A novel fluorescent chemosensor based on coumarin and quinolinyl-benzothiazole for sequential recognition of Cu2+ and PPi and its applicability in live cell imaging

Article abstract of DOI:10.1016/j.saa.2019.118022

In this study, a highly selective fluorescent sensor (E)-2-((2-(benzo[d]thiazol-2-yl)quinolin-8-yl)oxy)-N′-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)acetohydrazide (TQC) was synthesized from 2-methylquinolin-8-ol and 4-(diethylamino)-2-hydroxybenzaldehyde and its structure was characterized by ¹H NMR, ¹³C NMR, ESI-HR-MS and density functional theory (DFT) calculation. Sensor TQC showed an obvious “on-off-on” fluorescence response to Cu²⁺ and PPi in a DMSO/HEPES (3:2 v/v, pH = 7.4) buffer system. The detection limits of sensor TQC were 0.06 μM to Cu²⁺ and 0.01 μM to PPi. In addition, sensor TQC showed a 1:1 binding stoichiometry to Cu²⁺ and TQC-Cu²⁺ complex showed a 2:1 binding stoichiometry to PPi. The optimum pH range of sensor TQC and TQC-Cu²⁺ was 3–8. Further studies demonstrated that sensor TQC could be made into test paper strips for the qualitative of Cu²⁺ and PPi and showed sequentially “on-off-on” fluorescent bio-imaging of Cu²⁺ and PPi in HeLa cells.

Full text of DOI:10.1016/j.saa.2019.118022

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118022
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A novel fluorescent chemosensor based on coumarin and quinolinyl-
benzothiazole for sequential recognition of Cu2+ and PPi and its
applicability in live cell imaging
Shengling Li a, Duanlin Cao a, Xianjiao Meng c, Zhiyong Hua,b, Zhichun Li a, Changchun Yuan a, Tao Zhou a,
Xinghua Han a,b, Wenbing Ma a,b,
⁎
a
School of Chemical Engineering and Technology, North University of China, Taiyuan 030051, PR China
National Demonstration Center for Experimental Comprehensive Chemical Engineering Education, North University of China, Taiyuan 030051, PR China
College of Arts and Sciences, Shanxi Agricultural University, Taigu, Shanxi 030801, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a highly selective fluorescent sensor (E)-2-((2-(benzo[d]thiazol-2-yl)quinolin-8-yl)oxy)-N′-((7-
(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)acetohydrazide (TQC) was synthesized from 2-
Received 6 November 2019
Received in revised form 24 December 2019
Accepted 30 December 2019
Available online 03 January 2020
methylquinolin-8-ol and 4-(diethylamino)-2-hydroxybenzaldehyde and its structure was characterized by 1
H
NMR, 13C NMR, ESI-HR-MS and density functional theory (DFT) calculation. Sensor TQC showed an obvious
“on-off-on” fluorescence response to Cu2+ and PPi in a DMSO/HEPES (3:2 v/v, pH = 7.4) buffer system. The de-
tection limits of sensor TQC were 0.06 μM to Cu2+ and 0.01 μM to PPi. In addition, sensor TQC showed a 1:1 bind-
ing stoichiometry to Cu2+ and TQC-Cu2+ complex showed a 2:1 binding stoichiometry to PPi. The optimum pH
range of sensor TQC and TQC-Cu2+ was 3–8. Further studies demonstrated that sensor TQC could be made into
test paper strips for the qualitative of Cu2+ and PPi and showed sequentially “on-off-on” fluorescent bio-imaging
of Cu2+ and PPi in HeLa cells.
Keywords:
Coumarin
Sequential recognition
Fluorescent sensor
Cu2+
PPi
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
PPi is the hydrolysis product of adenosine triphosphate in living
cells, and it has many important biological functions and is involved in
As is known to all, fluorescent sensors can be utilized as an effective
tool for detection ions on the basis of their high selectivity and high sen-
sitivity [1–3]. Furthermore, sensors of sequential recognition of cations
and anions have caused more attention to do extensive research, and
dozens of fluorescent sensors have been reported, such as Zn2+/PPi
[4,5], Al3+/PPi [6–8], Al3+/H2PO4−/CN− [9], Fe3+/PPi [10–12], Cu2+/PPi
[13–15], Ag+/F− [16], Hg2+/S2− [17–18] and so on.
many energy conversions and metabolic processes [33–35]. A stable
metabolic process allows for the normal functioning of the organism
[36–38]. However, there often exist many phosphorus-containing an-
ions with the same charge and similar structure in the body, making it
very difficult to selectively identify PPi from various phosphoric acid de-
rivatives. Using a specific Cu2+ sensor to sequentially identify PPi which
can effectively eliminate the interference of other quenching agents in
the system on Cu2+ would be more cost-effective and convenient [39]
(Scheme 1).
Coumarin fluorophores are often applied in structural design of fluo-
rescent dyes due to their advantages of high fluorescence quantum
yield, large Stokes shift and superior light stability [40] and the nitrogen
and oxygen atoms in benzothiazole and quinoline have excellent coor-
dination ability to metal ions [41–44]. In this paper, we synthesized a
new fluorescent sensor TQC based on coumarin and quinolinyl-
benzothiazole which showed an obvious “on-off” fluorescence
quenching response toward Cu2+ with a quenching efficiency of 100%,
and TQC-Cu2+ complex showed an “off-on” fluorescence enhancement
response toward PPi in a DMSO/HEPES (3:2 v/v, pH = 7.4). It showed
higher selectivity, higher sensitivity than current fluorescent sensors
in sequential recognition of Cu2+ and PPi.
Cu2+ is an essential element in human body and moderate concen-
tration of Cu2+ is of great benefit to maintain the normal function of
body [19–21]. The data shows that the allowable limits of Cu2+ are
1.3 ppm (20 μM) [22] in drinking water and 15.7–23.6 μM [23] in
blood. Higher or lower concentration of Cu2+ can seriously interfere
with normal physiological activities which leads to various diseases
[24–26]. Therefore, it is vital to detect Cu2+ and track its role in life ac-
tivities. So far, the types of sensor that identify in relation to Cu2+ se-
quentially are Cu2+/PPi [13–15], Cu2+/S2− [27–30], Cu2+/H2PO−4
[22,31], and Cu2+/F− [32].
⁎
Corresponding author at: School of Chemical Engineering and Technology, North
University of China, Taiyuan 030051, PR China.
E-mail address: mawenbing@nuc.edu.cn (W. Ma).
https://doi.org/10.1016/j.saa.2019.118022
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
S. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118022
Scheme 1. Synthesis of sensor molecule TQC
2. Experimental
TMS): δ (ppm) 10.48 (s, 1H, NH), 8.69 (s, 1H, CH_N), 8.53 (s, 1H,
ArH), 8.53 (d, 1H, J = 8.4 Hz, ArH), 8.40–8.43 (m, 1H, ArH), 8.24 (d,
1H, J = 8.4 Hz, ArH), 8.05–8.08 (m, 1H, ArH), 7.45–7.51(m, 4H,
ArH), 7.37 (d, 1H, J = 8.8 Hz, ArH), 7.00 (q, 1H, J = 2.8 Hz, ArH),
6.62 (dd, 1H, J1 = 8.8 Hz, J2 = 2.4 Hz, ArH), 6.53 (d, 1H, J = 2.0 Hz,
ArH), 4.83 (s, 2H, COCH2O), 3.45 (q, 4H, J = 7.2 Hz, CH2CH3),
1.22–1.26 (m, 6H, CH3). 13C NMR (CDCl3, 100 MHz, TMS): δ (ppm):
169.0, 163.7, 161.6, 157.3, 154.1, 152.4, 151.7, 150.7, 144.0, 140.2,
139.3, 136.9, 136.3, 130.6, 130.1, 127.6, 126.3, 126.2, 123.4, 123.3,
121.1, 119.3, 112.3, 110.0, 109.6, 108.8, 97.2, 67.0, 45.1, 12.5. The
NMR spectra of sensor TQC were shown in Figs. S1 and S2. HRMS
(ESI): m/z calcd for C32H28N5O4S [(M + H)+]: 578.1864, found
578.1858; C32H27N5NaO4S [(M + Na)+]: 600.1682, found 600.1678
(Fig. S3).
2.1. Materials and methods
Compounds 1 and 2 were synthesized according to the reported
reference. [45, 46] The raw material was purchased from Aladdin
and Energy Chemical Reagents Ltd. All solvents were commercially
available AR and CP and were used without any treatment. The 1H
NMR and 13C NMR spectra were measured on a Bruker AVANCE III
spectrometer (Switzerland) in CDCl3 solution at 400 and 100 MHz,
respectively. The ESI-MS spectra of sensor TQC was measured on a
Bruker Solarix XR Fourier transform-ion cyclotron resonance (FT-
ICR) mass spectrometer. The UV-spectra and fluorescence spectra
of all samples were recorded on a UV-2602 spectrophotometer and
HITACHIF-2700 spectrophotometer. Transient photoluminescence
decay spectra were recorded on Edinburgh FLS1000 steady-state/
transient fluorescence spectrometer. The melting points of sensor
TQC was obtained on a WRS-C1 microcomputer melting point in-
strument. The pH of all solutions was making-up on a PHS-3C pH
meter. The bright field, fluorescence and confocal fluorescence mi-
croscope images of the cells were obtained using a fluorescence mi-
croscope (EVOS fl auto, life, America) at an excitation wavelength
of 365 nm.
2.3. General procedure for UV–vis absorption spectra and fluorescence
spectra experiments
All tests were carried out at room temperature (25 °C) in DMSO/
HEPES (3:2 v/v, pH = 7.4) buffer system. A stock solution of TQC was
prepared (c = 1 × 10−3 M) in DMSO. The stock solutions of various
metal ions and anions (1 × 10−2 M) were prepared from their chlo-
ride and sodium salts in deionized water, respectively. The concen-
tration of sensor TQC was 10 μM in all experiments. The UV–vis
absorption spectra and fluorescence spectra of sensor TQC at a con-
centration of 1 × 10−5 M were recorded at an excitation of 449 nm,
the slits of emission and excitation were 5 nm in all experiments.
In this study, K+, Na+, Cs+, Li+, Ag+, Mg2+, Ca2+, Zn2+, Cu2+, Hg2
2.2. Synthesis
2.2.1. Preparation of sensor TQC
245 mg of 1 mmol compound 1, 350 mg of 1 mmol compound 2
and 40 mL absolute ethanol was added to 100 mL and heated to re-
flux for 7 h and TLC monitoring. Then it was cooled to room temper-
ature, filtered, and washed with ice ethanol to afford a yellow solid
(550 mg, yield, 95%). mp: N221.4 °C. 1H NMR (CDCl3, 400 MHz,
+, Pb2+, Ba2+, Cd2+, Al3+, and Fe3+ cations and F−, Cl−, Br−, I−
,
HPO24−, H2PO−4 , PPi, OAc−, HCO3−, CO23−, HSO−3 , SO23−, SO24−, S2−
NO−3 and NO−2 anions were tested.
,

S. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118022
3
2.4. Fluorescence quantum yield experiment
3.2. The fluorescence recognition
According to formula (3), the relative fluorescence quantum yields
were calculated when quinine sulfate (∅ = 0.577) was used as the ref-
erence solution.
3.2.1. Fluorescence recognition of sensor TQC toward Cu2+
The fluorescence response behavior of sensor TQC was investi-
gated upon treatment with various metal ions, including K+, Na+
,
Cs+, Li+, Ag+, Mg2+, Ca2+, Zn2+, Cu2+, Hg2+, Pb2+, Ba2+, Cd2+, Al3
+, and Fe3+ in DMSO/HEPES (3:2 v/v, pH = 7.4) at an excitation of
449 nm. As shown in Fig. 3, only Cu2+ led to a remarkable fluores-
cence quenching at 505 nm (∅ changed from 0.286 to 0.009),
while the other ions caused a little or no fluorescence change. In
order to explore the effects of other ions on the recognition of Cu2
+, the interference tests were carried out. As shown in Fig. S4, the
fluorescence intensity of other metal ions solution showed obvious
fluorescence quenching after the addition of 10 equiv. of Cu2+. Evi-
dently, these metal ions did not interfere with the recognition of
Cu2+. In consequence, sensor TQC showed fine specificity.
2.5. General method for cell imaging
HeLa cells were cultured in culture medium in an in vitro incubator
with 5% CO2 at 37 °C. Cells were seeded onto two culture dishes at a den-
sity of 1.0 × 105 cells per well and then incubated at 37 °C for 12 h. After
washing with PBS (phosphate-buffered saline) buffer two times, the
cells were imaged using a fluorescence microscope. Then the cells
were incubated with three PBS buffers (containing 5 μM sensor TQC,
50 μM Cu2+ and 50 μM PPi, respectively) for 30 min successively. Each
incubation was completed, the dish was washed twice with PBS buffer
thoroughly and after that the cells were imaged.
3.2.2. Fluorescence titration of sensor TQC toward Cu2+
The sensitivity of sensor TQC for Cu2+ was investigated by fluores-
cence titration. The observed changes in the fluorescence emission
spectra with Cu2+ are shown in Fig. 4. The intensity of the fluorescence
peak at 505 nm decreased upon successive addition of the Cu2+ and sat-
urated at 2.0 equiv. and the quenching rate was 100%, indicating that
sensor TQC was highly sensitive to Cu2+. The Job curve at the upper
right corner showed that the inflection point was observed at a Cu2+
concentration of 0.55 equiv., indicating that the coordination ratio of
sensor TQC and Cu2+ was 1:1. The complexation constant Ka =
6.27 × 104 M−1 (Y = 6.43 × 10−9 × X + 4.03 × 10−4, R2 = 0.99901,
Fig. S5). Moreover, the minimum detection limit of sensor TQC was cal-
culated to be 0.06 μM (Y = −918.16 × X + 1190.88, R2 = 0.99200,
Fig. S6) by LOD = 3σ/m, indicating high sensitivity of sensor TQC.
3. Results and discussion
3.1. The UV–vis recognition
3.1.1. UV–vis recognition of sensor TQC toward Cu2+
The UV–vis spectrum of sensor TQC was recorded in DMSO/HEPES
(3:2 v/v, pH = 7.4) and shown in Fig. 1. After the addition (10 equiv.)
of various metal ions, namely K+, Na+, Cs+, Li+, Ag+, Mg2+, Ca2+, Zn2
+, Cu2+, Hg2+, Pb2+, Ba2+, Cd2+, Al3+, and Fe3+, the presence of Cu2+
induced a new band at 479 nm with a change in color from bright yel-
low to orange. The maximum absorption wavelength showed a signifi-
cant red shift of 30 nm from 449 nm to 479 nm, and the absorbance was
also significantly increased, indicating that sensor TQC could be used for
3.2.3. Fluorescence recognition of TQC-Cu2+ complex to PPi
naked eye recognition of Cu2+
.
The complex formed by sensor TQC and Cu2+ was used as a new
sensor TQC-Cu2+ for sequential recognition of anions, including: F−
,
3.1.2. UV–vis titration of sensor TQC toward Cu2+
Cl−, Br−, I−, HPO42−, H2PO−4 , PPi, OAc−, HCO−3 , CO23−, HSO3−, SO32−
,
As shown in Fig. 2, by successive addition of Cu2+ ions to sensor TQC
in DMSO/HEPES (3:2 v/v, pH = 7.4) up to 2.0 equiv., the absorption
gradually increased and the maximum absorption wavelength shifted
from 449 nm to 479 nm. Further addition of Cu2+ did not make further
significant spectral change. The two isosbestic points, observed at
383 nm and 447 nm, indicate that sensor TQC and the complex TQC-
Cu2+ were in a dynamic equilibrium.
SO24−, S2−, NO3− and NO−2 . As shown in Fig. 5, only the addition of PPi in-
stantly caused an obvious fluorescence enhancement but remained un-
changed after the addition of other anions. The response rate of H2PO4−
and HPO24− was 10% and 12%, respectively. Compared with H2PO−4 and
HPO24−, the fluorescence was recovered to a large extent after adding
PPi with a response rate of above 90% (∅recovered to 0.206), indicating
that the new probe could identify PPi specifically. The competitive
Fig. 1. UV–vis spectra of sensor TQC in DMSO/HEPES (3:2 v/v, pH = 7.4) with the addition of 10 equiv. of various metal ions and blank. Inset: the color of sensor TQC (10 μM) in the absence
and presence of Cu2+ (10 equiv.).

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S. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118022
Fig. 2. UV–vis spectra of sensor TQC with various concentrations [0–2.0 equiv. of Cu2+.] in DMSO/HEPES (3:2 v/v, pH = 7.4).
experiments were further carried out using a solution containing PPi
and all other anions. As shown in Fig. S7, the addition of PPi to TQC-
Cu2+ containing other anions still showing a fluorescent enhancement
effect without being interfered by other anions, which further indicated
that TQC-Cu2+ had good selectivity to PPi among other co-existing an-
ions especially phosphorus-containing anions.
3.3. Fluorescence lifetime studies
As shown in Fig. 7, the fluorescence lifetime of sensor TQC was calcu-
lated to be 1.13 ns, when Cu2+ was added, the fluorescence lifetime
shorted to 0.64 ns. Whereas, the addition of PPi resulted in a recovery
in fluorescence lifetime which was calculated to be about 1.13 ns,
which suggested that sensor TQC could be applied for sequential detec-
tion of Cu2+ and PPi.
3.2.4. Fluorescence titration of TQC-Cu2+ with PPi
As shown in Fig. 6, the intensity of the strongest fluorescence
emission peak gradually increased with increasing the PPi concen-
tration up to 10 equiv., after that the intensity remained largely un-
changed. Thus, TQC-Cu2+ had a high sensitivity to PPi. The Job
curve at the upper right corner showed that the inflection point
was observed at a PPi concentration of 0.3 equiv., indicating that
the coordination ratio between probe and PPi was 2:1. Furthermore,
the detection limit was 0.01 μM according to the formula LOD =3σ/
m (Fig. S8).
3.4. Reversibility of sensor TQC for Cu2+ and PPi
The stability of sensor TQC in identifying Cu2+ and PPi was
discussed with Cu2+ and PPi as input signals and fluorescence inten-
sity as output signals. As shown in Fig. 8, the fluorescence intensity
still remained stable after 6 cycles, indicating that the sensor TQC
could be used to continuously identify Cu2+ and PPi in practical
applications.
Fig. 3. Fluorescence spectra of sensor TQC in DMSO/HEPES (3:2 v/v, pH = 7.4) at an excitation of 449 nm with the addition of 10 equiv. of various metal ion and blank. Inset: the
fluorescence of sensor TQC (10 μM) in the absence and presence of Cu2+ (10 equiv.) under UV light.

S. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118022
5
Fig. 4. Fluorescence spectra of 10 μM sensor TQC with the addition of various concentrations [0–2.0 equiv.] of Cu2+ in DMSO/HEPES (3:2 v/v, pH = 7.4) at an excitation of 449 nm. The inset
plots showed that sensor TQC formed a 1:1 complex with Cu2+
.
3.5. DFT study
verified the experimental results and presumed complexation mode
of chemosensor TQC with Cu2+ and explained the PET (photo in-
duced electron transfer) process.
To gain further insight into the photophysical properties and the
nature of optical response of sensor TQC toward Cu2+, TQC and TQC-
Cu2+ were examined by density functional theory calculation by
using Gaussian 09 program with the methods of B3LYP/6-31G (d,
p) for TQC and B3LYP/LANL2DZ for TQC-Cu2+. The optimized struc-
tures and the highest occupied molecular orbital (HOMO) and the
lowest unoccupied molecular orbital (LUMO) of TQC and TQC-Cu2+
were shown in Fig. 9. The coumarin ring and the quinolinyl
benzothiazole in TQC were not in the same plane and presented a di-
hedral angle of about 20° while were coplanar after complexing with
Cu2+. Most electrons in the HOMO orbital of TQC were located on the
coumarin ring. After complexing with Cu2+, the electrons trans-
ferred toward Cu2+ and were distributed at the binding site of TQC
and Cu2+ and resulted in fluorescence quenching. Meanwhile, the
energy gaps between HOMO and LUMO of the TQC and TQC-Cu2+
were 2.83 eV and 2.64 eV, respectively. It was obvious that the en-
ergy gap of TQC-Cu2+ was lower than TQC which was in accordance
with the redshift of TQC-Cu2+ in UV–vis absorption. DFT calculations
3.6. The possible mechanism of sensor TQC for Cu2+ and PPi
In order to identify the possible mechanism between sensor TQC
for Cu2+ and PPi, we did the Job experiment. The Job curves showed
that the coordination ratio of sensor TQC to Cu2+ was 1:1 and that
to PPi was 2:1. The sensor TQC provided six coordination points
through O atom and N atom to form a copper complex by 1:1 bonding
with Cu2+. The coordination complex TQC-Cu2+ exhibited a fluores-
cence quenching response and the probe fluorescence was restored
after the addition of PPi. During this process, the sensor fluorescence
emission showed no red shift with the increase of Cu2+ concentra-
tion, and it showed a distinct “on-off” fluorescence response. Further
efforts were made to study the binding sites of TQC with Cu2+ by
means of the ESI-MS spectra (Fig. S10). The molecular ion peak
1200
1500
1000
800
600
only TQC
PPi
10 equiv
1200
900
400
200
only TQC-Cu2+, F-, Cl-, Br-, I-,
0.0
0.2
0.4
0.6
0.8
PPi
900
HCO3-, CO32-, HSO3-, SO32-
,
Mole Fraction of PPi
600
300
0
SO42-, S2-, NO2-, NO3-, OAC-
0
600
300
0
H2PO4-, HPO42-
500
550
600
475
500
525
550
575
600
Wavelength (nm)
Wavelength (nm)
Fig. 6. Fluorescence spectra of 10 μM TQC-Cu2+ in the presence of various concentrations
[0–10 equiv.] of PPi in DMSO/HEPES (3:2 v/v, pH = 7.4) at an excitation of 449 nm. The
inset plots indicated that TQC-Cu2+ formed a 2:1 complex with PPi.
Fig. 5. Fluorescence spectra of 10 μM TQC-Cu2+ with the addition of 10 equiv. of various
anions in DMSO/HEPES (3:2 v/v, pH = 7.4).

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S. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118022
Fig. 7. Transient photoluminescence decay spectra of sensor TQC (10 μM) upon addition of Cu2+ and PPi in DMSO/HEPES (3:2 v/v, pH = 7.4) buffer system.
(ESI-MS) at 639.09935 [sensor TQC-Cu2+; m/z calcd for 639.09905]
indicated an 1:1 complexation between sensor TQC and Cu2+. Base
on Job's plot analysis, mass spectrometry, DFT calculation and spec-
tral properties analysis, we speculated that the photoinduced elec-
tron transfer from the donor (coumarin rings) to the receptor
(quinolinyl-benzothiazole) was blocked at the beginning, then the
complexation of Cu2+ promoted the PET process to occur and caused
to quench the fluorescence emission due to the paramagnetic cation
of Cu2+, and there was no shift of emission spectra. On the contrary,
PET process was hindered when Cu2+ was complexed by PPi and
the fluorescence was recovered. So the possible coordination mode
was shown in Scheme 2.
color changes were observed in this process under both visible light
and UV irradiation, indicating that the sensor TQC had the potential
for naked-eye detection of Cu2+ and PPi in water.
3.8. Cell imaging
Fig. S9 showed that the fluorescence at pH = 3–8 was quenched
with the addition of Cu2+ and recovered with the addition of PPi. Be-
cause of the excellent selectivity at physiological pH levels, the applica-
tion of sensor TQC for detection of Cu2+ and PPi in liver cells was further
investigated. Fluorescence imaging was performed in HeLa cells, as
shown in Fig. 11. No fluorescence was observed in cells (Fig. 11a, a’,
a”). Upon the addition of sensor TQC, the cells exhibited highly intense
green fluorescence (Fig. 11b, b’, b”), indicating that the sensor was well
permeable. Cells incubated with Cu2+ also showed quenching of fluo-
rescence intensity (Fig. 11c, c’, c”). However, fluorescence was restored
for cells treated with PPi (Fig. 11d, d’, d”). The cellular studies clearly in-
dicated that sensor TQC had great cell permeability and could be sup-
plied as a useful sensor for mapping Cu2+ and PPi in live cells.
3.7. Practical application on test strips
A paper strip test was performed as described previously to illustrate
its practical utility [47], as shown in Fig. 10. Filter paper strips were put
into DMSO/HEPES (3:2 v/v, pH = 7.4) solution of TQC for 5 min and
dried with paper, and then put into Cu2+ aqueous solution and PPi
aqueous solution in succession and dried again with paper. Obvious
Fig. 8. The sequential reversible behavior of sensor TQC (10 μM) upon alternate addition of Cu2+ and PPi in DMSO/HEPES (3:2 v/v, pH = 7.4) (λex = 449 nm).

S. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 230 (2020) 118022
7
Fig. 10. Color changes on test paper in DMSO/HEPES (3:2 v/v, pH = 7.4) (a) TQC; (b) TQC-
Cu2+; (c) TQC-Cu2++PPi under visible light and (a’) TQC; (b’) TQC-Cu2+; (c’) TQC-Cu2+
+PPi under UV light.
fluorescence response toward Cu2+ and PPi. The sequential recognition
behavior could be repeated six times with little fluorescence efficiency
loss using Cu2+ and PPi. The sensor TQC showed a 1:1 binding stoichi-
ometry to Cu2+ with a complexation constant of 6.27 × 104 M−1 and
the detection limit for Cu2+ was 0.06 μM. The TQC-Cu2+ complex
showed a 2:1 binding stoichiometry to PPi and the detection limit for
PPi was calculated to be 0.01 μM. The stable pH range of the sensor
TQC to Cu2+ and TQC-Cu2+ to PPi was from 3 to 8. Moreover, the sensor
not only could be used to determine Cu2+ and PPi in test paper strip
qualitatively, but also could be successfully applied to detect Cu2+ and
PPi within living cells.
CRediT authorship contribution statement
Shengling Li:Methodology, Validation, Formal analysis, Investiga-
tion, Data curation, Writing - original draft, Writing - review & editing,
Project administration.Duanlin Cao:Resources, Funding acquisition.
Xianjiao Meng:Conceptualization.Zhiyong Hu:Funding acquisition.
Zhichun Li:Visualization.Changchun Yuan:Resources.Tao Zhou:Soft-
ware, Formal analysis.Xinghua Han:Resources.Wenbing Ma:Conceptu-
alization, Methodology, Resources, Writing - review & editing,
Supervision.
Fig. 9. The optimized molecular structure, HOMO and LUMO of TQC and TQC-Cu2+ by DFT
calculation.
Declaration of competing interest
4. Conclusions
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
In this study, the fluorescent sensor TQC was successfully synthe-
sized and its spectral performance for sequential recognition of Cu2+
and PPi was studied. The sensor TQC showed an obvious “on-off-on”
Scheme 2. The possible mechanism of TQC-Cu2+ with PPi

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microscope (a”, b”, c”, d”) images of HeLa cells: blank cell (a, a’, a”), cells treated with
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Acknowledgements
We gratefully acknowledge the financial support from the Science
Foundation of North University of China (No. 110121).
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Appendix A. Supplementary data
Supplementary data includes the 1H NMR spectra, 13C NMR spectra,
ESI-MS spectra and part of spectrums of sensor TQC. Supplementary
data to this article can be found online at https://doi.org/10.1016/j.saa.
2019.118022
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