A complementary palette of fluorescent silver nanoclustersw

Jaswinder Sharma, Hsin-Chih Yeh, Hyojong Yoo, James H. Werner and

Jennifer S. Martinez*

Received 24th December 2009, Accepted 9th March 2010

First published as an Advance Article on the web 26th March 2010

DOI: 10.1039/b927268b

We report the synthesis and photophysical properties of silver-

nanoclusters templated on DNA, with fluorescence excitation

and emission at distinct wavelengths that are tuned to common

laser excitation wavelengths.

The complementary base pairing and rapid enzymatic means

of replication have made DNA an attractive tool for the direct

detection of specific organisms and diseases.1 In addition,

many of the same features that make DNA useful in medical

diagnostics make DNA an attractive template for the synthesis

of two- and three-dimensional nanostructures and also for

templating and organizing inorganic nanoparticles.2–4 In the

medical diagnostics arena, the molecular specificity of DNA is

often used to accurately confirm the presence of one or many

DNA molecules. This simultaneous measurement of multiple

DNA sequences (multiplex analysis) necessitates numerous

fluorophores each with high quantum yield and with distinct

emission. Here, we exploit the chemical and structural speci-

ficity of DNA to not only template nanomaterials (fluorescent

nanoclusters), but to also enable their use as small fluorescent

tags for multiplex analysis of DNA or proteins.

Fluorescent metal nanoclusters (o1 nm) are collections of

two to tens of atoms of gold or silver. While for gold

nanoclusters the fluorescence emission is thought to scale as

a function of the number of atoms within the cluster, for silver

nanoclusters the relation of atom number to fluorescence

emission is less clear. Nevertheless, noble metal nanoclusters

are gaining much interest because of their desirable photo-

physical properties and smaller size than quantum dots. While

quantum dots have tremendous photostability and brightness,

their large size (larger than most proteins or protein complexes)

and potential toxicity remain challenges to be solved. As a

complement to quantum dots and molecular fluorophores,

fluorescent nanoclusters have been produced using templates

of dendrimers and polymers,5 small molecular ligands,6 or

within biological materials of interest, such as DNA.7 Among

the DNA bases, cytosine is well known for its ability to bind

Ag+ ions. Taking advantage of Ag+-base binding, Dickson,

Petty, and Fygenson’s groups have each developed nice

strategies to synthesize Ag-nanoclusters templated, most typi-

cally, in cytosine rich single-stranded DNA.7 While Dickson

and co-workers produced individual silver nanoclusters with

distinct emissions, most of the clusters were produced under

different buffer and salt conditions,7e which limits their practical

use in multiplex assays. Conversely, while Fygenson and

colleagues produced clusters under one reaction condition,

the resultant clusters had broad excitation and emission

profiles.7d Because most multiplex assays are performed under

one buffer condition (typically physiological pH), there is still

a need for a palette of nanoclusters that each emit at different

wavelengths in the same conditions (i.e. pH, buffer, temperature).

Here, we synthesize and photophysically characterize

Ag-nanoclusters, which were templated on DNA under one

reaction condition (near physiological pH), with distinct,

and more narrow, excitation and emission profiles tuned to

common laser excitation wavelengths.

In a typical synthesis protocol, 15 mM DNA (Integrated

DNA Nanotechnologies, HPLC purified) and 90 mM of

AgNO3 (Sigma Aldrich) were mixed by vortexing in an

Eppendorf tube, in phosphate buffer (20 mM, pH 6.6) or

water, and then kept at room temperature in the dark. After

20 minutes, 90 mMof freshly prepared NaBH4 (Sigma Aldrich)

was added, thoroughly mixed, and then stored without further

mixing at room temperature in the dark.7e Four DNA strands

of different sequence and length were selected after testing

numerous DNA sequences (Fig. 1). We find that sequence Ag1

gives Ag-nanoclusters (AG1) emitting in the green region

(lexc/lemi = 460 nm/550 nm) of the visible spectrum while

sequence Ag2 generates Ag-nanoclusters (AG2) emitting in the

orange region (lexc/lemi = 530 nm/600 nm) of the visible

spectrum. Similarly, Ag-nanoclusters (AG3), emitting in the

red region (lexc/lemi = 595 nm/650 nm), were produced by

using the sequence Ag3, while the use of sequence Ag4 resulted

in Ag-nanoclusters (AG4) emitting in the near-infrared region

(lexc/lemi = 640 nm/700 nm) of the spectrum. More synthesis

details are provided in Fig. S4, ESIw. Nearly identical spectra

are obtained when the clusters are synthesized in phosphate

buffer (Fig. 1) or in water (Fig. S3, ESIw). We note that the

nanoclusters produced can be excited at common laser excita-

tion wavelengths, enabling future multiplexed analysis. For

example, the green (AG1) clusters are readily excited by the

488 nm line of an argon ion laser, while the orange (AG2)

clusters are readily excited by 532 nm emission from a

Nd :YAG. In addition, the red (AG3) clusters are well excited

by the 594 nm line of a HeNe laser and the near-IR emitting

AG4 clusters can be excited by either the 632 nm line of a

HeNe or by a 635 nm laser diode.

While the AG1 nanoclusters (green-emitting) are not as

bright as the other clusters, and take longer to develop (12 h),

we find that they are stable at room temperature for over a

Center for Integrated Nanotechnologies, Los Alamos NationalLaboratory, Los Alamos, New Mexico, 87545, USA.E-mail: jenm@lanl.gov; Fax: +1505-665-9030; Tel: +1505-665-0045w Electronic supporting information (ESI) available: Fluorescencespectra in water, fluorescence correlation spectroscopy, stability ofnanoclusters, and non-specific binding of nanoclusters with variousproteins. See DOI: 10.1039/b927268bz J. S. and H.-C. Y. equally contributed to this article.

3280 | Chem. Commun., 2010, 46, 3280–3282 This journal is �c The Royal Society of Chemistry 2010

COMMUNICATION www.rsc.org/chemcomm | ChemComm

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week and at 4 1C for over a month. In contrast to AG1

nanoclusters, the near-infrared emitting clusters, AG4, are

very bright, develop within a few minutes, but are only stable

for four days at room temperature and two weeks at 4 1C.

AG2 nanoclusters (orange-emitting) develop quickly and

remain stable for weeks at 4 1C. AG3 nanoclusters (red-emitting)

develop within an hour, but unlike the other clusters oxidize

within 2–3 days, with a new peak developing (505 nm/575 nm),

resulting in a transition of nanocluster emission from red to

orange, and finally to yellow. The remaining clusters (AG1, 2,

and 4) convert to nonfluorescent solutions from one to four

weeks time. However, the fluorescence for every nanocluster

can be regained with the addition of another equivalent of

NaBH4 (Fig. S4, ESIw).To further study the stability of these nanoclusters in the

presence of high salt conditions, we added different molar

ratios of NaCl. Near-infrared emitting nanoclusters showed

more stability in the presence of NaCl as compared to the

red (AG3), orange (AG2), and green (AG1) emitting

Ag-nanoclusters (Fig. S5, ESIw). We assume that the enhanced

stability of AG4 is as a result of its longer nucleotide length,

which may help shield the nanocluster from salt induced

aggregation. Non-specific binding of nanoclusters with protein

molecules was also studied (Fig. S6, ESIw).The fluorescence quantum yields (ff) for each of the four

clusters were measured using the gradient method,8 with

fluorescein (ff = 0.79), rhodamine 101 (ff = 1.00), cresyl

violet (ff = 0.54), and zinc phthalocyanine (ff = 0.30)

as standard fluorophores, with known quantum yields,

respectively (Table 1). We find that the red and near-IR-

emitting species, AG3 and AG4, produced by longer DNA

sequences, had quantum yields greater than 50% and are the

most promising as biolabels for ultra-sensitive, potentially

single-molecule, multiplex analysis.

In addition to quantum yields, we performed time-

correlated single photon counting (TCSPC) on all of the four

nanoclusters synthesized, and fluorescence correlation spectro-

scopy (FCS) on one of the brightest clusters, AG4. TCSPC

was used to measure the fluorescence lifetimes of the nanoclusters

while FCS was used to measure cluster size, estimate cluster

concentration (which leads to an estimate of the extinction

coefficient), and measure the brightness per cluster at the single

molecule level. The lifetimes for all fluorescent clusters were in

the low ns, which is consistent with previous measurements of

gold and silver nanoclusters.5c The fluorescence decay of the

AG4 cluster is well fit by a single exponential (Fig. S1, ESIw).However, two exponential components are needed to fit the

fluorescence decays of AG1, AG2, and AG3 (Fig. S1, ESIw).Interestingly, excitation scans performed at differing emission

wavelengths for each cluster shift little, indicating a single

species dominates the fluorescence spectra for each cluster.

The fact that the fluorescent lifetimes are two-component for

three of the nanoclusters points to some type of heterogeneity

(static or dynamic) in the nanocluster population. We are

currently working on experiments to further explore the nature

of this heterogeneity.

As stated above, we used FCS to measure cluster size,

estimate cluster concentrations, and determine the brightness

per cluster on the single molecule level for one of the brightest

of the four clusters, AG4. For the AG4 cluster, we measured a

hydrodynamic radius of 3 nm (see ESIw), a size which indicates

the clusters are associated with a DNA complex. In addition to

a measure of cluster size, we used FCS to obtain cluster

concentration. Combined with standard UV-VIS absorption

measurements, we obtain an extinction coefficient of 4.2 �104 M�1 cm�1 for AG4.

The average fluorescence detection rate per nanocluster is

an important figure of merit for potential single molecule

biophysical and bioanalytical applications. We obtained the

average photon detection rate for the AG4 cluster from FCS

measurements by dividing the measured count rate on a single

photon counting detector by the average occupancy of

fluorescent clusters in the detection volume. This calculation

yielded a maximum detection rate of 32 kHz for a single

AG4 cluster, compared to a detection rate of 79 kHz for

Fig. 1 Excitation and emission spectra of Ag-nanoclusters templated

by different DNA strands (as indicated). (a) Nanoclusters fluorescing

green, (b) nanoclusters fluorescing orange, (c) nanoclusters fluorescing

red, and (d) nanoclusters fluorescing in the near-infrared region. All

DNA sequences shown 50–30.

Table 1 Photophysical properties of silver nanoclusters

NanoclusterEmissionpeak/nm

DNAsize/nt t/ns f (%) ff

AG1 550 22 2.5 38 0.002 � 0.00020.6 62

AG2 600 29 2.9 59 0.10 � 0.010.8 41

AG3 650 34 3.5 64 0.64 � 0.011.3 36

AG4 700 46 3.6 0.52 � 0.03

Cy5-DNA 670 15 2.1 51 0.271.2 49

t, f, and ff are the fluorescence lifetime, fractional intensity for each

component, and fluorescence quantum yield, respectively. The error

for lifetime measurements is �0.02 ns.

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a Cy5-DNA conjugate (Cy5-50-CTCTTCAGTTCACAG)

measured under the same excitation (635 nm pulsed laser,

68.4 kW cm�2) and emission collection conditions (Fig. 2a).

AG4 is brighter than Cy5 at low excitation power (o103W cm�2)

and dimmer at higher excitation power (>103 W cm�2) due to

significant differences in blinking dynamics under the two

excitation conditions.5c At the illumination density of

4.27 kW cm�2, the AG4 clusters blinked on and off with a

2.7 ms time scale, while the Cy5 on DNA blinked with a 14.2 mstime scale (see Fig. S2 and Table S1, ESIw). We note that the

faster blinking dynamics of AG4, as compared to Cy5, make

this nanocluster more useful than Cy5 for biophysical studies

that use fluorescence fluctuations to probe biomolecular

dynamics on tens of microsecond timescales—a temporal

range that often overlaps fast biomolecular structural inter-

conversions such as protein folding.9 Another important feature

of the Ag nanoclusters was their superior photostability

compared to that of organic dyes. Ten consecutive FCSmeasure-

ments of AG4 and Cy5-DNA are shown in Fig. 2b and c.

The autocorrelation curves of AG4 overlap well, while those of

Cy5-DNA gradually increase in amplitude after each measure-

ment run, due to photobleaching of Cy5 under the same

illumination conditions.

In summary, we report four DNA sequences that template

Ag-nanoclusters fluorescing at different wavelengths, syn-

thesized under the same solution conditions, that are tuned

to common laser excitation wavelengths. These Ag-nanoclusters

open up opportunities for in vivo and in vitro multiplex

bioassays. The ease of synthesis, small size, and high photo-

stability of these Ag-nanoclusters may make them fluorescent

probes of choice for biological studies. We expect that these

nanoclusters will also pave a path for further understanding of

the interaction of DNA bases with Ag and Ag-nanoclusters.

This research is supported by the Los Alamos National

Laboratory Directed Research and Development (LDRD)

and a Director’s Postdoctoral Fellowship to J.S. This work

was performed at the Center for Integrated Nanotechnologies,

a U.S. Department of Energy, Office of Basic Energy Sciences

user facility.

Notes and references

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2 (a) N. C. Seeman, Nature, 2003, 421, 427; (b) N. C. Seeman,Biochemistry, 2003, 42, 7259; (c) C. Lin, Y. Liu, R. Sherri andH. Yan, ChemPhysChem, 2006, 7, 1641; (d) C. Lin, Y. Liu andH. Yan, Biochemistry, 2009, 48, 1663.

3 (a) J. Sharma, R. Chhabra, Y. Liu, Y. Ke and H. Yan, Angew. Chem.,Int. Ed., 2006, 45, 730; (b) J. Sharma, R. Chhabra, A. Cheng,J. Brownell, Y. Liu and H. Yan, Science, 2009, 323, 112;(c) E. B. Andersen, M. Dong, M. Morten, Ku. Jahn, R. Subramani,W. Mamdouh, M. M. Golas, B. Sander, O. Holger, L. P. Cristiano,J. S. Pedersen, V. Birkedal, B. Victoria, F. Besenbacher, K. V. Gothelfand J. Kjems, Nature, 2009, 459, 73.

4 (a) G. Shemer, O. Krichevski, G. Markovich, T. Molotsky, I. Lubitzand A. B. Kotlyar, J. Am. Chem. Soc., 2006, 128, 11006; (b) N. Ma,E. H. Sargent and S. O. Kelley, Nat. Nanotechnol., 2009, 4, 121;(c) J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys.Chem., 2007, 58, 409.

5 (a) J. Zheng and R. M. Dickson, J. Am. Chem. Soc., 2002, 124,13982; (b) J. Zheng, J. T. Petty and R. M. Dickson, J. Am. Chem.Soc., 2003, 125, 7780; (c) T. Vosch, Y. Antoku, J. C. Hsiang,C. I. Richards, J. I. Gonzalez and R. M. Dickson, Proc. Natl. Acad.Sci. U. S. A., 2007, 104, 12616; (d) Y. Bao, C. Zhong, D. M. Vu,J. P. Temirov, R. B. Dyer and J. S. Martinez, J. Phys. Chem. C,2007, 111, 12194.

6 Y. Bao, H.-S. Yeh, C. Chang, S. Ivanov, J. Sharma, M. L. Neidig,D. M. Vu, A. P. Shreve, R. B. Dyer, J. H. Werner andJ. S. Martinez, J. Phys. Chem. C, 2010, DOI: 10.1021/jp909580Z.

7 (a) J. Zheng, C. Zhang and R. M. Dickson, Phys. Rev. Lett., 2004, 93,077402; (b) J. T. Petty, J. Zheng, N. V. Hud and R. M. Dickson, J. Am.Chem. Soc., 2004, 126, 5207; (c) C. M. Ritchie, K. R. Johnsen,J. R. Kiser, Y. Antoku, R. M. Dickson and J. T. Petty, J. Phys. Chem.C, 2007, 111, 175; (d) E. G. Gwinn, P. O’Neill, A. J. Guerrero,D. Bouwmeester and D. K. Fygenson, Adv. Mater., 2009, 20, 279;P. O’Neill, L. R. Velazquez, D. G. Dunn, E. G. Gwinn andD. K. Fygenson, J. Phys. Chem. C, 2009, 113, 4229; (e) C. I. Richards,S. Choi, J. C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y.-L. Tzengand R. M. Dickson, J. Am. Chem. Soc., 2008, 130, 5038.

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Fig. 2 (a) Brightness per AG4 nanocluster as determined by FCS,

along with reference Cy5 dye on a short 15 nt oligo. The emission rate

of both the AG4 nanoclusters and Cy5 on DNA reached a plateau at

an excitation intensity of 68.4 kW cm�2. (b) and (c) Ten consecutive

autocorrelation curves of AG4 and Cy5-DNA, respectively. Each FCS

measurement was 15 s and an excitation intensity of 68.4 kW cm�2 was

used. The excitation was provided by a 635 nm pulsed laser source

from PicoQuant.

3282 | Chem. Commun., 2010, 46, 3280–3282 This journal is �c The Royal Society of Chemistry 2010

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