Irreversible coumarin based fluorescent probe for selective detection of Cu2+ in living cells

Article abstract of DOI:10.1016/j.saa.2021.120313

Copper ion (Cu²⁺) is an essential part of the living organisms. Cu²⁺ ions play a vital role in many biotic processes. An abnormal amount of Cu²⁺ ions may result in serious diseases. Herein, a novel “fluorescent ON” probe NC-Cu to trace minute levels of Cu²⁺ ions in presence of various biological active species has been developed. Lysosomal cells targeting group (Morpholine) was added to the probe. The spectral properties of probe NC-Cu were recorded in HEPES buffer (0.01 M, pH = 7.4, comprising 50% CH₃CN, λ_ex = 430 nm, slit: 5 nm). The synthesized probe NC-Cu work based on copper promoted catalytic hydrolysis of hydrazone and shows remarkable fluorescence enhancement. The reaction of the probe with Cu²⁺ ions was completed within 20 min. An excellent linear relationship (R² = 0.9952) was found and the limit of detection (LOD, according to the 3σ/slope) for Cu²⁺ ions was calculated to be 5.8 μM. Furthermore, NC-Cu was effectively functional in the living cells (KYSE30 cells) to trace Cu²⁺ ions.

Full text of DOI:10.1016/j.saa.2021.120313

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 264 (2022) 120313
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Irreversible coumarin based fluorescent probe for selective detection of
Cu2+ in living cells
a
a,
Nadeem Ahmed a, Wajeeha Zareen a, Di Zhang b, , Xiaopeng Yang , Yong Ye
⇑
⇑
a Green Catalysis Center, and College of Chemistry, Zhengzhou University, Zhengzhou 450001, China
b Institute of Agricultural Quality Standards and Testing Technology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Probe NC-Cu displayed high selectivity, fast response (20 min), ultrasensitivity (0.65mM) and fluorescence
ꢀ
ꢀ
A selective Coumarin fluorophore
based probe was developed.
Newly synthesized probe shows
strong fluorescence induced by
Copper promoted catalytic hydrolysis
of hydrazone.
imaging of Cu2+ was attained in KYSE30 cells.
ꢀ
Imaging Cu2+ in KYSE30 cells was
attained with NC-Cu.
a r t i c l e i n f o
a b s t r a c t
Copper ion (Cu2+) is an essential part of the living organisms. Cu2+ ions play a vital role in many biotic
processes. An abnormal amount of Cu2+ ions may result in serious diseases. Herein, a novel ‘‘fluorescent
ON” probe NC-Cu to trace minute levels of Cu2+ ions in presence of various biological active species has
been developed. Lysosomal cells targeting group (Morpholine) was added to the probe. The spectral prop-
erties of probe NC-Cu were recorded in HEPES buffer (0.01 M, pH = 7.4, comprising 50% CH3CN, kex = 430-
nm, slit: 5 nm). The synthesized probe NC-Cu work based on copper promoted catalytic hydrolysis of
hydrazone and shows remarkable fluorescence enhancement. The reaction of the probe with Cu2+ ions
was completed within 20 min. An excellent linear relationship (R2 = 0.9952) was found and the limit
Article history:
Received 30 June 2021
Received in revised form 19 August 2021
Accepted 22 August 2021
Available online 26 August 2021
Keywords:
Fluorescent probe
Copper ions
KYSE30 cells
Catalytic hydrolysis
Fluorescence imaging
of detection (LOD, according to the 3
NC-Cu was effectively functional in the living cells (KYSE30 cells) to trace Cu2+ ions.
Ó 2021 Elsevier B.V. All rights reserved.
r
/slope) for Cu2+ ions was calculated to be 5.8 mM. Furthermore,
1. Introduction
ferent parts of the body [3]. The key amount of Cu2+ ion is present
in the liver. The average concentration of blood Cu2+ ion is found to
Copper is a transition metal that has four oxidation states Cop-
per0, Copper1, Copper11, and Copper111 [1]. Mostly copper in water
is present in divalent (Cu2+) form. Copper is considered the third
plentiful trace element in the human after zinc and iron [2]. It is
a vital trace element and is present in low deliberation in the dif-
be 15.7–23.6 l
M [4]. Limits of Cu2+ ions in drinking water were set
as 1.3 ppm by the U.S. environmental protection agency (EPA) [5].
Cu2+ ions play a fundamental role in physiological processes in liv-
ing organisms [6]. As Cu2+ ions have electrophilic nature it can be
used as a cofactor of many enzymes such as copper amine oxidase
[7], dopamine b-hydroxylase [8], Cu\Zn superoxide dismutase [9],
tyrosinase [10], ceruloplasmin and cytochrome c oxidase [11,12].
These cofactors are responsible for many functions in the body
such as handling of dietary amines, pigmentation, antioxidant
⇑
Corresponding authors.
E-mail address: yeyong03@tsinghua.org.cn (Y. Ye).
https://doi.org/10.1016/j.saa.2021.120313
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

N. Ahmed, W. Zareen, D. Zhang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 264 (2022) 120313
defense, neurotransmitter synthesis, and metabolism. On the
other-hand deficiency and excess concentration of Cu2+ ions leads
to many serious human genetic and metabolic disorders such as
Indian childhood cirrhosis (ICC), Indian prion disease, Huntington’s
disease, Alzheimer’s disease, Parkinson’s disease, Menkes disease,
Wilson disease, mitochondrial dysfunction to damage cells, obe-
sity, and diabetes [6,13–19]. Therefore, the development of effec-
tive and suitable methods for sensing Cu2+ ions in biological
samples is highly appreciated to understand its physiological func-
tions and uncontrolled events.
lane (TMS) as inner standard. UV–vis spectra were plaid on a
Lambda 35 UV–visible spectrometer, PerkinElmer. Fluorescence
saturation was done on a HITACHI F-4600 fluorescence spectrom-
eter, and the excitation and emission wavelength cuts were set at
5 nm, excitation voltage was 700 V. By means of methanol as
mobile phase mass spectra were recorded on a Q-Tof HR-MS spec-
trometer (Waters Micro mass). Fluorescence imaging by using
KYSE30 cells was studied under a LEICA TCS SP8 laser scanning
confocal microscope.
The conventional analytical methods for the detection of Cu2+
ions are mostly dependent upon instrumental techniques such as
atomic absorption spectroscopy (AAS), voltammetry, inductively
coupled plasma atomic emission (ICP-AES), and piezoelectric crys-
tals. These methods have some drawbacks such as required classy
instrumentation, time-consuming, and expensive [20–22]. Many
fluorescent probes have been reported for sensing copper ions
[23–29]. Most of them form complex with Cu2+ ions and as a result,
the fluorescence of the probe was quenched. Some probes showed
low water solubility and less selectivity. Recently, fluorescent
methods are getting the attention of the researchers. These meth-
ods are inexpensive, selective, and sensitive to trace copper ions.
Previously many synthesized probes work as ‘Turn Off’ mode
because of the paramagnetic nature of copper [30–32]. Now a
day, some ‘Turn On’ probes are synthesized for Cu2+ ions detection
[2,33–35].
Recently, our group has reported some fluorescent probes for
selective detection of Cu2+ions [36–38]. Herein, we have reported
a novel ‘fluorescence On’ probe NC-Cu for selective and sensitive
detection of Cu2+ ions among various biological active species
because of the hydrolysis of hydrazone derivatives by Cu2+ ions
(see Scheme 1). Carbonyl compounds can be protected by forming
hydrazone and these carbonyl compounds can be regenerated by
Cu2+ ions promoted hydrolysis of hydrazone [39]. To develop probe
NC-Cu we have used coumarin as a carbonyl source. Due to its high
water solubility, 1, 8-naphthalic anhydride derivative was used as
a source of the amino group. Also, we introduced a lysosomal tar-
geting group in our new design probe NC-Cu. Newly synthesized
probe NC-Cu work well in HEPES buffer solution having 50% CH3-
CN. Photo-physical properties of probe NC-Cu have been studied
in detail by some spectroscopic techniques such as UV–vis, fluores-
cence, and mass spectra. Cell imaging was taken by fluorescence
confocal microscopy.
2.3. Cell culture and imaging
KYSE30 cells were cultivated in Dulbecco’s Modified Eagle Med-
ium (DMEM) added with 10% fetal bovine serum, at 37° C, in 5%
CO2 incubator. For imaging, probe NC-Cu (10
KYSE30 cells and incubated at 37 °C for 30 min in 5% CO2 incubator.
PBS buffer (pH 7.4) solution was used to wash (three times) and
again treated with Cu2+ ions solution, and incubate them in 5%
CO2 incubator for 1 h. Cells were washed three times again with
PBS buffer (pH 7.4). Then the cells were imaged using Leica TCS
SP8 confocal microscope.
lM) was added to
2.4. Synthesis
Starting material (Naphthalic anhydride derivative 1 and Cou-
marin 2) were synthesised by previous reported method [40,41].
Synthesis of probe NC-Cu: probe NC-Cu was synthesized by
dissolving reactant
1 (0.131 g, 0.38 mmol) and 2 (0.10 g,
0.38 mmol) in absolute ethanol, catalytic amount of glacial acetic
acid (3 drops) was added and reflux for 12 h, reaction progress
was checked by thin layer chromatography (TLC). After consump-
tion of reactant as confirmed by TLC the solvent was removed
and crude product was subjected to column chromatography for
purification (eluant: CH2Cl2/MeOH = 50 /1, v/v) to get probe NC-
Cu as a red solid (112 mg) 1H NMR (400 MHz, CDCl3, TMS) d
(ppm): 1.26 (t, J = 7.4 Hz, 6H, CH2CH3), 2.53 (s, J = 13.76 Hz, 3H,
CH3), 2.71 (s, J = 5.73 Hz, 4H, NCH2CH2N), 3.47 (q, J = 6.30 Hz,
4H, CH2CH3), 3.73 (s, J = 8.13 Hz, 4H, OCH2), 4.39 (q, J = 3.43 Hz,
2H, NCH2), 6.50 (s, J = 3.25 Hz, 1H, PhH), 6.65 (s, J = 3.25 Hz, 1H,
PhH), 7.29 (s, J = 1.72 Hz, 1H, PhH), 7.41 (s, J = 2.60 Hz, 1H, PhH),
7.71 (s, J = 2.20 Hz, 1H, PhH), 7.81 (s, J = 2.53 Hz, 1H, PhH), 8.10
(s, J = 4.86 Hz, 1H, PhH), 8.19 (s, J = 2.15 Hz, 1H, PhH), 8.55 (s,
J = 5.18 Hz, 2H, PhH), 8.61 (s, J = 2.55 Hz, 1H, NH = N); 13C NMR
(CDCl3, 100 MHz): d = 164.17, 163.47, 160.56, 156.68, 151.42,
150.22, 147.48, 142.36, 134.59, 130.61, 130.10, 125.28, 122.25,
119.75, 118.80, 111.90, 109.86, 108.51, 108.34, 96.53, 66.64,
60.21, 59.72, 53.69, 44.63, 16.51, 14.67 ppm; HR-MS: m/z calcd
for [C33H35N5O5][H+] = 582.2711, Found: 582.2709.
2. Experimental
2.1. Materials
All chemicals and reagents including 1,8-Naphthalic anhydride
derivative, 2-morpholinoethan-1-amine, Hydrazine monohydrate
(80%), 7-(diethylamino) benzaldehyde, ethyl-3-oxobutanoate,
piperidine, acetic acid, ethanol, dichloromethane, and methanol
were used as such as provided by the seller. According to standard
procedures, solvents were purified for chemical synthesis and
analysis. Double purified water was used during the experimenta-
tion. Probe standard solution (1*10-3 M) was made in DMSO. The
solutions of ions and amino acid (10 mM) were made from con-
forming salts counting: NaCl, KCl, MgCl2, CuCl2, CaCl2, ZnCl2, Na2-
SO4, PbCl2, CdCl2, AgNO3, BaCl2, NiCl2, NaHS, HgCl2, NaNO3,
NaNO2, HSO-3, SO32-, NaOCl, Na2HPO4, Cysteine and Homo-cysteine.
3. Results and discussion
3.1. Plan and synthesis of NC-Cu
7-diethylaminocoumarin was used as a fluorophore due to its
good photostability, high emission effect, and large stoke shift
[42–45]. To increase water solubility hydrazine was attached with
1,8-Naphthalic anhydride derivative [46–48], and lysosomes tar-
geting group was added to newly synthesized probe NC-Cu. Probe
work in HEPES buffer solution having 50 % CH3CN. Cu2+ ions pro-
mote hydrolysis of hydrazone. We have planned and prepared a
new probe NC-Cu in a very easy method with a good yield. The
new probe NC-Cu was confirmed by 1H NMR, 13C NMR, and HR-
MS [Fig. S1-S3].
2.2. Apparatus
Nuclear magnetic resonance (NMR) spectra were noted on a
Bruker DTX-400 spectrometer in d-chloroform with tetramethylsi-
2

N. Ahmed, W. Zareen, D. Zhang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 264 (2022) 120313
Scheme 1. Synthetic route for title compound. Reagents and conditions: a) 1,8-Naphthalic anhydride derivative, 2-morpholinoethan-1-amine, Ethanol, 12 h reflux, b)
Hydrazine monohydrate (80%), EtOH, reflux 24 h c) 7-(diethyl amino) benzaldehyde, ethyl-3-oxobutanoate, piperidine, CH3COOH, EtOH, reflux 12 h. d) 1 and 2, acetic acid,
ethanol, reflux 12 h.
3.2. Spectral reaction of probe NC-Cu toward Cu2+ ions
of probe NC-Cu shows a high selectivity toward Cu2+ ions which
demonstrates that probe NC-Cu could used for Cu2+ ions detecting
by the absorption spectra method.
The Cu2+ ions tracing ability of newly synthesized probe NC-Cu
was confirmed by some spectroscopic techniques. The selectivity
of probe NC-Cu was studied by ultra-violet (UV) and fluorescence
spectroscopy. Firstly, UV selectively was performed. We patterned
experiments in HEPES buffer (0.01 M, pH = 7.4, containing 50%
CH3CN). The probe itself had an intense absorption band centered
at 492 nm. When the solution of probe NC-Cu was treated with
Cu2+, Zn2+, HS-, Cd2+, Pb2+, Ba2+, Mg2+, Hg2+, Ni2+, Ag+, Na+, K+,
NO2–, NO3–, SO23-, HSO-3, ClO-, HPO-4, Cys and Hcy. The only changes
were observed with Cu2+ ions. The absorption band centered at
492 nm was disappeared and two new absorption bands centered
at 444 nm and 497 nm appeared (as shown in Fig. 1) after the addi-
tion of Cu2+ ions. There was no change upon the addition of other
ions and amino acids to the solution (Fig. S4). UV selectivity result
Next, we achieved fluorescence selectivity of probe NC-Cu. The
probe had almost no fluorescence in absence of Cu2+ ions due to
C=N isomerization (protection of carbonyl group), however, a
strong emission peak centered at 495 nm emerged as Cu2+ ions
solution was added to the probe solution (Fig. 2). Cu2+ ions pro-
mote hydrolysis of hydrazone and free the highly fluorescent cou-
marin derivative. The quantum yields of probe NC-Cu is 0.007.
After its reaction with Cu2+, the quantum yield was calculated as
0.23. There was no change in fluorescent intensity when other ions
such as Zn2+, HS-, Cd2+, Pb2+, Ba2+, Mg2+, Hg2+, Ni2+, Ag+, Na+, K+,
NO–2, NO3–, SO32-, HSO-3, ClO-, HPO-4, and amino acid (Cys and Hcy)
were added to probe solution (Fig. S5). This result indicates that
probe NC-Cu has high discrimination toward Cu2+ ions.
3

N. Ahmed, W. Zareen, D. Zhang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 264 (2022) 120313
Fig. 4. The emission spectra of probe NC-Cu (10 mM) at 430 nm when treated with
Cu2+ ions (10 equiv.) with time in HEPES buffer (0.01 M, pH = 7.4, containing 50%
CH3CN, kex = 430 nm, slit: 5 nm).
Fig. 1. The UV–vis spectra of probe NC-Cu (10 mM) with Cu2+ ions (10 equiv.) in
HEPES buffer (0.01 M, pH = 7.4, containing 50% CH3CN).
Fig. 2. The emission spectra of probe NC-Cu (10 mM) with Cu2+ (10 equiv.) in HEPES
buffer (0.01 M, pH = 7.4, containing 50% CH3CN, kex = 430 nm, cuts: 5 nm).
Fig. 5. Emission spectra of probe NC-Cu (10 mM) in the existence of Cu2+ ions (0–
120 mM) in HEPES buffer (0.01 M, pH = 7.4, containing 50% CH3CN, kex = 430 nm, slit:
5 nm).
Next, we performed a competitive study to sightsee the func-
tionality of probe NC-Cu as a fluorescence sensor to trace Cu2+ ions
in the occurrence of interfering species. When probe NC-Cu was
treated with Cu2+ ions in presence of ions such as Zn2+, HS-, Cd2+
,
Pb2+, Ba2+, Mg2+, Hg2+, Ni2+, Ag+, Na+, K+, NO–2, NO–3, SO23-, HSO3- ,
ClO-, HPO-4, and amino acid (Cys and Hcy), only a slight change
was found with concomitant species, except for H2S that can attack
the double bond of hydrazone and prohibit the copper promoted
hydrolysis (Fig. 3). Competitive study with other metal ions and
amino acids show that fluorescence intensity of probe NC-Cu was
not changed by other ions and amino acids except H2S. Probe
NC-Cu shows a fast response to Cu2+ ions and complete hydrolysis
of probe NC-Cu with Cu2+ ions was materialized within 20 min
(Fig. 4 and Fig. S6). These results show that the probe NC-Cu can
be used for the detection of Cu2+ ions and cell imaging.
3.3. Fluorescent titration of probe NC-Cu with Cu2+ ions
Fig. 3. The emission spectra of NC-Cu (10 mM) at 495 nm changes upon
accumulation of ions such as Zn2+, HS-, Cd2+, Pb2+, Ba2+, Mg2+, Hg2+, Ni2+, Ag+, Na+,
K+, NO–2, NO3–, SO32-, HSO3- , ClO-, HPO4- , Cys and Hcy (10 equiv.) in the occurrence of
Cu2+ ions (10 equiv.) in HEPES buffer (0.01 M, pH = 7.4, containing 50% CH3CN,
kex = 430 nm, slit: 5 nm).
Next, fluorescence titration spectra of probe NC-Cu with Cu2+
ions were recorded. The probe itself had almost no emission, but
4

N. Ahmed, W. Zareen, D. Zhang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 264 (2022) 120313
3.4. The outcome of pH on the fluorescence
The consequence of pH on the probe respond to Cu2+ ions was
studied. The pH solutions 1 to 13 were prepared. The experiment
was performed with the fixed concentration of probe NC-Cu and
Cu2+ ions (10.0 mM). The result shows that probe NC-Cu in the
free-state has low fluorescence intensity. On the other hand, the
fluorescence intensity of the probe in presence of Cu2+ ions is
enhanced at pH 7 (Fig. 6). However, the fluorescence intensity at
lower than 7 or higher than 7 pH is decreased because of protona-
tion of the nitrogen atom at lower pH and deprotonation at higher
pH. The pH result shows that the probe NC-Cu can work at the
physiological pH range.
3.5. Sensing mechanism
According to the described literature [49], subsequently, an
anticipated reaction mechanism was labeled in Scheme 2. The
probe NC-Cu was hydrolyzed to 1 and 2. As a result, ICT process
was enhanced and strong emission showed. The product of probe -
NC-Cu with Cu2+ ions was confirmed by the HRMS spectrum. A
Fig. 6. The emission spectra of probe NC-Cu (10 mM) with pH (1–13) in the absence
and presence of Cu2+ ions (10 equiv.).
+
peak at m/z 768.88 consistent with [C33H41Cl2CuN5O8] was pre-
sent in the MS spectrum (Fig. S8). The intense peak at m/z 260.14
was related to the coumarin derivative [C15H18NO3]+ (Fig. S9).
The sensing mechanism was also proved by 1H NMR spectra. After
the addition of Cu2+ ions to the probe solution, the mixture was
the emission intensity of the probe was slowly increased upon
treatment with a low concentration of Cu2+ ions solution. The
inclusion of Cu2+ ion was sustained till a stage touched when there
were no more enhancements in fluorescence intensity (Fig. 5).
With the addition of Cu2+, probe NC-Cu exhibited a significant
and progressive emission enhancement at 495 nm. This enhance-
ment in fluorescence intensity was linearly associated with the
addition of Cu2+ solution in the range of 0–120 mM (R2 = 0.9952,
subjected to column chromatography and coumarin
1 was
obtained. The 1H NMR spectra confirmed it (Fig. S10). These results
demonstrated that Cu2+ could promote hydrolysis of hydrazone.
3.6. Cell cytotoxicity and imaging
Fig. S7). The limit of detection (LOD, conferring to the 3r/slope)
for Cu2+ ions was determined as low as 5.8 mM. This showed a fairly
To examine whether probe NC-Cu can be useful to screen intra-
high sensitivity for Cu2+ ions.
cellular Cu2+ ions levels in living cells, bioimaging investigation
Scheme 2. Proposed reaction mechanism of probe NC-Cu in presence of Cu2+.
5

N. Ahmed, W. Zareen, D. Zhang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 264 (2022) 120313
easily promote hydrolysis of probe NC-Cu. The outcome proposes
that probe NC-Cu is an effective indicator for monitoring the level
of Cu2+ ions in the living cells. This probe is promising as an effec-
tive lysosomal-targetable fluorescent probe for exploring Cu2+ ions
in biological samples.
4. Conclusions
Here, we have planned and prepared a new probe NC-Cu (cou-
marin based hydrazone) by a simple and easy method in a single
step. The newly synthesized hydrazone derivative showed high
selectivity and sensitivity toward Cu2+ ions in the presence of var-
ious coexisting species and amino acids. Hydrazone derivative is a
very effective fluorescent sensor and exhibits strong fluorescence
intensity in the presence of paramagnetic Cu2+. This fluorescence
enhancement is because of the catalytic hydrolysis of hydrazone
by Cu2+ ions. The probe is promising as an effective lysosomal-
targetable fluorescent probe for exploring Cu2+ ions in biological
samples. The synthesis and application of more precise fluorescent
probes for imaging in living cells, tissues and animals is our present
attention and target in future.
Fig. 7. Toxicity assay of probe NC-Cu at different concentration in KYSE30 cells.
was achieved in KYSE30 cells by confocal fluorescence microscopy.
Earlier to these tests, we confirm the toxicity of the probe NC-Cu in
living cells. MIT assay was done to confirm the toxicity of probe in
cells. As is clarified in Fig. 7, cell feasibility was greater than 80%
even when the concentration of probe NC-Cu was 20 mM. This rep-
resented the low toxicity of probe NC-Cu in live KYSE30 cells.
Due to the low cytotoxicity, high selectivity and sensitivity of
probe NC-Cu, we further exposed the probe to image Cu2+ in the
living cells. The cells (KYSE30) were nurtured with probe NC-Cu
(10 mM) for 30 min at 37 °C in 5% CO2. As shown in Fig. 8b, the
image of probe showed no emission in the blue region in absence
of Cu2+ ions. When the cells (KYSE30) were incubated with Cu2+
ions (10 equiv.), a blue emission was detected from the confocal
microscopic images (Fig. 8e). This indicated that Cu2+ ions could
5. Credit author statement
Mr. Nadeem Ahmed does the experiments and prepares the
manuscript.
Miss. Wajeeha Zareen helps to write the manuscript.
Dr. Di Zhang revises the manuscript.
Mr. Xiaopeng Yang helps to draw some schematics and analy-
sis the data.
Prof. Yong Ye helps to design experiments and to revise the
manuscript thoroughly.
Fig. 8. Confocal fluorescence images of Cu2+ ions with probe NC-Cu in KYSE30 cells. (a,b) KYSE30 cells pretreated with NC-Cu (10
lM) for 30 min. (d,e) KYSE30 cells were
pretreated with probe NC-Cu (10
l
M) for 30 min and then incubated with Cu2+ ions (10 equiv.) for 1 h. (c) and (f) are fused images of a with b, d with e.(excitation: 440 nm,
emission: 495 nm).
6

N. Ahmed, W. Zareen, D. Zhang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 264 (2022) 120313
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Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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This work was financially supported by the Natural Science
Foundation of China (21907025) and Scientific and Technological
Innovation Project of Henan Academy of Agricultural Sciences
(2020CX05).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.120313.
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