Silica nanoparticles functionalised with cation coordination sites and fluorophores for the differential sensing of anions in a quencher displacement assay (QDA)

Article abstract of DOI:10.1039/c1cc13039k

In conjunction with quenching metal ions, silica nanoparticles carrying terpyridine coordination sites and sulforhodamine B signalling units were employed for the differential fluorometric recognition of anions.

Full text of DOI:10.1039/c1cc13039k

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COMMUNICATION
Silica nanoparticles functionalised with cation coordination sites and
fluorophores for the differential sensing of anions in a quencher
displacement assay (QDA)w
c
ade
ade
Pilar Calero,ab Mandy Hecht, Ramon Martınez-Manez,* Felix Sancenon, Juan Soto,ad
´
´
´
´
´
Jose L. Vivancos and Knut Rurack*c
df
´
Received 24th May 2011, Accepted 11th August 2011
DOI: 10.1039/c1cc13039k
In conjunction with quenching metal ions, silica nanoparticles
carrying terpyridine coordination sites and sulforhodamine B
signalling units were employed for the differential fluorometric
recognition of anions.
Especially the independent functionalization of inorganic surfaces
with receptor and fluorophore units is an appealing approach
that overcomes the synthetic problems usually immanent
to the classical ‘‘binding site–signalling subunit’’ architecture.7
Moreover, signal amplification and cooperative binding effects
associated with the independent anchoring of receptors and
fluorophores in close proximity to each other and to the surface
of the support are noticeable features of this strategy, allowing
for instance intercommunication between both subunits without
the need for a direct covalent link between them. Employing silica
nanoparticles functionalised with fluorophores and suitable
coordination sites, this approach has recently been used for the
selective recognition of metal cations.8 In contrast, hybrid
nanoparticles for the optical recognition of anions are much
less explored.9
The development of chromo- and fluorogenic chemosensors
for anions has received growing interest during the past years
due to the fundamental roles that anions play in chemical and
biological processes.1 The majority of these chemosensors
incorporate molecular receptors of supramolecular chemistry
design. Traditionally, supramolecular anion sensing utilizes either
colour and/or fluorescence changes in probes with a ‘‘binding
site–signalling subunit’’ architecture or undergoing chemo-
dosimetrical reactions and indicator release in displacement
assays (IDAs) for signal transduction.2,3 However, recently a
novel trend in chemosensor development has emerged which
uses organic–inorganic hybrid materials, in many cases nano-
particles.4 These hybrid systems often show synergistic effects
which are hardly achievable with molecular systems or inorganic
solids alone.5
Our present hybrid system for the optical differential recog-
nition of anions is based on the competitive coordination to
a metal centre. Since one of our interests in such assays is
the facile regeneration of the sensing material,10 we modified
the conventional IDA concept. Instead of displacing an
indicator dye with the analyte from a binding site, which
requires a two-step regeneration procedure of analyte removal
and subsequent indicator reloading (and sometimes also the
spatial separation of an indicator from the host–guest complex
for detection), we attached a fluorophore (F) and a receptor
(R), which does not bind to the analyte but to a mediator,
separately to the support. A quenching metal ion (Q), which is
at the same time a good binder for the target anion, then
served as the mediator, i.e., as the displaceable species, arriving
at a quencher displacement assay (QDA, Scheme 1). In the
first step of the protocol, the R units of the highly fluorescent
receptor- and fluorophore-functionalised nanoparticles (RFNs)
are loaded with Q, switching off the hybrid’s fluorescence.
Second, addition of the target analyte, which shows a higher
affinity for Q than R does, displaces Q from R, leading
to revival of the fluorescence of the RFNs. In a third step,
regeneration of the material is simply accomplished by reloading
Q onto the RFNs. For the title material, terpyridine units as
R for the quencher metal ions Q and sulforhodamine B
as signalling units F were independently anchored onto the
surface of the silica nanoparticles.
Among different inorganic supports silica nanoparticles have
been extensively used for hybrid sensing materials due to their
straightforward preparation and surface functionalization and
high stability in aqueous suspensions.6 As an alternative to
chromo- and fluorogenic probes, self-assembled structures on
surfaces in particular have attracted increasing attention recently.
a
´
´
Departamento de Quımica, Universidad Politecnica de Valencia,
Camino de Vera s/n, 46022, Valencia, Spain.
E-mail: rmaez@qim.upv.es
b
´
Instituto Tecnologico de la Construccion (AIDICO), Avenida
Benjamın Franklin 17, 46980, Paterna (Valencia), Spain
¨
Richard-Willstatter-Straße 11, D-12489 Berlin, Germany.
¨
´
´
c Div. 1.5, BAM Bundesanstalt fur Materialforschung und -prufung,
¨
E-mail: knut.rurack@bam.de
d
´
Centro de Reconocimiento Molecular y Desarrollo Tecnologico
(IDM), Unidad Mixta Universidad de Valencia-Universidad
Polite´cnica de Valencia, Spain
e
f
´
CIBER de Bioingenierıa, Biomateriales y Nanomedicina
(CIBER-BBN), Spain
Departamento de Proyectos de Ingenierıa, Universidad Politecnica de
´
Valencia, Camino de Vera s/n, 46022, Valencia, Spain
´
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13039k
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10599–10601 10599

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quenching of 94–99% of the rhodamine emission was found,
suggesting that the average distance between R and F is
sufficiently short to activate the transduction channel. Depending
on the metal ion bound at the terpyridine unit, the process
responsible for the quenching of the dye’s fluorescence involves
an energy or electron transfer or enhanced spin–orbit coupling
because of the heavy atom effect.11 At saturation quenching,
the major lifetime components in the complexed hybrids
amount to 20–45 ps for TSNP–Fe3+
,
TSNP–Hg2+
,
.
TSNP–Pb2+ and TSNP–Cu2+ and 0.11 ns for TSNP–Ni2+
In contrast to metal ions bound directly at a fluorophore, for
which paramagnetic ions such as Cu2+ usually show static
quenching, i.e., a non-emissive complex, and heavy metal ions
like Hg2+ a complex with a reduced lifetime, here, due to the
separation of R and F, all metal ions lead to a reduction of the
lifetime of F, stressing the fact that a distance-dependent
process is active.
Scheme 1 Design concept of the quencher displacement assay (QDA)
involving terpyridine–sulforhodamine-functionalised nanoparticles
(TSNPs) and metal ion quenchers (Q).
The critical role of the terpyridine receptor was demonstrated
in parallel studies using the model SNPs that are only func-
tionalised with F but do not contain R. The addition of metal
ions to acetonitrile suspensions of SNPs induced negligible
changes in the emission of the dye, indicating that the presence
of the R units in TSNP is critical for the quenching mechanism.
Only when coordinated to terpyridine are the metal ions in a
spatial proximity with the fluorophore that is close enough to
induce quenching.
The synthesis of the reactive precursors 1 and 2 (Scheme 1)
and the functionalised nanoparticles is described in the ESI.
The contents of terpyridine and rhodamine amount to 30.84 and
3.9 mmol (mol[SiO2])À1 in the terpyridine–sulforhodamine-
functionalized nanoparticles (TSNP) and that of rhodamine to
3.9 mmol (mol[SiO2])À1 in the nanoparticles functionalized
only with 2 (SNPs). Both, TSNP and SNP show a homogenous
particle size of 20 Æ 2 nm (Fig. 1). As a rough estimate, each
coated TSNP carries up to 2100 attached molecules. The
average distance between two subunits thus amounts to ca. 8 A.
The first step toward a successful assay is evident from the
observation that the emission of the sulforhodamine fluorophore
was retained after the grafting of the signalling units onto
the surface of the nanoparticles. Excitation of acetonitrile
suspensions of TSNPs at 575 nm gave rise to an emission
band at 580–660 nm, similar to that obtained for sulforhod-
amine B chloride in acetonitrile (Fig. 1). Fluorescence lifetime
measurements revealed that the decay times of the dye in the
TSNPs (tf = 2.21 ns; and SNPs tf = 2.21 ns) is virtually
identical to that of the isolated molecule in solution (tf = 2.24 ns),
stressing the fact that the single dye molecules do not interact
with each other when attached to the surface and that grafting
has also no influence on the fluorophore’s properties.
Having established the signalling mechanism the corresponding
TSNP–Q hybrid materials (Q = Cu2+, Fe3+, Hg2+, Ni2+
and Pb2+) were tested as sensing systems for anions. The
underlying idea of our approach relies on the ability of the
anion to successfully compete with the terpyridine units for
the metal ion and displace the latter from the R units, resulting
in an enhancement of the fluorescence. This enhancement
should be a direct consequence of a delicate balance between
the binding strength of the metal ion with terpyridine and the
affinity of the cation for the added anion.
To test the ability of the TSNP–Q ensembles for differential
recognition of small monovalent anions, acetonitrile solutions
of TSNP–Q chemosensors were prepared by adding 1 equivalent
(with respect to the 2.0 Â 10À4 mmol R units contained, see
ESIw for details) of the corresponding metal ion to quench the
rhodamine emission. Then, 0.1–10 equivalenÀts of the anions
H2PO4À, HSO4À, FÀ, ClÀ, BrÀ, IÀ and NO3 were added to
the TSNP–Q suspensions. Except in one case (see below), no
uniquely selective fluorescence changes were found. However,
In the second step, various heavy and transition metal ions
were tested as quenchers in CH3CN. In a typical experiment,
suspensions of TSNP (2.5 mg in 20 mL) were titrated with
Fe3+, Hg2+, Cu2+, Ni2+ and Pb2+ (as perchlorates). As would
be expected for a rather non-selective ligand, terpyridine binds
all these metal ions. Moreover, in all the cases, a severe
as a general trend the addition of H2PO4 and FÀ induced a
À
remarkable fluorescence enhancement, addition of ClÀ, BrÀ
and HSO4À induced a moderate increase of the emission whereas
À
the addition of IÀ and NO3 induced only poor fluorescence
changes. In addition, despite this general observation a clear
modulation of the relative response in the presence of anions
was found, depending on the metal ion employed.12 This
behaviour is reminiscent of other probe–metal ion–anion
ternary complex systems13 and is a well-suited prerequisite for
differential recognition.14 Qualitative analysis of this differential
response for anions using PCA (Principal Component Analysis)
algorithms yielded the results summarized in Fig. 2. As can be
deduced from the figure, recognition patterns can be identified
for all the anions studied even for those that display a rather
Fig. 1 (a) TEM image of TSNPs, average diameter = 20 nm;
(b) fluorescence spectra of TSNP and SNP suspended and sulforhod-
amine B dissolved in acetonitrile.
c
10600 Chem. Commun., 2011, 47, 10599–10601
This journal is The Royal Society of Chemistry 2011

View Online
Coordination of metal ions at the terpyridine units induced
quenching of the neighbouring fluorophores. Upon addition
of anions, coordination to the metal centres and a partial
recovery of the fluorescence was observed. The final response
of a certain TSNP–Q system to a particular anion is a delicate
balance between the binding strength of the metal ion with the
terpyridine and the affinity of the cation for the added anion.
This allows differential recognition of small inorganic anions
in a quencher displacement assay. Furthermore, regeneration
of the system is possible in a straightforward manner. The
facile and independent functionalisation of silica surfaces with
various chemical entities and the possible use of a number of
different coordination units and metal ions make this approach
highly appealing for the search for new chemosensors for anions.
Financial support from the Spanish Government (project
MAT2009-14564-C04-01), the Generalitat Valencia (project
PROMETEO/2009/016) and the Innovationsfonds (BAM/
Fig. 2 Principal component analysis (PCA) score plot for the anions as
indicated using TSNP–Q (Q = Fe3+, Hg2+, Cu2+, Ni2+ and Pb2+
)
ensembles. Data shown from three different trials and 1 : 5 TSNP–Q to
anion ratios. PC axes are calculated to lie along lines of diminishing levels
of variance in the data set.
Bundesministerium fur Wirtschaft und Technologie) is gratefully
¨
acknowledged.
poor change in fluorescence. Fluorescence lifetime studies of
the ternary systems revealed that upon addition of anions, the
lifetime features of the TSNP–Mn+ ensembles are gradually
turned into the features of TSNP, i.e., the major 2.2 ns
component of the sulforhodamine is recovered, pointing to
an actual displacement of the metal ion from the terpyridine units.
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´
´
À
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´
´
´
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Fig. 3 Emission intensity of acetonitrile suspensions of TSNP–Pb2+
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c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10599–10601 10601