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IOP PUBLISHING ADVANCES IN NATURAL SCIENCES: NANOSCIENCE AND NANOTECHNOLOGY

Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 045017 (7pp) doi:10.1088/2043-6262/3/4/045017

Production of antibody labeled goldnanoparticles for influenza virus H5N1diagnosis kit development

Van Dong Pham1, Ha Hoang2, Trong Hoang Phan2,3, Udo Conrad3 andHoang Ha Chu2

1 Institute of Material Science, Vietnam Academy of Science and Technology (VAST),18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam2 Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST),18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam3 Leibniz-Institut fuer Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstraße 3,D-06466 Stadt Seeland, OT Gatersleben, Germany

E-mail: chuhoangha@ibt.ac.vn

Received 5 October 2012Accepted for publication 13 October 2012Published 7 December 2012Online at stacks.iop.org/ANSN/3/045017

AbstractPreparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its usein developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles(AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1(scFv7 and scFv24) were produced using pTI2+ vector and E. coli strain HB2151. Theseantibodies were purified by affinity chromatography technique employing HiTrap ChelatingHP columns pre-charged with Ni2+. The method for preparation of antibody–colloidal goldconjugate was based on electrostatic force binding antibody with colloidal gold. The effect offactors such as pH and concentration of antibody has been quantitatively analyzed usingspectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. Themorphological study by scanning electron microscopy (SEM) showed that the average size ofthe spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidalAuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum.The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity ofsynthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blotimmunological method. The results confirmed the ability in detection of the A/H5N1 virus ofthe prepared antibody labeled gold particles and opened up the possibility of using them inmanufacturing rapid detection kit for this virus.

Keywords: gold nanoparticles, nanoprobes, H5N1 bird flu diagnostic kit, scFv

Classification numbers: 2.04, 4.02, 5.00, 5.08

1. Introduction

An influenza pandemic is known as a global outbreak ofdisease that occurs when a new strain of influenza A virusappears in the human population, causes serious illness, andthen spreads easily from person to person worldwide [1].Highly pathogenic avian influenza viruses of the H5N1subtype are circulating in eastern Asia with unprecedented

epizootic and epidemic effects [2]. The three viral envelopeproteins of influenza A virus are most medically relevant.The hemagglutinin (HA), neuraminidase (NA) and M2 areessential viral proteins targeted by host antibodies or antiviraldrugs such as oseltamivir and rimantadine [3–5]. Influenza Abecame a topic of much discussion since its appearance inhumans, having caused deaths in the first 18 cases reportedby Hong Kong Special Administration Region (SAR), China,

2043-6262/12/045017+07$33.00 1 © 2012 Vietnam Academy of Science & Technology

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 045017 V D Pham et al

in 1997 [6]. Because of certain characteristics, H5N1 isof particular concern because it mutates rapidly and has apropensity to acquire genes from viruses that infect otheranimal species. Its ability to cause severe disease and deathin humans has been documented.

Single-chain fragment variable (scFv) antibody is a smallantibody engineered by connecting the gene fragments forthe variable regions of the heavy and light chains of animmunoglobin with a linker. The resulting scFv antibodyusually retains the affinity and specificity of its parentantibody [7]. The scFv antibody has great advantages indiagnostic applications because of its small molecule andtargeting drug-delivery agent potential [8].

The development of nanotechnology has resulted in newmethods and approaches in designing rapid, accurate andsensitive diagnostic techniques. Gold nanoparticles (AuNPs)have attracted a wide range of biomedical applicationsbecause of their unique surface chemistry, electronic andoptical properties [9–12]. Colloidal AuNPs exhibit a uniquephenomenon, known as surface plasmon resonance (SPR)in the visible wavelength range which depends on theirsize, shape, particle–particle distances and surroundingmedium [13]. AuNPs are nanomaterials that possess excellentbiocompatibility and ease of conjugation to various biologicalmolecules such as peptides, aptamers, antibodies etc. Theseproperties allow colloidal AuNPs to be a prominent candidatefor bioassay techniques; examples of such techniques arecolorimetric sensor [14–16], lateral flow test trip [17–20],dot blot immunoassay [21–24], which have been usedsuccessfully in biological and medical aims as well. Thus,binding of AuNPs to biomolecules offers a promisingapproach for facile tracking of desired targets in aqueoussamples.

In this study we developed, novel nanoprobes forH5N1 bird flu diagnosis by combining colloidal AuNPswith expressed antibody specific to HA surface antigen ofH5N1. AuNPs were synthesized through the reduction ofchloroauric acid (HAuCl4) by sodium citrate. Two kinds ofScFv (scFv7 and scFv24) antibodies specific to HA surfaceantigen of H5N1 virus were produced recombinantly inE. coli. The purified scFv7 was then labeled on AuNPsto produce antibody/AuNPs nanoprobes. The reactivity ofthese nanoprobes was qualitatively tested using dot blot in asandwich mechanism in which scFv24 was used as secondaryantibody for detection. As a result, these nanoprobes werehighly evaluated and proved to be a good material for thefurther development of H5N1diagnotic kit.

2. Materials and methods

2.1. Materials

Chloroauric acid (HAuCl4) and sodium citrate (C6H5O7Na3)were obtained from Merck. Deionized water was usedthroughout the experiment. Nitrocellulose membrane(BIO-RAD) and all other chemicals in this study were of highquality.

2.2. Synthesis of AUNPs

Citrate-stabilized AuNPs were prepared according toTurkevich et al [25]. Briefly, 100 µl of an aqueous HAuCl4(5%) was added into a flask containing 80 ml deionized waterand then the solution was brought to boil, with constantstirring. As quickly as possible, 3.5 ml of an aqueous sodiumcitrate (1%) solution was added. The solution was heated foranother 20 min until a deep-red solution was observed. Theparticles solution was centrifuged for 10 min at 3500 rpm andsupernatant was collected.

2.3. Characterization of the colloidal gold particles

The morphology and elemental information of AuNPswere characterized by scanning electron microscopy (SEM)(S-4800 FESEM equipped with energy dispersive x-ray(EDX) spectroscopy) in which SEM images were usedto physically measure the size of the AuNPs. UV-Visspectroscopy (Shimadzu, UV-1650PC) determined the opticalcharacteristics of colloidal AuNPs by SPR measurement.

2.4. Expression and purification of single chain antibodies(scFv) specific to H5N1

Human Single Framework scFv Libraries A + B(I. Tomlinson, MRC, University of Cambridge, UK) [26] wereused to screen specific recombinant antibodies againstE. coli-expressed HA1 protein. The screening procedurewas described by Gahrtz and Conrad [27]. DNA fragmentsencoding scFv7 and scFv24 were cloned into the pTI2+plasmid. The recombinant plasmid was then introducedinto E. coli strain HB2151. The expression of scFv7and scFv24 was induced using 1 mM of isopropyl-D-1-thiolgalactopyranoside (IPTG) for 5 h at 30 ◦C. The bacteriawere then collected and lyzed by lysozyme and sonication.The inclusion bodies were collected by centrifuging, followedby washing with buffer (50 mM Tris, 1% triton X-100 and100 mM NaCl, pH 7.5). The scFv7 and scFv24 proteins wererefolded and further purified by affinity chromatographyusing HiTrap Chelating HP columns pre-charged withNi2+. The expression and purity of the scFv were furthercharacterized by sodium dodecyl sulfate polyacrylamide gelelectrophotoresis (SDS-PAGE).

2.5. Preparation of binding antibody/gold probes

2.5.1. Determining the optimal pH. The colloidal AuNPsformed by citrate reduction were stable in a colloidal state by arepulsive force which exists along particles and maintained bya net negative charge on their surface. Typically, these chargedparticles were very sensitive to changes in solution dielectric.The presence of cations in salt solutions negated this chargerepulsion and caused these particles to agglomerate andeventually precipitate [28]. Thus, for typical citrate stabilizedparticles, the addition of aqueous NaCl covered the surfacecharge, decreased the interparticle distance and eventuallyinduced particles’ aggregation. Therefore, binding of proteinsto AuNPs or other stabilizing agents to surface of particleswould keep a suspended state by blocking the salt-inducedprecipitation of colloidal AuNPs.

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 045017 V D Pham et al

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Figure 1. (a) SEM micrograph of AuNPs, (b) size distribution histogram of AuNPs, (c) UV-Vis spectrum of colloidal gold solution and(d) EDX spectrum of the AuNPs.

The binding of scFv7 antibody to colloidal AuNPsdepends on the pH of the colloidal gold and the antibodysolutions. To determine the optimal pH for binding, the pHof 2 ml colloidal gold aliquots was adjusted from pH 5 to 11by 1 N NaOH. Purified scFv7 was diluted to a concentrationof 100 µg ml−1 in 3 mM of TRIS-HCl pH 8.0. Then, 100 µlof this stock solution was added to the 7 aliquots of adjustedgold solution and incubated for 15 min followed by additionof 100 µl of a 10% NaCl solution to each of the aliquots toinduce particle precipitation. The pH binding optimum wasdefined at the pH that allowed antibody to bind to AuNPs toprevent their agglomeration by NaCl.

2.5.2. Binding procedure. Binding protocol of scFv7 toAuNPs was carried out according to the method described byHorisberger and Leuvering et al [29]. The optimal concen-tration of antibody scFv7 for conjugation was determined bytitrating aliquots of diluted antibody with colloidal AuNPs.The purified scFv7 was diluted to a concentration of0.1 mg ml−1 in phosphate buffered saline (PBS) buffer(0.001 M, pH 7.4). The pH of colloidal gold solution and thediluted scFv7 was adjusted to pH 8.0 with 0.1 N NaOH. Tenaliquots of variable concentrations (0.01–0.1 mg ml−1) of thediluted antibodies were prepared in 0.1 ml PBS buffer, andadded separately to 1 ml of colloidal gold solution. The tubes

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 045017 V D Pham et al

Figure 2. Expression and purification of recombinant scFv7 and scFv24. The pTI2+ plasmids were transformed into E. coli HB2151 andthe expression of recombinant scFv was induced by IPTG. The expressed recombinant proteins were separated on 12% SDS–PAGE, stainedwith Coomassie blue R-250. (a) SDS-PAGE analysis of induced total bacteria proteins. M: protein marker; lane 1 and 2: the induced totalbacteria proteins; lane 2: the uninduced total bacteria proteins. (b) Characterization of the purified scFv7 (lane 1), scFv24 (lane 2).

were incubated for 15 min and 0.1 ml of 10% NaCl was added.The UV-visible absorbance was recorded and estimated atthe wavelength of around 523 nm. The least amount ofprotein required to sufficiently bind to the colloidal gold wasdetermined from the curves of the absorbance.

After determining the optimal concentration of bindingscFv7, the antibody/gold nanoprobes were prepared. Analiquot (50 µl) of scFv7 prepared in PBS (0.01 M, pH7.4) was added slowly to 2 ml colloidal gold solution pH8(adjusted by 1 N NaOH) followed by the addition of bovineserum albumin (BSA) (100 µl, 10%) under gentle stirringafter 45 min. The mixture was incubated for another 1 h at4 ◦C and then centrifuged (10 000 rpm for 15 min at 4 ◦C)to remove supernatant unconjugated antibody. The pelletobtained was washed with PBS once again. The pellet wasfinally redispersed in 0.5 ml PBS (pH7.4) containing 2% BSAand stored at 4 ◦C.

2.6. Dot blot immunoassay for rapid detection of H5N1surface antigen

The dot blot immunoassay was carried out by using sandwichreaction mechanism in which scFv24 was used as a secondaryantibody (figure 4(a)). A nitrocellulose membrane used fordot blotting test was drawn in a grid by pencil to indicate theregion of blot before spotting 2 µl of different concentrationsof scFv24 antibodies onto the membrane at the center of thegrid. Six areas on the membrane were spotted and allowedto dry in air. The membrane was soaked in a petri dish andimmersed in PBS buffer containing 5% BSA for another45 min to block all non-specific sites. Then, the membranewas washed three times with PBS buffer to remove all theremaining BSA and dried by air. For the dot blot test, themembrane was soaked into the diluted solution containingH5N1 viruses and antibody/gold nanoprobes for rapid specificinteraction.

Surface plasmon properties of red AuNPs were enhancedas the AuNPs moved to scFv24-spotted circular template

due to the sandwich specific interaction. As a result, thecircular templates were directly observed on the surface ofnitrocellulose membrane.

3. Results and discussion

3.1. Characterization of AuNPs

Colloidal AuNPs were synthesized according to standardwet chemical methods using sodium citrate as a reducingagent. Colloidal gold solution appears intensely red in color.A characteristic SPR band of AuNPs was shown in theUV-Vis spectrum (the typical peak at 523 nm) which confirmsthe presence of spherical AuNPs (figure 1(c)). An SEMimage (figure 1(a)) shows mostly monodispersed AuNPswith an average particle diameter of 23 nm (figure 1(b)). Inaddition, EDX spectrum (figure 1(d)) clearly confirms thepresence of pure gold in the sample with the prominentpeaks corresponding to Au element. The prepared colloidalgold solution was stable for months and this stability ofcolloids allowed AuNPs to be used for further conjugationwith antibody without other stabilizing agent.

3.2. Expression and purification of scFv7 and scFv24

The plasmid of pTI2+ was designed to carry DNA fragmentsencoded to generate protein scFv7 and scFv24. Theseplasmids were then transformed into E. coli strain HB2151,and the expression of recombinant scFv proteins was inducedby IPTG (figure 2(a)).

The recombinants scFv7 and scFv24, as expected,have the same molecular weight of approximate 25 kDa.Following sonication and washing with appropriate buffers,inclusion bodies were subjected to purification by affinitychromatography technique employing HiTrap ChelatingHP columns pre-charged with Ni2+, refolded and finallycharacterized by SDS-PAGE. The generated scFv have apurity of 90%. Approximately, 5 and 12.5 mg of refolded

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 045017 V D Pham et al

(a)

(b)

(c) (d)

Figure 3. (a) Photograph of different aliquots of colloidal gold supplemented with different concentrations of antibodies. (b) Spectralanalysis of AuNPs incubated with various concentrations of antibodies. Ten aliquots of variable concentrations of 0.01–0.1 mg ml−1 (curvesfrom (a) to (j), respectively) of the diluted antibodies were prepared in 0.1 ml PBS buffer, and added separately to 1 ml of colloidal goldsolution. SEM micrographs of (c) antibody stabilized AuNPs, compared to (d) citrate AuNPs without antibody.

scFv7 and scFv24 were yielded from 1 l of bacterialsuspension, respectively. The purified scFv7 and scFv24proteins provide useful reagents for further testing theirbioactivity.

3.3. Preparation of antibody labeled AuNPs

3.3.1. Optimal pH for binding. It is evident that opticallytransparent red-colored gold solution was obtained whenappropriate pH was applied. The color change from red to blueoccurred immediately after adding NaCl 1 N at low pH 4 andpH 5, causing the aggregation of the nanoparticles in solution.The optimal pH for binding antibody to colloidal gold wasdetermined between 8.0 and 10.0. At lower pH values, theaddition of the antibody caused the particles to agglomerate,while adjusting the pH of the colloidal gold above 10 resultedin the generation of unstable nanoprobes preparation, since the

addition of large quantities of NaCl caused agglomeration ofthe gold particles before the further addition of protein [30].

3.3.2. Determining optimal concentration of binding antibodyto AuNPs. Figure 3(a) illustrates the color change of goldsuspension containing scFv7 coupled AuNPs with differentratios compared with the dispersed particles. The color ofthe suspension of dispersed AuNPs changed into blue afteraddition of a low amount of antibodies in the presence ofNaCl. In contrast, at enough or excess amount of antibodies,AuNPs remained in a stable state even after addition of NaClto induce precipitation.

The binding of antibody to the colloidal AuNPs exhibitedsaturation kinetics. As shown in figures 3(a) and (b), at5 µg of antibody per 1 ml of gold solution, the antibody wasadequately bound to AuNPs. As the mass of antibody added to

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 045017 V D Pham et al

Figure 4. (a) Scheme of dot blot hybridization between scFv7 labeled AuNPs and H5N1 showing on nitrocellulose membrane. (b) Six dotswere experimentally assayed in serial dilution of antigen of 0.1–1 µg ml−1 (a) to (f), respectively).

the gold solution increased, more protein bound to the AuNPsuntil all of the available binding sites were occupied.

The plasmon peak depends on the extent of colloidaggregation [31]. To monitor stability of the silver colloid,we measured the absorption of the colloid after addition ofdifferent amounts of antibodies. The evolution of UV-Visspectra is shown in figure 3(b). The wavelength of absorptionpeak of citrate-stabilized AuNPs is 523 nm and the adsorptionpeak of stabilized AuNPs with scFv7 is red-shifted by severalnanometers. This is due to the fact that the scFv7 antibodybound to the surface of AuNPs has slightly increased the sizeof particles. As the particles increase in size, the absorptionpeak usually shifts toward the red wavelengths [32]. Theabsorptions curves of aggregated AuNPs are much broaderthan those of stabilized AuNPs and shifted to the largerwavelengths, while their absorption intensity drasticallydecreased. Spectra change in maximum adsorption and theintensity of surface plasmon band determines the optimalamount of antibodies binding to AuNPs. The high stabilityof antibody stabilized AuNPs was further confirmed by SEMcharacterization. As a control, the SEM micrograph of citrateAuNPs without antibody was also taken (figures 3(c) and3(d)). It is clearly seen that the antibody stabilized AuNPsremain as clusters of particles without aggregation. Theaddition of antibody into gold sol allows antibody to bind tothe surface of particles and cause the change in surface energy,as a consequence, this results in a tendency of nanoparticlesto clump together on the substrate in SEM.

3.4. Immunoblotting analysis

Figure 4(a) depicts the sandwich mechanism for detecting HAsurface antigen on the H5N1 virus. The scFv24-immobilizedmembrane was soaked into nanoprobes solution and specificinteraction between antibody stabilized AuNPs and HAsurface antigen was allowed; then this complex furtherinteracted with scFv24 on nitrocellulose membrane. ColloidalAuNPs solution shows a particular color due to collectiveoscillations of the surface electrons induced by visible light ofsuitable wavelength. The intensely red colored dots appeared

within 5–10 min as many AuNPs moved together on thesurface of membrane. The description of the test can bedescribed as follows. First, scFv7 labeled AuNPs specificallyinteracted with HA surface antigen of H5N1 virus to forma complex between nanoprobes and antigen. Second, thecomplex moved forward along the scFv24-spotted membrane,the scFv24 captured other HA surface antigens by specificinteraction and then formed the red-circular templates whichcould be directly observed by the naked eye. The intenselyred color, which was produced by the accumulation ofAuNPs in the dots, as a result, demonstrates the evidencefor specifying nanoprobes in detecting antigen (figure 4(b)).As the concentration of scF24 increases, the color of theAuNPs-blotted dot changes from pale to strong. Therefore,we can speculate that this method is very sensitive at very lowconcentration of H5N1.

4. Conclusion

In this work we have demonstrated a simple method toproduce gold nanoprobes by labeling scFv7 antibody toAuNPs. Spherical monodispersed AuNPs were synthesizedby citrate reduction method with an average size of 23 nm.The scFv antibodies specific to HA surface antigen of H5N1virus were successfully expressed by IPTG induction andpurified by affinity chromatography technique employingHiTrap Chelating HP columns pre-charged with Ni2+. Theoptimal binding concentration of scFv7 antibody to thesynthesized AuNPs occurring at 5 µg of scFv7 antibodyper 1 ml colloidal gold was determined by using UV-visiblespectra. Results showed that gold nanoprobes were tested withhigh activity by dot blot giving a rapid test result within 5 min.Gold nanoprobes demonstrated a well applicable material forfurther development of H5N1 diagnostic kit.

Acknowledgments

This work was supported by VAST’s project ‘Researchon production of rapid detection kit of influenza A virusby application of recombinant single chain antibody scFv’.

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 045017 V D Pham et al

Several experiments were done using equipment of NationalKey Laboratory for Gene technology of Institute ofBiotechnology, VAST.

References

[1] Ligon B L 2005 Semin. Pediatr. Infect. Dis. 16 326[2] World Health Organization Confirmed human cases of avian

influenza A(H5N1) since 28 January 2004 (on the Internet)2005 Jan 26 (cited 2005 Aug 1) Available fromhttp://www.who.int/csr/disease/avian influenza/country/cases table 2005 01 26/en/

[3] Nemchinov L G and Natilla A 2007 Protein Expr. Purif.56 153

[4] Welliver R, Monto A S, Carewicz O, Schatteman E, HassmanM, Hedrick J, Jackson H C, Huson L, Ward P and OxfordJ S 2001 J. Am. Med. Assoc. 285 748

[5] Hay A J, Wolstenholme A J, Skehel J J and Smith M H 1985EMBO J. 4 3021

[6] Claas E C, Osterhaus A D, van Beek R, De Jong J C,Rimmelzwaan G F, Senne D A, Krauss S, Shortridge K Fand Webster R G 1998 Lancet 351 472

[7] Zhang T, Wang C, Zhang W, Gao Y, Yang S, Wang T, ZhangR, Qin C and Xia X 2010 Vaccine 28 3949

[8] Yang X, Hu W, Li F, Xia H and Zhang Z 2005 Protein Expr.Purif. 41 341

[9] Li L, Li B, Cheng D and Mao L 2010 Food Chem.122 895

[10] Zhang X D, Wu D, Shen X, Chen J, Sun Y M, Liu P X andLiang X J 2012 Biomaterials 33 6408

[11] Raoof M, Corr S J, Warna D, Kaluarachchi, Katheryn L,Massey, Briggs K, Zhu C, Cheney M A, Wilson L J andCurley S A 2012 Nanomedicine: Nanotechnol. Biol. Med.8 1096–105

[12] Chang T L, Tsai C Y, Sun C C, Uppala R, Chen C C, Lin C Hand Chen P H 2006 Microelectron. Eng. 83 1630

[13] Rodriguez-Fernandez J, Perez-Juste J, Garcia de Abajo F J andLiz-Marzan L M 2006 Langmuir 22 32

[14] Ding N, Zhao H, Peng W, He Y, Zhou Y, Yuan L and Zhang Y2012 Colloids Surf. A: Physicochem. Eng. Aspects 395 16

[15] Lee H, Joo S, Lee S, Lee C, Yoon K and Lee K 2010 Biosens.Bioelectron. 26 730

[16] Liu M, Yuan M, Lou X, Mao H, Zheng D, Zou R, Zou N, TangX and Zhao J 2011 Biosens. Bioelectron. 26 4294

[17] Rastogi S K, Gibson C M, Branen J R, Eric Aston D, LarryBranen A and Hrdlicka P J 2012 Chem. Commun.48 7714

[18] Yang F, Duan J, Li M, Wang Z and Guo Z 2012 Anal. Sci.28 333

[19] Moon J, Kim G and Lee S 2012 Materials 5 634[20] Wang Y, Fill C and Nugen S R 2012 Biosensors 2 32[21] Walter J G, Petersen S, Stahl F, Scheper T and Barcikowski S

2010 J. Nanobiotechnol. 8 21[22] Pandey S K, Suri C R, Chaudhry M, Tiwari R P and Rishi P

2012 Mol. BioSyst. 8 1853[23] Hou S Y, Chen H K, Cheng H C and Huang C Y 2007 Anal.

Chem. 79 980[24] Thiruppathiraja C, Kumar S, Murugan V, Adaikkappan P,

Sankaran K and Alaga M 2011 Aquaculture 318 262[25] Turkevich J, Stevenson P C and Hillier J 1951 Discuss.

Faraday Soc. 11 55[26] de Wildt R M T, Mundy C R, Gorick B D and Tomlinson I M

2000 Nat. Biotech. 18 989[27] Gahrtz M and Conrad U 2009 Methods Mol. Biol. 483 289[28] Baptista P, Doria G, Henriques D, Pereira E and Franco R

2005 J. Biotechnol. 119 111[29] Horisberger M 1990 Progr. Colloid. Polym. Sci. 81 156[30] Paciotti G F, Myer L, Weinreich D, Goia D, Pavel N,

McLaughlin R E and Tamarkin L 2004 Drug Deliv.11 169

[31] Yamamoto S, Fujiwara K and Watari H S 2004 Anal. Sci.20 1347

[32] Xia Y and Halas N J 2005 MRS Bull. 30 338

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