A benzothiazole-rhodol based luminophor: ESIPT-induced AIE and an application for detecting Fe2+ ion

Article abstract of DOI:10.1016/j.saa.2019.05.019

Herein, we designed and synthesized a luminophor, Rh-F, which is an intergrant of rhodol and 2-hydroxy benzothiazole by introducing a benzothiazole unit onto the ortho-position of the phenolic hydroxy of rhodol. Rh-F exhibited excellent fluorescence properties such as a large Stokes shift (>180 nm) and the synergistic effect of aggregation-induced emission (AIE) and an excited state intramolecular proton transfer (ESIPT) feature. The AIE/ESIPT mechanism was thoroughly explored using X-ray single-crystal structures and photophysical determinations. Furthermore, Rh-F showed a sensitive fluorescence response to Fe²⁺ with low detection limits of 115.2 nM and high selectivity. Studies of its sensing mechanism indicated that the Fe²⁺-induced blue-green fluorescence-quenched at 525 nm originates from an irreversible Fe²⁺ chelate with the oxygen atom of the hydroxyl group and the N atom of the benzothiazole moiety. This blocked the ESIPT process of Rh-F which resulted in the quenching of the fluorescence sensor for Rh-F.

Full text of DOI:10.1016/j.saa.2019.05.019

Accepted Manuscript
A benzothiazole-rhodol based luminophor: ESIPT-induced AIE
and an application for detecting Fe2+ ion
Jianping Guan, Qin Tu, Long Chen, Mao-Sen Yuan, Jinyi Wang
PII:
DOI:
Reference:
S1386-1425(19)30495-0
https://doi.org/10.1016/j.saa.2019.05.019
SAA 17114
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date: 14 March 2019
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10 May 2019
Please cite this article as: J. Guan, Q. Tu, L. Chen, et al., A benzothiazole-rhodol based
luminophor: ESIPT-induced AIE and an application for detecting Fe2+ ion,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/
10.1016/j.saa.2019.05.019
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ACCEPTED MANUSCRIPT
A Benzothiazole-rhodol Based Luminophor: ESIPT-induced
AIE and an Application for Detecting Fe2+ Ion
Jianping Guan 1#, Qin Tu 1#, Long Chen1, Mao-Sen Yuan 1,2* & Jinyi Wang1
1College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, P. R.
China
2 State Key Laboratory of Crystal Materials, Shandong University, Jinan, 250100, China
# Authors contributed equally
* Corresponding author. Tel/fax: +86-29-87082520.
E-mail address: yuanms@nwsuaf.edu.cn (M.-S. Yuan).
Abstract
Herein, we designed and synthesized a luminophor, Rh-F, which is an intergrant of rhodol and 2-
hydroxy benzothiazole by introducing a benzothiazole unit onto the ortho-position of the phenolic
hydroxy of rhodol. Rh-F exhibited excellent fluorescence properties such as a large Stokes shift
(>180 nm) and the synergistic effect of aggregation-induced emission (AIE) and an excited state
intramolecular proton transfer (ESIPT) feature. The AIE/ESIPT mechanism was thoroughly explored
using X-ray single-crystal structures and photophysical determinations. Furthermore, Rh-F showed a
sensitive fluorescence response to Fe2+ with low detection limits of 115.2 nM and high selectivity.
Studies of its sensing mechanism indicated that the Fe2+-induced blue-green fluorescence-quenched
2
+
at 525 nm originates from an irreversible Fe chelate with the oxygen atom of the hydroxyl group
and the N atom of the benzothiazole moiety. This blocked the ESIPT process of Rh-F which resulted
in the quenching of the fluorescence sensor for Rh-F.
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Keywords: Luminophor, ESIPT, AIE, Detect, Fe2+
1. Introduction
In recent years, an increasing number of new fluorophores based on aggregation-induced emission
(AIE) or excited state intramolecular proton transfer (ESIPT) mechanisms have been used in organic
optoelectronic materials [1-3], molecular probes [4-6], and in various laser dyes [7-9]. As we know,
ESIPT is a photo-induced proton transfer through an intramolecular hydrogen bond. Its large Stokes
shift is beneficial for the prevention self-absorption of fluorophores and increases efficiency [10-11].
ESIPT-based illuminants display excellent signal performance on account of dual emission in
aqueous solution: enol-form emission and keto-form emission [9,12]. This possess excellent
photostability and spectral sensitivity to the surrounding medium [13]. In addition, an AIE
phenomenon was reported by Tang et al. in 2001 which is completely opposite to the aggregation-
caused quenching (ACQ) effect [14]. At present, most of the reported AIE molecules show high
solid-state luminous efficiency, high concentration of aggregation-induced emission, and background
signals that are easy to eliminate [15], so they have been widely used in the development of organic
optoelectronic materials and fluorescent biosensors.
To combine AIE and ESIPT is an effective strategy to improve the photophysical properties of the
illuminants. The unique advantages of AIE/ESIPT dual-mechanism fluorophores result from the
following two aspects. On the one hand, the AIE characteristics of the fluorophore facilitate the
induction of the ESIPT process with large Stokes shift emission by forming aggregates, especially in
very polar or strong hydrogen bonding solvents. On the other hand, the ESIPT process can also
enhance the AIE effect by intramolecular hydrogen bonding, resulting in enhanced fluorescence in
the aggregated state [16]. These advantages are not available with a single AIE or ESIPT fluorophore.
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Therefore, this kind of novel double-system (AIE/ESIPT) fluorophore with a large Stokes shift,
strong light stability, and no self-quenching at high concentrations has great potential for application
in the development of bioimaging sensors and organic photoelectric materials.
Iron is one of the essential trace elements of biological systems and it is an indispensable
substance in many physiological processes. Iron is generally present in the form of ferrous and
trivalent iron. Creatures mainly use ferrous iron because ferrous iron is easier to be assimilated than
trivalent iron. Iron deficiency or poor iron use will result in anemia, fatigue, decreased immunity and
it will eventually cause a variety of diseases. If iron ion concentration surpasses the normal level, it
may lead to liver and kidney damage, cardiovascular disease, and heart disorders. At the same time,
iron ion is also a major environmental pollutant. Based on the importance of iron ions in the function
of organisms and in the environment, it is of great significance to detect iron ions in clinical, medical,
health, environmental, and other aspects [17-20]. So far, many fluorescent probes have been
developed to detect iron ions, such as Schiff base, crown ether, imidazole, leptin, fluorescein,
naphthalimide, rhodamine, and so on [21-29]. However, most of these fluorescent sensors are applied
for the detection of Fe3+. There has been little research for detecting Fe2+ and many methods lack
selectivity and sensitivity. Therefore, it is still a great challenge to develop highly selective, sensitive,
and efficient chemosensors for detection of Fe2+.
Rhodol (or rhodafluor), which can be regarded as a mixture of rhodamine and fluorescein, exhibits
many excellent photophysical properties (i.e., high photostability, high fluorescence quantum yield,
and good solubility) [30-33]. In this study, we designed and synthesized a new benzothiazole-
modified rhodol luminophore, Rh-F, by introducing a benzothiazole unit onto the ortho-position of
the phenolic hydroxyl of rhodol dyes (Scheme 1). Rh-F possess excellent AIE and ESIPT
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fluorescence properties. Furthermore, in this molecular structure, the formative 2-hydroxy
benzothiazole can be used as the sensor site to recognize Fe2+ through blocking ESIPT responding
mechanisms. We deeply explored the new AIE/ESIPT-active luminophor and clarified the
AIE/ESIPT mechanism. As a fluorescent chemodosimeter, Rh-F was successfully demonstrated to
be useful for the discriminative detection of Fe2+ in aqueous solution. A significant decrease in the
blue-green fluorescence at 525 nm and a color change from colorless to yellow induced by Fe2+ was
observed.
2. Experimental Section
2.1. Materials
All chemicals in this study were purchased from Energy chemical or Aladdin Reagent Company
and used without any further purification. The liquid solvents used for the synthesis and analysis
were analytical grade. Column chromatography purification generally used silica gel (200-300 mesh)
for the separations. The metal salt solutions were all formulated using Milli-Q system purified water
(purified to 18.2 MΩ).
2.2. Instruments and methods
1H-nuclear magnetic resonance (NMR) and 13C NMR spectra of the products were recorded on a
Bruker 500 MHz/125 MHz NMR spectrometer using tetramethylsilane (TMS) as the internal
standard (chemical shifts in ppm). High-resolution mass spectral data of the product were obtained
using a Thermo LTQ-Orbitrap mass spectrometer. UV–vis absorption spectra were measured by
using a Shimadzu UV1780 spectrometer (Shimadzu, Japan), and fluorescence emission spectra
measurements were performed on a Shimadzu RF-5301 fluorescence spectrometer (Shimadzu,
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Japan). The single crystal of compound Rh-F was obtained by the slow diffusion of
THF/cyclohexane solutions over several days at room temperature. The data collection was done at
room temperature on a Bruker SMART APEX-II CCD area detector using graphite-mono-chromated
Mo Ka radiation (λ=0.71073 Å). Data reduction and integration together with global unit cell
refinements were done by the INTEGRATE program of the APEX2 software. Semi-empirical
absorption corrections were applied using the SCALE program for the area detector. The structure
was solved by direct methods and refined by full matrix least-squares methods on F2 using SHELX.
2.3. Synthetic procedures
2.3.1 Synthesis of compound 4.
To a suspended solution of 3 (0.52 g, 1 mmol) in ethanol (20 mL) was added an excess of
Ethylenediamine (3.2 mL, 90%) and the reaction mixture was refluxed under nitrogen for 24 h with
stirring. The reaction mixture was cooled to room temperature and concentrated under vacuum. The
resulting residue was finally purified by silica gel chromatography (CH2Cl2: CH3OH = 30:1, v/v) to
1
afford compound 4 as a pale white solid (0.31 g, 67.6% yield).: mp 247 – 248 °C. H NMR (500
MHz, CDCl3): δ H (500 MHz, CDCl3) 12.79 (1 H, s), 8.64 (1 H, s), 8.06 (1 H, d, J 5.0), 7.97 (1 H, d,
J 7.9), 7.83 (1 H, d, J 7.9), 7.59 (2 H, d, J 4.1), 7.51 (1 H, t, J 7.4), 7.41 (1 H, t, J 7.4), 8.90 – 6.06
(15 H, m), 7.31 (1 H, s), 7.17 (1 H, d, J 5.2), 6.98 (2 H, d, J 19.6), 6.50 (2 H, d, J 11.7), 6.38 (1 H, d,
J 8.8), 3.41 (5 H, d, J 7.1), 3.67 – 2.82 (8 H, m), 3.19 (2 H, dd, J 37.3, 8.9), 1.66 (2 H, s), 1.73 – 1.14
(7 H, m), 1.30 (1 H, s), 1.24 (6 H, t, J 6.6); 13C NMR (125 MHz, CDCl3 ): δ 169.81, 167.92, 159.32
155.16, 152.68, 151.35, 149.43, 133.58, 132.26, 129.06, 128.03, 127.97, 126.81, 125.70, 123.95,
123.41, 122.08, 121.54, 114.62 111.24 109.37, 104.89 97.99 77.32, 77.07, 76.81, 67.12, 49.18, 44.51,
40.23, 29.73,12.55; HRMS (ESI, m/z): Calcd. for [C33H30N4O3S - H]-, 563.22; Found, 563.209.
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2.3.2 Synthesis of compound Rh-F
To a suspended solution of 4 (54 mg, 0.1 mmol) in 1,4-dioxane (3 mL) was added an excess of
Sodium borohydride (25 mg, 0.6 mmol) and Trifluoroacetic acid (800 µL). Next , the solution was
heated at 80 °C under nitrogen for 8 h with stirring. The reaction mixture was cooled to room
temperature and concentrated under vacuum. The resulting residue was finally purified by silica gel
chromatography (CH2Cl2: CH3OH = 80:1, v/v) to afford compound Rh-F as a pale white solid (28.2
1
mg, 42.8% yield).mp 195 – 196 °C. H NMR (500 MHz, CDCl3): δ 12.85 (s, 1H), 8.61 (1 H, s),
8.02 (2 H, dd, J 47.1, 6.1), 7.82 (1 H, d, J 7.3), 7.62 – 7.44 (3 H, m), 7.41 (1 H, d, J 7.0), 9.01 – 6.21
(14 H, m), 7.31 (1 H, s), 7.17 (1 H, d, J 5.1), 6.98 (2 H, d, J 18.3), 6.46 (3 H, dd, J 54.5, 7.3), 3.65 –
2.81 (8 H, m), 1.68 (1 H, s), 1.26 (6 H, t, J 19.3);13C NMR (125 MHz, CDCl3 ): δ (126 MHz,
C
CDCl3) 170.56, 167.89, 159.27, 155.09, 153.19, 152.66, 151.57, 149.35, 133.59, 132.22, 129.66,
128.96, 128.02 (d, J 17.3), 126.83, 125.72, 123.87, 123.49, 122.10, 121.51, 114.51, 111.20, 109.10,
104.91, 103.35, 97.95, 77.33, 77.07, 76.82, 67.12, 65.41, 44.46, 41.45, 39.59, 12.54; HRMS (ESI,
m/z): Calcd. for [C35H29F3N4O4S-H]-, 659.20; Found, 659.199.
2.4. Fluorescence assay of Fe2+ ion
The metal salts used in this study included NaCl, CaCl2, AlCl3·6H2O, CuCl2·2H2O,
AgClO4·6H2O, ZnCl2, FeCl3·6H2O, CoCl2, KCl, HgCl2, FeCl2·4H2O, MgCl2, BaCl2, MnCl2, CrCl3,
CdCl2·H2O and PbCl2. These metal salts were dissolved in ultrapure water to make a stock solution
having a concentration of 100 μM for analysis. Rh-F was dissolved in THF to prepare a stock
solution, which was then diluted with THF/H2O to prepare an analytical solution (THF/H2O, v/v, 7/3).
The 100 μM metal ion solution and 10 μM Rh-F analysis solution could be directly used for
absorption and fluorescence spectroscopy. In addition, different metal ion solutions and different
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concentrations of Fe2+ analytical solutions were added dropwise to the Rh-F analysis solution for
selectivity and sensitivity experiments, respectively. Meanwhile, fluorescence absorption was
detected, and the fluorescence spectra were recorded.
Scheme 1. (a-e) The synthetic route of Rh-F; (f) Equilibrium between the enol and keto forms of Rh-F under the
ESIPT cycle.
2.5. Single crystal X-ray diffraction
Two single crystals were obtained through slow diffusion of their respective organic solutions
for several days at room temperature. Because all the title crystals are stable under ambient
conditions, the data collection was performed without any inert gas protection at room temperature
on a Bruker SMART APEX-II CCD area detector using graphite-monochromated Mo Kα radiation
(λ = 0.71073 Å). The data reduction and integration and global unit cell refinements were performed
using the INTEGRATE program of the APEX2 software package. Semiempirical absorption
corrections were applied using the SCALE program for the area detector. The structures were solved
by direct methods and refined using the full-matrix least squares methods on F2 using SHELX
3. Results and discussion
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3.1. Synthesis
The synthesis of the luminophor Rh-F is shown in Scheme 1. The intermediates 1 and 2 were
synthesized according to literature procedures using 3-hydroxy-N,N-diethylaniline and 2,4-
dihydroxybenzaldehyde as raw materials, respectively [34-36]. Intermediate 3 was synthesized from
intermediates 1 and 2 using methanesulfonic acid as solvent at 90 °C to give a dark purple solid in
56.7% yield. Intermediate 3 and ethylenediamine were refluxed in ethanol solution and purified by
column chromatography to give a yellowish solid compound 4 which was excited by ultraviolet light
to produce yellowish green fluorescence with a yield of 42%. Finally, compound 4 was dissolved in
1,4-dioxane solution and reacted with sodium borohydride and trifluoroacetic acid to obtain the
target product as a white powder Rh-F (enol form) in a yield of 78.2%. The details of the synthesis
1
13
and purification processes of the relevant compounds as well as the H and C NMR data and the
mass spectrometry data results are both given in the Supplementary Materials.
3.2. The ESIPT and AIE characteristics
The new benzothiazole-modified rhodol luminophor Rh-F is based on the AIE+ESIPT structure.
The ESIPT process of Rh-F was explored first. An excellent ESIPT between the phenolic hydroxyl
protons and the aromatic nitrogen in the benzothiazole unit takes place upon photoexcitation which
can convert the enol form to a ketone form in a short time through intramolecular hydrogen bonding
(Scheme 1f). The excited keto tautomer relaxes to the ground state with fluorescence emission and
then returns to its intensively favored ground state enol tautomers through ground state
intramolecular proton transfer (GSIPT).
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Figure 1. Normalized FL spectra of Rh-F in different solvents (λex = 350 nm); Inset: Images of Rh-F in different
solvents under a 365 nm UV lamp.
The dual emission of the enol-form type (high energy, low Stokes shift) and the keto-form type
(low energy, high Stokes shift) is a typical characteristic of ESIPT fluorophores. To verify the ESIPT
mechanism of Rh-F, we studied the emission behaviors of Rh-F in different solvents (Figure 1).
Under excitation at 350 nm in the highly polar solvents {ethanol, methanol, tetrahydrofuran (THF)
and dimethyl sulfoxide (DMSO)}, the double emission of enol and keto tautomers of Rh-F (about
384 and 520 nm) with low fluorescence quantum yields (ΦF) were observed, respectively. On the
basis of our previous research results, the emission at approximately 384 nm originates from the enol
form. The presence of hydrogen bonds between these highly polar solvents and the phenolic
hydroxyl groups of Rh-F can stabilize the enol form to obstruct the occurrence of the ESIPT process.
The emission band at approximately 520 nm originates from the keto form induced by the ESIPT
process. In non-polar or low-polar solvents {toluene, dichloromethane (DCM), ethyl acetate,
acetonitrile, and acetone}, only the keto-form emission occurred at about 520 nm, which showed a
large Stokes shift and relatively high fluorescence quantum yields.
In addition, we also analyzed the single crystal structure of Rh-F (Figure 2 and Table S1). It can
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be seen that there is a strong intramolecular OH···N hydrogen bond in the structure of Rh-F with the
corresponding distance of 1.92 Ǻ. Furthermore, the dihedral angle between xanthene plane and
benzothiazole ring plane is only 7.26 º, almost in the same plane. The pre-existing intramolecular
hydrogen bond and coplane of rhodol with benzothiazole will facilitate intramolecular proton
transfer and generate the photoinduced structural transformation of rhodol from the phenol (or enol)
form to the keto form. Moreover, from the crystal packing of Rh-F, we observed that the hyper-
conjugated planar construction of rhodol-benzothiazole results in the formation of intermolecular π–
π stacking of Rh-F with the interaction distance of 3.41 Å (Figure 2b). This will limit the rotation of
the C‒C single bond between rhodol and benzothiazole to promote the ESIPT process.
Figure 2. (a) X-ray single crystal structures of Rh-F showing the intramolecular OH···N hydrogen bonds; (b) The
crystal packing of Rh-F showing the intermolecular hydrogen bonding and the π–π stacking.
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Rh-F also exhibited AIE characteristics, and we have finished the following investigation. Rh-F
showed very faint fluorescence in pure THF solution with ΦF = 0.07, while Rh-F solid powder
showed strong fluorescence with ΦF = 0.56 (Figure S1). The above phenomenon indicates that Rh-F
has AIE characteristics. To further prove this view, we studied the fluorescence properties of Rh-F in
THF/water mixtures with different water fractions (fw) (Figure 3a), and Rh-F exhibited a very
interesting fluorescent spectral change. As the water fractions (fw) gradually increased from 0% to
70%, we observed that the keto emission at 524 nm (ESIPT) decreased gradually until almost fully
quenched, and simultaneously the enol emission at 384 nm was slightly enhanced. However, when
the water volume fraction reached 80%, the fluorescence intensity of the keto emission at 522 nm
exhibited a remarkable increase (Figure 3a inset). This can also be demonstrated by the fluorescent
image under UV illumination (Figure 3c). The UV–vis absorption spectra of Rh-F in THF/water
mixtures showed outstanding changes at fw ≥ 80%, which were consistent with the emission spectra.
It is not difficult to explain this phenomenon. In the THF/H2O solution with relatively low water
content (fw ≦ 70%), the intermolecular hydrogen bonds between the phenolic hydroxyl groups of
rhodol and H2O molecules are easier to form. An increase in the water content can stabilize the enol
form of Rh-F to restrain the ESIPT process upon being excited, resulting in the keto-form emission
(524 nm) decreasing and enol-form emission (384 nm) increasing. When the water content exceeds
this threshold concentration (≧80%), the solubility of Rh-F sharply decreased and Rh-F separated
from the solution rapidly and aggregated, thereby increasing the emission intensity of keto form due
to the ESIPT.
To gain more insight into the mechanism, PL decay measurements of the solid and the methanol
solution of Rh-F were employed, as shown in Figure 3b. Comparing the decay profiles of the solid
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powder and the methanol solution samples, the fluorescence lifetime of Rh-F in the solid powder
(5.90 ns) is longer than it is in methanol solution (1.03 ns), and that results from the facilitated
radiative decay in the aggregates emission. Furthermore, the PL decay curve of the methanol solution
of Rh-F can be fitted to a second-order exponential decay. The decay times of the fast and slow
components are respectively 0.92 and 6.56 ns. Evidently, the shorter lifetime (0.92 ns) should be
attributed to the enol form of Rh-F. The longer lifetime (6.56 ns) originates from the keto emission
of Rh-F (ESIPT), which conforms to the lifetime of the solid powder of Rh-F (5.90 ns). The results
are consistent with the dual emission of the methanol solution of Rh-F with both enol and keto
emission, as shown in Figure 1.
From the above results, we can conclude that the molecular aggregation of Rh-F through π–π
stacking can facilitate the ESIPT process, while the ESIPT process can also promote the AIE effect,
resulting in the enhanced fluorescent emission in the aggregated state. Based on the coupling of
ESIPT-AIE mechanisms, Rh-F could exhibit no self-extinguishing at high concentrations and strong
emission in the aggregated state with a large Stokes shift.
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Figure 3. (a) Emission spectra of Rh-F (10 μM) in THF/water mixtures with different water fractions (0 - 90%),
Inset: plots of the maximum emission intensity of Rh-F in THF/water mixtures with different water fractions (0 -
99%); (b) Fluorescence decay profiles of the Rh-F solid and the methanol solution of Rh-F; (c) Images of Rh-F in
THF/water mixtures with different water fractions (0 - 99%) under a 365 nm UV lamp.
3.3. Sensing for Fe2+
To gain insight into the fluorescence properties of the Rh-F probe towards metal ions, a sensing
experiment of Rh-F was carried out in a THF/H2O (7:3, v/v, pH = 7.4) solution. Various metal ion
solutions (100 μM) were added separately to the 10 μM Rh-F analysis solution. The fluorescent
spectra were recorded, and the results are shown in Figure 4a. The Rh-F solution (10 μM) showed a
high fluorescence intensity on excitation at 350 nm. There is almost no apparent change in
fluorescence intensity with adding Na+, K+, Pb2+, Mg2 +, Ca2 +, Cu2 +, Zn2 +, Co2 +, Ag+, Cd2+, Hg2+,
Mn2+, Fe3+, Cr3+, and Al3+ ion. However, only when Fe2+ was added to the Rh-F solution, the
fluorescence intensity at 384 nm and 525 nm showed a remarkable quenching (Figure 4a). At the
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same time, we can observe with naked eyes that the color of the fluorescent sensor Rh-F solution
changes from colorless (blank and with other metal ions) to yellow (after adding Fe2+) (Figure 5, top).
The corresponding Figure 5 (bottom) shows the fluorescence change under UV light in 10 μM Rh-F
solution with different metal ions. When adding Fe2+, the fluorescence of the Rh-F solution is
quenched. This indicates that Fe2+ complexed with Rh-F with its own specific paramagnetic nature
to participate in the electron/energy transfer leads to the closure of the ESIPT, the inactivation of the
radioactive channel of the fluorophore, and the loss of fluorescence. The occurrence of these
phenomenon confirmed that Rh-F has the capacity for “naked-eye” discrimination of Fe2 + in
aqueous solution.
Figure 4. (a) Fluorescence emission spectra of Rh-F probe (10 μM) treated with 10 equiv. of various metal ions in a
mixed solution of THF/H2O (7:3, v/v, pH = 7.4); (b) Fluorescence responses of probe Rh-F (10 μM) to various
metal ions (100 μM) at 525 nm in the absence and presence of Fe2+ in a mixed solution of THF/H2O (7:3, v/v, pH =
7.4). Red bar: probe Rh-F with various metal ions. Green bar: probe Rh-F with various metal ions in the presence
of Fe2+.
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Figure 5. Color changes of Rh-F probe (10 μM) solutions after the addition of various metal ions (10 equiv.) under
natural light (Top) and a 365 nm UV lamp (Bottom).
To further estimate the specificity of Rh-F, competitive experiments in the presence of Fe2+ mixed
with other competitive metal ions were carried out. Competitive ions were initially added into the
detection solution, then Fe2+ ion was added. The changes in fluorescence intensity at 525 nm before
and after the addition of Fe2+ are displayed in Figure 4b. The results demonstrated that the emission
of Rh-F at 525 nm was effectively quenched by Fe2+ ions with or without these metal ions. This
observation confirms that the competing ions do not interfere with the sensitivity of the probe for
Fe2+.
To further elicit the interactions between probe Rh-F and Fe2+, the sensitivity of Rh-F for Fe2+
was also investigated using UV-visible absorption (Figure S2) and fluorescence titration experiments
(Figure 6). As shown in Figure 6a, upon treatment with increasing concentrations of Fe2+, the
fluorescence intensity of Rh-F continuously decreases and no change of the maximum emission
wavelength was observed. The result indicates that when Fe2+ is combined with Rh-F, the
fluorophore and the receptor cannot be integrated under the excited state, resulting in no significant
change of the maximum emission wavelength. A good linear relationship between the fluorescence
intensity and Fe2+ concentration in range of 0-1.0 μM was determined (Figure 6b, R2 = 0.99099). In
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addition, the detection limit of the Rh-F probe for Fe2+ is about 115.2 nM, which is sufficiently low
for the detection of Fe2+ ions found in many environmental and biological systems.
To optimize the experimental conditions for the detection of Fe2+, the response time between Rh-F
and Fe2+ and the fluorescence intensity of probe Rh-F and Rh-F + Fe2+ complexes in different pH
were investigated. The result showed that the fluorescence intensity of Rh-F + Fe2+ (2 equiv) reduced
to a minimum value within 4 minutes (Figure S3a), indicating that the probe Rh-F could detect Fe2+
rapidly. In addition, as shown in Figure S3b, no significant changes in the fluorescence intensity at
525 nm was observed for both the free probe Rh-F and Rh-F + Fe2+ complex in the range of pH 2-
13. It indicated that the probe Rh-F and Rh-F + Fe2+ had good stability upon acidic and alkaline
conditions. Compared the fluorescence intensity of Rh-F and Rh-F + Fe2+, it showed that when the
probe Rh-F treated with 2 equiv of Fe2+, the fluorescence of the probe Rh-F is quenched in the
range of pH 2-13. The reslut indicated that the probe Rh-F is suitable for the detection of Fe2+ in any
pH condition.
Figure 6. (a) Fluorescence response of the Rh-F (10 μM) probe upon incubation with increasing concentrations of
Fe2+ (0 - 1.5 μM). Inset: plot of the emission intensity at 525 nm as a function of the concentrations of Fe2+. (b)
Linear plots of fluorescence intensity as a function of Fe2+ concentrations (0 - 1.0 μM).
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3.4. The proposed sensing mechanism
Further studies were carried out to explore the mechanism of sensing Fe2+. We proposed the
1
possible mechanism which is shown in Figure 7a. The HNMR titration of the free Rh-F shows the
characteristic phenolic hydroxyl proton signal at 12.836 ppm (Figure S4a), and the phenolic
hydroxyl proton signal disappeared when the 1.0 µM Fe2+ was added (Figure S4b). The mechanism
of fluorescence quenching when Rh-F combined with Fe2+ may be caused by the following two
factors. First, with the addition of Fe2+ to the Rh-F solution, Fe2+ chelated with the O atom of the
hydroxyl group and the N atom of the benzothiazole moiety (Figure 7a). This might cause the ESIPT
process of the Rh-F to be devastated and the keto-type emission decreased. The second reason may
result from the paramagnetic properties of Fe2+. Figure 7b showed the curve fitting of Rh-F receptor
fluorescence intensity against the reciprocal of the Fe2+ concentration with the Benesi-Hildebrand
equation based on fluorescence titration. The binding constant Ka of Rh-F with Fe2+ had been
established (Ka= 1.31 × 105, R2 = 0.98171). The linear curve fit provides evidence for the 1:1
complexation behavior of Rh-F to Fe2+. Simultaneously, we also carried out a Job’s plot (Figure 7c)
and the lowest intensity change point appeared at about 0.5, this provides further confirmation that
Rh-F formed a 1:1 stoichiometry complex with Fe2+. Furthermore, the binding mode of Rh-F and
Fe2+ was confirmed by the FT-IR (Figure S5) and HRMS (Figure S8). Comparing the FT-IR of Rh-F
with Rh-F-Fe2+, upon the addition of 1.0 equiv Fe2+, the absorption peak of the phenolic hydroxyl
group at 3218.14 cm-1 disappeared. The absorption peak of C=N valence vibration underwent a
redshift from 1473 to 1492 cm-1. These results indicated that the complexation between Rh-F and the
iron center resulted in the occurrence of a strong weakening of the C=N bond. In the HRMS of Rh-
F-Fe2+, the characteristic peak of [Rh-F + H]+ at m/z 659.19 completely disappeared (Figure S7) and
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a new peak arose at m/z 749.19 which was assigned to [Rh-F-Fe2+ + H]+. The above results
confirmed the binding mechanism proposed in Figure 7a. The destruction of the ESIPT process of
Rh-F was caused by the chelation of Fe2+ with the O atom of the hydroxyl group and the N atom of
the benzothiazole moiety, which resulted in the quenching of the Rh-F fluorescence sensor.
Figure 7. (a) Sensing mechanism of the Rh-F probe for Fe2+; (b) Benesi-Hildebrand plot for the determination of
the binding constant of Rh-F (10 µM) for Fe2+ in a mixed solution of THF/H2O (7:3, v/v, pH = 7.4) (λex = 350 nm);
(c) Job's plot for determining the stoichiometry of Rh-F with Fe2+ in a mixed solution of THF/H2O (8:2, v/v, pH =
7.4) (λex = 350 nm), XFe2+ = [Fe2+ ]/{[Fe2+] + [Rh-F]}.
4. Conclusions
In conclusion, we introduced the benzothiazole into the xanthene ring of Rhodol to design and
synthesize a new Rh-F fluorophore based on the dual mechanism of AIE-ESIPT. Due to the mutual
synergy between ESIPT and AIE mechanisms, it exhibited excellent photophysical properties such as
solvent dependent emission behavior, efficient aggregated emission around 525 nm, and a large
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Stokes shift (>180 nm). Furthermore, the Rh-F fluorophore exhibited outstanding identification
ability for Fe2+. It is a sensor with high selectivity and sensitivity, which mainly utilized the ESIPT
mechanism and the polymerization fluorescence method of Rh-F. This work can stimulate us to
easily develop fluorescent sensors for different analytes with the AIE/ESIPT mechanism by
modifying the phenolic hydroxyl functional groups in the Rhodol ring. The development of
AIE/ESIPT fluorophores will provide a viable design strategy for biosensors.
Acknowledgments
We gratefully acknowledge the National Natural Science Foundation of China (21202132,
21505105, and 21874108), China Postdoctoral Science Foundation (2016M592837), and the Open
Foundation of State Key Laboratory of Crystal Materials (KF1806) for funding.
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Graphical Abstract
A new Benzothiazole-rhodol based fluorescent probe was constructed facilely with ESIPT-
induced AIE and an application for Detecting Fe2+ Ion.
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Highlights:
> Rh-F exhibited excellent synergistic effect of AIE and ESIPT feature.
> Rh-F achieves "on-off" fluorescence and visual color change for detecting Fe2+.
> Rh-F showed a sensitive fluorescence response to Fe2+ with high selectivity.
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