SYNTHESIS OF SILVER NANOPARTICLES BY

CHEMICAL METHOD AND GREEN SYNTHESIS

AND TESTING ITS ANTIMICROBIAL PROPERTY

Thesis submitted by the partial fullfillment of the degree

Of
Bachelor of Technology in Biotechnology
By
CHANDRABALI SAHA
University Roll number-12615004006
Under the guidence of
Dr Soma Banerjee
Assistant Professor
Department of Biotechnology

DEPARTMENT OF BIOTECHNOLOGY
HERITAGE INSTITUTE OF TECHNOLOGY,
KOLKATA
INTRODUCTION
Nanoparticles are particles between 1 and 100 nanometres (nm) in size with a
surrounding interfacial layer. The interfacial layer is an integral part of nanoscale matter,
fundamentally affecting all of its properties. The interfacial layer typically consists of
ions, inorganic and organic molecules. Nanoparticles often possess unexpected optical
properties as they are small enough to confine their electrons and produce quantum
effects.

SIZE;-
That size range—from 1 to 100 nm—overlaps considerably with that previously
assigned to the field of colloid science—from 1 to 1,000 nm—which is sometimes
alternatively called the mesoscale. Thus, it is not uncommon to find literature that refers
to nanoparticles and colloidal particles in equal terms. The difference is essentially
semantic for particles below 100 nm in size.

PROPERTIES ;-
There are three major physical properties of nanoparticles, and all are interrelated:
(1) They are highly mobile in the free state (e.g., in the absence of some other
additional influence, a 10-nm-diameter nanosphere of silica has
a sedimentation rate under gravity of 0.01 mm/day in water);

(2) They have enormous specific surface areas (e.g., a standard teaspoon, or about
6 ml, of 10-nm-diameter silica nanospheres has more surface area than a dozen
doubles-sized tennis courts; 20 percent of all the atoms in each nanosphere will be
located at the surface);

(3) They may exhibit what are known as quantum effects. Thus, nanoparticles
have a vast range of compositions, depending on the use or the product.
MEDICAL USE OF NANOPARTICLES;-
DIFFERENT METALS WHICH ARE USUALLY USED FOR
MAKING NANOPATICLES;-

WHY WE
ARE

CHOOSING SILVER METAL FOR FORMATION OF NANO

PARTICLES?

Using silver nanoparticles for catalysis has been gaining attention in recent
years . Although the most common applications are for medicinal purposes or
antibacterial purposes , silver nanoparticles have been demonstrated to show
catalytic redox properties for dyes, benzene, carbon monoxide and likely
other compounds

DIFFERENT METHODS FOR FORMATION OF

NANOPARTICLES;-

Physical methods
Evaporation-condensation and laser ablation are the most important physical
approaches. The absence of solvent contamination in the prepared thin films and
the uniformity of NPs distribution are the advantages of physical synthesis
methods in comparison with chemical processes.

EVAPORATION;-The preparation of nanoparticles by emulsion solvent

evaporation is a very popular method. The purpose of the present study was
to clarify the mechanism by which nanoparticles of ethylcellulose (EC) and
poly(lactic acid) (PLA) are formed during the emulsion solvent evaporation
procedure. This study was mainly based on the measure of the variation of the
emulsion and nanoparticle surface charge and size during the solvent
evaporation process. From the data obtained and depending on the polymer
used (EC or PLA), two different models are proposed to explain the
nanoparticle formation. In the EC model, after shrinkage of the emulsion
droplets as the direct consequence of solvent evaporation, coalescence
occurred before stable and solvent-free nanoparticles were formed. On the
contrary, in the PLA model, no or limited coalescence was found to occur so
that the picture is that one PLA nanoparticle originated from one (or only a
few) PLA emulsion droplet after its shrinkage.

CONDENSATION

Gas condensation was the first technique used to synthesize nanocrystalline

metals and alloys. In this technique, a metallic or inorganic material is vaporized
using thermal evaporation sources such as a Joule heated refractory crucibles,
electron beam evaporation devices, in an atmosphere of 1-50 m bar. In gas
evaporation, a high residual gas pressure causes the formation of ultra fine
particles (100 nm) by gas phase collision. The ultrafiine particles are formed by
collision of evaporated atoms with residual gas molecules. Gas pressures greater
than 3 mPa (10 torr) are required. Vaporization sources may be resistive heating,
high energy electron beams, low energy electron beam and inducting heating.

LASER ABLATION METHOD

Silver NPs could be synthesized by laser ablation of metallic bulk materials in

solution . The ablation efficiency and the characteristics of produced nano-silver
particles depend upon many parameters, including the wavelength of the laser
impinging the metallic target, the duration of the laser pulses (in the femto-, pico-
and nanosecond regime), the laser fluence, the ablation time duration and the
effective liquid medium, with or without the presence of surfactants

CHEMICAL METHOD;-

CHEMICAL REDUCTION METHOD

The most common approach for synthesis of silver NPs is chemical reduction by
organic and inorganic reducing agents. In general, different reducing agents such
as sodium citrate, ascorbate, sodium borohydride (NaBH4), elemental hydrogen,
polyol process, Tollens reagent, N, N-dimethylformamide (DMF), and poly
(ethylene glycol)-block copolymers are used for reduction of silver ions (Ag+) in
aqueous or non-aqueous solutions. These reducing agents reduce Ag + and lead to
the formation of metallic silver (Ag0), which is followed by agglomeration into
oligomeric clusters. These clusters eventually lead to the formation of metallic
colloidal silver particles . It is important to use protective agents to stabilize
dispersive NPs during the course of metal nanoparticle preparation, and protect
the NPs that can be absorbed on or bind onto nanoparticle surfaces, avoiding
their agglomeration . The presence of surfactants comprising functionalities (e.g.,
thiols, amines, acids, and alcohols) for interactions with particle surfaces can
stabilize particle growth, and protect particles from sedimentation,
agglomeration, or losing their surface properties.

TOLLEN’S METHOD

A simple one-step process, Tollens method, has been used for synthesis of silver
NPs with a controlled size. This green synthesis technique involves reduction of
Ag(NH3)2+ (as Tollens reagent) by an aldehyde . In the modified Tollens procedure,
silver ions are reduced by saccharides in the presence of ammonia, yielding silver
nanoparticle films (50-200 nm), silver hydrosols (20-50 nm) and silver NPs of
different shapes. In this method, ammonia concentration and nature of the
reducing agent play an important role in controlling size and morphology of silver
NPs. It was revealed that the smallest particles were formed at the lowest
ammonia concentration. Glucose and the lowest ammonia concentration (5 mM)
resulted in the smallest average particle size of 57 nm with an intense maximum
of surface plasmon absorbance at 420 nm. Moreover, increase in NH 3 from 0.005
M to 0.2 M resulted in a simultaneous increase in particle size and polydispersity

. Silver NPs with controllable sizes were synthesized by reduction of [Ag(NH 3)2]+
with glucose, galactose, maltose, and lactose .

IRRADIATION METHOD

Silver NPs can be synthesized by using a variety of irradiation methods. Laser

irradiation of an aqueous solution of silver salt and surfactant can produce silver
NPs with a well defined shape and size distribution . Furthermore, laser was used
in a photo-sensitization synthetic method of making silver NPs using
benzophenone. At short irradiation times, low laser powers produced
silver NPs of about 20 nm, while an increased irradiation power produced NPs of
about 5 nm. Laser and mercury lamp can be used as light sources for production
of silver NPs . In visible light irradiation studies, photo-sensitized growth of silver
NPs using thiophene (sensitizing dye) and silver nanoparticle formation by
illumination of Ag(NH3)+ in ethanol has been done.

Electrochemical synthetic method

Electrochemical synthetic method can be used to synthesize silver NPs. It is

possible to control particle size by adjusting electrolysis parameters and to
improve homogeneity of silver NPs by changing the composition of electrolytic
solutions. Polyphenylpyrrole coated silver nanospheroids (3-20 nm) were
synthesized by electrochemical reduction at the liquid/liquid interface. This nano-
compound was prepared by transferring the silver metal ion from aqueous phase
to organic phase, where it reacted with pyrole monomer
UV-initiated photoreduction

A simple and effective method, UV-initiated photoreduction, has been reported

for synthesis of silver NPs in the presence of citrate, polyvinylpyrrolidone, poly
(acrylic acid), and collagen. For instance, Huang and Yang produced silver NPs via
photoreduction of silver nitrate in layered inorganic laponite clay suspensions
which served as stabilizing agent for prevention of NPs aggregation. The
properties of produced NPs were studied as a function of UV irradiation time.
Bimodal size distribution and relatively large silver NPs were obtained when
irradiated under UV for 3 h. Further irradiation disintegrated the silver NPs into
smaller sizes with a single distribution mode until a relatively stable size and size
distribution was obtained . Silver NPs (nanosphere, nanowire, and dendrite) have
been prepared by UV irradiation photoreduction technique at room temperature
using poly (vinylalcohol) (as protecting and stabilizing agent). Concentration of
both poly (vinylalcohol) and silver nitrate played significant role in the growth of
the nanorods and dendrites

BIOLOGICAL SYNTHESIS

Bio-based protocols could be used for synthesis of highly stable and well-
characterized NPs when critical aspects, such as types of organisms, inheritable
and genetical properties of organisms, optimal conditions for cell growth and
enzyme activity, optimal reaction conditions, and selection of the biocatalyst state
have been considered. Sizes and morphologies of the NPs can be controlled by
altering some critical conditions, including substrate concentration, pH, light,
temperature, buffer strength, electron donor (e.g., glucose or fructose), biomass
and substrate concentration, mixing speed, and exposure time. In the following
section, we discussed the synthesis of NPs using microorganisms and biological
systems
GREEN SYNTHESIS

. It was reported that Ocimum sanctum leaf extract could reduce silver ions into
crystalline silver NPs (4-30 nm) within 8 min of reaction time. These NPs were
stable due to the presence of proteins which may act as capping agents. O.
sanctum leaves contain ascorbic acid which may play an important role in
reduction of silver ions into metallic silver NPs.

Acalypha indica (Euphorbiaceae) leaf extracts have produced silver NPs (20-30
nm) within 30 min . These NPs had excellent antimicrobial activity against water
borne pathogens, E. coli and V. cholera (minimum inhibitory concentration
(MIC)=10 μg/ml).

Plant extracts from alfalfa (Medicago sativa), lemongrass (Cymbopogon

flexuosus), and geranium (Pelargonium graveolens) have served as green
reactants in silver nanoparticle synthesis. Moreover, a high density of extremely
stable silver NPs was rapidly synthesized by challenging silver ions (1with Datura
metel leaf extract. The leaf extracts of this plant contains biomolecules, including
alkaloids, proteins/enzymes, amino acids, alcoholic compounds, and poly-
saccharides which could be used as reductant to react with silver ions, and
therefore used as scaffolds to direct the formation of silver NPs in the solution.

ROLE OF REDUCING AGENT AND STABILIZING AGENT IN

PREPARATION OF NANOPARTICLES

Reducing agent is the chemical compound which performs reduction reaction.

Typically reducing agents are used in the synthesis of metal nanostructures.
They are generally reduce metal salts into pure metals, while surfactants are
polymers used in the wet chemical synthesis to stabilize nanoparticles against
aggregation by steric repulsion. The amount of reducing agent required in a
typical reaction is normally decided by the quantity of metal salt at the
starting point.During nanoparticles synthesis, reducing agents are used to
reduce the metal to zero oxidation state, is it necessary to change
nanoparticles to aero oxidation state, 

Stabilizing agent could be used to prevent agglomeration of the nanoparticles.

Here too, the final product could be either a solid or liquid.
DIFFERENT METHODS FOR CHARECTERISATION OF
NANOPARTICLES;-

U ltraviolet-Visible (UV-vis) Spectrum analysis .

X-Ray Diffraction Studies

. Transmission Electron Microscopy (TEM) analysis

. Scanning Electron Microscopy analysis

Fourier Transform Infra-Red Spectroscopy

PROTOCOL FOR CHEMICAL SYNTHESIS

COMBINATION 1;-

CHEMICALS REQUIRED;-

Silver nitrate,Trisodium citrate, were the main chemicals which were used
Mili-q water was used through out the experiments

CHEMICAL SYNTHESIS;-

The silver nanoparticles were prepared by using chemical reduction method.

All ingredients of reacting materials were prepared in Miliq water. In
solution, 5 ml of 1% trisodium citrate was included drop by drop which
results in the formation of yellow colour silver ions. Chemical reduction
method involves the reduction of AgNO3 in aqueous solution by an effective
reducing agent in the presence of appropriate stabiliser, which is necessary in
shielding the growth of silver particles through aggregation.

In this case we are using trisodium citrate as a reducing as well as a

stabilizing agent

COMBINATION 2;-

CHEMICALS REQUIRED;-

Sodium borohydrate,Silver nitrate,SDS

CHEMICAL SYNTHESIS;-

30Mml of Sodium borohydride sol was prepared. To this 1ml of silver nitrate
solution is added. The solution is stirred at 37.c for 30’.Colour changes from
light to dark yellow colour. SDS is added and stirred for 30 min.

In these case we are using SDS (Sodium Dodecyl Sulphate) as stabilizing agent
and Sodium Borohydride as reducing agent

QUALITATIVE ESTIMATION OF NANOPARTICLES ;-

SPECTROPHOTOMETRIC METHOD;-

Ultraviolet visible (UV-Vis) spectral analysis was done by using ELICO SL-159
Spectrophotometer in the range of 350-470 nm. The reduction of pure silver
ions of two samples synthesised by chemical means was monitored by
measuring the UV-Vis spectrum of the reaction mixture.

ANTIMICROBIAL SENSIVITY

MICRORGANISMS USED ARE;-Staphylococus Aureus,

Acinetobacter baumannii

Silver nanoparticles synthesised by chemical means were tested for

antimicrobial activity by taking Gram-positive and Gram-negative bacteria by
the agar well diffusion method. Twenty millilitre of nutrient agar medium was
poured into sterilised Petri plates. One day old bacterial culture was taken
and was used to prepare the bacterial lawns. With the help of a sterilised
borer, agar wells of 8 mm diameter were prepared. The wells were injected
with 60 ml of biologically synthesised silver nanoparticle solution, 60 ml of
chemically synthesised silver nanoparticle solution, 60 ml of 1 mM AgNO3 as a
negative control along with 60 ml of 30mg/ml of streptomycin as a positive
control. The plates were further incubated at 37 °C for 24 h and were then
examined for the presence of zones of inhibition. The inhibition zone was
measured and expressed in millimetre unit.

GREEN SYNTHESIS;-

In this present study silver nanoparticles were synthesized from aqueous

silver nitrate (1mM) through a simple and ecofriendly route using leaf broth
of Ocimum sanctum as reductant and stabilizer agent and leaf broth of
Alternanthera reineckii.

CASE1;-
PREPARATION OF Ocimum sanctum LEAF EXTRACT;-

The AR grade silver nitrate (AgNO3) was purchased from Sigma-Aldrich

chemicals and fresh Ocimum leaves were collected from surroundings of
Tirupati, Andhra Pradesh, India. The Ocimum fresh leaf extract used for the
reduction of Ag+ ions to Ag was prepared by taking 20g of thoroughly washed
finely cut leaves in 500 ml Erlenmeyer flask along with 100 ml of distilled
water and then boiling the mixture for 5 min. before decanting it. Further, the
extract was filtered with Whatman No. 1 filter paper and stored at 4°C and
used for further experiments.

SYNTHESIS OF SILVER NANOPARTICLES In a typical experiment,

the leaf extract (0.5 ml) was added to 10 ml of 1 mM AgNO3 aqueous solution.
The bioreduced aqueous component (0.5 ml) was used to measuring UV-Vis
spectra of the solution. The particle suspension was diluted 10 times with
distilled water to avoid the errors due to high optical density of the solution.

CASE 2;-

PRAPARATION OF Alternanthera bettzickiana . LEAF

EXTRACT

Fresh A. bettzickiana leaves were collected and thoroughly washed with

running tap water. Then, the leaves were washed with double distilled water
and shade dried up to 5 days. Dried leaves were powdered by using
mechanical grinder. 10 g of the fine leaf powder was mixed with 100 ml of
double distilled water. The mixture was boiled at 80°C for 10 min in boiling
water bath and filtered using Whatmann no.1 filter paper. The extract was
collected and stored in a refrigerator at 4°C for further studies

SYNTHESIS OF SILVER NANOPARTICLES

In a typical nanoparticle synthesis procedure, silver solution was prepared by

mixing 1 mM (10-3 M) of silver nitrate with 20 ml of double distilled water.
About 5 ml of leaf extract was added to 20 ml aqueous silver nitrate solution
which was heated up to 80ºC for 10 min. Resulted mixture became yellow to
red and finally dark brown in color after heating. This indicated the reduction
of silver nanoparticles . The mixture was kept dark condition at room
temperature to prevent further reduction of silver ions

QUALITATIVE ANALYSIS OF NANOPARTICLES

Synthesized silver nanoparticles was confirmed by sampling the aqueous

component of different time intervals and the absorption maxima was
scanned by UV-Vis spectrophotometer at the wavelength of 300 – 800 nm on
Perkin-Elmer Lambda 25 spectrophotometer.

ANTIMICROBIAL ACTIVITY ;- MICRORGANISMS USED ARE

Staphylococus Aureus, Acinetobacter baumannii

The antibacterial activity of biogenic synthesized Ag NPs was tested by the

standard agar well diffusion method. About four wells were made using sterile
borer (5mm) under aseptic condition. Different concentration of Ag NPs 10µl,
20µl, 30µl, and 40µl were added to the well and incubated for 24 h at 37°C.
The zone of inhibition was measured using a ruler and expressed in mm.

ADVANTAGES OF GREEN SYNTHESIS

OVER CHEMICAL METHOD
1) eco-friendly
2)low-cost
3)can be used in large scale synthesis
4)no need to use high pressure,temp,toxic chemicals,