Gold Nanoparticles Decorated with Sialic Acid Terminated Bi-antennary N-Glycans for the Detection of Influenza Virus at Nanomolar Concentrations

Article abstract of DOI:10.1002/open.201500109

Gold nanoparticles decorated with full-length sialic acid terminated complex bi-antennary N-glycans, synthesized with glycans isolated from egg yolk, were used as a sensor for the detection of both recombinant hemagglutinin (HA) and whole influenza A virus particles of the H1N1 subtype. Nanoparticle aggregation was induced by interaction between the sialic acid termini of the glycans attached to gold and the multivalent sialic acid binding sites of HA. Both dynamic light scattering (DLS) and UV/Vis spectroscopy demonstrated the efficiency of the sensor, which could detect viral HA at nanomolar concentrations and revealed a linear relationship between the extent of nanoparticle aggregation and the concentration of HA. UV/Vis studies also showed that these nanoparticles can selectively detect an influenza A virus strain that preferentially binds sialic acid terminated glycans with α(2→6) linkages over a strain that prefers glycans with terminal α(2→3)-linked sialic acids.

Full text of DOI:10.1002/open.201500109

DOI: 10.1002/open.201500109
Gold Nanoparticles Decorated with Sialic Acid Terminated
Bi-antennary N-Glycans for the Detection of Influenza
Virus at Nanomolar Concentrations**
[
a, b]
[d]
[d]
[a, b]
Vivek Poonthiyil,
Prashanth T. Nagesh, Matloob Husain, Vladimir B. Golovko,*
and
[a, c]
Antony J. Fairbanks*
Gold nanoparticles decorated with full-length sialic acid termi-
nated complex bi-antennary N-glycans, synthesized with gly-
cans isolated from egg yolk, were used as a sensor for the de-
tection of both recombinant hemagglutinin (HA) and whole in-
fluenza A virus particles of the H1N1 subtype. Nanoparticle ag-
gregation was induced by interaction between the sialic acid
termini of the glycans attached to gold and the multivalent
sialic acid binding sites of HA. Both dynamic light scattering
(DLS) and UV/Vis spectroscopy demonstrated the efficiency of
the sensor, which could detect viral HA at nanomolar concen-
trations and revealed a linear relationship between the extent
of nanoparticle aggregation and the concentration of HA. UV/
Vis studies also showed that these nanoparticles can selectively
detect an influenza A virus strain that preferentially binds sialic
acid terminated glycans with a(2!6) linkages over a strain
that prefers glycans with terminal a(2!3)-linked sialic acids.
Introduction
Constructs comprised of metal nanoparticles attached to a vari-
ety of chemical entities with specific binding properties have
as localized surface plasmon resonance (LSPR) and their high
[
5]
extinction coefficients.
[
1]
attracted significant interest in the fields of therapeutics, di-
The distinctive color change of a AuNP solution in the pres-
ence of a specific analyte, upon either aggregation (red to
purple/blue) or aggregate re-dispersion (purple/blue to red),
[
2]
[3]
agnostics, and sensing. Gold nanoparticles (AuNPs) are par-
ticularly well suited to such applications for a number of rea-
sons: there are well-established methods for the synthesis of
AuNPs with defined sizes and shapes, it is easy to modify their
surfaces, and they have excellent stability and biocompatibil-
[
6]
makes AuNPs an ideal choice as a colorimetric sensor. Such
sensors based on AuNP aggregation have been used for
a wide variety of analytes including low-molecular-weight spe-
cies, such as metal ions, anions, amino acids and small-mole-
cule pharmaceuticals, as well as larger species, such as pep-
[4]
ity. In particular, interest in the application of modified AuNPs
as colorimetric sensors has increased exponentially over the
last two decades due to some of their unique properties, such
[
7]
tides, proteins, DNA, and single cells.
Dynamic light scattering (DLS, also known as photon correla-
tion spectroscopy) is a noninvasive technique used to measure
[
a] V. Poonthiyil, Dr. V. B. Golovko, Prof. A. J. Fairbanks
Department of Chemistry, University of Canterbury
Private Bag 4800, Christchurch 8140 (New Zealand)
E-mail: antony.fairbanks@canterbury.ac.nz
[
8]
the size distribution of proteins, polymers, and nanoparticles.
AuNPs have strong light-scattering properties; for instance,
a AuNP with a diameter of 60 nm has a light-scattering cross-
section 200–300 times larger than that of a polystyrene bead
of the same size, and is 4–5 orders of magnitude larger than
vladimir.golovko@canterbury.ac.nz
[
b] V. Poonthiyil, Dr. V. B. Golovko
The MacDiarmid Institute for Advanced Materials and Nanotechnology
Wellington 6140 (New Zealand)
[
9]
that of a florescent dye, such as fluorescein. DLS has there-
fore been extensively used as a means of detection of AuNPs
[
c] Prof. A. J. Fairbanks
Biomolecular Interaction Centre, University of Canterbury
Private Bag 4800, Christchurch 8140 (New Zealand)
[
10]
as the basis of sensors for various analytes.
The influenza virus is a respiratory pathogen and causes an
acute febrile respiratory infection in humans and other mam-
mals. Influenza causes regular seasonal epidemics and occa-
[
d] P. T. Nagesh, M. Husain
Department of Microbiology and Immunology, University of Otago
PO Box 56, Dunedin 9054 (New Zealand)
[
11]
sional pandemics in the human population. Recent reports
[
**] This article is part of the Virtual Special Issue “Carbohydrates in the 21st
show that seasonal influenza epidemics result in ~300000
Century: Synthesis and Applications”.
[
12]
human deaths annually. The influenza virus particle contains
two types of surface glycoprotein: neuraminidase (NA) and he-
magglutinin (HA), and influenza A viruses are subtyped accord-
ing to the variations of these two glycoproteins, for example
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/open.201500109.
ꢀ
2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
[13]
as H1N1. Viral HA is an integral membrane glycoprotein and
has a trimeric binding pocket on the globular head of each
[
14]
monomer. HA from the influenza virus is made up of ~556
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amino acids, and accounts for more than 80% of the envelope
Results and Discussion
[
11,15]
proteins of the virus.
One of the essential steps in influen-
za virus infection is binding of the viral particles to host
Extraction of SGP and conversion into a thiol
[
16]
cells, a process mediated by specific interactions between
the HA of the virus and sialic acid residues present on the host
Extraction from 300 egg yolks, following the procedure of
[17]
[22]
cell. It has been found that human-adapted influenza viruses
Huang and colleagues, yielded 1.200 g of SGP 1 (Scheme 1).
preferentially bind to sialylglycans with terminal a(2!6) link-
Incubation of 1 with pronase (from Streptomyces griseus) in
Tris·HCl buffer (pH 7.5) at 378C then afforded the asparagine-
linked complex bi-antennary N-glycan 2. Reaction of thiogly-
[
16]
ages. Whilst the binding constant of HA for a single sialic
3
À1
[24]
acid residue is relatively weak (10 m ), the binding constant
between the multiple HAs of a viral particle with the multiple
sialic acids present on a host cell has been estimated to be
colic acid 3 with acetyl chloride and triethylamine in 1,4-diox-
[
25]
ane afforded S-acetylthioacetic acid 4. Reaction of 4 with N-
13
À1 [18]
1
0 m .
Thus, a multivalent effect significantly increases
hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) in
[
26]
binding and prevents dissociation of the virus from the host
THF gave the activated ester 5, which was then reacted with
2 in sodium phosphate buffer (pH 7.4) containing 20% aceto-
nitrile to give thioacetate 6. Deacetylation of 6 by treatment
with hydroxylamine hydrochloride followed by air oxidation af-
forded disulfide 7, which could typically be made on a multi-
hundred-milligram scale.
[18b]
cell.
Glycogold nanoparticles (gAuNPs, that is, AuNPs attached to
saccharides or glycoconjugates) have been used as sensors for
[19]
various biomolecules and toxins, including the detection of
[20]
pathogenic agents such as bacteria and viruses. Indeed, the
detection of both the influenza virus itself and viral HA using
[18b,21]
gAuNPs was reported recently.
The majority of previous
Synthesis and characterization of SG-gAuNPs
studies have involved AuNPs decorated with synthetic oligo-
saccharides of various lengths that were terminated with sialic
acid. Although none of these materials corresponded to full-
length N-glycans typically found on the mammalian cell sur-
face, their syntheses were nonetheless typically time-consum-
Citrate-capped AuNPs (diameter, Ø: 12 nm) were synthesized
[
27]
by using the Turkevich reaction. Ligand exchange with disul-
[
19c]
fide 7 then gave 12-nm-diameter SG-gAuNPs;
ligand ex-
change is driven by the higher binding affinity of gold for
a thiol than for citrate. The FTIR spectrum of the sialylglycan-
capped SG-gNPs was similar to that of the free disulfide 7, indi-
cating both the removal of citrate and the successful incorpo-
ration of sialylglycans on the surface of the NPs (see Support-
[18b,21b,d]
ing, expensive, and logistically demanding.
Additionally,
in the case where sialic acid itself was used as the ligand for
[
20a]
gAuNPs,
the selective detection of human-adapted influen-
za viruses that prefer a(2!6)-linked sialylglycans is not possi-
ble.
[
28]
ing Information Figures S5 and S6). The particle sizes and
size distributions (as demonstrated by TEM) and the UV/Vis ab-
sorption spectra of the AuNPs were essentially the same both
before and after ligand exchange, indicating that the AuNP
cores had not been affected. Analysis of the TEM images of
the SG-gAuNPs (at least 200 particles were measured) revealed
the particle sizes to be 12.1Æ1.6 nm (Figure 1a). The absorp-
tion maximum of the SG-gAuNPs in the UV/Vis spectrum was
at 523 nm (Figure S3), whilst ESI-MS also confirmed that the
sialylglycan had been successfully incorporated into the SG-
gAuNPs. The average molecular formula of the SG-gAuNPs was
determined by TGA (Figure S8, Supporting Information) to be
Au54464(C H N O S)12404, which was confirmed by elemental
In this study we isolated a sialylglycopeptide (SGP, a sialic
acid terminated bi-antennary complex-type N-glycan attached
to a short peptide) from egg yolks, following an improved pro-
[22]
cedure recently reported by Huang and co-workers. The SGP
was converted into a thiol-terminated sialylglycan, which was
then used for the development of a simple and rapid sialylgly-
can-capped gold nanoparticle (SG-gAuNP)-based sensor for HA
and virus detection, using both UV/Vis spectroscopy and DLS.
To our knowledge, this is the first report of the synthesis of
gAuNPs decorated with naturally occurring full-length sialic
acid terminated N-glycans and their application as sensors for
HA and the influenza virus. Whilst UV/Vis spectroscopy allowed
easy and rapid detection of HA by measuring the red shift of
the LSPR band, DLS provided quantitative information on the
size of the AuNP aggregates that were formed in the presence
90
29]
14
8
65
[
analysis.
[
23]
of HA. UV/Vis studies also showed that these nanoparticles
could selectively detect an influenza A virus that preferentially
binds sialic acid terminated glycans with a(2!6) linkages over
an influenza virus that prefers glycans with terminal a(2!3)-
linked sialic acids. The study reported herein opens a new
avenue for the development of an easy-to-use, inexpensive, se-
lective, highly sensitive, and rapid sensing platform for the de-
tection of the influenza virus.
Figure 1. Representative TEM images of the SG-gAuNPs a) before and
b) after the addition of HA.
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Scheme 1. Isolation and synthesis of the sialyloligosaccharide disulfide ligand 7.
SG-gAuNP aggregation in the presence of HA
A pictorial representation of the HA-induced aggregation of
SG-gAuNPs, based on previous reports on similar analyte-in-
duced aggregation-effected sensors, is depicted in Fig-
[
6,21d]
ure 2.
The performance of any gAuNP-based colorimetric
sensing system depends on how effectively the gAuNP disper-
sion is converted into gAuNP aggregates by the analyte of in-
terest. Typically, gAuNPs are stabilized against attractive
van der Waals forces by the steric effects of the surface cap-
ping ligands: in this study, the sialic acid terminated complex
[5a]
bi-antennary N-glycans. Aggregation of gAuNPs can be ach-
ieved in two ways, either by inter-particle crosslinking or by
[
5a]
non-crosslinking.
Inter-particle crosslinking aggregation is
presumed to be the basis of the sensing response reported
here; controlled aggregation of the SG-gAuNPs occurs through
binding of the multiple sialic acid binding sites of HA to the
Figure 2. Schematic representation of the mechanism of aggregation of SG-
gAuNPs in the presence of HA.
[
16]
sialic acid terminated ligands present on the SG-gAuNPs.
Indeed the addition of excess sialic acid (10 mm) to a solution
of SG-gAuNP aggregates formed in the presence of HA
(
564 nm) resulted in aggregate re-dispersion, as demonstrated
SPR peak maximum to l=523 nm. The dissociation of these
by a color change (from blue to red) and by a blue-shift in the
aggregates to individual particles in the presence of sialic acid
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showed that binding of the SG-gAuNPs to HA is reversible, Colorimetric detection of influenza HA using SG-gAuNPs
and also provides strong supporting evidence for the mecha-
nism of aggregation depicted in Figure 2. With respect to ag- When HA was added to the SG-gAuNPs (3 nm) to give a final
gregation, TEM analysis also clearly showed that, following the HA concentration of 71 nm, the LSPR band peak maximum of
addition of HA, the SG-gAuNPs were no longer present as indi- SG-gAuNPs centered at l=523 nm did not undergo any signif-
vidual well-separated particles (Figure 1a), but had assembled icant shift, either immediately or upon standing (Figure S3,
into networks of aggregated NPs (Figure 1b). HA detection Supporting Information). However, when the final concentra-
was also possible simply with the naked eye, as the color of tion of HA was increased to 141 nm, the SPR peak began to
the colloidal solution changed from red to purple upon SG- shift gradually in a time-dependent fashion, indicating aggre-
gAuNP aggregation (Figure 3).
gation of the SG-gAuNPs (Figure 4a). After 60 min no further
change in the absorbance spectrum was observed, and the
LSPR band peak maximum had shifted to l=531 nm. When
the HA concentration was further increased to 283, 424, and
5
64 nm, the LSPR band peak maximum underwent even more
pronounced shifts to 534, 539, and 544 nm, respectively, over
the same time period (60 min), indicating the formation of pro-
gressively larger SG-gAuNP aggregates as the HA concentra-
tion was increased (Figure 4b–d).
A further demonstration of the effect of the HA concentra-
tion on the LSPR band of the SG-gAuNPs, by plotting the ratio
of the absorbance intensity measured at l=650 nm both with
HA
0
and without HA [(I /I )650] against the HA concentration be-
tween 71 and 564 nm, is shown in Figure 5. These data show
a linear relationship between the concentration of HA added
and the degree of SG-gAuNP aggregation. This change in ab-
Figure 3. SG-gAuNP solutions a) before and b) after the addition of HA.
Figure 4. UV/Vis absorption spectra of SG-gAuNP with HA at concentrations of a) 141, b) 283, c) 424, and d) 564 nm.
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Figure 5. Plot of the ratio of absorbance intensity at 650 nm both with and
without HA versus HA concentration for SG-gAuNP. Data points are the aver-
ageÆSEM of three independent measurements.
sorbance can therefore be used to estimate the concentration
[
21d]
of HA in an unknown sample. Wei et al.
reported that at HA
Figure 6. Plot of the absorbance intensity at 650 nm versus time for SG-
gAuNPs with two different influenza virus strains as well as a negative con-
trol.
À1
concentrations greater than 4.2 mgmL (equivalent to 71 nm),
AuNPs stabilized with synthetic mimics of sialylglycans were
red shifted. Figure 5 demonstrates that the SG-gAuNPs report-
ed here are similar in terms of detection efficiency, and that
the sensitivity is similar to the results obtained from traditional
A further demonstration of the effect of the influenza virus
concentration on the LSPR band of the SG-gAuNPs is shown in
Figure 7, which plots the ratio of the absorbance intensity
measured at l=650 nm both with and without the New Cale-
[30]
methods such as ELISAs and glycan microarrays. However,
considering the logistically demanding and time-consuming
chemoenzymatic synthetic procedure required to make the sia-
[21d]
HA 0
lylglycan mimics in the report of Wei et al.,
the use of natu-
donia strain [(I /I )650] against the New Caledonia strain con-
centration between 1000 and 30000 PFU. These data show
a linear relationship between the concentration of virus added
and the degree of SG-gAuNP aggregation, which is similar to
the case of HA shown in Figure 5. Additionally, the binding of
the SG-gAuNPs to the virus was found to be faster than to HA.
The virus–SG-gAuNP interaction was complete within 30 min,
rally occurring full-length N-glycans reported herein offers
a simpler and more attractive method for the development of
an AuNP-based sensor for HA detection.
Colorimetric detection of the influenza virus
[
21d]
It is known that in general avian-adapted influenza A viruses
prefer sialylglycans with a(2!3) linkages that are present in
the intestinal epithelial cells of birds, whilst human-adapted in-
fluenza A viruses prefer a(2!6) linkages, which are commonly
in line with the report of Wei et al.
It is clear from Figure 7
that the SG-gAuNPs used here can readily detect the New Ca-
ledonia strain in amounts as low as 10000 PFU.
[16]
expressed in the upper respiratory tract of humans. To inves-
tigate any selectivity of the SG-gAuNPs produced in this study,
UV/Vis experiments were performed with influenza virus strains
A/Puerto Rico/8/34 (H1N1) and A/New Caledonia/20/1999
(
H1N1). The A/Puerto Rico/8/34 strain (hereafter referred as the
Puerto Rico strain) specifically binds a(2!3)-linked sialylgly-
cans, whilst the influenza A/New Caledonia/20/1999 strain
[
31]
(
hereafter referred as the New Caledonia strain) has specificity
[32]
for a(2!6)-linked glycans.
Upon the addition of 10000
plaque-forming units (PFU) of the New Caledonia strain to the
SG-gAuNPs (3 nm), there was a significant increase in the ab-
sorbance intensity at l=650 nm (Figure 6). However, addition
of the Puerto Rico strain at the same PFU number (and also at
3
0000 PFU; data not shown) did not result any increase in the
Figure 7. Plot of the ratio of the absorbance intensity at 650 nm with and
without the New Caledonia strain versus the New Caledonia viral concentra-
tion. Data points are the averageÆSEM of three independent measure-
ments.
absorbance intensity at l=650 nm, confirming that the SG-
gAuNPs selectively detected viral particles that have a(2!6)
specificity (Figure 6).
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DLS detection of HA using SG-gAuNPs
mean diameters of the SG-gAuNP aggregates formed corre-
spondingly increased to 183Æ5, 389Æ11, and 596Æ21 nm re-
spectively (Figure 8b–d). The larger standard deviation ob-
served for the larger aggregates indicates that the polydispers-
ity of the aggregates also increased with increasing HA con-
centration.
The aggregation of SG-gAuNPs by the addition of varying con-
centrations of viral HA was also investigated by DLS, a tech-
nique that can provide a quantitative estimate of the average
[33]
diameter of the AuNP aggregates produced. As the ability of
AuNPs to scatter light is significantly higher than that of bio-
molecules, DLS is selective for the AuNPs present in these solu-
A plot of the size of SG-gAuNP aggregates measured by DLS
versus HA concentration (Figure 9) was found to be linear, with
[
10a]
2
tions.
In the DLS assays carried out in this study, the HA-in-
an R value of 0.9748. This linear relationship can therefore be
duced SG-gAuNP aggregates were not physically separated
from any remaining non-aggregated NPs that may have been
present. However, the inherent bias of DLS toward larger parti-
cles and aggregates means that estimated diameters are repre-
used to estimate an unknown concentration of HA, in a similar
fashion to the change in absorbance intensity at l=650 nm
using UV/Vis (as shown in Figure 5). The proportionality con-
stants of the two corresponding linear equations, 0.007 and
1.143 for the colorimetric and DLS assays respectively, indicate
that the DLS assay is more sensitive toward nanoparticle ag-
[21c]
sentative of only the particle aggregates.
From the DLS measurements, the size of the “as made” SG-
gAuNPs was found to be 21Æ1 nm (Figure S4a, Supporting In-
formation). The diameter of the AuNPs in solution measured
by DLS is always larger than the AuNP core diameters mea-
[
8c,36]
gregation, in accordance with previous reports.
[
34]
sured by TEM because organic ligands on the Au surface are
transparent to electrons and so do not contribute to the size
[23]
of the AuNPs when measured by TEM.
In contrast, DLS
measures the hydrodynamic radius of the particles, which in
this case includes the sialylglycan ligands and any trapped
[
35]
water molecules.
When HA was added to SG-gAuNPs (3 nm) to obtain a final
HA concentration of 71 nm, a DLS assay gave a particle size of
2
1Æ1 nm, indicating that no aggregation had occurred at that
HA concentration (Figure S4b, Supporting Information). All
DLS measurements were carried out 60 min after the addition
of HA to the SG-gAuNP solution, as after that point the LSPR
band of the solutions remained constant. When the final HA
concentration was increased to 141 nm, DLS indicated that the
mean diameter of the SG-gAuNPs had increased to 97Æ4 nm,
clearly indicating aggregate formation in the presence of the
HA at this concentration (Figure 8a). When the final HA con-
centration was further increased to 283, 424, and 564 nm, the
Figure 9. Plot of the SG-gAuNP aggregate size versus concentration of HA
added. Data points are the averageÆSEM of three independent measure-
ments.
Figure 8. DLS measurements of the average diameters of the SG-gAuNP aggregates formed in the presence of HA at concentrations of a) 141, b) 283, c) 424,
and d) 564 nm.
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Selectivity of the SG-gAuNPs for HA
TOF mass spectrometer. Thermogravimetric analysis (TGA) was per-
formed with an Alphatech SDT Q600 TGA/DSC apparatus on 6–
8
mg of purified dry materials (alumina crucible was used as the
To demonstrate the selectivity of HA/virus detection by the SG-
gAuNPs, aliquots of a SG-gAuNP solution were mixed with
bovine serum albumin (BSA) and the lectins concanavalin A
À1
sample holder), under N (at a flow rate of 100 mLmin ), record-
ing data from 25 to 10008C at a heating rate of 108Cmin . Ele-
2
À1
mental analyses were performed by the Campbell Microanalytical
Laboratory at the University of Otago. TEM images of AuNPs were
obtained using a Philips CM200 TEM operating at 200 kV. Samples
for TEM imaging were prepared by dropping 2 mL of a freshly pre-
pared solution of nanostructured Au onto a holey carbon-coated
copper grid (300 mesh, SPI Supplies, USA) followed by drying at
room temperature. UV/Vis absorption spectra were recorded with
a Varian Cary 100 UV/Vis spectrophotometer or a multimode
reader (Varioskan Flash, Thermo Scientific). DLS measurements
were performed using a Malvern Zetasizer Nano ZS DLS system
(
Con A) and the B subunit of heat-labile enterotoxin (each at
00 nm). It was found that none of these species had any
6
effect on either the UV/Vis absorption band or the DLS meas-
urements, confirming that the SG-gAuNPs did not undergo
nonspecific aggregation, and that amongst the species investi-
gated detection was selective for HA and virus.
Conclusions
(
Malvern Instruments Ltd., UK). The DLS system is equipped with
a l=633 nm He–Ne laser and an avalanche photodiode detector
configured to collect backscattered light at 1738.
AuNPs capped with naturally occurring sialic acid terminated
complex bi-antennary N-glycans can be used for the simple
and highly selective detection of viral HA and the influenza
virus, by the use of both DLS and UV/Vis spectroscopy. A ho-
mogeneous sialylglycopeptide, isolated from egg yolks by
a simple procedure, was converted into a thiol and then used
in a ligand-exchange reaction with citrate-capped AuNPs to
obtain SG-gAuNPs. The presence of sialic acid binding sites on
viral HA, and their specific and multivalent binding to sialic
acid residues at the termini of the ligands on the SG-gAuNPs,
caused nanoparticle aggregation as the basis for HA/virus de-
tection. Both UV/Vis and DLS measurements revealed a linear
relationship between SG-gAuNP aggregation and the concen-
tration of HA that was added. Additionally, the SG-gAuNPs
used in this study selectively detected viral particles with bind-
ing specificity for a(2!6)-linked sialylglycans. The SG-gAuNPs
did not undergo nonspecific aggregation in the presence of
BSA or two lectins. The SG-gAuNP-based sensor reported
herein should allow the development of an easy-to-use, inex-
pensive, selective, and highly sensitive sensing platform for the
detection of the influenza virus.
Colorimetric detection of HA using SG-gAuNPs: Purified SG-
gAuNPs were freeze-dried and then re-suspended in PBS (pH 7.4)
to give a nanoparticle concentration of 3 nm. A solution of HA in
À1
PBS (0.25 mgmL , pH 7.4) was prepared. Aliquots of the HA solu-
tion (5, 10, 20, 30 and 40 mL) were then added (with mixing) to an
aliquot of the SG-gAuNP solution (200 mL), and in each case the so-
lution was made up to a total volume of 300 mL by adding PBS; re-
action progress was monitored by UV/Vis spectroscopy at various
time intervals. Additionally, an aliquot of the SG-gAuNP solution
(200 mL) was also mixed with BSA (600 nm) to confirm that the SG-
gAuNPs did not undergo nonspecific aggregation, and also with
Con A and B subunits of heat-labile enterotoxin (each at 600 nm)
to confirm that the particles were selective only toward HA.
Colorimetric detection of viral particles using SG-gAuNPs: Egg
allantoic fluid containing influenza strains A/New Caledonia/20/
5
À1
1999 (H1N1; 6”10 PFUmL ) or A/Puerto Rico/8/34 (H1N1; 2”
1
7
À1
0 PFUmL ) was prepared in PBS. Aliquots of the A/New Caledo-
nia/20/1999 (H1N1) solution (1.6, 8.0, 16.7, 33.4, and 50.1 mL) were
added (with mixing) to an aliquot of the SG-gAuNP solution (30 mL
of 20 nm solution) and formalin (15 mL of 10% stock solution). In
each case, the solution was made up to a total volume of 300 mL
by adding PBS. Reaction progress was monitored by UV/Vis spec-
troscopy at various time intervals. In the case of the A/Puerto Rico/
Experimental Section
8
/34 (H1N1) virus, 1.5 mL of the stock solution was added to the
SG-AuNP solution.
Reagents: Water used throughout this study was purified by
a Milli-Q system (Millipore). Au metal (99.99%), trisodium citrate,
and concanavalin A (Con A) isolated from jack bean (Canavalia en-
sifromis) were purchased from Sigma–Aldrich. Recombinant influ-
enza A virus H1N1 hemagglutinin (A/California/04/2009) was pur-
chased from Sino Biological Inc. (Beijing, China). The influenza virus
strains A/Puerto Rico/8/34 (H1N1) and A/New Caledonia/20/1999
DLS assay: The sample was equilibrated at 258C for 120 s before
analysis. Each measurement consisted of 12 scans, each 30 s in du-
ration. The mean particle hydrodynamic diameters reported were
calculated from intensity-based particle size distributions.
[
22]
Extraction of sialylglycopeptide (SGP) from egg yolks 1: Egg
yolks (300 total, 3.3 L) were stirred in H O (1.5 L) and then freeze-
dried to obtain 2.1 kg of yolk powder. This powder was washed
successively with Et O (2”6 L) and 70% aqueous acetone (6 L)
after thorough mixing. Aqueous acetone (40%, 2”3 L) was added
with vigorous mixing, and the solution was then filtered through
Celite. The filtrate was concentrated and freeze-dried to give 30 g
of yolk extract powder. This powder (30 g) was dissolved in H O
and purified by solid-phase extraction with an active carbon/Celite
(300 g:300 g) column (the column was sequentially washed with
2
(
H1N1) were propagated in 10-day-old embryonated chicken eggs,
and titrated on Madin–Darby canine kidney (MDCK) cells. The B-
subunit of heat-labile enterotoxin was purchased from Reagent
Proteins (San Diego, CA, USA). Tetrachloroauric(III) acid trihydrate
2
[37]
(
HAuCl ·3H O) was prepared following published procedures. All
4 2
other reagents and solvents were analytical grade and were used
without further purification.
2
Instrumentation: Infrared spectra were recorded on a PerkinElmer
1
Spectrum One instrument. H NMR (d ) spectra were recorded on
5% and 10% aqueous CH CN before elution of SGP with 30%
aqueous CH CN) to afford 1.2 g of sialylglycopeptide (SGP) 1 as
a white powder. H NMR (400 MHz, D O): d=0.84 (6H, d, gCH of
H
3
[
22]
Agilent Technologies 400 MR (400 MHz) or Varian VNMR500
3
1
(
500 MHz) spectrometers. All chemical shifts are quoted on the d
2
3
scale in ppm using residual solvent as internal standard. High-reso-
lution mass spectra were recorded with a BrukermaXis 3G UHR-
Val), 1.04 (3H, d, gCH of Thr), 1.25 (3H, d, bCH of Ala), 1.31 (2H,
3 3
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1
m, gCH of Lys), 1.52–1.64 (3H, m, dCH of Lys, H-3 NeuAc7, H-3ax
a white powder. H NMR (400 MHz, D O): d=1.80 (2H, at, J=
2
2
ax
2
NeuAc7’), 1.75 (2H, m, bCH of Lys), 2.54 (1H, m, H-3eq NeuAc7, H-
12.4 Hz, H-3ax NeuAc7, H-3ax NeuAc7’), 2.10–2.2 (18H, s, 6”
CH CO ), 2.30 (3H, s, CH COS), 2.53–2.67 (4H, m, bCH of Asn, H-
2
3eq NeuAc7’), 2.70 (2H, m, bCH of Asn), 2.87 (2H, t, eCH of Lys),
2
2
3
2
3
2
4
1
1
.33 (2H, d, H-1Gal-6, H-1Gal6’), 4.47 (3H, m, H-1GlcNAc2, H-
GlcNAc5, H-1GlcNAc5’), 4.63 (1H, m, H-1Man3), 4.82 (1H, m, H-
3eq NeuAc7, H-3eq NeuAc7’), 4.17 (1H, bd, H-2Man4’), 4.24 (1H, bd,
H-2Man4), 4.30 (1H, bs, H-2Man3), 4.49 (2H, d, J=7.6 Hz, H-1Gal6,
H-1Gal6’), 4.66 (3H, m, H-1GlcNAc2, H-1GlcNAc5, H-1GlcNAc5’),
4.86 (1H, s, H-1Man3), 5.15 (1H, d, J=9.5 Hz, H-1GlcNAc), 5.18 (1H,
Man4’), 4.91 (1H, d, H-1GlcNAc1), 5.00 (1H, m, H-1Man4); HRMS
+
(
ESI): calcd for C112H189N O [M+H] 2865.1764, found 2865.1734.
15
70
+
s, H-1Man4); HRMS (ESI) calcd for C H N O S [M+H]
[24]
92 148
8
66
Asparagine-linked complex bi-antennary N-glycan 2: Pronase
2
453.8264, found 2453.8191.
(
3 mg, from Streptomyces griseus) was added to a solution of SGP
[
22]
1
(20 mg) and NaN in Tris·HCl buffer (50 mm, pH 7.5, 1 mL), and
Disulfide 7: Hydroxylamine hydrochloride (51 mg, 0.73 mmol) was
added to a solution of thioacetate 6 (300 mg, 0.122 mmol) in
sodium phosphate buffer (100 mm, 80 mL, pH 7.4). The mixture
was stirred at room temperature for 2 h and then lyophilized. The
residue was purified by gel filtration on a Sephadex G-25 column
(8.7”1.7 cm), which was pre-equilibrated and eluted with 0.01%
3
the mixture was incubated at 378C. The pH of the solution was
checked regularly and adjusted to 7.5. After seven days, the mix-
ture was purified by gel filtration using a Sephadex G-25 column
(
8.7”1.7 cm) which was pre-equilibrated and eluted with 0.01%
aqueous NH . Fractions (8 mL) were collected at a flow rate of
0
3
À1
.5 mLmin and examined for their sugar content using the
aqueous NH followed with oxidation by bubbling air continuously
3
phenol/H SO test. The fractions that showed positive results were
through the solution for a total of 64 h to obtain sialylglycan disul-
2
4
[24]
1
pooled and lyophilized to obtain asparagine-linked N-glycan 2
fide 7 (210 mg, 71%) as a white powder. H NMR (400 MHz, D O):
2
1
(
(
10 mg, 57%) as a white powder. H NMR (400 MHz, D O): d=1.80
d=1.80 (2H, at, J=12.4 Hz, H-3ax NeuAc7, H-3ax NeuAc7’), 2.10–2.2
(18H, s, 6”CH CO ), 2.53–2.67 (4H, m, bCH of Asn, H-3 NeuAc7,
2
2H, at, J=12.4 Hz, H-3ax NeuAc7, H-3ax NeuAc7’), 2.10–2.2 (18H, s,
3
2
2
eq
6
”CH CO ), 2.76 (2H, m, H-3 NeuAc7, H-3eq NeuAc7’), 2.95 (1H,
H-3eq NeuAc7’), 4.17 (1H, bd, H-2Man4’), 4.24 (1H, bd, H-2Man4),
4.30 (1H, bs, H-2Man3), 4.49 (2H, d, J=7.6 Hz, H-1Gal6, H-1Gal6’),
4.66 (3H, m, H-1GlcNAc2, H-1GlcNAc5, H-1GlcNAc5’), 4.86 (1H, s, H-
1Man3), 5.15 (1H, d, J=9.5 Hz, H-1GlcNAc), 5.18 (1H, s, H-1Man4);
3
2
eq
dd, J=8, 16 Hz, bCH of Asn), 3.03 (1H, dd, J=4, 16 Hz, bCH of
2
2
Asn), 4.17 (1H, bd, H-2Man4’), 4.24 (1H, bd, H-2Man4), 4.30 (1H,
bs, H-2Man3), 4.49 (2H, d, J=7.6 Hz, H-1Gal6, H-1Gal6’), 4.66 (3H,
m, H-1GlcNAc2, H-1GlcNAc5, H-1GlcNAc5’), 4.86 (1H, s, H-1Man3),
2
+
HRMS (ESI) calcd for C H N O S [M+2H] 1205.9082, found
90
146
8
65
5
.15 (1H, d, J=9.5 Hz, H-1GlcNAc), 5.18 (1H, s, H-1Man4); HRMS
1205.9192.
+
(
ESI) calcd for C H N O [M+H] 2337.8390, found 2337.8340.
88 144 8 64
Sialylglycan-capped gold nanoparticles (SG-gAuNP): An aqueous
solution of trisodium citrate (1.7 mL, 39.0 mm) was added to a boil-
ing aqueous solution of tetrachloroauric acid (7.6 mL, 1 mm) with
constant stirring (500 rpm). After 15 min, the reaction mixture was
cooled to room temperature, and a solution of disulfide 7 (144 mg,
1 mmol) was added. The reaction was stirred for 48 h at RT. The re-
action mixture was concentrated in vacuo, the residue was dis-
[
25]
S-Acetylthioacetic acid 4: Acetyl chloride (3.8 mL, 0.054 mmol)
was added dropwise to a stirred mixture of Et N (15 mL, 0.108 mol)
and thioglycolic acid 3 (5.22 g, 0.054 mol) in 1,4-dioxane (100 mL)
at 08C under N , and the reaction was then warmed to room tem-
perature. After 12 h, TLC (EtOAc) showed the formation of a major
3
2
product (R =0) and the complete consumption of starting material
f
(Rf =0.5). The reaction mixture was filtered through Celite and con-
solved in Milli-Q H O (10 mL), and then purified by centrifugal fil-
2
centrated in vacuo. Aqueous HCl (1m, 30 mL) was added, and the
reaction was stirred for 1 h. The reaction mixture was extracted
with CH Cl (3”30 mL), dried (MgSO ), filtered, and concentrated in
tering (Amicon Ultra 10K, Millipore, MWCO=10 kDa, 1 h, 5000 g).
The residue was then dissolved in H O (2 mL) and lyophilized to
2
obtain SG-gAuNP. TEM: Ø=12.1Æ1.6 nm; IR (KBr): n˜ =3300 (broad
2
2
4
[25]
À1
vacuo to give S-acetylthioacetic acid 4 (4.2 g, 54%) as a yellow
band), 2933, 2873, 1062 cm ; UV: 523 nm; TGA: ligand 73.6%, Au
1
oil. H NMR (400 MHz, CD SOCD ): d=2.35 (3H, s, CH COS), 3.66
26.4%; Elemental analysis: found C 32.65, H 4.50, N 3.44, S 1.04,
3
3
3
(
2H, s, CH CO ), 12.75 (1H, brs, COOH).
calcd for Au54464(C H O S)
: C 32.96, H 4.47, N 3.42, S 1.01%;
12404
2
2
12 24
8
2
+
HRMS (ESI) calcd for C H N O S [M+2H] 1206.4116, found
[26]
[25]
90 146
8
66
N-Hydroxysuccinimide ester 5:
(
S-Acetylthioacetic acid 4
1
206.415.
2.6 g, 0.019 mol) and N-hydroxysuccinimide (2.2 g, 0.019 mol)
were dissolved in THF (50 mL), and the reaction mixture was stirred
at room temperature for 10 min before cooling to 08C. Dicyclohex-
ylcarbodiimide (DCC; 3.97 g, 0.019 mol) was dissolved in THF
(
5 mL) and then added to the reaction mixture. The reaction mix-
Acknowledgements
ture was warmed to room temperature and stirred under N . After
2
1
2 h, TLC (EtOAc/MeOH 4:1) showed the formation of a major
product (R =0.6) and the complete consumption of starting mate-
The authors thank Dr. Yusuke Tomabechi, Professor Milo Kral,
Mike Flaws (TEM), Alistair Duff (AAS), and Rayleen Fredericks
f
rial (R =0.4). The residue was purified by flash column chromatog-
f
[
26]
raphy (PE/EtOAc 2:1) to afford N-hydroxysuccinimide ester 5
1.9 g, 61%) as a yellow oil. H NMR (400 MHz, CD Cl ): d=2.43
3 3
3H, s, COCH ), 2.85 (4H, s, C(O)CH CH CO), 3.99 (2H, s, SCH CO ).
(
DLS) for technical assistance. Financial support is gratefully ac-
1
[26]
(
(
knowledged from the University of Canterbury (PhD scholarship
to V.P.), the University of Otago (PhD scholarship to P.T.N.), the
Health Research Council (Emerging Researcher Grant 12/614 to
M.H.), and the MacDiarmid Institute.
3
2
2
2
2
[26]
Thioacetate 6: A solution of N-hydroxysuccinimide ester 5
(
12 mg, 0.049 mmol) in CH CN (0.3 mL) was added to a solution of
3
[24]
asparagine-linked N-glycan 2
(7.6 mg, 0.003 mmol) in sodium
phosphate buffer (50 mm, 1 mL, pH 7.4) containing 20% CH CN.
3
The mixture was stirred at room temperature for 12 h and then
lyophilized. The residue was purified by gel filtration on a Sephadex
G-25 column (8.7”1.7 cm), which was pre-equilibrated and eluted
Keywords: carbohydrates
hemagglutinin · influenza · sensors · sialylglycan
·
gold
nanoparticles
·
with 0.01% aqueous NH to obtain thioacetate 6 (5 mg, 68%) as
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Received: April 20, 2015
Published online on July 14, 2015
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ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim