A bis-(rhodamine)-based off–on colorimetric and fluorescent probe for Fe3+ ion detection in serum and bioimaging

Article abstract of DOI:10.1007/s11696-018-0492-9

Abstract: An off–on colorimetric and fluorescent probe N¹, N⁴-bis (2-(3′, 6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl) ethyl) terephthalamide (Rp) for Fe³⁺ was designed and synthesized, which could selectively and sensitively respond to Fe³⁺ among other tested metal cations in THF–H₂O solution (v/v = 4/6, pH = 7.2) accompanied with a remarkable fluorescence enhancement and an obvious color change visible to “naked-eye”. The fluorescence intensity of the probe Rp showed a good linearity with the concentration of Fe³⁺ covering from 0.1 to 26?μM and the detection limit was 0.0231?μM. Meanwhile, the binding mode between Rp and Fe³⁺ was found to be a 1:2 stoichiometric formation. Moreover, the complexation of Rp–Fe³⁺ could be broken by carbonate, so this system might be utilized as a carbonate probe in the future. Most importantly, the probe Rp was not only applied to monitor Fe³⁺ level in human blood serum and water samples, but also was used for fluorescence imaging of Fe³⁺ in living cell. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11696-018-0492-9

Chemical Papers
https://doi.org/10.1007/s11696-018-0492-9
ORIGINAL PAPER
A bis‑(rhodamine)‑based of–on colorimetric and fuorescent probe
for Fe3+ ion detection in serum and bioimaging
Pengying Liu1 · Aoheng Luo1 · Yuyuan Wang1 · Jiangke Hu1 · Qiao Huang1 · Hongqing Wang1
Received: 12 December 2017 / Accepted: 27 April 2018
© Institute of Chemistry, Slovak Academy of Sciences 2018
Abstract
An of–on colorimetric and fuorescent probe N1, N4-bis (2-(3′, 6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-
2-yl) ethyl) terephthalamide (Rp) for Fe3+ was designed and synthesized, which could selectively and sensitively respond
to Fe3+ among other tested metal cations in THF–H2O solution (v/v = 4/6, pH = 7.2) accompanied with a remarkable
fuorescence enhancement and an obvious color change visible to “naked-eye”. The fuorescence intensity of the probe Rp
showed a good linearity with the concentration of Fe3+ covering from 0.1 to 26 μM and the detection limit was 0.0231 μM.
Meanwhile, the binding mode between Rp and Fe3+ was found to be a 1:2 stoichiometric formation. Moreover, the compl-
exation of Rp–Fe3+ could be broken by carbonate, so this system might be utilized as a carbonate probe in the future. Most
importantly, the probe Rp was not only applied to monitor Fe3+ level in human blood serum and water samples, but also
was used for fuorescence imaging of Fe3+ in living cell.
Graphical abstract
Keywords Fluorescent probe · Fe3+ · Rhodamine · Serum · Cell imaging
Introduction
The design and development of probes for the detection
of heavy and transition metal ions have attracted more and
more attention nowadays (Sahana et al. 2018; Zhou et al.
* Hongqing Wang
2018; Liao et al. 2017; Zheng et al. 2017), which is not
hqwang2015@126.com
only because of their inherent excellent properties includ-
1
ing operational simplicity, low cost and real-time monitor-
School of Chemistry and Chemical Engineering, University
of South China, Hunan 421001, China
ing, but also their advantages of signifcant application in
Vol.:(0123456789)
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biological and environmental systems (Li et al. 2015; Ucar
et al. 2017; Zhu et al. 2017; Gu et al. 2017; Karimi et al.
2016). Among these metal ions, Fe3+ is one of the important
targets to be monitored in the biological and environmen-
tal matrices. On the one hand, the defciency or overload
of Fe3+ in human and animal bodies can result in various
disorders and harmful impact on the health of living body
subsequently (Gao et al. 2015; Chen et al. 2013; Carlson
et al. 2008). For another, Fe3+ as one of the environmental
contaminants has serious efects on the yield and quality of
crops via various people’s activities (Jolly 2013; Hofmann
et al. 2015). It is known that Fe3+ is easily soluble in water
and the environment of the organism is an aqueous solu-
tion, but most of the reported Fe3+ probes are only used
in the detection system with low water content (Liu et al.
2014; Kagit et al. 2014). And though there is a popular belief
that turn-on probes are more efcient, most of the reported
probes for Fe3+ undergo a fuorescence quenching due to
the paramagnetic nature of Fe3+ (Fu et al. 2013; Wang et al.
2016). Hence, it is signifcant and challenging to develop
turn-on fuorescent probes for Fe3+ which can be used in the
system with high water content to meet the needs of biologi-
cal and environmental application.
Based on the above studies, herein, a bis-(rhodamine)-
based colorimetric and fuorescent probe Rp for Fe3+ was
reported by the means of changing the structure of the
probe to ameliorate the characteristic of fexibility and
rigidity and increase the conjugated structure of the probe.
As expected, the Rp exhibited an excellent selectivity and
sensitivity toward Fe3+ by forming a 1:2 complex in a high
water content detection system. Meantime, the Rp–Fe3+
complex was broken in the presence of carbonate, so it
had the value for detecting carbonate. Most importantly,
the probe Rp was used to analyze Fe3+ in human blood
serum and water samples and image Fe3+ in living SGC-
7901 cells.
Experimental
Materials and apparatus
All the reagents were analytical grade and used without
further purifcation unless otherwise noted. The pH was
measured using a Model pHs-3C meter (Shanghai, China).
The melting points were acquired from a X-4 microscopic
melting point apparatus (Shanghai, China). NMR spectra
were determined by a Bruker 400 MHz spectrophotometer
with CDCl3 as solvent. HRMS spectra were recorded using
a Thermo Fisher Scientifc LTQ FT Ultramass spectrometer.
UV–Vis spectra were obtained from a Hitachi U-3900 spec-
trophotometer (Tokyo, Japan). Fluorescence spectra were
measured at λex =550 nm on a Hitachi F-7000 spectrofuor-
imeter (Tokyo, Japan) with a 10 mm quartz cuvette. The
fuorescence image experiments were performed on a Nikon
TE2000 inverted fuorescence microscope.
Among the probes family, the rhodamine framework is
used as a platform to construct colorimetric and turn-on
fuorescent probes for detecting ions and has attracted con-
siderable interests because of its excellent photophysical
properties with large molar extinction coefcients and high
fuorescence quantum yields (Mandal et al. 2013; Jiao et al.
2016; Puangploy et al. 2014; Ramette and Sandell 1956).
In addition, the rhodamine derivatives undergo the form’s
conversion from spirolactam structure (nonfuorescence) to
ring-opening amide form (fuorescence) once interacting
with specifc metal ions (Zhang et al. 2015; Kim et al. 2008)
or some solvents (Sekar and Kothavale 2016) or protons (Xu
et al. 2017). However, most of the reported probes based on
rhodamine for Fe3+ still have some shortcomings in practical
application, such as relatively narrow detection range (Liu
et al. 2014), higher detection limit (Liu et al. 2014; Ji et al.
2013; Liu et al. 2015) and without application in biological
systems (Sivaraman et al. 2014; Hu et al. 2011). Moreo-
ver, the reversibility of a fuorescent probe is an important
feature because this feature can reduce the cost of analysis,
but only a few rhodamine-based Fe3+ probes are reversible
(Lee et al. 2015; Zhang et al. 2013; Sun et al. 2015). It is
believed that bis-(rhodamine)-based probes with appropri-
ate binding sites for metal ions would have better sensitivity
and selectivity than conventional mono-rhodamine-based
probes (Yan et al. 2015; Chereddy and Thennarasu 2011).
On the other hand, increasing the conjugation or rigidity of
the probe will lead to the changes in the optical properties
and responsiveness (Weerasinghe et al. 2010; Chen et al.
2012; Luo et al. 2016).
UV–Vis absorption and fuorescence measurement
Stock solutions (1.0×10−3–1.0×10−2 mol/L) of Li+, Na+,
K+, Ag+, Ni2+, Mn2+, Mg2+, Pb2+, Ca2+, Ba2+, Zn2+, Cu2+,
Cd2+, Co2+, Hg2+, Fe2+, Fe3+, Al3+ and Cr3+ were prepared
by dissolving their nitrate or chloride salts into double-
distilled water. Stock solution (1.0 × 10−3 mol/L) of Rp
was prepared by dissolving the appropriate amount of Rp
into DMSO, DMF or THF. 0.1 mL of the Rp stock solu-
tion (1.0×10−3 mol/L) and the appropriate amount of the
ion stock solution were added into a 10 mL brown volu-
metric fask, and diluted to 10 mL with DMSO/Tris–HCl,
DMF/Tris–HCl or THF/Tris–HCl aqueous bufer solutions,
respectively. After waiting for 20 min, the spectral data were
recorded. Fluorescence measurements were carried out with
excitation wavelength of 550 nm and excitation and emission
slit width of 5 nm.
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Testing Fe3+ in the reference materials or samples
Synthesis of the probe Rp
The reference materials: GBW(E)080915, GBW(E)080916
and GBW(E)080917 were purchased from Beijing Bohui
Innovation Optoelectronic Technology Co., Ltd. (Beijing,
China). The human blood serum was treated according to
the reported method (Olson and Hamlin 1969). After 4 mL
20% trichloroacetic acid (TCA) was added into the blood
serum (4 mL), the mixture was stirred and heated to 90 °C
for 15 min. Then, the mixture was cooled into room tem-
perature, sonicated for 2 min and centrifuged for 10 min at
10,000 rpm and then the supernatant was collected for analy-
sis. The water samples were collected locally and fltrated
through a 0.45 μm membrane before used. The concentration
of Fe3+ in the reference materials, water sample or human
blood serum was determined by the fuorescence intensity
and the linear relationship between the fuorescence intensity
of Rp–Fe3+ solution and Fe3+ concentration.
The synthetic route of the probe Rp was shown in Scheme 1.
2-(2-aminoethyl)-3′, 6′-bis(diethylamino)spiro[isoindoline-
1,9′-xanthen]-3-one (1) was easily synthesized according to
the procedure in the literature (Zhang et al. 2007). N1, N4-
bis(2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-
xanthen]-2-yl)ethyl)terephthalamide (Rp) was synthesized
according to the following procedure: The terephthaloyl
chloride (0.1 g, 0.49 mmol) dissolved in the dry dichlo-
romethane (10 mL) was added into the mixed solution of
compound 1 (0.58 g, 1.19 mmol) and triethylamine (400 μL,
3.0 mmol) in dry dichloromethane (25 mL) with vigorously
stirring at 0 °C. After adding, the solution was stirred for
8 h at 25 °C. Then, the reaction solution was washed three
times with water and the organic layer was dried over anhy-
drous Na2SO4, and the crude solid was obtained by removing
the organic solvent under reduced pressure. After purifed
by silica gel column chromatography (methanol/dichlo-
romethane=1/9, v/v), 0.42 g white solid (Rp) was obtained
in 79% yield, m.p.>320 °C. 1H NMR (400 MHz, CDCl3), δ
(ppm): 8.25 (s, 2H), 7.93–7.96 (m, 6H), 7.42–7.45 (m, 4H),
7.05–7.08 (m, 2H), 6.42 (d, 4H, J=8.85 Hz), 6.35–6.36 (m,
4H), 6.24 (d, 2H, J= 2.26 Hz), 6.22 (d, 2H, J= 2.25 Hz),
3.40–3.42 (m, 4H), 3.29 (q, 16H, J=7.12 Hz), 3.20 (m, 4H),
1.14 (t, 24H, J = 7.07 Hz); 13C NMR (100 MHz, CDCl3),
δ (ppm): 170.4, 166.4, 153.9, 153.3, 149.0, 136.6, 132.8,
130.4, 128.4, 128.2, 127.4, 123.9, 123.0, 108.4, 104.6, 97.8,
65.9, 44.4, 41.9, 40.0, 12.6; HRMS (MALDI) m/z calcd for
[C68H74O6N8 +H]+: 1099.5818, found: 1099.5824.
Fluorescence imaging
Gastric carcinoma cell lines (SGC-7901) were cultured with
10% (v/v) fetal bovine serum (FBS) in Dulbecco’s Modi-
fed Eagle Medium (DMEM) at 37 °C in an atmosphere of
5% CO2 for 24 h. Immediately the SGC-7901 cells were
incubated with 10 μM Rp at 37 °C for 30 min, washed three
times with phosphate bufered saline (PBS) and then imaged.
Following, the cells were cultured with 50 μM FeCl3 for
another 30 min, washed three times with phosphate bufered
saline (PBS) and imaged.
Scheme 1 The synthetic route of the probe Rp
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cells. Therefore, the probe Rp was more suitable for the
detection of Fe3+ in biological systems and real samples.
The pH efect on the fuorescence intensity of Rp and
Rp–Fe3+ was investigated (Fig. 2). Without Fe3+, the probe
Rp with spirolactam structure was colorless and exhibited
no fluorescence in the pH range of 4.0–14.0. However,
when pH value was less than 4.0, an obvious fuorescence
appeared due to the formation of merocyanine open struc-
ture induced by strong protonation. Once Fe3+ (5.0 equiv.)
was added, ring-opening forms induced by Fe3+ occured
with an obvious fuorescence enhancement in the pH range
of 2.0–10.0, especially 4.0–9.0. The fuorescence inten-
sity decreased sharply once the pH value was higher than
9.0, which could be ascribed to the formation of insoluble
hydroxide. In consideration of the physiological condition,
pH value was adjusted to 7.2 for the following experiments.
The efect of reaction time on the response of Rp to Fe3+
was studied (Fig. 3). Interestingly, when 5.0 equiv. Fe3+
was added into the solution of Rp (10 μM) in THF–water
(v/v=4/6, pH = 7.2), the color of the solution changed from
colorless to pale red within few minutes, then gradually
changed to red and remained unchanged after 20 min. Addi-
tionally, the fuorescence intensity of Rp–Fe3+ increased
within few minutes and reached a maximum value at 20 min
and then remained almost constant, while the fuorescence
intensity of blank solution almost unchanged during the test
time. Therefore, the spectral properties were measured after
waiting for 20 min in subsequent experiments.
Fig. 1 Efect of water content in diferent media on the fuorescence
intensity of Rp (10 μM) with and without Fe3+ (5.0 equiv.)
Results and discussion
The efect of diferent detection media on the fuorescence
spectral measurement of Fe3+ was investigated (Fig. 1).
The free Rp hardly exhibited fuorescence in DMSO–water
mixtures, DMF–water mixtures, or THF–water mixtures.
But upon adding same amount of Fe3+ (5.0 equiv., 5.0
equiv. of Fe3+ or other metal ions were used in the all
physical characteristics experiments of Rp to ensure that
Rp with spirolactam structure could convert to ring-
opening amide form as far as possible and the fuores-
cence intensity could reach the maximum value) into the
Rp solutions with diferent detection media, respectively,
the fuorescence intensity of all the solutions increased
obviously, particularly in THF–water mixed solution.
Furthermore, the content of water in the media had efect
on the fuorescence intensity. The fuorescence intensity
increased sharply and then decreased gradually along with
the decrease of water content in the detection media. Inter-
estingly, when the ratio of THF/water (v/v) was 4:6, the
fuorescence intensity reached the maximum. The results
showed that Rp exhibited a more sensitively response to
Fe3+ in THF–water (v/v=4:6) and the water content of the
test media was distinctly higher than that of some reported
Fe3+ fuorescent probes (Liu et al. 2014, 2015; Xu et al.
2017). And the solubility of Rp in THF/water solution
was examined by adding 10 mL THF/water solution with
diferent water content into Rp (0.0110 g, 0.01 mmol).
The Rp was easily dissolved in THF, but in THF–water
mixed solution, the Rp could not be dissolved completely
once the ratio of THF/water (v/v) was lower than 7:13. It
is well known that ions have better solubility in water than
that in organic solvent and it is just the aqueous system in
Selectivity is an important indicator to evaluate the per-
formance of a fuorescent probe. Therefore, the UV–Vis
absorption and fuorescence spectra of Rp with various
metal ions were explored under the same condition by add-
ing various metal ions (Li+, Na+, K+, Ag+, Ni2+, Mn2+
,
Fig. 2 Efect of pH on fuorescence intensity of Rp (10 μM) in the
absence and presence of Fe3+ (5.0 equiv.) in THF–water (v/v=4/6)
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Fig. 5 The color in daylight (top) and fuorescence color (bottom)
changes of Rp with addition of various metal ions in THF–water
(v/v=4/6, pH = 7.2). From the left to right: blank, Ag+, Ba2+, Ca2+
Fe3+, Al3+, Hg2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, K+, Li+, Mg2+
Mn2+, Na+, Ni2+, Pb2+, Zn2+
,
,
a UV lamp (365 nm), respectively (Fig. 5). The free Rp
solution was colorless and non-fuorescent. In the presence
of Fe3+, the Rp solution absorbed daylight and became red
(the upper part of Fig. 5) and a signifcant red fuorescence
was observed (the lower part of Fig. 5), whereas the other
tested metal ions did not induce the changes except for
Al3+ and Hg2+, which only resulted in a little bit pink color
and slight fuorescence emission.
Fig. 3 Efect of reaction time on the fuorescence intensity of Rp
(10 μM) in the absence and presence of Fe3+ (5.0 equiv.) in THF–
water (v/v=4/6, pH = 7.2)
Mg2+, Pb2+, Ca2+, Ba2+, Zn2+, Cu2+, Cd2+, Co2+, Hg2+
,
Fe2+, Fe3+, Al3+, Cr3+) into the solution of Rp (10 μM) in
THF–water (v/v=4/6, pH = 7.2) (Fig. 4). After adding (5.0
equiv.) Fe3+ into the Rp solution, a strong absorption peak
at 564 nm and fuorescence emission at 584 nm appeared,
whereas the other tested metal ions did not produce an obvi-
ous response to Rp except for Hg2+ and Al3+, which only
resulted in a little absorption peak and slight fuorescence
emission.
Competitive experiments were performed to evaluate
the possible interferences of other coexistent metal ions
on the detection of Rp to Fe3+ (Fig. 6). As expected, other
tested competitive metal ions hardly afected the recogni-
tion of Rp to Fe3+ even though their concentration were
twice that of Fe3+ except for Hg2+ and Al3+, which induced
the fuorescence intensity of the Rp and Fe3+ solution only
to increase by 1.49 and 0.89%, respectively. These results
indicated that the probe Rp could be employed to detect
Fe3+ with a high selectivity.
Meanwhile, the colorimetric and fuorescent properties
of Rp toward various metal ions in THF–water (v/v= 4/6,
pH = 7.2) were studied under irradiation with daylight and
Fig. 4 a UV–Vis absorption and b fuorescence spectra of Rp (10 μM) in the presence of various metal ions (5.0 equiv.) in THF–water
(v/v=4/6, pH = 7.2)
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added, a distinct absorption band at 564 nm occured and
enhanced with gradual addition of Fe3+. When the con-
centration of Fe3+ was greater than 2.6 equiv. (excess), the
absorbance did not increase further and became saturated
(Fig. 7 inset).
The fuorescence sensing behavior of Rp for Fe3+ was
explored by fuorescence titration in THF–water (v/v=4/6,
pH = 7.2) (Fig. 8). A signifcantly enhanced fuorescence
signal at 584 nm could be detected when Fe3+ was added.
Besides, the fuorescence intensity at 584 nm enhanced
gradually with the increasing concentration of Fe3+. The
results indicated that the fuorescence intensity of Rp–Fe3+
showed a good linearity with the concentration of Fe3+ cov-
ering from 0.1 to 26 μM. The linear regression equation was
F=20.75535+106.49896C and R2 was 0.9925. The detec-
tion limit was as low as 0.0231 μM calculated from 3ꢀ∕k
(where δ was the blank solution standard deviation and k was
the slope of the calibration curve) (Goswami et al. 2013).
These results implied that Rp could be used as a sensitive
probe for the detection of Fe3+.
Fig. 6 The fuorescence intensity of Rp (10 μM) in the presence of
various interfering ions (10 equiv.) and coexistence with Fe3+ (5.0
equiv.) in THF–water (v/v=4/6, pH = 7.2). The black bars represent
the fuorescence response of Rp and competing ions: 1, blank; 2, Li+;
To investigate the binding mode between Rp and Fe3+,
the Job’s plot analysis was adopted according to the absorb-
ance of Rp at diferent Fe3+ mole fraction in THF–water
(v/v=4/6, pH = 7.2) (Fig. 9). The absorption intensity of
Rp came to maximum when the mole fraction of Fe3+ was
around 0.67, which revealed that Rp and Fe3+ complexed at
a stoichiometric ratio of 1:2.
3, Na+; 4, K+; 5, Mg2+; 6, Ca2+; 7, Zn2+; 8, Pb2+; 9, Cu2+; 10, Cd2+
11, Ba2+; 12, Ni2+; 13, Mn2+; 14, Ag+; 15, Fe2+; 16, Cr3+; 17, Co2+
;
;
18, Hg2+; 19, Al3+; 20, Fe3+. The red bars represent the subsequent
addition of Fe3+ (5.0 equiv.) to the above solutions (λex =550 nm,
λem =584 nm)
The association constant K of Rp with Fe3+ was deter-
mined by the Benesi–Hildebrand equation (Wang and Yu
2007): (I
max
− I0)∕(I − I0) = 1 + (1∕K)(1∕[Mn]), where I
was the measured absorption intensity, I0 was the absorption
intensity of the free Rp, Imax was the absorption intensity of
the Rp with saturated Fe3+, [Fe3+] was Fe3+ concentrations.
Based on the 1:2 binding mode (n=2), a plot of (Imax−I0)/
(I−I0) vs. 1/[Fe3+]2 yielded the apparent binding constant
value as 6.54 × 109 M−2 (R2 = 0.991) (Fig. 10). And the
Benesi–Hildebrand plot’s excellent linearity further dem-
onstrated the fact that the probe Rp responded to Fe3+ by
the 1:2 binding mode. It’s interesting that the complex ratio
of Rp to Fe3+ was distinct from that of the probes reported
by Luo et al. (2016) or Weerasinghe et al. (2010) in which
the complex ratio was 1:1, though they have similar molecu-
lar structure. Compared with the present work, the probe
reported by Weerasinghe et al. (2010) had a higher rigidity
and the probe in our group’s previous work (Luo et al. 2016)
had a higher fexibility. That is to say, in this work, the rigid-
ity and fexibility of Rp fell in between them, leading to the
diferent spatial orientation of coordination atoms and thus
the binding mode of Rp–Fe3+ was diferent.
Fig. 7 The UV–Vis absorption spectra of Rp (10 μM) upon the addi-
tion of various amounts of Fe3+ in THF–water (v/v=4/6, pH = 7.2).
Inset: UV–Vis absorbance at 564 nm as a function of Fe3+ concentra-
tion
The binding mode of Rp and Fe3+ was further explored
using DFT calculations with the B3LYP method, where
6-31+G (d, p) used as the basis set (Bao et al. 2014; Yan
et al. 2017). The stoichiometry ratio of Rp–Fe3+ was found
UV–Vis titration experiments were conducted by add-
ing various concentrations of Fe3+ into the Rp solution in
THF–water (v/v = 4/6, pH = 7.2) (Fig. 7). Once Fe3+ was
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Fig. 8 a The fuorescence spectra of Rp (10 μM) in THF–water (v/v=4/6, pH=7.2) with increasing amounts of Fe3+. b The calibration curve in
the concentration range of 0.1–26 μM of Fe3+ (λex =550 nm, λem =584 nm)
Fig. 9 Job’s plot for the stoichiometry of Fe3+ and Rp at 564 nm in
Fig. 10 Benesi–Hildebrand plot for the formation of Rp–Fe3+ com-
THF–water (v/v=4/6, pH = 7.2)
plex
to be 1:2 based on the Job’s plot and Benesi–Hildebrand
plot and this Rp–Fe3+ complex with 1:2 binding mode
was modelled by DFT calculations. At the same time,
the probe Rp and the Rp–Fe3+ complex with 1:1 bind-
ing mode were also modelled as a comparison model. In
the optimized geometries model of the Rp–Fe3+ complex
with 1:2 binding mode (Fig. 11), the oxygen atoms and
nitrogen atoms on each side of terephthaloyl were oriented
in coordination with one Fe3+, that is, Rp and Fe3+ were
coordinated by the ratio of 1:2. The spatial distributions
and the energy gap (ΔE) of the HOMO and LUMO of
these three structures were also obtained from the DFT
calculations (Fig. 12). For Rp and the Rp–Fe3+ complex
with 1:1 binding mode, the energy gap (ΔE) between the
ELUMO and EHOMO were 92.3757 and 23.5861 kcal/mol,
respectively. However, for the Rp–Fe3+ complex with 1:2
binding mode, the energy gap (ΔE) was 3.2944 kcal/mol,
which was far less than that of Rp or the Rp–Fe3+ complex
with 1:1 binding mode. These results indicated that the
Rp–Fe3+ complex with 1:2 binding mode could converge
to a more stable state than that of the Rp–Fe3+ complex
with 1:1 binding mode and could play down the energy
gap and stabilize the system.
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Fig. 11 The optimized geometries model of ‘a’ (Rp), ‘b’ (the Rp–Fe3+ complex with 1:2 binding mode) and ‘c’ (the Rp–Fe3+ complex with 1:1
binding mode)
Fig. 12 The HOMO and LUMO orbital of ‘a’ (Rp), ‘b’ (the Rp–Fe3+ complex with 1:2 binding mode) and ‘c’ (the Rp–Fe3+ complex with 1:1
binding mode)
The fuorescence changes of Rp–Fe3+ complex were
investigated in the presence of various small anions, such
which produced more OH− and disrupted the complexation
of Rp with Fe3+ (He et al. 2015). Subsequently, a reversible
experiment of the probe Rp was also performed by alter-
nating addition of Fe3+ and Na2CO3 into the Rp solution
(Fig. 13). Upon addition of Fe3+ (5.0 equiv.), the solution
of Rp (10 μM) transmitted strong fuorescence and notice-
able color. Then, the addition of Na2CO3 (20 equiv.) into
the mixed solution of Rp and Fe3+ resulted in fuorescence
quenching and color fading, followed by adding Fe3+ (10
−
−
as CO32−, HPO42−, SO42−, F−, Cl−, Br−, I−, HCO3 , NO3 ,
−
CH3COO− and H2PO4 . In the presence of carbonate, the
fuorescence intensity of Rp–Fe3+ was obviously quenched
and hydrogen phosphate resulted in slight fluorescence
quenching, while other tested anions exhibited no signif-
cant infuence. The quenching may be ascribed to the high
alkalinity and large hydration-free energy of carbonate
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enhancement and color change. Then, the addition of car-
bonate disrupted the chelation of Rp–Fe3+, resulting in the
reversible re-formation of the non-fuorescence spirolactam.
To evaluate the accuracy of the developed analytical
method based on the probe Rp for detecting Fe3+, three
certifed reference materials (CRMs) (GBW (E) 080915,
GBW(E) 080916, and GBW(E) 080917) were used. The test
results agreed well with the standard values (Table 1). Fur-
thermore, the applicability of the probe Rp in human blood
serum and water samples was also conducted using standard
addition method. As presented in Table 2, the concentration
of Fe3+ in the serum was found to be 22.30 0.10 µM, which
coincided well with the value (21.1 0.8 µM) obtained from
human blood serum in previous study (Long et al. 2014).
The recoveries of Fe3+ were in the range from 99.1 to
100.6% in human blood serum and 98.0 to 99.2% in waste
water with good R.S.D. The results suggested that Rp had
the potential application for quantitative detection of Fe3+
in real samples.
Fig. 13 Reversibility of the probe Rp (10 µM) for the sequen-
2−
tial sensing of Fe3+ and CO3 in THF–water media (v/v=4/6, pH
= 7.2). Inset: the color change of Rp upon sequential addition of
2−
Fe3+ and CO3 in solution. (a)=Rp (10 µM); (b)=(a)+Fe3+
;
(c)=(b)+CO32−; (d)=(c)+Fe3+
Cell imaging experiments were carried out to test the
application of Rp in living cells (Fig. 14). After SGC-7901
equiv.) into the mixed solution, the color and the fuores-
cence appeared again (Fig. 13 inset). It was interesting that
in the reversible experiment of our group’s previous work
(Luo et al. 2016), CO32− was 5.0 equivalent of Fe3+, which
was more than that of the present work, and the fuores-
cence intensity of the probe-Fe3+ complex just decreased in
some degree. Thus, this fnding suggested that the complex
of Rp–Fe3+ had potential application value in the detection
of carbonate.
Table 1 Analytical results for the determination of Fe3+ in certifed
reference materials
Samples
Concentration of Fe3+ (mg/L)
Certifed
Measureda
GBW(E)080915
GBW(E)080916
GBW(E)080917
4.96 0.09
10.10 0.20
19.90 0.30
4.88 0.14
9.95 0.23
19.78 0.27
Based on the above results, the possible binding mode
of Rp and Fe3+ was proposed in Scheme 2. The probe Rp
interacted with Fe3+ in a 1:2 stoichiometry with fuorescence
aMean of three replicates SD
Scheme 2 The proposed binding mode between Rp with Fe3+
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Table 2 Determination of Fe3+
in human blood serum and
water samples
Sample
Fe3+ spiked (µM)
Fe3+ found (µM)
Recovery (%)
R.S.D.(%,
n=5)
Human blood serum
0.00
1.00
2.00
0.00
5.00
10.00
22.30 0.10
23.10 0.08
24.45 0.20
1.12 0.13
6.07 0.17
10.90 0.09
–
–
99.1
100.6
–
99.2
98.0
2.8
4.1
–
4.3
3.9
Waste water
Conditions: (10 μM) Rp in THF–water (v/v=4/6, pH = 7.2), excitation/emission was performed at
550/584 nm
Fig. 14 Fluorescence images of SGC-7901 cells. a bright-feld image:
cells stained by Rp (10 µM) for 30 min; b fuorescence image of “a”;
c merged images of “a” and “b”; d bright-feld image: cells stained
with Rp (10 µM) for 30 min and then incubated with Fe3+ (50 µM)
for another 30 min; e fuorescence image of “c”; f merged images of
“d” and “e”
cells were cultured with Rp (10 µM) for 30 min and
washed with phosphate bufered saline (PBS), no intra-
cellular fuorescence signal could be observed (Fig. 14b),
which was probably because the intracellular concentra-
tion of Fe3+ was too low to reach the detection limit of Rp.
When the cells were cultivated with FeCl3 (50 µM) for
another 30 min and washed with phosphate bufered saline
(PBS), some of the Fe3+ entered the cell. At this time,
the intracellular concentration of Fe3+ was higher than the
detection limit of Rp and thus a bright intracellular fuo-
rescence appeared (Fig. 14d). The results demonstrated
that the Rp had cell permeability and could be applied to
image Fe3+ in SGC-7901 cells.
The comparison of the present probe Rp over some
other pioneering fuorescent probes was listed in Table 3
(Liu et al. 2014; Wang et al. 2015; Liu et al. 2015; Luo
et al. 2016; Wang et al. 2014; Zhou et al. 2017; Xu et al.
2017). The previous reported Fe3+ fuorescent probes had
higher ratio of organic solvent in the detection system or
higher detection limit, or the probes were not used in bio-
logical, environmental sample or cell imaging. Therefore,
the present probe Rp had broader applications and higher
potential value.
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Table 3 The comparison of Rp with some other probes for Fe3+
Sensor
Solution
(v/v)
CNCH3 : H2O
=8:2
LOD
(μM)
0.32
Application
/
Linear
range (μM)
0-10
Reference
Liu et al., 2014
C2H5OH
0.418
1.8
Cell imaging
0.2-1.0
0-20
Wang et al., 2015
Liu et al., 2015
CH3OH : H2O
=6:4
/
CH3OH : H2O 0.0124
=7:3
Cell imaging
Cell imaging
0.3-14
0-20
Luo et al., 2016
CH3OH : H2O
=4:6
0.105
Wang et al., 2014
C2H5OH : H2O
=5:5
0.025
0.065
Cell imaging
/
/
Zhou et al., 2017
Xu et al., 2017
CNCH3 : H2O
=5:5
/
THF : H2O
=4:6
0.0231
Cell imaging
Water samples
Blood serum
0.1-26
This work
had a potential application in the detection of carbonate.
In addition, the binding mode of Rp–Fe3+ was proposed
and further verifed by DFT calculation. Most importantly,
the fuorescence imaging of intracellular Fe3+ in SGC-7901
cells and the determination of Fe3+ in human blood serum
and water samples indicated that the probe Rp had practical
application value in biological and environmental systems.
Conclusions
In conclusion, a colorimetric and fuorescent probe Rp
for Fe3+ was developed, which showed a highly selective
response to Fe3+ in THF–water (v/v=4/6, pH = 7.2) with
a low detection limit of 0.0231 μM. The complexation of
Rp with Fe3+ could be broken by carbonate, so this system
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Acknowledgements This work was supported by the National Natural
Science Foundation of China (11175080), the Natural Science Founda-
tion of Hunan Province, China (2016JJ5003), the Scientifc Research
Fund of the Hunan Provincial Education Department (12A117).
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