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L. Ding, J. Zheng, C. Zeng, Y. Wen, G. Liu, A. Aldalbahi, J. Shi, S. Song, X. Zuo and L. Wang, Nanoscale,
2017, DOI: 10.1039/C7NR00819H.

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ARTICLE
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Yolk-shell nanostructured Fe3O4@C magnetic nanoparticles with

enhanced peroxidase-like activity for label-free colorimetric
Received 00th January 20xx,
Accepted 00th January 20xx
detection of H2O2 and glucose
a b,* b b a c c d
Na Lu, Min Zhang, Lei Ding, Jing Zheng, Caixia Zeng, Yanli Wen, Gang Liu, Ali Aldahlbahi,

Nanoscale Accepted Manuscript

DOI: 10.1039/x0xx00000x
e e e e,*
Jiye Shi, Shiping Song, Xiaolei Zuo, and Lihua Wang
www.rsc.org/
Herein, we have developed a simple and facile method to synthesize yolk-shell nanostructured Fe3O4@C nanoparticles
(NPs) as a multifunctional biosensing platform for label-free colorimetric detection of H2O2 and glucose. It was
demonstrated that Fe3O4@C yolk-shell nanostructures (YSNs) retained the magnetic properties that can be used for
separation and concentration. Also importantly, the Fe3O4@C YSNs exhibited an intrinsic peroxidase-like activity that could
quickly catalyze the enzyme substrate in the presence of H2O2 and produce a blue color. Compared to other similar ferric
oxide-based NPs with different structures, Fe3O4@C YSNs exhibited greatly enhanced catalytic activities due to the unique
structural features. Moreover, steady-state kinetics manifested the catalytic behaviors in agreement with classic
Michealis-Menten models. Taking advantage of the high catalytic activity, Fe3O4@C YSNs were employed as a novel
peroxidase mimetics for label-free, rapid, sensitive, and specific colorimetric sensing of H2O2 and glucose, suggesting that
Fe3O4@C YSNs have the potential for construction of portable sensors in the application on point-of-care (POC) diagnosis
and on-site test.

6 7 8 9
CeO2 nanoparticles (NPs), V2O5 nanowires, Co3O4 NPs, AuNPs,
10, 11
1. Introduction and metallic alloy NPs, have been successfully formed and
Natural enzymes have been extensively studied and applied in applied in a variety of fields. Besides that, carbon-based
12
the fields of medicine, environment, food, and industry, owing to nanomaterials, e.g. graphene oxide (GO), single-walled carbon
13 14
their high catalysis efficiency and good substrate specificity. nanotubes (SWNTs), and carbon nanodots (C-Dots), have been
However, the wide applications of enzymes are restricted by high explored to mimic the functions of naturally occurring enzymes.
costs of purification and storage, as well as their catalytic activity Furthermore, a series of hybrid complexes composed of metal
can suffer from environmental conditions such as temperature, nanomaterials and other matrixes have been investigated as
15
inhibitors, acidity, and protease digestion. Therefore, considerable nanocatalysts, such as GO-Fe3O4 nanocomposites, Pt-MoO3 hybrid
16 17
attentions have been devoted to the development of artificial nanostructure, and Fe3O4@SiO2@Au@mSiO2. Nevertheless,
1 2
enzyme mimics, such as cyclodextrins, porphyrins, polymers, and
3 nanomaterials-based artifical enzymes still meets enormous
biomolecules.
4 challenges in the application of biocatalysis, including
uncontrollable synthesis, and untunable physical and chemical
Recently, a variety of inorganic nanosystems have been found to properties. Therefore, extensive efforts should be devoted to
5
show unexpected peroxidase- or oxidase-like activity. For example, designing and fabricating new nanosystems with excellent
peroxidase-like catalysis.
a
School of Materials Engineering and bCollege of Chemistry and Chemical
Engineering, Shanghai University of Engineering Science, Shanghai 201620, China. Recently, yolk-shell nanoparticles (YSNs), or so-called “nano-
E-mail: zhangmin@sues.edu.cn
c rattles”, have emerged as a unique class of special core/shell
Laboratory of Biometrology, Division of Chemistry and Ionizing Radiation
Measurement Technology, Shanghai Institute of Measurement and Testing structures with different core@void@shell architectures, generally
Technology, Shanghai 201203, China 18
d denoted as A@B. Owing to their appealing structures, YSNs have
Chemistry Department, King Saud University, Riyadh 11451, Saudi Arabia
e
Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation tunable physical and chemical properties. Specifically, the
Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, tailorability and functionality in both the cores and hollow shells
Shanghai 201800, China. E-mail: wanglihua@sinap.ac.cn 19, 20
make them attractive in various applications of nanoreactors,
Electronic Supplementary Information (ESI) available: [details of any 21, 22 23
lithium-ion batteries, and drug delivery. Xia and co-workers
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x firstly reported a practical route to synthesis of Au@polymer

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1

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24
YSNs. Up to now, a variety of YSNs with inorganic core and shell
19, 25-27
compositions have been synthesized and characterized.
28-
Interestingly, YSNs with noble metal cores (e.g. Au, Ag, Pt, and Ni)
30
have exhibited high catalytic activity, ascribing to the freely
movable core catalysts, the protective shells, and the void space
between the core and the shell, where could afford a homogenous
environment in catalytic reactions.
Published on 10 March 2017. Downloaded by Central Electrochemical Research Institute (CECRI) on 17/03/2017 07:00:27.
Diabetes, one of the spread modern lifestyle diseases, is among
the most leading causes of deaths with worldwide prevalence. As so
far, great efforts have thus been invested to develop glucose
biosensors that depend on various techniques, including
electrochemistry, optical technique, spectroscopy, and field-effect
31-35
transistors (FETs). Among them, colorimetric biosensing method
has drawn tremendous attention due to the portability, low cost,
and no need for complicated instrumentation. Especially important,
36-39
the signal change can be directly read out by our naked eyes.
40-43
Therefore, colorimetric sensors could be a promising tool in the
applications of point-of-care (POC) diagnosis and field test. Ever
since the first demonstration of Fe3O4 magnetic nanoparticles
(MNPs) possessing an intrinsic enzyme mimetic activity similar to
44
that of natural peroxidases, magnetic nanomaterials have been
15, 17, 45, 46
considered as an important nanocatalyst due to their
multifunctional properties of peroxidase-like acitivity, magnetism,
and good biocompatibility. Unfortunately, there still exist some
limitations in the design and fabrication of MNPs as nanozymes in
the colorimetric biosensing application. One is limited catalytic
activity of commonly used bare Fe3O4 MNPs may lead to limited
performance of biosensors, and the other is unprotected
nanocatalysts will decrease the catalytic activity and reduce the
stability. Consequently, a new type of magnetic nanomaterials with
enhanced peroxidase-like catalysis and good stability are urgently
desired for favorable biosensing platforms.
In this study, we prepared multifunctional yolk-shell
nanostructured Fe3O4@C MNPs with enhanced peroxidase-like
activities that are designed for label-free colorimetric detection of
H2O2 and glucose. Fe3O4@C YSNs are synthesized through a one-
step versatile and simple approach. As a result, it was found that
Fe3O4@C YSNs exhibited good aqueous dispersion, excellent
magnetism, and especially greatly enhanced peroxidase-like
activity, which can be used for biocatalysis, separation and
concentration. Based upon the high catalytic activity, Fe3O4@C YSNs
were firstly employed as a novel nanozyme for label-free, rapid,
high sensitive and selective colorimetric detection of H2O2 and
glucose.
2. Experimental section
2.1 Materials and reagents
3,3',5,5'-Tetramethylbenzidine (TMB) and 30% H2O2 were
obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai,
China). Tetraethoxysilane (TEOS, 95%) was purchased from Energy
Chemical Company (Shanghai, China). Dopamine hydrochloride was
obtained from the Alfa Aesar (China) Chemical Co., Ltd. Glucose
2 | J. Name., 2012, 00, 1-3
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Journal Name
oxidase (GOx) was purchased from Sangon Biotech (Shanghai) Co.,
View Article Online
Ltd. (Shanghai, China). Glucose, lactose, fructose, and sucrose were
DOI: 10.1039/C7NR00819H
obtained from Shanghai Aladdin Bio-Chem Technology Co., LTD
(Shanghai, China). All chemicals were used without further
purification.
2.2 Instrumentation
Scanning electron microscopy (SEM, JEOL-4800) and transmission
electron microscopy (TEM, JEOL-1011) were employed to
characterize the morphology and microstructures of the samples. X-
ray diffractometer (XRD) was performed on D/Max-2500
diffractometer using Cu Kα radiation (Shimadzu, Japan). Fourier
transform infrared (FTIR) spectra was measured on a PerkinElmer，
Spectrum Two IR spectrometer to obtain compound structure of
Nanoscale Accepted Manuscript
the samples in KBr pellets. The magnetic properties were studied by
a vibrating sample magnetometry (VSM, MicroSense EV7) at room
temperature. Absorption spectra were obtained on a UV-2450
spectrophotometer (Shimadzu, Japan).
2.3 Synthesis of Fe3O4@C YSNs
In a typical procedure, 0.10 g of Fe3O4 NPs was dispersed in an
ethanol solution (94%, v/v) in the presence of 2 mL of ammonium
hydroxide, and the mixed solution was stirred for several minutes at
room temperature. Then, an alcohol solution containing 300 L of
TEOS was dropwise added into the above mixture, stirring for 12 h
at room temperature. After that, 50 mg of dopamine was added
and the mixed solution was continued to be stirred for 24 h to coat
polydopamine (PDA) layer. The suspension of Fe3O4@SiO2@PDA
NPs was separated by an external magnet field and washed with
deionized water and ethanol for several times. Subsequently,
carbonized samples of Fe3O4@SiO2@C NPs were obtained by
annealing Fe3O4@SiO2@PDA NPs in a tube furnace under a nitrogen
atmosphere at 500 °C for 4 hours. Finally, the Fe3O4@C YSNs were
obtained by selective etching the SiO2 template using a solution
containing 25 mL of deionized water, 6 mL of ethanol and 10 mL of
NH3∙H2O at 150 °C for 24 h.
2.4 Catalytic activity of Fe3O4@C YSNs
The catalytic activities of Fe3O4@C YSNs were performed as the
following procedures. Firstly, 40 L of Fe3O4@C YSNs stocking
solutions were added to 360 L of HAc-NaAc buffer (0.2 M, pH 4.0)
solution containing 0.1 mM TMB and 0.5 mM H2O2. After the
mixture was incubated in a 60 ⁰C water bath for 10 min, Fe3O4@C
YSNs were then quickly removed by applying an external magnetic
field. Subsequently, the solution was transferred to a quartz cell
and UV-Vis spectra were measured using a wavelength-scan mode
under the optimal conditions, unless otherwise stated.
In the test of the effect of pH on the catalytic activities of
Fe3O4@C YSNs, 0.2 M sodium acetate solutions with different pH
(2.0-10.0) were used. In the study of influence of temperature on
peroxidase-like activity, experiments were carried out from 25 ⁰C to
65 ⁰C for at an interval of 5 ⁰C under the same conditions. The
assays of amount of nanocatalysts were performed in pH 4.0 buffer
solution at 60 ⁰C by varying the concentration of Fe3O4@C YSNs.
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In the steady-state kinetic assays, catalytic reactions of Fe3O4@C
YSNs were performed with varied concentrations of one of the
substrate of TMB or H2O2 under the optimized conditions. The key
kinetic parameters were calculated using the Lineweaver-Burk plots
of the double reciprocal of the Michaelis-Menten equation, where v
is the initial velocity, Vmax is the maximal reaction velocity, and Km is
the Michaelis-Menten constant, is the concentration of substrate.
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Vmax is determined by molar change from absorbance at 652 nm
based on Moore's law.
2.5 Colorimetric detection
Colorimetric detection of H2O2 was performed as follows. 4 L of
-1
TMB (10 mM), 8 L of as-prepared Fe3O4@C YSNs (1 mg mL ), and
8 L of H2O2 with varied concentrations (1 M to 2 mM) were
added into 380 L sodium citrate buffer (0.2 M, pH 4.0). After 10
min incubation at 60 ⁰C, the reacted solution was applied to the
absorption spectroscopy measurement.
Glucose sensing was realized through the following steps. 10 L
-1
of GOx (10 mg mL ), 10 L of glucose with different concentrations,
and 80 L of phosphate buffered saline (PBS) buffer solution (pH
7.0) were mixed and incubated at 37 ⁰C for 30 min. After that, 20 L
-1
of Fe3O4@C YSNs stock solution (1 mg mL ), 10 L of mM TMB (10
mM), and 380 L of acetate buffer (0.2 M, pH 4.0) were added into
the above glucose reaction solution, and then the mixed solution
reacted for 10 min at 60 ⁰C. Consequently, Fe3O4@C YSNs were
removed from the reaction solution by applying a magnetic field,
and the final reaction solution was recorded by a UV-Vis
spectrometer.
3. Results and Discussion
Scheme 1 illustrates the synthesis roadmap for Fe 3O4@C
yolk-shell nanostructures. First, Fe 3O4 MNPs were prepared
through a solvothermal reaction. Then, we employed an
extended stöber method for coating double layers of silicon
oxide (SiO2) and polydopamine (PDA) to construct
Fe3O4@SiO2@PDA nanospheres using one-step method. Next,
Fe3O4@SiO2@C NPs were formed by annealing
Fe3O4@SiO2@PDA in a tube furnace under nitrogen
atmosphere at 500°C. Finally, SiO2 template was selectively
removed by chemical etching to obtain Fe 3O4@C YSNs.
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ARTICLE
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DOI: 10.1039/C7NR00819H
Nanoscale Accepted Manuscript
Scheme 1. Schematic illustrations of the fabrication process of
Fe3O4@C YSNs by one-step synthesis approach.
Figure 1 shows the topologies and dispersions of the obtained
products in the process which were characterized by scanning
electron microscopy (SEM) and transmission electron microscopy
(TEM). Fe3O4 NPs were prepared by the reported solvothermal
47
approach. From the SEM image in Figure 1A, it can be seen that
Fe3O4 MNPs were dispersed and uniform nanospheres with an
average diameter of 150 nm. TEM image in Figure 1B indicates that
Fe3O4 NPs have a rough surface and consisted of numerous
nanocrystals, and the citrate groups were capping to endow
hydrophilic property of these Fe3O4 nanospheres. As a result, they
were well-dispersed in the water/ethanol solution. Because of the
similar synthetic conditions but different reaction rate of stöber
silica and polydopamine polymers, a one-pot stöber method for
PDA and SiO2 coating have been developed to synthesize nearly
monodispersed Fe3O4@SiO2@PDA spheres. Followed by calcination
in inert atmosphere, the Fe3O4@SiO2@C composites with double
shells composed of silica and carbon layer, which were show in
Figure 1C and 1D. Compared to Fe3O4 NPs, Fe3O4@SiO2@C NPs has
a larger diameter of 250 nm and smooth surfaces, attributing to
being coated with double layer shells. After hydrothermal reaction
with ammonium hydroxide solution, the Fe3O4@SiO2@C
transformed a yolk-shell magnetic carbon, which further confirmed
the double layered core-shell structure with inner silica shell and
outer carbon shell of Fe3O4@SiO2@C. Figure 1E-1G reveals the SEM
and TEM image of Fe3O4@C YSNs with an average diameter of ~220
nm (Figure S1), which clearly manifests that Fe3O4@C YSNs retained
the spherical shape of the original Fe3O4@SiO2@C, but included the
removable Fe3O4 core, hollow space between the core and the
carbon shell with void size of ~40 nm, as the structure of our initial
expectations.
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ARTICLE Journal Name

bare Fe3O4 MNPs and Fe3O4@C YSNs exhibit superparamagnetic

A B behavior and little hysteresis (Figure 2C). AsDOI:
View Article Online
illustrated in Table S1,
10.1039/C7NR00819H

the magnetic saturation (Ms) value of Fe3O4@C YSNs was a little

lower than that of the bare Fe3O4 MNPs, which might be resulted
from the decrease in the density of Fe3O4 (~83%) in the prepared
nanocomposites. The as-prepared multifunctional Fe3O4@C
200 nm 100 nm
nanomaterials not only has a good dispersion in aqueous solution,
Published on 10 March 2017. Downloaded by Central Electrochemical Research Institute (CECRI) on 17/03/2017 07:00:27.

but also it still exhibits strong magnetic nature, in which

C D nanocomposites quickly separated to the side of the sample vial
within a few seconds. Hence, the magnetic nanomaterials can be
used for effective separation and concentration of an analyte from
complicated matrices by applying a magnetic field.

200 nm 100 nm A (311)  Fe3O4 B


a

Nanoscale Accepted Manuscript

(220) (400)  1406
a   (422)

% Transmittance
 1620

Intensity (a.u.)
E F G
b 



(440)
(511) 
 b
3433
808

960 576
1206
50 nm 
1088

c  

 c

10 20 30 40 50 60 70 80 4000 3500 3000 2500 2000 1500 1000 500

-1
2 (degree) Wavenumbers (cm )
200 nm 100 nm
C 40 Fe3O4 MNPs D
Fe3O4@C YSNs
20
M (emu/g)

Figure 1. SEM and TEM images of Fe3O4 MNPs (A, B), 0

Fe3O4@SiO2@C (C, D), and Fe3O4@C YSNs (E, F), and inset (G) shows
-20
TEM image at higher magnification. b
a
-40
To further confirm the formation of the Fe3O4@C YSNs, Figure 2A -6000 -3000 0 3000 6000
H (Oe)
displays the X-Ray diffraction (XRD) patterns of as-prepared samples
at different stages. It is found that six characteristic peaks (2 =
Figure 2. (A) XRD patterns and (B) FTIR spectra of (a) Fe3O4 NPs, (b)
30.3°, 35.6°, 43.2°, 53.5°, 57.2°, and 62.9°, corresponding to (220),
Fe3O4@SiO2@C MNPs, and (c) Fe3O4@C YSNs, respectively. (C)
(311), (400), (422), (511), and (440) in the case of Fe3O4, which can
Magnetization curves of (a) Fe3O4 MNPs and (b) Fe3O4@C YSNs. (D)
be indexed to the face-centered cubic structure (curve a, Figure 2A).
A photograph of the magnetic separation of Fe3O4@C YSNs.
Curve b exhibits an XRD pattern of Fe3O4@SiO2@C NPs, which
presented a broad peak at 20~30° related to amorphous SiO 2 shell.
Moreover, the intensity of diffraction peaks of the Fe3O4 was also
increased which is due to the good crystallinity after high
temperature treatment. The XRD data of Fe3O4@C YSNs (curve c,
Figure 2A) reveals that the main peaks were all overlapped with the
diffraction peak of the Fe3O4 (curve a, Figure 2A), which fully
indicates that the Fe3O4 cores for Fe3O4@SiO2@C NPs was kept
stable in the hydrothermal reaction. FTIR spectra further confirm
the synthesis of obtained products. In Figure 2B, curve a of bare
Fe3O4 NPs indicates the main peaks appear at 3433, 1620, 1406, Figure 3. (A) Typical UV-Vis spectra and (B) Visual photograph of
-1 48
and 576 cm . After coating and carbonization, it can be clearly different reaction systems: (b) NaAc buffer, (I) TMB + H 2O2 +
-1
seen the stretching vibrations of Si-O-Si at 1206 and 1088 cm , the Fe3O4@C YSNs, (I) TMB + H2O2, and (III) TMB + Fe3O4@C YSNs.
-1
-1
stretching vibrations of the Si-OH groups at 960 cm , and the [TMB] = 0.1 mM; [H2O2] = 0.5 mM; [Fe3O4@C] = 40 g mL .
-1
symmetric stretching of the Si-O-Si group at 808 cm in the
The Fe3O4@C YSNs can catalytically oxidize the chromogenic
Fe3O4@SiO2@C samples (curve b, Figure 2B), proving that SiO2 shell
49, 50 substrate 3,3',5,5'-Tetramethylbenzidine (TMB) in the presence of
had been successfully coated on the surface of Fe3O4. Curve c
H2O2 to a blue-colored oxidized product. To study the peroxidase-
shows the FTIR spectrum of Fe3O4@C YSNs, and it is observed that
-1 like activity of the as-prepared Fe3O4@C YSNs, the catalysis of TMB-
three peaks at 1206, 1088, and 960 cm corresponding to the Si-O-
H2O2 reaction systems has been assayed. As shown in Figure 3A,
Si and Si-OH groups disappeared, suggesting the removal of middle
there is an obvious absorption peak at 652 nm originated from the
silica layer and successful conversion of Fe3O4@SiO2@C NPs to
oxidation of TMB in the presence of H2O2. It gave a 625% response
Fe3O4@C YSNs. Additionally, the magnetic hysteresis loops of the
compared to that in the absence of Fe3O4@C YSNs, indicating the

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nanocomposites indeed displayed an excellent catalytic behavior. (2.0-10.0), temperature (25⁰C -65⁰C), and nanomaterial amount (0-
View Article Online
-1
Meanwhile, the TMB-H2O2 reaction systems in the presence of 100 g mL ) dependent-activity of Fe3O4@C YSNs was studied,
DOI: 10.1039/C7NR00819H

Fe3O4@C YSNs produced a deep blue color, while no obvious
reactions occured in the control systems (Figure 3B). It again proves
the important role of yolk-shell structured Fe3O4@C nanozymes as
the case with horseradish peroxidase (HRP).
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respectively. The catalytic reaction of TMB with H2O2 was much
faster in acid solution than in neutral or basic solutions (Figure 5A
and 5B). The catalytic activity always increases in the temperature
ranged from 25 ⁰C to 60 ⁰C, whereas it dramatically decreases at a
temperature higher than 60 ⁰C (Figure 5C). The absorbance is

To investigate the structural advantages of Fe3O4@C YSNs, we
compared the catalytic activities with other four ferroferric oxide-
based nanomaterials with different structures. Figure 4A indicates
the UV-Vis spectrum of Fe3O4@C YSNs has the largest absorption
peak at 652 nm. Meanwhile, either Fe3O4@SiO2 or Fe3O4@SiO2@C
NPs displayed no obvious peroxidase-like activity toward TMB
oxidation, and bare Fe3O4 or Fe3O4@C NPs has a moderate peak,
monotonically increased with the amount of Fe3O4@C YSNs, and
-1
the intersection point of two curves at 20 g mL is considered as
the optimal concentration (Figure 5D). Hence, the optimized
experimental conditions were under the following conditions of pH
-1
4.0, 60 ⁰C, and 20 g mL of nanocomposites. Remarkably, the
catalytic activity of Fe3O4@C YSNs is less sensitive to temperature
than that of HRP, which would open up potential a variety of

Nanoscale Accepted Manuscript

respectively. It was further found that the relative activities of bare applications in harsh environment.
Fe3O4 and Fe3O4@C NPs give respective response of 68% and 64%,
when that of Fe3O4@C YSNs was set as 100% (Figure 4B). In
addition, the TMB-H2O2 reaction solutions catalyzed by different
nanomaterials can be visually observed through the typical
photograph in inset, in accordance with the foregoing results. The
greatly enhanced catalytic activity of Fe3O4@C YSNs was attributed
18
to the unique yolk-shell nanostructure. First, the hollow space
between the core and the shell endow it a nanoreactor of a
homogeneous micro-environment for heterogeneous catalysis,
which were highly favorable for locating the active species. And
then, since the mesoporous channels are shortened, it provides a
free and short diffusion pathway around the active catalyst for the
mass transfer of reactant molecules and product species. The last
but not the least, the freely movable Fe3O4 cores can provide more
exposed active sites, in favor of speeding up the catalytic reaction
rate. The Fe3O4 cores are protected by the outer carbon shell from
aggregation and poisoning, resulting in the increase of catalysis and Figure 5. Peroxidase activities under varied reactions. (A) A pH-
stability of the Fe3O4@C YSNs. dependent response curve measured at pH 2.0-10.0. (B) Image of
catalytic oxidation of TMB and H2O2 solutions incubating with
different pH solutions. (C, D) Dependence of the peroxidase-like
activity on (C) temperature, and (D) concentration of Fe3O4@C
YSNs.

In order to better understand the catalytic mechanism of Fe3O4@C

YSNs, steady-state kinetics with TMB and H2O2 as substrate was
investigated. A series of kinetic data were measured by changing
one substrate concentration while keeping the other substrate at a
constant concentration. As illustrated in Figure 6A and 6B, typical
Figure 4. (A) UV-Vis absorption spectra of TMB-H2O2 reaction
Michaelis-Menten curves were measured for TMB and H2O2 in the
solutions and (B) Comparison of catalytic activities of different
suitable range of concentrations, respectively. Plots of reciprocal
nanocomposites: (a) Fe3O4@C YSNs, (b) Fe3O4 NPs, (c) Fe3O4@C
initial velocity versus reciprocal substrate concentration were
NPs, (d) Fe3O4@SiO2@C NPs, and (e) Fe3O4@SiO2 NPs. The
obtained (Figure 6C and 6D). It is found that the oxidation catalyzed
maximum point was set as 100%. Inset shows photographs of TMB-
by Fe3O4@C YSNs toward both substrates, TMB and H2O2, follows
H2O2 reaction solutions catalyzed by different magnetic
the typical Michaelis-Menten model. From Lineweaver-Burk plots,
nanomaterials with different structures. [TMB] = 0.1 mM; [H2O2] =
-1 the key enzyme kinetic parameters such as the maximum initial
0.5 mM; [Fe3O4@C] = 20 g mL .
velocity (Vmax) and Michaelis-Menten constant (Km) were
15
determined using the Lineweaver-Burk equations and given in
Like other peroxidase-like artificial nanozymes, the catalytic
Table 1. It is well known that Km is commonly identified as an
activity of Fe3O4@C YSNs is also dependent on pH, temperature,
indicator of enzyme affinity to substrates. A lower Km represents a
and amount of nanocomposites. Thereupon, varied reaction of pH

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ARTICLE Journal Name

higher enzyme affinity and vice versa. Surprisingly, the Km value of detect H2O2 utilizing blue color variation of TMB oxidized
View Article Online
Fe3O4@C YSNs toward the substrate H2O2 was 106, 4400, and 20 production. A typical H2O2 concentration-absorbance profile is
DOI: 10.1039/C7NR00819H

times lower than that of HRP, bare Fe3O4 NPs, and GO-Fe3O4
composites, respectively. The results indicate that Fe 3O4@C YSNs
possess a much higher binding affinity towards H2O2, suggesting
that H2O2 could be detected at a lower concentration. The
enhancement of catalytic activity may be caused by effective
Published on 10 March 2017. Downloaded by Central Electrochemical Research Institute (CECRI) on 17/03/2017 07:00:27.
shown in Figure 7A. It is found that a wide dynamic range from
1 M to 2000 M has been detected. The inset of Figure 7A shows
evident color variation, indicating that H2O2 can be visually detected
using naked eyes. Through changing the H2O2 concentration, a
linear calibration plot (0-10 M) is seen with a limit of detection

trapping of substrate H2O2 in the void space, positive synergistic
effect of Fe3O4 and carbon, as well as the protection of Fe3O4 cores
by carbon shell. In consideration of the peroxidase mimics activity
and robust stability, the Fe3O4@C YSNs could be employed as an
excellent substitute of natural enzyme HRP.
(LOD) of 0.39 M H2O2, which is 8 times sensitive than other
45
reported H2O2 sensors. It is in good agreement with the previous
results that Fe3O4@C YSNs have a higher affinity for H2O2 than
other artificial nanozymes.

Nanoscale Accepted Manuscript

Figure 7. (A) The dose-response curve and (B) the linear calibration
plot for H2O2 detection using Fe3O4@C YSNs. Inset: photograph of
color solutions with varied concentration of H2O2. The error bars
indicate the standard deviation of three measurements.

Combination of glucose oxidase (GOx) oxidation of glucose and

the catalytic reaction of Fe3O4@C YSNs, we have established a label-
free, sensitive and selective colorimetric biosensor toward glucose
sensing (Figure 8A). Figure 8B demonstrates a concentration
Figure 6. Steady-state kinetic assays of Fe3O4@C YSNs. (A) The
response curve for glucose detection utilizing Fe3O4@C YSNs as
concentration of H2O2 was 0.1 mM and the TMB concentration was
peroxidase mimics. The inset of Figure 8A displays obvious blue
varied. (B) The concentration of TMB was 0.5 mM and the H 2O2
color can be directly distinguished by naked eyes as the glucose
concentration was varied. (C, D) The Lineweaver-Burk plots of the
concentration surpasses 10 M. As shown in Figure 8C, the linear
double reciprocal of the Michaelis−Menten equation, with the
range for glucose sensing is from 1 M to 10 M. A detection limit
concentration of one substrate fixed and the other varied. The
-1 of 1.12 M could be calculated for this assay, which is much more
velocity (v) of the reaction was measured by using 20 g mL
sensitive than other reported glucose sensors based on different
Fe3O4@C YSNs in 0.2 M NaAc (pH 4.0) at 60 ⁰C for 10 min. Error bars
peroxidase mimetics (Table S2). Figures of merit are listed in Table
shown represent the standard error derived from three repeated
2.
measurements.
In order to further investigate the specificity of this assay, the
Table 1. Comparison of the kinetic parameters of different catalysts other three glucose analogues including lactose, fructose, and
and HRP. sucrose were chosen as control samples. As expected, the
-8 -1 absorbance curve of glucose has an apparent peak at 652 nm,
Catalyst Substrate Km (mM) Vmax (10 M s )
whereas that of the three interferences was almost as low as the
TMB 0.27 12.0
Fe3O4@C YSNs blank sample (Figure 8D). The control samples give smaller
H2O2 0.035 3.34
42 TMB 0.434 10 response compared to that of glucose, even though the
HRP concentration (5 mM) of analogues is 25 times higher than that of
H2O2 3.7 8.71
42 TMB 0.098 3.44 glucose (0.2 mM) (Figure 8E). The color variation (Figure 8D, inset)
Fe3O4
H2O2 154 9.78 also can be observed by naked eyes. Thereupon, it further verifies
15 TMB 0.43 13.08 the selectivity of the glucose analysis, attributing to the specificity
GO-Fe3O4
H2O2 0.71 5.31 of glucose oxidase toward glucose. To evaluate the practical
application of the proposed method, the Fe3O4@C YSNs based
On the basis of peroxidase mimics activity of the Fe3O4@C YSNs, biomimetic sensor was used to determine glucose in human serum
we developed a facile and label-free colorimetric approach to sample. The results of the determination were shown in Figure S2.

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According to the linear calibration plot, we calculated that glucose applicable to glucose determination in medical diagnosis and
View Article Online
in the serum sample was 4.51 mM. The analytical result were quite biological analysis. DOI: 10.1039/C7NR00819H

close to the provided value of 4.33 mM from the hospital,

suggesting that this label-free colorimetric method can be
Published on 10 March 2017. Downloaded by Central Electrochemical Research Institute (CECRI) on 17/03/2017 07:00:27.

Nanoscale Accepted Manuscript

Figure 8. Sensitivity and selectivity analysis for glucose sensor utilizing Fe3O4@C YSNs. (A) Schematic illustration of a simple and label-free
colorimetric detection of glucose based on as-prepared Fe3O4@C YSNs. (B) The dose-response curve for glucose detection. Inset:
photograph of reaction solutions with varied concentration of glucose. (C) The linear calibration plot for glucose detection. (D) UV-Vis
spectra by using 0.2 mM glucose, 5 mM lactose, 5 mM fructose, and 5 mM sucrose. (E) Selective detection of glucose by monitoring the
relative absorbance at 652 nm. Inset: photograph of colorimetric response of different reaction systems. The error bars represent the
standard deviation of three measurements.

Table 2. Quantitative analysis of H2O2 and glucose in our method.

Regression equation
Correlation Linear range b
Analyte LOD (M)
a a coefficient (M)
Slope Intercept

H2O2 0.01005±0.00059 0.1271±0.0023 0.99289 1-20 0.39

Glucose 0.00422±0.00016 0.0751±0.0006 0.99767 1-10 1.12

a b
The standard deviation derives from three repeated measurements. LOD = 3σ/k, where σ is the standard deviation for target-blank
sample and k represents the slope of the calibration curve.

4. Conclusions typical Michaelis-Menton kinetics through catalytic oxidations of

Taken together, we have synthesized yolk-shell nanostructured
Fe3O4@C composites by utilizing a facile one-step approach and
selectively chemical etching. The unique structures of Fe3O4@C
YSNs endowed them with the enhanced intrinsic peroxidase mimics
activity and excellent superparamagnetic properties. The catalytic
activities of Fe3O4@C YSNs were dependent on pH, temperature,
amount of catalyst, and substrate concentration. Also, they showed
TMB and H2O2. Based upon that, we developed a label-free,
sensitive and selective colorimetric method for H2O2 and glucose
detection. Owing to the facile and ease of preparation, robust
storability, and magnetic properties, Fe3O4@C YSNs showed great
potential applications in environmental monitoring, biomedical
researches, and clinical diagnosis in the future.

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Acknowledgments
This work was financially supported by National Key Research and
Development Program (2016YFA0201204, 2013CB933802),
Shanghai Municipal Natural Science Foundation (17ZR1412100),
Key Laboratory of Interfacial Physics and Technology, Chinese
Academy of Sciences (CASKL-IPT1603), Startup Foundation for
Published on 10 March 2017. Downloaded by Central Electrochemical Research Institute (CECRI) on 17/03/2017 07:00:27.
Doctors of Shanghai University of Engineering Science, the National
Natural Science Fund of China (21305086, 21373260, 31470960),
Research Innovation Program of Shanghai Municipal Education
Commission (14YZ138), and the Special Scientific Foundation for
Outstanding Young Teachers in Shanghai Higher Education
Institutions (ZZGJD13016).
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