Materials Chemistry and Physics 241 (2020) 122360

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Materials Chemistry and Physics

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

Preparation and in vitro antibacterial properties of anodic coatings

co-doped with Cu, Zn, and P on a Ti–6Al–4V alloy
Yaping Wang a, b, Shuyue Zhao a, Guoqiang Li b, Shufang Zhang b, Rongfang Zhao b,
Anjie Dong a, c, *, Rongfa Zhang b, **
a
Department of Polymer Science and Technology, School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering (Ministry of Education),
Tianjin University, Tianjin, 300072, China
b
School of Materials and Electromechanics, Jiangxi Science and Technology Normal University, Nanchang, 330013, China
c
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300072, China

H I G H L I G H T S

� Anodic coating co-doped with Cu, Zn, and P is prepared on a titanium alloy.
� The entrance mechanisms of Cu and Zn into anodic coating are discussed.
� The competition of Cu ions with Zn ions decreases the Zn content.
� Anodic coatings co-doped with Cu and Zn achieve excellent antibacterial ability.
� Compared with Zn element, Cu does not readily enter anodic coating.

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

Keywords: In this study, copper (Cu) and zinc (Zn) as well as phosphorus (P) were co-doped into a Ti–6Al–4V titanium alloy
Titanium alloys via micro-arc oxidation (MAO). The entrance mechanisms of Cu and Zn into MAO coatings were investigated by
Micro-arc oxidation an orthogonal experiment. The results show that Cu and Zn in Cu–Zn–P coatings separately exist mainly in the
Copper
forms of Cu2O and Zn3(PO4)2. The Cu content in MAO coating increases with increasing EDTA-CuNa2 and KOH
Zinc
Mechanism
concentrations and decreasing phytic acid concentration but is nearly independent of EDTA-ZnNa2 concentra­
tion. The Zn content increases with increasing EDTA-ZnNa2 concentration and decreasing EDTA-CuNa2 con­
centration, indicating the competing effect between Cu and Zn for incorporation into Cu–Zn–P coatings.
Compared with Zn ions, Cu ions do not readily enter anodic coatings due to their simultaneous involvement in
coating formation on the anode and precipitation of metallic copper on the cathode. Cu–Zn–P coatings with
appropriate amounts of Cu and Zn achieve excellent antibacterial ability against Escherichia coli (E.coli), Staph­
ylococcus aureus (S.aureus), and Methicillin-resistant Staphylococcus aureus (MRSA). These antibacterial properties,
in addition to excellent cytocompatibility, make Cu–Zn–P coatings as promising materials for orthopedic
applications.

1. Introduction postoperative complications or traumatization may lead to surgical

Unique features such as good mechanical strength, high chemical
stability, excellent corrosion resistance and biocompatibility make ti­
tanium and its alloys appealing used as implant materials [1]. However,
microbial infections acquired during surgical implantation and ensuing
failure [2]. It is therefore crucial that Ti-based surgical implants are
improved in their antibacterial performance, or even their osteogenesis
and angiogenesis activities. Several examples from literatures demon­
strate that varied inorganic antibacterial agents [3], such as silver [4],
copper [5], zinc [6], Cu/Zn [7], and Zn/Ag [4], can modify an implant

* Corresponding author. Department of Polymer Science and Technology, School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengi­
neering (Ministry of Education), Tianjin University, Tianjin, 300072, China.
** Corresponding author.
E-mail address: ajdong@tju.edu.cn (A. Dong).

https://doi.org/10.1016/j.matchemphys.2019.122360
Received 16 August 2019; Received in revised form 14 October 2019; Accepted 22 October 2019
Available online 24 October 2019
0254-0584/© 2019 Published by Elsevier B.V.
Y. Wang et al. Materials Chemistry and Physics 241 (2020) 122360

surface to promote better antibacterial performance.

As a trace element in human body, Cu ions benefit cardiovascular
system formation by stimulating the proliferation of vascular endothe­
lial cells. Cu ions display several auspicious characteristics such as
antibacterial properties and low cytotoxicity at low concentrations [7].
Zn is also an important trace element in human body and can enhance
osteogenic function, cell proliferation, alkaline phosphatase (ALP) ac­
tivity, and osteoblast marker gene expression [8,9]. Together, Cu and Zn
are indispensable for human body, wherein they protect and support the
cardiovascular system [7], regulate metabolic activities, improve
osteogenesis, and promote osteointegration of metal implants with bone
tissue [8,9]. By coating a titanium implant with both Cu and Zn, we can
achieve excellent biocompatibility, good antibacterial and osteogenic
properties.
Micro-arc oxidation (MAO) is a simple and effective electrochemical
method for producing porous, relatively rough, and strongly adhering
titanium oxide coatings on titanium alloy surfaces [10,11]. We have
previously demonstrated that MAO can effectively deposit ceramic
coatings on magnesium [12,13] and titanium [14,15] alloys by using
phytic acid as the main electrolyte. In order to fabricate antibacterial
MAO coatings on titanium alloys, the used MAO electrolytes typically
contain one kind of cation [16], for example, copper [17] or zinc [9]. At
present, the entrance mechanism of co-implanted Cu and Zn ions is not
reported. Herein, a multi-functional Cu–Zn–P coating is deposited on
surface of Ti–6Al–4V via MAO in a special electrolyte system consisting
of EDTA-CuNa2, KOH, phytic acid, and EDTA-ZnNa2. The mechanism of
Cu and Zn into MAO coatings is discussed according to the influences of
four processing factors on coating characteristics. In addition, this work
further analyzes the synergistic effects of Cu and Zn contents on anti­ Fig. 1. The schematic diagram showing the coating formation during
microbial performance and evaluates in vitro cytocompatibility of MAO treatment.
MAO-treated samples by CCK-8 assay.

2. Experimental Table 1
Extreme difference analyses and the orthogonal experiment results, as measured
2.1. Sample preparation by Cu and Zn contents as function of four different factors.(As for K, the numbers
in parentheses refer to values for Zn, while the other numbers refer to Cu. A rank
of 1 corresponds to the factor with the greatest impact and 4 corresponds to the
Prior to MAO treatment, commercially available 30 � 30 � 3 mm3
factor with the lowest impact.)
Ti–6Al–4V plates were sequentially ground with SiC paper from 80 to
Process EDTA- KOH (g/ Phytic EDTA- Cu Zn
1000 grits, degreased with acetone, washed with distilled water, and
No. CuNa2 (g/ L) acid (g/ ZnNa2 (wt. (wt.
dried in a hot air stream. MAO coatings were deposited using a MAO5D L) L) (g/L) %) %)
power supply (Chengdu Tongchuang New Material Surface Engineering
No. 1 2 3 5 2 0 3.47
and Technology Center) under a constant current control mode. During
No. 2 2 6 8 6 0 9.84
MAO treatment, two Ti–6Al–4V samples separately served as the anode No. 3 2 9 11 10 0 7.90
and the cathode. The electrolyte solution consisted of phytic acid, KOH, No. 4 6 3 8 10 0.61 11.41
EDTA-CuNa2, and EDTA-ZnNa2. The electrical parameters were fixed at No. 5 6 6 11 2 0.98 4.42
No. 6 6 9 5 6 2.15 5.42
current density of 110 mA/cm2, treating time of 3 min, pulse frequency
No. 7 10 3 11 6 0 5.64
of 2000 Hz, and duty cycle of 35%. The schematic of the MAO experi­ No. 8 10 6 5 10 1.25 6.71
ment was depicted in Fig. 1. No. 9 10 9 8 2 4.18 2.89
Based on our previous work, the selected factors and levels of the K1 0 (21.21) 0.61 3.4 5.16
orthogonal experiment were listed in Table 1. According to the L9 (34) (20.52) (15.6) (10.78)
K2 3.74 2.23 4.79 2.15
table, nine different samples should be MAO-treated according to the set
(21.25) (20.97) (24.14) (20.9)
processing parameters. K3 5.43 6.33 0.98 1.86
(15.24) (16.21) (17.96) (26.02)
2.2. Surface characterization Difference 5.43 (6.01) 5.72 3.81 3.3
(4.76) (8.54) (15.24)
Rank 2 (3) 1 (4) 3 (2) 4 (1)
Surface morphologies and chemical compositions of the MAO-
treated Ti–6Al–4V were characterized by a field emission scanning
electron microscope (SEM, ΣIGMA, Germany) with an attached energy 2.3. Cu and Zn concentration measurements
dispersive X-ray spectrometer (EDS). Phase structure of the measured
samples was analyzed by X-ray diffraction (XRD, Shimadzu XRD-6100, During MAO, both ion migration in the electrolyte solution and ion
Japan). An X-ray photoelectron spectrometer (XPS, ESCALAB250, dispersion near the electrodes are strongly related with their entrance
Thermo VG, USA) with an Al Kα anode (λ ¼ 1486.6 eV) was used to mechanism into MAO coatings. To analyze the possible mechanism,
detect chemical states on surfaces of MAO coatings. three samples of the electrolyte solution were simultaneously collected
by syringes from the anode area, the cathode area, and the middle area.
Cu and Zn concentrations in the electrolyte solution were analyzed after

2
Y. Wang et al.
40 s and 120 s of MAO treatment by an inductively coupled plasma
optical emission spectrometer (ICP-OES) (Thermo 6300, American
Thermo Fisher Company, USA).
2.4. Antibacterial test
Prior to MAO treatment, Ti–6Al–4V alloy samples were machined
into cuboidal shapes (10 mm � 10 mm � 2 mm or
50 mm � 50 mm � 2 mm), progressively ground with sandpaper from 80
to 1000 grits, washed with distilled water, dried under hot air, and
stored in a desiccator. Then, Ti–6Al–4V samples were oxidized and
measured for their antibacterial activity. According to Chinese standards
QB/T 2591–2003, the antibacterial effects of the MAO samples against
Methicillin-resistant Staphylococcus aureus (MRSA) (ATCC43300),
Escherichia coli (E.coli) (CGMCC13373), and Staphylococcus aureus (S.
aureus) (CGMCC12465) were assayed using the plate coating method.
The tested samples were first soaked in a 75 vol% ethanol solution and
exposed to ultraviolet radiation for 30 min. Next, 0.2 mL of a 106 CFU/
mL bacterial solution was dripped onto the MAO samples and the control
material (slide). The bacterial solution was collected after incubation at
37 � 1 � C for 24 h and the active bacteria were counted in accordance
with the standard method. Finally, the antibacterial rate (AR) was
determined by the following equation:
N1 N2
ARð%Þ ¼ 100 � (1)
N1
where N1 and N2 are the bacterial counts on the plastic sheet control and
the MAO-treated sample, respectively. All experiments were conducted
using three replicates in separate samples.
2.5. Cell culture
Osteosarcoma cells (MG63) were cultured at 37 C in Dulbecco’s
�
modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal
calf serum (FBS, Gibco) and 100 units/mL penicillin and streptomycin.
The incubator was humidified with an atmosphere of 5% CO2. Prior to
the experiment, all samples were sterilized at 121 � C in a high temper­
ature and high pressure environment, then immersed in 75 vol% ethanol
for 30 min, and finally rinsed three times with sterile phosphate buffered
saline (PBS, Gibco).
2.6. Initial cell attachment and cell viability assay
To assess initial cell attachment and proliferation, MG63 cells were
seeded directly onto each of the Ti-6A1-4 V samples at a density of
2 � 104 cells/well in 24-well plates. The cells were allowed to settle for
4 h during the initial cell attachment. Then, the inoculated samples were
washed twice with PBS and fixed with 3.7% formaldehyde in PBS for
5 min. To rupture the cell membranes, we piped 1 mL of 0.1% Tritonx-
100 into the 24-well plates over the course of 5 min. The cytoskeletal
protein F-actin was then stained with phalloidin-fluorescein isothiocy­
anate (Sigma) for 40 min at room temperature, and the nuclei were
counterstained with DAPI (Sigma, USA). Cell images were captured by a
fluorescence microscope (Olympus-IX71, Japan), and cell proliferation
was assessed using CCK-8 (Beyotime, China) to examine the cell cultures
on the Ti-6A1-4 V samples after 1, 3, and 7 days. At each time interval,
the samples were rinsed with PBS and transferred to a new 24-well plate.
DMEM containing 10% CCK-8 was added to each well and incubation
proceeds for an additional 2 h at 37 � C before 100 μL solution was
transferred to a new 96-well plate. Absorbance was measured at 450 nm
on a microplate spectrophotometer (Thermo Fisher Scientific, USA).
2.7. Statistical analysis
All experiments were repeated three times and the statistical analysis
3
Materials Chemistry and Physics 241 (2020) 122360
results were expressed as the mean � one standard deviation (SD). A
value of p < 0.05 was considered to indicate a statistically significant
difference between groups.
3. Results
3.1. Surface characteristics of Cu–Zn–P coatings
According to the processing conditions listed in Table 1, nine Ti-6A1-
4 V samples were treated and their surface morphologies are shown in
Fig. 2. As demonstrated in Fig. 2a, no surface deposit is observed on
Sample No. 1, indicating that the processing conditions are not suitable
to form a Cu–Zn–P coating. The deposits observed in Fig. 2b–i exhibit
smooth and generally porous structures, indicating the successful
development of Cu–Zn–P coating on Samples No. 2–9, although the pore
size and the inter-pore distance varies from one sample to another.
Interestingly, the MAO coatings on Samples No. 4, No. 5, and No. 7
(Fig. 2d, e, and 2g) exhibit both larger pore sizes and larger inter-pore
distances relative to the other samples. Together, the electrolyte com­
positions listed in Table 1 and the SEM morphologies shown in Fig. 2
imply that high KOH concentration (Fig. 2f and i) and low phytic acid
concentration (Fig. 2a, f and 2h) are not conducive to the formation of a
Cu–Zn–P coating with uniform pore size distribution and high pore
density.
In addition to surface morphology, chemical compositions of
Cu–Zn–P coatings are affected by MAO processing parameters, so the
fabricated anodic coatings are analyzed by EDS. Table 2 lists the coating
compositions, which mainly consist of C, O, P, Cu, Zn, Ti, and a small
amount of K, indicating that the substrate and used electrolytes partic­
ipate in coating formation.
3.2. XRD analysis
According to Table 2, Samples No. 9 and No. 4 separately achieve the
highest Cu and Zn contents, while Samples No. 6 and No. 2 separately
have the second highest Cu and Zn contents. Therefore, Samples No. 2,
No. 4, No. 6, and No. 9 serve as representative samples for further sys­
tematic characterization of chemical composition, phase structure, and
the chemical state of elements in MAO coatings.
The XRD spectra of Samples No. 2, No. 4, No. 6, and No. 9 are pre­
sented in Fig. 3. Substances containing Cu and Zn are not identified. It
appears that Zn and Cu may exist in amorphous states. Therefore, the
elemental existing states in MAO coatings will be further analyzed using
high-resolution XPS spectrum.
3.3. XPS analysis
According to Fig. 4a, Sample No. 9 contained C, O, Cu, Zn, P, and Ti
elements. More specifically, the C 1s peak with a binding energy of
284.6 eV indicates the presence of C–C or C–H bonds (Fig. 4b). The Cu
2p spectrum (Fig. 4c) exhibits two peaks at 932.5 eV and 952.6 eV,
which correspond to Cu 2p3/2 and Cu 2p1/2, respectively. The lack of
shake-up satellite peaks indicate that Cu mainly exists in the form of
Cu2O [17–19]. The Zn 2p spectrum (Fig. 4d) depicts two peaks at
1022.5 eV and 1045.6 eV, which are attributed to Zn3(PO4)2 [20]. The P
2p spectrum (Fig. 4e) displays two peaks at 132.9 eV and 133.8 eV,
corresponding to PO4 3- and HPO4 2-, respectively [21]. Finally, the Ti 2p
spectrum (Fig. 4f) includes peaks at 458.3 eV and 464.0 eV, which are
attributed to typical binding energies of TiO2 [21].
3.4. The orthogonal results
The measured Cu content and Zn content are set as the objective
parameters, and the intuitionistic analysis is used to analyze the
experimental data. K1, K2, and K3 listed in Table 1 separately represent
the summation of the objective parameters corresponding to Level 1,
Y. Wang et al. Materials Chemistry and Physics 241 (2020) 122360

a b c

10 um

d e f

g h i

Fig. 2. Surface morphologies of anodic coatings developed under different processing parameters: (a) No. 1; (b) No. 2; (c) No. 3; (d) No. 4; (e) No. 5; (f) No. 6; (g) No.
7; (h) No. 8; (i) No. 9.

decreases with increasing EDTA-ZnNa2 concentration. The overall trend

Table 2
indicates that Cu ions bind with phytate to form a negatively-charged
Elemental compositions of anodic coatings developed on nine MAO treated
chelate and migrate toward the anode.
samples (wt.%).
According to Fig. 5, the dependence of the Zn content on each factor
Process No. C O P K Ti Cu Zn
is distinct from and more complicated than that of the Cu content,
No. 1 5.91 32.46 19.93 0.26 37.97 – 3.47 namely that the Zn content curves exhibit “turning points” wherein the
No. 2 4.78 30.40 18.35 0.85 35.79 – 9.84 trend of increasing or decreasing Zn is reversed. More specifically, the
No. 3 3.95 29.64 16.02 0.78 41.70 7.90
Zn content (1) increases with increasing EDTA-ZnNa2; (2) reaches a
–
No. 4 5.59 30.82 21.13 0.34 30.10 0.61 11.41
No. 5 – 33.61 23.84 0.45 36.70 0.98 4.42 maximum at Level 2 of KOH concentration; (3) reaches a maximum at
No. 6 – 29.33 10.26 0.60 52.24 2.15 5.42 Level 2 of phytic acid concentration; and (4) decreases with increasing
No. 7 3.25 33.12 22.93 – 35.05 – 5.64 EDTA-CuNa2. This is consistent with the combination of Zn ions with
No. 8 4.15 29.85 16.96 0.59 40.48 1.25 6.71
phytate cleavage products to form negatively charged chelates and
No. 9 5.70 29.80 18.46 2.16 36.09 4.18 2.89
migrate toward the anode, where they bond with PO4 3- from phytic
cleavage to form Zn3(PO4)2 on anode surface.
Level 2, and Level 3 for each processing factor. The maximum difference
between K1, K2, and K3 at two levels of each factor indicates the general
3.5. Cu and Zn concentration
influence of that factor. A greater difference denotes that a factor has a
more significant impact on the objective parameters. Based on the
Sample No. 8, which is processed in electrolyte solution comprised of
values of the differences listed in Table 1, the influencing sequence of
10 g/L EDTA-CuNa2, 6 g/L KOH, 5 g/L phytic acid, and 10 g/L EDTA-
the four factors on the Cu content is KOH concentration > EDTA-CuNa2
ZnNa2 as summarized in Table 2, is selected for further study as a
concentration > phytic acid concentration > EDTA-ZnNa2 concentra­
representative MAO coating due to its relatively high Cu and Zn con­
tion. However, the trend for the Zn content is EDTA-ZnNa2 concentra­
tents. Of particular interest are the migration of Cu and Zn ions in the
tion > phytic acid concentration > EDTA-CuNa2 concentration > KOH
electrolyte solution, their distribution near the electrodes, and the
concentration.
mechanism of their entrance into the MAO coating.
The achieved results in Table 1 are plotted in Fig. 5 to exhibit the
A general phenomenon observed during the MAO process is the
changing trends of the Cu content and Zn content with the levels of the
absence of sparking on the Ti–6Al–4V anode surface sample during the
four processing factors. The qualitative variation of Cu content with
first 40 s of MAO treatment. However, by 120 s of treatment, many white
each component can be briefly summarized as follows: (1) the Cu con­
sparks appear. Fig. 6 illustrates the Cu and Zn concentrations in the
tent increases with increasing EDTA-CuNa2 concentration, first
electrolyte solution collected after 40 s and 120 s of MAO treatment from
dramatically from Level 1 to Level 2, but only slightly from Level 2 to
the anode area, the cathode area, and the middle area (Fig. 6). ICP
Level 3, indicating that diffusion plays an important role on Cu into
analysis reveals that at 40 s the Cu concentrations in the anode, middle,
anodic coatings; (2) the Cu content increases with increasing KOH
and cathode regions are 1.3959 � 0.0101, 1.3839 � 0.0061, and
concentration; (3) the Cu content increases first but then decreases with
1.3983 � 0.0047 g/L, respectively, and that at 120 s they are
the increase of phytic acid concentration; and (4) the Cu content
1.3889 � 0.0226, 1.3923 � 0.0093, and 1.3886 � 0.2122 g/L,

4
Y. Wang et al.
2000
--Titanium
1800
1600
1400
1200
Intensity(a.u.)
1000 No. 9
800 No. 6
600
No. 4
400 No. 2
200
Ti-6Al-4V
0
0 10 20 30
2 (degree)
Fig. 3. The X-ray diffraction patterns of Ti–6Al–4V alloy and MAO coatings developed on No. 2, No. 4, No. 6 and No. 9.
respectively. Zn concentrations at 40 s in the anode, middle, and cathode
regions are 1.4532 � 0.0232, 1.5072 � 0.2730, and 1.5029 � 0.0106 g/
L, respectively, and at 120 s they are 1.5139 � 0.0017, 1.4736 � 0.2250,
and 1.4522 � 0.0100 g/L, respectively.
3.6. Cathode characteristics after MAO treatment
When a stainless steel container served as the cathode, we found that
a red metal-like substance was developed on the cathode surface after
MAO treatment. In order to reveal reaction process, a plastic barrel is
used in this study to contain MAO electrolytes and a Ti–6Al–4V sample is
connected with the cathode. By using the processing conditions for
preparation Sample No. 9, the corresponding cathode characteristics are
shown in Fig. 7. It is clear that the untreated sample exhibits metallic
sheen (Fig. 7a), whereas the cathode becomes deep red (Fig. 7b). The
SEM image in Fig. 7c shows clustered petal deposits on the cathode
surface. XRD and EDS indicate that the deposits are mostly comprised of
metallic copper (Fig. 7d and e), which accounts for the color change of
the titanium alloy cathode to a reddish color after MAO treatment
(Fig. 7b).
3.7. Antibacterial properties
Fig. 8 shows typical MRSA, E.coli, and S.aureus colonies cultured on
control and MAO samples for 24 h. Fig. 8a1-a4 reveal that many MRSA
colonies occupy the control, but no colony appeares on Sample No. 2,
which has high zinc content, or Sample No. 9, which has high copper
content. A few colonies appear on Sample No. 6. Similarly, Fig. 8b1
demonstrates that many S.aureus colonies grow on the control, but
virtually none appeares on the MAO-treated samples (Fig. 8b2-8b4). For
E.coli, a similar trend appears on the control and MAO-treated samples,
namely, growing freely on the control and practically absent on the
MAO-treated samples (Fig. 8c1-8c4). Overall, these results clearly
demonstrate that the MAO-treated samples possess superior antibacte­
rial properties compared to the control.
3.8. Initial cell attachment
Fig. 9 shows that after 4 h incubation, there are more MG63 cells on
5
Materials Chemistry and Physics 241 (2020) 122360
--Anatase
40 50 60 70 80 90
the surfaces of Samples No. 2, No. 6, and No. 9 than on the control. For
all samples, the cells are polygonally- or spindle-shaped and display
evident filose pseudopodiums and flat membranes. Compared to Sam­
ples No. 2, the cells on Samples No. 6 and No. 9 exhibit more spreading
and superior filopodia extension, which can promote initial cell
attachment.
3.9. In vitro cytocompatibility
Cell proliferation gradually increases throughout the culturing
period, as measured at 1, 3, and 7 days (Fig. 10). The cells on Samples
No. 2, No. 6, and No. 9 exhibit lower proliferation rates than that of the
untreated control on day 1 (p < 0.05; Fig. 10). This trend still persists at
day 3 (p < 0.05, 0.05, and 0.01, for Samples No. 2, No. 6, and No. 9,
respectively). However, at day 7, the proliferation rates of the MAO-
treated samples exceed that of the control (p > 0.05, Fig. 10).
4. Discussion
In a typical electrochemical system, cations move towards the
cathode and anions move towards the anode. By contrast, both anions
and cations in MAO electrolytes arrive first at the anode-electrolyte
interface and then enter into the MAO coating. The movement of an­
ions towards the anode is aided by diffusion and electromigration, while
cation movement is typically driven by diffusion alone [22–24].
Nevertheless, our previous research verified that in an electrolyte solu­
tion containing a chelating agent, e.g., phytic acid, cations first combine
with the chelating agent to develop negatively charged complexes and
then move towards the anode by diffusion or electromigration [1].
Therefore, it is important to identify the formation process of MAO
coatings and the entrance mechanism of Cu and Zn into anodic coating.
4.1. Mechanism of Cu ion entrance into MAO coatings
ICP analysis indicates that, regardless of whether MAO treatment
proceeds for 40 s or 120 s, the Cu ion concentration near the anode
approximates that near the cathode. According to EDS, XRD, and XPS
analysis, Cu2þ in the electrolyte is ultimately deposited on the anode and
cathode in the form of Cu2O and metallic Cu, respectively. It can
Y. Wang et al. Materials Chemistry and Physics 241 (2020) 122360

300000 45000

250000
a O 1s 40000 b 284.6eV
35000

200000
30000

Counts(/s)
150000 Ti 2p 25000

Zn 2p
Cu 2p 20000
100000
15000
Zn LMM

50000 P 2s 10000
P 2p C 1s
5000
0
0
-200 0 200 400 600 800 1000 1200 1400 270 275 280 285 290 295 300
Binding energy(eV) Binding energy(eV)

54000
Cu 2p
50000
c 932.5eV 52000 d Zn 2p

50000 1022.5eV
45000
48000
Counts(/s)

40000

Counts(/s)
46000 1045.6eV
952.6eV
44000
35000
42000

30000 40000

38000
25000
36000

920 930 940 950 960 970 1010 1020 1030 1040 1050 1060
Binding energy(eV) Binding energy(eV)

P 2p Ti 2p
f
70000
e
9000

132.9eV 60000
458.3eV
8000

50000
7000
Counts(/s)
Counts(/s)

40000 464.0eV
6000
133.8eV 30000
5000
20000

4000
10000

3000
0
440 445 450 455 460 465 470 475 480 485
120 125 130 135 140 145
Binding energy(eV)
Binding energy(eV)

Fig. 4. XPS spectra of anodic coatings obtained in the solution containing 8 g/L phytic acid, 10 g/L EDTA-CuNa2, 2 g/L EDTA-ZnNa2, and 9 g/L KOH: (a) survey; (b)
C 1s; (c) Cu 2p; (d) Zn 2p; (e) P 2p; (f)Ti 2p.

therefore be inferred that some Cu2þ ions from EDTA-CuNa2 combine described by the following chemical reaction equations:
with the chelating agent, phytic acid (e.g., IP6), or its hydrolysis prod­
IP6 þ H2O ¼ IP5 þ H3PO4 (2)
ucts (e.g., IP5, IP4, IP3, etc.) to form a negatively charged complex
anion, which then travels to the anode via electromigration, and is
HO OH
finally deposited as Cu2O in MAO coatings. Meanwhile, some Cu2þ ions HO OH
P
P O O OH
in the electrolyte solution simply migrate toward the cathode and are O O OH
P OH O P O
OH O HO
deposited on the cathode surface as metallic Cu. As a result, the MAO HO
P O OOH + H-OH P O OH
+ HO P OH
O O OH
coating on the anode always exhibits a Cu content lower than that of Zn, O O O O O OH
P HO O O
HO O O HO OH P
which is confirmed in Table 2. P
O O P
P HO
The deposition of Cu ions onto the anodic MAO coating can be HO HO OH HO OH

6
Y. Wang et al. Materials Chemistry and Physics 241 (2020) 122360

IP5 þ H2O ¼ IP4 þ H3PO4 (3)

HO OH
P O OH O OH
O
OH O P OH OH P O
HO OH HO OH
P O + H-OH P O + HO P OH
O OH O OH
O O O O OH
HO O O HO O O
P P
O P O P
HO HO HO OH
HO OH

based substance. According to ICP analysis, after 40 s of MAO treatment,

Cu 2þ
þ IPn–
6–n ¼ [Cu(IPn)]
n 2
Cu2þ þ 2OH ¼ Cu(OH)2
Cu(OH)2 þ 2e– ¼ Cu þ 2OH
2Cu(OH)2 þ 2e ¼ Cu2O þ H2O þ 2OH –
The “turning points” in Cu content as a function of KOH and phytic
acid concentrations, which were discussed above, may be attributed to
the ability of the KOH-based alkaline electrolyte system to ionize EDTA-
CuNa2 and hydrolyze phytic acid (IP6). Together, these actions promote
Cu2þ migration towards the anode in the form of a complex anion,
consequently increasing Cu content in the MAO coating. As a result,
KOH concentration is a key factor for controlling the Cu content of
anodic coatings. By contrast, of the four electrolyte components studied,
EDTA-ZnNa2 concentration plays the smallest effect on the Cu content,
which may be resulted from the relatively high stability constant of the
Cu2þ complex than that of Zn2þ complex [25]. Consequently, Zn2þ does
not seem to compete with Cu2þ to form negatively charged complex
Zn concentration is lower near the anode (1.45 g/L) than the cathode
(4) (1.50 g/L), which may be attributed to the inability of the small electric
(5) field to induce electromigration of Zn ions. Therefore, diffusion could be
expected to play a major role in the movement of Zn ions toward the
(6) cathode. Furthermore, Zn2þ cations migrate toward the cathode more
readily and quickly than negatively charged Zn complexes migrate to­
(7)
wards the anode.
Interestingly, after 120 s of MAO treatment, Zn concentration is
higher near the anode (1.51 g/L) than near the cathode (1.45 g/L). Since
Zn exists as Zn3(PO4)2 in the MAO coating, we can infer that Zn2þ
migration towards the anode is induced mainly by electromigration.
Therefore, the entrance mechanism of Zn ions into MAO coating can be
described as follows: Zn2þ ions from EDTA-ZnNa2 first combines with
1.8
Anode region
1.6
The middle area
a 1.4
Cathode region
Cu ion concentration(g/L)

which favor migration towards the anode. 1.2

1.0

4.2. Mechanism of Zn ion entrance into MAO coatings

0.8

EDS, XRD, and XPS analysis results indicate that Zn2þ in the elec­ 0.6

trolyte is ultimately deposited onto the anodic MAO coating as 0.4

Zn3(PO4)2, and that the cathodic MAO coating is virtually free of any Zn-
0.2

30 0.0
Cu 40 s 120 s
Zn Treating time(s)
25
a b c 2.0
d
Element content(wt.%)

Anode area
The middle area
b
1.8
20
Cathode area
1.6
Zn ion concentration(g/L)

15 1.4

1.2
10 1.0

0.8
5
0.6

0.4
0
0.2

1 2 3 1 2 3 1 2 3 1 2 3 0.0
40 s 120 s
Factors(Level) Treating time(s)

Fig. 5. Effects of factors on the content of Zn and P elements in anodic coatings: Fig. 6. The copper concentration at 40 s and 120 s in anode area, the middle
(a) EDTA-CuNa2 concentration; (b) KOH concentration; (c) phytic acid con­ area and cathode area (a). The zinc concentration at 40 s in anode area, the
centration; (d) EDTA-ZnNa2 concentration. middle area, and cathode area (b).

7
Y. Wang et al. Materials Chemistry and Physics 241 (2020) 122360

a b c

10mm
Before MAO

1600
500 nm
1400
d --Titanium
--Cu

1200

1000
e
Intensity(a.u.)

800
The cathode sample
600

400
Ti-6Al-4V
200

0 keV
0 10 20 30 40 50 60 70 80 90

2 (degree)

Fig. 7. Pictures of (a) the substrate and (b) the cathode after MAO treatment, and (c) the SEM image, (d) X-ray diffraction patterns, and (e) EDS spectrum of the
cathode after MAO treatment in the solution of 10 g/L EDTA-CuNa2, 9 g/L KOH, 8 g/L phytic acid and 2 g/L EDTA-ZnNa2.

MRSA

S.aureus

E.coli

Ti-6Al-4V No. 2 No. 6 No. 9

Fig. 8. Typical growth of MRSA, S.aureus and E.coli colonies after 24 h incubation on a titanium substrate control, No. 2, No. 6, and No. 9.

8
Y. Wang et al. Materials Chemistry and Physics 241 (2020) 122360

4h
24h

Ti-6Al-4V No. 2 No. 6 No. 9

Fig. 9. Fluorescent images of MG63 cells after culturing for 4 h and 24 h on titanium substrate and the MAO samples of No. 2, No. 6, No. 9.

optimal concentrations of both KOH and phytic acid to maximize Zn

1.8
Ti-6Al-4V content in the MAO coating.
1.6 No. 2
No. 6
1.4 No. 9 4.3. Biocompatibility and antibacterial ability of Cu–Zn–P coatings

As necessary trace elements for human body and inorganic anti­

Absorbance at 450nm

1.2
bacterial agents, Cu and Zn are introduced into titanium alloy surface by
1.0
some treatment methods [26]. For coatings doped with Cu and Zn, the
0.8
biocompatibility has become a hot topic. However, the relationship
between Zn and Cu content and biocompatibility has rarely been studied
0.6 [27]. Zinc-containing coatings are biocompatible for rat bone marrow
stromal cells and present an increased proliferation and ALP expression
0.4 [26]. Cu can stimulate the activity of several enzymes and plays a role in
the cross-linking of collagen and elastin of bones [28]. In our work, we
0.2
used MAO method to co-dope Cu and Zn into MAO coatings. Samples
0.0 No. 9 and No. 4 separately achieve the highest Cu and Zn contents, while
Day1 Day3 Day7 Samples No. 6 and No. 2 separately have the second highest Cu and Zn
contents. According to Table 2, Sample No. 2 contained 9.84 wt% Zn,
Culture time while Samples No. 6 and No. 9 separately contain 2.15 wt% Cu and
Fig. 10. Proliferation of the MG63 cell cultured after 1, 3 and 7 days of growth 5.42 wt% Zn, and 4.18 wt% Cu and 2.89 wt% Zn. As shown in Fig. 10, no
on a titanium substrate control, as well as MAO-treated samples of No. 2, No. 6, cytotoxicity is found on Samples No. 2, No. 6 and No. 9. Cu is an effective
and No. 9. inorganic antibacterial agent, while Zn has a weak antibacterial effect
[29]. The results show that Sample No. 2 and No. 9 achieve the anti­
the chelating agent phytic acid (e.g., IP6) or its hydrolysis products (e.g., bacterial ability of 100% both against E.coli, S.aureus and MRSA
IP5, IP4, IP3, etc.) to form negatively charged complexes. Next, these Zn- (Fig. 8). However, for Sample No. 6, the antibacterial rate is very close to
containing complexes move towards the anode mainly by electro­ 100% but several negligible small colonies appear on agar plates. The
migration. Finally, Zn2þ ions bind with PO34 hydrolyzed from phytic reason is that the antibacterial effect of Zn element is smaller than that of
acid to develop Zn3(PO4)2 into MAO coatings. copper. This phenomenon also confirms the importance of the syner­
The process of Zn ion deposition onto the MAO coating can be gistic effect of Cu and Zn on coating antibacterial ability. The fabricated
described by the following chemical reaction equations: Cu–Zn–P coatings can achieve satisfactory antibacterial effect and
ensure the Cu and Zn contents within the safe range. These results show
Zn2þ þ IPn–
6–n ¼ [Zn(IPn)]
n 2
(8) that the MAO sample achieved good antibacterial property and has
H3PO4 ¼ 3Hþ þ PO34 (9) powerful potential in orthopedic applications.

3Zn 2þ
þ 2PO34 ¼ Zn3(PO4)2 (10) 5. Conclusions
Therefore, the following reactions may take place at the cathode:
In this study, Cu–Zn–P MAO coatings were successfully fabricated on
2Hþ þ2e ¼ H2 φΘ H þ = H ¼ 0jV Ti–6Al–4V alloy and the effects of EDTA-CuNa2, KOH, phytic acid, and
EDTA-ZnNa2 on coating compositions were studied by using the
Cu2þ þ 2e ¼ Cu φΘCu2þ =Cu ¼ 0:34V orthogonal experiment of four factors with three levels. In vitro cyto­
compatibility and antimicrobial performance of Cu–Zn–P coatings were
Zn2þ þ 2e þ 2e ¼ Zn φΘZn2þ =Zn ¼ 0:762V evaluated. The conclusions of this study are as follows:

These equations highlight two important phenomena: (1) Cu2þ can (1) The sequence of the four processing factors on the Cu content in
evidently compete with Zn2þ to form complex anions which migrate the Cu–Zn–P coatings is KOH concentration > EDTA-CuNa2 con­
toward the anode and therefore EDTA-CuNa2 concentration is the third centration > phytic acid concentration > EDTA-ZnNa2 concen­
most important factor on the Zn content (Table 1); and (2) there may be tration, while the order on the Zn content is EDTA-ZnNa2

9
Y. Wang et al.
concentration > phytic acid concentration > EDTA-CuNa2 con­
centration > KOH concentration.
(2) The Cu content in MAO coating increases with increasing EDTA-
CuNa2 and KOH concentrations and decreasing phytic acid con­
centration but is nearly independent of EDTA-ZnNa2 concentra­
tion. The Zn content in anodic coating increases with increasing
EDTA-ZnNa2 concentration and decreasing EDTA-CuNa2 con­
centration, indicating the competing effect between Cu and Zn for
incorporation into Cu–Zn–P coatings.
(3) Compared with Zn ions, Cu ions are difficult to enter into anodic
coatings due to their simultaneous involvement in coating for­
mation on the anode and precipitation of metallic copper on the
cathode.
(4) Because of excellent cytocompatibility and antibacterial ability,
Cu–Zn–P coatings with appropriate amounts of Cu and Zn con­
tents are promising used as implant material.
Acknowledgment
The study was financially supported by the National Natural Science
Foundation of China (51661010).
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