Organic &Biomolecular Chemistry

PAPER

Cite this: Org. Biomol. Chem., 2013, 11,7101

Received 20th August 2013,Accepted 23rd August 2013

DOI: 10.1039/c3ob41703d

www.rsc.org/obc

Glyconanoparticles for the plasmonic detection anddiscrimination between human and avian influenzavirus†

María J. Marín,‡a Abdul Rashid,‡b Martin Rejzek,b Shirley A. Fairhurst,b

Stephen A. Wharton,c Stephen R. Martin,c John W. McCauley,c Thomas Wileman,d

Robert A. Field*b and David A. Russell*a

A plasmonic bioassay for the specific detection of human influenza virus has been developed based on

gold nanoparticles functionalised with a designed and synthesised thiolated trivalent α2,6-thio-linkedsialic acid derivative. The glyconanoparticles consist of the thiolated trivalent α2,6-thio-linked sialic acid

derivative and a thiolated polyethylene glycol (PEG) derivative self-assembled onto the gold surface.

Varying ratios of the trivalent α2,6-thio-linked sialic acid ligand and the PEG ligand were used; a ratio of

25 : 75 was found to be optimum for the detection of human influenza virus X31 (H3N2). In the presence

of the influenza virus a solution of the glyconanoparticles aggregate following the binding of the triva-

lent α2,6-thio-linked sialic acid ligand to the haemagglutinin on the surface of the virus. The aggregation

of the glycoparticles with the influenza virus induces a colour change of the solution within 30 min.

Non-purified influenza virus in allantoic fluid was successfully detected using the functionalised glyco-

nanoparticles. A comparison between the trivalent and a monovalent α2,6-thio-linked sialic acid functiona-

lised nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the

trivalent ligand for the detection of the X31 virus. Importantly, the glyconanoparticles were able to dis-

criminate between human (α2,6 binding) and avian (α2,3 binding) RG14 (H5N1) influenza virus high-

lighting the binding specificity of the trivalent α2,6-thio-linked sialic acid ligand.

Introduction

Seasonal influenza is the cause of tens to hundreds of thou-sands of human deaths each year.1 However, of particularconcern is the threat of a pandemic caused by the influenzavirus crossing from animal species. A recent example of suchan influenza pandemic is the influenza A (H1N1) ‘swine flu’outbreak of 2009 which caused high morbidity and, in somecases, severe disease and mortality. The influenza virus hastwo types of surface glycoproteins, haemagglutinin (HA) andneuraminidase (NA). The HA recognises sialic acids present onthe surface of host cells and binds to these carbohydrates in

order to infect the cell and the NA releases progeny virus fromthe infected cell.2 Measures to prevent a new influenza viruspandemic involve both vaccination and antiviral drugs, thelatter ideally administered within 48 h of the infection.3,4 Theeffective use of antivirals requires rapid and early diagnosis.Current methods for the detection of influenza include: mole-cular identification of influenza isolates including reverse-tran-scription PCR, immunofluorescence antibody staining, virusisolation in cell culture or in embryonated chicken eggs, andserological diagnosis by haemagglutination inhibition or bymicroneutralisation.4 All of these methods are time-consum-ing, taking several hours or even days for results to beobtained, and also require specialised equipment and trainedanalysts. It is clear that a rapid, diagnostic test that is simpleto perform, works on unpurified samples, and is ideally ableto discriminate between human influenza and emerginganimal strains, such as the avian H5N1 ‘bird flu’ virus thatgenerated considerable concern following its re-emergence in2003–2004 or the avian H7N9 transmitted to humans in 2013,is essential.

The need for a simple diagnostic test encouraged us tofocus on practical and inexpensive gold nanoparticle-based

†Electronic supplementary information (ESI) available: Materials, methods andsupporting results. See DOI: 10.1039/c3ob41703d‡These authors contributed equally to this study.

aSchool of Chemistry, University of East Anglia, Norwich Research Park, Norwich,

Norfolk NR4 7TJ, UK. E-mail: d.russell@uea.ac.ukbDepartment of Biological Chemistry, John Innes Centre, Norwich Research Park,

Norwich, Norfolk NR4 7UH, UK. E-mail: rob.field@jic.ac.ukcMRC National Institute of Medical Research, Mill Hill, London, NW7 1AA, UKdNorwich Medical School, University of East Anglia, Norwich Research Park,

Norwich, Norfolk NR4 7TJ, UK

This journal is © The Royal Society of Chemistry 2013 Org. Biomol. Chem., 2013, 11, 7101–7107 | 7101

colorimetric assays.5 Gold nanoparticles (ca. 16 nm in dia-meter) in aqueous suspension exhibit an intense red colourdue to their surface plasmon absorption band. This opticalproperty is distance-dependent and upon aggregation of themetal nanoparticles the solution changes colour. The colourchange, readily observed with the naked eye, is due to thecoupling interactions between the surface plasmon fields ofthe particles. Gold nanoparticle-based colorimetric assays havebeen reported for the detection of a variety of species, includ-ing oligonucleotides, metal ions, anions, small organic mole-cules and proteins, a field reviewed recently by Rotello et al.6

By functionalising metal nanoparticles with specifically syn-thesised carbohydrate ligands, glyconanoparticles can becreated.7 Previously we have developed glyconanoparticle-based colorimetric assays for the detection of lectins, calciumions, and cholera toxin.8–12

Recently, gold nanoparticles have been used for the inhi-bition and detection of influenza virus. Papp et al. employed14 nm gold nanoparticles functionalised with a sialic-acid-terminated glycerol dendron to inhibit X31 influenza virus (areassortant H3N2 influenza virus carrying the HA and NAgenes of A/Aichi/2/68).13 In addition, gold nanoparticlescoated with a phosphonate ester analogue of the influenzatherapeutic Oseltamivir,14 with mercaptoethanesulfonate andmercaptosuccinic acid,15 and gold nanorods functionalisedwith ssRNA16 have also been used for the inhibition of influ-enza virus. Influenza A/Puerto Rico/8/34 (PR8) (H1N1) virushas been detected using antibody-functionalised gold nano-particles and dynamic light scattering.17 Gold nanoparticlesfunctionalised with a chemically unmodified monomer ofsialic acid have been used to colorimetrically detect influenzaB viruses of the B/Victoria and B/Yamagata lineages throughthe interaction between the sialic acid and the HA on thevirus.18 The reported methods highlight the utility of gold

nanoparticles for the detection of influenza virus in a rapidand easy to perform assay. However, none of the ligands usedto functionalise the nanoparticles has been demonstrated todiscriminate between human and avian strains of influenzavirus.

There is potential to discriminate between human andavian influenza virus strains by virtue of the sialic acid linkagespecificity of the corresponding HAs.2 Human influenza virusbinds preferentially to sialic acid α2,6 galactose sequenceswhile avian influenza virus binds preferentially sialic acidα2,3 galactose sequences.19,20 Additionally, given that viral HAis trivalent in nature,21 we considered trivalent ligands for HAthat could be used as a basis for flu virus detection and typing.

Here we describe the synthesis of glyconanoparticles pres-enting a trivalent α2,6-thio-linked sialic acid (trivalent ligand 1)that can detect strains of human influenza within 30 min ina simple colorimetric assay (Fig. 1). The glyconanoparticlesconsist of the thiolated trivalent ligand 1 and a thiolated poly-ethylene glycol derivative (PEG ligand 2) both self-assembledonto the surface of gold nanoparticles (Fig. 2). Varying ratiosof the trivalent ligand 1 and PEG ligand 2 were used; a ratio of25 : 75 was found to be optimum for the detection of humaninfluenza virus X31 (H3N2). A comparison between gold nano-particles functionalised with the trivalent ligand 1 and par-ticles functionalised with a monovalent α-thio-linked sialicacid (monovalent ligand 3, Fig. S1†) confirmed that the tri-valent ligand 1 provided significantly superior results for thedetection of the human influenza virus. To show the utility ofthe trivalent ligand 1 : PEG (25 : 75) functionalised gold nano-particles for unpurified samples, the particles were used todetect the X31 virus from allantoic fluid (AF) at clinically rele-vant concentrations. Importantly, the α2,6-binding trivalentsialic acid glyconanoparticles were shown to specifically detecthuman rather than avian influenza virus.

Fig. 1 Schematic representation of the aggregation of the glyconanoparticles in the presence of the influenza virus. The trivalent ligand 1 on the surface of thegold nanoparticles binds to the haemagglutinin on the surface of the virus inducing aggregation.

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Results and discussion

Analysis of the crystal structures of influenza virus HA suggestsa distance of ca. 4 nm between the carbohydrate binding sitesof the HA monomers within the HA trimer complex.13,22 Topresent 3 ligands to the 3 HA binding sites without introdu-cing strain into the system, tether lengths of ca. 2–2.5 nm(22–25 bond lengths) between the sialic acid anomeric centreand the middle of the tripodal core are required. Based onthese distance considerations, trivalent ligand 1 was syn-thesised as outlined in Scheme 1. To facilitate the later optimi-sation of ligand presentation, a modular synthetic approachbased on dipolar cycloaddition click chemistry23,24 wasdevised that enabled modification of tether length, compo-sition and rigidity. A related synthetic approach to divalentsialic acid has been previously reported by Kale et al.25 Full

Fig. 2 Schematic representation of trivalent ligand 1 : PEG functionalised goldnanoparticles: gold nanoparticles functionalised with trivalent ligand 1 (black)and PEG ligand 2 (blue).

Scheme 1 Synthesis of trivalent sialic acid derivative (trivalent ligand 1). Reagents and conditions: (a) 10% Pd-C, EtOAc; (b) azidohexanoic acid NHS ester,Et3N, CH2Cl2; (c) NaOMe-MeOH; (d) TBDMS-Cl/DMF; (e) Ac2O/Pyr; (f ) 10% TFA in 80% aq. AcOH; (g) Tf2O/Pyr; (h) Et2NH/DMF; (i) NaOMe/MeOH; ( j) CuSO4-NaAsc-tBuOH/H2O 1 : 1; (k) 1 M aq. NaOH; (l) 80% aq. TFA; and (m) γ-thiobutyrolactone/DTT/0.5 M aq. NaHCO3/EtOH 1.5 : 1.

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experimental details concerning the chemical synthesis oftrivalent ligand 1, PEG ligand 2 and monovalent ligand 3 maybe found in the ESI.† Briefly, known azidopropyl galactoside4 was reduced and acylated with known 6-azidohexanoic acidNHS ester.26 Protecting groups were manipulated, via tert-butyldimethylsilyl (TBDMS) ether 5, to provide access to theunstable triflate 6, which reacted directly with known sialicacid thioacetate 727 in order to access a chemically and biologi-cally stable thioglycoside.28 The resulting α2,6-thio-linkedsialic acid derivative 8 was subjected to CuI-catalysed 1,3-dipolar cycloaddition23,29,30 with tripropargyl ether 9, whichwas prepared through standard transformation of knownN-Boc-tri-O-propargyl-trishydroxymethylaminomethane.31 Globaldeprotection of trivalent compound 10 and finally derivatisationof the resulting primary amine with thiobutyrolactone gave thetrivalent α2,6-thio-linked sialic acid (trivalent ligand 1) for immo-bilisation on gold nanoparticles.

The synthesis of the gold nanoparticles (ca. 16 nm diameter)was achieved using citrate as both the reducing agent and thestabiliser of the gold core.32 Citrate-reduced gold nanoparticleswere functionalised with a mixed monolayer (Fig. 2) consistingof the thiolated trivalent α2,6-linked sialic acid (trivalent ligand 1)and a thiolated polyethylene glycol derivative (PEG ligand 2).To establish whether the binding of the influenza virus to thetrivalent ligand 1 on the surface of the gold nanoparticles wasaffected by ligand density, varying ratios of the trivalent ligand1 : PEG (50 : 50, 25 : 75, 10 : 90, 5 : 95 and 2 : 98) were used tofunctionalise the particles. TEM images of the functionalisedgold nanoparticles showed disperse nanoparticles with anaverage size of 16.4 ± 1.6 nm (Fig. S2†). The nanoparticle solu-tions, with varying ligand density, were all deep red in colourand exhibited a surface plasmon absorption band at ca.525 nm. The surface plasmon absorption band was red-shiftedby ca. 5 nm, as compared to that observed for the citrate-coated nanoparticles, due to the assembly of the monolayer onthe gold surface. Human influenza virus X31 (2.55 µg mL−1)was added to the various trivalent ligand 1 : PEG function-alised gold nanoparticles. Citrate coated gold nanoparticleswere used as a control. The UV-Vis extinction spectrum of eachsample was measured before and 0, 15, 30, 60 and 240 minafter addition of the virus (Fig. S3†). The greatest change in thesurface plasmon absorption band was observed when thenanoparticles functionalised with trivalent ligand 1 : PEG of aratio 25 : 75 interacted with the human influenza virus X31 for240 min (Fig. 3a). The UV-Vis extinction spectrum in Fig. 3ahighlights the broadening of the surface plasmon absorptionband of the 25 : 75 ratio trivalent ligand 1 : PEG gold nanoparticlesindicating significant interaction with the influenza virus. Thesame experiment was performed using gold nanoparticlesfunctionalised with different ratios of monovalent ligand3 : PEG ligand 2 (50 : 50, 25 : 75, 10 : 90, 5 : 95 and 2 : 98). Theresults obtained suggest that a 25 : 75 ratio of the monovalentligand 3 : PEG was also the optimum ligand density (Fig. S4†).

The optimised trivalent ligand 1 : PEG glyconanoparticleswere used to colorimetrically detect increasing concentrationsof the X31 influenza virus. As shown in Fig. 3b, upon addition

of the human influenza virus X31 the surface plasmon absorp-tion band red-shifted (from 525 to 536 nm) and decreased inintensity with increasing concentration of the virus (Fig. 3band Fig. S5a†). The results suggest that the influenza virusinduces aggregation of the glyconanoparticles as schematicallyshown in Fig. 1. The aggregation of the optimised glyconano-particles was spectroscopically measured 30 min followingaddition of increasing virus concentration. Changes of thesurface plasmon absorption band due to the addition of thevirus led to changes of solution colour, from the initial deepred to lighter red (Fig. S5b†). A comparison of the bindingaffinity of the trivalent ligand 1 : PEG (25 : 75) and monovalentligand 3 : PEG (25 : 75) functionalised gold nanoparticlestowards human influenza virus X31 was made. The addition ofhuman influenza virus X31 to a solution of the monovalentligand 3 : PEG (25 : 75) functionalised gold nanoparticles pro-duced a small decrease and red-shift (from 525 to 534 nm) ofthe surface plasmon absorption band intensity with anincrease in the extinction at ca. 620 nm (Fig. 3c and Fig. S5a†).These results indicate the initiation of the aggregation of thenanoparticles. However, a significantly greater concentrationof virus was required when the monovalent ligand 3 was usedto produce the same change of the extinction spectrum as thatobserved with the trivalent ligand 1 : PEG functionalised goldnanoparticles. The control experiment of gold nanoparticlesfunctionalised only with PEG ligand 2 (Fig. S6†) induced negli-gible changes of the extinction spectrum following addition ofincreasing concentrations of the influenza virus X31 (Fig. 3dand Fig. S5a†). These results highlight the increased affinity ofthe trivalent sialic acid ligand 1 : PEG glyconanoparticlestowards the human influenza virus X31 as compared to themonovalent sialic acid ligand 3 : PEG functionalised particles.Further, the control experiments show that the presence of asialic acid derivative is essential to bind the glyconanoparticlesto the virus via HA binding sites to facilitate plasmonic detec-tion of the virus.

To mimic clinical samples of unknown concentration andpurity, colorimetric detection of the human influenza virusX31 from influenza allantoic fluid (AF) was achieved using thetrivalent ligand 1 : PEG (25 : 75) functionalised gold nanoparti-cles. The addition of increasing volumes of X31 influenza AFto a sample of the functionalised gold nanoparticles inducedthe aggregation of the optimised glyconanoparticles. Uponincreasing volume of the X31 influenza AF, a decrease in theextinction intensity of the surface plasmon absorption band atca. 525 nm with a consequent solution colour change fromdeep red to colourless was observed (Fig. 4a). A controlexperiment, with increasing volumes of Tris buffer added tothe trivalent ligand 1 : PEG (25 : 75) functionalised gold nano-particles, confirmed that the changes observed were due to thepresence of the X31 AF and not to a dilution effect (Fig. S7†).

The primary intention of synthesising the α2,6-configuredtrivalent sialic acid functionalised gold nanoparticles was tocreate sensors that would discriminate between human andavian influenza virus. Trivalent ligand 1 was synthesised con-taining three α2,6-thio-linked sialic acids. Human influenza

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Fig. 4 UV-Vis extinction spectra of: (a) trivalent ligand 1 : PEG (25 : 75) functionalised gold nanoparticles following addition of increasing volumes of influenza AFX31 (H3N2) (from 0 to 43.1 µL), inset: cuvettes containing trivalent ligand 1 : PEG (25 : 75) functionalised gold nanoparticles before (left) and following (right)addition of AF X31 (43.1 µL); and (b) trivalent ligand 1 : PEG (25 : 75) functionalised gold nanoparticles following addition of increasing concentrations of avian virusRG14 (H5N1) (from 0 to 3.1 µg mL−1).

Fig. 3 (a) UV-Vis extinction spectra of gold nanoparticles functionalised with citrate only (black) and with trivalent ligand 1 : PEG ratio of: 2 : 98 (red), 5 : 95 (green),10 : 90 (blue), 25 : 75 (magenta) and 50 : 50 (orange) measured 240 min after addition of influenza virus X31 (2.55 µg mL−1). Variation of the UV-Vis extinction spec-trum of gold nanoparticles functionalised with: (b) trivalent ligand 1 : PEG (25 : 75), (c) monovalent ligand 3 : PEG (25 : 75) and (d) PEG, following addition of increas-ing concentrations of purified influenza virus X31 (from 0 to 3.0 µg mL−1); measurements were made 30 min following virus addition.

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virus binds preferentially to α2,6 residues while avian influ-enza virus binds to α2,3 residues.19,20 Consequently, the opti-mised glyconanoparticles should not aggregate in the presenceof avian influenza virus. A reassortant influenza virus, RG14with HA and NA genes from an avian virus A/Vietnam/1194/2004 (H5N1), was added at increasing concentrations (from0 to 3.1 µg mL−1) to a solution of trivalent ligand 1 : PEG (25 : 75)functionalised gold nanoparticles. Changes in the surfaceplasmon absorption band were monitored 30 min afteraddition of the virus. As shown in Fig. 4b, no changes in thesurface plasmon absorption band of the functionalised goldnanoparticles were observed after addition of 3.1 µg mL−1 ofthe avian virus RG14. With a similar concentration of humaninfluenza virus X31, the spectroscopic characteristics of thesurface plasmon absorption band of the functionalised goldnanoparticles substantially changed following binding andaggregation of the glyconanoparticles (Fig. 3b). To furtherdemonstrate that the synthesised glyconanoparticles bindspecifically to human influenza, the concentration of avianinfluenza RG14 was increased to 6.8 µg mL−1 in a solution offunctionalised gold nanoparticles. Negligible changes of thesurface plasmon absorption band of the particles wereobserved (Fig. S8a†). The avian virus was incubated with thenanoparticles for a period of 6 days, after which the UV-Visextinction spectrum of the particles was measured. Only asmall decrease in the extinction intensity was observed(Fig. S8b† – red) as compared to the change of the surfaceplasmon absorption band caused by the presence of the sameconcentration, and same incubation time, of human influenzavirus X31 (Fig. S8b† – green). From these results it is apparentthat the designed trivalent ligand 1 : PEG (25 : 75) functiona-lised gold nanoparticles can readily distinguish betweenhuman and avian influenza virus strains.

Conclusions

In summary, we have achieved the synthesis of a thiolated tri-valent α2,6-thio-linked sialic acid derivative to functionalisegold nanoparticles. The optimised glyconanoparticles consistof the thiolated trivalent α2,6-thio-linked sialic acid derivativeand a thiolated PEG derivative self-assembled onto the goldsurface in a 25 : 75 ratio. These glyconanoparticles were usedfor the plasmonic detection of influenza virus. The trivalentligand 1 : PEG (25 : 75) functionalised gold nanoparticles wereused to detect the human influenza virus X31 (H3N2) within30 min. Non-purified, influenza virus in allantoic fluid wassuccessfully detected by the functionalised nanoparticles. Acomparison between the trivalent and a monovalent α2,6-thio-linked sialic acid functionalised nanoparticles confirmed thatmore rapid results, with greater sensitivity, were achievedusing the trivalent ligand for the detection of the X31 virus.Importantly, the trivalent ligand 1 : PEG (25 : 75) functionalisedgold nanoparticles were able to discriminate between human(α2,6 binding) and avian (α2,3 binding) influenza. Since thedominant strain of human influenza varies seasonally, and

with the possible threat of influenza virus crossing betweenanimal species and thereby potentially initiating a pandemic,the ability to distinguish between human and avian influenzavirus strains is exceptionally important. The synthesis of a tri-valent α2,6-thio-linked sialic acid derivative to functionalisegold nanoparticles provides an innovative bioassay for thespecific recognition and detection of influenza virus strains inclinical samples.

Acknowledgements

We thank Dr Milton Kiefel (Griffith University) for invaluableadvice on sialic acid chemistry, Dr Lionel Hill (John InnesCentre) for assistance with mass spectrometry analyses and DrColin McDonald (University of East Anglia, UEA) for assistancewith the TEM images. MJM acknowledges the School of Chem-istry, UEA, for her studentship. MJM and DAR, AR and RAF,thank the UEA and PBL, respectively, for ‘Proof of Concept’financial support. Elements of this work were underpinnedthrough the JIC MET programme (ISPG ref number BB/J004561/1) from BBSRC, and the John Innes Foundation.

Notes and references

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