Turn on fluorescent chemosensor containing rhodamine B fluorophore for selective sensing and in vivo fluorescent imaging of Fe3+ ions in HeLa cell line and zebrafish

Article abstract of DOI:10.1016/j.jphotochem.2019.112060

Rhodamine dyes are utilized effectively as a chemosensor to monitor biologically important metal ions and effectively applied for fluorescent imaging studies in living systems. Herein, rhodamine-B armed fluorescent chemosensor (RhBNC) was designed and synthesized by condensation reaction between rhodamine B hydrazide and naphthyl chromone with ONO binding site in good yield. Upon sensing study towards metal ions, the probe showed selective turn-on fluorescent change for Fe³⁺ over other cations even in the presence of competing trivalent metal ions as well. The fluorescence switch is “on” when Fe³⁺ ions binds to RhBNC with orange fluorescence. The probe can detect lower concentration of Fe³⁺ ions with detection limit of 0.16 μM. Due to its lower detection limit; it can be applied to monitor Fe³⁺ ion presence in a living system by fluorescent imaging technique. The probe was employed effectively to ascertain the presence of Fe³⁺ and to image Fe³⁺ ions in HeLa cell line and zebrafish.

Full text of DOI:10.1016/j.jphotochem.2019.112060

Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112060
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Turn on fluorescent chemosensor containing rhodamine B fluorophore for
selective sensing and in vivo fluorescent imaging of Fe3+ ions in HeLa cell
line and zebrafish
a
b
a,⁎
Natarajan Vijay , Shu Pao Wu , Sivan Velmathi
a
Organic and Polymer Synthesis Laboratory, Department of Chemistry, National Institute of Technology, Tiruchirappalli-620 015, India
Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Rhodamine dyes are utilized effectively as a chemosensor to monitor biologically important metal ions and
effectively applied for fluorescent imaging studies in living systems. Herein, rhodamine-B armed fluorescent
chemosensor (RhBNC) was designed and synthesized by condensation reaction between rhodamine B hydrazide
Turn on fluorescence
3+
Fe
ions
Selectivity
Fluorescence imaging
Zebrafish
and naphthyl chromone with ONO binding site in good yield. Upon sensing study towards metal ions, the probe
3+
showed selective turn-on fluorescent change for Fe
over other cations even in the presence of competing
3
+
trivalent metal ions as well. The fluorescence switch is “on” when Fe
ions binds to RhBNC with orange
3
+
fluorescence. The probe can detect lower concentration of Fe ions with detection limit of 0.16 μM. Due to its
3
+
lower detection limit; it can be applied to monitor Fe ion presence in a living system by fluorescent imaging
3
+
3+
technique. The probe was employed effectively to ascertain the presence of Fe and to image Fe ions in HeLa
cell line and zebrafish.
1. Introduction
handling as well as being cheaper to use. Utilizing the fluorescent probe
for heavy metal ion detection is a reliable technique compared to so-
Selective and sensitive detection of heavy metal ions in aqueous
phisticated instrumental technique. Basically, colorimeric or fluoro-
metric receptors were designed with chromophore / fluorophore with
binding unit and employed as a receptor for sensing of targeted analytes
[5,6]. In this decade, rhodamine dyes were applied effectively as a
heavy metal ion sensor due to their very good photophysical properties,
higher fluorescence quantum yield and tuneable selectivity nature
[7,8]. Rhodamine based chemosensor showed turn on fluorescence
change for metal ions when it binds with metal ions. This is because of
the spirolactam ring opening/closing mechanism of the receptor with
addition of metal ions [9]. Rhodamine dye is a good fluorophore with
emission maximum of 580 nm. Rhodamine derived receptors were ap-
plied widely for bioimaging of particular analyte in a living cell, which
gives promising results due to its photophysical properties and the
minimal background noise from the living system [10,11]. Iron is found
to be a predominant microelement, play a number of biologically and
pathologically important roles to maintain a normal physiological
function in a system. It is a crucial element for oxygen sensing in living
system where metabolism is been linked with oxygen metabolism [12].
It is also important to maintain redox balance of labile iron species
which can generate iron catalyzed reactive oxygen species (ROS)
solution attracts enormous importance in monitoring and studying the
physiological functions of these ions in living organisms [1,2]. Con-
ventional spectroscopic methods failed due to their complicated ex-
perimental procedure and requirement of sophisticated instrument for
the detection. So, there is a requirement of smart and simple detection
method to monitor the amount of trace level metal ions in a living
system under its physiological condition. Recently, monitoring the
biologically important heavy metals by optical methods grabbed much
attention due to their relatively lower cost compared to instrumental
methods [3]. Nowadays, Small molecule fluorescent probes are utilized
effectively for monitoring the particular analyte in a living system.
Those possesses high sensitivity and selectivity with simple detection
procedure. Hence, this decade several studies focused on the con-
struction of selective and sensitive chemosensors using spectral
methods includes both spectrophotometric and spectrofluorometric
methods for the detection [4]. In case of chemosensor, the sensing study
is based on quenching/growing of emission/absorption peaks of the
receptor induced by the analyte provides the unique changes in de-
tection. Fluorescent signalling supplies high sensitivity and easy
⁎
Corresponding author.
E-mail address: velmathis@nitt.edu (S. Velmathi).
https://doi.org/10.1016/j.jphotochem.2019.112060
Received 21 June 2019; Received in revised form 21 August 2019; Accepted 23 August 2019
Available online 24 August 2019
1010-6030/ © 2019 Elsevier B.V. All rights reserved.

N. Vijay, et al.
Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112060
[
13–15]. It is important to prevent the ROS generation for survival.
Both inadequacy and excess of iron concentration in our body can cause
the cellular homeostasis and can cause various biological diseases
[
16–20]. We are living in world where we are surrounded with iron and
its derivatives, either direct or indirect manner. We are consuming iron
in our daily life even through food and water. Thus it is important to
monitor it’s amount in living system by simple method. Utilizing the
3
+
organic receptor as a selective probe to trace out Fe
ions in a livin+g
system is an easy and reliable way. To discover the presence of Fe3
ions, number of chemosensors were reported in recent days [21–24].
Some rhodamine derived fluorescent probes were also reported for
3
+
sensing of Fe
by fluorescent change and colorimetric change
[
10,25–27]. But, most of the probes showed either limited selectivity
over other trivalent ions, or poor solubility and higher detection limit.
Particularly, most of the reported Fe3+ probes failed in selectivity over
existing trivalent ions. Moreover, paramagnetic fluorescence quenching
3
+
of Fe ions becomes major obstacle in designing fluorescent probe for
3
+
Fe
probe for Fe is still in need. Herein, we have designed and developed
rhodamine B derived fluorescent probe for selective detection of Fe3+
ions sensing [28,29]. So the requirement of selective fluorescent
3
+
.
The probe was constructed with ONO (via Oxygen-Nitrogen-Oxygen)
Scheme 1. Synthesis route of probe (RhBNC).
binding unit with rhodamine B hydrazide and naphthyl chromone. The
3
+
probe shows excellent selectivity for Fe
over other metal ions and
reaction mixture was cooled to room temperature and filtered using
funnel and washed with ethanol to get the pure product as a yellow
solid (Scheme 1). Yield: 80% (202 mg). Melting point: 195 ⁰C. FT-IR (ν/
3
+
trivalent ions as well. It exhibits turn on fluorescence for Fe with 20
fold fluorescence enhancement. It can even detect Fe
3
+
in lower con-
centration as well with the detection limit of 0.16 μM. Thus, it can be
−1
1
cm ): H NMR (CDCl
3
, 500 MHz, ppm): 9.29 (s, 1 H), 8.3 (d, 1H,
3
+
applied for the detection of Fe
in a living system. Hence, we have
J:17 Hz), 8.1 (d, 1H, J: 18 Hz), 7.9 (d, 1H, J:16 Hz), 7.7 (t, 1H, J: 15 Hz),
performed fluorescent imaging of Fe3 ions in HeLa cell line and zeb-
+
13
7
(
,6 (t, 1H, J: 15 Hz), 7.4 (d, 1H, J: 18 Hz), 2.788 (s, 3 H). C NMR
CDCl , 125 MHz, ppm): 195.63,
59.47, 156.19, 143.34, 136.34, 130.23, 129.87, 129.25, 126.68,
22.45, 121.72, 116.52, 112.80 and 30.72.
3
+
rafish and the probe shows red emission for Fe in both zebra fish and
3
HeLa cell line.
1
1
2. Experimental Section
2.1. Instrumental techniques and methods
2.3. Synthesis of probe (RhBNC)
All the reagents, chemicals and solvents were obtained from com-
Rhodamine-B-hydrazide can be synthesized by reaction between
rhodamine B and hydrazine hydrate by following the previously re-
ported procedure [30,31]. Rhodamine-B hydrazide (0.5 mmol, 228 mg)
and naphthylchromone (0.5 mmol, 119 mg) were taken in RB flask and
dissolved in 0.5 mL of DMSO. The reaction mixture was heated to
120 °C for 6 h in an oil bath. The reaction completion was monitored
thorough TLC and the reaction mixture was poured to ice-cold water.
The resulting precipitate was filtered, washed with cold water and dried
over vacuum to get desired product as greenish yellow solid (Scheme
mercially available sources (Sigma and Merck) and used without ad-
ditional purification throughout the studies. Double distilled water is
utilized for doing all the experiments. The product formation was
1
13
confirmed by the characterization techniques like FT-IR, H & C NMR,
ESI-Mass analysis and the complete photophysical studies were carried
out using UV/Vis and Fluorescence spectrophotometer. Shimadzu UV-
2
600 spectrophotometer was used to get the absorption spectra using
quartz cell having cm path length. RF-5301PC spectro-
1
−
1
fluorophotometer was utilized to get the emission spectra with scanning
1). Yield: 81% (275 mg). Melting point: 208 °C. FT-IR (ν/cm ):
−1
−1
−1
−1
−1 1
rate of 500 nm/min. Bruker AV-500 spectrometer (Bruker, Switzerland)
3449 cm , 3060 cm , 1735 cm , 1612 cm , 1513 cm
. H NMR
1
13
was used to obtain H (500 MHz) and C (125 MHz) NMR spectra
using CDCl as a solvent. Nicolet IS 5 FTIR spectrometer was used to
(CDCl , 500 MHz, ppm): 8.67 (s, 1 H), 8.1 (d, 1H, J: 16 Hz), 8.0 (d, 1H,
3
3
J: 8 Hz), 7.7 (q, 2 H), 7.6 (t, 1H, J: 15 Hz), 7.5 (d, 2H, 6), 7.5 (t, 1H, J:
record the IR spectra by KBr pressed disc method. The stock solutions of
metal ions that applied for the sensing studies were prepared by dis-
solving the manganese acetate and other metal chloride salts of corre-
14 Hz), 7.2 (d, 1H, J: 10 Hz), 7.2 (d, 1H, J: 17 Hz), 6.6 (d, 2H, J: 18 Hz),
6.443 (s, 2 H), 6.3 (d, 2H, J:17 Hz) 3.3 (d, 8H, J: 13 Hz), 2.347 (s, 3 H)
and 1.1 (t, 12H, J: 17 Hz). 13C NMR (CDCl , 125 MHz, ppm): 153.9,
3
3
+
3+
2+
2+
2+
2+
sponding metal salts such as (Cr , Fe , Co , Ni , Cu , Zn
,
150.8, 148.8, 139.5, 134.1, 132.5, 131.3, 130.1, 129.4, 128.9, 128.5,
128.2, 126.0, 124.1, 124.0, 123.2, 121.6, 116.3, 113.0, 107.9, 106.8,
97.6, 44.3, 19.9, 12.6. ESI Mass: 676.3, m + 1 677.3 (ESI: Fig. 1–9).
2
+
2+
3+
Cd , Hg
lutions and the stock solutions of anions were prepared by dissolving
and Al ) in double distilled water to afford 1.5 mM so-
−
−,
−
tetra-n-butylammonium salts of corresponding anions (CN , F Cl
,
−
−
−
2 4 3
Br , I , AcO , H PO4−, HSO , NO
and –OH) in double-distilled
−
−
water to get 1.5 mM solutions. 1.5 mM solution of the probe RhBNC
stock solution was prepared in DMSO and kept in normal day light
condition for several months; no characteristic change was observed.
2.4. Physical constants determination
Stoichiometric ratio (R:M) of RhBNC with Fe3+ ions were calculated
by Job’s plot method. To find out the stoichiometry; emission at 580 nm
was plotted against the mole ratio of the ([Fe3+] / [Fe ] + RhBNC)
3+
2.2. Synthesis of naphthylchromone
(
Job’s plot). Benesi-Hildebrand equation was used to calculate the
2
-Hydroxy-1-naphthaldehyde 172.00 mg (1 mmol) was taken in
binding constant (Ka) and limits of detection (LODs) of the receptor
RhBNC using the fluorescence incremental titration data. For that,
fluorescence incremental titration experiment was performed with
gradual increment of Fe3+ ion concentration (0–2eq.).
1
0 mL absolute ethanol with ethylacetoactate 150 μL (1.2 mmol) in a
round bottom flask. The reaction mixture was refluxed overnight in an
oil bath. The reaction completion was monitored through TLC and the
2

N. Vijay, et al.
Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112060
2.5. Cytotoxicity assay
Cell viability experiment for RhBNC in HeLa cell lines was measured
by using the MTT (methyl thiazolyl tetrazolium) assay experiment. For
this experiment the desired cell line were cultured in multi well cell-
culture plate and different concentrations (5, 10, 15, 20, 25 μM) of
receptor were treated with the cells. After that, the cells were allowed
for incubation for 24 h at 37 °C under 5% CO
2
. Then, to each of the well
−
1
10 μL MTT (5 mg mL ) was added and allowed for incubation again
for 4 h with same condition. After 4 h incubation the media were re-
moved and 200 μL DMSO and 25 μL Sorensen’s glycine buffer (0.1 M
glycine and 0.1 M NaCl) was added to that. Then the absorbance was
measured by single time Multiskan GO microplate reader (at 570 nm).
The cell viability was calculated using the given equation:
Fig. 1. Color change experiment for RhBNC (50 μM receptor in THF upon ad-
dition of 2 eq. of cations in H O; a) Day light image; b) Fluorescence image.
2
designed and synthesized in very good yield (Scheme 1). The product
obtained as a yellow solid with melting point 208 °C. Synthesized probe
was well characterized and the product formation was confirmed by FT-
IR, 1H NMR, C NMR and ESI-Mass spectroscopic methods (Fig. ESI
Cell viability (%) = Mean absorbance (Treated cell) / Mean absorbance
(
Control cell).
13
1–9).
2.6. Cell culture for HeLa cell line
For bioimaging studies the HeLa cell line was obtained from the
3.2. Visual sensing studies
Food Industry Research and Development Institute (Taiwan). The de-
sired HeLa cell lines were cultured in DMEM which is having 10% fetal
Things, that seeing visually will make more effect and will be re-
membered for long time. Visual color change experiment is a key fact in
sensing field. The visual color change of probe (RhBNC) with cations
bovine serum (FBS) in addition and is kept at 37 °C under 5% CO
mosphere. After the culturing, the cells were treated with 20 μM probe
RhBNC) in 2 mL of DMSO and incubated for 30 min at 37 °C. Then the
media was removed and the cells were washed with 1 M PBS twice to
2
at-
3+
2+
3+
2+
2+
2+
2+
2+
2+
3+
(
(Cr , Mn , Fe , Co , Ni , Cu , Zn , Cd , Hg
and Al
)
were observed by naked eye experiment. The experiments were carried
out in different solvents such as ACN, DMSO, ethanol, MeOH and THF
to find out the selectivity of the probe over biologically existing cations.
The probe was taken in glass vial having 3 mL THF solvent with a final
concentration of 50 μM. To that solution 2 eq. of metal ions were added
which was dissolved in double distilled water. The images were taken in
normal day light and under UV light (Fig. 1). The probe (RhBNC) shows
excellent selectivity towards Fe3+ over other cations in THF solvent
medium with a colour change from colourless to appearance of pale
pink colour.
3
+
remove excess probe. Then, 20 μM Fe
(in DMSO) was added to the
cell and incubated, allowed for 30 min and the media were removed.
Finally, the cell lines were plated on 18 mm glass coverslips and al-
lowed to adhere for 24 h before imaging. Fluorescence imaging was
performed with Leica TCS SP5 X AOBS Confocal Microscope (Germany)
and a 63-oil-immersion lens has been utilized as an objective lens. The
excitation wavelength was fixed at 540 nm and emission was recorded
at 580 nm ± 10 nm. Institutional Animal Care and Use Committee
(
IACUC) of NCTU accepted National Institute of Health, Taiwan
guidelines was followed for imaging of zebrafish and maintained the
condition according to the guideline. The fish was maintained at am-
bient temperature with good condition. The both gender fishes (Male
and female) are maintained in same container at 28 °C with 10 h/14 h
cycle (dark/light respectively) for mating purpose, and then the egg
fertilization was induced by sun light. Egg fertilization was good in this
condition; most off the eggs were fertilized. The zebrafish culturing was
carried out in 6 well cell culturing plate with addition of 5 mL fish
medium along with 1-phenyl-2-thiourea (PTU) in each well at 30 °C for
3
.3. Photophysical studies
In continuation of colorimetric studies, the sensing action was
monitored through photophysical experiments using UV–vis and
fluorescence spectrophotometer. To investigate the efficiency of the
probe as a metal ion sensor the photophysical changes of probe with
various metal ions were verified in THF solvent medium. The absorp-
tion spectrum of the probe exhibit bands in the range of 350–400 nm
corresponding to π-π* and n- π* transition in chromone and rhodamine
moiety. Noticeable change was not observed after adding 2 eq. metal
ions, in absorption spectra because not much colour change in naked
eye experiment as well. For Fe3+ very small change was found in ab-
sorption spectra in the range of 350–400 nm (Fig. 2(a)). The fluorescent
change of the probe towards various metal ions was checked in THF
solvent medium using fluorescent spectrometer. In a fluorescence
spectrometer the probe was excited at 520 nm and the emission was
collected at 540 nm. The probe alone doesn’t exhibit any fluorescence
in 550–600 nm range, after addition of metal ions, Fe3 ions alone
1day. Followed by this, zebrafish were anaesthetized using 50 mg/L
tricaine and treated with 20 μM probe at 28 °C for 30 min. Then, the
remaining probe which is present in the outside of the fish was removed
by washing it with 1 M PBS and then the fish was treated with 20 μM of
3
+
Fe
ions in DMSO and kept for incubation at 28 °C for 30 min. Then
3+
again the PBS wash has been carried out to remove the excess Fe
present in the fish. Then finally, zebrafish imaging was performed with
Leica TCS SP5 X AOBS Confocal Fluorescence Microscope.
+
3. Results and discussions
causes 20 fold fluorescence enhancement with
λmaxat 580 nm
(
Fig. 2(b)). Only the addition of Fe3+ causes turn on fluorescence
3.1. Synthesis and characterization
change with distinct colour change. This is due to the ring opening of
the spirolactam present in rhodamine dye triggered by the addition of
Fe
The real application of the fluorescent probe can be achieved when
it shows exclusive sensitivity towards particular analyte with distinct
3+
ions [32,33].
change. In order to achieve that, herein the fluorescent probe RhBNC
3
+
was designed for the detection of Fe ions. The probe was constructed
3.4. Incremental addition and selectivity
by utilizing Rhodamine B as a fluorophore, with naphthyl chromone as
3
+
Fluorescence titration experiment was performed with Fe3+ to find
out its binding ability with probe. Initially, 50 μM of probe was taken in
binding unit for Fe . In case of rhodamine based probe with ONO as
3
+
binding unit will selectively detect Fe . In that motive, the probe was
3

N. Vijay, et al.
Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112060
Fig. 2. a) UV–vis spectrum of RhBNC (50 μM) with different cations (100 μM) in THF; b) Fluorescence spectrum of RhBNC (50 μM) with different cations (100 μM) in
THF.
3
+
3+
Fig. 3. a) Fluorescence spectrum of RhBNC (50 μM) with Fe
(0–2 eq.) in THF; b) Fluorescence intensity of RhBNC (50 μM) with Fe
(2 eq.) at 580 nm in the
presence of other cations (2 eq.) in THF (Black bar: Intensity of RhBNC with cations; Red bar: Intensity of RhBNC with cations and Fe3+).
THF and Fe3+ was added gradually (0–2 eq.). The fluorescence in-
tensity at 580 nm increases gradually with increasing the Fe3+ con-
centration (Fig. 3(a)). The complete application of the probe will be
fulfilled, if it works even in the presence of the other metal ions. Be-
cause, in real time application the probe should work inside the bio-
logical medium with the presence of other competing metal ions. In
the fluorescence titration experiment. During fluorescence titration, the
intensity at 580 nm gradually increases while increasing the con-
centration of Fe3+ ions. From the above titration, we have plotted the
graph according to Benesi-Hildebrand equation. Binding constant of the
probe with Fe3+ was calculated according to the equation (1).
) [Fe3+] + 1/ (Imax-I
)
(1)
3+
1/(I-I
0
) = 1/Ka (Imax - I
0
0
order to ensure the selectivity of the probe (RhBNC) towards Fe with
the presence of other metal ion such as Cr , Mn , Co , Ni2+, Cu2+
3
+
2+
2+
,
I and I were fluorescence intensity of RhBNC at 580 nm in presence
0
2
+
2+
2+
3+
3+
Zn , Cd , Hg
and Al
competition experiment was performed
and absence of Fe
ions respectively and Imax is the saturated fluor-
3
+
with the presence of above mentioned metal ions. Initially 50 μM of
probe taken in THF solvent medium and 2 eq. of various metal ions
were added to that and the fluorescence spectrum was recorded with
similar excitation (520 nm) and the emission spectrum was collected
from 540 nm. Then the intensity at 580 nm was plotted (Fig. 3(b))
escence intensity with the presence of high concentration of Fe
ions
and [Fe3+] is concentrations of iron added to the probe. The calculated
binding constant (Ka) is 7450. The U.S. Environmental Protection
3+
Agency (EPA) permitted amount of Fe
ions in drinking water was
−
1
0.3 mg L (equivalent to 5.4 μM). To detect iron even at lower con-
centration, we have calculated the limit of detection of the probe to-
wards Fe3+ ions detection using fluorescent titration experiment. We
have followed the equation that based on definition provided by IUPAC.
The IUPAC provided equation was,
(
Black bar). To that 2 eq. of Fe3+ was added and the fluorescence
spectrum was taken and the intensity at 580 nm was monitored and
plotted again (red bar). The probe shows 20 fold fluorescence en-
3
+
hancement for Fe with the presence of other metal ions as well. The
3
+
probe shows excellent selectivity for Fe
with ‘turn on’ fluorescence
Limit of Detection = 3*Standard deviation of blank / slope between
intensity and concentration
even in the presence of higher concentration of competitive metal ions.
The detection limit of the probe for the detection of Fe3+ ions was
3
.5. Stoichiometry, binding constant and detection limit calculation
very low, 0.16 μM. It is very much lower than EPA permitted level of
3
+
Fe ions concentration in drinking water. So, the probe can be applied
To understand the clear sensing mechanism and to carry out further
3+
for detection of Fe
ions in practical applications.
3
+
applications, stoichiometry between probe and Fe
need to be ver-
ified. It is calculated using mole ratio plot methods and Job’s plot
method. The fluorescence change at 580 nm was plotted against the
3.6. Proposed sensing mechanism
3
+
concentration of Fe
the binding ratio is 1:1 between probe and Fe
Furthermore, the binding constant and limit of detection of the probe
ions added to the probe. The result reveals that
3
+
The significant colour change and fluorescence change for Fe3+
with the probe need to be analyzed and the clear sensing mechanism
should be proposed. The results reveal that, the probe followed general
(Fig. ESI: 13).
3
+
towards Fe
was calculated using Benesi-Hildebrand equation from
4

N. Vijay, et al.
Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112060
Fig. 4. Proposed sensing mechanism of the probe for Fe3+ ions sensing event.
3
+
rhodamine-B mechanism for the sensing event. The metal ion induces
spirolactam ring opening and results with selective colour change and
fluorescence change in the range 550–600 nm (Fig. 4). Rhodamine-B
(Fig. 5 channel A). After that cell line was treated with 20 μM Fe ions
dissolved in DMSO and incubated for 30 min. After the incubation the
cell was imaged using the microscope. It gives bright red emission when
excited at 560 ± 10 nm.
3
+
dye with ONO binding mostly utilized as Fe
sensor with distinct
colour change and fluorescence change [25,34,35]. The probe worked
To account for the cell permeability and the fluorescence in the
cytoplasm, initially fluorescent image was captured only with nuclear
3
+
as we expected, it makes ONO binding with Fe
ions and gives se-
lective change over the other cations. The binding was confirmed with
staining dye. It showed blue emission in the nucleus. After the treat-
3
+
3+
stoichiometry ratio between probe and Fe
by Jobs plot method. It
ment of probe with Fe
it showed red emission in cytoplasm only
3
+
3+
showed 1:1 binding mode with Fe . To confirm further, RhBNC-Fe
complex was synthesized and formation of the product was confirmed
by ESI mass analysis (Fig. ESI: 9 & 10). Calculated mass of the probe
(Fig. 5 channel B). So the probe possesses very good cell permeability
with less cell death, further it applied for in vivo fluorescent imaging of
3+
Fe . This experiment was performed with zebrafish. Three days old
zebrafish was treated with probe (20 μM) for 30 min and fluorescent
image was captured using the microscope; no characteristic emission
(
RhBNC) was 676.3 and mass peak obtained at 677.3 (m + 1) and
3
+
calculated mass for RhBNC-Fe
complex is 855.2 and obtained mass
peak at 856.2 (m + 1), confirms the 1:1 stoichiometry between probe
was observed (Fig. 6, panel A). Then the fish was allowed to treat with
3
+
3+
RhBNC and Fe
.
Fe
for half an hour and the emission image was captured with mi-
croscope. It exhibited bright red emission in the fish stomach as like
3.7. Cell imaging and zebrafish imaging
HeLa cell line (Fig. 6, panel B).
Once, the primary photophysical studies were completed with the
4. Conclusion
probe, it is employed in real time application studies. The predominant
3
+
application of this study is to monitor the presence of Fe
ions in
In summary, rhodamine-B derived fluorescent probe was designed
and synthesized for selective detection of Fe3+ ions in aqueous
medium. The receptor shows turn on fluorescent change selectively for
living system. In prior to carry out the bioimaging studies, the cell
viability experiments were performed with the probe in order to find
out the toxicity of the probe towards living cells. MTT assay experiment
was performed with HeLa cell line with various concentration of RhBNC
< 25 μM). In MTT assay experiment the probe shows more than 80%
cell viability towards HeLa cell line (Fig. ESI: 12). So the probe can be
applied for biological studies as it shows less cell death. Fluorescence
3+
3+
Fe
ions over other cations. It can detect the Fe
in lower con-
centration as well with the detection limit of 0.16 μM. The receptor
(
shows less cell death in cell viability test towards HeLa cell line, hence
3
+
it is applied successfully for monitoring the intracellular Fe
ions in
living cells. This experiment demonstrated by fluorescent imaging of
3
+
3+
imaging of HeLa cell line towards Fe
was performed using Leica
Fe
in HeLa cell line using fluorescence microscope. Finally, in vivo
3+
fluorescence microscope. The probe RhBNC (20 μM) treated with HeLa
cell line and incubated for 30 min and imaged using the microscope. It
doesn’t show any emission in cell line before it treated with Fe3+ ions
fluorescent imaging studies were performed for Fe
using zebrafish.
The probe can be applied to monitor the Fe3+ in a living system with
selectively over other trivalent ions and other metal ions.
Fig. 5. Fluorescence images of HeLa cell line. (First)
Bright field; (Second) DAPI (4′,6-diamidino-2-pheny-
lindole, nuclear stain); (Third) Fluorescence; (Fourth)
merged image. (A) The cells were treated with Probe
(
20 μM) for 30 min. (B) Cells were incubated with
probe (RhBNC) (20 μM) for 30 min and then treated
with Fe
3
+
(20 μM).
5

N. Vijay, et al.
Journal of Photochemistry & Photobiology A: Chemistry 384 (2019) 112060
Fig. 6. Confocal Fluorescence images of zebrafish embryos (3 days old). (A) The fish was treated with probe (20 μM) for 30 min. (B) The zebrafish were treated with
3+
probe (20 μM) and then treated with Fe
(20 μM) for 30 min.
Declaration of Competing Interest
Cancer Lett. 266 (2008) 21–29.
[
15] V. Natarajan, N. Thirumalaivasan, S.-P. Wu, V. Sivan, A far-red to NIR emitting ultra-
sensitive probe for the detection of endogenous HOCl in zebrafish and the RAW 264.7 cell
line, Org. Biomol. Chem. 17 (2019) 3538–3544.
There is no conflict of interest to declare in this submission.
[
[
[
16] J.D. Haas, T. Brownlie IV, Iron deficiency and reduced work capacity: a critical review of
the research to determine a causal relationship, J. Nutr. 131 (2001) 676S–690S.
17] T. Thum, S.D. Anker, Nutritional iron deficiency in patients with chronic illnesses, Lancet
Acknowledgements
370 (2007) 1906.
18] H. Zheng, L.M. Weiner, O. Bar-Am, S. Epsztejn, Z.I. Cabantchik, A. Warshawsky,
M.B. Youdim, M. Fridkin, Design, synthesis, and evaluation of novel bifunctional iron-
chelators as potential agents for neuroprotection in Alzheimer’s, Parkinson’s, and other
neurodegenerative diseases, Biorg. Med. Chem. 13 (2005) 773–783.
One of the Authors N. Vijay thanks the support of TEEP@India
Program (Taiwan) for an internship. SV acknowledges CSIR for the
research project Ref. No: 01(2949)/18/EMR-II dated 01-05-2018.
[
19] D.J. Bonda, H.-g. Lee, J.A. Blair, X. Zhu, G. Perry, M.A. Smith, Role of metal dysho-
meostasis in Alzheimer’s disease, Metallomics 3 (2011) 267–270.
[20] A.S. Pithadia, M.H. Lim, Metal-associated amyloid-β species in Alzheimer’s disease, Curr.
Opin. Chem. Biol. 16 (2012) 67–73.
Appendix A. Supplementary data
[
21] L. Qiu, C. Zhu, H. Chen, M. Hu, W. He, Z. Guo, A turn-on fluorescent Fe 3+ sensor derived
from an anthracene-bearing bisdiene macrocycle and its intracellular imaging applica-
tion, Chem. Commun. 50 (2014) 4631–4634.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2019.
[
22] J. Li, Q. Wang, Z. Guo, H. Ma, Y. Zhang, B. Wang, D. Bin, Q. Wei, Highly selective
112060.
3
+
fluorescent chemosensor for detection of Fe based on Fe
3558.
[23] Z. Li, Y. Zhou, K. Yin, Z. Yu, Y. Li, J. Ren, A new fluorescence “turn-on” type chemosensor
3 4
O @ ZnO, Sci. Rep. 6 (2016)
2
References
3
+
for Fe
based on naphthalimide and coumarin, Dyes Pigments 105 (2014) 7–11.
[
24] A. Kuwar, R. Patil, A. Singh, S.K. Sahoo, J. Marek, N. Singh, A two-in-one dual channel
[
[
[
1] M. Dutta, D. Das, Recent developments in fluorescent sensors for trace-level determina-
tion of toxic-metal ions, TrAC Trends Anal. Chem. 32 (2012) 113–132.
2] D.W. Domaille, E.L. Que, C.J. Chang, Synthetic fluorescent sensors for studying the cell
biology of metals, Nat. Chem. Biol. 4 (2008) 168.
3] S. Aoki, D. Kagata, M. Shiro, K. Takeda, E. Kimura, Metal chelation-controlled twisted
intramolecular charge transfer and its application to fluorescent sensing of metal ions and
anions, J. Am. Chem. Soc. 126 (2004) 13377–13390.
chemosensor for Fe3+ and Cu2+ with nanomolar detection mimicking the IMPLICATION
logic gate, J. Mater. Chem. C 3 (2015) 453–460.
[25] Y. Xiang, A. Tong, A new rhodamine-based chemosensor exhibiting selective FeIII-am-
plified fluorescence, Org. Lett. 8 (2006) 1549–1552.
[26] H. Wu, L. Yang, L. Chen, F. Xiang, H. Gao, Visual determination of ferric ions in aqueous
solution based on a high selectivity and sensitivity ratiometric fluorescent nanosensor,
Anal. Methods 9 (2017) 5935–5942.
[
[
[
4] D.T. Quang, J.S. Kim, Fluoro-and chromogenic chemodosimeters for heavy metal ion
detection in solution and biospecimens, Chem. Rev. 110 (2010) 6280–6301.
5] B. Valeur, I. Leray, Design principles of fluorescent molecular sensors for cation re-
cognition, Coord. Chem. Rev. 205 (2000) 3–40.
6] K. Kaur, R. Saini, A. Kumar, V. Luxami, N. Kaur, P. Singh, S. Kumar, Chemodosimeters: an
approach for detection and estimation of biologically and medically relevant metal ions,
anions and thiols, Coord. Chem. Rev. 256 (2012) 1992–2028.
[27] A.J. Weerasinghe, C. Schmiesing, S. Varaganti, G. Ramakrishna, E. Sinn, Single-and
multiphoton turn-on fluorescent Fe3+ sensors based on bis (rhodamine), J. Phys. Chem. B
114 (2010) 9413–9419.
[28] W. He, Z. Liu, A fluorescent sensor for Cu 2+ and Fe 3+ based on multiple mechanisms,
RSC Adv. 6 (2016) 59073–59080.
[29] L. Fu, J. Mei, J.T. Zhang, Y. Liu, F.L. Jiang, Selective and sensitive fluorescent turn‐off
chemosensors for Fe3+, Luminescence 28 (2013) 602–606.
[
7] V. Dujols, F. Ford, A.W. Czarnik, A long-wavelength fluorescent chemodosimeter selec-
tive for Cu (II) ion in water, J. Am. Chem. Soc. 119 (1997) 7386–7387.
8] M. Kumar, N. Kumar, V. Bhalla, P.R. Sharma, T. Kaur, Highly selective fluorescence turn-
on chemodosimeter based on rhodamine for nanomolar detection of copper ions, Org.
Lett. 14 (2011) 406–409.
[30] X.-F. Yang, X.-Q. Guo, Y.-B. Zhao, Development of a novel rhodamine-type fluorescent
probe to determine peroxynitrite, Talanta 57 (2002) 883–890.
[
[31] N. Vijay, G. Balamurugan, P. Venkatesan, S.P. Wu, S. Velmathi, A triple action chemo-
sensor for Cu2+ by chromogenic, Cr3+ by fluorogenic and CN
−
by relay recognition
methods with bio-imaging of HeLa cells, Photochem. Photobiol. Sci. 16 (2017)
1441–1448.
[
9] J.Y. Kwon, Y.J. Jang, Y.J. Lee, K.M. Kim, M.S. Seo, W. Nam, J. Yoon, A highly selective
fluorescent chemosensor for Pb2 , J. Am. Chem. Soc. 127 (2005) 10107–10111.
+
[32] Y.-K. Yang, K.-J. Yook, J. Tae, A rhodamine-based fluorescent and colorimetric chemo-
[
10] A. Kundu, S.P. Anthony, Triphenylamine based reactive coloro/fluorimetric chemo-
sensors: structural isomerism and solvent dependent sensitivity and selectivity,
Spectrochim. Acta Part. A: Mol. Biomol. Spectrosc. 189 (2018) 342–348.
11] P. Puangploy, S. Smanmoo, W. Surareungchai, A new rhodamine derivative-based che-
mosensor for highly selective and sensitive determination of Cu2 , Sens. Actuators B:
Chem. 193 (2014) 679–686.
dosimeter for the rapid detection of Hg2+ ions in aqueous media, J. Am. Chem. Soc. 127
(2005) 16760–16761.
[33] J.-S. Wu, I.-C. Hwang, K.S. Kim, J.S. Kim, Rhodamine-based Hg2+ selective chemodosi-
[
meter in aqueous solution: fluorescent OFF− ON, Org. Lett. 9 (2007) 907–910.
+
[34] L.-F. Zhang, J.-L. Zhao, X. Zeng, L. Mu, X.-K. Jiang, M. Deng, J.-X. Zhang, G. Wei, Tuning
with pH: the selectivity of a new rhodamine B derivative chemosensor for Fe3+ and Cu2+
,
[
[
[
12] J.F. Collins, J.R. Prohaska, M.D. Knutson, Metabolic crossroads of iron and copper, Nutr.
Sens. Actuators B Chem. 160 (2011) 662–669.
Rev. 68 (2010) 133–147.
[35] B. Wang, J. Hai, Z. Liu, Q. Wang, Z. Yang, S. Sun, Selective detection of Iron (III) by
13] B. D’autréaux, N.P. Tucker, R. Dixon, S. Spiro, A non-haem iron centre in the transcription
factor NorR senses nitric oxide, Nature 437 (2005) 769.
3 4
rhodamine‐modified Fe O nanoparticles, Angew. Chem. Int. Ed. 49 (2010) 4576–4579.
14] D. Galaris, V. Skiada, A. Barbouti, Redox signaling and cancer: the role of “labile” iron,
6