Green Biosynthesis of Silver Nanoparticle via Calamansi (Citrofortunella microcarpa)

extracts and their Antibacterial Activity, Enhanced DPPH Free Radical Scavenging
Activity, and Metal Sensing Capacity

Submitted as Partial Fulfillment for the Requirements of Research I

Of Science Technology and Engineering Curriculum of

Jacinto P. Elpa National High School

Researchers

Irvino Carolino E. Bendanillo

Wilman A. Zozobrado

Razenne Erika A. Maquiling

IX-Berzelius

Advisers

Joseph Romeo P. Patan

Mary Rossane Callote

Diane Omas-as

Earl Kaizzer A. Maquiling

X-Graham Bell

X-Feynman
 DEFINITION OF KEY TERMS

This study contains some words that may be unfamiliar for others. These words are defined here,
for better understanding of the study.

Antibacterial- Anything that destroys bacteria or suppresses their growth or their ability to
reproduce.

Antibacterial assay- An assay utilized to test and screen the inhibitory effects of myriad
compounds against microorganisms before establishing their inhibitory spectra (broad vs.
narrow).

Antibiotic resistance- Happens when germs like bacteria and fungi develop the ability to defeat
the drugs designed to kill them.

Antibiotics- Include a range of powerful drugs that kill bacteria or slow their growth. They treat
bacterial infections, not viruses.

Antioxidant- Either man-made or natural substances that may prevent or delay some types of
cell damage

Bacterial Infection – Infection caused by bacteria.

Biosynthesis of silver nanoparticles- Is one of the methods for synthesizing silver nanoparticles
with the use of plants and other organic substances

Colorimetric assay - Colorimetric analysis is a method of determining the concentration of a

chemical element or chemical compound in a solution with the aid of a color reagent.

Phytochemicals- Are chemical compounds produced by plants and have been used as traditional
medicine.

Silver Nanoparticles (AgNPs)- Silver nanoparticles are nanoparticles of silver of between 1 nm

and 100 nm in size. They can induce cell apoptosis through double-strand DNA breaks, oxidative
stress, and chromosomal instability.
 CHAPTER I

INTRODUCTION

A. RATIONALE OF THE STUDY

Antibiotics are widely used as agents to treat bacterial infection; however frequent
utilization of these agents lead to severe side effects in human body including digestive
problems, fungal infections, photosensitivity, anaphylaxis and more (J. Huizen, 2018).
Moreover, one global crisis involving these agents portrays bacteria being resistant to antibiotics
due to the ability of the bacteria to develop resistance towards the antibiotic; therefore, causing it
to be the most urgent threats to public’s health.

Antibiotic-resistance of the bacteria may remain untreatable which may reach to

dangerous infections; in addition, these strains are more difficult to eliminate and more
expensive to treat for the development of new antibiotics specified for this antibiotics-resistant
strain is urgently required. Moreover, according to the World Health Organization (WHO),
around 700,000 people die annually as a result of antibiotic resistance. In such cases, antibiotic-
resistant infections yield to extended clinical checks, hospital stays, and additional follow-up
doctor visits which are costly (Centers for Disease Control and Prevention, 2018) wherein the
only way of possible medication is the development of new antibiotics for these specific strains
of bacteria (World Health Organization, 2011).

Yet due to all these being financially demanding, an alternative for an effective and
cheaper termination of these bacteria is in great need. Henceforth, the manipulation versatile
applications of Silver Nanoparticles specifically the biosynthesized ones including having
antimicrobial effect and antibiotics (Khan et al., 2014; Kumar et al., 2014) come as a potential
alternative in this case.

The green synthesis of silver nanoparticles is widely used in the synthesis of metallic
nanoparticles for the conventional methods of synthesizing Silver Nanoparticles have various
limitations in which processes are slow, have high cost, and toxic to the environment.
Biosynthesized Silver Nanoparticles in contrast being an eco-friendly method by utilizing the
different parts of any selected plant have come to be the best option to be utilized for the
synthesis (R. Prasad, 2014). Also, other advantages of green synthesis include having well-
defined and control size of the silver nanoparticles.

Calamansi (Citrofortunella microcarpa) being a plant of choice is considered to have

antibacterial and antioxidant properties and has various important phytochemicals. Various
biomolecules present in the plant extract are enzymes, proteins, flavonoids, terpenoids and
cofactors which act both the reducing and capping agent. Other than that, the phytochemical
analysis of Calamansi peel has depicted the high percentage of phenolic acids; in addition, it also
contains caffeic, p-coumaric, ferulic and sinapic acids, the leaves exhibited the presence of
alkaloids, total phenolics, total flavonoids, tannins, saponins and ascorbic acid according on the
conducted research of J. Senguttuvan et al., 2014, and the juice contains volatile and non-volatile
components such as Phenolic acids and Flavedo (M. Cheong et al., 2012). Hence, we
hypothesize that the aqueous extract of Calamansi contains rich amount of phytochemicals that
can aid as the antibacterial agent through the synthesis of Silver Nanoparticles (M, Cheong, Z.
Chong, & S. Liu, 2012). Therefore, the utilization of biosynthesis of silver nanoparticle through
Calamansi extracts can possibly hinder the bacterial formation.

Aside from that issue, AgNPs can also be manipulated to have an enhanced DPPH free
radical scavenging activity. involving green biosynthesis of silver nanoparticles through
Calliandra haematocephala’s leaf extract. Hydrogen peroxide (H2O2) is a strong oxidizing
agent utilized in pharmaceutical, foods, cosmetics, and wood and pulp industries; nevertheless,
the slightest presence and exposure of this compound in process streams can lead to
environmental and biological hazards because of its toxicity (Tagad et al., 2013). Henceforth,
there is an upmost essentiality to form accurate and fast methods in determining H2O2.

A study by Aadil et al. focused on acacia synthesized silver nanoparticles and its
Hydrogen Peroxide sensing capabilities. Recently, interest in the development of new methods
for the determination of hydrogen peroxide has significantly increased. Silver nanoparticles
(AGNPs) became a promising candidate for the recognition of ultrasensitive chemical and
biological molecules, such as glucose, folic acid, and of course H2O2. This is particularly due to
its low cost, high catalytic activity and notable optical properties (Aadil et al., 2015).
 According to previous related studies from Liu et al., 2013 developed a H2O2 sensor
through the biosynthesis of silver nanoparticles by B. subtilis and recent study of Mohan et al.,
2014 produced silver nanoparticles synthesized via dextrose for an optical sensing of H2O2;
however, a cost-effective, dependable and specific methods in determining one is still sought in
these recent researches.

To the full extent of our knowledge there has not been a study yet conducted that showed
the use of Calamansi (Citrofortunella microcarpa) extracts to synthesize Silver Nanoparticle to
assess their antibacterial activity and Hydrogen Peroxide sensing capability. Therefore, this study
aims to fill that gap and further increase our knowledge of silver nanoparticles and its
antibacterial agents and Hydrogen Peroxide sensors.

B. STATEMENT OF THE PROBLEM

This study aims to depict whether the antibacterial activity of Green Synthesized Silver
Nanoparticles via Calamansi (Citrofortunella microcarpa) extracts can be a potential
antibacterial agents against Escherichia coli and Staphylococcus aureus bacteria, as well as a
more reliable and accurate sensor for determining Hydrogen peroxide. Furthermore, this study
seeks to answer the following questions:

A. Which Calamansi (Citrofortunella microcarpa) extract can synthesize more Silver

Nanoparticles (AgNPs)?
B. What are the effects of the green synthesized silver nanoparticles from qualified
Calamansi extract on the bacterial samples?

C. In what way can the green synthesized AgNPs of qualified Calamansi extract combat
the bacterial infection?

E. How can the green-synthesized Silver Nanoparticles via Calamansi extract portray its
Hydrogen Peroxide sensing capacity?

C. RESEARCH OBJECTIVES
To carry out this study, the following objectives are formulated. Specifically, this study
aims to:
 1. Synthesize the Silver Nanoparticles (AgNPs) with AgNO3 and Calamansi extracts as
its reducing and stabilizing agent.

2. Determine which part of Calamansi extracts has the capability to produce more
synthesized silver nanoparticles.

3. Assess if the Green Synthesized AgNPs exhibit high antimicrobial activity against the
different gram class bacteria.

4. Evaluate the effects of the biosynthesized Silver nanoparticles via Calamansi extracts
on the E. coli and S. Aureus samples.

5. Determine the effects of synthesized silver nanoparticles as it contact with Hydrogen

Peroxide at a fixed concentration.

D. HYPOTHESES

Ho: There will be no significant difference of effects between the Green-synthesized

AgNPs from Calamansi (Citrofortunella microcarpa) in inhibiting bacterial infections
and sensing Hydrogen Peroxide and the known methods of treating the bacterial
infections and conventional methods in determining H2O2.

Ha: There will be a significant difference of effects between the Green-synthesized

AgNPs from Calamansi (Citrofortunella microcarpa) in inhibiting bacterial infections and
sensing Hydrogen Peroxide and the known methods of treating the bacterial infections
and conventional methods in determining H2O2.

E. SIGNIFICANCE OF THE STUDY

To the World Health Organization (WHO) and To the Department of Health

(DOH), the result of the study may give additional information and beneficiary in developing a
low-cost alternative anti-bacterial agent.

To the Eco-researchers, the result of this study may give a more accurate and reliable
sensory method in determining Hydrogen peroxide in processed streams or even the
surroundings.
 To the future researchers, the result of the study may provide a source of information
about the significant use of biosynthesized silver nanoparticles in the application of it’s an
antibacterial activity and hydrogen peroxide sensing capabilities.

The purpose of this study is to analyze the effect of bio-synthesized silver nanoparticles
in order to formulate an anti-bacterial agent which serves as a cheaper alternative for known
bacterial infection treatments such as antibiotics for S. aureus and E. coli. Furthermore, this
study also seeks the AgNPs capability as Hydrogen peroxide sensor with lower cost and high
catalytic activity.

F. SCOPE AND LIMITATION

This study will help prove that the biosynthesis of silver nanoparticles via Calamansi
extracts can be manipulated as an effective yet inexpensive way to treat the bacterial infection
through testing in E. coli and S. aureus bacteria. In addition to this, the AgNPs hydrogen sensing
capabilities are also tested.

The research will be limited to the biosynthesis of silver nanoparticles using AgNO3 and
the Calamansi extracts, the resources and equipment used, and the knowledge and skill of the
researchers.

E. CONCEPTUAL FRAMEWORK

The research focuses on the antibacterial effect of the biosynthesized silver nanoparticles
on the bacteria sample E. coli and S. aureus; moreover, the sensing capability of green
synthesized Silver Nanoparticles on determining the presence of Hydrogen Peroxide (H2O2) shall
be evaluated as well. The methods and techniques will be performed in the laboratory. The
methods include the collection, processing, and preperation of Calamansi peel, leaves, and juice
extracts, synthesis of Silver Nanoparticles via Calamansi extracts and AgNO3, the
characterization of biosynthesized SNPs, Antibacterial assay, and the Hydrogen peroxide-
sensing capacity of silver nanoparticles utilizing the protocol developed by these researchers
Bera and Raj, 2013 (S. Raja et al., 2015).

The paradigm presented in figure 1 explains the conceptual framework of this study.
 INPUT
TREATMENT
a.) Biosynthesis of Silver
Nanoparticles (AgNPs) via
Calamansi (Citrofortunella
microcarpa) extracts
b.) Antibacterial activity of
biosynthesized Silver
Nanoparticle on bacteria
samples E. coli and S. aureus
c.) Hydrogen Peroxide sensing
activity of biosynthesized SNPs
Figure1: Conceptual paradigm presenting the independent and dependent variables of the current study.
PROCESS OUTPUT
METHODS RESULTS
a.) Collection, processing, and
preperation of Calamansi peel, a.) Characterization Results
leaves, and juice extracts
b) Particle size analysis Results
b.) Synthesis of Silver
c.) Antibacterial effect of
Nanoparticles via Calamansi
biosynthesized SNPs
(Citrofortunella microcarpa)
extracts and AgNO3 d.) Colorimetric effect of
biosynthesized AgNPs when in
c.) Characterization of
contact with Hydrogen Peroxide
biosynthesized SNPs
(H2O2)
d.) Antibacterial assay
e.) Hydrogen peroxide-sensing
capacity of silver nanoparticles
 CHAPTER II

REVIEW OF RELATED LITERATURE

The application of antibiotics is the most common approach in treating bacterial

infection. However, ever since the rise of resistant strains, the use of routine antibiotics has been

rendered ineffective. To make it worse, bacterium can rapidly form biofilm on the implant

surface, making antibiotics unable to penetrate and develop antibacterial function (Qing et al.,

2018). Thus, many urgent studies have been made to find an antibacterial agent that can destroy

drug-resistant bacteria and inhibit its spread.

The antibacterial property of silver has long been discovered, even dating back to the

ancient Greeks. Additionally, research to date has shown that bacteria have been unable to

develop any immunity to silver. Silver can be produced into nanoparticles (AgNPs) to better suit

its antibacterial activity. The extremely small size of AgNPs allows them to exhibit enhanced

and different properties when compared with the material in bulk. The study of Quing et al.

stated that the size of nanoparticles results in them having a large surface area relative to their

volume. This allows them to easily interact with the bacterial cells and increases their

antibacterial efficiency.

All bacteria use an enzyme as a form of ‘chemical lung’ in order to metabolise oxygen.

Silver ions cripple the enzyme and stop the take up of oxygen. This effectively suffocates any

bacteria, killing it within 6 minutes and leaving surrounding tissue or material unaffected. This is

supported by the study of Salomoni et al., where it stated that AgNPs enter cells and inhibit

enzymatic systems in some bacteria, thereby altering their DNA synthesis.

 Across the years, there have been a variety of methods for synthesizing AgNPs. Methods

include chemical, physical, and, recently improved biological methods. Among these however,

biological methods or green synthesis using plants or microorganisms is proven to be easier,

more efficient, and ecofriendly. Many plant extracts have been recognized as a potent natural

reducing agent, because of the presence of rich functional molecules, such as phenolic

compounds with high antioxidant activity. Greener way of synthesizing nanoparticles has

emerged as an alternative method, as it is eco- friendly and cost effective (M. Reenaa and

Aathira S. Menon, 2017).

The benefits of using plants for the synthesis of the nanoparticles are that the plants are

easily available and possess a large variety of active functional groups that can promote the

reduction of silver ions. Most of the plant parts like leaves, roots, latex, bark, stem, and seeds are

being used for the nanoparticle synthesis. Furthermore, the use of medicinal plants in the

synthesis of Ag-NPs is not only used for size and shape control, but also for providing plant

antimicrobial properties to Ag-NPs (Remziye Güzel and Gülbahar Erdal, 2017). Citrus fruits are

the largest contributors to the fruit production around the world. In addition to their nutritional

value and special flavor citrus fruits are known to contain bio active compounds such as

phenoliccompounds, that result in antibacterial activity (Aruoma et al., 2012).

Calamansi (Citrofortunella microcarpa) is commonly known to have antibacterial and

antioxidant properties and has various important phytochemicals. A study by Cheong MW, et al.,

2012 determined the presence of four common hydroxycinnamic acids, the caffeic, p-coumaric,

ferulic and sinapic acids wherein Philippines Calamansi peel contained the highest amount of

total phenolic acids in which these various biomolecules present in the plant extract may be both
the reducing and capping agent. Thus, combining the Calamnsi (Citrofortunella microcarpa)

antibacterial properties with the AgNPs, an even more potent antibacterial agent is on the work.

With Calamansi (Citrofortunella microcarpa) having the highest phenolic content of

309.38 mg AAE/g sample compared to Dalandan (Citrus aurantium), and Pomelo (Citrus

maxima) according to the study of Rich Milton R. Dulay and Ma. Ellenita G. De Castro of De La

Salle University, there is a promising potential of calamansi in performing antioxidant activity in

which is depicted in the antioxidant activity of this citrus which displayed the highest scavenging

activity as the output of the conducted study. In this told, calamansi alone has exhibited great

antioxidant activity; therefore, utilizing calamansi extract as the reducing and stabilizing agent in

synthesizing Silver as nanoparticles with its promising DPPH free radical scavenging activity,

this may result to an enhanced DPPH free radical scavenging activity of the biosynthesized

AgNPS.

Aside from that, with the wide use of lead in various industrial fields, lead has become

one of the leading pollutants in the world, and specifically, it is present in tap waters because

majority of the household plumbing systems contain lead even the Polyvinyl chloride (PVC)

pipes which resulted to high lead concentration in drinking-water according to the World Health

Organization. A study Li Qui et al stated that increased levels of lead caused muscle paralysis,

memory loss, irritability, and anemia; moreover, more than 80% of daily intake of lead is derived

from the food, dust, and dirt while 5 µg/l is the daily intake of lead from water, and in addition,

10% of the lead contained food is absorbed by adults but it is alarming to children to whom

absorb 4-5 times as much than adults (World Health Organization, 2011). Nevertheless, there are

many methods developed for detecting lead and other metals. According to a study by Ja Young

Cheon and Won Ho Park the most common of these methods include atomic/molecular
absorption spectrometry, and many others. However these methods demand a high cost and

adcanced laboratory equipment and skills. Thus, the use of AgNPs as a lower costing and

effective alternative is being researched. The study of Cheon and Park also stated that metal

nanoparticles have been used extensively for detection of heavy metal ions, owing largely to

their localized surface plasmon resonance (SPR) absorption and unique optical properties.

Therefore, using calamansi as the synthesizing agent can be beneficial due to its availability in

the country, the low cost and adding to that its antibacterial and DPPH free radical scavenging

properties.

AgNPs are able to detect heavy metals such as Lead and in testing; this will result in a

change in the AgNP solution from dark yellow to colourless (Teerasong et al, 2015) which is

depicted in colorimetric assay. With Calamansi being readily available, and adding to that its

antimicrobial properties, enhanced DPPH free radical scavenging activity, and heavy metal

sensing capability (Lead) this study aims to find an even better alternative in green synthesizing

AgNPs and in maximizing its antibacterial properties and essential applications.

 CHAPTER III

MATERIALS AND METHODS

This chapter discusses about the methodologies and materials utilized in the study. It
gives details for the development of biosynthesized silver nanoparticles (AgNPs) via Calamansi
(Citrofortunella microcarpa) utilized as antibacterial agent, DPPH free radical scavenging
activity determiner, and metal sensor due to its capability to be manipulated in these applications.
This mainly includes the following: research design, research locale, data gathering procedures,
sampling technique, and experiment procedures.

Research Design

This research follows an experimental and examination research design. Calamansi

(Citrofortunella microcarpa) as the stabilizing and reducing agent will be collected from Tandag
City, Surigao del Sur while the synthesis, characterization of the biosynthesized Silver
Nanoparticles, and as well the assessment of DPPH free radical scavenging activity sensing
capability, and metal sensing capacity of green synthesized SNPs will be performed in the
laboratories of University of Immaculate Concepcion (UIC) and Ateneo de Davao University
(ADDU). Moreover, various lab tools will also be needed for the study.

Research Locale

The researchers will conduct the experiment at the University of Immaculate Concepcion
(UIC) and Ateneo de Davao University (ADDU). The tools used in the study include net bag,
electric blender, sealed container, rotary evaporator, Erlenmeyer flask, and centrifuge which are
used for the preparation of plant extract and synthesis of the Silver Nanoparticles. Furthermore,
Ultraviolet (UV)-visible spectroscopy, and Scanning Electron Microscopy (SEM)-Energy
Diffraction X-ray (EDX) are for the characterization of the biosynthesized silver nanoparticles.
In addition, Antibacterial assay is for evaluating the anti-microbial activity of the green
synthesized silver nanoparticles while DPPH Free Radical Scavenging Activity Sensing
Capability and Metal Sensing Capacity via colorimetric assay.
 Data Gathering Procedures

A. Chemicals

Silver Nitrate and Hydrogen Peroxide (H2O2) shall be procured in the laboratory of University of

Immaculate Concepcion as well as the nutrient agar and other media components.

B. Collection of Plant Material

Fresh leaves and the Calamansi fruit itself which initially weighed 2 kilograms were collected in

the month of May in the vicinity of Tanabog, Tandag City, Surigao del Sur.

C. Processing of Calamansi Leaves, peels, and Juice extract

The Calamansi leaves and peels were thoroughly washed up through tap water to in order to

remove dust particles present on the surface of the plant material and then after were rinsed with

distilled water. The cleaned Calamansi leaves and peels were dried through air drying at an

ambient temperature for 72 hours. Moreover, the remaining pulps of Calamansi were squeezed,

strained, and filtered by pressing using strainer and then was stored in an air-tight sealed

container.

D. Preparation of Calamansi Extracts

The air-dried peel and leaves are then powdered using an electric blender for powdered samples

have a more homogenized and smaller particle, leading to better surface contact with extraction

solvents (Azwanida NN, 2015). The 100 grams air dried plant of each sample will be placed into

a container and will be soaked in 200mL of 95% ethanol solution for maceration for 2-3 days

then will undergo in a rotary evaporation afterwards. The clear leaf, peel and juice extracts of
Calamansi (Citrofortunella microcarpa) obtained will be used for synthesis of silver

nanoparticles.

E. Synthesis of Silver Nanoparticles

For the biosynthesis of AgNPs, 10 mL of each leaf, peel, and juice extract will be mixed with 90

mL of 1mM Silver nitrate (AgNO3) solution separately and will be water bathed at 80 degrees

Celsius for 10 minutes. Then, a color change from yellow to darkish brown depicts the formation

of these colloidal SNPs.

F. Characterization of Silver Nanoparticles

(Cited: S. Rashid, M. Azeem, S. A. Khan, M.M. Shah & R. Ahmad, 2019)

UV-Vis spectroscopy

The Ultraviolet-Visible absorption spectrophotometer with a corresponding resolution of 1 nm

within the points of range of 200 and 600 nm will be utilized to determine the rate of reduction

of the pulverized AgNPs. The reduction of silver ions will be monitored by measuring the

spectrum of the reaction mixture in UV-Visible spectrophotometer with the equal amount of

suspension that is 1 ml of the sample will be pipetted in a cuvette and will be diluted with 1 ml of

distilled water and then analyzed at room temperature. From the results of UV-Vis spectroscopy

on the 3 aqueous extracts, the qualified Calamansi extract which has most synthesized silver

nanoparticles and has the best results shall be used in the preceding methods and processes of the

study.

Scanning Electron Microscopy (SEM)-Electron Diffraction X-ray

Morphological and chemical analysis of synthesized AgNPs will be carried out through Scanning

Electron Microscopy (SEM) which will be equipped with EDX detector.

Scanning Electron Microscope (SEM) coupled with Energy Dispersive X-ray Spectroscopy

(EDX) will be used to assess the shape and percentage of the synthesized silver nanoparticles

respectively. The samples shall be prepared by fixing the powdered Silver nanoparticles to

microscope holder through a conducting carbon strip. Preparing the sample will require the

separation of single silver particles from the powder delivered Silver Nanoparticles. Due to this,

1 mg of silver powder in 5 ml of hexane will be dissolved. The solution will be subjected to the

process of particles selection.

X-ray Diffraction (XRD) Analysis

The crystalline structure of powdered AgNPs will further be determined and confirmed by the

utilization of X-Ray diffraction technique. The centrifuged and powdered AgNPs will be then

placed on microscopic glass slide and then be analyzed using XRD. The average crystallite size

of synthesized silver nanoparticles will be determined via calculation through Scherrer’s -

formula:

In which D is for the average crystallite size, K is constant, 𝛌 is incident wavelength, β is full

width at half maximum and is Bragg angle. X-Ray diffraction analysis will be performed using

(PAN analytical X pert PRO Model) to depict the dimension of the synthesized AgNPs with h, K

and I values.
Dynamic Light Scattering (DLS)

The particle size distribution and zeta potential analysis of powdered AgNPs will be evaluated

via dynamic light scattering (DLS). Dynamic Light Scattering can probe the size distribution of

small particles a scale ranging from submicron down to one nanometer in solution or suspension,

especially in the range of 2–500 nm. Usually, DLS measures the light scattered from a laser that

passes through a colloid, and mostly relies on Rayleigh scattering from the suspended

nanoparticles. Next, the modulation of the scattered light intensity as a function of time is

analyzed, and the hydrodynamic size of particles can be then determined.

Fourier-transform Infrared Spectrometry

For FTIR analysis, the pulverized AgNPs will be centrifuged at 12,000 rpm for 30 mins at 0 °C

and then Supernatant will be discarded and pallet of AgNPs washed with deionized water, where

the freeze-dried (lyophilization) powder will be pelletized with potassium bromide (KBr) powder

and then subjected to Fourier-transform infrared spectroscopic (FTIR) analysis at a wavelength

ranging from 4000 to 400 cm−1.

G. Antibacterial activity

The green synthesized silver nanoparticles via qualified Calamansi extract will be tested for its

antibacterial activity through standard agar-well diffusion method. Escherichia coli and

Staphylococcus aureus will be utilized as the test specimens. The pure culture of E. coli and S.

aureus will be sub-cultured in nutrient broth and the strains will be equally spread on sterilized

petri plates. In addition, three circular wells, A, B, and C of 6mm diameter will be developed via

sterile cork-borer. Well C shall be filled with a chemical antibiotic (can be chloramphenicol in 50

uL) as a representative of positive control while wells A and B will be loaded with SNPs for the
assessment of its antibacterial activity. Other than that, the plates will be incubated at 37 degrees

Celsius overnight and the zones of inhibition will be observed furthermore.

G. Hydrogen Peroxide-sensing capability of silver nanoparticles

In evaluating the H2O2 sensing capacity of these biosynthesized AgNPs, the protocol developed

by the researchers Bera and Raj, 2013 shall be performed. The initial spectrum of the diluted

AgNPs solution at 3 mL will be assessed by spectrophotometer. In the protocol, 1 mL of 20 mM

of H2O2 will be added to the SNPs solution and will be then thoroughly mixed. The spectrum

readings will be taken at regular intervals and the change in spectra shall be thoroughly noted.

Flowchart of the Study

Procuration of Chemicals utilized

Collection, processing, and preparation of Calamansi Extracts

Synthesis of Silver Nanoparticles

Characterization of Silver Nanoparticles

UV-vis spectroscopy

Scanning Electron Microscopy (SEM)-Electron Diffraction X-ray

X-ray Diffraction (XRD) Analysis

Dynamic Light Scattering (DLS)

 Fourier-transform Infrared Spectrometry

Antibacterial Assay

Hydrogen Peroxide-sensing capability of silver nanoparticles

Assessment of the Results

Figure3. The flowchart shows the sequence of methods applied in the study.

Calamansi (Citrofortunella microcarpa) will be collected in the vicinity of Tandag City,

Surigao del Sur. The plant sample is then prepared for extraction. Then follows the synthesis of
silver nanoparticles using the leaf, peel, and juice extract of calamansi as stabilizing and reducing
agent and silver nitrate as synthesizing agent where it will be performed in University of
Immaculate Conception. After the synthesis, it will then characterized via Ultraviolet-Visible
spectrophotometry (UV-Visible analysis), Scanning Electron Microscopy (SEM)- Electron
Diffraction X-ray, X-ray Diffraction (XRD) analysis, Dynamic Light Scattering (DLS), and
Fourier transform Infrared Spectrophotometer (FT-IR),. Afterwards, the SNPs will be assessed
on its antibacterial activity and Hydrogen peroxide-sensing capability.
 Experimental Design

Experimental Set-up ————–———————— Control Group

Independent Controlled Variable Dependent Negative

Variable Variables Controls

Biosynthesis of Silver Escherichia coli Characterization Results For the assessment of

Nanoparticles (AgNPs)
via Calamansi
(Citrofortunella
microcarpa) extracts
Antibacterial activity of
biosynthesized Silver
Nanoparticle on bacteria
samples E. coli and S.
aureus
Hydrogen Peroxide
sensing activity of
biosynthesized SNPs
(E.coli) antibacterial activity:
Particle size analysis Results Without the presence
Staphylococcus of any antibacterial
aureus (S. aureus) Antibacterial effect of agents
biosynthesized SNPs
Hydrogen
Colorimetric effect of For the Hydrogen
Peroxide (H2O2)
biosynthesized AgNPs when Peroxide-sensing
in contact with Hydrogen Capability:
Peroxide (H2O2) Absence of
biosynthesized Silver
Nanoparticles

Figure4. Experimental design of the study portrays all the variables that will be utilized in the experimental
process of the research study

The figure above shows the variables used in the recent research study. The
biosynthesis of Silver Nanoparticles (AgNPs) via Calamansi (Citrofortunella microcarpa)
extracts, the antibacterial activity of biosynthesized Silver Nanoparticles on bacteria samples E.
coli and S. aureus, and the Hydrogen Peroxide sensing activity of biosynthesized SNPs serve as
the independent variable. The dependent were the characterization results, particle size analysis
results, antibacterial effect of biosynthesized SNPs, and the colorimetric effect of biosynthesized
AgNPs when in contact with Hydrogen Peroxide (H2O2). Without the presence of any
antibacterial agents is for the assessment of antibacterial activity while for the Hydrogen
Peroxide-sensing capability is the absence of biosynthesized Silver Nanoparticles which are
utilized as the negative controls of the study.