An unusual OFF-ON fluorescence sensor for detecting mercury ions in aqueous media and living cells

Article abstract of DOI:10.1039/c3cc47915c

A novel azo derivative sensor (BDAA) based on alkynes was designed and utilized to direct detection of Hg²⁺ in aqueous solution and living cells. The new strategy achieved off to on switchable fluorescence. BDAA permits the highly selective and

Full text of DOI:10.1039/c3cc47915c

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An unusual OFF–ON fluorescence sensor for
detecting mercury ions in aqueous media and
living cells†
Cite this: Chem. Commun., 2014,
50, 2055
Received 21st October 2013,
Accepted 20th December 2013
Maozhong Tian,*ab Libing Liu,a Yongjun Li,a Ruifeng Hu,a Taifeng Liu,a Huibiao Liu,a
Shu Wanga and Yuliang Li*a
DOI: 10.1039/c3cc47915c
www.rsc.org/chemcomm
A novel azo derivative sensor (BDAA) based on alkynes was designed
and utilized to direct detection of Hg2+ in aqueous solution and living
cells. The new strategy achieved off to on switchable fluorescence.
BDAA permits the highly selective and sensitive detection of Hg2+
.
This sensor can be used for imaging of Hg2+ in living cells.
The design and synthesis of functionalized organic small molecules
with controlled properties on the molecular level remains a challen-
ging and attractive target of chemistry, materials science and biology.1
The design of highly selective and sensitive fluorescence sensors
based on organic conjugated molecules that are capable of detecting
heavy- and transition-metal ions has been the subject of strong
interest, because of the widespread use of these metal ions and their
subsequent pollution triggering serious environmental and health
problems.2 Mercury is one of the most toxic and dangerous heavy
metal elements in the environment, and can be easily bioaccumulated
in the body.3 Mercury compounds exhibit severe side effects on the
nervous system, the endocrine system and kidneys.4
Scheme 1 Recognition mechanism of BDAA toward Hg2+
.
This sensor system exhibits significant advantage that the fluorescence
output can be modulated from ‘‘off’’ to ‘‘on’’ in response to Hg2+ in
aqueous media and living cells with little background interference. It
also shows excellent selectivity for Hg2+ ions over relevant competing
metal ions, and a 168-fold turn-on response was achieved with the
sensitivity of ppb levels in aqueous solutions. The fluorescent response
is insensitive to media pH. To the best of our knowledge, this is the
first fluorescence sensor having the feature of chelation-enhanced
fluorescence (CHEF) based on alkynes for Hg2+ ions.
Mercury is a soft Lewis acid, and a lot of indicators for Hg2+ ions
reported typically use sulfur as a soft donor atom to tightly bind the
metal as part of fluorescence probes.5 Only a few probes for Hg2+
based on alkynes have been developed according to the p electro-
philicity of mercury ions.5i,6 Furthermore, the recognition mechan-
isms of them were all attributed to irreversible chemical reactions of
alkynes rather than to reversible interactions. Lee and co-workers
reported a selective chemodosimeter for Hg2+ ions which were
detected by a colorimetric channel in neutral aqueous solutions.6b
Herein, we describe a new, highly selective and sensitive OFF–ON
fluorescent Hg2+ sensor BDAA in aqueous solution (Scheme 1).
To indeed obtain a highly sensitive and selective OFF–ON sensor
for Hg2+ in aqueous solution, we introduced orthoethynyl and other
yne groups onto the aryl ring of a m-phenylenediamine derived
skeleton to define crescent-shaped ligand donor arrays as in BDAA.
The introduction of amino groups can help to improve the solubility
and chelation ability in aqueous solution. As shown in Scheme 2,
BDAA can be easily synthesized (see ESI† for its synthesis, Table S1
for its crystal structure data).
The aqueous solubility of the BDAA has been determined to be
25 mM at least (Fig. S1, ESI†).7 The BDAA in aqueous media does
not exhibit fluorescence (Fig. S2 in ESI†) as typical azo dyes.8
a Beijing National Laboratory for Molecular Science, Key Laboratory of Organic
Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190,
P. R. China. E-mail: ylli@iccas.ac.cn; Tel: +86-010-62587552
b College of Chemistry and Environmental Engineering, Shanxi Datong University,
Datong 037009, P. R. China. E-mail: tmz1994@@iccas.ac.cn
† Electronic supplementary information (ESI) available: Synthetic details, UV-Vis
and fluorescence spectra and fluorescence cell images. CCDC 961622 (BDAA). For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3cc47915c
Scheme 2 Synthesis of BDAA (right, crystal structure of BDAA).
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Hg2+ in a linear manner (linearly dependent coefficient: R2 = 0.9969).
This indicated that the BDAA can be used to sensitively detect Hg2+
with a detection limit of ca. 46.5 nM (Fig. S7, ESI†). A Job’s plot
indicated that BDAA chelated Hg2+ ions with 1 : 1 stoichiometry
(inset of Fig. 1). The association constant K was determined to be
3.96 Â 106 MÀ1 (Fig. S8, ESI†), which is inferred from the Hg2+ titration
curves.11 The BDAA–Hg2+ solution was subsequently treated with
excess Na2S, the strong fluorescence of the BDAA–Hg2+ was
almost quenched, indicating that the BDAA is a reversible sensor
Fig. 1 (a) Absorption spectra of BDAA (10 mM) in HEPES (50 mM) solution (Fig. S9, ESI†).
(0.1 M KNO3, pH = 7.4) upon addition of Hg2+. (b) Emission spectra
In order to elucidate the binding mode of BDAA and Hg2+, we
(excitation at 430 nm) of BDAA (10 mM) in the presence of Hg2+ (0, 2, 4,
6, 8, 10, 15, 20, 25, 30, 35, 40, 50, 60 mM). Inset: Job’s plot of BDAA.
Excitation and emission slit widths were 3 and 6 nm, respectively.
initially compared the 1H NMR spectra of BDAA and its Hg2+ complex
(Fig. S10, ESI†). When 0–1.1 equiv. of Hg2+ was added to a solution of
sensor BDAA in DMSO-d6, the signals of the six methyl protons
adjacent to one oxygen atom were shifted downfield (Dppm = 0.34)
In addition, the acid titration control experiments revealed that more than that of the methyl protons of the tert-butyl group (Dppm =
no obvious fluorescence change of BDAA could be observed from 0.11) and those of the other six methyl protons near the other oxygen
pH 3 to 11 (Fig. S3, ESI†). This phenomenon attested that no PET atoms (Dppm = 0.19). This indicated that one oxygen atom of the
process emerged in this sensor. However, 3 equiv. of Hg2+ induced sensor was coordinated in the first coordination sphere of the Hg2+
large increases of fluorescence intensity of BDAA in the pH range of complex in the solution. It should be noted that the downfield shifts
5–9. These results indicated that the BDAA can be utilized under of the aromatic protons for the BDAA–Hg2+ complex suggested a
physiological pH conditions for detection of Hg2+.
stronger metal–nitrogen interaction in the BDAA–Hg2+ complex. This
We investigated the spectroscopic characteristics and Hg2+ response might improve the efficiency of the fluorescence enhancement.
of the BDAA under physiological conditions (Fig. 1). It was found that However, further insights into 1H NMR titration spectra revealed that
free BDAA displayed very weak fluorescence (quantum yield: 0.008). The no further changes in 1H NMR signals were observed at higher
coordination of Hg2+ with the receptors blocked cis–trans transforma- equivalents of Hg2+. The 1H NMR titration results also indicated
tion of azobenzene at the excited state upon Hg2+ binding, which lead 1 : 1 binding stoichiometry of BDAA with Hg2+ which supported the
to strong fluorescence. At the same time, the removal of intermolecular Job’s plot results from fluorescence titration (inset of Fig. 1b).
hydrogen bonding upon Hg2+ binding also contributes to the turn-on
To obtain additional information about the binding mode of the
response.9 The colorimetric and fluorescence responses of BDAA to BDAA with Hg2+, the 13C NMR spectroscopic analyses in DMSO-d6
Hg2+ in aqueous solution could be visible even with the naked eye solution were performed (Fig. 2). The chemical shifts of BDAA were
(Fig. S4, ESI†). The fluorescence intensity at 525 nm dramatically assigned by HSQC and HMBC experiments (Fig. S11–S14, ESI†). The
enhanced by about 168-fold and the quantum yield increased 13C signals of one alkynyl group dramatically shifted downfield upon
B52-fold (up to 0.419). Upon the addition of Hg2+, a new absorption the addition of Hg2+, which confirmed the coordination of the
peak at 263 nm appeared with the concomitant distinct decrease of the acetylenic group with Hg2+. The peak of the C(t) atom and the C(g)
peak at 446 nm. Furthermore, the fluorescence studies of the BDAA– atom also displayed an apparent downfield shift due to the binding of
Hg2+complex were carried out in different solvents (Fig. S5, ESI†). The N, O atoms with Hg2+. The binding mode of the sensor with Hg2+ was
complex was sensitive to solvent effects, with the lem shifting from further confirmed through FT-IR spectroscopic analysis (Fig. S15,
461 nm in CH2Cl2 to 525 nm in water. In this molecular system, the ESI†). The characteristic alkyne stretching frequency of the sensor
amino groups functioned as the strong donors which led to more appeared at 2219 cmÀ1. In the BDAA–Hg2+ complex, it red shifted to
pronounced ICT in the excited state.10 Standard density functional 2013 cmÀ1. In addition, the IR spectra of the BDAA confirmed the
theory (DFT) was used to investigate the ICT effect. The spacial distri- presence of an azo group (1420–1355 cmÀ1). However, upon binding
butions of HOMO and LUMO are calculated at the B3LYP/6-31G (d) Hg2+, the stretching frequency signal of the azo group weakened
level (Fig. S6, ESI†). The calculated spatial distributions indicated that clearly. A weak band appeared in the vicinity of 461 cmÀ1 which
the density in the HOMO is concentrated in the m-phenylenediamine was attributed to Hg2+–N bonds in the complex. A strong band of
moiety, and the density in the LUMO is delocalized among the azo 631 cmÀ1 should be assigned to Hg2+–O bonds. Therefore, the
units, therefore, the aromatic amines work as the donating group for fluorescence enhancement of the BDAA was mainly due to the
the D–A system, while the azo group acts as the acceptor. Meanwhile, blockage of cis–trans transformation of azobenzene at the excited
the Stokes shift of BDAA–Hg2+ in CH2Cl2 was 55 nm, whereas the
Stokes shift in water was increased to 118 nm. The very large Stokes
shifts observed in protic solvents (water) were related to their ability to
form hydrogen bonds. The large Stokes shift of BDAA–Hg2+ is a great
advantage for fluorescence imaging, because a small Stokes shift can
cause self-quenching and measurement error due to noise from the
excitation and scattered light. More importantly, the enhancement of
fluorescence intensity of BDAA corresponds to the concentration of
Fig. 2 13C NMR spectra of the free sensors BDAA and BDAA + Hg2+
.
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Fig. 3 Histogram showing selectivity of BDAA (10 mM) for Hg2+ in HEPES buffer
(50 mM, containing 0.1 M KNO3, pH = 7.4) solution. The pillars in the front row
from left represent the I/I0 value in the presence of various metal ions (5 equiv.).
The pillars in the back row from left indicate the change in the emission intensity
upon subsequent addition of Hg2+ (3 equiv.) to the solution containing BDAA
and the metal ions of interest. For all measurements, lex = 430 nm; T = 298 K.
Excitation and emission slit widths were 3 and 6 nm, respectively.
Fig. 4 Confocal fluorescence images of live HT-29 cells. (a) Cells incubated
with 10 mM BDAA for 30 min. (b) Cells incubated with 30 mM Hg2+ for 30 min,
washed two times, and then further incubated with BDAA for 30 min.
sciences, with great potential to produce new structure fluores-
cence sensor devices.
This study was supported by the National Basic Research
state upon the chelating of Hg2+ with one of the hydroxyl groups, azo 973 Program of China (2011CB932302 and 2012CB932901) and
group, one of the alkyne bonds and the amino group next to the azo the National Natural Science Foundation of China (201031006
group (Scheme 1). Further corroborative evidence for the mercury and 91227113).
complex was observed in the MALDI-TOF mass spectrum which
showed a mass peak corresponding to [BDAA + Hg(ClO4)2Á3H2O +
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2Na+–H+]+ at m/z 931.3 (Fig. S16, ESI†).
The specificity of the sensor BDAA toward Hg2+ was determined
by fluorescence screening. Nearly no fluorescence intensity changes
were observed in emission spectra with various metal ions (Fig. 3) as
À
well as anions such as ClO4À, CO32À, SO42À, FÀ, ClÀ, BrÀ, H2PO4
and AcÀ (Fig. S17, ESI†). However, under identical conditions,
fluorescence intensity was enhanced significantly in the presence
of Hg2+. When 3 equivalents of Hg2+ were added into the solution
of BDAA in the presence of 5 equivalents of other metal ions
(10 equivalents of anions), the emission spectra displayed a similar
pattern at near 525 nm to that with Hg2+ ions only, whereas Fe3+ and
Ni2+ slightly quenched the fluorescence. These results clearly demon-
strated that the BDAA sensor was highly specific for Hg2+ ions.
To further demonstrate the practical application of the BDAA,
fluorescence imaging for Hg2+ was carried out in living cells using
scanning confocal microscopy (Fig. 4). HT-29 cells were incubated
with 10 mM of BDAA and 30 mM of Hg2+ ions (Fig. S18, ESI†) for
30 min at 37 1C, respectively. Both of them did not show intra-
cellular fluorescence. However, after the addition of Hg2+ (30 mM),
cells stained with BDAA were incubated for another 0.5 h, a marked
increase in intracellular fluorescence was observed. These experi-
ments indicated that BDAA is cell permeable and can respond to
Hg2+ ions within living cells.
In summary, we have demonstrated a new strategy to direct
detection of Hg2+ in aqueous solution and living cells which
achieved ‘‘off’’ to ‘‘on’’ switchable fluorescence. The fluorescence
intensity of the unusual sensor was significantly enhanced about
168-fold. Such a fluorescence sensor of the new structure expanded
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