Sensors and Actuators B 203 (2014) 452–458

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Sensors and Actuators B: Chemical

journal homepage: [Link]/locate/snb

A rhodamine-based fluorescent probe for Hg2+ and its application

for biological visualization
Jiwen Hu a , Zhangjun Hu a,b,∗ , Yang Cui a , Xuanjun Zhang b,∗∗ ,
Hong-Wen Gao a , Kajsa Uvdal b
a
State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092,
PR China
b
Division of Molecular Surface Physics & Nanoscience, Department of Physics, Chemistry and Biology, Linköping University, Linköping 58183, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: A new visible light excitable fluorescent probe (1) is synthesized by appending a hydroxymethyl-pyridine
Received 31 March 2014 to rhodamine B hydrazide. The probe displays very specific Hg2+ -induced colour change and fluorescent
Received in revised form 16 June 2014 enhancement in the aqueous systems. The “turn-on” response of fluorescence is based on a binding-
Accepted 24 June 2014
induced ring-opening process from the spirolactam (nonfluorescent) to acyclic xanthene (fluorescent)
Available online 10 July 2014
in rhodamine B. The coordinating atoms • O–N–N–O• from the hydroxymethyl-pyridine and rhodamine
B hydrazide play dominant role in the formation of a complex with 1:1 stoichiometry of Hg2+ to 1. It
Keywords:
exhibits a linear response in the range of 0.1–5 ␮M with the limit of detection (LOD) of 15.7 nM (3/slope),
Mercury
Rhodamine
while the calculated value of the association constant of Hg2+ /1 is 0.70 × 105 M−1 . Furthermore, confocal
Fluorescence microscopy imaging experiment demonstrates the probe 1 can be applied as a fluorescent probe for
Probe visualization of Hg2+ in living HeLa cells.
Fluorescent imaging © 2014 Elsevier B.V. All rights reserved.

1. Introduction advised to apply, because “turn-off” detection can always be

interfered by various factors, giving poor sensitivity and selectiv-
The toxicity of mercury ions (Hg2+ ), even at very low concentra- ity [10]. However, mercury ion is known to cause fluorescence
tion, has long been recognized as the healthy and environmental quenching through the spin–orbit coupling effect [11]; and many
problem of primary concern [1]. Mercury poisoning [2] can result “turn-off” fluorogenic Hg2+ probes have been reported [12–16].
in several diseases such as acrodynia, Alzheimer disease and Mina- Therefore, Hg2+ reactive “turn-on” mechanisms attract more atten-
mata disease. It is extremely important to explore sensitive and tions for exploring Hg2+ fluorogenic probes [17]. Among them,
selective Hg2+ detection methods that are simple, rapid and realis- rhodamine-based reactive probes are often reported ones due to
tic. Many available approaches have been devoted to monitor Hg2+ , its fascinating structure-dependent spectral properties [18–36].
including spectrophotometry [3], atomic absorption spectroscopy This unique structural feature of rhodamine is that the spirocyclic
[4], gas chromatography and liquid chromatography, etc. [5]. How- form is nonfluorescent and colorless, whereas H+ or metal ions
ever, these methods usually have drawbacks such as expensive induced spirolactam ring-opening leads to strong fluorescence and
instruments, sophisticated and multi-step sample pretreatments significant color changes [37]. In addition, rhodamine with acyclic
[6]. In contrast, the fluorogenic detection has excellent simplic- xanthene have long emission wavelength (>550 nm), which is supe-
ity, sensitivity and real-time monitoring with fast response, works rior for the detection or imaging with minimizing the influence of
both in the environmental and biological samples as well [7–9]. background fluorescence (<500 nm). Thus, rhodamine substrates
It is considered to be one of the most convenient tools for trace could be taken as ideal choices for the molecular construction of
level detecting and visualizing in different systems. For the relevant “turn-on” Hg2+ probes by using appropriate design and synthesis.
applications, “turn-on” fluorogenic probes are very particularly Herein, a new rhodamine-based probe 1 was synthesized
(Scheme 1). In probe 1, a 2 -hydroxymethyl-2-picolyl moiety was
appended to rhodamine B hydrazide forming a • O–N–N–O• che-
lating receptor. The • O–N–N–O• chelating site in probe 1 was found
∗ Corresponding author: Tel.: +86 21 6598 8598; fax: +86 21 6598 8598.
∗∗ Corresponding author: Tel.: +46 13 28 5826; fax: +46 13 28 8969. to have high affinity towards Hg2+ by showing apparent spec-
E-mail addresses: huzjun@[Link], zhahu@[Link] (Z. Hu), tral response. In addition, S2− induced fluorescence quenching of
xuazh@[Link] (X. Zhang). Hg2+ /1 complex served as an experimental evidence to support the

[Link]
0925-4005/© 2014 Elsevier B.V. All rights reserved.
 J. Hu et al. / Sensors and Actuators B 203 (2014) 452–458 453

Scheme 1. Chemical structure and synthetic route of probe 1.

spirolactam ring-opening mechanism [38–40]. The sensitivity and 3.32 (q, J = 7.05 Hz, 8H), 3.91 (d, J = 5.32 Hz, 2H), 4.62 (s, 2H), 4.83
selectivity of 1 for the detection of Hg2+ in the presence of other (s, 1H), 6.20 (d, J = 2.64 Hz, 1H), 6.23 (d, J = 2.64 Hz, 1H), 6.37 (d,
competing species were established. The compatibility and fluo- J = 2.58 Hz, 2H), 6.40 (d, J = 8.83 Hz, 2H), 6.96 (dd, J = 15.28, 7.63 Hz,
rescence characteristics of 1 in physiologically relevant conditions 2H), 7.13–7.06 (m, 1H), 7.42–7.46 (m, 3H), 7.89–7.92 (m, 1H); 13 C
were exploited for the visualization of living HeLa cells exposed to NMR (75 MHz, CDCl3 ) ı (ppm) = 166.60, 157.65, 157.01, 153.76,
Hg2+ ions. 151.53, 148.72, 136.61, 132.58, 130.36, 128.56, 128.08, 123.98,
122.76, 121.05, 118.36, 107.80, 105.80, 97.88, 65.62, 63.80, 56.14,
2. Experimental 44.29, 12.59. MS (ESI): m/z (%): 578.2 (100) [M+ +H]. Anal. Calcd for:
C, 72.76; H, 6.80; N, 12.12. Found: C, 72.28; H, 7.13; N, 12.23.
2.1. Chemicals and instruments
2.3. General procedures of ions
Most of the chemicals are analytical grade. Rhodamine B
(AR), hydrazine hydrate (85%), 2,6-pyridinedimethanol (AR), 4- Stock solution of probe 1 (1 × 10−3 M) was prepared by
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were dissolving 1 in acetonitrile. Stock solutions of cations and
purchased from Aladdin ([Link] All the anions (1 × 10−3 M) were prepared by dissolving appropriate
aqueous solutions were prepared with deionized water. amount of metal salts in deionized water. The cation solu-
Proton nuclear magnetic resonance (1 H NMR) and carbon tions were prepared from KNO3 , NaNO3 , FeCl2 ·4H2 O, FeCl3 ·6H2 O,
nuclear magnetic resonance (13 C NMR) was recorded on a Varian Mg(NO3 )2 , Ca(NO3 )2 , Ba(NO3 )2 , MnCl2 ·4H2 O, Ni(NO3 )2 , Cu(NO3 )2 ,
300 MHz spectrometer. Electrospray ionisation mass spectra (ESI- Pb(NO3 )2 , Zn(NO3 )2 , Co(NO3 )2 , Cr(NO3 )3 , Cd(NO3 )2 , Al(NO3 )3 ,
MS) were recorded on a Thermo Finnigan Surveyor MSQ. Elemental La(NO3 )3 ·nH2 O, Hg(NO3 )2 , and anions solutions were obtained
analysis was performed with a Perkin-Elmer 240 analyzer. The from NaNO2 , NaNO3 , NaF, NaCl, NaBr, NaI, NaOAc, NaOH,
pH was measured on a PHS-3 C meter. UV–vis absorption spectra NaHCO3 , Na2 CO3 , Na2 SO4 , Na2 SiO3 , NaH2 PO4 ·2H2 O, Na2 HPO4 ,
and fluorescent spectra measurements were performed on S-4100 Na3 PO4 ·12H2 O and Na2 S·9H2 O in the deionized water. The solu-
UV–vis Spectrophotometer and F-4500 fluorescence spectrometer, tion of HEPES–MeOH is mixed with 1 M of HEPES and MeOH with
respectively. All fluorescence measurements were carried out at ratio of 4:6.
room temperature with excitation and emission slit widths of 5 nm, Test solution of probe 1 (5 ␮M) was prepared by placing 10 ␮L
respectively. of the stock solution in a 2 mL test tube, and then diluted to 2 mL
with HEPES–MeOH (0.4 M, pH = 7, 4:6, v/v). The complex solution
2.2. Synthesis of probe 1 of Hg2+ /probe 1 was prepared by addition of the stock solution of
probe 1 (10 ␮L) and Hg(NO3 )2 solution (100 ␮L, 50 ␮M) in a 2 mL
Rhodamine-B hydrazide [41] and 6-hydroxymethyl-pyridine- test tube. Then, the mixture was diluted to 2 mL with HEPES–MeOH
2-carboxaldehyde [42] were synthesized by following reported (0.4 M, pH = 7, 4:6, v/v) buffer solution. Other metal ions were pre-
methods, and characterized by NMR and mass spectra. Rhodamine- pared in the same procedure.
B hydrazide (1.0 g, 2.2 mmol) and 6-hydroxymethyl-pyridine-2- The solutions of different concentration of Hg2+ and probe 1
carboxaldehyde (0.318 g, 2.3 mmol) were dissolved in 30 mL of were prepared by addition of stock solution of probe 1 (10 ␮L) and
anhydrous methanol and the mixture was heated to reflux for Hg(NO3 )2 solutions (0.2–300 ␮L, 0.1–150 ␮M) in 2 mL test tubes.
6 h to give intermediate Schiff base product (see in the supple- Then, the mixture was diluted to 2 mL with HEPES–MeOH (0.4 M,
mentary data). Subsequently, the mixture was cooled to 0 ◦ C and pH = 7, 4:6, v/v) buffer solution.
then solid sodium borohydride (0.38 g, 10.0 mmol) was carefully
added in portions under stirring. The resulting mixture was stirred 2.4. Cytotoxicity assays
at room temperature for 48 h and was quenched with 1 M HCl
(10 mL) at 0 ◦ C. After stirring at room temperature for another 2 h, To test the cytotoxic effect of the probe 1 in the cells over a 24 h
the reaction mixture was adjusted to neutral with 1 M NaHCO3 . period, an MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium
Methanol was removed by rotary evaporation, and the aqueous bromide) assay was performed as previously reported [43]. HeLa
phase was extracted three times with CH2 Cl2 (3 × 20 mL). The com- cells were passed and plated to ca. 70% confluence in 96-well
bined organic phases were dried over anhydrous sodium sulfate, plates overnight before treatment. Before the treatment of probe
filtered and evaporated under vacuum. The residue was purified 1 to cells, DEME (Dulbecco’s modified eagle medium, KeyGEN
by silica flash chromatography (EtOAc:MeOH:NH3 ·H2 O = [Link]), BioTECH) with 10% FBS (fetal bovine serum, KeyGEN BioTECH)
yielding a pale foamy solid 1 (0.80 g, 63%). M.p.: 126–127 ◦ C. was removed and replaced with fresh DMEM, and were added
1 H NMR (300 MHz, CDCl ): ı (ppm) = 1.15 (t, J = 7.06 Hz, 12H), probe 1 with the final concentrations of 10, 20, 30, 40, and 50 ␮M
3
454 J. Hu et al. / Sensors and Actuators B 203 (2014) 452–458

Fig. 1. Fluorescence intensity at 574 nm (ex = 515 nm) of free 1 (5 ␮M) and in the Fig. 2. Fluorescence spectra (ex = 515 nm) of probe 1 (5 ␮M) and 1 (5 ␮M) in the
presence of Hg2+ (25 ␮M) as function of different pH values. presence of various metal ions (50 ␮M). Inset image: the visual color and fluores-
cence under illumination at 365 nm of probe 1 (10 ␮M) before (top line) and after
addition of 50 ␮M of metal ions (bottom line).
which were diluted from stock solution (1 mM). The treated cells
were incubated for 24 h at 37 ◦ C under 5% CO2 . 24 h later, the
2+
medium was removed before 50 ␮L of MTT (KeyGEN BioTECH) 600 without Hg
Fluorescence Intensity(a.u.) with Hg
2+
solution (1:5 diluted in dilution buffer from 20 mg/mL stock solu-
tion) was added and incubated for 4 h at 37 ◦ C under 5% CO2 .
500
Then the solution was removed and 150 ␮L DMSO was added
to the cells. After agitated on the orbital shaker for 5 min, the
absorbance of each well at 570 nm was recorded. The cell viability 400
(%) was calculated according to the following equation: cell viabil-
ity % = OD570 (sample)/OD570 (control) × 100, where OD570 (sample) 300
represents the optical densities of the wells treated with various
concentrations of probe 1 and OD570 (control) represents that of
200
the wells treated with DMEM plus 10% FBS. The percentage cell
survival values are relative to untreated control cells.
100

2.5. Fluorescence imaging

0
g 2+

g 2+
n 2+

u 2+

o 2+
a 2+

d 2+
+

+
+

+
a 2+

2+

r +
i +

l +
a+

Fe 3

Zn 2
Fe 2

La 3
C 2
N 2

A 3
The living HeLa cells (human cervical cancer cell) were provided
K+

Pb
M

H
N

C
C

C
B

by the School of Medicine, Tongji University (Shanghai, China) and

cultured in DEME supplemented with 10% FBS. One day before Fig. 3. The fluorescence spectra (ex = 515 nm) of probe 1 (5 ␮M) and Hg2+ (5 ␮M)
imaging, cells were seeded on confocal glass dishes. The next day, in the presence of various anions (50 ␮M).
the HeLa cells were incubated with 0 ␮M Hg2+ and 10 ␮M Hg2+ for
30 min at 37 ◦ C under 5% CO2 and then washed with PBS buffer
An important feature of 1 is its high selectivity towards the Hg2+
solution (phosphate buffered saline, KeyGEN BioTECH) three times
over other species. Affinities of probe 1 towards various ions K+ ,
before incubating with 5 ␮M probe 1 for another 20 min. The cells
Na+ , Fe2+ , Fe3+ , Mg2+ , Ca2+ , Ba2+ , Mn2+ , Ni2+ , Cu2+ , Pb2+ , Zn2+ , Co2+ ,
were rinsed with PBS three times again, after which the confo-
Cr3+ , Cd2+ , Al3+ , La3+ and Hg2+ were firstly checked by monitor-
cal images were performed under a leica TCS SP5 Laser Scanning
ing its absorbance (Fig. S1) and fluorescence changes (Fig. 2) in
Confocal Microscope (excited at 543 nm).
HEPES–MeOH medium. The results showed that only Hg2+ induced
significant absorbance and fluorescence changes in the presence of
3. Results and discussion 10 equiv. of ions. The other ions did not promote obvious changes
except for Cu2+ , which indicates that Cu2+ has a slight capacity to
To study the practical applicability, the effects of pH values induce the spirolactam ring-opening of 1 to some extent. A compe-
on fluorescence response were investigated. Fluorescence spectral tition experiment was then carried out. The comparison between
studies for 1 in HEPES–MeOH medium at different pH values were the fluorescence response of probe 1 with each metal ion before and
evaluated (Fig. 1). At acidic condition (pH < 6.0), 1 shows an obvi- after the addition of Hg2+ was measured (Fig. 3). The results show
ous fluorescence due to the formation of an acyclic xanthene form that the enhancement in fluorescence intensity resulting from the
because of the protonation. In the pH range from 6.0 to 10.0, neither addition of Hg2+ is not affected by coexistence of other metal ions
color nor fluorescence was observed for free 1. This suggests that even Cu2+ , which displays a slight capacity to induce the spirolac-
the spirocyclic form of probe 1 is stable in this pH range. How- tam ring-opening of 1. In other words, the selective fluorescence
ever, in the presence of Hg2+ , an obvious different fluorescence response of probe 1 towards Hg2+ is much better than other com-
enhancement was observed under different pH values from 3.0 to petitive metal ions.
8.0. The maximum fluorescence response toward Hg2+ was cen- To assess the performance of probe 1 in complicated systems,
tered at pH 7.0. Therefore, further studies were carried out in pH another comparative experiment with interfering anions was also
7.0 HEPES–MeOH medium (0.4 M, 4:6, v/v). carried out. As shown in Fig. 4, with addition of 50 ␮M (10 equiv.)
 J. Hu et al. / Sensors and Actuators B 203 (2014) 452–458 455

Fig. 4. Fluorescence intensity at 574 nm (ex = 515 nm) of probe 1 (5 ␮M) with Na+ ,
Fig. 5. Fluorescence spectra (ex =515 nm) of probe 1 (5 ␮M) upon addition of Hg2+
K+ , Mg2+ , Ca2+ (0.5 mM) and other various metal ions (25 ␮M) in the absence and
(0–30 equiv.). Inset: (a) fit linear and (b) fluorescence intensity of probe 1 (5 ␮M)
presence of Hg2+ (5 ␮M).
upon addition of Hg2+ .

anions to Hg2+ /1 (5 ␮M Hg2+ and 5 ␮M 1), only S2− significantly

quenched the fluorescence with the color changed from pink to a ring-opened amide form. These results indicated that 1 could
colorless. It indicates that the spirolactam ring-opening rhodamine serve as a naked-eye chemosensor for Hg2+ . Similarly, an emis-
returned to the spirocyclic form. It is because that S2− has very high sion peaked at around 574 nm was significantly increased in the
affinity with Hg2+ compared to probe 1, and can competitively sub- intensity upon the gradual addition of Hg2+ (Fig. 5), which cor-
stitute 1 from Hg2+ /1 complex. And I− quenched the fluorescence responds to the emission of ring-opened xanthene. The emission
a little bit. It indicates that the affinity of 1 towards Hg2+ is obvi- intensity stabilized after the amount of added Hg2+ ions reached
ous stronger than those probes system that can be reversible by 30 equiv. with a defined emission point. The linear fluorescence
I− [44]. In addition, influence of two common amino acids con- enhancement of probe 1 upon addition of Hg2+ was obtained in the
taining sulphur on both probe 1 and Hg2+ /1 was also evaluated. range of 0.1–5 ␮M (R2 = 0.9979). And the limit of detection (LOD)
As shown in Fig. S2, l-methionine have no influence on the flu- was determined to be 15.7 nM (3/slope), which was comparable
orescence of Hg2+ /1, but l-cysteine quenched the fluorescence a to that of the reported rhodamine-based probes (Table 1). There-
little bit, similar as I− does. This is because l-cysteine has mercapto fore, it indicates that LOD of probe 1 is in the normal range and also
group, which has stronger binding capacity to Hg2+ compared to the probe 1 can be sensitive enough to detect the concentration
thioether moiety in l-methionine. These results further indicate of Hg2+ in the environment samples and apply in the quantitative
that the probe 1 is a highly selective probe for the detection of free analysis.
Hg2+ ions. To find out the stoichiometry of the Hg2+ /1 complex, Job’s
The UV–vis and fluorescence titration experiments were car- method was applied. The ratio of combination using the method
ried out (Figs. S3 and 5). Upon the gradual addition of Hg2+ ions, a of continuous variations (Job’s plot) was measured. As shown in
new absorption peak at 560 nm emerged with increasing intensity, Fig. 6, the result of fluorescence intensity being a maximum value
and the solution changed from colorless to pink simultaneously, at a molar fraction of about 0.5 indicates that probe 1 and Hg2+ form
which can be ascribed to the variation from a spirocyclic form to complex with molar ratio of 1:1. As shown in Fig. S4, a plot of 1/F

Table 1
The comparison of Ka and LOD with some reported rhodamine-based Hg2+ probes.

Refs. Ka (M−1 ) LOD Solution system

[6] 2.78 × 104 5.9 nM CH3 CN–PBS (pH 7.5, 1:1)

[18] – 0.22 ␮M CH3 CN–HEPES (10 mM, pH 7.4, 4:1)
[19] – 39 nM DMF–PBS (25 mM, pH 7.2, 1:4)
[20] 1.44 × 106 0.434 ␮M C2 H5 OH–H2 O (pH 7.0, 1:1)
[21] – 3 nM MeOH–HEPES (pH 7.0, 1:1)
[23] – 20 nM 1,4-Dioxane–PBS (20 mM, pH 7.0, 0.5:99.5)
[24] 1.26 × 106 8.25 nM MeOH–HEPES (20 mM, pH 7.0, 3:7)
[25] (5.9 ± 0.23) × 104 – HEPES (1 mM, pH 7.1)
[26] (1.82 ± 0.04) × 105 ppb level DMF–H2 O (1:1)
[28] 1.86 × 106 1.72 nM EtOH–HEPES (50 mM, pH 7.0, 1:1)
[29] – 50 nm PIPES (50 mM) KCl (100 mM) (pH 7.0)
[31] (1.45 ± 0.1) × 104 0.1 ppm CH3 CN–H2 O (1:1)
[32] 7.08 × 104 2 ␮M THF–HEPES (pH 7.0, 9.5:0.5)
[33] (6.5 ± 0.55) × 104 – MeOH–HEPES (1 mM, pH 7.2, 1:1)
[34] (1.01 ± 0.05) × 105 0.35 ppb CH3 CN–HEPES (1.0 mM, pH 7.2, 1:1)
[35] – 27.5 nM CH3 CN–PBS (25 mM, pH 7.0, 2.5:97.5)
[43] 1.21 × 105 – MeOH–HEPES (20 mM, pH 7.0, 1/99, 50 mM NaNO3 )
[44] (6.31 ± 0.74) × 105 – CH3 CN–TRIS (10 ␮M, pH 7.0, 4:1)
[45] 2.1 × 107 25 nM MeOH–HEPES (10 mM, pH 7.0, 4:6)
4.4 × 105 42 nM
This work 0.94 × 105 15.7 nM MeOH–HEPES (0.4 M, pH 7.0, 6:4)
456 J. Hu et al. / Sensors and Actuators B 203 (2014) 452–458

Fig. 6. Job’s plot analysis of 1 and Hg2+ at a total concentration of 10 ␮M. Fig. 8. Fluorescence spectra (ex = 515 nm) of probe 1 (5 ␮M) only, probe 1 (5 ␮M)
in the prescence of Hg2+ (5 ␮M) and mix soulution of probe 1 (5 ␮M), Hg2+ (5 ␮M)
versus 1/[Hg2+ ] shows a linear relationship, from which one can and 4 equiv. of S2− (20 ␮M). Inset: fluorescence intensity of 1 (5 ␮M)/Hg2+ (5 ␮M)
estimate that 1 is bound with Hg2+ in a 1:1 binding stoichiometry, upon addition of S2− .
and the association constant (Ka ) is calculated to be 0.70 × 105 M−1
according to the B–H plot. As the results, according to the binding
mode between 1 and Hg2+ , 1 is most likely to be chelated with Hg2+ displayed apparent downfield shifts of 0.15 ppm, along with pro-
via its O and N atoms (Scheme 1) [45]. The comparative informa- tons (h, i) on pyridine displaying down-filed shifts of 0.21 and
tion from some studies on rhodamine-based Hg2+ probes is given 0.29 ppm, which indicated the coordination of the N and pyri-
in Table 1. It shows that the • O–N–N–O• receptor from carbonyl, dine to Hg2+ . Protons (b, f) displayed up-field shifts of 0.05 and
amido, pyridine and hydroxyl is favorable to coordinate with Hg2+ 0.25 ppm, which was caused by coordination of hydroxyl group.
under a suitable geometry conformation and switch the spirocyclic Furthermore, the proton (j) position showed an up-field shift of
form to the ring-opened form. 0.12 ppm, which was attributed to the increase in electron density
To further elucidate the binding mode, 1 H NMR and 1 H–1 H cor- arising from Hg2+ ion induced ring-opening of the spirolactam in 1.
related spectroscopy (COSY) experiments were conducted. Fig. 7 The aromatic 1 H–1 H COSY spectra were applied to identify spectra
shows the 1 H NMR change of 1 in the absence and presence of Hg2+ (Figs. S5 and S6). Taken together, Hg2+ was possibly coordinated
ions. After addition of Hg2+ , the protons (a) beside the N atoms with the constructed • O–N–N–O• receptor.

1
Fig. 7. H NMR spectra of 1 (a); and 1 + 1 equiv. of Hg2+ (b) in CD3 OD.
 J. Hu et al. / Sensors and Actuators B 203 (2014) 452–458 457

Fig. 9. Confocal images of Hg2+ using probe 1 in HeLa cells: (a) brightfield image of living HeLa cells treated with probe 1 (5 ␮M) for 20 min; (b) fluorescence image of (a) in
red channel at 580 ± 10 nm, ex = 543 nm; (c) merged image of (a) and (b); (d) brightfield image of living HeLa cells treated with Hg2+ (10 ␮M) for 30 min, then treated with
probe 1 (5 ␮M) for 20 min; (e) fluorescence image of (d) in red channel at 580 ± 10 nm, ex = 543 nm; (f) merged image of (d) and (e).

Reversibility experiment is also important for the chemical 4. Conclusion

sensors applicable to detection of intracellular analysis. As men-
tioned above, fluorescence of Hg2+ /1 complex can be quenched by In summary, a new rhodamine B-based probe 1 for Hg2+ ions was
S2− . Thus the reversibility of probe 1 binding was investigated by synthesized, which displayed highly selective and sensitive fluores-
a simple titration methodology. With addition of 4 equiv. of S2− cent enhancement and color change upon the addition of Hg2+ over
(20 ␮M) into the Hg2+ /1 complex system, the fluorescence inten- other metal ions. The spectral studies show a “turn-on” fluores-
sity decreased gradually (Fig. 8) due to stronger binding capability cence response towards Hg2+ as a 1:1 binding mode at physiological
of S2− towards Hg2+ [46]. And the solution returned to the origi- pH. Furthermore, the “turn-on” response mechanism was studied
nal colorless. This result reveals that the binding of 1 and Hg2+ is a by 1 H NMR spectra and S2− -induced reversibility experiment. The
reversible spirocycle open–close mechanism rather than a metal- living cell imaging experiments demonstrated that visualizing abil-
induced hydrolysis reaction. The binding S2− and Hg2+ releases the ity of probe 1 towards trace Hg2+ in the biological systems. These
• O–N–N–O• site from complex form along with reforming color- entire results show probe 1 is a potential candidate for the detection
less and non-fluorescent spirolactam. It was concluded that the of Hg2+ in chemical, environmental and biological systems.
free probe was in the spirocyclic form, whereas the coordination
of Hg2+ induced the N atom of spirolactam to attack the C atom of Acknowledgements
carbonyl, resulting in a ring-opening of the spirolactam (Scheme 1),
which is reversible under the addition of S2− . We thank the National Key Technology R&D Program of China
Owing to the high sensitivity and selectivity, probe 1 showed (2013BAC01B01), the National Natural Science Foundation of China
excellent performance in the detection of Hg2+ in biological sam- (Grant No. 21007046) and Swedish Government Strategic Faculty
ples. HeLa cells were incubated with Hg2+ (0 ␮M and 10 ␮M) for Grant in Material Science (SFO, MATLIU) in Advanced Functional
20 min at 37 ◦ C and washed with PBS buffer solution three times; Materials (Faculty Grant SFO-Mat-LiU#2009-00971) for financially
then incubated with probe 1 (5 ␮M) for another 20 min. Subse- supporting this work.
quently, the cells were rinsed with PBS buffer solution again, and
their fluorescence images were recorded under a leica TCS SP5
Laser Scanning Confocal Microscope (Fig. 9). No detectable fluores- Appendix A. Supplementary data
cence in living cells was collected for the cells pre-incubated with
0 ␮M Hg2+ (Fig. 9b). In contrast, the cells pre-incubated with 10 ␮M Supplementary data associated with this article can be found, in
Hg2+ show a remarkable enhanced fluorescence (Fig. 9e). More- the online version, at [Link]
over, the MTT assay demonstrates that the cell viability remains
more than 98.6% after treatment with 10 ␮M probe 1 after 24 h References
of incubation (Fig. S7). These results demonstrate that probe 1
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imaging in live cells based on a rhodamine–thioamide–alkyne scaffold, Chem.
Commun. 46 (2010) 3529–3531. Jiwen Hu is a Ph.D. candidate majoring in environmental science at Tongji Uni-
[20] K.H. Chu, Y. Zhou, Y. Fang, Rhodamine-pyrene conjugated chemosensors for versity. Her current research interests focus on novel fluorescent probes for
ratiometric detection of Hg2+ ions: different sensing behavior between aspiro- environmental detection and biomedical imaging.
lactone and a spirothiolactone, Dyes Pigm. 98 (2013) 339–346.
[21] Y.J. Gong, X.B. Zhang, An efficient rhodamine thiospirolactam-based fluores- Zhangjun Hu earned his Ph.D. degree in chemistry from Anhui University in 2008,
cent probe for detection of Hg2+ in aqueous samples, Analyst 137 (2012) respectively. From 2008 to 2014, he worked as a postdoctoral researcher at Tongji
932–938. University and Linköping University successively. Since 2014, he has been an assis-
[22] X.L. Zhang, Y. Xiao, A ratiometric fluorescent probe based on FRET for imaging tant professor at Linköping University. His research interests focus on the fluorescent
Hg2+ ions in living cells, Angew. Chem. Int. Ed. 47 (2008) 8025–8029. molecules, coordination complex and nano-materials for biomedical imaging and
[23] W. Shi, H.M. Ma, Rhodamine B thiolactone: a simple chemosensor for Hg2+ in environmental sensing.
aqueous media, Chem. Commun. 13 (2008) 1856–1858.
[24] F.Y. Yan, D.L. Cao, N. Yan, A selective turn-on fluorescent chemosensor based Yang Cui earned his Ph.D. degree in 2014 in environmental science from Tongji Uni-
on rhodamine for Hg2+ and its application in live cell imaging, Sens. Actuators versity. Since 2014, he worked as a postdoctoral researcher at University of Science
B 162 (2012) 313–320. & Technology of China. His research interests focus on the fluorescent molecules
[25] S. Saha, M.U. Chhatbar, P. Mahato, Rhodamine–alginate conjugate as self indi- and nano-materials for environment-related applications.
cating gel beads for efficient detection and scavenging of Hg2+ and Cr3+ in
Xuanjun Zhang obtained his PhD degree in 2004 from the University of Science
aqueous media, Chem. Commun. 48 (2012) 1659–1661.
& Technology of China in chemistry. From 2004 to 2011, he worked as a postdoc-
[26] W. Huang, C.X. Song, C. He, Recognition preference of rhodamine thiospiro-
toral researcher and visiting scientist at Shantou University, National University
lactams for mercury (II) in aqueous solution, Inorg. Chem. 48 (2009)
of Singapore, Linköping University, University of Washington, Seattle. Since 2011,
5061–5072.
he has been an assistant professor at Linköping University and obtained Docent in
[27] A.K. Mandal, M. Suresh, P. Das, Recognition of Hg2+ ion through restricted imine
2014. His research interest focuses on the fluorescent molecules, coordination poly-
isomerization: crystallographic evidence and imaging in live cells. B thiolac-
mers, and nanoparticles for applications in light harvesting, biomedical imaging and
tone: a simple chemosensor for Hg2+ in aqueous media, Org. Lett. 14 (2012)
sensing.
2980–2983.
[28] Y. Zhou, X.Y. You, Y. Fang, J.Y. Li, K. Liu, C. Yao, A thiophen–thiooxorhodamine Hong-Wen Gao is a full professor and the head of EMSL lab at college of environmen-
conjugate fluorescent probe for detecting mercury in aqueous media and living tal science and engineering in Tongji University. His interests focus on the trace level
cells, Org. Biomol. Chem. 8 (2010) 4819–4822. detection of pollutants, toxicity of environmental pollutants, and functionalized
[29] E.M. Nolan, S.J. Lippard, Turn-on and ratiometric mercury sensing in water with materials for environment-related applications.
a red-emitting probe, J. Am. Chem. Soc. 129 (2007) 5910–5918.
[30] X. Chen, S.W. Nam, M.J. Jou, Y. Kim, S.J. Kim, S. Park, J. Yoon, Hg2+ selective Kajsa Uvdal received her PhD in 1991 from Linköping University. From 1993 to
fluorescent and colorimetric sensor: its crystal structure and application to 1994 she did postdoctoral work at University of Washington, Seattle. From 1994
bioimaging, Org. Lett. 10 (2008) 5235–5238. she worked as an assistant professor at Linköping University and obtained Docent
[31] M. Suresh, S. Mishra, S.K. Mishra, E. Suresh, A.K. Mandal, A. Shrivas- in 1999. Since 2007 she has been a full-professor and division head of Molecular
tav, Resonance energy transfer approach and a new ratiometric probe Surface Physics and Nanoscience. Her research interest focuses on surface physics
for Hg2+ in aqueous media and living organism, Org. Lett. 11 (2009) and magnetic and fluorescent nanoparticles for applications in biomedical imaging
2740–2743. and sensing.