A novel “off-on” rhodamine-based colorimetric and fluorescent chemosensor based on hydrolysis driven by aqueous medium for the detection of Fe3+

Article abstract of DOI:10.1016/j.saa.2019.117951

A novel “off-on” colorimetric and fluorescent chemosensor N²,N⁶-bis(2-(rhodamine B amido)ethyl)pyridine-2,6-dicarboxamide (RhBEP) has been designed and synthesized, which can selectively detect the presence of Fe³⁺ with about 75-fold enhancement in fluorescence emission intensity at 585 nm over various environmentally relevant metal cations such as Hg²⁺, Cu²⁺, Cr³⁺, Li⁺, Na⁺, K⁺, Ag⁺, Zn²⁺, Mg²⁺, Ca²⁺, Ba²⁺, Fe²⁺, Cd²⁺, Ni²⁺, Mn²⁺, Al³⁺, Bi³⁺ and Au³⁺ in PBS reaction media. The remarkable color change from UV–Vis titration experiments indicates that RhBEP can be used as a colorimetric chemosensor for Fe³⁺. The ultrasensitive detection limit for Fe³⁺ in the fluorescence measurement is down to 2.0 × 10^{? 8} mol·L^{? 1}. The recognition mechanism of RhBEP to Fe³⁺ was analyzed by Job's plot and mass spectrometry analysis. In addition, the data from fluorescent cell imaging experiments confirmed that the chemosensor RhBEP has a promising application for the detection of Fe³⁺ in biological systems.

Full text of DOI:10.1016/j.saa.2019.117951

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A novel “off-on” rhodamine-based colorimetric and fluorescent
chemosensor based on hydrolysis driven by aqueous medium for
the detection of Fe3+
Xiaochun Wang, Tiechun Li
PII:
S1386-1425(19)31342-3
https://doi.org/10.1016/j.saa.2019.117951
SAA 117951
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 16 October 2019
Revised date:
10 December 2019
0 December 2019
Accepted
date:
1
Please cite this article as: X. Wang and T. Li, A novel “off-on” rhodamine-based
colorimetric and fluorescent chemosensor based on hydrolysis driven by aqueous medium
for the detection of Fe3+, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy(2018), https://doi.org/10.1016/j.saa.2019.117951
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A novel “off-on” rhodamine-based colorimetric and
fluorescent chemosensor based on hydrolysis driven by
3+
aqueous medium for the detection of Fe
Xiaochun Wang *, Tiechun Li
School of Chemistry and Life Science, Anshan Normal University, Anshan 114016, China
ABSTRACT
novel
A
“off-on”
colorimetric
and
fluorescent
chemosensor
2
6
N ,N -bis(2-(rhodamine B amido)ethyl)pyridine-2,6-dicarboxamide (RhBEP) has
3
+
been designed and synthesized, which can selectively detect the presence of Fe with
about 75-fold enhancement in fluorescence emission intensity at 585 nm over various
2
+
2+
3+
+
+
+
+
environmentally relevant metal cations such as Hg , Cu , Cr , Li , Na , K , Ag ,
2
+
2+
2+
2+
2+
2+
2+
2+
3+
3+
3+
Zn , Mg , Ca , Ba , Fe , Cd , Ni , Mn , Al , Bi and Au in PBS reaction
media. The remarkable color change from UV-Vis titration experiments indicates that
3
+
RhBEP can be used as a colorimetric chemosensor for Fe . The ultrasensitive
3
+
-8
-1
detection limit for Fe in the fluorescence measurement is down to 2.0 × 10 mol·L .
3
+
The recognition mechanism of RhBEP to Fe was analyzed by Job's plot and mass
spectrometry analysis. In addition, the data from fluorescent cell imaging experiments
confirmed that the chemosensor RhBEP has a promising application for the detection
3
+
of Fe in biological systems.
3
+
Keywords: Chemosensor; Rhodamine B; Fe ; Fluorescence enhancement; Living
cell imaging
1

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1
. Introduction
3
+
Fe plays an important role in living organisms since it is an important
component of hemoglobin, myoglobin and various other enzymes, which can promote
3
+
the metabolism of vitamin B [1]. In cellular biochemical reaction, Fe is an
indispensable ion for most organisms due to its role in biological signal transmission
and material transportation [2-4]. As one of the most abundant transition metal ions in
3
+
the human body, both the lack and the excess of Fe can cause various dysfunctions
3
+
of organisms. For instance, the lack of Fe can affect the synthesis of hemoglobin
and myoglobin and reduce the activities of cytochrome C and ribonucleotide
reductase, etc [5-7]. Because these enzymes are closely linked to tissue respiration,
3
+
bio-oxidation and neurotransmitter‟s decomposition and synthesis, the lack of Fe
can cause the decreases of immunity, intelligence and anti-infective capacity, as well
as can affect the body‟s ability in regulating temperature, thereby can lead to
3
+
neurological disorders [8, 9]. However, excessive intake of Fe can cause an
imbalance of iron ion in blood, which can lead to oxidative damage to cells and
reduced blood circulation to the heart [10-12]. Therefore, it is important to find a fast
3
+
and sensitive method to identify the distribution of Fe in living organisms.
3
+
Although some methods have been developed for the detection of Fe in the past
few decades, these methods have some disadvantages, including low specificity,
complicated sample preparation and operation, requirement of skilled personnel, and
3
+
costly instruments [13-19]. Thus, they are not ideal for the detection of Fe in living
organisms. In recent years, fluorescence method has been rapidly developed and
turned into one of the most extensively used techniques in the field [20-27]. It has
various advantages, such as high sensitivity and selectivity, rapid analysis, and
simplicity in instrument operation. It does not cause damage to cell while can generate
2

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visually observed signals. Actually, Fluorescence quenching due to the paramagnetic
3
+
3+
properties of Fe when binding of fluorescent chemosensors to Fe may result in the
3
+
difficulty in the detection of Fe
[28-32]. Compared with the quenchable
chemosensors, the enhanced fluorescent chemosensors (“turn-on” or “off-on”) have
stronger signal intensity, cannot be easily interfered by background interference, and
can be used for the detection in vivo [33-36]. Thus, it is of great significance to
develop an “off-on” fluorescent chemosensors with high sensitivity, selectivity and
3
+
rapid response for Fe .
3
+
As we know from the hard-soft acids and bases theory, Fe is a hard acid and O
and N are hard bases, which can be relatively easily combined. In this paper, we
reported the design and synthesis of an “off-on” rhodamine-based chemosensor
2
6
N ,N -bis(2-(rhodamine B amido)ethyl)pyridine-2,6-dicarboxamide (RhBEP), which
3
+
demonstrates an ultrasensitive, selective colorimetric and fluorescent response to Fe
3
+
and can realize the visual recognition of Fe in biological systems.
2
. Experimental details
2
.1 Synthesis of RhBEP
The synthetic route of the chemosensor RhBEP was illustrated in Fig. 1. The
compound RhBE was synthesized using rhodamine B as a raw material according to
the previously described method [37, 38]. Chemosensor RhBEP was synthesized as
the follow: pyridine-2,6-dicarbonyl dichloride (0.20 g, 1 mmol) was mixed with 0.55
mL (4 mmol) of triethylamine and 10 mL of CH
stirred at 0°C for 30 min. After that, 20 mL of a solution containing 1.0 g of RhBE
2.1 mmol) was added dropwise at 0°C. The mixture was then stirred for 12 h at room
2
Cl
2
in a 100-mL flask and then
(
temperature. After the reaction was completed, the reaction solution was dried and
washed three times with 100 mL of cold 95% EtOH. Finally, 0.78 g of white powder
3

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1
was obtained with a yield of 71%. ESI-MS: m/z 1100.31 (Fig. S1). H NMR (300
MHz, Chloroform-d) δ 9.65 (s, 2H), 8.18 (d, J = 7.7 Hz, 2H), 8.04 (d, J = 7.2 Hz, 2H),
7
4
1
1
1
.93–7.84 (m, 1H), 7.57-7.39 (m, 4H), 7.07 (d, J = 7.1 Hz, 2H), 6.52 (d, J = 8.7 Hz,
H), 6.38 (s, 4H), 6.25 (s, 4H), 3.58 (s, 4H), 3.38 (s, 4H), 3.29 (d, J = 7.0 Hz, 16H),
1
3
.15 (t, J = 6.9 Hz, 24H) (Fig. S2). C NMR (75 MHz, Chloroform-d) δ 169.55,
63.91, 153.90, 153.30, 148.67, 138.13, 132.49, 130.62, 128.65, 128.21, 123.84,
23.68, 123.22, 108.16, 105.23, 97.90, 65.54, 44.30, 40.25, 39.82, 12.58 (Fig. S3).
Fig. 1 Synthesis of the chemosensor RhBEP
2
.2 Fluorescence measurements
To express concisely, CH
3
CN: PBS solution (3:7, v/v, 50 mM PBS, pH = 7.4)
was defined as the PBS reaction media. Nine-hundred and fifty microliters of PBS
-3
reaction media and 10 μL of 1.0×10 M RhBEP in DMSO stock solution were
mixed in a 1-mL centrifuge tube. After that, different concentrations of metal
perchlorate salt solution were added and final volume of the solution was adjusted to
1
mL with PBS reaction media. After the reaction was allowed to incubate at room
temperature for 20 min, a series of tests including fluorescence selectivity, UV
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titration and fluorescence titration tests were performed to investigate the effect of pH
and fluorescence imaging on living cells.
2
.3 Cell culture and fluorescence imaging
HepG2 cells were cultured in DMEM medium containing 10% bovine serum,
00 units/mL penicillin and 100 mg/L streptomycin in an incubator saturated with 5%
1
CO
10 μM) for 0.5 h prior to the observation under a confocal fluorescence microscope.
Prior to fluorescence imaging, cells in experimental group were subjected to
2
and 95% air at 37 °C for 24 h. Cells in control group were incubated with RhBEP
(
3
+
exogenous test with different concentrations of Fe . The first group was implemented
as follow: cells were incubated with RhBEP (10 μM) for 0.5 h. The second group was
implemented as follow: cells were incubated with 100 μM Fe(ClO
RhBEP (10 μM) was added for 0.5 h. The third group was implemented as follow:
cells were incubated with 20 μM Fe(ClO for 3h and then RhBEP (10 μM) was
added for 0.5 h. Prior to fluorescence imaging test, RhBEP and Fe(ClO that did not
4
)
3
for 3h and then
)
4 3
)
4 3
enter into the cells were washed with PBS buffer so that they do not interfere with cell
imaging.
3
. Results and discussion
3
.1 Selectivity towards cations
Due to its significance in the evaluation, the selectivity of chemosensor RhBEP
towards various metal cations was examined by fluorescence spectroscopy. First, the
selectivity of RhBEP to metal ions in pure acetonitrile was investigated, and the
results are shown in Fig. 2 (black bars). After different common metal ions, each at a
3
+
2+
2+
3+
concentration of 50 μM were added, only Fe , Hg , Cu and Cr led to the
3
+
enhancement of fluorescent signal, among which the enhancement caused by Fe was
2
+
2+
3+
1
0 times higher that RhBEP signal, while that caused by Hg , Cu and Cr was
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about 5-8 times. This indicates that the chemosensor RhBEP has poor specificity
towards metal ions in in pure acetonitrile medium. After that, the selectivity of the
chemosensor RhBEP was examined in PBS reaction media. The results were shown in
3
+
Fig. 2 (red bars). Fe caused fluorescence enhancement by 75 times and other metal
ions only led to slight enhancement. These data indicate that higher selectivity for
3
+
Fe of the chemosensor RhBEP could be achieved in PBS reaction media. In the
addition, the competition experiments have also been carried out. As shown in Fig.S4,
the addition of 5 equiv of other competitive metal ions to RhBEP solution scarcely
2
+
2+
3+
+
+
+
+
2+
2+
showed fluorescence enhancement for Hg , Cu , Cr , Li , Na , K , Ag , Zn , Mg ,
2
+
2+
2+
2+
2+
2+
3+
3+
3+
3+
Ca , Ba , Fe , Cd , Ni , Mn , Al , Bi and Au except for Fe (black bar).
However, the fluorescence intensity of the mixtures increased after the subsequent
3
+
addition of the Fe (red bar), which suggests that the fluorescent selectivity of
3
+
RhBEP for Fe over other coexisting metal ions is remarkably high (Fig. S4).
Fig. 2 Fluorescence intensity of RhBEP (10 μM) in 100% CH
3 3
CN (black bars) and 30% CH CN
(
0.1 M PBS, pH 7.4, red bars) at 585 nm, in the absence and presence of different metal ions (50
6

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3+
2+
2+
3+
+
+
+
μM). Excitation wavelength was 520 nm. From 1 to 20: none, Fe , Hg , Cu , Cr , Li , Na , K ,
+
2+
2+
2+
2+
2+
2+
2+
2+
3+
3+
3+
3+
Ag , Zn , Mg , Ca , Ba , Fe , Cd , Ni , Mn , Al , Bi , and Au . Inset: The proposed Fe
3+
recognition mechanism of the RhBEP, and ESI-MS spectra of RhBEP upon addition of Fe in the
absence and presence of H
2
O
3
+
3
.2 Fe -recognition mechanism of the chemodosimter RhBEP
First, we conducted a Job‟s plot experiment of the reaction between RhBEP and
3
+
Fe in different media. The results showed that the binding ratio between RhBEP and
3
+
Fe was 1:1 irrespective of the types of media (Fig. S5). The mass spectrum showed
3
+
a peak at m/z = 1153.21 associated with a 1:1 ratio of RhBEP and Fe in pure
acetonitrile medium, further indicating that the RhBEP-Fe complex was formed.
When PBS reaction media was used, the peak of RhBEP-Fe disappeared, but two new
peaks were observed at m/z = 485.01 and 167.75, which correspond to RhBE-H and
dipicolinic acid, respectively (Fig. 2, Inset). To further confirm the structure of the
new product, the product was separated by column chromatography (silica gel,
1
CH
2
Cl
2
: MeOH = 15:1), and its structure was characterized by H NMR (Fig. S6).
The result showed that the product had the structure of RhBE, which further
confirmed that the complex RhBEP-Fe hydrolyzed completely in PBS reaction media.
Based on the results, a recognition mechanism was proposed as shown in Fig. 2. In
3
+
the mechanism, after Fe is added, the fluorescence intensity of RhBEP varies with
the types of solvents or media. In pure acetonitrile medium, the RhBEP-Fe complex is
formed and causes the quenching of fluorescence. By contrast, in the PBS reaction
media, the RhBEP exists as RhBE-H with an opened spiro ring that leads to high
fluorescence signal.
3
.3 Optimization of experimental conditions
The selectivity experiment demonstrated that the presence of water could affect
3
+
the fluorescence intensity of RhBEP when bound to Fe . Therefore, the fluorescence
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responses of RhBEP in different ratios of CH
3
CN: PBS solution (50 mM, pH = 7.4)
were investigated. As shown in Fig. 3a, when the proportion of water increased from
3
+
1
0% to 70%, the fluorescence signal significantly increased (after Fe was added).
This indicates that the increase of the proportion of water may improve the amount of
3
+
hydrolysis of the complex RhBEP-Fe . When the water content was increased to
higher than 70%, the fluorescence signal decreased, indicating that the decreased
solubility of RhBEP leads to the quenching of fluorescence [39, 40]. Therefore,
CH
medium.
The effect of pH on the fluorescent signal was also investigated. As illustrated in
3
CN: PBS solution (3:7, v/v, 50 mM PBS, pH = 7.4) was adopted as the reaction
Fig. 3b, the fluorescence intensity of RhBEP remained unchanged between a pH
range of 5 to 12. At pH < 5, the fluorescence intensity increased with the increase of
+
pH since H can open the spiro ring of rhodamine B, thereby led to higher emission of
3
+
fluorescence. When Fe was added to RhBEP solution with different pH, RhBEP
emitted strong fluorescent signal within the pH range of 2 to 8, which indicated that
3
+
the complex between RhBEP and Fe underwent hydrolysis at this pH range. At
pH > 8, the fluorescence intensity of RhBEP was drastically reduced, which suggested
3
+
that Fe was hydrolyzed into its hydroxide derivative that could not form complex
with RhBEP [41]. Therefore, it can be concluded that the optimal pH range for the
3
+
detection of Fe using RhBEP is 5 - 8. The physiological pH (e.g. in the human body)
3
+
is between 7.35 and 7.45, which lies within the optimal pH range of the Fe -specific
3
+
chemosenor RhBEP. Thus, pH 7.4 was used in subsequent study, in which Fe in
living cells was detected.
In addition, the effect of reaction time was also investigated. As shown in Fig. 3c,
3
+
the fluorescence intensity of RhBEP without the addition of Fe maintained stable.
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3
+
After Fe was added, the fluorescence intensity of RhBEP rapidly increased within
the first 5 min, and the rate of increase was declined from 6 to 15 min. The intensity
remained stable after 20 min. Therefore, 20 min was selected as the optimum reaction
time.
3
.4 UV and fluorescence titration tests
Under the optimal conditions, UV-Vis and fluorescence titration tests were
3
+
carried out. The UV-Vis titration spectra (Fig. 4a) showed that in the absence of Fe ,
the solution was colorless and had a very weak absorption peak at 560 nm, which
indicates that the spiro structure of the chemosensor RhBEP is in a closed state. When
RhBEP
a
1
1
600
200
3
+
RhBEP+Fe
8
4
00
00
0
0
10
20
30
40
50
60
70
80
90
Water Content (%)
2
1
1
000
500
000
1800
1600
b
RhBEP
RhBEP+Fe
RhBEP
c
3
+
3+
RhBEP+Fe
1
1
1
8
400
200
000
0
5
00
7
0
0
40
0
1
2
3
4
5
6
7
8
9
10 11 12 13
10
15
20
25
30
pH
Time (min)
3+
Fig. 3 Fluorescence intensity of RhBEP (10 μM) in the absence and presence of Fe (50 μM) at
585 nm. (a) effect of water content. The reaction was performed at room temperature for 20 min
with different water content. (b) effects of pH. The reaction was performed at room temperature
for 20 min in 0.1 M PBS buffer solution with different pH values adjusted by HCl or NaOH. (c)
effect of time. The reaction was conducted at room temperature for different times in 0.1 M PBS
buffer solution (pH 7.4).
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3
+
Fe was gradually added, the intensity of the absorption peak at 560 nm was
gradually increased, and the solution color turned pink. This indicates that the spiro
ring is in open state. This remarkable color change implies that RhBEP can be used as
3
+
a colorimetric chemosensor to detect Fe , and the outcomes can be observed by the
naked eye. Fluorescence titration test was also carried out (Fig. 4b). In the absence of
3
+
3+
Fe , no fluorescent emission at 585 nm was observed. When Fe was gradually
added, the fluorescent emission at 585 nm gradually became intensified, which is in
agreement with the UV-Vis titration results. The results also showed that the
3
+
fluorescence intensity of RhBEP had a linear relationship with Fe concentration in
3
+
the concentration range of 0 to 50 μM ( F = 29.92 [Fe ] + 11.19, r = 0.9987). The
detection limit was found to be 20 nM from ten blank solution measurements, based
on the definition by IUPAC (DL = 3S/ρ, S: standard deviation of the measurement of
the blank; ρ: the slope of linear equation) (Fig. 4c), which was lower than that
obtained by previously reported fluorescent probes (Table S1).
3
+
Fig. 4 (a) UV-Vis titration and (b) Fluorescence titration of RhBEP (10 μM) upon addition of Fe
3
+
(
0-100 μM). (c) The curve of fluorescence intensity versus Fe concentration, fitted with a linear
3
+
regression equation over Fe concentrations of 0-50 μM
3
.5 Fluorescent imaging of living cell
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The designed chemosensor can play an important role in biological applications
if it can be applied to selective detection of target analytes in biological systems [42].
To verify the assumption, cytotoxicity assay and fluorescence imaging were carried
out using HepG2 cells. As shown in Fig. S7, at least 85% of HepG2 cells remained
viable within 12 h after being treated with RhBEP at a concentration of up to 50 μM.
This indicates that RhBEP has low cytotoxicity, thus good biocompatibility. As
depicted in Fig.5a, the fluorescent red channel of cells in the control group was not
observed under the microscope although after being treated with RhBEP. The results
from exogenous test (Fig. 5b-5c) showed that bright red fluorescence was observed in
3
+
the cells, and the signal intensities varied with the concentration of Fe . Meanwhile,
nuclear staining with DAPI indicates that the cells are viable throughout the imaging
experiments. These results indicate that RhBEP is able to penetrate cell membranes
and causes fluorescence signal observed in the cells.
3+
Fig. 5 Fluorescence imaging of Fe in HepG2 cells. The cells are treated with DAPI (0.1 mM)
nuclear counter-stain (control). (a) cells incubated with RhBEP (10 μM) for 0.5 h; (b) cells
4 3
incubated with Fe(ClO ) (100 μM) for 3 h, and then RhBEP (10 μM) for 0.5 h. (c) cells incubated
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with Fe(ClO
4
)
3
(20 μM) for 3 h, and then RhBEP (10 μM) for 0.5 h. Blue channel: λex= 405 nm,
λem= 425-475 nm; red channel: λex=532 nm, λem= 560-660 nm.
4
. Conclusions
In this paper, a novel“off-on” rhodamine-based colorimetric and fluorescent
chemosensor RhBEP was designed and synthesized, which showed colorimetric and
3
+
fluorescent selectivity to Fe over other competing ions and “off-on” UV-vis and
fluorescence response with high sensitivity due to the hydrolysis of the
chemosensor-target complex in PBS reaction media. The recognition mechanism of
RhBEP was confirmed by Job‟s plot and mass spectrometry. In addition, the
3
+
exogenous tests were applied to investigate the ability of RhBEP in detecting Fe in
human hepatocarcinoma cells. This study offers an effective analytical method for
3
+
real-time detection of Fe in biological systems.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(
(
21775005, 41430641), Liaoning Provincial Natural Science Foundation of China
201602001), Liaoning BaiQianWan Talents Program, Key Scientific Research
Program of Anshan Normal University (16kyxm01) and Research Fund for the
Doctoral Program (2016B03) from Anshan Normal University.
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Author contribution statement
Xiaochun Wang: Conceptualization, Methodology, Validation,
Investigation, Writing-Original Draft, Funding acquisition.
Tiechun Li: Resources, Supervision, Project administration.
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Declaration of interests
The authors declare that they have no known competing financial
☒
interests or personal relationshipsthat could have appeared toinfluence
the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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Graphical Abstract
A novel “off-on” colorimetric and fluorescent chemosensor RhBEP was designed and
3
+
synthesized, which exhibits not only high sensitivity and selectivity for Fe over
other coexistent metal ions, but also cell permeability and suitability for detecting Fe
in the living cells by confocal microscopy.
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Highlight
2
6
A novel “off-on” chemosensor N ,N -bis(2-(rhodamine B
amido)ethyl)pyridine-2,6-dicarboxamide (RhBEP) was developed
3
+
Chemosensor RhBEP exhibits high sensitivity and selectivity for Fe over other
coexistent metal ions
3
+
Chemosensor RhBEP is cell permeable and suitable for detecting Fe in the living
cells by confocal microscopy
22