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Jentashapir J Cell Mol Biol. 2023 June; 14(2):e136410.
Published online 2023 July 1.
https://doi.org/10.5812/jjcmb-136410.
Research Article
Green Production and Characterization of Silver Selenide
Nanoparticles Using the Cladosporium Species ssf15 Cell-free Extract
Morahem Ashengroph 1, * and Asal Barshidi 1
1Department of Biological Sciences, Faculty of Sciences, University of Kurdistan, Sanandaj, Iran
*Corresponding author: Department of BiologicalSciences, Faculty of Sciences, University of Kurdistan, Sanandaj, Iran. Email: m.ashengroph@uok.ac.ir
Received 2023 March 19; Revised 2023 May 17; Accepted 2023 May 20.
Abstract
Background: Chalcogenide nanoparticles (NPs), such as silver selenide NPs (Ag2Se NPs), are used as quantum dots in pharmaceuti-
cal formulations for the detection of cancer cells, as well as in solar cells, sensors, and electrical, optical, and magnetic instruments.
Objectives: The physical-chemical production of metal NPs is expensive and difficult and pollutes the environment due to the use
of dangerous chemicals and by-products. Hence, green chemistry-based processes must be developed as a reliable alternative. This
study investigated indigenous aquatic fungal isolates for Ag2Se NP synthesis.
Methods: Visual examination, UV-Vis spectroscopy, scanning electron microscopy (SEM), dynamic light scattering (DLS) analysis,
and energy-dispersive X-ray spectroscopy (EDX) tests confirmed the synthesis of Ag2Se NPs. Basedon the characteristics of thecolony,
the microscopic features, and the polymerase chain reaction (PCR) amplification of the ITS1-5.8S-ITS2-ITS4 areas, the selected fungal
isolate’s identity was established.
Results: The SEM analysis revealed that Cladosporium species ssf15 produced spherical Ag2Se NPs with an average diameter of 37.84
nm. The average size of NPs synthesized by the fungal isolate was determined to be 40.92 nm using the DLS analysis, and the polydis-
persity index (PDI) was determined to be 0.26. Based on the results, regular spherical Ag2Se NPs with correct dispersion distribution
were produced using 2 mM AgNO3and 1mM selenious acid (H2SeO3).
Conclusions: The results of this research have the potential to contribute to the development of a biocompatible approach and
innovative research methods for investigating the green synthesis of Ag2Se NPs using fungal isolates.
Keywords: Aquatic Fungus, Cell-free Extract, Cladosporium, Silver Selenide Nanoparticle, Spectroscopy
1. Background
The transition from microparticles to nanoparticles
(NPs) involves several noteworthy modifications, includ-
ing a dramatic increase in the surface area relative to vol-
ume and quantum phenomena (1). The increase in the
surface-to-volume ratio, which occurs gradually as the par-
ticle size decreases, causes the behavior of the atoms on
the particle’s surface to take precedence over the behav-
ior of the internal atoms. This phenomenon affects the
particle’s properties and its interaction with other mate-
rials; due to the significantly larger number of molecules
or atoms on the surface compared to those in the sam-
ple’s mass, the particles tend to accumulate (2). Silver se-
lenide (Ag2Se) is a semiconducting substance that occurs
naturally as a mineral. With an optical band gap of 1.8
- 2.1 eV, this material belongs to compounds I - VI. As a
semiconductor, Ag2Se can be used in a variety of applica-
tions, including infrared sensors, photolithography layers,
electrochemical batteries, and electrochemical potential
memory devices (3). Ag2Se NPs are chalcogenide NPs that
are employed in solar cells, sensors, and electrical, opti-
cal, and magnetic instruments, as well as in pharmaceu-
tical formulations such as quantum dots for cancer cell
detection (4). Many physicochemical processes, includ-
ing chemical reduction, electrochemistry, chemical depo-
sition, thermal evaporation, and hydrothermal reaction,
are used to create Ag2Se NPs. However, besides being ex-
pensive and requiring multiple steps, the physiochemical
synthesis of the aforementioned NPs also pollutes the en-
vironment because of the usage of harmful chemicals and
the creation of hazardous by-products. Thus, developing
a green chemistry-based method is crucial as an effective
and trustworthy substitute (5). Minerals can be synthe-
sized by microorganisms, both unicellular and multicel-
lular. Biosynthesis is a bottom-up strategy in which NPs
are formed by the reduction/oxidation of metal ions via bi-
ological substances such as enzymes, carbohydrates, and
proteins released by microorganisms. However, the incli-
Copyright © 2023, Jentashapir Journal of Cellular and Molecular Biology. This is an open-access article distributed under the terms of the Creative Commons
Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in
noncommercial usages, provided the original work is properly cited.
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Ashengroph M and Barshidi A
nation of microbes to behave differently and interact with
various metal ions differs depending on the type of organ-
ism (6). In the last 2 decades, there has been little study
on the synthesis of Ag2Se NPs using physicochemical ap-
proaches such as thermal degradation (7,8) and coprecip-
itation (9), as well as biological methods based on the uti-
lization of plant extracts (10). Fungi have distinct proper-
ties for the creation of metal NPs as compared to their bi-
ological equivalents. Fungi produce huge amounts of pro-
teins and enzymes per unit of biomass, resulting in the ex-
tracellular synthesis of large numbers of NPs. Recent stud-
ies have revealed that fungi have a high intracellular metal
absorption volume, and the produced particles are typi-
cally smaller (11).
2. Objectives
It is vital to develop methods based on green chemistry
as an efficient and trustworthy alternative to the synthesis
of NPs. It was attempted in this study to harness the po-
tential of aquatic fungal isolates for the synthesis of Ag2Se
NPs. This is the first study to report on the green synthe-
sis of Ag2Se NPs in the Cladosporium fungus. Given the nov-
elty of using Cladosporium sp. fungus for extracellular syn-
thesis of Ag2Se NPs via the cell-free extract (CFE) method,
the findings of this study could be used as a biocompati-
ble method in the field of green synthesis of Ag2Se NPs by
other fungal and bacterial strains.
3. Methods
3.1. Sampling and Enrichment Conditions
Surface water samples from Saral Divandareh, Kurdis-
tan Province, western Iran, were obtained from a depth of
15 to 30 cm. Water samples were collected in sterile con-
tainers and stored at 4°C in the laboratory until they were
used. The collected water samples (25 mL) were transferred
to sterile 45-mL falcon tubes and centrifuged at 5000g for
10 minutes. To screen fungal isolates, 500 µL of liquid
was added to potato dextrose agar (PDA) culture media
(200 g/L of potato infusion, 20 g/L of glucose, and 15 g/L
of agar) supplemented with 0.5mM stocks of sodium se-
lenite (Na2SeO3) and silver nitrate (AgNO3). Streptomycin,
at a final concentration of 35 mg/L, was used as a selective
agent to inhibit bacterial growth (12). The culture plates
were incubated for 7 to 14 days at 25°C. On the PDA culture
medium, fungal isolates were purified using the single
spore and hyphal-tipping technique (13). The tolerance pat-
tern of fungal isolates was examined by the agar dilution
technique (14). To do this, 250-mL Erlenmeyer flasks con-
taining 20 mL of PDA culture media were separately placed
into 10-cm diameter Petri dishes with particular concentra-
tions of Na2SeO3and AgNO3(1 mM, 2.5 mM, 5 mM, 5 mM,
7 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM, 20 mM, and 22.5
mM). To examine the tolerance of fungal isolates, a 5-mm
disc was placed on the PDA culture medium at the edge of
a 7- to 14-day culture of fungal isolates. The plates were ex-
amined after 2 weeks of incubation at 25°C in the dark. As a
control, we used culture media incubated with fungal iso-
lates without adding stock. All trials were conducted twice
consecutively.
3.2. Isolation of Ag2Se NPs-Producing Fungi
The CFE technique was applied to the extracellular pro-
duction of Ag2Se NPs (15). To prepare a CFE of fungal
cells, fungal isolates with high tolerance to Na2SeO3and
AgNO3were grown in potato dextrose broth (PDB) liquid
culture medium in a shaker incubator set to 150 rpm at
25°C. Following 7 to 14 days of incubation, the mycelium
was centrifuged (4000g for 30 minutes) and washed sev-
eral times with sterile deionized water to purge the culture
media. Then, under the same conditions, 10 g of the fun-
gal biomass was added to 50 mL of sterile deionized water
and incubated for 120 hours. Following this interval, the
gained fungal extract was centrifuged and passed through
0.45-µm membrane filters to separate it from the fungal
biomass. Ag2Se NPs were created using precursor salts and
a coating agent. For this purpose, 50 mL of the CFE pro-
duced in 125 mL Erlenmeyer flasks was added. The conver-
sion reaction mixture was then supplemented with silver
nitrate stock at a concentration of 2 mM and sodium selen-
ite stock at a concentration of 1 mM. Because the reaction is
light sensitive, the inoculated Erlenmeyer flasks were kept
in a shaker incubator for 48 hours at 25°C and 150 rpm.
Meanwhile, control samples containing biological extracts
but no stock salts were cultured under the same culture
conditions (10). Visual observations and UV-Vis light spec-
troscopy were used to examine the characteristics of Ag2Se
NPs produced in the conversion reaction mixture (16,17).
3.3. Ag2Se NPs Characterization
The optical properties of Ag2Se NPs, such as absorbance
and bandgap energy, were determined using UV-Vis spec-
troscopy (Specord 210, Germany). Field emission scanning
electron microscopy (FESEM; TESCAN Mira 3-LMu, Czech Re-
public) was used to determine the size and shape of the
NPs. It provides high-resolution photographs of individual
particles and allows for the study of particle size distribu-
tion, size, and shape of NPs. Dynamic light scattering (DLS,
SZ-100, HORIBA, Japan) was used to determine the hydrody-
namic diameter of NPs in solution. The elemental compo-
sition of the NPs was determined using energy-dispersive
2 Jentashapir J Cell Mol Biol. 2023; 14(2):e136410.
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Ashengroph M and Barshidi A
X-ray spectroscopy (EDS; Mira 3-LMu). To precipitate and
purify Ag2Se NPs produced by fungal extracts in the re-
action mixture, the CFE was first filtered through 0.2-µm
membrane filters, and then the samples were centrifuged
at high speed (13000g for 30 minutes at 4°C). The precip-
itate was rinsed 3 times with sterile deionized water and
96% ethanol. A freeze dryer was used to dry the samples.
The morphology, distribution size, and elemental content
of the produced Ag2Se NPs were studied using a field emis-
sion scanning electron microscope equipped with X-ray
energy diffraction spectroscopy. The images were taken us-
ing SEM and then processed using ImageJ software to build
a histogram of particle size dispersion and estimate the av-
erage size of the NPs. Dynamic light scattering was used to
detect the size of particles in liquids and suspensions rang-
ing from several nanometers to several microns. An ultra-
sonic instrument was used to disperse the powdered sam-
ples of synthetic NPs in sterile distilled water until a homo-
geneous colloid was produced. The colloidal samples were
then examined using a DLS machine to determine the av-
erage of NPs and the polydispersity index (PDI).
3.4. Isolate Characterization
The morphological examination was performed on
cultures grown on PDA at 25°C for 14 days. After 14
days, colony diameters were measured, and mean colony
growth was calculated. The color of the mycelia on the
PDA and the development pattern were also documented.
The genomic DNA of selected fungal isolates was extracted
using the phenol/chloroform method and glass beads
(18). The forward primer ITS1 (5-TCCGTAGGTGAACCTGCGG-
3) and reverse primer ITS4 (5-TCCTCCGCTTGATATG-3) re-
ported by White et al. were used for polymerase chain reac-
tion (PCR) amplification (19). The purified PCR product was
sequenced bidirectionally using an automated sequencer
(Macrogen Inc, Seoul, Korea). The BLASTN software was
used to match the sequences to identical sequences. The
genome sequence was registered in GenBank using BankIt
(a web-based application), and accession numbers were as-
signed.
4. Results
4.1. Biosynthesis of Ag2Se NPs Under Cell-free Extract Strategy
Using an enrichment cultivation technique in PDA cul-
ture media, 19 fungal isolates (named ssf1-ssf19) with dif-
ferent morphological characteristics were isolated and pu-
rified with sodium selenite and silver nitrate salts at a
final concentration of 0.5 mM. Then, the tolerance pat-
tern of fungal isolates was examined using the agar di-
lution method to get fungal isolates with high tolerance
to sodium selenite oxyanion and silver nitrate salt. Based
on the results, the tolerance of selected fungal isolates to
sodium selenite oxyanion was determined to be between 1
mM and 20 mM and between 0.5 mM and 15 mM to silver
nitrate salt. Among these, 4 fungal isolates (ssf8, ssf9, ssf13,
and ssf15) with the highest resistance to selenite oxyanion
(tolerance greater than 15 mM) and silver nitrate salt (tol-
erance greater than 10 mM) were chosen as superior fun-
gal isolates for the biosynthesis of Ag2Se NPs. Visual inspec-
tion and UV-Vis light spectroscopy were used to assess the
synthesis of Ag2Se NPs produced by the CFEs of selected
fungal isolates. Visual observations revealed that after 48
hours of treating the CFEs of 4 fungal isolates (ssf8, ssf9,
ssf13, and ssf15) with solutions of 2mM AgNO3and 1mM se-
lenious acid (H2SeO3), the color of the bioconversion reac-
tion mixture changed from colorless to light brown (Fig-
ure 1a), showing the possible synthesis of Ag2Se NPs (20). A
UV-Vis spectrometer was used to confirm the possible pro-
duction of Ag2Se NPs in the colloidal solution. Figure 1b
depicts the UV-Vis spectra obtained after treating the CFEs
of the selected fungal isolates 48 hours after the reaction,
which is consistent with the visual observations of the iso-
lates. Fungi ssf8, ssf9, ssf13, and ssf15 showed unique ab-
sorption peaks at wavelengths ranging from 359 to 378 nm,
indicating that Ag2Se NPs were present in the reaction mix-
ture. According to reliable sources, the maximum absorp-
tion peak of Ag2Se NPs is between 359 and 385 nm (16). In
the control solution (CFEs of fungal isolates not treated
with selenious acid and silver nitrate stocks), there was no
absorption peak at wavelengths between 300 and 400 nm.
4.2. Validation of the Production of Ag2Se NPs by Microscopy
and Spectroscopy Analyses
Fungal isolates ssf8, ssf9, ssf15, and ssf13 are based on
the evaluation results of visual observations and UV-Vis
spectroscopy. Since they could synthesize Ag2Se NPs, they
were chosen for SEM observations. In the studied isolates,
SEM micrograph images showed a uniform distribution of
spherical and rod-shaped Ag2Se NPs ranging in size from 37
to 182 nm (Figures 2a, 2b, 2c, and 2d). Figure 3a depicts the
SEM images and DLS analysis of Ag2Se NPs generated with
CFE from isolate ssf15. As illustrated in the image, spherical
NPs with a mean diameter of 40.92 nm and PDI of 0.26were
produced. The PDI for a homogenous colloidal sample of
NPs is between 0.01 and 0.70. The PDI for non-uniform
samples with improper dispersion is greater than 0.7. (21).
Thus, the PDI of the Ag2Se NPs produced by fungal isolate
ssf15 is appropriate. The SEM and DLS analyses of the sur-
face structure of Ag2Se NPs produced by the fungal isolate
ssf8 revealed the synthesis of spherical NPs with an average
particle size of 59.69 nm, and their PDI was found to be 0.28
based on the results of the DLS study (Figure 2b). Ag2Se NPs
Jentashapir J Cell Mol Biol. 2023; 14(2):e136410. 3
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Ashengroph M and Barshidi A
Figure 1. (a) Visible observations (color changed from colorless to light brown) and (b) spectrophotometric absorption spectra of the cell-free extract of the selected fungal
isolates (2mM silver nitrate and 1mM selenious acid) after 48 hours incubation on a shaker (150 rpm) at 25°C
synthesized by fungal isolate ssf9 revealed rod-shaped NPs
with an average diameter of 169.51 nm, and PDI was 0.46
(Figure 2c). Then, SEM was used to analyze the morphol-
ogy and size distribution range of the NPs generated by the
CFE of fungal isolate ssf13. The synthesis of rod-shaped NPs
with an average particle size of 191.66 nm and a PDI of 0.25
is presented in Figure 2d. Based on the findings, fungal iso-
late ssf15 was identified as the most suitable candidate for
further analysis due to its favorable morphology and size,
as well as its high monodispersity index. The effect of com-
bined concentrations of the aforementioned stocks on the
size and morphology of the Ag2Se NPs was explored using
the CFE strategy to improve the biological reduction of se-
lenious acid and silver nitrate stocks on Ag2Se NPs. The for-
mation of spherical Ag2Se NPs with an average size of 26.6
nm and a PDI of 0.32 was reported in a reaction mixture
containing 1mM AgNO3and 0.5mM H2SeO3(Figure 4a). In
the presence of 2mM AgNO3and 1mM H2SeO3, Ag2Se NPs
with an average size of 37.8 nm and a PDI of 0.28 were iden-
tified (Figure 4b). Based on the results, Ag2Se NPs with uni-
form spherical morphologies and correct dispersion dis-
tribution were generated in the presence of 2mM AgNO3
and 1mM H2SeO3.Figure 3 depicts the energy-dispersive X-
ray spectroscopy (EDX) analysis of Ag2Se NPs generated by
fungal isolate ssf15 to evaluate constituent elements and
product purity. The significant absorption peaks were as-
sociated with silver (70.37%) and selenium (29.63%), respec-
tively, showing the creation of Ag2Se NPs as Ag2Se. The
gained percentages, as observed, were compatible with the
stoichiometric values of Ag2Se. The existence of minor oxy-
gen and carbon absorption peaks is most likely due to trace
levels of organic compounds in the fungal extract or coat-
ing agents involved in the stability of produced NPs.
The effect of incubation time (24, 48, 72, 96, and 120
hours) was examined to improve the biological efficiency
of Ag2Se NPs under the CFE of fungal isolate ssf15 in the re-
action mixture containing 2 mM AgNO3and 1mM H2SeO3
(Figure 5). The synthesis of Ag2Se NPs began after 24 hours
of reaction and reached its peak efficiency after 96 hours.
A steady decline in the absorbance spectrum of Ag2Se NPs
was detected between 96 and 120 hours. The effect of in-
cubation time (24, 48, 72, 96, and 120 hours) was examined
4 Jentashapir J Cell Mol Biol. 2023; 14(2):e136410.
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Ashengroph M and Barshidi A
Figure 2. Field emission scanning electron microscopy images and dynamic light scattering analysis of extracellular Ag2Se NPs produced under the strateg y of cell-free extract
isolates ssf15 (a), ssf8 (b), ssf9 (c), and ssf13 (d)
Figure 3. The energy-dispersive X-ray spectroscopy spectrum of Ag2Se NPs generated in a biotransformation reaction mixture, including 2 mM AgNO3and 1mM H2SeO3by the
cell-free extract of fungal isolate ssf15
Jentashapir J Cell Mol Biol. 2023; 14(2):e136410. 5
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Ashengroph M and Barshidi A
Figure 4. Field emission scanning electron microscopy images and a size distribution histogram of extracellular Ag2Se NPs synthesized using cell-free extract ssf15 in the
presence of various amounts of silver nitrate and selenious acid precursor salts
to improve the biological efficiency of Ag2Se NPs under the
CFE of fungal isolate ssf15 in the reaction mixture contain-
ing 2 mM AgNO3and 1 mM H2SeO3(Figure 5). The synthesis
of Ag2Se NPs began after 24 hours of reaction and reached
its peak efficiency after 96 hours. A steady decline in the ab-
sorbance spectrum of Ag2Se NPs was detected between 96
and 120 hours.
4.3. Characterization of Isolate ssf15
Based on the characteristics of the colony and micro-
scopic features, the chosen fungal isolate was identified
morphologically. After 14 days, the fungal colony on PDA
culture media at 25°C has grown by 3.8 to 4.2 cm, which is
visible as a black-green color. Conidia with dark pigment
is produced, along with one to several cells that are orga-
nized into simple or branched chains. To precisely iden-
tify the chosen fungal isolate, the ITS1-5.8S-ITS2 sections
were subsequently amplified by PCR using the universal
ITS1 and ITS4 primers. Cladosporium species strain ssf15
was identified as isolate ssf15 (GenBank accession number
OP242915) by BLAST analysis, which revealed 99% similarity
with strains from the genus Cladosporium.
5. Discussion
Recent studies have revealed that the synthesis of NPs
through green chemistry-based methods using microbes
and plants is a safe, economical, and environmentally-
friendly option Plants and microbes have long shown the
ability to absorb and accumulate inorganic metal ions
from their surroundings. Because of this characteristic,
many microorganisms have effective biofactories capable
of considerably reducing environmental pollution and re-
covering metals from industrial waste. To date, microbes’
capability to interact with, remove, and accumulate metal
elements from their surroundings has been exploited in a
variety of biotechnology applications, such as bioremedi-
ation and NP production (22). Fungi are currently used for
the biosynthesis of nanoparticles because of their ability
to tolerate toxins, vast storage capacity, effective biomass
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Ashengroph M and Barshidi A
Figure 5. The effect of incubation time on the creation of Ag2SeNPs in a reaction involving the cell-free extract of fungal isolate ssf15 treated with 2 mM AgNO3and 1mM H2SeO3
as measured by spectrophotometric absorption spectra
management, ease of manipulation and large-scale ex-
ploitation, and their capacity to produce a variety of com-
pounds that support preservation and homeostasis. They
may therefore thrive in harsh environments with poor nu-
trition and toxic substances. (23). The major objectives of
this study were to isolate and identify aquatic fungal iso-
lates resistant to sodium selenite oxyanion and silver ni-
trate, as well as to produce Ag2Se NPs. In this context, the
production of spherical Ag2Se NPs with an average size of
40.92 nm and a PDI of 0.26 was reported for the first time
in fungal isolate ssf15 belonging to the genus Cladosporium
under CFE conditions. The chemicals and substances ex-
creted by the fungi outside the cell are used as an effec-
tive factor in the production of the desired NPs. Because of
the release of reducing compounds from the microbial cell
and their presence in the reaction mixture, this method
enhances the production of NPs (15). Although several Cla-
dosporium species have been described in previous studies
because of the synthesis of Ag NPs (24), Au NPs (25), and
ZnO NPs (26), this is the first study to describe the produc-
tion of Ag2Se NPs in the genus Cladosporium. Throughout
the last 2 decades, little research has been conducted on
the synthesis of Ag2Se NPs, which has primarily relied on
physicochemical and biological techniques based on plant
extracts (10). TackáNg et al. were successful in synthesiz-
ing Ag2Se NPs via thermal degradation. In this approach,
the precursor molecule (PPh3)3Ag2(SeC(50)Ph) was ther-
mally decomposed in a solution containing trioctylphos-
phine and hexadecylamine for 2 hours at 180°C, and Ag2Se
NPs were produced (7). Khanna et al. synthesized Ag2Se
NPs by coprecipitation at 130°C for 5 hours using cyclohep-
tane 1, 2, and 3-selenadiazole as a source of selenium and
silver nitrate as a silver source in the presence of dimethyl-
formamide as a solvent (9). Under nitrogen gas, Ag2Se NPs
were produced using selenium powder as a source of sele-
nium, silver nitrate as a source of silver, and trioctylphos-
phine, 1-octadecylamine, oleic acid, and 1-octadecine as sur-
factants. The disadvantage of this process is that it requires
expensive materials, an enormous volume of raw materi-
als, and a long reaction time (8). Mirzaei et al. explored
the biological synthesis of Ag2Se nanochalcogens using an
aqueous extract of the plant Melilotus officinalis (yellow al-
falfa) with biological activity. The antibacterial, antibiotic,
antioxidant, and cytotoxic properties of biosynthesized
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Ashengroph M and Barshidi A
Ag2Se NPs were promising (10). In fact, a low-cost, non-
toxic, and environmentally friendly synthesis technique
based on fungal extracts for the synthesis of Ag2Se NPs
with acceptable size and high monodispersity index was
reported in this study.
5.1. Conclusions
Due to the tremendous diversity and environmental
adaptability of aquatic fungal isolates, they can be ex-
ploited as potential biological nanofactories for the extra-
cellular synthesis of different metal NPs. Cladosporium is
one of the most common types of fungi, and its species
have been used in the production of antibiotics, bioreme-
diation, biofuels, and biopesticides. This species has been
used in the nanobiotechnology field for the biosynthesis
of metal NPs, such as silver, gold, and zinc oxide. The pro-
duction of Ag2Se NPs in the genus Cladosporium was first
documented in this study. This study’s approach is based
on using an extract free of fungal cells and carefully select-
ing a solvent medium, environmental regenerating agent,
and non-toxic materials to maintain the stability of NPs in
the reaction medium. By following the principles of green
chemistry, our method reduces environmental concerns,
making it a promising option for numerous applications,
particularly in the field of biomedicine.
Footnotes
Authors’ Contribution: Morahem Ashengroph: Concept
development, project management, finance acquisition,
formal analysis, and supervision. Asal Barshidi: Data col-
lection, visualization, and writing (first draft). All authors
reviewed and approved the final version of the manuscript
for publication.
Conflict of Interests: Regarding the research, author-
ship, and/or publication of this paper, the author(s) state
that they have no potential conflicts of interest.
Data Reproducibility: The dataset presented in the study
is available on request from the corresponding author dur-
ing submission or after publication.
Funding/Support: The authors would like to thank the
Research Vice Chancellor of the University of Kurdistan for
their financial support of this study.
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