An easily prepared hypersensitive water-soluble fluorescent probe for mercury(ii) ions

Article abstract of DOI:10.1039/b907386h

A hypersensitive water-soluble fluorescent probe, dansyl-l-tryptophan methyl ester (1), was easily prepared for the detection of Hg²⁺ with a significantly improved detection limit (5 nM vs. 500 nM) in buffered aqueous solution. The Royal Societ

Full text of DOI:10.1039/b907386h

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An easily prepared hypersensitive water-soluble fluorescent probe
for mercury(II) ionsw
Hong-Wei Li, Yue Li, Yong-Qiang Dang, Li-Jun Ma, Yuqing Wu,* Guangfeng Hou
and Lixin Wu*
Received (in Cambridge, UK) 14th April 2009, Accepted 27th May 2009
First published as an Advance Article on the web 12th June 2009
DOI: 10.1039/b907386h
A hypersensitive water-soluble fluorescent probe, dansyl-L-
tryptophan methyl ester (1), was easily prepared for the detection
of Hg2+ with a significantly improved detection limit (5 nM vs.
500 nM) in buffered aqueous solution.
non-covalent complexation between the probe via its carboxyl
group and the protein weakened the bond strength of the N–H
in the sulfonamide moiety and further generated an affinity
binding site for Hg2+. Therefore, covalent modification of the
carboxyl group by a methyl ester may favor the deprotonation
of the amide group and strengthen the binding between the
amino group and the metal ions. We extended our studies to
the design and synthesis of dansyl-L-tryptophan methyl ester
(1) (Fig. 1a), as a more efficient and selective fluorescent Hg2+
probe (Fig. S1, ESIw). This compound displayed a rapid and
specific response to Hg2+ in buffered aqueous solution with a
significant improvement of detection limit (5 nM vs. 500 nM),
which was also effective in live cells.
Mercury pollution, which mainly stems from mercury(II) ion
(Hg2+) contaminated natural water, has become a worldwide
environmental problem since Hg2+ can easily pass through
biological membranes, causing serious damage to the central
nervous and endocrine systems.1 To ensure human health, the
upper limit for Hg2+ in drinking water has been decreased to
10 nM.2 Therefore, new mercury detection methods that are
effective, rapid, facile, and applicable to environmental
and/or biological systems have become a significant and
insistent goal.
Compound 1 was easily synthesized in 81.7% yield from the
reaction of dansyl chloride and L-tryptophan methyl ester and
its structure was confirmed by spectroscopic data (see ESIw).
This fluorescent probe displayed remarkable advantages
including: (i) simple and high-yielding synthesis; (ii) turn-on
fluorescence properties upon Hg2+ addition; (iii) rapid and
specific response to Hg2+; (iv) high sensitivity at the nano-
molar concentration range; and (v) suitable detection both in
buffered aqueous solution and in live cells. In addition, the
crystal structure of 1–Hg2+ offers a suggestion for the
Hg2+-induced blue-shift and enhancement of the fluorescence
emission intensity.
As opposed to traditional instrumental techniques,
small-molecule probes are well-suited for quick detection of
Hg2+ in the field and for in vivo studies in biological
systems.1,3–7 However, most small-molecule probes require
the use of organic3 or mixed aqueous–organic solutions,4
features that may be problematic for real-world samples. In
addition, since the emission of many fluorophores is easily
quenched by heavy-metal ions, probes for Hg2+ normally
display a turn-off response,5 although a turn-on response has
proven to be more effective. Therefore, the search for new
probes with the characteristics of high selectivity and affinity,
fast and sensitive turn-on response to Hg2+, as well as good
water-solubility, pH-stability, and cell-permeability has been
the focus of extensive investigation. Such probes that demonstrate
practical application in aqueous solution6 and/or live cells7
have recently attracted much attention.
Compound 1 itself has a very weak emission band at 550 nm
in aqueous solution (Fig. 1b). Upon addition of small amounts
of Hg2+, a significant enhancement of the emission band at
487 nm occurred, resulting in a 63 nm blue-shift and a large
increase in the intensity (saturated at 2.5 mM Hg2+ with a
35-fold enhancement) of the fluorescence emission (Fig. S2,
ESIw), and the fluorescent color of the solution turned from
brown to green (inset in Fig. 1b). The effect can be compared
To improve the water-solubility of fluorescent probes,
hydrophilic amino acids have been introduced into their design.8
A fluorescent probe derived from pyrene and tryptophan
showed a detection limit of 0.15 mM for lead ion and good
solubility in aqueous solution.8a In a recent approach to Hg2+
detection, we have designed a protein-supported fluorimetric
assay using dansyl-L-aspartic acid whose fluorescence emission
spectrum underwent an obvious blue-shift and an enhancement
induced by Hg2+ in aqueous solution.8b We proposed that the
State Key Laboratory of Supramolecular Structure and Materials,
Jilin University, No. 2699, Qianjin Street, Changchun, 130012, China.
E-mail: yqwu@jlu.edu.cn, wulx@jlu.edu.cn; Fax: +86-431-85193421;
Tel: +86-431-85168730
w Electronic supplementary information (ESI) available: Synthetic
details and spectroscopic data. CCDC 719421. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/b907386h
Fig. 1 (a) Chemical structure and atomic numbering of dansyl-L-
tryptophan methyl ester (1). (b) Fluorescence titration spectra of 1
(5.0 mM) in 10.0 mM HEPES solution (pH 7.5) which is blue-shifted
and enhanced upon gradual addition of Hg2+ ions from 5.0 nM to
7.0 mM (lex = 355 nm).
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to the results previously obtained with dansyl-containing
probes.9 Coordinated with a metal ion, the amino group loses
its ability to donate an electron to the dansyl moiety.
Consequently, the involved internal charge transfer (ICT)
between the amide group and the dansyl moiety is inhibited
and the emission band shifts toward the blue region of the
emission spectrum (Fig. 1b). More importantly, the fluores-
cence intensity of 1 corresponded to the concentrations of
Hg2+ in a linear manner at low concentrations (o150 nM)
(Fig. S3, ESIw). Thus, 1 can be used to quantitatively detect
Hg2+ at low levels. A detection limit of 5.0 nM for Hg2+ was
estimated for 1, which is only 1% of that achieved in the
previous research (500 nM)8b and is lower than the EPA
mandate of 10 nM for Hg2+ in drinking water.2 To the best
of our knowledge, 1 displays the lowest detection limit of all
the small-molecule fluorescent probes used for Hg2+ detection
in pure water.
Fig. 3 (a) Region of the 1H NMR spectra of (a) 1 (0.5 mM) and
(b) 1–Hg2+ (1 : 1) in CD3OD : D2O = 1 : 2. (b) View of the X-ray
crystal structure of complex 1–Hg2+ (H-atoms were omitted for
clarity).
Characteristic structural changes can be observed upon inter-
action with Hg2+ (Fig. 3a, Table S1, ESIw). Chelation of
Hg2+ with the nitrogen atoms of the sulfonamide groups
reduced the charge transfer between it and the dansyl moiety
and resulted in large chemical shifts of protons in the naphtha-
lene ring. In addition, coordination between Hg2+ and oxygen
in the carboxylic ester induced a large chemical shift in H(21)
(DdH21 = 0.058 ppm). The splitting of the chemical shift of
H(18) after binding with Hg2+ suggested an asymmetric
binding model between Hg2+ and two molecules of 1.
Meanwhile, the minor chemical shifts of H(12), H(13),
H(14), and H(15) may be related to the weak binding of the
Over the pH range tested, compound 1 alone was not
sensitive to pH as illustrated by fluorescence intensity at
487 nm (I487) (Fig. S4, ESIw). In the presence of Hg2+
,
however, the complex of 1–Hg2+ had a strong pH-dependence
which showed a stable and sensitive fluorescence response
in the pH range of 7.5–10.0, affording the possibility for
detection of Hg2+ by 1 under weakly basic conditions. Thus,
in the current study, all the determinations of Hg2+ and other
metal ions were carried out in the presence of 5.0 mM 1 in
10.0 mM HEPES buffer solution (pH 7.5).
indole rings to Hg2+
.
Slow evaporation of solvent from the sample which contained
a complex of 1–Hg2+ in ethanol and water at room temperature
yielded buff-colored, strongly fluorescent crystals (Fig. S8, ESIw),
which were analyzed by single crystal X-ray diffraction.z The
complex comprises two molecules of 1 and one Hg2+ (Fig. 3b),
in accordance with the data revealed by Job’s plot and metal-
binding fluorescence titrations. The diagram also reveals that the
deprotonated amino group resulted in a super-strong interaction
of nitrogen and Hg2+ defined by an averaged bond distance of
2.049(8) A, and that the two sulfur and four oxygen atoms
stabilize the complex through weak interactions. In addition, the
weak interaction between Hg2+ and C(10), C(11) (for numbering
see Fig. 1a) suggests that the two indole rings may play a role in
stabilizing the 1–Hg2+ complex (Fig. S9, ESIw). The crystallo-
graphic data thus supply solid evidence and confirm the
1H NMR spectral data obtained in solution (Fig. 3a).
We also examined the fluorescence response of 1 to 15 other
metal ions under the same conditions used to test Hg2+
(Fig. 2). No obvious fluorescence changes were observed for
1 in the presence of these metal ions, even at concentrations as
high as 20.0 mM. Perhaps incompatible ion size or binding
affinity between these metal ions and 1 prevented such an
interaction. To test the practical application of 1 as a Hg2+
probe, we conducted competitive experiments in mixing
2.0 mM Hg2+ with other metal ions at 10.0 mM. Little
interference was observed by these metal ions in the detection
of Hg2+ (Fig. S5, ESIw), confirming that 1 can be used for the
practical detection of Hg2+ in aqueous solution.
In addition, the continuous variation (Job’s plot, Fig. S6,
ESIw) and metal-binding fluorescence titrations indicated that
a 2 : 1 stoichiometric ratio of 1 : Hg2+ was the most stable
species in solution with an overall Ka of 3.98 ꢁ 1013 L2 molꢂ2
(Fig. S7, ESIw). Using NMR experiments, we examined the
On the basis of these results, we confirmed that the blue-
shift and enhancement of the emission band of 1 (Fig. 1b)
originated from the strong binding between Hg2+ and the
deprotonated amino group, which induced the disruption of
ICT between the amino group and the dansyl moiety. The high
stability of 1–Hg2+ complex in aqueous solution could be
attributed to the enhanced chelation of the nitrogen atom and
Hg2+, various weak interactions provided by multiple atoms,
and also by the fixed placement in space of Hg2+ by the two
indole rings.
coordination mechanism of
1
and Hg2+ in solution.
Finally, 1 was used for imaging in HeLa cells to confirm that
our novel fluorescent probe can detect Hg2+ in live cells
(see ESIw). HeLa cells were incubated for 15 min with 1 (5.0 mM)
to allow the probe to permeate into the cell. The increases in
the fluorescence intensity in live cells were observed upon
further addition of Hg2+ (5.0 mM) into the medium and
incubation for another 15 min at 37 1C. The cells displayed
Fig. 2 (a) The fluorescence intensity of 1 (5.0 mM) in 10.0 mM
HEPES solution (pH 7.5) in the presence of 2.0 mM of Hg2+ and
20.0 mM of the other indicated ions (lex = 355 nm). (b) The intensity
ratio of the fluorescence emission bands at 487 nm in the presence of
each indicated ion.
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Fig. 4 Bright field (a, c) and confocal fluorescence microphotographs
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to 5.0 mM 1; and (c, d) 15 min exposure to 5.0 mM 1 and further 15 min
exposure to 5.0 mM HgCl2. The emission was measured over the range
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In summary, we have developed a hypersensitive fluorescent
probe that can selectively detect low levels of Hg2+ in buffered
aqueous solution. The novel probe exhibits characteristics of
high affinity, fast and turn-on response to Hg2+, and
especially good water-solubility when compared to other
reported probes. The detection of Hg2+ by 1 is evidence that
specific properties can strongly influence the emission behavior
of 1 both in aqueous solution and in live cells. Further
investigations in live cells demonstrated the potential applica-
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bioactivity of Hg2+ in live cells.
The authors are grateful to the projects of the Natural
Science Foundation of China (No. 20773051), the National
Basic Research Program (2007CB808006), the Programs for
New Century Excellent Talents in University (NCET) and the
111 Project (B06009).
Notes and references
z Crystal data for the complex 1–Hg2+ (C48H48HgN6O8S2):
Mw = 1101.63, crystal dimensions: 0.22 ꢁ 0.10 ꢁ 0.05 mm3, triclinic,
space group: P1, a = 9.694(2) A, b = 14.896(4) A, c = 16.783(5) A,
a = 87.210(11)1, b = 87.610(11)1, g = 85.290(11)1, V = 2410.8(12) A3,
Z = 2, rcalcd = 1.518 g cmꢂ3, m(MoKa) = 3.337 mmꢂ1, F(000) = 1108,
T = 291(2) K, 2ymax = 55.02. 18 562 reflections measured, of which
13763 were unique (Rint = 0.0565). Final R1 = 0.0453 and wR2 = 0.0789
for 10 458 observed reflections with I 4 2s(I), Flack parameter = 0.000(5).
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Chem. Commun., 2009, 4453–4455 | 4455