A novel turn-on colorimetric and fluorescent sensor for Fe3+ and its application in living cells

Article abstract of DOI:10.1016/j.jphotochem.2016.09.006

A novel turn-on colorimetric and fluorescent sensor 1 based on rhodamine B was developed as a colorimetric and fluorescent chemosensor for Fe³⁺. Upon addition of Fe³⁺ in aqueous ethanol, the 1 displayed a distinct color change from c

Full text of DOI:10.1016/j.jphotochem.2016.09.006

Accepted Manuscript
Title: A novel turn-on colorimetric and fluorescent sensor for
Fe3+ and its application in living cells
Author: Yang Hu Fang Zhao Shengli Hu Yingying Dong
Duanzhuo Li Zhenhong Su
PII:
S1010-6030(16)30664-5
DOI:
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http://dx.doi.org/doi:10.1016/j.jphotochem.2016.09.006
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9-9-2016
10-9-2016
Please cite this article as: Yang Hu, Fang Zhao, Hu Shengli, Yingying Dong, Duanzhuo
Li, Zhenhong Su, A novel turn-on colorimetric and fluorescent sensor for Fe3+ and its
application in living cells, Journal of Photochemistry and Photobiology A: Chemistry
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A novel turn-on colorimetric and fluorescent sensor for
Fe3+ and its application in living cells
Yang Hua, Fang Zhaoa, Shengli Hua*, Yingying Dong a,
Duanzhuo Lib, Zhenhong Sub*
a
Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Hubei
Collaborative Innovation Center for Rare Metal Chemistry, College of Chemistry
and Chemical Engineering, Hubei Normal University, Huangshi, P. R. China
b Hubei Provincial Key Laboratory of Occurrence and Intervention of Kidney
Diseases, Medical College, Hubei Polytechnic University, Huangshi, Hubei, P. R .
China
*Corresponding author: E-mail：hushengli168@126.com Tel.: +86 0714 6515602 Fax: +86 0714
6515602
*Corresponding author: E-mail：szh7977@163.com Tel.: +86 027 67867773 Fax: +86 027
88661729
1

Graphical Abstract
Highlights
 A novel rhodamine-based colorimetric and fluorescent sensor 1 was
synthesized.
 Sensor 1 displayed a distinct color change from colorless to pink and a
significant fluorescence enhancement after binding of Fe3+ .
 Fluorescent microscopy experiments established that 1 could be used for
sensing Fe3+ in living cells.
2

Abstract
A novel turn-on colorimetric and fluorescent sensor 1 based on rhodamine B was
developed as a colorimetric and fluorescent chemosensor for Fe3+. Upon addition of
Fe3+ in aqueous ethanol, the 1 displayed a distinct color change from colorless to pink
and a significant fluorescence enhancement, which can be directly detected by the
naked eye. The stoichiometry of 1 to Fe3+ complex was found to be 1:1 and the
detection limit was determined as low as 7.68× 10-7M. The results suggest that the
sensor 1 may provide a conveniently method for visual detection of Fe3+ with high
sensitivity. Furthermore the sensor has been utilized for fluorescence imaging of
Fe3+ in living cells.
Key words
Rhodamine B, colorimetric, fluorescent sensor, Fe3+, bioimaging
3

1. Introdution
The development of fluorescent chemosensors for a variety of metal ions have
received increasing attention because of their fundamental role in medical,
environmental and biological applications [1,2]. Among the metal ions, Iron performs
a crucial role in a wide range of biochemical processes [3,4], several biological
functions depend directly or indirectly on the proper concentration and oxidation
states of iron to maintain the homoeostatic mechanism of biosystem. Deficiency or
overdose of iron can lead to serious diseases, including Alzheimer’s, Huntington’s
and Parkinson’s disease [5-7]. Therefore, design of fluorescent chemosensors for
detecting Fe3+ is of great importance. However, Fe3+ is a well-known fluorescence
quencher due to its paramagnetic nature, which makes it difficult to develop a
sensitive turn-on fluorescent sensor. Only limited number of turn-on fluorescent
chemosensors is available in literature [8-19]. Moreover, among these fluorescent
sensors, only a few sensors have been applied in bioimaging [15-19]. Therefore,
design of turn-on fluorescent sensor for Fe3+ imaging in living cells remains a
challenging work.
Rhodamine-based fluorogenic and chromogenic probes have received increasing
interest in recent years by virtue of their properties of long-wavelength emission, high
fluorescence quantum yield and large molar extinction coefficient, and many
fluorescent probes based on metal induced spiroring opening have been developed
[20–33], because they can perform not only great fluorescence intensity enhancement
toward some specific cations, but also a strong colour development against the
4

colourless blank during the sensing event. In continuing of our study on developing
molecular sensors for Fe3+ detection [34, 35], herein, we introduce a novel and simple
method to prepare rhodamine B-based chemosensor by three steps reaction. The
sensor 1 showed “off–on” type chromogenic behavior toward Fe3+ over other metal
ions. Moreover, fluorescent microscopy experiments indicated that 1could be used as
fluorescent probe for sensing Fe3+ in living cells.
2. Experimental
2.1. Reagents
All the reagents were purchased from commercial suppliers and used without
further purification. The salts used in stock solutions of metal ions were CoCl2·6H2O,
ZnCl2, MnCl2·4H2O, KCl, NaCl, CuCl2·2H2O, NiCl2·6H2O, CdCl2·2H2O, HgCl2,
MgCl2·6H2O, CrCl3·6H2O, Pb(NO3)2, FeCl3·6H2O, FeSO4·7H2O, Al(NO3)3·9H2O.
2.2. Apparatus
NMR spectra were measured on a Varian Mercury 300 spectrometer operating at
300 MHz for 1H and 75 MHz for 13C relative to tetramethylsilane as internal standard.
MS spectra were obtained on a Finnigan Trace MS spectrometer. IR spectra were
recorded on a Perkin-Elmer PE-983 infrared spectrometer as KBr pellets with
absorption reported in cm-1. Absorption spectra were determined on UV-2501 PC
spectrophotometer. Fluorescence spectra measurements were performed on a
FluoroMax-P spectrofluorimeter equipped with a xenon discharge lamp, 1 cm quartz
cells at room temperature (about 298K).
5

2.3. Synthesis of rhodamine derivate (1)
The synthetic route of the compound 1 is shown in Scheme 1. 2-(methylthio)-1,4-
diphenylbut-2-ene-1,4-dione (2) [36] and rhodamine B ethylenediamine (3) [37] was
prepared according to literatures. To a 100 mL flask, rhodamine B ethylenediamine 3
(0.48g, 1.0 mmol) was dissolved in 50 mL ethanol. After the addition of compound 2
(0.28g, 1.0 mmol), the stirred mixture was heated to reflux for 24h. The residue was
recrystallized from ethanol to afford pure compound 1 (0.51g, 71%) as a yellow solid.
M.p.: 124-125oC. IR (KBr, cm-1): 3449, 2969, 1681, 1624, 1514, 1384, 1221, 1113,
1
700; H NMR (CDCl3, 300MHz): δ10.6(s,1H),7.94-7.97(m,2H), 7.76- 7.79(m,1H),
7.50-7.63 (m,2H), 7.32-7.47(m,8H),7.03(m,1H), 6.41-6.44(m,3H), 6.22-6.34(m,2H),
5.63(s,1H), 3.22-3.34(m,9H) ,3.01-3.04 (m,2H), 1.62(s,2H), 1.15(t, 12H); 13C NMR
(75 MHz, CDCl3):191.2, 189.5, 168.3, 160.6, 153.7, 152.9, 148.6, 139.4, 134.5, 134.4,
132.4, 131.1, 130.5, 130.1, 128.7, 128.4, 128.1, 127.0, 123.6, 122.8, 108.1, 104.8,
97.6, 90.9, 64.7, 53.3, 44.2, 42.7, 40.9, 12.5; ESI-MS: m/z 719.46 [M+H]+. Anal.calcd
for C46H46N4O4: C76.85 H 6.45 N7.79; found C76.63 H 6.25 N7.64.
(Scheme 1)
2.4. Analytical procedure
The stock solutions of 1(1.0×10-5 M) were prepared by dissolving 1 in EtOH/
water (9:1, v/v) containing HEPES buffer (10 mM, pH =7.2). The cationic stocks
were all in H2O with a concentration of 3.0×10-3 M for UV–vis absorption and
fluorescence spectra analysis. For metal ion absorption and fluorescence titration
6

experiments, each time 3 mL solution of 1 filled in a quartz cell of 1 cm optical path
length, and we increased concentrations of metal ions by stepwise addition of
different equivalents using a micro-syringe. After each addition of Fe3+ ion, the
solution was stirred for 3 min. The volume of cationic stock solution added was less
than 100 μL with the purpose of keeping the total volume of testing solution without
obvious change. For all measurements of fluorescence spectra of 1, the excitation was
at 510 nm.
2.5. Cell culture
Cell line Hela was purchased from ATCC (American Type Culture Collection,
Rockville, MD, USA), and maintained in the Dulbecco’s Modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS, Gibico, USA) in a
humidified incubator with 5% CO2 at 37°C. Hela cells plated on 18mm glass
coverslips in 6-well plates and allowed to adhere for 24h.
2.6. Fluoreszence imagine
1×10-2M FeCl3 were prepared in PBS (phosphate-buffered saline, PH =7.2), and
sensor 1 was dissolved in DMSO. The cells plated on glass cover lips cultured in
DEME were treated with final concentration 1×10-5M FeCl3 solution and incubated at
37°C for 30 min. The treated cells were washed with PBS 3 times to remove
remaining metal ions. 2ml DMEM was added to the cell culture, which was then
treated with final concentration 1×10-5 M solution of sensor 1, and the sample were
incubated at 37°C for 30 min. the culture medium with sensor 1 was removed, and
washed 3 times with PBS before observation. Fluorescence imaging was performed
7

under an Olympus SZX16 microscope with an Olympus DP72 digital camera with
cell Sens version1.6 software, and processed with Adobe Photoshop CS4 software.
3. Results and discussion
3.1. Synthesis and structural characteristics of 1
The compound 1 was obtained by the reaction of rhodamine B ethylenediamine (3)
with 2-(methylthio)-1,4-diphenylbut-2-ene-1,4-dione (2) in EtOH under reflux. The
yield of 1 was 71%. The structures of 1 were identified by using 1H NMR, 13C NMR,
ESI-MS (Figures S1-S3, in the ESI).
3.2. Spectral characteristics
To examine the binding properties of 1 with metal ions, the absorption spectra of
1(10μM) in EtOH/water (9:1, v/v) containing HEPES buffer (10 mM, pH =7.2) was
first explored in the presence of 10 equivalent of different metal ions (K+, Na+, Cr3+,
Zn2+, Cd2+, Co2+, Hg2+, Mn2+, Mg2+, Ni2+, Fe3+, Cu2+ , Pb2+ , Fe2+ , and Al3+), and the
results were depicted in Figure 1. The sensor 1 exhibited c abroad band at 319 nm
(log  =5.04) and no absorption was observed in the visible range, suggesting that the
closed spirolactone form of Rhodamine B presented. Upon binding of metal ions it
was found that coordination of Fe3+ to 1 resulted in the enhancement of its absorbance
band at 319 nm (log  =5.28) and a new absorption band was appeared at 552 nm (log
 = 5.10). At the same time, the solution changed from colorless to pink (Inset Figure
1). The new absorption band at 552 nm was ascribed to the ring opened rhodamine
moiety, which indicated that the interaction of 1 with Fe3+ can triggered the formation
of the ring-opened form of 1 from the spirolactam form. Other metal ions did not
8

cause this change under the same conditions. Accordingly, 1 can serve as a
“naked-eye” Fe3+ indicator in aqueous media.
(Figure 1)
The fluorescence spectra of 1 with respective metal cations are shown in Figure 2.
The sensor 1 without Fe3+ showed no apparent fluorescence emission at 582nm. In
fact, 1 also did not give any observable response for many metal ions such as K+, Na+,
Cr3+, Zn2+, Cd2+, Co2+, Hg2+, Mn2+, Mg2+, Ni2+, Cu2+, Pb2+, Fe2+, and Al3+. However,
addition of Fe3+ created a remarkable fluorescence enhancement at 582nm. In
agreement to the fluorescent spectra, the emission color change from colorless to
yellow can be clearly observed under UV light in presence of Fe3+, while other ions
did not induce any significant emission color change. The specific response of 1
towards Fe3+ was assumed to be based on the opening function of the spirolactam ring.
Meanwhile, the reaction of Fe3+ with a chelating agent induces rigidity in the resulting
molecule and tends to produce a large chelation enhancement of the fluorescence
(CHEF) which induces the large enhancement of fluorescence. These results
demonstrated that 1 can be considered as a new “off-on” chemosensor for detecting
Fe3+.
To get an insight into the sensing properties of 1 to Fe3+, fluorescence titration
experiments were then performed (Figure 3). The fluorescence intensity of 1 (10 μM)
at 582 nm was gradually increased with the addition of increasing amount of Fe3+ (10
μM-100 μM). The enhanced intensity of 1-Fe3+ displayed a good linear regression
9

relationship (R2 = 0.9916) (Inset Figure 3).
(Figure 2)
The fluorescence spectra of 1 upon titration with Fe3+ were then performed
(Figure 3). The fluorescence intensity of 1 (10 μM) at 588 nm was gradually increased
with the addition of increasing amount of Fe3+ (10 μM-100 μM). The enhanced
intensity of 1-Fe displayed a good linear regression relationship (R2 = 0.994) (Figure
3, inset). The fluorescence quantum yield (Фu) of 1 in the present of 100 μM Fe3+ was
calculated to be 0.61 with respect to rhodamine B in ethanol solution (Фs=0.89) [38].
(Figure 3)
To investigate the binding stoichiometry between 1 and the Fe3+ ion, the Job’s plot
experiment[39]was carried out by keeping the total concentration of 1 and Fe3+ions at
10 μM and changing the molar ration of Fe3+ ([Fe3+]/[1+ Fe3+]) from 0 to 1. As shown
in Figure 4, the result shows that a maximum at a molar fraction of 0.5, indicating the
formation of 1:1complex of 1 and Fe3+. The 1:1 complexation is also supported from
ESI-MS spectra analyses (Fig. S4), which show a prominent peak at 844.16
(calculated value, 844.22) due to [1+ Fe3++2Cl-)]+.
(Figure 4)
The association constant (Ka) of 1–Fe3+ complexes was determined by the
Benesi–Hildebrand Eq. (1) [40]:
10

1
1
1
=
+
(1)
Fmax _ Fo
_
3+
F _ Fo
}
Fo) x [Fe ]
K x (F
{
a
max
Where F is the fluorescence intensity at 588 nm at any given Fe3+concentration,
F0 is the fluorescence intensity at 588 nm in the absence of Fe3+, and Fmax is the
maxima fluorescence intensity at 588 nm in the presence of Fe3+ in solution. The
association constant Ka was evaluated graphically by plotting 1/ (F−F0) against
1/[Fe3+] (Figure 5). Data were linearly fitted according to Eq. (1) and the Ka value
was obtained from the slope and intercept of the line. The Ka value of 1- Fe3+
complexe was 5.43× 103 M-1. The detection limit, based on the definition by IUPAC
(CDL =3Sb/m) [41], was found to be 7.68× 10-7 M from 10 blank solutions, which
is allowed for the detection of micromolar concentration range of Fe3+.
All of these suggested that the receptor can be used as a selective fluorescent sensor
for Fe3+ in environmental analysis and analytical chemistry.
(Figure 5)
To check the practical applicability of 1 as a selective fluorescent sensor for Fe3+,
competition experiment, the systems of other metal ions and Fe3+ coexisted was
examined in aqueous ethanol. As shown in Figure 6, we found that all the coexistent
metal ions had no obvious interference with the detection of Fe3+.
(Figure 6)
To investigate the binding mechanism, IR spectra experiments of 1 and 1-Fe3+
complexs were measured (Figure 7). The IR spectra were primarily characterized by
11

band in the double-bond region. Two bands, 1681cm-1 and 1624 cm-1, were associated
with the amide carbonyl and ketone carbonyl absorption in chemosensor 1,
respectively. Upon addition of Fe3+, the carbonyl groups IR peak at 1681cm-1 and
1624 cm-1 disappeared and a new IR peak appeared at 1631 cm-1. This suggests that
the ketone and amide carbonyl O of 1 is actually involved in the coordination with
Fe3+.
(Figure 7)
According to the above experiments, a proposed binding mechanism of Fe3+ with
1 was shown in Scheme 2. As it would be expected, the Fe3+ binds with 1, and opens
the spirolactam ring that results in the fluorescence enhancement and development of
pink color.
(Scheme 2)
To access the feasibility of Fe3+ detection in living cells, the fluorescence
imaging was recorded using confocal fluorescence microscopy. Figure 8 shows the
results of confocal fluorescence imaging of Fe3+in live HeLa cells. As expected, HeLa
cells incubated with 1 gave no intracellular background fluorescence (Figure 8a). By
contrast, when 1 deposited HeLa cells were incubated with 10μM Fe3+ for 30 min,
intense red fluorescence was observed (Figure 8c). It is well known that the Fe3+
could be taken up into the living cells by diffusion through porins, and transport by
transferrin and lactoferrin[42]. So, we conclude the remarkable fluorescence
12

enhancement was because of the complexation of 1 with Fe3+ in the living cells. An
overlay of fluorescence and bright-field images (Figure 8d) shows that the
fluorescence signals is localized in the intracellular area, indicating a subcellular
distribution of Fe3+ and good cell-membrane permeability of chemosensor 1.
(Figure 8)
4. Conclusion
In summary, we demonstrated a novel fluorescent chemosensor 1 for Fe3+ and its
bioimaging applications. 1 exhibited a turn-on fluorescent response for detecting Fe3+
with excellent selectivity over other metal ions. Moreover, fluorescent microscopy
experiments indicated that 1could be used as fluorescent probe for sensing Fe3+ in
living cells.
Acknowledgments
This study was supported by the Science Technology Foundation for Creative
Research Group of HBDE (T201311) and the Hubei Province Education Ministry
Foundation of China (D20112507).
13

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[42] E. L. Mackenzie, K. Iwasaki, Y. Tsuji, Antioxid. Redox Signaling 10 (2008)
997–1014.
19

Captions:
Figure 1. Absorption spectral changes of compound 1 (10 μM) in EtOH/water (9:1,
v/v) containing HEPES buffer (10mM, pH =7.2) upon additions of various metal ions
(100 μM). Inset: the color changes of 1 before and after addition of Fe3+.
Figure 2. fluorescence spectral changes of compound 1 (10μM) in EtOH/water (9:1,
v/v) containing HEPES buffer (10mM, pH =7.2) upon additions of various metal ions
(50 μM ). λex = 510 nm. Inset: color of 1 and 1+Fe3+ system under UV lamp.
Figure 3. Emision spectra of 1 (10μM) in EtOH/water (9:1, v/v) containing HEPES
buffer (10 mM, pH =7.2) upon the addition of Fe3+ (0–10eq) at 25oC.
Figure 4. Job’ plot of 1 and Fe3+. The total concentration of 1 and Fe3+ was kept at a
fixed 10μM.
Figure 5. Benesi–Hildebrand plot of sensor 1 with Fe3+.
Figure 6. Competitive experiments in the 1+Fe3+ system with interfering metal ions. [1] = 10 μM, [Fe3+] = 100
μM, and [Mn+] = 100 μM. λex = 510 nm.
Figure 7. Infrared spectra (KBr) of free 1 and 1-Fe3+ complex at room temperature.
Figure 8. Fluorescence images of Fe3+ in HeLa cells. (a) Fluorescence images of
HeLa cells treated with 1; (b) bright-field image of cells shown in panel; (c)
fluorescence images after presence of Fe3+; (d) overlay image of (b) and (c)
20

Figure 1
21

Figure 2
22

600
500
400
300
200
100
0
500
400
300
200
100
0
y = 48.87x-12.31
R = 0.9916
10eq
Fe3+
0
-1
0
1
2
3
4
5
6
7
8
9
10 11
[Fe3+] / [1]
550
575
600
625
650
675
700
Wavelength (nm)
Figure 3
23

Figure 4
24

0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
y = 0.03724x-0.0022
R = 0.9967
Kass = 5.43103M-1
0.0
0.2
0.4
0.6
0.8
1.0
1/[Fe3+]105M-1
Figure 5
25

1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.11
1.12
1.07
1.06
1.05
1.05
1.01
1.02
1.00
0.99
0.98
0.97
0.96
0.96
0.90
+
2+
2+
2+
y
l
2+
2+
2+
2+
2+
3+
l
3+
+
2+
2+
i
a
r
u
o
n
Z
d
b
g
e
F
A
+
on
N
+
N
+
C
C
+
C
+
C
+
P
H
+
+K
Mg
3+
Mn
+
3+
+
+
+
3+
3+
3+
+
3+
+
3+
3+
3+
3+
3+
3+
3+
3+
3+
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Figure 6
26

1+Fe3+
1
3000
2500
2000
1500
1000
500
Wavenumbers(cm-1)
Figure 7
27

Figure 8
28

Scheme 1. Synthesis of rhodamine derivate 1
Scheme 2. Proposed binding mechanism for 1 with Fe3+.
O
O
O
I2, CuO
DMSO
C CH C C
SCH3
O
O
2
H
N
EtOH
reflux
N
O
O
NH2
COOH
NH2
N
H2N
Et2N
O
NEt2
EtOH
1
Et2N
O
NEt2
CI
Et2N
O
NEt2
3
Scheme 1
Cl
Cl
O
O
Fe3+
O
O
H
Fe3+
N
N
N
N
H
O
O
Et2N
O
NEt2
Et2N
O
NEt2
[M+H]+ = 719.78
[M+Fe3++2Cl- ]+ = 844.16
Scheme 2.
29