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This article is part of the
Surface Enhanced Raman
Spectroscopy web themed issue
Guest editors: Professors Duncan Graham,
Zhongqun Tian and Richard Van Duyne
All articles in this issue will be gathered together online at www.rsc.org/sers.
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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 4225–4227 4225
Cite this: Chem. Commun., 2011, 47, 4225–4227
Magnetic separation and immunoassay of multi-antigen based on surface
enhanced Raman spectroscopywzShuai Chen, Yaxian Yuan, Jianlin Yao,* Sanyang Han and Renao Gu*
Received 1st December 2010, Accepted 9th February 2011
DOI: 10.1039/c0cc05321j
A novel and highly sensitive immunoassay method based on
surface enhanced Raman spectroscopy (SERS) and magnetic
particles has been developed. This method exhibits great
potential application in bio-separation and immunoassay.
Recently, magnetic nanoparticles have attracted considerable
interest due to their widespread applications in various areas
such as ferrofluids, medical imaging, targeted drug delivery,
environmental remediation, and catalysis.1–3 In particular,
gold-coated magnetic core–shell nanoparticles have shown
promise as magnetic biomaterials for bioseparation and
immunoassay because of their well established surface
chemistry and biocompatibility.4–6 Moreover, the gold shell
exhibited tuned optical properties and well established surface
modification and fuctionalization. For example, the gold shell
with appropriate thickness could contribute giant surface-
enhanced Raman scattering (SERS) activity. Therefore, the
preparation of core–shell magnetic materials with the rich
surface chemistry and optical properties of the gold shell
together with the magnetic properties in controllable and
convenient ways still is a challenge.
So far, the SERS technique has proved to be a very effective
and general tool because of its non-destructive, ultrasensitive
characterization up to single molecular level, high selectivity,
and fluorescence-quenching properties.7,8 Another distinct
advantage, especially because biological studies are becoming
increasingly important, is the suitability of this technique for
analysis performed on molecules in an aqueous environment
due to the extremely weak SERS signal from water.9,10 Very
recently, SERS was extended widely as general tool due to the
innovative method of shell-isolated nanoparticle-enhanced
Raman spectroscopy (SHINERS).11 Immunoassay is a common
and useful means of biochemical analysis. The strong specific
binding of an antibody to its antigen has been widely exploited
in biochemical studies, clinical diagnostics, sensor design,
and environmental monitoring. In the past, many different
approaches have been developed for a direct measurement of
antibody–antigen binding. SERS immunoassay, as an indirect
approach, has been deeply investigated between qualitative
and quantitative.12–14 Mirkin and his coworkers reported two
methods for the detection on DNA and RNA at a high
sensitivity based on the SERS and magnetic composition.15,16
In qualitative aspects, the SERS immunoassay technique
offers high sensitivity, showing the ability to detect antigens
in the range of 100 fg/ml to 1 fg/ml.5,17 Typically, this
technique often uses a complex protocol of a standard
sandwich immunoassay structure. In our previous studies,
two antigens were effectively separated by the magnetic
Fe2O3/Au core/shell nanoparticles and rapid detection was
performed based on sandwich assembly for SERS immuno-
assay.5 Here, we report a direct immunoassay based on the
magnetic nanoparticles without requiring the assembly of
sandwich structure onto a solid surface. The separation
efficiency, the sensitivity and the specificity for the separation
and detection were evaluated by SERS.
The SERS-based immunoassay was carried out according to
a new method called magnetic separation immunoassay, as
shown in Scheme 1. The immuno-g-Fe2O3/Au, MBA-labeled
immunogold, and antigens were mixed and incubated in a little
weighing bottle. The corresponding target antigens would
link them with three structures (Scheme 1) due to the fact
that antibody molecules interact highly specifically with corres-
ponding antigens. However, immuno-g-Fe2O3/Au linked with
MBA-labeled immunogold (second structure) and a little of
the third structure with immuno-g-Fe2O3/Au were sedimented
via application of an external magnetic field and only the
second structure showed mainly two peaks at about 1081 and
1594 cm�1 in SERS spectra, which correspond to n8a and
n12 (vibrational modes) aromatic ring vibrations of MBA,
respectively. In the target antigen solutions, the second
structure was also mainly observed from the TEM images
(see supporting information). The test solutions were made by
serial dilution of a 1 mg/ml rabbit IgG standard to cover the
range from 10 pg/ml to 0.1 fg/ml and detected by magnetic
separation immunoassay. Fig. 1 presents a set of SERS spectra
of the sediment from the solution with different concentrations
of target antigen together with the calibration curve. One
could find that the detection limit of rabbit antigen was down
to 0.1–1 fg/ml. This was higher than the SERS immunoassay
based on the assembly of sandwich structure.12,14,15 This might
be due to enormous SERS ‘‘hot spots’’ emerging among Au
Department of Chemistry, Soochow University, No. 199 Renai Road,Suzhou, China. E-mail: jlyao@suda.edu.cn, ragu@suda.edu.cn;Fax: +86 512 65880089; Tel: +86 512 65880359w This article is part of a ChemComm web-based themed issue onSurface Enhanced Raman Spectroscopy.z Electronic supplementary information (ESI) available. See DOI:10.1039/c0cc05321j
ChemComm Dynamic Article Links
www.rsc.org/chemcomm COMMUNICATION
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4226 Chem. Commun., 2011, 47, 4225–4227 This journal is c The Royal Society of Chemistry 2011
and g-Fe2O3/Au nanoparticles aggregates formed on the edges
between the metal layers and also between the junctions of
metal nanoparticles in the metal layer.18,19 This dramatically
enhances the SERS signal produced by MBA marker molecules,
thus improving the detection limit of SERS. As a comparison,
immuno-g-Fe2O3/Au and MBA-labeled immunogold capped
with goat anti-rabbit IgG were added into the solution with
the rabbit IgG or mouse IgG respectively after the same
magnetic enrichment protocol; the SER spectra from the
sedimented nanoparticle solution are presented in Fig. 2. Very
strong SERS signals of the marker were detected from the
sediment extracted from the former case, while no obvious
peaks were observed from the latter case with mouse IgG. It
was mainly due to the high specificity between corresponding
antibody–antigen pairs. Therefore, one can conclude that the
present magnetic separation immunoassay exhibited high
sensitivity and specificity without the requirement for the
complicated assembly procedure.
In order to verify the separation capability, the immuno-
g-Fe2O3/Au nanoparticles capped with goat anti-rabbit
IgG were immersed in the antigen solutions containing two
components of rabbit IgG and mouse IgG with the same
concentration. After being incubated at room temperature, the
corresponding specific antigen (rabbit IgG) was immobilized
onto the immuno-g-Fe2O3/Au particles and it induced the
aggregation of immuno-g-Fe2O3/Au particles. This provided
an effective means for the separation of the rabbit IgG via
application of a magnetic field. The upper solution was
detected by the magnetic immunoassay protocol, after the
separation no obvious change in the intensity of the SERS
signal was observed for the mouse IgG case, and the SERS
signal disappeared for the rabbit IgG case. This indicates that
the rabbit IgG was sedimented together with the immuno-
g-Fe2O3/Au nanoparticles, i.e. was removed from the solution,
while the mouse IgG remained in the solution and did not
affect the bioseparation protocol.
In our previous work, antigen mixtures of mouse IgG
and rabbit IgG (1 mg/ml) have been successfully separated.
However, the separation on the lower concentrations of antigen
mixtures is highly desired. In order to verify whether antigen
mixtures with lower concentrations can be separated by this
method, various concentrations of antigen mixtures (mouse
IgG and rabbit IgG) from 1 ng/ml to 0.01 pg/ml were
separated by g-Fe2O3/Au nanoparticles. By applying the same
protocol for the separation and immunoassay, the separation
limitation was monitored.
Fig. 3 presents the SER spectra contributed by the sedimented
nanoparticles from upper solution by adding goat anti-rabbit
IgG immuno-Fe2O3/Au nanoparticles and the corresponding
MBA labelled immuno Au nanoparticles. As can be seen, for
the rabbit IgG case, no obvious peaks in SERS spectra
(a0,b0,c0,d0) were detected after the separation. For the
mouse IgG case, no obvious SERS signal was detected at a
concentration of 0.01 pg/ml (d), although it was located in the
sensitivity of this method (about 0.1–1 fg/ml). This may be
due to the nonspecific adsorption of immuno-g-Fe2O3/Au
Scheme 1 Schematic illustration of SERS immunoassay.
Fig. 1 SER spectra and calibration curve of immunoassay for rabbit
IgG of various concentrations. 10 pg/ml (a); 1 pg/ml (b); 100 fg/ml (c);
10 fg/ml (d); 1 fg/ml (e); 0.1 fg/ml (f); blank (g).
Fig. 2 SER spectra of immunoassay for rabbit IgG (a) and mouse
IgG (b) by immuno-g-Fe2O3/Au and MBA-labeled immunogold
capped with goat anti-rabbit IgG.
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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 4225–4227 4227
nanoparticles to mouse IgG, causing the separation of mouse
IgG as well. Thus, the separation limitation by this method
was above 0.1 pg/ml. The results above clearly demonstrate
the success of this separation method when using two antigens.
In summary, a novel magnetic separation immunoassay
method was demonstrated with various concentrations of
rabbit IgG assays. The detection limit of this immunoassay
method was as low as 1–0.1 fg/ml. The antigens have been
separated by g-Fe2O3/Au magnetic nanoparticles capped with
corresponding antibodies from the dual antigen solutions. The
result of the magnetic separation immunoassay demonstrated
that the magnetic bioseparation program used by Fe2O3/Au
nanoparticles could separate almost all of the corresponding
specific antigens in the two component antigen solutions. Even
0.1 pg/ml antigen mixtures can be successfully separated. This
primary study shows that the magnetic separation immuno-
assay method based on SERS might hold promising potential
for immunoassay.
We gratefully acknowledge the support from the Natural
Science Foundation of China (NSFC) (20773091, 20976120)
and the Program of Innovative Research Team of Soochow
University. We also thank Miss Morag Clark-Heptinstall for
her revision.
Notes and references
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Fig. 3 SER spectra of immunoassay for different concentrations of
antigens mixture which contains mouse IgG (a,b,c,d) and rabbit
IgG (a0,b0,c0,d0). a,a0 = 1 ng/ml; b,b0= 1 pg/ml; c,c0 = 0.1 pg/ml;
d,d0 = 0.01 pg/ml.
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