A naphthalimide-rhodamine ratiometric fluorescent probe for Hg2+ based on fluorescence resonance energy transfer

Article abstract of DOI:10.1016/j.dyepig.2011.07.020

On the basis of fluorescent resonance energy transfer from 1,8-naphthalimide to rhodamine B, a new fluorophore dyads (4) containing rhodamine B and a naphthalimide moiety was synthesized as a ratiometric fluorescent probe for detecting Hg²⁺ wit

Full text of DOI:10.1016/j.dyepig.2011.07.020

Dyes and Pigments 92 (2012) 909e915
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A naphthalimideerhodamine ratiometric fluorescent probe for Hg2þ based on
fluorescence resonance energy transfer
*
Yunlong Liu, Xin Lv, Yun Zhao, Maliang Chen, Jing Liu, Pi Wang, Wei Guo
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of fluorescent resonance energy transfer from 1,8-naphthalimide to rhodamine B, a new
fluorophore dyads (4) containing rhodamine B and a naphthalimide moiety was synthesized as a ratio-
metric fluorescent probe for detecting Hg2þ with a broad pH range 5.7e11.0. The selective fluorescence
response of 4 to Hg2þ is due to the Hg2þ-promoted desulfurization of the thiocarbonyl moiety, leading to
Received 25 June 2011
Received in revised form
24 July 2011
Accepted 25 July 2011
Available online 4 August 2011
the ring-opening of rhodamine B moiety of 4. When 4 was employed at 0.1 mM with the slit size being
20 nm/20 nm, a low level of Hg2þ (up to 3 ꢀ 10ꢁ8 M) can be detected using the system.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probe
Ratiometric
Naphthalimide
Rhodamine
Desulfurization
Mercury ion
1. Introduction
probe concentration can interfere with the signal output. To elim-
inate those effects, a ratiometric fluorescent measurement is
desirable [10e14]. This technique uses the ratio of the fluorescent
intensities at two different wavelengths, and provides a built-in
correction for environmental effects, and stability under illumina-
tion, allowing precise and quantitative analysis and imaging even in
complicated systems.
Mercury is the third most frequently found and second most
common toxic heavy metal in the list of the Agency for Toxic
Substances and Disease Registry (ATSDR) of the U.S. Department of
Health and Human Services [1,2]. The extreme toxicity of mercury
and its derivatives results from its high affinity for thiol groups
in proteins and enzymes, leading to the dysfunction of cells
and consequently causing health problems [3e5]. Unfortunately,
mercury contamination can occur through a variety of natural and
anthropogenic sources including oceanic and volcanic emissions,
gold mining, and combustion of fossil fuels. Thus, the health
concerns over exposure to mercury have motivated the exploration
of selective and efficient methods for the monitoring of mercury in
biological and environmental samples.
Fluorescence spectroscopy has become a powerful tool for
sensing and imaging trace amounts of species such as metal ions
because of its simplicity and high sensitivity [6e9]. The most
reported fluorescent sensors display an increase or decrease in the
emission intensity upon binding to species of interest. As the
change in fluorescence intensity is the only detection signal, factors
such as instrumental efficiency, environmental conditions, and the
Several signaling mechanisms, such as intramolecular charge
transfer (ICT) [15e18], excimer/exciplex formation [19e22], and
fluorescence resonance energy transfer (FRET) [23e28], can be
employed for the design of ratiometric measurement. Among them,
FRET is a nonradiative energy transfer process in which the exci-
tation energy of the donor is transferred to the nearby acceptor via
long-range dipoleedipole interaction and/or short-range multi-
polar interaction. Although the efficiency of energy transfer is
affected by the distance between the donor and the acceptor as well
as the relative orientation of transition dipoles of both the donor
and acceptor, it is mainly determined by the extent of the spectral
overlap between the donor emission and acceptor absorption [29].
Therefore, it would be possible to fabricate a ratiometric probe
based on the FRET mechanism if a molecule could generate a suit-
able fluorescent energy acceptor by the interaction with target
analyte. In addition, because the pseudo-Stokes shifts of FRET-
based probes are larger than the Stokes shifts of either the donor
or acceptor dyes, thus, the possible self-quenching as well as
fluorescence detection errors due to backscattering effects from the
* Corresponding author. Tel./fax: þ86 351 7011600.
E-mail address: guow@sxu.edu.cn (W. Guo).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.07.020

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Y. Liu et al. / Dyes and Pigments 92 (2012) 909e915
excitation source will be efficiently avoided [30]. However, despite
many advantages, examples of the ratiometric fluorescence probes
for Hg2þ based on FRET are considerably limited [31e34].
On the basis of the spirolactam (nonfluorescent) to ring-open
amide (fluorescent) equilibrium of rhodamine, rhodamine-based
dyes have been demonstrated to be excellent OFF/ON-type fluores-
cence probes [35,36]. On the other hand, 1,8-naphthalimide deriva-
tives are excellent fluorophores due to their high stability and
quantum yield as well as convenient functionalization at 4-position or
imine site, and have been widely exploited as fluorescent probes
[37e44]. Recently, two independent energy transfer dyads, both of
which combine the properties of 1,8-naphthalimide and rhodamine
dyes in a donoreacceptor chemical architecture for excited state
energy transfer, were exploited as fluorescence ratiometric and turn-
on probes for Cr3þ [25] and Hg2þ [45], respectively. Inspired by these
results as well as the Hg2þ-induced desulfurization reactions [46e49],
in this paper we hope to report a new naphthalimideerhodamine
ratiometric fluorescent probe for Hg2þ based on FRET. The strategy for
FRET detection of Hg2þ is based on modulating the FRET process in
a fluorophore dyads (4) comprising a 1,8-naphthalimide donor and
a rhodamine-thiosemicarbazide acceptor linked by a rigid phenyl
spacer (Fig. 1). In the absence of Hg2þ, the rhodamine moiety adopts
a closed, non-fluorescent spirolactam form, thus, FRET is suppressed,
and only the yellow emission of the donor is observed upon excitation
Fig. 2. Spectral overlap of the donor 3 emission with the acceptor rhodamine B
absorption.
2.2. Synthesis
of the 1,8-naphthalimide donor. In the presence of Hg2þ, the Hg2þ
-
2.2.1. Compound 4
promoted reaction of thiosemicarbazide to oxadiazole will induce
opening of the rhodamine moiety [46], and produces intense
rhodamine absorption in the 1,8-naphthalimide emission region.
Therefore, excitation of the 1,8-naphthalimide chromophore results in
strong red emission of rhodamine due to FRET.
A solution of 4-morpholin-1,8-naphthalic anhydride (1, 0.1 g,
0.35 mmol) and p-phenylenediamine (0.76 g, 0.70 mmol) in
ethanol (40 mL) was heated under reflux for 24 h. After left to cool,
the precipitated crystals were filtered and washed with ethanol to
give 2 as yellow solid (96 mg, 73%). Mp: 306e308 ꢂC. 1H NMR
(300 MHz, DMSO-d6)
d
8.49 (d, J ¼ 9.0, 1H), 8.44 (d, J ¼ 6.9, 1H), 8.37
(d, J ¼ 8.1, 1H), 7.80 (t, J ¼ 7.8, 1H), 7.35 (d, J ¼ 8.1, 1H), 6.88 (d, J ¼ 7.2,
2H), 6.62 (d, J ¼ 8.1, 2H), 5.19 (b, 2H), 3.90 (b, 4H), 3.21 (b, 4H).
A solution of 2 (0.1 g, 0.26 mmol) in CH2Cl2 (10 mL) was added
2. Experiment
2.1. Materials and general methods
slowly to a solution of thiocarbonyl chloride (75 mL, 0.1 mmol) in
CH2Cl2 (12 mL) and NEt3 (1 mL), and the mixture was stirred for 5 h
at room temperature. Then the reaction mixture was washed with
water, dried over Na2SO4, and evaporated under reduced pressure.
The solid was purified on flash silica gel using CH2Cl2 as eluent to
4-Morpholin-1,8-naphthalic anhydride (1) [50] and rhodamine
B hydrazine [51] were prepared by literature procedures. All other
reagents and solvents were purchased from commercial sources
and were of the highest grade. Solvents were dried according to
standard procedures. All reactions were magnetically stirred and
monitored by thin-layer chromatography (TLC). Flash chromatog-
raphy (FC) was performed using silica gel 60 (230e400 mesh).
Absorption spectra were taken on an Agilent 8453 spectropho-
tometer. Fluorescence spectra were taken on HITACHI F-2500
fluorescence spectrometer. The 1H NMR and 13C NMR spectra were
recorded at 300 and 75 MHz, respectively. The following abbrevi-
ations were used to explain the multiplicities: s ¼ singlet;
d ¼ doublet; t ¼ triplet; q ¼ quartet; m ¼ multiplet; br ¼ broad.
High resolution mass spectra were obtained on a Varian QFT-ESI
mass spectrometer.
give 3 as yellow solid (97 mg, 90%). 1H NMR (300 MHz, CDCl3)
d 8.68
(d, J ¼ 6.9,1H), 8.63 (d, J ¼ 8.1,1H), 8.54 (d, J ¼ 8.4,1H), 7.82 (t, J ¼ 8.1,
1H), 7.46e7.32 (m, 5H), 4.09 (b, 4H), 3.36 (b, 4H).
Fig. 3. Changes in absorption spectra of 4 (10
(10 mM TriseHCl, pH 7.0) with various amounts of Hg2þ ions. Inset: Job’s plot for
determining the stoichiometry of 4 and Hg2þ
mM) in 2:1 (v/v) MeOH/water solution
Fig. 1. The response mechanism of 4 to Hg2þ
.
.

Y. Liu et al. / Dyes and Pigments 92 (2012) 909e915
911
Fig. 4. Fluorescence titration spectra of 4 [10
m
M, 1
mM, and 0.1
m
M for (a), (b) and (c), respectively] in 2:1 (v/v) MeOH/water solution (10 mM TriseHCl, pH 7.0) upon gradual
addition of Hg2þ. Inset: Fluorescence intensity ratio changes (I585/I545) of 4 upon gradual addition of Hg2þ
.
lex ¼ 400 nm. Slits: 10 nm/10 nm for (a), 20 nm/20 nm for (b) and (c).
A
stirred of solution of rhodamine B hydrazide (0.11 g,
(b, 1H), 7.53e7.70 (m, 5H), 7.19e7.28 (m, 4H), 7.05e7.10 (m, 3H),
6.50 (d, J ¼ 8.1, 2H), 6.44 (s, 1H), 6.32 (d, J ¼ 8.1, 2H), 4.02 (b, 4H),
3.27e3.34 (m, 12H), 1.15 (t, J ¼ 6.6, 12H). 13C NMR (75 MHz,
CDCl3): 165.0, 164.6, 156.6, 154.9, 150.8, 150.0, 140.9, 138.5, 135.0,
133.6, 132.7, 132.2, 131.1, 130.9, 129.8, 129.3, 128.3, 126.8, 126.6,
125.4, 124.6, 124.0, 117.7, 115.7, 109.1, 104.8, 99.0, 67.6, 54.1, 46.5,
45.1, 13.3. TOF MS calcd. for (MþH)þ 872.3516, found 872.3539.
0.24 mmol) and compound 3 (0.1 g, 0.24 mmol) in DMF was
heated at 60 ꢂC for 36 h. The solvent was evaporated at reduced
pressure, and the residue was purified on flash gel using CH2Cl2/
MeOH (10:1, v/v) as eluent to give 4 as yellow solid (95 mg, 45%).
Mp: 242e244 ꢂC. 1H NMR (300 MHz, CDCl3)
d 8.50 (m, 4H), 8.01
2.2.2. Compound 5
A mixture of compound 4 (87.2 mg, 0.1 mmol) and Hg(NO3)2$H2O
(34.2 mg, 0.1 mmol) was stirred in MeOH for 10 min at room
temperature. The solvent was evaporated under reduced pressure
and the crude product was purified by column chromatography on
silica gel using CH2Cl2/MeOH (20:1, v/v) as eluent to give 5 as red solid
(77 mg, 91%). 1H NMR (300 MHz, CDCl3):
d 11.14 (b, 1H), 8.40e8.52
(m, 3H), 8.22 (b, 1H), 7.89 (d, J ¼ 8.4, 2H), 7.67 (m, 3H), 7.08e7.27 (m,
6H), 6.72e6.81 (m, 4H), 3.99 (b, 4H), 3.52 (b, 8H), 3.24 (b, 4H), 1.25 (b,
12H). 13C NMR (75 MHz, CDCl3): 165.0, 161.3, 158.9, 156.3, 139.7, 133.6,
132.3, 131.7, 131.2, 131.0, 129.8, 126.8, 124.4, 119.4, 118.1, 115.8, 115.0,
114.7, 97.3, 67.8, 54.3, 46.9, 13.5. TOF MS calcd. for (M)þ 838.3717,
found 838.3722.
2.2.3. Compound 6
Compound 6 was synthesized according to the similar procedure
to compound 4 except that phenyl isothiocyanate was used. 1H NMR
(300 MHz, DMSO-d6)
d 8.03 (d, 1H), 7.66e7.54 (m, 3H), 7.48 (s, 2H),
7.15(d, J ¼ 7.2, 2H), 7.07 (d, J ¼ 2.1, 3H), 6.95(d, J ¼ 3.6,1H), 6.75 (s,1H),
6.48 (m, 3H), 6.31 (s, 1H), 3.33 (q, 8H), 1.15 (t, J ¼ 6.6, 12H). 13C NMR
(75 MHz, CDCl3): 183.2, 167.7, 154.8, 150.7, 149.8, 138.2, 134.8, 129.5,
128.8, 126.6, 125.6, 125.3, 124.4, 108.8, 104.7, 98.8, 67.7, 44.9, 13.1.
Fig. 5. Normalized excitation, absorption and emission spectra of 5 in 2:1 (v/v) MeOH/
water solution (10 mM TriseHCl, pH 7.0). The emission data were collected at 585 nm.
The excitation wavelength is 400 nm.

912
Y. Liu et al. / Dyes and Pigments 92 (2012) 909e915
O
NH2
NCS
O
O
O
CSCl2
CH2Cl2
N
O
N
EtOH, reflux
O
N
O
O
1
N
N
2
3
O
O
O
O
S
NH
NH
N
NH2
NCS
DMF
N
3
Hg(NO3)2
MeOH
5
4
DMF
N
O
N
N
O
6
N
rhodamine B hydrazide
Scheme 1. Synthesis of compounds 4, 5 and control compound 6.
2.3. Procedures of ions sensing
(see: Experimental section). As we know, the spectra overlap is
the main factor that determines the efficiency of FRET. In the
construction of FRET system of 4, 1,8-naphthalimide fluorophore
was chosen as an energy donor because it has strong emission in
the visible range and its broad emission (450e650 nm) covers
a part of rhodamine’s absorption, fulfilling a favorable condition for
FRET (Fig. 2). A 2:1 (v/v) MeOH/water solution (10 mM TriseHCl,
pH 7.0) was selected as a testing system to investigate the chemical
response of 4 to Hg2þ at room temperature (about 20 ꢂC). A time
course of the absorption response of 4 upon addition of Hg2þ
revealed the recognizing event could complete in 10 min, and
remains quite stable from 10 to 50 min. Thus, a reaction time of
10 min was chosen for the present studies.
Deionized water was used throughout all experiments. Solutions
of Ca2þ and Au3þ were prepared from their chloride salts; solutions
of Naþ, Kþ, Agþ, Mg2þ, Cu2þ, Cd2þ, Co2þ, Mn2þ, Ni2þ, Zn2þ, Hg2þ
,
Pb2þ, Cr3þ, and Fe3þ were prepared from their nitrate salts. Solution
of PdCl2 was prepared in 95:5 MeOH/Brine. A stock solution of 4
(1.25 ꢀ10ꢁ2 M) was prepared in DMF. The stock solution of 1 was
then diluted to the corresponding concentration (10 mM and 1 mM)
with the 2:1 (v/v) MeOH/water solution (10 mM TriseHCl, pH 7.0).
The Hg2þ stock solution of 5.0 ꢀ 10ꢁ2 M was diluted to 1.0 ꢀ 10ꢁ2 M,
1.0 ꢀ 10ꢁ3 M and 1.0 ꢀ 10ꢁ4 M with deionized water for spectra
titration studies. In the titration experiments, a 2.5 mL solution of 4
(10 mM and 1 mM) was poured into a quartz optical cell of 1 cm
optical path length each time, and Hg2þ solution was added into the
quartz optical cell gradually by using a micro-pipette. Spectra data
were recorded in an indicated time after the addition.
3.1. UV/Vis titration investigation
The UV/Vis spectrum of 4 showed only the absorption profile of
the donor (1,8-naphthalimide), which has a maximum at 405 nm.
Addition of Hg2þ ions induced an increase in the absorption intensity
at 565 nm (Fig. 3), which corresponds to the absorption of rhodamine.
The absorption stabilized after the amount of added Hg2þ ions
3. Results and discussion
Compound 4 and its ring-opened product 5, as well as a control
compound 6 were efficiently synthesized and well characterized
Fig. 7. Changes in absorption (565 nm) of 4 (10 mM) in 2:1 (v/v) MeOH/water solution
Fig. 6. Fluorescence titration spectra of 4 and 6 (both: 1
m
M) in 2:1 (v/v) MeOH/water
measured with and without Hg2þ (1 equiv) as a function of pH. 4 þ Hg2þ (red), 4
(black). (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
solution (10 mM TriseHCl, pH 7.0) upon addition of Hg2þ (1 equiv). lex ¼ 400 nm. Slits:
20 nm/20 nm.

Y. Liu et al. / Dyes and Pigments 92 (2012) 909e915
913
reached 1 equiv and a significant color change from colorless to red
3.2. Fluorescence titration investigation
could be observed easily by eye, indicating that the addition of Hg2þ
ions can promote the formation of the ring-opened compound 5.
Further evidence for the formation of 5 comes from the independent
synthesis of 5 from 4 by using 1 equiv of Hg(NO3)2 in MeOH at room
temperature (see: Experimental section). Job plot analysis of the
UVeVis titrations revealed a maximum at about 0.5 mole fraction
(Fig. 3, inset), indicating 1:1 binding stoichiometry. Noteworthy is that
in the titration process the 1,8-naphthalimide absorption at 405 nm
was almost unchanged, and the absorption spectrum obtained after
adding 1 equiv Hg2þ ions reflects the presence of the separate 1,8-
naphthalimide and rhodamine B units, indicating that there are
very weak electronic interactions between the donor and the acceptor
in the ground state.
As shown in Fig. 4a, the free 4 (10
m
M) displayed a weak 1,8-
naphthalimide emission band centered at 540 nm when excited at
400 nm (excitation of 1,8-naphthalimide moiety). The weak fluo-
rescence emission of naphthalimide unit of 4 is probably due to
photo-induced electron transfer (PET) from the nitrogen atom of
thiosemicarbazide moiety to the photoexcited naphthalimide
moiety. Upon addition of Hg2þ the 1,8-naphthalimide emission
decreased, and a new emission band centered at 585 (emission of
rhodamine B moiety) appeared and gradually increased in intensity.
The emission intensity stabilized after the amount of added Hg2þ
ions reached 1 equiv with a well-defined isoemission point. The
emission intensities at 585 nm and 545 nm (F585/F545) exhibited
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
a
2+
Hg
0.8
0.6
0.4
0.2
+
Others
Ag
0.0
500
550
600
650
700
Wavelength/nm
300
250
300
250
200
150
100
50
b
2+
Hg
200
150
100
50
0
+
Ag
Others
0
500
550
600
Wavelength/nm
650
700
Fig. 8. The absorption (a) and fluorescence (b) spectra of 4 (10
m
M and 1 m ,
M, respectively) upon addition of 1 equiv of Hg2þ and various other metal ions, including of Naþ, Kþ, Mg2þ
Fe2þ, Fe3þ, Cr3þ, Ca2þ, Zn2þ, Co2þ, Pb2þ, Cd2þ, Ni2þ, Agþ, Au3þ, Pd2þ (10 equiv) and Cu2þ (1 equiv), in 2:1 (v/v) MeOH/water solution (10 mM TriseHCl, pH 7.0). Inset: spectra
response of 4 to Hg2þ containing various metal ions.

914
Y. Liu et al. / Dyes and Pigments 92 (2012) 909e915
a big change from 0.44 in the absence of Hg2þ to 28.7 when the
fluorescence and absorption changes (Fig. 8, inset). In addition,
because the excess of Cu2þ can partly quenches the fluorescence of
4 þ Hg2þ due to its paramagnetic property, thus, only 1 equiv of
Cu2þ was used in the selectivity experiment.
amount of Hg2þ ions added reached 1 equiv of 4. A good linear
working range from 2 to 10 mM was observed in the titration
experiment. In addition, we also measured the sensitivity of 4 for
monitoring the lower concentration of Hg2þ by changing the slit
size. When 4 was employed at 1
20 nm, the good linear working range from 0.3 to 1
(Fig. 4b). Further, when 4 was employed at 0.1 M with the same slit
size (20 nm/20 nm), the good linear working range from 0.03 to
m
M with the slit size being 20 nm/
4. Conclusions
mM is obtainable
m
In summary, we have developed a new ratiometric fluorescent
probe 4 for Hg2þ based on an intramolecular FRET with an excellent
selectivity over other metal ions. It exhibits a clear Hg2þ-induced
change in the intensity ratio of the two emission bands of naph-
thalimide and rhodamine. The selective absorption and fluores-
cence response of 4 is due to the Hg2þ-promoted desulfurization of
the thiocarbonyl moiety, leading to the ring-opening of rhodamine
B moiety of 4. We expect that the method will serve as practical tool
for environmental samples analysis and biological studies.
0.08 mM can still be obtained (Fig. 4c), suggesting a lower level of
Hg2þ can be detected using the system.
It was clear that the FRET process was switched on by Hg2þ ions
as excitation of 1,8-naphthalimide at 400 nm resulted in the
emission of rhodamine with a maximum of 585 nm. This can also
be corroborated by the excitation spectra of the ring-opened
product 5. The excitation spectra obtained by collecting emission
data of 5 at 585 nm shows both the donor and the acceptor bands
with good correspondence with the absorption spectra (Fig. 5),
indicating that both the two transitions participate in the emission
process, and an efficient energy transfer can occur from the 1,8-
naphthalimide donor to the rhodamine acceptor. The efficiency of
energy transfer (EET) was calculated to be 86.3% based on the
Acknowledgments
We would like to thank the Natural Science Foundation of China
(NSFC Nos. 20772073 and 21072121) and the Natural Science
Foundation of Shanxi Province (2008011015-2) for financial
support.
equation [52]: hEET ¼ 1 ꢁ FF(donor in FRET system)
/FF(free donor), in which
hEET is the efficiency of energy transfer, FF(donor in FRET system) is the
fluorescence quantum yields of the donor part in FRET system (1,8-
naphthalimide part in 5 in this investigation), and FF(free donor) is the
fluorescence quantum yields of the donor when not connected to
the acceptor (1,8-naphthalimide part in 4 in this investigation).
Further, to evaluate the role of the 1,8-naphthalimide donor
moiety, a control compound 6 (with the 1,8-naphthalimide group
in 4 being replaced by a phenyl group) was synthesized (Scheme 1)
and its fluorescent response performances were investigated and
compared with that of 4 in the same conditions. The free 6 dis-
played no any emission when excited at 400 nm (excitation of 1,8-
naphthalimide). Upon addition of 1 equiv Hg2þ, only a weaker
emission compared with 4 þ Hg2þ was observed at 585 nm (Fig. 6).
Obviously, the 1,8-naphthalimide donor plays an important role for
FRET of 4 þ Hg2þ. The property is especially important because
a large Stokes shift (185 nm) can be realized in this system, which
can eliminate any influence of excitation backscattering effects on
the fluorescence assay and facilitate the practical application.
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