Avinash M.phil Thesis

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Characterization and application of silver

nanoparticles biosynthesized under control and
clinorotation...
Thesis · August 2013

DOI: 10.13140/RG.2.1.3076.5600
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Avinash Jagannath Aher

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CHARACTERIZATION AND APPLICATION OF SILVER
NANOPARTICLES BIOSYNTHESIZED UNDER CONTROL AND
CLINOROTATION CONDITION
A THESIS SUBMITTED
TO THE UNIVERSITY OF PUNE
FOR THE AWARD OF THE DEGREE OF
MASTER OF PHILOSOPHY (M. Phil)
IN
PHYSICS
BY
AVINASH JAGANNATH AHER
UNDER THE GUIDANCE OF
PROF. PANDIT B. VIDYASAGAR
DEPARTMENT OF PHYSICS
UNIVERSITY OF PUNE
PUNE, INDIA
MAY 2013
i
“If you do not hope, you will not find what is
beyond your hopes.”
-St. Clement of Alexandra
Dedicated to....
My Family!!
i
CERTIFICATE OF THE GUIDE
This is to certify that the work presented in this thesis entitled,

“Characterization and application of silver nanoparticles biosynthesized
under control and clinorotation condition”, submitted by Mr. Avinash
Jagannath Aher to the University of Pune for the award of Master of Philosophy
in Physics, and was carried out by the candidate under my supervision. Material
obtained from other sources has been duly acknowledged in the thesis.
Prof. Pandit B. Vidyasagar

(Supervisor)
Biophysics Laboratory
Department of Physics
University of Pune
PunE-411007
India.
ii
DECLARATION BY THE CANDIDATE
I hereby declare that the work in the present thesis entitled,

“Characterization and application of silver nanoparticles biosynthesized
under control and clinorotation condition” is as account of the original work
carried out by me. This work or part of this work thereof has not been previously
submitted to any other university or Institute for the award of any degree or
diploma.
Mr. Avinash Jagannath Aher

(Candidate)
Forwarded through:
Prof. Pandit B. Vidyasagar

(Supervisor)
iii
ACKNOWLEDGEMENTS
During the past two years as a research student, I have been inspired, assisted and
guided by a number of people. I take opportunity to express my heartfelt feelings at this
stage of my career.
Prof. Pandit B. Vidyasagar deserves my highest and sincere regards as a guide and
philosopher. He has always made himself available for very frank, open and illuminating
discussions. His constant encouragement and inspiration, deep insight and practical
approach have played a vital role in showing me the right path and seeing me to the end
of it.
It is with great pleasure that I express my gratitude to my co-guide Dr. Suresh W.
Gosavi, who through his inspiring guidance helped me in taking up this problem and
completing the thesis work within the stipulated time.
I express my sincere thanks to Dr. Gauri kulkarni, Director, School of basic
medical Sciences for fruitful suggestions during my M.Phil work. I am also thankful to
Department faculty members especially Prof. R. N Karekar, Prof. S. K. Date, Prof. V. N.
Boraskar, Dr. A. G. Banpurkar, Dr. N. B. Chaure for the encouragement and support.
I am thankful to Prof. Kisan M. Kodam and Mr. Vijay L. Markad from
Biochemistry division, Department of Chemistry, University of Pune for their
cooperation during our collaborative work.
I would like to thanks the present and former Head of the Department of Physics
for availing all the facilities at department. I am also thankful to non-teaching staff of
department for their help in characterization especially Mr. Jagtap, Mr. Shinde, Mr.
Lolage and Mr. Krishna.
I owe sincere thank to all my friends Laxman Tatikondewar, Shankar Kekade,
Prashant Gaikwad, Nilesh Kanhe and Dinesh Mali for their support and encouragement. I
would like to thank all my lab mates Sandhya Singh, Jyotsana Dixit, Sarika Hinge, Mr. S.
M. Kamble, Mr. Atul Khire, Mr. Vijay Ghodake and all my seniors Dr. Sagar Jagtap, Dr.
Vivek Jadhav, Dr. Santosh Bhaskaran, and Dr. Vijay Ghadage who helped me in many
ways.
iv
No words would be appropriate to express my gratitude towards my family who
inspired me to dream and pursue my dreams to reality. My elder brothers Sandip and
Santosh have shouldered all responsibility of our family allowing me to concentrate on
my work. This thesis would not have been possible without constant encouragement,
love, patience and understanding of my family. I appreciate the affection and concern
shown by my family towards my academic pursuit.
Last, but not the least, financial support from Centre for Nanomaterials and
Quantum Systems (CNQS) in form of Project Assistant Fellowship is greatly
acknowledged.
Avinash Jagannath Aher
v
Table of Contents
Chapter 1. General Information ................................................................................... 1
1.1 Introduction .......................................................................................................... 1

1.2 Methods for synthesis of nanomaterials ............................................................... 1
1.3 Bio-nanotechnology ............................................................................................. 2
1.4 Toxic effects of Nanomaterials ............................................................................ 2
1.5 Microgravity ......................................................................................................... 4
1.6 Objective of thesis ................................................................................................ 4
1.7 Plan of thesis ........................................................................................................ 5
1.8 Bibliography ......................................................................................................... 6
Chapter 2. Novel route for biosynthesis of silver nanoparticles using mushroom P.

badius and assessment of their cyto-genotoxicity ........................................................... 8
2.1 Introduction .......................................................................................................... 8

2.2 Methodology: ....................................................................................................... 9
2.3 Results and Discussion: ...................................................................................... 10
2.4 Conclusion:......................................................................................................... 18
2.5 Bibliography: ...................................................................................................... 19
Chapter 3. Green synthesis of subnano sized silver atomic clusters using fruit of
medicinal herb Rauwolfia serpentina ............................................................................ 21
3.1 Introduction ........................................................................................................ 21

3.2 Methodology ...................................................................................................... 22
3.2.1 Synthesis of silver clusters and nanoparticles ............................................. 22

3.2.2 Characterization of silver clusters and nanoparticles .................................. 23
3.3 Result and Discussion ........................................................................................ 23
3.3.1 UV-Visible and Photoluminescence spectroscopy ..................................... 23

3.3.2 Energy dispersive spectroscopy .................................................................. 24
3.3.3 Transmission electron microscopy ............................................................. 25
3.4 Conclusion .......................................................................................................... 27

3.5 Bibliography ....................................................................................................... 27
vi
Chapter 4. Effect of clinorotation on biosynthesis of silver nanoparticles. ............ 29
4.1 Introduction ........................................................................................................ 29

4.2 Methodology ...................................................................................................... 31
4.3 Result and Discussion ........................................................................................ 35
4.3.1 UV-Visible Spectroscopy analysis.............................................................. 35

4.3.2 X-Ray Diffraction (XRD) Analysis ............................................................ 36
4.3.3 Energy Dispersive Spectrometer (EDS) Analysis ...................................... 37
4.3.4 Photoluminescence (Pl) Analysis ............................................................... 38
4.3.5 Transmission Electron Microscopy (TEM) Analysis ................................. 39
4.3.6 Cyto-genotoxicity assesment ...................................................................... 42
4.4 Conclusion .......................................................................................................... 46

4.5 Bibliography ....................................................................................................... 46
Chapter 5. Conclusion and Future scope ................................................................... 50
5.1 Summary ............................................................................................................ 50
5.1.1 Studies with P.badius.................................................................................. 51

5.1.2 Studies with Rauwolfia Serpentina fruit ..................................................... 51
5.1.3 Studies with Clinostat ................................................................................. 52
5.2 Future Scope ....................................................................................................... 52
vii
List of Figures
Figure 1.1: Different methods for synthesis of nanomaterials. .......................................... 2

Figure 1.2: Optical micrograph of human glioblastma cell (U251). A) Untreated B)
treated with silver nanoparticles [11]. ................................................................................. 3
Figure 1.3: TEM image of ultrathin section of cell showing silver nanoparticles
accumulated in different parts of cells [11]. ....................................................................... 3
Figure 2.1: Mushroom Polyporus badius ........................................................................... 9
Figure 2.2: UV-Vis absorption spectra of silver nanoparticles synthesized by P. badius
with concentration from 2 to 12 mM of AgNO3. .............................................................. 11
Figure 2.3- Represents X-ray diffraction pattern of silver nanoparticles synthesized using
P.badius. ........................................................................................................................... 12
Figure 2.4: Represents EDS profile of silver nanoparticles synthesized using P.badius. 12
Figure 2.5: Representative FTIR Spectrum of silver nanoparticles synthesized using
P.badius ............................................................................................................................ 13
Figure 2.6: Representative TEM image of silver nanoparticles synthesized from P.
badius. ............................................................................................................................... 14
Figure 2.7: Particles size distribution............................................................................... 14
Figure 2.8: The Selected Area Electron Diffraction (SEAD) pattern of silver
nanoparticle. ...................................................................................................................... 14
Figure 2.9: HRTEM Image of silve nanoparticle synthesized using P.badius. ............... 14
Figure 2.10: DNA damage (% Tail DNA) in cervical cancer cells (HeLa) (A), and
human skin keratinocytes (B) exposed to 10, 25, 50 and 100µg/ml of synthesized silver
nanoparticles. (C) and (D) presents representative comets images of HeLa and HaCaT
cells. .................................................................................................................................. 15
Figure 2.11: Viability of blood lymphocytes when exposed with different concentration
of AgNPs ........................................................................................................................... 16
Figure 2.12: % Tail DNA damage in blood lymphocytes incubated with different
concentrations of AgNPs .................................................................................................. 16
Figure 2.13: %ROS generation in blood lymphocytes incubated with different
concentrations of AgNPs. ................................................................................................. 17
Figure 3.1: Sarpagandha Plant ......................................................................................... 22
viii
Figure 3.2: UV-Visible absorption spectra of reaction mixture. ..................................... 24
Figure 3.3: Photoluminescence spectra of reaction mixture (λ-ex is 220 nm). ............... 24
Figure 3.4: Representative EDS profile of silver nano and subnano atomic clusters. ..... 25
Figure 3.5: TEM images of sub-nano silver atomic clusters at different magnifications.26
Figure 3.6: Two major size distributions observed in reaction mixture after synthesis
analysed from TEM images. ............................................................................................. 26
Figure 3.7: a) SAED pattern and b) HRTEM image of silver nanoparticles and atomic
clusters synthesized from Sarpagandha fruit extract......................................................... 27
Figure 4.1: Experimental setup ........................................................................................ 32
Figure 4.2: a) Under 1 g conditions, particles will sediment and take a spatial position
that is determined by their weight (G) and buoyancy. b) Under free fall conditions,
particles distribute homogenously as sedimentation is abolished. c) A homogenous
particle distribution can also be achieved on ground by rotating a suspension. ............... 33
Figure 4.3: The path of fall of particles within the fluid during clinorotation. Forces other
than gravity have been neglected. [13] ............................................................................. 34
Figure 4.4: UV-Vis spectra: recorded from the aqueous 5mM reaction mixture after 24
hrs of reaction in controlled (1g) and Clinorotated (simulated microgravity) conditions.
Inset figure shows the color of reaction mixtures at the time of absorption measurement
(A- Reaction mixture in simulated microgravity, B- Reaction mixture in 1-g, C- Luria
Broth and D- AgNO3 Solution). ........................................................................................ 36
Figure 4.5: X-ray diffraction pattern of silver nanoparticles synthesized in clinorotation
(Black) and1g condition (Red). ......................................................................................... 37
Figure 4.6: EDS profile for silver nanoparticles synthesized in 1g. ................................ 38
Figure 4.7: EDS profile for silver nanoparticles synthesized in simulated microgravity
condition. .......................................................................................................................... 38
Figure 4.8: The emission spectra of silver nanoparticles synthesized in 1g (in red) and
simulated microgr condition (in black). ............................................................................ 39
Figure 4.9: Representative images of silver nanoparticles synthesized in 1 g condition.
Inset figures (a) represents electron diffraction pattern and figure (b) represent HRTEM
image. ................................................................................................................................ 40
ix
Figure 4.10: Representative images of silver nanoparticles synthesized in simulated
microgravity condition. Inset figures (a) represents electron diffraction pattern and figure
(b) represent HRTEM image. ........................................................................................... 40
Figure 4.11: Particles size distribution for silver nanoparticles synthesized in 1 g
condition ........................................................................................................................... 41
Figure 4.12: Particles size distribution for silver nanoparticles synthesized in simulated
microgravity condition. ..................................................................................................... 41
Figure 4.13: Toxic effects on Human Skin Keratinocytes ............................................... 42
Figure 4.14: Toxic effects on Cervical cancer cells ......................................................... 43
Figure 4.15: Possible reduction mechanism .................................................................... 44
x
Chapter 1. General Information
1.1 Introduction
Nanotechnology refers to the technology of matter manipulation at atomic level. It

is an area in which traditional disciplines converge. Nowadays nanotechnology has made
remarkable contribution in field of technology by means of its vital applications.
Nanotechnology involves synthesis and characterization of nanomaterials. Nanomaterials
have proven to be more effective in many fields as compare to their bulk form such as
nano metals, nano semiconductors, nano inorganic and organic materials are the few
examples. Bottom up and top down approaches of synthesis nanomaterials provides
variety in synthesis methods which leads to variety in products. It is well accepted that
properties of nanomaterials are size and shape dependent which keeps research busy is
finding new protocols/methods to achieve fine control over size and shape of
nanomaterials [1].
1.2 Methods for synthesis of nanomaterials
The large numbers of techniques are available to synthesize different types of

nanomaterials in the form of particles, colloids, thin film, tubes, rods etc. The Figure 1.1
shows different methods for synthesis of nanomaterials. The technique to be used
depends upon the materials of interest and type of nanostructures viz. quantum dots, nano
wires, nano rods and nano plates as per requirement [2].
1
Figure 1.1: Different methods for synthesis of nanomaterials.
1.3 Bio-nanotechnology
The nanostructures in biological systems are not new as bio-molecules function and
interact at nanoscale since evolution [3]. The biological world, animal kingdom, plants
and microorganisms make use of materials in their metabolic processes and produces
different derivatives of the same. In many situations the inter conversion of materials or
interaction of bio-molecules with materials (organic and inorganic both) degrade or
reduces the materials to nanoscale [4–8]. These biologically reduced nanoscale materials
have distinct properties than chemically synthesized nanomaterials. The green route of
synthesis makes them more biocompatible than other methods which are helpful for
further application in medical field.
1.4 Toxic effects of Nanomaterials
It has been shown that humans have always been exposed to tiny particles via dust
storms, volcanic ash and other natural processes. But human body is well adapted to
protect us from these foreign particles. Body has developed special kind of mechanism to
neutralize and eliminates these contaminations. The tiny particles existed since
millennium which have been produced due to human activity e.g. smoke from
combustion and lint from garments but the technological advancement has significantly
changed the characteristics of this particulate pollution [9], [10].
The important aspect of nanoparticle toxicity is their size which is smaller than the
size of cell and sub cellular particles which allows them to penetrate these biological
structures and disrupting their normal function. These toxic effects include tissue
inflammation, alteration of cellular pathways and causing imbalance in redox balance
inside the cell which further results in abnormal cell death [11]. The following are few
results showing penetration of nanoparticles in cells and effects on cell morphology after
nanoparticle exposure [11].
The work done by Asharani and co-workers has shown that interaction of
nanoparticles with cells changes the morphology of cells. The Figure 1.2A shows healthy
cells of human glioblastma (U251) which were not treated with silver nanoparticles on
2
the other hand Figure 1.2B shows the changed morphology in treated cells [11].
Nanoparticle treated cells appeared to be clustered with a few cellular extensions, and cell
spreading patterns were restricted as compared to control cells. This could be due to
disturbances in cytoskeletal functions as a consequence of nanoparticle treatment [11].
The Figure 1.3 shows TEM image of ultrathin section of cancer cell treated with silver
nanoparticles. Silver nanoparticles were found distributed throughout the cytoplasm [11].
Figure 1.2: Optical micrograph of human glioblastma cell (U251).

A) Untreated B) treated with silver nanoparticles [11].
Figure 1.3: TEM image of ultrathin section of cell showing silver

nanoparticles accumulated in different parts of cells [11].
3
1.5 Microgravity
Earth creates a gravitational force which attracts objects with force inversely
proportional to the square of the distance between the centre of object and centre of earth.
The acceleration acted by only earth’s gravitational field on object is commonly referred
as 1g or earth’s gravity and this acceleration is approximately 9.8 m/s2.
Microgravity environment is one in which acceleration imparted on objects is 10-6
times the measured at Earth’s surface. Microgravity environment can be created in two
ways. Gravitational field of earth diminishes with distance, by travelling away from earth
one can achieve microgravity environment. But to reach the point where gravitational
force is reduced to one millionth of that at the surface, we would have to travel into space
a distance of 6.37 million kilometres from the earth but this approach is impractical. To
overcome this problem condition of free fall certainly helps us to create or simulate
microgravity environment using drop towers, parabolic aircraft flights, and sounding
rockets. The common problem with these techniques is that, the microgravity
environment created by these laboratories lasts for few seconds to minutes. Space shuttle
provides solution over this problem which orbits earth and its centrifugal acceleration of
revolution counterbalances the earth’s gravitational pull. The space shuttle is therefore in
a state of free fall around the earth for months to years. Clinostats and random positioning
machines are also a good gravity simulators used nowadays in microgravity research.
These machines mimics free fall condition in ground based laboratories and provides the
simulated microgravity or zero gravity condition for months to years [12].
Since from the earth appeared as solid planet, gravitational acceleration of earth’s
surface has been constant. The existence of life on planet earth is thought to be of 4
billion years formed by spontaneous aggregation of molecules [13]. Life has evolved in
more complex form in further billion years and during evolution gravity has been only
constant factor. Not only in processes involved in living world, gravity also played a vital
role in solidification and, aggregation and convective transports in materials [14], [15].
1.6 Objective of thesis
o To find out novel routes for biosynthesis of silver nanoparticles.
4
o To investigate effects of clinorotation (simulated microgravity) on biosynthesis of
silver nanoparticles.
o To assess cytogenotoxicity of silver nanoparticles against human peripheral blood
lymphocytes, Human skin keratinocytes (HaCaT) and cervical cancer cells
(HeLa) synthesized using Rauwolfia serpentina and P.badius extract.
o To investigate toxicity of silver nanoparticles synthesized under clinorotation and
normal gravity condition.
The chapters in the thesis are written in the form of concise manuscripts and
detailed literature survey is given in respective chapters.
1.7 Plan of thesis
Chapter 1 is having idea about nanomaterials and different methods for

synthesizing nanomaterials. It also includes detailed discussion about toxic effects of
nanoparticles. In the chapter 1 the general information about microgravity, simulation of
microgravity and application also included. The chapter 1 ends with providing objective
and plan of thesis.
Chapter 2 gives detail information about biosynthesis of silver nanoparticles using
extract of mushroom P. badius. The silver nanoparticles characterization using UV-
visible spectroscopy, XRD, TEM, EDS and Photoluminescence spectroscopy also
described in the chapter. Furthermore the cytogenotoxicity of silver nanoparticles against
human peripheral blood lymphocytes, human skin keratinocytes and cervical cancer cells
estimated using alkaline comet assay is given at the end of the chapter.
Chapter 3 provides literature survey about synthesis and application of metallic
clusters. Moreover the novel green route for synthesis of silver clusters and nanoparticles
using fruit extract of medicinal herb Rauwolfia serpentina and its characterization also
give in this chapter.
Chapter 4 shades light on effects of clinorotation (simulated microgravity) on
biosynthesis of silver nanoparticles. Chapter also gives detailed information about
simulation of microgravity using clinostat, experimental setup for nanoparticles synthesis
in clinorotation and characterization done after synthesis. At the end of the chapter
5
toxicity investigation of silver nanoparticles synthesized in normal and simulated
microgravity is given.
Chapter 5 connects the work presented in thesis and provides summary and future
scope of the thesis.
1.8 Bibliography
[1] T. Pradeep, “Nano: The expanding Horizon,” in A textbook of Nanoscience and

nanotechnology, New Delhi: The MacGraw Hill, 2012, pp. 3–21.
[2] T. Pradeep, Nanoscience and Nanotechnology. 2012.
[3] N. Vigneshwaran and P. Jain, “Biomolecules-Nanoparticles Interaction in Nano
scale,” in Metal Nanoparticles in Microbiology, N. D. Mahendra Rai, Ed. Springer
Berlin Heidelberg, 2011, pp. 135–150.
[4] N. Jain, A. Bhargava, S. Majumdar, J. C. Tarafdar, and J. Panwar, “Extracellular
biosynthesis and characterization of silver nanoparticles using Aspergillus flavus
NJP08: a mechanism perspective.,” Nanoscale, vol. 3, no. 2, pp. 635–41, Feb.
2011.
[5] H. J. Bai, Z. M. Zhang, Y. Guo, and G. E. Yang, “Biosynthesis of cadmium sulfide
nanoparticles by photosynthetic bacteria Rhodopseudomonas palustris.,” Colloids
and surfaces. B, Biointerfaces, vol. 70, no. 1, pp. 142–6, Apr. 2009.
[6] D. Philip and C. Unni, “Extracellular biosynthesis of gold and silver nanoparticles
using Krishna tulsi (Ocimum sanctum) leaf,” Physica E: Low-dimensional Systems
and Nanostructures, vol. 43, no. 7, pp. 1318–1322, May 2011.
[7] S. Ashraf, A. Z. Abbasi, C. Pfeiffer, S. Z. Hussain, Z. M. Khalid, P. R. Gil, W. J.
Parak, and I. Hussain, “Protein-mediated synthesis, pH-induced reversible
agglomeration, toxicity and cellular interaction of silver nanoparticles.,” Colloids
and surfaces. B, Biointerfaces, vol. 102, pp. 511–8, Feb. 2013.
[8] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M. I. Khan, R. Kumar, and M.
Sastry, “Extracellular biosynthesis of silver nanoparticles using the fungus
Fusarium oxysporum,” Colloids and Surfaces B: Biointerfaces, vol. 28, no. 4, pp.
313–318, May 2003.
6
[9] K. Batsungneon and T. Kulworawanichpong, “Effect of Dust Particles in Local
Rice Mills on Human Respiratory System,” pp. 260–265, 2011.
[10] C. Buzea, I. I. Pacheco, and K. Robbie, “Nanomaterials and nanoparticles: sources
and toxicity.,” Biointerphases, vol. 2, no. 4, pp. 17–71, Dec. 2007.
[11] P. V Asharani, G. Low, K. Mun, M. P. Hande, and S. Valiyaveettil, “Cytotoxicity
and Genotoxicity of Silver,” ACS Nano, vol. 3, no. 2, pp. 279–290, 2009.
[12] Gilles Clement, “Microgravity,” in Fundamentals of space medicine, EI Segundo,
California: Springer US, 2005, p. 4.
[13] S. J. William, “Microfossils of the Early Archean Apex Chert: New Evidence of the
Antiquity of Life,” Science, vol. 260, no. 5108, pp. 640–646, 1993.
[14] S. Bhaskaran, J. S. S., and V. P. B., “Life and Gravity,” Biophysical Reveiews and
Letters, vol. 4, no. 4, pp. 299–318, 2009.
[15] H. Ahari, R. L. Bedard, C. L. Bowes, T. Jiang, N. Coombs, G. A. Ozin, S. Petrov,
and I. Sokolov, “Effect of microgravity on the crystallization of a self- assembling
layered material,” vol. 388, no. August, pp. 857–860, 1997.
7
Chapter 2. Novel route for biosynthesis of silver nanoparticles using
mushroom P. badius and assessment of their cyto-genotoxicity
2.1 Introduction
In recent years, biologically synthesized silver nanoparticles have drown

considerable attention of researchers because of their bio-compatibility and wide range of
applications in medicinal field for combating microbes, bio-labelling and as an
antimicrobial agent[1]. Several biological systems such as bacteria, fungi and plant
biomass or extract are reported for synthesis of AgNPs [1]. In this letter, we demonstrate
a novel and rapid route for the synthesis of silver nanoparticles using aqueous extract of
Polyporus badius (P. badius) (Figure 2.1). The Table 2.1 gives scientific classification
of P. badius. Literature survey has shown that polyporus mushrooms have not been
investigated for the synthesis of silver nanoparticles. Since from ancient times,
mushrooms have long been associated with human life as a food and medicine because of
their agreeable medicinal properties and nutritious values. Mushroom belong to the this
genus viz. P. gilvus, P. sulphureus, P. annosus, P. radiatus, P. pinicola, P. volvatus, P.
fomentarius, P. stevenii and P. badius are well known for antioxidant activity and they
also play a major role in protection from oxidative damage [2]. Given increasing use of
AgNPs in medicine and food industry; their effects on environmental health is of
increasing concern because AgNPs have been reported for toxic effects on different cell
lines [3]. This unevenness in properties motivated us to screen the cyto-genotoxicity of
synthesized AgNPs on cervical cancer (HeLa) cell lines, human skin keratinocytes
(HaCaT) and Human peripheral blood lymphocytes using alkaline comet assay [4].
Table 2.1: Scientific Classification of P. badius
Kingdom Fungi
Division Basidiomycota
Class Agaricomycetes
Order Polyporales
Family Polyporaceae
Genus Polyporus
Species P. badius
8
Figure 2.1: Mushroom Polyporus badius
2.2 Methodology:
The mushroom P. badius was washed with distilled water, dried, and crushed to
fine powder. For the aqueous extraction, 8 g of crushed powder was added to 100 ml
distilled water, boiled for 15 min, and then filtered through Whatman filter paper (No.1).
Filtrate was collected and used for biosynthesis of nanoparticles. A stock solution of 1M
AgNO3 was added to 10 ml of fungal extract to get final concentration of 2, 4, 6, 8, 10,
and 12mM and placed in boiling water bath for 5 min. The colour of reaction mixture
changed from pale yellow to dark brown indicates the synthesis of silver nanoparticles
[5]. The nanoparticles were characterized using UV-visible spectrophotometer (JASCO
V-670), X-ray diffractometer (BRUKER AXS D8) with a Cu kα (λ=1.54 A°),
Transmission Electron Microscopy (TEM-TECNAI G2 20U-TWIN), EDS (JEOL JSM
6360A) and FTIR (JASCO FT/IR 6100).
The synthesized nanoparticles were investigated for their cyto-genotoxic effects on
human skin keratinocytes (HaCaT), cervical cancer (Hela) cell lines and human
peripheral blood lymphocytes. The HaCaT and HeLa cells were obtained from National
centre for Cell Sciences, Pune, India and human peripheral blood lymphocytes were
isolated using lymphocyte separation medium (Himedia, India) from the blood sample
collected from healthy volunteers. Cells were washed thrice with phosphate buffered
saline (PBS) and finally suspended in RPMI 1640 medium (Hyclone, Thermo Scientific)
supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100
µg/ml streptomycin (Sigma, St. Louis, USA).
9
Cell viability, DNA damage, and ROS generation was measured in the cells
exposed to the varying concentrations of AgNPs. To measure the DNA damage, the cell
suspension was mixed with 90µL of 0.55% low-melting agarose, and added to slides pre-
coated with 1.0% normal-melting agarose and kept on ice. After solidification of the
agarose, slides were covered with another 90µL of 0.5% low-melting agarose and then
immersed in lysis buffer (2.5M NaCl, 100mM EDTA, 10mM Tris, 1% Triton X-100, and
10% DMSO) for 2 h at 4°C. The slides were placed for 25 min into an electrophoresis
tank containing freshly prepared, chilled alkali buffer (300mM NaOH, 10mM Na2EDTA,
pH> 13.0) for unwinding of the DNA. Electrophoresis was carried out at 1 V/cm and 300
mA for 25 min at 4°C. Slides were washed thrice with neutralizing buffer (0.4 M Tris,
pH 7.5) for 5 min at 4°C to neutralize the excess alkali. Slides were then stained with
75µL of ethidium bromide and observed under the fluorescence microscope (Olympus
CX41, Japan) equipped with CCD camera. Cell viability was measured by Trypan blue
exclusion assay [6]. Intracellular ROS generation was measured by using a fluorescent
probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA). The assay is based on the ROS-
dependent oxidation of non-fluorescent DCFH-DA to fluorescent dichlorofluorescein
(DCF). After the exposure to AgNPs, cells were washed with PBS and further incubated
with DCFH-DA (10 µM) for 30 min in dark and fluorescence generated was measured on
spectrofluorometer (PERKIN ELMER LS-55) [7]. The fluorescence intensity was
recorded at an excitation wavelength of 485 nm and emission wavelength of 525 nm. The
alkaline comet assay was used to study whether exposure to silver nanoparticles damages
cellular DNA. The comet assay was carried out according to the method of Singh et. al.
[4]. A total 150 cells per concentrations were scored to calculate the amount of DNA in
Tail region using computer-assisted image analysis system, CASP software.
2.3 Results and Discussion:
The extract obtained from P. badius was pale yellow in colour with pH 6.3. When
aqueous solution of AgNO3 was added in extract to make desired final concentration,
initial pale yellow colour of the mixture was changed to brown in 5 minutes of incubation
in boiling water bath. The appearance of brown colour was due to the surface plasmon
resonance of silver nanoparticles [8]. AgNPs were synthesized for different
10
concentrations of AgNO3 ranging from 2 to 12 mM and monitored by UV-visible
spectrophotometer to understand the effect of silver salt concentration on synthesis
mechanism as shown in Figure 2.2. Literature shows that the surface plasmon resonance
of the nanoparticles depends on its size and shape [8]. The 2 mM reaction mixture
showed absorption at 429 nm, 4 mM at 433 nm, 6 mM at 440 nm, 8 mM at 448 nm, 10
mM at 449 nm and 12 mM at 453nm, respectively. The surface plasmon resonance
shifted towards higher wavelength with increasing concentration of AgNO3. It indicates
that size of the silver nanoparticles increases with increasing concentration of silver salt,
which may be due to the higher concentration of Ag ions in reaction mixture that
facilitate the aggregation of silver and growth of silver AgNPs [8].
The XRD pattern of silver nanoparticles is shown in Figure 2.3. The diffraction at
2θ = 38.1°, 44.2°, 64.4° and 77.4° shows intense peak corresponding to Bragg’s
reflection of silver atomic planes (111), (200), (220) and (311), respectively. X-ray
diffraction pattern illustrate that the silver nanoparticles formed are crystalline in nature
with face centred cubic structure (JCPDS- File no. 04-0783). The average size of silver
nanoparticles calculated by Debye-Scherrer’s formula and is found to be ~13 nm. The
distinct signal around 3KeV energy in EDS profile as shown in Figure 2.4, indicates the
presence of atomic silver in the substance with signal for oxygen, these results are
consistent with earlier report on synthesis of silver nanoparticles by fusarium oxysporum
[1].
Figure 2.2: UV-Vis absorption spectra of silver nanoparticles synthesized by P.

badius with concentration from 2 to 12 mM of AgNO3.
11
Figure 2.3- Represents X-ray diffraction pattern of silver nanoparticles
synthesized using P.badius.
Figure 2.4: Represents EDS profile of silver nanoparticles synthesized

using P.badius.
FTIR analysis has carried out in order to investigate the role of functional group
present in extract of P. badius for capping and stabilization of Ag nanoparticles.
Representative FTIR spectrum is shown in Figure 2.5. The spectra shows absorption
peak located at 3341 cm-1 that revealed the presence of secondary amides (N-H
stretching), where as peaks at 1643 and 1588 cm-1 show their corresponding bending
vibrations. The peaks at 2926 cm-1 and 2352 cm-1 show the presence of alkanes (-CH2)
and charged amines (NH stretching), respectively. The absorption peaks at 1755, 1340
and 1046 cm-1 revealed the presence of carbonyl stretching, sulphonic chloride (S=O
12
stretching) and C-N stretching, respectively. The presence of these functional groups on
AgNPs suggests that the mushroom biomolecules may be responsible for the reduction of
silver salt to Ag0 and stabilization of the Ag NPs.
Figure 2.6 shows representative TEM image of silver nanoparticles. The
nanoparticles were found to be spherical with size range of 4-23 nm. The size of silver
nanoparticles estimated from XRD pattern by using Debye-Scherer formula are fairly in
good agreement with the size measured by TEM analysis. Due to the capping of proteins
present on surface of nanoparticles they are not segregated [9]. Figure 2.7 and Figure 2.8
show the particles size distribution and electron diffraction pattern of silver nanoparticles.
The narrow size distribution has been observed in silver nanoparticles synthesized by this
route. Distinct rings in electron diffraction pattern confirmed the crystalline nature of
silver nanoparticles. Figure 2.9 shows HRTEM image of one of AgNPs. The separation
in inter atomic planes was observed to be 2.25Å. With this merits, P. badius becomes a
promising candidate for green synthesis of silver nanoparticles.
Figure 2.5: Representative FTIR Spectrum of silver nanoparticles

synthesized using P.badius
13
Figure 2.7: Particles size distribution.
Figure 2.6: Representative TEM image of
silver nanoparticles synthesized from P.
badius.
Figure 2.9: HRTEM Image of silver

Figure 2.8: The Selected Area Electron
nanoparticle synthesized using P.badius.
Diffraction (SEAD) pattern of silver
nanoparticle.
14
Figure 2.10: DNA damage (% Tail DNA) in cervical cancer cells (HeLa) (A), and
human skin keratinocytes (B) exposed to 10, 25, 50 and 100µg/ml of synthesized silver
nanoparticles. (C) and (D) presents representative comets images of HeLa and HaCaT
cells.
15
100
* *
80
**
Cell viability (%)

60 ***
40
20
0
Control 10 25 50 100 200
AgNPs concentration (µg/ml)
Figure 2.11: Viability of blood lymphocytes when exposed with

different concentration of AgNPs
30
***
25
***
***
20
***
% Tail DNA
15
**
10
0
Control 10 25 50 100 H 2O 2
Figure 2.12: % Tail DNA damage in blood lymphocytes incubated

with different concentrations of AgNPs
16
200 *
180
160
140
% ROS generation
120
100
80
60
40
20
0
Control 10 25 50 100 H2O2
Figure 2.13: %ROS generation in blood lymphocytes incubated with
different concentrations of AgNPs.
Silver nanoparticles are known to have cytotoxic and genotoxic potential in various
model systems viz. human lung fibroblast cells (IMR-90), human glioblastoma cells
(U251), human change liver cells, and human peripheral blood cells [3], [10], [11]. In the
present study, cyto-genotoxic effects of the synthesized AgNPs were investigated on
cervical cancer (HeLa) cells, human skin keratinocytes (HaCaT) and human peripheral
blood lymphocytes using array of bioassay viz. cell viability, DNA damage, and ROS
generation. The DNA damaging potential of the synthesized nanoparticles was evaluated
using alkaline comet assay as it detects several types of damages viz. alkali labile sites,
strand breaks (single and double), and incomplete excision repair sites [12], [13]. The cell
viability of blood lymphocytes exposed to the Ag NPs was found to be decreased with
increasing concentrations of Ag NPs and cell survival rate decreased to about 60% for
200µg/ml (Figure 2.11), Therefore, based on our cell viability data four concentrations of
Ag NPs, viz.10, 25, 50 and 100µg/ml were chosen for further analysis. As shown in
Figure 2.10 and Figure 2.12, a clear dose dependent DNA damage (% tail DNA) was
17
observed in the human skin keratinocytes, cervical cancer cells and human peripheral
blood lymphocytes exposed to Ag NPs when compared with untreated cells. Figure 2.10
also shows representative comet images of (C) HeLa and (D) HaCAT, respectively.
Similar observations were reported by Flower and co-workers in the blood cells exposed
to silver nanoparticles for 3h [11]. A possible mechanism of cytotoxicity involves
disruption of the mitochondrial respiratory chain by Ag NP; leading to increased ROS
production and affects ATP synthesis, which in turn damages cellular DNA [3], [11]. To
understand the role of ROS in DNA damage, ROS generation was determined using
dichloroofluorescein diacetate based fluorescence assay [14]. A significant (p<0.05)
concentration-dependent increase in ROS generation in blood lymphocytes was observed
after exposure to Ag NPs (11, 26, 34, 44 and 64% at 10, 25, 50, and 100µg/ml
respectively) as shown in Figure 2.13. It was reported that Ag NPs induced ROS
generation and suppression of reduced glutathione; resulted in damage to various cellular
components, DNA breaks, lipid membrane peroxidation, and protein carbonylation [10].
The skin keratinocytes and cervical cancer cell lines also shows a significant (p<0.05)
concentration-dependent increase in ROS generation after exposure to Ag NPs (data not
shown).
2.4 Conclusion:
To conclude, mushroom P. badius, which have long been associated with human as
part of diet and medicine, have used first time for rapid, eco-friendly and cost-effective
method of green synthesis of silver nanoparticles. Synthesis of AgNPs in five minutes by
this route opens up new possibilities for mass production of AgNPs. These bio-inspired
AgNPs showed cyto-genotoxic effects at concentration of 10µg ml−1 on cervical cancer
(HeLa), human skin keratinocytes and human peripheral blood lymphocytes. Therefore,
P.badius has proven to be promising candidate in nanoparticles synthesis with their
application in the medicinal field.
18
2.5 Bibliography:
[1] M. S. G. Ratnika Varshney, Seema Bhadauria, “Review Nano Biomed Eng A

Review : Biological Synthesis Of Silver,” Nano Biomed Eng, vol. 4, no. 2, pp. 99–
106, 2012.
[2] R. M. V. A. Isabel C F R Ferreira, Lillian Barros, “Antioxidants in wild
mushrooms.,” Current Medicinal Chemistry, vol. 16, no. 12, pp. 1543–1560, 2009.
[4] S. E. Singh NP, McCoy MT, Tice RR, “A simple technique for quantitation of low
levels of DNA damage in individual cells.,” Experimental Cell Research, vol. 175,
no. 1, pp. 184–91, 1988.
[5] A. S. R. S. Murali Sastry ,* Vijaya Patil, “Electrostatically Controlled Diffusion of
Carboxylic Acid Derivatized Silver Colloidal Particles in Thermally Evaporated
Fatty Amine Films,” J. Phys. Chem. B, vol. 102, no. 8, pp. 1404–1410, 1998.
[6] Strober W., “Trypan blue exclusion test of cell viability.,” Curr Protoc Immunol,
vol. 3, no. 3B, 2001.
[7] T. I. Jian Huang, Lijun Wu, Shin-ichi Tashiro, Satoshi Onodera, “Reactive oxygen
species mediates oridonin-induced HepG2 apoptosis through p53, MAPK and
mitochondrial pathways,” J Pharmacol Sci, vol. 107, no. 4, pp. 370–379, 2008.
[8] J. J. Mock, M. Barbic, D. R. Smith, D. A. Schultz, and S. Schultz, “Shape effects in
plasmon resonance of individual colloidal silver nanoparticles Shape effects in
plasmon resonance of individual colloidal silver nanoparticles,” Chemical Physics,
vol. 116, no. 15, pp. 6755–6759, 2002.
[9] M. I. K. S Anil Kumar, Majid Kazemian Abyaneh, S W Gosavi, Sulabha K
Kulkarni, Renu Pasricha, Absar Ahmad, “Nitrate reductase-mediated synthesis of
silver nanoparticles from AgNO3.,” Biotechnology Letters, vol. 29, no. 3, pp. 439–
445, 2007.
[10] J. W. H. Mei Jing Piao, Kyoung Ah Kang, In Kyung Lee, Hye Sun Kim, Suhkmann
Kim, Jeong Yun Choi, Jinhee Choi, “Silver nanoparticles induce oxidative cell
damage in human liver cells through inhibition of reduced glutathione and
19
induction of mitochondria-involved apoptosis.,” Toxicology Letters, vol. 201, no.
1, pp. 92–100, 2011.
[11] N. A. L. Flower, B. Brabu, M. Revathy, C. Gopalakrishnan, S. V. K. Raja, S. S.
Murugan, and T. S. Kumaravel, “Mutation Research / Genetic Toxicology and
Environmental Mutagenesis Characterization of synthesized silver nanoparticles
and assessment of its genotoxicity potentials using the alkaline comet assay,”
Mutation Research - Genetic Toxicology and Environmental Mutagenesis, vol.
742, no. 1–2, pp. 61–65, 2012.
[12] A. N. J. T S Kumaravel, Barbara Vilhar, Stephen P Faux, “Comet Assay
measurements: a perspective.,” Cell Biology and Toxicology, vol. 25, no. 1, pp.
53–64, 2009.
[13] R. R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y.
Miyamae, E. Rojas, J. Ryu, and Y. F. Sasaki, “Single Cell Gel / Comet Assay :
Guidelines for In Vitro and In Vivo Genetic Toxicology Testing,” Environmental
and Molecular Mutagenesis, vol. 221, pp. 206 –221, 2000.
[14] An WF, “Fluorescence-based assays,” Methods Molecular Biology, vol. 489, pp.
97–107, 2009.
20
Chapter 3. Green synthesis of subnano sized silver atomic clusters
using fruit of medicinal herb Rauwolfia serpentina
3.1 Introduction
The sub-nano metallic clusters are well known for their applications due to their
extraordinary properties. Highly stable nanoclusters can play a vital role as building
blocks for synthesis of new materials and for manufacture of nano-devices [1]. Size-
dependent properties of metallic clusters also open a possibility for tailoring properties of
nanoclusters by controlling the formation process [1]. However advances in this field
mainly inhibited by the difficulty in preparing clusters with mono-disperse size in large
quantity. Synthesis of clusters has becomes also challenging job for researchers because
they are highly unstable than nanoparticles. The usual capping techniques used in
nanoparticles synthesis such as thiol capping, use of surfactant for stabilization of
particles are also applicable for metallic clusters [2]. Clusters are very sensitive for
reaction conditions like nanoparticles hence search for the techniques which can be
operated at ambient conditions are desirable [3]. Green synthesis of sub-nano
particles/clusters provides a solution for this difficulty. Biosynthesis methods have been
used for synthesis of metal and semiconductor nanoparticles because of nontoxic
chemical processing and precise control over particles morphology [4], [5].
In this basis, the present study deals with green synthesis of sub-nano silver clusters
using medicinal plant Rauwolfia serpentina. Rauwolfia serpentina is commonly known
as sarpagandha in India (Figure 3.1). This plant has been widely used in medicine in
western countries and in ayurveda, unani and folk medicine. Sarpagandha is an important
medicinal plant distributed in the foot-hills of Himalaya range, up to the elevation of
1300- 1400 m and almost all over the country. It is used in traditional medicine in India,
China, Africa and many other countries. The chemical analysis of serpentina has proven
the richness of this plant in minerals, vitamins and phytochemicals [6]. The major
alkaloid present in roots, stem and leaves of sarpentina is Reserpine, which is used in
treatment for high blood pressure. Apart from this Rauwolfia herb has been used for relief
of various nervous systems disorders, insomnia and schizophrenia. The scientific
classification of Sarpagandha is given in Table 3.1.
21
Figure 3.1: Sarpagandha Plant
(http://www.dexaprine.org/ingredients/rauwolfia-serpentina)
Table 3.1: Scientific Classification of Medicinal herb Rauwolfia Serpentina

Plantae
Kingdom
Division Magnoliophyta
Class Magnoliopsida
Order Gentianales
Family Apocynaceae
Genus Rauwolfia
Species R. serpentina
Binomial name Rauwolfia serpentina
3.2 Methodology
3.2.1 Synthesis of silver clusters and nanoparticles

The fresh fruits of Rauwolfia serpentina were collected and one fruit have been
crushed in 10 ml distilled water. Filtrate were collected after filtration and used for
synthesis of silver clusters. The pH of filtrate was 6.3. The aqueous solution of silver
nitrate was used as silver source. Silver nitrate solution was added in 10 ml filtrate so as
to get final concentration of 2mM. Reaction mixture which maintained at room
temperature showed the colour change from faint purple to brown after five hours
confirmed the synthesis of silver nanoparticles and clusters. Synthesized silver colloidal
solution was taken for further characterization.
22
3.2.2 Characterization of silver clusters and nanoparticles
The synthesized sub-nano silver clusters initially characterized using UV-Visible
spectroscopy (JASCO UV-VIS-NRI Spectrometer, Model Name-V-670) in the range
200-800 nm. The fluorescent nature of silver clusters was determined by using
photoluminescence spectrum (PERKIN ELMER LS-55). The presence of silver was
confirmed by Energy dispersive spectrometer (JEOL LSM 6360A). The shape and size
distribution of silver clusters were analyzed from TEM micrographs (TEM-TECNAI G2-
20U-TWIN).
3.3 Result and Discussion
3.3.1 UV-Visible and Photoluminescence spectroscopy

UV–visible spectra of a silver clusters recorded as a function of time and
absorbance spectra are shown in Figure 3.2. The increase in absorbance at 439 nm as a
function of time can be seen. Plasmon absorption band with a λmax at 439 nm is
commonly presented as the characteristic of spherical faceted Ag-nanoparticles synthesis
[7]. The band gap estimated (~570 nm i.e. 2.17 eV) from absorption spectra shows good
agreement with band gap of silver clusters [8]. The Figure 3.3 depicted the fluorescence
spectra of silver clusters recorded at excitation wavelength of 220 nm. When the size of
metal nanoparticles is much smaller that wavelength of light, they behave as a dipole in
optical field and they absorb and emit the radiation near to its surface excitation energy
[9]. The clusters display strong fluorescence emission band centred at 352 nm (3.52 eV).
The fluorescence in the range of UV confirms that the presence of clusters in sample [8].
The another absorption band centred at 258 nm is clearly visible and is attributed to
aromatic amino acids of proteins and it arises due to electronic excitations in tryptophan
and tyrosine residues in the proteins [10]. The broad emission band around 521 nm which
may due to larger size particles also present in sample along with clusters.
As stated earlier, Rauwolfia Serpentina herb is good source of Alkaloids,
Flavonoids, Phenols and tannins [6]. It is also good source of Plastohydroquinones or
quinols which may have played major role in reduction of silver nitrate to silver
nanoparticle and silver subnano atomic clusters. The absorption peak around 200 nm to
23
300 nm in absorption spectra confirmed the presence of these biological reducing agents
in extract of Sarpagandha fruit.
Figure 3.2: UV-Visible absorption spectra of reaction mixture.
Figure 3.3: Photoluminescence spectra of reaction mixture (λ-ex is 220 nm).
3.3.2 Energy dispersive spectroscopy

The EDS of silver sub-nano clusters were taken by drop casting a sample on silicon
wafer shown in Figure 3.4. The distinct signal and high atomic percent values for silver
were obtained. Metallic silver nano-crystals generally show typical optical absorption
24
peak approximately at 3 keV due to surface plasmon resonance. There is also strong
signal of silicon (Si) in the EDS spectra, which is a signature of silicon substrate used in
sample preparation. The signals of other elements are also observed in EDS spectra
which may due to the enzymes and proteins present in filtrate of Sarpagandha fruit.
Figure 3.4: Representative EDS profile of silver

nano and subnano atomic clusters.
3.3.3 Transmission electron microscopy

TEM images were recorded from subnano silver clusters drop coated on copper
grid. The Figure 3.5 a & b shows the silver nanoparticles at magnification of 200 nm and
50 nm respectively. At magnification of 200 nm, silver clusters are not visible but they
can be seen in magnification of 50 nm (Highlighted in oval shape). The clear image of
silver clusters is shown in c and d .The silver clusters are homogeneously distributed with
silver nanoparticles in the mixture. Two major size distributions are observed in TEM
images of silver clusters having mean size of 9.5 nm and 0.4 nm as shown in Figure 3.6
a & b respectively. The HRTEM image and selected area electron diffraction (SAED)
patterns confirms the crystalline nature of silver clusters having 0.276 nm fringe
separation as shown in Figure 3.7 a and b respectively. In the SAED pattern the rings
and bright spots are observed which may due to the two types of size distributions present
in solution. The fringe separation of 0.276 nm obtained from HRTEM of Ag NPs shows
good agreement with literature.
25
Figure 3.5: TEM images of sub-nano silver atomic clusters at different magnifications.
Figure 3.6: Two major size distributions observed in reaction mixture after synthesis analysed
from TEM images.
26
Figure 3.7: a) SAED pattern and b) HRTEM image of silver nanoparticles and atomic
clusters synthesized from Sarpagandha fruit extract.
3.4 Conclusion
The subnano silver clusters have been successfully synthesized through green
approach using Ayurvedic medicinal herb Rauwolfia Serpentina (Sarpagandha). To the
best of Authors knowledge the use of Sarpagandha fruits for synthesis of silver
nanoparticles/clusters has not been reported earlier. The application of metallic clusters in
the field of medicine and industry are well known. The biological synthesis of these
metallic clusters certainly becomes alternative route for hazardous and expensive
chemical ways used for their synthesis.
3.5 Bibliography
[1] J. Wang and X. C. Zeng, Nanoscale Magnetic Materials and Applications. Boston,
MA: Springer US, 2009.
[2] S. Wu, H. Zeng, and Z. a Schelly, “Growth of uncapped, subnanometer size gold
clusters prepared via electroporation of vesicles.,” The journal of physical
chemistry. B, vol. 109, no. 40, pp. 18715–8, Oct. 2005.
[3] T. Linnert, P. Mulvaney, A. Henglein, and H. Weuer, “Long-Lived Nonmetallic
Silver Clusters in Aqueous Solution: Preparation and Photolysis,” no. 8, 1990.
[4] C. Krishnaraj, E. G. Jagan, S. Rajasekar, P. Selvakumar, P. T. Kalaichelvan, and N.
Mohan, “Synthesis of silver nanoparticles using Acalypha indica leaf extracts and
27
its antibacterial activity against water borne pathogens.,” Colloids and surfaces. B,
Biointerfaces, vol. 76, no. 1, pp. 50–6, Mar. 2010.
[5] H. J. Bai, Z. M. Zhang, Y. Guo, and G. E. Yang, “Biosynthesis of cadmium sulfide
nanoparticles by photosynthetic bacteria Rhodopseudomonas palustris.,” Colloids
and surfaces. B, Biointerfaces, vol. 70, no. 1, pp. 142–6, Apr. 2009.
[6] R. Harisaranraj, K. Suresh, and S. Saravanababu, “Evaluation of the Chemical
Composition Rauwolfia serpentina and Ephedra vulgaris,” vol. 3, pp. 174–178,
2009.
[7] Z. Khan, S. A. Al-Thabaiti, A. Y. Obaid, and a O. Al-Youbi, “Preparation and
characterization of silver nanoparticles by chemical reduction method.,” Colloids
and surfaces. B, Biointerfaces, vol. 82, no. 2, pp. 513–7, Feb. 2011.
[8] J. Zheng and R. M. Dickson, “Individual water-soluble dendrimer-encapsulated
silver nanodot fluorescence.,” Journal of the American Chemical Society, vol. 124,
no. 47, pp. 13982–3, Nov. 2002.
[9] O. Varnavski, R. G. Ispasoiu, L. Balogh, D. Tomalia, and T. Goodson, “Ultrafast
time-resolved photoluminescence from novel metal–dendrimer nanocomposites,”
The Journal of Chemical Physics, vol. 114, no. 5, p. 1962, 2001.
[10] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M. I. Khan, R. Kumar, and M.
Sastry, “Extracellular biosynthesis of silver nanoparticles using the fungus
Fusarium oxysporum,” Colloids and Surfaces B: Biointerfaces, vol. 28, no. 4, pp.
313–318, May 2003.
28
Chapter 4. Effect of clinorotation on biosynthesis of silver
nanoparticles.
4.1 Introduction
Since form discovery of nanotechnology the various routes for the synthesis of
nanoparticles have been developed. Various techniques like, chemical and physical
means such as chemical reduction [1], Electrochemical reduction [2], Photochemical
reduction [3], heat evaporation [4] and so on to control the size and shape of
nanoparticles which is a crucial in tuning physical, chemical and optical properties.
Nowadays nanoparticles of different size and shapes are extensively used in Industry and
medical applications [5]. Among all metal nanoparticles silver nanoparticles are
important materials that have been studied extensively. The most widely used and known
application of silver and silver nanoparticles are in medical industry. These includes
topical ointments and creams containing silver to prevent infection of burns and open
wounds [6]. More over the medical devices and implants are also prepared with silver
impregnated polymers and silver containing consumer products such as colloidal silver
gels and silver embedded fabrics are now used in sporting equipments [7].
From past several years microgravity has been used as a tool in the field of
biological and physical processes [8]. Microgravity environment provides a unique
window to gain a better understanding of how gravity driven phenomena like
sedimentation, buoyancy driven convection, solidification and crystal growth get affected
[8]. Microgravity allows researchers to study underlying events free from these effects.
Materials science research in microgravity can lead to a better understanding of how
materials are formed and how the properties of material are influenced by their formation
in microgravity. The number of applications of microgravity is expected to increase
dramatically, for pharmaceutical industry and structural biology is a major participant in
the frame of the commercialization of space [9]. The investigation of protein and virus
crystallization in microgravity opens up wide perspective for structural biology. The
atomic and subatomic resolutions are indispensable to know the relationship of structure
to function which is heart of macromolecular interaction [9]. Microgravity considerably
29
affects the growth of crystals and registry of atomic planes in crystals which affects the
conductivity [10].
In case of nanoparticle synthesis, gravity induced perturbations such as flow
induced shear, sedimentation, convection, and hydrostatic pressure causes the alteration
in synthesis process because these materials has weak collective interaction [11].
Microgravity environment created using parabolic space flights has affected the size
distribution of gold nanoparticles generated by ultrasonic reduction [12]. Microgravity
environment enhanced the reduction rate of gold chloride which further results in
reduction of nanoparticles size [12]. Microgravity environment also suppresses the
coagulation of sub particles in formation of silica stober particles by favouring the
diffusion limited aggregation [11]. Apart from these evidences which has been done in
real microgravity or microgravity simulated using parabolic flight experiments, no report
has been published on effects of microgravity simulated in ground based laboratories on
synthesis of nanoparticles.
In this regard, present chapter deals with studying the effects of simulated
microgravity environment on synthesis of silver nanoparticles. As silver nanoparticles are
extensively used and large number of methods are available in the literature for synthesis
of silver nanoparticles, in present study silver nanoparticles have been synthesized in
simulated microgravity condition created by one dimensional horizontal axis clinostat
[13]. The synthesis of silver nanoparticles has been carried out using the protocol
reported by Sangiliyandi gurunathan and co-workers with slight modification [14]. The
protocols described in chapter 1 and chapter 2 has not been tried for experiment in
simulated microgravity condition because these protocols are rapid. In fast reaction, one
cannot observe and record the effects of gravity induced perturbations on synthesis
mechanism hence the well reported protocol for the synthesis of silver nanoparticles
using bacterium E.coli has been used. Silver nanoparticles synthesized in earth’s gravity
and clinorotation condition were further characterized using UV-visible spectroscopy,
XRD, EDS, Photoluminescence spectrometer and TEM. The synthesized silver
nanoparticles also investigated for cyto-genotoxicity using alkaline comet assay [15] on
human skin keratinocytes and cervical cancer cell lines.
30
4.2 Methodology
The Biosynthesis of silver nanoparticles was carried out as described by

Sangiliyandi gurunathan and co-workers with slight modifications [14]. E.coli (DH5α)
was first grown for 24 hrs in 1g (Earth’s gravity) condition aerobically at 37°C in Luria
Broth (here after L.B) (Yeast extract-2gm/lit, Nacl-2gm/lit and Tryptone-0.5gm/lit)
medium. The cells were harvested by centrifugation and washed twice with phosphate-
buffered saline (pH-7.3). The harvested cells were re-suspended in Erlenmeyer flasks
containing L.B without NaCl (pH-10) and incubated for again 24 hrs at 37°c in an orbital
shaker (120 rpm) until they reached the stationary phase. After incubation period, the
cultures were centrifuged at 10,000 rpm and the supernatant was used for the synthesis of
silver nanoparticles. The aqueous solution of AgNO3 was added in supernatant to make
final concentration 5mM of reaction mixture.
To assess the effects of clinorotation on synthesis of silver nanoparticles, 1-
dimensional horizontal axis clinostat has been used [13]. This 1-d clinostat was designed
and developed in our laboratory, where rpm can be selected by adjusting voltage of the
DC motor. Rotation speed of clinostat was kept at 2 rpm. The final reaction mixture of
concentration 5mM containing culture supernatant and silver nitrate solution was divided
in two equal volumes and was transferred in two air tight bottles having diameter of 1.5
cm. One bottle placed at fixed holder in horizontal manner which acts as a control in the
experiment and another bottle on rotating holder (2rpm) which was a condition of
clinorotation (simulated microgravity) as shown in Figure 4.1. The whole experimental
setup was fixed in oven which was mentioned at 60°C. To the best of our ability the
reaction condition was maintained identical for control and clinorotating reaction
mixtures.
31
Figure 4.1: Experimental setup
Figure 4.2 shows the schematic representation of idea behind the function of
clinostat. All circles in the figure represent the fluid chambers having suspended
particles. In sub figure a) the chamber is in earth’s gravity where all particles are
sediment due to attractive force of gravity and particles agglomerated at bottom. If the
chamber is in free fall condition, the suspended particles are remains homogeneously
distributed in fluid inside the chamber as shown in figure b). During the free fall
condition there is no gravitational force pulling the suspended particle to the bottom. It is
possible to simulate the free fall condition my mechanical rotation of fluid chamber about
its axis. As shown in figure c) the chamber is rotated about a central axis with a specific
revolution per minute. The revolutions of chamber to achieve homogeneous distribution
of particles inside the chamber (Like in figure b)) depends on many factors such as mass
of particles, density of particles, viscosity of fluid etc.
32
Figure 4.2: a) Under 1 g conditions, particles will sediment and take a spatial position
that is determined by their weight (G) and buoyancy. b) Under free fall conditions,
particles distribute homogenously as sedimentation is abolished. c) A homogenous
particle distribution can also be achieved on ground by rotating a suspension.
Consider one such a chamber filled with fluid and particles inside it. Due to the
force of gravity in normal condition particles get sediment at the bottom of chamber. If
this chamber is subjected to clinorotation, the particles inside behaves as shown in Figure
4.3. The mechanism has been proposed by Dedolph and co-workers [13] for the sub
cellular particles inside the cells which have subjected to clinorotation. In this experiment
the concept of cytoplasm and sub cellular particles has extrapolated for fluid and particles
in airtight chamber. If this chamber filled with fluid and particles subjected to
gravitational force and constant rotation. When particles moves with rotation from
position 1 to position 2 (Figure 4.3), particles fall vertically down along the path as
shown by s1. In further rotations through position 2, 3, 4 and ultimately to again1
produces further vertical displacement of particles along s2 and s4. In one complete
rotation though the particles falling vertically, the path traced by particles become quasi
circular and particle remains at the same position. This quasi circular trajectory keeps the
particles in still falling condition which has been observed in real microgravity condition
[13].
33
Figure 4.3: The path of fall of particles within the fluid during clinorotation. Forces other than
gravity have been neglected. [13]
The nanoparticles synthesized in 1g and clinorotation condition were investigated

for their cyto-genotoxic effects on human skin keratinocytes (HaCaT) and cervical cancer
(Hela) cell lines, obtained from National Centre for Cell Sciences, Pune, India. Cell
viability, DNA damage, and ROS generation was measured in the cells exposed to the
varying concentrations of AgNPs. Cell viability was measured by Trypan blue exclusion
assay [16]. Intracellular ROS generation was measured by using a fluorescent probe 2’,
7’-dichlorofluorescin diacetate (DCFH-DA). The alkaline comet assay was used to study
whether exposure to silver nanoparticles damages cellular DNA. The comet assay was
carried out according to the method of Singh et. al. [15]. A total 150 cells per
concentrations were scored to calculate the amount DNA in Tail region using computer-
assisted image analysis system, CASP software.
34
4.3 Result and Discussion
4.3.1 UV-Visible Spectroscopy analysis

The experimental setup in oven was monitored continuously. The synthesis of
silver nanoparticles by supernatant was analyzed by visual inspection and UV-Vis
absorption spectroscopy. The colour change of reaction mixture from pale yellow to
brown is a visual indication for synthesis of silver nanoparticles in solution [17]. The
absorption spectra for both reaction mixtures are shown in Figure 4.4 which is recorded
on JASCO UV-VIS-NIR Spectrometer, Model Name-V-670 after 24 hrs of incubation.
The nanoparticles synthesized in 1 g and clinorotation condition exhibited maximum
absorption at wavelength 458 nm and 426 nm respectively corresponding to the surface
Plasmon resonance (SPR) of silver nanoparticles. During incubation, the reaction mixture
under clinorotating condition appears brown at 8th hr from time of incubation whereas at
14th hr under 1g condition. This is an indication of reduction of reaction time during
clinorotation. The inset figure shows the colours of reaction mixtures at the time of
absorption spectra measurement. Clinorotated reaction mixture (Bottle A) appears to be
dark brown than reaction mixture in 1g condition (Bottle B) and the absorption spectra
(Figure 4.4) shows higher absorption in simulated microgravity condition than 1g
condition. Figure 4.4 also shows two absorption spectra for L.B and aqueous solution of
AgNO3 and their corresponding solution colours are given in bottle C and bottle D
respectively for comparison.
35
Figure 4.4: UV-Vis spectra: recorded from the aqueous 5mM reaction mixture after 24
hrs of reaction in controlled (1g) and Clinorotated (simulated microgravity) conditions.
Inset figure shows the color of reaction mixtures at the time of absorption measurement
(A- Reaction mixture in simulated microgravity, B- Reaction mixture in 1-g, C- Luria
Broth and D- AgNO3 Solution).
4.3.2 X-Ray Diffraction (XRD) Analysis

The crystal structure and average size of the Ag nanoparticles was analyzed by
XRD pattern carried on BRUKER AXS D8 with a Cu kα (λ=1.54 A°). Figure 4.5 shows
the XRD pattern for clinorotated and controlled samples obtained from the drop-coated
film of colloidal Ag nanoparticles on glass substrate. The Ag NPs synthesized in
clinorotation condition has showed broader Bragg’s reflection peaks of (111), (200) and
(220) planes (JCPDS- File no. 01-1164) than those synthesized in 1g condition. The
average size of Ag nanoparticles synthesized in 1g and clinorotation condition was found
to be 14.5 nm and 7.7 nm respectively as calculated by Scherer formula at (111) plane
which indicates the smaller size of Ag NPs under clinorotation condition. This result has
shown good agreement with the results obtained by Reed and co-worker in parabolic
flight experiment [12]. The peaks observed in diffraction pattern under both conditions
reveal the face centred cubic (FCC) structure of Ag NPs.
36
Figure 4.5: X-ray diffraction pattern of silver nanoparticles
synthesized in clinorotation (Black) and1g condition (Red).
4.3.3 Energy Dispersive Spectrometer (EDS) Analysis

Energy Dispersive Spectrometer (EDS) analysis was carried out using JEOL JSM
6360A to investigate chemical composition of silver nanoparticles synthesized during
clinorotation and 1 g conditions. The EDS profile was recorded by drop coating of 100µl
each colloidal silver nanoparticle on silicon substrate. Figure 4.6 and Figure 4.7
represents the EDS spectrum for 1 g and clinorotated samples respectively. The presence
of intense peak around 3KeV energy indicates the presence of silver in both samples [18].
Out of total mass present in volume of 100 µl coated on silicon substrate, 6.51% (Figure
4.6) of metallic silver have been found in silver nanoparticles synthesized under 1g
condition. Whereas 11.91% of metallic silver was found in clinorotated sample (Figure
4.7). There was no change observed in mass percent of other elements like O, Na, P, S,
Cl, K and Ca except Carbon. The mass% of C in controlled sample was found to be
59.26% and in clinorotated sample it was found to be 54.70%.
37
Figure 4.6: EDS profile for silver nanoparticles synthesized in 1g.
Figure 4.7: EDS profile for silver nanoparticles synthesized in

simulated microgravity condition.
4.3.4 Photoluminescence (Pl) Analysis

The Photoluminescence (PL) emission spectra of clinorotated and control samples
were recorded on PERKIN ELMER LS-55 spectrofluorometer with an excitation
38
wavelength of 220 nm. The emission spectra shows intense peak around 448 nm in both
samples as shown in Figure 4.8. The higher intensity of fluorescence has been observed
from silver nanoparticles synthesized in clinorotation condition as compared to 1g.
Figure 4.8: The emission spectra of silver nanoparticles

synthesized in 1g (in red) and simulated microgravity
condition (in black).
4.3.5 Transmission Electron Microscopy (TEM) Analysis

The Transmission electron microscopy was employed to analyze the structure of
the nanoparticles. Figure 4.9 and Figure 4.10 show representative TEM images of silver
nanoparticles synthesized in 1g and clinorotation condition respectively. The inset figure
a and b shows electron diffraction pattern and HRTEM image for selected silver
nanoparticle for both conditions. The mean particle size was observed to be 14.9 nm in 1g
and 8.8 nm in clinorotation condition. Nanoparticles synthesized during clinorotation
shows small particles are large in number as compared to the 1 g condition. Figure 4.11
and Figure 4.12 represent the particle size distribution of Figure 4.9 and Figure 4.10
respectively. Along with smaller particles in clinorotated condition, the particles size
distribution is also narrow than 1 g condition. In electron diffraction pattern of silver
nanoparticles synthesized during clinorotation has shown diffused rings along with faint
spots on it, where as the silver nanoparticles synthesized in 1 g condition shown bright
39
spots on ring. The fringe spacing has been calculated as 0.268 nm and 0.253 nm in
controlled and clinorotated silver nanoparticles respectively.
Figure 4.9: Representative images of silver nanoparticles synthesized in 1 g condition.

Inset figures (a) represents electron diffraction pattern and figure (b) represent HRTEM
image.
Figure 4.10: Representative images of silver nanoparticles synthesized in simulated

microgravity condition. Inset figures (a) represents electron diffraction pattern and figure
(b) represent HRTEM image.
40
Figure 4.11: Particles size distribution for silver nanoparticles
synthesized in 1 g condition
Figure 4.12: Particles size distribution for silver nanoparticles

synthesized in simulated microgravity condition.
41
4.3.6 Cyto-genotoxicity assesment
The silver nanoparticles synthesized in clinorotation and normal gravity condition
were further investigated for their toxic effects against Human skin keratinocytes
(HaCaT) and cervical cancer (HeLa) cell lines.
The HaCaT and HeLa cells were obtained from National Centre for Cell Sciences,
Pune, India. Cells were washed thrice with phosphate buffered saline (PBS) and finally
suspended in RPMI 1640 medium (Hyclone, Thermo Scientific) supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin
(Sigma, St. Louis, USA). The alkaline comet assay was used to study whether exposure
to silver nanoparticles damages cellular DNA. The comet assay was carried out according
to the method of Singh et. al. [15]. A total 150 cells per concentrations were scored to
calculate the amount of DNA in Tail region using computer-assisted image analysis
system, CASP software. Details of protocol have given in Chapter 2.
Figure 4.13: Toxic effects on Human Skin Keratinocytes
42
Figure 4.14: Toxic effects on Cervical cancer cells
The clear distinction has been observed in silver nanoparticles synthesized in

clinorotation condition as compared to 1 g investigated by visual inspection. As reported
by Gurunathan and co-workers the bacterial supernatant contains Nicotinamide Adenine
Dinucleotide Hydrogen (NADH) dependant Nitrate Reeducates Enzymes secreted by
bacteria in medium extracellularly. The conversion of Ag+ in to Ag0 was done by electron
transported through the reduction of Nitrate through the reduction of NADH to NAD+
which probably acts as an electron shuttle in synthesis process of silver nanoparticles
[19], [20]. The pictorial representation of this possible reduction mechanism is given in
Figure 4.15. Early appearance of brown color in reaction mixture clearly indicates the
increased rate of reduction of silver nitrate in clinorotation condition than 1 g. Increased
reduction rate has enhanced the reaction yield as depicted by absorption spectra and color
of reaction mixture as shown in Figure 4.4. The blue shift of SPR in clinorotated sample
clearly indicates the reduction in size of silver nanoparticles. These observations are also
supported by EDS and Pl analysis of silver nanoparticles synthesized in both conditions.
43
The increased mass percent of silver (Figure 4.7) and increased fluorescence intensity in
Pl spectra (Figure 4.8) confirms the enhanced reaction yield in clinorotation condition
than 1 g. At initial time of reaction Ag+ interact with enzyme present in supernatant
(Figure 4.15). The increased rate and reduced time of reaction could be explained on
basis of reeducates enzyme activity which may have increased during clinorotation.
Figure 4.15: Possible reduction mechanism
In initial stage of reduction reaction the silver atoms comes together and forms
clusters. In early stage of reduction these clusters are the main product of reaction. In
further process number of silver atoms gets attached to these clusters and clusters achieve
crystalline nanoparticle nature [21]. This aggregation of clusters can be understood on the
basis of diffusion phenomenon. According to Stokes-Einstein law of diffusion, the
diffusion coefficient has indirect dependence of gravity. The diffusion coefficient Dm of
particle having mass m undergoing random diffusion is given by
1 1 Equation 4-1
𝐷𝑚 ∝ = 1
𝑟𝑒𝑓𝑓
𝑚𝑑(𝑔)
In Equation 4-1 reff is effective radius, d(g) is mass fractal dimension of particles.
Since diffusion phenomenon is present at level of gravity, it may affect the growth
44
mechanism of silver nanoparticles in clinorotation. The extensive work has been done
both experimental and computer simulation to understand the role of diffusion limited
conditions in crystal growth. They have shown that in diffusion limited conditions
cluster-cluster aggregation produce more extended structure and low fractal dimensions
[22]. The XRD analysis of silver nanoparticles synthesized in clinorotation and control
supports this discussion. In Figure 4.5 the diffraction pattern of clinorotated sample
shows higher full width at half maxima which indicate smaller crystal size in
clinorotation. TEM analysis of these nanoparticles supports XRD crystal size estimation.
As shown in Figure 4.9 and Figure 4.10 the 40.9% decreased in particle size was
observed in clinorotated sample than normal. This investigation supports the diffusion
limited aggregation of particles which suppresses the coagulation of sub particles and
reduces the size of particles. The results obtained are in good agreement with previous
results reported by Reed et.at. and Smith et.al. [11], [12]. The particles size distribution of
silver nanoparticles given in Figure 4.11 and Figure 4.12 also support this possible
mechanism.
Due to suppression of coagulation of sub particles in clinorotation condition, the
registry of silver atoms during cluster growth also gets affected. As depicted from
selected area electron diffraction pattern given in inset figure of Figure 4.9 and Figure
4.10, the combination of spots and diffuse rings is may resulted due to alteration in
plane/atom registry during nanoparticles growth [10]. The slight variation observation in
fringe spacing, but difference is not considerable.
The size and shape of nanoparticles play major role in their applications as
medicine and catalyst. As stated earlier the clinorotation has reduced 40.9% of particles
size in silver nanoparticles, hence these nanoparticles must have taken for assessment of
their medicinal values. The toxicity of these nanoparticles was tested using alkaline
comet assay. The Figure 4.13 and Figure 4.14 represent percentage DNA damaged in
human skin keratinocytes and cervical cancer cell lines respectively for both control and
clinorotated nanoparticles. In both cell types with increasing concentration toxicity has
increased. In comparison to control silver nanoparticles synthesized in clinorotation
condition has shown high toxic effects of cells. The increased toxicity of silver
nanoparticles can be interpreted in terms of particles size. The small particles size and
45
number of small particles are higher in clinorotated sample these small particles are more
permeable through cell wall which further result in higher damage of cellular DNA. The
obtained results are statistically significant and showing good agreement with early
reports on toxicity of silver nanoparticles [23]. As reported in several studies, toxicity of
nanoparticles is not only dependant on particles size. The surface morphology, capping
agents and chemical homogeneity of capping on surface of nanoparticles also plays vital
role. In this study the toxicity only understand in terms of variation in particles size.
During growth of silver nanoparticles surface morphology and chemical homogeneity of
capping proteins may also got affected in clinorotation. This would be a new perspective
to look at the incorporation of simulated microgravity in functional nanomaterials
synthesis methods.
4.4 Conclusion
To conclude, the effects of clinorotation (simulated microgravity) on biosynthesis

of silver nanoparticles have been successfully investigated. Simulated microgravity
environment considerably affects the reaction time and rate of reaction in synthesis of
silver nanoparticles. As evident by TEM analysis the growth mechanism of silver
nanoparticles could be affected during clinorotation which results in reduction of particles
size, narrow size distribution and change in electron diffraction pattern with slight
variation in fringe spacing. The results obtained in clinostat are consistent with the earlier
results obtained in space environment. The toxicity assessment has shown that silver
nanoparticles synthesized in clinorotation condition are more toxic as compare to normal
gravity condition. In simulated microgravity condition nanoparticles of smaller size and
uniform distribution can be obtained, so this study provides insight into effects of
simulated microgravity of growth of nanoparticles and in future it will be helpful to
incorporate microgravity in field of Materials Science and Nanotechnology for the
production of smart, ultrafine and ultrapure materials for benefits of mankind.
4.5 Bibliography
[1] D.-G. Yu, “Formation of colloidal silver nanoparticles stabilized by Na+-

poly(gamma-glutamic acid)-silver nitrate complex via chemical reduction
46
process.,” Colloids and surfaces. B, Biointerfaces, vol. 59, no. 2, pp. 171–8, Oct.
2007.
[2] Y. C. Liu and L. H. Lin, “New pathway for the synthesis of ultrafine silver
nanoparticlesfrom bulk silver substrateinaqueous solution bysonoelectrochemical
methods,” Electrochemical communication, vol. 6, pp. 1163–1168, 2004.
[3] G. D. S. Keki, J. Torok, “Silver nanoparticles by PAMAM-assisted photochemical
reduction of Ag+,” Journal of colloid interface science, vol. 229, pp. 550–553,
2000.
[4] S. M. P. C. H. Bae, S. H. Nam, “Formation of Silver nanoparticlesby laser ablation
of a silver target in NaCL solution.,” Appl. Surf. Sci., vol. 197, pp. 628–634, 2002.
[5] D. F. Jiang H, manolache S, Wong ACL, “Plasma-enhanced deposition of silver
nanoparticles onto polymer and metal surface for the generation of antimicrobial
characteristics,” Jurnal applied polymer science, vol. 93, pp. 1411–1422, 2004.
[6] B. RO, “Silver ions in the treatment of local infections.,” Met based drugs, vol. 6,
no. 4–5, pp. 311–4, 1999.
[7] Silver S, “bacterial silver resistance: molecular biology and uses and misuses of
silver compunds,” FEMS microbial Rev, vol. 27, pp. 341–353, 2003.
[8] M. L. Pusey, “Continuing adventures in lysozyme crystal growth NaCI Tempera,”
vol. 122, pp. 1–7, 1992.
[9] B. Lorber, “The crystallization of biological macromolecules under microgravity: a
way to more accurate three-dimensional structures?,” Biochimica et biophysica
acta, vol. 1599, no. 1–2, pp. 1–8, Sep. 2002.
[10] H. Ahari, R. L. Bedard, C. L. Bowes, T. Jiang, N. Coombs, G. A. Ozin, S. Petrov,
and I. Sokolov, “Effect of microgravity on the crystallization of a self- assembling
layered material,” vol. 388, no. August, pp. 857–860, 1997.
[11] D. D. Smith, L. Sibille, R. J. Cronise, A. J. Hunt, S. J. Oldenburg, D. Wolfe, and N.
J. Halas, “Effect of Microgravity on the Growth of Silica Nanostructures,”
Langmuir, vol. 16, no. 26, pp. 10055–10060, Dec. 2000.
[12] J. a Reed, A. Cook, D. J. Halaas, P. Parazzoli, A. Robinson, T. J. Matula, and F.
Grieser, “The effects of microgravity on nanoparticle size distributions generated
47
by the ultrasonic reduction of an aqueous gold-chloride solution.,” Ultrasonics
sonochemistry, vol. 10, no. 4–5, pp. 285–9, Jul. 2003.
[13] R. R. Dedolph and M. H. Dipert, “The physical basis of gravity stimulus
nullification by clinostat rotation.,” Plant physiology, vol. 47, no. 6, pp. 756–64,
Jun. 1971.
[14] S. Gurunathan, K. Kalishwaralal, R. Vaidyanathan, D. Venkataraman, S. R. K.
Pandian, J. Muniyandi, N. Hariharan, and S. H. Eom, “Biosynthesis, purification
and characterization of silver nanoparticles using Escherichia coli.,” Colloids and
surfaces. B, Biointerfaces, vol. 74, no. 1, pp. 328–35, Nov. 2009.
[15] S. E. Singh NP, McCoy MT, Tice RR, “A simple technique for quantitation of low
levels of DNA damage in individual cells.,” Experimental Cell Research, vol. 175,
no. 1, pp. 184–91, 1988.
[16] Strober W., “Trypan blue exclusion test of cell viability.,” Curr Protoc Immunol,
vol. 3, no. 3B, 2001.
[17] A. S. R. S. Murali Sastry ,* Vijaya Patil, “Electrostatically Controlled Diffusion of
Carboxylic Acid Derivatized Silver Colloidal Particles in Thermally Evaporated
Fatty Amine Films,” J. Phys. Chem. B, vol. 102, no. 8, pp. 1404–1410, 1998.
[18] D. S. P Magudapatty, P Gangopadhyayrans, B.K Panigrahi, Nair K.G.M,
“Electrical transport studies of Ag nanoparticles embedded in glass matrix,”
Physica B, vol. 299, pp. 142–146, 2001.
[19] M. I. K. S Anil Kumar, Majid Kazemian Abyaneh, S W Gosavi, Sulabha K
Kulkarni, Renu Pasricha, Absar Ahmad, “Nitrate reductase-mediated synthesis of
silver nanoparticles from AgNO3.,” Biotechnology Letters, vol. 29, no. 3, pp. 439–
445, 2007.
[20] N. Durán, P. D. Marcato, O. L. Alves, G. I. H. De Souza, and E. Esposito,
“Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium
oxysporum strains.,” Journal of nanobiotechnology, vol. 3, p. 8, Jan. 2005.
[21] X. C. Jiang, W. M. Chen, C. Y. Chen, S. X. Xiong, and a. B. Yu, “Role of
Temperature in the Growth of Silver Nanoparticles Through a Synergetic
Reduction Approach,” Nanoscale Research Letters, vol. 6, no. 32, pp. 1–9, Sep.
2011.
48
[22] P Meakin, “Models for Colloidal Aggregation,” Annual Review of Physical
Chemistry, vol. 39, pp. 237–267, 1988.
49
Chapter 5. Conclusion and Future scope
5.1 Summary
In the present work, new routes for biosynthesis of silver nanoparticles are
demonstrated. These nanoparticles are further assessed for their cyto-genotoxicic
potential against Human peripheral blood lymphocytes, Human skin keratinocytes and
cervical cancer cell lines. Moreover a unique alteration has also been done in well
reported nanoparticles synthesis protocol by altering gravity during synthesis. This
modification is only of its kind because first time in the scientific research, clinostat
(Microgravity simulator) and synthesis of nanoparticles have been clubbed together. The
silver nanoparticles synthesized under clinorotation and normal gravity condition have
been employed for their cyto-genotoxicity.
The bio-compatibility is always a major concern in biomedical nanotechnology.
Extensive research has been done to use different nano-products for medical application.
Nanoparticles are successfully incorporated with drugs and implemented for development
of new drug delivery systems. Chemically synthesized metal nanoparticles like Ag and
Au had shown appreciable antibacterial and antifungal activities. Nowadays they are used
as good anti fungal/bacterial agents. But due to toxic chemicals involved in synthesis
process, chemically synthesized nanoparticles are not preferred in vivo applications
without any purification. The purification and removal of toxic capping need expensive
techniques which will not remain cost effective.
The green nanotechnology provides a promising solution over this problem and
biologically synthesized nanoparticles are preferred in medical application as they are not
capped with toxic substances and also due to their less production cost. Apart from vital
application of nanoparticles in cancer and tumour treatment, they are reported for causing
damages to normal cells. This bimodal behaviour of nanoparticles inspired us to evaluate
cyto-geno toxicity of silver nanoparticles synthesized in our work for both cancer cells
like cervical cancer (HeLa) cells and normal cell like Skin keratinocytes (HaCaT) and
human peripheral blood lymphocytes. Blood lymphocytes and skin keratinocytes were
intentionally selected for our study because nanoparticles are widely used in cosmetics
and drug delivery systems.
50
5.1.1 Studies with P.badius
In this chapter, new route for green synthesis of silver nanoparticles has been
investigated. P.badius mushrooms are well known for their medicinal values and for the
first time used for nanoparticle synthesis. The well dispersed silver nanoparticles have
been synthesized in just five minutes from extract of P. badius. These nanoparticles were
further investigated for their toxicity potential measurement against peripheral blood
lymphocytes, cervical cancer cells and skin keratinocytes. Nanoparticles have shown
ROS mediated concentration dependent anticancer activity against cancer cell lines.
Apart from anticancer activity, these nanoparticles are also toxic for normal blood
lymphocytes and skin keratinocytes. When interacted with normal cells they have shown
same toxic mechanism. Present investigation indicates that, before using nanoparticles for
medical application their toxicity assessment should be done.
5.1.2 Studies with Rauwolfia Serpentina fruit

In this chapter the ancient Indian medicinal herb Rauwolfia serpentina commonly
known as sarpagandha has been used for synthesis of silver nanoparticles and sub nano-
sized atomic clusters. This plant has been widely used in medicine in western countries
and in ayurveda, unani and folk medicine. Sarpagandha is an important medicinal plant
distributed in the foot-hills of Himalaya range, up to the elevation of 1300- 1400 m and
almost all over the country. After synthesis the nanoparticles were characterized and
interestingly we found silver clusters along with silver nanoparticles. The synthesis of
metallic clusters is always a challenging job for researchers because of their low stability.
The extensive research has been done on chemical synthesis of silver and gold atomic
clusters. To the best of our knowledge this is first report on biological synthesis of silver
metallic clusters. The co-existence of cluster and nanoparticles of silver after reduction of
silver nitrate using extract of Rauwolfia serpentina fruit, confirmed the presence of same
special types of enzymes/proteins in the extract which have played vital role in stabilizing
clusters. As clusters have wide range of applications than nanoparticles, green synthesis
of clusters provides alternative for fast and low cost synthesis of metallic clusters.
51
5.1.3 Studies with Clinostat
In this chapter the well reported protocol for synthesis of silver nanoparticles using
E.coli (Dh5α) has been used with slight modification. According to the protocol, silver
nanoparticles were synthesized in normal and simulated microgravity condition using
clinostat. We were interested in looking at the effects on clinorotation of synthesis
mechanism of silver nanoparticles. So far clinostat has been used from past several
decades to study effects of gravity nullification in plant systems. For the first time,
clinostat has been clubbed with nanotechnology to investigate the effects of microgravity
environment on synthesis of nanoparticles. The results obtained have shown that
clinorotation reduces time required for reaction and enhance the rate of reaction.
Interestingly, the size of silver nanoparticles synthesized in clinorotation got reduced by
40.9% than silver nanoparticles synthesized in normal gravity condition. The electron
diffraction studies have confirmed alteration of atoms/planes registry in clinorotation
during growth of crystals. These nanoparticles were further investigated for their cyto-
genotoxicity. Nanoparticles synthesized in clinorotation have shown higher toxicity
against cancer cells and skin keratinocytes as compared to normal gravity.
5.2 Future Scope
Biosynthesized metal nanoparticles have been a source of attraction as it has given

a new avenue especially in the field of medical science and nanotechnology. The
protocols reported in present thesis will be useful in future for rapid and mass synthesis of
silver nanoparticles and silver subnano metallic clusters. The toxicity investigation will
help the researcher in future for better implementation of metallic nanoparticles in
medicinal field.
The effects of clinorotation on synthesis process will serve as fundamental results
for further space materials science research. It has also opened a new window for use of
clinostat in nanotechnology and materials science.
52
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Characterization and application of silver

Thesis · August 2013

Avinash Jagannath Aher

PROF. PANDIT B. VIDYASAGAR

This is to certify that the work presented in this thesis entitled,

Prof. Pandit B. Vidyasagar

I hereby declare that the work in the present thesis entitled,

Mr. Avinash Jagannath Aher

Prof. Pandit B. Vidyasagar

Avinash Jagannath Aher

Chapter 1. General Information ................................................................................... 1

1.1 Introduction .......................................................................................................... 1

Chapter 2. Novel route for biosynthesis of silver nanoparticles using mushroom P.

2.1 Introduction .......................................................................................................... 8

3.1 Introduction ........................................................................................................ 21

3.2.1 Synthesis of silver clusters and nanoparticles ............................................. 22

3.3 Result and Discussion ........................................................................................ 23

3.3.1 UV-Visible and Photoluminescence spectroscopy ..................................... 23

3.4 Conclusion .......................................................................................................... 27

4.1 Introduction ........................................................................................................ 29

4.3.1 UV-Visible Spectroscopy analysis.............................................................. 35

4.4 Conclusion .......................................................................................................... 46

Chapter 5. Conclusion and Future scope ................................................................... 50

5.1 Summary ............................................................................................................ 50

5.1.1 Studies with P.badius.................................................................................. 51

5.2 Future Scope ....................................................................................................... 52

Figure 1.1: Different methods for synthesis of nanomaterials. .......................................... 2

Nanotechnology refers to the technology of matter manipulation at atomic level. It

1.2 Methods for synthesis of nanomaterials

The large numbers of techniques are available to synthesize different types of

1.4 Toxic effects of Nanomaterials

Figure 1.2: Optical micrograph of human glioblastma cell (U251).

Figure 1.3: TEM image of ultrathin section of cell showing silver

1.6 Objective of thesis

o To find out novel routes for biosynthesis of silver nanoparticles.

1.7 Plan of thesis

Chapter 1 is having idea about nanomaterials and different methods for

[1] T. Pradeep, “Nano: The expanding Horizon,” in A textbook of Nanoscience and

In recent years, biologically synthesized silver nanoparticles have drown

Table 2.1: Scientific Classification of P. badius

2.3 Results and Discussion:

Figure 2.2: UV-Vis absorption spectra of silver nanoparticles synthesized by P.

Figure 2.4: Represents EDS profile of silver nanoparticles synthesized

Figure 2.5: Representative FTIR Spectrum of silver nanoparticles

Figure 2.9: HRTEM Image of silver

Cell viability (%)

Figure 2.11: Viability of blood lymphocytes when exposed with

Figure 2.12: % Tail DNA damage in blood lymphocytes incubated

[1] M. S. G. Ratnika Varshney, Seema Bhadauria, “Review Nano Biomed Eng A

Table 3.1: Scientific Classification of Medicinal herb Rauwolfia Serpentina

3.2.1 Synthesis of silver clusters and nanoparticles

3.3 Result and Discussion

3.3.1 UV-Visible and Photoluminescence spectroscopy

Figure 3.2: UV-Visible absorption spectra of reaction mixture.

Figure 3.3: Photoluminescence spectra of reaction mixture (λ-ex is 220 nm).

3.3.2 Energy dispersive spectroscopy

Figure 3.4: Representative EDS profile of silver

3.3.3 Transmission electron microscopy

The Biosynthesis of silver nanoparticles was carried out as described by

The nanoparticles synthesized in 1g and clinorotation condition were investigated

4.3.1 UV-Visible Spectroscopy analysis

4.3.2 X-Ray Diffraction (XRD) Analysis

4.3.3 Energy Dispersive Spectrometer (EDS) Analysis

Figure 4.7: EDS profile for silver nanoparticles synthesized in

4.3.4 Photoluminescence (Pl) Analysis