Self-assembly and selective exchange of oligoanions on the surface of monolayer protected Au nanoparticles in water

Article abstract of DOI:10.1039/c2cc16926f

Self-assembled monolayers on Au nanoparticles terminating with TACN·Zn^II head groups are attractive scaffolds for the formation of multivalent supramolecular structures at submicromolar concentrations in water.

Full text of DOI:10.1039/c2cc16926f

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COMMUNICATION
Self-assembly and selective exchange of oligoanions on the surface of
monolayer protected Au nanoparticles in waterw
Gregory Pieters, Alessandro Cazzolaro, Renato Bonomi and Leonard J. Prins*
´
Received 8th November 2011, Accepted 15th December 2011
DOI: 10.1039/c2cc16926f
Self-assembled monolayers on Au nanoparticles terminating
II
with TACNꢀZn head groups are attractive scaffolds for the
formation of multivalent supramolecular structures at submicromolar
concentrations in water.
Multivalency is a key concept in biorecognition and a strong
interest exists in the development of synthetic multivalent
1
,2
systems able to interact with biological targets. Monolayer
protected Au clusters (Au MPCs) have emerged as highly attractive
multivalent systems because of their ease of preparation,
3
stability and compatibility with physiological conditions.
Although heterofunctionalized surfaces on Au MPCs can be
simply prepared by using two or more different thiols for
nanoparticle coverage, the obtained mixed self-assembled
4
Fig. 1 (a) Schematic representation of the self-assembly of oligoanionic
probes on the surface of Au MPCs and fluorescence quenching. (b)
Chemical structures of the Au MPCs and probes.
monolayers (SAMs) have generally only a limited complexity.
Challenges are the purification of the resulting Au MPCs and the
characterization of the mixed SAMs both in terms of composition
and morphology. Here, we propose an alternative strategy
towards heterofunctionalized multivalent surfaces relying on the
Previously, we have shown that Au MPCs 3 highly efficiently
5
catalyze the transphosphorylation of 2-hydroxypropyl-4-nitro-
10,11
phenyl phosphate (HPNPP).
self-assembly of small molecules on the surface of Au MPCs.
It was observed that Au MPCs
We show that the cationic surface of Au MPCs 1 can bind quantit-
atively up to around 18 oligoanions even at low mM concentrations
in water (Fig. 1a and b). The surface composition can be controlled
simply by changing the ratio of added oligoanions. Seminal studies
by Rotello et al. have shown that Au MPCs containing terminal
cationic ammonium groups have a very high affinity for polyanions,
3
have a high affinity for oligoanions, such as ATP or Ac-DDD.
This permitted a modulation of the catalytic activity of the
Au MPCs through competition with the HPNPP-substrate,
1
which led to the development of a new enzyme assay. In
2
F
addition, the fluorescence quenching of ATP (2-aminopurine
0
6
7
riboside-5 -O-triphosphate, lex = 305 nm, lem = 370 nm)
upon binding to the Au MPC surface permitted an assessment of
such as proteins and polymers. This property has been used
8
for the development of a series of highly innovative bioassays.
13
the morphology of mixed monolayers. The fact that all these
binding studies were performed under physiological conditions
using even submicromolar concentrations of both nano-
particles and oligoanions made us consider these Au MPCs
as potential scaffolds for the self-assembly of multivalent
structures in water.
Nonetheless, binding studies with small peptides revealed relatively
low surface saturation concentrations with a maximum of around
9
–6 peptides bound per Au MPC. Here, it will be shown that the
5
presence of Zn(II)-metal ions on the surface of Au MPCs 1 offers
significant advantages for the interaction with small molecules:
a higher binding affinity, a higher surface saturation concen-
tration, and the possibility to discriminate between phosphate
and carboxylate binding. It is shown that the latter property
can be used to selectively displace one of the surface bound
components, which is an attractive feature for the development
of responsive surfaces.
14,15
Au MPCs 2 were prepared according to literature procedures
using a new thiol 5, which is accessible in only 4 steps from
commercially available components. Au MPCs 1 were prepared
in situ by adding Zn(NO
stoichiometric ratio compared to the number of triazacyclononane
TACN)-head groups. In addition, Au MPCs 4 containing an
3 2
) to a solution of Au MPCs 2 in a
(
ammonium-head group were prepared as a reference cationic
scaffold. Both Au MPCs 2 and 4 were characterized by NMR,
TEM, TGA, UV-Vis and DLS (ESIw). All analyses were con-
sistent with a diameter of the Au core equal to 1.8 ꢁ 0.5 nm
Department of Chemical Sciences, University of Padova,
via Marzolo 1, 35131 Padova, Italy. E-mail: leonard.prins@unipd.it
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc16926f
1
916 Chem. Commun., 2012, 48, 1916–1918
This journal is c The Royal Society of Chemistry 2012

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Fig. 2 (a) Fluorescence intensity at 370 nm (a.u.) as a function of the
amount of ATP added to a solution of Au MPCs 1 (’), 2 (&), and 4
J) with a headgroup concentration of 10 mM. (b) Fluorescence
intensity (370 nm) as a function of the amount of ATP added to
Au MPCs 1 at different concentrations of TACNZn (’: 1 mM; J:
.5 mM; &: 0.1 mM). Experimental conditions: [HEPES] = 10 mM,
F
(
F
II
Fig. 3 (a) Schematic representation of the sequential addition of two
proves for the controlled formation of a heterofunctionalized surface
on Au MPCs 1. (b) Fluorescence intensity at 545 nm (a.u.) as a
function of the amount of NBD-GDDD added to Au MPCs 1 in the
0
pH 7.0, T = 25 1C. Solid lines are obtained by fitting the data to a
model (ESIw).
presence of different amounts of ATP
32% (J), 53% (m), 66% (n), 80% (*). (c) Surface composition as a
function of the amount of ATP present. Experimental conditions:
F
: 0% (’), 13% (&), 26% (K)
and 1.8 ꢁ 0.4 nm, respectively, implying that around 70 thiols
1
6
are present on the surface of each nanoparticle.
F
The affinity of the cationic surface of Au MPCs 1 for ATPF
was studied by measuring the fluorescence intensity as a
function of the amount of ATPF added to a solution of
Au MPCs 1 (Fig. 2a). This titration relies on the ability of
Au nanoparticles to highly efficiently quench the fluorescence
[HEPES] = 10 mM, pH 7.0, T = 25 1C.
was prepared, in which the NBD (7-nitro-2,1,3-benzoxadiazole)
fluorophore (lex = 484 nm, lem = 545 nm) was attached to the
N-terminus of the tetrapeptide GDDD. The advantage of this
probe is, first, that its fluorescence can be measured indepen-
dently from the ATPF probe and, second, that the negative
charges originate from carboxylate rather than phosphate
groups. A titration of the NBD-GDDD probe to Au MPCs 1
reveals a saturation curve which is practically superimposable
on the curve obtained for ATPF (Fig. 3b, ’). Curve fitting
yielded a surface saturation concentration of 2.7 ꢁ 0.1 mM
indicating that both ATPF and NBD-GDDD saturate the
surface to the same extent. Next, the NBD-GDDD probe
was titrated to Au MPCs 1 ‘preloaded’ with increasing
1
7
of bound fluorophores. The resulting graph is characteristic
of complex formation under saturation conditions: ATP is
F
quantitatively bound to the surface up to the saturation
concentration and remains free in solution afterwards. A surface
saturation concentration of 2.5 ꢁ 0.1 mM was calculated by
fitting of the data to a simplified model assuming the formation
of a 1 : 1 complex between ATPF and a fictitious binding
site (ESIw). The saturation concentration of 2.5 ꢁ 0.1 mM
corresponds to a total of around 18 molecules of ATP bound
F
to the surface at saturation. Strikingly, the surface saturation
concentration dropped to just 0.5 ꢁ 0.1 mM when the fluores-
cence titration was performed on Au MPCs 2, i.e. the identical
system but in the absence of Zn(II) (Fig. 2a). Likewise, a surface
saturation concentration of 1.7 ꢁ 0.1 mM was determined for
Au MPCs 4 equipped with cationic ammonium groups (Fig. 2a). In
addition, the shallow saturation curve for the latter nanoparticles is
amounts of ATP (ranging from 0 to 2.5 mM). Interestingly,
F
the observed surface saturation concentration of NBD-GDDD
decreases regularly as the amount of ATPF on the surface
increases (Fig. 3a). No fluorescence emission originating from
a displacement of ATPF (see below) from the surface was
observed. Importantly, summing the concentrations of bound
indicative of a lower binding affinity of ATP for the surface.
F
NBD-GDDD and ATP probes gave a constant concentration
F
These results clearly demonstrate that the presence of the
II
Zn -metal ion on the surface of Au MPCs 1 drives the
of 2.6 ꢁ 0.2 mM over the complete range of probe ratios
studied (Fig. 3c). This shows that the surface composition can
be varied simply by changing the ratio of added oligoanions
without affecting the valency of the complex.
formation of a multicomponent complex of significantly higher
valency compared to the positively charged surfaces of either
Au MPCs 2 or 4. The impressive ability of Au MPCs 1 to bind
The noncovalent nature of the multivalent complexes provides
the additional possibility of exchanging the surface bound
components. This is highly attractive for the development of
F
ATP is further evidenced by the fact that binding could be
II
detected at a concentration of TACNꢀZn down to 0.5 mM,
1
8
which corresponds to a nanoparticle concentration of around
responsive multivalent surfaces. The dynamic nature of the
system is illustrated by the displacement of the ATPF probe
(2.0 mM) from the surface of Au MPCs 1 upon the addition of
increasing amounts of competing ATP.z The displacement can
be simply monitored by measuring the fluorescence intensity
1
0 nM. From these measurements an apparent binding constant,
8
ꢃ1
K
app, of 2.4 ꢂ 10 M , was calculated (Fig. 2b).
Working under saturation conditions implies that all added
oligoanions are localized on the surface of Au MPC 1. We
investigated the possibility of creating a heterofunctionalized
multivalent surface through the self-assembly of different oligo-
anions (Fig. 3a). For that purpose, a second probe NBD-GDDD
originating from free ATP (Fig. 4a). A displacement of 50%
F
of ATP requires the addition of 3.3 mM ATP, corresponding
F
to a relative binding affinity of 3.3 (K
,
app ATP
: K
,
app ATP
).
F
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1916–1918 1917

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displacement curves confirm that the NBD-GDDD probe is
released at lower concentrations compared to ATP
F
and the
6
.7 ratio between the relative binding affinities (120 and 18,
respectively) indicates that the two probes exchange indepen-
dently (Fig. 5c).
In conclusion, our results show that Au MPCs 1 are highly
attractive supramolecular building blocks for the self-assembly
of multivalent nanostructures. The high affinity of both oligo-
phosphates and oligocarboxylates for the multivalent TACNꢀ
II
Zn surface allows the complexation of up to 18 probe
Fig. 4 Displacement of ATP
b) as a function of the concentration of competitor (’: ATP, &: ADP,
: Ac-DDD) added. Experimental conditions: (a) [ATP ] = 2.0 mM,
b) [ATP ] = 1.4 mM, [HEPES] = 10 mM, pH 7.0, T = 25 1C.
F
from the surface of Au MPCs 1 (a) and 4
molecules at low micromolar concentrations in water. The
surface composition can be modulated in a straightforward
manner simply by changing the ratio of the anionic probes.
The surface of Au MPCs 1 has a higher affinity for phosphates
compared to carboxylates and this can be used for the selective
exchange of surface bound components. Financial support from
the ERC (StG-239898) and COST (CM0703) is acknowledged.
(
*
F
(
F
In the same manner, relative binding affinities of 120 and 580 were
determined for ADP and Ac-DDD, respectively. Remarkably,
the same displacement studies performed on ammonium-
terminated Au MPCs 4 gave nearly identical relative binding
affinities of 10 and 14 for ATP and Ac-DDD, respectively
Notes and references
(
Fig. 4b).z This strong difference between the two systems
z All displacement studies were performed at 80% of the surface
saturation concentrations in order to avoid the presence of even a
minimal amount of fluorescent probe in solution. The weaker binding
II
illustrates the importance of the Zn -metal ion in differentiating
between phosphate and carboxylate binding. For the Au MPCs
F
of the ATP probe to Au MPCs 4 renders determination of the relative
1
presented here, this creates an attractive opportunity to
binding affinities less precise.
selectively exchange a surface bound carboxylate in the presence
of a phosphate (Fig. 5a). In order to verify that hypothesis, the
1
2
3
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F
3
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(
Fig. 5b). Next, the ability to selectively exchange one of the
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5
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F
and
NBD-GDDD in a 1 : 1 ratio (1.0 mM each). The obtained
7
8
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918 Chem. Commun., 2012, 48, 1916–1918
This journal is c The Royal Society of Chemistry 2012