Materials Science and Engineering C 33 (2013) 2020–2024

Contents lists available at SciVerse ScienceDirect

Materials Science and Engineering C

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

Synthesis and concentration dependent antibacterial activities of CuO nanoflakes

T. Pandiyarajan a, R. Udayabhaskar a, S. Vignesh b, R. Arthur James b, B. Karthikeyan a,⁎
a
Department of Physics, National Institute of Technology, Tiruchirappalli 620 015, India
b
Department of Marine Science, Bharathidasan University, Tiruchirappalli 620 024, India

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

Article history: We report, synthesis and antibacterial activities of CuO nanoflakes. CuO nanoparticles are prepared at
Received 27 September 2012 room temperature through sol–gel method. X-ray diffraction studies show the particles are monoclinic
Received in revised form 24 November 2012 (crystalline) in nature. Scanning electron microscopy (SEM) images clearly show that the prepared particles
Accepted 11 January 2013
are flake like in structure. Fourier transform infrared (FTIR) spectra exhibits three different bands that
Available online 17 January 2013
correspond to the Au and Bu modes. Antibacterial studies were performed on Shigella flexneri, Staphylococcus
Keywords:
aureus, Staphylococcus epidermidis, Salmonella typhimurium, Bacillus subtilis, Escherichia coli, Vibrio cholera,
Sol–gel method Pseudomonas aeruginosa and Aeromonas liquefaciens bacterial strains. Among these bacterial strains, S. flexneri
CuO nanoflakes and B. subtilis are most sensitive to copper oxide nanoparticles than the positive control (Penicillin G) and S.
X-ray diffraction typhimurium strain shows the less sensitive. Results show that sensitivity is highly dependent on the concen-
Antimicrobial activities trations of CuO nanoflakes.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction affected with the metal nanoparticles. Perelshtein et al. [18] prepared
CuO–cotton nanocomposite through sonochemical irradiation technique
Recently considerable attention has been paid for the synthesis of and studied their formation, morphology and antibacterial activities.
nanostructured materials. Understanding the physical and chemical Mahapatra et al. [19] prepared ultrafine CuO nanoparticles through wet
properties [1] gain much interest due to their applications in the chemical process and studied their antibacterial activity. Recently, Azam
field of optoelectronics [2], nanoelectronics [3], nanosensors [4], storage et al. [14] have studied the size-dependent antimicrobial properties of
materials [5], bio-labeling, nano-medicine and catalysis [6,7]. Among CuO nanoparticles against Gram-positive and negative bacterial strains
the various metal oxide nanostructures, copper oxide nanostructures and their results show that the microbial strain is highly affected with
have attracted more attention because of its wide range of applica- the size of the CuO nanoparticles. Similarly, Ramyadevi et al. [20] have
tions such as high-Tc superconductors [8], sensors [9,10], catalytic reported the antimicrobial activity of CuO nanoparticles and they found
[11], optical [12], electrical [13] and antimicrobial [14] applications. that the copper nanoparticles showed more inhibitory activity in bacteria
Naturally, CuO is a p-type semiconductor with the band gap of than the fungus.
1.7 eV [15]. To date there are several preparation techniques Gaining the importance of CuO nanostructures, this report contains
like sol–gel, hydrothermal, laser ablation and colloidal chemical preparation of CuO nanoparticles through sol–gel method at room
synthesis technique have been developed for the synthesis of CuO temperature and explains their antibacterial activities against various
nanostructures. Among these, so–gel method is the simplest tech- bacterial organisms.
nique and has the ability to control the morphology and size.
In day to day life, microbial contamination on water poses a major 2. Materials and methods
impact to the human life. Since the ancient times silver and copper
ions are widely used for the antimicrobial agents of many diseases 2.1. Synthesis and characterization of CuO nanoflakes
like burn wounds, dental work and catheters [16]. In the past few
decades with the help of nanoscience and nanotechnology, metal All the chemicals used in the experiment are of high purity reagents
nanoparticles (silver and copper) and semiconductor nanoparticles purchased from Sigma-Aldrich, U.S.A. CuO is prepared by adding
have been used for the antifungal/antibacterial materials. For drop-wise 120 ml of 0.125 M of NaOH which is dissolved in doubly
instance, Ruparelia et al. [17] have studied the antimicrobial activity distilled water into 100 ml of 0.05 M of copper sulfate pentahydrate
of silver and copper nanoparticles using disk diffusion method and (CuSO4.5H2O) dissolved in doubly distilled water. Initially, bluish
found that Escherichia coli and Staphylococcus aureus are highly green colored gel was formed and after 3 h, it became black colored
one. This precipitate was kept at room temperature for over 12 hour
⁎ Corresponding author. Tel.: +91 431 2503612; fax: +91 431 2500133. period, and then washed five times using doubly distilled water. It is
E-mail address: [email protected] (B. Karthikeyan). then filtered and dried at hot air oven at 60 °C for 4 h. Similar procedure

0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.msec.2013.01.021
 T. Pandiyarajan et al. / Materials Science and Engineering C 33 (2013) 2020–2024 2021

was done for 0.150 M of NaOH molar concentrations. The resultant Table 1
powders are named as CuO125 and CuO150. To identify the crystal Name of the bacterial strains with code name.

structure of the CuO nanostructures, X-ray diffraction (XRD) measure- S. no. Name of the bacteria Code
ments are carried out using Rigaku Dmax 2000. The power of the XRD
1. Shigella flexneri MTCC 1457 B1
(Cu-Kα radiation) was fixed at 40 kV and 30 mA. The diffraction angles 2. Staphylococcus aureus NCIM 5021 B2
(2θ) were fixed between 20° and 80°. The morphology and elemental 3. Staphylococcus epidermidis NCIM 2493 B3
composition of the prepared particles was characterized by energy dis- 4. Salmonella typhimurium NCIM 2501 B4
5. Bacillus subtilis NCIM 2920 B5
persive spectroscopy (EDS) attached with SEM instrument (Carl Zeiss
6. Escherichia coli NCIM 2931 B6
MA15/EVO 18). FTIR absorption measurements were done using Shimdzu 7. Vibrio cholerae MTCC 3906 B7
FTIR spectrometer through KBr pellet technique in the range of 650 to 8. Pseudomonas aeruginosa NCIM 5029 B8
400 cm−1. 9. Aeromonas liquefaciens MTCC 2645 B9

2.2. Antibacterial studies

‘θ’ is the diffraction angle and ‘λ’ is the wavelength of X-ray used.
2.2.1. Preparation of culture media Calculated ‘d’ spacing values are listed in Table 2.
1.3 g of nutrient broth powder is dissolved in 100 ml of water by Morphology and elemental composition is analyzed through SEM
gently boiling in a 250 ml of conical flask stoppered by cotton plug. and EDS. Fig. 2 shows that the prepared materials are in flake like
The broth is sterilized by autoclaved at 15 lbs of pressure in 121 °C for structure and the presence of Cu and O atoms in the nanoflakes are
about 15 min. Then it is allowed to cool in a laminar hood. The laminar confirmed by EDS measurement.
hood is disinfected by cleaning thoroughly with absolute alcohol
followed by UV irradiation for 20 min. A disinfected wire loop size 3.1.1. FTIR spectra
stock bacterial strain is transferred into the cold medium under laminar To investigate the local structure and crystallinity of the particles,
flow. After transferring the cultured microorganism, the conical flask is analysis is done through infrared spectroscopy. FTIR spectra of
replugged and kept in an incubator oven for 37± 1 °C for 24–48 h. The prepared CuO nanostructures in the range of 400 to 650 cm −1 are
cultures are obtained from, Chandigarh, India and Pune, India. depicted in Fig. 3. The spectra clearly show that three different
bands correspond to the Cu\O bond vibrations. CuO has monoclinic
2.2.2. Testing of antibacterial activity symmetry with space group C2/c and it has 12 phonon branches
Bacterial sensitivity to antibiotics is tested using a disk diffusion because of 4 atoms in the primitive cell. Group theory predicts
method, employing antibiotic impregnated disk [21]. A similar test vibrational modes at zone center ΓVib = Ag + 2Bg + 4Au + 5Bu, among
with nanoparticle laden disk is used in this study. 3.80 g of Muller them 3Au + 3Bu modes are infrared active. The infrared active
Hinton Agar (MHA) powder is dissolved in 100 ml of water and modes involved the motion of both the Cu and O atoms. The induced
sterilized by autoclaving at 15 lbs of pressure at 121 °C for about dipole moment is along the b-axis of the unit cell for the Au modes
15 min. After adequate level of cooling, nearly 20 ml of the above and perpendicular to it for the Bu modes. The vibrational bands
medium is added into all sterilized Petri dishes, separately after tak- which appeared at 503 and 602 cm −1 correspond to the Bu modes
ing all precautions to avoid any contamination. The dishes are cooled and the band at 427 cm −1 is assigned to the Au modes. The obtained
for sufficient time (25–35 min) to solidify the agar medium then a values are in good agreement with the literature [15].
sterile cotton swab is used to inoculate the culture on the surface of
Muller Hinton Agar (MHA) plate and rotating the plate every 60 °C 3.2. Antibacterial activities
to ensure homogeneous growth. With the help of sterilized forceps
the nanoparticle coated disks (6-mm diameter) are placed in each CuO nanoflakes are investigated with respect to potential antimi-
dish. Two different concentration samples of test solution are poured crobial applications. Microbial sensitivity to the nanoparticles is
in each disk in three dishes. Each such dish contained 0.75 mg and found to vary depending on the microbial species and also CuO con-
1.50 mg concentrations of same copper oxide (CuO 0.125 M) sample centrations. The bactericidal effect of the nano-crystalline CuO flakes
disks. Similar process is carried out for 0.15 M of copper oxide is tested against harmful microorganisms. The two tested concentra-
nanoparticles. The dishes are wrapped well by a paraffin film to seal tions of 0.75 and 1.50 mg/disk produce zone of inhibition on MHA.
them. The entire handling is done inside the laminar hood in front Antimicrobial activity of copper nanoflakes is scrutinized with various
of a flame of a spirit lamp. The plates were incubated at 37 ± 1 °C
for 24–48 h for bacteria, after which the average diameter of the
inhibition zone surrounding the disk is measured with help of a
ruler. The mean reported for each concentration of nanoparticles
and with each microbial strain are based on three replicates. Penicillin
G (10 mcg/disk — for bacteria) is used as a positive control. All the
media and disks were purchased from Hi-Media (Mumbai, India).
Name of the bacterial strains and corresponding code names are
listed in Table 1.

3. Results and discussion

3.1. Structural and morphological analysis

The XRD pattern of 0.125 and 0.15 M of CuO is shown in Fig. 1. All
the diffraction patterns are well indexed to monoclinic structure of
CuO (JCPDC no.: 801916). The patterns show no impurity peaks like
Cu2O and Cu clusters which confirms that the prepared particles are
in high purity. ‘d’ spacing is calculated from the XRD patterns by
using Bragg's law: 2d sin θ = nλ where, ‘d’ is the interplanar spacing, Fig. 1. X-ray diffraction patterns of CuO nanoflakes.
2022 T. Pandiyarajan et al. / Materials Science and Engineering C 33 (2013) 2020–2024

Table 2
Calculated values of d-spacing.

(hkl) planes 2θ (degrees) d spacing (Å)

(110) 32.56 2.748

(−111) 35.58 2.521
(111) 38.82 2.318
(−202) 48.95 1.860
(020) 53.55 1.710
(202) 58.36 1.580
(−113) 61.61 1.504
(331) 66.14 1.412
(220) 68.16 1.374

microorganisms using the (measure the inhibition zone) disk diffu-

sion test. The inhibition zone reflects the magnitude of susceptibility
of the microorganism. The strains susceptible to the disinfectants ex-
hibit larger inhibition zone, whereas resistant strains exhibit smaller
inhibition zone (see Fig. 4). In the present study, 1.5 mg of concen-
trated samples showed greater sensitivity than 0.75 mg of concentra-
tion against all the tested microorganisms and both CuO samples
were most effective against two pathogens such as Shigella flexneri Fig. 3. FTIR spectra of different concentrations of CuO nanoflakes in the range of 400 to
MTCC 1457 (B1) and Bacillus subtilis NCIM 2920 (B5), while smaller ef- 650 cm−1.
fect is noticed from Salmonella typhimurium NCIM 2501 (B4) and
Aeromonas liquefaciens MTCC 2645 (B9). All the organisms are highly af-
fected by CuO (0.125 M) when compared to CuO (0.150 M), except A.
liquefaciens MTCC 2645 (B9) and Staphylococcus epidermidis NCIM concentration (1.5 mg/disk) for both CuO nanoparticles when com-
2493 (B3). Interestingly, no effect was observed in E. coli NCIM 2931 pared to the positive control, except S. aureus NCIM 5021 (B2), S.
(B6) and Pseudomonas aeruginosa NCIM 5029 (B8) with positive con- typhimurium NCIM 2501 (B4) and A. liquefaciens MTCC 2645 (B9).
trol. All the bacterial strains depict higher sensitivity to the higher Among all the strains in this study, S. flexneri MTCC 1457 (B1) and B.

Fig. 2. Scanning electron microscopic images and EDX spectra of prepared nanoflakes.
 T. Pandiyarajan et al. / Materials Science and Engineering C 33 (2013) 2020–2024 2023

Fig. 4. The pictures show disk diffusion (zone of inhibition) results of (a) Escherichia coli (higher sensitive effect), (b) Staphylococcus epidermidis and (c) Salmonella typhimurium
NCIM 2501 (less sensitive effect).

subtilis (B5) show most sensitivity to copper oxide nanoparticle which Indirect effects through changes in the surrounding charge environment
is comparable with the positive control (Penicillin G). S. typhimurium also have an impact on the effectiveness of nanoparticulate metals against
NCIM 2501 (B4) strain shows the least sensitivity. In this present microorganisms [25]. Another proposed mechanism is, there will be
study the antibacterial effect of copper oxide nanoparticles showed copper ions released from the nanoparticles that may attach to the
good effects against microorganisms as shown in Fig. 5. negatively charged bacterial cell wall and rupture it, thereby leading to
protein denaturation and cause cell death [26]. However, similar kind
of mechanism was proposed by Azam et al. [14], Line et al. [27] and
4. Discussions Nawaz et al. [28].

There are very few reports available for the mechanism behind the
antimicrobial activities. In the present case S. flexneri and B. subtilis 5. Conclusions
show greater sensitivity to Cu nanoparticles. Naturally, S. flexneri and
B. subtilis have abundance of amine and carboxyl groups [22] on cell In summary, antibacterial activities of CuO nanoflakes were studied
surface and high affinity of copper towards these groups [23]. Schematic which are prepared through sol–gel method at room temperature.
representation shows that Cu2+ is attached with the S. flexneri and X-ray diffraction studies show that the particles are monoclinic
B. subtilis species (shown in Fig. 6). Copper ions released subsequently (crystalline) in nature without any impurity phases. SEM images
may bind with DNA molecules and lead to disorder of the helical clearly show the prepared flake like structures. FTIR spectra exhibit
structure by cross-linking within and between the nucleic acid strands. three different bands which correspond to the Au and Bu modes.
Copper ions inside bacterial cells also disrupt the biochemical processes Antibacterial studies were performed against a set of bacterial
[24]. Cu 2+ and Ag+ ions are also small enough to disrupt the bacterial strains. Among them, S. flexneri and B. subtilis show most sensitivity
cell membranes and gain entry in order to disrupt enzyme function. to copper oxide nanoparticles which are powerful than the positive

Fig. 5. Zone of inhibition graph against bacterial strains. Result shows for the positive control (PC), 0.75 mg of CuO (0.75 mg) and 1.5 mg of CuO (1.5 mg). It is clear that CuO sam-
ples were most effective against two pathogens such as Shigella flexneri MTCC 1457 (B1) and Bacillus subtilis NCIM 2920 (B5), while smaller effect was noticed from Salmonella
typhimurium NCIM 2501 (B4) and Aeromonas liquefaciens MTCC 2645 (B9).
2024 T. Pandiyarajan et al. / Materials Science and Engineering C 33 (2013) 2020–2024

Fig. 6. (a) Schematic representation of Cu2+ attached with the carboxyl group present in the B. subtilis species. (b) Cu2+ attached with the amine group present in the B. subtilis
species. CuO nanoparticles have been attached with the greater abundance of amine and carboxyl groups on their cell surface and greater affinity of copper towards these groups.
Attachment of Cu2+ with the bacterial cell wall leads to the chemical changes in the cell wall which give rise to the cell wall damage.

control (Penicillin G). Salmonella typhimurium strain shows the least [14] A. Azam, A.S. Ahmed, M. Oves, M.S. Khan, A. Memic, Int. J. Nanomedicine 7 (2012)
3527–3535.
sensitivity. [15] L. Debbichi, M.C. Marco de Lucas, J.F. Pierson, P. Krüger, J. Phys. Chem. C 116
(2012) 10232–10237.
References [16] J.S. Kim, Nanomedicine 3 (2007) 95–101.
[17] J.P. Ruparelia, A.K. Chatterjee, S.P. Duttagupta, S. Mukherji, Acta Biomater. 4
[1] M.F. Garcıa, A.M. Arias, J.C. Hanson, J.A. Rodriguez, Chem. Rev. 104 (2004) (2008) 707–716.
4063–4104. [18] I. Perelshtein, G. Applerot, N. Perkas, E.W. Sigl, A. Hasmann, G. Guebitz, A. Gedanken,
[2] C.H. Aaronson, H. Amekura, Y. Sato, N. Kishimoto, J. Appl. Phys. 109 (2011) Surf. Coat. Technol. 204 (2009) 54–57.
024506–024507. [19] O. Mahapatra, M. Bhagat, C. Gopalakrishnan, K.D. Arunachalam, J. Exp. Nanosci. 3
[3] M.J. Deen, F. Pascal, J. Mater. Sci: Mater. Electron. 17 (2006) 549–575. (2008) 185–188.
[4] N. Tessler, V. Medvedev, M. Kazes, S.H. Kan, U. Banin, Science 295 (2002) [20] J. Ramyadevi, K. Jeyasubramanian, A. Marikani, G. Rajakumar, A.A. Rahuman,
1506–1508. Mater. Lett. 71 (2012) 114–116.
[5] S. Kim, H. Moon, D. Gupta, S. Yoo, Y.K. Choi, IEEE Trans. Electron. Devices 56 [21] C.L. Case, T.R. Johnson, Laboratory Experiments in Microbiology, Benjamin
(2009) 696–699. Cummings Publisher Inc., California, 1984, pp. 126–129.
[6] C.J. Lin, T. Liedl, R.A. Sperling, M.T.F. Arguelles, J.M. Costa-Fernandez, R. Pereiro, [22] T.J. Beveridge, R.G.E. Murray, J. Bacteriol. 141 (1980) 876–887.
A. SanzMedel, W.H. Chang, W.J. Parak, J. Mater. Chem. 17 (2007) 1343–1346. [23] R.J. Doyle, T.H. Matthews, U.N. Streips, J. Bacteriol. 143 (1980) 471–480.
[7] S.M. Moghimi, A.C. Hunter, J.C. Murray, FASEB J. 19 (2005) 311–330. [24] J. Kim, H. Cho, S. Ryu, M. Choi, Arch. Biochem. Biophys. 382 (2000) 72–80.
[8] S.K. Yip, J.A. Sauls, Phys. Rev. Lett. 69 (1992) 2264–2267. [25] S.J. Stohs, D. Bagchi, Free Radic. Biol. Med. 18 (1995) 321–336.
[9] Y.S. Kim, I.S. Hwang, S.J. Kim, C.Y. Lee, J.H. Lee, Sensors Actuators B 135 (2008) [26] I. Sondi, B.S. Sondi, J. Colloid Interface Sci. 275 (2004) 177–182.
298–303. [27] Y.E. Lin, R.D. Vidic, J.E. Stout, C.A. Mccartney, V.L. Yu, Water Res. 32 (1998)
[10] A. Umar, M.M. Rahman, A. Al-Hajry, Y.B. Hahn, Electrochem. Commun. 11 (2009) 1997–2000.
278–281. [28] M. Nawaz, M.Y. Han, T. Kim, U. Manzoor, M.T. Amin, Sci. Total Environ. 431 (2012)
[11] S. Yang, C. Wang, L. Chen, S. Chen, Mater. Chem. Phys. 120 (2010) 296–301. 20–25.
[12] T. Yu, F.C. Cheong, C.H. Sow, Nanotechnology 15 (2004) 1732–1736.
[13] P. Podhajecky, B. Klapste, P. Novak, J. Mrha, R. Moshtev, V. Manev, A. Nassalevska,
J. Power. Sources 14 (1985) 269–275.