Chemosphere 306 (2022) 135389

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Synthesis of two different zinc oxide nanoflowers and comparison of

antioxidant and photocatalytic activity
Ozan Eskikaya a, Sadin Ozdemir b, Gülsah Tollu c, Nadir Dizge a, **, Rameshprabu Ramaraj d,
Arthi Manivannan e, Deepanraj Balakrishnan f, *
a
Department of Environmental Engineering, Mersin University, Mersin, 33343, Turkey
b
Food Processing Programme, Technical Science Vocational School, Mersin University, Mersin, 33343, Turkey
c
Department of Laboratory and Veterinary Health, Technical Science Vocational School, Mersin University, Mersin, 33343, Turkey
d
School of Renewable Energy, Maejo University, Chiang Mai, 50290, Thailand
e
Saveetha School of Engineering, Saveetha Institute of Medical and Technical Sciences, Chennai, 602105, India
f
College of Engineering, Prince Mohammad Bin Fahd University, Al Khobar, 31952, Saudi Arabia

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• The antibacterial activity and the pho­

tocatalytic effect of ZnO nanoflowers
was compared.
• The hydrothermal and precipitation
methods were used to prepare ZnO
nanoflowers.
• Photocatalytic properties of the synthe­
sized ZnO nanoflowers were also
investigated.
• The color was completely removed for
BR18 and RR180 dyes for 90 min using
1.5 g/L photocatalyst amount using
ZnO–NF1.
• 90.00% dye removal efficiency was
detected for 90 min using 2 g/L
ZnO–NF2 for RR180 dye.

A R T I C L E I N F O A B S T R A C T

Handling Editor: Derek Muir Nanoflowers are a newly developed class of nanoparticles that show flower-like structures and attract much
attention due to their simple preparation methods, high stability, and increased efficiency. The aim of the study is
Keywords: to investigate a strong alternative to reduce the severity of infection and increase the treatment of wastewater by
Zinc oxide nanoflowers exhibiting biofilm inhibition in medical and environmental applications of the ZnO-NFs with two different
Photocatalysis
shapes. ZnO-NFs were synthesized by two different processes hydrothermal method (named ZnO–NF1) and the
Antioxidant
precipitation method (named ZnO–NF2). ZnO-NFs produced by two different synthesis methods were compared
Antimicrobial
Biofilm inhibition for the photocatalytic and antioxidant efficiency. The effects of Reactive Red 180 (RR180) and Basic Red 18
DNA cleavage Ability (BR18) dyes concentration, photocatalyst amount, and reaction time were investigated on dye removal efficiency
Microbial cell viability for photocatalytic experiments. The color was completely removed for 25 mg/L BR18 and RR180 dyes for 75 min
Photodynamic activity and 90 min, respectively, using 1.5 g/L photocatalyst amount using ZnO–NF1. However, 59.18% dye removal
efficiency was obtained for 90 min by using a 1.5 g/L ZnO–NF2 photocatalyst for 25 mg/L BR18 dye removal,

* Corresponding author.
** Corresponding author.
E-mail addresses: ndizge@mersin.edu.tr (N. Dizge), dbalakrishnan@pmu.edu.sa (D. Balakrishnan).

https://doi.org/10.1016/j.chemosphere.2022.135389
Received 4 February 2022; Received in revised form 30 May 2022; Accepted 14 June 2022
Available online 16 June 2022
0045-6535/© 2022 Elsevier Ltd. All rights reserved.
O. Eskikaya et al.
while a dye removal efficiency of 90.00% was detected for 90 min using 2 g/L ZnO–NF2 for 25 mg/L RR180 dye.
Then, comparison of general properties such as antibacterial, antibiofilm, microbial cell viability, DNA frag­
mentation, antioxidant activities, and antimicrobial photodynamic therapy of ZnO-NFs were investigated. The
antioxidant activity of ZnO–NF2 was found to be higher than ZnO–NF1 at each concentration (82.32% and
87.18% for ZnO–NF1 and ZnO–NF2, respectively, at 200 mg/mL).
1. Introduction
Nanotechnology is defined as the science that enables nanoscale
materials with different properties according to their size and structure
(Luo et al., 2020). Recent developments in the field of nanotechnology
have made good progress on both economic growth and public interest.
Such remarkable developments in nanotechnology are promising addi­
tional studies for the development of advanced nanotechnology and its
applications in different fields (Talebian et al., 2021). Nanotechnology is
used in wide range of sectors including medicine and therapeutic health
field (Oroojalian et al., 2020), textile industry (Krifa and Prichard,
2020), energy (Serrano et al., 2009), food safety (Sahani and Sharma,
2021), environmental science (Cheriyamundath and Vavilala, 2021),
information technology (Li et al., 2009), technological applications (Ali
et al., 2020), and transportation (Shafique and Luo, 2019).
The application areas of nanotechnology are very wide and nano­
scale materials such as nanoparticles, nanosensors, and nanotubes are
synthesized for every purpose. Nanomaterials have features such as easy
functionality, high surface area, and small size. NPs which have an
important place in nanoscale materials, are generally obtained from
carbon, graphene, polymer, and metallic materials (Arslan et al., 2022;
Kirtane et al., 2021). Nanoparticles (NPs), which are frequently used in
the field of nanotechnology, have proven themselves in many areas. NPs
was used to fabricate electrochemical biosensor. For example, a novel
DNA-based electrochemical biosensor was fabricated for sensing of
idarubicin. The results showed that the fabricated biosensor performed
well in determining idarubicin in concentrations ranging from 1.0 nM to
65 μM, with a detection limit of 0.8 nM (Karimi-Maleh et al., 2002b,
2022a). NPs are also used for synthesis of nanocomposite as an adsor­
bent. For example, chitosan/Al2O3/Fe3O4 nanocomposite was synthe­
sized for removal of Cd(II), Cu(II) and Zn(II) metal ions from aqueous
solution (Karimi et al., 2022). The maximum adsorption efficiency was
achieved at pH of 5.3 with 99.98%, 93.69%, and 83.81% maximum
adsorption efficiency, respectively, for the removal of Cu, Cd and Zn
ions. In recent years, the use of both natural and synthetic biodegradable
polymers and their nano-composites can be seen as promising adsor­
bents for the removal of endocrine-disrupting chemicals (EDCs) from
wastewater (Sharabati et al., 2021). A high-performance monolithic
adsorbent was developed for methyl orange (MO) adsorption (Karaman
et al., 2022). Jain et al. (2021) emphasized that nanoparticles provide
advantages in wastewater treatment due to their excellent properties.
Shweta et al. (Shweta Sood et al., 2021) obtained efficient results with
nanoparticles in agriculture application by using less pesticides due to
the high surface area-to-volume ratio of nanoparticles and their high
reactivity. As a result of the study, they emphasized the importance of
using nanotechnology in traditional vegetable production.
Among different nanoparticles species, nanoflower is gaining much
attention because of its simple method of synthesis from organic, inor­
ganic materials and showing structural similarity to plant flowers.
Nanoflowers (NFs) are of interest besides it being an innovative species
and distinctive flower shape that increased their active area. NFs are
used in several applications such as biosensor, controlled drugs delivery,
enzyme purification, dye and heavy metal removal from water, and
catalysis (Shende et al., 2018; Gonca et al., 2022). NFs, an innovative
species in the nanoparticle class, have recently been applied especially
in the field of health and wastewater treatment (Gao et al., 2021;
Yabalak et al., 2022). Their flower-like shapes increase the surface
area/volume ratio, thus increasing the active area even more (Aggarwal
2
Chemosphere 306 (2022) 135389
et al., 2017; Kumar et al., 2021). The structure of heavy metal-based
nanoflowers increases the surface area and light absorption, thus
increasing the degradation efficiency (Fadillah et al., 2021). For
example, the zinc oxide nanoflowers (ZnO-NFs) have a higher number of
adsorption sites which increase adsorption efficiency. Zinc oxide (ZnO)
nanomaterials have a tremendous potential in the biomedical field,
ranging from anticancer therapeutics to antibacterial agents, and to
tissue engineering scaffolds (Racca et al., 2018). ZnO is a semiconductor
compound with wide band-range dielectric properties. It has also a high
electron mobility compared to other metals. It provides photocurrent
effectively and can transfer electrons quickly (Wang et al., 2021a; Nie
et al., 2021). The structure of ZnO-NFs has recently attracted interest
because of its excellent performance in many areas. It is preferred for its
physicochemical properties thanks to its flower-like morphology (Das
et al., 2021). Zou et al. (2022) synthesized ZnO-NFs material with
different ratios of cetyltrimethyl ammonium bromide using hydrother­
mal method and nano-sized ZnO-NFs were tested for Rhodamine B dye
removal using the photocatalytic method. Vinayagam et al. (2021)
produced photocatalytic ZnO-NFs using peltophorum pterocarpum pod
extract as a green synthesis method. The photocatalytic efficiency of the
ZnO-NFs was examined for the removal of methylene blue (MB) dye
under sunlight and 88.39% of MB dye was photodegraded within 180
min. Adhikari et al. (2021) investigated the antibacterial properties of
non-toxic ZnO nanomaterial as a membrane filter for the face mask. The
purpose of the investigation was to eliminate the potential spread threat
of COVID-19 disease with a mask produced using ZnO-NFs nano­
material. In their study, Pseudomonas aeruginosa with a similar corona­
virus spike protein was used and ZnO-NFs provided ~79% lower
bacterial growth in the colony number compared to control group.
The shape of nanoparticles is as important as their functionality. The
purpose of this study is to investigate and compare the antimicrobial
ability as well as the photocatalytic effect of ZnO-NFs. Two different
methods were used to obtain different shape of nanoparticles. NFs ob­
tained by the hydrothermal method was named as ZnO–NF1 and NFs
produced by precipitation method was named as ZnO–NF2.
2. Material and methods
2.1. Materials
Zinc acetate dihydrate (Zn(CH3COO)2.2H2O, >%98), sodium hy­
droxide (NaOH, >%99), ethanol (C2H6O, >%99), acetic acid (C2H4O2,
>%98) were bought from Sigma-Aldrich. Distilled water (DI) was ob­
tained using the two-stage Millipore Direct-Q3.
2.2. Synthesis of nanoflowers
A schematic diagram showing the preparation of ZnO-NFs for the
two different methods is shown in Fig. 1. 2.19 g of Zn(CH3COO)2 2H2O
(0.1 M) was thoroughly dissolved in 100 mL of distilled water (Kaur
et al., 2020). 1.0 M NaOH solution prepared with 50 mL of distilled
water was added dropwise into the prepared solution and mixed to
obtain ZnO–NF1 using hydrothermal method (Fig. 1A). The mixture was
allowed to stir for 1 h until a white precipitate which indicated the
presence of ZnO was obtained. After the mixture was placed in a water
bath at 65 ◦ C for 6 h, it was left to cool at room temperature in the dark.
The obtained powder was washed thoroughly with ethanol and distilled
water. It was left to dry overnight at 70 ◦ C (Ya et al., 2017).
O. Eskikaya et al.
Fig. 1. A schematic diagram revealing the preparation of ZnO-NFs (A) Hy­
drothermal method for ZnO–NF1 preparation and (B) Precipitation method for
ZnO–NF2 preparation.
The precipitation method was performed to synthesize of ZnO–NF2
(Fig. 1B). First, 0.5 g of NaOH and 0.5 mL of acetic acid were added into
50 mL of distilled water. 0.47 g of Zn(CH3COO)2 chemical was mixed
thoroughly until dissolved in 50 mL of distilled water. The zinc-
containing solution was slowly added to the other solution prepared
beforehand. The resulting mixture was stirred at 90 ◦ C for 15 min.
Finally, the mixture was centrifuged at 1200 rpm for 15 min. The pre­
pared powder was washed thoroughly with ethanol and distilled water.
The powder was left to dry at 70 ◦ C (Zhang et al., 2020).
2.3. Characterization of nanoflowers
Scanning Electron Microscopy (SEM, Zeiss Supra 55, Germany) with
Cu-Kα radiation were used for the morphology of nanoflowers. X-ray
Diffraction Spectroscopy (XRD, Bruker, D8 Venture) with 2θ scan car­
ried out from 10◦ to 90◦ was used to determine the chemical structure of
the nanoflowers and phase purity. Pore volume and specific surface area
were determined using the Brunauer–Emmett and Teller (BET) appa­
ratus (Mi-croActive for TriStar II Plus 2.00).
2.4. Photocatalytic activity
Zinc oxide (ZnO), one of the metallic nanoparticles with proven
effectiveness, is known to produce hydroxyl radical (OH•) which is a
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Chemosphere 306 (2022) 135389
strong oxidizer in photocatalytic systems (Rupa et al., 2019). In this
study, ZnO-NFs used as a catalyst were tested to remove two industrial
dyes (BR18 and RR180) from aqueous solution using photocatalytic
process. Reactive Red 180 (RR180) and Basic Red 18 (BR18) containing
azo group (-N– – N-) dyes are frequently used in textile industry and
printing ink production. In order to determine the effectiveness of the
prepared catalysts in the photocatalytic system, the effect of some pa­
rameters such as catalyst amount, reaction time, and dye concentration
were studied. A column-shaped Pyrex photoreactor (500 mL) and six
absorbing daylight lamps (UVA, Philips TL8W Actinic BL) were used
with an aluminum-coated tube for uniform reflection. An air-pump was
used to supply the ambient air (150 mL/min) from the bottom of the
reactor to the system to obtain homogenous mixing of the catalysts. All
samples were centrifuged at 6000 rpm for 5 min before color measure­
ments of the samples. Maximum absorbance was measured by using a
UV–VIS spectrophotometer (HACH-DR3900) to calculate color removal
efficiency. All experiments were performed in duplicate. The color
removal efficiency was measured using Eq. (1):
(C0 − Ce )
Color Removal Efficiency(%) : x100 (1)
C0
where C0 was the initial dye concentration and Ce was the dye con­
centration after defined reaction time t (min).
2.5. DPPH activity
The free radical scavenging activity (RSA) of ZnO–NF1 and ZnO–NF2
were measured using the DPPH method in the literature with minor
differences (Shabaaz Begum et al., 2020). Samples of various concen­
trations were prepared. They were then mixed with DPPH solution for
30 min incubation at 20–24 ◦ C in a dark environment. The reduction of
DPPH was measured by taking the absorbance of the control and test
samples at 517 nm. Ascorbic acid and Trolox were used as positive
controls and each measurement was made in triplicate. The RSA (%) was
calculated using the following Eq. (2):
( )
Abs(control) − Abs(sample)
Capacity (%) = × 100 (2)
Abs(control)
2.6. DNA cleavage ability
The DNA cleavage ability of ZnO–NF1 and ZnO–NF2 on plasmid DNA
was measured (Jawoor et al., 2018). E. coli plasmid DNA was used to
investigate the effect of newly synthesized ZnO–NF1 and ZnO–NF2.
pBR322 DNA was treated with ZnO–NF1 and ZnO–NF2 (100, 250, and
500 μg/mL) and then ZnO–NF1 and ZnO–NF2 were incubated with DNA
molecule in the dark at 37.5 ◦ C for 60 min. The mixture was loaded onto
1% agarose gel and electrophoresed for 1 h at 120 V in 1XTAE buffer.
After electrophoresis DNA bands were viewed under UV trans
illuminator.
2.7. Antimicrobial activity
The antimicrobial activity of ZnO–NF1 and ZnO–NF2 was investi­
gated against different microorganisms. These different microorganisms
(Pseudomonas aeruginosa (ATCC 9027), Legionella pneumophila subsp.
pneumophila (ATCC 33152), Escherichia coli (ATCC 10536), Enterococcus
faecalis (ATCC 29212), Enterococcus hirae (ATCC 10541) and Staphylo­
coccus aureus) were examined for antibacterial activities (Elbeshehy
et al., 2015; Vasantharaj et al., 2017). Also, the antifungal effect was
tested against Candida tropicalis (ATCC 750) and Candida parapisilosis
(ATCC 22019). Minimum inhibitory concentrations (MICs) of ZnO–NF1
and ZnO–NF2 were appreciated by the standard broth microdilution
method. The microorganisms were cultured by inoculation in Nutrient
Broth (NB) for 24 h at 37 ◦ C and shaking. The microorganisms were
diluted and inoculated into a medium containing different
O. Eskikaya et al.
concentrations of ZnO–NF1 and ZnO–NF2. Antimicrobial activity was
evaluated with MIC values, defined as the lowest concentration that
inhibits microbial growth at the end of 24 h.
2.8. Bacterial cell viability inhibition test and antimicrobial photodynamic
therapy
In this study, E. coli (ATCC 25922) was used as a model microor­
ganism for bacterial cell viability inhibition test and antimicrobial
photodynamic therapy. E. coli was inoculated into NB medium and
incubated for 24 h at 37 ◦ C. Next to incubation, the bacterial cell was
centrifuged and the bacterial pellet was washed with sterile saline so­
lution to remove the residue of fermentation medium. By adding 10 mL
of NaCl, a microbial solution was prepared to use in both cell viability
inhibition and antimicrobial photodynamic therapy studies. This mi­
crobial suspension was treated with different concentrations (125, 250,
and 500 mg/L) of ZnO–NF1 and ZnO–NF2 at 37 ◦ C for 60 min for mi­
crobial cell viability assay. After 60 min, it was diluted in different ratios
and inoculated into the medium. The same application was also studied
with the control group that didn’t contain ZnO–NF1 and ZnO–NF2. In
addition to these, the method in the microbial cell viability study was
applied after the compounds were exposed to LED light irradiation for
Fig. 2. Formation of ZnONFs (A) Hydrothermal method to prepare ZnO-NP1 and (B) Precipitation method to prepare ZnO–NF2.
4
Chemosphere 306 (2022) 135389
20 min in the antimicrobial photodynamic therapy study. After that
incubation duration, the colonies were counted and the cell viability
inhibition calculated using with Eq. (3)
[( )/ ]
Cell viability inhibition (%) = Acontrol − Asample Acontrol x 100 (3)
2.9. Biofilm inhibition
ZnO–NF1 and ZnO–NF2’s ability to inhibit biofilm production was
evaluated using S. aureus and P. aeruginosa as test microorganisms.
Different concentrations of ZnO–NF1 and ZnO–NF2 (125, 250, and 500
mg/L) was incubated with test microorganisms in a 24 well plate at
37 ◦ C for 3 days. Then, the well plates filtered by gentle inversion and
the plates were washed twice with distilled water. Plates then dried at
80 ◦ C for 20 min. Crystal violet after drying (CV) was added to the stain
biofilms for 60 min. The CV was then removed and the plates slowly
washed away. Later ethanol was added and it was waited for 15 min for
the absorbed CV to be recovered. The microbial fermentation media,
which was not contained ZnO–NF1 and ZnO–NF2, used as controls for
both microorganisms and biofilm inhibition was calculated by spectro­
photometer at 595 nm as follows: Eq. (4):
O. Eskikaya et al. Chemosphere 306 (2022) 135389

( )
Abs(control) − Abs(sample)
Biofilm Inhibition (%) = × 100 (4)
Abs(control)
3. Results and discussion
3.1. Characterization of ZnO-NFs
Colorless sodium hydroxide was mixed with the zinc acetate solution
to obtain ZnO–NF1 using hydrothermal method (Fig. 2A). This domi­
nates to the formation of a colloidal ZnO-NFs suspension upon heating.
The subsequent addition of NaOH leads to a white precipitate in due
course. The same observation was seen to obtain ZnO–NF2 using pre­
cipitation method (Fig. 2B). Under the basic environment, metal hy­
droxide forms zinc oxide nanoparticles while heating.
In this study, SEM images were used to determine the morphology of
the synthesized flower-shaped nanoparticles (Fig. 3). It was clearly seen
that flower-like structures were formed in both of ZnO–NF1 and
ZnO–NF2. Some irregular particles were found on the surface of two
ZnO-NFs catalysts and the flowers showed densely packed petals. It was
noticed that the structure obtained from SEM images reminded to the
Mullein Grass (Verbascum Thapsus) plant for both nanoflower structures
of ZnO–NF1 (Fig. 3A and B) and ZnO–NF2 (Fig. 3C and D).
All the detected peaks confirmed the ZnO cubic structure from the
XRD analyses (Fig. 4). The XRD peaks of ZnO–NF1 and ZnO–NF2 are
dominated by ZnO and suitable with h,k,l plane. The XRD peaks of the
ZnO–NF1 material are given in detail in Fig. 4A The crystallographic
peaks observed at 2θ = 33.6◦ , 38.9◦ , 56.3◦ , 67.2◦ , 70.6◦ , and 83.7◦
corresponded to (111), (002), (022), (113), (222), and (004) directions,
respectively. The XRD results of ZnO–NF2 are shown in Fig. 4B, there are
diffraction peaks at 2θ = 36.4◦ , 42.3◦ , 61.4◦ , 73.5◦ , and 77.4◦ are
attributing to the growth along the (111), (002), (022), (113), and (222)
directions, respectively (Raj et al., 2022; Zaimbashi et al., 2021).
It is well-known that ZnO-NFs have more specific surface area as
compared to other morphological forms. BET method was used to
determine the specific surface area and pore volume. ZnO–NF1 and
ZnO–NF2 showed a specific surface area of 15.65 and 13.89 m2/g. It was
reported that commercial ZnO nanoparticles showed specific surface
area of 3.23 m2/g (Chakrabarti and Dutta, 2004). Moreover, the pore
volumes of ZnO–NF1 and ZnO–NF2 were 0.0659 and 0.0585 cm3/g
which confirmed the mesoporous structure of pores (Vinayagam et al.,
2021). Therefore, the high surface area as well as mesoporous structure
of ZnONFs make them vital representatives for photocatalytic and
antioxidant applications.
Based upon the SEM analysis, the proposed growth mechanism of
ZnO nanoflowers can be offered as depicted in Fig. 5. A similar diagram
was offered by Singh et al. (2022) for hydrothermal synthesis of MoS2
nanoflowers. Zn+2 and OH− precursors were mixed and formed a ho­
mogenous solution. In the hydrothermal process, initially, Zn(OH)2 were
formed and after that the formed Zn(OH)2 is dissolved to considerable
extent in water to form Zn(OH)−2 4 ions. When the concentrations of Zn
(OH)−2 4 ions reached the saturated concentration, ZnO nanoflowers are
occurred. The relevant chemical reactions can be written as follows
(Kaur et al., 2020):
Zn+2 + 2OH − → Zn(OH)2 ↓
Zn(OH)2 + 2OH − → Zn(OH)−2 4
Zn(OH)−2 4 → ZnO + H2 O + 2OH −
3.2. Photocatalytic experiments
Photocatalytic degradation of dye-containing pollutants using metal
oxide nanomaterials is the process of absorbing photons from the sun or
UV–Vis source by the photocatalyst and then the bonds between dye
molecules in wastewater break with radicals produced by nanoparticles.
In this study, the photocatalytic degradation performances of BR18 and
RR180 dyes using two different nanoflowers were investigated by
advanced oxidation process contained photo-oxidation reactions under
UV-A light. The effects of catalyst amount, reaction time, and dye

Fig. 3. SEM images of (A–B) ZnO–NF1 and (C–D) ZnO–NF2.

5
O. Eskikaya et al. Chemosphere 306 (2022) 135389

Fig. 4. XRD patterns of (A) ZnO–NF1 and (B) ZnO–NF2.

Fig. 5. Schematic diagram for proposed growth mechanism of ZnO nanoflowers.

Fig. 6. The effect of photocatalyst amount on photocatalytic color removal efficiency using ZnO-based nanoflowers (A) BR18 dye removal efficiency for ZnO–NF1,
(B) RR180 dye removal efficiency for ZnO–NF1, (C) BR18 dye removal efficiency for ZnO–NF2, (D) RR180 dye removal efficiency for ZnO–NF2. (For interpretation
of the references to color in this figure legend, the reader is referred to the Web version of this article.)

6
O. Eskikaya et al.
concentration were investigated in order to determine the optimum
conditions for the efficient use of two types of nanoflowers as the
photocatalyst.
3.2.1. Effect of ZnO-NFs amount on photocatalytic treatment
The optimum amount of ZnO-NFs used as the photocatalyst was
determined at 25 mg/L initial dye concentration of both BR18 and
RR180 dyes. 100 mL of dye-containing water was used during the all
experiments at original pH of the solutions. All samples were taken from
the reactor every 15 min for 90 min during the experiments under UV
light. After the photocatalytic treatment, color measurements were done
using the spectrophotometer for BR18 and RR180 dyes at wavelengths
of 484 nm and 542 nm, respectively. The amount of photocatalysts were
chosen as 1.0, 1.5, and 2.0 g/L. The effect of ZnO–NF1 and ZnO–NF2
photocatalysts amount on dyes removal are given in Fig. 6. The results
belong to BR18 dye removal efficiency using different catalyst amounts
of ZnO–NF1 are given in Fig. 6A. Obviously, the concentration of BR18
dye in aqueous solution gradually decreased versus time. Approximately
60% removal efficiency was determined for all catalyst amounts in the
first 30 min. The photocatalytic color removal efficiency was obtained as
100% at 90 min and 75 min of reaction time for 1.5 g/L and 2 g/L,
respectively. A trace amount of dye (97.40% dye removal efficiency)
remained at 90 min for 1.0 g/L. It was observed that there was accept­
able difference when the removal efficiencies of 1.0 g/L and 1.5 g/L for
75 min were compared in the dose studies. Therefore, the optimum
amount for ZnO–NF1 photocatalyst was determined as 1.5 g/L for BR18
dye removal.
The results belong to RR180 dye removal efficiency using different
catalyst amounts of ZnO–NF1 are given in Fig. 6B. RR180 dye removal
efficiencies were determined as 82.41%, 99.60% and 100% for 1.0 g/L,
Fig. 7. The effect of dyes concentration on photocatalytic color removal efficiency using ZnO-based nanoflowers (A) BR18 dye removal efficiency for ZnO–NF1, (B)
RR180 dye removal efficiency for ZnO–NF1, (C) BR18 dye removal efficiency for ZnO–NF2, (D) RR180 dye removal efficiency for ZnO–NF2. (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of this article.)
7
Chemosphere 306 (2022) 135389
1.5 g/L, and 2.0 g/L for 90 min. Detectable color removal was observed
for RR180 dye with increasing of irradiation time in the presence of
semiconductor photocatalyst powder. 1.5 g/L was also determined as
the optimum amount for the removal of RR180 dye using ZnO–NF1.
The color removal efficiency of BR18 dye using ZnO–NF2 photo­
catalyst are given in Fig. 6C when different catalyst amounts are used. In
the first 30 min, around 45% removal efficiency was determined for all
catalyst amounts. Even after 90 min, a complete color removal efficiency
could not be achieved. It was observed that color removal efficiency of
BR18 dye was 63.04% for the amount of 2.0 g/L of ZnO–NF2 photo­
catalyst. The reason can be explained as the adsorbing of dye molecules
on the catalyst surface eliminate the active sites by dye molecules and it
reduces the formation of hydroxyl radicals.
The results of the removal efficiency of RR180 dye by photocatalytic
treatment are given in Fig. 6D using ZnO–NF2. The removal efficiency of
RR180 dye increased with the increasing amount of photocatalyst.
Considering the color removal efficiency of RR180 dye, the highest ef­
ficiency was obtained as 90.00% at the end of 90 min for ZnO–NF2.
Increasing the amount of catalyst increases the active sites on the
catalyst surface and increases the photocatalytic removal efficiency. The
acceptable photocatalyst amount of ZnO–NF2 was determined as 2 g/L
for RR180 dye. Consequently, it can be observed there is a positive
correlation between amount of added ZnO-NFs and both dyes removal
efficiency was higher with ZnO–NF1 performing better than ZnO–NF2.
However, ZnO–NF2 supplied better performance for RR180 dye removal
efficiency compared to BR18 dye.
3.2.2. Effect of dyes concentration on photocatalytic treatment
One of the most significant factors affecting the dye removal effi­
ciency in photocatalytic processes is the concentration of pollutants. The
O. Eskikaya et al.
experiments were carried out under UVA light by adding the pre-
determined optimum amount of photocatalyst into a 100 mL sample
with dyes concentration of 10 mg/L, 25 mg/L, and 50 mg/L (Fig. 7). A
complete dye removal efficiency was obtained for 10 mg/L and 25 mg/L
BR18 dye concentration at 1.5 g/L ZnO–NF1 photocatalyst for 90 min
(Fig. 7A). However, when 50 mg/L dye concentration was used, 86.38%
removal efficiency was achieved for 90 min.
The results for the removal efficiency of RR180 dye are given in
Fig. 7B. It was determined that the color removal efficiency was 100% at
10 mg/L dye concentration for 60 min and 100% at 25 mg/L dye con­
centration for 90 min. However, the dye removal efficiency was only
70.48% at 50 mg/L dye concentration.
The data on the effect of dyes concentration at the optimum photo­
catalyst amount for ZnO–NF2 are shown in Fig. 7C. The removal effi­
ciency decreased as the concentration of BR18 increased. The color
removal efficiency reached 100% within 60 min at 10 mg/L BR18 dye
concentration. However, when the dye concentration increased from 25
mg/L to 50 mg/L at the end of 90 min, the dye remove efficiency
decreased from 59.18% to 40.45%.
The results of the experiments in which ZnO–NF2 was used as a
photocatalyst for the removal of RR180 dye are given in Fig. 7D. 100%
color removal efficiency was achieved at 10 mg/L dye concentration and
2 g/L photocatalyst amount for 45 min However, when the dye con­
centration was increased from 25 mg/L to 50 mg/L, the dye removal
efficiency decreased from 90.00% to 57.21%, respectively, for 90 min.
The decrease in dye removal efficiency at high dye concentration is
attributed to the fact that dye adsorption causes a reduction in OH•
formation. In addition, the amount of photons entering the solution may
decrease with increasing dye concentration.
3.3. DPPH cleavage ability
Since the DPPH test is an easy, fast and useful method, it is frequently
used to investigate the potential of scavenging free radicals. In the
treatment of many diseases, it is of great importance to identify anti­
oxidants that can inhibit free radicals. Identifying potential antioxidants
to inhibit free radicals is now becoming one of the new effective ther­
apeutic targets (Sharmila et al., 2019). In this study, ZnO-NFs were
performed for theirs antioxidant capacity. Trolox and Ascorbic acid were
used as standard for comparison of percentage inhibition of DPPH
radical by ZnO-NFs. DPPH radical scavenging activity of ZnO–NF1 and
ZnO–NF2s is presented in Fig. 8. ZnO-NFs exhibited excellent inhibition
of the DPPH radical. The antioxidant activity of ZnO–NF2 was found to
Fig. 8. DPPH scavenging ability.
8
Chemosphere 306 (2022) 135389
be higher than ZnO–NF1 at each concentration.
When the concentrations of ZnO–NF1 and ZnO–NF2 raised from 12.5
to 100 mg/L, the antioxidant activities raised from 52.7% to 60.61%,
and from 55.63%to, 64.29%, respectively. The highest radical inhibition
ability achieved with ZnO–NF2 as 87.18%. Saif et al. (2021) examined
an enhanced photocatalytic degradation and infectious treatment of
phyto-reflected ZnO-NFs synthesis in terms of biological and chemical
differences. They found that antioxidant activity of ZnO-NFs on DPPH
radicals increased with concentration and the synthesized ZnO itself had
antioxidant activities. Arumugam et al. (2021)) evaluated the DPPH
scavenging activity of green synthesized of ZnO-NFs using Syzygium
cumini. They found that ZnO-NFs indicated antioxidant activity. Khan
et al. (2021a) investigated the antioxidant activity properties of
Ag2S–ZnO/GO, Ag2S–ZnO and ZnO nanocomposites by the DPPH
scavenging test. They found that Ag2S–ZnO and ZnO separately showed
the least antioxidant activity, and Ag2S–ZnO and ZnO displayed 58%
and 52% DPPH radical inhibition ability. In addition to these, they
indicated that synergistic effect of the Ag2S–ZnO/GO nanocomposite is
responsible for its notable radical scavenging properties. The results
from the antioxidant activity showed that Ag2S–ZnO/GO was in agree­
ment with earlierdata reported information, which is a clearly explains
of the elevated effect of Ag2S–ZnO/GO equated with ZnO and Ag2S–ZnO
nanocomposite. Zinc is main micronutrient element that plays a key role
in antioxidant defense, growth and strengthening of the immune system,
and studies have shown that it has strong antioxidant power in both
extracellular systems and intracellular conditions (Khan et al., 2021b).
The newly synthesized and characterized ZnO–NF1 and ZnO–NF2 seem
to be the best candidate due to theirs good antioxidant activity. As a
result, the evaluation of antioxidant ability for ZnO–NF1 and ZnO–NF2
can be very significant and essential before theirs used in animal model
or in vivo investigations and human applications.
3.4. DNA cleavage ability
In this investigation, agarose gel electrophoresis was performed for
determination of the DNA cleavage activity of ZnO–NF1 and ZnO–NF2.
The view of DNA cleavage activity of ZnO–NF1 and ZnO–NF2 is pre­
sented in Fig. 9. According to the obtained results, DNA was completely
degraded by ZnO–NF1 and ZnO–NF2 at all tested concentrations. De
et al. (2021) studied the synthesis and DNA fragmentation activities of
ZnO-NPs. As a result of DNA fragmentation analysis, they found that
ZnO-NPs were able to digest approximately 55% of the E. coli DNA
sample under corrected terms. They concluded that these results were
because of the electronic environment and small size of ZnO-NPs. Santos
et al. (2020) conducted a study on diclofenac and ibuprofen zinc
(II)-nicotinamide triple complexes on their characterization and DNA
Fig. 9. DNA Cleavage activity of ZnONPs. Lane 1, pBR 322 DNA; Lane 2, pBR
322 DNA +50 mg of ZnONF-1; Lane 3, pBR 322 DNA +100 mg of ZnONF-1;
Lane 4, pBR 322 DNA +50 mg of ZnONF-2; Lane 5, pBR 322 DNA +100 mg/
L ZnONF-2.
O. Eskikaya et al. Chemosphere 306 (2022) 135389

interaction. They found that there was no DNA cleavage activity and
both compounds had pharmacological potential to be applied as an
anti-inflammatory agent with enhanced properties. Zinc is an essential
element for life. Zinc is the second most abundant transition metal in
living systems after iron. Many enzymes require the zinc ion as a
cofactor for their catalytic activity. In addition, zinc complexes are often
used as medicine. Zinc prevents oxidative DNA damage by protecting
cells from oxidative stress and is a significant mechanism for main­
taining cellular integrity. The interactions of ZnO nanoparticles with
DNA molecules at different concentrations were investigated. They
informed that ZnO nanoparticle cause DNA damage. Results attributed
to DNA fragmentation show their ability to inhibit the pathogen
microorganism growth by fragmenting their genome.
3.5. Antimicrobial activity
The antimicrobial effect of ZnO–NF1 and ZnO–NF2 were determined
by serial dilution method (Table 1). It was demonstrated that antimi­
crobial activities showed different activities according to microorgan­
isms. The changing rates of the antimicrobial activity of the particles at
the nanoscale are thought to be due to the distinction in the microbial
cell metabolism and morphology of the tested microorganisms. The
minimum inhibition concentration of ZnO–NF1 was found as 16 mg/L
for L. pneumophila, E. hirae, S. aureus and E. fecalis, 32 mg/L for E. coli, C.
parapisilosis and P. aeruginosa, 64 mg/L for C. tropicalis. The MIC of
ZnO–NF2 was also found as 64 mg/L for E. coli, E. hirae, P. aeruginosa, C.
parapisilosis, and S. aureus, 128 mg/L for L. pneumophila, E. fecalis and
C. tropicalis. When the results of antimicrobial ability of ZnO-NFs
compared, it was seen that the antibacterial activity of ZnO–NF1 was
much better than the antibacterial activity of ZnO–NF2. Khan et al.
(2021c) examined the antibacterial activities of ZnO, Ag2S–ZnO and
Ag2S–ZnO/GO nanocomposites. They found that higher antibacterial
inhibition was demonstrated as the concentration increased. They were
also found that Ag2S–ZnO and Ag2S–ZnO/GO nanocomposites showed
important toxic impacts against bacterial strains compared to pure ZnO
sample under same conditions. They also found a lower effect on
Gram-negative bacteria strains than Gram-positive bacteria. Rambabu
et al. (2021) examined the antimicrobial ability of prepared DP-ZnO-NPs
against pathogenic Gram (− ) and Gram (+) bacteria and they also found
that different antimicrobial activity observed with DP-ZnO-NPs against
different microbial strains. The mechanism of antimicrobial ability of
ZnO-NPs may be due to their specific morphology and involvement via
receptor-ligand interactions with the negatively charged cell membrane.
This can cause irreversible membrane damage due to weak electrostatic
interactions (Doan Thi et al., 2020). ZnO–NF1 and ZnO–NF2 can be used
as a resource of new potential bactericidal agents for biological appli­
cations. ZnO–NF1 and ZnO–NF2 demonstrated reducing and effective
antimicrobial activity against the tested microbial strains. The newly
synthesized nanoflowers can be safely used for the control of microor­
ganisms in an environmentally friendly and sustainable way after
further studies.
3.6. Bacterial cell viability inhibition test and antimicrobial photodynamic
therapy
The microbial cell viability inhibition activity of ZnO–NF1 and
ZnO–NF2 was also tested against bacterial strain of E. coli. The results
are presented in Fig. 10. It was determined that the newly synthesized
nanoflowers caused very effective E. coli growth inhibition. As seen in
Fig. 10, ZnO–NF1 and ZnO–NF2 inhibited the E. coli growth as 95.32%
and 92.78% at 100 mg/L and 99.67% and 98.74% at 200 mg/L,
respectively. The results suggested that ZnO–NF1 and ZnO–NF2
exhibited a potent inhibitory effect, therefore further research is
required to evaluate the antibacterial efficacy of ZnO–NF1 and ZnO–NF2
using in-vivo animal models. The chemical agents which reduce cell
proliferation is a significant field of drug discovery and cell biology
investigation. Khan et al. (2021c) used contact lenses coated with
multifunctional triple nanocoatings for the treatment of antibacterial
and fungal keratitis. In these study, antimicrobial photodynamic therapy
study was also performed by exposing the compounds prepared as
mentioned above to LED light for 20 min. As a result of antimicrobial
photodynamic therapy, it was determined that ZnO–NF1 and ZnO–NF2
inhibited bacterial growth as 100% at 100 and 200 mg/L (Fig. 11).
Antimicrobial photodynamic therapy is an emerging non-antibiotic
approach to eradication of antibiotic-resistant microorganisms (Mor­
ton et al., 2013). According to cell viability and antimicrobial photo­
dynamic therapy results, newly synthesized ZnO–NF1 and ZnO–NF2
seems to be quite effective on E. coli bacteria. After further studies, we
can say that NP can be used as antimicrobial agent. In addition, in the
light of our results, we can complete that ZnO–NF1 and ZnO–NF2 can
prevent irritation in hospitals, wastewater, local human infections in a
short time and at very low concentrations.
3.7. Biofilm inhibition
Biofilms are the most common form of life in a well-organized bac­
terial community, where they attach to each other and to other wet
surfaces, and as noted by the National Institutes of Health, over 75% of
infections are caused by microbial biofilms (Davies, 2003). Because
microbial biofilms retain many unmatched properties that confer resis­
tance to antimicrobial agents, antibacterial coating has become an
exciting and very important research topic for protection against bac­
teria and related infections. In this investigation, the biofilm inhibition
abilities of ZnO–NF1 and ZnO–NF2 were tested, which are known as
biofilm forming microorganisms. Biofilm inhibition of S. aureus and

Table 1
The minimum inhibition concentration (MIC) of test microorganisms.
Microorganisms ZnONF-1 ZnONF-2

E. coli 32 64
P. aeruginosa 32 64
L. pneumophila subsp. pneumophila 16 128
E. hirae 16 64
E. fecalis 16 128
S. aureus 16 64
C. parapisilosis 32 64
C. tropicalis 64 128
Fig. 10. Microbial cell viability inhibition.

9
O. Eskikaya et al.
Fig. 11. Photodynamic antimicrobial activity.
Fig. 12. Biofilm inhibition of S. aureus.
P. aeruginosa are represented in Fig. 12 and Fig. 13. It was determined
that the biofilm inhibition ability was concentration dependent when
using ZnO–NF1 and ZnO–NF2. It can be obviously seen that ZnO–NF1
exhibited higher biofilm inhibition activity than ZnO–NF2 against both
test microorganisms at 250 and 500 mg/L. The biofilm inhibition ac­
tivities of ZnO–NF1 and ZnO–NF2 were found as 70.25% and 34.54% for
P. aeruginosa and 25.05% and 38.68% for S. aureus at 125 mg/L,
respectively. When concentration of ZnO–NF1 and ZnO–NF2 raised up
from 250 mg/L to 500 mg/L, the biofilm inhibition activity raised up
from 82.36% to 87.63% and from 51.43% to 79.64% for P. aeruginosa,
respectively. When the concentration of ZnO–NF1 and ZnO–NF2 also
raised up from 250 to 500 mg/L the biofilm inhibition raised up from
78.42% to 84.56% and from 57.65% to 81.78% for S. aureus, respec­
tively. Jasima et al. (2020) investigated whether ZnO-NPs could reduce
the biofilm produced by vancomycin resistant Staphylococcus aureus
(VRSA) isolates. They calculated the mean inhibition rates produced by
each ZnO-NP concentration against VRSA isolates and found that
ZnO-NPs significantly inhibited biofilm production in a
concentration-dependent manner. They found that 10 mg/mL ZnO-NPs
10
Chemosphere 306 (2022) 135389
Fig. 13. Biofilm inhibition of P. aeruginosa.
concentration demonstrated the highest inhibitory activity against
production of biofilm (73.95 ± 2.17%), while the lowest inhibition rate
(19.08 ± 1.32%) was obtained at 0.625 mg/mL. Wang et al. (2021b)
conducted a study to determine the inhibitory effect of nanoparticles on
S. aureus biofilms. They observed a significant dose-dependent effect for
samples treated with ZnO or SPIO/ZnO NPs. They concluded that the
prepared SPIO/ZnO Nps or ZnO could disrupt the preformed biofilm of
the test microorganisms. In other published reports, it was demonstrated
that ZnO-NPs had a powerful activity to inhibit biofilm production
(Al-Shabib et al., 2018; Xu et al., 2018). It can be concluded that the
newly synthesized ZnO–NF1 and ZnO–NF2 can be used as a strong
alternative to reduce the severity of infection and increase the treatment
efficiency of wastewater with biofilm inhibition in medical and envi­
ronmental applications.
Overall, a summary of the ZnO-NFs preparation and investigations
on the photocatalytic efficiency and antioxidant performance is shown
in Table 2 and Table 3, respectively. In Table 2, the optimum conditions
and removal efficiencies were compared with the ZnO-NFs synthesized
with different production conditions. Rupa et al. (2019) investigated the
dye removal of nanoflower material containing ZnO, which they pro­
duced from organic material in four different dye solutions. Dye removal
efficiencies were obtained at different reaction times with the nano­
materials. When other studies were compared with this study, high
removal efficiency was obtained at lower dye concentrations. Hemala­
tha et al. (2016) synthesized ZnO nanoflower with lanthanum (La) using
microwave assisted sol-gel technique. In their study, they stated that the
La-doped material enhanced high photocatalytic properties when
compared with the pure ZnO sample. However, according to the results
obtained, a very large amount of photocatalyst should be used to purify
20 mg/L dye. It is also known that La reduces the chlorophyll content in
plants (Hu et al., 2016). For this reason, an additional environmental
problem may arise by producing a material that is more harmful to the
environment. According to Vinayagam et al. (2021), ZnO nanoflower
was synthesized with green synthesis. As a result of their studies, they
achieved a dye removal efficiency of 88% for 270 min using 500 mg/L
photocatalyst. It is thought that the long reaction time may cause
additional costs in real-scale studies. Mohammad et al. (2016) synthe­
sized nanoflowers to use in the purification of organic dyes. They ach­
ieved high efficiency by using a photocatalyst in the amount of 20 mg/L.
Vinayagam et al. (2021) synthesized ZnO nanoflower using leaf extract.
In their study, they were able to obtain high efficiency by using 400
mg/L photocatalyst to remove the RhB dye. The use of photocatalyst in
large quantities increases the production cost and it is predicted that it
O. Eskikaya et al. Chemosphere 306 (2022) 135389

Table 2
Comparison of photocatalytic activity of ZnO–NF1 and ZnO–NF2 with other studies.
Photocatalytic Activity

Photocatalyst Catalyst amount (mg/L) Dye Concentration of dye (mg/L) Time (min) Efficiency (%) Ref.

ZnO–NF1 1.5 BR18 25 75 100 This study

RR180 90 100 This study
ZnO–NF2 2.0 BR18 90 60 This study
RR180 90 90 This study
SBT-ZnO/NF 150 MG 15 70 100 Rupa et al. (2019)
MB 70 99
CR 80 100
EY 90 99
3 mol% 1800 MB 20 60 90 Hemalatha et al. (2016)
La-doped ZnO
CH-ZONPs 500 MB 20 270 88 Vinayagam et al., 2021
ZnONF 20 MO 15 50 99 Mohammad et al. (2016)
CR 80 99
CSB 80 96
EY 80 95
BR–ZnONFs 400 RhB 13 165 94 Vinayagam et al., 2021.

CSB: Chicago Sky Blue Dye, MO: Methyl Orange Dye, MG: Malachite Green Dye, CR: Congo Red Dye, MB: Methylene Blue Dye, EY: Eosin Y Dye.

Table 3
Comparison of antibacterial activities of Zn–ONF1 and ZnO–NF2 with other studies.
Antibacterial Activity

Material Microorganisms Minimum inhibitory conc. (mg/ Antioxidant dosage (mg/ Antioxidant activities (%) (E. Ref.
L) L) coli)

ZnO–NF1 L. pneumophila 16 100 95 This study

E. hirae
S. aureus
E. fecalis
E. coli 32 200 99
C. parapisilosis
P. aeruginosa
C. tropicalis 64
ZnO–NF2 E. coli 64 100 92 This study
E. hirae
P. aeruginosa
C. parapisilosis
S. aureus 200 98
L. pneumophila 128
E. fecalis
C. tropicalis
5 wt% Ag/ZnO E. coli 1000 100 99 Lam et al. (2018)
ZnONF with A. E. coli – 500 60 Khan et al., 2021
membranaceus
ZnO-1 E. coli – 10 38 Thakur and Mandal
– 100 88 (2020)
– 150 98

may pose a problem both in storage and disposal after use. In our study,
higher dye removal efficiencies were obtained at higher dye concen­
tration at low photocatalyst amount compared to other studies. This can
be stated among the advantages of the nanomaterial produced within
the scope of the study.
Table 3 shows the comparison of antibacterial effect of ZnO–NF1 and
ZnO–NF2 with other studies. Lam et al. (2019) investigated the anti­
bacterial activity of silver (Ag)-containing ZnO nanoflower material.
Within the scope of the experiments carried out for the minimum
inhibitory concentration of the material containing 5% Ag by weight,
the highest efficiency was obtained in the amount of 1000 mg/L. In our
study, it was determined that the produced nanoflowers were effective
at lower concentrations. Khan et al. (2020) investigated the antioxidant
effect on E. coli with nanoflower containing ZnO, which they synthesized
together with organic material. In order to prevent E. coli reproduction
and to reduce the number of colonies, 500 mg/L nanoflowers were
added into petri dishes. According to the results, it was determined that
the organic doped ZnO nanoflower supplied only 60% efficiency for 24
h. In this case, they stated that the produced material showed a
moderate antioxidant effect. Thakur and Mandal (2020) examined both
the photocatalytic treatment and antibacterial activity of
ZnO-containing nanoflower material in their work. They examined the
effect on bacteria at three different doses. It was found that the efficiency
increased with the increasing amount of antioxidants. In experiments
with 10 and 100 mg/L doses, lower yields were obtained at the same
dose compared to our study.
4. Conclusion
In this study, we examined the photocatalytic efficiency of two
different ZnO nanoflowers which were synthesized using hydrothermal
and precipitation methods. Moreover, antioxidant, antibacterial, anti­
microbial photodynamic therapy, antibiofilm, DNA cleavage activity,
the bacterial cell viability inhibition was also tested. The color was
completely removed for 25 mg/L BR18 and RR180 dyes in 75 min and
90 min, respectively, using a 1.5 g/L photocatalyst amount for
ZnO–NF1. However, 59.18% dye removal efficiency was obtained in 90
min by using 1.5 g/L photocatalyst amount for 25 mg/L BR18 dye, while

11
O. Eskikaya et al.
90.00% removal efficiency was achieved using 2.0 g/L photocatalyst for
25 mg/L RR180 dye in the same reaction time for ZnONF-2. The results
showed that antioxidant and antimicrobial activities of ZnO–NF1 and
ZnO–NF2 were very effective. The maximum antioxidant activities of
ZnO–NF1 and ZnONF-2 were found as 82.3% and 87.1%, respectively,
for 200 mg/mL concentration. ZnO-NFs exhibited excellent inhibition of
the DPPH radical, which increased as the ZnO-NFs concentration
increased. When the results were examined, it was seen that the anti­
bacterial activity of ZnO–NF1 was much better than the antibacterial
activity of ZnO–NF2. The microbial cell viability inhibition activity of
ZnO–NF1 and ZnO–NF2 was also tested against bacterial cell strain
E. coli. According to these results, it was determined that the synthesized
of ZnO–NF1 and ZnO–NF2 caused very effective E. coli growth inhibi­
tion. Also, antimicrobial photodynamic therapy study was performed by
exposing the compounds prepared as mentioned above to LED light for
20 min. As a result, inhibition rates of antimicrobial photodynamic
therapy were determined as 100% at 100 and 200 mg/L for both syn­
thesized ZnO-NFs. It was also determined that the antibiofilm activity
was concentration dependent and ZnO–NF1 and ZnO–NF2 displayed
significant biofilm inhibition activities.
It can be concluded that the synthesized ZnO–NF1 and ZnO–NF2 can
be used as a strong alternative to reduce the severity of infection and
increase the treatment efficiency of wastewater with biofilm inhibition
in medical and environmental applications. ZnO-NP1 and ZnO-NP2 can
be used for microbial cell viability inhibition and photodynamic anti­
microbial activity in the future. Moreover, a dual photosensitizer
coupled nanoscintillation capable of producing type I and type II ROS
can be developed for next-generation photodynamic therapy using ZnO-
NFs.
Credit author statement
Ozan Eskikaya: concepts, design, data collection and experimenta­
tion. Sadin Özdemir: concepts, design, data collection and experimen­
tation. Gülsah Tollu: concepts, design, data collection and
experimentation. Nadir Dizge: concepts and design, draft, technical
correction and proof read, Rameshprabu Ramaraj: draft, technical
correction and proof read, Arthi Manivannan: draft, technical correction
and proof read, Balakrishnan Deepanraj: draft, technical correction and
proof read.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
References
Adhikari, A., Pal, U., Bayan, S., Mondal, S., Ghosh, R., Darbar, S., Pal, S.K., 2021.
Nanoceutical fabric prevents COVID-19 spread through expelled respiratory
droplets: a combined computational, spectroscopic, and antimicrobial study. ACS
Appl. Bio Mater. 4, 5471–5484.
Aggarwal, N., Krishna, S., Sharma, A., Goswami, L., Kumar, D., Husale, S., Gupta, G.,
2017. A highly responsive self-driven UV photodetector using GaN nanoflowers.
Adv. Electron. Mater. 3 (5), 1700036.
Al-Shabib, N., Husain, F., Hassan, I., Khan, M., Ahmed, F., Qais, F., Khan, R., 2018.
Biofabrication of zinc oxide nanoparticle from Ochradenus baccatus leaves: broad-
spectrum antibiofilm activity, protein binding studies, and in vivo toxicity and stress
studies. J. Nanomater. https://doi.org/10.1155/2018/8612158.
Ali, J.A., Kalhury, A.M., Sabir, A.N., Ahmed, R.N., Ali, N.H., Abdullah, A.D., 2020.
A state-of-the-art review of the application of nanotechnology in the oil and gas
industry with a focus on drilling engineering. J. Petrol. Sci. Eng. 191, 107118.
Arslan, H., Eskikaya, O., Bilici, Z., Dizge, N., Balakrishnan, D., 2022. Comparison of Cr
(VI) adsorption and photocatalytic reduction efficiency using leonardite powder.
Chemosphere 300, 134492.
Arumugam, M., Manikandan, D., Dhandapani, E., Sridhar, A., Balakrishnan, K.,
Markandan, M., 2021. Green Synthesis of Zinc Oxide Nanoparticles (ZnO NPs) Using
Syzygium Cumini: Potential Multifaceted Applications on Antioxidants, Cytotoxic
and as Nanonutrient for the Growth of Sesamum indicum. Environmental
Technology & Innovation, 101653.
12
Chemosphere 306 (2022) 135389
Chakrabarti, S., Dutta, B.K., 2004. Photocatalytic degradation of model textile dyes in
wastewater using ZnO as semiconductor catalyst. J. Hazard Mater. 112, 269–278.
Cheriyamundath, S., Vavilala, S.L., 2021. Nanotechnology-based wastewater treatment.
Water Environ. J. 35 (1), 123–132.
Das, A., Kumar, P.M., Bhagavathiachari, M., Nair, R.G., 2021. Shape selective flower-like
ZnO nanostructures prepared via structure-directing reagent free methods for
efficient photocatalytic performance. Mater. Sci. Eng., B 269, 115149.
Davies, D., 2003. Understanding biofilm resistance to antibacterial agents. Nat. Rev.
Drug Discov. 114–122.
De, A., Das, R., Kaur, H., Jain, P., 2021. Synthesis, physicochemical investigations, DNA
cleavage activity of biogenic zinc oxide nanoparticles and their interaction with Calf-
Thymus DNA. Mater. Today Proc. 2214–7853.
Doan Thi, T., Nguyen, T., Thi, Y., Ta Thi, K., Phan, B., Pham, K., 2020. Green synthesis of
ZnO nanoparticles using orange fruit peel extract for antibacterial activities. RSC
Adv. 23899–23907.
Elbeshehy, E., Elazzazy, A., Aggelis, G., 2015. Silver nanoparticles synthesis mediated by
new isolates of Bacillus spp., nanoparticle characterization and their activity against
Bean Yellow Mosaic Virus and human pathogens. Front. Microbiol. 6.
Fadillah, G., Saleh, T.A., Munawaroh, H., Wahyuningsih, S., Ramelan, A.H., 2021. Flow
photocatalysis system-based functionalized graphene oxide-ZnO nanoflowers for
degradation of a natural humic acid. Environ. Sci. Pollut. Control Ser. 1–9.
Gao, L., Yan, H., Zhu, S., Wang, X., Tan, Y., Du, J., Gu, Z., 2021. Targeted delivery of
Bi2Se3 Nanoflowers to orthotopic liver tumor via transarterial infusion for enhanced
microwave ablation sensibilization. Nano Today 41, 101314.
Gonca, S., Ozidemir, S., Isik, Z., M’barek, I., Shaik, F., Dizge, N., Balakrishnan, D., 2022.
Synthesis of silver nanoparticles from red and green parts of the pistachio hulls and
their various in-vitro biological activities. Food Chem. Toxicol. 165, 113170.
Hemalatha, P, et al., 2016. La-doped ZnO nanoflower as photocatalyst for methylene
blue dye degradation under UV irradiation. J. Mater. Sci., Mater. Electron. 27,
2367–2378.
Hu, H., Wang, L., Li, Y., Sun, J., Zhou, Q., Huang, X., 2016. Insight into mechanism of
lanthanum (III) induced damage to plant photosynthesis. Ecotoxicol. Environ. Saf.
127, 43–50.
Jain, K., Patel, A.S., Pardhi, V.P., Flora, S.J.S., 2021. Nanotechnology in wastewater
management: a new paradigm towards wastewater treatment. Molecules 26 (6),
1797.
Jasima, N., Al-Gasha’a, F., Al-Marjini, M., Al-Rahal, A., Abide, H., Al-Kadhmi, N., 2020.
ZnO nanoparticles inhibit growth and biofilm formation of vancomycin-resistant
S. aureus (VRSA). Biocatal. Agric. Biotechnol. 101745.
Jawoor, S., Patil, S., Kumbar, M., Ramawadgi, P., 2018. Green synthesis of nano sized
transition metal complexes containing heterocyclic Schiff base: structural and
morphology characterization and bioactivity study. J. Mol. Struct. 378–385.
Karaman, C., Karaman, O., Show, P.L., Orooji, Y., Karimi-Maleh, H., 2022. Utilization of
a double-cross-linked amino-functionalized three-dimensional graphene networks as
a monolithic adsorbent for methyl orange removal: equilibrium, kinetics,
thermodynamics and artificial neural network modeling. Environ. Res. 1 (207),
112156.
Karimi, F., Ayati, A., Tanhaei, B., Sanati, A.L., Afshar, S., Kardan, A., Dabirifar, Z.,
Karaman, C., 2022. Removal of metal ions using a new magnetic chitosan nano-bio-
adsorbent; A powerful approach in water treatment. Environ. Res. 203, 111753.
Karimi-Maleh, H., Karimi, F., Fu, L., Sanati, A.L., Alizadeh, M., Karaman, C., Orooji, Y.,
2002. Cyanazine herbicide monitoring as a hazardous substance by a DNA
nanostructure biosensor. J. Hazard Mater. 423 (Part A), 127058.
Karimi-Maleh, H., Khataee, A., Karimi, F., Baghayeri, M., Fu, L., Rouhi, J., Karaman, C.,
Karaman, O., Boukherroub, R., 2022. A green and sensitive guanine-based DNA
biosensor for idarubicin anticancer monitoring in biological samples: a simple and
fast strategy for control of health quality in chemotherapy procedure confirmed by
docking investigation. Chemosphere 291 (Part 3), 132928.
Kaur, J., Singh, H., Singh, A., 2020. Fabrication and investigation of zinc oxide
nanoflowers-based piezoelectric nanogenerator. IET Circuits, Devices Syst. 14 (Iss.
4), 477–483.
Khan, A., Arooj, A., Tahir, K., Ibrahım, M., Jevtovic, V., AL-Abdulkarim, H., Lia, B.,
2021a. Facile fabrication of novel Ag2S-ZnO/GO nanocomposite with its enhanced
photocatalytic and biological applications. J. Mol. Struct., 131991
Khan, F., Khan, Z., Ma, J., Khan, A., Sohail, M., Chen, Y., 2021b. An Astragalus
membranaceus based eco-friendly biomimetic synthesis approach of ZnO nanoflowers
with an excellent antibacterial, antioxidant and electrochemical sensing effect.
Mater. Sci. Eng. C 118, 111432.
Khan, S., Shahid, S., Mahmood, T., Lee, C., 2021c. Contact lenses coated with hybrid
multifunctional ternary nanocoatings (Phytomolecule-coated ZnO nanoparticles:
Gallic Acid:Tobramycin) for the treatment of bacterial and fungal keratitis. Acta
Biomater. 262–276.
Kirtane, A.R., Verma, M., Karandikar, P., Furin, J., Langer, R., Traverso, G., 2021.
Nanotechnology approaches for global infectious diseases. Nat. Nanotechnol. 16 (4),
369–384.
Krifa, M., Prichard, C., 2020. Nanotechnology in textile and apparel research–an
overview of technologies and processes. J. Textil. Inst. 111 (12), 1778–1793.
Kumar, B., Smita, K., Borovskikh, P., Shchegolkov, A., Debut, A., Cumbal, L., 2021.
Spectroscopic and morphological characterization of Nephelium lappaceum peel
extract synthesized gold nanoflowers and its catalytic activity. Inorg. Chem.
Commun. 133, 108868.
Lam, S.M., Quek, J.A., Sin, J.C., 2018. Mechanistic investigation of visible light
responsive Ag/ZnO micro/nanoflowers for enhanced photocatalytic performance
and antibacterial activity. J. Photochem. Photobiol. A: Chem. 353, 171–184.
Li, X., Hu, D., Dang, Y., et al., 2009. Nano Mapper: an Internet knowledge mapping
system for nanotechnology development. J. Nano Res. 11, 529–552.
O. Eskikaya et al.
Luo, Y., Wang, Q., Zhang, Y., 2020. Biopolymer-based nanotechnology approaches to
deliver bioactive compounds for food applications: a perspective on the past,
present, and future. J. Agric. Food Chem. 68 (46), 12993–13000.
Mohammad, A., Kapoor, K., Mobin, S.M., 2016. Improved photocatalytic degradation of
organic dyes by ZnO-nanoflowers. Chemistry 1, 3483–3490. https://doi.org/10.
1002/slct.201600476.
Morton, C., Szeimies, R., Sidoroff, A., Braathen, L., 2013. European guidelines for topical
photodynamic therapy part 1: treatment delivery and current indications - actinic
keratoses, bowen’s disease, basal cell carcinoma. J. Eur. Acad. Dermatol. Venereol.
536–544.
Nie, Y., Xie, Y., Zheng, Y., Luo, Y., Zhang, J., Yi, Z., Wu, P., 2021. Preparation of ZnO/
Bi2O3 composites as heterogeneous thin film materials with high photoelectric
performance on FTO base. Coatings 11 (9), 1140.
Oroojalian, F., Charbgoo, F., Hashemi, M., Amani, A., Yazdian-Robati, R.,
Mokhtarzadeh, A., Hamblin, M.R., 2020. Recent advances in nanotechnology-based
drug delivery systems for the kidney. J. Contr. Release 321, 442–462.
Racca, L., Canta, M., Dumontel, B., Ancona, A., Limongi, T., Garino, N., Laurenti, M.,
Canavese, G., Cauda, V., 2018. 12 - zinc oxide nanostructures in biomedicine, editor
(s): gianni ciofani. In: Micro and Nano Technologies, Smart Nanoparticles for
Biomedicine. Elsevier, pp. 171–187.
Raj, V.J., Ghosh, R., Girigoswami, A., Girigoswami, K., 2022. Application of zinc oxide
nanoflowers in environmental and biomedical science. BBA Adv. 2, 100051.
Rambabu, K., Bharath, G., Banat, F., Show, P., 2021. Green synthesis of zinc oxide
nanoparticles using Phoenix dactylifera waste as bioreductant for effective dye
degradation and antibacterial performance in wastewater treatment. J. Hazard.
Mater., 123560
Rupa, EJ, Kaliraj, L, Abid, S, Yang, D-C, Jung, S-K, 2019. Synthesis of a zinc oxide
nanoflower photocatalyst from sea Buckthorn fruit for degradation of industrial dyes
in wastewater treatment. Nanomaterials 9 (12), 1692. https://doi.org/10.3390/na
no9121692.
Sahani, S., Sharma, Y.C., 2021. Advancements in applications of nanotechnology in
global food industry. Food Chem. 342, 128318.
Saif, M., Zafar, A., Waqas, M., Hassan, S., Haq, A., Tariq, T., Shu, X., 2021. Phyto-
reflexive Zinc Oxide Nano-Flowers synthesis: an advanced photocatalytic
degradation and infectious therapy. J. Mater. Res. Technol. 2375–2391.
Santos, P., Pich, C., Back, D., Smiderle, F., Dumas, F., Moura, S., 2020. Synthesis,
chemical characterization and DNA interaction study of new diclofenac and
ibuprofen zinc (II)-nicotinamide ternary complexes as cyclooxygenase inhibitor
prototypes. J. Inorg. Biochem., 111046
Serrano, E., Rus, G., García-Martínez, J., 2009. Nanotechnology for sustainable energy.
Renew. Sustain. Energy Rev. 13 (9), 2373–2384.
Shabaaz Begum, J., Manjunath, K., Pratibha, S., Dhananjaya, N., Sahu, P., Kashaw, S.,
2020. Bioreduction synthesis of zinc oxide nanoparticles using Delonix regia leaf
extract (Gul Mohar) and its agromedicinal applications. J. Sci. Adv. Mater. Devices
468–475.
Shafique, M., Luo, X., 2019. Nanotechnology in transportation vehicles: an overview of
its applications. Environ. Health Safety Concerns Mater. (Basel) 12 (15), 2493.
Sharabati, M.A., Abokwiek, R., Al-Othman, A., Tawalbeh, M., Karaman, C., Orooji, Y.,
Karimi, F., 2021. Biodegradable polymers and their nano-composites for the removal
of endocrine-disrupting chemicals (EDCs) from wastewater: a review. Environ. Res.
202, 111694.
13
Chemosphere 306 (2022) 135389
Sharmila, G., Muthukumaran, C., Saraswathi, H., Sangeetha, E., Soundarya, S.,
Kumar, N., 2019. Green synthesis, characterization and biological activities of
nanoceria. Ceram. Int. 12382–12386.
Shende, P., Kasture, P., Gaud, R.S., 2018. Nanoflowers: the future trend of
nanotechnology for multi-applications, Artificial Cells. Nanomed. Biotech. 46
(Suppl. 1), 413–422.
Shweta, Sood, S., Sharma, A., Chadha, S., Guleria, V., 2021. Nanotechnology: a cutting-
edge technology in vegetable production. J. Hortic. Sci. Biotechnol. 1–14.
Singh, J., Rishikesh, Kumar S., Soni, R.K., 2022. Synthesis of 3D-MoS2 nanoflowers with
tunable surface area for the application in photocatalysis and SERS based sensing.
J. Alloys Compd. 849, 156502.
Talebian, S., Rodrigues, T., Das Neves, J., Sarmento, B., Langer, R., Conde, J., 2021. Facts
and Fig.s on materials science and nanotechnology progress and investment. ACS
Nano 15 (10), 15940–15952.
Thakur, S., Mandal, S.K., 2020. Morphology engineering of ZnO nanorod arrays to
hierarchical nanoflowers for enhanced photocatalytic activity and antibacterial
action against Escherichia coli. N. J. Chem. 44 (27), 11796–11807.
Vasantharaj, K., Jerold, M., Deepanraj, B., Velan, M., Sivasubramanian, V., 2017.
Assessment of a sulfidogenic system utilizing microalgal biomass of Chlorella
pyrenoidosa as an electron donor: taguchi based grey relational analysis. Int. J.
Hydrogen Energy 42, 26545–26554.
Vinayagam, R., Pai, S., Varadavenkatesan, T., Pugazhendhi, A., Selvaraj, R., 2021.
Characterization and photocatalytic activity of ZnO nanoflowers synthesized using
Bridelia retusa leaf extract. Appl. Nanosci. 1–10.
Wang, J., Jia, Z., Liu, X., Dou, J., Xu, B., Wang, B., Wu, G., 2021a. Construction of 1D
heterostructure NiCo@ C/ZnO nanorod with enhanced microwave absorption.
Nano-Micro Lett. 13 (1), 1–16.
Wang, J., Zhua, X., Peia, W., Zhou, L., Cai, L., Jiang, H., Chen, J., 2021b. ZnO nanocluster
loaded superparamagnetic iron oxide nanocomposites as recyclable antibacterial
agent. Colloid Inter. Sci. Commun., 100510
Xu, S., Sun, T., Xu, Q., Duan, C., Dai, Y., Wang, L., Song, Q., 2018. Reparation and
antibiofilm properties of zinc oxide/porous anodic alumina composite films.
Nanoscale Res. Lett. https://doi.org/10.1186/s11671-018-2568-4.
Ya, Y., Jiang, C., Li, T., Liao, J., Fan, Y., Wei, Y., Xie, L., 2017. A zinc oxide nanoflower-
based electrochemical sensor for trace detection of sunset yellow. Sensors 17 (3),
545.
Yabalak, E., Al-Nuaimy, M.N.M., Saleh, M., Isik, Z., Dizge, N., Balakrishnan, D., 2022.
Catalytic efficiency of raw and hydrolyzed eggshell in the oxidation of crystal violet
and dye bathing wastewater by thermally activated peroxide oxidation method.
Environ. Res. 212 (Part A), 113210.
Zaimbashi, R., Mostafavi, A., Shamspur, T., 2021. ZnO nanoflower based electrochemical
sensor for the selective determination of venlafaxine. J. Iran. Chem. Soc. 1–9.
Zhang, C., Cao, Z., Zhang, G., Yan, Y., Yang, X., Chang, J., Wei, J., 2020. An
electrochemical sensor based on plasma-treated zinc oxide nanoflowers for the
simultaneous detection of dopamine and diclofenac sodium. Microchem. J. 158,
105237.
Zou, X., Ke, J., Hao, J., Yan, X., Tian, Y., 2022. A new method for synthesis of ZnO
flower-like nanostructures and their photocatalytic performance. Phys. B Condens.
Matter 624, 413395.