A fluorescent chemosensor for relay recognition of Fe3+ and PO43? in aqueous solution and its applications

Article abstract of DOI:10.1016/j.tet.2017.07.018

A novel fluorescent probe XDS based on 4-methylumbelliferone and 2-picolylamine platforms has been designed and synthesized, which behaves fast relay recognition of Fe³⁺ and PO₄^3? via a fluorescence “on?off?on” response signal. Probe XDS exhibited very high sensitivity and unique selectivity for Fe³⁺ over other common metal ions, and the detection limit of was 3.2 × 10^?7 M. Moreover, the addition of the PO₄^3? ions could cause the recovery of fluorescence. This relay recognition feature of probe XDS has potential applications in the determination of trace amount of Fe³⁺ and PO₄^3? in environmental systems. Interestingly, fluorescence imaging experiments demonstrated that the probe XDS can also be used to monitoring the intracellular Fe³⁺ in RAMOS cells.

Full text of DOI:10.1016/j.tet.2017.07.018

Tetrahedron xxx (2017) 1e10
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A fluorescent chemosensor for relay recognition of Fe3þ and PO43ꢀ in
aqueous solution and its applications
,
Renjie Liu a, Xu Tang a, Yun Wang a **, Juan Han b, Huiqin Zhang c, Chen Li a,
,
Wenli Zhang a *, Liang Ni a, Haoran Li d
a School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, 212013, PR China
b School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, PR China
c Zhenjiang Entry-exit Inspection Quarantine Bureau, State Key Laboratory of Food Additive and Condiment Testing, Zhenjiang, Jiangsu Province, 212013,
China
d Department of Chemistry, ZJU-NHU United R&D Center, Zhejiang University, Hangzhou, 310027, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel fluorescent probe XDS based on 4-methylumbelliferone and 2-picolylamine platforms has been
designed and synthesized, which behaves fast relay recognition of Fe3þ and PO43ꢀ via a fluorescence
“onꢀoffꢀon” response signal. Probe XDS exhibited very high sensitivity and unique selectivity for Fe3þ
over other common metal ions, and the detection limit of was 3.2 ꢁ 10ꢀ7 M. Moreover, the addition of
the PO34ꢀ ions could cause the recovery of fluorescence. This relay recognition feature of probe XDS has
potential applications in the determination of trace amount of Fe3þ and PO43ꢀ in environmental systems.
Interestingly, fluorescence imaging experiments demonstrated that the probe XDS can also be used to
monitoring the intracellular Fe3þ in RAMOS cells.
Received 4 May 2017
Received in revised form
10 July 2017
Accepted 11 July 2017
Available online xxx
Keywords:
Fluorescent probe
Ferri ion
© 2017 Elsevier Ltd. All rights reserved.
PO34ꢀ
Relay recognition
Cell imaging
1. Introduction
spectrophotometric and spectrofluorometric methods.15,16
It is well known that iron is one of the most essential trace el-
The design and development of chemosensors for the recogni-
tion of cations and anions have attracted noticeable attention due
to their important roles in the wide range of environmental, clin-
ical, and biological systems.1e4 With the comparisons to other
instrumental methods such as inductively coupled plasma atomic
emission or mass spectroscopy (TCP-AES, ICP-MS), atomic absorp-
tion spectroscopy (AAS), electro-chemical methods, etc,5e11 fluo-
rescent detections have more practical value for sensing and
detecting trace amounts ions owing to its favorable superiorities
including non-destructive character, high selectivity and sensi-
tivity, fast response and economical method for the detection
without any tedious sample pretreatment.12e14
ements in human body. It is an indispensable ion for most organ-
isms by exhibiting a crucial role in various biological and chemical
processes at the cellular level such as oxygen uptake, oxygen
metabolism, enzyme catalysis.17e19 However, its deficiency or
excess may lead to hypoferremia or hyperferremia, accordingly. In
addition, it has recently been discovered that iron is another key
limiting factor for the primary productivity of phytoplankton be-
sides elemental nitrogen, phosphorus and silicon.20,21 In contrast,
phosphates and its derivatives (eg, adenosine triphosphate (ATP))
play a crucial role in signal transduction and energy storage in
biological systems.22,23 Phosphate anions are ubiquitous in bio-
logical systems and it plays vital functional roles in cell signaling,
membrane integrity, bone mineralization, muscle function and
other important biological processes.24e26 Unfortunately, only a
small number of fluorescent anion sensors can effectively distin-
guish Fꢀ, AcOꢀ, phosphates and their derivatives.27e31
Therefore, considerable studies have concentrated on con-
struction of highly efficient and convenient ion probe using
Thus, it is highly desirable to develop a highly selective and
sensitive analytical method for the detection of Fe3þ and PO43ꢀ for
further study of their physiological and pathophysiological
* Corresponding author.
** Corresponding author.
E-mail addresses: yunwang@ujs.edu.cn (Y. Wang), wlzhang@ujs.edu.cn
(W. Zhang).
http://dx.doi.org/10.1016/j.tet.2017.07.018
0040-4020/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Liu R, et al., A fluorescent chemosensor for relay recognition of Fe3þ and PO43ꢀ in aqueous solution and its
applications, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.07.018

2
R. Liu et al. / Tetrahedron xxx (2017) 1e10
functions in living organisms.
K2CO3(1.1 g) and KI (1.3 g) at room temperature under N2 over a
period of 30 min and the resulting mixture was stirred for an
additional 12 h. Then the resulting mixture was cooled to room
temperature, filtered over gravity and the solvent was removed
under reduced pressure to afford solid product, which was purified
by recrystallization from ethanol to get analytically pure compound
XDS (4-methyl-2-oxo-2H-chromen-7-yl 2-((pyridin-2-ylmethyl)
amino)acetate) (1.1 g) in 86% yield (Scheme 1). The product is
verified by 1H NMR, 13C NMR, MS (Figs. S1e3). 1H NMR (400 MHz,
Herein, we have prepared a simple and efficient fluorescent
probe XDS based on 4-methylumbelliferone and 2-picolylamine
platforms for relay recognition of Fe3þ and PO43ꢀ. We chose
coumarin as the fluorescent group because of its good fluorescence
properties and water solubility. Meanwhile, as a recognition group,
2-aminomethylpyridine can provide the N atoms as excellent
coordinating site. Thus, we chose Chloroacetyl chloride as a linker
to effectively combine the 4-methylumbelliferone and 2-
picolylamine to form a compound XDS with strong fluorescence
emission. In this study, we also further investigated in detail the
fluorescence performances of the probe XDS. The investigated re-
sults demonstrated that the probe XDS exhibits high selectivity,
sensitivity and rapid fluorescence response to Fe3þ ion over other
metal cations in aqueous solution. Moreover, we also explore
antion-sensing properties of the in situ-formed [XDS-Fe3þ] com-
plexes in the aqueous environment and test trips. As anticipated,
the complex exhibited excellent properties for PO34ꢀ detection ac-
cording to the strong binding capacity of PO34ꢀ to Fe3þ. At the same
time, the performance of XDS for the fluorescence imaging in living
cells was also evaluated.
DMSO):
d
¼ 8.87 (t, J ¼ 5.7 Hz, 1H), 8.51 (d, J ¼ 4.5 Hz, 1H), 7.74 (dd,
J ¼ 12.8, 5.1 Hz, 2H), 7.35e7.21 (m, 2H), 7.09e6.97 (m, 2H), 6.24 (s,
1H), 4.75 (s, 2H), 4.46 (d, J ¼ 5.9 Hz, 2H), 2.37 (d, J ¼ 29.8 Hz, 3H)
ppm; 13C NMR (101 MHz, DMSO):
39.75, 39.58, 39.37, 18.61 ppm.
d
¼ 40.62, 40.41, 40.20, 39.89,
MS: [M þ H]þ calcd for C18H16O4N2: 325.33, found: 325.60.
2.3. Fluorescence spectroscopy
Unless otherwise noted, materials were of analytical grade from
commercial suppliers and were used without further purification.
Deionized water was used throughout all experiments.
Stock solutions (10 mM) of the various anions of Fꢀ, Clꢀ, Brꢀ, Iꢀ,
SO24ꢀ, SCNꢀ, ClO4ꢀ, CO32ꢀ, NOꢀ3 , H2POꢀ4 , HPO42ꢀ, NO2ꢀ, AcO - and CNꢀ in
deionized water, were prepared. Stock solutions (10 mM) of various
metal ions were prepared from NaCl, CsCl, PbCl2, CoCl2, ZnCl2,
CuCl2, NiCl2, HgCl2, CdCl2, CrCl3, FeCl2,FeCl3, LiCl, MgCl2, CaCl2, AlCl3,
SrCl2 and MnCl2.
Stock solution of probe XDS (1 mM) was also prepared in
deionized water and diluted to prepare the analytical solution
(10 mM). For fluorescence measurements, both the excitation and
emission slit widths were 5 nm. The excitation wavelength was set
at 320 nm.
2. Experimental
2.1. Materials and instruments
Unless otherwise noted, all reagents and solvents employed for
synthesis were purchased from Aladdin Chemical Reagent Ltd., and
used without further purification. All the metal chlorate salts (Al3þ
,
,
Sr2þ, Liþ, Csþ, Ca2þ, Cd2þ, Cr3þ, Co2þ, Cu2þ,Fe2þ, Fe3þ,Kþ, Hg2þ
Mg2þ, Mn2þ,Ni2þ,Naþ, Pb2þ and Zn2þ.) and all anionic sodium salt
(PO34ꢀ,Fꢀ, Clꢀ, Brꢀ, Iꢀ,SO24ꢀ, SCNꢀ, ClO4ꢀ, CO32ꢀ, NOꢀ3 , NO2ꢀ, H2PO4ꢀ,
HPO24ꢀ, AcO - and CNꢀ.) were purchased from Sinopharm Chemical
Reagent Ltd.,
2.4. Cell culture and imaging studies
1H NMR and 13C NMR spectra were measured using an AVANCE
Ⅱ 400 MHz spectrometer (Bruker, Switzerland). Mass spectra were
obtained using a Thermo LXQ Liquid chromatography ion trap mass
spectrometer (USA). Fluorescence measurements were taken at a
Cary Eclipse fluorescence spectrophotometer (Variance. LTD,
Australia). Absorption spectra were recorded with a UV-2450
UVeVis spectrophotometer (Shimadzu, Japan) at room tempera-
ture. Cell experiments were applied on an inverted fluorescence
microscope (Leica DMI4000B, Germany).
The RAMOS cells (Human B lymphocyte tumor cells) were
seeded on a 24-well plate and were incubated in medium (sup-
plementing with RPMI 1640, 10% FBS) at 37 ꢂC for 24 h. Subse-
quently, the cells were incubated with 20 m
M of probe XDS at 37ꢂ C
for 0.5 h and then washing with three times to remove the
remaining probe XDS. The cells were then incubated with
Fe3þ(20 M) at 37ꢂ C for an additional 0.5 h, then 20 M of PO34ꢀ
m m
were added and incubated for another 0.5 h. The treated cells were
rinsed three times with buffered saline. The cell imaging in
different stages were obtained by an inverted fluorescence
microscope.
2.2. Synthesis of XDS
2.2.1. Compound R: 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)
acetyl chloride
2.5. Computational methodology
Chloroacetyl chloride (0.35 mL) was dissolved in dry CH2Cl2 at
0
ꢂC, and then added dropwise to a cooled stirred mixture of 4-
The density functional theory (DFT) calculations were perused
to get the theoretical aspects of the coordinating mode of the probe
XDS and Fe3þ. The DFT optimizations of probe XDS and XDS-Fe3þ
complex were carried out using the Gaussian 09 program,32 in
which the B3LYP function was used. The 6-311 þ G* and LanL2DZ
basis sets were used for the probe XDS and the metal ions,
respectively.33 Besides, the distribution of electronic clouds on both
HOMO and LUMO of probe XDS and XDS-Fe3þ complex were also
studied.
MethylmMbelliferone (0.70 g, 4 mmol) and triethylamine (Et3N)
(0.3 mL) in CH2Cl2 under N2 within 1 h, After being stirred over
night at room temperature, the reaction mixture was quenched
with distilled water and then was extracted three times with 20 mL
of CH2Cl2. The combined organic layer was dried over anhydrous
Na2SO4 and removed under reduced pressure to obtain a white
solid product. The crude product was purified by recrystallization
from ethanol (methanol) to give analytically pure compound R
(0.93 g) in 93% yield.
3. Results and discussion
2.2.2. Compound XDS: 4-methyl-2-oxo-2H-chromen-7-yl 2-
((pyridine-2-ylmethyl) amino)acetate
3.1. Fluorescence characterization and selectivity of XDS toward
Fe3þ
R (1.006 g, 4 mmol) was dissolved in anhydrous CH3CN and the
solution was added dropwise to
aminomethylpyridine (0.86 g,
a
stirred mixture of 2-
8
mmol), NaHCO3 (0.69 g),
The effect of metal ions on the fluorescence properties of XDS
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R. Liu et al. / Tetrahedron xxx (2017) 1e10
3
Scheme 1. Synthesis of probe XDS.
was investigated through the naked eye detection in aqueous so-
lution. As shown in Fig. 1, only the mixture of XDS and Fe3þ showed
a fluorescence quenching phenomenon, the emission color change
from blue to colorless can be clearly observed in the presence of
Fe3þ under UV irradiation (365 nm), while no significant emission
color change can be induce by the addition of other metal ions.
Additionally, the response of the probe XDS toward different
metal ions was evaluated for affinity toward metal ions through
naked eye in the further investigation, another interesting phe-
nomenon was found that the addition of Fe3þ not only caused
obvious fluorescence change of the probe solution but also a slight
color change from colorless to yellow, while no change in color was
observed with the addition of other metal ions (Fig. S4).
In the subsequent fluorescence studies, the fluorescence prop-
erties of the mixture of XDS and Fe3þ were therefore carried out in
aqueous solution by fluorescence spectrophotometer. As can be
seen from Fig. 2, the free XDS (10 mM) showed a degree of fluo-
rescence intensity with emission maxima around 380 nm, sur-
prisingly, a significant reduction in the fluorescence intensity was
observed by the addition of Fe3þ, while minor fluorescence changes
can be noticed in the presence of other metal ions. The results
indicated that XDS can response to Fe3þ with high specifity by
obvious fluorescence quenching due to the formation of a new
Fig. 2. The fluorescence characterization of XDS and metal ions: the fluorescence
response of XDS (10
solution.
mM) by mixing with different metal ions (10 equiv.) in aqueous
complex between XDS and Fe3þ
.
characterizing the performance of the fluorescence probe, thus the
effect of the respond time on fluorescence intensity of this system
was also calculated by an experimental determination of the re-
action rate constant in aqueous solution. As shown in Fig. 3a, we
concluded that the complexation reaction of XDS and Fe3þ belongs
to the zero-order reaction. It is generally known that the zero-order
reaction rate is independent of the reactant concentration. To
provide a complete combination with the probe, we used 10 eq. of
Fe3þ ions in previous experiments. We also calculated its complete
reaction time of about 100 s from its linear equation, less than
2 min. The further experimental results displayed that the fluo-
rescence intensity of XDS was quenched rapidly, reaching a stable
value within 2 min after the addition of Fe3þ to XDS. This phe-
nomenon proved evidently that the interaction between XDS and
3.2. The stability of XDS and the respond time on sensing Fe3þ
In order to evaluate the stability of XDS and [XDS-Fe3þ], first,
XDS was irradiated under 365 nm for more than 60 min using the
LED source while the fluorescence intensity was investigated by the
fluorescence spectrophotometer. Fig. S5 shown that no apparent
fluorescence intensity change was observed during this period,
which suggested XDS is very stable. Thereafter, once 20 equivalents
of Fe3þ solution was added to the XDS solution, the fluorescence
intensity sharply decreased. The fluorescence signal is then main-
tained at a steady level for more than 60 min. These results indi-
cated evidently that both of XDS and [XDS-Fe3þ] complex are very
stable under the test conditions.
The response time is an extremely important factor in
Fig. 1. The luminescence change of the mixture of XDS (20
mM) and various metal ions (10 equiv.) under the UV lamp (
l
¼ 365 nm).
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4
R. Liu et al. / Tetrahedron xxx (2017) 1e10
Fig. 3. (a) The determination of the reaction rate constant of the complexation reaction of XDS and Fe3þ; (b) Fluorescence quenching profile of addition Fe3þ (10 equiv.) to XDS
(10 M) in aqueous solution from 1 to 60 min.
m
Fe3þ can occur directly at room temperature with excellent sensi-
tivity. The rapid, stable complexation provided XDS a potential
utility for an on-site rapid detection method for monitoring Fe3þ by
portable device in field.
3.3. Fluorescence titration
In order to further evaluate the response properties of probe
XDS to Fe3þ, fluorescence titration experiments with incremental
addition of a solution of Fe3þ ions were performed. It can be seen
from Fig. 4, fluorescence intensity was gradually weakened with
the increasing the concentration of Fe3þ. As Fig. S6 shown, a good
liner relationship (R ¼ 0.989) was noted between (F0-F) and [Fe3þ
]
at Fe3þ concentration from 1
mM to 10
m
M. The detection limit was
calculated to be 3.2 ꢁ 10ꢀ7 M (L ¼ 3
s
/K, K ¼ 1.49 ꢁ 107,
s
¼ 1.58),
much lower than most of the reported in literature. The job's plot
revealed 1:1 stoichiometry for the binding between XDS and Fe3þ
(Fig. 5). The association constant of XDS with Fe3þ in aqueous so-
lution was accordingly calculated to be 1.895 ꢁ 104
M
ꢀ1 (Fig. S7).
Fig. 5. Job's plot of XDS and Fe3þ, which indicated that the stoichiometry of the XDS-
Fe3þ complex is 1:1.
On the other hand, it must be noted that the probe XDS
possessed a large advantage over most reported fluorescent probes.
The probe XDS can show the excellent determination for Fe3þ in
pure water solution, and most of the previously reported fluores-
cent probes can only be applied in pure organic36,37 or the organic
solvent modified water solution.34,38e44 The comparisons about the
detection limit, linearity range and detection media between the
present method and those reported in literature were shown in
Table 1.
In addition, in order to check the stability and repeatability of
the quantitative determination of Fe3þ, we repeated the test for 26
times in 4 days. Our results suggested the established method is
very stable for Fe3þ determination with RSD of 2.2% (Fig. S8).
3.4. Competition experiments with other metal ions
Selectivity or anti-interference ability is a very important factor
in the evaluation of fluorescent probes or sensors. It determines
whether the probe or sensor can be performed to an actual sample
Fig. 4. Fluorescence spectra of probe XDS in 100% aqueous solution upon the addition
of Fe3þ ions (Ex ¼ 320 nm).
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R. Liu et al. / Tetrahedron xxx (2017) 1e10
5
Table 1
Comparison of the detection limit, detection media and response time for Fe3þ between the present method and those reported in literature.
Probes
Detection limit (
mM)
Detection media
Response time
References
34
35
37
38
39
40
42
43
44
45
46
47
Rhodamine derivatives
Rhodamine derivatives
Rhodamine derivatives
Fluorescent dye
Rhodamine derivatives
Ratiometric fluorescent
Pyrene derivatives
Rhodamine derivatives
Polypyrrole nanowires
Rhodamine derivatives
Silver nanoclusters
Rhodamine derivatives
XDS
0.396
20
5
0.469
0.105
2
2.5
2.2
2
1.5
MeOH/H2O (1/1)
CH3CN/H2O
MeOH
DMF/H2O (2/3)
CH3OH/H2O (4/6)
DMSO/H2O (1/99)
CH3OH/H2O (6/4)
MeOH/H2O (1/9)
EtOH/H2O
Aqueous
Aqueous
Aqueous
Aqueous
200 min
30 min
e
e
1 min
e
e
e
e
10 min
10 min
e
0.12
0.3
0.32
2 min
This study
with a complex matrix. To further investigate the selectivity of XDS
to Fe3þ against other metal ions, competition experiments were
therefore carried out, where the probe was first treated with 10 eq.
of various metal ions respectively, including Hg2þ, Al3þ, Sr2þ,Csþ,
fluorescence of XDS and [XDS-Fe3þ], we also carried out those ex-
periments in a series of solutions with pH ranging from pH 2 to 12
maintained by the respective buffers.
The results are shown in Fig. 7, as can be seen that the emission
behavior of sensor XDS almost remained constant in the pH range
4.0e7.0. However, the fluorescence intensity of the sensor
decreased and slightly increased when the pH value of solution is
below 4.0 and above 8.0 due to protonation and deprotonation of
urea eNH function, respectively. Similarly, upon the addition of
Fe3þ (10 equiv.), the fluorescence intensity was slightly increased
by the pH value in pH > 8 solution (Fig. 7 red spot). This is possibly
attributed to the hydrolysis reaction of Fe3þ when pH value is above
9. Thus, the investigated results demonstrated that sensor XDS is
suitable for the detection of Fe3þ in the pH range from 4.0 to 7.0.
Ca2þ, Cd2þ, Co2þ, Cr3þ, Cu2þ, Fe2þ, Liþ, Kþ, Mg2þ, Mn2þ, Ni2þ, Pb2þ
,
Naþ and Zn2þ, followed by adding 10 eq. of Fe3þ ions. As shown in
Fig. 2, the fluorescence intensities of these mixtures were almost as
same as that of free XDS solution with the minor significant change.
However, a noticeable fluorescence quenching has occurred obvi-
ously with the addition of Fe3þ into the above mixed solutions of
XDS and the competitive metal ions (Fig. 6a).
Meanwhile, in order to check the ability of using XDS as a
practical ion selective fluorescent chemosensor for Fe3þ in real
complicated samples, we also investigated the interference of
mixed 18-reference metal ions on the interaction between XDS and
Fe3þ. In this experiment, XDS aqueous solution (10
m
M) was treated
with 10 eq. of 18 reference metal ions, the concentration of each
metal ion was 100 M. As expected, the fluorescence of XDS solu-
3.6. Competition experiments with common anions
m
Considering practical application, we have not only discussed
the interference of common metal ions on the detection of XDS
tion with the presence of the mixture of 18 metal ions showed
almost no obvious change (Fig. 6b, yellow line). In contrast, the
fluorescence reduced greatly when Fe3þ solution was added into
the above mixture (Fig. 6b, red line), which revealed that Fe3þ
displays an effective fluorescence quenching effect to XDS. From
these distinct observations, it is confirmed that XDS can be used as
a practical ion selective fluorescent probe for Fe3þ detection in the
presence of most competing metal ions.
towards Fe3þ
, but also investigated the fluorescence signal
response of probe XDS toward Fe3þ in the presence of various
coexistent anions such as Fꢀ, Clꢀ, Brꢀ, Iꢀ, SO42ꢀ, SCNꢀ, ClO4ꢀ, PO34ꢀ
,
H2POꢀ4 , HPO42ꢀ, CO32ꢀ, NO3ꢀ, NO2ꢀ, AcO and CNꢀ. Interestingly, a
remarkable fluorescence recovery from colorless to blue can be
clearly noted with the addition of 10 equiv. of PO34ꢀ to the [XDS-
Fe3þ] complex. At the same time, a minor fluorescence recovery of
the XDS-Fe3þ complex system can be noticed with the addition of
H2POꢀ4 . However, its effect on the fluorescence recovery of the XDS-
Fe3þ complex system is minimal compared to PO34ꢀ(Fig. 8).
-
3.5. Effect of pH
In addition, in order to explore the effect of pH value on the
As Fig. 9a shown, an intense fluorescence emission at 378 nm
Fig. 6. Fluorescence intensity for probe XDS (10
presence of mixed competitive metal ions (b); Ex ¼ 320 nm.
m m
M) with Fe3þ in the presence of single competitive metal ions (a); Fluorescence intensity for probe XDS (10 M) with Fe3þ in the
Please cite this article in press as: Liu R, et al., A fluorescent chemosensor for relay recognition of Fe3þ and PO43ꢀ in aqueous solution and its
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6
R. Liu et al. / Tetrahedron xxx (2017) 1e10
Fig. 10. Fluorescence spectra of [XDS-Fe3þ] complex (10
mM) upon the progressive
Fig. 7. The influence of pH value of solution on the fluorescence of XDS and [XDS-
Fe3þ].
addition of PO34ꢀ in various concentration ranges.
was observed when excited at 320 nm with the addition of PO43ꢀ
and a weak fluorescence emission recovery of the XDS-Fe3þ com-
plex system can be noticed with the addition of H2POꢀ4 . In contrast,
no significant response was observed when the [XDS-Fe3þ] com-
plex was mixed with 10 equiv. of the other various anions of Fꢀ, Clꢀ,
[XDS-Fe3þ] complex can function as a fluorescent probe for PO34ꢀ
via a fluorescence “turn-on” mechanism.
The fluorescence titrations of PO34ꢀ were also conducted using a
10
m
M solution of [XDS-Fe3þ] complex (10
m
M þ 10
mM) in aqueous
solution upon progressive addition of PO34ꢀ in various concentra-
tion ranges to the solution. An obvious increase in the fluorescence
intensity at 378 nm was observed when excited at 320 nm (Fig. 10).
-
Brꢀ, Iꢀ,SO24ꢀ, SCNꢀ, ClO4ꢀ, CO23ꢀ, NOꢀ3 , NO2ꢀ, HPO24ꢀ, AcO and CNꢀ
respectively. The selective recognition of [XDS-Fe3þ] complex to-
wards PO34ꢀ was hardly affected by these commonly coexistent
anions (Fig. 9b). These experimental results indicated that the
Further increase in the concentration of PO34ꢀ (>10
mM) led to no
further fluorescence increase (Fig. 10, inset), which clearly
Fig. 8. The luminescence change of the mixture of [XDS-Fe3þ] (10
mM) and various anions (10 equiv.) under the UV lamp (
l
¼ 365 nm).
Fig. 9. (a) The fluorescence spectra changes of [XDS-Fe3þ] complex system (10
m
M XDS þ 10 equiv. Fe3þ) in the presence of various anions (Fꢀ, Clꢀ, Brꢀ, Iꢀ, AcOꢀ, SO24ꢀ, H2POꢀ4
,
,
HPO24ꢀ, CO32ꢀ, ClO4ꢀ, SCNꢀ, NOꢀ3 , NOꢀ2 and CNꢀ (10 equiv., respectively)) in aqueous solutions; (b) the influence of single anions to the interaction between [XDS-Fe3þ] and PO34ꢀ
(Ex ¼ 320 nm).
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R. Liu et al. / Tetrahedron xxx (2017) 1e10
7
addition of PO34ꢀ could induce the flocculent precipitation in the
high concentration experiments, the results are shown in Fig. S9.
The addition of PO34ꢀ could also cause the fluorescence recovery of
[XDS-Fe3þ] complex system, and the fluorescence of the system
could quench again with the addition of Fe3þ ions. These phe-
nomena further confirmed that the binding capacity of PO34ꢀ to Fe3þ
is stronger than that of the probe XDS. Thus, it is reasonable to
surmise that the bound Fe3þ in [XDS-Fe3þ] complex can be sepa-
rated by PO34ꢀ. The orthophosphate (PO34ꢀ) anion can induce the
precipitation of ferric ion with the regeneration of the probe XDS.
As shown in Fig. 11, the addition of 10 equiv of PO34ꢀ to the so-
lution containing XDS (10 m
M) and Fe3þ (10 equiv.) immediately
restored the emission to the original level of XDS. The alternate
additions of Fe3þ and PO43ꢀ to the probe XDS solution caused the
obvious changes in the fluorescence intensity. This “on-off-on”
switching process could be repeated several times with little
fluorescent efficiency loss.
Upon the addition of PO34ꢀ, the recovery of fluorescence was
observed with short response time. These results clearly revealed
that XDS-Fe3þ can serve as a relay probe to detect phosphate (PO34ꢀ
anion with high selectivity and sensitivity in pure water.
)
Fig. 11. Reversible switching cycles of fluorescence intensity by alternate addition of
Fe3þ and PO34ꢀ ions.
3.7. Quantum mechanical calculations
demonstrated a 1:1 stoichiometry between the [XDS-Fe3þ] com-
plex and PO34ꢀ.Based on the fluorescence titration data (Fig. 10), we
calculated the detection limit is 8.0 ꢁ 10ꢀ7 M.
The optimized structures of probe XDS and XDS-Fe3þ complex
were shown in Fig. 12, The results showed that metal ions and
probe XDS are combined with a ratio of 1:1 and the Fe3þ ion was
chelated through three coordination sites, which is pyridine ni-
trogen, amino nitrogen and oxygen atoms of coumarin and the
bond length are 1.96161 Å, 1.75464 Å, and 2.07515 Å. The UV
spectrum of XDS-Fe3þ complex has also been made by time-
dependent density functional method (TD-DFT). The solvation
model is Polarizable Continuum Model (PCM) and water as the
solvent. As shown in Fig. 13, the theory maximum absorption
It is also noteworthy that PO34ꢀ induced emission characteristics,
which are almost identical to XDS in the absence of any guest
species, suggest that the observed fluorescence response should be
derived from the regeneration of the acceptor XDS, possibly due to
a reduction of Fe3þ to FePO4 to regenerate the XDS. It is generally
known that the FePO4 (Ksp ¼ 1.3 ꢁ 10ꢀ22) is indissoluble in aqueous
solutions.48 In the further investigations, we also found that the
Fig. 12. The optimized geometry of XDS and the XDS-Fe3þ complex at the B3LYP level of theory (the green atoms is chlorine).
Fig. 13. (a) The UVeVis spectra of XDS-Fe3þ complex at the B3LYP/LanL2DZ level of theory; (b) the UVeVis spectra of XDS-Fe3þ complex in aqueous solution, the corresponding
molar extinction coefficient (ε ¼ A/bc) is 3.33 ꢁ 104 L molꢀ1 cmꢀ1
.
Please cite this article in press as: Liu R, et al., A fluorescent chemosensor for relay recognition of Fe3þ and PO43ꢀ in aqueous solution and its
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8
R. Liu et al. / Tetrahedron xxx (2017) 1e10
spectrum obtained by theoretical calculations is indeed strikingly
similar to the experimentally measured spectra, which is beyond
our expectation.
The frontier molecular orbital and energy level of the free XDS
and the XDS-Fe3þ complex were shown in Fig. 14. For free XDS, the
localized charge density of HOMO of free XDS localized on the
pyridine group end and the LUMO is localized on the coumarin
group. There is an obvious intramolecular charge transfer and the
fluorescence being emitted from the coumarin group, consistent
with our experimental observations. But the molecular orbital
structure of XDS-Fe3þ complex is distinctly different from that of
XDS. The charge density of the HOMO of XDS-Fe3þ complex is
localized on coumarin group and the LUMO is mainly localized on
the metal ions and nearby groups. The introduction of Fe3þ ions has
destroyed the original charge transfer mode. The change of elec-
tronic structure led to the change of optical properties. In addition,
the paramagnetic and unfilled d-orbital of the Fe3þ ions are other
reasons for fluorescence quenching.49
3.8. Determinations in test strips
Fig. 14. Molecular orbitals plots of HOMO and LUMO of free XDS and XDS-Fe3þ
complex, and the orbital plots is generated by the VESTA software.50
To investigate the practical application of probe XDS, test strips
were prepared by immersing a silica gel plate into solutions of XDS
(10 mM), and then drying in air. The test strips containing probe XDS
were used to sense iron ions. For the iron ion solution, different test
strips were immersed for 10 s, and then a noticeable emission color
change was observed under UV lamp (365 nm). As the iron ion
concentration increased, the fluorescence quenched gradually
(Fig. 15). However, an apparent fluorescence recovery from color-
less to blue can be noticed immediately when these test strips were
immersed into 10
convenient application of XDS for the relay recognition of Fe3þ and
PO34ꢀ
m
M of PO34ꢀ(Fig.16). These results demonstrated a
.
Fig. 15. Photographs of the test strips with XDS (10ꢀ5 mol/L) for the detection of Fe3þ
in the aqueous solution with different concentrations.
3.9. Fluorescence imaging in RAMOS cells
In order to examine the ability of XDS to track the level of
intracellular Fe3þ and PO43ꢀ, RAMOS cells were used for this
experiment. Firstly, the cells were incubated with probe XDS
(20 m
M) for 30 min at 37 ꢂC and then washed 3 times to remove
excess probe XDS. The Fig. 17a and b showed bright-field and
fluorescence images of RAMOS cells after treatment with XDS. The
cells loaded with XDS showed obvious fluorescence. 20 m
M of Fe3þ
were supplemented to the cells and the cells were incubated at
37 ꢂC for another 30 min. The fluorescence from the intracellular
almost complete disappearance of fluorescence was observed in
Fig. 17c. Obvious changes of fluorescence indicated that probe XDS
is cell membrane permeable and capable of imaging of Fe3þ in the
Fig. 16. Photographs of the test strips with [XDS-Fe3þ] (10
mM) for the detection of
PO43ꢀ in aqueous solution. (AeC): XDS, XDS þ Fe3þ, [XDS-Fe3þ] þ PO34ꢀ
.
living cells. The cells then were incubated with PO34ꢀ(20
mM) for
another 30 min. The cells showed fluorescence recovered after the
wavelength (lmax) is 305 nm. There is a negligible error to the
experimental value (lmax ¼ 308 nm). The UVevis absorption
addition of PO34ꢀ(Fig. 17d). The results suggested that the XDS can
be used for the detection of the level of intracellular Fe3þ and PO43ꢀ
.
Fig. 17. Fluorescence images of probe XDS in RAMOS cells: bright-field images (a) and fluorescence images of RAMOS cells incubat with probe XDS (b), 20
Fe3þ and 20 M of PO34ꢀ (d), respectively.
m mM of
M of Fe3þ (c), 20
m
Please cite this article in press as: Liu R, et al., A fluorescent chemosensor for relay recognition of Fe3þ and PO43ꢀ in aqueous solution and its
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R. Liu et al. / Tetrahedron xxx (2017) 1e10
428e432.
9
4. Conclusion
15. Legin AV, Kirsanov DO, Babain VA, Borovoy AV, Herbst RS. Cross-sensitive rare-
earth metal sensors based on bidentate neutral organophosphorus compounds
and chlorinated cobalt dicarbollide. Anal Chim Acta. 2006;572:243e247.
16. Kirsanov DO, Legin AV, Babain VA. Polymeric sensors based on extraction
systems for determination of rare-earth metals. Russ J Appl Chem. 2005;78:
568e573.
17. Crabtree RH. Metal ions at work. Science. 1994;266:1591e1592.
18. Cairo G, Pietrangelo A. Iron regulatory proteins in pathobiology. Biochem J.
2000;352:241e250.
19. Eisenstein RS. Iron regulatory proteins and the molecular control of mamma-
lian iron metabolism. Annu Rev Nutr. 2000;20:627e662.
20. Geider RJ. Biological oceanographydcomplex lessons of iron uptake. Nature.
1999;400:815e816.
In conclusion, we have designed and synthesized a novel fluo-
rescent probe XDS based on 4-methylumbelliferone and 2-
picolylamine platforms. It displayed high selectivity and sensi-
tivity for Fe3þ in 100% aqueous solution with a significant fluores-
cence quenching effect to form [XDS-Fe3þ] complex. The addition
of PO34ꢀ could cause the recovery of fluorescence. So the [XDS-Fe3þ
]
complex could relay recognize the PO34ꢀ. This “on-off-on” switching
process could be repeated at several times and the detection of Fe3þ
and PO34ꢀ was not disrupted by the presence of other competing
21. Tyrrell T. The relative influences of nitrogen and phosphorus on oceanic pri-
mary production. Nature. 1999;400:525e531.
ions. The low detection limit (0.32 mM) makes it possible to detect
trace amounts of iron ions. Also, probe XDS was successfully
22. Gaidamauskas E, Parker H, Kashemirov BA, et al. Complexation of bisphosph-
onates with ytterbium(III): application of phosphate and ATP detection assay
based on Yb3þ-pyrocatechol violet. J Inorg Biochem. 2009;103:1652e1657.
23. Shervedani RK, Pourbeyram S. Zirconium immobilized on gold-
mercaptopropionic acid self-assembled monolayer for trace determination of
phosphate in blood serum by using CV, EIS, and OSWV. Biosens Bioelectron.
2009;24:2199e2204.
applied in fluorescence imaging of living cells.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Nos. 31470434, 21576124, 21507047 and
21676124), China Postdoctoral Science Foundation funded project
(No. 2017M610308), Zhenjiang Social development project (No.
SH2016019), and the Project supported by the Science Foundation
of Jiangsu Entry-exit Inspection Quarantine Bureau (Nos. 2016KJ51
and 2017KJ47).
24. Chen CC, Chen YZ, Huang YJ, Sheu JT. Using silicon nanowire devices to detect
adenosine triphosphate liberated from electrically stimulated HeLa cells. Bio-
sens Bioelectron. 2011;26:2323e2328.
25. Gaidamauskas E, Saejueng K, Holder AA, et al. Metal complexation chemistry
used for phosphate and nucleotide determination: an investigation of the
Yb3þ-pyrocatechol violet sensor. J Biol Inorg Chem. 2008;13:1291e1299.
26. Samanta A, Vendrell M, Das R, Chang YT. Development of photostable near-
infrared cyanine dyes. Chem Commun. 2010;46:7406e7408.
27. Song KC, Kim H, Kang ML, Lee YS, Do YK, Lee MH. Ratiometric fluorescence
sensing of fluoride ions by triarylborane-phenanthroimidazole conjugates. Sens
Actuat B Chem. 2013;176:850e857.
28. Kim SK, Seo D, Han SJ, et al. A new imidazolium acridine derivative as fluo-
rescent chemosensor for pyrophosphate and dihydrogen phosphate. Tetrahe-
dron. 2008;64:6402e6405.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2017.07.018.
29. Kondo S, Takai R. Selective detection of dihydrogen phosphate anion by fluo-
rescence change with tetraamide-based receptors bearing isoquinolyl and
quinolyl moieties. Org Lett. 2013;15:538e541.
30. Cao X, Lin W, Yu Q, Wang J. Ratiometric sensing of fluoride anions based on a
BODIPY-coumarin platform. Org Lett. 2015;13:6098e6101.
References
31. Jadhav JR, Ahmad MW, Kim HS. Selective recognition of H2PO-4 by a cholestane-
imidazole-zinc ensemble. Tetrahedron Lett. 2012;53:2627e2631.
32. Frisch MJ, Trucks GW, Schlegel HB, et al. Gaussian, Inc. Wallingford CT. 2013.
33. Tang X, Han J, Wang Y, et al. A fluorescent chemosensor for Cu2þ ions and its
application in cell imaging. Tetrahedron. 2017;73:1367e1373.
34. Bao XF, Cao XW, Nie XM, et al. A new selective fluorescent chemical sensor for
Fe3þ based on rhodamine B and a 1,4,7,10-tetraoxa-13-azacyclopentadecane
conjugate and its imaging in living cells. Sens Actuat B Chem. 2015;208:54e66.
35. Chai MM, Zhang D, Wang M, Hong HJ, Ye Y, Zhao YF. Four Rhodamine B-based
fluorescent chemosensor for Fe3þ in aqueous solution. Sens Actuat B Chem.
2012;174:231e236.
1. Cort AD, Forte G, Schiaffino L. Anion recognition in water with use of a neutral
uranyl-salophen receptor. J Org Chem. 2011;76:7569e7572.
2. Li YP, Yang HR, Zhao Q, Song WC, Han J, Bu XH. Ratiometric and selective
fluorescent sensor for Zn2þ as an “off-on-off” switch and logic gate. Inorg Chem.
2012;51:9642e9648.
3. Zhao L, Ren P, Zhang Z, Fang M, Shi W, Cheng P. Synthesis, crystal structures
and fluorescence properties of four mixed-ligands Zn(II) and Cd(II) coordina-
tion compounds. Sci China Ser B. 2009;52:1479e1484.
4. Yang Y, Wang Y, Peng Y. A bifunctional, colorimetric and fluorescent probe for
recognition of Cu2þ and Hg2þ and its application in molecular logic gate. Sci
China Chem. 2013;57:289e295.
36. Li P, Zhao Y, Yao L, Nie HR, Zhang M. A simple, selective, fluorescent iron(III)
sensing material based on peripheral carbazole. Sens Actuat B Chem. 2014;191:
332e336.
37. She MY, Yang Z, Yin B, et al. A novel rhodamine-based fluorescent and color-
imetric “off-on” chemosensor and investigation of the recognizing behavior
towards Fe3þ. Dyes Pigments. 2012;92:1337e1343.
5. Nomngongo PN, Ngila JC. Determination of trace Cd, Cu, Fe, Pb and Zn in diesel
and gasoline by inductively coupled plasma mass spectrometry after sample
clean up with hollow fiber solid phase microextraction system. Spectrochim
Acta B. 2014;98:54e59.
6. Caldas LF, Brum DM, de Paula CE, Cassella RJ. Application of the extraction
induced by emulsion breaking for the determination of Cu, Fe and Mn in used
lubricating oils by flame atomic absorption spectrometry. Talanta. 2013;110:
21e27.
7. Shervedani RK, Hatefi-Mehrjardi A, Asadi-Farsani A, Shervedani RK, Hatefi-
Mehrjardi A, Asadi-Farsani A. Sensitive determination of iron(III) by gold
electrode modified with 2-mercaptosuccinic acid self-assembled monolayer.
Anal Chim Acta. 2007;601:164e171.
38. Yang L, Yang W, Xu DM, Zhang ZY, Liu AF. A highly selective and sensitive Fe3þ
fluorescent sensor by assembling three 1,8-naphthalimide fluorophores with a
tris(aminoethylamine) ligand. Dyes Pigments. 2013;97:168e174.
39. Wang CC, Zhang D, Huang XY, et al. A fluorescence ratiometric chemosensor for
Fe3þ based on TBET and its application in living cells. Talanta. 2014;128:69e74.
40. Wang M, Wang JG, Xue WJ, Wu AX. A benzimidazole-based ratiometric fluo-
rescent sensor for Cr3þ and Fe3þ in aqueous solution. Dyes Pigments. 2013;97:
475e480.
8. Hosseini MS, Raissi H, Madarshahian S. Synthesis and application of a new
chelating resin functionalized with 2, 3-dihydroxy benzoic acid for Fe(III)
determination in water samples by flame atomic absorption spectrometry.
React Funct Polym. 2006;66:1539e1545.
41. Casanueva Marenco MJ, Fowley C, Hyland BW, Galindo-Riano D, Sahoo SK,
Callan JF. A new fluorescent sensor for the determination of Iron(III) in semi-
aqueous solution. J Fluoresc. 2012;22:795e798.
€
9. Weber G, Neumann G, Romheld V. Speciation of iron coordinated by phytosi-
42. Fegade U, Tayade S, Chaitanya GK, Attarde S, Kuwar A. Fluorescent and chro-
mogenic receptor bearing amine and hydroxyl functionality for iron(III)
detection in aqueous solution. J Fluoresc. 2014;24:675e681.
43. Liu SR, Wu SP. New aqueous-soluble highly selective fluorescent chemosensor
for Fe(III) ions and its application to living cell imaging. Sens Actuat B Chem.
2012;171e172:1110e1116.
44. Wang ML, Meng GW, Huang Q, Xu QL, Liu GD. A GBI@PPyNWs-based prototype
of reusable fluorescence sensor for the detection of Fe3þ in aqueous solution.
Anal Methods. 2012;4:2653e2656.
derophores by use of HPLC with pulsed amperometric detection and AAS.
Analytical and Bioanalytical Chemistry. Anal Bioanal Chem. 2002;373:767e771.
10. Huang C, Hu B. Silica-coated magnetic nanoparticles modified with g-mer-
captopropyltrimethoxysilane for fast and selective solid phase extraction of
trace amounts of Cd, Cu, Hg, and Pb in environmental and biological samples
prior to their determination by inductively coupled plasma mass spectrometry.
Spectrochim Acta A. 2008;63:437e444.
11. Zamani HA, Faridbod F. Liquid membrane potentiometric sensor for determi-
nation of Fe3þ ion. J Anal Chem. 2014;69:1073e1078.
45. Wang JH, Zhang D, Liu YQ, et al. A N-stabilization rhodamine-based fluorescent
chemosensor for Fe3þ in aqueous solution and its application in bioimaging.
Sens Actuat B Chem. 2014;191:344e350.
12. Liu Z, He W, Guo Z. Metal coordination in photoluminescent sensing. Chem Soc
Rev. 2013;42:1568e1600.
13. Silva AP, Gunaratne HQ, Gunnlaugsson T, et al. Signaling recognition events
with fluorescent sensors and switches. Chem Rev. 1997;97:1515e1566.
14. Hu ZQ, Feng YC, Huang HQ, et al. Fe3þ-selective fluorescent probe based on
Rhodamine B and its application in bioimaging. Sens Actuat B Chem. 2011;156:
46. Chen Z, Lu DT, Zhang GM, Yang J, Dong C, Shuang SM. Glutathione capped silver
nanoclusters-based fluorescent probe for highly sensitive detection of Fe3þ
Sens Actuat B Chem. 2014;202:631e637.
.
Please cite this article in press as: Liu R, et al., A fluorescent chemosensor for relay recognition of Fe3þ and PO43ꢀ in aqueous solution and its
applications, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.07.018

10
R. Liu et al. / Tetrahedron xxx (2017) 1e10
47. Du YY, Chen M, Zhang YX, et al. Determination of iron(III) based on the fluo-
rescence quenching of rhodamine B derivative. Talanta. 2013;106:261e265.
48. Scaccia S, Carewska M, Bartolomeo AD, Prosini PP. Thermoanalytical investi-
gation of iron phosphate obtained by spontaneous precipitation from aqueous
solutions. Thermochim Acta. 2002;383:145e152.
49. Li ZX, Zhang LF, Zhao WY, Li XY, Guo YK. Fluoranthene-based pyridine as
fluorescent chemosensor for Fe3þ. Inorg Chem Commun. 2011;14:1656e1658.
50. Momma K, Izumi F. VESTA 3 for three-dimensional visualization of crystal,
volumetric and morphology data. J Appl Crystallogr. 2011;44:1272e1276.
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