Spirolactone and spirothiolactone rhodamine-pyrene probes for detection of Hg2+ with different sensing properties and its application in living cells

Article abstract of DOI:10.1016/j.saa.2016.01.024

Two new rhodamine B-based fluorescent probes PyRbS and PyRbO containing pyrene moiety were designed and synthesized. Both of the probes showed colorimetric and fluorometric sensing abilities for Hg²⁺ with high selectivity over other metal ions.

Full text of DOI:10.1016/j.saa.2016.01.024

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 209–218
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spirolactone and spirothiolactone rhodamine-pyrene probes for detection
of Hg2+ with different sensing properties and its application in living cells
Qing-Qing Rui a, Yi Zhou a, Yuan Fang a, Cheng Yao a,b,
⁎
a
College of Science, Nanjing Tech University, Nanjing 211816, China
b
State Key Laboratory of Coordination Chemistry, Nanjing University, Jiangsu 210093, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 April 2015
Received in revised form 12 January 2016
Accepted 16 January 2016
Available online 20 January 2016
Two new rhodamine B-based fluorescent probes PyRbS and PyRbO containing pyrene moiety were designed and
synthesized. Both of the probes showed colorimetric and fluorometric sensing abilities for Hg2+ with high selec-
tivity over other metal ions. The binding analysis using Job's plot suggested 1:1 stoichiometry for the complexes
formed for Hg2+. Compared with PyRbO, the PyRbS showed higher selectivity and sensitivity due to the
thiophilic property of Hg2+ ion. The PyRbS exhibited the linear fluorescence quenching to Hg2+ in the range
of 0.3 to 4.8 μM (λex = 365 nm) and 0.3 to 5.4 μM (λex = 515 nm), and the detection limit was 0.72 μM. More-
over, ratiometric changes of PyRbS with Hg2+ in absorption spectrum were observed, which could not be obtain-
ed in the combination of PyRbO with Hg2+. In addition, the methyl thiazolyl tetrazolium (MTT) assay
demonstrated that RbPyS had low cytotoxicity and was successfully used to monitor intracellular Hg2+ levels
in living cells.
Keywords:
Fluorescent probe
Hg2+ ion
Rhodamine B
Live-cell imaging
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
limitations that were mentioned above. Recently, colorimetric and fluo-
rometric probes for the detection of Hg2+ have attracted considerable
Mercury pollution is a ubiquitous problem due to the inorganic
mercury releasing into the environment through varieties of anthro-
pogenic behavior (like coal and gold mining, combustion of fossil
fuels and chemical industry) and natural sources (like volcanic erup-
tion and forest fires) [1–3]. Mercury is harmful to photosynthesis and
transpiration of plants, and it would pollute the environment [4,
5].The inorganic mercury also can be transformed to CH3HgX species
(X = halogen) by some prokaryotes that live in the underwater hab-
itats [6]. Ultimately, methylmercury would be ingested by humans
through mercury bioaccumulation [7]. To make things worse, mer-
cury intoxication can lead to manifold neurological problems, such
as prenatal brain damage, cognitive and motion disorders, vision
and hearing loss, and death [8–10]. Therefore, the detection of mer-
cury ion is of great significance.
attention, because of their operational simplicity, high selectivity, sensi-
tivity, rapidity, low cost of equipment and direct visual perception
[19–21].
In this paper, we developed two rhodamine-based probes, PyRbS
and PyRbO consisting of pyrene fluorophore (Scheme 1). Both of
them could be used for the detection of Hg2+ with high sensitivity
and selectivity, and showed colorimetric and fluorometric response in
a short time. Because PyRbS contained sulfur atom, the thiophilic nature
of Hg2+ resulted in the advantageous properties of PyRbS. Moreover,
pyrene moiety served successfully as a source of ratiometric absorption
changes of PyRbS with Hg2+. The low cytotoxicity and cell-membrane
penetrability of PyRbS were confirmed by MTT assay and cell imaging,
respectively.
In recent years, many conventionally quantitative approaches to the
detection of Hg2+ have been reported, such as chromatography
[11–13], capillary electrophoresis [14–16], atomic absorption spectros-
copy [17,18], etc. Because the above analysis methods require multistep
sample pretreatment and/or sophisticated instrumentation, and they
are not well-suited for the rapid detection or studies of Hg2+ in vivo.
The application of Hg2+-sensitive small-molecule ligands provides re-
markable optional response immediately, which can overcome the
2. Experimental section
2.1. Equipment and reagent
1H NMR and 13C NMR were measured on a Bruker AV-400 or Bruker
AV-300 spectrometer with chemical shifts reported in ppm (in CDCl3;
TMS as internal standard). Electrospray ionization mass spectra (ESI-
MS) were analyzed on a LCTTM system. UV–visible spectra were record-
ed on a Perkin–Elmer 35 spectrometer and fluorescence spectra were
measured on Perkin–Elmer LS 50B fluorescence spectrophotometer.
All the reagents and solvents (analytic grade) were purchased from
commercial suppliers. Doubly distilled water was used in all of the
⁎
Corresponding author at: College of Science, Nanjing Tech University, Nanjing 211816,
China.
E-mail addresses: yaocheng@njtech.edu.cn, yaochengnjut@126.com (C. Yao).
http://dx.doi.org/10.1016/j.saa.2016.01.024
1386-1425/© 2016 Elsevier B.V. All rights reserved.

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Scheme 1. Synthetic route of PyRbO and PyRbO.

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211
experiments. Metal ions were prepared by Hg(ClO4)2·3H2O, NaNO3,
KNO3, Mg(NO3)2, Ca(NO3)2, Ni(NO3)2·6H2O, Ba(NO3)2, AgNO3,
Zn(NO3)2·2H2O, MnCl2, CuSO4, CdCl2·H2O, Pb(NO3)2, CrCl3·6H2O,
Co(NO3)2·6H2O, Al(NO3)3·9H2O and FeCl2·4H2O.
solution was adjusted with sodium hydroxide aqueous solution to 9–
10. The appeared precipitate was filtered and washed with cold ethanol
3 times. After drying, the reaction afforded 1.44 g pink solid in a yield of
70%. 1H NMR (400 MHz, CDCl3) δ 7.93 (t, J = 4.4 Hz, 1H, Ar-H), 7.45 (t,
J = 4.2 Hz, 2H, Ar–H), 7.11 (t, J = 4.2 Hz, 1H, Ar–H), 6.46 (d, J = 8.8 Hz,
2H, Ar–H), 6.42 (d, J = 2.5 Hz, 2H, Ar–H), 6.29 (dd, J = 8.8, 2.6 Hz, 2H,
Ar–H), 3.61 (s, 2H, NH2), 3.34 (q, J = 7.1 Hz, 8H, CH2), 1.17 (t, J =
7.0 Hz, 12H, CH3). TOF-MS: m/z 457.3[M + H]+, 479.3[M + Na]+.
2.2. Synthesis for probes
2.2.1. Synthesis of 1-pyrenemethanol 8
1-pyrenecarboxaldehyde (0.35 g, 1.50 mmol) was dissolved in
15 mL methanol, and NaBH4 (0.08 g, 2.10 mmol) was added slowly
into the reaction mixture at 0 °C for 30 min. After stirring at room tem-
perature overnight, 20 mL 5% HCl was added in the reaction to quench
the excess NaBH4, and the solution changed to milk-white suspension.
The organic solvent was removed and then the solid was extracted
with ethyl acetate (3 × 15 mL). The organic fractions were washed
with saturated NaHCO3 aqueous solution. The collected organic solution
was dried with sodium sulfate and was concentrated to give compound
8 in an 81% yield. 1H NMR (400 MHz, CDCl3) δ 8.30–7.96 (m, 9 H, Ar–H),
5.33 (s, 2 H, CH2). TOF-MS: m/z 231.1[M–H]−.
2.2.4. Synthesis of compound 5
Rhodamine hydrazide (6, 0.12 g, 0.16 mmol) was added in 40 mL dry
methyl alcohol. Salicylic aldehyde (0.063 g, 0.52 mmol) was added and
then the reaction was refluxed at 50 °C. After 12 h, the appeared pink
precipitate was filtered and washed 3 times with cold methyl alcohol.
After drying, the reaction afforded 0.09 g solid in a 62% yield. 1H NMR
(400 MHz, CDCl3) δ 10.83 (s, 1H, OH), 9.23 (s, 1H, N_CH), 7.98 (dd,
J = 6.4, 1.6 Hz, 1H, Ar–H), 7.58–7.48 (m, 2H, Ar–H), 7.20–7.14 (m, 2H,
Ar–H), 7.10 (dd, J = 7.7, 1.3 Hz, 1H, Ar–H), 6.86 (d, J = 8.2 Hz, 1H,
Ar–H), 6.78 (t, J = 7.1 Hz, 1H, Ar–H), 6.49 (d, J = 9.1 Hz, 4H, Ar–H),
6.26 (dd, J = 8.9, 2.5 Hz, 2H, Ar–H), 3.32 (q, J = 7.0 Hz, 8H, CH2), 1.15
(t, J = 7.0 Hz, 12H, CH3). TOF-MS: m/z 583.3[M + Na]+.
2.2.2. Synthesis of compound 7
Compound 7 was synthesized according to the reported method
[22]. 1H NMR (400 MHz, CDCl3) δ 8.30–7.99 (m, 9 H, Ar–H), 5.86 (s,
2H, CH2), 3.44 (t, J = 6.5 Hz, 2 H, CH2), 2.58 (t, J = 7.2 Hz, 2 H, CH2),
2.2.5. Synthesis of probe 1 (PyRbO)
Compound 5 (0.28 g, 0.5 mmol), compound 7 (0.57 g, 1.5 mmol), po-
tassium carbonate (0.69 g, 5 mmol) and small amount of potassium io-
dide were mixed in 30 mL dry acetone and stirred under reflux
conditions for 12 h. The suspension was filtered, and then the solution
was evaporated. The residue was purified by flash chromatography
(EA/PE, 1:2, v/v) as eluant to afford 0.13 g light yellow product in a
yield of 30%. 1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H, N_CH), 8.32 (d,
J = 9.2 Hz, 1H, Ar–H), 8.20 (dd, J = 13.7, 6.4 Hz, 2H, Ar–H), 8.16–8.10
(m, 3H, Ar–H), 8.08 (d, J = 6.7 Hz, 2H, Ar–H), 8.04 (d, J = 7.7 Hz, 1H,
2.20 (t, J = 6.8 Hz, 2 H, CH2). TOF-MS: m/z 403.1, 405.1 [M + Na] +
.
2.2.3. Synthesis of rhodamine B hydrazide 6
Rhodamine B (2.00 g, 4.5 mmol) was dissolved in 150 mL dry etha-
nol, and 4.0 mL (excess) hydrazine hydrate (80%) was added dropwise
in the reaction suspension with stirring at room temperature. Then, the
mixture was refluxed at 85 °C for 2 h and evaporated to get orange solid.
The solid was dissolved in diluted hydrochloric acid. The pH of the
Fig. 1. (a) and (b) Fluorescence spectra of PyRbS (5 μM) in EtOH/H2O (2:1, v/v) with various amount of Hg2+ (0–6 μM). Inset: the fluorescence intensity at 592 nm. (a) λex = 365 nm.
(b) λex = 515 nm. (c) and (d) Fluorescence spectra of PyRbO (10 μM) in EtOH with various amount of Hg2+ (0–16 μM). Inset: the fluorescence intensity at 583 nm. (c) λex
365 nm. (d) λex = 515 nm.
=

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Fig. 2. (a) Fluorescence changes of 5 μM PyRbS in the presence of various metal ions in EtOH/H2O (2:1, v/v) at 592 nm. Bars represent the final fluorescence intensity (Ff) over the initial
fluorescence intensity (Fi) at λex = 515 nm. Black bars represent the fluorescence intensity of 5 μM PyRbS with various competing metal ions (4.0 equiv) and red bars represent the change
of fluorescence signal after the introduction of Hg2+ (1.0 equiv) to the above solution. (b) Fluorescence changes of 10 μM PyRbO in the presence of various metal ions in EtOH at 583 nm.
Bars represent the final fluorescence intensity (Ff) over the initial fluorescence intensity (Fi) at λex = 515 nm. Black bars represent the fluorescence intensity of 10 μM PyRbO with various
competing metal ions (2.0 equiv) and red bars represent the change of fluorescence signal after the introduction of Hg2+ (1.0 equiv) to the above solution. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
Ar–H), 8.02–7.97 (m, 2H, Ar–H), 7.48–7.40 (m, 2H, Ar–H), 7.07–7.00 (m,
2H, Ar–H), 6.79 (t, J = 7.5 Hz, 1H, Ar–H), 6.52 (d, J = 8.9 Hz, 3H, Ar–H),
6.39 (d, J = 2.5 Hz, 2H, Ar–H), 6.15 (dd, J = 8.9, 2.5 Hz, 2H, Ar–H), 5.95
(s, 2H, CH2), 3.77 (t, J = 5.7 Hz, 2H, CH2), 3.10 (q, J = 7.1 Hz, 8H, CH2),
2.66 (t, J = 7.4 Hz, 2H, CH2), 2.13–2.03 (m, 2H, CH2), 0.97 (t, J = 7.0 Hz,
12H, CH3). 13C NMR (75 MHz, CDCl3) δ 173.37, 157.08, 152.47, 148.81,
140.59, 128.22, 127.91, 127.79, 127.71, 127.21, 125.44, 124.52, 123.43,
122.83, 111.25, 108.01, 105.26, 97.85, 66.69, 64.79, 43.96, 30.80, 24.51,
12.38. TOF-MS: m/z 861.4 [M + H]+, 883.6 [M + Na]+.
2.2.7. Synthesis of probe compound 3
Thiooxorhodamine B hydrazide 4 (0.41 g, 0.87 mmol) in 40 mL dry
methyl alcohol was added. Salicylic aldehyde (0.13 g, 1.04 mmol) was
added and then the reaction was refluxed at 50 °C. After 12 h, the ap-
peared pink precipitate was filtered and washed 3 times with cold
methyl alcohol. After drying, the reaction afforded 0.30 g solid in 74%
yield. 1H NMR (400 MHz, CDCl3) δ 11.25 (s, 1H, OH), 8.70 (s, 1H,
N_CH), 8.20–8.04 (m, 1H, Ar–H), 7.47–7.42 (m, 2H, Ar–H), 7.34–7.27
(m, 2H, Ar–H), 7.18–7.12 (m, 1H, Ar–H), 6.95 (d, J = 8.2 Hz, 1H, Ar–
H), 6.90 (t, J = 7.4 Hz, 1H, Ar–H), 6.75 (d, J = 8.8 Hz, 2H, Ar–H), 6.40–
6.23 (m, 4H, Ar–H), 3.32 (q, J = 7.0 Hz, 8H, CH2), 1.15 (t, J = 7.0 Hz,
12H, CH3). TOF-MS: m/z 577.3 [M + H]+, 599.2 [M + Na]+.
2.2.6. Synthesis of thiooxorhodamine B hydrazide 4
Lawesson's reagent (0.30 g, 0.75 mmol) was added in 5.0 mL tolu-
ene, and then the suspension was stirred under the atmosphere at
30 °C for 30 min. Compound 6 (0.57 g, 1.25 mmol) dissolved in 20 mL
toluene was added to the reaction, and the reaction was refluxed at
85 °C. After 12 h, organic solvent was removed. 20 mL saturated solution
of potassium carbonate was added to the residue, stirring for 2 h at
room temperature, and then extracted by CH2Cl2. The organic phase
was collected and removed. The crude product was purified by neutral
alumina column chromatography (ethyl acetate/petroleum, 3:2, v/v)
to afford compound 4 (0.23 g, yield: 39%). 1H NMR (400 MHz, CDCl3)
δ 8.09 (d, J = 4.7 Hz, 1H, Ar–H), 7.47 (m, 2H, Ar–H), 7.12 (d, J =
6.5 Hz, 1H, Ar–H), 6.43 (d, J = 2.5 Hz, 2H, Ar–H), 6.35 (d, J = 8.8 Hz,
2H, Ar–H), 6.27 (dd, J = 8.9, 2.6 Hz, 2H, Ar–H), 4.82 (s, 2H, NH2), 3.34
(q, J = 7.1 Hz, 8H, CH2), 1.16 (t, J = 7.1 Hz, 12H, CH3). TOF-MS: m/z
473.2 [M + H]+.
2.2.8. Synthesis of probe 2 (PyRbS)
Compound 3 (0.12 g, 0.2 mmol), compound 7 (0.23 g, 0.6 mmol),
potassium carbonate (0.27 g, 2 mmol) and small amount of potassi-
um iodide were mixed in 60 mL dry acetone and the mixture was
stirred under reflux conditions for 12 h. The suspension was filtered,
and then the solution was evaporated. The residue was purified by
flash chromatography (EA/PE, 1:3, v/v) as eluant to afford 0.06 g
light yellow product in a yield of 34%. 1H NMR (400 MHz, CDCl3) δ
8.97 (s, 1H, N_CH), 8.30 (d, J = 9.2 Hz, 1H, Ar–H), 8.19 (dd, J =
7.5, 3.0 Hz, 2H, Ar–H), 8.14 (dd, J = 8.6, 6.5 Hz, 3H, Ar–H), 8.07
(dd, J = 8.5, 6.6 Hz, 3H, Ar–H), 8.01 (dt, J = 12.9, 3.9 Hz, 2H, Ar–
H), 7.43–7.32 (m, 2H, Ar–H), 7.20–7.14 (m, 1H, Ar–H), 7.11 (dd,
J = 7.5, 0.8 Hz, 1H, Ar–H), 6.83 (t, J = 7.6 Hz, 1H, Ar–H), 6.76 (d,
Fig. 3. (a) Fluorescence intensity of the response time of 5 μM PyRbS with Hg2+ (1.0 equiv) in EtOH/H2O (2:1, v/v) at λem = 592 nm. (b) Fluorescence intensity of the response time of
10 μM PyRbO with Hg2+ (1.0 equiv) in EtOH at λem = 583 nm.

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213
Fig. 4. (a) and (b) Reversibility of Hg2+ to PyRbS (5 μM) by Na2S in EtOH/H2O (2:1, v/v). Red line: free PyRbS (5 μM), blue line: PyRbS + Hg2+ (2.0 equiv), black line: PyRbS + Hg2+ (2.0
equiv) + S2−(4.0 equiv). (a) λex = 365 nm. (b) λex = 515 nm. (c) and (d) Reversibility of Hg2+ to PyRbO (10 μM) by Na2S in EtOH. Red line: free PyRbO (10 μM), blue line: PyRbO + Hg2+
(2.0 equiv), black line: PyRbO + Hg2+ (2.0 equiv) + S2− (4.0 equiv). (c) λex = 365 nm. (d) λex = 515 nm.
J = 8.9 Hz, 2H, Ar–H), 6.70 (d, J = 8.3 Hz, 1H, Ar–H), 6.31 (d, J =
2.6 Hz, 2H, Ar–H), 6.25 (dd, J = 8.9, 2.6 Hz, 2H, Ar–H), 5.90 (s, 2H,
CH2), 4.01 (t, J = 6.0 Hz, 2H, CH2), 3.30 (q, J = 7.0 Hz, 8H, CH2),
2.66 (t, J = 7.1 Hz, 2H, CH2), 2.20 (p, J = 6.6 Hz, 2H, CH2), 1.13 (t,
J = 7.0 Hz, 12H, CH3). 13C NMR (75 MHz, CDCl3) δ 173.05, 171.23,
157.83, 155.17, 154.3, 151.71, 148.09, 131.96, 130.20, 128.19,
125.40, 124.55, 111.57, 110.55, 108.16, 97.37, 66.97, 64.76, 62.77,
44.26, 30.94, 24.57, 12.53. TOF-MS: m/z 877.5 [M + H]+, 899.5
2.3. Fluorescence and UV–vis spectra experiments
Stock solutions of 10 mM PyRbS and PyRbO were prepared by dis-
solving in dimethyl sulphoxide. Stock solutions of 10 mM metal ions
were prepared by dissolving in water. In our detecting experiments,
the fluorescence and UV–vis response of PyRbO would be prevented
with a certain amount of water in the experimental system. It was as-
cribed to the strong hydrolysis between the water and Hg2+, which
prohibited the ring opening of rhodamine B. However, the introduction
[M + Na]+
.
Fig. 5. (a) UV–vis absorption spectra of PyRbS (5 μM) in EtOH/H2O (2:1, v/v) with various amount of Hg2+ (0–6 μM). Inset: The absorbance ratio of PyRbS at A560 nm/A325 nm and A560 nm
/
A345 nm as functions of the Hg2+ concentration (0.3–5.1 μM) in EtOH/H2O (2:1, v/v) (top); the color change after the Hg2+ introduction of PyRbS (bottom). (b) UV–vis titrition of PyRbO
(30 μM) in EtOH with various amount of Hg2+ (0–35 μM). Inset: the absorbance at 558 nm of PyRbO (30 μM) as a function of the Hg2+ concentration (2–24 μM) in EtOH (top); the color
change after the Hg2+ introduction of PyRbO (bottom).

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Fig. 6. (a) Absorbance of PyRbS in the different concentration of Hg2+ in EtOH/H2O (2:1, v/v) at 560 nm, normalized between the minimum absorbance and the maximum absorbance
intensity. The detection limit was calculated to be 0.72 μM. (b) Absorbance of PyRbO in the different concentration of Hg2+ in EtOH at 558 nm, normalized between the minimum
absorbance and the maximum absorbance intensity. The detection limit was calculated to be 5.3 μM.
of sulfur improved the complexation ability of PyRbS toward Hg2+
.
2.5. Cytotoxicity experiments
Therefore, experiments of PyRbS were carried out in aqueous ethanol
solution (EtOH/H2O, 2:1, v/v), and experiments of PyRbO were in
pure ethanol.
Appropriate amounts of different metal ions solution and PyRbS or
PyRbO were added in 10 mL volumetric flasks by pipette, and then di-
luted with EtOH/H2O (v/v, 2:1) or ethanol to the calibration tail. The to-
tally mixed resulting solutions were detected in a quartz optical cell
with a 1 cm optical path length. For the fluorescence measurements,
the excitation wavelength was set at 365 nm and 515 nm.
The MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium
bromide) assay was performed using A549 cells (human lung adenocar-
cinoma cells) to ascertain the cytotoxicity of PyRbS. A549 cells were
seeded in 96-well cell-culture plate at 37 °C under 5% CO2 before treat-
ment. The cytotoxicity experiment was determined by MTT assay (Cell
Proliferation Kit; keygen biological products, Nanjing, China), following
the instructions of the kit. Briefly, A549 cells were treated with various
concentrations (1, 5, 25, 50, 100 μM) of PyRbS and incubated 37 °C
under 5% CO2 for 24 h. Subsequently, MTT (5 mg/mL) was added to
each well and incubated for 4 h. A microplate reader (SPECTRA SLT;
Labinstruments, Salzburg, Austria) was used to measure the absor-
bance. The excitation wavelength was at 492 nm, and the emission
was read at 690 nm. Each treatment was done in six wells, and the ex-
periments were replicated three times. The control experiments were
conducted under the identical conditions treated with DMEM plus
10% FBS and no-PyRbS added.
2.4. Confocal fluorescence imaging
MCF-7 cells (human breast carcinoma cells) were cultured in
Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented
with 10% fetal bovine serum (FBS, Sigma), and penicillin
(100 μg/mL) and streptomycin (100 μg/mL, Invitrogen) at 37 °C
under 5% CO2. One day before imaging, the cells were passed and
plated on 24-well plates to 70% confluence. Subsequently, PyRbS
(5 μM) in the culture media containing ethanol/PBS (1:49, v/v) was
added to MCF-7 cells, which were incubated for 30 min. After the
removal of extracellular extra PyRbS by washing for three times
with PBS (phosphate buffered saline, pH = 7.2, Gibco), the cells
were incubated with 5 μM Hg2+ for 30 min at 37 °C and imaged by
fluorescence microscopy (BX51, olympus, Japan).
3. Results and discussion
3.1. The fluorescence spectra of probes
As shown in Fig. 1, the fluorescence properties of PyRbS (5 μM) and
PyRbO (10 μM) were studied. PyRbS showed weak fluorescence with-
out Hg2+ in aqueous ethanol solution (EtOH/H2O = 2:1, v/v) due to
Fig. 7. (a) The Job's Plot of PyRbS with a total concentration of 10 μM ([PyRbS]+[Hg2+]) in EtOH/H2O (2:1, v/v) at 560 nm. (b) The Job's Plot of PyRbO with a total concentration of 60 μM
([PyRbO]+[Hg2+]) in EtOH at 558 nm.

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215
Fig. 8. (a) Benesi–Hilderbrand plot of PyRbS with Hg2+. (b) Benesi–Hilderbrand plot of PyRbO with Hg2+
.
the spirothiocyclic formation. When the Hg2+ was introduced, signifi-
cant bands at 592 nm (λex = 365 nm and 515 nm) (Fig. 1a and
b) were gradually appeared. The emission color was changed from col-
orless to bright orange, which could be observed by naked eye. This was
also attributed to the ring opening process of the rhodamine derivatives
[23]. The fluorescence signal at 592 nm increased with Hg2+ concentra-
tion, and reached a maximum with 5 μM of Hg2+. A nearly linear rela-
tionship (R2 = 0.999) was observed with Hg2+ concentrations from
0.3 to 4.8 μM (λex = 365 nm). Similarly, linear fluorescent response
(R2 = 0.997) to Hg2+ ranging from 0.3 to 5.4 μM was also obtained
when emission wavelength was 515 nm. In the case of PyRbO, remark-
able enhancement of fluorescence intensity at 583 nm (λex = 365 nm
and 515 nm) (Fig. 1c and d) and the obvious change of color which is
from colorless to reddish-orange were observed. The linear relation-
ships between fluorescence intensity and Hg2+ concentrations
(λex = 365 nm, 2–16 μM Hg2+, R2 = 0.984; λex = 515 nm, 6–16 μM
Hg2+, R2 = 0.990) were also obtained. Taken together, PyRbS had bet-
ter linear relationship with low concentration Hg2+, compared with
PyRbO.
result in small fluorescence intensity changes. To further evaluate the
selectivity of the probes for Hg2+, metal ions competition experiments
(including Hg2+ and other ions) were also investigated under identical
conditions. The addition of only Hg2+ (1.0 equiv) caused obvious fluo-
rescence change in the solution of PyRbS or PyRbO and metal ions. As
shown in bar graphs, there was no obvious fluorescence intensity vari-
ation of probes/Hg2+ complex comparing the results with/without
other metal ions. These results clearly demonstrated that these metal
ions could not interfere with the detection of Hg2+. Thus, PyRbS and
PyRbO could be used as selective Hg2+ probes.
Response time experiments of PyRbS/Hg2+ and PyRbO/Hg2+ sys-
tem were also studied. Hg2+ (1.0 equiv) was separately added to two
optical quartz cells with 1 cm path length containing 5 μM PyRbS in
EtOH/H2O (2:1, v/v) and 10 μM PyRbO in EtOH. Excitation wavelength
was 515 nm and emission wavelengths were 592 nm (PyRbS) and
583 nm (PyRbO), respectively. The changes of fluorescence intensity
were recorded. As shown in Fig. 3, both PyRbS and PyRbO completed
the complexation progress with Hg2+ within a short time. Fluorescence
signal could be detected immediately after the introduction of Hg2+ to
probe solution. Therefore, PyRbS and PyRbO did not need long time
for equilibrium before detecting, and they could be used in real time
Moreover, the fluorescence spectra of PyRbS (5 μM) and PyRbO
(10 μM) in the presence of various metal ions (Na+, K+, Ca2+, Mg2+
,
Ni2+, Ba2+, Ag+, Zn2+, Mn2+, Cu2+, Cd2+, Pb2+, Cr3+, Co2+, Al3+
and Fe2+) were separately studied (Fig. 2). Free PyRbS showed almost
no fluorescence at 592 nm (λex = 515 nm) in ethanol aqueous solution
(EtOH/H2O = 2:1, v/v). Then these metal ions (4.0 equiv) were intro-
duced. In the solution of PyRbS, alkali metal ions (K+, Na+), alkali-
detection of Hg2+
The reversibility is an important property of chemosensor, and mer-
cury is sulphophile element (the Kd value of [HgS2− 2− is 10−50 M2)
.
]
[24]. Then Na2S-addition experiments were conducted to examine the
reversibility of PyRbS and PyRbO with Hg2+ (Fig. 4). The results
earth metal ions (Mg2+, Ca2+) had negligible influence and Ag+
,
displayed that the fluorescence enhancement caused by Hg2+
-
Fe2+, Cr3+ and Al3+ caused slight fluorescence variation. Similarly,
free PyRbO also showed very low fluorescence in pure ethanol and a
few metal ions (Ag+, Zn2+, Cu2+ Cr3+, Al3+, Fe2+) (2.0 equiv) could
addition (2.0 equiv) declined to the initial fluorescence intensity after
the addition of Na2S solution (4.0 equiv) (λex = 365 nm and 515 nm),
and the color of them faded to the pink color. The fluorescence spectra
Scheme 2. Proposed binding mechanism of PyRbO and PyRbO with Hg2+
.

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Q.-Q. Rui et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 209–218
Fig. 9. Confocal fluorescence images in MCF-7 cells. (a) Brighe-field image. (b) Cells stained with PyRbS (5 μM) for 30 min. (c) subsequently exposed to Hg2+ for 30 min. (d) cell nucleus
labeled with Hoechst 33342.
were observed, indicating that PyRbS had a better reversibility. There-
fore, both PyRbS and PyRbO were reversible of detecting Hg2+
spirolactam rhodamine form was the dominant species. Upon the addi-
.
tion of Hg2+, there were obvious absorption peaks at 560 nm (PyRbS)
and 558 nm (PyRbO) respectively (PyRbS,
ε = 6.71 ×
104
L mol−1 cm−1; PyRbO, ε = 1.06 × 104 L mol−1 cm−1) and their color
changed from colorless to pink, suggesting the formation of ring-opened
rhodamine species. Additionally, ratiometric changes in the absorption
spectra of PyRbS were observed. Free PyRbS showed pyrene absorptions
at 324 nm and 345 nm, which decreased gradually after the addition of
Hg2+. A near linear dependence of the ratio of A560 nm/A325 nm (blue
3.2. The UV–vis spectra of probes
The changes of the UV–vis spectroscopy titration for PyRbS (5 μM)
with the increasing amounts of Hg2+ in aqueous solution (EtOH/
H2O = 2:1, v/v) were recorded in Fig. 5a. Besides, analog compound
PyRbO (30 μM) was detected in absolute ethanol in the same way
(Fig. 5b). The absorption of both PyRbS and PyRbO in the absence of
Hg2+ was weak from 450 to 600 nm, which indicated that the
line, R2 = 0.991) and the ratio of A560 nm/A345 nm (green line, R2
=
0.998) as functions of Hg2+ ion concentration (0.3–5.1 μM) were obtain-
ed. The ratios changed from 0.21 to 2.69 and 0.20 to 1.93 respectively. This
indicated the capability of PyRbS for quantitatively detecting Hg2+ by
being a ratiometric probe, which has the important feature in that it
permits signal rationing and thus increases the dynamic range and
provides built-in correction for environmental effects [25]. A nearly linear
relationship (R2 = 0.995) of PyRbO was observed with Hg2+ concentra-
tions from 2 to 24 μM.
Besides, the UV–vis titration data were used to calculated the detec-
tion limit according to a reported method [26,27]. Absorbance of PyRbS
and PyRbO in the different concentration of Hg2+ at the maximum
absorption wavelength was normalized between the minimum absor-
bance and the maximum absorbance intensity. Then the normalized ab-
sorbance was linearly proportional to log[Hg2+] (y = A + Bx) (Fig. 6).
The detection limit value was obtained from 10−A/B. The detection limit
of PyRbS was calculated to be 0.72 μM (Fig. 6a), while that of PyRbO
was 5.3 μM (Fig. 6b). These observations clearly indicated that both
PyRbS and PyRbO could be used for quantitative detection of Hg2+
.
Obviously, PyRbS had advantages over PyRbO not only because of its
higher molar absorption coefficient and lower detection limit, but also
the capability of PyRbS for ratio measurements, which could eliminate
the errors caused by the concentration of probe molecules, surrounding
environment and other factors.
Fig. 10. Cell viability (%) of A549 cells cultured in the presence of PyRbS (0–100 μM) at
37 °C for 24 h.

Q.-Q. Rui et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 209–218
217
Job's Plot analysis was used to figure out the stoichiometry for
PyRbS/Hg2+ and PyRbO/Hg2+ complex. The Job's Plot was conducted
by using absorption of the probes and Hg2+ at the maximum absorption
wavelength plotted against continuous variation with a total concentra-
tion of 10 μM ([PyRbS]+[Hg2+]) and 60 μM ([PyRbO]+[Hg2+])
(Fig. 7). The results of the both Job's Plots exhibited the same maximum
absorbance when the molar fraction of [Hg2+]/([Hg2+]+[probe]) was
0.5, indicating a 1:1 stoichiometry. On the basis of the 1:1 stoichiometry
and UV–vis titration date, the association constants (Ka) of the two
probes with Hg2+ were calculated using the Benesi–Hildebrand meth-
od [28], which was derived as the following formula [29]:
cell survival values are relative to untreated control cells. As shown in
Fig. 10, the cell viability was estimated to be more than 85% upon
PyRbS treatment after 24 h, which indicated the low cytotoxicity even
at concentration of PyRbS as high as 100 μM. Both cell imaging and
cell viability examination suggested that PyRbS was biocompatible
with living cells. It was noticeable that the concentration of PyRbS
(5 μM) used for cell imaging was much lower than that for cell viability
testing (100 μM), suggesting its potential for biological imaging
application.
4. Conclusion
h
ꢀ
h
iꢁi
1=ðA−A0Þ ¼ ½a=ða−bÞꢀ 1 þ 1= Ka Hg2þ
In summary, we have successfully prepared two colorimetric and
fluorometric probes PyRbS and PyRbO based on rhodamine B and
pyrene for the detection of Hg2+. These two probes showed high selec-
tivity to Hg2+. The stoichiometric ratio of probes and Hg2+ was 1:1, and
changes of color were obvious under visible under visible and UV light,
which could be used for naked-eye detection. Compared with PyRbO,
PyRbS exhibited better spectral properties, including higher selectivity,
advantageous reversibility, and lower detection limit, due to the
thiophilic property of Hg2+ ion. Furthermore, pyrene moiety served
successfully as a source of ratiometric absorption changes of PyRbS
with Hg2+, which showed the capability of PyRbS for calibrating and
determining Hg2+ by absorption ratio measurement. Additionally, con-
focal fluorescence images and cytotoxicity experiment indicated that
PyRbS could be potential candidate for detecting Hg2+ in biological
system.
where A is the obtained absorbance of probes at the maximum absorp-
tion wavelength after the addition of Hg2+, and A0 stands for the initial
absorbance of free probes. A linear relationship was obtained
(R2PyRbS = 0.997, R2PyRbO = 0.999), when the 1/(A−A0) was plotted
as a function of 1/[Hg2+] (y = A + Bx) (Fig. 8), which further proved
the 1:1 stoichiometry. The association constant value was calculated
as 2.74 × 105 M−1 (PyRbS/Hg2+) and 1.56 × 105 M−1 (PyRbOHg2+
)
respectively from A/B.
From the foregoing conclusions, the spectral response of PyRbS
or PyRbO to Hg2+ elicited by the reversible binding events were
likely due to the chelation-induced ring-opening of the rhodamine
spirolactam. The possible structures of this reversible process were
shown in Scheme 2.
Taking all the aforementioned in consideration, it was found that
PyRbS was superior to PyRbO owing to its higher molar absorption
coefficient, higher association constant and lower detection limit in
aqueous solution. Moreover, PyRbS could be used for detecting Hg2+
quantitatively by ratiometric absorption measurement. These superior
properties of PyRbS were presumably ascribed to the thiophilic nature
of Hg2+, which had been confirmed from reported references [30].
Owing to its favorable molecular properties, PyRbS should be suited
for Hg2+ monitoring in living cells. Therefore, imaging and cytotoxicity
experiments were performed.
Acknowledgments
This work is supported by the Open Fund of the State Key Laboratory
of Materials-Oriented Chemical Engineering (KL13-07), the Open Fund
of the State Key Laboratory of Coordination Chemistry, the Postgraduate
Education and Innovation Project of Jiangsu Province (KYLX-0772), the
Specialized Research Fund for the Doctoral Program of Higher Education
(20123221110012), and the China Postdoctoral Science Foundation
(2014M550287).
Appendix A. Supplementary data
3.3. Live-cell imaging
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.saa.2016.01.024.
To investigate the property of the probe PyRbS for detecting Hg2+ in
living cells, fluorescence images of MCF-7 cells observed under the con-
focal microscope (Fig. 9). MCF-7 cells were incubated with PyRbS
(5 μM) for 30 min at 37 °C showed very weak intracellular fluorescence
(Fig. 9b). However, a much brighter red fluorescence from the intracel-
lular area was observed after the introduction of Hg2+ (5 μM) in
(Fig. 9c). Bright-field image after Hg2+ and PyRbS treatment confirmed
the viability of the cells over the course of the imaging experiments
(Fig. 9a). Furthermore, the image of fluorescence microscopy of cells
stained with PyRbS/Hg2+ and Hoechst 33342 was shown in Fig. 9d.
The result indicated that PyRbS was cell-membrane permeable, and
Hg2+ predominantly existed in the perinuclear area.
References
[1] E.M. Nolan, S.J. Lippard, Chem. Rev. 108 (2008) 3443–3480.
[2] A. Renzoni, F. Zino, E. Franchi, Environ. Res. 77 (1998) 68–72.
[3] D. Zhang, J. Su, X. Ma, H. Tian, Tetrahedron 64 (2008) 8515–8521.
[4] D.L. Godbold, A. Hüttermann, Physiol. Plant. 74 (1988) 270–275.
[5] I. Terashima, K. Ono, Plant Cell Physiol. 43 (2002) 70–78.
[6] E.G. Malcolm, J.K. Schaefer, E.B. Ekstrom, C.B. Tuit, A. Jayakumar, H. Park, B.B. Ward,
F.M.M. Morel, Mar. Chem. 122 (2010) 11–19.
[7] J.K. Schaefer, F.M.M. Morel, Nat. Geosci. 2 (2009) 123–126.
[8] S.A. Counter, L.H. Buchanan, Toxicol. Appl. Pharmacol. 198 (2004) 209–230.
[9] P.M. Bolger, B.A. Schwetz, N. Engl. J. Med. 347 (2002) 1735–1736.
[10] J.F. Nyland, S.B. Wang, D.L. Shirley, E.O. Santos, A.M. Ventura, J.M. de Souza, E.K.
Silbergeld, Environ. Res. 111 (2011) 584–589.
3.4. Cytotoxicity experiments
[11] M. Lombardo, I. Vassura, D. Fabbri, C. Trombini, J. Organomet. Chem. 690 (2005)
588–593.
[12] L. Liu, W.-Y. Wong, Y.-W. Lam, W.-Y. Tam, Inorg. Chim. Acta 360 (2007) 109–121.
[13] L. Liu, Y.-W. Lam, W.-Y. Wong, J. Organomet. Chem. 691 (2006) 1092–1100.
[14] I. Medina, E. Rubí, M.C. Mejuto, R. Cela, Talanta 40 (1993) 1631–1636.
[15] S. Saito, S. Sasamura, S. Hoshi, Analyst 130 (2005) 659–663.
[16] P. Kubáň, P. Houserová, P. Kubáň, P.C. Hauser, V. Kubáň, J. Sep. Sci. 30 (2007)
1070–1076.
Thiazolyl Blue Tetrazolium Bromide (MTT) assay was applied to
measure the cytotoxicity of PyRbS in human pulmonary adenocarcino-
ma cell (A549 cells). The cell viability (%) was calculated according to
the following equation:
[17] R. Cervenka, Zelinkov, Aacute, Hana, Konecn, Aacute, Marie, Kom, Aacute, J. Rek,
cell viability (%) = ODsample / ODcontrol × 100
Anal. Sci. 26 (2010) 989–993.
[18] E. Stanisz, J. Werner, H. Matusiewicz, Microchem. J. 110 (2013) 28–35.
[19] R. Martínez-Máñez, F. Sancenón, Chem. Rev. 103 (2003) 4419–4476.
[20] D.T. Quang, J.S. Kim, Chem. Rev. 110 (2010) 6280–6301.
[21] R. Álvarez, B. Vaz, H. Gronemeyer, Á.R. de Lera, Chem. Rev. 114 (2013) 1–125.
[22] Z. Zeng, A.A.J. Torriero, A.M. Bond, L. Spiccia, Chem. Eur. J. 16 (2010) 9154–9163.
where ODsample is the mean of optical densities of the wells treated with
different concentrations of probe PyRbS, and ODcontrol represents the
mean of optical densities of the control group. The reported percent

218
Q.-Q. Rui et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 209–218
[23] X. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Chem. Rev. 112 (2011) 1910–1956.
[24] R.D. Armstrong, D.F. Porter, H.R. Thirsk, J. Phys. Chem. 72 (1968) 2300–2306.
[25] Z. Xu, Y. Xiao, X. Qian, J. Cui, D. Cui, Org. Lett. 7 (2005) 889–892.
[26] W. Lin, L. Yuan, Z. Cao, Y. Feng, L. Long, Chemistry 15 (2009) 5096–5103.
[27] Y.S. Mi, Z. Cao, Y.T. Chen, Q.F. Xie, Y.Y. Xu, Y.F. Luo, J.J. Shi, J.N. Xiang, Analyst 138
(2013) 5274–5280.
[28] X.-B. Yang, B.-X. Yang, J.-F. Ge, Y.-J. Xu, Q.-F. Xu, J. Liang, J.-M. Lu, Org. Lett. 13 (2011)
2710–2713.
[29] M. Zhu, M. Yuan, X. Liu, J. Xu, J. Lv, C. Huang, H. Liu, Y. Li, S. Wang, D. Zhu, Org. Lett.
10 (2008) 1481–1484.
[30] K.-H. Chu, Y. Zhou, Y. Fang, L.-H. Wang, J.-Y. Li, C. Yao, Dyes Pigments 98 (2013)
339–346.