Article

Green Synthesis of Ag/Chitosan Nanoparticles using Avocado Leaves

Bioreductor (Persea americana Mill.) as a Nitrite Colorimetry

Detector

Nani Lestari¹, Ratih Dyah Puspitasar¹* , Nindita Clourisa Amaris Susanto², Indra Lasmana

Tarigan¹, Nelson Nelson³

¹Department of Chemistry, Faculty of Science and Technology, Universitas Jambi, Indonesia

²Department of Pharmacy, Vocational School, Universitas Sebelas Maret, Surakarta, Indonesia

³Department of Chemical Analyst, Faculty of Science and Technology, Universitas Jambi, Indonesia

Abstract

Urinary tract infection (UTI) is one of the most common bacterial infections, characterized by the

proliferation of microorganisms within the human urinary tract. Nitrite serves as an important diagnostic

indicator of UTI because it is produced through the bacterial reduction of nitrate. Various analytical

techniques—including spectroscopy, electrochemistry, chemiluminescence, chromatography, capillary

electrophoresis, and flow injection analysis—have been employed to detect nitrite. However, these

methods generally require lengthy procedures involving bacterial incubation and extensive sample

preparation; therefore, they are less suitable for rapid screening. This limitation highlights the need for

a fast, reliable, and cost-effective detection approach. Colorimetric sensors, particularly those based on

nanoparticles, offer a promising alternative due to their rapid response, low cost, and ability to provide

visually observable results. In this context, the present study aims to synthesize silver nanoparticles

(AgNPs) using avocado leaf extract (Persea americana Mill.) as a natural bio-reductant and chitosan as a

stabilizing agent, thereby reducing reliance on hazardous and environmentally unfriendly inorganic

chemicals. The synthesized AgNPs were characterized using UV–Vis spectroscopy, Fourier Transform

Infrared (FT-IR) spectroscopy, and Particle Size Analyzer (PSA) measurements. The synthesis was

optimized by varying the bioreductant volume, reaction time, and chitosan concentration. The optimal

synthesis time was determined to be 4 h, yielding a surface plasmon resonance (SPR) peak at 428 nm

with an absorbance of 2.112 and nanoparticle size within the desirable range. Furthermore, a chitosan

concentration of 2.5% produced the most stable nanoparticles, indicated by an SPR peak at 435 nm and

an absorbance of 1.341. The resulting AgNPs/chitosan system functioned effectively as a colorimetric

nitrite sensor, exhibiting a visible color change to purple, with a limit of detection (LOD) of 0.1303 µM and

a limit of quantification (LOQ) of 0.4345 µM. All measurements were conducted in triplicate to ensure

reproducibility.

Keywords: Ag Nanoparticles; Avocado Leaves; Colorimetry; Nitrite.

*

Corresponding author

Email addresses: ratihdyah@unja.ac.id (Ratih Dyah Puspitasari)

DOI: https://doi.org/10.22437/chp.v9i2.46583

Received July 04^th2025; Accepted October 08^th2025; Available online November 25^th2025

(https://creativecommons.org/licenses/by/4.0 )

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Graphical Abstract

Introduction

screening method to diagnose UTIs based on

a nitrite colorimetric sensor. Nitrite is an

indicator of UTI. Under normal circumstances,

nitrites are not found in urine, but the

presence of bacteria can convert nitrates in

urine into nitrites, which indicates the

presence of bacteria and diagnoses UTI [6-7].

The nitrate reductase enzyme is useful for

reducing nitrate to nitrite. In the urinary tract,

pathogenic bacteria contain nitrate enzymes

that can reduce nitrates to nitrites [8]. Nitrate

reductase that occurs in leaves and roots

comes from photosynthesis and respiration,

which causes the transfer of electrons. In the

nitrate reductase enzyme, nitrate is converted

into nitrite, nitrate (NO₃) will be reduced to

nitrite (NO₂) [9].

Health problems in the world are increasing

along with the development of disease [1].

Infectious diseases are risky, especially in

developing countries [2]. One of the most

common infections is Urinary Tract Infection

(UTI), which is a disease that can attack

women and men of all ages. Urinary tract

infection is a common contagion among men

and women but the incidence is quite high

among women due to their physiology [3].

Urinary tract infection (UTI) is an infection

caused by the growth of microorganisms and

bacteriuria

in

the

urinary

tract.

This

bacteriuria includes Escherichia coli, Klebsiella

sp, Proteus sp, Providensiac, P. aeruginosa,

Acinetobacter, and Enterococu faecali, but 90%

is caused by Escherichia coli [4]. Currently,

general techniques for diagnosing urinary

tract infections (UTI) tend to take quite a long

time, so they are not suitable for rapid

screening because they require adequate

Several analytical methods used to determine

nitrite

include

colorimetric

methods,

spectrophotometric

chromatographic

electroanalytical

methods,

methods.

and

Urine

bacterial

incubation

time

and

sample

examination as an indicator of health can be

done macroscopically, microscopically, and

preparation [5]. There is a need for a rapid

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Chempublish Journal, 9(2) 2025, 230-246

chemically [9-10]. Macroscopic examination

of urine consists of assessing color, clarity,

odor, specific gravity, and pH. Microscopic

examination to see the presence of urine

sediment such as erythrocytes, leukocytes,

epithelial cells, crystals, cylinders, bacteria,

fungi, parasites, and spermatozoa. To detect

NO₂sensitively and selectively, sophisticated

instruments and highly trained operators are

needed. Colorimetric detection is attractive

because of its simplicity and low cost and can

be used for on-site visual analysis. NO₂can

also be detected indirectly in a colorimetric

assay by utilizing a highly selective diazo

reaction [11].

steroids, phenolics, saponins, and flavonoids,

which basically can reduce silver ions into

silver atoms and form silver nanoparticles

[16]. Currently, there is a new method, namely

the biosynthesis of plant- based nanoparticles

as a bioreductant. The use of plant organic

compounds for nanoparticle synthesis is an

environmentally friendly and simpler method.

Apart from that, the types of plants that

contain

this

reducing

agent

are

quite

abundant and easy to obtain in Indonesia [16].

Several

studies

have

succeeded

in

synthesizing silver nanoparticles using plant

extracts, such as using bamboo leaf extract as

a reducer of silver ions from the AgNO₃

compound to become silver nanoparticles at

a temperature of 659C which obtained less

than 100 nm [17]. Synthesis of silver

nanoparticles using tapak dara leaf extract,

with an average nanoparticle size of 35-55 nm

in random cubic shape [18]. Synthesis of silver

nanoparticles using extracts Lantara camara

leaf produced nanoparticles with an average

size 1.6 to 25 nm [19]. Synthesis of silver

nanoparticles using strawberry leaf extract

obtained spherical silver nanoparticles with a

size of 9-15 nm [20]. Synthesis of silver

nanoparticles using methanol extract from

green tea leaves obtained a size of 157.8 nm

[21]. Bioactive compounds contained in plants

such as antioxidant compounds and certain

secondary metabolite compounds, such as

the group of terpenoid and flavonoid

compounds which are thought to play a role

in the metal ion reduction process [22].

Various types of plant groups contain

secondary metabolites as written above, one

of which is the avocado plant.

Silver nanoparticles are one of the most

researched nanoparticles and their most

common application is the use of silver

nanoparticles as antibacterial, antimicrobial,

anti-inflammatory, anti-angiogenesis, anti-

fungal, antiviral, and anti-platelet activity [12].

Currently, various types of nanoparticles have

been synthesized, such as gold, silver, iron,

zinc, and metal oxide nanoparticles [13]. Silver

nanoparticles

(NPP)

have

advantages

compared to gold nanoparticles because the

optical properties of NPP are better [14], so

they can be used as a detector and also as a

coloring indicator (colorimetry). In addition,

silver nanoparticles have been widely used in

clothing,

footwear,

paint,

bandages,

household appliances, cosmetics, and plastics

because they have antibacterial properties

[15].

Silver nanoparticles can be synthesized by

physical, chemical, and biological methods.

Although physical and chemical methods

produce pure particles, they are expensive

and not environmentally friendly. Recently,

silver nanoparticle synthesis techniques have

been developed that are simpler, cost-

The secondary metabolite content in avocado

leaves

includes

saponins,

alkaloids,

flavonoids,

and

tannins [23].

efficient,

and

environmentally

research stated that the flavonoid content

contained in avocado leaves has antifungal,

antiviral, and antibacterial activity [24].

friendly, one of which is using plant extracts.

This plant extract contains alkaloids, tannins,

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Avocado leaves have high antioxidant

activity compared to other parts of this plant

(fruit skin, flesh, and fat). From the results of

the FTIR spectrum on avocado leaves, strong

peaks appeared at 3300-3284 cm^-1and

1671- 1611 cm^-1, which indicates the

buffer, Ammonia (NH₃), Drangendorff's

reagent, Mg powder, Ferric chloride (FeCl₃),

Liebermann's reagent- Burchard, Naphthyl

ethylenediamine,

Nitric

acid

(HNO₃),

Phosporic acid (H₃PO₄), Sulfuric acid (H₂SO₄)

and sodium carbonate (Na₂CO₃) were

presence

of

polyphenolic

compounds

purchased

from

Merck

Sigma-Aldrich

(flavonoids) [25].

Reagent Pte, Singapore.

Silver nanoparticles have low stability and

can form aggregations so that the size

becomes non-uniform and large. Therefore,

a capping agent is needed to prevent the

formation of aggregates between the

Preparation of Ethanol Extract

Extraction method refers to the research by

[20]. 50 grams of avocado leaves were

washed cleaned and dried by airing at room

temperature. The dried leaves are weighed

again to determine the final water content.

After that, cut it into small pieces and then

puree it with a blender. Avocado leaf

surfaces

of

silver

nanoparticles.

The

stabilization of silver nanoparticles can be

enhanced by polymers. One polymer that

can be used is chitosan [26]. Chitosan has a

- NH₂group which can interact with the

surface of silver nanoparticles.

extraction

is

carried

out

using

the

maceration method. Fifty (50) grams of dry

simpilicia powder was soaked with 500 mL

of ethanol solvent (ratio 1:10 w/v). The

maceration process lasts for 2 days. The

mixture is soaked for the first 6 hours,

stirring occasionally, then left for 18 hours.

The macerate is separated with filter paper.

Next, it is evaporated at a temperature

below 40℃ and evaporated until a thick

extract is obtained. The extract obtained

was then characterized using a UV-Vis and

FT-IR spectrophotometer which will be used

for the next process.

Further research on green synthesis of silver

nanoparticles (Ag/Chitosan) using avocado

leaves as a reducing agent for colorimetric

detection of nitrite has not been carried out.

However, based on previous reported that

stabilized Chitosan AgNPs are believed to be

AgNPs composites, which are simple, strong,

and cheap, and have great potential for

-

application in sensitive low-level NO

detection. and low [27]. This research aim²s

to synthesize Ag/Chitosan Nanoparticles

using Avocado Leaves bioreductor (Persea

americana Mill.) as a Colorimetric Nitrite

Detector for urine.

Phytochemical Analysis.

Phytochemical analysis method refers to the

research by [16]. Phytochemical screening

was carried out; a) Alkaloid Test: A total of 1

mL of the sample was dissolved in several

drops of 2N sulfuric acid, and then tested

with Dragendorff's reagent, Meyer's reagent

and Wagner's reagent. The test result is

declared positive if the Dragendorff reagent

forms a red-orange precipitate, in the Meyer

reagent a yellowish-white precipitate forms,

and in the Wagner reagent a brown

precipitate forms.b) Flavonoid Test: A few

Materials and Methods

Materials

Avocado Leaves were collected from Muaro

Jambi Regency, Jambi Province, South

Sumatera, Indonesia. Laboratory grade

Silver Nitrate (AgNO₃), Sodium nitrate

(NaNO₃), Sodium nitrite (NaNO₂), Ethanol,

Distilled water, Hydrochloric acid (HCl) 2N, 4-

Aminothiophenol (4-ATP), chitosan, pH 9

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drops of concentrated HCl were added to

several samples and then Mg powder was

added. The positive results of the HCl

reagent and Mg powder were indicated by

the formation of foam and the color change

of the solution to orange. c) Phenolic Test:

Several samples were added with FeCl₃and

homogenized. Positive results from the

FeCl₃reagent are indicated by the formation

were added with the Liebermann-Burchad

reagent. If a blue/green color forms, it

indicates the presence of steroids. If a

purple or orange color forms, it indicates the

presence of triterpenoids. e) Saponin Test:

Saponins can be detected by the foam test

in hot water. Stable foam that can last a long

time and does not disappear when adding 1

drop of 2N HCl indicates the presence of

saponin.

of

a

blackish-purple

color.

d)

Triterpenoid/Steroid Test: Several samples

[A]

[B]

[C]

Figure 1. Simplicia Preparation [A] avocado leaves; [B] dried avocado leaves; [C] avocado leaves

simplicia powder.

[A]

[B]

[C]

Figure 2. Maceration Process: [A] Simplicia is added; [B] Ethanol is added; [C] the resulting

maceration extract is filtered

Nanocomposite Biosynthesis

colloid was dried using a freeze-dryer. The

silver nanoparticles obtained were

characterized using PSA spectrophotometer.

Synthesis time optimization. 0.01 M AgNO₃

into a glass beaker, add 5 mL of 0.5%

avocado leaf extract, add NH₃solution until

pH 9, then stir using a magnetic stirrer with

varying times of 1, 2, 4, and 6 hours. The

solution was centrifuged at 3500 rpm for 10

minutes, and the solution was taken for

analysis using a UV-Vis spectrophotometer

and FTIR (Fabiani et al., 2018). The resulting

Chitosan

dissolved

Concentration.

in 1% acetic

Chitosan

acid

was

a

with

concentration of 1.5, 2, 2.5% (w/v). Put 0.01

M AgNO₃into a glass beaker, add 5 mL of

0.5% avocado leaf extract, add NH₃solution

until pH 9, then stir using a magnetic stirrer

for the optimal time obtained in the previous

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process. A total of 2 mL of chitosan was

added to the synthesized solution. NH₃was

added until it reached pH 9. The solution was

centrifuged at 3500 rpm for 10 minutes, and

the solution was taken for analysis by UV-Vis

and FTIR spectrophotometer; the resulting

colloid was dried using a freeze dryer. The

optimal results of adding chitosan were

observed within 0, 3, and 6 days to see the

calculated using equation (1) and LOQ using

equation (2).

LOD = 3× ^푆퐷

(1)

(2)

푏

LOQ = 10× ^푆퐷

푏

SD: standard deviation of absorbance values

from measurement results; b: slope of the

calibration curve equation

stability

Ag/Chitosan nanoparticle colloids produced

under optimal conditions were

of

silver

nanoparticles

[28].

Result and Discussion

characterized using particle size analyzer.

Preparation of Ethanolic Extract

Nitrite Colorimetric Sensor

Avocado leaves are used as an organic

compound in the green synthesis process.

Then, it is dried by air-drying. After drying,

the avocado leaves are then separated

between the leaf veins and the leaf stalks.

Once separated, the avocado leaves are

blended until they become simple (Figure 1).

Avocado leaves simplicia powder is stored in

a clean container and protected from light to

avoid damage and loss of quality. The

maceration method is used to make avocado

leaf extract. The maceration method is a

method commonly used to extract active

compounds from natural materials by

soaking the material in a solvent that is

suitable for the active compound to be taken.

This method is used because it is more

straightforward, relatively easy, and cheap. It

can be carried out on a large scale without

going through a heating process, thereby

minimizing the possibility of damage to the

chemical compound components in the

sample. Process of making the extract can be

seen in Figure 2.

AgNPs/Carrageenan was added with 1M

H₂SO₄

until

pH

3,

then

the

AgNPs/carrageenan solution was mixed with

10 mM 4-ATP at a ratio of 8:1 (v/v). A total of

0.5 ml of nitrite with a concentration of 0; 0.5;

1; 1.5; 2 and 2.5 μM were added with 0.6 ml

of 1M H₂SO₄to create an acidic atmosphere.

Then 1 ml of 4-ATP-AgNPs/Carrageenan was

added. Then 1 ml of 1 M CH₃COOH was

added and 0.5 ml of 20 mM NED was added

[29]. The solution was left for 5 minutes at

room temperature and the color changes

were

recorded

using

a

camera.

A

colorimetric selectivity test was carried out

-

with the addition of NO₃, PO₄, SO₄, CO₃, and

Cl^-ions using a method that has been carried

out on nitrite [30].

Method Validation

Method validation was carried out for

selectivity tests, linearity tests, LOD (Limit of

Detection), and LOQ (Limit of Quantity) tests.

The absorbance value of the test solution

was

measured

using

a

UV-Vis

Simplicia maceration was carried out for 2 x

24 h using ethanol (96%), stirring in a closed

place and calculated at 6 h, 18 h and 48 h of

maceration time. The longer the extraction

time, the greater the amount of material

extracted because the opportunity for

contact between the material and the

spectrophotometer to obtain a calibration

curve and r value. Meanwhile, determining

the LOD and LOQ values is carried out

statistically through linear regression and

calibration

curves.

The

LOD

can

be

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Chempublish Journal, 9(2) 2025, 230-246

solvent is more significant. Maceration time

that exceeds the optimum time will cause

the extracted components to decrease.

Maceration times that exceed the optimum

time will damage the dissolved substances in

the material and have the potential to

increase the process of loss of compounds in

the extraction solution due to evaporation.

Ethanol's polarity value allows compounds

that dissolve in polar solvents to attract each

other through the formation of hydrogen

contained in avocado leaf extract. Based on

the results of the phytochemical screening

carried out, the secondary metabolite

compounds contained in avocado leaf

extract can be seen in Table 1. The positive

results obtained in this phytochemical test

indicate the presence of flavonoid, phenolic,

steroid and saponin compounds in avocado

leaf extract. Flavonoid compounds are the

largest group of phenolic compounds found

in nature. These compounds are red, purple

and blue dyes, as well as yellow dyes found

in plants. One of the secondary metabolite

contents that can be derived is flavonoid

compounds. Flavonoid compounds have

hydroxyl (-OH) and carbonyl (-CO) functional

groups as stabilizers and form a two-layer

silver electrical layer. The size of silver

bonds

and

dipole-dipole

interactions

between the ethanol hydroxyl group and the

polar groups in the compound. The extract

was evaporated using a Rotary evaporator at

a speed of 32 rpm and a temperature of 50⁰C.

Phytochemical Profiling.

nanoparticles

concentration of the stabilizing compound is

higher.

will

be

smaller

if

the

The phytochemical test was carried out as a

preliminary test to screen the compounds

Table 1. Phytochemical Screening of Avocado Leaves Extract

Compound

Indetification

Reaction

Result

Avocado Leaves

Reference*

extract

Alkaloids

Flavonoids

Phenolic

Steroids

Terpenoids

Saponins

Meyer's reagent

Concentrated HCl + Mg powder

FeCl₃

Liebermann-Burchad’s reagent

-

+

-

+

Hot water + 2N HCl

Information: (+) There are groups of compounds/ (-) There are no compound classes

Synthesis Time-Dependent Optimization

Time optimization is carried out

nanoparticles

environmental

can

conditions.

form

in

alkaline

pH

Alkaline

to

causes the functional groups of the extract

to be deprotonated so that its ability to

chelate metals is stronger, as previous

research reported that alkaline pH mediates

the rapid synthesis of AgNPs, where

immediately after adding NH₃as a function

of the pH, it becomes more alkaline causing

an accelerated reduction in the mixture of Ag

determine the optimal time for the synthesis

to be carried out. At this stage, the avocado

leaf extract is used as a bioreductant to

make nanoparticles. Then, functions are

added to provide an alkaline atmosphere.

Ag⁺ions interact more effectively with the

phytochemicals in avocado leaf extract at

higher

pH

conditions

(>pH-7).

Silver

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and extract. This is caused by changes in the

electrical charge of the biomolecules in the

reducer to become more reactive [31]. This

is also proven by research conducted by et

al.; the synthesis was carried out under

alkaline conditions (pH 9), which has been

optimized for the synthesis of SC bark at

has taken place and silver nanoparticles

have been formed. The formation of AgNPs

is characterized by a change in the color of

the solution to yellow as time increases.

Indications of the formation of nanoparticles

with color changes were proven using a UV-

Vis spectrophotometer [19]. Then, the

synthesis results were centrifuged at 3500

rpm for 45 minutes, and the filtrate was

taken for analysis, and it could settle so that

in the UV-Vis spectrophotometer analysis,

there were no impurities that interfered with

the reading.

room

temperature.

The

synthesis

of

nanoparticles obtained with an absorption

peak of 420 nm at pH 9 resulted in the

formation of more extensive nanoparticles.

The color change to yellow indicates that Ag

nanoparticles have been formed. This color

change indicates that the reduction reaction

Table 2. UV-Vis Spectrophotometry Results of Ag Chitosan Nanoparticles

Chitosan

Concentration (%)

SPR (Surface Plasmone

No.

Absorbance

Responance)

1.

2.

3.

1.5

2.0

2.5

414

440

435

2.028

0.753

1.341

Table 3. UV-Vis Spectrum Characterization of the Stability of Ag/Chitosan Nanoparticles

No.

Storage Time (Days)

SPR (Surface Plasmone

Absorbance

Responance) (nm)

1.

2.

3.

0

3

6

435

427

430

1.41

1.290

1.249

Figure 3. UV-Vis Spectrophotometric Characterization of Ag Nanoparticles with Various Time of

Synthesis

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Figure 4. UV-Vis Spectrophotometry Results for Chitosan Concentration Optimization

Figure 5. Results of UV-Vis Spectrophotometric Characterization of the Stability of Ag/Chitosan

Nanoparticles

The optimal time obtained was obtained at a

synthesis time of 4 h, as shown by the

highest absorption peak at a synthesis time

of 4 hr with the highest absorbance (Figure

3). As seen in Table 3, the absorbance value

was high at 4 hr. Ag nanoparticles can be

seen from the characteristic absorption

characteristic of silver nanoparticles with a

wavelength of 400-450 nm [18]; apart from

that, the number of silver nanoparticles

formed can be determined from the

absorption peak time increases, and the

absorbance increases. However, the

variation in synthesis time of 6 h decreased,

which seems influenced by the length of

stirring, which causes the nanoparticles to

re-aggregate into clusters

Based

on

the

results

of

the

PSA

characterization test, the nanoparticle size

obtained was 129 nm. It can be said to be

nano if the particle size is 1-100 nm.

However, some literature states that the size

of nanoparticles ranges from 1-1000 nm.

Nanoparticles are particles with a size of 10-

1000 nm. The size of the nanoparticles can

absorbance

value.

value,

The

the

higher

the

nanoparticles formed. Based on the results

of UV-Vis characterization in Figure 4, the

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be influenced by the stability of the solution.

Apart from that, agglomeration is the main

cause of increasing nanoparticle size. Where

color of the solution changes from brownish-

yellow to transparent, the formation of

nanoparticles in the solution stops. Based on

Table 2, the results of 1% chitosan have the

lowest absorption peak of 414 nm and

absorbance of 2.028. 2% chitosan has an

absorption peak of 440 nm and an

absorbance of 0.753. Chitosan 2.5% has an

absorption peak of 435 nm with an

absorbance of 1.341.

the

agglomeration

event

can

be

characterized by a change in the color of the

solution to ash and the absorbance of the

solution increases or decreases, which

means the AgNPs are unstable.

Optimization of Chitosan Concentration.

AgNO3,

which

was

reacted with

the

The results of measuring the stability of

Ag/Chitosan nanoparticles in Figure 6 show

that the length of storage affects the stability

of silver nanoparticles. Increasing storage

time shows the formation of particles with

larger sizes. The absorbance value continues

to increase as the absorption peak shifts to a

bioreactor, experienced a color change from

clear greenish to brownish yellow. This

change indicates that silver nanoparticles

are starting to form. Apart from color

changes, the characteristic absorption peaks

of the nanoparticles can also be seen [29]. As

seen

in

Figure

4,

have

all

variations

in

larger

wavelength, indicating that the

concentration

characteristic

number of silver nanoparticles formed

increases with the length of contact time.

Characterize Fourier Transform Infra-Red

(FTIR), which aims to show the functional

groups contained in the sample. The

occurrence of the oxidation process as a

result of the reduction process of silver

nanoparticles will result in a shift in the wave

number, which indicates that there has been

an interaction between the functional

groups and the silver nanoparticles [33].

Specific groups in the IR spectrum are

presented in Table 4. Detected functional

groups will appear in the presence of peaks

in the FT-IR spectrum. Each functional group

has a different wave number according to

the functional group's ability to absorb

infrared energy.

absorption peaks of nanoparticles. 1.5%

chitosan has the lowest absorption peak of

414 nm and absorbance of 2.028. 2%

chitosan has an absorption peak of 440 nm

and an absorbance of 0.753 (Table 4).

Chitosan 2.5% has an absorption peak of 435

nm with an absorbance of 1.341. The higher

the absorbance, the smaller the resulting

nanoparticles, and the higher the absorption

peak obtained, the more nanoparticles will

be obtained. The stability of AgNP colloidal

solutions based on time can also be analyzed

based on changes in absorption peaks [32].

The shift in the absorption peak to a larger

wavelength indicates that the stability of

silver nanoparticles is less, and they tend to

experience agglomeration [28]. The results

of

determining

the

stability

of

the

synthesized Ag/Chitosan Nanoparticles are

shown in Figure 5. Stability was measured on

days 0, 3, and 6 after synthesis.

From Figure 6 it can be seen that the FTIR

graph looks the same at each intensity,

showing a wide and strong band in the O-H

group at the wave number frequency cm-1

which is typical of the alcohol functional

group. A reduced frequency value indicates

The stability of Ag/Chitosan nanoparticles

can

be

measured

using

UV-Vis

Spectrophotometry and can be seen from

their color. The higher the absorbance, the

more the nanoparticles agglomerate. If the

a

downward

shift

in

intensity.

The

decreasing O-H and C-O-C groups are due to

contributing ions to reduce Ag+ to Ag0.

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Hydroxide ions are needed to accelerate the

Ag+ ion reduction process in the synthesis of

experienced a shift in their absorption peaks

from the data obtained. A wave number shift

silver

mechanism between biomolecules and

bioreduction influences the reduction

process of Ag+ ions undergoing stabilization

into AgNPs. The functional groups

nanoparticles.

The

possible

shows

an

interaction

between

Ag

nanoparticles and secondary metabolite

compounds contained in the bioreduction,

indicating the presence of metabolite

compounds that react Ag+ ions into Ag0 [34].

Figure 6. FTIR characterization results

nanoparticles [28]. The slightly clumped

chitosan affects the size of the nanoparticles.

It coats the nanoparticles thicker. This event

is supported by the results of particle size

characterization using the Particle Size

Analyzer

Ag/Chitosan nanoparticles with PSA aims to

determine the size distribution and

(PSA).

Characterization

of

uniformity of the particles. The results of the

size analysis of Ag/Chitosan Nanoparticles

with the optimal concentration of chitosan

can be seen in Figure 7. Based on Figure 7,

the size of AgNPs/Chitosan is 479.3 nm.

Figure 7. Particle size of AgNPs/Chitosan

Nitrite Colorimetric Sensor

The size of the nanoparticles can be

influenced by several factors, such as

solution temperature, pH, stirring speed,

reductant, and reaction time. Temperature

variations were carried out during the

synthesis of Ag/Chitosan nanoparticles. This

results in the formation rate of silver

nanoparticles taking longer to produce more

Detection preparation begins by reacting 8

ml of AgNPs/Chitosan with 4-ATP added with

H₂SO₄so that the solution is in an acidic

condition. Then, a standard NaNO₂solution

was prepared with a concentration of 0 µM,

0.5 µM, 1 µM, 1.5 µM, 2 µM and 2.5 µM,

240

N. Lestari et al.

Chempublish Journal, 9(2) 2025, 230-246

which was made from a stock solution with a

concentration of 50 µM. At each nitrite

concentration, sulfuric acid was added, and

addition of NED aims to lengthen the

conjugated double bonds, which are based

on the diazotation reaction of aromatic

primary amine compounds coupled with

naphthylethylenediamine. The presence of

AgNPs/Chitosan-4ATP

was

added.

The

solution changed color to yellowish following

the color of AgNPs/Chitosan-4ATP. Then

acetic acid is a weak acid. Then, the addition

of NED functions as a coupling agent. Where

diazonium salts are unstable in water due to

their tendency to lose diazonium groups,

this can be stabilized through the formation

of azo dyes, which cause bathochromic shifts

in the SPR (Surface Plasmon Response)

absorption of AgNP/chitosan bands as a

result of charge transfer interactions on the

nanoparticle surface. Apart from that, the

nitrite

will

produce

a

reddish-purple

compound, which can be measured using a

spectrophotometer, where the number of

moles of nitrite reacting is the same as the

number of azo compounds produced by the

reaction (Sinaga et al., 2013). The response

time to reach 90% signal change was less

than 5 minutes. Calibration curves were

obtained from triplicate measurements to

ensure reproducibility

.

[B]

[A]

Figure 8. Coloring changes; [A] Addition of AgNPS/Chitosa; [B] after addition of NED

The working principle of the colorimetric

sensor is based on changing the color of the

silver nanoparticle reagent and NED from

yellow to purple. Nitrite is reacted with

H₂SO₄to provide an acidic atmosphere

because the diazotization-coupling process

can take place in acidic conditions. Because

at higher pH conditions, the diazotization-

coupling reaction does not run perfectly due

to the lack of acid [35]. Nitrite reacts with

AgNPs/chitosan-4ATP to produce diazonium

salt. Under acidic conditions, 4-ATP is

converted into the cationic conjugate acid

form in the presence of nitrite and acid, and

aromatic amines change to diazonium salts

[30]. Figure 8 shows that when given NED,

nitrite can be detected. The higher the nitrite

concentration, the more concentrated it is,

and the color of the resulting solution.

Nitrite reacts with 4-ATP−AgNPs to produce

NED-coupled diazonium salts to form

nanoparticle-modified silver chromophores,

as shown in Figure 9. Under acidic

conditions,

aminothiophenol-

the

amine

modified

groups

of

4-

are

AgNPs

converted into cationic groups. A conjugate

acid (ammonium) forms, which stabilizes the

nanoparticles by electrostatic repulsion.

Nitrites and acid minerals, primary aromatic

amines, turn into diazonium salts. Although

diazonium salts are unstable in aqueous

solutions due to their tendency to lose

diazonium groups, they are stabilized

through the formation of azo-dyes with the

addition of NED, causing a bathochromic

shift in the local SPR absorption band of

AgNPs as a result of charge transfer

interactions at the nanoparticle surface.

241

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Chempublish Journal, 9(2) 2025, 230-246

Surface

complexation

between

organic

proven to be superior to the application of

the (4-ATP+NED) binary combination without

the involvement of AgNPs due to its much

better sensitivity and linearity concerning

nitramine concentration.

molecules having active functional groups

and AgNPs is reported to play an important

role in both the stabilization and formation

of new chromophores of nanoparticles. The

proposed method (4-ATP−AgNP+NED) was

Figure 9. Reaction mechanism for NO ^-detection with Ag/Chitosan Nanoparticles + 4-ATP and NED

Validation Method

test using colorimetry, 4-ATP was first

reacted with Chitosan AgNPs at pH 2 to form

-

2-

-

a complex matrix. NO₃, PO₄, SO₄, CO₃, Cl^-

The selectivity test was carried out using

-

2-

-

NO₃, PO₄, SO₄, CO₃, Cl^-and NO₂, 2.5 µM

ions as a comparison. This selectivity test

was carried out based on colorimetry. Where

color changes are the main observation. If

the sample does not change color, it can be

said that the sample cannot detect anything

other than nitrite. In observing the selectivity

-

and NO₂, 2.5 µM ions were added with

sulfuric acid to form an acidic atmosphere.

The addition of complex matrix and acetic

acid was carried out sequentially. Then the

addition of NED is used to form a diazonium

reaction.

[A]

[B]

-

Figure 10. NO₃, PO₄, SO₄, CO₃, Cl^-and NO₂ion selectivity test (a). Before the addition of NED (b). After

adding NED

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Chempublish Journal, 9(2) 2025, 230-246

Based on the observations made, it can be

seen in Figure 10. Before adding NED, the

sample containing ions will follow the color

of the complex matrix. When NED was

Determination of linearity is determined to

determine the ability of an analytical method

to obtain results that are appropriate to the

analyte concentration in the sample. This

measurement is to obtain the equation of

the regression line from the calibration curve

created. The linearity test is expressed as a

correlation coefficient (r). The following is the

calibration curve for a standard nitrite

solution using AgNP/chitosan. Based on

statistical calculations, it can be seen in

Figure 10 that the regression equation y =

0.2488x + 0.2741 is obtained with a

regression value of R2 = 0.9882. The

requirement for the correlation coefficient

(r) value is that it must be greater than 0.99

according to SNI. The r value obtained is

0.994 so it can be said that the linearity data

is declared valid and there is a correlation

between concentration and intensity

-

added to the sample containing NO₂ions,

-

the color changed to purple, while the NO₃,

-

PO₄, SO₄, CO₃, and Cl^-ions did not change

color. Based on the selectivity test, it can be

stated that the analytical method is selective

for nitrite. The selectivity test is carried out

based on colorimetry or color observation.

Figure 11. Nitrite calibration curve

Table 5. LOD and LOQ Test Results

Concentration (µM) (x)

Absorbance (y)

0.401

(Ŷ)

(Y-Ŷ)

0.0025

-0.0159

0.0037

0.0333

-0.0221

(Y-Ŷ)²

0.000006

0.000253

0.000014

0.001109

0.000488

0.0019

0.5

1

1.5

2

0.3985

0.5229

0.6473

0.7717

0.8961

0.507

0.651

0.805

0.874

2.5

Total

Sy(Y/X)²

Sy(Y/X)

LOD

0.0001

0.0108

0.1304

LOQ

0.4345

LOD is the smallest amount of analyte in a

sample that can be detected which still

provides a significant response compared to

a blank. LOQ is an analytical parameter that

is defined as the smallest amount of analyte

in a sample that can still meet the precision

the calibration curve. The research results

show that the developed colorimetric sensor

can detect nitrite in silver nanoparticles in

the concentration range of 0.5-2.5 µM and

produces detection limits and quantitation

limits of 0.130 µM and 0.434 µM respectively

(Table 5). This value shows the amount of

analyte that can still be measured by

AgNPs/chitosan. The nitrite concentration in

and

quantitation

statistically via a linear regression line from

accuracy

criteria.

Detection

and

limits

can

be calculated

243

N. Lestari et al.

Chempublish Journal, 9(2) 2025, 230-246

the

analyte

can

be

detected

if

the

extended to Muhammad Ghazy Fernandes

for preparing and providing the coating

samples used in this study. This research

would not have been successfully completed

without the collective effort of all involved.

concentration is greater than the detection

limit, namely 0.1304 µM. If the analyte

concentration is less than 0.1304 µM then

AgNPs/chitosan cannot detect the analyte.

Author Contributions

Conclusions

All authors have read and agreed to the

Based on the research that has been carried

out, the following conclusions can be

obtained that the optimal time for the

synthesis of silver nanoparticles using

avocado leaf bioreduction is 4 h with an SPR

(Surface Plasmon Response) value of 428 nm

and an absorbance value of 2.112 with a

nanoparticle size of 129 nm. The addition of

chitosan can affect the stability of chitosan.

Chitosan has amine (-NH2) and hydroxyl (-

OH) groups so chitosan can act as a capping

agent for silver nanoparticles using avocado

leaf reductants. The limit of detection (LOD)

published

version

of

the

manuscript.

Conceptualization: RDP., NCAS., and ILT;

research design, RDP., NCAS., and ILT;

methodology: RDP., NL., ILT., and NN.

Validation: RDP., ILT., NN. All authors have

read and agreed to the published version of

the manuscript.

Conflict of Interest

The authors declare that there are no

conflicts of interest.

and

quantitation

(LOQ)

values

of

Ethical Standards

Ag/Chitosan nanoparticles as a colorimetric

nitrite detector respectively obtained LOD

values of 0.01303 µM and LOQ 0.0434 µM.

Limitations and Future Work: While PSA was

used to measure particle size, more accurate

characterization such as SEM/TEM, DLS

(hydrodynamic diameter, zeta potential),

and XRD (crystallinity) will be included in

future studies. The current sensor was

single-use; regeneration and reusability will

be addressed in ongoing work. Biosafety was

considered, and the small amount of AgNPs

used poses minimal risks when disposed

according to laboratory standards.

This article does not contain any studies

involving human or animal subjects.

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