Study on the photochromism, photochromic fluorescence switch, fluorescent and colorimetric sensing for Cu2+ of naphthopyran-diaminomaleonitrile dyad and recognition Cu2+ in living cells

Article abstract of DOI:10.1016/j.saa.2020.118191

A well-designed naphthopyran-diaminomaleonitrile dyad (sensor 1) has been synthesized successfully, its molecular structure was well characterized by NMR and mass spectrometry. Sensor 1 exhibits excellent photochromic and photochromic fluorescence switch

Full text of DOI:10.1016/j.saa.2020.118191

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Study on the photochromism, photochromic fluorescence switch,
fluorescent and colorimetric sensing for Cu2+ of naphthopyran-
diaminomaleonitrile dyad and recognition Cu2+ in living cells
1
1
⁎
Heyang Zhang , Tianyuan Zhong , Nan Jiang, Zhuo Zhang, Xue Gong, Guang Wang
Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Faculty of Chemistry, Northeast Normal University, Changchun 130024, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A well-designed naphthopyran-diaminomaleonitrile dyad (sensor 1) has been synthesized successfully, its mo-
lecular structure was well characterized by NMR and mass spectrometry. Sensor 1 exhibits excellent photochro-
mic and photochromic fluorescence switch performance with reversible color change and good fatigue resistance
upon alternating ultraviolet irradiation and thermal bleaching. In addition, sensor 1 displayed excellent fluores-
cent and colorimetric sensing ability towards Cu2+ ions with high selectivity and sensitivity. The addition of 5.0
equiv. of Cu2+ ions into sensor 1 (1 × 10−5) in CH3CN solution significantly quenched the fluorescence of sensor 1
by 80.0%. Furthermore, the addition of Cu2+ ions also caused the complete disappearance of the absorbance band
at 350–450 nm in absorbance spectra of sensor 1 and accompanied by the distinct color change form yellow to
colorless. Job's plot, mass spectrometry, 1H NMR titration and DFT calculations proved that sensing performance
was attributed to the formation of 1:1 sensor 1-Cu2+complexes. Sensor 1 can monitor the existence of Cu2+ ions
in living cells via the fluorescence images. Sensor 1 showed great potential applications as chemosensor and pho-
tochromic materials.
Received 10 December 2019
Received in revised form 21 February 2020
Accepted 22 February 2020
Available online 24 February 2020
Keywords:
Photochromism
Photochromic fluorescence switch
Fluorescent and colorimetric chemosensor
Copper ions
Naphthopyran
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
for a wide variety of enzymes in all living organisms and in various redox
processes and pigments [14,15]. On the other hand, excess existence of Cu2
Photochromic materials have potential applications in optical
switches, optical storage, optoelectronic devices and ultraviolet
protecting due to their reversible photo-induced molecular isomeriza-
tion between two isomers whose absorption spectra are distinguishably
different [1–3]. Naphthopyran with excellent photochromic perfor-
mance such as the breadth of color generated, excellent thermal revers-
ibility, absence of background color, easy control over fading kinetics,
and good resistance to photochemical fatigue has been extensively
studied in the field optical storage, controlled release materials,
chemosensors and fluorescent switches [4–7]. So exploring the new
molecular naphthopyran with multiple functions is significant.
In recent years, fluorescent and colorimetric chemosensors are becom-
ing increasingly important in recognition and detection of metal ions due
to their fast recognition process, in-situ detection, precise accurateness
and low-cost [8–11]. All heavy metal ions play an important role in organ-
isms, in which, Cu2+ ions perform a dual effect on physiology of human
beings [12,13]. On the one hand, Cu2+ ion is essential trace metal ion to
sustain normal human health, for examples, Cu2+ ions sever as a cofactor
+ in living body can cause some serious diseases, such as prion disease,
neurodegenerative diseases, irritation of nose and throat resulting in nau-
sea, vomiting, and diarrhea others [16–18]. Therefore, it is necessary to
monitor and detect copper ions in water and other environmental sam-
ples. Although the traditional detection methods for Cu2+ ions such as
atomic absorption spectroscopy (AAS) [19], inductively coupled plasma
atomic emission spectrometry (ICP-AES) [20] and electrochemical
methods (EM) [21] display high sensitivity and accuracy, their analysis
process requires sophisticated equipment, tedious sample preparation
procedure and trained operators [22]. Due to the easy detection operation,
up to now, a variety of fluorescent and colorimetric chemosensors of Cu2+
ions including small organic molecules, polymers, nanoparticles were ex-
plored [23–26]. Even so, there is a need to explore new high sensitive
and selective fluorescence chemosensor for Cu2+ ions is still necessary.
Schiff base, condensation of reactive aldehydes and amines, is a
family of important compounds with various potential applications.
Some of Schiff base compounds were used as electron donor to
coordinate with metal ions in fluorescent chemosensors [27,28].
Diaminomaleonitrile has special steric structure and containing four ni-
trogen atoms, our research work had proved that it can selectively coor-
dinate with Cu2+ ion and further induce the obvious fluorescence
change of the whole molecule [29].
⁎
Corresponding author.
E-mail address: wangg923@nenu.edu.cn (G. Wang).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.saa.2020.118191
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
In this paper, a new Schiff base composed of naphthopyran and
was washed with water for three times and dried with anhydrous so-
dium sulfate. The product was purified by silica gel column chromatog-
raphy using CH2Cl2:petroleum = 7:1 as eluent to give faint yellow
powder, 0.7 g, yield, 62.3%. M.p. 184–186 °C. 1H NMR (DMSO,
500 MHz), δ 8.33 (d, J = 7.6 Hz, 2H), 8.27 (d, J = 10.5 Hz, 1H), 8.13
(d, J = 8.9 Hz, 1H), 7.96 (d, J = 12.2 Hz, 2H), 7.85 (d, J = 8.8 Hz, 1H),
7.54 (d, J = 9.8 Hz, 1H), 7.48 (d, J = 7.3 Hz, 4H), 7.35 (dd, J = 16.1,
8.5 Hz, 5H), 7.25 (t, J = 7.3 Hz, 2H), 6.63 (d, J = 10.0 Hz, 1H). 13C
NMR (151 MHz, CDCl3) δ 159.01, 157.15, 155.28, 144.51, 133.23,
130.36, 128.23, 126.98, 124.20, 122.36, 119.45, 118.92, 114.31, 113.72,
112.36, 108.95. HRMS Calcd. for C30H20N4O [M + H+]: 452.1637,
Found: 453.1717.
diaminomaleonitrile has been developed (Scheme 1). The designed
Schiff molecule not only displayed good photochromic performance
and photochromic fluorescence switch but also presented fluorescent
and colorimetric sensing ability for Cu2+ ions in CH3CN solution with
high selectivity and sensitivity.
2. Experimental
2.1. General methods
Diaminomaleonitrile and 1,1-diphenyl-2-propyn-1-ol was pur-
chased from Sinopharm Chemical Reagent Co., Ltd. p-Toluenesulfonic
acid (PTSA) was recrystallized from ethanol. Solvents were purified by
normal procedures and handled under moisture free atmosphere. The
other materials were commercial products and used without further
purification. The salts used in stock solutions of metal ions are PbCl2,
Zn(NO3)2·6H2O, Cd(NO3)2·4H2O, Cu(NO3)2·3H2O, Co(NO3)2·6H2O,
SrSO4, HgCl2, Cr(NO3)3·9H2O, FeSO4·7H2O, Ni(NO3)2·6H2O, AgNO3,
GaCl3, BaCl2·2H2O, Al(NO3)3·9H2O, MnCl2·4H2O, InCl3·4H2O. All the
stock solutions of metal ion were prepared in water.
3. Results and discussion
3.1. Design and synthesis of sensor 1
Our group has extensively studied on naphthopyran as photochro-
mic materials in fluorescence switching and sensor, and also used
diaminomaleonitrile as coordination unit to fabricate chemosensor for
Cu2+ and Fe3+ ions [4–6,29]. In this paper, the naphthopyran and
diaminomaleonitrile were combined by Schiff base to prepare a bifunc-
tional compound (sensor 1) with photochromic properties and sensing
ability. The molecular structure of sensor 1 was well characterized with
1H NMR, 13C NMR and mass spectroscopic methods (Figs. S1 and S2). It
is interesting that sensor 1 not only displayed excellent photochromic
property but also presented selective fluorescence and colorimetric
sensing ability for Cu2+ ions.
1H and 13C NMR spectra were recorded on a Varian Unity Inova
Spectrometer at room temperature using d-chloroform and
dimethylsulfoxide-d6 as solvent. CE-MS spectra were obtained on CESI
8000 Plus-Triple TOF4600 mass spectrometer. UV–Vis absorption and
fluorescence spectra were measured on Varian Cary 500 spectropho-
tometer and Cary Eclipse Fluorescence Spectrophotometer, respec-
tively. The ultraviolet light source for irradiation was CHF-XM35
parallel light system (Beijing Changming Technology Co. Ltd) with a
500 W xenon lamp and monochromatic filter (360 nm). The irradiating
light reached to the samples was parallel and its intensity was 1.2 mW/
cm2.
3.2. Photochromic performance and photochromic fluorescence switch of
sensor 1
3.2.1. Photochromic performance
2.2. Synthesis of sensor 1
The absorbance spectra of sensor 1 (1 × 10−5 M) in CH3CN solution
were shown in Fig. 1. The absorption band was at between 338–410 nm
with two maximum absorption peak at 363 nm and 382 nm, which was
ascribed to the π~π* electron transition of naphthopyran [6,7]. After UV
light irradiation, the new absorbance band between 405–600 nm with a
peak at 487 nm appeared and gradually increased with the irradiation
time indicating the occurrence of ring-open reaction of naphthopyran
ring. The absorbance reached the maximum value after UV irradiation
for 5 min accompanied by color change of the solution from colorless
to light brown (insert in Fig. 1b). The naphthopyran unit on sensor 1
underwent an electrocyclic pyran-ring opening with cleavage of the C
(sp3)-O bond. The photochromic reaction of naphthopyran molecule is
illustrated in Fig. 1a, the more planar structure (the so called ‘open
form’ including TT and TC structures) with greater conjugation formed,
which is responsible for the increased absorption band in the visible
range of the spectrum. After ceasing of UV irradiation and the solution
was put in the dark, the open-form of naphthopyran converted back
to the closed-form accompanied by the gradually disappearing of the
absorbance band in visible range and the color of solution via the ther-
mal effect [30,31]. The absorption intensity at 487 nm gradually de-
creased with time and dropped down near to the original level after
1 h, the fading curve of the absorption intensity at 487 nm as the func-
tion of time was displayed in Fig. 1c. The fading kinetic was evaluated by
2.2.1. Synthesis of 3,3-diphenyl-[3H]naphtho[2,1-b]pyran-8-aldehyde
(compound a)
1,1-Diphenyl-2-propyn-1-ol (1.668 g, 8 mmol), 6-Hydroxy-2-
naphthaldehyde (2.096 g, 12 mmol) and p-toluenesulfonic acid
(PTSA) (0.1 equiv.) were added into 60 mL dry CH2Cl2 and stirred
under nitrogen atmosphere at room temperature for 40 h. The reaction
mixture was washed with water and dried with anhydrous sodium sul-
fate. The crude product was purified by column chromatography on sil-
ica gel (eluent: CH2Cl2:petroleum ether = 1:2) to give white powder,
1.64 g, yield 56.3%. 1H NMR (500 MHz, CDCl3), δ: 10.05 (s, 1H), 8.16
(s, 1H), 8.01 (d, 1H, J = 9.0 Hz), 7.92–7.90 (m, 1H), 7.78 (d, 1H, J =
8.5 Hz), 7.47 (d, 4H, J = 7.5 Hz), 7.34–7.30 (m, 4H), 7.28–7.24 (m,
4H), 6.30 (d, 1H, J = 10 Hz).
2.2.2. Synthesis of sensor 1
A solution of diaminomaleonitrile (0.40 g, 3.2 mmol) in 20 mL meth-
anol was added into the solution of 3,3-Diphenyl-[3H]naphtho[2,1-b]
pyran-8-aldehyde (0.89 g, 2.5 mmol) in 30 mL methanol. The reaction
mixture was refluxed for 24 h and then cooled to room temperature.
After the solvent was evaporated under reduced pressure, the crude
product was dissolved into 100 mL CH2Cl2. The obtained organic solvent
CHO
OH
HO
NH2
O
H2N
NC
O
OHC
NH2
CN
C
N
N
CH3OH, 70 o
C
CN
Sensor 1
a
Scheme 1. The synthetic route of sensor 1.

H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
3
Fig. 1. a) The photochromic reaction of sensor 1; b) the changes of the absorbance spectra and color of sensor 1 in (1 × 10−5 M) in CH3CN solution before (black) and after (red) UV light
irradiation; c) the kinetic curve of sensor 1 in (1 × 10−5 M) in CH3CN solution monitored at 487 nm. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
fittingthefollowingbiexponentialequation[32]:A(t)=A1e−k1t +A2e−-
k2t + Ath with the experimental data. The fitting curve derived from Or-
igin 8.0 computer program was almost superimposed over the
experimental plot and the correlation coefficient of the curve fitting
(R) was greater than 0.999, which indicated that biexponential model
is suitable for photo-kinetic analysis of sensor 1. All the fading kinetic
constants were shown in Table 1. The fading speed was slower and
the half-life period 600 s was obvious longer than those in other reports
[4,31,33], which may be due to the formation of large conjugation with
diaminomaleonitrile moiety via Schiff base unit.
The fatigue resistance is an important parameter for practical appli-
cation. Upon the alternative UV irradiation for 5 min and putting in the
dark for 1 h, the colorless solution of sensor 1 in CH3CN changed alter-
nately between colorless and orange. In the meantime, the absorption
band of open-form naphthopyran centered at 487 nm rose and decayed
alternately, which indicated that the ring-open and ring-close reactions
alternately proceeded upon the alternative UV irradiation and putting in
the dark. The reproducibility of absorbance at 487 nm after each color-
ation/fading sequence was shown in Fig. 2a and no marked absorption
degradation was observed after 6 cycles (Fig. 2b). All above results dem-
onstrated that sensor 1 had an excellent photochromic performance
and great potential application value as a photochromic candidate.
isomerization of naphthopyran on the fluorescence of sensor 1 was
also investigated. Fig. 1 demonstrated that UV irradiation induced the
ring-open reaction of naphthopyran accompanied by the appearance
of the new absorbance band. The new absorbance band overlapped
with the fluorescence band at a large extent, it is reasonable to speculate
that the energy transfer could take place and the fluorescence of sensor
1 will be quenched. However, it was unexpected that the fluorescence of
sensor 1 (1 × 10−5 M) in CH3CN solution after UV light irradiation was
not quenched but enhanced gradually upon the UV irradiation time, as
shown in Fig. 3a. The fluorescence intensity at 520 nm was enhanced
by 19.6% [(I-I0)/I0] after UV irradiation for 15 min, and would not further
increase if the irradiation time was increased. This result illustrated the
fluorescence emission of sensor 1 can't be absorbed by the open-ring
form of naphthopyran. The fluorescence of sensor 1 came from its
whole molecule, so it is reasonable to deduce that the fluorescence en-
hancement was caused by the enlarged conjugation system of the
whole molecule due to the planar structure of open-ring naphthopyran.
After UV irradiation, the solution was put in the dark for 1 h, the fluores-
cence band decreased to the original location before UV irradiation,
which was due to the ring-close isomerization of pyran ring. This result
demonstrated that sensor 1 had the property of the fluorescence switch
and the fluorescence intensity could be modulated in the range of
0–19.6%. The fatigue resistance is an important factor for a fluorescence
switch, so the fluorescence intensity at 520 nm was measured upon the
alternative UV irradiation for 15 min and put in the dark for 1 h, the re-
sult was shown in the Fig. 3b. The reproducibility can be seen after each
UV irradiation/put in the dark sequence and the fluorescence degrada-
tion was not observed after 6 cycles. All the above result demonstrated
that sensor 1 possessed the excellent photochromic fluorescence switch
performance. In addition, this result also illustrated that the fluores-
cence emission can't be absorbed by its open-ring structure.
3.2.2. Photochromic fluorescence switch
The sensor 1 also displayed strong fluorescence emission (Fig. 3a)
and emission band was between 417 and 680 nm with maximum emis-
sion peak at 520 nm (λex = 385 nm). Our previous research results
demonstrated naphthopyran unit only emitted very weak fluorescence,
and diaminomaleonitrile almost had no fluorescence emission, there-
fore the fluorescence emission of sensor 1 came from the conjugation
system of whole molecule of sensor 1. Naphthopyran moiety has been
used to fabricate the photochromic fluorescence switches via the energy
transfer mechanism [5,6,34], therefore the modulation effect of
3.3. Fluorescent sensing of sensor 1 towards Cu2+ ion and fluorescence im-
ages of live cells
Table 1
3.3.1. Fluorescent sensing of sensor 1 towards Cu2+ ions
Kinetic data of sensor 1 in CH3CN solution.
Diaminomaleonitrile can selectively coordinated with metal ions
and further influence the photophysical properties of the whole com-
pound in which it existed [29,35]. Therefore, the effect of metal ions
on the photophysical properties was investigated.
λmax
(nm)
A1
A2
Ath
k1 × 10−2
(s−1
k2 × 10−2
t1/2
(s)
R
)
(s−1
)
487
0.0214
0.1237
0.00786
14.09
6.9
600
0.9997

4
H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
Fig. 2. a) The changes of the absorbance spectra of sensor 1 (1 × 10−5 M) in CH3CN solution upon the UV irradiation and thermal fading at room temperature. b) the photochromic cycles of
sensor 1 (1 × 10−5 M) in CH3CN solution monitored at 487 nm before and after sequential steps of UV irradiation and being put in the dark. (For interpretation of the reference to color in
this figure, the reader is referred to the web version of this article.)
The first factor to consider for a chemosensor is high selectivity.
Many factors, such as the polarity, solubility, pH value and protophilia
affected the selectivity of sensor. Therefore, the pure solvents DMSO,
CH3CN and THF, the mixed solvents containing Tris buffer (10 mM,
pH = 7.2), DMSO:H2O (1:1, V:V), THF:H2O (1:1, V:V), THF:H2O (4:1,
V:V) and CH3CN:H2O (1:1, V:V) were used to investigate the absorbance
and fluorescence responses of sensor 1 to metal ions. In the most above
solvent systems, sensor 1 displayed irregular response to different metal
ions. Only in pure CH3CN, sensor 1 showed satisfactory high selective
absorbance and fluorescence response towards Cu2+ ions.
Sensor 1 in this work displayed high fluorescent emission capacity,
so we try to explore its availability as a sensitive and selective fluores-
cence chemosensor. Fig. 4a showed the fluorescence spectra of sensor
1 (1 × 10−5 M) in CH3CN solution and its mixed solution with different
metal ions (5 equiv. of sensor 1). Sensor 1 itself in CH3CN solution emit-
ted strong fluorescence with an emission peak at 520 nm under the ex-
citation of UV light of 385 nm. Naphthopyran is less fluorescence and
diaminomaleonitrile itself hardly emits fluorescence, so it is reasonable
to speculate that the fluorescence of sensor 1 originated from the whole
molecule of sensor 1. Expect for Cu2+ ion, the addition of the following
metal ions including Zn2+, Cd2+, Hg2+, Co2+, Al3+, Cr3+, In3+, Pb2+, Sr2
+, Ag+, Ni2+, Ca3+, Fe2+, Mn2+ and Ba2+ ions did not give obvious ef-
fect on the fluorescence spectra of sensor 1 and just caused a small fluc-
tuation up and down. On the contrary, the addition of 5 equiv. Cu2+ ions
caused the remarkable fluorescence quenching by 80.0% calculated
from the fluorescence intensity at 520 nm and the fluorescence changed
from “on” to “off”. The fluorescence photographs of the above solutions
further illustrated the selective quenching effect of Cu2+ ion on the
fluorescence of sensor 1. Under the irradiation of 360 nm light, the sen-
sor 1 solution displayed bright yellow fluorescence, the addition of the
abovementioned metal ions did not cause the obvious fluorescence
changes, as shown in Fig. 4b. The yellow fluorescence of the solution
with Cu2+ ions only disappeared completely indicating the strong fluo-
rescence quenching effect of Cu2+ ion on the sensor 1. These results in-
dicated that sensor 1 had the high selective fluorescence response
towards Cu2+ ions. Therefore, based on the changes of fluorescence
spectra and fluorescence color, it is safe to say that sensor 1 could be
used as a selective fluorescent sensor to recognize and monitor the ex-
istence of Cu2+ ions.
The fluorescence quenching of sensor 1 by Cu2+ ion can be ascribed
to its coordination with Cu2+ ion. Cu2+ ion and sensor 1 are tightly com-
bined after complexation, the ligand to metal charge transfer (LMCT)
from the excited states of sensor 1 to the d-orbitals of copper ions in
the complex molecule occurred due to the strong paramagnetic nature
of Cu2+ ion, which thus quenched the fluorescence. The other compet-
itive metal ions did not form stable complexes and just cause the negli-
gible fluorescence change, which may be due to the following two
reasons. One was the unsuitable spatial coordination geometry confor-
mation of diaminomaleonitrile, another was the inappropriate ion ra-
dius and insufficient binding energy of these metal ions for ligand.
This kind of the fluorescence quenching mechanism of chemosensors
by Cu2+ ion have been explained in previous reports [36,37].
The stoichiometry of sensor 1-Cu2+ complex was tested by Job's plot
method, a constant total concentration (2 × 10−5 M) of sensor 1 and Cu2
+ and their molar ratio varied. And then, the fluorescence spectra of so-
lutions were measured and the emission intensity at 520 nm was
Fig. 3. a) The changes of the fluorescence spectra of sensor 1 in (1 × 10−5 M) in CH3CN solution after UV irradiation. b) The fluorescence fatigue resistance upon the alternative UV
irradiation for 15 min and put in the dark for 1 h.

H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
5
Fig. 4. Fluorescence spectra (a) and fluorescence photographs (b) of sensor 1 (1 × 10−5 M) in CH3CN solution and its mixed solution with different metal ions (5 equiv. of sensor 1,
respectively).
plotted against the fraction of Cu2+ ions and the maximum value ap-
peared at very close to 0.5 M fraction on the Job's plot pointing out 1:1
binding stoichiometry between sensor 1 and Cu2+ ion (Fig. 5a). The
stoichiometry was then confirmed by positive ESI-HRMS. After adding
the excess Cu2+ salt into the sensor 1 solution in DMSO and shaking
for enough time to finish the complexing reaction, the supernatant
was taken out to be measured for MS spectrum (Fig. S4). The mass spec-
trum of [sensor 1 + Cu2+] showed a peak at m/z = 515.0439, which
equiv. of Cu2+ ion into the sensor 1 (1.0 × 10−5 M) in CH3CN solution,
the fluorescence intensity at 520 nm was monitored, the result was
shown in Fig. S5. The fluorescence intensity gradually decreased with
time and reached the lowest level after 15 min, which indicated sensor
1 had a fast fluorescence response towards Cu2+ ion and was suitable in
the real-time test.
The fluorescence titration was also used to investigate the sensing
properties. Here, the fluorescence titration experiment of sensor 1
(1 × 10−5 M) in CH3CN solution upon the incremental addition of Cu2
+ ion from 0.5–5 equiv. The fluorescence spectra gradually decreased
with the increase of Cu2+ ion (Fig. 6a) and the fluorescence intensity
at 520 nm presented a very good linear relationship with the concentra-
tion of Cu2+ ions in the range of 0.01–0.1 mM with a correlation coeffi-
cient of R2 = 0.9813 (Fig. 6b). The wide linear range is beneficial for
practical quantitative measurements. The limit of detection (LOD) was
also calculated to be 4.03 μM by the equation of LOD = 3σ/k, here k
and σ were the slope of the calibration curve and standard deviation
of the blank solutions, respectively. The obtained limit of detection
was much lower than the limit of copper (~20 μM) in drinking water
permitted by US Environment Agency (EPA) [38] and general concen-
was attributed to [sensor 1 + Cu2+–H+ +
] (calcd. 514.0855). These re-
sults confirmed the formation of sensor 1-Cu2+ complex with 1:1
stoichiometry.
Another necessary consideration is the anti-interference ability of
sensor 1 towards the competitive metal ions. The competitive experi-
ments could further confirmed the selectivity of sensor 1 to Cu2+ and
evaluate the interference effect of the coexistence metal ions on the de-
tection of sensor 1 for Cu2+ ions. Therefore, the competitive experiment
was carried out by measuring the fluorescence intensity at 520 nm of
the series of the solutions of sensor 1 in CH3CN (1 × 10−5 M) containing
5 equiv. of Cu2+ ion and 20 equiv. of the other metal ions. From the re-
sult as shown in Fig. 5b, all the fluorescence under the coexistence of Cu2
+ ion and any one of the above test metal ions was quenched to the sim-
ilar level of that only existence of Cu2+ ion. This result demonstrated
that the coexistence of other metal ions did not interfere with fluores-
cent sensing of sensor 1 for Cu2+ ion and further indicated sensor 1 pos-
sessed the practical application potential of sensing Cu2+ ions.
The fast fluorescence response of sensor 1 towards Cu2+ ion was
more favorable for the real-time detection. After the addition of 5
tration of blood copper in normal individuals (1.57
× –
10−5
2.36 × 10−5 M) defined by the U.S. Environmental Protection Agency
[39]. This result indicated that sensor 1 possessed the practical sensing
ability of quantitative detection of Cu2+ concentration in solution.
The affinity of sensor 1 with Cu2+ ion can be estimated using the
data from fluorescence titration experiment. The association constant
(Ka) for the sensor 1-Cu2+ complex was evaluated using the Benesi-
Fig. 5. a) Job's plot from the fluorescence intensity at 520 nm of sensor 1 and Cu2+ ion in CH3CN solution with the total concentration of 20 μM. b) Selectivity of sensor 1 (1 × 10−5 M) in
CH3CN solution with 5 equiv. of Cu2+ ions over other competitive metal ions (20 equiv. of sensor 1).

6
H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
Fig. 6. a) The changes of fluorescence spectra of sensor 1 in CH3CN solution (1 × 10−5 M) upon successive addition of Cu2+ (0.5–5 equiv.). (b) The changes of the fluorescence intensity at
520 nm of sensor 1 with the increase of Cu2+ concentration (R2 = 0.9813).
Hildebrand (B-H) plot according to the following equation [30].
F0 and F are the fluorescence intensities of sensor 1 at 520 nm in the
absence and presence of Cu2+ ions, respectively. Ksv is the Stern-Volmer
quenching constant and [M] is the concentration of Cu2+ ion. As shown
in Fig. 7b, within the range of test concentration, F0/F (blue line) was not
linear with Cu2+ concentration, which indicated the fluorescence
quenching of sensor 1 upon the coordination by Cu2+ ions did not con-
form to dynamic quenching process. On the contrary, the lnF0/F values
displayed approximate linear relationship with Cu2+ concentration,
the fitting linear relationship R2 was 0.9507. This result was in agree-
ment with the Perrin model of static quenching (black line), which fur-
ther supports that fluorescence quenching in this systems occurs
intramolecularly and also indicate that all the added Cu2+ ions were
bound to sensor 1.
1=ðF0−FÞ ¼ 1=fKaðF0−FminÞCg þ 1=ðF0−FminÞ
where F0 and F are the fluorescence intensities of sensor 1 only and in
the presence of Cu2+ ions, C is the concentration of Cu2+ in the solution.
Fmin is the minimum fluorescent intensity at 520 nm in the presence of
excess Cu2+ ions. The fitted curve based on the 1:1 binding stoichiome-
try of sensor 1 and Cu2+ was linear and almost superimposed over the
experimental plot with a correlation coefficient over 0.9881 (Fig. 7a),
which further confirmed the 1:1 binding stoichiometry of sensor 1
with Cu2+. Ka was calculated from the slope and intercept of the linear
plot be 8287 M−1. The above results indicated that sensor 1 was a
good candidate as a selective “on–off” fluorescent chemosensor for
Cu2+ ions.
3.3.3. Fluorescence imaging for live cells
In view of the fluorescent sensing performance of sensor 1 to Cu2+
ions, we investigated its practical application to monitor copper ion in
living cells. The fluorescent cell imaging experiments were conducted
using liver cancer HepG2 cells. First, the solutions of sensor 1
(1 × 10−5 M) in DMSO and Cu2+ ions (1 × 10−4 M) in the empty me-
dium solution were prepared. Then, in each of well 1 and 2, 500 μL of
cell suspension and 10 μL of Cu2+ solution were added. And after, the
cells were incubated for 30 min at 37 °C and then washed three times
with PBS. At last, well 1 was added 10 μL solution of sensor 1 and was
further incubated for 50 min at 37 °C and washed three times with
PBS. Finally, the cells were washed for three times with PBS solution
and then were fixed, stained and mounted onto glass slides and the
fluorescence images were taken under the excitation wavelength at
3.3.2. Stern-Volmer analysis
Stern-Volmer analysis was often used to confirm the fluorescence
quenching nature of a fluorophore in the complexation process with
metal ions. The plots of the fluorescence intensity (F0/F or lnF0/F) at
520 nm of sensor 1 against the concentration of Cu2+ ion and showed
a linear graph [34].
F0
F
¼ 1 þ KSV ½Mꢀ dynamic quenching
F0
F
ln ¼ eK ½Mꢀ static quenching
SV
Fig. 7. a) Benesi–Hildebrand plot of sensor 1 upon gradual addition of Cu2+ ions. B) Stern-Volmer quenching plots for dynamic quenching and static quenching of sensor 1 by Cu2+ ion.

H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
7
The above obvious color change and absorption decrease of sensor 1
solution upon the addition of Cu2+ ions were also ascribed to the com-
plexation between the electron-donating diaminomaleonitrile and ac-
ceptor Cu2+ ion. The other competitive metal ions did not form stable
complexes and not cause the negligible change of the absorption
spectra.
The next necessary consideration is anti-interference ability of sen-
sor 1 towards the competitive metal ions. The competitive experiments
could further confirmed the high selectivity of sensor 1 to Cu2+ ions.
Therefore, the competitive experiment was carried out by measuring
the absorbance spectra of the series of sensor 1 (1 × 10−5 M) in
CH3CN solutions containing 5 equiv. of Cu2+ ion and 5 equiv. of the
other metal ions, respectively. As shown in Fig. 10a, the ordinate values
are the absorbance at 382 nm, the absorption intensity of sensor 1-Cu2+
at 382 nm in the presence of the other competitive metal ions was not
influenced by the competitive metal ions. This result indicated that sen-
sor 1 possessed the high colorimetric selectivity for Cu2+ ion and the
other coexistent competitive cations did not interfere in the colorimet-
ric sensing of sensor 1 for Cu2+ ion.
Fig. 8. Confocal fluorescence images of HepG2 cells. (a) fluorescence image of HepG2 cells
pretreated just with sensor 1. (b) fluorescence image of HepG2 cells pretreated with Cu2+
(1 × 10−4 M) for 30 min and then incubated with sensor 1 (1 × 10−5 M).
405 nm. The fluorescence images were shown in Fig. 8, the cells just
treated only with sensor 1 showed bright blue fluorescence. Whereas,
the fluorescence of the cells incubated with Cu2+ and sensor 1 almost
disappeared, the residual blue color was the result of cell stain. These re-
sults indicated that sensor 1 can permeate the cell and coordinated with
Cu2+ ions, which was accompanied by the fluorescence quenching.
Based on these fluorescence images, it is safe to say that sensor 1 can
be used to monitor the existence of Cu2+ ions in living cells.
The UV–visible absorbance titration of sensor 1 by Cu2+ ions was
also carried out in CH3CN (1 × 10−5 M) to evaluate the colorimetric
sensing sensitivity. The absorbance spectra of sensor 1 solution after ad-
dition of the different concentration of Cu2+ ions were recorded and
shown in Fig. S6. The UV–vis absorption band at 330–450 nm decreased
gradually with the increasing of Cu2+ ion. The absorption intensity at
382 nm showed good linear relationship with the Cu2+ concentration
3.4. Selective colorimetric sensing of sensor 1 towards Cu2+ ions
from 0 to 5 equiv. of sensor 1 with a correlation coefficient of R2
=
0.98951 (Fig. S7). The better linear was in favor of quantitative detection
of Cu2+ ions. In addition, the detection limit (LOD) was also calculated
to be 5.39 × 10−4 mol/L from the calibration curve according to the
equations LOD = 3σ/K defined by IUPAC. Here, σ is the standard devia-
tion of the blank measurement and K is the slope of the calibration curve
of the absorption intensity as a function of Cu2+ concentration [32,33].
The associate constants (Ka) for the interaction of sensor 1 with Cu2+
ion was also estimated from the titration data using the Benesi–
Hildebrand plot according to the following equation [10].
Previous studies on fluorescence spectra showed that sensor 1 could
selectively coordinated with Cu2+ ions and further influenced the fluo-
rescence properties of sensor 1. Therefore, the changes of absorbance
spectra and color of sensor 1 in CH3CN solution upon the addition of
Cu2+ ions was also investigated.
The most direct proof for selective colorimetric sensing ability of
sensor 1 towards Cu2+ ion was that the yellow color of solution
(3 × 10−5 M) in CH3CN straightway turned to colorless after addition
of Cu2+ ion (5.0 equiv. of sensor 1) (Fig. 9a). On the contrary, the addi-
tion of the other metal ions including Zn2+, Cd2+, Hg2+, Co2+, Cu2+, Al3
+, Cr3+, In3+, Pb2+, Sr2+, Ag+, Ni2+, Ga3+, Fe2+, Mn2+and Ba2+ ions
(5.0 equiv. of sensor 1) did not induce the solution color change. This re-
sult directly indicated that the sensor 1 can selectively recognize Cu2+
ions and visually confirm the existence of Cu2+ ions.
The selective color change of sensor 1 solution upon the addition of
Cu2+ ions was further confirmed by the absorbance spectra, shown in
Fig. 9b. The addition of 5 equiv. Cu2+ ions reduced the absorbance
band in wavelength range of 350 nm~450 nm nearly to vanished. Sim-
ilar with the color change, the addition of the other metal ions did not
cause the obvious changes, which further indicated the high selectivity
and colorimetric sensing ability of sensor 1 towards Cu2+ ions.
1
A0
1
Ka
1
ꢁ ðA0–AminÞ ꢁ C þ –Amin
A0
−A ¼
where A and A0 are the absorption intensities at 382 nm in the present of
any given concentration and in the absence of Cu2+ ion, respectively.
Amin is the lowest value of the absorption intensity at 382 nm in the
presence of excess Cu2+ ions. The Benesi-Hildebrand plot was drawn
using 1/(A0 − A) as function of 1/[Cu2+] and showed a linear plot
(Fig. 10b). The fitting plot based on 1:1 stoichiometry (sensor 1:Cu2+
)
is almost superimposed over the experimental curve with a correlation
coefficient over 0.99934 indicating the 1:1 binding mode of sensor 1-
Cu2+ complex. The associate constant Ka of sensor 1 with Cu2+ was
Fig. 9. a) The photographs of sensor 1 (3 × 10−5 M) in CH3CN solutions containing different metal ions (5 equiv. of sensor 1). (b) The absorbance spectra of sensor 1 (1 × 10−5 M) in CH3CN
solution and different metal ions (5 equiv. of sensor 1). (For interpretation of the reference to color in this figure, the reader is referred to the web version of this article.)

8
H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
Fig. 10. a) Absorbance response at 382 nm of sensor 1 (1 × 10−5 M) in CH3CN solution upon the addition of different metal ions (5.0 equiv. of sensor 1). b) Benesi-Hildebrand plot of sensor
1 in CH3CN solution (1 × 10−5 M) in the presence of Cu2+ (0–5 × 10−5 M) (R2 = 0.99934). (For interpretation of the reference to color in this figure, the reader is referred to the web
version of this article.)
calculated from the slope and the intercept of the fitting line to be
5370 M−1, this value was close to Ka obtained from fluorescence titra-
tion. The large affinity also verified sensor 1 was sensitive enough to
sense Cu2+ ions in solution over the other metal ions. Although the
sensing performance of sensor 1 was carried out in non-aqueous sol-
vent, the metal ions were in pure water solutions. Therefore, sensor 1
could be useful for the detection of Cu2+ ions in chemical and environ-
mental samples.
naphthaline on sensor 1 showed slight downshift and widening with
the gradual addition of Cu2+ ions, which was ascribed to the coordina-
tion of diaminomaleonitrile moiety with Cu2+ ion. The result of 1H NMR
indicated the existence of interaction of sensor 1 with Cu2+ ion.
To further clarify the coordination mode of sensor 1 with Cu2+ ions,
density functional theory (DFT) calculations with the Gaussian 09 pro-
gram were carried out to provide a theoretical explanation [40]. The
CYL view program was used to generate the 3D molecular structures
[41]. All studied complexes were optimized at the B3LYP level [42] of
density functional theory, together with vibrational frequency calcula-
tions to obtain minima of energy. The LANL2DZ was employed for the
Cu atom with effective core potentials (ECPs) for its core electrons
[43,44]. The 6-31G(d) basis set was used to describe the C, H, O and N
atoms [45].
3.5. Proposed binding mode
The above results of Job's plot, mass spectra and Benesi-Hildebrand
plot had confirmed the 1:1 complexing mode of sensor 1 with Cu2+
ion. To further study the binding mode of sensor 1 with Cu2+ ions in
greater detail, 1H NMR titrations of sensor 1 by Cu2+ ion in DMSO-d6
and the theoretical calculations were conducted to interpret and ascer-
tain the complex mode of sensor 1 with Cu2+ ion.
As shown in Fig. 11, the optimized molecular modeling structures of
sensor 1 and sensor 1-Cu2+ ion disclosed the 1:1 stoichiometry of sen-
sor 1-Cu2+ complex via the coordination of nitrogen atoms of cyan with
copper atom. On the free sensor 1, the highest occupied orbit (HOMO)
was mainly distributed on naphthaline unit and benzene ring. Different
The 1H NMR spectral changes of sensor 1 upon the incremental ad-
dition of Cu2+ ions were shown in Fig. S8. The proton singles of
Fig. 11. Optimized ground-state geometries and frontier orbital shapes of free sensor 1 and sensor 1 + Cu2+ complex. Color code: C (gray), N (blue), H (white), O (red), Cu (orange). (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

H. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118191
9
Scheme 2. The complex mode and sensing mechanism of sensor 1 towards Cu2+ ion.
from HOMO, the lowest unoccupied orbit (LUMO) was mainly located
on the naphthaline unit and diaminomaleonitrile moiety. Their band
gap between HOMO (−5.55 eV) and LUMO (−2.45 eV) was calculated
as 3.08 eV. When copper ion coordinated with the diaminomaleonitrile
moiety, obvious changes had taken place in the orbital distribution and
band gaps. The change of ɑ-HOMO of complex was very little, however,
the β-HOMO remarkably focus on the benzene ring from pyran and
naphthaline units. Compared with LUMO of free sensor 1, ɑ-LUMO of
mechanism was the complex formation of sensor 1 with Cu2+ ion
with 1:1 stoichiometry, in which, the diaminomaleonitrile was the coor-
dination unit. Sensor 1 displayed great potential to be used as photo-
chromic material, colorimetric and fluorescent sensor for Cu2+ ions, so
we hope these results will promote the rational design of more excel-
lent functional materials in the future.
CRediT authorship contribution statement
complex
was
mainly
situated
on
naphthopyran
and
diaminomaleonitrile units and hardly on the benzene rings. β-LUMO
was also mainly on the naphthopyran unit and few on the benzene
rings. The band gaps both ɑ orbits (2.82 eV) and β (1.29 eV) orbits re-
duced significantly compared with that of free sensor 1 (3.08 eV),
which indicated the complex was more stable. Generally, a molecule
with low HOMO energy level will be more stable and consequently
more un-reactive. In this case, all the ɑ orbits and β orbits energy levels
of complex were more lower than those of free sensor 1, respectively,
which further confirmed higher stability of sensor 1 + Cu2+ complexes.
The above DFT results had further explained the formation of sensor
1 + Cu2+ complexes with 1:1 stoichiometry.
Heyang Zhang:Investigation.Tianyuan Zhong:Investigation, Formal
analysis.Nan Jiang:Formal analysis.Zhuo Zhang:Investigation.Xue
Gong:Investigation.Guang Wang:Methodology, Writing - original draft.
Declaration of competing interest
The authors confirm that this manuscript has been read and ap-
proved by all named authors, there are no known conflicts of interest as-
sociated with this publication and there has been no significant financial
support for this work that could have influenced it's outcome.
Based on all the results of Job's plot, mass spectra and Benesi-
Hildebrand plot, 1H NMR titration and theoretical calculation, it was
confirmed the selective colorimetric and fluorescent responses of sen-
sor 1 towards Cu2+ ions could be ascribed to the selective coordination
of sensor 1 with Cu2+ via the two nitrogen atoms of -C≡N on the
diaminomaleonitrile unit. The complex mode and sensing mechanism
of sensor 1 towards Cu2+ ion was illustrated in Scheme 2.
Acknowledgement
This work was supported by the National Natural Science Founda-
tion of China (no. 50873019) and Research Fund for the Doctoral Pro-
gram of Higher Education of China (20120043110007).
Appendix A. Supplementary data
4. Conclusions
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.118191.
In this paper, a naphthopyran-diaminomaleonitrile dyad (sensor 1)
as multifunction materials was designed and prepared. Sensor 1
displayed excellent photochromic property and photochromic fluores-
cence switch performance with well fatigue resistance. The fluores-
cence intensity of sensor 1 can be modulated reversibly via the
alternative UV irradiation and put in the dark. Sensor also 1 displayed
the high selective absorbance and fluorescence response towards Cu2
+ ions accompanied by the obvious color change from yellow to color-
less. In addition, both the absorbance and fluorescence intensities pre-
sented good linear relationship with the Cu2+ concentration. Sensor 1
can be used as colorimetric and fluorescent sensing for Cu2+ ion to
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