A comparative study on

antibacterial activity of green

synthesized silver nanoparticles

Summer Internship

Presented by-
Abhijit Lodh
 AIM & OBJECTIVES

AIM: Green synthesis of silver nanoparticles using and its

antibacterial activity
OBJECTIVE
Green synthesis of Metal nanoparticles viz., silver nanoparticles

 Characterization by UV- visible Spectrophotometer and FTIR

analysis

To check antibacterial activity at different ratio 1:1,1:2, 1:5 as well as

at different RPM on different time period
 INTRODUCTION
• Nanotechnology, the manipulation and manufacture of materials and devices
on the scale of atoms or small groups of atoms.
• Nanotechnology is highly interdisciplinary, involving physics,
chemistry, biology, materials science, and the full range of the
engineering disciplines.
• There are two principal reasons for qualitative differences in material
behaviour at the nanoscale:
First, quantum mechanical effects come into play at very small dimensions
and lead to new physics and chemistry.
Second, a defining feature at the nanoscale is the very large surface-to-
volume ratio of these structures.
 APPLICATIONS

Source: https://www.researchgate.net/figure/Various-applications-of-nanotechnology_fig1_273217334
NANOPARTICLES
• Nanoparticles (NPs) are wide class of
materials that include particulate
substances, which have one dimension
less than 100 nm at least (Laurent et al.,
2010).

Different sized and shaped nanoparticles

 METAL NANOPARTICLES
Metal nanoparticles are submicron scale entities made of pure metals (e.g., gold,
platinum, silver, titanium, zinc, cerium, iron, and thallium) or their compounds (e.g.,
oxides, hydroxides, sulfides, phosphates, fluorides, and chlorides).

AuNPs AgNPs RuNPs & PtNPs CuNPs

Source: https://www.googleimages.com
 Methods of synthesis

Physical Method Chemical Method Biological Method

1. Mechanical 1. Co-precipitation 1. Using Plant Extracts

2. Vapour deposition 2. Sol-gel 2. Agriculture Wastes
3. Sputter deposition 3. Microemulsions 3. Enzymes
4. Electric arc deposition 4. Hydrothermal 4. Micro-organisms
5. Ion beam technique 5. Sonochemical (Bacteria, Fungi,
6. Molecular beam 6. Microwave Yeasts, etc.)
epitaxy
• Physical method: Vapour deposition method

o Utilizes the physical energies to produce

nanoparticle of narrow size distribution

o It is time and energy consuming,

o Synthesis occurs at high temperature & pressure

• Chemical method:
o Synthesis of nanoparticles by chemical reduction
using organic and inorganic reducing agents

o Simple, inexpensive & low temperature synthesis

method
Hydrothermal method
o Use of toxic reducing & stabilizing agents makes
it harmful
• Biological method:
o Using natural reducing agents such as
polysaccharides, plant extracts, or biological
micro-organisms such as bacteria and fungi

o Easy, efficient, and eco-friendly Using plant Using bacteria (E.coli) Using Fungi

Source: https://www.googleimages.com
 GREEN SYNTHESIS
 It is the use of biological material like plant Principles of green synthesis :
extracts, bacteria, fungi, etc which acts as the  Prevent waste
reducing agent for production of
 Less hazardous chemical synthesis
nanoparticles
 It reduces hazards to the global efforts and  Designing safer chemicals
helps in implantation of a sustainable  Safer solvents
process.
 Design for energy efficiency
 Green synthesis relys on the different process
parameters and reaction conditions such as  Use of renewable feedstock
temperature, pH, solvent medium, stirring  Reduce derivatives
time and reducing agents etc.  Catalysis
 Green synthesis method can be done via
three ways:  Design for degradation
• Using plants  Real time analysis for pollution prevention
• Using bacteria  Inherently safer chemistry for accident prevention
• Using fungi
 Advantages Disadvantages
Cost effective Time consuming process
Environment friendly Not much of manipulations can be
done
Easily scaled up for large scale
low yield produced
synthesis
Difficult to have control over shape,
There is no need to use high
size and crystallinity
pressure, temperature and energy
Low rate of synthesis
No toxic and hazardous by products
released
 SILVER NANOPARTICLES
• Size: 10nm-100nm
• Chemical formula: Ag
• Silver nanoparticles are ultra fine particles of silver and
they differ from the bulk silver as they have different
colors such as yellow, as opposed to the silver.
• Silver nanoparticles have a surface plasmon resonance in
the range of 400nm – 500nm.
• Incident light rays create oscillation in free electrons on
the surface of nanoparticles, causing them to absorb
electromagnetic radiation, creating different colors
reflected.
• AgNPs have unique properties (e.g., size and shape
depending optical, electrical, and magnetic properties)
which can be incorporated into antimicrobial
applications, biosensor materials, etc.
Source: Silver Nanoparticles: Synthesis, Characterization, Properties,
Applications, and Therapeutic Approaches; Xi-Feng Zhang et el; Int. J. Mol.
Sci. 2016
 Antimicrobial Activity

• Many studies suggest that AgNPs is involved in the

direct damage of the cell membrane which is the
principal mechanism of action of AgNPs.
• Silver nanoparticle adhere on the microbial cell wall
and thus this induces
o structural changes in the cell membrane and
increases cell permeability ,
o leading to a powerful toxic effect that is related
to the uncontrolled transport across cytoplasmic
membrane
• The electrostatic interactions are responsible in the
binding of the nanoparticles & the bacterial cell
membranes.
• The effect though is directly dependent on the size,
shape and concentration of AgNPs.
Mechanism of action
AgNPs
Respiratory
enzymes
1. Increases permeability
and disrupts peptidoglycan ROS
layer
4. ATP3. Attaches to
2. deletion
5. Destabilizes
Causes 30s
Phosphorus &
causesRibosomal
mitochondrial
cell Deathsubunit and
damages DNA
disfunction
inhibits protein synthesis

Bacterial
cell
Increases permeability of membrane.
Binds to thiol groups(-SH) in respiratory and
deactivates the enzyme.
Interacts with respiratory enzymes and generate
reactive oxygen species oxidative stress
apoptosis.
Interacts with DNA (phosphorus-containing
compound) and inhibits its replication.
Destructs peptidoglycan layer in cell wall.
Structural changes in bacterial cell wall and
nuclear membrane.
Binds to 30s ribosomal subunit and inhibits Microscopic image showing damaged (a) E.coli and
(b) S.aureus cells
protein synthesis.

Source: A progressive approach on inactivation of bacteria using silver–

titania nanoparticles; Jingbo Louise Liu et.al; Biomaterials Science; Issue 2,
2013
 Advantages Disadvantages
Easily available and does not require Argyria most common outcome of
rigorous processing. exposure to AgNPs(The most dramatic
Available Option for waste management. symptom of argyria is that the skin
turns purple or purple-grey).
Fast and cost effective approach.
Does not induce toxic NP.
AgNPs are the most powerful weapons
against the multidrug resistant bacteria
such as ampicillin-resistant Escherichia
coli, erythromycin–resistant
streptococcus pyogenes, methicillin-
Paul karason
resitant staphylococcus aureus.
Source: https://www.googleimages.com
 MATERIALS & METHODS
Sample collection

Extraction of plant extract

Formation of nanoparticles at different ratio with plant extract and Agno3 solution

Centrifugation of solution to isolate Nanoparticles

Antibacterial assay
Source: Biotechnological Applications of Green Synthesized Silver Nanoparticles; Nafeesa Khatoon,
Jahirul Ahmed Mazumder and Meryam Sardar ; Journal of Nanosciences: Current
Research 2017
 Synthesing of AgNPs

Silver
nitrate

Silver nitrate
AgNPs
solution
(Dark brown colour)

. ₃
 Optimization

(a) (b) (c) (a) (b) (c)

(a) Plant Extract with Distilled water. (a) Plant Extract : Silver nitrate( 1 : 1 )
(b) Plant Extract with methanol. (b) Plant Extract : Silver nitrate( 1 : 2 )
(c) Plant Extract with Acetone. (c) Plant Extract : Silver nitrate( 1 : 5 )
 CHARACTERIZATION
UV-Vis Spectrophotometry

Characterization

Fourier-transform infrared
AgNPs spectroscopy (FTIR)

Antibacterial Assay &

Antifungal Assay
 UV-Visible Spectrophotometry
1.4
4
1.2
3.5

3 1

Absorbance
2.5 0.8
Absorbance

2 0.6
1.5
0.4
1
0.2
0.5
0
0 300314328342356370384398412426440454468482496510524538552566580594
300315330345360375390405420435450465480495510525540555570585600
Wavelength, nm

a Wavelength, nm

Figure 2: UV-Vis spectra of :

(a) Synthesized AgNPS from

moringa oliefera
b)Synthesised AgNPs from water
hyacinth
 FTIR Analysis
• FTIR spectrum were obtained for the plant extract and the nanoparticles of
AgNPs. For FTIR the samples were prepared, the nanoparticle solution was
centrifuged and oven dried for the analysis and the plant extract was placed
under 40c. FTIR spectra of silver nanopartivles formed shows prominent
peak at 2924cm-, 1638cm-1 ,1384cm-, the peak at 1638cm- corresponds to
stretching vibration of (NH)(C=O).The peak at 1384cm _ corresponds to C-
C and C-N stretching vibrations while the peak at 2924 cm - showed
stretching vibrations of C‑H of methoxy compounds, the peak at 3421cm -
corresponds to OH stretching.(fig-2). FTIR spectra showed the
biomolecules responsible for capping and stabilization of metal
nanoparticles synthesized by plants extract. Similar results were also
obtained from nanoparticles formed by moringa oliefera extract.
 FTIR Analysis

FTIR analysis of silver nanoparticles made using plant

FTIR analysis of plant extract(water hyacinth)
extract(water hyacinth)
 FTIR Analysis

FTIR Analysis of silver nanoparticles made using plant

FTIR Analysis of plant extract (DRUMSTICK)
extract(DRUMSTICK)
 Anti bacterial Assay
• AntIbacterial studies were done using two different types of
bacteria E. coli and Pseudomonas Aeruginosa. Each of the
bacteria was grown in its selective media, E. coli was grown
using EMB and Mackonkey broth and was plated in
mackonkey agar and Pseudomonas was grown in citrimide
broth and was plated in citrimide agar. After that wells were
made using 1 ml tips and in each well 100µL of nanoparticle
solution was placed to check there anti-bacterialproperties
 ANTI-BACTERIAL ASSAY

Anti-bacterial studies of NP synthesized Anti bacterial studies of NP

by using Moringa extract against synthesis by using Water hyacinth
Pseudomonas.A against Pseudomonas.A
 Comparative study
Changes in Antibacterial effect of Ag nanoparticle Changes in Antibacterial effect of Ag nanoparticle
extracted from moringa leaves with respect to variation extracted from water hyancith leaves with respect to
of speed of centrifugation variation of speed of centrifugation:

Centrifu Zone of
gation Zone of Zone, Centrifug inhibition(mm)
speed inhibition Pseudomona ation Zone of ,
(RPM ) Time Ratio (mm) , E coli s aeruginosa speed inhibition, E Pseudomona
(RPM) Time Ratio coli(mm) s aeruginosa
10 1:2 15 20
6000 10 1:2 13 11
6000 10 1:5 13 19
10 1:5 14 11
15 1:2 17 16 9000 15 1:2 16 13
9000 15 1:5 13 23 15 1:5 16 10
20 1:2 12 16 20 1:2 14 13
12000 20 1:5 15 17 12000 20 1:5 13 11
 Acknoledgement
• The completion of any inter-disciplinary project depends upon cooperation, coordination and combined efforts of several
sources of knowledge. This was an excellent experience for me to undergo my summer training at an esteemed facility such
as Defence Research Laboratory, Tezpur and get acquainted with the research environment.
• I would like to forward my special sense of gratitude to Dr. Sanjai K. Dwivedi, Director, DRL Tezpur, who provided me this
opportunity to undertake my internship in this prestigious institution. I would also like to thank Dr. Nitin Desi, Director
Amity institute of Biotechnology , for his encouragement and support towards me for undertaking this project.
• I am heavily indebted to Dr. Soumya Chatterjee, Scientist ‘E’ and Dr. Rashmi Rekha Devi, Scientist ‘E’ for helping me
with their scientific knowledge in every step and his ever willingness to give me valuable advice and direction whenever I
approached him with a problem. I am also grateful to Mohan G Vairale, Scientist ‘D’ and Dr. Sonika Sharma, Scientist
‘C’ for planning and constructing the feasibility of the project and guiding me further at each and every step in order to
smoothly execute the project.
• I would specially like to thank Dr. Rama Dubey, Scientist ‘E’, Mr. Ajit Kumar Das, Technical officer ‘C’HRD officer for
taking out their precious time to help me out in the training program and familiarizing me with the rules and code of
conduct of DRL.
• I would also like to thank the Biodegradation Technology Division for allowing me to use their instruments whenever
required. Furthermore, I would like to convey my thanks to Dr. Sampriti Kataki (Research Associate) for helping me out in
the intricacies of research work and providing assistancefor making me learn better, Mr. Manoj and Mr. Sunil from
Biodegradtaion Technology Division, DRL for extending their helping hand in whenever and wherever required.
• Thanking the staff , for helping me with a part of my project work.
• I would like to thank my parents for their love, social, moral support and encouragement throughout my career.
Abhijit Lodh
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