Enhanced fluorescence of silver nanoclusters stabilized with branched oligonucleotides

Article abstract of DOI:10.1039/c3cc40446c

DNA stabilized silver nanoclusters (AgNCs) are promising optical materials, whose fluorescence properties can be tuned by the selection of the DNA sequence employed. In this work we have used modified oligonucleotides in the preparation of AgNCs. The fluorescent intensity obtained was 60 times higher than that achieved with standard oligonucleotides. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc40446c

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Enhanced fluorescence of silver nanoclusters stabilized
with branched oligonucleotides†
Cite this: Chem. Commun., 2013,
49, 4950
a
ab
ab
a
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´
Alfonso Latorre,z Romina Lorca,z Felix Zamora* and Alvaro Somoza*
Received 18th January 2013,
Accepted 7th April 2013
DOI: 10.1039/c3cc40446c
www.rsc.org/chemcomm
DNA stabilized silver nanoclusters (AgNCs) are promising optical clusters.9 Moreover, the emission of DNA-AgNCs depends
materials, whose fluorescence properties can be tuned by the strongly on the DNA sequence,10 allowing the preparation of
selection of the DNA sequence employed. In this work we have a wide palette of colours by changing the nucleotide sequence.
used modified oligonucleotides in the preparation of AgNCs. The Interestingly, some of these fluorescent materials have shown
fluorescent intensity obtained was 60 times higher than that good pH, temperature and oxidation resistance, preserving
achieved with standard oligonucleotides.
31% of the initial emission intensity after 10 months.11
The environment of AgNCs can also affect their fluorescent
The use of fluorescent materials in biology has allowed the properties. In this sense, the structural changes promoted by
discovery of many biological processes in living cells. Structures the interaction with a given analyte have been exploited to
commonly employed in cells and animals are fluorescent proteins,1 develop sensors for different entities such as ions,12 small
organic dyes2 and quantum dots.2a,3 Fluorescent proteins have molecules,13 DNA,14 microRNAs,15 proteins16 or tumour cells.17
been successfully used in many applications such as protein
On the other hand, it is well known that DNA is affected in
expression or protein–protein interaction studies.4 However the hybrid materials modified with multiples strands, due to an
size of these structures may interfere with some internal cell interaction between the neighbouring DNA ion clouds. This
processes, and aggregation events cause cellular toxicity. The use behaviour is typically observed in hybrids such as nanoparticles18
of organic dyes is limited by their poor photostability and toxicity.5 or polymers19 with a great number of strands, but it is also possible
In the case of quantum dots, their size and toxicity are important in hybrid materials with only three20 or two21 strands around a
drawbacks that prevent their use in live cells and animals.3 In this rigid and small molecule core. However, the effect of neighbouring
context, DNA-stabilized silver nanoclusters (DNA-AgNCs) are being strands on the fluorescent properties of DNA stabilized silver
intensively studied as alternative fluorescent probes.6 AgNCs have nanoclusters in this kind of hybrids is still unexplored.
shown good quantum yields, and possess high photostability and
In this work, we report the synthesis of a family of branched DNA
small size7 (o2 nm). The use of DNA as a template enhances the structures that can be used in the preparation of DNA-AgNCs. The
biocompatibility and water solubility of silver clusters. What is fluorescent intensities obtained with these derivatives are higher
more, the specific binding properties of aptamers make these compared with those obtained in the case of AgNCs stabilized with
materials promising candidates for specific biolabeling.8
a single stranded DNA. To the best of our knowledge this is the first
The fluorescence properties of AgNCs depend on their report of AgNCs prepared with modified oligonucleotides. The
structural features and other parameters such as buffer, pH, derivatives reported herein are composed of two or three ssDNA
concentration of reagents, size and oxidation state of the silver attached to a small hydrophobic benzene core at different positions.
The dimers (1 and 2) contain DNA strands at ortho and meta
positions (Fig. 1). On the other hand, the trimers (3 and 4) have
the strands at alternating positions.
a
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Instituto Madrileno de Estudios Avanzados en Nanociencia (IMDEA Nanociencia),
´
& CNB-CSIC-IMDEA Nanociencia Associated Unit ‘‘Unidad de Nanobiotecnologıa’’
Particularly, the hybrid structure 3 contains a benzene ring
where the DNA strands are placed at positions 1, 3 and 5, which
should give rise to a planar DNA trimer structure. In the case of
hybrid 4, the benzene ring is further substituted with three ethyl
groups at positions 2, 4 and 6. The presence of the ethyl groups in 4
should yield a more rigid structure and probably with a different
orientation of the DNA strands (Fig. 1). The benzene scaffold with
three ethyl groups has been previously used to force the groups
Cantoblanco, 28049 Madrid, Spain. E-mail: alvaro.somoza@imdea.org;
Fax: +34 91 2998725; Tel: +34 912998856
b
´
´
´
Universidad Autonoma de Madrid, Departamento de Quımica Inorganica,
28049 Madrid, Spain. E-mail: felix.zamora@uam.es; Fax: +34 91 497 4833;
Tel: +34 91 497 3962
† Electronic supplementary information (ESI) available: Synthesis of organic
molecules and oligonucleotides, characterization data, AFM images, fluores-
cence, UV and CD spectra. See DOI: 10.1039/c3cc40446c
‡ Both authors contributed equally to this work.
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4950 Chem. Commun., 2013, 49, 4950--4952
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should be due to the disposition of the strands. Particularly, in
the case of the dimers, the ortho derivative (1) showed higher
fluorescence than the meta derivative (2). This result suggests
that the close proximity between strands better promotes the
formation of fluorescent AgNCs.
The most striking results were obtained with the trimers
where the fluorescence obtained was around 60 times higher
than that obtained with the PolyC12 (Fig. 2). Among the trimers,
4 showed the highest fluorescence values compared with all the
samples tested, including single strands of DNA with 24 and 36
cytosines (ESI,† Fig. S2). Interestingly, its fluorescence did not
decay significantly during the first 24 h as in the single stranded
samples (ESI,† Fig. S3). What is more, the fluorescence of the
trimers was maintained after 20 days in the dark (ESI,† Fig. S4).
Due to the good results obtained using the trimers we decided
to further study these materials. First, a UV analysis of the samples
revealed a peak at 440 nm, which is characteristic of silver
nanoparticles, and a peak at 560 nm, indicating the presence of
AgNCs. In the case of the single stranded PolyC12 the signals were
significantly less intense than those of the trimers (Fig. 3). AFM
analysis of the samples revealed small particles (ca. 2 nm height) in
the case of samples 3 and 4, which could be the AgNCs responsible
for the absorbance band at 560 and the intense fluorescence (ESI,†
Fig. S13 and S14). In the case of PolyC12 larger structures of 12 nm
height were observed, which could be due to the formation of
silver nanoparticles during the reduction step (ESI,† Fig. S12).
In addition, we monitored the fluorescence and absorbance
during the first few hours to better understand the kinetics of
AgNCs formation (ESI,† Fig. S5–S8). We observed that the
highest fluorescence is achieved after 10 hours, and then it
decreases slowly. In the UV spectrum, the band at 440 nm, that
could be related to Ag nanoparticles, decreases overtime, mean-
while the band associated to AgNCs (560 nm) increases.
We believe that the high fluorescence as well as the stability
observed in the trimers is due to the spatial arrangement of the
AgNCs and the DNA strands. In these cases the AgNCs could be
better protected from quenching and also the close proximity of
the strands could result in a cooperative effect, leading to the
Fig. 1 Schematic representation of the derivatives prepared in this work. The
spatial arrangement presented is arbitrary.
placed at alternating positions to be oriented at different sides of
the benzene ring.22 In our case the arrangement of the groups
is difficult to predict, but taking into account the sensitivity of
DNA-AgNCs to the changes in the surrounding media, we expect
variations in the fluorescence properties of trimers 3 and 4 as well in
the dimers 1 and 2.
For our studies we selected a DNA strand composed of
twelve cytosines (C12) because it is known that cytosines
stabilize AgNCs better than guanines and much better than
adenines and thymines. In addition, this sequence has been
previously employed in the preparation of AgNCs, affording
fluorescent AgNCs with a maximum absorption at 560 nm, and
its corresponding maximum emission at 630 nm.14b
The derivatives 1–4 were synthetized using a ‘‘click’’ reac-
tion23 between a C12 oligonucleotide modified with an alkyne
moiety at the 30 end and the corresponding azide derivative.24
The required DNA strand (C12 sequence) was synthetized using
a MerMade4 DNA synthesizer, a modified CPG prepared in our
laboratory, and commercial cytosine phosphoramidites (ESI†).
The Cu-catalyzed Huisgen reaction afforded the corresponding
branched derivatives without significant DNA degradation (ESI†).
Then, the compounds 1–4 were employed in the preparation
of silver nanoclusters. These derivatives were incubated with
silver nitrate (6 equiv. per oligonucleotide strand) for 10 min at
room temperature and then reduced with a fresh solution of
sodium borohydride.
The fluorescence of the silver nanoclusters generated using
compounds 1–4 was measured after 4 hours. The fluorescence
obtained with the branched derivatives was higher than the
fluorescence obtained with a single strand of DNA (PolyC12)
(Fig. 2). Since the effect of the benzene ring on the fluorescence
intensity is not relevant (ESI,† Fig. S2) the enhanced fluorescence
Fig. 3 UV spectra of the three samples: PolyC12, tris (3) and tris-ethyl (4). The
peak at 440 nm indicates the presence of silver nanoparticles, meanwhile the
peak at 560 nm arises due to AgNCs. The inset is a zoomed area of an AFM
topographic image showing small particles of 2 nm height.
Fig. 2 Fluorescence intensity (ex. 560 nm, em. 630 nm) of AgNCs prepared with
PolyC12, ortho (1), meta (2) tris (3) and tris-ethyl (4) measured after 4 h.
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significant change in the structure after the generation of AgNCs.
high fluorescence. In this sense, minimization of the structures
using molecular mechanics suggests a globular structure for
the trimers. This structure could better protect the AgNCs from
quenching, compared with the standard oligonucleotides,
which have linear structures (ESI,† Fig. S15).
We also monitored the structural changes in the oligonucleo-
tides promoted by AgNCs formation using circular dichroism (CD).
Interestingly, a significant change was observed before and after
AgNCs formation where the positive band at 280 nm is completely
inverted after AgNCs formation (Fig. 4 and ESI† Fig. S9–S11). What
is more, this change in the structure is related to the fluorescence
intensity since when the fluorescence of AgNCs decreases the
positive band at 280 nm is recovered, whereas the negative band
at 270 nm decreases. These data suggest that the structure of the
oligonucleotides stabilizing fluorescent AgNCs is different from the
structure of the oligonucleotides alone and also that the original
DNA structures can be recovered over time leading to an efficient
quenching of the fluorescence of AgNCs.
We have prepared fluorescent silver nanoclusters using
novel trimers of oligonucleotides connected to a benzene
molecule. The fluorescence exhibited by these derivatives is
60 times higher than that obtained with a single strand. We
believe that the high fluorescence obtained using the trimers is
due to a cooperative effect between vicinal strands, which could
better stabilize the AgNCs and prevent their quenching. This
work illustrates the use of modified oligonucleotides in the
preparation of AgNCs with improved fluorescence properties.
Spanish Ministry of Science and Innovation (Grants: SAF2010-
15440, ACI2009-0969, MAT2011-15219-E and MAT2010-20843-
C02-01) and IMDEA Nanociencia are acknowledged for financial
support.
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