This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 1961–1965 1961

Cite this: New J. Chem., 2012, 36, 1961–1965

A bis(rhodamine)-based highly sensitive and selective fluorescent

chemosensor for Hg(II) in aqueous mediaw

Rui Han,aXian Yang,

aDi Zhang,

aMin Fan,

aYong Ye*

acand Yufen Zhao

abc

Received (in Gainesville, FL, USA) 17th March 2012, Accepted 23rd August 2012

DOI: 10.1039/c2nj40638a

A new bis(rhodamine)-based fluorescent probe 3 was syn-

thesized, and it exhibited high selectivity for Hg2+

over other

commonly coexistent metal ions in aqueous media with a broad

pH span (3–9).

Hg2+ is one of the most hazardous components in the

environment. Its frequent and fruitful use can result in a high

level of residues, which may result in the contamination of

water systems and soil, and therefore cause health hazards.1

Governmental restrictions on the levels of residual heavy

metals in end products are very strict. Therefore, Hg detection

has attracted tremendous attention. The design and develop-

ment of fluorescent Hg2+ probes has therefore attracted a

great deal of attention. A lot of Hg2+ probes have been

proposed so far;2 however, most of the probes act only in

organic media. Hg2+ probes capable of acting in aqueous

media have also been proposed; however, many of these

probes show insufficient selectivity for Hg2+.3 The design of

Hg2+ probes with high selectivity in aqueous media is there-

fore of current focus. In addition, Hg2+ exists in various

places such as in living organisms, soil, rivers, and seas.4

Versatile Hg2+ probes must therefore be applicable to samples

with a broad pH range. Several Hg2+ probes showing high

selectively in aqueous media have been proposed; however,

most of these probes act only at neutral pH,5 and only a few

probes act at an acidic–neutral pH6 or at a relatively broader

pH range.7 On the basis of the well-known spirolactam (non-

fluorescent) to ring-open amide (fluorescent) equilibrium,

rhodamine frameworks have been considered as an ideal mode

for the construction of the OFF-ON systems that have fre-

quently been utilized to design fluorescence-enhanced probes

for metal ions.8 Here, we report a new bis(rhodamine)-based

fluorescent probe that exhibited high selectivity for Hg2+ over

other commonly coexistent metal ions in aqueous media and

in a broad pH range.

As shown in Scheme 1, compound 2 was facilely synthesized

from rhodamine B derivative 1 and Lawesson reagent in

moderate yield in toluene. Compound 3 was prepared by the

reaction of compound 2 and benzenedicarbonyl dichloride in

the presence of triethylamine. Their structures have been

confirmed using 1H NMR, 13C NMR, ESI mass spectrometry,

and elemental analysis (see ESIw). Although 3 is a derivative of

rhodamine B, it forms a nearly colorless solution in either

water or absolute ethanol, indicating that the spirocyclic

form exists predominantly. The characteristic peak near

65.6 ppm (9-carbon) in the 13C NMR spectrum of 3 also

supports this consideration. Besides, neither the color nor

the fluorescence (excited at 580 nm) characteristics of

rhodamine could be observed for 3 in water or ethanol,

suggesting that the spirocyclic form was still preferred in this

range. Addition of mercuric ion to the solution of 3 causes

instantaneous development of a pink color and a strong

fluorescence (Fig. 1). This observation shows that the mercury-

induced ring-opening reaction takes place rapidly at room

temperature.

To further investigate the interaction of Hg2+ and 3, an

ultraviolet photometric titration experiment was carried out

(Fig. 2). A linear increase of absorption intensity of 3 could be

observed with increasing Hg2+ concentration accompanied by

color changes from colorless to pink. To determine the

stoichiometry of the Hg–ligand complex, Job’s method for

absorbance measurement was applied.10 Keeping the sum of

Scheme 1 The synthesis of the title compound 3.

a Key Lab of Chemical Biology and Organic Chemistry of HenanProvince, Department of Chemistry, Zhengzhou University,Zhengzhou 450052, China. E-mail: yeyong03@tsinghua.org.cn;Fax: +86 371 67767051; Tel: +86 371 67767050

bDepartment of Chemistry and Key Laboratory for Chemical biologyof Fujian Province, Xiamen University, Xiamen 361005, China

c Key Laboratory of Bioorganic Phosphorus Chemistry & ChemicalBiology (Ministry of Education), Department of Chemistry,Tsinghua University, Beijing 100084, Chinaw Electronic supplementary information (ESI) available. See DOI:10.1039/c2nj40638a

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1962 New J. Chem., 2012, 36, 1961–1965 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

the initial concentration of Hg2+ and 3 at 100 mM, the molar

ratio of Hg2+ was varied from 0 to 2. A plot of [Hg2+]/

{[Hg2+] + [3]} versus the molar fraction of Hg2+ is provided

in Fig. S5 (ESIw). It showed that the [Hg2+]/{[Hg2+] + [3]}

value went through a maximum at a molar fraction of

0.36, indicating a 1 : 2 stoichiometry of Hg2+ to 3 in the

complex.

Further evidence for this stoichiometry comes from the

fluorescence experiment. A fluorescence titration of Hg2+

was conducted using 80 mM solution of 3 in water–ethanol

(20/80 v/v) at pH 7. The observed fluorescence intensity was

nearly proportional to the Hg2+ concentration. The satura-

tion behavior of the fluorescence intensity after 2 equiv. of

Hg2+ reveals that the Hg2+ chemodosimeter has a 1 : 2

stoichiometry (Fig. 3).

Fig. 4 shows the absorption spectra of 3 in the presence of

various metal ions in ethanol–water. When no metal ion was

added to the solution of 3 (10 mM), almost no absorption

above 562 nm could be observed, whereas a significant

enhancement of the characteristic absorption of rhodamine B

emerged soon after Hg2+ was injected into the solution. There

was a large enhancement factor (500-fold) of absorbance at

lmax = 562 nm upon the addition of 20 equiv. of Hg2+. Other

cations of interest gave no response (Fig. 5).

Changes in the fluorescence properties of 3 caused by other

metal ions, including Ag+, Na+, Cr3+, Pb2+, Mg2+, K+,

Co2+, Mn2+, Ba2+, Ca2+, Al3+, Li+, Cu2+, Fe2+, Fe3+,

Ni2+, Zn2+, Cd2+, were also measured. The fluorescence

spectra of solutions of 3 (10 mM), recorded within 5 min after

the addition 2 equiv. of each of these metal ions, are displayed

in Fig. 6. Only Ag+ ions promote small fluorescence intensity

changes, while other metal ions did not cause any significant

changes under identical conditions. The selectivity observed

for Hg2+ over other ions is remarkably high. In addition, the

enhancement in fluorescence intensity resulting from the addi-

tion of Hg2+ is not influenced by subsequent addition of other

metal ions. Finally, while the colorless to pink color change

associated with the reaction of 3 with Hg2+ is visually readily

detectable, no significant color changes are promoted by other

metal ions (Fig. S6, ESIw). This interesting feature reveals that

3 can serve as a selective ‘‘naked-eye’’ chemosensor for Hg2+.

Fig. 1 The color (top) and the fluorescent (bottom) changes of 3 to

Hg2+ in EtOH–water solution (80 : 20, v/v) [3] = 40 mM, [Hg2+] =

40 mM.

Fig. 2 Absorption spectra of 3 in C2H5OH–H2O (80/20, v/v) upon

addition of different amounts of Hg2+ ions.

Fig. 3 Fluorescence intensities of 3 with gradual addition of different

amounts of Hg2+ (from bottom 0–8 eq. Hg2+).

Fig. 4 Changes in the absorption spectra of 3 in the presence of

different metal ions in ethanol–water (80 : 20, v/v).

Fig. 5 Change in the absorbance at 562 nm of 3 (10 mM) in presence

of 20 eq. of various different metal ions in ethanol–water (80 : 20, v/v).

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This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012 New J. Chem., 2012, 36, 1961–1965 1963

To check the practical ability of compound 3 as a Hg2+

selective fluorescent sensor, we carried out competitive experi-

ments in the presence of Hg2+ at 10 equiv. mixed with Mg2+,

K+, Co2+, Cu2+, Mn2+, Ba2+, Na+, Ca2+, Pb2+, Cr3+,

Ag+, Ni2+, Cd2+, Zn2+, Fe2+, Fe3+ and Li+ (10 equiv.

each). No significant variation in the fluorescence emission

was observed by comparison, with or without the other metal

ions (Fig. S7, ESIw). The fluorescence enhancement of 3 with

HgCl2 has almost no change with the addition of different

anions, such as CH3COO�, NO3�, SO4

2�, F�, ClO4� and Br�

(Fig. S8, ESIw).To investigate the practical applicability of 3, the detection

limit of this new chemodosimeter system was evaluated. The

fluorescence titration profile of 3 (10 mM) with Hg2+, shown in

Fig. 7, demonstrates that the detection of Hg2+ is at the parts

per billion level. Under these conditions, the fluorescence

intensity of the solution of 3 was nearly proportional to the

amount of Hg2+ added (Fig. 7b). The detection limit was

measured to be 0.128 ppm. Therefore, the proposed probe 3

was sensitive enough to detect Hg2+ in industrial wastewater,

which has a discharge limit of 0.25 mM (50 ppb) defined by the

Standardization Administration (SA) of the People’s Republic

of China.11

For practical applicability, the optimized pH conditions of

this new probe was also evaluated (Fig. S9, ESIw). For free 3,at acid conditions (pH o 3), the ring opening of rhodamine

took place because of the strong protonation. When pH > 3,

no significant ring opening was observed. However, in the

presence of Hg2+ ions, there was an obvious fluorescence

OFF-ON change between pH 3 and 9. Thus, probe 3 can

detect Hg2+ ions with a wide pH span (3–9), because in this

region, 3 with the Hg2+ ions induces a remarkable fluores-

cence OFF-ON, whereas 3 without the Hg2+ ions does not

lead to such a change.

Both UV-vis and fluorescence data lead to a significant

OFF-ON signal. From the molecular structure and spectral

results of 3, it is concluded that the reaction mechanism should

involve two steps. First, the addition of the Hg2+ ions induces

the N atom of the amide to attack the C atom of the CQS

bond, and thus a ring opening of the spirolactam of rhodamine

took place. Secondly, water-promoted hydrolysis took place.

After the removal of HgS and m-phthalic acid, an intra-

molecular guanylation took place. Finally, a cyclic product 5

was formed through an irreversible hydrolysis desulfurization

reaction, as depicted in Scheme 2.

In order to prove the reaction mechanism of the present

system, the reaction products of 3 with Hg2+ were subjected to

electrospray ionization mass spectral analyses. In the ESI-MS

spectra, a major ion peak was detected at m/z 471.4 (Fig. S10,

ESIw). It was characterized to be the ring opening product of

Fig. 6 Fluorescence spectra of 3 (10 mM) in ethanol–water (80 : 20,

v/v) in the presence of 2 equiv. of Hg2+, Ag+, Na+, Cr3+, Pb2+,

Mg2+, K+, Co2+, Mn2+, Ba2+, Ca2+, Al3+, Li+, Cu2+, Fe2+, Fe3+,

Ni2+, Zn2+, Cd2+. Ex: 560 nm, Em: 580 nm, slit: 5.

Fig. 7 (a) Fluorescence emission changes of 3 (10 mM) upon addition

of Hg2+ in water–ethanol (20/80 v/v) at 25 1C. (b) The fluorescence

intensities at 560 nm.

Scheme 2 The proposed mechanism for the fluorescence OFF-ON.

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1964 New J. Chem., 2012, 36, 1961–1965 This journal is c The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2012

rhodamine, indicating the generation of 5 as a final product.

According to ESI-MSn, the fragment ion at m/z 443, derived

from the precursor ion at m/z 471, can be further fragmented

into the ions at m/z 399. The fragmentation pathway of

compound 5 is shown in Fig. S11. High-resolution FTICR-

MS indicated that the exact mass of the ion at m/z 471 is

471.3136, corresponding to the formula C30H39N4O (calcu-

lated 471.3118).

In conclusion, we synthesized a new bis(rhodamine)-based

fluorescent probe for Hg2+ detection. The colorimetric and

fluorescent response to Hg2+ can be conveniently detected

even by the naked eye, which provides a facile method for

visual detection of Hg2+. The selectivity of this system for

Hg2+ over other metal ions is excellent, and the detection of

Hg2+ at the 0.128 ppm level is still available. There are two

good features of this system: (i) a remarkably high selectivity

toward Hg2+ ions over miscellaneous competitive cations, and

(ii) a wide pH span (3–9). These features make it a promising

candidate for the determination of Hg2+ ions in aqueous

solution for practical analysis. The cell-permeable property

and synthesis of a series of similar compounds were con-

ducted. The results will be published elsewhere.

Experimental

General

All the materials for the syntheses were purchased from

commercial suppliers and used without further purification.

The solutions of metal ions were prepared from their nitrate

salts, except for FeCl2, CrCl3 and MnCl2. The use of Fe(NO3)3and FeCl3 yielded nearly the same results. Rhodamine B

derivative (1) was synthesized according to the literature.9 A

Hitachi F-4500 spectrofluorimeter was used for fluorescence

measurements. The absorption spectra were recorded with a

Techcomp UV-8500 spectrophotometer (Shanghai, China).

NMR spectra were measured on a Bruker AMX-400 spectro-

meter at 400 MHz in CDCl3. Elemental analyses were carried

out with a Flash EA 1112 instrument. Mass spectra were

acquired in positive ion mode using a Bruker ESQUIRE 3000

ion trap spectrometer equipped with a gas nebulizer probe,

capable of analyzing ions up to m/z 6000.

Synthesis of 2

Lawesson reagent (0.5055 g, 1.25 mmol) was added in three

portions to a solution of 1 (1.21 g, 2.5 mmol) in 30 mL dry

toluene under argon. The mixture was refluxed for 12 h. After

removal of the solvent, the residue was purified by silica gel

column chromatography with CH2Cl2/MeOH (35/1, v/v),

yielding the desired product 2 as a red solid. Yield 45.7%,

ESI-MS: m/z 500.3 [M + H]+.

Synthesis of 3

A solution of benzenedicarbonyl dichloride (0.202 g, 1 mmol)

in 20 mL CH2Cl2 was slowly added to a mixture of 2 (0.500 g,

1 mmol) and triethylamine (0.5 mL) in 15 mL CH2Cl2 at

0 1C. After the addition, the mixture was stirred at room

temperature for 4 h. The solvent was removed and the residue

was purified by silica gel column chromatography with

CH2Cl2/MeOH (20/1, v/v) as eluent to afford 3, yield 83.5%.

M.p. 174 1C–176 1C. 1H NMR (400 MHz, CDCl3, d ppm):

8.16–8.20 (m, 3H), 7.93 (d, 2H, J = 8.4 Hz), 7.76 (s, 2H),

7.45–7.50 (m, 5H), 7.10 (d, 2H, J = 7.6 Hz), 6.35–6.41

(m, 8H), 6.25 (d, 4H, J = 8.4 Hz), 4.32 (bs, 4H), 3.30–3.34

(m, 20H), 1.16–1.19 (m, 24H); 13C NMR (100 MHz, CDCl3,

d ppm): 192.8, 166.3, 153.2, 151.4, 149.3, 137.5, 134.3, 132.8,

130.9, 130.1, 128.8, 128.6, 128.4, 125.9, 125.4, 123.3, 108.4,

103.2, 97.9, 65.6, 44.4, 43.3, 41.0, 12.6; ESI-MS: m/z 1130.5

[M + H]+; Anal. calcd for C68H74N8O4S2: C 72.18, H 6.59,

N 9.90, found: C 72.22, H 6.71, N 9.87.

Acknowledgements

This work was financially supported by the National

Science Foundation of China (nos. 20972143, 20972130) and

Program for New Century Excellent Talents in University

(NCET-11-0950).

Notes and references

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