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Surface Enhanced Raman

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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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9 C. L. Haynes, A. D. McFarland and R. P. Van Duyne, Anal.Chem., 2005, 77, 338A.

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11 J. F. Li, Y. F. Huang, Y. Ding, Z. L. Yang, S. B. Li, X. S. Zhou,F. R. Fan, W. Zhang, Z. Y. Zhou, D. Y. Wu, B. Ren, Z. L. Wangand Z. Q. Tian, Nature, 2010, 464, 392.

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14 Y. Cui, B. Ren, J. L. Yao, R. A. Gu and Z. Q. Tian, J. Phys.Chem. B, 2006, 110, 4002.

15 Y. Cao, R. Jin and C. A. Mirkin, Science, 2002, 297, 1536.16 S. I. Stoeva, F. Huo, J. Lee and C. A. Mirkin, J. Am. Chem. Soc.,

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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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