Nanoscale
View Journal
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. Obliosca, C. Liu and H. Yeh,
Nanoscale, 2013, DOI: 10.1039/C3NR01601C.
This is an Accepted Manuscript, which has been through the RSC Publishing peer
review process and has been accepted for publication.
www.rsc.org/nanoscale
Nanoscale
Accepted Manuscripts are published online shortly after acceptance, which is prior
to technical editing, formatting and proof reading. This free service from RSC
Publishing allows authors to make their results available to the community, in
citable form, before publication of the edited article. This Accepted Manuscript will
be replaced by the edited and formatted Advance Article as soon as this is available.
To cite this manuscript please use its permanent Digital Object Identifier (DOI),
which is identical for all formats of publication.
More information about Accepted Manuscripts can be found in the
Information for Authors.
ISSN 2040-3364
Pages 1156
COVER ARTICLE
Graham et al.
Mixed metal nanoparticle assembly
and the effect on surface-enhanced
Raman scattering
REVIEW
Lin et al.
Progress of nanocrystalline growth
kinetics based on oriented attachment
2040-3364(2010)2:1;1-T
Please note that technical editing may introduce minor changes to the text and/or
graphics contained in the manuscript submitted by the author(s) which may alter
content, and that the standard Terms & Conditions and the ethical guidelines
that apply to the journal are still applicable. In no event shall the RSC be held
responsible for any errors or omissions in these Accepted Manuscript manuscripts or
any consequences arising from the use of any information contained in them.
www.rsc.org/nanoscale
Registered Charity Number 207890
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 1 of 33
REVIEW
Nanoscale
www.rsc.org/nanoscale | Nanoscale
DOI: 10.1039/C3NR01601C
1. Introduction
15
20
25
30
35
40
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.
10
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.
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
DOI: 10.1039/C3NR01601C
20
25
30
35
40
10
Page 2 of 33
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 3 of 33
Nanoscale
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.
DOI: 10.1039/C3NR01601C
+
10
15
20
25
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
30
35
40
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
NaBH4
Normalized intensity
ABCD
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
10
15
20
25
30
35
40
45
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
Page 4 of 33
DOI: 10.1039/C3NR01601C
Nanoscale
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 5 of 33
Nanoscale
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.
DOI: 10.1039/C3NR01601C
10
15
20
25
30
35
40
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
Page 6 of 33
3.2 Activatable silver nanocluster probes for DNA detection NanoCluster Beacons
3.2.1 Background of DNA detection
10
15
20
25
30
35
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
DOI: 10.1039/C3NR01601C
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 7 of 33
Nanoscale
A BC
In absence of target
NCB alone
Dark Ag NC
Braf 1:1
View Article Online
DOI: 10.1039/C3NR01601C
NC-bearing
strand
Dark Ag NC
Enhancer
5 m
NC-nucleation sequence
In presence of target
NCB binds a target
Bright red Ag NC
NC probe
G-rich enhancer
10
15
20
25
Enhancer
strand
no target
Enhancer probe
5 m
Target DNA
30
35
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
[journal], [year], [vol], 0000 | 7
This journal is The Royal Society of Chemistry [year]
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
10
15
20
25
30
Page 8 of 33
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
35
40
45
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)
DOI: 10.1039/C3NR01601C
Nanoscale
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 9 of 33
Nanoscale
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
DOI: 10.1039/C3NR01601C
10
15
20
25
30
35
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
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
10
15
20
25
30
35
40
45
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
10 | [journal], [year], [vol], 0000
This journal is The Royal Society of Chemistry [year]
Page 10 of 33
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 11 of 33
Nanoscale
10
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.
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.
15
..
-3
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
Page 12 of 33
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
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
12 | [journal], [year], [vol], 0000
This journal is The Royal Society of Chemistry [year]
20
15
Relativefluo.
10
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
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
10
15
20
25
30
35
40
45
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
DOI: 10.1039/C3NR01601C
Page 13 of 33
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
10
15
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
C C
C
C
C C
A
A
Target
C C
C
C
C C
Quencher
25
30
A
T
35
40
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
This journal is The Royal Society of Chemistry [year]
Page 14 of 33
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
10
15
20
25
30
35
40
45
DOI: 10.1039/C3NR01601C
Page 15 of 33
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
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.
Page 16 of 33
DOI: 10.1039/C3NR01601C
15
20
25
30
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
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 17 of 33
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.
DOI: 10.1039/C3NR01601C
10
15
20
25
30
35
40
45
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
15
DOI: 10.1039/C3NR01601C
20
25
30
35
40
45
10
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.
Page 18 of 33
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 19 of 33
Nanoscale
DOI: 10.1039/C3NR01601C
20
25
30
35
40
45
10
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
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.
Page 20 of 33
DOI: 10.1039/C3NR01601C
10
15
20
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.).
6. Conclusion
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
10
15
20
25
40
References
(1)
45
fluorescent probe design in medical diagnostic imaging Chem Rev, 2010, 110, 2620.
(2)
nanodot fluorescence Journal of the American Chemical Society, 2002, 124, 13982.
(3)
DOI: 10.1039/C3NR01601C
Page 21 of 33
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
(4)
Page 22 of 33
nanocluster formation using a cytosine oligonucleotide template J Phys Chem C, 2007, 111, 175.
5
(6)
DOI: 10.1039/C3NR01601C
dependent fluorescence of DNA-hosted silver nanoclusters Adv Mater, 2008, 20, 279.
(8)
Optically modulated fluorophores for selective fluorescence signal recovery Journal of the
American Chemical Society, 2009, 131, 4619.
(10)
probe that fluoresces upon hybridization Nano Letters, 2010, 10, 3106.
(12)
molecular probe for homogeneous detection of nucleic acid targets IEEE Nanotechnology Magazine,
2011, 5, 28.
(14)
K-edge EXAFS analysis of DNA-templated fluorescent silver nanoclusters: insight into the
structural origins of emission tuning by DNA sequence variations Journal of the American Chemical
30
In situ generation of intrinsically fluorescent recognition ligands for protein detection Chemical
communications, 2011, 47, 2294.
(16)
35
fluorescence via the DNA bases J Phys Chem C, 2011, 115, 24061.
(17)
Sharma, A. P. Shreve, J. S. Martinez, T. Goodson III, Bright two-photon emission and ultra-fast
relaxation dynamics in a DNA-templated nanocluster investigated by ultra-fast spectroscopy
45
(9)
15
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 23 of 33
Nanoscale
(21)
S. Choi, J. Yu, S. A. Patel, Y.-L. Tzeng, R. M. Dickson, Tailoring silver nanodots for
DOI: 10.1039/C3NR01601C
(25)
22, 1078.
(26)
10
application in fuel cells and analytical sensors Nano Today, 2011, 6, 240.
B. Han, E. Wang, DNA-templated fluorescent silver nanoclusters Analytical and
Dickson, Optically enhanced, near-IR, silver cluster emission altered by single base changes in the
20
25
(32)
(33)
Non-metallic oligomeric clusters and metallic particles Faraday Discussions, 1991, 92, 31.
(34)
G. Schmid, Large clusters and colloids. Metals in the embryonic state Chem Rev, 1992,
92, 1709.
(36)
30
in solid argon, krypton, and xenon The Journal of chemical physics, 1968, 49, 5209.
(38)
35
and excitation spectra of Ag< sub> 4</sub> in an argon matrix Chemical Physics Letters, 1999, 313,
105.
(39)
fluorescence spectra of Ar-matrix-isolated Ag< sub> 3</sub> clusters Chemical Physics Letters,
2000, 320, 59.
40
(40)
W. Schulze, I. Rabin, G. Ertl, Formation of lightemitting Ag2 and Ag3 species in the
Broer, Excited-state relaxation of Ag_ {8} clusters embedded in helium droplets Physical Review
Letters, 2004, 92, 173403.
45
(42)
(28)
15
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
(43)
Page 24 of 33
DOI: 10.1039/C3NR01601C
multiarm star poly(acrylic acid) as "molecular hydrogel" templates Adv Mater, 2007, 19, 349.
(46)
synthesis and in situ immobilization of fluorescent silver nanoclusters on DNA nanoscaffolds by use
of the tollens reaction Angewandte Chemie International Edition, 2011, 50, 4176.
(48)
T. Udaya Bhaskara Rao, T. Pradeep, Luminescent Ag7 and Ag8 clusters by interfacial
Kitaev, Chiral thiol-stabilized silver nanoclusters with well-resolved optical transitions synthesized
by a facile etching procedure in aqueous solutions Langmuir, 2009, 25, 5840.
(51)
20
encapsulated fluorescent silver nanoclusters Angewandte Chemie International Edition, 2007, 46,
2028.
(52)
living HeLa cells with fluorescent poly-cytosine encapsulated Ag nanoclusters Photochemical &
Photobiological Sciences, 2010, 9, 716.
(53)
25
nanoclusters stabilized by glutathione: a promising fluorescent label for bioimaging Nano Research,
2012, 5, 379.
(54)
Bouwmeester, E. Gwinn, Evidence for rodshaped DNAstabilized silver nanocluster emitters Adv
30
exhibit strong two-photon-induced fluorescence Journal of the American Chemical Society, 2008,
130, 11602.
(56)
35
fluorophores with data from 690 to 1050 nm Journal of the Optical Society of America B, 1996, 13,
481.
(57)
poly-C loops stabilize four types of fluorescent Ag-n:DNA J Phys Chem C, 2009, 113, 4229.
(58)
40
clusters assemble at programmed sites on DNA nanotubes Nano Letters, 2012, 12, 5464.
(59)
and their DNA analogs: C, G versus A, T (U) dichotomy Chemical communications, 2011, 47, 4715.
(60)
labeling with fluorescent Ag nanocluster conjugates Photochemistry and Photobiology, 2008, 84,
45
1435.
(61)
(49)
15
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 25 of 33
Nanoscale
(62)
fluorescence probes for the highly selective and sensitive detection of the Hg2+ ion Chemical
communications, 2009, 3395.
(63)
5
G. Y. Lan, C. C. Huang, H. T. Chang, Silver nanoclusters as fluorescent probes for DOI: 10.1039/C3NR01601C
selective and sensitive detection of copper ions Chemical communications, 2010, 46, 1257.
(64)
assay using aptamer-functionalized silver nanocluster DNA probes Analytical chemistry, 2012, 84,
5170.
(65)
10
fluorescence turn-on aptasensor for protein detection via target-induced silver nanoclusters
formation Analytica chimica acta, 2012, 749, 70.
(66)
(67)
15
Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin Nature, 1997,
388, 882.
(68)
reporters of protein tyrosine kinase activities in living cells Proceedings of the National Academy of
Sciences of the United States of America, 2001, 98, 15003.
(69)
20
kinase A activity reveal impact of substrate tethering Proceedings of the National Academy of
Sciences of the United States of America, 2001, 98, 14997.
(70)
assembled nanoscale biosensors based on quantum dot FRET donors Nature Materials, 2003, 2,
25
630.
(71)
conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution
30
35
engineered self-assembling fragments of green fluorescent protein Nature Biotechnology, 2005, 23,
102.
(77)
in live bacterial cells using fluorescent protein complementation Nature Methods, 2007, 4, 421.
(79)
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
(80)
Page 26 of 33
Llopis, R. Y. Tsien, New biarsenical ligands and tetracysteine motifs for protein labeling in vitro
and in vivo: synthesis and biological applications Journal of the American Chemical Society, 2002,
124, 6063.
5
(81)
DOI: 10.1039/C3NR01601C
triphenylmethane dyes Journal of the American Chemical Society, 2003, 125, 14716.
(82)
15
fluorophores: a mechanism for activatable, in vivo optical molecular imaging ACS Chemical
Biology, 2009, 4, 535.
(86)
20
typing through cooperative binding of DNA probes carrying a metal chelator for luminescent
lanthanide ions Analytical Biochemistry, 2006, 359, 259.
(87)
accompanied with excimer formation from two pyrene-labeled probes Photochemistry and
Photobiology, 1995, 62, 836.
(89)
30
probe nucleic acid hybridization method with pyrene as a fluorophore Nucleic Acids Research,
1998, 26, 5409.
(90)
monomer-excimer fluorescence color change Nucleic Acids Research, 1998, 26, 3789.
(91)
35
excimer beacon assays for ribonuclease H kinetic study Chembiochem : a European journal of
chemical biology, 2008, 9, 355.
(92)
probes for rapid protein monitoring in complex biological fluids Proceedings of the National
Academy of Sciences of the United States of America, 2005, 102, 17278.
40
(93)
Hobartner, T. Ha, S. K. Silverman, M. S. Strano, Multimodal optical sensing and analyte specificity
45
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 27 of 33
Nanoscale
nanotube fluorescence as a chaperone sensor for nitroaromatics Proceedings of the National
Academy of Sciences of the United States of America, 2011, 108, 8544.
(96)
(97)
DOI: 10.1039/C3NR01601C
reactivity and readout strategy for amplified signaling and sequence selectivity Chemistry - A
European Journal, 2009, 15, 6723.
(98)
transfer as a probe for nucleic acid structures and sequences Nucleic Acids Research, 1994, 22, 920.
(99)
25
30
35
C. Chen, Y. Zhai, High resolution analysis of DNA copy number variation using comparative
genomic hybridization to microarrays Nature Genetics, 1998, 20, 207.
(110) G. Sozzi, D. Conte, L. Mariani, S. L. Vullo, L. Roz, C. Lombardo, M. A. Pierotti, L.
Tavecchio, Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung
cancer patients Cancer Research, 2001, 61, 4675.
40
(111) H. C. Fan, W. Gu, J. Wang, Y. J. Blumenfeld, Y. Y. El-Sayed, S. R. Quake, Noninvasive prenatal measurement of the fetal genome Nature, 2012, 487, 320.
(112) C. A. Heid, J. Stevens, K. J. Livak, P. M. Williams, Real time quantitative PCR Genome
Research, 1996, 6, 986.
(113) T. Hunkapiller, R. Kaiser, B. Koop, L. Hood, Large-scale and automated DNA sequence
45
(100) D. M. Kolpashchikov, Binary probes for nucleic acid analysis Chem Rev, 2010, 110,
15
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
Page 28 of 33
(115) A. Raj, P. van den Bogaard, S. A. Rifkin, A. van Oudenaarden, S. Tyagi, Imaging
individual mRNA molecules using multiple singly labeled probes Nature Methods, 2008, 5, 877.
(116) S. Tyagi, D. P. Bratu, F. R. Kramer, Multicolor molecular beacons for allele
discrimination Nature Biotechnology, 1998, 16, 49.
5
DOI: 10.1039/C3NR01601C
10
15
Antoku,
Fluorescent
polycytosine-encapsulated
silver
nanoclusters
Ph.D.
25
30
35
40
(130) H.-C. Yeh, C. M. Puleo, Y.-P. Ho, V. J. Bailey, T. C. Lim, K. Liu, T.-H. Wang, Tunable
blinking kinetics of Cy5 for precise DNA quantification and single-nucleotide difference detection
Biophysical Journal, 2008, 95, 729.
(131) T. Heinlein, J.-P. Knemeyer, O. Piestert, M. H. M. Sauer, Photoinduced electron transfer
between fluorescent dyes and guanosine residues in DNA-hairpins J Phys Chem B, 2003, 107, 7957.
45
fluorescent silver nanoclusters in hybridized DNA duplexes for single nucleotide mutation
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 29 of 33
Nanoscale
(133) K. Nakatani, Chemistry challenges in SNP typing Chembiochem : a European journal of
chemical biology, 2004, 5, 1623.
(134) P. Y. Kwok, Methods for genotyping single nucleotide polymorphisms Annual Review of
Genomics and Human Genetics, 2001, 2, 235.
(135) A.
C.
Syvanen,
Accessing
genetic
DOI: 10.1039/C3NR01601C
variation:
genotyping
single
nucleotide
20
25
30
National Academy of Sciences of the United States of America, 2005, 102, 11606.
(145) A. Okamoto, K. Kanatani, I. Saito, Pyrene-labeled base-discriminating fluorescent DNA
probes for homogeneous SNP typing Journal of the American Chemical Society, 2004, 126, 4820.
(146) A. Okamoto, Y. Saito, I. Saito, Design of base-discriminating fluorescent nucleosides
Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 2005, 6, 108.
35
(147) Q. Li, G. Luan, Q. Guo, J. Liang, A new class of homogeneous nucleic acid probes based
on specific displacement hybridization Nucleic Acids Research, 2002, 30, e5.
(148) W. J. Kim, Y. Sato, T. Akaike, A. Maruyama, Cationic comb-type copolymers for DNA
analysis Nature Materials, 2003, 2, 815.
(149) A. H. Buck, C. J. Campbell, P. Dickinson, C. P. Mountford, H. C. Stoquert, J. G. Terry,
40
45
unambiguous detection and symbolic display of single nucleotide polymorphisms on DNA origami
Nano Letters, 2011, 11, 910.
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
Page 30 of 33
(152) D. Y. Zhang, S. X. Chen, P. Yin, Optimizing the specificity of nucleic acid hybridization
Nature Chemistry, 2012, 4, 208.
(153) K. Nakatani, S. Sando, H. Kumasawa, J. Kikuchi, I. Saito, Recognition of guanineView Article Online
guanine mismatches by the dimeric form of 2-amino-1,8-naphthyridine Journal of the American DOI: 10.1039/C3NR01601C
5
10
20
25
30
genotyping of single nucleotide polymorphisms using novel minor groove binding DNA
oligonucleotides (MGB probes) Human Mutation, 2002, 19, 554.
(164) D. M. Kolpashchikov, Binary malachite green aptamer for fluorescent detection of
nucleic acids Journal of the American Chemical Society, 2005, 127, 12442.
(165) Y. Xiao, K. J. I. Plakos, X. Lou, R. J. White, J. Qian, K. W. Plaxco, H. T. Soh,
35
40
45
2183.
(169) G. Y. Lan, W. Y. Chen, H. T. Chang, One-pot synthesis of fluorescent oligonucleotide
Ag nanoclusters for specific and sensitive detection of DNA Biosens Bioelectron, 2011, 26, 2431.
6915.
15
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Page 31 of 33
Nanoscale
(170) P. Shah, A. Rrvig-Lund, S. B. Chaabane, P. W. Thulstrup, H. G. Kjaergaard, E. Fron, J.
Hofkens, S. W. Yang, T. Vosch, Design aspects of bright red emissive silver nanoclusters/DNA
probes for microRNA detection ACS Nano, 2012, 6, 8803.
(171) S. He, B. Song, D. Li, C. Zhu, W. Qi, Y. Wen, L. Wang, S. Song, H. Fang, C. Fan, A DOI: 10.1039/C3NR01601C
5
graphene nanoprobe for rapid, sensitive, and multicolor fluorescent DNA analysis Advanced
Functional Materials, 2010, 20, 453.
(172) K. P. Loh, Q. Bao, G. Eda, M. Chhowalla, Graphene oxide as a chemically tunable
platform for optical applications Nature Chemistry, 2010, 2, 1015.
(173) Y. Tao, Y. Lin, Z. Huang, J. Ren, X. Qu, DNA-templated silver nanoclusters-graphene
10
oxide nanohybrid materials: a platform for label-free and sensitive fluorescence turn-on detection of
multiple nucleic acid targets The Analyst, 2012, 137, 2588.
(174) K. Ma, Q. Cui, G. Liu, F. Wu, S. Xu, Y. Shao, DNA abasic site-directed formation of
fluorescent silver nanoclusters for selective nucleobase recognition Nanotechnology, 2011, 22,
(175) S. W. Yang, T. Vosch, Rapid detection of microRNA by a silver nanocluster DNA probe
Analytical chemistry, 2011, 83, 6935.
(176) Y. Q. Liu, M. Zhang, B. C. Yin, B. C. Ye, Attomolar ultrasensitive microRNA detection
by DNA-scaffolded silver-nanocluster probe based on isothermal amplification Analytical
chemistry, 2012, 84, 5165.
20
(177) H. Dong, S. Jin, H. Ju, K. Hao, L. P. Xu, H. Lu, X. Zhang, Trace and label-free
microRNA detection using oligonucleotide encapsulated silver nanoclusters as probes Analytical
chemistry, 2012, 84, 8670.
(178) L. Stryer, R. P. Haugland, Energy transfer - a spectroscopic ruler Proceedings of the
National Academy of Sciences of the United States of America, 1967, 58, 719.
25
30
35
40
Liu, J. Gould, L. Hood, Integrated barcode chips for rapid, multiplexed analysis of proteins in
microliter quantities of blood Nature Biotechnology, 2008, 26, 1373.
(186) K. Shiroguchi, T. Z. Jia, P. A. Sims, X. S. Xie, Digital RNA sequencing minimizes
sequence-dependent bias and amplification noise with optimized single-molecule barcodes
Proceedings of the National Academy of Sciences, 2012, 109, 1347.
45
(187) J.-M. Nam, C. S. Thaxton, C. A. Mirkin, Nanoparticle-based bio-bar codes for the
ultrasensitive detection of proteins Science, 2003, 301, 1884.
305502.
15
CREATED USING THE RSC REPORT TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
Nanoscale
Page 32 of 33
(188) M. Xiao, A. Phong, C. Ha, T.-F. Chan, D. Cai, L. Leung, E. Wan, A. L. Kistler, J. L.
DeRisi, P. R. Selvin, Rapid DNA mapping by fluorescent single molecule detection Nucleic Acids
Research, 2007, 35, e16.
(189) M. Xiao, E. Wan, C. Chu, W.-C. Hsueh, Y. Cao, P.-Y. Kwok, Direct determination of DOI: 10.1039/C3NR01601C
5
10
15
20
kidney targeting and biomedical imaging Journal of the American Chemical Society, 2011, 133,
8617.
(196) S. Pal, R. Varghese, Z. Deng, Z. Zhao, A. Kumar, H. Yan, Y. Liu, Site-specific synthesis
and in situ immobilization of fluorescent silver nanoclusters on DNA nanoscaffolds by use of the
Tollens reaction Angewandte Chemie, 2011, 123, 4262.
25
(197) L. Feng, Z. Huang, J. Ren, X. Qu, Toward site-specific, homogeneous and highly stable
fluorescent silver nanoclusters fabrication on triplex DNA scaffolds Nucleic Acids Research, 2012,
40, e122.
(198) D. Schultz, E. G. Gwinn, Silver atom and strand numbers in fluorescent and dark
Ag:DNAs Chemical communications, 2012, 48, 5748.
30
35
40
45
Page 33 of 33
Nanoscale
View Article Online
DOI: 10.1039/C3NR01601C
Table of Contents
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.