A novel ratiometric fluorescent probe for detection of Fe3+ by rhodamine-quinoline conjugate

Article abstract of DOI:10.1016/j.jphotochem.2015.05.010

In this study, we have developed a novel ratiometric fluorescence probe (HL) which contains rhodamine and quinoline moiety. In the absence of Fe³⁺, the sensor displays fluorescence emission at 425 nm, in the presence of Fe³⁺, the sensor shows a large fluorescence enhancement at 550 nm in methanol. More importantly, the reversibility of the recognition process of HL is performed by adding a Fe³⁺ bonding agent Na₂EDTA.

Full text of DOI:10.1016/j.jphotochem.2015.05.010

Accepted Manuscript
Title: A novel ratiometric fluorescent probe for detection of
Fe3+ by rhodamine- quinoline conjugate
Author: Jing-can Qin Zheng-yin Yang Guan-qun Wang
PII:
S1010-6030(15)00161-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2015.05.010
JPC 9897
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Journal of Photochemistry and Photobiology A: Chemistry
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Accepted date:
6-3-2015
6-5-2015
9-5-2015
Please cite this article as: Jing-can Qin, Zheng-yin Yang, Guan-qun Wang,
A novel ratiometric fluorescent probe for detection of Fe3+ by rhodamine-
quinoline conjugate, Journal of Photochemistry and Photobiology A: Chemistry
http://dx.doi.org/10.1016/j.jphotochem.2015.05.010
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A novel ratiometric fluorescent probe for detection of Fe3+
by rhodamine- quinoline conjugate
Jing-can Qin, Zheng-yin Yang* Guan-qun Wang
College of Chemistry and Chemical Engineering, State Key Laboratory of Applied
Organic Chemistry, Lanzhou University, Lanzhou 730000, P.R. China
**Corresponding author. Tel.: +86 931 8913515; Fax: +86 931 8912582; e-mail:
yangzy@lzu.edu.cn (Z.Y. Yang)
Graphical abstract
Upon addition of Fe3+, the fluorescence of rhodamine (at 550 nm) and quinoline
(at425 nm) enhanced simultaneously. Fluorescence resonance energy transfer process
(FRET), coupled with photoinduced electron transfer (PET) process are proposed to
explain the observed spectral.

Highlights
1. The probe displayed selectivity toward Fe3+ even coexistent with other metal ions.
2. FRET, coupled with PET process are proposed to explain the observed spectral
3. The detection limit of Fe3+ reached at 8.3×10-7 m
Abstract: In this study, we have developed a novel ratiometric fluorescence probe
(HL) which contains rhodamine and quinoline moiety. In the absence of Fe3+, the
sensor displays fluorescence emission at 425 nm, in the presence of Fe3+, the sensor
shows a large fluorescence enhancement at 550 nm in methanol. More importantly,
the reversibility of the recognition process of HL is performed by adding a Fe3+
bonding agent Na2EDTA.
Keywords: fluorescence sensor; rhodamine; quinoline; Fe3+; reversibility

1. Introduction
Fluorescent probes with high selectivity, sensitivity and real-time detection for
various metal ions have attracted considerable attention because of their fundamental
roles in environmental research and analytical fields [1-5]. Fe3+ is one of the most
essential metal ions and plays a pivotal role for many living organisms by
participating in a wide range of metabolic processes, such as cellular metabolism,
enzyme catalysis and as an oxygen carrier in hemoglobin. Less iron in the body has
been reported to be linked to diabetes, anemia, liver and kidney damages, and heart
diseases, On the other hand, the superfluous ingestion of Fe3+ is responsible for an
increased incidence of certain cancers and organ dysfunction [6-15] Hence, in order to
protect the human health, it is important to monitor the concentration levels of iron
ions in the environment and many scientific fields.
. Rhodamine and its derivatives are a kind of classic fluorescent dye, which is
extensively suitable to act as fluorescent chromophores because of their excellent
photo physical properties such as high extinction coefficients, excellent quantum
yields and relatively long emission wavelengths in the visible region [16-18]. Thus,
rhodamine-based probes have been attracting the interest of scientists and widely used
to the detection of various metal ions [19-22]. In general, the design of
rhodamine-based probe is based primarily on the ring-opened reaction of the
rhodamine spirolactam which is triggered by metal ion. More specifically, the free
rhodamine-based probe in which the rhodamine core is in the ring closed isomeric
form is colorless and non-fluorescent, whereas, upon addition of metal ions, the

ring-opening of the spirolactam induced by the analyte gives rise to pink color and a
strong characteristic fluorescence emission. Thus, the sensor base on the special
optical signal which is emitted from rhodamine or its derivatives would develop a
fluorescent probe for detection of metal ion [23-27].
Out of consideration of these circumstances, we have designed and synthesized a
novel rhodamine-based probe (HL) for Fe3+ in which rhodamine and quinoline
moietiesare linked by glyoxal. In the absence of Fe3+ the sensor exhibits fluorescence
,
emission at 425 nm, which belong to quinoline moieties, upon addition of Fe3+, the
sensor shows a large fluorescence enhancement at 550 nm, which was ascribed to the
ring opened rhodamine moiety. Moreover, the addition of Na2EDTA to a mixture of
HL and Fe3+ result in diminution of the fluorescence intensity at 550 nm.
2. Experimental
2.1. Apparatus and materials
All chemicals were obtained from commercial suppliers and used without further
1
purification. H NMR spectra were measured on the JNM-ECS 400MHz instruments
using TMS as an internal standard. Bruker esquire 6000 spectrometer, Perkin Elmer
Lambda 35 UV-Vis spectrophotometer, Hitachi RF-5301 spectrophotometer equipped
with quartz cuvettes of 1 cm path length, Beijing XT4-100x microscopic melting
point apparatus. IR spectra were obtained in KBr discs on a Therrno Mattson FT-IR
spectrometer in the 4000–400cm-1 region.

2.2. Analysis
The solutions of metal ions (5mM) were prepared from NaNO3, KNO3, LiNO3,
Ca(NO3)2·4H2O, Mg(NO3)2·6H2O, Al(NO3)3·9H2O, CrCl3·6H2O, FeCl3·6H2O,
FeCl2·4H2O, Co(NO3)2·6H2O, Ni(NO3)2·6H2O, Cu(NO3)2·3H2O, Pb(NO3)2,
Zn(NO3)2·6H2O, Cd(NO3)2·4H2O, Mn(NO3)2·4H2O, BaCl2·2H2O respectively, and
were dissolved in methanol or water. A stock solution of HL (1 mM) was prepared in
DMSO, and then the stock solution was diluted 100 times under test. For fluorescence
measurements, the excitation slit width was 5 nm, the emission slit width was 3 nm.
2.3. Synthesis
Rhodamine 6G hydrazide (Fig.S1) was synthesized according to the method
reported [28]. The Schiff base was synthesized according to the route as shown in
Scheme 1.
2.2.1. Synthesis of compound RG1 [29-30]
Rhodamine hydrazide (0.86 g, 2 mmol) was dispersed in 20 mL ethanol, excess
of glyoxal (40%, 1.16 g, 8 mmol) was added, and the mixture was stirred for 8 h at
room temperature. The precipitate produced was filtered and washed 3 times with 10
mL cold ethanol. After drying under reduced pressure, the reaction yielded 0.77g RG1
1
as yellow solid. Yield: 76.3%, HNMR (400 MHz; CDCl3) (Fig.S2) δ(ppm) = 9.40 (d,
1H, J=7.5 Hz, CH=O), 8.05 (dd, 1H, Ar--H), 7.54 (dd, 2H, Ar--H), 7.27 (d, 1H, J=7.5
Hz, CH=N), 7.18 (m, 1H, Ar--H), 6. 37 (s, 2H, xanthene--H), 6.24 (s, 2H, xanthene--H),
3.54 (s, NHCH2CH3), 3.24-3.29 (q, J=7.1 Hz, 4H, NCH2CH3), 1.87 (s, 6H,
xanthene--CH3) 1.29-1.32 (t, J=7.1 Hz, 6H, NCH2CH3).

2.2.2. Synthesis of 2-(Quinolin-8-yloxy) acetohydrazide [31-32]
To a solution of 8-hydroxy quinoline (0.5 mol, 7.26g) in dry acetone (150 mL),
ethyl chloroacetate (0.60 mol, 6.4mL) were added followed by anhydrous K2CO3 (1.0
mol, 13.8g), After 48 hours, the reaction mixture was filtered and the filtrate was
evaporated to give liquid ester. The ester was directly used for next step without
further purification. Hydrazine hydrate (80%, 10 equiv.) was added dropwise to an
ethanol solution (40 mL) of Ethyl (quinolin-8-yloxy) acetate. The mixture was
refluxed under stirring for 24 h. Then the solvent was concentrated under reduced
pressure and let stand overnight in refrigerator. A white needle crystal was observed.
The final product was filtered, dried and recrystallized from ethanol. Yield: 55%, Mp:
1
140-141°C, H NMR (Fig.S3): (400 MHz; CDCl3) δ= 3.98 (s, 2H9), 4.84 (s, 2H7),
7.15 (d, J=7.2 Hz, H1), 7.44~7.52 (m, H2, H3, H5), 8.91 (d, J=4.2 Hz, H6), 8.17 (d,
J=8.3Hz, H4), 9.65 (s, H8).
2.2.3. Synthesis of 1-(Rhodamine6G)-2-(α-(8-quinolyoxy) acetic) ethlyendihydrazide
(HL)
An ethanol solution (20 mL) of 2-(Quinolin-8-yloxy) acetohydrazide (1 mmol,
0.22 g) was added to another ethanol (20 mL) containing RG1 (1 mmol, 0.46 g). Then
the solution was refluxing for 12 h under stirring, the precipitate produced was filtered
and washed 3 times with 10 mL hot ethanol. After drying under reduced pressure, the
reaction afforded HL as pale yellow solid. 1H NMR (400 MHz; CDCH3) (Fig.S4)
δ=12.44 (s, 1H), 9.16~ 8.98 (m, 1H), 8.17~8.22 (m.1H), 7.91~8.01 (m, 2H),
7.37~7.61 (m, 6H), 7.22~7.24 (m, 1H), 6.95~7.05(m, 1H), 6.37(s, 2H), 6.31(s, 2H),

4.85(s, 2H), 3.47(s, 2H), 3.22 (q, 7.1 Hz, 4H), 1.80 ~ 1.97 (s, 6H),1.26~ 1.39 (t, J=7.1
Hz, 6H,). IR (KBr, cm-1) (Fig.S5): 3426, 1703, 1620, 1573, ESI/MS (Fig.S6) [M+1]+
668.23, Calcd : 668.29, Elemental analysis: C39H37N7O4, Found: C, 70.22; H, 5.29;
N, 14.76. Calcd: C, 70.15; H, 5.58; N, 14.68.
3. Results and discussion
3.1 General information
The detection limit was calculated according to the following the equation:
detection limit 3σ/λ, where σ is the standard deviation of blank measurements, and λ
is the slope between intensity versus sample concentration [33-34].
The binding constant value was determined from the emission intensity data
following the modified Benesi–Hildebrand equation [35-36] (A):
where F0, Fx, and Fmax were the emission intensities (at 550 nm) of the organic moiety
considered in the absence of metal ion, at an intermediate metal ion concentration,
and at a concentration of complete interaction, respectively, and where K was the
binding constant concentration.
3.2 UV-Vis titrations
To investigate the binding property of HL toward Fe3+, we initially measured the
UV-vis spectra of HL as a function of the concentration of Fe3+ in methanol. As
shown in Fig.1, the spectra of free HL only showed an absorption band centered at
300 nm, which should be assigned to the quinoline group, and that the characteristic
absorption band of Rhodamine 6G at about 530 nm was not observed which

demonstrated that the rhodamine core was in spirolactam form in the absence of Fe3+.
However, upon addition of Fe3+ to solution of HL, the absorption band at 300 nm
slightly increased while two new absorption bands appeared at 350 nm, 530 nm with
increasing intensity. The new absorption band at 530 nm was ascribed to the ring
opened rhodamine moiety [37-38], which indicated that the interaction of HL with
Fe3+ can triggered the formation of the ring-opened form of HL from the spirolactam
form.
3.3. Selectivity studies and effects of metal ions
The fluorometric behaviour of HL was further investigated upon addition of
several metal ions such as Na+, K+, Mg2+, Ca2+, Pb2+, Cr3+, Mn2+, Fe2+, Ni2+, Co2+,
Fe3+, Cu2+, Ba2+, Cd2+, Li+ and Zn2+ in methanol. As shown in Fig.2, the sensor
without Fe3+ displayed fluorescence emission at 425 nm and the characteristic
emission of rhodamine at 550 nm not appeared, which also demonstrated the
rhodamine core was in the ring closed isomeric form. Upon addition of Fe3+, the
fluorescence emission of the sensor showed slight enhancement at 425 nm and
remarkable fluorescence enhancement at 550 nm, the former emission was ascribed to
quinoline moieties, the latter emission should belong to the characteristic signal of the
ring-opening of the spirolactam which further confirmed that the addition of Fe3+ ions
promoted the ring-opened reaction of the rhodamine spirolactam [29, 37-39]. In
addition, the sensor did not give any observable response for other relevant metal ions,
it meant the sensor HL, having predominant recognition and selectivity, is of a
good potential in the detection of Fe3+.

In order to further validate the conjugation of L-Fe, Job's method for absorbance
measurement was carried out in methanol. The total concentration of HL and Fe3+ was
100μM. XL= ([HL]/ ([Mn+] + [HL]. As shown in Fig. 3, the maximum point appeared
at a mole fraction of 0.5, the result suggested it was possible a 1:1 stoichiometry of
the binding mode of HL and Fe3+ which was further confirmed by the appearance of a
peak at m/z 757.16 assignable to [HL+Fe3++Cl--H+]+ in the ESI/MS (Fig.S7). Since
the formation of a 1: 1 ligand-metal complex was confirmed by Job’s plot analysis
and ESI/MS, in combination with the fluorescence titration (Fig. 4), the binding
constant of HL with Fe3+ also could be estimated using the Benesi–Hildebrand
equation (A), the slope of the line of best fit give the Kas 1.67×104 (Fig. S8).
3.5. The practical applicability of HL
To check the practical applicability of HL as a selective fluorescent sensor for
Fe3+, two experiments should be carried out. One was competition experiment, the
systems of other metal ions and Fe3+ coexisted was examined in methanol. As shown
in Fig. 5, we found that all the coexistent metal ions had no obvious interference with
the detection of Fe3+ except in the case of Cu2+, although the fluorescence intensity is
low but detectable. The other was the investigation of reversibility which also a
prerequisite in developing fluorescent probe for practical application. The reversibility
of the recognition process of HL was performed by adding Na2EDTA (a bonding
agent). As shown in Fig. 6, the addition of Na2EDTA to a mixture of HL and Fe3+
resulted in diminution of the fluorescence intensity at 550 nm, which indicated the

regeneration of the free sensor HL. Upon the addition of Fe3+, the fluorescence
intensity of HL showed significant fluorescence enhancement at 550 nm again.
Additionally, the theoretical detection limit of Fe3+ reached at 8.3×10-7 m which
was calculated based on the fluorescence titration (Fig.S9). Thus, all of these
suggested that the receptor can be used as a selective fluorescent sensor for Fe3+ in
environmental analysis and analytical chemistry.
3.5. The sensing mechanism
According to the theory of FRET, the fluorescence emission of quinoline should
decrease when such energy transfer takes place, however, the fluorescence intensity
(at 425nm) of HL showed slightly increased instead of decreased as shown in Fig. 4.
In order to explain the phenomenon, Based on the above experiments and some
reported fluorescent probes which shared some similarities to HL [40-48], the sensing
mechanism is schematically illustrated in Scheme 2. For quinoline moieties, the N
atom of quinoline ring with an unshared electron pair partly quenched the
fluorescence emission of quinoline in the absence of Fe3+ due to a photoinduced
electron transfer (PET) process. Up addition of Fe3+, the coordination of N atom and
Fe3+ resulted in the efficient inhibition for the PET process, as a result, the
fluorescence increased. For rhodamine moieties, the fluorescence intensity of HL also
increased which was attributed to the FRET from conjugated quinoline to rhodamine
by the addition of Fe3+. Just because when such energy transfer takes place, the
inhibited PET process also occured in quinoline moieties, the fluorescence intensity at
425nm was enhancement, not diminished comparing with the fluorescence intensity

of the free sensor. And, in short, Fluorescence resonance energy transfer process
(FRET), coupled with photoinduced electron transfer (PET) process are proposed to
explain the observed spectral that the fluorescence of rhodamine (at 550 nm) and
quinoline (at425 nm) enhanced simultaneously.
4. Conclusion
In summary, we have designed and characterized a novel ratiometric
chemosensor for Fe 3+ based on an asymmetric bis-Schiff base in which rhodamine
and quinoline moietieswere linked by glyoxal. The probe displayed a high sensitivity
and selectivity toward Fe3+ even coexistent with other metal ions. In addition, because
of its good reversibility and lower detection limit, the sensor may find potential
applications to detect micromolar concentrations of Fe 3+ in both biological system
and the environment.
Acknowledgments
This work is supported by the National Natural Science Foundation of China
(81171337), Gansu NSF (1308RJZA115).

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Figure captions
Scheme 1 Reagents and conditions: (a) ethyl chloroacetate, acetone, anhydrous
K2CO3, reflux, 48 h; (b) EtOH, N2H4·H2O, 80℃,24h (c) EtOH, N2H4·H2O, reflux,
20h; (d) EtOH, glyoxal, 40%, at room temperature, 8 h (e) EtOH, reflux, 12h
Scheme 2 Proposed mechanism for detection of Fe3+ by HL
Fig.1. Absorption spectra of HL (10.0 μM) as a function of the concentration of Fe3+
(0-7.5 equiv.) in methanol at room temperature
Fig.2. Fluorescence spectra of HL (10 μM) upon the addition of metal salts (2.5 equiv.)
of Li+, Na+, K+, Ca2+, Mg2+, Cu2+, Co2+, Mn2+, Ni2+, Zn2+, Ba2+, Fe2+, Cd2+, Hg2+, Pb2+,
Fe3+ and Cr3+ in methanol. Excitation wavelength was 350nm. Inset: color of HL and
HL+Fe3+ system under UV lamp.
Fig. 3 Job’s plot for determining the stoichiometry of HL and Fe3+ in methanol (XL=
[HL]/([Fe3+] + [HL]), the total concentration of HL and Fe3+ was 100 μM, the
absorption band centered at530nm).
Fig.4. Fluorescence spectra of HL (10 μM) in methanol, upon the addition of Fe3+ (0-2
equiv.) with an excitation of 350nm. Inset: color of HL and HL+Al3+ system under
UV lamp.
Fig. 5 Fluorescence intensity ( at 550 nm) of HL and its complexation with Fe3+ in the
presence of various metal ions in methanol, Red bar: HL (10.0 μM) and HL with 2.5
equiv. of Li+, Na+, K+, Ca2+, Mg2+, Cu2+, Co2+, Mn2+, Ni2+, Zn2+, Ba2+, Fe2+, Cd2+,
Hg2+, Pb2+ and Cr3+ stated. Green bar: 10.0 μM of HL and 2.5 equiv. of Fe3+ with
2.5equiv of metal ions stated (λex = 350 nm)

Fig. 6 Fluorescence intensity (at 550 nm) of HL (10.0 μM) as a function of Fe3+
concentration in methanol 1: HL, 2: 3:
HL+Fe3++EDTANa2,
HL+Fe3++EDTANa2+Fe3+, 4: HL+Fe3+ (Ex =350 nm)

Scheme1

Scheme 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5
Fig. 6