An aqueous fluorescent probe for Hg2+ detection with high selectivity and sensitivity

Article abstract of DOI:10.1002/bio.2893

An aqueous fluorescent probe, 1, was developed for the rapid detection of Hg²⁺ with high sensitivity and excellent selectivity. Upon the addition of Hg²⁺ in pure aqueous media, the Hg²⁺-mediated hydrolysis of vinyl ether and subsequent cyclization reactions converted probe 1 into the corresponding iminocoumarin dye, which is strongly fluorescent when excited. The application of this probe for the detection of intracellular Hg²⁺ was successfully demonstrated in living cells.

Full text of DOI:10.1002/bio.2893

Research article
Received: 29 August 2014,
Revised: 23 January 2015,
Accepted: 28 January 2015
Published online in Wiley Online Library: 11 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2893
2+
An aqueous fluorescent probe for Hg
detection with high selectivity and sensitivity
Qian Fang,a Qian Liu,b Xiangzhi Songa,c,d* and Jian Kange*
ABSTRACT: An aqueous fluorescent probe, 1, was developed for the rapid detection of Hg2+ with high sensitivity and excellent
selectivity. Upon the addition of Hg2+ in pure aqueous media, the Hg2+-mediated hydrolysis of vinyl ether and subsequent cycli-
zation reactions converted probe 1 into the corresponding iminocoumarin dye, which is strongly fluorescent when excited. The
application of this probe for the detection of intracellular Hg2+ was successfully demonstrated in living cells. Copyright © 2015
John Wiley & Sons, Ltd.
Keywords: fluorescent probe; Hg2+; hydrolysis reaction; vinyl ether; water-soluble
effectively improve their water solubility (27). Herein, we set out to
Introduction
Mercury is an extremely toxic element that is naturally occurring
design and synthesize a new water-soluble fluorescent dye, probe
1, for the rapid and selective detection of Hg2+. The two cyano
or introduced by human activities. Owing to the widespread
groups in probe 1 provide good water solubility, making this probe
distribution of mercury in water and soil, the mercury pollution
applicable in aqueous environments. When treated with Hg2+ in
has become a serious problem in the past several decades
100% aqueous solution (20 mm phosphate-buffered saline [PBS],
(1–3). Mercury is strongly thiophilic to enzymes and proteins,
pH 7.4), probe 1 rapidly generates the iminocoumarin, reference
which can cause severe damage to the brain, kidney, nervous
dye 2, which is highly fluorescent upon excitation. The proposed
and digestive systems even at low-level exposure (4–9). Conse-
mechanism of probe 1 with Hg2+ was presented in Scheme 1. Hg2
quently, mercury pollution has become a serious threat to
+ induced the hydrolysis of the vinyl ether in probe 1 to generate
humans and wildlife. Generally, the mercury pollutant exists as
a hydroxy intermediate, which was quickly transformed into refer-
ence dye 2 via an intramolecular nucleophilic reaction.
Hg2+. Therefore, it is necessary to develop effective methods
for the qualitative and quantitative detection of Hg2+ in living
species and the environment (10,11).
Fluorescence analysis has been widely used in molecular rec-
ognition owing to its great selectivity, high sensitivity, easy
Experimental
operation and non-destructiveness. Thus, a variety of fluorescent
probes for Hg2+ detection has been developed in the past
Materials and instruments
decade (12–15). Early on, most Hg2+ fluorescent probes were
Unless otherwise noted, all reagents were used as received from
designed based on their coordinative property with
heteroatom-based ligands. However, coordination-based fluores-
cent probes suffer from low selectivity to Hg2+ over other metal
ions, particularly for intracellular detection in living cells. As a
result, Hg2+-promoted chemical reactions, including desulfuriza-
tion, hydrolysis, cyclization and elimination have been employed
in the design of fluorescent probes for Hg2+ detection to achieve
good selectivity and high sensitivity (16–22). As Ando and Koide
commercial suppliers without further purification. Silica gel
(200–300 mesh) and thin-layer chromatography plates were
purchased from Yantai Chemical Industry Research Institute
* Correspondence to: X. Song, College of Chemistry & Chemical Engineering,
Central South University, Changsha, Hunan Province, Peoples Republic of
China 410083. E-mail: xzsong@csu.edu.cn
* J. Kang, The Third Xiangya Hospital, Central South University, Changsha,
Hunan Province, Peoples Republic of China, 410013. E-mail: kangjian@csu.edu.cn
reported a turn-on Hg2+ fluorescent probe based on Hg2+
-
mediated hydrolysis of vinyl ether, this strategy has gained great
interest and has become a general approach in the design of
Hg2+ fluorescent probes (23).
a
College of Chemistry & Chemical Engineering, Central South University,
Changsha, Hunan Province, Peoples Republic of China
Iminocoumarin dyes are good fluorophores with relatively high
fluorescent quantum yield, good water solubility and excellent
photostability. Owing to the above-mentioned advantages of
iminocoumarin dyes, recently they were used to construct fluores-
cent probes for the detection of F–, Cu2+, Zn2+ and thiophenol
(24–26). In addition, Cho and Ahn developed a coumarin-based
probe for the detection of Hg2+ with good selectivity and
sensitivity (1). However, this probe can only function in organic co-
solvent systems because of its poor water solubility and it is known
that the introduction of cyano groups into organic compounds can
b
College of Chemical Engineering and Modern Materials, Shangluo University,
Shangluo, Shanxi Province, Peoples Republic of China
c
State Key Laboratory for Powder Metallurgy, Central South University,
Changsha, Hunan Province, Peoples Republic of China
d
State Key Laboratory of Fine Chemicals, Dalian University of Technology,
Dalian, Liaoning Province, Peoples Republic of China
e
The Third Xiangya Hospital, Central South University, Changsha, Hunan
Province, Peoples Republic of China
Luminescence 2015; 30: 1280–1284
Copyright © 2015 John Wiley & Sons, Ltd.

An aqueous fluorescent probe for Hg2+ detection
Scheme 1. Sensing mechanism of probe 1 with Hg2+
.
(China). All the ultraviolet-visible and fluorescence spectra were
measured by Agilent Technologies (USA) and HITACHI F-4600
spectrometers ( Japan), respectively. The pH was test by pHs-
3B meter (Shanghai Precision & Scientific Instrument Co. Ltd,
Shanghai, China). 1H NMR spectra in CDCl3 was measured by
Bruker 400M NVANCE-III spectrometer (Switzerland) with chem-
ical shifts reported as p.p.m. (tetramethylsilane as the internal
standard). The accurate mass spectrometric experiment was per-
formed on a micrOTOF-Q II mass spectrometer (BrukerDaltonik,
Germany). Cell experiment were applied on an inverted fluores-
cence microscope (Olympus IX-70) connected with a digital
camera (Olympus, c-5050).
Results and discussion
Proposed mechanism for the reaction of probe 1 with Hg2+
First, we investigated the absorption and emission spectra of
probe 1, reference dye 2 and the reaction product of probe 1 with
Hg2+. The solution of probe 1 displays an absorption maximum at
447 nm and is essentially non-fluorescent (shown in Fig. 1). The ref-
erence dye 2 absorbs at 438 nm and was strongly fluorescent with
a maximum at 486 nm (shown in Supplementary Fig. 2). Upon
treatment with Hg2+ in aqueous PBS buffer, the solution of probe
1 showed a blue-shift to 438 nm in its absorption spectrum and
exhibited a strong blue fluorescent signal with a maximum at
486 nm (Fig. 1). The optical spectra studies clearly suggested that
Hg2+ induced the hydrolysis and generated reference dye 2, which
is highly fluorescent upon excitation.
Absorption and fluorescence analysis
The stock solution of probe 1 was prepared at 1 mm in dimethyl
sulfoxide (DMSO). Stock solutions of various cations, including
Na+, Ca2+, Cd2+, Fe3+, Ag+, Co2+, Pb2+, Cu2+, Zn2+, Cr3+, Mg2+, Ba2
+, Al3+, Mn2+ and Ni2+ were prepared in PBS buffer (20 mm, pH
7.4). The test samples for absorption and emission spectra mea-
surements were prepared by placing appropriate amounts of stock
solutions containing various cations into the corresponding solu-
tion of probe 1. Unless otherwise noted, the excitation wavelength
was 438 nm and the excitation and emission slit widths were set at
5 nm for all fluorescence measurements.
To understand further the reaction mechanism of probe 1 with
1
Hg2+, H NMR titration analysis was conducted for a mixture of
probe 1 and Hg2+ in DMSO-d6/D2O (V/V, 4:1) solution (Fig. 2). Upon
addition of Hg2+ to the solution of probe 1, the protons at 4.68 p.p.
m. (H1), 4.96 p.p.m. (H2), 6.98 p.p.m. (H3) assigned to CH=CH2 and
7.94 p.p.m. (H4) assigned to CH=(CN)2 disappeared and a new 1H
NMR spectrum was obtained, which is identical to that of reference
dye 2. Moreover, mass spectrum analysis on the reaction product
of probe 1 with Hg2+ showed a peak at m/z = 242.1290
(Supplementary Fig. 9, Supporting Information), which is nearly
Synthesis of probe 1 and reference dye 2
The synthesis of reference dye 2 was obtained using the method
in the literature (28). The synthetic route of probe 1 is shown in
Scheme 2. Compound b was synthesized according to the method
in the literature with 4-(diethylamino)salicylaldehyde as the
starting material (1). Piperdine (0.3 mmol, 30.6 mg) in 2 mL of eth-
anol was added dropwise to a solution of compound b (1 mmol,
220 mg) and malononitrile (2 mmol, 130 mg) in 15 mL of anhy-
drous ethanol, while stirring at room temperature. The resulting
mixture was kept at room temperature for 5 h. After removing
the solvent, the residue was purified by silica gel column chroma-
tography using petroleum ether/dichloromethane (v/v = 10: 1) as
eluent to give pure probe 1 as a light yellow powder (180 mg, yield
71%). 1H NMR (400 MHz, CDCl3): δ1.25 (t, J = 7.6 Hz, 6H), 3.47 (q, J =
8.8 Hz, 4H), 4.65 (dd, J = 6.8, 1.6 Hz, 1H), 4.92 (dd, J = 1.6, 1.6 Hz, 1H),
6.22 (d, J = 2.4 Hz, 1H), 6.66 (dd, J = 2.4, 2.4 Hz, 1H), 6.95 (dd, J = 6.4,
5.6 Hz, 1H), 7.09 (s, 1H), 8.06 (d, J = 9.6 Hz, 1H). MS (ESI): found: m/z =
268.18 (M+1)+, calcd. for C16H17N3O = 268.14.
Figure 1. Ultraviolet-visible absorption (solid line) and emission (dot line) of probe 1
in the absence (black lines) and presence (red lines) of Hg2+ in phosphate-buffered
saline (20 mm, pH 7.4). Inset: Emission images of probe 1 before (a) and after (b) addi-
tion of Hg2+ under a 365 nm ultraviolet lamp.
Scheme 2. The synthetic route of probe 1. (i) BrCH2CH2Br, K2CO3, acetone; (ii) tert-BuOK, dimethyl sulfoxide; (iii) malononitrile, piperidine, EtOH.
Luminescence 2015; 30: 1280–1284
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Q. Fang et al.
Figure 2. Partial 1H NMR spectra of (a) probe 1 (20 mm) in DMSO-d6, (b) probe 1 (20 mm) with Hg2+ (60 mm) in DMSO-d6/D2O (v/v=4:1) and (c) reference dye 2 in DMSO-d6.
DMSO, dimethyl sulfoxide.
Figure 3. The time-dependent fluorescence of probe 1 (10 μm) with Hg2+ (20 μm) in
Figure 5. Fluorescence responses of probe 1 (10 μm) with various cations (10 μm) in
phosphate-buffered saline (20 mm, pH 7.4) within 30 min.
phosphate-buffered saline (20 mm, pH 7.4) measured after 30 min mixing.
Figure 4. The changes of fluorescence intensity at 486 nm of probe 1 (10 μm) upon
the addition of Hg2+ (0.0–20 equiv.) in phosphate-buffered saline (20 mm, pH 7.4). In-
set: (top) the dependence of the fluorescence intensity (at 486 nm) on the concentra-
tions of Hg2+; (bottom) fluorescence images of probe 1 (10 μm) in the absence (a) and
presence (b) of Hg2+ in phosphate-buffered saline (20 mm, pH 7.4).
Figure 6. Interfering effect of various analytes on the fluorescence intensity at 486
nm of probe 1 (10 μm) with Hg2+ (20 μm) in phosphate-buffered saline (20 mm, pH
7.4) after incubated for 30 min.
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Luminescence 2015; 30: 1280–1284

An aqueous fluorescent probe for Hg2+ detection
identical to the theoretical molecular mass of reference dye 2
([M + 1] = 242.2884). These data provide strong support for our
proposed mechanism for the manner in which Hg2+ reacts with
probe 1 to generate the strong fluorescent reference dye 2.
Fig. 4, a good linear relationship was observed between the fluo-
rescence intensity and the concentration of Hg2+. The detection
limit of probe 1 for the detection of Hg2+ is calculated to be 20
nm. In the presence of 2.0 equivalents of Hg2+, probe 1 (10 μm)
produced a strong fluorescence enhancement (68-fold) (Fig. 4).
These results evidently indicated that probe 1 could effectively
detect Hg2+ qualitatively and quantitatively.
Fluorescence studies
To evaluate the selectivity of probe 1, the fluorescence experi-
ments were carried out on probe 1 in response to other common
To obtain an appropriate reaction time, the time-dependent fluo-
rescence studies of probe 1 with Hg2+ were conducted. It can be
seen in Fig. 3 that a pronounced fluorescence enhancement was
observed within 5 min and the fluorescence intensity became
saturated after 30 min, indicating probe 1 could make a rapid
detection of Hg2+.
metal ions, including Fe3+, Cr3+, Ca2+, Mg2+, Co2+, Mn2+, Ni2+, Pb2+
,
Zn2+, Ba2+, Cd2+, Cu2+, Ag+, Al3+ and Na+ in PBS buffer (20 mm, pH
7.4). As seen in Fig. 5, Hg2+ induced a prominent fluorescence en-
hancement whereas all other metal ions only trigger a faint fluo-
rescence signal. Moreover, the coexistence of other interfering
metal ions showed little effect on the detection of Hg2+ for probe
1 (Fig. 6). The above results implied that probe 1 exhibited high
selectivity for Hg2+ detection.
To gain insight into the analytic performance of probe 1 for Hg2
+
detection, the fluorescence behavior of probe 1 (10 μm) in
response to varying concentrations of Hg2+ was investigated. In
It is known that Hg2+-mediated hydrolysis is pH dependent.
Thus, we also investigated the pH effect on the fluorescence be-
havior of probe 1 in response to Hg2+. As shown in Fig. 7, the fluo-
rescence of probe 1 is negligible under pH values from 4.0 to 9.0,
indicating probe 1 has a good stability within a wide pH range.
The solution of probe 1 with Hg2+ produced a strong fluorescence
signal between pH 6.0 and 8.0, suggesting that this probe is
suitable for the detection of Hg2+ in physiological conditions.
Cell imaging
Encouraged by the good selectivity, high sensitivity and good
water solubility of probe 1, we set out to evaluate the capability
of this probe for the detection of Hg2+ in living cells. When
HeLa cells were incubated with the 10 μm probe 1 for 30
min at 37°C and then washed with PBS buffer three times, only
little fluorescence was produced, as shown in Fig. 8. In contrast, a
strong green fluorescence signal was observed inside HeLa cells
Figure 7. pH effect on the fluorescence intensity at 486 nm of probe 1 (10 μm) only
(black line) and probe 1 (10 μm) with Hg2+ (10 μm) (red line).
Figure 8. Images of HeLa cells: (a) bright-field and (b) fluorescent images of HeLa cells incubated with probe 1 (10 μm); (c) bright-field and (d) fluorescent images of HeLa cells
treated with probe 1 (10 μm) for 30 min and then incubated with 30 μm Hg2+ for another 30 min.
Luminescence 2015; 30: 1280–1284
Copyright © 2015 John Wiley & Sons, Ltd.
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Conclusions
We have developed a fluorescent probe for the detection of
Hg2+ with high sensitivity and good selectivity. In the presence
of Hg2+, this probe produced a 90-fold fluorescence enhance-
ment. Owing to its good water solubility, this probe can detect
Hg2+ in pure aqueous solution with a fast response time.
Importantly, the successful imaging of Hg2+ in living cells was
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The research was supported by State Key Laboratory of Fine
Chemicals (KF1202) and Hunan Nonferrous Metals Holding Group
Co. Ltd.
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sion of this article at the publisher’s web site.
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