DECLARATION

The project entitled “Comparative Study of Antimicrobial Efficacy of

Chemically synthesized and Green synthesized Nanoparticles“ was
carried out by us from the period of as a part of final year project at Heritage Institute
of Technology, Kolkata under the supervision of (prof) Dr .Soma Bannerjee,
Department of Biotechnology .The content and source derived from existing
literature has been indicated at appropriate places throughout the report. The project is
original and has not submitted as apart or whole for any degree or diploma in any other
university or organization.

CHANDRAMA GHOSH (1556030)

CHANDRABALI SAHA (1556055)

Department of Biotechnology (4th year)

Heritage Institute of Technology

CERTIFICATE OF APPROVAL

Certified that the thesis entitled “Synthesis of Silver Nanoparticles

and Checking its Antimicrobial Property” submitted by Ms
Chandrabali Saha (12615004006) to & Ms Chandrama Ghosh
(12615004007) Heritage Of Institute of Technology for the award of the degree
of Bachelor of Technology in Biotechnology has been accepted by the external
examiners and the students have successfully defended the thesis in viva voice
examination held to today.

(Supervisor) (HOD)
ACKNOWLEDGEMENT

I would like to express my heartfelt gratitude to prof Dr Soma Banerjee Mam,

Department of biotechnology, Heritage Institute of Technology, Kolkata for
providing me this golden opportunity to work in such an inspiring and motivating
environment. I would like to thank ma’am for her valuable suggestions, constant
support and inspiration without which this project would have been impossible.

I would also like to thank Ms Anindita Chatterjee, PhD scholar, Biotechnology,

Heritage Institute of Technology, Kolkata.

My sincere regards and thanks to HOD mam Professor Dr Srabanti Basu,

Department of Biotechnology, Heritage Institute of Technology, and Kolkata. I
would also like to thank all my respected faculty members for their constant
support and guidance.

I would like to express my gratitude to other lab members Mrs. Sharmishtha

Mukherjee, friends and classmates who were a constant source of support
throughout the project.

I owe my gratitude to all my family members and loved ones for their constant
motivation which helped me to complete the project.

My final words of acknowledgement to the almighty God for giving me the

strength for performing my task well.
Chandrama Ghosh

Chandrabali Saha
ABSTRACT:
Nanotechnology is an emerging field in the area of interdisciplinary research,
especially in biotechnology. The synthesis of silver nanoparticles is extensively
studied by used chemical and physical methods but the development of reliable
environment friendly technology to produce nanoparticles is an important aspect of
nanotechnology that does not involve the usage of toxic chemicals in the process of
synthesis .The bonding reaction between antibiotic and nanoparticles enhances the
inhibition effect against the test organisms. The antibiotic molecules contain many
active groups such as hydroxyl and amide groups which react easily with nano
silver by chelation and helps in effective inhibition. In the present investigation we
examined the formation of silver nanoparticles following two methods
Chemical synthesis and Green synthesis.
The characterization of these was confirmed by UV spectroscopy analysis.
Comparative analysis of antimicrobial susceptibility against Staphylococcus aurous
and Ecoli .The antimicrobial activity of synthesized silver nanoparticles was also
studied to ensure the contribution of biologically synthesized silver nanoparticles to
nanomedicine. A visible color change from yellow to brown in the silver nitrate
contained in testubes indicated the formation of silver nanopaticles whereas no
color change was observed in control and blank tubes respectively.

Keywords- Nanotechnology, nonmaterial’s, AgNPs, chemical synthesis , green

synthesis, biosynthesis, antibacterial activity, Staphylococcus aurous ,Ecoli, UV
spectroscopy nanomedicine.
INTRODUCTION AND LITERATURE REVIEW:

NANOTECHNOLOGY :
In recent years nanotechnology has become the most important aspect in fields of
physics chemistry and biology. It shows great promise for providing us in the near
future with many breakthroughs that will change the direction of technological
advances in the wide range of applications. The transition of nanoparticles to micro
particles can lead to a number of changes to physical properties. Two of the major
factors in this are:- The increase in ratio of the surface area to volume ratio and size
of the particle moving into the realm of quantum effects predominate[1]. The
increase in the surface area to volume ratio which is as gradual progression as the
particle gets smaller leads to an increasing dominance of behavior of the atoms on
the surface of the particle over that of those of the interior of the particle [2]. This
affects both the properties of the particle in insulation and its interaction with other
material.
In recent years noble metal nanoparticles have been subject of the focused research
due to their unique, optical, magnetic, electrical and chemical properties that are
significantly different from those of bulk materials. The special and unique
properties could be attributed to their small sizes and large surface area. For these
reasoned metallic nanoparticles found uses in different fields such as catalysis,
electronics, and photonics [3].
NANOPARTICLE:
Nanoparticles are particles between 1 and 100 nanometers (nm) in size
with a surrounding interfacial layer. In nanotechnology, a particle is
defined as a small object that behaves as a whole unit with respect to its
transport and properties [4].
Nanotechnology is one of the key technologies of the 21st century.
Needless to say, the production and use of the tiniest particles invisible
to the naked eye are not a latter-day invention. Examples of the earlier
use of nonmaterial are the Lycurgus Cup from the 4th century AD on
display in the British Museum in London, some late medieval church
windows and also the famous Damascene Swords: When light shines
from the outside on the antique Roman cup the cup looks olive green,
when illuminated from the inside it shines ruby red and the mythological
king depicted on it turns lilac. Colloidal nanoparticles of silver and gold
contained in the glass are responsible for this phenomenon [5][6].
Nanoparticles (NPs) have wide range of applications in areas such as
health care, cosmetics, food and feed, environmental health, mechanics,
optics, biomedical sciences, chemical industries, electronics, space
industries, drug-gene delivery, energy science, optoelectronics, catalysis,
single electron transistors, light emitters, nonlinear optical devices, and
photo-electrochemical applications[7].
HISTORY OF NANOTECHNOLOGY:
In December of 1959, Richard Feynman gave a talk called “There's
Plenty of Room at the Bottom” at an annual meeting of the American
Physical Society at Caltech. In this famous lecture, Feynman laid the
conceptual foundations for the field now called nanotechnology when he
imagined a day when things could be miniaturized -- when huge
amounts of information could be encoded onto increasingly small
spaces, and when machinery could be made considerably smaller and
more compact[8][9]. Although some have questioned the degree to
which Feynman influenced the rise of nanotechnology, his lecture is still
seen as a seminal event in the short history of the nano field. The term
"nano-technology" was first used by Norio Taniguchi in 1974, though it
was not widely known[10][11].

Richard Feynman
https://myvintagephotos.com/product/richard-feynman-2/
Inspired by Feynman's concepts, K. Eric Drexler used the term
"nanotechnology" in his 1986 book Engines of Creation: The Coming
Era of Nanotechnology, which proposed the idea of a nanoscale
"assembler" which would be able to build a copy of itself and of other
items of arbitrary complexity with atomic control. Also in 1986, Drexler
co-founded The Foresight Institute (with which he is no longer
affiliated) to help increase public awareness and understanding of
nanotechnology concepts and implications [10][11].
Thus, emergence of nanotechnology as a field in the 1980s occurred
through convergence of Drexler's theoretical and public work, which
developed and popularized a conceptual framework for nanotechnology,
and high-visibility experimental advances that drew additional wide-
scale attention to the prospects of atomic control of matter. Since the
popularity spike in the 1980s, most of nanotechnology has involved
investigation of several approaches to making mechanical devices out of
a small number of atoms.
In the 1980s, two major breakthroughs sparked the growth of
nanotechnology in modern era. First, the invention of the scanning
tunneling microscope in 1981 which provided unprecedented
visualization of individual atoms and bonds, and was successfully used
to manipulate individual atoms in 1989. The microscope's developers
Gerd Binnig and Heinrich Rohrer at IBM Zurich Research Laboratory
received a Nobel Prize in Physics in 1986. Binnig, Quate and Gerber
also invented the analogous atomic force microscope that year [10][12].
APPLICATIONS OF NANOTECHNOLOGY
The 2000s have seen the beginnings of the applications of
nanotechnology in commercial products, although most applications are
limited to the bulk use of passive nonmaterials. Examples include
titanium dioxide and zinc oxide nanoparticles in sunscreen, cosmetics
and some food products; silver nanoparticles in food packaging,
clothing, disinfectants and household appliances such as Silver Nano;
carbon nanotubes for stain-resistant textiles; and cerium oxide as a fuel
catalyst.[1] As of March 10, 2011, the Project on Emerging
Nanotechnologies estimated that over 1300 manufacturer-identified
nanotech products are publicly available, with new ones hitting the
market at a pace of 3–4 per week.[11][13]
Nanotechnology is being used in developing countries to help treat
disease and prevent health issues. The umbrella term for this kind of
nanotechnology is Nanomedicine.
Nanotechnology is also being applied to or developed for application to
a variety of industrial and purification processes. Purification and
environmental cleanup applications include the desalination of water,
water filtration, wastewater treatment, groundwater treatment, and other
nanoremediation. In industry, applications may include construction
materials, military goods, and nano-machining of nano-wires, nano-rods,
few layers of grapheme [14][15] etc. Recently a new field arisen from
the root of Nanotechnology is called Nanobiotechnology.
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.

Image 1: Sizes of nanomaterial

https://commons.wikimedia.org/wiki/File:Comparison_of_nanomaterials_sizes.jpg

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.[16][17]
SILVER NANOPARTICLES

Image 2: Electron micrograph of silver

nanoparticleshttps://www.bing.com/images/search

Silver nanoparticles are nanoparticles of silver (image 2) of between

1 nm and 100 nm in size.[1] While frequently described as being 'silver'
some are composed of a large percentage of silver oxide due to their
large ratio of surface-to-bulk silver atoms. Numerous shapes of
nanoparticles can be constructed depending on the application at hand.
Commonly used are spherical silver nanoparticles but diamond,
octagonal and thin sheets are also popular [18].
APPLICATIONS OF SILVER NANOPARTICLES
Image (3): Application of AgNPs
https://www.slideshare.net/gowtham10/applications-of-nanotechnology-
in-eor
HISTORY OF SILVER NANOPARTICLES
Nanosilver is one nonmaterial that is currently under a lot of scrutiny.
Much of the discussion is based on the assumption that nanosilver is
something new that has not been seen until recently and that the
advances in nanotechnology opened completely new application areas
for silver. However, we show in this analysis that nanosilver in the form
of colloidal silver has been used for more than 100 years and has been
registered as a biocidal material in the United States since 1954. Fifty-
three percent of the EPA-registered biocidal silver products likely
contain nanosilver.[19] Most of these nanosilver applications are silver-
impregnated water filters, algaecides, and antimicrobial additives that do
not claim to contain nanoparticles. Many human health standards for
silver are based on an analysis of agrarian occurrence (discoloration of
the skin, a cosmetic condition) from the 1930s and include studies that
considered nanosilver materials.[20] The environmental standards on the
other hand are based on ionic silver and may need to be re-silver form, is
present in the form of small clusters or nanoparticles. The implications
of this analysis for policy of nanosilver is that it would be a mistake for
regulators to ignore the accumulated knowledge of our scientific and
regulatory heritage in a bid to declare nanosilver materials as new
chemicals, with unknown properties and automatically harmful simply
on the basis of a change in nomenclature to the term “nano”.[21]

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 types of nanometals and their antibacterial activity:

No. Name of Size Concentration Antimicrobial
nanometal Activity

1 Silver (Ag) 40nm 0 ug/ml No inhibition of

bacterial
growth
2 ug/ml 25% inhibition.

6 ug/ml 100%
inhibition.

12.5 ug/ml 100%

inhibition.

100nm 0 ug/ml No inhibition of

bacterial
growth.
2 ug/ml 10% growth
inhibition.

6 mg/ml 60% inhibition.

12.5 mg/ml 100%

inhibition.

2 Gold (Au) 30-60nm 1.5 ug/ml 26% growth

reduction.
 3.5 ug/ml 37% growth
reduction

6.1 ug/ml 48% growth

reduction

15 ug/ml 99% growth

reduction

3 Zinc (Zn) 100-200 nm 10 ug/ml No growth

reduction

30 ug/ml 10.5% growth

reduction

50 ug/ml 31% growth

reduction

70 ug/ml 47% growth

reduction

4 Platinum (Pt) 20-40 nm 2 ug/ml No inhibition of

bacterial
growth.
6ug/ml 25% growth
reduction

12 ug/ml 100% growth

reduction

20 ug/ml 100% growth

reduction
5 Palladium (Pd) 50nm 2 ug/ml No inhibition.

12 ug/ml 50% inhibition.

Role of physical agents:
Temperature (degree C) pH Effect

Room temperature Neutral Normal absorbance spectra

and narrow peak.
40 Neutral Intensity of absorbance
increases.
60 Neutral More narrow peak. More
stable nanoparticles.
100 Neutral Intensity decreases. Unstable
nanoparticle.
Room temperature 5 Intensity of absorbance
spectra decreases.
Room temperature 7 Intensity increases. Stable
nanoparticles.
Room temperature 8.5 No peak. Very unstable
particle/ no formation of
particles.
Medicinal Plants used in nanoparticle synthesis:
No. Name of the Scientific Active Compound Medicinal Use
plant name
1 Haldi Curcuma longa Curcuminoids Turmeric Contains Bioactive
Compounds With Powerful
Medicinal Properties.
Curcumin Is a Natural Anti-
Inflammatory Compound.
Turmeric Dramatically
Increases the Antioxidant
Capacity of the Body.

2 Tulsi Ocimum Ocimumtenuiflorum Fights acne. Protects against

sanctum diabetes. Helps fight
cancer. Balances hormones
and lowers stress.
Relieves fever.
Helps improve respiratory
disorders.
Good source of vitamin K.
Dental care and oral health.
3 Neem Azadirachta Gedunin and Quercetin and ß-sitosterol,
indicia azadirachtin. polyphenolic flavonoids,
were purified from neem
fresh leaves and were known
to have antibacterial and
antifungal properties [6] and
seeds hold valuable
constituents.
4 Nayantara Catharanthus vinblastine and Many of the vinca alkaloids
roses vincristin were first isolated from
Catharanthus roseus,
including vinblastine and
vincristine used in the
treatment of leukemia and
Hodgkin's lymphoma.
5 Ginger Zingier zingiberene and Ginger Contains Gingerol, a
officinal bisabolene, Substance With Powerful
Medicinal Properties.
Ginger Can Treat Many
Forms of Nausea, Especially
Morning Sickness. Ginger
May Reduce Muscle Pain
and Soreness. The Anti-
Inflammatory Effects Can
Help With Osteoarthritis.

DIFFERENT METHODS FOR

SYNTHESIS OF NANOPARTICLES:
Silver nanoparticles (NPs) have been the subjects of researchers because
of their unique properties (e.g., size and shape depending optical,
antimicrobial, and electrical properties). A variety of preparation
techniques have been reported for the synthesis of silver NPs; notable
examples include, laser ablation, gamma irradiation, electron irradiation,
chemical reduction, photochemical methods, microwave processing, and
biological synthetic methods. The process of silver nanoparticle
preparation can be carried out by physical, chemical, green and
biological synthesis methods.
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. Physical synthesis of silver NPs using a tube furnace at
atmospheric pressure has some disadvantages, for example, tube furnace
occupies a large space, consumes a great amount of energy while raising
the environmental temperature around the source material, and requires
a lot of time to achieve thermal stability. Moreover, a typical tube
furnace requires power consumption of more than several kilowatts and
a preheating time of several tens of minutes to reach a stable operating
temperature. The small ceramic heater was used to evaporate source
materials. The evaporated vapor can cool at a suitable rapid rate, because
the temperature gradient in the vicinity of the heater surface is very steep
in comparison with that of a tube furnace.[21][22]

This makes possible the formation of small NPs in high concentration.

The particle generation is very stable, because the temperature of the
heater surface does not fluctuate with time. This physical method can be
useful as a nanoparticle generator for long-term experiments for
inhalation toxicity studies, and as a calibration device for nanoparticle
measurement equipment. The results showed that the geometric mean
diameter, the geometric standard deviation and the total number
concentration of NPs increase with heater surface temperature. Spherical
NPs without agglomeration were observed, even at high concentration
with high heater surface temperature. The geometric mean diameter and
the geometric standard deviation of silver NPs were in the range of 6.2-
21.5 nm and 1.23-1.88 nm, respectively.[22][23]

Chemical methods:
Chemical reduction: 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.[23] 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.[23]

Silver nitrate and tri-sodium citrate were used for preparation of silver
nanoparticles where the silver-nitrate as precursor and the tri-sodium
citrate as reducing agent. The silver colloid was prepared by using
chemical reduction method. All solutions of reacting materials were
prepared in distilled water. In typical experiment, 100 ml of 0.02 M
AgNO3 was heated and vibrated in an ultrasonic mixing bath for15 min,
with this solution 25 ml of 0.2 M tri-sodium citrate was added drop by
drop. During the process, solutions were mixed vigorously and heated
until change of color was evident pale yellow.[24] The mechanism of
reaction could be expressed as follows:
4Ag+ + C6H5O7Na3 + 2H2O → 4Ago + C6H5O7H3 + 3Na+ +
H+ + O2↑

Silver nitrate (AgNO3), Hydrazine hydrate, Citrate of sodium and

Sodium Dodecyl Sulphate (SDS) were purchase from Merck Peruana.
All chemicals were used as received. Double-distilled deionised water
was used. For the preparation of silver nanoparticles two stabilizing
agents, Sodium Dodecyl Sulphate (SDS) and Citrate of sodium were
used. For the synthesis of silver nanoparticles, silver nitrate solution
(from 1.0 mM to 6.0 mM) and 8 %(w/w) . Sodium Dodecyl Sulphate
(SDS) was used as a metal salt precursor and a stabilizing agent,
respectively. Hydrazine hydrate solution with a concentrate ranging
from 2,0 mM to 12 mM and Citrate of sodium solution (1,0 mM to 2,0
mM) were used as a reducing agents. Citrate of sodium was also used as
stabilizing agent at room temperature. The transparent colorless solution
was converted to the characteristic pale yellow and pale red color, when
citrate of sodium was used as stabilizing agent. The occurrence of color
was indicated the formation of silver nanoparticles. The silver
nanoparticles were purified by centrifugation. To remove excess silver
ions, the silver colloids were washed at least three times with deonized
water under nitrogen stream. A dried powder of the nanosize silver was
obtained by freeze-drying.[23][24]

Silver nitrate (AgNO3), trisodium citrate (Na3C6H5O7), and tannic acid

(C76H52O46) were purchased from Sigma-Aldrich. All chemicals were
used as received without further purification. Distilled water passed
through a Millipore system (ρ =18.2 MΩ) was used in all experiments.
All glassware was first rinsed with acetone and then with Millipore
water before use Methods. The typical procedure for the production of
Ag NPs is given below. Synthesis of Silver Seeds. A 100 mL volume of
aqueous solution containing sodium citrate (SC) (5 mM) and tannic acid
(TA) was prepared and heated with a heating mantle in a three-neck
round-bottomed flask for 15 min under vigorous stirring. A condenser
was used to prevent the evaporation of the solvent. After boiling had
commenced, 1 mL of AgNO3 (25 mM) was injected into this solution.
The solution became bright yellow immediately. The size of the Ag
seeds was controlled by adjusting the concentration of tannic acid. Thus,
by increasing the TA concentration from 0.025 mM to 0.1 mM, 0.25
mM, mM and 5 mM, the size of Ag NPs increased from 10.1 ±0.9 nm,
to 14.8 ± 1.4 nm, 23.4 ± 5.0 nm, 36.9 ± 6.2 nm, and 46.1 ±8.3 nm,
respectively. Resultant Ag NPs were purified by centrifugation(10000g
to 18000g, depending on its size) in order to remove the excess of TA
and further redispersed in Milli-Q-water or SC 2.2 mM before sample
characterization.[25]

Biological Methods:
A number of reports prevailed in the literatures indicate that synthesis of
nanoparticles by chemical approaches are eco-unfriendly and expensive.
Thus, there is a growing need to develop environmentally and
economically friendly processes, which do not use toxic chemicals in the
synthesis protocols. This has conducted researchers to look at the
organisms. The potential of organisms in nanoparticle synthesis ranges
from simple prokaryotic bacterial cells to eukaryotic fungi and plant.[23]
[26]
https://www.researchgate.net/publication/270594670_Synthesis_of_silv
er_nanoparticles_with_different_shapes

Green Method:
Neem: Silver nanoparticles (AgNPs) were synthesized using aqueous
extract of Neem (Azadirachta indicia) leaves and silver salt. XRD, SEM,
FTIR, optical absorption and photoluminescence (PL) were measured
and analysed. The synthesized AgNPs exhibits lowest energy absorption
band at 400 nm.[24][25] The effects of various parameters i.e., extract
concentration, reaction pH, reactants ratio, temperature and interaction
time on the synthesis of AgNPs were studied. It was found that the
formation of AgNPs enhanced with time at higher temperature and
alkaline pH. The AgNPs formed were found to have enhanced
antimicrobial properties and showed zone of inhibition against isolated
bacteria (Escherichia coli) from garden soil sample. Based on the results
obtained, it can be concluded that the resources obtained from plants can
be efficiently used in the production of AgNPs and could be utilized in
various fields such as biomedical; nanotechnology etc .Silver nitrate was
obtained from Sigma-Aldrich chemical Co. All the glassware were
washed with distilled water and dried in oven. The Petri-plates and agar
were autoclaved before use.20 g of finely cut Neem leaves were boiled
in 100 ml water for10 min and filtered to obtain Neem leaves extract.
The extract of Neem leaves (5 ml) were mixed with 45 ml of 1 mM
silver nitrate (AgNO3) and color change was observed indicating the
formation of AgNPs.[26]

5 g home grown Curcuma Longa or Zingier Officinal rhizomes were

washed several times with distilled water to remove the dust particles,
cleaned and cut in to small pieces. It was then boiled in a 500ml glass
beaker along with 250 ml sterile distilled water for 30 minutes. The
aqueous extract was separated by filtration with Whatman 1 filter paper.
20ml of the respective rhizome extract was added to 5ml of 0.5mM,
1mM and 5mM aqueous silver sulphate solutions respectively, stirring
magnetically at room temperature. The extract acted as the reducing
agent as well as capping agent. The reduction of pure silver ions was
observed by recording the UV-Visible spectra of the samples withdrawn
from the reaction mixture periodically on a Perkin Elmer
spectrophotometer.[25][26]

AgNO3 was purchased. Leaves of O. sanctum were collected.

Lyophilized cultures of Escherichia coli, Staphylococcus aurous were
pro-cured. Nutrient media used here was supplied by Hi-Media
Laboratories. Experiment was performed thrice to check the accuracy
and repeatability of the result. 20 g fresh leaves of O. sanctum were
collected and washed three times with distilled water to remove dust
particles. Leaves were finely chopped and added to 100 mL of D/W and
stirred at 60 C for 1 h. After boiling, the mixture was cooled and filtered
with Whatman paper No. 1. Filtrate was collected and was stored at 4 C
for further analysis. 5 mL of leaf extract was added to 45 mL of 10-3 M
AgNO3 solution for bioreduction process at 30 C.[27]
ANALYSIS OF NANOPARTICLES:
U ultraviolet-Visible (UV-vis) Spectrum analysis. - UV-Visible
Nanoparticle Analysis UV-Visible spectroscopy (UV-Vis) measures the
extinction (scatter + absorption) of light passing through a sample.
Nanoparticles have unique optical properties that are sensitive to the
size, shape, concentration, agglomeration state, and refractive index near
the nanoparticle surface, which makes UV-Vis a valuable tool for
identifying, characterizing, and studying nanomaterials.[18]
X-Ray Diffraction Studies- As a primary characterization tool for
obtaining critical features such as crystal structure, crystallite size, and
strain, x-ray diffraction patterns have been widely used in nanoparticle
research. The randomly oriented crystals in nanocrystalline materials
cause broadening of diffraction peaks. This has been attributed to the
absence of total constructive and destructive interferences of x-rays in a
finite sized lattice. Moreover, inhomogeneous lattice strain and
structural faults lead to broadening of peaks in the diffraction patterns
[16]. The size calculated from x-ray diffraction peak broadening is a
measure of the smallest unfaulted regions or coherently scattering
domains of the material. In fact, this is the size of regions bounded by
defects and grain boundaries and separated from surrounding by a small
disorientation, typically one or two degrees [27].
The scanning electron microscope (SEM) uses a focused beam of high-
energy electrons to generate a variety of signals at the surface of solid
specimens. The signals that derive from electron-sample interactions
reveal information about the sample including external morphology
(texture), chemical composition, and crystalline structure and orientation
of materials making up the sample. In most applications, data are
collected over a selected area of the surface of the sample, and a 2-
dimensional image is generated that displays spatial variations in these
properties. Areas ranging from approximately 1 cm to 5 microns in
width can be imaged in a scanning mode using conventional SEM
techniques (magnification ranging from 20X to approximately 30,000X,
spatial resolution of 50 to 100 nm). The SEM is also capable of
performing analyses of selected point locations on the sample; this
approach is especially useful in qualitatively or semi-quantitatively
determining chemical compositions (using EDS), crystalline structure,
and crystal orientations (using EBSD). The design and function of the
SEM is very similar to the EPMA and considerable overlap in
capabilities exists between the two instruments.

. Transmission Electron Microscopy (TEM) analysis - The TEM can

yield information such as particle size, size distribution and morphology
of the nanoparticles. In particle size measurement, microscopy is the
only method in which the individual particles are directly observed and
measured [18]. Typically, the calculated sizes are expressed as the
diameter of a sphere that has the same projected area as the projected
image of the particle. Manual or automatic techniques are used for
particle size analysis. Manual technique is usually based on the use of a
marking device moved along the particle to obtain a linear dimensional
measure of the particle added up and divided by the number of particles
to get a mean result [17]. TEM images can also be used to judge whether
good dispersion has been achieved or whether agglomeration is present
in the system. Electron microscopy requires elaborate sample
preparation and is slow and few particles are examined. In combination
with diffraction studies, microscopy becomes a very valuable aid to the
characterization of nanoparticles.
.
 Image(4): Particle size analysis by Electron Microscopy

https://www.researchgate.net/figure/DLS-particle-size-analysis-of-green-synthesised-silver-
nanoparticles-having-a-size-range_fig4_261548685
DIFFERENT TYPES OF STABILIZING AND REDUCING
AGENT USED FOR SYNTHESIS OF NANOPARTICLES

https://www.bing.com/images/search?view=detailV2&ccid=Rg
%2bAapFt&id=EE0FD53A8DF6432D9783F472
DEFINITION AND ROLES OF REDUCING AGENT AND
STABILIZING AGENT IN FORMATION OF
NANOPARTICLES:
REDUCING AGENT: Gives electrons ions to form atoms. Then these atoms will
develop a particle by combing together.
Role of reducing agent if formation of AgNP: Reducing agent takes away
electrons from Ag3+ and makes it Ag0, which is metallic silver.
STABILIZING AGENT: Stabilizes the nanoparticles for long time effect.
Capping agent /surfactant /stabilizing agent is responsible for....
 prevent uncontrollable growth of particles
 prevent particle aggregation
 controls growth rate
 controls particle size
 allows particle solubility in various solvents
 allows particle solubility in various solvents
These possibilities can be achieved by-
 Electrostatic stabilization: Adsorption of ions to the surface creates an
electrical double layer which results in a Columbic repulsion force
between individual particles.
 Steric Stabilization: Surrounding the metal centre by layers of material
that are sterically bulky. Examples: polymers, surfactants, etc.[27][28]
 Image (5): Reduction and Stabilization of AgNP
https://pubs.rsc.org/en/content/articlehtml/2014/ra/c3ra44507k

ROLE OF NANOPARTICLES AS AN ANTIMICROBIAL

AGENT
The antibacterial properties of a variety of nanostructures have been
investigated. In understanding the antibacterial properties of these
nanostructures, it is important to recognize that while some metals, such as zinc,
silver, and copper, exhibit antibacterial mechanisms in their bulk form, other
materials, such as iron oxide, are not antibacterial in their bulk form but may
exhibit antibacterial properties in nanoparticulate form.[29]
MODE OF ACTION:
The mechanism of this antibacterial activity varies from nanoparticle to
nanoparticle. For all varieties of nanoparticles, the antibacterial mechanism is not
fully understood. While some proposed mechanisms relate to the physical
structure of the nanoparticles (ie, membrane-damaging abrasiveness of the
nanoparticle), others relate to the enhanced release of antibacterial metal ions
from nanoparticle surfaces. The specific surface area of a dose of nanoparticles
increases as the particle size decreases, allowing for greater material interaction
with the surrounding environment. Thus, for inherently antibacterial materials,
such as zinc and silver, increasing the surface to volume ratio enhances the
antibacterial effect. A nanoparticle of an inherently antibacterial material may,
therefore, have multiple mechanisms of antibacterial activity, such as the release
of antibacterial metal ions from the particle surface and the antibacterial physical
properties of a nanoparticle related to cell wall penetration or membrane
damage.[29][30]
Comparing results across a greater number of studies allows for the identification
of nanoparticle parameters which are most relevant in designing the ideal
antibacterial particle. Chemistry, particle size, particle shape, and zeta potential
are among the most relevant variables affecting antibacterial activity.
TOXICITY OF NANOPARTICLRS

Nano toxicology is the study of the toxicity of nonmaterial’s.[31] Because of

quantum size effects and large surface area to volume ratio, nonmaterial’s have
unique properties compared with their larger counterparts that affect their
toxicity. Of the possible hazards, inhalation exposure appears to present the most
concern, with animal studies showing pulmonary effects such as inflammation,
fibrosis, and carcinogenicity for some nanomaterial’s. Skin contact and ingestion
exposure are also a concern
Nanotoxicology is a sub-specialty of particle toxicology. Nanomaterials appear to
have toxicity effects that are unusual and not seen with larger particles. For
example, even inert elements like gold become highly active at nanometer
dimensions. Nanotoxicological studies are intended to determine whether and to
what extent these properties may pose a threat to the environment and to
human beings.[31] Nanoparticles have much larger surface area to unit mass
ratios which in some cases may lead to greater pro-inflammatory effects in, for
example, lung tissue. In addition, some nanoparticles seem to be able to
translocate from their site of deposition to distant sites such as the blood and the
brain.
Nanoparticles can be inhaled, swallowed, absorbed through skin and deliberately
or accidentally injected during medical procedures. They might be accidentally or
inadvertently released from materials implanted into living tissue.[32][33][34]
One study considers release of airborne engineered nanoparticles at workplaces,
and associated worker exposure from various production and handling activities,
to be very probable.
Size is a key factor in determining the potential toxicity of a particle. However it is
not the only important factor. Other properties of nanomaterials that influence
toxicity include: chemical composition, shape, surface structure, surface charge,
aggregation and solubility, [35] and the presence or absence of functional groups
of other chemicals. The large number of variables influencing toxicity means that
it is difficult to generalize about health risks associated with exposure to
nanomaterials – each new nanomaterial must be assessed individually and all
material properties must be taken into account.[33][35]
OBJECTIVE:
 Synthesis of Silver Nanoparticles- Chemical and Green Methods with
the help of different stabilizing and reducing agents
 Characterization of Ag nanoparticles by UV spectrophotometer.
 Comparative analysis of antibacterial susceptibility using Chemical
and Green Methods.

MATERIALS FOR THE EXPERIMENT

CHEMICAL SYNTHESIS
 Trisodium citrate
 5Mm Silver nitrate
 Double distilled water

GREEN SYNTHESIS
 Collection of plant parts
Dried plant parts of Neem and Tulsi were purchased in local
supermarket from Kolkata and evaluated for antimicrobial activity
 5Mm of Silver Nitrate solution
 Double distilled water

ANTIBACTERIAL STUDY

 Organisms : Staphylococcus aureus ,E.coli

 MHA media
 Sterile petriplates ,cotton swabsticks, micropippertes,
 Borer for agar well diffusion technique
 Parafilm for sealing
 INSTRUMENTS AND APPARATUS
 Test Tubes, Conicals, beakers{sterilized by
autoclaving}
 Falcons,Eppendorfs,
 Autoclave
 Sterile petriplates ,cotton swabsticks, micropippertes,
 Laminar air flow hood with vertical hepafilter
 Magnetic stirrer with hotplate,
 Research centrifuge
 U.V Double beam Spectrophotometre

METHODOLOGY OF EXPERIMENT:-
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

COMBINATION 3:-

Sodium borohydrate,Silver nitrate

CHEMICAL SYNTHESIS:-

A 0.002M NABH4 Solution was prepared 10ml. The soloution was stirred on a
magnetic stirrer kept on a ice bath .4ml of 0.001m of Agno3 was added
dropwise .Stir the solution vigorously on a magnetic stirrer.Solution turned light
yellow on addition of 2ml of Agno3 and bright yellow on addition of entire Agno3
solution.At this stage the clear yellow colloidal silver is stable at room temperature

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 neem
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 UVVIS 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:-
PREPARATION OF NEEM EXTERACT:-
2gM of finely cut neem leaves boiles in 10ml h20 for 10 mins.The solution is
filtered through whatmann paper.1.1ml of neem leaves extract were mixed with 10
ml of 0.001 M Agno3.Colour change was observed
RESULTS:

1) 24 hr Reading of Nanoparticle synthesis by Chemical Method on E.coli:

 Chemical (24 hr) E.coli
3

2.5

2
MIC (cm)

1.5

0.5

0
10 20 40 80 160

Concentration (mcg)

TCA+TA 2mM 5mM TA, TCA NABH4+SDS Only TSC

graph (1): Chemical (24 hr) E.coli

figure(1): zones of inhibition of ecoli under the influence of synthesized AgNPs (5mM TA+TSC)

(24)CHEMICAL
   10 20 40 80 160

TSC+TA 0.8 0.8 0.7 1.4 1.5

2mM

5mM TA, 0 1.1 1.2 1.4 1.5

TSC

NaBH4+SDS 0 0 0 1.4 0

Only TSC 0 0 0 0 2.6

table(1) zones of inhibition by Chemical AgNP

 table(1) ,graph(1) Silver nanoparticles of 4 combinations( TSC+TA 2mM, 5mM

TA + TSC, NaBH4+SDS , Only TSC). TSC+TA (2mM) showing zone of
inhibition 0.8 cm at 10 mcg concentration where as for TSC+TA (5mM) MIC was
formed at 20 mcg conc (fig 1) and for NaBH4 the MIC was found at 80 mcg conc.

2) 24 hr Readings for Green Synthesis of Nanoparticles on E.coli:

Green (24 hr) E.coli

2.5
2
MIC (cm)

1.5
1
0.5
0
10 20 40 80 160

Concentration (mcg)

TULSI Haldi Neem

Graph(2): Green (24 hr) E.coli

 fig(2): MIC on Haldi (E.coli)

(24)GREEN

  10 20 40 80 160

TULSI 0 0 0 1.2 1.2

Haldi 1.4 1.5 2 2.1 2.2

Neem 0 0 0 1.5 1.6

Table(2): MIC by Green AgNP.

 table(2) ,graph(2): Silver nanoparticles of 3 combinations(Tulsi, Neem, Haldi). gTulsi

showing zone of inhibition 1.2 cm at 80 mcg concentration where as for Haldi MIC
was formed at 10 mcg conc (fig 2) and for Neem the MIC was found at 80 mcg conc.
3) 48 hr Reading of Nanoparticle synthesis by Chemical Method on E.coli:

Chemical (48 hr) E.coli

3
2.5
2
MIC (cm)

1.5
1
0.5
0
10 20 80 160
Concentration (mcg)

5mM TA, TCA TSC+TA 2mM ONLY TSC

graph (3): Chemical (48 hr) E.coli
 fig(3): MIC on E.coli plate

CHEMICALS(48)

  10 20 80 160

5mM 0 0 1.4 1.6

TA,
TCA

TSC+T 0.8 0.8 1.4 1.5

A 2mM

ONLY ------ ------ ------ 2.6

TSC
Table(3): table(1) zones of inhibition by Chemical AgNP

 table(3) ,graph(3) Silver nanoparticles of 3 combinations( TSC+TA 2mM, 5mM TA + TSC,

Only TSC). TSC+TA (2mM) showing zone of inhibition 0.8 cm at 10 mcg concentration where as
for TSC+TA (5mM) MIC was formed at 80 mcg conc and for only TSC the MIC was found at 10
mcg conc.
4) 48 hr Reading of Nanoparticle synthesis by Green Method on E.coli:

Green (48 hr) Staph

2.5

1.5
MIC (cm)

0.5

0
20 40 80 160
Concentration (mcg)

TULSI Haldi
Graph(4): Green (48 hr) Staph

fig(4): ): zones of inhibition of ecoli

GREEN

  20 40 80 160

TULSI 0 0 1.2 1.2

 Haldi 1.5 1.6 2 2
Table(4): MIC by Green AgNP (48 hrs) E.coli

 table(4) ,graph(4) Silver nanoparticles of 2 combinations (Tulsi, Neem), Tulsi showing zone of
inhibition 1.2 cm at 80 mcg concentration where as for Haldi MIC was formed at 20 mcg conc (fig
4).

5) 72 hours reading on E.coli plates using AgNPs:

72 hr E.coli
1.8

1.6

1.4

1.2

1
MIC(cm)

0.8

0.6

0.4

0.2

0
10 20 40 80 160
TSC+TA NABH4+AGNO3 ONLY TSC NEEM
concentration (mcg)
Graph(5): 72 hr E.coli
 fig(5): Zone of inhibition in E.coli plates (72 hr)

72 hr Ecoli

  10 20 40 80 160

TSC+TA 0.2 0.2 0.2 0.5 0.5

NABH4+AGNO ------ ------ 0.5 1.2 1.2

3

ONLY TSC ------ ------ ------ 1.2 1.2

NEEM 0.4 0.4 0.8 1.4 1.6

table(5): MIC on E.coli plate by AgNPs.

 table(5) ,graph(5) Silver nanoparticles of 4 combinations( TSC+TA, Only TSC, NaBH4 +

AgNO3, Neem). TSC+TA showing zone of inhibition 0.2 cm at 10 mcg concentration where
as for NaBH4 MIC was formed at 40 mcg conc and for Neem the MIC was found at 10 mcg
conc.
6) Readings of MIC in Staphylococcus plates by AgNPs (24 hr):

24 hr (Staph)
1.8
1.6
1.4
1.2
MIC (cm)

1
0.8
0.6
0.4
0.2
0
Combination 2 Combination 3 Combination 4 Combination 5
Concentration (mcg)

40 80 160
Graph(6): 24 hr (Staph)
 fig (6): Zone of inhibition in Staphylococcus by AgNPs (24 hr)

staph

Combination
Combination 2 Combination 3 Combination 4 5

40 0 0 1.2 0

80 1.2 1.2 1.2 1.3

160 1.5 1.5 1.3 1.6

Table(6): MIC result on Staphylococcus (24 hr)

 table(6) ,graph(6) Silver nanoparticles of 5 combinations(Combination 1= NaBH4, SDS

Combination 2= 2mM TA, TCA, Combination 3= 5mM TA, TCA, Combination 4= Haldi,
Combination 5= Neem). Combination 2 showing zone of inhibition 1.2 cm at 80 mcg concentration where
as for combination 3 MIC was formed at 40 mcg conc (fig 6) and for combination 5 the MIC was found at
80 mcg conc.

7) Readings of MIC in Staphylococcus plates by AgNPs (24 hr):

1.4
48 hrs staph

1.2

1
MIC (cm)

0.8

0.6

0.4

0.2

0
C2 C3 C4
combination
40mcg 80mcg 160mcg

Graph(7): 48 hrs Staphylococcus

 fig(7): MIC on Staphylococcus by AgNPs (48 hrs)

Staphylococcus

C2 C3 C4

40mcg 0 0 1.1

80mcg 0 1.2 1.2

160mcg 1.2 1.3 1.2

Table(7): Zone of inhibion on Staphs (48 hrs)

 table(7) ,graph(7) Silver nanoparticles of 3 combinations( Combination 2= 2mM TA + TSC,

Combination 3= 5mM TA+TSC, Combination 4= Haldi ). C2 showing zone of inhibition 1.1 cm
at 40 mcg concentration where as for C3 MIC was formed at 80 mcg conc (fig 7) and for NaBH4
the MIC was found at 40 mcg conc.

8) Readings of MIC for pH4 (E.coli):

pH 4 (E.coli)
2.5
2
MIC (cm)

1.5
1
0.5
0
Concentration (mcg)

10 20 40
Graph (8): pH 4 (E.coli)
 fig (8): MIC on E.coli plate at pH4

Combination 1 Combination 2 Combination 3 Combination 4

10 1.1 1.3 1.8 0

20 1.3 1.3 1.9 0

40 1.3 1.5 2.2 2

table(8): MIC on E.coli pH4

 table(8) ,graph(8) Silver nanoparticles of 3 combinations(Combination 1= NaBH4, Combination

2= 2mM TA + TSC, Combination 3= 5mM TA+TSC, Combination 4= Haldi ). C1 showing
zone of inhibition 1.1 cm at 10 mcg concentration where as for C2 MIC was formed at 10 mcg
conc (fig 8) and for C4 the MIC was found at 40 mcg conc.

9) Readings of MIC for pH4 (Staphylococcus):

 pH 4 ( Staph)
3
2.5
2
MIC (cm)

1.5
1
0.5
0
10 20 40 80 160
Concentration (mcg)

Combination 1 Combination 2 Combination 3 Combination 4

Graph (9): pH 4 (Staph)

fig (9): MIC on Staphylococcus plate at pH4

Combination Combination
2 3 Combination 4

10 1.2 1.7 0

20 1.4 1.9 0
 40 1.6 2.3 0

80 2 2.6 1.3

160 2.1 2.6 2.4

table(9): MIC on Staph pH4

 table(9) ,graph(9) Silver nanoparticles of 3 combinations(Combination 1= NaBH4, Combination

2= 2mM TA + TSC, Combination 3= 5mM TA+TSC, Combination 4= Haldi ). C1 showing
zone of inhibition 1.1 cm at 10 mcg concentration where as for C2 MIC was formed at 10 mcg
conc (fig 8) and for C4 the MIC was found at 40 mcg conc.

10) Readings of MIC for pH13 (E.coli):

pH13 E.coli
3
2.5
MIC (cm)

2
1.5
1
0.5
0
10 20 40 80 160

concentration (mcg)

TSC+TA 2mM TA, TCA 5mM TA, TCA NABH4+AGNO3

Graph (10): pH 13 (E.coli)

 fig (10): MIC on E.coli plate at pH 13

10 20 40 80 160

2mM TA, TSC 1.2 1.7 1.7 1.6 2.4

(C1)

5mM TA, TSC 0 0 0 1.3 1.4

(C2)

NABH4+AGNO ------ ------ ------ 0.5 0.8

3 (C3)

table(10): MIC on E.coli pH13

 table(10) ,graph(10) Silver nanoparticles of 3 combinations( TSC+TA 5mM,TSC+TA 2mM, NaBH4

). C1 showing zone of inhibition 1.2 cm at 10 mcg concentration where as for C2 MIC was
formed at 80 mcg conc and for C3 the MIC was found at 80 mcg conc.

11) Readings of MIC for pH13 (Staphylococcus):

 Chemical pH 13 (Staph)
2.5
MIC (cm) 2
1.5
1
0.5
0
10 20 40 80 160

Concentration (mcg)

2mM TA, TCA 5mM TA, TCA NABH4+SDS NABH4+AGNO3

Graph (10): pH 13 (Staph)

fig (10): MIC on E.coli plate at pH 13

10 20 40 80 160

2mM TA, TCA 1.2 1.5 1.4 1.2 2.1

5mM TA, TCA 0 0 1.3 1.4 1.4

 NABH4+SDS 0 0 0 0.2 0.2

NABH4+AGNO 0 0 0 0.5 0.4

3

table(11h): MIC on Staph pH13

 table(11) ,graph(11) Silver nanoparticles of 4 combinations( TSC+TA 5mM,TSC+TA 2mM, NaBH4+

SDS, NaBH4+AgNO3 ). 2mM TA+TSC showing zone of inhibition 1.2 cm at 10 mcg
concentration where as for 5mM TA+TSC MIC was formed at 40 mcg conc and for NaBH$ the
MIC was found at 80 mcg conc.

12) Comparison 24 hrs vs 72 hrs:

 24 vs 72 hrs ( TCA+TA 2mM)
1.6
1.4
1.2
1
MIC (cm)

0.8
0.6
0.4
0.2
0
10 20 40 80 160
Concentration (mcg)

TCA+TA 2mM(24) TSC+TA 2mM (72)

fig(12): hrs. Comparison of MIC at 24 hrs vs 48

  10 20 40 80 160

TSC+TA 0.8 0.8 0.7 1.4 1.5

2mM(24
)

TSC+TA 0.2 0.2 0.2 0.5 0.5

2mM
(72)

Table(12): Comparison of MIC at 24 hrs vs 48 readings

 table(12) ,graph(12) Silver nanoparticles combination 2mM TA + TSC. CThe 24 hr reading

showing zone of inhibition 0.8 cm at 10 mcg concentration where as for 48 hr MIC was formed
at 10 mcg conc which is 0.2cm.

13) pH4 E.coli Chemically Synthesized AgNPs

 pH4 E.coli Chemically Synthesized AgNPs
2.5
2
ZONE OF INHIBITION (cm)

1.5
1
0.5
0
1 2 3
on on on
nati n ati n ati
bi bi bi
o m om om
C C C

CONCENTRATION (mcg)

10 20 40

Graph (13)

E.coli (pH 4)

Combination 1 Combination 2 Combination 3

10 1.1 1.3 1.8

20 1.3 1.3 1.9

40 1.3 1.5 2.2

Table (13) MIC result for pH4 in E.coli

 table(13) ,graph(13) Silver nanoparticles of 3 combinations( NaBH4+SDS,TSC+TA 2mM, TSC+TA

5mM). C1 showing zone of inhibition 1.1 cm at 10 mcg concentration where as for C2 MIC
was formed at 20 mcg conc and for C3 the MIC was found at 20 mcg conc.
14) pH 4 Staphylococcus aureus Chemically Synthesized AgNPs

pH 4 Staphylococcus aureus Chemically Synthesized

AgNPs
3
ZONE OF INHIBITION (cm)

2.5
2
1.5
1
0.5
0
10 20 40 80 160
CONCENTRATION (mcg)

Combination 1 Combination 2 Combination 3

Graph (14)

Staphylococcus (pH 4)

Combination 1 Combination 2 Combination 3

10 0 1.2 1.7

20 0 1.4 1.9

40 0 1.6 2.3

80 0 2 2.6

160 0 2.1 2.6

Table (14) MIC of pH4 in Staph

 Table(14) ,graph(14) Silver nanoparticles combination (NaBH4+SDS, TSC+TA 2Mm, TSC+TA

5Mm) showing zone of inhibition 1.2 cm at 10 mcg and 1.7 cm at 10 mcg
 15)COMPARISON ANALYSIS OF EFFECT OF GREEN SYNTHESIZED pH4 on
Staphylococcus aureus AND E. coli

COMPARISON ANALYSIS OF EFFECT OF GREEN

SYNTHESIZED pH4 on Staphylococcus aureus AND E. coli
2.5
2
zone of inhibition (cm)

1.5
1
0.5
0
1 n1 2 2 3
n
tio on on on
tio a ti ati ti
na
bi
n na bi
n na
bi m bi m bi
m m m
Co Co Co Co Co

CONCENTRATION(mcg)

10 20 40 80 160

Graph( 15)

COMPARISON OF pH 4 OF Staphylococcus and E. coli

Combination 1 Combination1 Combination 2 Combination2 Combination 3

10 1.1 0 1.3 1.2 1.8

20 1.3 0 1.3 1.4 1.9

40 1.3 0 1.5 1.6 2.2

80 0 0 0 2.1 0
 160 0 0 0 0 0

Table (15) Comparison of Ph 4 (e.coli vs Staph)

 Table(15) ,graph(15) Silver nanoparticles combination (NaBH4+SDS, TSC+TA 2Mm, TSC+TA

5Mm) showing zone of inhibition 1.1 cm at 10 mcg and 1.2 cm at 10 mcg

16) pH 13 Staphyloccocus aureus Chemically Synthesized AgNPs

pH 13 Staphylococcus aureus CHEMICALLY

SYNTHESIZED AgNPs
2.5
ZONE OF INHIBITION (cm)

2
1.5
1
0.5
0
10 20 40 80 160
CONCENTRATION (mcg)

2mM TA, TCA 5mM TA, TCA NABH4+SDS NABH4+AGNO3

Graph 16

CONCENTRATION (STAPH)

10 20 40 80 160
 2mM TA, TCA 1.2 1.5   1.4   1.2   2.1

5mM TA, TCA 0 0   1.3   1.4   1.4

NABH4+SDS 0 0   0   0.2   0.2

NABH4+AGNO 0 0   0   0.5   0.4

3

Table (16) MIC showing Ph13 Staph (chemical)

 Table(16) ,graph(16) Silver nanoparticles combinations (NaBH4+SDS, TSC+TA 2Mm, TSC+TA

5Mm) showing zone of inhibition 1.2 cm at 10 mcg and 1.5 cm at 20 mcg

17) pH 13 Staphylococcus aureus Green Synthesized AgNPs

pH 13 Staphylococcus aureus Green Synthesized AgNPs

2
1.8
ZONE OF INHIBITION (cm)

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
TULSI NEEM NEEM
CONCENTRATION (mcg)

80 160

Graph (17)
 CONCENTRATION (STAPH)

80 160

TULSI 0.3   0.5

NEEM 0   1.9

NEEM 0.2   0.2

Table (17) showing MIC at Ph 13 Staph (Green)

 Table(17) ,graph(17) Silver nanoparticles combinations (NaBH4+SDS, TSC+TA 2Mm, TSC+TA

5Mm) showing zone of inhibition 0.3 cm at 80 mcg and 0.5 cm at 160 mcg

18) pH 13 E. coli Chemically Synthesized AgNPs

pH 13 E. coli Chemically Synthesized AgNPs

3
ZONE OF INHIBITION (cm)

2.5
2
1.5
1
0.5
0
10 20 40 80 160
CONCENTRATION (mcg)

TSC+TA 2mM TA, TCA 5mM TA, TCA NABH4+AGNO3

Graph (18)

CONCENTRATION (e. coli)

10 20 40 80 160

TSC+TA 0   0   0   0.2   0.2

2mM TA, TCA 1.2   1.7   1.7   1.6   2.4

5mM TA, TCA 0   0   0   1.3   1.4

NABH4+AGNO ------   ------   ------   0.5   0.8

3

Table (18) showing MIC of Staph at Ph 13 (chemical)

 Table(18) ,graph(18) Silver nanoparticles combinations (NaBH4+SDS, TSC+TA 2Mm, TSC+TA

5Mm) showing zone of inhibition 1.2 cm at 10 mcg and 1.7 cm at 10 mcg

18) pH 13 E. coli Green Synthesized AgNPs

pH 13 E. coli Green Synthesized AgNPs

3
ZONE OF INHIBITION (cm)

2.5
2
1.5
1
0.5
0
10 20 40 80 160
CONCENTRATION (mcg)

Haldi TULSI NEEM

Graph (18)

CONCENTRATION (E. coli)

10 20 40 80 160

Haldi 2   2   2.2   2.5   2.6

TULSI ------   ------   ------   ------   1.2

NEEM ------   ------   ------   0.2   0.2

Table (18)

\
DISCUSSION
Chemical and Green synthesis of silver nanoparticle synthesis were being
followed over the decades, but their formation was found to be expensive and
the use of various toxic chemicals for their synthesis makes the green synthesis
the more preferred option. Different methods are suggested for nanoparticle
synthesis; the chemical reduction method and physical synthesis method were
widely studied due to its advantage in controlling particle size and morphology
very effectively .In the case of chemical synthesis methods, a stabilizer
(surfactant) is added to the first solution to prevent the agglomeration of silver
nanoparticles , whereas in Green synthesis there is no need to add a stabilizing
agent as plant extract itself acts as a stabilizing agent and reducing agent .
Environmental pollution is a disadvantage of the chemical method and the
chemical reduction methods are energy-intensive. Green synthesis methods are
carried out in environmental conditions and they are safe enough, and consume
no energy. Although the time required to synthesize Ag NPs is longer compared to
chemical methods; in fact, the synthesis time has recently reduced with finding
suitable organisms.
For leaf extract of O. sanctum can be used efficiently for the bioreduction of
AgNO3 into silver nanoparticles of average diameter of 14.31 ± 2.5 nm. The
synthesized silver nanoparticles were seen to be stable due to the presence of
proteins which may act as a capping agent, yet further research is needed in this
area to explore the possible bimolecular responsible for bioreduction process.
These nanoparticles were crystalline in nature and possess antimicrobial property.
The biological mechanism of nanoparticles synthesis using leaf extract is not yet
fully understood and needs to be explored. Some reports suggest that the
reduction occurs due to NADH-dependent reductase released into the solution.
AgNP characterisation.
Silver nanoparticles (AgNPs) appear yellowish brown in colour in aqueous
medium as a result of surface Plasmon vibrations [18]. As the different leaf
extracts were added to aqueous silver nitrate solution, the colour of the solution
changed from faint light to yellowish brown to reddish brown and finally to
colloidal brown indicating AgNP formation. Similar changes in colour have also
been observed in previous studies [21] and hence confirmed the completion of
reaction between leaf extract and AgNO3. Absorption spectra of AgNPs formed in
the reaction media has absorption maxima in the range of 425 to 475 nm due to
surface Plasmon resonance of AgNPs.

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