Colloids and Surfaces B: Biointerfaces 81 (2010) 600606

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Colloids and Surfaces B: Biointerfaces

j o u r n a l h o m e p a g e : w w w.e l s e v i e r.c o m / l o c a t e / c o l s u r f b

Surface plasmon resonance biosensor based on Fe3 O4 /Au nanocomposites

Jian Wang, Ying Sun, Liying Wang, Xiaonan Zhu, Hanqi Zhang, Daqian Song

College of Chemistry, Jilin University, Qianjin Street No. 2699, Changchun 130012, PR China

a r t i c l e

i n f o

Article history:
Received 29 March 2010
Received in revised form 16 July 2010
Accepted 4 August 2010
Available online 10 August 2010
Keywords:
Core/shell structure
Fe 3 O4 /Au nanocomposites
Surface plasmon resonance
(SPR) Human IgM

a b s t r a c t
In this paper, the core/shell Fe3 O4 /Au nanocomposites modied with 3-mercaptopropionic acid (MPA)
were prepared and applied in the surface plasmon resonance (SPR) biosensor. And the detailed investigation of Fe3 O4 /Au nanocomposites was separately performed by UVvis spectroscopy and transmission
electronic microscopy. As the magnetic property and exceptional optical properties, the Fe3 O4
/Au nanocomposites were used as the solid support for the goat anti-human IgM, which could be
immo- bilized on the surface of SPR biosensor chip by a magnetic pillar. This novel method of
immobilizing goat anti-human IgM simplied experimental procedures and facilitated the
regeneration of the sens- ing membrane. In addition, the different diameter of Fe3 O4 /Au
nanocomposites could be obtained with the different amount of MPA in the solution. And the effect of
Fe3 O4 /Au nanocomposites with different diameters on the sensitivity of SPR biosensor was also
explored. As a result, the SPR biosensor exhibits a satisfactory response for human IgM in the
concentration range of 0.3020.00 g ml1 and the increasing nanocomposite diameter is in favor of the
sensitivity enhancement of SPR biosensor.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Recently, magnetic nanomaterials have proved promising
appli- cations in various elds, such as cancer diagnosis [1], drug
delivery [2], biological separation and detection [3]. Due to the
super param- agnetic property and high ratio of surface area to
volume, magnetic nanoparticles could disperse in blood and
direct to a specic tar- get with an external magnetic eld,
which has
great signicance for medical development [4].
However, the magnetic nanoparticles are easy to aggregate and
lose magnetic properties when applied in
complex
environmental system. And due to the lack of func- tional
groups, the magnetic nanoparticles are difcult to couple with
biomolecules directly, which limits the application of mag- netic
nanomaterials. These disadvantages could be improved by
coating magnetic nanomaterials with metal or metal oxide
shell that could protect magnetic nanomaterials and provide a
platform for
surface modication. Gu
et al.
deposited
amorphous CdS on the surface of FePt nanoparticles to form a
metastable core/shell structure, in which the CdS transformed
into a crystalline state upon heating [5]. Xu et al. presented a
facile synthesis of Au- and Ag-coated magnetic nanoparticles
with controlled plasmonic and magnetic properties, and proved
that the multifunctional nanopar- ticles have great potentials for
nanoparticle-based diagnostic and therapeutic application [6]. By
virtue of its exceptional optical prop- erties, good stability and
facility of surface functionality, Au was

Corresponding author. Tel.: +86 431 85168399; fax: +86 431 85112355.
E-mail address: songdq@[Link] (D. Song).

considered as an excellent material to coat magnetic

nanomaterials [7]. Due to the protection of Au shell for magnetic
core from oxida- tion, the stabilization of Fe3 O4 /Au
nanocomposites (Fe3 O4 /AuNPs) was enhanced obviously. And the
Au shell also
provides the mag- netic core with surface
plasmonic
properties, which makes the nanocomposites
extremely interesting for optical and biomedical applications. In
addition, the Au shell can be readily functionalized with thiol
groups, such as 3-mercaptopropionic acid (MPA). The Au
nanoparticles modied with MPA have been widely used to bind
to proteins [8]. However, the Au nanoparticles have a shortcoming.
When Au nanoparticles were used in the separation of
proteins from a sample solution, centrifugation is generally
required, which is tedious and time-consuming. The Fe3 O4 /Au
nanocomposites, in which core is Fe3 O4 and shell is Au, overcome
this limit. Due to the magnetic property of Fe3 O4 , the Fe3 O4 /Au
nanocomposites coupled with proteins can be separated from
the sample solution easily and then immobilized on the
sensing lm by a magnetic pillar. In conclusion, the Fe3 O4 /Au
nanocomposites combine the advan- tages of Fe3 O4 and Au
nanoparticles, and broaden the application of magnetic
nanomaterials.
The preparation and application of Fe3 O4 /Au nanocomposites
have been synthesized in several ways [911]. In most of the
papers, Fe3 O4 /Au nanocomposites were synthesized and then
the alkanethiolate was self-assembled on Au shell by immersing
the nanocomposites overnight. In this paper, Fe3 O4 /Au
nanocompos- ites modied with MPA were synthesized in one
step. The MPA could be adsorbed on the surface of Au shell to
control the growth of Au shell. By altering the amount of MPA, the
Fe3 O4 /Au nanocom- posites with different diameters were
obtained [12].

0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/[Link].2010.08.007

J. Wang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 600

606

Surface plasmon resonance (SPR)

biosensor could
be
applied for label-free detection of biomolecule at the metal layer
surface, such as Au or Ag [13]. Owing to many advantages
such as real- time, label-free and non-destructive detection, the
SPR biosensor has been applied for studying various interaction
partners, includ- ing proteins [14,15], nucleic acids [16], hormone
[17], cells [18] and especially antigens [19]. The way of
immobilizing biomolecules on the surface of biosensor chip is
crucial in enhancing the sensitivity of SPR biosensor and
simplifying the immunoassay procedures. The biomolecules have
been immobilized on sensor surfaces by vari- ous techniques,
such as sandwich assays [20,21], the interaction between avidin
and biotin [22], the label of inorganic nanoparticles [13] and the
chemical bond [23]. In most of these methods, proteins were
immobilized on the surface of biosensor by chemical method with
the binding of many chemical reagents, which is complex in the
immunoassay. The magnetic nanoparticles have great advan- tages
in the immobilization of proteins in the SPR biosensor [24].
Compared to traditional antibody immobilization on the sensing
lm, there is no covalent link between the biosensor chip surface
and the antibody. With the magnetic property and good
biocompat- ibility, the Fe3 O4 /Au nanocomposites could be one of
the excellent candidates as carrier for protein immobilization. The
conjugates of Fe3 O4 /Au nanocomposites coupled with antibody
can be immobi- lized on the surface of the biosensor by a
magnetic pillar easily. And with the exceptional optical
properties, the Fe3 O4 /Au nanocompos- ites can play an important
role in the sensitivity enhancement of the biosensor [25].
IgM is a large (950 kDa) pentamer which consists of ve IgGlike monomers connected by a polypeptide chain rich in disulde
bonds and characterized by the presence of a disulde-rich J
chain. As a consequence of its pentameric structure, IgM
antibodies are good in clumping microorganisms for eventual
elimination from the body, and their presence in sera is a
valuable indicator of running infections [26]. Therefore, rapid,
accurate and sensitive analytical method for detection of IgM is
important in the disease testing.
In this paper, the core/shell Fe3 O4 /Au nanocomposites
modi- ed with MPA were applied in the SPR biosensor as solid
support for the goat anti-human IgM. The goat anti-human IgM
was bound to Fe3 O4 /Au nanocomposites by MPA and the
conjugates were immobilized on the surface of biosensor chip by
magnetic pillar. Then human IgM was detected by the novel SPR
biosensor. Inter- estingly, with the increasing amount of MPA, the
thickness of Au shell deposited on the Fe3 O4 nanoparticles
decreases. By this way, the different diameter Fe3 O4 /Au
nanocomposites
were
successfully
obtained
and
the
characteristics of nanocomposites were investi- gated by UVvis
and transmission electronic microscopy. And the effect of
different diameter Fe3 O4 /Au nanocomposites on the sen- sitivity
of SPR biosensor was also explored.
2. Materials and methods
2.1. Reagents
Human
IgM
and
goat
anti-human
IgM
(antihuman IgM)
were purchased from Beijing Biosynthesis
Biotechnol- ogy
Company.
Tetramethylammonium
hydroxide (TMAOH) and 3-mercaptopropionic acid
(MPA)
were
purchased
from Jkchemical.
N-hydroxysuccinimide
(NHS) and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide
(EDC) were purchased from
Pierce. Hydrogen tetrachloroauratehydrate (HAuCl4 3H2 O)
was
purchased from Acrosl. Ferric chloride hexahydrate (FeCl3 6H2 O),
ferrous sulfate tetrahydrate (FeSO4 4H2 O) and all other chemicals
were of analytical reagent grade.
Human IgM and anti-human IgM were stored at 20 C, and
all other biological reagents were stored at 4 C. PBS buffer

(0.01 mol l1 , pH 7.4) was used as running buffer.

89

90

2.2. SPR equipment

J. Wang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 600

606
of EDC for

In this paper, the wavelength-modulation SPR biosensor

was installed in our laboratory. It is working with Kretschmann
con- guration to achieve the resonant condition by attenuated
total reection (ATR) in a prism [27]. The prism was vacuumdeposited with approximately 2 nm adhesive layer of Cr and 50
nm Au lm followed, which formed the biosensor chip. The light
emitted from a lamp passes through the optical prism and excites
surface plasmon at the interface between the biosensor chip
surface and analytes. The output light is guided into the
optical ber and then enters the spectrophotometer (Ocean
Optics, Inc., USA). A 2048 element charge-coupled device (CCD)
linear array detector was used. A
100
l ow cell was used for the reaction and magnetic pillar
was immobilized under the optical prism. The diameter and
height of the magnetic pillar are 25 and 12 mm, respectively. The
sensor sur- face was at a distance of 13 mm from the magnetic
pillar and in the magnetic eld of 900 G that detected with Tesla
meter.
2.3. Assay procedure
2.3.1.
Preparation of Fe3 O4 /Au
nanoparticles
The Fe3 O4
nanoparticles were prepared by chemical
copre- cipitation methods as reported [10]. A stock solution
containing FeCl3 6H2 O (2.6 g), FeSO4 4H2 O (1.0 g) and 12 mol l1
HCl
(0.425 ml) was prepared at rst. Then the stock solution
was
dropped into 125 ml NaOH solution (1.5 mol l1 ) under
nitrogen gas protection and vigorous stirring at 80 C. After the
reaction, the
obtained Fe3 O4
nanoparticles were washed with deionized
water four times and the precipitate was dispersed in 100 ml
TMAOH
(0.125 mol l1 ) solution for further
use.
The
Fe3 O4 /Au
nanocomposites were prepared as
followed: sodium citrate (0.024 g) and a certain amount of MPA
were dis- solved in 100 ml water. The MPA solution should be
adjusted to the neutral by adding NaOH solution. Under
nitrogen gas pro- tection and vigorous stirring, the solution
was heated up to boil and 0.24 ml of Fe3 O4 nanoparticles was
added quickly. After boil- ing for 5 min, 0.012 mmol of HAuCl4
was added immediately into the solution. The color of the
solution changed from black to red, which suggests that the Au
shell has been performed on the Fe3 O4 nanoparticles successfully.
After the reaction, the obtained solution was precipitated by a
magnetic pillar, and the supernatant solution was removed. By
this way, the pure Au nanoparticles formed can be removed.
Then the solution was washed with deionized water
and 5 ml of HCl (2 mol l1 ) was added to remove the
uncoated
Fe3 O4 . After
the nanocomposites were washed with
deionized water, the Fe3 O4 /Au nanocomposites were
precipitated by a mag- netic pillar and the supernatant solution
was removed. 100 ml of
NaOH (0.05 mol l1 ) was added to the precipitation and the
colloid solution was obtained again. When the amounts of MPA are
0.012, 0.009, 0.006, 0.003 and 0 mmol, respectively, the Fe3 O4 /Au
nanocomposites show different colors such as orange, light red,
red, deep red and purple. The UVvis absorption spectra and
transmis- sion electronic microscopy (TEM) images were used to
characterize the nanocomposites.
2.3.2. Preparation of Fe3 O4 /Au nanocomposites coupled
with antibody
At rst, the carboxyl group of MPA on the Fe3 O4 /Au
nanocom- posites was activated with NHS under the catalysis

20 min. The activated carboxyl can react to amino group of protein, while the un-activated carboxyl is hard to bind amino
group at room temperature. Then 500 l of Fe3 O4 /Au colloid
solution were precipitated by a magnetic pillar and the
precipitation was washed with PBS buffer solution twice. A
certain concentration of goat anti-human IgM (1 ml) dissolved
with PBS buffer solution was

group of antigen and lead to the non-specic binding of antigen.

So ethanolamine hydrochloride was used to bind to the un-reacted
carboxyl groups, and the non-specic binding sites on the
biosensor surface can be blocked. After 10 min, PBS buffer was
injected to the ow cell and the Fe3 O4 /AuNPs/anti-human IgM
sensing membrane was formed.

Fig. 1. Schematic diagram of the preparation of Fe3 O4 /Au

coupled with antibody.

nanocomposites

added into the Fe3 O4 /Au colloid solution. After 5 h, the

conjugates of Fe3 O4 /Au nanocomposites coupled with antihuman IgM were obtained. Then the solution was precipitated by
a magnetic pillar, and the supernatant was poured away to
remove the un-reacted antibody. 1 ml PBS solution was added
into the precipitation. The experimental procedures of preparing
the conjugates of Fe3 O4 /Au nanocomposites coupled with
anti-human IgM are shown in Fig. 1.
2.3.3. Preparation of Fe3 O4 /AuNPs/anti-human IgM
sensing membrane
The conjugates of Fe3 O4 /Au nanocomposites coupled with
anti- human IgM were introduced into the ow cell equipped
with a magnetic pillar under the biosensor chip. The conjugates
can be immobilized on the biosensor chip surface by a magnetic
pillar, which greatly simplify the procedures of anti-human IgM
immobi- lization. The immobilization of conjugates on the
biosensor chip surface could produce change in refractive index
prole that is observed as a shift in the SPR resonant
wavelength. After 2 h, PBS buffer was injected to the ow cell to
stabilize the baseline and
then 1 mol l1 ethanolamine hydrochloride (pH 8.0) was injected
into the ow cell. As mentioned in Section 2.3.2, after the
anti- body added, the amino group of protein can react to
activated carboxyl group of MPA. While there may be some unreacted bind- ing sites of activated carboxyl group, which can
react to the amino

Fig.

2. TEM images of Fe3 O4 /Au

O4 /Au

2.3.4. Preparation of MPA/anti-human IgM sensing membrane

As a contrast, the MPA/anti-human IgM sensing membrane was
also constructed. PBS buffer was used as the baseline solution,
and
10 mmol l1 MPA was injected into the ow cell. After 2 h,
carboxyl group of MPA was activated 1
with NHS under the
catalysis
of EDC
20 min. anti-human
25.0
g ml IgManti-human
IgM was
injected. And
thefor
redundant
can be removed
by
1
PBS. 1 mol l
ethanolamine hydrochloride (pH 8.0) was used to block the nonspecic binding sites on the biosensor surface. After 10 min,
the MPA/anti-human IgM sensing membrane was formed.
2.3.5. Immunoassay
At room temperature, human IgM at different concentrations
were separately injected into the ow cell of the SPR
biosensor system. The shifts of resonant wavelength due to
antibodyantigen immunoreactions were measured by SPR
biosensor. After 40 min, PBS was injected into the ow cell. Then
another sample solution was injected into the ow cell.
To evaluate the selectivity of the proposed method, human
serum albumin and bovine serum albumin were determined by
the procedure mentioned above.
2.3.6. Regeneration
After
a series of human IgM
immunoassays were
performed, the sensing membrane of the SPR biosensor was
regenerated. The magnetic pillar was taken away from the
prism and water was introduced into the ow cell to wash away
the Fe3 O4 /Au nanocom- posite coupled with anti-human IgM.

nanocomposites at different diameters. (a) 8 nm, (b) 12 nm, (c) 17 nm, (d) 20 nm, (e) 30 nm and (f) a typical 20 nm Fe3

nanocomposite.

3. Results and discussion

3.1. Preparation and characterization of Fe3 O4
/Au nanocomposites
Fe3 O4
nanoparticles were rst prepared as seeding
materials, and then Au was deposited on the Fe3 O4 nanoparticles
in the pres- ence of MPA to form the core/shell Fe3 O4 /Au
nanocomposites. The MPA plays an important role in the synthesis
of Fe3 O4 /Au nanocom- posites with different diameter. As shown
in Fig. 1, when the Au shell began to generate, the MPA can be
adsorbed on the Au sur- face to restrain Au shell from growing on
the Fe3 O4 nanoparticles. So the decrease of MPA concentration
in the solution results in the increase of thickness of Au shell
on the Fe3 O4 nanoparticles. Thus, the Fe3 O4 /Au nanocomposites
with different diameter were obtained by adding different
amounts of MPA. Fig. 2 shows the transmission electron
micrographs of Fe3 O4 /Au
nanocomposites with different
diameter. When the amount of MPA in the solution is 0.012,
0.009, 0.006, 0.003 and 0 mmol, the diameter of Fe3 O4 /Au
nanocomposites is
about 8,
12,
17,
20
and 30 nm,
respectively. Fig.
2f
shows the transmission
electron
micrographs of a typi- cal 20 nm Fe3 O4 /Au nanocomposite. The
TEM images showed that there are some smaller and lighter
particles, which signify bare Fe3 O4 nanoparticles. The Au shell
exhibits topographical rough- ness on the nanometer scale. It can
be hypothesized that the Au shell grows by nucleating from
small nanoparticles on the Fe3 O4 nanoparticles surface and then
develops the shell structure [28].
As the thickness of Au shell increases, the Fe3 O4 /Au
nanocom- posites show gradually light colors as orange, light red,
red, deep red and purple. Fig. 3 shows UVvis spectra of Fe3 O4
nanoparti- cles and Fe3 O4 /Au nanocomposites with different
diameter. The UVvis spectra of Fe3 O4
nanoparticles show
that no absorption appears. When the amount of MPA added into
the solution is 0.006,
0.003
and 0 mmol, Fe3 O4 /Au
nanocomposites show
maximum absorption at 518, 522 and 546 nm, respectively. The
maximum absorption wavelength is red shifted due to
the
increasing diam- eter of Fe3 O4 /Au nanocomposites. When the
amount of MPA is
0.009 and 0.012 mmol, the Au shell deposited is so thin that the
absorption peaks of Fe3 O4 /Au nanocomposites are not
evident.
3.2. Preparation of SPR biosensor based on Fe3 O4
/Au nanocomposites coupled with anti-human IgM
In order to select the optimal concentration of anti-human
IgM
to conduct the next experiment, Fe3 O4 /Au nanocomposites
coupled

Fig. 3. UVvis absorption spectra of Fe3 O4 nanoparticles (a) and Fe3 O4 /Au
nanocom- posites at different diameters: (b) 8 nm, (c) 12 nm, (d) 17 nm, (e)
20 nm and (f)
30 nm.

30 nm, the shifts of resonant wavelength are 0.5, 1.0, 2.3, 3.5 and
5.2 nm, respectively. The fact is not surprising that the increasing
nanocomposites diameter leads to
the large red-shift of
resonant wavelength. With the increase

Fig. 4. The kinetic adsorption curves of Fe3 O4 /Au nanocomposites coupled

with anti-human IgM at different concentrations. Error bars = S.D. and n = 3.

with anti-human IgM at different concentrations were studied.

The Fe3 O4 /Au nanocomposites were prepared with 0.003 mmol of
MPA, and the conjugates of Fe3 O4 /Au nanocomposites coupled
with anti- human IgM at different concentrations were injected
into the ow cell and the conjugates can be immobilized on the
biosensor chip surface by the magnetic pillar.
Fig. 4 shows the kinetic adsorption curves of immobilizing
Fe3 O4 /Au nanocomposites with anti-human IgM at different
con- centrations. The resonant wavelength shifts signicantly at
rst and tends to be stable in 100 min, which indicates that the
immobiliza- tion of conjugates on the biosensor chip surface
was completed. Due
to
the increase of anti-human IgM
concentration, more anti- bodies were immobilized on the
biosensor chip surface, which
resulted in the resonant wavelength moving to the longer wavelength. When the concentrations of anti-human IgM are 15.0,
20.0,
25.0,
30.0
and 35.0
g ml1 , the shifts of resonant
wavelength
are 10.7, 14.6, 23.9, 23.9 and 24.4 nm, respectively (Fig. 4).
With the increase of the concentration of anti-human IgM, the
amount of antibody immobilized increases at rst and reaches
saturation gradually, which results in no change of the resonant
wavelength. It means that the immobilization of anti-human IgM
can reach saturation when the concentration of anti-human IgM is 25.0 g ml1
.
3.3.
Effect of
nanocomposites

different

sizes

of

Fe3 O4 /Au

In this paper, the effect of different diameters of Fe3 O4

/Au nanocomposites on the sensitivity enhancement of SPR
biosensor was also studied. The conjugates of Fe3 O4 /Au
nanocomposites cou- pled with anti-human IgM were separately
injected into the ow cell. Fig. 5 shows the kinetic adsorption
curves of the conjugates. It can be seen from Fig. 5, when the
diameter of nanocomposite is 8, 12, 17 and 20 nm, the shifts of
resonant wavelength are 16.3,
17.3, 20.3 and 23.9 nm, respectively. When the 30 nm of Fe3 O4
/Au nanocomposites coupled with anti-human IgM without MPA
were immobilized on the biosensor chip surface, the
corresponding max- imum shift of the resonant wavelength is
only 15.8 nm. In order to identify the amount of anti-human IgM
immobilized, the bare Fe3 O4 /Au nanocomposites were also
injected. The shifts of reso- nant wavelength tend to be stable in
15 min. When the diameter of nanocomposite is 8, 12, 17, 20 and

Fig.
5.
The kinetic adsorption
curves of conjugates of
Fe3 O4 /Au
nanocomposites coupled with anti-human IgM at different diameters. Error
bars =
S.D. and n = 3.

of Fe3 O4 /Au nanocomposites diameter, the thickness of the

sens- ing membrane of SPR biosensor increases. SPR biosensor is
highly sensitive to the minimal change of sensing membrane,
which can lead to the shift of the SPR resonant wavelength [29].
As a result, it can be expected that the conjugates with larger
nanocomposite immobilized on the sensing membrane, the
longer shift of reso- nant wavelength in the SPR curve obtained.
In addition, the amount of proteins immobilized on the surface
of biosensor also has an important inuence on the shift of the
SPR resonant wavelength. The proteins can couple with Fe3 O4
/Au nanocomposites through MPA or electrostatic adherence.
When there is MPA attached on the nanocomposites, the MPA can
link with proteins through chem- ical bond, which is rm and
prior to react. When there is no MPA, the Fe3 O4 /Au
nanocomposites can adsorb proteins through elec- trostatic
adherence [20], which is much weaker compared with the
chemical bond between the protein and MPA. So the amount of
proteins immobilized with 30 nm Fe3 O4 /Au nanocomposites is
less than that immobilized with other nanocomposites. As a
result, the immobilization of 20 nm Fe3 O4 /Au nanocomposites
coupled with anti-human IgM leads to the largest red-shift of
resonant wave- length.
Fig. 6 shows the relationship between the concentration of
human IgM and the shifts of the resonant wavelength (
eq
). When the Fe3 O4 /Au nanocomposites (8, 12, 17 and 20 nm)
were immobilized on the biosensor chip surface, the biosensor
shows a satisfactory response to
human IgM in the
concentration range
of 0.3020.00 g ml1 (Fig. 6). And with the increase of Fe3 O4
/Au
nanocomposites diameter, the maximum shift of resonant wavelength due to antibodyantigen immunoreaction increases. The
SPR biosensor shows highest sensitivity when the 20 nm Fe3 O4
/Au nanocomposites coupled with anti-human IgM was
immobilized on the biosensor chip surface. As the diameter of
nanocompos- ite immobilized in the SPR biosensor increases,
the SPR curve can be expected to shift to longer wavelength,
which has been
proved to be benecial to enhance the sensitivity of SPR
biosensor [30]. It can also be seen from Fig. 6 that the lowest
concentra- tion that could be detected obtained by the biosensor
with 30 nm
nanocomposites is 1.25
g ml1 , which is 4 times higher than
that
obtained by the biosensor with 20 nm nanocomposites. As mentioned before, the amount of anti-human IgM immobilized
with

30 nm Fe3 O4 /Au nanocomposites without MPA is less than

that immobilized with other nanocomposites, which results in
the low sensitivity of SPR biosensor. These results demonstrate
that both the diameter of Fe3 O4 /Au nanocomposites and the
amount of pro-

Fig. 6. The relationship between the concentration of human IgM and the shifts
of the resonant wavelength (
eq ) with biosensor based on Fe3 O4 /Au
nanocomposites
at different diameter. Error bars = S.D. and n =
3.

teins immobilized on the sensing membrane play important

roles in enhancing sensitivity of SPR biosensor. As a result, the
20 nm Fe3 O4 /Au nanocomposite that prepared with 0.003 mmol
MPA is the best candidate to immobilize proteins on the
surface of SPR biosensor.

3.4.
IgM

Determination

of human

In order to study the effect of Fe3 O4 /Au nanocomposites in

enhancing biosensor sensitivity, the different biosensors based on
Fe3 O4 /AuNPs/anti-human IgM sensing membrane and MPA/antihuman IgM sensing membrane were constructed. The sensitivity
of a biosensor chip greatly depends on the surface density
of the antibodies immobilized, so it is important to select the
opti- mum concentration of antibody. In theory, as the larger
surface area of Fe3 O4 /Au nanoparticles, the needed amount of
anti-human IgM immobilized by Fe3 O4 /Au nanoparticles is
more than that immobilized by MPA. In order to provide
enough amount for MPA/anti-human IgM sensing membrane,
the same concentration of anti-human IgM was used, which can
compare the sensitivity
of two different biosensors effectively. So the 25.0
g ml1
antihuman IgM was used to prepare the MPA/anti-human IgM
sensing membrane.
Fig. 7 shows the kinetic adsorption curves of conjugates on the
biosensor and anti-human IgM on the MPA monolayer. The
amount of anti-human
IgM immobilized by Fe3 O4 /Au
nanocomposites is signicantly more than that by MPA. Fig. 8
shows the relationship between the concentration of human IgM
and the shifts of resonant wavelength with the biosensor based
on Fe3 O4 /Au nanocompos- ites and MPA. It can be seen from
Fig. 8a that the SPR biosensor based on Fe3 O4 /AuNPs/antihuman IgM sensing membrane shows a good response to
human IgM in the concentration range of
0.3020.00 g ml1 . The minimum shift of resonant wavelength
is
0.5 nm at 0.30 g ml1 and the maximum shift of resonant wavelength is 5.3 nm at 20.00 g ml1 . The SPR biosensor based
on
MPA/anti-human
sensing
membrane
shows
good response
to 1human IgM inIgMthe
concentration
range
of a 1.2520.00
g
ml
(Fig. 8b). The minimum shift of resonant wavelength is 0.5 nm at
1.25
g ml1 and the maximum shift of resonant wavelength
is
4.1 nm at 20.00 g ml1
.

Fig. 9. The SPR response curve of a whole cycle.

3.5. Regeneration of biosensor

Fig. 7. The kinetic adsorption curves of conjugates of Fe3 O4 /Au
nanocomposites coupled with anti-human IgM (a) and anti-human IgM (b).

These experimental results indicated that the biosensor

used Fe3 O4 /Au nanocomposites is more sensitive. The exceptional
elec- tromagnetic effect of the Au surface plasmon is mostly
responsible for improving the biosensor sensitivity. Meanwhile,
the Au shell has its own localized surface plasmon due to the
collective oscilla- tion of its conduction electrons [31], which
would create a stronger local electromagnetic eld near the Fe3 O4
/Au nanocomposites. The
coupling between the local
electromagnetic eld of the nanocom- posites and Au lm could
inuence the plasmon mode. By this way, the SPR response
propagation at the surface of Au lm can be increased and the
sensitivity of SPR biosensor is enhanced. On the other hand,
when the Fe3 O4 /Au nanocomposites were immo- bilized on the
biosensor chip surface, the thickness of the sensing membrane
increases and the resonant wavelength moves to longer
wavelength, which results in the sensitivity enhancement of the
wavelength-modulation SPR biosensor. In conclusion, due to magnetic property and plasmonic property, Fe3 O4 /Au nanocomposites
can simplify the immunoassay and improve the sensitivity of SPR
biosensor.

Fig. 8. The relationship between the concentration of human IgM and the shifts
of resonant wavelength with the biosensor based on Fe3 O4 /Au nanocomposites (a)
and
MPA (b). Error bars = S.D. and n = 3.

The regeneration of biosensor is also an important parameter to

affect its practical application. The human IgM at low concentration was detected rst. Due to a large amount of ions in the PBS
buffer, the antigen could be washed by impulsive injection of PBS
buffer [32]. When the concentration is low, the antigen is easy to
be removed. The antigen at high concentration, such as 20 g ml1
,
cannot be removed completely (Fig. 9). As the human IgM
was detected from low concentration to high concentration, it has
little effect on the experimental results.
After the magnetic pillar was taken away from the prism,
water was introduced into the ow cell, and the conjugates could
be dis- sociated from the biosensor chip surface. The SPR
response curve of the whole cycle is shown in Fig. 9, including the
immobilization of conjugates, the binding of antigen at
different concentrations and the regeneration of the biosensor.
As shown in Fig. 9, the shift of the resonant wavelength of
conjugates immobilization is about 24 nm, while the shift of
the resonant wavelength of conjugates dissociation is about 20
nm. As the biosensor chip sur- face is not absolutely smooth,
some Fe3 O4 /Au nanocomposites
may be immobilized in these pits of surface, which is hard
to
remove. The dissociation rate of biosensor chip can come up
to
83%, which can meet the demand of regeneration. And the
biosen- sor chip can be used repetitive. Traditionally, strong acid
or alkali solutions, such as NaOH or HCl solution, were used as
regenera- tion solution to inject the ow cell. After 40 min, the
antibodies can be dissociated from the biosensor chip surface.
However, the NaOH solution would damage the Au lm and
reduce the service life of biosensor chip. The use of Fe3 O4 /Au
nanocomposites can overcome this limit. In conclusion, the Fe3 O4
/Au nanocomposites are useful to simplify the sensing membrane
regeneration proce- dures.
The evaluation of the selective binding of anti-human IgM was
also performed by determining bovine serum albumin and
human
serum albumin in the concentration range of 5.0020.00 g ml1
.
The shifts of the resonant wavelength of bovine serum
1 albumin
were
not and human serum albumin at 5.0010.00 g ml
observable (Fig. 10). When the concentration of bovine
serum
albumin
and human serum albumin is 20
g ml1 , the
resonant wavelength shifted to 0.5 nm.
There may be gaps
between the conjugates immobilized on the biosensor chip. As
a result, the proteins have chance to be immobilized in the
gaps, which would induce the non-special adsorption. The shift
of the resonant wavelength of 20.00
g ml1 human IgM is 5.3 nm,

which
is
more obvious than that of non-special adsorption.
These experimental results indicated the selective binding of
human IgG.

References
[1]

Fig. 10. The shift of resonant wavelength measured by SPR biosensor for
different antigens at different concentrations. Error bar = S.D. and n = 3.

4. Conclusions
The
Fe3 O4 /Au
nanocomposites modied with MPA,
owning good biocompatible and exceptional optical properties,
are ideal carriers to bind proteins. And the nanocomposites
coupled with proteins can be immobilized on the biosensor chip
surface by mag- netic pillar easily, which simplies the procedures
of immobilizing proteins and
regenerating the sensing
membrane. Furthermore, the Fe3 O4 /Au nanocomposites can
play an important role in the sensitivity enhancement of SPR
biosensor. In this paper, Fe3 O4 /Au nanocomposites coupled with
anti-human IgM were immobilized on the surface of SPR
biosensor by magnetic pillar to detect human IgM. The
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the sensitivity enhancement of SPR biosensor. In addition, the
amount of proteins immobilized on the sensing membrane also
plays important roles in enhancing the sensitivity of SPR
biosensor.
Acknowledgements
This
work was supported by National Natural Science
Foun- dation of China (No. 20727003) and Science and
Technology Developing Foundation of Jilin Province (No.
20070551).

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