Surface & Coatings Technology 422 (2021) 127553

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Surface & Coatings Technology

journal homepage: www.elsevier.com/locate/surfcoat

Mechanism of protein biofilm formation on Ag-DLC films prepared for

application in joint implants
P.P. Jing , Y.H. Su , Y.X. Li , W.L. Liang , Y.X. Leng *
Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu
610031, China

A R T I C L E I N F O A B S T R A C T

Keywords: Silver-doped diamond-like carbon (Ag-DLC) films offer low residual stress, good adhesion, and excellent wear
Diamond-like carbon resistance, and thus are promising materials for surface modification of joint prostheses. Further, as proteins are
Silver the most abundant component of joint fluid, the wear performance of Ag-DLC films in protein environments
Biofilm
warrants investigation. We prepared DLC and 10.0 at.% Ag-DLC films using hybrid deposition technique by
Protein adsorption
Protein denaturation
combining high-power pulsed magnetron sputtering and high-power pulsed plasma-enhanced chemical vapor
deposition. The wear performance of the films was tested using bovine serum albumin (BSA) solution. A biofilm
of denatured proteins formed at the friction interface of the Ag-DLC film, which improve wear resistance.
Subsequent protein adsorption, Ag+ ions release, and spectroscopic evaluation of the interaction between Ag+
ions and BSA molecules revealed the mechanism of biofilm formation. Ag doping promoted the protein
adsorption on film surface and friction interface of Ag-DLC films. Meanwhile, Ag-DLC films released Ag+ ions
when exposed in physiological solutions. The released Ag+ ions break the hydrogen bonds and disulfide bonds in
proteins and transform the α-helix structure to β-sheet and β-turn structure, thus unfolding the protein, exposing
the inner hydrophobic groups, and inducing protein deposition and biofilm formation. The study elucidates the
biofilm formation mechanism at the friction interface of Ag-DLC films and counterparts and can aid in design of
hardwearing joint implants.

1. Introduction fluid, composed of proteins (10–30 mg/ml), hyaluronic acid (2.5–5.0

Joint replacement is an effective treatment for joint disease and
advanced arthritis, and the joint replacement market occupies a key
position in the biomedical field [1]. Various materials with specific
advantages and disadvantages have been used for treating joints over
the years [2–6]. Particularly, promising candidates for joint prostheses
include materials with functional films, such as diamond-like carbon
(DLC), because of their chemical inertness, suitable mechanical prop­
erties, biocompatibility, and excellent tribological properties [5,7–9].
However, low toughness and adhesion of pure DLC films result in their
failure and exposure to base materials [10,11]. To address this issue,
DLC films are doped with elements such as Cr [12], Ti [13], Cu [14], and
Ag [10]. Specifically, Ag doping improves DLC films' toughness and
adhesion and enhances their wear resistance and antibacterial proper­
ties [15–17]. This makes Ag-DLC films a promising candidate for the
surface modification of artificial joints.
The service environment in an articular cavity contains the joint
mg/ml), lipids (0.2–0.3 mg/ml), and inorganic salts [18,19]. Proteins
are the most abundant component in joint fluids and play an essential
role in the friction process of artificial joint materials. The exposed
surface of metal joints forms a protein layer due to friction, resulting in
reduced wear and improved corrosion resistance of the joint prosthesis
[20–22]. Although metal ions contribute to the formation of protein
layers, the formation mechanism remains unclear and requires further
study. For exploring the potential application of Ag-DLC films in joint
prostheses, wear tests must be performed in protein solutions to simulate
the joint fluid composition. Furthermore, as the service process of Ag-
DLC films causes the release of Ag+ ions, the interaction between Ag+
ions and proteins deserves an in-depth study.
In this study, DLC and Ag-DLC films were prepared using a hybrid
deposition technique combining high-power pulsed magnetron sput­
tering (HPPMS) and high-power pulsed plasma-enhanced chemical
vapor deposition (HPP-PECVD). A previous study systematically inves­
tigated the effects of Ag doping on the microstructure, chemical

* Corresponding author.
E-mail address: yxleng@263.net (Y.X. Leng).

https://doi.org/10.1016/j.surfcoat.2021.127553
Received 30 June 2021; Received in revised form 22 July 2021; Accepted 22 July 2021
Available online 24 July 2021
0257-8972/© 2021 Elsevier B.V. All rights reserved.
P.P. Jing et al.
Fig. 1. (a) Friction coefficient and (b) wear depth and wear rate of DLC and Ag-DLC films in BSA solutions.
bonding, mechanical properties, and adhesion stability of DLC films
[23]. Accordingly, the current study investigates the wear properties of
DLC and Ag-DLC films in bovine serum albumin (BSA) solutions and
discusses the interaction mechanism between the released Ag+ ions and
BSA molecules.
2. Materials and methods
2.1. Film deposition
DLC and 10.0 at.% Ag-DLC films were deposited on Si (100) wafer
and Co-Cr-Mo alloy substrates by a hybrid deposition technique
combining HPPMS and HPP-PECVD [23]. Si wafer was used as a sub­
strate for the film thickness measurement because of low surface
roughness. Co-Cr-Mo alloy is a commonly used material for artificial
joints, which was used as the substrate for subsequent properties test. In
the hybrid system, HPPMS was primarily used to produce Ag species,
and HPP-PECVD was utilized to ionize acetylene for DLC deposition
[23]. This combined method can effectively increase the deposition rate
of DLC films. Argon (purity ~99.99%, 40 sccm) and acetylene (purity
~99.9%, 10 sccm) were the precursor and reactant gases, respectively.
Next, the DLC films were prepared using a graphite target and Ag-DLC
films using a mosaic silver-graphite target. The dimensions of the
graphite target were 170 × 134 × 6 mm. For silver-graphite, 16 Ag rods
(purity ~99.99%, Φ = 5 mm, L = 6 mm) were inserted into the graphite
target racetrack in a mosaic configuration.
Before sputtering, the surface impurities were removed by
bombardment of the targets with Ar+ at 0.6 Pa and DC power of 500 W
for 10 min. The substrates were first cleaned ultrasonically in anhydrous
ethanol, followed by Ar+ bombardment at 3.0 Pa with a pulse bias of
− 1500 V for 30 min. The substrates were placed on a holder, and the
target-to-substrate distance was 80 mm. The base pressure of the vac­
uum chamber was 2.0 × 10− 3 Pa. The DLC and Ag-DLC films were
deposited using an HPPMS power source (voltage~800 V, pulse width
~ 80 μs, and frequency ~ 250 Hz) for 30 min at a constant working
pressure of 0.5 Pa with a DC bias voltage of − 75 V. The thickness of the
prepared films on Si (100) wafer was approximately 1 μm.
2.2. Characterization methods
The wear performance of DLC and Ag-DLC films deposited on Co-Cr-
Mo alloy substrates were evaluated using ball-on-disk tribometry
(CSEM, Switzerland). The experiment employed an alumina ball with a
diameter of 6 mm as the counterpart. A relative sliding speed of 1.885
cm/s and loading force of 0.5 N were applied for 100,000 cycles. The
transverse shear force was determined using a force sensor because the
friction coefficient is a ratio of the shear force to the applied load. In
2
Surface & Coatings Technology 422 (2021) 127553
addition, the BSA solution used as the wear medium at a concentration
of 20 mg/ml simulated the articular fluid environment. Subsequently,
we observed the morphologies of the wear tracks and their counterparts
through an optical microscope (Zeiss Axio A1, Germany) and measured
the wear track profiles using a stylus profiler (XP-2, AMBIOS, USA). The
V
wear rate of the films was calculated using μ=LS [22], where V, L, and S
represent the wear volume of the film, load, and grinding distance,
respectively.
We analyzed the structure and composition of the deposits at the
friction interface using a Fourier-transform infrared (Micro-FTIR,
Thermo iN10, USA) spectrometer, comprising a microscope module,
with a testing range of 1700–1100 cm− 1 and resolution of 2 cm− 1.
For estimating the release of Ag+ ions, the Ag-DLC films deposited on
Co-Cr-Mo alloy substrates were immersed in 10 ml of phosphate buffer
solution (PBS) and stored at 37 ◦ C for 30 min, 1, 3, 7, 15, 30, 90, and 300
days, respectively. The Ag+ ion concentration was determined using
inductively coupled plasma mass spectrometry (ICP-MS, iCAP Q,
Thermo Fisher, USA).
The static adsorption capacity of the proteins on the films was esti­
mated using the bicinchoninic acid (BCA) assay (Thermo Fisher, USA).
For the assay, the DLC and Ag-DLC films deposited on Co-Cr-Mo alloy
substrates with an area of 1.33 cm2 were immersed in a BSA solution (2
mg/ml) at 37 ◦ C for 24 h, followed by cleaning with deionized water.
Next, the films were submerged in BCA solution at 37 ◦ C for 2 h. Finally,
the extent of protein adsorption on the films was estimated by measuring
the optical density of the solution at 562 nm using a microplate analyzer
(Biotek, Synergy H1, USA).
The protein adsorption in the friction process was characterized by
conducting a wear test in fluorescein isothiocyanate-labeled BSA (FITC-
BSA, Beijing Bersee, China) solution for 10,000 cycles. Subsequently, the
morphologies of the wear tracks were observed under a fluorescence
microscope (OLYMPUS-IX 51, Japan).
Considering the enrichment of Ag+ ions during long-term friction,
the concentrations of Ag+ ions and the BSA solution was 1 mM and 20
mg/ml, respectively. The interaction between Ag+ ions and proteins was
investigated by mixing Ag+ ion solution and BSA solution in a ratio of
1:1 (v/v). The conformational changes of BSA molecules were detected
using a UV–visible spectrophotometer (Agilent, Cary UV–Vis Compact
Peltier, USA), with a detection range of 190–400 nm and resolution of 1
nm. The changes in the secondary structure of BSA molecules were
measured by attenuated total reflection Fourier-transform infrared
(ATR-FTIR) spectroscopy (Nicolet iS 50, USA), with a detection range
and resolution of 2000–1000 cm− 1 and 2 cm− 1, respectively. In addi­
tion, circular dichroism (CD, J-1500, JASCO, Japan) was used to eval­
uate the effect of Ag+ ions on the secondary structure of BSA molecules.
The detection wavelength range was 190–300 nm, with a resolution of 1
nm, and the nitrogen flow rate was 3 l/min. The results were expressed
P.P. Jing et al.
Fig. 2. Morphology of the wear track for (a) DLC and (b) Ag-DLC films, and morphology of the counterpart for (c) DLC and (d) Ag-DLC films.
in terms of the mean residue ellipticity (MRE) [24,25], and the α-helix
content of BSA was obtained from the MRE using a previously reported
method [24,26]. Raman spectroscopy (Horiba, LabRAM, Japan) was
used to analyze the binding between the Ag+ and BSA molecules.
Because the signals for metal-ligand bonds usually appear at relatively
low frequencies, the detection range was 4000–100 cm− 1.
3. Results and discussion
3.1. Wear properties of films in BSA solutions
Fig. 1 demonstrates the friction coefficient, wear depth, and wear
rate of the DLC and Ag-DLC films in BSA solution. Fig. 1a shows a
3
Surface & Coatings Technology 422 (2021) 127553
running-in period in the initial stage of friction, accompanied by a
relatively high friction coefficient, after which the coefficient decreases
and stabilizes. The friction coefficients of the DLC and Ag-DLC films
were ~0.19 and ~0.14, respectively. Furthermore, several saltation was
observed in the DLC film curve, whereas the Ag-DLC film curve was
relatively smooth. Fig. 1b revealed wear track depths of ~50 nm and
~30 nm for the DLC and Ag-DLC films, respectively. Further, Fig. 1b
presents the wear rate of the films, as calculated using the formula
presented in Section 2.2. The wear rates of the DLC and Ag-DLC films
were 3.7 × 10− 8 mm3/(N⋅m) and 1.8 × 10− 8 mm3/(N⋅m), respectively.
Thus, the incorporation of Ag reduced the friction coefficient and
maintained stable friction, improving the wear performance of DLC
films in BSA solutions.
P.P. Jing et al.
Fig. 3. Micro-FITR spectra of deposits on the counterpart of DLC and Ag-
DLC films.
0.7
0.6
Optical Density at 562 nm
0.5
0.4
0.3
0.2
0.1
0.0
DLC 10.0 at.% Ag-DLC
Samples
Fig. 4. Amounts of protein adsorbed on DLC and Ag-DLC films.
3.2. Formation of biofilm
Fig. 2 shows the morphology of the wear track and its counterpart for
DLC and Ag-DLC films after wear in the BSA solution. Fig. 2a and b
illustrate colorful deposits observed inside the wear track for both DLC
and Ag-DLC films. The deposits on the surface covered the worn area
almost entirely for the Ag-DLC film, and only partially for the DLC film.
Deposits were also observed on the surface of the counterparts, as shown
in Fig. 2c and d. Such deposits are reported on the surface of simulator
and explant hip joints and are identified as proteins, where the deposits
act as solid boundary films to protect the sliding surface and reduce wear
[21,22]. In the present study, a continuous biofilm composed of protein
deposits was found at the friction interface of the Ag-DLC film, resulting
in improved wear resistance.
Fig. 3 illustrates the micro-FTIR spectra of the deposits on the
counterparts of DLC and Ag-DLC films, indicating the composition of
protein biofilms. The relationship between the FTIR absorption band
and protein conformation is well established [22,27,28]. Multiple IR
absorption peaks were observed for the deposits on the counterpart in
the 1700–1100 cm− 1 region, such as the amide I band at 1650 cm− 1, CH2
stretching band at 1450 cm− 1, and CH3 stretching band at 1388 cm− 1.
4
Surface & Coatings Technology 422 (2021) 127553
Moreover, the amide III band was between 1400 cm− 1 and 1200 cm− 1,
while the bands at 1300 cm− 1 and 1250 cm− 1 correspond to the α-helix
and random coil structures of proteins, respectively. The position of the
amide I band shifts down with increasing helix length [27,29]. For the
deposits on the Ag-DLC film counterpart, the position of the amide I
band shifted from 1650 cm− 1 to 1655 cm− 1 relative to the DLC film,
indicating a decrease in the helix length. Meanwhile, the band at 1300
cm− 1 was almost undetectable for the deposits on the counterpart of the
Ag-DLC film, representing a loss in the α-helix structure of the protein
biofilm. The results indicate that Ag doping promotes protein denatur­
ation during the wear of films in BSA solutions. The biofilm which is
composed of denatured proteins, forms on the friction interface and
plays a vital role in the wear process. However, the mechanism of bio­
film formation on Ag-DLC films is poorly studied and requires further
study.
3.3. Protein adsorption
Fig. 4 depicts the extent of protein adsorption on the DLC and Ag-
DLC films. The Ag-DLC films showed higher protein adsorption than
DLC film, indicating better protein affinity of the Ag-DLC material. The
DLC films with a larger sp2/sp3 ratio possess more unpaired electrons
and have high reactivity with proteins to form covalent binding [30].
Our previous studies show that the incorporation of Ag increases the
sp2/sp3 ratio of DLC films [23], enhancing the ability of the Ag-DLC film
to adsorb proteins.
To characterize the adsorption of proteins in the friction process,
fluorescein isothiocyanate (FITC) was used to label BSA molecules.
Fig. 5 presents the fluorescence images of wear tracks of DLC and Ag-
DLC films after wear. The FITC-BSA molecules were observed only at
the edge of wear track for DLC film and inside the wear track for Ag-DLC
film. The result indicates that the friction interface of the Ag-DLC film
can significantly adsorb proteins during the friction process. The static
BSA adsorption of the Ag-DLC film was relatively strong, thus the
adsorbed protein accumulated inside the wear track of Ag-DLC films
under the action of friction.
3.4. Ag+ ion release
Notably, metal-doped implants release metal ions when exposed in
physiological solutions [31], and the released metal ions affect the
behavior of proteins. The long-term Ag+ ion release from Ag-DLC films
was quantified until 300 days of immersion, as shown in Fig. 6. The
release rate of Ag ions was relatively fast in the first 3 days and then
gradually leveled off. After immersion for 300 days, the concentration of
Ag+ ions was 1.965 μM. Considering the actual amount of joint fluid
(1–5 ml) [6] and typical size of the prosthesis (ϕ 16 mm to ϕ 55 mm)
[32,33], the concentration of Ag+ ions in the articular cavity is
0.01–0.70 mM.
3.5. Protein denaturation
We investigated the structural changes of BSA molecules in an
enriched Ag+ ion solution using UV, ATR-FTIR, CD, and Raman spec­
troscopy. The solution was diluted to meet the different detection limits
of each instrument.
Fig. 7 shows the UV–visible absorption spectra of BSA and Ag+/BSA
solutions. Two absorbance bands were observed in the range of
200–300 nm for pure BSA. The strong absorbance band at ~206 nm
corresponds to the Π − Π* transition of the polypeptide backbone
structure of BSA [34,35], whereas the relatively weak peak at ~278 nm
originates from aromatic residues and disulfide bonds in BSA [34,36].
Notably, the peak at ~278 nm can provide information about the metal-
BSA association [35]. As shown in Fig. 7, the intensity of the absorption
band at ~278 nm increased after the addition of Ag+ ions. Further, the
addition of metal ions altered the tertiary structure of BSA and exposed
P.P. Jing et al. Surface & Coatings Technology 422 (2021) 127553

Fig. 5. Fluorescence images of wear tracks of (a) DLC and (b) Ag-DLC films after wear in FITC-BSA solutions.

2.5
Ag ion concentration
2.0
Ag ion concentration ( M)

1.5

1.0

0.5

0.0

0 60 120 180 240 300

Immersion time (days)
Fig. 7. UV–visible spectra of BSA and Ag+/BSA solutions.
Fig. 6. Concentration of Ag ions released from 10.0 at.% Ag-DLC film
after immersion.
decreased, and the intensity of β-sheet band at 1236 cm− 1 increased. The
changes in the amide I and III regions confirmed that Ag+ ions promote
the aromatic heterocyclic hydrophobic group of tryptophan and tyrosine
the transformation of α-helix structure to β-sheet and β-turn structures in
residues, resulting in increased absorbance at ~278 nm [37]. Therefore,
BSA molecules, resulting in the loss of helix structure.
the addition of Ag+ ions can promote the exposure of hydrophobic
Further, Fig. 9 shows the secondary structure evaluation of the BSA
groups of BSA molecules. Furthermore, the absorbance maximum at
molecule by comparing the CD spectra for the BSA and Ag+/BSA solu­
~206 nm shifted to a longer wavelength, accompanied by an increase in
tions. The CD spectra of BSA presents two negative minima at 208 and
the peak intensity. The shift was attributed to the disruption of the
222 nm, representing Π − Π* and n − Π* transitions of α-helix,
secondary structure of BSA molecules [24,34].
respectively [34,43]. In this study, the two negative peaks were located
Fourier transform infrared (FTIR) spectroscopy is a valuable tool for
at 213 and 222 nm, while the peak intensity decreased after the addition
investigating protein structure [27]. The amide I and III bands in the
of Ag+ ions. The α-helix content of BSA molecules was calculated using a
infrared spectra, as the superposition of several individual components
previously published method [24–26]. The α-helix content in pure BSA
of the different secondary structure elements, are generally used to
was 65.66%, whereas that of Ag+/BSA was 59.51%, suggesting a loss of
interpret the structure of proteins [38–40]. Characteristic bands and
the α-helix structure induced by Ag+ ions.
corresponding band positions for proteins are widely reported
Fig. 10 presents the Raman spectra of BSA and Ag+/BSA solutions.
[28,41,42]. Fig. 8 shows the characteristic FTIR spectra of BSA and Ag+/
The higher wavenumber region shows two prominent broad bands at
BSA solutions with the amide I and amide III ranges. The addition of Ag+
~3200 and ~3400 cm− 1, corresponding to the hydrated water mole­
ions shifted the amide I band from 1647 to 1653 cm− 1 (Fig. 8a), rep­
cules of the protein [44]. The intensities of these two bands weakened
resenting a decrease in the helix length. Furthermore, the addition of
after the addition of Ag+ ions. Studies show that hydrogen bonding in
Ag+ ions increased the intensities of characteristic β-sheet band at 1630
water molecules changes with an increase in the solution viscosity
cm− 1 and β-turn bands at 1666 cm− 1 and 1681 cm− 1. Contrastingly, in
[44–46], while the OH stretching band is sensitive to the geometry of
the amide III region (Fig. 8b), the intensity of α-helix band at 1295 cm− 1

5
P.P. Jing et al. Surface & Coatings Technology 422 (2021) 127553

Fig. 8. FTIR spectra of BSA and Ag+/BSA solutions in (a) amide I and (b) amide III ranges.

hydrogen bonds [47,48]. The BSA solution became cloudy and the
213 220 fluidity declined after the addition of Ag+ ions (inset image). Thus, we
50
BSA speculated that the addition of Ag+ ions increased the viscosity of the
Ag+/BSA protein solution, modifying the hydrogen bonding, and decreased the
0 intensity of the corresponding water peaks. In the lower wavenumber
region, there was an evident band at approximately 224 cm− 1, assigned
-50 to the Ag-S stretching vibration [49,50]. The peak observed in the low-
frequency region reveals that covalent bonds are formed between the
CD (mdeg)

Ag+ and S atoms. The S atom exists as a component of disulfide bonds

-100
that maintain the spatial configuration of the peptide chain. The Ag–S
bonds indicate that Ag+ ions break the disulfide bonds, loosening the
-150 compact spatial configuration and unfolding the protein structure.

-200
3.6. Mechanism of biofilm production
-250
Based on the above results, we propose a mechanism for biofilm
190 200 210 220 230 240 250
production on the interface of the Ag-DLC films, as shown in Fig. 11. The
Wavelength (nm) incorporation of Ag promoted the protein adsorption on the surface and
friction interface of Ag-DLC films. Furthermore, the Ag+ ions released
Fig. 9. CD spectra of BSA and Ag+/BSA solutions. from Ag-DLC films when exposed in physiological solutions. The
released Ag+ ions break the hydrogen bonding and disulfide linkages in
BSA, promoting the transformation of α-helix to β-sheet and β-turn
structures. This induced protein unfolding with exposure of the inner
hydrophobic groups, leading to protein deposition and formation of
biofilms. The newly formed biofilm covers the friction interface and
plays a crucial role in improving lubrication and reducing wear.

4. Conclusions

DLC and 10.0 at.% Ag-DLC films were prepared by hybrid deposition
technique using graphite and mosaic silver-graphite targets, respec­
tively. Further, friction in BSA solutions caused formation of continuous
deposits on the counterpart of the Ag-DLC film. The deposit, comprising
denatured proteins, formed a layer of biofilm on the friction interface,
thereby improving lubrication and reducing wear. On investigating the
mechanism of biofilm formation, we found that the incorporation of Ag
promoted the protein adsorption on the surface and friction interface of
Ag-DLC films. Thereafter, the released Ag+ ions from Ag-DLC films de­
natured the hydrogen bonding and disulfide linkages of proteins,
causing conformational transformation from α-helix to β-sheet and
β-turn; this induced unfolding of the protein and exposure of inner hy­
Fig. 10. Raman spectra and appearance (inset image) of BSA and Ag+/ drophobic groups. Eventually, the denatured proteins readily deposited
BSA solutions.
on the surface of Ag-DLC film, forming a layer of biofilm. The study
compared the wear properties of DLC and Ag-DLC films in protein so­
lutions and expounded the mechanism for biofilm formation at the

6
P.P. Jing et al.
Fig. 11. Mechanism of biofilm production on the interface of Ag-DLC films.
friction interface of Ag-DLC films. The findings aid the application of Ag-
DLC films in the surface modification of artificial joints. Moreover, metal
doping of internal joint implants can promote the formation of biofilms,
enhancing wear resistance and joint lubrication.
CRediT authorship contribution statement
P.P. Jing: Conceptualization, Methodology, Investigation, Formal
analysis, Writing – original draft, Writing – review & editing. Y.H. Su:
Investigation, Writing – original draft. Y.X. Li: Investigation, Formal
analysis. W.L. Liang: Methodology, Investigation. Y.X. Leng: Concep­
tualization, Formal analysis, Supervision, Funding acquisition.
Declaration of competing interest
No conflict of interest is associated with the publication of this
manuscript.
Acknowledgment
The study was supported by the National Natural Science Foundation
of China (52072312), Sichuan Science and Technology Program
(2020YFH0044, 2018HH0025), and Chengdu Science and Technology
Bureau Program (2020-GH02-00050-HZ).
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