A Readily-Synthesized Fluorescent Probe Based on N, N-Bis (Pyridin-2-Ylmethyl) Aniline for Copper(II) Detection in Aqueous Solution

Article abstract of DOI:10.1007/s10895-016-1922-0

In this paper, probe 1 based on N, N-bis (pyridin-2-ylmethyl) aniline is prepared as an effective fluorescent probe for Cu²⁺. It exhibits good sensitivity and selectivity for Cu²⁺ over other metal ions in aqueous solution. The detect

Full text of DOI:10.1007/s10895-016-1922-0

J Fluoresc
DOI 10.1007/s10895-016-1922-0
ORIGINAL ARTICLE
A Readily-Synthesized Fluorescent Probe Based on N, N-Bis
(Pyridin-2-Ylmethyl) Aniline for Copper(II) Detection
in Aqueous Solution
Xiaoyu Liu1 & Yuan Zhang2
Received: 22 June 2016 /Accepted: 26 August 2016
#
Springer Science+Business Media New York 2016
Abstract In this paper, probe 1 based on N, N-bis (pyridin-2-
ylmethyl) aniline is prepared as an effective fluorescent probe
for Cu2+. It exhibits good sensitivity and selectivity for Cu2+
over other metal ions in aqueous solution. The detection limit
of probe 1 is 56 nM. Furthermore, probe 1 is pH-insensitive
under near-neutral conditions and can work well in environ-
mental samples.
and more significant. So far, many analytical technologies have
been developed to detect Cu2+, including Cu2+-specific-
DNAzyme based fluorescence probes [10], atomic absorption
spectrometry [11], quantum-dot based measurements [12] and
electrochemical probes [13]. Although these technologies show
advantages of good sensitivity and selectivity, their application
is affected by the need for complicated equipment and difficult
synthesis procedure. Different from other analytical techniques,
fluorescent probes for the detection of Cu2+ have had great
popularity due to specificity, high sensitivity, low cost, conve-
nient, and real-time monitoring in biological and environmental
samples [14]. In this terms, the design and synthesis of Cu2+
probes have attracted considerable attentions in recent years and
demand is still high [15–17]. In the course of practical applica-
tion, different environmental samples were measured, for exam-
ple, tap, lake and river water or human hair, serum and urine,
which have been widely reported [18–20]. It can be found that
many factors can have an impact on measuring environmental
samples, such as pH, coexisting ions interference, temperature,
and so on. In view of the above questions, we designed a simple,
water-soluble fluorescent probe, probe 1, based on N, N-bis
(pyridin-2-ylmethyl) aniline, applied to the detection Cu2+ in
pure water system. It exhibits good sensitivity and selectivity
for Cu2+ over other metal ions in aqueous solution. Furthermore,
probe 1 is pH-insensitive under near-neutral conditions and can
work well in environmental samples.
.
.
Keywords Fluorescentprobe Copperion Aqueoussolution
Introduction
Copper has received great attention in chemical, environmental,
and biological systems in that it is a transition metal essential for
plants, animals and humans [1, 2]. At higher level, however, it is
very harmful to organisms such as bacteria, viruses, and humans
[3, 4]. Variety of copper ions was investigated to be associated
with some serious diseases such as prion diseases and
Alzheimer’s disease [5–7]. In addition, copper deficiency may
cause Menkes disease, colon cancer, and so on [8, 9]. Therefore,
the measurement and detection of copper ions has become more
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-016-1922-0) contains supplementary material,
which is available to authorized users.
* Xiaoyu Liu
shtxiaoyu@buu.edu.cn
Experimental
* Yuan Zhang
zhangyuan333@126.com
Materials and Instruments
1
College of Applied Science and Technology, Beijing Union
University, Beijing 102200, China
All materials were purchased from commercial suppliers and
used without further purification. Silica gel (200–300 mesh,
Qingdao Haiyang Chemical Co.) was used for column
2
College of Biochemical Engineering, Beijing Union University,
Beijing 100023, China

J Fluoresc
chromatography. 1H NMR spectra were acquired on a Bruker
AMX400 spectrometer. Chemical shifts (δ) were reported in
ppm relative to a Me4Si standard. Electrospray ionization
(ESI) mass spectra were measured with an LC-MS 2010 A
(Shimadzu) instrument. Fluorescence measurements were ob-
tained on a Hitachi F-2500 fluorescence spectrometer with a
10 mm quartz cuvette. The pH measurements were made with
a Sartorius basic pH-meter PB-10.
A
0.3
0.2
0.1
0.0
1
+
2
+
1 Cu
300
350
400
450
500
550
wavelength (nm)
Synthesis of Compound 1
B
The POCl3 (9 mL, 97 mmol) was added to DMF (30 mL), the
mixture was cooled to 0 °C in an ice-water bath. After stirring
for 0.5 h, a mixture of N, N-bis (pyridin-2-ylmethyl) aniline
(5.4 g, 20 mmol) and DMF (10 ml) was added. The reaction
solution was heated to 95 °C for 5 h, then the reaction was
terminated. The reaction solution was adjusted to a neutral pH
value by a saturated K2CO3 solution, extracted with CH2Cl2,
dried over anhydrous MgSO4, finally Compound 1 was iso-
lated by silica gel column (Fig. 1) (VPE / VEA = 5/1). NMR
of Compound 1 is shown in Supporting Information, Fig. S1.
1
2000
1500
1000
500
0
+
2
+
1 Cu
500
600
700
wavelength (nm)
Fig. 2 a Absorption spectra and b fluorescence spectra of probe 1 (5 μM)
before and after addition of Cu2+ (15 μM), measured in PBS buffer
(5 mM, pH = 7). The spectrum was acquired after Cu2+ addition for
0.5 h at room temperature. Λex = 385 nm
Synthesis of Probe 1
(4-methyl-n-methyl-pyridinium hydriodide) (2.36 g,
10 mmol), compound 1 (3.03 g, 10 mmol) and 30 mL
morpholine (morpholine) was sequentially added to 50 ml of
n-butanol, and refluxed 2.5 h. Then, the reaction mixture was
cooled to room temperature, orange solid was precipitated,
suction filtration, and further separated by silica gel column
(CH2Cl2 as eluent). ESI -MS calcd for C26H24N4 [M]+
393.2074, found 393.2067. Probe 1 was finally collected
(Fig. 1) (2.5 g, 49.3 %). HRMS of Probe 1 is shown in
Supporting Information, Fig. S2.
corresponding orange-colored fluorescence maximum at
575 nm (Figs. 2a, b). After Cu2+ addition, the maximum absorp-
tion peak underwent a 81 nm blue shift from 426 to 345 nm,
coupled with the fluorescence intensity decreased to original 8 %
(Fig. 2b).
The fluorescence quenching phenomenon was attributed to
the strong chelation of Cu2+ with probe 1, which decreases the
electron-donating ability of the central nitrogen atom of probe
1 and thereby results in a decrease in intramolecular charge
transfer (ICT) efficiency [21]. In addition, the paramagnetic
nature of Cu2+ can also be a factor for the quenching.
According to Job plot (Fig. S3), probe 1 and Cu2+ formed a
1:1 stoichiometry complex [22].
Results and Discussion
Spectral Response of Probe 1 to Cu2+
Sensitivity of Probe 1 toward Cu2+
The spectral response of probe 1 to Cu2+ was studied in PBS
buffer (5 mM, pH 7). In the absence of Cu2+, probe 1 displayed
one major absorption band centered at 426 nm with a
Probe 1 showed high sensitivity for Cu2+ detection in PBS
solution (5 mM, pH =7). As seen in Fig. 3a, upon an increasing
Fig. 1 The schematic synthesis
route of probe 1

J Fluoresc
A
2500
2000
1500
1000
500
0
500 550 600 650 700 750
wavelenght (nm)
B
Fig. 4 The black bar: fluorescence responses of probe 1 (5 μM) toward
various metal ions (15 μM for all ions); The red bar: fluorescence
responses of probe 1 (5 μM) toward Cu2+ in the presence of various
metal ions (15 μM for all ions). All experiments were performed after
reacting with metal ions for 0.5 h in PBS solution (10 mM, pH = 7).
2500
2000
1500
1000
500
+
y=-182.578 x 2161.308
2
R
=0.9969
λex = 385 nm
Mg2+, Ca2+, Mn2+, Co2+, Zn2+, Cd2+, Pb2+, Al3+ and Fe3+.
Under the same conditions (5 mM PBS, pH = 7), probe
1 exhibited no apparent fluorescence change in the pres-
ence of other metal ions (Fig. 4). Moreover, the interfer-
ence of the various metal ions on monitoring Cu2+ was
also studied. These results showed probe 1 possesses
high selectivity toward Cu2+ even in the presence of
other metal ions.
0
0
2
4
6
8
10 12
+
2
Cu ( M)
Fig. 3 a Fluorescence responses of probe 1 (5 μM) toward Cu2+ (final
concentration: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 μM). b the linear
relationship between fluorescence intensity and Cu2+ concentrations.
All experiments were performed after reacting with metal ions for 0.5 h
in PBS solution (5 mM, pH = 7). λex = 385 nm
addition of Cu2+ (final concentration: 0 ~ 11 μM), the
solution containing probe 1 (5 μM) presented a gradual
decrease in fluorescent intensity at 575 nm. There was a
good linearity (R2 = 0.9969) between the fluorescence
intensity and Cu2+ concentrations in the range of
0 ~ 11 μM (Fig. 3b). The detection limit of probe 1 is
56 nM (Detection limit =3σ/k [26], Where σ is the stan-
dard deviation of blank measurement, k is the slope of the
plot of fluorescence intensity versus Cu2+ concentration.).
As shown in Table 1, the detection limit of several tech-
niques was listed and compared [23–25].
pH-Dependent Fluorescence of Probe 1
Finally, the effect of pH changes on the fluorescence detection
of Cu2+ was tested. As shown in Fig. 5, probe 1 presented
limited changes toward pH in the range of 3–11, which proves
that probe 1 is pH-insensitive under near-neutral conditions.
The same result was obtained when Cu2+ was added in the
presence of the probe. Therefore, probe 1 could work well in
environmental samples.
2500
2000
The Selectivity of Probe 1 to Cu2+
To evaluate the selectivity of probe 1, it was tested with other
environmentally relevant metal ions, including K+, Na+, Ag+,
1500
1
+
2
1000
500
0
+
1 Cu
Table 1 The comparison of detection limit of several techniques
Technique
Detection limit Reference
Electrochemical sensor
0.03 μM
23
3
4
5
6
7
8
9
10 11
Cu2+-dependent DNA ligation DNAzyme 5 μM
24
pH
Colorimetric methods
Fluorescent probe
0.9 μM
25
Fig. 5 The fluorescence intensity at 575 nm responds to various PBS
solution with different pH values = 3, 4, 5, 6, 7, 8, 9, 10, 11; the black line:
probe 1 (5); the red line: probe 1(5 μM) + Cu2+ (15 μM); λex = 385 nm
0.056 μM
This work

J Fluoresc
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Conclusions
In summary, We have successfully developed a readily-
synthesized fluorescent probe for copper ions (Cu2+) in aque-
ous solution. It displayed several adventages because of high
sensitivity, selectivity, low-cost and operational simplicity.
The detection limit of probe 1 is 0.056 μM, which was far
below the WHO acceptable limit (31.5 μM) in drinking water.
In our further study, practical applications using real environ-
mental samples will be carried out, and the proposed method
may have potential use in many application areas such as
biological and ecological samples.
Acknowledgments We gratefully acknowledge assistance from Dr.
Hailiang Nie.
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Ultrasensitive detection of Cu2+ with the naked eye and application
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