A novel “turn-on” fluorescence probe based on azoaniline-arylimidazole dyad for the detection of Cu2+

Article abstract of DOI:10.1016/j.dyepig.2017.03.051

A novel fluorescence probe TPI-AD bearing azoaniline and arylimidazole moieties is conveniently synthesized for the detection of Cu²⁺. A “naked-eye” visible color change from colorless to lake-blue together with a 220-fold fluorescence enhancem

Full text of DOI:10.1016/j.dyepig.2017.03.051

Accepted Manuscript
A novel “turn-on” fluorescence probe based on azoaniline-arylimidazole dyad for the
2+
detection of Cu
Yongpeng Wang, Shuzhi Liu, Hongbiao Chen, Yijiang Liu, Huaming Li
PII:
S0143-7208(17)30115-8
10.1016/j.dyepig.2017.03.051
DYPI 5878
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 18 January 2017
Revised Date: 23 March 2017
Accepted Date: 23 March 2017
Please cite this article as: Wang Y, Liu S, Chen H, Liu Y, Li H, A novel “turn-on” fluorescence probe
2+
based on azoaniline-arylimidazole dyad for the detection of Cu , Dyes and Pigments (2017), doi:
0.1016/j.dyepig.2017.03.051.
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A novel “turn-on” fluorescence probe TPI-AD based on azoaniline-arylimidazole
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dyad was synthesized for colorimetric and fluorometric detection of Cu through
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Cu -catalyzed oxidation cyclization reaction.

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A novel “turn-on” fluorescence probe based on azoaniline-
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arylimidazole dyad for the detection of Cu
a
a
a
a
a, b
Yongpeng Wang , Shuzhi Liu , Hongbiao Chen *, Yijiang Liu *, Huaming Li
a
College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province,
China
b
Key Laboratory of Polymeric Materials & Application Technology of Hunan
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Province, Key Laboratory of Advanced Functional Polymeric Materials of College of
Hunan Province, and Key Laboratory of Environment-Friendly Chemistry and
Application in Ministry of Education, Xiangtan University, Xiangtan 411105, Hunan
Province, China
*Corresponding author. Tel.: +86 731 58298572; Fax: +86 731 58293264.
E-mail address: liuyijiang84@163.com, chhb606@163.com

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Abstract:
A novel fluorescence probe TPI-AD bearing azoaniline and arylimidazole moieties is
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conveniently synthesized for the detection of Cu . A “naked-eye” visible color
change from colorless to lake-blue together with a 220-fold fluorescence enhancement
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can be achieved by Cu -catalyzed oxidative cyclization, which transforms the
non-emissive TPI-AD into strongly fluorescence benzotriazole derivative (TPI-BTA).
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The TPI-AD probe can be applied for quantitative detection of Cu with good linear
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relationship between its fluorescence intensity and Cu concentration. Moreover,
TPI-AD shows high selectivity over other common metal ions as well as excellent
sensitivity with a detection limit of 14.4 nM. In addition, the fluorescence imaging of
living cells MCF-7 reveals the potential application of TPI-AD in biological system.
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Keywords: fluorescence probe; azoaniline-arylimidazole dyad; fluorescence
enhancement; oxidative cyclization; bioimaging.

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1. Introduction
Copper as the third essential trace metal element in human body plays a crucial role in
numerous physiological processes including cellular energy generation, oxygen
transport and activation, and signal transduction [1-3]. However, excessive or
deficient intake of copper will do harm to human health and result in severe
neurodegenerative diseases such as Wilson’s disease and Alzheimer’s disease [4-6].
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On the other hand, copper ion (Cu ) is a toxic species and seriously pollutes
environment and water because of its widespread application in industry and in our
daily life. According to the Environmental Protection Agency (EPA), the highest
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acceptable level of Cu in drinking water is about 20 ꢀM [7]. Therefore, it is very
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significant to detect Cu both in biological and environmental systems.
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Recently, fluorescence probes for Cu detection have become more popular due
to their high sensitivity and selectivity, simplicity, low cost and real-time analysis
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[8-14]. Owing to the intrinsic paramagnetic property of Cu , many of the reported
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fluorescence probes showed fluorescence-quenching behavior upon binding with Cu ,
therefore interference resulting from background and other closely relevant metal ions
was still unavoidable [15-20]. An alternative strategy is applying the reactivity-based
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Cu detection, which utilizes the irreversible chemical reactions to improve the
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sensitivity and selectivity of these probes. Typical reactions include Cu -promoted
hydrolysis of amide [21], hydrazine [22-25], imine [26], ether [27], picolinate [28-30],
dansylhydrazine [31], and oxidation of N-acylhydrazones [32], thiosemicarbazone

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[33-34], phenothiazine [35], aniline [36], o-phenylenediamine [37], dihydrorosamine
[38]. Recently, the oxidative cyclization of o-(phenylazo)aniline has been considered
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as an efficient strategy for Cu fluorescence probes because of the non-emissive azo
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group can be converted to strong emissive triazole group in the presence of Cu . For
example, Lee et al. have synthesized a series of azoanilines with different electron-
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donating units, which can be applied for the detection of Cu with above 80 fold
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“turn-on” fluorescence due to the Cu -catalyzed cyclization reaction, resulting in the
transformation of azoanilines into benzotriazoles [39]. Analogous azo derivatives
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have also been developed as “turn-on” fluorescence probes for Cu [40-44].
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Considering that arylimidazole is a classical fluorophore with large π-conjugated
structure, high molar absorption coefficient, moderate fluorescence quantum yields
and good photochemical stability, it is highly desirable to construct novel “turn-on”
fluorescence probes by the combination of arylimidazole and azoaniline cyclization
reaction-induced fluorescence enhancement. Actually, arylimidazole-derived
fluorescence probes have already been reported for the detection of metal ions [45-47].
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In this paper, we designed and synthesized a novel fluorescence probe for Cu
detection based on azoaniline-arylimidazole dyad (TPI-AD). A “turn-on” fluorescence
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and a good selectivity were envisioned via the Cu -promoted oxidative cyclization
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reaction. The performance for Cu detection and the bioimaging of the probe TPI-AD
were investigated in detail.

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2. Experimental
2.1. Reagents and apparatus
4-(4,5-Diphenyl-1H-imidazol-2-yl)aniline (TPI-NH ) was synthesized according to a
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literature procedure [44]. All reagents were purchased from commercial source and
used without further purification unless otherwise noted. The NMR spectra were
recorded with a Bruker AV-400 NMR spectrometer. MALDI-TOF mass spectra were
recorded on a Bruker BIFLEXeIII mass spectrometer using a nitrogen laser (337 nm)
and an accelerating potential of 20 kV. UV-Vis spectra were recorded with an Agilent
Cary 100 UV-Vis spectrometer. Photoluminescence emission spectra were recorded
with an Agilent QM 100 luminescence spectrometer. Bioimaging of probe was
measured through a FV 1000-IX81 confocal laser scanning microscope (CLSM,
Olympus, Japan) with an excitation wavelength of 383 nm.
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2.2. Synthesis of probe TPI-AD
Under stirring, conc. HCl (1.0 mL) was slowly added to a MeOH/EtOH (1/1, v/v, 14
mL) solution of TPI-NH (1.56 g, 5.00 mmol). The mixture was stirred at 0 ºC for 15
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min. An aqueous solution (1.0 mL) of NaNO (379 mg, 5.50 mmol) was then added
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dropwise and the reaction mixture was further stirred for 10 min, yielding an azonium
intermediate. In a separate reaction vessel, a solution of m-phenylenediamine (0.65 g,

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6.00 mmol) and sodium hydroxide (1.20 g) in MeOH/H O (1/1, v/v, 40 mL) was
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prepared and stirred in an ice bath. With stirring, the above generated azonium
intermediate was added dropwise. After stirring for 3 h, 200 mL of water was added
and the solution was adjusted to neutral to accelerate the formation of a reddish-black
solid, which was separated by filtration, washed with water, and dried under vacuum.
The target product TPI-AD was obtained by column chromatography with ethyl
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acetate and petroleum ether as eluent (1.63 g, 76%). H NMR (DMSO-d , 400 MHz)
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δ (ppm): 12.99 (s, 1H, NH), 8.11-8.14 (d, 2H, ArH), 7.56-6.89 (m, 13H, ArH),
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6.02-6.00 (d, 1H, ArH), 5.86 (s, 1H, ArH). C NMR (100 MHz, DMSO-d ) δ (ppm):
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154.0, 153.1, 150.1, 147.0, 145.8, 133.0, 130.0, 129.3, 128.9, 128.3, 127.6, 127.3,
126.3, 121.6, 117.3, 114.0, 106.6. MALDI-TOF MS(C H N ) m/z: calcd for 430.19,
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found: 431.20 [M+H] .
2.3 General spectroscopic procedures
All spectroscopic measurements were performed in MeOH/HEPES solution (8/2, v/v,
pH = 7.2). All the metal cationic solutions were prepared from their chloride salts,
sulfate salts or nitrate salts. Then 3.0 mL of probe solution (5 ꢀM) was placed in a
quartz cell (10.0 mm width) and the absorption and fluorescence spectra were
recorded. The CuCl and other cationic aqueous solutions were introduced in portions
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and the fluorescence and absorption changes were recorded at room temperature each
time.

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2.4 Stoichiometry between probe and Cu
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The stoichiometry between probe and Cu was determined by Job’s titration. For the
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Job’s plot analyses, a series of solutions with varying mole fraction of Cu were
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measured while kept the same total concentration of probe and Cu unchanged.
2.5 Cell culture and fluorescence imaging
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The bioimaging of probe was study through cell imaging experiment of human breast
cancer cells (MCF-7). The MCF-7 cells were culture under the condition of
Dulbecco’s modified Eagle medium (DMEM) and 10% fetal bovine serum (FBS) at
37 ºC under an atmosphere of 5% CO . For fluorescence imaging, the MCF-7 cells
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were firstly treated with 20 ꢀM probe in culture media for 30 min at 37 ºC and
washed three times with phosphate-buffered saline (PBS, pH = 7.2). Then, the cells
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were further incubated with 80 ꢀM Cu in culture media for 30 min at 37 ºC and
washed three times with PBS. Finally, the cells were observed by CLSM.
3. Results and discussion
3.1 Synthesis and characterization of probe TPI-AD
The probe TPI-AD was synthesized by the simple and convenient routes as shown in

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Scheme 1. 4-(4,5-Diphenyl-1H-imidazol-2-yl)aniline (TPI-NH ) was synthesized
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according to the reported literature [48]. The target compound, TPI-AD, was
generated through the coupling reaction between TPI-NH and m-phenylenediamine.
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The structure of TPI-AD was fully characterized by H NMR spectrum (Fig. S1), C
NMR spectrum (Fig. S2) and MALDI-TOF mass spectrum (Fig. S3).
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Scheme 1. The synthetic route for probe TPI-AD.
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3.2 UV-Vis absorption response of TPI-AD towards Cu
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Spectroscopic property of probe TPI-AD (10 µM) and its response to Cu were
investigated in the mixture of MeOH/HEPES solution (8/2, v/v, pH = 7.2) at room
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temperature. Upon the addition of Cu , the characteristic absorption band of
azoaniline compound TPI-AD at about 470 nm was decreased and accompanied by a
new peak at about 383 nm, indicating a benzotriazole ring was formed under the
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catalysis of Cu (Fig. S4). As a typical azo dye, the TPI-AD does not exhibit
fluorescence due to the energy consumption in the cis-trans isomerism [49-50].
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However, a strong fluorescence peak at 490 nm was observed when Cu was added,

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suggesting the cis-trans isomerism was blocked and an efficient charge transfer from
arylimidazole to benzotrizazole was occurred (Fig. S5). Simultaneously, an apparent
color change from colorless to lake-blue could be easily observed by the naked-eye
under UV light (inset, Fig. S4). This phenomenon demonstrated a good response of
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TPI-AD towards Cu .
Next, the UV-Vis absorption titration experiment of TPI-AD with various
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concentrations of Cu in MeOH/HEPES solution was performed. As shown in Fig. 1a,
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with increasing Cu concentration, the absorption peak at 470 nm gradually
decreased and the new absorption peak at 383 nm gradually enhanced with an
isosbestic point at about 403 nm. The absorption spectra remained unchanged when
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more than 4.0 eq of Cu was added, indicating the Cu -promoted azoaniline
cyclization reaction was completed and a new benzotriazole ring was generated. A
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plot of absorbance at 470 nm (A ) of TPI-AD vs Cu concentration (0-40 ꢀM)
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showed an excellent linear relation with an linearly dependent coefficient R = 0.9917
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(Fig. 1b). Thus, the quantitative detection of Cu via UV-Vis absorption is available.
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Fig. 1. (a) Absorption spectra of TPI-AD solution responding to various concentrations of Cu (0,
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0.5 eq, 1.0 eq, 1.5 eq, 2.0 eq, 2.5 eq, 3.0 eq, 3.5 eq, 4. 0 eq, 5.0 eq; 1 eq = 10 µM); (b) A plot of
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absorbance at A470 of TPI-AD as a function of the concentration of Cu . (TPI-AD, 10.0 µM, in
MeOH/HEPES, 8/2, v/v, pH = 7.2).
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3.3 Fluorescence titration of TPI-AD towards Cu
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Fluorescence titration of TPI-AD towards various amounts of Cu was also carried
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out with an excitation wavelength of 383 nm. The fluorescent intensity was recorded
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every 30 min after the addition of Cu . The pure probe TPI-AD showed almost no
fluorescence at 490 nm (Φ = 0.007, Coumarin 30 as a standard). As shown in Fig. 2a,
an apparent enhancement of fluorescent intensity at 409 nm was observed with
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increasing amounts of Cu . The fluorescent intensity remained unchanged upon
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addition of 4.0 eq of Cu , in which the total fluorescent intensity was 220-fold higher
than that of pure TPI-AD (Φ = 0.344, Coumarin 30 as a standard). In addition, the
gradual change in color of the probe solution could be seen by the naked-eye under

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UV lamp (inset of Fig. 2a). Good linear relationship between the fluorescent intensity
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at 490 nm (I ) and the concentration of Cu in the range of 0-20 µM was observed
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with a linearly dependent coefficient R = 0.9920 (Fig. 2b). According to the equation
DL = 3σ/S, the detection limit was determined to be 14.4 nM, which is much lower
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than the maximum permissive level of Cu in drinking water [7]. These results
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demonstrated that TPI-AD can be used to quantitative detection of Cu with high
sensitivity.
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Fig. 2. (a) Fluorescence spectra (λ , 383 nm) of TPI-AD solution responding to various
ex
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concentrations of Cu (0, 0.4 eq, 0.8 eq, 1.2 eq, 1.6 eq, 2.0 eq, 2.4 eq, 2.8 eq, 3.2 eq, 3.6 eq, 4.0
eq, 5.0 eq; 1 eq = 5 µM); (b) A plot of fluorescent intensity at I490 of TPI-AD as a function of the
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concentration of Cu . (TPI-AD, 5.0 µM, in MeOH/HEPES, 8/2, v/v, pH = 7.2).
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3.4 Selectivity and interference study
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To evaluate the selectivity of TPI-AD towards Cu , the fluorescence emission of
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TPI-AD towards various metal ions including Mg , Mn , Fe , Zn , Ni , Na , Ag ,
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3+
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Ba , Al , Ca , Hg , and Cu were measured. As mentioned previously, the probe
TPI-AD showed very weak fluorescence emission peak excited at 383 nm, and there
were no obvious variations for the fluorescence emission spectra of TPI-AD when the
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above mentioned metal ions were added (Fig. S6). Conversely, only Cu can
remarkably increase the fluorescence of TPI-AD, confirming its high selectivity
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towards Cu (Fig. 3a). Considering that there are many co-existed metal ions in
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practical Cu sample determination, it is necessary to investigate the fluorescence

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response of TPI-AD towards Cu in the presence of commonly co-existed metal ions.
As shown in Fig. 3b, there were weak fluorescence for TPI-AD with other metal ions,
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but the fluorescent intensity was dramatically increased with the addition of Cu to
the mixture of TPI-AD and other metal ions, which demonstrated that the
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fluorescence enhancement of Cu to TPI-AD was no effected by other co-existed
metal ions. This result clearly revealed the high anti-interference ability of TPI-AD.
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Fig. 3. (a) Fluorescence response of TPI-AD towards metal ions (each 20 µM); (b) Fluorescence
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response of TPI-AD towards Cu in the presence of other co-existed metal ions (each 20 µM),
(TPI-AD, 4.0 µM, MeOH/HEPES, 8/2, v/v, pH = 7.2)

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3.5 Fluorescence kinetic behavior of TPI-AD and Cu
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Based on the spectroscopic analysis, the addition of Cu resulted in the formation of
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a benzotriazole ring. Hence, the reaction kinetic behavior between TPI-AD and Cu
was investigated through time-dependent fluorescence spectra. As shown in Fig. 4, the
fluorescent intensity at 490 nm of the tested solution increased quickly within 15 min.
Then, the enhancement rate slowed down and finally remained almost unchanged
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after 30 min. Therefore, the detection of Cu in MeOH/HEPES solution through
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Cu -catalyzed cyclization reaction could be accomplished within 30 min at room
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temperature.
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Fig. 4. Reaction-time profile of TPI-AD (5.0 ꢀM, MeOH/HEPES, 8/2, v/v, pH = 7.2) in the
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presence of Cu (40.0 ꢀM).

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3.6 The detection mechanism of TPI-AD towards Cu
According to the reported literatures, azoaniline is a typical recognition group for
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Cu detection [35-40]. In this study, the Cu -catalyzed oxidative cyclization
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mechanism was also proposed for TPI-AD. After the addition of Cu to TPI-AD, a
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new benzotriazole compound (TPI-BTA) was formed, which was confirmed by the H
NMR spectrum (Fig. S7) and MALDI-TOF mass spectrum (Fig. S8). Fig. 5a showed
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the H NMR spectra of TPI-AD and TPI-BTA. The chemical shifts of protons H and
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H were not changed before and after the addition of Cu . Proton H was shifted
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downfield due to the formation of benzotriazole. H , H and H of amino-substituted
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benzene ring were also shifted downfield and the corresponding chemical shifts were
noted as H ’, H ’ and H ’. This phenomenon was ascribed to the electron-withdrawing
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property of the benzotriazole moiety. Further, the MALDI-TOF mass spectrum
showed a peak at m/z 429.2, which was assigned to the cyclization product of
TPI-BTA (Fig. 5b). The mole ratio of the cyclization reaction of between TPI-AD and
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Cu was confirmed to be 1:4 derived from the Job’s titration of fluorescent spectra
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(Fig. S9). Therefore, the Cu -catalyzed oxidation cyclization was verified to be the
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detection mechanism of probe TPI-AD towards Cu .

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Fig. 5. (a) H NMR spectra of TPI-AD (black line) and TPI-BTA (red line) in DMSO-d ; (b)
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MALDI-TOF mass spectrum of TPI-AD (black line) and TPI-BTA (red line).
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3.7 Biological application
To further demonstrate the practical utility of probe in biological samples, the
bioimaging experiment was carried out in living cells of MCF7. Firstly, MCF-7 cells
were treated with TPI-AD (20 ꢀM) for 30 min at 37 ºC, but there was almost no
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fluorescence, which is consist with the fluorescence titration results. Further, the
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MCF-7 cells were incubated with Cu (80 ꢀM) for another 30 min at 37 ºC. As
expected, the CLSM image showed obvious fluorescence, indicating probe TPI-AD is
cell permeable and the fluorescence enhancement behavior is not affected within
biological system (Fig. 6a). Therefore, the probe TPI-AD has the potential application
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for the detection Cu in biological system.
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Fig. 6. Fluorescence images of MCF-7 cells treated with: (a) TPI-AD; (b) TPI-AD + Cu . (Left)
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Bright field images; (Middle) Fluorescence images; (Right) Merged images.
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4. Conclusion
In summary, a novel fluorescence probe TPI-AD bearing azoaniline and arylimidazole
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moieties was designed and synthesized for the detection of Cu . The probe TPI-AD
exhibited a naked-eye visible color change from colorless to lake-blue together with a
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220-fold fluorescence enhancement upon the addition of Cu . The colorimetric and

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fluorescent dual channel response of TPI-AD towards Cu was ascribed to the
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Cu -catalyzed oxidative cyclization. The “reactive” probe TPI-AD possessed the
high sensitivity and selectivity over other common metal ions, and the detection of
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TPI-AD was calculated to be 14.4 nM. Furthermore, the intracellular Cu imaging
demonstrated that probe TPI-AD is available in biological systems.
Acknowledgements
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The authors gratefully thank Prof. XiaoZhong Qu of University of Chinese Academy
of Sciences (UCAS) for helping the bioimaging experiments.
This study was financially supported by National Natural Science Foundation of
China (NSFC 51273170) and the General Project of the Education Department of
Hunan Province (11C0705).

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References and notes
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A novel fluorescence probe TPI-AD bearing azoaniline and arylimidazole
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moieties was synthesized and applied for Cu detection.
The probe TPI-AD exhibited 220 fold fluorescence enhancement and a
“naked-eye” visible color change.
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The detection limit was down to 14.4 nM in aqueous media.
The application of probe in living cells was successfully realized.