ISSN: 2320-5407 Int. J. Adv. Res.

4(8), 1563-1568

Journal Homepage: -www.journalijar.com

Article DOI:

Article DOI:10.21474/IJAR01/1362
DOI URL: http://dx.doi.org/10.21474/IJAR01/1362

RESEARCH ARTICLE

GREEN SYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES.

Gonsalves Chrislyn1, Salanke Prachi2, Shetty Sangeeta4* and Sundarrajan Priya3.

Department of Life Science and Biochemistry, St. Xavier’s College-Autonomous, Mumbai.
……………………………………………………………………………………………………....
Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History Nanoparticles have altered properties because of their size and result
of which they have found enormous applications. Silver nanoparticles
Received: 12 June 2016 have been known to have antimicrobial properties. Biological
Final Accepted: 16 July 2016 synthesis of nanomaterials are not only ecofriendly but also the
Published: August 2016
nanoparticles synthesized have superior properties than those
Key words:- synthesized using physical and chemical methods. In the present
Green synthesis, Silver nanoparticles, study, silver nanoparticles (AgNPs) were synthesized using various
Antimicrobial activity, Disinfection of plant extracts like fern, jackfruit, and onion. The effect of varying pH
water. of these extracts on the synthesis of AgNPs was studied, and alkaline
medium was found to decrease the time taken for synthesis. The
synthesized AgNPs were found to have absorption maxima close to
420 nm, and mean size less than 50 nm. The antimicrobial activity of
AgNPs was checked against pathogens like, E. coli, S. aureus, S.
pyogenes, S. typhi, S. paratyphi A, S. paratyphi B, Shigella and
Proteus. The MIC of these AgNPs against E. coli and S. aureus was
determined. It was found that these AgNPs when adsorbed on to
activated carbon could be used to disinfect water contaminated with E.
coli.
Copy Right, IJAR, 2016,. All rights reserved.
……………………………………………………………………………………………………....
Introduction:-
Nanotechnology, is a modern technology in science that deals with the synthesis, design, and application of
nanomaterials by controlling their shape and size. Nanoparticles are defined as discrete objects having all of their
three Cartesian dimensions in the nano range i.e. 1-100 nm. The properties of these nanomaterials are different than
those of their bulk materials due to their higher surface to volume ratio. Hence, the nanomaterials are used in various
applications due to their improved properties. Nanotechnology finds its application in the fields of electronics, food
industry, packaging industry, biomedical devices, drug delivery systems, medical diagnosis, biological tissue
regeneration, space industry, optical devices, water purification, environment monitoring, and many more [Hirsch et
al., 2013] [Koria et al., 2011] [Wang et al., 2011].

Significant amount of research is being done on the various ways to synthesize silver nanoparticles, like physical,
chemical and biological/green methods. Green methods have gained more importance over the years as it is more
eco-friendly than the other two methods. It does not make use of toxic reducing agents that are employed in
chemical methods and the energy input is comparatively much less than that used during physical methods of
nanoparticle synthesis [Iravani et al., 2014].

Corresponding Author:- Shetty Sangeeta

There has been equal contribution of 1 and 2 towards this paper.
Address:-Department of Life Science and Biochemistry, St. Xavier’s College-Autonomous, Mumbai. 1563
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Since AgNPs have antimicrobial activity, they are used to coat various medico-surgical instruments like catheters,
ventilators and surgical implants like artificial joints, pacemakers, etc. to prevent colonization of bacteria [Furno et
al., 2004]. AgNPs have been impregnated on the dressings and bandages to prevent infections in the wounds. The
antimicrobial property is also made use of in water disinfection [Hameed et al., 2013].

The aim of this study was to synthesize silver nanoparticles by the green synthesis method using Fern, Jackfruit and
Onion extracts and to check for its antimicrobial activity against pathogenic microorganisms.

Materials and methods:-

Synthesis of Silver Nanoparticles:-
Fern (Nephrolepis exaltata), Jackfruit (Artocarpus heterophyllus), and Onion (Alium cepa) were obtained from local
market. 1% (w/v) extract of Fern leaves and 10% extracts (w/v) of sundried Jackfruit pulp and onion bulb in
deionised water (DIW) were prepared by boiling for two minutes. After cooling, the extracts were centrifuged at
8,000 rpm for 10 minutes to separate out particulate matter. To these extracts Silver Nitrate solution was added to
make final concentration of 10 mM. The mixture was incubated in the presence of sunlight until a stable colour
change was obtained.

AgNPs were also synthesized using chemical method to compare the two methods of nanoparticle synthesis, i.e.
green method and chemical method. 50 ml of 1 mM AgNO3 was boiled and 5 ml of 10% trisodium citrate was
added drop wise. The mixture was constantly shaken till a stable colour was observed [Rashid et al., 2013].

Effect of pH on the synthesis of Silver Nanoparticle:-

The extracts prepared using the aforementioned green protocol were found to have pH of 4.5. The pH of the extracts
was adjusted to 7.0 and 10.0 using 10N NaOH. To these extracts at varying pH (4.5, 7.0, and 10.0) silver nitrate
solution was added, and the mixture was incubated in the presence of sunlight until a stable colour change was
obtained. [Darroudi et al., 2010]

Characterization of synthesized Silver nanoparticles:-

Once a stable colour change was obtained, the solution was first centrifuged at 5000 rpm for 10 minutes to remove
any plant preciptate. The supernatant obtained was further centrifuged at 12000 rpm for 30 minutes. The pellet
obtained was washed thrice with DIW to remove residual plant extract. The final pellet obtained was resuspended in
DIW and was used for characterization.

Spectrophotometric analysis:-
A spectrum scan from 300-800 nm was carried out using UV-Visible spectrophotometer to determine the absorption
maxima of synthesized Silver nanoparticles. [Manivasagan et al., 2013]

Nanoparticle tracking system:-

The synthesized AgNPs were characterized using nanoparticle tracking system (Nanosight LM20) to determine the
size of the nanoparticles. [Filipe et al., 2010]

Antimicrobial studies of the isolated AgNPs:-

Agar cup method:-
Antimicrobial activity of the synthesized AgNPs was tested against pathogens like, E. coli, S. aureus, S. pyogenes, S.
typhi, S. paratyphi A, S. paratyphi B, Shigella and Proteus using agar cup method. Sterile DIW was used as a
control. The plates were incubated overnight at 370C. [Elbeshehy et al., 2015]

Broth dilution method:-

The minimum inhibitory (MIC) of Ag-NPs was determined by two-fold broth dilution method using Nutrient Broth
(NB). AgNPs were dried overnight at 600C to make the stock in sterile DIW from which a two-fold serial dilutions
were made in NB. Overnight grown culture of approximately 106 cfu/ml E. coli and S. aureus were inoculated in the
tubes containing NB alone (Positive control), NB containing AgNPs at a final concentration ranging from 1.5 to 200
µg/ml. Whereas, sterile uninoculated NB was used as a negative control. The tubes were incubated overnight at
370C. The MIC values corresponded to the AgNPs dose that inhibited bacterial growth (when compared to the
positive control). [Elbeshehy et al., 2015]

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Water disinfection:-
Activated carbon (AC) was incubated at 110˚C, for three hours. 1.6 g of this activated carbon and 10 ml of AgNPs
(2mg/ml) (obtained from jackfruit and onion at pH 7.0) were mixed and kept on a magnetic stirrer overnight, to
bring about adsorption of the nanoparticles onto the activated carbon under aseptic conditions [Karimi et al., 2012].

One ml of the activated charcoal adsorbed with nanoparticles (AC-AgNPs) was centrifuged at 7,000 rpm for 5
minutes to separate the non-adsorbed SNPs from the AC-AgNPs. The pellet obtained was washed twice with sterile
deionized water to remove any loosely adsorbed AgNPs. The pellet obtained after washing was resuspended in
sterile distilled water.

E. coli cells of density 106 CFU/ml suspended in sterile distilled water (19 ml), were exposed to 1 ml of the above
AC-AgNPs. 106 CFU/ml of E. coli suspended in sterile distilled water was exposed to 1 ml of activated carbon, to
serve as a charcoal control. Whereas, 106 CFU/ml of E. coli suspended in sterile distilled water was used as a culture
control. At different time intervals (0, 2, 4, 8, and 12 minutes), aliquots were filtered using sterile Whattman filter
paper. The filtrate was surface spread on sterile nutrient agar plates, in duplicates and the plates were incubated
overnight at 37˚C.

Results and discussions:-

Synthesis of Silver Nanoparticles:-
The extracts of Fern (Nephrolepis exaltata), Jackfruit (Artocarpus heterophyllus), and Onion (Alium cepa) were
used for synthesis of silver nanoparticles. On addition of silver nitrate a colour change to brown was observed for
all the three extracts used. A change in colour of the mixture, indicates the reduction of silver ions (Ag +) to atomic
silver (Ag0) which coalesce to form SNPs [Saxena et al., 2010], [Krishnaraj et al., 2010]. The time required for the
colour change varied for each biological extract, ranging from 22, 56 and 57 minutes for Onion, Fern and Jackfruit
extract respectively.

Effect of pH on the synthesis of Silver Nanoparticle:-

Effect of pH on the synthesis of SNPs was studied. It was found that at pH 10 the time taken for sythesis of the
SNPs was the least for all three extracts (Table 1). This could be because alkaline conditions might enhance the
efficiency of the reducing and stabilizing agents present in the extracts, thus resulting in faster synthesis [Mostafa et
al., 2014].
Table 1:- Effect of varying pH of the extracts on the rate of synthesis of AgNPs
Extract Fern Jackfruit Onion
pH 4.5 7.0 10.0 4.5 7.0 10.0 4.5 7.0 10.0
Time taken 56 36 29 57 57 31 22 11 4
for
synthesis
(min)

Characterization of synthesized Silver nanoparticles:-

Spectrophotometric analysis:-
The absorption maxima of the AgNPs synthesized using green method with varying pH and chemical method were
found to be in the range of 418 to 427 (Table 2). This is close to the characteristic absorption maxima of the AgNPs,
i.e. 420 nm [Manivasagan et al., 2013].
Table 2:-Spectrophotometric analysis of the synthesized AgNPs
Extract Fern Jackfruit Onion Chemical
synthesis
pH 4.5 7.0 10.0 4.5 7.0 10.0 4.5 7.0 10.0
Absorption 421 424 427 423 427 418 419 425 421 418
maxima (nm)

Nanoparticle tracking system::-

Nanoparticle tracking system (Nanosight LM20, Manuf*) was used to determine the size of the nanoparticles. The
synthesized AgNPs were found to have mean sizes ranging from 22-44 nm (Table 3). The AgNPs showed a large
standard deviation which proves that AgNPs are polydispersed. This is consistent with the size distribution of

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AgNPs synthesized using green method without any external addition of capping or stabilizing agents [Raut et al.,
2010].
Table 3: Representative size distribution obtained from the Nanoparticle Tracking System of the synthesized
AgNPs at different pH values.
Extract Fern Jackfruit Onion Chemical
synthesis
pH 4.5 7.0 10.0 4.5 7.0 10.0 4.5 7.0 10.0
Size Mean 28 31 33 44 36 26 37 40 36 22
(nm) Mode 19 22 22 30 26 17 26 33 29 16
Standard 17 17 25 32 25 20 23 27 26 11
deviation

Antimicrobial studies:-
Agar cup method:-
The AgNPs synthesized using green method showed antimicrobial activity against various microorganisms which
were tested in this study (Table 4). However, the chemically synthesized AgNPs showed no antimicrobial activity
against any of the cultures tested. This could be attributed to the shape of the chemically synthesized AgNPs as
compared to the green synthesis method [Pal et al., 2007].
Table 4:-Qualitative agar cup assay for determination of antimicrobial activity of synthesized AgNPs.
Culture Fern (pH 4.5, 7.0, Jackfruit (pH 4.5, Onion (pH 4.5, 7.0, Chemical
10.0) 7.0, 10.0) 10.0) synthesis
E. coli + + + -
S. typhi - - + -
S. paratyphi A - - - -
S. paratyphi B - - + -
Proteus + + - -
Shigella + + + -
S. aureus + + + -
S. pyogenes + + + -

Broth dilution method:-

The minimum inhibitory (MIC) of Ag-NPs was determined by two-fold broth dilution method using Nutrient Broth
(NB). It was observed that the MIC of synthesized AgNPs, for S. aureus is higher than that for E. coli (Table 5).
Since S. aureus is a Gram positive microorganism containing a thicker peptidoglycan layer in its cell wall than that
in E. coli, probably AgNPs are not able to cross this barrier as efficiently as they can cross the thinner peptidoglycan
layer in E. coli.
Table 5:-Minimum Inhibitory Concentrations of synthesized AgNPs
Extract Fern Jackfruit Onion
pH 4.5 7.0 10.0 4.5 7.0 10.0 4.5 7.0 10.0
MIC E. coli - 20 20 25 25 - 25 25 50
(µg/ml) S. aureus - 20 40 100 100 - 50 50 -

Water disinfection:-
To determine the efficacy of AgNps for water disinfection, AgNPs were adsorbed on to Activated carbon under
aseptic conditions. The AC control itself had a 10-fold reduction in cell count as AC is known to adsorb organic
matter. However, AgNP-AC showed a further 1000-fold reduction in the cell count of the contaminated water
(Table 6). Therefore, AgNPs adsorbed onto AC together showed a 10,000-fold reduction in the cell count. Zero cell
count after zero-minute exposure could be attributed to the time lag during filtration of the aliquots during which
AgNP-AC could have acted for extended time. Hence, this method can be used to disinfect water contaminated with
sewage sample, having microbial load as high as 106 cfu/ml.

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Table 6:-Results for water disinfection assay.

Time (min) CFU/ml
Culture control AC control SNP-AC Jackfruit SNP-AC Onion
0 8500 1115 0 0
2 28000 845 0 0
4 10000 2155 0 0
8 28200 2775 0 0
12 15000 1445 0 0

Figure 1:-Fern and Onion extracts before adding AgNO3

Figure 2:-Fern and Onion extracts after reduction of silver.

Conclusion:-
The green synthesis of silver nanoparticles was carried out by using Fern, Jackfruit and Onion extracts. The effect of
pH on the synthesis of AgNPs was studied, and it was observed that in an alkaline medium the synthesis of AgNPs
was faster. The size of the synthesized AgNPs was determined using a nanoparticle tracking system, and the
synthesized AgNPs were found to have a mean size of 22-44 nm. Antimicrobial studies were carried out using the
AgNPs synthesized by biological and chemical synthesis. Where the chemically synthesized AgNPs did not show
antimicrobial activity against any of the eight cultures used, the biologically synthesized SNPs showed antimicrobial
activity against some of the eight cultures used. The MIC of the synthesized AgNPs against E. coli and S. aureus
was determined, using the broth dilution method. The antimicrobial property of these AgNPs was used to disinfect
water containing a known number E. coli CFU/ml. We can hence conclude that AgNPs adsorbed to AC can be used
effectivelyin water purifiers to reduce microbial load of contaminated water.

Acknowledgment:-
The authors are thankful to Dr. Rajesh Raut from the Institute of Science, Mumbai; for helping with the
characterization of nanoparticles using the Nanoparticles Tracking System.

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