Synthesis and catalytic evaluation of Fe3O4/MWCNTs nanozyme as recyclable peroxidase mimetics: Biochemical and physicochemical characterization

Article abstract of DOI:10.1002/aoc.4018

In this research, the nanocomposite of multiwalled carbon nanotubes and magnetic metal oxide nanoparticles (Fe₃O₄/MWCNTs), as enzyme mimetic, was synthesized using an in situ chemical reduction method. The structure, composition and

Full text of DOI:10.1002/aoc.4018

Received: 28 April 2017
DOI: 10.1002/aoc.4018
Revised: 4 July 2017
Accepted: 6 July 2017
F U L L P A P E R
Synthesis and catalytic evaluation of Fe O /MWCNTs
3
4
nanozyme as recyclable peroxidase mimetics: Biochemical
and physicochemical characterization
1
1
2
Navvabeh Salarizadeh
| Minoo Sadri
| Reza H. Sajedi
1
Department of Biochemistry and
Biophysics, Education and Research
Center of Science and Biotechnology,
Malek Ashtar University of Technology,
Tehran, Iran
Abstract
In this research, the nanocomposite of multiwalled carbon nanotubes and mag-
netic metal oxide nanoparticles (Fe O /MWCNTs), as enzyme mimetic, was
3
4
synthesized using an in situ chemical reduction method. The structure, compo-
sition and morphology of the prepared Fe O /MWCNT nanocomposite mate-
2
Department of Biochemistry, Faculty of
Biological Sciences, Tarbiat Modares
University, Tehran, Iran
3 4
rials were characterized using X‐ray diffraction, FT‐IR and scanning electron
microscopy with energy dispersive X‐ray spectroscopy, respectively. The mag-
netic properties of the nanocomposite were investigated by the vibrating sample
magnetometer. A colorimetric system involving nanozyme, phenol/4‐
aminoantipyrine and H O was utilized for the determination of peroxidase
Correspondence
Minoo Sadri, Department of Biochemistry
and Biophysics, Education and Research
Center of Science and Biotechnology,
Malek Ashtar University of Technology,
Tehran, Iran.
2
2
mimetic catalytic assay. The obtained results confirmed that the synthesis of
Fe O /MWCNTs nanostructures was successful. It was found that Fe O /
Email: sadri.mn313@gmail.com
3
4
3 4
MWCNTs nanohybrid exhibited peroxidase‐like activity without any pH limita-
tion. Colorimetric data demonstrated that the prepared nanocatalyst had higher
catalytic activity toward H O than MWCNTs. The kinetic parameters of the
2
2
−
1
nanozyme, Km and Vmax, were estimated to be 8.3 mM and 1.4 mM min ,
respectively. The Fe O /MWCNTs nanostructures were also successfully
3
4
applied for glucose detection. In addition, peroxidase‐like activity of the
nanozyme increased in the presence of butyl‐imidazolium bromide ionic liquid.
These biomimetic catalysts have some advantages, such as simplicity, stability,
reusability and cost effectiveness, which makes them great candidates to be
used in various fields of biotechnology applications.
KEYWORDS
3 4
4‐aminoantipyrine, characterization, Fe O /MWCNTs nanostructure, nanozyme, peroxidase‐like
activity
1
| INTRODUCTION
hydroperoxide. This enzyme was widely applied in the
synthesis of polymers and the bioremediation of toxic
organic compounds like phenolic structures in wastewa-
Peroxidases have wide biotechnological applications in
environment bioremediation, immunoassay, bimolecular
detections and industrial catalysts.
idase (EC 1.11.1.7) catalyzes the oxidation process of aro-
matic compounds by hydrogen peroxide or alkyl
[
1,2,6]
ters.
Despite the importance of natural enzymes,
[1–5]
Horseradish perox-
due to their sensitivity against environmental changes,
low stability, high costs of purification and storage, their
[
7–9]
usages are not cost‐effective.
Therefore, artificial
Appl Organometal Chem. 2017;e4018.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 10
https://doi.org/10.1002/aoc.4018

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SALARIZADEH ET AL.
catalysts can be designed and synthesized as enzyme
mimetics, including oxidase, peroxidase, dehydrogenases,
catalase, nuclease and proteases with sufficient stabil-
energy dispersive X‐ray spectroscopy (SEM/EDX), X‐ray
diffraction (XRD) and FT‐IR. Finally, biochemical charac-
terizations of the nanocatalysts were evaluated using phe-
nol/4‐aminoantipyrine (4‐AAP) as a new colorimetric
assay for peroxidase mimetics. A wide range of substrates,
such as phenol, aminophenols, indophenols, diamines
and a number of other compounds have been used for
[10–14]
ity.
been investigated by some researchers, recently.
These peroxidase mimetics have been used for glucose
Peroxidases, as one of the enzyme mimics, have
[
15–19]
[20]
[16]
and H O detection.
DNAzymes,
nanozymes and
2
2
[
38]
complexes of organic nanostructures such as heme,
hemin, porphyrine and cyclodextrines as peroxidase
HRP spectrophotometric assay.
In most studies, H O
2 2
was determined by peroxidase artificial enzymes on
[4,15,21]
mimetics have been studied.
Nanozymes are
3,3′,5,5′‐tetramethylbenzidine (TMB) and ABTS as sub-
[
17]
nano‐based materials with similar activity to natural
strate.
Also, a combination of the peroxidase‐like cata-
[4,9]
enzymes.
Compared with natural enzymes,
lytic activity of the Fe O /MWCNTs nanohybrids with
3
4
nanozymes possess advantages, including thermal stabil-
glucose oxidase (GOX) allowed the system of a sensitive
colorimetric assay for glucose monitoring. Fe O /
ity, low cost, easy production and different range pH‐tol-
3
4
[22]
erable.
It has been found that iron and iron oxide
MWCNT nanocatalysts exhibited good peroxidase‐like
catalytic activity. These magnetic nanocatalysts can be
suitable candidates in future enzyme‐based applications,
such as glucose and H O detection.
nanoparticles possess intrinsic peroxidase and oxidase‐
[
2,4,23,24]
like activities.
Research has been reported where
[
9,25]
magnetic nanostructures have enzyme‐like activity,
2
2
but the self‐aggregation of magnetic nanoparticles
decreases the catalytic activity of nanoparticles. There-
fore, loading these nanoparticles on nanostructures, with
large specific surface areas, like carbon nanotubes, graph-
eme, and their combination with surfactants reduces their
2
| EXPERIMENTAL
.1 | Materials
2
[24,26–29]
self‐aggregation and dissolution.
Recently, carbon
Multiwalled CNTs (MWCNTs) with diameters of 5–20
nm, lengths of 6–15 mm and purities of 97% were pro-
vided from Research Institute of Petroleum Industry of
Iran (RIPI). Ammonium iron (II) sulfate, sodium acetate,
4‐AAP, GOX and hydrazine hydrate were purchased from
Sigma‐Aldrich (USA). Phenol, TMB, dimethyl sulfoxide,
H O , acetic acid, KH PO and K HPO were obtained
nanotubes (CNTs) have been extensively considered by
researchers due to their exceptional mechanical, thermal
[30]
and electrical properties.
The combination of CNTs
with metal oxide nanoparticles magnetic types has
attracted much attention due to their unique properties,
including electrical and chemical conductivity, mechani-
cal and structural properties of CNTs, and magnetic
properties and chemical stability of metal oxide nanopar-
2
2
2
4
2
4
from Merck (Germany).
[24,31–33]
ticles.
CNTs have been used in imaging tech-
2.2 | Synthesis of Fe O /MWCNT
3 4
niques, such as Raman spectroscopy, near‐infrared
nanocomposites
[30,31]
fluorescence and ultrasonography.
A high ratio of
surface area to weight in nanotubes is an important factor
for designing of nanocomplexes. These carbon‐based
nanocomposites have been used in various fields, like
drug delivery, biosensing, catalysis, fuel cells, capacitors
In order to synthesis the nanocomposites; MWCNTs were
modified with a mixture of nitric acid/sulfuric acid as oxi-
dative reagents at 150 °C for 2 h. Then, the treated
MWCNTs were washed, filtered and sonicated for 15
min. In order to synthesis the Fe O /MWCNTs nanocom-
[7,32,34]
and decontamination purposes.
Peroxidase‐like cat-
3
4
alytic activities of Fe O , CNTs and carbon‐based nano-
posites, 0.57 g ammonium ferrous sulfate (hexahydrate)
was dissolved in 10 ml of deionized water. Next, 0.1 g of
the modified MWCNTs was added to the previous solu-
tion, and they were sonicated with 3 min intervals for
15 min. Then, 1.25 ml of hydrazine hydrate was added
to the mixture, which was followed by adding step by step
of 2 M NaOH solution under ultrasonic stirring for 15
min. When the pH of suspension reached 11–12, the sus-
pension was refluxed at the boiling temperature of the sol-
vent. The magnetic hybrid product was separated with a
magnet, and then washed with deionized water and etha-
nol several times, and the final product was dried in the
vacuum oven at 50 °C for 24 h.
3
4
[32–37]
structures have been approved.
The synthesis of
CNT‐coated Fe O nanocomplex was reported by some
3
4
researchers, and their applications for H O detection
2
2
[2,9,35]
and Orange II degradation were investigated.
The
earth‐abundant element‐based nanoparticles are low‐cost
materials in the design of novel enzyme mimetics.
Because of the disadvantages of natural peroxidases, this
research is focused on the artificial peroxidase mimics.
In this research, Fe O /MWCNTs nanocomposites
3
4
were prepared via a novel method and they were utilized
as catalysts. The characterization of the nanocomposites
was achieved using scanning electron microscopy with

SALARIZADEH ET AL.
3 of 10
2
.3 | Characterization of Fe O /MWCNTs
2.7 | Effect of ionic liquids on catalytic
activity of nanozyme
3
4
nanocomposites
The morphology and elemental composition of nanocom-
posites were investigated by SEM/EDX (Zeiss Sigma 500
VP SEM/Oxford Instrument Detector). XRD [Philips
Ionic liquids, known as green catalysts, have been used in
bio‐electrochemistry and biocatalysis as stable sol-
[
39]
vents.
Because peroxidase‐like activity of these cata-
[
40]
PW1730, with Cu K radiation (λ = 1.540598 Å)] was
lysts has been demonstrated,
the synergistic effect of
α
used for determination of nanomaterial structures. FT‐IR
imidazolium‐based ionic liquid (butyl‐imidazolium bro-
(Bruker Equinox, Germany) was applied to study the
mide) with Fe O /MWCNTs nanocatalysts was investi-
3
4
changes of chemical structure. The magnetic properties
of Fe O /MWCNTs nanocomposites were investigated
gated. For this purpose, 200 μl butyl‐imidazolium
−
1
bromide was mixed with 2 mg ml
Fe O /MWCNTs
3 4
3
4
by the vibrating sample magnetometer (VSM; BHV‐55,
Riken, Japan).
and the obtained mixture was stirred for 20 min. Then,
catalytic activity assay was measured at 510 nm and the
results were compared with those obtained from ionic
liquid and nanocatalyst.
2
.4 | Colorimetric determination of
peroxidase‐like activity of Fe O /MWCNTs
nanozyme
2.8 | Glucose detection
3
4
Detection of glucose was performed with reaction mixture
−
1
Colorimetric evaluation of the nanocomposites was per-
consisting of 100 μl of 1 mg ml GO and 1 ml of different
concentrations of glucose solutions, which were incu-
bated (pH 7.0) at 37 °C for 20 min. Then, 1 ml of 4‐AAP/
phenol solution and 50 μl of nanocatalyst were added into
the above mixture. Finally, the absorption spectrum of the
obtained solution was recorded by the UV–Vis spectro-
photometer at the wavelength range from 300 to 700 nm.
−
1
formed by adding 2 mg ml nanozyme to 2.5 mM 4‐
AAP, 25 mM phenolic compound in 200 mM potassium
phosphate buffer (pH 7.0) and H O at room temperature.
2
2
The absorbance changes were recorded at 510 nm using a
μQuant™ microplate spectrophotometer (BioTek Instru-
ments, Winooski, USA). This reaction mixture was cho-
sen as nanozyme standard assay. Also, the colorimetric
assay of nanocomposites was investigated in the presence
of TMB‐H O substrates in acetate buffer (pH 4.5) at room
3
| RESULTS AND DISCUSSION
2
2
temperature, and the absorbance was measured at 652
nm. The control experiments were also performed under
the same conditions to compare relative catalytic activity
of nanocatalysts.
3
.1 | Characterization of Fe O /MWCNTs
3
4
nanostructure
Physicochemical characterization of Fe O /MWCNTs
3
4
nanocatalyst is shown in Figure 1. The morphology of
synthesized nanostructures was investigated with SEM.
It was clearly obvious that the Fe O nanoparticles were
3
4
2
.5 | Kinetic measurements of peroxidase‐
well attached and distributed on the surface of MWCNTs.
The average size of nanocomposites was estimated to be
about 25 nm using SEM analysis. The EDX spectrum in
Figure 1b shows the presence of carbon, iron and oxygen
in magnetic Fe O /MWCNTs. Also, the weight ratios of
like activity of nanozymes
The kinetic measurements were performed at appropriate
concentrations of H O substrate according to the assay
conditions (like standard assay conditions) as described
2
2
3
4
above. The nanozyme's kinetic parameters, Michaelis–
Fe, O and C were obtained as 19.9%, 15% and 65%, respec-
tively, by EDX analyses. The carbon, iron and oxygen
signals corresponded to CNTs and Fe O nanoparticles,
Menten constant (K ) and Vmax, were determined using
m
Lineweaver–Burk plot.
3
4
respectively.
FT‐IR spectroscopy of Fe O /MWCNTs, Fe O and
3
4
3 4
−
1
MWCNTs throughout the range of 400–4000 cm was
2
.6 | Optimum pH determination
also performed to confirm the structural changes. The
position of bands at 584 and 1618 cm can be attributed
−
1
Because the enzyme's activity is affected by environmen-
tal conditions, the dependency of peroxidase‐like activity
of nanozyme on pH value was investigated at the pH
range of 4.0–9.0. The pH profile of nanozyme was deter-
mined under standard assay reaction.
to the Fe–O–Fe stretching and bending modes in Fe O
3
4
[
17]
nanoparticles.
It was observed that the bands at 1100,
−
1
1412, 1631, 2928 and 3424 cm were assigned to the
stretching modes of C–O, C–C, C = O, C–H and –OH in

4
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SALARIZADEH ET AL.
the functional groups of the MWCNTs, respectively. In
−
1
Fe O4 graph, the peak at 3420 cm
related to the
3
hydroxyl group (−OH) of adsorbed water. In the FT‐IR
spectra of Fe O /MWCNTs, apart from the main peaks
3
4
of Fe O , additional peaks were also observed that were
3
4
attributed to the functional groups of the MWCNTs but,
as expected, the intensity of the peaks decreased signifi-
cantly. The reduction of –OH peak intensity in Fe O /
3
4
−
1
MWCNTs spectrum at 3423 cm demonstrated that the
binding of Fe O nanoparticles to MWCNTs was success-
3
4
ful. These observations clearly confirm that Fe O nano-
3
4
particles have successfully supported onto the surface of
the MWCNTs.
The crystalline structures of the synthesized Fe O /
3
4
MWCNTs nanohybrids and MWCNTs were confirmed
with powder XRD measurements, and the main peaks of
Fe O /MWCNTs crystals were clearly presented. The
3
4
reduction of peak intensity at 2θ = 26.6° that related to
MWCNTs indicated that the MWCNTs structure was
changed in the synthesis process. Peaks at 2θ = 30.48°,
3
(
5.82°, 43.54°, 53.86°, 57.40° and 62.98° are devoted to
220), (311), (400), (422), (511) and (440) crystal planes,
respectively, which are the major peaks of Fe O /
3
4
MWCNTs. These characteristic peaks originated from
Fe O nanoparticles. These results are consistent with
3
4
[
17]
previous studies.
Also, no diffraction peak due to any
other new phase was observed. The average crystallite size
of nanohybrids calculated by the Debye–Scherer equation
was about 18 nm.
The hysteresis curves of the Fe O ‐coated MWCNTs
3
4
were recorded at room temperature with a VSM. The sat-
uration magnetization Ms, the remanent magnetization
Mr and the coercivity Hc were the main technical param-
eters to characterize the magnetism of ferromagnetic
materials. These nanocomposites were magnetically sepa-
rated and reused after the completion of reaction. With
magnetic separation, it was not required to recover the
catalyst using filtration and centrifugation methods. The
resulting data showed that the saturation magnetization
of Fe O /MWCNTs nanohybrids was lower than Fe O
3
4
3 4
[
41,42]
nanoparticles.
This was due to the low content of
magnetic nanoparticles in the carbon‐based nanocompos-
ites, as the MWCNTs exhibited no magnetic characteris-
tics (Figure 2).
3
.2 | Colorimetric assay of peroxidase‐like
activity of Fe O /MWCNTs nanozyme
3
4
FIGURE 1 (a, b) typical SEM/EDX images of Fe
nanocomposites. (c) FT‐IR spectra of Fe /MWCNT nanohybrids,
Fe and MWCNT. (d) XRD patterns of Fe /MWCNT, Fe
and MWCNT
3 4
O /MWCNT
3 4
O
In this study, the peroxidase‐like catalytic activity of
3
O
4
3
O
4
3 4
O
Fe O /MWCNTs nanocomposites, prepared according to
3
4
the described method, was investigated using a different
process compared with the peroxidase mimetics reported
in previous studies. The colorimetric assay of the

SALARIZADEH ET AL.
5 of 10
exhibited no catalytic activity (Figure 4). 4‐
Aminoantipyrine/phenol/nanozyme system showed a
weak color variation, but significant color variations were
observed in the presence of nanozyme and two substrates.
Therefore, the nanocatalyst in the absence of two sub-
strates exhibited no significant absorption peak in the
range of 350–700 nm (Figure 4). These data demonstrated
that both the substrates were required for reaction prog-
ress. Also, colorimetric evaluation was performed with
TMB substrate (Figure 3b). TMB is a chromogenic sub-
strate for peroxidase enzymes, and it is used as a hydrogen
donor and converts to oxidized TMB product with a blue
color solution that can be read at 652 nm. According to
obtained data from two reaction systems, no such absorp-
tion spectrum was observed from a solution in the
absence of the nanocatalysts. When nanocatalysts were
added to the solution, the maximum absorption was
obtained at 510 and 652 nm as a strong response.
Figure 4b shows the catalytic activity of nanozymes at
510 nm in different situations.
3 4
FIGURE 2 Hysteresis loops of Fe O /MWCNTs nanocomposite
nanocomposites was performed in the presence of 4‐AA/
phenol and H O as substrates at room temperature. A
2
2
solution of Fe O /MWCNTs could catalyze the oxidation
3
4
of 4‐AAP in the presence of H O to produce a pink‐col-
ored quinoneimine dye resulting from oxidation of 4‐
AAP, which showed its maximum absorbance at 510 nm
The catalytic activity of the reaction system also
depends on the amount of nanocatalyst (Figure 5). It
was found that the peroxidase‐like catalytic activity was
increased by increasing the amount of nanocatalyst. Our
findings reveal that the prepared Fe O /MWCNTs can
2
2
(Figure 3a).
4
‐Aminoantipyrine/phenol/H O2 reaction without
2
3 4
nanocatalysts and H O /nanozyme system without AAP
be used as a peroxidase mimetic with high activity
2
2
2 2 3 4
FIGURE 3 Colorimetric investigations of H O concentrations with 4‐AAP/phenol and TMB substrates using Fe O /MWCNTs nanozyme

6
of 10
SALARIZADEH ET AL.
FIGURE 5 Typical absorption spectra of phenol/4‐AAP reaction
solutions (90 μl phenol/4‐AAP + 90 μl H
2
O
2
) catalytically
oxidized by different concentrations of Fe
3 4
O –MWCNTs nanozyme
exhibited lower catalytic activity. Because the catalytic
activities of nanocatalysts could be shape‐ and size‐depen-
[
2]
dent, these properties may be associated with the spread
graphene sheet and special surface morphology of nano-
composites. The preparation of Fe O –MWCNTs nano-
3
4
composites for the purpose of diarylpyrimidinones
[
43]
[9]
synthesis
and orange II degradation has previously
been done (Figure 6).
Peroxidase mimetics activity of MWCNTs and Fe O /
3
4
MWCNTs nanocatalysts was compared in the presence
of TMB and 4‐AAP/phenol substrates. It was found that
the peroxidase‐like catalytic activity of Fe O /MWCNTs
3
4
FIGURE 4 (a) UV–vis absorption spectra of the oxidized 4‐AAP/
phenol in phosphate buffer for different systems. (b) the relative
was higher than MWCNTs in both reaction systems.
Moreover, both nanostructures exhibited higher catalytic
3 4
activity of Fe O /MWCNTs nanocatalyst in different situations
activity in the TMB/H O2 reaction mixture compared
2
with the 4‐AAP/phenol/H O system. It can be concluded
2
2
[
2,20]
that the affinity of nanocatalysts to TMB substrate is
greater than that of 4‐AAP/phenol.
compared with similar systems.
According to the per-
oxidase‐like activity of Fe O and CNT nanomaterial, as
3
4
shown in previous studies, the combination of Fe O
3
4
and CNT nanostructures results in a peroxidase mimetic
3.3 | Kinetic parameters
with noticeably higher efficiency compared with Fe O4
3
and CNT.
The kinetic parameters of nanozyme were calculated
based on Michaelis–Menten and Lineweaver–Burk plots.
The Fe O /MWCNTs nanozyme indicated Michaelis–
Peroxidase‐like activities of Fe O /MWCNTs in the
degradation of methylene blue,
3
4
[17]
GOCNT–Pt nanocom-
3
4
posites (platinum nanocatalysts loaded on graphene
Menten‐type kinetics. The kinetic parameters of Km and
Vmax were determined to be 8.3 mM and 1.4 mM min ,
[8]
−1
oxide‐carbon nanotubes), Fe O nanoparticles loaded
3
4
[2]
on GO‐dispersed carbon nanotubes, CNTs and Fe O4
respectively. In comparison with HRP, which was
3
[
44]
have been reported using TMB and ABTS as substrates
by other researchers. Prepared nanocomposite exhibited
higher catalytic activity compared with pure CNTs and
Fe O nanostructures. Compared with GOCNT–Pt and
reported,
Fe O /MWCNTs exhibited good peroxidase‐
3
4
like activity. The kinetic parameters of Fe/CNT‐based
similar nanocatalysts in previous studies for H O sub-
2
2
strate are shown in Table 1. The Km value of this
3
4
GCNT–Fe O4 nanocomposites, our nanocatalysts
nanocatalyst is smaller than that one for Fe O /
3 4
3

SALARIZADEH ET AL.
7 of 10
The kinetic parameters were measured for the various
concentrations of H O with a constant concentration of
2
2
4
‐AAP/phenol and Fe O /MWCNTs nanozyme. In
3
4
another study, the kinetics assay was performed in the
presence of TMB substrate and kinetic parameters were
obtained. In this experiment, it was shown that the
nanozyme catalytic activity had a direct relation with
the concentrations of TMB and H O (the data are not
2
2
shown; Figure 7).
3.4 | Effect of ionic liquids in catalytic
activity of nanozyme
Ionic liquids have good properties, such as catalytic
activity, high thermal stability and ionic conductivity.
Ionic liquids have been used in catalytic reactions as
solvents, catalysts and catalyst supports. Recently, many
researchers have been interested in magnetic
nanohybrids, especially CNT–ionic liquid nanostructures,
[
45]
for magnetic separation processes.
In this research, the
effect of butyl‐imidazolium bromide ionic liquid on the
catalytic activity of Fe O /MWCNTs was investigated. As
3
4
shown in Figure 10, the absorbance of Fe O /MWCNTs
3
4
FIGURE 6 UV–vis absorption spectra of: (a) oxidized 4‐AAP in
phosphate buffer pH 7; and (b) oxidized TMB in acetate buffer for
3 4
MWCNT and Fe O /MWCNTs
MWCNTs, which suggests that the Fe O /MWCNTs have
3
4
a lower affinity for H O2 than the other mentioned
2
FIGURE
7
3 4
Michaelis–Menten curve for Fe O /MWCNTs
nanocatalysts according to assay conditions. Colorimetric
assay and Vmax value showed that the catalytic rate of
Fe O /MWCNTs to 4‐AAP oxidation was much greater
nanozyme. The experiment was performed in phosphate‐buffered
saline (0.2 M, pH 7.0) at a fixed concentration of nanozyme, 4‐AAP/
phenol and different concentrations of H O . Inset: Lineweaver–
3
4
2 2
than the reported nanocatalysts.
Burk plot for Fe
3 4
O /MWCNTs
3 4 2 2
TABLE 1 Kinetic parameters of Fe O /MWCNT nanozyme for H O substrate in comparison with HRP and other Fe/CNT‐based similar
peroxidase mimetics
−
1
Catalyst
Fe /MWCNT
GCNT‐Fe
V
max (m s
)
K
m
(mM)
Temperature
Ref.
−
5
3
O
4
2.3 × 10
8.3
Room temperature
Present study
−
8
3
O
4
0.387 × 10
2.52
0.08
3.7
Room temperature
2
−
8
Hemin–SWCNT
4.79 × 10
37 °C
22
2
−
8
HRP
8.7 × 10
—

8
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SALARIZADEH ET AL.
FIGURE 8 The effect of imidazolium‐
based ionic liquid on Fe
nanozyme
3 4
O /MWCNTs
nanozyme increased in the presence of ionic liquid.
According to our data and previous studies, for a synergis-
tic effect and high efficiency of nanocatalysts, it can be
proposed that the ionic liquids are immobilized on mag-
netic nanostructures. Also, ionic liquids can improve elec-
2.5, 5, 10 and 20 mM under standard assay conditions
(Figure 10). According to Equation 2, at a wavelength of
[
45]
trochemical responses due to their ionic conductivity,
therefore it is expected that the deposition of ionic liquids
and nanostructures on the electrode surface can play an
effective role in the design of glucose sensors. In this
study, for the first time, the combination of ionic liquids
and nanomaterials as peroxidase mimetics was examined
(Figure 8).
3.5 | Effect of pH on nanozyme activity
To determine the optimum pH for the Fe O /MWCNTs
3
4
assay method, peroxidase‐like catalytic activity was
investigated in the pH range of 4.0–9.0, and the
nanozyme activity was measured according to the men-
tioned procedure. As shown in Figure 9, there is not a
considerable difference in peroxidase activity rate at
the different pH values. Compared with synthesized
Fe O /MWCNTs nanocatalysts in reported studies, this
3
4
nanozyme exhibited catalytic activity over a wide pH
range. Similar results were obtained from Wang
[17]
et al.
Because GOX is more stable at pH 7–8, it is
suggested that this nanozyme can be a suitable peroxi-
dase mimetics for designing non‐enzymatic sensors for
glucose detection.
3.6 | Detection of glucose
H O is the main product of the GOX‐catalyzed reaction;
2
2
therefore, the nanocatalysts with peroxidase‐like activity
were also applied in the colorimetric and quantitative
detection of glucose with final concentrations of 1.25,
FIGURE 9 The effect of pH on activity of Fe O /MWCNTs
nanozyme in phosphate solution with 4‐AAP substrate
3
4

SALARIZADEH ET AL.
9 of 10
4
| CONCLUSIONS
In summary, in situ assembly of Fe O on MWCNTs
3
4
nanostructures was performed by chemical reduction with
hydrazine as peroxidase mimetics. This nanozyme exhib-
ited higher peroxidase mimic behavior than MWCNTs.
Colorimetric assays and kinetic parameters revealed that
peroxidase mimetic activity of prepared nanocatalysts to
TMB and 4‐AAP oxidation was very high in comparison
with other reported systems. The synergistic effect of
Fe O and MWCNTs enhances the enzyme‐like activity
3
4
of nanostructure. On the other hand, CNT supports the
improvement of dispersivity of Fe O nanoparticles
3
4
and decreases agglomeration. These magnetic enzyme
mimics exhibit some advantages over natural protein
enzymes due to their characteristics, such as magnetic
separation and reusability, multi‐functionality, and
direct electrochemistry to substrates.
The nanozyme showed a good sensitivity for glucose
detection. Also, in the TMB/nanozyme colorimetric assay
system, without H O , TMB oxidation was observed. We
2
2
further explored the investigation and application of mag-
netic nanostructures in biocatalysis and biotechnology.
This nanocatalyst with H O ‐induced oxidation can play
2
2
an important role in reducing environmental pollution.
It is suggested that this nanozyme with high catalytic
activity can catalyze other organic substrates such as
aromatic compounds even in the absence of H O . Also,
2
2
the combination of nanocatalysts with ionic liquids and
deep eutectic solvents can be applied in the design of
new peroxidase mimetic systems.
FIGURE 10 (a) absorption spectrum of glucose detection by
Fe O /MWCNTs nanocatalyst in the presence of glucose at various
3 4
ACKNOWLEDGEMENTS
Financial support provided by the Research Council of
the Malek‐Ashtar University of Technology is gratefully
appreciated. The authors would like to thank Dr Hassan
Hosseini from Nanoscience Center of Imam Hossein
University for the helpful comments.
concentrations (1.25, 2.5, 5, 10 and 20 mM). (b) the absorbtion
intensity at 510 nm against different concentrations of glucose
5
10 nm, the absorbance has a direct relation with the glu-
cose concentration.
ORCID
Navvabeh Salarizadeh
832
Minoo Sadri http://orcid.org/0000-0001-8540-7464
http://orcid.org/0000-0002-8243-
FAD‐GOX
3
Glucose þ O ƒƒƒ ƒƒ ! H O þ δ‐D‐gluconolactone (1)
2
2
2
Fe3O4=MWCNT
Phenol þ 4‐AAP þ 2H2O2 ƒƒƒƒƒƒƒƒ! quinoneimine dye þ 4H2O
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