A novel core@shell magnetic molecular imprinted nanoparticles for
selective determination of folic acid in different food samples
Sajjad Hussain
a,b
, Sabir Khan
a
,SaimaGul
c
, Maria Isabel Pividori
d
, Maria Del Pilar Taboada Sotomayor
a,
⁎
a
Department of Analytical Chemistry, Institute of Chemistry, State University of São Paulo (UNESP), 14801-970 Araraquara, SP, Brazil
b
Faculty of Material and Chemical Engineering,GIK Institute of Engineering Sciences and Technology, Topi, KPK 23460, Pakistan
c
Department of Chemistry University of Swabi for Woman Gulo-Deri, Swabi, KPK, Pakistan
d
Group de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona (UAB), 08193, Bellaterra, Barcelona, Spain
abstractarticle info
Article history:
Received 11 February 2016
Received in revised form 7 June 2016
Accepted 22 July 2016
Available online 25 July 2016
In this work, magnetic molecularly imprinted polymers (MMIPs) were synthesized and tested for the determina-
tion of folic acid (FA) in different food samples. The MMIPs were polymerized at the surface of Fe
3
O
4
@SiO
2
mag-
netic nanoparticles (MNPs) using acrylonitrile (functional monomer), ethylene glycol dimethacrylate (EGDMA)
as cross-linking agent and azobiisobutyronitrile (AIBN) as an radical initiator. The morphological, topological and
chemical char acteristics of the MMIPs were inve stigated by field emission scanning elec tron microscop y
(FESEM), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared (FTIR) tech-
niques. The physico-chemical characterization, such as adsorption capacities and selectivity of MMIPs was inves-
tigated and compared with the respective MNIPs. The adsorption experimental data demonstrate that maximum
adsorption capacity of MMIP at equilibrium was 8 mg g
−1
and than the adsorption process of FA over MMIPs fol-
lows Freundlich adsorption isotherm model and pseudo-first-order reaction kinetic. For evaluation of this new
proposed material, the recovery studies were carried out in spiked samples at different concentration levels
and the obtained values were in the range of 95–104% for orange and for spinach the recoveries were between
99.5 and 102.5%. The relative standard deviations (RSD) for the recoveries were b 0.5% for both samples. These
results demonstrate that this novel MMIP material can be efficiently used for the selective extraction of folic
acid from different food complex matrices.
© 2016 Elsevier B.V. All rights reserved.
Keywords:
Molecularly imprinted polymer
Folic acid
Magnetic nanoparticles
Food
1. Introduction
Folic-acid (or folate) supplementation offers substantial and well-
defined health benefits [1]. Numerous chronic diseases like gigantocytic
anemia, leucopoenia, mental ity devolution, psychosis; heart attack,
malformation and neural tube defects during pregnancy are related
due to the deficiency of folic acid (FA) [2]. Folic acid is found naturally
in a wide variety of foods , including vegetables, fruits, poultry and
meat [3]. It is necessary to monitor the quantity of FA in various food
samples for their safety reasons. However, analysis of FA is not an easy
task because of its presence in extremely lower concentration in biolog-
ical systems, due to its lower stability under acidic conditions and its
sensitiveness against light and high temp erature [4]. Numerous
methods have been used for the determination of FA including, spectro-
photometry [5], flow injection chemiluminescence [6,7] fluorimetric
[8], high-perfor mance li quid chromatography (HPLC) [9,10],LC–MS
[11,12], electrochemical [13,14] and capillary electrophoresis [15].On
the other hand microbiological assay of FA has widely been used, but
it is lengthy, time consuming, need extreme care and skill for its analysis
[16].
Among the chromatographic techniques the LC-MS is believed to be
the most efficient [17,18]. However, methods those based on chroma-
tography, involve complexes pre-treatment, pre-concentration and ex-
traction procedure s [19]. Some of these methods used to determine
folates in biological fluids and foods, using solid phase extraction
(SPE) [20] for the pre-treatment of the samples, in which the adsorbent
material is the unspecified organic functionalized silica C8 or C18.
In addition there is also exist a most cost-effective procedure for FA
analysis, which is based on the enzyme protein-binding assay [21],but
although the measurement is not influenced by the matrix effect, the
corresponding kits have short self-life, due to the biological material
used in the detection.
In this sense, aim at more easy, rapid and efficient pre-treatment of
complex samples, our research group have proposed the development
of magnetic nanoparticles coated with MIPs for different analytes than
can be used to separate and concentrate chemicals conveniently using
a simple external magnetic field from a Neodimium magnet. Therefore,
a combination of molecular imprinting technology would provide a
powerful analytical tool with all desir ables characteristics, besides
being more selective than traditional silica adsorbent materials [22,
Reactive and Functional Polymers 106 (2016) 51–56
⁎ Corresponding author.
E-mail addresses: mpilar@iq.unesp.br, mpilarts@hotmail.com (M. Del Pilar Taboada
Sotomayor).
http://dx.doi.org/10.1016/j.reactfunctpolym.2016.07.011
1381-5148/© 2016 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Reactive and Functional Polymers
journal homepage: www.elsevier.com/locate/react
23]. Thus, continuing the dissemination of the results of our research
group, in this article we report the successful synthesis, characterization
and application of a novel magnetic material based on magnetic nano-
particles coated with MIP selective to folic acid molecule.
2. Materials and methods
2.1. Reagents
Folic acid, FeCl
2
·4H
2
O (98%), FeCl
3
·6H
2
O (97%), tetraethoxysilane
(TEOS), acrylonitrile (≥ 99%),
[3(methacryloyloxy)propyl]trim ethoxysilane (MPS), methylacrylic
acid (MAA), ethylene glyc ol dimetha crylate (EGDMA), 2,2′-
azobisisobutyronitrile (AIBN), were provided by Sigma-Aldrich.
NH
4
OH, NaOH and H
2
SO
4
were obtained from Synth-Brazil. Acetonitrile
(ACN), ethanol, methanol (HPLC grade) and acetic acid (glacial, 100%),
were purchased from Panreac Quimica. All the reagents used without
prior purification and solutions were prepared in deionized wa ter
(18 MΩ at 25 °C) obtained from Milli-Q Direct-0.3 (Millipore) purifica-
tion system.
2.2. Preparation of magnetic nanoparticles (Fe
3
O
4
)andFe
3
O
4
@SiO
2
Magnetic nanoparticles were prepared by co-precipitation method
as described by Lu et al. [24] A known amount of both FeCl
2
·4H
2
Oand
FeCl
3
·6H
2
O were dissolved in 80 mL of water with vigorous stirring in
nitrogen atmosphere. NH
4
OH (10 mL) drop wise was added in the sys-
tem and the reaction was maintained at 80 °C for 30 min. The black
Fig. 3. Transmission electron microscopy (TEM) of magnetic molecular non-imprinted
polymer (MNIP).
Fig. 2. Transmission electron microscopy(TEM) of magnetic molecular imprinted polymer
(MMIP) for folic acid at two different scales.
(a)
(b)
Fig. 1. Scanning electron microscopy images of MMIPs (a) and MNIPs, with their
respective particles sizes.
52 S. Hussain et al. / Reactive and Functional Polymers 106 (2016) 51–56
precipitation of Fe
3
O
4
nanoparticles was separated with a permanent
magnet and washed thoroughly with deionized water to remove the
unreacted chemicals, and then dried in the vacuum. 300 mg Fe
3
O
4
nanoparticles were dispersed in 40 mL of ethanol (EtOH) and 4 mL of
deionized water by ultra-sonication for 15 min and then followed by
the addition of 5 mL of NH
4
OH and 2 mL of tetraethoxysilane (TEOS).
The mixtu re was left to react for 12 h at the room temperature. The
products were collected by magnetic separation and was washed with
deionized water three times and then dried in the vacuum. In the next
step Fe
3
O
4
@SiO
2
nanoparticles were modified by 3-
metac riloxipropiltrimetoxissilano (MPS) by taking 250 mg of the
Fe
3
O
4
@SiO
2
nanoparticles was dispersed in 50 mL of anhydrous toluene
containing 5 mL of 3-metacriloxipropiltrimetoxissilano (MPS) and the
mixture was allowed to react for 12 h in dry nitrogen atmosphere. The
obtained product was separate d by an external magnet and dried in
vacuum.
2.3. Preparation of MMIPs and MNIPs
MMIPs were prepared by polymerization of 0.2 mmol of folic acid
(FA), 0.8 mmol of monomer (Acrylonitrile, Supplementary data) in eth-
anol (30 mL). The mixture was shaken in a water bath at 25 °C for 12 h,
then 200 mg Fe
3
O
4
@SiO
2
–C_C were added into the reaction system
and was shaken for more 3 h. Furthermore, 4.0 mmol of EGDMA and
0.05 mmol of AIBN were added into the system and the mixture was
sonicated for 5 min in nitrogen atmosphere, and the reaction mixture
was left at 60 °C under nitrogen gas protection for 24 h. After polymer-
ization, the template molecule was leached out by Soxhlet extraction
using methanol: acetic acid (9:1, v/v) as eluent, and the eluent was re-
placed every 12 h. The template (Folic acid) had been determined
using HPLC in eluent at the interval of 12 h. After complete removal of
template molecule the products (MMIPs) were dried at 40 °C under
vacuum. Similarly magnetic non-molecular imprinted polymers
(MNIPs) were prepared under the same conditions as described above
without analyte.
2.4. Folic acid solution and food samples
A stock solution of FA (pH 6.5) was prepared by dissolving 10 mg FA
in 100 mL of deionized water with a few drops of 0.1 mol L
−1
NaOH, the
prepared solution stored in a dark flask and kept in a refrigerator to pre-
vent degradation. All working solutions of FA were prepared daily by
appropriate dilution and the pH was adjusted to 6.5 with the help of
0.1 mol L
−1
NaOH.
The spinach, and orange were purchased from a local supermarket in
Araraquara in the state of São Paulo - Brazil. The spinach samples were
prepared as previously described [25]. A 50.0 g of real sample (spinach)
was cut into small pieces using a razor blade, and was boiled with water
under reflux for 50 min. The mixture was cooled and filtered through a
membrane filter. A suitable aliquot of the filtrate was used to determine
folic acid. The orange juice was obtained by squeezing fresh orange by
manual orange juice extractor. The orange juice was first filtered using
semi-analytical whatman paper, after this it was filtered using Millipore
membrane of 0.45 mm and the filtrate was stored in refrigerator for
using it in further analysis [26].
2.5. Binding and selective adsorption experiments
To evaluate the binding capacity of prepared MMIPs and MNIPs ad-
sorption tests were carried out, by adding 10 mg of MMIPs and MNIPs in
a separated 20 mL glass vial and adds 10.0 mL of 10 mg L
−1
of the folic
acid solutions. The mixture was shaken in a rotating shaker for 120 min
and after being shaken the magnetic polymer suspensions were sepa-
rated by magnetic bar and filtered with a 0.45 μm membrane for HPLC
2 4 6 8 10 12 14 16 18 20
2
3
4
5
6
7
8
q
e
(mg g
-1
)
Initial Concentration (mg L
-1
)
Fig. 5. Binding capacity of folic acid onto MMIPs (■ ) and MNIPs (•) at different initial
concentrations (C
i
).
Table 1
The rate constant and binding capacity for the sorption of ametryne.
Pseudo first order kinetic model Pseudo second order kinetic model
Sorbents q
e
(mg g
−1
)R
2
k
1
(mg g
−1
min
−1
)q
e
(mg g
−1
)R
2
k
2
(mg g
−1
min
−1
)
MIP 7.510 0.9936 0.0252 9.432 0.9756 0.00379
NIP 5.575 0.9902 0.0236 10.20 0.9506 0.00133
020406080100120
0
2
4
6
8
q
e
(mg g
-1
)
Time (min)
Fig. 4. Binding capacity of folic acid vs time on to MMIPs (■) and MNIPs (•).
53S. Hussain et al. / Reactive and Functional Polymers 106 (2016) 51–56
analysis. The binding capacity is calculated using by the Eqs. (1) and (2)
[27,28].
Q
t
¼
C
o
−C
e
ðÞV
m
ð1Þ
Q
e
¼
C
t
−C
e
ðÞ
V
m
ð2Þ
Q
t
(mg g
− 1
) is the experimental adsorption quantity at time t, Q
e
(mg g
− 1
) equilibrium adsorption quantity, C
o
(mg L
− 1
) is the initial
concentration of FA, Ce (mg L
−1
) is the equilibrium concentration, Ct
is the final concentrations (mg L
−1
), V (mL) is the volume of FA solution
and m (g) is the weight of MMIPs and MNIPs. All the experiments were
performed in triplicate.
2.6. Instruments and analytical methods of analysis
The chromatographic analyses were performed using a Shimadzu
Model 20A liquid chromatograph, coupled to an SPD-20A UV/Vis detec-
tor, a SIL-20A autosampler and a DGU-20A5 degasser. The chromatogra-
phy system was controlled by a microcomputer and the C18 column
(250 mm × 4.6 m) was used in the analysis. The chromatographic con-
ditions used were, mobile phase composed of a mixture of
acetinitonitryle:water (90:10, v/v), flow rate of 1.0 mL min
−1
,sample
injection volume of 20 μL and wavelength of 280 nm for folic acid
detection.
FTIR–VERTEX 70 spectrometer (BRUKER) with spectr al range of
4000 to 370 cm
−1
and a detector of DLaTGS was employed to examine
the FTIR spectra of mag-MIPs and Mag-NIPs.
The size and surface morphology of nanoparticle samples were ana-
lyzed using field emission gun scanning electron microscope (FEG-SEM;
JEOL model 7500F) (Germany) GmbH. TEM analysis were performed
using high resolution transmission electron microscope HR-TEM
(Philips- model CM200 supertwin with resolution of 1.9 A.
3. Result and discussion
3.1. Morphologic characterization
The structures of MMIPs and MNIPS were well characterized by Field
Emission Scanning electron microscopy (FESEM) and high-resolution
transmission electron microscope (HRTEM). It could be seen from the
Fig. 1 that the MMIPs were all spherical shape and the size of the parti-
cles was in the range of 50–100 nm (Fig. 1a). However, in Fig. 1b, corre-
sponding to MNIP could be observ ed irregular spheres with a high
degree of agglomeration and a roughness in the surface.
The TEM analysis further reveals that magnetic-nanoparticles were
of spherical shape with approximately 100 nm in diameter, such sug-
gest for the SEM images. It is evident in Fig. 2b than the core-shell struc-
ture of MMIPs was successfully constructed, since the double circle of
microsphere indicates that in fact was obtained a core-shell particle of
Fe
3
O
4
@MIP (MMIP), in wich can be clearly seen the SiO
2
-MIP shell uni-
formly coating the Fe
3
O
4
dark core, which for their small size was very
helpf ul for more recognition sites and higher adsorption capacity of
the template molecule, due to the high active area.
On the other hand, TEM images of MNIPs show aggregates of th e
nanoparticles (Fig. 3) from which it can be clearly distinguished a rela-
tively large mesosphere of aggregates of fine particles with sizes c.a.
200 nm, which also confirms the SEM results.
3.2. Effect of contact time on the adsorption of folic acid onto MMIPs and
MNIPs
The effect of contact time on the adsorption of FA was studied. As
shown in Fig. 4 the adsorption increases with time, and was rapid in
the initial 60 min and then continued slowly. The 90 min was taken as
equilibrium time. Initially a large numbers of active sites are expected
to be available on the surface of MMIPs and MNIPs. As the adsorption
progresses the sites become saturated and rate of adsorption decreased
and get covered and there is no free space is available to the adsorbate
molecules. The initial rapid uptake of (upto 60 min) FA from solution
was likely due to more expos ed binding sites and the slow sorption
phase likely resulted from more internal binding sites. Due to high sur-
face area of MMIPs shows maximum adsorption than MNIPs for the FA.
3.3. Adsorption kinetics
In order to investigate the kinetics of adsorption of FA on MMIPs and
MNIPs, the pseudo-first-order and pseudo-second-order equations [29]
were applied. The pseudo-first-order expression is given by Eq. (3).
log q
e
−q
t
ðÞ¼−
k
1
t
2:303
þ logq
e
ð3Þ
Where q
e
is the amount of adsorption of FA (mg g
−1
) adsorbed at
equilibrium and at time qt (minutes) respectively. k
1
is overall rate con-
stant. Straight lines were obtained by plotting log(q
e
− q
t
) against t, as
shown in Fig. S2. The values of rate constant k
1
were calculated from the
slopes of straight lines of Fig. S2 and the values are reported in Table 1.
Table 2
Data obtained used the Langmuir and Freundlich isotherm models applied in our experimental results.
Langmuir isotherm equation Freundlich isotherm equation
Sorbents q
e
(mg g
−1
)
⁎
R
2
K
1
(L mg
−1
)R
2
1/n K (mg g
−1
)(Lmg
−1
)
1/n
MIP 8.757 0.9639 0.646 0.9910 0.3357 3.557
NIP 5.938 0.9861 0.380 0.9886 0.3738 1.940
⁎
q
e
(mg g
−1
) is calculated from Langmuir isotherm equation (Eq. 5).
02468101214
0,0
0,3
0,6
0,9
1,2
1,5
1,8
2,1
2,4
2,7
C
e
/q
e
Ce (mg L
-1
)
Fig. 6. Langmuir model fit for the adsorption of folic acid onto MMIPs (■)and MNIPs (•)
material.
54 S. Hussain et al. / Reactive and Functional Polymers 106 (2016) 51–56
The pseudo-second order kinetics model in Eq. (4).
t
q
t
¼
1
k
2
q
2
e
þ
1
q
e
t ð4Þ
where q
e
is the maximum adsorption capacity (mol g
−1
) for the pseu-
do-second-order adsorption, q
t
the amount of the FA adsorbed at time
t (mol g
− 1
), k
2
the equilibrium rate constant of pseudo-second-order
adsorption (mg g
−1
min
− 1
). The values of k
2
and q
e
were calculated
from the plot of t/q
t
against t (Fig. S3). The calculated q
e
values agree
with experimental q
e
values, and also, the correlation coefficients (R
2
)
for the pseudo-first-order kinetic plots at all the studied concentrations
were above 0.9936 for MMIPs and 0.9902 for MNIPs (Table 1). These re-
sults imply that the adsorption system studied obeys to the pseudo-
first-order-kinetic model.
3.4. Study of the folic acid onto MMIPs and MNIPs by adsorption isotherms
In order to study the effect of the FA adsorption in the MMIP and
their corresponding MNIP, experiments were conducted at a fixed ad-
sorbent dosage of 10 mg at different initial concentration (C
i
). The re-
sults obtained from these expe riments are given in Fig. 5 in which
possible to observe that the adsorption increases as the FA concentra-
tion increases which mainly due to the availability of large number of
site over the surface at certain level and after that the sorption capacity
slightly decreases because the adsorption site become saturated with FA
molecules. It's also obvious that quantity adsorbed by MMIP was more
than the MNIP, which proposed that imprinted sites molded due to
the reaction of imprinting and crosslinking. This also demonstrates
that initial FA concentration plays an important role in the adsorption
process.
Since the determination of adsorption isotherm is important in de-
signing the nature of adsorption system, two widely used isother m
models have been applied. The data were evaluate d by applying the
Langmuir (5) and Freundlich (6) isotherms.
For the Langmuir model was used the Eq. (5) follow:
C
e
q
e
¼
1
K
1
X
m
þ
C
e
X
m
ð5Þ
where C
e
is th e equili brium concentration of FA (mg L
− 1
), q
e
is th e
amount (mg g
−1
) of FA adsorbed; X
m
(mg g
−1
) is adsorption capacity
(amount od adsorbate adsorbed per unit mass of the FA) and K
1
is Lang-
muir constants, representing the adsorption capacity (mg g
−1
) respec-
tively [30] [31].
Straight lines were obtained by plotting C
e
/q
e
against C
e
as shown in
Fig. 6 the linear plots indicate the applicability of Langmuir adsorption
isotherm, consequently the formation of monolayer surface of the ad-
sorbate on the surface of the adsorbent. Langmuir constant k
1
and X
m
maximum adsorption capacity were calculated from the slopes and in-
tercepts of plots and are given in Table 2.
The Freundlich isotherm is an empirical equation employed to de-
scribe the heterogeneous system. The linear form of Freundlich iso-
therm was also applied to the adsorption of FA.
ln
x
m
¼ lnK þ
1
n
lnC
e
ð6Þ
where K (mol g
−1
)and1/n(gL
−1
) are Freundlich constants, indi-
cating the adsorption capacity and adsorption intensity respectively.
x/m adsorption per gram of adsorbent which is obtained by dividing
the amount of adsorbate (x) by the weight of the adsorbent (m).
Straight lines were obtained by plotting ln x/m against lnC
e
and K and
1/n were calculated from the slope and intercept of these lines (Fig.
7). The values of Freundlich constants are given in Table 2 For favorable
adsorption, the value of 1/n is smaller than 1 and when the unfavorable
adsorption takes place, the adsorption bond becomes weak and the
value of 1/n is N 1, so adsorption decreases. As shown in Table 2 that
the value of 1/n for FA adsorption is less than unity, which shows high
adsorption intensity. The correlation coefficient is more close to 1 for
the Freundlich isotherm that indicates good agreement with experi-
mental data and isotherm parameters, however it is important to em-
phasize that the MAC value obtained by the Langmuir isotherm
(8.757 mg g
− 1
) is very close that ex perim ental value of Fig. 5
(8.1 mg g
− 1
) indicating that thi s adjustment despite not being the
best is also valid.
3.5. Application of the material
In order to applied the material to the samples, MMIPs was used as a
adsorbent material used 10 mg of MMIP on a SPE system. For this,
100.0 mL of the filtrate of orange or spinach were spiked with 2–
12 mg L
−1
of FA solution and passed through the packed-bed column
at 1 mL min
− 1
flow rate. After preconcentration step, the MMIPs
retained on the column wa s eluted with 10 mL of CH
3
OH at
0.5 mL min
−1
flow rate followed by injection of eluent in the HPLC sys-
tem. As it is observed that preconcentration procedure enhances the
chromatographic peak of MMIPs and promotes a cleanup of the sample.
The recovery values were obtained in the range of 95–104% for orange
samples and for spinach sample good recovery were obtained in the
range of 99.5–102.5. The recoveries obtained are shown in the Table 3.
This excellent results can be attributed to than in the MMIP the
imprinted sites are situated on the surface of imprinting materials and
this property allows them have a higher sorption ability. In addition
the magnetic property allows will perform the steps of pre-
Table 3
Recovery results obtained for the analysis of folic acid collected from the supermarkets of
the city of Araraquara-SP.
FA add (mg
L
−1
)
Orange juice FA
found
Recovery
(%)
Spinach FA
found
Recovery
(%)
0 0.15 ± 0.05 – 0.09 ± 0.03 –
2 1.90 ± 0.03 95.0 2.05 ± 0.04 102.5
4 4.10 ± 0.11 102.5 4.07 ± 0.06 101.8
6 6.13 ± 0.17 102.2 6.10 ± 0.02 101.7
8 8.33 ± 0.20 104.1 7.96 ± 0.06 99.5
10 9.55 ± 0.36 95.6 10.1 ± 0.1 101.1
12 11.96 ± 0.41 99.7 12.1 ± 0.1 101.1
-1,0 -0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
2,2
ln q
e
ln C
e
Fig. 7. Freundlidch model fit for the adsorption of folic acid onto MMIPs (■)andMNIPs(•)
materials.
55S. Hussain et al. / Reactive and Functional Polymers 106 (2016) 51–56
