Visualizing Hg2+ ions in living cells using a FRET-based fluorescent sensor

Article abstract of DOI:10.1016/j.saa.2012.12.092

A novel FRET fluorescent sensor for Hg²⁺ imaging in living cells is rationally designed based on a coumarin-rhodamine platform. RBC1 exhibit high selectivity and excellent sensitivity in both absorbance and fluorescence detection of Hg²⁺ in aqueous solution. After addition of increasing concentrations of Hg²⁺, it result in the decrease of coumarin emission at 467 nm and a new emission profile of rhodamine at 590 nm gradually increased. The response time to Hg²⁺ is less than 2 min, and other metal ions including Fe²⁺, Mn²⁺, Ni²⁺, Co ²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Pb ²⁺, and Cr³⁺ had no interference. In addition, fluorescent imaging of Hg²⁺ in A375 cells is also successfully demonstrated. The design strategy of two fluorophores switching in this work would help to extend the development of FRET fluorescent sensors.

Full text of DOI:10.1016/j.saa.2012.12.092

Accepted Manuscript
Visualizing Hg2+ ions in living cells using a FRET-based Fluorescent Sensor
Yi Zhou, Kaihui Chu, Haifu Zhen, Yuan Fang, Cheng Yao
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http://dx.doi.org/10.1016/j.saa.2012.12.092
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Please cite this article as: Y. Zhou, K. Chu, H. Zhen, Y. Fang, C. Yao, Visualizing Hg2+ ions in living cells using a
FRET-based Fluorescent Sensor, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2013),
doi: http://dx.doi.org/10.1016/j.saa.2012.12.092
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Visualizing Hg2+ ions in living cells using a FRET-based Fluorescent Sensor
Yi Zhou, Kaihui Chu, Haifu Zhen, Yuan Fang, Cheng Yao*
State Key Laboratory of Materials-Oriented Chemical Engineering and College of Science, Nanjing
University of Technology, Nanjing 210009, P. R. China
*Corresponding author. Tel: +86-25-8358-7433; Fax: +86-25-8358-7433
E-mail: yaochengnjut@126.com (C. Yao)
Abstract
A novel FRET fluorescent sensor for Hg2+ imaging in living cells is rationally designed based
on a coumarin-rhodamine platform. RBC1 exhibit high selectivity and excellent sensitivity in both
absorbance and fluorescence detection of Hg2+ in aqueous solution. After addition of increasing
concentrations of Hg2+, it result in the decrease of coumarin emission at 467 nm and a new emission
profile of rhodamine at 590 nm gradually increased. The response time to Hg2+ is less than 2 mins,
and other metal ions including Fe2+, Mn2+, Ni2+, Co2+, Cu2+, Zn2+, Cd2+, Pb2+ and Cr3+ had no
interference. In addition, fluorescent imaging of Hg2+ in A375 cells is also successfully demonstrated.
The design strategy of two fluorophores switching in this work would help to extend the
development of FRET fluorescent sensors.
Key words: coumarins; rhomamine; fluorescent sensors; FRET; mercury; imaging agents
1

1. Introdction
Mercury is considered as a prevalent toxic and dangerous heavy metal element because of its high
affinity for thiol group in proteins and enzymes, leading to the dysfunction of cells and consequently
causing many health problems in the brain, kidney, central nervous, mitosis and endocrine system
[1].Thus, developing new methods for imaging Hg2+ ions in living cells is crucial for the elucidation of
their biological effects [2]. Up until now, most reported fluorescent sensors for sensing Hg2+ ions in
living cells were designed by the enhancement of fluorescence signals [3]. Generally, the single
enhancement signal output is often influenced by instrumental efficiency, environmental conditions and
the sensor concentration [4]. The FRET method can theoretically eliminate the above-mentioned
problems by self-calibration of two emission bands.
The fluorescence resonance energy transfer (FRET) is drawing particular interests due to potential
benefits in drug discovery, elucidation of gene [5] and protein function (protein conformational change),
examination of protein interactions with ligands, genes, or other proteins, and use in high-throughput
assays [6]. The two independent fluorophores of FRET-based sensors are able to tune by the controlling
FRET process in response to external analytes, so the corresponding molecular-level signals
communication device can be constructed. FRET process is known to be sensitive, selective, and
adaptable to a wide variety of biological systems [7], but only few cases can clarify the interconvert
structure of the FRET molecules. In addition, most of FRET processes have been used to monitor
biomacromolecules, and the examples of FRET-based sensors for low-weight molecular species
(especially metal cations) are not common in the literature [8].
Tae and co-workers used Hg2+-promoted reaction of thiosemicarbazides to form 1,3,4-oxadiazoles
in order to serve as the foundation strategy for novel Hg2+ sensors [9]. By means of spirolactam
2

ring-open and oxadiazole formation, thiosemicarbazide-rhodamine could be monitored accurately in
vitro detection and in vivo bioimaging. Then, Qian and co-workers exploited a BODIPY-Rhodamine
architecture FRET sensor that could ratiometric detect amounts of Hg2+ ions on the ppb scale under
physiological conditions [13a]. Inspired by these results [13] [14], we designed a sensor RBC1
embracing two fluorophores coumarin and rhodamine, which were expected to exhibit FRET, that is,
7-diethylcoumarin served as an energy donor and rhodamine acted as an energy acceptor. This design
also produced a large emission wavelength shift (around 123 nm; larger than Qian’s sensor, around 75
nm) between the coumarin emission and rhodamine emission, meanwhile it achieved a lower detection
limit (~0.42 ppb) compared to the previously reported FRET sensors [13]. In the absence of Hg2+, the
thiosemicarbazide-protecting groups forced the acceptor to adopt a closed form, and only the green
emission of the donor was observed upon excitation of the coumarin chromophore. Upon interaction
with Hg2+ ions, the Hg2+ promoted reaction of thiosemicarbazide to oxadiazole would induce opening
of the rhodamine moiety. The change of FRET efficiency was reflected in a fluorescence increase of the
acceptor rhodamine and a fluorescence decrease of the donor 7-diethylcoumarin. As a result, the
acceptor (opened-cyclic form) moiety showed a strong red emission of rhodamine owing to FRET,
which the changes in [Hg2+] could be detected by measuring the ratio of green and red fluorescence
intensities.
2. Experimental
2.1. Reagents and Apparatus
All the solvents were of analytic grade. The salts used in stock solutions of metal ions were
NaNO3, KNO3, Mg(NO3)2, Ca(NO3)2, FeCl2·4H2O, MnCl2, Ni(NO3)2·6H2O, Co(NO3)2·6H2O, CuSO4,
Zn(NO3)2·2H2O, CdCl2·H2O, AgNO3, Hg(ClO4)2, Pb(NO3)2 and CrCl3·6H2O. Thiophosgene was
3

purchased from Shanghai TCI Reagents Ltd. The other reagents were purchased from Taiyuan RHF
1
Reagents Ltd. Rhodamine B hydrazide was synthesized according to the reference [3d]. The H-NMR
and 13C-NMR spectra were measured on a BrukerAV-500 or BrukerAV-300 spectrometer with
chemical shifts reported in ppm (in CDCl3 or DMSO-d6). UV-visible spectra were recorded on a
Perkin-Elmer 35 spectrometer. Electrospray ionization mass spectra (ESI-MS) were measured on a
Micromass LCTTM system. All pH measurements were made with a Sartorius basic pH-Meter PB-10.
Fluorescence measurements were performed at room temperature on a Perkin-Elmer LS 50B
fluorescence spectrophotometer. Melting points were determined on a hot-plate melting point apparatus
XT4-100A and uncorrected.
2.2. Cell Culture and Fluorescence Imaging
A375 cells (human malignant melanoma cells) were passed and plated on a 24-well plate at a
density of 2×103 cells per well in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented
with 10% fetal bovine serum (FBS, Sigma), penicillin (100 μg mL-1), and streptomycin (100 μg mL-1)
at 37 °C in a humidified atmosphere with 5% CO2 and 95% air for 24 h prior to staining. Experiments
to assess Hg(II) uptake were performed in the same media supplemented with 5 μM RBC1 for 30 min.
After washing twice with PBS (phosphate buffered saline, pH=7.2, Gibco) to remove the remaining
sensor, the treated cells were imaged by fluorescence microscopy (BX51, Olympus, Japan). Then, 10
μM Hg2+ in the culture media containing CH3CN/PBS (2:48, v/v) was added to the cells, which further
incubated for 60 min. After washing twice with PBS (phosphate buffered saline, pH=7.2, Gibco) to
remove the remaining Hg2+, the treated cells were imaged by fluorescence microscopy.
2.3. Synthesis of intermediates and Mercurysensor (RBC1)
2.3.1 Synthesis of compound 1
4

To a 100 mL flask, 1.93 g (10.0 mmol) of 4-diethylaminosalicyldehyde, 1.05 ml (10.3 mmol) of
ethylnitroacetate, 0.2 ml piperidine and 5 ml glacial acetic acid were added in 40 ml of n-BuOH. After
heated to 100 oC for 18 h, the mixture changed from light orange to dark brown and became clear. Then
the orange solids were formed while cooling. The solids were filtered and washed with cold n-BuOH (3
x 10 ml) and finally dried in vacuum, which afforded an orange solid (2.24 g, yield: 86%). M.p.
169.4~170.7 °C. 1H-NMR (500 MHz, CDCl3): δ = 8.71 (s, 1H, Ar-H), 7.44 (d, J = 9.1 Hz, 1H, Ar-H),
6.70 (dd, J = 7.7, 2.5 Hz, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 3.50 (q, 7.2 Hz, 4H, -CH2-), 1.28 (t, 7.2 Hz, 6H,
-CH3). Anal. Calcd for C13H14N2O4: C, 59.54; H, 5.38; N, 10.68%. Found: C, 59.30; H, 5.47; N,
10.72%.
2.3.2 Synthesis of compound 2
To a mixture of 1 1.05 g (4.0 mmol) and SnCl2·2H2O 2.25g (10 mmol) was added in 15 mL
concentrate HCl solution in 8 mL water dropwise. The resulting solution was heated to reflux for 6 h
and became clear. After neutralizing the excess acid with 5M NaOH solutions, the reaction mixture was
filtered and extracted with ethyl acetate (3 x 30 ml). The organic layer was dried with anhy. Na2SO4 and
evaporated to dryness. The residues were purified by silica gel column chromatography using CH2Cl2
1
as eluent to afford an orange solid (0.66 g, yield: 71%). H-NMR (500 MHz, CDCl3): δ = 7.13 (d, J =
8.5 Hz, 1H, Ar-H), 6.70 (s, 1H, Ar-H), 6.57 (s, 2H, Ar-H), 3.91 (s, 2H, -NH2), 3.40 (q, 7.8 Hz, 4H,
-CH2-), 1.20 (t, 7.2 Hz, 6H, -CH3).
2.3.3 Synthesis of compound 3
A solution of 2 (232 mg, 1 mmol) in dichloromethane (8 mL) was added slowly to a solution of
thiocarbonyl chloride (338 mg, 3 mmol) in dichloromethane (15 mL) and triethylamine (1.5 mL) and
the mixture was stirred for 30 min. Then the reaction mixture was washed with water, dried with
5

MgSO4, and evaporated to dryness. The crude product was purified on flash silica gel using CH2Cl2
/petroleum (3:1) as eluent. The crude product was purified by flash chromatography (CH2Cl2 /
petroleum, 3:1, Rf = 0.6) as eluent to afford 3 (227 mg, yield: 83%). M.p. 178.2~179.3 °C. Rf = 0.6
(SiO2; CH2Cl2 / petroleum, 3:1). 1H-NMR (500 MHz, DMSO-d6): δ = 7.89 (s, 1H, Ar-H), 7.42 (d, J =
9.0 Hz, 1H, Ar-H), 6.76 (dd, J = 7.4, 2.5 Hz, 1H, Ar-H), 6.60 (d, J = 2.5 Hz, 1H, Ar-H), 3.45 (q, 7.8 Hz,
4H, -CH2-), 1.13 (t, 7.2 Hz, 6H, -CH3). 13C-NMR (75 MHz, CDCl3): 158.77, 154.96, 150.81, 145.24,
133.47, 129.03, 113.62, 109.64, 107.18, 97.42, 44.90, 12.40. Anal. Calcd for C14H14N2O2S: C, 61.30; H,
5.14; N, 10.21. Found: C, 61.24; H, 5.06; N, 10.32. TOF-MS: m/z 297.1 [M+Na]+.
2.3.4 Synthesis of compound 4 (RBC1)
To a solution of 3 (274 mg, 1.0 mmol) in dry DMF (20 mL) was added Rhodamine-B hydrazide
(473 mg, 1.0 mmol). The mixture was heated at 70 °C for 6 h and a dark yellow solution was obtained.
After removing the solvent under reduced pressure, the residue was purified by flash chromatography
(CH2Cl2 / EA, 98:2, Rf = 0.4) as eluent to afford 4 (314 mg, yield: 43%) as a yellow soild. M.p.
138.7~140.2 °C. Rf = 0.4 (SiO2; CH2Cl2 / EA, 98:2). 1H-NMR (300 MHz, CDCl3): δ = 9.33 (s, 1H,
-NH), 8.61 (s, 1H, -NH), 8.03 (d, J = 6.7 Hz, 1H, Ar-H), 7.61 (m, 2H, Ar-H), 7.58 (m, 2H, Ar-H), 7.20
(t, 9.3 Hz, 1H, Ar-H), 7.25 (d, 6.6 Hz, 1H, Ar-H), 6.81 (s, 1H, Ar-H), 6.58 (m, 2H, Ar-H), 6.54 (d, 2.4
Hz, 1H, Ar-H), 6.42 (d, 2.3 Hz, 1H, Ar-H), 6.36 (d, 2.5 Hz, 2H, Ar-H), 6.26 (dd, 2.5 Hz, 8.9 Hz, 2H,
Ar-H), 3.39 (q, 7.2 Hz, 4H, -CH2-), 3.24 (q, 6.9 Hz, 8H, -CH2-), 1.17 (t, 6.9 Hz, 6H, -CH3), 1.08 (t,
6.9Hz, 12H, -CH3). 13C-NMR (75 MHz, CDCl3): 178.90, 167.14, 159.27, 153.91, 152.53, 150.40,
149.33, 149.12, 143.28, 134.11, 128.81, 128.77, 128.71, 128.33, 125.16, 124.54, 123.98, 118.98,
109.36, 108.41, 107.75, 103.68, 97.97, 97.20, 67.19, 44.64, 44.22, 12.39. Anal. Calcd for C42H46N6O4S:
C, 69.02; H, 6.34; N, 11.50. Found: C, 69.14; H, 6.26; N, 11.62. TOF-MS: m/z 731.4 [M+H]+, 753.4
6

[M+Na]+.
2.3.5 Reaction between RBC1 and Hg2+
To 20 mL acetonitrile solution containing 4 (731 mg, 1.00 mmol) and Hg(ClO4)2·3H2O (500 mg,
1.10 mmol) was stirred for 30 min at room temperature, the solvent was removed in vacuum to give a
dark purple solid. The crude product was purified by flash column chromatograph (CH2Cl2 / MeOH,
4:1) as eluent to afford 5 (652 mg, yield: 96%). m.p. 165.4~168.1 °C. Rf = 0.3 (SiO2; CH2Cl2 / MeOH,
4:1). 1H-NMR (500 MHz, CDCl3): δ = 8.17 (t, J = 8.9 Hz, 2H, Ar-H), 7.82 (s, 1H, -NH), 7.78 (d, 7.7 Hz,
2H, Ar-H), 7.38 (s, 1H, Ar-H), 7.18 (d, 8.6 Hz, 1H, Ar-H), 7.13 (d, 9.4 Hz, 2H, Ar-H), 6.85 (dd, 2.3 Hz,
8.9 Hz, 2H, Ar-H), 6.80 (s, 2H, Ar-H), 6.58 (dd, 2.3 Hz, 8.9 Hz, 1H, Ar-H), 6.45 (s, 1H, Ar-H), 3.60 (q,
7.2 Hz, 4H, -CH2-), 3.37 (q, 7.3 Hz, 8H, -CH2-), 1.27 (t, 6.9 Hz, 12H, -CH3), 1.16 (t, 6.9Hz, 6H, -CH3).
13C-NMR (125 MHz, CDCl3): 158.92, 158.69, 157.93, 156.94, 156.54, 155.54, 152.27, 149.21, 131.27,
131.11, 130.69, 130.58, 128.45, 128.13, 122.62, 122.53, 117.84, 114.28, 113.68, 109.78, 107.85, 97.24,
96.47, 45.98, 44.61, 12.41. Anal. Calcd for C42H45N6O4: C, 72.29; H, 6.50; N, 12.04. Found: C, 72.44;
H, 6.76; N, 11.92. TOF-MS: m/z 697.4 [M]+.
3. Results and discussion
3.1 Synthesis of RBC1
RBC1 combines a coumarin moiety having desirable optical properties [10] with a
thiosemicarbazide receptor to favor selective and stable binding of Hg2+ to form HgS. Reaction of
4-diethylaminosalicyldehyde and ethylnitroacetate affords 3-nitro coumarin 1 in 86% yield. Reduction
of 1 with SnCl2 proceeds smoothly to generate the corresponding amino coumarin 2 in 71% yield.
Base-mediated condensation of thiophosgene with 2 furnishes isothiocyanato coumarin 3 in 83% yield.
RBC1 was prepared by the nucleophilic addition of 3 and rhodamine-B hydrazide in DMF for 6 h with
7

a yield of 43% (Scheme 1).
Scheme 1. Synthesis of Mercurysensor 4 (RBC1) and proposed mechanism
3.2 Optical investigation
3.2.1 UV/Vis titration investigation
The UV/Vis spectrum of RBC1 in CH3CN/water 80:20 exhibits an absorption maximum at 404 nm
(ε = 1.08 × 105 M-1 cm-1), which can be assigned to the donor (coumarin) transition band (Figure 1)
[11]. When titrated by Hg2+, a new absorption band centered at 563 nm (ε = 1.43 × 105 M-1 cm-1)
accompanying a remarkable increasing intensity, corresponds to the absorption of rhodamine, which
induced a clear color change from pale yellow to pink (Figure 2). This confirms that RBC1 was
induced to the ring-opened structure from the spirolactam by Hg2+ which possessed a long absorption
wavelength and high molar extinction coefficient.
8

Figure 1. Absorption spectra of RBC1 (2 μM) in aqueous acetonitrile (HEPES/CH3CN, v/v=20:80; pH
7.0) obtained by adding aliquots of 300 μL Hg(ClO4)2 (0.1 mM) solution. The [Hg2+] total increases
from 0.0 to 3.0 μM along the direction of the arrow.
Figure 2. Photos and fluorescence Photos of RBC1 (20 μM) in in aqueous acetonitrile
(HEPES/CH3CN, v/v=20:80; pH 7.0) before (left) and after (right) addition of 20 μM of Hg2+ by the
naked eye.
3.2.2 Fluorescence titration investigation
Fluorescence studies of RBC1 revealed the profile of the FRET sensor with a high response to the
presence of Hg2+ ions (Figure 3). The ion-free form of RBC1 exhibited the strong emission profile of
coumarin at 467 nm (Фf =0.31) and was close to non-emission at 590 nm with an excitation at 365 nm.
While addition of increasing concentrations of Hg2+ ions, it resulted in the decrease of coumarin
9

emission and a new emission profile of rhodamine at 590 nm gradually increased. The ratio of emission
intensities of rhodamine and coumarin (F590/F467) varied from 0.02 to 4.68, corresponding to a
240-fold enhancement. As a result, the color of the fluorescence clearly changed from green to red. The
titration reaction curve shows a steady and smooth increase until a plateau reached (1.20 equiv Hg2+
ions) with the quantum yield Фf = 0.13, suggesting the formation of 5 (opened-cyclic form) consumed
one equiv Hg2+ ions. Moreover, RBC1 could be used in real-time determination of Hg2+ ions in
environmental and biological conditions (equilibrium time less than 2 mins, Figure 4). The 1:1 reaction
mode was also supported by the peak at m/z 697.4 (calc. 697.2) corresponding to 5 in ESI-MS spectrum
(Figure S1, see Supplementary data) with Hg2+ ions 1:1 desulfurization of RBC1. The Förster energy
transfer efficiency and distance R0 (the distance at which 50% energy transfer takes place between
coumarin and rhodamine) were also estimated to be 84.2% and 68.7 Å, obtained by steadystate
theoretical calculation [12]. This thiosemicarbazide was stable in CH3CN-water solutions and solid
state for over 2 weeks. The acid-base titration experiments revealed that RBC1/RBC1-Hg2+ remained
unaffected between pH 5.81 and 9.04 (excitation at 365 nm) in fluorescence intensity (Figure 5),
suggesting that it was insensitive to pH near 7.0 and could work in approximate physiological
conditions.
10

Figure 3. Emission spectra of RBC1 (2 μM) in aqueous acetonitrile (HEPES/CH3CN, v/v=20:80; pH
7.0) upon addition of different amounts of Hg2+ ion. Inset, the titration profile based on the emission
ratio at 467 and 590 nm with excitation at 365 nm.
Figure 4. Time evolution of RBC1 (2 μM) in in aqueous acetonitrile (HEPES/CH3CN, v/v=20:80;
pH 7.0) in the presence of 1.5 equiv Hg2+.
11

Figure 5. Fluorescence responses of probe RBC1 (2 μM) and RBC1-Hg2+ at different pH.
3.2.3 Selectivity investigation
Figure 6 depicts the fluorescence responses of a 2 μM solution of RBC1 to the presence of various
environmentally relevant metal ions. Transition metal ions, including Fe2+, Mn2+, Ni2+, Co2+, Cu2+, Zn2+,
Cd2+, Pb2+ and Cr3+, produce no discernible changes in emission intensities for the apo sensor. However,
with Ag+ ions, caused a significant decrease of fluorescence at 467 nm but only weak enhancement of
fluorescence at 590 nm (consistent with the earlier reported BODIPY-rhodamine sensor [13a]), and the
change in F590/F467 ratio increased 31-fold upon adding 2 μM Ag+ (Figure 7). The discrimination
between two similar responding Ag+ ions and Hg2+ ions could be realized ratiometrically by using the
large changes (240-fold, 7.7 times) with Hg2+ ions to trigger the FRET switching of RBC1. Moreover,
alkali and alkali-earth metal ions, which are abundant in water and living cells, do not result in distinct
change in F590/F467 ratio even at the mM level. In addition, the competitive experiments were
conducted in the presence of 1.0 equiv of Hg2+ ions mixed with 1.0 equiv of various cations and no
significant variation in F590/F467 ratio was found by comparison with Hg2+ ions added in RBC1
solution.
12

Figure 6. Fluorescence responses of 2 μM RBC1 to various 2 μM transition-metal ions (1 mM for
alkali and alkali-earth metal ions). Bars represent the integrated ratiometric fluorescence response from
590nm (F590) over the 467 nm (F567) integrated emission. Spectra were acquired in aqueous
acetonitrile (HEPES/CH3CN, v/v=20:80; pH 7.0) with excitation at 365 nm. The red bars represent the
addition of the competing metal ions to a 2 μM solution of RBC1. The blue bars represent the change
of the emission that occurs upon the subsequent addition of 2 μM Hg2+ to the above solution.
13

Figure 7. Relative fluorescence intensities of RBC1 (2 μM) (a) RBC1 only (black); (b) RBC1 + 1.5
equiv Hg2+ (red); (c) RBC1 + 1.5 equiv Ag+ (blue) with excitation at 365 nm.
3.2 Applications in living cells
The application of RBC1 to detect intracellular Hg2+ ions in living cells was observed with
dual-channel fluorescence images. The A375 cells (human malignant melanoma cell line) were
incubated with RBC1 (5 μM) of Hg2+-AM for 30 min showing a strong green intracellular fluorescence
(Figure 8a) and negligible red fluorescence (Figure 8b), which suggested that RBC1 was cell
penetrable. When cells stained with RBC1 were further treated with 10 μM Hg(ClO4)2 in PBS for 60
min, a strong quenching of the green fluorescence (Figure 8d) and a remarkable enhancement of the red
fluorescence (Figure 8c) were observed. These preliminary experimental results demonstrate that
RBC1 could be used for monitoring intracellular Hg2+ ions in biological samples with FRET methods.
14

Figure 8. Fluorescent images of A375 cells when stained by RBC1 solution (5.0 μM in PBS) at room
temperature; (a) with blue light excitation; (b) with green light excitation; and then further incubated
with 10.0 μM Hg2+ for 60 min. (c) with green light excitation; (d) with blue light excitation.
4. Conclusion
In summary, we have successfully developed a ratiometric FRET sensor RBC1 for Hg2+ ions
based on the coumarin-rhodamine platform. RBC1 exhibits the dual-responsive colorimetric and
fluorescent detection for Hg2+ ions in the presence of other competing cations. The significant changes
in the fluorescence color could be used for naked-eye detection. Moreover, fluorescence imaging shows
RBC1 can be used for ratiometric detecting changes in intracellular Hg2+ levels. The design strategy of
two fluorophores switching in our work would help to extend the development of FRET fluorescent
sensors.
Acknowledgements
This work was supported by the Open Fund of the State Key Laboratory of Materials-Oriented
Chemical Engineering (KL09-9), the postgraduate practice innovation fund of Jiangsu province
(CXZZ12-0444), and the doctor thesis innovation fund of Nanjing University of Technology.
15

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Highlights
 A novel sensor RBC1 is developed based on the coumarin-rhodamine platform.
 The sensor produce a large emission wavelength shift (123 nm).
 RBC1 achieve a lower detection limit (~0.42 ppb).
 The sensor exhibit both colorimetric and fluorescent response to Hg2+.
 It can be used for ratiometric detecting changes in intracellular Hg2+ levels.