Applied Surface Science 341 (2015) 157–165

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Applied Surface Science

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

Antibacterial efficiencies of TiO2 nanostructured layers prepared in

organic viscous electrolytes
Cristina Dumitriu a , Marian Popescu a,b , Camelia Ungureanu a , Cristian Pirvu a,∗
a
University Politehnica of Bucharest, 313 Splaiul Independentei, 060042 Bucharest, Romania
b
National Institute for Research and Development in Microtechnologies, 126A, Erou Iancu Nicolae Street, 077190 Bucharest, Romania

a r t i c l e i n f o a b s t r a c t

Article history: Using easy and cheap potential step anodization in electrolytes with different molar mass and water
Received 17 October 2014 content, a Ti substrate was covered with a nanostructured TiO2 layer. Surface characterization of the
Received in revised form 27 February 2015 prepared samples was conducted using scanning electron microscopy (SEM), Fourier transform infrared
Accepted 28 February 2015
spectroscopy (FT-IR) and contact angle analysis. The formation mechanism and surface morphologies are
Available online 14 March 2015
very different, depending on the molar mass and water percent of electrolyte solutions used for anodizing
Ti substrate. The electrochemical behavior of the samples was studied using Tafel plots, and electrochem-
Keywords:
ical impedance spectroscopy recorded in a simulated body fluid. All used anodizing treatments have
Titanium nanostructures
Anodization
conducted to samples with increased corrosion protection. The paper illustrates the antibacterial effi-
Viscous electrolytes ciencies of TiO2 nanostructured layers (shielded nanotubes, nanoporous oxide layer and some remaining
Antibacterial effect PEG electrolyte) quantitatively estimated using gram-negative bacterium Escherichia coli ATCC 8738.
Polyethylene glycol © 2015 Elsevier B.V. All rights reserved.

1. Introduction electrochemical anodization is strongly affected by the electro-

Titanium and its alloys are of great importance being used in
many applications due to their properties such as: high-quality
mechanical properties, excellent biocompatibility and capacity of
bio-reaction with human plasma and tissue, as well as enhanced
corrosion resistance – they form a surface native titanium dioxide
(TiO2 ) layer as a protective coating [1,2]. Titania (TiO2 ) as a wide-
bandgap semiconductor, non-toxic, environmentally friendly, with
almost unique ionic and electronic properties [3] has also received
extensive attention. The TiO2 nanoscale geometry (characterized
by high surface-to-volume ratio) allows better control of the chem-
ical or physical behavior, and has the ability to provoke a greater
degree of biological plasticity [1,3]. Titania nanostructures are a
unique combination between these functional features of TiO2 with
controllable nanoscale geometry [3].
A method of obtaining nanoscale architectures of metal oxides
is electrochemical anodization. This is among the easiest, cost-
effective, and versatile techniques to obtain nano-organized
TiO2 from inorganic or organic fluoride containing electrolytes
[4–6]. The morphology of TiO2 nanostructures prepared by
∗ Corresponding author at: General Chemistry Department, Faculty of Applied
Chemistry and Materials Science, University Politehnica of Bucharest, 1-7 Polizu,
011061 Bucharest, Romania. Tel.: +40 214023930; fax: +40 213111796.
E-mail address: c pirvu@yahoo.com (C. Pirvu).
chemical conditions [7–9] and post-elaboration treatment [10,11].
By controlling electrochemical conditions, it is easy to prepare
nanoporous, bamboo-shaped structures, self-organized nanotubu-
lar TiO2 or titania nanochannels [12,13]. For the morphology of the
obtained nanostructures, the electrolyte chemical composition also
has a critical role [14]. Thus, the morphology is influenced in the
main stages of formation mechanism such as: the formation rate of
nanotubes, as well as the dissolution rate of the resulting oxide [14].
Organic electrolytes have a small amount of oxygen compared to an
aqueous solutions and, that is why, the oxide chemical dissolution
depends on the water content [5]. So, for anodization in organic
electrolytes, the electrolyte viscosity and the H2 O content are also
important. The diffusion constant decreases in those electrolytes.
Research studies showed that nanostructured TiO2 exhibits pos-
itive effect on cell behavior. So this material can be used as a
future implant material [1]. Implant-related microbial infection is
a serious threat. Subsequently, new strategies to reduce bacterial
adhesion and to prevent biofilm formation, both in vitro and in
vivo, are under evaluation [15]. TiO2 was used for antibacterial
applications having incorporated components (such as Ag or Zn)
with biocidal activity [16,17]. Also, ultraviolet (UV) light on tita-
nium dioxide can be used to generate oxidative effect by formation
of reactive oxygen species or highly reactive hydroxyl radicals,
which are strong enough for lysis of microbes [16,18]. Other
studies show that addition of Cu-doped TiO2 nanoparticles, sig-
nificantly reduced the Mycobacterium smegmatis growth rate, and

http://dx.doi.org/10.1016/j.apsusc.2015.02.183
0169-4332/© 2015 Elsevier B.V. All rights reserved.
158 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165
the antibacterial effect increased with the Cu content in nanopar-
ticles [19]. Moreover, CuO/TiO2 composite nanorods fabricated by
sol–gel electrospinning method demonstrated antimicrobial activ-
ity against Escherichia coli and Staphylococcus aureus [20]. Recently,
TiO2 –ZrO2 nanotubes with different diameters, prepared by two-
step anodization of Ti50Zr alloy, exhibited efficient antibacterial
behavior against E. coli bacteria [21]. Another compound with sig-
nificant antibacterial activity against various pathogenic bacteria,
including E. coli is concentrated polyethylene glycol 400 (PEG 400)
solution. It was shown that PEG has antibacterial activity since
1983. Its antibacterial effect might be attributed to the property of
PEG 400 in lowering water activity and, besides this, to the specific
action of PEG-400 molecules on bacterial cells. The antibacterial
effect of PEG 400 is much lower compared to traditional disinfect-
ing agents, but PEG 400 is nonirritating for the skin, its toxicity being
low in case of acute oral administration or topical application [22].
Composite coatings based on polypyrrole (PPy) with various per-
centages of polyethylene glycol 400 (PEG 400) electrodeposited on
TiAlZr alloy showed antibacterial properties as well [23].
The importance of TiO2 nanostructures and evaluation of TiO2
antibacterial efficiencies were the motivation for this study. In pre-
vious works we successfully obtained TiO2 nanotubes in organic
electrolytes (EG and PEG with average molecular weights 400 and
600) with addition of NH4 F and small water content [24,25]. Thus,
in this work, TiO2 nanostructured surfaces were obtained in very
viscous PEG 1000. But in this case, much more water content was
needed in order to solubilize it. It has been studied as well, how
water content influences the morphologies and corrosion resis-
tance of those samples. On other hand, FT-IR analyses proved that
those samples prepared in viscous electrolytes have the surface
covered with a shielding layer composed from TiO2 and some
remaining PEG electrolyte. As literature data report the antibac-
terial activity of PEG 400, we tested if the presence of PEG, with
different molecular weight in the shielding layer improves the
antibacterial activity of those samples against E. coli bacteria.
2. Experimental
2.1. Materials
The following organic reagents were used for anodization: ethy-
lene glycol (EG) from Reanal Finomvegyszergyár, polyethylene
glycols with average molecular weight 400 (PEG 400), 600 (PEG
600) and, respectively, 1000 (PEG 1000) from Fluka Analytical.
Ammonium fluoride (NH4 F) from Sigma–Aldrich constituted the
source for fluoride ions. All those reagents were analytical grade
and used as received. Purified water was obtained with a Millipore
Direct-Q UV3 water purification system.
2.2. Methods
2.2.1. Sample elaboration via potential-step anodizing treatment
Small pieces sliced from a 99.7% pure, commercially avail-
able titanium foil, having 2 mm thickness (Sigma–Aldrich), were
anodized in order to obtain TiO2 nanostructures. The surface pre-
treatment was first conducted by abrasion with silicon-carbide
sandpaper with increasing grit size (from 800 to 4000 grit size),
and alumina paste successively. For degreasing the surface prior
to experiment, the polished samples were cleaned with purified
water, ethanol and acetone, 15 min each, by ultra-sonication. The
cleaned samples were immediately used.
Freshly conditioned samples were mounted like an anodic
electrode (contacted and then pressed against an O-ring) in a one-
compartment two-electrode electrochemical cell. A Pt foil was used
as counter electrode. A MATRIX MPS-7163 electrochemical source
Table 1
Electrolyte composition.
Electrolyte Electrolyte composition Resulting sample name
1 EG + 3% v/v H2 O + 0.5 wt% NH4 F Ti/EG 3%w
2 EG + 54% v/v H2 O + 0.5 wt% NH4 F Ti/EG 54%w
3 PEG 400 + 3% v/v H2 O + 0.5 wt% NH4 F Ti/PEG 400 3%w
4 PEG 400 + 54% v/v H2 O + 0.5 wt% NH4 F Ti/PEG 400 54%w
5 PEG 600 + 3%v/v H2 O + 0.5 wt% NH4 F Ti/PEG 600 3%w
6 PEG 600 + 54% v/v H2 O + 0.5 wt% NH4 F Ti/PEG 600 54%w
7 PEG 1000 + 54% v/v H2 O + 0.5 wt% NH4 F Ti/PEG 1000
and a digital multimeter (type VA18B, from V&A International)
were also connected to the electrochemical setup. PC-Link acquisi-
tion software was used for recording anodizing current vs. time. In
all cases, synthesis of TiO2 nanostructures was achieved applying
a voltage raising from 0 to 50 V, with a 2 V/10 s step, and then kept
constant at 50 V. The entire process of anodization of Ti substrate
lasted 2 h at room temperature.
The electrolytes composition is described in Table 1.
2.2.2. Samples characterization
The nanostructures morphology, including pore size of the
anodized samples was observed through high-resolution field
emission scanning electron microscope (SEM) QUANTA INSPECT
F with a 1.2 nm field emission gun. Thickness information was
obtained by SEM cross-sectional observation on samples that were
mechanically fractured, in order to crack and partially lift-off the
TiO2 layers.
Surface roughness was performed with an atomic force micro-
scope in contact mode (AFM) from APE Research, Italy. The
microscopy data were processed with Gwyddion 2.9 software.
Fourier transform infrared spectroscopy (FT-IR) spectra were
taken from the nanostructure layers on Ti substrate using a Spec-
trum 100 Series FT-IR Spectrometer from PerkinElmer, USA. All
samples were measured, as they were, with ATR mode.
The wettability of samples has been assessed by static con-
tact angle measurements, carried out with an accuracy of ±1◦ at
room temperature with CAM 100 Optical Contact Angle Meter
(KSV Instruments). Reported contact angles are the average of at
least three consecutive measurements. Purified water was used for
determinations.
Electrochemical behavior characterization was conducted with
an Autolab (PGSTAT 302N) potentiostatic assembly of three elec-
trodes in a single compartment. Samples have been used as working
electrodes, platinum as an auxiliary electrode, and all potentials
reported were measured vs. an Ag/AgCl/Cl− (3 M) reference elec-
trode. All experiments were done in Hank bio liquid, with the
following composition (g/L): 8 NaCl, 0.4 KCl, 0.35 NaHCO3 , and 0.25
NaH2 PO4 ·H2 O, 0.06 Na2 HPO4 ·2H2 O, 0.19 CaCl2 ·2H2 O, 0.19 MgCl2 ,
0.06 MgSO4 ·7H2 O, 1glucose [26]. The area of the electrode in con-
tact with solution was 0.28 cm2 .
2.2.3. Antibacterial activity assays
The obtained titanium nanostructures antibacterial activity was
evaluated against E. coli ATCC 8738 strain. The gram-negative bac-
terium E. coli was chosen for our study because this one has
recently aroused more scientific interest, having new forms and
causing various diseases [27]. E. coli bacterium was inoculated into
a Luria Bertani (LB) medium (which contained peptone 10.0 g/L,
NaCl 5.0 g/L and yeast extract 5.0 g/L) as the test strain and kept on
an incubator shaker (Laboshake Gerhardt) overnight at 37 ◦ C and
300 rpm [28].
The samples were sterilized by autoclave at 121 ◦ C for 15 min.
No visual or morphological changes were observed after this pro-
cess. The culture was obtained from a volume of 10 mL sterile
culture medium, which was inoculated with 100 ␮L of E. coli. Once
 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165 159

Fig. 1. Current density–time (j–t) characteristics after a voltage step for anodic film growth in: (A) in EG; (B) in PEG 400; (C) in PEG 600 and (D) in PEG 1000.

obtained, 0.5 mL of culture was placed over the samples. Finally, the
bacterial broth suspensions were incubated at 37 ◦ C for 18 h in the
dark conditions. Samples were properly covered to ensure absence
of visible light.
The bacterial growth was determined by measuring optical den-
sity for the samples and control (bacterium culture without sample)
at 600 nm, using UV-VIS spectrophotometer (Jenway Spectropho-
tometer) [29]. The antibacterial activities of the samples were
determined by calculating the percentage inhibition of growth
using the formula proposed by Jaiswal et al. [30].
The total charge density calculated by integrating the area under
the current density vs. time curve is presented in Fig. 2.
It is visible that the total charge density is higher for samples
with 54% water content. So, increasing water content in the elec-
trolyte used for anodization increases the charge density. If we
consider that the total charge density is used for oxide formation,
a thicker layer is formed for samples with 54% water. In cross-
sectional SEM images presented for Ti/PEG 600 3%w sample and
Ti/PEG 600 54%w sample it is visible a thicker layer is formed for
samples with 54% water. This is also the case for Ti/PEG 400 3%w

3. Results and discussions

3.1. Samples anodization

The changes of current densities in time between Ti substrate

and the Pt electrode were recorded during anodization, in order to
obtain electrochemical information, as shown in Fig. 1. In all cases, it
is visible that current densities are smaller for the samples prepared
in electrolyte with small percent of water, compared to those with
high amount of water. For PEG 1000, its viscosity does not allow the
preparation of an anodizing solution with 3% water. In the first min-
utes of anodization, the current density increases with the potential
increase. Two cases can be observed. In EG (Fig. 1A), the current den-
sity is successively increasing and decreasing, because of the oxide
layer formation and dissolution. In PEG electrolytes, regardless of
average molar mass of PEG or water percentage in the anodization
solution, the current density continuously increases, suggesting a
faster consolidation of the oxide layer in viscous electrolytes. Then,
when the potential was kept constant at 50 V, the current density
begins to decrease. For sample Ti/EG 54%w (Fig. 1A), the current
density fall is higher compared to other samples and regardless of
water percentage. The current density begins to go to constants at
different time points. Fig. 2. Total charge density during anodic film growth.
160 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165
Fig. 3. SEM images for: (A) Ti/EG 3%w; (B) Ti/EG 54%w; (C) Ti/PEG 400 3%w; (D) Ti/PEG 400 54%w; (E) Ti/PEG 400 54%w; (F) Ti/PEG 600 54%w; (G) Ti/PEG 1000.
sample and Ti/PEG 400 54%w sample (not shown). Increasing of
average molecular weight of PEG leads to a charge increasing both
in case of samples with 54%w, and in the case of samples with 3%w.
The charge density value in the case of Ti/EG 3%w sample is between
the one for Ti/PEG 400 3%w and Ti/PEG 600 3%w. So, increasing
the average molecular weight and water content have an influence
on formation and grow of nanotubes or nanoporous layers on Ti
substrate.
3.2. Surface analysis
SEM images, top view and cross-section for all anodized sam-
ples, are presented in Table 1 and Fig. 3. In the case of samples
Ti/EG 3%w, and Ti/PEG 600 3%w we obtain nanotubes, as we
have previously reported for those electrolytes with 2% of water
[24,25]. Nanotubes have different diameters; in EG around 50 nm
and between 117 nm and 208 nm in case of PEG 600 electrolyte.
In PEG 400, with 2% water, we have obtained nanotubes [24] com-
pared to the present study, in which, for Ti/PEG 400 3%w sample,
we have obtained nanotubular layer, but with nanotubes not very
well defined and arranged. So, it seems that in this electrolyte the
water content is very important for obtaining nanotube structures,
and small differences between the amounts of water can radi-
cally change the morphology of the sample. Anodization does not
result in formation of a nanotubular layer for samples prepared
in 54% water electrolyte, despite the fact that we kept other elec-
trochemical parameters constant and those electrolytes have the
same amount of fluoride salt. The anodization in all those cases
which have higher amounts of water leads to nanoporous layers
morphologies similar with a sponge, with irregularities. Despite
the fact that we used potentiostatic step anodization, these mor-
phologies are very similar with those obtained by Schmuki group,
during galvanostatic anodization of Ti in 1 M (NH4 )2 SO4 + 0.5 wt%
NH4 F electrolyte [31]. Using electrolytes with different viscosities
(EG, PEG 400, PEG 600, PEG 1000) and also changing the water con-
tent of the electrolyte solution caused a corresponding change in
the morphology of the surfaces. It is clear that the morphology of
the film is strongly dependent on the electrolyte.
A method for surface characterization of the oxide films was
water drop contact angle, which gives a measure of the hydrophilic
or hydrophobic nature of the surface. For Ti substrate and anodized
samples, the values of the contact angle are presented in Fig. 4.
The uncoated titanium substrate is less hydrophilic, having a water
contact angle close to 90◦ , while the nanotubes and nanoporous
structures showed a hydrophilic behavior. For samples prepared
in EG, the contact angle value for the one with 3%w (unshielded
nanotubes) is lower, compared to the one for the sample with 54%w
(nanoporous oxide layer). This difference between contact angles
can be associated with different surface morphology, very well
organized in the first case and pretty rough in the second case (as
 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165
Fig. 4. Contact angle for all prepared samples and Ti substrate.
can be seen in SEM presented in Fig. 3) not allowing water droplet
to spread too much. For samples prepared in PEG 400 or PEG 600,
wetting behavior is different for different electrolytes and water
content in the electrolyte composition, the morphologies being also
different. In these cases, contact angle value for Ti/PEG 400 3%w
sample (shielded nanotubes) > Ti/PEG 400 54%w (nanoporous oxide
layer) and Ti/PEG 600 3%w sample (shielded nanotubes) > Ti/PEG
600 54%w (nanoporous oxide layer). Samples prepared in PEG 600
with higher water content and in PEG 1000 are very hydrophilic,
having the lowest contact angles values.
In the previous works, we showed that some remaining viscous
electrolyte is still present on the surface of the anodized samples,
despite the fact that those were carefully rinsed with purified water
before recording FT-IR spectra [24,25]. The surface of anodized Ti
was analyzed using the FT-IR transmission spectra, in order to probe
if this observation is also valid for the samples anodised in those
electrolytes with more water content. These spectra were recorded
for the pure ethylene glycol and polyethylene glycols, and also for
the TiO2 nanoporous layers at room temperature, in the region
4000–600 cm−1 . In Fig. 5 characteristic IR vibrations can be found
in the transmission spectra. The peak for stretching vibration of
O–H group is visible: at around 3290 cm−1 in pure EG spectrum,
3442 cm−1 in pure PEG 400, 3450 cm−1 in pure PEG 600 spectrum
and, respectively, 3390 cm−1 in the spectrum of pure PEG 1000.
The sharp band corresponding to stretching vibration of CH2
is visible: at around 2870 cm−1 (for pure EG), at 2860 cm−1 (for
pure PEG 400 and PEG 600) and at 2880 cm−1 (for pure PEG 1000).
The band corresponding to binding vibration of CH2 is at around
1450 cm−1 (for pure EG, pure PEG 400 and PEG 600), and 1460 cm−1
(for pure PEG 1000) The corresponding band from the stretching
vibration of C O C bond can be found at around 1340 cm−1 (for
pure PEG 400, PEG 600 and for pure PEG 1000). A very sharp and
intense peak appears at around 1080–1100 cm−1 in the pure EG and
pure polyethylene glycols spectrum corresponding to C C stretch-
ing. In the case of Ti/EG 3%w sample, there are some remaining
Table 2
Corrosion characteristics from Tafel polarization curves obtained in Hank bioliquid.
Sample jcorr (A/cm2 )
Ti substrate 19.2 × 10−8
Ti/EG 3%w 5.1 × 10−8
Ti/EG 54%w 5.3 × 10−8
Ti/PEG 400 3%w 17.9 × 10−8
Ti/PEG 400 54%w 17.2 × 10−8
Ti/PEG 600 3%w 3.21 × 10−8
Ti/PEG 600 54%w 46.13 × 10−8
Ti/PEG1000 0.32 × 10−8
161
of the used electrolyte, because low intensity peaks characteris-
tic for pure EG are visible. In the spectrum of anodized samples
Ti/EG 54%w and, respectively, Ti/PEG 600 54%w these peaks men-
tioned above are not visible. So, anodizing in these solutions with
increased water content, does not lead to electrolyte incorporation
on the surface of the samples. For these two samples the differences
between measured contact angles could be attributed to surface
morphology, noticing major differences between porosity (as can
be observed from SEM images) and roughness of the two samples
estimated from AFM analysis (rms, 137 nm for Ti/EG 54%w and rms,
771 nm for Ti/PEG 600 54%w).
For a liquid droplet on a flat homogeneous surface, contact angle
established could be described by Young’s relation. But when sur-
face features’ dimensions are much smaller than the droplet size,
there could be Cassie–Baxter model or the Wenzel state or a tran-
sition between them. In first case the droplet sits on top of the
textured surface with trapped air underneath while in the second
case, the liquid is in contact with the entire exposed surface of the
solid. In the Wenzel state, the substrate becomes more hydrophilic
with a higher surface roughness [32]. These two hydrophilic sam-
ples (Ti/EG 54%w and Ti/PEG 600 54%), where the contact angle
decrease with the increasing of the roughness, appear to be in the
Wenzel state.
In the case of anodised sample Ti/PEG400 54%w and Ti/PEG1000
(Fig. 5B and D), characteristic peaks of pure PEG 400 and, respec-
tively, PEG 1000 are present (more or less intense or shifted),
despite the fact that these samples were also prepared from solu-
tions with increased water content. In the case of Ti/PEG 400 3%w
and Ti/PEG 600 3%w samples there are also traces of the used
anodizing electrolytes, so, in the shielding covering the nanotubes,
visible in the SEM images, the electrolytes used are also incorpo-
rated.
3.3. Electrochemical analysis
Fig. 6 compares polarization curves for freshly cleaned titanium
surface with those for anodized samples in Hank’s naturally aer-
ated solution at room temperature. Curves have been registered
between ±300 mV vs. OCP, with a 2 mV/s scan rate, at initial immer-
sion time. The quantification of electrochemical stability in terms
of corrosion parameters such as jcorr – corrosion current, Ecorr – cor-
rosion potential, vcorr – corrosion rate and polarization resistance
are summarized in Table 2. Most of samples showed an increase
corrosion resistance compared to Ti substrate.
For the Ti/PEG 600 54%w sample, nanostructured layer does
not protect Ti substrate against corrosion, probably because of
the increased porosity of the layer (as it can be seen from the
SEM image). If we compare samples prepared in the same elec-
trolyte, but with different amount of water, in the case of EG and
PEG 400, the values are similar, although there are very different
morphologies, 4.41 × 10−4 mm/y and 4.63 × 10−4 mm/y for Ti/EG
3%w and Ti/EG 54%w and 15.6 × 10−4 mm/y and 14.98 × 10−4 mm/y
for Ti/PEG 400 3%w and Ti/PEG 400 54%w, respectively. Only in the
Ecorr vcorr (mm/y) Polarization resistance ()
V vs. Ag/AgCl/3 M KCl
−0.41 16.7 × 10−4 2.83 × 106
−0.07 4.41 × 10−4 2.84 × 106
0.09 4.63 × 10−4 6.82 × 106
−0.12 15.6 × 10−4 0.77 × 106
0.08 14.98 × 10−4 1.97 × 106
0.07 2.8 × 10−4 3.50 × 106
0.003 40.15 × 10−4 0.82 × 106
−0.06 0.2799 × 10−4 62.3 × 106
162 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165

Fig. 5. FT-IR spectra for anodic film obtained in electrolytes with composition given in Table 1.

case of PEG 600, it is a big difference (one order of magnitude)
between the samples with different amount of water. Samples
Ti/PEG 600 3%w and Ti/PEG 1000 have showed the lowest corro-
sion rates. Both samples were prepared in the viscous electrolyte
solutions and is expected that the PEG content to be highest on the
surface of these samples. In a previous study it was concluded that
molecules sequestered within the PEG-doped composites are less
responsive to O2 indicating that they are rendered inaccessible to
O2 as a result of the PEG doping [33]. This can be an explanation that
the presence of PEG on the surface of Ti/PEG 600 3%w and Ti/PEG
1000 can leads to reduced corrosion rate due to the influence of
cathodic reaction.
Generally, corrosion resistance for prepared samples is higher
compared to Ti substrate for all samples because of the nanotubes
or nanoporous layers, except Ti/PEG 600 54%w.
The electrochemical impedance spectroscopy (EIS) data were
acquired in the frequency range of 10−1 –105 Hz at open circuit
potential. The voltage perturbation amplitude of the AC potential
 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165 163

Fig. 6. Polarization curves in Hank’s naturally aerated solution at room temperature for freshly cleaned titanium surface and for anodized samples in: (A) electrolytes with
3% v/v H2 O and (B) electrolytes with 54% v/v H2 O.

Fig. 7. Nyquist diagram obtained in Hank bioliquid vs. Ag/AgCl 3 m KCl reference for freshly cleaned titanium surface and for anodized samples in: (A) electrolytes with 3%
v/v H2 O and (B) electrolytes with 54% v/v H2 O. Full lines show the fitting to the equivalent circuits in Fig. 8.

was 10 mV. Nyquist diagram obtained in Hank bioliquid vs. Ag/AgCl
3 M KCl reference is presented in Fig. 7. EIS data were fitted with
NOVA 1.7 software, establishing the most probable electrical equiv-
alent circuit for tested samples. Those are drawn in Fig. 8. For freshly
cleaned titanium surface, a system with one time constant elec-
trical equivalent circuit is satisfactory (Fig. 7A). In this circuit, Rs
is the solution resistance, and time constant represents the oxide
barrier layer (layer pseudo-capacitance CPE1 and layer resistance
R1 ). For prepared samples, a more complex circuit was used, taking
into account the nanotube and the shielding layer. Parameters of
equivalent electrical circuits are presented in Table 3. Resistance R1
value (assigned to barrier oxide layer) is lower for TI/EG 3%w sam-
ple compared to the one for Ti/EG 54%w sample. This means that
a large part of the total charge density, used in anodization, is for
oxide layer consolidation. But in the case of samples prepared in
PEG 400, PEG 600 and PEG 1000, R1 values are lower for cases with
higher water content, compared to the ones with lower water con-
tent. For samples Ti/PEG 400 54%w, Ti/PEG 600 54%w, and Ti/PEG
1000, the charge density used in anodization is higher, but R1 val-
ues for those samples are lower, this meaning that a large part of

Fig. 8. Equivalent circuit used for fitting the EIS data (A) for titanium surface, (B) for Ti/EG 54%w sample and (C) for Ti/EG 3%w; Ti/PEG 400 3%w, Ti/PEG 400 54%w, Ti/PEG
600 3%w, Ti/PEG 600 54%w, Ti/PEG 1000.
164 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165

Table 3
Parameters of equivalent electrical circuits used for fitting the EIS data.

Element Sample

Ti substrate Ti/EG 3%w Ti/EG 54%w Ti/PEG 400 3%w Ti/PEG 400 54%w Ti/PEG 600 3%w Ti/PEG 600 54%w Ti/PEG 1000

Rs () 242.2 186.50 96.59 112.26 57.76 68.23 322.39 85.656

R1 () 6.6 × 104 11.03 × 105 20.93 × 105 63.87 × 105 4.88 × 105 11.89 × 105 2.51 × 105 6.81 × 105

CPE1
Y01 (S sn ) 0.6 × 10−5 1.07 × 10−5 0.25 × 10−5 5.89 × 10−5 0.54 × 10−5 3.45 × 10−5 1.27 × 10−5 4.54 × 10−5
n1 0.85 0.95 0.97 0.85 0.883 0.99 0.89 0.72

R2 () – 0.30 × 105 1.07 × 105 0.04 × 105 0.28 × 105 3.0 × 105 0.89 × 105 1.52 × 105

CPE2
Y02 (S sn ) – 17.19 × 10−6 4.98 × 10−6 10.24 × 10−6 6.01 × 10−6 6.37 × 10−6 2.58 × 10−6 2.71 × 10−6
n2 – 0.87 0.87 0.84 0.88 0.93 0.99 0.99

R3 () – – 336.01 121.88 367.48 132.3 110.84 296.05

CPE3
Y03 (S sn ) – – 0.09 × 10−7 1209 × 10−7 0.0036 × 10−7 106.1 × 10−7 133.5 × 10−7 261.5 × 10−7
n3 – – 0.90 0.33 1 0.39 0.49 0.19

2 0.07 0.005 0.02 0.002 0.04 0.0008 0.02 0.04

the charge density is used for nanoporous layer formation. Val-
ues for R3 resistance, assigned to the shielding layer are hundreds
of ohms showing that this layer is not responsible for corrosion
resistance.
n1 is in the range between 0.8 and 0.99 for most of samples,
this indicating a pseudo-capacitive behavior. N1 for Ti/PEG 1000
sample is 0.72, suggesting that in this case the barrier oxide layer is
not compact. However, this sample has the lowest corrosion rate,
meaning that the corrosion resistance in this case is due to the thick
nanoporous oxide layer. Ti/PEG 1000 sample has also the highest
charge density used during anodizing process.
3.4. Percentage inhibition of growth
The bactericidal effects of Ti substrate and anodized sam-
ples were investigated against E. coli strain. The bacteriological
experiments performed in vitro demonstrated the effectiveness of
anodized samples in inhibiting the growth of this bacterium.
Fig. 9 shows percentage inhibition of bacteria growth in the
experiments conducted according to the above description for the
anodized samples and Ti coated with EG, PEG 400, PEG 600 and
PEG 1000, respectively. Samples prepared in EG solution (which
has the highest value for contact angle of anodized samples)
have antibacterial activity similar with Ti substrate. For samples
Fig. 9. Percentage inhibition of bacteria growth for titanium surface, anodized sam-
ples in electrolytes with composition described in Table 1 and also for titanium
substrate coated with EG, PEG 400, 600 and 1000, respectively.
anodized in PEG 400 and 600, values of antibacterial activity are the
highest.
As it has been mentioned in the introduction part, PEG 400 was
found to have bactericidal effect. As expected, samples prepared
in polyethylene glycols have higher values probably because they
have some remaining electrolyte incorporated (according to FT-IR
analysis). Samples Ti/PEG 400 3%w and Ti/PEG 600 3%w with nano-
tubes layers have higher antibacterial activity compared to samples
Ti/PEG 400 54%w, Ti/PEG 600 54%w, Ti/PEG 1000 with nanoporous
layers. This behavior might be attributed to two effects: lowering of
water activity, and the specific action of PEG 400 on bacterial cells.
Chirife et al. [22] showed that PEG 400 caused immediate clump-
ing of the bacterial cells. Meanwhile, Gothwal and Srivastava [34]
reported the antibacterial activity of certain compounds with PEG
600. The killing effect of Ti/PEG 400 3%w and Ti/PEG 600 3%w is
much lower than that of traditionally disinfecting agents. But its
merits should be considered, as PEG 400 and PEG 600 meets many
of the physicochemical requirements for an ideal disinfectant for
topical use. At present, PEG 400 and PEG 600 have several pharma-
ceutical applications, due to their antibacterial properties [35]. At
the same time, TiO2 nanotubes possess antimicrobial activity. This
mechanism involves an instantaneous and reversible physical first
phase, and a cellular second phase [36]. After implantation, a com-
petition between microbial colonization and tissue integration of
the implant is taking place [37]. For Ti/EG 54%w sample, the antimi-
crobial activity is not improved compared to the one for Ti and Ti
with EG. Sample Ti/EG 3%w has antimicrobial activity a little higher
compared with the one for Ti and Ti with EG. So in this case, this little
improvement may be caused by the nanotubes seen also in the SEM
image.
For all samples obtained by anodization in PEG electrolytes,
the antimicrobial activity was higher compared with the one
for Ti. This behavior was also similar for Ti covered with PEG
compared with Ti samples showing the beneficial effect of both
surface nanostructuring and the antibacterial activity of this
PEG.
In SEM images it is visible that in the case of Ti/PEG 400 3%w
and Ti/PEG 600 3%w samples, anodization leads to the forma-
tion of nanotubes. For those samples, with a higher contents of
PEG on the surface, the antimicrobial activity is higher compared
with the one for Ti/PEG 400 54%w and PEG 600 54%w, and also
higher compared with Ti sample (non-anodized) coated with PEG
400 and PEG 600. This behavior suggests a synergic effect due to
both nanotube structures and the presence of PEG in the outer
layer.
 C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165
The results obtained in this paper suggest that Ti/PEG 600 3%w
and Ti/PEG 400 3%w may have a potential value as an antibacterial
agent.
4. Conclusions
Titanium plates anodised in ethylene glycol and polyethylene
glycols with different average molecular weight, having higher
amount of water (Ti/EG 54%w, Ti/PEG 400 54%w, Ti/PEG 600
54%w and Ti/PEG 1000) showed similar nanoporous morphologies.
Despite this fact, these samples have different wetting behavior,
most hydrophilic being Ti/PEG 600 54%w and Ti/PEG 1000 sam-
ples. They have also different electrochemical behaviors, some with
improved corrosion protection.
Samples prepared in polyethylene glycols showed increased
values for antibacterial activity, higher for those with shielded
nanotubes layers. It seems that the nanotubes with shielding
layer (which have incorporated polyethylene glycol) give stronger
antibacterial effect, compared to the uncovered long nanotubes
prepared in ethylene glycol.
Titanium surface modification by anodization in viscous elec-
trolytes with controlled water content leads to a change in
morphology and surface chemistry materialized with increased
corrosion stability and also improved bactericidal properties.
Acknowledgement
This work was supported by a grant from the Romanian National
Authority for Scientific Research, CNCS – UEFISCDI, project PN2-
253/2014 – NANOCOAT.
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