Fe3O4-Au@mesoporous SiO2 microspheres: An ideal artificial enzymatic cascade system

Article abstract of DOI:10.1039/c3cc40622a

An artificial enzymatic cascade system with high activity and stability is engineered based on Fe₃O₄-Au@mesoporous SiO₂ (MS) microspheres, which possess both intrinsic glucose oxidase (GOx)- and peroxidase-mimic activities

Full text of DOI:10.1039/c3cc40622a

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Fe3O4–Au@mesoporous SiO2 microspheres: an ideal
artificial enzymatic cascade system†
Xiaolong He,ab Longfei Tan,a Dong Chen,*c Xiaoli Wu,ab Xiangling Ren,a
Yanqi Zhang,a Xianwei Menga and Fangqiong Tang*a
Cite this: Chem. Commun., 2013,
49, 4643
Received 24th January 2013,
Accepted 29th March 2013
DOI: 10.1039/c3cc40622a
www.rsc.org/chemcomm
An artificial enzymatic cascade system with high activity and linking GOx and horseradish peroxidase (HRP) to the DNA
stability is engineered based on Fe3O4–Au@mesoporous SiO2 scaffolds for biocatalysis.11 The organized enzyme cascades were
(MS) microspheres, which possess both intrinsic glucose oxidase found to proceed more effectively than homogeneous mixtures
(GOx)- and peroxidase-mimic activities.
of GOx and HRP, due to a high local concentration of the reactive
components confined in the vicinity of biocatalysts.12 This
Enzyme mimics have attracted sustained attention because ordered enzyme system also inspired the synthesis of other
they possess many advantages compared with natural enzymes, organized biosystems for bio-applications.13 Natural enzymatic
such as low cost of preparation and purification, high stability cascade systems also have the intrinsic drawbacks similar to
to environmental conditions, and difficulty in degeneration enzymes. Therefore, engineering of enzyme-mimic NPs for arti-
and inactivation.1 Recently, Fe3O4 magnetic nanoparticles (NPs), ficial enzymatic cascade systems is a logical implementation to
which were generally considered to be biologically inert, have simulate the biocatalysis system, which will be significant in
been discovered to possess intrinsic peroxidase-mimic activity.2 bionics, biosensing and biomedicine applications. However,
Afterwards, peroxidase-mimic activities were reported for CeO2 enzyme mimics have not been used to simulate complex enzyme
NPs,3 FeS nano-sheets,4 V2O5 nanowires5 and single walled systems, especially cascade enzyme-catalytic reactions. In addi-
carbon nanotubes.6 Very recently, small Au NPs were found to tion, the enzyme-mimic NPs reported are limited by low activity
exhibit glucose oxidase (GOx)-mimic activity. In the presence of and stability due to their heterogeneous distribution and easy
oxygen, they can catalytically oxidize glucose and produce aggregation during catalytic reactions.14
H2O2.7 Enzyme mimics possess some properties similar to
Herein, we report novel, discrete and monodisperse Fe3O4–
natural enzymes such as selectivity, specificity and high activity, Au@mesoporous SiO2 (MS) microspheres with both high GOx-
and have been used to replace enzymes in biomedical applica- and peroxidase-mimic activities. The superparamagnetic Fe3O4
tions, such as immuno-assays,3 detection of DNA,8 and other cores provide high peroxidase-mimic activity and make the
biological applications as well.9
artificial enzymatic system easily recyclable. On the surface of
Exploring the ‘‘static’’ enzyme mimics is not enough to Fe3O4 cores, ultrafine Au NPs with high GOx-mimic catalytic
simulate intelligent architectures, just like combining protein, activity are dispersed. Furthermore, Fe3O4–Au particles are
nucleic acid and lipid alone is not sufficient to form a functional encapsulated in MS shells to hinder the aggregation and keep
cell.10 One desirable but challenging topic is the assembly of them stable even under harsh conditions. Meanwhile, small
various nanomaterials together into ordered functional systems, active molecules are allowed to diffuse in and out of the MS
a common phenomenon in nature, but much more difficult to shells.15 Based on these functional units, the Fe3O4–Au@MS
achieve with artificial components. To mimic complex enzyme microspheres as robust nanoreactors can catalyze a self-organized
systems in nature, Willner et al. fabricated enzyme cascades by cascade reaction, which includes oxidation of glucose by
oxygen to yield gluconic acid and H2O2, and then oxidation of
3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to produce a colour
change. To the best of our knowledge, it is reported for the first
a Laboratory of Controllable Preparation and Application of Nanomaterials,
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
Beijing 100190, P.R. China. E-mail: tangfq@mail.ipc.ac.cn; Fax: +86-10-62554670; time that both glucose oxidase- and peroxidase-mimic activities
Tel: +86-10-82543521
are combined in one nanoparticle to simulate the enzymatic
cascade system without the help of natural enzymes.
b University of Chinese Academy of Sciences, Beijing 100049, P.R. China
c Beijing Creative Nanophase Hi-Tech Company, Limited, Beijing 100086,
The preparation of Fe3O4–Au@MS microspheres is described
P.R. China. E-mail: creative.nanophase@gmail.com
in Scheme 1. Firstly, the water dispersible superparamagnetic
Fe3O4 particles were synthesized via a hydrothermal method
† Electronic supplementary information (ESI) available: Experimental details and
relevant figures. See DOI: 10.1039/c3cc40622a
c
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Scheme 1 The synthesis route to Fe3O4–Au@MS microspheres.
described in the literature with minor modifications (see
Experimental section in the ESI†).16 Secondly, Fe3O4–Au parti-
cles with highly dispersed Au NPs on the surfaces were
prepared by reducing auric chloride ions with NaBH4 in the
presence of Fe3O4 particles. Finally, through a surfactant-
assembled sol–gel process with TEOS as the SiO2 source and
cetyltrimethylammonium bromide (CTAB) as the structure-
directing agent, a mesostructured CTAB–SiO2 composite layer
was formed on the Fe3O4–Au particles. After the removal of the
CTAB template by acetone extraction, Fe3O4–Au@MS micro-
spheres were obtained.
Fig. 1 (a) TEM image of Fe3O4–Au particles. (b) High-resolution TEM image of
Fe3O4–Au particles. (c) TEM image of Fe3O4–Au@MS microspheres. (d) SEM
image of Fe3O4–Au@MS microspheres.
Scanning electron microscopy (SEM) images show that the
Fe3O4 particles have a nearly spherical shape and uniform size
with a rough surface (Fig. S1a, ESI†). Transmission electron
microscopy (TEM) images indicate that the obtained magnetite
particles are loose clusters composed of nanocrystals with a size
of about 7–10 nm (Fig. S1d, ESI†). Fourier transform infrared
(FT-IR) spectra show that Fe3O4 particles are capped by citrate
groups exhibiting excellent dispersibility in polar solvents such
as water and ethanol, which favour the subsequent coating or
modification with other NPs or polymers (Fig. S2, ESI†). For the
preparation of Fe3O4–Au particles, the citrate groups on the
surface of the magnetite particles act as stabilizing agents by
providing anchoring sites to allow the formation of highly
dispersed AuNPs. The TEM images of Fe3O4–Au particles
obtained clearly show that their surfaces are evenly covered
by ultrafine Au NPs (Fig. 1a and b). A wide-angle XRD pattern of
the Fe3O4–Au microspheres shows the characteristic broad
diffraction peaks corresponding to the cubic-phase Au and
spinel Fe3O4 NPs, which further prove the attachment of Au
NPs and the well-retained magnetite phase (Fig. S3, ESI†).
According to the Debye–Scherrer formula, the sizes are calcu-
lated to be 4.2 and 7.2 nm for Au and magnetite nanocrystals
respectively, in good agreement with the TEM observations. We
have analyzed the content of Au in Fe3O4–Au particles using an
energy dispersive spectrometer (Fig. S4, ESI†). The Au content is
6.54% (wt%).
as 251.8 m2 gꢀ1 (Fig. S5, ESI†). In addition, Fe3O4–Au@MS
microspheres show a superparamagnetic behaviour according to
the hysteresis loops (Fig. S6, ESI†). No remanence or coercivity
was detected at room temperature as the Fe3O4 particles are
composed of ultrafine magnetite nanocrystals, which have been
proved by TEM and XRD. Fe3O4–Au@MS microspheres with
superparamagnetic property can remain homodispersed in
solution instead of aggregating, which is beneficial for diffusing
substrates to interact with catalysts. By simply using a magnet,
the Fe3O4–Au@MS microspheres can be completely separated
from mixture within 1 min because of the high magnetite
content (Fig. S6, ESI†). The excellent dispersibility, high stability
and superparamagnetic properties of the Fe3O4–Au@MS micro-
spheres make them an ideal material for liquid-phase catalysis.
In our study, we proved that Fe3O4–Au@MS microspheres
possessed both intrinsic GOx- and peroxidase-mimic activities,
and engineered an artificial enzymatic cascade system with
high activity and stability based on this nanostructure. The
peroxidase-mimic activity of Fe3O4–Au@MS microspheres in
solution was evaluated first. It was found that the Fe3O4–
Au@MS microspheres could catalytically oxidize TMB in the
presence of H2O2, producing H2O and the oxidation of TMB
(oxTMB) with blue color [eqn (1)]:
Fe3O4ꢀAu@MS
To stabilize these Fe3O4–Au particles and keep their high
enzyme-mimic activity during enzyme-catalyzed reactions, we
H O þ TMB
H O þ oxTMB
(1)
ƒƒƒƒƒƒƒƒ!
2
2
2
intended to induce a MS layer on the surface of Fe3O4–Au As shown in Fig. S7 (ESI†), a typical blue colour reaction was
particles in our study. The obtained Fe3O4–Au@MS micro- observed after the Fe3O4–Au@MS microspheres were added
spheres (Fig. 1d) exhibit a more regular spherical shape with into the TMB–H2O2 solution (pH = 4.0) for 10 min. Removing
smooth surfaces due to the MS shells of the Fe3O4–Au@MS the microspheres using an external magnetic field, we found
microspheres, compared with Fe3O4 particles (Fig. S1b, ESI†). that the major absorbance peaks of the reaction mixture
N2 sorption–desorption isotherms of the Fe3O4–Au@MS micro- appeared at 370 and 652 nm, which originated from oxTMB.
spheres show representative type-IV curves, suggesting cylin- Additional control experiments showed that neither H2O2–TMB
drical pores with a narrow pore size distribution at 3.8 nm nor Fe3O4–Au@MS microspheres/TMB could introduce any
(Fig. S5, ESI†). The BET surface area is calculated to be as high colour change (Fig. S7, ESI†). It proved that Fe3O4–Au@MS
c
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130 mM (Fig. 2c) with a detection limit of 0.5 mM (Fig. S9, ESI†),
after 30 min of reaction. It is proved that the activity and
stability of Fe3O4–Au@MS are much higher than the mixture
of Fe3O4 and Au particles (Fig. S10, ESI†). The activity and
structure of Fe3O4–Au@MS microspheres can be maintained
after 6 cycles of reaction. As control, serious aggregation of Au
NPs on the surface of Fe3O4–Au particles occurred after only
one cycle of reaction (Fig. S11, ESI†).
In summary, we have prepared Fe3O4–Au@MS micro-
spheres, which possess both intrinsic glucose oxidase (GOx)-
and peroxidase-mimic activities and can mimic the complexity
and function of the enzymatic cascade system. Based on the
color reaction caused by the artificial enzymatic cascade system,
Fe3O4–Au@MS microspheres can be used to target and visualize
tumor tissues as cell probes, detect biomolecules and catalyze
biochemical reactions. This work can also inspire the studies for
constructing various nanocomponents together into organized
Fig. 2 (a) The principle of the artificial enzymatic cascade system based on
Fe3O4–Au@MS microspheres, (b) typical H2O2 concentration response curves functional systems.
(pH = 4.0; reaction time was 10 min), (c) typical glucose concentration response
This work was supported by the National Natural Science
Foundation of China (no. 51202260, 81171454, 61171049,
61178035, 81000667).
curves (pH = 4.0; reaction time was 30 min). The linear range for glucose was
from 1 ꢁ 10ꢀ5 to 1.3 ꢁ 10ꢀ4 mol Lꢀ1
.
Notes and references
microspheres exhibited intrinsic peroxidase-mimic activity.
In addition, the relationship between the absorbance of oxTMB
and H2O2 concentration was further studied. From Fig. 2b, we
can see that the absorbance of the reaction mixture intensified
with increasing amounts of H2O2.
It was reported that small Au NPs possessed intrinsic GOx-
mimic activity and could catalyze the oxidation of glucose with
the cosubstrate oxygen (O2), producing gluconate and hydrogen
peroxide (H2O2).7 In our study, we suggested that Au NPs of
Fe3O4–Au@MS microspheres would act as GOx [eqn (2)]:
1 (a) Y. Song, K. Qu, C. Zhao, J. Ren and X. Qu, Adv. Mater., 2010, 22,
2206–2210; (b) M. I. Kim, Y. Ye, B. Y. Won, S. Shin, J. Lee and
H. G. Park, Adv. Funct. Mater., 2011, 21, 2868–2875; (c) K. Fan,
C. Cao, Y. Pan, D. Lu, D. Yang, J. Feng, L. Song, M. Liang and X. Yan,
Nat. Nanotechnol., 2012, 7, 459–464; (d) J. Mu, Y. Wang, M. Zhao and
L. Zhang, Chem. Commun., 2012, 48, 2540–2542.
2 L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang, J. Feng,
D. Yang, S. Perrett and X. Yan, Nat. Nanotechnol., 2007, 2, 577–583.
3 A. Asati, S. Santra, C. Kaittanis, S. Nath and J. M. Perez, Angew.
Chem., Int. Ed., 2009, 48, 2308–2312.
4 Z. Dai, S. Liu, J. Bao and H. Ju, Chem.–Eur. J., 2009, 15, 4321–4326.
´
´
5 R. Andre, F. Natalio, M. Humanes, J. Leppin, K. Heinze, R. Wever,
¨
H. C. Schroder, W. E. G. Mu¨ller and W. Tremel, Adv. Funct. Mater.,
Fe3O4ꢀAu@MS
2011, 21, 501–509.
Glucose þ O2
Gluconic acid þ H O
(2)
ƒƒƒƒƒƒƒƒ!
2
2
6 R. Cui, Z. Han and J. J. Zhu, Chem.–Eur. J., 2011, 17, 9377–9384.
7 (a) P. Beltrame, M. Comotti, C. Della Pina and M. Rossi, Appl.
Catal., A, 2006, 297, 1–7; (b) W. Luo, C. Zhu, S. Su, D. Li, Y. He,
Q. Huang and C. Fan, ACS Nano, 2010, 4, 7451–7458.
8 M. Liu, H. Zhao, S. Chen, H. Yu and X. Quan, Chem. Commun., 2012,
48, 7055–7057.
9 (a) L. Su, J. Feng, X. Zhou, C. Ren, H. Li and X. Chen, Anal. Chem.,
2012, 84, 5753–5758; (b) C. W. Lien, C. C. Huang and H. T. Chang,
Chem. Commun., 2012, 48, 7952–7954; (c) Z. Chen, J.-J. Yin,
Y.-T. Zhou, Y. Zhang, L. Song, M. Song, S. Hu and N. Gu, ACS Nano,
2012, 6, 4001–4012.
10 Y. Lu and J. Liu, Acc. Chem. Res., 2007, 40, 315–323.
11 (a) Z.-G. Wang, O. I. Wilner and I. Willner, Nano Lett., 2009, 9,
4098–4102; (b) O. I. Wilner, Y. Weizmann, R. Gill, O. Lioubashevski,
R. Freeman and I. Willner, Nat. Nanotechnol., 2009, 4, 249–254.
12 O. Idan and H. Hess, Nat. Nanotechnol., 2012, 7, 769–770.
13 (a) M. You, R.-W. Wang, X. Zhang, Y. Chen, K. Wang, L. Peng and
W. Tan, ACS Nano, 2011, 5, 10090–10095; (b) J. Fu, M. Liu, Y. Liu,
N. W. Woodbury and H. Yan, J. Am. Chem. Soc., 2012, 134,
5516–5519; (c) S. Schoffelen and J. C. M. van Hest, Soft Matter,
2012, 8, 1736–1746.
We have already proved that the Fe3O4–Au@MS microspheres
could act as effective peroxidase mimics. And H2O2 is the main
product of the glucose oxidase (GOx)-catalyzed reaction. There-
fore, a self-organized cascade reaction could be realized using
Fe3O4–Au@MS microspheres only. The proposed principle for
the artificial enzymatic cascade system is described as follows
(Fig. 2a). In Fe3O4–Au@MS microspheres, glucose was oxidized
by the cosubstrate oxygen (O2) in solution catalyzed by Au NPs,
yielding gluconic acid and H2O2. H2O2 was confined in the
microsphere, and formed high local concentration. It was
directly catalyzed by Fe3O4 to oxidize TMB, and had no time
to diffuse. Owing to the oxidation of TMB, the blue color
reaction occurred, demonstrating that an effective artificial
enzymatic cascade system had been formed in solution.
Control experiments indicated that neither glucose/TMB nor
Fe3O4–Au@MS microspheres/TMB can lead to any color change
(Fig. S7, ESI†). The effect of pH on the catalytic activity of this
artificial enzymatic cascade system was investigated. The velo-
city of the cascade reaction was the highest at pH 4.0 (Fig. S8,
ESI†). At this value of pH, it can be seen that the absorbance of
the reaction mixture became stronger with the increasing
concentration of glucose in a linear relationship from 10 to
14 X. Zheng, Q. Liu, C. Jing, Y. Li, D. Li, W. Luo, Y. Wen, Y. He,
Q. Huang, Y. T. Long and C. Fan, Angew. Chem., Int. Ed., 2011, 50,
11994–11998.
15 (a) S. H. Joo, J. Y. Park, C.-K. Tsung, Y. Yamada, P. Yang and
G. A. Somorjai, Nat. Mater., 2009, 8, 126–131; (b) L. Tan, D. Chen,
H. Liu and F. Tang, Adv. Mater., 2010, 22, 4885–4889; (c) X. He,
L. Tan, X. Wu, C. Yan, D. Chen, X. Meng and F. Tang, J. Mater.
Chem., 2012, 22, 18471–18478.
16 J. Liu, Z. Sun, Y. Deng, Y. Zou, C. Li, X. Guo, L. Xiong, Y. Gao, F. Li
and D. Zhao, Angew. Chem., Int. Ed., 2009, 48, 5875–5879.
c
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