Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues

Article abstract of DOI:10.1016/j.tetlet.2019.04.025

A Fe³⁺-specific probe (N-TC) based on Rhodamine B was designed and synthesized. N-TC has a good spectral response to Fe³⁺ in the EtOH/H₂O solution (1:1, v/v, HEPES, 0.5 mM, pH = 7.38) with low detection limits and high binding constants. N-TC displays the reversible “turn-off-on” fluorescence response with 1:1 binding stoichiometry. It is further proven to be practical in sensitively monitoring trace Fe³⁺ in environmental water specimens. Biological experiments demonstrated that N-TC can be respectively used as a probe for detection of Fe³⁺ in living cells, animals and plant tissues.

Full text of DOI:10.1016/j.tetlet.2019.04.025

Accepted Manuscript
Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine
B based probe in vivo and plant tissues
Fan Song, Xiaotao Shao, Jing Zhu, Xiaofeng Bao, Lei Du, Chun Kan
PII:
S0040-4039(19)30362-4
https://doi.org/10.1016/j.tetlet.2019.04.025
TETL 50740
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 January 2019
9 April 2019
13 April 2019
Please cite this article as: Song, F., Shao, X., Zhu, J., Bao, X., Du, L., Kan, C., Reversible “turn-off-on” fluorescence
response of Fe(III) towards Rhodamine B based probe in vivo and plant tissues, Tetrahedron Letters (2019), doi:
https://doi.org/10.1016/j.tetlet.2019.04.025
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reversible “turn-off-on” fluorescence response of Fe(III) towards Rhodamine B
based probe in vivo and plant tissues
Fan Song a, Xiaotao Shao a, Jing Zhu b, Xiaofeng Bao c, Lei Du a, Chun Kan a,*
a College of Science, Nanjing Forestry University, 159 LongpanRoad, Nanjing 210037, China
b
Department of Pharmacy, Jiangsu Key Laboratory for Pharmacology and Safety Evaluation
of Chinese Materia Medica, Nanjing University of Chinese Medicine, 138 XianlinDadao,
Nanjing 210023, China
c School of Environmental and Biological Engineering, Jiangsu Key Laboratory of Chemical
Pollution Control and Resources Reuse, Nanjing University of Science and Technology, 200
Xiaolinwei, Nanjing 210094, China
ABSTRACT
A Fe3+-specific probe (N-TC) based on Rhodamine B was designed and synthesized. N-TC
has a good spectral response to Fe3+ in the EtOH/H2O solution (1:1, v/v, HEPES, 0.5mM,
pH=7.38) with low detection limits and high binding constants. N-TC displays the reversible
“turn-off-on” fluorescence response with 1:1 binding stoichiometry. It is further proven to be
practical in sensitively monitoring trace Fe3+ in environmental water specimens. Biological
experiments demonstrated that N-TC can be respectively used as a probe for detection of Fe3+ in
living cells，animals and plant tissues.
Keywords: Fluorescent probe; Water sample; Imaging; In vivo
Introduction
Metal ions are found everywhere in nature and play a key role in maintaining the
physiological balance of living organisms [1-3]. As a result of human production and activities, a
large amount of metal ions is generated. Thus, the atmosphere, water and soil in the environment
begin to be seriously polluted, which in turn poses a serious hazard to the health of the organism
[4-11]. Therefore, designing a chemical sensor that can detect metal ions is imminent. Because of
its excellent light stability and relatively large molar absorption coefficient, Rhodamine B has
become an important material for designing fluorescent probes and plays an extremely important
role in the detection of metal ions in the environment. In addition, Rhodamine B also possesses
excellent biocompatibility and permeability, as well as low cytotoxicity, allowing its detection
range to extend from the living organism to the organism [12-16]. Now Rhodamine B-type probes
have been widely used in medical diagnostics [17,18], environmental chemistry [19], materials
science and other fields [20-26].
Iron is abundant metal elements in the earth and plays an important role in the development of
1

modern society [27-30]. More importantly, iron is the most important more than 10 kinds of trace
elements necessary for the human body in. Iron is widely distributed in the human body and is
found in almost all organs, including blood, muscle, spleen, liver and bone marrow [31-37]. Iron
is an important component of hemoglobin, myoglobin and various enzymes. It is also an essential
trace element in human physiological processes, such as cell metabolism, oxygen metabolism,
electron transfer and DNA and RNA synthesis, proton transfer, maintenance of cell osmotic
pressure and acid-base balance in the regulation of physiological activity [38-44]. When the body
iron content deviates from normal or abnormal distribution, human health will be endangered.
Iron deficiency or excessive iron content in the human body can impair normal physiological
function [45-48]. Therefore, real-time detection of iron ions plays an important role in biological
and human health.
Herein, we designed a reversible “turn-off-on” fluorescent probes based on Rhodamine B for
Fe3+, which was named N-TC. N-TC was successfully used as a selective and sensitive
fluorescent and colorimetric probe for Fe3+. N-TC is further proven to be practical in sensitively
monitoring trace Fe3+ in environmental water specimens. Most importantly, biological
experiments showed that N-TC can be used as a fluorescent probe for the detection of Fe3+ in
living cells, zebrafish and plant tissues.
Results and discussion
Synthesis and characterization
The N-TC synthesis procedure is illustrated in Scheme 1. The intermediates 1 was
synthesized by using rhodamine B and ethylenediamine as reactants. Reaction of 1 with
pyridine-3-sulfonyl chloride afforded the probe N-TC. The structures of compound 1 and N-TC
1
were confirmed by H NMR, 13C NMR and ESI-MS (Supplementary Material). Taking into
account the solubility of probe in component solvent system, EtOH/H2O (1:1, v/v, HEPES,
0.5mM, pH=7.38) for N-TC in sensing Fe3+ was chosen as a test solution. N-TC was compared
with Fe3+ by UV-vis absorption and fluorescence emission to get the detailed concentration of
probes used in sensing Fe3+ was 20μM. Due to induction of probe by Fe3+, the structure of
spironolactam is constantly destroyed, the five-membered ring opens and produces strong
fluorescence. Evaluation of pH response of N-TC by acid-base titration (Fig. 1A). The results
show that N-TC was insensitive to pH from 6.0 to 12.0 in the EtOH/H2O (1:1, v/v, HEPES,
0.5mM, pH=7.38) solution and may have been able to sense Fe3+ under approximately
physiological conditions with very low background fluorescence. Moreover, the response time of
N-TC in sensing Fe3+ was investigated (Fig. 1B). In fluorescence emission spectra, with the
increase of time, the fluorescence intensity of N-TC at 582 nm gradually increased and until
maintained stable, which indicates that N-TC are sensitive probes for Fe3+. In order to further
understand the interaction between N-TC and Fe3+, the Fe3+ titration against N-TC was also
monitored by the fluorescence spectra (Fig. S5). In absence of Fe3+, N-TC had no clear
2

fluorescent intensity at 582 nm (λex = 560nm). With the increasing concentration of Fe3+, the
fluorescence is obviously enhanced. The measurement solution underwent color change from
colorlessness to the light pink and reach saturation thereafter. The change of N-TC towards Fe3+
can be ascribed to the formation of the ring-opened amide form of N-TC upon Fe3+ binding. Thus,
N-TC can act as quantitative determination methods of measuring Fe3+ concentrations. The
association constant Ka of N-TC/Fe3+ complex could be calculated based on the revised
Benesi-Hildebrand equation as 1.15×104 M-1 (Fig. S6A). On basis of fluorescence titration, the
detection limit of N-TC for Fe3+ also could be determined to be 0.201μM (Fig. S6B). Thus, N-TC
has higher association constant and lower detection limit.
Scheme 1. Synthesis of N-TC.
Fig. 1. (A) Effect of pH on the fluorescence of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES,
0.5mM, pH=7.38) in the absence and presence of Fe3+ (100μM). (B) Fluorescence spectra of
N-TC (20μM) with Fe3+ (100μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM, pH=7.38) solution.
The excitation and emission wavelengths were 560 nm and 582 nm, respectively. Inset: (B) Plot
of the fluorescence intensities at 582 nm over a period of 100 min.
Ions selectivity
To evaluate the selectivity of N-TC, common metal cations (Fe3+, Fe2+, Al3+, Ba2+, Ca2+, Co2+,
Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+) was tested in aqueous solutions by
UV–vis absorption spectroscopy and fluorescence spectrometry. The UV–vis absorption
spectroscopy of N-TC showed best strongest absorption behavior to Fe3+ along with significant
color changes of test solution (Fig. 2A). However, under the same condition, no significant
response was observed after other metal ions were implanted. As far as N-TC is concerned, only
3

absorption behavior to Al3+ of other cations induced a much weaker change than Fe3+. Without the
addition of metal ions in the fluorescence spectrum, N-TC displayed no obvious fluorescence and
showed the form of spirolactam in the experimental system. When Fe3+ was added, the
fluorescence of N-TC was significantly enhanced. When other metal ions were added
respectively, no obvious fluorescence enhancement was revealed. Only other few cations (Al3+)
induced a much weaker fluorescent intensity change than Fe3+ (Fig. 2B). The results indicated
that the interaction between N-TC and Fe3+ resulted from the formation of the strongly
fluorescent ring-opened N-TC- Fe3+ complex. Thus, N-TC had distinctive selectivity to Fe3+ and
N-TC could be served as highly specific-Fe3+ sensing probe.
To further confirm the selectivity of N-TC to Fe3+, the fluorescence competition experiments
were also conducted by additionally mixing Fe3+ to N-TC solution in presence of other metal ions
(Fig. S7). No significant variation in the emission of the N-TC-Fe3+ complex was observed when
compared with the results obtained with or without other metal ions. The detection of Fe3+ is not
interfered with other metal ions. The above results show that N-TC could be used as selective
Fe3+ fluorescent probe.
We further studied the effects of various anions on the rupture of the N-TC-Fe3+ complex to
regenerate N-TC. Fluorescence spectroscopic experiments of the N-TC-Fe3+ complex was
2-
-
2-
-
-
performed in the presence of different anions such as SO4 , Br-, BrO3 , B4O7 , F-, H2PO4 , NO3 ,
S2O8 and IO3 (Fig. S8). The results showed that the fluorescence intensity of the N-TC-Fe3+
complex didn’t change significantly after adding various anions to the solution containing
N-TC-Fe3+ complex, thus these anions failed to produce any discernible spectral change under
the identical conditions. The above conclusion indicates that the N-TC-Fe3+ complex can still
exist stably when anions are present.
2-
-
Fig. 2. (A) UV–vis absorbance spectra of N-TC (20μM) in EtOH/H2O (1:1, v/v, HEPES, 0.5mM,
pH=7.38) in the absence and presence of 5 equiv. of various metal ions: Fe3+, Fe2+, Al3+, Ba2+,
Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+. The absorption
wavelength of N-TC was 558 nm. (B) Fluorescence spectra of N-TC (20μM) in EtOH/H2O (1:1,
v/v, HEPES, 0.5mM, pH=7.38) in the absence and presence of 5 equiv. various metal ions. The
excitation and emission wavelengths were 560 nm and 582 nm, respectively.
4

Proposed sensing mechanism
The reversibility between N-TC and Fe3+ was determined with the Na4P2O7-adding
experiment by UV-vis (Fig. 3A) and fluorescence method (Fig. 3B). The addition of Na4P2O7 can
restore the color and fluorescence to the initial state of N-TC. Then, an excess of Fe3+ is
respectively added to the above solution again, and N-TC would return to the “On-state” in both
absorption and fluorescence signals. Re-titrating both N-TC with Fe3+ and Na4P2O7 for four
cycles could still obtained good “Off-On” fluorescence signal response, which indicated that the
sensing process between N-TC and Fe3+ was reversible. The experiment of the relationship
between reversibility and pH was also tested. It can be seen that pH is less than 9 all the time after
N-TC with Fe3+ and Na4P2O7 was retitrated for four cycles. According to Fig. 1A, the
fluorescence intensity of its complex is strong and can remain stable at pH<9 after the addition of
Fe3+. Thus, the measured experimental results are effective. Job’s plot experiments were
conducted that the maximum value of absorption of N-TC/Fe3+ appeared at 0.5 (Fig. S9). Thus,
the binding stoichiometry between N-TC and Fe3+ was 1:1 in EtOH/H2O (1:1, v/v, HEPES,
0.5mM, pH=7.38).
1H NMR titration experiments were carried out in CD3OD:D2O (1:1, v/v), Fe3+ and their
complex with N-TC can affect the proton signals that are close to the Fe3+ binding site. As shown
in Fig. S10, the proton signals of He, Hf, Hg displayed apparent downfield shifts due to the Fe3+
ion induced ring-opening process of the rhodamine B spirocycle. The peaks of Hh and Hi protons
also show significant shifts. This change is due to the coordination of N-TC with Fe3+. The
hydrogen on pyridine ring also shifts to a certain extent, and the peak pattern changes from
multiple peaks to a wide single peak. Thus, we coordinated the N atom on the pyridine ring with
Fe3+. In this way, a stable six-membered ring and a seven-membered ring can be formed.
From above conclusions, a stable sensing mechanism of N-TC-Fe3+ could be summarized as
Scheme 2. To further confirm the mechanism, the ESI–MS result of the N-TC/Fe3+ species was
also provided (Fig. S11).
Fig. 3. Reversibility of Fe3+ to N-TC by Na4P2O7. Examined (A) N-TC by UV-vis absorption
titration. Pink line: free probe (20μM), purple line: probe + Fe3+ + Na4P2O7 (20μM), black line:
probe + Fe3+ + Na4P2O7 + Fe3+. (B) Retitrated N-TC with Fe3+ and Na4P2O7 for four cycles by
5

fluorescence method and the effect of pH on retitrating N-TC with Fe3+ and Na4P2O7 for four
cycles.
Scheme 2. Proposed sensing mechanism of N-TC with Fe3+.
Performance in environmental water samples
Samples of pure water and lake water were both collected to study the performance of N-TC
in the detection of Fe3+ in environmental water samples. To determine the recovery of Fe3+, the
linear plotting of fluorescence intensity of N-TC as a function of Fe3+ concentration (0~200μΜ)
in pure water was firstly carried out. (Fig. 4A). The lake specimens were spiked with Fe3+ of
different concentration, and the recovered Fe3+ concentrations were measured by fluorescent
method (red for Zixi Lake or green for Zixia Lake) (Fig. 4B). Considering the complexity of the
environmental water sample, the recovery of Fe3+ obtained by N-TC was little bit lower than
that in pure water, but both still keep high recovery, which indicated that N-TC can be used for
monitoring trace Fe3+ in environmental water.
Fig. 4. (A) Plotting the fluorescence spectra of N-TC (20μM) as a function of Fe3+ concentration
(0~200μM) in pure water. (B) Recovered Fe3+ concentration in Zixi Lake and Zixia Lake
determined by the low-concentration linear plotting of N-TC.
Cell imaging
N-TC and its complex with Fe3+ remain stable in the range of 6~12 pH wide, which implied
that N-TC would have the potential to visualize Fe3+ in living cells. MTT assay was used to
6

detect the cytotoxicity of N-TC in MCF-7 cells (Fig. S12). The result of MTT test showed that
N-TC had no cytotoxicity at low concentrations, but had a relatively high cytotoxicity when the
concentration of N-TC reached 10μM and still could have more than 80% cell viability.
Owing to their favorable molecular properties, N-TC should be suited for fluorescence
imaging in living cells. According to MTT test, 10μM was then chosen as the suitable
concentration for N-TC in the further confocal fluorescence imaging experiment. After culturing
with N-TC, no fluorescence was observed by confocal fluorescence microscopy from the red
channel (Fig. 5C1). Remarkable red fluorescence was observed in N-TC medium, when 20μM
Fe3+ were extra added (Fig. 5C2). Fluorescence captured from the blue channel implied that
MCF-7 cells still stayed in good cell states after staining with N-TC (Fig. 5B). The merged
images indicated that the areas of red fluorescence and blue fluorescence could complement with
each other, and also revealed that Fe3+ mainly existed in the cytoplasm of the cells sensing by
N-TC (Fig. 5D). The bright-field images confirmed that the cells were viable throughout the
imaging experiments (Fig. 5A). Thus, N-TC had good cell-membrane permeability and could
both be applied in fluorescence imaging Fe3+ in living cells.
Fig. 5. Confocal fluorescence images of N-TC in MCF-7 cells. Cells pretreated with N-TC
(10μM) incubated with Fe3+ (20μM) for 2h. (A) bright field, (B) blue channel, (C) red channel, (D)
the overlay images. Scale bar: 50μM. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
Animal and plant tissue imaging
Zebrafish is a highly valuable model for in vivo imaging. We then examined the feasibility of
detecting Fe3+ by N-TC in zebrafish. As shown in Fig. 6, the zebrafish itself did not show
fluorescence, and there is almost no fluorescence after incubation for 30 min in an aqueous
solution containing N-TC. Then, significant fluorescence was observed after incubation of
zebrafish for 30 min with Fe3+. These results indicate that N-TC can detect Fe3+ in living
zebrafish. In plant tissue imaging, soybean root samples showed low fluorescence before
7

immersion in N-TC. After soaking with N-TC for 30 min, the samples of soybean root showed
obvious red fluorescence (Fig. 7). All the results proved histocompatibility of N-TC which could
be applied to detect Fe3+ in plant tissues.
Fig. 6. Fluorescence images of Fe3+ in zebrafish using N-TC (λex=560 nm). (1) Merged image of
zebrafish incubated with probe (20μM) for 30 min, (2) merged image of probe-loaded zebrafish
incubated with Fe3+ (20μM) for 30 min, (A) bright field, (B) fluorescent images, (C) the overlay
images.
Fig. 7. Fluorescence images of soybean root tissues treated with N-TC (λex=560 nm). (1) Merged
image of soybean incubated with Fe3+ (20μM) for 30 min, (2) merged image of soybean roots
incubated with probe (10μM) for 30 min, (A) bright field, (B) fluorescent images, (C) the overlay
images.
Conclusion
8

In summary, we developed a new reversible “turn-off-on” fluorescence probes (N-TC) for
sensing Fe3+ based on rhodamine B derivative. N-TC exhibited the excellent selectivity and
sensitivity for Fe3+ in aqueous media. N-TC exhibited sensing mechanism towards Fe3+ with 1:1
binding stoichiometry. We found N-TC has higher association constant and lower detection limit.
Moreover, N-TC was successfully applied in detecting trace Fe3+ in lake water with good
recovery efficiency. Besides, Biological experiments demonstrated their potential value of
applications in living cells, animals and plant tissues.
Experimental
Materials and instruments
For details, see the Supplementary Material. Compound 1 was synthesized in Supplementary
Material.
Synthesis of N-TC
To a solution of compound 1 (160mg, 0.33mmol) in 30mL of dry CH2Cl2 was added
pyridine-3-sulfonyl chloride (56mg, 0.32mmol) and triethylamine (40μL, 0.3mmol). The mixture
was stirred at room temperature for 3h, and the solvent was removed in a vacuum. The residue
was dissolved in CH2Cl2 and then washed with H2O. The organic phase was dried with
Magnesium sulfate and then concentrated. The residue was purified by column chromatography
1
using CH2Cl2/ethyl acetate (97:3, v/v) as an eluent to afford pink solid (104mg, 48%). H NMR
(CDCl3), δ8.95 (d, J=2.1Hz, 1H), 8.72 (dd, J=5.0, 4.7Hz, 1H), 8.09 (m, 1H), 7.88 (dd, J=6.6,
5.3Hz, 1H), 7.46 (t, J1=4.7Hz, J2=3.9Hz, 2H), 7.36 (dd, J=8.0, 7.9Hz, 1H), 7.04 (dd, J=6.6, 5.1Hz,
1H), 6.46 (s, 1H), 6.35 (s, 2H), 6.19 (s, 4H), 3.33 (dd, J=7.0, 6.9Hz, 8H), 3.20 (t, J1=5.2Hz,
J2=5.1Hz, 2H), 2.80 (t, J1=5.2Hz, J2=4.6Hz, 2H), 1.19 (m, 12H) ppm. 13C NMR (CDCl3), δ169.83,
153.43, 153.28, 152.83, 149.01, 148.06,136.94, 134.80, 133.02, 130.30, 128.37, 128.29, 123.93,
123.59, 122.93, 108.32, 104.28, 97.76, 65.72, 44.41, 43.93, 40.08, 29.73, 29.35, 12.59 ppm.
ESI-MS (M+H)+ found, 626.27; calculated for C35H39N5O3S2, 625.79.
Spectral properties test
The stock solution of N-TC (1mM) was prepared in EtOH/H2O (1:1, v/v, HEPES, 0.5mM,
pH=7.38) solution. The stock solutions (1mM) of the chloride or nitrate salts of Fe3+, Fe2+, Al3+,
Ba2+, Ca2+, Co2+, Cd2+, Cr3+, Cu2+, Hg2+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+ in water were
prepared as well. Working solutions of N-TC was freshly prepared by diluting the highly
concentrated stock solution to the desired concentration prior to the spectroscopic measurements.
In all of the spectroscopy experiments, the spectral data of N-TC were recorded 60 min after the
addition of the ions. To investigate the metal ion selectivity, the test samples were prepared by
placing 10 equiv. of the cation stock solution in 3 mL of the N-TC solution (20μM). For the
9

fluorescence measurements, excitation was provided at 560nm, and the emission was collected
from 564 to 700nm. Na4P2O7 was respectively chosen as the chelating agent with Fe3+ to confirm
the reversibility of N-TC in both UV–vis absorption and fluorescence methods. Pure water
samples were used directly, and lake water samples were collected from Zixi Lake of Nanjing
Forestry University and Zixia Lake of Nanjing University of Science and Technology which were
passed through the microfiltration membrane before use.
Cell culture and fluorescence imaging
MCF-7 cells were purchased from the American type culture collection (ATCC) and 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 a 5% CO2/95% air
incubator at 37℃. MCF-7 cells were seeded at 1.5×105 cells per well in 24-well flat-bottomed
plates in an atmosphere of 5% CO2/95% air at 37℃ and incubated for 24h before treatment. For
living cell imaging, cells were incubated with 10μM N-TC in culture medium for 5h in an air
incubator at 37℃. In the control experiment, cells were pretreated with 100μM Fe3+ for 1h and
then incubated with 10μM N-TC in culture medium for an additional 2h. Fluorescence imaging
was performed after washing the cells with PBS 3 times and was recorded using the red channel,
blue channel and merged channel under an inverted fluorescence microscope.
Animal and plant tissue imaging
Zebrafish was purchased from Kaiji Biotechnology Co., Ltd. (Nanjing, China) and
respectively incubated in aqueous solution containing N-TC for 30 min and were imaged by
confocal microscopy. After incubation with Fe3+ for 30 min, imaging was performed.
Fluorescence imaging was recorded using the red channel and merged channel under a confocal
microscopy. The seeds of soybean disinfected by 70% ethanol and 3% sodium hypochlorite
solution were cultured on Petri dishes at room temperature and 70% relative humidity. The
control group was supplied with distilled water; the experimental groups were treated with Fe3+
(20μM). After a week, the roots were collected and immersed in solution containing N-TC
(10μM) for 30 min. After three times washing with PBS, the samples were imaged by confocal
microscopy. The control group was incubated with 10μM probe N-TC for 30 min.
Cytotoxicity assay
The cytotoxicity of N-TC was evaluated by an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,
5-diphenyl-2-H-tetrazolium bromide) assay using MCF-7 cells. Cytotoxicity was determined by
an MTT colorimetric method using four parallel wells at each time point. MCF-7 cells were
cultured in 96-well plates at a density of 5×103 cells per well and were incubated at 37℃ in 5%
CO2/95% air for 24h.Then, cells were incubated with different concentrations of N-TC (0-90μM)
for 24h, followed by further incubation with 5 mg/mL of MTT for 4h at 37℃. The supernatant
10

was then discarded, and DMSO (150μL/well) was added to dissolve the formazan. Finally, the
absorbance was measured at 550 nm with a microplate reader (Infinite M200 Pro).
Acknowledgement
This work was supported by the National Natural Science Foundation of China (50772048,
81371616), Jiangsu Six Talent Peak Award (2015SWYY013), Natural Science Fund for Colleges
and Universities in Jiangsu Province (09KJD430007). This research was also supported by Key
Project of Jiangsu Province for Fundamental Research and Development (BE2018717), Scientific
Research Foundation of Nanjing Forestry University, Undergraduate Scientific and Technological
Innovation Project of Nanjing Forestry University, Practical Innovation Training Program for
College Students of Nanjing Forestry University (2018NFUSPITP582). We thank the testing
instruments and technical support provided by Advanced analysis and testing center of Nanjing
Forestry University.
References
[1] M. Garcíacaballero, A. Reyesortiz, Nutr. Hosp. 29 (2014) 1088–1094.
[2] X. Huang, Y. Xu, N.C. Partridge, Bone. 55 (2013) 458–460.
[3] X. Huang, Mutat. Res. Fundam. Mol. Mech. Mutagen. 533 (2003) 153–171.
[4] M. Sandroni, L. Favereau, A. Planchat, H. Akdas-Kilig, N. Szuwarski, Y. Pellegrin, E. Blart,
H.L. Bozec, M. Boujtita, F. Odobel, J. Mater. Chem. (2014) 9944–9947.
[5] M.Y. Yang, J. Li, P.R. Chen, Chem. Soc. Rev. 43 (2014) 6511–6526.
[6] B.L. Sui, S. Tang, T.H. Liu, B. Kim, K.D. Belfield, ACS Appl. Mater. Interfaces. 6 (2014)
18408–18412.
[7] M. Zheng, H.Q. Tan, Z.G. Xie, L.G. Zhang, X.B. Jing, Z.C. Sun, ACS Appl. Mater.
Interfaces. 5 (2013)1078–1083.
[8] S.W. Zhang, R. Adhikari, M.X. Fang, N. Dorh, C. Li, M. Jaishi, J.T. Zhang, A. Tiwari, R.
Pati, F.T. Luo, H.Y. Liu, ACS. Sens. 1 (2016) 1408–1415.
[9] V. Bhalla, N. Sharma, N. Kumar, M. Kumar, Sens. Actuators B.178 (2013) 228–232.
[10] L.Z. Zhang, J.Y. Wang, J.L. Fan, K.X. Guo, X.J. Peng, Bioorg. Med. Chem. Lett. 21(2011)
5413–5416.
[11] F. Yu, L.Y. Qin, Z.B. Shang, Z.M. Dong, Y. Wang, W.J. Jin, Chem. Res. Chin. Univ. 31
(2015)919–924.
[12] F. Song, C. Yang, X.T. Shao, L. Du, J. Zhu, C. Kan, Dyes. Pigms. 165 (2019) 444–450.
[13] M. Khalili, M.A. Ebrahimzadeh, M. Kosaryan, A. Abbasid, M. Azadbakht, RSC Adv. 5
(2015) 4804–4810.
[14] T.C. Duan, J.Z. Kang, H.T. Chen, X.J. Zeng, Spectrochim. Acta Part B. 58 (2003)
1679–1685.
[15] X.P. Hu, D.W. Pan, M.Y. Lin, H.T. Han, F. Li, Microchim. Acta. 183 (2016) 855–861.
[16] Y. Wang, F. Song, J. Zhu, Y. Q. Zhang, L. Du, C. Kan, Tetrahedron Lett. 59 (2018)
3756–3762.
11

[17] J. Widengren, V. Kudryavtsev, M. Antonik, S. Berger, M. Gerken, C.A.M. Seidel, Anal.
Chem. 78 (2006) 2039–2050.
[18] A.B. Chinen, C.M. Guan, J.R. Ferrer, S.N. Barnaby, T.J. Merkel, C.A. Mirkin, Chem. Rev.
115 (2015) 10530–10574.
[19] H.G. Lv, X.F. Yang, Y.G. Zhong, Y. Guo, Z. Li, H. Li, Anal. Chem. 86 (2014) 1800–1807.
[20] H.J. Xu, Z.D. Liu, L.Q. Sheng, M.M. Chen, D.Q. Huang, H. Zhang, C.F. Song, S.S. Chen,
New J. Chem. 37 (2013) 274-277.
[21] U. Diwan, A. Kumar, V. Kumar, K.K. Upadhyay, Dalton Trans. 42 (2013) 13889-13896.
[22] N.R. Chereddy, M.V.N. Raju, P. Nagaraju, V.R. Krishnaswamy, P.S. Korrapati, P.R. Bangal,
V.J. Rao, Analyst 139 (2014) 6352-6356.
[23] Y.B. Zhang, S. Xia, M.X. Fang, W. Mazi, Y.B. Zeng, T. Johnston, A. Pap, R.L. Luck, H.Y.
Liu, Chem. Comm. 54 (2018) 7625–7628.
[24] X.F. Bao, Q.S. Cao, X.L. Wu, H. Shu, B.J. Zhou, Y.L. Geng, J. Zhu, Tetrahedron Lett. 57
(2016) 942–948.
[25] L. He, J.N. Li, J.H. Xin, Biosens. Bioelectron. 70 (2015) 69–73.
[26] X. Han, D.E. Wang, S. Chen, L.L. Zhang, Y.D. Guo, J.Y. Wan, Anal. Methods. 7 (2015)
4231–4236.
[27] X.H. Yang, S. Li, Z.S. Tang, X.D. Yu, T. Huang, Y. Gao, Chin. Chem. Lett. 26 (2015)
129–132.
[28] G.Y. Ciftci, E. Senkuytu, M. Durmus, F. Yuksel, Dalton Trans. 42 (2013) 14916-14926.
[29] S.H. Li, Y.C. Li, J. Cao, J. Zhu, L.Z. Fan, X.H. Li, Anal. Chem. 86 (2014) 10201-10207.
[30] X.Y. Qu, Q. Liu, X.N. Ji, H.C. Chen, Z.K. Zhou, Z. Shen, Chem. Commun. 48 (2012)
4600-4602.
[31] X.D. Yang, X.L. Chen, X.D. Lu, C.G. Yan, Y.K. Xu, X.D. Hang, J.Q. Qu, R.Y. Liu, J. Mater.
Chem. C. 4(2016) 383-390.
[32] G. Mun, S.H. Jung, A. Ahn, S.S. Lee, M.Y. Choi, D.H. Kim, J. Kim, J.H. Jung, RSC Adv. 6
(2016) 53912-53918.
[33] L. Yuan, W.Y. Lin, K.B. Zheng, S.S. Zhu, Acc. Chem. Res. 46(2013) 1462–1473.
[34] M. Homma, Y. Takei, A. Murata, T. Inoue, S. Takeoka, Chem. Commun. 51 (2015)
6194–6197.
[35] E.M.S. Stennett, M.A. Ciuba, M. Levitus, Chem. Soc. Rev. 43 (2014) 1057–1075.
[36] D.T. Zhang, Y.T. Ma, R.B. An, Res. Chem. Intermed. 41 (2015) 5059–5069.
[37] L.Q. Li, L.P. Meng, Spectrochim. Acta Part A. 122 (2014) 772–775.
[38] R.Y. Wei, Z.W. We, L.N. Sun, J.Z. Zhang, J.L. Liu, X.Q. Ge, L.Y. Shi, Appl. Mater.
Interfaces. 8 (2016) 400–410.
[39] J.H. Bi, M.X. Fang, J.B. Wang, S. Xiao, Y.B. Zhang, J.T. Zhang, G. Vegesna, S.W. Zhang, M.
Tanasova, F.T. Luo, H. Liu, Inorg. Chim. Acta. 468 (2017) 140–145.
[40] F.Y. Yan, T.C. Zheng, S.S. Guo, D.C. Shi, Z.Y. Han, S.Y. Zhou, L. Chen, Spectrochim. Acta
Part A, Mol. Biomol. Spectrosc. 151 (2015) 881–887.
[41] R. Bhowmick, A.M. Islam, U. Saha, G.S. Kumar, M. Ali, New J. Chem. 42 (2018)
3435–3443.
12

[42] Q. Chen, Z.J. Fang, Spectrochim. Acta, Part A. 193 (2018) 226–234.
[43] H.L. Chen, X.F. Bao, H. Shu, B.J. Zhou, R.L. Ye, J. Zhu, Sens. Actuators B. 242 (2017)
921–931.
[44] L. Dong, C. Wu, X. Zeng, L. Mu, S.F. Xue, Z. Tao, J.X. Zhang, Sens. Actuators B. 145 (2010)
433–437.
[45] X.F. Bao, X.W. Cao, X.M. Ni, Y. Xu, W.H. Guo, B.J. Zhou, L.Y. Zhang, H. Liao, T. Pang,
Sens. Actuators B. 208 (2015) 54–66.
[46] J.C. Qin, Z.Y. Yang, G.Q. Wang, J. Photochem. Photobiol. A: Chem. 310 (2015) 122–127.
[47] S.Z. Ji, X.M. Meng, W.P. Ye, Y. Feng, H.T. Sheng, Y.L. Cai, J.S. Liu, X.F. Zhu, Q.X. Guo,
Dalton Trans. 43 (2014) 1583–1588.
[48] N.R. Chereddy, K. Saranraj, A.K. Barui, C.R. Patra, V.J. Rao, S. Thennarasu, RSC Adv. 4
(2014) 24324–24327.
13

Highlights
 A Fe3+-specific probe based on Rhodamine B was designed and synthesized.
 The probe displays the reversible “turn-off-on” fluorescence change.
 The probe is feasible for monitoring trace Fe3+ in environmental water samples.
 The probe can be used for detection of Fe3+ in cells, animals and plant tissues.
14

Graphical Abstract
15