- Email: [email protected]
Effects of silver segregation on sputter deposited antibacterial silver-containing diamond-like carbon films
Accepted Manuscript Effects of silver segregation on sputter deposited antibacterial silver-containing diamond-like carbon films
L.J. Wang, F. Zhang, A. Fong, K.M. Lai, P.W. Shum, Z.F. Zhou, Z.F. Gao, T. Fu PII: DOI: Reference:
S0040-6090(18)30102-0 https://doi.org/10.1016/j.tsf.2018.02.015 TSF 36477
To appear in:
Thin Solid Films
Received date: Revised date: Accepted date:
8 August 2017 7 February 2018 8 February 2018
Please cite this article as: L.J. Wang, F. Zhang, A. Fong, K.M. Lai, P.W. Shum, Z.F. Zhou, Z.F. Gao, T. Fu , Effects of silver segregation on sputter deposited antibacterial silver-containing diamond-like carbon films. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tsf(2017), https://doi.org/10.1016/j.tsf.2018.02.015
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 Effects of silver segregation on sputter deposited antibacterial silver-containing diamond-like carbon films
L.J. Wang1, F. Zhang1, A. Fong2, K.M. Lai2, P.W. Shum2, Z.F. Zhou3, Z.F. Gao2, T. Fu1, 1. Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China
PT
2. Asahi Group Co. Ltd, Kwun Tong, Hong Kong, China
3. Department of Mechanical and Biomedical Engineering, City University of Hong Kong,
RI
Kowloon, Hong Kong, China
SC
Abstract: Silver-containing diamond-like carbon (DLC) films for antibacterial applications were prepared on 316L stainless steel and slide glass substrates by
NU
magnetron sputtering at different silver target current. Scanning electron microscopy and atomic force microscopy analyses show that the film surface becomes rough due
MA
to the formation of nanoparticles/clusters and even a surface layer with the increase of silver target current. The Ag content was measured as 0, 2.1, 6.1 and 14.3 at.%,
D
respectively by energy dispersive X-ray analysis. X-ray diffraction and X-ray
PT E
photoelectron spectroscopy reveal the dominant metallic nature of silver for the Ag-containing films. Silver segregation from the carbon matrix to film surface is confirmed by the above analyses. Microhardness indentation, contact angle
CE
measurement and potentiodynamic polarization test in a Ca-free Hank’s balanced salt solution indicate that the addition of silver has decreased hardness, elastic modulus,
AC
H/E and H3/E2 ratios, wettability, surface energy and corrosion resistance of the DLC films, respectively. The antibacterial tests by agar disk diffusion assay and agar plate counting method against Escherichia coli demonstrate long-lasting and reusable antibacterial activity of the Ag-containing films. The study shows that silver segregation plays a crucial role in microstructure, mechanical, chemical and biological properties of the antibacterial Ag-containing DLC films.
*
Corresponding author. E-mail address: [email protected] (T. Fu) 1
ACCEPTED MANUSCRIPT Keywords: Diamond-like carbon; thin films; Silver; Sputtering; Corrosion; Antibacterial 1. Introduction Vapor deposited diamond-like carbon (DLC) films have been widely applied in protective, biocompatible and other functional surface treatments due to their
PT
excellent mechanical properties (high hardness, low coefficient of friction, high wear
RI
resistance, etc), surface smoothness, good chemical inertness and biocompatibility. The doping of different metallic elements (Cr [1,2], Ti [1,3], W [4,5], Mo [5], the
SC
non-carbide-forming elements of Ag [6,7], Cu [8], Al [9], Ni [10], etc) can reduce residual stress [2,4,6-9], improve electric conductivity [3,11] and enable new
NU
functions of the DLC films.
Currently one of the greatest threats in the clinical practice is the difficulties in
MA
the treatment of implant-related infections due to the bacterial drug resistance [12]. The hospital-acquired infections have been a major public health concern in hospitals
D
and healthcare units worldwide, and it is of great importance to reduce the spread of
PT E
unwanted bacteria in clinical applications. Silver has been known as an effective antimicrobial material since the ancient times, with the broad spectrum against multiple drug-resistant bacteria as well as the limited toxicity towards mammalian
CE
cells [13-15]. Silver-containing DLC (Ag-DLC) and other silver doped coatings
AC
prepared by magnetron sputtering [11], pulsed laser deposition [13], ion deposition [16], plasma enhanced chemical vapor deposition [6], etc have been widely studied for antibacterial surface modification of biomedical implants and environmental surfaces [12,16-18]. For vapor deposited Ag-DLC films, silver usually segregates from carbon matrix and results in composite structures during the relatively energetic film deposition [16,19] or the ageing process after deposition [11,12, 20,21] due to its low solubility with carbon and its inability to form carbides. Silver segregation can increase silver content at the film surface, but its influence on mechanical, chemical and biological 2
ACCEPTED MANUSCRIPT properties of the Ag-DLC films have rarely been studied. In this study DLC films with different silver content are prepared by magnetron sputter method. Austenitic 316L stainless steel is selected as the substrate material for its wide applications in biomedical and environmental fields, and slide glass is also coated for analysis. Silver segregation is evidenced by structural and compositional analyses, and its effects on
PT
hardness, wettability, corrosion resistance and antibacterial property of the Ag-DLC
RI
coated samples are investigated.
SC
2. Experimental 2.1. Sample preparation
NU
The Ag-DLC films were deposited on polished 316L stainless steel disks (101 mm2) and glass slides (size 75251 mm3) by an industrial closed field unbalanced
MA
magnetron sputtering system (UDP650, Teer Coatings Ltd). The substrates were degreased, ultrasonically cleaned, and subsequently blown dry in flowing nitrogen gas. Surface roughness of the stainless steel disks was Ra= 17.7 nm measured by a stylus
D
profilometer (Ambios XP-2) with the test length of 800 m. The computer-controlled
PT E
deposition system comprised of six rectangular cathodes (two chromium and three graphite targets, as well as one silver target). The size of the targets was 345×145×8
CE
mm3, with a purity of >99.9 %. The six magnetrons were driven by DC power suppliers in current mode. The cleaned substrates were mounted on the substrate
AC
holder that was rotated at a speed of 10 rpm, and the target-to-substrate distance was 17 cm. Prior to deposition the vacuum chamber was evacuated to a background pressure of 410-4 Pa. Pure Ar working gas (99.999% purity) was introduced into the chamber via a mass flow controller at constant flow rate (30 sccm), corresponding to a working gas pressure of 0.18 Pa. For plasma ion etching, the substrates were biased with pulse DC at a frequency of 250 kHz and a voltage of -450 V. In film deposition a thin Cr-C interlayer of ~0.4 µm thickness was prepared, and then the Ag-DLC top layers were produced by co-sputtering the graphite targets (currents fixed at 3.5 A) and the silver target for 30 min. The Cr-C interlayer was deposited to improve
3
ACCEPTED MANUSCRIPT adhesion and accommodate stresses of the top DLC film. The Ag target current was selected as 0, 0.2, 0.4 and 0.6 A, respectively to vary silver content in the films. During the deposition stages the bias voltage was reduced to -60 V. The substrates were heated due to ion bombardment, but the temperature was estimated to be lower than 200C. The coated steel/glass samples were noted as M00/G00, M02/G02,
PT
M04/G04 and M06/G06, respectively, in which the numbers indicated the silver target
RI
current.
2.2. Material characterization and tests
SC
Surface morphology of the samples aged in ambient conditions for different time
NU
(1, 4 and 10 months) was observed by scanning electron microscopy (SEM, FEI Quanta 600F) and atomic force microscopy (AFM, Veeco diInnova, tapping mode,
MA
scanning area 55 m2). The scanning electron microscope was equipped with energy dispersive X-ray analysis (EDX, operating voltage 15 kV). Phase structure of the samples was analyzed by X-ray diffraction (XRD, X’Pert PRO, -2 configuration,
D
CuK source, 40 kV and 40 mA). Elemental composition and chemical bonding state
PT E
of the samples were examined by X-ray photoelectron spectroscopy (XPS, VG K-Alpha, AlK source, 12 kV and 6 mA). XPS spectra of the surface layer were
CE
acquired with an elliptical detection area (long axis ~400 m, short axis ~300 m) at a detection angle of 45 without and with 30 s sputter etching. For the sputter etching
AC
to remove surface contaminants, the angle, current and energy of the Ar+ ion gun was 30, 10 mA and 2 keV, respectively. Wettability of the samples was assessed by measuring water and glycol contact angles with a contact angle goniometer under ambient conditions. Surface free energy of the samples was determined from the above contact angles with the method described in [20]. The dispersive ( Sd ) and polar ( Sp ) components of surface energy of a material can be calculated with the following equation [22,23]:
L (1 cos ) 2( Sd rLd )1 / 2 2( Sp rLp )1 / 2
(1)
4
ACCEPTED MANUSCRIPT where L is the liquid surface energy containing dispersive ( Ld ) and polar ( Lp ) components, and is the contact angle. Based on the well known polar and dispersive components of surface energy of water and glycol [22], the polar and dispersive components of surface energy of the samples were calculated from the above measured contact angles using a Matlab program written according to Eq. 1.
PT
Hardness of the films was measured with a microhardness tester (Fischerscope H100C, Vicker’s diamond pyramid indenter, test load 20 mN). Corrosion resistance of
RI
the coated stainless steel samples was evaluated by potentiodynamic polarization test
SC
with an electrochemical workstation (Corrtest CS150) under ambient conditions. The electrolyte was a Ca-free Hank’s balanced salt solution (HBSS, NaCl 8.00 g/L, KCl
NU
0.40 g/L, NaHCO3 0.34 g/L, KH2PO4 0.06 g/L, Na2HPO412H2O 0.12 g/L), and the counter and reference electrodes were platinum electrode and saturated calomel
MA
electrode (SCE), respectively. In the test the potential sweep rate was 0.50 mV/s and
PT E
2.3. Antibacterial test
D
the sampling rate was 2.0 Hz.
Antibacterial property of the samples was assessed by the qualitative evaluation using agar disk diffusion assay and the quantitative evaluation using agar plate
CE
counting method against Escherichia coli. As a typical kind of Gram-negative bacteria, E. coli has been found in biomaterial-related infection sites and is one of the main
AC
sources responsible for hospital-acquired infections. For agar disk diffusion assay, 20 ml of viscous Luria-Bertani agar was poured into sterile Petri dishes, and 1.5 ml of bacteria culture fluid (106 colony forming units, CFU/ml) was mixed with the agar. After the agar was cooled down, the sterilized samples were placed face down on the inoculated agar plates, with effective contact between them. The seeded agar plates were cultured aerobically at 37C in an incubator for 24 h. The photos of the samples were taken by a digital camera. For agar plate counting method, 10 l of E. coli suspension (105 CFU/ml) was
5
ACCEPTED MANUSCRIPT dropped onto the sample surface, and then covered quickly with a filter membrane. After placed in dark at room temperature for 3 h, the samples were rinsed with 3 ml of sterilized water to collect the survival bacteria. After stirring, 700 l of the suspension was mixed with viscous Luria-Bertani agar. The inoculum was spread and incubated aerobically at 37C for 24 h. The photos of colonies were taken by a digital camera. In
PT
order to evaluate the long-lasting and reusable antibacterial property, the tested samples were cleaned, soaked in deionized water for two weeks, tested and cleaned
SC
RI
for two cycles, and tested again.
3. Results and discussion
NU
3.1. Morphological, compositional and structural analyses SEM surface images and optical photos of the samples aged for different time
MA
are shown in Fig. 1a-g. The films are black, dense and uniform for samples M00-M04, but the film is gray for sample M06. Nanoparticles with the size of about 80-100 nm are observed by SEM for sample M00, which reflect fine columnar structure of the
D
film. The total thickness of the Cr-C interlayer is about 0.38 m, and the top carbon
PT E
layer is about 0.37 µm thick (Fig. 1h). Similar nanoparticles with a few white tiny dots are observed for sample M02. There are many white particles and clusters
CE
(50-100 nm) at the surface of sample M04, which have higher Ag content than the whole film revealed by EDX analysis. For sample M06, the nanoparticles are about
AC
60-180 nm in size at 1 month, a loose surface layer composed of big particles and agglomerations (120-400 nm) is formed at 4 months, and the film becomes compact at 10 months of aging. Correspondingly, the surface roughness Ra of sample G06 measured by AFM (test area 55 m2) increases from ~8 nm at 1 month to ~20 nm at 4 and 10 months of ageing. The Ag-DLC layer is about 0.47 µm thick at the cross-section (Fig. 1f). Elements of C, Cr and Ag are detected at the surface by areal EDX analysis (test area around 128 m2), and the Ag content increases with silver target current, being 0, 2.1, 6.1 and 14.3 at.% for G00-G06, respectively (Table 1). The measurement of Ag content is influenced by the gradient structure and different 6
ACCEPTED MANUSCRIPT thickness of the films. The EDX analysis and the following tests were carried out on the samples with an intermediate aging time (4 months). In XRD patterns (Fig. 2) only steel substrate peaks are present for samples M00 and M02. Weak silver diffraction peaks are observed for sample M04, and the silver peaks are strong for sample M06. The silver grain size of sample M06 is calculated to
PT
be 28 nm by the Ag(111) peak located at 2=38.13 according to the Debye-Scherrer equation. The XRD and the above EDX analyses may suggest that the white
RI
nanoparticles/clusters in Fig. 1b,c and the rough surface layer in Fig. 1f are composed
SC
of metallic silver.
Surface elemental composition of sample G06 was further examined by XPS
NU
analysis, and elements of Ag, C and O are detected by survey scan. The Ag 3d5/2 peak is positioned at 368.2 eV and it moves to slightly higher binding energy after 30 s
MA
sputtering (Fig. 3a). The Ag 3d5/2 peak for metallic silver is located at higher binding energy (368.4 eV) than those for its oxides (367.8 eV for Ag2O, 367.4 eV for AgO) [24]. This indicates that silver is present mainly in metallic state, agreeing with the
D
above XRD analysis, but silver is slightly oxidized at the topmost surface when
PT E
exposed to ambient air. In O 1s spectra the peak at 530.4 eV ascribed to silver oxide is nearly removed by 30 s sputtering (Fig. 3b). The silver oxide top surface layer was
CE
revealed by continuous sputtering etching in other vapor deposited Ag-DLC films [12,19,24]. The nonpassive silver oxide thin film is essential for the release of
AC
antimicrobial Ag+ ions [25,26]. The atomic ratio of Ag/C is 260% after 30 s sputter cleaning, much higher than that measured by areal EDX (20%) for sample G06, which indicates the enrichment of silver at the film surface. Due to its low solubility with carbon and its inability to form carbides, silver can segregate from carbon matrix and result in composite structures during the relatively energetic film deposition [19] and the ageing process after deposition [11,12,20]. The post-deposition segregation of silver nanostructures is especially obvious at high silver contents (samples M04 and M06 in Fig. 1, 20 at.%
7
ACCEPTED MANUSCRIPT Ag in Ref. [21]). The segregation induced silver-rich surface layer will influence mechanical, chemical and biological properties of the Ag-DLC films.
3.2. Hardness, wettability and corrosion resistance The addition of silver has decreased microhardness and elastic modulus of the
PT
DLC films, from 13.3 GPa and 165 GPa for sample M00 to 6.1 GPa and 124 GPa for sample M06 (Table 1). This is consistent with the report that the surface hardness and
RI
electrical resistivity of DLC film decreased with the increase of Ag content [11,20,27].
SC
The hardness reduction is attributed to the incorporation of soft/compliant silver phase and the subsequent graphitization of amorphous carbon matrix [28]. However,
NU
hardness of the Ag-DLC films is still higher than that of stainless steel substrate (~2.5 GPa). The H/E and H3/E2 ratios are two factors to assess wear-resistant properties and
MA
plastic deformation resistance of hard coatings, respectively [29,30], and they are much higher for samples M00-M04 (0.081-0.083, 0.077-0.086 GPa) than those for sample M06 (0.049, 0.015 GPa). This indicates better mechanical properties of the
D
DLC films of samples M00-M04 compared with those of sample M06, which has the
PT E
highest silver content (14.3 at.%).
Water contact angle increases continuously from 81.6 for sample G00 to 111.9
CE
for sample G06 (Table 2). Usually, a hydrophobic surface has a contact angle higher than 70°, while a hydrophilic surface has a contact angle lower than 70°. Thus, the
AC
addition of silver increases hydrophobicity of the DLC film. The increased hydrophobicity of Ag-DLC film can be attributed to the chemical structure change of DLC film [11,23], the hydrophobic nature of silver nanoparticles/clusters and the increased surface roughness. According to Wenzel’s model describing the contact angle at a rough surface, the surface roughness enhances hydrophilicity of hydrophilic surfaces and enhances hydrophobicity of hydrophobic surfaces [31,32]. The glycol contact angle also increases from 59.7 for sample G00 to 91.9 for sample G06 (Table 2). As the result, the surface energy decreases from 27.4 mJ/m2 for sample G00 to 14.2 mJ/m2 for sample G06, with polar component being the minor. 8
ACCEPTED MANUSCRIPT Thus, the addition of silver has decreased wettability and surface energy of the DLC films. The hydrophobic surface with low surface energy is beneficial for the biomedical applications of anti-fouling, antimicrobial and blood contacting materials [11,16,20,23]. Moreover, the fibroblast cell test with titanium and titanium alloys indicated that a low polar component (or low fractional polarity) was the major
PT
parameter for good cell proliferation [33]. Corrosion resistance of the Ag-DLC coated stainless steel samples was evaluated
RI
by potentiodynamic polarization test (Fig. 4). Samples M00 and M02 exhibit similar
SC
polarization plots, with a long passivation zone followed by pitting at the potentials above 0.76 V (Table 3), which are much higher than that of the polished stainless
NU
steel sample (~0.4 V) . The pitting potential of sample M04 is still high (0.67 V), but there are two current peaks within the passivation zone. The first current peak at 0.08
MA
V is related to dissolution of metallic silver particles at the film surface [34,35]. Similar current peaks at 0.08-0.12 V were found in the polarization plots of sputter prepared DLC films containing 2.97-8.37 at.% silver that were tested in 3.5% NaCl
D
solution [36]. The second current peak is likely relevant to corrosion of the Cr-C
PT E
interlayer and the steel substrate. SEM observation of the tested sample shows that there is a corrosion pit (diameter ~200 m) at the sample surface, and the steel
CE
substrate at the bottom of the pit is seriously corroded (Fig. 5). It is postulated that more structural defects are generated in the film due to the increased silver content
AC
(e.g. voids, pores formed by silver segregation), and the defects increase the pitting probability of the coated steel sample. It was documented that the free corrosion potential of titanium alloys in vivo lies in the range of 450-550 mV SHE (or 206-306 mV SCE [37]). The coated stainless steel samples M00-M04 were stable up to 0.4 V according to the polarization test; however, their corrosion behavior in vivo still needs a further study. For sample M06, pitting occurs at only 0.05 V, which is caused by significant dissolution of the silver surface layer of the sample. Thus, sample M06 is especially unstable in the simulated body fluid. With the increase of silver content in
9
ACCEPTED MANUSCRIPT the DLC film, the corrosion resistance (Rcorr) is gradually decreased, and the corrosion current density (Icorr) and passive current density (Ipass) are slowly increased (Table 3).
3.3. Antibacterial test Antibacterial property of the coated samples was first qualitatively tested by agar
PT
disk diffusion assay (Fig. 6). There is no inhibition zone for samples G00 and G02. The inhibition zone is obvious for sample G04 and it is wide (~5.4 mm) for sample
RI
G06. This reveals the silver dependent antibacterial activity of the Ag-DLC films. Due
SC
to the oxidation of metallic silver by oxygen, the Ag-containing films can release silver ions of different concentration to the surrounding agar plate and kill bacteria
NU
there. Two mechanisms have been proposed for bactericidal property of silver ions. First, silver ions can bind at the negatively charged bacterial cell membrane or enter
MA
the cells, and then impair the structure and functions of bacterial cells. Second, silver ions can catalyze molecule oxygen in water and produce ROS, and ROS will induce bacteria death [38]. Since silver of high contents is harmful to adhesion and
D
biocompatibility of a material [12], the antibacterial property should be guaranteed at
PT E
the lowest silver content in a specific application. The antibacterial property of the samples was then quantitatively evaluated by
CE
agar plate counting method (Fig. 7). The un-doped sample G00 favors bacteria growth when tested for 3 h. The bacterial colonies are markedly reduced for sample G02, and
AC
no colonies are observed for samples G04 and G06. Therefore, DLC films with 2.1 at.% or more silver can release enough Ag+ ions to kill E. Coli, resulting in an antibacterial rate of >95%. The atomic absorption spectroscopy analysis demonstrated that DLC coating with 2.4 at.% Ag had displayed slow release kinetics, with a total silver ion release in the sub-ppb range after 4 h in solution [24]. The limited diffusion of biocidal silver ions with a slow and continuous release is mandatory to ensure a lasting and localized antibacterial effect of the Ag-DLC films. Furthermore, the tested films were cleaned with 75% ethanol, soaked in deionized water for two weeks and tested and cleaned for two cycles, and tested again. Similar results were obtained for 10
ACCEPTED MANUSCRIPT samples G02-G06 in the last test (Fig. 7f-h), which demonstrates their long-lasting and reusable antibacterial property. The above results and the previous studies show that the addition of silver in DLC films can enhance antibacterial property, but decrease hardness, H/E and H3/E2 ratios, wettability, corrosion resistance and biocompatibility of the coated samples
PT
[20]. Proper content of silver should be chosen for the balance of mechanical, chemical and biological properties of the coated samples in a specific situation. For
RI
example, the optimal combination with reasonable physical-chemical properties,
SC
efficient protection against microbial colonization and beneficial effects on human MG63 cells was found for carbon films containing 2-7 at.% silver [13]. In another
NU
case, increasing silver content of Ag-DLC films to above 2 at.% improved bactericidal properties against E. coli, but decreased the viability of osteoblast cells [12].
MA
Apart from the silver content, the distribution [19,39] and chemical state of sliver [25] can also influence silver ion release and antibacterial activity of Ag-DLC and Ag-O films. Obvious silver segregation at the film surface has been observed in
D
Ag-DLC films prepared by different deposition methods [11-13,19,28,39]. Silver
PT E
content at the film surface will be much increased due to silver segregation from the carbon matrix. Since bacteria only interact with materials at the surface, Ag-DLC
CE
films with even lower average silver content would have the enhanced antibacterial activity due to silver segregation (e.g. antibacterial rate >95% for G02 with 2.1 at.%
AC
Ag) in comparison with the films without obvious silver segregation (e.g. a high antibacterial rate for DLC film with 9.3 at.% Ag [40]). The average silver content in a specific application can be lowered while keeping the high antibacterial activity due to silver segregation for Ag-DLC films.
4. Conclusions Ag-DLC films with the silver contents of 0, 2.1, 6.1 and 14.3 at.% were prepared by magnetron sputter method. The surface roughness increases with silver content due to silver segregation at the film surface from the carbon matrix. The addition of silver 11
ACCEPTED MANUSCRIPT decreases hardness, elastic modulus, H/E and H3/E2 ratios, wettability, surface energy and corrosion resistance of the DLC films. The antibacterial property of the silver-containing DLC films is long-lasting and reusable, and the antibacterial activity is high even at 2.1 at.% Ag due to silver segregation at the film surface. The effects of silver segregation on structure and properties of Ag-DLC films have to be considered
PT
for their antibacterial applications.
RI
Acknowledgements
SC
This work was supported by Asahi Group Co. Ltd, the Fundamental Research Fund for the Central Universities (xjj2015072, xjj2017163) and the Science and
NU
Technology Program of Chongqing Municipal Education Commission (No.
MA
KJ1600926).
References
[1] M. Jelinek, J. Zemek, J. Remsa, J. Miksovsky, T. Kocourek, P. Pisarik, M. Travnickova, E.
PT E
Science 417 (2017) 73-83.
D
Filova, L. Bacakova, Hybrid laser technology and doped biomaterials, Applied Surface
[2] M.Y. Ming, X. Jiang, D.G. Piliptsou, Y. Zhuang, A.V. Rogachev, A.S. Rudenkov, A.
CE
Balmakou, Chromium-modified a-C films with advanced structural, mechanical and corrosive-resistant characteristics, Applied Surface Science 379 (2016) 424-432.
AC
[3] N.R. Lee, Y.S. Jun, K.I. Moon, C.S. Lee, Ti-doped hydrogenated diamond like carbon coating deposited by hybrid physical vapor deposition and plasma enhanced chemical vapor deposition, Japanese Journal of Applied Physics 56 (2017) 035506. [4] X.M. Wang, W.D. Wu, S.Y. Li, S.L. Chen, Y.J. Tang, L. Bai, H.P. Wang, Properties of W doped diamond-like carbon films prepared by pulsed laser deposition, Rare Metal Materials and Engineering 39 (2010) 1251-1255. [5] I.C. Muller, J. Sharp, W.M. Rainforth, P. Hovsepian, A. Ehiasarian, Tribological response and characterization of Mo-W doped DLC coating, Wear, 376 (2017) 1622-1629. [6] F.R. Marciano, L.F. Bonetti, L.V. Santos, N.S. Da-Silva, E.J. Corat, V.J. Trava-Airoldi,
12
ACCEPTED MANUSCRIPT Antibacterial activity of DLC and Ag-DLC films produced by PECVD technique, Diamond and Related Materials 18 (2009) 1010-1014. [7] C. Wang, X. Yu, M. Hua, Microstructure and mechanical properties of Ag-containing diamond-like carbon films in mid-frequency dual-magnetron sputtering, Applied Surface Science 256 (2009) 1431-1435.
PT
[8] L. Sun, P. Guo, P. Ke, X. Li, A. Wang, Synergistic effect of Cu/Cr co-doping on the wettability and mechanical properties of diamond-like carbon films, Diamond and Related
RI
Materials 68 (2016) 1-9.
[9] X. Pang, L. Shi, P. Wang, Y. Xia, W. Liu, Effects of Al incorporation on the mechanical and
SC
tribological properties of Ti-doped a-C:H films deposited by magnetron sputtering, Current Applied Physics 11 (2011) 771-775.
NU
[10] N.W. Khun, E. Liu, G.C. Yang, Structure, scratch resistance and corrosion performance of nickel doped diamond-like carbon thin films, Surface & Coatings Technology, 204 (2010)
MA
3125-3130.
[11] W.-C. Lan, S.-F. Ou, M.-H. Lin, K.-L. Ou, M.-Y. Tsai, Development of silver-containing
D
diamond-like carbon for biomedical applications. Part I: Microstructure characteristics,
4099-4104.
PT E
mechanical properties and antibacterial mechanisms, Ceramics International 39 (2013)
[12] D. Bociaga, W. Jakubowski, P. Komorowski, A. Sobczyk-Guzenda, A. Jedrzejczak, D. Batory,
CE
A. Olejnik, Surface characterization and biological evaluation of silver-incorporated DLC coatings fabricated by hybrid RF PACVD/MS method, Materials Science & Engineering C
AC
63 (2016) 462-474.
[13] I.N. Mihailescu, D. Bociaga, G. Socol, G.E. Stan, M.-C. Chifiriuc, C. Bleotu, M.A. Husanu, G. Popescu-Pelin, L. Duta, C.R. Luculescu, I. Negut, C. Hapenciuc, C. Besleaga, I. Zgura, F. Miculescu, Fabrication of antimicrobial silver-doped carbon structures by combinatorial pulsed laser deposition, International Journal of Pharmaceutics 515 (2016) 592-606. [14] M.G. Raucci, K. Adesanya, L. Di Silvio, M. Catauro, L. Ambrosio, The biocompatibility of silver-containing Na2OCaO2SiO2 glass prepared by sol-gel method: in vitro studies, Journal of Biomedical Materials Research Part B 92B (2010) 102-110. [15] M. Bellantone, H.D. Williams, L.L. Hench, Broad-spectrum bactericidal activity of 13
ACCEPTED MANUSCRIPT Ag2O-doped bioactive glass, Antimicrobial Agents and Chemotherapy 46 (2002) 1940-1945 [16] K. Baba, R. Hatada, S. Flege, W. Ensinger, Y. Shibata, J. Nakashima, T. Sawase, T. Morimura, Preparation and antibacterial properties of Ag-containing diamond-like carbon films prepared by a combination of magnetron sputtering and plasma source ion implantation, Vacuum 89 (2013) 179-184.
PT
[17] J.L. Endrino, A. Anders, J.M. Albella, J.A. Horton, T.H. Horton, P.R. Ayyalasomayajula, M. Allen, Antibacterial efficacy of advanced silver-amorphous carbon coatings deposited using
RI
the pulsed dual cathodic arc technique, Journal of Physics: Conference Series 252 (2010) 012012.
SC
[18] N. Harrasser, S. Jussen, A. Obermeir, R. Kmeth, B. Stritzker, H. Gollwitzer, R. Burgkart, Antibacterial potency of different deposition methods of silver and copper containing
NU
diamond-like carbon coated polyethylene, Biomaterials Research 20 (2016) 181-190. [19] M. Cloutier, S. Turgeon, Y. Busby, M. Tatoulian, J.J. Pireaux, D. Mantovani, Controlled
MA
distribution and clustering of silver in Ag-DLC nanocomposite coatings using a hybrid plasma approach, ACS Applied Materials & Interfaces 8 (2016) 21020-21027.
D
[20] L. Swiatek, A. Olejnik, J. Grabarczyk, A. Jedrzejczak, A. Sobczyk-Guzenda, M. Kaminska,
PT E
W. Jakubowski, W. Szymanski, D. Bociaga, Multi-doped diamond like-carbon coatings (DLC-Si/Ag) for biomedical applications fabricated using the modified chemical vapour deposition method, Diamond and Related Materials 67 (2016) 54-62.
CE
[21] N.K. Manninen, R.E. Galindo, S. Carvalho, A. Cavaleiro, Silver surface segregation in Ag-DLC nanocomposite coatings, Surface & Coatings Technology, 267 (2015) 90-97.
AC
[22] T. Zhao, Y. Li, Y. Gao, Y. Xiang, H. Chen, T. Zhang, Hemocompatibility investigation of the NiTi alloy implanted with tantalum, Journal of Materials Science-Materials in Medicine 22 (2011) 2311-2318. [23] H.W. Choi, R.H. Dauskardt, S.-C. Lee, K.-R. Lee, K.H. Oh, Characteristic of silver doped DLC films on surface properties and protein adsorption, Diamond and Related Materials 17 (2008) 252-257. [24] M. Cloutier, R. Tolouei, O. Lesage, L. Levesque, S. Turgeon, M. Tatoulian, D. Mantovani, On the long term antibacterial features of silver-doped diamondlike carbon coatings deposited via a hybrid plasma process, Biointerphases 9 (2014) 029013. 14
ACCEPTED MANUSCRIPT [25] R. Rebelo, S.V. Calderon, R. Fangueiro, M. Henriques, S. Carvalho, Influence of oxygen content on the antibacterial effect of Ag-O coatings deposited by magnetron sputtering, Surface & Coatings Technology 305 (2016) 1-10. [26] I. Ferreri, S.V. Calderon, R. Escobar Galindo, C. Palacio, M. Henriques, A.P. Piedade, S. Carvalho, Silver activation on thin films of Ag-ZrCN coatings for antimicrobial activity,
PT
Materials Science & Engineering C 55 (2015) 547-555. [27] N.K. Manninen, F. Ribeiro, A. Escudeiro, T. Polcar, S. Carvalho, A. Cavaleiro, Influence of
RI
Ag content on mechanical and tribological behavior of DLC coatings, Surface & Coatings Technology 232 (2013) 440-446.
SC
[28] M. Constantinou, M. Pervolaraki, P. Nikolaou, C. Prouskas, P. Patsalas, P. Kelires, J. Giapintzakis, G. Constantinides, Microstructure and nanomechanical properties of pulsed
Coatings Technology, 309 (2017) 320-330.
NU
excimer laser deposited DLC:Ag films: Enhanced nanotribological response, Surface &
MA
[29] N. Olah, Z. Fogarassy, A. Sulyok, J. Szivos, T. Csanadi, K. Balazsi, Ceramic TiC/a:C protective nanocomposite coatings: Structure and composition versus mechanical properties
D
and tribology, Ceramics International 42 (2016) 12215-12220.
PT E
[30] Q.Z. Wang, F. Zhou, M. Callisti, T. Polcar, J.Z. Kong, J.W. Yan, Study on the crack resistance of CrBN composite coatings via nano-indentation and scratch tests, Journal of Alloys and Compounds 708 (2017) 1103-1109.
CE
[31] R.N. Wenzel, Surface roughness and contact angle, Journal of Physical and Colloid Chemistry 53 (1949) 1466-1467.
AC
[32] M. Miyauchi, H. Tokudome, Super-hydrophilic and transparent thin films of TiO2 nanotube arrays by a hydrothermal reaction, Journal of Materials Chemistry, 17 (2007) 2095-2100. [33] L. Ponsonnet, K. Reybier, N. Jaffrezic, V. Comte, C. Lagneau, M. Lissac, C. Martelet, Relationship between surface properties (roughness, wettability) of titanium and titanium alloys and cell behaviour, Materials Science & Engineering C, 23 (2003) 551-560. [34] T. Fu, Y.G. Shen, Z. Alajmi, S.Y. Yang, J.M. Sun, H.M. Zhang, Sol-gel preparation and properties of Ag-TiO2 films on surface roughened Ti-6Al-4V alloy, Materials Science and Technology, 31 (2015) 501-505. [35] Z. Alajmi, T. Fu, Y.T. Zhao, S.Y. Yang, J.M. Sun. Ag-enhanced antibacterial property of MgO 15
ACCEPTED MANUSCRIPT film. Materials Science Forum, 859 (2016) 90-95. [36] V.S. Dhandapani, E. Thangavel, M. Arumugam, K.S. Shin, V. Veeraraghavan, S.Y. Yau, C. Kim, D.-E. Kim, Effect of Ag content on the microstructure, tribological and corrosion properties of amorphous carbon coatings on 316L SS, Surface & Coatings Technology, 240 (2014) 128-136.
PT
[37] M.H. Wong, F.T. Cheng, H.C. Man, Characteristics, apatite-forming ability and corrosion resistance of NiTi surface modified by AC anodization, Applied Surface Science, 253 (2007)
RI
7527-7534.
[38] K. Loza, J. Diendorf, C. Sengstock, L. Ruiz-Gonzalez, J.M. Gonzalez-Calbet, M. Vallet-Regi,
SC
M. Koller, M. Epple, The dissolution and biological effects of silver nanoparticles in biological media, Journal of Materials Chemistry B, 2 (2014) 1634-1643.
NU
[39] N.K. Manninen, S. Calderon, I. Carvalho, M. Henriques, A. Cavaleiro, S. Carvalho, Antibacterial Ag/a-C nanocomposite coatings: The influence of nano-galvanic a-C and Ag
MA
couples on Ag ionization rates, Applied Surface Science, 377 (2016) 283-291. [40] P. Pisarik, M. Jelinek, J. Remsa, J. Miksovsky, J. Zemek, K. Jurek, S. Kubinova, J. Lukes, J.
D
Sepitka, Antibacterial, mechanical and surface properties of Ag-DLC films prepared by dual
AC
CE
PT E
PLD for medical applications, Materials Science & Engineering C, 77 (2017) 955-962.
16
ACCEPTED MANUSCRIPT
Fig. 1. SEM secondary electron surface images and optical photos of the Ag-DLC coated steel samples aged for different time: (a) M00, 4 months, (b) M02, 4 months, (c) M04, 4 months, (d) M04, 10 months, (e) M06, 1 month, (f) M06, 4 months, (g) M06, 10 months. The back-scattered electron cross-sectional images of samples G00
Fig. 2. XRD patterns of the Ag-DLC coated steel samples.
PT
and G06 (4 months) with glass substrate are shown in (h) and (i), respectively.
RI
Fig. 3. XPS high-resolution Ag 3d and O 1s spectra of sample G06 (glass substrate) before and after 30 s sputtering.
SC
Fig. 4. Potentiodynamic polarization plots of the Ag-DLC coated steel samples. Fig. 5. SEM images of the corrosion pit of sample M04 with steel substrate after
NU
polarization test observed at (a) low and (b) high magnifications. Fig. 6. Antibacterial test results of the Ag-DLC coated glass samples by agar disk
MA
diffusion assay. The inhibition zones shown between the white and yellow dash lines are around 2.2 and 5.4 mm in width for samples G04 and G06, respectively.
D
Fig. 7. Typical antibacterial test results of the Ag-DLC coated glass samples G00-G06
PT E
(a-d) by agar plate counting method. The tested samples were cleaned by 75% ethanol, soaked in deionized water for two weeks and tested and cleaned for two cycles, and tested again to get the results shown sequentially in (e-h). The number of bacterial
CE
colonies is 1220, 48, 1152, 55 in (a), (b), (e), (f), respectively.
AC
Table 1 The Ag content ([Ag]), thickness (T), hardness (H), elastic modulus (E), H/E and H3/E2 ratios of the Ag-DLC coated glass samples. Samples
[Ag] (at.%)
T (m)
H (GPa)
E (GPa)
H/E
H3/E2 (GPa)
G00
0
0.75
13.3
165
0.081
0.086
G02
2.1
0.76
12.4
150
0.083
0.085
G04
6.1
0.78
11.8
146
0.081
0.077
G06
14.3
0.85
6.1
124
0.049
0.015
17
ACCEPTED MANUSCRIPT
Table 2 Contact angle and surface energy of the Ag-DLC coated glass samples. Surface energy (mJ/m2)
Water
Glycol
Dispersive
Polar
Sum
G00
81.60.8
59.75.2
18.0
9.4
27.4
G02
84.62.6
62.90.8
17.8
8.0
25.8
G04
100.21.3
82.54.4
12.4
3.6
G06
111.91.4
91.91.3
13.6
0.6
PT
Contact angle ()
16.0 14.2
SC
RI
Samples
Table 3 Corrosion data of the Ag-DLC coated steel samples. Ecorr (V SCE)
Icorr (A/cm2)
M00
-0.24
0.012
M02
-0.24
0.014
M04
-0.22
0.036
M06
-0.20
0.063
Rcorr (cm2)
Epit (V SCE)
Ipass (A/cm2)
9.65105
0.76
0.090(*)
9.00105
0.79
0.129(*)
4.73105
0.67
0.197(*)
2.52104
0.05
2.483(*)
MA
NU
Sample
Highlights
AC
CE
PT E
D
(*) current density at middle potential of the passivation zone.
DLC films with 0-16.7 at% Ag were sputter deposited with an industry scaled coater
Silver segregation and corrosion are serious for the film with 16.7 at% Ag
Hardness, hydrophilicity and surface energy decrease with the addition of silver
Antibacterial property of the Ag-containing films is long-lasting and reusable
18
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
L.J. Wang, F. Zhang, A. Fong, K.M. Lai, P.W. Shum, Z.F. Zhou, Z.F. Gao, T. Fu PII: DOI: Reference:
S0040-6090(18)30102-0 https://doi.org/10.1016/j.tsf.2018.02.015 TSF 36477
To appear in:
Thin Solid Films
Received date: Revised date: Accepted date:
8 August 2017 7 February 2018 8 February 2018
Please cite this article as: L.J. Wang, F. Zhang, A. Fong, K.M. Lai, P.W. Shum, Z.F. Zhou, Z.F. Gao, T. Fu , Effects of silver segregation on sputter deposited antibacterial silver-containing diamond-like carbon films. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tsf(2017), https://doi.org/10.1016/j.tsf.2018.02.015
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 Effects of silver segregation on sputter deposited antibacterial silver-containing diamond-like carbon films
L.J. Wang1, F. Zhang1, A. Fong2, K.M. Lai2, P.W. Shum2, Z.F. Zhou3, Z.F. Gao2, T. Fu1, 1. Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China
PT
2. Asahi Group Co. Ltd, Kwun Tong, Hong Kong, China
3. Department of Mechanical and Biomedical Engineering, City University of Hong Kong,
RI
Kowloon, Hong Kong, China
SC
Abstract: Silver-containing diamond-like carbon (DLC) films for antibacterial applications were prepared on 316L stainless steel and slide glass substrates by
NU
magnetron sputtering at different silver target current. Scanning electron microscopy and atomic force microscopy analyses show that the film surface becomes rough due
MA
to the formation of nanoparticles/clusters and even a surface layer with the increase of silver target current. The Ag content was measured as 0, 2.1, 6.1 and 14.3 at.%,
D
respectively by energy dispersive X-ray analysis. X-ray diffraction and X-ray
PT E
photoelectron spectroscopy reveal the dominant metallic nature of silver for the Ag-containing films. Silver segregation from the carbon matrix to film surface is confirmed by the above analyses. Microhardness indentation, contact angle
CE
measurement and potentiodynamic polarization test in a Ca-free Hank’s balanced salt solution indicate that the addition of silver has decreased hardness, elastic modulus,
AC
H/E and H3/E2 ratios, wettability, surface energy and corrosion resistance of the DLC films, respectively. The antibacterial tests by agar disk diffusion assay and agar plate counting method against Escherichia coli demonstrate long-lasting and reusable antibacterial activity of the Ag-containing films. The study shows that silver segregation plays a crucial role in microstructure, mechanical, chemical and biological properties of the antibacterial Ag-containing DLC films.
*
Corresponding author. E-mail address: [email protected] (T. Fu) 1
ACCEPTED MANUSCRIPT Keywords: Diamond-like carbon; thin films; Silver; Sputtering; Corrosion; Antibacterial 1. Introduction Vapor deposited diamond-like carbon (DLC) films have been widely applied in protective, biocompatible and other functional surface treatments due to their
PT
excellent mechanical properties (high hardness, low coefficient of friction, high wear
RI
resistance, etc), surface smoothness, good chemical inertness and biocompatibility. The doping of different metallic elements (Cr [1,2], Ti [1,3], W [4,5], Mo [5], the
SC
non-carbide-forming elements of Ag [6,7], Cu [8], Al [9], Ni [10], etc) can reduce residual stress [2,4,6-9], improve electric conductivity [3,11] and enable new
NU
functions of the DLC films.
Currently one of the greatest threats in the clinical practice is the difficulties in
MA
the treatment of implant-related infections due to the bacterial drug resistance [12]. The hospital-acquired infections have been a major public health concern in hospitals
D
and healthcare units worldwide, and it is of great importance to reduce the spread of
PT E
unwanted bacteria in clinical applications. Silver has been known as an effective antimicrobial material since the ancient times, with the broad spectrum against multiple drug-resistant bacteria as well as the limited toxicity towards mammalian
CE
cells [13-15]. Silver-containing DLC (Ag-DLC) and other silver doped coatings
AC
prepared by magnetron sputtering [11], pulsed laser deposition [13], ion deposition [16], plasma enhanced chemical vapor deposition [6], etc have been widely studied for antibacterial surface modification of biomedical implants and environmental surfaces [12,16-18]. For vapor deposited Ag-DLC films, silver usually segregates from carbon matrix and results in composite structures during the relatively energetic film deposition [16,19] or the ageing process after deposition [11,12, 20,21] due to its low solubility with carbon and its inability to form carbides. Silver segregation can increase silver content at the film surface, but its influence on mechanical, chemical and biological 2
ACCEPTED MANUSCRIPT properties of the Ag-DLC films have rarely been studied. In this study DLC films with different silver content are prepared by magnetron sputter method. Austenitic 316L stainless steel is selected as the substrate material for its wide applications in biomedical and environmental fields, and slide glass is also coated for analysis. Silver segregation is evidenced by structural and compositional analyses, and its effects on
PT
hardness, wettability, corrosion resistance and antibacterial property of the Ag-DLC
RI
coated samples are investigated.
SC
2. Experimental 2.1. Sample preparation
NU
The Ag-DLC films were deposited on polished 316L stainless steel disks (101 mm2) and glass slides (size 75251 mm3) by an industrial closed field unbalanced
MA
magnetron sputtering system (UDP650, Teer Coatings Ltd). The substrates were degreased, ultrasonically cleaned, and subsequently blown dry in flowing nitrogen gas. Surface roughness of the stainless steel disks was Ra= 17.7 nm measured by a stylus
D
profilometer (Ambios XP-2) with the test length of 800 m. The computer-controlled
PT E
deposition system comprised of six rectangular cathodes (two chromium and three graphite targets, as well as one silver target). The size of the targets was 345×145×8
CE
mm3, with a purity of >99.9 %. The six magnetrons were driven by DC power suppliers in current mode. The cleaned substrates were mounted on the substrate
AC
holder that was rotated at a speed of 10 rpm, and the target-to-substrate distance was 17 cm. Prior to deposition the vacuum chamber was evacuated to a background pressure of 410-4 Pa. Pure Ar working gas (99.999% purity) was introduced into the chamber via a mass flow controller at constant flow rate (30 sccm), corresponding to a working gas pressure of 0.18 Pa. For plasma ion etching, the substrates were biased with pulse DC at a frequency of 250 kHz and a voltage of -450 V. In film deposition a thin Cr-C interlayer of ~0.4 µm thickness was prepared, and then the Ag-DLC top layers were produced by co-sputtering the graphite targets (currents fixed at 3.5 A) and the silver target for 30 min. The Cr-C interlayer was deposited to improve
3
ACCEPTED MANUSCRIPT adhesion and accommodate stresses of the top DLC film. The Ag target current was selected as 0, 0.2, 0.4 and 0.6 A, respectively to vary silver content in the films. During the deposition stages the bias voltage was reduced to -60 V. The substrates were heated due to ion bombardment, but the temperature was estimated to be lower than 200C. The coated steel/glass samples were noted as M00/G00, M02/G02,
PT
M04/G04 and M06/G06, respectively, in which the numbers indicated the silver target
RI
current.
2.2. Material characterization and tests
SC
Surface morphology of the samples aged in ambient conditions for different time
NU
(1, 4 and 10 months) was observed by scanning electron microscopy (SEM, FEI Quanta 600F) and atomic force microscopy (AFM, Veeco diInnova, tapping mode,
MA
scanning area 55 m2). The scanning electron microscope was equipped with energy dispersive X-ray analysis (EDX, operating voltage 15 kV). Phase structure of the samples was analyzed by X-ray diffraction (XRD, X’Pert PRO, -2 configuration,
D
CuK source, 40 kV and 40 mA). Elemental composition and chemical bonding state
PT E
of the samples were examined by X-ray photoelectron spectroscopy (XPS, VG K-Alpha, AlK source, 12 kV and 6 mA). XPS spectra of the surface layer were
CE
acquired with an elliptical detection area (long axis ~400 m, short axis ~300 m) at a detection angle of 45 without and with 30 s sputter etching. For the sputter etching
AC
to remove surface contaminants, the angle, current and energy of the Ar+ ion gun was 30, 10 mA and 2 keV, respectively. Wettability of the samples was assessed by measuring water and glycol contact angles with a contact angle goniometer under ambient conditions. Surface free energy of the samples was determined from the above contact angles with the method described in [20]. The dispersive ( Sd ) and polar ( Sp ) components of surface energy of a material can be calculated with the following equation [22,23]:
L (1 cos ) 2( Sd rLd )1 / 2 2( Sp rLp )1 / 2
(1)
4
ACCEPTED MANUSCRIPT where L is the liquid surface energy containing dispersive ( Ld ) and polar ( Lp ) components, and is the contact angle. Based on the well known polar and dispersive components of surface energy of water and glycol [22], the polar and dispersive components of surface energy of the samples were calculated from the above measured contact angles using a Matlab program written according to Eq. 1.
PT
Hardness of the films was measured with a microhardness tester (Fischerscope H100C, Vicker’s diamond pyramid indenter, test load 20 mN). Corrosion resistance of
RI
the coated stainless steel samples was evaluated by potentiodynamic polarization test
SC
with an electrochemical workstation (Corrtest CS150) under ambient conditions. The electrolyte was a Ca-free Hank’s balanced salt solution (HBSS, NaCl 8.00 g/L, KCl
NU
0.40 g/L, NaHCO3 0.34 g/L, KH2PO4 0.06 g/L, Na2HPO412H2O 0.12 g/L), and the counter and reference electrodes were platinum electrode and saturated calomel
MA
electrode (SCE), respectively. In the test the potential sweep rate was 0.50 mV/s and
PT E
2.3. Antibacterial test
D
the sampling rate was 2.0 Hz.
Antibacterial property of the samples was assessed by the qualitative evaluation using agar disk diffusion assay and the quantitative evaluation using agar plate
CE
counting method against Escherichia coli. As a typical kind of Gram-negative bacteria, E. coli has been found in biomaterial-related infection sites and is one of the main
AC
sources responsible for hospital-acquired infections. For agar disk diffusion assay, 20 ml of viscous Luria-Bertani agar was poured into sterile Petri dishes, and 1.5 ml of bacteria culture fluid (106 colony forming units, CFU/ml) was mixed with the agar. After the agar was cooled down, the sterilized samples were placed face down on the inoculated agar plates, with effective contact between them. The seeded agar plates were cultured aerobically at 37C in an incubator for 24 h. The photos of the samples were taken by a digital camera. For agar plate counting method, 10 l of E. coli suspension (105 CFU/ml) was
5
ACCEPTED MANUSCRIPT dropped onto the sample surface, and then covered quickly with a filter membrane. After placed in dark at room temperature for 3 h, the samples were rinsed with 3 ml of sterilized water to collect the survival bacteria. After stirring, 700 l of the suspension was mixed with viscous Luria-Bertani agar. The inoculum was spread and incubated aerobically at 37C for 24 h. The photos of colonies were taken by a digital camera. In
PT
order to evaluate the long-lasting and reusable antibacterial property, the tested samples were cleaned, soaked in deionized water for two weeks, tested and cleaned
SC
RI
for two cycles, and tested again.
3. Results and discussion
NU
3.1. Morphological, compositional and structural analyses SEM surface images and optical photos of the samples aged for different time
MA
are shown in Fig. 1a-g. The films are black, dense and uniform for samples M00-M04, but the film is gray for sample M06. Nanoparticles with the size of about 80-100 nm are observed by SEM for sample M00, which reflect fine columnar structure of the
D
film. The total thickness of the Cr-C interlayer is about 0.38 m, and the top carbon
PT E
layer is about 0.37 µm thick (Fig. 1h). Similar nanoparticles with a few white tiny dots are observed for sample M02. There are many white particles and clusters
CE
(50-100 nm) at the surface of sample M04, which have higher Ag content than the whole film revealed by EDX analysis. For sample M06, the nanoparticles are about
AC
60-180 nm in size at 1 month, a loose surface layer composed of big particles and agglomerations (120-400 nm) is formed at 4 months, and the film becomes compact at 10 months of aging. Correspondingly, the surface roughness Ra of sample G06 measured by AFM (test area 55 m2) increases from ~8 nm at 1 month to ~20 nm at 4 and 10 months of ageing. The Ag-DLC layer is about 0.47 µm thick at the cross-section (Fig. 1f). Elements of C, Cr and Ag are detected at the surface by areal EDX analysis (test area around 128 m2), and the Ag content increases with silver target current, being 0, 2.1, 6.1 and 14.3 at.% for G00-G06, respectively (Table 1). The measurement of Ag content is influenced by the gradient structure and different 6
ACCEPTED MANUSCRIPT thickness of the films. The EDX analysis and the following tests were carried out on the samples with an intermediate aging time (4 months). In XRD patterns (Fig. 2) only steel substrate peaks are present for samples M00 and M02. Weak silver diffraction peaks are observed for sample M04, and the silver peaks are strong for sample M06. The silver grain size of sample M06 is calculated to
PT
be 28 nm by the Ag(111) peak located at 2=38.13 according to the Debye-Scherrer equation. The XRD and the above EDX analyses may suggest that the white
RI
nanoparticles/clusters in Fig. 1b,c and the rough surface layer in Fig. 1f are composed
SC
of metallic silver.
Surface elemental composition of sample G06 was further examined by XPS
NU
analysis, and elements of Ag, C and O are detected by survey scan. The Ag 3d5/2 peak is positioned at 368.2 eV and it moves to slightly higher binding energy after 30 s
MA
sputtering (Fig. 3a). The Ag 3d5/2 peak for metallic silver is located at higher binding energy (368.4 eV) than those for its oxides (367.8 eV for Ag2O, 367.4 eV for AgO) [24]. This indicates that silver is present mainly in metallic state, agreeing with the
D
above XRD analysis, but silver is slightly oxidized at the topmost surface when
PT E
exposed to ambient air. In O 1s spectra the peak at 530.4 eV ascribed to silver oxide is nearly removed by 30 s sputtering (Fig. 3b). The silver oxide top surface layer was
CE
revealed by continuous sputtering etching in other vapor deposited Ag-DLC films [12,19,24]. The nonpassive silver oxide thin film is essential for the release of
AC
antimicrobial Ag+ ions [25,26]. The atomic ratio of Ag/C is 260% after 30 s sputter cleaning, much higher than that measured by areal EDX (20%) for sample G06, which indicates the enrichment of silver at the film surface. Due to its low solubility with carbon and its inability to form carbides, silver can segregate from carbon matrix and result in composite structures during the relatively energetic film deposition [19] and the ageing process after deposition [11,12,20]. The post-deposition segregation of silver nanostructures is especially obvious at high silver contents (samples M04 and M06 in Fig. 1, 20 at.%
7
ACCEPTED MANUSCRIPT Ag in Ref. [21]). The segregation induced silver-rich surface layer will influence mechanical, chemical and biological properties of the Ag-DLC films.
3.2. Hardness, wettability and corrosion resistance The addition of silver has decreased microhardness and elastic modulus of the
PT
DLC films, from 13.3 GPa and 165 GPa for sample M00 to 6.1 GPa and 124 GPa for sample M06 (Table 1). This is consistent with the report that the surface hardness and
RI
electrical resistivity of DLC film decreased with the increase of Ag content [11,20,27].
SC
The hardness reduction is attributed to the incorporation of soft/compliant silver phase and the subsequent graphitization of amorphous carbon matrix [28]. However,
NU
hardness of the Ag-DLC films is still higher than that of stainless steel substrate (~2.5 GPa). The H/E and H3/E2 ratios are two factors to assess wear-resistant properties and
MA
plastic deformation resistance of hard coatings, respectively [29,30], and they are much higher for samples M00-M04 (0.081-0.083, 0.077-0.086 GPa) than those for sample M06 (0.049, 0.015 GPa). This indicates better mechanical properties of the
D
DLC films of samples M00-M04 compared with those of sample M06, which has the
PT E
highest silver content (14.3 at.%).
Water contact angle increases continuously from 81.6 for sample G00 to 111.9
CE
for sample G06 (Table 2). Usually, a hydrophobic surface has a contact angle higher than 70°, while a hydrophilic surface has a contact angle lower than 70°. Thus, the
AC
addition of silver increases hydrophobicity of the DLC film. The increased hydrophobicity of Ag-DLC film can be attributed to the chemical structure change of DLC film [11,23], the hydrophobic nature of silver nanoparticles/clusters and the increased surface roughness. According to Wenzel’s model describing the contact angle at a rough surface, the surface roughness enhances hydrophilicity of hydrophilic surfaces and enhances hydrophobicity of hydrophobic surfaces [31,32]. The glycol contact angle also increases from 59.7 for sample G00 to 91.9 for sample G06 (Table 2). As the result, the surface energy decreases from 27.4 mJ/m2 for sample G00 to 14.2 mJ/m2 for sample G06, with polar component being the minor. 8
ACCEPTED MANUSCRIPT Thus, the addition of silver has decreased wettability and surface energy of the DLC films. The hydrophobic surface with low surface energy is beneficial for the biomedical applications of anti-fouling, antimicrobial and blood contacting materials [11,16,20,23]. Moreover, the fibroblast cell test with titanium and titanium alloys indicated that a low polar component (or low fractional polarity) was the major
PT
parameter for good cell proliferation [33]. Corrosion resistance of the Ag-DLC coated stainless steel samples was evaluated
RI
by potentiodynamic polarization test (Fig. 4). Samples M00 and M02 exhibit similar
SC
polarization plots, with a long passivation zone followed by pitting at the potentials above 0.76 V (Table 3), which are much higher than that of the polished stainless
NU
steel sample (~0.4 V) . The pitting potential of sample M04 is still high (0.67 V), but there are two current peaks within the passivation zone. The first current peak at 0.08
MA
V is related to dissolution of metallic silver particles at the film surface [34,35]. Similar current peaks at 0.08-0.12 V were found in the polarization plots of sputter prepared DLC films containing 2.97-8.37 at.% silver that were tested in 3.5% NaCl
D
solution [36]. The second current peak is likely relevant to corrosion of the Cr-C
PT E
interlayer and the steel substrate. SEM observation of the tested sample shows that there is a corrosion pit (diameter ~200 m) at the sample surface, and the steel
CE
substrate at the bottom of the pit is seriously corroded (Fig. 5). It is postulated that more structural defects are generated in the film due to the increased silver content
AC
(e.g. voids, pores formed by silver segregation), and the defects increase the pitting probability of the coated steel sample. It was documented that the free corrosion potential of titanium alloys in vivo lies in the range of 450-550 mV SHE (or 206-306 mV SCE [37]). The coated stainless steel samples M00-M04 were stable up to 0.4 V according to the polarization test; however, their corrosion behavior in vivo still needs a further study. For sample M06, pitting occurs at only 0.05 V, which is caused by significant dissolution of the silver surface layer of the sample. Thus, sample M06 is especially unstable in the simulated body fluid. With the increase of silver content in
9
ACCEPTED MANUSCRIPT the DLC film, the corrosion resistance (Rcorr) is gradually decreased, and the corrosion current density (Icorr) and passive current density (Ipass) are slowly increased (Table 3).
3.3. Antibacterial test Antibacterial property of the coated samples was first qualitatively tested by agar
PT
disk diffusion assay (Fig. 6). There is no inhibition zone for samples G00 and G02. The inhibition zone is obvious for sample G04 and it is wide (~5.4 mm) for sample
RI
G06. This reveals the silver dependent antibacterial activity of the Ag-DLC films. Due
SC
to the oxidation of metallic silver by oxygen, the Ag-containing films can release silver ions of different concentration to the surrounding agar plate and kill bacteria
NU
there. Two mechanisms have been proposed for bactericidal property of silver ions. First, silver ions can bind at the negatively charged bacterial cell membrane or enter
MA
the cells, and then impair the structure and functions of bacterial cells. Second, silver ions can catalyze molecule oxygen in water and produce ROS, and ROS will induce bacteria death [38]. Since silver of high contents is harmful to adhesion and
D
biocompatibility of a material [12], the antibacterial property should be guaranteed at
PT E
the lowest silver content in a specific application. The antibacterial property of the samples was then quantitatively evaluated by
CE
agar plate counting method (Fig. 7). The un-doped sample G00 favors bacteria growth when tested for 3 h. The bacterial colonies are markedly reduced for sample G02, and
AC
no colonies are observed for samples G04 and G06. Therefore, DLC films with 2.1 at.% or more silver can release enough Ag+ ions to kill E. Coli, resulting in an antibacterial rate of >95%. The atomic absorption spectroscopy analysis demonstrated that DLC coating with 2.4 at.% Ag had displayed slow release kinetics, with a total silver ion release in the sub-ppb range after 4 h in solution [24]. The limited diffusion of biocidal silver ions with a slow and continuous release is mandatory to ensure a lasting and localized antibacterial effect of the Ag-DLC films. Furthermore, the tested films were cleaned with 75% ethanol, soaked in deionized water for two weeks and tested and cleaned for two cycles, and tested again. Similar results were obtained for 10
ACCEPTED MANUSCRIPT samples G02-G06 in the last test (Fig. 7f-h), which demonstrates their long-lasting and reusable antibacterial property. The above results and the previous studies show that the addition of silver in DLC films can enhance antibacterial property, but decrease hardness, H/E and H3/E2 ratios, wettability, corrosion resistance and biocompatibility of the coated samples
PT
[20]. Proper content of silver should be chosen for the balance of mechanical, chemical and biological properties of the coated samples in a specific situation. For
RI
example, the optimal combination with reasonable physical-chemical properties,
SC
efficient protection against microbial colonization and beneficial effects on human MG63 cells was found for carbon films containing 2-7 at.% silver [13]. In another
NU
case, increasing silver content of Ag-DLC films to above 2 at.% improved bactericidal properties against E. coli, but decreased the viability of osteoblast cells [12].
MA
Apart from the silver content, the distribution [19,39] and chemical state of sliver [25] can also influence silver ion release and antibacterial activity of Ag-DLC and Ag-O films. Obvious silver segregation at the film surface has been observed in
D
Ag-DLC films prepared by different deposition methods [11-13,19,28,39]. Silver
PT E
content at the film surface will be much increased due to silver segregation from the carbon matrix. Since bacteria only interact with materials at the surface, Ag-DLC
CE
films with even lower average silver content would have the enhanced antibacterial activity due to silver segregation (e.g. antibacterial rate >95% for G02 with 2.1 at.%
AC
Ag) in comparison with the films without obvious silver segregation (e.g. a high antibacterial rate for DLC film with 9.3 at.% Ag [40]). The average silver content in a specific application can be lowered while keeping the high antibacterial activity due to silver segregation for Ag-DLC films.
4. Conclusions Ag-DLC films with the silver contents of 0, 2.1, 6.1 and 14.3 at.% were prepared by magnetron sputter method. The surface roughness increases with silver content due to silver segregation at the film surface from the carbon matrix. The addition of silver 11
ACCEPTED MANUSCRIPT decreases hardness, elastic modulus, H/E and H3/E2 ratios, wettability, surface energy and corrosion resistance of the DLC films. The antibacterial property of the silver-containing DLC films is long-lasting and reusable, and the antibacterial activity is high even at 2.1 at.% Ag due to silver segregation at the film surface. The effects of silver segregation on structure and properties of Ag-DLC films have to be considered
PT
for their antibacterial applications.
RI
Acknowledgements
SC
This work was supported by Asahi Group Co. Ltd, the Fundamental Research Fund for the Central Universities (xjj2015072, xjj2017163) and the Science and
NU
Technology Program of Chongqing Municipal Education Commission (No.
MA
KJ1600926).
References
[1] M. Jelinek, J. Zemek, J. Remsa, J. Miksovsky, T. Kocourek, P. Pisarik, M. Travnickova, E.
PT E
Science 417 (2017) 73-83.
D
Filova, L. Bacakova, Hybrid laser technology and doped biomaterials, Applied Surface
[2] M.Y. Ming, X. Jiang, D.G. Piliptsou, Y. Zhuang, A.V. Rogachev, A.S. Rudenkov, A.
CE
Balmakou, Chromium-modified a-C films with advanced structural, mechanical and corrosive-resistant characteristics, Applied Surface Science 379 (2016) 424-432.
AC
[3] N.R. Lee, Y.S. Jun, K.I. Moon, C.S. Lee, Ti-doped hydrogenated diamond like carbon coating deposited by hybrid physical vapor deposition and plasma enhanced chemical vapor deposition, Japanese Journal of Applied Physics 56 (2017) 035506. [4] X.M. Wang, W.D. Wu, S.Y. Li, S.L. Chen, Y.J. Tang, L. Bai, H.P. Wang, Properties of W doped diamond-like carbon films prepared by pulsed laser deposition, Rare Metal Materials and Engineering 39 (2010) 1251-1255. [5] I.C. Muller, J. Sharp, W.M. Rainforth, P. Hovsepian, A. Ehiasarian, Tribological response and characterization of Mo-W doped DLC coating, Wear, 376 (2017) 1622-1629. [6] F.R. Marciano, L.F. Bonetti, L.V. Santos, N.S. Da-Silva, E.J. Corat, V.J. Trava-Airoldi,
12
ACCEPTED MANUSCRIPT Antibacterial activity of DLC and Ag-DLC films produced by PECVD technique, Diamond and Related Materials 18 (2009) 1010-1014. [7] C. Wang, X. Yu, M. Hua, Microstructure and mechanical properties of Ag-containing diamond-like carbon films in mid-frequency dual-magnetron sputtering, Applied Surface Science 256 (2009) 1431-1435.
PT
[8] L. Sun, P. Guo, P. Ke, X. Li, A. Wang, Synergistic effect of Cu/Cr co-doping on the wettability and mechanical properties of diamond-like carbon films, Diamond and Related
RI
Materials 68 (2016) 1-9.
[9] X. Pang, L. Shi, P. Wang, Y. Xia, W. Liu, Effects of Al incorporation on the mechanical and
SC
tribological properties of Ti-doped a-C:H films deposited by magnetron sputtering, Current Applied Physics 11 (2011) 771-775.
NU
[10] N.W. Khun, E. Liu, G.C. Yang, Structure, scratch resistance and corrosion performance of nickel doped diamond-like carbon thin films, Surface & Coatings Technology, 204 (2010)
MA
3125-3130.
[11] W.-C. Lan, S.-F. Ou, M.-H. Lin, K.-L. Ou, M.-Y. Tsai, Development of silver-containing
D
diamond-like carbon for biomedical applications. Part I: Microstructure characteristics,
4099-4104.
PT E
mechanical properties and antibacterial mechanisms, Ceramics International 39 (2013)
[12] D. Bociaga, W. Jakubowski, P. Komorowski, A. Sobczyk-Guzenda, A. Jedrzejczak, D. Batory,
CE
A. Olejnik, Surface characterization and biological evaluation of silver-incorporated DLC coatings fabricated by hybrid RF PACVD/MS method, Materials Science & Engineering C
AC
63 (2016) 462-474.
[13] I.N. Mihailescu, D. Bociaga, G. Socol, G.E. Stan, M.-C. Chifiriuc, C. Bleotu, M.A. Husanu, G. Popescu-Pelin, L. Duta, C.R. Luculescu, I. Negut, C. Hapenciuc, C. Besleaga, I. Zgura, F. Miculescu, Fabrication of antimicrobial silver-doped carbon structures by combinatorial pulsed laser deposition, International Journal of Pharmaceutics 515 (2016) 592-606. [14] M.G. Raucci, K. Adesanya, L. Di Silvio, M. Catauro, L. Ambrosio, The biocompatibility of silver-containing Na2OCaO2SiO2 glass prepared by sol-gel method: in vitro studies, Journal of Biomedical Materials Research Part B 92B (2010) 102-110. [15] M. Bellantone, H.D. Williams, L.L. Hench, Broad-spectrum bactericidal activity of 13
ACCEPTED MANUSCRIPT Ag2O-doped bioactive glass, Antimicrobial Agents and Chemotherapy 46 (2002) 1940-1945 [16] K. Baba, R. Hatada, S. Flege, W. Ensinger, Y. Shibata, J. Nakashima, T. Sawase, T. Morimura, Preparation and antibacterial properties of Ag-containing diamond-like carbon films prepared by a combination of magnetron sputtering and plasma source ion implantation, Vacuum 89 (2013) 179-184.
PT
[17] J.L. Endrino, A. Anders, J.M. Albella, J.A. Horton, T.H. Horton, P.R. Ayyalasomayajula, M. Allen, Antibacterial efficacy of advanced silver-amorphous carbon coatings deposited using
RI
the pulsed dual cathodic arc technique, Journal of Physics: Conference Series 252 (2010) 012012.
SC
[18] N. Harrasser, S. Jussen, A. Obermeir, R. Kmeth, B. Stritzker, H. Gollwitzer, R. Burgkart, Antibacterial potency of different deposition methods of silver and copper containing
NU
diamond-like carbon coated polyethylene, Biomaterials Research 20 (2016) 181-190. [19] M. Cloutier, S. Turgeon, Y. Busby, M. Tatoulian, J.J. Pireaux, D. Mantovani, Controlled
MA
distribution and clustering of silver in Ag-DLC nanocomposite coatings using a hybrid plasma approach, ACS Applied Materials & Interfaces 8 (2016) 21020-21027.
D
[20] L. Swiatek, A. Olejnik, J. Grabarczyk, A. Jedrzejczak, A. Sobczyk-Guzenda, M. Kaminska,
PT E
W. Jakubowski, W. Szymanski, D. Bociaga, Multi-doped diamond like-carbon coatings (DLC-Si/Ag) for biomedical applications fabricated using the modified chemical vapour deposition method, Diamond and Related Materials 67 (2016) 54-62.
CE
[21] N.K. Manninen, R.E. Galindo, S. Carvalho, A. Cavaleiro, Silver surface segregation in Ag-DLC nanocomposite coatings, Surface & Coatings Technology, 267 (2015) 90-97.
AC
[22] T. Zhao, Y. Li, Y. Gao, Y. Xiang, H. Chen, T. Zhang, Hemocompatibility investigation of the NiTi alloy implanted with tantalum, Journal of Materials Science-Materials in Medicine 22 (2011) 2311-2318. [23] H.W. Choi, R.H. Dauskardt, S.-C. Lee, K.-R. Lee, K.H. Oh, Characteristic of silver doped DLC films on surface properties and protein adsorption, Diamond and Related Materials 17 (2008) 252-257. [24] M. Cloutier, R. Tolouei, O. Lesage, L. Levesque, S. Turgeon, M. Tatoulian, D. Mantovani, On the long term antibacterial features of silver-doped diamondlike carbon coatings deposited via a hybrid plasma process, Biointerphases 9 (2014) 029013. 14
ACCEPTED MANUSCRIPT [25] R. Rebelo, S.V. Calderon, R. Fangueiro, M. Henriques, S. Carvalho, Influence of oxygen content on the antibacterial effect of Ag-O coatings deposited by magnetron sputtering, Surface & Coatings Technology 305 (2016) 1-10. [26] I. Ferreri, S.V. Calderon, R. Escobar Galindo, C. Palacio, M. Henriques, A.P. Piedade, S. Carvalho, Silver activation on thin films of Ag-ZrCN coatings for antimicrobial activity,
PT
Materials Science & Engineering C 55 (2015) 547-555. [27] N.K. Manninen, F. Ribeiro, A. Escudeiro, T. Polcar, S. Carvalho, A. Cavaleiro, Influence of
RI
Ag content on mechanical and tribological behavior of DLC coatings, Surface & Coatings Technology 232 (2013) 440-446.
SC
[28] M. Constantinou, M. Pervolaraki, P. Nikolaou, C. Prouskas, P. Patsalas, P. Kelires, J. Giapintzakis, G. Constantinides, Microstructure and nanomechanical properties of pulsed
Coatings Technology, 309 (2017) 320-330.
NU
excimer laser deposited DLC:Ag films: Enhanced nanotribological response, Surface &
MA
[29] N. Olah, Z. Fogarassy, A. Sulyok, J. Szivos, T. Csanadi, K. Balazsi, Ceramic TiC/a:C protective nanocomposite coatings: Structure and composition versus mechanical properties
D
and tribology, Ceramics International 42 (2016) 12215-12220.
PT E
[30] Q.Z. Wang, F. Zhou, M. Callisti, T. Polcar, J.Z. Kong, J.W. Yan, Study on the crack resistance of CrBN composite coatings via nano-indentation and scratch tests, Journal of Alloys and Compounds 708 (2017) 1103-1109.
CE
[31] R.N. Wenzel, Surface roughness and contact angle, Journal of Physical and Colloid Chemistry 53 (1949) 1466-1467.
AC
[32] M. Miyauchi, H. Tokudome, Super-hydrophilic and transparent thin films of TiO2 nanotube arrays by a hydrothermal reaction, Journal of Materials Chemistry, 17 (2007) 2095-2100. [33] L. Ponsonnet, K. Reybier, N. Jaffrezic, V. Comte, C. Lagneau, M. Lissac, C. Martelet, Relationship between surface properties (roughness, wettability) of titanium and titanium alloys and cell behaviour, Materials Science & Engineering C, 23 (2003) 551-560. [34] T. Fu, Y.G. Shen, Z. Alajmi, S.Y. Yang, J.M. Sun, H.M. Zhang, Sol-gel preparation and properties of Ag-TiO2 films on surface roughened Ti-6Al-4V alloy, Materials Science and Technology, 31 (2015) 501-505. [35] Z. Alajmi, T. Fu, Y.T. Zhao, S.Y. Yang, J.M. Sun. Ag-enhanced antibacterial property of MgO 15
ACCEPTED MANUSCRIPT film. Materials Science Forum, 859 (2016) 90-95. [36] V.S. Dhandapani, E. Thangavel, M. Arumugam, K.S. Shin, V. Veeraraghavan, S.Y. Yau, C. Kim, D.-E. Kim, Effect of Ag content on the microstructure, tribological and corrosion properties of amorphous carbon coatings on 316L SS, Surface & Coatings Technology, 240 (2014) 128-136.
PT
[37] M.H. Wong, F.T. Cheng, H.C. Man, Characteristics, apatite-forming ability and corrosion resistance of NiTi surface modified by AC anodization, Applied Surface Science, 253 (2007)
RI
7527-7534.
[38] K. Loza, J. Diendorf, C. Sengstock, L. Ruiz-Gonzalez, J.M. Gonzalez-Calbet, M. Vallet-Regi,
SC
M. Koller, M. Epple, The dissolution and biological effects of silver nanoparticles in biological media, Journal of Materials Chemistry B, 2 (2014) 1634-1643.
NU
[39] N.K. Manninen, S. Calderon, I. Carvalho, M. Henriques, A. Cavaleiro, S. Carvalho, Antibacterial Ag/a-C nanocomposite coatings: The influence of nano-galvanic a-C and Ag
MA
couples on Ag ionization rates, Applied Surface Science, 377 (2016) 283-291. [40] P. Pisarik, M. Jelinek, J. Remsa, J. Miksovsky, J. Zemek, K. Jurek, S. Kubinova, J. Lukes, J.
D
Sepitka, Antibacterial, mechanical and surface properties of Ag-DLC films prepared by dual
AC
CE
PT E
PLD for medical applications, Materials Science & Engineering C, 77 (2017) 955-962.
16
ACCEPTED MANUSCRIPT
Fig. 1. SEM secondary electron surface images and optical photos of the Ag-DLC coated steel samples aged for different time: (a) M00, 4 months, (b) M02, 4 months, (c) M04, 4 months, (d) M04, 10 months, (e) M06, 1 month, (f) M06, 4 months, (g) M06, 10 months. The back-scattered electron cross-sectional images of samples G00
Fig. 2. XRD patterns of the Ag-DLC coated steel samples.
PT
and G06 (4 months) with glass substrate are shown in (h) and (i), respectively.
RI
Fig. 3. XPS high-resolution Ag 3d and O 1s spectra of sample G06 (glass substrate) before and after 30 s sputtering.
SC
Fig. 4. Potentiodynamic polarization plots of the Ag-DLC coated steel samples. Fig. 5. SEM images of the corrosion pit of sample M04 with steel substrate after
NU
polarization test observed at (a) low and (b) high magnifications. Fig. 6. Antibacterial test results of the Ag-DLC coated glass samples by agar disk
MA
diffusion assay. The inhibition zones shown between the white and yellow dash lines are around 2.2 and 5.4 mm in width for samples G04 and G06, respectively.
D
Fig. 7. Typical antibacterial test results of the Ag-DLC coated glass samples G00-G06
PT E
(a-d) by agar plate counting method. The tested samples were cleaned by 75% ethanol, soaked in deionized water for two weeks and tested and cleaned for two cycles, and tested again to get the results shown sequentially in (e-h). The number of bacterial
CE
colonies is 1220, 48, 1152, 55 in (a), (b), (e), (f), respectively.
AC
Table 1 The Ag content ([Ag]), thickness (T), hardness (H), elastic modulus (E), H/E and H3/E2 ratios of the Ag-DLC coated glass samples. Samples
[Ag] (at.%)
T (m)
H (GPa)
E (GPa)
H/E
H3/E2 (GPa)
G00
0
0.75
13.3
165
0.081
0.086
G02
2.1
0.76
12.4
150
0.083
0.085
G04
6.1
0.78
11.8
146
0.081
0.077
G06
14.3
0.85
6.1
124
0.049
0.015
17
ACCEPTED MANUSCRIPT
Table 2 Contact angle and surface energy of the Ag-DLC coated glass samples. Surface energy (mJ/m2)
Water
Glycol
Dispersive
Polar
Sum
G00
81.60.8
59.75.2
18.0
9.4
27.4
G02
84.62.6
62.90.8
17.8
8.0
25.8
G04
100.21.3
82.54.4
12.4
3.6
G06
111.91.4
91.91.3
13.6
0.6
PT
Contact angle ()
16.0 14.2
SC
RI
Samples
Table 3 Corrosion data of the Ag-DLC coated steel samples. Ecorr (V SCE)
Icorr (A/cm2)
M00
-0.24
0.012
M02
-0.24
0.014
M04
-0.22
0.036
M06
-0.20
0.063
Rcorr (cm2)
Epit (V SCE)
Ipass (A/cm2)
9.65105
0.76
0.090(*)
9.00105
0.79
0.129(*)
4.73105
0.67
0.197(*)
2.52104
0.05
2.483(*)
MA
NU
Sample
Highlights
AC
CE
PT E
D
(*) current density at middle potential of the passivation zone.
DLC films with 0-16.7 at% Ag were sputter deposited with an industry scaled coater
Silver segregation and corrosion are serious for the film with 16.7 at% Ag
Hardness, hydrophilicity and surface energy decrease with the addition of silver
Antibacterial property of the Ag-containing films is long-lasting and reusable
18
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7