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DOI: 10.1039/C5AN00452G
1
A highly selective fluorescent probe for in vitro and in
2
vivo detection of Hg2+
3
Quan Zhou†, Zeming Wu†, Xiaohua Huang†, Fenfen Zhong†, Qingyun Cai†*
4
†
5
Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China.
6
Abstract
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of
7
In this paper, a simple fluorescent probe, rhodamine B derivatives (RS), was
8
designed and prepared for sensitive detection of Hg2+ in CH3CN/H2O (5/5, v/v). RS
9
exhibits high selectivity and sensitivity toward Hg2+ over other common metal ions,
10
displaying a significant color change from colorless to pink in the presence of Hg2+.
11
The fluorescence responses remain stable over abroad pH range (5.0 to 9.0) and
12
suitable for detections under physiological conditions. Experiments results of Hela
13
cells and zebrafish show that RS is cell and organism permeable. We also demonstrate
14
the acquistition of imaging of Hg2+ in the Hela cells and zebrafish by using a simple
15
fluorescence confocal imaging technique.
16
Keywords: Rhodamine B, Hg , Fluorescent Probe, In vitro, In vivo
2+
*
Corresponding author. Tex.: +86-73188821848. E-mail:
[email protected],
[email protected].
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DOI: 10.1039/C5AN00452G
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1. Introduction
18
As one of the most toxic metal elements, mercury brings an extremely pollutional
19
impact on our surviving environment.1-5 The gradual accumulation of Hg2+ through
20
food-chain in human body results in a wide variety of diseases, including acrodynia,
21
Hunter-Russell syndrome, Alzheimer and Minamata disease.6-8 Thus, quantification of
22
Hg2+ is of great interest for monitoring and preventing its contamination toward the
23
environment and the living world. Conventional analytical techniques towards Hg2+
24
are mainly consist of cold-vapor atomic fluorescence spectrometry (CV-AFS),9
25
cold-vapor atomic absorption spectrometry (CV-AAS),10 inductively coupled
26
plasma-mass spectrometry (ICPMS),11 ultraviolet visible spectrometry and X-ray
27
absorption spectroscopy.12 Most of them have been extensively used for the assay of
28
Hg2+ in water samples. However, high cost and tedious sample preparation procedures
29
are often required for these measurements, which are especially not suitable for in
30
vitro or in vivo monitoring of intracellular mercury. In recent years, chemical sensors
31
based on chromophores, fluorophores, polymers, DNAzymes,13-19 oligonucleotides,20
32
and functionalized nanomaterials
33
Especially, some organic small molecule-based ones have attracted many attentions
34
by opening the spirolactam ring of rhodamine derivatives with the appropriate
35
recognition units.
36
bearing a squaraine moiety as a selective fluorescent chemosensor for Hg2+.30 Yoon et
37
al. reported a rhodamine hydrazone derivatives bearing thiol and carboxylic acid
38
groups as selective fluorescent and colorimetric chemosensors for Hg2+.31 Although
26-29
21-25
have been developed for detection of Hg2+.
For example, Son et al. reported a rhodamine-6G derivative
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these reported ones have realized fluorescence imaging of Hg2+ in living cells,32-39
40
only a few have been developed for detection of Hg2+ in vivo.40 Therefore, the
41
construction of a simply prepared, selective probe with fast response to quantification
42
of Hg2+ in environmental samples, and image dynamic changes of Hg2+ in biological
43
samples including living cells and organism is still highly desirable.
44
Herein, we have designed a novel rhodamine derivative RS (shown in Scheme 1),
45
which can be prepared by two steps synthesis from rhodamine B in about 10 h and
46
respond to the presence of Hg2+ with high selectivity and sensitivity. The probe (RS)
47
was synthesized by treating rhodamine B with N2H4·H2O followed by 3-(methylthio)-
48
propionaldehyde. After column chromatogramphy using acetate/petroleum ether (1/5,
49
v/v) as eluent, RS was obtained with 73% yield. We speculated that introduction of
50
the 3-(methylthio)- propionaldehyde receptor to the probe RS would have the follow
51
advantages compared with other probes: (1) increasing affinity for Hg2+ in
52
competitive aqueous media through anchoring of the thioether bond with Hg2+; (2)
53
leading to its quick fluorescence and color responses through subsequent coordination
54
of the spirolactum oxygen with Hg2+; and (3) improving selectivity toward other
55
interference ions. The large fluorescence enhancements as well as colorimetric
56
changes from colorless to pink were observed for the probe RS in the presence of
57
Hg2+, which can be attributed to the structural conversion of the spirolactam ring into
58
the xanthene form, due to breaking the conjugation of C-N bond by coordination with
59
Hg2+. Furthermore, the probe RS were successfully applied to image Hg2+ in vitro and
60
in vivo.
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2. Experimental section
62
2.1. Reagents and apparatus
63
All chemicals were obtained from commercial suppliers and used without further
64
purification. Water used in all experiments was doubly distilled and purified by a
65
Milli-Qsystem (Millipore, USA). Mass spectra were performed using a LCQ
66
Advantage ion trap mass spectrometer (Thermo Finnigan). NMR spectra were
67
recorded on a BrukerDRX-400 spectrometer using TMS as an internal standard. The
68
pH was measured with a Mettler-Toledo Delta 320 pH meter. Fluorescence
69
measurements were carried out on a Hitachi-F4600 fluorescence spectrometer with
70
excitation and emission slits set at 5.0 nm and 10.0 nm respectively. Fluorescence
71
imaging of Hela cells and Zebrafish were obtained using OLYMPUS FV-1000
72
inverted fluorescence microscope.
73
2.2. Synthesis of probe RS
74
Synthesis of Rhodamine-Probe (RS) Rhodamine-NHNH2 was prepared following a
75
literature method.41 AcOH (two drops) was added to a solution of Rhodamine-NHNH2
76
(223.0 mg, 0.5 mmol) and 3-(methylthio)-propionaldehyde (78.0 mg, 0.75 mmol) in
77
EtOH (10 mL). Then the reaction mixture was stirred at 80 °C for 6 h. The solvent of
78
the mixture was removed under reduced pressure, and the resulting residue was
79
purified on a silica gel column (acetate/petroleum ether =1:5) to afford compound RS
80
as a white solid (197.8 mg, isolated yield: 73%). 1H NMR (400 MHz, CDCl3): δ 7.96
81
(d, J = 8.0 Hz, 1H),7.60 (s, 1H), 7.42-7.40 (m, 2H),7.04 (d, J = 8.0Hz, 1H), 6.51-6.49
82
(m, 2H), 6.40-6.39 (m, 2H), 6.26-6.24 (m, 2H), 3.33 (q, J = 8.0 Hz, 8H), 2.44-2.35 (m,
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4H), 1.90 (s, 3H) 1.15 (t, J = 8.0 Hz, 12H); 13CNMR (100 MHz, CDCl3): δ 165.17,
84
152.58, 152.44, 149.83, 148.78, 133.25, 128.14, 128.02, 127.72, 123.45, 123.39,
85
107.98, 105.50, 97.97, 65.16, 44.29, 33.54, 30.65, 15.17, 12.53; ESI-MS: m/z: 543.3
86
[M+H]+.
87 88 89
Scheme 1 Synthesis of the compund Rhodamine-Probe (RS)
2.3 Spectrophotometric Experiments.
90
Both the fluorescence and UV-vis absorption experiments were conducted in
91
CH3CN/H2O (5:5, v/v). The fluorescence emission spectra were recorded at excitation
92
wavelength of 500 nm with emission wavelength range from 550 to 700 nm. A
93
solution of RS (1 × 10-3 M) was prepared by dissolving RS in CH3CN. The solution of
94
RS was prepared by adding 100 µL of the stock solution of RS in a 10 mL volumetric
95
flask, and the solution was diluted to 10 mL with CH3CN and H2O (CH3CN/H2O =5:5,
96
v/v).
97
2.4 Cytotoxicity of Probe RS.
98
The cytotoxic effect of compound RS and RS-Hg complex was determined by an
99
MTT assay following the manufacturer instruction (Sigma-Aldrich, MO). HeLa cells
100
were initially propagated in a 25 cm2 tissue culture flask in Dulbecco’s Modified
101
Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS),
102
penicillin (100 µg/mL), and streptomycin (100 µg/mL) in a CO2 incubator. For
103
cytotoxicity assay, cells were seeded into 96-well plates (approximately 104 cells per
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well), and various concentrations of compound RS and RS-Hg complex (10, 20, 40,
105
80, and 100 µM) made in DMEM were added to the cells and incubated for 24 h.
106
Solvent control samples (cells treated with DMSO alone) and cells treated with
107
Hg(ClO4)2 alone were also included in parallel sets. Following incubation, the growth
108
media was removed, and fresh DMEM containing MTT solution was added. The plate
109
was incubated for 3-4 h at 37 °C. Subsequently, the supernatant was removed, the
110
insoluble colored formazan product was solubilized in DMSO, and its absorbance was
111
measured by Benchmark Plus (Bio-Rad Instruments Inc., Japan) at 550 nm. The assay
112
was performed in seven sets for each concentration of compound RS and RS-Hg
113
complex.
114
2.5 Cell Cultures and Imaging Experiments.
115
HeLa cells were obtained from the biomedical engineering center of Hunan
116
University (Changsha, China). The cells were propagated in Dulbecco’s Modified
117
Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, penicillin
118
(100 µg/mL), and streptomycin (100 µg/mL). Cells were maintained under a
119
humidified atmosphere of 5% CO2 and at 37 °C incubator as mentioned before. For
120
cell imaging studies, cells were seeded into a Confocal dish and incubated at 37 °C in
121
a CO2 incubator for one day. After one day cells were washed three times with
122
phosphate buffered saline (pH 7.4) and incubated with 10 µM RS in DMEM at 37 °C
123
for 1 h in a CO2 incubator and observed under Olympus FV1000 laser confocal
124
microscope. The cells were again washed thrice with PBS (pH 7.4) to remove the free
125
RS, and then incubated in phosphate buffered saline with 10 µM Hg(ClO4)2 for 1 h.
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Again, images were taken using confocal microscope.
127
2.6 Imaging of Zebrafish
128
Zebrafishes were obtained from the school of life sciences of Hunan normal
129
University (Changsha, China). The Zebrafishes were maintained in E3 embryo media
130
(15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO4, 1.0 mM CaCl2, 0.15 mM KH2PO4, 0.05
131
mM Na2HPO4, 0.7 mM NaHCO3, 5-10% methylene blue, pH 7.5). In fluorescence
132
imaging experiments, three-day-old zebrafishes were incubated with RS 20 µM in E3
133
embryo media for 1 h at 28 °C and washed with PBS to remove the remaining RS.
134
The treated zebrafish was then incubated in a solution containing 20 µM Hg2+ for 1 h
135
at 28 °C. Confocal fluorescence image were observed under an Olympus FV1000
136
laser confocal microscope.
137
3. Results and Discussion
138
3.1. Spectroscopic Studies of RS in Presence of Hg2+.
139
According to previous studies, certain transition-metal ions can bind selectively
140
with suitable derivatives of rhodamine, leading to the opening of the spirolactam ring
141
and generation of the xanthene form.42,43 UV-vis spectra were recorded for RS in
142
10 µM CH3CN/H2O in the presence of various cations including Hg2+ (Fig.1A). Fig.
143
1A shows a significant change in the UV-vis absorption spectrum pattern at 558 nm in
144
the presence of Hg2+ among all the tested ions including Na+, Ca2+, Mg2+, Cu2+, Zn2+,
145
Fe2+, Fe3+, Co2+, Ni2+, Ba2+, Mn2+ Ag+ , Cd2+ and ClO4- in CH3CN/H2O (5:5, v/v),
146
indicating a high selectivity of RS toward Hg2+. The UV-vis absorption peak around
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558 nm increased with increasing of the Hg2+ concentration from 0 to 100µM
148
(Fig.1B). The solution turned from colorless to pink with addition of Hg2+ (The inset
149
of Fig.1B).
150 151
Figure 1. (A) UV-vis absorption spectra of receptor RS (10 µM) observed upon addition of 100
152
µM ions (Na+, Ca2+, Mg2+, Hg2+, Cu2+,Zn2+, Fe2+, Fe3+, Co2+, Ni2+, Ba2+, Mn2+, Ag+, Cd2+ and
153
ClO4-) in a CH3CN/H2O (5:5, v/v).(B) UV-vis titration spectra of RS (10µM) upon incremental
154
addition of 0 to 100 µM Hg2+ in CH3CN/H2O (5:5, v/v). Inset: the color changes of RS to Hg2+.
155
The fluorescence spectrum was recorded in 10 µM RS in CH3CN (5:5, v/v) in the
156
absence and presence of Hg2+ (50 µM) and other ions (100 µM) (Fig.2A). The
157
metal-ligand binding induces ring-opening of RS and the generation of xanthene
158
moiety that was highly selective toward Hg2+, hence a significant fluorescent response
159
was observed in the presence of Hg2+ (Fig.2A, and Fig.S1, see ESI†). There are not
160
any noticeable spectral change for other tested ions (Na+, Ca2+, Mg2+, Cu2+, Zn2+, Fe2+,
161
Fe3+, Co2+, Ni2+, Ba2+, Mn2+, Ag+, Cd2+ and ClO4-), confirming again the high
162
selectivity of RS to Hg2+. Excitation of the initial solution of probe RS at 500 nm
163
wavelength did not show any significant emission over the range from 550 to 700 nm
164
(Fig.2A). This supported the facts that in the absence of Hg2+, the receptor remained
165
in the spirolactam form. The nonexistence of highly conjugated xanthene form
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166
resulted in the suppression of emission in near 587 nm. To gain an insight into the
167
properties of RS as a receptor for Hg2+, the titration was performed with increasing
168
concentration of Hg2+. As described in Fig.2B, the fluorescence intensity of RS was
169
enhanced with about 80-fold increase upon addition of only 50 µM Hg2+ ions,
170
exhibiting a high sensitivity of RS toward Hg2+. The plot of emission intensity vs
171
concentration of Hg2+ was linear from 0.1 to 10 µM (Fig.S2 and Fig.S3, see ESI†), the
172
limit of detection was calculated as 1.6 × 10−8 M (equal to 3.2 µg/kg) from the 3σ
173
method (the limit of detection= 3σ/slope), which is lower than the maximum
174
allowable levels of Hg2+ regulated by the USFDA (1mg/kg) and Health Canada
175
(3mg/kg).[44] To study the stability of the probe RS in different pH values, the
176
fluorescence spectra of RS response toward Hg2+ in different pH conditions were
177
evaluated (Fig.S4, see ESI†). These results clearly explain that this probe can be used
178
in a broad range of pH 5.0-9.0.
179 180
Figure 2. (A) Changes of the fluorescence emission of receptor RS (10 µM) observed upon
181
addition of ions (Na+, Ca2+, Mg2+, Hg2+, Cu2+,Zn2+, Fe2+, Fe3+, Co2+, Ni2+, Ba2+, Mn2+, Ag+, Cd2+
182
and ClO4-) (100 µM) in CH3CN/H2O (5:5, v/v). (B) Fluorescence titration spectra of RS (10 µM)
183
upon incremental addition of 0-50 µM of Hg2+ in CH3CN/H2O (5:5, v/v), λex = 500 nm. Inset: the
184
visual fluorescence color changes of RS to Hg2+.
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Fig.3 shows the naked eye and UV illuminated color of RS (10 µM)in the
186
presence of different ions (50 µM). Common cations viz. Na+, Ca2+, Mg2+, Cu2+,Zn2+,
187
Fe2+, Fe3+, Co2+, Ni2+, Ba2+, Mn2+, Ag+, Cd2+ and ClO4- couldn’t induce color change
188
of free RS. However, the addition of Hg2+ to the RS solution induces an obvious
189
color change, indicating RS can selectively sense Hg2+ through colorimetric method.
190
Above the results illuminated that the introduction of the 3-(methylthio)-
191
propionaldehyde receptor to a rhodamine-based probe could increase its affinity for
192
Hg2+ in competitive aqueous media through anchoring of the thioether bond with
193
Hg2+, and improving selectivity toward other interference ions.
194 195
Figure 3. Color of RS (10 µM) in the presence of different ions 50 µM: naked eye (top); under
196
UV lamp (bottom) in CH3CN-water (5:5, v/v).
197
3.2 Sensing mechanism
198
The sensing mechanism was studied using FTIR technique (Fig.S5, see ESI†).
199
The addition of 2 equiv Hg2+ resulted in the characteristic stretching frequency of the
200
C=O amide bond at 1692 cm−1 of the rhodamine unit shifting to 1650 cm−1. Such a
201
shift was reported to be due to the binding of the rhodamine unit in RS with a metal
202
ion,45 the amide carbonyl group was involved in the interactions with Hg2+. This is a
203
key factor to the spiro ring-opening and fluorescence recovery of the rhodamine dye.
204
In order to further confirm the above mechanism, 1H NMR titration (Fig.S6, see
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ESI†) was performed by concomitant addition of Hg2+ to a RS solution in CDCl3.
206
Significant spectral changes of RS were observed upon addition of Hg2+ from 5 min
207
to 30 min. The a and b proton peaks (-SCH3 and HC=N) were shifted downfield from
208
1.91 to 2.03 and 7.60 to 11.32 ppm, respectively, which was attributed to the
209
Hg2+-induced spirolactam ring opening of RS. The c and d proton peaks were shifted
210
downfield from 3.33 to 3.60 and 1.16 to 1.29 ppm, respectively, which was ascribed
211
to the positive charge’s nitrogen atom [=N+(CH2CH3)2] generating by the
212
rearrangement of rhodamine B after Hg2+-induced spirolactam ring opening of RS.
213
Furthermore, we studied the mass spectrum of the RS-Hg system to verify
214
Hg2+-triggered spiro ring-opening process. The Job’s plot showed the reaction of RS
215
with Hg2+ at a mole ratio of 1:1 (Fig.S8, see ESI†). Furthermore, we studied the
216
proposed mechanism by ESI-MS spectrum. In an ethanol solution of RS (100 mg,
217
0.18 mmol), 2 mL solution of Hg(ClO4)2 (40 mg, 0.1mmol) in water was added
218
drop-wise and shaked for 30 minutes. The solution was kept for several days to finally
219
obtain a deep pink compound. ESI-MS: [M+H+] = 443.2. We speculated that it may
220
be caused by the hydrolysis of probe RS following the mechanism, which was showed
221
in the second step of scheme 2 , the similar mechanism has been reported by the
222
references.[46] Taken these results together, a likely sensing mechanism based on the
223
Hg2+-triggered spiro ring-opening process is proposed in Scheme 2. Based on the
224
relationship between the fluorescence intensity and concentration of Hg2+, the binding
225
constant was calculated to be logK = 7.5 (Fig. S9, see ESI†).
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226 227 228 229
Scheme 2. Proposed mechanism for the fluorescent changes of sensor RS upon the addition of Hg2+
3.3 Fluorescence Imaging Experiments of RS in living cell
230
To demonstrate the potential use of the probe RS in bioimaging applications, we
231
tested the cytotoxicity of RS toward HeLa cells, by the reduction activity of
232
methylthiazolyltetrazolium (MTT) assay (Fig.S10, see ESI†). The viability of
233
untreated cells was assumed to be 100%. Upon incubation of 0-100 µM RS for 6 and
234
12 h, no significant difference in the proliferation of the cells was observed.
235
Specifically, cell viabilities of about 80% even at a high-dose concentration of 100
236
µM Hg2+ were observed after 6 and 12 h. These data indicated the satisfactory
237
biocompatibility of the Hg2+ fluorescent probe at all dosages, thus enabling the RS to
238
serve as a potential probe for fluorescence bioimaging.
239
In order to extend the application of RS in more complex metrics, we examined
240
the imaging characteristics of RS to cultured living cells in vitro (Hela, human
241
cervical cancer cell) by fluorescence microscopy (Fig. 4). The cells were incubated
242
with 10 µM RS (suspended in phosphate-buffered saline; PBS) for 1 h at 37 °C. Then
243
the cells were washed with PBS for three times and mounted on a microscope stage.
244
As shown in Fig.4A, the cells display modest intracellular staining after incubation
245
with RS for 1 h, suggesting that RS was efficiently taken up by the cells. Upon
246
incubation with 10 µM Hg2+ for 1 h, a striking turn-on fluorescence is observed inside
247
HeLa cells, indicating the formation of the RS-Hg2+ complex (Fig.4B), which
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248
conformed with the studies observed in solution. Essentially, the fluorescence
249
microscopic analysis strongly suggested that the probe RS could readily cross the
250
membrane barrier, permeated into HeLa cells, and rapidly sense intracellular Hg2+. It
251
is significant to mention that bright field imaging of treated cells did not reveal any
252
gross morphological perturbations, which suggested that HeLa cells were viable. In
253
addition, we have compared the probe RS with the previously reported probe 1[47]
254
imging in living cell, the results showed that the probe RS was similar to the reported
255
probe 1 imaged in vivo, and it could detect the Hg2+ in a low concentration in vitro
256
(Fig.S11, see ESI†). This finding encouraged us for in vivo biomedical applications of
257
the probe RS.
258 259
Figure 4. Fluorescence microscopic images of HeLa cells: (A) after treating with 10 µM RS
260
(under green light); (B) after addition10 µM of Hg2+ (under green light) to the RS treated cells.
261
3.4 Fluorescence Imaging Experiments of RS in Zebrafish.
262
Whole-organism experiments were also carried out to examine whether the probe
263
can be used to image Hg2+ in living organisms. A 3-days-old zebrafish was incubated
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with RS (20 µM) in E3 embryo media for 1 h at 28 °C and washed with PBS to
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remove the remaining RS. The treated zebrafish was then incubated in a solution
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DOI: 10.1039/C5AN00452G
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containing 20 µM Hg2+ for 1 h (Fig.5). In a reverse experiment, the zebrafish was first
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incubated with 20 µM of HgCl2 in E3 embryo media for 1 h at 28 °C, washed with
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PBS to remove the remaining mercury ions, and then incubated in a solution
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containing 20 µM of RS for 1h. The results of fluorescence microscope analysis of
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these specimens showed that Hg2+ in zebrafish are fluorescently detected by RS
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(Fig.S11, see ESI†). The zebrafish remained alive throughout the imaging
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experiments. These results indicate that the RS proble is useful for the study of the
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toxicity or bioactivity of Hg2+ in living organisms.
A
B
C
D
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Figure 5. Bright-field (A, C) and Fluorescence microscopic (B, D) images of three-day-old
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zebrafish incubated with RS (20 µM) in the absence (A, B) and presence (C, D) of Hg2+ (20 µM).
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4. Conclusions
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In conclusion, a novel fluorescent probe was well designed, synthesized, and
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applied for Hg2+ imaging. After careful evaluation, this probe paraded high selectivity
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and sensitivity toward Hg2+ in vitro and in vivo. The reasonable histocompatibility and
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stability of this probe contribute to the feasible applications in animals. Overall, all
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experimental results suggest that this probe stipulates a selective and sensitive method
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DOI: 10.1039/C5AN00452G
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for imaging of Hg2+ in living zebrafish. Furthermore, the influence of the levels of
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Hg2+ on health, aging, and disease may be researched in the upcoming future by
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utilizing such a fluorescence microscopic images approach. In addition, multiplexed
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detections of more than one heavy metals by the spirocyclic derivatives of rhodamine
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dye senors will be further researched in our lab.
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Acknowledgements
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We gratefully acknowledge the National Science Foundation of China (grant 21235002, 21175038) for financial support.
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Figure captions
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Scheme 1. Synthesis of the compund Rhodamine-Probe (RS)
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Scheme 2. Proposed mechanism for the fluorescent changes of sensor RS upon the addition of
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Hg2+
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Figure 1. (A) UV-vis absorption spectra of receptor RS (10 µM) observed upon addition of 100
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µM ions (Na+, Ca2+, Mg2+, Hg2+, Cu2+,Zn2+, Fe2+, Fe3+, Co2+, Ni2+, Ba2+, Mn2+, Ag+, Cd2+ and
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ClO4-) in a CH3CN/H2O (5:5, v/v). (B) UV-vis titration spectra of RS (10µM) upon incremental
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addition of 0 to 100 µM Hg2+in CH3CN/H2O (5:5, v/v). Inset: the color changes of RS to Hg2+.
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Figure 2. (A) Changes of the fluorescence emission of receptor RS (10 µM) observed upon
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addition of ions (Na+, Ca2+, Mg2+, Hg2+, Cu2+, Zn2+, Fe2+, Fe3+, Co2+, Ni2+, Ba2+, Mn2+ and Ag+,
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Cd2+ and ClO4-) (100 µM) in CH3CN/H2O (5:5, v/v). (B) Fluorescence titration spectra of RS (10
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µM) upon incremental addition of 0-50 µM of Hg2+ in CH3CN/H2O (5:5, v/v), λex = 500 nm.
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Inset:the visual fluorescencecolor changes of RS to Hg2+.
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Figure 3. Color of RS (10 µM) in the presence of different ions 50 µM: naked eye (top); under
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UV lamp (bottom) in CH3CN-water (5:5, v/v).
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Figure 4. Fluorescence microscopic images of HeLa cells: (A) after treating with 10 µM RS
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(under green light); (B) after addition10 µM of Hg2+ (under green light) to the RS treated cells.
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Figure 5. Bright-field (A, C) and Fluorescence microscopic (B, D) images of three-day-old
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zebrafish incubated with RS (20 µM) in the absence (A, B) and presence (C, D) of Hg2+ (20 µM).
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Scheme 1
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Scheme 2
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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B
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