A rhodamine-based colorimetric and fluorescent probe for dual sensing of Cu2+ and Hg2+ ions

Article abstract of DOI:10.1016/j.jphotochem.2015.10.027

A fluorescent sensor 'RhAsP' prepared from rhodamine hydrazine and 2-hydroxy-acetophenone was designed and synthesised. The characterisation of 'RhAsP' was also accomplished by using FT-IR, ¹H NMR, ¹³C NMR, ESI-MS, elemental analysis

Full text of DOI:10.1016/j.jphotochem.2015.10.027

Accepted Manuscript
Title: A rhodamine-based colorimetric and fluorescent probe
2
+
2+
for dual sensing of Cu and Hg ions
Author: Mecit Ozdemir
PII:
S1010-6030(15)00424-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2015.10.027
JPC 10068
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
23-7-2015
26-10-2015
31-10-2015
Please cite this article as: Mecit Ozdemir, A rhodamine-based colorimetric and
fluorescent probe for dual sensing of Cu2+ and Hg2+ ions, Journal of Photochemistry
and Photobiology A: Chemistry http://dx.doi.org/10.1016/j.jphotochem.2015.10.027
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2
+
2+
A rhodamine-based colorimetric and fluorescent probe for dual sensing of Cu and Hg ions
Mecit Ozdemira*
Department of Food Processing, Vocational High School, Kilis 7 Aralik University, Kilis,
TURKEY
*
Corresponding author. Tel.: +90 348 0 348 814 26 66; fax: +90 348 814 26 69.
E-mail address: mecitozdemir@kilis.edu.tr
1

Graphical Abstract
A highly selective and sensitive ‘off-on’ fluorescent sensor RhAsP based on rhodamine
2
+
2+
derivative for the detection of Hg and Cu in aqueous medium
2

Highlights


Synthesis and characterization of ‘RhAsP’.
The spectroscopic studies were carried out for investigating the selectivity of ‘RhAsP’
2
+
toward Hg .


Addition of mercury ion into ethanol-water solution of sensor caused a strong fluorescent
emission at 586 nm.
2
+
The limit of detection was found to be 150 nM for Hg .
3

ABSTRACT
A fluorescent sensor ‘RhAsP’ prepared from rhodamine hydrazine and 2-hydroxy-acetophenone
was designed and synthesised. The characterisation of ‘RhAsP’ was also accomplished by using
1
13
FT-IR, H NMR, C NMR, ESI-MS, elemental analysis, fluorescence and UV/Vis spectroscopy.
For the purpose of investigating optical sensing behaviour of RhAsP’ toward various metal ions
+
+
+
2+
2+
3+
3+
including alkali, alkaline earth and transition metal ions (Na , K , Ag , Ca , Mg , Cr , Fe ,
2
+
2+
2+
2+
2+
2+
2+
2+
3+
2+
Hg , Cu , Pb , Zn , Fe , Co , Ni , Cd , Al and Mn ) in aqueous medium under
physiological pH conditions, fluorescence and absorption spectroscopic techniques were
carefully conducted. Thereby, ‘RhAsP’ showed both colorimetric and fluorometric selective
2
+
recognition of Hg from colorless to purple with a reversible ‘off-on’ fluorescence response. In
addition, a nearly 100-fold increase in emission intensity was determined upon the addition of
2
+
two equivalents Hg ions.
2
+
2+
Keywords: chemosensor, selective, rhodamine, fluorometric, Hg and Cu .
4

1
. Introduction
In the last few decades, heavy transition metal ions (HTMs) have been an important
environmental issue in the global society due to the industrial and agricultural activities that
contaminate aquatic ecosystems with various toxic ion species [1]. Copper and Mercury are two
important transition metal ions. Beside Fe2+ and Zn , the copper ion—an essential trace
element in the human body—is the third most abundant soft transition metal ion and plays
crucial roles in various biological processes, including metalloenzymes, superoxide dismutase,
cytochrome c oxidase and tyrosinase. Once the exceeding level of Cu2+ ions for the cellular
needs, this leads to neurodegenerative disorders, including Alzheimer’s, Parkinson’s disease,
Wilson’s disease, prion disease, amyloidal precipitation and toxicity [2–4]. However, unlike this
situation, the deficiency of copper in the human body can increase the risk of coronary heart
diseases [4].
2+
On the other hand, one of the most poisonous and hazardous contaminations is Mercury that
2
+
is released through natural events or human activities. A very little amount of Hg can affect the
human health with a various aspects. Therefore, a number of severe harmful effects on human
health have been related to mercury-induced toxicity, which leads to many health problems in
the brain, digestive system, kidneys and central nervous system. Thus, for all these reasons, the
2
+
2+
synthesis and development of the highly selective and sensitive chemosensors for Cu and Hg
ions are still desired [5–8].
There are various conventional detection techniques such as atomic absorption spectrometry
AAS), atomic fluorescence spectrometry (AFS), inductively coupled plasma-mass spectrometry
ICP-MS), electrochemical sensing, and high performance liquid chromatography (HPLC).
(
(
Among the well-known and widely used many conventional methods, owing to their low cost,
applicability, simplicity, low detection limit and useful application in environmental, biological,
and industrial processes, the most promising unconventional techniques based on optical
detection methods via colorimetric or fluorescence changes have presently attracted the attention
of many researchers worldwide [9-11]. Therefore, new approaches in the design and
development of synthetic fluorescent receptors that are capable of recognising biologically and
environmentally important ion species, especially HTMs in aqueous media, have received a
widespread interest by chemists, biologists, clinical biochemists and environmentalists recently.
5

[
12–19]. As is well known, rhodamine-based dyes are useful for selective detection of metal ions
leading to strongly fluorescent colorful open ring form [20]. With all these aspects, since their
characteristics such as high quantum yields, good photostability, simplicity, low detection limit,
capability for special recognition and excellent spectroscopic properties, rhodamine derivatives
have commonly been employed as fluorescent sensors for detection of metal cations in water or
organic solvents and labelling them in living cells without causing cell damage [21].
In this work, we developed and described a fluorescent sensor ‘RhAsP’ based on a
rhodamine framework. To the best of our knowledge, rhodamine derivatives with the spirolactam
form are colorless and non-fluorescent, whereas the ring-opened amide form induced by metal
ions causes an appearance of a purple color and strong fluorescence intensity at a comparatively
long wavelength [22-25]. Likewise, the spirocyclic structure of ‘RhAsP’ was non-fluorescent
2
+
and colorless, while the ring-open form of this chemosensor induced by Hg ions was purple
and fluorescent. From this point of view, we speculated that ‘RhAsP’ may be used as a rapid and
2
+
highly selective “off-on” fluorescent probe for Hg detection with significant changes in color
and fluorescence.
2
. Experimental
2
.1. Materials and methods
All of the chemicals and solvents used in this study were the best commercial reagents and
they were used without further purification. Rhodamine hydrazine was synthesised according to
the methods outlined in the previous reports [26–30]. Ethanol (EtOH), 2-hydroxy-acetophenone,
methanol, dichloromethane (DCM), acetic acid (AcOH) and metal salts (nitrate or chloride) were
purchased from Sigma-Aldrich and Merck. The characterisation of the synthesised fluorescent
1
13
sensor ‘RhAsP’ was based on the analysis of FT-IR, H NMR, C NMR, ESI-MS, elemental
analysis, fluorescence spectra and UV/Vis spectra. NMR spectra were recorded on a Bruker
AVANCE III HD 400 using CDCl as the solvent and tetramethylsilane (TMS) as an internal
3
standard. LC-MS/MS analyses were performed on a Varian 325-MS. The absorption spectra
were recorded on a Varian cary 100 bio spectrophotometer at 298 K. Fluorescence spectra were
recorded on a Varian cary eclipse fluorescence spectrophotometer at 298 K. The pH
6

measurements were carried out on a Corning pH meter equipped with a Sigma-Aldrich micro
combination electrode calibrated with a standard buffer solution.
+
+
2+
To prepare the solutions of metal ions, the nitrate or chloride salts such as Na , K , Ca ,
2
+
3+
3+
2+
2+
2+
2+
2+
2+
2+
2+
3+
2+
+
Mg , Cr , Fe , Hg , Cu , Pb , Zn , Fe , Co , Ni , Cd , Al , Mn and Ag were
dissolved in double-distilled water to afford 1 mM aqueous solutions. A 1 mM stock solution of
‘RhAsP’ was prepared in ethanol. Once the stock solution of ‘RhAsP’ was stored for more than
several months under the daylight condition, no change was observed. These solutions were
further diluted to the micro-molar (µM) level for fluorescence and absorption spectrometry
measurements with an EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2).
2
.2. Synthesis of the fluorescent sensor ‘RhAsP’
The synthetic pathway of ‘RhAsP’ was shown in Scheme 1. Rhodamine B hydrazide was
firstly synthesised according to the literature methods by a one-step reaction of rhodamine B
with hydrazine hydrate (80%) in ethanol [31-32]. The fluorescent sensor ‘RhAsP’ was prepared
by following procedure [33]. Briefly, 2-hydroxy-acetophenone (272 mg, 2 mmol) in 20 mL
ethanol with a few drops of acetic acid was slowly added to the stirred ethanolic solution of
rhodamine hydrazide (912 mg, 2 mmol). The mixture was stirred and heated to reflux overnight.
The crude product evaporated under the reduced pressure, and the mixture was purified by silica
gel column chromatography using CH
The yield was 823 mg (72 %).
2
Cl
2
/CH OH (20:1, v/v) as an eluent to obtain ‘RhAsP’.
3
Elemental Analysis (theoretical/actual): % C (75.24/74.12), % H (6.66/6.26), % N (12.51/11.88).
-1
FT-IR (ν/cm ): 2970 (Ar-H); 2901-2932 (-C-H); 1699 (C=O, amide); 1614 (C=N, azomethine);
1
596 (C=N, pyridine); 1548-1413 (C=C); 1306, 1218, 1117 (C-O); 785, 752) (Supplementary
1
Fig. S1). H NMR (CDCl
3
-d, 400 MHz) δ(ppm): 11.79 (s, 1H), 7.95 (d, 1H), 7.48-7.52 (m, 3H),
6
.80-7.20 (m, 4H), 6.29-6.33 (s, 2H), 6.41-6.59 (d, 4H), 3.32 (q, 8H), 2.33 (s, 3H), 1.16 (t, 12H)
1
3
(
Supplementary Fig. S2). C NMR (CDCl
azomethine C=N), 160.3, 153, 151, 149, 133, 132, 130, 129, 128.5, 128.3, 124, 123, 119, 118,
08, 105, 98, 77 (solvent), 44, 18, 12 (Supplementary Fig. S3). LC-MS/MS: The tandem mass
3
-d, 400 MHz) δ(ppm): 173 (amide C=O), 161.8
(
1
spectrum of the ‘RhAsP’ was represented in Supplementary Fig. S4. Fragment at m/z = 575
+
(base peak) was attributed to molecular ion peak [C36
H
38
N
4
O
3
+ H] . The other one at m/z = 597
+
that is appeared in the mass spectra was related to [C36
H
38
N
4
O
3
+ Na] . In addition to those
7

peaks, there are also other fragmentation products of molecular ion such as 497, 326, 179, and
01.
1
2
.3. Determination of complexation ratio (S:M), binding constant (K
detection (LODs)
a
) and limits of
2
+
2+
The stoichiometry of ‘RhAsP’ with Hg and Cu was determined by employing Job’s plot
and mole ratio plot methods. The absorption enhancement observation at 560 nm was plotted
2
+
2+
2+
against the molar value of the [Hg ]/([Hg ] + RhAsP) (Job’s plot) and [Hg ]/RhAsP (mole
2
+
2+
2+
ratio plot) and also [Cu ]/([Cu ] + RhAsP) (Job’s plot) and [Cu ]/RhAsP (mole ratio plot).
The binding constants of the fluorescent sensor ‘RhAsP’ were determined by the Benesi-
Hildebrand equation by using the UV-vis and fluorescence titration data (Figs. 2 and 3). For the
2
+
practical applicability of ‘RhAsP’, limits of detection (LODs) of this chemical probe to Hg and
2
+
Cu ions were calculated using the equation given by Zheng et al. (Figs. 4 and 5) [34].
3
. Result and discussion
The chemosensor ‘RhAsP’ was synthesised according to the literature procedures in ethanol
with a good yield as given in Scheme 1 [26, 33]. Then, this fluorescent sensor was successfully
1
13
characterised by elemental, FT-IR, H NMR, C NMR and LC-MS/MS spectroscopic methods.
Therefore, all spectroscopic studies confirmed the structure of ‘RhAsP’, which is a colorless
powder that is highly stable in ethanol. Furthermore, the geometry of ‘RhAsP’ was optimised by
the density functional theory (DFT) method with the 6-31G (d, p) basis set in the ground state.
Molecular electrostatic potential (MEP) and frontier molecular orbitals (FMOs) of fluorescent
molecule synthesised in this work were investigated by using the theoretical calculations as
shown in Figs. S5 and S6 [16, 35]. To better understand the sensing behaviour of ‘RhAsP’
toward mercury and copper ions in an EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2),
fluorescence and UV/vis spectroscopy experiments were carried out according to the literature
reports [36-38]. The stock solution of ‘RhAsP’ and metal ions were prepared at a concentration
of 1 mM and diluted to the micro-molar (µM) level [39–41]. An EtOH-water (2:1, v:v) buffer
solution (10 mM, HEPES, pH 7.2) of ‘RhAsP’ was used as a testing system to conduct the UV-
2
+
2+
vis and fluorescence spectral studies. As shown in Fig. 1, upon the addition of Hg and Cu
ions that are HTMs to the fluorescent sensor in HEPES buffered solution, apparent changes in
8

color from colorless to purple could be observed, permitting by direct observation with “naked-
eye” detection. However, the addition of other competing metal ions did not lead to changes in
the solution color of ‘RhAsP’ in an EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2)
during the experimental studies.
3
.1. Possible sensing mechanism and Effect of pH
To further investigation of the sensing performance of chemosensor, fluorometric and
2
+
2+
colorimetric titration of Hg and Cu ions were conducted using a solution of ‘RhAsP’ in an
2
+
2+
EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2). Upon the addition of Hg or Cu to the
colorless solution of ‘RhAsP’, the fluorescent and colorimetric characteristics of rhodamine
appeared as shown in Scheme 2, and Fig. 1. This indicated that the oxygen atom of amide group
2
+
on the chemosensor ‘RhAsP’ plays an important role in the course of binding with Hg and
2
+
Cu . Likewise, these changes may also be related to the structural transformation of ‘RhASP’
from spirolactam (non-fluorescent) to ring-opened (fluorescent) form due to its complexation
2
+
2+
behaviour with Hg and Cu ions [42-43]. The optimum pH conditions for the successful
application of the fluorescent sensor ‘RhAsP’ and ‘RhAsP-Hg2+’ complex were also
investigated. For the purpose of this assay, as depicted in Fig. 2, the effects of pH on the
2
+
fluorescence intensity of the sensor were measured in the absence and presence of Hg in an
EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2). As is well known, rhodamine-based
2
+
spirolactam fluorescent sensors generally display pH-dependent response to Hg ion. Similar to
many reported rhodamine spirolactam-based fluorescent chemosensors, it was found that the
2
+
fluorescence intensity of RhAsP-Hg complex decreased with the acidic conditions (pH<6).
Besides, there were no important observations of emission intensity of free ‘RhAsP’ in a wide
2
+
range value of pH from 4 to 11. However, in the presence of Hg (1 equiv.), there was an
obvious difference in fluorescence intensity between pH=6 and pH=11 [26, 34]. Meanwhile, as
depicted in Fig. S7, the effects of pH on the absorbance of sensor ‘RhAsP’ in the absence and
2
+
presence of Cu were also investigated. The experiments were carried out at a pH range from
.0 to 11.0. The free chemosensor in EtOH-water (2:1, v:v) buffer (10 mM, HEPES) did not
4
show characteristic color of rhodamine between pH 7.0 and 11.0. But, there were observed both
color changes and increases in the absorption of free ‘RhAsP’ between pH 4.0 and 7.0. Also, the
2
+
RhAsP-Cu complex solutions exhibited strong absorbance enhancements with apparent color
9

changes from colorless to purple at different pH values ranging from 4.0 to 11.0. Thereby, the
experimental results revealed that the fluorescent chemosensor ‘RhAsP’ may further be used for
2
+
high-selectivity detecting Hg ions under physiological pH conditions.
Figs. 1 and 2
3
.2.
a
K , limits of detection (LODs) and binding ratio (S:M) calculations of ‘RhAsP’ with
2
+
2+
Hg and Cu
2
+
2+
The stoichiometry of RhAsP-Hg and RhAsP-Cu complexes was calculated using the Job’s
plot and mole ratio plot methods as shown in Figs. S8-11[44]. To determine the association
constants, the UV-VIS titrations of different amounts of Hg2+ and Cu in the ethanol-water
solution of the chemosensor were carried out. As shown in Figs. S10 and S11, upon the addition
2+
2
+
2+
of various concentrations of Hg and Cu ions in the range of 0–100 μM caused changes in the
absorption spectrum of ‘RhAsP’. Upon addition of Hg2+ and Cu2+ ions, the absorbance of
chemosensor gradually increased with color changes from colorless to purple [45]. In
consequence, we estimated the binding constants by using the absorption titration data and
employing the Benesi-Hildebrand equation [16]. According to the Job’s plot assay assuming a
2
+
2+
1
:1 binding ratio between ‘RhAsP’ and Cu or Hg ions, the association constants ‘K
chemosensor with these metal ions were determined using Eqs. 1 and 2 as follows, respectively
46–48].
a
’ of the
[
A/F and A
0
/F
0
are the absorbance or fluorescence intensity of the ‘RhAsP’ solution in the
2
+
2+
presence and absence of Hg or Cu ions, respectively; the saturated absorbance or the intensity
2
+
2+
of the sensor in the presence of too much Cu (II) and Hg (II) is Amax/Fmax; [Hg ] and [Cu ] are
the concentrations of mercury and copper ions added to the solution of ‘RhAsP’ (µM). Based on
2
+
2+
the Benesi-Hildebrand equation, calculated association constants ‘K
a
’ for Cu and Hg from
3
-1
4
-1
UV-vis and fluorescence data were 2.2x10 L.mol and 1.3x10 L.mol plotting of 1/(A-A
0
) or
2
+
2+
1
/(F-F ) versus 1/[Cu ] or 1/[Hg ], respectively, exhibiting a linear relationship as shown in
0
2
+
Figs. 3 and 4 [49]. To better understand the selectivity of ‘RhAsP’ toward Hg , protocols for
determination of limits of detection were performed using fluorescence titration data with the
1
0

equation, based on the definition by IUPAC (LOD= 3S
b
/m), where S
b
is the standard deviation of
blank measurements and m represents the slope between fluorescence intensity and mercury
2
+
metal ion concentrations as shown in Fig. 5. So, the detection limit for Hg was found to be 150
2
+
nM. In addition, the LOD of Cu was calculated using the absorption titration data of ‘RhAsP’
2
+
with Cu (LOD=3.37 µM) as shown in Fig. 6. Meanwhile, when the obtained limits of detection
2
+
2+
for Hg and Cu were compared to previous studies, herein, they were quiet acceptable range of
results under the similar conditions [31, 41]. Indeed, the standard for the maximum allowable
2
+
2+
level of Hg in drinking water is only 10 nM, and the permitted limit for Cu in drinking water
is only < 20 µM according to the United States Environmental Protection Agency (EPA) [35,
4
6–47, and 50–51].
Figs. 3, 4, 5, and 6
3
.3. Selectivity studies
To shed some light on the metal-binding properties of ‘RhAsP’, its sensitivity and selectivity
for Hg2+ and Cu2+ ions were investigated by using UV-vis and fluorescence spectroscopy
techniques over various possible interfering metal ions and as shown in Figs. 7 and 8. The free
fluorescent sensor ‘RhAsP’ showed almost no absorption between 450 nm and 600 nm. In fact,
this was related to the closed (spirolactam) forms of fluorescent sensor molecules in the solution.
2+
Upon the addition of Cu to the solution of ‘RhAsP’, a strong absorption band was appeared at
+
+
2+
2+
3+
3+
3+
2+
2+
5
60 nm. When other competing metal ions (Na , K , Ca , Mg , Cr , Fe , Al , Hg , Mn ,
2
+
2+
2+
2+
2+
2+
+
Zn , Pb , Fe , Co , Ni , Cd and Ag ) were added to the solution of ‘RhAsP’, they did not
cause any changes in the absorption spectrum of the chemosensor except there was an absorption
2
+
2+
increase with Hg , which is smaller than with Cu . Moreover, as shown in Scheme 2 and Fig.
2
+
2+
1
, only the Cu and Hg induced a notable color change in the HEPES buffered from colorless
to purple under natural light, which can be assigned to the spirolactam bond (amide group)
cleavage of the chemosensor ‘RhAsP’, while the other metal ions did not proceed any obvious
changes under identical conditions [52–57]. Thereby, the finding of the colorimetric recognition
of Cu2+ supported that ‘RhAsP’ shows a very high sensitivity by important enhancement of
+
+
2+
2+
3+
3+
3+
2+
absorption band. On the other hand, the addition of Na , K , Ca , Mg , Cr , Fe , Al , Mn ,
2
+
2+
2+
2+
2+
2+
2+
+
Zn , Cu , Pb , Fe , Co , Ni , Cd and Ag ions exerted little or no effect upon the emission
of ‘RhAsP’, while remarkable fluorescence enhancement was detected only upon the addition of
1
1

Hg2+ ions by excitation of the chemosensor at 530 nm. However, the colorful solution of
2
+
‘
RhAsP’ induced by Cu did not show any significant fluorescence emission band around 586
9
nm because of fluorescence quenching by the paramagnetic effect of the d electronic system of
2
+
Cu as shown in Fig. 8 [58]. Unlike copper metal ions, ‘RhAsP’ displayed high fluorescence
selectivity toward Hg2+ over other metal ions, except for a very small enhancement of
fluorescence intensity with Cr3+ and Al . For better understanding the sensing property of
3+
2
+
2+
‘
RhAsP’ to Hg , fluorescence titration experiments of the sensor solution with Hg were
gradually carried out. As shown in Fig. S10, the absorption band and fluorescence intensity
2
+
steadily increased with the progressive addition of Hg (in the range of 0-200 µM) to the
‘
RhAsP’ solution (100 µM). From the titration profiles, the binding constants ‘K
a
’ and LODs of
‘RhAsP’ for mercury and copper ions were determined as depicted in Figs. 4 and 5. For practical
applications, reversibility that is the ability to reproduce the free sensors from the complex is a
significant parameter for fluorescent sensors. As depicted in Fig. S12, and Scheme 2, once the
2
+
colorful solution of the fluorescent sensor-Hg complex was finally treated with 0.1 mM Na S,
2
the fluorescence turned off gradually with changes in color from purple to colorless form.
Therefore, these results demonstrated that coordination occurring between ‘RhAsP’ and Hg2+
was reversible by chemical processes.
In addition to the above-mentioned studies, the competitive experiments of mercury over
other various metal ions were investigated by performing the fluorescence response of ‘RhAsP’
2
+
+
+
2+
2+
+
3+
3+
3+
toward Hg in the presence of metal ions such as Na , K , Ca , Mg , Ag , Cr , Fe , Al ,
2
+
2+
2+
2+
2+
2+
2+
2+
Mn , Zn , Cu , Pb , Fe , Co , Ni and Cd as shown in Fig. S13. A reduced selectivity for
Hg2+ showed no changes with the addition of the other metal ions; only Fe (II) and Cu (II)
showed a very small decrease in fluorescence intensity because of the their paramagnetic effects.
Moreover, as shown in Fig. S13, some alkaline, alkaline-earth or transition metal ions used in
2
+
this study did not interfere with the Hg -induced fluorescence increase, showing that ‘RhAsP’
2
+
may also serve as a selective fluorescent probe for Hg detection.
Figs. 1, 7 and 8
4
. Conclusion
In this paper, we report a novel rhodamine hydrazone derivative as a fluorescent “off-on”
2
+
sensor ‘RhAsP’ for Hg in aqueous medium. The experimental results revealed that ‘RhAsP’
1
2

2
+
behaves both as a colorimetric and selective fluorescent sensor for Hg , whereas it displays
colorimetric and absorbance (at 560 nm) responses toward Cu2+ as colorless to purple. In
addition, ‘RhAsP’ showed high fluorescent selectivity toward mercury in the coexistence of
various metal ions as depicted in Fig. S12, representing a nearly 100-fold increase in
fluorescence emission intensity in an EtOH-water buffer under physiological pH conditions.
Meanwhile, the binding mode of the sensor to Hg2+ ion was found to be at a 1:1 ratio
2
+
(
RhAsP/Hg ). Similarly, the LOD was calculated as 150 nM. Consequently, a highly selective
and sensitive fluorescent sensor ‘RhAsP’ may be further performed a promising candidate for
2
+
detecting of Hg species for real time applications in drinking water and human serums.
Acknowledgements
In this study, theoretical calculations using Gaussian 09W was supported by Kilis 7 Aralik
University with BAP project 2010/02/08.
1
3

References
2
+
[
1] M. Saleem, K.H. Lee, Selective fluorescence detection of Cu in aqueous solution and living
cell, J. Lumin. 145 (2014) 843-848.
2] N. Kumari, N. Dey, S. Bhattacharya, Remarkable role of positional isomers in the design of
sensors for the ratiometric detection of copper and mercury ions in water, RSC Adv. 4 (2014)
230.
3] J. Wang, H. Li, L. Long, G. Xiao, D. Xie, Fast responsive fluorescence turn-on sesnsor for
[
4
[
2
+
Cu and its application in live cell imaging, J. Lumin. 132 (2012) 2456-2461.
4] M. Wang, F. Yan, Y. Zou, L. Chen, N. Yang, X. Zhou, Recognition of Cu and Hg in
2
+
2+
[
physiological conditions by a new rhodamine based dual channel fluorescent probe, Sens. and
Actuators B: Chem. 192 (2014) 512-521.
[
5] W. Wang, Y. Zhang, Q. Yang, M. Sun, X. Fei, Y. Song, Y. Zhang, Y. Li, Fluorescent and
2
+
colorimetric magnetic microspheres as nanosensors for Hg in aqueous solution prepared by a
sol-gel grafting reaction and host-guest interaction, Nanoscale 5 (2013) 4958.
[
6] T. Li, Z. Yang, Y. Li, Z. Liu, G. Qi, B. Wang, A novel fluorescein derivative as a
colorimetric chemosensor for detecting copper (II) ion, Dyes Pigm. 88 (2011) 103-108.
7] Y. Zhou, J. Zhang, H. Zhou, Q. Zhang, T. Ma, J. Niu, A new rhodamine B-based “off-on”
[
2
+
fluorescent chemosensor for Cu in aqueous media, J. Lumin. 132 (2012) 1837-1841.
8] D. Guo, Z. Dong, C. Luo, W. Zan, S. Yan, X. Yao, A rhodamine B-based “turn-on”
[
2
+
fluorescent sensor for detecting Cu and sulfur anions in aqueous media, RSC Adv. 4 (2014)
718-5725.
9] Z. Sun, H. Li, D. Guo, Y. Liu, Z. Tian, S. Yan, A novel piperazine-bis(rhodamine-B)-based
5
[
2+
chemosensor for highly sensitive and selective naked-eye detection of Cu and its application as
an INHIBIT logic device, J. Lumin.167 (2015) 156–162.
[
10] F. Wang, S.W. Nam, Z. Guo, S. Park, J. Yoon, A new rhodamine derivative bearing
benzothiazole and thiocarbonyl moieties as a highly selective fluorescent and colorimetric
2
+
chemodosimeter for Hg , Sens. and Actuators B: Chem. 161 (2012) 948-953.
11] J. Ni, Q. Li, B. Li, L. Zhang, A novel fluorescent probe based on rhodamine B derivative for
[
highly selective and sensitive detection of mercury (II) ion in aqueous solution, Sens. and
Actuators B: Chem. 186 (2013) 278-285.
[
12] D.S. Tira, M. Focsan, S. Ulinici, D. Maniu, S. Astilean, Rhodamine B-Coated Gold
Nanoparticles as Effective “Turn-on” Fluorescent Sensors for Detection of Zn II Ions in Water,
Spectrosc Lett. 47 (2014) 153-159.
[
13] R. Balamurugan, C.C. Chien, K. M. Wu, Y.H. Chiu, J.H. Liu, A depropargylation-triggered
fluorescence "turn-on" probe for the detection of Pd2+ based on a bispropargylamine-rhodamine
conjugate, Analyst 138 (2013) 1564.
[
14] T. Hirayama, K. Okuda, H. Nagasawa, A highly selective turn-on fluorescent probe for
iron(II) to visualize labile iron in living cells, Chem. Sci. 4 (2013) 1250.
1
4

[
15] Y. Wei, Z. Aydin, Y. Zhang, Z. Liu, M. Guo, A Turn-on Fluorescent Sensor for Imaging
3
+
Labile Fe in Live Neuronal Cells at Subcellular Resolution, ChemBioChem. 13 (2012)1569 –
573.
16] L. Zhang, J. Fan, X. Peng, X-ray crystallographic and photophysical properties of
1
[
3
+
rhodamine-based chemosensor for Fe , Spectrochim. Acta A 73 (2009) 398-402.
17] K. Dai, B. Xu, J. Chen , A rhodamine-based “off-on” colorimetric and fluorescent
chemosensor for Cu (II) in aqueous and non-aqueous media, J Fluoresc, 24 (2014) 1129-1136.
[
2
+
[
18] Y. Kai, S. Yang, X. Gao, Y. Hu, Colorimetric and “turn-on” fluorescent for Hg based on
rhodamine-3,4-etylenedioxythiophene derivative, Sens. and Actuators B: Chem. 202 (2014) 252-
56.
19] N. Wanichacheva, O. Hanmeng, S. Kraithong, K. Sukrat, Dual optical Hg -selective
2
[
2
+
sensing through FRET system of fluorescein and rhodamine B fluorophores, J. Photochem.
Photobiol. A Chem. 278 (2014) 75-81.
[
20] Z. Sun, D. Guo, H. Li, L. Zhang, B. Yang, S. Yan, Multifunctional Fe3O4@SiO2
nanoparticles for
selective detection and removal of Hg ion in aqueous solution, RSC Adv. 5 (2015) 11000.
2
+
th
[
21] R.P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, 2002, 9 ed.,
Molecular Probes, Eugene, OR.
22] S. Erdemir, O. Kocyigit, S. Malkondu, Detection of Hg2+ ion in aqueous media by new
[
fluorometric and colorimetric sensor based on triazole–rhodamine, J. Photochem. Photobiol. A
Chem. 309 (2015) 15–21.
[
23] J.C. Qin, Z.Y. Yang, G.Q. Wang, A novel ratiometric fluorescent probe for detection of Fe3+
by rhodamine–quinoline conjugate, J. Photochem. Photobiol. A Chem. 310 (2015) 122–127.
2
+
[
24] N. Wanichacheva, O. Hanmeng, S. Kraithong, K. Sukrat, Dual optical Hg -selective
sensing through FRET system of fluorescein and rhodamine B fluorophores, J. Photochem.
Photobiol. A Chem. 278 (2014) 75–81.
2
+
[
25] C.Y. Li, Y. Zhou, Y.F. Li, C.X. Zou, X.F. Kong, a fluorescent chemosensor for Hg based
on a rhodamine derivative in an aqueous solution, Analytical Sciences 29 (2013) 899-903.
26] V. Dujols, F. Ford, A.W. Czarnik, A Long- Wavelength Fluorescent Chemodosimeter
Selective for Cu(II) Ion in Water , Chem Soc. 119 (1997) 7386.
27] L. Xu, Y. Xu, W. Zhu, B. Zeng, C. Yang, B. Wu, X. Qian, Versatile trifunctional
[
[
2
+
2+
chemosensor of rhodamine derivative for Zn , Cu and His/Cys in aqueous solution and living
cell, Org. Biomol. Chem. 9 (2011) 8284-8287.
[
28] Y. Zhao, B. Zheng, J. Du, D. Xiao, L. Yang, A fluorescent “turn-on” probe for the dual-
channel detection of Hg (II) and Mg (II) and its application of imaging in living cells, Talanta 85
(2011) 2194-2201.
[
29] Y. Mao, M. Hong, A. Liu, D. Xu, Highly selective and sensitive detection of Hg (II) from
HgCl
2
by a simple rhodamine-based fluorescent sensor, J. Fluoresc, 2015; DOI 10.1007/s10895-
0
15-1564-7.
1
5

[
30] J. Ni, B. Li, L. Zhang, H. Zhao, H. Jiang, A fluorescence turn-on probe based on rhodamine
2
+
derivative and its functionalized silica material for Hg -selective detection, Sens. and Actuators
B: Chem. 215 (2015) 174-180.
[
31] Y. Kai, S. Yang, X. Gao, Y. Hu, Colorimtric and “turn-on” fluorescent for Hg2+ based on
rhodamine-3,4-ethylenedioxythiophene derivative, Sens. and Actuators B: Chem. 202 (2014)
52–256.
32] J. Zhang, B. Li, L. Zhanga, H. Jiang, An optical sensor for Cu(II) detection with
upconverting luminescent nanoparticles as an excitation source, Chem. Commun. 48 (2012)
860–4862.
33] L. Liu, P. Guo, L. Chai, Q. Shi, B. Xu, J. Yuan, X. Wang, X. Shi, W. Zhang, Fluorescent
and colorimetric detection of pH by a rhodamine-based probe, Sens. and Actuators B: Chem. 194
2
[
4
[
(2014) 498–502.
[
34] A. Zheng, J. Chen, G. Wu, G. Wu, Y.G. Zhang, A novel fluorescent distinguished probe for
Cr(VI) in aqueous solution, Spectrochim. Acta A 74 (2009) 265-270.
35] M. Shellaiah, Y.C. Rajan, P. Balu, A. Murugan , Apyrene based Schiff base probe for
[
2
+
selective fluorescence turn-on detection of Hg ions with live cell application, New J. Chem 39
(2015) 2523-2531.
[
36] F. Yan, D. Cao, M. Wang, N. Yang, Q. Yu, L. Dai, L. Chen, A new Rhodamine-Based
“
Off-On” Fluorescent Chemosensor for Hg (II) Ion and its Application in Imaging Hg (II) in
Living Cells, J Fluoresc 22 (2012) 1249-1256.
37] X. Chen, X. Meng, S. Wang, Y. Cai, Y. Wu, Y. Feng, M. Zhu, Q Guo, A rodamine-based
[
2
+
fluorescent probe for detecting Hg in a fully aqueous environment, Dalton Trans. 42 (2013)
4819-14825.
38] S. Mandal, A. Banerjee, S. Lohar, A. Chattopadhyay, B. Sarkar, S.K. Mukhopadhyay, A.
1
[
2
+
Sahana, D. Das, Selective sensing of Hg using rhodamine-thiophene conjugate: Red light
2
+
emission and visual detection of intracellular Hg at nanomolar level, J. Haz. Mat. 261 (2013)
98-205.
1
[
[
39] M. P. Walkees, Cadmium carcinogenesis, Mutat. Res. 533 (2003) 107-120.
40] M. Waisberg, P. Joseph, B. Hale, D. Beyersmann, Molecular and cellular mechanisms of
cadmium carcinogenesis, Toxicology. 192 (2003) 95-117.
41] C. Wang, D. Zhang, X. Huang, P. Ding, Z. Wang, Y. Zhao, Y. Ye, A ratiometric
[
2
+
fluorescent chemosensor for Hg based on FRET and its application in living cells, Sens. and
Actuators B: Chem. 198 (2014) 33-40.
[
42] S. Tong, L. Zhang, L. Bing, L. Yi, Two rhodamine derived fluorescence turn-on
chemosensors for Hg(II) recognition and sensing: synthesis, characterization and sensing
performance, Sens. and Actuators B: Chem. 203 (2014) 157–164.
[
43] J. Xu, Y. Hou, Q. Ma, X. Wu, S. Feng, J. Zhang, Y. Shen, A highly selective fluorescent
probe for Cu2+ based on rhodamine B derivative Spectrochim. Acta A 124 (2014) 416–422.
44] B. Valeur, S. Berberan, N. Mário, Molecular Fluorescence: Princeples and Applications, 2.
Edition, 2002, Wiley-VCH. Weinheim.
[
1
6

[
45] J.T. Hou, Q.F. Zhang, B.Y. Xu, Q. Liu, J. Zhang, X.Q. Yu, A novel BINOL-based
cyclophane via click chemistry: synthesis and its applications for sensing silver ions,
Tedrahedron Lett., 52 (2011) 4927-4930.
[
46] Y. Sun, S. Fan, D. Zhao, L. Duan, A Fluorescent Turn-On Probe Based on Benzo [E]
Indolium for Cyanide Ion in Water With High Selectivity, J Fluoresc, 23 (2013) 1255-1261.
47] K.C. Tayade, U.A. Kuwar, A.S. Fegade, H. Sharma, N. Singh, U.D. Patil, S.B. Atarde,
Dessign and Synthesis of a Pyridine Based Chemosensor: Highly Selective Fluorescent Probe
[
2
+
For Pb , J Fluoresc 24 (2014) 19-26.
48] R. Tang, K. Lei, K. Chen, H. Zhao, J. Chen, A Rhodamine-Based Off-On Fluorescent
Chemosensor for Selective Sensing Cu(II) in Aqueous Solution, J Fluoresc 21 (2011) 141-148.
49] R. Tang, K. Lei, K. Chen, H. Zhao, A Rhodamine-Based Off-On Fluorescent Chemosensor
for Selectively Sensing Cu(II) in Aqueous Solution, J Fluoresc 21 (2011) 141-148.
[
[
2
+
[
50] Z. Wang, J.H. Lee, Y. Lu, Highly sensitive “turn-on” fluorescent sensor for Hg in
aqueous solution based on structure-switching DNA, Chem Commun 45 (2008) 6005-6007.
51] L. Tang, J. Guo, N. Wang, A new rhodamine B hydrazide hydrazone derivative for
[
colorimetric and fluorescent “off-on” recognition of copper (II) in aqueous media, Bull. Korean
Chem. Soc. 34 (2013) 159.
[
52] H. Sasaki, K. Hanaoka, Y. Urano, T. Terai, T. Nagano, Design and synthesis of a novel
2
+
fluorescence probe for Zn based on the spirolactam ring-opening process of rhodamine
derivatives, Bioorg Med Chem 19 (2011) 1072-1078.
[
53] P. Puangploy, S. Smanmoo, W. Surareungchai, A new rhodamine derivative-based
2
+
chemosensor for highly selective and sensitive determination of Cu , Sens. and Actuators B:
Chem. 193 (2014) 679-686.
[
2
+
2+
54] M. Wang, F. Yan, Y. Zou, L. Chen, N. Yang, X. Zhou, Recognition of Cu and Hg in
physiological conditions by a new rhodamine based dual channel fluorescent probe, Sens. and
Actuators B: Chem. 192 (2014) 512-521.
[
55] J. Hu, Z. Hu, Y. Cui, X. Zhang, H.W. Gao, K. Uvdal, A rhodamine-based fluorescent probe
2
+
for Hg and its application for biological visualization, Sens. and Actuators B: Chem. 203
(2014) 452-458.
[
56] S.K. Kempahanumakkagari, R. Thippeswamy, P. Malingappa, A new rhodamine B based
2
+
fluorometric chemodosimeter for Cu ion in aqueous and cellular media, J. Lumin. 146 (2014)
1-17.
57] M. Saleem, K.H. Lee, Selective fluorescence detection of Cu2+ in aqueous solution and
living cells, J. Lumin. 145 (2014) 843-848.
58] J.S. Kim, D.T. Quang, Calixarene-derived fluorescent probes, Chem. Rev. 107 (2007) 3780-
799.
1
[
[
3
1
7

FIGURES
Fig. 1 Colorimetric changes of chemical sensor ‘RhAsP’ (50 µM) in absence and presence of
+
+
2+
2+
3+
+
2+
2+
2+
2+
2+
2+
2+
3+
3+
2+
2+
Na , K , Mg , Ca , Al , Ag , Zn , Hg , Fe , Cd , Mn , Ni , Cu , Fe , Cr , Pb , Co
100 µM) in EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2) under natural light (a) and
UV lamp at 365 nm (b).
(
1
8

1
200
000
RhAsP + Hg2+
RhAsP
1
800
600
400
200
0
4
5
6
7
8
9
10
11
12
pH
Fig. 2 Fluorescent intensity (586 nm) of ‘RhAsP’ (100 µM) in the absence and presence of Hg2+
ions (200 µM) in EtOH-water (2:1, v:v) buffer (10 mM, HEPES, λex= 530 nm) with different pH
conditions. The pH values of solution were adjusted by using an appropriate volume of 0.1 M
HCl or NaOH stock solution.
1
9

2+
RhAsP-Cu
2.0
1.6
1.2
0.8
0.4
y = 9.20998*x + 0.17838
2
R = 0.99491
0.00
0.05
0.10
0.15
0.20
1
/ [Cu2+] (M-1)
2
+
Fig. 3 Benesi-Hildebrand plot from absorbance (at 560 nm) of 1/ΔA versus 1/[Cu ] to determine
2
+
the binding constant (K
a
) of Cu with ‘RhAsP’ in EtOH-water (2:1, v:v) buffer (10 mM,
HEPES, pH 7.2, absorption method).
2
0

0.30
0.25
0.20
0.15
0.10
0.05
0.00
RhAsP - Hg2+
y = 1.53209*x - 0.02872
2
R = 0.99622
0.00
0.05
0.10
0.15
0.20
2+
-1
1
/[Hg ] (M )
2
+
Fig. 4 Benesi-Hildebrand Plot from fluorescence intensity (at 586 nm) of 1/ΔF versus 1/[Hg ] to
determine the binding constant (K ) of ‘RhAsP’ (50 µM, λex= 530 nm, fluorescence method).
a
2
1

RhAsP-Hg2+
250
200
150
100
y = 5.7225*x - 98.3914
2
R = 0.95712
50
0
0
10
20
30
40
50
60
[
Hg2+] ()
Fig. 5 Fluorescence intensity of ‘RhAsP’ (50 µM, at 586 nm) upon continuous addition of
2
+
increasing amount of Hg (0-55 µM) in EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2,
ex = 530 nm).
λ
2
2

RhAsP-Cu2+
1
0
9
8
7
6
5
4
3
2
y = 0.17865*x + 2.01984
2
R = 0.97864
0
10
20
30
40
50
60
[
Cu2+] (M)
Fig. 6 Absorption intensity of ‘RhAsP’ (50 µM, at 560 nm) upon continuous addition of
2+
increasing concentration of Cu (0-55 µM) in EtOH-water (2:1, v:v) buffer (10 mM, HEPES,
pH 7.2).
2
3

Fig. 7 UV-vis absorption spectra of chemical sensor ‘RhAsP’ (50 µM) in the presence of Cu2+
and Hg2+ with other metal ions (100 µM) (a), and the bar graph represents the changes of
absorbance value of ‘RhAsP’ upon addition of various competing metal ions (100 µM) (b) in
EtOH-water (2:1, v:v) buffer (10 mM, HEPES, pH 7.2).
2
4

Fig. 8 Fluorescence response of RhAsP (100 µM) to various metal ions (200 µM) (a), bar graph
represents the change of relative emission intensity of RhAsP upon addition of various
2
+
competing metal ions (b), and inset: color changes of RhAsP-Hg complex (100 µM) under
natural light and portable UV lamp at 365 nm in EtOH-water (2:1, v:v) buffer (10 mM, HEPES,
pH 7.2, λ ex = 530 nm).
2
5

Scheme 1. The synthesis route of fluorescent sensor ‘RhAsP’
2
6

Scheme 2. Binding mode of ‘RhAsP’ (100 µM) with Hg2+ (2 equiv.), “off-on” situation:
2
+
photograph of ‘RhAsP’ in absence and presence of Hg in EtOH-water (2:1, v:v) buffer (10
mM, HEPES, pH 7.2).
2
7