Magnetic Nanoparticles Coated With Polyaniline To Stabilize Immobilized Trypsin

Download as pdf or txt
Download as pdf or txt
You are on page 1of 13

Hyperfine Interact (2016) 237:3

DOI 10.1007/s10751-016-1264-y

Magnetic nanoparticles coated with polyaniline


to stabilize immobilized trypsin

J. C. Maciel1 A. A. D. Merces2 M. Cabrera2 W. T. Shigeyosi3


S. D. de Souza4 M. Olzon-Dionysio4 J. D. Fabris4 C. A. Cardoso3
D. F. M. Neri5 M. P. C. Silva2 L. B. Carvalho Jr.2

Springer International Publishing Switzerland 2016

Abstract It is reported the synthesis of magnetic nanoparticles via the chemical co-
precipitation of Fe3+ ions and their preparation by coating them with polyaniline. The
electronic micrograph analysis showed that the mean diameter for the nanoparticles is
15 nm. FTIR, powder X-ray diffraction and Mossbauer spectroscopy were used to under-
stand the chemical, crystallographic and 57 Fe hyperfine structures for the two samples. The
nanoparticles, which exhibited magnetic behavior with relatively high spontaneous mag-
netization at room temperature, were identified as being mainly formed by maghemite
( Fe2 O3 ). The coated magnetic nanoparticles (sample labeled mPANI) presented a real
ability to bind biological molecules such as trypsin, forming the magnetic enzyme derivative
(sample mPANIG-Trypsin). The amount of protein and specific activity of the immobi-
lized trypsin were found to be 13 5 g of protein/mg of mPANI (49.3 % of immobilized
protein) and 24.1 0.7 U/mg of immobilized protein, respectively. After 48 days of stor-
age at 4 C, the activity of the immobilized trypsin was found to be 89 % of its initial
activity. This simple, fast and low-cost procedure was revealed to be a promising way

This article is part of the Topical Collection on Proceedings of the International Conference on the
Applications of the Mossbauer Effect (ICAME 2015), Hamburg, Germany, 13-18 September 2015

 J. C. Maciel
jackeline [email protected]

1 Universidade Federal de Roraima, Campus Paricarana, 69301000 Boa Vista, Roraima, Brazil
2 Laboratorio de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Recife, PE,
Brazil
3 Departamento de Fsica, Universidade Federal de Sao Carlos, Sao Carlos, SP, Brazil
4 Universidade Federal dos Vales de Jequitinhonha e Mucuri, Diamantina, MG, Brazil
5 Universidade Federal do Vale do Sao Francisco, Petrolina, PE, Brazil
3 Page 2 of 13 Hyperfine Interact (2016) 237:3

to prepare mPANI nanoparticles if technological applications addressed to covalently link


biomolecules are envisaged. This route yields chemically stable derivatives, which can be
easily recovered from the reaction mixture with a magnetic field and recyclable reused.

Keywords Magnetic nanoparticles PANI Trypsin Biomedical applications

1 Introduction

Magnetic nanomaterials as magnetite, maghemite and metal ferrites has been widely used
in biomedicine and in heterogeneous catalysis, due to their unique magnetic properties and
multifunctional surface that can be chemically modified [1]. The functionalization of the
surface of magnetic iron oxides nanoparticles by organic molecules has been giving rise to
intensive searches addressed to biomedical applications such as magnetic drug targeting,
hyperthermia and enzyme immobilization [2]. The main interests are due to their unique
catalytic, magnetic and electrical behaviors [3]. Within this scope, magnetite (Fe3 O4 ) and
maghemite ( -Fe2 O3 ) have become the main magnetic iron oxides nanoparticles present-
ing real potentiality for different technological applications. The main features of magnetite
nanoparticles are based on the chemical functionality of their active surface, their biocom-
patibility, low-cost and the relatively high saturation magnetization [4, 5]. The need for
combining the magnetic and electrical conduction properties leads to design and to develop
new materials such as magnetic particles anchoring a conductor polymer [6]. Polyaniline
(PANI) may be a special focus, among other conductor polymers, taking into account that it
may be more easily synthesized, starting from low-cost materials. PANI has good chemical
stability and significant ability to be electrically switched between its conductive and resis-
tive states [7]. The electric and magnetic properties of polyaniline can be modified by adding
inorganic fillers, as by the inclusion of magnetic particles, intended to improve the magnetic
and dielectric properties of host materials [8]. The production of monodisperse nanopow-
ders and the assessment of their composition and sizes distribution are therefore key-points
on those applications [9]. The main objective of this work was to produce magnetic nanopar-
ticles coated with PANI to be used as a support for enzyme immobilization, using trypsin
as molecule model. For this, magnetic nanoparticles were prepared via the co-precipitation
Fe2+ and Fe3+ ions in aqueous solution. The catalytic performance of the enzyme deriva-
tive was then evaluated. These magnetic nanoparticles were also characterized by electronic
micrographic observations, FTIR, X-ray diffraction analysis, 57 Fe Mossbauer spectroscopy
and magnetization measurements.

2 Materials and methods

2.1 Materials

Trypsin (E.C.3.4.21.4) from porcine pancreas, bovine serum albumin (BSA), N -benzoyl-D-
L-arginine-p-nitroanilide (BAPNA), aniline (ACS reagent), potassium permanganate (ACS
reagent), Folin-Ciocalteus phenol reagent and, 25 % glutaraldehyde were purchased from
Sigma-Aldrich (USA). Dimethylsulfoxide (DMSO) and sodium hydroxide were from Vetec
Chemical (Brazil). Ferric chloride hexahydrate and ferrous chloride tetrahydrate were from
Merck (Germany). All other reagents were of analytical grade.
Hyperfine Interact (2016) 237:3 Page 3 of 13 3

2.2 Magnetic particles preparation

Solutions of 1.1 mol L1 FeCl3 .6H2 O (5 mL) and of 0.6 mol L1 FeCl2 .4H2 O (5 mL)
were added to 50 mL of distilled water, under magnetic agitation, and 5.0 mol L1 NaOH
was added dropwise up to pH 10 when black particles precipitated. The mixture was heated
at 50 C for 30 min with vigorous stirring. The magnetic nanoparticles were thoroughly
washed with distilled water until neutral pH. The material was dried up and kept at 25 C.

2.3 Coating with PANI

Oxidative polymerization of aniline was carried out in the presence of magnetic nanoparti-
cles (0.5 g), then treated with 0.1 mol L1 KMnO4 (50 mL) solution at 25 C for 1 h and
washed with distilled water. Then magnetic-KMnO4 nanoparticles were immersed into 50
mL of 0.5 mol L1 aniline solution (in 1.0 mol L1 HNO3 ). Polymerization was allowed
to occur at 4 C for 1 h and after that the magnetic nanoparticles coated with polyaniline
(mPANI) were successively washed with distilled water, 0.1 M citric acid and rewashed
with distilled water, and finally the material was dried up and kept at 25 C.

2.4 Trypsin immobilization

mPANI (0.01 g) was incubated with 2.0 % w/v glutaraldehyde (1.0 mL) at 25 C for 2 h
under mild stirring, washed with distilled water and Tris-HCl buffer (0.1 mol L1 , pH 8.0)
to remove the glutaraldehyde excess. Then, it was activated with glutaraldehyde (mPANIG),
kept in buffer at 4 C and finally incubated with trypsin (200 g/mL) for 16 h at 4 C
under mild stirring. The enzymatic derivative (mPANIG-Trypsin) was collected by a mag-
netic field (Ciba corning; 0.6 T) and the supernatant and washings were used for protein
determination.

2.5 Activity assay

Amidase activity of trypsin was measured by using the artificial substrate BAPNA (4.0 mM
in DMSO). The mixture of reaction was incubated at 25 C for 15 min. The hydrolyzed p-
nitroanilide was monitored in spectrophotometer at 440 nm and the activity of enzyme was
calculated. All assays were carried out in replicates and the results were expressed as mean
SD. The activity of trypsin (unit) was defined as mol BAPNA hydrolyzed per minute
using an absorption coefficient of 9.100 M1 cm1 . The specific activity was calculated by
dividing the enzymatic activity (U) by the amount of protein (mg). For the residual specific
activity the specific activity of free enzyme was considered as 100 %.

2.6 Protein determination

The amount of immobilized protein was estimated by the difference between the offered
protein and that found in the supernatant and washing solutions measured according to the
method Lowry et al. [10] using bovine serum albumin (BSA) as a standard.

2.7 Magnetic nanoparticles characterization

The morphology and particle size of the nanoparticles were characterized using a Jeol
1200EXII 80 transmission electron microscope (TEM). The Fourier transform infrared
3 Page 4 of 13 Hyperfine Interact (2016) 237:3

(FTIR) spectra were obtained using an IFS66 FTIR spectrometer (Bruker, Billerica, MA,
USA) in the range of 4000-400 cm1 using KBr pellets. Typically, 100 scans were recorded
with a 4 cm1 resolution.
The magnetic particles structural properties were characterized by X-ray powder diffrac-
tion, which was carried out in a ray-X diffractometer Shimadzu model XDR 7000, using Cu
K radiation ( = 0.154 nm) at a scanning speed of 0.1 s1 in the range of 2 = 1090
with a 0.02 step.
Mossbauer spectra were recorded with a constant acceleration transmission mode setup,
at room temperature, using a 10 mCi 57 Co (Rh-matrix) source. The Doppler velocity scale
of the Mossbauer spectrometer (model MB500) was calibrated by using a thin -Fe foil as
absorber. Data analysis was performed using the computer program WinNormos for IGOR
Pro 6.36 assuming Lorentzian functions, through the least-square convergence fitting.
Magnetization measurements were obtained at 298 K and 323 K in 5 T applied field
using a SQUID magnetometer (Quantum Design Model MPMS-5S).

2.8 Chemical stability during storage and reuse

The mPANIG-Trypsin was stored in Tris-HCl buffer at 4 C. The remaining activity of


immobilized trypsin (item 2.5) was evaluated for 6 subsequent cycles during 55 days mea-
suring its activity followed by Tris-HCl buffer washing and storage again in the buffer at
4 C.

3 Results and discussion

3.1 Synthesis of magnetic nanoparticles and PANI coating

According to Sun et al. [11], the synthesis of magnetic particles with sizes around 10 nm
was achieved using a reaction temperature of 50 C, a solution at pH 1011 and a high
stirring rate (above 800 rpm). According to those authors, temperatures above 50 C pro-
duce larger particles. At pH values between 10 and 11, smaller magnetic nanoparticles are
obtained. This can be explained by means of the chemical mechanism to form the magnetic
particles [12]. In order to obtain nanoparticles, the synthesis protocol of magnetic parti-
cles [11], which is generally used in our laboratory was modified so to use the incubation
time of 30 min, at 50 C, with the final pH of the mixture reaching 10, and NaOH as the
co-precipitation agent.
NaOH was chosen as the precipitating agent instead of NH4 OH as used by Carneiro
Leao et al. [11], as according to Hong et al. [13] the magnetite nanoparticles shows minor
agglomeration and smaller diameter if NaOH is used. The magnetic nanoparticles produced
in this work (mean diameter, 15 nm) have dimensions much smaller than those obtained
by Neri et al. [14] (10100 m) who followed the protocol reported by Carneiro Leao et al.
[11]. Roth et al. [15] performed the synthesis of superparamagnetic nanoparticles by the
co-precipitation of Fe2+ and Fe3+ in aqueous alkaline environment (NaOH) and obtained
nanoparticles between 3 and 17 nm, a particle size range comparable to that found in this
study (Fig. 1).
The magnetic nanoparticles produced were coated with PANI (mPANI) by the oxida-
tion of aniline with potassium permanganate, producing PANI in form of emeraldine, that
is the most stable and conductive [7]. It is well known that, under certain synthesis condi-
tions, PANI forms a sub-micrometer film on the surface of the objects immersed into the
Hyperfine Interact (2016) 237:3 Page 5 of 13 3

Fig. 1 Electron microscopic images. TEM image of magnetic nanoparticles and mPANI. The circle on the
figures indicate isolated magnetic nanoparticle

polymerization mixture. These films are formed on various types of substrates: conducting,
insulating, hydrophobic, and hydrophilic. The parameters of the film (thickness, uniformity,
and morphology) depend both on the chemical nature of the coated surface and on the syn-
thesis conditions [16]. While hydrophobic materials are covered by a more or less uniform
PANI film, the film relief on hydrophilic microparticles has often a discontinuous patchy
character [17]. In this work it was observed that besides the covering process a PANI pre-
cipitate was produced. This residue was easily removed from the magnetic nanoparticles
surface by washing under a magnetic field. The ratio between the amounts of precipitate
and film depends on the synthesis conditions [18]. Previous mechanism [14] was used to
produce particles composed of a magnetic core (iron oxide) and an organic shell (PANI).
Here, the optimal conditions of the coating process were established by using an experi-
mental design (results not shown). These conditions were: 0.1 M KMnO4 , 0.5 M aniline,
contact time of KMnO4 of 1 h, polymerization time of 1 h and polymerization tempera-
ture at 4 C. The concentrations of the oxidizing agent and aniline were identical to the
protocol; however, the contact time of KMnO4 was reduced from overnight to 1 h, the
polymerization time from 2 to 1 h and, the polymerization temperature from 25 to 4 C.
The morphology, shape and size distribution of magnetic nanoparticles and mPANI were
examined by transmission electron microscopy (TEM) and typical images are shown in
Fig. 1. The magnetic nanoparticles exhibited agglomerates with apparently regular mor-
phology, nearly spherical/ellipsoidal shape, dimensions around 15 nm, and narrow particle
size distribution (Fig. 1).

3.2 FTIR spectra

The emeraldine salt is the partly oxidized form of polyaniline where six benzene rings
and two quinoid rings are present in an eight-ring repeating unit. The conductivity of the
emeraldine form of polyaniline can be changed by about 10 orders of magnitude by doping
[19]. The FTIR spectra of magnetic nanoparticles and mPANI are shown in Fig. 2. It was
found that mPANI had characteristic peaks at around 1494 cm1 (C = C stretching of
benzenoid ring), 1398 cm1 (Q = NB stretching deformation, Q refers to the quinoid ring
3 Page 6 of 13 Hyperfine Interact (2016) 237:3

Fig. 2 FTIR spectra (a) and absorption bands of 750 400 cm1 (b) of magnetic nanoparticles and mPANI

and B to the benzenoid ring), 1305 and 1260 cm1 (CN stretching benzenoid ring) and
805 cm1 (out-of-plane deformation of CH in the 1, 4-disubstitued benzene ring), which
was similar with polyaniline [14, 20, 21].
Polymerization proceeds initially on the surface of these oxide particles in the presence
of KMnO4 . The adsorption of polymer to the magnetic nanoparticles results in constrained
chain growth around the particles. Such adsorption and constrained motion of the chains
will restrict the modes of vibration in polyaniline, which in turn lead to the reduction in
intensity in the FTIR spectra [19, 22]. The presence of FeO bond in particles can be seen
by two strong absorption bands at around 635 and 590 cm1 . These bands result from split
of the 1 band at 570 cm1 , which corresponds to the FeO bond of bulk magnetite [23
26]. The absorption bands of FeO bond were observed around 631 and 588 cm1 for
magnetic nanoparticles and 630 and 580 cm1 for mPANI. A principal effect of finite size
of nanoparticles is the breaking of large number of bonds for surface atoms, resulting in the
Hyperfine Interact (2016) 237:3 Page 7 of 13 3

(311)
Magnetic nanoparticles

(440)
(220)
(511)
Intensity (a.u.)

(400)
(422)

mPani

20 30 40 50 60 70
2 (degree) ( Cu K )

Fig. 3 XRD patterns of magnetic nanoparticles and mPANI

rearrangement of electrons on the particle surface [23]. The FTIR spectra in the range 750
450 cm1 for magnetic nanoparticles and mPANI are shown in the Fig. 2b. These spectra
are compared with the spectrum of maghemite (not shown) confirming the oxidation and its
increase after coating (Fig. 2b). The spectra exhibit absorption bands around the 570 cm1
characteristic of magnetite [9]. Moreover, the bandwidth increases, and small shoulders
in the 600750 cm1 range as well as a band at 450 cm1 appear with greater intensity
after coating of the magnetic particles. The appearance of the characteristic maghemite
peaks seems to promote coating. So, FTIR spectra indicate the presence of magnetite and/or
maghemite in the samples.

3.3 XRD analysis

Colors of iron oxide may be very dependent on grain size and chemical composition and
this feature is a first auxiliary attribute in their identification [27]. The magnetic nanopar-
ticles and mPANI were very dark and magnetic, suggesting that magnetite could be the
dominant phase, although maghemite may not be excluded. Figure 3 shows XRD patterns
for magnetic nanoparticles and mPANI. The 2 peaks at 18.44, 30.30, 35.67, 43.37, 53.80,
57.35, 62.97, 71.43 and 74.48 [3, 2831] are attributed to the crystal planes at (111), (220),
(311), (400), (422), (511), (440), (620) and (533) [32], which can refers to both, magnetite
and maghemite, according to the International Center for Diffraction Data (reference code:
ICDD 019-0629). As observed by Andrade et al. [27] no clear reflection peak due to other
crystalline phase, which could occur as impurity, was observed, indicating that final prod-
ucts were pure enough, from this point of view, consisting essentially of binary mixture of
the two spinel magnetic iron oxides, meaning magnetite and maghemite. In this way, XRD
results confirm FTIR results. Furthermore, the results show broadened lines that can be
3 Page 8 of 13 Hyperfine Interact (2016) 237:3

Fig. 4 298 K-Mossbauer spectra for the (a) magnetic nanoparticles and (b) mPANI samples

due to the magnetization relaxation from superparamagnetism and matrix constritions for
nanosized particles [33].

3.4 Mossbauer spectroscopy (MS)

Magnetite ([Fe3+ ]{Fe2+ Fe3+ }O4 , where [ ] and { } stand for tetrahedral and octahe-
dral symmetries
 of the coordination polyhedral Fe-O) and maghemite (ideal formula,
[Fe3+ ] Fe3+
5 1 O4 , where = cation vacancy; stoichiometrically, it corresponds to the
3 3
Fe2 O3 ) have cubic unit cells with close dimensions. These circumstances render very sim-
ilar XRD patterns. On the other hand, maghemite contains only Fe3+ whereas magnetite
has Fe3+ and Fe2+ in the spinel structure. Mossbauer spectroscopy can thus give a direct
assessment of the chemical state of iron, which may distinguish between the two iron oxides
nanoparticles. The used fitting model for these Mossbauer patterns (Fig. 4), with relatively
asymmetric resonance lines, was mainly based on two sextets (subspectral areas of 81.4
and 82.9 % for the sample containing the sole magnetic nanoparticles and for the mag-
netic nanoparticles coated with with polyanilinethe mPANI sample, respectively) with
Lorentzian lines (fitted spectra are shown in Fig. 4; corresponding Mossbauer parameters
are presented in Table 1). The remaining area of the two spectra (18.6 and 17.1 % for the
sole magnetic and for the polyaniline coated magnetic nanoparticles, respectively) were
completed by fitting them with model-independent hyperfine field distributions. Figures
Hyperfine Interact (2016) 237:3 Page 9 of 13 3

Table 1 Fitted hyperfineparameters for the 298 K-Mossbauer spectra

Sample 57 Fe site /mm s 1 2Q /mm s 1 /mm s 1 Bhf /T RA/%

Magnetic nanoparticles [Fe3+ ] 0.329(2) 0.010(5) 0.86(2) 45.45(5) 47.0


{Fe3+ } 0.319(2) 0.001(1) 0.57(1) 48.49(3) 34.4
mPANI [Fe3+ ] 0.321(2) 0.0075(5) 0.85(2) 45.28(5) 45.6
{Fe3+ } 0.319(2) 0.001(1) 0.60(1) 48 (3) 37.3

= isomer shift relative to Fe; 2Q = quadrupole shift; = resonance linewidth; Bhf = hyperfine field;
RA = relative subspectral area. [ ] and { } denotes tetrahedral and octahedral symmetries, respectively, for
the Fe-O coordination sites. Numbers in parentheses are uncertainties over the last significant digit of the
numerical value, as output by the least squares fitting computer program

of the corresponding hyperfine field probability profiles are not shown. The found isomer
shift values are characteristic of high spin Fe3+ . The fitted values of quadrupole shift are
consistent with those expected for iron spinels. The hyperfine field relative to the two sex-
tets for each sample correspond to the tetrahedral and octahedral Fe3+ sites in nanosized
maghemite. The decrease in the fitted hyperfine field Bhf for both sites (45.3 and 48.4 T)
relatively to pure oxide (nearly 50 T) is consistent with the expected effect due to the collec-
tive magnetic excitations [34]. From these Mossbauer data, the occurrence of magnetite can
be safely discarded in these two samples. Moreover, Mossbauer results indicate that coating
with mPANI does not significantly alter the chemical nature of the produced oxide [35].

3.5 Magnetization measurements

The magnetic properties of the magnetic nanoparticles and mPANI were investigated in the
applied magnetic field sweeping from 60 to 60 kOe at 298 K and 323 K as shown in Fig. 5.
It is well-known that the size [36], structure and shape may affect the magnetic proper-
ties of the products [37]. Under applied magnetic field, magnetic nanoparticles and mPANI
showed the positive magnetizations and there is no hysteresis (Fig. 5). It indicates the super-
paramagnetic behavior [25]. The difference between the magnetic behavior of small and
medium sized particles can also be observed in their hysteresis loops [9].
Figure 5 shows the field dependent magnetization curves of magnetic nanoparticles and
mPANI. The saturation magnetization (Ms ) for the magnetic nanoparticles has value equal
to 60.51 emu g1 , which is lower than that of bulk magnetite (Ms = 92 emu g1 ) [38],
and mPANI has value equal to 59.05 emu g1 (Fig. 5). The reduction in the saturation
magnetization values signifies the decrease in the particles size [39] and can be attributed
to surface effects such as magnetically inactive layer containing spins that are not collinear
with the magnetic field. These discrepancies in the saturation values of magnetization may
be explained by variations in the methods employed to synthesize magnetite, which can
generate different particle sizes, magnetite surfaces and chemical compositions [40].

3.6 Stability during storage and reuse

Data of operational stability showed that after 5 cycles combining storage and reuse,
mPANIG-Trypsin maintained 89 % of its initial activity (Fig. 6). An 11 % decrease in
activity was detected during this period. The immobilized trypsin on polyaniline supports
retained 50 % its original activity after 21 days when stored at 4 C in phosphate buffer
3 Page 10 of 13 Hyperfine Interact (2016) 237:3

Fig. 5 Magnetization curves of magnetic nanoparticles and mPANI at 298 and 323 K

Fig. 6 Shelf life of mPANIG-Trypsin at 4 C stored in Tris-HCl buffer

[41], 58 % its original activity after 49 days when stored at 4 C in glycine buffer contain-
ing CaCl2 [42] and 80 % its original activity after 44 days (approximately) when stored
in glycine buffer containing CaCl2 [43]. Sun et al. [44] conducted the immobilization of
trypsin onto the superparamagnetic carboxymethyl chitosan and the immobilized enzyme
exhibited stability for six times reuse, holding 76.3 % of its initial activity. In our study, we
observed better results. Moreover, Chellapandian [45] reported that immobilized trypsin on
vermiculite retained 84 % its original activity after 60 days when stored in borate buffer.
The enzymatic derivative obtained this study was more stable during the period of 48 days,
however its activity after 55 days was 77 % of initial value. This result indicates that the
mPANIG-Trypsin may be stored in buffer only (no stabilizer, e.g. CaCl2 ) for approximately
50 days, retaining about 90 % of its initial activity.
Hyperfine Interact (2016) 237:3 Page 11 of 13 3

4 Conclusions

In conclusion, magnetic nanoparticles were obtained from modifications of the experimen-


tal protocol used routinely by our research group. These nanoparticles showed dimensions
smaller than those obtained by original the protocol, corroborating the advantages of the
optimization process. The FTIR, XRD and Mossbauer spectroscopy analysis of the nanopar-
ticles confirmed the presence of maghemite. Furthermore, after PANI coating, nanoparticles
preserved good magnetization, an increase in the surface area and showed to be an excellent
support for trypsin immobilization. The major benefits of the magnetic enzymatic deriva-
tive (mPANIG-Trypsin) are its operational stability, high activity, simple method and low
cost of production.

Acknowledgments The authors wish to acknowledge financial support from the Brazilian agency CNPq
(Conselho Nacional de Desenvolvimento Cientfico e Tecnologico) and FACEPE (Fundacao de Amparo a
Ciencia e Tecnologia de Pernambuco). The authors would like to thank Prof. Dr. Marcia Attias (Laboratorio
de Ultraestrutura Celular Herta Meyer, UFRJ, Brazil) for the transmission electron microscopy analysis,
Prof. Dr. Adilson J.A. de Oliveira (UFSCar, SP, Brazil) for the magnetization measurements. CAPES (Brazil)
grants the Visiting Professor PVNS fellowship to JDF at UFVJM.

References

1. Reddy, L.H., Arias, J.L., Nicolas, J., Couvreur, P.: Magnetic nanoparticles: design and characterization,
toxicity and biocompatibility, pharmaceutical and biomedical applications. Chem. Rev. 112, 58185878
(2012)
2. Daou, T.J., Greneche, J.M., Pourroy, G., Buathong, B., Derory, A., Ulhaq-Bouillet, C., Donnio, B.,
Guillon, D., Begin-Colin, S.: Coupling agent effect on magnetic properties of functionalized magnetite-
based nanoparticles. Chem. Mater. 20, 58695875 (2008)
3. Gu, H., Huang, Y., Zhang, X., Wang, Q., Zhu, J., Shao, L., Haldolaarachchige, N., Young, D.P., Wei, S.,
Guo, Z.: Magnetoresistive polyaniline-magnetite nanocomposites with negative dielectrical properties.
Polymer 53, 801809 (2012)
4. Salado, J., Insausti, M., Lezama, L., de Muro, I.G., Goikolea, E., Rojo, T.: Preparation and characteri-
zation of monodisperse Fe3 O4 nanoparticles: na electron magnetic resonance study. Chem. Mater. 23,
28792885 (2011)
5. Belaabed, B., Wojkiewicz, J.L., Lamouri, S., Kamchi, N.E., Lasri, T.: Synthesis and characteriza-
tion of hybrid conducting composites based on polyaniline/magnetite fillers with improved microwave
absorption properties. J. Alloy Compd. 527, 137144 (2012)
6. Khafagy, R.M.: Synthesis, characterization, magnetic and electrical properties of the novel conductive
and magnetic Polyaniline/MgFe2 O4 . J. Alloy Compd. 509, 98499857 (2011)
7. Balint, R., Cassidy, N.J., Cartmell, S.H.: Conductive polymers: towards a smart biomaterial for tissue
engineering. Acta Biomater. 10, 23412353 (2014)
8. Gandhi, N., Singh, K., Ohlan, A., Singh, D.P., Dhawan, S.K.: Thermal, dielectric and microwave
absorption properties of polyanilineCoFe2 O4 nanocomposites. Compos. Sci. Technol. 71, 17541760
(2011)
9. Salazar, J.S., Perez, L., Abril, O., Phuoc, L.T., Ihiawakrim, D., Vazquez, M., Greneche, J.-M., Begin-
Colin, S., Pourroy, G.: Magnetic iron oxide nanoparticles in 1040 nm range: composition in terms of
magnetite/maghemite ratio and effect on the magnetic properties. Chem. Mater. 23, 13791386 (2011)
10. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol
reagent. J. Biol. Chem. 193, 265275 (1951)
11. Carneiro Leao, A.M.A., Oliveira, E.A., Carvalho Jr., L.B.: Immobilization for protein on ferromagnetic
Dacron. Appl. Biochem. Biotech. 32, 5358 (1991)
12. Sun, J., Zhou, S., Hou, P., Yang, Y., Weng, J., Li, X., Li, M.: Synthesis and characterization of
biocompatible Fe3 O4 nanoparticles. J. Biomed. Mater. Res. A 80A, 333341 (2007)
13. Hong, R., Li, J., Wang, J., Li, H.: Comparison of schemes for preparing magnetic Fe3 O4 nanoparticles.
China Part 5, 186191 (2007)
3 Page 12 of 13 Hyperfine Interact (2016) 237:3

14. Neri, D.F.M., Balcao, V.M., Dourado, F.O.Q., Oliveira, J.M.B., Carvalho Jr., L.B., Teixeira, J.A.: Immo-
bilized -galactosidase onto magnetic particles coated with polyaniline: support characterization and
galactooligosaccharide production. J. Mol. Catal. B-Enzym. 70, 7480 (2011)
15. Roth, H.-C., Schwaminger, S.P., Schindler, M., Wagner, F.E., Berensmeier, S.: Influencing factors in the
co-precipitation process of superparamagnetic iron oxide nanoparticles: a model based study. J. Magn.
Magn. Mater. 377, 8189 (2015)
16. Stejskal, J., Sapurina, I., Prokes, J., Zemek, J.: In-situ polymerization polyaniline films. Synthetic Met.
105, 195202 (1999)
17. Kohut-Svelko, N., Reynaud, S., Dedryevre, R., Martinez, H., Gonbeau, D., Francois, J.: Study of a
nanocomposite based on a conducting polymer: polyaniline. Langmuir 21, 15751583 (2005)
18. Stejskal, J., Trchova, M., Fedorova, S., Sapurina, I., Zemek, J.: Surface polymerization of aniline on
silica gel. Langmuir 19, 30133018 (2003)
19. Nagaraja, N., Pattar, J., Shashank, N., Manjanna, J., Kamada, Y., Rajanna, K., Mahesh, H.M.: Electrical,
structural and magnetic properties of polyaniline/pTSA-TiO2 nanocomposites. Synthetic Met. 159, 718
722 (2009)
20. Xiao, Q., Tan, X., Ji, L., Xue, J.: Preparation and characterization of polyaniline/nano-Fe3 O4 composites
via a novel Pickering emulsion route. Synthetic Met. 157, 784791 (2007)
21. Lin, Y.-W., Wu, T.-M.: Synthesis characterization of externally doped sulfonated polyaniline/multi-
walled carbon nanotube composites. Compos. Sci. Technol. 69, 25592565 (2009)
22. Feng, W., Bai, X.D., Lian, Y.Q., Liang, J., Wang, X.G., Yoshino, K.: Well-aligned polyaniline/carbon-
nanotube composite films grown by in-situ aniline polymerization. Carbon 41, 15511557 (2003)
23. Ma, M., Zhang, Y., Yu, W., Shen, H., Zhang, H., Gu, N.: Preparation and characterization of magnetite
nanoparticles coated by amino silane. Colloid Surface A 212, 219226 (2003)
24. Xu, X.Q., Shen, H., Xu, J.R., Li, X.J.: Aqueous-based magnetite magnetic fluids stabilized by surface
small micelles of oleolysarcosine. Appl. Surf. Sci. 221, 430436 (2004)
25. Yamaura, M., Camilo, R.L., Sampaio, L.C., Macedo, M.A., Nakamura, M., Toma, H.E.: Preparation
and characterization of (3-aminopropyl) triethoxysilane-coated magnetite nanoparticles. J. Magn. Magn.
Mater. 279, 210217 (2004)
26. Klokkenburg, M., Hilhorst, J., Erne, B.H.: Surface analysis of magnetite nanoparticles in cyclohexane
solutions of oleic acid and oleylamine. Vib. Spectrosc. 43, 243248 (2007)
27. Andrade, A.L., Valente, M.A., Ferreira, J.M.F., Fabris, J.D.: Preparation of size-controlled nanoparticles
of magnetite. J. Magn. Magn. Mater. 324, 17531757 (2012)
28. Reddy, K.R., Lee, K.-P., Gopalan, A.I., Kang, H.-D.: Organosilane modified magnetite
nanoparticles/poly(aniline-co-o/m-aminobenzenesulfonic acid) composites: synthesis and
characterization. React. Funct. Polym. 67, 943954 (2007)
29. Yu, W., Zhang, T., Zhang, J., Qiao, X., Yang, L., Liu, Y.: The synthesis of octahedral nanoparticles of
magnetite. Mater. Lett. 60, 29983001 (2006)
30. Xu, F., Ma, L., Hu, Q., Gan, M., Tang, J.: Microwave absorbing properties and structural design of
microwave absorbers based on polyaniline and polyaniline/magnetite nanocomposite. J. Magn. Magn.
Mater. 374, 311316 (2015)
31. Vandenberghe, R.E., Barrero, C.A., da Costa, G.M., Van San, E., De Grave, E.: Mossbauer characteri-
zation of iron oxides and (oxy)hydroxides: the present state of the art. Hyperfine Interact 126, 247259
(2000)
32. Klug, H.P., Alexander, L.E. X-ray Diffraction Procedures for Polycrystalline and Amorphous Materials,
2nd edn. Willey-Interscience, New York (1974)
33. Quin, X.Y.: The exothermal and endothermal phenomena in nanocrystalline aluminum. Nanostruct.
Mater. 2, 99106 (1993)
34. Mrup, S., Frandsen, C., Hansen, M.F.: Magnetic interactions between nanoparticles. Beilstein J.
Nanotechnol. 1, 4854 (2010)
35. Jaramillo-Tabares, B.E., Isaza, F.J., Cordoba de Torresi, S.I.: Stabilization of polyaniline by the
incorporation of magnetite nanoparticles. Mater. Chem. Phys. 132, 529533 (2012)
36. Wen, X., Yang, J., He, B., Gu, Z.: Preparation of monodisperse magnetite nanoparticles under mild
conditions. Curr. Appl. Phys. 8, 535541 (2008)
37. Xu, H., Shao, M., Chen, T., Zhuo, S., Wen, C., Peng, M.: Magnetism-assisted assembled porous Fe3 O4
nanoparticles and their electrochemistry for dopamine sensing. Micropor. Mesopor. Mat. 153, 3540
(2012)
38. Cullity, D.: Introduction to Magnetic Materials. Addison-Wesley, Reading, MA (1972)
39. Xu, L., Du, J., Li, P., Qian, Y.: In situ synthesis, magnetic property, and formation mechanism of Fe3 O4
particles encapsulated in ID bamboo-shaped carbon microtubes. J. Phys. Chem. B 110, 38713875
(2006)
Hyperfine Interact (2016) 237:3 Page 13 of 13 3

40. Grigorova, M., Blythe, H.J., Blaskov, V., Rusanov, V., Petkov, V., Masheva, V., Nihtianova, D., Martinez,
L.M., Munoz, J.S., Mikhov, M.: Magnetic properties and Mossbauer spectra of nanosized CoFe3 O4
powders. J. Magn. Magn. Mater. 183, 163172 (1998)
41. Caramori, S.S., Fernandes, K.F.: The use of poly(ethylene terephthalate)-poly(aniline) composite for
trypsin immobilization. Mater. Sci. Eng. C 28, 11591163 (2008)
42. Purcena, L.L.A., Caramori, S.S., Mitidieri, S., Fernandes, K.F.: The immobilization of trypsin onto
polyaniline for protein digestion. Mater. Sci. Eng. C 29, 10771081 (2009)
43. Caramori, S.S., Faria, F.N., Viana, M.P., Fernandes, F.K., Carvalho, J.r., L.B.: Trypsin immobilization on
discs of polyvinyl alcohol glutaraldehyde/polyaniline composite. Mater. Sci. Eng. C 31, 252257 (2011)
44. Sun, J., Hu, K., Liu, Y., Pan, Y.: Novel superparamagnetic nanoparticles for trypsin immobilization and
the application for efficient proteolysis. J. Chromatography B 942943, 914 (2013)
45. Chellapandian, M.: Preparation and characterization of alkaline protease immobilized on vermiculate.
Process Biochem. 33, 169173 (1998)

You might also like