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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;
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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 2000’s, 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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Nanoscale

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DOI: 10.1039/C3NR01601C

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 system’s 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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Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.1039/C3NR01601C + 10 + Excitation wavelength (nm) 15 20 25 Figure 1.17 and (3) the ability to switch the fluorescence of silver clusters on and off11.1) . and (3) stabilize clusters in the solution phase. Addition of NaBH4 reduces these ions.22.48 lipoic acid.16. including PAMAM.11.36 the optical transitions of silver clusters encapsulated by templating ligands vary not only with stoichiometry. Reproduced from ref.49 thiol ligands. (A) Illustration depicting how silver nanoclusters are formed on single-stranded DNA.13.46 sugar molecules. charge. 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). and geometry.44. NaBH4 + Normalized intensity A                         B                                 C                                     D .RSC.ORG/ELECTRONICFILES FOR DETAILS Page 3 of 33 Nanoscale 5 encapsulation agents or templates are necessary as they (1) offer initial nucleation sites for the cluster formation.17 through interactions with a nearby DNA sequence (Figures 1D and 2A).7.13 and tune their color13. 00–00 | 3 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript Published on 06 June 2013. [year].2.45 poly(NIPAM-AA-HEA) microgels.CREATED USING THE RSC REPORT TEMPLATE (VER. (2) prevent clusters from overgrowth. 2.10 Among those different silver clusters synthesized. [vol].2 Synthesis and Intriguing Properties 30 35 40 Compared to gold 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. lower red strand). Dickson’s group first reported the synthesis of highly fluorescent silver nanoclusters in aqueous solution using amine-rich dendrimers as templates. Reprinted from ref.50 peptides51 and DNA.43 polyacrylate. 3. ssDNA-templated silver nanoclusters (DNA/Ag NCs. In the early 2000’s. Silver ions preferentially associate with cytosines on the DNA strand.42 PMMA.17.8. This provides the basis for the creation of complementary palettes of silver cluster fluorophores using different DNA sequences as encapsulation [journal]. a wide variety of organic materials have been tested as templates to synthesize fluorescent silver nanoclusters in solution. 16 with the permission from American Chemical Society.2 Ever since. As pointed out by many groups.4. close to the absorption maxima of nucleobases. (C) All DNA/Ag NCs share a common excitation peak between 260 and 270 nm. leading to the formation of silver nanoclusters. Reprinted from ref.13 or tune their color13.10 (2) the ability to use a single UV source to excite all silver cluster species templated on DNA (Figure 1C).17 through interactions with nearby DNA sequences (called enhancers.4. 13 with the permission from IEEE. 8 with the permission from American Chemical Society. one can excite all DNA/Ag NCs with a single UV excitation source. opening up opportunities for silver clusters as biological labels. silver nanoclusters are brighter31 and can be easily synthesized by using a number of ligands as encapsulation agents . Through energy transfer from nucleobases. but also with interactions with their encapsulating ligands.47 mercaptosuccinic acid. (D) It is possible to switch the fluorescence of silver clusters on and off11.SEE WWW. View Article Online DOI: 10.

17 This common excitation peak. followed by adding 6-12 equivalents of sodium borohydride (NaBH4) to reduce the silver ions. the synthesis starts with mixing micromolar DNA with 6-12 equivalents of silver nitrate (AgNO3) for 15 min in sodium phosphate buffer (pH 6. regardless of the location of their visible excitation peaks.12 (e.17 Other than DNA templates. The highest quantum yield ever reported on a purified DNA/Ag NC species is 0. Phosphate buffer5. 1:357 as compared to 1:14 or 2:17). Other than the above advantages.RSC.21 The longest lifetime reported is 4.60 molecular logic devices. however. Gwinn has demonstrated the use of ssRNA to nucleate the growth of fluorescent silver clusters. including small size (hydrodynamic diameter. the common UV excitation feature can greatly facilitate the use of DNA/Ag NCs as reporters in multiplexed detection.12 DNA/Ag NCs can also be brighter6.0).4.10 The on/off switching and color-tuning capabilities.57 The faster onset of fluorescence was coupled with reduced chemical lifetimes for some emitters.16 Nevertheless. which sits close to the absorption maxima of nucleobases (around 260-270 nm). Other than the programmable and switchable fluorescence properties.4 while the latter believed this UV excitation peak is due to energy transfer between nucleobases and silver clusters.16.9 strong two-photon-induced fluorescence (two-photon absorption cross-section was calculated to be 3.59 While DNA/Ag NCs have demonstrated applications in single-molecule microscopy. all DNA/Ag NCs share a similar UV excitation feature.20. was previously reported by Petty/Dickson4 and Fygenson.17 Interestingly. leading to new biosensing opportunities especially in DNA detection which we detail below.21.6.52. The mixture is vortexed for one minute and left in the dark for 12 hours.52.53 and are less prone to blinking on long (> 1 ms) timescales.8. 10 15 20 25 30 35 40 45 ligands. on the other hand.g. one emitter showed a 10-fold photostability improvement over Cy3 and Cy5 dyes8).SEE WWW. 3.35 ns.11. the photophysical properties of as-synthesized DNA/Ag NCs were found quite consistent. Proper selection of buffer.5 to 7.8 Other useful optical properties include fluorescence recovery with low-energy secondary excitation. and the reaction molar ratio enables the preferential formation of Ag nanoclusters (2-30 atoms) over larger plasmonic Ag nanoparticles.57. and fluorescence recovery upon nanocluster transfer.g.58 (compatible with electrospray ionization mass spectroscopy) is most commonly used.8.57 With fixed preparation conditions.13.11 cellular imaging.ORG/ELECTRONICFILES FOR DETAILS 5 Published on 06 June 2013. one emitter was 20 times brighter than a Cy3 dye20) and more photostable than organic dyes6.9.61 metal ion 4 | [journal]. a faster onset of fluorescence and a larger maximum intensity were observed in the reactions.CREATED USING THE RSC REPORT TEMPLATE (VER. [vol].6. ranging from 3 to 6 nm).8.21 (e.12. In general. 00–00 This journal is © The Royal Society of Chemistry [year] Page 4 of 33 View Article Online DOI: 10. allow DNA/Ag NCs to be used not only as fluorescent tags but as fluorescent sensors.000~4.g.1) .5105 M-1cm-1.1039/C3NR01601C Nanoscale Accepted Manuscript Nanoscale .10.to Ag+ was used (e. silver salt.21 or greater55. [year]. when a lower ratio of BH4.51.6.11 or acetate buffer7. where one UV source should excite all silver cluster species. about 100fold larger than that of fluorescein (37 GM)56).12 low cellular toxicity. DNA/Ag NCs have more benefits. buffer pH.9354 and the largest extinction coefficient is 9.16 The former suggested that the UV excitation peak may be due to higher lying excited states accessible in the UV region.18. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.10.20 The synthesis of DNA/Ag NCs is carried out at room temperature via sequential mixing of a cytosine-rich oligonucleotide. and reducing reagent in buffer solution. as the fluorescence emission of DNA/Ag NCs upon UV excitation is highly depolarized.000 GM (Goppert-Mayer units)19. analogous to previously studied DNA/Ag NCs. which includes the layer of DNA.

97 and FRET binary probes.1) . a number of fluorescence color-switching methods (upon probe-target binding) have also been demonstrated. Nanomaterials that “communicate” with their surroundings and “transduce” specific responses into unique emission spectra therefore hold great promise to enable new devices for diagnostics.63 and protein detection. and bioimaging. where silver nanoclusters were proved superior to organic dye or quantum dot alternatives. for fluorescent nanoclusters.72 electron transfer-based probes.1 Kolpashchikov. However.ORG/ELECTRONICFILES FOR DETAILS Page 5 of 33 Nanoscale 5 sensing. 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). [year].105 In general. such as microarrays.66-70 intercalating dyes.88-92 carbon nanotube sensors.79. where removal of unbound probes is difficult. Activatable and color-switchable silver nanocluster probes Published on 06 June 2013.85 and lanthanide complexes. Extended from their special environmental sensitivities. DNA/Ag NCs can possess environmental sensitivities not commonly seen among existing fluorophores. activatable and color-switchable probes that enable “detection without separation” are particularly desirable. In other words. split fluorescence protein).96.g. fluorescence activation mechanisms of existing fluorophores. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.74 environment-sensitive Aequorea fluorescent proteins. and geometry. (3) constraint on molecular movement (e. View Article Online DOI: 10.64. activatable probes eliminate the wash steps. As mentioned above. Since the activatable probes only turn on in specific environments but otherwise remain undetectable.13.CREATED USING THE RSC REPORT TEMPLATE (VER.62. a whole new type of fluorescence activation mechanisms could exist for DNA/Ag NCs.71. other than cluster’s stoichiometry.15. therapeutics.102 Li. [journal].g.1039/C3NR01601C 3.84. Until now.17 NCBs are new molecular probes that fluoresce upon hybridization with DNA targets but do not use Förster resonance energy transfer (FRET) as the fluorescence on/off switching mechanism. the development of new activatable and multi-color probes is often hindered by the use of the known. they often achieve high signal-tobackground ratios in target detection.81-83 H-dimer formation. activation strategies have been used to develop so-called “separation-free probes” or “turn-on probe”.98.11. For in vitro studies.73. charge.65 the most significant advance and impact were made in DNA detection by the introduction of NanoCluster Beacons (NCBs). intercalating dyes and some aptamer systems). which is detailed below.103 Ranasinghe and Brown. 00–00 | 5 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 3. 10 15 20 25 30 35 40 Among all fluorescent probes developed to detect and image biomolecules or metabolites.80 aptamer systems.SEE WWW. While activatable and multi-color probes have enabled many detection schemes.1 Current activatable and color-switching probes for biological detection .75 split fluorescent proteins.RSC.86. greatly simplifying the process.93-95 quencher transfer/detachment. and (4) combinatorial assembly of fluorescent complex (e. 3. activation mechanisms can be categorized as follows: (1) FRET. including FRET.104 and Ihara.76-78 biarsenicical-tetracysteine systems.87 Similarly. (2) charge transfer. [vol]. especially for intracellular studies. but limited.100 Vendrell. a number of non-enzymatic. researchers still need more activatable and multi-color probes in order to push fundamental biology research and medical diagnostic imaging to the next level. environmental sensing.101 Kool.99 Readers are recommended to refer to recent reviews in fluorescent probe development by Kobayashi. such as excimer formation.

2 Activatable silver nanocluster probes for DNA detection – NanoCluster Beacons 3. 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. low-abundance DNA sequences is the goal for DNA detection.110 The number of specific alleles found in the maternal circulating DNA reflects the haplotype of fetus genome. In contrast to DNA microarrays. forming a donor-acceptor FRET pair. For instance. [vol].111 A number of methods have been developed for the detection and quantification of specific DNA.109 The amount of circulating DNA in plasma can discriminate between cancer patients and healthy individuals and is related to disease progression.1039/C3NR01601C Nanoscale Accepted Manuscript 5 Nucleic acid detection is an enabling technology for a number of fields. as a surface immobilization technique. microarrays. a sophisticated thermodynamic analysis is often needed for the stem-loop sequence selection. which is never 100%. eliminating the wash steps and facilitating the clinical use.106 the recognition of genetic mutations. Moreover.107 and forensic analysis. While the FRET-based probes and methods have been successfully commercialized. including pathogen identification.17. One widely used turn-on probe for the homogeneous detection of DNA is the molecular beacon (MB).RSC.112 DNA sequencing.93. deletions of tumorsuppressor genes and copy number increases of oncogenes were found in many forms of cancer. While FRET probes can provide better signal-tobackground (S/B) ratios in detection (as compared to singly labeled probe115).114 As mentioned above.1) . Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.2. specific DNA can also be directly detected in solution using aforementioned turn-on probes (often termed homogeneous detection). their S/B ratios are also bound by quenching (either Förster or contact quenching) efficiency. DNA/Ag NCs have also attracted attention as a new probe for homogenous detection of DNA hybridization. such as real-time quantitative polymerase chain reaction (qPCR). require multiple wash steps and can only be run at central laboratories. 3. In particular.CREATED USING THE RSC REPORT TEMPLATE (VER.ORG/ELECTRONICFILES FOR DETAILS Nanoscale Page 6 of 33 3. with recent results showing the capability to differentiate DNA targets with only single-nucleotide differences.SEE WWW.94 As an emerging class of nanomaterial fluorophores.120 6 | [journal].113 and DNA microarrays. FRET probes that contain either the fluorophore or the quencher (socalled singly labeled impurities) contribute to a high fluorescence background or a reduced signal.1 Background of DNA detection Published on 06 June 2013.30. In addition.116 . therefore serving as indicators for possible inherited diseases. including gold nanoparticles117-119 and carbon nanotubes. 00–00 This journal is © The Royal Society of Chemistry [year] View Article Online DOI: 10.66 which is a hairpinshaped oligonucleotide labeled with a fluorophore and a quencher at the two ends. FRET probes are expensive since they require costly processes to attach two moieties to the same DNA strand.108 While identification of specific. precise quantification of the amount of nucleic acids present in the samples can be just as important in many applications. they have drawbacks. [year].111.

00–00 | 7 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 5 .RSC.121. [vol].2. Photographs of the resulting emission are taken under UV 365 nm light. NC probe G-rich enhancer 10 15 20 25 Enhancer strand no target Enhancer probe 5 µm Target DNA Figure 2. recently found “guanine proximity” can also trigger reversible transformations among different silver cluster species. [year]. Reproduced from ref. A non-emissive silver cluster is first prepared on a NC-nucleation sequence.ORG/ELECTRONICFILES FOR DETAILS Page 7 of 33 Nanoscale A                                      B                                             C In absence of target NCB alone Dark Ag NC Braf 1:1 View Article Online DOI: 10.5. 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. temperature.10. dark silver clusters can be transformed into bright red-emitting clusters when [journal].122 Other than these factors. in the appropriate context. oxygen and salt content. 3. (B) NanoCluster Beacon (NCB) detection scheme. 11 with the permission from American Chemical Society. a strong red fluorescence emission is observed from the solution. with a bulk enhancement ratio greater than 500 fold. Schematic showing the guanine-proximity-induced fluorescence activation phenomenon of DNA-templated silver nanoclusters and the associated DNA detection probe. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.SEE WWW. While these conversions can be viewed as a drawback. Once a guanine-rich enhancer sequence is brought close to the silver cluster through hybridization. NCBs were non-specifically adsorbed to a coverslip and images were taken with a total internal reflection fluorescence microscope. they can be used as new signal transduction modes for molecular sensing.CREATED USING THE RSC REPORT TEMPLATE (VER. including time.10. 3. Reproduced from ref. (A) Guanine proximity can dramatically increase the red fluorescence emission of DNA/Ag NCs.11 As shown in Figure 2A. 13 with the permission from IEEE. 11 with the permission from American Chemical Society. Reprinted from ref. transformations among silver cluster species (which have distinct emission colors) can depend upon a number of factors. DNA/Ag NCs can sometimes change color5.11 Often reversible.1) . Yeh et al. but remain dark in the absence of targets. NanoCluster Beacon (NCB). (C) Fluorescence images of individual NCBs from samples with (upper) and without (lower) DNA targets.122 – a dynamic conversion process neither well understood nor generally shared by existing fluorophores.1039/C3NR01601C NC-bearing strand Dark Ag NC Enhancer 5 µm NC-nucleation sequence In presence of target NCB binds a target Bright red Ag NC Published on 06 June 2013.2 NanoCluster Beacons for DNA detection 30 35 While DNA/Ag NCs are promising as small and photostable fluorescent labels.121.

11 Taking advantage of this guanine-proximity-induced fluorescence activation phenomenon.11 When varying the number of guanine bases in proximity to silver clusters.RSC. Interestingly. the NC probe) and the other having a guanine-rich tail (i. which cycled the temperature between 95C (no fluorescence) and 25C (strong red fluorescence). 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. one bearing a non-emissive silver cluster (i. transforming the cluster from a non-emissive species to a highly fluorescent species. A                                            B                                      C Change NC-nucleation sequence Change enhancer sequence Change alignment between sequences 35 40 45 Figure 3.11 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. (A) Using the same enhancer sequence (lower strands) but different NC-nucleation sequences (upper strands). The resulting reaction solution had weak green fluorescence emission. 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.1) . The two probes were designed to bind in juxtaposition to a target DNA. The reversibility of the fluorescence enhancement was demonstrated using a real-time PCR thermal cycler. 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. As illustrated in Figure 2B. with a bulk enhancement greater than 500 fold (Figure 2A). Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.e. as compared to the sample without target.13 (B) Using the same NC-nucleation sequence (upper strands) 8 | [journal]. Not relying on FRET as the fluorescence activation mechanism.SEE WWW. Silver clusters were first prepared on an NCbearing strand having a cytosine-rich nucleation sequence (called NC-nucleation sequence). and was further verified in binding competition experiments. termed a NanoCluster Beacon (NCB). [year].1039/C3NR01601C Nanoscale Accepted Manuscript Nanoscale . Three different strategies to tune the emission color of NanoCluster Beacons. 10 15 20 25 30 Page 8 of 33 placed in proximity to guanine bases.e. [vol]. a new turn-on probe. a typical form of binary probes.CREATED USING THE RSC REPORT TEMPLATE (VER. Fluorescence occurred only when a specific DNA target was present in the sample. the extent of red fluorescence enhancement was found to exponentially increase with an increasing number of guanine bases. 00–00 This journal is © The Royal Society of Chemistry [year] View Article Online DOI: 10. was designed for DNA detection. 3.ORG/ELECTRONICFILES FOR DETAILS 5 Published on 06 June 2013. an NCB consisted of two linear probes. the enhancer probe).100 Such a juxtaposition binding enabled the enhancer to interact with the silver cluster. when brought close to the same silver clusters. 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).

reducing oxidized-NC species (in this case.e. used NCB for DNA detection. but changing the alignment between sequences (by adding or deleting nucleotides at the end of the hybridization sequences).ORG/ELECTRONICFILES FOR DETAILS Page 9 of 33 Nanoscale but different enhancer sequences (lower strands). 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. but rather by the existence of sparse NCBs that are red emitting in the absence of target.13 (C) Using the same enhancer and NC-nucleation sequences. and reliable methods to identify [journal].2. There is no need to remove excess silver ions or borohydride ions from the solution after cluster formation is completed. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. performed an experiment where the guanines on an enhancer were replaced by 7-deazaguanines. opening opportunities for NCBs in multiplexed assays. While the initial work by Yeh et al.1039/C3NR01601C Published on 06 June 2013.125-130 In the fluorescence activation of NCBs.132 would not only achieve the largest therapeutic effect. The advantages of NCBs lie in their simple.11 3. non-emissive clusters) into bright red-emitting clusters. This important feature. 10 15 20 25 30 35 In another report by Yeh et al.SEE WWW. NCBs have shown an S/B ratio five times better than that of a molecule beacon.1) .e. The ability to tailor drug treatment for individuals in order to avoid adverse side effects. Another experimental result in the report that weakened the electron-transfer hypothesis is that thymine-proximity produced a green fluorescence enhancement. referred to as personalized medicine.CREATED USING THE RSC REPORT TEMPLATE (VER. as these cluster precursors are essentially non-fluorescent. Yeh et al. cheap. the best electron donor)125 and can often alter the emission rates of organic dyes. 00–00 | 9 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 5 . While future medicine clearly requires rapid.3 Background of SNP detection 40 Single-nucleotide polymorphisms (SNPs). 3. To further test this electron-transfer hypothesis. with thymine being a worse electron donor than adenine (adenine proximity did not generate any measurable fluorescence enhancement).17 View Article Online DOI: 10. [vol]. the most common genetic variations between individuals. one-step preparation process (i.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). no red fluorescence emission was seen after hybridization. low-cost. Guanine has the lowest oxidation potential among the four nucleobases (i. weakening the electron transfer hypothesis. but also allow pharmaceutical companies to bring many more drugs to market. the fluorescence background of NCBs is not limited by Förster quenching.74. guanines may serve as electron donors.. having multiple activation colors from the same origin.11 Without taking any special steps to reduce the background emission (such as purification or prephotobleaching123). other groups have modified NCBs to detect proteins64 and small molecules.11. silver cluster nucleation process on the NC probe) and their potential to achieve an ultrahigh S/B ratio in homogenous detection. Unlike conventional MBs. is not commonly shared by existing fluorophores.131 Surprisingly. [year].RSC. One possibility is that the red fluorescence emission is due to electron transfer from the guanine bases to the silver clusters. which are stronger electron donors than guanines.124 The physical mechanism driving the increase in red fluorescence emission is not well understood at this moment.

Allele-specific hybridization is the most common method in non-enzymatic SNP typing. thermodynamic discrimination is not likely to be a reliable basis for large-scale and multiplexed SNP typing. an ideal SNP probe can quantitate the amount of DNA target using fluorescence intensity and identify four SNP variants with unique fluorescence colors.SEE WWW. [year].146. no target DNA) is also “off”.g. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. As a result. 3. Current methods for SNP detection (e.133 To circumvent the issues of allele-specific hybridization.CREATED USING THE RSC REPORT TEMPLATE (VER.144 base-discriminating fluorescent nucleosides145.142). a single-base mismatch only generates a small difference in duplex stability. 00–00 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 5 Page 10 of 33 . and cleavage. However. However. Allele-specific hybridization has been implemented in two different detection platforms – on a solid surface (e. but displays four different fluorescent colors when interacting with the four DOI: 10.1) .e. it is difficult to discriminate all four SNP variants using a single turn-on probe. the development of new SNP probes is often challenging. Therefore. For instance. making SNP scoring ambiguous. SNP detection is mainly carried out in central laboratories. 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. similar to the signal level of mismatched alleles.g. Ideally.ORG/ELECTRONICFILES FOR DETAILS Nanoscale Published on 06 June 2013.143.139 To reduce the cost and assay time.g. ASO binds preferably to the fully matched allele rather than the single-base mismatched alleles.153-155 While overcoming the issues of small differences in thermodynamic stability. where totally four different enzymes are needed for SNP identification. sophisticated ASO design algorithms are often required. C. T) at a polymorphic site. kinetic schemes. DNA microarrays140) and in homogeneous solution (e. the signal level (either optical or electrical) of the “absence-of-allele” condition (i. where allele-specific oligonucleotides (ASO) are designed to hybridize with polymorphic target alleles. such an ideal multi-color SNP probe does not exist today. 10 15 20 25 30 35 40 45 SNPs in patients’ genomes. ideally one would like to 10 | [journal]. using molecular beacons141.133-136 The major advantage of enzymatic methods is the high fidelity of the base sequence recognition.138 the requirements of primer extension and fluorescent ddNTPs add to the assay time and cost. While the array platform is suitable for large-scale SNP typing. one would like to have a multi-color SNP probe that is non-fluorescent in View Article Online solution. ligation.133 Additionally. [vol]. genotyping of known SNPs) typically require enzymatic reactions such as primer extension.RSC. SNPs have been detected by charge transport through the π-stack of DNA duplexes. A.1039/C3NR01601C allelic variants (G. several non-enzymatic SNP-typing methods (methods that do not use enzymes except for PCR amplification) have been developed. new SNP-typing methods are currently being developed that do not rely on the differences in thermodynamic stability for discrimination.137. As mentioned above.133 Currently. Other methods employ even more enzymatic reaction steps for SNP typing (such as molecular inversion probes. the allelic discrimination is based on the differences in thermodynamic stability of the ASO/target duplexes. it is far more challenging to set up a single hybridization and washing condition that is applicable for all ASOs used on the microarray. In other words.147-152 and mismatch-binding ligands. making it difficult to choose optimal hybridization conditions that best discriminate the fully matched allele from the singlebase mismatched alleles. which are often characterized by the melting temperatures of duplexes. Moreover. While four-color SNP typing using single-base extension and four differently fluorescently labeled dideoxynucleotides (ddNTPs) has been demonstrated and commercialized.

(B) Emission spectra of the eleven samples under 365 nm excitation. one would need four single-color. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. Consequently. A change greater than 70 nm in ESGM is observed upon a two-nucleotide frame shift alignment from position -1 to +1.CREATED USING THE RSC REPORT TEMPLATE (VER. View Article Online DOI: 10. 17 with the permission from American Chemical Society. which sets a simple criterion for color-switching probe design) of the eleven samples.RSC. The colors of positions +1. While researchers continue to develop various kinds of non-enzymatic methods that do not rely on thermodynamic stability for SNP typing.1039/C3NR01601C A                                                                                                       B Position 1 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 2 T A 6 C T 3 A T 7 C G 4 3’ 5’ G G G T G 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 G G 3' 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 G G 3' …. (D) Emission-spectrum-global-maximum (ESGM. [vol]. Gray: Hybridization sequences. have been explored to create multi-color probes for SNP detection. as we detail below.. A common NC probe is hybridized with one of the eleven enhancer probes. -3 . (A) Schematic showing three alignment states (positions -3. on/off probe. +2.ORG/ELECTRONICFILES FOR DETAILS Page 11 of 33 Nanoscale 5 10 have a probe that not only can differentiate all four allelic variants at once. DNA/Ag NCs. Red: Enhancer sequence. on/off probes but only one multi-color. The resulting eleven alignment-shifting samples are named position -3 to position +7. which differ by advancement of the enhancer sequence along the NC-nucleation sequence one nucleotide at a time. (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 effect of repositioning of the enhancer sequence with respect to the NCnucleation sequence.. the oligonucleotide synthesis and handling cost is much lower for the multi-color probe SNP assays. Reproduced from ref. D C 0 -1 20 25 30 35 +3 +4 305 nm excitation +5 +6 -2 +7 -3 control ESGM (nm)   +1 +2 365 nm excitation 700 660 620 580 Figure 4. +2. 00–00 | 11 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 15 …. Published on 06 June 2013. [year]. +2 +4 5’ G 3' Upper strand: a common NC probe which carries Ag NCs. Blue: NC-nucleation sequence. which possess many interesting and unique environmental sensitivities. less effort is put into the development of multi-color SNP probes due to challenges in chemistry. and +4) between the enhancer sequence (red fill) and the NCnucleation sequence (blue fill).SEE WWW. [journal]. but also can indicate the amount of DNA target present in the sample (as could be used in digital PCR156). 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. To fully identify the SNP type at a specific polymorphic site. Lower strand: individual enhancer probe. and +3 are clearly blue shifted.1) . 3.

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.4 Chameleon NanoCluster Beacons for SNP detection A 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 D A 0.6 View Article Online DOI: 10. [year].g.5 0. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. Schematic and results of chameleon NanoCluster Beacon (cNCB) SNP typing. Without knowing the exact separation distance between the silver cluster and the enhancer sequence in each alignment. 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. The ability to obtain measurably different ensemble activation colors using the same enhancer sequence shifted only by a single nucleotide is unique to NCBs.SEE WWW.17 They exploited this new property for the specific detection and identification of a number of disease-related SNPs. The SNP under investigation is GGT (wild-type) to GTT (mutant-type) mutation on Kras oncogene codon number 12.u.) 5 Yeh et al.17 As shown in Figure 4A. it was found subtle repositioning of the enhancer sequence relative to the NC-nucleation sequence (e.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. 3. However. [vol].55 0.RSC. defined as the wavelength where the emission intensity is the highest) was used as a gross indicator of color for each sample (Figure 4D).16 differences in the emission color could be clearly visualized among the eleven alignment-shifting samples under UV 365 nm excitation (Figure 4C). Emission-spectrum-global-maximum (ESGM. where the emission spectra are plotted. from position -1 to 0 or from position +3 to +4) can shift the ESGM by as much as 60~70 nm (Figure 4D). As illustrated here.CREATED USING THE RSC REPORT TEMPLATE (VER.4. 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. frame-shift basepairing will lead to an alignment 12 | [journal]. Due to the common UV excitation peak of all DNA/Ag NCs. position -1 (sample A) is generated by mixing cNCB with Kras mutant-type target. as shown on the wild-type target in sample B.2. if the thymine base on codon 12 (shown in red letter) is replaced by a guanine base.45 30 40 50 60 70 80 90 100 Temperature (deg C) C E A_GTT_"‐1" B_GGT_"+1" C_GAT D_GCT 1 0. 10 Intensity (a. 00–00 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 20 Absorbance (A260 nm) 15 Relative  fluo.1) . Published on 06 June 2013.ORG/ELECTRONICFILES FOR DETAILS Nanoscale Page 12 of 33 3.1039/C3NR01601C cNCB‐wild type target complex cNCB‐mutant target complex 0.17 . terming the resulting multi-color SNP probes chameleon NanoCluster Beacons (cNCBs). The color variation is more clearly seen in Figure 4B. 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 The alignment-change-induced ESGM shifts were used to directly and quantitatively identify SNPs. potentially direct contact.151. [vol]. have previously used a three-way-junction design. sample B (wild-type. These four samples resulted in three distinguishable emission spectra. between the enhancer bases and the silver cluster determined the emission color of silver cluster. bind both wild-type and mutant-type targets with nearly equal thermodynamic stability (Figure 5D). 10 15 20 25 30 35 40 45 state of position +1 after beacon-target hybridization.130 which agreed well with the three distinguishable emission spectra that were obtained using the cNCB detection method. B_GGT. (C) Emission spectra of the cNCB mixing with the four SNP targets. As predicted.17 When mixing with all four allelic variants (A. C. C_GAT. and T).140-146. having only cNCB) under 365 nm light. pointed out that a short-range interaction. 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). 00–00 | 13 This journal is © The Royal Society of Chemistry [year] View Article Online DOI: 10. Another attractive feature of cNCBs lies in their capability for a two-dimensional analysis (Figure 5E) – the fluorescence intensity [journal]. and D_GCT) and the notarget control (E. (D) The melting curves showing that cNCB binds both wild-type and mutant-type targets with nearly equal thermodynamic stability. chameleon NanoCluster Beacons (cNCBs). as predicted from Figure 4D. position -1) (Figure 5B).RSC. Yeh et al. 17 with the permission from American Chemical Society. resulting in a different turn-on color. (B) Color photo of the cNCB mixing with all four SNP targets (A_GTT. Sample A (position -1) was clearly the most red of the four. it is the environmental sensitivity of DNA/Ag NCs that enables SNP discrimination. as a whole. 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).SEE WWW. In Figure 5A. As shown in Figure 5A. for single-nucleotide variation detection. in contrast to currently employed SNP probes.ORG/ELECTRONICFILES FOR DETAILS Nanoscale 5 Published on 06 June 2013. cNCB’s 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. Yeh et al. This frame-shift basepairing moved the enhancer sequence relative to the NC-nucleation sequence by two nucleotides. requires advanced detection tools to measure the change of Cy5 blinking dynamics at microsecond timescale. cNCB displayed three distinguishable and reproducible emission spectra (Figure 5C). therefore only two differentiable results from the four allelic variants120. showed that the four allelic variants gave three distinguishable blinking times.CREATED USING THE RSC REPORT TEMPLATE (VER. 3.130 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. Yeh et al.1039/C3NR01601C Nanoscale Accepted Manuscript Page 13 of 33 .17 The Cy5 blinking detection method.1) . were based on a three-way-junction (3WJ) design.130 Using the blinking time as an indicator. Further.160-165). however. combined with monitoring Cy5 blinking dynamics by fluorescence correlation spectroscopy. position +1) did appear more orange than sample A (mutant-type. While a distinguishable color for each allelic variant is the ultimate goal. As previously determined (Figure 4D). Reproduced from ref. 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). [year].158. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. the SNP probes. (E) The fluorescence intensity of cNCB can be used to quantify the amount of target (as shown here 2~16 M). G. cNCBs. The change of fluorescence color could be observed both spectroscopically and by the naked eye. Rather.

not seen in existing fluorophores. distance of a single nucleotide) is unique to cNCBs. that lays the foundation for multi-color probe development using DNA/Ag NCs. 00–00 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 5 Page 14 of 33 . In terms of general applicability.1 Detection of DNA targets 35 40 View Article Online DOI: 10. fluorescence is enhanced by a proportional increase in the number of emissive clusters in the sample. in other words. Again. this feature is not shared by existing SNP probes. (A) Schematic of a quencher-mediated turn-on probe.166 This method eliminates the need of a guanine-rich enhancer to turn on fluorescence. 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. As displayed in Figure 6A.120 4. where events without a target cannot be easily differentiated from events with a mismatched target (both give a low signal. [vol]. It is this environmental sensitivity. A 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. 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]. However.166 (B) Schematic of hybridized DNA structures with a six-cytosine loop as nucleation site for silver cluster synthesis and SNP differentiation. the enhancer) that is physically shifted by only a small distance (e. 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). the ability to obtain measurably different ensemble activation colors using the same activator (e. cNCBs have successfully been validated on three SNPs with all four allelic variants. the wild-type duplex produced strong yellow fluorescence emission.g. Sequences around the SNP sites were not particularly chosen and all alleles studied produced effective cNCBs.17 As mentioned above.CREATED USING THE RSC REPORT TEMPLATE (VER. both are “off”).SEE WWW. No fluorescence was observed for the mutant-type duplex after the nucleation process. 10 15 can be used to quantify the amount of target (cNCB is a turn-on probe).ORG/ELECTRONICFILES FOR DETAILS Nanoscale Published on 06 June 2013. and on six SNPs with two allelic variants (which covered all six possible single-nucleotide substitution scenarios). but yields a mere 2-3 fold enhancement of fluorescence upon target binding. After the quencher (black) is displaced by a target (red).g. Other DNA probes based on fluorescent silver nanoclusters 4. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. [year]. the DNA target binds to the NC probe through a competitive process known as toehold displacement.1039/C3NR01601C Petty reported a turn-on nanocluster probe with silver clusters initially quenched via hybridization with a quencher strand.RSC.1) . 3.

11 The proposed on/off switching mechanism involves the invasion of the quencher strand into the 3' region of the NC-nucleation sequence. In a recent study by Ren. the adsorbed probes hybridize with the targets.173 demonstrating the compatibility between DNA/Ag NCs and fullerenes. DNA/Ag NCs were used as reporters in a GO-based sensor for multiplexed detection of DNA.1) . A similar observation of the self-dimer formation was reported by Yang and Vosch in their turn-off probe design. 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).2. The key advantage of this method lies in the emitter’s near-infrared electronic transition. this method differentiates single-nucleotide variations by the level of fluorescence intensity. NC-nucleation loop. thus changing the electronic environment around the silver cluster and resulting in a fluorescence quenching effect. Petty's new probe relies on silver clusters to condense the template. Wang was able to identify the SNPs responsible for sickle cell anemia using a DNA probe with a six-cytosine.171. In the presence of DNA targets.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.166 It has yet to be determined if the detection scheme displayed in Figure 6A is applicable to longer DNA targets.SEE WWW.170 A possible mechanism for dimer formation is the non-Watson-Crick basepairing mediated by silver ions. 00–00 | 15 This journal is © The Royal Society of Chemistry [year] View Article Online DOI: 10. the method requires silver clusters to be [journal]. rather than the spectrum of emission. 10 15 20 25 30 35 40 45 (5’-C3AC3AC3GC3A). forming rigid duplexes which are then released from the GO due to conformational changes. which enables DNA detection in serum-containing buffers (whose endogenous background fluorescence is low in the near-infrared spectral region). Additionally. Petty found the probe/target duplexes formed dimers.120 As show in Figure 6B.ORG/ELECTRONICFILES FOR DETAILS Nanoscale 5 Published on 06 June 2013. the probe was first hybridized with the wild-type and the mutant-type targets (T to A substitution). Petty found that fluorescence was enhanced through a proportional increase in the number of emissive clusters.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.RSC. For instance. Despite this success. the reporters labeled on DNA are quenched by GO.CREATED USING THE RSC REPORT TEMPLATE (VER.172 This releasing mechanism reverses the quenching effect and restores the fluorescence.122 which links cytosine homopolymers via cytosine-Ag+-cytosine interactions. 3.58 As an effective universal quencher that can initially switch off the fluorescence of reporters in turn-on detection. Detection of SNPs Several methods other than cNCBs have been proposed to differentiate SNPs using DNA/Ag NCs as indicators. forming two different duplexes. graphene oxide (GO) has been recently explored for DNA detection.1039/C3NR01601C Nanoscale Accepted Manuscript Page 15 of 33 . thereby creating an environment favoring the non-active state of the silver clusters. 4. whereas the wild-type duplex produced a strong yellow fluorescence emission. as is the case with cNCBs. hosting twice the amount of silver atoms in a dimer. Introduction of DNA targets and subsequent hybridization alter the cluster environment to restore fluorescence of the silver clusters. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. After NCnucleation process was completed on these duplexes. [year]. Interestingly.166 similar to the NCB results.167 This stoichiometry was determined via inductively coupled plasma atomic emission spectroscopy (ICP-AES). [vol]. little fluorescence was observed from the mutant-type duplex.

While researchers are eager to find more real-world use of fluorescent nanoclusters. [vol]. Ye used DNA/Ag NCs as fluorescent indicators for the amplicons in an isothermal amplification process. 3.1039/C3NR01601C 15 20 25 30 Fluorescent silver nanocluster probes have also been used for RNA detection. 5 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. Lastly. a large number of silver clusters were brought close to the electrode’s surface. Also for miRNA detection. While this method can differentiate CA. Zhang developed a simple miRNA sensor using DNA/Ag NCs to catalyze the reduction of hydrogen peroxide. The detected current was found directly proportional to the logarithm of the miRNA target concentration. Outlook 35 40 Fluorescent nanoclusters are interesting fluorophores that are currently being explored by many groups around the world. it may be possible to turn environmentally sensitive nanoclusters into a short-range. Page 16 of 33 View Article Online DOI: 10. it is not generally applicable to any other type of substitutions. enabling quantitative detection of target miRNA. the cytosine-facing abasic site produced a strong fluorescence emission. fluorescent distance reporter – a whole new 16 | [journal]. with these hairpin probes having binding sites for both miRNA targets and NC-nucleation probes. thiol-functionalized DNA hairpin probes were immobilized on the surface of a gold electrode. Detection of RNA targets . Here we outline some future directions in this exciting research area. The fluorescence of the DNA/Ag NC probe was quenched when it hybridized with a complementary miRNA target. 00–00 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript Published on 06 June 2013.1 Molecular rulers based on fluorescent silver nanoclusters With proper design and characterization. CG.SEE WWW.CREATED USING THE RSC REPORT TEMPLATE (VER. neurological diseases and viral infections. Fluorescence quenching was shown to follow a linear Stern-Volmer relationship over a range of target concentrations (from 20 nM to 1.ORG/ELECTRONICFILES FOR DETAILS Nanoscale synthesized after the probe/target hybridization is completed.177 In their method. it has yet to be shown that the method is applicable to a wide variety of target sequences with similar discriminatory results. Upon completion of NC-nucleation process. 4.176 In their approach. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.170. 5. Upon sequential binding of miRNA and NC-nucleation probes (which carry silver clusters). electrochemical methods have also been used.5 M).175 MicroRNA (miRNA) are small non-coding ribonucleic acids that can regulate the genes associated with cancer. [year].174 The nucleobases opposite and next to the abasic site (a tetrahydrofuran residual) control the formation of emissive silver clusters.1) .3. which will be discussed below. whereas the non-cytosine-facing abasic sites produced little fluorescence. basic questions remain and great efforts are needed to address these challenges.RSC. 5. Fluorescence measurements are not the only means by which DNA/Ag NCs have been used for the detection of miRNA. leading to a longer assay time. enabling catalytic reduction of hydrogen peroxide.e. and CT mutations. the amplicons are single-stranded DNA that can template the growth of highly emissive silver clusters. Yang and Vosch developed a microRNA detection probe using fluorescence quenching of silver clusters as the basis of detection (i. a turn-off probe).

180 switching dynamics-based.181 or plasmon coupling-based rulers. interactions between silver and cytosines4). but their labeling cost will be significantly lower (the nucleation precursors are cheap and essentially nonfluorescent).CREATED USING THE RSC REPORT TEMPLATE (VER.182 and can be an economic solution to study biomolecules’ small conformational changes due to various molecular interactions. fluorescent silver clusters can also be assembled at specific sites with great specificity.179 electron transfer-based.195-197 For example. but they give no information on the ligand-cluster binding geometry nor the location of metal cluster. To specifically identify and precisely quantify a large number of distinct molecular species present in complex biological systems. 3.g.193 In particular.SEE WWW. 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.178 nanometal surface energy transfer-based. NMR spectroscopy. silver ions were reported to interact specifically with the CG.189 branched DNA-based barcodes. Electrospray ionization mass spectrometry (ESIMS)5. including metallic barcodes.ORG/ELECTRONICFILES FOR DETAILS Page 17 of 33 Nanoscale 5 type of molecular ruler.3 In-situ or site-specific synthesis of fluorescent nanoclusters 30 35 40 45 One challenge in the development of metal cluster-based biosensors is that we don’t know exactly where the clusters are templated on the encapsulation agents. [year].187 direct-labelling barcodes.190 M13 DNA-based barcodes. fluorescence programmable nanotubes have the potential to realize low-cost barcodes in the near future. Fygenson has started to construct DNA nanotubes with spatially distributed hairpin protrusions for in-situ growth of fluorescent silver nanocluster. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. only indicates direct interactions between metal clusters and surrounding ligands (e.188.184 barcode chips.192. several barcode strategies have been developed.C+ base triplet in a triplex DNA.1) .2 Biological barcodes using fluorescent silver nanoclusters .196 More recently. [vol].191 While achieving excellent quantification results. the costly fluorescence labelling remains a challenge.185 sequencing-based barcodes. Several different strategies have been recently reported to achieve in-situ or site-specific synthesis of metal clusters on protein or DNA templates. This is particularly a problem when we are designing nanocluster sensors to monitor their surrounding microenvironments.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.197 leading to a method [journal].58 Although currently suffering from aggregation issues due to cytosine-Ag+-cytosine interactions.186. Yin recently used DNA origami techniques to construct structurally rigid DNA nanotubes that act as geometrically encoded fluorescent barcodes. To stiffen the DNA barcodes.7.1039/C3NR01601C Published on 06 June 2013. such as energy transfer-based.57 and inductively coupled plasma atomic emission spectroscopy (ICP-AES)167 have revealed the number of metallic atoms on a template molecule. Compared to organic dye labelling. 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. 5. M13 barcodes are soft (a stretch step is required191) and expensive (dense labelling with organic dyes and the required purification processes). View Article Online DOI: 10. on the other hand.183 polymer particle barcodes. 10 15 20 25 Another potential application of fluorescent silver nanoclusters is in biological barcodes as substitutes for conventional fluorophores.194 However. Such a ruler will be complementary to the existing spectroscopic rulers. 00–00 | 17 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 5.191 and quantum dot-based barcode beads.RSC.

ORG/ELECTRONICFILES FOR DETAILS Nanoscale 5 15 View Article Online DOI: 10. HPLC-MS can reveal whether or not HPLC separation succeeds in isolating pure emitters. Alternatively. the ability to limit silver cluster growth to hairpins on DNA nanotubes provided a unique means of estimating the chemical yield.CREATED USING THE RSC REPORT TEMPLATE (VER. a chemical yield of ~45% was obtained.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.167.14. especially the DNA/Ag NCs.198 Using ICP-AES to analyze DNA/Ag NCs separated by HPLC.31.g.198 making it unlikely to obtain TEM or AFM images of highly emissive. In the most recent review by Dickson.57.57 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.RSC. precluding interrogation of cluster species responsible for specific fluorescence emission. some cluster emitters still cannot be studied since they are not stable enough to be purified (e. 00–00 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript Published on 06 June 2013. [year].58 While the nucleation site was not controlled at the atomic level. followed by solvent evaporation prior to mass spectral analysis.32. Liquids are charged and aerosolized during electrospray ionization. 18 | [journal]. As a result. 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.58 By counting individual emitters along a nanotube with a known linear density of hairpins under a fluorescence microscope. even if a chemically pure cluster solution is used.1) . High performance liquid chromatography (HPLC) is often used to purify the fluorescent metal clusters. 5. This is important since distinct emitter species can have similar HPLC retention times. clusters that could easily convert into others due to oxidation. [vol].198 Even with HPLC-MS. single few-atom metal clusters. While the strong environmental sensitivity of some metal clusters has raised great interests in sensing applications. the images indicated the presence of crystalline nanoparticles (roughly 2 nm in diameter).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). losing their original emission signatures). 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. Fygenson used DNA hairpins for the sitespecific synthesis of fluorescent silver clusters on long DNA nanotubes. Petty reported ten silver atoms encapsulated on a specific DNA strand. suggesting each metal particle consisted of hundreds of gold or silver atoms.198 Compared to ICP-AES.22 ESI-MS was described as a problematic approach to study poly-disperse silver cluster populations and their associated optical properties. While these reports showed TEM195-197 or AFM196 images as proof of site-specific cluster synthesis. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.SEE WWW.167 O’Neil also identified silver atom/DNA ratios through the correlation of separately measured fluorescence and mass spectra. 10 of employing CG. Page 18 of 33 .1039/C3NR01601C While we anticipate a broader impact of fluorescent nanoclusters in chemical and biological sensing in the next few years.C+ base triplet for site-specific synthesis of silver clusters on DNA. 3. highly fluorescent metal clusters should consist of less than 30 atoms. Nevertheless.

10 it is questionable to use gas-phase data to infer sizes of solution-phase emitters. Gwinn identified both the charge and the number of silver atoms contained in a cluster using mass spectra of compositionally pure DNA/Ag NCs. the strong polarization dependence of single emitters. conformation and geometrical structure determination. [year]. DFT calculations still can provide some qualitative insights into the DNA/Ag NC systems and the observations from experiments. 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.200 size. [vol]. thiolate ligands form Au(SR)2 and Au2(SR)3 motifs that bind to the gold core. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32.32 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.RSC. showing agreement with the X-ray crystal structure determination.12.202 and photophysical characterization at the single-molecule level6.ORG/ELECTRONICFILES FOR DETAILS Page 19 of 33 Nanoscale 5 View Article Online DOI: 10. which is necessary for the electronic structure investigations. despite the great potential of silver clusters in biomedical applications. known properties at this moment.1039/C3NR01601C 5.54 The variation of charge with the number of silver atoms.54.54 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. This is mainly due to a large number of possible ligandcluster binding geometries in DNA/Ag NC structures. stoichiometry determinations of specific emissive species remain technically challenging. The ligand orientation can have a significant effect on the electronic structures and optical properties of metal clusters.54.203 are also critically important for bringing the metal cluster research to the next level.199 not to mention the prediction of optical absorption of DNA/Ag NCs. Recently.1) .36. and single-molecule characterization of fluorescent nanoclusters 15 20 25 30 35 40 45 Other than advances in synthesis and characterization.SEE WWW.202 Knowledge of these binding motifs led to theoretical predictions of the geometric structure of Au25(SR)18. because some of the silver atoms on DNA may not even participate in the visible emission process. 00–00 | 19 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript Published on 06 June 2013. As pointed out by Jin. suggested that all chemically stable DNA/Ag NCs are rodshaped emitters.30.CREATED USING THE RSC REPORT TEMPLATE (VER. It is challenging to make truly mono-disperse metal clusters with desired. together with the evidence from theoretical calculations205 and cryogenic spectra of single-molecule emitters.199.204 On the other hand. structure determination. . Strand interactions may add or remove silver atoms from a nanorod or disrupt [journal]. as synthetic methods yield multiple absorption bands associated with distinct types of clusters.22 Even though we are able to obtain metal atom/template ratios for most of the purified emissive species.6.5 Theoretical modelling. having eleven silver atoms on a DNA showing green fluorescence may not necessarily mean that Ag11 is green. we don’t even know the geometric structure and optical properties of even the simplest DNA/Ag NCs. fundamental understanding of metal clusters through quantum chemical calculations.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. and the fluorescence activation and color switch observed in NCBs may be due to changes in rod length. Precise control of metal clusters at the atomic level is needed to enable such a predictive design of metal clusters.22 In addition. we still cannot claim that we know the correlation between the cluster stoichiometry and the emission.201. spectral purity in the emission spectrum does not necessarily mean chemical purity.22. For instance.167 Thus. 3.8.

including mono-disperse and sitespecific synthesis. [vol]. theoretical modelling. To bring the noble metal nanocluster research to the next level.Y. 10 15 20 In this review we have discussed the recent progress of fluorescent silver nanoclusters and their applications in DNA detection. 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. Acknowledgements 25 We thank Drs. Welch Foundation (F-1833 to H.RSC. and single-molecule characterization of fluorescent nanoclusters.). the activatable and color-switchable properties of DNA-templated silver nanoclusters (DNA/Ag NCs) have led to the invention of NanoCluster Beacons (NCBs).1) . Conclusion . This work was financially supported by Robert A. Page 20 of 33 View Article Online DOI: 10. while they hold great promise for cost-effective detection compatible with DNA nanotechnologies. Batson and M. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. 3. James Werner and Jennifer Martinez at Los Alamos National Laboratory for providing great assistance and advice in our research. Babin for proofreading. In particular. While more characterization evidence is needed to valid this theory. it is exciting to see a possible mechanism be proposed.22 This review is driven by the humbling realization that a simple metal cluster still holds profound questions and rich possibilities.A. but because a tremendous amount of science is not yet understood and needs to be explored. DNA/Ag NCs are excellent systems to investigate size-dependent emission and understand unique photophysics in metal clusters.1039/C3NR01601C Published on 06 June 2013. 20 | [journal]. [year]. We also thank the group members R.SEE WWW. NanoCluster Beacons are promising molecular probes with great potential.CREATED USING THE RSC REPORT TEMPLATE (VER. In their view. 00–00 This journal is © The Royal Society of Chemistry [year] Nanoscale Accepted Manuscript 6.ORG/ELECTRONICFILES FOR DETAILS Nanoscale 5 it into disjoint pieces. researchers need to simultaneously tackle a number of challenges.-C. not only because their signal transduction mechanism is fundamentally different from those of the existing molecular probes. structure determination.

00–00 | 21 This journal is © The Royal Society of Chemistry [year] View Article Online DOI: 10. Y. M. (2) J. [journal]. As an undergraduate research assistant under the supervision of Professor Zhenghui Hu. he and coworkers invented NanoCluster Beacons. 15 20 Judy M. Zheng. carbon nanotubes and graphene. His graduate research is focused on fluorescence nanomaterials characterization and 3D single-molecule tracking. [vol]. R. Urano.1) . Individual water-soluble dendrimer-encapsulated silver nanodot fluorescence Journal of the American Chemical Society. 2010. Dickson. 124. water-soluble. Alford. size-tunable gold quantum dots Physical Review Letters. At LANL. 2004. Zheng. R.1039/C3NR01601C Nanoscale Accepted Manuscript Page 21 of 33 . At Johns Hopkins. L. Ogawa.ORG/ELECTRONICFILES FOR DETAILS Nanoscale 5 10 Published on 06 June 2013. which won a 2011 R&D 100 Award. Yeh joined the Biomedical Engineering Department at the University of Texas at Austin in 2012 as an assistant professor. Highly fluorescent. 35 Dr. 13982. [year]. Kobayashi. During her graduate study. 25 40 Professor Tim Yeh (Hsin-Chih Yeh) obtained his BS degree from National Taiwan University. References (1) 45 H. and PhD from Johns Hopkins University. R.RSC. Zhang. Cong Liu obtained his BS degree in Optical Engineering at Zhejiang University. P. 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. (3) J. M. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. she worked on the synthesis and optical properties characterization of noble metal nanostructures. developing MEMS-based photonic switches for telecommunications. he joined Professor Tim Yeh's group at University of Texas at Austin as a PhD student in the Biomedical Engineering Department. in the Center for Integrated Nanotechnologies. BioMEMS and new biosensing techniques based on fluorescence dynamics. In 2012. Choyke. 2002. Los Angeles. cancer biomarker detection. all in mechanical engineering. New strategies for fluorescent probe design in medical diagnostic imaging Chem Rev. Based on these findings. M. 077402. 93. Her current research is to develop new molecular probes based on fluorescent noble metal nanoclusters for biomedical applications. His research interests include nanobiosensor development. in San Diego from 98-03 as an R&D engineer. Yeh received his postdoctoral training at Los Alamos National Laboratory from 09-12. After graduation from 30 UCLA. 110. he worked on hemodynamic modelbased fMRI BOLD signal analysis.CREATED USING THE RSC REPORT TEMPLATE (VER. 3. his research focused on single-molecule spectroscopy. Dr. she joined Professor Tim Yeh’s group at University of Texas at Austin as a Postdoctoral Associate.SEE WWW. 2620. MS degree from University of California. China in 2012. Hangzhou. In 2012. he worked at Optical Micro Machines Inc. C. he discovered controlled fluorogenic and color-switching phenomena on DNA-templated silver nanoclusters. 3D molecular tracking and super-resolution imaging. Dickson.

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utexas.yeh@austin.: +1-512-471-7931. University of Texas at Austin. 1. Colour graphic 2. Tel. Cong Liu and Hsin-Chih Yeh* .1039/C3NR01601C Table of Contents Fluorescent silver nanoclusters as DNA probes Department of Biomedical Engineering. Cockrell School of Engineering.edu. Text Fluorescent silver nanoclusters have been used to create a new class of DNA probes that are not only activatable and programmable. Nanoscale Accepted Manuscript Published on 06 June 2013. USA * Author to whom correspondence should be addressed. Austin.Page 33 of 33 Nanoscale View Article Online DOI: 10. E-mail: tim. but can also be excited by a single UV source. Downloaded by University of Texas Libraries on 12/06/2013 18:04:32. Fax: +1-512-471-0616. Obliosca. TX 78712. Judy M.