A highly sensitive and selective fluorescent probe for Fe3+ containing two rhodamine B and thiocarbonyl moieties and its application to live cell imaging

Article abstract of DOI:10.1016/j.tet.2019.01.029

A novel turn-on rhodamine B-based fluorescent chemosensor (RBCS) was designed and synthesized by reacting N-(rhodamine B)lactam-1,2-ethylenediamine and carbon disulfide. Upon addition of Fe³⁺ in EtOH/H₂O solution (2:1, v/v, HEPES buffer, 0.6 mM, pH 7.20), the RBCS displayed a significant fluorescence enhancement at 582 nm and a dramatic color change from colorless to pink, which can be detected by the naked eye. Significantly, the RBCS exhibited a highly selective and sensitive ability toward Fe³⁺. The detection limit of the probe was 2.05 × 10^?7 M. Job's plot indicated the formation of 1:1 complex between the RBCS and Fe³⁺. Moreover, the practical use of the RBCS is demonstrated by its application in the detection of Fe³⁺ in HeLa cells.

Full text of DOI:10.1016/j.tet.2019.01.029

Tetrahedron xxx (xxxx) xxx
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A highly sensitive and selective fluorescent probe for Fe3þ containing
two rhodamine B and thiocarbonyl moieties and its application to live
cell imaging
,
, *
Zhigang Gao a, Chun Kan c, Haibo Liu a, Jing Zhu b **, Xiaofeng Bao a
a School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei, Nanjing, 210094, PR China
b Department of Pharmacy, Nanjing University of Chinese Medicine, 138 Xialin Dadao, 210023, PR China
c College of Science, Nanjing Forestry University, Nanjing, 210037, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel turn-on rhodamine B-based fluorescent chemosensor (RBCS) was designed and synthesized by
reacting N-(rhodamine B)lactam-1,2-ethylenediamine and carbon disulfide. Upon addition of Fe3þ in
EtOH/H2O solution (2:1, v/v, HEPES buffer, 0.6 mM, pH 7.20), the RBCS displayed a significant fluores-
cence enhancement at 582 nm and a dramatic color change from colorless to pink, which can be detected
Received 18 November 2018
Received in revised form
11 January 2019
Accepted 15 January 2019
Available online xxx
by the naked eye. Significantly, the RBCS exhibited a highly selective and sensitive ability toward Fe3þ
.
The detection limit of the probe was 2.05 ꢀ 10ꢁ7 M. Job's plot indicated the formation of 1:1 complex
between the RBCS and Fe3þ. Moreover, the practical use of the RBCS is demonstrated by its application in
the detection of Fe3þ in HeLa cells.
Keywords:
Rhodamine B
Fe(Ⅲ) ions
© 2019 Elsevier Ltd. All rights reserved.
Fluorescent chemosensors
Cell imaging
1. Introduction
fluorescence intensity, and only a few probes have been applied in
biological systems [21e24]. Therefore, the development of new
Fe3þ is the most of essential metal ion in biological systems and
plays a crucial role in numerous physiological and pathological
processes, such as in cellular metabolism, in DNA and RNA syn-
theses, in enzymatic catalysis involved in the mitochondria, in
carrying oxygen by heme [1e8]. Both overloading and deficiency of
Fe3þ can induce serious diseases in the living body, such as liver and
kidney damage, Parkinson's and Alzheimer's diseases, heart failure,
and diabetes [9e12]. Therefore, design of fluorescent chemsensors
with sensitivity and selectivity for detecting Fe3þ is of great
importance. However, applying fluorescent techniques to detect
Fe3þ is very difficult, because Fe3þ possesses fluorescence
quenching ability. Fluorescence enhancement through chelation of
Fe3þ with any fluorophore is a challenging mission in vitro and vivo.
Recently, several groups have developed some xanthene-based
fluorescent Fe3þ probes [13e20]. However, a lot of Fe3þ probes
selective Fe3þ fluorescent chemsensors for imaging in living cells is
still an interesting and challenging work.
Since rhodamine was first synthesized by Noelting and Dzie-
wonsky in 1905, the rhodamine-based fluorescent chemsensors
have received increasing interest due to their excellent photo-
physical properties, such as high fluorescence quantum yield, long-
wavelength and large molar extinction coefficient [25e28]. More-
over, it is well known that rhodamine derivatives with a spi-
rolactam structure are non-fluorescent [29e31]; however, when
rhodamine derivatives bind to metal ions or protons, ring-opened
amides give rise to strong fluorescence emission [32e35]. On the
basis of the previous works, we are attempted to chose rhodamine
B as the fluorescent group because of its good fluorescence prop-
erties and water solubility. Meanwhile, as a recognition group,
carbon disulfide can provide the S atoms as excellent coordinating
site. Thus, we chose thiocarbonyl as a linker to effectively combine
two rhodamine B moieties to form a compound RBCS with strong
fluorescence emission. In this paper, we designed and synthesized a
novel turn-on fluorescent chemsensor RBCS (Fig. 1). The probe
exhibited a high selectivity and sensitivity toward Fe3þ ions.
Moreover, the fluorescence microscopy experiments also indicated
have several limits, such as poor selectivity to Fe3þ
, weak
* Corresponding author.
** Corresponding author.
E-mail addresses: zhujing1227@hotmail.com (J. Zhu), baoxiaofeng@njust.edu.cn
(X. Bao).
https://doi.org/10.1016/j.tet.2019.01.029
0040-4020/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Z. Gao et al., A highly sensitive and selective fluorescent probe for Fe3þ containing two rhodamine B and thiocarbonyl
moieties and its application to live cell imaging, Tetrahedron, https://doi.org/10.1016/j.tet.2019.01.029

2
Z. Gao et al. / Tetrahedron xxx (xxxx) xxx
The pH levels of stock solutions were measured using a PHS-25C
Precision pH/mV Meter (Aolilong, Hangzhou, China). UVevis and
fluorescence spectra were obtained on a UV-3600 UVeviseNIR
spectrophotometer (Shimadzu, Japan) and an Edinburgh FLS920
fluorescence spectrophotometer (Livingston, UK), respectively, at
room temperature.
2.3. Preparation and purification of RBCS
Fig. 1. Chemical structure of RBCS.
The procedure of synthesis of RBCS was shown in Scheme 1.
that RBCS can be used as a fluorescent chemsensor for the detec-
2.3.1. Synthesis of compound N-(rhodamine B)lactam-1,2-
ethylenediamine (1)
tion of Fe3þ in living cells.
A
mixture of rhodamine B (4.78 g, 10 mmol) and 1,2-
2. Experimental section
ethylenediamine (3.25 mL, 50 mmol) in 100 mL ethanol was stir-
red at 80 ꢂC for 12 h, when TLC analysis indicated completion of
reaction. After cooling to room temperature, the organic solvent
was evaporated under reduced pressure. The residue was dissolved
in CH2Cl2 and then washed with H2O and brine. The organic layer
was dried with anhydrous magnesium sulfate. After removal of the
solvent, the crude product was purified by column chromatography
with MeOHeCH2Cl2 (5:95, v/v) to afford the pure product 1 (pink
2.1. General information
All reagents and organic solvents used were of ACS grade or
higher and were used without further purification. Unless other-
wise noted, all chemicals were purchased from J＆K Scientific
(Shanghai, China) and were used as received. All solvents were of
analytical grade, and double-distilled water was used in all of the
experiments. The solutions of metal ions were prepared from their
chloride or nitrate salts. The reactions were performed under an
argon atmosphere using standard Schlenk techniques. The chemi-
solid, 4.11 mg, 85%). 1H NMR (CDCl3),
d
7.90 (dd, J ¼ 5.6, 3.0 Hz, 1H),
7.61e7.38 (m, 2H), 7.09 (dd, J ¼ 5.6, 2.9 Hz, 1H), 6.43 (d, J ¼ 8.8 Hz,
2H), 6.37 (d, J ¼ 2.5 Hz, 2H), 6.27 (dd, J ¼ 8.9, 2.6 Hz, 2H), 3.38e3.27
(m, 8H), 3.19 (t, J1 ¼6.65 Hz, J2 ¼ 6.6 Hz, 2H), 2.41 (t, J1 ¼6.65 Hz,
J2 ¼ 6.6 Hz, 2H), 1.16 (t, J1 ¼7.0 Hz, J2 ¼ 7.1 Hz, 12H) ppm. 13C NMR
cal shifts are reported in
d units (ppm) downfield relative to the
chemical shift of tetramethylsilane. The abbreviations br, s, d, t and
m denote broad, singlet, doublet, triplet and multiplet, respectively.
Thin-layer chromatography was performed on a HAIYANG silica gel
F254 plate, and compounds were visualized under UV light
(CDCl3), d 168.59, 153.35, 148.79, 132.38, 131.20, 128.64, 128.02,
123.80, 122.71, 108.13, 105.65, 97, 64.91, 44.36, 44.06, 40.73,
12.56 ppm.
(
l
¼ 254 nm). Column chromatography was performed using
2.3.2. Synthesis of compound RBCS
HAIYANG silica gel (type: 200e300 mesh ZCX-2).
To a stirred solution of compound 1 (200 mg, 0.42 mmol) in
EtOH (10 mL) was added carbon disulfide at 0 ꢂC for 40 min, then
heated at 78 ꢂC for 6 h, when TLC analysis indicated completion of
reaction. After removal of the solvent under reduced pressure, the
crude product was purified by column chromatography with
MeOHeCH2Cl2 (1:99, v/v) to yield a pink solid RBCS (167.9 mg,
2.2. Instruments
1H NMR (500 MHz) and 13C NMR (126 MHz) spectra were
recorded on an Avance 500 spectrometer (Bruker: Billerica, MA,
USA). Mass spectra were obtained with a Finnigan TSQ Quantum
LC/MS Spectrometer. High-resolution mass spectra (HRMS) were
acquired under electron ionization conditions with a double-
focusing high-resolution instrument (Autospec; Micromass Inc.).
79%). 1H NMR (CDCl3)
d
7.86 (d, J ¼ 6.08 Hz, 2H), 7.46e7.38 (m, 4H),
7.08e7.04 (m, 2H), 6.45 (d, J ¼ 8.74 Hz, 4H), 6.37 (d, J ¼ 2.17 Hz, 4H),
6.28 (dd, J ¼ 8.93, 2.55 Hz, 4H), 3.40e3.27 (m, 20H), 3.22 (s, 4H),
1.26 (s, 2H), 1.15 (t, J ¼ 7.25 Hz, 24H) ppm.13C NMR (CDCl3)
d 181.08,
Scheme 1. Synthesis of RBCS.
Please cite this article as: Z. Gao et al., A highly sensitive and selective fluorescent probe for Fe3þ containing two rhodamine B and thiocarbonyl
moieties and its application to live cell imaging, Tetrahedron, https://doi.org/10.1016/j.tet.2019.01.029

Z. Gao et al. / Tetrahedron xxx (xxxx) xxx
3
channel under Fluorescent Inverted microscope.
3. Results and discussion
3.1. Design and synthesis of RBCS
The synthesis procedure of RBCS is illustrated in Scheme 1.
Reaction of rhodamine B with 1,2-ethylenediamine in EtOH affor-
ded compound 1 in 85% yield, as described previously [36]. It was
then used for the next reaction with carbon disulfide in EtOH at 0 ꢂC
for 40 min, then heated at 78 ꢂC for 6 h to give RBCS in a 79% yield.
The structure of RBCS was confirmed by 1H NMR, 13C NMR and
HRMS (Supporting materials).
3.2. pH stability of RBCS
To determine a suitable pH range for Fe3þ sensing in EtOH/H2O
(2:1, v/v) solutions, we performed pH titration of probe RBCS. The
Fig. 2. The effect of the pH on the fluorescence of RBCS (20 mM) in EtOH/H2O solutions
(2:1, v/v). The excitation and emission wavelengths were 558 nm and 582 nm,
respectively.
fluorescence intensities of RBCS were measured at lex em
/
¼ 558/
582 nm in different pH values solutions, range of 2.0e11.0. As
shown in Fig. 2, In the pH range from 5.0 to 11.0, there was no in-
fluence on the fluorescence emission. However, at the pH between
3.0 and 5.0, fluorescence intensity increased significantly at
582 nm, which meant that ring-opening process of the spirocyclic
moiety of rhodamine B was formed (Scheme 2). Moreover, the
fluorescence intensities of RBCS at 582 nm reached maximum at
169.11, 153.91, 153.22, 148.90, 132.65, 130.48, 128.53, 128.04, 123.18,
122.94, 108.36, 104.15, 91.83, 77.31, 77.12, 76.86, 65.61, 44.35, 39.69,
29.68, 12.64 ppm. HRMS [Mþ1]þ found, 1011.5319, calculated for
C
61H71N8O4Sþ, 1010.5319.
2.4. Cell studies and fluorescence imaging
HeLa cells were cultured in Dulbecco's Modified Eagle's Medium
(High Glucose) supplemented with 10% FBS (Fetal Bovine Serum),
100 units/mL penicillin and 100 units/mL streptomycin in an at-
mosphere of 5% CO2/95% air incubator at 37 ꢂC. Cytotoxicity of RBCS
was determined by an MTT assay. Briefly, HeLa Cells were cultured
in 96-well plates at a density of 9 ꢀ 103 cells per well and incubated
in an atmosphere of 5% CO2/95% air at 37 ꢂC for 12 h. Then the cells
were incubated with different concentrations of RBCS for 24 h.
After that, cells were incubated with 5 mg/mL of MTT for 4 h at
37 ꢂC, the supernatant was then discarded, and DMSO (150
mL/well)
was added to dissolve the formazan. The absorbance was then
measured at 550 nm with a microplate reader (Tecan Infinite 200
PRO). Before fluorescence imaging, cells (1 ꢀ 105/mL) were seeded
in 24-well flat-bottomed plates in the incubator and allowed to
adhere for 2 h. For cell imaging, cells were incubated with
10
control experiment, cells were pretreated with 100
100 min, and then incubated with 10 M RBCS in culture medium
for 5 h. Cell imaging was then carried out after washing cells for 3
times. Finally, fluorescent imaging was recorded using the red
m
M RBCS in culture medium for 5 h in the incubator. In the
mM FeCl3 for
Fig. 3. Fluorescence spectra of RBCS (20 m mM, 5 equiv.) in an EtOH/
M) with Fe3þ (100
H2O (2:1, v/v, HEPES buffer, 0.6 mM, pH 7.20) solution. The excitation and emission
wavelengths were 558 and 582 nm, respectively. Inset: Plot of the fluorescence in-
tensities at 582 nm over a period of 250 min.
m
Scheme 2. Mechanism of the response of RBCS to pH changes.
Please cite this article as: Z. Gao et al., A highly sensitive and selective fluorescent probe for Fe3þ containing two rhodamine B and thiocarbonyl
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4
Z. Gao et al. / Tetrahedron xxx (xxxx) xxx
the pH 3.0 and became stable. All the above results suggested that
the RBCS was stable in the pH range of 5.0e11.0 with very low
background fluorescence. Thus, all photochemical experiments
were carried out in an EtOH/H2O (2:1, v/v, HEPES buffer, 0.6 mM, pH
7.20) solution.
3.4. UVevis spectral and fluorescence titration of RBCS
To evaluate the Fe3þ-selectivity, the UVevis absorption study of
the probe RBCS toward other metal ions (Agþ, Ba2þ, Ca2þ, Co2þ
,
Cr3þ, Cu2þ, Fe3þ, Hg2þ, Kþ, Mg2þ, Mn2þ, Naþ, Ni2þ, Pb2þ,Sn2þ and
Zn2þ) were explored. As shown in Fig. 4, the probe RBCS (20
M)
m
did not show any observable absorption in the solution. No obvious
changes in spectra were observed upon the addition of other metal
ions. However, upon the addition of 5 equiv. Fe3þ, a new absorption
peak was observed at 558 nm under the identical condition.
To further evaluate the selectivity of RBCS for Fe3þ, fluorescence
titration of RBCS with metal ions was performed. As shown in Fig. 5,
3.3. Response time of RBCS
To evaluate the sensitivity of RBCS towards Fe3þ, the response
time of RBCS with Fe3þ was detected in the solution. The change in
the fluorescence intensity of RBCS over a period of 2 h was recor-
ded. As shown in Fig. 3, the fluorescence signal of the system at
when no metal ion was added to the solution of RBCS (20 mM),
582 nm increased rapidly after Fe3þ (100
(20
mM) was added into RBCS
almost no fluorescence signal was observed in the range from
570 nm to 700 nm. Upon the addition of 5 equiv. Fe3þ, a remarkable
fluorescence intensity was observed at 582 nm, which the excita-
tion wavelength was 558 nm, and the solution changed from
colorless to pink. In contract, no any apparent changes in
mM) solution in the first 40 min. The fluorescence intensity of
RBCS reached maximum 20 min later and stabilized thereafter.
Therefore, a 60 min reaction time was set in subsequent experi-
ments to ensure that the Fe3þ had enough time to sufficiently
chelate with the RBCS.
Fig. 6. Metal ions selectivity profiles of RBCS (20 mM) in the presence of various metal
ions in an EtOH/H2O solution (2:1, v/v, HEPES buffer, 0.6 mM, pH 7.20). Black bars
Fig. 4. UVevis absorbance spectra of RBCS (20 mM) in EtOH/H2O (2:1, v/v, HEPES
represent the fluorescence intensity (582 nm) of RBCS in the presence of 5 equiv. of the
buffer, 0.6 mM, pH 7.20) in the absence and presence of 5 equiv. of various metal ions:
Agþ, Ba2þ, Ca2þ, Co2þ, Cr3þ, Cu2þ, Fe3þ, Hg2þ, Kþ, Mg2þ, Mn2þ, Naþ, Ni2þ, Pb2þ and
Zn2þ. The absorption wavelength for RBCS was 558 nm.
metal ions: Agþ, Ba2þ, Ca2þ, Zn2þ, Kþ, Naþ, Mg2þ, Hg2þ, Cu2þ, Liþ, Mn2þ, Pb2þ, Ni2þ
,
Cr3þ and Sn2þ in the absence of Fe3þ. The red bars represent the fluorescence intensity
(582 nm) in the presence of various metal ions after the addition of Fe3þ. The excitation
wavelength is 558 nm.
Fig. 5. Fluorescence spectra of RBCS (20 mM) in an EtOH/H2O solution (2:1, v/v, HEPES
buffer, 0.6 mM, pH 7.20) in the absence and presence of 5 equiv. of various metal ions:
Agþ, Ba2þ, Ca2þ, Co2þ, Cr3þ, Cu2þ, Fe3þ, Fe2þ, Hg2þ, Kþ, Mg2þ, Mn2þ, Naþ, Ni2þ, Pb2þ and
Zn2þ. The excitation and emission wavelengths were 558 nm and 582 nm, respectively.
Fig. 7. Fluorescence spectra of RBCS (20 mM) in an EtOH/H2O solution (2:1, v/v, HEPES
buffer, 0.6 mM, pH 7.20) upon the addition of different amounts of Fe3þ. The excitation
and emission wavelengths were 558 and 582 nm, respectively.
Please cite this article as: Z. Gao et al., A highly sensitive and selective fluorescent probe for Fe3þ containing two rhodamine B and thiocarbonyl
moieties and its application to live cell imaging, Tetrahedron, https://doi.org/10.1016/j.tet.2019.01.029

Z. Gao et al. / Tetrahedron xxx (xxxx) xxx
5
hardly influenced by other commonly co-existent metal ions. It
meant that RBCS could be used as a selectivity Fe3þ fluorescent
chemosensor.
In order to address the sensitivity of RBCS towards Fe3þ, the
fluorescence titration of RBCS (20
shown in Fig. 7, the fluorescence spectrum of probe RBCS (20
m
M) with Fe3þ was carried out. As
mM) in
the absence of Fe3þ in the solution displayed very weak fluores-
cence intensity at 582 nm. With continuous addition of FeCl3
(0e100 equiv.), the fluorescence intensity at 582 nm increased
smoothly and gradually peaked, which resulted in a color change
from colorless to pink. The results were attributed to the Fe3þ
induced the formation of the ring-opened amide form of RBCS. The
emission intensity reached a steady state after the addition of 45
equiv. of Fe3þ. Over a 300-fold fluorescence enhancement was
observed under saturation conditions.
3.5. Stability of RBCS-Fe3þ complex over other anions
Fig. 8. Changes in the fluorescence spectra of the RBCS-Fe3þ complex (20
mM þ 5
equiv. Fe3þ) at 582 nm in an EtOH/H2O solution (2:1, v/v, HEPES buffer, 0.6 mM, pH
The binding ability of RBCS-Fe3þ in the presence of different
anions was investigated using fluorescence spectroscopic. As
shown in Fig. 8, upon addition of 5 equiv. of various other anions
(Iꢁ, Fꢁ, Brꢁ, H2PO4ꢁ, CO32ꢁ, S2ꢁ, NO3ꢁ, BrO3ꢁ, SO24ꢁ, S2O82ꢁ), the fluo-
7.20) solution upon the addition 5 equiv. of different anions (lex ¼ 558 nm).
rescence intensity of the RBCS-Fe3þ (20
mM) complex didn't
decrease significantly. The results indicated that the detection of
Fe3þ will not be interfered by the anions under the same conditions.
3.6. Job's plot, binding constant and detection limit
The binding stoichiometry was proven with Job's plot, which
Fig. 9. Job's plot of RBCS in an EtOH/H2O solution (2:1, v/v, HEPES buffer, 0.6 mM, pH
7.20) with a total concentration of [RBCS] þ Fe3þ ¼ 20
mM. The excitation and emission
wavelengths were 558 and 582 nm, respectively.
fluorescence intensity at 582 nm upon the addition of 5 equiv. of
other metal ions. All the results revealed that the RBCS has high
selectivity in the presence of other competing metal ions. In addi-
tion, a competitive experiment of coexisting ions was performed
under the identical condition. As shown in Fig. 6, no significant
changes in emission of the obtained with or without other metal
ions. The results revealed that the recognition of Fe3þ by RBCS was
Fig. 10. BenesieHildebrand plot (lem ¼ 582 nm) of 1/(F-F0) versus 1/[Fe3þ] based on a
1:1 association stoichiometry between RBCS and Fe3þ
.
Scheme 3. Proposed complexation mechanism of RBCS with Fe3þ
.
Please cite this article as: Z. Gao et al., A highly sensitive and selective fluorescent probe for Fe3þ containing two rhodamine B and thiocarbonyl
moieties and its application to live cell imaging, Tetrahedron, https://doi.org/10.1016/j.tet.2019.01.029

6
Z. Gao et al. / Tetrahedron xxx (xxxx) xxx
was generated by continuously varying the mole fraction of Fe3þ
The detection limit was calculated with the following equation
detection limit ¼ 3SD/S, where SD is the standard deviation of the
blank and S is the slope of the calibration curve. The fluorescence
emission spectrum of RBCS was measured 10 times, and the stan-
dard deviation of the blank measurement was then calculated
(Fig. 11). To obtain the slope, the ratio of the fluorescence intensity
at 582 nm was plotted verses the concentration of Fe3þ. The
from 0 to 1 in a solution of [Fe3þ] þ [RBCS] with a total concen-
tration of 20 mM. Job's plot analysis indicated host-guest molecular
complexation, the RBCS with Fe3þ ions form 1:1 stoichiometry
(Fig. 9). Therefore, we hypothesized that a possible binding
mechanism between RBCS and Fe3þ, as Scheme 3 shows.
Using the BenesieHildebrand expression: F-F0 ¼ △F ¼ [
Fe3þ](Fmax-F0)/(1/K
a
þ[ Fe3þ]) based on a 1:1 stoichiometry [37]. A
detection limit of Fe3þ ions with the probe was 0.205
mM.
good
linear
regression
equation
was
obtained
as
y ¼ 9.95477 ꢀ 10ꢁ5þ3.61508 ꢀ 10ꢁ9 x based on the fluorescence
titration curves of RBCS with Fe3þ ion (Fig. 10). Thus, the binding
constant was calculated to be 2.75 ꢀ 104 Mꢁ1 in the solution, which
indicated that Fe3þ with RBCS had a strong binding capacity.
3.7. 1H NMR study of RBCS
To further elucidate the binding mode of RBCS with Fe3þ 1H
,
NMR spectra of RBCS in the presence and absence of Fe3þ were
studied. Fe3þ is paramagnetic metal ion, and its complex with RBCS
can affect the proton signals that are close to the Fe3þ binding site.
As shown in Fig. 12, upon 0.2 equiv. of Fe3þ was added to RBCS in
Fig. 11. Fluorescence intensity at 582 nm of RBCS (20
m
M) in an EtOH/H2O solution
Fig. 13. Cytotoxicities of the probe RBCS in HeLa cells. Cells were treated with different
concentrations of probe RBCS for 24 h, and the cell viabilities were determined by the
MTT assay. Data are expressed as the mean SD.
(2:1, v/v, HEPES buffer, 0.6 mM, pH 7.20) with different amounts of Fe3þ. The excitation
wavelength is 558 nm.
Fig. 12. 1H NMR spectra (500 MHz, 298k, CD3OD/D2O (5:1, v/v)) of (A) RBCS and (B) RBCSþ0.2 equiv. Fe3þ
.
Please cite this article as: Z. Gao et al., A highly sensitive and selective fluorescent probe for Fe3þ containing two rhodamine B and thiocarbonyl
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Z. Gao et al. / Tetrahedron xxx (xxxx) xxx
7
Fig. 14. Fluorescence images of HeLa cells: (AeC): the bright field; (DeF): fluorescence images; (D) Cells treated with no added probe RBCS or FeCl3; (E): Cells treated with only
10 M probe RBCS for an additional 5 h; (F): Cells were incubated with 100 M FeCl3 for 2 h followed by treated with 10 M probe RBCS for an additional 5 h. Condition: an at-
mosphere of 5% CO2, 95% air at 37 ꢂC.
m
m
m
CD3OD:D2O (5:1, v/v) solution, the proton signals of Hb and Hc,
showed apparent downfield shifts from multiplet peaks centered at
fluorescent response to Fe3þ with high selectivity and sensitivity
and the detection limit of RBCS was 2.05 ꢀ 10ꢁ7 M. The 1:1 coor-
dination mode was proposed based on Job's plot experiment. In
addition, RBCS was successfully applied to detect Fe3þ in living
cells. Based on the experimental results, RBCS could potentially
serve as an effective tool for investigation of Fe3þ in biosystems.
7.54 ppm and 7.50 ppm to board peaks centered at 7.71 (△
d
¼ 0.17
and 0.21 ppm, respectively.). These shifts originate from the Fe3þ
-
induced ring-opening process of the rhodamine B spirocycle.
What's more, the proton signals of Hh displayed significant shifts
from the multiplet peak centered at 3.13 ppm to the broad peak
centered at 3.21 ppm (△
d
¼ 0.08 ppm), because of the coordination
Acknowledgements
of RBCS with Fe3þ. Slight changes in the signals from the protons on
the imidazole ring were also observed. The above results indicated
the mode of interaction proposed in Scheme 3 between RBCS and
Fe3þ, in which Fe3þ may be coordinated with the secondary amine
nitrogen atoms of RBCS and the oxygen atom of the amide and the
sulfur atom of the thiocarbonyl.
This research was supported by NSF China (81371616), the Key
Project of Jiangsu Province for Fundamental Research and Devel-
opment (BE2018717) and the Six Talent Peak Award
(2015SWYY013). We thank the NJUST Sensor Lab for providing fa-
cilities for UVevis studies.
3.8. Cell imaging studies of RBCS
Appendix A. Supplementary data
The cytotoxicity assays was evaluated using MTT assays. As
shown in Fig. 13, no apparent loss of cell viability was found for
HeLa cells after 24 h of exposure to the RBCS at concentrations
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2019.01.029.
below 20 mmol/L. This result indicates that the RBCS has low
cytotoxicity and good biocompatibility under the experimental
conditions.
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Please cite this article as: Z. Gao et al., A highly sensitive and selective fluorescent probe for Fe3þ containing two rhodamine B and thiocarbonyl
moieties and its application to live cell imaging, Tetrahedron, https://doi.org/10.1016/j.tet.2019.01.029

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