Colloids and Surfaces A: Physicochem. Eng.

Aspects 212 (2003) 219 /226

www.elsevier.com/locate/colsurfa

Preparation and characterization of magnetite nanoparticles

coated by amino silane
Ming Ma
, Yu Zhang, Wei Yu, Hao-ying Shen, Hai-qian Zhang, Ning Gu
National Laboratory of Molecular and Biomolecular Electronics, Southeast University, Nanjing 210096, PR China

Received 13 January 2002; received in revised form 21 May 2002; accepted 18 June 2002

Abstract

Magnetite nanoparticles were prepared by coprecipitation of Fe2 and Fe3 with NH4OH, and then, amino silane
was coated onto the surface of the magnetite nanoparticles. Transmission electronic microscopy shows the average size
of 7.5 nm in diameter. Powder X-ray diffraction and electronic diffraction measurements show the spinel structure for
the magnetite nanoparticles. FT /IR spectra indicate that amino silane molecules have been bound onto the surface of
the magnetite nanoparticles by Fe/O/Si chemical bonds. Energy dispersive X-ray spectroscopy (SEM /EDS) indicates
atomic ratio of 96.75:3.25 for Fe:Si, implying a nearly monolayer coating of amino silane on the magnetite particle
surface according to a rough calculation. By an enzyme-linked assay, it was proved that the amino silane-coated
magnetite nanoparticles could significantly improve the protein immobilization.
# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Magnetite nanoparticles; Core-shell structure; Surface coating; Amino silane; Protein immobilization

1. Introduction generally of core /shell type: biological species

cells, nucleic acids, proteins are connected to the
Magnetic particles (microspheres, nanospheres magnetic core through an organic or polymeric
and ferrofluids) are widely studied for their shell. The shells are either biocompatible in general
applications in various fields in biology and (such as dextran, PEG, etc.), or possessing active
medicine such as enzyme and protein immobiliza- groups which can be conjugated to biomolecules
tion, genes, radiopharmaceuticals, magnetic reso- such as proteins and enzymes. Therefore, the
nance imaging MRI, diagnostics, immunoassays, investigation of magnetic nanoparticles with or-
RNA and DNA purification, magnetic cell separa- ganic coating is of significance for applications.
tion and purification, magnetically controlled In this work we prepared magnetite nanoparti-
transport of anti-cancer drugs as well as hyperther- cles coated with a near monolayer of amino silane,
mia generation [1 /3]. These magnetic beads are which has active group of /NH2 that can connect
biomolecules, drugs and so on. And the morphol-
ogy, structure and composition of the coated

Corresponding author magnetite nanoparticles were characterized by
E-mail address: [email protected] (M. Ma). TEM, ED, XRD, FT-IR and SEM /EDS.
0927-7757/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 7 - 7 7 5 7 ( 0 2 ) 0 0 3 0 5 - 9
220 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226

Furthermore, to prove that amina silane-coated magnetite particles were measured by energy
magnetite nanoparticles can be conjugated to dispersive X-ray spectroscopy (SEM /EDS,
some biomolecule, an enzyme-linked colorimetric EDAX, PV9100). Fourier transform infrared
assay was carried out after the enzyme horseradish spectroscopy (FT-IR, Nicolet, 750) of the samples
peroxidase (HRP) of different concentrations was were used to study the chemical bonds between
used to interact with the coated and the uncoated Fe3O4 and APTS.
magnetite nanoparticles.
2.4. HRP-immobilized and its activity assays

2. Experimental The APTS-coated magnetite nanoparticles and

uncoated magnetite nanoparticles as control were
2.1. Synthesis of magnetite nanoparticles all dispersed in phosphate-buffered saline (PBS,
0.01 M, pH 7.4) with identical concentrations of 2
Magnetite was made according to the method of g l 1. Enzyme horseradish peroxidase HRP of
Molday [4]. Typically, a solution of mixture of different concentrations were added into 200 ml
FeCl3 (0.01 M) and FeSO4 (0.006 M) at pH 1.7 magnetite-PBS solutions. The mixtures were in-
was prepared under N2 protecting. Then, ammo- cubated in 37 8C for 1 h, and then retracted in
nia aqueous solution (1.5 M) was dropped into it 4 8C refrigeratory for 4 h. Then the mixtures were
with violently stirring until the pH of the solution washed carefully by PBS for 4 times and shifted to
raised to 9. The obtained magnetite was washed other vessels to remove the dissociated enzymes.
immediately with water for 5 times and ethanol for Developed by addition of substrates, namely
2 times by magnetic separation. Finally, part of 3,3?,5,5?-tetramethylbenzidine and hydrogen per-
magnetite nanoparticles were dispersed in ethanol oxide (TMB-H2O2), for 10 min, the reaction was
with concentration of 0.0128 M, and the others stopped by 2 mol l 1 H2SO4. The optical density
were dried into powder at room temperature under at 450 nm was read immediately in an automatic
vacuum. plate reader (Stat fax-2100, Beiken Company). All
samples were tested in duplicate, arithmetic means
2.2. APTS-coated magnetite nanoparticles and standard deviations of absorbance values were
calculated (x9/s).
25 ml magnetite colloid ethanol solution pre-
pared above was diluted to 150 ml by ethanol and
1 ml H2O. The solution was then treated by 3. Results and discussion
ultrasonic wave for 30 min. 35 ml 3-aminopropyl-
triethoxysilane ( NH2(CH2)3Si(OC2H5)3, APTS) Fig. 1 is the TEM and ED photography of the
was added into it with rapid stirring for 7 h. The magnetite nanoparticles coated with APTS, which
result solution was washed with ethanol for 5 shows that most of the particles are quasi-spherical
times, and then dried into powder at room and with an average diameter of 7.5 nm. The
temperature under vacuum. distribution of particle diameters is shown in Fig.
2.
2.3. Characterization According to the ED pattern, the d -spacing can
be calculated in the following eqution [5],
The particle size and morphology of the samples
LldR (1)
were determined by transmission electronic micro-
scopy (TEM, JEOL, JEM-200CX, 200 kV). Pow- where L is the distance between the test sample
der X-ray diffraction (XRD, Rigaku, D/Max-RA, and the film ( L /137 cm), l is the wavelength of
Cu Ka) and electronic diffraction ( ED) were used electron beam (l /0.0251 Å), R is the radius of
to determine the crystal structure of the samples. the diffraction ring. The calculation results are
The elemental analysis and APTS loading on shown in Table 1, which accord with the XRD
 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 221

Fig. 2. Diameter distribution of magnetite nanoparticles coated

Fig. 1. TEM and ED photography of the magnetite nanopar- with APTS.
ticles coated with APTS.
possess absorption bands in 2971.8 and 2925.5
pattern of the sample shown in Fig. 3 and indicate cm 1 due to stretching vibration of C /H bond,
the inverse cubic spinel structure of Fe3O4. Com- band in 1091.5 cm 1 due to the stretching vibra-
pared with the theoretical values, the reduction of tion of C /N bond, band in 1051.0 cm 1 due to the
d -spacing obtained experimentally is due to lattice stretching vibration of Si /O bond, band in 885.2
constrictions for nanosized particles [7]. cm 1 due to the bending vibration of /NH2
Since there are large surface-to-volume atomic group. All of these reveal the existence of APTS.
ratio, high surface activity, and amount of dan- In addition, in Fig. 5(a) and (b) the absorption
gling bonds on nanoparticle surface, the atoms on bands near 3400 and 1630 cm 1 refer to the
the surface are apt to adsorb ions or molecules in vibration of remainder H2O in the samples. And
solution. For Fe3O4 nanoparticles dispersed in a there also exists the contribution of /NH2 for the
neutral aqueous solution, the bare atoms of Fe and band near 3400 cm 1 in Fig. 5(a).
O on the particle surface would adsorb OH  and Previously, it was reported that the character-
H  respectively, so that there is OH-rich surface. istic absorption bands of the Fe /O bond of bulk
The /OH on the surface can react with APTS as Fe3O4 were in 570 and 375 cm 1 [8]. However, in
the process shown in Fig. 4. Therefore, the Fig. 5(b) these two bands shift to high wavenum-
magnetite nanoparticles can be coated with bers of about 600 and 440 cm 1 respectively, and
APTS molecules by chemical bond. The fact was the band near 600 cm 1 is split into two peaks of
proven by comparison of FT-IR spectra of the 631.4 and 582.9 cm 1. A principal effect of finite
coated and uncoated Fe3O4 nanoparticles shown size of nanoparticles is the breaking of a large
in Fig. 5. It can be seen that, compared with the number of bonds for surface atoms, resulting in
uncoated sample, the coated Fe3O4 nanoparticles the rearrangement of inlocalized electrons on the
222 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226

Table 1
ED and XRD data for the magnetite nanoparticles coated with APTS

1 2 3 4 5 6 7
R (cm) 0.73 1.19 1.39 1.67 2.04 2.19 2.37
ED results-d (Å) 4.74 2.89 2.47 2.06 1.69 1.57 1.47
XRD results-d (Å) 4.78 2.83 2.50 2.08 1.70 1.60 1.48
Theory values-d (Å) 4.85 2.97 2.53 2.10 1.71 1.62 1.48
Crystalline plane (hlk) [6] (111) (220) (311) (400) (422) (511) (440)

Fig. 3. XRD pattern of the magnetite nanoparticles coated with APTS.

Fig. 4. The procedure of the coating reaction of APTS with magnetite nanoparticles.
M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 223

Fig. 5. FT-IR spectra of the coated (a) and uncoated (b) magnetite nanoparticles.
224 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226

Fig. 6. SEM /EDS elemental analysis of APTS-coated Fe3O4 nanoparticles on Au substrate.

particle surface [9]. And the lattice constrictions ticles can be observed. In addition, the split of the
have been observed as indicated in Table 1. As a bands is attributed to the split of the energy levels
result, the surface bond force constant increases as of the quantized Fe3O4 nanoparticles[10].
Fe3O4 is reduced to nanoscale dimension, so that It is also found that the characteristic absorp-
the absorption bands of IR spectra shift to higher tion bands of the Fe /O bond of APTS-coated
wavenumbers. So the blue-shift of absorption Fe3O4 shift to high wavenumbers of 636.4 and
bands of the Fe /O bond of the Fe3O4 nanopar- 590.1 cm 1 compared with that of uncoated

Fig. 7. HRP-linked colorimetric assays of APTS-coated and uncoated Fe3O4 nanoparticles.

 M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 219 /226 225

Fe3O4(in 631.4 and 582.9 cm 1). The phenom- the APTS-coated magnetite nanoparticles is 1.4 /
enon can be explained according to the formation 2.0 times higher than that of the uncoated
of Fe /O /Si bonds where Fe /O /H groups on the magnetite nanoparticles according to the measured
surface of the Fe3O4 particles are replaced by Fe / absorbance value. The increase of absorption is
O /Si(O /)2 /R as shown in Fig. 4. More electro- attributed to the contribution of APTS whose
negativity of /Si(O /)2 / than H leads to the active group of /NH2 can be conjugated to HRP
enhancement of bond force constant for Fe /O by chemical band, and the uncoated magnetite
bonds[11], so that the absorption bands shift to nanoparticles connect the HRP by static adsorp-
high wavenumbers. tion only.
Since we know the mean diameter of the
magnetite nanoparticles is 7.5 nm, the number of
Fe atoms in every Fe3O4 particle can be calculated 4. Conclusions
by means of following formula,
APTS-coated magnetite nanoparticles with 7.5
4
pR3 Na nm average diameter were prepared and charac-
3 terized by TEM, ED, XRD, FT-IR, and SEM /
NFe  3 8945 (2)
V Fe3 O4 EDS. Especially, FT-IR spectra were utilized to
prove the formation of Fe /O /Si chemical bonds.
where V̄ Fe3 O4/refers to the molar volume of bulk
A near monolayer APTS-coating on the particle
Fe3O4, R is the mean radius of Fe3O4 nanoparti-
surface was also indicated according to the com-
cles, Na is Avogadro’s number. If there is a
parison of the experimental analysis by SEM /
monolayer of APTS molecules coated on the
EDS with a simple calculation.
Fe3O4 particle, the number of APTS molecules
By an enzyme-linked assay, it has been proved
on the surface of every Fe3O4 nanoparticle can be
that these APTS-coated magnetite nanoparticles
calculated by following formula,
could significantly improve the protein immobili-
SFe3 O4 4pR2 zation.
NAPTS   442 (3)
SAPTS SAPTS
where SFe3 O4 is the surface area of Fe3O4 particle, Acknowledgements
SAPTS is the area of surface coverage of about 40
Å2 per APTS molecule reported in the literature This work was supported by the National
[12]. So the atomic ratio of Fe/Si is/NFe =NAPTS  Natural Science Foundation of China
20:2: Fig. 6 shows a typical SEM /EDS elemental (No.69890220, No.60171005) and the High Tech-
analysis of APTS-coated magnetite nanoparticles. nology Research Subject of Jiangsu Province in
From the peak area of Fe and Si, the atomic ratio China (BG2001006). I am also very grateful to
of Fe/Si is obtained to be 96.75/3.25 /30. This Prof. Hong Jian-min of Center of Analysis and
indicates that the surface APTS coverage ratio of Test, Nanjing University for his helping in TEM
Fe3O4 nanoparticles is about 67.3%. Probably, the experiments.
incompleteness (a near monolayer) of the surface
coating is owing to the incompleteness of surface
hydroxylation and the existence of the spatial References
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