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ORIGINAL PAPER
Immunoassay using surface-enhanced Raman scattering
based on aggregation of reporter-labeled
immunogold nanoparticles
Ji-Wei Chen &Yong Lei &Xiang-Jiang Liu &
Jian-Hui Jiang &Guo-Li Shen &Ru-Qin Yu
Received: 7 May 2008 /Revised: 30 May 2008 /Accepted: 6 June 2008 / Published online: 4 July 2008
#Springer-Verlag 2008
Abstract A one-step homogenous sensitive immunoassay
using surface-enhanced Raman scattering (SERS) has been
developed. This strategy is based on the aggregation of
Raman reporter-labeled immunogold nanoparticles induced
by the immunoreaction with corresponding antigens. The
aggregation of gold nanoparticles results in a SERS signal
increase of the Raman reporter. Therefore, human IgG
could be directly determined by measuring the Raman
signal of the reporter. The process of aggregation was
investigated by transmission electron microscopy (TEM)
and UV–Vis absorption spectroscopy. The effects of the
temperature, time, and size of gold nanoparticles on the
sensitivity of the assay were examined. Using human IgG
as a model protein, a wide linear dynamic range (0.1–
15 μgmL
−1
) was reached with low detection limit
(0.1 μgmL
−1
) under optimized assay conditions. The
successful test suggests that the application of the proposed
method holds promising potential for simple, fast detection
of proteins in the fields of molecular biology and clinical
diagnostics.
Keywords Immunoassay .Surface-enhanced Raman
scattering .Aggregation .Gold nanoparticles .Human IgG
Introduction
Since Singer and Plotz first reported aggregation-based
immunoassays in 1956 [1], there have been extensive
efforts devoted to the development and the application of
this technique [1–9]. Generally, antigens were determined
by the aggregation resulting from the reaction of the
antibody-coated particles with the corresponding antigens.
Recently attention has turned towards the application of
gold nanoparticles in aggregation-based immunoassays
owing to the excellent physical and chemical characteristics
of these nanoparticles. The proteins bound to colloidal gold
particles are known to better retain biological activity in the
immunoassay [6,7]. Therefore, several advantages can be
obtained using gold nanoparticles in aggregation-based
immunoassays such as simple preparation, easy readout,
and good stability. A pioneering study on the application of
gold nanoparticles in aggregation-based immunoassays was
published by Thanh and Rosenzweig [8]. In their work,
gold nanoparticles coated with protein A were used to
determine anti-protein A in aqueous and serum solutions.
Aggregation in the presence of protein A was detected by
measuring the absorption of the gold colloid suspension
and a limit of detection of 1 μgmL
−1
of anti-protein A was
obtained. Recently, Du et al. reported an immunoassay
based on aggregation of antibody-functionalized gold
nanoparticles coupled with light-scattering detection [9].
The strategy was performed with one-step operation in
homogeneous solution followed by the measurement of
light scattering with a common spectrofluorimeter. To
expand the scope of aggregation-based immunoassay, it is
interesting to explore the possibility of using some new
detection methods such as surface-enhanced Raman scat-
tering (SERS).
In recent years, SERS has been emerging as an important
method for ultrasensitive chemical analysis owing to
inherent richness of Raman signatures and the single-
molecule-level detection sensitivity. It is generally agreed
that the large enhancement is predominantly from electro-
magnetic (EM) fields at some “hot spots”, most often
Anal Bioanal Chem (2008) 392:187–193
DOI 10.1007/s00216-008-2237-z
J.-W. Chen :Y. Lei :X.-J. Liu :J.-H. Jiang :G.-L. Shen :
R.-Q. Yu (*)
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering,
Hunan University,
Changsha 410082, China
e-mail: rqyu@hnu.cn
consisting of nanoscale junctions and interstices in interact-
ing metal nanostructures such as nanoparticle dimers or
aggregates [10–12]. According to theoretical calculations,
Raman scattering intensity of a molecule at nanocrystal
junctions can be several orders of magnitude higher than
that of the same molecule on the surface of single particles
[13,14]. Therefore, the aggregates of colloidal silver or
gold particles were often used as SERS-active substrates to
provide great enhancement in trace analysis. For example,
Nie and Emory confirmed the existence of Raman enhance-
ment factors on the order of 10
14
–10
15
for rhodamine 6G
molecules on small salt-induced aggregates of colloidal
silver nanoparticles [15]. Recently, Han et al. reported a
SERS-based immunoassay on a microtiter plate in which
strong signals were obtained from silver aggregates result-
ing from the addition of salts and surfactants [16]. On the
other hand, metal colloidal aggregates can also be utilized
as Raman tags for the detection of biomolecules [17,18].
Unlike other Raman tags based on individual gold particles,
these kinds of particles are clusters coalesced from metal
nanoparticles in the presence of organic Raman labels or
positively charged polymer. For example, Su and co-workers
synthesized a new type of Raman tag called composite
organic–inorganic nanoparticles (COINs) for multiplex and
ultrasensitive immunoassays [18], in which a variety of
organic Raman compounds were embedded at the particle
junctions of silver particles to achieve an optimal surface
enhancement effect.
In the present study, we developed an alternative approach
for aggregation-based immunoassays using Raman reporter-
labeled immunogold nanoparticles as probes coupled with
SERS detection. As shown in Scheme 1A, gold nano-
particles were functionalized with Raman reporter and
antibody successively in a two-step process before the
detection of target. Scheme 1Bdepictstheimmunoassay
protocol based on SERS. The reporter-labeled immunogold
nanoparticles should be monodispersed in the absence of
target. When the antigen was introduced, aggregation of
gold nanoparticles was induced by the immunoreaction
between the antigen and the antibody modified on the
surface of nanoparticles. As a result, SERS signals of the
reporter on the surface of gold nanoparticles would be
greatly enhanced when the particles aggregated. Therefore,
the content of human IgG could be directly determined by
measuring the Raman signal of the reporter. Unlike
previously reported SERS-based immunoassays in hetero-
geneous formats [19–21], the proposed immunoassay was
performed with a one-step operation in homogeneous
solution, which can avoid the multiple steps of immuno-
reactions and washings in the heterogeneous immuno-
assays. The process was also investigated by transmission
electron microscopy (TEM) and UV–Vis absorption spec-
troscopy. The experimental conditions for the detection of
human IgG were optimized. On the basis of this strategy,
human IgG has been detected with a relatively low
detection limit as well as a wider linear dynamic range.
Materials and methods
Materials
Hydrogen tetrachloroaurate(III) trihydrate, trisodium citrate,
and rhodamine B isothiocyanate (RBITC) were purchased
from Aldrich. Goat anti-human IgG antibody, human IgG,
and bovine serum albumin (BSA) were purchased from
Scheme 1 Schematic of the
immunoassay procedure for hu-
man IgG (see text for details)
188 J.-W. Chen et al.
Beijing Dingguo Biotechnology Development Center (Bei-
jing, China). All chemicals and materials were of analytical
grade and used as received. Buffers used in this work
included borate buffer (2 mM, pH 9) and sodium phosphate-
buffered saline (PBS, 0.05 M, pH 7.4). All solutions were
prepared with deionized water (18.32 MΩ) purified by a
Nanopure Infinity Ultrapure Water System (Barnstead/
thermolyne Corp, Dubuque, IA).
Instrumentation
Raman spectra were collected using a Jobin Yvon micro-
Raman spectrometer (RamLab-010), comprising an integral
Olympus BX40 microscope with a ×10 objective that
focuses the laser on the sample and collects the back-
scattered radiation, a notch filter to cut the exciting line, a
holographic grating (1,800 g/mm) offering a spectral
resolution of 2 cm
−1
, and a semiconductor-cooled 1,024×
256 pixels charge-coupled device detector. A laser of
632.8 nm with a power of ca. 5 mW was used as the
excitation source. The slit and pinhole were set at 100 μm
and 300 μm, respectively. Under these settings, the sampl-
ing area was about 10 μm in diameter on the substrate sur-
face. All SERS spectra were acquired with 15-s integration
time and processed with the software from Jobin Yvon
(Labspec4.0).
UV–Vis spectra were recorded on MultiSpec-1501 UV–
Vis spectrometer (Shimadzu, Japan) coupled with Hyper
UV software. Transmission electron micrographs (TEM)
were obtained by using a JEM-3010 electron microscope
(JEOL, Japan) with Digitalgraph software at an accelerating
voltage of 100 KV. The prepared substrate was analyzed by
scanning electron microscopy (SEM) using a JSM-5600LV
microscope (JEOL, Ltd., Japan).
Preparation of Raman reporter-labeled immunogold
nanoparticles
Raman reporter-labeled immunogold colloids were pre-
pared in a three-step process according to the developed
procedures with slight modifications [19,22]. In the first
step, gold nanoparticles with 25 ± 1 nm diameters were
synthesized by adding 1.5 mL of 34 mM sodium citrate to a
100 mL aqueous solution of boiling 0.24 mM hydrogen
tetrachloroaurate(III) trihydrate under vigorous stirring [23].
After appearance of a deep red color, boiling and stirring
were continued under reflux for 10 min and then cooled to
room temperature. The second step of the preparation
involved the immobilization of the Raman dye on the
prepared colloids through the spontaneous adsorption of
isothiocyanate (–N=C=S) group onto gold [24]. Typically,
2.5 μL of 1 mM RBITC was slowly added to 1 mL of
colloidal gold under vigorous stirring and the resultant
mixture was allowed to react for 12 h. The reporter-labeled
colloids were then separated from solution by centrifuga-
tion at 12,000 rpm for 12 min. The clear supernatant was
discarded, and the loosely packed red gold sediment was
resuspended in 1 mL of borate buffer (2 mM, pH 9). In the
third step, the reporter-labeled colloids were immobilized
with antibody. Under gently agitation, 12 μL of 2 mg mL
−1
antibody was added to 1 mL of the reporter-labeled gold
colloids. This amount of antibodies is ca. 50% more than
the minimum amount for coating the unmodified portion of
the nanoparticle surface. The mixture was incubated at
room temperature for 1 h. To block the bare sites on the
surface of gold nanoparticles, 100 μL of 10% BSA was
added to the prepared RBITC-labeled immunogold colloids.
After 30 min, the mixture was centrifuged at 10,000 rpm for
10 min. Next, the loose sediment of reporter-labeled
immunogold was rinsed by resuspending in 2 mM borate
buffer and collecting after a second centrifugation at
10,000 gfor 10 min. Finally, the complex was resuspended
in 50 μL of 0.05 M PBS. The prepared suspensions were
stored at 4 °C before use.
Immunoassay protocol
Under the ambient temperature, 5-μL aliquots of a series of
dilutions of human IgG were pipetted to 50 μL as-prepared
reporter-labeled immunogold colloids (for blank sample,
5μL PBS buffer was added to 50 μL of as-prepared
reporter-labeled immunogold colloids) and the resultant
mixture was allowed to react for 1 h. Before SERS
detection, the mixture was diluted 20-fold with 0.05 M
PBS to avoid nonspecific aggregation, then 10 μL of the
dilution was dropped onto glass microscope slide. Raman
spectra were collected randomly on different spots in a 50 ×
50 μm
2
area when the slide was dry. The signal intensity of
the band at 1,643 cm
−1
was recorded versus different
concentrations of human IgG.
Results and discussion
Aggregation process
To characterize the aggregation process of nanoparticles,
the TEM images and UV–Vis spectra were recorded at
different conditions. Figure 1shows the TEM images of (a)
pure gold nanoparticles, (b) RBITC/gold/antibody compos-
ite nanoparticles, and (c) RBITC/gold/antibody composite
nanoparticles after the addition of antigen (10 μgmL
−1
).
One can observe that gold colloids are almost monodis-
persed nanoscale particles, which indicates a 25 ± 1 nm
diameter based on a sampling of approximately 100
particles (Fig. 1a). After the modification of RBITC and
Immunoassay using surface-enhanced Raman scattering 189
antibody successively, the nanoparticles are still monodis-
persed in solution (Fig. 1b), which demonstrates that no
aggregates have been formed through the preparation
process of the RBITC/gold/antibody composite nanopar-
ticles. When human IgG is added in the composite
nanoparticles, as shown in Fig. 1c, the gold particles
aggregate to form larger particles induced by the immuno-
reaction. The UV–Vis absorption spectra of solutions
during the aggregation process are shown in Fig. 2.
Spectrum a is typical of a pure gold nanoparticles solution
showing a plasmon resonance peak at 525 nm. As shown in
Fig. 2b, the modification of gold nanoparticles with RBITC
and antibody results in a decrease in absorbance, but the
plasmon resonance peak is still at 525 nm. Figure 2c shows
the spectrum of RBITC/gold/antibody composite nano-
particles in the presence of 10 μgmL
−1
antigen. One can
observe the broadening of the absorption band toward the
long-wavelength region, which indicates the formation of
gold nanoparticles aggregates in the presence of human
IgG. These experimental observations using TEM and UV–
Vis spectroscopy consistently demonstrate that aggregation
of gold nanoparticles has taken place induced by the
immunoreaction between the human IgG and the labeled
antibodies. As a result, the SERS signals of RBITC would
increase in the presence of target in this method for im-
munoassay. This assumption was subsequently confirmed
by SERS experiments.
Response performance in SERS
Figure 3depicts the SERS spectra in response to PBS
buffer and 0.5 μgmL
−1
human IgG as targets respectively
in the immunoassay. In the absence of target, weak SERS
signals were obtained for RBITC in the region from 1,700
to 1,100 cm
−1
(curve a). When human IgG was added, the
SERS signals exhibited an obvious enhancement with a
clear observation of main bands of RBITC in the same
region (curve b). The peaks at ca. 1,354, 1,508, 1,523, and
Fig. 1 TEM images of pure gold nanoparticles (a), RBITC/gold/
antibody composite nanoparticles (b), and RBITC/gold/antibody
composite nanoparticles after the addition of antigen (10 μgmL
−1
)(c)
Wavelength (nm)
300 400 500 600 700 800
Absorbance
-.2
0.0
.2
.4
.6
.8
1.0
a
b
c
Fig. 2 UV–Vis absorption spectra of pure gold nanoparticles (a),
RBITC/gold/antibody composite nanoparticles (b), and RBITC/gold/
antibody composite nanoparticles after the addition of antigen
(10 μgmL
−1
)(c)
190 J.-W. Chen et al.
1,643 cm
−1
are assigned to aromatic C–C stretching and
those at ca. 1,196 and 1,276 cm
−1
are assigned to aromatic
C–H bending and C–C bridge band stretching, respectively
[25]. The results indicate that SERS signals of RBITC were
efficiently enhanced by the aggregation of immunogold
nanoparticles resulting from the introduction of human IgG,
which is in good agreement with the assay principle
mentioned above. In the absence of target, the Raman
signals of the reporter were enhanced by the isolated single
gold nanoparticles. According to the size-dependent optical
properties of colloidal gold nanoparticles reported by Krug
et al. [26], the isolated single gold nanoparticles with an
average particle size of 25 nm are not efficient in enhancing
Raman signals. In contrast, the immunogold nanoparticles
tend to form aggregates due to the reaction between human
IgG and antibodies upon the addition of the target, thus
resulting in strong SERS signals of the Raman reporter.
Optimization of immunoassay conditions
In this work, the particle size played a crucial role in SERS
response. We investigated the response performance of
13-nm, 25-nm, and 50-nm gold colloids in the immuno-
assay. Herein, I
Target
/I
No target
(the target/blank intensity
ratio) was used as the measure to determine the optimum
conditions, where I
Target
and I
No target
were the intensities at
1,643 cm
−1
in response to 0.5 μgmL
−1
human IgG and
PBS buffer under the same conditions, respectively. It
should be noted that the application of I
Target
/I
No target
could
quite efficiently mitigate the effect of instrumental compli-
cations. As shown in Fig. 4a, the biggest SERS signal
enhancement was obtained when 25-nm gold colloid was
chosen in the immunoassay. It can be assumed that the
aggregation of small nanoparticles is still insufficient to
enhance the Raman signals of the reporter strongly. In
Raman Shift (cm-1)
1000 1200 1400 1600
SERS Signal Signal (a.u.)
-200
-100
0
100
200
300
400
a
b
164315231508
13541196 1276
Fig. 3 SERS spectra of RBITC from the immunoassay in response to
PBS buffer (a) and 0.5 μgmL
−1
human IgG (b)
25
13 50
Size of Gold Nanoparticle (nm)
ITarget / INo target
0.0
.5
1.0
1.5
2.0
2.5
ITarget / INo target
0.0
.5
1.0
1.5
2.0
2.5
ITarget / INo target
0.0
.5
1.0
1.5
2.0
2.5
a
Time (min)
10 30 60 90 120 150
b
Temperature (°C)
4 25303745
c
Fig. 4. RatioofSERSsignalat1,643cm
−1
in response to
0.5 μgmL
−1
human IgG (denoted I
Target
) and PBS buffer (denoted
I
No target
) plotted versus the size of gold nanoparticles (a), immuno-
reaction time (b), and immunoreaction temperature (c). The selected
particle size of gold nanoparticles is thus 25 nm. Each data point
represents an average of four measurements. The error bars are
relative standard deviations
Immunoassay using surface-enhanced Raman scattering 191
contrast, much bigger gold nanoparticles would result in
great SERS signals without any aggregation, thus leading to
high background in the immunoassay. Both cases would
deteriorate the sensitivity of the strategy. Consequently, the
25-nm gold colloids were used throughout the work.
The effects of time and temperature on the sensitivity of
the assay have also been investigated. As shown in Fig. 4b,
I
Target
/I
No target
was strongly dependent on the incubation
time between human IgG and Raman reporter-labeled
immunogold colloids, and the peak is achieved at after
1 h. According to Thanh and Rosenzweig [8], a short time
for immunoreaction would result in incomplete aggregation
and a very long time would induce precipitation of large
aggregates with a clear supernatant. As a result, the sensitivity
of the immunoassay would be deteriorated in both situations.
The effect of temperature on the SERS response is shown in
Fig. 4c. One can observe that I
Tar ge t
/I
No target
decreases with
increasing temperature above 25 °C. This may be attributed
to the increase in free energy ΔGat higher temperature, thus
resulting in the instability of the aggregates formed [8].
Nevertheless, weak response is found when the temperature
decreases to 4 °C, which is assumed to be related to increased
nonspecific aggregation at a relatively low temperature, thus
leading to high background in SERS detection.
Therefore, 1 h and 25 °C were selected as optimum im-
munoreaction time and temperature, respectively, through-
out subsequent experiments.
Method specificity
To investigate the selectivity of the sensing system, we
compared the SERS signal changes (I
Target
/I
No target
)at
1,643 cm
−1
brought by human IgG and other four proteins
human IgM, human IgA, human IgE, and Thrombin. As
shown in Fig. 5, an obvious increase of SERS signal could
be observed in response to 0.5 μgmL
−1
human IgG, while
human IgM, human IgA, human IgE, and Thrombin are
less than 5.0% under the same experimental conditions,
which suggests that the developed method is very selective
for the detection of human IgG.
SERS-based detection of human IgG
The relationship between the SERS response and the
concentration of human IgG was investigated under
optimized experimental conditions mentioned above. As
shown in Fig. 5,I
Target
/I
No target
increases with increasing
target concentration and a linear correlation between
I
Target
/I
No target
(y) and the logarithmic concentration of
human IgG (x) is achieved in the concentration range from
I
g
MI
g
AI
g
E Thrombin I
g
G
ITarget / INo target
0.0
.5
1.0
1.5
2.0
2.5
Fig. 5 Ratio of SERS signal at 1,643 cm
−1
in response to
0.5 μgmL
−1
human IgG, human IgM, human IgA, human IgE, and
thrombin. The error bars are relative standard deviations
Human I
g
G Concentration ( g/mL)
0246810121416
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Log ( human IgG concentration / g/mL)
-1.0 -.5 0.0 .5 1.0 1.5
ITarget
/ INo target
1.0
1.5
2.0
2.5
3.0
3.5
ITarget / INo target
Í
Í
Fig. 6 Response curve for the immunoassay of human IgG in PBS
buffer. Inset A linear relationship between SERS response and
logarithmic concentration of human IgG. Each data point represents
an average of four measurements. The error bars are relative standard
deviations
The proposed immunoassay (
µg
/mL)
.5 1.0 1.5 2.0 2.5 3.0 3.5
ELISA (µg/mL)
0.0
.5
1.0
1.5
2.0
2.5
3.0
3.5
Fig. 7 Correlation between ELISA and the proposed immunoassay
192 J.-W. Chen et al.
0.1 to 15 μgmL
−1
. At higher concentration, the aggregation
process is inhibited due to the blocking of the active sites,
which would decrease the sensitivity of immunoassay. The
calibration equation is y¼2:272 þ1:049x, with a correla-
tion coefficient of 0.9929 (Fig. 6, inset). The detection
limit is estimated to be 0.1 μgmL
−1
based on the 3σrule
(where σis the standard deviation of a blank), indicating a
high sensitivity for protein detection. The obtained sensi-
tivity demonstrates the competency of SERS in comparison
with those reported approaches by UV–Vis absorption [8]
or light-scattering techniques [9] for aggregation-based
immunoassay.
Analytical application
To investigate the applicability and reliability of the present
method for clinical diagnostics, a series of human serum
samples were analyzed simultaneously with the proposed
immunoassay and ELISA. As shown in Fig. 7, a good
correlation of determination results of human IgG in the
serum samples between the proposed immunoassay and
ELISA is obtained. The correlation equation is y¼0:14 þ
1:04x(where yand xare the determination results of ELISA
and the proposed method, respectively), and the correlation
coefficient r=0.9811.
Conclusions
In present study, a homogenous immunoassay has been
developed using SERS based on the aggregation of
antibodies and Raman reporter co-functionalized gold
nanoparticles. The target can be detected by monitoring
the SERS signal change of the Raman reporter. The process
of aggregation was investigated by TEM and UV–Vis
absorption spectroscopy. Utilizing human IgG as a model
protein, SERS response linearly correlated with the loga-
rithmic concentration of the target over a range from 0.1 to
15 μgmL
−1
with a detection limit of 0.1 μgmL
−1
, i.e., our
method exhibited good competency in comparison with
analogous immunoassay based on UV–Vis absorption or
light-scattering techniques. Our proposed approach also has
several advantages over conventional SERS-based hetero-
geneous immunoassays with respects to good reproducibil-
ity, short assay time, and one-step operation. Therefore, it is
expected that the proposed approach might hold promising
potential for protein assay in the fields of molecular biology
and clinical diagnostics.
Acknowledgments This work was supported by “973”National
Key Basic Research Program (2007CB310500), the National NSF of
China (No. 20435010, 20575020, 20675028, 20605007, 20775023)
and Ministry of Education (NCET-04–0768).
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Immunoassay using surface-enhanced Raman scattering 193
