Green Synthesis of Zinc Oxide Nanoparticles Using Peel

Extract of Calamansi (Citrus x microcarpa) as Reducing and

Stabilizing Agents to Test Photodegradation of Methylene

Blue

And ZnO NPs Antibacterial Property

Submitted to:

Oliver Lahorra
Research Teacher

Submitted by:

Basco, Anne Penelope M.

Caro, Chenise Leigh E.

Castillo, Mica Joy M.

Cortez, Juan Miguel R.

Lim, Darius G.

Salvador, Astrea Elysia A.

Tancinco, Charleen Gail G.

Vicente, Daniel Adrian M.

CHAPTER 1
 THE PROBLEM AND ITS BACKGROUND

INTRODUCTION

As technological advances coincide with the development in different areas of

science, the increasing interest in the vision of possible breakthroughs on human life

using nanoscience and nanotechnology has been expanding and further researches that

focus on developing systems of nanotechnology using green synthesis to eliminate the

adverse effects observed on people and in the environment when using non-biological

methods of nanosynthesis. Nanoscience is the field of study that deals with novel

researches using minuscule structures measured at nanoscale which consequently has led

to the beginning of the expansion of knowledge and breakthroughs on commercial

application of nanotechnology (i.e. practical usage of technology using devices in

nanoscale ranging between 1 to 100 nanometers) in different fields of science including

biology, physics, material science, chemistry, computer science, and engineering (Bayda

et. al, 2019). On December 1959, the American physicist Richard Feynman was the first

to present the ideas on nanotechnology in a lecture entitled “There’s Plenty of Room at

the Bottom,” without using the term “nanotechnology” he described the process that

would enable scientists to control and manipulate individual particles (Lozano, 2014); the

nascency of nanoscience from Feynman’s idea focused on the vision of using machines

to produce smaller machines down at a molecular scale (Bayda et. al, 2019). In addition,

his hypotheses related to his idea have been proven correct, thus making him the pioneer

and is considered as the father of modern nanotechnology.

 The expansion of knowledge about nanoscience after Feynman had presented the

concept of nanotechnology led to the development of different practicable approaches on

the synthesis of nanostructures. The two manufacturing methods that have been proven

successful in synthesizing nanostructures are under the categories: top-down, and bottom-

up. The methods on top-down approach include the application of physical processing of

the solid bulk material into smaller particles, by milling, crushing, and grinding of

materials (Khan, 2020). Top-down approach is applied on synthesizing zinc oxide

nanoparticles: in physical method, for instance through thermal evaporation, while in

chemical method, zinc oxide NPs, for instance, can be synthesized through precipitation

technique (Jin & Jin, 2019). On the contrary to the top-down approach, the bottom-up

method involves the synthesis of nanoparticles by building up the material atom-by-atom

and molecule-by-molecule (Cele, 2019). Furthermore, the self-assembly of particles in a

bottom-up approach enables it to form nanomaterials with uniform size and shape that is

very difficult to acquire the similar result when top-down approach is used (Khan, 2020).

Under bottom-up approach, zinc oxide NPs can be produced by applying chemical

methods, for instance the sol-gel technique through heating stoichiometric reagents to

obtain the solid state product.

The application of nanosynthesis on metallic particles particularly in zinc oxide

has posed breakthroughs, for instance in biomedical applications of nanomedicine due to

its therapeutic properties and drug delivery (Jin & Jin, 2019). The US Food and Drug

Administration categorized the zinc oxide nanosynthesis as “considered safe,” however,

they still recognized the toxicity issues accompanying the production of zinc oxide NPs.

Although, the conventional approaches on nanosynthesis, particularly the physical and

chemical techniques applied in the production of nanoparticles from metal oxides have

resulted to effective and large scale synthesis of nano products, these approaches

consequently observed and proven to cause global concerns on public health and

associated environmental impacts due to chemical (Khalafi & Buazar, 2019).

Furthermore, by setting up efficient, safe, and eco-friendly synthesis methods, 'Green

synthesis' is needed to prevent the creation of unnecessary or hazardous by-products. To

attain this aim, the need and use for ideal solvent systems and natural materials and

resources like organic systems is important. Basically, green material/nanomaterial

synthesis, generated via the process of control, monitoring, sanitation, and restoration,

would directly contribute to enhance their environmental sustainability and friendliness.

Some fundamental concepts of 'green synthesis' can thus be clarified by multiple

components such as waste prevention or minimization, mitigation of derivative products

or pollution, and the use of cleaner and safer (or non toxic) solvents or auxiliaries as well

as reusable feedstocks (Singh, 2018).

One of the popular metallic nanoparticles with major uses in many sectors and

research facilities is zinc oxide nanoparticles or ZnO NPs. Multiple processes and

techniques have been introduced in the development of ZnO NPs so that it can satisfy its

high demand in the fierce market. The environmental effects and economic difficulties

associated with so many of the means of synthesizing zinc oxide nanoparticles synthesis
have contributed to the search for other solutions with economic and environmental

benefits. Intriguingly, because of its various nutritional, economic, environmental, and

medicinal effects, the biological approach of synthesis utilizing plant products has been

considered to be suitable for the development of ZnO NPs. The distinctive characteristics

of zinc oxide synthesized with plant/bio extracts have increased their use in agricultural

production for the manufacture of pesticide, fertilizer and fumigant. Phyto Synthesized

zinc oxides have achieved exceptional use in the manufacture of disinfectants,

antifungals, antidiabetics, antioxidants, anti-inflammatories and anticancer agents in the

medication and pharmaceutical sectors. While the advantages of biosynthesized ZnO NPs

have been recognized, the difficulties and complexities associated with the elucidation of

the process of development and reaction persist (Folorunso, 2020).

At the molecular and cellular levels, nanobiotechnology and organic/biomedical

sciences open up the opportunity for a broad range of biological scientific subjects and

medicinal applications. As a cost effective and environmentally friendly and sustainable

solution to physical and chemical approaches, the biosynthesis of nanomaterials has been

suggested. A green chemistry/synthetic solution linking nanotechnology and plants is

plant mediated synthesis of nanostructures. Therefore, innovative strategies of preferably

synthesizing NPs are assumed to be produced at ambient conditions, neutral pH range,

cheap price and environmental friendly style. Nanomaterials were synthesized through

diverse approaches to hold these targets in mind. The best alternative appears to be plant

extracts and plants themselves, among the biological substitutes. Since, plants are the
"food and chemical factories" of nature and life. They are budget-effective and only

require minimum maintenance (Parveen, 2016).

CONCEPTUAL FRAMEWORK

HYPOTHESES

1. The expected size range of the synthesized zinc oxide nanoparticles is 100-500

nm. The procedure in which the ZnO NPs are to be synthesized requires high

temperatures which will affect the size of the synthesized ZnO NPs as high

temperatures making the ascorbic acid present in the calamansi extract to become

unstable causing a poor reduction process.

 2. The degradation rate of MB of the synthesized zinc oxide nanoparticles is at least

40% in 3 hours for ZnO NPs greater than 13 nm. Because of the nature of ZnO to

be non-toxic and have high photosensitivity, it is already considered to be one of

the best photocatalysts in the degradation of organic pollutants. Furthermore, the

size of the particle involved is a major factor in catalytic degradation as suggested

by the results from the experiment by Luque et al.

SCOPE AND LIMITATIONS OF THE STUDY

This study focuses on the use of calamansi extract as a reducing and stabilizing

agent in the synthesis of Zinc oxide nanoparticles. The study will only use the extract of

the calamansi fruit, thus will not use the rest of the calamansi plant. This study will focus

on the observation of synthesized Zinc oxide nanoparticles and their characteristics such

as their size range and antimicrobial activity.

SIGNIFICANCE OF THE STUDY

The findings of this study will prove to be beneficial to the following:

a. BODY OF KNOWLEDGE. The results of this study will be able to expand the past

knowledge on the topic of green synthesis using plant materials, thus opening the topic to

more research and studies,

b. INDUSTRY. The findings of this study will be able to provide a cheaper and more

environmentally-friendly alternative to synthesizing Zinc oxide nanoparticles, thus

reducing the cost and environmental impact of said synthesis of commercial industries

such as in cosmetics, storage, optical devices, etc.

c. PEOPLE. Due to its cheaper cost and environmentally-friendly approach, the findings

of this study will be able to help in making more affordable products made with Zinc

oxide nanoparticles for the community, as well as reduce the impact the synthesis of the

nanoparticles has on the environment.

CHAPTER III

RELATED LITERATURE

General Background
 In the field of bionanotechnology, green synthesis is an emerging concept that brings

economic and environmental advantages as a solution to physical and chemical approaches. In

this process, non-toxic reagents that are environmentally-friendly and biosafe are being used. For

the synthesis of metal oxide nanoparticles, numerous natural resources present in the natural

world have been researched, such as plant extracts, cyclodextrin, chitosan, and several others. As

an approach, the use of plant extracts in the synthesis of metal oxide nanoparticles attracted and

garnered much attention (Sangeetha et al., 2018).

Green synthesis has received significant attention in materials science as a reliable,

viable, and environmental-friendly procedure for the synthesis of a vast array of materials, which

include nanomaterials of metal/metal oxides, composite materials, and biomaterials. As such,

green synthesis is known to be an effective way of reducing the destructive results of

conventional nanoparticle synthesis techniques widely used in laboratories and industry.

Through setting up efficient, safe, and eco-friendly synthesis procedures, 'Green synthesis' is

needed to prevent any creation of unnecessary or hazardous by-products. To attain this purpose,

the use of ideal solvent systems and organic systems is important. To manage different biological

materials (e.g., microbes, fungus, algae, and flower extracts), green synthesis of metal oxides has

been introduced. The use of plant materials is a relatively basic and easy way of generating

nanomaterials on a wide scale compared to bacteria/fungi-mediated synthesis, among the

available green synthesis methods for metal/metal oxide nanoparticles. Such materials are

generally termed organic nanoparticles (Singh et al., 2018).

Nanotechnology
 Nanotechnology is science and technology-based on nanoparticles which, based on its

name, are particles that are less than 100 nanometers. It may not seem so impressive that there

are particles that small but nanostructures exhibit new properties that are different from the

particles they come from. This is due to the fact that particles smaller than a particular length

differ in their chemical and physical behavior which is based on size. To reduce the size of a

particle also changes its chemical, structural, and other such properties. This gives scientists and

researchers the power to modify its properties to make its application in many certain fields such

as nanomedicine and bionanotechnology possible.

There has already been significant interest in certain properties of nanoparticles, such as

the study on the antibacterial activity of zinc oxide nanoparticles. Zinc oxide in itself is used in

our everyday lives, usually in treating skin irritations such as rashes and burns, and is even

approved by the Food and Drug Administration as a food additive. Nano-sized zinc oxide

particles have shown significant antibacterial activity as proven by researchers and are still being

studied as an antibacterial agent. Additionally, even if the interaction between the nanoparticles

and bacterial surface and core are toxic, reports of several studies have found that zinc oxide

nanoparticles are non-toxic and even compatible with human cells.

Nanoparticles have been evaluated constantly and have been used for a generation in so

many commercial applications. Due to different properties, such as Ultraviolet filter

characteristics and photochemical, antifungal, high catalyst, and antimicrobial activities, the

function of zinc oxide nanoparticles has received considerable attention particularly. The green

methods for the production of nanomaterials were introduced using fungi, plants, bacteria, and

algae due to the extreme rate of toxic chemicals and the harsh environment used for the physical

and chemical processes (Kalpana, V., & Devi Rajeswari, V., 2018).
 Nanoparticles (NPs) are chemical substances or materials having one of the dimensions

within the range of 1-100nm (Hussain et al., 2015). The wide variety of approaches in

synthesizing nanoparticles presents novel and vast development, especially in the field of

medicine, leads to the emergence of a new field in nanotechnology and nanomedicine (Guvva et

al., 2008).

The usual approaches in the synthesis of nanoparticles are achieved by physical and

chemical methods, both require high-energy inputs and the use of toxic chemicals (Benelli,

2019). Advancements in nanotechnology lead to the development of the biological method in the

production of nanoparticles, called green synthesis, as a possible alternative to physical and

chemical methods, which generate hazardous by-product and particle toxicity that might harm

the environment and human health (Li et al., 2011).

Green Synthesis

Green synthesis determines a "Bottom-Up" biogenic reductive approach by replacing a

chemical agent with natural products such as plant bacteria, fungi, and by peptides (Duran et al.,

2011). The biological synthesis of nanoparticles allows the recycling of expensive and limited

resources of salts like gold and silver contained in waste streams (Wang et al. 2009).

Green synthesis of particles requires the use of secure biological agents as an eco-friendly

and cost-effective alternative to chemical synthesis. In one study, silver particles were

biosynthesized using silver nitrate and aqueous Citrus microcarpa peel extract as a reducing and

stabilizing agent. Synthesized silver particles have been confirmed and characterized by UV-Vis

spectroscopy, electron scanning microscopy (SEM), energy-dispersive X-ray spectroscopy

(EDX), and dynamic light scattering (DLS). The UV-Vis spectrum revealed a surface plasmon

resonance (SPR) with a maximum peak intensity of approximately 450 nm. The DLS research

shows that silver particles have an average Z-diameter value of 235 nm and a polydispersity

index of 0.363, indicating the presence of agglomeration. Reaction parameters have a substantial

impact on silver particle formation. The highest absorbance reported was 1.42 obtained under

conditions of 72 hours of reaction time, using 5 wt. percent of the peel extract that reacted with 8

mM of silver nitrate solution, while the ratio of the peel extract to silver nitrate solution was set

at 1:5. Silver particles have been successfully synthesized by the extract of Citrus microcarpa

peel, which has the potential to replace the chemical process. (Jie et al., 2020).

Citrofortunella microcarpa

Citrofortunella microcarpa (Calamondin) extract is used for the synthesis of Copper

Oxide Nanoparticles (CuO-NPs) as a green and environmentally friendly reducing agent as well

as for capping agents. The optical, structural, and morphological characteristics of CuO-NPs are

investigated using UV-vis spectroscopy, XRD, SEM, and FTIR. EDS and SEM identified the

purity and spherical-like shape of the CuO-NPs with a size range of 54–68 nm. The synthesis

was confirmed by UV – vis spectroscopy with a surface plasmonic resonance at 270−300 nm,

while XRD showed a monoclinic phase of prepared NPs with a space group C2 / c. In addition,

the existence of functional groups responsible for synthesis was monitored using FTIR. The

synthesized CuO-NPs were used as a photocatalyst for the purification of Rhodamin B (RhB)

dye contaminated water and represented 98% degradation efficiency under UV light exposure.

Therefore, this study shows a promising cost-effective, simple and green biosynthesis of CuO

photocatalyst, which is a possible candidate for the remediation of dye contaminated water

(Rafique et al., 2020).

Synthesis Process Of Zinc Oxide Nanoparticles

Biosynthesis processes of zinc oxide nanoparticles with the use of plant extracts are

preferred as sources of NPs synthesis of stable and large-scale production of NPs with varied

shapes and sizes (Agarawal et al., 2017). To produce zinc oxide nanoparticles, the following are

the reactants used in the reduction process: zinc nitrate hexahydrate [Zn(NO3)2·6H2O] ( 99%

purity) is used as zinc source, ethylene glycol anhydrous (, 99.8% purity) is used as solvent,

polyvinyl alcohol (MM = 85 kDa) as dispersant and sodium hydroxide ( 99% purity) as Zn(OH)2

forming agent(Biron et al., 2020).

Moreover, sodium hydroxide (NaOH) as reactant, ensures the process of producing

nanoscale zinc oxide particles with uniformity on its structure: the removal of NaOH in the

process prohibits the formation of ZnO nanoparticles because its function ascertains the

formation and obtaining of nanostructures. Furthermore, the first equation determines the

formation of zinc hydroxide due to the double exchange that occurred between zinc nitrate and
sodium hydroxide. In the second equation, it determines the formation of ZnO nanoparticles due

to heating of solution that causes decomposition of zinc hydroxide into nanoparticulate zinc

oxide.

RELATED STUDIES

Green Synthesis

Luque, et al., (2018) conducted a study about the green synthesis of Zinc oxide

nanoparticles using Citrus sinensis (orange peel) extract. The extract was obtained by placing a

mixture of dried orange peel powder and de-ionized water in a water bath at 90°C. Zinc nitrate
was then placed in the mixture, stirred for 1 hour, and then placed in a water bath at 60°C until

the consistency of the mixture is thick. The sample was then heat treated at a temperature of

400°C for an hour and then pulverized. The sample then resulted with nanoparticles with better

degradation ratio for photo-catalytic activity than other nanoparticles synthesized with other

methods.

Nanotechnology

Ramesh, Rajendran, & Subramanian, (2014) conducted an experiment in which the

researchers synthesized Zinc oxide nanoparticles by boiling the fruit extract of Citrus

aurantifolia and adding Zinc Nitrate to the solution. The solution was boiled until the color has

turned into a yellow-colored paste. The paste was then collected and heated for 2 hours. The

resulting material was then mashed in a mortar-pestle to get finer materials. The findings

indicated that the resulting Zinc oxide nanoparticles were moderately stable and roughly

spherical.

Kumar, Smita, Cumbal, & Debut, (2014) made a study about fabricating Zinc oxide

nanoparticles using Citrus paradisi (grapefruit) peel extract. The researchers made the peel

extract by boiling the peel with 40 mL of double distilled water for 30 minutes. 1-1 mL of the

extract was then added to a 10 mL solution of 3 mM ZnSO4·7H2O with pH adjusted to 8. The

mixture was stirred for 3 hours until the change of the mixture’s color from yellowish green to

light yellow, as evidence of Zinc oxide nanoparticles forming. The Zinc oxide nanoparticles

were then separated, washed, and dried. The Zinc oxide nanoparticles were then suspended in

deionized water for further observation. The results indicated that the formed Zinc oxide

nanoparticles were highly stable and had significant photocatalytic and antioxidant activity.
Citrofortunella microcarpa

Citrofortunella microcarpa, better known as calamansi or calamondin, is a fruit member

of the citrus genus and widely prominent in the Southeast region of Asia, particularly in the

Philippines(Titong, 2020). As a citrus, it has the capability to reduce and stabilize the synthesis

process of many nanoparticles including Copper oxide nanoparticle(Rafique, et al., 2019) . The

citrus genus has a high number of phenolic compounds and flavonoids which aid in the metallic

ion reduction process(Luque, et al., 2018).

Photocatalytic Activity and of Zinc Oxide Nanoparticles

Blazeka et. al (2020) conducted a study about the photocatalytic activity of zinc oxide

nanoparticles wherein it has shown high photocatalytic activities dependent on the UV light

transmission. In addition, due to low cost, high photocatalytic efficiency, and non-toxicity of

ZnO nanoparticles, is one of the widely researched and promising photocatalytic semiconductor

materials. Moreover, the radicals produced from electron transfer to oxygen molecule

producing superoxide with a top valence band at 2.7V that as a result, allows hole transfer and

are main contributors to the degradation of chemical pollutants that are absorbed by the catalyst

at their production rate which is proportional to electron or proton excitation rate in

photocatalytic material. Additionally, the potential of ZnO in redox reaction is the main reason

for its degradation mechanisms.

Photodegradation of Methylene Blue

According to a study conducted by Blazeka et. al (2020), wherein they examined the

photocatalytic degradation of UV-irradiated Methylene Blue as well as Rhodamine B solutions

of varying concentrations while incorporating ZnO catalyst masses to assess its effect on the rate

of photodegradation. The study was brought to the conclusion that the photodegradation rate is

approximately 40% higher for Methylene Blue compared to that of Rhodamine B. However, the

degradation rate growth whilst increasing the concentration of the catalyst is more apparent in

Rhodamine B than that of Methylene Blue. In the case of the latter, the saturation of the

degradation rate was obtained from the concentration range of the observed catalyst.

SYNTHESIS

Nanotechnology was discovered because of mankind’s continuous pursuit of innovation

and constant search for solutions to the problems we face on a day to day basis. Nanoparticles

come in different variants and among them are zinc oxide nanoparticles (ZnO). ZnO has

incredible physical and chemical properties to its advantage. It is one of the most utilized

materials in different industries such as cosmetics, nanofertilizers, electronics, bioimaging, and

many more because of its affordability. (Jiang, 2018)

The majority of industrial areas have been revolutionized by nanoparticles or NPs,

ranging from medicine, to agriculture, and the food industry. NPs are synthesized by chemical

means. The process includes various methods such as emulsion, solvent extraction, double

emulsion and evaporation, salting out, emulsion diffusion, and solvent precipitation. However, it

is evident that the industrial scale production of NPs has introduced a new kind of pollution to

the environment. (Jie, 2010)

 Despite all the wonders that this substance brings, the toxicity of nanoparticles is

naturally present. However, it continues to be synthesized because it is essential to many

industries. This process emits environmental contaminants such as excessive energy and water

consumption, generation of heat, and chemical waste. Therefore, eco-friendly techniques to be

used for the synthesis of ZnO NPs must be developed. As of the moment, the goal is to reduce

the massive amounts of chemical pollution on the environment by developing new and

convenient methods not only to deal with the impact of chemical methods, but also to improve

the yield, and to lower the production cost. (Kalpana, 2018)

Numerous parts of plants such as the leaf, stem, root, fruit, and seed have been used for

the synthesis of ZnO NPs due to their unique ability of producing phytochemicals. The usage of

natural extracts of these plant parts is a cheap and very eco-friendly process. In addition, it takes

less time, does not involve the use of expensive equipment, and results in a product that is

quantity enriched free of impurities. Furthermore, plants are the most preferred source of

nanoparticle synthesis because they lead to a large-scale production of stable, and diverse

nanoparticles in terms of size and shape. (Kalpana, 2018)

In this study, the researchers will be utilizing Citrofortunella microcarpa. It is more

commonly known as calamansi, a citrus fruit that is widely available in the Philippines. The said

fruit is capable of reducing and stabilizing the synthesis process of various nanoparticles

including Copper oxide nanoparticles. Due to the fact that it is a member of the citrus genus, it

has a high number of phenolic compounds and flavonoids that aid in the metallic ion reduction

process. Hence, it would be ideal to incorporate it in the process of synthesizing ZnO

nanoparticles.
 CHAPTER III

METHODOLOGY

INTRODUCTION

This chapter introduces the research design as well as the data-gathering procedure for

this descriptive research of examining the calamansi extract as a viable green alternative

stabilizer for nanoparticle synthesis. The researchers take a nuance approach in collecting data by

cross-examining different literature of the same background to make the foundation necessary

for the right assumptions of verifying the hypothesis.

RESEARCH DESIGN

The researchers implemented a pure experimental research design. In an experimental

research design, researchers are able to execute experiments in a controlled environment in order

to get the desired results. The researchers can compare the output of a controlled group and an

experimental group where the variables can be manipulated on a trial-and-error basis to prove or

disprove a hypothesis. It is specific and shows high accuracy as it relies on the statistical analysis

of collected data.
PRODUCTION OF ZNO NPs USING CALAMANSI EXTRACT

The procedure of the experiment will be as follows:

First, the necessary materials zinc nitrate, calamansi (Citrus microcarpa) peels and de-

ionized water will be prepared by the researchers. Second, the calamansi peels and de-ionized

water gathered will be mixed to form an aqueous solution of a calamansi extract. Third, for one

hour, the mixture of the previous step is then put in a water bath at 90 °C. This is to ensure the

formation of the calamansi extract will be completed. Fourth, 2 grams of zinc nitrate will be

placed in the extract. Fifth, after completing the third and fourth steps, the mixture will be stirred

(Luque et. al, 2018). The formation of the Zinc oxide nanoparticles is the result of the Zinc ions

reacting with the polyphenols present in the calamansi extract. This will be followed with the

process of hydrolysis as a result of the presence of hydroxyl groups in the polyphenols in the

extract, this hydrolysis reaction will then result in Zinc hydroxide. The produced Zinc hydroxide

will then undergo calcination in a box furnace at 400 ͒ Cwhich will then form the Zinc oxide

nanoparticles (Ogunyemi et al., 2019). To determine if Zinc oxide nanoparticles are really

produced, the Zinc oxide nanoparticles will be characterized using X-ray diffraction analysis

(XRD). XRD will be able to provide information regarding the Zinc oxide nanoparticles such as

the structure of the crystalline, nature of the phase, size of the crystalline, etc. This will help the

researchers determine if Zinc oxide nanoparticles are produced (Mourdikoudis et al.,

2018).

DATA-GATHERING PROCEDURE
 The first step the researchers will have to take before undergoing official testing is to

make a letter indicating the request to use the laboratory and the necessary equipment. Upon

approval, the researchers will retrieve the letter, make the necessary preparations, travel to the

laboratory, and start the synthesis of the Zinc oxide nanoparticles.

Once finished, the researchers will test and observe specific characteristics of the

synthesized nanoparticles specifically, the size range, the photocatalytic activity, and the

antibacterial activity of the synthesized nanoparticles.

SIZE RANGE

To observe the size ranges of the synthesized nanoparticles, the researchers will use

transmission electron microscopy (TEM) to observe the formed size ranges, using a high voltage

electron beam to pass through the nanoparticles to create a high-resolution image of the

nanoparticles (Cheadle Center for Biodiversity & Ecological Restoration, n.d.). The Zinc oxide

nanoparticles will be prepared for TEM imaging by applying a droplet of the nanoparticles onto

a TEM grid. The grid will then be rinsed twice, blotted, and then finally measured using the

TEM (Microscopy Innovations LLC, 2015). TEM imaging is to be used for detailed imaging of

the nanoparticles, in which nanomaterials with atomic dimensions (< 1 nm) can still be viewed at

high-resolution (Narain, 2020).

PHOTOCATALYTIC ACTIVITY
 The photocatalytic activity of the synthesized nanoparticles will be tested via Methylene

Blue Degradation with the Zinc oxide nanoparticles acting as catalysts for the photocatalytic

reaction between the UV light and the Methylene Blue dye. Zinc oxide nanoparticles will then be

added to a 200 ml sample solution of Methylene Blue with a concentration of 15 mg/L. The

solution will be stirred continuously for 30 minutes and then exposed to a 10 W UV light bulb at

18 ml/cm2. For 3 hours every 30 minutes, 2 ml of the solution will then be tested for Methylene

Blue concentration using a UV-Vis spectrophotometer with a particular wavelength value

(Nava et al., 2017). The rate of degradation will be calculated using the following formula

(Ramli et al., 2014):

C0 −C
Degradation Rate = × 100
C0

Where C 0is the initial concentration of Methylene Blue at time = 0, while C is the concentration during

irradiation. Both C and C 0 values will be obtained from the UV-Vis spectrophotometer

ANTIBACTERIAL ACTIVITY

The antibacterial activity of the synthesized nanoparticles will be tested using the disk

diffusion test against two pathogenic bacteria, one gram-positive and one gram-negative, namely

S. aureus and E. coli. Zinc oxide nanoparticles are able to inhibit the growth and kill off bacteria

via electrostatic forces. The positive charge of the zinc oxide nanoparticles allows the

nanoparticles to bind to the negatively charged surface of the bacterial cell walls. This results in

the distortion of the overall structure of the bacteria, allowing its contents to leak, eventually

killing the bacteria.

 The bacteria will be grown on an agar plate with the synthesized Zinc oxide nanoparticles

with concentrations ranging from 50 µg/mL to 1000 µg/mL. To see the antibacterial activity of

the Zinc oxide nanoparticles, the zone of inhibition formed around the nanoparticles will be

measured and compared against the zone of inhibition of standard drugs used against both S.

aureus and E. coli, which are around 16-40 mm and 15-20 mm respectively.

DATA PROCESSING

The collected data will be gathered from two sample groups namely Group A and Group B,

where Group A is the control group wherein the citrus x sinensis extract is synthesized with ZnO

NPs, and Group B is the group with changing variable amounts of citrofortunella microcarpa

extract, is synthesized with ZnO NPs. The statistical treatment will show if the green synthesis of

ZnO nanoparticles to calamansi extract are equivalent to the green synthesis of ZnO

nanoparticles of the control group, or if it has a significant difference. (Luque, P. et al. 2020).

STATISTICAL TREATMENT OF DATA

The researchers will use a Two-Sample T-test statistical treatment method. The two

sample t-test is used to determine if there is a significant difference between the means of two

individual sample groups. The statistical treatment will be used on 3 samples from Group A and

Group B, and will compare their results to know if the means are equal or different. The average

mean of the the size range, the mean of the degradation rate of methylene blue, and the mean of

the size of zone of inhibition of the synthesized ZnO NP’s of Group A, the Citrus x sinensis
green synthesized ZnO NP’s, and Group B, the Citrofortunella microcarpa green synthesized

ZnO NP’s, will be gathered and tested. The results therefore of Group B must be equivalent to

the results of Group A. The statistical testing will conduct a two-tailed test and will use a

significance level of 0.05. The values will find the critical region of the test and will determine if

the null hypothesis will be rejected or not. The researchers will use the following formulas for

finding the mean of the two groups, the standard deviation and the t-value:

μ 1−μ2
x 1 +...+ x n Σ( x i−μ)2 t=
π=Σ
n
s=
√ n
√ s1❑ s2
+
n n

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