Analytica Chimica Acta 644 (2009) 78–82

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Analytica Chimica Acta

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DNA based gold nanoparticles colorimetric sensors for sensitive and selective
detection of Ag(I) ions
Bingling Li 1 , Yan Du 1 , Shaojun Dong ∗
Graduate School of the Chinese Academy of Sciences, Beijing, 100039, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In this work, we reported both unlabeled and labeled sensing strategies for Ag(I) ions detection by using
Received 3 March 2009 the DNA based gold nanoparticles (AuNPs) colorimetric method. In the unlabeled strategy, C-base riched
Received in revised form 14 April 2009 single strand DNA (C-ssDNA) enwinded onto AuNPs to form AuNPs/C-ssDNA complex. In the labeled
Accepted 16 April 2009
method, sulfhydryl group modified C-ssDNA (HS-C-ssDNA) was covalently labeled on AuNPs to produce
Available online 23 April 2009
AuNPs-S-C-ssDNA complex. In both strategies, C-ssDNA or HS-C-ssDNA could enhance the AuNPs stability
against the salt-induced aggregation. However, the presence of Ag(I) ions in the obtained AuNPs/C-ssDNA
Keywords:
or AuNPs-S-C-ssDNA complex would decrease such stability to display purple even blue colors due to the
Colorimetric sensors
Gold nanoparticles
formation of Ag(I) ions mediated C-Ag(I)-C base pairs. Through this phenomenon, Ag(I) ions could be
Ag(I) ions detected qualitatively and quantitatively using both unlabeled and labeled sensing strategies. Compared
Deoxyribonucleic acid (DNA) with the labeled method, the unlabeled strategy avoided the label and separation steps in common
Cytosine base sensors, which may thus save both the time and cost for the detection. Nevertheless, the labeled strategy
provided more sensitive, stable and controllable sensing results compared with the unlabeled method. By
the labeled strategy, 12 nM Ag(I) ions could be observed directly by naked eyes, and the lowest detectable
concentration of 0.59 nM was gotten under by the UV–vis spectra measurement, which was one of the
most sensitive results among DNA based AuNPs colorimetric sensors for metal ions.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Recently, colorimetric sensors have drawn more and more atten-
tion to decrease the experimental cost, simplify the sensing process,
and even only utilize naked eyes [1,2] instead of the complex instru-
ments. One of the most popular colorimetric techniques makes use
of gold nanoparticles (AuNPs) as reporting probes [3–5]. As metallic
nanoparticles, AuNPs are easy to synthesize and display high extinc-
tion and strong size- and distance-dependence characters [6]. Once
the AuNPs are induced to aggregate, their plasmon peaks at 520 nm
decrease (the ones at 650 nm increase) and the color embodies
a visible change from red to purple even blue [6]. Such obvious
color change is sensitive, thus it is considered ideal in designing
colorimetric sensing platforms. It has been proved that using either
unlabeled or labeled AuNPs as colorimetric reporters, unfolded sin-
gle strand deoxyribonucleic acid (ssDNA) can be well discriminated
from the folded ssDNA or the double strand DNA (dsDNA) [6,7]. A
lot of DNA based AuNPs colorimetric sensors are thus developed,
∗ Corresponding author at: State Key Laboratory of Electroanalytical Chem-
istry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun, Jilin, 130022, PR China. Tel.: +86 431 85262101; fax: +86 431 85689711.
E-mail address: dongsj@ciac.jl.cn (S. Dong).
1
These authors contribute equally to this paper.
including DNA sensors [6–9], DNAzyme sensors [10] and aptasen-
sors [11–13]. And the targets cover the metal ions [10,14–16], small
molecules [11,17] and proteins [18,19], significantly extending the
applications of AuNPs based biosensing strategies.
Ag(I) ions are reported to be greatly toxic to a lot of bacteria,
viruses, algae and fungi [20], and such antibacterial activity of Ag(I)
ions has been employed into many application fields (such as toi-
letry, timbering, clinical material). However, once the concentration
of Ag(I) ions is high enough, they can still bring harmful side-effects
to the environments. Additionally, Ag(I) ions are considered to be
one of the heavy metal ions that can bring environmental pollutant
for water resources [20]. Therefore, the recognition of Ag(I) ions
should be an important part of chemical research. But until now
the effective methods to test and standardize Ag(I) ions are still
limited and relatively complex [20–22].
In a recent report, Ono et al. found a novel Ag(I) ions mediated
base pair in DNA duplexes [23]. As reported, around neutral pH,
cytosine–cytosine (C–C) mismatches selectively capture Ag(I) ions
by forming strong C–Ag(I)–C base pairs in DNA duplexes and such
C–Ag(I)–C base pairs can stabilize the duplexes in turn. Our sensor
is designed based on this property. In this work, we report simple
and sensitive DNA based colorimetric sensors for Ag(I) ions using
the unlabeled or labeled AuNPs as probes for the first time. In the
unlabeled strategy (Fig. 1, Strategy A), the C-base riched sin-
gle strand DNA (C-ssDNA) enwind onto the AuNPs to form the

0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2009.04.022
 B. Li et al. / Analytica Chimica Acta 644 (2009) 78–82
Fig. 1. The strategies of DNA based unlabeled (A) and labeled AuNPs colorimetric sensors (B) for Ag(I) ions detection.
AuNPs/C-ssDNA complexes. In the labeled strategy (Fig. 1, Strat-
egy B), sulfhydryl group modified C-ssDNA (i.e., HS-C-ssDNA) are
covalently labeled onto the AuNPs to produce the AuNPs-S-C-ssDNA
complexes. In both strategies, C-ssDNA or HS- C-ssDNA can enhance
the AuNPs stability against the salt-induced aggregation. While,
the addition of Ag(I) ions could decrease such stability, due to
the formation of Ag(I) ions mediated C–Ag(I)–C base pairs. There-
fore, when enough salt exists, the AuNPs colloidal solutions with
Ag(I) ions will aggregate to display purple even blue colors, from
which Ag(I) ions can be recognized. This sensing platform not only
enlarges the target library of the DNA based AuNPs colorimetric
sensors, but also provides a new simple and economical method to
detect Ag(I) ions successfully.
2. Experimental
2.1. Materials
The C-ssDNA (5 CTC TCT CCA ACC TCT CTC 3 ) and the HS-
C-ssDNA, (5 HS-(CH2 )6 -CTC TCT CCA ACC TCT CTC 3 ) were
synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai,
China). Chloroauric acid (HAuCl4 ) was purchased from Shang-
hai Chemical Reagent Company (Shanghai, China). Sodium citrate
and sodium chloride were purchased from Beijing Chemical
Reagent Company (Beijing, China). Tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) was purchased from Bio Basic Inc. (Markham
Ontario, Canada). Gold nanoparticles (AuNPs) were synthesized
with the procedure as shown in the supporting information (SI).
HS-C-ssDNA modified AuNPs (AuNPs-S-C-ssDNA) complex was fab-
ricated by the method indicated in SI. Other reagents and chemicals
were at least analytical reagent grade. All the stocks and buffer solu-
tions were prepared by using autoclaved double-deionized water.
Oligonucleotides mentioned in this work were all dissolved in
the HEPES buffer (10 mM HEPES–NaOH, 140 mM NaNO3 , pH 7.4),
and stored at 4 ◦ C before use. The concentrations of oligonucleotides
were determined using the 260 nm UV absorbance.
2.2. Instrumentation
Absorption spectra were recorded on a Cary 500 Scan
UV–vis–NIR spectrophotometer (Varian, Harbor City, CA, USA) at
room temperature (15–18 ◦ C). Transmission electron microscopy
79
(TEM) measurements were made on a Hitachi H-8100 transmission
electron microscope which operated at an accelerating voltage of
200 kV.
2.3. Procedure
2.3.1. Unlabeled strategy for the colorimetric detection of
Ag(I)ions
As illustrated schematically in Fig. 1A for the unlabeled detection
of Ag(I) ions, AuNPs (130 ␮L) were first mixed with of 4 ␮M C-ssDNA
(20 ␮L) to form the AuNPs/C-ssDNA complex. Then, 20 ␮L of Ag(I)
ions was added. The obtained mixture was incubated overnight to
offer enough time for reaction, after which AuNPs/C-ssDNA/Ag(I)
colloidal solution was formed. Afterward, 1 M NaNO3 (20 ␮L) was
added to produce color change. After two min, 330 ␮L of H2 O was
injected into the mixture before UV–vis measurement. Calibration
curve was made based on the data collected on the first minute
after the injection of water.
2.3.2. Labeled strategy for the colorimetric detection of Ag(I) ions
As schematically shown in Fig. 1B for the labeled detection
of Ag(I) ions, AuNPs-S-C-ssDNA complex (200 ␮L) and Ag(I) ions
(30 ␮L) were first mixed and kept for ∼30 min. Then, 1 M NaNO3
(20 ␮L) was added into the mixture. After two min, 260 ␮L of
H2 O was added before UV–vis measurement. Calibration curve was
made based on the data collected on the first minute after the
injection of water.
2.4. The selectivity of the colorimetric sensors
In control experiments for both unlabeled and labeled strate-
gies, Ag(I) ions were simply displaced or mixed by other metal
ions/other metal ions with ethylene diamine tetraacetic acid (EDTA)
(i.e., Cu(II), Pb(II), K(I), Li(I), Mg(II), Na(I), Mn(II), Ni(II), Ba(II), Cd(II),
Zn(II), Co(II), Hg(II) or Ca(II) in this work).
3. Result and discussion
3.1. Unlabeled colorimetric strategy to detect Ag(I) ions
To get a simplified sensing process, we preferentially chose unla-
beled strategy to realize the colorimetric detection of Ag(I) ions.
80 B. Li et al. / Analytica Chimica Acta 644 (2009) 78–82

ing result. As shown in Fig. 2B, the ratio of AuNPs surface plasma
absorption at 650–520 nm (A650 /A520 ) is enhanced with increase of
Ag(I) ions, illustrating the aggregation of the AuNPs/C-ssDNA/Ag(I)
solution.
Through the experimental result and mechanism mentioned
above, Ag(I) can be quantitatively detected. According to UV–vis
spectra measurements (Fig. 2), a linear range from 38.4 nM to
1.94 ␮M is obtained, with the lowest detectable concentration
of 19.2 nM. When the concentration of Ag(I) ions is as low as
52 nM (Fig. 2A, 52 nM), a slight color change from Ag(I) ions-free
AuNPs/CRS colloidal solution can be observed by naked eyes.

3.2. The selectivity of the unlabeled strategy

The selectivity of the unlabeled strategy is investigated in the

control experiments. As shown in the photographs of Fig. 3 and S1A,
in the presence of 2.1 ␮M Ag(I) ions, AuNPs/C-ssDNA/Ag(I) solu-
tion turned purple after 0.12 M NaNO3 was added. When Ag(I)
ions were replaced by other ions, such as 6.3 ␮M of Cu(II),
Pb(II), K(I), Li(I), Mg(II), Na(I), Mn(II), Ni(II), Ba(II), Cd(II), Zn(II),
Co(II) or Ca(II), the obtained AuNPs/C-ssDNA/Metal ions solution
still kept red after the salt was added. Meanwhile, by adding
2.1 ␮M Ag(I) ions into AuNPs/C-ssDNA/Metal ions solution, the
obtained mixture turned purple, which showed similar color com-
Fig. 2. (A) Colorimetric responses of the sensor to different concentrations of Ag(I)
ions using unlabeled strategy. (B) Plot of the absorption ratio (A650 /A520 ) of different pared with the AuNPs/C-ssDNA/Ag(I) solution. These indicate that
concentrations of Ag(I) ions (from 0 ␮M to 19.2 ␮M) using unlabeled strategy. Inset: through color observation, the metal ions mentioned above can not
Plot of the absorption ratio (A650 /A520 ) of Ag(I) ions from 38.4 nM to 1.94 ␮M. bring interferences to Ag(I) ions detection in the unlabeled strat-
egy.
However, when Hg(II) ions (6.3 ␮M) were present, AuNPs/C-
C-ssDNA was directly mixed with AuNPs to form the AuNPs/C- ssDNA/Hg(II) solution also turned purple when the salt was added.
ssDNA complexes, which did not display obvious color changes This indicates Hg(II) ions may interfere Ag(I) ions detection, which
compared with the original AuNPs (red, data not shown) and were could be attributed to the formation of strong T-Hg(II)-T in strands
employed as the signal producer for the unlabeled colorimetric with thymine bases [14,15,25]. Additionally, in the UV–vis spectra,
detection of Ag(I) ions in the next sensing step. After adding Ag(I) Cu(II) ions also displayed a little interference. As reported, Cu(II)
ions or 0.12 M NaNO3 (final concentration) into AuNPs/C-ssDNA ions can also stabilize the dsDNA with C-C mismatches, but the
solution, the color of the obtained solution still kept red (Fig. 2A, strength is much less than Ag(I) ions [23]. However, the interfer-
0 nM). However, after Ag(I) ions and 0.12 M NaNO3 were both added
into AuNPs/C-ssDNA solution, the obtained solution turned purple
(Fig. 2A, 52 nM, 5.2 ␮M and 10.4 ␮M) even dark blue with the pres-
ence of higher concentration of Ag(I) ions (Fig. 2A, 20.8 ␮M and
52 ␮M) in 1 min.
The color changes described above are interesting and can be
interpreted by the mechanism of colorimetric effects between
AuNPs/C-ssDNA and AuNPs/C-ssDNA/Ag(I) [7,12,24]: The as-
prepared AuNPs are stabilized by negative capping agents’ (citrate
in this work) with electrostatic repulsion against van der Waals
attraction between AuNPs. Once enough salt is added, the repulsion
between the negative-charged AuNPs is screened and the AuNPs
are induced to aggregate, leading to a decreased plasmon peak
around 520 nm. As previous report [7], there is stronger coordina-
tion interaction between the N atoms of the unfolded ssDNA and the
AuNPs than eletctrostatic repulsion between the negative-charged
phosphate backbone and the negative-charged AuNPs. Thus, in this
system, the unfolded C-ssDNA can adsorb onto the AuNPs through
Au–N interaction and greatly increase the density of the negative-
charges on the AuNPs, consequently enhancing the AuNPs’ stability
against salt-induced aggregation. With the increase amount of Ag(I)
ions, the C-ssDNA tends to gradually form a Ag(I) ions mediated
folded structure or a kind of double strand combined by C–Ag(I)–C
base pairs (Fig. 1A) [23]. Both the folded and double strand struc-
tures are much rigider than the unfolded ones, which ultimately
prevent the exposure of the DNA bases to the AuNPs. Thus the
C-ssDNA can no longer adsorb on AuNPs and lose the ability of pro- Fig. 3. (A) Control experiments of different ions using unlabeled strategy. The con-
centration of Ag(I) ions was 2.1 ␮M, and the concentration of all the other ions was
tecting the AuNPs. Therefore, in the presence of enough salt, the
6.3 ␮M. (B) Eliminating interference from Hg(II) ions using EDTA. The y-axis was
AuNPs/C-ssDNA/Ag(I) system is easier to aggregate. UV–vis spectra normalized by using A650 /A520 of the mixed solution of Ag(I) ions and Hg(II) ions as
also testify this conclusion, and provide the quantitative detect- 1.
 B. Li et al. / Analytica Chimica Acta 644 (2009) 78–82
Fig. 4. (A) Colorimetric responses of the labeled sensor to different concentrations of Ag(I) ions. (B) UV–vis absorption spectra for different concentrations of Ag(I) ions
obtained from Fig. 3A. (C) Plot of the absorption ratio (A650 /A520 ) of different concentrations of Ag(I) ions. Inset: Plot of the absorption ratio (A650 /A520 ) of Ag(I) ions from
0.59 nM to 59 nM.
ences from Hg(II) ions and Cu(II) ions can be obviously decreased
by adding EDTA to the metal ions solutions directly. That is because
the conditional stability constant of EDTA-Hg(II) and -Cu(II) are
more than 1010 -fold higher than EDTA–Ag(I) (pH 7.4). Taking Hg(II)
as an example, as shown in Fig. 3B, the AuNPs/C-ssDNA colloidal
solution with 0.76 ␮M Ag(I) ions and 2.3 ␮M Hg(II) ions aggre-
gated much more seriously than that in the condition with only
0.76 ␮M Ag(I) ions. However, in the presence of no less than 1.53 ␮M
EDTA, the response of the mixture of 0.76 ␮M Ag(I) ions and 2.3 ␮M
Hg(II) ions was almost the same with that of only 0.76 ␮M Ag(I)
ions. At the same time, the effect of EDTA on Ag(I) ions detection
is faint. According to our previous work, binding ability of Ag(I)
ions with EDTA was less than that of Ag(I) ions with C bases [26].
Therefore, even in presence of EDTA, Ag(I) ions detection could not
be interfered. It was observed that when Ag(I) ions were mixed
with both EDTA and other metal ions, the sensing responses were
in coincident with that of only Ag(I) ions (Fig. S2). As shown, in
this unlabeled Ag(I) ions sensor, even though some metal ions can
bring interferences, strong chelating agent to bi- or multi-valent
ions (such as EDTA) can be used to eliminate the interferences
from these metal ions. The interference from Hg(II) ions can also
be directly avoided by replacing C-ssDNA by other sequences with-
out T bases. Here C-ssDNA is chosen with two reasons. Firstly, it
is a relatively free coil without possible folded structures. Sec-
ondly, there is a purpose to investigate that if certain metal ions
can combine two bases such as Hg(II) ions, whether the inter-
ferences could be solved by simply adding chelated agent to the
samples.
3.3. Labeled colorimetric strategy to detect Ag(I) ions
The scheme of the labeled method is illustrated in Fig. 1B.
According to previous reports [27], due to Au–S bond is more sta-
ble than Au–N bond, the AuNPs labeled with HS-ssDNA can provide
better stability against high salt than the unlabeled ssDNA adsorbed
AuNPs. While such stability can still be obviously destroyed by
the hybridization of HS-DNA tagged on the AuNPs with its com-
plementary strand, just like the unlabeled AuNPs/ssDNA system.
That is because the repulsive interactions from phosphate back-
bones on ssDNA are greatly decreased by formation of the rigid
double strands. As shown in Fig. 1B, the HS-C-ssDNA is covalently
81
labeled on the AuNPs to form the AuNPs-S-C-ssDNA complexes.
Such AuNPs-S-C-ssDNA colloidal solutions display well stability
against the salt. When Ag(I) ions are added, each Ag(I) ion com-
bines two C-bases in the same C-ssDNA strand or the neighboring
strands on the same AuNPs, forming the folded or the double
strand structure. So with enough amount of salt (0.12 M NaNO3,
final concentration), the AuNPs-S-C-ssDNA/Ag(I) colloid aggregates
more easily (in 1 min) than Ag(I) ions-free colloid, and displays
different colors. Here, the color change is also highly dependent
on the concentrations of Ag(I) ions (Fig. 4A), which is consistent
with the condition of the unlabeled strategy. After the optimiza-
tion, the concentration of Ag(I) ions of 12 nM can be discriminated
by naked eyes (Fig. 4A, 12 nM). Additionally, the UV–vis spec-
tra provide concentration dependence response from 0 ␮M to
29.5 ␮M, with the lowest detectable concentration of 0.59 nM
(Fig. 4B and C). It should be noted that in this system, we sup-
pose the formation of C–Ag(I)–C between C-ssDNA strands on
different AuNPs is difficult. Otherwise, without salt, the aggrega-
tion can take place slowly in the presence of enough Ag(I) ions.
However, even when 60 ␮M Ag(I) ions were added, the AuNPs-S-C-
ssDNA colloid still kept red. Therefore, according to the previous
report for Hg(II) ions [14], we consider that the C–Ag(I)–C base
pairs were formed preferentially between the two C-bases in the
same C-ssDNA strand or the neighboring strands on the same
AuNPs.
The successful detection of Ag(I) ions has been also confirmed by
the TEM measurements. As shown in Fig. S2, in the absence of Ag(I)
ions, the AuNPs-S-C-ssDNA colloid is mono-dispersed even after
the salt is added (Figs. S2A and S2C). However, in the presence of
Ag(I) ions, the AuNPs-S-C-ssDNA colloid aggregated together after
the salt was added (Fig. S2D), which illuminates the sensor strategy
intuitionisticly. Note that only in the presence of Ag(I) ions (no salt),
the AuNPs-S-C-ssDNA colloid kept mono-dispersed (Fig. S2B). This
further testifies that the formation of C–Ag(I)–C between C-ssDNA
strands on different AuNPs is difficult.
3.4. The selectivity of the labeled strategy
The selectivity (Fig. S1B in SI) and the method to eliminate inter-
ferences from Hg(II) ions and Cu(II) ions are similar with those of
the unlabeled strategy discussed in Section 3.2.
82 B. Li et al. / Analytica Chimica Acta 644 (2009) 78–82
3.5. Comparison between the unlabeled colorimetric strategy and
the labeled colorimetric strategy
Unlabled and labeled strategies are two commonly used meth-
ods for fabricating DNA based AuNPs colorimetric sensors. But most
reported work only achieved unlabled or labeled strategy without
the comparision of them [10–12].
In this work, we achieve both unlabeled and labeled strategies
for Ag(I) ions colorimetric detection, which gives us the opportu-
nity to compare the properties of both sensors. For the performance
of sensors, both strategies can selectively detect Ag(I) ions suc-
cessfully. But the labeled strategy provides much lower the lowest
detectable concentration and extends the linear detection range
to 100-fold lower than the unlabeled one. Additionally, in the
labeled strategy, HS-C-ssDNA used in unlabeled method provides
better stability to the AuNPs than the C-ssDNA strands applied in
unlabeled method, thus the repeatability of the sensor would be
enhanced and the sensing process and the result are more con-
trollable. In terms of the preparation and handling of sensors, any
label or separation steps are avoided in the unlabeled strategy,
which obviously simplifies the sensing process and would be more
convenient and economical than the labeled one. However, once
the AuNPs-S-C-ssDNA complexes are ready to use, the chromo-
meric step in labeled strategy can be carried out in a few minutes
after Ag(I) ions are added, which is much faster than the condi-
tion in unlabeled strategy (no less than 5 h reaction of Ag(I) ions
and AuNPs/C-ssDNA is required before chromomeric step). This is
attributed to that in the labeled strategy, Ag(I) ions can directly
combine the C-bases in the C-ssDNA, while in the unlabeled strat-
egy, Ag(I) ions need to compete C-bases with AuNPs.
In summary, the unlabeled and labeled strategies can provide
different advantages and disadvantages for Ag(I) detection. Our
work may supply a potential choice for the operators according to
their concrete needs.
4. Conclusions
In this work, unlabeled and labeled DNA based sensing strate-
gies by using AuNPs as colorimetric probes are both developed
for the first time to selectively detect Ag(I) ions. The unlabeled
strategy avoids the label and separation steps in common sen-
sors, while the labeled strategy provides more sensitive, stable
and controllable sensing results. Through careful optimization (vol-
ume of AgNO3 , concentration of DNA and salt), existence of low as
∼10−8 M target can be observed directly by naked eyes. And the
lowest detectable concentration with labeled strategy is 0.59 nM.
This sensitivity is higher than that of previous reports using fluores-
cence (10 nM) [23], DNAzyme based colorimetry (2.5 nM) [26], and
chromogenic method (130 nM) [28]. The result is even comparable
to the graphite-furnace atomic absorption spectrometry (0.46 nM)
[29]. The advantages of the sensors are summarized as follows. On
the one hand, new sensitive and selective colorimetric methods are
provided for Ag(I) ions detection. The complex instruments for the
measurements can be replaced by naked eyes, which simplifies the
whole experimental process. On the other hand, the designed sen-
sor enlarges the applications of both DNA based AuNPs colorimetric
sensors and the Ag(I) ions mediated C-base pairs.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China no. 20675076 and National Key Technology R&D
Program no. 2006BAE03B08.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.aca.2009.04.022.
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