Research article

Received: 27 January 2015, Revised: 01 March 2015, Accepted: 08 March 2015 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bio.2915

A ratiometric fluorescence sensor for Fe3+

based on FRET and PET processes
Yongjun Hu,b Jiaoliang Wang,a,b,c* Liping Longb and Xiaoming Xiaod
ABSTRACT: A rhodamine/coumarin-based ratiometric fluorescent Fe3+ sensor has been designed and synthesized. The sensor
exhibits a good response to Fe3+ ions with high sensitivity, selectivity and a large shift in the emission spectra (>100 nm), which
shows Fe3+-induced FRET OFF–ON and PET ON–OFF behavior. Copyright © 2015 John Wiley & Sons, Ltd.

Keywords: sensor; Fe3+; FRET; rhodamine/coumarin

Introduction emission wavelength changed from 465 to 573 nm and the ratio
between 465 and 573 nm increased 75-fold. Studies have shown
Metals, an absolute requirement for all life, play critical roles in that Fe3+ ion induced FRET OFF–ON and PET ON–OFF behavior.
fundamental processes, including osmotic regulation, metabolism,
catalysis, biomineralization and signaling. Iron is the most abun-
dant transition metal in the human body and has significant roles Experimental
in physiological processes (1). However, an iron imbalance in the
human body, particularly an iron overload, leads to some serious General procedures
diseases, such as Alzheimer’s and Parkinson’s (2). Therefore, the Unless otherwise stated, all reagents were purchased from com-
detection of trace amounts of Fe3+ ions is crucial to track its intra- mercial suppliers and used without further purification. Solvents
cellular metabolism, clarify its roles in living systems and identify were purified and dried by standard methods prior to use.
therapeutic strategies for related diseases. Double-distilled water was used throughout. Melting points were
Recently, optical fluorescent imaging has received wide atten- determined with a Beijing Taike XT-4 microscopy (Beijing Taike
tion in metal ions sensing because of its non-ionizing radiation Instrument Co. LTD., Beijing, China) and were uncorrected.
damage, simple operation and high sensitivity and selectivity Electronic absorption spectra were recorded with a U-3010
(3,4). At the same time, some fluorescent probes for the detection spectrometer. Emission spectra were measured on a Hitachi F4500
of Fe3+ have been successfully prepared (5–7). However, most of fluorescence spectrophotometer. 1H NMR spectra were measured
these probes do not show the level of accuracy required for quan- on an INOVA-400 spectrometer (Varian, Inc.) using TMS as an
titative determination due to factors such as illumination intensity, internal standard. Thin-layer chromatography (TLC) analyses
autofluorescence and instrumental factors. Förster resonance were performed on silica gel plates and column chromatography
energy transfer (FRET), a non-radiation energy transfer process was conducted over silica gel (mesh 200–300), both of which
from an excited donor fluorophore to an acceptor fluorophore, were obtained from Qingdao Ocean Chemicals.
has been widely applied in biological experiments because of its
specific advantages for analyst quantification (8,9). By analyzing
the ratio between the emission intensities of two fluorophores in General procedures for metal ion binding studies
the presence and absence of analytes, the concentration can be Metal chlorate (Hg2+, Ni2+, Pb2+, Ca2+, Mg2+, Cd2+, Cu2+, Co2+, Zn2+,
determined more accurately because there is an in-built correction Na+, K+), nitrate (Ag+, Fe3+) or sulfate (Fe2+, Mn2+) stock solutions
for environmental effects (i.e. canceling out of artifacts due to var-
iations in probe concentration) (10–14). Therefore, a FRET-based
fluorescent probe is more suitable for tracking and quantifying * Correspondence to: J. Wang, College of Chemistry and Chemical Engineering,
University of South China, Hengyang, Hunan, People’s Republic of China.
Fe3+ in living systems. Nevertheless, only a few ratiometric fluores- E-mail: [email protected]
cence sensors for Fe3+ ions have been reported (11,15–18), and the
a
use of most is limited in live systems due to the narrow emission College of Chemistry and Environment Engineering, Hunan City University,
shift, short excitation wavelength and non-biocompatibility (19). Yiyang, Hunan, People’s Republic of China
Therefore, the development of a fluorescence sensor for detecting b
College of Chemistry and Chemical Engineering, University of South China,
and quantifying Fe3+ with high accuracy and sensitivity remains Hengyang, Hunan, People’s Republic of China
challenging.
c
Here, we report a FRET-based coumarin–rhodamine derivative State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan Univer-
(1) by an ethylenediamine spacer as a sensitive ratiometric fluores- sity, Changsha, Hunan, People’s Republic of China
cence sensor for Fe3+. In the absence of Fe3+, the solution of sensor d
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Re-
1 showed blue–green fluorescence from the excited donor search (Hunan Normal University), Ministry of Education, Changsha, Hunan,
fluorophore. Whereas after addition of the Fe3+, the maximum People’s Republic of China

Luminescence 2015 Copyright © 2015 John Wiley & Sons, Ltd.

 Y. Hu et al.

Scheme 1. Synthesis of sensor 1.

were prepared in MeOH. Sensor 1 was dissolved in MeOH at room Synthesis of sensor 1
temperature to give the stock solution (100 μM). Test solutions
Scheme 1 shows the synthetic route for sensor 1.
were prepared by placing 0.5 mL of the sensor stock solution
7-(Diethylamino)-coumarin-3-carboxylic acid 2 (195.6 mg, 0.7
and an appropriate aliquot of each metal stock into a 5 mL volu-
mmol), Dicyclohexylcarbodiimide(DCC) (144.4 mg, 0.7 mmol) and
metric flask, and diluting to 5 mL with methanol. The resulting so-
4-dimethylaminopyridine (DMAP) (5.8 mg, 0.05 mmol) were mixed
lution was shaken well before recording the absorption and
in dried CH2Cl2 (20 mL), and the reaction mixture was stirred for
emission spectra. Unless otherwise noted, for all measurements,
30 min at 0–5°C, then N-(rhodamine-B)lactam-ethylenediamine
the excitation wavelength was at 420 nm, and both the excitation
(340.0 mg, 0.7 mmol) in 2 mL dried CH2Cl2 was added to the reaction
and emission slit widths were 2.5 nm.
mixture; the mixture continue to react for another 3 h at 0–5°C. After
filtration of the precipitate, the solvent was removed in vacuo. The
mixture was dissolved in 25 mL CH2Cl2 and the CH2Cl2 layer was
washed twice with saturated NaCl solution, followed by drying over
anhydrous Na2SO4, then filtered, and the solvent removed in vacuo.
The filtrate was concentrated under reduced pressure, and the
product was obtained by purification using flash chromatography
on silica gel (petroleum ether/ethyl acetate eluent, 5 : 2, v/v). The
appropriate fractions were combined, and the solvents were removed
under vacuum to yield sensor 1 as a yellow solid (313.0 mg, 0.43
mmol) in 61.4% yield. mp: 249–251°C; 1H NMR (400 MHz, CD3OH): δ
= 1.12 (t, 12H), 1.25 (t, 6H), 3.25–3.31 (m, 8H), 3.33–3.53 (m, 4H),
6.28–6.31 (m, 2H), 6.37–6.42 (m, 4H), 6.53 (s, 1H) , 6.78 (t, 1H), 7.06–
7.08 (m, 1H), 7.48–7.51 (m, 3H), 7.86–7.88 (m, 1H), 8.48 (s, 1H), 8.76
(t, 1H); 13C NMR (100 MHz, CD3OH): δ = 13.50, 13.66, 39.54, 41.38,
45.88, 46.60, 49.16, 49.37, 49.59, 49.80, 50.01, 50.23, 50.44, 67.52,
79.24, 79.57, 79.89, 97.90, 99.62, 106.66, 110.07, 110.12, 110.99,
112.08, 124.13, 125.68, 129.96, 130.17, 132.46, 133.07, 134.58, 149.47,
150.68, 154.81, 155.15, 155.57, 159.53, 164.45, 165.77, 171.32 ppm.
ESI-MS m/z: 728.3 [M+H]+. Anal. Calcd for C21H20N2O3: C 72.60,
H 6.79, N 9.62, Found: C, 72.37, H, 6.92; N, 9.58.

Figure 1. UV–vis spectra (a) and fluorescence spectra (b) of sensor 1 (10 μM) in the
absence and presence of different metal ions (20 equiv.). The excitation wavelength Figure 2. The emission spectra of sensor 1 (10 μM) (○) and coumarin 3 (10 μM) (■)
was 420 nm. excited at 420 nm.

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Ratiometric fluorescence sensor for Fe3+ based on FRET and PET

Figure 3. (a) Photographs of solutions of sensor 1 (10 μM) in the absence and presence of different metal ions (20 equiv.). (b) Visual fluorescence changes of 1 (10 μM) in the
absence and presence of different metal ions (20 equiv.). The photos were taken under a handheld UV (365 nm) lamp.

Results and discussion

Design and synthesis
It is well known that rhodamine derivatives with a spirolactam
structure show no absorption and fluorescence at long wave-
lengths (>500 nm), whereas the spiro-ring opening of rhodamine
dye gives rise to a strong new absorption and fluorescence emis-
sion band (20–22). Moreover, the rhodamine fluorophore has a
longer emission wavelength (>550 nm), which is often preferred
as a reporting group for analytes in order to avoid the influence
of background fluorescence (<500 nm) (23,24). To date, some
rhodamine-modified chemosensors or chemodosimeters for
Hg2+, Cu2+, Pb2+, Cr3+ and Fe3+ ions have been developed (23,24).

Figure 4. (a) Absorption and (b) emission spectra of 1 (10 μM) with the addition of Figure 5. Fluorescence ratiometrict response (I573/I465) of sensor 1 (10 μM) upon
3+
Fe (0–10 equiv.). The excitation wavelength was 420 nm. addition of various metal ions (20 equiv.). The excitation wavelength was 420 nm.

Luminescence 2015 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/luminescence
 Y. Hu et al.

(>100 nm) compared with the rhodamine emission band (around

570 nm). Therefore, coumarin and rhodamine dyes are of potential
use in the design of a FRET-based ratiometric fluorescence sensor
with large emission shift. Sensor 1 was readily synthesized in one
step (Scheme 1).

UV–vis/fluorescence spectra of sensor 1

The photophysical properties of sensor 1 in methanol solution were
determined. The absorption spectrum of sensor 1, shown in Fig. 1(a),
closely resembles that of the reference compound 3, and displayed
an absorption band centered at 420 nm, which is attributed to the
coumarin moiety, suggesting that the rhodamine moiety remains
in a ring-closed spirolactam form. Upon irradiation at 420 nm, strong
emission at 465 nm is observed (Fig. 1b), which is attributed to the
fluorescence emitted from the coumarin donor unit; no intramolec-
ular FRET phenomenon can be observed in free sensor 1. However,
Figure 6. Fluorescence ratiometrict response (I573/I465) of sensor 1 (10 μM) upon ad-
3+
dition of Fe ions in the prescent of various metal ions (20 equiv.). The excitation
compared with compound 3, the fluorescence intensity of sensor 1
wavelength was 420 nm. is strongly quenched (Fig. 2), which is attributed to a photoinduced
electron transfer (PET) process from amide to coumarin.

Selectivity studies
The sensing properties of sensor 1 toward various metal ions were
examined using visual and fluorescence spectra. Various metal
ions such as Fe3+, Ag+, Hg2+, Na+, K+, Mg2+, Ca2+, Pb2+, Mn2+, Co2+,
Ni2+, Cu2+, Fe2+, Cd2+ and Zn2+ were used. All titrations studies were
conducted using 10 μM of the sensor and 20 equiv. of metal ions.
Most of the metal ions had no effect on the absorption spectra of
sensor 1 (Fig. 1a), and no color change was observed (Fig. 3a).
However, upon addition of Fe3+, a new absorption band at 555
nm was observed (Fig. 1a), and the color of the solution changed
to red (Fig. 3a), indicative of the formation of the ring-opening state
of rhodamine. The fluorescence spectra (Figs. 1b and 3b) also show
a similar result, which is consistent with that of UV–vis spectra.
Addition of 20 equiv. of Fe3+ results in an obviously enhanced
Figure 7. Job plots for the determination of the stoichiometry of sensor 1 to Fe .
3+
fluorescence at 573 nm (OFF–ON), whereas other ions induce much
The total concentrations of sensor 1 and metal ions were kept constant (100 μM). smaller or no increases in fluorescence.
The excitation wavelength was 420 nm.

UV–vis and fluorescence spectra of sensor 1 titrated with Fe3+

Figure 4 details changes in the absorption and fluorescence of
sensor 1 upon gradual titration of Fe3+. As expected, the addition

3+
Figure 8. Benesi–Hildebrand plots for Fe -bound sensor 1.

Diethylamine–coumarin (25–27) dyes have been used widely as

fluorophores in probe designs owing to their excellent photophysical
properties. More importantly, diethylamine–coumarin dyes have a 3+
Figure 9. (a) The fluorescence spectra of sensor 1 (■), sensor 1 + Fe (▲), and sensor
3+
strong emission band at around 465 nm, with a large spectra shift 1 + Fe + ethylenediamine (○). Excitation at 420 nm.

wileyonlinelibrary.com/journal/luminescence Copyright © 2015 John Wiley & Sons, Ltd. Luminescence 2015
Ratiometric fluorescence sensor for Fe3+ based on FRET and PET

3+
Figure 10. Fe -induced FRET OFF–ON and PET ON–OFF.

of a small amount of Fe3+ induces a new absorption band at 555 absorptivity leads to a large ratiometric fluorescence response at
nm, as well as a visual color change from yellow to pink (Figs. 3a two wavelengths.
and 4a), which was attributed to the absorption peak of the ring- To obtain further insight into the above-proposed FRET signal
opening rhodamine moiety. Upon addition of increasing concentra- mechanism, control experiments were performed. Compound 2
tions of Fe3+, there is a slight enhancement in the fluorescence can also open the fluorescent rhodamine moiety by Fe3+; when
around 465 nm due to inhibition of the quenching PET process. In excited at 500 nm, sensor 1–Fe3+ and compound 2–Fe3+ show
addition, a new emission band peaking at 573 nm appears with similar emission at 573 nm (Fig. 12), however, when excited at
increasing intensity due to promotion of an FRET process from
coumarin to rhodamine when excited 420 nm. There is a shift of
up to 110 nm in the two emission spectra, which enabled almost
complete separation of the emission peaks before and after treat-
ment with Fe3+, so that ratiometric titration can be utilized rather
than mere intensity changes. Indeed, sensor 1 showed remarkable
enhancement of the emission intensity ratio (I573/I460) from 0.014
to 1.05 (75-fold) on Fe3+ treatment (Fig. 5). More importantly, changes
in the visual color and fluorescence in the presence of Fe3+ are also
shown in Fig. 3. Further experiments for Fe3+-selective sensing were
performed with 10 μM sensor 1 in the presence of Ag+, Hg2+, Na+,
K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Fe2+, Cd2+ and Zn2+ (20 equiv.,
respectively). Upon addition of 20 equiv. of Fe3+ ion, the above
solution still displays a distinctly enhanced fluorescence (Fig. 6) .

Sensing mechanism
To confirm the stoichiometry between sensor 1 and Fe3+, Job’s
method (28) was used keeping the total concentrations of Fe3+ 3+
Figure 11. Increasing spectral overlap on Fe binding. The emission of donor cou-
and sensor 1 at 50 μM, but varying the molar fraction of Fe3+ from 3+
marin 3 (■) and the acceptor absorption of 2 (▼) and 2 + Fe (○) were normalized
3+
0.1 to 0.9 (Fig. 7), which gave solid evidence for the formation of a while keeping the peak ratios between free 2 and 2 + Fe unchanged.
1 : 1 complex; the association constant of sensor 1 with Fe3+ ion in
CH3OH was found to be 0.99 × 104 M 1 (Fig. 8). As expected, the
binding of Fe3+ to sensor 1 is reversible, as evidenced by the com-
plete reversal of the fluorescence signal to free sensor 1 when ex-
cess ethylenediamine was added to Fe3+-bound sensor 1 (Fig. 9).
The proposed binding mode of sensor 1 with Fe3+ is shown in
Fig. 10. This proposed mechanism is consistent with the dual chan-
nel emissions of the complex at 465 and 573 nm. Coordination of
Fe3+ with nitrogen atoms may effectively decrease their electron
donating ability, so the quenching PET process from the nitrogen
atoms to the coumarin rings is inhibited, which should result in a
large increase in the fluorescence at 465 nm. However, binding
to Fe3+ induces opening of the fluorescent rhodamine moiety, cor-
responding to intense absorption in the coumarin emission region.
Thus, the extent of the spectral overlap between the energy donor
emission and acceptor absorption is enhanced, due to enhanced
rhodamine energy acceptor molar absorptivity upon Fe3+ ion
binding (Fig. 11). Thus, the dramatic modulation of FRET efficiency Figure 12. The emission spectra of sensor 1 excited 420 nm (▽), 500 nm (▲) respec-
by Fe3+-induced variation in the energy acceptor molar tively, and compound 2 excited 420 nm (■), 500 nm (○) respectively.

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 Y. Hu et al.

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