A protein-supported fluorescent reagent for the highly-sensitive and selective detection of mercury ions in aqueous solution and live cells

Article abstract of DOI:10.1039/b815281k

A fluorescent sensor, dansyl-l-aspartic acid (1), coupled with BSA was used to specifically detect Hg²⁺ in a neutral aqueous solution as well as in live cells; the fluorescence emission spectrum underwent an obvious blue shift with an enhancement in fluorescence intensity, and these effects were evident as color changes in fluorescence imaging pictures. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b815281k

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A protein-supported fluorescent reagent for the highly-sensitive and
selective detection of mercury ions in aqueous solution and live cellsw
Li-Jun Ma, Yue Li, Lei Li, Jian Sun, Chunjuan Tian and Yuqing Wu*
Received (in Cambridge, UK) 2nd September 2008, Accepted 2nd October 2008
First published as an Advance Article on the web 30th October 2008
DOI: 10.1039/b815281k
A fluorescent sensor, dansyl-L-aspartic acid (1), coupled with
BSA was used to specifically detect Hg2+ in a neutral aqueous
solution as well as in live cells; the fluorescence emission
spectrum underwent an obvious blue shift with an enhancement
in fluorescence intensity, and these effects were evident as color
changes in fluorescence imaging pictures.
solubility, and in the present case, it may also enhance the
electrostatic interaction of 1 with proteins. The molecular
structure of 1 (Fig. 1), was confirmed by its spectroscopic data
(ESIw). Bovine serum albumin (BSA), with a well character-
ized hydrophobic cavity,9 was chosen as the support protein to
change the polarity of the dansyl group environment. The
addition of Hg2+ to an aqueous solution of 1 alone resulted in
strong fluorescence quenching, while in the presence of BSA,
the addition of Hg2+ induced a blue shift and enhanced the
fluorescence emission, suggesting the potential for the highly-
sensitive and selective detection of Hg2+ in aqueous solution
and in live cells.
The recent development of selective and sensitive fluorescent
tools capable of detecting heavy- or transition-metal ions has
attracted considerable attention due to the widespread use of
these metals and their subsequent impact on the environment
and nature.1 Hg2+ is a highly toxic heavy metal ion and, even
at very low concentrations, its toxicity has long been recog-
nized as a problem in environmental and health systems.2 For
the selective detection of Hg2+, several optical techniques have
been developed, based on Hg2+-induced changes in the
UV-Vis or fluorescence spectra of the indicators.3 However,
the design of sensors that give fluorescent enhancement (FE)
upon Hg2+ binding is a particular challenge because, like many
other heavy metals, Hg2+ often causes fluorescence quenching
via enhanced spin–orbit coupling associated with the heavy
atom effect.4 This limitation may be overcome by coupling
of the fluorophore to a protein within its hydrophobic region
as has been reported for the coupling between an environ-
ment-sensitive probe and protein, either in a covalent or
non-covalent manner, which induced dramatic changes of
fluorescent properties.5 The complexation of a fluorophore in
the hydrophobic area of a water-soluble protein has the added
advantage of facilitating the probe’s solubility in aqueous
media, as low water solubility is another often encountered
obstacle in the development of fluoroionophores.1e,4a,6
Herein, we report a fluorometric assay for Hg2+ as proof of
principle for protein-supported fluorescence enhancement of
sodium dansyl-L-aspartic acid (1) (Fig. 1) with a signification
emission wavelength shift in a neutral aqueous solution. We
chose the dansyl group as the fluorophore of 1 due to its
characteristic photophysical properties.7 Its emission proper-
ties are strongly dependent upon the nature of the environ-
ment; in particular, it exhibits a large blue shift on going from
a polar to a nonpolar environment.8 In addition, as we
demonstrated earlier with a water-soluble Pb2+ probe,1e the
incorporation of an amino acid in 1 mainly enhanced its water
The addition of BSA to a solution of 1 changed the fluores-
cence spectrum (Fig. 2). 1 itself had a very weak emission in
solution. The addition of 5 equiv. BSA to 1 solution led to a
pronounced blue shift (Dlmax = 44 nm) and a significant increase
in the intensity (about 10.7 times) of the fluorescence emission
(Fig. S1w). These spectral changes were caused by a complexa-
tion, in which 1 is strongly bound to the hydrophobic region of
the BSA through hydrophobic interaction.9a,d The fluorometric
titration measurement shows a binding constant (K1/BSA) of
(6.35 Æ 0.03) Â 104 MÀ1 (R2 = 0.995) with a 1 : 1 stoichiometry
between 1 and BSA (Fig. S1w),10 indicating one BSA can occupy
a sensor in its hydrophobic cavity.
The addition of Hg2+ to an aqueous solution containing the
complex of 1 and BSA (1/BSA) induced additional spectral
shifts toward shorter wavelengths and augmented the fluores-
cence emission. As shown in Fig. 2 (from (b) to (k)), 1/BSA
could easily detect Hg2+ at low concentration (Fig. 2(c)) and a
detection limit of 0.5 mM can be estimated (Fig. 3 and S2w) as
displaying changes in fluorescence properties upon the recog-
nition of Hg2+ in aqueous solution (pH 6.7). During titration,
the emission band was further blue-shifted by about 35 nm
and the intensity was enhanced 1.5 times maximally. The
changes in the enhanced ratio of fluorescence intensity
(I À I0)/I0 (I0 is the fluorescence intensity of 1/BSA) at the maxi-
mal emission peak (lmax) were measured upon the addition of 15
different metal ions (Fig. 3). The additions of Na+, Mg2+
,
State Key Lab for Supramolecular Structure and Material, Jilin
University, No. 2699, Qianjin Street, Changchun, 130012, China.
E-mail: yqwu@jlu.edu.cn; Fax: +86-431-85193421;
Tel: +86-431-85168730
w Electronic supplementary information (ESI) available: Experimental
procedures on the prepared compounds, fluorescence photographs
and Fig. S1–S11. See DOI: 10.1039/b815281k
Fig. 1 Chemical structures of sodium dansyl-L-aspartic acid (1).
Chem. Commun., 2008, 6345–6347 | 6345
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confirmed that a ratio of 2 : 1 of 1:BSA was best for Hg2+
detection (Fig. S6w). Meanwhile, a study of the effect of pH on
the (I À I0)/I0 and Dlmax of 1/BSA with the addition of Hg2+
indicated that a pH between 6.0–7.0 was the optimum condi-
tion (Fig. S7w). Therefore, in the current study, all the
determinations of Hg2+ and other metal ions are carried out
in the presence of 30.0 mM 1 and 15.0 mM BSA in 50.0 mM
NaAc solution (pH 6.7). We also investigated the potential
interference of competing metal ions on Hg2+ detection. The
presence of a 1-fold molar excess of Cu2+ reduced the
fluorescence intensity response for Hg2+ detection, but accen-
tuated the blue-shift of fluorescent peak. No obvious interfer-
ences by other metal ions (Na+, Ca2+, Fe2+, Zn2+, Co2+
,
Fig. 2 Fluorescence emission spectra of (a) 1 (30.0 mM), (b) 1/BSA
(30.0/15.0 mM) and (c–k) 1/BSA/Hg2+ with different concentrations of
Hg2+ from 1.0 to 150.0 mM in 50.0 mM NaAc solution (pH 6.7). Inset
shows fluorescence photographs of (a), (b) and (i) (here [Hg2+] is
90.0 mM) under illumination with 365 nm light.
Cr3+ etc.) were observed (Fig. S8w).
Upon substituting the naphthalenyl group for the dansyl
moiety in 1, we noted that the addition of Hg2+ to the new
complex and BSA induced only detectable fluorescence
quenching instead of the enhanced and blue-shifted response
(Fig. S9w). When dansylated derivatives of glutamic acid,
serine, phenylalanine or tryptophan were prepared, similar
phenomenon as for the aspartyl variant 1 were achieved (data
not shown). These results demonstrate that on the platform of
BSA, the dansyl moiety of 1 plays a crucial role while aspartic
acid is not an exclusive amino acid required for the specific
recognition of Hg2+
.
NMR experiments were performed to explore the coordina-
tion mechanism between 1 or 1/BSA and Hg2+. Comparison
1
of the H NMR spectra of 1 before and after the addition of
BSA (0.01 equiv.) and Hg2+ (from 0.5 to 1.5 equiv.) (Fig. 4
and S10w) revealed that all the protons shifted upfield upon
BSA binding, while obvious shifts downfield were observed
with gradual addition of Hg2+. Chelation of Hg2+ with
5-dimethylamino and –COOÀ close to C(9) led to large
Fig. 3 (I À I0)/I0 ratios of 1/BSA (30.0 mM/15.0 mM) in the presence
of various metal ions (60.0 mM) in 50.0 mM NaAc solution (pH 6.7).
chemical shifts of H(5) (DdH5 = 0.087 ppm), H(7) (DdH7
=
0.161 ppm) and H(9) (DdH9 = 0.064 and À0.057 ppm) in 1 (the
detailed mechanistic investigations that would be required to
probe the observed prompting effect of BSA are beyond the
scope of the present study).
K+, Ca2+ Cr3+ Mn2+ Fe2+ Co2+ Ni2+ Cu2+ Zn2+
, , , , , , , ,
Ag+, Cd2+ or Pb2+ did not have similar results as Hg2+, i.e.,
either they displayed no further blue-shift or there was no intensity
enhancement other than a certain extent quenching of fluorescence
of 1/BSA (Fig. S3w). Therefore, 1/BSA exhibited specific selectivity
for Hg2+ over the other examined metal ions in aqueous solution.
The specific recognition for Hg2+ required the co-contributions
of 1 and BSA. While the addition of Hg2+ enhanced fluorescence
emission of the complex of 1/BSA, strong fluorescence quenching
occurred when Hg2+ was mixed with either BSA or 1 separately
(Fig. S4 and S5w). The fluorometric titration measurements
showed binding constants for BSA/Hg2+ and 1/Hg2+ with 1 : 1
stoichiometry of KBSA/Hg = (5.47 Æ 0.70) Â 103 MÀ1 (R2 =
0.998) and K1/Hg = (1.71 Æ 0.12) Â 105 MÀ1 (R2 = 0.991),
respectively (Fig. S4 and S5w). Comparison of the binding
constants show that K1/Hg 4 K1/BSA 4 KBSA/Hg, revealed that
the interaction of 1 and Hg2+ is evidently stronger than those
of 1/BSA and BSA/Hg2+ and, consequently, supply a possibility
of Hg2+ detection after the formation of a complex between
1 and BSA.
Finally, 1/BSA was used for imaging in fetal bovine serum
(Fig. S11w) and B16–F10 cells to confirm that our fluoroprobe
can detect Hg2+ in live cells. To observe the entry of free 1 and
Hg2+ into mammalian cells, B16–F10 cells were dyed with 1
The concentration of BSA affects the response of 1 to Hg2+
using the BSA platform. The equiv. ratio between 1 and BSA
strongly influenced the response of 1/BSA to Hg2+, which
Fig. 4 Selected region of the 1H NMR spectra of (a) 1 (10.0 mM), (b)
1/BSA (1 : 0.01), (c) 1/BSA/Hg2+ (1 : 0.01 : 0.5), (d) 1/BSA/Hg2+
(1 : 0.01 : 1) and (e) 1/BSA/Hg2+ (1 : 0.01 : 1.5) in D2O.
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supported Hg2+ probe, which was used to specifically detect
Hg2+ in high sensitivity (0.5 mM) over other 14 metal ions in
neutral aqueous solution and in live cells by monitoring the
changes in the fluorescence emission wavelength and intensity.
This is the first report of a protein-supported probe for the
detection of heavy metal ions in live cells. The detection of
Hg2+ by 1/BSA is proof that specific properties of the support
protein strongly influence the emission behavior of 1 both in
aqueous solution and in live cells. Although we have used this
system to demonstrate the detection of Hg2+ ion only, this
approach holds potential application to other metal ions,
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fluorescent reagent and a compatible protein platform.
The authors are grateful to the projects of NSFC (No.
20773051), the Major State Basic Research Development
Program (2007CB808006), the Programs for New Century
Excellent Talents in University (NCET), Jilin Province
Natural Science Foundation (20070926-01) and the 111
project (B06009).
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