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Non-doped and transition metal-doped CuO nanopowders: structure-physical properties and antiadhesion activity relationship
N. Khlifi,ac S. Mnif,b F. Ben Nasr,a N. Fourati,c C. Zerrouki,c M. M. Chehimi,d
H. Guermazi, *a S. Aifab and S. Guermazia
Bacterial contamination and biofilm formation generate severe problems in many fields. Among these biofilmforming bacteria, Staphylococcus epidermidis (S. epidermidis) has emerged as a major cause of nosocomial
infection (NI). However, with the dramatic rise in resistance toward conventional antibiotics, there is
a pressing need for developing effective anti-biofilms. So, fabrication of copper oxide nanoparticles (NPs) is
one of the new strategies to combat biofilms. Notably, doped CuO NPs in anti-biofilm therapy have
become a hot spot of attention in recent years due to their physicochemical properties. In this context, the
present work deals with the investigation of undoped and transition metal (TM)-doped CuO NPs (TM ¼ Zn,
Ni, Mn, Fe and Co), synthesized via the co-precipitation method. The synthesized CuO NPs are
characterized using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, field-emission
scanning electron microscopy (FE-SEM), energy dispersive spectroscopy (EDS) and X-ray photoelectron
spectroscopy (XPS). Results consistently revealed the successful formation of CuO NPs using the coprecipitation method and confirmed that TM ions are successfully inserted into CuO crystal lattice. We
found that doping changes the morphology of the CuO NPs and increases their crystallite size. The XPS
results show a non-uniform distribution of the doping concentration, with a depletion or an enrichment of
the NP surface depending on the element considered. Furthermore, the anti-adhesive potential of CuO NPs
Received 15th April 2022
Accepted 31st July 2022
against S. epidermidis S61 biofilm formation is evaluated in this study by crystal violet and fluorescence
microscopy assays. All synthesized NPs exhibit considerable anti-adhesive activity against S. epidermidis S61
biofilm. Interestingly, compared to undoped CuO, Fe and Ni-doped oxides show an improved activity when
DOI: 10.1039/d2ra02433k
rsc.li/rsc-advances
used at high concentrations, whereas Mn-doped CuO is the most efficient at low concentrations. This
makes TM-doped CuO a promising candidate to be used in biomedical applications.
1. Introduction
Nanotechnology is an interdisciplinary eld that is based on
fundamental sciences such as physics, chemistry, and biology
and is growing with current developments. Particularly, metal
oxide nanoparticles (MONPs) possess special physical and
chemical properties due to their nite size and large surface area.
These properties make metal oxides an interesting project for
scientists because of their multidisciplinary applications.1–3
Particularly, copper oxide nanoparticles (CuO NPs) have been
widely studied and are receiving more attention from many
Laboratory of Materials for Energy and Environment, and Modeling (LMEEM), Faculty
of Sciences, University of Sfax, B.P: 1171, 3038, Tunisia. E-mail: hajer.guermazi@
gmail.com
a
Laboratory of Molecular and Cellular Screening Processes, Centre of Biotechnology of
Sfax, P.O. Box 1177, 3018 Sfax, Tunisia
b
Laboratory of Information and Energy Technology Systems and Applications (SATIE),
UMR 8029, CNRS, ENS Paris-Saclay, CNAM, 292 Rue Saint-Martin, 7503 Paris, France
c
Université Paris Cité, CNRS, ITODYS (UMR 7086), 75013 Paris, France
d
© 2022 The Author(s). Published by the Royal Society of Chemistry
scientists and engineers thanks to their motivating properties
and high potential applications in various elds.4–6 In the energy
domain for example, CuO NPs have been considered as electrode
materials for the next-generation rechargeable lithium-ion
batteries, owing to their abundance, environmental compatibility and eco-friendliness, high theoretical capacity, and nontoxicity.7 Moreover, both morphology and size remarkably
affected their physical properties. As a p-type semiconductor of
narrow band gap in visible region, CuO NPs are expected to be
good candidate for high potential applications as solar cells,
storage devices, and sensors, as well as super capacitors and
especially it acts as a good catalyst in some of the chemical
reactions.8–10 In fact, several researchers have been directed
towards the photo-catalytic activities of CuO NPs, for the degradation of organic pollutants.11,12 Recently, CuO NPs have been
largely prepared for various applications in elds ranging from
semi-conductors to biomedical. For the latter, many studies
involving CuO NPs have been carried out on anti-bacterial,13,14
anti-biolm,14 anti-cancer,15 anti-microbial,16 and anti-fungal17
aspects. Among these studies, biolms were explored several
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centuries ago by researching the link between its formation and
the persistence of the disease.
Bacterial biolms are a growing problem as it is a major cause
of nosocomial infection (NI) affecting hospitals worldwide and
one of the health issues among all societies. Biolms, derived
from microbe colonized on surfaces, cause persistent infections
and are an issue of considerable concern to healthcare
providers.18,19 They caused serious problems to society from both
health concerns and economical perspectives.20 One of the most
potentially successful strategies may be the use of nano-materials.
Nowadays, the use of NPs is considered one of the future challenges that govern the biomedical applications.21 Among many
NPs, the most promised and widely studied is CuO NPs.
Numerous researches indicated that CuO had higher anti-biolm
activity against clinically isolated, biolm forming, multi-drug
resistant micro-organisms such as Escherichia coli, Staphylococcus aureus22–24 and Streptococcus mutans.25 However, knowledges about the effect of TM doping on CuO's anti-bacterial
activities, is still in its juvenile phase. Likewise, doping CuO with
various TM has been considered to enhance the physical/
chemical properties of the host material CuO without modication of its structure.26 Indeed, several researches showed that
physicochemical properties of CuO NPs can be modied by
doping with a suitable element to meet the pre-determined needs.
In particular, the effect of doping of CuO NPs by 3d TM such as
Mn2+/3+, Co2+/3+, Fe2+/3+, Ni2+/3+ and Zn2+, on their electrical,
optical, and magnetic properties were largely considered.27–31 The
control of the synthesis process and the understanding of its
mechanism allowed elaborating undoped and TM-doped CuO
NPs with specic properties, according to the envisaged applications. The use of TM-doped CuO as anti-biolm agents has
recently attracted considerable attention in biomedical elds.32,33
In addition, the results show benets in the use of undoped and/
or doped CuO NPs that could work synergistically to provide an
anti-biolm effect against various bacterial strains. Among these
bacterial strains, Staphylococcus epidermidis strain (S. epidermidis)
is a major nosocomial pathogen with a remarkable ability to
persist on indwelling medical devices through biolm formation.
Unfortunately, S. epidermidis strains have become the most
common cause of nosocomial infections (NIs) and infections on
indwelling medical devices, which typically involve biolms.34,35 It
is one of the major human pathogen cause's mild supercial
infections to severe life-threatening invasive infections to the
human world resulting in signicant disease and mortality.36,37
Several works reported on CuO NPs synthesized by physical,
chemical, and biological methods such as sol–gel,38 co-precipitation,39 solvo-thermal,40 microwave irradiation,41 laser ablation,42 bio-synthesis using Verbascum thapsus leaves extract,43 and
biological methods using the cyanobacteria Phormidium.44
Among these various synthesis methods, co-precipitation is of
interest since it is considered as an economical and eco-friendly
approach. Moreover, using this technique, shape and particle
size of NPs can be tuned by varying different parameters such as
pH value, precursor's concentrations, and reaction temperature.
A comprehensive study of the already published reports has
thrown light on the fact that no work has been done so far to
determine the anti-adhesion activities of different TM-doped
23528 | RSC Adv., 2022, 12, 23527–23543
Paper
CuO NPs compared to pure CuO NPs against biolm forming
by S. epidermidis bacteria. In the present work, undoped and TMdoped CuO NPs are successfully synthesized via the coprecipitation method. The purpose of the current study is to
investigate TM doping of CuO on its potential activity as inhibitors of biolm formation. The anti-biolm effect of the prepared
samples (TM-doped and undoped CuO NPs) will be correlated to
physical and structural changes induced by doping.
The incorporation of anti-adhesive MONPs within or on the
surface of materials, or by coatings, to prevent microbial
adhesion or kill the microorganisms aer their attachment to
biolms, represents an important strategy in an increasingly
challenging eld.45 Consequently, this study aimed to evaluate
the effectiveness of incorporating 3d TM (TM ¼ Mn, Fe, Co, Ni,
and Zn) into the physicochemical properties of CuO NPs and
assess its anti-biolm activity effects against S. epidermidis S61.
2.
Experimental details and materials
2.1. Materials and synthesis
All chemical reagents, doping precursors either chlorides as
manganese chloride tetra-hydrate (MnCl2$4H2O), cobalt chloride hexa-hydrate (CoCl2$6H2O), nickel chloride hexa-hydrate
(NiCl2$6H2O), zinc chloride (ZnCl2), or sulfates as iron sulfate
hepta-hydrate (FeSO4$7H2O), and sodium hydroxide (NaOH),
distilled water and ethanol are supplied by Sigma Aldrich
(purity >99%) and used without any further purication.
Undoped (CuO) and TM-doped CuO, TM ¼ Fe, Mn, Co, Zn,
and Ni (will be noted as CuO:Fe, CuO:Mn, CuO:Co, CuO:Zn and
CuO:Ni respectively), are synthesized through co-precipitation
method, using copper sulfate pentahydrate [CuSO4$5H2O] as
primary precursor.
Firstly, aqueous solution (1 M) of copper sulfate is prepared
under magnetic stirring. Subsequently, 50 ml of 1 M NaOH
solution is added drop-wise to the homogeneously mixed
solution every 15 min under constant stirring, and the pH was
maintained between 11 and 12. Then, the solution is kept at
25 C for 3 hours under constant stirring, until a blue precipitate is formed. This precipitate is ltered, and then washed
several times with distilled water and ethanol to remove any
residue. Finally, the precipitate is dried, ground and annealed at
500 C for 4 hours to obtain a pure CuO phase and enhance the
crystalline quality of the synthesized powder.
The TM-doped samples are also prepared by co-precipitation
method. To prepare TM-doped CuO NPs, doping precursors are
taken respectively and added separately into the (CuSO4$5H2O)
solutions. The remaining process is the same as the synthesis of
undoped CuO NPs.
The obtained samples with an expected doping rate of
2 mol% are used for further characterization.
2.2. Characterization techniques
The powder X-ray diffraction (XRD) measurements were carried
out at room temperature using a D8 Advance Bruker diffractometer with a Lynx eye accelerator using CuKa1 radiation (l ¼
1.5406 Å) at 40 kV and 40 mA. The angular range 2q is between
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5 and 70 with a step of 0.02 , and a scan rate of 1 step per
second. Morphology analysis is undergone using scanning
electron microscope (SEM, HITACHIS-3400 N, Japan) at 20 kV.
The elemental composition of the samples is analyzed using
energy dispersive spectroscopy (EDS) analysis. The chemical
signature (molecular vibration energies) of the samples is
provided via Fourier transform infrared (FTIR) experiments,
investigated with a BRUKER spectrophotometer in the 400–
4000 cm 1 range. X-ray photoelectron spectra (XPS) were
acquired using a K Alpha+ machine (Thermo, East Grinsted,
UK) tted with monochromatic Al K alpha source (hn ¼ 1486.6
eV). The pass energy was set to 80 eV for the narrow regions and
200 eV for recording the survey spectra. A ood gun was used
during the surface analysis to compensate for static charge
build-up. The surface composition was determined using the
manufacturer's sensitivity factors.
3.
2.3. Anti-biolm activity
The anti-biolm activities of TM-doped and undoped CuO NPs
are performed against S. epidermidis S61, a biolm forming
strain in our Laboratory collection.46 Anti-adherence activities
are tested in 96-well at bottom microtiter plates as described
by Nostro et al.47 with some modications. Nanoparticle solutions (CuO, CuO:Fe, CuO:Mn, CuO:Co, CuO:Zn and CuO:Ni),
dissolved in Tryptone Soy Broth (TSB),are subjected to an
ultrasonic bath for 1 h at 25 C. Solutions (100 ml) are then
added into the rst column of plates and used to perform
a serial two-fold dilution. This is followed by the addition of 50
ml of glucose at a nal concentration of 2.25% in each well. An
overnight culture of S. epidermidis S61 is performed in TSB
medium at 30 C and diluted with TSB. 50 mL of bacterial
suspension are served to inoculate all wells in order to reach
a nal optical density (OD) of 0.1 at a wavelength of 600 nm. Cell
culture without NPs is served as a control. Following incubation
for 24 h at 30 C, TSB and plank tonic cells are discarded by
reversing the plate and all wells are washed twice with 200 mL of
sterile phosphate buffer saline (PBS) (pH 7.2). The plate is then
dried at 60 C for 1 h. Each well is stained with 150 mL of crystal
violet 0.2% (w/v of ethanol 20%) for 15 min. The wells contents
are removed and the remaining of crystal violet, which stained
the attached bacteria, is rinsed thrice with water. A volume of
200 mL of glacial acetic acid (33% (v/v)) is added in all wells and
the solubilisation of biolm is obtained following the incubation for 1 h at room temperature. Finally, the optical density is
measured with the micro-plate reader at a wavelength of
570 nm. Percentage of biolm inhibition is calculated by the
comparison between the absorbance of untreated and treated
biolms according to the following formula:
% biofilm inhibition ¼
ðODðgrowth controlÞ OD ðsampleÞÞ
100
ODðgrowth controlÞ
Results and discussion
Discovering the key factors involved in biolm inhibition will
allow exploring the utilization of undoped and TM-doped CuO
NPs in biomedical applications. So, it is necessary to determine
the relevant physico-chemical parameters such as crystallite
size, shape, surface area, composition, and morphology, which
could be advantageously, involved in the anti-biolm activities.
3.1
Structural studies by X-ray diffraction (XRD)
XRD patterns of the undoped and TM-doped CuO NPs are
shown in Fig. 1. The diffraction peaks in all samples matched
well with the typical CuO monoclinic structure with C2/c
symmetry according to the International Center for Diffraction
Data (ICDD card no. 01-089-2529)48 and were assigned to the
(110), (002), (111), ( 202), (020), (202), ( 113), ( 311) and (113)
planes. Besides the absence of signicant change in the crystal
structure, no secondary oxide phases are detected in samples,
indicating pure CuO phase formation, and the successful
insertion of the doping elements in CuO lattice during the
growth process. However, micro-changes undoubtedly occur in
terms of characteristic parameters and/or in terms of microstrain, and therefore need to be analyzed more closely. Lattice
constants (a, b, c, and b) of the monoclinic structure and unit
cell volume (V) of prepared samples are estimated from the
diffraction peaks, using the following equations:49
2dhkl sin q ¼ nl
2
1
1
h
k2 sin2 b l 2
¼
þ
þ 2
c
b2
dhkl 2
sin2 b a2
V ¼ abc sin b
(1)
The effect of TM-doped CuO NPs on biolm inhibition is
conrmed by the uorescence microscopy. To visualize the
biolm inhibition, cultures of S. epidermidis (in TSB plus 2.25%
© 2022 The Author(s). Published by the Royal Society of Chemistry
glucose) are grown on round cover slips (diameter 1 cm)
immersed in a 24-well polystyrene plate with or without
(control) NPs addition. Incubation of plates for 24 h at 30 C, is
followed by removal of the medium and subsequent cover slips
washing with sterile PBS (1X, pH 7). The uorescent dye Acridine orange, at a concentration of 0.1% w/v in PBS 1X, is used to
stain the formed biolm in cover slips during 15 min. Biolms
were imaged with a OLYMPUS uorescent microscope BX50
equipped with a digital camera OLYMPUS DP70 using an
appropriate lter.
All experiments are done in triplicate. The obtained results
are expressed as mean values with the standard error. The
statistical analyses are performed using Student's t-test to
compare the controls and treatment conditions at a signicance
level of 5%.
(2)
2hl cos b
ac
(3)
(4)
where q is the Bragg's diffraction angle, l is the wavelength of
the used Cu Ka radiation, dhkl is the (hkl) interplanar spacing.
The obtained values of lattice parameters for the synthesized
NPs are gathered in Table 1. As expected, we notice some
changes of crystal lattice parameters, in consequence of doping
with transition metals.
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XRD patterns of CuO, CuO:Fe, CuO:Mn, CuO:Co, CuO:Zn and
CuO:Ni samples.
Fig. 1
Table 1
Structural parameters of undoped and M-doped CuO NPs
Lattice parameters
Synthesized
NPs
a (Å)
b (Å)
c (Å)
b ( )
V (Å3)
CuO
CuO:Fe
CuO:Zn
CuO:Mn
CuO:Co
CuO:Ni
4.863
4.881
4.791
4.700
4.701
4.644
3.433
3.427
3.430
3.432
3.431
3.454
5.136
5.138
5.137
5.133
5.131
5.136
99.309
99.455
99.546
99.549
99.470
99.309
84.638
84.776
83.291
81.660
81.656
81.327
The Gaussian fit to the (a) (002) and (b) (111) diffraction peaks of
undoped and TM-doped CuO NPs.
Fig. 2
The unit cell volume of CuO NPs is sensitive to TM doping, as
shown by the values gathered in Table 1. A reduction between
1.6 and 3.9% is observed depending on the elements considered, except for Fe for which we notice a very slight increase
(of 0.2%).
The relative differences between the ionic radii, extracted
from the reference database (Ni2+: 0.69 Å, Mn2+: 0.67 Å, Co2+:
0.65 Å, Cu2+: 0.73 Å, Zn2+: 0. 74 Å and Fe2+: 0.78 Å),50 do not allow
correlating the effects of the dopants in the volume to their
ionic radius alone, as one could be tempted to do at rst
approach. The dispersion of results proves that other intrinsic
parameters (electro negativity for example) or experimental
ones, act in a correlated way on the properties of the obtained
products including their mesh parameters. However, the
difference between the synthesized NPs (increase of the unit cell
volume in case of iron and decrease for Ni, Zn, Co and Mn), let
us expect a sufficient difference in their physical properties and,
consequently, in their potential anti-biolm activities.
To further investigate the effect of doping, we focused on the
two most intense Bragg's peaks corresponding to the (002) and
(111) plans, which we tted by a Gaussian model (Fig. 2(a)
and (b)).
For the peak (002) (Fig. 2(a)), we observe a slight shi
towards the high angles for Fe and Mn doping. For the other
elements and given the accuracy of about 0.01 , the observed
23530 | RSC Adv., 2022, 12, 23527–23543
shi is too small to be judged. For the peak (111) (Fig. 2(b)), we
still observe a slight shi towards the high angles for Fe and Mn
doping, but also for Co this time. As for the Ni doping, it
produces a shi in the opposite direction.
In Fig. 3, we gathered the intensity ratio I(002)/I(111) of the
main peaks, for all NPs oxides.
By considering the I(002)/I(111) peak intensity ratio as
discriminating parameter (Fig. 3), we notice that this parameter
decreases for all doped oxides. Nevertheless, its value remains
slightly superior to the unit for all oxides except for CuO:Zn
sample. This means that the crystalline growth of undoped and
Ni, Mn, Fe and Co-doped CuO NPs occurred preferentially
according to the (002) direction while an inversion is observed
for the crystallites of CuO:Zn NPs, which preferentially grow in
the (111) direction. From these results, we can note that the
insertion of transition metals within the CuO matrix induces
some structural changes and moderately affects the preferential
growth direction.
To complete this comparative structural study, microstructural parameters such as crystallite size (D), specic
surface area (SSA), micro-strain (3) and dislocation density (d)
were investigated for all the synthesized samples.
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d¼
1
D2
(8)
The SSA is intimately linked to particle size, but other
parameters, such as the microstructure and morphology of the
materials, can greatly affect its value. Particle size determination was considered as an indirect estimate of the SSA values.
This extremely important parameter greatly affects the properties of the NPs, and inuences their mode of interaction with
the surrounding media.
Assuming that the particles have a spherical shape, we can
estimate the SSA from the crystallite size determined by
Scherrer equation, using formula eqn (6):57,58
Fig. 3
S¼
6
Dr
(9)
r¼
nM
N V
(10)
I(002)/I(111) peak intensity ratio of undoped and TM-doped CuO
NPs.
The average crystallite size and lattice strain were deduced
from the broadening of the XRD peak by the relation:51
b2(hkl) ¼ b2size + b2strain
(5)
where bsize and bstrain are the broadening contributions due to
crystallite size and strain, respectively.
Aerward, Debye–Scherrer (D–S) method52–55 is used to estimate the average crystallite size from the equation:
D¼
kl
bsize cos q
(6)
where l is the wavelength of X-ray radiation (1.5406 Å), k ¼ 0.9 is
the shape factor, D is the average crystallite size, and q is the
Bragg's diffraction angle.
However, the effect of strain and imperfections on the line
broadening differs from the effect of crystalline size. The microstrain induced in the prepared powders is calculated using
eqn (7).
3¼
bstrain
4 tan q
(7)
Moreover, the dislocation density (d) of the crystal is evaluated using the following formula:56
Table 2
S is the SSA [m2 g 1], r is the density of the particles [g cm 3], M
is the molar weight of substance (MCuO ¼ 79.545 g mol 1), n is
the number of formula units in the unit cell (n ¼ 4 for CuO
according to the ICDD data no. 01-089-2529), V design the
volume of the unit cell (eqn (2)) and N is the Avogadro's number
(N ¼ 6.02214 1023 mol 1). The calculated values of SSA and
the density (r) are added in Table 2.
These values clearly show that the structural parameters
exhibit slight variations, as consequence of the incorporation of
TM ion within CuO host lattice. Moreover, it is found that the
micro-strain decreases with doping compared to the undoped
CuO NPs, except for CuO:Fe. An increase in the average crystallite
size is thus clearly observed for all TM-doped CuO samples
compared to undoped CuO. However, the CuO:Fe shows the
smallest crystallite size and the highest micro-strain of the alldoped samples, while the largest crystallite size associated with
the lowest micro-strains are obtained with Zn doping.
Fig. 4 depicts the evolution of the SSA of the synthesized NPs
as a function of the average crystallite size for undoped CuO and
TM-doped oxide with different metal transition elements.
As expected, and inversely to the crystallites size, the highest
SSA value of 70 m2 g 1, has been obtained for the undoped CuO
NPs. For all the TM-doped CuO NPs, SSA values are smaller, and
the lowest one is associated to CuO:Zn sample (26 m2 g 1). All
estimated SSA values are higher than the typical specic surface
Micro structural parameters of undoped and M-doped CuO NPs
Samples
Crystallite
size, D (nm)
Micro-strain
3 (10 2)
Dislocation density,
d (1015 m 2)
Density of the
particles r (g cm 3)
Specic surface
area, S (m2 g 1)
CuO
CuO:Zn
CuO:Ni
CuO:Mn
CuO:Fe
CuO:Co
13.712
36.132
22.243
19.043
14.474
19.666
0.75
0.47
0.57
0.71
0.84
0.61
5.318
0.765
2.021
2.757
4.773
2.585
6.242
6.343
6.496
6.470
6.232
6.470
70.101
26.179
41.525
48.698
66.517
47.155
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Relationship between the SSA and the average crystallite size
(D) of undoped and TM-doped CuO NPs.
Fig. 4
area values of high purity commercially available CuO nanopowders (i.e. $99.99%), in the range of 10 to 15 m2 g 1 (for
example Get Nano Materials, American Research Nano-materials.). Obviously, the estimated SSA values are dependent on
several assumptions, mainly a homogenous spherical shape,
and are therefore subject to uncertainties. Nevertheless, they
remain interesting because they are high despite uncertainties,
and they are different one from the other. These are two
important reasons which prompt us to test the NPs, we
successfully synthesized, against bacterial activity.
It is noted that the specic surface area and surface to
volume ratio increase dramatically as the size of materials
decreases. The surface states will play an important role in the
NPs, due to their large surface to volume ratio with a decrease in
particle size.59 It has a more importance in case of adsorption,
heterogeneous catalysis and reactions on surfaces. Also, in
biomedical eld, NPs are preferred over other agents because of
their small size, large surface area and highly reactive nature.60
Then, the consistence between surface area and crystallite size
values estimated from XRD pattern data using Scherer method
indicates that both undoped and-doped CuO NPs has surface
activities. The reactivity of the NPs varies according to its
surface function, particle size, shape, and state of aggregation.61
Hence, exact knowledge around the surface chemistry of NPs
is important before their applications. Fourier transform
infrared (FTIR) technique can be effectively used to gain information about the surface chemistry of our samples.
3.2. Fourier transform infrared (FTIR) study
The Fourier transform infrared spectroscopy (FTIR) is carried
out to identify the main chemical bonds in the synthesized CuO
samples. Fig. 5(a) and (b) shows the FTIR spectra of prepared
samples in the range (400–4000 cm 1). However, for better
clarity of region of interest, FTIR spectra are enlarged in
a selective range of 400–1300 cm 1 (Fig. 5(b)). In the range of
400–700 cm 1 (Fig. 5(a) and (b)) IR spectra display vibrational
bands ascribed to stretching mode of CuO bonds.62–65 In addition, a broad band in the region 3200–3600 cm 1 (Fig. 5(a)) can
23532 | RSC Adv., 2022, 12, 23527–23543
Fig. 5 FTIR spectra of CuO and TM-doped CuO NPs (a) in 400–
4000 cm 1 range, and (b) enlarged spectra from 400 cm 1 to
1300 cm 1.
be attributed to the stretching vibration of the O–H bonds,66–69
due to adsorbed water molecules on the CuO NPs surface.
Moreover, all samples have the absorption bands in the 1434–
1643 cm 1 range that may be assigned to O–H bending vibrations, combined with copper atoms.70 As shown in FTIR spectra
(Fig. 5(b)), new bands appear between 750 cm 1 and 990 cm 1,
which can be attributed to degenerate and non-degenerate
modes of SO4 groups respectively.71 In this way, the broad
band around 1112 cm 1 is the evidence of the existence of
SO42 in the all analyzed samples.72–74 Indeed, the precursors of
copper is the sulfate type (CuSO4$5H2O).
No other active modes corresponding to other species like
Cu2O or Cu (OH)2 or other peaks from doping elements were
detected, agreeing with the XRD analysis which conrms the
purity of prepared samples. In addition, no bands related to
TM–O vibrations have observed, which conrms the successful
substitution of doping ions in CuO structure, and the purity of
CuO phase. All observed bands are found to be shied towards
higher wave-number side for all doped CuO NPs. This shi can
be due to the perturbation of CuO bonding and the difference in
the bond lengths as a result of the incorporation of doping ions
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in the CuO host lattice.62–65 Indeed, TM-doping can induce
strong interactions between the 3d TM ions and the CuO
network. Also, it can originate from size induced lattice variations and crystal defects such as the surface unsaturated coordination sites and edge dislocations induced by doping
elements.75
3.3. Scanning electron microscope (SEM) and energy
dispersive spectroscopy (EDS) analysis
The surface morphology and elemental composition of the
synthesized samples are investigated using SEM – EDS analysis.
The SEM images of undoped and TM-doped CuO NPs are
shown in Fig. 6(a)–(f). It is noted high agglomerated particles. The
observed agglomeration is mainly due to the OH groups (as
shown in FTIR spectra) present at the surface of NPs. It is obvious
Fig. 6
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that doping cause morphological changes of grains that affect
their SSA (Fig. 4). The variation in the shape and size of NPs is, in
fact, the consequence of added doping element in reactional
mixture. It may be due to weak inter-intra particle forces. This can
inuence the potential of anti-biolm activity of the CuO NPs.
EDS analysis is performed at different locations on each
sample, to examine its purity and elemental chemical
composition. From the EDS spectra, variation of the intensity
[cps/eV] as a function of the energy [keV] (Fig. 7), we identify
the chemical elements present in the synthesized samples.
The intense peak, located at 0.99 keV, is assigned to the L peak
of Cu.76 Oxygen characteristic peak is present at 0.520 keV.76
Carbon (C) is detected around 0.270 keV, it originates from
the substrate. Thus, the EDS spectra show only the elements
copper (Cu), oxygen (O) and TM doping elements (¼ Zn, Ni,
Mn, Fe and Co) and no other elements were observed for
High magnification SEM images of (a) undoped CuO, (b) CuO:Zn, (c) CuO:Ni, (d) CuO:Co, (e) CuO:Fe, and (f) CuO:Mn.
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Fig. 7 EDX spectra of (a) undoped CuO, (b) CuO:Zn, (c) CuO:Ni, (d) CuO:Mn, (e) CuO:Fe, and (f) CuO:Co NPs.
undoped and TM-doped copper oxide NPs. So, undoped CuO
sample contains characteristic peaks of Cu (Ka, Kb and La), O
(ka) and C (Ka) and no signicant impurities (Fig. 7(a)).
Whereas TM-doped CuO NPs spectra show a signature (La and
Lb) of each doping element, besides those of Cu (La and Lb),
and O (ka) (Fig. 7(b–f)). The presence of the Carbon (C) in the
spectra of all samples is due to the substrate based on
conductive carbon glue, used as a support during the analysis.
The quantitative analysis obtained from the EDS experiment
must be considered with caution because the spectra are
convolved, and the deconvolution is even more precise as the
gap between the uorescence lines is high (compared to the
resolution of the detector).
For Mn and Fe, whose characteristic lines are relatively
separated from those of Cu, 290 eV and 220 eV respectively, the
doping rates are estimated at 1.8% and 1.5% respectively. These
rates are relatively close to the expected 2%, in contrast with
those of Ni (9%) and Zn (0.4%) whose characteristic lines are
only 80 eV from Cu (by default for Ni and by excess for Zn). In
23534 | RSC Adv., 2022, 12, 23527–23543
the case of Co, the doping rate is estimated at 5%. This value is
two and a half times higher than expected and shows that the
doping technique is not yet fully mastered. Of course, this point
deserves to be further elucidated in the future, to control the
doping rate on the one hand and to quantify it with precision on
the other. But at this stage what is important is to have succeeded in doping with all the elements considered, allowing
considering the biological tests in a comparative way with the
non-doped CuO. Nevertheless, we have considered XPS analyses
to conrm in a complementary way the presence of dopant
atoms within the CuO matrix.
3.4. Surface chemical analysis by XPS
X-ray photoelectron spectroscopy (XPS) was used to examine
the chemical state of the host and doping elements in samples.
The XPS survey and narrow regions are displayed in Fig. 8. For
the sake of clarity, survey regions are displayed for CuO
(Fig. 8a) and CuO:Zn (Fig. 8b) only. Indeed, all other doped
CuO particles did not show any striking differences in the
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survey regions; all other survey spectra resemble that of pure
CuO. Cu2p in the 930–965 eV range is clearly visible together
with the prominent Auger peaks. O1s is centred at 529–530 eV,
and C1s from adventitious hydrocarbon contamination is
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placed at 285 eV. The survey region from CuO:Zn exhibits
clearly the Zn 2p doublet. No such clear appearance on the
survey scan was noted for Mn, Fe, Co and Ni dopants. These
elements could be probed only aer acquisition of their
Fig. 8 Survey regions from (a) CuO and (b) CuO:Zn; as well as high resolution spectra of (c) Cu 2p from undoped CuO, (d) Mn 2p from CuO:Mn,
(e) Fe 2p from CuO:Fe, (f) Co 2p from CuO:Co, (g) Ni 2p from CuO:Ni, and (h) Zn 2p3/2 from CuO:Zn.
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Table 3 Comparison of dopant to copper atomic ratio determined by
XPS and EDS
Technique
Mn/Cu
Fe/Cu
Co/Cu
Ni/Cu
Zn/Cu
XPS
EDS
0.33
0.018
0.25
0.015
0.057
0.051
0.038
0.093
0.12
0.004
corresponding main 2p core level spectra. Fig. 8c displays the
peak tted Cu 2p3/2 region from pure CuO. There is one
prominent peak at 934 eV and its satellite doublet with two
peaks at 941.3 and 943.8 eV, assigned to the so-called shake-up
satellites. This is the mark of Cu(II) chemical state. The rst
main peak is tted with three components centred at 932.7
(Cu(I), 933.9, and 936 eV, assigned to Cu(I), Cu(II) from CuO,
and possibly Cu(OH)2, respectively. It is to note that the in situ
reduction of Cu(II) is very likely to occur in XPS, and this noted
even if the whole set of recording of the core level peaks starts
with copper. The difference between the peak position of Cu(I)
and Cu(II) is in line with the literature.77 All other Cu 2p peaks
from the CuO:M series look the same, for the simple reason
that doping at such low level does not alter the spectral shape
of Cu 2p from the major CuO material. Mn 2p, Fe 2p, Co 2p, Ni
2p and Zn 2p3/2 spectra are displayed in Fig. 8((d)–(h). The Mn
2p doublet (Fig. 8(d) displays two peaks at 641.2 and 653.5 eV,
assigned to Mn 2p3/2 and Mn 2p1/2, respectively. It is difficult to
further exploit the Mn 2p doublet for quantication as it is
convoluted with a strong Auger peak from copper. Nevertheless, the Mn 2p region could possibly be assigned to oxidized
manganese in the form of Mn3O4.78
Fig. 8(e) shows the Fe 2p region, another one which is
convoluted with Cu L3M23M23 Auger transition.79 Fe 2p3/2 is
located at 713.0 eV, and could be assigned to Fe2O3. This is
conrmed by the occurrence of Fe 2p1/2 positioned at
724.2 eV.80
Fig. 8(f) displays the Co 2p doublet from CuO:Co with Co 2p3/
and
Co 2p1/2 centred at 781.5 and 797.4 eV, respectively. These
2
values are in line with Co(OH)2.81 The binding energy position of
Co 2p3/2 is not in line with neither Co3O4, nor CoO.78 One can
also note the presence of satellites reported for Co(OH)2 in line
with those reported in the literature (satellites Sat 1 and Sat 2 at
786.2 and 790.2 eV, respectively).82
Ni 2p doublet is shown in Fig. 8(g); Ni 2p3/2 is centered at
855.0 eV, whilst the Ni 2p3/2 satellite is located at 860.8 eV,
both peak positions are consistent with nickel(II) oxides/
hydroxides NiO/Ni(OH)2.78
Fig. 8(h) shows the Zn 2p3/2 peak from CuO:Zn, centered at
1021.7 eV. It is well known from the XPS literature that it is
difficult to use Zn 2p core electron level to distinguish Zn from
ZnO. For this reason, we have recorded the Zn Auger peak
(L3M45M45) and determined a, the Zn Auger parameter (a ¼ BE
(Zn 2p3/2) + KE (Zn Auger); KE ¼ kinetic energy of the Auger
electron). Herein, a ¼ 2010.8 eV and ts in with ZnO of which
a values are reported to be in the 2009.5–2011 eV range.78
The Auger parameter obtained in this work is much lower
than that of metallic zinc (2013.4–2014.4 eV) implying that zinc
is in the oxidized state.
We have determined the transition metal (TM) dopant to
copper atomic ratio (TM/Cu) and compared to the same ratios
determined by EDS (Table 3).
Anti-adhesive activity of doped and un-doped CuO NPs applied at different concentrations expressed as biofilm inhibition. The results are
presented as means SD of three independent experiments. *p < 0.05, **p < 0.01 or ***p < 0.001 versus control values (undoped CuO) and only
significant improvements are presented.
Fig. 9
23536 | RSC Adv., 2022, 12, 23527–23543
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With the exception of cobalt for which the Co/Cu atomic
ratio determined by XPS is matching that obtained by EDS, all
other ratios indicate gradient distribution of the dopant. Zinc is
most likely to be depleted to the surface as the XPS determined
Zn/Cu ratio (surface) is higher than that of the obtained by EDS
(bulk). Nickel shows 3-fold lower surface Ni/Cu ratio, indicating
depletion from the surface and preferential existence of copper
at the surface. For Mn and Fe, obviously the ratios are overestimated as the core electron peaks Mn 2p and Fe 2p are
convoluted with Auger lines from copper. Nevertheless, these
values indicate depletion in volume of these two elements,
probably due to diffusion to the surface.
As far as interactions are concerned, XPS is an ideal technique
for probing interactions via electron transfer. However, all values
reported for CuO and their dopants fall within the binding
energy ranges reported for the probed transition metal
compounds. Attempts to relate the trends to the electronegativity of oxidized transition metals M2+ are not successful
due to very close values of electro-negativity: Mn2+ (1.303) < Fe2+
(1.341) < Co2+ (1.349) < Ni2+ (1.367) < Cu2+ (1.372) > Zn2+ (1.336).83
3.5. Anti-biolm activities of undoped and doped CuO NPs
Anti-adherence activity using crystal violet quantication
method is summarized in the following graph (Fig. 9). All doped
and undoped CuO NPs show signicant anti-adhesive activities
on the tested concentrations from 7.81 mg ml 1 to 4 mg ml 1.
Generally, the activity is dose dependent. The activity is significantly improved with NPs-doped with Fe and Ni at high
concentrations in comparison with undoped CuO at the same
concentrations. In fact, at 4 mg ml 1 of CuO:Fe the activity is
signicantly improved from 67.56% (for undoped) to 89.75%
(CuO:Fe), and the p-value is <0.001. The activity is signicantly
improved also with Ni at concentrations of 2 and 4 mg ml 1
with p-value of 0.008 and 0.009, respectively (Fig. 9). The activity
is not signicantly improved with Mn doping at low concentrations from 7.81 to 31.25 mg ml 1 (p-value> 0.05). Doping with
Zn and Co seems to be inefficient which is not in agreement
with previous studies showing the effect of doping with Zn in
improvement of antibacterial activity against Streptococcus
mutants.25 In previous studies, Pugazhendhi et al.33 showed that
CuO:Fe could inhibit S. epidermidis biolm formation of
approximately 88% which is slightly in agreement with our
study in which the inhibition of biolm formation is close to
70% using CuO:Fe at 125 mg ml 1.
All the tested oxides showed activity, of which the importance depends on the nature of the doping element. This is
probably due to their high capacity of absorbing oxygen, giving
them a semi-conducting p-type behavior. Excess oxygen
increases NPs bacterial activity. The doping of TM into the CuO
structure causes distortion, lattice strain, and defect generation
in the base structure, as well as ROS generation, leading to the
anti-biolm activity of NPs.
To highlight the benet of CuO doping, results are expressed
in terms of the enhancement of the anti-adhesive activity of
some TM-doped CuO NPs, compared to that of undoped CuO
(Fig. 10(a) and (b)). For concentrations below 25 mg ml 1, Mn
© 2022 The Author(s). Published by the Royal Society of Chemistry
Fig. 10 Enhancement rate of anti-adhesive activity of NPs-doped with
(a) Fe and Mn at lower concentrations and (b) Fe and Ni at higher
concentrations, in comparison with undoped CuO.
doping leads to an increase in adhesion inhibition efficiency of
more than 30% compared to undoped CuO. This improvement
even exceeds 75% for the lowest concentrations (lower than 16
mg ml 1). An improvement is also observed in the case of Fe
doping with an increase exceeding 40% for concentrations
above 100 mg ml 1 (Fig. 10(a)). For the highest concentrations,
between 500 mg ml 1 and 4 mg ml 1, the improvements
compared to undoped CuO are brought by Fe and Ni doping. In
the case of Fe, the efficiency increase is between 32 and 52%,
while for Ni it is about 30% on average (Fig. 10(b)).
This suggests that the efficiency could be further improved,
considering not only one type of doped NPs but a mixture of
them. By combining low concentration CuO:Mn NPs with high
concentration CuO:Fe NPs, we could achieve a better inhibition
of biolm adhesion. This will be investigated in our future work.
3.6. Biolm imaging aer treatment with doped CuO NPs
Fluorescent microscopy was used to conrm the anti-adherence
activity of doped or undoped CuO NPs. Images obtained for
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Fig. 11 Fluorescence microscopy with biofilm cover slips. Biofilms were stained with acridine orange. Bar equals 100 mm. Images obtained with
control (untreated biofilm) and treated biofilm with doped NPs (at 400 mg ml 1) are presented.
untreated control and doped CuO NPs conrmed the activity,
obtained aer crystal violet quantication, as showed in the
following gure (Fig. 11).
All treatments resulted in a signicant reduction in biolm
adhesion, conrming the NPs anti-adhesive activity, already
assessed by crystal violet analysis. Anti-adhesion properties of
MONPs against biolm formation are not fully understood.
Researchers proposed multiple mechanisms for the damage
caused by CuO NPs to bacteria.
3.7. The mechanism of interaction CuO NPs – bacteria in
biolms
Researchers have demonstrated that the mechanism of interaction between NPs and bacteria depends on several factors (e.g.
physic-chemical properties of NPs).54,84,85 In fact, the toxicity
strength of MONPs depends on the natural toxic properties of
heavy metals. It seems that there is a quantitative relationship
between average size, surface area and the concentration of
MONPs, as described in the last discussions. It is well known
that the mechanisms of oxide NPs toxicity against biolm
bacteria are complex and depend on several factors such as
composition, surface modication, intrinsic properties (average
size, surface area, .), and the bacterial species (Gram-positive
or Gram-negative bacteria). A simplied mechanism is
proposed in this paper.
Several studies report that the nanotoxicity of NPs is generally triggered by the induction of oxidative stress by free radical
formation, following the administration of NPs.86,87 The mechanisms underlying the anti-adhesion and antibacterial effects of
23538 | RSC Adv., 2022, 12, 23527–23543
NPs are not completely understood and vary from the productions of oxidative and/or free radical formation (e.g., reactive
oxygen species; ROS) stressors to bacteria damage. ROS are ions
or small molecules that contain free radicals (cOH), peroxide
(e.g., hydrogen peroxide; H2O2), or oxygen ions (e.g., superoxide;
cO2 ).88–92 These ROS are formed during the normal metabolism
of oxygen.93 Nevertheless, uncertainties remain about their
mechanism of toxic action, which is supposed to be exerted
mainly through oxidative stress induced by ROS generation,
disruption of cell wall and membrane, disruption of mitochondria, enzymatic inhibition protein deactivation, DNA
damage or changes in gene expression as well as bacteria wall
disruption.54,94
Various researches reported that the anti-bacterial activity of
CuO NPs is generally attributed to both the released Cu2+ ions
and the particles CuO themselves.95,96 The interaction between
CuO NPs and biolm can be considered as a stage process:
transfer of NPs in the vicinity of the biolm, attachment to the
biolm surface, migration in biolms and biolm disruption
(Fig. 12(a)).
Metal oxide NPs and their ions (e.g., copper, zinc and iron.)
can produce free radicals, resulting in induction of oxidative
stress. Bacterial contact killing by CuO NPs is preceded by
a sequence of specic actions: cell membrane damage, copper
inux into the cells, oxidative damage and DNA damage.54,97 The
targeting of DNA by copper leads to rapid DNA fragmentation,
resulting in bacterial death,98 as presented in Fig. 12(b).
Moreover, the addition of 3d transition metal ions as a doping
(e.g., Fe; Zn; Co; .) to the metal oxides (MO) lattice results in the
increasing of structural defects, which, in turn, enhance their
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Fig. 12 (a) Illustration of the inhibition of biofilm formation on surfaces coated by CuO NPs. Metal ions (Cu2+) or CuO NPs penetrate the bacteria,
the dual functions of nanopowders in killing bacteria and inhibiting biofilm result in biofilm disruption. (b). The mechanism (intra-bacteria)
includes cell membrane disruption, disruption of electron transport chains, ROS production, release of metal ions, damage of DNA contents,
disruption of mitochondria and protein synthesis.
antibacterial activity due to more ROS generation.99 Nevertheless,
the detailed mechanisms of metal oxide NPs toxicity against
various bacteria still not completely understood.
4. Conclusions
In this work, undoped and TM-doped CuO NPs have been
successfully synthesized via co-precipitation method. The
synthesized NPs were characterized by XRD, FT-IR, SEM, EDX
and XPS techniques. The X-ray diffraction and EDS analysis
conrmed the pure single-phase formation of CuO for all
samples. The crystalline size was estimated via Debye–Scherrer's law, to be dependent on TM-doping element. The XPS
measurements highlight the effective doping, in addition to
showing a concentration gradient, with either depletion or an
enrichment of the surface in doping element. A difference in
morphology of M-doped CuO NPs was also showed by SEM
analysis. All these disparities in properties inuence the interaction between NPs and the surrounding media.
© 2022 The Author(s). Published by the Royal Society of Chemistry
Besides, all the synthesized samples showed signicant antiadhesive activity against S. epidermidis S61 biolms. At high
concentrations, CuO:Fe and CuO:Ni exhibit signicant
improvement over undoped CuO. In contrast, low concentration of CuO:Mn NPs exhibited the strongest anti-adhesive
activity. In summary, our results suggest that the synthesized
powders can be used as a promising anti-adhesive agent in the
biomedical treatment.
In the other hand, it is well known that the mechanisms of
oxide NP's toxicity against biolm bacteria are very complicated
and depend on several factors such as composition, surface
modication, intrinsic properties (average size, surface area,
.), and the bacterial species (Gram-positive or Gram-negative
bacteria). Nevertheless, a simplied mechanism is proposed
in this paper.
Finally, the obtained results are promising, and pave the way
for the synthesis of effective nanopowders to reduce or even
eliminate the dangerous effects of biolms formed by bacteria,
and which can nd potential applications in the medical eld.
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Conflicts of interest
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There are no conicts to declare.
Acknowledgements
Authors gratefully thank the nancial support of the Tunisian
Ministry of Higher Education And Scientic Research, and
Campus France, within program (PHC Utique project No
17G1143). Mr P. Decorse (experimental officer) is acknowledged
for technical assistance with XPS measurements.
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