An azo-phenol derivative probe: colorimetric and "turn-on" fluorescent detection of copper(II) ions and pH value in aqueous solution

Article abstract of DOI:10.1039/c7ra01109a

The optical properties of a novel, rhodamine-based derivative, synthesized by reacting rhodaminehydrazide and an azo-phenol derivative in ethanol, were investigated in ethanol-water solution. The novel sensor displayed selectivity for Cu²⁺ ions, as evidenced by a colourless to dark red colour change, which was characterized using UV visible spectroscopy and which also allowed visual detection of Cu²⁺. In contrast, selectivity towards pH was determined from changes in the absorption spectra in the micromolar range. This represents the first reported rhodamine-based sensor capable of detecting both Cu²⁺ and pH using two different modes. In addition, the rhodamine-based sensor has been successfully applied in the biomedical research field to the fluorescence imaging of Cu²⁺ in HeLa cells.

Full text of DOI:10.1039/c7ra01109a

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An azo-phenol derivative probe: colorimetric and
“turn-on” fluorescent detection of copper(II) ions
and pH value in aqueous solution
Cite this: RSC Adv., 2017, 7, 20537
b
c
Yanchun Li,a Xiaojun Han
and Yan Song
*
The optical properties of a novel, rhodamine-based derivative, synthesized by reacting rhodaminehydrazide
and an azo-phenol derivative in ethanol, were investigated in ethanol–water solution. The novel sensor
displayed selectivity for Cu2+ ions, as evidenced by a colourless to dark red colour change, which was
characterized using UV visible spectroscopy and which also allowed visual detection of Cu2+. In contrast,
selectivity towards pH was determined from changes in the absorption spectra in the micromolar range.
This represents the first reported rhodamine-based sensor capable of detecting both Cu2+ and pH using
two different modes. In addition, the rhodamine-based sensor has been successfully applied in the
biomedical research field to the fluorescence imaging of Cu2+ in HeLa cells.
Received 25th January 2017
Accepted 31st March 2017
DOI: 10.1039/c7ra01109a
rsc.li/rsc-advances
intracellular uctuations of metal ions by using microscopy
techniques to allow real-time local imaging.6–11 Up to now, many
1. Introduction
uorescent sensors for Cu2+ have been reported,12,13 but only
a few of them are applicable to cellular imaging,14,15 their
practical application is still restrained due to their poor water
solubility, UV-excitation and pH-dependent uorescence in
physiological environments. To date, it is still a tremendous
challenge to design Cu2+-selective sensors, in particular, uo-
rescent chemosensor for the accurate detection of Cu2+ in
aqueous solutions and biological environments.
A practical uorescent chemosensor must produce a strong
uorescent response upon binding the analyte. Rhodamine, as
a dye, is an ideal mode for construction of uorogenic-labeling
and uorescent chemosensors because of their particular spi-
rolactam structures and excellent photophysical properties,
including high absorption coefficient, high uorescence
quantum yield and high photostability.16,17 By virtue of these
fascinating properties, the excellent examples of rhodamine-
based uorescent chemosensor have been designed and
successfully applied for detecting metal ions.18–23 And most
rhodamine-based chemosensors are uorescence enhancement,
which is of great importance for metal ions detection.24,25
Nevertheless, there is a relatively few reports concerned the
chemosensor based on rhodamine derivative for selective detec-
tion of metal ions in aqueous and application in cell imaging.26–28
In this paper, we report the synthesis and uoroionophoric
properties of a novel uorescent sensor aimed at the selective
recognition of Cu2+ ions in aqueous solution and living cells.
Sensor 1 is composed of an rhodamine uorophore and
Cu2+ has caused serious harm to the environment and human
health. It can cause serious injury to human kidneys, lungs,
bone and the nervous system, which results in renal dysfunc-
tion, calcium metabolism disorders and an increased incidence
of certain forms of cancer.1 However, the mechanisms involved
in Cu2+-uptake and carcinogenesis remain undened.2,3 The
EPA (United States Environmental Protection Agency) gives an
enforceable drinking water standard for Cu of 5 ppb to prevent
kidney damage and other related diseases, while the WHO
(World Health Organization) provides a more strict guideline
value for Cu of 3 ppb for drinking water. Therefore, developing
reliable methods for Cu2+ trace quantication in environmental
samples and in living cell/tissue samples is of great signicance
for clarifying Cu2+-carcinogenesis and other biological effects.
In recent years, the development of uorescent chemo-
sensors for sensing and reporting heavy transition-metal ions
has been receiving considerable attention.4,5 Fluorescence
techniques have become powerful tools for sensing and
imaging metal ions in trace amounts because of its simplicity,
high sensitivity and real-time monitoring with in short response
time. Sensitive and reliable uorescent molecular sensors seem
to be the ideal tool for evaluating and dynamically mapping the
aDepartment of Chemistry and Pharmaceutical Engineering, Jilin Institute of Chemical
Technology, Jilin, 132022, People's Republic of China
bState Key Laboratory of Urban Water Resource and Environment, School of Chemistry
and Chemical Engineering, Harbin Institute of Technology, 92 West Da-Zhi Street,
Harbin, 150001, China
a
receptor of the N,N0-bis(salicylidene)-diethylenetriamine
attached via a space linker. Sensor 1 exhibits Cu2+-selective
TURN-ON and Cu2+-selective TURN-OFF type signaling behaviors
that can be used to distinguish Cu2+ ions. Moreover, to the best of
cDepartment of Chemical and Materials Engineering, Jilin Institute of Chemical
Technology, Jilin, 132022, People's Republic of China. E-mail: songyan199809@163.
com
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our knowledge, this is the rst example of a cation-induced Na2CO3 (18 g Na2CO3 + 120 mol H2O). Then the solution of
TURN-ON and TURN-OFF type of uorescent signal control salicylaldehyde was added dropwise to the bright yellow solution
behavior which was applied to intracellular imaging of Cu2+.
for 1 h. Aer stirring for 4 h, the reaction mixture was neutralized
with HCl, the brown crude solid was ltered and recrystallized
from ethanol to afford a pure yellow product. Yield: 62.8%.
2. Experiment section
2.1 Materials
3.2 Synthesis of intermediate (2)
Rhodamine B 98% purity, (Beijing Hengyue Zongyuan Chem-
ical CO., LTD). N,N-Dimethylform amide (DMF) was distilled
and dried by anhydrous magnesium sulfate. Aniline (99%
purity), salicylaldehyde, hydrazine hydrate, and all other
reagents were purchased from Sinopharm Chemical Reagent
Co., Ltd., and used as received. The solutions of metal ions were
prepared from NaCl, KCl, CaCl2, MgSO4, FeCl3, Mn(NO3)2-
$6H2O, CoCl2$6H2O, NiCl2$6H2O, Zn(NO3)2, CuCl2, CuCl2-
$2H2O, Hg(NO3)2, AgNO3, Pb(NO3)2, respectively, and were
Rhodamine B (5.0 g, 10.46 mmol) was dissolved in 150 mL of
anhydrous ethanol; 10 mL (excess) hydrazine hydrate (85%) was
then added. Aer the addition, the stirred mixture was heated to
reux in an air bath for 2 h. The solvent was evaporated under
reduced pressure, 1 M NaOH was added slowly with stirring
until the pH of the solution reached.30,31 The resulting precipi-
tate was ltered and washed 3 times with water. The crude
material was puried by ash column chromatography to give
white product RHB-hydrazine (CH2Cl2/EtOH/Et3N, 5 : 1 : 0.1),
yield: 50.8%.
dissolved in deionized water. Aqueous Tris–HCl (0.1 mol Lꢀ1
)
solution was used as buffer to keep pH value (pH ¼ 7.14), and to
maintain the ionic strength of all solutions in experiments.
3.3 Synthesis of sensor 1
To 30 mL anhydrous ethanol containing (2) (0.50 g, 1.1 mmol),
an excessive (1) (0.34 g, 1.5 mmol) was added and the mixture
was stirred vigorously at 90 ꢁC for 24 h. Aer completion of the
reaction, the formed precipitate was ltered, washed with cold
ethanol (3 ꢂ 10 mL) and then dried in vacuum, affording 0.65 g
crude product, which was puried by ash column chroma-
tography to give white product RHB-hydrazine. (CH2Cl2/EtOH/
Et3N, 5 : 1 : 0.1), yield: 75.6%. 1H NMR (300 MHz, CDCl3, d):
8.06 (d, J ¼ 7.6 Hz, 6H), 7.89 (s, 7H), 7.86 (d, J ¼ 2.2 Hz, 7H), 7.80
(s, 5H), 7.60 (d, J ¼ 5.1 Hz, 10H), 7.52 (d, J ¼ 7.7 Hz, 8H), 7.48 (d,
J ¼ 6.9 Hz, 5H), 7.28–7.17 (m, 6H), 7.02 (d, J ¼ 8.7 Hz, 6H), 6.56
(s, 19H), 6.35 (s, 10H), 3.39 (d, J ¼ 7.0 Hz, 40H), 1.33–1.12 (m,
59H); 13C NMR (300 MHz, CDCl3, d): 168.67, 159.93, 152.70,
151.89, 151.09, 148.56, 146.93, 145.00, 134.01, 130.81, 129.29,
128.85, 128.48, 127.57, 125.42, 123.83, 123.34, 123.06, 122.17,
120.06, 117.28, 108.19, 104.91, 97.42, 65.54, 43.61, 12.27; IR
(KBr, cmꢀ1): n ¼ 3437, 2972, 2929, 2893, 1692, 1615, 1548, 1515,
1481, 1466, 1428, 1401, 4135, 1375, 1307, 1267, 1220, 1120,
1079, 814, 788, 769, 759, 700 and 690; HRMS (ESI, m/z): [M + H]+
cal. Cu for C41H40N6O3, 664.3162; found, 665.3459.
2.2 Characterization
The synthesis of sensor 1 is shown in Scheme 1. The interme-
diate, compound (1), was synthesized from salicylaldehyde and
aniline following a literature procedure.29 Compound (2) was
prepared in 50.8% yield of pure product by coupling Rhoda-
mine B with hydrazine hydrate. Sensor 1 was easily synthesized
by compound (1) and (2) in 75.6% yield, and characterized by
1
ESI-MS, H-NMR and 13C-NMR.
1H NMR spectra were measured on a Bruker AV-300 spec-
trometer with chemical shis reported as parts per million (in
CDCl3, TMS as internal standard). IR spectra were recorded on
a Bruker Vector-22 spectrometer. The pH values of the test
solutions were measured with a glass electrode connected to
a Mettler-Toledo Instruments DELTA 320 pH meter (Shanghai,
China) and adjusted if necessary. Fluorescence spectra were
determined on a Hitachi F-4500. UV-Vis spectra were recorded
on a Hitachi U-3010 UV-Vis spec.
3. Synthesis
3.1 Synthesis of intermediate (1)
4. Results and discussion
To a solution of aniline (5 mL, 0.05 mol) in a small quantity of
water was slowly added 20 mL of 50% HCl solution at 0–5 ꢁC with
stirring. Then, the solution of NaNO2 (4 g NaNO2 + 20 mL H2O)
was added to the above-mentioned mixture and the resulting
solution was stirred for 1 h to give a bright yellow solution.
Salicylaldehyde (5 mL, 0.05 mol) was dissolved in the solution of
4.1 pH-titration and spectral responses
The effects of pH on the absorption of sensor 1 in the absence
and presence of Cu2+ are determined over the range 4–14. The
pH dependence of absorption spectra is shown in Fig. 1 and 2.
As we can see the sensor and the complex are stable in the wide
range of pH 4–10, suggesting that it could be used for the
estimation of Cu2+ under complex physiological conditions for
biological applications.
4.2 Cu2+-titration and spectral responses
In order to gain an insight into the signalling properties of the
sensor 1 toward Cu2+, uorescence titration was conducted.
Fluorescence titration of the sensor 1 was investigated in
Scheme 1 Synthesis and structure of sensor 1.
20538 | RSC Adv., 2017, 7, 20537–20541
ethanol–water (1 : 10, v/v) solution ([Cu2+] ¼ 2.0 ꢂ 10ꢀ6 mol Lꢀ1
,
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Fig. 4 Curve of fluorescence intensity at 525 nm of sensor 1 versus
increasing concentrations of Cu2+
.
Fig. 1 Absorption of sensor 1 with different pH.
retained. The reaction responsible for these changes reached
completion well within the time frame (<30 s) of the measure-
ment. Signicantly, the enhancement of uorescence intensity of
sensor 1 corresponded to the concentration of Cu2+ in a linear
manner (linearly dependent coefficient: R ¼ 0.9929) (Fig. 4),
which indicated that sensor 1 had potential use for the quanti-
tative determination of Cu2+. It should be noted that this switch
on sensing process could be readily detected not only by uo-
rescence spectroscopy but also by the naked eye. Moreover,
a Job's plot, which exhibited a maximum at 0.5 mole fraction of
sensor 1, indicated the 1 : 1 stoichiometry between Cu2+ and
sensor 1 (Fig. 5). Therefore, on the basis of 1 : 1 stoichiometry
and uorescence titration data, the detection limit was found to
be 7.2 ꢂ 10ꢀ7 mol Lꢀ1 (based on S/N ¼ 3).
Fig.
2 Color changes of sensor 1 in ethanol–water (1 : 10, v/v)
measured as a function of pH.
Fig. 6 shows the result of absorption titration of sensor 1
with Cu2+ ions. When no Cu2+ ions were added to the solution of
sensor 1, free 1, as expected, exhibited almost no absorption
near 500–600 nm, indicating that sensor 1 exists as a spirocyclic
form. Upon addition of increasing concentrations of Cu2+ ions
to the solution, two absorption bands centered at 380 nm and
560 nm appeared with increasing intensity, which can be
ascribed to the formation of the ring-opened amide form of
sensor 1 upon Cu2+ ions binding. Moreover, the titration solu-
tion exhibited an obvious and characteristic colour change from
0.1 M Tris–HCl buffer at pH ¼ 7.14, lex ¼ 500 nm, lem ¼ 565 nm).
Upon addition of Cu2+, about a 14-fold increase in uorescence
intensity was observed at 575 nm (Fig. 3). When more than 1.0
equiv. Cu2+ was added, the maximum uorescence intensity was
Fig. 3 The fluorescence intensity change profile of sensor 1 (1 mM)
to different metal ions (1 mM) in 0.1 M Tris–HCl solution (ethanol–
water ¼ 1 : 10, v/v, pH 7.14, lex ¼ 500 nm). Inset: corresponding Fig. 5 Job's plot for sensor 1 (forms 1 : 1 complexes) in 0.1 M Tris–HCl
fluorescent spectra of 1 in the presence of different concentrations solution (ethanol–water ¼ 1 : 10, v/v, pH ¼ 7.14). The total concen-
Cu2+ ions.
tration of sensor 1 and Cu2+ is 2 mM.
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Fig. 9 The fluorescence intensity of sensor 1 (mM) with 1.5 equiv. of
Cu2+, followed by 10 equiv. of Mn+. (Tris–HCl, pH 7.14, ethanol–water
¼ 1 : 10, v/v, lex ¼ 500 nm).
Fig. 6 The absorption change profile of sensor 1 (1 mM) to different
metal ions (2 mM) in 0.1 M Tris–HCl solution (ethanol–water ¼ 1 : 10,
v/v, pH 7.14).
Fig. 10 Images of HeLa cells. (a) Bright fields image of HeLa cells
incubated sensor 1; (b) fluorescence image of (a); (c) fluorescence
image of HeLa cells incubated with sensor 1 and Cu2+
.
negligible effects on the uorescence behavior of sensor 1
(Fig. 8). In order to further test the interference of other
common cations in the determination of Cu2+, competition
experiments were performed in which the uorescent sensor
was added to a solution of Cu2+ in the presence of other metal
ions (Fig. 9). The competition experiments were conducted in
the presence of Cu2+ ion mixed with Na+, K+, Ca2+, Mg2+, Mn2+,
Ni2+, Fe3+, Hg2+, Co2+, Pb2+, Zn2+, respectively. Experimental
results indicated that these ions showed no obvious interfer-
ence in the Cu2+ detection. Thus, the sensor 1 exhibited excel-
lent selectivity toward Cu2+, which its practical application.
Fig. 7 Change in color (left) and fluorescence (right) of sensor 1 (10
mM) in buffered (Tris–HCl, pH 7.14, ethanol–water ¼ 1 : 10, v/v) upon
addition of 1 mM Cu2+ ions.
colourless to red (Fig. 7), suggesting that sensor 1 can serve as
a “naked-eye” indicator for Cu2+ ions.
4.3 Response of sensor 1 to various metal ions and
competition experiments
Other representative metal ions, such as Na+, K+, Ca2+, Mg2+,
Mn2+, Ni2+, Fe3+, Hg2+, Co2+, Pb2+, Zn2+ ions, showed almost
4.4 Potential application of sensor 1 for Cu2+ uorescence
imaging in HeLa cells
To investigate the potential biological application of sensor 1,
uorescence imaging experiments were attempted on HeLa cells
(Fig. 10). The cells were incubated with 10 mM of sensor 1 for
30 min at 37 ꢁC, only slightly uorescence was observed owing to
a small quantity of Cu2+ in cancer cells (Fig. 10b), which was
conrmed by previous research.32–34 However, the cells were
added with 20 mM of Cu2+ for another 30 min, an obviously
uorescence response was observed (Fig. 10c). The uorescence
imaging experiments conrmed that sensor 1 had good cell-
permeability and could be used for detecting Cu2+ in living cells.
5. Conclusions
In conclusion, we have synthesized a simple and easy-to-
prepare azo-phenol derivative optical chemosensor 1 for the
Fig. 8 The fluorescence intensity of sensor 1 (1 mM) in the presence of
different metal ions (10 mM).
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detection of Cu2+ ions. The chemosensor 1 displayed 1 : 1 15 M. Yu, M. Shi, Z. Chen, F. Li, X. Li, Y. Gao, J. Xu, H. Yang,
complex formation with Cu2+ ions. Cu2+-specic binding
Z. Zhou and T. Yi, Chemistry, 2008, 14, 6892–6900.
enabled the opening of the spirolactam ring and consequently 16 W. Wang, Q. Yang, L. Sun, H. Wang, C. Zhang, X. Fei, M. Sun
successfully exhibited a obvious absorption and uorescence and Y. Li, J. Hazard. Mater., 2011, 194, 185–192.
enhancement response toward Cu2+ ions over other metal ions. 17 X. Q. Chen, T. Pradhan, F. Wang, J. S. Kim and J. Yoon,
Thus the chemosensor 1 behaves as a highly sensitive and Chem. Rev., 2012, 112, 1910–1956.
selective uorescent probe for Cu2+ ions detection. Moreover, 18 X. Zhang, X. J. Huang and Z. J. Zhu, RSC Adv., 2013, 3, 24891–
we have demonstrated the application of sensor 1 by imaging 24895.
intracellular Cu2+ in HeLa cells, which demonstrated the 19 A. Dhara, A. Jana, N. Guchhait, P. Ghosh and S. K. Kar, New J.
potentialvalue in living system.
Chem., 2014, 38, 1627–1634.
20 Y. Zhou, F. Wang, Y. Kim, S.-J. Kim and J. Yoon, Org. Lett.,
2009, 11, 4442–4445.
21 Z. Xu, L. Zhang, R. Guo, T. Xiang, C. Wu, Z. Zheng and
F. Yang, Sens. Actuators, B, 2011, 156, 546–552.
22 C. Yu, J. Zhang, R. Wang and L. Chen, Org. Biomol. Chem.,
2010, 8, 5277–5279.
Acknowledgements
This work was supported by the Scientic and Technological
Development Plan Project of Jilin Province (Grant No.
20160101311JC).
23 Y. Zhao, X.-B. Zhang, Z.-X. Han, L. Qiao, C.-Y. Li, Li-X. Jian,
G.-L. Shen and R.-Q. Yu, Anal. Chem., 2009, 81, 7022–7030.
24 Y. X. Wu, J. B. Li, L. H. Liang, D. Q. Lu, J. Zhang, G. J. Mao,
L. Y. Zhou, X. B. Zhang, W. H. Tan, G. L. Shen and R. Q. Yu,
Chem. Commun., 2014, 50, 2040–2042.
25 Y. Yuan, S. G. Sun, S. Liu, X. W. Song and X. J. Peng, J. Mater.
Chem. B, 2015, 3, 5261–5265.
26 B. Zhu, C. Gao, Y. Zhao, C. Liu, Y. Li, Q. Wei, Z. Ma, B. Du and
X. Zhang, Chem. Commun., 2011, 47, 8656.
27 L. M. Zhang, L. E. Guo, X. M. Li, Y. G. Shi, G. F. Wu, X. G. Xie,
Y. Zhou, Q. H. Zhao and J. F. Zhang, Tetrahedron Lett., 2014,
55, 6131–6136.
28 J. F. Zhang, S. Kim, J. H. Han, S. J. Lee, T. Pradhan, Q. Y. Cao,
C. Kang and J. S. Kim, Org. Lett., 2011, 13, 5294–5297.
29 M. R. Maurya, S. J. J. Titinchi and S. Chand, J. Mol. Catal. A:
Chem., 2003, 193, 165–176.
Notes and references
1 K. C. Jones, Environ. Pollut., 1993, 81, 301.
2 C. C. Bridges and R. K. Zalups, Toxicol. Appl. Pharmacol.,
2005, 204, 274–308.
3 J. H. Lee and G. F. Birch, Environ. Monit. Assess., 2016, 188, 1–
17.
4 J. Tian, Q. Liu, A. M. Asiri, A. O. Al-Youbi and X. Sun, Anal.
Chem., 2013, 85, 5595–5599.
5 W. Lu, X. Qin, S. Liu, G. Chang, Y. Zhang, Y. Luo, A. M. Asiri,
A. O. Al-Youbi and X. Sun, Anal. Chem., 2012, 84, 5351–5357.
6 L. Zeng, E. W. Miller, A. Pralle, E. Y. Isacoff and C. J. Chang, J.
Am. Chem. Soc., 2010, 128, 10–11.
7 S. P. Wu, Z. M. Huang, S. R. Liu and P. K. Chung, J. Fluoresc.,
2012, 23, 1139–1145.
8 B. Sen, M. Mukherjee, S. Pal, S. Sen and P. Chattopadhyay,
RSC Adv., 2015, 5, 50532–50539.
30 H. Yang, Z. Zhou, K. Huang, M. Yu, F. Li, T. Yi and C. Huang,
Org. Lett., 2007, 9, 4729–4732.
9 P. Chattopadhyay, B. Sen, M. Mukherjee, S. Pal and S. Sen,
RSC Adv., 2015, 5, 50532–50539.
31 H. S. Jung, P. S. Kwon, J. W. Lee, J. I. Kim, S. H. Chang,
J. W. Kim, S. Yan, Y. L. Jin, J. H. Lee and T. Joo, J. Am.
Chem. Soc., 2009, 131, 2008–2012.
32 M. C. Linder and M. Hazegh-Azam, Am. J. Clin. Nutr., 1996,
63, 797S–811S.
33 D. Chen, Q. C. Cui, H. Yang, R. A. Barrea, F. H. Sarkar,
S. Sheng, B. Yan, G. P. Reddy and Q. P. Dou, Cancer Res.,
2007, 67, 1636–1644.
34 R. An, D. Zhang, Y. Chen and Y.-Z. Cui, Sens. Actuators, B,
2016, 222, 48–54.
10 M. Yang, H. Wang, J. Huang, M. Fang, B. Mei, H. Zhou, J. Wu
and Y. Tian, Sens. Actuators, B, 2014, 204, 710–715.
11 Y. Wang, H. Q. Chang, W. N. Wu, X. J. Mao, X. L. Zhao,
Y. Yang, Z. Q. Xu, Z. H. Xu and L. Jia, J. Photochem.
Photobiol., A, 2017, 335, 10–16.
12 X. Wang, J. Zhao, C. Guo, M. Pei and G. Zhang, Sens.
Actuators, B, 2014, 193, 157–165.
13 S. Li and S. Dong, J. Mater. Chem., 2008, 18, 4636–4640.
14 Q. Qu, A. Zhu, X. Shao, G. Shi and Y. Tian, Chem. Commun.,
2012, 48, 5473–5475.
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