Demonstrating the use of bisphenol a-functionalised gold nanoparticles in immunoassays

Article abstract of DOI:10.1071/CH13043

Spherical gold nanoparticles (5-nm diameter) were modified with a small-molecule thiolated bisphenol A (BPA) ligand to achieve an estimated coverage of ～3.3×10-10molcm-2, or 180 ligands per particle. The modified particles were tested in an enzyme-linked immunosorbent assay (ELISA) format to measure functionality and were shown to bind specifically to anti-BPA antibody while resisting the non-specific adsorption of an antibody with no affinity for BPA. It was found that the use of 10% ethanol as a co-solvent was required in the ELISA as aqueous buffers alone resulted in poor binding between anti-BPA antibody and the functionalised nanoparticles. This is likely due to the hydrophobic nature of the BPA ligand limiting its solubility, and therefore its availability for antibody interactions, in purely aqueous environments. To our knowledge, this is the first example of a nanoparticle modified with a small organic molecule being used in an ELISA assay.

Full text of DOI:10.1071/CH13043

CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 613–618
Full Paper
http://dx.doi.org/10.1071/CH13043
Demonstrating the Use of Bisphenol A-functionalised
Gold Nanoparticles in Immunoassays
A
B
A C
,
B E
,
Joshua R. Peterson, Yang Lu, Erwann Luais,
Nanju Alice Lee,
A D E
, ,
and J. Justin Gooding
A
School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia.
School of Chemical Engineering, University of New South Wales, Sydney,
NSW 2052, Australia.
B
C
Current address: GREMAN, University Franc¸ois Rabelais, Parc de Grandmont,
37200 Tours, France.
D
Australian Centre for NanoMedicine, University of New South Wales, Sydney,
NSW 2052, Australia.
E
Corresponding authors. Email: justin.gooding@unsw.edu.au, alice.lee@unsw.edu.au
Spherical gold nanoparticles (5-nm diameter) were modified with a small-molecule thiolated bisphenol A (BPA) ligand to
achieve an estimated coverage of ,3.3 ꢀ 10ꢁ10 mol cmꢁ2, or 180 ligands per particle. The modified particles were tested
in an enzyme-linked immunosorbent assay (ELISA) format to measure functionality and were shown to bind specifically
to anti-BPA antibody while resisting the non-specific adsorption of an antibody with no affinity for BPA. It was found that
the use of 10 % ethanol as a co-solvent was required in the ELISA as aqueous buffers alone resulted in poor binding
between anti-BPA antibody and the functionalised nanoparticles. This is likely due to the hydrophobic nature of the BPA
ligand limiting its solubility, and therefore its availability for antibody interactions, in purely aqueous environments.
To our knowledge, this is the first example of a nanoparticle modified with a small organic molecule being used in an
ELISA assay.
Manuscript received: 25 January 2013.
Manuscript accepted: 5 March 2013.
Published online: 8 April 2013.
Introduction
used in the production of polycarbonate plastics, and produced
on the billions of kg per year scale, BPA is ubiquitous in the
environment.[6] Although human exposure is predominantly the
result of its use in food packaging, both the US Environmental
Protection Agency and Food and Drug Administration have
taken steps to reduce human exposure to BPA, as have the
relevant agencies in many other countries around the world.[7]
As a result, a great deal of research has been directed at the
detection of BPA in a variety of media including human
serum,[8] food products,[9] and environmental samples.[10] In
addition, a variety of techniques have been developed for the
extraction and detection of BPA such as HPLC-mass spectro-
scopy (MS), gas chromatography (GC)-MS, fluorescence, elec-
trochemical detection, and immunochemistry methods.[11]
Recent advancements in the detection of many analytes,
including BPA, have combined immunochemistry with nano-
particles, resulting in assays that have the specificity of anti-
bodies but increased sensitivity compared with traditional
enzyme-linked immunosorbent assay (ELISA). Modification
of nanoparticles with antibodies has been shown to increase
sensitivity in ELISA for cancer biomarkers.[12] Magnetic
nanoparticles have been shown in the ELISA format to pre-
concentrate analyte, lowering the limit of detection by an order
of magnitude,[13] and to reduce ELISA assay time from hours to
a matter of minutes.[14] Graham and coworkers have used
modified gold nanoparticles in an ELISA with surface-enhanced
A dizzying array of uses for gold nanoparticles has been reported
in recent years and spans an impressive variety of research areas
from energy[1] to healthcare,[2] food,[3] and chemical and bio-
logical analysis.[4] The use of nanoparticles in sensing appli-
cations is of particular interest as they have many advantageous
properties that make them suitable for incorporation into sensing
devices: they are easily synthesised across a range of sizes, they
have a high surface area-to-volume ratio, it is relatively
straightforward to functionalise their surfaces with biocompat-
ible ligands, and they possess unique optical and electronic
properties that can be exploited in several different analytical
formats. As a result, there has been a large output of research
aimed at using nanoparticles for the detection of analytes
ranging from malignant cells to metal ions and small organic
compounds.[4] In several studies, nanoparticles have demon-
strated the potential for improved analyte capture, detection, and
sensitivity, making nanoparticles an attractive option when
considering analytes that are present in low levels or with lim-
ited water solubility, for example small organic molecule
contaminants.
Bisphenol A (BPA) is one such contaminant that has gar-
nered a great deal of attention over the past decade owing to its
activity as an endocrine disrupter, capable of interfering with
male and female reproductive development in utero, and also its
association with increased risk of certain cancers.[5] A monomer
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614
J. R. Peterson et al.
resonance Raman scattering (SERRS) to increase sensitivity.[15]
Most recently, nanoparticles modified with anti-BPA aptamers
have been used for the visual detection of BPA down to
Results and Discussion
Preparation and Characterisation of Functionalised
Gold Nanoparticles
0.1 mg Lꢁ1 [16]
.
Synthesis of the thiol-modified BPA ligand followed a common
amide coupling procedure to first activate the acid group of
BPA-valeric acid with N,N0-dicyclohexylcarbodiimide/
N-hydroxysuccinimide (DCC/NHS), followed by reaction with
cysteamine (Fig. 2). Although the yield of purified cysBPAv
ligand was low, it was sufficient to allow functionalisation of a
large number of nanoparticles because so little is used. For
example, 1 mg of cysBPAv ligand is theoretically sufficient to
prepare 145 mL of functionalised nanoparticles, enough to
perform more than 14000 ELISA assays.
Although the use of modified gold nanoparticles in detection
systems, including ELISA assays, has been reported, the nano-
particles have been almost exclusively modified with antibodies
or large proteins. In the few studies that have reported the use of
small-molecule-functionalised nanoparticles, great potential
has been shown in both medical[17] and biosensor applica-
tions.[18] To our knowledge, there have been no studies in which
gold nanoparticles modified with a small organic molecule have
been tested in an ELISA. The combination of ELISA and
nanoparticles is potentially useful in developing detection
methods for many analytes, including BPA. There are, however,
broader implications for this research than using them in ELISA.
Demonstrating the functionality of a nanoparticle sensing ele-
ment could be invaluable in generating immunogenic species as
a substitute to hapten–protein conjugates and also in designing
new electrochemical immunosensors that incorporate nanopar-
ticles.[19] For these reasons, we decided to test BPA-functiona-
lised nanoparticles in a sandwich ELISA assay to assess their
functionality and their suitability for future incorporation into
immunosensors. In this paper, we discuss the use of nanoparti-
cles modified with the small organic molecule bisphenol A in an
ELISA assay (Fig. 1), the need for an organic co-solvent, and the
specific binding exhibited by the modified nanoparticles.
The cysBPAv ligand was added to the nanoparticle solution,
which, after overnight incubation, was centrifuged and decanted
to remove any cysBPAv not bound to the nanoparticle surface.
The addition of cysBPAv to the nanoparticle surface significant-
ly increases the hydrophobicity of the nanoparticles themselves
even though BPA has been reported as a ‘moderately soluble’
compound, with a water solubility in the range of 120 to 300 mg
L .
ꢁ1 [20] In this case, the cysBPAv ligand is not sufficiently water
soluble to allow nanoparticle functionalisation and redispersion
in pure water (which would be the most desirable conditions
considering that immunochemistry reactions in the ELISA are
optimised for aqueous conditions). However, the resulting mod-
ified nanoparticles were highly dispersible in absolute ethanol,
OH
avidin-HRP
HO
O
HO
O
NH
NH
S
S
OH
HO
Ab∝BPA-biotin
OH
AuNP
S
H
N
cysBPAv-NPs
Ab∝BPA
N
S
O
O
H
OH
HO
S
OH
HN
O
OH
Fig. 1. Schematic of BPA nanoparticle sandwich ELISA (NP, nanoparticle).
O
H
N
O
OH
O
O
O
SH
N
i
ii
O
HO
OH
HO
OH
OH
HO
BPA-valeric acid
BPA-valeric–NHS
cysBPAv
Fig. 2. Synthesis of cysBPAv from BPA-valeric acid via NHS activation and amide coupling with cysteamine: (i) DCC,
NHS, dry THF, 22 h, rt, 36 % yield; (ii) cysteamine, DIPEA, dry THF, 3 h, rt, 11 % yield.

BPA-Nanoparticles
615
and the precipitated particles could be resuspended in a small
volume of ethanol before dilution with water to the final mixture
of 10 % ethanol in water. In this ethanol/water mixture, the
cysBPAv-NP suspension was stable for several months.
displayed in Fig. 4c. For gold nanoparticles synthesised with
citrate present, as was done in the present study, the carbon
material on the gold nanoparticle surface is the citrate coating,
and the C 1s signal can be fitted in three components. The first
component, at a binding energy value of 285.6 eV, is attributed
to aliphatic carbon whereas at higher binding energies, two other
components are present resulting from carbon bound to more
electronegative elements, in this case oxygen. The second
component, at a binding energy of 286.7 eV, corresponds to
the carbon–hydroxyl functional group whereas the third com-
ponent at 288.9 eV is for carbon atoms in a carboxylic environ-
ment. After the functionalisation with the cysBPAv ligand, the
C 1s signal is very different and is fitted with four components.
The first component, at 284.6 eV, corresponds to carbon atoms
in an sp2 hybridisation state from the two phenyl rings in the
ligand structure. At a binding energy value of 285.3 eV, the
second component is for aliphatic carbon atoms. For carbon in a
more oxidised state, a component at 286.3 eV is attributed to
carbon atoms bound to sulfur, nitrogen, and oxygen (phenol
group). The last component, at a binding energy of 288.1 eV,
corresponds to carbon atoms engaged in the amide bond.[24] The
presence of amide is also confirmed through the XPS N 1s signal
(Fig. 4d) where the N 1s signal is fitted with a single component
at a binding energy of 400.9 eV.[24] Evidence that the citrate
capping is removed by the self-assembled monolayer ligand on
the gold nanoparticle surface is also supported by the decrease in
atomic percentage of Na 1s, which drops from 4.05 atom-% in
the unmodified particles to 0.83 atom-% in the cysBPAv-NPs.
The modified particles used for these studies were prepared
by the addition of 20 mL of a 1 mM solution of cysBPAv to 1 mL
of nanoparticle solution. Assuming spherical bare nanoparticles
of 5.3-nm diameter (s.d. ¼ 1.6 nm, n ¼ 107, based on transmis-
sion electron microscopy (TEM) measurements), and a concen-
tration of 3.2 ꢀ 1013 nanoparticles mLꢁ1 calculated using the
methods of Haiss and coworkers,[21] the ligand added is suffi-
cient to achieve a theoretical surface coverage of 7.1 ꢀ 10ꢁ10
mol thiol ligand cmꢁ2 (equivalent to ,380 cysBPAv ligands per
particle). This is slightly less than the maximum coverage of
close-packed alkanethiols on gold that was reported by Porter
and coworkers[22] as 9.3 ꢀ 10ꢁ10 mol cmꢁ2 (equivalent to 500
cysBPAv ligands per particle). In fact, the bulkiness of the
cysBPAv ligand would not allow close packing on the surface as
one would expect for straight-chain alkanethiols, so the surface
density of cysBPAv ligand would likely be even less than
7.1 ꢀ 10ꢁ10 mol cmꢁ2. Basic modelling shows the footprint of
2
˚
the cysBPAv ligand to be ,50 A . Using this figure, a surface
coverage of 3.3 ꢀ 10ꢁ10 mol cmꢁ2 can be calculated, which
equates to ,180 ligands per nanoparticle.
The modified nanoparticles (cysBPAv-NPs) were charac-
terised by TEM, X-ray photoelectron spectroscopy (XPS), and
UV-vis. TEM confirmed that the centrifuged and resuspended
particles were on average 5.4 nm in diameter (s.d. ¼ 1.4 nm,
n ¼ 228), essentially unchanged compared with the unmodified
particles (Fig. 3a). UV-vis showed a shift in the surface plasmon
resonance (SPR) peak from 517 nm to 529 nm (Fig. 3b), which is
consistent with shifts predicted for particles coated with an
organic compound.[21]
Use of Functionalised Gold Nanoparticles
in the Sandwich ELISA Assay
The cysBPAv-NPs were tested in an ELISA assay to ascertain
the binding characteristics to anti-BPA antibodies. Polyclonal
antibodies to BPA-valerate (AbpBPA) were raised as
described previously.[9b] The assay was run in a sandwich
ELISA format (Fig. 1) where the surface of the ELISA plate was
first coated with AbpBPA, followed by subsequent incubations
in cysBPAv-NPs, AbpBPA–biotin and then avidin–horseradish
peroxidase (HRP). The resulting complex could then be mea-
sured using standard ELISA colorimetric methods.
Initial ELISA results showed poor binding between the
cysBPAv-NPs and AbpBPA in phosphate buffered solution
(PBS) buffer (Fig. 5). This is likely due to the hydrophobic
nature of the cysBPAv ligand, which could strongly associate
with the nanoparticle surface and adjacent ligands rather than
extending out into the solution phase where it would be available
as an antibody binding site. This is even more likely in an
The functionalisation of gold nanoparticles was demonstrated
through XPS characterisation of gold nanoparticles before
and after modification with the cysBPAv ligand. Comparing the
survey spectra (Fig. 4a), new XPS signals appear for sulfur and
for nitrogen. These XPS signals are from the thiolated cysBPAv
ligand, which contains an amide bond in addition to the sulfur.
Evidence that the cysBPAv ligand is bound to the gold nano-
particles surface is provided by the S 2p XPS signal for
functionalised nanoparticles (Fig. 4b), fitted with two compo-
nents corresponding to the XPS S 2p splitting signal for S 2p3/2
and S 2p1/2 at respective binding energies of 162.1 and
163.4 eV. These binding energy values match with bound thiol
on gold surfaces whereas free or uncomplexed thiol typically
exhibits values at higher energies.[23] XPS C 1s scans are
(b)
1.0
0.9
0.8
0.7
(a)
0.6
0.5
0.4
0.3
0.2
0.1
0
300
400
500
600
700
20 nm
λ [nm]
Fig. 3. (a) TEM of cysBPAv-NPs. Particle diameter ¼ 5.4 nm (s.d. 1.4 nm, n ¼ 228); and (b) UV-vis spectra showing
shift in SPR peak from 517 nm (dotted line, unmodified particles) to 529 nm (solid line, cysBPAv-NPs) when particles are
modified with cysBPAv ligand.

616
J. R. Peterson et al.
(b)
(a)
N 1s
S 2p
cysBPAv-NPs
cysBPAv-NPs
O 1s
Au 4f
Na 1s
C 1s
NPs
CI 2p
200
NPs
1200
1000
800
600
400
0
294
292
290
288
286
284
282
280
278
Binding energy [eV]
Binding energy [eV]
(c)
(d)
410
407
404
401
398
395
392
172
170
168
166
164
162
160
158
156
154
Binding energy [eV]
Binding energy [eV]
Fig. 4. (a) XPS survey of unmodified gold nanoparticles and of cysBPAv-modified gold nanoparticles; (b) XPS C 1s scans of unmodified particles and
cysBPAv-NPs; (c) XPS N 1s spectra of cysBPAv-NPs; and (d) XPS S 2p spectra of cysBPAv-NPs.
0.5
considered a typical ELISA binding curve. As shown in Fig. 5,
significant binding between AbpBPA and cysBPAv-NPs in
10 % ethanol can be measured at nanoparticle concentrations as
0.4
low as 10 nM cysBPAv-NP with the colorimetric response
signal being saturated at 100 nM cysBPAv-NP. In contrast,
0.3
without ethanol the binding cannot be reliably measured below
a concentration of 100 nM cysBPAv-NP and the colorimetric
0.2
response is not saturated even at a nanoparticle concentration of
5000 nM. In other words, it takes a great deal fewer nanoparti-
0.1
cles to achieve a colorimetric response in the ELISA when the
ELISA is carried out in 10 % ethanol. As noted above, this is
likely due to the small amount of organic co-solvent being
sufficient to allow some of the surface-bound cysBPAv ligands
to be available to the bulk solution where they can interact with
the AbpBPA antibodies.
To ensure that the observed binding was due to specific
interactions betweencysBPAv-NP and AbpBPA and not simply
hydrophobicinteractions, the ELISAassay wasruncomparing the
response of AbpBPA with an antibody with no affinity for BPA
(AbpHareI#3, which was raised against 17a-ethinylestradiol).
In the ELISA assay, the cysBPAv-NPs showed specific binding
with an anti-BPA antibody (AbpBPA) with the maximum
absorbance value difference of 0.58 in the range of 3 ꢀ 10ꢁ1 to
1 ꢀ 10ꢁ10 M cysBPAv-NPs (Fig. 6). The antibody with no
affinity for BPA (AbpHareI#3) showed no binding to the
modified nanoparticles across a wide range of concentrations.
It is clear from the data that incorporation of 10 % ethanol is
sufficient to alter the environment at the surface of the modified
particles to an extent that allows specific interactions with the
0
10Ϫ2
10Ϫ1
100
101
102
103
104
Concentration of cysBPAv-NPs [nmol LϪ1
]
Fig. 5. Dose–response between cysBPAv-NPs and AbpBPA with and
without ethanol present: ꢂ¼ cysBPAv-NPs measured in PBS/10 % EtOH;
’ ¼ cysBPAv-NPs measured in PBS alone. The results demonstrate the
need for 10 % EtOH as a co-solvent to allow efficient binding of the antibody
to the cysBPAv ligand on the modified nanoparticles. The data for unmodi-
fied nanoparticles measured in PBS/10 % EtOH (¢) show that no binding
occurs when the cysBPAv ligand is absent.
aqueous environment with high salt content such as PBS, so
in the standard ELISA method, there is probably very little
surface-bound BPA ligand available in solution for the antibody
to bind. To make the bulk solution more favourable for the
cysBPAv ligand, an organic co-solvent was added to the PBS.
It was found that the addition of 10 % (v/v) ethanol to the PBS
resulted in much better binding between the AbpBPA and
cysBPAv-NPs, and the data were on par with what would be

BPA-Nanoparticles
617
0.6
Technologies, Inc.). N,N-dimethylformamide (DMF, Ajax Fine-
chem) was dried with alumina (Sigma–Aldrich, activated,
˚
neutral, Brockmann I) and stored over 4-A molecular sieves
(Ajax Finechem) before use. Deionised water was prepared with
a Millipore Milli-Q Academic system (18.2 MO cm). Silica
chromatography medium was purchased from Grace Davison
(Davisil LC60A, 40–63 mm). Maxisorp polystyrene 96-well
plates were obtained from Nunc (ThermoFisher Scientific,
Hvidovre Denmark).
0.4
0.2
NMR measurements were performed on a 300 MHz Bruker
Avance III. UV-vis measurements were performed on a Varian
Cary 50 Bio UV-visible spectrophotometer. TEM measure-
ments were performed on a Phillips CM200 fitted with an SIS
CCD camera and image analysis carried out using ImageJ
software (version 1.42q, National Institutes of Health, USA).
Centrifugation was performed using a Sigma 1–14 Laboratory
Table-top Microcentrifuge (14800 rpm ¼ 16163g). An ELISA
plate reader (SpectraMax M2) was obtained from Molecular
Devices (Sunnyvale, CA, USA).
The chemical environments of gold nanoparticles before and
after functionalisation with the cysBPAv ligand were analysed
by X-ray photoelectron spectroscopy using an EscaLab 250-
IXL spectrometer with a monochromated Al Ka source
(1486.6 eV). Take-off angle was normal (908) to the surface.
Survey and narrow scans were acquired with a pass energy of
100 and 20 eV respectively. The sample preparation consisted of
drop-casting a solution of gold nanoparticles onto a silicon chip
and letting it dry until the solvent evaporated.
0
10Ϫ10
10Ϫ5
100
105
Concentration of cysBPAv-NPs [nmol LϪ1
]
Fig. 6. Dose–response between cysBPAv-NPs and specific and non-
specific antibodies in PBS with 10 % (v/v) EtOH: ꢂ¼ cysBPAv-NPs/
AbpBPA; ’ ¼ cysBPAv-NPs/AbpHareI#3. AbpBPA is an anti-BPA
antibody, whereas AbpHareI#3 is an antibody with no affinity for BPA.
The results demonstrate a selective interaction between BPA antibody and
the cysBPAv-modified gold nanoparticles.
anti-BPA antibodies. This demonstration of selective interaction
between the anti-BPA antibody and the cysBPAv-NPs is prom-
ising for their use in an electrochemical immunosensor.
Conclusions
Gold nanoparticles ,5 nm in diameter were synthesised and
modified with a thiol-bisphenol A ligand. The modified parti-
cles were characterised by TEM, XPS, and UV-vis and were
estimated to have a surface coverage of 3.3 ꢀ 10ꢁ10 mol cmꢁ2
Synthesis of BPA-valeric–NHS
A mixture of BPA-valeric acid (1.51 g, 5.26 mmol), DCC
(1.30 g, 6.30 mmol), and NHS (0.732 g, 6.36 mmol) in dry THF
(40 mL) was stirred for 22 h at room temperature. The mixture
was filtered, the solvent removed under vacuum, the residue
dissolved in dry DCM, dried over Na2SO4 and filtered, and the
solvent removed under vacuum. The product was purified by
column chromatography (silica, 8 : 2 DCM/MeOH, Rf ¼ 0.8) to
give the product as a white solid (0.719 g, 36 %). dH (300 MHz,
CDCl3) 7.06–7.02 (m, 4H), 6.78–6.73 (m, 4H), 4.90 (br s, 2H),
2.83 (s, 4H), 2.51–2.34 (m, 4H), 1.58 (s, 3H). m/z (HRMS-ESI)
406.1255 ([M þ Na]þ requires 406.1261).
,
or an average of 180 ligands per particle. The modified particles
were characterised by ELISA assay and demonstrated func-
tionality with specific binding to anti-BPA antibody while
resisting the non-specific adsorption of an antibody with no
affinity for BPA. A key feature of the ELISA assay was the need
to use 10 % ethanol as a co-solvent, presumably owing to the
hydrophobic nature of the BPA ligand, which may limit its
availability for antibody interactions at the nanoparticle surface
in purely aqueous environments. Future work will focus on
additional characterisation of interactions of modified nano-
particles with specific and non-specific antibodies and incor-
poration of these nanoparticles as sensing elements in
electrochemical immunosensors.
Synthesis of CysBPAv
To a stirred solution of BPA-valeric–NHS (0.439 g, 1.14 mmol)
in dry THF (30 mL) under a nitrogen atmosphere was added a
suspension of cysteamine (90.3 mg, 1.17 mmol) and DIPEA
(0.2 mL, 1.2 mmol) in dry THF (5 mL). The mixture was stirred
at room temperature for 3 h, concentrated under vacuum, then
ethyl acetate added and the precipitate removed by filtration.
The solvent was removed under vacuum and the crude product
purified twice by column chromatography (silica, first column
EtOAc (Rf ¼ 0.5); second column 2 : 1 EtOAc/hexane
(Rf ¼ 0.25)) to afford the product as a white solid (42 mg, 11 %).
dH (300 MHz, CD3OD) 7.03–6.98 (m, 4H), 6.72–6.65 (m, 4H),
3.28–3.23 (t, 2H), 2.56–2.52 (t, 2H), 2.36–2.31 (m, 2H),
1.99–1.96 (m, 2H), 1.54 (s, 3H). m/z (HRMS-ESI) 346.1467
([M þ H]þ requires 346.1471), 368.1285 ([M þ Na]þ requires
368.1291).
Experimental
Chemicals and Instruments
4,4-Bis(4-hydroxyphenyl)valeric acid (BPA-valeric acid),
DCC, NHS, N,N-diisopropylethylamine (DIPEA), hydrogen
tetrachloroaurate(III) hydrate, sodium borohydride, thimerosal
and 3,30,5,50-tetramethylbenzidine (TMB) were purchased from
Aldrich. Trisodium citrate, potassium phosphate (dibasic), sul-
furic acid, hydrochloric acid, nitric acid, and hydrogen peroxide
were purchased from Ajax Finechem. Avidin-HRP was pur-
chased from Sigma. The preparation of anti-BPA antibody
(AbpBPA) has been described previously.[9b]
Methanol (MeOH, HPLC grade), and absolute ethanol
(EtOH, Ajax Finechem) were used as received. Dichloro-
methane (DCM), ethyl acetate (EtOAc), and hexane (drum
grade, Ajax Finechem) were distilled before use and tetra-
hydrofuran (HPLC grade, preservative-free, Fisher Scientific)
was dried by a solvent purification system (Innovative
Preparation of AbpBPA-biotin Conjugate
Biotin (0.2 g, 0.08 mmol), DCC (0.025 g, 0.12 mmol), and NHS
(0.014 g, 0.12 mmol) were dissolved in anhydrous THF (10 mL).
Subsequently, the mixture was stirred overnight at room

618
J. R. Peterson et al.
temperature, filtered to remove the precipitate, and then con-
centrated under reduced pressure to yield the biotin active ester.
The active ester dissolved in DMF was added slowly in
droplets into AbpBPA solution with a biotin-antibody molar
ratio of 75 to form AbpBPA–biotin. The reaction was allowed
to proceed for 2 h at room temperature followed by 16 to 24 h at
48C. Finally, the crude conjugate was dialysed with 50 mM
K2HPO4 for 1.5 days with three buffer changes. After the
addition of thimerosal, the antibody–biotin conjugate was stored
at 48C until use.
[5] C. A. Richter, L. S. Birnbaum, F. Farabollini, R. R. Newbold, B. S.
Rubin, C. E. Talsness, J. G. Vandenbergh, D. R. Walser-Kuntz, F. S.
vom Saal, Reprod. Toxicol. 2007, 24, 199. doi:10.1016/J.REPROTOX.
2007.06.004
[6] P. Fu, K. Kawamura, Environ. Pollut. 2010, 158, 3138. doi:10.1016/
J.ENVPOL.2010.06.040
[7] (a) US EPA Bisphenol A Action Plan 2010 Available at: http://www.
epa.gov/oppt/existingchemicals/pubs/actionplans/bpa_action_plan.pdf
(Verified 16 October 2012)
(b) US FDA, Bisphenol A (BPA): Use in Food Contact Application
2010. Available at: http://www.fda.gov/newsevents/publichealthfocus/
ucm064437.htm – current (Verified 16 October 2012)
[8] H. Ohkuma, K. Abe, M. Ito, A. Kokado, A. Kambegawa, M. Maeda,
Analyst 2002, 127, 93. doi:10.1039/B103515K
Preparation of Functionalised Gold Nanoparticles
All glassware was cleaned with piranha (H2SO4/H2O2 3 : 1 v/v)
followed by aqua regia (HCl/HNO3 1 : 1 v/v), then rinsed with
copious amounts of distilled water before nanoparticle synthe-
sis. A solution of 0.25 mM HAuCl4 and 0.25 mM trisodium
citrate in 100 mL deionised water was stirred vigorously during
the addition of 1 mL of an ice-cold solution of 100 mM NaBH4.
The resulting mixture was stirred vigorously for an additional
10 min, resulting in a suspension of 5-nm diameter spherical
gold nanoparticles.
To 1 mL of the above gold nanoparticle solution, 20 mL of
1 mM cysBPAv solution (in absolute ethanol) was added and the
mixture left overnight at room temperature. After centrifugation
at 14800 rpm (16163g) for 15 min, the solution was decanted
and the precipitated particles were redispersed in 100 mL of
absolute ethanol followed by the addition of 900 mL of distilled
water.
[9] (a) Z. Brenn-Struckhofova, M. Cichna-Markl, Food Add. Contam.
2006, 23, 1227. doi:10.1080/02652030600654382
(b) Y. Lu, J. Peterson, J. Gooding, N. Lee, Anal. Bioanal. Chem. 2012,
403, 1607. doi:10.1007/S00216-012-5969-8
(c) D. Podlipna, M. Cichna-Markl, European Food Research and
Technology Zeitschrift Lebensmittel A. 2007, 224, 629. doi:10.1007/
S00217-006-0350-9
[10] (a) H. Fromme, T. Ku¨chler, T. Otto, K. Pilz, J. Mu¨ller, A. Wenzel,
Water Res. 2002, 36, 1429. doi:10.1016/S0043-1354(01)00367-0
(b) G. Gatidou, N. S. Thomaidis, A. S. Stasinakis, T. D. Lekkas, J.
Chromatog. A. 2007, 1138, 32. doi:10.1016/J.CHROMA.2006.10.037
(c) L. Patrolecco, S. Capri, S. De Angelis, S. Polesello, S. Valsecchi,
J.Chromatog. A. 2004, 1022, 1. doi:10.1016/J.CHROMA.2003.09.050
(d) Y. Watabe, T. Kondo, M. Morita, N. Tanaka, J. Haginaka, K.
Hosoya, J. Chromatogr. A 2004, 1032, 45. doi:10.1016/J.CHROMA.
2003.11.079
[11] A. Ballesteros-Go´mez, S. Rubio, D. Pe´rez-Bendito, J. Chromatogr. A
2009, 1216, 449. doi:10.1016/J.CHROMA.2008.06.037
[12] (a) A. Ambrosi, F. Airo`, A. Merkoc¸i, Anal. Chem. 2010, 82, 1151.
doi:10.1021/AC902492C
Nanoparticle ELISA
Wells were modified with anti-BPA antibodies (AbpBPA) by
adding 110 mL of a 10 mg mLꢁ1 antibody solution and incubat-
ing overnight. After washing three times with washing solution,
1 % soy bean protein in PBS (SBP-PBS) was incubated in the
wells for 1 h. Following the blocking step, the plate was washed
three times with the washing solution (0.05 % Tween-20/reverse
osmosis (RO) water). CysBPAv-NPs diluted in 10 % EtOH/
PBS were added to each well and incubated for 30 min. There-
after, the plate was washed another three times, and then
1.17 mg mLꢁ1 AbpBPA-biotin in 10 % EtOH/PBS was added
over 1 h. After three washes, avidin-HRP conjugate was added
to the plate and incubated for 30 min. The plate was washed five
times before the TMB substrate solution was incubated (150 mL
per well) in all the microwells for 20 min to develop the colour.
Stop solution (1.25 M sulfuric acid) was added (50 mL per well)
to the wells to stop further colour reaction and measurement of
the absorbance was conducted using a microplate reader with a
dual wavelength mode at 450 and 650 nm.
(b) C.-P. Jia, X.-Q. Zhong, B. Hua, M.-Y. Liu, F.-X. Jing, X.-H. Lou,
S.-H. Yao, J.-Q. Xiang, Q.-H. Jin, J.-L. Zhao, Biosens. Bioelectron.
2009, 24, 2836. doi:10.1016/J.BIOS.2009.02.024
[13] C.-L. Mao, K. D. Zientek, P. T. Colahan, M.-Y. Kuo, C.-H. Liu, K.-M.
Lee, C.-C. Chou, J. Pharm. Biomed. Anal. 2006, 41, 1332.
doi:10.1016/J.JPBA.2006.03.009
[14] (a) A. Radoi, M. Targa, B. Prieto-Simon, J. L. Marty, Talanta 2008,
77, 138. doi:10.1016/J.TALANTA.2008.05.048
(b) K. Chuah, L. M. H. Lai, I. Y. Goon, S. G. Parker, R. Amal, J. J.
Gooding, Chem. Commun. 2012, 3503. doi:10.1039/C2CC30512G
[15] F. M. Campbell, A. Ingram, P. Monaghan, J. Cooper, N. Sattar,
P. D. Eckersall, D. Graham, Analyst 2008, 133, 1355. doi:10.1039/
B808087A
[16] Z. Mei, H. Chu, W. Chen, F. Xue, J. Liu, H. Xu, R. Zhang, L. Zheng,
Biosens. Bioelectron. 2013, 39, 26. doi:10.1016/J.BIOS.2012.06.027
[17] R. Weissleder, K. Kelly, E. Y. Sun, T. Shtatland, L. Josephson, Nat.
Biotechnol. 2005, 23, 1418. doi:10.1038/NBT1159
[18] L. M. H. Lai, I. Y. Goon, K. Chuah, M. Lim, F. Braet, R. Amal,
J. J. Gooding, Angew. Chem. Int. Ed. 2012, 51, 6456. doi:10.1002/
ANIE.201202350
Acknowledgements
[19] (a) G. Z. Liu, S. G. Iyengar, J. J. Gooding, Electroanalysis. 2012, 24,
The authors would like to thank the CSIRO Flagship Collaboration Fund
research cluster program for financial support. Yang Lu is grateful for a
CSIRO Flagship Scholarship.
1509. doi:10.1002/ELAN.201200233
(b) G. Z. Liu, S. G. Iyengar, J. J. Gooding, Electroanalysis 2012,
in press.
[20] C. A. Staples, P. B. Dome, G. M. Klecka, S. T. Oblock, L. R. Harris,
Chemosphere 1998, 36, 2149. doi:10.1016/S0045-6535(97)10133-3
[21] W. Haiss, N. T. K. Thanh, J. Aveyard, D. G. Fernig, Anal. Chem. 2007,
79, 4215. doi:10.1021/AC0702084
References
[1] S. E. Lohse, C. J. Murphy, J. Am. Chem. Soc. 2012, 134, 15607.
doi:10.1021/JA307589N
[2] E. C. Dreaden, A. M. Alkilany, X. Huang, C. J. Murphy, M. A.
El-Sayed, Chem. Soc. Rev. 2012, 41, 2740. doi:10.1039/C1CS15237H
[3] M. Cao, Z. Li, J. Wang, W. Ge, T. Yue, R. Li, V. L. Colvin, W. W. Yu,
Trends Food Sci. Technol. 2012, 27, 47. doi:10.1016/J.TIFS.2012.
04.003
[22] C. A. Widrig, C. Chung, M. D. Porter, J. Electroanal. Chem. 1991,
310, 335. doi:10.1016/0022-0728(91)85271-P
[23] D. G. Castner, K. Hinds, D. W. Grainger, Langmuir 1996, 12, 5083.
doi:10.1021/LA960465W
[24] E. Luais, C. Thobie-Gautier, A. Tailleur, M. A. Djouadi, A. Granier, P.
Y. Tessier, D. Debarnot, F. Poncin-Epaillard, M. Boujtita, Electro-
chim. Acta 2010, 55, 7916. doi:10.1016/J.ELECTACTA.2010.02.070
[4] K. Saha, S. S. Agasti, C. Kim, X. Li, V. M. Rotello, Chem. Rev. 2012,
112, 2739. doi:10.1021/CR2001178