Accepted Manuscript Novel Rapid Synthesis of Zinc Oxide Nanotubes via Hydrothermal Technique and Antibacterial Properties Nadia Abdel Aal, Faten Al-Hazmi, Ahmed A. Al-Ghamdi, Attieh A Alghamdi, Farid El-Tantawy, F. Yakuphanoglu PII: DOI: Reference:

S1386-1425(14)01180-9 http://dx.doi.org/10.1016/j.saa.2014.07.099 SAA 12519

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

4 June 2014 10 July 2014 29 July 2014

Please cite this article as: N.A. Aal, F. Al-Hazmi, A.A. Al-Ghamdi, A.A. Alghamdi, F. El-Tantawy, F. Yakuphanoglu, Novel Rapid Synthesis of Zinc Oxide Nanotubes via Hydrothermal Technique and Antibacterial Properties, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/ 10.1016/j.saa.2014.07.099

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Novel Rapid Synthesis of Zinc Oxide Nanotubes via Hydrothermal Technique and Antibacterial Properties 1

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Nadia Abdel Aal1, Faten Al-Hazmi2, Ahmed A. Al-Ghamdi2, Attieh A Alghamdi3, Farid El-Tantawy4, F. Yakuphanoglu2,5

Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia, Egypt Department of Physics, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia 3 Centre of nanotechnology, King AbdulAziz University, Jeddah, Saudi Arabia 4 Department of Physics, Faculty of Science, Suez Canal University, Ismailia, Egypt 5 Department of Physics, Faculty of Science, Firat University, Elazig 23169, Turkey

Abstract ZnO nanotubes with the wurtzite structure have been successfully synthesized via simple hydrothermal solution route using zinc nitrate, urea and KOH for the first time. The structural, compositions and morphology architectures of the as synthesized ZnO nanotubes was performed using X - ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), energy dispersive X-ray spectroscopy (EDS) and high resolution transmission scanning electron microscopy (HRTEM). TEM showed that ZnO nanotubes exhibited a wall thickness of less than 2 nm, with an average diameter of 17 nm and the length is 2 µ m .

In addition, the antibacterial activity of ZnO nanosheets was carried out in vitro against two kinds of bacteria: gram - negative bacteria (G -ve) i.e. Escherichia coli (E. coli) and gram - positive bacteria (G +ve) i.e. Staphylococcus aureus. Therefore, this work demonstrates that simply synthesized ZnO nanotubes have excellent potencies, being ideal antibacterial agents for many biomedical applications. Keywords: ZnO, nanotubes, hydrothermal, antibacterial properties Corresponding author:[email protected] (F.Yakuphanoglu) Tel:+90 424 2370000-3792 Fax:+90 424 2330062

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1. Introduction

This study is part of an on-going research project aiming to develop high performance nanoscale inorganic metal oxides for antibacterial with high aspect ratio and good antibacterial efficacy. In fact, the re-emergence of infectious diseases and the continuous development of antibiotic resistance among a variety of disease, microbial pollution and its contamination induced by microorganisms 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, stability, 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 different 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 nanocrystals with controlled dimensionality (e.g., 0D dots, 1D rods and wires, 2D sheets and disks and 3D flowers are versatile building 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, nanotechnology, biology, and medicine [8, 19]. In particular, zinc oxide (ZnO) is an important class of inorganic material, which processes unique

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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.

2. Experimental Details 2.1. Synthesis of ZnO Nanotubes

The raw materials are analytic grade reagents and purchased without further treatment. First, for the preparation of ZnO nanotubes, an aqueous solution of 50 mL, which contained 0.35 mol of zinc nitrate hexahydrate Zn( NO3 )2 ⋅ 6 H 2O and 0.7 mol urea was prepared 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 0C 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 0C overnight. After that, 1 gm of as - prepared ZnO powder was added to 33 ml of 12 mol KOH and additionally backed in hydrothermal cell at 220 0C for 10 h.

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2.2 Characterization of as-Synthesized ZnO Nanotubes

The structure of the as-synthesized ZnO nanotubes was analyzed by X-ray powder diffraction (X-ray) using a Shimatzou X-ray diffractometer (Shimatzou, XRD6000), with Cukα 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 spectometer (EDS). The particle size and orientation of synthesized ZnO was analyzed by the highresolution transmission electron 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 1ml of ethanol, and then a drop of the solution was placed on a Cu grid covered with carbon film.

2.3. 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 oC, 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 µ l

of the bacterial suspension of the tested material using

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micropipette [1,2]. Ampicilin (5 lg/ml) was used as positive control and water was used as negative 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 0C for 24 h, followed by the measurement 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 average value was taken. All experiments were carried out in triplicate and the reported data represents average values.

2.4. 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 0C. The bacterial growth was determined by measuring the absorbance at 595 nm. Control flasks without ZnO nanomaterial were prepared as control.

2.5. Mode of Action of the Tested Nanomaterials

After incubation of the tested bacterial in nutrient broth medium, 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 consumed/min) was determined using Oxigraphe.

The quantity of K+ flowed from the treated and untreated cells were

determined using atomic adsorption [1,2].

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3. Results and Discussion 3.1 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 patterns of the as-synthesized ZnO

nanotubes prepared by hydrothermal method which were vertically normalized for clarity is depicted in Fig. (1). Several well-defined diffraction reflections were appeared in the pattern at 2θ (degrees) of 31.72 0 , 34.410 , 36.210 , 47.49 0 , 56.520 , 62.810 , 67.86 0 and 68.990 , which correspond to the lattice planes of (100 ) , ( 002 ) , (101) ,

(102 ) , (110 ) , (103) , (112 )

and ( 201) , 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 lattice constants of a = 0.325 nm and c = 0.524 nm [9-11]. No diffraction 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 (002) 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 (101) planes by using the Scherrer’s equation [3]:

D=

kλ β cosθ

(1)

where k is a constant (k = 0.9), λ is the wavelength of X-ray used, β is the full-width at the half maximum (FWHM) of the diffraction peak and θ is the 6

Bragg

angle. Interestingly, to check the preferential crystallite orientation of as

synthesized ZnO, we calculated the texture coefficient

(TC ) ( hkl )

using the

following equation [10]: TC ( hkl ) =

I ( hkl ) / I 0 ( hkl ) ×100 ( % ) ∑ I ( hkl ) / I 0 ( hkl )

(2)

where I ( hkl ) is the measured relative intensity of a plane ( hkl ) , and I 0 ( 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 ( 002 ) 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 ( 002 ) plane [26,27]. The chemical composition and quality of as-synthesized ZnO nanotubes was performed by Fourier transform infrared (FTIR) spectroscopy in the range of 400 – 4000 cm-1. The typical FTIR spectrum 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 cm-1 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 cm

-1

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

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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 confirming the high purity of the as synthesized ZnO nanotubes. The element 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. (4 b,c,d). The low and high magnification FESEM images in Fig. (3 a-c), respectively clearly exhibits that the obtained products are synthesized in large quantity and possessing nanotube-like morphologies. Each tube is symmetrically formed with hexagonal cross sections for both the inner and outer walls. The typical diameter of the nanotubes is on average around 17 nm and the length is 2 µ m . 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 nanotubes exhibited a wall thickness of less than 2 nm, with an average diameter of 17 nm and the length is 2 µ m . The average particle diameter obtained from the Scherrer formula 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 nanotube-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

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nanostructures. The distance between lattice fringes along the longitudinal axis direction, corresponding to the d-spacing of ZnO indexed to (002) crystal planes, is measured 51 nm supporting that the grown ZnO nanotubes are highly crystalline and possessing hexagonal ZnO structure [13,31].

3.3 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 Bacillus. Interestingly enough, the antibacterial activity of E. coli is higher

than that of Bacillus sp., with increasing ZnO concentration. This implies that ZnO nanotubes showed high sensitivity against E-coli and the zone of E. coli bacterial inhibition increased. There are two possible mechanisms for the antibacterial activity of zinc oxide nanotubes towards E. coli baterial. First, formation of increase levels of reactive oxygen species mainly hydroxyl radical and singlet oxygen, in turns to damage the bacterial cell wall. Second, the ZnO nanotubes release ions which react with the thiol groups of protein present in the cell wall, inactivate the protein and decrease the cell permeability which leads to cellular death [32,33]. To support the above facts, we examined the SEM of E. Coli bacterial 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,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

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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 characteristics of bacterial species [1-3]. The antimicrobial activity of the ZnO nanotubes compared to ampicillin, as a positive control for different bacterial species, is listed in Table (1). It is clear that, Gram-positive bacteria (such as Bacillus sp., Micrococcus sp., Staphylococcus aureus, Staphylococcus epiderimdis, 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 Gramnegative 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. coli have higher respiration of oxygen consumed and flow of potassium compared with Bacillus sp. This demonstrates that E. coli is more sensitive to ZnO nanotubes than Bacillus sp. One explanation for the higher respiration of oxygen consumed and flow of potassium in E. coli compared to Bacillus sp. is attributed to differences in the polarity of their cell membrane [7,8]. This reflects that the ability of

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ZnO nanotubes to inhibit growth by generation of radical oxygen species is well suggested. In addition, electrostatic attachment, this leads to adhere a large number of bacteria on the surface 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 bacteria's listed in Table (3). This activity might be due to the size, surface morphology and particle morphology of ZnO nanotubes. The possible 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 antibacterial agent for E. coli bacterial.

4. Conclusions

New ZnO nanotubes have been successfully synthesized by simple hydrothermal method from the reaction between zinc nitrate, urea and KOH at 220 0C for 12 h. X-ray and FESEM analysis coincidentally 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 µ m . 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 bacteria is strong and capable of inhibition. The ZnO nanotubes have good antibacterial ability

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against E-coli which makes ZnO very useful due to their relatively non-toxic profile, chemical stability and efficient antibacterial efficacy.

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Fig. (1): Typical X-Ray pattern of synthesized ZnO nanotubes and the inset is the calculated TC vs. ZnO plane (hkl).

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Fig. (2): FTIR spectra of as synthesized ZnO nanotube.

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Fig. (3): SEM-EDS profile of the ZnO nanotubes.

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Fig. (4): Typical (a) low-magnification and (b and c) high-resolution FESEM images of the synthesized ZnO nanotubes.

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Fig. (5): low (a) and (b) high-resolution TEM images of as-synthesized ZnO nanotubes

hexagonal nanostructures.

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1.2

E. coli Bacillus sp

Bacterial Growth

1

0.8

0.6

0.4

0.2

0 100

200

300

400

500

600

700

800

900

1000

Concentration (µg/ml)

Fig. (6): Bacterial growth against concentration of ZnO nanotubes for E.coli and Bacillus sp bacterial.

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Fig. (7): (a): SEM image of normal cells of E. coli grown in nutrient broth medium after 24h of growth at 37°C, (b): Treated cells of E. coli grown in nutrient broth medium supplemented with 0.7 g/l ZnO after 24h of growth at 37°C.

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Table (1): Antibacterial activity (Diameter of inhibition zone, mm) of the ZnO nanotubes compared to Ampicillin as a positive control. Tested bacteria

Gram

ZnO

reaction

Positive control (Ampicillin)

Acinetobacter sp.

- ve

11.3+0. 62

15+1.0

Bacillus sp.

+ ve

13.2+0.54

30+1.5

Escherichia coli

- ve

14.8+0.38

25+4.5

Klebsiella

- 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

Pseudomonas

- ve

10.8+0.56

19+2.50

Salmonella sp.

- ve

11.8+4.59

20+0.56

Staphylococcus

+ ve

10.0+2.56

32+0.36

+ ve

12.7+1.57

34+0.34

+ ve

ND

30+0.54

11.69

28.7

pneumonia

aeruginosa

aureus Staphylococcus epiderimdis Streptococcus pneumonia Antibacterial index

ND: Not detected inhibition zone, + ve: Gram positive, - ve: Gram negative.

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

Tested bacteria

Control

ZnO

Flow of

Respiration

Flow of

Respiration

potassium

(Quantity of

potassium

(Quantity of O2

(µg /g cells)

O2 consumed

(µg /g cells)

consumed µg/h.mg

µmol/h.mg

cells)

cells)

E. coli

14.0 x10 -7

29.50

13.14x10 -7

24.53

Bacillus sp

14.0 x10 -7

22.80

12.98x10 -7

20.84

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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 epiderimdis

0.60±0.04

Streptococcus pneumonia

0.60±0.04

30

31

1. ZnO nanotubes were been successfully synthesized via simple hydrothermal solution route 2. ZnO nanotubes exhibited a wall thickness about 2 nm, with an average diameter of 17 nm. 3. ZnO nanotubes have excellent potencies for many biomedical applications.

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