Applied Physics A (2020) 126:824

https://doi.org/10.1007/s00339-020-04007-1

In‑vitro antibacterial and anti‑biofilm efficiencies

of chitosan‑encapsulated zinc ferrite nanoparticles
Rashmi P. Sharma1 · Siddheshwar D. Raut2 · Ambadas S. Kadam3 · Ramjan M. Mulani1 · Rajaram S. Mane2

Received: 18 July 2020 / Accepted: 17 September 2020 / Published online: 30 September 2020
© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Recently, nanoparticle (NP)-encapsulated surfaces have received remarkable attention as a promising antimicrobial alternate.
Thereby, the present investigation focuses to develop chitosan-encapsulated zinc ferrite nanoparticles (CT-ZnFe2O4 NPs) for
antibacterial and anti-biofilm efficiencies against the range of pathogens. In this study, ­ZnFe2O4 NPs synthesized by a sol–gel
auto-combustion method are coated with a natural CT polymer. Agar well diffusion, growth kinetics and colony-forming unit
measurement studies demonstrated that the CT-ZnFe2O4 NPs behave excellent antibacterial agent against both Gram-positive
and Gram-negative bacteria. Subsequently, their inhibitory effect on biofilm formation and removal of established biofilm
are also been evaluated. Obtained results demonstrated that the CT-ZnFe2O4 NPs inhibit the biofilm formation more than
65% and reduce established biofilm up to 50% at a respective minimum inhibitory concentration (MIC). Promising findings
of this study suggest an exciting opportunity in antimicrobial therapy like antibacterial coatings that wound care and target
drug delivery in biofilm treatment.

Keywords Antibacterial activity · Anti-biofilm activity · Chitosan · CT-ZnFe2O4 NPs · Sol–gel auto-combustion method

1 Introduction intense research [9]. As on date, most conventional metal

and metal oxide NPs (like silver, zinc, copper, zinc oxide and
Ineffective response of conventional antimicrobial drugs and copper oxide) have massively been screened for anti-biofilm
rapid spread of drug resistance raise a serious concern in activity [10–12], although some of these NPs are being criti-
the global public health [1, 2]. Additionally, their biofilm cised for their toxic behaviour for mammalian cells [13, 14].
mediated pathogenesis exacerbate the healthcare system Despite toxicity, high surface energy of these NPs is another
especially the indwelling medical devices [3, 4]. Therefore, major drawback because they tend to agglomerate quickly in
an innovative antimicrobial approach is on high demand [5]. a biological medium and thereby hamper antimicrobial per-
Over the years, nanoparticles (NPs) have received much formance [15]. To overcome these limitations, surface engi-
attention to deal with the antibiotic mediated crisis [6, 7]. neered NPs are turned out to be more secure and effective
Their exceptional properties and multi-mode actions have antimicrobials which are strongly related to the inclusion of
demonstrated a great advantage over the antibiotics [8]. different functional moieties over the surface of NPs [16].
More recently, targeting the biofilm with NPs is a topic of In this context, chitosan (CT) polymer is the most prefer-
able surface coating agent as it serves excellent therapeutic
* Rashmi P. Sharma properties along with remarkable biocompatibility [17, 18].
[email protected] Till date, several attempts were made to avail the benefits
* Rajaram S. Mane of CT polymer for significant antimicrobial activity [19,
[email protected] 20]. From this perspective, Arakha et al., proposed surface
modulation of iron oxide NPs for strengthening antimicro-
1
School of Life Sciences, Swami Ramanand Teerth bial response. This improved antimicrobial efficiency can
Marathwada University, Nanded, Maharashtra 431606, India
largely be credited to positively charged CT which facilitates
2
School of Physical Sciences, Swami Ramanand Teerth a better interaction with negatively charged microorganisms
Marathwada University, Nanded, Maharashtra 431606, India
[21]. In another study, the antibacterial properties of CT-
3
Department of Botany, DSM’S ACS, College, District coated superparamagnetic iron oxide NPs appears to be
Parbhani, Jintur, Maharashtra 431509, India

13
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824 Page 2 of 9 R. P. Sharma et al.

superior upon surface-coating. In this study, we showed that 2.2 Synthesis and measurements
the CT provides greater stability to NPs by preventing the
agglomeration and, ultimately, which supports antimicro- 2.2.1 Synthesis of ­ZnFe2O4 NPs and chitosan polymer
bial potency [22]. Similarly, Nehra et al., [23] examined the surface‑coating
antimicrobial activity of CT-coated iron oxide NPs against
a diverse range of bacteria and fungi including Escherichia Initially, ­ZnFe2O4 NPs were synthesized by a conventional
coli, Bacillus subtilis, Candida albicans, Aspergillus niger sol–gel auto-combustion method in presence of citric acid
and Fusarium solani. Their results also supported a strong as reducing agent [36]. Surface-coating steps were carried
antimicrobial activity of NPs over uncoated counterparts. out according to a procedure described preciously with some
Moreover, numerous reports evidenced that the CT-based modifications [37]. Briefly, stoichiometric amounts of chem-
NPs hold a great potential in anti-biofilm activities [24, 25]. ical solutions (presented in Table 1) including zinc nitrate,
Mainly, polycationic CT plays important role in biofilm ferric nitrate and citric acid were prepared in distilled water,
eradication by penetrating the negatively charged biofilm followed by a slow addition of liquid ammonia to adjust the
matrix followed damaging the biofilm embedded cells [26]. pH of the solution close to 7 while constant stirring. The
It also helps to reduce the bacterial adhesion during early mixture was magnetically stirred for ~ 3 h at 80–90 °C until a
stages of biofilm formation [27]. Therefore, CT-based NPs wet gel of the metal nitrates was obtained which was allowed
frequently reported as an efficient drug carrier to achieve to burn till it turned out to be an ash. Ash was collected and
complete eradication of biofilm [28]. Overall, these studies calcined in a muffle furnace at 500 °C for 5 h. As-obtained
showed that the CT polymer demonstrates several promises NPs were surface-coated using CT-polymer. For that 0.5 g
for practicing in antimicrobial applications. Since few years, CT was suspended in 2% (V/V) glacial acetic acid solu-
­ZnFe2O4 and its composites have received considerable tion and stirred for 20 min at 65 °C. After stirring, 0.5 g of
popularity in various fields such as electronics, chemical, the as-synthesized Z ­ nFe2O4 NPs were added into an above
optical and biogenic sensors and photocatalysts on account solution and then vigorously stirred for 20 min following
of their unique magnetic and electrical properties, and good 15 min room-temperature (27 °C) sonication. Finally, 1 ml
chemical stability and mechanical robustness [29, 30]. A low of 2% glutaraldehyde solution was added into this solution
saturation magnetization, coercivity, and conductivity along and kept on a magnetic stirrer for 15–20 h. Thereafter, the
with biocompatibility have made them as an excellent choice above solution was centrifuged at 2000 rpm for 20 min, and
in biomedical applications [31, 32]. However, agglomera- the pellet was collected and washed twice in deionised water.
tion tendency of the Z­ nFe2O4 NPs hinders putative applica- Then, the pellet was dried in a hot air oven at 65 °C for 1 h.
tions to some level which can be restrained via CT coating.
Thereby, CT-ZnFe2O4 NPs are being widely researched for 2.3 Characterizations
theranostic applications including bio-imaging, hyperther-
mia therapy and drug delivery [33–35]. Notably their anti- The structural elucidation, surface morphology analy-
microbial potential has not been, till date, addressed well. sis and elemental scrutiny were confirmed from various
Considering all these aspects, the present investigation is physical instruments. The X-ray diffraction pattern of
intended to explore antibacterial and anti-biofilm properties ­ZnFe2O4 NPs was recorded on X-ray diffractometer (XRD,
of the CT-ZnFe2O4 NPs. D8-Discovery Bruker, Cu Kα, 40 kV, 40 mA) which was
scanned from 10 to 100°. Scanning electron microscopy
(SEM, Hitachi, S-4800, 15 kV) and transmission electron
2 Experimental details microscopy (TEM) digital surface images were recorded to
confirm surface-type, shape and size of obtained NPs. The
2.1 Materials surface area of the as-prepared NPs was recorded from the
Brunauer–Emmett–Teller (BET) measurement. The func-
Selected pathogenic strains Gram-positive bacteria Staphy- tionality of the as-prepared CT-ZnFe2O4 NPs was monitored
lococcus epidermidis (MTCC-435), Staphylococcus aureus
(MTCC-3160), and Gram-negative bacteria Escherichia
coli (MTCC-40), Pseudomonas aeruginosa (MTCC-424)
were purchased from the Institute of Microbial Technology Table 1  Chemical details used while preparing ­ZnFe2O4 NPs
(IMTECH), Chandigarh, India. All bacterial cultures were Zinc nitrate Ferric nitrate Citric acid
grown overnight on Luria–Bertani agar slants and main- Zn(NO3)2·6H2O Fe(NO3)3·9H2O C6H8O7
tained at 4 °C for further experiments. All inorganic materi- (g) (g) (g)
als used in the present work were purchased from the Sigma
6.273 17.04 13.30
Aldrich and used as received.

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In‑vitro antibacterial and anti‑biofilm efficiencies of chitosan‑encapsulated zinc ferrite… Page 3 of 9 824

using Fourier transform infrared (FTIR) spectroscopy analy- previously by Barros et al., [39] with some modifications.
sis. Surface elements and their proportions were examined The remaining culture was decanted from the wells of the
from the energy-dispersive X-ray spectroscopy (EDX) bacterial growth kinetics experiment and the wells were
measurement. washed three times with sterile phosphate buffer saline
(PBS) and stained with 0.1% crystal violet for 15 min.
2.4 Antibacterial evaluation Excess stain was rinsed off by thorough washing with ster-
ile PBS and plate was kept for drying. After drying, stained
2.4.1 Agar well diffusion method adherent cells were re-dissolved in 30% (v/v) glacial acetic
acid and optical density (OD) was determined at 600 nm.
Antimicrobial activity of the CT-ZnFe2O4 NPs was deter- The experiment was performed in triplicate. The inhibitory
mined by Kirby-Bauer method [38]. Briefly, overnight effect of the CT-ZnFe2O4 NPs on biofilm formation was cal-
grown culture of test bacteria was used to prepare standards culated using the following relation;
inoculums by adjusting turbidity equal to the standard 0.5
Inhibition (%)
McFarland solution at 600 nm. This inoculum suspension of
bacteria was swabbed uniformly on a nutrient agar plate. In {OD of untreated control − OD of treated sample}
= (1)
each agar plate, wells of 6 mm diameter were made by flame OD of untreated control
sterilized cork borer. Using a micropipette, a 100 μL solution × 100.
of the NPs was poured in each well of all plate and all plates
were kept for overnight incubation at 37 °C. After incuba-
tion, plates were envisaged for antimicrobial activity and 2.8 Inhibition of established biofilm inhibition
obtained results were recorded as a zone of inhibition (ZOI).
Anti-biofilm effect of the ­ZnFe2O4 NPs was evaluated as
2.5 Bacterial growth kinetic analysis the method described above. Biofilms were cultivated as
described previously for 24 h. After incubation, the wells
Growth kinetics studies were performed in presence and were washed three times with sterile PBS to remove non-
absence of various concentrations of CT-ZnFe 2O4 NPs adherent cells. Wells containing attached biofilms were
(range from 0 to 256 μg/mL) incubated at 37 °C. The reac- treated with various concentrations of CT-ZnFe 2O4 NPs
tion mixtures without NPs served as control. Briefly, inocu- and the plates were incubated at 37 °C for 24 h; untreated
lum suspensions of all bacteria were prepared (as described cells were treated as a control. After incubation, wells were
in the above assay) and about 100 μL of these bacterial sus- washed again with sterile PBS and stained with crystal violet
pensions were seeded in 96-well plates. Afterwards, respec- as per the inhibition of biofilm-forming assay and 0D was
tive concentrations of NPs were added into the respective measured at 600 nm while the percentage of biofilm inhibi-
reaction mixtures. Growth rates of bacterial species were tion was calculated using above relation.
assessed at regular time intervals by measuring absorbance
at 600 nm using a plate reader.
3 Results and discussion
2.6 Colony‑forming units (CFU) measurement
3.1 Structure and morphology analyses
To quantify the CFU, 50 μL of the sample was taken from
the stationary phase of the bacterial growth kinetics experi- The XRD peaks of various intensities as seen in Fig. 1a
ment which was serially diluted in sterile water. Following were in accord to (111), (220), (311), (222), (400), (522),
which, 10 μL of the final dilution was distributed onto nutri- (511), (440), (533) and (553) reflection planes which are
ent agar plates and incubated at 37 °C. After 24 h incubation, well-matched with the JCPDS card no. 22-1012. Obtained
colonies were counted and compared with positive control. data illustrates the sharp diffraction peaks of high intensity
This experiment was performed in triplicate as reconfirma- without any impurity, confirming the formation of phase-
tion test. pure and polycrystalline ­ZnFe2O4. The crystallite size is a
very important aspect that influences the physicochemical
2.7 Anti‑biofilm study properties of ferrites which is controlled by annealing tem-
perature. In this study, the product material was annealed
2.7.1 Inhibition of biofilm formation to 500 ºC because below this annealing temperature, fer-
rite particles were amorphous or of lower crystallinity.
The inhibitory effect of the CT-ZnFe2O4 NPs on biofilm Conversely with increasing annealing temperature a better
formation was determined through a method reported

13
824 Page 4 of 9 R. P. Sharma et al.

Fig. 1  a XRD, b SEM, c TEM, d ­N2 adsorption–desorption, e pore-size distribution, and f EDX measurements of ­ZnFe2O4 NPs

crystallinity but enlarged the particle size could be obtained

[30, 40]. The average crystallite size calculated using the
Scherrer equation was ~ 28 nm. The SEM image as shown in
Fig. 1b confirmed a random distribution of NPs in an aggre-
gated form due to which noting a precise size and shape
of individual nanoparticle was tedious. The TEM image as
shown in Fig. 1c presents the formation of different sized
NPs (20–60 nm). In addition, this image endowed 0.25 nm
interplanar spacing which is in good agreement to the inter-
planar reflection of (311) plane. The difference between the
two measurements was assigned to the presence of more
than one crystal boundaries on the surface of NPs. Since the
XRD cannot distinguish between the two boundaries; there-
fore, the actual size (examined form TEM) could be greater
than that calculated from the Scherrer equation [41]. The ­N2
adsorption–desorption curves of the Z ­ nFe2O4 are shown in
Fig. 1d with corresponding pore-size distribution (Fig. 1e)
where type IV isotherm with 24.59 m2g−1 specific surface Fig. 2  FTIR spectrum of the CT-ZnFe2O4 NPs
area of the Z
­ nFe2O4 NPs is evidenced. The pore-size distri-
bution curve of the ­ZnFe2O4 NPs has suggested mesoporous
character [42]. Figure 1f shows the EDX elemental analysis spinel ferrites (Fig. 3). Particularly, the absorption bands
where surfaces of the as-synthesized ­ZnFe2O4 NPs revealed at 454–467 cm−1 and 568–606 cm−1 were ascribed to the
1:2:4 stoichiometric ratios, which is well in accordance with presence of metal-oxygen stretching vibration in octahedral
the chemical formula proposed through a structural analysis. sites and tetrahedral sites, respectively. In ­ZnFe2O4 NP, ­Zn2+
In FTIR spectrum of the as-obtained CT-ZnFe2O4 NPs occupies the tetrahedral sites, while the ­Fe3+ occupies both
(Fig. 2) confirmed absorption peak at 543 cm−1, which is octahedral and tetrahedral sites. Hence, the absorption peak
in association with 600–400 cm−1, commonly observed for at 543 cm−1 could be associated with Zn–O stretching in

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Fig. 3  Antimicrobial activity of the CT-ZnFe2O4 NPs against; a S. epidermidis, b S. aureus, c E. coli, and d P. aeruginosa

Table 2  Antimicrobial activity (zone of inhibition) of the CT- concentration 32 μg/mL) of CT-ZnFe2O4 NPs, for nearly all
ZnFe2O4 NPs against microbial pathogen (The data is the mean ± SD studied bacteria. Therefore, we excluded them in the rest
<0.05)
of the studies. The CFU measurement (Fig. 5) clearly indi-
Microbial strain Zone of cated a viability of all bacterial cells which is significantly
inhibition reduced with increasing concentration, confirming a strong
(mm)
antimicrobial activity of CT-ZnFe2O4 NPs against all studied
Staphylococcus epidermidis (MTCC-435) 17 ± 1.20 bacteria. In addition, CFU measurement was used to evalu-
Staphylococcus aureus (MTCC-3160) 16 ± 0.50 ate the MIC of CT-ZnFe2O4 NPs. MIC was considered as
Escherichia coli (MTCC-40) 19 ± 0.20 the lowest concentration that inhibited ≥ 50% of microbial
Pseudomonas aeruginosa (MTCC-424) 22 ± 0.10 growth. The CFU data demonstrated that the lowest amount
of concentrations 64 and 128 μg/mL can successfully be
reduced the cell growth of Gram-negative bacteria and
Gram-positive bacteria, respectively.
tetrahedral sites [42, 43]. The absorption peak at 1047 cm−1
suggested an existence of C–O bonds. The peak located at 3.3 Antibiofilm efficacy of the CT‑ZnFe2O4 NPs
1383 cm−1 was due to a stretching vibration of C=O. The
peak at 2854 cm−1 could be due to the stretching vibrations Biofilm formation is the most crucial step of microbial life-
of the C–H groups. The bands at 3433 cm−1 and 1629 cm−1 style which is attributed to drug resistance and contributes
were in support of the stretching vibration of OH group. to almost 80% infectious disease alone [45]. Given to fact
The FTIR data collected here were almost similar to those that researcher opined, targeting biofilm is an important
reported previously for CT-ZnFe2O4 NPs [37, 42], suggest- tactic to get effective control over the pathogens [9], hence
ing a successful encapsulation of chitosan over Z
­ nFe2O4 NP anti-biofilm activity of the CT-ZnFe2O4 NPs was evaluated.
surfaces. Data of present study as shown in Fig. 6a revealed that the
biofilm formation is reduced by almost 30–87% with the
3.2 Antibacterial efficacy of CT‑ZnFe2O4 NPs increase of concentration of the CT-ZnFe2O4 NPs as com-
pared to untreated samples. About 66–75% reductions in
The as-synthesized CT-ZnFe2O4 NPs were utilized to access biofilm formation were achieved at their respective MIC. In
antimicrobial efficiency and their results were expressed addition, the effects of CT-ZnFe2O4 NPs on already estab-
as a zone of inhibition (mm) shown in Fig. 3. Results of lished biofilms were also determined. As shown in Fig. 6b,
the present study clearly demonstrated excellent antibac- about 65–78% of established biofilms were prominently
terial activity of the CT-ZnFe2O4 NPs against all bacteria inhibited at the highest concentration (256 μg/mL) of the
as summarized in Table 2. The maximum zone of inhibi- CT-ZnFe2O4 NPs. About 51–57% inhibitions were found
tion was recorded for the P. aeruginosa (22 ± 0.10 mm) for their respective MIC concentration. However, lower
and the least zone of inhibition was recorded for S. aureus concentration i.e. 32 μg/mL did not exert effective response
(16 ± 0.50 mm). Obtained results revealed a strong antimi- in anti-biofilm studies. Present study inferred that the MIC
crobial efficiency than previously reported studies [29, 44]. of the CT-ZnFe2O4 NPs not only significantly restricts the
The bacterial growth kinetic studies were performed with biofilm formation but also supports the inhibition of estab-
different concentrations of CT-ZnFe2O4 NPs. All studied lished biofilms.
bacteria (Fig. 4) confirmed the significant growth inhibition Results of the present study were very close to previ-
as compared to their respective control. This effect, how- ous findings, when CT based NPs were more promising
ever, was not so efficient at lower concentrations (below the against Gram-negative bacteria [46]. However, the exact

13
824 Page 6 of 9 R. P. Sharma et al.

Control Control
1.2 8 µg/ml 1.2 8 µg/ml
16 µg/ml 16 µg/ml
O. D. (600 nm) 32 µg/ml 32 µg/ml

O. D. (600 nm)
0.9 64 µg/ml 0.9 64 µg/ml
128 µg/ml 128 µg/ml
256 µg/ml 256 µg/ml
0.6 0.6

0.3 0.3
(a) (b)
0.0 0.0
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)
Control Control
1.2
1.2 8 µg/ml 8 µg/ml
16 µg/ml 16 µg/ml
32 µg/ml 32 µg/ml
O. D. (600 nm)

O. D. (600 nm)
64 µg/ml 0.8 64 µg/ml
0.8 128 µg/ml 128 µg/ml
256 µg/ml 256 µg/ml

0.4
0.4

(c) 0.0 (d)

0.0
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)

Fig. 4  Bacterial growth kinetic analysis in presence and absence of the CT-ZnFe2O4 NPs for; a S. epidermidis, b S. aureus, c E. coli and d P.
aeruginosa. Triplicate experiments were performed for each reaction. Error bar represents the standard error of the mean

mechanism behind antimicrobial activity has not yet clearly

been defined. The most accepted one illustrated that the pos-
Control
100 32 µg/ml itively charged CT-based NPs get attracted toward negatively
64 µg/ml charged microorganisms by electrostatic interaction on alter-
Bacterial cell viability (%)

128 µg/ml ing membrane permeability of bacteria which leads to loss

80 of the cellular activity and eventually causes the cell death
256 µg/ml
[47]. Even though it seems to be, Gram-negative bacteria are
60 more susceptible to CT-based NPs probably due to the pres-
ence of higher negative charge on the cell membrane that
40 can facilitate higher affinity for positively charged CT based
NPs [47]. In addition to this, one more possibility could
be pointed that, Gram-negative bacteria enclosed with thin
20 lipopolysaccharides layer are likely to rupture more easily as
compared to thick peptidoglycan layer of the Gram-positive
0 bacteria [48]. Meanwhile, some reported studies highlighted
S. epidermidis S. aureus E. coli P. aeruginosa --
that the CT molecule can bind to the cell surface and affect
the nutrient exchange between microbial cells and their sur-
Fig. 5  Colonies forming unit measurements were quantified and rep- rounding environment [49]. In the present investigation, the
resented as the percentage of viable bacterial cells in comparison to
control. Error bars are expressed in terms of standard error from the CT-ZnFe2O4 NPs exhibited a better antimicrobial and anti-
mean biofilm efficiencies than previous reports. Interestingly, this

13
In‑vitro antibacterial and anti‑biofilm efficiencies of chitosan‑encapsulated zinc ferrite… Page 7 of 9 824

Fig. 6  a Effect of the CT- 120 120

Inhibition of established bioilm (%)

Inhibition of bioilm formation (%)
ZnFe2O4 NPs on biofilm forma- 32 µg/ml
64 µg/ml
(a) 32 µg/ml
64 µg/ml
(b)
tion, and b established biofilms. 100 128 µg/ml 100 128 µg/ml
Error bars are expressed in 256 µg/ml 256 µg/ml
terms of standard error from 80 80
the mean
60 60

40 40

20 20

0 0
S. epidermidis S. aureus E. coli P. aeruginosa S. epidermidis S. aureus E. coli P. aeruginosa

study observed a quite similar microbial sensitivity pattern to develop and established biofilms causes a decrease in the
for NPs to those found in the literature [29, 50, 51]. From viable cells count which has made them unputdownable
extensive studies, it has been reported that the antibacterial material for biofilm treatment. The present findings present
activity of nano-ferrites is highly associated with the produc- an importance of ferrite products in anti-bacterial and anti-
tion of reactive oxygen species (ROS) while susceptibility biofilm activities. The antifungal activity of the CT-ZnFe2O4
pattern of microbes for NPs depends on dissimilarity in bac- NPs as an extended target is the upcoming study.
terial cell membrane composition [52]. In recent studies, the
semiconductor nature of Z ­ nFe2O4 NPs with narrow bandgap Acknowledgements The authors would like to thanks Director of
UGC-DAE-CSR for allowing the EDAX facility. Thanks to Dr. D. M.
for photo-catalytic activity [29] has been put forward. Gener- Phase and V. K. Ahire for allowing time slot for performing the experi-
ally, presence of holes in valence band and electrons in the ments and providing data.
conduction band of Z ­ nFe2O4 NPs are responsible for redox
reactions, resulting in the production of a large number of Funding None.
electron–hole pairs system which generates the free radi-
cals through series of reactions. Holes react with water to Compliance with ethical standards
produce a hydroxyl radical while the lone electrons in the
conduction band react with dissolved oxygen molecules to Conflict of interest The authors declare that they have no conflict of
interests.
form a superoxide anion which also can be applicable for
antimicrobial activity [53]. Moreover, several investigators
revealed that the antimicrobial activity of such NPs can be
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