An Application of a Schiff-Base Type Reaction in the Synthesis of a New Rhodamine-Based Hg(II)-Sensing Agent

Article abstract of DOI:10.1007/s10895-019-02462-5

A facile synthesis procedure, whereby 9-Anthraldehyde (AA) is coupled to aminated rhodamine (AR) via a Schiff base-type reaction, is reported. The applicability and performance of the obtained material (AA-AR) as a sensing agent was studied towards 16 metal cations (i.e. Li⁺, Na⁺, Ag⁺, Ca²⁺, Ba²⁺, Co²⁺, Cs⁺, Cu²⁺, Mg²⁺, Hg²⁺, Mn²⁺, Pb²⁺, Ni²⁺, Sr²⁺, Zn²⁺, Al³⁺). Among the studied metals, an extraordinary selectivity was observed for Hg²⁺, and the observed selectivity was found not to be influenced by the presence of other cations and some common anions (i.e. Br^?, Cl^?, I^?, HPO₄ ^2?, H₂PO₄ ^?, NO₃ ^?, NO₂ ^?, ClO₄ ^?, AcO^?, HSO₄ ^?, SO₄ ^2?, Cr₂O₇ ^?, CO₃ ^2?, OH^? and HCO₃ ^?). The material, AA-AR, exhibited such a high selectivity and sensitivity towards Hg²⁺ that it could be detected even by naked eyes. The Hg²⁺-sensing property of AA-AR was found not to be limited to colorimetric detections so that a high fluorescent nature of the compound was also observed upon binding Hg²⁺ ion. The detection limit, which is correspondent to fluorescence emission intensity, was found as 0.87 μM. The underlying mechanism of sensing property was studied by using some spectroscopic techniques such as FT-IR, ¹H-NMR, ¹³C-NMR, and UV-Vis. (Job-plot). In the final course of the experiments, the performance of AA-AR in cell-imaging was also studied, and even trace amounts of Hg²⁺ in living cells could be detected by the studied probe. Thus, the applicability of a new synthesis approach in producing a highly efficient new fluorescence sensor for the detection of Hg²⁺ ions is discussed in detail.

Full text of DOI:10.1007/s10895-019-02462-5

Journal of Fluorescence
https://doi.org/10.1007/s10895-019-02462-5
ORIGINAL ARTICLE
An Application of a Schiff-Base Type Reaction in the Synthesis
of a New Rhodamine-Based Hg(II)-Sensing Agent
1
1
1
2
Fulya Cicekbilek & Bahar Yilmaz & Mevlut Bayrakci & Orhan Gezici
Received: 8 July 2019 /Accepted: 5 November 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A facile synthesis procedure, whereby 9-Anthraldehyde (AA) is coupled to aminated rhodamine (AR) via a Schiff base-type
reaction, is reported. The applicability and performance of the obtained material (AA-AR) as a sensing agent was studied towards
+
+
+
2+
2+
2+
+
2+
2+
2+
2+
2+
2+
2+
2+
3+
1
6 metal cations (i.e. Li , Na , Ag , Ca , Ba , Co , Cs , Cu , Mg , Hg , Mn , Pb , Ni , Sr , Zn , Al ). Among the
2
+
studied metals, an extraordinary selectivity was observed for Hg , and the observed selectivity was found not to be influenced by
the presence of other cations and some common anions (i.e. Br , Cl , I , HPO , H PO , NO , NO , ClO , AcO , HSO ,
SO4 , Cr O , CO , OH and HCO ). The material, AA-AR, exhibited such a high selectivity and sensitivity towards Hg
that it could be detected even by naked eyes. The Hg -sensing property of AA-AR was found not to be limited to colorimetric
−
−
−
2−
−
−
−
−
−
−
4
2
4
3
2
4
4
2−
−
2−
−
−
2+
2
7
3
3
2
+
2
+
detections so that a high fluorescent nature of the compound was also observed upon binding Hg ion. The detection limit, which
is correspondent to fluorescence emission intensity, was found as 0.87 μM. The underlying mechanism of sensing property was
1
13
studied by using some spectroscopic techniques such as FT-IR, H-NMR, C-NMR, and UV-Vis. (Job-plot). In the final course
2
+
of the experiments, the performance of AA-AR in cell-imaging was also studied, and even trace amounts of Hg in living cells
could be detected by the studied probe. Thus, the applicability of a new synthesis approach in producing a highly efficient new
2
+
fluorescence sensor for the detection of Hg ions is discussed in detail.
.
.
.
.
.
Keywords Anthracene Cell imaging Fluorescence sensor Mercury Rhodamine Schiff base
Introduction
The sensing agent (i.e. fluorescence sensor) is a chemical
that is prepared by combining “a receptor” with “a
fluorophore” by chemical means, and it is, actually, the key
factor that determines the overall success of a fluorometric
sensing technique. For this reason, nowadays, greater empha-
sis is given to the development of new types of sensing agents
exposing high selectivity and sensitivity toward the chemical
species under consideration. With this regard, intelligent de-
sign and synthesis of a sensing agent is, admittedly, an art of
science that requires a collective knowledge of chemistry and
spectroscopy. In the past decades, many different fluorescence
sensors were developed due to the potential of this technique
in different fields ranging from environmental sciences to
medicine [4–6].
Detection of heavy metal ions in various environments is
one of the most widely studied fields where the fluorescence
sensing techniques applied successfully. With this respect,
simplicity, high selectivity, low operation costs, and
(sometimes) the possibility of naked-eye detection are some
advantages of fluorescence sensing techniques over much
more sophisticated instrumental techniques. Among the heavy
Fluorescence sensing techniques have received great interest
from different disciplines over the past decades. Together with
the selectivity and sensitivity achieved with these techniques,
they offer easy-to-use, inexpensive, fast, flexible, and innova-
tive analytical perspectives [1–3]. Hence, the fluorescence
sensing techniques comprise a series of detection manners that
are, today, indispensable for analytical chemists when fast,
selective and sensitive detection of a chemical or a
biologically-important species is necessary.
*
*
Mevlut Bayrakci
mevlutbayrakci@gmail.com
Orhan Gezici
ogezici@gmail.com; ogezici@ohu.edu.tr
1
2
Faculty of Engineering, Department of Bioengineering,
Karamanoglu Mehmetbey University, Karaman, Turkey
Faculty of Science and Arts, Chemistry Department, Nigde Ömer
Halisdemir University, 51240 Nigde, Turkey

J Fluoresc
metals, mercury is a highly toxic one since it can readily pen-
etrate through the cell membranes because of its solubility in
the lipid bilayer [7, 8]. Thus, mercury ions are recognized as a
primary source of health problems for living things even at
trace levels [9, 10]. For this reason, selective, sensitive, and
rapid detection of mercury ions in various media is deemed
important. Several analytical techniques such as voltammetry,
atomic-absorption spectroscopy (AAS), inductively coupled
plasma emission spectroscopy (ICP-AES), and potentiometric
and spectrophotometric sensors have been utilized to detect
trace levels of mercury ions [11–15]. Despite their widespread
use, some of the mentioned techniques are generally labor-
intensive, and expensive when compared to fluorescence
sensing techniques. Moreover, their selectivity is usually less
than that achieved with the fluorometric techniques [16].
Rhodamine-based sensors have been widely used in
sensing applications of metal ions. This is, basically, due
to high quantum yields and high molar extinction coeffi-
cients achieved with rhodamine-based agents. Good
photostability, and relatively long absorption and emission
wavelengths are some other advantages of this class of
sensors [17]. Because of these properties, rhodamine-
based sensors exhibit strong color shifts upon interaction
with chemical species which sometimes provide “naked-
eye” detection of ions. As it is seen in the literature, the
applicability of rhodamine-based agents as fluorescence
sensors for the determination of mercury ions is an exten-
sively studied topic [5, 6, 17]. It should, however, be
noticed that only a limited number of the studied sensors
have exhibited high sensitivity and selectivity toward
mercury ions. In order to increase the efficiency and the
stability of rhodamine-based sensors, some researchers
have studied the incorporation of sulfur-containing groups
into the rhodamine framework [18–20]. However, the
presence of sulfur in the prepared sensors can constitute
serious problems for living things, and therefore, alterna-
tive –safer– strategies are necessary to prepare rhodamine-
based sensors.
Materials and Methods
Chemicals and Instruments
Rhodamine B, 9-Anthracenecarboxaldehyde, and the studied
metal salts were of analytical grade and used as received
(Sigma-Aldrich). Nitrate salts of some common cations (i.e.
+
+
+
2+
2+
2+
+
2+
2+
2+
Li , Na , Ag , Ca , Ba , Co , Cs , Cu , Mg , Hg ,
2
+
2+
2+
2+
2+
2+
Mn , Pb , Ni , Sr , Zn and Hg ), and tetra butyl am-
−
−
−
monium salts of some common anions (i.e. Br , Cl , I ,
2
−
−
−
−
−
−
−
2
HPO , H PO , NO , NO , ClO , AcO , HSO , SO
4
4
2
4
3
2
4
−
4
−
−
2−
−
, Cr O , CO , OH and HCO ) were used to prepare
2
7
3
3
synthetic test solutions in DMF (Merck).
1
13
H-NMR and C-NMR spectra were generated by an
Agilent™ Premium Compact spectrometer operating at
600 MHz. A Bruker™ Vertex FTIR spectrophotometer with
an ATR compartment was used for recording FTIR spectra.
UV-Vis. absorption spectra were collected on a Shimadzu™
UV-1800 instrument, and the fluorescence measurements
were conducted on a Hitachi™ F-7100 instrument.
Fresh ultrapure water produced on a Millipore™ Milli-Q
Plus water purification system was used throughout the study.
Synthesis and Characterization of the Sensing Agent
(AA-AR)
Aminated-rhodamine (AR) was synthesized according to a
known procedure [22]. A 75 mM solution of AR was prepared
in ethanol, and 1.0 eq of 9-Anthraldehyde (AA) was added to
20.0 mL of this solution while stirring the mixture, continu-
ously. The final mixture was stirred overnight under reflux.
Afterward, the solvent was evaporated under vacuum, and the
solid residue was, subsequently, washed with 1.0 M HCl,
brine and water. The crude product (AA-AR) was crystallized
from hot methanol. The product, which is a dark-red solid,
was obtained with a 56% yield, and the molecular structure
was confirmed with FTIR, NMR, and elemental analysis tech-
niques. A summary of the synthesis procedure is given in
Scheme 1.
Schiff-base type reactions offer easy, mild and some-
times one-step manners in the synthesis of functional ma-
terials. Therefore, a Schiff-base type reaction might be a
Absorption and Fluorescence Measurements
2
+
method of choice when preparing Hg -selective rhoda-
mine-derivatives. Except few attempts [21], the applica-
bility of this approach is seen to be lacking in the litera-
ture whereas it is promising to build highly selective, sen-
Before spectrophotometric measurements, the stock solutions
which comprise (i) a 1.0 mM AA-AR solution, (ii) 1.0 mM
solutions of metal cations, and (iii) 1.0 mM solutions of anions
were prepared (in DMF). Test solutions were prepared by the
addition of 20 eq cation or anion to 0.010 mM AA-AR solu-
tion, and the final volume was fixed at 2.0 mL. The absorption
spectra of AA-AR with and without studied ions were record-
ed in the range of 200–600 nm using a UV-Vis. spectropho-
tometer. The emission spectra were recorded at room temper-
ature and the instrument parameters were set as follows:
2
+
sitive and biocompatible rhodamine-based Hg sensors.
Thus, in the present study, we focus on the applicability
of a straightforward Schiff-base-type condensation reac-
tion between aminated rhodamine (AR) and antraldehyde
(
AA) to prepare a rhodamine-anthracene chemosensor for
the first time. Both characterization of the prepared mate-
rial and its sensor properties have been critically drawn
throughout the work.
Excitation wavelength (λ : 520 nm; scan speed: 1200 nm/
ex

J Fluoresc
Scheme 1 A schematic view for the synthesis of AA-AR probe
1
min; PMT voltage: 400 V; Emission wavelength scan range:
&
&
&
&
&
2964 cm− (Aromatic C–H stretching);
−1
5
40–750 nm; and slit width: 10 nm (both for excitation and
2900–2925 cm (Aliphatic C–H stretching);
−1
emission). During the fluorescence intensity measurements,
1683 cm (C=O stretching vibration for amides);
−1
the excitation (λ ) and the emission (λ ) wavelengths were
1631 cm (C=N, for azomethine moiety);
ex
em
−1
set at 520 and 592 nm, respectively.
1548–1413 cm (C=C vibrations).
Cell Imaging
1
In order to have more details about the structure, H-NMR
1
3
and C-NMR analyses were also conducted, and the results
are listed below (Fig. 2):
The living cells of MCF7 and MIA PaCa-2 were supplied
from ATCC (American Type Culture Collection, Rockville,
2
+
MD, USA). The cells were incubated in a Hg solution of
.010 mM in the culture medium at 37 °C for 1 h. After
washing the cells with phosphate-buffered saline (PBS),
.020 mM AA-AR was added to the medium just before
1
&
H NMR (600 MHz CDCl ): δ 8.31 (s, 1H, CH=N), 8.06
s, 1H, Ar-H), 8.00–7.99 (d, J = 8.6 Hz, 2H, Ar-H), 7.88–
.86 (d, J = 8.2 Hz, 2H, Ar-H), 7.53–7.50 (m, 3H, Ar-H),
.36–7.30 (m, 2H, Ar-H), 7.29–7.25 (m, 2H, Ar-H), 7.21–
.20 (d, 1H, J = 8.4 Hz, Ar-H), 6.77–6.75 (d, 2H,
3
0
(
7
7
7
0
cell-imaging. Bright-field and fluorescent images were taken
by using a Leica™ DM3000 fluorescence microscopy.
J = 8.7 Hz, Ar-H), 6.47 (s, 2H, Ar-H), 6.36–6.35 (d, 2H,
J = 8.8 Hz, Ar-H), 3.34–3.33 (m, 8H, CH -CH ), 1.15–
2
3
1
.14 (m, 12H, CH -CH ).
2 3
1
3
Results and Discussion
&
C NMR (CDCl ): δ 165.12, 152.97, 152.22, 149.02,
3
147.08, 133.45, 131.15, 129.92, 128.76, 128.38, 127.92,
Characterization of AA-AR
127.69, 126.18, 125.53, 125.01, 123.81, 123.53, 108.26,
05.95, 98.29, 65.82, 44.35, 12.64.
1
The studied approach, which is based on a Schiff-base-type
reaction, yielded a dark-red solid, i.e. AA-AR, with a 56%
yield. The molecular structure of the product was studied by
1
various techniques like elemental analysis, FTIR, H-NMR
1
3
and C-NMR spectroscopy analyses.
Absorption and Emission Behavior of AA-AR
The results of elemental analysis perfectly fitted to the the-
oretical atomic percentages we expected for AA-AR, and thus
confirming the molecular formula C H O N :
2
+
The Impact of Hg
4
3 40 2 4
2
+
Since developing a new sensing agent for Hg consti-
tutes the objective of the present work, after synthesis
and characterization of AA-AR, we directly focused on
&
&
Theoretical atomic percentages ➔ C: 80.10%; H: 6.25%;
N: 8.69%.
Experimental atomic percentages ➔ C: 80.13%; H:
2
+
the effect of Hg on absorption and emission spectra of
AA-AR, as spectral changes could guide us to have some
intuitions about the potential of the new material as a
sensor. For this purpose, a solution of AA-AR
(0.010 mM; in DMF) was spiked with different amounts
6
.29%; N: 8.62%.
Further information about the molecular structure was
gathered from FTIR spectroscopy analysis. The following ba-
sic vibration bands, which confirm the molecular structure,
were seen in the FTIR spectrum of AA-AR (Fig. 1):
2
+
of Hg ranging from 0.0 to 20.0 eq, and both absorption
and emission spectra were recorded (Figs. 3 and 4).

J Fluoresc
2
+
2+
Fig. 1 FTIR spectra for AA-AR (i.e. 0.0 eq Hg ) and AA-AR-Hg (0.5 eq and 1.0 eq)
As seen in Fig. 3, the absorption spectrum of AA-AR
comprises two broad absorption bands at around 315 and
The Impact of some Common Cations and Anions
2
+
4
00 nm. Intra-ligand π − π* charge transfer electronic
Single-solute experiments performed with Hg revealed a
nice sensor property toward this highly toxic ion. However,
without analyzing the effect of some common cations and
anions, this observed sensor property would remain as un-
proven and most of the discussions would be speculative.
With this sense, the spectral pattern of ion-interacted AA-
transitions resulting from anthracene moiety (coupled to
rhodamine molecule) was thought to be the main source
of these bands. Though very weak, a band centered at
around 560 nm was also observed in the spectrum of
AA-AR. Surprisingly, the intensity of this band was ob-
served to be increased immediately after interacting AA-
+
AR was studied with respect to 16 metal cations (i.e. Li ,
2
+
2+
+
+
2+
2+
2+
+
2+
2+
2+
2+
AR molecule with Hg ion, and higher Hg concentra-
tions resulted in greater intensities at this wavelength.
Despite such an obvious change in the absorption inten-
Na , Ag , Ca , Ba , Co , Cs , Cu , Mg , Hg , Mn ,
2
+
2+
2+
2+
3+
−
Pb , Ni , Sr , Zn , Al ) and 15 common anions (i.e. Br ,
−
−
2−
−
−
−
−
−
−
Cl , I , HPO , H PO , NO , NO , ClO , AcO , HSO ,
4
2
4
3
2
4
4
2
+
2−
−
2−
−
−
sity at 560 nm, the addition of Hg ion has no significant
impact on the intensities of the bands at 315 and 400 nm
as is seen in Fig. 3. Thus, AA-AR was found to be ab-
sorbing in the UV and visible regions, and the intensity of
the absorption at 560 nm was understood to be affected by
SO4 , Cr O , CO , OH and HCO ).
2 7 3 3
2
+
Among the studied cations, Hg was the only one that had
a considerably high impact on the absorption spectra of AA-
AR. As it is discussed above, the greatest change in the spec-
trum of AA-AR was recorded at 560 nm after its interaction
2
+
2+
the concentration of Hg .
with Hg . Hence, in the case of other studied cations and
When we come to the emission spectra recorded for AA-
AR, up to 37-fold increment in fluorescence intensity could be
anions, no considerable change was observed in the spectrum
of AA-AR regardless of the type of ion. Note that some weak
hyperchromic and/or hypochromic changes centered at
around 315 nm and 400 nm could be observed with some ions
(Figures not shown).
2
+
achieved when the probe interacted with Hg ion (Fig. 4).
This revealed a high fluorescence character of the AA-AR-
2
+
Hg complex at 592 nm when it is excited at 520 nm. All
these observations implied the potential of AA-AR as a sens-
As it is well known, absorption and emission spectros-
copies are complementary techniques in evaluating the sensor
property of a probe. It should, however, be noticed that the
fluorescence emission spectroscopy offers much better selec-
tivity as well as sensitivity, and thus becoming an indispens-
able technique in sensor applications. In this regard, the fluo-
rescence emission spectra for ion-interacted AA-AR were also
analyzed, and the spectra recorded for single-solute samples
2
+
ing agent for Hg . However, most of the discussions would
remain as speculative, without studying the selectivity of AA-
2
+
AR toward Hg . Thus, after studying the general absorption
and emission behavior of AA-AR probe with respect to the
2
+
spectral changes after its interaction with Hg ion, in the
second part of the study, we, basically, focused on the influ-
ence of various metal cations and some common anions on the
absorption and the emission behavior of the probe.
2
+
were compared with that of AA-AR-Hg (Fig. 5).

J Fluoresc
1
13
2+
Fig. 2 H-NMR and C-NMR spectra for AA-AR-Hg (a and c) and AA-AR (b and d)
2
+
The fluorescence intensity of AA-AR-Hg was set at 1.0
and the intensities of other species were normalized accord-
ingly; Concentration of metal ion: 20 eq; Concentration of
AA-AR: 10 μM; Excitation and emission wavelengths: λEx/
λEm = 520 nm/592 nm.
As it is seen in the emission spectrum of AA-AR and the
studied metal complexes of AA-AR (Fig. 4), 592 nm is a
critical wavelength at which AA-AR emits light which is
2
+
mostly specific to Hg ion when excited at 520 nm. At the
same excitation and emission wavelengths, most of the
studied anions and cations did not induce a significant
2
+
emission when compared to what Hg ion did. Among
2
+
3+
the studied metal cations, Cu and Al exhibited some-
how emissions when interacted with the probe AA-AR.
However, the fluorescence emission intensity observed
2
+
Fig. 3 UV–Vis. absorption spectra recorded for AA-AR at different Hg
2
+
2+
with Hg was approximately 4–8 times greater than that
concentrations Hg concentration: 0.0–20.0 eq; AA-AR concentration:
1
0 μM
we observed with these cations. Despite a somehow

J Fluoresc
Fig. 4 Emission spectra recorded
2
+
for AA-AR at different Hg
2+
concentrations Hg concentra-
tion: 0.0–18.0 eq; Concentration
of AA-AR: 10 μM; λex/ λem
20 nm/592 nm
=
5
−
−
quenching effect of some anions (e.g. CN and HSO ) on
The Impact of Coexisting Ions
4
the emission of AA-AR, very similar results were obtained
from the experiments conducted with anions. Hence, it was
2
+
As it is discussed above, the Hg -sensing feature of AA-
AR has been proven through single-solute experiments.
However, the selectivity of a probe cannot be thoroughly
evaluated on the basis of the results of single-solute ex-
periments. This is true because the selectivity of a probe
is reflected by its capability to discern the analyte in a
mixture despite the presence of possible interferences that
may come from matrix constituents. For this purpose, an
2
+
concluded that Hg had an “overwhelming” fluorescence
emission intensity among the studied ions, and the mole-
cule was found to be fluorescing only in the presence of
2
+
Hg . This is a typical behavior observed with “on-off”
sensing agents. Besides, the “naked-eye” detection was on-
2
+
ly achieved with Hg ion, indicating a great selectivity
toward this cation. Finally, owing to the observed efficient
2
+
2+
2+
“
turn-on rate”, Hg ion could be quickly detected under
AA-AR-Hg solution, which contains 20 eq Hg , was
prepared and this solution was spiked with 20 eq of
both (i) day-light and (ii) UV-light as is seen in Fig. 6.
Fig. 5 Normalized fluorescence intensities for AA-AR and metal-interacted AA-AR

J Fluoresc
−
4 2 4 3
I , (21) AA-AR + HPO PO , (23) AA-AR + NO
2−, (22) AA-AR + H
−
−,
Fig. 6 The response of AA-AR to different ions under day-light (a and b)
+
+
−
−
−
and UV-light (c and d) # Test tubes: (1) AA-AR + Li , (2) AA-AR + Na ,
(24) AA-AR + NO
AA-AR + HSO
AA-AR + CO
2
, (25) AA-AR + ClO
4
, (26) AA-AR + AcO , (27)
, (29) AA-AR + Cr
, (31) AA-AR + OH and (32) AA-AR + HCO
+
2+
2+
−
2−
−
(
(
3) AA-AR + Ag , (4) AA-AR + Ca , (5) AA-AR + Ba , (6) AA-AR,
4
, (28) AA-AR + SO
4
2
O
7
, (30)
2
+
+
2+
2−
−
−
7) AA-AR + Co , (8) AA-AR + Cs , (9) AA-AR + Hg , (10) AA-
3
3
.
2+
2+
2+
AR + Cu , (11) AA-AR + Mg , (12) AA-AR + Mn , (13) AA-AR +
Pb , (14) AA-AR + Ni , (15) AA-AR + Sr , (16) AA-AR + Zn , (17)
AA-AR + Al , (18) AA-AR + Br , (19) AA-AR + Cl , (20) AA-AR +
Concentration of ions: 20 eq; Concentration of AA-AR: 10 μM; λEx
/
2+
2+
2+
2+
λEm = 520 nm/592 nm
3+
−
−
different cations and/or anions. Fluorescence intensities of
Possible Interaction Mechanism
Spectroscopic results given in previous sections supported the
the final mixtures were measured at 520 nm (λ )/592 nm
Ex
(
λEm) and graphed against the type of mixtures (Figs. 7
2
+
and 8). As is seen in Fig. 7, almost all the studied metal
cations did not have a significant effect on the emission
fast interaction capability of AA-AR with Hg ion.
2
+
According to the results, Hg binding to AA-AR was evalu-
ated as a process that is basically governed by a ring-opening
reaction of spirolactam moiety in AA-AR. The process was
2
+
signal of AA-AR-Hg complex except some metal cat-
3
+
2+
+
ions (i.e. Al , Cu , and Li ). As for the studied anions
−
−
−
−
2+
(
Fig. 8), some of them (i.e. NO , CN , Br , H PO ,
found to be resulting in a net color change. Hg binding to
2
2
4
−
−
SCN , and I ) were found to have greater quenching ef-
fects on the fluorescence intensity of the complex AA-
AA-AR was supported by the recorded UV-Vis. spectra (Fig.
3) so that the absorption band at around 560 nm was observed
2
+
2+
AR-Hg . Nevertheless, the synthesized probe, i.e. AA-
AR, was found to exhibit a nice Hg -sensing property
among many different cations and anions.
only in the case of AA-AR-Hg . The absorption intensity at
2
+
this wavelength was found to be increasing with increasing
2
+
Hg concentration from 0 to 20 eq, and further increments in
Fig. 7 The effect of interfering
metal cations on the fluorescence
2
+.
intensity of AA-AR-Hg
Concentration of interfering
2+
cation and Hg : 20 eq;
Concentration of AA-AR:
1
5
0 μM; λEx/ λEm = 520 nm/
92 nm

J Fluoresc
Fig. 8 The effect of interfering
anions on the fluorescence
2
+
intensity of AA-AR-Hg .
Concentration of interfering anion
2+
and Hg : 20 eq; Concentration of
AA-AR: 10 μM; λEx/ λEm
20 nm/592 nm
=
5
5
−1
concentration did not cause further changes in the spectrum.
Likewise, the absorption intensities at 315 and 400 nm tended
to increase gradually, indicating a possible role of intra-ligand
π − π* transitions [23]. Very similar results were observed
with the fluorescence emission spectrum of AA-AR (Fig. 4)
so that the band at 592 nm was arisen in the spectrum only
calculated as 2.4 × 10 M . Such a high binding constant
might indicate the presence of a high affinity between AA-
2
+
AR and Hg . To have further information about the mecha-
2
+
nism of Hg binding, FTIR and NMR spectra were analyzed.
FTIR analyses (Fig. 1) confirmed the results of UV-Vis.
spectroscopy analyses. For example, a strong band appeared
at around 1631 cm in the spectrum of AA-AR shifted to a
lower frequency at 1608 cm when AA-AR has interacted
2
+
−1
after interacting AA-AR with Hg ion. The fluorescence in-
tensity at this wavelength was found to be increased gradually
−1
2
+
2
when the AA-AR solution was titrated with Hg . The obtain-
with Hg . This band is attributed to the azomethine group, and
2
+
ed data were used to calculate the detection limit for Hg and
found as 0.87 μM for the linear signal to the concentration
range. This value is lower than (or comparable to) those ob-
the spectral shift indicates the possible role of imine function-
ality in the mercury complexation process. Furthermore, a
band related to the carbonyl group in the FTIR spectrum of
2
+
−1
tained with some Hg -selective chemosensors [23].
Moreover, the detection limit achieved with AA-AR for
AA-AR (at around 1683 cm ) was disappeared when it was
2
+
interacted with Hg , revealing the contribution of the carbon-
yl group in complexation. Another indication about the role of
a possible spirolactam ring-opening reaction was a new band
arisen at 1585 cm in the spectrum of AA-AR after binding
of Hg , which is consistent with the previous findings in the
literature [28].
Much more details about the complexation mechanism
were obtained from the NMR titration experiments performed
with an AA-AR solution in DMSO-d . For this purpose,
1.0 eq Hg was added to this solution, and H-NMR and
C-NMR analyses were conducted for the complex AA-
AR-Hg . As is seen in Fig. 2, the signal ascribed to imine
proton at 8.31 ppm shifts to 8.60 ppm in the case of AA-AR-
Hg complex. This behavior is most probably due to the de-
shielding of azomethine group after the complexation of Hg
with AA-AR. Furthermore, the signals attributed to benzene
protons were observed to be broadened upon the addition of
2
+
Hg was found to be approximately 60 times less than that
achieved with another rhodamine-based sensor prepared
through a Schiff base-type reaction between rhodamine and
quinoline-2-aldehyde [24]. Better sensitivity observed in the
case of AA-AR is attributed to higher conjugation in the case
of anthracene (compared to quinoline). Finally, when the spec-
tral patterns of these two sensors compared, a bathochromic
shift was obvious in the case of AA-AR. Hence, AA-AR
−1
2
+
6
2
+
2+
1
exhibited excellent sensor properties for Hg detection, and
2
+
13
this led to fast Hg detection even at sub-micromolar concen-
tration levels.
2
+
The results obtained from UV-Vis and fluorescence spec-
troscopy analyses were, also, used to estimate the progress of
2
+
2
+
2+
Hg coordination by AA-AR as well as the stoichiometry of
the reaction. For this purpose, the recorded spectra were ana-
lyzed on the basis of the Job’s plot method [25] and the
Benesi-Hildebrand method [26, 27] (figures not shown).
Both the methods evidenced a 1:1 stoichiometry for mercury
complexation by AA-AR. Besides, from the Benesi-
2
+
Hg ion, and this was attributed to the ring-opening reaction
1
3
of spirolactam moiety. The results of C-NMR supported this
phenomenon so that a characteristic peak appeared (at
65.82 ppm) in the spectrum of AA-AR shifted to 142.6 ppm
2
Hildebrand plot (r = 0.983), the binding constant was

J Fluoresc
2
+
Fig. 9 Bright-field and fluorescence images recorded for MIA PaCa-2
Bright-field image of MCF7 cells treated with AA-AR and Hg ; (e)
2+
and MCF7 living cells (a) Bright-field image of MIA PaCa-2 cell incu-
Fluorescence image of MCF7 cells treated with AA-AR and Hg ; (d);
2+
2+
bated with AA-AR and Hg ; (b) Fluorescence image of MIA PaCa-2
(f) Merged image of (d) and (e). Concentration of Hg : 0.010 mM;
2+
cells treated with AA-AR and Hg ; (c) Merged image of (a) and (b); (d)
Concentration of AA-AR: 0.020 mM
2
+
in the case of AA-AR-Hg complex (Fig. 2). Very similar
This conclusion proves the successful design and synthesis of
AA-AR.
results have been reported in previous studies [29]. Thus, the
observed color change upon formation of the AA-AR-Hg
2
+
complex was ascribed to a ring-opening reaction of
spirolactam moiety in the rhodamine framework.
Conclusions
In conclusion, a low-cost and efficient fluorescent probe (i.e.
AA-AR) containing rhodamine and anthracene units has been
developed for the first time. The probe exhibited excellent selec-
tivity and sensitivity towards a toxic and mutagenic metal ion,
Cell-Imaging
Owing to the accumulation of mercury ions in plants, animals,
and microorganisms [2], the design and synthesis of new
chemosensors for the detection of mercury ions in biological
samples are deemed important. Therefore, a simple experiment
was organized whereby Hg ions are allowed to penetrate
through living-cells (i.e. MCF7 and MIA PaCa-2), and afterward,
the probe, AA-AR, was used to detect Hg and thus to record
cell images in a biological sample by a fluorescence microscope.
In fluorescence images (Fig. 9), Hg -smeared zones within the
cells were visualized as bright fields after the addition of the AA-
AR probe to the medium. Thus, Hg stains in living cells could
be detected by using a very dilute AA-AR solution (i.e. 20 μM),
and thus the performance of the probe AA-AR was proven by
detecting this toxic metal ion in living cells. At this point, the role
of anthracene moiety in penetration of AA-AR through the cell
membranes should be mentioned. This hydrophobic moiety is
believed to facilitate penetration of the probe, which is water-
soluble, through the lipid membranes of cells having hydropho-
bic character [30]. Hence, both receptor (i.e. anthracene) and
fluorophore (i.e. rhodamine) parts of the newly designed sensor,
AA-AR, were observed to be functioning in an expected way.
2+
Hg , over various metal ions and anions. AA-AR exhibited a
2+
prominent Hg -induced fluorescence response followed by an
apparent color change under both day-light and UV-light. For
example, under day-light, a net color change from light yellow
2
+
2+
2+
to pink made a naked-eye detection possible for Hg . The de-
2+
termination of Hg in aqueous samples was found to be possible
even at trace levels. The emission signals were found to be useful
2
+
2+
to detect Hg at trace concentration levels with a detection limit
−7
2+
of 8.73 × 10 M. Finally, the probe, which was synthesized in a
new manner based on a facile Schiff-base type reaction, exhibited
good penetrating performance through the cell membranes and
thus it was successfully utilized in intracellular mercury detection
and imaging. Thus, the reaction protocol presented here offers a
straightforward and sulfur-free (and thus environmentally-
2+
friendly) manner for the design and synthesis of Hg -selective
probes.
Acknowledgments Authors wish to thank Karamanoglu Mehmetbey
University (Karaman, Turkey), and Nigde Ömer Halisdemir University
(Nigde, Turkey) for the facilities provided.

J Fluoresc
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