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Nano Today (2014) xxx, xxx—xxx

Available online at www.sciencedirect.com

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journal homepage: www.elsevier.com/locate/nanotoday

REVIEW

Metal nanoclusters: New fluorescent probes

for sensors and bioimaging
Libing Zhang a,b, Erkang Wang a,b,∗

a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese
Academy of Sciences, Changchun 130022, Jilin, China
b
University of Chinese Academy of Sciences, Beijing 100049, China

Received 14 December 2013; received in revised form 11 February 2014; accepted 12 February 2014

KEYWORDS Summary Fluorescent metal nanoclusters (NCs) as a new class of fluorophores have attracted
Metal nanocluster; more and more attention due to their unique electronic structures and the subsequent unusual
Fluorescent probe; physical and chemical properties. The size of metal NCs approaches the Fermi wavelength
Biolabels; of electrons, between metal atoms and nanoparticles, resulting in molecule-like properties
Sensor; including discrete energy levels, size-dependent fluorescence, good photostability and bio-
Imaging compatibility. These excellent properties make them ideal fluorescent probes for biological
application. Up to now, significant efforts have been devoted to the synthesis, property and
application studies of gold and silver NCs. Recently, a growing number of studies on copper and
other metal clusters have also been reported. In this review article, we focus on summariz-
ing recent advances in controllable synthesis strategies, chemical and optical properties, and
sensing and imaging applications of metal NCs (mainly including Au, Ag, Cu, etc.). Finally, we
conclude with a look at the future challenges and prospects of the future development of metal
NCs.
© 2014 Elsevier Ltd. All rights reserved.

Introduction

Nanomaterials have been recognized as the most modish

∗
Corresponding author at: State Key Laboratory of Electroanalyt-
ical Chemistry, Changchun Institute of Applied Chemistry, Chinese
Academy of Sciences, Changchun 130022, Jilin, China.
Fax: +86 431 85689711.
E-mail addresses: ekwang@ciac.ac.cn, ekwang@ciac.jl.cn
(E. Wang).
research topics in the past decades [1—3]. Nobel metal
nanomaterials with interesting size-dependent electrical,
optical, magnetic, and chemical properties have been inten-
sively pursued, not only for their fundamental scientific
interest, but also for their many technological applications
[4—6]. Especially, metal nanoclusters (NCs) have attracted
special attention due to their attractive features and

http://dx.doi.org/10.1016/j.nantod.2014.02.010
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2 L. Zhang, E. Wang

molecule-like properties [7—16]. Metal NCs usually consist
of a few to a hundred atoms, and the sizes are comparable
to the Ferimi wavelength of electrons [7], which endows
them an important role-the missing link between single
metal atoms and plasmonic metal nanoparticles. In this
size regime, the continuous density of states breaks up into
discrete energy levels [16,17]. Due to the electrons of metal
atoms confined in molecular dimensions and the special dis-
crete energy levels, metal NCs exhibit dramatically different
optical, electronical and chemical properties, including
strong photoluminescence, excellent photostability, good
biocompatibility and sub-nanometer size. Such novel prop-
erties make metal NCs an ideal nanomaterial for promise
applications in biological analysis and imaging, environmen-
tal monitoring, industrial catalysis and electronic devices.
Fluorescent metal NCs have been developed as a new
class of fluorophores. Current fluorescence applications
mostly involve organic fluorophores, semiconductor quan-
tum dots (QDs) [18] or fluorescent proteins [19]. Organic
fluorophores exist in a wide range of chemical struc-
tures and spectral properties; however, they are prone to
photobleaching, which may limit their many applications.
Semiconductor QDs are fluorescence-tunable and photo-
stable; however, their large physical size may hinder their
use as fluorescent reporters of binding events, which may
compromise their use for in vivo applications. Fluores-
cent proteins are genetically encodable, which can be
produced by the cells and organisms themselves. Conse-
quently, there is no need for additional labeling and/or
other chemical procedures in studies of live cells and organ-
isms. Metal NCs show strong photoluminescence, combined
with good photostability and high emission rates, and have
a sub-nanometer size and size or scaffold-dependent tun-
able fluorescence. They have an appealing set of features
that complements the conventional fluorophores. These
properties establish them as a new class of ultra-small, bio-
compatible fluorophores for applications as biological labels
or optoelectronics emitters.
It should be pointed out that several excellent review
papers have been dedicated to the metal NCs [8—14]. How-
ever, these previous reviews focused on either one kind of
metal or metal NCs with actually a wide size range and
different focusing aspects. In this article, we will mainly
summarize recent advances in the synthesis, special prop-
erties and the applications of metal NCs (Au, Ag, Cu, etc.)
as new fluorescent probes for analytical sensing and biologi-
cal imaging. In the final section, we will give a brief outlook
on the challenges and opportunities for future metal NCs
research.
Energy levels: from bulk metals to
nanoclusters
The physical and chemical properties of metals depend
greatly on their size. With the varying size, their behaviors
go through several noticeable transitions (Fig. 1) [17]. Bulk
metals are good optical reflectors and electrical conductors.
The electronic situation in bulk metals is characterized by
the existence of energy bands. They result from the com-
bination of an infinite number of energetically very similar
orbitals. The valence band contains the relevant valence
electrons. The conduction band of metals overlaps to some
extent with the valence band and so becomes partially

Figure 1 The effect of size on metals. Whereas bulk metal and metal nanoparticles have a continuous band of energy levels,
the limited number of atoms in metal nanoclusters results in discrete energy levels, allowing interaction with light by electronic
transitions between energy levels. Metal nanoclusters bridge the gap between single atoms and nanoparticles.
Reprinted from Ref. [17] with permission by Springer-Verlag.

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Metal nanoclusters 3

occupied with electrons. These electrons are finally respon- method, Au3+ ions are reduced in the presence of a suitable
sible for the electric conductivity of metals. In contrast to ligand to form the clusters. Through this strategy, fluores-
the electrons in a filled band, those in the conduction band cence emission of formed Au NCs could be effectively tuned
are fully mobile and make conductivity possible. In bulk from blue to near-IR regimes. A group of water-soluble
metals, the energy levels of the electrons are continuous. glutathione-protected Au clusters with defined chemical
When the size of metals reduces to become metal compositions have been synthesized by reducing Au3+ ions
nanoparticles, the motion of electrons becomes limited by in the presence of glutathione [21,22,26]. In addition, the
the size of the nanoparticle and interactions are expected alkanethiol-bound Au NCs with various emission wavelengths
to be mostly with the surface. Metal nanoparticles will dis- also have been prepared in the presence of different chain
play intense colors due to surface plasmon resonance effect, lengths of alkanethiols [27—29]. In similar approaches, many
in which the optical properties are determined by the col- other thiols such as tiopronin [30], phenylethylthiolate [31],
lective oscillation of conduction electrons upon interaction and D-penicillamine [32] all have been used to prepare
with light. Usually, metal nanoparticles absorb light strongly, monolayer-protected Au NCs.
but they do not or only show weak luminescence. Jin group developed a kinetically controlled method for
Metal nanoparticles further reduced in size, they will synthesizing high purity Au25 (SR)18 (where, SR = thiolate)
change to metal NCs. At this stage, the properties of parti- NCs in high yield [33]. An interesting ‘‘size focusing’’
cles disappear and the bands turn into more or less discrete method was discovered [34], which has been demonstrated
energy levels. Metal NCs are not conductors any more, as to be versatile for different thiolate ligands, including 2-
the energy levels are too far separated. Thus, the collec- phenylethanethiol (—SC2 H4 Ph), dodecanethiol (—SC12 H25 ),
tive oscillation of electrons is obstructed and metal NCs do and glutathione (—SG) [34], as well as for the different-sized
not give rise to surface plasmon resonance effect. However, Aun (SR)m NCs [35]. The fluorescence of Au25 (SR)18 clusters
they will follow quantum mechanical rules. Through interac- (R = C6 H13 (hexanethiol), C2 H4 Ph, glutathiolate, etc.) was
tion with light and then electronic transitions between the found to be affected by the ligands and metal core charge
energy levels, they will show bright luminescence. state [36]. Bigioni et al. observed photoluminescence in the
near-infrared region (1100—1600 nm) for 1.1 and 1.7 nm par-
Synthesis of metal nanoclusters ticles [37]. Wu et al. found that the surface of Au25 (SR)18
played a major role in the fluorescence generation [36].
Specifically, the ligands with electron rich atoms (e.g., O,
The controllable synthesis of metal NCs with high quality
N) or groups (e.g., —COOH, —NH2 ) can considerably enhance
is of paramount importance and highly desirable. To date,
fluorescence. Recently, Jin group reported a one-pot synthe-
in order to obtain high-quality metal NCs, some key factors
sis of Au NCs using captopril (Capt) as protected ligand [38].
should be taken into consideration. (1) The ligand should
The obtained Au25 (Capt)18 exhibited high thermal stability,
have strong interaction with metal NCs. (2) The reducing
which should be attributed to the ligand stability of capto-
condition should be strict. Generally, strong reducing agents
pril. The chiral ligands (Capt) give rise to distinct chiroptical
or light irradiation or sonication should be employed to
features of Au NCs. Those excellent properties of Au25 (SR)18
improve the quantum yield (QY) of NCs. (3) Long aging time
NCs render this material quite promising for biological appli-
is also important for obtaining high-quality NCs. Consider-
cations.
ing these crucial factors, different approaches have been
devised to synthesize high-quality metal NCs with various
scaffolds in a controllable manner. The scaffolds typically
have multiple groups that allow strong interaction with
metal ions, which plays a crucial role in determining some
of the properties of the clusters. The scaffolds influence the
geometry of the clusters and also provide suitable conditions
to avoid aggregation of the clusters into large nanoparti-
cles [20]. They potentially alter the electronic structure
and hence significantly influence their properties. The used
scaffolds for the synthesis of clusters usually include small
thiol molecules, DNA, peptides and proteins, dendrimers and
polymers. To date, significant efforts have been devoted to
the studies of gold and silver NCs. More recently, a grow-
ing number of studies on copper and other metal NCs have
also been reported. Accordingly, we will discuss the vari-
ous synthesis strategies for Au, Ag, Cu and other metal NCs,
respectively.

Au nanoclusters
In the aspects of Au NCs, small thiol molecules have firstly
been employed to synthesize Au NCs via chemical reduc-
tion of Au precursor in the presence of NaBH4 owing to
the strong interaction between Au and S [21—25]. In this
Figure 2 Excitation (dashed) and emission (solid) spectra of
different gold nanoclusters. Excitation and emission maxima
shift to longer wavelength with increasing nanocluster size.
Reprinted from Ref. [40] with permission by the American Phys-
ical Society.

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Dendrimers are repetitively branched molecules with
well-defined molecular weights and small cavities, which
have been employed to synthesize Au NCs. For example,
poly(amidoamine) dendrimer (PAMAM) was used as tem-
plates to encapsulate few-atom Au NCs [39]. After gold ions
were introduced into the solution of PAMAM, an equivalent
of NaBH4 was added into the solution and some reduced
gold atoms aggregated within the dendrimers to form very
small dendrimer-encapsulated Au NCs. Once the large NPs
were removed through centrifugation, luminescent Au NCs
were obtained, which exhibited bright blue emission and
two-order-of-magnitude improved fluorescence QY (42%) in
aqueous solution. Subsequently, to further understand size-
dependent emission from Au NCs, Zheng et al. tuned molar
ratios between dendrimer and gold ions as well as the
amount of NaBH4 used in the reactions, and a class of Au
NCs with different emission maxima ranging from UV to IR
were obtained [40]. Using electrospray ionization (ESI) mass
spectrometry, they were able to identify the sizes of differ-
ent emissive Au NCs. As shown in Fig. 2, emissions at 3.22,
2.72, 2.43, 1.65, and 1.41 eV correspond to Au5 , Au8 , Au13 ,
Au23 and Au31 , respectively.
Fluorescent Au NCs could be prepared through etch-
ing metallic nanoparticles using large amounts of suitably
selected ligands. Pradeep group has presented two possi-
ble mechanisms regarding the formation of clusters [41].
In the first route, Au atoms are removed from the surface
of nanoparticles by excess ligands as a gold(I)—ligand com-
plex, which may undergo strong aurophilic interaction and
then to form the clusters. In another route, ligand may
etch the surface gold atoms of the nanoparticles leading to
reduction in the size of the nanoparticles in steps, resulting
in the formation of clusters. For example, two fluorescent
Au NCs have been synthesized from mercaptosuccinic acid
(MSA)-protected gold nanoparticles by etching with excess
glutathione [41]. Etching at pH 3 was yielded Au25 , while

Figure 3 (a) General strategy to fabricate water-soluble fluorescent Au nanoclusters. DDAB-stabilized gold nanoparticles (Au
NP@DDAB) are etched by the addition of Au precursors (HAuCl4 or AuCl3 ) to smaller nanoclusters (Au NC@DDAB). By the addition of
reducing agent (TBAB) the Au NCs grow again reversibly into bigger Au NPs. The hydrophobic Au NC@DDAB become water soluble
upon ligand exchange with dihydrolipoic acid (Au NC@DHLA); (b) TEM images of Au NP@DDAB, Au NC@DDAB, and Au NC@DHLA; 100
particles were randomly selected for measuring the size distribution, resulting in 5.55 ± 0.68 nm, 3.17 ± 0.35 nm, and 1.56 ± 0.3 nm
in diameter, respectively. (c) Pictures of particle solutions under daylight. Contrary to Au NP@DDAB solution which features the
red color of surface plasmon absorption, Au NC@DDAB and Au NC@DHLA display a colorless and brown translucent solution without
plasmon absorption, respectively. (d) Pictures of the same particle solutions under UV excitation. The Au NC@DHLA solution shows
red photoluminescence.
Reprinted from Ref. [43] with permission by American Chemical Society.

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Metal nanoclusters
that at pH 7—8 it was Au8 . Nie group reported a green
fluorescent Au NCs was synthesized by using multivalent
polyethylenimine (PEI) to etch colloidal gold nanoparti-
cles [42]. In addition, didodecyldimethylammonium bromide
(DDAB) stabilized Au nanoparticle was brought to aqueous
phase upon ligand exchange with dihydrolipoic acid (DHLA)
[43], resulting in the formation of brightly red emitting
Au NCs (Fig. 3). The resulting Au NC@DHLA particles have
a QY of around 1—3%, reduced photobleaching compared
to organic fluorophores, and very good colloidal stability.
Interestingly, an already synthesized Au NCs also could be
converted to another Au NCs through the ligand exchange
reaction. A phosphine-stabilized Au11 cluster was treated
with water soluble thiol glutathione, the glutathione pro-
tected Au25 were obtained [44] and the subsequent research
confirms that Au25 have the more extraordinary thermody-
namic stability [45]. Similarly, bright-red-emitting Au23 NCs
were synthesized from Au25 NCs [46] and Au75 NCs were
obtained through the reaction of phosphine-protected Au55
NCs with hexanethiol and other thiol [47].
Biologically important molecules also can act as tem-
plates for the synthesis of highly fluorescent Au NCs. The
use of bio-ligand can effectively reduce the possible toxicity
of Au NCs and further achieve good biological applica-
tions. An important result was given by Ying et al. [48],
who reported a simple, one-pot and ‘‘green’’ synthetic
route for the preparation of Au NCs at the physiologi-
cal temperature (37 ◦ C) with red emission using bovine
serum albumin (BSA) as a bio-ligand (Fig. 4). This process
is similar to the bio-mineralization behavior of organisms
in nature. Upon addition of Au ions to the BSA solution,
the protein molecules sequestered Au ions and entrapped
them. The reduction ability of BSA molecules was then acti-
vated by adjusting the reaction pH to 12 and entrapped
ions underwent progressive reduction to form Au NCs in
situ. Notably, BSA functions both as a stabilizing agent
and a reductant in this process. This work is remarkable
not only for providing an alternative approach of syn-
thesizing highly fluorescent Au NCs, but also providing a
successful paradigm for exploring other proteins and met-
als. Later, Lu’s group [49] found that lysozyme could also
act as a good reducing and stabilizing agent for the prepa-
ration of highly fluorescent Au NCs. Recently, Pradeep group
Figure 4 Schematic of the formation of Au NCs in BSA solution.
Reprinted from Ref. [48] with permission by American Chemical Society.
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5
demonstrated the synthesis of AuAg NCs clusters with tune-
able compositions in a BSA template starting from Au and
Ag clusters [50]. The results revealed that the alloy cluster
core of ∼1.2 nm diameter is composed of nearly zero-valent
Au and Ag atoms that exhibit distinctly different steady
state and time resolved excited state luminescence profiles
compared to the parent clusters. They believe that alloy
clusters with the combined properties of the constituents
in versatile protein templates would have potential appli-
cations in the future. In addition, horseradish peroxidase
[51], insulin [52], pepsin [53] and transferrin protein [54,55]
all have been used as scaffolds for the preparation of Au
NCs, respectively. These functionalized Au NCs all exhibit
excellent properties and great potential for biological appli-
cations.
Recently, DNA has also been used as template for the
preparation of fluorescent Au NCs. DNA template synthesis
has several advantages. First, DNA is non-toxic with good
biocompatibility. Second, DNA possesses functional roles
for molecular recognition (e.g. aptamers), it is possible to
directly couple analyte binding with fluorescence signal-
ing through designing appropriate DNA sequences. Third,
chemical synthesized DNA can be easily modified, which
facilitating fundamental studies and practical applications.
Liu group [56] successfully prepared blue fluorescent Au NCs
in the presence of poly-cytosine DNAs at low pH and poly-
adenine at neutral pH using citrate as the reducing agent,
and they explored the affecting factors for the synthesis
of Au NCs in detail. Subsequently, Shao group [57] synthe-
sized fluorescent Au NCs using various DNA as template.
The emission behavior of the hairpin (HP)-DNA-stabilized Au
NCs is dependent on the loop sequence. The cytosine loop
is the most efficient host to produce fluorescent Au NCs.
However, the loop composed of thymine as well as adenine
creates Au NCs with a much weaker emission than the cyto-
sine and guanine loops. The cytosine-favored Au NC growth
was further confirmed using the sing-strand DNA (ssDNA)
having an identical base composition as the HP-DNA. Addi-
tionally, the fully matched DNAs seem to be less efficient
than the corresponding HP-DNA and ssDNA structures. The
DNA-stabilized Au NCs can be used as probes to in situ ana-
lyze the DNA hybridization and cytosine-related mutation.
This DNA sequence-dependent growth of fluorescent Au NCs
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6 L. Zhang, E. Wang
will hold a great promise for developing biological labels,
biosensors, and DNA-related nanomaterials.
Ag nanoclusters
Compared with Au NCs, Ag NCs are easily synthesized via
designing different ligands as stabilizing agents. However,
the stability of Ag NCs was more easily affected by many
factors. Except for their vulnerability to atmospheric oxy-
gen and other electron acceptors in solution, reduced silver
atoms have a strong tendency to agglomerate, it is necessary
to prevent stable NCs formation in favor of thermodynami-
cally favored large silver nanoparticle generation.
The pioneering reported water-soluble fluorescent Ag
NCs were prepared in the presence of dendrimers, by Dick-
son and co-workers [58]. OH-terminated dendrimers PAMAM
G4 and G2 (fourth- and second-generation) were used as
scaffolds to synthesize fluorescent Ag NCs through direct
photoreduntion. The Ag NCs exhibited distinct fluorescence
peaks ranging from 533 to 648 nm with high photostability.
Latterly, Balogh and coworkers also utilized PAMAM G5 to
prepare dendrimer templated Ag NCs by irradiating the solu-
tions of silver(I)—dendrimer complexes with UV light [59].
Polymers containing amino and carboxylic groups that can
form complexes with Ag ions have been used for the prepa-
ration of fluorescent Ag NCs. Dong’s group [60] reported a
new approach for the synthesis of fluorescent and water-
soluble Ag NCs, using the common polyelectrolyte PMAA
as the template under UV illumination, with an absorp-
tion peak near 510 nm that overlaps with the excitation
spectrum for 620 nm emission. The Ag NCs show lumines-
cence lifetimes of 2.3 ns and a high QY of 18.6%. They found
that pH of solution has an important role on the fluores-
cent emission of obtained Ag NCs. The maximum intensity
was obtained at pH 4.5 whereas either lower or higher pH
value led to weaker fluorescent intensity. Instead of ultra-
violet light, Ras’s group [61] found that visible light also
could initiate the reduction of silver ion in the presence
of PMAA and further lead to the formation of silver NCs in
an environmentally friendly manner. Their results indicate
a phenomenon that higher Ag concentration leads to the
generation of Ag NCs with emission at the redder end. An
increase in the Ag/PMAA molar ratio resulted in red-shifts
of the absorption peaks and the corresponding emission
peaks. Later, Suslick et al. [62] presented a new and eas-
ily controlled sonochemical strategy for the synthesis of
water-soluble fluorescent Ag NCs also in the presence of
PMAA. The evolution of absorption and fluorescence of Ag
NCs is quite similar to that of photoactivation, the obtained
Ag NCs exhibit red emission with a maximum excitation
of 510 nm. In addition, poly(acrylic acid) derivatives [63]
and poly(N-isopropylacrylamide-acrylic acid-2-hydroxyethyl
acrylate) [64] both were used for the synthesized fluorescent
Ag NCs under UV irradiation (365 nm). Recently, Mattoussi
and coworkers [65] developed one phase growth reaction to
prepare a series of Ag nanoparticles and luminescent Ag NCs
using NaBH4 as a reducing agent in the presence of polyethy-
lene glycol (PEG) appended with lipoic acid (LA) groups
at one end and reactive (—COOH/—NH2 ) or insert (—OCH3 )
functional groups at the other end. The size and properties
of Ag NCs were primarily controlled by varying the Ag-to-
ligand (Ag:L) molar ratios and the molar amount of NaBH4 .
Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
Luminescent Ag NCs formed at lower molar ratio of Ag:L,
while nonluminescent silver nanoparticles were produced
at higher molar ratio (Fig. 5). Interestingly, nonluminescent
silver nanoparticles could be converted into luminescent Ag
NCs, via a process referred to as ‘‘size focusing’’ in the pres-
ence of added excess ligands and reducing agent. Another
type of polyelectrolyte polyethyleneimine (PEI) was utilized
as scaffolds for the synthesis of fluorescent Ag NCs through
the strong interaction between N atom and Ag atom [66].
The PEI capped Ag NCs showed a blue emission at 455 nm
and a high stability which made it kept at room temperature
for at least 30 days.
Small molecules containing carboxylic groups or thiols
also can be used as templates for the synthesis of fluorescent
Ag NCs. Pradeep group [67] synthesized red- and blue-green-
emitting clusters Ag8 (H2 MSA)8 and Ag7 (H2 MSA)7 (H2 MSA:
mercaptosuccinic acid) through an interfacial etching reac-
tion starting from H2 MSA-protected silver nanoparticles as
precursor. The result of X-ray photoelectron spectroscopy
(XPS) showed that the Ag:S atomic ratio for each band
was 1:1 and that the silver was fully reduced. The detec-
tion of [Ag8 (H2 MSA)4 (HMSA)4 ]− and [Ag7 (H2 MSA)5 (HMSA)2 ]−
by the mass spectrometry was further used as strong evi-
dence to assign the Ag8 as a red emitter and Ag7 as a
blue emitter. Recently, Chen group presented a facile one-
pot sonochemical approach to prepare highly blue-emitting
Ag NCs using glutathione as a stabilizing agent in aqueous
solution [68]. The results reveal that it is a rapid and envi-
ronmentally friendly method for preparing fluorescent Ag
NCs. The Ag NCs consist of 12 silver atoms and the exper-
imental conditions have been optimized to derive Ag NCs
with high emission. The Ag NCs exhibit excellent water
solubility, high stability, and have an excellent sensitivity
and high selectivity toward S2− , with a detection limit of
2 nM.
Proteins and peptides have been used for the synthesis
of Ag NCs. Proteins possess abundant binding sites that can
potentially bind and some of them can further reduce Ag
ion, thus offering better scaffolds for template-driven for-
mation of fluorescent Ag NCs. Cell matrices were considered
to be rich libraries of various proteins for the production
of luminescent Ag NCs. The proteins of nucleolus, such as
nucleolin, are known to have high affinity to silver ions due
to their amino-terminal domain, was employed as the major
scaffold for the formation of fluorescent Ag NCs by Dickson
and coworkers [69]. The fluorescent Ag NCs can be gener-
ated by photoactivation of cell fed with silver salt in vivo
ambient-temperature condition, which opens a new path
for the application of Ag NCs in biological systems. Besides
the formation of Ag NCs in fixed cells, the fluorescent Ag
NCs can also be synthesized in the presence of proteins.
Narayanan and Pal [70] reported on the use of an enzyme,
bovine pancreatic ␣-chymotrypsin (CHT) as scaffold through
the chemical reduction of silver ions to produce well dis-
persed and stable fluorescent Ag NCs. The protein-stabilized
Ag NCs show emission at 680 nm when excited at 500 nm.
Later, denatured bovine serum albumin (dBSA) also was used
as scaffold for the synthesis of fluorescent Ag NCs [71]. The
dBSA had 35 free cysteine residues which could contribute
to polyvalent interactions with the Ag NCs and serve as
effective stabilizing agents for the Ag NCs. More recently,
Chen and Yang group [72] developed a facile approach to
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Metal nanoclusters 7

synthesize red-emitting fluorescent Ag NCs using lysozyme
as scaffold and NaBH4 as a reducing agent at room tem-
perature. The water-soluble Ag NCs have a positive surface
charge and good stability, which is still kept through adjust-
ing the pH using acetic acid. This is the first report of the
synthesis of fluorescent Ag NCs with positive surface charge
using a protein as scaffold.
Peptides contain carboxylic acids with high silver affinity
and a variety of amino acid sequences also offer potential
tenability in the local site to improve silver NCs stability.
Wright group [73] reported a new approach for the synthesis
of fluorescent Ag NCs using the histidine-rich AHHAHHAAD as
template. Later, inspired by the protein, uncleolin, Dickson
and coworkers [69] design several short peptides used for the
synthesis of Ag NCs. KECDKKECDKKECDK (P1), incorporating
the specific amino acids most prevalent in nucleolin: glu-
tamic acid (15.6%), lysine (12.7%), and aspartic acid (8.5%),
was designed for the synthesized of Ag NCs. However, the
Ag NCs were only moderately stable at room temperature
(chemical lifetime of 3 days) due to the short length of
peptide. The incorporation of several hydrophobic amino
acids, another two peptides HDCHLHLHKCHLHLHCDH (P2)
and HDCNKDKHDCNKDKHDCN (P3) were designed to synthe-
size Ag NCs. The chemical lifetimes of P2- or P3-stabilized
Ag NCs were extended to two weeks in deionized water and
5 weeks in PBS, respectively. More recently, Gao group [74]
has utilized an artificial peptide with amino acid sequence
CCYRGRKKRRQRRR to biomineralize a series of Ag NCs, which
show blue and red emission under different alkaline condi-
tions.
DNA is also an effective template for the preparation
of fluorescent Ag NCs mainly because the cytosine base
exhibits strong affinity for silver ions. The presence of
both carbonyl oxygens and doubly bonded ring nitrogens
in heterocyclic bases plays an important role in determin-
ing their interaction strength with Ag ions [75]. At present,
more investigations have focused on the use of DNA as
scaffolds for the synthesis of Ag NCs [76—89]. These stud-
ies reveal that the generation of these novel fluorescent
NCs is highly DNA-sequence-dependent [77,79,80] and their
photoluminescence emission band could be easily tuned
throughout the visible and near-IR range by simply changing
the sequence of the oligonucleotide.
The first DNA-protected Ag NCs was reported by Dick-
son and coworkers in 2004 [90]. They use ssDNA containing
the 12-base 5 -AGGTCGCCGCCC-3 as templates for synthe-
sizing Ag NCs in a phosphate buffer. Mass spectroscopy
demonstrated the fluorescent Ag NCs with 1—4 atoms were
formed, which show distinct absorption and fluorescence
spectral features. Their results confirm that it may be fea-
sible to control the formation of NCs using DNA strands with
specific sequences, and cytosine is the most favored base
for the formation of Ag NCs. Subsequently, they use the
12-mer cytosine as template for the synthesis of Ag NCs.
The obtained Ag NCs show red and blue/green emission.
Base sequence tuning has been found to result in distinct

Figure 5 (A) Chemical structures of the ligands, (B) synthetic strategy of NPs/NCs of various core sizes. (C) Table showing the
Ag:L ratios used for the synthesis. (D, E) Photographs of aqueous dispersions of the particles synthesized at various Ag:L ratios under
room light and UV light irradiation, respectively. Images were collected from a set of freshly prepared dispersions, without filtration
or adjustment of the nanoparticle or cluster concentrations.
Reprinted from Ref. [65] with permission by American Chemical Society.

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emission properties. Richards et al. [79] reported on five dis-
tinct Ag NCs obtained based on five different sequences DNA
as scaffolds with tunable fluorescence emission throughout
the visible and near IR. Blue and green emitting species
were only created in unbuffered solutions, and yellow,
red and near-IR emitting species formed best in appropri-
ately buffered solutions. Their results demonstrate that the
photostability of Ag NCs was drastically increased with these
longer wavelength emitters.
The sequence and secondary structure of DNA both affect
the fluorescence properties of DNA-encapsulated Ag NCs.
Gwinn and coworkers [80] used six 19-mer DNA oligomers
as templates for the synthesis of Ag NCs. They found that
both C-rich and G-rich single-strand DNA (ssDNA) produced
brightly fluorescent Ag NCs, while the duplex formed by
C-rich and G-rich DNA was an invalid template for the forma-
tion of fluorescent Ag NCs, accompanied by negligible visible
fluorescence. Thus, the spectral properties of DNA-attached
few-atom Ag clusters are sensitive to the secondary struc-
ture of DNA. Furthermore, a set of hairpin structures named
C-loop, G-loop, A-loop and T-loop were designed to compare
the relative capacities of C, G, A, and T homopolymers to
host fluorescent species. C-loop and G-loop solutions exhibit
fluorescence of similar brightness, while A-loop solutions
fluoresce weakly. T-loop solutions even produce no fluores-
cence for excitation at visible wavelengths. Later, Petty and
coworkers [91] first explored pH-dependent i-motif DNAs as
templates for the synthesis of Ag NCs. Two C-rich sequences
(dTA2 C4 )4 and (dC4 A2 )3 C4 with a common C4 i-motif core are
chosen as model systems. They obtained red and green emit-
ting fluorescent Ag NCs. Their results indicate that protons
dominate DNA folding for the red emissive species, while the
green emissive clusters themselves determine the shape of
their DNA matrix. These studies provide the basis for under-
standing how specific base arrangements and environmental
factors influence the formation of fluorescent NCs.
Recently, our group is amazing to find that hybridized
DNA duplexes could also be developed as capping scaffolds
for the generation of fluorescent Ag NCs [92]. And, the
formation of fluorescent Ag NCs in hybridized DNA duplex
scaffolds was highly sequence-dependent, which could iden-
tify a typical single-nucleotide mutation, the sickle cell
mutation (Fig. 6). Afterwards, Li and coworkers further
investigated the effect of DNA templates with different
secondary structures, such as i-motif, G-quadruplex and
Watson—Crick duplex [93]. Polymorphic DNA templated Ag
NCs show distinct fluorescent properties. With DNA template
adopting i-motif or duplex structure, silver atoms tend to
aggregate inside the encapsulated spaces of nucleobases,
and the formed Ag NCs are positively charged with high flu-
orescent spectral features. While with G-quadruplex DNA
as template, silver atoms favor to aggregate outside of the
G-tetrad, which results in the formation of larger silver crys-
tals without fluorescence property. More recently, Qu and
coworkers have demonstrated site-specific, homogeneous,
and bright Ag NCs with triplex DNA as template [94]. By
reasonable design of DNA sequence, homogeneous Ag2 clus-
ter was obtained in the predefined position of CG.C+ site of
triplex DNA. This strategy was also explored for controlled
alignment of Ag NCs on the DNA nanoscaffold.
Cu nanoclusters
Compared with Au and Ag NCs, reports on the synthesis of Cu
NCs are still scarce primarily because of their susceptibility
to oxidation and the difficulty in preparing extremely tiny
particles. Up to now, several methods have been success-
fully developed to synthesize Cu NCs with unique optical
and catalytic properties. Crooks and coworkers [95] used
OH-terminated dendrimers PAMAM G4 (fourth-generation)
as scaffolds to synthesize fluorescent Cu NCs. The PAMAM
dendrimers have an ethylenediamine core (G4-OH) contain-
ing interior tertiary amines that can strongly interact with
Cu ions, after the reduction by sodium borohydride to form
Cu NCs. Almost at the same time, Balogh and Tomalia [96]
also successfully synthesized stable Cu NCs using PAMAM den-
drimers as scaffolds.
Later, Vazquez-Vazquez et al. [97] reported the prepa-
ration of a series of small atomic copper clusters by the
microemulsion technique. Their size can be controlled by

Figure 6 Use of two different DNA duplexes with inserted cytosine loops working as synthetic scaffolds to generate fluorescent
silver NCs for the identification of the sickle cell anemia gene mutation (black dots represent hydrogen bonds formed in base pairing
and black dashed lines the sugar-phosphate backbone).
Reprinted from Ref. [92] with permission by American Chemical Society.

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Figure 7 Schematic representation of detection strategy on
the basis of the formation of fluorescent Cu NCs (Y: SNP site).
Reprinted from Ref. [102] with permission by American Chemi-
cal Society.
adjusting the percentage of the reducing agent. Photolu-
minescent copper clusters (Cun , n ≤ 13) can be obtained
using very low percentages of the reducing agent (<10% of
the stochiometric amount), while photoluminescent clusters
disappear for larger percentages of reducing agent. Vilar-
Vidal and coworkers [98] reported a simple electrochemical
approach for the synthesis of fluorescent Cun (n ≤ 14) NCs
stabilized by tetrabutylammonium nitrate. Copper ions pro-
duced during the electrolysis from the Cu anode were
reduced to NCs on the cathode. The prepared Cu NCs are
very stable over years. They show photoluminescence in the
visible range with unusual high QY of 13% and exhibit an
amphiphilic character and can be dispersed in both polar
and apolar solvents, which are suitable for applications in
biological system.
Recently, Kawasaki et al. [99] reported the synthesis of
fluorescent Cu NCs via a microwave-assisted polyol method
without using additional protective and reducing agents.
Their results demonstrated that the synthesized Cu NCs
were highly stable and exhibited strong blue fluorescence.
Chen group [100] reported a simple on-pot wet chemical
reduction method for the synthesis of fluorescent Cu NCs.
The synthesized stable Cun (n ≤ 8) NCs use 2-mercapto-
5-n-propylpyrimidine (MPP) as protecting ligands, which
exhibited interesting photoluminescence with dual emis-
sions at 423 and 593 nm, respectively.
DNA can also be used as an effective template for
the preparation of fluorescent Cu NCs. Mokhir group [101]
reported a method for the selective formation of fluorescent
Cu NCs on double-stranded DNA in solution. The synthe-
sized Cu NCs show emission range 587—600 nm when excited
at 340 nm. The size of copper NCs can be controlled by
the use of a double-stranded DNA (dsDNA) template of a
selected length. The metallization is highly selective for
dsDNA compared to ssDNA, which can be used for the detec-
tion of dsDNA by fluorescence. Subsequently, our group [102]
reported that the fluorescence of DNA-hosted Cu NCs is
very sensitive to base type located in the major groove of
dsDNA (Fig. 7). The research results have shed some light
on the luminescent mechanism of Cu NCs, owing to its high
specificity and easy operation without rigorously controlled
temperature and arduous probe DNA design. Recently, Liu
et al. [103] successfully synthesized fluorescent Cu NCs
using ssDNA as templates and to identify the critical DNA
sequences. They found DNA thymine base plays a dominant
Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
9
role in producing red-emissive fluorescent Cu NCs on ssDNA
templates. The thymine-dependent growth of the fluores-
cent Cu NCs is confirmed by Hg2+ mediated T—T base pair
in comparison with the other non-specific metal ions, which
could be developed into a practical sensor for turn-on fluo-
rescence detection of Hg2+ with a high selectivity.
Other metal nanoclusters
Except for Au, Ag and Cu NCs, other metal materials such as
Pt [104—106] and Pd [107], could also been made into fluo-
rescent NCs. For instance, Kawasaki group [105] and Obora
group [107] demonstrated simple, one-pot strategies for the
synthesis of Pt and Pd NCs in N, N-dimethylformamide (DMF)
solution in the absence of any capping agents such as surfac-
tant, polymer, or thiolate-organic compounds, respectively.
The synthesis process is similar except for the different pre-
cursors (H2 PtCl6 for Pt clusters and PdCl2 for Pd clusters)
and modified reaction time (8 h for Pt and 6 h for Pd). The
as-prepared Pt and Pd NCs are highly stable in organic, aque-
ous, or salt solutions and they were readily dispersible in a
variety of solvents. The property will greatly facilitate their
use in metal catalysis and bioimaging applications.
Recently, Inouye and Jin group [106] reported a simple
method for the synthesis of water soluble and fluorescent
Pt NCs using the fourth-generation PAMAM (G4-OH) den-
drimer as the template by reducing chloroplatinic acid with
NaBH4 . The synthesized Pt NCs emitted a strong blue fluores-
cence under UV light (365 nm) irradiation. However, simple
oxidation of PAMAM (G4-OH) with (NH4 )2 S2 O8 can also pro-
duce species that emit blue fluorescence [108]. In order to
avoid the possible fluorescence interference from oxidation
species of the dendrimer, they applied mercaptoacetic acid
(MAA) to replace PAMAM (G4-OH) as the ligand for the Pt NCs
through a ligand exchange reaction. Atomically monodis-
persed Pt5 (MAA)8 NCs were obtained by using size-exclusion
high performance liquid chromatography (HPLC) and the Pt5
NCs show blue photoluminescence with an 18% quantum
yield in water.
Properties of metal nanoclusters
Absorption properties
The absorption properties of metal nanoparticles are mainly
dependent on the surface plasmon resonance of conduc-
tion electrons. Generally, metal nanoparticles show intense
colors due to the collective oscillation of conduction elec-
trons upon interaction with visible light. While, as for metal
NCs, the continuous density of states breaks up into dis-
crete energy levels [16], they no longer exhibit plasmonic
properties, but they still interact with light through elec-
tronic transitions between energy levels, leading to sharp
absorption. For instance, Collings et al. [109] reported that
the optical absorption spectra of a series of Aun (n = 7, 9,
11, 13) and their cations located between 1.9 and 5.6 eV.
Recently, Jin group [110] found that glutathione-protected
Au25 showed characteristic absorption features in the range
400—1000 nm, which are believed to arise from intraband
(sp←sp) or interband (sp←d) transitions (Fig. 8). Tsukuda
group [21] found that the spacing between the discrete
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Figure 8 (A) Kohn—Sham orbital energy level diagram for a
model compound Au25 (SH)18 − . Each KS orbital is drawn to indi-
cate the relative contributions (line length with color labels)
of the atomic orbitals of Au (6sp) in green, Au (5d) in blue,
S (3p) in orange, and others in gray (those unspecified atomic
orbitals, each with a <1% contribution). The left column of the
KS orbitals shows the orbital symmetry (g, u) and degeneracy (in
parentheses); the right column shows the HOMO and LUMO sets.
(B) The theoretical absorption spectrum of Au25 (SH)18 − . Peak
assignments: peak a corresponds to 1.8 eV (observed), peak b
corresponds to 2.75 eV (observed), and peak c corresponds to
3.1 eV (observed).
Reprinted from Ref. [110] with permission by American Chemi-
cal Society.
states in each band increased with the size decrease of
glutathione-protected Au NCs, leading a blue shift in the
absorption peaks. Dickson group [90] reported the absorp-
tion peaks of fluorescent Ag NCs with 1—4 atoms at 357 nm
and 440 nm. In recent work, DHLA-protected Ag4 and Ag5
show absorption peaks at 435 nm and 335 nm, respectively,
with another shoulder peak at 500 nm [111].
Fluorescence properties
Bulk metals normally show very weak luminescence owning
to the efficient nonradiative decay and the absence of an
energy gap. As the sizes of metals gradually decrease to the
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L. Zhang, E. Wang
nanometer level, the efficiency of luminescence obviously
enhances. In particular, the sizes approach to the Ferimi
wavelength of electrons [7], metal NCs will show strong
luminescence properties. The luminescence of metal nan-
oclusters is generally assigned to the electronic transitions
between occupied d bands and states above the Fermi level
(ca. sp bands) or the electronic transitions between the
highest occupied orbital and the lowest unoccupied orbital
(HOMO—LUMO) [12]. With the development of the synthesis
methods for metal NCs, their fluorescence properties
get more attention and more in-depth development. For
instance, Dickson group synthesized a series of Ag NCs with
tunable fluorescence emission throughout the visible and
near IR [79]. They also reported DNA-encapsulated Ag nan-
oclusters yield highly stable and bright fluorescence in the
near IR [78]. The very high emission rates without blinking
on experimentally relevant time scales (0.1 to > 1000 ms).
Ying group [48] reported the synthesis of water-soluble and
fluorescent Au25 NCs with good biocompatibility and the Au25
NCs show emission peak at 640 nm. Recently, Jin group [36]
reported that the surface ligand play a major role in the
fluorescent of thiol-protected Au NCs. These studies suggest
that there are two major sources for explaining the emission
properties of metal NCs, the fluorescence may arise from (i)
the metal core through a manifestation of intrinsic quanti-
zation effects and (ii) the particle surface due to interaction
of the metal core and surface ligands.
Two-photon absorption
Two-photon absorption (TPA) is the simultaneous absorp-
tion of two photons of identical or different frequencies
in order to excite a molecule from one state (usually
the ground state) to a higher energy electronic state.
Compared with one-photon excitation, two-photon excita-
tion in the near-infrared region increases the penetration
depth, spatial resolution due to lower scattering, and min-
imizes autofluorescence [112], which is suitable for in vivo
imaging and photodynamic therapy. Goodson group [113]
reported that two-photon emission of Au25 NCs is observed
at 830 nm by exciting at 1290 nm, and the two-photon
absorption cross-section was measured to be 42,700 GM
(Göpert-Mayer unit, 10−50 cm4 s), which is superior to the
TPA cross-sections of many organic chromophores. Recently,
glutathione-protected Au NCs was also observed strong two-
photon emission, and the TPA cross-section was determined
to be 189,740 GM [114]. In addition, DNA-protected Ag NCs
also show two-photon emission and possess high TPA cross-
sections, the cross section of the 660 nm emitter peaks is
35,000 GM, while the 680 nm emitter peaks is 34,000 GM, and
the 710 nm emitter reaches 50 000 GM [115]. More recently,
Goodson group [84] reported that two-photon excited fluo-
rescence was detected for Ag NC on dsDNA at 630 nm when
excited at 800 nm. The two-photon absorption cross-section
was calculated to be ∼3000 GM.
Electrochemiluminescence
Electrochemiluminescence (ECL) is the process whereby
species generated at electrodes undergo high-energy
electron-transfer reactions to form excited states that emit
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Metal nanoclusters
light [116], has received considerable attention in the ana-
lytical field due to its high sensitivity, low background noise
and cost. In the ECL system of metal NCs, the NCs usu-
ally show strong ECL in the presence of co-reactant. A
co-reactant is a species that produces a highly reactive
intermediate upon oxidation or reduction, which reacts with
a luminescent species to either produce an excited state
or commence an excitation pathway by a one-electron oxi-
dation or reduction. The most common excitation pathway
in ECL is the ox-red excitation pathway, in which the lumi-
nescent species is first oxidized by a cathodically produced
oxidizing radical and then reduced by one energetic elec-
tron to the excited form of its original oxidation state. Ras
group [61] first reported that PMAA-protected Ag NCs exhib-
ited ECL. Recently, Chen group [117] found BSA-stabilized
Au NCs also showed ECL using triethylamine as the co-
reactant on the Pt electrode surface. At the same time,
Zhu group [118] observed ECL from BSA-stabilized Au NCs
on a modified indium tin oxide electrode surface, where
K2 S2 O8 was used as the co-reactant. More recently, our group
[119] reported that organic-soluble fluorescent Au8 NCs
synthesized through a unique heterophase ligand-exchange
induced etching of gold NPs, and the fluorescent Au8 NCs
exhibited both the annihilation ECL in organic solution and
the co-reactant ECL in aqueous solution.
Solvatochromic properties
As we all know, metal NPs exhibit obvious solvatochromic
effect due to the surface plasmon phenomenon [120].
Recent studies demonstrated that metal NCs also dis-
play similar solvatochromic properties. Chen group [121]
observed solvent-dependent photoluminescence properties
of Au8 NCs when exposed to different organic solvents.
They also investigated the solvatochromic effect of Au8
NCs protected by different ligands, and they attributed the
solvatochromic effect of Au NCs to the rich surface prop-
erties of the clusters. In addition, variously capped organic
soluble and aqueous Ag NCs also exhibit spectral shifts as sol-
vent polarity is changed [61,122]. DNA-encapsulated Ag NCs
were found to exhibit solvatochromic properties, absorption
and emission bands red-shift as the content of methanol in
aqueous solution increased. This may result from structural
changes of the cluster—ligand system or from high frequency
dielectric response in response to ground vs. excited state
dipole moment changes, but the reversible solvatochromism
suggests that the optical shifts are not from cluster size
changes. Those studies indicate that the change in chem-
ical environment may induce electronic energy splitting and
electron redistribution on the cluster surface, leading to a
variation of the optical properties of the metal NCs.
Fluorescence lifetimes
As for the fluorescence lifetime of metal NCs, most metal
NCs exhibit monoexponential nanosecond fluorescence life-
times. Some polymer- and peptide-protected metal NCs
show biexponential lifetime decays, with a hundreds
of picoseconds component and the other 2—3 nanosec-
onds component [10]. However, thiol-protected metal NCs
present extremely short picoseconds fluorescence lifetimes
[67,123]. Microsecond lifetimes were also detected from
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11
some reported metal NCs. Such metal NCs with lifetimes
from sub-microsecond to microsecond are ideal for the
utilization in fluorescence detection and imaging, which
greatly eliminates autofluorescence from biological tis-
sue. For example, DHLA- and lipoic acid-protected Au
NCs both exhibit sub-microsecond fluorescence lifetimes
[29,124]. While, lipoic acid-stabilized Ag NCs show a life-
time of 37 ␮s, with 652 nm emission at 425 nm excitation
[111]. Multiple lifetime components longer than 20 ␮s were
observed in PMAA-encapsulated Ag NCs as well [61]. These
metal NCs have great potential applications in biological
imaging.
Polarized emmission
Recently, Raut and coworkers [125] found BSA-protected
Au25 clusters exhibited polarization properties. They studied
steady state and time resolved fluorescence and polariza-
tion behavior in water, propylene glycol and glycerol. The
major observation is the presence of polarization behavior
in these long fluorescence lifetime clusters. We expect that
the polarization properties of metal NCs can be utilized in
polarization based sensing applications and other exciting
applications such as the study of large molecular weight
protein complexes.
Applications of metal nanoclusters
The unique physicochemical properties of metal NCs make
them attractive for use in various fields including both fun-
damental science studies and technological exploration. In
this section, we summarize recent advances in the applica-
tion of metal NCs as new fluorescent probes for analytical
sensors and biological imaging.
Metal ion sensors
The determination of heavy metal ions in biological sys-
tems and environment is of tremendous significance due to
their hazardous effects on the ecosystem and the human
health. In recent years, numerous of types of fluorescent
metal NCs have been applied to detect heavy metal ions. Our
group [126] has developed a new strategy for the selective
detection of Hg2+ using DNA-stabilized Ag NCs as fluorescent
probes. The DNA-stabilized Ag NCs were found to be good
indicators for the selective identification of Hg2+ based on
quenching the fluorescence of Ag NCs by Hg2+ (Fig. 9). The
unchanged fluorescence lifetime implies that the quenching
of the Ag NCs by Hg2+ ions obeyed a simple static quenching
mechanism. Thus, the Hg2+ ions and DNA-stabilized Ag NCs
may form a kind of non-fluorescent complex, which results
in quenching of the fluorescence. The proposed detection
protocol is environmentally-friendly, sensitive and selective,
and a detection limit of 5 nM was obtained. Subsequently,
Dong group [127] developed a label-free method for ‘‘turn-
on’’ detection of Hg2+ using DNA duplex stabilized Ag NCs as
fluorescent probes. The strategy utilizes the Hg2+ -mediated
T-T formation to strengthen the DNA duplexes and influ-
ence the formation of fluorescent Ag NCs, which shows high
sensitivity and selectivity. In another study, Wei et al. [49]
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Figure 9 The ‘‘turn-off’’ fluorescent mechanism for Hg2+
sensing with high selectivity.
Reprinted from Ref. [126] with permission by Royal Society of
Chemistry.
synthesized lysozyme-stabilized fluorescent Au NCs, which
can be used as a sensor for sensitive and selective Hg2+
detection with a detection limit of 10 nM. Inspired by this
finding, Zhou et al. [72] synthesized positively charged and
red-emitting lysozyme-stabilized Ag NCs, which also could
be applied in the highly selective determination of Hg2+
based on their fluorescence quenching due to the 5d10 (Hg2+ )-
4d10 (Ag+ ) metallophilic interaction. Recently, Shang et al.
[128] reported a rapid synthesis of fluorescent DHLA-capped
Au NCs through a microwave-assisted strategy. This method
could enhance the fluorescence QY of Au NCs by about 5-
fold and shorten the reaction time from hours to several
minutes. The DHLA-Au NCs were capable of sensing Hg2+
through quenching the fluorescence of the Au NCs. The fluo-
rescence decay of Au NCs became faster in the presence
of Hg2+ , with the average lifetime decrease. This result
implies that fluorescence quenching results from interac-
tions between Hg2+ and Au+ on the nanoparticle surface due
to the specific and strong d10 —d10 metallophilic interaction
(e.g., 5d10 (Hg2+ )—5d10 (Au+ )). And the limit of detection for
Hg2+ was 0.5 nM. Moreover, they have successfully demon-
strated the utilization of DHLA-Au NCs for monitoring Hg2+
in living cells.
Copper is essential to all living systems for its key role
in cooperating with certain proteins to produce numer-
ous enzymes. However, Cu2+ ions are toxic when its level
exceeds cellular needs, which will cause serious diseases
[129]. Considerable efforts have been devoted to detecting
Cu2+ using fluorescent metal NCs as probes. Dong group [130]
has developed a simple and sensitive fluorescent sensor for
Cu2+ detection based on fluorescent PMAA protected Ag NCs.
A detection limit of 8 nM was obtained, which is much lower
than the U.S. Environmental Protection Agency (EPA) limit
for Cu2+ in drinking water (20 ␮M). Chang and coworkers
have developed a novel, simple and turn-on method for the
detection of Cu2+ using water-soluble DNA/Ag NCs [131].
The fluorescent Ag NCs were prepared with a reported DNA
sequence as synthesis template. The obtained DNA/Ag NCs
display yellow emission with a QY of 11.5%. The introduction
Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
L. Zhang, E. Wang
of Cu2+ induced a quite significant fluorescence enhance-
ment, which could be developed for selective and sensitive
detection of Cu2+ . The limit of detection (LOD) for Cu2+
was estimated to be 8 nM. In another protocol, they devel-
oped DNA-Cu/Ag NCs-based fluorescent assay for ‘‘turn-on’’
detection of Cu2+ [132]. The fluorescence of DNA-Cu/Ag NCs
was quenched by 3-mercaptopropionic acid (MPA), which
was recovered after the addition of Cu2+ . The LOD reached as
low as 2.7 nM for Cu2+ . The practicality of the present strat-
egy for the detection of Cu2+ in environmental samples was
validated through analysis of soil and pond water samples.
Ag(I) ions are reported to be greatly toxic to a lot of
bacteria, viruses, algae and fungi, and such antibacterial
activity of Ag(I) ions has been employed into many appli-
cation fields (such as toiletry, timbering, clinical material)
[133]. However, once the concentration of Ag(I) ions is high
enough, they can still bring harmful side-effects to the envi-
ronments and the human health. Jin [134] group developed
a novel type of nanosensor for silver ion detection utiliz-
ing glutathionate (SG)-capped Au25 NCs as fluorescent probe
based on the fluorescence enhancement. The Ag+ detection
limit is approximately 200 nM in combination with the good
selectivity against other cations. Further experiments reveal
three important factors responsible for the unique fluores-
cence enhancement caused by silver ions. Among them, the
oxidation (leading to a higher charge state of Au25 ) con-
tributes most, with the other two contributions coming from
the interactions of Ag+ with Au25 and Ag0 with Au25 . This work
indicates another potential application of Au NCs, offers
new strategies for nanocluster-based chemical sensing, and
reveals a new way to influence nanocluster chemistry for
potential applications.
In aqueous solutions, chromium mainly exists in two
valence states: Cr(III) and Cr(VI). Cr(III) in trace amounts is
an essential nutrient required for sugar and fat metabolism,
its high concentration is believed to induce oxidative DNA
damage, causing cancer. While Cr(VI) is approximately 100
times more toxic and is considered a human carcinogen with
adverse impact on human skin, stomachs, lung, liver and
kidneys [135]. Zhu group [136] prepared highly fluorescent
Ag NCs via a rapid microwave-assisted green approach and
used them as novel fluorescence probes for the sensitive
and selective determination of Cr3+ ions based on quench-
ing the fluorescence of Ag NCs. The detection limit is found
to be 28 nM. Recently, Liu group [137] developed a novel
method for selective determination of Cr(III) and Cr(VI) in
environmental water samples based on target-induced fluo-
rescence quenching of glutathione-stabilized gold NCs. The
obtained detection limits are found to be 2.5 ␮g/L for Cr(III)
and 0.5 ␮g/L for Cr(VI), respectively.
In addition, Banerjee [138] developed Au cluster-based
fluorescent sensor for ‘‘turn-on’’ detection of AsIII with high
sensitivity and selectivity. The Au NCs synthesized through
a wet-chemical approach using a dipeptide L-cysteinyl-L-
cysteine, which show the emission maximum at 410 nm with
a QY of 41.3%. The addition of AsIII induced a quite signifi-
cant fluorescence enhancement of Au NCs, which should be
attributed to the fact that positive charged AsIII ions interact
with the negatively charged gold clusters and the electrons
can flow from the electron rich gold clusters system to the
electron deficient AsIII ion, resulting in an increase in the
radiative decay rate Au NCs. The LOD reached as low as
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Metal nanoclusters
Figure 10 Schematic illustrations of the analysis of ATP using
DNA/Ag NCs-G-quadruplex/Hemin conjugates.
Reprinted from Ref. [143] with permission by American Chemi-
cal Society.
53.7 nM, which is far below the permissible limit (133 nM) of
AsIII in drinking water permitted by U.S. EPA.
Small molecule sensors
Adenosine triphosphate (ATP) is generally acknowledged as
‘‘energy currency’’ in most animate beings, and plays an
important role in most enzymatic activities [139]. The con-
centration and dissipative rate of ATP have been found to be
closely related to many diseases. Dong group [140] designed
a simple and convenient method for turn-on detection of ATP
using DNA protected Ag NCs as fluorescence probe, which
shows high sensitivity and selectivity, and a detection limit
of 0.2 ␮M was obtained. In another study, they use dsDNA-
templated Cu NCs as fluorescent probe for ATP detection
based on quenching the fluorescence of Cu NCs [141], and
a detection limit of 28 nM was obtained. Subsequently, Ye
group [142] demonstrated that the DNA/Ag NCs can be used
as a fluorescent molecular beacon for simple and selec-
tive detection of ATP using the guanine-rich (G-rich) DNA
sequence as a signal transducer, which can induce the flu-
orescent enhancement of Ag NCs enabling the detection of
ATP. Recently, our group [143] has reported a novel DNA/Ag
NCs-based photoinduced electron transfer (PET) system that
enables the specific detection of ATP with high sensitivity. As
shown in Fig. 10, the designed A-G4-ATP DNA assembles into
a hairpin structure consisting of the G-quadruplex sequence
(black) and the aptamer part against ATP (blue). This hairpin
composition ensures that parts of the aptamer and G-
quadruplex sequences are caged in the stem of the hairpin.
Formation of the ATP-aptamer complex opens the hair-
pin structure and deprotects the G-quadruplex sequence,
accompanied by self-assembling of the G-quadruplex/hemin
complex. The photoinduced electrons of DNA/Ag NCs were
transferred to the G-quadruplex/hemin complex, leading
to the quenching of the fluorescence of DNA/Ag NCs. This
approach allowed the detection limit of 8.0 nM for ATP based
Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
13
on the signal-to-noise ratio and exhibited excellent selec-
tivity for ATP detection over other ATP analogs GTP, CTP,
and UTP. More recently, Willner and coworkers [144] have
reported a hybrid system consisting of nucleic-acid func-
tionalized Ag NCs and grapheme oxide (GO) are used for
the development of fluorescent ATP sensors. The designed
nucleic acid consisting of the anti-ATP aptamer sequence
and conjugated to the nucleic-acid-protected Ag NCs acted
as functional probe. The Ag NCs were adsorbed onto GO
to yield the quenched Ag NCs. In the presence of ATP, the
resulting ATP-aptamer complex is desorbed from GO, lead-
ing to the fluorescence of the Ag NCs. This strategy shows
high sensitivity and selectivity for ATP detection.
Cocaine is a powerfully addictive stimulant drug that
increases the level of dopamine, abusing cocaine has a vari-
ety of adverse effects on the body. Simple and sensitive
detection of cocaine is very important for law enforcement
and clinical diagnostics. Dong group [140] designed a sim-
ple and convenient method for turn-on detection of cocaine
using DNA protected Ag NCs as fluorescence probe. The
design contains two DNA strands which are both chimeric
conjugates of the aptamer sequence fragment and G-rich
sequence fragment. The formed Ag NCs show weak fluores-
cence emission. In the presence of cocaine, however, the
two aptamer fragments bind cocaine, which in turn puts the
two G-rich sequence fragments in proximity and the fluores-
cent intensity of DNA/Ag NCs enhances greatly. The strategy
shows high sensitivity and selectivity, and a detection limit
of 0.1 ␮M was obtained. In another study, they use dsDNA-
templated Cu NCs as fluorescent probe for cocaine detection
based on quenching the fluorescence of Cu NCs [141], and a
detection limit of 0.1 ␮M was obtained.
Dopamine (DA), as an important catecholamine neu-
rotransmitter, plays a vital role in the function of the
central nervous, renal, hormonal, and cardiovascular sys-
tems. Abnormal DA concentrations in the brain may result
in serious diseases. Qu group [145] developed an easy pre-
pared fluorometric and colorimetric dual channel probe for
DA detection with high sensitivity and selectivity by use
of BSA-stabilized Au NCs (Fig. 11). The BSA-Au NCs exhibit
strong fluorescence emission, while upon addition of DA,
the Au NCs show a dramatic decrease of the fluorescence
intensity as a result of the photoinduced electron transfer
process from the electrostatically attached DA to the BSA-
Au NCs. The detection limit of DA can be as low as 10 nM.
In addition, the assay for DA can also be easy to implement
for visual detection due to the observed inhibition of the
peroxidase-like activity of Au NCs in the presence of DA,
with a detection limit of 10 nM. Both fluorometric and col-
orimetric methods exhibit excellent selectivity toward DA
over interfering substances.
In addition, other important small molecules, such as
biothiols [146,147] including cysteine, homocysteine and
glutathione, and hydrogen peroxide [148,149], can also be
detected by fluorescent metal NCs, which has been reviewed
in detail by shang et al. [11].
Protein sensors
The selective detection of proteins with fluorescent metal
NCs as probes often needs to combine selective recogni-
tion molecules (e.g. antibodies and aptamers). Considerable
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Figure 11 (A) Schematic illustration of the fluorescence response of the BSA-stabilized Au nanoclusters to dopamine. (B) Schematic
representation of peroxidase-like catalytic color reaction for sensitive sensing of dopamine.
Reprinted from Ref. [145] with permission by Elsevier.

effort has been devoted to constructing metal NCs-based form G-quadruplex structure, and released from the duplex
fluorescent protein sensors. Thrombin is a coagulation pro- structure. Then, G-rich DNA could enhance the fluorescence
tein in the bloodstream, which converts soluble fibrinogen intensity of DNA/Ag NCs, realizing the detection of throm-
into insoluble strands of fibrin as well as catalyzing many bin, and a detection limit of 10 nM was obtained. More
other coagulation-related reactions. Martinez and cowork- recently, Willner and coworkers [144] reported a hybrid sys-
ers [150] developed a new strategy for the detection of tem consisting of DNA functionalized Ag NCs and GO are
thrombin that combines the bright DNA-templated Ag NCs used for the development of fluorescent thrombin sensors.
with the specificity and strong binding affinity of DNA The designed nucleic acid consisting of the anti-thrombin
aptamers for thrombin. The used DNA sequence with two aptamer sequence and conjugated to the DNA-protected Ag
active domains: one domain was an aptamer for throm-
bin, and the other was a sequence able to generate Ag
NCs. The fluorescence of Ag NCs was quenched considerably
after the binding of thrombin with its aptamer. Through this
approach, nanomolar concentrations of thrombin could be
detected. Subsequently, Zhu and coworkers [151] presented
a binding-induced fluorescence turn-on assay of thrombin
using DNA/Ag NCs as probes. Key features of this assay
include aptamer binding-induced DNA hybridization and flu-
orescent enhancement of Ag NCs with G-rich DNA sequences.
When thrombin binds with its two aptamer, hybridization
process would be initiated between the complementary
sequences attached to each aptamer, thereby making the
G-rich overhang in proximity with Ag NCs and resulting in a
significant fluorescence enhancement. With this approach,
a detection limit of 1 nM was obtained for thrombin detec-
tion. Recently, our group [152] developed a new DNA/Ag
NCs-based beacon for turn-on detection of thrombin through
specific G-rich DNA sequences enhance the fluorescence
intensity of DNA/Ag NCs. This process could be modu- Figure 12 Schematic analysis of thrombin by the light-up
lated by the binding-induced strand displacement reactions DNA/Ag NCs system through binding-induced strand displace-
(Fig. 12). Initially, aptamer of thrombin inhibit the G-rich ment reactions.
DNA to enhance the fluorescence. However, in the presence Reprinted from Ref. [152] with permission by Royal Society of
of thrombin, thrombin could combine with its aptamer to Chemistry.

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Metal nanoclusters
NCs acted as functional probe. The Ag NCs were adsorbed
onto GO to yield the quenched Ag NCs. Desorption of the
probe upon formation of the probe-thrombin aptamer com-
plex leads to the recovery of fluorescence, achieving the
detection of thrombin.
Proteases are a major class of enzymes that catalyze
the hydrolysis of peptide bonds to break down proteins into
smaller pieces in a process known as proteolysis [153]. Ubiq-
uitous in nature, proteases are present in all living cells
and organisms, and are known to play a pivotal role in the
development and control of many biological processes. Xia
group [154] demonstrated a new nanoscale platform based
on protein-protected Au NCs for the fluorescence detection
of proteases with high sensitivity and selectivity. The detec-
tion is based on the concept that the protein shell can serve
as a protective layer to prevent the fluorescent Au NCs from
being exposed to the O2 from ambient air. When the pro-
tein shell is degraded by a protease, the O2 molecules can
quickly access the Au NCs and thus quench the fluorescence.
The advantages of this new detection system lies in its sim-
plicity, low cost and ‘‘one-step’’ of preparation, as well as
the potential use with a wide range of protein substrates.
Plateletderived growth factor B-chain homodimer (PDGF-
BB) is a protein growth factor that is concerned with
controlling the proliferation or differentiation of cells
including fibroblasts, smooth muscle cells and glial cells. Its
overexpression has been reported in some human tumors.
As a potential protein marker for cancer diagnosis, sensitive
and rapid detection of PDGF-BB could help early diagnosis,
treatment, and prognosis of cancers. Yang and coworkers
[155] reported a turn-on and homogeneous aptasensor for
PDGF-BB detection, which relies on target induced forma-
tion of Ag NCs. The aptasensor contains two hairpin DNA
probes, one consists of the aptamer sequence of PDGF-BB
and the other contains the Ag NCs nucleation sequence,
which is blocked by the hairpin stem region. They can co-
exist metastably in the absence of PDGF-BB and maintain
hairpin structure. However, in the presence of PDGF-BB, the
binding of PDGF-BB with aptamer will result in the hybridiza-
tion of the two hairpin DNA structures, and release the
Ag NCs nucleation sequence. In this case, Ag NCs can be
formed via the reduction of Ag+ by NaBH4 . By monitoring
the increase in fluorescence intensity, a detection limit of
0.37 nM was obtained for PDGF-BB detection.
SsDNA-binding protein (SSB) is an important protein in
cells of all living organisms. It binds selectively and cooper-
atively to single-strand region of DNA to prevent premature
annealing that is essential for DNA replication, recombi-
nation, and repair [156]. Chang and coworkers [157] have
reported a DNA-Cu/Ag NCs-based approach for the highly
sensitive and selective detection of SSB.110 SSB can strongly
and specifically interact with ssDNA template, and thus
change the conformation of the template, leading to the
decreased fluorescence of ssDNA-stabilized Cu/Ag NCs. The
approach provided a limit of detection of 0.2 nM for SSB
detection.
Cystatin C (Cys C) is a significant cysteine protease
inhibitor in human bodies, and is proposed as a fascinating
novel marker of glomerular filtration rate for kidney injury
detection. Huang group [158] report a simple, immune-
independent and label-free method for Cys C detection using
BSA-stabilized Au NCs as fluorescent probes. This method
Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
15
relies on the BSA scaffold degradation caused by the cys-
teine protease activity of papain and the specific inhibition
of papain activity by Cys C. The fluorescence of BSA-Au NCs
can be effectively quenched by papain, and restored by the
coexistence of Cys C. This method enables sensitive and
selective measurement of Cys C with the detection limit of
4.0 ng/mL.
DNA and miRNA sensors
The detection and quantification of DNA are important
for in vivo real-time monitoring of cellular processes and
for in vitro biosensing and clinical diagnosis. By virtue of
the highly sequence-dependent generation of fluorescent
DNA/Ag NCs, our group [92] explored Ag NCs-based assay
for the detection of single nucleotide mutation, in partic-
ular the mutation associated with sickle cell anemia. In
this study, DNA duplexes with an inserted cytosine loop
have been developed as capping scaffolds for the gener-
ation of fluorescent Ag NCs. The obtained results showed
that even a single-nucleotide mismatch located two bases
away from the nanocluster formation site would prohibit
the generation of fluorescent Ag NCs. Subsequently, Ren and
coworkers [159] reported the site-specific growth of flu-
orescent Ag NCs by using a mismatched dsDNA template.
Few-atom, molecular-scale Ag NCs are found to localize at
the mismatched site and this formed Ag NCs can be utilized
as functional biological probes to identify single-nucleotide
polymorphisms. In addition, our group [102] also utilized
DNA-hosted Cu NCs for fluorescent identification of single
nucleotide polymorphisms. The fluorescence of Cu NCs is
very sensitive to base type located in the major groove of
dsDNA. This intriguing finding provides a sensitive fluori-
metric diagnostic of the mismatch type in a specific DNA
sequence.
Based on the ‘‘light up’’ of DNA/Ag NCs in proximity to
G-rich DNA sequences, DNA/Ag NCs have been designed for
the analysis of DNA. Yeh et al. [160] designed a nanoclus-
ter beacon (NCB) consisting consists of two short linear DNA
probes (a NC probe and a G-rich probe). They were brought
into proximity through hybridization with the target DNA.
The turn-on of red emission indicated the presence of tar-
get DNA, a sequence from influenza A virus was detected
with a signal/background (S/B) ratio of 175. Similar detec-
tion based on the moleclular beacon yields a S/B ratio of
32, illustrating the advantages of this NCB. Subsequently,
they reported a chameleon NCB that lights up into different
colors upon binding single-nucleotide polymorphisms (SNP)
targets [161]. They found the fluorescence emission color of
a NCB can change substantially depending upon the align-
ment between the Ag NCs and the DNA enhancer sequence.
Chameleon NCBs exploit this color shift to directly detect
SNPs, and this method has been validated on all single-
nucleotide substitution scenarios in three synthetic DNA
targets, in six disease-related SNP targets (Fig. 13), and
in two clinical samples taken from patients with ovarian
serous borderline tumors. Samples with single-nucleotide
variations can be easily identified by the naked eye under
UV excitation, making this method a reliable and low-cost
assay with a simple readout format. Recently, our group
[152] achieved to modulate the fluorescent enhancement
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16 L. Zhang, E. Wang

Figure 13 Chameleon NCBs (cNCBs) was applied to detect a wide variety of disease-related SNPs, which covered all 6 types of
single-nucleotide substitution scenarios (C→G: Set 1, G→A: Set 2, C→T: Set 3, A→C: Set 4, T→A: Set 5, and G→T: Set 6). (A)
Design of cNCBs and nomenclature of the samples. (B) Detection results of the 6 cNCBs. As expected, all SNP targets were clearly
differentiated based on their emission spectra (for position +1 samples always being blue-shifted), proving the general use of cNCBs.
The integrated emission spectrum from 420 to 800 nm (normalized to unity) was divided into three elements: blue, 420—510 nm;
green, 510—640 nm; and red, 640—800 nm The percentiles of the 3 color elements in each detection sample are shown in the stacked
columns on the right, serving as a simple criterion for SNP scoring.
Reprinted from Ref. [161] with permission by American Chemical Society.

of DNA/Ag NCs through toehold-mediated strand displace-
ment reactions to place DNA/Ag NCs in proximity to G-rich
DNA sequences. This approach could be used to develop a
new fluorescent sensing platform for the detection of tar-
get DNA with high selectivity and sensitivity. Furthermore,
to improve the sensitivity, exonuclease III-catalyzed ampli-
fication was applied in the present DNA detection system,
a very low detection limit of 0.1 nM was obtained, which is
much lower than that of the traditional unamplified strategy.
In addition, our group [143] also found a new phe-
nomenon of photoinduced electron transfer (PET) between
DNA/Ag NCs and G-quadruplex/hemin complexes, accompa-
nied by a decrease in the fluorescence of the DNA/Ag NCs.
Based on this mechanism, we developed a new nanocluster-
based molecular beacon for the detection of target DNA
with high sensitivity and selectivity. In this PET process, a
parallel Gquadruplex and the sensing sequences are blocked
by a duplex. The specific combination of target DNA with the
sensing sequence triggers the release of the G-quadruplex
and allows it to fold properly and bind hemin to form a stable
G-quadruplex/hemin complex, which promotes the electron
transfer from the DNA/Ag NCs to the hemin FeIII center, thus

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Metal nanoclusters 17

Figure 14 Multiplexed analysis of the HBV gene (5) and of the HIV gene (8) using the near-infrared- and red-emitting Ag NCs/GO
hybrid system.
Reprinted from Ref. [144] with permission by American Chemical Society.

resulting in a decrease in the fluorescence intensity of the DNA-12nt-RED-172 probe to increase formation of secondary
DNA/Ag NCs. structure. The redesigned DNA-GG172-12nt-RED showed a
More recently, Willner group [144] reported on the inte- dramatic increase in red emission, and the presence of tar-
gration of DNA/Ag NCs with GO, and the implementation of get RNA-miR172 could efficiently quench its fluorescence,
these hybrid materials for the development of DNA sensors. providing an approach for RNA-miR172 detection.
As shown in Fig. 14, the nucleic acid sequence protecting the In addition, Zhang and coworkers [164] developed a
Ag NCs is elongated with the nucleic acid probe sequence simple, sensitive, and label-free method for miRNA detec-
that is complementary to the target DNA. The adsorption of tion using DNA/Ag NCs as effective electrochemical probes
the Ag NCs-functionalized DNA to GO induced the quenching (Fig. 15). The functional DNA probe integrates both recog-
of the fluorescence of Ag NCs. In the presence of target DNA, nition sequence for hybridization and template sequence
the formation of the duplex between Ag NCs-functionalized
DNA and target DNA triggers the desorption from the sur-
face of GO, leading to the regeneration of fluorescence.
More important, they implemented the sensing platform to
analyze the hepatitis B virus (HBV) gene, the human immun-
odeficiency virus (HIV) gene, and the syphilis (Treponema
pallidum) gene.
Detection of microRNAs (miRNAs) is also significant
because they play important roles in the regulation of gene
expression. The individual levels of miRNAs can be useful
biomarkers for cellular events or disease diagnosis. Yang and
Vosch [162] have designed a DNA/Ag NC probe that can mon-
itor the presence of target miRNA. The red fluorescence of
the DNA/Ag NC probe is diminished in the presence of target
miRNA, and high specificity toward detecting specific miRNA
sequences is performed. Subsequently, they investigated the
influence of nucleic acid secondary structure on the fast for-
mation of bright red emissive Ag NCs in a DNA sequence
(DNA-12nt-RED-160), and designed for the detection of a
microRNA sequence (RNA-miR160) [163]. Their results indi- Figure 15 Illustration of electrochemical detection of MiRNA
cate that the reduction in secondary structures led to the using oligonucleotide encapsulated Ag-NCs
decreased amount of red emissive Ag NCs. On the basis of the Reprinted from Ref. [164] with permission by American Chemi-
finding, they rearranged the sequence of the low-emissive cal Society.

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18 L. Zhang, E. Wang

for in situ synthesis of Ag NCs, which appears to possess
exceptional metal mimic enzyme properties for catalyzing
H2 O2 reduction. The miRNA assay employs gold electrodes
to immobilize the molecular beacon (MB) probe. After the
MB probe subsequently hybridizes with the target and func-
tional probe, the DNA-encapsulated Ag NCs are brought to
the electrode surface and produce a detection signal, in
response to H2 O2 reduction. A detection limit of 67 fM was
obtained for target miRNA with high selectivity. This is the
first application of the electrocatalytic activity of Ag NCs in
bioanalysis, which would be attractive for genetic analysis
and clinic biomedical application.
Biological labeling and imaging
Metal NCs possess an attractive set of features, such
as ultrasmall size, good biocompatibility, brightness and
photostability, which renders them attractive alternatives
as fluorescent probes for biological labeling and imaging
[165,166]. Moreover, large Stoke shifts of metal NCs can pre-
vent spectral cross-talk and, thus, enhance the detection
signal. In recent years, a number of works have reported
biological labeling and imaging applications based on flu-
orescent metal NCs. Lin group [167] reported a galvanic
replacement route to prepare fluorescent Au NCs using
presynthesized and size-controlled Ag NCs as templates. The
obtained Au NCs show strong fluorescence and favorable bio-
compatibility. They used the Au NCs as fluorescent probes
for cellular marking in CAL-27 cancer cells and MC3T3-E1
normal cells, respectively. The results indicated that the flu-
orescence signals were not only distributed in the cytoplasm
but most came into the cellular nucleus for both CAL-27 and
MC3T3-E1. Later, Wang group [168] reported a novel strat-
egy for synthesis of Au NCs and applied them in fluorescence
imaging in vivo. The fluorescent Au NCs were spontaneously
biosynthesized by cancerous cell (i.e., HepG2, human hep-
atocarcinoma cell line; K562, leukemia cell line) through
Au(III) reduction inside cells cytoplasms, and ultimately con-
centrated around their nucleoli, thus affording precise cell
imaging (Fig. 16). Importantly, this does not occur in non-
cancerous cells, as evidenced with human embryo liver cells
(L02) used as controls. This dichotomy was exploited for a
new strategy for specific fluorescent self-bio-marking of the
tumors, which opens up promising opportunities for biomed-
ical applications requiring specific and sensitive imaging of
tumors without direct injection of vectorized nanoparticles
or molecular fluorescent probes. Recently, Qu and Ren group
[169] report the synthesis of the polyethyleneimine (PEI)-
templated Au NCs (PEI-Au NCs) as an efficient carrier for
gene delivery. The PEI-Au NCs integrate the advantages of
PEI and Au NCs: the presence of Au NCs can effectively
decrease the cytotoxicity of PEI, making it possible to apply
them in biological systems, while the cationic polymer layer
PEI with positive charges is essential for enhanced gene
transfection efficiency. In addition, with excellent photo-
luminescent properties, the Au NCs also endow the system
with the versatility of fluorescent imaging, indicating a great
potential as an ideal fluorescent probe to track the trans-
fection behavior.
In addition, multifunctional nano-conjugates based on
Au NCs have been applied in tumor imaging. Gu and
coworkers [170] investigated the utilization of BSA pro-
tected Au NCs as fluorescent probes for tumor imaging.
Au NCs were conjugated with methionine (Met) and MPA,
a near-infrared fluorescent dye, giving a probe, Au-Met-
MPA. They investigated the internalization of fluorescent
Au-BSA and Au-Met-MPA by L02 normal human cells and
MCF-7 tumor cells. The fluorescence microscopy images
were shown in Fig. 17. With regard to the images of
L02 cells, only weak red fluorescence emitted by the Au
NCs in cells could be observed indicating that little Au-
BSA or Au-Met-MPA had entered normal cells. By contrast,
Au NCs in MCF-7 tumor cells showed strong red fluores-
cence. The fluorescence of cells incubated with Au-Met-MPA
which was found to have entered cytoplasm and nucleus
of MCF-7 tumor cells could obviously eclipse that of the
cells treated with Au-BSA. This clearly demonstrated that
the presence of Met on the surface of Au NCs endowed
the tumor targeting capability, which increased its cellular

Figure 16 Schematic illustration of in situ biosynthesis of gold nanoclusters in cells and tumor imaging. HAuCl4 solution was
incubated in vitro with target cells or subcutaneously injected in vivo near a tumor. The sequestration and reduction of AuCl4 − anions
inside cells give rise to the progressive formation of gold nanoclusters. After incubation for 24—48 h, fluorescent gold nanoclusters
were observed inside the tumor cells or accumulated around the tumor.
Reprinted from Ref. [168] with permission by Nature Publishing Group.

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Metal nanoclusters 19

Figure 17 The fluorescence microscopy images of L02 normal human cells and MCF-7 tumor cells after incubated with Au-BSA
and Au-Met-MPA.
Reprinted from Ref. [170] with permission by Elsevier.

uptake by MCF-7 tumor cells. Chen et al. [171] synthesized
multifunctional no-conjugated amphiphilic copolymers (Au
NCs-PLAGPPS-FA) base on BSA protected Au NCS for tumor-
targeted drug delivery, which consisted by Au NCs as core
and folate (FA)-conjugated amphiphilic hyperbranched block
copolymer as shell based on poly(L-lactide) (PLA) inner arm
and FA-conjugated sulfated polysaccharide (GPPS—FA) outer
arm. The nanocarriers displayed strong red emission at
620 nm when excited at 480 nm and good biocompatibility.
They used the nanocarriers as fluorescent probes for cel-
lular imaging in HeLa cells. The results indicated that the
nanocarriers provided high anticancer activity and showed
the specificity to target cancer cells due to the enhanced cell
uptake mediated by FA moiety. Furthermore, the nanocar-
riers could be tracked at the cellular level for advance
therapy.
Two-photo excitation confocal microscopy was used
to observe biological imaging, which can afford some
advantages, such as the ability of deeper imaging inside
tissues and the reduced phototoxicity of NIR light. Shang
et al. [32] investigated the internalization of fluorescent
D-penicillamine conjugated Au NCs (DPA-Au NCs) in the endo-
somal vesicles of human cervical cancer (HeLa) cells via
two-photo fluorescence imaging. They could easily observe

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Figure 18 Microscopic observation of internalization of the insulin-Au nanoclusters (NCs). Differentiated C2C12 myoblasts were
treated with insulin-Au NCs for 2 h. (a) Cell nucleus stained with 4 ,6-diamidino-2-phenylindole (DAPI, blue). (b) Actin fiber stained
with Alexa Fluor 488 phalloidin to confirm the cell boundary (green). (c) Insulin-Au NCs exhibit red luminescence. (d) Fluorescence
image overlay of the three images.
Reprinted from ref. [52] with permission by Wiley-VCH.

the bright fluorescence of Au NCs inside the cells. The
images of 3D reconstruction further indicate that DPA-Au
NCs inside the cells as well as attached to the outside of the
plasma membrane. Recently, Liu et al. [52] reported the
synthesis of fluorescent Au NCs by using insulin as protected
ligand. The as-prepared insulin-Au NCs show excellent bio-
compatibility and retain the insulin bioactivity in reducing
blood glucose. The uptake efficiency of insulin-Au NCs for
C2C12 cells may serve as a biomarker to distinguish the dif-
ferentiated versus undifferentiated C2C12 myoblasts. The
confocal images depicted in Fig. 18 clearly show that the red
fluorescence of Au NCs is dispersed in the fully differentiated
C2C12 mouse myoblasts. A further detailed two-photon z-
stacking study confirms that insulin-Au NCs entered into the
cell and were distributed in the cytoplasm but not simply
adhered on the surface of the membranes. Moreover, they
also presented that the fluorescent Au NCs could be used in
X-ray computed tomography (CT) imaging.
Fluorescence lifetime imaging (FLIM) is also an alter-
native technique for cell imaging that takes advantages
of the longer lifetime (>100 ns) of metal NCs than that
of auto-fluorescence of cellular organelles. Shang et al.
[29] reported a facile strategy to synthesize water-soluble
fluorescent Au NCs stabilized with the bidentate ligand
DHLA. The Au NCs shows many attractive features includ-
ing ultrasmall size, bright near-infrared luminescence, high
colloidal stability, and good biocompatibility. Moreover,
their fluorescence lifetime is longer than 100 ns. These
properties make them attractive as labels in FLIM appli-
cations. They investigated the internalization of DHLA-Au
NCs by live HeLa cells. The results indicated that the
distributions of Au NCs was in the intracellular and mem-
brane regions and the Au NCs exhibited bright fluorescence
luminescent with long fluorescence lifetimes (500—800 ns),
which could avoid significant autofluorescence emitted by
the cells. FLIM images not only reveal the uptake of Au
NCs by the cells but also provide information on their
changed local environment. Subsequently, they synthesized
lipoic acid-protected Au NCs, which emit bright fluores-
cence in the near-IR region and show the pronounced
temperature dependence of the steady-state fluorescence
emission spectra, the fluorescence intensity decreases up
raising the temperature [124]. Moreover, the Au NCs exhibit
excellent stability in biological media and display a char-
acteristic, nonexponential fluorescence decay that can be
fitted with a sum of three exponentials, with a short
( 1 ≈ 24 ns), a medium ( 2 ≈ 130 ns), and a long lifetime
(600 ns <  3 < 750 ns). Those properties endow the Au NCs

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Metal nanoclusters
Figure 19 Typical FLIM images of HeLa cells with internalized Au NCs at four different temperatures.
Reprinted from Ref. [124] with permission by Wiley-VCH.
with the capability for temperature sensing in HeLa human
cancer cells using FLIM. Lifetime maps were calculated on a
pixel-by-pixel basis by fitting the fluorescence decay of each
pixel with a tri-exponential function. As shown in Fig. 19,
these images provide clear evidence that the long fluo-
rescence lifetime components in the range of 600—1000 ns
arise from internalized Au NCs rather than cellular autoflu-
orescence. With increasing temperature, the fluorescence
lifetime decreased markedly from 970 ns at 14 ◦ C to 670 ns
at 43 ◦ C.
In addition to Au NCs, functional Ag NCs have been
employed for biological imaging. Dickson and coworkers [82]
synthesized Ag NCs with the linear poly(-acrylic acid) (PA)
as stabilizer. They found that the formed PA-Ag NCs readily
transfer NCs to high-affinity ssDNA sequences, which results
in the loss of PA-Ag NCs fluorescence and generation of char-
acteristic DNA-encapsulated Ag NCs emission. Concomitant
with the spectral shifts, cluster transfer from low-quantum-
yield PA-Ag NCs to high-quantum-yield ssDNA/Ag NCs results
in a 10-fold increase in cluster brightness. Using this flu-
orogenic NCs shuttle, they fluorescently labeled cellular
components by first conjugating C12 DNA to antibodies, stain-
ing with these nonfluorescent DNA-conjugated antibodies,
and transferring NCs as the final step. The results demon-
strate that the emission image from the NCs (Fig. 20A)
colocalized well with that of the fluorescein isothiocyanate
(FITC) (Fig. 20B), thus revealing antibody location and
indicating actin staining with NCs through NCs transfer
(Fig. 20C). Furthermore, they have successfully stained
microtubules and labeled live cell with the same manner.
Vosch and coworkers used C24 -encapsulated fluorescent Ag
NCs to monitor the transfection of DNA/Ag NCs inside liv-
ing HeLa cells [172]. The C24 /Ag NCs were complexed with a
Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
21
commercially available transfection reagent Lipofectamine,
and the internalization of C24 /Ag NCs was followed with
confocal fluorescence microscopy. Bright near-infrared flu-
orescence was observed from inside the transfected HeLa
cells, which opens up the possibility for the utilization of
Ag NCs for biological labeling and imaging of living cells and
for monitoring the transfection process with limited harm to
the living cells.
Combining the specific recognition of aptamer toward
CCRF-CEM live cells, Gao and coworkers have successfully
achieved the specifically marking the nucleus of live cells
by DNA/Ag NCs [173]. Sgc8c aptamer, selected by cell-
SELEX, has been shown to be a specific aptamer for the
endosomes of CCRF-CEM cells [174]. Eight cytosine bases
were inserted at the 5 end of the sgc8c aptamer to form
Msgc8, which was used for the synthesis of Ag NCs. The
formed cluster—aptamer hybrids could specially target the
nucleus of CCRF-CEM cells after incubation with the cells for
2 hours at room temperature. Confocal microscope observa-
tion showed that the cluster—aptamer hybrids were mainly
located in the nucleus of the live cells.
AS1411 is an antiproliferative G-rich phosphodiester DNA
and currently an anticancer agent under phase II clinical
trials. Its function as an aptamer to nucleolin has led to
the identification of nucleolin as a new molecular target for
cancer therapy [175]. Zhu and coworkers [176] presented a
strategy to synthesize AS1411-functionalized Ag NCs through
a facile one-pot process. The obtained Ag NCs emitted
red color and the fluorescence quantum yield could reach
40.1%, which was beneficial to the applications of biological
imaging. AS1411 retained its anticancer nature within the
complex, and unexpectedly, functionalized Ag NCs demon-
strated enhanced efficiency of growth inhibition compared
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22 L. Zhang, E. Wang

Figure 20 (A) Fluorescence images of BPAEC from Ag NCs and (B) FITC tagged to actin C12 to indicate the location of actin, with
colocalization in (C).
Reprinted from Ref. [82] with permission by Wiley-VCH.

Figure 21 Schematic illustration and confocal fluorescence images of HeLa cells upon incubation with DHLA-AgNCs (25 ␮g/mL)
for 2 h in DMEM medium without (A—C) and with 100 ␮mol/L HSA (D—F). Cell membranes were stained with CellMaskTM DeepRed (B
and E).
Reprinted from Ref. [177] with permission by Springer-Verlag.

with naked AS1411. In addition, such functionalized Ag NCs reduced in the presence of HSA, as seen from the much lower
could be internalized into MCF-7 human breast cancer cells fluorescence intensity (Fig. 21D). The results indicate that
and stain the nucleus via receptor-mediated endocytosis. protein adsorption has a significant effect on the biological
The obtained results could improve the permeability of DNA response to Ag NCs exposure.
protection groups for live cell imaging and help understand
the therapeutic mechanism of AS1411. Conclusion and perspective
Recently, Shang et al. [177] rationally connected DHLA-
Ag NCs with a model protein, human serum albumin
In summary, we provide recent advances of the research
(HSA), which has been have been systematically investi-
progress on metal NCs, from the challenging synthesis,
gated by using a variety of techniques. The results show
unique properties to the promising application in analyt-
that the physicochemical properties of both proteins and
ical sensors and biological imaging. Now, metal NCs have
Ag NCs undergo changes upon their interactions. However,
been developed as a new class of fluorophores, their excel-
it appears that the overall conformation of HSA remains
lent properties endow them attractive fluorescent probes
essentially unaffected in the complex. Furthermore, they
for biological applications. These research results reveal
investigated the biological implications of protein adsorp-
that metal NCs can open many good opportunities in an
tion by evaluating responses of HeLa cells to Ag NC exposure
extremely multidisciplinary environment for promoting the
through live-cell fluorescence microscopy. As shown in
rapid developments of different research fields. Despite
Fig. 21, intense fluorescence emission from Ag NCs was
substantial progress in metal NCs, a variety of challenges
observed from inside the cells, suggesting an efficient inter-
remains and needs to be addressed.
nalization of these luminescent particles by the cells. In
At present, one of the major challenges for the deeper
contrast to the pronounced uptake of bare Ag NCs, the
development of metal NCs has been lack of an efficient
amount of Ag NCs internalized by the cells is substantially
route for preparing size precisely controlled metal NCs

Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
+Model
NANTOD-359; No. of Pages 26 ARTICLE IN PRESS
Metal nanoclusters
with high quality and high yield due to the properties of
metal NCs are strongly dependent on the size and the cur-
rently reported metal NCs possess relatively low QY. In
addition, the high monodispersity and purity are also the
critical points of the synthesis of metal NCs. Thus, how to
synthesize size-controlled metal NCs and how to purify the
synthesized metal NCs will be challenging work. Up to now,
many researches have been concentrated on Au and Ag NCs.
Only in recent years, the studies on Cu and other metal NCs
emerged gradually, and the emerging metal NCS also show
unique and excellent properties. Those properties make
them potentially useful in a wide variety of applications in
fluorescent detection and other fields. Therefore, it is nec-
essary to develop methods for the synthesis of other new
metal NCs, including the size-controlled synthesis, structure
characterization and their property studies. The new metal
NCs may exhibit surprising properties, which will broaden
the applications of metal NCs.
Many reports have suggested that these fluorescent metal
NCs are promising tools for sensing and intracellular imag-
ing. It could be useful to simultaneously endow metal NCs
with other properties, such bi- or multifunctional probes
bear a lot of promise for applications. Although many unique
optical and electronic properties have been discovered, the
structures and fundamental understanding of fluorescent
metal NCs are still lacking. The sensitivity and stability of
metal NCs to their environments offer the opportunity for
the applications in sensors and imaging. However, the coor-
dination nature of the NCs-protection group complex implies
that the stability of metal NCs is affected by the presence
of other strong competing ligands in aqueous solution. The
intracellular stability of metal NCs is also a challenging issue.
Until the issues are fully resolved, metal NCs will be more
useful in vitro/in vivo imaging under one-photon or two-
photon excitation.
Overall, significant progress has been achieved in the
field of metal NCs in the past decades. Many challenges have
to be faced and resolved in the future work. With contin-
ued development, we believe that metal NCs will find wide
applications in addressing important issues related to more
different fields.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Nos. 21190040 and 91227114) and 973
Project (Nos. 2010CB33600 and 2011CB911000).
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NANTOD-359; No. of Pages 26 ARTICLE IN PRESS
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Nano Res. 5 (2012) 531—542.
Libing Zhang was born in Shandong Province,
China. He received his B.S. degree from
Liaocheng University in 2007 and M.S. degree
from Changchun University of Science and
Technology in 2010. Then, he moved to
Changchun Institute of Applied Chemistry as a
Ph.D. student under the direction of Professor
Erkang Wang, and received his Ph.D. degree in
2013. He is currently a Postdoctoral Research
Associate in Biodesign Institute at Arizona
State University. His scientific interests focus
on functional nucleic acids and nanomaterials for analytical and
biological applications.
Please cite this article in press as: L. Zhang, E. Wang, Nano Today (2014), http://dx.doi.org/10.1016/j.nantod.2014.02.010
L. Zhang, E. Wang
Erkang Wang, Professor of Chemistry at
Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences (CAS), and an
advisor in the State Key Laboratory of Elec-
troanalytical Chemistry. He received his Ph.D.
degree in 1959 from Czechoslovak Academy
of Sciences directly under Professor J. Hey-
rovsky, the Nobel Prize Laureate. He is
academician of both the CAS and the Academy
of Sciences for the Developing World. He has
been on the Editorial and Advisory Board of
nine international journals. His research interests lie in the fields of
nanomaterials/nanotechnology, biosensors, electrochemistry and
electrochemiluminescence. He has published over 900 papers and
monographs in international journals with the SCI over 17,000 times
cited.