Short-peptide-based hydrogel: A template for the in situ synthesis of fluorescent silver nanoclusters by using sunlight

Article abstract of DOI:10.1002/chem.201001240

N-terminally Fmoc-protected dipeptide, Fmoc-Val-Asp-OH, forms a transparent, stable hydrogel with a minimum gelation concentration of 0.2 % w/v. The gelation property of the hydrogel was investigated by using methods such as transmission electron microscopy, field-emission scanning electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy. The silver-ion-encapsulating hydrogel can efficiently and spontaneously produce fluorescent silver nanoclusters under sunlight at physiological pH (7.46) by using a green chemistry approach. Interestingly, in the absence of any conventional reducing agent but in the presence of sunlight, silver ions were reduced by the carboxylate group of a gelator peptide that contains an aspartic acid residue. These clusters were investigated by using UV/Vis spectroscopy, photoluminescence spectroscopy, high-resolution transmission electron microscopy (HR-TEM), atomic force microscopy (AFM) and X-ray diffraction (XRD) studies. Mass spectrometric analysis shows the presence of a few atoms in nanoclusters containing only Ag₂. The reported fluorescent Ag nanoclusters show excellent optical properties, including a very narrow emission profile and large Stokes shift (>100 Nm). The reported fluorescent Ag nanoclusters within hydrogel are very stable even after 6 Months storage in the dark at 4 °C. The as-prepared hydrogel-nanocluster conjugate could have applications in antibacterial preparations, bioimaging and other purposes. Silver nanoparticles: An N-terminally protected dipeptide-based hydrogel has been used to make fluorescent silver nanoclusters in the presence of sunlight at room temperature and at physiological pH by using a green chemistry approach. The fluorescent silver nanoclusters exhibit interesting fluorescence properties with a very narrow emission profile and large Stokes shift that may be useful in future applications. Copyright

Full text of DOI:10.1002/chem.201001240

DOI: 10.1002/chem.201001240
Short-Peptide-Based Hydrogel: A Template for the In Situ Synthesis of
Fluorescent Silver Nanoclusters by Using Sunlight
Bimalendu Adhikari and Arindam Banerjee*[a]
Dedicated to Professor Animesh Chakravorty on the occasion of his 75th birthday
Abstract: N-terminally Fmoc-protected
dipeptide, Fmoc-Val-Asp-OH, forms a
ly, in the absence of any conventional
reducing agent but in the presence of
sunlight, silver ions were reduced by
the carboxylate group of a gelator pep-
tide that contains an aspartic acid resi-
due. These clusters were investigated
by using UV/Vis spectroscopy, photolu-
minescence spectroscopy, high-resolu-
tion transmission electron microscopy
(HR-TEM), atomic force microscopy
(AFM) and X-ray diffraction (XRD)
studies. Mass spectrometric analysis
shows the presence of a few atoms in
nanoclusters containing only Ag2. The
reported fluorescent Ag nanoclusters
show excellent optical properties, in-
cluding a very narrow emission profile
and large Stokes shift (>100 nm). The
reported fluorescent Ag nanoclusters
within hydrogel are very stable even
after 6 months storage in the dark at
48C. The as-prepared hydrogel–nano-
cluster conjugate could have applica-
tions in antibacterial preparations, bio-
imaging and other purposes.
transparent, stable hydrogel with
a
minimum gelation concentration of
0.2% w/v. The gelation property of the
hydrogel was investigated by using
methods such as transmission electron
microscopy, field-emission scanning
electron microscopy, atomic force mi-
croscopy and Fourier transform infra-
red spectroscopy. The silver-ion-encap-
sulating hydrogel can efficiently and
spontaneously produce fluorescent
silver nanoclusters under sunlight at
Keywords: fluorescence
·
gels
·
green chemistry · nanoparticles ·
peptides
physiological pH (7.46) by using
a
green chemistry approach. Interesting-
Introduction
need for the development of environmentally friendly meth-
ods for the synthesis of nanomaterials targeted for biomedi-
cal applications. An eco-friendly reducing and capping
agent, an environmentally suitable solvent system and
room-temperature synthesis are essential criteria for an ab-
solutely green nano-structured synthesis.[3]
Noble metal nanoclusters have attracted considerable atten-
tion due to their marvellous optical and chemical properties
and interesting applications in single-molecular spectrosco-
py, catalysis, biological/chemical sensing and biological
imaging.[1] Few-atom gold/silver clusters are even smaller
than the corresponding plasmon-supporting metal nanopar-
ticles. Interestingly, they show molecule-like electronic tran-
sitions within the conduction band, which results in fluores-
cence properties.[2] These fluorescent nanoclusters are at-
tractive candidates for biolabelling and bioimaging due to
their bright fluorescence, ultrafine size, very low fluores-
cence intermittency and nontoxicity. There is a growing
Of the noble metals, fluorescent gold nanoclusters have
been extensively studied because of their chemical stability
and facile synthetic procedure.[1d,f,2c,4] However, relatively
few studies have been focused on the silver clusters because
these clusters are generally not very stable in water and
have a natural tendency to aggregate to form larger nano-
particles. There are several methods to prepare fluorescent
silver nanoclusters, which include radiolytic,[5] chemical re-
duction[6–10] and photochemical (using UV light) approach-
AHCTUNGTRENNUNG
es.[2a,11–15] Dickson and co-workers made a pioneering contri-
[a] B. Adhikari, Dr. A. Banerjee
Department of Biological Chemistry
Indian Association for the Cultivation of Science
Jadavpur, Kolkata, 700032 (India)
Fax : (+91)332473-2805
bution to the preparation of silver clusters by using different
methods.[1a,b,6] There are many reports for the synthesis of
fluorescent silver clusters by using DNA as a scaffold in
aqueous solution.[7,8] The role of sequence-dependent DNA
templates in the formation of fluorescent Ag nanoclusters
has also been reported recently.[8] Jin and co-workers recent-
E-mail: bcab@iacs.res.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem201001240.
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FULL PAPER
ly reported Ag7 clusters by using 2,3-mercaptosuccinic acid
as a ligand.[10] Formation of silver nanoclusters have also
been reported within silver-exchanged zeolites formed by
heat treatment[16] and within glass by using femtosecond
laser irradiation.[17] Kumacheva and co-workers reported the
successful photogeneration of fluorescent Ag nanoclusters
in poly(N-isopropylacrylamide-acrylic acid-2-hydroxyethyl
acrylate) copolymer-based hydrogel microspheres by UV ir-
radiation.[12] Recently, the formation of fluorescent Ag nano-
particles in p(NIPAM-AA-AAm) microgels by using NaBH4
was reported.[9c] However, environmentally harmful UV ir-
radiation/gamma irradiation or toxic sodium borohydride
has been used as a reducing agent in all above-mentioned
cases. To the best of our knowledge, there is only one report
on an absolutely green synthesis of fluorescent gold nano-
clusters by using a commercially available protein, bovine
serum albumin (BSA), at physiological temperature.[18]
Herein we report the green synthesis of fluorescent Ag
nanoclusters within a dipeptide-based supramolecular hy-
drogel by using sunlight irradiation (for a few minutes) at
physiological pH and room temperature.
Figure 1. The change in Tgel with respect to the gelator concentration.
Inset: A photograph of the hydrogel at 0.2% w/v at physiological pH.
(AFM). The TEM image (Figure 2a) of a slightly diluted so-
lution of this peptidic hydrogel in water shows an entangled
three-dimensional network structure of fibrous aggregates.
The width of the gel fibres is within the range of 35 to
Results and Discussion
Preparation and characterisation of the dipeptide-based hy-
drogel: The N-terminally protected dipeptide Fmoc-Val-
Asp-OH forms a stable, transparent hydrogel in the pH
range 1–12 (Figure 1, inset). The minimum gelation concen-
Figure 2. a) TEM and b) FE-SEM images of the hydrogel.
42 nm and each fibre is a few micrometers in length. Indi-
vidual junctions between these fibres were also observed,
which are probably responsible for stabilising the supra-
molecular hydrogel network. The FE-SEM image (Fig-
ure 2b) of the freeze-dried hydrogel shows a dense inter-
twined fibrous aggregate. The width of these fibres remains
30 to 45 nm. It has been found from this study that the
width of these gel fibres is much higher than that of the mo-
lecular dimension of the gelator peptide. This clearly indi-
cates that several molecular chains are self assembled to
form the supramolecular aggregated fibrilar structure. The
AFM image (Figure 3a) of the hydrogel also shows a uni-
form three-dimensional nanofibrillar network structure with
an average diameter of 50 nm in the gel state. The uniform
nature of the aggregates observed in these morphological
studies suggests that this peptide molecule self-assembles in
a hierarchical fashion to form a one-dimensional supra-
molecular polymer of intertwined fibres to give a larger net-
work of superstructures in a gel. An FT-IR study (Figure 3b)
of freeze-dried hydrogel was carried out to understand the
intermolecular interaction between the gelator peptides in
the gel state. The presence of bands at n˜ =3294, 1647 and
tration is 0.2% w/v at pH 7.46, that is, a single peptide mole-
cule can gelate almost 12611 water molecules. This indicates
that the gelator peptide is very efficient at forming hydro-
gels. The gelator peptide was first dissolved in a minimum
volume of dimethyl sulfoxide with gentle heating, and then
cooled to room temperature. Upon addition of the required
amount of water to the solution, a transparent thermorever-
sible hydrogel was immediately produced. The temperature
at which the gel-to-sol transition occurs is called the gel-
melting temperature (Tgel) and it increases as the concentra-
tion of the gelator peptide increases, until the plateau region
is reached (Figure 1). This suggests that the formation of the
supramolecular gel network is essentially complete at the
higher concentration region.
The morphology of the supramolecular structure formed
by the hydrogel was investigated by using field-emission
scanning electron microscopy (FE-SEM), transmission elec-
tron microscopy (TEM) and atomic force microscopy
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A. Banerjee and B. Adhikari
producing nanoparticles because hydrogels are generally
nontoxic, biocompatible materials. A controlled deposition
of silver nanoparticles into the polymer microgel has been
achieved by Kumacheva and co-workers by using UV light
or in presence of an external reducing agent, such as
NaBH4.[24] To the best of our knowledge, there is no report
of the synthesis of fluorescent Au/Ag nanoclusters by using
a supramolecular hydrogel in the presence of sunlight.
Herein, we have successfully demonstrated the in situ prepa-
ration of fluorescent Ag nanoclusters within a short-peptide-
based supramolecular hydrogel in presence of only sunlight.
In a typical experiment, the solid peptide Fmoc-Val-Asp-
OH (4.5 mg, 10À2 mmol) was dissolved in dimethyl sulfoxide
(200 mL) by gentle heating, and then cooled to room temper-
ature. Freshly prepared dilute aqueous AgNO3 (3.8 mL,
pH 7.46) was added to this solution in a molar ratio of 1:1
[Ag+]/COOH to give a final gelator concentration of 0.2%
w/v (2.5 mm) and [Ag+]=5 mm. Note that the each gelator
peptide has two free COOH groups. An Ag+-encapsulating
transparent hydrogel is immediately formed. A light violet
colouration started to appear if the gel was exposed to
bright sunlight (Figure 4a) and the reaction process was
Figure 3. a) AFM image of hydrogel (scale bar: 1 mm) and b) FT-IR spec-
trum of the dried gel.
1537 cmÀ1 can be assigned as the hydrogen-bonded N H
À
À
stretching (amide A), C=O stretching (amide I) and N H
bending (amide II) bands, respectively.[19] The amide I and
amide II bands suggest the presence of an intermolecularly
hydrogen-bonded structure in the gel state. Another peak at
n˜ =1719 cmÀ1 appeared; this is characteristic of the C=O
stretching band of carboxylic acid.[19]
Synthesis of fluorescent silver nanoclusters within the hydro-
gel: Preparation of nanoparticles in the gel phase is a fasci-
nating research area. Gels in the swollen stage provide a
large free space between the three-dimensional cross-linked
networks that acts as a nanoreactor for the nucleation and
growth of nanoparticles. Stability, longevity and controlled
growth of nanoparticles are easily achieved within the gel
phase. There are several examples of the formation of metal
nanoparticles in gel-phase materials. The entrapment of pre-
synthesised metal nanoparticles in gels is relatively
common.[20] However, examples of the in situ synthesis of
metal/semiconductor nanoparticles in gels by using the cor-
responding gel as a reaction medium are limited.[21–24] We
previously reported the in situ synthesis of Au/Ag nanopar-
ticles in an oligopeptide-based supramolecular organogel by
using the redox-active tyrosine moiety of the gelator pep-
tide.[22] Other reports include the reduction of Ag+ in an or-
ganogel by using DMF as a gelling solvent and hydroqui-
none as a reducing agent.[23] However, the three-dimensional
network provided by hydrogels is an important template for
Figure 4. a) Photograph of silver ions encapsulated by the hydrogel after
irradiation by bright sunlight for a few minutes. b) Time-dependent UV/
Vis spectra during Ag nanocluster formation within the hydrogel.
complete within a few (3–4) minutes. The violet colouration
indicates the reduction of silver ions to silver clusters (Ag0);
this type of colour change was not observed in the absence
of sunlight or in the absence of silver or gelator peptide.
This suggests that both the peptide molecule and sunlight
have a role in silver-ion reduction. Transiently stable silver
clusters were formed in an aqueous solution of gelator pep-
tide (not in the gel state) that contained AgNO3 in the pres-
ence of sunlight; these clusters aggregate to form bigger par-
ticles and are ultimately precipitated out from solution
within 2 h. Thus, it can be envisaged that the three-dimen-
sional gel network structure provides good stability for
silver nanoclusters and prevents further growth of nanoclus-
ters to form larger non-fluorescent nanoparticles.
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Short-Peptide-Based Hydrogel
FULL PAPER
It is important to note that peptide-controlled mineralisa-
tion of silver nanoparticles has so far been achieved by
redox-active peptides based on tyrosine or tryptophan.[25]
However, herein we present the spontaneous reduction of
silver ions by using a short-peptide-based hydrogel (contain-
ing aspartic acid residue) in the presence of bright sunlight
to facilitate the production of templated fluorescent silver
nanostructures. Interestingly, in the absence of any conven-
tional reducing agent in medium, silver ions were (bio)re-
duced in the presence of bright sunlight by the carboxylate
group of the gelator peptide that contained an aspartic acid
residue. Generally, the deprotonated carboxylic acid groups
in block copolymer systems have been utilised for silver-ion
reduction because carboxylic acid has excellent binding af-
finity towards Ag+ ions.[9c,12,13,26] However, in all these
above-mentioned cases, the presence of a reducing agent,
such as H2 gas,[26] NaBH4,[9c] UV radiation[12,13] or g radia-
nucleophilic, their binding with silver ions induces partial
electron transfer that displaces the Fermi level of silver clus-
ter toward more negative potentials. We also believe that
water could be a source of electrons to initiate the reduction
process, and it is evident that the presence of solvated elec-
trons in water are possible.[30] The carboxylate radical acti-
vated by visible light could possibly be involved in photo-
chemical reductions of FeIII complexes of polycarbox
ates.[31] Considering the effect of complexation, silver ions
can be photoreduced by the carboxylic acid group of the
gelator peptide even under sunlight.
ACHTUNGTRENNUNGyl-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
Characterisation of the hydrogel–nanocluster composite
Optical study: We have investigated the spontaneous reduc-
tion of silver ions by using UV/Vis absorption spectroscopy.
At first, the Ag+-encapsulating hydrogel is colourless and
transparent and the UV/Vis spectrum showed no absorption
within the range of l=300 to 800 nm. Subsequent exposure
to bright sunlight resulted in a gradual colour change from
colourless to violet, and after 1 min of the exposure to sun-
light a broad surface plasmon resonance band appeared
around l=530 nm with a shoulder around l=390 nm (Fig-
ure 4b). With increased exposure time of up to 3 min, the in-
tensities of these peaks gradually increased. However, no
significant change in the peak intensity or wavelength was
observed upon further irradiation. Fluorescent silver clusters
in the polymer microgel template exhibit a shoulder at l=
330 to 360 nm with an intense peak at l=490 to 520 nm in
UV/Vis absorption spectra.[12] Herein, the obtained absorp-
tion band may be responsible for in situ formation of Ag
clusters within the hydrogel.
It is important to note that in our investigation, hydrogel
alone (without any silver ions/Ag clusters) gives an emission
maximum at l=415 nm upon the excitation at l=300 nm
(Figure S1 in the Supporting Information). This photolumi-
nescence (PL) emission comes from self-assembled gelator
peptides that contain an aromatic Fmoc group. It can be
stated that no other PL emission was obtained in the range
of l=300 to 800 nm. PL and photoluminescence excitation
(PLE) spectra of the Ag+-encapsulating hydrogel after irra-
diation with bright sunlight are shown in Figure 5. The PL
spectrum shows an emission maximum at l=634 nm upon
excitation at l=530 nm. As the reaction progresses, the
fluorescence intensity steadily increases with the increase in
irradiation time and is ultimately saturated after 3 min (Fig-
ure S2 in the Supporting Information). This emission is due
to the formation of fluorescent silver nanoclusters within
the hydrogel and it does not come from the gelator mole-
cules. Other researchers have reported that the oxidised
form of dendrimers, which are used as templates in the for-
mation of Au/Ag nanoclusters,[4j] are fluorescent.[32] To ex-
plore the possibility of whether the fluorescence originates
from metal-assisted oxidation of the peptide, experiments
were carried out in which AgNO3 was replaced by oxidising
agents such as H2O2, FeIII or persulfate under similar condi-
tions.[4k,32] However, no fluorescence emission around l=
ACHTUNGTRENNUNG
tion,[5a] was required. There is a single report for the reduc-
tion of silver ions into non-fluorescent nanoparticles by the
polycarboxylic acid group of peptides (E6 or D6) that con-
tain glutamic acid (E) or aspartic acid (D) and by using am-
bient light.[27] Herein we have used the aspartic acid residue
for the reduction of silver ions into nanoclusters (Ag0)
within a hydrogel medium, and the carboxylate groups of
the asparACHTUNGTRENNUNGtic acid unit play a vital role in the reduction under
bright sunlight. Sunlight-induced reduction of Ag+ to Ag
nanoparticles by using DNA templates has been recently re-
ported.[28] Another report includes the formation of silver
nanoparticles in the presence of light by using lysozyme as a
template.[29]
Herein we have attempted to determine the mechanism
involved in this reduction. We conducted several extensive
experiments to probe whether any structural change in the
gelator peptide occurs during the process of reduction. FT-
1
IR, H NMR and 13C NMR spectroscopies were performed
for the peptide before and after silver mineralisation (given
in the Supporting Information). However, no structural
change in the gelator peptide (or aspartic acid residue) was
observed during the reduction. It is necessary to judge the
possibility of Ag+ ion complexation during reduction. Ag+
ions are known to complex with the carboxylic acid groups
of the aspartic acid residues. The standard electrode poten-
tial of the Ag+/Ag redox system is +0.8 V for bulk material.
However, this value can be drastically different when the
Ag+ ions are involved in complexation. Usually, complexa-
tion decreases the redox potential and in that condition it is
easier to reduce the Ag+ ions. Examples include the reduc-
tion potential of the Ag+/Ag system in Ag halides (AgBr)
used in photography (+0.07 V), in Ag diamine complexes
[AgACHTUNGTRENNUNG(NH3)2] (+0.37 V), in Ag benzoate (+0.52 V), in
Ag2CO3 (+0.47 V) and in AgOH (+0.24 V).[25a] Herein, the
energy barrier for silver-ion reduction is significantly de-
creased when silver ions are complexed with the carboxylic
groups of the aspartic acid residue present in the gelator
peptide. The combination of carboxylic-acid-containing di-
peptide and sunlight make it possible to reduce silver ions
to Ag0 to form nanoclusters. Because carboxylic groups are
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A. Banerjee and B. Adhikari
Figure 6. a) HR-TEM image of small Ag nanoclusters within the hydro-
gel. b) Enlarged version of the HR-TEM image of the Ag nanoclusters,
clearly showing the lattice fringes.
shows the presence of a (111) plane for silver (Figure S6 in
the Supporting Information). This result of SAED and HR-
TEM are in consistent with that of X-ray diffraction study.
Atomic force microscopic (AFM) images of hydrogel–Ag
cluster nanocomposite exhibited the presence of very small
spherical nanoclusters with an average diameter of 1.53 nm,
and these nanoclusters are attached to the larger gel fibres
of average diameter 50 nm (Figure 7). Thus, this hydrogel
has been shown to be an excellent medium for the forma-
tion of fluorescent silver nanoclusters.
The hydrogel is stable at this high concentration of silver
(a ratio of COOH/Ag+ =1:1). Morphological studies (HR-
TEM, AFM) and FT-IR (Figure S7 in the Supporting Infor-
mation) suggest that the supramolecular structure of the hy-
drogel is similar before and after silver mineralisation. In
this study it is clear that the hydrogel can endure the pres-
ence of a considerable amount of silver nanoclusters, with-
out having any adverse effect on gelation and its supra-
molecular structure. This may be due to the fact that the
size of nanoclusters is very small compared to the gel fibres
and this is evident from TEM and AFM images.
Figure 5. Normalised a) PL and b) PLE spectra of the silver nanocluster
within the hydrogel.
634 nm was observed from the peptide hydrogel in the pres-
ence of any of these above-mentioned oxidants upon excita-
tion at l=530 nm (Figure S3 in the Supporting Informa-
tion). UV/Vis spectra also show a small shoulder at l=
390 nm. Excitation at this wavelength did not produce any
emission maximum at l=634 nm, which indicates that the
absorption band at l=530 nm is responsible for the PL
property. The quantum yield (Q) of the reported fluorescent
Ag nanoclusters was found to be 1.3% in water by using the
Atto 520 dye (Q=90% in water) as a reference. Our fluo-
rescent Ag nanocluster–hydrogel composite shows good op-
tical properties. Firstly, it exhibits an emission band width
with a full width at half maximum (FWHM) of 36Æ1 nm,
which is narrower than all previously reported Ag nanoclus-
ters and indicates the narrow size distribution of Ag nano-
clusters.[1d] Secondly, the as-prepared Ag nanoclusters within
the hydrogel have a large Stokes shift (104 nm); this is a
consequence of a small overlap between the absorption and
emission spectra. The sunlight-induced formation of fluores-
cent Ag clusters within hydrogel is very stable. Both PL in-
tensity and wavelength of the Ag clusters do not change
even after storage of this nanomaterial for 6 months in the
dark at 48C.
X-ray diffraction studies: The XRD pattern for the hydro-
gel–Ag nanocomposite showed diffraction peaks at 2q=
38.2, 44.2, 64.5, 77.4 and 81.68, all of which are consistent
with those for Ag (Figure 8). These diffraction peaks corre-
spond to the (111), (200), (220), (311) and (222) Miller indi-
ces of fcc Ag, respectively. Generally the intensity ratio be-
tween the peaks corresponding to (111) versus (200) planes
is 0.40 versus 0.24.[33] However, in this study the intensity
ratio was found to be 2.92 versus 0.73; this is higher than
the conventional value. This observation suggests that the
silver nanoclusters are primarily dominated by (111) facets.
The reduction of silver ions growing along particular facets
(111) may be explained by using a model suggested by Naik
and co-workers.[34] According to this model, peptides modify
crystal growth by allowing accumulation of the silver atoms
on the lowest-surface-energy face. Furthermore, the surface
energy of different crystal faces in face-centred-cubic metals
is lower for {111} faces than that of other planes.[34] As a
result, there is a preferential interaction of peptide gel fibres
with the {111} faces of Ag clusters. This is demonstrated by
the TEM and SAED data. We determined the silver cluster
Electron microscopy studies: The size of the nanoclusters
has been investigated through high-resolution transmission
electron microscopy (HR-TEM). HR-TEM images of hy-
drogel–Ag nanocomposite showed the formation of silver
nanoclusters, with the majority of nanoclusters being within
1 and 3 nm (Figure 6). The particle size distribution from
HR-TEM (Figure S4 in the Supporting Information) shows
that a significant amount of particles are below 2 nm, which
is responsible for the fluorescence property. Interestingly,
most of the nanoclusters are on the gel fibres (Figure S5 in
the Supporting Information). These nanoclusters are crystal-
line in nature. The HR-TEM image (Figure 6b) indicates
the presence of a lattice plane that has an interfringe dis-
tance of 2.34 ꢁ, which corresponds to the (111) plane of fcc
Ag. A selected-area electron diffraction study (SAED)
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Short-Peptide-Based Hydrogel
FULL PAPER
value obtained from the HR-TEM measurement. It is evi-
dent from the HR-TEM data that >2 nm particles are also
present. It is possible that XRD picks up larger particles be-
cause larger particles scatter X-rays much more strongly
than smaller clusters do.
MALDI mass spectrometric study: The formation of nano-
clusters within the hydrogel medium was also confirmed by
MALDI mass analysis. The spectrum (Figure 9) shows a
Figure 9. MALDI mass spectrum of silver nanoclusters within the hydro-
gel.
peak at 215.93 that corresponds to nanoclusters that contain
only Ag2. A peak at 454.77 is also present and is assigned to
the gelator peptide. By using MALDI mass spectrometry,
Dickson and co-workers reported the presence of Ag1 to
Ag5 clusters that results in the observation of a broad emis-
sion band.[1e] Ras et al. showed the presence of clusters con-
taining a small number of Ag atoms, such as Ag1, Ag2 and
some Ag5, from the MALDI-TOF mass spectrum reported
in the literature.[15] Mass analysis also confirms the relatively
narrower size distribution of our reported Ag nanoclusters
within the hydrogel and this result is in good agreement
with the relatively smaller emission band width obtained
from the photoluminescence spectrum (Figure 5).
Figure 7. AFM images of a) the Ag nanocluster–hydrogel composite,
b) an enlarged image showing a few clusters clearly and c) a 3D image of
clusters.
Conclusion
A short-peptide-based hydrogel has been successfully uti-
lised to make and stabilise fluorescent silver nanoclusters.
Interestingly, in the absence of any conventional reducing
agent in the medium, silver ions can complex with the car-
boxylate group of the aspartic acid residue of the gelator
peptide and are spontaneously reduced under bright sun-
light. The three-dimensional network structure of the hydro-
gel facilitates the stabilisation of the newly formed silver
clusters within the hydrogel. The as-prepared hydrogel–
nanoclusters are stable for a long time in the gel phase. The
fluorescent silver clusters have interesting fluorescent prop-
erties with a narrow emission profile and large Stokes shift.
Figure 8. XRD pattern of dried silver clusters, with the lattice planes indi-
cated.
size from XRD peaks by using the Debye–Scherrer equa-
tion. By considering the (111), (200), (220) and (311) planes,
particle diameters of 8.9, 5.17, 4 and 6.2 nm, respectively,
were obtained. These values are higher than the average
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A. Banerjee and B. Adhikari
CuKa X-ray radiation (l=1.54 ꢁ) source and Bragg diffraction setup
(Seifert 3000P).
The peptide-based hydrogel template is a biocompatible and
eco-friendly system because this peptide is only composed
of protein amino acid residues. The synthetic procedure for
fluorescent silver clusters is a green chemistry approach be-
cause the preparation takes place at physiological pH and
room temperature and does not require any toxic reducing
agent. Moreover, silver is considered to be a non-toxic and
environmentally friendly antibacterial agent.[29] Therefore,
our fluorescent silver cluster–hydrogel conjugate may be uti-
lised as an antimicrobial agent and for bioimaging purposes.
MALDI mass spectrometry: Mass spectrum of silver nanoclusters encap-
sulated hydrogel was recorded on a MALDI-TOF mass spectrometer
(Applied Biosystems 4700 Proteomics Analyser 170).
Acknowledgements
B.A. thanks the CSIR, New Delhi, India, for financial assistance.
[1] a) T. Vosch, Y. Antoku, J.-C. Hsiang, C. I. Richards, J. I. Gonzalez,
R. M. Dickson, Proc. Natl. Acad. Sci. USA 2007, 104, 12616–12621;
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Experimental Section
Materials: l-Valine and l-aspartic acid were purchased from Aldrich.
HOBt (1-hydroxybenzotriazole), DCC (dicyclohexylcarbodiimide),
Fmoc-chloride, silver nitrate (AgNO3), hydrogen peroxide and ferric
chloride were purchased from Merck. The fluorescent dye Atto 520 was
purchased from Sigma-Aldrich. The water used in all experiments was of
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Peptide synthesis: Peptide Fmoc-Val-Asp-OH was synthesised by con-
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Deprotection of the methyl ester was performed by using the sa
ification method. Deprotection of the Boc group was conducted by treat-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
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UV/Vis spectroscopy: UV/Vis absorption spectra of the Ag+-containing
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13704
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Chem. Eur. J. 2010, 16, 13698 – 13705

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Received: May 8, 2010
Published online: October 13, 2010
Chem. Eur. J. 2010, 16, 13698 – 13705
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
13705