Review of Literature

Chapter 2 Review of Literature
Nanotechnology is the next-generation technology having key significance in the

advancement of various fields, such as medicine, engineering, electronic,
pharmaceuticals, agriculture and food industry. It has attracted immense interest
during the last few decades. “Nanotechnology mainly consists of the processing,
separation, consolidation, and deformation of materials by one atom or one molecule”
(Zhang and Webster, 2008). Nanotechnology deals with physical, chemical,
biological and engineering sciences at the nanoscale (Ataee-Esfahani et al., 2013; Li
et al., 2019; Mahendra et al., 2017). It is rapidly developing by producing
nanoproducts that can have novel size related physicochemical properties differing
notably from larger matter (Nam and Lead, 2008). The term “Nanotechnology” has
been incorporated in many disciplines and there are huge applications of
nanotechnology are shown in Figure 2.1. Nanotechnology is quickly picking up
importance in various zones, for example, medicinal services, cosmetics, nourishment
and feed, natural wellbeing, mechanics, biomedical sciences, energy science,
optoelectronics, substance enterprises, single electron transistors, nonlinear optical
devices, catalysis, space industries, chemical industries, gadgets, light producers, and
photoelectrochemical applications (Li et al., 2011; Veiseh et al., 2015). Tools
developed through nanotechnology may be able to detect disease in a very small
amount of cells or tissue (Sahoo and Labhasetwar, 2003).
Figure 2.1: Different applications of nanotechnology.
Faculty of Applied Sciences and Biotechnology 6

Nanobiotechnology opens up new areas of research and applications such as

neutraceutical and pharmaceutical applications. It provides technology for higher
resolution materials and devices for the purification of biomolecules. Nanotechnology
can help to reproduce or to repair damaged tissue (Tarafdar et al., 2013). The most
recorded examples of nanotechnology in history are medieval stained glass (Giuseppe
et al., 2013).
One of the major developments in nanotechnology is the production and application
of nanoparticles. Nanoparticles are being considered the fundamental building blocks
of nanotechnology. New methods to produce nanoparticles are constantly being
studied and developed. Several kinds of nanoparticles exist in nature and they have
remarkable applications in their own respective fields. The term “nanoparticle” is a
combined name for both nanocapsules and nanospheres. Nanospheres are matrix
system where the drug is distributed uniformly, while nanocapsules are the system
where the drug is surrounded by a special polymeric membrane. Nanoparticles are the
smallest microscopic particles or ultrafine particles having size in the range of 1-100
nm (Saxena et al., 2010).
Nanoparticles with one or more dimensions of the order of 100nm or less, attracted
considerable attraction due to their unusual and fascinating properties over their bulk
counterparts, with several applications (Daniel and Astruc, 2004; Kato, 2011). The
novel properties of nanoparticles have been exploited in a wide range of potential
applications in biomedical devices, renewable energies, medicine, environmental
remediation, antimicrobials, manufacturing biological sensors (Ameen et al., 2020;
De et al., 2010; Fu and Fu, 2015; Ghosh et al., 2011; Lu et al., 2008; Manimaran and
Kannabiran, 2017; Tran et al., 2013; Tanwar et al., 2020; Ying et al., 2019).
2.1 Types of nanoparticles
Nanoparticles (NPs) can be broadly classified into two groups namely, organic
nanoparticles and inorganic nanoparticles. Nano-sized organic and inorganic particles
have attracted much attention owing to their unique properties that vary from their
bulk materials.
2.1.1 Organic nanoparticles
Organic nanoparticles (NPs) have an upper edge in terms of biodegradability over the
inorganic nanoparticles. Most of the organic NPs are formed by several organic
molecules which are driven together by self-organization or chemical binding. The
organic nanoparticles are also known as polymeric nanoparticles. The most known
shape of organic or polymeric nanoparticles is nanosphere or nanocapsule (Mansha et

al., 2017).
For the synthesis of organic NPs, with the exception of dendrimers and some other
polymer NPs, we first need organic molecules that can arrange themselves three-
dimensionally. Organic NPs are templated upon synthetic or natural organic
molecules. Nature provides a wide range of examples of organic NPs such as protein
aggregates, lipid bodies, milk emulsions, or more complex organized structures such
as viruses, to name a few. Organic NPs differ conceptually from inorganic NPs in
terms of the principles of fabrication. Organic NPs can be fabricated both by “top-
down” and by “bottom-up” approaches. Organic nanoparticles have gained
widespread attention for various biomedical applications, such as bioimaging, cancer
therapy, drug delivery and disease diagnosis (Fang et al., 2020).
2.1.2 Inorganic nanoparticles
There is a growing interest in inorganic nanoparticles i.e., of noble metal
nanoparticles as they provide superior material properties with functional versatility.
Inorganic nanoparticles are one of the most widely studied materials due to their
exclusive chemical and physical properties that originate from their nanoscale
dimensions (Giljohann et al., 2010). Some of the inorganic nanoparticles include
magnetic nanoparticles, noble metal nanoparticles and semi-conductor nanoparticles.
Metal nanoparticles synthesis are controlled by facet, size and shape (Dreaden et al.,
2012). Due to their size features and advantages over available chemical imaging drug
agents and drugs, inorganic particles have been examined as potential tools for
medical imaging as well as for treating diseases. Inorganic nanomaterials have been
widely used for cellular delivery due to their versatile features like wide availability,
rich functionality, good compatibility, and capability of targeted drug delivery and
controlled release of drugs (Xu, 2006). In general, inorganic nanoparticles are more
stable than organic nanostructures, which still face unsolved problems, such as their
limited chemical and mechanic stability and swelling. Inorganic nanoparticles are
highly stable, hydrophilic, biocompatible, and non-toxic compared to organic
materials (Paul and Sharma, 2020). Inorganic nanoparticles such as quantum dots,
ceramic, magnetic, polystyrene and metallic nanoparticles have a central core
composed of inorganic materials that defines their magnetic, optical and electronic
properties.

2.1.3 Metallic nanoparticles

Metallic nanoparticles are submicron scale entities made of pure metals (e.g.,
platinum, thallium, silver, zinc, iron, gold, titanium and cerium). The development of
highly uniform and biocompatible metallic nanoparticles with optimized functional
properties is critical to bring new biotechnological innovations. Another important
category of metallic nanoparticles includes simple metal oxides such as zinc oxide
(ZnO), magnesium oxide (MgO), iron oxide (Fe 3O4 and Fe2O3), aluminium oxide
(Al2O3), titanium oxide (TiO2), copper oxide (CuO) and manganese oxide (MnO2). In
present study, we synthesized zinc oxide and silver doped zinc oxide nanoparticles.
2.1.3.1 Zinc oxide nanoparticle
Zinc oxide is a distinct and unique material that exhibits unique properties such as
piezoelectric, semiconductor and pyroelectric properties and has versatile applications
in transparent electronics, ultraviolet (UV) light emitters, piezoelectric devices,
chemical sensors, photonic devices, gas sensors, spin electronics, paints, solar cell
materials, catalysis and energy conversion (Wang et al., 2004; Gao and Wang, 2003;
Hongsith et al., 2010; Stan et al., 2015). Zinc oxide nanoparticles (ZnONPs) are one
of the maximum interests generating particles because they are inexpensive to
produce, safe and can be synthesized easily (Jayaseelan et al., 2012). It normally
appears as a white powder and is almost insoluble in water, under UV light and high-
temperate, it is non-toxic and chemically stable (Rajiv et al., 2013).
The different morphology of ZnO nanoparticles like nanoflake, nanoflower, nanobelt,
nanorod and nanowire. USFDA (United State Food and Drug Admininistration)
regarded zinc nanoparticles as GRAS (generally recognised as safe) metal oxide
(Pulit-prociak et al., 2016). The ZnONPs have extensive biological applications i.e,
antifungal, anticancer, antidiabetic, antimicrobial, antioxidant, antibacterial, acaricidal
and also show results in drug delivery (Martinkova et al., 2009; Costenaro et al.,
2013; Movahedi et al., 2014; Jain et al., 2014; Bala et al., 2015; Hameed et al., 2016).
ZnO nanoparticles have exhibited a strong bacterial growth inhibiting character
(Sawai, 2003). ZnONPs also contain UV filtering properties, anti-inflammatory and
wound healing properties (Elumalai and Velmurugan, 2015; Mirzaei and Darroudi,
2017; Sangeetha et al., 2011; Sherly et al., 2014; Stan et al., 2015). The ZnO
nanoparticles can be prepared by Biological methods using milky latex of Calotropis
procera, Arya sativa and leaf extract of Acaphyla indica, Coriandrum sativum (Sabir

et al., 2014). In 2017, Agarwal et al., synthesized ZnO nanoparticles using fruit
extract of Rosa canina Trifolium and flower extract of Trifolium pretense.
2.1.3.2 Silver doped zinc oxide nanoparticle
Silver (Ag) nanoparticles have wide application in the field of medical devices, in
topical preparation, in textile and in dental materials. Ag nanoparticles are mostly
used as inorganic antibacterial agents. Ag nanoparticles release Ag + ions in aqueous
solution and these Ag ions showed a broad spectrum of antibacterial activities
(Kawashita et al., 2000; Lin et al., 2010; Pal et al., 2007). These nanoparticles are
also used to saturate bandages, which resist the growth of bacteria on injury (Kassaee
et al., 2008). Thus, Ag nanoparticles with ZnO may have antibacterial properties
according to their different particle size (Shahid et al., 2018). Nano ZnO with a lot of
transition metal ions is a non-toxic and a favourable semiconductor material with the
property of ferromagnetism at ambient temperature (Herng et al., 2007).
Doping of ZnO assists in getting good quality crystals with enhanced optical,
electrical as well as ferromagnetic properties. In practical devices, Mn, Co, Ag, V, Ni
introduced as dopants into doped ZnO and as a result diluted magnetic semiconductor
(DMS) formed (Shahid et al., 2018). Currently, different researchers are engaged in
improving the efficiency of photocatalysts by using metal dopants like Ag which is
the most effective due to its high stability and good electrical/thermal conductivity.
Furthermore, Ag doping on the surface of metal oxides employed to enhance
photocatalytic activity by preventing fast recombination processes (Bechambi et al.,
2015; Elango et al., 2017; Hastir et al., 2017) also, this mechanism could lead to the
generation of good antibacterial properties. According to Zhang et al., 2007, the
deposition of silver on the surface of ZnO improves the photocatalysis and UV
emission performance of ZnO. In particular, Ag doped ZnO has been frequently
explored for biomedical applications such as drug delivery, toxic dye absorber, dye
degradation reagent, wound treatment, cancer treatment, etc. due to its high
antimicrobial activities (Dias et al., 2019; Gouthaman et al., 2019; Khatami et al.,
2018).
2.2 Nanoparticles synthesis
Nanotechnology is fundamentally changing the way in which materials are
synthesized and devices are fabricated. The important aspect of nanotechnology and
nanoscience is the synthesis of nanomaterials. Synthesis of nanoparticles to have a
better control over particles size distribution, morphology, purity, quantity and
quality, by employing environment friendly economical processes has always been a

challenge for the researchers (Hahn, 1997). Nanoparticles exist in a variety of shapes,
such as circular, triangular, hexagonal or other such as chain like structures (Sajanlal
et al., 2011; Xie et al., 2012).
Traditionally, there are two approaches for the synthesis of nanoparticle (Bellah et al.,
2012), which can be categorized according to their assembly:
1. Top-down approach
2. Bottom-up approach
Both approaches play a significant role in the device industry and in nanoproducts.
The top-down or bottom-up approaches are schematically illustrated in Figure 2.2.
Figure 2.2: Schematic representation of ‘top-down approach’ and 'bottom-up

approach’ for nanoparticles synthesis.
2.2.1 Top-down approach
The top-down approach refers to a set of fabrication technologies starting with a block
bulk material which share the same material with the base substrate. This route is
based on the bulk material and makes it smaller by using physical processes like
crushing, milling or grinding to breakup large particles (Chokkareddy et al., 2018).
Top-down approach, where synthesis is initialized with the bulk counterpart that

leaches out systematically bit-after-bit leading to the generation of nanosized

structures or fine particles.
Top-down approaches are generally simpler and rely on either eliminating or
separating bulk material or on miniaturization of bulk fabrication processes to
produce the desired structure with appropriate properties.
Top-down synthesis techniques are extension of those that have been used for
producing micron-sized particles. The biggest problem with the top-down approach is
the imperfection of surface structure. Such imperfection would have a significant
impact on physical properties and surface chemistry of nanostructures and
nanomaterials. It is well known that the traditional top-down technique can cause the
significant crystallographic damage to the processed patterns. This route is typically
not appropriate for preparation of uniformly formed materials, and even with high
energy consumption it is very difficult to realize very small particles. Presently, the
top-down approach is dominantly used in industry for the fabrication of many man-
made materials (Kim et al., 2010; Xia and Whitesides, 2006). Top-down approaches
use larger initial structures, which can be externally controlled in the processing of
nanostructures, for example ball milling and plastic deformation (Rajput, 2015).
2.2.2 Bottom-up approach
The bottom-up approach is a phenomenon of self-assembly of materials in nano-
architecture. The integration of nanoscale building blocks into functional assemblies
and further into multifunctional devices can be accomplished by means of a “bottom-
up approach”. Bottom-up approaches include the miniaturization of materials
components with further self-assembly process leading to the formation of
nanostructures (Winterer and Metallkd, 2003). This alternative approach is more
economical and has the potential of creating less waste. The bottom-up approach
refers to build-up of material from bottom: from cluster-to-cluster, molecule-to-
molecule or atom-to-atom. This path is used more often to prepare much of the nano-
scale materials with the potential to generate a consistent shape, size and distribution.
This essentially includes chemical synthesis and regulates the reaction to prevent
further growth of particles. The main concern in bottom-up approach is adhesion of
the surface layers to base substrate. Organometallic chemical route, revere-micelle
route, sol-gel synthesis, colloidal precipitation, hydrothermal synthesis, template
assisted sol-gel, electrode position are some of the well-known bottom-up techniques
reported for the preparation of luminescent nanoparticles (Kumar et al., 2018).
Research on the synthesis of nanosized materials is of great interest because of their

unique properties which differs from bulk (Atul, 2010).
2.3 Methods for synthesis of nanoparticles
Currently, a large number of physical, chemical, biological methods are available to
synthesize various types of nanoparticles as shown in Figure 2.3 (Edison et al., 2020;
Liu, 2011; Mohanpuria et al., 2008; Tiwari, 2008).
Though physical and chemical methods are more popular for nanoparticle synthesis,
the use of toxic compounds limits their applications and gives a low yield (Malik et
al., 2014; Sriram et al., 2010). The development of safe eco-friendly methods for
biogenetic production is now of more interest due to simplicity of the procedures and
versatility (Popescu, 2010).
Figure 2.3: Different methods of nanoparticles synthesis.

2.3.1 Physical methods of nanoparticles synthesis
Traditionally, nanoparticles were produced by physical methods. To generate
nanoparticles, physical methods use high energy radiations, mechanical pressure,
thermal energy to cause condensation, material abrasion, melting and evaporation.
Physical methods provide advantages over chemical approaches in terms of the
absence of solvent contamination in thin films and the homogeneity of nanoparticle
distribution (Kruis et al., 2000). Physical methods primarily use a top-down approach
and are desirable because they are solvent-free and create consistent mono-disperse

nanoparticles. Inert gas condensation, High energy ball milling, laser ablation,
physical vapor deposition, laser pyrolysis, etc are some of the most regularly used
physical methods to generate nanoparticles.
2.3.1 (a) Inert gas condensation (IGC)
Inert gas condensation also known as ‘Inert gas evaporation’ is used on a commercial
scale for a wide range of materials.
Fabrication of size-controlled copper nanoparticles was performed using IGC method
combined with glow discharge sputtering as a source of copper nanoparticles (Pinilla
et al., 2010). In 2006, Ward et al., used this method to synthesize manganese
nanoparticles. Raffi et al., (2007) reported the synthesis of silver nanoparticles by
IGC method and found a strong dependence of the evaporation temperature and inert
gas pressure on the crystallinity, size distribution and morphology. Inert gas
condensation was found to be highly efficient method for the synthesis of good
quality silver and platinum nanoparticles (Maicu et al., 2014). In 2015, Benelmekki et
al., used an inert gas condensation method to make metallic dielectric multi-core-shell
nanoparticles.
2.3.1 (b) Laser ablation
Laser ablation (LA) method is also known as pulse laser deposition (PLD). It is the
process of removing material from a solid surface by using a laser beam to irradiate it.
Laser ablation method utilizes a high-power laser beam that evaporates particles from
a solid source (Chen et al., 2002). PLD is another vacuum based PVD (Physical vapor
deposition) process that employs laser energy to remove the material from the target.
The high-powered laser pulses impact the target's surfaces, causing melting,
evaporation, and ionisation. In 2005, Amoruso et al., fabricated Ni NPs by
femtosecond PLD, which was confirmed by atomic force microscopy (AFM). Singh
et al., (2007) have synthesized colloidal zinc metallic NPs using the LA technique.
Dhlamini et al., (2008) used pulse laser deposition to synthesise lead sulphide (PbS)
nanoparticles in an amorphous SiO2 matrix on a Si substrate with good
photoluminescence characteristics. In 2009, Andrea et al., used PLD for the synthesis
Ag NPs arrays for surface enhanced Raman scattering (SERS). In another study, PLD
used to deposit Ag NPs on nickel hydroxide nanosheet arrays for use in surface
enhanced Raman scattering (Jing et al., 2014). The particles synthesized through laser
ablation method depend upon the wavelength of the laser. The drawbacks of this
method are high cost and low yield, and difficulty in controlling the morphology of
the synthesized nanomaterials. Carbon nanotubes can also be produced by this

method.
2.3.1 (c) Physical vapour deposition (PVD)
Physical vapour deposition is a vaporisation coating technique that involves atomic
level of material transfer. Physical vapour deposition is a term that refers to a group of
procedures that are often used to make nanoparticles and deposit thin layers of
material (Swihart, 2003). Inorganic materials, such as alloys, metals and organic
compounds are also deposited using the physical vapour deposition technique.
Most widely used physical vapour deposition technique for nanoparticles synthesis
are Sputtering. Sputtering is a vacuum-based PVD method for depositing thin films
and nanoparticles. In 2012, Asanithi et al., optimized the process conditions for
deposition of uniformly sized silver nanoparticles by DC (Direct current) magnetron
sputtering. Bouchat et al., (2013) reported the synthesis of variety of metallic and
non-metallic nanoparticles by using this technique. Ichida et al., (2014) synthesized
Germanium nanoparticles and quantum dots by sputtering method for their use in
electronic and optoelectronic industry. Physical vapour deposition coatings are
commonly used to increase the hardness, oxidation resistance and wear resistance of
metals.
2.3.1 (d) Laser pyrolysis
Laser pyrolysis is a powerful method for synthesizing non-expensive and magnetic
nanoparticles. The laser pyrolysis technique is a vapor phase synthesis process
(Amato et al., 2013; Daraio et al., 2012). In this process, a salt solution is sprayed into
a series of reactors, where the solvent evaporates and the solute concentration within
the droplet increases. The particles that form as a result of this process are dried and
converted at a high temperature. Laser pyrolysis allows scalability for mass
production, but they require sophisticated and expensive equipment (Morjan et al.,
2003; Martelli et al., 2000, Morales et al., 1999). Laser pyrolysis has also created
molybdenum disulphide, silicon carbide and silicon nanoparticles (Swihart, 2003). In
2005, Marino et al., synthesized carbon nitride NPs with varying nitrogen
incorporation between 2% and 20% using laser pyrolysis.
Llamas-Jansa et al., (2007) used pulsed laser pyrolysis to generate carbon
nanoparticles. Using tetraethyl orthosilicate, silane and titanium isopropoxide as
precursor materials, and the laser pyrolysis process has been used to synthesize a
range of ceramic nanoparticles, such as silicon dioxide and titanium dioxide for
cultural heritage protection and energy applications (Amato et al., 2013). Laser
pyrolysis method can be used to synthesize NPs from a wide range of ternary
composites, oxide and non-oxide.
2.3.1 (e) High energy ball milling (HEBM)
The early nano materials were made by a simple method called ball milling. In this
process, kinetic energy of the moving balls is transferred to the milled material. Type
of milling (wet or dry), milling speed, ball-to-powder weight ratio, milling duration,
type of high energy ball mill, milling atmosphere Milling media, all influence the
amount of energy transferred between the balls and the material during the process,
affecting the morphological and physical properties of the nanomaterials (Dhand et
al., 2015).
Carbon black was employed as a precursor and phosphate ester as a surfactant in
aqueous media to manufacture graphite nanoparticles with sizes ranging from 1 to 30
nm using surfactant aided high energy ball milling (Chen et al., 2007). Salah et al.,
(2011) synthesized ZnO NPs of 30 nm size from ZnO microcrystalline powder using
HEBM process. In 2012, Islam et al., used surfactant assisted HEBM method to
prepare pure Calcium carbonate nanoparticles from cockle shells using dodecyl
dimethyl betaine. In 2013, Kar et al., optimized various milling parameters to prepare
uniform Lithium niobate nanoparticles. Recently researchers established HEBM as
the powerful method for large-scale production of nitrogen doped carbon NPs for
catalytic applications (Xing et al., 2013). In 2014, Chen and co-workers utilized
microwave assisted high energy ball milling method to prepare pure and well
crystallized cobalt ferrite nanoparticles.
2.3.2 Chemical methods of nanoparticles synthesis
Chemical method is the most commonly used method for the synthesis of
nanoparticles by using organic and inorganic reducing agents. The most widely used
reducing agents are sodium citrate, sodium borohydride, ascorbate, polyol process,
hydrazine hydrate, elemental hydrogen, poly (ethylene glycol), potassium auro
chlorate, Tollens reagent, sodium citrate, N, N-dimethylformamide (DMF) etc. The
aforementioned reducing agents reduce ions and lead to the formation of metal which
is followed by agglomeration into oligomeric clusters. These clusters eventually lead
to formation of metallic colloidal particles (Evanoff, 2004; Merga, 2007; Wiley,
2005). During the preparation of metal nanoparticles, it is important to use protective

agents to stabilize dispersive nanoparticles Protective agents can be absorbed on or

bind to surfaces of nanoparticles, thus avoiding agglomeration (Oliveira, 2005).
Poly vinyl pyrolidone (PVP), polyethylene glycol (PEG), polymethacrylic acid
(PMAA) and polymethylmethacrylate (PMMA) have been widely used as protecting
agents. The function of the protective agent is to protect the nanoparticles from
agglomeration (Oliveira, 2005). Various chemical methods used for the synthesis of
nanoparticles are: Sol-gel method, micro emulsion technique, hydrothermal synthesis,
polyol synthesis, Solvothermal synthesis, Microwave assisted synthesis, chemical
vapor synthesis and plasma enhanced chemical vapor deposition technique. These
techniques come under the bottom-up category of nanoparticle synthesis.
2.3.2 (a) Chemical vapor synthesis
Chemical vapor synthesis (CVS) is also known as chemical vapor precipitation
(CVP), chemical vapor deposition (CVD), chemical vapor condensation (CVC) and
chemical vapor reaction (CVR).This process is used for the deposition of solid films
from vapor phase via chemical reactions occurring at very high temperature
conditions. The quality of the deposited materials strongly depends on the reaction
temperature, reaction rate, and concentration of the precursors (Kim et al., 2004). This
process is often used in the semiconductor industry to produce high-purity, and high-
performance thin films. Thin films produced by CVD process at certain conditions
also contain ultrafine particles. In a typical CVD process, the substrate is exposed to
volatile precursors, which react and/or decompose on the substrate surface to produce
the desired film. In this synthesis method, the precursors that exist in three different
states (solid, liquid, gas) are produced in the form of vapor in the reactor under an
environment that requires particles to undergo the process of nucleation. In 2004, Cao
et al., and Gracia et al., synthesized nanoparticles by using this method and found that
more defects were present on the surface.
2.3.2 (b) Hydrothermal synthesis
Hydrothermal synthesis method has been recently proposed as a promising route for
the synthesis of either hydrophilic or hydrophobic magnetic nanoparticles with
controlled shape and size (Wang, 2005; Yan, 2009). It can be performed in two types
of systems, the continuous hydrothermal and batch hydrothermal process.
Nanoparticles less than 10 nm wide have been obtained at 150 °C by using the
hydrothermal method (Drofenik, 2007; Primc, 2009). Hydrothermal synthesis of
nanoparticles of diverse materials is a simple and quick procedure.
The hydrothermal method can be useful to control grain size, composition, particle
morphology, crystalline phase and surface chemistry through regulation of the
solution composition, reaction temperature, pressure, solvent properties, additives and
aging time (Carp et al., 2004). The hydrothermal synthesis can be carried out above or
below the supercritical point of water. A suitable reactor or sealed reaction vessel is
needed to control the temperature and the pressure during the reaction. This synthesis
route is able to produce mono-disperse particles with good crystallinity; however, in
some cases, it may be hardly scalable. Hydrothermal synthesis is a simple and fast
process for the synthesis of nanoparticles of various other materials such as ZnO,
CdS, CoFe2O4, Zr, Ag, FeWO4, etc (Behbahani et al., 2012; Choi et al., 2013;
Maryanti et al., 2014; Yang and Pan, 2012; ). Inorganic nanoparticles have been
obtained also by hydrothermal treatments or electrochemically (Cabrera, 2007;
Marques, 2008; Teja, 2009). The advantage of using this method includes the
capability to synthesize a huge number of NPs with an optimized size, morphology,
composition and surface chemistry that is rationally inexpensive.
2.3.2 (c) Microwave assisted synthesis
During the recent years, interest in the microwave assisted organic synthesis has
grown. Many nanomaterials were synthesized using radiation from microwave.
Microwave techniques eliminate the use of high temperature calcination over
extended periods of time and allow for fast, reproducible synthesis of nanomaterials.
Microwave-enhanced chemistry is based upon the effects of “microwave dielectric
heating” on effective heating of materials. This phenomenon depends on the ability of
a given material (reagents or solvent) to absorb and convert microwave energy into
heat. Recent reports have shown that microwave heating can be very convenient for
use in a large number of organic synthetic methods. Microwave heating is
instantaneous and very specific and there is no contact required between the energy
source and the reaction vessel. In 2005, Corradi et al., prepared colloidal TiO2
nanoparticle suspensions within 5 minutes using microwave radiation. Microwave
dielectric heating is a non-quantum mechanical effect, and it leads to volumetric
heating of the samples (Dasand Das, 2012).
2.3.2 (d) Sol-Gel method
Sol-gel method is used for the synthesis of metal oxides from a chemical solution
which acts as a precursor for integrated network of discrete particles or polymers. . In
material chemistry, Sol-gel is a technique for producing small particles. The initial
step of this process is converting monomers or the starting material into a sol, i.e., a
colloidal solution which is the precursor for further formation of a gel. The precursor
sol can be either deposited on the substrate to form a film cast into an appropriate
container having desired shape or can be used to synthesize powder. It is mainly used
for the metal oxides synthesis. This gel is made up of discrete particles or polymers.
Most commonly used precursors are chlorides or metal alkoxides. These precursors
are hydrolyzed and poly-condensed for the formation of colloids. Due to its economic
feasibility and the low-temperature process which gives the control over the
composition of the achieved product, sol-gel process is preferred. Sol-gel contains
two main reactions: hydrolysis of the alcoholic group and its condensation. The
obtained precursor sol can be given a desired shape using the appropriate casting
container. It can also be deposited on a substrate to form a film by dip coating or spin
coating or used to synthesize microsphere or nanosphere powders. The steps involved
in the sol-gel synthesis are mixing, casting, gelation, aging, drying and densification
(Kumar et al., 2015; Schmidt, 2006).
2.3.2 (e) Plasma assisted chemical vapor deposition (PECVD)
Plasma assisted chemical vapor deposition, also known as plasma assisted enhanced
chemical vapor deposition (PACVD), is a popular chemical vapor deposition process
which is widely used for the deposition of thin films. In the production of
microelectronic circuits, plasma-assisted thin film deposition is frequently used (Cote
et al., 1999). In 2010, Shimada et al., reported the synthesis of gallium nitride
nanoparticles by microwave plasma enhanced chemical vapor deposition (MPECVD).
Microwave plasma enhanced chemical vapor deposition (MPECVD) and radio
frequency plasma enhanced chemical vapor deposition PECVD (RFPECVD)
procedures were used to synthesize hydrogenated silicon nanoparticles and carbon
nanoparticles, respectively (Vach et al., 2006; Wang et al., 2001). Researchers and
their co-workers used PECVD method to synthesize hard carbon nanoparticle-based
films by tuning the self-bias, introducing different doping strategies and using
metal/carbon bi- and multilayer designs; where nitrogen was used as foreign atoms
while Ti, Cu and Ag were used as metal layer in metal/carbon bi- and multilayer
structures (Dwivedi et al., 2011; 2012; 2013). Yun et al., (2012) used PECVD process
for the synthesis of silica NP array on flexible polymer substrate. However, at higher
power, the plasma reactivity and chemical reaction got enhanced, leading to formation
of more nuclei and disappearance of unreacted species. Under this condition, the
coagulational growth can be prevented and formation of small size NPs would be
possible.
2.3.2 (f) Solvothermal synthesis
Solvothermal synthesis method has been found to be an adaptable route for the
synthesis of a wide variety of nanoparticles with narrow size distributions,
particularly when polar solvents under pressure and at temperatures above their
boiling points are chosen. Under the solvothermal conditions, the reaction of the
reagents increases significantly, thus enabling the reaction to take place at lower
temperature. The Solvothermal method is similar to the hydrothermal method, except
that it can be carried out using a variety of solvents other than water. The
solvothermal method normally has better control of the size and shape distributions
and the crystallinity than the hydrothermal method and has been employed to
synthesize nanoparticles and nanorods with/without the aid of surfactants (Byun et al.,
(2011).
2.3.2 (g) Polyol synthesis
Polyol synthesis or thermal decomposition was firstly developed to produce noble
metal particles and was later extended to produce magnetic nanoparticles (Fievet,
1989).
Polyol process is a method for synthesis of metal-containing compounds that employs
poly(ethylene glycol)s as a reaction medium that serves as a solvent, reducing agent
and complexing agent (Rahman and Green, 2009). The polyol synthesis designates
the liquid-phase synthesis in high-boiling, multivalent alcohols and is primarily
directed towards nanoparticles.
The metal precursor is dissolved in a polyol solution that is brought to near reflux
temperatures. In addition, the polyol acts as the reducing agent for metal reduction
and, at the relatively high temperatures for the reactions described herein, is likely
oxidized to various aldehyde and ketone species. Polyol method can also be used in
producing nanoparticles in large scale (Meshesha et al., 2009). The polyol technique
was also used to develop low-temperature ways for producing magnetic nanoparticles
of magnetite, Fe3O4 and other materials (Cheng et al., 2011; Songvorawit et al.,
2011). This process was used to synthesize a wide range of metal oxide nanoparticles,
metal hybrid nanoparticles, metal-based nanoparticles and magnetic nanoparticles
(Dhand et al., 2015).

2.3.2 (h) Micro emulsion technique

The term micro emulsion was first assigned by Schulman et al., in 1959. Micro
emulsion technique was used for the production of inorganic nanoparticles (Dong et
al., 2010). The synthesis of inorganic nanomaterials including composite
nanoparticles, semiconducting metal sulphite nanoparticles, metal nanoparticles,
metal salt nanoparticles, magnetic nanoparticles, metal oxide nanoparticles (Dhand et
al., 2015).
Micro emulsions are dispersions that are macroscopically homogeneous, optically
transparent, isotropic and thermally stable. Micro emulsion system comprises of
mono-dispersed spherical droplets of oil-in-water (o/w) and water-in-oil (w/o)
depending on the surfactant used. Martinez-Rodriguez et al., 2014, have reported
fast, easy and scalable production of Pt cubic NPs by reducing hexachloroplatinic acid
(H2PtCl6) with sodium borohydride using w/o microemulsion process. The water-in-
oil reverse micellar system is an ideal reaction site for the synthesis of nanoparticles
(Vincenzo, 2006). Well-crystallized nanoparticles can be obtained by this method, but
the versatility in terms of size, shape and nature of the nanoparticles is relatively
limited (Chatzidaki et al., 2017). Metal NPs were easily prepared using
microemulsion technique using the reduction strategy.
2.3.3 Biological methods of nanoparticles synthesis
Although chemical and physical methods are very successful to produce well-defined
nanoparticles, they have certain limitations such as long synthesis time, increase in
production costs, difficulty in purification and release of hazardous by-products
(Nagajyothi and Lee, 2011). Chemical synthesis methods also lead to the appearance
of certain adsorbed toxic chemical species that may have adverse effects in medical
applications (Parashar, 2009). Nowadays, the need to develop eco-friendly processes
that do not use toxic chemicals in the synthesis protocols is increasing. Green
synthesis approaches have advantages over conventional methods involving chemical
agents linked with environmental toxicity. The most important issues that must be
considered in green synthesis of NPs are selection of solvent medium and selection of
eco-friendly nontoxic reducing and stabilizing agents. The development of safe and
eco-friendly methods for biogenetic processing is now of great interest, due to the
simplicity of the processes and versatility. It has received increased attention due to
growing need to develop environmentally benign technologies in material synthesis.
This has motivated the researchers to use this route to synthesize the NPs, allowing
for better control of shape and size for various applications (Li et al., 2011; Popescu
et al., 2010).
Biosynthesis of nanoparticles is a kind of bottom-up method where
oxidation/reduction is the main reaction occurring. Bio-assisted methods provides an
environmentally benign, low-toxic, cost-effective and efficient protocol to synthesize
nanoparticles. These methods employ biological systems like bacteria, fungi, viruses,
yeast, actinomycetes, plant extracts, etc. for the synthesis of metal and metal oxide
nanoparticles. Biological methods of nanoparticle synthesis can be generally
categorized into three categories: i) Biological synthesis of nanoparticles using
biomolecules, ii) Biological synthesis of nanoparticles using microorganisms, and iii)
Biological synthesis of nanoparticles using different plants.
2.3.3.1 Biological synthesis of nanoparticles using microorganisms
Microbes are small in size like bacteria, fungi and viruses. Microorganisms grab
target ions from their environment and then turn the metal ions into the element metal
through enzymes generated by cellular activities. There are some microorganisms that
can survive on metal ion and can also grow under different conditions, and this
phenomenon is due to their resistance to that metal. The mechanisms involved in the
resistance are efflux systems, alteration of solubility and toxicity via reduction or
oxidation, biosorption, bioaccumulation, extracellular complex formation or
precipitation of metals, and lack of specific metal transport systems (Husseiny et al.,
2006).
Various category of microbes such as bacteria, virus, actinomycetes, fungi, algae and
yeast are extensively used as bioreactors for the synthesis of nanoparticles (Dhas et
al., 2014a, b; Fariq et al., 2017; Narayanan and Sakthivel, 2010; Shedbalkar et al.,
2014) as shown in Figure 2.4. Microbial synthesis of NPs has actually merged
different disciplines such as biotechnology, microbiology and nanotechnology into a
new field of nanobiotechnology (Roychoudhury, 2020).

Figure 2.4: Different microorganisms used for synthesis of nanoparticle.

2.3.3.1 (a) Bacteria
Bacteria are unicellular organism which grows very rapidly under warmth and
moisture. Many bacteria can synthesize nanoparticles like gold nanoparticles, zinc
oxide nanoparticles, silver nanoparticles, arsenic nanoparticles, magnesium
nanoparticles, iron oxide nanoparticles etc. Bacteria utilize a number of anionic
functional groups, proteins and enzymes, reducing sugars, etc. in bacterial biomass to
reduce interacting metal ions. Both the gram-positive bacteria and gram-negative
bacteria are used for the production of nanoparticles. The presence of anionic
functional group rich cell wall around the gram-positive bacteria constituting of
peptidoglycan, teichoic acids, lipoteichoic acids, proteins, and polysaccharides acts as
sites for the biosorption and subsequent reduction of cations. In 2000, Fu et al., have
reported that dried cells of Bacillus megaterium, Lactobacillus sp. could reduce silver
ions where the processes of bio-reduction were probably non-enzymatic.
There silver ions were reduced by the interaction of silver ions with the groups on the
cell wall of microorganisms. Nair et al., (2002) and Shahverdi et al., (2007) reported
the synthesis of metallic nanoparticles of Ag using the cultural supernatants of
Klebsiella pneumonia, Escherichia coli and Enterobacter cloacae. Most of the metal
ions have toxic effect on bacteria so the reduction of ions or the formation of water

insoluble complexes is a defense mechanism developed by the bacteria to overcome

such toxicity (Sastry et al., 2003). The most widely accredited mechanism for the
biosynthesis of silver nanoparticles is the presence of the enzyme nitrate reductase
which converts nitrate into nitrite. During in vitro synthesis of silver using bacteria,
the presence of alpha-nicotinamide adenine dinucleotide phosphate reduced
(NADPH)-dependent nitrate reductase would remove the downstream processing step
thus during the reduction, nitrate is converted into nitrite and the electron is
transferred to the silver ion causing the silver ion to reduced silver. This has been
observed in Bacillus licheniformis which is known to secrete NADPH and NADPH-
dependent enzymes like nitrate reductase that effectively converts Ag + to Ag0
(Vaidyanathan et al., 2010). Bacterial system was also employed to synthesize
semiconducting CdS nanocrystals (wurtzite crystal structure) (Sweeney et al., 2004).
In 2007, Prasad et al., used Lactobacillus bacterium as the bio-factories for the
synthesis of TiO2 NPs. In 2009, Nanda and Saravanan used bacterium S. aureus for
the synthesis of bioactive Ag nanoparticles. Sintubin et al., (2009) described non-
enzymatic production of Ag NPs using biomatrix of the bacteria both as the capping
and reducing agent. In 2009, Babu and Gunasekaran reported the isolation of new
hyper metal resistant Bacillus cereus bacterial strain that form intracellular crystalline
Ag NPs. Many studies have been reported on the use of microorganisms for the
synthesis of biosynthesis of silver nanoparticles using Streptococcus sp., Proteus sp.,
Pseudomonas sp., and its antimicrobial activity against various pathogens (Nithya et
al., 2014) was concluded that the silver nanoparticles synthesized with Streptococcus
sp. have greater effectiveness against S. typhi. Bacteria are a particularly important
tool for fabricating NPs due to their variety and high adaptability to extreme
conditions (Geng et al., 2009; Wang et al., 2018). In the literature, various studies of
the biosynthesis of NPs by bacteria have been reported, including elementary
compounds and substances (Reverberi et al., 2017). Due to bacterial resistance, NPs,
including single metal, metalloid and related compounds, can be formed and
harvested by biosynthesis (Mekkawy et al., 2017; Timoszyk et al., 2017).
Therefore, bacteria used as nanofactories can provide a novel approach to the removal
of metal or metalloid ions and fabricating materials with unique properties (Fang et
al., 2019).

2.3.3.1 (b) Fungus

Several fungi have been used as novel and assuring tool for the development of
nanoparticles both intracellularly and extracellularly. Fungal-mediated green
chemistry approach for NPs synthesis has many benefits such as economic viability,
higher bioaccumulation, easily scaled up method of synthesis due to easy downstream
processing and handling of biomass. Fungi can produce larger amounts of
nanoparticles in comparison to bacteria because they can secrete larger amounts of
proteins which directly translate to higher productivity of nanoparticles (Mohanpuria
et al., 2008).The advantage of the production of nanoparticles from fungi is that a
large quantity of enzyme which are in pure state and free from cellular protein can be
easy to apply for the simple downstream process.
The possible mechanism for the synthesis of silver nanoparticle by fungi is said to
follow the following steps: trapping of Ag+ ions at the surface of the fungal cells and
the subsequent reduction of the silver ions by the enzymes present in the fungal
system (Mukherjee et al., 2001).There are several reports on the NPs biosynthesis of
magnatite, TiO2 and ZrO2 using various fungal species (Bharde et al., 2006; Bansal et
al., 2005; Bansal et al., 2004). In 2006, Vigneshwaran and coworkers reported the
biomimetics of silver nanoparticles by using Phaenerochaete chrysosporium
commonly known as White rot fungus. Sadowski et al., (2008) demonstrated that the
Penicillium strain is isolated from the soil, capable of reducing silver ion and thus
producing silver nanoparticles extracellularly. Varshney et al., (2009) reported the
synthesis of Ag nanoparticles using novel fungi. Biosynthesis of Ag NPs was also
reported by using Aspergillus terreus and herein the speculated mechanism is reported
to be NADH dependent enzyme catalyzed extracellular reaction process (Li et al.,
2012). There are number of other reports available on the biosynthesis of Ag NPs
using versatile fungal species including Bryophilous rhizoctoni, Aspergillus flavus,
Pleurotusostreatus, etc. (Devika et al., 2012; Raudabaugh et al., 2013). Numerous
studies have also shown the mycosynthesis of silver nanoparticles extracellularly
through the reduction of Ag+ ions using different species belonging to genus
Aspergillus, such as A. niger (Kumar et al., 2008), A. flavus (Ranjbar et al., 2010), A.
terreus (Jaidev et al., 2010), A. clavatus (Li et al., 2011; Verma et al., 2009), A.
fumigatus (Saravanan et al., 2010), and A. tamari (Raliya et al., 2014).
Various Fusarium species have been reported to produce metallic nanoparticles for
silver nanoparticles intra- and extracellularly (Bawaskaret al., 2010; Duran et
al.,2010; Deepa et al., 2014; Ingle et al., 2008; Khosraviet al.,2009; Sadowski et
al.,2008). For the synthesis of metallic nanoparticles, some filamentous fungi such as
Penicillium were also studied. For this reason, various studies have been performed
using different species of Penicillium such as, P. brevicompactum (Shaligram et al.,
2009), P. fellutanum (Kathiresan et al., 2009), Penicillium sp. (Singh et al., 2014), P.
citrinum (Honary et al., 2013), and P. nalgiovense (Maliszewska et al., 2014). Fungi
can produce a considerable amount of extracellular enzymes such as chitinases,
glucanases, proteases, glycosyl hydrolases, xylanases, cellulases, and mannanases
under suitable conditions (Elgorban et al., 2016). Most of the fungi have a large range
of growth for sodium chloride, temperature and pH, facilitating the change in culture
conditions to produce homogeneous nanoparticles (Guilger-Casagrande and Lima,
2019).
Ramos et al., (2020), used Trichoderma sp. to synthesize AgNPs and also test their
antibacterial activity and it was observed that their antibacterial activity in Gram-
negative bacteria was superior as compare to gram-positive bacteria. The isolation of
the endophytic fungi, Fusarium solani from the Chonemorpha fragrans plant, used
for the biosynthesis of gold nanoparticles. The gold synthesized nanoparticles showed
cytotoxic activity against human breast cancer cells and cervical cancer cells
(Clarance et al., 2020)
2.3.3.1 (c) Yeast
Yeast is a eukaryotic microorganism belonging to the kingdom fungi. In 2003,
Kowshik et al., reported bulk extracellular synthesis of silver nanoparticles using
silver tolerant yeast strain MKY3.Torres-Chavolla et al., 2008, reported the synthesis
of comparatively larger monodispersed Au NPs (30-60 nm) in the presence of
Thermomonospora curvata, Thermomonospora fusca and Thermomonospora
chromogena species. Yarrowia lipolytica was also used as efficient reducing agent for
AuCl4 to produce gold NPs (Pimprikaret al., 2009). In 2011, Sathish et al., shown the
applicability of Hanensula anomala species to reduce gold salt generating AuNPs.
Yeast was also reported to be useful in the synthesis of Cd and PbS nanoparticles
using Candida glabrata and Rhodosporidium diobovatum sp., respectively (Seshadri
et al., 2011; Dameron et al., 1989). Enriched with various enzymes, actinomycetes
have been used for the reduction of AuCl4 to generate Au NPs by Thermomonospora
sp. (Ahmad et al., 2003). In 2011, Chauhan et al., synthesized Au NPs employing
Candida albicans mediated bio-reduction strategy.
According to Kumar et al., 2011, Yeast cells can act as a template that induces
biomineralization, which is the major mechanism for NP formation. In 2017,
Ganbarov et al., showed the synthesis of Ag-NPs by yeast strain, BDU-XR1 isolated
from spontaneous yogurt used in Azerbaijan. Li et al., (2018) produced Ag-NPs both
at the cell wall and inside cells, depending on the preparation methods.
Horstmann et al., (2019) observed several differentially expressed genes via RNAseq
based transcriptome analyses in yeast cells, treated with 20 nm spherical citrate-
coated silver NPs. Biosynthesized Ag-NPs which were characterized by fourier-
transform infrared, which revealed notable peaks corresponding to the binding of the
Ag-NPs to active biomolecules, namely, aromatic amines, phenols, carboxylic acids
and alcohols (Wilfred and Akin-Osanaiye, 2019). In 2020, Shu et al., produced well-
dispersed silver nanoparticles of uniform spherical shape using yeast extract as
reducing and capping agents.
2.3.3.1 (d) Actinomycetes
Actinomycetes are microorganisms that share some of the important characteristics of
fungi and bacteria. Due to their ability to produce secondary metabolites such as
antibiotics, actinomycetes are now getting focus for the synthesis of metallic
nanoparticles. Sastry et al., (2003) reported the synthesis of Au nanoparticles by using
the extremophilic actinomycete, Thermomonospora sp. which yielded polydispersed
Au nanoparticles. Ahmad et al., (2003) reported the intracellular synthesis of Au
nanoparticles by using alkalotolerant Rhodococcus sp. They observed that the
concentration of nanoparticles was more on the cytoplasmic membrane than on the
cell wall. This could be due to reduction of the metal ions by enzymes present in the
cell wall and on the cytoplasmic membrane but not in the cytosol. Although the
intracellular synthesis of metal and semiconductor nanoparticles has now been
reported only from bacteria and fungi, the use of actinomycetes in the intracellular
synthesis of nanoparticles is yet to be investigated. Actinomycetes have received a
considerable attention as they are least explored and stand as an efficient candidate for
the synthesis of metal nanoparticles (Golinska et al., 2014).
Actinomycetes are regarded as superior groups among microbial species that are of
commercial interest for their saprophytic behavior and for the production of diverse
bioactive secondary metabolites and extracellular enzymes (Kumar et al., 2016; Yu et
al., 2015).

Biological synthesis of metallic NPs has been successfully reported by actinobacteria

and on this basis actinomycetes have been considered for the synthesis of NPs (Silva-
Vinhote et al., 2017; Aswani et al., 2019). The biosynthesized copper oxide
nanoparticles from S. pseudogriseolus Acv-11 and S. zaomyceticus Oc-5 exhibits
antimicrobial activity against different pathogenic bacteria, antioxidant activity,
cytotoxicity activity, antifungal property and in-vitro larvicidal activity (Hassan et al.,
2019b).
2.3.3.1 (e) Algae
Algae are a diverse group within the plant kingdom that isbeing studiedin
nanotechnologyfor their application. In 2019, Sharma et al., reported platinum NPs
(PtNPs) from S. myriocystum, and used as biosensors for detecting the adrenaline
level in the body, which is a hormone-based drug used to treat asthma, heart attacks
and allergies. Gracilaria edulis was used for the synthesis of spherical AgNPs and
octahedral ZnONPs (Madhiyazhagan et al., 2017; Priyadharshini et al., 2014). In
2015, Sirelkhatim et al., used brown algae for biosynthesizing metal oxide NPs, such
as titanium oxide nanoparticles (TiO2NPs) and zinc oxide nanoparticles (ZnONPs).
According to another study, S. muticum was used to synthesize ZnONPs and
synthesized ZnONPs were hexagonal in shape, ranging from 35 to 57 nm in size, and
were capped by bioactive functional groups like hydroxyl, amines, carbonyl and
sulfate (Azizi et al., 2014 and 2017).
A variety of shapes, like, rectangular, polydispersed, triangular and spherical AuNPs
were generated from T. conoides by extracellular biosynthesis (Khodashenas and
Ghorbani, 2015). AgNPs biosynthesized from green algae Ulva latica and red algae
Hypneamusciformis have been effective in retarding the growth of A. niger, C.
albicans, and Candida parapsilosis fungal strains (Dhavale et al., 2019). Similarly,
AuNPs and AgNPswere synthesized from Neodesmuspupukensis and tested for their
antifungal potential. The antifungal potency of AgNPs was confirmed with mycelial
inhibition of 57.1%, 65.4%, 69.8%, 79.4%, and 80.6%, against A. fumigatus, F.
solani, and C. albicans A. flavus and A. niger, respectively, while AuNPs had 44.3%,
54.9%, 66.4%, 75.4%, and 79.4% against A. fumigatus, F. solani, C. albicans, A.
flavus and A. niger, respectively (Omomowo et al., 2020). The AgNPs synthesized
from Microchaete showed great de-colorization ability against methyl red azo dyes as
compared to cyanobacterial extract (Husain et al., 2019). In a recent study, the
Chlorella ellipsoidea-mediated biomatrix-loaded AgNPs exhibited high
photocatalytic activity for the degradation of the hazardous pollutant dyes methylene
blue and methyl orange (Borah et al., 2020).
2.3.3.2 Biological synthesis of nanoparticles using biomolecules
Biomolecules are sophisticated nanostructures, programmed by sequence information
and adapted by evolution. Numerous biomolecules such as viruses, diatoms, DNA
(Deoxyribonucleic acid), RNA (Ribonucleic acid), proteins are powerful tool and
have been used as templates for the synthesis of nanoparticles (Dhand et al., 2015).
The first use of cytoskeletal proteins for nanotechnology was the use of actin
filaments as templates for formation of nanowire (Patolsky et al., 2004). DNA is
commonly known as an admirable biomolecular template and a promising candidate
that has strong attraction with transition metal ions.
Many functional biological assemblies represent genuine systems and devices of
nanotechnology (Sarikaya et al., 2003; Zhang et al., 2003). A further use of DNA is
the use of protein and peptide fibers for nanoparticles synthesis (Hamada et al., 2004;
Scheibel et al., 2003; Scheibel, 2005). Different researcher reported that DNA used as
templates or organic scaffolds to synthesis a wide range of nanoparticles and
nanoparticle assemblies (Anantharaj et al., 2014; Ede et al., 2015; Kundu, 2013;
Kundu and Jayachandran, 2013; Kundu and Nithiyanantham, 2014; Nithiyanantham
et al., 2014). Lipid bilayer modified nanomaterials like silica nanoparticles,
semiconductor quantum dots, carbon nanotubes, noble metal nanoparticles have
attracted a lot of scientific attention because of their biocompatibility, water solubility
and prospective applications (Li and Yang, 2011).
2.3.3.3 Biological synthesis of nanoparticles using plants
Various microorganisms such as bacteria, algae, fungi and yeasts are used for the
biosynthesis of nanoparticles but recently a new trend has come to force i.e., use of
plants for the fabrication of nanoparticles because of its spontaneous, economical,
eco-friendly protocol, suitable for large scale production and single step technique for
the biosynthesis process (Huang et al., 2007). While fungi and bacteria require a
comparatively longer incubation time for the reduction of metal ions, water soluble
phytochemicals do it in a much lesser time. Therefore, compared to bacteria and
fungi, plants are better candidates for the synthesis of nanoparticles. Taking use of
plant tissue culture techniques and downstream processing procedures, it is possible
to synthesize metallic as well as oxide nanoparticles on an industrial scale once issues
like the metabolic status of the plant etc. are properly addressed. Plants are having
various applications else than acting as medicines like, food supplements,

nutraceuticals etc. The medicinal properties of plants are known since human
civilization started.
According to Sharfkandy in 1367, Rigveda shows the medicinal use of plants and
known to be the oldest literature of human knowledge. Also, their medicinal
properties hold a great economic value across the world. We are blessed with rich
botanical resources composing a variety of plants across country. In Ayurveda, Unani
and Siddha system, about 1500 plants are used in indigenous medical system.
However, the findings of medical efficacy of plants have already been tried by
ethnopharmacologists, botanists, microbiologists and natural-product chemists.
Although there are drugs that are in use for treating infectious diseases but there is a
major drug safety concern regarding them. It is reported that approximately 2.22
million patients with adverse drug reactions are hospitalized and 106,000 die every
year in U.S.A. In case of natural herbal products, in spite of being used over years,
there have been very few instances of such adverse reactions (Ekor, 2014). Apart
from the property of being herbal and inexpensive, there is very less research on these
beneficial plants (Nair et al., 2005). Plants are one of the most important resource for
drugs, food supplements and nutraceutical (Tiwari and Bimleshkumar, 2011). A
number of plants and their different parts are being currently investigated for their
role in the synthesis of nanoparticles and used for different applications as shown in
Figure 2.5. Certain plants are known to accumulate higher concentrations of metals
compared to others and such plants are termed as hyperaccumulators.

Figure 2.5: Plant mediated synthesis of nanoparticles and their different

applications.
The main mechanism considered for the synthesis of nanoparticles mediated by the
plants is due to the presence of phytochemicals. The major phytochemicals
responsible for the spontaneous reduction of ions are flavonoids, terpenoids,
carboxylic acids, quinones, aldehydes, ketones, and amides (Prabhu and Bhute, 2012).
The advantage of using plants for the synthesis of nanoparticles is that they are easily
available, safe to handle and possess a broad variability of metabolites that may aid in
reduction. Various researchers screened different plants species to be used as potential
candidate(s) for biosynthesis of nanoparticles. Many natural biomolecules in plants
such as phenols, proteins, enzymes, amino acids, polysaccharides, alkaloids, alcoholic
compounds, terpenoids, polyphenols, phenolic acids and vitamins play important role
in bioreduction, formation, capping and stabilization of NPs (Kuppusamy et al., 2016;
Makarov et al., 2014). The main water-soluble phytochemicals are flavones, organic
acids and quinones which are responsible for immediate reduction (Mallikarjuna et
al., 2014).
2.4 Selection of medicinal plants for synthesis of nanoparticles
Moringa oleifera and Trillium govanianum were selected in this study for the
preparation of nanoparticles and exhibition of their antimicrobial, antioxidant and
anticancer activities.

2.4.1 Moringa oleifera

Moringa oleifera is a genus of 14 species of flowering plant in the family
Moringaceae. It is also called “nebeday” or the tree that never dies. Moringa oleifera
is a multipurpose and fast-growing tree. It tolerates a wide range of soils and rainfall
conditions. It easily reaches three meters in height, just ten months after planting. It is
now extensively cultivated and has become naturalized in many locations in the
tropics (James, 1983).Moringa oleifera tree is the most popular underutilized tropical
crops. It is drought-tolerant and must be cut back several times to make it branch out
more. M. oleifera pods contain round-shaped seeds (Figure 2.6). M. oleifera seeds
have 30-40 % of oil which is edible for human consumption and its roots are
relatively short, tough and fibrous.
Moringa is one of the most useful trees in the world, with a huge amount of benefits.
Moringa is used as a micronutrient to treat many diseases. Every part of Moringa
oleifera are used in a variety of traditional medicines as shown in Figure 2.7. The
multiple functions and numerous values of Moringa oleifera tree indicates its
significant for commercial application and income generator to the places where the
species are available.
Figure 2.6: Moringa oleifera pods and seed kernels.

In fact, M. oleifera is said to provide 7 times more vitamin C than oranges, 10 times
more vitamin A than carrots, 17 times more calcium than milk, 9 times more protein
than yoghurt, 15 times more potassium than bananas and 25 times more iron than
spinach (Rockwood et al., 2013).

Figure 2.7: Different parts of Moringa oleifera and their medicinal importance.
The seeds are edible and its medicinal properties are well documented and supported
by the experience of the traditional Ayurvedic practioners (Amagloh and Benang,
2009). The seeds extract is a natural coagulant, mostly used at household for water
purification in some villages in Africa such as Sudan. As reported by Hegazy et al.,
2011; the seeds show similar effect as alum and less effective in low turbidity water.
The seeds contain proteins which are essential in antioxidant properties and water
purification.
The National Charity for organic growing reported that Moringa oleifera seeds are
proficient medical treatment to rheumatism, sexually transmitted diseases, urinary
infections, epilepsy and gout (Amagloh and Benang, 2009; Sengupta et al., 2012).
2.4.1.1 Taxonomical classification of Moringa oleifera tree
The Moringa oleifera also known as drumstick tree, is one of the forteen species
belonging to the monogeneric family Moringaceae (Jahn et al., 1986). The
Moringaceae family are related to Brassicaceae and other fifteen families that
produce mustard oil (Chase et al., 1996).
Kingdom - Plantae
Subkingdom - Viridiplantae
Infrakingdom - Streptophyta – land plants
Superdivision - Embryophyta

Division - Tracheophyta – vascular plants, tracheophytes

Subdivision - Spermatophytina – spermatophytes, seed plants
Class - Magnoliopsida
Superorder - Rosanae
Order - Brassicales
Family - Moringaceae - moringas
Genus - Moringa
Species - Moringa oleifera Lam. – horseradish tree
2.4.1.2 Distribution
Moringa oleifera is indigenous of Himalayas of North-Western India. This species is
located in the sub-Himalayan region of Uttar Pradesh and Himachal Pradesh in India.
Later, this species has been cultivated to other parts of the continent such as Malaysia,
Philippines, Singapore, Sri-lanka, Cuba, Burma and Cuba. In Africa, the tree has been
distributed in Nigeria, Egypt and Sudan; also spread to Central and South America,
Peru to Mexico, Paraguay and other cities of the world (Ramachandran et al., 1980 and
Mishra et al., 2011).
2.4.1.3 Traditional uses of Moringa oleifera
a) Antioxidant properties
The strong antioxidant properties of medicinal plants may improve the capability of
plants to survive under polluted conditions. Such natural materials may provide exact
advantages over synthetic ones, because they contain some essential compounds.
Therefore, it is significant to verify the antioxidant compounds of Moringa oleifera
seed extract. Siddhuraju and Becker, (2003) reported that antioxidant compounds are
presence in diverse quantities from Moringa oleifera tree. Antioxidative properties of
phenolic acid in Moringa oleifera seeds arises from its great reactivity as electron or
hydrogen donors from the ability to maintain, delocalize the unpaired electron (chain-
breaking function) and chelate metal-ions (Fenton reaction terminator).
While, flavonoids antioxidative power are resulted from distinct mechanisms includes
scavenging of free radicals and chelation of metal-ions (Chanda and Dave, 2009). M.
oleifera is a rich mine of antioxidant. The antioxidant properties in the aqueous
extracts of leaf, fruit and seed of MO are already presented by Singh et al., 2009. In
addition to this information, Bajpai et al., 2005; Siddhuraju et al., 2003 concluded
that the major bioactive compounds of phenolics, like quercetin and kaempferol are
attributes for antioxidant activity. Quercetin and kaempferol are also reported to have
good antioxidant activity on hepatocyte growth factor (HGF) induced Met

phosphorylation. A comparison between palm oil with MO seeds against their
antioxidant potential concluded that MO seed can act as chief radical scavengers
(Ogbunugafo et al., 2011).
b) Anticancer activity
M. oleifera has other characteristic which make it a good compliment to a cancer
preventive or treatment plan. It is loaded with potassium, protein, iron, calcium,
vitamin A and C, and as many more properties which promote a healthy body that has
the tools to fight cancer.
c) Analgesic activity
Moringa oleifera has been reported for their analgesic activity. Hot plate and tail
immersion method (Sutar et al., 2008) carried using alcoholic extract of the leaves
and seeds of MO shown an evidence about analgesic activity of Moringa.
d) Anti-inflammatory activity
Inflammation is a result of the body’s own natural immune response and is usually
caused by the increased presence of plasma white blood cells in the affected area.
This response involves the autoimmune response as well as the vascular systems and
is a necessary part of the healing process, but can cause severe problems when the
inflammation becomes chronic or is extreme in its duration or extent. Inflammation is
usually categorized as either chronic or acute and treatment for the condition is
dependent in the part on the cause of the inflammation and its chronic or acute status.
M. oleifera leaves possess antinociceptive and anti-inflammatory activities (Sulaiman
et al., 2008).
According to Padmalochana, (2018) the ethanol extracts of M. oleifera can be a
potential source of anti-inflammatory agents compared to standard drug.
e) Anti-asthmatic activity
Alkaloids present in Moringa plant acts like ephedrine and can serve to treat asthma.
These Alkaloids relaxes bronchioles. Bronchial asthma is effectively treated using
seed kernels of MO, shown in a study carried out to check the efficacy and safety of
these kernels with respect to asthmatic patients proved a decreased severity of asthma
symptoms and also improved respiratory functions (Agrawal and Mehta, 2008).
f) Antibacterial and antifungal activities
Pterygosperminis a powerful antibacterial and fungicidal and also reported to be a
part of Moringa's contributions (Ruckmani et al., 1998). Moringa roots are known for
their antibacterial activity (Rao et al., 1996) and are counted as rich source of
antimicrobial agents. The fresh leaf juice is helpful to inhibit the growth of
microorganisms that are pathogenic (Caceres et al., 1991). The aqueous extracts of
MO are reported to have inhibition potential against many pathogenic bacteria (Eilert
et al., 1981; Saadabi et al., 2011). In addition to antibacterial activity of MO oils, it
also exhibits anti-fungal activity (Chuang et al., 2007).
A study to compare relative antimicrobial activity of seed extracts against fungi
(Fusarium solani and Rhizopussolani) and bacteria (Pasturellamultocida, E. coli, B.
subtilis and S. aureus) concluded P. multocida and B. subtilis as the most sensitive
strains, and activity of these strains are also influenced by cations (Na+, K+, Mg 2+ and
Ca2+) (Jabeen et al., 2008). MO steam distillate was again processed into another
relative comparison for antibacterial and antifungal ability (Prashith, 2010). There are
several components of Moringa that are reported with antibacterial activity such as; 4-
(a-L-rhamnopyranosyloxy) benzyl isothiocyanate, 4- (4'-O-acetyl-a-L-
rhamnopyranosyloxy) benzyl isothiocyanate, niazimicin, benzyl isothiocyanate, and
4- (a-L-rhamnopyranosyloxy) benzyl glucosinolate (Fahey, 2005).
2.4.2 Trillium govanianum
One of the Himalayan plants with immense potential in the pharmaceutical industry is
Trillium govanianum, an indigenous medicinal herb belongs to the
genus Trillium (family: Melanthiaceae alt. Trilliaceae). The T. govanianum herb
grows up to 30 cm height and has three leaves in one whorl at the apex of the stem
and a central purplish brown flower. Leaves are broadly acute and ovate with a
conspicuous petiole. Plant composed of 3 petals, 3 sepals and 6 stamens (Fukuda,
2001).
Flowering in this plant occurs during May to July followed by seed setting from
September to October. Rhizome is elongated, prolong and creeping grayish to brown
in color having rough external surface (Rahman et al., 2015a). It is also known as
Himalayan trillium or Nagchhatri with important phytoconstituents named as steroid
saponins which are extensively used in the pharmaceutical industry. Trillium
govanianum plant are used in a variety of traditional medicines as shown in Figure
2.8. Medicinal and therapeutic importance of this plant is due to the occurrence of
glycosides, steroidal saponins, tannins, sterols and flavonoids (Hufford et al., 1988;
Ono et al., 1986; Rahman et al., 2015b). Secondary metabolites such as steroids,
saponins, and glycosides have been reported from rhizomes of Trillium genus by
phytochemical analysis (Akihito and Yoshihiro, 2008; Jiang et al., 2014). T.

govanianum contains three times diosgenin content in contrast with other investigated
species of medicinal plants (Sharma et al., 2016).
Figure 2.8: Medicinal uses of Trillium govanianum plant.

Rhizomes of T. govaninum contain trillarin, which upon hydrolysis yields diosgenin.
Traditional medicines, Rhizomes of T. govanianum plant are utilized as a part of
treatment for neurasthenia, sepsis, dysentery, cancer, giddiness, arthritis,
inflammation, hypertension, reproductive disorders (Pant and Samant, 2010; Sharma
and Samant, 2014). It is also used in treating skin boils, wounds, sexual disorders,
infections, stomach and joint pains (Rani et al., 2013; Rahman et al., 2015a; Sharma,
2017; Sharma et al., 2018). The dried rhizome of Trillium species is being used
traditionally for immuno-regulation, also as anti-inflammatory, anti-aging agents and
have antitumor properties (Khan et al., 2016; Luo et al., 2006; Wang et al., 2013).
2.4.2.1 Taxonomical classification of Trillium govanianum tree
The Trillium govanianum also known as Nagchatri, widely distributed from the
western Himalayas. Trillium genus consists of 31 species, some of the Trillium
species viz., T. tschonoskii, T. erectum, T. govanianum, and T. grandiflorum around
the world are of great medicinal importance (Luo et al., 2006; Zomlefer et al., 2001).
Kingdom - Plantae
Phylum - Tracheophyta
Sub Kingdom - Tracheobionta

Class - Liliopsida
Sub class - Liliidae
Order - Liliales
Family - Trilliaceae
Genus - Trillium
Species - Trillium govanianum
2.4.2.2 Distribution
T. govanianum usually prefers shady areas of temperate regions. In temperate Asia,
the plant is distributed in Tibet, Afghanistan, Western Asia and China while in
Tropical Asia the plant is distributed in the Indian Subcontinent, Pakistan, East
Himalaya, West Himalaya, Nepal (Hara et al., 1978; Karthikeyan et al., 1989;
Rechinger et al., 1990; Zhengyi and Raven, 2000). In the Himalayan mountain region,
T.govanianum grows throughout the region along with the related species T.
tschonoski. The morphological characters and features of both species are common
and sometimes get confused with each other (Fukuda, 2001).
2.4.2.3 Therapeutic uses of Trillium govanianum
a) Antioxidant activity
T. govanianum have highest scavenging capacity, the antioxidant action of extract was
lesser than that of butylated hydroxytoluene (BHT) and ascorbic acid. The lower
scavenging ability was accredited to the existence of bigger sized fats constituents
(Rahman et al., 2015b).
b) Antibacterial activity
According to various researchers, the species of Trillium from North America are
known to have antibacterial properties (Huang and Zou, 2011; Ono, 2007; Yokosuka
and Mimaki, 2008). The antibacterial activity of different extracts of T. govanianum
rhizome was studied invitro using three human pathogenic bacteria i.e.,
Staphylococcus aureus, Yersinia pestis and Escherichia coli (Sagar et al., 2017).
c) Analgesic and Anti-inflammatory activity
The crude extract of T. govanianum shown analgesic and anti-inflammatory activity
(Sharma, 2017 and Sharma et al., 2018). The rhizomes could serve as potential novel
source of compounds effective for inflammation and alleviating pain (Rahman et al.,
2016).
d) Anticancerous activity

Sharma et al., (2016) reported less toxicity of T. govanianum extract towards normal
cells but significant antiproliferative activity against cancer cells and reduced their
viability. Cytotoxicity of diosgenin has been reported on V79 fibroblast and K562
cells (Lui, 2005; Melo et al., 2004). The cytotoxicity activity against human
carcinoma cells (urinary bladder, liver, lung and breast) with methanol extract was
reported by Khan et al., (2016). According to Rahman et al., (2015b), the occurrence
of diosgenin along with other steroidal saponin compounds in T. govanianum is
accountable for their anticancerous activity.
2.5 Applications of green synthesized silver and zinc nanoparticles
Various plants extract has acted as precursors for the synthesis of NPs with potential
applications (Akhtar et al., 2013). The most common approach for green synthesis of
nanoparticles is environmental friendly routes and biocompatibility (Kumar et al.,
2014; Parashar et al., 2009). The biocompatibility of biosynthesized nanoparticles
offers very interesting applications in biomedicine and related fields (Huang et al.,
2015).
2.5.1 Antioxidant property
The prefix “anti” means opposition to or corrective in nature. Oxidants are the
substances which oxide the other substances. “Antioxidant is a substance that when
present in low concentration relative to the oxidisable substances significantly delays
or reduces oxidation of substrate (Halliwell, 1999). Oxidation is a chemical reaction
that causes loss of electrons or transfer of electrons from a substance to an oxidizing
agent. The mechanism of antioxidant activity depends on the scavenging of reactive
oxygen species (ROS) that terminates the oxidation chain reaction. Generally, free
radicals occur in various molecular structures composed of one or two unpaired
electrons that are extremely unstable, creating further harm to neighbouring molecules
by withdrawing electrons from them to achieve equilibrium.
This free radical formation within the living system is highly reactive and it damages
the short-lived chemical organisms. While highly reactive, free radicals should be
generated continuously in the living environment as they have a crucial role to play
detoxification, energy supply, chemical signaling and even immune function. The
phenolic compounds such as gallate, catechins, ferulic acid, epigallocatechine,
flavonoids, proanthocyanidins and tannins possessing antioxidant activity are
primarily due to their redox potential, which has important role in absorbing and

neutralizing the free radical formation which in turn absorbs singlet and triplet oxygen
or it decomposes peroxides (Calabrese et al., 2012).
There are various antioxidant methods to determine the scavenging capability of the
synthesized nanoparticles by ABTS (2, 2‟-Azino- bis (3-ethyl benzothiazoline-6-
sulphonic acid), DPPH (2, 2‟- diphenyl -1-picrylhydrazyl) solution, and
hydrogenperoxide (H2O2) assays. The findings obtained for free radical scavenging
activity performed at various concentrations were determined and it was compared
with IC50 values corresponding to the standard antioxidant (Chahal et al., 2019).
ABTS radical scavenging activity consists of radicals, produced chemically,
decolorizing of its non-radical form, which is also used to screen the complex
mixtures such as plant extracts, biological fluids. DPPH is known as a stabilized free
radical that accepts an electron or hydrogen radical to obtain stable type of molecule
due to its ability to give hydrogen. Comparatively, DPPH radical scavenging is a
rapid method which is widely used to evaluate antioxidant activity. Absorption
decreased to calculate the free radical scavenging effects. Caroling et al., (2013)
invesigated that silver nanoparticles of aqueous extract of Brassica oleracea var.
Capitata showed the antioxidant activity.
The plant extract showed higher levels of ascorbic acid and polyphenols antioxidant
compound in the quantitative antioxidant assay and the scavenging activity was
confirmed by the DPPH free radical scavenging activity. Silver nanoparticles showed
the higher activity at 150 µg/ml concentration. In 2016, Satya et al., studied the
comparative antioxidant activity of Acacia sinuata crude extract and their synthesized
silver nanoparticles. Antioxidant activity was estimated by the scavenging effect of
DPPH radical and FRAP assay. Silver nanoparticles showed the higher antioxidant
activity than plant extract. In DPPH assay, silver nanoparticles made from Acacia
sinuata showed 65.78% antioxidant activity and plant extract showed 58.7% activity
at 500 µg/ml concentrations. FRAP assay also showed their increased activity in 500
µg/ml concentrations with 62.20% and 49.8% for silver nanoparticles and plant
extract respectively.
Sudha et al., (2017) demonstrated the antioxidant activity in Lippianodiflora silver
nanoparticles. The antioxidant activity of silver nanoparticles was confirmed by
reducing power assay and it showed the higher scavenging effect against DPPH,
superoxide, hydrogen peroxide and hydroxyl radical in 500 µg/ml concentration with
67%, 70%, 69 and 71.1% respective scavenging percentage. Patel et al., (2017)
demonstrated the antioxidant activity in Melaleuca leucadendra plant extract and

their silver nanoparticles by the DPPH and reducing power assay. Both plant extract
and silver nanoparticles exhibited the dose dependant antioxidant activity. Lingaraju
et al., (2016) investigated green synthesis of ZnO nanoparticles by aqueous stem
extract of Rutagraveolens (L.), evaluated for its free radical scavenging effect. The
Rutagraveolens (L.) derived ZnO nanoparticles proved efficient antioxidant potential
of IC50 value 9.34 mg/mL. Rehana et al., (2017) phytofabricated zinc oxide (ZnO)
nanoparticles with different leaf extracts namely Azadirachta indica, Hibiscus
rosasinensis, Murrayakoenigii, Moringa oleifera and Tamarindus indica for various
bioapplications.
The antioxidant activity was evaluated using five different radical scavenging assays.
The antioxidant activity was observed as higher in the extracts of M. oleifera and
Tamarindus indica comparatively than other extracts.
2.5.2 Anticancer studies of nanoparticles
Cancer is a big threat to human health worldwide, especially in developed countries,
where it is a leading cause of death due to cardiac and cerebrovascular diseases
(Velusamy et al., 2016). Cancer can be described as group of many diseases with the
spread of somatic clone and autonomous expansion (Underwood, 2020). The need of
drugs that could target cancer cells specifically without affecting the normal cells
gave birth to nanooncology (Riggio et al., 2011). Nanoparticles can be utilised as
drug carriers of chemotherapeutics, targeting them directly to the tumor without
distressing the healthy tissues (Tao et al., 2014). Nanotechnology-based therapies for
cancer have minimal side effects, high specificity and are taken as self by the immune
system. Small size, larger surface area to volume ratio allows the nanoparticles to
bind easily and carry DNA, RNA and proteins with high efficiency. Many
nanoparticles have been synthesized using different biological entities. The anti-
cancer activity of biogenic synthesized nanoparticles was resolved against MCF-7
breast malignant growth cell lines (Sivaraj et al., 2014). There are few reports are
available that show an increase in in-vitro cytotoxicity using zinc oxide nanoparticles
for a variety of cancer, including breast, leukemias, bone, lymphomas colon and
glioma (Hanley et al., 2008; Yamaki et al., 2009). In 2018, Raajshree and Brindha,
evaluate the anticancer effect of zinc oxide (ZnO-NPs) nanoparticles synthesized
from Turbinaria conoides against a murine model of Dalton's lymphoma ascites
(DLA) and ZnO-NPs possess more significant anticancer activities. Pei et al., (2019)
synthesized silver nanoparticles by aqueous extract of Coptis chinensis, revealed a

significant decline in cell viability.
2.5.3 Antifungal studies of nanoparticles
Fungi molds or mildew are complex organisms with slow growth rate stain and
deteriorate the performance properties of the fabrics. Algae belongs to either fungal or
bacteria groups require continuous source of water and sunlight to grow and develop
darker stains on the fabrics. Fungi are desirable agents for biogenic synthesis of
nanoparticles, since they give high metals tolerance and are simple to handle. They
also secrete significant amount of extracellular proteins that contribute to the stability
of the nanoparticles (Balaji et al., 2009; Du et al., 2010; Netala et al., 2016).
Hassan et al., (2019a) examined that the biosynthesized ZnO NPs from leaf extracts
of Olea europaea and Origanum majorana plants were evaluated for antifungal
activitiy These NPs reduced the appearance of grey and black mould disease on
artificial inoculation with Botrytis cinerea and Alternaria alternata in test pepper
plants compared to synthesized ZnONPs and untreated plants. The antimicrobial and
antifungal efficacy of silver nanoparticles synthesized using the filtrate
from Aspergillus flavus. These nanoparticles were effective in controlling the
bacteria Bacillus subtilis, Bacillus cereus, Enterobacter aerogenes, Escherichia coli,
and Staphylococcus aureus, with B. subtilis and E. coli being most sensitive (Fatima
et al., 2016).
Elumalai et al., (2014) showed that the antimicrobial activities of the samples were
enhanced by various microbial and fungal organisms such as P. mirabilis, C. albicans
and C. tropicalis using the disk diffusion method. The concentration of zinc oxide
decreased the growth of microbes. Bhat et al., (2015) evaluated that synthesized silver
nanoparticles using Candida albicans and their effects when used alone or in
combination with the antibiotic. It was found that the activity the antibiotic increased
when it was used with nanoparticles. He et al., (2011) showed the antifungal effect of
ZnO NPs and their mode of action on two pathogenic postharvest fungi: Penicillin
expansum and Botrytis cinerea. The results obtained showed that at concentrations
greater than 3mmol L-1, the growth of these pathogens was significantly inhibited.
Sharma et al., (2010) studied the antifungal activity of ZnO nanoparticles, on the
strains of Fusarium sp. under various conditions.

The antifungal activity of these NPs was compared with fungi under conventional
antifungal treatment using copper sulphate. The finding shows that the antifungal
activity of the ZnO NPs depends on the concentration and size of the nanoparticles.
2.5.4 Anti-bacterial studies of nanoparticles
Now a- days broad spectrum antibiotics became multidrug resistant towards human
pathogenic organisms. Recently, numerous antibacterial activities of green
synthesized nanoparticles were conducted against gram positive and gram- negative
bacterial population. The focus on the toxicity of biogenic nanoparticles on
pathogenic species has opened a new variety of antimicrobial agents. Silver and zinc
oxide nanoparticles are considered to be the new generation antimicrobials because of
their tremendous antimicrobial property. These biogenic NPs could be utilized as
nano-pesticide for enhancing use of agribusiness. There are many reports of
antimicrobial property of green synthesized zinc oxide, silver, silver doped zinc
nanoparticles using different medicinal plants. Some of them are reported in Table
2.1.
Table 2.1: Antibacterial activity of synthesized nanoparticles ZnO, Ag, Ag-ZnO using
various plants extracts against different bacterial strains
Plant Name NPs Bacterial Organisms References
Adhathodavasica ZnO Salmonella typhimurium and Nazeruddin et al., 2014

Bacillus subtilis
Coptidis ZnO Escherichia coli, B. Nagajyothi et al., 2014
megaterium
Mimusopselengi Ag S. aureus, Escherichia coli Kumar et al., 2014
Hibiscus sabdariffa ZnO Escherichia coli Bala et al., 2015
Centella asiatica, ZnO Pseudomonas aeruginosa, Logeswari et al., 2015

Citrus sinensis, Escherichia coli,
Ocimumtenuiflorum, Staphylococcus aureus
Syzygiumcumini, Klebsiella pneumonia
Solanum tricobatum
Azadirachta indica ZnO Staphylococcus aureus Bhuyan et al., 2015

Althaea officinalis Ag S. aureus Jafari et al., 2015
Psidium guajava Ag Pseudomonas aeruginosa Bose and Chatterjee,

2016
Cymbopogon Ag S. aureus, Pseudomonas Gupta and Ganjewala,

flexuosus aeruginosa 2015
Aloe vera ZnO Escherichia coli, Ali et al., 2016

Pseudomonas aeruginosa
Rosa canina ZnO Staphylococcus aureus, Jafarirad et al., 2016
Escherichia coli
Carica papaya ZnO Klebsiella pneumoniae, Kokila et al., 2016

Bacillus subtilis,
Staphylococcus
aureus,Escherichia coli
Capsicum sp. Ag Staphylococcus aureus, Singh et al., 2016
Escherichia coli
Passiflora caerulea ZnO Klebsiella pneumoniae, Santhoshkumar et al.,

Bacillus subtilis, Escherichia 2017
coli, Serratia sp., and
Streptococcus sp.
Ficus religiosa ZnO Bacillus subtilis, Salmonella Nakkala et al., 2017
typhi, Pseudomonas
fluorescens, Escherichia coli,
Diospyros sylvatica ZnO Escherichia coli, Pethakamsetty et al.,
Staphylococcus aureus, 2017
Klebsiella pneumoniae,
Bacillus subtilis,
Pseudomonas aeruginosa,

Proteus vulgaris, Bacillus

pumilis, Streptococcus
pyogenes
Tamarindus indica ZnO Bacillus subtilis Jayaprakash et al., 2017
Staphylococcus aureus,
Salmonella typhi, Bacillus
cereus, Micrococcus luteus,
Escherichia coli, Klebsiella
pneumonia
Zea mays Ag B. cereus, L. Patra et al., 2017
monocytogenes, S. aureus, E.
coli and S. typhimurium
Cassia auriculata ZnO Bacillus cereus, Bacillus Padalia et al.,2018

subtilis, Staphylococcus
aureus, Salmonella
typhimurium,
Corynebacterium rubrum,
Escherichia coli, Klebsiella
pneumoniae and Salmonella
typhimurium
Costus pictus ZnO Bacillus subtilis and S. Suresh et al., 2018
paratyphi
Ag Escherichia coli Shehzad et al., 2018
Rhazya stricta
and Bacillussubtilis
pu-erh tea Ag Escherichia coli, Klebsiella Loo et al., 2018

pneumoniae,
Salmonella typhimurium
and Salmonella Enteritidis

Prunus cerasifera Ag- L. theobromae, A. fumigatus, Ahmad et al., 2018

ZnO X. citri, A. terreus, P.
chrysogenum, P. syringae, A.
flavus F. solani, A. niger
Albizia lebbeck ZnO S. aureus, E. coli, B. Umar et al., 2019

cereus and S. typhi and K.
pneumoniae
Pandanus odorifer ZnO Bacillus Hussain et al., 2019

subtilis and Escherichia coli
Silybum marianum ZnO Escherichia coli Arvanag et al., 2019
Impatiens Ag Staphylococcus aureus and Aritonang et al., 2019

balsamina and Lantan Escherichia coli
a camara
Phyllanthus emblica Ag Acidovoraxoryzae Masum et al., 2019
Clitoriaternatea Ag- Escherichia coli Chan et al., 2019

ZnO
Colocasia esculenta Ag- Escherichia coli and Bacillus Kumar et al., 2019
ZnO subtilis
Sambucus ebulus ZnO Bacillus cereus, S. aureus, Alamdari et al., 2020

and Escherichia coli
lippiaadoensis ZnO Escherichia coli, Klebsiella Demissie et al., 2020

pneumonia, Staphylococcus
aureus, and Enterococcus
faecalis
Cassia ZnO Escherichia coli and S. Minha et al., 2020

fistula and Melia aureus

azadarach
Lysilomaacapulcensis Ag Escherichia coli and Garibo et al., 2020

Carallumatuberculata Ag Escherichia coli, Zarei et al.,2020
and Bacillus cereus
Cannabis sativa Ag- Pseudomonas aeruginosa, Chauhan et al., 2020
ZnO Klebsiella pneumonia,
MRSA, Escherichia coli,
Salmonella typhi
Tridax procumbens Ag- Bacillus subtilis, Jabeen et al., 2020
ZnO Staphylococcus aureus,
Klebsiella pneumonia,
Streptococcus pyogenes,
Escherichia coli
Hence, we aimed to synthesize green metal nanoparticles i.e., Zinc oxide (ZnO) and
Silver doped zinc oxide (AgZnO) using aqueous extract of Moringa oleifera and
Trillium govanianum. Characterization of these green synthesized ZnO and AgZnO
bimetallic nanoparticles was performed by UV-Vis spectroscopy, FTIR, XRD, FE-
SEM-EDS mapping and TEM. The antioxidant, anticancer and antimicrobial
activities of the plant extracts and these nanoparticles were also analysed. The
antimicrobial activity of ZnO and AgZnO nanoparticles on different fungi and
bacteria are studied. The anticancer activity of plant extract and synthesized ZnO and
AgZnO nanoparticles are also studied on different cell lines.

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Chapter 2 Review of Literature

Nanotechnology is the next-generation technology having key significance in the

Figure 2.1: Different applications of nanotechnology.

Faculty of Applied Sciences and Biotechnology 6

Nanobiotechnology opens up new areas of research and applications such as

shape of organic or polymeric nanoparticles is nanosphere or nanocapsule (Mansha et

Faculty of Applied Sciences and Biotechnology 8

2.1.3 Metallic nanoparticles

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quality, by employing environment friendly economical processes has always been a

Figure 2.2: Schematic representation of ‘top-down approach’ and 'bottom-up

Faculty of Applied Sciences and Biotechnology 11

leaches out systematically bit-after-bit leading to the generation of nanosized

Research on the synthesis of nanosized materials is of great interest because of their

Figure 2.3: Different methods of nanoparticles synthesis.

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the synthesized nanomaterials. Carbon nanotubes can also be produced by this

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agents to stabilize dispersive nanoparticles Protective agents can be absorbed on or

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2.3.2 (h) Micro emulsion technique

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Figure 2.4: Different microorganisms used for synthesis of nanoparticle.

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insoluble complexes is a defense mechanism developed by the bacteria to overcome

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2.3.3.1 (b) Fungus

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Biological synthesis of metallic NPs has been successfully reported by actinobacteria

various applications else than acting as medicines like, food supplements,

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Figure 2.5: Plant mediated synthesis of nanoparticles and their different

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2.4.1 Moringa oleifera

Figure 2.6: Moringa oleifera pods and seed kernels.

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Division - Tracheophyta – vascular plants, tracheophytes

good antioxidant activity on hepatocyte growth factor (HGF) induced Met

phytochemical analysis (Akihito and Yoshihiro, 2008; Jiang et al., 2014). T.

Figure 2.8: Medicinal uses of Trillium govanianum plant.

Faculty of Applied Sciences and Biotechnology 37

Faculty of Applied Sciences and Biotechnology 38

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demonstrated the antioxidant activity in Melaleuca leucadendra plant extract and

synthesized silver nanoparticles by aqueous extract of Coptis chinensis, revealed a

Faculty of Applied Sciences and Biotechnology 42

Adhathodavasica ZnO Salmonella typhimurium and Nazeruddin et al., 2014

Hibiscus sabdariffa ZnO Escherichia coli Bala et al., 2015

Centella asiatica, ZnO Pseudomonas aeruginosa, Logeswari et al., 2015

Faculty of Applied Sciences and Biotechnology 43

Althaea officinalis Ag S. aureus Jafari et al., 2015

Psidium guajava Ag Pseudomonas aeruginosa Bose and Chatterjee,

Cymbopogon Ag S. aureus, Pseudomonas Gupta and Ganjewala,

Aloe vera ZnO Escherichia coli, Ali et al., 2016

Carica papaya ZnO Klebsiella pneumoniae, Kokila et al., 2016

Passiflora caerulea ZnO Klebsiella pneumoniae, Santhoshkumar et al.,

Faculty of Applied Sciences and Biotechnology 44

Proteus vulgaris, Bacillus