Article

pubs.acs.org/ac

“Turn-On” Fluorescent Sensor for Hg2+ Based on Single-Stranded

DNA Functionalized Mn:CdS/ZnS Quantum Dots and Gold
Nanoparticles by Time-Gated Mode
Dawei Huang,† Chenggang Niu,*,† Xiaoyu Wang,†,‡ Xiaoxiao Lv,† and Guangming Zeng*,†
†
College of Environmental Science and Engineering, Key Laboratory of Environmental Biology and Pollution Control, Ministry of
Education, Hunan University, Changsha 410082, China
‡
College of Chemistry and Chemical Engineering, Xinxiang University, Xinxiang 453003, China

ABSTRACT: An ultrasensitive “turn-on” fluorescent sensor

was presented for determination of Hg2+. This method is
mainly based on Hg2+-induced conformational change of a
thymine-rich single-stranded DNA. The water-soluble long-
lifetime fluorescence quantum dot (Mn:CdS/ZnS) acted as
the fluorophore, which was labeled on a 33-mer thymine-rich
single-stranded DNA (strand A). The gold nanoparticles
(GNPs) functionalized 10-mer single-stranded DNA (strand
B) is selected as the quencher to quench the fluorescence of
Mn:CdS/ZnS. Without Hg2+ in the sample solution, strands A and B could form hybrid structures, resulting in the fluorescence
of Mn:CdS/ZnS being decreased sharply. When Hg2+ is present in the sample solution, Hg2+-mediated base pairs induced the
folding of strand A into a hairpin structure, leading to the release of GNPs-tagged strand B from the hybrid structures. The
fluorescence signal is then increased obviously compared with that without Hg2+. The sensor exhibits two linear response ranges
between fluorescence intensity and Hg2+ concentration. Meanwhile, a detection limit of 0.18 nM is estimated based on 3α/slope.
Selectivity experiments reveal that the fluorescent sensor is specific for Hg2+ even with interference by high concentrations of
other metal ions. This sensor is successfully applied to determination of Hg2+ in tap water and lake water samples. This sensor
offers additional advantage to efficiently reduce background noise using long-lifetime fluorescence quantum dots by a time-gated
mode. With excellent sensitivity and selectivity, this sensor is potentially suitable for monitoring of Hg2+ in environmental
applications.

H eavy metal ion pollution has become an important

worldwide issue for years due to the severe risks in
human health and the environment. As one of the most toxic
heavy metals, mercury with the feature of strong toxicity and
bioaccumulation can cause serious human health problems
even at very low concentrations.1,2 The exposure to mercury
can cause a number of toxicological effects such as brain
damage, kidney failure, and various cognitive and motion
disorders.3,4 The solvated Hg2+, one of the most stable
inorganic forms of mercury, is well-known to be highly toxic
due to its good water solubility.5 Because of these environ-
mental and health problems of Hg2+, it is obviously of great
necessity to obtain new and efficient Hg2+ detection methods
that are cost-effective, rapid, sensitive, and selective. Traditional
methods for Hg2+ quantification include atomic absorption/
emission spectroscopy, selective cold vapor atomic fluorescence
spectrometry, inductively coupled plasma mass spectrometry
(ICPMS), and so forth. These methods are very sensitive and
selective but require complicated sample preparation and
sophisticated instruments which limit their application in
routine Hg2+ monitoring.6−8 Thus, it is still of great challenge
to develop a method which is sensitive, selective, and
environmentally friendly for aqueous Hg2+ detection.
It has been previously reported that Hg2+ can selectively link
T−T pairs to form stable T−Hg2+−T base pairs.9,10 The Hg2+-
mediated T−Hg2+−T pair is even more stable than the
Watson−Crick A−T pair.11 Hg2+ can be incorporated into
DNA duplex without altering the double-helical structure
because the van der Waals radius of mercury (≈1.44 Å) is
smaller than the base pair spacing of DNA duplex (≈3.4 Å).9 A
large number of fluorescent, colorimetric, and electrochemical
sensors were designed for Hg2+ based on T−Hg2+−T
coordination chemistry.12−14 As far as fluorescent sensors are
concerned, most of them can be suitable for Hg2+ detection in
drinking water because detection limits are lower than the toxic
level reported by the U.S. Environmental Protection Agency.
However, many fluorescent sensors work in a “turn-off”
mode.15−17 As we know well, the sensors working in “turn-
off” mode may produce false positive results because of a
number of other quenchers.18 As a result, a “turn-on” sensor is
preferred.12,14,19−21
Recently, researchers have devoted a lot of efforts to the
fluorescent approach that involves the use of nanometer
Received: October 23, 2012
Accepted: December 20, 2012

© XXXX American Chemical Society A dx.doi.org/10.1021/ac303084d | Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
materials, especially semiconductor quantum dots (QDs) or/
and gold nanoparticles (GNPs), for the detection of Hg2+ based
on this T−Hg2+−T coordination chemistry.22,23 QDs have
many unique photophysical properties such as high fluores-
cence quantum yields, narrow emission bands, high Stokes
shifts, and stability against photobleaching. Because of these
excellent properties, QDs were used as fluorescence labels to
develop fluorescent sensors.24−26 GNPs have been of great
interest because of their high extinction coefficient and a broad
absorption spectrum in a visible light. GNPs are unique
quenchers for organic dyes or QDs through both energy-
transfer and electron-transfer processes.27,28 A lot of sensors
have been fabricated based on GNPs as quenchers for DNA,
small molecules, or protein detection.29−32 Up to now,
fluorescent sensors based on QDs and GNPs are still a good
choice for the detection of different analytes.
No matter whether it is “turn-on” or “turn-off” mode, most
fluorescent sensors for Hg2+ are based on organic dyes or QDs
that usually have short-lifetime fluorescence. The background
signals might interfere with the fluorescence of organic dyes or
QDs, affecting the sensitivity of the fluorescent sensors.
Therefore, it should be desirable to develop a novel method,
which uses a long-lifetime fluorophore to decrease the
background noises on the basis of time-gated fluorescence
assay. The authors found that Mn-doped QDs are good choices
which have high quantum yield and long-lifetime fluores-
cence.33−37
In this work, the authors designed a simple “turn-on”
fluorescent sensor for Hg2+ in aqueous solution that utilized the
T−Hg2+−T coordination chemistry and its induced displace-
ment of the quencher-labeled oligonucleotides. Water-soluble
Mn-doped CdS/ZnS core/shell QDs [the QDs have long-
lifetime fluorescence (∼4.8 ms) and excellent stability in
aqueous solution] and GNPs are selected as the fluorophore
and quencher, respectively. This fluorescent sensor has a
detection limit below the U.S. Environmental Protection
Agency limit of acceptable Hg2+ concentration in drinking
water. The sensor also exhibits superior selectivity toward Hg2+
even in the presence of other competitive metal ions.
Furthermore, the sensor was employed to detect Hg2+ spiked
in tap water and lake water samples to demonstrate its potential
for practical applications.
■ EXPERIMENTAL SECTION
Chemicals and Apparatus. All oligonucleotides used in
the present study were synthesized and HPLC-purified by
Shanghai Sangon Biological Engineering Technology &
Services Co., Ltd. (Shanghai, China) and dissolved in ultrapure
water (18.3 MΩ·cm) produced by a Millpore water purification
system. The sequences were shown as follows: 5′SH−C6−
TGAAA CTGTA-3′; 5′SH−C6−TACAG TTTCA CCTTT
TCCCC CGTTT TGGTG TTT-3′. AuCl3·HCl·4H2O was
purchased from Shanghai Institute of Fine Chemical Materials
(Shanghai, China). 3-Mercaptopropionic acid (MPA, 99+%)
was purchased from Sigma-Aldrich. The chemicals were used as
received without further purification. All other chemicals used
were of analytical grade and were used without further
purification. Ultrapure water was used throughout the experi-
ments.
The time-gated fluorescence intensities were measured and
recorded with a Perkin-Elmer LS-55 spectrofluorimeter
(United Kingdom). UV−vis absorption spectra were recorded
by using a Shimadzu UV spectrophotometer (UV-2550, Kyoto,
B dx.doi.org/10.1021/ac303084d | Anal. Chem. XXXX, XXX, XXX−XXX
Article
Japan). Atomic fluorescence measurements were performed on
an atomic fluorescence spectrometer (AFS-9700) (Beijing,
China).
PBS buffer (100 mM) was prepared by mixing an appropriate
content of 200 mM Na2HPO4 and 200 mM NaH2PO4. The
composition of hybridization buffer, incubation buffer, and
washing buffer was 10 mM PBS buffer (pH = 7.4), 100 mM
NaNO3. In addition, 2 M NaCl was also prepared.
Synthesis of Mn-Doped CdS/ZnS Core/Shell QDs. Mn-
doped CdS/ZnS core/shell QDs (Mn:CdS/ZnS QDs) were
prepared according to a three-step synthesis method.33,34 The
resulting Mn:CdS/ZnS QDs were dispersed in hexane. The
lack of water solubility of the prepared QDs hindered their
reaction with the water-soluble alkylthiol-capped oligonucleo-
tides. Therefore, the authors used 3-mercaptopropionic acid to
prepare the water-soluble QDs according to the literature.38
The resulting water-soluble QDs also have long-lifetime
fluorescence (∼4.8 ms) and exhibit high stability in aqueous
solutions. The water-soluble QDs should be an excellent
fluorescence label and could play an important role in a number
of QDs-based biochemical and biomedical applications.
Synthesis of GNPs. All glassware and mechanical stirrers
used for the synthesis were thoroughly cleaned in aqua regia (3
parts HCl, 1 part HNO3), rinsed with ultrapure water, and then
oven-dried prior to use. The colloidal solution of GNPs was
synthesized by means of citrate reduction of
AuCl3·HCl·4H2O.39 An amount of 5 mL of 38.8 mM sodium
citrate was rapidly added to a boiled 50 mL of 1 mM HAuCl4
solution with vigorous stirring in a 250 mL round-bottom flask
equipped with a condenser. The color changed from light
yellow to wine red. Boiling was continued for 10 min; the
heating mantle was then removed, and stirring was continued
for an additional 15 min. After the solution was cooled to room
temperature, the prepared GNPs solution was stored in the 4
°C refrigerator before use. The size of the nanoparticles was
typically ∼13 nm in average diameter. The concentration of the
GNPs was ∼17 nM, which was determined according to the
Beer’s law by using UV−vis spectroscopy, based on the
extinction coefficient of 2.7 × 108 M−1 cm−1 at λ = 520 nm for
13 nm particles.40
Preparation of DNA-Functionalized GNPs. According to
literature with slight modifications,41 8.0 mL of 17 nM GNPs
were incubated with 20 μL of 0.1 mM oligonucleotides
overnight. After standing for 16 h at 50 °C, the mixed solution
was changed into 0.1 M NaCl, 10 mM phosphate buffer (pH =
7.4) by addition of the necessary salts and was kept at 50 °C for
40 h. To remove unreacted oligonucleotides, the oligonucleo-
tide-conjugated GNPs were purified three times by centrifuga-
tion at 13 200 rpm for 30 min. The final product was
redispersed into 1.2 mL of PBS buffer (10 mM, pH = 7.4) to
make a stock solution and stored at 4 °C for future usage. The
number of oligonucleotides probes immobilized on the GNPs
was estimated by measuring the absorbance difference at 260
nm before and after modification with oligonucleotides. The
average oligonucleotide loadings were about 10 oligonucleo-
tides per GNP, and the final concentration of GNPs was 95
nM.
Preparation of DNA-Functionalized Mn:CdS/ZnS QDs.
DNA-functionalized QDs were prepared according to a
previously published protocol with minor modifications.38
The QDs solution and oligonucleotides solution were mixed
together at a ratio of 11 oligonucleotides per QD (100 μL of
1.8 μM QDs mixed with 20 μL of 0.1 mM oligonucleotides).
Analytical Chemistry Article

Scheme 1. Schematic Description of the “Turn-On” Fluorescent Sensor for Hg2+ Based on the Hg2+-Mediated Formation in
DNA Duplexesa

a
The 33-mer single-stranded DNA (strand A) with a Mn:CdS/ZnS quantum dot attached at the 5′ end was hybridized with a 10-mer single-stranded
DNA (strand B) with a gold nanoparticle attached at the 5′ end, which resulted in energy transfer from the Mn:CdS/ZnS quantum dot to the gold
nanoparticle, leading to a decrease in the time-gated fluorescence intensity of the Mn:CdS/ZnS quantum dot. In the presence of Hg2+, the folding of
strand A releases strand B and increases the fluorescence of the Mn:CdS/ZnS quantum dot. The drawing of QDs and GNPs modified single-
stranded DNA is only a graphic presentation.

After standing for 12 h, the mixed solution was brought to 0.15
M NaCl and the particles were aged for an additional 12 h. The
NaCl concentration was then raised to 0.3 M, and the mixture
was allowed to stand for a further 40 h before centrifugalization
using centrifugal filter devices (Amicon Ultra-0.5). Finally, the
QDs were redispersed into 4.0 mL of PBS buffer (10 mM, pH
= 7.4) by vortex and stored at 4 °C for future usage. The
Mg2+, Zn2+, Al3+, Fe3+, Pb2+, Ag+, and Au3+. The lake water
samples were taken from Taozi lake in Hunan University. The
time-gated fluorescence signal was measured and recorded by a
Perkin-Elmer LS-55 spectrofluorimeter. The parameters of the
spectrofluorimeter are set as λex = 400 nm; λem = 609 nm; delay
time, 0.1 ms; gate time, 1.0 ms; excitation slit, 15 nm; emission
slit, 20 nm.

number of oligonucleotides probes immobilized on the QDs
was also estimated by measuring the absorbance difference at
260 nm before and after modification with oligonucleotides.
The average oligonucleotide loadings were about six
oligonucleotides per QD, and the final concentration of QDs
was 45 nM.
Procedures for Hg2+ Detection. To detect Hg2+ or other
metal ions in buffer or real environmental water samples, 30 μL
of 95 nM DNA/GNPs, 70 μL of 45 nM DNA/QDs, and 140
μL of 0.01 M PBS buffer were mixed uniformly by vortex and
hybridized for 35 min first. Then, various concentrations of
Hg2+ (20 μL) were added into the mixture solution and
incubated for 16 min to form T−Hg2+−T coordination
chemistry. Finally, the time-gated fluorescence spectra of
different concentrations of Hg2+ were monitored after the
completion of the reaction. For the sensitivity experiment, the
concentrations of Hg2+ were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0,
6.0, 8.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 nM,
respectively. For the optimizing experiment, 1.0 μM was
selected as the Hg2+ concentration to determine the optimum
hybridization time and incubation time. Various metal ions of
10 μM were used in the selectivity experiments. The metal ions
are as follows: Mn2+, Ba2+, Ni2+, Cu2+, Ca2+, Cr2+, Co2+, Cd2+,
C dx.doi.org/10.1021/ac303084d | Anal. Chem. XXXX, XXX, XXX−XXX
■
RESULTS AND DISCUSSION
Experimental Principle of the Proposed Sensor. The
“turn-on” fluorescent sensor for Hg2+ is outlined in Scheme 1.
The sensor system comprises two single-stranded DNAs
(strand A and strand B) with an alkanethiol moiety at their
5′-terminus. Strand A is a 33-mer thymine-rich oligonucleotide,
and strand B is a 10-mer oligonucleotide complementary with
strand A. Strand A contains two major parts: the first part (in
red) is a five-base segment close to the 5′-terminal that could
hybridize with strand B close to the 3′-terminal; five self-
complementary base pairs separated by seven thymine−
thymine mismatches are introduced to the second part (in
blue). There are five-base segments in the second part which
could hybridize with five-base segments of strand B close to the
5′-terminal. Strand A was functionalized with ∼5 nm sized
Mn:CdS/ZnS QDs, and strand B was prepared by function-
alization of ∼13 nm diameter GNPs, according to the 5′-
terminal −SH reaction. The QDs with ∼5 nm in diameter
showed the fluorescence emission at 609 nm under light
excitation at 400 nm. In our experiment, the average distance
between QD and GNP in hybridized structures was estimated
to be 5.25 nm. In the absence of Hg2+, strands A and B could
Analytical Chemistry
hybridize each other because of the 10 complementary base
pairs. Under these conditions, the QDs and GNPs are close to
each other, resulting in fluorescence quenching due to
fluorescence resonance energy transfer. On the contrary,
when Hg2+ was present in the sensor solution, mercury-
mediated base pairs (T−Hg2+−T) induce the folding of strand
A into a hairpin structure. Consequently, there are only five
base pairs remaining between strands A and B, which is not
stable enough under the conditions we used in the proposed
method. As a result, strand B will be released from the hybrid
structure, and the time-gated fluorescence of QDs will be
observed upon light excitation. The fluorescence spectra of the
sensor before and after the addition of 1.0 μM Hg2+ is shown in
Figure 1A. Furthermore, the authors used a control experiment
with the results of no fluorescence intensity changed, in which
Hg2+ was added to the solution only containing strand A. The
results indicated that Hg2+ makes negligible contribution to
quench the fluorescence of QDs (Figure 1B).
Optimization of the Experimental Conditions. In the
present strategy, the hybridization and incubation times,
Figure 1. (A) Time-gated fluorescence emission spectra of the sensor
without and with 1.0 μM Hg2+. The measure conditions are given
below: hybridization and incubation times, operational temperature,
and media pH are 60 min, 30 min, 25−28 °C, and 7.4, respectively.
The parameters of the spectrofluorimeter are set as λex = 400 nm; λem
= 609 nm; delay time, 0.1 ms; gate time, 1.0 ms; excitation slit, 15 nm;
emission slit, 20 nm. (B) Fluorescence emission spectra of Mn:CdS/
ZnS quantum dots modified strand A in the absence and presence of
10, 100, and 500 nM Hg2+, indicating that Hg2+ has negligible effect on
the quenching of fluorescence.
D dx.doi.org/10.1021/ac303084d | Anal. Chem. XXXX, XXX, XXX−XXX
Article
operational temperature, and media pH played crucial roles
in the detection sensitivity. The hybridization time between
strands A and B was investigated as it may influence the
hybridization efficiency. Although longer hybridization time
may yield more stable fluorescence signal, it is unnecessary if
the reaction attained the equilibrium. The fluorescence signal
was recorded along with the hybridization time increasing
(Figure 2). It is found that the fluorescence signal decreased
Figure 2. Optimization experiments of hybridization and incubation
time; 1.0 μM was selected as the Hg2+ concentration to determine the
optimum hybridization and incubation time.
with increasing hybridization time and became stable over 32
min, then continued at an almost constant value. To ensure the
completeness of hybridization, the authors chose 35 min as the
optimum hybridization time.
The incubation time after the introduction of Hg2+ into the
solution to prompt the T−Hg2+−T formation was also
investigated, and the results are shown in Figure 2. The
fluorescence intensity increased when Hg2+ was introduced and
then tended to stabilize after more than 16 min. On the basis of
this, the authors chose 16 min as the optimum incubation time.
Furthermore, taking into account operational convenience,
room temperature (25−28 °C) was selected as the operational
temperature for all experiments. In order to facilitate the
hybridization reaction, the media pH for all the experimental
steps was 7.4. Herein, 1.0 μM was used as the Hg 2+
concentration to optimize the experimental conditions.
Sensitivity of the Sensor. On the basis of the above
standard procedures and optimized assay conditions, various
concentrations of Hg2+ were introduced to the buffer to
evaluate the sensitivity of the “turn-on” fluorescent sensor. The
various concentrations of Hg2+ were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0,
4.0, 6.0, 8.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0
nM. As shown in Figure 3 parts A and B, higher concentrations
of Hg2+ resulted in more fluorescence intensity enhancement. It
is worth noting that, even at very low concentration of Hg2+,
the time-gated fluorescence intensity exhibited perceptible
change which indicated that the Hg2+ could be detected with
high sensitivity in this proposed fluorescent sensor.
The time-gated fluorescence intensity was found to be linear
with the concentration of Hg2+ in the range from 0 to 1 × 10−9
M and from 1 × 10−9 to 1 × 10−8 M (Figure 3C). The
equations for the resulting calibration plot were y = 5.75x +
0.43 (eq 1) and y = 1.71x + 5.40 (eq 2) (x was the
Analytical Chemistry
Figure 3. (A) Time-gated fluorescence emission spectra of the sample
solution after addition of various concentrations (0−1000 nM) of
Hg2+ in buffer. (B) Plot of time-gated fluorescence intensity as a
function of the concentration of Hg2+. (C) Linear region of panel B.
The concentrations of Hg2+ were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0,
8.0, and 10 nM (C). Every data point was the mean of three
measurements. The error bars are the standard deviation.
concentration of Hg2+, y was the time-gated fluorescence
intensity) with correlation coefficient of 0.9939 and 0.9967,
respectively. According to the standard deviation of 0.35 for the
blank signal with 20 parallel measurements and eq 1, a
detection limit of approximately 0.18 nM was estimated based
on a 3α/slope. This is an exceptionally low detection limit for
E dx.doi.org/10.1021/ac303084d | Anal. Chem. XXXX, XXX, XXX−XXX
Article
Hg2+ detection using fluorescent methods, which is lower than
the standards of the U.S. Environmental Protection Agency
(EPA). The EPA regulates the maximum allowable level of
Hg2+ in drinking water to be 10 nM. In the present method,
higher concentrations of Hg2+ resulted in more free GNPs in
the sample solution, and free GNPs have a quenching effect on
the fluorescence of QDs. As shown in Figure 3, parts B and C,
the proposed method exhibits nonlinear character in the range
from 0 to 1000 nM; such behavior may be caused by the free
GNPs.
For examination of the repeatability of this sensor, three
sample solutions with different concentrations of Hg2+ were
prepared, and the relative standard deviations (RSDs) are about
6.2%, 3.8%, and 2.5% for three independent determinations of
0.8, 6.0, and 50 nM Hg2+ under the optimum conditions,
respectively.
Selectivity of the Sensor. To determine the selectivity of
this protocol, two control experiments were conducted. First,
1.0 μM of Hg2+ and 10 μM of other metal ions including Mn2+,
Ba2+, Ni2+, Cu2+, Ca2+, Cr2+, Co2+, Cd2+, Mg2+, Zn2+, Al3+, Fe3+,
Pb2+, Ag+, and Au3+ were added to the sample solution, and
then the time-gated fluorescence intensity was recorded. As
indicated in Figure 4, only Hg2+ exhibits significant response.
Figure 4. Selectivity of the “turn-on” fluorescent Hg2+ sensor. The
concentrations of Hg2+ and other metal ions are 1.0 and 10 μM. Every
data point was the mean of three measurements. The error bars are the
standard deviation.
Second, 1.0 μM of Hg2+ and 10 μM of other metal ions were
mixed together to form a mixture solution as a sample for
selectivity testing (Figure 4). The fluorescence intensity was
obviously higher than that of other samples without Hg2+.
These results clearly indicated that the approach is not only
insensitive to other metal ions but also selective toward Hg2+ in
their presence.
Assay of Hg2+ Concentrations in Environmental Water
Samples. With excellent sensitivity and selectivity in buffer
solution, the proposed method was further tested with real
environmental water samples to demonstrate its practical
application. The environmental water samples used in the
study were tap water and lake water samples. These samples
spiked with Hg2+, with concentrations of 0, 1.0, 2.0, and 10.0
nM, were tested using the proposed method and AFS (atomic
fluorescence spectrometer). The lake water samples were
Analytical Chemistry

■
Article

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■
CONCLUSIONS
The authors have developed a “turn-on” fluorescent sensor for
determination of Hg2+ in aqueous media with excellent
sensitivity and selectivity by using Hg2+-mediated T−Hg2+−T
pairs, long-lifetime fluorescence QDs, and GNPs. It combines
the advantages of specific and stable binding interactions
between Hg2+ and thymine, the unique photophysical proper-
ties and long-lifetime fluorescence of QDs, and the excellent
quenching performance of GNPs. The detection limit (LOD,
0.18 nM) is much lower than the EPA limit of Hg2+ in
drinkable water (<10 nM). In addition, the fluorescent sensor
showed outstanding selectivity for Hg2+ against other metal
ions. The sensor also can be used for the detection of Hg2+ in
real environmental water samples. The authors believed that
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