Proposal Final Version
Proposal Final Version
Proposal Final Version
By
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.
2
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
DNA……………………………………………….Deoxyribonucleic Acid
UV-Vis…………………………………………………Ultra visible
5
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
6
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
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,
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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].
1.3 Objectives
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2 LITERATURE REVIEW
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].
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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].
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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].
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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.
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
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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].
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
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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.
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.
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
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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.
It should be nontoxic.
It should not react with food or container.
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
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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)
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 ).
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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.
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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
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:
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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.
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.
6 Antibacterial test x
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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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