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2066

Ionic liquid modied magnetic microspheres for

isolation of heme protein with high binding capacity
Yun Wei,* Yan Li, Ailin Tian, Yuntian Fan and Xiong Wang
The imidazolium cation was chosen as protein selective anity group and a kind of ionic liquid magnetic
microspheres was developed by immobilizing imidazolium cations onto the surface of silica-coated
magnetic

microspheres

to

form

imidazolium

ionic

liquid

modied

magnetic

microspheres

(Fe3O4@SiO2@IL). Elemental analysis data conrmed and quantied the immobilization of the ionic
liquid. The most distinct feature of the magnetic microspheres is the high hemoglobin binding capacity
(more than 2 g of hemoglobin per gram of beads) owing to the covalent coordination between the
imidazolium cation and the iron atom in the heme group. Moreover, imidazolium ionic liquid modied
Received 26th December 2012
Accepted 6th February 2013

magnetic microspheres (Fe3O4@SiO2@IL) have selective anity to proteins with dierent charges due
to their own isoelectric point. Therefore, the magnetic microspheres can selectively capture protein
under an appropriate solution pH. Furthermore, the short processing time (15 min) is suitable for

DOI: 10.1039/c3tb00576c

purifying unstable proteins. In addition, the separating and recycling of the magnetic microspheres by

www.rsc.org/MaterialsB

using a magnet is quite convenient.

Introduction

Protein separation by functionalized magnetic particles is based

on the surface modication of ion exchange groups or anity
ligands. So many protein-purication methods employ a separation step through electrostatic adsorption or specic interactions between immobilized ligands and anity tags on the
protein,1,2 and the most common anity tag is polyhistidine,
which binds to immobilized Ni2+ complexes.35 The methods are
attractive for their high protein loading, mild elution conditions, and easy regeneration of the metal complexes on the
resin.
Surface polymerizing has been the most popular method to
coat nanomaterials for achieving the desired surface properties.611 However, slow intraparticle biomolecule diusion to
binding sites in porous micrometer-sized beads can lead to
relatively long processing times which can be problematic when
purifying unstable proteins. In principle, addition of more
beads to a sample can overcome low binding capacities, but the
greater volume of the particle slurry will lead to increased
nonspecic adsorption or sample loss. Several studies have
examined modied nanoparticles for protein binding. These
extremely small particles (diameters less than 3 nm) have a
large surface area to volume ratio and a high binding capacity
(23 g of protein per gram beads),12,13 but such particles may be

dicult to collect and some nonspecic protein adsorption

occurred. A surface functionalized magnetic material with low
diusion limitation and high binding capacity would show
great advantages.
Several papers have reported the synthesis of ionic liquid
modied magnetic nanoparticles and their applications in
controlled catalyst and dye removal.1416 Recent studies show
that the ferrous atom in the heme group of heme-proteins
provides a vacant coordinating position, which oers possibilities for covalent coordination or interaction between the
ferrous atom and the imidazolium cation in the ionic liquid.17
So the imidazolium cation was chosen as the anity ligand to
form ionic liquid modied magnetic microspheres (Fe3O4@
SiO2@IL) in the present study. This work shows that Fe3O4@
SiO2@IL with a diameter of about 300 nm leads to a binding
capacity of 2.15 g hemoglobin per gram of beads due to the
covalent coordination binding with hemoglobin as well as the
high surface area. Moreover, Fe3O4@SiO2@IL has selective
anity to proteins with dierent charges in terms of its own
isoelectric point, and direct isolation of a relatively high amount
of hemoglobin can be achieved in 15 minutes, which displayed
great eciency compared with previous reports requiring as
long as several hours.

Results and discussion

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China.
E-mail: weiyun@mail.buct.edu.cn; Fax: +86 10 64442928; Tel: +86 10 64442928

2.1 Fabrication strategy for ionic liquid modied magnetic

microspheres

Electronic supplementary
10.1039/c3tb00576c

The synthesis procedure is shown in Scheme 1. Firstly, the

magnetic microspheres (Fe3O4) were synthesized through a

information

(ESI)

2066 | J. Mater. Chem. B, 2013, 1, 20662071

available.

See

DOI:

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Journal of Materials Chemistry B

Scheme 1 Schematic representation of the preparation procedure for

Fe3O4@SiO2@IL and its anity interaction with the heme protein.

typical solvent-thermal reaction described previously.18

Secondly, a solgel approach was applied to synthesize core/
shell-structured magnetic silicaFe3O4 microspheres (Fe3O4@
SiO2).19,20 Thirdly, Fe3O4@SiO2@chloropropyl microspheres was
obtained through the silylation reaction with 3-chloropropyltrimethoxysilane.21 Fourthly, the chemically bonded chloropropyl group reacted with N-methylimidazole to obtain the
imidazolium ionic liquid modied magnetic microspheres
(Fe3O4@SiO2@IL).22
Fig. 1A shows a representative transmission electron
microscopy (TEM) image of the synthesized Fe3O4 microspheres, suggesting that magnetic microspheres were spherical.
Additionally, it was found from the TEM observation that these
magnetite microspheres have a mean diameter of about 270 nm
and consist of microspheres with an average size of about 18 nm
calculated by the Scherrer equation from the X-ray diraction
(XRD) results as described in our previous work.23 The TEM
image shown in Fig. 1B and C revealed that core/shell-structured silica coated magnetic microspheres with a silica layer of
about 20 nm were successfully prepared (Fig. S1).
Magnetic characterization at 300 K with a vibrating sample
magnetometer showed that the saturation magnetization values
of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@IL microspheres were

Fig. 1

TEM images of (A) Fe3O4 particles, (B and C) Fe3O4@SiO2 microspheres.

This journal is The Royal Society of Chemistry 2013

73.7, 48.8 and 46.5 emu g
1 (Fig. 2A), respectively. The
Fe3O4@SiO2@IL microspheres can be dispersed in water by
vigorous shaking or sonication. Fast aggregation of the microspheres from their homogeneous dispersion was observed in
the presence of an external magnetic eld (Fig. 2B). These
results showed that Fe3O4@SiO2@IL microspheres possess
excellent magnetic responsivity, which is important in terms of
their practical manipulation.
In the infrared spectrum of the functionalized microspheres,
one small important band at 1540 cm
1 is attributed to the
characteristic frequencies of cationic imidazolium ring group
(Fig. S2).24
The elemental analysis data conrmed the successful
immobilization of ionic liquid onto the surface. In Fe3O4@
SiO2@chloropropyl: C 3.87%, H 0.72%; in Fe3O4@SiO2@IL: C
4.59%, H 0.770%, N 0.693%. The bonding quantity was calculated to be 1.08  10
3 mol g
1 for Fe3O4@SiO2@chloropropyl
and 2.48  10
4 mol g
1 for Fe3O4@SiO2@IL. The results suggested that about 26% of chloropropyl reacted with the Nmethylimidazolium in the nal reaction step.
The isoelectric point of Fe3O4@SiO2@IL is pH 6.7 (Fig. 3). As
the solution pH increased from 2 to 11, zeta potential decreased
from 40 mV to
40 mV linearly. The results indicated that the
magnetic microspheres have the potential to be a pH-dependent adsorbent,25,26 which could be used to optimize the
absorption and elution of heme proteins at suitable pH.

2.2

Protein adsorption performance

2.2.1 Inuence of pH. To explore the adsorption performances inuenced by pH, a series of adsorption experiments
with single component protein [Hb (pI 6.9) and Lys (pI 11)]
under dierent pH solution were investigated. As shown in
Fig. 4, for hemoglobin, the maximum adsorption eciency was
achieved at pH 6.8. When the pH increased or decreased, the
adsorption eciency of Hb decreased rapidly. The phenomenon must be attributed to the electrostatic interactions
between the protein species and the surfaces of the

Fig. 2 (A) Room-temperature (300 K) magnetic hysteresis loops of (a) Fe3O4, (b)
Fe3O4@SiO2 and (c) Fe3O4@SiO2@IL microspheres. (B) Strong magnetic response
to an applied magnetic eld.

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Fig. 3 Zeta potentials of Fe3O4@SiO2@IL in dierent pH solutions, pH 6.7

turned out to be the isoelectric point of Fe3O4@SiO2@IL.

Fig. 5 Adsorption isotherm for (a) the adsorption of Hb on Fe3O4@SiO2@IL at

pH 6.8 and (b) the adsorption of Lys on Fe3O4@SiO2@IL at pH 8.0 [phosphate]
1/15 mol L
1, 15  C. q is the equilibrium adsorption amount of the proteins (Hb or
Lys) (mg mg
1), Ce is the protein concentration in solution (mg mL
1).
Fig. 4 pH dependence of the adsorption eciencies of 0.1 mg mL
1 proteins in
3 mL 1/15 mol L
1 phosphate buer (pH 4.010.0) by 3 mg Fe3O4@SiO2@IL. (a)
Hb (b) Lys.

microspheres, which can be further demonstrated by the variation of zeta potential on the surfaces of the microspheres as a
function of pH. When pH is outside the range of 6.7 to 6.9, they
are repelling each other due to having the same charge. Despite
this, the remaining adsorption eciency proved that the
complexation between the Fe3O4@SiO2@IL and the Hb played a
dominant role in adsorption. Compared with the low adsorption eciency of Lys, that is the reason why there is a huge
distinction of binding capacity between the two proteins. For
Lys (pI 11), the maximum adsorption eciency was achieved
at pH 8.0, when the solution pH increased or decreased, the
adsorption eciency of Lys decreased to zero. Above all, selective adsorption of Hb in a binary protein mixture of Hb and Lys
can be achieved in a pH 6.0 phosphate buer solution.
Additionally, the result of recycling experiments is shown in
Fig. S3.
2.2.2 Adsorption isotherms of proteins. The adsorption
isotherms for Hb and Lys have been obtained based on the
Langmuir model.27,28 As shown in Fig. 5, the binding capacity
for the adsorption of Hb can be estimated to be 2.15 g g
1 and
0.02 g g
1 for the adsorption of Lys from the slope and intercept.
Table 1 demonstrates the contrasting capture ability of several
Hb immobilization resins, some of them used to bind the Hb

2068 | J. Mater. Chem. B, 2013, 1, 20662071

group as a catalyst. Table S1 shows the separation eciency of

the IL resin compare to some commercial resins. As expected,
the binding capacity for the Hb is greater than those previously
reported. This is mainly due to the complexation between the
imidazolium cation and the iron atom in the heme group of Hb.
Table S2 shows the comparison of size, molecular weight and
maximum adsorption number between Hb and Lys. As seen
from the results, the ionic liquid modication improved the
selectivity and greatly enhanced the adsorption capacity of
proteins with heme groups. It is very favorable for the use of
Fe3O4@SiO2@IL in separation and loading of proteins with
heme group.
2.2.3 Separation of a binary protein mixture. To further
explore the separation, a binary protein mixture was used for
testing (Fig. 6).32,33 The spectra revealed that the remaining
protein solution aer adsorption by Fe3O4@SiO2@IL contained
little Hb, indicating essentially complete removal of Hb from
the binary protein mixture, which may be a good microsphere
for the removal of highly abundance protein, and enrichment of
the proteins with low abundance in proteomics analysis.34
2.2.4 Practical isolation of hemoglobin from human whole
blood. The practical applicability of the Fe3O4@SiO2@IL was
demonstrated by selective adsorption of hemoglobin from
human whole blood. A human whole blood sample was diluted
1000-fold with 1/15 mol L
1 phosphate buer (pH 6.8), then
ultrasonic and ltrated. 2 mL diluted sample was subjected to

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Table 1

Journal of Materials Chemistry B

Properties of dierent resins for Hb capture

Capacity

Size

Capture time

Mesoporous silica29

300 mg Hb per g silica

Gently stirred for 30 min

Mesoporous TiO2SiO230

301.8 mg Hb per g TiO2SiO2

Polyvinyl chloride31

No reported data

50 and 10 mm (pore diameters from

6 to 20 nm)
Wall thickness 5 nm (pore
diameters from 5.8 to 7.25 nm)
>150 mm

Fe3O4@SiO2@IL (this work)

2.15 g Hb per g microspheres

270 nm

15 min

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Resin

Experimental section

3.1

Materials and instruments

12 h continuous stirring
Shaken vigorously for 10 min

Lysozyme from chicken egg white (Lys, L8120, and isoelectric

point pI 11), bovine hemoglobin (Hb, H8020, pI 6.9) was
purchased from Solarbio; N-methylimidazole was purchased
from the Centre for Green Chemistry and Catalysis, LICP, CAS.
Other chemicals employed were at least of analytical reagent
grade and commercially available, including ferric chloride
hexahydrate (FeCl3$6H2O), sodium acetate (NaAc), ethylene
glycol (EG), ammonium hydroxide (NH3$H2O, 28%), tetraethyl
orthosilicate (TEOS), concentrated hydrochloric acid (HCl,
37.5%), triethylamine (TEA). All solvents used in the preparation process were of analytical reagent grade. The sizes and
morphologies of microspheres were tested using a transmission
electron microscope (TEM, Philip Tecnai 20, Netherland),
Fourier transform infrared (FT-IR) spectra were obtained using
Nicolet 8700 (Thermal Fisher, USA) with the KBr method.
Elemental analysis (Elementar, Vario EL cube, Germany) was
utilized to measure the relative C, H, N composition of
Fe3O4@SiO2@IL. In addition, the surface charge properties
were investigated by measuring the zeta potential within pH 2
11, by using a Nano Zetasizer (Malvern, England).

Fig. 6 (a) UV-vis spectra for standard solutions, (a) Hb and (b) Lys; (b) UV-vis
spectra of a mixture of Hb and Lys in 2 mL of pH 10.0 phosphate solution before
and after adsorption by 5 mg Fe3O4@SiO2@IL. (a) Without adsorption; (b)
remaining protein after adsorption of Fe3O4@SiO2@IL, and (c) desorbed protein
solution.

the adsorption procedure by 5 mg Fe3O4@SiO2@IL. Aer

elution, the eluate was assayed by SDS-PAGE. The electrophoretogram is shown in Fig. 7. It can be seen that hemoglobin was
eectively isolated from the coexisting protein species in a
complex biological sample matrix. At the same time, a certain
extent of pre-concentration of hemoglobin was achieved. This
observation illustrated the practical applicability of the present
system for the eective isolation of hemoglobin from biological
samples with complex matrix components compared with
previous reports.35,36

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Fig. 7 The standard SDS-PAGE of proteins. The samples were subjected to

electrophoresis on a 30% acrylamide gel at 150 V. (1) 1000-fold diluted human
whole blood; (2) 1000-fold diluted human whole blood after pretreated by the
magnetic microspheres; (3) hemoglobin isolated from human whole blood with
the present procedure; (4) pure hemoglobin solution.

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3.2 Preparation of the imidazolium ionic liquid modied

magnetic microspheres
3.2.1 Synthesis of Fe3O4 microspheres. The magnetic
microspheres were synthesized through a typical solventthermal reaction described previously. In brief, 1.35 g of
FeCl3$6H2O was rst dissolved in 40 mL of ethylene glycol
whilst stirring to obtain a clear yellow solution. Then 3.60 g of
NaAc (sodium acetate) were added to this solution with
continuous stirring for 10 min. The obtained homogenous
solution was transferred into a Teon-lined stainless-steel
autoclave with a capacity of 50 mL. The autoclave was sealed
and heated at 200  C for 8 h, and cooled down to room
temperature. The obtained black magnetite particles were
washed several times with ethanol and water with the help of a
magnet, and kept in ethanol.18
3.2.2 Synthesis of silica coated magnetic microspheres
(Fe3O4@SiO2). A solgel approach was applied to synthesize
core/shell-structured magnetic silica nanospheres. It had been
proposed that a pre-treatment of the magnetite microspheres
with an aqueous solution of hydrochloride was necessary before
silica coating, otherwise, a mixture of naked magnetite microspheres and blank silica microspheres would form during the
reaction process. Herein, 0.1 g synthesized Fe3O4 particles were
treated with 1 mol L
1 HCl aqueous solution (50 mL) by ultrasonication. Aer treatment for 10 min, the magnetite particles
were separated and washed with deionized water, and then
homogeneously dispersed in the mixture of ethanol (160 mL)
and deionized water (40 mL), followed by the addition of
concentrated ammonia aqueous solution (6.0 mL, 28 wt%).
300 mL of TEOS (tetraethyl orthosilicate) diluted in 20 mL
ethanol was added dropwise into the reaction system. Aer
agitating at room temperature for 5 h, rapid neutralization of
the silica-coating solution by addition of HCl is critical to avoid
the formation of large clusters during collection of the particles
with a magnet. The product was collected with a magnet and
washed repeatedly with ethanol and water, and then dried in a
vacuum at 70  C for 12 h.19,20
3.2.3 Synthesis of the chloropropyl-silica coated magnetic
microspheres (Fe3O4@SiO2@chloropropyl). The Fe3O4@SiO2
(0.4 g) was suspended in 150 mL of dry toluene and then an
excess of 3-chloropropyltrimethoxysilane (4 mL) was added,
followed by 1 mL of triethylamine (added as a catalyst). The
suspension was mechanically stirred and reuxed for 48 h
under inert conditions using N2 gas. Aer reuxing, the reaction was stopped and the modied Fe3O4@SiO2 was cooled to
room temperature, the product was collected with a magnet and
washed with toluene (200 mL), ethanolwater (1 : 1, v/v) mixture
(500 mL), deionized water (500 mL) in turn and nally with
methanol (100 mL). Chloropropyl silica, SilprCl, was dried
under vacuum at 60  C for 8 h prior to the reaction with
N-methylimidazole.21
3.2.4 Preparation of the imidazolium ionic liquid modied
magnetic microspheres (Fe3O4@SiO2@IL). The chemically
bonded chloropropyl group on the Fe3O4@SiO2@chloropropyl
surface was reacted with N-methylimidazole. In brief, 0.25 g of
dry Fe3O4@SiO2@chloropropyl was placed in a reaction ask

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containing 150 mL of anhydrous toluene and a large excess of Nmethylimidazole was used (5 mL). The mixture was reuxed
with stirring for 48 h. Aer reuxing, the reaction was stopped
and the modied silica was cooled to room temperature,
transferred to a vacuum glass lter, and washed with methanol
(350 mL), water (300 mL), and again with methanol (150 mL).
The silica chemically bonded with N-methylimidazolium,
SilprMim, was dried under vacuum at 50  C for 8 h prior to
characterization or protein separation.22
3.3

Characterization of magnetic microspheres

3.3.1 Protein adsorption by the Fe3O4@SiO2@IL. The pH

eects on the adsorption performance of the magnetic microspheres for proteins were initially investigated. Hb and Lys were
used as adsorbates. The adsorption of Hb and Lys by magnetic
microspheres was investigated in 1/15 mol L
1 phosphate
buer (pH 4.010.0). Before adsorption, the magnetic microspheres were washed by phosphate buer with corresponding
pH. 3 mg Fe3O4@SiO2@IL was used to extract proteins in 2 mL
of aqueous solution (0.1 mg mL
1). Aer a vibration at room
temperature for 10 min by vortex, it was placed for adsorption
for ve minutes, and the supernatant was collected under the
help of a magnet within 30 s. The concentrations of proteins in
the aqueous phase before and aer adsorption were obtained by
measuring the absorbance at their characteristic absorption
wavelengths (408 nm for Hb, 280 nm for Lys) with a UV-vis
spectrophotometer. The adsorption eciency (E) was thus
calculated based on the protein concentrations in the aqueous
solution before and aer adsorption, respectively.
The elution of Hb aer adsorption was performed by using
Na2CO3NaHCO3 buer (pH 10) aer washed by 2 mL 1/15 mol
L
1 phosphate buer twice, as well as 0.5% (m/v) SDS aqueous
solution leading to a favourable elution eciency of up to 86%,
without SDS the elution eciency was still 57%.
3.3.2 Adsorption isotherm. The adsorption of Hb at pH 6.8
and the adsorption of Lys on magnetic microspheres at pH 8.0
were carried out in 1/15 mol L
1 phosphate buer at 15  C.
Generally, 3.0 mg of microspheres were added to a 2 mL Hb
solution (0.130 mg mL
1) and 1 mL Lys solution (0.15.0 mg
mL
1). Aer the adsorption and desorption process, the
amount of Hb and Lys adsorbed on the Fe3O4@SiO2@IL was
calculated from the concentration change of Hb (or Lys) in the
solution aer adsorption by a spectrophotometric method at
408 nm or at 280 nm.
3.3.3 Separation of binary protein mixture. To demonstrate
the utility of these beads for protein separation, a binary protein
mixture of Hb (0.1 mg mL
1, pI 6.9) and Lys (0.2 mg mL
1,
pI 11) was separated by Fe3O4@SiO2@IL. 5.0 mg of Fe3O4@
SiO2@IL was added to the binary protein mixture at pH 6.0,
Aer a vibration at room temperature for 10 min by vortex and a
static adsorption for 5 min, the supernatant was collected with
the help of a magnet within 60 s, aer being washed with the
corresponding pH solution three times. The beads were desorbed in a pH 10.0 phosphate solution by sonication for 10 min.
The desorbed protein solution, remaining protein solution aer
adsorption and the stock binary protein solution were examined
using a UV-visible spectrophotometer.

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Conclusions

The immobilization of imidazolium cations onto the surface of

the silica coated magnetic microspheres enhanced the separation capacity and selectivity in protein separation. In addition,
high hemoglobin binding capacity was achieved owing to high
specic surface area of the microspheres. The surface modication of ionic liquid monolayer reduces diusion limitations
to allow protein to be captured within 15 min. Moreover, the
target protein can be directly isolated from a blood sample via
these particles with high purity. Simultaneously, the microspheres can be separated quickly and easily using a magnet due
to the magnetite core. This technology will result in quick and
easy operation compared to other approaches and has good
selectivity towards heme proteins.

Acknowledgements
This work was supported by National Natural Science Foundation of China (NSFC, Grant no. 21075007), Program for New
Century Excellent Talents in University (NCET-11-0563), and
Special Fund for Agro-scientic Research in the Public Interest
(project 200803022 & 201103027).

Notes and references

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