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Sensors and Actuators B 191 (2014) 396– 400

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locate /snb

ighly sensitive carcinoembryonic antigen detection using Ag@Auore–shell nanoparticles and dynamic light scattering

iangmin Miaoa,∗,1, Seyin Zouc,1, Hong Zhangb, Liansheng Lingb,∗

School of Life Science, Jiangsu Normal University, Xuzhou 221116, PR ChinaSchool of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, PR ChinaDepartment of Laboratory Medicine, the Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, PR China

r t i c l e i n f o

rticle history:eceived 17 May 2013eceived in revised form8 September 2013

a b s t r a c t

High level of carcinoembryonic antigen (CEA) can signal the presence of cancer directly. Thus simple andsensitive detection of CEA is of great importance. Here, sensitive CEA detection in human serum wasrealized by using Ag@Au core–shell nanoparticles (CSNPs) and dynamic light scattering (DLS) based onmonitoring the average diameter change of Ag@Au CSNPs after the specific bind between CEA antibody

ccepted 2 October 2013vailable online 15 October 2013

eywords:arcinoembryonic antigenynamic light scattering

(anti-CEA) and CEA. Under optimal conditions, CEA could be detected linearly in the range of 60 pg/mLto 50 ng/mL, with a detection limit of 35.6 pg/mL. Moreover, satisfactory results were obtained when theassay was used in human serum CEA detection.

© 2013 Elsevier B.V. All rights reserved.

g@Au core–shell nanoparticles

. Introduction

Tumor markers are molecules occurring in blood or tissue, theevel of them directly related to the state of cancer. Thus, earlyetection of them will be valuable for clinical research and earlyiagnosis, accordingly initiate appropriate treatment and poten-ially avoid a fatal outcome [1,2]. Thereinto, carcinoembryonicntigen (CEA), as a glycoprotein with a molecule mass of 200 kDa,irectly associate with colorectal, gastric, pancreatic, lung, breastnd medullary thyroid cancers [3–8]. The normal level of CEA inealthy adults is about 3.0–5.0 ng/mL [9], and high level of it canignal the presence of cancer. Therefore, it is urgent to develop aensitive and rapid method for the detection of CEA at nanogramer milliliter level or lower.

Immunoassay, which including fluorescence [10,11], colorime-ry [12,13] electrochemistry [14,15], chemiluminescence [16,17],ynamic light scattering (DLS) [18,19] and surface-enhancedaman scatting (SERS) [20,21], is one of the most important ana-

ytical techniques in quantitative detection of CEA. Thereinto,

uorescence, colorimetry and DLS methods are typical homoge-eous immunoassay. In such methods, tumor markers can beetected directly in solution based on monitoring the signal change

∗ Corresponding authors. Tel.: +86 516 83403171.E-mail addresses: mxm0107@jsnu.edu.cn (X. Miao), cesllsh@mail.sysu.edu.cn

L. Ling).1 These authors contributed equally to this work.

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.10.016

of probes after highly specific recognition interaction between anti-gen and antibody.

Metal nanoparticles have been extensively used in biosensorpreparation, among which gold nanoparticles (AuNPs) are oftenused as signal probes while very little work on silver nanoparticles(AgNPs), due to the difficulty in the methods used for synthesis ofAgNPs, accordingly make achieving repeatable size and character-istics problematic compared to AuNPs synthesis [22]. Additionally,AgNPs is not considered biocompatible [23]. However, the molarextinction coefficient of AgNPs is 100× greater than that of AuNPsin the same size, so it can increase the sensitivity of optical sen-sors [24]. In recent years, Ag core Au shell nanoparticles (Ag@AuCSNPs) were used in immunoassay to combine the advantagesof AuNPs and AgNPs simultaneously. On one hand, core–shellnanoparticles possess more excellent physical and chemical prop-erties than individual metallic counterparts, due to a localizedelectric field enhancement in the core–shell structure [25]. On theother hand, these kinds of core–shell nanoparticles can make upfor the deficiencies of both AuNPs and AgNPs [26–28]. In addition,the underneath Ag core may enhance the surface of AuNPs.

Herein, a DLS based sensor for CEA detection was constructedby using Ag@Au CSNPs as the signal probe, anti-CEA was modifiedonto Ag@Au CSNPs surface firstly, and then the specific recognitionbetween anti-CEA and CEA in solution formed a sandwich com-

plex, accordingly induced the aggregation of Ag@Au CSNPs and theaverage diameter increase of them. Such diameter increasing ofAg@Au CSNPs could then be correlated quantitatively to CEA con-centration, and higher concentration of CEA would lead to more

X. Miao et al. / Sensors and Actuators B 191 (2014) 396– 400 397

tion o

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Fig. 1. The schematic illustra

ggregation and larger size increase of the whole nanoparticles,hich could be measured by DLS analysis (Fig. 1).

. Experimental

.1. Chemicals and materials

Anti-CEA and CEA were obtained from Biocell Co., Ltd.Zhengzhou, China). Gold chloride (HAuCl4), sodium citrate,ydroxylamine hydrochloride (NH2OH·HCl) and bovine serumlbumin (BSA) were purchased from Sigma–Aldrich Co. 10 mM ofhosphate buffer saline (PBS, pH 7.4, with 100 mM NaCl) was used

n our experiments. Nanopure water (18.1 M�) was obtained from 350 Nanopure water system (Guangzhou Crystalline Resdurceesalination of Sea Water and Treatment Co. Ltd.) and used for allxperiments. All chemicals were of analytical reagent grade.

.2. Apparatus

Dynamic light scattering (DLS) measurements were performedy using Zetaplus/90plus Dynamic Light Scattering instrumentBrookhaven Instrument Co., USA). The DLS instrument was oper-ted under the following conditions: temperature 25 ◦C, detectorngle 90◦, incident laser wavelength 683 nm and laser power00 mW. The distribute status of nanoparticles were learned fromransmission electron microscope (TEM) (JEM-2010HR, Japan). AU-1901 UV–visible absorption spectrometer was used to obtainhe absorption spectrum. Centrifugation experiments were con-tructed by using a high speed Anke GL-20G-IIcentrifuge (Antingcientific Instrument Factory, Shanghai, China).

.3. Preparation of Ag@Au/anti-CEA

Ag@Au CSNPs were prepared according to literature [29].hen, Ag@Au/anti-CEA probes were prepared by adding 100 �L of40 ng/mL anti-CEA to 2 mL of Ag@Au CSNPs solution and incu-ated for 2 h at room temperature. After that, 1 mL of 0.25% BSAolution was employed to block the unspecified sites and prevent

he non-specific adsorption. Excess reagents were then removedy centrifuging at 6000 rpm for 3 times (each time for 10 min andashed by PBS, pH 7.4). Finally, the remained red oily precipitate

ontaining Ag@Au/anti-CEA was collected and resolved in PBS for

Fig. 2. (A) TEM images of Ag@Au CSNPs; (B) UV–vis absorption sp

f CEA detection by using DLS.

our experiments, and the concentration of anti-CEA in solution was4.0 �g/mL.

2.4. Immunoassay of CEA by DLS

CEA detection was constructed by adding different concentra-tion of CEA antigen solution to a series of 100 �L Ag@Au/anti-CEAsolutions (PBS, pH = 7.4) and incubated for 15 min at 37 ◦C. Afterthat, sample solutions were prepared by diluting 20 �L of the assaysolution into 200 �L nanopure water for DLS analysis. All sizesreported here were based on the intensity average, and the resultfor each sample was the average of three measurements.

3. Results and discussion

3.1. TEM, UV and XPS assay of Ag@Au CSNPs

TEM images provided the information about the morphology ofAg@Au CSNPs. From the TEM images in Fig. 2A, it could be seenthat the average size of them was about 58–63 nm, and a cen-tral bright and outer dark core–shell nanostructure images werealso observed. Meantime, the core–shell nanoparticles are almostspherical in morphology. Such results were in accord with litera-ture reports [25,26]. Meantime, UV/vis absorption spectra providedadditional evidence about the formation of Ag@Au CSNPs in Fig. 2B,and a characteristic absorption peak of pure AgNPs was obtained at393 nm (curve a). Then, after the formation of Ag@Au CSNPs, twoabsorption peaks of AgNPs and AuNPs appeared simultaneously,only with AgNPs a slight shift of the absorption peak and a slightdecline of the absorption intensity (curve b). To further analyzethe formation of Ag@Au/anti-CEA probes, XPS characterization wasemployed in Fig. 2C, XPS peaks at 166.84 eV, 284.78 eV, 399.67 eVand 532.55 eV showed the positions of S2p, C1s, N1s and O1s ofCEA antibody, and the XPS signature of Au 4f doublet at 84.08 eVand 87.8 eV illustrated the presence of metallic gold Au0, while theXPS signature of Ag 3d doublet at 371.95 and 368.1eV representedsilver Ag0.

3.2. CEA detection by using DLS

The characteristics of the immunosensor monitored by DLSexperiments were shown in Fig. 3A, the average diameter of

ectra of Ag@Au CSNPs; (C) XPS analysis of Ag@Au/anti-CEA.

398 X. Miao et al. / Sensors and Actuators B 191 (2014) 396– 400

Fig. 3. The size distribution (A) and TEM images (B) of 4.0 �g/mL of Ag@Au/anti-CE

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Fig. 4. The effect of anti-CEA concentration (A), pH value (B) and incubation time

Table 1, the sensitivity of some electrochemical and SERS methodswere higher than the proposed method, but they need com-plicated procedure for preparing sensor or nanoparticles, whilethe proposed method could be constructed in solution directly.

EA probes in the presence of 0 ng/mL (a), 1.0 ng/mL (b) and 10.0 ng/mL (c) of CEA.xperiments were conducted in 10 mM of PBS buffer (pH 7.4).

g@Au/anti-CEA probes was about 65.3 nm (a). Subsequently, uponddition of 1.0 ng/mL and 10.0 ng/mL CEA, the average diameterf them increased to 138.7 and 201.6 nm. Such results obviouslyemonstrated that the successful recognition of anti-CEA with CEAriggered the aggregation of Ag@Au CSNPs, and resulted in thencrease of their average diameter. Meantime, a direct evidenceor anti-CEA and CEA recognition induced aggregation of suchanoparticles could be further supported by TEM images (Fig. 3B).

.3. The optimization of experimental conditions

Since the assembly of Ag@Au/anti-CEA probes depends on thepecific recognition of anti-CEA and CEA, the concentration of anti-EA that modified onto Ag@Au CSNPs surface directly affect theharacteristics of the sensor. As shown in Fig. 4A, the averageiameter of nanoparticles increased with the increase of anti-CEAoncentration in the range of 1.0–3.0 �g/mL, and then decreasedetween 4.0 �g/mL and 6.0 �g/mL. Such result indicated thatigher concentration of Ag@Au/anti-CEA probes was propitiouso the assembly of nanoparticles, while too high concentration ofhem would affect the sensitivity of the sensor. So, 4.0 �g/mL ofnti-CEA was chosen in the further study.

Meantime, the effect of pH value was studied in Fig. 4B, the aver-ge diameter of Ag@Au/anti-CEA probes was proportional to the pHalue from 5.5 to 7.5 and then decreased over the range from 7.5 to.0. The reason for that might be highly acidic or alkaline surround-

ngs would damage the activity of protein, especially in alkalinity30]. Due to the pH of body fluid is close to 7.4. Thus, the optimalH of 7.4 was chosen for our research.

It is well known to all that the antigen–antibody recognition isignificantly affected by the incubation time. Seen from Fig. 4C, theverage diameter of nanoparticles increased with the incubationime up to 15 min and then reached a plateau. Result illustrated that

nti-CEA and CEA could be effectively recognized within 15 min.hus, the optimal incubation time of 15 min was taken for all of thexperiments.

(C) on the average diameter of Ag@Au/anti-CEA probes in the presence of 6.0 ng/mLCEA. The error bars represent the standard deviation from three measurements ateach point.

3.4. Performance of the immunosensor

The specific recognition between anti-CEA and CEA occurredand was detected by DLS, which would induce the assembly ofAg@Au/anti-CEA probes, and triggered the increase of their averagediameter. It could be seen from Fig. 5A that the average diame-ter of nanoparticles was linear to CEA concentration in the rangeof 60 pg/mL to 50 ng/mL, with a low detection limit of 35.6 pg/mL(3�/slope). Such wide linear range mainly attributed to the effec-tive combination of highly molar extinction coefficient of AgNPsand stability of AuNPs, and improved the sensitivity of the sensoraccordingly. Meantime, the characteristics of the proposed assaywere compared with those reported in literature. As shown in

Fig. 5. (A) The calibration curve of CEA by using the proposed method; (B) assayresults of serum samples using the proposed method and the reference ELISA meth-ods; (C) diameter change (�D) of nanoparticles before and after the addition of1.0 ng/mL CEA, AFP, HBsAg, CA15-3, CA 19-9 and BSA.

X. Miao et al. / Sensors and Actuators B 191 (2014) 396– 400 399

Table 1The analytical performance of our method compared to literature methods.

Sensing element Transducer used Linear range Detection limit References

AuNPs Colorimetric 50–300 ng/mL 20 ng/mL [24]Hollow AuNPs SERS 10 pg to 100 �g/mL 0.1 pg/mL [20]Hollow AuNPs SERS 1–10 ng/mL – [21]Gold nanorod Electrochemical 0.05–20 pM [14]AuNPs Electrochemical – 0.8 fg/mL [15]Capillary Chemiluminescent 12.5 ng to 4.0 mg/mL [16]Lanthanide Fluorescent 4.9 ng to 1.25 �g/mL [10]AuNPs Fluorescent – 0.7 ng/mL [11]AuNPs DLS 0.5–50 ng/mL – [19]

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oreover, only 20 �L of sample was used for each step detection,hich decreased the cost of the assay greatly. For further evalu-

ting the properties of the proposed method in clinical analysis,ight serum samples from the third affiliated hospital of Guangzhouedical University were analyzed. The accuracy of CEA detec-

ion was examined by comparing the results with those from thenzyme-linked immunosorbent assay (ELISA) analysis (Fig. 5B). Theegression equation of the concentration of CEA obtained from theroposed method (y) and ELISA technique (x) was y = 0.195x + 1.19ith a coefficient of 0.993, which revealed that the proposed sensor

s in good agreement with the conventional ELISA method.Moreover, to estimate the selectivity of this method for CEA

etection, the response from a number of other interfering sub-tances in human serum such as a-fetoprotein (AFP), HBsAg, cancerntigen 15-3(CA 15-3), cancer antigen 19-9(CA 19-9) and (BSA) wasnvestigated. As shown in Fig. 5C, compared to that of blank solu-ions, the addition of 1.0 ng/mL CEA resulted in an obvious increasef the average diameter while little change could be observed uponddition of other interfering components (10 ng/mL). Such resultslearly indicated the high selectivity of the proposed immunosen-or.

.5. Analysis of spiking serum samples

To investigate the practicability of the assay, six serum samplesith the concentration of 3.5, 7.8, 12.6, 21.9, 30.2 and 40 ng/mLere obtained from the third affiliated hospital of Guangzhou Med-

cal University. Our proposed method was used to evaluate theoncentration of such samples and the results were 3.4, 7.5, 12.7,1.3, 31.0 and 41.7 ng/mL, the recovery for six serum samples wasaried within the range from 96.1% to 104.3%. Such results revealedhat the assay could be considered for the clinical diagnosis of CEA.

. Conclusions

Here, a DLS based CEA detection was constructed by detectinghe average diameter change of Ag@Au/anti-CEA probes in solu-ion. The effective combination of AuNPs and AgNPs can make upor the deficiencies of both AuNPs and AgNPs, accordingly improvehe sensitivity of the sensor, and a detection limit of 35.6 pg/mL wasbtained. Moreover, only small volumes of sample were neededor each measurement (20 �L), which correspondingly reduced theubstantial cost of the research. Besides, the assay results wereatisfactory in clinical CEA detection of human serum samples.

cknowledgements

This work was supported by the National Natural Scienceoundation of China (21305053 and 20975116), the Natural Sci-nce Fund for Colleges and Universities in Jiangsu Province13KJB150015), the Natural Science Fund in Jiangsu Province

[

0.4 pM [18]

0 ng/mL 35.6 pg/mL Our method

(BK20130227), the Scientific Research Support Project for Tea-chers with Doctor’s Degrees (Jiangsu Normal University, China, No.12XLR022), and the Project Funded by the Priority Academic Pro-gram Development of Jiangsu Higher Education Institutions.

References

[1] A. Szilvas, A. Blazovices, G. Szekely, E. Dinya, J. Feher, G. Mozsik, Comparativestudy between the free radicals and tumor markers in patients with gastroin-testinal tumors, Journal of Physiology 95 (2001) 247–256.

[2] D. Faraggi, A. Kramar, Methodological issues associated with tumor markerdevelopment. Biostatistical aspects, Urologic Oncology: Seminars and OriginalInvestigations 5 (2000) 211–213.

[3] N. Laboria, A. Fragoso, W. Kemmner, D. Latta, O. Nilsson, M.L. Botero, K. Drese,C.K. O’Sullivan, Amperometric immunosensor for carcinoembryonic antigenin colon cancer samples based on monolayers of dendritic bipodal scaffolds,Analytical Chemistry 82 (2010) 1712–1719.

[4] M.J. Duffy, A. van Dalen, C. Haglund, L. Hansson, R. Klapdor, R. Lamerz, O. Nilsson,C. Sturgeon, O. Topolcan, Clinical utility of biochemical markers in colorec-tal cancer: European group on tumor markers (EGTM) guidelines, EuropeanJournal of Cancer 39 (2003) 718–727.

[5] S.P. Prete, L. Rossi, P.P. Correale, M. Turriziani, S. Baier, G. Tamburrelli, L.D. Vec-chis, E. Bonmassar, A. Aquino, Combined effects of protein kinase inhibitors and5-fluorouracil on CEA expression in human colon cancer cells, PharmacologicalResearch 52 (2005) 167–173.

[6] L. Hernandez, A. Espasa, C. Fernandez, A. Candela, C. Martin, S. Romero, CEAand CA 549 in serum and pleural fluid of patients with pleural effusion, LungCancer 36 (2002) 83–89.

[7] F. Naghibalhossaini, P. Ebadi, Evidence for CEA release from human coloncancer cells by an endogenous GPI-PLD enzyme, Cancer Letters 234 (2006)158–167.

[8] K. Bremer, S. Micus, G. Bremer, CEA, CA 15-3 and MCA: comparative clinicalrelevance in breast cancer, European Journal of Cancer 31 (1995) S262.

[9] S. Hammarström, The carcinoembryonic antigen (CEA) family: structures, sug-gested functions and expression in normal and malignant tissues, Seminars inCancer Biology 9 (1999) 67–81.

10] O. Kavanagh, M.K. Estes, A. Reeck, R.M. Raju, A.R. Opekun, M.A. Gilger, D.Y. Gra-ham, R.L. Atmar, Serological responses to experimental Norwalk virus infectionmeasured using a quantitative duplex time-resolved fluorescence immunoas-say, Clinical and Vaccine Immunology 18 (2011) 1187–1190.

11] S. Mayilo, M.A. Kloster, M. Wunderlich, A. Lutich, T.A. Klar, A. Nichtl, K.Kürzinger, F.D. Stefani, J. Feldmann, Long-range fluorescence quenching by goldnanoparticles in a sandwich immunoassay for cardiac tropponin T, Nano Letters9 (2009) 4558–4563.

12] Y. Yuan, J. Zhang, H.C. Zhang, X.R. Yang, Label-free colorimetric immunoassayfor the simple and sensitive detection of neuroge-nin3 using gold nanoparticles,Biosensors and Bioelectronics 26 (2011) 4245–4248.

13] Y. Yuan, J. Zhang, H.C. Zhang, X.R. Yang, Silver nanoparticle based label-freecolorimetric immunosensor for rapid detection of neurogenin 1, Analyst 137(2012) 496–501.

14] D. Du, L.M. Wang, Y.Y. Shao, J. Wang, M.H. Engelhard, Y.H. Lin, Multiplexedelectrochemical immunoassay of phosphorylated proteins based on enzyme-functionalized gold nanorod labels and electric field-driven acceleration,Analytical Chemistry 83 (2011) 746–752.

15] J. Tang, D.P. Tang, B.L. Su, J.X. Huang, B. Qiu, G.N. Chen, Enzyme free electro-chemical immunoassay with catalytic reduction of p-nitrophenol and recyclingof p-aminophenol using gold nano-particles-coated carbon nanotubes asnanocatalysts, Biosensors and Bioelectronics 26 (2011) 3219–3226.

16] D.A. Michels, A.W. Tu, W. McElroy, D. Voehringer, O. Salas-Solano, Chargeheterogeneity of monoclonal antibodies by multiplexed imaged capillary iso-

electric focusing immunoassay with chemiluminescence detection, AnalyticalChemistry 84 (2012) 5380–5386.

17] Q. Liu, M. Han, J.C. Bao, X.Q. Jiang, Z.H. Dai, CdSe quantum dots as labelsfor sensitive immunoassay of cancer biomarker proteins by electrogeneratedchemiluminescence, Analyst 136 (2011) 5197–5203.

4 ctuat

[

[

[

[

[

[

[

[

[

[

[

[

[

00 X. Miao et al. / Sensors and A

18] X. Liu, Q. Huo, A washing-free and amplification-free one-step homogeneousassay for protein detection using gold nano-particle probes and dynamic lightscattering, Journal of Immunological Methods 349 (2009) 38–44.

19] X. Liu, Q. Dai, L. Austin, J. Coutts, G. Knowles, J.H. Zou, H. Chen, Q. Huo, AOne-Step Homogeneous immunoassay for cancer biomarker detection usinggold nanoparticle probes coupled with dynamic light scattering, Journal of theAmerican Chemical Society 130 (2008) 2780–2782.

20] M. Lee, S. Lee, J. Lee, H. Lim, G.H. Seong, E.K. Lee, S. Chang, C.H. Oh, J. Choo, Highlyreproducible immunoassay of cancer markers on a gold-patterned microar-ray chip using surface-enhanced Raman scattering imaging, Biosensors andBioelectronics 26 (2011) 2135–2141.

21] H. Chon, C. Lim, S.M. Ha, Y. Ahn, E.K. Lee, S.I. Chang, G.H. Seong, J. Choo, On-chip immunoassay using surface-enhanced Raman scattering of hollow goldnanospheres, Analytical Chemistry 82 (2010) 5290–5295.

22] D.G. Thompson, A. Enright, K. Faulds, W.E. Smith, D. Graham, UltrasensitiveDNA detection using oligonucleotide–silver nanoparticle conjugates, Analyti-cal Chemistry 80 (2008) 2805–2810.

23] K.C. Grabar, K.J. Allison, B.E. Baker, R.M. Bright, K.R. Brown, R.G. Freeman, A.P.Fox, C.D. Keating, M.D. Musick, M.J. Natan, Two-dimensional arrays of colloidalgold particles: a flexible approach to macroscopic metal surfaces, Langmuir 12(1996) 2353–2361.

24] Y. Wang, F. Yang, X.R. Yang, Colorimetric detection of mercury(II) ion usingunmodified silver nanoparticles and mercury-specific oligonucleotides, ACSApplied Materials & Interfaces 2 (2010) 339–342.

25] S. Guha, S. Roy, A. Banerjee, Fluorescent Au@Ag core–shell nanoparticles withcontrolled shell thickness and HgII sensing, Langmuir 27 (2011) 13198–13205.

26] L. Wu, Z.Y. Wang, S.F. Zong, Z. Huang, P.Y. Zhang, Y.P. Cui, A SERS-based

immunoassay with highly increased sensitivity using gold/silver core–shellnanorods, Biosensors and Bioelectronics 38 (2012) 94–99.

27] P.P. Dong, Y.Y. Lin, J.J. Deng, J.W. Di, Ultrathin Gold-shell coated silver nanopar-ticles onto a glass platform for improvement of plasmonic sensors, ACS AppliedMaterials & Interfaces 5 (2013) 2392–2399.

ors B 191 (2014) 396– 400

28] X. Guo, Z.Y. Guo, Y. Jin, Z.M. Liu, W. Zhang, D.P. Huang, Silver–gold core–shellnanoparticles containing methylene blue as SERS labels for probing and imag-ing of live cells, Microchimica Acta 178 (2012) 229–236.

29] Y. Cui, B. Ren, J.L. Yao, R.A. Gu, Z.Q. Tian, Synthesis of Ag coreAu shell bimetallic nanoparticles for immunoassay based on surface-enhanced Raman spectroscopy, Journal of Physical Chemistry B 110 (2006)4002–4006.

30] D.P. Tang, R. Yuan, Y. Chai, X. Zhong, Y. Liu, J. Dai, Ultrasensitive potentio-metric immunosensor based on SA and OCA techniques for immobilization ofHBsAb with colloidal Au and polyvinyl butyral as matrixes, Langmuir 20 (2004)7240–7245.

Biographies

Xiangmin Miao have received a PhD from Sun-Yat Sen University in June 2012.Now he is working in Jiangsu Normal University and the current fields of interestwere mainly biosensors, optical and electrochemical nano-biological analysis andimmunoassay.

Seyin Zou has been received a Master Degree Candidate from Sun-Yat Sen Univer-sity in June 2011. Now she is working in the third affiliated hospital of GuangzhouMedical University and the current field of interest was immunoassay.

Hong Zhang has been received a Master Degree Candidate from Sun-Yat SenUniversity in June 2011. Now she is studying in Sun-Yat Sen University and thecurrent fields of interest were mainly biosensors, optical nano-biological analysis

and immunoassay.

Liansheng Ling has been received a PhD from WuHan University in June 2000. Nowhe is working in Sun-Yat Sen University and the current fields of interest were mainlybiosensors, optical nano-biological analysis and immunoassay.