Вы находитесь на странице: 1из 9

Applied Surface Science 341 (2015) 157–165

Contents lists available at ScienceDirect

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-


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


Electrolyte composition.
ticles [19]. Moreover, CuO/TiO2 composite nanorods fabricated by
sol–gel electrospinning method demonstrated antimicrobial activ- Electrolyte Electrolyte composition Resulting sample name
ity against Escherichia coli and Staphylococcus aureus [20]. Recently, 1 EG + 3% v/v H2 O + 0.5 wt% NH4 F Ti/EG 3%w
TiO2 –ZrO2 nanotubes with different diameters, prepared by two- 2 EG + 54% v/v H2 O + 0.5 wt% NH4 F Ti/EG 54%w
step anodization of Ti50Zr alloy, exhibited efficient antibacterial 3 PEG 400 + 3% v/v H2 O + 0.5 wt% NH4 F Ti/PEG 400 3%w
behavior against E. coli bacteria [21]. Another compound with sig- 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
nificant antibacterial activity against various pathogenic bacteria,
6 PEG 600 + 54% v/v H2 O + 0.5 wt% NH4 F Ti/PEG 600 54%w
including E. coli is concentrated polyethylene glycol 400 (PEG 400) 7 PEG 1000 + 54% v/v H2 O + 0.5 wt% NH4 F Ti/PEG 1000
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 and a digital multimeter (type VA18B, from V&A International)
action of PEG-400 molecules on bacterial cells. The antibacterial were also connected to the electrochemical setup. PC-Link acquisi-
effect of PEG 400 is much lower compared to traditional disinfect- tion software was used for recording anodizing current vs. time. In
ing agents, but PEG 400 is nonirritating for the skin, its toxicity being all cases, synthesis of TiO2 nanostructures was achieved applying
low in case of acute oral administration or topical application [22]. a voltage raising from 0 to 50 V, with a 2 V/10 s step, and then kept
Composite coatings based on polypyrrole (PPy) with various per- constant at 50 V. The entire process of anodization of Ti substrate
centages of polyethylene glycol 400 (PEG 400) electrodeposited on lasted 2 h at room temperature.
TiAlZr alloy showed antibacterial properties as well [23]. The electrolytes composition is described in Table 1.
The importance of TiO2 nanostructures and evaluation of TiO2
antibacterial efficiencies were the motivation for this study. In pre- 2.2.2. Samples characterization
vious works we successfully obtained TiO2 nanotubes in organic The nanostructures morphology, including pore size of the
electrolytes (EG and PEG with average molecular weights 400 and anodized samples was observed through high-resolution field
600) with addition of NH4 F and small water content [24,25]. Thus, emission scanning electron microscope (SEM) QUANTA INSPECT
in this work, TiO2 nanostructured surfaces were obtained in very F with a 1.2 nm field emission gun. Thickness information was
viscous PEG 1000. But in this case, much more water content was obtained by SEM cross-sectional observation on samples that were
needed in order to solubilize it. It has been studied as well, how mechanically fractured, in order to crack and partially lift-off the
water content influences the morphologies and corrosion resis- TiO2 layers.
tance of those samples. On other hand, FT-IR analyses proved that Surface roughness was performed with an atomic force micro-
those samples prepared in viscous electrolytes have the surface scope in contact mode (AFM) from APE Research, Italy. The
covered with a shielding layer composed from TiO2 and some microscopy data were processed with Gwyddion 2.9 software.
remaining PEG electrolyte. As literature data report the antibac- Fourier transform infrared spectroscopy (FT-IR) spectra were
terial activity of PEG 400, we tested if the presence of PEG, with taken from the nanostructure layers on Ti substrate using a Spec-
different molecular weight in the shielding layer improves the trum 100 Series FT-IR Spectrometer from PerkinElmer, USA. All
antibacterial activity of those samples against E. coli bacteria. 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
2. Experimental
room temperature with CAM 100 Optical Contact Angle Meter
(KSV Instruments). Reported contact angles are the average of at
2.1. Materials
least three consecutive measurements. Purified water was used for
determinations.
The following organic reagents were used for anodization: ethy-
Electrochemical behavior characterization was conducted with
lene glycol (EG) from Reanal Finomvegyszergyár, polyethylene
an Autolab (PGSTAT 302N) potentiostatic assembly of three elec-
glycols with average molecular weight 400 (PEG 400), 600 (PEG
trodes in a single compartment. Samples have been used as working
600) and, respectively, 1000 (PEG 1000) from Fluka Analytical.
electrodes, platinum as an auxiliary electrode, and all potentials
Ammonium fluoride (NH4 F) from Sigma–Aldrich constituted the
reported were measured vs. an Ag/AgCl/Cl− (3 M) reference elec-
source for fluoride ions. All those reagents were analytical grade
trode. All experiments were done in Hank bio liquid, with the
and used as received. Purified water was obtained with a Millipore
following composition (g/L): 8 NaCl, 0.4 KCl, 0.35 NaHCO3 , and 0.25
Direct-Q UV3 water purification system.
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-
2.2. Methods tact with solution was 0.28 cm2 .

2.2.1. Sample elaboration via potential-step anodizing treatment 2.2.3. Antibacterial activity assays
Small pieces sliced from a 99.7% pure, commercially avail- The obtained titanium nanostructures antibacterial activity was
able titanium foil, having 2 mm thickness (Sigma–Aldrich), were evaluated against E. coli ATCC 8738 strain. The gram-negative bac-
anodized in order to obtain TiO2 nanostructures. The surface pre- terium E. coli was chosen for our study because this one has
treatment was first conducted by abrasion with silicon-carbide recently aroused more scientific interest, having new forms and
sandpaper with increasing grit size (from 800 to 4000 grit size), causing various diseases [27]. E. coli bacterium was inoculated into
and alumina paste successively. For degreasing the surface prior a Luria Bertani (LB) medium (which contained peptone 10.0 g/L,
to experiment, the polished samples were cleaned with purified NaCl 5.0 g/L and yeast extract 5.0 g/L) as the test strain and kept on
water, ethanol and acetone, 15 min each, by ultra-sonication. The an incubator shaker (Laboshake Gerhardt) overnight at 37 ◦ C and
cleaned samples were immediately used. 300 rpm [28].
Freshly conditioned samples were mounted like an anodic The samples were sterilized by autoclave at 121 ◦ C for 15 min.
electrode (contacted and then pressed against an O-ring) in a one- No visual or morphological changes were observed after this pro-
compartment two-electrode electrochemical cell. A Pt foil was used cess. The culture was obtained from a volume of 10 mL sterile
as counter electrode. A MATRIX MPS-7163 electrochemical source 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 The total charge density calculated by integrating the area under
bacterial broth suspensions were incubated at 37 ◦ C for 18 h in the the current density vs. time curve is presented in Fig. 2.
dark conditions. Samples were properly covered to ensure absence It is visible that the total charge density is higher for samples
of visible light. with 54% water content. So, increasing water content in the elec-
The bacterial growth was determined by measuring optical den- trolyte used for anodization increases the charge density. If we
sity for the samples and control (bacterium culture without sample) consider that the total charge density is used for oxide formation,
at 600 nm, using UV-VIS spectrophotometer (Jenway Spectropho- a thicker layer is formed for samples with 54% water. In cross-
tometer) [29]. The antibacterial activities of the samples were sectional SEM images presented for Ti/PEG 600 3%w sample and
determined by calculating the percentage inhibition of growth Ti/PEG 600 54%w sample it is visible a thicker layer is formed for
using the formula proposed by Jaiswal et al. [30]. 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 in 54% water electrolyte, despite the fact that we kept other elec-
average molecular weight of PEG leads to a charge increasing both trochemical parameters constant and those electrolytes have the
in case of samples with 54%w, and in the case of samples with 3%w. same amount of fluoride salt. The anodization in all those cases
The charge density value in the case of Ti/EG 3%w sample is between which have higher amounts of water leads to nanoporous layers
the one for Ti/PEG 400 3%w and Ti/PEG 600 3%w. So, increasing morphologies similar with a sponge, with irregularities. Despite
the average molecular weight and water content have an influence the fact that we used potentiostatic step anodization, these mor-
on formation and grow of nanotubes or nanoporous layers on Ti phologies are very similar with those obtained by Schmuki group,
substrate. during galvanostatic anodization of Ti in 1 M (NH4 )2 SO4 + 0.5 wt%
NH4 F electrolyte [31]. Using electrolytes with different viscosities
3.2. Surface analysis (EG, PEG 400, PEG 600, PEG 1000) and also changing the water con-
tent of the electrolyte solution caused a corresponding change in
SEM images, top view and cross-section for all anodized sam- the morphology of the surfaces. It is clear that the morphology of
ples, are presented in Table 1 and Fig. 3. In the case of samples the film is strongly dependent on the electrolyte.
Ti/EG 3%w, and Ti/PEG 600 3%w we obtain nanotubes, as we A method for surface characterization of the oxide films was
have previously reported for those electrolytes with 2% of water water drop contact angle, which gives a measure of the hydrophilic
[24,25]. Nanotubes have different diameters; in EG around 50 nm or hydrophobic nature of the surface. For Ti substrate and anodized
and between 117 nm and 208 nm in case of PEG 600 electrolyte. samples, the values of the contact angle are presented in Fig. 4.
In PEG 400, with 2% water, we have obtained nanotubes [24] com- The uncoated titanium substrate is less hydrophilic, having a water
pared to the present study, in which, for Ti/PEG 400 3%w sample, contact angle close to 90◦ , while the nanotubes and nanoporous
we have obtained nanotubular layer, but with nanotubes not very structures showed a hydrophilic behavior. For samples prepared
well defined and arranged. So, it seems that in this electrolyte the in EG, the contact angle value for the one with 3%w (unshielded
water content is very important for obtaining nanotube structures, nanotubes) is lower, compared to the one for the sample with 54%w
and small differences between the amounts of water can radi- (nanoporous oxide layer). This difference between contact angles
cally change the morphology of the sample. Anodization does not can be associated with different surface morphology, very well
result in formation of a nanotubular layer for samples prepared organized in the first case and pretty rough in the second case (as
C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165 161

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-
Fig. 4. Contact angle for all prepared samples and Ti substrate. 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
can be seen in SEM presented in Fig. 3) not allowing water droplet solid. In the Wenzel state, the substrate becomes more hydrophilic
to spread too much. For samples prepared in PEG 400 or PEG 600, with a higher surface roughness [32]. These two hydrophilic sam-
wetting behavior is different for different electrolytes and water ples (Ti/EG 54%w and Ti/PEG 600 54%), where the contact angle
content in the electrolyte composition, the morphologies being also decrease with the increasing of the roughness, appear to be in the
different. In these cases, contact angle value for Ti/PEG 400 3%w Wenzel state.
sample (shielded nanotubes) > Ti/PEG 400 54%w (nanoporous oxide In the case of anodised sample Ti/PEG400 54%w and Ti/PEG1000
layer) and Ti/PEG 600 3%w sample (shielded nanotubes) > Ti/PEG (Fig. 5B and D), characteristic peaks of pure PEG 400 and, respec-
600 54%w (nanoporous oxide layer). Samples prepared in PEG 600 tively, PEG 1000 are present (more or less intense or shifted),
with higher water content and in PEG 1000 are very hydrophilic, despite the fact that these samples were also prepared from solu-
having the lowest contact angles values. tions with increased water content. In the case of Ti/PEG 400 3%w
In the previous works, we showed that some remaining viscous and Ti/PEG 600 3%w samples there are also traces of the used
electrolyte is still present on the surface of the anodized samples, anodizing electrolytes, so, in the shielding covering the nanotubes,
despite the fact that those were carefully rinsed with purified water visible in the SEM images, the electrolytes used are also incorpo-
before recording FT-IR spectra [24,25]. The surface of anodized Ti rated.
was analyzed using the FT-IR transmission spectra, in order to probe
if this observation is also valid for the samples anodised in those 3.3. Electrochemical analysis
electrolytes with more water content. These spectra were recorded
for the pure ethylene glycol and polyethylene glycols, and also for Fig. 6 compares polarization curves for freshly cleaned titanium
the TiO2 nanoporous layers at room temperature, in the region surface with those for anodized samples in Hank’s naturally aer-
4000–600 cm−1 . In Fig. 5 characteristic IR vibrations can be found ated solution at room temperature. Curves have been registered
in the transmission spectra. The peak for stretching vibration of between ±300 mV vs. OCP, with a 2 mV/s scan rate, at initial immer-
O–H group is visible: at around 3290 cm−1 in pure EG spectrum, sion time. The quantification of electrochemical stability in terms
3442 cm−1 in pure PEG 400, 3450 cm−1 in pure PEG 600 spectrum of corrosion parameters such as jcorr – corrosion current, Ecorr – cor-
and, respectively, 3390 cm−1 in the spectrum of pure PEG 1000. rosion potential, vcorr – corrosion rate and polarization resistance
The sharp band corresponding to stretching vibration of CH2 are summarized in Table 2. Most of samples showed an increase
is visible: at around 2870 cm−1 (for pure EG), at 2860 cm−1 (for corrosion resistance compared to Ti substrate.
pure PEG 400 and PEG 600) and at 2880 cm−1 (for pure PEG 1000). For the Ti/PEG 600 54%w sample, nanostructured layer does
The band corresponding to binding vibration of CH2 is at around not protect Ti substrate against corrosion, probably because of
1450 cm−1 (for pure EG, pure PEG 400 and PEG 600), and 1460 cm−1 the increased porosity of the layer (as it can be seen from the
(for pure PEG 1000) The corresponding band from the stretching SEM image). If we compare samples prepared in the same elec-
vibration of C O C bond can be found at around 1340 cm−1 (for trolyte, but with different amount of water, in the case of EG and
pure PEG 400, PEG 600 and for pure PEG 1000). A very sharp and PEG 400, the values are similar, although there are very different
intense peak appears at around 1080–1100 cm−1 in the pure EG and morphologies, 4.41 × 10−4 mm/y and 4.63 × 10−4 mm/y for Ti/EG
pure polyethylene glycols spectrum corresponding to C C stretch- 3%w and Ti/EG 54%w and 15.6 × 10−4 mm/y and 14.98 × 10−4 mm/y
ing. In the case of Ti/EG 3%w sample, there are some remaining for Ti/PEG 400 3%w and Ti/PEG 400 54%w, respectively. Only in the

Table 2
Corrosion characteristics from Tafel polarization curves obtained in Hank bioliquid.

Sample jcorr (A/cm2 ) Ecorr vcorr (mm/y) Polarization resistance ()


V vs. Ag/AgCl/3 M KCl

Ti substrate 19.2 × 10−8 −0.41 16.7 × 10−4 2.83 × 106


Ti/EG 3%w 5.1 × 10−8 −0.07 4.41 × 10−4 2.84 × 106
Ti/EG 54%w 5.3 × 10−8 0.09 4.63 × 10−4 6.82 × 106
Ti/PEG 400 3%w 17.9 × 10−8 −0.12 15.6 × 10−4 0.77 × 106
Ti/PEG 400 54%w 17.2 × 10−8 0.08 14.98 × 10−4 1.97 × 106
Ti/PEG 600 3%w 3.21 × 10−8 0.07 2.8 × 10−4 3.50 × 106
Ti/PEG 600 54%w 46.13 × 10−8 0.003 40.15 × 10−4 0.82 × 106
Ti/PEG1000 0.32 × 10−8 −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) the presence of PEG on the surface of Ti/PEG 600 3%w and Ti/PEG
between the samples with different amount of water. Samples 1000 can leads to reduced corrosion rate due to the influence of
Ti/PEG 600 3%w and Ti/PEG 1000 have showed the lowest corro- cathodic reaction.
sion rates. Both samples were prepared in the viscous electrolyte Generally, corrosion resistance for prepared samples is higher
solutions and is expected that the PEG content to be highest on the compared to Ti substrate for all samples because of the nanotubes
surface of these samples. In a previous study it was concluded that or nanoporous layers, except Ti/PEG 600 54%w.
molecules sequestered within the PEG-doped composites are less The electrochemical impedance spectroscopy (EIS) data were
responsive to O2 indicating that they are rendered inaccessible to acquired in the frequency range of 10−1 –105 Hz at open circuit
O2 as a result of the PEG doping [33]. This can be an explanation that 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 equivalent electrical circuits are presented in Table 3. Resistance R1
3 M KCl reference is presented in Fig. 7. EIS data were fitted with value (assigned to barrier oxide layer) is lower for TI/EG 3%w sam-
NOVA 1.7 software, establishing the most probable electrical equiv- ple compared to the one for Ti/EG 54%w sample. This means that
alent circuit for tested samples. Those are drawn in Fig. 8. For freshly a large part of the total charge density, used in anodization, is for
cleaned titanium surface, a system with one time constant elec- oxide layer consolidation. But in the case of samples prepared in
trical equivalent circuit is satisfactory (Fig. 7A). In this circuit, Rs PEG 400, PEG 600 and PEG 1000, R1 values are lower for cases with
is the solution resistance, and time constant represents the oxide higher water content, compared to the ones with lower water con-
barrier layer (layer pseudo-capacitance CPE1 and layer resistance tent. For samples Ti/PEG 400 54%w, Ti/PEG 600 54%w, and Ti/PEG
R1 ). For prepared samples, a more complex circuit was used, taking 1000, the charge density used in anodization is higher, but R1 val-
into account the nanotube and the shielding layer. Parameters of 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- anodized in PEG 400 and 600, values of antibacterial activity are the
ues for R3 resistance, assigned to the shielding layer are hundreds highest.
of ohms showing that this layer is not responsible for corrosion As it has been mentioned in the introduction part, PEG 400 was
resistance. found to have bactericidal effect. As expected, samples prepared
n1 is in the range between 0.8 and 0.99 for most of samples, in polyethylene glycols have higher values probably because they
this indicating a pseudo-capacitive behavior. N1 for Ti/PEG 1000 have some remaining electrolyte incorporated (according to FT-IR
sample is 0.72, suggesting that in this case the barrier oxide layer is analysis). Samples Ti/PEG 400 3%w and Ti/PEG 600 3%w with nano-
not compact. However, this sample has the lowest corrosion rate, tubes layers have higher antibacterial activity compared to samples
meaning that the corrosion resistance in this case is due to the thick Ti/PEG 400 54%w, Ti/PEG 600 54%w, Ti/PEG 1000 with nanoporous
nanoporous oxide layer. Ti/PEG 1000 sample has also the highest layers. This behavior might be attributed to two effects: lowering of
charge density used during anodizing process. water activity, and the specific action of PEG 400 on bacterial cells.
Chirife et al. [22] showed that PEG 400 caused immediate clump-
3.4. Percentage inhibition of growth ing of the bacterial cells. Meanwhile, Gothwal and Srivastava [34]
reported the antibacterial activity of certain compounds with PEG
The bactericidal effects of Ti substrate and anodized sam- 600. The killing effect of Ti/PEG 400 3%w and Ti/PEG 600 3%w is
ples were investigated against E. coli strain. The bacteriological much lower than that of traditionally disinfecting agents. But its
experiments performed in vitro demonstrated the effectiveness of merits should be considered, as PEG 400 and PEG 600 meets many
anodized samples in inhibiting the growth of this bacterium. of the physicochemical requirements for an ideal disinfectant for
Fig. 9 shows percentage inhibition of bacteria growth in the topical use. At present, PEG 400 and PEG 600 have several pharma-
experiments conducted according to the above description for the ceutical applications, due to their antibacterial properties [35]. At
anodized samples and Ti coated with EG, PEG 400, PEG 600 and the same time, TiO2 nanotubes possess antimicrobial activity. This
PEG 1000, respectively. Samples prepared in EG solution (which mechanism involves an instantaneous and reversible physical first
has the highest value for contact angle of anodized samples) phase, and a cellular second phase [36]. After implantation, a com-
have antibacterial activity similar with Ti substrate. For samples 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
Fig. 9. Percentage inhibition of bacteria growth for titanium surface, anodized sam-
400 and PEG 600. This behavior suggests a synergic effect due to
ples in electrolytes with composition described in Table 1 and also for titanium both nanotube structures and the presence of PEG in the outer
substrate coated with EG, PEG 400, 600 and 1000, respectively. layer.
C. Dumitriu et al. / Applied Surface Science 341 (2015) 157–165 165

The results obtained in this paper suggest that Ti/PEG 600 3%w [13] D. Kowalski, D. Kim, P. Schmuki, TiO2 nanotubes, nanochannels and
and Ti/PEG 400 3%w may have a potential value as an antibacterial mesosponge: self-organized formation and applications, Nano Today 8 (2013)
235–264.
agent. [14] H.Y. Hwang, A.A. Prabu, D.Y. Kim, K.J. Kim, Influence of the organic electrolyte
and anodization conditions on the preparation of well-aligned TiO2 nanotube
4. Conclusions arrays in dye-sensitized solar cells, Sol. Energy 85 (2011) 1551–1559.
[15] A. Simchi, E. Tamjid, F. Pishbin, A.R. Boccaccini, Recent progress in inorganic and
composite coatings with bactericidal capability for orthopaedic applications,
Titanium plates anodised in ethylene glycol and polyethylene Nanomedicine 7 (2011) 22–39.
glycols with different average molecular weight, having higher [16] H. Cao, X. Liu, Activating titanium oxide coatings for orthopedic implants, Surf.
Coat. Technol. 233 (2013) 57–64.
amount of water (Ti/EG 54%w, Ti/PEG 400 54%w, Ti/PEG 600 [17] A. Roguska, M. Pisarek, M. Andrzejczuk, M. Lewandowska, Synthesis and
54%w and Ti/PEG 1000) showed similar nanoporous morphologies. characterization of ZnO and Ag nanoparticle-loaded TiO2 nanotube compos-
Despite this fact, these samples have different wetting behavior, ite layers intended for antibacterial coatings, Thin Solid Films 553 (2014)
173–178.
most hydrophilic being Ti/PEG 600 54%w and Ti/PEG 1000 sam-
[18] Y. Xing, X. Li, L. Zhang, Q. Xu, Z. Che, W. Li, Y. Bai, K. Li, Effect of TiO2 nanoparticles
ples. They have also different electrochemical behaviors, some with on the antibacterial and physical properties of polyethylene-based film, Prog.
improved corrosion protection. Org. Coat. 73 (2012) 219–224.
[19] B. Wu, R. Huang, M. Sahu, X. Feng, P. Biswas, Y.J. Tang, Bacterial
Samples prepared in polyethylene glycols showed increased
responses to Cu-doped TiO2 nanoparticles, Sci. Total Environ. 408 (2010)
values for antibacterial activity, higher for those with shielded 1755–1758.
nanotubes layers. It seems that the nanotubes with shielding [20] M.S. Hassan, T. Amna, H.Y. Kim, M.S. Khil, Enhanced bactericidal effect of novel
layer (which have incorporated polyethylene glycol) give stronger CuO/TiO2 composite nanorods and a mechanism thereof, Compos. B Eng. 45
(2013) 904–910.
antibacterial effect, compared to the uncovered long nanotubes [21] S. Grigorescu, C. Ungureanu, R. Kirchgeorg, P. Schmuki, I. Demetrescu, Various
prepared in ethylene glycol. sized nanotubes on TiZr for antibacterial surfaces, Appl. Surf. Sci. 270 (2013)
Titanium surface modification by anodization in viscous elec- 190–196.
[22] J. Chirife, L. Herszage, A. Joseph, J.P. Bozzini, N. Leardini, E.S. Kohn, In vitro
trolytes with controlled water content leads to a change in antibacterial activity of concentrated polyethylene glycol 400 solutions,
morphology and surface chemistry materialized with increased Antimicrob. Agents Chemother. 24 (1983) 409–412.
corrosion stability and also improved bactericidal properties. [23] C. Ungureanu, C. Pirvu, M. Mindroiu, I. Demetrescu, Antibacterial polymeric
coating based on polypyrrole and polyethylene glycol on a new alloy TiAlZr,
Prog. Org. Coat. 75 (2012) 349–355.
Acknowledgement [24] C. Dumitriu, C. Pirvu, I. Demetrescu, The electrochemical formation and
shielding mechanism of TiO2 nanotubes in organic electrolytes with different
viscosity, J. Electrochem. Soc. 160 (2013) G55–G60.
This work was supported by a grant from the Romanian National [25] C. Dumitriu, C. Pirvu, Electrochemical studies on TiO2 nanotubes modified Ti
Authority for Scientific Research, CNCS – UEFISCDI, project PN2- electrodes, U.P.B. Sci. Bull. B 74 (2012) 93–106.
253/2014 – NANOCOAT. [26] M. Mindroiu, C. Pirvu, R. Ion, I. Demetrescu, Comparing performance of nanoar-
chitectures fabricated by Ti6Al7Nb anodizing in two kinds of electrolytes,
Electrochim. Acta 56 (2010) 193–202.
References [27] S. Viazis, F. Diez-Gonzalez, Chapter one – enterohemorrhagic Escherichia coli:
the twentieth century’s emerging foodborne pathogen: a review, Adv. Agron.
[1] A.W. Tan, B. Pingguan-Murphy, R. Ahmad, S.A. Akbar, Review of titania nano- 111 (2011) 1–50.
tubes: fabrication and cellular response, Ceram. Int. 38 (2012) 4421–4435. [28] M.A. Ansari, H.M. Khan, A.A. Khan, A. Malik, A. Sultan, M. Shahid, F. Shu-
[2] K.S. Brammer, C.J. Frandsen, S. Jin, TiO2 nanotubes for bone regeneration, Trends jatullah, A. Azam, Evaluation of antibacterial activity of silver nanoparticles
Biotechnol. 30 (2012) 315–322. against MSSA and MRSA on isolates from skin infections, Biol. Med. 3 (2011)
[3] P. Roy, S. Berger, P. Schmuki, TiO2 nanotubes: synthesis and applications, 141–146.
Angew. Chem. Int. Ed. 50 (2011) 2904–2939. [29] D. Ionita, M. Grecu, C. Ungureanu, I. Demetrescu, Antimicrobial activity of the
[4] J.M. Macak, H. Tsuchiya, A. Ghicov, K. Yasuda, R. Hahn, S. Bauer, P. Schmuki, TiO2 surface coatings on TiAlZr implant biomaterial, J. Biosci. Bioeng. 112 (2011)
nanotubes: self-organized electrochemical formation, properties and applica- 630–634.
tions, Curr. Opin. Solid State Mater. Sci. 11 (2007) 3–18. [30] S. Jaiswal, B. Duffy, A.K. Jaiswal, N. Stobie, P. McHale, Enhancement of the
[5] S. Minagar, C.C. Berndt, J. Wang, E. Ivanova, C. Wen, A review of the application antibacterial properties of silver nanoparticles using ␤-cyclodextrin as a cap-
of anodization for the fabrication of nanotubes on metal implant surfaces, Acta ping agent, Int. J. Antimicrob. Agents 36 (2010) 280–283.
Biomater. 8 (2012) 2875–2888. [31] L.V. Taveira, J.M. Macak, K. Sirotna, L.F.P. Dick, P. Schmuki, Voltage oscillations
[6] K.M. Deen, A. Farooq, M.A. Raza, W. Haider, Effect of electrolyte composition and morphology during the galvanostatic formation of self-organized TiO2
on TiO2 nanotubular structureformation and its electrochemical evaluation, nanotubes, J. Electrochem. Soc. 153 (2006) B137–B143.
Electrochim. Acta 117 (2014) 329–335. [32] V.C. Anitha, J.H. Lee, J. Lee, A.N. Banerjee, S.W. Joo, B.K. Min, Biofilm forma-
[7] P. Schmuki, H. Tsuchiya, J.M. Macak, L. Taveira, A. Ghicov, E. Balaur, S. Bauer, tion on a TiO2 nanotube with controlled pore diameter and surface wettability,
Growth and properties of self-organized TiO2 nanotube layers, ECS Trans. 1 Nanotechnology 26 (2015).
(2006) 335–341. [33] C. Pirvu, C.C. Manole, A.B. Stoian, I. Demetrescu, Understanding of electrochem-
[8] I. Demetrescu, D. Ionita, C. Pirvu, D. Portan, Present and future trends in TiO2 ical and structural changes of polypyrrole/polyethylene glycol composite films
nanotubes elaboration, characterization and potential applications, Mol. Cryst. in aqueous solution, Electrochim. Acta 56 (2011) 9893–9903.
Liq. Cryst. 521 (2010) 195–203. [34] P. Gothwal, Y.K. Srivastava, Microwave induced synthesis of some substituted
[9] Y. Li, Q. Ma, J. Han, L. Ji, J. Wang, J. Chen, Y. Wang, Controllable preparation, 2-amino thiadiazines as a prospective antimicrobial agents, Der Chem. Sin. 3
growth mechanism and the properties research of TiO2 nanotube arrays, Appl. (2012) 318–322.
Surf. Sci. 297 (2014) 103–108. [35] T. Zhou, X. Xiao, G. Li, Z.W. Cai, Study of polyethylene glycol as a green solvent in
[10] C.C. Manole, C. Pirvu, I. Demetrescu, Evaluation of TiO2 nanotubes changes after the microwave-assisted extraction of flavone and coumarin compounds from
ultrasonication treatment, Mol. Cryst. Liq. Cryst. 521 (2010) 84–92. medicinal plants, J. Chromatogr. A 1218 (2011) 3608–3615.
[11] C.C. Manole, C. Pirvu, Surface and electrochemical analysis for the understand- [36] C.M.N. Chan, A.M.C. Ng, M.K. Fung, H.S. Cheng, M.Y. Guo, A.B. Djurišić, F.C.C.
ing of TiO2 nanopores/nanotubes changes in post-elaboration treatment, Surf. Leung, W.K. Chanc, Antibacterial and photocatalytic activities of TiO2 nano-
Interface Anal. 43 (2011) 1022–1029. tubes, J. Exp. Nanosci. 8 (2013) 859–867.
[12] T.-H. Kim, J.-W. Lee, B.-S. Kim, H. Cha, Y.-C. Nah, Morphological investigation of [37] M. Katsikogianni, Y.F. Missirlis, Concise review of mechanisms of bacterial
anodized TiO2 nanotubes fabricated using different voltage conditions, Micro- adhesion to biomaterials and of techniques used in estimating bacteria-
porous Mesoporous Mater. 196 (2014) 41–45. material interactions, Eur. Cell. Mater. 8 (2004) 37–57.