A Schiff-based fluorescence sensor for the detection of Cu2+ and its application in living cells

Article abstract of DOI:10.1016/j.jphotochem.2019.112326

A novel fluorescent and colorimetric probe FL was designed and synthesized for selective Cu²⁺ detection in aqueous solutions. The detection limit of probe FL for Cu²⁺ was determined to be 1.09 μM. Furthermore, probe FL was successfully used to recognize Cu²⁺ in living cells, indicating its high potential in biological imaging applications.

Full text of DOI:10.1016/j.jphotochem.2019.112326

Journal Pre-proof
A Schiff-based fluorescence sensor for the detection of Cu2 and its
+
application in living cells
Yi-Ru Chiou, Hongbin Yan, Chin-Feng Wan, Chi Yen Huang, An-Tai
Wu
PII:
S1010-6030(19)31890-8
DOI:
https://doi.org/10.1016/j.jphotochem.2019.112326
JPC 112326
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
6 November 2019
16 December 2019
17 December 2019
Please cite this article as: Chiou Y-Ru, Yan H, Wan C, Yen Huang C, Wu A-Tai, A Schiff-based
fluorescence sensor for the detection of Cu2+ and its application in living cells, Journal of
Photochemistry and amp; Photobiology, A: Chemistry (2019),
doi: https://doi.org/10.1016/j.jphotochem.2019.112326
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2019 Published by Elsevier.

2
+
A Schiff-based fluorescence sensor for the detection of Cu and its application in
living cells
a
b
c
a
d*
Yi-Ru Chiou , Hongbin Yan , Chin‐Feng Wan , Chi Yen Huang , An-Tai Wu
a. Graduate institute of photonics, National Changhua University of Education,
Changhua 50058, Taiwan
b. Department of Chemistry, Brock University, 1812 Sir Isaac Brock Way, St.
Catharines, ON L2S 3A1, Canada
c. Department of Medical Applied Chemistry, Chung Shan Medical University,
Taichung,Taiwan
d. Department of Chemistry, National Changhua University of Education, Changhua
5
0058, Taiwan
*
Corresponding author.
E-mail address: antai@cc.ncue.edu.tw
Graphic abstract
+
+
+
2+
2+
2+
2+
2+
2+
2+
3+
2+
2+
2+
3+
2+
FL
Li
Na
K
Ca
Mn
Hg
Fe
Cu
Co
Ni
Fe
Pb
Cd
Zn
Al
Mg
Pb2+
Highlight:
+2
1
. Probe FL can display selectivity toward Cu over other ions in DMSO/water (2/8,
v/v) and showed an obvious color change.
2
. The living cell image experiments demonstrate probe FL could be used to detect
2
+
intracellular Cu in living cells.

Abstract
A novel fluorescent and colorimetric probe FL was designed and synthesized for
selective Cu2+ detection in aqueous solutions. The detection limit of probe FL for
2
+
Cu was determined to be 1.09 μM. Furthermore, probe FL was successfully used to
recognize Cu2+ in living cells, indicating its high potential in biological imaging
applications.
2
+
Keywords: Colorimetric; Fluorescent; Living cells; Cu ions
1
. Introduction
Among various crucial analytes, heavy metal ions tend to be highly toxic and
harmful [1]. Cu2+ is an essential trace element and plays a crucial role in various
physiological processes in the human body [2]. However, the high accumulation of
copper can damage the central nervous system and increase the risk of Alzheimer’s
2
+
and Parkinson’s diseases [3, 4]. In addition, Cu is an environmental pollutant that
can hinder animal and plant growth when present in excessive amounts [5]. Therefore,
2
+
developing probes for Cu recognition in environmental applications is highly crucial.
2
+
Numerous fluorescent probes for Cu have been developed in the past decades [6–
2
+
1
5]. For rapid analysis and environment-friendly reasons, a probe that detects Cu by

simple methods and color changes is highly desirable.
In this study, a probe FL (Scheme 1) was synthesized and its ion recognition ability
was examined for a series of metal ions. Probe FL exhibited a high sensitivity and
2
+
selectivity toward Cu . In addition, fluorescence microscopy experiments indicated
2
+
that probe FL can be used to detect Cu present in living cells.
2
2
. Experimental section
.1 Materials and general methods
All reagents and solvents were obtained commercially and used without further
purification. Thin-layer chromatography (TLC) was performed using TLC plates
1
13
precoated with silica gel 60 F254. The H and C NMR spectra were recorded using a
Bruker AM300 spectrometer and referenced to the solvent signals. High-resolution
mass spectroscopy (HRMS) and electrospray ionization (ESI) spectra were obtained
using a mass spectrometer and Orbitrap mass spectrometer (Thermo Scientific™),
respectively. UV–visible spectra were measured using
a
Jasco V360
spectrophotometer with a diode array detector, and the resolution was set at 1 nm.
Fluorescence spectra were recorded using a Jasco FP-8300 fluorescence spectrometer.
Melting points were measured using a melting point apparatus without calibration. pH
was measured using a digital pH meter. Fluorescence microscopy was performed

using an IX-71 fluorescence microscope (Olympus, Center Valley, PA, USA).
2
.1.1 Synthesis of (9H-fluoren-9-ylidene)hydrazine (FL-1)
A solution of 9-fluorenone (0.50 g, 2.78 mmol) was dissolved in 10 mL of
ethanol, and 98% aqueous N
2
4
H (0.8 mL) was added into the resulting solution. The
reaction mixture was heated under reflux for 10 h. The reaction mixture was then
cooled to room temperature (RT), and the precipitate was filtered and dried to give a
1
yellow solid (0.49 g, 90%). H NMR (supporting information, Fig. S1) (300 MHz,
DMSO-d ): δ/ppm 8.18 (d, J = 6.9 Hz, 1H), 7.98 (s, 2H), 7.88 (d, J = 1.2 Hz, 1H),
6
1
3
7
.86–7.76 (m, 1H), 7.68–7.65 (m, 1H), 7.45–7.37 (m, 2H), 7.35–7.28 (m, 2H); C
NMR (supporting information, Fig. S2) (75MHz, DMSO-d ): δ/ppm 139.7, 139.6,
6
1
38.4, 137.3, 129.8, 129.1, 128.1, 128.0, 127.8, 125.5, 120.7, 120.2, 120.1.
.1.2 Synthesis of probe FL
2
(E)-2-(((9H-Fluoren-9-ylidene)hydrazono)methyl)-5-(diethylamino)phenol
(FL)
Compound FL-1 (0.50 g, 2.576 mmol) was dissolved in 20 mL ethanol followed by
addition of 4-(diethylamino)salicylaldehyde (0.497 g, 2.576 mmol). The resulting
solution was then heated under reflux for 12 h. After the reaction mixture was cooled
to RT, the solvent was removed using rotary evaporation and the residue was purified
by column chromatography on silica. The appropriate fractions, which were eluted
with ethyl acetate–hexane (1:4, v/v), were combined to evaporated under reduced
1
pressure to give probe FL as a red solid (0.76 g, 80%). H NMR (supporting

information, Fig. S3) (300 MHz, DMSO-d ): δ/ppm 11.18 (s, 1H), 8.87 (s, 1H),
6
8
6
.41(d, J = 7.5 Hz, 1H), 7.92–7.81 (m, 3H), 7.63–7.34 (m, 5H), 6.44–6.39 (m, 1H),
1
3
.21 (d, J = 2.4 Hz, 1H), 3.45–3.38 (m, 4H), 1.14 (t, J = 2.4 Hz, 6H); C NMR
(supporting information, Fig. S4) (75 MHz, DMSO-d
6
):δ/ppm 163.8, 161.6, 157.4,
1
1
52.6, 142.0, 140.7, 137.1, 132.9, 131.9, 131.3, 131.0, 128.8,128.7, 128.5, 122.5,
+
21.2, 120.9, 107.6, 105.3, 97.3, 44.5, 13.0; HRMS (EI) (m/z): [M+H] calcd for
C
24
H
23ON 369.1841, found: 369.1843.
3
2
.1.3 Cell culture and imaging
The MCF-7 cell line was routinely cultured in Dulbecco’s modified Eagle
medium (DMEM) supplemented with fetal bovine serum (10%) and antibiotic
penicillin/streptomycin (1%). All cells were cultivated under a humidified atmosphere
with 5% CO and 95% air at 37 °C. For fluorescence imaging of the probe in the cell
2
environment, MCF-7 cells were incubated with FL (20 µM) and phosphate buffer
solution (PBS) for 1 h at 37 °C. The cells were then washed twice with PBS to
remove the excess probe FL, and PFA was added to fix the cells for 30 min. The
fluorescence images were captured using an Olympus IX-71 fluorescence microscope.
2
+
2
.1.4 Detection of Cu in real water samples
To evaluate the practicality of the present method, lake water samples were examined.
The lake water samples were obtained from the White Sand Lake of Changhua

University of Education, Changhua, Taiwan. The lake water samples were filtered
through a 0.20 μm filtered membrane and then centrifugedat 12,000 rpm for 10 min.
Cu(ClO
4
2
) (10 equiv) were first dissolved in water of the above source, followed by
the addition of probe FL into each water sample.
3
3
. Results and discussion
.1. UV/vis and Fluorescence spectra of the probe FL for various metal ions
We used by UV/vis and fluorescence measurements to investigate the chemosensor
behavior of probe FL with metal ions in DMSO/water (2/8, v/v). The solution of
probe FL showed a major absorption band at 475 nm (Fig. 1). In the presence of Cu2+
3
+
and Al , the absorption spectra of probe FL showed a major band at 480 and 525 nm
with a red-shift, respectively. However, in the fluorescence spectra of probe FL (Fig.
2
) indicated that the probe FL alone and other cations except for Cu2+ exhibited a
considerably strong single fluorescence emission band at 565 nm when excited at 420
2
+
nm. On adding Cu , probe FL exhibited prominent fluorescence quenching. A color
2+
change from yellow to orange was observed only in the probe FL solution with Cu ,
which can be easily detected with the naked eye, as depicted in Fig. 3. The observed
color change and fluorescent quenching may be attributed to the binding of probe FL
with Cu2+ through the hydroxyl oxygen and imine nitrogen that chelate Cu . The
fluorescence quenching and color change were not observed when other ions were
2+

added to probe FL. The results indicated that probe FL is a sensitive probe that can be
2
+
used to detect Cu with the naked eye in environmental analyses.
3
.2. Fluorescence titration and binding studies
The fluorescence titration profiles (Fig. 4) indicate that the addition of 0.5
2
+
equivalents of Cu instantly led to a decrease of the fluorescence intensity at 565 nm
by more than 80%. The association constant for the probe FL-Cu2+ complex in
5
−2
DMSO/water (2/8, v/v) was determined to be 4.61 × 10 M by using the Stern–
2
+
Volmer plot (Fig. S5). The detection limit of probe FL for the analysis of Cu ions
was estimated to be 1.09 μM by using the expression 3σ/S, where σ is the standard
deviation of the control sample and S is the slope of the calibration curve (Fig. S6). A
2
+
Job plot indicated a 2:1 stoichiometric complexation of probe FL with Cu (Fig. S7).
2
+
In addition, the formation of 2:1 complex between probe FL and Cu was further
2
+
confirmed by the appearance of a peak at m/z 860, assignable to [2probe FL + Cu +
+
2
H
2
O + Na ] (Fig. S8).
3
.3. Application in real samples
For biological applications, probe FL can operate in a wide range of pH (Fig. S9).
The time course of fluorescence changes of probe FL in the presence of 10.0
2
+
equivalents of Cu in DMSO/water (2/8, v/v) was examined, and the result indicated
2
+
that the recognition interaction was nearly completed within 1 min of Cu addition

2
+
(
Fig. S10). The practical application of probe FL for the selective detection of Cu in
different water sources, such as lake, ground and tap water, was also demonstrated.
2
+
Cu was introduced to the water of different sources (200 μM). When probe FL was
added to each water sample solution, the fluorescence intensity was quenched
immediately (Fig. 5). This result indicated that probe FL is a sensitive sensor that can
be used in environmental analyses.
3
.4. Reversibility and competition experiments
The reversibility of probe FL toward Cu2+ was analyzed in the presence of EDTA
200 µM). As expected, the fluorescence intensity of probe FL, which was quenched
(
2
+
in the presence of Cu , recovered to approximately 75% of its original value upon the
addition of EDTA (Fig. 6). This reversibility is essential for the fabrication of devices
2
+
for detecting Cu .
1
To confirm the binding site, NMR titration experiments were performed. The H
NMR spectra of probe FL indicated that an enamine proton signal at 8.81 ppm
broadened and shifted to 8.79 ppm. Other proton signals also exhibited similar
marginal shifts (Fig. 7). These observations suggested the formation of the probe
2
+
FL-Cu complex.
Competition experiments were performed to study the influence of other ions on the
2
+
binding ability of the probe FL-Cu complex. Some interference was observed for

2
+
2+
+3
the detection of Cu in the presence of Hg and Fe (10.0 equiv). The fluorescence
2
+
2+
2+
quenching of probe FL-Cu may be due to the displacement of Cu by Hg and
+
3
2+
Fe from probe FL-Cu complex. (Fig. S11).
3
.5. Cell viability and fluorescence imaging of FL in live cells
To demonstrate the practical applications of probe FL, experiments were
2
+
conducted to examine its ability in detecting Cu in MCF-7 cells. As displayed in Fig.
2
+
8
, incubation of probe FL with MCF-7 cells without Cu gave yellow fluorescence
(Fig.8b). However, the intensity of intracellular yellow fluorescence was quenched in
2
+
the presence of Cu (Fig. 8d). Furthermore, the cytotoxicity of probe FL on MCF-7
cells was evaluated using the MTT assay. As displayed in Fig. 9, the results indicated
a cell viability of approximately 90% after treatment with probe FL and 48-h
incubation. Thus, probe FL can be used for detecting Cu2+ in living cells with
minimal cytotoxicity. Taken together, probe FL demonstrated advantages in practical
applications in biological systems.
4
. Conclusion
In summary, a simple probe FL was designed and synthesized as a selective
2
+
2+
fluorescent sensor for Cu . Probe FL exhibited selectivity toward Cu over other
2
+
ions in DMSO/water (2/8, v/v) and demonstrated an obvious color change with Cu .
The living cell image experiments indicated that probe FL can be used to detect

2
+
intracellular Cu ions in living cells.
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgment: The authors acknowledge the Ministry of Science and Technology,
R. O. C., for financially supporting this study (grant number: 107-2113-M-018-006).
Supporting information: The supplementary data associated with this article can be
found.
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Biographies
Yi-Ru Chiou is currently a PhD student in Graduate institute of photonics at National
Changhua University of Education. Her current research interests are sensors
chemistry.
Hongbin Yan is currently a professor in Department of Chemistry at Brock University
major in Natural and modified nucleic acids research.
Chin‐Feng Wan is currently an associate professor in Department of Medical Applied
Chemistry, Chung Shan Medical University major in biochip and sensors
research.
Chi Yen Huang is currently a professor in Graduate institute of photonics at National
Changhua University of Education major in liquid crystals research.
An-Tai Wu is currently a professor in Department of Chemistry at National
Changhua University of Education major in organic chemistry. Her current
research interests are sensors chemistry and carbohydrate research.

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Host + other cations
Host+Cu
+2
+
3
Host+Al
300
350
400
450
500
550
600
650
700
Wavelength (nm)
Fig. 1: Absorbance responses of FL (20 μM) with various metal ions (10 eq.) in
DMSO/water (2:8, v/v).
2400
2200
2000
1800
1600
1400
1200
1000
host and other cations
800
600
400
200
0
+2
Cu
450
500
550
600
650
700
Wavelength (nm)
Fig. 2: Fluorescence emission spectra of probe FL (20 μM) recorded in DMSO/water
2/8, v/v) (λem = 565 nm) after addition of 10 equiv. of various metal ions.
(
+
+
+
2+
2+
2+
2+
2+
2+
2+
3+
2+
2+
2+
3+
2+
FL
Li
Na
K
Ca
Mn
Hg
Fe
Cu
Co
Ni
Fe
Pb
Cd
Zn
Al
Mg
Fig. 3: The color changes observed by naked eye of probe FL (20 μM) upon addition
of 10 equiv. of various metal ions in DMSO/water (2/8, v/v).

2500
2000
1500
1000
0
eq.
(a)
3
0 eq.
500
0
450
500
550
600
650
700
750
Wavelength (nm)
(b)
2000
1500
1000
500
0
0
5
10
15
+
20
25
30
35
2
[
Cu ]/[FL]
Fig. 4: (a) Fluorescence spectra of probe FL (20 μM) in DMSO/water (2/8, v/v) upon
2
+
addition of increasing concentrations of Cu ; (b) Intensity (λem = 565 nm) of probe
2
+
FL as a function of Cu equiv.
2500
2000
1500
1000
500
0
r
r
r
r
r
ke
La
in
nk
dri
ate
ate
ate
d w
un
gro
ate
tch w
ate
w
HOST
w
g w
DI
ng
ni
di
run
2
+
Fig. 5: Fluorescence responses of FL (20 μM; λem = 565 nm) with Cu (10 eq.) in
different water source.

2500
2000
1500
1000
HOST
EDTA
EDTA
+2
Cu
+
Cu
2
500
0
+
Cu
2
1
2
3
4
5
6
Cycle index
2
+
Fig. 6: Fluorescence responses of FL (20 μM; λem = 565 nm) with Cu (10 eq.) and
EDTA in DMSO/water (2/8, v/v).
2
.0 eq.
1
.5 eq.
1
0
.0 eq.
.5 eq.
0
eq.
1
2+
Fig. 7: H NMR titration plots of probe FL with Cu in DMSO-d .
6

Fig. 8: (a) Bright field images of MCF-7 cells incubated with probe FL, (b) confocal
fluorescence microscopy images of MCF-7 cells incubated with probe FL, (c) bright
field images of MCF-7 cells incubated with probe FL-Cu2+ and (d) confocal
2
+
fluorescence microscopy images of MCF-7 cells incubated with probe FL-Cu .
100
50
0
0
5
10
Concentration (uM)
Fig. 9: Cytotoxicity assay of FL toward MCF-7 cells. MCF-7 cells were incubated
with 5 and 10 μM of probe FL for 48 h.

Scheme 1. Synthesis of probe FL.