Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 871–877

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Novel rapid synthesis of zinc oxide nanotubes via hydrothermal

technique and antibacterial properties
Nadia Abdel Aal a, Faten Al-Hazmi b, Ahmed A. Al-Ghamdi b, Attieh A Al-Ghamdi c, Farid El-Tantawy d,
F. Yakuphanoglu b,e,⇑
a
Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia, Egypt
b
Department of Physics, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
c
Centre of Nanotechnology, King AbdulAziz University, Jeddah, Saudi Arabia
d
Department of Physics, Faculty of Science, Suez Canal University, Ismailia, Egypt
e
Department of Physics, Faculty of Science, Firat University, Elazig 23169, Turkey

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 ZnO nanotubes were been

successfully synthesized via simple
hydrothermal solution route.
 ZnO nanotubes exhibited a wall
thickness about 2 nm, with an
average diameter of 17 nm.
 ZnO nanotubes have excellent
potencies for many biomedical
applications.

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

Article history: ZnO nanotubes with the wurtzite structure have been successfully synthesized via simple hydrothermal
Received 4 June 2014 solution route using zinc nitrate, urea and KOH for the first time. The structural, compositions and mor-
Received in revised form 10 July 2014 phology architectures of the as synthesized ZnO nanotubes was performed using X-ray diffraction (XRD),
Accepted 29 July 2014
Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM),
Available online 8 August 2014
energy dispersive X-ray spectroscopy (EDS) and high resolution transmission scanning electron micros-
copy (HRTEM). TEM showed that ZnO nanotubes exhibited a wall thickness of less than 2 nm, with an
Keywords:
average diameter of 17 nm and the length is 2 lm. In addition, the antibacterial activity of ZnO nanotubes
ZnO
Nanotubes
was carried out in vitro against two kinds of bacteria: gram - negative bacteria (G ve) i.e. Escherichia coli
Hydrothermal (E. coli) and gram – positive bacteria (G +ve) i.e. Staphylococcus aureus. Therefore, this work demonstrates
Antibacterial properties that simply synthesized ZnO nanotubes have excellent potencies, being ideal antibacterial agents for
many biomedical applications.
Ó 2014 Elsevier B.V. All rights reserved.

Introduction

This study is part of an on-going research project aiming to

develop high performance nanoscale inorganic metal oxides for
⇑ Corresponding author. Tel.: +90 424 2370000 3792; fax: +90 424 2330062. antibacterial with high aspect ratio and good antibacterial efficacy.
E-mail address: fyhanoglu@firat.edu.tr (F. Yakuphanoglu). In fact, the re-emergence of infectious diseases and the continuous

http://dx.doi.org/10.1016/j.saa.2014.07.099
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
872 N.A. Aal et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 871–877
development of antibiotic resistance among a variety of disease,
microbial pollution and its contamination induced by microorgan-
isms causing bacterial pose a serious and produced various
problems in living conditions, global health, and industrial fields
[1–5]. Furthermore, infection of artificial organs can lead to life-
threatening complications resulting in significant morbidity and
mortality [6–10]. To solve these problems, many new antibacterial
agents and techniques have been studied and applied for example,
organic antibacterial agents, inorganic antibacterial agents, natural
antibacterial agents, and physical sterilizing methods [11–15]. In
general, inorganic antibacterial agent features long lasting, stabil-
ity, safety, and broad-spectrum antibacterial activity, overcoming
the drawbacks of organic ones and having excellent antibacterial
activity against both bacteria and funguses [13,16]. Thereof there
is an urgent need to produce the new antibacterial agents from dif-
ferent sources alternative to organic materials. Currently, the use
of inorganic antibacterial bio-ceramic materials is highlighted for
the control of bacteria [17,18]. More recently, metal oxides nano-
crystals with controlled dimensionality (e.g., 0D dots, 1D rods
and wires, 2D sheets and disks and 3D flowers are versatile build-
ing blocks for constructing diverse superstructures, functional
mesocrystals, and new nano devices, which are scientifically
important and technologically useful in multidisciplinary fields of
chemistry, physics, materials science, nanoscience, nanotechnol-
ogy, biology, and medicine [8,19]. In particular, zinc oxide (ZnO)
is an important class of inorganic material, which processes unique
properties such as transparent semiconducting oxide, catalysts,
sensors, and UV – shielding materials, biomaterials and more
[20–25]. The advantages of inorganic biomaterials like ZnO are
nontoxicity, heat resistance and suitable for biological applications,
safety, durability, antiseptic effect and stability compared with
organic antibacterial biomaterials [1,2].
The present study is focused mainly with the rapid and facile
synthesized ZnO nanotubes by hydrothermal method for the first
time in the open literatures. Finally, the antibacterial activity to
cope with the increasing needs for protection against different
kinds of bacterial was subsequently examined.
Experimental details
Synthesis of ZnO nanotubes
The raw materials are analytic grade reagents and purchased
without further treatment. First, for the preparation of ZnO nano-
tubes, an aqueous solution of 50 mL, which contained 0.35 mol of
zinc nitrate hexahydrate Zn(NO3)26H2O and 0.7 mol urea was pre-
pared at room temperature. The mixture was magnetically stirred
vigorously for 10 min and then transferred into Teflon-lined steel
autoclave of 50 mL capacity. The steel autoclave was sealed and
kept at 220 °C for 5 h. After the cooling of the autoclave to room
temperature naturally white precipitates solid product was filtered
and rinsed with water up to pH 7. Obtained powders were dried in
oven at 90 °C overnight. After that, 1 g of as  prepared ZnO pow-
der was added to 33 ml of 12 mol KOH and additionally backed in
hydrothermal cell at 220 °C for 10 h.
Characterization of as-synthesized ZnO nanotubes
The structure of the as-synthesized ZnO nanotubes was ana-
lyzed by X-ray powder diffraction (X-ray) using a Shimatzou X-
ray diffractometer (Shimatzou, XRD-6000), with Cu ka radiation
and wavelength is 0.15147 nm, working at 30 mA and 20 kV. The
phases were identified using the JCPDS database. Fourier transform
infrared spectra (FTIR) were obtained on KBr pellets at room
temperature using a Bruker FTIR spectrometer (TENSOR 37). The
morphology and the elemental purity of the as synthesized ZnO
nanotubes were characterized by field emission scanning electron
microscope (FSEM) equipped with an energy dispersive X-ray
spectrometer (EDS). The particle size and orientation of synthe-
sized ZnO was analyzed by the high-resolution transmission elec-
tron microscopy (HRTEM) and selected area electron diffraction
(SAED) (JEOL EM-2100F) operated at 200 kV. For TEM, the samples
were separated by ultrasonically dispersion in 1 ml of ethanol, and
then a drop of the solution was placed on a Cu grid covered with
carbon film.
Antibacterial activities in solid agar medium
This test was carried out according to the method described in
references, which was performed in sterile Petri-dishes with
90 mm diameter containing sterile Nutrient agar medium
(15 ml). After the renewal of cultures bacterial for 24 h at 25 °C,
the freshly prepared bacterial inoculums were swabbed over the
entire surface of the medium three times, rotating the plate 60
after each application by using sterile cotton swab, to ensure the
spread of bacteria on the surface of the plates. One well of 6 mm
diameter was pored in the medium for each plates with the help
of sterile cork-borer and was filled with 50 ll of the bacterial sus-
pension of the tested material using micropipette [1,2]. Ampicilin
(5 lg/ml) was used as positive control and water was used as neg-
ative control. Plates were left for 45 min at room temperature to
allow proper diffusion of the extract to occur in the medium. All
plates were incubated at 37 °C for 24 h, followed by the measure-
ment of the diameters of inhibition zones. Inhibition of bacterial
growth was measured as zone diameters (mm) at 3-equidistant
points taken from the center of the inhibition zone, and the aver-
age value was taken. All experiments were carried out in triplicate
and the reported data represents average values.
Antibacterial activities in liquid medium
The various concentrations of the tested ZnO nanotubes were
prepared and added to 48 ml of nutrient broth medium in
250 ml Erlenmeyer flasks to give 100–900 lg/ml. Each flask was
inoculated with 2 ml of the tested bacterium, containing 5x105/
ml, and the flasks were incubated at 37 °C. The bacterial growth
was determined by measuring the absorbance at 595 nm. Control
flasks without ZnO nanomaterial were prepared as control.
Mode of action of the tested nanomaterials
After incubation of the tested bacterial in nutrient broth med-
ium, containing different concentration of the ZnO ranging from
100–900 lg/ml, bacterial cells were collected by centrifugation at
3000 rpm and were washed several times with sterile distilled
water. The collected cells were re-suspended in sterile distilled
water (OD. at 550 = 0.65). Cell respiration (quantity of O2 con-
sumed/min) was determined using Oxigraphe. The quantity of K+
flowed from the treated and untreated cells were determined using
atomic adsorption [1,2].
Results and discussion
Structural, morphological and elemental compositional properties of
synthesized ZnO nanotubes
X-ray analysis was performed in order to identify the crystal
structure and phases of as-synthesized ZnO nanotubes. X-ray pat-
terns of the as-synthesized ZnO nanotubes prepared by hydrother-
mal method which were vertically normalized for clarity is
 N.A. Aal et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 871–877
Fig. 1. Typical X-ray pattern of synthesized ZnO nanotubes and the inset is the
calculated TC vs. ZnO plane (hkl).
depicted in Fig. 1. Several well-defined diffraction reflections were
appeared in the pattern at 2h (degrees) of 31.72°, 34.41°, 36.21°,
47.49°, 56.52°, 62.81°, 67.86° and 68.99°, which correspond to
the lattice planes of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3),
(1 1 2) and (2 0 1), respectively for the wurtzite hexagonal phase
pure ZnO (space group: P63mc) [5–8]. All the diffraction peaks in
the pattern is well matched with the available Joint Committee
on Powder Diffraction Standards for bulk ZnO (JCPDS 36-1451)
and are indexed as the wurtzite structures phase of ZnO with lat-
tice constants of a = 0.325 nm and c = 0.524 nm [9–11]. No diffrac-
tion peaks from impurities and metallic Zn are detected in the
pattern. Additionally, higher intensity and narrower spectral width
of ZnO peaks in the spectrum affirmed that the nanoparticles are
well crystalline. The strongest diffraction peak corresponding to
the (0 0 2) crystal plane of ZnO revealed the preferred orientation
along the c-axis.
The crystallite sizes (D) were estimated based on the width of
the peak due to (1 0 1) planes by using the Scherrer’s equation [3]:
kk
D¼
b cos h
where k is a constant (k = 0.9), k is the wavelength of X-ray used, b is
the full-width at the half maximum (FWHM) of the diffraction peak
and h is the Bragg angle. Interestingly, to check the preferential
crystallite orientation of as synthesized ZnO, we calculated the tex-
ture coefficient (TC) (hkl) using the following equation [10]:
IðhklÞ=I0 ðhklÞ
TCðhklÞ ¼ P  100ð%Þ
IðhklÞ=I0 ðhklÞ
where I(hkl) is the measured relative intensity of a plane (hkl), and
I0(hkl) is the standard intensity of the plane taken from the JCPDS
Card No. 36-1451.
The estimated average crystallite size is found to be 17 nm.
The estimated values of TC versus (hkl) plane of as synthesized
ZnO are inset in Fig. 1. It is worth noting that the texture coefficient
(TC) of (0 0 2) plane direction is higher. This indicates that the
growth rate of c-axis direction increases, which was favorable for
the formation of the ZnO samples with tube-like shape and the
preferred growth orientation, is (0 0 2) plane [26,27].
873
Fig. 2. FTIR spectra of as synthesized ZnO nanotube.
The chemical composition and quality of as-synthesized ZnO
nanotubes was performed by Fourier transform infrared (FTIR)
spectroscopy in the range of 400–4000 cm1. The typical FTIR spec-
trum of as-synthesized ZnO nanotubes is depicted in Fig. 2. One
can see that the appearance of a sharp and strong two bands at
690 and 830 cm1 is attributed to the formation of the stretching
vibration of metal–oxygen (Zn–O) bonds which confirms that the
synthesized products are pure ZnO [22,23]. In addition to this,
one band located at 3370 cm1 is mainly due to characteristic
absorption of hydroxyls group (O–H) stretching and bending mode
of vibrations [3,4].
The energy dispersive X-ray spectroscopy (EDS) spectra of as –
synthesized ZnO nanotubes are displayed in Fig. 3. One can see that
the EDS spectrum reveals that the products compose of zinc and
oxygen, only. The molar ratio of as-synthesized ZnO nanotubes is
51.82 at.% of zinc and 48.18 at.% of oxygen, confirming the real
stoichiometry of ZnO [1.2]. No other elements were found confirm-
ing the high purity of the as synthesized ZnO nanotubes. The ele-
ment of carbon was from the coating layer used for SEM imaging
of the products.
The morphologies of the synthesized ZnO nanotubes were
examined by FESEM and demonstrated in Fig. 4b–d. The low and
high magnification FESEM images in Fig. 3a–c, respectively clearly
exhibits that the obtained products are synthesized in large quan-
tity and possessing nanotube-like morphologies. Each tube is sym-
metrically formed with hexagonal cross sections for both the inner
ð1Þ
ð2Þ
Fig. 3. SEM–EDS profile of the ZnO nanotubes.
874 N.A. Aal et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 871–877
Fig. 4. Typical (a) low-magnification and (b and c) high-resolution FESEM images of
the synthesized ZnO nanotubes.
and outer walls. The typical diameter of the nanotubes is on aver-
age around 17 nm and the length is 2 lm.
The clear morphologies of the synthesized ZnO nanotubes were
performed by transmission electron microscopy (TEM) equipped
with high-resolution TEM (HRTEM). As shown in Fig. 5a TEM
micrograph indicates that the ZnO possesses uniform nanotubes
and are grown in large scale. In addition, TEM showed that ZnO
Fig. 5. Low (a) and (b) high-resolution TEM images of as-synthesized ZnO
nanotubes hexagonal nanostructures.
nanotubes exhibited a wall thickness of less than 2 nm, with an
average diameter of 17 nm and the length is 2 lm.
The average particle diameter obtained from the Scherrer for-
mula is 28 nm, in good agreement with the value obtained from
analysis of transmission electron microscope images. Furthermore,
the nanotube is composed of well defined crystals with a nano-
tube-like wurtzite hexagonal ZnO structure [28–30].
To further check the structural properties of the as-synthesized
ZnO nanotubes were done by high-resolution TEM (HRTEM) and
are demonstrated in Fig. 5b. It is clear that, very well defined lattice
images are appeared from a single tube-shape of ZnO nanostruc-
tures. The distance between lattice fringes along the longitudinal
axis direction, corresponding to the d-spacing of ZnO indexed to
(0 0 2) crystal planes, is measured 51 nm supporting that the grown
ZnO nanotubes are highly crystalline and possessing hexagonal
ZnO structure [13,31].
Antibacterial activity
The bacterial growth in liquid medium of E. coli and Bacillus sp.
against concentration of ZnO nanotubes is depicted in Fig. 6. It is
clear that the bacterial growth decreases with increasing ZnO
nanotubes concentration into bacterial species of E. coli and Bacil-
lus. Interestingly enough, the antibacterial activity of E. coli is
higher than that of Bacillus sp., with increasing ZnO concentration.
 N.A. Aal et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 871–877 875

1.2 Table 1
Antibacterial activity (Diameter of inhibition zone, mm) of the ZnO nanotubes
E. coli compared to Ampicillin as a positive control.

Tested bacteria Gram ZnO Positive control

1 Bacillus sp
reaction (Ampicillin)
Acinetobacter sp. ve 11.3 ± 0. 62 15 ± 1.0
Bacillus sp. +ve 13.2 ± 0.54 30 ± 1.5
0.8 Escherichia coli ve 14.8 ± 0.38 25 ± 4.5
Bacterial Growth

Klebsiella pneumonia ve 9.8 ± 0.30 24 ± 0.5

Micrococcus spp. +ve 11.0 ± 0.82 30 ± 2.5
Proteus mirabilis ve 12.2 ± 1.61 16 ± 1.5
0.6
Pseudomonas ve 10.8 ± 0.56 19 ± 2.50
aeruginosa
Salmonella sp. ve 11.8 ± 4.59 20 ± 0.56
Staphylococcus aureus +ve 10.0 ± 2.56 32 ± 0.36
0.4
Staphylococcus +ve 12.7 ± 1.57 34 ± 0.34
epidermidis
Streptococcus +ve ND 30 ± 0.54
0.2 pneumonia
Antibacterial index 11.69 28.7

ND: Not detected inhibition zone, +ve: Gram positive, ve: Gram negative.
0
100 200 300 400 500 600 700 800 900 1000
singlet oxygen, in turns to damage the bacterial cell wall. Second,
Concentration (µg/ml) the ZnO nanotubes release ions which react with the thiol groups
Fig. 6. Bacterial growth against concentration of ZnO nanotubes for E. coli and
of protein present in the cell wall, inactivate the protein and
Bacillus sp. bacterial. decrease the cell permeability which leads to cellular death
[32,33].
To support the above facts, we examined the SEM of E. coli bac-
terial sample before and after inhibition test. SEM images of E. coli
before and after treatment for 24 h with 0.7 g/l ZnO nanotubes, is
depicted in Fig. 7a and b), respectively. It is clear that treatment
of the E. coli bacteria with ZnO nanotubes has led to considerable
damage to E. coli which caused the breakdown of the bacterial cell
wall [34,35]. Furthermore, because particle size of ZnO nanotubes
is about 17 nm, the cluster effect becomes very significant due to
the very high aspect ratio of the synthesized ZnO nanotubes.
Thereof, the ZnO nanotubes activate the bacterial grovel and spores
so that bactericidal efficiency becomes higher [2]. The antibacterial
power of ZnO nanotubes may be associated with some characteris-
tics of bacterial species [1–3]. The antimicrobial activity of the ZnO
nanotubes compared to ampicillin, as a positive control for differ-
ent bacterial species, is listed in Table 1. It is clear that, Gram-posi-
tive bacteria (such as Bacillus sp., Micrococcus sp., Staphylococcus
aureus, Staphylococcus epidermidis, Streptococcus pneumonia) are
less susceptible to Zn ions than Gram-negative bacteria (such as
Acinetobacter sp., E. coli, Klebsiella pneumonia, Proteus mirabilis,
Pseudomonas aeruginosa, Salmonella sp.) due to differences in their
membrane structure [1,2]. The Gram-positive bacteria have more
peptidoglycan than Gram-negative bacteria because of their
thicker cell wall, and because peptidoglycan are negatively charged
and ZnO are positively charged hole (h+). Thus, more Zn ions may
get trapped by peptidoglycan in Gram-positive bacteria than in
Gram-negative bacteria. These holes react with hydroxyl groups
and adsorb water to create hydroxyl radical (OH)1 and the lone
electron of ZnO creates a superoxide ion. The derivatives of this
active oxygen damage the bacterial cell. The dependence of ZnO
nanotubes on the respiration of oxygen consumed and the flow
of potassium for E. coli and Bacillus sp. bacteria is listed in Table 2.
By taking a closer look at the data in Table 2, it is seen that the E.
Fig. 7. (a): SEM image of normal cells of E. coli grown in nutrient broth medium
coli have higher respiration of oxygen consumed and flow of potas-
after 24 h of growth at 37 °C, (b): treated cells of E. coli grown in nutrient broth sium compared with Bacillus sp. This demonstrates that E. coli is
medium supplemented with 0.7 g/l ZnO after 24 h of growth at 37 °C. more sensitive to ZnO nanotubes than Bacillus sp. One explanation
for the higher respiration of oxygen consumed and flow of potas-
This implies that ZnO nanotubes showed high sensitivity against E- sium in E. coli compared to Bacillus sp. is attributed to differences
coli and the zone of E. coli bacterial inhibition increased. There are in the polarity of their cell membrane [7,8]. This reflects that the
two possible mechanisms for the antibacterial activity of zinc ability of ZnO nanotubes to inhibit growth by generation of radical
oxide nanotubes towards E. coli baterial. First, formation of increase oxygen species is well suggested. In addition, electrostatic attach-
levels of reactive oxygen species mainly hydroxyl radical and ment, this leads to adhere a large number of bacteria on the surface
876 N.A. Aal et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 871–877

Table 2
Effect of ZnO nanotubes (500 lg/ml) on both respiration and flow of potassium from the plasma membranes of E. coli and Bacillus sp.

Tested bacteria Control ZnO

Flow of potassium Respiration (Quantity of O2 consumed Flow of potassium Respiration (Quantity of O2 consumed
(lg/g cells) lmol/h mg cells) (lg/g cells) lg/h mg cells)
E. coli 14.0  107 29.50 13.14  107 24.53
Bacillus sp. 14.0  107 22.80 12.98  107 20.84

properties of mixed Co3O4/CoO nanorods by microwave hydrothermal

Table 3
MIC of ZnO nanotubes for different tested bacteria.
Tested bacterium MIC (mg/ml)
Acinetobacter baumannii. 0.55 ± 0.04
Escherichia coli 0.45 ± 0.04
Klebsiella pneumonia 0.55 ± 0.04
Proteus mirabilis 0.45 ± 0.04
Pseudomonas aeruginosa 0.55 ± 0.04
Salmonella typhi. 0.65 ± 0.04
Bacillus subtilis. 0.65 ± 0.04
Micrococcus luteus. 0.75 ± 0.04
Staphylococcus aureus 0.75 ± 0.04
MRSA 0.75 ± 0.04
Staphylococcus epidermidis 0.60 ± 0.04
Streptococcus pneumonia 0.60 ± 0.04
and direct interaction of Zn ions with microorganism. Furthermore,
the slow releasing of antibacterial of Zn ions into medium prevents
bacterial multiplication and/or kills bacteria [14,15]. The minimum
inhibition concentration (MIC) of ZnO nanotubes for different
tested bacteria is recorded in Table 3. From the result in Table 3,
the antibacterial effect of ZnO shows better antibacterial effect
was observed against E. coli and slight effect towards other bacte-
ria’s listed in Table 3. This activity might be due to the size, surface
morphology and particle morphology of ZnO nanotubes. The possi-
ble mechanism for the cell lyses is, the ZnO nanotubes release ions
which react with the thiol (–SH) groups of protein present in the
cell wall, inactivate the protein and decrease the cell permeability
which leads to cellular death. It is concluded from the present
study that, the ZnO nanotube could be used as an effective antibac-
terial agent for E. coli bacterial.
Conclusions
New ZnO nanotubes have been successfully synthesized by sim-
ple hydrothermal method from the reaction between zinc nitrate,
urea and KOH at 220 °C for 10 h. X-ray and FESEM analysis coinci-
dentally indicate that the ZnO nanotube is crystalline. Most of the
crystallites have nanotube morphology with a wall thickness of
less than 2 nm, with an average diameter of 17 nm and the length
is 2 lm. The bactericidal growth decreased with increasing ZnO
nanotubes concentration. It is proposed that due to the extremely
high aspect ratio of ZnO nanotubes, their interaction with the bac-
teria is strong and capable of inhibition. The ZnO nanotubes have
good antibacterial ability against E-coli which makes ZnO very use-
ful due to their relatively non-toxic profile, chemical stability and
efficient antibacterial efficacy.
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