New rhodamine-based turn-on and colorimetric probe for copper(II) ion with high selectivity and sensitivity

Article abstract of DOI:10.1016/j.ica.2014.04.020

A new rhodamine-based derivative fluorescent probe (L) was synthesized as a highly selective and turn-on fluorescent probe for quantitative detection of Cu²⁺ exhibiting an almost 200-fold fluorescence increase. Moreover, the X-ray single crystal structure of the probe had also been measured. Besides, the fluorescent probe can also be considered as a good OFF-ON switch for Cu²⁺ with naked-eye colour change from colorless to pink. The 1:1 binding ratio between chemosensor L and Cu²⁺ was investigated using Job's plot method, ¹H NMR titration and ESI-MS. The probe exhibited excellent selectivity and sensitivity toward Cu²⁺ with little interference, meanwhile the detection limit of Cu²⁺ was at a quite lower micromolar level.

Full text of DOI:10.1016/j.ica.2014.04.020

Accepted Manuscript
New rhodamine-based turn-on and colorimetric probe for copper(II) ion with
high selectivity and sensitivity
Yan Fu, Qiao-Feng Tian, Yan-Qin Guo, Shuang-Quan Zang
PII:
S0020-1693(14)00225-4
http://dx.doi.org/10.1016/j.ica.2014.04.020
ICA 15959
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 December 2013
15 April 2014
19 April 2014
Please cite this article as: Y. Fu, Q-F. Tian, Y-Q. Guo, S-Q. Zang, New rhodamine-based turn-on and colorimetric
probe for copper(II) ion with high selectivity and sensitivity, Inorganica Chimica Acta (2014), doi: http://dx.doi.org/
10.1016/j.ica.2014.04.020
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New rhodamine-based turn-on and colorimetric probe
for copper(II) ion with high selectivity and sensitivity
Yan Fua, Qiao-Feng Tianb, Yan-Qin Guoa, Shuang-Quan Zanga*
aCollege of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, P. R.
China
bSias International University, Zhengzhou 450001, P. R. China
*Corresponding authors: Tel: (+86)-371-67780136; Fax: (+86)-371-6778 0136.
E-mail: zangsqzg@zzu.edu.cn (S.-Q. Zang).
A new rhodamine-based derivative fluorescent probe (L) was synthesized as a highly
selective and turn-on fluorescent probe for quantitative detection of Cu2+ exhibiting an
almost 200-fold fluorescence increase. Moreover, the X-ray single crystal structure of the
probe had also been measured. Besides, the fluorescent probe can also be considered as a
good OFF–ON switch for Cu2+ with naked-eye colour change from colorless to pink. The
1:1 binding ratio between chemosensor L and Cu2+ was investigated using Job’s plot
1
method, H NMR titration and ESI-MS. The probe exhibited excellent selectivity and
sensitivity toward Cu2+ with little interference, meanwhile the detection limit of Cu2+ was
at a quite lower micromolar level.
Key words: rhodamine, turn on, fluorescent probe, naked-eye detection, Cu2+
1. Introduction
In recent years, the design and synthesis of highly selective fluorescent probe for
detecting transition and heavy metal ions is one of the most important research areas in
chemistry because of their potential applications in clinical biochemistry and
environmental research [1]. Among various transition and heavy metal ions, Cu2+ ranks
the third most abundant in the human body besides zinc and iron, and plays an important
role in the areas of biological and environmental systems [2–6]. However, copper is
1

highly toxic to some organisms such as many bacteria and viruses if their levels exceed
cellular needs, elevated concentrations of copper hamper the self-purification capability
of the sea or rivers and destroy the biological reprocessing systems in water. Alteration in
the cellular homeostasis of copper ions was reported to be connected with some serious
neurodegenerative diseases such as Alzheimer’s disease and prion diseases [7–10].
Accordingly, monitoring the concentration of Cu2+ in environmental samples is of
considerable significance for environment protection and human health.
In the past decade, considerable attention has been focused on the design of fluorescent
chemosensors for Cu2+ due to the highly sensitive, quick, and nondestructive advantages
of the fluorescent method [11, 12]. Most of the classic and early-reported cation probe,
however, generally undergo fluorescence quenching upon the binding of Cu2+ due to the
fluorescence quenching nature of paramagnetic Cu2+ and give a “turn-off” signal [13–19].
Moreover, the selectivity for Cu2+ over other metal ions, such as Fe3+, Pb2+ and Hg2+, is
not very satisfactory for some of the probes [20–25]. To overcome these disadvantages,
fluoroionophores which show a selective response to Cu2+ by a copper-amplified
fluorescence emission have been well developed in recent years [26, 27].
Rhodamine framework seems to be an ideal model to construct “turn-on” fluorescent
probe because of it’s a spirolactam ring-opening system [28–32]. Moreover, rhodamine
derivatives have excellent spectroscopic properties such as long absorption and emission
wavelength, large molar extinction coefficient, and high fluorescence quantum yield [33,
34]. Recently, a spirolactam to ring-opening amide process was utilized for the detection
of metal ions [35]. In this paper, we reported the design and synthesis of a new
rhodamine spirolactam-based chemosensor (L). It showed a “turn-on” fluorogenic and
colorimetirc recognition functions toward Cu2+ in N, N-dimethylformamide (DMF)
aqueous solution with remarkably high sensitivity and selectivity. This fluorescent probe
may play a critical role in the detection of Cu2+ in environmental pollution.
2. Experimental
2.1 Materials and measurement
2

All the materials for the syntheses were commercially available and used as received
without further purification. DMF was of analytical reagent grade. All aqueous solution
was prepared using freshly deionized water. The solutions of the metal ions were
1
prepared from their chloride salts except that Ag+ was prepared from its nitrate salts. H
13
NMR and C NMR spectra were recorded on a Bruker DRX spectrometer operating at
400 MHz. UV-visible absorption spectra were recorded on a TU-1900 double-beam
UV−Vis Spectrophotometer. Fluorescence emission spectra were recorded on a
FluoroMax-4 Spectrofluorometer (Horiba Jobin Yvon Inc., France), excitation and
emission slits of 5.0 nm were used for the measurements of fluorescence. Elemental
analyses (C, H, N) was conducted using a Perkin-Elmer 240 elemental analyzer.
Electrospray ionization time-of-flight mass spectrometry (MS) spectra were measured on
a LC‐MSD‐Trap‐XCT instrument. Melting points were determined on an X-4
microscope electron thermal apparatus.
2.2 Synthesis of the fluorescent probe L
The rhodamine 6G hydrazide was prepared according to the literature method [36]. To
a solution of methanol (15 mL) containing rhodamine 6G hydrazide (214.0 mg, 0.5 mmol)
and 3-formyl-2-hydroxy-benzoic acid methyl ester (90.1 mg, 0.5 mmol), a few drops of
glacial acetic acid were added into the mixture. The resulting solution was stirred at
68 °C refluxed for 2 h, and light pink precipitation was obtained after the solution cooled
to room temperature. The precipitation was filtered off, washed with a mixture of
methanol and diethyl ether (1:1, v/v) several times and dried under vacuum. The desired
1
compound L was obtained in 86 % yield (253.8 mg). m.p: 246-248 °C. H NMR (400
MHz, CDCl3, ppm) δ = 11.10 (s, 1H), 8.74 (s, 1H), 8.05 (m, 1H), 7.91, (d, 1H), 7.75 (m,
1H), 7.50 (m, 2H), 7.07 (m, 1H), 6.81 (t, 1H), 6.44 (s, 2H), 6.36 (s, 2H), 3.91 (s, 3H),
3.52 (s, 2H), 3.23 (m, 4H), 1.90 (s, 6H), 1.33 (t, 6H). 13C NMR (400 MHz, CDCl3, ppm)
δ = 169.58, 165.05, 159.87, 152.47, 151.34, 147.57, 142.80, 133.55, 132.91, 131.66,
128.52, 128.26, 127.64, 123.77, 123.44, 122.76, 118.61, 118.01, 113.86, 105.80, 96.84,
65.75, 52.24, 38.36, 16.71, 14.74. Anal. Calcd for C35H34N4O5: C, 71.17, H, 5.80, N, 9.49.
Found: C, 71.21, H, 5.76, N, 9.51,. ESI-MS: [M + H]+ 591.3.
3

2.3 X-ray crystallographic data collection and refinement of L
Single-crystal X-ray diffraction data for L were conducted on a Bruker SMART APEX
CCD diffractometer [37] with graphite-monochromatized Mo Kα radiation (λ = 0.71073
Å) at room temperature using the ω-scan technique. Lorentz polarization and absorption
corrections were applied. The structures were solved by direct methods with SHELXS-97
[38] and refined with the full-matrix least-squares technique using the SHELXL-97 [39]
program. All nonhydrogen atoms were refined anisotropically. The hydrogen atoms of
the organic ligand were included in the structure factor calculation at idealized positions
using a riding model and refined isotropically. The hydrogen atoms of the solvent water
molecules were located from the difference Fourier maps, then restrained at fixed
positions and refined isotropically. Crystallographic data for L was provided in Table S1.
Crystallographic data has been deposited with the Cambridge Crystallographic Data
Center. CCDC reference number: 973784.
3. Results and Discussion
Our research involved the design, synthesis, and evaluation of the fluorescent probe L.
As shown in Scheme 1 (Supporting Information), L was easily synthesized by simple
1
reaction, and the characterization of the new compound H NMR (Fig. S1), 13C NMR
(Fig. S2) was presented in Supporting Information. Single crystals of L suitable for X-ray
diffraction were obtained by slow evaporation from a solvent mixture of tetrahydrofuran
and DMF undisturbed at room temperature after about two weeks. X-ray single crystal
structure for L was shown in Fig. 1. L crystallized in a monoclinic system with space
group P21/c.
[inset Fig. 1]
3.1. The fluorescence emission spectroscopy of L to Cu2+
To investigate the fluorescence selectivity toward special metal ions, the fluorescence
selectivity experiments of various metal (Cu2+, Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Zn2+,
Al3+, Cd2+, Ba2+, Ag+, Hg2+, Fe2+, Ni2+ and Pb2+) were carried out in DMF/H2O (95:5). As
4

shown in Fig. 2, only Cu2+ has been successful to perturb the emission signal of L, but
other cations can’t affect the emission profiles. Upon the addition of 10 equiv of Cu2+, a
‘‘turn-on’’ ratio over 200-fold was triggered because of the rhodamine ring-opening
process (Scheme 2). In addition, according to the histogram (Fig. 2, inset), the high
selectivity of L for Cu2+ over other metal ions were indicated obviously.
[inset Fig. 2 and scheme 2]
Fluorescence titration experiment (Fig. 3) using L and Cu2+ was performed in
DMF/H2O (95:5) at room temperature. Upon binding with ever-increasing Cu2+, the
fluorescence intensity was obviously enhanced. The emission spectrum of L, which was
excited at 521 nm, exhibited an emission maximum at 559 nm with a low quantum yield
(Φ = 0.0024) by using rhodamine 6G (Φ = 0.95) in ethanol as the reference [40]. Upon
coordinating to Cu2+ (50 µM), the fluorescence intensity of emission band at 559 nm with
a quantum yield (Φ = 0.473) was largely enhanced. From the changes in Cu2+ dependent
fluorescence intensity the detection limit was estimated to be 9.12 × 10−7 M−1 (Fig. S3).
Therefore, the probe L was sensitive enough to detect Cu2+.
[inset Fig. 3]
3.2. The UV–Vis absorption spectroscopy response of L to Cu2+
To get further insight into the binding of Cu2+ with L, the absorption spectra of L upon
titration with Cu2+ was recorded (Fig. 4). Upon addition of Cu2+, a new absorption band
at 528 nm appeared with increasing intensity, which induced a clear color change from
colorless to pink. Similar to other rhodamine spirolactam derivatives, L formed a nearly
colorless and fluorescence inactive in DMF/H2O (95:5), indicated that the spirolactam
form predominantly existed [41]. The characteristic peak of the spirocycle carbon of L
near 66 ppm in the 13C NMR spectrum (Fig. S2) [42] and X-ray single crystal structure of
L (Fig. 1) also supported this consideration. Upon interaction with Cu2+, the spirolactam
ring of L was opened and caused a significant enhancement of absorbance. Moreover,
other metal cations exerted a negligible effect on the absorption response for L. The
5

association constant (K) for L binding to Cu2+ was determined from the absorption
titration data following the reported method [43]. The association constant (K) of L with
Cu2+ was (2.439 0.456) × 104 (R = 0.999) from linear least-squares analysis obtained by
Benesi-Hilderbrand plot (Fig. S4), which unambiguously demonstrated that L had a
potent binding affinity for Cu2+.
[inset Fig. 4]
3.3. Coordination mode between L and Cu2+
Job’s method for the fluorescence intensity was applied to determine the coordination
mode of L to Cu2+, by keeping the sum of the initial concentration of copper ion and L at
10 µM and the molar ratio of copper ion changing from 0 to 1 [44, 45]. The results
showed the 1:1 stoichiometry of the complex between Cu2+ and L (Fig. 5). To further
1
confirm the stoichiometry between L and Cu2+, the H NMR titration (Fig. S5) and
ESI-MS spectrometry (Fig. S6) were both undertaken. Upon the addition of 1 equiv Cu2+,
the signals for these active protons (‒OH and ‒CH=N‒) shifted to downfield in certain
degrees in comparison to those of L. The proton of ‒OH shifted from 10.99 to 11.10 due
to the coordination “O” to “Cu2+”and the proton of ‒CH=N‒ shifted from 8.86 to 8.92
due to the coordination “N” to “Cu2+”, the spirolactam ring of L was opened, which
associated with the enhancement of fluorescent intensity in complex L + Cu2+. And these
active protons stopped changing after addition of another 1 equiv Cu2+, the result also
supported that L and Cu2+ formed a 1:1 ratio complex. ESI-MS measurements were
carried out on by using methanol as mobile phase, a main peak at m/z 591.3
corresponding to [L + H]+. After the addition of 1 equiv of CuCl2, a peak at m/z 755.3
appeared, coinciding exactly with that for the species of [L + Cu2+ + 2Cl− + CH3OH +
H]+ which confirming the 1:1 formation of L−Cu2+ complex, too.
[inset Fig. 5]
3.4. Competition experiment
A very important parameter to evaluate the performance of a fluorescence probe is the
ability to detect a specific cation in the vicinity of other competing ions. To further testify
the specific selectivity of L for Cu2+, the fluorescence intensity of L in the presence of
6

Cu2+ mixed with 10 equivalents other metal ions was carried out in DMF/H2O (95:5), as
shown in Fig. 6, no significant fluorescence intensity changes were observed.
Furthermore, the anion responses to the detection systems were also investigated.
Sufficient fluorescence enhancement was observed for probe L after the addition of Cu2+
−
−
−
in the presence of different anions (F−, Cl−, Br−, I−, AcO−, ClO4 , H2PO4 , HSO4 ) (Fig.
S7). All these results indicated that the probe L can be used as a good selective
fluorescent probe for Cu2+ in the presence of a wide range of the environmentally
relevant ions.
[inset Fig. 6]
3.5 The influence of solvent
The fluorescence behaviour of chemosensors L in different ratios of DMF/H2O
solution was investigated as shown in Fig. S8 in Supporting Information. Upon adding of
7 equiv Cu2+ and photoexcitation at 521 nm, the dilute aqueous solution of L hardly
showed any fluorescence and then sharply enhanced when the DMF fraction was over
95%. In this work, DMF/H2O (95:5) was used throughout the experiment.
3.6. The reversibility of the binding of Cu2+ by L
Reversibility of target ion binding is a momentous character to a fluorescent probe.
Consequently, we studied the chemical reversibility of the binding of L to Cu2+ in
DMF/H2O (95:5). As seen by the naked eye, the pink color of the solution of L-Cu2+
immediately disappeared upon the addition of excess Na2S, the fluorescent intensity
almost recovered to the original level (Fig. 7). The phenomenon could also be attributed
to the fact that Cu2+ can coordinate with sulfide anions to form the stable species, CuS,
with the low solubility product constant (KSP = 1.27 × 10−36) [46], which indicated that
addition of sulfide anions to the L-Cu2+ ensemble resulted in the release of the free L.
[inset Fig. 7]
4. Conclusion
In summary, we have synthesized a new fluorescent probe (L) derived from rhodamine
6G, which showed “turn-on” response to Cu2+ based on rhodamine ring-opening approach,
the X-ray single crystal structure of the probe has also been measured. The complex of
7

L-Cu2+ with a 1:1 stoichiometry was formed on the treatment with Cu2+. L exhibited an
excellent selectivity for Cu2+ with nearly 200-fold fluorescence enhancement compared
with other metal ions. In addition, the fluorescence response of the probe could be
switched “ON” and “OFF” by adding Cu2+ and Na2S alternately with obvious colour
change observed by naked eye. Furthermore, the chemosensor L has highly sensitive
property for Cu2+ with a low detection limit. This fluorescent probe L may make
contribution to solving the problem of environmental pollution caused by Cu2+.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
20901070 and 21371153), the Program for Science & Technology Innovation Talents in
Universities of Henan Province (No. 13HASTIT008) Key Scientific, Technological
Project of Henan Province (132102210411) and Zhengzhou University (P. R. China).
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Fig. 1. X-ray single crystal structure for L.
Fig. 2. Fluorescence emission spectra of L (5 µM) in the presence of various metal ions
(10 equiv for 1. Cu2+, 2. Na+, 3. K+, 4. Mg2+, 5. Ca2+, 6. Mn2+, 7. Co2+, 8. Zn2+, 9. Al3+, 10.
Cd2+, 11. Ba2+, 12. Ag+, 13. Hg2+, 14. Fe2+, 15. Ni2+ and 16. Pb2+) in DMF/H2O (95:5). λ
ex
= 521 nm.
Fig. 3. Fluorescence spectra of L (5 µM) in the presence of increasing concentration of
Cu2+ (0–7 equiv). The excitation wavelength was 521 nm. Inset: (a) Linear relationship
between the emission intensity at 559 nm and Cu2+ concentration from 0 to 15 µM; (b)
Fluorescence change of L (5 µM) in the absence and presence of Cu2+ (50 µM) under UV
light with an excitation of 365 nm.
Fig. 4. Absorption spectra of L (5 µM) with addition of different concentrations of Cu2+
(0–5 equiv) in DMF/H2O (95:5). The inset showed the colour change under visible light,
in the absence and presence of Cu2+ (25 µM) respectively.
Fig. 5. Job’s plot method between chemosensor L and Cu2+.
Fig. 6. The fluorescent response of L (5 µM) upon addition of different metal ions at 559
nm. The black bars represented the addition of various metal ions (50 µM), the red bars
represented the change in the emission that occured upon the subsequent addition of Cu2+
(50 µM) to the above solution.
Fig. 7. Reversible fluorescence response of L to Cu2+ in DMF/H2O (95:5). Green: 5 µM
L; Blue: 5 µM L with 7 equiv Cu2+; Red: 5 µM L with 7 equiv Cu2+ and then addition of
20 equiv sulfide ion (Na salt). Excitation was performed at 521 nm.
Scheme 2. Possible mechanism of the response of L to Cu2+.
11

Fig. 1. X-ray single crystal structure for L. (All hydrogen atoms were omitted for clarity).
Fig. 2. Fluorescence emission spectra of L (5 µM) in the presence of various metal ions
(10 equiv for 1. Cu2+, 2. Na+, 3. K+, 4. Mg2+, 5. Ca2+, 6. Mn2+, 7. Co2+, 8. Zn2+, 9. Al3+, 10.
Cd2+, 11. Ba2+, 12. Ag+, 13. Hg2+, 14. Fe2+, 15. Ni2+ and 16. Pb2+) in DMF/H2O (95:5). λ
ex
= 521 nm.
12

Fig. 3. Fluorescence spectra of L (5 µM) in the presence of increasing concentration of
Cu2+ (0–7 equiv). The excitation wavelength was 521 nm. Inset: (a) Linear relationship
between the emission intensity at 559 nm and Cu2+ concentration from 0 to 15 µM; (b)
Fluorescence change of L (5 µM) in the absence and presence of Cu2+ (50 µM) under UV
light with an excitation of 365 nm.
Fig. 4. Absorption spectra of L (5 µM) with addition of different concentrations of Cu2+
(0–5 equiv) in DMF/H2O (95:5). The inset showed the colour change under visible light,
in the absence and presence of Cu2+ (25 µM) respectively.
13

Fig. 5. Job’s plot method between chemosensor L and Cu2+.
Fig. 6. The fluorescent response of L (5 µM) upon addition of different metal ions at 559
nm. The black bars represented the addition of various metal ions (50 µM), the red bars
represented the change in the emission that occured upon the subsequent addition of Cu2+
(50 µM) to the above solution.
14

Fig. 7. Reversible fluorescence response of L to Cu2+ in DMF/H2O (95:5). Green: 5 µM
L; Blue: 5 µM L with 7 equiv Cu2+; Red: 5 µM L with 7 equiv Cu2+ and then addition of
20 equiv sulfide ion (Na salt). Excitation was performed at 521 nm.
Scheme 2. Possible mechanism of the response of L to Cu2+.
15

New rhodamine-based turn-on and colorimetric probe for
copper(II) ion with high selectivity and sensitivity
Yan Fua, Qiao-Feng Tianb, Yan-Qin Guoa, Shuang-Quan Zanga*
New rhodamine-based turn-on and colorimetric probe for
copper(II) ion with high selectivity and sensitivity
Yan Fua, Qiao-Feng Tianb, Yan-Qin Guoa, Shuang-Quan Zanga*
A new rhodamine-based derivative fluorescent probe (L) was synthesized as a highly
selective and turn-on fluorescent probe for quantitative detection of Cu2+ exhibiting an
almost 200-fold fluorescence increase. Moreover, the X-ray single crystal structure of the
probe had also been measured. Besides, the fluorescent probe can also be considered as a
good OFF–ON switch for Cu2+ with naked-eye colour change from colorless to pink. The
1:1 binding ratio between chemosensor L and Cu2+ was investigated using Job’s plot
1
method, H NMR titration and ESI-MS. The probe exhibited excellent selectivity and
sensitivity toward Cu2+ with little interference, meanwhile the detection limit of Cu2+ was
at a quite lower micromolar level.
16

►A new fluorescent probe (L) exhibited significant fluorescence
enhancement upon binding with Cu2+ over 200-fold.
►L showed naked-eye detection of Cu2+ from colorless to pink.
►L exhibited good selectivity for Cu2+ over other metal ions with little
interference.
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