Water-soluble rhodamine-based chemosensor for Fe3+ with high sensitivity, selectivity and anti-interference capacity and its imaging application in living cells

Article abstract of DOI:10.1016/j.dyepig.2017.03.057

Fe³⁺ plays a crucial role in many vital cell functions and its detection has attracted considerable attention. In this work, a water-soluble rhodamine-based chemosensor RL has been designed and synthesized as an “off-on” chemosensor for Fe

Full text of DOI:10.1016/j.dyepig.2017.03.057

Accepted Manuscript
3+
Water-soluble rhodamine-based chemosensor for Fe with high sensitivity,
selectivity and anti-interference capacity and its imaging application in living cells
Feng Zhou, Tao-Hua Leng, Ya-Jing Liu, Cheng-Yun Wang, Ping Shi, Wei-Hong Zhu
PII:
S0143-7208(17)30188-2
10.1016/j.dyepig.2017.03.057
DYPI 5884
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 29 January 2017
Revised Date: 28 March 2017
Accepted Date: 28 March 2017
Please cite this article as: Zhou F, Leng T-H, Liu Y-J, Wang C-Y, Shi P, Zhu W-H, Water-soluble
3+
rhodamine-based chemosensor for Fe with high sensitivity, selectivity and anti-interference capacity
and its imaging application in living cells, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.03.057.
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Graphical Abstract
Water-soluble rhodamine-based chemosensor for Fe3+ with
high sensitivity, selectivity and anti-interference capacity
and its imaging application in living cells
Feng Zhou,a Tao-Hua Leng, b Ya-Jing Liu, c Cheng-Yun Wang,*a Ping Shi*c and Wei-Hong Zhu*a
A water-soluble rhodamine-based chemosensor RL with high sensitivity, selectivity
and anti-interference capacity and low detection limit for Fe3+ was fabricated and
successfully used for living cell imaging.

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Water-soluble rhodamine-based chemosensor for Fe3+
with high sensitivity, selectivity and anti-interference
capacity and its imaging application in living cells
Feng Zhou a, Tao-Hua Leng b, Ya-Jing Liuc, Cheng-Yun Wang a, , Ping Shi c,*, Wei-Hong Zhu a,*
a Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced
Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East
China University & Technology, Shanghai 200237, P. R. China.
bNational Food Quality Supervision and Inspection Center (Shanghai)/Shanghai Institute of
Quality Inspection and Technical Research, Shanghai 200233, P. R. China.
cState Key Laboratory of Bioreactor Engineering, East China University of Science & Technology,
Shanghai 200237, P. R. China.
Abstract
Fe3+ plays a crucial role in many vital cell functions and its detection has attracted
considerable attention. In this work, a water-soluble rhodamine-based chemosensor
RL has been designed and synthesized as an “off-on” chemosensor for Fe3+ detection.
Upon the addition of Fe3+, RL displayed obvious color change, quick fluorescence
enhancement, high sensitivity and anti-interference capacity over other metal ions
(Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+,
Cu2+, Ag+, Zn2+, Cd2+, Hg2+). The detection limit for Fe3+ was calculated to be 0.28
Corresponding author. Tel./fax: +86-021-64252967; e-mail: cywang@ecust.edu.cn; ship@ecust.edu.cn;
whzhu@ecust.edu.cn
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ꢀM with the binding constant Ka to be 4.67 × 108 M-2. Furthermore, an application of
RL in the imaging of HeLa cells exposed to Fe3+ was also successfully demonstrated.
Key words
Rhodamine dye, Chemosensors, Fe3+, Naked-eye detection, Living cell imaging
1. Introduction
Selective detection of biologically important metal ions has gained
tremendous importance because of their important roles in a variety of
fundamental biological processes in cells. Among these metal ions, iron is of
indispensable importance in the metabolism and energy transfer mechanisms of
numerous biological systems. The deficiency or overload of iron can lead to
damage to cells, tissues and organs as well as occurrence of certain diseases
such as anemia and breathing problems or some cancers [1-5]. Therefore, it is
extremely important for physiological concerns to develop efficient
chemosensors with good biocompatibility for detection of iron in biological
samples.
Fluorogenic methods in conjunction with suitable chemosensors are
preferable solutions to the measurement of iron in consequence of their features
- rapidly performed, non-destructive, highly sensitive and can afford real
information on the localization and quantity of the targets of interest [6-11]. So
far, there have been many successful fluorescent chemosensors for iron based
on the fluorophores such as naphthalimide [12,13], fluoranthene [14], coumarin
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[15-19], BODIPY [20,21], cyanine [22] and so on. Currently, the main
shortcoming of Fe3+ chemosensors is their low water solubility. At the same
time, rhodamine dye is also an ideal material to construct chemosensors
because of their excellent photophysical properties such as high extinction
coefficients, excellent quantum yields, high photostability and relatively long
emission wavelength in the visible region [23-26]. As a result, considerable
attention has been paid to the development of rhodamine-based chemosensors
[27-32], whose designs are primarily based on the fact that its spirolactam form
is colorless and non-fluorescent while its ring-opened form induced by the
analyte gives rise to pink or purple color and a strong fluorescence emission
[23,33,34]. Thus, the chemosensor based on the special optical signal which is
emitted from rhodamine or its derivatives could be developed for detection of
iron [35-40].
Herein, a new water-soluble rhodamine-based colormetric and fluorescent
chemosensor RL for Fe3+ detection was designed and synthesized, which
provided high sensitivity, selectivity and anti-interference capacity in DMSO/H2O
PBS
solution (1:1, v/v,
buffer, pH=7.04, 20 mM) towards Fe3+ over other metal
ions and might be used for characteristic ‘naked-eye’ detection. The detection
limit could reach as low as 0.28 ꢀM. Furthermore, chemosensor RL was
successfully applied in the imaging of Fe3+ in HeLa cells without significant
cytotoxic effect.
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2. Experimental
2.1 General
All the starting materials, reagents and solvents were AR grade purchased
from J&K Chemical Co. and used without further purification. The salts used to
prepare metal ion stock solutions were FeCl3, AlCl3, SnCl·2H2O, PbCl2, KCl,
MgCl2, BiCl3, CrCl3·6H2O, MnCl2·4H2O, CaCl2, CoCl2·6H2O, NiCl2·6H2O,
PdCl2, CuCl2·2H2O, LiCl, BaCl2, AgNO3, NaCl, ZnCl2. CdCl2·2.5H2O, HgCl2.
Thin-layer chromatography was performed on a HAIYANG silica gel F254 plate.
Column chromatography was performed using HAIYANG silica gel.
1H and 13C NMR spectra were measured on a Bruker AM-400 spectrometer
at room temperature. The chemical shifts were reported in d units (ppm)
downfield relative to the chemical shift of tetramethylsilane (TMS). The
abbreviations br, s, d, t and m denoted broad, singlet, doublet, triplet and
multiplet, respectively. Mass spectra (MS) were obtained with a Waters
Micromass LCT Premier TOF Mass Spectrometer. High-resolution mass
spectra (HRMS) were acquired under electron ionization conditions with a
(UV-vis)
double-focusing high- resolution instrument. The ultraviolet-visible
spectra were recorded on a Nicolet CARY 100 and the Fluorescence spectra
were measured on a CARY Eclipse at room temperature.
2.2 Synthesis
2.2.1
2-amino-3',6'-bis(diethylamino)spiro[iso-indoline-1,9'-xanthen]-3-
one (R1)
.
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R1 was synthesized according to the literature[41]. Rhodamine B (1.0 g, 2.09
mmol) was dissolved in absolute ethanol (30 mL), then excess hydrazine
hydrate (80 wt%, 6 mL) was added dropwise to the solution. The mixture was
refluxed in an oil bath for 8 h with vigorous stirring. The solution color
changed from dark purple to orange. Under reduced pressure, the solvent was
removed mostly and then a large quantity of water was added, and the resulting
precipitate was filtered and washed by water for several times. After drying in
vacuo, the light pink solid product was obtained (0.76 g, 80%). 1H NMR (400
MHz, CDCl3) δ 7.97-7.90 (m, 1H), 7.49-7.41 (m, 2H), 7.14-7.07 (m, 1H), 6.41-6.47 (m,
4H), 6.29 (dd, J = 8.8, 2.6 Hz, 2H), 3.61 (s, 2H), 3.34 (q, J = 7.1 Hz, 8H), 1.17 (t, J = 7.0
Hz, 12H).
2.2.2 2-(((3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthen]-2-yl)
imino)methyl)benzonitrile (RL)
.
R1 (0.46 g, 1.0 mmol) and 2-Formylbenzonitrile (0.13 g, 1.0 mmol) were
dissolved in absolute ethanol (30 mL). After refluxing for 12 h with vigorous
stirring, the solvent was removed mostly under reduced pressure. The crude
product was purified by column chromatography on a silicagel column
(
petroleum ether: ethyl acetate = 6:1, v/v) to give RL as light yellow powder (0.48
g, 85%).1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.03 (d, J
= 6.6 Hz, 1H), 7.56-7.43 (m, 4H), 7.27-7.31 (m, 1H), 7.13 (d, J = 7.0 Hz, 1H), 6.49-6.51
(m, 4H), 6.25 (dd, J = 8.9, 2.6 Hz, 2H), 3.37 (q, J = 7.1 Hz, 8H), 1.15 (t, J = 7.0 Hz,
12H); 13C NMR (400 MHz, DMSO) δ 164.27, 152.60, 151.36, 148.49, 140.92, 136.96,
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134.40, 133.44, 133.31, 130.46, 128.92, 128.01, 127.46, 124.55, 123.91, 123.22,
116.14, 111.07, 108.08, 104.70, 97.55, 65.53, 43.64, 12.33. HRMS [M+H]+ found:
+
570.2864; calcd for C36H36N5 O2
:
570.2869.
2.3 Stock solution preparation
10-2 M for Fe3+ and 5.0×10-2 M for the other metal ions
)
Stock solutions (1.0
×
of the chloride or nitrate salts of Fe3+, Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+,
Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+ and Hg2+ were
prepared in deionized water. The stock solution of RL (1.0×
10-3 M) was prepared in
DMSO. Working solutions of RL were freshly prepared by diluting the high
concentrated stock solution to the desired concentration prior to spectroscopic
measurements.
2.4 Absorption and fluorescence studies
PBS
All experiments were carried out in DMSO/H2O solution (1:1, v/v,
buffer,
pH=7.04, 20 mM) unless otherwise specified. In each titration experiment, a 10 ꢀM
solution of RL was placed in a quartz optical cell with a 1-cm optical path length, and
the appropriate amount of the metal ion stock solution was added to the quartz optical
cell using a micropipette. Spectral data were recorded after 1 min on the addition of
the metal ions. In the selectivity and anti-interference experiments, the test samples
were prepared by placing an appropriate amount of the metal ion stock solution in 3
mL of the chemosensor RL solution (10 ꢀM). In the fluorescence measurements,
λ =
nm
) and emission spectra were collected
excitation was provided at 520 nm (
520
ex
from 525 to 720 nm.
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2.5 Cell studies
Vitro experiments were performed using HeLa cells. HeLa cells were cultured in
o
RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 C.
Cytotoxicity of RL was determined by the MTT assay. Cells were incubated with
different concentrations of RL for 24 h. Cell viability was evaluated by incubating
with 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
o
for 24h at 37 C. For fluorescence microscopy images, cells were incubated with 10
o
ꢀM Fe3+ for 30 min at 37 C and washed with PBS for three times, followed by
o
incubation with 10 ꢀM RL at 37 C for additional 30 min. The incubated cells were
washed with PBS for three times again and mounted onto a glass slide. Fluorescence
images in the living cell were acquired on a Nikon A1 Laser Confocal Microscope.
3. Results and discussion
3.1. Synthesis
The synthesis of RL was shown in Scheme 1. Compound R1 was prepared in
80% yield from commercially available Rhodamine B and hydrazine hydrate, as
described previously [41]. Compound RL was prepared in 85% yield by the reaction
of R1 with 2-Formylbenzonitrile in absolute ethanol for 12 h. The structures of R1
13
and RL were confirmed by 1H NMR, C NMR and HRMS (Supporting Information
(SI), Fig. S1-S4).
Insert Scheme 1 here
3.2 Response time of RL towards Fe3+
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The response time of RL towards Fe3+ in the fluorescence intensity was
investigated as shown in Fig. 1.
With the addition of Fe3+, the fluorescence
intensity increased rapidly and reached a maximum without 1 min, which
indicated that the reaction system was stable after 1 min. These results indicated that
RL was a sensitive chemosensor for Fe3+. Therefore, a 1-min reaction time was
selected in subsequent experiments to ensure that the metal ions had enough time to
sufficiently chelate with the chemosensor.
Insert Fig.1 here
3.3 pH response
DMSO/H O solution
Acid-base titration experiments were performed in
(1:1,
2
v/v) by using PBS buffer solutions of different pH. The fluorescence intensities of RL
at 589 nm at different pH values (2.02, 3.01, 3.98, 5.00, 6.02, 6.98, 7.04, 8.00, 9.01,
10.03, 11.01 and 11.98, respectively) were recorded as shown in Fig. 2. Without the
addition of Fe3+, RL did not emit any fluorescence in the pH range of 4.0-12.0, which
indicated that the spirolactam form of RL was the predominant species. When the pH
was adjusted to 2.0, the fluorescence intensity at 589 nm was sharply enhanced due to
the strong protonation which led to the ring-opening process of the spirocyclic moiety
of Rhodamine (Scheme 2). Upon the addition of Fe3+, RL emitted fluorescence in the
pH range of 4.0-11.0 between which RL did not emit any fluorescence without Fe3+,
which indicated that the addition of Fe3+ led to the opening of the spirocyclic moiety
of Rhodamine. At the same time, there was no obvious fluorescence change in the pH
range of 4.0-8.0. However, when the pH was over 8, the fluorescence intensity
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gradually decreased with the increase of pH so that fluorescence almost completely
disappeared when pH reached 12, which might be attributed to the formation of
Fe(OH)3 which prevented the combination of Fe3+ and RL [42,43]. For further spectral
analysis and potential practical applications, pH value should be adjusted to 7.04.
Insert Fig.2 here
Insert Scheme 2 here
3.4 Absorption and fluorescence studies of RL towards Fe3+
To gain insight into the
property of RL with Fe3+, UV-vis spectra of
optical
RL upon titration with Fe3+ in DMSO/H2O solution were recorded as shown in
Fig. 3(a). Without addition of Fe3+ to the solution of RL, almost no absorption
above 500 nm could be observed. However, with the addition of the Fe3+ (0-180
0-18 e ), a
quiv of RL new absorption band centered at 562 nm with a
µM,
shoulder peak at 520 nm was observed and gradually increased, resulting in a
color change from colorless to pink as shown in the insert picture of Fig. 3(a).
These phenomena might be ascribed to the formation of the ring-opened form
of RL after the addition of Fe3+ (Scheme 3).
Insert Fig.3 here
Insert Scheme 3 here
Fig. 3(b) displayed the fluorescence spectra of RL in the presence of different
amounts of Fe3+. Like most spirocycle Rhodamine B derivatives, the free RL (10 ꢀM)
λ =
520
nm
). However, the titration of
exhibited very weak fluorescence at 589 nm (
ex
Fe3+ (0-180 µM) led to a significant increase in the fluorescence emission intensity.
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Over 50-fold fluorescence enhancement was observed under saturation conditions.
The insert picture of Fig. 3(b) displayed the fluorescence change of RL on the
addition of Fe3+ under a 365 nm ultraviolet lamp
3.5 Selectivity and anti-interference capacity
To evaluate the selectivity of RL for Fe3+, the
.
property of RL towards
optical
different metal ions was also studied by UV-vis and
spectra. All of
fluorescence
the measurements were performed according to the following process. Test
Fe3+ in place of Fe3+ (180
metal ions (900
were added to 3
ꢀM)
ꢀM, 5 equiv of
)
mL of 10 M RL in DMSO/H2O solution, and the UV-vis and
ꢀ
fluorescence
spectra were measured 1 min later. As shown in Fig. 4(a) , other metal ions
(b)
(Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+,
Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+)
possessed very little absorption and very
weak
. These results indicated that RL exhibited excellent selectivity
fluorescence
towards Fe3+,
fl
and
which in fact endowed the chemosensor with “naked eye”
uorescence detection of Fe3+ (Fig. 4(c)(d)).
Insert Fig.4 here
Besides, anti-interference capacity of RL for Fe3+
(180 ꢀM) in the presence of
Fe3+
), including Li+, Na+, K+, Mg2+, Ca2+,
many other metal ions (900
ꢀ
M, 5 equiv of
Ba2+, Al3+, Sn2+, Pb2+, Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+ and
2+
by
Hg , was also studied
fluorescence spectra. As shown in Fig. 5, compared with
Fe3+, no obvious fluorescence was detected upon the addition of above metal ions
(black bars), which indicated that the Fe3+ induced the formation of the strongly
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fluorescent, ring-opened RL-Fe3+ complex (Scheme 3). When Fe3+ was added intoRL
solution in the presence of these competitive metal ions, no significant variationswere
observed compared to the spectra when only Fe3+ exited (red bars). These results
indicated that the detection of Fe3+ was not interfered by other metal ions, and RL
could be used as a selective Fe3+ chemosensor, which might be a result of the
simultaneous effect of a series of factors, such as the apposite combination ratio, the
suitable coordination geometry conformation of the receptor and the radius of the Fe3+
(
Scheme 3) [44].
Insert Fig.5 here
3.6 Detection limit calculations
The detection limit is calculated with the following equation[45]:
Detection limit= 3SD/S
where SD is the standard deviation of the blank and S is the slope of the calibration
curve.
The detection limit of RL for Fe3+ was also calculated based on the fluorescence
µM
titration. The fluorescence spectra of free RL (10
) were measured 20 times, and
the standard deviation of fluorescence intensities was then calculated. To obtain the
slope, the ratio of the fluorescence intensity at 589 nm was plotted versus the
concentration of Fe3+ (Fig. S5). The detection limit was thus calculated to be 0.28 ꢀM.
3.7 Quantum yield measurements
The fluorescence quantum yield is determined by using rhodamine B as a
reference with a known ΦR value of 0.89 in ethanol [46,47]. The area of the
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fluorescence spectra is integrated using the software available in the instrument and
the quantum yield is calculated according to the following equation [47]:
where ΦS and ΦR are the fluorescence quantum yields of the sample and the reference,
respectively; FS and FR are the area under the fluorescence spectra of the sample and
the reference, respectively; AS and AR are the corresponding optical densities of the
sample and the reference solution at the wavelength of excitation; ηS and ηR are the
refractive indexes of the sample and the reference, respectively.
The fluorescence quantum yield of RL was calculated to be 0.27 in ethanol.
3.8 Binding mechanism analysis
To explore the binding mechanism of RL to Fe3+, Job’s plot of fluorescence titration
of Fe3+ was carried out as shown in Fig. 6(a). Total concentration of RL and Fe3+ was 50
3+
µM
and kept constantly when the mole fraction of Fe changed from 0 to 1. Maximum
emission was observed when Fe3+
fraction was between 0.3 and 0.4, indicating
mole
that 2:1 stoichiometry should be the most possible binding stoichiometry for RL and
Fe3+.
Insert Fig.6 here
This binding stoichiometry was further confirmed by Benesi-Hildebrand plot
[48-50]. When assuming a 2:1 stoichiometry between RL and Fe3+, the following
equation is obtained:
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where F0, F, and Fmax are the emission intensities in the absence of, at a series of
intermediate concentrations of and at an infinite concentration of Fe3+, respectively;
Ka is the binding constant.
When 1/(F-F0) was plotted as a function of 1/[Fe3+]2, a linear relationship was
obtained (y = A + Bx), and Ka was calculated from A/B. As shown in Fig. 6(b), the
plot showed a good linear relationship (R2 = 0.9996), which further confirmed the 2:1
stoichiometry between RL and Fe3+ and the binding constant Ka was calculated to be
4.67 × 108 M-2, from which it is inferred that RL possessed high affinity towards Fe3+.
Besides, MS of RL on the addition of Fe3+ was acquired. As shown in Fig. S6,
without the combination of Fe3+, the peak m/z 570.3 corresponded to that of [RL+H]+
(the calculated value was m/z 570.2864; with the combination of RL and Fe3+, a peak
appears at m/z 398.2, which might be attributed to the three-charged complex
[2RL+Fe]3+ (Scheme 3) ( the calculated value was m/z = 1194.4914/3 = 398.1638).
These results also confirmed the 2:1 stoichiometry between RL and Fe3+.
3.9 Cell imaging studies
Further applicability of RL as a chemosensor for Fe3+ in vitro cell studies was also
evaluated. HeLa cells were chosen as the sample. The cells were incubated with RL
o
(10 ꢀM) in the growth medium for 30 min at 37 C, which led to very weak
fluorescence when determined by laser scanning confocal microscopy (Fig. 7(b)); in
contrast, a bright fluorescence was detected for those cells incubated with Fe3+ (10 ꢀM)
o
for 30 min at 37 C, followed by incubation with RL (10 ꢀM) under the same
conditions (Fig. 7(e)). The phase-contrast images in the bright fields of the cells (Fig.
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7(a) (d)) showed that the cells were viable during the experiments, which indicated
that RL might be cell-permeable and non-toxic to the living HeLa cells.
Insert Fig.7 here
To further evaluate the cytotoxicity of RL, cell viability was determined by an
MTT assay in HeLa cells incubated with different concentrations of RL (10, 20, 50,
100 ꢀM). As shown in Fig. 8, even at a reasonably high concentration of RL (20 ꢀM)
for 24 h, the cell viability value still remained more than 80%. These results suggested
that RL might be suitable to be used as a potential chemosensor for detecting Fe3+ in
biological samples.
Insert Fig.8 here
4. Conclusions
In summary, the synthesis of a water-soluble chemosensor RL was presented,
which showed excellent labeling and monitoring of Fe3+ in cellular systems with high
selectivity, sensitivity and anti-interference capacity. RL displayed an apparent and
quick response to Fe3+ with significant changes in UV-vis and fluorescence spectra
while other metal ions have nearly no interference on this specific recognition of Fe3+.
The 2:1 stoichiometry between RL and Fe3+ was proposed based on a Job’s plot and
MS of [2RL+Fe]3+. Besides, the binding constant between RL and Fe3+ was
calculated by the Benesie-Hildebrand equation. In addition, the cell imaging of RL
with Fe3+ was studied in HeLa cells, the results revealed that the chemosensor RL
was highly effective tools for Fe3+ labeling and could detect Fe3+ sensitively at the
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cellular level, which will be critically important to understand the functions of Fe3+ in
related cells and biological organs in the future.
Acknowledgments
This work is sponsored by the Natural Science Foundation of Shanghai (No.
16ZR1408000), and the National Key Program of China (No.
2016YFA0200302).
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Scheme captions:
Scheme 1 Synthesis procedure for the chemosensor RL
.
Scheme 2 Proposed mechanism of the RL response to pH changes.
Scheme 3 Proposed complexation mechanism of RL with Fe3+.
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ACCEPTED MANUSCRIPT
Scheme 1 Synthesis procedure for the chemosensor RL
.
Scheme 2 Proposed mechanism of the RL response to pH changes.
Scheme 3 Proposed complexation mechanism of RL with Fe3+.
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ACCEPTED MANUSCRIPT
Figure captions:
Fig. 1 Time-dependent fluorescence intensity change
of RL (10ꢀM)
at 589 nm
with Fe3+ (180 ꢀM) in
, λ = nm.
520
DMSO/H2O solution
ex
(10ꢀM)
Fig. 2 Variation of fluorescence intensity at 589 nm of RL solution
with and
without Fe3+
as a function of pH,
(180 ꢀM) in DMSO/H2O solution (1:1, v/v)
λ =
520
ex
nm
.
Fig. 3 UV-vis spectra (a) and fluorescence spectra (b) of RL (10 µM) in DMSO/H2O
3+
λ =
nm
solution with gradual increase of Fe (0-180 µM) ,
520 . Inset: the photos of
ex
RL solution before and after the addition of Fe3+ (180 µM) under the visible light
(a) and a 365 nm ultraviolet lamp (b).
(λ =
520
nm)
, colorimetric
Fig. 4 UV-vis spectra (a) and fluorescence spectra (b)
ex
erformance (c) and fluorescence performance (d) (
p
under a 365 nm ultraviolet
lamp of RL (10 ꢀM) in DMSO/H2O solution in the presence of di erent metal
From left to
)
ff
ions (180
right(c,d):
ꢀ ꢀM for the other metals ions).
M for Fe3+ and 900
+
+
+
2+
2+
2+
3+
2+
2+
3+
3+
Free, Li , Na , K , Mg , Ca , Ba , Al , Sn , Pb , Bi , Fe ,
Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+.
µM
Fig. 5 Anti-interference capacity of RL (10
in DMSO/H2O solution: 180
) in the presence of various metal ions
ꢀ
M for Fe3+ and 900
ꢀM for the other metal ions.
3+
+
+
+
2+
2+
2+
3+
2+
2+
From left to right:
Free, Fe , Li , Na , K , Mg , Ca , Ba , Al , Sn , Pb ,
Bi3+, Cr3+, Mn2+, Co2+, Ni2+, Pd2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+. λex=520 nm.
3+
µM and
Fig. 6 (a) Job’s plot with a total concentration of [RL] and [Fe ] was 50
(b)
λex=520
Benesi-Hilderbrand plot in DMSO/H2O solution (Fluorescence spectra,
25

ACCEPTED MANUSCRIPT
nm
).
Fig. 7 Fluorescence images of HeLa cells treated with RL (10 ꢀM, Top) and RL/ Fe3+
(10 ꢀM/10 ꢀM, Bottom): Bright field image (Left); fluorescence image (Middle); and
overlay of bright field and fluorescence image (Right).
Fig. 8 Cell viability values (%) estimated by MTT proliferation test versus different
incubation concentrations of RL. HeLa cells were cultured in the presence of RL (10,
20, 50, 100 ꢀM).
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ACCEPTED MANUSCRIPT
Fig. 1 Time-dependent fluorescence intensity change
of RL (10ꢀM)
at 589 nm
with Fe3+ (180 ꢀM) in
, λ = nm.
520
DMSO/H2O solution
ex
(10ꢀM)
Fig. 2 Variation of fluorescence intensity at 589 nm of RL solution
with and
without Fe3+
as a function of pH,
(180 ꢀM) in DMSO/H2O solution (1:1, v/v)
λ =
520
ex
nm
.
27

ACCEPTED MANUSCRIPT
Fig. 3 UV-vis spectra (a) and fluorescence spectra (b) of RL (10 µM) in DMSO/H2O
3+
λ =
nm
solution with gradual increase of Fe (0-180 µM),
520 . Inset: the photos of
ex
RL solution before and after the addition of Fe3+ (180 µM) under the visible light
(a) and a 365 nm ultraviolet lamp (b).
(λ =
520
nm)
, colorimetric
Fig. 4 UV-vis spectra (a) and fluorescence spectra (b)
ex
erformance (c) and fluorescence performance (d) (
p
under a 365 nm ultraviolet
lamp of RL (10 ꢀM) in DMSO/H2O solution in the presence of di erent metal
From left to
)
ff
ions (180
ꢀ
M for Fe3+ and 900
ꢀ
M for the other metals ions).
28