Materials Letters 65 (2011) 34993501

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Materials Letters
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t l e t

Immobilization of albumin on magnetite nanoparticles

Esra Maltas a,, Mustafa Ozmen a, Hasibe Cingilli Vural b, Salih Yildiz a, Mustafa Ersoz a
a
b

Department of Chemistry, Selcuk University, Konya 42075, Turkey

Department of Biology, Selcuk University, Konya 42075, Turkey

a r t i c l e

i n f o

Article history:
Received 3 May 2011
Accepted 18 July 2011
Available online 22 July 2011
Keywords:
Magnetic materials
APTES
Immobilization
Biomaterials
SDS-PAGE

a b s t r a c t
The magnetite (Fe3O4) nanoparticles were prepared by the co-precipitation of ferrous and ferric salts with
NH4OH, and then modied with 3-aminopropyltriethoxysilane (APTES) by silanization reaction and
subsequent reaction with glutaraldehyde (GA) to obtain functional groups on their surface. The inuence
of different terminated groups on protein binding was studied with bare and modied magnetite
nanoparticles. Amine terminated magnetite nanoparticles were shown the highest binding ability for
immobilization process compared to Fe3O4 NPs and GA bonded NPs. This binding ability was shown by using
sodium dodecyl polyacrylamide gel electrophoresis technique (SDS-PAGE). Albumin attached magnetite
nanoparticles were also examined by Scanning Electron Microscopy (SEM).
2011 Elsevier B.V. All rights reserved.

1. Introduction
In recent years, separation and purication of target protein and
elucidation of protein function are some of the major tasks facing
researchers. Thus, development of tools to enhance protein studies is
critical. Many tools have been developed to purify or separate
individual proteins from biological matrices. One of the tools that
are magnetite particles (microspheres, nanospheres and ferrouids)
is widely used in purication, separation and immobilization of
protein and enzymes. They are also used in the biomedical eld as a
solid support for immunoassays, DNA sequencing, and cell analysis
and magnetically controlled transport of anti-cancer drugs [15]. Iron
oxides magnetite particles are a group of the paramagnetic nanoparticles modied with various functional group such as epoxy, amine
and aldehyde that give better results for immobilization or binding.
The application for biomolecules immobilization mainly based on the
solid-phase magnetic feature which has the advantages of quick, easy,
and gentle separation of biological compounds using an external
magnetic eld gradient [712]. For taking advantage of magnetic
properties, many multifunctional materials have been reported for
use in bioapplications [1315]. Recently, some magnetic bead-based
materials have been developed for cancer markers via immunoassay
studies like antibodyantigen interactions by electrochemical analysis
as an alternative method to HPLC or western blotting system [16,17].
Magnetic particles are also used in recombinant DNA technology.
The production of recombinant fusion proteins having appropriate
afnity tags like GST (glutathione-S-transferase) or His-tag (Histi-

Corresponding author. Tel.: + 90 332 223 38 96; fax: + 90 332 241 24 99.
E-mail address: esramaltas@gmail.com (E. Maltas).
0167-577X/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.matlet.2011.07.045

dine) labeled proteins is expressed by the specic genes in different

host cells. This afnity tags make fast and easy purication of the
target proteins from the complex biological samples. Magnetic batch
separations, employing magnetic afnity and ion exchange particles,
have been shown to be very useful for protein separations [18].
In this paper, magnetic Fe3O4 nanoparticles were prepared by the
chemical co-precipitation of Fe(III) and Fe(II) ions. Then, the nanoparticles were modied by 3-aminopropyltriethoxysilane (APTES) to
introduce reactive groups onto the particles surface, and subsequently
with GA was grafted onto modied nanoparticles by surface. The
functionalized magnetic particles were used for protein binding.
2. Materials and methods
Superparamagnetic magnetite nanoparticles were prepared via
improved chemical co-precipitation method [6]. APTES and subsequently GA modication on magnetite nanoparticles was prepared as
before [21]. Prepared NPs were mixed with 2 mg/mL albumin in 1
PBS, pH 7.4 and thumbled overnight at 4 C at a particle concentration
range of 530 mg/mL. Supernatant was removed and kept at 4 C for
further protein analysis after BSA bounded Fe3O4 particles separated
magnetically. Albumin bounded particles were also washed with PBS
and ethanol for chemical characterization. Protein concentration was
determined using Bradford (1976) method [19] according to the
manufacturer's instructions. Remaining protein after albumin binding
on NPs was diluted to appropriate concentrations. 3 mL of Bradford
reagent was mixed 100 L of each mixture and absorbance was
recorded at 595 nm. Unbounded protein concentration was calculated
from standard curve equation of the BSA (y = 0.7725x). Samples were
examined via SDS-PAGE as described by Laemmli [20]. Briey, albumin
immobilized particles were mixed with 3 SDS sample buffer and

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E. Maltas et al. / Materials Letters 65 (2011) 34993501

Table 1
Amount of BSA coupled to Fe3O4, Fe3O4APTES and Fe3O4APTESGA at different amounts of nanoparticles.
Nanoparticles,
mg
5
10
15
20
25
30

Fe3O4APTES

Fe3O4

Fe3O4APTESGA

Protein, mg

Bounding, %

Recovery, %

Protein, mg

Bounding, %

Recovery, %

Protein, mg

Bounding, %

Recovery, %

0
0
0
0
0.062
0.067

0
0
0
0
3.12
3.38

100
100
100
100
96.88
96.72

0.074
0.270
0.405
0.595
0.859
1.070

3.72
13.52
20.24
29.76
42.96
53.52

97.28
86.48
79.76
70.24
57.04
46.48

0
0.016
0.079
0.164
0.192
0.256

0
0.81
3.98
8.19
9.60
12.76

100
99.19
96.02
92.81
90.40
87.24

binding percentage of albumin to bare NPs, APTES coated and GA

modied magnetite NPs with PBS buffer solution. BSA was immobilized on bare and modied NPs at pH 7.4 (1 PBS buffer). Unbounded
albumin was determined by Bradford method after the removal of
albumin from particles. Amount of albumin binding to magnetic NPs
was measured in terms of absorbance reduction of commassie
brilliant blue in Bradford reagent at 595 nm. Results showed that
maximum percentage of albumin binding was observed for APTES
modied magnetite NPs (53.52%, Table 1) due to amine groups at a

Fig. 1. Percentage of bounded protein to Fe3O4, Fe3O4APTES and Fe3O4APTESGA at

different amounts of nanoparticles.

boiled for 25 min to cleavage of albumin from magnetic nanoparticles. Then, the samples were loaded onto an SDS-PAGE gel, run and
stained according to the standard protocol.

3. Results and discussion

Recent work in our laboratory has shown that with different buffer
solutions, immobilization capacity of albumin to bare and modied
magnetite NPs was different. The characterization of bare magnetite
NPs, APTES coated NPs and subsequently GA modied magnetite NPs
was examined by Fourier transform infrared spectroscopy (FTIR),
transmission electron microscopy (TEM), thermal gravimetric analysis (TGA) and magnetization measurement [21]. We now outline the

Fig. 2. SDS page gel image of BSA: 1 BSA; 2 BSA bounded to Fe3O4; 3 supernatant
from Fe3O4; 4 BSA; 5 BSA bounded to Fe3O4APTS; 6 supernatant from Fe3O4
APTES; 7 supernatant from BSA bounded to Fe3O4APTESGA; 8 BSA bounded to
Fe3O4APTESGA; 9 BSA.

Fig. 3. SEM images of immobilized BSA on (A) Fe3O4, (B) Fe3O4 + APTES and (C) Fe3O4 +
APTES + GA nanoparticles.

E. Maltas et al. / Materials Letters 65 (2011) 34993501

concentration of 30 mg/mL compared with bare and GA modied NPs

(Fig. 1).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) has been one of the commonly used methods for the
determination of protein molecular weight for over three decades
[22,23]. The combination of isoelectric focusing in two dimensional
gel (2D-gel) format has made SDS-PAGE a key tool for protein
research. The aim of this study is to improve a protein separation
method by using magnetic nanoparticles. Albumin immobilization in
this study is a kind of model system to show protein binding on the
surface of magnetite NPs via albumin composed of basic twenty
amino acids. These amino acids provide similar chemical properties to
all proteins which consist of various sequences and numbers of amino
acids, resulting in different types of proteins such as enzyme, receptor,
glycoprotein and lipoprotein with specic functions in cell. Albumin
binding onto NPs was visualized on SDS-PAGE gel by using electrophoresis technique. For this purpose, the particles were washed with
PBS buffer and boiled with Laemmli buffer for albumin cleavage and
loaded to SDS gel and run at 200 V for 1 h. The gel was stained with
commassie brilliant blue solution. It can be seen that albumin
bounded at different amounts onto bare and modied NPs depends
on visually band thickness in gel image (Fig. 2).
The morphology of the products was characterized by a Zeiss LS-10
eld emission SEM. Fig. 3 shows the SEM micrographs of immobilized
albumin on bare, APTES coated and GA modied magnetite NPs.
Magnied SEM images have clearly shown that albumin adsorbed
more on APTES coated magnetite NPs compared with bare and GA
modied magnetite NPs.
4. Conclusions
In this study, a facile and an effective method of preparation and
modication of magnetite nanoparticles with APTES and subsequently with GA in order to produce a superparamagnetic material for
attachment to biomolecules with the required properties is described.
Their use in protein immobilization was investigated using albumin as
a model protein. The maximum value of albumin immobilization
capacity was obtained with APTES modied magnetic NPs in phosphate buffer saline solution. Displaying immobilization of albumin to
modied magnetic NPs, the superparamagnetic albumin modied
Fe3O4 NPs are of signicance for magnetic applications in various
bioprocesses, biomedical devices and biomedicine.
Acknowledgement
We would like to thank the Research Foundation of Selcuk
University (BAP) for the nancial support of this work.
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