Accepted Manuscript

Title: Assisted green synthesis of copper nanoparticles using

Syzygium aromaticum bud extract: Physical, optical and
antimicrobial properties

Authors: K.M. Rajesh, B. Ajitha, Y. Ashok Kumar Reddy, Y.

Suneetha, P. Sreedhara Reddy

PII: S0030-4026(17)31287-1
DOI: https://doi.org/10.1016/j.ijleo.2017.10.074
Reference: IJLEO 59810

To appear in:

Received date: 13-5-2017

Revised date: 17-10-2017
Accepted date: 18-10-2017

Please cite this article as: K.M.Rajesh, B.Ajitha, Y.Ashok Kumar Reddy,
Y.Suneetha, P.Sreedhara Reddy, Assisted green synthesis of copper
nanoparticles using Syzygium aromaticum bud extract: Physical, optical and
antimicrobial properties, Optik - International Journal for Light and Electron
Optics https://doi.org/10.1016/j.ijleo.2017.10.074

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Assisted green synthesis of copper nanoparticles using Syzygium aromaticum
bud extract: Physical, optical and antimicrobial properties

K.M. Rajesha†, B. Ajithab†, Y. Ashok Kumar Reddyc*, Y. Suneethad, P. Sreedhara Reddya*

a
Department of Physics, Sri Venkateswara University, Tirupati-517502, India
b
Department of Nano-Structured Materials Research, National NanoFab Center at KAIST, 291
Daehak-ro, Yuseong-gu, Daejeon-34141, Republic of Korea
c
Department of Electrical Engineering, Korea Advanced Institute of Science and Technology,
291 Daehak-ro, Yuseong-gu, Daejeon-34141, Republic of Korea
d
Department of Zoology, Sri Venkateswara University, Tirupati-517502, India

ABSTRACT

In the present study copper nanoparticles (CuNPs) were prepared using Syzygium aromaticum
(clove) bud extract through simple and eco-friendly green route. The synthesized nanoparticles
were subjected to structural, morphological, optical and antimicrobial studies. The high crystalline
nature of CuNPs with a face centered cubic phase is evident from the X-ray diffraction (XRD)
pattern. Morphological studies were used to study the shape and size of the synthesized
nanoparticles. Energy dispersive spectroscopy (EDS) attests the high intense metallic peak of
copper (Cu) and low intense peaks of carbon (C), oxygen (O), chlorine (Cl) and phosphorus (P)
elements due to the capping action of biomolecules of bud extract in CuNPs formation. The zeta
(ζ) potential of the CuNPs attests the stability of the nanoparticles. Ultraviolet-visible (UV-Vis)
absorption spectrum shows the characteristic absorption peak of CuNPs. Fourier transform
infrared spectroscopy (FTIR) analysis shows the presence of different functional groups at various
positions. The antimicrobial activity was investigated against the selected pathogens using bio-
CuNPs. The positive test results of zone of inhibitions of 8 mm and 6 mm were attained against
Bacillus spp. and Penicillium spp., respectively.
Keywords: Biological synthesis; Copper nanoparticles; Syzygium aromaticum; Morphology;
Absorbance; Antimicrobial studies.
† Both authors contributed equally to this work.
*Corresponding akreddy111@gmail.com (Y. Ashok Kumar Reddy) and
psreddy4@gmail.com (P. Sreedhara Reddy)
1
1. Introduction

During the last two decades, nanostructured materials were extensively studied because of

their unique properties and applications in various fields [1, 2]. In particular, metal nanoparticles

have received a great attention because of their optical, electronic and magnetic properties, which

are different from their bulk materials [3, 4]. The synthesis of novel nanoparticles is a significant

focal point, where the continuous efforts are being made by the researchers for both technological

and industrial advancements. It has been reported that metal nanoparticles (Cu, Ag, Au and Pt)

exhibit a wide spectrum of antimicrobial activity against different species of microorganisms

including the gram negative and gram positive bacteria and as well as fungi [5-7]. Among all metal

nanoparticles, copper nanoparticles (CuNPs) have drawn a much attention owing to their versatile

applications for instance of electrical, catalytic, optical, antibacterial and antifungal purposes [8,

9]. In addition, the availability and cost of Cu made it as a better choice comparing to the Ag, Au

and Pt metals for manifold applications. The usefulness of copper as an antimicrobial agent has

been known for a long time. It is an effective agent with low toxicity, which is especially important

in the biomedical field.

Earlier, many synthesis methods were employed for the synthesis of Cu nanoparticles such

as chemical [10], solvothermal [11], sono-chemical reduction [12], electrochemical [13] thermal

reduction [14] and biosynthesis [15, 16] routes. However, the green syntheses of nanoparticles

through natural seed extracts have a significant potential to boost the nanoparticles production

without the use of toxic and expensive chemicals. Due to an effective nature of low toxicity, Cu

metal is an especially important in the typical antibacterial treatment. Nowadays, many reports

confirm the antibacterial, antifungal, and antiviral properties of spice plants [17]. In particular,

Syzygium aromaticum (Clove) is one of the most valuable spices and has attracted in the much

2
attention due to the potent antioxidant and antimicrobial activities standing out among the other

spices [18]. In the present study, the copper nanoparticles (CuNPs) through Syzygium aromaticum

bud extract were synthesized and the bio-CuNPs could be subjected to the structural,

morphological, optical and antimicrobial studies.

2. Experimental

2.1. Materials

Cupric acetate (monohydrate) ((CH3COO)2Cu.H2O) was purchased from Molychem. All

the chemicals were of analytical reagent grade and used without any further purification. Syzygium

aromaticum buds were collected from the local market. Milli-Q water was used as a solvent

throughout the experiment.

2.2. Preparation of bud extract

Syzygium aromaticum buds (10 g) were washed and cleaned with distilled water and dried

with water absorbent paper. The buds were crushed with the help of mortar and pestle dispensed

in 10 mL of sterile distilled water and heated for 5 minutes at 80 oC. The extract was then filtered

using Whatman’s No.1 filter paper. The filtered extract was collected in a clean and dried conical

flask by standard sterilized filtration method and was stored for further usage in CuNPs synthesis.

2.3. Synthesis of bio-CuNPs

For the synthesis of CuNPs, 50 mL of 0.001 M of copper acetate solution was first prepared

in a reaction vessel of 100 mL volume at 30oC of temperature. Later, 10 mL of aqueous extract of

Syzygium aromaticum bud was added and mixed homogeneously on magnetic stirrer for 15 min

so as to ensure the formation of CuNPs. Within 10-15 min, the blue colour solution changes to

pale bluish green colour due to the formation of CuNPs. The schematic synthesis procedure of bio-

CuNPs is depicted in Fig. 1. The obtained nanoparticles were purified by centrifuging the pale

3
bluish green solution at 10,000 rpm for 10 min, and the resulting pellets were re-dispersed in water

and subjected to further studies.

2.4. Characterization of the bio-CuNPs

The bio-synthesized CuNPs were subjected to various characterization techniques at room

temperature. The structural properties of the synthesized nanoparticles were analyzed by Seifert

3003TT X-Ray diffractometer, using Cu Kα radiation (λ=0.1546 nm). The morphology of the

biosynthesized CuNPs was observed from field emission scanning electron microscopy (FESEM)

of an FEI model FESEM (Sirion) and transmission electron microscopy (TEM, JEOL, JEM-2100F

HR). Elemental composition of the synthesized nanoparticles were examined through energy

dispersive spectroscopy (EDS) using Oxford Inca Penta FETx3 EDS instrument attached to Carl

Zeiss EVO MA 15 scanning electron microscopy. The zeta potential analysis was done through

the Zetasizer Nano series (Model: ZEN3600). UV-Visible absorption study was assessed using a

UV-visible spectrophotometer (Model: Mecasys Optizen Pop) with a quartz cell having an optical

length of 1 cm. Fourier transform infrared spectroscopy (FTIR) spectra of the freeze-dried samples

were recorded with ATR-FTIR using Bruker Vettex-80 spectrometer.

2.5. Antimicrobial assay

The bio-synthesized CuNPs were made to adsorb on the Whatman’s No. 1 paper discs and

loaded at required volumes. The antibacterial activity of biosynthesized CuNPs was tested against

different pathogenic bacteria. In order to assess the antibacterial activity of bio-reduced CuNPs,

the gram-positive Staphylococcus spp., Bacillus spp. and gram-negative Pseudomonas spp.,

Escherichia coli test organisms were selected. Similarly, Aspergillus niger, Aspergillus flavus and

Penicillium spp. test organisms were selected for antifungal activity. The pure cultures of all

4
specified pathogens were procured from the department of Microbiology, S.V. University,

Tirupati, India.

The antimicrobial activity was studied through Kirby-Bauer disc diffusion method against

selected pathogenic organisms [19].The bacterial test organisms were grown in nutrient broth for

24 h and used for further study. Nutrient agar plates were prepared, sterilized and solidified. After

solidification, 100 µl overnight culture of each organism was spreaded on the petriplates using a

sterile glass rod to prepare bacterial lawns. Sterile discs (diameter of 3 mm) were placed on these

plates and CuNPs were loaded at required volumes of 4 µl, 8 µl, 12 µl and 16 µl on respective

discs and incubated at 37oC for 24 h. After the incubation period, clear zone of inhibition around

each disc could be observed. The fungi were grown on a potato dextrose broth (PDB) for 72 h and

200 µl of respective fungi was spread on potato dextrose agar (PDA) plates with sterile L glass rod

followed by the lawn culture preparation. Next, the discs were placed on agar plates and CuNPs

(4 µl, 8 µl, 12 µl and 16 µl) were loaded on to each disc in each petri plates of respective pathogens

under the incubation at 37oC for 72 h. The diameter of the zone of inhibition was measured with

the meter ruler around each disk and the mean value of inhibition was calculated and expressed in

millimeter.

3. Results and discussion

3.1. Structural analysis

X-ray diffraction (XRD) is used to determine the crystalline characteristics of the

synthesized nanoparticles. The XRD profile of the bio-synthesized CuNPs is shown in Fig. 2. All

the peaks correspond to the face centered cubic (fcc) structure and were in consistence with the

standard JCPDS (No. 04-0836) data. From the spectra, we perceived three diffraction peaks

corresponding to (111), (200), and (220) orientations of which, the highest intensity peak suggests

5
the (111) orientation. The peak positions are in good agreement with literature values of metallic

copper [20, 21]. The average size of the crystallites has been estimated from the most prominent

(111) orientation using the Debye-Scherrer’s formula:

D= Kλ/βcosθ (1)

Where K denotes the Scherrer’s constant (K = 0.94), D is the average crystallite size, β is the full

width at half maximum (FWHM) of a Gaussian fit, λ is the wavelength of the Cu Kα radiation

(λ =0.1546 nm) and θ is the half diffraction angle. The crystallite size was found to be ~12 nm.

The lattice constant calculated from (111) plane was 4.078 Å for the CuNPs which is in close

agreement with the standard data (a=4.0862 Å; JCPDS No: 04-0836) [22].

3.2. Morphological studies

The FESEM images of bio-synthesized CuNPs were recorded at different magnifications

(Fig. 3). The formation of homogeneous and relatively spherical nanoparticles with an average

particle size of ~20 nm was observed in FESEM images. To get the further deep insight on the

morphology, size and crystalline nature of the CuNPs, TEM analysis was employed. TEM

micrographs show that the particles are monodispersed, almost spherical in nature and well

segregated without any agglomeration with an average diameter of ~15 nm (Fig. 4a,b). Typical

high resolution TEM (HR-TEM) and the selected area electron diffraction (SAED) pattern of a

nanoparticle are displayed in Fig. 4 (c,d). The HR-TEM clearly visualizes the presence of equi-

spaced interplanar spacing (d). The "d" spacing was estimated to 0.235 nm and well correlates

with the "d" spacing of the (111) orientation of the fcc structure of metallic copper (JCPDS No:

04-0836) [23]. The SAED pattern depicts the circular fringes corresponding to (111), (200), and

(220) planes of fcc structure of Cu which is in accordance with XRD results.

3.3. Compositional and particle stability studies

6
 The EDS spectrum of biologically synthesized CuNPs is shown in Fig. 5(a). This spectrum

shows a high intense major peak of elemental Cu with the atomic% of 71.8%, which is in

accordance with the main identification line of metallic copper nanoparticles. The presence of

weak signals of C, O, Cl and P elements are having the atomic% of 5.8%, 9.6%, 10.5% and 2.3%,

respectively. The presence of addition elements can be caused by the existence of biomolecules

that are bound to the surface of CuNPs.

Zeta (ζ) potential analysis generally assesses the stability of nanoparticles. The ζ- potential

measurement elicits the appearance of maximum peak at 21.3 mV (Fig. 5b), which was evidence

for the stability of the nanoparticles. Particles that have a large negative or positive zeta potential

are likely to repel each other, and are therefore relatively stable. The higher negative value

indicates that a strong repulsive force is in between the particles leading in enhancement of

stability. The observed negative ζ-potential suggesting a very good stability of the CuNPs and was

consistent with the previous reports [24].

3.4. UV-Vis absorption studies

The formation of CuNPs was also monitored through the UV-Vis spectroscopy. The

reduction of copper acetate to copper was monitored by recording UV-Vis spectrum of the reaction

mixture after diluting a small aliquot of the sample with deionised water. In general, CuNPs exhibit

the surface plasmon resonance peak in the range of 550-650 nm [25, 26]. The surface plasmon

resonance (SPR) is the absorption of the visible electromagnetic radiation of the collective

oscillation of surface electrons. According to Mie’s theory, only one single SPR band will arise

for spherical nanoparticles, while two or more will result depending on the shape of the

nanoparticles [27]. The addition of S. aromaticum bud extract to copper acetate solution resulted

in color change of the solution from blue to pale bluish green due to the formation of CuNPs (Fig.

7
1). The surface plasmon vibrations of copper nanoparticles produced a peak at ~580 nm (Fig. 6)

and which can be attributed from the CuNPs [28]. The only single curve of SPR attributes to the

spherical NPs on confirmation with FESEM and TEM images.

3.5. FTIR Spectroscopy

FTIR measurements were carried out to identify the possible biomolecules liability for

capping and efficient stabilization of the metal nanoparticles. The obtained FTIR spectrum of bio-

synthesized CuNPs is shown in Fig. 7. The band at 3301 cm-1 corresponds to hydroxyl groups of

alcohols, asymmetric stretching of C–H, –C–N groups and the secondary amines are located at

wavenumber of 2918 cm-1. The presence aromatic stretching vibration of –C=C– and carboxyl

(C=O) stretching vibrations of amide linkages of proteins in addition to amide-I vibrations located

at wave number of 1639 cm-1. In the spectrum, the –C–H bending vibration of alkanes band and –

C–O stretching mode bands are located at 1470 cm-1 and 1363 cm-1, respectively [29, 30]. The

differences in the peak locations indicate that the proteins obligation for the synthesis of CuNPs is

diverse. These protein molecules act as surface coating molecules that keep away from the internal

agglomeration of the particles [31]. The small variations in the peak position from original bonding

indicate that the some of the metabolites such as tannins, flavonoids, alkaloids, and carotenoids

are abundant in the bud extract that can produce the CuNPs [32].

3.6. Antimicrobial activity

In general, smaller nanoparticles with higher surface area interact more with bacteria

compare to bigger particles thereby furnishing higher antibacterial activity. Copper used as an

antimicrobial agent for decades has revealed a strong antimicrobial activity and was able to

decrease the microorganism concentration by 99.9%. Bio-synthesized CuNPs have shown

prominent bactericidal activity against Staphylococcus spp., Escherichia coli, Pseudomonas spp.

8
and Bacillus spp. test pathogens. The variations of zone of inhibitions at different pathogenic

bacteria and fungi as a function of bio-copper volumes are depicted in Table 1. Anti-bacterial

studies elucidates that the Bacillus spp. is more effective bacterial activity and it has been

eliminated potentially with a zone of inhibition of 8 mm at 16 µl of CuNPs volume. The

bactericidal property of CuNPs is mainly due to the release of copper cations (Cu +) and these Cu+

ions are attach to the bacterial cell wall due to electrostatic attraction. Moreover, the metal ions are

not only interacting with the surface of a membrane but can also penetrate inside the bacteria [33].

Antifungal properties of bio-CuNPs were also studied against Aspergillus niger, Aspergillus flavus

and Penicillium spp. fungal test pathogens. These fungal organisms are found to affect in certain

fruits and vegetables by forming the mold. Among all the tested fungal organisms, CuNPs

exhibited pronounced fungicidal activity against Penicillium spp. with a zone of inhibition of 6

mm at 16 µl of CuNPs volume (Table 1). Finally, the current study clearly indicates that the S.

aromaticum bud extract mediated CuNPs exhibited the excellent antimicrobial activity.

4. Conclusions

In conclusion, copper nanoparticles were synthesized using Syzygium aromaticum bud

extract at room temperature (30oC) through an easy, rapid and eco-friendly pathway. The bio-

reduction of aqueous Cu+ ions by the bud extract has been demonstrated. The phytoconstituents

present in the S. aromaticum bud extract were responsible for the reduction and stabilization of

CuNPs. The shape and size of the CuNPs were analyzed from the morphological studies. The

elemental analysis confirms the presence of major copper element with minor functional bio-

molecules (C, O, Cl and P) of bud extract. The antimicrobial studies of bio-reduced CuNPs

exhibited an excellent bactericidal effect against Bacillus spp. and pronounced fungicidal activity

9
against Penicillium spp. of microorganisms. As a result, the bio-synthesized CuNPs can be used

as a futuristic effective antimicrobial agent in biomedical field.

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14
Table caption:

Table 1 The variation of zone of inhibitions at different bacterial and fungal pathogens as a

function of bio-CuNPs volumes.

Figure captions:

Fig. 1. Schematic synthesis procedure of bio-CuNPs using Syzygium aromaticum bud extract.

Fig. 2. X-ray diffraction profile of green synthesized CuNPs using S. aromaticum bud extract.

Fig. 3. FESEM images of biosynthesized CuNPs at different magnifications.

Fig. 4. (a, b) TEM images at different magnifications, (c) HR-TEM and (d) SAED pattern of

copper nanoparticles.

Fig. 5. (a) EDS spectrum and (b) Zeta potential analysis of bio-synthesized copper nanoparticles.

Fig. 6. UV-Vis absorbance spectra of S. aromaticum bud extract and bio-synthesized CuNPs

(Inset: Sample solutions of S. aromaticum bud extract and bio-CuNPs).

Fig. 7. FTIR spectrum CuNPs using S. aromaticum bud extract.

15
Figures

Fig. 1. Schematic synthesis procedure of bio-CuNPs using Syzygium aromaticum bud extract.

(111)
Intensity (a.u)

(200)

(220)

20 30 40 50 60 70 80
2degree

Fig. 2. X-ray diffraction profile of green synthesized CuNPs using S. aromaticum bud extract.

16
Fig. 3. FESEM images of biosynthesized CuNPs at different magnifications.

(a) (b)

(c) (d)

Fig. 4. (a,b) TEM images at different magnifications, (c) HR-TEM and (d) SAED pattern of copper
nanoparticles.

17
 (a)

(b)

Fig. 5. (a) EDS spectrum and (b) Zeta potential analysis of bio-synthesized copper nanoparticles.

18
 Absorbance (a.u)

CuNPs
Bud extract

200 300 400 500 600 700 800 900

Wavelength (nm)

Fig. 6. UV-Vis absorbance spectra of S. aromaticum bud extract and bio-synthesized CuNPs
(Inset: Sample solutions of S. aromaticum bud extract and bio-CuNPs).

1363
1470
2918
T%

1639
3301

4000 3500 3000 2500 2000 1500 1000

-1
Wavenumber (cm )

Fig. 7. FTIR spectrum CuNPs using S. aromaticum bud extract.

19
Table

Table 1 The variation of zone of inhibitions at different bacterial and fungal pathogens as a
function of bio-CuNPs volumes.
Zone of inhibitions (±1.0 mm)
CuNPs
Bacterial pathogens Fungal pathogens
volume
(µl) Staphylococcus E. Pseudomonas Bacillus A. A. Penicillium
spp. coli spp. spp. flavus niger spp.
4 3 4 5 6 3 4 4
8 4 5 6 7 3 4 5
12 5 5 6 8 4 5 6
16 5 6 7 8 5 5 6

20