C-C and C-H bonds, located at 285 eV. Curve fitting of the spectra was performed

AC

with the Thermo Electron software “Avantage”. The inelastic mean free path values were calculated by the TPP2M formula [22], and the photoemission cross-sections were taken from Scofield [23].

Results and discussion

QCM study of BSA adsorption/desorption: influence of the film characteristics

Examples of total frequency variation ( f ) during BSA adsorption on the silver nanocomposite film, the polymeric matrix and massive Ag, deposited on quartz crystals are 8

ACCEPTED MANUSCRIPT shown in Fig. 1(a). For studying the desorption process, the BSA-free solution was switched back to the flow-cell after 60 min for massive Ag and the polymeric matrix, and after 100 min for the nanocomposite film. During step I, f stabilization in the BSA-free solution was

T

quite fast for massive Ag and the polymeric matrix (except a possible drift), whereas in the

IP

case of the silver nanocomposite film, f linearly increased with time with a slope of ~ 0.5

SC R

Hz.min-1 corresponding to mass loss at constant rate. It can be noted that no mass loss was observed for massive Ag. For step II, f started decreasing a few seconds after BSA

NU

introduction into the cell (t = 0 min) for the three materials under study. Then, f reached a plateau after about 50 min for massive Ag and 30 min for the polymeric matrix whereas, for

MA

the silver nanocomposite film, f started increasing linearly with time ~ 20 min after the switch with a slope of ~ 0.3 Hz.min-1, corresponding to mass loss at constant rate. For

TE

D

massive Ag and the polymeric matrix, the frequency decrease after BSA introduction is associated to BSA adsorption on the surface, as further confirmed by XPS (see below). In the

CE P

case of the silver nanocomposite film, the reasons for such an increase in f could be manifold, as reported by Wang et al. [13]: overshoot adsorption followed by re-orientation of

AC

adsorbed proteins, desorption of trapped water within the adsorbed layer and/or release of silver from the substrate. Consistent with the results depicted in step I and owing to our XPS results (see below), we assumed that the release of Ag nanoparticles and/or Ag dissolution (as Ag+ ions) from the coating into the surrounding solution was most probably the reason for the observed linear increase in f . The total frequency variation is thus the sum of the frequency variation corresponding to BSA adsorption (fBSA < 0) and the frequency variation corresponding to silver release/dissolution into the solution (fAg > 0) as follows:

f  f BSA  f Ag

(2)

where f Ag  K  t with K = 0.3 Hz.min-1 and t corresponds to the elapsed time since BSA introduction. In Fig. 1(b), data for the silver nanocomposite film after BSA introduction have 9

ACCEPTED MANUSCRIPT been corrected for Ag release/dissolution (fBSA vs t; Eq. (2)); fBSA reaches a plateau after approximately 30 min like for the polymeric matrix whereas f reached a plateau after about

IP

T

50 min for massive Ag.

SC R

Altogether, these results show that BSA adsorption is observed regardless of the chemical composition of the quartz crystal coating. The amount of BSA adsorbed on the different surfaces can be estimated by application of Eq. (1). For the silver nanocomposite film, fBSA

NU

reaches -35 ± 7 Hz on the adsorption plateau, which corresponds to a mass variation mBSA of

and

mBSA

=

650

±

MA

620 ± 120 ng. cm-2. For massive Ag, the results are the following: f = fBSA = -37 ± 1 Hz, 20

ng.

cm-2;

and

for

the

polymeric

matrix,

D

f = fBSA = -19 ± 1 Hz, and mBSA = 340 ± 20 ng. cm-2. From the QCM data, it can be

TE

concluded that the amount of adsorbed protein is similar on pure Ag sample and on the silver

CE P

nanocomposite film, and is lower on the polymeric matrix.

Once a steady-state was reached, the introduction of the BSA-free solution (step III) did not

AC

lead to any significant increase in frequency regardless of the coating material. This means that BSA adsorption is an irreversible process or at least that the kinetics of desorption is very slow, which is fully consistent with previous findings [13].

XPS characterization of the films before and after BSA adsorption

The elemental and chemical composition of the coatings, before (native films) and after BSA adsorption, was determined by XPS. Fig. 2 shows typical XPS spectra recorded for the same silver nanocomposite film as the one studied by QCM in Fig. 1. From the survey spectrum (Fig. 2(a)), it was found that the native film was composed of silver, silicon, carbon and oxygen, consistent with previous findings [11]. As expected, N 1s was detected after BSA 10

ACCEPTED MANUSCRIPT adsorption. The intensity in the Ag 3d region was markedly lower after BSA adsorption compared to native conditions (Fig. 2(b)). In a first attempt, this signal attenuation was explained by the presence of the adsorbed protein layer and/or a significant loss of silver from

T

the coating during the QCM experiment. The Ag 3d5/2 peak binding energy is 368.9 eV. The

IP

silver chemical state is difficult to determine using the Ag 3d5/2 peak because the

SC R

corresponding binding energy varies with the particle size and may be shifted due to charging effect. Instead, the modified Auger parameter (α’) was used [24-25]. It is the sum of the

NU

binding energy of an intense photoemission peak (Ag 3d5/2) and the kinetic energy of the sharpest Auger line (M4N45N45), shown in Fig. 2(c). The modified Auger parameter for silver

MA

was found to be 726 eV, in good agreement with published data for metallic silver [24-25]. Thus, silver is not oxidized in the native film, as already shown by Saulou et al. [11], but also

D

after BSA adsorption. In Fig. 2(d), the Si 2p peak at 102.2 eV corresponds to Si bonded to

TE

oxygen and hydrocarbon (R), such as R3-Si-O (101.5 eV), R2-Si-O2 (102.1 eV) and R-Si-O3

CE P

(102.8 eV) [26]. The peak at 97.8 eV in Fig. 2(d) is attributed to Ag 4s; its intensity was lower after the QCM experiment, confirming the presence of an adsorbed protein layer and/or the loss of silver from the coating. The N 1s peak, centred at 400.1 eV as shown in Fig. 2(e), is

AC

symmetric as expected for the amine or amide groups of BSA [27]. Fig. 2(f) shows the C 1s spectra before and after BSA adsorption. For the native film, the main C 1s core level component is located at 285 eV and is associated to C-C and C-H bonds. After BSA adsorption, the C 1s signal was fitted with three contributions corresponding to well-identified carbon bonds present in the BSA molecules: C1, at a binding energy of 285 eV, attributed to C-C and C-H; C2, at a binding energy of 286.5 eV, attributed to C-N and C-O single bonds; C3, at a binding energy of 288.5 eV, assigned to O=C-O and O=C-N (peptidic bond) bonds [14, 21, 28]. The atomic N/(C2+C3) ratio was 0.5, in good agreement with the theoretical value for the BSA molecule (0.49) [21]. Such agreement between experimental and theoretical values provides a fingerprint of the protein, and allows us to conclude that BSA is

11

ACCEPTED MANUSCRIPT adsorbed on the surface. From the XPS data, the Ag/Si atomic ratio was calculated, using the Ag 3d and Si 2p peak intensities. Its value decreases from 1.8 for the native film to 0.6 after BSA adsorption, confirming silver release/dissolution from the silver nanocomposite film

IP

T

during the QCM experiment.

SC R

The polymeric matrix and the pure silver sample were also characterized by XPS before and after BSA adsorption. The recorded spectra for Si 2p, Ag 3d, N 1s and C 1s core levels are

NU

shown in Fig. 3. The intensities in the Si 2p and Ag 3d regions were markedly lower after BSA adsorption compared to native conditions (Fig. 3(a and b)). This signal attenuation was

MA

explained by the presence of the adsorbed protein layer respectively on the polymeric matrix and the pure silver sample.

D

In both cases, after BSA adsorption, the N 1s peak at 400.1 eV, attributed to amine or amide

TE

groups of the protein, was detected, as well as the three C1, C2 and C3 components of the C

CE P

1s signal, associated with the protein as described above. The N/(C2+C3) atomic ratio is 0.47 for the pure silver sample and 0.45 for the polymeric matrix, confirming the presence of BSA. No modification of the Si 2p or Ag 3d peak position was observed after BSA adsorption,

AC

meaning that the film composition was not modified by the adsorbed protein.

Modelling the surface coverage by BSA and its consequences on silver release

The presence of adsorbed protein after the QCM experiment was confirmed by XPS for pure silver samples and for the plasma-mediated films, with or without embedded nanosilver. Therefore, the frequency decrease measured after BSA introduction into the switch-flow cell can be assigned to BSA adsorption on the quartz crystal surface. The next step to estimate the surface coverage by the protein [15] for all the coatings under study. In a previous work [14], adsorption of BSA on chromium was studied by XPS and EQCM. 12

ACCEPTED MANUSCRIPT The equivalent thicknesses of a continuous BSA adsorbed layer was estimated in situ by EQCM and ex situ by XPS and the results were in excellent agreement. In the submitted paper, the BSA thickness estimated by XPS with the continuous layer is markedly different

T

from the BSA thickness obtained by EQCM. To account for this result, a more general model

IP

with a discontinuous layer of adsorbed protein on the surface was considered, using a

SC R

procedure previously established [15]. This approach is analogous to the one adopted by Lhoest et al. [29], who combined the data characterizing the adsorbed protein (fibronectin) on

NU

polymer (polystyrene) obtained from both radiolabeling (3Hlabeled fibronectin) and XPS (N 1s core level peak). However this result was not been checked by direct imaging technique

MA

and is merely the results of calculations using the recorded experimental data. For that purpose, a model, based on the formation of a discontinuous layer of adsorbed

D

protein, was considered. In this “island” model, two parameters were introduced: the height of

TE

the protein islands (h) and their surface coverage (). For the estimation of these two

CE P

parameters, it was assumed that the mass variation obtained by QCM (mBSA) and the N 1s core level peak located at 400.1 eV corresponded exclusively to adsorbed BSA [14]. The amount of water possibly trapped in the BSA layer, also sensed by QCM [14-15], was

AC

neglected, as well as the amount of loosely bound BSA molecules that may be removed during dipping into milli-Q grade water before XPS analysis. These assumptions may lead to a slight overestimation of the adsorbed protein mass.

BSA

Calculations were performed using the I N

BSA

f

/ I Ag, Si ratio, where I N

is the N 1s peak

f

intensity corresponding to nitrogen in the adsorbed BSA layer and I Ag,Si the Ag 3d5/2 (silver nanocomposite film or pure silver sample) or Si 2p (polymeric matrix) peak intensity corresponding to silver or silicon in the deposited film:

13

ACCEPTED MANUSCRIPT BSA N f Ag , Si

I I

 

T  D  T  D 

BSA BSA N N N N f f Ag , Si Ag , Si Ag , Si Ag , Si

   1  exp   BSAh    sin     N        1      exp   BSA h   sin  Ag , Si  

(3)

IP

T

where TX and σX are the transmission factor and the photoionisation cross-section of

SC R

photoelectrons emitted by X, respectively, D XBSA, f the atomic concentration of X in the ,f adsorbed BSA layer or in the deposited film, BSA the attenuation length of photoelectrons X

emitted by X in the BSA layer or in the film, and  the take-off angle of photoelectrons with

MA

NU

respect to the sample surface ( = 90° in this work).

The height of the protein islands is given by: mBSA

D

   BSA

(4)

TE

h

using the density of adsorbed BSA (  BSA = 1.15 g.cm-3 [30]).

CE P

Equations (3) and (4) form a set of two equations with two unknowns,  and h, that can be solved. The values of  and h obtained for the silver nanocomposite film, the polymeric matrix

AC

and the pure silver sample are summarized in Table 1.

On the polymeric matrix, the surface coverage by adsorbed BSA was low ( = 0.16 ± 0.01), whereas on silver it was much higher and similar for the pure silver sample and nano-sized forms ( = 0.53± 0.08). BSA interacted more with silver at the nanocomposite film surface than at the polymeric matrix surface. This is consistent with separate BSA molecules because BSA size (~8 nm) has the same order of magnitude as Ag nanoparticles (3 to 15 nm) and BSA interacts more with silver at the nanocomposite film surface than at the polymeric matrix surface. The height of the protein islands was in the range of 8 to 15 nm, with quasi-similar values for 14

ACCEPTED MANUSCRIPT the silver nanocomposite film and pure silver sample silver. As BSA molecules exhibit an elliptic shape at neutral pH with ~ 3×8 nm2 dimensions [31], a height of about 9 nm would correspond to three ”side-on” (i.e. oriented parallel to the surface) protein monolayers.

T

This”side-on” elongated orientation has already been described for BSA adsorption on silver

IP

surface [13]. However there may be a small amount of BSA on the polymeric matrix so the

SC R

thickness of BSA islands on silver nanoparticles would amount to two or three BSA layers. This result was not been checked by direct imaging technique, but is the result of reliable

MA

NU

calculations using the QCM and XPS data.

Adsorption of BSA to different surfaces, including charged, hydrophilic/hydrophobic surfaces, has been widely investigated [32-37]. In the conditions used in this work,

TE

D

electrostatic interactions were strongly inhibited by the high ionic strength of the suspending medium (NaCl 0.15 M) which screened the charges of the quartz crystal surface and BSA.

CE P

Concerning the contribution of other non-electrostatic interactions, like hydrophobic ones, it was previously shown that the polymeric matrix yielded a water contact angle of 83.8 ± 2.4

AC

indicating a hydrophobic character [12]. On hydrophobic surfaces, BSA adsorption is driven by the attraction with the nonpolar parts of protein molecules. We could reasonably assume that such mechanisms occurred on the polymeric matrix. In presence of silver at the sample surface, the strong binding of BSA to silver might be related to the formation of sulphur-silver complexes as BSA contains 17 intrachain disulfide bridges and one free thiol group at residue 34 [42]. Metallic nanoparticles, such as silver, are also likely to interact with amine groups of BSA [15].

Concerning the relationship between protein adsorption and conformation, the adsorbed protein molecules may undergo some structural transformation and often denaturation to 15

ACCEPTED MANUSCRIPT maximize their favourable interactions with the surface and the solvent [40]. For instance, it has been postulated that the -helical content in the adsorbed BSA molecules is decreased, whereas the -structure and random coil content is increased, compared to that of BSA

IP

T

molecules in solution [41]. In another study [42], the authors observed that, upon adsorption onto polystyrene particles, the relatively hydrophobic core of the dissolved BSA molecule

SC R

could open up in order to expose hydrophobic amino acid residues at the hydrophobic polystyrene surface. The authors concluded that structural rearrangements in the BSA

NU

molecule, like in other “soft” proteins, played a major role in the adsorption process, which was a direct consequence of its adaptability to changes in the environment. Although beyond

MA

the scope of this study, it should be valuable in further work to evaluate such putative conformational changes in the BSA molecule, in relation with adsorption rate and coating

D

properties. Owing to the high ionic strength of the suspending medium, the role of salts on

CE P

TE

water exclusion and protein arrangement could not be ruled out.

As previously mentioned, BSA preferentially adsorb on the silver nanocomposite film due to its high affinity for silver. As the diameter of the silver nanoparticles is comprised between 3

AC

and 15 nm, they exhibit a size of the same order of magnitude as that of a BSA molecule that presents an elliptic shape of 8 nm diameter. For the QCM experiment with the silver nanocomposite film (Fig. 1), if silver nanoparticles were released together with adsorbed BSA molecules, then the mass loss rate during steps II and III would be the same as the one obtained during step I (i.e. ~ 0.5 Hz.min-1 in the BSA-free solution), and the amount of adsorbed BSA detected by XPS after the QCM experiment would be negligible. However, the mass loss rate is lower during steps II and III (~ 0.3 Hz.min-1) than that achieved during step I. This may indicate that, once adsorbed, the BSA molecules may prevent, at least to some extent, the release of silver nanoparticles from the film into the surrounding solution. Thus, the adsorbed protein is assumed to exert a "blocking" effect and decrease the "active" surface 16

ACCEPTED MANUSCRIPT area for silver release, corresponding to the surface not covered by BSA. If a constant mass flux density (expressed in ng.cm-2.min-1) is assumed for silver nanoparticle release during the whole QCM experiment: (5)

IP

T

M II / III M I  S I * t S II / III * t

SC R

then the ratio of active surface areas is equal to the ratio of mass loss rates (in ng.min-1), which is also equal to the ratio of slopes (in Hz.min-1) between steps II/III and I: (6)

NU

M II / III / t S II / III f II / III / t   M I / t SI f I / t

MA

wheref, M, t are a frequency variation (in Hz), a mass variation (in ng) and a time variation (in min), respectively, and S is the surface not covered by the protein islands.

D

From the example given in Fig. 1, the active surface ratio is found to be equal to 0.3/0.5 = 0.6,

TE

corresponding to a surface coverage by the protein islands of 1-0.6 = 0.4. Therefore, considering that all the BSA molecules only adsorb on the silver nanoparticles, the BSA

CE P

surface coverage should be 0.4. From combined QCM and XPS data, the surface coverage is estimated to be 0.57 (Table 1). This difference probably means that some BSA molecules also

AC

interact with the matrix (in agreement with data obtained for the polymeric matrix) and/or that some silver nanoparticles in contact with adsorbed protein may dissolve into the solution as Ag+ ions through the discontinuous layer. Fig. 4 presents a scheme that proposes the possible mechanisms behind the silver release/dissolution from the nanocomposite film in the presence of adsorbed BSA as a discontinuous layer, according to the “island’ model. Due to a higher affinity for silver compared to that for the polymeric matrix, BSA molecules preferentially adsorb on silver nanoparticles. In this case, nanoparticle release from the film into the surrounding solution is prevented due to the blocking effect of adsorbed protein; however, silver dissolution as Ag+ ions may still occur. For silver nanoparticles not covered by BSA (i.e. voids in the BSA layer), silver dissolution takes place at the periphery of the nanoparticles. A decohesion zone 17

ACCEPTED MANUSCRIPT is hence created at the particle-matrix interface, triggering silver release in the aqueous media, as previously described for the silver nanocomposite film after ageing [12]. Altogether, our results demonstrate that adsorption of BSA inhibits rather than promotes the release of silver

T

from the silver nanocomposite film, in contrast with previous findings for pure silver sample

IP

surfaces in 0.15 M sodium nitrate solution . Wang et al. [13] shown that protein adsorption

SC R

itself influences the metal release process of metallic materials [43, 44]. Depending on the material and its surface properties, the type of protein and the prevailing physico-chemical

NU

conditions, the release of metals can be either enhanced or inhibited. To date, data on silver surfaces are scarce [13,45]. Recently, it was shown for pure silver sample surfaces that

MA

adsorption of bovine serum albumin (BSA) promoted the release of silver from the substrate through a dual mechanism [13]: (i) complexation of BSA with silver surface and the dynamic

D

equilibrium between surface adsorbed BSA and BSA molecules in the bulk solution and (ii)

TE

direct release of silver ions through voids in the BSA layer. In our work, no silver release was

CE P

here detected in 0.15 M NaCl for the pure-silver deposited on quartz crystals. The differences

AC

observed were probably related to the type of electrolyte solution.

Conclusions

In this work, nanocomposite thin films, with silver nanoparticles embedded in a polymeric matrix, were obtained by combination of silver sputtering and plasma decomposition of HMDSO. Silver-free polymeric matrix films were also synthesized. Adsorption of bovine serum albumin (BSA) on the different coatings was investigated in situ using a quartz crystal microbalance (QCM) coupled to a switch-flow cell and ex situ by X-ray Photoelectron Spectroscopy (XPS). For comparison, BSA adsorption was also studied on pure silver sample. Both techniques showed that BSA adsorption systematically occurred, regardless of the 18

ACCEPTED MANUSCRIPT chemical composition of the solid surface. QCM results also demonstrated that BSA adsorption was a fast and irreversible process. For the adsorbed BSA layer characterization, a general island model was considered. The height of the protein islands (h) and their surface

T

coverage () were estimated from combined QCM and XPS data. On the polymeric matrix,

IP

the surface coverage was low whereas on pure silver sample and on the nanocomposite film,

SC R

it was significantly increased. BSA interacts more with silver at the nanocomposite film surface than at the polymeric matrix surface. Moreover, adsorbed BSA prevented the release

NU

of silver nanoparticles by promoting a blocking effect. However, silver dissolution as Ag+ ions could still occur. Therefore, it would be interesting to take benefit from protein

MA

adsorption on nanocomposite films in order to regulate silver release/dissolution in solution. By controlling the surface coverage by the protein, it would be possible to adjust the silver

D

amount released/dissolved into the surrounding medium. This dual strategy of silver-

TE

nanocomposite film deposition followed by protein adsorption may then influence microbial

CE P

adhesion and biofilm formation.

Acknowledgements

AC

The research leading to these results has received funding from the French National Research Agency (ANR-07-BLAN-0196-01; ”Bio-Pleasure” project).

19

ACCEPTED MANUSCRIPT References

J. Fu, J. Ji, W. Yuan and J. Shen, Construction of anti-adhesive and antibacterial multilayer films via layer-by-layer assembly of heparin and chitosan, Biomaterials 26 (2005) 6684-6692.

[2]

K. Glinel, P. Thebault, V. Humblot, C.M. Pradier, T. Jouenne, Antibacterial surfaces developed from bio-inspired approaches, Acta Biomaterialia, 8 issue 5 (2012) 16701684. S.K. Hendricks, C. Kwok, M. Shen, T.A. Horbett, B.D. Ratner and J.D. Bryers, Plasmadeposited membranes for controlled release of antibiotic to prevent bacterial adhesion and biofilm formation, J. Biomed. Mater. Res. 50 (2000) 160-170.

SC R

NU

[3]

IP

T

[1]

C.A. Collinge, G. Goll, D. Seligson and K.J. Easley, Pin tract infections: silver vs. uncoated pins, Orthopedics 17 (1994) 445.

[5]

M. Bosetti, A. Massè, E. Tobin and M. Cannas, Silver coated materials for external fixation devices: in vitro biocompatibility and genotoxicity, Biomaterials 23 (2002) 887-892.

[6]

H. Kumon, H. Hashimoto, M. Nishimura, K. Monden and N. Ono, Catheter-associated urinary tract infections: impact of catheter materials on their management, J. Antimicrob. Ag. 17 (2001) 311-316.

[7]

P. Favia, M. Vulpio, R. Marino, R. d’Agostino, R.P. Mota and M. Catalano, Plasmadeposition of Ag-containing polyethyleneoxide-like coatings, Plasmas Polym. 5 (2000) 1-14.

[8]

E. Sardella, P. Favia, R. Gristina, M. Nardulli, and R. d’Agostino, Plasma-aided microand nanopatterning processes for biomedical applications, Plasma Process Polym. 3 (2006) 456-469.

[9]

H. Jiang, S. Manolache, A.C. Lee Wong and F.S. Denes, Plasma-enhanced deposition of silver nanoparticles onto polymer and metal surfaces for the generation of antimicrobial characteristics, J. Appl. Polym. Sci. 93 (2004) 1411-1422.

AC

CE P

TE

D

MA

[4]

[10] B. Despax and P. Raynaud, Deposition of “polysiloxane” thin films containing silver particles by an RF asymmetrical discharge, Plasma Process. Polym. 4 (2007) 127-134. [11] C. Saulou, B. Despax, P. Raynaud, S. Zanna, A. Seyeux, P. Marcus, J.-N. Audinot and M. Mercier-Bonin, Plasma-mediated nanosilver-organosilicon composite films deposited on stainless steel: synthesis, surface sharacterization, and evaluation of antiadhesive and anti-microbial properties on the model yeast Saccharomyces cerevisiae, Plasma Process. Polym. 9 (2012) 324-338. [12] A. Allion-Maurer, C. Saulou-Bérion, R. Briandet, S. Zanna, N. Lebleu, P. Marcus, P. Raynaud, B. Despax, M. Mercier-Bonin, Plasma-deposited nanocomposite polymersilver coating against Echerichia coli and Staphylococcus aureus : Antibacterial 20

ACCEPTED MANUSCRIPT properties and ageing, Surface&Coatings Technology 281 (2015) 1-10. X. Wang, G. Herting, I. Odnevall Wallinder, E. Blomberg, Adsorption of bovine serum albumin on silver surfaces enhances the release of silver at pH neutral conditions, Phys.Chem.Chem.Phys. 17 (2015) 18524-18534.

[14]

I. Frateur, J. Lecœur, S. Zanna, C.-O. A. Olsson, D. Landolt and P. Marcus, Adsorption of BSA on passivated chromium studied by a flow-cell EQCM and XPS, Electrochim. Acta 52 (2007) 7660-7669.

[15]

A. Ithurbide, I. Frateur, A. Galtayries and P. Marcus, XPS and flow-cell EQCM study of albumin adsorption on passivated chromium surfaces: Influence of potential and pH, Electrochim. Acta 53 (2007) 1336-1345.

[16]

V. Payet, S. Brunner, A. Galtayries, I. Frateur and P. Marcus, Cleaning of albumincontaminated Ti and Cr surfaces: An XPS and QCM study, Surf. Interface Anal. 40 (2008) 215-219.

NU

SC R

IP

T

[13]

MA

[17] E.M. Pinto, D.M. Soares, C.M.A. Brett, Interaction of BSA protein with copper evaluated by electrochemical impedance spectroscopy and quartz crystal microbalance,

D

Electrochim. Acta 53(2008) 7460-7466.

TE

[18] N. Li: Ph.D. Thesis, University of Paris 6, France, 2012.

CE P

[19] C. Saulou, B. Despax, P. Raynaud, S. Zanna, P. Marcus and M. Mercier-Bonin, Plasma deposition of organosilicon polymer thin films with embedded nanosilver for prevention of microbial adhesion, Appl. Surf. Sci. (2009) 256S, S35–S39. [20] G. Sauerbrey Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung Z., Phys. 155 (1959) 206-222.

AC

[21] P.G. Rouxhet, N. Mozes, P.B. Dengis, Y.F. Dufrêne, P.A. Gerin and M.J. Genet, Application of X-ray photoelectron spectroscopy to microorganisms Colloids and Surfaces, B: Biointerfaces 2 (1994) 347-369. [22] S. Tanuma, C.J. Powell and D.R. Penn, Calculations of electron inelastic mean free paths. II. Data for 27 elements over the 50-2000 eV range, Surf. Interface Anal 17 (1991) 911-926. [23] J.H. Scofield, Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV, J. of Electron Spectrosc. Relat. Phenom. 8 (1976) 129-137. [24] S. Bera, P. Gangopadhyay, K.G.M. Nair, B.K. Panigrahi and S.V. Narasimhan, Electron spectroscopic analysis of silver nanoparticles in a soda-glass matrix, J. Electron. Spectrosc. and related phenomena 152 (2006) 91-95. [25] G. Schon, ESCA Studies of Ag, Ag2O and AgO, Acta Chem. Scand. 27 (1973) 26232633. [26] M.R. Alexander, R.D. Short, F.R. Jones, W. Michaeli and C.J. Blomfield, A study of HMDSO/O2 plasma deposits using a high-sensitivity and -energy resolution XPS 21

ACCEPTED MANUSCRIPT instrument: curve fitting of the Si 2p core level, Appl. Surf. Sci. 137 (1999) 179-183. [27] Y.F. Dufrêne, T.G. Marchal and P.G. Rouxhet, Probing the organization of adsorbed protein layers: complementarity of atomic force microscopy, X-ray photoelectron spectroscopy and radiolabeling, Appl. Surf. Sci. 144-145 (1999) 638-643.

IP

T

[28] C.-M. Pradier, D. Costa, C. Rubio, C. Compère and P. Marcus, Role of salts on BSA adsorption on stainless steel in aqueous solutions. I. FT-IRRAS and XPS characterization, Surf. Interface Anal. 34 (2002) 50-54.

SC R

[29] J.-B. Lhoest, E. Detrait, P. van den Bosch de Aguilar, P. Bertrand, Fibronectin adsorption, conformation, and orientation on polystyrene substrates studied by radiolabeling, XPS, and ToF SIMS, J. Biomed.Mater. Res. 41 (1998) 95-103.

NU

[30] J. Vörös, The Density and Refractive Index of Adsorbing Protein Layers, Biophys. J. 87 (2004) 553-561.

MA

[31] D.C. Carter and J.X. Ho, Structure of Serum Albumin, Adv. Protein Chem., 45 (1994) 153. [32] C.E. Giacomelli, M.J. Avena, C.P. De Pauli, Adsorption of Bovine Serum Albumin onto TiO2 Particles, Colloid Interface Sci. 188 issue 2 (1997) 387-395.

TE

D

[33] J. Kim, G.A. Somoejai, Molecular packing of lysozyme, fibrinogen and bovine serum albumin on hydrophilic and hydrophobic surfaces studied by infrared-visible sum frequency generation and fluorescence microscopy, J. Am. Chem. Soc. 125 (10) (2003) 3150-3158.

CE P

[34] C.M. Pradier, F. Karman, J. Telegdi, E. Kalman, P. Marcus, Adsorption of Bovine Serum Albumin on chromium and molybdenum surfaces investigated by Fouriertransform infrared reflection-adsorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy, J. Phys. Chem. B 107(28) (2003) 6766-6773.

AC

[35] R. Ishiguro, Y. Yokoyama, H. Maeda,A. Shimamura, K. Kameyama, K. Hiramatsu, Modes of conformational changes of proteins adsorbed on a planar hydrophobic polymer surface reflecting their adsorption behaviors, J. Colloid Interface Sci. 290 issue 1 (2005) 91-101. [36]

R. Tantipolphan, T. Rades, A.J. McQuillan, N.J. Medlicott, Adsorption of bovine serum albumin (BSA) onto lecithin studied by attenuated total reflectance Fourier transform infrared (ATR_FTIR), Int. J. Pharm. ), 337 Issue 1-2 (2007) 40-47.

[37]

Y.L. Jeyachandran, E. Mielczarski, B. Rai, J.A. Mielczarski, Quantitative and qualitative evaluation of adsorption/desorption of bovine serum albumin on hydrophilic and hydrophobic surfaces, Langmuir 25(19) (2009) 11614-11620.

[38]

S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5A resolution, Protein Eng. 12 (6) (1999) 439-46.

[39]

S.Y. Liau, D.C. Read, W.J. Pugh, J.R. Furr and A.D. Russell, Interaction of silver nitrate with readily identifiable groups: relationship to the antibacterialaction of silver ions, Lett. Appl. Microbiol. 25 (1997) 279-283.

22

ACCEPTED MANUSCRIPT L. Lensun, T. A. Smith, M. L. Gee, Partial denaturation of silica-adsorbed bovine serum albumin determined by time-resolved evanescent wave-induced fluorescence spectroscopy, Langmuir 18 (25) (2002) 9924–9931.

[41]

H. Zeng, K.K. Chittur, W.R. Lacefield, Analysis of bovine serum albumin adsorption on calcium phosphate and titanium surfaces, Biomaterials 20 (4) (1999) 377-84.

[42]

W. Norde, C.E. Giacomelli, BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states, J. Biotechnol. 79 (2000) 259-268.

[43]

M. Lundin, Y. Hedberg, T. Jiang,G. Herting, X. Wang, E. Thormann, E. Blomberg, I.O. Wallinder, Adsorption and protein-induced metal release from chromium metal and stainless steel, J Colloid Interface Sci. 366 (1) (2012) 155-64.

[44]

Y. Hedberg, X. Wang, J. Hedberg, M. Lundin, E. Blomberg, I.O. Wallinder, Surfaceprotein interactions on different stainless steel grades: effects of protein adsorption, surface changes and metal release, J. Mater. Sci. Mater. Med. 24(4) (2013) 1015-33.

[45]

X. Wang, G. Herting, I. Odnevall Wallinder, E. Blomberg, Adsorption of Lysozyme on silver and its influence on silver release, Langmuir 30(46) (2014) 13877-13889.

AC

CE P

TE

D

MA

NU

SC R

IP

T

[40]

23

ACCEPTED MANUSCRIPT Figure captions

T

Figure 1: BSA adsorption on the silver nanocomposite film (red), the polymeric matrix

IP

(blue), and massive Ag (black) deposited on quartz crystals, followed by QCM. (a) Raw data,

SC R

and (b) data corrected for Ag release/dissolution in the case of the silver nanocomposite film. 0.15 M NaCl without (Steps I and III) and with 20 mg.L-1 of BSA (Step II). For the sake of

NU

clarity, all signals were graphically offset to zero at BSA introduction.

MA

Figure 2: XPS spectra recorded for the silver nanocomposite film. (a) Survey spectrum, (b) Ag 3d, (c) Ag Auger peaks, (d) Si 2p and Ag 4s, (e) N 1s, and (f) C 1s core level spectra

D

before (native surface) and after BSA adsorption followed by QCM. Solid lines: experimental

CE P

TE

spectra; dashed lines: peak decomposition.

Figure 3: XPS spectra recorded for the polymeric matrix (a,c,e) and massive silver (b,d,f). (a) Si 2p, (b) Ag 3d, (c,d) N 1s, and (e,f) C 1s core level spectra before (native surface) and after

AC

BSA adsorption followed by QCM. Solid lines: experimental spectra; dashed lines: peak decomposition.

Figure 4: Scheme of the mechanisms proposed for silver release/dissolution in the case of the nanocomposite film covered with a discontinuous layer of adsorbed BSA (“island” model).

24

ACCEPTED MANUSCRIPT 0 10 0

-10

-30

-20 -30

-40 Step I

-60 -120 -90 -60 -30

Step II

Step III

SC R

-40 -50

-50

0

30 60 90 120 150

NU

t (min)

0

Step II

30

Step III

60

90

120

150

t (min)

(b)

MA

(a)

T

-20

IP

fBSA (Hz)

f (Hz)

-10

AC

CE P

TE

D

Figure 1

25

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Figure 2

26

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Figure 3 27

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Figure 4

28

ACCEPTED MANUSCRIPT Tables Table I: Protein island model: surface coverage () and height (h) of the protein islands,

T

calculated from combined QCM and XPS data, for the silver nanocomposite film, the

SC R

IP

polymeric matrix and massive silver.

0.57

h (nm)

8

0.16

0.53

16

9

TE

D

MA



NU

Silver nanocomposite film Polymeric matrix Massive Ag

CE P

Table II : Three steps monitoring used for QCM experiments

Step II

Step III

(stabilization

BSA solution

0.15M NaCl

duration)

(20 mg.L-1)

(BSA free solution)

120 min

60 min

100 min

Polymeric matrix

120 min

60 min

60 min

Bulk silver

45 min

60 min

60 min

AC

Step I

0.15M NaCl (BSA free solution)

Silver nanocomposite film

29

ACCEPTED MANUSCRIPT Highlights ● BSA adsorption on silver nanocomposite films is a fast and irreversible process. ● BSA interacts more with silver at the nanocomposite film surface than at the polymeric matrix surface.

T

● BSA adsorption controls silver release

AC

CE P

TE

D

MA

NU

SC R

IP

● Mechanisms of adsorption of protein and release of silver from nanoparticles are presented

30