A Rhodamine-Benzimidazole Based Chemosensor for Fe3+ and its Application in Living Cells

Article abstract of DOI:10.1007/s10895-015-1696-9

A rhodamine-benzimidazole based chemosensor was designed and prepared for Fe³⁺ via opening of the spiro-ring to give fluorescent and colored species. L exhibited high selectivity and excellent sensitivity in both absorbance and fluorescence det

Full text of DOI:10.1007/s10895-015-1696-9

J Fluoresc
DOI 10.1007/s10895-015-1696-9
ORIGINAL ARTICLE
3+
A Rhodamine-Benzimidazole Based Chemosensor for Fe
and its Application in Living Cells
1
2
2
2
Gongchun Li & Jun Tang & Peigang Ding & Yong Ye
Received: 30 July 2015 /Accepted: 14 October 2015
#
Springer Science+Business Media New York 2015
Abstract A rhodamine-benzimidazole based chemosensor
haemoglobin and a cofactor in many enzymatic reactions in-
volved in the mitochondrial respiratory chain [3–6]. Besides
the beneficial effects, less iron in the body has been reported
linked to diabetes, anemia, liver and kidney damages, and
heart diseases [7]. While, deposition of iron in the central
nervous system has been involved in a number of diseases,
such as Parkinson’s and Alzheimer’s disease, associated with
an increased quantity of iron [8]. Much effort has been fo-
3
+
was designed and prepared for Fe via opening of the
spiro-ring to give fluorescent and colored species. L exhibited
high selectivity and excellent sensitivity in both absorbance
3
+
and fluorescence detection of Fe in aqueous solution with
comparatively wide pH range (5.8–7.4). The detection
limit of this newly developed probe was shown to be
up to 2.74 μM. The reversibility establishes the poten-
3
+
3+
tial of both probes as chemosensors for Fe detection.
cused on the development of fluorescent Fe indicators, es-
3
+
3+
Fluorescence imaging experiments of Fe in living
MGC803 cells demonstrated its value of practical applications
in biological systems.
pecially those that exhibit selective Fe -amplified emission
[9–12].
Fluorescent sensors for metal ions have consistently
demonstrated their potential in a variety of fields, such
as biological probes, environmental sensors [13–15].
The rhodamine framework is an ideal mode to construct
fluorescent chemosensors due to its excellent photophysical
properties such as long absorption and emission wave-
length, large absorption coefficient and high fluores-
cence quantum yield [16]. To data, some rhodamine
3
+
.
.
Keywords Rhodamine Fluorescence Fe
Introduction
Among transition metal ions, iron is the most abundant essen-
tial trace element for both plants and animals. It plays an
important role in enzyme catalysis, cellular metabolism,
DNA, RNA synthesis [1, 2] and as an oxygen carrier in
3
+
based probes for Fe have been reported [17–21],
however, an organic cosolvent was needed to guarantee
3
+
a high probe affinity for Fe and there were only a
few successful examples of fluorescent probes for de-
3
+
tecting Fe ions being used in natural water samples
Electronic supplementary material The online version of this article
3+
for Fe .
(doi:10.1007/s10895-015-1696-9) contains supplementary material,
Herein, a rhodamine-benzimidazole chemosensor was pre-
pared by a new method which is different to Han’s [22]. The
title compound could sensitively and selectively detect
which is available to authorized users.
*
Yong Ye
3
+
yeyong03@tsinghua.org.cn
Fe in Tris-HCl buffer solution. It also displayed en-
hanced fluorescence intensities and clear color changes
upon recognition. Moreover, fast response (in 10 s) and
1
2
School of Chemistry and Chemical Engineering, Xuchang
University, Xuchang 461000, China
3
+
neutral aqueous medium for Fe made it possible to be
practical application. The probe could be applied in biological
Phosphorus Chemical Engineering Research Center of Henan
Province, The College of Chemistry and Molecular Engineering,
Zhengzhou University, Zhengzhou 450052, China
3
+
systems for the detection of Fe through confocal laser
scanning microscopy experiments.

J Fluoresc
Cl
Scheme 1 Synthetic route of
target compound
O
N
N
O
N
N
POCl
3
1,2-diaminobenzene
COOH
NEt3, CH CN
ClCH
2
CH
2
Cl
COCl
3
N
O
N
N
O
N
Lawesson
toluene,reflux,24h
N
O
N
N
2
H N
1
L
Experimental
solutions of metal ions were prepared from their nitrate salts,
except for FeCl , FeCl , CrCl , AlCl and MnCl . The metal
3
2
3
3
2
Apparatus Reagents and Chemicals
ions were prepared as 10.00 mM in water solution.
Fluorescence spectra measurements were performed on the
F-4500 FL Spectrophotometer, and the excitation and
emission wavelength band passes were both set at
Synthesis
Synthesis of Compound L
5
2
.0 nm. Absorption spectra were measured on a UV-
102 double-beam UV/VIS spectrometer, Perkin Elmer
As shown in Scheme 1, compound 1 was synthesized accord-
ing to the literature [23]. The concrete synthesis way of com-
pound L was described as follows: Compound 1 (266 mg,
0.5 mmol) and Lawesson’s reagent (243 mg, 0.6 mmol) were
dissolved in dry toluene, and the reaction mixture was stirred
precisely. NMR spectra were recorded on a
BrukerDTX-400 spectrometer in CDCl , using TMS as
internal standard. Mass spectral determination was carried
on a HPLC Q-T of HR-MS.
3
All the materials for synthesis were purchased from com-
mercial suppliers and used without further purification. The
at 110 °C for 24 h under N atmosphere. After removing the
solvent under reduced pressure, the residue was purified by
2
Fig. 1 Absorbance spectra of L
(10 μM) in Tris-HCl buffer solu-
tion (2.5 mM, pH = 7.0) with the
presence of 10 equiv. of various
species

J Fluoresc
Fig. 2 Absorption spectra of L
(
10 μM) with gradual addition of
3+
various amounts of Fe (from
bottom 0–10 equiv) in Tris-HCl
buffer solution (2.5 mM,
pH = 7.0)
column chromatography using EA/PE = 1:1 as eluent to afford
L (202 mg, 78.6 %). H-NMR(400 MHz, CDCl3): 1.14 (t,
148.5, 148.9, 153.0, 156.1, 156.4. HR-MS: C H N O [M +
34 35 4
H] , calculated for 515.2805. Found: 515.2807. (Supporting
1
+
1
2 H, J = 7 Hz), 3.31 (q, 8 H, J = 7 Hz), 6.15 (q, 2 H,
Information, Figs. S1–S3).
J = 3.7 Hz), 6.31 (d, 2 H, J = 8.8 Hz), 6.49 (d, 2 H,
J = 2.4 Hz),6,94 (d, 1 H, J = 8 Hz), 7.02 (t, 1 H,
J = 7.4 Hz), 7.16 (t, 1 H, J = 7.4 Hz), 7.22 (d, 1 H,
J = 7.6 Hz), 7.38 (q, 1 H, J = 5 Hz), 7.49 (q, 1 H, J = 5 Hz),
Results and Analysis
1
3
1
7
.8 (d, 1 H, J = 8.4 Hz), 8.09 (s, 1 H). C NMR (100 MHz,
CDCl3) δ (ppm): 12.6, 44.3, 97.7, 106.2, 108.1, 110.2, 120.0,
21.4, 121.9, 122.5, 124.8, 127.8, 128.3, 128.6, 130.5, 130.9,
The structure of compounds L was characterized by H NMR,
1
3
C NMR, and HR-MS. The results were in good agreement
with the structure showed in Scheme 1. Fluorescence and
1
Fig. 3 Fluorescence spectra of L
10 μM) in the presence of
00 μM different metal ions in
(
1
Tris-HCl buffer solution (2.5 mM,
pH = 7.0). λex = 520 nm, scan
range 550–700 nm, slit width
5
nm

J Fluoresc
Fig. 4 Fluorescence responses of
L to various cations in Tris-HCl
buffer solution (2.5 mM,
pH = 7.0). [L] =10 μM,
λex = 520 nm, λem = 600 nm
3
+
UV–vis studies were performed using a 10 μM solution of L
in a Tris-HCl (2.5 mM) buffer solution with appropriate
amounts of metal ions. Solutions were shaken for 15 min be-
fore measuring the absorption and fluorescence in order to
make the metal ions chelate with the sensors sufficiently.
Adding of 10 equiv. Fe into solution immediately
resulted in a significant enhancement of absorbance at
about 554 nm, and simultaneously accompanying with
the color changes from colorless to red. Under the iden-
tical condition, no obvious response could be observed
2
+
3+
upon the addition of other ions including Zn , Cr ,
2
+
2+
2+
3+
2+
2+
2+
2+
2+
UV–vis Spectral Responses of L
Mg , Ca , Cd , Al , Pb , Hg , Ba , Ni , Fe ,
2
+
+
+
+
2+
+
2+
Mn , K , Li , Ag , Co and Na except for Cu
(Fig. 1), which caused a mild effect compared to
As shown in Fig. 1, UV–vis spectrum of compound L
3
+
(
10 μM) exhibited only very weak bands over 450 nm.
Fe . The results demonstrated that L was characteristic
Fig. 5 Fluorescence responses of
L to various anions in Tris-HCl
buffer solution (2.5 mM,
pH = 7.0). [L] =10 μM,
λex = 520 nm, λem = 600 nm

J Fluoresc
Fig. 6 Effect of reaction time on
the fluorescence intensity (at
6
00 nm) of L (10 μM) in the
absence and presence of 10 equiv.
3+
Fe in Tris-HCl buffer solution
(
2.5 mM, pH = 7.0).
ex = 520 nm, slit =5 nm)
(λ
3
+
of high selectivity toward Fe over other competitive
metal ions.
Fluorescence Spectral Responses of L
3
+
3+
To further investigate the interaction of Fe and L, an
ultraviolet titration experiment was carried out (Fig. 2). A
linear increasing of absorption intensity of L could be ob-
served accompanying with color changes from colorless to
red along with the increasing concentrations of Fe (Fig. 1,
inset). To determine the stoichiometry of the iron–ligand com-
plex, Job’s method for absorbance measurement was applied
The selectivity of L for Fe was further observed in the fluo-
rescent spectra. As shown in Fig. 3, L exhibited a very weak
fluorescence in the absence of metal ions. When 10 equiv.
3
+
Fe was introduced in a 10 uM solution of L in Tris-HCl
buffer solution (2.5 mM, pH = 7.0), a remarkably enhance-
ment of fluorescence spectra was observed. Competition ex-
periments were carried out to explore the use of L as an ion-
3
+
3
+
[
24] (Fig. S4). The absorbance reached a maximum when the
ratio being 0.5, indicating a 1:1 stoichiometry of the Fe to L
selective fluorescent probe for Fe . L (10 uM) was treated
3
+
3+
with 10 equiv. Fe in the presence of other metal ions (10
in the complex.
equiv.). As shown in Fig. 4, the competing metal ions showed
Fig. 7 Fluorescence intensity (at
3+
6
00 nm) of L (10 μM) to Fe in
Tris-HCl buffer solution (2.5 mM,
pH = 7.0). (1) Baseline: 10 μM L
only; (2) red line: 10 μM L with
3
+
1
1
0 equiv. Fe ; (3) green line:
3
+
0 μM L with 10 equiv. Fe and
then addition of 10 equiv.
PO ;(4) blue line: 10 μM L
with 10 equiv. Fe and 10 equiv.
PO , then addition of 10
K
3
4
3+
K
3
4
3+
equiv.Fe (λex = 520 nm,
slit =5 nm)

J Fluoresc
Scheme 2 Possible sensing
3+
mechanism of L with Fe
N
O
N
N
O
N
FeCl3
N
N
2
H O
N
NH
Cl
O
Fe
Cl
2
H O
3
+
very low interference with the detection of Fe . Moreover,
the competitive experiments also confirmed that the back-
ground metal ions showed very low interference with the de-
In order to investigate the influence of the different acid
concentration on the spectra of L and found a suitable pH span
3
+
in which L could selectively detect Fe efficiently, the acid
titration experiments were performed. As shown in Fig. S6,
the fluorescent titration curve of free sensor L in Tris-HCl
buffer solution did not show obvious characteristic color of
rhodamine between pH 5.8 and 7.4, suggesting that
spirolactam tautomer of L was insensitive to the pH changes
3
+
tection of Fe in water solution. The fluorescence response of
3
+
L toward Fe in the presence of various coexistent anions
was also investigated, and it was gratifying to notice that all
the tested anions had low interference (Fig. 5).
To further investigate the interaction of chemosensor L
with Fe , a fluorescence titration experiment was conducted.
3
+
3+
in this range. However, the addition of Fe led to the fluores-
As shown in Fig. S5, the fluorescence intensity of L was
cence enhancement over a comparatively wide pH range (5.8–
3
+
enhanced with the increasing concentration of Fe
.
7.4), which was attributed to opening of the rhodamine ring.
3
+
Moreover, the time-dependence fluorescence of probe L was
Consequently, L might be used to detect Fe in approximate
physiological conditions. Generally, the detection limit of the
fluorescence sensor was one of the most important and useful
application. Under optimal conditions, the linear response for
the fluorescence intensity response was between 0 and 1 μM
3
+
also evaluated in the presence of Fe ions (Fig. 6). The kinet-
ics of fluorescence enhancement at 600 nm by the probe L
were recorded, and the results indicated that the recognizing
event could be completed in 10 s (T = 25 °C). These results
also demonstrated that compound L was a selectivity and
3
+
(Fig. S7), and the detection limit of Fe was measured to be
3
+
rapidly sensor for Fe over various other metal ions.
2.74 μM.
Fig. 8 Fluorescence images of
3+
Fe in MGC-803 cells with
1
2
0 μM solution of L in H O for
3
0 min at 37 °C, bright-field
transmission images (a, c) and
fluorescence images(b, d) of
MGC-803 cells incubated with
3+
0
μM, 10 μM of Fe for 15 min,
respectively

J Fluoresc
Mechanism
The significant changes in the fluorescence color could be
used for naked-eye detection. L might be used to detect Fe
3
+
Further, it was of great interest to investigate the reversible
binding nature of the sensor (shown in Fig. 7). Upon addition
in some environmental regions in a wide pH range with a
detection limit up to 2.74 μM. Moreover, it was applied for
imaging in MGC803 cells to confirm that it could be used as a
3
+
of 10 equiv. K PO to the solution of 10 μM L with Fe (10
3
4
3
+
equiv.), the fluorescence intensity at 600 nm was quenched
fluorescent sensor for monitoring Fe in living cells.
3
+
(
green line) due to the competitive binding of Fe from L by
3
+
Acknowledgments This work was supported by the National Science
Foundation of China (Nos. 21572209) and Program for New Century
Excellent Talents in University (NCET-11- 0950).
K PO . Further addition of 10 equiv. Fe could recover the
3
4
strong fluorescence again (blue line). These indicated that the
3
+
coordination of compound L with Fe was reversible.
3
+
According to the 1:1 stoichiometry of the Fe to L in the
complex (Fig.S4), a possible sensing mechanism was postu-
lated (Scheme 2). It was supported by the HR-MS spectra. A
directly evidence was obtained by comparing the HR-MS of L
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1
1
1
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In summary, an efficient rhodamine-based fluorescent
Chemosensor L was synthesized. The probe exhibited selec-
tivity and sensitivity in Tris-HCl buffer solution (2.5 mM,
pH = 7.0) with dramatic enhanced fluorescence intensities.
3
2
2