A colorimetric and turn-on fluorescent chemosensor for selectively sensing Hg2+ and its resultant complex for fast detection of I- over S2-

Article abstract of DOI:10.1016/j.dyepig.2016.01.008

A novel bipyridine-functionalized turn-on fluorescent chemosensor was successfully synthesized and fully characterized by ¹H NMR, ¹³C NMR and MS, UV-vis and fluorescence spectroscopies. The sensor specifically binds to Hg²⁺ over other competing ions with a significant fluorescence enhancement as well as a visual colour change under physiological conditions. The detection limit of Hg²⁺ was as low as 32 nM, confirming very high sensitivity toward Hg²⁺. Moreover, the fluorescence intensity and colour change of the sensor-Hg²⁺ was quenched by I^- or S^2- and was proportional to their concentrations with a detection limit of 0.37 μM and 0.43 μM, respectively. The reaction of I^- grabbing Hg²⁺ from the sensor-Hg²⁺ finished in 10 s due to a stronger binding force, much faster than that of S^2-, which allowed fast detection of I^- over S^2- even in a competent environment. In addition, the sensor was successfully used for the highly sensitive detection of Hg²⁺ in living cells.

Full text of DOI:10.1016/j.dyepig.2016.01.008

Accepted Manuscript
2+
A colorimetric and turn-on fluorescent chemosensor for selectively sensing Hg and
‒
2‒
its resultant complex for fast detection of I over S
Ximing Huang, Zhengliang Lu, Zhuo Wang, Chunhua Fan, Wenlong Fan, Xiaomin
Shi, Haitao Zhang, Meishan Pei
PII:
S0143-7208(16)00011-5
10.1016/j.dyepig.2016.01.008
DYPI 5061
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 27 October 2015
Revised Date: 5 January 2016
Accepted Date: 6 January 2016
Please cite this article as: Huang X, Lu Z, Wang Z, Fan C, Fan W, Shi X, Zhang H, Pei M, A colorimetric
2+
and turn-on fluorescent chemosensor for selectively sensing Hg and its resultant complex for fast
‒
2‒
detection of I over S , Dyes and Pigments (2016), doi: 10.1016/j.dyepig.2016.01.008.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
A colorimetric and turn-on fluorescent chemosensor for selectively sensing Hg2+
and its resultant complex for fast detection of I‒ over S2‒
Ximing Huang,† Zhengliang Lu,*,† Zhuo Wang,‡ Chunhua Fan,† Wenlong Fan,† Xiaomin Shi†,
Haitao Zhang, † Meishan Pei†
†Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of
Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China
‡ Faculty of Science, Beijing University of Chemical Technology, Beijing, 100029, China.
Abstract
A novel bipyridine-functionalized turn-on fluorescent chemosensor was successfully
1
synthesized and fully characterized by H NMR, 13C NMR and MS, UV-Vis and
fluorescence spectroscopies. The sensor specifically binds to Hg2+ over other
competing ions with a significant fluorescence enhancement as well as a visual colour
change under physiological conditions. The detection limit of Hg2+ was as low as 32
nM, confirming very high sensitivity toward Hg2+. Moreover, the fluorescence
intensity and colour change of the sensor-Hg2+ was quenched by I‒ or S2‒ and was
proportional to their concentrations with a detection limit of 0.37 ꢀM and 0.43 ꢀM,
respectively. The reaction of I‒ grabbing Hg2+ from the sensor-Hg2+ finished in 10
seconds due to a stronger binding force, much faster than that of S2‒, which allowed
fast detection of I‒ over S2‒ even in a competent environment. In addition, the sensor
was successfully used for the highly sensitive detection of Hg2+ in living cells.
Keywords: colorimetric, chemosensor, mercury, iodide, sulphide
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1. Introduction
The developments of multifunctional chemosensors for heavy and transition
metal cations as well as various anions have attracted considerable attention due to
their potential applications in biological and environmental systems [1-4]. As one of
the most toxic metals mercury (Hg) can significantly destroy human central nervous
system and endocrine system [5-7]. Even a very low level of Hg2+ can disturb a series
of cellular processes and consequently trigger serious health disorders in the human
body, such as Minamata, edema and anemia [8-11]. Moreover, mercury ion (Hg2+)
from artificial and mineral processes such as gold mining, fossil fuel combustion, and
chemical manufacturing can be released into the natural environment, accumulated in
food chains, and finally entered into higher trophic biological systems [12]. The
World Health Organization (WHO) has strictly stipulated that the level of Hg2+ is not
more than 0.001 mg·L‒1 in drinking water [13]. Among various chemically and
biologically important anions, I‒ is one of dispensable elements in human body and
the heaviest element commonly needed by living organisms [14-16]. Iodide
deficiency leads to mental retardation and thyroid gland dysfunction and increases the
risk of breast and stomach cancer [17]. Release of excessive I‒ ions and iodine can
damage environment and physical systems.[18]Also H2S from industrial processes
such as sewage plants and petroleum refining results in serious environmental
pollution. Moreover, endogenic sulfide ions from microbial reduction of sulfate and
sulfur-containing amino acids can destroy mucous membranes and brain tissues,
which are correlated with Alzheimer’s disease, Down’s syndrome, and diabetes
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[19-24]. So it is urgent to develop novel analytical methods with high sensitivity and
selectivity to detect I‒ and S2‒ ions as well as Hg2+ in either aqueous or non-aqueous
media.
Fluorescence methodology has been widely used as a great tool for detection of
metal ions and anions due to its operational simplicity, high sensitivity and selectivity,
and ease of observation over traditional methods such as inductively coupled plasma
mass spectrometry, atomic absorption, and chemiluminescence [25-28]. Thus,
numerous colorimetric and fluorescent chemosensors have been reported in the recent
literatures for selective and sensitive sensing of Hg2+ [29-35], I‒ [36-38] and S2‒
[39-41] ions, respectively. As a matter of fact, few multifunctional probes sensing
metal ions and anions have been reported [42, 43]. Without doubt, it is of challenge to
rationally design simple, practical multifunctional chemosensors in aqueous media.
Usually, the construction of fluorescent sensors depends on rational
combinations of recognition sites and signalling subunits [44]. It is well known that
each signalling subunit has its own distinct emission spectrum and each recognition
group could detect a wide array of ions. Even the subtle changes of the linker such as
incorporation of either a single- or double-bond could dramatically influence the
detection efficiency of designed probes. For example, Duan’s group reported the
probe P1 constructed through the condensation of rhodamine 6G hydrazide and
2-pyridinecarboxaldehyde (Scheme 1a), which can detect Hg2+ in 1:1 (v/v) H2O/DMF
solution with very high efficiency [45]. Later, Zhang’s group reported that the probe
P2 (Scheme 1b), obtained through reducing probe P1, could rapidly sense Cu2+ within
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one minute in buffered H2O/EtOH (8:2, v/v, Tris-HCl, pH 7.1) [46]. Probe P3
containing thiophene tailed pyridine as the recognition group could detect Zr4+ in
CH3OH-H2O (4:1, 1/1, HEPES, 10 ꢀM, pH 7.4) (Scheme 1c) [47]. Furthermore,
ligand-metal ensembles tend to selectively and sensitively recognize anions in
aqueous solutions [48-52].
Encouraged by these excellent studies and speculations, a multifunctional
chemosensor RBP (Scheme 2) was constructed based on the rhodamine fluorescence
platform combining a 2,2`-bipyridyl recognition site. As anticipated, the binding of
RBP with Hg2+ triggered the opening of the spirolactam ring of the rhodamine moiety
in neutral aqueous solutions with remarkably high sensitivity and selectivity.
Interestingly, the ensemble RBP-Hg2+ could sense I‒ over S2‒. Moreover, RBP can be
successfully applied to bioimaging and detecting Hg2+ in living cells.
2. Experimental
2.1 Material and apparatus
All starting materials were used as received without further purification. All
solvents were purified according to standard procedures unless stated otherwise.
1
Doubly purified water used in all experiments was from Milli-Q systems. H and 13C
NMR were performed on a Bruker DRX-400 spectrometer operating at 400 and 100
MHz, respectively, using TMS as an internal standard. Mass spectrometric data were
collected on a PE Sciex API 3000 mass spectrometer. Elemental analyses (C, H and
N) were carried out using a Perkin-Elmer 240 elemental analyser. UV-Vis absorption
and fluorescence spectra were measured on a Shimadzu UV-2100 spectrophotometer
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and
an
F-7000
spectrofluorophotometer,
respectively.
(3) was
2-amino-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one
synthesized according to the literature [53].
2.2 Synthesis
2.2.1 Synthesis of 6-methyl-2,2'-bipyridine (1)
The title compound was synthesized using a revised procedure [54].
Methyllithium (1.3 M, 33.80 mmol) in THF (26 mL) was added dropwise to a
solution of 2,2'-bipyridine (5.30 g, 34.00 mmol) in diethyl ether (100 mL) at 0oC
under N2 atmosphere. The reaction mixture was stirred for 2 h, and then refluxed for 3
h. Water (10 mL) was added when the mixture cooled to room temperature. The
organic layer was separated and the aqueous layer was extracted three times with
ether. The combined organic layer was dried by anhydrous Na2SO4. The solvent was
removed by evaporation. The resulting orange oil was oxidized with saturated
KMnO4/acetone (300 mL) and stirred for 1 h. The filtrate was placed in a flask and
acetone was removed by evaporation. The resulting dark oil was distilled under
vacuum and finally gave pure 6-methyl-2,2'-bipyridine as colourless oil (3.73 g,
1
63.8%). H NMR (400 MHz, CDCl3) δ ppm : 8.65 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H),
8.15 (d, J = 7.9 Hz, 1H), 7.72-7.82 (m, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.21-7.30 (m,
1H), 7.13 (d, J = 7.7 Hz, 1H), 2.61(s, 3H). 13C NMR (100 MHz, CDCl3) δ ppm: 157.7,
156.3, 155.4, 149.0, 136.9, 136.7, 123.6, 123.2, 121.02, 117.9, 24.5. ESI-MS (m/z):
calcd for C11H10N2 [M+H]+ 171.22, found 171.61.
2.2.2 Synthesis of 2,2'-bipyridine-6-carbaldehyde (2)
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Compound 2 was obtained using a revised procedure [55]. A mixture of
compound 1 (3.31g, 19.45 mmol) and selenium dioxide (1.27 g, 11.5 mmol) in
dioxane (50 mL) containing H2O (0.21 mL) was refluxed for 3 h. After the foregoing
mixture was cooled to room temperature, additional selenium dioxide (1.27 g, 11.5
mmol) and H2O (0.2 mL) was added and then the mixture was refluxed for 27 h. The
hot reaction mixture was filtered and the insoluble material was washed with warm
dioxane and ethyl acetate (20 mL × 3). the crude product was purified by column
chromatography to give the title compound (1.28 g, 34.1%) as colourless oil. 1H NMR
(400 MHz, CDCl3) δ ppm: 10.18 (s, 1H), 8.74 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 8.65 (dd,
J = 7.1, 1.95 Hz, 1H), 8.56 (d, J = 7.9 Hz, 1H), 7.97-8.02 (m, 2H), 7.90 (td, J = 7.9,
1.8 Hz, 1H), 7.40 (ddd, J = 7.1, 4.8, 1.1 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ ppm:
193.7, 156.6, 155.0, 152.3, 149.3, 138.0, 137.1, 125.3, 124.4, 121.5, 121.4. ESI-MS
(m/z): calcd for C11H8N2O [M+H]+ 185.20, found 185.15.
2.2.3
Synthesis of 2-(2,2'-bipyridin-6-ylmethyleneamino)-3',6'-bis (diethylamino)
spiro[isoindoline-1,9'-xanthen]-3-one (RBP)
A mixture of Rhodamine B hydrazide (0.547 g, 1.14 mmol) and
2,2'-bipyridine-6-carbaldehyde (0.184 g, 1.00 mmol) in ethanol (20 mL) was refluxed
for 6 h. The pure RBP as a yellow solid (0.36 g, 50.1%) was obtained by column
chromatography. 1H NMR (400 MHz, CDCl3) δ ppm: 8.84 (s, 1H), 8.31-8.60 (m, 2H),
7.70-8.03(m, 3H), 7.31-7.62 (m, 4H), 7.15 (d, J = 8.3 Hz, 1H), 6.55 (t, J = 8.4 Hz,
1H), 6.46 (d, J = 7.7 Hz, 2H), 6.42 (d, J = 7.8 Hz, 2H), 6.23-6.32 (m, 2H), 3.33 (q, J =
13
7.0 Hz, 8H), 1.15 (t, J = 6.9 Hz, 12H). C NMR (100 MHz, CDCl3) δ ppm: 166.2,
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153.8, 149.0, 148.9, 148.8, 136.9, 136.8, 132.5, 128.1, 127.9, 120.8, 120.5, 108.0,
105.9, 104.5, 97.9, 97.9, 66.2, 65.9, 44.3, 12.6. ESI-MS (m/z): calcd for C39H38N6O2
[M+H]+ 623.3134, found 623.3174.
2.2.4
Synthesis of 2-(benzylideneamino)-3',6'-bis(diethylamino)spiro[isoindoline-
1,9'-xanthen]-3-one (RBB)
RBB was synthesized using excess benzaldehyde in an identical procedure to
1
RBP as a yellow solid (0.71 g, 65.4%). H NMR (400 MHz, CDCl3) δ ppm: 8.65 (s,
1H), 7.98 (s, 1H), 7.54-7.58 (m, 2H), 7.39-7.50 (m, 2H), 7.22-7.26 (m, 3H), 7.11 (s,
1H), 6.52 (dd, J = 8.8, 2.3 Hz, 2H ), 6.43 (d, J = 2.3 Hz, 2H), 6.24 (dd, J = 8.8, 2.3 Hz,
2H), 3.31 (q, J = 7.1 Hz, 8H), 1.15 (t, J = 7.1 Hz, 12H). 13C NMR (100 MHz, CDCl3)
δ ppm : 164.3, 153.3, 151.6, 149.0, 148.4, 135.1, 134.4, 130.8, 129.4, 128.1, 127.3,
124.4, 123.5, 108.5, 106.0, 97.8, 66.0, 44.1, 12.9. Anal. calc. for C35H36N2O2: C,
77.18; H, 6.66; N, 10.29; found: C, 77.24; H, 6.62; N, 10.32.
2.3 Cell culture and confocal imaging
MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM,
Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), and maintained in
5% CO2 at 37oC. 12 hours before imaging, the cells were cultured on confocal culture
dishes in 1 mL of DMEM without FBS the cells were treated and incubated with 10
ꢀM RBP in EtOH-H2O stock solution at 37oC under 5% CO2 for 30 min, and were
provided with fresh medium that contained Hg2+ (50 ꢀM). The cells were incubated
for another 10 min under the above conditions. The images were taken after the cells
were rinsed three times with phosphate buffered saline (PBS).
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3. Results and discussion
3.1. Synthesis
The synthesis of compound RBP is shown in Scheme 2. Briefly, 2 was prepared
by methyl group oxidation of 2,2'-bipyridine according to a previously reported
procedure [54, 55]. Thereafter RBP was obtained in good yields by heating a mixture
of 2 and 3 to reflux in methanol for 6 h. A reference compound
2-(benzylideneamino)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one
(RBB), was synthesized in good yields using the same procedure as RBP from
1
benzaldehyde. RBP was characterized by H NMR, 13C NMR, and MS (SI, Figs.
S1-S3). Comparison of the fluorescence emission spectra of RBP and RBB further
confirmed the important role of the binding ability of bipyridyl group.
3.2. UV-vis and fluorescence studies
First, the UV-Vis titration experiments of RBP with Hg2+ were performed in 1:4
(v/v) EtOH/H2O solution at pH 7.0 (HEPES 20 mM). As shown in Fig. 1a, the
solution of free RBP in EtOH/H2O (1:4, v/v) was colourless and exhibited no
absorption, which is ascribed to the spirolactam form of RBP. Addition of Hg2+
immediately induced a new absorption band at 568 nm which is proportional to the
Hg2+ concentration ranging from 0‒33 equiv. (Inset of Fig. 1a). The maximum
absorption appeared upon addition of 33 equiv. of Hg2+. An obvious colour response
of RBP from colourless to pink was stimulated by coordination of Hg2+ (Fig. 1b and
1c). Such a characteristic colour change and absorption response indicates that RBP
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can be used as a sensitive and colorimetric “naked-eye” chemosensor for Hg2+ in
environmental and biological samples.
To further examine the sensitivity, fluorescence properties of RBP (20 ꢀM) with
Hg2+ were investigated in EtOH/H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0) (Fig.
2). As anticipated, a new fluorescence emission peak at 584 nm was observed upon
incremental addition of Hg2+ and finally reached the maximum in the presence of 33
equiv. of Hg2+, an 18-fold increase over that with 1 equiv. of Hg2+, whereas free RBP
displayed almost no fluorescence emission upon excitation at 520 nm. This
remarkable enhancement was reasonably attributed to the existing conjugated
xanthene tautomer of the rhodamine moiety of RBP. The fluorescence quantum yield
was 0.386 with Rhodamine B as a reference. In the inset of Fig. 2a, there is a good
linear relationship between the emission intensity at 584 nm and the concentration of
Hg2+ ranging from 0 – 33 equiv. (R2 = 0.997), which was further confirmed by the
linearity of UV-Vis data. The detection limit for Hg2+ was evaluated to be 32 nM
using the equation LOD = K*Sb/S (where K = 3, Sb is the standard deviation of the
blank solution and S is the slope of the calibration curve of fluorescence emission)
(Fig. S4), which was much lower than the permissible limit of 0.001 mg/L (tolerable
value for mercury in drinking water by the World Health Organization (WHO)) [13].
A Job’s plot indicated a 1:1 binding stoichiometry with a maximum emission change
observed at a mole ratio of 1:1 for RBP and Hg2+ (Fig. S5). With the absorption data,
the association constant Ka was evaluated to be 0.95 × 103 M‒1 using the
Benesi-Hildebrand equation [56]. Thereby, our supposed fluorescence method to
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detect Hg2+ provides excellent sensitivity comparable to most turn-on sensors reported
in the literature (Table S1).
3.3. pH stability studies
To explore RBP applications in biological systems, the fluorescence response of
RBP, with or without Hg2+ at 584 nm at different pH values, was investigated (Fig. S6)
in EtOH/H2O (1:4, v/v). In the absence of Hg2+ the obvious fluorescence emission of
the free RBP was detected only in a more acidic environment (pH < 5.0), indicating
the susceptibility of the spirolactam ring. There was no any obvious change of
complex RBP-Hg2+ when pH was not more than 10. That is because RBP-Hg2+
would decompose at strong basic conditions. This good fluorescence response of RBP
to Hg2+ in a wide pH range of 5 ‒ 10 indicated that it can act as a sensitive
chemosensor under physiological conditions.
3.4. Metal Ion Competition and Anion Recognition Studies
Competition experiments to study the selectivity of chemosensor RBP toward
Hg2+ over other competitive metal cations were performed and the respective
fluorescence intensities are displayed in Fig. 2b. When the titration was conducted in
EtOH/H2O solution (1:4, v/v, HEPES 20 mM, pH 7.0), respective addition of other
competitive metal ions such as Ag+, Al3+, Ba2+, Cd2+, Fe3+, K+, Li+, Mn2+, Na+, Ni2+,
Pb2+, and Zn2+ did not cause any absorption and fluorescence response of RBP even at
a concentration of 50 equiv. of metal ions under physiological conditions. Only Hg2+
induced a significant fluorescence enhancement; the colour change from colourless to
red although Cu2+ and Cr3+ induced a weak fluorescence change. As shown in Fig. S7,
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addition of Hg2+ into a solution of RBP with other competitive metal ions together
induced significant fluorescence emission. In fact, RBP also exhibited satisfactory
selectivity toward Hg2+ in a mixture of all competitive metal ions. These results
indicate that RBP is highly selective chemosensor for Hg2+ by direct visual
observation in aq. ethanol solution under physical conditions.
To further verify the selectivity, the fluorescence emission of complex RBP-Hg2+
2‒
3‒
was investigated with some representative anions such as SO4 , PO4 , CH3COO‒,
2‒
‒
CO3 , NO3 F‒, Cl‒, Br‒, I‒ and S2‒ in EtOH-H2O (1:4, v/v) solution at pH 7 (HEPES
20 mM). As shown in Fig. 3a, only I‒ and S2- could dramatically quench the
fluorescence intensity of the solution of RBP (10 ꢀM) and Hg2+ (30 equiv.), and
induce a colour change from red to colourless. Other anions did not perturb any
marked fluorescence emission of the RBP-Hg2+ complex in solution except a weak
fluorescence-quenching from Br‒. The fluorescence intensity of RBP-Hg2+ decreased
upon gradual addition of I‒ (Fig. 3b). That also indicated that Hg2+ prefers binding
with I‒ than RBP. A good linear relationship between the fluorescence change and the
I‒ concentration was obtained during the I‒ range from 0-2 equiv. compared to the
Hg2+ concentration (inset of Fig. 3c). Based on LOD = K*Sb/S, the limit of detection
(LOD) of RBP-Hg2+ to I‒ is calculated to be 0.75 ꢀM. The titration of S2‒ to
RBP-Hg2+ also was carried out and gave rise to good linearity of the fluorescence
change and the S2‒ concentration with the detection limit of 0.43 ꢀM (Fig. S8).
Response time and reversibility are fundamental parameters for most
coordination-based chemosensors, and the kinetic profiles of the reaction of RBP and
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Hg2+, RBP-Hg2+ and I‒, and RBP-Hg2+ and S‒, respectively, at room temperature was
examined. The fluorescence emission reached maximum within 70 minutes (Fig. S9).
Surprisingly the binding of I‒ to Hg2+ from the RBP-Hg2+ complex was over in ca. 10
seconds and the fluorescence emission was unchanged over the subsequent 120 s (Fig.
3d). However, the reaction between S2‒ and Hg2+ from RBP-Hg2+ almost finished in
almost 30 minutes, which is much longer than that of I‒ (Fig. S10). Therefore,
RBP-Hg2+ still can rapidly sense I‒ in few seconds even in the presence of S2‒
interference. The recyclability experiments of RBP upon the addition of Hg2+ and
subsequent I‒/S2‒ were carried out and confirmed the high stability of RBP in
EtOH/H2O solution although a slightly attenuation was found, which fully support the
reversible spirolactam ring-opening mechanism of rhodamine derivatives (Fig. S11).
These results indicated that RBP can be a selective and sensitive Hg2+ chemosensor
and its resultant complex can fast detect I‒.
3.5. The proposed sensing mechanism
Up to now turn-on fluorescent chemosensors are still preferable due to high
selectivity, sensitivity and ease of observation compared to turn-off ones. The
fluorescence enhancement of RBP toward Hg2+ is supposed to arise from the spiro
ring-opening mechanism rather than an ion-catalysed hydrolysis reaction. So
a
plausible response mechanism of RBP to Hg2+ is shown in Scheme S1. Based on
this, RBP was rationally designed containing the rhodamine B platform as the
potential strong fluorophore, and a bipyridyl fragment as a specific binding receptor
of Hg2+. The bipyridyl fragment binding with Hg2+ induced opening of the
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spirolactam ring of rhodamine moiety, which caused the enhancement of fluorescence
and colour changes. Addition of I‒ led to the regeneration of the no-fluorescence
spirolactam ring of rhodamine moiety of RBP because of the strong binding ability
between I‒ and Hg2+ which causes the formation of HgI2. Although compared with I‒,
S2‒ binds Hg2+ much more slowly, the reaction still can finish about 30 minutes. A
second addition of Hg2+ also recovered the fluorescence emission. Thus, the
reversibility evidently ruled out the possibility of hydrolysis mentioned in the
literature [57]. In order to confirm the importance of the bipyridyl fragment, RBB was
synthesized and studied by fluorescence emission spectra in EtOH/H2O (1:4, v/v). As
shown in Fig. S12, RBB did not generate any fluorescence emission even with excess
addition of 100 equiv. of Hg2+, which confirmed the crucial role of the bipyridyl
group of RBP. Job’s plot also ascertained a 1:1 stoichiometry of RBP and Hg2+.
3.6. Cytotoxicity and application of RBP in living cells
The excellent absorbance and fluorescence spectroscopic properties of RBP
inspired us to carry out the Hg2+ bioimaging studies using MCF-7 cells with a
confocal microscope Zeiss LSM710 (Fig. 4). MCF-7 cells were cultured with RBP
(10 µM) in DMEM for 30 min at 37°C and washed with PBS buffer. No intracellular
fluorescence was detected (Figs. 4a and 4b), indicating that the probe RBP
maintained its spirolactam form in cells. When the MCF-7 cells pre-incubated with
RBP and further treated with 50 µM Hg2+ for 10 min, an intense red fluorescence was
observed (Figs. 4c and 4d). This phenomenon illustrated RBP was cell permeable,
and could sense Hg2+ in living cells. We further performed a conventional MTT assay
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to examine the cytotoxicity of RBP on MCF-7 cells, in which no cytotoxicity was
observed, even at RBP concentrations up to 100 µM. These results indicated that
probe RBP could viably sense Hg2+ both in vitro and in vivo cells.
4. Conclusions
In conclusion, we have developed a colorimetric and fluorescent chemosensor
RBP for Hg2+ detection with high selectivity and sensitivity under physiological
conditions. Moreover, the ensemble RBP-Hg2+ can be an excellent sensory system for
fast detection I‒ over S2‒ with reversibility, indicating that the metal-based complex is
a promising tool to selectively and sensitively detect anions. The confocal
fluorescence image confirmed that RBP owes high cell permeability and low toxicity
for sensing Hg2+ in vivo cells. We believe that the proposed strategy can be applied to
construct other multifunctional fluorescent probes owing wide potential applications
in environmental and biological analysis, or in vivo cells.
Acknowledgements
We thank the national Natural Science Foundation of china (Grant No. 21101074,
21222502, 21575032), Shandong Provincial Natural Science Foundation of China
(Grant No. ZR2013BQ009), and the Doctor’s Foundation of University of Jinan
(Grant No. XBS1320) for funding. Dr. Srinivas Abbina from Center for Blood
Research, Department of Pathology and Laboratory Medicine, University of British
Columbia, Canada is kindly acknowledged for his constructive comments and
suggestions to improve the manuscript significantly.
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Scheme and Figure Captions
Scheme 1. The structures of formerly reported probes.
Scheme 2. Structures and the syntheses of the RBP and RBB.
Fig. 1. (a) UV-Vis absorption spectra of probe RBP (10 ꢀM) upon addition of Hg2+ (0
- 36 equiv.) in EtOH/H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). Inset:
Calibration plot of absorption and concentration of Hg2+. (b) Colour change of RBP
upon interaction with different metal ions (Hg2+, Ag+, Al3+, Ba2+, Ca2+, Cd2+, Fe3+, K+,
Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Zn2+). (c) Colour change of RBP upon addition of
Hg2+ (0 - 36 equiv.).
Fig. 2. (a) Fluorescence spectra of RBP (10 ꢀM) upon addition of Hg2+ (0 - 36 equiv.)
in EtOH-H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). Inset: Calibration plot of
fluorescence intensity and concentration of RBP. (b) Fluorescence intensity at 584 nm
of RBP (10 ꢀM) upon addition of 15 equiv. of various metal ions (Red bars: RBP
with other metals, green bars: RBP with other metals and Hg2+) in EtOH/H2O (1:4,
v/v) solution (HEPES 20 mM, pH 7.0).1, blank; 2, Ag+; 3, Al3+; 4, Ba2+; 5, Ca2+; 6,
Cd2+; 7, Cr3+; 8, Cu2+; 9, Fe3+; 10, K+; 11, Li+; 12, Mg2+; 13, Mn2+; 14, Na+; 15,
Ni2+;16, Pb2+; 17, Zn2+.
Fig. 3. (a) Fluorescence spectra of RBP-Hg2+ (10 ꢀM) upon addition of various
anions (60 equiv.) in EtOH-H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). (b)
Fluorescence intensity of RBP-Hg2+ (10 ꢀM) upon addition of I‒ (0-2 equiv. of Hg2+)
in EtOH-H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). (c) Calibration plot of
fluorescence intensity and concentration of I‒. (d) Fluorescence intensity of
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RBP-Hg2+ (10 ꢀM) upon adding 2 equiv. of I‒ in EtOH/H2O (1:4, v/v) solution
(HEPES 20 mM, pH 7.0) as a function of the time.
Fig. 4. Images of MCF-7 cells treated with RBP (20 µM) in the absence or
presence of Hg2+ (100 ꢀM). (a) Confocal fluorescence image of MCF-7 cells
with RBP. (b) Bright field image of (a). (c) Confocal fluorescent image of
MCF-7 cell with Hg2+ and RBP. (d) Bright field image of (c). Excitation
wavelengths of RBP and RBP-Hg2+ are 543 nm. The scale bar of all figures is 20
μm.
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Scheme 1. The structures of formerly reported probes.
Hg2+
N
O
O
N
N
(a)
N
N
N
Hg2+
H
N
H
O
N
H
N
H
O
N
H
P1
N
Cu2+
O
O
N
(b)
N
H
N
N
NH
Cu2+
H
N
H
O
N
H
N
H
O
N
H
P2
S
S
(c)
O
Zr4+
O
N
N
N
N
N
Zr4+
N
H
N
O
N
N
O
N
P3
Scheme 2. Structures and the syntheses of the RBP and RBB.
N
O
O
N
N
NH2
N
N
N
N
N
N
N
N
CH3Li
SeO2
N
O
N
3
N
O
N
CHO
2
1
RBP
O
O
N
NH2
CHO
N
N
N
O
N
3
N
O
RBB
N
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Fig. 1. (a) UV-Vis absorption spectra of probe RBP (10 ꢀM) upon addition of Hg2+ (0
- 36 equiv.) in EtOH/H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). Inset:
Calibration plot of absorption and concentration of Hg2+. (b) Colour change of RBP
upon interaction with different metal ions (Hg2+, Ag+, Al3+, Ba2+, Ca2+, Cd2+, Fe3+, K+,
Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Zn2+). (c) Colour change of RBP upon addition of
Hg2+ (0 - 36 equiv.).
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Fig. 2. (a) Fluorescence spectra of RBP (10 ꢀM) upon addition of Hg2+ (0 - 36 equiv.)
in EtOH-H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). Inset: Calibration plot of
fluorescence intensity and concentration of RBP. (b) Fluorescence intensity at 584 nm
of RBP (10 ꢀM) upon addition of 15 equiv. of various metal ions (Red bars: RBP
with other metals, green bars: RBP with other metals and Hg2+) in EtOH/H2O (1:4,
v/v) solution (HEPES 20 mM, pH 7.0).1, blank; 2, Ag+; 3, Al3+; 4, Ba2+; 5, Ca2+; 6,
Cd2+; 7, Cr3+; 8, Cu2+; 9, Fe3+; 10, K+; 11, Li+; 12, Mg2+; 13, Mn2+; 14, Na+; 15,
Ni2+;16, Pb2+; 17, Zn2+.
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Fig. 3. (a) Fluorescence spectra of RBP-Hg2+ (10 ꢀM) upon addition of various
anions (60 equiv.) in EtOH-H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). (b)
Fluorescence intensity of RBP-Hg2+ (10 ꢀM) upon addition of I‒ (0-2 equiv. of Hg2+)
in EtOH-H2O (1:4, v/v) solution (HEPES 20 mM, pH 7.0). (c) Calibration plot of
fluorescence intensity and concentration of I‒. (d) Fluorescence intensity of
RBP-Hg2+ (10 ꢀM) upon adding 2 equiv. of I‒ in EtOH/H2O (1:4, v/v) solution
(HEPES 20 mM, pH 7.0) as a function of the time.
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Fig. 4. Images of MCF-7 cells treated with RBP (20 µM) in the absence or
presence of Hg2+ (100 ꢀM). (a) Confocal fluorescence image of MCF-7 cells
with RBP. (b) Bright field image of (a). (c) Confocal fluorescent image of
MCF-7 cell with Hg2+ and RBP. (d) Bright field image of (c). Excitation
wavelengths of RBP and RBP-Hg2+ are 543 nm. The scale bar of all figures is 20
μm.
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A colorimetric multifunctional sensor RBP was synthesized.
The sensor exhibited a selective fluorescence enhancement response to Hg2+.
The resultant sensor-Hg2+ complex rapidly detected I‒ over S2‒.
The sensor was successfully used to selectively detect Hg2+ in living cells.