Synthesis of Zinc Oxide /PVA nanocomposite for Antibacterial activities

A Thesis Proposal Submitted to the Department of Applied Chemistry

School of Applied Natural Science

Adama Science and Technology University

By

Ayana Darge (UGR/17894/11)

Jan, 2022

Adama, Ethiopia
Acknowledgement
First, I give all glory and honor to the supreme God, the source of all wisdom, knowledge and
understanding I would like to thank and praise my God for giving life and wisdom to achieve
this feat. Then and foremost, the completion of this seminar-1, work will not be possible without
the excellent guidance of my advisor Dr. Eneyew Amare. I acknowledge all his encouragement
and valuable time given to me with deep gratitude and appreciation.

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Contents
Acknowledgement 1
List Acronyms2
List of figures and List of tables 3
Abstract 4
1 INTRODUCTION 5
1.1 Background of the study..............................................................................................................5
1.2 Statement of the problem.............................................................................................................6
1.3 Objectives....................................................................................................................................7
1.3.1 General objective.................................................................................................................7
1.3.2 Specific objective.................................................................................................................7
1.3.3 Significance of the study......................................................................................................7
1.3.4 Scope of the study................................................................................................................7
2 LITERATURE REVIEW 8
2.1 Nano Particles (NPs)....................................................................................................................8
2.2 Zinc Oxide Nanoparticles (ZnO NPs)..........................................................................................8
2.3 Synthesis method of ZnO NPs.....................................................................................................9
2.3.1 Physical Method................................................................................................................10
2.3.2 Chemical Method...............................................................................................................10
2.4 Polyvinyl alcohol (PVA)...........................................................................................................11
2.4.1 Chemistry of polyvinyl alcohol..........................................................................................12
2.5 Polymer-Coated Nanoparticles..................................................................................................13
2.6 Effect of Particle Size And Shape of Polymer-Coated Nanoparticles On Antibacterial Activity
13
2.7 Antibacterial activity of ZnO NPs..............................................................................................14
3 MATERIALS AND METHODS 17
3.1 Chemicals and Reagents............................................................................................................17
3.2 Materials and Instruments..........................................................................................................17
3.3 Method.......................................................................................................................................18
3.3.1 ZnO Nanoparticle synthesis by sol gel process..................................................................18
3.3.2 ZnO-Polyvinyl alcohol (PVA) nano composites................................................................18
3.3.3 Characterization Techniques of ZnO Nanoparticles...........................................................18

3
 3.3.4 Antibacterial Activity study...............................................................................................19
4 RESEARCH WORK PLAN 19
5 BUDGET PLAN 20
6 REFERENCES 20

4
List Acronyms
AFM……………………………………………… Atomic Force Microscope

CVD………………………………………………. Chemical Vapour Deposition

DNA……………………………………………….Deoxyribonucleic Acid

FTIR…………………………………………….... Fourier transmission infrared

LB…………………………………………………. Lysogeny Broth

NP…………………………………………………. Nano Particles

PEG………………………………………………… polyethylene glycol

PGA………………………………………………… l-glutamic acid

PQQ………………………………………………… pyrroloquinoline quinone

PVA………………………………………………… Polyvinyl alcohol

PVP………………………………………………… polyvinyl pyrolidone

ROS…………………………………………………Reactive oxygen species

SEM………………………………………………… scanning electron microscopy

TEM………………………………………………… transmitted electron microscopy

UV-Vis…………………………………………………Ultra visible

XRD………………………………………………… X-Ray diffraction

ZnO NPs……………………………………………………………………….. Zinc Oxide Nanoparticles

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 List of figures and List of tables
Figure 2 1: Antibacterial activity and/or zone of inhibition produced by zinc oxide
nanoparticles against gram-positive and gram-negative bacterial strains namely a)
Escherichia coli, b) Staphylococcus aureus, c) Pseudomonas aeruginosa, and d) Bacillus
subtilis [54]...........................................................................................................................16

Table 2 1: The toxicity (30-min EC50, EC20 and NOEC, and MIC) of metal oxide aqueous
suspensions CuSO4 and ZnSO4·7H2O to bacteria Vibrio fischeri [59].......................................15

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 Abstract
Nanoscience, nanotechnology, nanomaterials or nanochemistry are only a few of the new nano-
containing terms that occur frequently in scientific reports, in popular books as well as in
newspapers and that have become familiar to a wide public, even of non-experts. Nanoparticles
are the building blocks of nanotechnology. Zinc oxide nanoparticles have received considerable
attention due to their antimicrobial, UV blocking, high catalytic and photochemical activities.
Although zinc oxide nanoparticles are stable, they have been further stabilized by coating them
with different polymers such as polyvinyl pyrolidone (PVP), polyvinyl alcohol (PVA), poly (α,
γ, l-glutamic acid) (PGA), polyethylene glycol (PEG), chitosan, and dextran. Zinc oxide
nanoparticles are more active against gram-positive bacteria relative to other NPs of the same
group of elements. The crystal structures of the ZnO nanoparticles and nanocomposite film were
studied by X-ray diffraction (XRD) technique.

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1 INTRODUCTION

1.1 Background of the study

Prefix “nano” has found in last decade an ever-increasing application to different fields of the
knowledge. Nanoscience, nanotechnology, nanomaterials or nanochemistry are only a few of the
new nano-containing terms that occur frequently in scientific reports, in popular books as well as
in newspapers and that have become familiar to a wide public, even of non-experts. The prefix
comes from the ancient Greek να̃νος through the Latin nanus meaning literally dwarf and, by
extension, very small. Within the convention of International System of Units (SI) it is used to
indicate a reduction factor of 10_9 times. So, the nanosized world is typically measured in
nanometers (1nm corresponding to 10-9 m) and it encompasses systems whose size is above
molecular dimensions and below macroscopic ones (generally > 1 nm and < 100 nm).
Nanotechnology is the science of the small; the very small. It is the use and manipulation of
matter at a tiny scale. At this size, atoms and molecules work differently, and provide a variety of
surprising and interesting uses. Nanotechnology and Nanoscience studies have emerged rapidly
during the past years in a broad range of product domains. It provides opportunities for the
development of materials, including those for medical applications, where conventional
techniques may reach their limits. Nanotechnology should not be viewed as a single technique
that only affects specific areas. Although often referred to as the ‘tiny science’, nanotechnology
does not simply mean very small structures and products. Nanoscale features are often
incorporated into bulk materials and large surfaces. Nanotechnology represents the design,
production and application of materials at atomic, molecular and macromolecular scales, in order
to produce new nanosized materials [1]. Pharmaceutical nanoparticles are defined as solid,
submicron-sized (less than 100 nm in diameter) drug carrier that may or may not be
biodegradable. The term nanoparticle is a combined name for both nanosphares and
nanocapsules. Nanospheres are matrix system in which drug is uniformly dispersed, while
nanocapsules are the system in which the drug is surrounded by a unique polymeric membrane.
This systemic review focuses on Classification, method of preparation, Characterization,
application, health prospective and Pharmacological aspects of nanoparticles [1].

8
Many kinds of Nano materials have been used to prepare organic/inorganic Nano composites
among these inorganic fillers ZnO Nanoparticles have obtained an exceptional spot because of
their fine stability, superior refractive index, hydrophobicity, capability to absorbed UV,

9
nontoxicity, as well as magnificent transparency for the perceptible light. These materials have
increased in the field of basic and applied sciences due to their many applications in
microelectronic and optoelectronic devices [2]. In order to achieve desirable properties, two or
more oxides are combined. This makes it possible to change the composition ratio of two oxides
and produce a new material with special preset characteristics [3]. Zinc Oxide powder dampen
the development of gram positive bacteria keenly unless gram-negative bacteria [4]. This report
register was localized on metal oxides like ZnO and CuO as antibacterial factor to stop bacterial
development [5].

Metal oxide nanoparticles (NPs) are viewed as a potential next generation or disinfecting agents,
which are finding applicable in the field of clinical concern, consumer products and in other
industrial applications [6]. Zinc oxide nanoparticles (ZnO NPs) also have considerable attention
to their unique antibacterial, antifungal, UV filtering properties, high catalytic and photochemical
activity [7]. However, most ZnO NPs are produced synthetically and has the advantage of low
cost and white appearance over the silver nanoparticles [8]. The antibacterial activities of
synthesized ZnO NPs have also been studied. Antibacterial activities of ZnO, NPs have been
assessed against the microorganisms Staphylococcus aureus, Escherichia coli, Salmonella typhi,
Pseudomonas aeruginosa, and Streptococcus pyogenesis, as well as antifungal microorganism
Aspergillus Niger, by disc diffusion method. [9].

PVA is leading water-soluble clear polymer, and is extensively applied in industries due to the
good chemical and physical properties, non-toxicity, better film formation capacity, better
chemical resistance, biodegradability and high crystal modulus. PVA is applied here in
hydrolyzed form with the point of 85% hydrolysis [10]. It is a polymer with many
pharmaceutical, biomedical applications and technological [11]. It can be applied for casing of
ZnO [12].

1.2 Statement of the problem

Microbes, such as bacteria, viruses, fungi, and parasites, are living organisms that evolve over
time. Their primary function is to reproduce, thrive, and spread quickly and efficiently.
Therefore, microbes adapt to their environments and change in ways that ensure their survival. If
something stops their ability to grow, such as an antimicrobial, genetic changes can occur that
enable the microbe to survive. With the increasing resistance of antibiotics to bacteria, new and
effective methods are needed to transform existing antibiotics to solve the problem of long
development cycles for new drugs. The antibiotic nano delivery system has proven to be a
promising strategy. Research indicated that the problem of antibiotic resistance can be
effectively solved by {developing zinc oxide nanoparticle-based antibacterial therapies are well
known in recent years} using new nanomedicine carriers to effectively deliver antibiotics.

1.3 Objectives

1.3.1 General objective

The general objective of this proposed research work is to synthesis and characterizes ZnO /PVA
nanocomposite to investigate its antibacterial activity.

1.3.2 Specific objective

The specific objectives of this research are to:

 Synthesis of ZnO/PVA nanoomposites.

 Characterize ZnO/PVA nanocomposite using XRD, FTIR, UV-Vis, and SEM.
 Examine the activities of ZnO NPs to bacterial species.

1.3.3 Significance of the study

In this research, ZnO nanoparticles are examined that have several advantages, like high
antibacterial activity at low concentrations, activity against several strains of bacteria, and
comparatively low cost, among others. The study will also provide the community with
information about synthesizing and using ZnO nanoparticles as antimicrobials at a low cost and
environmental friendly manner. Additionally, the study presents significant information
regarding a variety of characterization techniques employed to synthesize ZnO NPs.

1.3.4 Scope of the study

The study includes the preparation of ZnO nanoparticles by co-precipitation method using PVA
as the capping agent. To obtain a better understanding of the nature of the synthesized ZnO
nanoparticles, the characterization of the study will be performed by XRD, SEM, FTIR and UV-
Vis techniques. Finally, the antibacterial activities of synthesized ZnO nanoparticles are studied.

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2 LITERATURE REVIEW

2.1 Nano Particles (NPs)

Nanoparticles are the building blocks of nanotechnology. Nanotechnology is the study of
manipulating matter on a nuclear and sub atomic scale levels. Nanotechnology refers to the
production and usage of material with nanoscale dimension. It is a multidisciplinary scientific
field undergoing explosive development and one of the most active research areas in modern
material science. Because of Specific characteristics such as size distribution and morphology
nanoparticles exhibit completely new or improved property.

Nanoparticles are particles in the nanoscale size between 1nm and 100nm with surrounding
interfacial layer. They are very small particles with improved thermal conductivity, catalytic
reactivity, nonlinear optical performance, and chemical stability due to their larger surface area –
to- volume ratio [13]. The interfacial layer is an integral part of nanoscale matter, fundamentally
affecting all of its properties. In nanotechnology, a particle is defined as a small object that
behaves as a whole unit with respect to its transport and properties [14].

NPs are the great scientific interest as they are, in effect a bridge between bulk material and
atomic or molecular structures. A bulk material should have constant physical properties
regardless of its size, but at nanoscale size dependent properties are often observed [15].

2.2 Zinc Oxide Nanoparticles (ZnO NPs)

Zinc oxide nanoparticles have received considerable attention due to their antimicrobial, UV
blocking, high catalytic and photochemical activities. Sharma et al., (2010) [16] have reported
that ZnO nanoparticles posses antibacterial and antifungal activities even at lower concentrations
hence suitable for thin coating applications. Further, antifungal activity of ZnO nanoparticles
does not affect soil fertility compared to the conventional antifungal agents. Feris et al., (2010)
[17] have concluded that the bacterium and fungal lipid bilayers get ruptured due to cytotoxic
behavior of ZnO nanoparticles resulting in the drainage of the cytoplasmic contents. Raghupathi
et al., (2011) [18] have investigated the antibacterial effect of ZnO nanoparticles and developed
antibacterial agents against a wide range of microorganisms to control the bacterial infections.
Jayaseelan et al., (2012) [19] have reported significant antimicrobial activity of biosynthesized

11
ZnO NPs. The green synthesis of ZnO nanoparticles and the studies on their antimicrobial
activities are still in the infancy stage and limited number of works have been reported [20].

Over the past of few years, more interest is drawn towards ZnO NPs since it has wider varieties
of application particularly in the field of biomedical system, optics and electronics. Among all
these of metal oxides ZnO NPs attract much attention because of their stimulating properties
such as the high direct bandwidth 3.3eV at room temperature and high excitation energies at
60meV, optical properties, high catalytic activities, anti-inflammatory wound healing and UV
filtering properties. ZnO is the biosensors for cholesterol, enzyme biochemistry and other bio-
sensing application [21].

Zinc oxide is an inorganic, nontoxic, crystalline, nonhygroscopic, and nontoxic material, which
is very cheap, safe, and readily available, which has aroused great interest in the field of organic
transformations, sensors, and transparent conductors. [22]. The ZnO NP is an exclusive material
that has semiconducting, piezoelectric and has versatile applications in transparent electronics,
UV light emitters, chemical sensors, spin electronics, personal care products, catalysts, coating
and paints Due to these unique properties, ZnO NPs find application in antireflection coatings,
transparent electrodes in solar cells and UV light emitters, diode lasers [23].

2.3 Synthesis method of ZnO NPs

The preparation of nanoscale structures and devices can be accomplished through “bottom-up”
or “top-down” methods. In the bottom-up approach, small building blocks are assembled into
larger structures; chemical synthesis is a good example of bottom-up approach in the synthesis of
nanoparticles. In the top-down approach, large objects are modified to give smaller features,
attrition or milling is a good example of top-down approach. Both approaches play very
important roles in modern industry and most likely in nanotechnology. Methods to produce
nanoparticles from atoms are chemical processes based on transformations in solution e. g. sol-
gel processing, chemical vapour deposition (CVD), plasma or flame spraying synthesis, and laser
pyrolysis, atomic or molecular condensation. These chemical processes rely on the availability of
appropriate “metal-organic” molecules as precursors [24, 25, 26, 27]. The synthesis approaches

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are also classified into physical, chemical and biological synthesis depending on the processes or
materials involved in the production process [27].

2.3.1 Physical Method

In the physical method, physical forces are involved in the attraction of nano scale particles and
formation of large, stable, well-defined nanostructures. The physical method often called top-
down approach. It uses mechanical forces to break bulk particles or beams of atoms for the
formation of nanoparticles. Physical method includes nanoparticle synthesis through colloidal
dispersion, vapor condensation, amorphous crystallization, physical fragmentation and
evaporation, pulse laser deposition, sputtering. Physical synthesis methods are highly useful in
producing ultra-thin and highly pure nanostructures and Nano alloys. The main shortcoming of
physical method is the use of costly equipment, high temperature and pressure, large space area
for setting up of machines [26]. Physical methods of ZnO nanoparticles synthesis include high
energy ball milling, melt mixing, physical vapor deposition, laser ablation, sputter deposition,
electric arc deposition, and ion implantation [27].

2.3.2 Chemical Method

The chemical method involves the use of toxic chemicals which can prove to be hazardous for
the environment and the person handling it. These methods may result in toxic and harsh
products which may pose biological risks to the environment; due to high surface charge and
high surface area of nanoparticles, harsh chemicals may remain adsorbed onto nanoparticles.
Releasing these chemicals into the environment may cause adverse effects on organisms
including microorganisms, plants, invertebrates, and vertebrates including humans at various
trophic levels. These drawbacks have led to the emergence of green synthesis methods based on
the use of environmentally friendly and biocompatible materials such as plants and
microorganisms. Therefore, it is essential to optimize green methods for nanoparticle synthesis
[26, 28]. Chemical methods need to use of toxic chemical solvents such as reducing agents and
these methods are expensive. Recently in nanotechnology, scientists wish for green synthesis
with plant extracts, because this method has been useful and plays a very important role which is
cheap, clean, safe, eco-friendly, non-toxic and without any dangerous substance.[27] Metal

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nanoparticles are usually synthesized using various chemical method such as chemical reduction,
solvo- thermal reduction, electrochemical techniques (Krishna and Goia Dan, [29]; Saxena et al.
[29] and photochemical reaction in reverse micelles (Taleb et al. [30].Among them, chemical
reduction is the most frequently applied method. Previous studies showed that the use of a
chemical reducing agent resulted in generation of larger particles and consume more energy. It
was also reported that more side produts were formed by chemical approaches which are not
eco-friendly. Moreover, the chemically synthesized nanoparticles were reported to show less
stability and more agglomeration (Mukherjee et al. [31]. Hence there is a need to develop an eco-
friendly protocol that could produce stable and dispersible nanoparticles of controllable size by
consuming less energy, environmentally friendly and cost effective.

2.4 Polyvinyl alcohol (PVA)

Polyvinyl alcohol (PVA) is a vinyl polymer joined by only carbon-carbon linkages. The linkage
is the same as those of typical plastics such as polyethylene, polypropylene, and polystyrene, and
of watersoluble polymers such as polyacrylamide and polyacrylic acid. Among the vinyl
polymers produced industrially, PVA is the only one known to be mineralized by
microorganisms. PVA is water soluble and biodegradable; hence it is used to make water-soluble
and biodegradable carriers, which may be useful in the manufacture of delivery systems for
chemicals such as fertilizers, pesticides, and herbicides. PVA is completely degraded and utilized
by a bacterial strain, Pseudomonas 0-3, as a sole source of carbon and energy. However,
PVAdegrading microorganisms are not ubiquitous within the environment. Almost all the
degrading strains belong to the genus Pseudomonas, although some do belong to other genera
[32]. Among the PVAdegrading bacteria reported so far, a few strains showed no requirement for
pyrroloquinoline quinone (PQQ). From a PVA-utilizing mixed culture, Pseudomonas sp.
VM15C and P. putida VM15A were isolated. Their symbiosis is based on a syntrophic
interaction. VM15C is a PVA-degrading strain that degrades and metabolizes PVA, while
VM15A excretes a growth factor that VM15C requires for PVA utilization.

Polyvinyl alcohol (PVA), a degradable polymer, is easily dissolved in water, and combination of
ZnO nanoparticles and PVA results in improved electrical, mechanical and optical properties
(Roy et al. 2013). In a study, the effect of ZnO nanoparticles on the physical, mechanical and
antibacterial properties of pediocin nanofilm was examined, which indicated that the addition of

14
ZnO nanoparticles can enhance the properties of the film [32]. In a similar research, it was
revealed that the insertion of ZnO nanorods resulted in improvements of physical, chemical and
antibacterial properties of sago starch films [33]. In another study, the mechanical, thermal and
antibacterial properties of waterborne polyurethane with addition of flower-like ZnO were
recovered [34].

Although several reports on ZnO-based nanocomposite biopolymers have appeared in the

literature, it seems that a few of them focused on PVA, which is a degradable polymer suitable
for food packaging. Thus, in the present study, ZnO nanoparticles were synthesized via sol-gel
method. Furthermore , the microstructure, mechanical, antibacterial, physical and optical
properties of PVA nanocomposites were compared as a function of nanoreinforcements in filler
loading contents and under the same preparation conditions.

2.4.1 Chemistry of polyvinyl alcohol

PVA full form is polyvinyl alcohol. Polyvinyl alcohol is a man-made or synthetic polymer. PVA
polymer molecule contains vinyl and alcohol groups. It is a very useful polymer due to its
adhesive properties. PVA is water soluble, crystalline and flammable in nature. Presence of
alcohol group in it makes it flammable.

i. PVA Structure or Polyvinyl Alcohol Structure: PVA monomer unit is vinyl acetate. It
means, it is formed by the polymerization of vinyl acetate. Vinyl acetate formula is H3C-
CO2-CH=CH2. PVA is not prepared by direct polymerization of vinyl acetate. Instead, it
is prepared by hydrolysis of polyvinyl acetate (PVAc) through an alcohol (generally
methanol) in presence of an alkaline catalyst. 
ii. Physical Properties of PVA:  PVA shows high tensile strength and flexibility. It is
soluble in water and has no odor. PVA molecular weight or polyvinyl molecular weight
ranges between 26,000-30,000. Its melting point is 185°C. It is insoluble in organic
solvents but slightly soluble in ethanol. 
iii. Chemical Properties of PVA:  Polyvinyl alcohol can react with butyraldehyde and
formaldehyde. It is atactic type material and resistant to oil and grease. 
iv. Polyvinyl Alcohol Uses: PVA polymer is used in many fields. It is used as main
component in many drugs. Many of its applications are based on its adhesive property.
PVA is a type of synthetic adhesive. PVA is used as PVA glue in many fields. Meaning

15
 of PVA glue is a glue or fixative made up of polyvinyl alcohol. PVA films are used in
packaging of different materials such as detergents, chemicals, disinfectants etc. in many
industries. As PVA is water soluble, PVA packaging material is biodegradable. 

2.5 Polymer-Coated Nanoparticles

Many bacterial infections are transmitted by contact with door knobs, key boards, water taps,
bath tubs, and telephones; therefore, it is essential to develop and coat such surfaces with
inexpensive advanced antibacterial substances so that their growth is inhibited. It is important to
use such concentrations of antibacterial substances that they may kill the pathogens but spare the
human beings. It may happen only if they are coated with a biocompatible hydrophilic polymer
of low cost. Schwartz et al. [35] have reported the preparation of a novel antimicrobial composite
material hydrogel by mixing a biocompatible poly (N-isopropylacrylamide) with zinc oxide
nanoparticles. The SEM image of the composite film showed uniform distribution of zinc oxide
nanoparticles. It exhibited antibacterial activity against E. coli at a very low zinc oxide
concentration (1.33 mM). Also, the coating was found to be nontoxic toward mammalian cell
line (N1H/3T3) for a period of 1 week. Zinc oxide/hydrogel nanocomposite may safely be used
as biomedical coating to prevent people from contracting bacterial infections.

Although zinc oxide nanoparticles are stable, they have been further stabilized by coating them
with different polymers such as polyvinyl pyrolidone (PVP), polyvinyl alcohol (PVA), poly (α,
γ, l-glutamic acid) (PGA), polyethylene glycol (PEG), chitosan, and dextran [36, 37]. The
antibacterial activity of engineered zinc oxide nanoparticles was examined against gram-negative
and gram-positive pathogens, namely E. coli and S. aureus and compared with commercial zinc
oxide powder. The polymer-coated spherical zinc oxide nanoparticles showed maximum
bacterial cell destruction compared to bulk zinc oxide powder [38]. Since nanoparticles coated
with polymers are less toxic due to their low solubility and sustained release, their cytotoxicity
can be controlled by coating them with a suitable polymer.

2.6 Effect of Particle Size And Shape of Polymer-Coated Nanoparticles On Antibacterial

Activity

E. coli and S. aureus exposed to different concentrations of poly ethylene glycol (PEG)-coated
zinc oxide nanoparticles (1–7 mM) of varying size (401 nm–1.2 μm) showed that the

16
antimicrobial activity increases with decreasing size and increasing concentration of
nanoparticles. However, the effective concentration in all these cases was above 5 mM. There
occurs a drastic change in cell morphology of E. coli surface which can be seen from the SEM
images of bacteria before and after their exposure to zinc oxide nanoparticles [39]. It has been
nicely demonstrated by Nair et al. [40] that PEG-capped zinc oxide particles and zinc oxide
nanorods are toxic to human osteoblast cancer cells (MG-63) at concentration above 100 μM.
The PEG starch-coated nanorods/nanoparticles do not damage the healthy cells.

2.7 Antibacterial activity of ZnO NPs

Zinc oxide nanoparticles are more active against gram-positive bacteria relative to other NPs of
the same group of elements. Ready to eat food is more prone to infection by Salmonella,
Staphylococcus aureus, and E. coli which pose a great challenge to food safety and quality. The
antimicrobial compounds are incorporated in the packed food to prevent them from damage.
Antimicrobial packaging contains a nontoxic material which inhibits or slows down the growth
of microbes present in food or packaging material [41]. An antimicrobial substance for human
consumption must possess the following properties.

 It should be nontoxic.
 It should not react with food or container.

 It should be of good taste or tasteless.

 It should not have disagreeable smell.

Zinc oxide nanoparticle is one such inorganic metal oxide which fulfills all the above
requirements, and hence, it can safely be used as medicine, preservative in packaging, and an
antimicrobial agent [42, 43]. It easily diffuses into the food material, kill the microbes, and
prevent human being from falling ill. In accordance with the regulations 1935/2004/EC and
450/2009/EC of the European Union, active packaging is defined as active material in contact
with food with ability to change the composition of the food or the atmosphere around it [44].
Therefore, it is commonly used as preservative and incorporated in polymeric packaging material
to prevent food material from damage by microbes [45]. Zinc oxide nanoparticles have been
used as an antibacterial substance against Salmonella typhi and S. aureus in vitro. Of all the

17
metal oxide nanoparticles studied thus far, zinc oxide nanoparticles exhibited the highest toxicity
against microorganisms [46].

It is universally known that zinc oxide nanoparticles are antibacterial and inhibit the growth of
microorganisms by permeating into the cell membrane. The oxidative stress damages lipids,
carbohydrates, proteins, and DNA [47]. Lipid peroxidation is obviously the most crucial that
leads to alteration in cell membrane which eventually disrupt vital cellular functions [48]. It has
been supported by oxidative stress mechanism involving zinc oxide nanoparticle in Escherichia
coli [49]. However, for bulk zinc oxide suspension, external generation of H 2O2 has been
suggested to describe the anti-bacterial properties [50]. Also, the toxicity of nanoparticles,
releasing toxic ions, has been considered. Since zinc oxide is amphoteric in nature, it reacts with
both acids and alkalis giving Zn2+ ions.

The free Zn2+ ions immediately bind with the biomolecules such as proteins and carbohydrates,
and all vital functions of bacteria cease to continue. The toxicity of zinc oxide, zinc
nanoparticles, and ZnSO4·7H2O has been tested (Table ) against Vibrio fischeri. It was found that
ZnSO4·7H2O is six times more toxic than zinc oxide nanoparticles and zinc oxide. The
nanoparticles are actually dispersed in the solvent, not dissolved, and therefore, they cannot
release Zn2+ ions. The bioavailability of Zn2+ ions is not always 100% and may invariably change
with physiological pH, redox potential, and the anions associated with it such as Cl− or SO42−.

Table 2 1: The toxicity (30-min EC50, EC20 and NOEC, and MIC) of metal oxide aqueous
suspensions CuSO4 and ZnSO4·7H2O to bacteria Vibrio fischeri [59]

Chemical Toxicity to Vibrio fischeri, EC50, EC20, NOEC, and MIC (mg l− 1)
EC50 ± SD EC20 ± SD NOEC MIC
ZnO 1.8 ± 0.1 (1.4 ±  1.0 ± 0.4 (0.8 ± 0.3) 1.0 (0.8) 200 (160)
0.08)
Nano-ZnO 1.9 ± 0.2 (1.5 ±  0.9 ± 0.4 (0.7 ± 0.3) 0.75 (0.6) 100 (80)

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 0.16)
ZnSO4·7H2O 1.1 ± 0.25 (0.25 ±  0.8 ± 0.3 (0.2 ± 0.1) 0.5 (0.11) 10 (2.0)
0.06)
CuO 3811 ± 1012 903 ± 457 (722 ±  313 (250) 20,000
(3049 ± 819) 366) (16,000)
Nano-CuO 79 ± 27 (63 ± 22) 24 ± 5 (19 ± 4) 16 (12) 200 (160)

CuSO4 1.6 ± 0.29 (0.64 ±  0.9 ± 0.3 (0.36 ±  0.63 (0.25) 2.5 (1.0)

0.12) 0.12)

Solubility of zinc oxide (1.6–5.0 mg/L) in aqueous medium is higher than that of zinc oxide
nanoparticles (0.3–3.6 mg/L) in the same medium [51] which is toxic to algae and crustaceans.
Both nano-zinc oxide and bulk zinc oxide are 40–80-fold less toxic than ZnSO 4 against V.
fischeri. The higher antibacterial activity of ZnSO4 is directly proportional to its solubility
releasing Zn2+ ions, which has higher mobility and greater affinity [52] toward biomolecules in
the bacterial cell due to positive charge on the Zn2+ and negative charge on the biomolecules.

Since zinc oxide and its nanoparticles have limited solubility, they are less toxic to the microbes
than highly soluble ZnSO4·7H2O. However, it is not essential for metal oxide nanoparticles to
enter the bacterial cell to cause toxicity [53]. Contact between nanoparticles and the cell wall is
sufficient to cause toxicity. If it is correct, then large amounts of metal nanoparticles are required
so that the bacterial cells are completely enveloped and shielded from its environment leaving no
chance for nutrition to be absorbed to continue life process. Since nanoparticles and metal ions
are smaller than the bacterial cells, it is more likely that they disrupt the cell membrane and
inhibit their growth.

In a study, Azam et al. [54] have reported that the antimicrobial activity against both gram-
negative (E. coli and P. aeruginosa) and gram-positive (S. and Bacillus subtilis) bacteria
increased with increase in surface-to-volume ratio due to a decrease in particle size of zinc oxide
nanoparticles. Moreover, in this investigation, zinc oxide nanoparticles have shown maximum
(25 mm) bacterial growth inhibition against B. subtilis (Fig: 2.1 ).

19
Figure 2 1: Antibacterial activity and/or zone of inhibition produced by zinc oxide nanoparticles
against gram-positive and gram-negative bacterial strains namely a) Escherichia coli, b)
Staphylococcus aureus, c) Pseudomonas aeruginosa, and d) Bacillus subtilis [54]

It has been reported that the smaller size of zinc oxide nanoparticles exhibits greater antibacterial
activity than microscale particles [55]. It is known that antibacterial activity of zinc oxide
nanoparticle is inversely proportional to their size and directly proportional to their concentration
[56]. It has also been noticed that it does not require UV light for activation; it functions under
normal or even diffused sunlight. Cytotoxic activity perhaps involves both the production of
ROS and accumulation of nanoparticles in the cytoplasm or on the outer cell membrane.
However, the production of H2O2 and its involvement in the activation of nanoparticles cannot be
ignored. Raghupathi et al. [56] have synthesized zinc oxide nanoparticles from different zinc
salts and observed that nanoparticles obtained from Zn(NO3)2 were smallest in size (12 nm) and
largest in surface area (90.4). Authors have shown that the growth inhibition of S. aureus at a
concentration of 6 mM of zinc oxide nanoparticles is size dependent. It has also been indicated
from the viable cell determination during the exposure of bacterial cells to zinc oxide
nanoparticles that the number of cells recovered decreased significantly with decrease in size of
zinc oxide nanoparticles.

3 MATERIALS AND METHODS

3.1 Chemicals and Reagents

The reagents and chemicals that will be required and used under this study will includes: zinc
acetate salt, ethanol absolute, ethanol reagent, distilled water, molten Hilton agar medium, HCl
and NaOH, Glycerol, PVA, yeast, agar and tryptone

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3.2 Materials and Instruments
The materials that will be required consist of: Beaker, pipette, magnetic stirrer, P H meter, micro-
oven, and bottom rounded flask bottle, filter paper, aluminum foil, dropper, centrifuge, furnace,
micro grinder, hot plate and refrigerator. Some of the instruments that will be used under this
study include, XRD and FTIR.

3.3 Method

3.3.1 Zno Nanoparticle synthesis by sol gel process

The ZnO nanoparticles will be prepared using the sol–gel method. Initially, a solution of 5 g
zinc acetate in 150 mL EtOH is dissolved, stirred vigorously for 1 h. In another beaker, 9 g
H2C2O4 is mixed with 100 mL ethanol, stirred for 1 h at 50 °C, and added slowly to the
previous solution. A white gelatinous precipitate will be obtained, which will dry at 90 °C for 2 h
and then calcined at temperatures of 400 °C.

3.3.2 ZnO-Polyvinyl alcohol (PVA) nano composites

To prepare different percentage of ZnO/PVA nanoparticles, a solution of PVA (0.125, 0.25, and
0.5 g, respectively) in 35 mL ethanol will be added to a zinc acetate solution (5 g in 150 mL
ethanol), and a precipitate will be obtained by adding H2C2O4- ethanol solution. The product
will be obtained as a powder after drying in oven at 50°C for 24 hrs followed by calcination at
the same temperatures mentioned earlier [57].

3.3.3 Characterization Techniques of ZnO Nanoparticles

3.3.3.1 Characterization using XRD

The crystal structures of the ZnO nanoparticles and nanocomposite film will be studied by X-ray
diffraction (XRD) technique. The patterns will be taken using X-ray diffractometer (Cu Kα line
λ = 0.15406 nm). The intensity is determined in the range of 10° < 2θ < 70° with 0.04° step size.
The Scherrer equation will be sed to determine the average crystallite size as:

21
 where D, K, λ, β and θ are the average crystallite (nm), constant factor, X-ray wavelength, full
width at half height and scattering angle, respectively.

3.3.3.2 Chemical Compound Study

The Fourier transmission infrared (FTIR) spectrophotometer (FTIR AVATAR) in the

wavelength range of 400–4,000 cm−1 is used to study the chemical bonding of samples.

3.3.3.3 Morphological Analysis

The morphology of ZnO nanoparticles and film surface will be examined by transmitted
electron microscopy (TEM) and scanning electron microscopy (SEM), respectively. In addition,
a particle size analyzer will be employed for the determination of particle size distribution.

3.3.4 Antibacterial Activity study

The bactericidal activity of the films will be evaluated by a typical agar diffusion test. The film’s
antibacterial effect will be assessed by the inhibition zone against Escherichia coli. The culture
containing lysogeny broth (LB) with sodium chloride for sterilization is autoclaved at 121C for
20 min to reach this object. Then bacteria will be cultured with lawn method and incubated at
37C to initiate the bacteria growth. Nanofilms added to the culture containing the bacteria are
incubated at 37C for 48 h to achieve bacteria disintegration interval. The zone area surrounding
the nanofilm will be determined by use of ARCGIS 9.3.

4 RESEARCH WORK PLAN

Time schedule
No. Activities Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug
1 Proposal writing
x
2 Proposal submission and x
defense
3 Sample collection and x
preparation
4 Sample preparation x
5 Sample characterization x x

6 Antibacterial test x

22
7 Data analysis and thesis x
write-up
8 Submission of the thesis
9 Thesis defense

5 BUDGET PLAN
No Materials and Price (birr) Quantity Unit Total price
other tasks
1 Zinc acetate salt 1000 250kg 1 1000
2 Ethanol 250 liter 1 250
3 Distilled water 100 liters 4 400
and ethanol
4 Oxalic acid 500 Liter 1 500
5 Bacterial test 300 Sample 5 1500
6 XRD 500 Sample 2 1000
7 FTIR 200 Sample 3 600
8 UV-Vis 200 Sample 5 1000
Total 6000

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