Environmental Research 188 (2020) 109555

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Environmental Research
journal homepage: www.elsevier.com/locate/envres

Ag-doped magnetic metal organic framework as a novel nanostructured T

material for highly efficient antibacterial activity
Ziba Rahmatia,b, Jafar Abdic, Manouchehr Vossoughia,b,∗, Iran Alemzadeha,b
a
Department of Chemical and Petroleum Engineering, Sharif University, Tehran, Iran
b
Institute for Biotechnology and Environment, Sharif University of Technology, Tehran, Iran
c
Faculty of Chemical and Material Engineering, Shahrood University of Technology, Shahrood, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: In the last decades, numerous attempts have been made to prevent microbial pollution spreading, using anti-
Metal organic framework bacterial agents. Zeolitic imidazolate framework-8 (ZIF-8) belongs to a subgroup of metal organic frameworks
ZIF-8 (MOFs) merits of attention due to the zinc ion clusters and its effective antibacterial activity. In this work, Ag-
Magnetic nanostructured material doped magnetic microporous γ-Fe2O3@SiO2@ZIF-8-Ag (FSZ-Ag) was successfully synthesized by a facile
Silver doping
methodology in room temperature and used as an antibacterial agent against the growth of the Gram-negative
Antibacterial activity
Escherichia coli and Gram-positive Staphylococcus aureus bacteria. Several characterization methods were applied
to analyze the properties of the materials, and the results confirmed the accuracy of the synthesis procedure.
Silver ions have employed to enhance the efficiency of antibacterial activity. As the results illustrated, FSZ-Ag
nanostructured material had superior performance to inactive E. coli and S. aureus in growth inhibition test in
liquid media. The best antibacterial activity as minimum inhibitory concentration (MIC) was 100 mg/L of FSZ-
Ag against both bacteria. Leaching rates of silver ions showed that 80% of Ag released in the solutions, which
was responsible for inhibiting the growth of bacteria. Also, fluorescence microscopy was used to investigate
bacterial viability after 20 h contacting FSZ-Ag to distinguish live and dead bacteria by staining with DAPI and PI
fluorescence stains. This novel magnetic nanostructured material is an excellent promising candidate to use in
biological applications as high potential bactericidal materials.

1. Introduction been mostly considered in various applications such as medical and

Microbial infection is recognized as one of the most threatening
factors for human health (Wyszogrodzka et al., 2016); therefore, re-
searchers have investigated different remedies to overcome its irre-
parable damages. Generally, three waves of disinfectant are used
against bacteria. The first wave is chemical disinfectants, which can be
defined as chemicals that inhibit and inactivate microbes (Brudzynski,
2006; Odlaug, 1981) (such as halogens, peroxides and phenols). The
second wave is characterized by the use of metals and metal oxides (Jan
et al., 2014) (such as CuO, Cu2O, ZnO and CoO). The third wave is the
use of one-dimensional (colloids), two-dimensional (Graphene oxide
(Liu et al., 2011), MXenes (Chen et al., 2020) and MoS2 based nano-
materials (Zeng et al., 2018b)) or three-dimensional (MOFs (Alavijeh
et al., 2018)) compounds, either without supports or with structured
supports such as nanotubes, chitosan (Song et al., 2015), silica nano-
fibers (Min et al., 2007), and biopolymers (Huang et al., 2018a).
Amongst these materials the antimicrobial property of metal ions has
dental therapies. Metals and metal oxides will show an efficient anti-
bacterial activity when they are very tiny in size. Among these, silver,
zinc, and copper have noticeable antibacterial property (Besinis et al.,
2014). So far, silver nanoparticles have been widely used in different
products such as medicine, surgical instruments, cosmetics, electrical
machines, etc. (Jung et al., 2007). There is not an exclusive mechanism
for antibacterial activity of silver ions; nonetheless, some researchers
have presented different approaches such as the interaction between
silver and bacteria's thiol group which results in decomposition of
bacteria enzymes and proteins (Jung et al., 2008; Spadaro et al., 1974).
Besides, silver nanoparticles go through the cell wall and transform
bacteria DNA, which impedes cell division (Sondi and Salopek-Sondi,
2004). Therefore, silver has employed in multiple methods to act as an
antibacterial agent. Some approaches emphasize preparation of Ag-
based materials by doping or encapsulating silver ions as a supple-
mentary particle into the precursor carriers to enhance the intended
activity along with providing more efficiency of antibacterial effect

∗
Corresponding author. Department of Chemical and Petroleum Engineering, Sharif University, Tehran, Iran.
E-mail address: vosoughi@sharif.edu (M. Vossoughi).

https://doi.org/10.1016/j.envres.2020.109555
Received 1 February 2020; Received in revised form 13 April 2020; Accepted 17 April 2020
Available online 21 April 2020
0013-9351/ © 2020 Elsevier Inc. All rights reserved.
Z. Rahmati, et al.
(Alexander, 2009; Jung et al., 2008; Lu et al., 2014; Panáček et al.,
2018; Tang et al., 2016).
Recently, a new class of porous materials, nominated metal-organic
frameworks (MOFs), have widely used in multiple applications because
of their advantageous properties. These nanoscale pore materials due to
the tailorable and designability of their topological structures were
applicable in so many fields, including adsorption (aqueous and gas-
eous phase), storage (gas and liquid components), catalysis (UV/Vis
light photocatalyst), ion exchange, sensing (chemical sensors), device
fabrication, biomedicine (drug delivery, antimicrobial applications),
and others (Abdi et al., 2019b; Mahmoodi and Abdi, 2019a, 2019b;
Mahmoodi et al., 2019a, 2019b). Furthermore, MOFs properties can be
modified utilizing several ways like supplementing of active groups or
particles for target plans (Ahmed and Jhung, 2014; Furukawa et al.,
2013; Maya et al., 2015; Pang et al., 2015; Quirós et al., 2015). Mod-
ification processes involve three methods, including in-situ ion doping,
reformation of organic ligand, and encapsulation of active elements
(Mao et al., 2014). Moreover, many types of researches have shown the
capability of MOFs antibacterial activity against both kinds of bacteria
according to gram staining standard (gram-negative and positive) and
some yeast (Aguado et al., 2014; Au-Duong and Lee, 2017; Lu et al.,
2014; Quirós et al., 2015). The antibacterial performance of MOFs is
attributed to their structure characteristic. Normally, MOFs are un-
stable in aqueous media, and the metal ions leave the construction and
act as an antibacterial agent (Wu et al., 2018). Zeolitic imidazolate
frameworks (ZIFs), are a classified branch of MOFs with high surface
area and excellent thermal and chemical stability in a variety of organic
solvents which lead ZIFs as well-candidate materials for abundant
functionalities. The tetrahedral structure of ZIFs consists of Zn2+ or
Co2+ salts bonding with N atoms in 2-methyl imidazolate ligands or-
ganic source. Among ZIFs, zeolitic imidazolate framework-8 (ZIF-8) is a
more attractive choice than other relatives in many applications due to
its chemical and physical properties with different usages (Abdi et al.,
2017a; Cravillon et al., 2009; Lee et al., 2015; Maya et al., 2015).
Furthermore, related to zeolites, framework flexibility of ZIF-8 pro-
motes surface modification (Fairen-Jimenez et al., 2011). So, this MOF
can be used as a good substrate candidate for doping Ag nanoparticles
and also, the Zn2+ cations in ZIF-8 structure can be improve the anti-
bacterial activity of silver ions via synergetic effect.
In spite of the special properties of MOFs, separation of these par-
ticles is a common problem when these materials are dispersed in a
solution through various applications. This concern can be resolved by
using magnetically separable nanoparticles which facilitate recycling
when a magnetic field is applied externally. Different magnetic supports
(such as Fe3O4 (Chi et al., 2013) and Fe2O3 (Wu et al., 2014)) have been
recruited so far in some reports. Iron oxides are such renowned mag-
netic nanoparticles (MNPs) and have acquired more attention. Fe3O4@
ZIF-8 structure has been reported in several works for different appli-
cations such as removal of toxic pollutant from water, dye degradation,
and adsorption, selective separation of target product from solutions,
etc. (Huo et al., 2018; Pang et al., 2015; Zhang et al., 2013a; Zheng
et al., 2015). These studies prove the functional advantages of magnetic
supports in the separation process.
Till now, different methods have been emerged and utilized for the
fabrication and surface modification of materials through layer by layer
assembly, self-assembly monolayer formation, hydrolysis of functional
silanes, Langmuir–Blodgett deposition, plasma surface modification,
etc. (Liu et al., 2016). However, many of these existing strategies have
limitations, including chemical specificity, the size and shape of the
substrates, requirement of complex instruments and multistep proce-
dures. Mussel-inspired chemistry has recently emerged as one of the
most important and universal method for the surface modification of
different inorganic and organic materials owing to its mild experiment
conditions and high modification efficiency. The mussel-inspired
chemistry is mainly inspired by the composition of adhesive proteins of
mussel feet and it is based on the adhesion of dopamine (DA) toward
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Environmental Research 188 (2020) 109555
surfaces via simple dip-coating of objects in an aqueous solution (Lee
et al., 2007). The simple operation and versatility of mussel inspired
chemistry make it to attract great attention from various fields such as
biomedical applications (Liu et al., 2015; Shi et al., 2017; Zeng et al.,
2018a; Zhang et al., 2013b) (Photothermal therapy, biological imaging,
drug delivery, biosensing, tissue engineering and antibacterial activity)
and environmental applications (Dou et al., 2019; Gan et al., 2020;
Huang et al., 2017a, 2018b, 2018c, 2018d; HYPERLINHuang etal.,
2017b; Liu et al., 2018; Zhang et al., 2015, 2017) (oil/water separation,
removal of heavy metal ions, organic dyes and catalytic reduction of
environmental pollutants). Considering the various advantages of
mussel inspired chemistry, it is has a great potential to be applied for
the fabrication of antibacterial applications. In fact Ag nanoparticles
can be deposited in situ on different supports (GO sheets, magnetic
nanoparticles, films, MOFs, etc.) taking advantage of the adhesion of
dopamine.
According to these all considerations, in this work, γ-Fe2O3 mag-
netic nanoparticles were synthesized and covered by an amorphous
SiO2 layer to prevent physical or chemical degradation of MNPs in
various solutions. Then, γ-Fe2O3@SiO2 MNPs were surrounded by a
layer of ZIF-8 particles in room temperature to obtain γ-Fe2O3@SiO2@
ZIF-8 (FSZ) nanostructure. Thereafter, the surface of the nanostructures
was modified by immobilizing silver particles due to their antibacterial
activity. Use of MNPs facilitated the separation process of nanos-
tructured materials from experimental environment readily. Several
analyses such as FTIR, SEM, XRD, EDX, ICP-OES, and VSM were studied
to characterize the prepared materials. The synthesized materials were
employed in antibacterial experiments against two different types of
bacteria strains.
2. Materials and methods
2.1. Materials and instruments
Iron (III) chloride (FeCl3), sodium acetate, ethylene glycol, zinc
nitrate (Zn(NO3)2.4H2O), 2-methylimidazole, methanol, ammonia aqu-
eous solution (25%), ethanol, tetraethylorthosilicate (TEOS), silver ni-
trate (AgNO3) and the other consuming materials were purchased from
Merck company (Germany). XRD patterns were measured using a
Bruker D8 advanced X-ray diffractometer with CuKα irradiation
(λ = 0.15406 nm) and FTIR spectra obtained by applying NB series
spectrometer. The morphology of the structures was investigated using
a scanning electron microscopy (SEM, XL30 model) and energy dis-
persive spectroscopy (EDS) on the same instrument was conducted. The
magnetic properties of the prepared materials were measured by VSM
analysis. Inductively coupled plasma optical emissions (ICP-OES) was
performed on a Varian 730-OES. The optical density of bacteria sus-
pension was detected at 600 nm applying a UV spectrophotometer for
determination of bacterial turbidity.
2.2. Synthesis of γ-Fe2O3 MNPs
The solvothermal method was employed to synthesize of γ-Fe2O3
magnetic nanoparticles. Briefly, 0.74 g of Iron (III) chloride was dis-
solved in 60 mL of ethylene glycol, followed by stirring for 20 min to
obtain a transparent solution. After that, 2.16 g of sodium acetate was
added to the previous solution, and the mixture was stirred con-
tinuously for an hour. Eventually, the final mixture was transferred to a
Teflon-lined stainless steel autoclave which was sealed and heated at
160 °C for 12 h. At the end of the reaction, the autoclave was cooled to
the ambient temperature, and the final product was gathered by a
magnet. Finally, γ-Fe2O3 MNPs was washed several times by ethanol
and deionized water and dried in the oven at 60 °C for 24 h.
Z. Rahmati, et al. Environmental Research 188 (2020) 109555

2.3. Synthesis of γ-Fe2O3@SiO2

The γ-Fe2O3 magnetic nanostructured were covered through an

amorphous SiO2 layer of using the modified Stöber procedure (Abdi
et al., 2019a; Chi et al., 2013). To achieve this goal, a mixture, in-
cluding pure ethanol, aqueous ammonia solution with 25% purity and
deionized water (40 mL, 1.2 mL, 20 mL, respectively), was prepared.
Then, 0.15 g of γ-Fe2O3 was dispersed into the mixture, and they were
intermingled together by stirring for 60 min. Next, tetra-
ethylorthosilicate (0.6 mL) was added to the mixture dropwise. After
mixing for 6 h, the substance was separated by applying the external
magnetic field and washed three times with pure ethanol, followed by
drying at 60 °C.

2.4. Synthesis of γ-Fe2O3@SiO2@ZIF-8 (FSZ)

The ZIF-8 crystals were formed on the surface of γ-Fe2O3@SiO2

nanostructure to synthesis γ-Fe2O3@SiO2@ZIF-8 nanocomposite. After
dissolving 0.41 g of Zn(NO3)2.4H2O in 30 mL of methanol, 0.1 g of γ-
Fe2O3@SiO2 was added and dispersed in the mixture by stirring. Then a
clear solution containing 1 mL of n-butylamine and 0.81 g of 2-me- Fig. 1. XRD patterns of the nanostructured materials: (a) ZIF-8, (b) γ-Fe2O3, (c)
thylimidazole (MIM) ligand in 30 mL of methanol was prepared. γ-Fe2O3@SiO2, (d) FSZ and (e) FSZ-Ag.
Afterward, the solution comprising organic ligand was added to another
solution dropwise. Then, white solid appeared in the mixture which
confirmed the formation of ZIF-8 crystals. The n-butylamine entered to
the ligand solution as a modulator to prevent overgrowing the ZIF-8
particles. Subsequently, the mixture was remained in room temperature
for 24 h to construct FSZ. The final product was collected magnetically
and washed, as mentioned in previous sections, then dried at 60 °C.
Pure ZIF-8 nanoparticles were prepared using the same procedure
without the presence of γ-Fe2O3@SiO2 MNPs (Abdi et al., 2017b).

2.5. Doping Ag ions on the surface of FSZ

A simple method was conducted to deposit silver ions on the surface

of FSZ nanostructure. Briefly, 22 mL of 5 mM AgNO3 solution con-
taining 20 mL pure ethanol and 2 mL deionized water was prepared.
After that, 0.1 g of FSZ powder was added to the prepared solution and
stirred for 2 h. When the reaction ran out, the magnetic FSZ-Ag na-
nostructured compound was collected by a magnet and rinsed three
times with ethanol and DI water and dried at 50 °C (Yang et al., 2018).

2.6. Antibacterial experiments Fig. 2. FTIR spectra of the nanostructured materials: (a) ZIF-8, (b) γ-Fe2O3, (c)
γ-Fe2O3@SiO2, (d) FSZ and (e) FSZ-Ag.

Antibacterial activity assessment was done to prevent the growth of

Escherichia coli (ATCC 1105) and Staphylococcus aureus (BBRC 10050)
bacteria. The microorganisms were kept in nutrient agar stocks, stored
at −4 °C for the long term until use. E. coli and S. aureus cells were
separately incubated in 100 mL of nutrient broth media to reactivate
them, and tracked by measuring optical density (OD) at 600 nm. Then,
growth inhibition assay and the zone of inhibition method were carried
out. Also, bacterial viability was evaluated using fluorescence micro-
scopy.
2.6.1. Inhibition zone on the agar plate
Agar plate disk diffusion method was accomplished to measure in-
hibition zone according to the standard method described by Bauer
et al. (1966). Sterile nutrient agar culture media was poured into the
separated sterile petri dishes before. The optical density of bacteria in
suspension was adjusted approximately at OD600 = 1 in comparison
with culture media. 100 μL of each microbial suspension were trans-
ferred to plates and dissipated uniformly to inoculation occur and let
dry at ambient temperature. Then, small amounts (~5 mg) of the
magnetic nanostructured materials were placed straightly onto the in-
oculated agar surface in powder form; also, ZIF-8 and AgNO3 were used
as the positive control. Then, plates were incubated at 37 °C, and
inhibition zones were measured after 24 h. Incubated plates without
antibacterial agents were also examined as a control to evaluate correct
microbial growth and the results accuracy. Sterile plates without any
bacteria suspension were incubated to investigate contamination
probability. The experiments were performed in triplicate and repeated
three times.
2.6.2. Growth inhibition in liquid culture media
In order to perform this test, at first, the colony of bacteria was
inoculated from a solid slant to 50 mL of sterile liquid media, kept at
37 °C and 150 rpm for 24 h until the bacteria reached OD600 = 1. Then,
an equal amount (1 mL) of suspension containing bacteria was added to
100 mL sterile nutrient broth in several separate Erlenmeyer. Then,
certain amounts of the compounds were added to the culture medium
containing the bacteria and the Erlenmeyer were placed in the shaking
incubator at 37 °C and 150 rpm. This experiment was carried out during
24 h, and optical density of each sample was measured using UV–vis
spectrometer at 2, 4, 6, 12, and 24 h after inoculation of the bacteria.
Thereby, the growth curve of the bacteria in the presence of the anti-
microbial materials and the absence of them were studied. The ex-
periments were performed in triplicate and repeated three times.

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Z. Rahmati, et al. Environmental Research 188 (2020) 109555

Fig. 3. SEM images of (a) γ-Fe2O3, (b) γ-Fe2O3@SiO2, (c) FSZ and (d) FSZ-Ag.

Fig. 4. EDS spectra of FSZ-Ag.

2.6.3. Live and dead bacteria staining

Fluorescence microscopy was accomplished to investigate bacterial
viability. E. coli and S. aureus were incubated at 37 °C and 150 rpm until
the concentration of bacteria suspension reached around 8 × 108 cell/
mL, then 1 mL of each cell suspension were transferred to 9 mL of
sterile culture media. Afterward, 10 mg of FSZ-Ag antimicrobial ma-
terial was added to the bacteria suspension; then, the mixture incubated
at 37 °C and 150 rpm overnight. Also, there were blank samples without
any antibacterial component. For bacteria staining, 1 mL of E. coli and
S. aureus suspensions were placed in an Eppendorf tube separately.
Next, 15 μL of the stain was transferred into the tube to stain bacteria
cells followed by incubating in the dark condition for 15 min. The stain
solution was made of a concentration of DAPI (5 μg/mL), and PI (3 μg/
mL) in phosphate-buffered saline (PBS) (Williams et al., 1998). In this
staining procedure, all bacteria (viable and nonviable) exhibited blue
fluorescence (DAPI), while only nonviable cells would be detected by
displaying red fluorescence (PI). DAPI is cell wall permeable, and stains
both live and dead bacteria, whereas PI stains only a cell with a da-
maged membrane; therefore, cells with an intact membrane are not
stained by PI. After 15 min incubation time, 10 μL of each sample was
properly implanted to a glass slide and sealed with a glassy cover sheet.
Confocal fluorescence microscopy was used to observe viable and
Fig. 5. Magnetization curves of γ-Fe2O3 and FSZ-Ag.
nonviable stained bacteria cells. For DAPI excitation and emission
wavelengths were 357 and 457 nm respectively, and also for PI
staining, excitation and emission wavelengths were 561 and
570–620 nm respectively. Fluorescence microscopy images were taken
using a Leica TCS-SPE Confocal fluorescence microscope.
2.6.4. Silver ion release test
To estimate released silver concentration in the solution, 10 mg of
the FSZ-Ag compound was dispersed in 100 mL distilled water at the
same conditions of growth inhibition test in the liquid phase. After 0, 4,
8, 12, and 24 h from the beginning, about 2 mL of the solution was
separated, and the concentrations of the silver ion were measured by
ICP-OES, and the release pattern was traced. The ICP-OES analysis were
done twice and the results were approximately oneness.

4
Z. Rahmati, et al.
Fig. 6. Antibacterial activity of 1) γ-Fe2O3, 2) γ-Fe2O3@SiO2, 3) FSZ-Ag, 4) FSZ, 5) ZIF-8, 6) AgNO3 by measuring the inhibition zones: a) E. coli, b) S. aureus.
Fig. 7. Inhibition zones diameter against E. coli and S. aureus for the synthe-
sized materials: 1) γ-Fe2O3, 2) γ-Fe2O3@SiO2, 3) FSZ-Ag, 4) FSZ, 5) ZIF-8, 6)
AgNO3. The experiments were performed in triplicate and repeated three times.
Fig. 8. Growth curves of (a) E. coli, (b) S. aureus (10 mg of each compound in 100 mL liquid media): 1) Blank sample, 2) γ-Fe2O3, 3) γ-Fe2O3@SiO2, 4) FSZ, and 5)
FSZ-Ag. The experiments were performed in triplicate and repeated three times.
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Environmental Research 188 (2020) 109555
3. Results and discussion
3.1. XRD and ICP-OES analysis
The XRD patterns of ZIF-8, γ-Fe2O3, γ-Fe2O3@SiO2, FSZ, and FSZ-Ag
are displayed in Fig. 1, to assess the structure and crystallinity of the
compounds. Based on Fig. 1, all diffraction patterns clearly show the
distinct peaks at 2θ = 18.1°, 30.25°, 35.5°, 37.2°, 43.2°, 53.6°, 57.13°,
62.7° and 74.2° attributed to the complete crystallization of γ-Fe2O3
MNPs without any transition in the crystalline phase. As shown in
Fig. 1, XRD pattern of γ-Fe2O3@SiO2 virtually corresponds to the XRD
pattern of pure γ-Fe2O3; however, a weak peak at 2θ = 22° shows the
appearance of amorphous SiO2 (Chi et al., 2013; Cui et al., 2013). The
specific peaks at 2θ = 7.3°, 10.43°, 12.75°, 14.75°, 16.52°, 18.08°,
19.56°, 22.18°, 24.5° and 29.65° are related to the (011), (002), (112),
(022), (013), (222), (123), (114), (233), and (044) reflection of ZIF-8
crystals in FSZ structure (Yang et al., 2018). The distinct diffraction
peaks of Ag are not observed in FSZ-Ag X-ray pattern because the small
amount of Ag component may not be determined by X-ray diffraction.
ICP-OES analysis was accomplished to determine the composition of
Fe, Zn, Ag, and Si elements in FSZ-Ag structure. According to this
analysis, each gram of the sample contained 26.8% Fe, 11.3% Zn, 7.6%
Si, and only 1.6% Ag. Therefore, these results stressed that why distinct
peaks of Ag were not identified at XRD pattern.
Z. Rahmati, et al.
Fig. 9. Growth curves of (a) E. coli, (b) S. aureus with different concentration of FSZ-Ag. The experiments were performed in triplicate and repeated three times.
Fig. 10. Concentration of Ag+ released in the experiment environment. The
ICP-OES analysis were done twice and the results were approximately oneness.
3.2. FTIR
The FTIR spectra of the prepared materials including γ-Fe2O3, γ-
Fe2O3@SiO2, FSZ, and FSZ-Ag are displayed in Fig. 2. The observed
peaks at 587, 888 and 1087 cm−1 in each pattern are attributed to the
γ-Fe2O3 MNPs. An absorption peak at 800 cm−1 was presented as
symmetric Si–O–Si vibration and wide bands from 1080 to 1100 cm−1
represented the asymmetric Si–O–Si stretching vibration in γ-Fe2O3@
SiO2 structure (Cui et al., 2013). The visible peak at 421 cm−1 reveals
Zn–N stretching bond and the 1588 cm−1 band illustrates the C]N
stretch, which confirms the appearance of the Zn–N band (Ordoñez
et al., 2010). The characteristic peaks placed between 1350 and
1550 cm−1 are assigned to the stretching vibration of the imidazole
ring; also, the peaks in the region of 900–1350 cm−1 are ascribed to the
outward bending of imidazole ring (Song et al., 2018). The peaks at
2883 and 3134 cm−1 admit the presence of aromatic and aliphatic
stretches. The broad peak at 3410 cm−1 is attributed to water and
absorption of hydroxyl groups (Bustamante et al., 2014). In addition,
the results showed that the structure of FSZ did not change after the
stabilizing of Ag nanoparticle.
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Environmental Research 188 (2020) 109555
3.3. SEM and EDS
The morphological and structural properties of all structures are
displayed in Fig. 3, taken by -scanning electron microscopy. The SEM
images confirm that γ-Fe2O3 particles have a spherical shape. Also, the
morphology of γ-Fe2O3@SiO2 has preserved the spherical shape while a
layer of SiO2 covered γ-Fe2O3 MNPs. After the formation of FSZ na-
nostructure, it is observed that the exterior surface of the γ-Fe2O3@SiO2
particles reveal coarseness and the spherical structure altered obviously
with the tetrahedral structure of ZIF-8. After stabilization of Ag parti-
cles, the structure of ZIF-8 become curled, mentioned by Yang and co-
workers (Yang et al., 2018). Energy dispersive spectroscopy (EDS) was
used to analyze the elemental characterization of the FSZ-Ag. Energy
peaks correspond distinct elements such as Fe, Si, Zn, Ag, O, N, and C,
which are present in the sample (Fig. 4).
3.4. VSM
The magnetic properties of γ-Fe2O3 and FSZ-Ag are displayed in
Fig. 5. The apparent difference between the two patterns showed that
FSZ-Ag had smaller magnetization saturation (Ms) value (37.8 emu/g)
than the γ-Fe2O3 particles (63.2 emu/g). This difference is related to the
increase in the mass of FSZ-Ag, which consists of the SiO2 layer, ZIF-8
crystals, and Ag ions with dielectric property. In spite of a lower Ms
value for the final product, magnetic separation process was accom-
plished in solutions properly through experiments in less than 1 min.
Therefore, nanoparticles were quickly collected using an external
magnetic field, which resulted in a transparent solution without sus-
pended solids. This ameliorated conducting the growth inhibition assay
because nanoparticles were not present in samples to interfere mea-
suring of OD600 in bacteria suspension.
3.5. Antibacterial activity
The antibacterial activity of the prepared antimicrobial materials
was evaluated by measuring the inhibition zone emerged on agar plates
as a result of nanostructured performance. As shown in Fig. 6, the
greatest inhibition zone belongs to FSZ-Ag nanostructure for both E. coli
and S. aureus strains. Moreover, the antibacterial ability of FSZ without
silver ions, γ-Fe2O3, γ-Fe2O3@SiO2, pure ZIF-8, and AgNO3 were eval-
uated. ZIF-8 and AgNO3 were studied as the positive control. The in-
hibition zone diameters for each compound were compared against the
growth of both bacteria strains in Fig. 7. No inhibition zone could be
observed for γ-Fe2O3 and γ-Fe2O3@SiO2, but the other compounds
showed significant antibacterial activity. The inhibition zone diameters
Z. Rahmati, et al. Environmental Research 188 (2020) 109555

Fig. 11. Fluorescence images of E. coli: (a, b) Blank sample and (c, d) treated with FSZ-Ag.

Fig. 12. Fluorescence images of S. aureus: (a, b) Blank sample and (c, d) treated with FSZ-Ag.

were 26.4 and 25.6 mm for E. coli and S. aureus, respectively, which
belonged to FSZ-Ag, and the inhibition zone diameters were nearly
21 mm for both bacteria belonging to FSZ. Although we expected that
the FSZ-Ag had to show far larger inhibition zone diameter compared
with FSZ, this result, which has been displayed in multiple experiments
with different dosage, can be related to weak permeability of silver ions
in solid media.
In addition, the growth inhibition function of the compounds was
assessed in liquid culture media by measuring the bacteria concentra-
tion in the presence of each compound. The results were compared to
the blank sample without any antibacterial material as control
according to the procedure mentioned before. As can be seen in Fig. 8,
the antibacterial activity of different compounds with the same con-
centration (100 ppm) showed that in the presence of FSZ-Ag, ap-
proximately no bacteria could multiply. In addition, different amounts
of FSZ-Ag (3, 5, 10, 15, and 20 mg) were used to obtain the minimum
inhibitory concentration (MIC) against the growth of E. coli and S.
aureus in the bacteria suspension. According to the results in Fig. 9, all
curves for the concentrations of 100, 150, and 200 ppm showed the
same trend and almost corresponded with each other for both bacteria.
Therefore, 100 ppm was evaluated as the MIC point. Although ZIF-8
showed efficient antibacterial activity due to Zn2+ in its structure, the

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Z. Rahmati, et al.
higher antibacterial activity of FSZ-Ag is assigned to the presence of
silver ions relative to the other antimicrobial materials in this study.
In order to investigate the quantity of silver ions in the solution
during the growth inhibition test, an additional experiment was per-
formed to calculate the amount of released silver ions tracked by ICP-
OES analysis versus time (Fig. 10). The result of elemental analysis
showed that 1.6% of the sample contained silver ions. Additionally, it
could be concluded from the findings in Fig. 10 that about 80% of silver
ions existing in 10 mg of FSZ-Ag was released after 24 h. Therefore, this
high amount of released silver ions is responsible for inhibiting the
growth of bacteria in the solutions. Although the toxicity of silver-based
materials has been questioned for potential applications in health-care
activities, in some reported works, the toxicity of Ag-MOFs has been
studied. According to these reports, white blood cell count (WBC) of
treated mice compared with normal mice has not changed significantly.
Thereby, regarding the low concentration of silver ions in Ag-MOF of
the present study, significant cytotoxicity cannot be exhibited (Lu et al.,
2014).
The results of fluorescence staining to distinguish live and dead
bacteria are displayed in Fig. 11 and 12. Because the excitation/emis-
sion wavelengths boundaries of DAPI and PI do not conform at the same
region, the staining results are displayed in different images in Figs. 11
and 12. (a, b, c, d). It could be observed that both bacteria blank
samples had no dead bacteria because in the PI staining images, there
were not any red-stained bacteria while there were many blue-stained
bacteria at the same sample stained with DAPI (Figs. 11 and 12 a, b). In
the samples containing treated bacteria suspension, almost all bacteria
existed in the DAPI staining represented red-stained in the PI staining
image (Figs. 11 and 12 c, d). The results confirmed the superior anti-
bacterial activity of FSZ-Ag due to the presence of Ag in its structure.
3.6. Antibacterial mechanism
As mentioned before, ZIF-8 has antibacterial activity due to the
released Zn2+ and the presence of organic ligands in the structure; for
example, zinc ions can interact with the hydroxyl groups formed bac-
teria cell walls and destroy them, which causes the bacteria death. By
adding the silver ions, its antibacterial activity increases extra-
ordinarily. Since silver has recognized as an antimicrobial component,
so researchers have reported some mechanisms to concede its anti-
bacterial activity. For example 1) penetrating across a bacteria cell wall
and interaction with the thiol group of distinct protein, 2) damaging the
bacteria cell wall, 3) inhibition of cell division by inactivation of its
enzymes, etc. (Ma et al., 2018). Thereby, in this study, the principal
purpose was to examine the antibacterial property of ZIF-8 in the pre-
sence and lacking silver nanoparticles. Thus, it can be concluded that
silver has improved the antibacterial activity of the FSZ significantly
because the bacterial culture media including FSZ-Ag remained trans-
parent after approximately ten days, while the other samples turned
into the turbid condition. In other words, it could be asserted that FSZ
loses its antibacterial activity before FSZ-Ag; therefore, adding silver to
the FSZ prolonged the time of antibacterial activity.
Additionally, some researchers have examined the Reactive oxygen
species (ROS) formation in the presence of light radiation to inactivate
bacteria using semiconductors. In this study, the experiments were
carried out in the dark. Presented antibacterial activity in the dark
outweighs the advantage of the antibacterial performance of FSZ-Ag to
eschew using further equipment for illumination, which can be utilized
in cost-effective applications (Cui et al., 2013; Feilizadeh et al., 2015;
Malik et al., 2018).
4. Conclusion
In summary, γ-Fe2O3@SiO2@ZIF-8-Ag as a novel magnetic nanos-
tructured material was successfully synthesized, and its antibacterial
activity was evaluated toward E. coli and S. aureus bacteria. The
8
Environmental Research 188 (2020) 109555
characteristic analysis, such as XRD, FTIR, SEM, EDX, VSM, and ICP-
OES confirmed the accuracy of the synthesized materials. The disc
diffusion method and growth inhibition assay in nutrient broth de-
monstrated the remarkable antibacterial activity of zeolitic imidazolate
framework-8. Moreover, the results from growth inhibition assay
showed that antibacterial activity of FSZ tremendously improved by
doping silver ions due to its superior antimicrobial function. The anti-
bacterial mechanism was explained in terms of bacteria membrane
damaging via slight amounts of zinc and silver ions release. The anti-
bacterial property of FSZ-Ag was displayed that no bacteria grew in the
liquid culture media at least with a concentration of 100 mg/L. In
consequence, the FSZ-Ag nanostructure is capable of being employed in
antibacterial applications.
Declaration of competing interest
The authors declare that there is no conflict of interest about this
work.
Acknowledgments
The present work was accomplished and supported under super-
vision of Chemical and Petroleum Engineering Department, Sharif
University of Technology.
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