A Schiff base probe for competitively sensing Cu2+ and cysteine through hydrolysis, complexation, and cyclization

Article abstract of DOI:10.1016/j.jphotochem.2020.113065

The form of a single response to a single target analyte limits the development of fluorescent probes. Here, a probe (HBTA) based on HBT (2-(2-Hydroxyphenyl)-benzothiazole) was synthesized to achieve a concept about nested probe, completing continuous det

Full text of DOI:10.1016/j.jphotochem.2020.113065

Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
A Schiff base probe for competitively sensing Cu2+ and cysteine through
hydrolysis, complexation, and cyclization
,
,
Fanyong Yana *,1, Xiaodong Suna 1, Yan Zhanga, Yingxia Jianga, Li Chena, Tengchuang Mab,
Liang Chenc
a State Key Laboratory of Separation Membranes and Membrane Processes/National Center for International Joint Research on Separation Membranes, School of
Chemistry and Chemical Engineering, Tiangong University, Tianjin, 300387, PR China
b Department of Nuclear Medicine, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, 150081, PR China
c Graduate School of Life Science, Hokkaido University, Sapporo, 0010024, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
The form of a single response to a single target analyte limits the development of fluorescent probes. Here, a
probe (HBTA) based on HBT (2-(2-Hydroxyphenyl)-benzothiazole) was synthesized to achieve a concept about
nested probe, completing continuous detections. HBTA through low concentration Cu2+ gives birth to a new
probe (HBT-CHO) that can detect Cu2+ by complexation and Cys by cyclization (HBT-Cys). These processes are
accompanied by different fluorescent signal changes and can quantitatively detect Cys and Cu2+. Furthermore,
HBT-CHO, HBT-Cys, and Cys can bind Cu2+, so competitive fluorescent response of Cu2+ can take place through
alternate addition of Cu2+ and Cys. At this stage, the response becomes too complex to accurately quantify, but it
is still dependable to predict changes in the content of the mixture system through competitive fluorescent
changes. In summary, it is because of the existence of the nested pattern that HBTA completes multiple forms of
responses to Cu2+ and Cys.
Fluorescent probe
Multistage logical response
Copper ion (II)
Cysteine
Biological imaging
1. Introduction
probe based on the hydrolysis of ester bonds caused by Cu2+. According
to the idea, Gu et al. [7] synthesized a chromene-based probe to detect
Fluorescent probes play vital roles in many areas [1–4], and are
attractive to many researchers for their unique sensitivity and selec-
tivity, convenient application, practicality, and economy [5–7]. How-
ever, compared to the traditional detection methods, there are
drawbacks such as the single-narrow response range due to the limit of
their own concentration. Most fluorescent probes have only one
response site, which makes them only respond to one analyte and pro-
duce enhanced, ratio or quenching fluorescence signals [8–11]. Through
designing the structure of fluorescent probes, either by increasing
response sites [12–15] or by synergistic interactions with other sub-
stances [16], researchers achieved multiple response ranges. In this
work, we proposed a nested probe model that can continue to act as a
new probe for two or even multiple responses, which provided a way for
the problem to some degree.
Cu2+. Zhang et al. [18] designed a reactive probe catalyzed by Cu2+ to
achieve a bilinear response to Cu2+. Li et al. [25] synthesized a
double-responsive probe for detecting Cu2+ by using the instability of
the imine structure. Additionally, the affinity of imine for Cu2+ was
discussed in our previous work [26]. In addition, according to literature
reports, HBT analogs can complex with Cu2+ to form a 2:1 complex [27,
28]. Therefore, the idea that Cu2+ can act as a trigger to disintegrate
probes to play other roles is feasible. Therefore, HBTA (ΦHBTA = 0.09)
with a double imine structure based on different Cu2+ concentrations as
triggered signals was designed and synthesized. Detection processes
show the corresponding fluorescence signals include enhanced, ratio,
and quenching with the continuous increase of the Cu2+ concentration.
At the same time, the product (HBT-CHO, ΦHBT-CHO = 0.16) obtained by
the reaction of HBTA with 0.1 eq (mole equivalent) of Cu2+ carries a
formyl group. It is a Cys recognition group and the mechanism has also
been proven [29–32]. The respond of HBT-CHO to Cys shows additional
ratio-fluorescent changes (ΦHBT-Cys = 0.13). Moreover, Cys can
Many fluorescent probes based on Cu2+ have been discovered
[17–21]. Complexation were often used to detect Cu2+ [22,23], and
probes based on the reaction are few. Zhang et al. [24] designed a NIR
* Corresponding author.
E-mail address: yanfanyong@tiangong.edu.cn (F. Yan).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.jphotochem.2020.113065
Received 10 September 2020; Received in revised form 15 November 2020; Accepted 30 November 2020
Available online 4 December 2020
1010-6030/© 2020 Elsevier B.V. All rights reserved.

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
competitively complex Cu2+ to achieve off-on alternation [33–35],
which further adds to functionality and application value of HBTA.
Design ideas and the main response methods involved are shown in
Scheme 1.
E. coli bioimaging was obtained through an upright fluorescence mi-
croscope (Olympus bx 53). All cells were observed by the Leica SP5
confocal microscopy. Reaction intermediates was determined though
UPLC-MS data by UPLC H-CLASS Xevo TQ-D MS.
On the other hand, Cu2+ as a common cation widely is found in soil
[36,37], water [38,39], and organism organisms [40–43]. 20 μM of
2.2. Quantum yield measurement
Cu2+ in drinking water was provided by the EPA (US Environmental
Protection Agency) [44]. Extra Cu2+ in human body affects the nervous
system and causes neurological diseases like Parkinson’s, Alzheimer’s,
Quinine sulfate as a reference (Φ = 0.54), the fluorescence quantum
yield (Φ) is calculated according to Eq. (1). Specifically, Quinine sulfate
was tested in 0.1 mol Lꢀ 1 H2SO4 solution, and probes (HBTA, HBT-CHO,
and HBT-Cys) in this experiment was dissolved in Tris/MeOH (v/v = 9/
1, 50 mM, pH = 7.4). Five concentration gradients of probes and qui-
nine sulfate were made. The absorbance in the 10 mm cuvette was kept
under 0.05 for quinine sulfate and 0.1 for probes to minimize the re-
absorption effects. The integrated fluorescence intensity is the integral
area of the fluorescence spectrum at the optimal excitation wavelength.
Then the graph was plotted using the integrated fluorescence intensity
against the absorbance and the intercept is zero.
Menkes’ and Wilson’s disease [45–48]. As
a
fundamental
thiol-containing amino, Cys acid is concerned with physiological pro-
cesses such as detoxification, metabolism, protein synthesis and
post-translational modification [49,50]. Anomalous Cys levels in human
body can cause a variety of diseases including lethargy, edema, slow
growth, hair depigmentation, skin lesions, liver damage, Alzheimer’s
and cardiovascular diseases [51,52]. HBTA realized the continuous
detection to Cu2+ and Cys and logical fluorescence output according to
concentration change and order of addition. Anyway, we hope to pro-
vide a new way of designing probes to detect interrelated analytes to
play a more important role in environmental, bioimaging, disease
treatment, etc.
(
)
2
Gradx ŋx
Φx = Φ
(1)
stGradst ŋst
Where Φ is the quantum yield, Grad is the gradient from the plot of
2. Experiment
integrated fluorescence intensity vs absorbance,
η is refractive index of
the solvent. The subscript “st” refer to quinine sulfate and “x” refer to the
2.1. Materials and equipment
sample.
Analytical grade 2-aminobenzenethiol was purchased from Aladdin
Reagent Co., LTD. Trifluoroacetic Acid (TFA) was purchased from
Tianjin Kemiou Chemical Reagent Co., LTD. Hexamethylenetetramine
(HMTA) was purchased from Tianjin Guangfu Technology Development
Co., LTD. Other solvents and test agents were purchased from Tianjin
fengchuan chemical reagent technology Co., LTD. All chemicals were
used without further purification. Thin layer chromatography (TLC) and
column chromatography (200–300 mesh silica gel) were purchased
from Qingdao ocean chemical Co., LTD and was used to monitor re-
actions and purify the products.
2.3. The cell imaging
MDA-MB-231 cells were incubated in an in-vitro incubator under
humidified 5% CO2 at 37 ℃ containing 10 % FBS and Penicillin-
Streptomycin Solution. First, Nystatin was added to MDA-MB-231 cells
in a 35 mm glass-bottomed petri dish for 30 min, then washed 3 times
with PBS, and then 10 μL of HBTA (1 mM,) and nystatin were added for
another 30 min. The Cells were divided into three groups as blank, 5 μL
of Cu2+ (1 mM) + 20
μL of NEM (1 mM), 10 μL of Cys (1 mM) for
IR spectra were obtained by a TENSOR37 FT-IR through KBr disks.
1H NMR were measured with a Bruker Advance III HD 400 MHz spec-
trometer in DMSO-d6 (TMS as an internal standard). The UV–vis spectra
were obtained by Purkinje General TU-1901 UV–vis spectrophotometer.
The fluorescence spectra were obtained by F-380 fluorescence spectro-
photometer. HRMS was obtained by Bruker micro TOF-QII instrument.
Standard quartz cuvettes of 1 cm path length were used in all optical
measurement. The pH was carried out on a Mettler Toledo pH meter.
another 60 min. The extra 10 μL of Cys (1 mM) was added to the group
of Cu2+ + NEM for 60 min. After washing three times with PBS, fluo-
rescence imaging was performed by a fluorescence confocal microscope.
2.4. Synthesize of 2-(benzo[d]thiazol-2-yl) phenol (HBT)
HBT was synthesized according to a reported method [53]. EtOH
solution
with
salicylaldehyde
(2.0 mL,
19.2 mmol)
and
Scheme 1. The design idea based on response types. a. Cu2+ caused hydrolysis of imine; b. Cyclization between fluorophore containing formyl and Cys; c. HBT
analogue complexing Cu2+; d. Structure of HBTA based on the above reaction mechanisms.
2

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
2-aminobenzenethiol (2.0 mL, 18.7 mmol) was stirred for 30 min at rt.
Then 30 % H2O2 (2.3 mL, 74.8 mmol) and 38 % HCl (1.1 mL,
37.3 mmol) was added with stirring for 2 h under N2. The reaction
mixture was poured into ice water and the pale-yellow solid was
precipitated out. The suspension was extracted with ethyl acetate
(EtOAc) and water. The organic layer was dried under vacuum after
treatment with anhydrous sodium sulphate. The crude product was
recrystallized in ethanol to afford a almost white to light yellow crystal
(3.5 g, 81.2 % yield).
1 H), 2.44 (s, 3 H), 2.32 (s, 3 H). 13C NMR (101 MHz, DMSO-d6) δ
163.34 (s), 161.93 (s), 161.44 (s), 151.45 (s), 140.47 (s), 137.24 (s),
135.94 (s), 135.53 (s), 132.70 (s), 131.39 (d, J =19.6 Hz), 127.80 (s),
126.17 (s), 124.63 (s), 122.18 (s), 121.97 (s), 121.40 (s), 119.04 (s),
117.76 (d, J =5.8 Hz), 20.62 (s), 17.73 (s). HRMS (ESI) m/z: calcd for
C
22H19N2OS [M + H]+ 359.1213, found: 359.1215 and C22H18N2OSNa
[M + Na]+ 381.1032, found: 381.1035. FTIR (KBr) νmax/cm-1 3344,
2918, 2851, 1615, 1478, 1427, 1296, 1202, 1109, 1036, 756, 617. M.
p. = 138–141 ℃. (Scheme S1)
3. Results and discussion
2.5. Synthesize of 3-(benzo[d]thiazol-2-yl)-2-hydroxybenzaldehyde
(HBT-CHO)
3.1. The segment detection of HBTA on Cu2+
HBT-CHO was synthesized according to Duff reaction. HBT (1.0 g,
4.4 mmol) was added to 15 mL TFA solution cooled in ice-bath. HTMA
(1.0 g, 7.1 mmol) was added to the solution in 30 min then heated up to
80 ℃ for 9 h. Then 20 mL 4 N HCl was added to the solution refluxing
for 30 min. The hot solution was cooled to RT and extracted with
dichloromethane (DCM). The organic layer was dried under vacuum
after treatment with anhydrous sodium sulphate to afford a yellow solid.
The crude product was purified by column chromatography (silica gel,
100–200 mesh, petroleum ether: ethyl acetate = 5:1, v/v) as yellow
solid (0.5 g, 46 % yield).
Titration experiments of Cu2+ were carried out at Tris buffer/MeOH
(v/v = 9/1, 50 mM, pH = 7.4). With Cu2+ concentration from
0.2–160 μM, the spectra change was displayed at Fig. S5. At different
concentration ranges of Cu2+, HBTA displayed different fluorescence
signal change. Fluorescent color changed from orange to green with the
addition of a small amount of Cu2+. A ratio response of a linear range
between I525/I590 and concentration of 0.2–1.6 μM was acquired (I525/
I590 = 0.234[Cu2+] + 0.69; R2 = 0.985). A detection limit of 86.9 nM
(3σ/k) was obtained in the concentration range (Fig. 1a and d). At the
concentration of 6–12 μM, green fluorescence at 525 nm was continu-
ously enhanced to obtain a linear range (I525 = 101.61[Cu2+] + 60.29;
2.6. Synthesize of 2-(benzo[d]thiazol-2-yl)-6-(((2,4-dimethylphenyl)
imino) methyl) phenol (HBTA)
R2 = 0.979). The limit of detection in the range was calculated to be
2.6 nM (3
σ
/k) (Fig. 1b and e). In Cu2+ concentration of 40–160
μM, the
green fluorescence was quenched and detection limit was calculated as
HBT-CHO (0.2 g, 0.8 mmol) and 2,4-dimethyl aniline (174.6 μL,
87.5 nM (3
σ
/k) (I525 = ꢀ 3.04[Cu2+] + 731.4; R2 = 0.997) (Fig. 1c and
1.4 mmol) was added to 40 mL methanol solution. One drop of acetic
acid was added to the solution. Then the reaction solution was heated to
70 ℃. The reaction was monitored by TLC until the material point dis-
appears (10 h). Then the solution was dried under vacuum to afford a
red solid. The crude product was purified by column chromatography
(silica gel, 100–200 mesh, petroleum ether: ethyl acetate = 100:1, v/v)
to afford a red powder (0.15 g, 55 % yield) 1H NMR (400 MHz, DMSO-
d6) δ 9.19 (s, 1 H), 8.59 (d, J =7.7 Hz, 1 H), 8.16 (d, J =7.9 Hz, 1 H),
8.05 (d, J =8.1 Hz, 1 H), 7.82 (d, J =7.5 Hz, 1 H), 7.57 – 7.49 (m, 2 H),
7.43 (t, J =7.5 Hz, 1 H), 7.18 (d, J =13.0 Hz, 2 H), 7.10 (t, J =7.6 Hz,
f). The linearity in the transition phase of these ranges was instable, thus
the quantitative detection failure (Fig. S5). This phenomenon of fluo-
rescence signal variation at different Cu2+ concentration ranges are in
line with our expectation, and these processes will interact with each
other.
The prominent lone pair electron and the small steric hindrance of
the imine structure makes imine have affinity for Cu2+ [26]. Therefore,
selective experiment was carried out. A stock solution (2.0 mM) of
HBTA was prepared in MeOH. Stock solutions (2.0 mM, high
Fig. 1. Fluorescence titration of Cu2+ to HBTA (Tris/MeOH v/v = 9/1, 50 mM, pH = 7.4, incubation time: 30 min). a. Fluorescence spectra of HBTA in a Cu2+ range
of 0.2 – 1.8
μ
M (λex =350 nm); b. Fluorescence spectra of HBTA in a Cu2+ range of 6 – 12
μ
M (λex =400 nm); c. Fluorescence spectra of HBTA in a Cu2+ range of 40 –
160
μ
M (λex =400 nm); d. Linearity in a Cu2+ range of 0.2 – 1.6
μ
M; e. Linearity in a Cu2+ range of 6 – 12
μ
M ; f. Linearity in a Cu2+ range of 60 – 160
μM.
3

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
concentration) of cation (Na+, Mg2+, Al3+, K+, Ca2+, Cr3+, Mn2+, Fe2+
,
detection limit was calculated to be 5.41 μM (3σ/k) (Fig. 4c and d). HBT-
Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Ba2+, Hg2+, and Pb2+) were
CHO can cyclize with Cys to form HBT-Cys (MS m/z: HBT-Cys, calcd:
358.04, found: 357.21, Fig. S15) and achieve a fluorescent color from
green to blue.
prepared in distilled water. 200 μL HBTA solution and 200 μL cationic
solution were added to a 10 mL volumetric flask with a stock solution of
Tris buffer/MeOH (v/v = 9/1, 50 mM, pH = 7.4). All ions except Cu2+
had no effect on fluorescence of HBTA, and only Cu2+ quenched its
fluorescence at 590 nm (Fig. 2a). Then competition experiments showed
that HBTA had good selectivity to Cu2+ (Fig. 2b).
3.4. Mechanism study of HBTA for Cu2+ and Cys
To get insight into the reaction mechanism of HBTA with Cu2+ and
verify the conjecture, nuclear magnetic experiment was carried out as
3.2. Effect of Time and pH on detecting Cu2+
shown in Fig. S7. After adding 0.1 eq of Cu2+
,
1H NMR showed three
distinct new peaks: at 10.3 ppm of formyl groups, at 8.3 ppm and
7.8 ppm of benzene ring, and peak at 3.9 ppm of aniline (2,4-dimethyl
aniline, MS m/z: [C8H10N]ꢀ calcd: 120.08, found: 120.49, Fig. S11).
Peaks of 1H NMR belonging to benzene ring were brought together by
influence of Cu2+. 1H NMR results showed that HBTA was disappeared
under the action of Cu2+ to form its precursor (HBT-CHO, MS m/z:
[C14H8NO2S]- calcd: 254.03, found: 254.03, Fig. S12) with green
fluorescence.
The effect of time on probe performance was studied to provide a
basis for detection conditions and preliminary mechanism judgment.
Three conditions including without Cu2+, 0.1 eq Cu2+, and 2 eq Cu2+
were test. In the case of no Cu2+, fluorescence at 525 nm and 590 nm
was synchronously reduced for the first 20 min and then remained sta-
ble. When 0.1 eq Cu2+ was added, fluorescent intensity at 525 nm first
increased and then slowly decreased. The fluorescence spectrum re-
mains stable after 30 min. Fluorescent intensity at 590 nm dropped in
the first 30 min and then remained stable (Fig. 3b). In Fig. 3c, fluores-
cence almost disappeared within 50 min and remained stable when 2 eq
Cu2+ were added. It was as expected that low concentrations of Cu2+ can
cause decomposition [25] of HBTA and the decomposition product can
further complex Cu2+ [28].
In FT-IR spectrum (Fig. S8), hydroxyl peak at 3434 cmꢀ 1 gradually
broadened and formed multiple peaks at 3486 cmꢀ 1, 3357 cmꢀ 1, and
3167 cmꢀ 1 eventually as concentration of Cu2+ increases. Emerging
doublets indicated the formation of primary amines. The movement of
hydroxyl peak to high wave number indicated that it participated in
complexation of Cu2+. The broadening of these peaks indicated that
The effect of pH on the test was carried out as shown in Fig. 3d.
Fluorescence intensity at 525 nm and 590 nm were recorded at different
pH values. In pH of 7–11, HBTA showed a high degree of quenching and
stability for Cu2+. Blank control sample showed a change from orange to
green with the change of pH and the fluorescence was green at lower pH
(<6.5). The reason for this change in fluorescence under strong acid
conditions should be due to the instability of the Schiff base structure on
HBTA.
Cu2+ caused an association effect on some groups. The “
C
–
– –
N ”
–
stretching vibration peak at 1618 cmꢀ 1 moved to 1641 cmꢀ 1, which
indicated the involvement in complexation of Cu2+. Peak at 1668 cm-1 is
a characteristic peak of formyl group indicating disappearance of HBTA
in presence of 2 eq Cu2+. The intensity of stretching vibration peak of
ꢀ 1
–
–
and stretching vibration peak of “C S” at
“C O” at 1109 cm
617 cmꢀ 1 were enhanced with increasing Cu2+ concentration, which
reflected the participation of Cu2+
.
From 1H NMR titration and IR titration spectrum, decomposition of
HBTA and formation of HBT-CHO can be inferred. These conclusions can
also be obtained by using the changes in UV–vis spectrum (Fig. S9) to
3.3. Detection of Cys after HBTA completes a response to 0.1 eq Cu2+
The formyl group hidden in HBTA was exposed by low concentration
of Cu2+. Subsequently, the selective experiment after this response was
carried out. Only Cys can cause a new peak at 473 nm among 17 kinds of
amino acids (Pro, Trp, Met, Phe, Ala, His, Thr, Arg, Gly, Tyr, Ser, Glu,
Asp, GSH, Hcy, Ile, Val), and S2ꢀ , SO32ꢀ (Fig. 4a). Affected by the
complicated solution, bad competitive (Fig. S6) and long response time
(Fig. 4b) was obtained. The color of the solution no longer changed
almost after adding Cys 40 h. A liner relationship between I473/I525 and
Cys was obtained to be I473/I525 = 0.00363[Cys] + 0.250 (R2 = 0.987)
give mutual confirmation. Shoulder peak at 282 nm belongs to π-π*
transition (benzenoid bands) on HBTA benzene ring conjugated system.
The peak is inconspicuous due to influence of dimethylaniline. It was
blue-shifted after Cu2+ indicating that dimethylaniline fall off. The fine
structure is to some extent similar with HBT-CHO. Due to formation of
formyl group, absorption peak caused by n-π* transition was red shifted
from 372 nm to 435 nm according with the UV–vis spectrum of HBT-
CHO. Formyl as a chromophore can cause R band to red-shift. The
phenomenon is also caused by the generation of the resonance structure
belonging to HBTCHO structure with ESIPT process (Fig. S9 Inset). The
in concentration range of Cys in 25–200 μM, and the corresponding
Fig. 2. a. Fluorescence spectra of HBTA (40 μM) upon addition of different cation (40 μM) in Tris buffer/MeOH (v/v = 9/1, 50 mM, pH = 7.4, λex =350 nm); b. The
F/F0 value of HBTA + other ions and HBTA + other ions + Cu2+ in Tris buffer/MeOH (v/v = 9/1, 50 mM, pH = 7.4, λex =350 nm). (incubation time: 30 min).
4

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
Fig. 3. a. Effect of time on fluorescence of HBTA at 525 nm and 590 nm; b. Effect of time on fluorescence of HBTA at 525 nm and 590 nm after adding 0.1 eq Cu2+; c.
Effect of time on fluorescence of HBTA at 525 nm and 590 nm after adding 2 eq Cu2+; d. Effect of pH (6 – 11) on fluorescence of HBTA at 525 nm and 590 nm after
adding 2 eq Cu2+ and blank control. (Tris/MeOH v/v = 9/1, 50 mM, pH = 7.4, λex =350 nm).
peak at 435 nm shifted to 400 nm indicating a formation of complex
after adding 2 eq Cu2+. By comparing UV–vis spectra of HBTA, HBT-
CHO and HBTA with Cu2+, changes of absorption peak were consis-
tent with previous validation and expectations.
reported methods [26] (see ESI). Fitting curve was y = 184.35x – 47.24
(R2 = 0.987) (Fig. 6c) and corresponding binding constant was 256.3
Mꢀ 0.5. Confirmation of complexing process and binding constant is a
prerequisite for subsequent competition of Cys for binding to Cu2+
.
B3LYP/6-31 g(d) was choose to optimize the ground state structure
of the molecule (Fig. S10), and then use the optimized result as the
initial value of the excited state to calculate the excited state, and finally
get the TD-DFT result. TD-DFT of HBTA and its decomposition product
(HBT-CHO) was calculated and shown in Fig. 5. Energy gap was changed
from 1.200 eV to 6.595 eV, which indicated that HBT-CHO has a shorter
emission wavelength and accords with experimental phenomenon. The
narrow band gap of HBTA makes electrons in the ground state more
easily excited. Therefore, HBTA molecules exhibit a certain degree of
instability. TD-DFT calculation provided a theoretical basis for experi-
mental phenomena and interpretation.
In summary, suggested plausible mechanism of HBTA detection of
Cu2+ and Cys can be summarized as follows: at low concentrations, Cu2+
will first combine with imine of HBTA (HBTA@Cu2+, MS m/z:
[C22H18NO2SCu]2+, calcd: 421.04, found: 422.40, Fig. S14), and then
HBTA initiate decomposition resulting in fluorescence color change
[55]. As Cu2+ concentration increases, decomposition products begin to
complex with Cu2+ and dominate whole process, causing fluorescence
quenching. On the other way, the decomposition product can detect Cys
and generate blue fluorescence (HBT-Cys). The Above process is shown
in Scheme 2 and further verified by mass spectrometry. Continuously
reaction and complexation of Cu2+-responsive probe (HBTA) shows
three-stage linear response ranges and three different signal changes
[56,57], which indicates its advantages and potentials compared to
some Cu2+ probes discovered in recent years (Table S1) [58–62].
Complexation between HBT-CHO and Cu2+ was further studied by
Job’s and B-H experiments. Detection process of HBTA on Cu2+ had
been confirmed, then Job’s titration was carried out in order to inves-
tigate the complex ratio of HBT-CHO to Cu2+. A series of solutions,
decomposed HBTA and Cu2+ with a total concentration of 40
μM were
3.5. HBTA imaging in E. coli and MDA-MB-231 cells
prepared. As proportion of Cu2+ concentration increases, the value of F0
- F reached maximum at abscissa of 0.32 as showed in Fig. 6a. Ratio of
HBT-CHO complexing Cu2+ was determined to be 2:1 by Job’s experi-
ment (2HBT-CHO@Cu2+, MS m/z: [C28H17N2O4S2Cu]+, calcd: 571.99,
found: 572.14, Fig. S13). In B-H experiment, absorption value at 435 nm
was continuously decreased and absorption value at 400 nm was
increased with the concentration of Cu2+, and the isoabsorbation point
at 410 nm (Fig. 6b). This indicated that HBT-CHO was continuously
complexed with Cu2+ to form a new complex product. Absorption peak
at 400 nm was selected to fit the B-H equation according to previously
In order to preliminarily verify whether HBTA can be applied to
organism, E. coli imaging experiments were carried out. Slides immo-
bilized with E. coli were immersed in HBTA solution for 30 min, and
then observed with a fluorescent upright microscope. Rod-shaped E. coli
was observed in bright and red channels (Fig. S16a and S16b). Slide was
removed and placed in a solution containing a low concentration of
Cu2+ for 30 min. Green and blue fluorescence were observed in the
microscope (Figs. S16c and S16d). For comparison, another piece of
E. coli-loaded slide was immersed in a solution of HBTA treated with
5

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
Fig. 4. a. Fluorescence spectra of HBT-CHO (tested after HBTA reacted with 0.1 eq Cu2+ in Tris buffer) (10
μM) upon addition of different amino acids (Cys 120 μM)
in Tris buffer/MeOH (v/v = 9/1, 50 mM, pH = 7.4, λex =385 nm, after 27 h); b. Value of I473/I525 versus time after adding 3 eq Cys; c. Fluorescence changes of HBTA
in Cu2+ concentration range of 25 – 200
μM (λex =385 nm, after 10 h); d. Linear relationship of Cys in 25 – 200 μM.
Fig. 5. HOMO-LUMO energy levels and orbitals of HBTA and HBT-CHO calculated from CAM-B3LYP/6-31G* level of theories.
6

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
Fig. 6. a. Job’s plot of HBT-CHO and Cu2+ in solution of Tris/MeOH (v/v = 9/1, 50 mM, pH = 7.4, λex =440 nm); b. UV–vis spectra change from decomposition
product of HBTA (20
μM) upon addition of 0 – 11
μ
M of Cu2+; c. B-H plot based on a 2:1 association stoichiometry between the decomposition product and Cu2+
.
(incubation time: 30 min).
Scheme 2. Suggested plausible reaction process of HBTA to Cu2+
.
Fig. 7. Confocal fluorescence images at bright, blue channel, green channel, red channel, and merge (left – right, respectively). (a – e). MDA-MB-231 cells incubated
with HBTA; (f – j). MDA-MB-231 cells incubated with Cu2+ and NEM; (k – o). MDA-MB-231 cells incubated with Cys; (p – t). MDA-MB-231 cells incubated with Cu2+
and Cys.
7

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
Cu2+. Fluorescent image was observed after 30 min as shown in
Fig. S16e and S16f. The results indicated that HBTA can stain E. coli and
respond to external Cu2+, which revealed that it has potential applica-
tions in biological detection.
3.6. Competitive fluorescent response
After HBTA was hydrolyzed by Cu2+, HBT-CHO continuedly as a new
probe response to Cu2+ and Cys achieving different fluorescence signal.
Therefore, HBTA can be regard as a secondary probe. Because of the
detoxification of Cys to Cu2+ in vivo, it can adjust the concentration of
heavy metal ions. This had been reported in literatures that Cys can
complex Cu2+. Based on this, we thought that HBTA could be used as a
three-stage even multistage probe. Cu2+ and Cys were alternately added
to the solution of HBT-CHO. The result showed that green fluorescence
was continuously quenched and recovered (Fig. S18a). On the other
hand, Cu2+ and Cys were alternately added to the solution of HBT-Cys
and the blue fluorescence was changed continuously (Fig. S18b).
In above experiments, we had accomplished color change from or-
ange to green to colorless and from orange to green to blue as the signal
response. Based on the effect of Cys on Cu2+, we further achieved the
color change from orange to green to colorless to green to blue and
displayed by fluorescence spectroscopy (Fig. 8a). In addition, changing
order of addition achieved the color change from orange to green to blue
to colorless to blue (Fig. 8b). The logical relationship diagram be ob-
tained in Fig. 8c and d, which shows the relationship between adding
order and amount and fluorescence. Mechanisms and processes are
illustrated in Scheme S2 (Scheme 3).
Then HBTA was used to detect Cu2+ and Cys in breast cancer cells
(MDA-MB-231). As shown in Fig. S17, cell viability was assessed by MTT
assay, and HBTA had low cytotoxicity to the cells. The results of HBTA
imaging in cells were shown in the Fig. 7a–e. It can be observed that the
cells mainly showed red fluorescence in different channels, which
indicated that the cell environment will not cause its decomposition. In
order to remove the effect of thiol in cells on the detection of Cu2+ by
HBTA, NEM together with Cu2+ were added to the culture medium. As
shown in Fig. 7f–j, the fluorescence in the red channel was weakened,
and the green fluorescence was dominant. Therefore, Cu2+-induced
decomposition had already taken place. As comparison, the results of
culturing Cys with cells without adding Cu2+ were shown in Fig. 7k–o.
The results were similar to blank experiments, which revealed that only
Cys did not cause change color of HBTA in cells. Finally, in order to show
that Cys can cause the cyclization process after Cu2+ response, Cu2+ and
Cys were used to culture cells together. As shown in Fig. 7p–t, the blue
fluorescence was mainly shown. In summary, HBTA can be used in cells
based a nested mode. This logical response made HBTA a potential tool
for studying the intrinsic relationship between Cu2+ and Cys in organ-
isms [63].
Fig. 8. Fluorescence spectrum changes after adding Cu2+ and Cys. a. Changes of fluorescence spectrum based on the addition order of 0.1 eq Cu2+, 2 eq Cu2+, 6 eq
Cys and 9 eq Cys (inset: photos of fluorescent change); b. Changes of fluorescence spectrum based on the addition order of 0.1 eq Cu2+, 3 eq Cys, 9 eq Cu2+ and 12 eq
Cys (inset: photos of fluorescent change); c. Color change according to the addition order of 0.1 eq Cu2+, 2 eq Cu2+, 6 eq Cys and 9 eq Cys; d. Color change according
to the addition order of 0.1 eq Cu2+, 3 eq Cys, 9 eq Cu2+ and 12 eq Cys.
8

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
Scheme 3. Suggested plausible response processes of HBTA with Cu2+ and Cys.
4. Conclusion
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgements
Inspired by instability and affinity of imine structure to Cu2+, a
nested fluorescence probe was designed for breaking the single response
defect of current fluorescence probes. According to design ideas, HBTA
had achieved a three-stage response to Cu2+ including ratio, enhance-
ment, and quenching based on hydrolysis and complexation. The LOD of
responses are 86.9 nM, 2.6 nM, and 87.5 nM. The binding ratio of hy-
drolysate (HBT-CHO) to Cu2+ is 2: 1 and the binding constant is 256.3
The work was supported by the National Natural Science Foundation
of China (51678409, 51638011, 51578375), Tianjin Research Program
of Application Foundation and Advanced Technology (19JCYBJC19800,
18JCYBJC87500, 15ZCZDSF00880), State Key Laboratory of Separation
Membranes and Membrane Processes (Z1-201507), and the Program for
Innovative Research Team in University of Tianjin (TD13-5042).
M
ꢀ 0.5. In addition, HBT-CHO can selectively detect Cys through cycli-
zation with a formyl group to achieve fluorescent change from green to
blue and the LOD is 5.4
μ
M. Based on the affinity of Cys to Cu2+, HBTA
has achieved a logical response of color change corresponds to con-
centration change of Cu2+ and Cys, verifying the feasibility and rich
variability of nested probes.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2020.
113065.
CRediT authorship contribution statement
Fanyong Yan: Conceptualization, Methodology, Software, Software,
Validation, Writing - review & editing. Xiaodong Sun: Conceptualiza-
tion, Methodology, Software, Data curation, Writing - original draft,
Writing - review & editing. Yan Zhang: Data curation, Writing - original
draft. Yingxia Jiang: Data curation, Writing - original draft. Li Chen:
Supervision, Software, Validation. Tengchuang Ma: Software, Valida-
tion, Writing - review & editing, Resources, Visualization. Liang Chen:
Writing - review & editing.
References
[1] B. Jiang, Y. Yu, X. Guo, Z. Ding, B. Zhou, H. Liang, X. Shen, White-emitting carbon
dots with long alkyl-chain structure: effective inhibition of aggregation caused
quenching effect for label-free imaging of latent fingerprint, Carbon 128 (2018)
12–20.
[2] H. Feng, Z. Zhang, Q. Meng, H. Jia, Y. Wang, R. Zhang, Rapid response
fluorescence probe enabled in vivo diagnosis and assessing treatment response of
hypochlorous acid-mediated rheumatoid arthritis, Adv. Sci. 5 (2018) 1800397.
[3] W. Geng, S. Jia, Z. Zheng, Z. Li, D. Ding, D. Guo, A noncovalent fluorescence turn-
on strategy for hypoxia imaging, Angew. Chem. Int. Ed. 58 (2019) 2377–2381.
9

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
[4] X. Han, R. Wang, X. Song, F. Yu, L. Chen, Evaluation selenocysteine protective
effect in carbon disulfide induced hepatitis with a mitochondrial targeting
ratiometric near-infrared fluorescent probe, Anal. Chem. 90 (2018) 8108–8115.
[5] L. Chang, Q. Gao, S. Liu, C. Hu, W. Zhou, M. Zheng, Selective and differential
detection of Hg2+ and Cu2+ with use of a single rhodamine hydrazone-type probe
in the absence and presence of UV irradiation, Dye. Pigment. 153 (2018) 117–124.
[6] W. Cheng, Y. Xie, Z. Yang, Y. Sun, M. Zhang, Y. Ding, W. Zhang, General strategy
for in situ generation of a Coumarin-Cu2+ complex for fluorescent water sensing,
Anal. Chem. 91 (2019) 5817–5823.
[33] J. Zong, X. Yang, A. Trinchi, S. Hardin, I. Cole, Y. Zhu, C. Li, T. Muster, G. Wei,
Carbon dots as fluorescent probes for "off-on" detection of Cu2+ and L-cysteine in
aqueous solution, Biosens. Bioelectron. 51 (2014) 330–335.
[34] J. Sun, F. Yang, D. Zhao, C. Chen, X. Yang, Integrated logic gate for fluorescence
turn-on detection of histidine and cysteine based on Ag/Au bimetallic
nanoclusters-Cu2+ ensemble, ACS Appl. Mater. Inter. 7 (2015) 6860–6866.
[35] J. Li, J. Ge, Z. Zhang, J. Qiang, T. Wei, Y. Chen, Z. Li, F. Wang, X. Chen, A cyanine
dye-based fluorescent probe as indicator of copper clock reaction for tracing Cu2+
catalyzed oxidation of cysteine, Sensor. Actuat. B-Chem. 296 (2019) 126578.
-
[7] B. Gu, L. Huang, Z. Xu, Z. Tan, H. Meng, Z. Yang, Y. Chen, C. Peng, W. Xiao, D. Yu,
H. Li, A reaction-based, colorimetric and near-infrared fluorescent probe for Cu2+
and its applications, Sensor. Actuat. B-Chem. 273 (2018) 118–125.
[8] N. Yan, S. Xie, B. Tang, W. Wang, Real-time monitoring of the dissolution kinetics
of silver nanoparticles and nanowires in aquatic environments using an
aggregation-induced emission fluorogen, Chem. Commun. 54 (2018) 4585–4588.
[9] L. Wu, Y. Wang, T.D. James, N. Jia, C. Huang, A hemicyanine based ratiometric
fluorescence probe for mapping lysosomal pH during heat stroke in living cells,
Chem. Commun. 54 (2018) 518–521.
[36] C.M. Borkert, F.R. Cox, M.R. Tucker, Zinc and copper toxicity in peanut, soybean,
rice, and corn in soil mixtures, Commun. Soil Sci. Plant Anal. 29 (1998)
2991–3005.
[37] J. Xu, L. Yang, Z. Wang, G. Dong, J. Huan, Y. Wang, Toxicity of copper on rice
growth and accumulation of copper in rice grain in copper contaminated soil,
Chemosphere 62 (2006) 602–607.
[38] D. Chen, M. Xu, W. Wu, S. Li, Multi-color fluorescent carbon dots for wavelength-
selective and ultrasensitive Cu2+ sensing, J. Alloys. Compd. 701 (2017) 75–81.
[39] Y. Shi, Q. Liu, W. Yuan, M. Xue, W. Feng, F. Li, Dye-assembled upconversion
nanocomposite for luminescence ratiometric in vivo bioimaging of copper ions,
ACS Appl. Mater. Inter 11 (2019) 430–436.
[10] F. Yan, Z. Bai, Y. Chen, F. Zu, X. Li, J. Xu, L. Chen, Ratiometric fluorescent
detection of copper ions using coumarin-functionalized carbon dots based on
FRET, Sensor. Actuat. B-Chem. 275 (2018) 86–94.
[40] F. Chen, F. Xiao, W. Zhang, C. Lin, Y. Wu, Highly stable and NIR luminescent Ru-
LPMSN hybrid materials for sensitive detection of Cu2+ in vivo, ACS Appl. Mater.
Interfaces 10 (2018) 26964–26971.
[11] F. Yan, Z. Bai, F. Zu, Y. Zhang, X. Sun, T. Ma, L. Chen, Yellow-emissive carbon dots
with a large Stokes shift are viable fluorescent probes for detection and cellular
imaging of silver ions and glutathione, Microchim. Acta 186 (2019) 113.
[12] P. Wang, Q. Wang, J. Huang, N. Li, Y. Gu, A dual-site fluorescent probe for direct
and highly selective detection of cysteine and its application in living cells,
Biosensor Bioelectron. 92 (2017) 583–588.
[41] M.L. Desai, B. Deshmukh, N. Lenka, V. Haran, S. Jha, H. Basu, R.K. Singhal, P.
K. Sharma, S.K. Kailasa, K. Kim, Influence of doping ion, capping agent and pH on
the fluorescence properties of zinc sulfide quantum dots: sensing of Cu2+ and Hg2+
ions and their biocompatibility with cancer and fungal cells, Spectrochim. Acta A
210 (2019) 212–221.
[13] L. He, X. Yang, K. Xu, X. Kong, W. Lin, A multi-signal fluorescent probe for
simultaneously distinguishing and sequentially sensing cysteine/homocysteine,
glutathione, and hydrogen sulfide in living cells, Chem. Sci. 8 (2017) 6257–6265.
[14] Y. Yue, F. Huo, P. Ning, Y. Zhang, J. Chao, X. Meng, C. Yin, Dual-site fluorescent
probe for visualizing the metabolism of cys in living cells, J. Am. Chem. Soc. 139
(2017) 3181–3185.
[42] Z. Guo, Q. Niu, T. Li, E. Wang, Highly chemoselective colorimetric/fluorometric
dual-channel sensor with fast response and good reversibility for the selective and
sensitive detection of Cu2+, Tetrahedron 75 (2019) 3982–3992.
[43] P. Zhu, Y. Shang, W. Tian, K. Huang, Y. Luo, W. Xu, Ultra-sensitive and absolute
quantitative detection of Cu2+ based on DNAzyme and digital PCR in water and
drink samples, Food Chem. 221 (2017) 1770–1777.
[15] Y. Yue, F. Huo, F. Cheng, X. Zhu, T. Mafireyi, R.M. Strongin, C. Yin, Functional
synthetic probes for selective targeting and multi-analyte detection and imaging,
Chem. Soc. Rev. 48 (2019) 4155–4177.
[44] T. Lv, Y. Xu, H. Li, F. Liu, S. Sun, A Rhodamine B-based fluorescent probe for
imaging Cu2+ in maize roots, Bioorg. Med. Chem. 26 (2018) 1448–1452.
[45] Z. Guo, Q. Niu, T. Li, T. Sun, H. Chi, A fast, highly selective and sensitive
colorimetric and fluorescent sensor for Cu2+ and its application in real water and
food samples, Spectrochim. Acta A 213 (2019) 97–103.
[16] Y. Guo, L. Yang, W. Li, X. Wang, Y. Shang, B. Li, Carbon dots doped with nitrogen
and sulfur and loaded with copper(II) as a “turn-on” fluorescent probe for cystein,
glutathione and mocysteine, Microchim. Acta 183 (2016) 409–416.
[17] M. hang, L. Gong, C. Sun, W. Li, Z. Chang, D. Qi, A new fluorescent-colorimetric
chemosensor based on a Schiff base for detecting Cr3+, Cu2+, Fe3+ and Al3+ ions,
Spectrochim. Acta A 214 (2019) 7–13.
[46] H. Jiang, Z. Li, Y. Kang, L. Ding, S. Qiao, S. Jia, W. Luo, W. Liu, A two-photon
fluorescent probe for Cu2+ based on dansyl moiety and its application in
bioimaging, Sensor. Actuat. B-Chem. 242 (2017) 112–117.
[18] Z. Zhang, Y. Liu, E. Wang, A highly selective “turn-on” fluorescent probe for
detecting Cu2+ in two different sensing mechanisms, Dye. Pigment. 163 (2019)
533–537.
[47] A.I. Said, N.I. Georgiev, V.B. Bojinov, The simplest molecular chemosensor for
detecting higher pHs, Cu2+ and S2ꢀ in aqueous environment and executing various
logic gates, J. Photochem. Photobiol. A 371 (2019) 395–406.
[19] S. Erdemir, S. Malkondu, Dual-emissive fluorescent probe based on
phenolphthalein appended diaminomaleonitrile for Al3+ and the colorimetric
recognition of Cu2+, Dye. Pigment. 163 (2019) 330–336.
[48] B. Tripathi, S. Mehta, A. Amar, J. Gaur, Oxidative stress in Scenedesmus sp. during
short- and long-term exposure to Cu2+ and Zn2+, Chemosphere 62 (2006) 538–544.
[49] F. Yan, X. Sun, F. Zu, Z. Bai, Y. Jiang, K. Fan, J. Wang, Fluorescent probes for
detecting cysteine, Methods Appl. Fluoresc. 6 (2018) 042001.
[20] K.S. Mani, R. Rajamanikandan, B. Murugesapandian, R. Shankar, G. Sivaraman,
M. Ilanchelian, S.P. Rajendran, Coumarin based hydrazone as an ICT-based
fluorescence chemosensor for the detection of Cu2+ ions and the application in
HeLa cells, Spectrochim. Acta A 214 (2019) 170–176.
[50] M. Jia, L. Niu, Y. Zhang, Q. Yang, C. Tung, Y. Guan, L. Feng, BODIPY-based
fluorometric sensor for the simultaneous determination of Cys, Hcy, and GSH in
human serum, ACS Appl. Mater. Inter 7 (2015) 5907–5914.
[21] F. Huo, C. Yin, Y. Yang, J. Su, J. Chao, D. Liu, Ultraviolet-Visible Light (UV–Vis)-
reversible but fluorescence-irreversible chemosensor for copper in water and its
application in living cells, Anal. Chem. 84 (2012) 2219–2223.
[51] S. Shahrokhian, Lead phthalocyanine as a selective carrier for preparation of a
cysteine-selective electrode, Anal. Chem. 73 (2001) 5972–5978.
[52] M. Liu, N. Li, Y. He, Y. Ge, G. Song, Dually emitting gold-silver nanoclusters as
viable ratiometric fluorescent probes for cysteine and arginine, Microchim. Acta.
185 (2018) 147.
[22] J. Li, C. Yin, Cu2+ biological imaging probes based on different sensing
mechanisms, Curr. Med. Chem. 24 (2017) 3958–4002.
[23] F. Huo, L. Wang, C. Yin, Y. Yang, H. Tong, J. Chao, Y. Zhang, The synthesis,
characterization of three isomers of rhodamine derivative and their application in
copper (II) ion recognition, Sensor. Actuat. B-Chem. 188 (2013) 735–740.
[24] H. Zhang, L. Feng, Y. Jiang, Y. Wong, Y. He, G. Zheng, J. He, Y. Tan, H. Sun, D. Ho,
A reaction-based near-infrared fluorescent sensor for Cu2+ detection in aqueous
buffer and its application in living cells and tissues imaging, Biosens. Bioelectron.
94 (2017) 24–29.
[53] S. Sahana, G. Mishra, S. Sivakumar, P.K. Bharadwaj, A 2-(2’-hydroxyphenyl)
benzothiazole (HBT)-quinoline conjugate: a highly specific fluorescent probe for
Hg2+ based on ESIPT and its application in bioimaging, Dalton Trans. 44 (2015)
20139–20146.
[55] D. Udhayakumari, V. Inbaraj, A review on schiff base fluorescent chemosensors for
cell imaging applications, J. Fluoresc. 30 (2020) 1203–1223.
[56] D. Udhayakumari, S. Naha, S. Velmathi, Colorimetric and fluorescent
chemosensors for Cu2+. A comprehensive review from the years 2013–2015, Anal.
Methods 9 (2017) 552–578.
[25] C. Li, Z. Yang, S. Li, 1,8-Naphthalimide derived dual-functioning fluorescent probe
for “turn-off” and ratiometric detection of Cu2+ based on two distinct mechanisms
in different concentration ranges, J. Lumin. 198 (2018) 327–336.
[57] B. Kaur, N. Kaur, S. Kumar, Colorimetric metal ion sensors – a comprehensive
review of the years 2011–2016, Coord. Chem. Rev. 358 (2018) 13–69.
[58] Y. Guo, L. Wang, J. Zhuo, B. Xu, X. Li, J. Zhang, Z. Zhang, H. Chi, Y. Dong, G. Lu,
A pyrene-based dual chemosensor for colorimetric detection of Cu2+ and
fluorescent detection of Fe3+, Tetrahedron Lett. 58 (2017) 3951–3956.
[59] S. Sakunkaewkasem, A. Petdum, W. Panchan, J. Sirirak, A. Charoenpanich,
T. Sooksimuang, N. Wanichacheva, Dual-analyte fluorescent sensor based on [5]
helicene derivative with super large stokes shift for the selective determinations of
Cu2+ or Zn2+ in buffer solutions and its application in a living cell, ACS Sens. 3
(2018) 1016–1023.
[26] F. Yan, X. Sun, R. Zhang, Y. Jiang, J. Xu, J. Wei, Enhanced fluorescence probes
based on Schiff base for recognizing Cu2+ and effect of different substituents on
spectra, Spectrochim. Acta A 222 (2019) 117222.
[27] S. Erdemir, B. Tabakci, M. Tabakci, A highly selective fluorescent sensor based on
calix[4]arene appended benzothiazole units for Cu2+, S2ꢀ and HSO4- ions in
aqueous solution, Sensor Actuat. B-Chem. 228 (2016) 109–116.
[28] Y. Li, M. Sun, K. Zhang, Y. Zhang, Y. Yan, K. Lei, L. Wu, H. Yu, S. Wang, A near-
infrared fluorescent probe for Cu2+ in living cells based on coordination effect,
Sensor. Actuat. B-Chem. 243 (2017) 36–42.
[29] Y. Li, A ratiometric fluorescent chemosensor for the detection of cysteine in
aqueous solution at neutral pH, Luminescence 32 (2017) 1385–1390.
[30] S. Goswami, A. Manna, S. Paul, A.K. Das, P.K. Nandi, A.K. Maity, P. Saha, A turn on
ESIPT probe for rapid and ratiometric fluorogenic detection of homocysteine and
cysteine in water with live cell-imaging, Tetrahedron Lett. 55 (2014) 490–494.
[31] C. Yin, K. Xiong, F. Huo, J. Salamanca, R. Strongin, Fluorescent probes with
multiple binding sites for the discrimination of Cys, Hcy, and GSH, Angew. Chem.
Int. Ed. 56 (2017) 13188–13198.
[60] N. Wang, Y. Liu, Y. Li, Q. Liu, M. Xie, Fluorescent and colorimetric sensor for Cu2+
ion based on formaldehyde modified hyperbranched polyethylenimine capped gold
nanoparticles, Sensor Actuat. B-Chem. 255 (2018) 78–86.
[32] C. Yin, F. Huo, J. Zhang, R. Martínez, Y. Yang, H. Lv, S. Li, Thiol-addition reactions
and their applications in thiol recognition, Chem. Soc. Rev. 42 (2013) 6032–6059.
10

F. Yan et al.
Journal of Photochemistry & Photobiology, A: Chemistry 407 (2021) 113065
[61] X. Wei, H. Xu, W. Li, Z. Chen, Colorimetric visualization of Cu2+ based on Cu2+
catalyzed reaction and the signal amplification induced by K+-aptamer-Cu2+
complex, Sensor Actuat. B-Chem. 241 (2017) 498–503.
-
[63] Y. Zhang, X. Zeng, L. Mu, Y. Chen, J.-X. Zhang, C. Redshaw, G. Wei, Rhodamine-
triazine based probes for Cu2+ in aqueous media and living cells, Sensor Actuat. B-
Chem. 204 (2014) 24–30.
[62] J. Qiu, S. Jiang, B. Lin, H. Guo, F. Yang, An unusual AIE fluorescent sensor for
sequentially detecting Co2+-Hg2+-Cu2+ based on diphenylacrylonitrile Schiff-base
derivative, Dye. Pigment. 170 (2019) 107590.
11