Magnetic nanoparticles coated with immobilized alkaline phosphatase for enzymolysis and enzyme inhibition assays

Article abstract of DOI:10.1039/c3tb00562c

Magnetic nanoparticles are potentially useful as supports for biomacromolecules because of their biocompatibility, low toxicity and easy separation. In this study, alkaline phosphatase (ALP) was used as a model enzyme, and a new type of immobilized ALP wa

Full text of DOI:10.1039/c3tb00562c

Journal of
Materials Chemistry B
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PAPER
Magnetic nanoparticles coated with immobilized
alkaline phosphatase for enzymolysis and enzyme
Cite this: J. Mater. Chem. B, 2013, 1,
1749
inhibition assays†
*
Siming Wang, Ping Su, Jun Huang, Jingwei Wu and Yi Yang
Magnetic nanoparticles are potentially useful as supports for biomacromolecules because of their
biocompatibility, low toxicity and easy separation. In this study, alkaline phosphatase (ALP) was used as
a
model enzyme, and a new type of immobilized ALP was prepared on superparamagnetic
nanoparticles and confirmed by various characterization techniques. X-ray diffraction (XRD), scanning
electron microscopy (SEM) and vibrating sample magnetometry (VSM) results present that the
synthesized nanoparticles possess a clear three-dimensional core–shell architecture with an average
diameter of about 390 nm and a high saturation magnetization of 86.7 emu gꢀ1. Fourier-transform
infrared spectra (FTIR) and thermogravimetric analysis (TGA) results show that ALP was successfully
attached to the surface of magnetic nanoparticles via a crosslinking technique. An enzyme inhibition
study was performed on the immobilized ALP magnetic nanoparticles using theophylline, L-tryptophan
and D-tryptophan as model inhibitors. The enzyme kinetics indicate that DL-tryptophan possesses chiral
discrimination inhibition and L-tryptophan exhibits uncompetitive inhibition for ALP, compared with no
Received 21st December 2012
Accepted 29th January 2013
obvious inhibition observed with its enantiomer. The results also show that theophylline is
a
noncompetitive inhibitor and has a markedly higher inhibitory effect than L-tryptophan. The protocol
described allows easy manipulation, reduces procedural time and can be adapted to high-throughput
screening of enzyme reactions and inhibitors.
DOI: 10.1039/c3tb00562c
www.rsc.org/MaterialsB
magnetic nanoparticles are compatible with multiple
approaches, allowing the coupling of various analytical tech-
1 Introduction
Magnetic nanoparticles are advanced materials that have
potential use as supports for biomacromolecules, such as
nucleic acids, antibodies, peptides and enzymes.1–5 Magnetic
nanoparticles with immobilized enzymes or proteins possess
several notable advantages due to their novel nanostructures
and magnetic properties. Such nanoparticles can provide a
biocompatible and inert microenvironment that does not
interfere with the native properties of enzymes or proteins, thus
retaining their biological activities.6 Their nanoscale structures
reduce diffusion limitations and offer a large specic surface
area, resulting in a low mass transfer resistance and high
binding capacity. In addition, sensitive magnetic responses
enable easy separation of immobilized enzymes or proteins
from reaction mixtures simply by applying a magnetic eld,
with no need for centrifuges, lters or other equipment, which
signicantly shortens procedural time and simplies the
experimental process.7,8 Furthermore, these functionalized
niques such as mass spectrometry and capillary electropho-
resis.9–11 Because of these unique properties and functions,
immobilized enzymes or proteins on magnetic nanoparticles
show great promise for applications in liquid-phase reactions,
especially in the elds of biocatalysis,12 biosensors,13 biological
recognition14 and biological screening.15
Alkaline phosphatase (ALP) is a nonspecic phosphomono-
esterase found widely in many mammalian tissues.16 It is
involved in various metabolic functions such as fat absorption,
mucosal defense and skeletal mineralization, to catalyze the
hydrolysis of phosphomonoesters, releasing free inorganic
phosphate and alcohol.17,18 ALP is oen used as a major target in
analytical chemistry, biochemistry and medical chemistry to
probe causative disease mechanisms. Studies of ALP activity and
screening of its inhibitors are required in clinical diagnostics to
develop potential drug therapies for osteoarthritis, idiopathic
infantile arterial calcication and end-stage renal diseases.19,20
However, many ALP assays use free enzyme in a homogeneous
solution, which may be susceptible to denaturation and is diffi-
cult to reuse or recover, leading to high cost and sample
complexity. Moreover, free enzyme is possibly limited through
spectrophotometric interferences arising from self-absorption.
Immobilized enzymes can not only retain a major enzymatic
College of Science, Beijing University of Chemical Technology, Beijing 100029, P.R.
China. E-mail: yangyi@mail.buct.edu.cn; Fax: +86-10-64434898; Tel: +86-10-
64454599
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3tb00562c
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activity but also improve stability compared with free Thermo Scientic Nicolet 8700 (Waltham, MA, USA). Ther-
enzymes.21,22 They have potential applications in the elds of mogravimetric analysis (TGA) was performedꢀ1for powdered
biochemistry, food, clinical diagnosis and environmental engi- samples with a heating rate of 10 ꢂC min
from room
neering.23–26 Therefore, a simple, efficient and economic immo- temperature to 800 ꢂC under a nitrogen atmosphere using a
bilized ALP is needed for enzymatic and inhibitory assays of ALP. HCT-2 thermo analysis system (Beijing Henven Scientic
This current study focuses on developing a new type of Instrument Corporation, Beijing, China).
immobilized ALP using superparamagnetic nanoparticles. Ami-
nated silica-coated Fe3O4 magnetite particles were synthesized 2.3 Preparation of core–shell structured Fe3O4@SiO2@APES
through a solvent-thermal method, characterized by various nanoparticles
analytical techniques, and applied to immobilization of ALP.
Aminated silica-coated magnetic nanoparticles were synthe-
This immobilized enzyme offers the advantages of easy isolation,
sized sequentially through a solvothermal reaction, sol–gel
low cost and high efficiency. Optimal enzymolysis conditions for
coating approach and silanization process.27,28 In brief, 1.35 g of
FeCl3$6H2O as a single ferric source and 3.6 g of sodium acetate
were dissolved in 40 ml of glycol under vigorous stirring for 30
min. The obtained homogenous yellow solution was transferred
applying the immobilized ALP magnetic nanoparticles, including
temperature, incubation time, pH and substrate concentration,
were identied by high performance liquid chromatography
(HPLC). The other enzymatic parameters of the immobilized
ALP, such as the Lineweaver–Burk plot and Michaelis constant as
well as repeatability and stability, were also evaluated. Finally,
theophylline, L-tryptophan and D-tryptophan were used as model
inhibitors, and an enzyme inhibition study was performed on the
immobilized ALP magnetic nanoparticles to conrm their
performance in enzyme assays.
into a Teon-lined stainless-steel autoclave, sealed, and heated
ꢂ
at 200 C for 8 h. The resulting black magnetite particles were
separated with an external magnet, washed with ethanol and
water several times, and vacuum dried for 4 h. Next, the sol–gel
approach was applied to preparing core–shell structured
Fe3O4@SiO2. The Fe3O4 nanobeads were dispersed in a solution
of ethanol (150 ml), deionized water (50 ml) and concentrated
ammonia aqueous solution (5 ml, 28 wt%), followed by drop-
wise addition of 50 ml of ethanol containing 2 ml of tetrae-
thoxysilane with stirring for 6 h. Subsequently, the silica-coated
Fe3O4 was collected with a magnet and washed sequentially
with 1 M HCl, water and ethanol. Aer vacuum drying,
Fe3O4@SiO2 was mixed with a 10% (v/v) APES toluene solution
at 100 ꢂC for 24 h under inert conditions using nitrogen. Finally,
the Fe3O4@SiO2@APES beads were separated, washed with
ethanol, and dried in a vacuum.
2 Materials and methods
2.1 Chemicals and materials
ALP (EC 3.1.3.1 from bovine intestinal mucosa, 10 U mgꢀ1) and 4-
nitrophenylphosphate were purchased from Sigma-Aldrich (St
Louis, MO, USA). Theophylline and 4-nitrophenol were obtained
from AccuStandard (New Haven, CT, USA). g-Amino-
propyltriethoxysilane (APES), sodium cyanoborohydride and
D-tryptophan were obtained from Acros Organics (Geel, Belgium).
L-Tryptophan, tetraethoxysilane and methanol (HPLC grade) were
purchased from J&K Scientic (Beijing, China). Magnesium
chloride, zinc chloride, iron(III) chloride hexahydrate, sodium
acetate and glycol were supplied by Beijing Yili Chemical (Beijing,
China). All other chemical reagents used in the experiment were
of analytical grade.
2.4 ALP immobilization
Aer washing with 20 mM pH 8.5 carbonate buffer, the
Fe3O4@SiO2@APES nanoparticles were reacted with 5%
glutaraldehyde solution for 1 h and then washed with carbonate
buffer. Thereaer, the magnetic nanoparticles were immersed
in a 2.5 mg mlꢀ1 ALP and sodium cyanoborohydride carbonate
solution (pH 8.5) and incubated for 2 h. Sodium cyanoborohy-
dride reduces the Schiff base and promotes the formation of
stable secondary amine linkages between the immobilized
enzyme and magnetite. Finally, the immobilized ALP magnetic
nanoparticles, rinsed with carbonate buffer, were stored at 4 ꢂC
until use. In this way, ALP was covalently attached to the surface
of the magnetic nanoparticles.
2.2 Instrumentation
HPLC analyses were performed on an Agilent 1100 Series HPLC
system (Agilent, Santa Clara, CA, USA) equipped with an Agilent
Eclipse XDB-C18 column (150 ꢁ 4.6 mm, i.d., 5 mm particle) at
room temperature and an ultraviolet detector operated at
311 nm wavelength. The mobile phase was methanol : water
(40% : 60%, v/v) with a ow rate of 1.0 ml minꢀ1. Agilent LC
ChemStation soware was used for system control, data
2.5 Immobilized enzyme activity assay and inhibition study
collection and processing. X-ray diffraction patterns (XRD) were An enzymatic hydrolysis reaction was chosen for the immobilized
collected on a D/max-Ultima (Rigaku, Tokyo, Japan) using Cu ALP magnetic nanoparticles based on 4-nitrophenylphosphate as
˚
Ka radiation (40 kV, 40 mA, l ¼ 1.5418 A) with a scan step of a substrate. The corresponding enzymolysis product was 4-nitro-
0.02ꢂ. The sizes and morphologies of magnetic nanoparticles phenol.29 Due to strong ultraviolet absorbance, both the substrate
were tested using a transmission electron microscope (TEM, and product can be monitored spectrophotometrically without
Hitachi H800, Tokyo, Japan). The magnetic properties of the requiring derivation. The immobilized ALP magnetic nano-
prepared nanoparticles were measured on a vibrating sample particles (10 mg) and substrate, in the presence or absence of
magnetometer (VSM, Lake Shore 7410, Westerville, OH, USA). inhibitors, were mixed and incubated in an enzyme assay buffer
Fourier-transform infrared spectra (FTIR) were obtained using a (20 mM carbonate buffer containing 5 mM MgCl2 and 1 mM
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ZnCl2) at a nal volume of 10 ml. A magnet was used to gather and
isolate the nanospheres, and 20 ml of supernatant was injected
into the HPLC. The optimal enzymolysis conditions were
obtained according to the peak area of 4-nitrophenol. For the
inhibition study, the inhibition percentage was calculated
according to the reduction of the product peak area. The proce-
dures for preparing immobilized ALP magnetic nanoparticles and
carrying out the enzyme assay are shown schematically in Fig. 1.
Fig. 2 Transmission electron microscopy images of Fe3O4 (A), Fe3O4@SiO2 (B)
and immobilized ALP magnetic nanoparticles (C).
3 Results and discussion
3.1 Characterization of immobilized ALP magnetic
nanoparticles
literature.30 The core–shell structure of Fe3O4@SiO2 with a shell
layer of 33 nm is obviously presented in Fig. 2B, and the
thickness of the shell increased to approximately 39 nm aer
ALP immobilization (Fig. 2C).
The crystalline structure and phase purity were determined by
power X-ray diffraction (ESI, Fig. S1†). The diffraction peaks in
curves can be assigned to the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1)
and (4 4 0) planes, and conrmed that the products were well
crystallized and indexed as the typical cubic iron oxide Fe3O4
(JCPDS card no. 65-3107). There was no obvious change aer
coating with the SiO2 layer and silanization, indicating the
amorphous nature of the silica shell.
The size and internal structure of magnetic nanoparticles
were investigated by TEM. Fig. 2A shows that these Fe3O4
magnetite particles had a mean diameter of about 350 nm and
consisted of many nanoparticles with an average crystallite size
of 16 nm, calculated from the X-ray diffraction results according
to the Scherrer equation, similar to the results reported in the
FTIR spectroscopy was performed to further reveal evidence
of the immobilized ALP present on the surface of magnetic
nanoparticles (ESI, Fig. S2†). The peak at 1086 cmꢀ1 is assigned
to the silica layer vibrations. The intense band between 3300
and 3400 cmꢀ1 results from the stretching vibration of the
hydroxyl group. Compared with Fe3O4@SiO2, the absorption
peaks at 2926, 2865, 1632 and 1456 cmꢀ1 could be observed in
the spectrum of immobilized ALP magnetic nanoparticles,
which correspond to the vibrations of methylene and amide
groups arose from enzyme side chains on the surface of nano-
particles. The FTIR results are closely similar to previous results
described in the literature,7,31 and suggest that ALP was
Fig. 1 Schematic of the synthesis of immobilized ALP magnetic nanoparticles and their use in an enzyme assay. Insets: typical HPLC chromatograms for the immo-
bilized enzyme assay with no inhibition (a) and sample containing theophylline (b). Peaks (1) 4-nitrophenylphosphate and (2) 4-nitrophenol. Conditions: column,
Agilent Eclipse XDB-C18 (150 ꢁ 4.6 mm i.d., 5 mm particle); detection wavelength, 311 nm; mobile phase, methanol : water (40 : 60%, v/v); flow rate, 1.0 ml minꢀ1
injection volume, 20 ml.
;
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3.2 Optimization of enzymolysis conditions
Control experiments were performed on the silanized magnetite
(Fe3O4@SiO2@APES) and the immobilized ALP magnetic
nanoparticles to validate the effect of the immobilized enzyme.
In the absence of immobilized ALP, only the substrate
(4-nitrophenylphosphate) was observed by HPLC. In contrast,
the colorless sample solution turned to yellow aer treatment
with immobilized ALP magnetic nanoparticles and, meanwhile,
another peak could be detected and identied by its retention
time as the enzymolysis product, 4-nitrophenol. This compar-
ison indicates that the ALP immobilized on magnetic nano-
particles and not the Fe3O4 core catalyzed the hydrolysis of the
substrate. The results further demonstrate the feasibility of the
protocol for using the immobilized ALP magnetic nanoparticles
and that the prepared magnetite could provide a biocompatible
microenvironment that retains the major activities of enzymes.
Temperature is one of the most important factors in enzy-
matic hydrolysis. The results of the experiments performed
between 20 and 60 ꢂC are presented in Fig. 5A, which show that
the enzymolysis efficieꢂncy initially increased with increasing
temperatures up to 40 C and that ALP was deactivated as the
temperature continued to rise, leading to a dramatic decline in
Fig. 3 TGA analysis of Fe3O4@SiO2 (a) and immobilized ALP magnetic nano-
particles (b).
successfully attached to the magnetic nanoparticles. The rela-
tive enzyme binding capacity was estimated to be about 115.5 mg
mgꢀ1 from the TGA result (Fig. 3).
The magnetic properties of samples were measured by
applying an external magnetic eld at room temperature by VSM,
and the hysteresis curves are presented in Fig. 4, which conrms
the superparamagnetism of these magnetite. The results showed
that the saturation magnetization values of Fe3O4, Fe3O4@SiO2
and immobilized ALP magnetic nanoparticles were 86.7, 57.2 and
47.9 emu gꢀ1 respectively. A dramatic decline in magnetic
response implied an increase in the thickness of the shell layer,
in agreement with the results obtained above. The immobilized
ALP magnetic nanoparticles could be dispersed in water by
sonication or vigorous shaking, resulting in a black suspension,
and were also easily aggregated from a homogeneous dispersion
by an external magnet, as shown in the inset of Fig. 4. Moreover,
re-dispersion occurred quickly with gentle shaking aer the
magnetic eld was removed. These results show that the
prepared nanoparticles possess remarkable magnetic respon-
siveness and redispersibility, which is an advantage in the
separation and reuse of the immobilized enzyme in practical
applications.
ꢂ
the enzymolysis efficiency. The optimal temperature was 40 C.
The effect of incubation time on the yield of the reaction product
is shown in Fig. 5B. A longer incubation time allows the substrate
to react more completely with the immobilized ALP magnetic
nanoparticles. On the other hand, longer enzymolysis increases
the duration of experiments. An optimal incubation time of 30
min was selected for the subsequent experiments. The effect of
pH on the activity of immobilized ALP was determined from pH
8.0 to 11.0, showing a maximum at pH 9.5 (Fig. 5C).
Based on the results obtained above, the optimal substrate
concentrations and the kinetic behavior of immobilized ALP
were investigated. The results for magnetic nanoparticles
incubated with a range of 4-nitrophenylphosphate concentra-
tions are shown in Fig. 5D. The enzymolysis efficiency increased
with increasing substrate concentrations up to 1 mg mlꢀ1
.
Fig.
4
Magnetization curves of Fe3O4, Fe3O4@SiO2 and immobilized ALP
Fig. 5 (A) Effect of temperature on enzymolysis efficiency. (B) Effect of incuba-
tion time on enzymolysis efficiency. (C) Effect of pH on enzymolysis efficiency. (D)
Effect of substrate concentration on enzymolysis efficiency.
magnetic nanoparticles. Inset: magnetic separation and re-dispersion of immo-
bilized ALP magnetic nanoparticles.
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However, further increases in concentration did not increase (maintaining a constant substrate concentration), as shown in
the enzymolysis efficiency signicantly because the enzyme shown in Fig. 6. The results show that the IC50 value for
binding capacity and enzymolysis ability were saturated due to theophylline was 0.71 mM and clearly smaller than that for
the xed amount of ALP immobilized on the surface of the L-tryptophan (10.32 mM), which suggests that theophylline has
nanoparticles. The most important kinetic constant of the a markedly stronger inhibitive effect. In contrast to the absence
enzyme reaction, the Michaelis constant, can be determined by of inhibitor (inset (a) of Fig. 1), inset (b) of Fig. 1 represents an
the Lineweaver–Burk method.32 The Lineweaver–Burk plot was ALP inhibition assay in which theophylline was added, showing
linear and gave a Michaelis constant of 67.14 mM, similar to the a dramatic reduction in the product peak area. The inhibition
results reported in the literature.20 In addition, X-ray diffraction constant Ki for the two inhibitors at IC50 was also determined to
patterns of ALP show that the diffraction peak angles are similar be 0.276 mM (theophylline) and 6.07 mM (L-tryptophan). The Ki
before and aer immobilization (ESI, Fig. S3†), which indicates values reported here are higher than those obtained with free
that the ALP conformation has no obvious change. These ALP in solution,35 reecting improved stability and resistance to
results demonstrate that no signicant change in enzyme inhibition by immobilized ALP. Compared with L-tryptophan, D-
properties was observed when using the proposed method.
tryptophan did not display obvious inhibition, which suggests
The repeatability of the immobilized ALP magnetic nano- that DL-tryptophan possesses a chirally dependent inhibition for
particles was assessed by carrying out ve sequential enzymatic ALP. This may be because the inhibitor recognition site is
hydrolyses using the same immobilized enzyme. The results commonly located within a groove or crack in the enzyme
show that the relative standard deviation (RSD) was 5.06%. The structure. Thus, only an inhibitor that possesses a suitable
storage stability was assessed by measuring activity aer storage spatial structure can bind to the enzyme and exhibit an inhib-
in buffer at 4 ꢂC for 30 days, and the activity remained at itory effect.36
80.15%. Compared with free ALPs in solution that are more
The enzyme inhibitive kinetics was investigated for the
susceptible to denaturation, the immobilized ALP on magnetic immobilized ALP magnetic nanoparticles using the optimal
nanoparticles lost some activity aer storage for 30 days but still enzymolysis conditions described above. The Lineweaver–Burk
retained a major enzymatic activity, enough for the subsequent plots were obtained at different inhibitor concentrations for
experiments. Moreover, the immobilized ALP on magnetic both inhibitors (theophylline and L-tryptophan). Fig. 7A shows
nanoparticles allows easy manipulation and separation from
reaction mixtures simply by applying a magnetic eld, with no
need for centrifuges or lters, which signicantly removes
interferences, shortens procedural time and simplies the
experimental process.
3.3 Immobilized ALP magnetic nanoparticles for enzyme
inhibition study
Abnormal activity or expression of enzymes occurs with the
emergence and metastasis of diseases. Enzymes are a useful
class of indicators for disease diagnosis and play an important
role during drug discovery processes.33,34 One of the most useful
applications of immobilized enzyme magnetic nanoparticles is
in enzyme inhibition assays. In this study, three inhibitor
candidates, theophylline, L-tryptophan and D-tryptophan, were
used to evaluate the function of the immobilized ALP magnetic
nanoparticles.
The inhibition efficiency curves for theophylline and L-tryp-
tophan were obtained by varying the inhibitor concentration
Fig. 7 (A) Lineweaver–Burk plot for the immobilized ALP magnetic nano-
particles in the presence of theophylline at various concentrations. Conditions:
(-) 0 mM; (C) 0.1 mM; (:) 1 mM. (B) Lineweaver–Burk plot for the immobilized
Fig. 6 Inhibition efficiency curves for the immobilized ALP magnetic nano-
ALP magnetic nanoparticles in the presence of L-tryptophan at various concen-
particles in the presence of inhibitors. (A) Theophylline and (B) L-tryptophan.
trations. Conditions: (-) 0 mM; (C) 5 mM; (:) 10 mM.
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that the maximum enzymolysis velocity declined with the
increasing concentration of theophylline, whereas the Michae-
lis constant barely changed. This corresponds to the charac-
teristics of noncompetitive inhibition and demonstrates that
8 E. Ranjbakhsh, A. K. Bordba, M. Abbasi, A. R. Khosropour
and E. Shams, Chem. Eng. J., 2012, 179, 272–276.
9 H. M. Chen, S. S. Liu, H. L. Yang, Y. Mao, C. H. Deng,
X. M. Zhang and P. Y. Yang, Proteomics, 2010, 10, 930–939.
theophylline is a noncompetitive inhibitor for ALP. The Line- 10 S. Lin, D. Yun, D. W. Qi, C. H. Deng, Y. Li and X. M. Zhang,
weaver–Burk curves for L-tryptophan exhibit that as the inhib- J. Proteome Res., 2008, 7, 1297–1307.
itor concentration increased, the maximum enzymolysis 11 X. Y. Yan and S. D. Gilman, Electrophoresis, 2010, 31, 346–
velocity and Michaelis constant both declined but their ratio 354.
was constant, showing a series of parallel straight lines in 12 A. H. Lu, E. L. Salabas and F. Schuth, Angew. Chem., Int. Ed.,
Fig. 7B. These results indicate that L-tryptophan displayed an
uncompetitive inhibition behavior.
2007, 46, 1222–1244.
13 R. Villalonga, M. L. Villalonga, P. Diez and J. M. Pingarron,
J. Mater. Chem., 2011, 21, 12858–12864.
14 L. S. Qing, Y. Xue, Y. Zheng, J. Xiong, X. Liao, L. S. Ding,
B. G. Li and Y. M. Liu, J. Chromatogr., A, 2010, 1217, 4663–
4668.
15 F. L. Hu, H. Y. Zhang, H. Q. Lin, C. H. Deng and X. M. Zhang,
J. Am. Soc. Mass Spectrom., 2008, 19, 865–873.
16 J. P. Lalles, Nutr. Rev., 2010, 68, 323–332.
17 T. D. Y. Chung, E. Sergienko and J. L. Millan, Molecules,
2010, 15, 3010–3037.
18 F. G. Sanchez, A. N. Diaz, M. C. R. Peinado and C. Belledone,
Anal. Chim. Acta, 2003, 484, 45–51.
19 S. Sidique, R. Ardecky, Y. Su, S. Narisawa, B. Brown,
J. L. Millan, E. Sergienko and N. D. P. Cosford, Bioorg.
Med. Chem. Lett., 2009, 19, 222–225.
20 J. Iqbal, Anal. Biochem., 2011, 414, 226–231.
21 S. M. Wang, P. Su and Y. Yang, Anal. Biochem., 2012, 427,
139–143.
22 S. M. Wang, P. Su, E. Hongjun and Y. Yang, Anal. Biochem.,
2010, 405, 230–235.
23 J. B. Zhang, H. H. Yan, T. Yang and Y. R. Zong, J. Environ.
Manage., 2011, 92, 53–58.
24 T. B. Goriushkina, A. P. Soldatkin and S. V. Dzyadevych,
J. Agric. Food Chem., 2009, 57, 6528–6535.
4 Conclusions
A new strategy using ALP immobilized onto magnetic nano-
particles for enzymolysis and enzyme inhibition assays has
been developed. The formation of the prepared immobilized
ALP magnetic nanoparticles was conrmed by various charac-
terization techniques and exhibited a clear three-dimensional
core–shell architecture and high saturation magnetization. A
practical application of the enzymatic hydrolysis and enzyme
inhibition assay was performed to evaluate the immobilized
ALP magnetic nanoparticles. The results demonstrate that they
possess high enzymolysis efficiency and satisfactory repeat-
ability. This method combines the advantages of immobilized
enzymes and magnetic media so that it not only offers low cost
through re-use of enzyme but also allows fast and simple
separation using magnets. The protocol provides a facile and
efficient approach to the fabrication of magnetic core/func-
tionalized ALP shell hierarchical structures, which can be easily
adapted to immobilization of other enzymes and potentially
realize high-throughput screening of enzyme reactions and
enzyme inhibitors.
25 L. Yang, C. Y. Chen, Y. F. Chen, J. Shi, S. D. Liu, L. P. Guo and
H. F. Xu, Anal. Chim. Acta, 2010, 683, 136–142.
26 Z. Liu, H. S. Wang, B. Li, C. Liu, Y. J. Jiang, G. Yu and
X. D. Mu, J. Mater. Chem., 2012, 22, 15085–15091.
27 H. Deng, X. L. Li, Q. Peng, X. Wang, J. P. Chen and Y. D. Li,
Angew. Chem., Int. Ed., 2005, 44, 2782–2785.
28 H. L. Wang, J. J. Cao, Y. P. Bi, L. Chen and Q. H. Wan,
J. Chromatogr., A, 2009, 1216, 5882–5887.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (no. 21075008 and 90713013).
Notes and references
1 F. X. Qie, G. X. Zhang, J. X. Hou, X. M. Sun, S. Z. Luo and 29 E. Akyilmaz and M. Turemis, Electrochim. Acta, 2010, 55,
T. W. Tan, Talanta, 2012, 93, 166–171.
5195–5199.
2 R. Hao, R. J. Xing, Z. C. Xu, Y. L. Hou, S. Gao and S. H. Sun, 30 Y. Wei, A. L. Tian, Y. Li, X. Wang and B. Cao, J. Mater. Chem.,
Adv. Mater., 2010, 22, 2729–2742. 2012, 22, 8499–8504.
3 H. M. Chen, C. H. Deng and X. M. Zhang, Angew. Chem., Int. 31 K. Ashtari, K. Khajeh, J. Fasihi, P. Ashtari, A. Ramazani and
Ed., 2010, 122, 617–621. H. Vali, Int. J. Biol. Macromol., 2012, 50, 1063–1069.
4 C. E. Probst, P. Zrazhevskiy and X. H. Gao, J. Am. Chem. Soc., 32 J. Saevels, K. Van den Steen, A. Van Schepdael and
2011, 133, 17126–17129.
J. Hoogmartens, J. Chromatogr., A, 1996, 745, 293–298.
5 M. Kalantari, M. Kazemeini, F. Tabandeh and A. Arpanaei, 33 S. M. Fang, H. N. Wang, Z. X. Zhao and W. H. Wang, J. Pharm.
J. Mater. Chem., 2012, 22, 8385–8393.
Anal., 2012, 2, 83–89.
6 S. A. Ansari and Q. Husain, Biotechnol. Adv., 2012, 30, 512– 34 Z. M. Tang and J. W. Kang, Anal. Chem., 2006, 78, 2514–2520.
523.
35 A. R. Whisnant and S. D. Gilman, Anal. Biochem., 2002, 307,
226–234.
7 K. Khoshnevisan, A. K. Bordbar, D. Zare, D. Davoodi,
M. Noruzi, M. Barkhi and M. Tabatabaei, Chem. Eng. J., 36 C. W. Lin, H. G. Sie and W. H. Fishman, Biochem. J., 1971,
2011, 171, 669–673.
124, 509–516.
1754 | J. Mater. Chem. B, 2013, 1, 1749–1754
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