Vietnam Journal of Chemistry - 2018 - Nam - Synthesis Characterization and Antimicrobial Activity of Copper Doped

Cite this paper: Vietnam J. Chem.
, 2018, 56(6), 672-678 Article

DOI: 10.1002/vjch.201800068
Synthesis, characterization and antimicrobial activity of copper doped

hydroxyapatite
Pham Thi Nam1*, Nguyen Thi Thom1, Nguyen Thu Phuong1, Nguyen Thi Xuyen1,
Nguyen Song Hai2, Nguyen Tuan Anh3, Pham Tien Dung4, Dinh Thi Mai Thanh1,5,6
1
Institute for Tropical Technology, Vietnam Academy of Science and Technology, Viet Nam
2
Military Institute of Traditional Medicine, 442 Kim Giang, Hoang Mai, Hanoi, Viet Nam
3
Viet Tri Central Preparatory School for Ethnic Groups, 19 Tran Phu, Viet Tri, Phu Tho, Viet Nam
4
Basic Science Faculty, Hanoi University of Mining and Geology, Viet Nam
5
Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Viet Nam
6
University of Science and Technology of Hanoi, Vietnam Academy of Science and Technology, Viet Nam
Received March 13 2018; Accepted for publication June 16 2018
Abstract
Copper hydroxyapatite coatings (CuHAp) were synthesized on 316L stainless steel by electrodepositon and ion
exchange methods. Effect of Cu2+ initial concentration and contact time to ion exchange process was investigated. The
FTIR, SEM, XRD and EDX measurements of the synthesized samples showed that CuHAp coatings were single
crystalline phase of HAp with %Cu of 0.08 (% mass) in Cu 2+ initial solution of 0.05 M. The presence of Cu2+ reduced
the HAp crystalline size and enabled to obtain samples with more uniform surface. The antimicrobial activity of
CuHAp coatings was tested. CuHAp coatings had good resistance to E. coli and S. aureus that antibacterial ratio
reached 89 % and 98 %, respectively.
Keywords. Copper hydroxyapatite coatings, antimicrobial activity, ion exchange.
1. INTRODUCTION growth of bacteria.[7] In addition, the small amount

of Cu also acts as an agent to enhance metabolism
Hydroxyapatite (HAp) is the main inorganic and stimulate bone growth.[8]
component of bones and teeth of vertebrate. So, There are many researches on the synthesis of
HAp has high bioactive and biocompatible. A thin CuHAp by different methods. But these synthetic
layer of HAp covering on the implant surface will methods have an effect on toxicity of CuHAp. Kim
provide the required osteointegration.[1] The et al. reported that by co-precipitation method,
postoperative infection is a major problem for the CuHAp synthesized in solution containing Ca(OH)2,
bone implant material.[2] This infection becomes H3PO4 and Cu(NO3)2 did not show any antibacterial
dangerous and must be revision of surgeries if it is effect on E. coli.[9] While, by ion exchange method
not treated properly. Therefore, the biomedical and using Ca(NO3)2, Na3PO4, NaOH and CuSO4 as
devices with antimicrobial property are very starting materials, CuHAp was successfully
significant to avoid infections after surgery. With its synthesized with strong antibacterial activity on E.
own stability and safety, inorganic antimicrobial coli and S. aureus by Li et al.[10]
agents such as silver, copper, zinc and cerium have In this study, the influence of key experimental
gained the attention of the researchers in the recent conditions (such as Cu2+ precursor concentration and
years.[3] These inorganic antimicrobial agents contact time) on the characterization and
substituted hydroxyapatite compounds have more antibacterial activity of the CuHAp coatings on
the antimicrobial activity than that of unsubstituted 316LSS substrates was investigated. The
hydroxyapatite.[4-6] morphology, structure and the composition of the
Research results show that Cu2+ doped HAp coating were thoroughly characterized by X-ray
(CuHAp) has the effect of reducing bacterial diffraction (XRD), Energy-Dispersive X-ray (EDX),
adhesion on the material surface and inhibiting the Scanning electron microscopy (SEM) and Fourier
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Vietnam Journal of Chemistry Pham Thi Nam et al.
transform infrared (FTIR) spectroscopy. The 2.4. Antimicrobial activity test

antibacterial activity of CuHAp coatings for E. coli
and S. aureus was tested by bacteriological plate Plate counting method was used in this study. Gram-
counting method. negative E. coli (ATCC25922) and Gram-positive S.
aureus (ATCC25923) were employed to test the
2. MATERIALS AND METHODS antimicrobial ability of HAp or CuHAp.
The first, all samples were sterilized in an
2.1. Electrodeposition HAp coatings autoclave at 150 °C for 30 min. The E. coli or S.
aureus bacterial solution concentration was prepared
316L stainless steel (316LSS) was selected as the 104 colonyforming unit (CFU)/mL. Second, 30 μL
substrate material in this study and the composition of bacterial solutions was dripped on the surface of
was listed in our previous report.[11] The electrolyte samples (control samples, HAp and CuHAp with the
solution was prepared from the mixture of 3×10-2 M different concentration). The next, the antimicrobial
Ca(NO3)2, 1.8×10-2 M NH4H2PO4 and 6×10-2 M test system was covered with an aseptic
NaNO3. The HAp coating was deposited by cathodic polyethylene film and incubated at 37 °C for 24 h.
scanning potential method in a three-electrode cell After 24 h, these samples were placed into a tube
and using the Autolab PGSTAT 30 equipment. The with 10 mL of sterilized phosphate-buffered saline
HAp deposited condition is scanning potential (PBS) (pH = 7.2), and shaked at 200 rpm for 5 min.
ranges of 0÷-1.7 V/SCE; 5 mV/s; 5 times at 50 oC. Then, a 100 μL shaken bacterial solution was
respectively spread evenly on Luria–Bertani medium
2.2. Synthesis of CuHAp coatings agar plates. The samples contained on the agar plates
which incubated at 37 °C for 24 h. Finally, the
The HAp coatings doped with ion Cu2+ were visible cells of each plate were counted by
prepared by ion exchange method. The coating of quantifying the CFUs. The results were an everage
HAp was immersed in 5 mL Cu(NO3)2 solution values of three times. The antimicrobial ratio (K) of
with different concentrations of 0.005, 0.01, 0.02, the coatings was calculated using the equations
0.05 and 0.1 M and different times of 5, 10, 20, 30, (3):[12]
60 and 80 minutes at 30 oC.
The obtained solution after immersion was (3)
determined the remaining concentration of ion Cu2+
where A is the average number of colonies in the
by atomic absorption spectroscopy (AAS) method.
control and B is the average number of bacteria
The ion-exchange capacity (Q (mg/g)) and
colonies in the CuHAp or HAp coatings.
efficiency (H (%)) were calculated according to
equations (1) and (2):
3. RESULTS AND DISCUSSION
H
 Co  Ci  100 (%) 3.1. Effect of contact time
(1)
Co
The variation of the Cu2+ ion exchange capacity and
C0  C
Q .V . 1000 (2) efficiency according to the contact time is presented
m in figure 1. During the 5 to 80 minutes period, the
where: C0 is the initial concentration of Cu2+ ion exchange capacity increases rapidly in the first
(mol/L), C is the remaining Cu2+ concentration after 30 minutes, then it increases slowly with contact
immersion (mol/L), V is the volume of solution (L), time from 30 to 80 minutes. The efficiency has the
m is mass of HAp coatings (g). similar change rule with capacity. The exchange
efficiency increases with contact time. However, the
2.3. Coatings characterization efficiency tends to stabilize and reach about 0.53 %
with contact time more 30 minutes. So, the 30
The characterizations of CuHAp coatings were minutes was selected for CuHAp coatings synthesis.
analyzed using Fourier transform infrared The CuHAp coatings are synthesized with the
spectrometry (FTIR), filed-emission scanning different contact times from 5 to 80 minutes in 0.02
electron microscopy (FE-SEM), Energy Dispersive M Cu2+ solution. The functional groups of CuHAp
X-ray spectrometry (EDX) and X-ray diffraction coatings were analyzed by FTIR spectra. Figure 2
(XRD) methods with the same measurement presents FTIR spectra of HAp and CuHAp coatings
conditions as in our previous report.[11] recorded in the wave number range from 4000 to
© 2018 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.vjc.wiley-vch.de 673
Vietnam Journal of Chemistry Synthesis, characterization and antimicrobial...
400 cm-1. The results show that all samples have a 0), (2 2 2) and (0 0 4) planes. [15] These results show
similar shape and characteristic picks of HAp. The that obtained CuHAp coatings have crystal structure
absorption peaks at 1034; 962; 602 and 565 cm-1 are and single phase of HAp. The structure and phase
assigned to PO43-.[13,14] In particular, the asymmetric component of the HAp coatings are not affected
stretching vibration of P–O bond was characterized by the Cu2+ ion exchange process.
by a band at 1034 cm-1 and 962 cm-1. The
asymmetric O–P–O bending mode was clearly found 2
at 602 cm-1 and 565 cm-1. The peaks of OH- group 1. HAp; 2. CrO.FeO.NiO; 3. Fe 3
2
are observed at 3430 and 1643 cm-1.[13] IR spectra 1 11 1
also indicate the presence of CO32- at 1390 cm-1.[14]
This can be explained by the fact that CO2 from in CuHAp
Intensity
the air can be dissolved in the electrolyte and reacts
with OH- to form the CO32- ions.
12 0.65
HAp
0.60
11
Q (mg Cu /g HAp)
0.55
10 20 30 40 50 60
10 2 (degree)
H (%)
2+
0.50
Figure 3: XRD patterns of HAp and CuHAp

9 0.45
coatings with contact time of 30 minutes
0.40
8
3.2. Effects of initial Cu2+ concentration
0.35
0 10 20 30 40 50 60 70 80 90
t (minutes) Figure 4 presents ion exchange capacity and
efficiency of HAp coatings in 5 mL Cu2+ solution
Figure 1: The variation of the Cu2+ ion exchange with the different initial concentrations of Cu2+ and
capacity and efficiency of HAp according to the contact time of 30 minutes. One observes that the
contact time in 0.02 M Cu2+ solution ion exchange capacity increases with the initial Cu2+
concentration. So, the Cu2+ content in CuHAp
g
OH
- CO3
2-
PO4
3-
PO4
3-
coatings increases.
H2O
f
Transmittance
e 16
d 0.8
14
Q (mg Cu /g HAP)
12 0.6
b
2+
H (%)
a 10
1643 1390
3430 0.4
602 565
1034 8
4000 3000 2000 1000
-1
Wavenumber (cm ) 6 0.2
4
Figure 2: FTIR spectra of HAp (a) and CuHAp 0.0
coatings synthesized at different contact times: 5 (b); 0.00 0.02 0.04
2+
0.06 0.08 0.10
10 (c); 20 (d); 30 (e); 60 (f) and 80 minutes (g) Co( Cu /M)
Figure 4: The variation of the Cu2+ ion exchange

Figure 3 presents the XRD patterns of HAp and capacity and efficiency of HAp as a function of Cu2+
CuHAp coatings. Both XRD patterns exhibit the
solution concentration with contact time of 30
HAp phase as well as to the 316LSS substrate. minutes
Two characteristic peaks of HAp were clearly
demonstrated at 2 ≈ 26o and 32o corresponding to The initial concentration of Cu2+ increased from
(0 0 2) and (2 1 1) planes. The different 0.005 to 0.02 M, the ion exchange capacity increases
characteristic peaks of HAp with less peak rapidly from 4.18 to 10.66 mg/g. Then, the capacity
intensity at 2  33, 46, 54 o corresponding to (3 0 increases slightly from 13.06 to 14.63 mg/g
corresponding to Cu2+ concentration increasing from XRD patterns means that the coatings are quite
0.05 to 0.1 M. This result is obtained when the ion thin. The obtained CuHAp coatings have crystal
exchange process tends to be in equilibrium. The structure and single phase of HAp. The structure
ion exchange efficiency decreases from 0.8 % to and phase composition of the HAp coatings are
0.18 %. The efficiency is low due to the exchange also unaffected by the Cu2+ ion exchange process.
only occurs at outermost of HAp coatings that does The morphology of CuHAp coatings synthesis
not occur in the whole HAp coatings. with the different initial concentration of Cu2+ is
present in figure 6. The SEM images show that with
2
1. HAp; 2. CrO.FeO.NiO; 3. Fe 3
2
1
Cu2+ presence, the CuHAp morphology has not
1 1
1
11 changed much. The HAp coatings are comprised of
cylindrical grains sizing from 100 to 400 nm. After
f the ion exchange with Cu2+ solution, the CuHAp size
is smaller and its surface is more uniform. At
e different concentrations of Cu2+, it does not
Intensity
d significantly affect the morphology of the CuHAp

c
coatings.
b 3.3. Cu2+ content in CuHAp coatings

a
The CuHAp coatings are synthesized in 0.05 M
10 20 30 40 50 60
(CuHAp-0.05 M) and 0.1 M (CuHAp-0.1 M) Cu2+
2 (degree) solutions which are determined for Cu2+ content by
EDX method (figure 7 and table 1). The results
Figure 5: XRD patterns of HAp (a) and CuHAp show the presence of three main elements Ca, P and
coatings synthesis at different concentrations of Cu2+ O that enter in HAp composition. Besides, Cu
solution: 0.005 M (b); 0.01 M (c); 0.02 M (d); 0.05 element is also presented for both CuHAp coatings.
M (e) and 0.1 M (f) This result confirms the Cu2+ ions were successful
doped into HAp coatings.
Figure 5 presents the XRD patterns of CuHAp Cu/Ca and (Ca+Cu)/P atomic ratios are
coatings that are synthesized with the different initial calculated from the percentage of elements
concentrations of Cu2+. The results show that presented in the CuHAp coatings. With CuHAp-0.05
CuHAp coatings have characteristic peaks of HAp M coatings, Cu/Ca and (Ca+Cu)/P ratios are 1×10-3
phase and have no other phase of Ca-P. The and 1.62, respectively. With CuHAp-0.1 M coatings,
characteristic peaks of HAp were at 2 ≈ 26o, 32o, Cu/Ca and (Ca+Cu)/P ratios are 3×10-3 and 1.69,
33o, 46o and 54o corresponding to (0 0 2), (2 1 1), respectively. While, the natural bone has ratio of
(3 0 0), (2 2 2) and (0 0 4) planes. [15] Besides, the Cu/Ca ≤ 1.5×10-4 and Ca/P = 1.67.[17] So, the 0.05 M
other peaks at 2θ ≈ 44°, 45° and 51° were Cu2+ solution was selected for the synthesis of
attributed to 316LSS substrate and its oxides. [11,16] CuHAp coatings in order to obtain the desired
The peak related to 316LSS substrate in these composition similar with the natural bone.
Table 1: Chemical composition of CuHAp-0.05 M and CuHAp-0.1 M coatings
Coatings Element O P Ca Cu C Cu/Ca (Ca+Cu)/P
%w 42.01 18.67 39.05 0.08 0.19

CuHAp-0.05 M 1×10-3 1.62
%a 62.20 14.27 23.13 0.03 0.04
%w 42.94 17.86 38.89 0.18 0.13

CuHAp-0.1 M 3×10-3 1.69
%a 63.21 13.57 22.90 0.07 0.03
The natural bone ≤ 1.5×10-4 1.67
676
Synthesis, characterization and antimicrobial...
www.vjc.wiley-vch.de
Figure 6: SEM images of HAp (a) and CuHAp (b) coatings synthesis at different concentrations
Figure 7: EDX spectra of CuHAp-0.05 M and CuHAp-0.1 M coatings
© 2018 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
of Cu2+ solution
Vietnam Journal of Chemistry
3.4. Antimicrobial assessment coli and S. aureus rised corresponding to the

increasing of Cu2+ concentration.
Antimicrobial activity of the HAp and CuHAp was E. coli antimicrobial ratio of CuHAp increased
studied by bacterialogical plate counting method sharply from 22 to 89 % when the Cu2+
against two microorganisms E. coli and S. aureus. concentration increased from 0.005 to 0.05 M. Then,
The results of the quantitative antibacterial tests it increased slightly and reached about 91% with
were shown in figures 8 and 9. Antimicrobial ratio increasing of Cu2+ concentration to 0.1 M. With S.
(K) was counted from the number of colonies aureus, the antimicrobial ratio of CuHAp also
presented in table 2. The results showed that both increased strongly from 29 to 98 % with increasing
HAp and CuHAp were resistant to the two Cu2+ concentration from 0.005 to 0.05 M. This ratio
microbials. The presence of copper enhanced the reached about 99 % for obtained CuHAp coating in
activity against E. coli and S. aureus in comparison 0.1 M Cu2+ solution. The results were clearly that
with pure HAp. The antimicrobial activity of HAp CuHAp-0.05 M and CuHAp-0.1 M coatings have
coatings on E. coli and S. aureus was about 20 % good antibacterial activity against to E. coli and S.
and 24.6 % compared to the control, respectively. aureus strains.
For CuHAp coatings, the antimicrobial activity on E.
Figure 8: E. coli antibacterial test photographs of control, HAp and CuHAp coatings
Figure 9: S. aureus antibacterial test photographs of control, HAp and CuHAp coatings
Vietnam Journal of Chemistry Synthesis, characterization and antimicrobial...
Table 2: The antimicrobial ratio of HAp and CuHAp coatings on E. coli and S. aureus
CuHAp- CuHAp- CuHAp- CuHAp- CuHAp-
Control HAp
0.005 M 0.01 M 0.02 M 0.05 M 0.1 M
E. coli 0 20 22 25 31 89 91
K (%)
S. aureus 0 24.6 29.4 36.5 61.3 98.3 98.9
4. CONCLUSION Mater. Sci. Eng. C, 2011, 31, 1320-1329.

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and zinc-doped hydroxyapatite nanopowders, Appl.
investigation about the effect of contact time and
Surf. Sci., 2010, 256(20), 6083-6089.
initial Cu2+ concentration showed that they do not 8. F. Bir, H. Khireddine, A. Touati, D. Sidane, S. Yala,
affect to the phase composition but affect the crystal H. Oudadesse. Electrochemical depositions of
size, morphology and elemental components of the fluorohydroxyapatite doped by Cu2+, Zn2+, Ag+ on
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11. Dinh Thi Mai Thanh, Pham Thi Nam, Nguyen Thu
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Corresponding author: Pham Thi Nam

Institute for Tropical Technology, Vietnam Academy of Science and Technology
18, Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam. E-mail: ptnam@itt.vast.vn.

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Cite this paper: Vietnam J. Chem.

, 2018, 56(6), 672-678 Article

Synthesis, characterization and antimicrobial activity of copper doped

1. INTRODUCTION growth of bacteria.[7] In addition, the small amount

transform infrared (FTIR) spectroscopy. The 2.4. Antimicrobial activity test

Figure 3: XRD patterns of HAp and CuHAp

Figure 4: The variation of the Cu2+ ion exchange

d significantly affect the morphology of the CuHAp

b 3.3. Cu2+ content in CuHAp coatings

Table 1: Chemical composition of CuHAp-0.05 M and CuHAp-0.1 M coatings

Coatings Element O P Ca Cu C Cu/Ca (Ca+Cu)/P

%w 42.01 18.67 39.05 0.08 0.19

%w 42.94 17.86 38.89 0.18 0.13

The natural bone ≤ 1.5×10-4 1.67

Figure 7: EDX spectra of CuHAp-0.05 M and CuHAp-0.1 M coatings

3.4. Antimicrobial assessment coli and S. aureus rised corresponding to the

4. CONCLUSION Mater. Sci. Eng. C, 2011, 31, 1320-1329.

Corresponding author: Pham Thi Nam

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