International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
Enhance Antioxidant Property of Silver Nanoparticle
Decorated Graphene Oxide Nanocomposite
Chetan Ramesh Mahajan, Lalit Bhanudas Joshi, Vijay Raman Chaudhari*
Department of Nanoscience and Technology, University Institute of Chemical Technology,
North Maharashtra University, Umavi Nagar, Jalgaon 425001, Maharashtra, India
*Corresponding author
Vijay Raman Chaudhari, Department of Nanoscience and Technology, University Institute of Chemical
Technology,
North Maharashtra University, Umavi Nagar, Jalgaon 425001, Maharashtra, India
Abstract-Report
discussed
the
preparation
of
radicals inside human body that responsible for the
graphene oxide-silver (GO-AgNPs) nano-composite
damage of healthy cells, also known as oxidative
by eco-friendly approach and their antioxidant
stress [3-5]. Silver is efficient bactericidal metal
properties. AgNPs were prepared by clove extract
because it is non-toxic to animal cells and highly toxic
assisted reduction method and deposited on GO
to bacteria [5-6]. Silver nanoparticles (AgNPs) are
solution mixing. Characterization by UV-Visible,
most commonly used due to its antioxidant and
Fourier Transform Infrared (FTIR) spectroscopy, X-
antimicrobial properties [7]. In addition to their
ray diffraction (XRD) and field emission microscope
medical uses, AgNPs are also used in clothing, food
(FE-SEM) suggests anchoring of AgNPs on GO sheet.
industry, paints, electronics and other fields [8-11].
Further, the antioxidant activities of AgNPs, GO and
Graphene, a single layer of sp2-hybridized carbon
GO-AgNPs were evaluated using DPPH assay by
atoms arranged in a honeycomb two dimensional (2-D)
comparing with ascorbic acid as standard. GO-
crystal lattice, possess remarkable physico–chemical
AgNPs shows higher antioxidant properties compared
properties, including a high Young’s modulus, high
to GO and AgNPs which is attributed to the
fracture strength, large specific surface area with
synergistic effect of Ag and GO in nano-composites.
provision to tune the surface chemistry, not-toxic and
Keywords: Silver nanoparticles, Graphene oxide,
biocompatible
GO-AgNPs nano-composites, Antioxidant activity.
decorated with oxygen-containing functional groups
[12-13].
GO
is
graphene
sheet
such as hydroxyl and epoxy groups on the basal planes
I. INTRODUCTION
Antioxidants property plays an essential role as health
shielding factor. Antioxidant behavior minimizes the
risk for cancer and heart related disease [1]. Naturally
antioxidants are widely occurring in whole grains,
fruits and vegetables with active constituents of
vitamin C, vitamin E, carotenes, phenolic acids etc [24]. Antioxidant reduces the rate of oxidation reactions.
Oxidation is a chemical reaction or process through
which electrons are transferred between molecules by
an oxidizing agent. Oxidation process generates free
and carbonyl and carboxylic groups at the edges.
Graphene oxide (GO) also shows bactericidal activity
through various possible mechanisms viz. damage of
cell membrane due to interaction with of sharp edges
of GO, increase in oxidative stress because of its
oxidative nature, releasing reactive oxygen species
(ROS) which oxidizes lipids [14-18]. GO exhibits
significant antioxidant activity due to the hydroxyl and
superoxide moieties which also acts as radical
scavenger, and can protect a variety of biomolecular
target molecules from oxidation [19]
ISSN: 2231-5381
http://www.ijettjournal.org
Page 546
International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
Therefore,
we
aimed
to
combine
these
two
Silver nanoparticles were prepared by using biological
components through composites formation so as to
source, clove extract as reducing agent. Clove extract
achieve higher antioxidant property. In present study
was prepared by soaking 10 g of clove in 100 ml
AgNPs were prepared using biological reducing
doubled distilled water for 24 h. Red brown color
agents ie. clove extract and deposited on GO sheet by
solution was obtained due to oozing out of active
mixing the dispersion of AgNPs into GO suspension.
constituents. Filtrate of extract was used as reducing
Antioxidant property of GO-AgNPs were observed by
agent for silver nanoparticle synthesis. 1% freshly
performing assay α,α- Diphenyl-β-Picrylhydrazyl
prepared suspension of clove extract was added into
(DPPH) radical scavenging method. DPPH is widely
freshly prepared AgNO3 and kept aside for 3 h for
used to test the ability of compounds to act as free
complete reduction. Color of solution was slowly
radical scavengers or hydrogen donors and to evaluate
changes from light yellowish to brown.
antioxidant activity.
II. EXPERIMENTAL
A. MATERIAL AND METHODS:
Graphite powder was procured from Loba chemicals
Pvt. Ltd. Potassium paramagnet was purchased from
D. Preparation of GO-Ag Nano-composite:
Qualigans fine Chemicals. Sulphuric acid, hydrogen
GO-AgNPs nanocomposite was prepared by ex-situ
peroxide, ether, hydrochloric acid and silver nitrate,
method. Aqueous disperson of GO was prepared by
ascorbic acid, DPPH were purchased from Merck
dispersing 35mg of GO in 50ml double distilled water
Specialties Private limited.
through sonication. Freshly prepared AgNPs was
B. Preparation of Graphene Oxide:
added drop-wise to GO dispersion using pressure
Synthesis of graphene oxide was done by improved
equalizing funnel with continuous stirring. After
hummer method [20]. The mixture of concentrated
complete addition sol was stored at room temperature.
H2SO4 and H3PO4 in 9: 1 ratio which provides strong
E. DPPH radical scavenging activity:
acidic condition was added in reaction assembly to a
The scavenging of (DPPH) radical by nanoparticles
mixture of graphite powder (3gm). KmnO4 (6 time wt
and nanocomposites was analysed by modifying the
equivalent to Graphite powder) was added slowly to
method of Shimada et al. [21]. A 0.8 ml of GO-AgNPs
the above mixture and heated to 50 °C with constant
(1420
stirring for 12 h. The reaction was cooled to room
prepared DPPH solution (0.2 mM in Methanol) were
temperature and poured onto ice (400 mL) containing
added to the phosphate buffer solution (PBS, 7.3 pH)
30% H2O2. Suspension was filtered through polyester
and allowed to react for 30 minutes in dark. Blank
fibre cloth and filtrate was centrifuged at 4000 rpm.
samples contained PBS (pH 7.3) with ascorbic acid as
The supernatant solution was decanted and remaining
standard. The scavenged DPPH was then monitored
solid material was washed in succession with copious
spectrophotometrically by measuring the absorbance
amount of double distilled water, 30% HCl, ethanol
at 517 nm. The % scavenging ability was determined
(200ml). Finally, it was coagulated with 200 mL of
as follows;
g/mL) nano-composites and 1 ml of freshly
)
ether and obtained solid material was vacuum-dried
overnight at room temperature.
×100
C. Preparation of Silver Nanoparticle:
ISSN: 2231-5381
http://www.ijettjournal.org
Page 547
International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
III. CHARACTERIZATION
Synthesis of AgNPs and GO-AgNPs nano-composites
were checked by recording UV-Visible (UV-Vis)
spectra using spectrophotometer (Cary60 UV-VIS
spectrophotometer) on respective sol. Crystallographic
phase of obtained products were determined using Xray powder diffractometer (XRD), Bruker, D8
ADVANCE (Bruker Corporation, Tokyo, Japan)
having monochromatic CuKα radiation (λ = 1.5406 Å)
at 40 kV and 40 mA. Scan rate was 50/ min between
the angles 50-800. Surface morphology was observed
using Field Emission Scanning Electron Microscope
(FE-SEM), HITACHI S-4800, operated at 5 to 15 Kv.
Suspension was drop casted onto carbon tape and
dried at room temperature. Energy Dispersive X-Ray
Spectroscopy (EDS) attached to FE-SEM was used for
element analysis.
Crystallographic nature and purity of prepared GO,
AgNPs and GO-Ag nanocomposite were investigated
by XRD and shown in Fig 2. In case of GO diffraction
line was observed at 8.4o due to the (001) plane.
Particle size and zeta potential of AgNPs observed by
particle size analyzer (Malvern Instruments, UK).
Chemical nature of prepared composite material was
investigated using Fourier Transform Infra-red (FTIR)
spectroscopy
Fig 1. UV-Visible spectra of GO, AgNPs and GO-AgNPs nanocomposites
using
Shimadzu-8400
spectrometer
within the frequency range 4000 to 400 cm-1. Reported
spectra are averaged of 100 scan with the resolution of
2 cm-1.
Determined d-spacing (1.04 nm) indicates the
exfoliated graphitic sheet due to oxidative treatment
[20, 23-24]. In case of AgNPs and GO-Ag, diffraction
lines are observed at 37.5o, 43.7 o, 63.5oand 77.1o due
to (111), (200), (220) and (311) planes of cubic phase
of metallic Ag (JCPDS No. 04-0783), respectively.
Disappearance of GO diffraction line for nanocomposites is attributed to the increase in exfoliation
IV. RESULTS AND DISCUSSION:
Figure 1 depicts the UV- visible spectra of GO,
of GO sheets due to encapsulated or sandwiched
AgNPs in between sheets.
AgNPs and GO-AgNPs. GO shows absorbance band
ca.230 nm along with small hump at 310 nm. Former
band is due to the - * transition of C=C skeleton and
hump is attributed to the n- * transition of C=O
functionalities on GO. AgNPs shows absorbance band
at 434 nm, a characteristics to the surface Plasmon
resonance for silver nanoparticles [22]. GO-AgNPs
suspension
also
exhibits
the
well
determined
absorbance band at 434 nm and suggest the presence
of AgNPs along with GO.
Fig 2. X-ray diffractogram of AgNPs, GO and GO-AgNPs nanocomposites.
ISSN: 2231-5381
http://www.ijettjournal.org
Page 548
International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
FTIR spectrum of AgNPs shows vibration band at
-1
AgNPs stabilized through the formation of protein
-1
3470 cm and 1637 cm due to O-H and COOH group, layer on the surface of nanoparticle and play important
respectively. These bands arise due to presence of
role for binding with oxygen functionality of GO [26].
volatile oils Eugenol and Flavonoid originated from
clove extract. Additional bands at 1383 cm-1 and 1071
cm-1 represents germinal methyl and ether linkage
respectively [25]. In case of GO, vibration bands were
observed at 3400 cm-1, 1715 cm-1, 1510 cm-1 and
1145 cm-1 corresponds to the O-H, C=O
of
carboxylic acid and ester functionalities [20,23] .
While for GO-AgNPs, O-H vibration band get
disappeared.
Moreover,
vibration band
due
Fig 4 . FE-SEM images of GO and GO-AgNPs nano-composites.
to
carboxylic acid became sharp with decrease in height
than C=C aromatic carbon (positioned at 1510 cm-1).
Antioxidant property:
Stretching frequency of carboxylic ester also diapered.
The antioxidant activity of AgNPs, GO, GO-AgNPs
All these modifications in FTIR spectrum of Ag-GO
was evaluated using DPPH scavenging activity. The
suggests the interaction between Ag and oxygenated
DPPH assay method is based on the reduction of
functionalities of GO and acts as an anchoring sites for
DPPH, a stable free radical [27]. The free radical
AgNPs.
DPPH with an odd electron gives a maximum
absorption
at
517nm
(purple
colour).
When
Antioxidants react with DPPH, it becomes paired off
and reduced to the DPPHH with decrease in
absorbance due to the de-colorization [28-29]. This
test has been the most accepted model for evaluating
the free radical scavenging activity of any new drug.
As shown in Fig. 5 significant difference in the
antioxidant activity was observed. AgNPs, GO and
GO-AGNPs shows 74.81 ± 2 %, 48.66 ± 2 % and
Fig 3. FTIR spectra of GO and GO-AgNPs nano-composites.
84.76 ± 2 % DPPH scavenging activity, respectively.
Result shows prepared GO-AgNPs composites gives
FE-SEM images of GO and GO-AgNPs are depicted
in Fig 4. Sheet like structure with wrinkled surface is
observed for GO. An approximate sheet dimension is
about few micrometers. AgNPs seems to be uniformly
distributed and anchored on GO sheet. Oxygenated
functionalities on GO may act as anchoring sites for
the AgNPs deposition. Fine size stability of AgNPs
may be due to the formation of protecting layer of
highest antioxidant activity which may be due to
synergistic effect of AgNPs and GO. Antioxidant
activity of tested compound was compared with
standard
ie.
Ascorbic
acid
with
different
concentrations (as shown in supporting information)
and it was found to be equivalent to 70 M standard
ascorbic acid.
active constituents present in clove ie. Eugenol,
flavonoids eugenin, kaempferol, rhamnetin etc. Faria
et al. also reported that biologically synthesized
ISSN: 2231-5381
http://www.ijettjournal.org
Page 549
International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
[5]
[6]
[7]
[8]
[9]
Fig 5. % Antioxidant activity of AgNPs, GO and GO-AgNPs nanocomposites.
[10]
V. CONCLUSION
GO-AgNPs nano-composites were prepared through
[11]
eco friendly and ex-situ reduction of AgNO3 using
clove extract followed by solution mixing of AgNPs
[12]
and GO dispersion. Presence of oxygen functionalities
on GO surface helps to anchored AgNPs. Antioxidant
assay was done on GO-AgNPs nano-composite with
DPPH and compared with Ascorbic acid as standard.
[13]
[14]
Results show enhanced antioxidant property of GOAgNPs due to synergetic effect on addition of two
[15]
antioxidant materials.
ACKNOWLEDGEMENT
[16]
Authors thanks to Dr. B.L Chaudhari and J.S. Pardeshi
(Dept. of Microbiology, SOLS, NMU Jalgaon) for
[17]
Antioxidant study. Authors also thanks to University
Grant Commission (UGC) New Delhi for financial
[18]
support under BSR scheme for newly recruited faculty.
CRM thanks to TEQIP program for fellowship.
[20]
REFERENCES
[1]
[2]
[3]
[4]
A.V.Badarinath, K. Mallikarjuna RAo, C.Madhu Sudhana
Chetty, S. Ramkanth, T.V.S Rajan, K.Gnanaprakash, A
Review on In-vitro Antioxidant Methods: Comparisions,
Correlations and Considerations, Int.J. PharmTech Res, 2
(2010) 1276-1285.
C.S. Tailor, A. Goyal, Antioxidant Activity by DPPH
Radical Scavenging Method of Ageratum conyzoides
Linn. Leaves, American Journal of Ethnomedicine, 1
(2014) 244-249.
A.Prakash, F.Rigelhof, E.Miller, Antioxidant Activity,
9000 Plymouth Ave North, Minneapolis, Minnesota
55427.
B.N Ames, M.K. Shigenega, T.M. Hagen, Oxidants and
the degenerative diseases of ageing Proc Nati Acad Sci ,
90(1993)7915 –7922.
ISSN: 2231-5381
[19]
[21]
[22]
[23]
[24]
R Ghosh, A Study on Antioxidant Properties of different
bioactive compounds, Journal of Drug Delivery &
Therapeutics; 4 (2014), 105-115.
U. Klueh, V. Wagner, S. Kelly, A. Johnson, J.D. Bryers,
Efficacy of silver-coated fabric to prevent bacterial
colonization and subsequent device-based biofilm
formation, J. Biomed. Mater. Res. 53 (2000) 621–631.
C. M-Jones, E.M.V. Hoek, A review of the antibacterial
effects of silver nanomaterials and potential implications
for human health and the environment, J. Nanopart. Res.,
12 (2010) 1531–1551.
K.M. Abou El-nour, A. Eftaiha, A. Al-Warthan, R.A.
Ammar, Synthesis and applications of silver nanoparticles,
Arab. J. Chem, 3 (2010) 135–140.
M.S. Cohen, J.M. Stern, A.J. Vanni, R.S. Kelley, E.
Baumgart, D. Field, J.A. Libertino, I.C. Summerhayes, In
vitro analysis of a nanocrystalline silver-coated surgical
mesh, Surg. Infect. (Larchmt.) 8 (2007) 397–403.
Q. Li, S. Mahendra, D. Lyon, L. Brunet, M. Liga,
Antimicrobial nanomaterials for water disinfection and
microbial control: potential applications and implications,
Water Res. 42 (2000) 4591–4602.
N. Vigneshwaran, A.A. Kathe, P.V. Varadarajan, R.P.
Nachane, R.H. Balasubramanya, Functional finishing of
cotton fabrics using silver nanoparticles, J. Nanosci.
Nanotechnol. 7 (2007) 1893–1897.
Y. Zhu, S. Murali, W. Cai , X. Li , J. Won Suk , J. R.
Potts, R. S. Ruoff, Graphene and Graphene Oxide:
Synthesis, Properties, and Applications, Adv. Mater. 22
(2010) 3906–3924.
S. Goenka, V. Sant, S. Sant, Graphene-based
nanomaterials for drug delivery and tissue engineering, J.
Controlled Release, 17 (2014) 375–388.
F. Perreault, A.F. de Faria, M .Elimelech.
Environmental
Applications
of
Graphene-Based
Nanomaterials. Chem. Soc. Rev. 44 (2015) 5861-5896.
J.Chen, H. Peng , X. Wang, F. Shao, Z.Yuan, H. Han.
Graphene oxide exhibits broad-spectrum antimicrobial
activity against bacterial phytopathogens and fungal
conidia by intertwining and membrane perturbation.
Nanoscale, 6 (2014)1879–1889.
O. Akhavan, Ghaderi E. Toxicity of graphene and
graphene oxide nanowalls against bacteria. ACS Nano 4
(2010) 5731–5736.
Y. Tu, M. Lv, P Xiu, T. Huynh, M. Zhang, M. Castelli,
Z.Liu, Q.Huang, C.Fan, H. Fang. Destructive extraction of
phospholipids from escherichia coli membranes by
graphene nanosheets. Nat. Nanotechnol. 8 (2013) 594–601.
F-O Perreault, A.F de Faria, S. Nejati, M. Elimelech,
Antimicrobial properties of graphene oxide nanosheets:
why size matters. ACS Nano. 9(2015) 7226–7236.
Y Qiu, Z Wang, A C. E. Owens, I Kulaots, Y Chen, A. B.
Kanec, R H. Hurt, Antioxidant chemistry of graphenebased materials and its role in oxidation protection
technology, Nanoscale 6 (2014) 11744-117455.
D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A.
Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu, J. M.
Tour, Improved Synthesis of Graphene Oxide, ACS Nano,
4 (2010) 4806-4814.
K Shimada, K Fujikawa, K Yahara, T Nakamur,
Antioxidative properties of xanthan on the autoxidation of
soybean oil in cyclodextrin emulsion, J Agric Food Chem
40 (1992) 945–948.
M.M Wadkar, V.R Chaudhari, S.K Haram, Synthesis
and characterization of stable organosols of silver
nanoparticles by electrochemical dissolution of silver in
DMSO, J. Phys. Chem. B 110(2006) 20889-20894.
J. Chen, B. Yao, C. Li, G. Shi, J. Chen, B. Yao, C. Li, G.
Shi, An Improved Hummers method for eco-friendly
synthesis of graphene oxide, carbon, 64 (2013) 225-229.
B. Paulchamy, G. Arthi, B. D. Lignesh, A Simple
Approach to Stepwise Synthesis of Graphene Oxide
Nanomaterial, J Nanomed Nanotechnol. 6 (2015) 1-4.
http://www.ijettjournal.org
Page 550
International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
[25]
[26]
[27]
[28]
[29]
A.K. Mittal, A. Kaler, U.C. Banerjee, Free Radical
Scavenging and Antioxidant Activity of Silver
Nanoparticles Synthesized from Flower Extract of
Rhododendron dauricum, Nano Biomed. Eng. 4
(2012)118-124.
A.F. de Faria, D.S.T. Martinez, S.M.M. Meira, A.C.M.
de Moraes, A. Brandelli, A.G.S. Filho, O.L. Alves. Antiadhesion and antibacterial activity of silver nanoparticles
supported on graphene oxide sheets. Colloids and
Surfaces B: Biointerfaces; 113(2014)115-124.
T Sravani, P.M Paarakh, Antioxidant activity of
Hedychium spicatum Buch. Ham Rhizomes, Indian
journal of natural products & resources, 3(2012) 354- 358.
K.R Kirtikar, B.D. Basu, Indian medicinal plants,
International book distributors, Dehradun, (2006), 993994.
P.K Warrier, VPK Nambier, R. Kutty, Indian medicinal
plants- A compendium of 500 species, Orient longman
Ltd, Madras, Vol-I (1994) 95-97.
Fig S3: Images of % DPPH Scavenging Activity of AgNPs, GO and
GO-AgNPs nanocomposite
Figure caption:
Fig 1: UV-Visible spectra of GO, AgNPs and GO-AgNPs nanocomposites
Fig 2: X-ray diffractogram of AgNPs, GO and GO-AgNPs nanocomposites.
Fig 3: FTIR spectra of GO and GO-AgNPs nano composites.
Fig 4: FE-SEM images of GO and GO-AgNPs nano-composites.
Fig5: % Antioxidant activity of AgNPs, GO and GO-AgNPs nanocomposites.
Supporting Information:
Fig S1. % DPPH Scavenging Activity of standard Ascorbic acid
Fig S2: Particle size silver nanoparticles
ISSN: 2231-5381
http://www.ijettjournal.org
Page 551