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Fluorescent silver nanoclusters as DNA probes


Judy M. Obliosca, Cong Liu and Hsin-Chih Yeh*

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DOI: 10.1039/C3NR01601C

Department of Biomedical Engineering, University of Texas at Austin, Austin, TX 78712, USA


*Author to whom correspondence should be addressed; E-mail: tim.yeh@austin.utexas.edu;
5

Fax: +1-512-471-0616; Tel: +1-512-471-7931

DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT]

1. Introduction
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Owing to the prior effort in the development of new fluorescent probes, biologists
and chemists are now equipped with an arsenal of fluorescent tools to identify and
quantify biomolecules of interest, especially DNA. However, perfect fluorophores (in
terms of size, toxicity, cost, emission line-width, brightness, chemical and photo stability,
bioconjugate friendliness, environmental sensitivity, fluorescent activability, and multicolor capability) have not yet been developed.1 Organic dyes are often dim and lack of
photostability, while commercial semiconductor quantum dots are expensive, large, blink
on all time scales, and have toxic cores. In the early 2000s, aqueous syntheses of stable
few-atom gold or silver nanoclusters (typically 2-30 atoms, size < 1 nm) with high
fluorescence quantum yields were reported,2,3 raising great interests to use these
fluorescent nanomaterials as new biolabels. Among those water-soluble gold or silver
nanoclusters developed in the past decade, ssDNA-templated silver nanoclusters
(DNA/Ag NCs)4-21 have received increasing attention due to a number of useful
properties. Compared to organic dyes, DNA/Ag NCs can be brighter6,9,12,20 and more
photostable.6,12,21 Compared to semiconductor quantum dots, DNA/Ag NCs are smaller
in size and less prone to blinking on long timescales.6,8,12 The preparation of DNA/Ag
NCs is carried out at room temperature via sequential mixing of three inexpensive
components in a buffer: a cytosine-rich oligonucleotide, silver salt, and reducing reagent.
There is no need to remove excess precursors as these precursors are essentially nonfluorescent, thus waiving the purification cost. To better understand these exciting
organic/inorganic composite nanomaterials, readers are also recommended to refer to
recent reviews by Dickson,22 Yu,23 Ras,24 Suslick,25 Nienhaus,26 Wang,27,28 and Somoza29
on DNA/Ag NCs.
Other than the benefits mentioned above, DNA/Ag NCs have three important
advantages when used as fluorescent labels. First, the fluorescence emission of DNA/Ag
NCs can be tuned throughout the visible to near-IR range (shortest emission peak at 485
nm8 and longest at 900 nm30) by simply programming the template sequences.

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Fluorescent silver nanoclusters (few atoms, quantum sized) have attracted much attention
as promising substitutes for conventional fluorophores. Due to their unique
environmental sensitivities, new fluorescent probes have been developed based on silver
nanoclusters for the sensitive and specific detection of DNA. In this review we overview
the recent discoveries of activatable and color-switchable properties of DNA-templated
silver nanoclusters and discuss the strategies to use these new properties in DNA sensing.

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2. Fluorescent nanoclusters background


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2.1 Physics and History


Fluorescent nanoclusters are collections of a small number, typically 2-30, of gold
or silver atoms.31 These nanoclusters provide the missing link between atomic and
nanoparticle behaviors in noble metals exhibiting dramatically different optical,
electronic, and chemical properties as compared to those of much larger nanoparticles or
bulk materials.31,32 With respect to optical properties, when being shrunk from a bulk
material to an atom, metals experience several different size regimes governed by
different length-scale parameters such as (1) the wavelength of light (400-700 nm for
visible light), (2) the electron mean free path of the bulk metal (52 nm for silver33), and
(3) the de Broglie wavelength of electrons at the Fermi energy (i.e. the Fermi
wavelength, ~0.5 nm for silver31).34 When the metal size approaches the wavelength of
excitation, the free electrons respond coherently to the electric field of light. As the metal
size approaches the electron mean free path of the bulk metal, the collisions at the surface
dominate the optical response, making the dielectric constant of the system not only
frequency dependent but also size dependent. When the metal size approaches the Fermi
wavelength, all electronic wavefunctions become overlapped and the systems density of
states becomes discrete,35 giving the system molecular behaviors. As the energy levels
are sufficiently spaced to allow visible energy transitions and eliminate non-radiative
dissipation pathways between energy levels, size-dependent fluorescence emission starts
to appear in the system. While the exact size at which the transition from collective
excitations (plasmon resonance) to molecular electronic transitions (fluorescence) occurs
has not been resolved in the scientific community,36 more interesting and potentially
useful optical properties are expected to be discovered within this relatively unexplored
size range.
Silver nanoclusters were initially prepared within rare gas matrices,37 where fewatom silver clusters were formed through a co-condensation process of metal vapor with
rare gas and their aggregation was prevented due to restricted diffusion in the matrices.
While rare gas matrices have provided the most detailed view of electronic transitions of
isolated silver clusters with defined stoichiometry (e.g. Ag1 to Ag4),38-41 these trapped,
bare clusters have no biological applications. To use silver clusters in solution,
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Complementary palettes of DNA/Ag NC fluorophores have been produced.8,10 Second, all


DNA/Ag NCs share a similar UV excitation feature, regardless of the location of their
visible excitation peaks.4,16,17 In other words, it is possible to use a single UV source to
excite all silver cluster species templated on DNA for multiplexed detection. Third,
DNA/Ag NCs possess unique environmental sensitivities. For instance, fluorescence of
silver clusters can be switched on and off11,13 or color tuned13,17 through interactions with a
nearby DNA sequence (called an enhancer, see the section 3.2.2). These activatable and
color-switchable properties allow DNA/Ag NCs to be used not only as fluorescent labels
but as sensors, leading to a variety of new biosensing opportunities. Many groups are
currently exploring and taking advantage of the environmental sensitivities of DNA/Ag
NCs in creating new tools for DNA detection and single-nucleotide polymorphism (SNP)
identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs
for developing DNA sensors.

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encapsulation agents or templates are necessary as they (1) offer initial nucleation sites
for the cluster formation, (2) prevent clusters from overgrowth, and (3) stabilize clusters
in the solution phase. In the early 2000s, Dicksons group first reported the synthesis of
highly fluorescent silver nanoclusters in aqueous solution using amine-rich dendrimers as
templates.2 Ever since, a wide variety of organic materials have been tested as templates
to synthesize fluorescent silver nanoclusters in solution, opening up opportunities for
silver clusters as biological labels.

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DOI: 10.1039/C3NR01601C

+
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Excitation wavelength (nm)

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Figure 1. (A) Illustration depicting how silver nanoclusters are formed on single-stranded
DNA. Silver ions preferentially associate with cytosines on the DNA strand. Addition of
NaBH4 reduces these ions, leading to the formation of silver nanoclusters. (B) DNA
microarrays (upper picture) have proved to be a useful screening tool to find DNA
sequences capable of nucleating silver clusters of different emission colors. Reprinted
from ref. 8 with the permission from American Chemical Society. (C) All DNA/Ag NCs
share a common excitation peak between 260 and 270 nm, close to the absorption
maxima of nucleobases. Through energy transfer from nucleobases, one can excite all
DNA/Ag NCs with a single UV excitation source. Reprinted from ref. 16 with the
permission from American Chemical Society. (D) It is possible to switch the fluorescence
of silver clusters on and off11,13 and tune their color13,17 through interactions with nearby
DNA sequences (called enhancers, lower red strand). Reproduced from ref. 13 with the
permission from IEEE.
2.2 Synthesis and Intriguing Properties

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Compared to gold nanoclusters, silver nanoclusters are brighter31 and can be easily
synthesized by using a number of ligands as encapsulation agents , including PAMAM,2,42
PMMA,43 polyacrylate,44,45 poly(NIPAM-AA-HEA) microgels,46 sugar molecules,47
mercaptosuccinic acid,48 lipoic acid,49 thiol ligands,50 peptides51 and DNA.4,7,10 Among
those different silver clusters synthesized, ssDNA-templated silver nanoclusters (DNA/Ag
NCs, Figure 1A) have become the research focus due to three key advantages: (1) the
ability to tune the fluorescence emission throughout the visible to near-IR range by simply
programming the template sequences (Figure 1B),8,10 (2) the ability to use a single UV
source to excite all silver cluster species templated on DNA (Figure 1C),4,16,17 and (3) the
ability to switch the fluorescence of silver clusters on and off11,13 or tune their color13,17
through interactions with a nearby DNA sequence (Figures 1D and 2A). As pointed out by
many groups,11,13,17,22,36 the optical transitions of silver clusters encapsulated by templating
ligands vary not only with stoichiometry, charge, and geometry, but also with interactions
with their encapsulating ligands. This provides the basis for the creation of complementary
palettes of silver cluster fluorophores using different DNA sequences as encapsulation

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NaBH4

Normalized intensity

ABCD

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ligands.8,10 The on/off switching and color-tuning capabilities, on the other hand, allow
DNA/Ag NCs to be used not only as fluorescent tags but as fluorescent sensors, leading to
new biosensing opportunities especially in DNA detection which we detail below. Other
than the programmable and switchable fluorescence properties, all DNA/Ag NCs share a
similar UV excitation feature, regardless of the location of their visible excitation
peaks.4,16,17 This common excitation peak, which sits close to the absorption maxima of
nucleobases (around 260-270 nm), was previously reported by Petty/Dickson4 and
Fygenson.16 The former suggested that the UV excitation peak may be due to higher lying
excited states accessible in the UV region,4 while the latter believed this UV excitation
peak is due to energy transfer between nucleobases and silver clusters, as the fluorescence
emission of DNA/Ag NCs upon UV excitation is highly depolarized.16 Nevertheless, the
common UV excitation feature can greatly facilitate the use of DNA/Ag NCs as reporters
in multiplexed detection, where one UV source should excite all silver cluster species.
Other than the above advantages, DNA/Ag NCs have more benefits, including small
size (hydrodynamic diameter, which includes the layer of DNA, ranging from 3 to 6
nm),6,8,12 low cellular toxicity,18,52,53 and are less prone to blinking on long (> 1 ms)
timescales.6,8,12 DNA/Ag NCs can also be brighter6,9,12 (e.g. one emitter was 20 times
brighter than a Cy3 dye20) and more photostable than organic dyes6,12,21 (e.g. one emitter
showed a 10-fold photostability improvement over Cy3 and Cy5 dyes8). The highest
quantum yield ever reported on a purified DNA/Ag NC species is 0.9354 and the largest
extinction coefficient is 9.5105 M-1cm-1.21 The longest lifetime reported is 4.35 ns.8 Other
useful optical properties include fluorescence recovery with low-energy secondary
excitation,9 strong two-photon-induced fluorescence (two-photon absorption cross-section
was calculated to be 3,000~4,000 GM (Goppert-Mayer units)19,21 or greater55, about 100fold larger than that of fluorescein (37 GM)56), and fluorescence recovery upon
nanocluster transfer.20
The synthesis of DNA/Ag NCs is carried out at room temperature via sequential
mixing of a cytosine-rich oligonucleotide, silver salt, and reducing reagent in buffer
solution. Proper selection of buffer, buffer pH, and the reaction molar ratio enables the
preferential formation of Ag nanoclusters (2-30 atoms) over larger plasmonic Ag
nanoparticles. Phosphate buffer5,10,11 or acetate buffer7,57,58 (compatible with electrospray
ionization mass spectroscopy) is most commonly used. In general, the synthesis starts with
mixing micromolar DNA with 6-12 equivalents of silver nitrate (AgNO3) for 15 min in
sodium phosphate buffer (pH 6.5 to 7.0), followed by adding 6-12 equivalents of sodium
borohydride (NaBH4) to reduce the silver ions. The mixture is vortexed for one minute
and left in the dark for 12 hours.10,11,13,17 Interestingly, when a lower ratio of BH4- to Ag+
was used (e.g. 1:357 as compared to 1:14 or 2:17), a faster onset of fluorescence and a
larger maximum intensity were observed in the reactions.57 The faster onset of
fluorescence was coupled with reduced chemical lifetimes for some emitters, however.57
With fixed preparation conditions, the photophysical properties of as-synthesized
DNA/Ag NCs were found quite consistent.17 Other than DNA templates, Gwinn has
demonstrated the use of ssRNA to nucleate the growth of fluorescent silver clusters,
analogous to previously studied DNA/Ag NCs.59
While DNA/Ag NCs have demonstrated applications in single-molecule
microscopy,6,11 cellular imaging,20,21,51,52,60 molecular logic devices,61 metal ion

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sensing,62,63 and protein detection,15,64,65 the most significant advance and impact were
made in DNA detection by the introduction of NanoCluster Beacons (NCBs), where silver
nanoclusters were proved superior to organic dye or quantum dot alternatives.11,13,17 NCBs
are new molecular probes that fluoresce upon hybridization with DNA targets but do not
use Frster resonance energy transfer (FRET) as the fluorescence on/off switching
mechanism, which is detailed below.

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3. Activatable and color-switchable silver nanocluster probes

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Among all fluorescent probes developed to detect and image biomolecules or


metabolites, activatable and color-switchable probes that enable detection without
separation are particularly desirable, especially for intracellular studies, where removal
of unbound probes is difficult. Since the activatable probes only turn on in specific
environments but otherwise remain undetectable, they often achieve high signal-tobackground ratios in target detection. For in vitro studies, such as microarrays,
activatable probes eliminate the wash steps, greatly simplifying the process. Until now, a
number of non-enzymatic, activation strategies have been used to develop so-called
separation-free probes or turn-on probe, including FRET,66-70 intercalating dyes,71,72
electron transfer-based probes,73,74 environment-sensitive Aequorea fluorescent
proteins,75 split fluorescent proteins,76-78 biarsenicical-tetracysteine systems,79,80 aptamer
systems,81-83 H-dimer formation,84,85 and lanthanide complexes.86,87 Similarly, a number
of fluorescence color-switching methods (upon probe-target binding) have also been
demonstrated, such as excimer formation,88-92 carbon nanotube sensors,93-95 quencher
transfer/detachment,96,97 and FRET binary probes.98,99 Readers are recommended to refer
to recent reviews in fluorescent probe development by Kobayashi,1 Kolpashchikov,100
Vendrell,101 Kool,102 Li,103 Ranasinghe and Brown,104 and Ihara.105 In general, activation
mechanisms can be categorized as follows: (1) FRET, (2) charge transfer, (3) constraint
on molecular movement (e.g. intercalating dyes and some aptamer systems), and (4)
combinatorial assembly of fluorescent complex (e.g. split fluorescence protein). As
mentioned above, for fluorescent nanoclusters, other than clusters stoichiometry, charge,
and geometry, encapsulation ligands can also have a strong influence on the fluorescence
properties of DNA/Ag NCs (through ligand-cluster binding or just ligand orientation
change36). In other words, DNA/Ag NCs can possess environmental sensitivities not
commonly seen among existing fluorophores. Extended from their special environmental
sensitivities, a whole new type of fluorescence activation mechanisms could exist for
DNA/Ag NCs. While activatable and multi-color probes have enabled many detection
schemes, researchers still need more activatable and multi-color probes in order to push
fundamental biology research and medical diagnostic imaging to the next level.
However, the development of new activatable and multi-color probes is often hindered
by the use of the known, but limited, fluorescence activation mechanisms of existing
fluorophores. Nanomaterials that communicate with their surroundings and
transduce specific responses into unique emission spectra therefore hold great promise
to enable new devices for diagnostics, therapeutics, environmental sensing, and bioimaging.

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3.1 Current activatable and color-switching probes for biological detection

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3.2 Activatable silver nanocluster probes for DNA detection NanoCluster Beacons
3.2.1 Background of DNA detection

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To overcome the limitations of FRET and to gain better photostability, many groups
are working on homogenous detection methods that use nanomaterials for sensing DNA
hybridization, including gold nanoparticles117-119 and carbon nanotubes.93,94 As an
emerging class of nanomaterial fluorophores, DNA/Ag NCs have also attracted attention
as a new probe for homogenous detection of DNA hybridization, with recent results
showing the capability to differentiate DNA targets with only single-nucleotide
differences.17,30,120

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Nucleic acid detection is an enabling technology for a number of fields, including


pathogen identification,106 the recognition of genetic mutations,107 and forensic
analysis.108 While identification of specific, low-abundance DNA sequences is the goal
for DNA detection, precise quantification of the amount of nucleic acids present in the
samples can be just as important in many applications. For instance, deletions of tumorsuppressor genes and copy number increases of oncogenes were found in many forms of
cancer.109 The amount of circulating DNA in plasma can discriminate between cancer
patients and healthy individuals and is related to disease progression.110 The number of
specific alleles found in the maternal circulating DNA reflects the haplotype of fetus
genome, therefore serving as indicators for possible inherited diseases.111 A number of
methods have been developed for the detection and quantification of specific DNA, such
as real-time quantitative polymerase chain reaction (qPCR),112 DNA sequencing,111,113
and DNA microarrays.114 As mentioned above, microarrays, as a surface immobilization
technique, require multiple wash steps and can only be run at central laboratories. In
contrast to DNA microarrays, specific DNA can also be directly detected in solution
using aforementioned turn-on probes (often termed homogeneous detection), eliminating
the wash steps and facilitating the clinical use. One widely used turn-on probe for the
homogeneous detection of DNA is the molecular beacon (MB),66 which is a hairpinshaped oligonucleotide labeled with a fluorophore and a quencher at the two ends,
forming a donor-acceptor FRET pair. While the FRET-based probes and methods have
been successfully commercialized, they have drawbacks. In particular, FRET probes are
expensive since they require costly processes to attach two moieties to the same DNA
strand. In addition, FRET probes that contain either the fluorophore or the quencher (socalled singly labeled impurities) contribute to a high fluorescence background or a
reduced signal. Moreover, a sophisticated thermodynamic analysis is often needed for the
stem-loop sequence selection. While FRET probes can provide better signal-tobackground (S/B) ratios in detection (as compared to singly labeled probe115), their S/B
ratios are also bound by quenching (either Frster or contact quenching) efficiency,
which is never 100%.116

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A BC
In absence of target
NCB alone

Dark Ag NC

Braf 1:1
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NC-bearing
strand

Dark Ag NC

Enhancer

5 m

NC-nucleation sequence
In presence of target
NCB binds a target

Bright red Ag NC

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NC probe

G-rich enhancer

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strand

no target

Enhancer probe

5 m

Target DNA

Figure 2. Schematic showing the guanine-proximity-induced fluorescence activation


phenomenon of DNA-templated silver nanoclusters and the associated DNA detection
probe, NanoCluster Beacon (NCB). (A) Guanine proximity can dramatically increase the
red fluorescence emission of DNA/Ag NCs. A non-emissive silver cluster is first
prepared on a NC-nucleation sequence. Once a guanine-rich enhancer sequence is
brought close to the silver cluster through hybridization, a strong red fluorescence
emission is observed from the solution, with a bulk enhancement ratio greater than 500
fold. Photographs of the resulting emission are taken under UV 365 nm light.
Reproduced from ref. 11 with the permission from American Chemical Society. (B)
NanoCluster Beacon (NCB) detection scheme. An NCB is composed of an NC probe
(carrying a non-emissive silver cluster) and an enhancer probe (having an enhancer
sequence). NCBs light up in the presence of DNA targets, but remain dark in the absence
of targets. Reproduced from ref. 13 with the permission from IEEE. (C) Fluorescence
images of individual NCBs from samples with (upper) and without (lower) DNA targets.
NCBs were non-specifically adsorbed to a coverslip and images were taken with a total
internal reflection fluorescence microscope. Reprinted from ref. 11 with the permission
from American Chemical Society.
3.2.2 NanoCluster Beacons for DNA detection

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While DNA/Ag NCs are promising as small and photostable fluorescent labels,
DNA/Ag NCs can sometimes change color5,10,121,122 a dynamic conversion process
neither well understood nor generally shared by existing fluorophores. While these
conversions can be viewed as a drawback, in the appropriate context, they can be used as
new signal transduction modes for molecular sensing.11 Often reversible, transformations
among silver cluster species (which have distinct emission colors) can depend upon a
number of factors, including time, temperature, oxygen and salt content.5,10,121,122
Other than these factors, Yeh et al. recently found guanine proximity can also
trigger reversible transformations among different silver cluster species.11 As shown in
Figure 2A, dark silver clusters can be transformed into bright red-emitting clusters when
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placed in proximity to guanine bases. Silver clusters were first prepared on an NCbearing strand having a cytosine-rich nucleation sequence (called NC-nucleation
sequence). The resulting reaction solution had weak green fluorescence emission.11 Upon
hybridization with a complementary enhancer strand having a guanine-rich tail (called
enhancer sequence), a strong red fluorescence emission was observed from the solution,
with a bulk enhancement greater than 500 fold (Figure 2A). The reversibility of the
fluorescence enhancement was demonstrated using a real-time PCR thermal cycler,
which cycled the temperature between 95C (no fluorescence) and 25C (strong red
fluorescence), and was further verified in binding competition experiments.11 When
varying the number of guanine bases in proximity to silver clusters, the extent of red
fluorescence enhancement was found to exponentially increase with an increasing
number of guanine bases. Interestingly, when brought close to the same silver clusters, a
polythymine enhancer was found able to enhance the green fluorescence of the silver
clusters by ~ 16 fold while a polycytosine enhancer could induce an irreversible cluster
transfer and generated moderate red fluorescence enhancement.11
Taking advantage of this guanine-proximity-induced fluorescence activation
phenomenon, a new turn-on probe, termed a NanoCluster Beacon (NCB), was designed
for DNA detection. As illustrated in Figure 2B, an NCB consisted of two linear probes,
one bearing a non-emissive silver cluster (i.e. the NC probe) and the other having a
guanine-rich tail (i.e. the enhancer probe). The two probes were designed to bind in
juxtaposition to a target DNA, a typical form of binary probes.100 Such a juxtaposition
binding enabled the enhancer to interact with the silver cluster, transforming the cluster
from a non-emissive species to a highly fluorescent species. Fluorescence occurred only
when a specific DNA target was present in the sample. Not relying on FRET as the
fluorescence activation mechanism, one NCB design showed a detection S/B ratio (175)
five times better than that of a conventional molecular beacon (32) on the same target.
Single-molecule NCB imaging taken on a total internal reflection fluorescence (TIRF)
microscope showed a significant increase in the number of red emitting NCBs with target
(Figure 2C), as compared to the sample without target. The single-molecule imaging
results demonstrated that NCBs could be used for both quantitative ensemble
measurements and single-molecule-based digital analysis of specific DNA targets.

A B C
Change NC-nucleation
sequence

Change enhancer
sequence

Change alignment
between sequences

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Figure 3. Three different strategies to tune the emission color of NanoCluster Beacons.
(A) Using the same enhancer sequence (lower strands) but different NC-nucleation
sequences (upper strands).13 (B) Using the same NC-nucleation sequence (upper strands)

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but different enhancer sequences (lower strands).11,13 (C) Using the same enhancer and
NC-nucleation sequences, but changing the alignment between sequences (by adding or
deleting nucleotides at the end of the hybridization sequences).17

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In another report by Yeh et al.,13 multi-color NCBs were achieved using (1) the
same enhancer sequence but different NC-nucleation sequences (Figure 3A) or using (2)
the same NC-nucleation sequence but different enhancer sequences (Figure 3B). This
important feature, having multiple activation colors from the same origin, is not
commonly shared by existing fluorophores, opening opportunities for NCBs in
multiplexed assays.
The advantages of NCBs lie in their simple, low-cost, one-step preparation process
(i.e. silver cluster nucleation process on the NC probe) and their potential to achieve an
ultrahigh S/B ratio in homogenous detection. There is no need to remove excess silver
ions or borohydride ions from the solution after cluster formation is completed, as these
cluster precursors are essentially non-fluorescent. Unlike conventional MBs, the
fluorescence background of NCBs is not limited by Frster quenching, but rather by the
existence of sparse NCBs that are red emitting in the absence of target.11 Without taking
any special steps to reduce the background emission (such as purification or prephotobleaching123), NCBs have shown an S/B ratio five times better than that of a
molecule beacon. While the initial work by Yeh et al. used NCB for DNA detection,
other groups have modified NCBs to detect proteins64 and small molecules.124
The physical mechanism driving the increase in red fluorescence emission is not
well understood at this moment. One possibility is that the red fluorescence emission is
due to electron transfer from the guanine bases to the silver clusters. Guanine has the
lowest oxidation potential among the four nucleobases (i.e. the best electron donor)125
and can often alter the emission rates of organic dyes.74,125-130 In the fluorescence
activation of NCBs, guanines may serve as electron donors, reducing oxidized-NC
species (in this case, non-emissive clusters) into bright red-emitting clusters. To further
test this electron-transfer hypothesis, Yeh et al. performed an experiment where the
guanines on an enhancer were replaced by 7-deazaguanines, which are stronger electron
donors than guanines.131 Surprisingly, no red fluorescence emission was seen after
hybridization, weakening the electron transfer hypothesis. Another experimental result in
the report that weakened the electron-transfer hypothesis is that thymine-proximity
produced a green fluorescence enhancement, with thymine being a worse electron donor
than adenine (adenine proximity did not generate any measurable fluorescence
enhancement).11
3.2.3 Background of SNP detection

40

Single-nucleotide polymorphisms (SNPs), the most common genetic variations


between individuals, often serve as biological markers to pinpoint diseases on the human
genome map and to understand a person's potential response to a drug therapy. The
ability to tailor drug treatment for individuals in order to avoid adverse side effects,
referred to as personalized medicine,132 would not only achieve the largest therapeutic
effect, but also allow pharmaceutical companies to bring many more drugs to market.
While future medicine clearly requires rapid, cheap, and reliable methods to identify
[journal], [year], [vol], 0000 | 9
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SNPs in patients genomes, the development of new SNP probes is often challenging.133
Currently, it is difficult to discriminate all four SNP variants using a single turn-on probe.
Ideally, one would like to have a multi-color SNP probe that is non-fluorescent in
View Article Online
solution, but displays four different fluorescent colors when interacting with the four DOI: 10.1039/C3NR01601C
allelic variants (G, C, A, T) at a polymorphic site. In other words, an ideal SNP probe can
quantitate the amount of DNA target using fluorescence intensity and identify four SNP
variants with unique fluorescence colors. However, such an ideal multi-color SNP probe
does not exist today.
SNP detection is mainly carried out in central laboratories. Current methods for
SNP detection (e.g. genotyping of known SNPs) typically require enzymatic reactions
such as primer extension, ligation, and cleavage.133-136 The major advantage of enzymatic
methods is the high fidelity of the base sequence recognition. While four-color SNP
typing using single-base extension and four differently fluorescently labeled
dideoxynucleotides (ddNTPs) has been demonstrated and commercialized,137,138 the
requirements of primer extension and fluorescent ddNTPs add to the assay time and cost.
Other methods employ even more enzymatic reaction steps for SNP typing (such as
molecular inversion probes, where totally four different enzymes are needed for SNP
identification.139 To reduce the cost and assay time, several non-enzymatic SNP-typing
methods (methods that do not use enzymes except for PCR amplification) have been
developed. Allele-specific hybridization is the most common method in non-enzymatic
SNP typing, where allele-specific oligonucleotides (ASO) are designed to hybridize with
polymorphic target alleles. ASO binds preferably to the fully matched allele rather than
the single-base mismatched alleles. Therefore, the allelic discrimination is based on the
differences in thermodynamic stability of the ASO/target duplexes, which are often
characterized by the melting temperatures of duplexes. However, a single-base mismatch
only generates a small difference in duplex stability, making it difficult to choose optimal
hybridization conditions that best discriminate the fully matched allele from the singlebase mismatched alleles. Allele-specific hybridization has been implemented in two
different detection platforms on a solid surface (e.g. DNA microarrays140) and in
homogeneous solution (e.g. using molecular beacons141,142). While the array platform is
suitable for large-scale SNP typing, it is far more challenging to set up a single
hybridization and washing condition that is applicable for all ASOs used on the
microarray.133 Additionally, sophisticated ASO design algorithms are often required. As
a result, thermodynamic discrimination is not likely to be a reliable basis for large-scale
and multiplexed SNP typing.133
To circumvent the issues of allele-specific hybridization, new SNP-typing methods
are currently being developed that do not rely on the differences in thermodynamic
stability for discrimination. For instance, SNPs have been detected by charge transport
through the -stack of DNA duplexes,143,144 base-discriminating fluorescent
nucleosides145,146, kinetic schemes,147-152 and mismatch-binding ligands.153-155 While
overcoming the issues of small differences in thermodynamic stability, these methods
produce so-called on/off probes that can only differentiate one allelic variant (probe
signal on) from all other variants (probe signal off) at a given polymorphic site.
Moreover, the signal level (either optical or electrical) of the absence-of-allele
condition (i.e. no target DNA) is also off, similar to the signal level of mismatched
alleles, making SNP scoring ambiguous. As mentioned above, ideally one would like to
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have a probe that not only can differentiate all four allelic variants at once, but also can
indicate the amount of DNA target present in the sample (as could be used in digital
PCR156). To fully identify the SNP type at a specific polymorphic site, one would need
four single-color, on/off probes but only one multi-color, on/off probe. Consequently, the
oligonucleotide synthesis and handling cost is much lower for the multi-color probe SNP
assays. While researchers continue to develop various kinds of non-enzymatic methods
that do not rely on thermodynamic stability for SNP typing, less effort is put into the
development of multi-color SNP probes due to challenges in chemistry. DNA/Ag NCs,
which possess many interesting and unique environmental sensitivities, have been
explored to create multi-color probes for SNP detection, as we detail below.

View Article Online

DOI: 10.1039/C3NR01601C

AB
Position

0 1 2 3

A
T

T
A

C
A

C
T

C
A

C
G

T
G

A
G

A
T

T
G

T
G

C
G

5
5'
G

T
A

6
C
T

A
T

7
C
G

3
5

3
5

A
T

T
A

A
G

T
G

C
G

C
T

C
G

C
G

T
G

A
G

A
T

T
G

T
G

C
G

C
G

C
T

5'
G

G 3'

A
G

T
G

A
G

T
T

C
G

C
G

C
G

C
G

T
T

A
G

A
G

T
G

T
G

C
T

C
G

C 5'
G G

G 3'

..
+2

+4 5

G 3'

Upper strand: a common NC probe which carries Ag NCs. Lower strand: individual enhancer probe.
Gray: Hybridization sequences. Blue: NC-nucleation sequence. Red: Enhancer sequence.

C
0
-1
20

25

30

35

+3

+4

305nmexcitation

+5
+6

-2

+7

-3

control

ESGM(nm)

+1 +2

365nmexcitation

700
660
620
580

Figure 4. The effect of repositioning of the enhancer sequence with respect to the NCnucleation sequence. A common NC probe is hybridized with one of the eleven enhancer
probes, which differ by advancement of the enhancer sequence along the NC-nucleation
sequence one nucleotide at a time. The resulting eleven alignment-shifting samples are
named position -3 to position +7. (A) Schematic showing three alignment states
(positions -3, +2, and +4) between the enhancer sequence (red fill) and the NCnucleation sequence (blue fill). A cartoon of silver clusters is drawn, showing a red turnon color for positions -3 and +4 and a yellow/orange turn-on color for position +2. (B)
Emission spectra of the eleven samples under 365 nm excitation. (C) Color photo of the
eleven alignment-shifting samples (position -3 to +7) and the control sample (NC probe
without a hybridization partner) under UV 365 nm light. The colors of positions +1, +2,
and +3 are clearly blue shifted. (D) Emission-spectrum-global-maximum (ESGM, which
sets a simple criterion for color-switching probe design) of the eleven samples. A change
greater than 70 nm in ESGM is observed upon a two-nucleotide frame shift alignment
from position -1 to +1. Reproduced from ref. 17 with the permission from American
Chemical Society.

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3.2.4 Chameleon NanoCluster Beacons for SNP detection

B
Sample A
(position -1)
Mutant-type target

25

5
3

T G
A C

NC
probe

30

35

T
A
G
A
G
C
C
C
C
T
A
A
T
T
C
C
C
5'

Sample B
(position +1)
Wild-type target

T G G
3 5 T G
A C C
5 3 A C
A
C
G
T
A
C
G
T
Enhancer
C
G
C
G
probe
C
G
C
T
T
G
A
G
A
G
T
G
T
T
C
G
C
G
C
G
5'
G
T
G
G
G
the nanocluster
G
3' arm

G
C
T
C
T
G
G
G
T
G
G
G
G
T
G
G
G
G
T
G
G
G
G
3'

C
D
E
GGT GAT GCT
None
GTT
+1
1
B

3
5

T G
A C

0.6

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DOI: 10.1039/C3NR01601C

cNCBwildtypetargetcomplex
cNCBmutanttargetcomplex

0.55

0.5

0.45
30 40 50 60 70 80 90 100
Temperature(degC)

E
A_GTT_"1"
B_GGT_"+1"
C_GAT
D_GCT

0.5

0
450 500 550 600 650 700 750 800
Wavelength(nm)

16 M

16 M

GTT target

GGT target

8 M
4 M
2 M

8 M
4 M
2 M

Figure 5. Schematic and results of chameleon NanoCluster Beacon (cNCB) SNP typing.
The SNP under investigation is GGT (wild-type) to GTT (mutant-type) mutation on Kras
oncogene codon number 12.157-159 (A) Schematic showing how different alignment states
between the NC-nucleation sequence (blue fill) and the enhancer sequence (red fill) can
be generated by mixing cNCB with different targets. As illustrated here, position -1
(sample A) is generated by mixing cNCB with Kras mutant-type target. However, if the
thymine base on codon 12 (shown in red letter) is replaced by a guanine base, as shown
on the wild-type target in sample B, frame-shift basepairing will lead to an alignment
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Absorbance (A260 nm)

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Intensity (a.u.)

Yeh et al. recently demonstrated an entirely new phenomenon the activation color
(or the turn-on color) of the silver cluster in an NCB can change substantially depending
upon its position relative to an enhancer sequence (Figure 3C).17 They exploited this new
property for the specific detection and identification of a number of disease-related SNPs,
terming the resulting multi-color SNP probes chameleon NanoCluster Beacons
(cNCBs).17 As shown in Figure 4A, eleven alignment-shifting samples (named position
-3 to position +7) were created by sliding the enhancer sequence along the NCnucleation sequence one nucleotide at a time. Due to the common UV excitation peak of
all DNA/Ag NCs,4,16 differences in the emission color could be clearly visualized among
the eleven alignment-shifting samples under UV 365 nm excitation (Figure 4C). The
color variation is more clearly seen in Figure 4B, where the emission spectra are plotted.
Emission-spectrum-global-maximum (ESGM, defined as the wavelength where the
emission intensity is the highest) was used as a gross indicator of color for each sample
(Figure 4D). Without knowing the exact separation distance between the silver cluster
and the enhancer sequence in each alignment, it was found subtle repositioning of the
enhancer sequence relative to the NC-nucleation sequence (e.g. from position -1 to 0 or
from position +3 to +4) can shift the ESGM by as much as 60~70 nm (Figure 4D). The
ability to obtain measurably different ensemble activation colors using the same enhancer
sequence shifted only by a single nucleotide is unique to NCBs. It is an environmental
sensitivity not seen in existing fluorophores that opens the door to the creation of a new
type of multi-color probe that can sense small variations in DNA sequences.17

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state of position +1 after beacon-target hybridization. (B) Color photo of the cNCB
mixing with all four SNP targets (A_GTT, B_GGT, C_GAT, and D_GCT) and the notarget control (E, having only cNCB) under 365 nm light. Sample A (position -1) was
clearly the most red of the four, as predicted from Figure 4D. (C) Emission spectra of the
cNCB mixing with the four SNP targets. These four samples resulted in three
distinguishable emission spectra. (D) The melting curves showing that cNCB binds both
wild-type and mutant-type targets with nearly equal thermodynamic stability. (E) The
fluorescence intensity of cNCB can be used to quantify the amount of target (as shown
here 2~16 M), whereas fluorescence color (represented by the ESGM) can be used to
identify the SNP type of the target (as shown here G to T mutation). Reproduced from
ref. 17 with the permission from American Chemical Society.
Yeh et al. pointed out that a short-range interaction, potentially direct contact,
between the enhancer bases and the silver cluster determined the emission color of silver
cluster.17 The alignment-change-induced ESGM shifts were used to directly and
quantitatively identify SNPs. As shown in Figure 5A, the SNP probes, chameleon
NanoCluster Beacons (cNCBs), were based on a three-way-junction (3WJ) design,130
where a single-nucleotide substitution on the DNA target (positioned at the branch point
of the 3WJ) caused frame-shift basepairing in the nanocluster arm. This frame-shift
basepairing moved the enhancer sequence relative to the NC-nucleation sequence by two
nucleotides, resulting in a different turn-on color. In Figure 5A, two alignment states
(position -1 and position +1) were generated by mixing a cNCB with the Kras mutanttype target (sample A) and the wild-type target (sample B). As previously determined
(Figure 4D), the turn-on color for position +1 sample (wild-type target) should appear
more yellow/orange as compared to the color for position -1 sample (mutant-type target).
As predicted, sample B (wild-type, position +1) did appear more orange than sample A
(mutant-type, position -1) (Figure 5B). The change of fluorescence color could be
observed both spectroscopically and by the naked eye.17
When mixing with all four allelic variants (A, C, G, and T), cNCB displayed three
distinguishable and reproducible emission spectra (Figure 5C). Yeh et al. have
previously used a three-way-junction design, combined with monitoring Cy5 blinking
dynamics by fluorescence correlation spectroscopy, for single-nucleotide variation
detection.130 Using the blinking time as an indicator, Yeh et al. showed that the four
allelic variants gave three distinguishable blinking times,130 which agreed well with the
three distinguishable emission spectra that were obtained using the cNCB detection
method.17 The Cy5 blinking detection method, however, requires advanced detection
tools to measure the change of Cy5 blinking dynamics at microsecond timescale. While a
distinguishable color for each allelic variant is the ultimate goal, cNCBs ability to
differentiate the four allelic variants into three groups surpasses the state-of-the-art in
non-enzymatic SNP detection (current methods only differentiate one matched target
from three mismatched targets, therefore only two differentiable results from the four
allelic variants120,140-146,151,158,160-165). Further, in contrast to currently employed SNP
probes, cNCBs, as a whole, bind both wild-type and mutant-type targets with nearly
equal thermodynamic stability (Figure 5D). Rather, it is the environmental sensitivity of
DNA/Ag NCs that enables SNP discrimination. Another attractive feature of cNCBs lies
in their capability for a two-dimensional analysis (Figure 5E) the fluorescence intensity

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can be used to quantify the amount of target (cNCB is a turn-on probe), whereas
fluorescence color (represented by the ESGM) can be used to identify the SNP type of
the target (cNCB is also a multi-color probe). Again, this feature is not shared by existing
SNP probes, where events without a target cannot be easily differentiated from events
with a mismatched target (both give a low signal; in other words, both are off). In
terms of general applicability, cNCBs have successfully been validated on three SNPs
with all four allelic variants, and on six SNPs with two allelic variants (which covered all
six possible single-nucleotide substitution scenarios). Sequences around the SNP sites
were not particularly chosen and all alleles studied produced effective cNCBs.17
As mentioned above, the ability to obtain measurably different ensemble activation
colors using the same activator (e.g. the enhancer) that is physically shifted by only a
small distance (e.g. distance of a single nucleotide) is unique to cNCBs. It is this
environmental sensitivity, not seen in existing fluorophores, that lays the foundation for
multi-color probe development using DNA/Ag NCs. New probes that fluoresce when
recognizing target molecules but fluoresce differently when sensing subtle differences in
target molecules would enable numerous applications in biosensing in the near future.

B
Quencher

NC-nucleation
sequence

Recognition site

20

Mutant type Mismatch

C C
C
C
C C

A
A

Target

Wild type Match

C C
C
C
C C
Quencher

25

30

A
T

Figure 6. (A) Schematic of a quencher-mediated turn-on probe. After the quencher


(black) is displaced by a target (red), fluorescence is enhanced by a proportional increase
in the number of emissive clusters in the sample.166 (B) Schematic of hybridized DNA
structures with a six-cytosine loop as nucleation site for silver cluster synthesis and SNP
differentiation. No fluorescence was observed for the mutant-type duplex after the
nucleation process. However, the wild-type duplex produced strong yellow fluorescence
emission.120

4. Other DNA probes based on fluorescent silver nanoclusters


4.1 Detection of DNA targets

35

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DOI: 10.1039/C3NR01601C

Petty reported a turn-on nanocluster probe with silver clusters initially quenched via
hybridization with a quencher strand. As displayed in Figure 6A, the DNA target binds to
the NC probe through a competitive process known as toehold displacement.166 This
method eliminates the need of a guanine-rich enhancer to turn on fluorescence, but yields
a mere 2-3 fold enhancement of fluorescence upon target binding. Petty and Dickson
have previously characterized a near-infrared-emitting silver cluster species (emission
maxima centered around 770nm) composed of 10 silver atoms per DNA template strand
14 | [journal], [year], [vol], 0000
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Nanoscale Accepted Manuscript

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(5-C3AC3AC3GC3A).167 This stoichiometry was determined via inductively coupled


plasma atomic emission spectroscopy (ICP-AES). Petty found that fluorescence was
enhanced through a proportional increase in the number of emissive clusters,166 similar to
the NCB results.11 The proposed on/off switching mechanism involves the invasion of
the quencher strand into the 3' region of the NC-nucleation sequence, thus changing the
electronic environment around the silver cluster and resulting in a fluorescence
quenching effect. The key advantage of this method lies in the emitters near-infrared
electronic transition, which enables DNA detection in serum-containing buffers (whose
endogenous background fluorescence is low in the near-infrared spectral region).166 It has
yet to be determined if the detection scheme displayed in Figure 6A is applicable to
longer DNA targets. Continued work by Petty has evolved the turn-on probe into a new
form that no longer requires a quencher strand168 (similar results were also achieved by
another group169). Petty's new probe relies on silver clusters to condense the template,
thereby creating an environment favoring the non-active state of the silver clusters.
Introduction of DNA targets and subsequent hybridization alter the cluster environment
to restore fluorescence of the silver clusters. Interestingly, Petty found the probe/target
duplexes formed dimers, hosting twice the amount of silver atoms in a dimer. A similar
observation of the self-dimer formation was reported by Yang and Vosch in their turn-off
probe design.170 It is suggested that the dimer formation of the silver cluster probes is
responsible for the creation and stabilization of highly emissive silver clusters.170 A
possible mechanism for dimer formation is the non-Watson-Crick basepairing mediated
by silver ions,122 which links cytosine homopolymers via cytosine-Ag+-cytosine
interactions.58
As an effective universal quencher that can initially switch off the fluorescence of
reporters in turn-on detection, graphene oxide (GO) has been recently explored for DNA
detection.171,172 GO can stably adsorb single-stranded DNA through -stacking
interactions between the hexagonal cells of the graphene and the ring structures in the
nucleobases. Upon adsorption, the reporters labeled on DNA are quenched by GO. In the
presence of DNA targets, the adsorbed probes hybridize with the targets, forming rigid
duplexes which are then released from the GO due to conformational changes.172 This
releasing mechanism reverses the quenching effect and restores the fluorescence. In a
recent study by Ren, DNA/Ag NCs were used as reporters in a GO-based sensor for
multiplexed detection of DNA,173 demonstrating the compatibility between DNA/Ag
NCs and fullerenes.
4.2. Detection of SNPs
Several methods other than cNCBs have been proposed to differentiate SNPs using
DNA/Ag NCs as indicators. For instance, Wang was able to identify the SNPs
responsible for sickle cell anemia using a DNA probe with a six-cytosine, NC-nucleation
loop.120 As show in Figure 6B, the probe was first hybridized with the wild-type and the
mutant-type targets (T to A substitution), forming two different duplexes. After NCnucleation process was completed on these duplexes, little fluorescence was observed
from the mutant-type duplex, whereas the wild-type duplex produced a strong yellow
fluorescence emission. Despite this success, this method differentiates single-nucleotide
variations by the level of fluorescence intensity, rather than the spectrum of emission, as
is the case with cNCBs. Additionally, the method requires silver clusters to be

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synthesized after the probe/target hybridization is completed, leading to a longer assay
time. Lastly, it has yet to be shown that the method is applicable to a wide variety of
target sequences with similar discriminatory results.

10

Shao demonstrated that an abasic site in the DNA duplex can serve as a nucleation
site for the synthesis of emissive silver clusters and used this property to differentiate
single-nucleotide variants.174 The nucleobases opposite and next to the abasic site (a
tetrahydrofuran residual) control the formation of emissive silver clusters. Upon
completion of NC-nucleation process, the cytosine-facing abasic site produced a strong
fluorescence emission, whereas the non-cytosine-facing abasic sites produced little
fluorescence. While this method can differentiate CA, CG, and CT mutations, it
is not generally applicable to any other type of substitutions.

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Fluorescent silver nanocluster probes have also been used for RNA detection. Yang
and Vosch developed a microRNA detection probe using fluorescence quenching of
silver clusters as the basis of detection (i.e. a turn-off probe).170,175 MicroRNA (miRNA)
are small non-coding ribonucleic acids that can regulate the genes associated with cancer,
neurological diseases and viral infections. The fluorescence of the DNA/Ag NC probe
was quenched when it hybridized with a complementary miRNA target. Fluorescence
quenching was shown to follow a linear Stern-Volmer relationship over a range of target
concentrations (from 20 nM to 1.5 M), enabling quantitative detection of target
miRNA. Also for miRNA detection, Ye used DNA/Ag NCs as fluorescent indicators for
the amplicons in an isothermal amplification process.176 In their approach, the amplicons
are single-stranded DNA that can template the growth of highly emissive silver clusters.
Fluorescence measurements are not the only means by which DNA/Ag NCs have
been used for the detection of miRNA; electrochemical methods have also been used.
Zhang developed a simple miRNA sensor using DNA/Ag NCs to catalyze the reduction
of hydrogen peroxide.177 In their method, thiol-functionalized DNA hairpin probes were
immobilized on the surface of a gold electrode, with these hairpin probes having binding
sites for both miRNA targets and NC-nucleation probes. Upon sequential binding of
miRNA and NC-nucleation probes (which carry silver clusters), a large number of silver
clusters were brought close to the electrodes surface, enabling catalytic reduction of
hydrogen peroxide. The detected current was found directly proportional to the logarithm
of the miRNA target concentration.

5. Outlook
35

40

Fluorescent nanoclusters are interesting fluorophores that are currently being


explored by many groups around the world. Here we outline some future directions in
this exciting research area. While researchers are eager to find more real-world use of
fluorescent nanoclusters, basic questions remain and great efforts are needed to address
these challenges, which will be discussed below.
5.1 Molecular rulers based on fluorescent silver nanoclusters
With proper design and characterization, it may be possible to turn environmentally
sensitive nanoclusters into a short-range, fluorescent distance reporter a whole new
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4.3. Detection of RNA targets

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Nanoscale

type of molecular ruler. Such a ruler will be complementary to the existing spectroscopic
rulers, such as energy transfer-based,178 nanometal surface energy transfer-based,179
electron transfer-based,180 switching dynamics-based,181 or plasmon coupling-based
rulers,182 and can be an economic solution to study biomolecules small conformational
changes due to various molecular interactions.

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Another potential application of fluorescent silver nanoclusters is in biological


barcodes as substitutes for conventional fluorophores. To specifically identify and
precisely quantify a large number of distinct molecular species present in complex
biological systems, several barcode strategies have been developed, including metallic
barcodes,183 polymer particle barcodes,184 barcode chips,185 sequencing-based
barcodes,186,187 direct-labelling barcodes,188,189 branched DNA-based barcodes,190 M13
DNA-based barcodes,191 and quantum dot-based barcode beads.192,193 In particular, the
M13 DNA-based barcodes have successfully been commercialized by NanoString
Technologies (the nCounter system) for mRNA identification and quantification at the
single-molecule level.191 While achieving excellent quantification results, M13 barcodes
are soft (a stretch step is required191) and expensive (dense labelling with organic dyes
and the required purification processes). To stiffen the DNA barcodes, Yin recently used
DNA origami techniques to construct structurally rigid DNA nanotubes that act as
geometrically encoded fluorescent barcodes.194 However, the costly fluorescence
labelling remains a challenge. Compared to organic dye labelling, fluorescent silver
clusters can also be assembled at specific sites with great specificity, but their labeling
cost will be significantly lower (the nucleation precursors are cheap and essentially nonfluorescent). Fygenson has started to construct DNA nanotubes with spatially distributed
hairpin protrusions for in-situ growth of fluorescent silver nanocluster.58 Although
currently suffering from aggregation issues due to cytosine-Ag+-cytosine interactions,
fluorescence programmable nanotubes have the potential to realize low-cost barcodes in
the near future.
5.3 In-situ or site-specific synthesis of fluorescent nanoclusters

30

35

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One challenge in the development of metal cluster-based biosensors is that we dont


know exactly where the clusters are templated on the encapsulation agents. This is
particularly a problem when we are designing nanocluster sensors to monitor their
surrounding microenvironments. Electrospray ionization mass spectrometry (ESIMS)5,7,57 and inductively coupled plasma atomic emission spectroscopy (ICP-AES)167
have revealed the number of metallic atoms on a template molecule, but they give no
information on the ligand-cluster binding geometry nor the location of metal cluster.
NMR spectroscopy, on the other hand, only indicates direct interactions between metal
clusters and surrounding ligands (e.g. interactions between silver and cytosines4). Several
different strategies have been recently reported to achieve in-situ or site-specific
synthesis of metal clusters on protein or DNA templates.195-197 For example, the
enzymatic active sites of ferritin (whose heavy chain and light chain form a 12 nm wide
nanocage) was used to direct the synthesis of gold clusters.195 Another example used
small sugar moieties linked to DNA at the 5 end as a specific nucleation site for silver
cluster synthesis via Tollens reaction.196 More recently, silver ions were reported to
interact specifically with the CG.C+ base triplet in a triplex DNA,197 leading to a method
[journal], [year], [vol], 0000 | 17
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Nanoscale Accepted Manuscript

5.2 Biological barcodes using fluorescent silver nanoclusters

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15

View Article Online

DOI: 10.1039/C3NR01601C

While we anticipate a broader impact of fluorescent nanoclusters in chemical and


biological sensing in the next few years, major advances in the in-situ synthesis of these
exciting nanomaterials and better characterization methods are desperately required in
order to optimize the cluster sensor design.
5.4 Mono-disperse fluorescent nanoclusters with desired properties

20

25

30

35

40

45

Another major challenge in metal cluster synthesis/characterization lies in a


reaction typically containing mixtures of many different fluorescent products having
various numbers of metal atoms in the clusters, precluding interrogation of cluster
species responsible for specific fluorescence emission. While the strong environmental
sensitivity of some metal clusters has raised great interests in sensing applications, the
origin and extent of this sensitivity remain obscure due to the difficulties of acquiring and
testing pure clusters with known ratios of metal atoms to template molecules. High
performance liquid chromatography (HPLC) is often used to purify the fluorescent metal
clusters, especially the DNA/Ag NCs.57,167,198 Using ICP-AES to analyze DNA/Ag NCs
separated by HPLC, Petty reported ten silver atoms encapsulated on a specific DNA
strand.167 ONeil also identified silver atom/DNA ratios through the correlation of
separately measured fluorescence and mass spectra.57 Most recently Gwinn used tandem
HPLC-mass spectrometry (HPLC-MS) with in-line absorbance and fluorescence
spectroscopy for cluster analysis and reported the silver atom/DNA ratios for several
purified fluorescent and dark species.198 Compared to ICP-AES, HPLC-MS can reveal
whether or not HPLC separation succeeds in isolating pure emitters. This is important
since distinct emitter species can have similar HPLC retention times.198 Even with
HPLC-MS, some cluster emitters still cannot be studied since they are not stable enough
to be purified (e.g. clusters that could easily convert into others due to oxidation, losing
their original emission signatures).
In the most recent review by Dickson,22 ESI-MS was described as a problematic
approach to study poly-disperse silver cluster populations and their associated optical
properties. Liquids are charged and aerosolized during electrospray ionization, followed
by solvent evaporation prior to mass spectral analysis.22 This ionization and evaporation
process leads to a problem where species that are most stable in the gas phase (measured
by mass spectrometry) may not necessarily be the most stable species in solution
(measured by fluorometry), even if a chemically pure cluster solution is used. As a result,
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of employing CG.C+ base triplet for site-specific synthesis of silver clusters on DNA.
While these reports showed TEM195-197 or AFM196 images as proof of site-specific cluster
synthesis, the images indicated the presence of crystalline nanoparticles (roughly 2 nm in
diameter), suggesting each metal particle consisted of hundreds of gold or silver atoms.
Nevertheless, highly fluorescent metal clusters should consist of less than 30
atoms,14,31,32,198 making it unlikely to obtain TEM or AFM images of highly emissive,
single few-atom metal clusters. Alternatively, Fygenson used DNA hairpins for the sitespecific synthesis of fluorescent silver clusters on long DNA nanotubes.58 While the
nucleation site was not controlled at the atomic level, the ability to limit silver cluster
growth to hairpins on DNA nanotubes provided a unique means of estimating the
chemical yield.58 By counting individual emitters along a nanotube with a known linear
density of hairpins under a fluorescence microscope, a chemical yield of ~45% was
obtained.

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Nanoscale

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5.5 Theoretical modelling, structure determination, and single-molecule


characterization of fluorescent nanoclusters
15

20

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Other than advances in synthesis and characterization, fundamental understanding


of metal clusters through quantum chemical calculations,36,199,200 size, conformation and
geometrical structure determination,6,8,12,54,201,202 and photophysical characterization at
the single-molecule level6,54,203 are also critically important for bringing the metal cluster
research to the next level. As pointed out by Jin,32 the fluorescence of metal clusters is
intriguing but not quite clear yet with respect to the origin of the fluorescence. A key to
understanding and predicting the properties of metal clusters lies in the elucidation of the
geometric structures of the metal clusters, which is necessary for the electronic structure
investigations. One example is the X-ray crystal structure determination of Au102(SR)44
[SR: thiolate ligands] which indicates that instead of simply passivating a large gold core,
thiolate ligands form Au(SR)2 and Au2(SR)3 motifs that bind to the gold core.202
Knowledge of these binding motifs led to theoretical predictions of the geometric
structure of Au25(SR)18, showing agreement with the X-ray crystal structure
determination.204 On the other hand, despite the great potential of silver clusters in
biomedical applications, we dont even know the geometric structure and optical
properties of even the simplest DNA/Ag NCs,199 not to mention the prediction of optical
absorption of DNA/Ag NCs. This is mainly due to a large number of possible ligandcluster binding geometries in DNA/Ag NC structures. The ligand orientation can have a
significant effect on the electronic structures and optical properties of metal clusters,36
but it is difficult to use density function theory (DFT) to simulate the DNA templates
used in the experiments (DNA strands are too large and solvent/ions have to be included
in calculations).199 Nevertheless, DFT calculations still can provide some qualitative
insights into the DNA/Ag NC systems and the observations from experiments. Recently,
Gwinn identified both the charge and the number of silver atoms contained in a cluster
using mass spectra of compositionally pure DNA/Ag NCs.54 The variation of charge with
the number of silver atoms, the strong polarization dependence of single emitters,
together with the evidence from theoretical calculations205 and cryogenic spectra of
single-molecule emitters, suggested that all chemically stable DNA/Ag NCs are rodshaped emitters.54 Gwinn also further suggested that any DNA strand that can stabilize a
silver nanorod with a given length and charge will produce similar emission colors, and
the fluorescence activation and color switch observed in NCBs may be due to changes in
rod length. Strand interactions may add or remove silver atoms from a nanorod or disrupt
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it is questionable to use gas-phase data to infer sizes of solution-phase emitters.22 In


addition, spectral purity in the emission spectrum does not necessarily mean chemical
purity, as synthetic methods yield multiple absorption bands associated with distinct
types of clusters.22,30,167 Thus, stoichiometry determinations of specific emissive species
remain technically challenging.22 Even though we are able to obtain metal atom/template
ratios for most of the purified emissive species, we still cannot claim that we know the
correlation between the cluster stoichiometry and the emission. For instance, having
eleven silver atoms on a DNA showing green fluorescence may not necessarily mean that
Ag11 is green, because some of the silver atoms on DNA may not even participate in the
visible emission process. It is challenging to make truly mono-disperse metal clusters
with desired, known properties at this moment. Precise control of metal clusters at the
atomic level is needed to enable such a predictive design of metal clusters.

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it into disjoint pieces. In their view, the significantly different affinities of silver ions to
the different nucleobases play a major role in determining lengths of the nanorods that
could form on different strands, and thereby the colors of DNA/Ag NCs. While more
characterization evidence is needed to valid this theory, it is exciting to see a possible
mechanism be proposed.

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In this review we have discussed the recent progress of fluorescent silver


nanoclusters and their applications in DNA detection. In particular, the activatable and
color-switchable properties of DNA-templated silver nanoclusters (DNA/Ag NCs) have
led to the invention of NanoCluster Beacons (NCBs). NanoCluster Beacons are
promising molecular probes with great potential, not only because their signal
transduction mechanism is fundamentally different from those of the existing molecular
probes, but because a tremendous amount of science is not yet understood and needs to
be explored. To bring the noble metal nanocluster research to the next level, researchers
need to simultaneously tackle a number of challenges, including mono-disperse and sitespecific synthesis, theoretical modelling, structure determination, and single-molecule
characterization of fluorescent nanoclusters. DNA/Ag NCs are excellent systems to
investigate size-dependent emission and understand unique photophysics in metal
clusters, while they hold great promise for cost-effective detection compatible with DNA
nanotechnologies.22 This review is driven by the humbling realization that a simple metal
cluster still holds profound questions and rich possibilities.

Acknowledgements

25

We thank Drs. James Werner and Jennifer Martinez at Los Alamos National
Laboratory for providing great assistance and advice in our research. We also thank the
group members R.A. Batson and M. Babin for proofreading. This work was financially
supported by Robert A. Welch Foundation (F-1833 to H.-C.Y.).

20 | [journal], [year], [vol], 0000


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Nanoscale Accepted Manuscript

6. Conclusion

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20

Judy M. Obliosca completed her PhD at National Tsing Hua


University under Taiwan International Graduate ProgramNanoScience and Technology (TIGP-NST) hosted by Academia
Sinica Research Institute in Taiwan in 2012. During her graduate
study, she worked on the synthesis and optical properties
characterization of noble metal nanostructures, carbon nanotubes
and graphene. In 2012, she joined Professor Tim Yehs group at
University of Texas at Austin as a Postdoctoral Associate. Her
current research is to develop new molecular probes based on
fluorescent noble metal nanoclusters for biomedical applications.
Cong Liu obtained his BS degree in Optical Engineering at
Zhejiang University, Hangzhou, China in 2012. As an
undergraduate research assistant under the supervision of
Professor Zhenghui Hu, he worked on hemodynamic modelbased fMRI BOLD signal analysis. In 2012, he joined Professor
Tim Yeh's group at University of Texas at Austin as a PhD
student in the Biomedical Engineering Department. His graduate
research
is
focused
on
fluorescence
nanomaterials
characterization and 3D single-molecule tracking.

25

40

Professor Tim Yeh (Hsin-Chih Yeh) obtained his BS degree


from National Taiwan University, MS degree from University of
California, Los Angeles, and PhD from Johns Hopkins
University, all in mechanical engineering. After graduation from
30 UCLA, he worked at Optical Micro Machines Inc. in San Diego
from 98-03 as an R&D engineer, developing MEMS-based
photonic switches for telecommunications. At Johns Hopkins, his
research focused on single-molecule spectroscopy, BioMEMS
and new biosensing techniques based on fluorescence dynamics.
35 Dr. Yeh received his postdoctoral training at Los Alamos
National Laboratory from 09-12, in the Center for Integrated
Nanotechnologies. At LANL, he discovered controlled fluorogenic and color-switching
phenomena on DNA-templated silver nanoclusters. Based on these findings, he and coworkers invented NanoCluster Beacons, which won a 2011 R&D 100 Award. Dr. Yeh
joined the Biomedical Engineering Department at the University of Texas at Austin in
2012 as an assistant professor. His research interests include nanobiosensor development,
cancer biomarker detection, 3D molecular tracking and super-resolution imaging.

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View Article Online

DOI: 10.1039/C3NR01601C

Table of Contents

Fluorescent silver nanoclusters as DNA probes

Department of Biomedical Engineering, Cockrell School of Engineering, University of Texas at


Austin, Austin, TX 78712, USA
* Author to whom correspondence should be addressed; E-mail: tim.yeh@austin.utexas.edu;
Tel.: +1-512-471-7931; Fax: +1-512-471-0616.
1. Colour graphic

2. Text
Fluorescent silver nanoclusters have been used to create a new class of DNA probes that are
not only activatable and programmable, but can also be excited by a single UV source.

Nanoscale Accepted Manuscript

Published on 06 June 2013. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.

Judy M. Obliosca, Cong Liu and Hsin-Chih Yeh*