A dual-functional probe for sensing pH change and ratiometric detection of Cu2+

Article abstract of DOI:10.1016/j.saa.2020.118318

A series of benzothiazole-based compounds were synthesized and characterized. Among them, probe Z showed significant dual-functional performance which was capable of sensing pH change and Cu²⁺. Probe Z displayed fluorescent turn-on under alkaline conditions due to deprotonation of the hydroxyl group along with the obviously color change from colorless to mint green. Interestingly, it further achieved in ratiometric detection of Cu²⁺ through absorbance or fluorescence signals in strong alkaline condition. The limit of detection was calculated correspondingly as 0.37 μM and 1.35 μM, respectively. Especially, the combination of the XNOR and INHIBIT logic gates could be used to confirm that one medium was in neutrality or alkalinity condition. Moreover, Z was successfully used in real water samples and test paper for fast identification of Cu²⁺, respectively.

Full text of DOI:10.1016/j.saa.2020.118318

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A dual-functional probe for sensing pH change and ratiometric detection
of Cu2+
b
b
b
c,
Jiang-Xu Wang a, Zhi-Yong Xing b, , Zhen-Nan Tian , Ding-Qi Wu , Yuan-Yuan Xiang , Jin-Long Li
⁎
⁎
a
College of Life Science, Northeast Agricultural University, Harbin 150030, PR China
b
Department of Applied Chemistry, College of Arts and Sciences, Northeast Agricultural University, Harbin 150030, PR China
School of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar 161006, PR China
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzothiazole-based compounds were synthesized and characterized. Among them, probe Z showed
significant dual-functional performance which was capable of sensing pH change and Cu2+. Probe Z displayed
fluorescent turn-on under alkaline conditions due to deprotonation of the hydroxyl group along with the obvi-
ously color change from colorless to mint green. Interestingly, it further achieved in ratiometric detection of
Cu2+ through absorbance or fluorescence signals in strong alkaline condition. The limit of detection was calcu-
lated correspondingly as 0.37 μM and 1.35 μM, respectively. Especially, the combination of the XNOR and
INHIBIT logic gates could be used to confirm that one medium was in neutrality or alkalinity condition. Moreover,
Z was successfully used in real water samples and test paper for fast identification of Cu2+, respectively.
© 2020 Elsevier B.V. All rights reserved.
Received 23 December 2019
Received in revised form 27 March 2020
Accepted 29 March 2020
Available online 01 April 2020
Keywords:
Benzothiazole
pH sensing
Cu2+
Ratiometric detection
Logic gate
1. Introduction
adversely affect various microorganisms and further undermines eco-
system balance [15]. Furthermore, the excessive accumulation of Cu2+
The pH value plays critical role in many fields including agricultural
production, sewage treatment procedures, industrial production, envi-
ronmental protection and many physiological processes [1–5]. There-
fore, the development of a fast, convenient and accurate method for
monitoring the pH of an environmental or the biological system is
very important for environmental and biomedical research. Compared
with traditional electrochemical-based pH meters, the fluorescent
probes has the advantages of high sensitivity, non-destructive, without
frequent calibrations, rapid measurement, continuous monitor the
changes of pH and no corrosion in strongly alkaline conditions [6–10],
thus more and more researchers are devoted to the development of
pH probes for a particular environment. However, among those probes
which were reported in recent years, there were still some defects such
as complicated synthesis steps, poor water solubility, slow detection
time, weak fluorescent signal, and low sensitivity [11–14]. Therefore,
the development of an excellent pH fluorescent probe, which is pro-
vided with such virtues (including simple procedures, sensitive fluores-
cence signal changes, rapid detection, and excellent water solubility),
will be the motivation to many researchers.
in human body can promote the formation of reactive oxygen species
(ROS) which can poison the normal cells, thus leading to severe neuro-
degenerative diseases such as Menkes syndrome, Parkinson's, Wilson's
and Alzheimer's diseases [16–20]. Considering the close association of
Cu2+ with environmental protection and human health, many re-
searchers pay more attention to design and synthesis of fluorescent
probes which can be used in quantity and quality detection of Cu2+
.
Up to now, many fluorescent probes in sensing Cu2+ were achieved
by the change in fluorescence intensity (fluorescence turn-on or turn-
off) as the only detection signal [16,21–26]. However, these signals are
inevitably influenced by several factors, including instrumental effi-
ciency, environmental conditions, and probe concentration. Compared
with the fluorescent turn-on (off) probes, the development of
ratiometric probe is more appealing because it can eliminate the inter-
ference caused by the physical or chemical method through the ratio
of two intensities of absorption or emission wavelength [27–30].
Some ratiometric fluorescence probes for Cu2+ have been reported
[31–36], but the synthesis of that type probe is still full of challenge
due to the paramagnetism of Cu2+ which often quenches fluorescence
[37–42]. In addition, 2-(2′-Hydroxyphenyl) benzothiazole (HBT) as a
fluorophore have been widely used in construction chemosensors by
using the strategy that which could inhibit excited-state intramolecular
proton transfer (ESIPT) to alter the fluorescent properties through the
coordination of oxygen atoms on a phenolic hydroxyl with a metal ion
[23,43,44].
Among many metal ions, Cu2+ plays an important role in the envi-
ronment and the human body. As one of the well-known toxic metal
ions, the excessive presence of Cu2+ in natural ecosystems can
⁎
Corresponding authors.
E-mail addresses: zyxing@neau.edu.cn (Z.-Y. Xing), jinlong141@163.com (J.-L. Li).
https://doi.org/10.1016/j.saa.2020.118318
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
J.-X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
Taking above discussion into consideration, in this article, we had
for 6 h. After completion of the reaction (detected by TLC), the mixture
was cooled to room temperature and then was poured into distilled
water (200 mL). The white precipitate was filtered and washed with
distilled water for 5 times to get the compound 1. Yield: 1.58 g, yield:
88.5%. 1H NMR (600 MHz, DMSO-d6) (Fig. S1) δ (ppm) 12.37 (s, 1H),
8.19 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.8 Hz,
1H), 7.59 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.36 (d, J =
7.4 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H), 2.29 (s, 3H). 13C NMR (151 MHz,
DMSO-d6) (Fig. S2) δ (ppm) 169.34, 155.69, 151.64, 134.37, 132.97,
127.48, 126.73, 126.62, 126.27, 122.79, 122.39, 120.08, 116.46, 16.26.
designed and synthesized a novel dual-functional fluorescent probe Z
using 2-(2′-Hydroxyphenyl) benzothiazole (HBT) as the fluorophore
(Scheme 1) to detect physiological pH and Cu2+, respectively. Probe Z
displayed fluorescent turn-on under alkaline conditions due to depro-
tonation of the hydroxyl group, and the Cu2+ could be further subjected
to trace detection by ratiometric fluorescence. To the best of our knowl-
edge, the ratiometric fluorescent probe for Cu2+ under strongly alkaline
conditions has not been reported. Moreover, the sensing mechanism of
Z to Cu2+ was further confirmed by the investigation of the properties
of compound Z-1, Z-2 and Z-3, which had the similar structure to com-
pound Z through changing the position of the hydroxyl group. More-
over, the application of Z in logic gate, real samples, and test paper
were all conducted.
2.2.2. Synthesis of compound 2
Hexamethylenetetraamine (3.12 g, 22.26 mmol) was added to a so-
lution of compound 1 (1.0 g, 4.15 mmol) in trifluoroaceticacid (15 mL),
the mixture was refluxed for 7 h. When the compound 1 was consumed
which was monitored by TLC, the distilled water was added to the mix-
ture. After cooling to the room temperature, the precipitate was filtered
to obtained compound 2. Yield: 1.03 g, yield: 77%. 1H NMR (600 MHz,
DMSO-d6) (Fig. S3) δ (ppm) 13.16 (s, 1H), 9.93 (s, 1H), 8.30 (s, 1H),
8.22 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.84 (s, 1H), 7.61 (t,
J = 7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 2.34 (s, 3H). 13C NMR
(151 MHz, DMSO-d6) (Fig. S4) δ (ppm) 191.52, 167.99, 160.66, 151.26,
133.44, 133.22, 130.06, 128.92, 127.91, 127.62, 126.58, 122.90, 122.60,
116.88, 16.28.
2. Experimental
2.1. Materials and instruments
Solvents and reagents (analytical or spectrophotometric grade) in-
volved in the experiments were all purchased from commercial sup-
pliers and were not purified for use unless otherwise stated. Metal
ions were prepared by NaClO4, KClO4, Mg(ClO4)2, Ba(ClO4)2, Fe(ClO4)3.
xH2O, Zn(ClO4)2. 6H2O, Cu(ClO4)2. 6H2O, Ca(NO3).24H2O, Al(NO3)3. 9H2O,
Co(NO3).26H2O, Ni(NO3)2. 6H2O, Cd(NO3)2, AgNO3, Pb(NO3)2, CuCl2,
HgCl2, FeCl.24H2O and MnSO4. H2O. The 1H NMR spectrum and 13C NMR
spectrum of the synthesized compounds were measured in DMSO-d6
on a Bruck AV-600 spectrometer. The pH of the solution was measured
by the PHS-3C meter (Shanghai, China). The data of absorption spectra
and fluorescence spectra were obtained with Shimadzu UV-2700 UV–
vis spectrometer and Perkin Elmer LS55 fluorescence spectrometer at
room temperature, respectively. The mass spectra were performed on
a Waters Xevo UPLC/G2-SQ Tof MS spectrometer.
2.2.3. Synthesis of sensor Z
Compound 2 (100 mg, 0.37 mmol) was firstly dissolved in ethanol
(20 mL), then salicylhydrazide (58.1 mg, 0.38 mmol) was added and
the mixture was refluxed for 4 h. After completion of reaction, the pre-
cipitate was filtered to get the light yellow probe Z. Yield: 134.9 mg,
yield: 89.2%. 1H NMR (600 MHz, DMSO-d6) (Fig. S5) δ (ppm) 12.63 (s,
1H), 11.90 (s, 1H), 11.85 (s, 1H), 8.47 (s, 1H), 8.22 (d, J = 8.4 Hz, 1H),
8.14 (d, J = 10.8 Hz, 2H), 7.92 (d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.61 (t,
J = 6.6 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.00
(d, J = 8.0 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 2.35 (s, 3H). 13C NMR
(151 MHz, DMSO-d6) (Fig. S6) δ (ppm) 168.32, 165.08, 159.45, 157.39,
151.53, 148.36, 134.24, 133.27, 132.47, 129.07, 127.58, 127.55, 126.41,
126.27, 125.83, 122.86, 122.57, 119.45, 117.75, 117.13, 116.46, 16.36.
HRMS (m/z) (TOF MS ES−) (Fig. S7): calcd for C22H17N3O3S: 402.0912
[M-H+]−, found: 402.0919.
2.2. Synthesis
The intermediates (1 and 2) and final sensor Z (Scheme 1) were syn-
thesized according to reported method [45].
2.2.1. Synthesis of compound 1
To a solution of 2-aminobenzenethiol (925 mg, 7.40 mmol) in DMF
(20 mL), 3-methylsalicylaldehyde (1.01 g, 7.43 mmol) and Na2S2O5
(1.64 g, 8.63 mmol) were added. The reaction mixture was refluxed
Synthetic procedures and characterization of compounds Z-1, Z-2
and Z-3 were illustrated in Supplementary Information (Fig. S8–16).
Scheme 1. Synthesis of compounds Z, Z-1, Z-2 and Z-3.

J.-X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
3
2.3. General information
2.6. Determination of quantum yield (QY)
The stock solutions (0.1 mM) of Z, Z-1, Z-2, and Z-3 were all pre-
pared in DMF, and the corresponding test solutions were all prepared
in HEPES buffer solution (10 mM, with 30% DMF, v/v) and was used to
measure spectra of absorption and fluorescence, respectively. The pH
of the solution was adjusted by using HCl and NaOH. The cationic salts
The Quantum Yield (QY) of a sample was calculated according to the
equation: QY(s) = QY (r) (Ar Fs/As Fr) (ns/nr)2. Where r and s represent
the reference and the sample, respectively, A, F and n refer to the absor-
bance intensity, fluorescence intensity and the refractive index of sol-
vent, respectively. The fluorescence quantum yield was calculated
using quinine sulfate (QY(r) = 0.55) as reference [49].
such as Zn+, K+, Mn2+, Fe2+, Fe3+, Hg2+, Ni2+, Pb2+, Ca2+, Ba2+
,
Cr3+, Cu2+, Al3+, Ag+, Na+, Cd2+, Co2+ and Mg2+ were prepared
with ultrapure water (50 mL) to obtain aqueous solution (10 mM).
The excitation wavelength used in the fluorescence examination was
390 nm, and the slits width of excitation and emission were all 10 nm.
3. Results and discussion
3.1. Spectrum studies of Z with different pH buffer solutions
2.4. Determination of basicity constant pKb values
The fluorescence spectra of probe Z in different pH buffer solutions
were firstly measured. In the solution of DMF-H2O (3/7, v/v, 0.01 M
HEPES) (Fig. 1a), the probe Z had almost no fluorescence emission
(QY = 0.00032) in the range from acidic (pH 2) to neutral (pH 7) me-
dium. However, in the alkaline (pH 8–13) medium, the fluorescence in-
tensity at 498 nm was gradually enhanced with the increasing of pH,
especially the QY data was reached 0.098 in stronger alkaline (pH =
13) medium. This result might be due to the deprotonation of phenolic
hydroxyl in basic environment and the enhancement of intramolecular
charge transfer (ICT) effect of Z. It could be found that in the pH range
from 7.5 to 9.5 (Fig. 1b), a good linear relationship between the emis-
sion intensity at 498 nm and the data of pH (R2 = 0.9899) was ob-
served, which indicated that the probe Z could be used for pH
detection in alkaline medium. The pKb was calculated according to the
above emission titration and the value was calculated as 7.43
(Fig. S17), which was essential for turn on fluorescence probe as pH sen-
sor in a basic medium. In addition, as shown in Fig. 1c, probe Z also could
achieved in pH sensing by naked eye detection along with the obviously
color change from colorless to mint green under 365 nm UV light.
In order to investigate the response of Z to different pH, the absorp-
tion spectra titration of Z with different pH buffer solutions was also
measured (Fig. S18). The absorption spectra at pH = 3.0–13.0 were spe-
cifically selected for comparison. As shown in Fig. 2a, the absorbance
centered at 319 nm of Z was observed when the pH was 7.0. However,
the absorption peak (319 nm) was almost disappeared and meanwhile
Henderson-Hasselbalch equation was used to determine the basicity
constant pKb values based on the previously report as followed [46,47].
pKb ¼ pOH þ log ½ðImax−IÞ=ðImin−IÞꢀ
ð1Þ
where; I is the observed fluorescence intensity at a fixed wavelength;
Imax and Imin are the corresponding maximum and minimum intensity,
respectively.
2.5. Determination of binding constant
The binding constant was calculated based on the modified Benesi–
Hildebrand equation according to the reported literature [48].
1
1
1
¼
−
ð2Þ
Â
Ã
n
KðFmax−F0Þ Mþ
F−F0
Fmax−F0
where, Fmax, F and F0 are the fluorescence intensities ratio (F451/F498) of
Z in the presence of Cu2+ at saturation, at an intermediate Cu2+-
concentration, and absence of Cu2+, respectively. K is the binding con-
stant. [M+] is the concentration of Cu2+, and n is the binding
stoichiometry of Z and Cu2+
.
Fig. 1. (a) Fluorescence spectra of Z (10 μM) in different pH buffer solutions; (b) The linear responses of pH with Z; (c) The fluorescence change of probe Z with different pH under 365 nm
UV light. (Error bar was represented as mean standard deviation, n = 3).

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the absorption peak at 284 nm was formed when the pH was 3.0, which
could be attribute to the formation of Z + H+ (Fig. 2a insert) by proton-
ation of nitrogen atom of thiazole ring of probe Z in the acidic medium.
When the pH data was increased from 7.0 to 13.0, two new absorption
peaks (302 nm and 315 nm) were detected and the absorbance inten-
sity decreased gradually, indicating the occurrence of deprotonation
on hydroxyl group of probe Z. Meanwhile, the absorption peak at
319 nm was gradually red-shifted to 374 nm, indicated the enhance-
ment of ICT effect from the deprotonated oxygen atom of hydroxyl
group to the benzothiazole core of probe Z.
measured (Fig. S20), respectively. The result showed that the typical
peaks of 3258 cm−1 and 3059 cm−1 of hydroxyl groups of compound
Z were all disappeared after addition of NaOH compared with that of
compound Z itself, indicating the deprotonation on hydroxyl groups of
compound Z indeed happened. Moreover, the carbonyl group peak of
compound Z turned into weak and the characteristic absorption of –
C=N- (1435 cm−1) was much more stronger after addition of NaOH, in-
dicating the enolation of amide group and further was deprotonated in
alkaline condition which was illustrated in Fig. 6 (insert) .
Moreover, to clear the deprotonation on which site of probe Z, 1
H
Furthermore, the compound Z-1, Z-2 and Z-3 were synthesized and
characterized for confirmation that the ICT enhancement caused by the
deprotonation of phenolic hydroxyl was the main process in pH sensing
in the alkaline medium. The fluorescence of Z-1, Z-2 and Z-3 with differ-
ent pH were measured in DMF/H2O (3/7, v/v, 0.01 M HEPES), respectively.
It was noticed that only under alkaline conditions, the fluorescence of
compounds Z-1, Z-2 and Z-3 were all turned on (Figs. 2b, 3c and d), but
the extent of enhancement in intensity among compounds Z-1, Z-2 and
Z-3 was entirely different in the condition of pH = 13.0. The maximum
fluorescence intensity of compounds Z-1 and Z-3 were significantly
lower than that of compounds Z and Z-2 at pH 13.0 (Fig. 2e). The fluores-
cence spectra of compound Z-2 was similar to the probe Z, and the com-
pound Z-2 also displayed a good linear relationship between the emission
at 494 nm and pH in the range of 8.0 to 11.0 (Fig. S19), which indicated
that the compound Z-2 also has potential of pH sensing in alkaline me-
dium. According to the above results, we found that the key factors caus-
ing the significant turn-on of probe Z were as followed. The primary factor
was the existence of hydroxyl whatever its position was in the ortho- or
para- of hydrazide group on benzene ring, and the other was the depro-
tonation of hydroxyl group in alkaline medium.
Thus, a probable sensing mechanism of Z to pH was proposed in Fig. 3.
In neutral medium, the conversion of probe Z between keto and enol form
was mainly due to the ESIPT mechanism which led to nonradiative decay
and fluorescence quenching [46,50,51]. As the acidity of the solution in-
creased, the protonation of Z was promoted and the benzothiazole
group was twisted, which resulted in non-emission of fluorescence
under acidic conditions reported previously by other group [52]. More-
over, the significant fluorescence turn-on of probe Z in alkali condition
was ascribed to the co-contributions as followed. The first factor was
the inhibition of ESIPT process from hydroxyl group to benzothiazole
fluorophore, the other was the occurrence of intramolecular charge trans-
fer (ICT) process from the oxygen atom to the benzothiazole core.
NMR spectrum of probe Z was measured before and after addition of
NaOD in DMSO-d6 (Fig. S21), respectively. The result obviously showed
that the proton signals (Ha and Hk of hydroxyl groups and Hj of amide
group) of probe Z were all disappeared after addition of NaOD. More-
over, the proton signals in the region of 6.8–8.6 ppm were almost all
up-field shifted compared with that of probe Z without addition of
NaOD, which was attributed to the increased electron cloud density
after the deprotonation on the two hydroxyl groups and amide group
of probe Z.
3.3. Spectra characteristics of Z for metal ions
The probe Z showed no significant fluorescence changes upon addi-
tion of different metal ions in DMF/H2O (3/7, v/v, 0.01 M HEPES, pH =
7.0), but its fluorescence turn-on under alkaline condition prompted
us to further study the spectra characteristics of Z (10 μM) for different
metal ions in DMF/H2O (3/7, v/v, 0.01 M HEPES, pH = 13.0). Interest-
ingly, as shown in Fig. 4a, the addition of Cu2+ (50 μM) led to a signifi-
cant color change from mint green to dark blue while other added metal
ions almost had no response under UV light (365 nm). Moreover, the
fluorescence spectrum of Z displayed an obvious blue-shift from
498 nm to 451 nm accompany with the decreased fluorescent intensity
with lower quantum yield (QY = 0.033) (Fig. 4b). Similarly, the absorp-
tion spectrum was also significantly blue shifted and decreased, and a
new absorption peak at 374 nm appeared (Fig. 4c). This obviously phe-
nomenon of blue-shift might attribute to the inhibition of ICT after the
coordination of oxygen atom of hydroxyl with Cu2+ [53]. The above re-
sults displayed that probe Z had high selectivity and sensitivity for Cu2+
and could be used as a fluorescent probe for Cu2+ under alkaline
medium.
In order to investigate the quantitative detection ability of probe Z to
Cu2+, the fluorescence and absorption titration of Z with Cu2+ were
measured, respectively. After adding different concentrations of Cu2+
(0–20 μM) to the solution of Z (10 μM), the color of the solution gradu-
ally changes from mint green to blue along with the increased concen-
tration of Cu2+ under 365 nm UV light (Fig. 5a), which indicated that
Z could rapid detection of Cu2+ concentration by colorimetric analysis.
3.2. Mechanism investigation of Z for pH sensing
In order to confirm the deprotonation of Z in alkaline condition, FT-
IR spectra of compound Z before and after addition of NaOH were firstly
Fig. 2. (a) Absorption spectra of Z (10 μM) in different pH buffer solutions. Insert: The structure of Z in different pH buffer solutions; fluorescence spectra of (b) Z-1, (c) Z-2 and (d) Z-3 in
different pH buffer solutions, respectively; (e) the maximum fluorescence intensity of compounds Z, Z-1, Z-2 and Z-3 at pH 13.0. (Error bar was represented as mean standard deviation,
n = 3).

J.-X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
5
Fig. 3. Sensing mechanism of Z to pH.
In addition, upon gradual addition of Cu2+ (0–40 μM), the fluorescence
intensity of Z at 498 nm was decreased while the maximum fluores-
cence peak was gradual blue-shift to 451 nm (Fig. 5b), and then reached
a platform when the amount of Cu2+ added was 20 μM. The fluores-
cence intensity ratio (F451/F498) versus the concentration of Cu2+
(0–20 μM) displayed a good relationship (Fig. S22). Furthermore, as
shown in Fig. 5c, along with the Cu2+ concentration (0–20 μM) in-
creased, the absorption peak at 374 nm was gradual decrease and
meanwhile the absorption peak at 283 nm was constantly increased.
the sensitivity of the detection, comparative experiments were per-
formed by the measurement of fluorescence spectrum of probe Z before
and after addition of different cupric salt (Fig. S24), respectively. The re-
sult showed that the change on fluorescence spectrum of probe Z was
almost the same after adding different cupric salt, indicating copper
salts with different anions (ClO−4 , Cl− and SO24−) had no disturbance
on the selectivity detection of Cu2+. These results further confirmed
that Z had strong anti-interference ability in the process of identifying
Cu2+ in the complex environment.
The good relationship between the absorbance intensity ratio (A283
A
/
374) and the concentration of Cu2+ (0–20 μM) was also detected
3.4. Binding stoichiometry and sensing mechanism
(Fig. S23). All these results showed that probe Z could be applied as a
Cu2+ probe in ratiometric detection. The limit of detection was calcu-
lated as 1.35 × 10−6 M and 3.75 × 10−7 M for fluorescence and absor-
bance detection, respectively [54–56].
In order to understand the binding mode and sensing mechanism of
Z with Cu2+, the Job's method for fluorescent titration was determined.
As shown in Fig. 7, when the molar fraction of [Z]/[Cu2++Z] was 0.3, the
fluorescence intensity ratio (F451/F498) reached maximum, which indi-
cated that the ratio of the Z to the Cu2+ was 1:2. This result was also
consistent with the fluorescence intensity ratio (F451/F498) reached plat-
form when the addition of Cu2+ was 2 equiv. in the fluorescence titra-
tion experiment. Accordingly, the binding constant between probe Z
and Cu2+ was calculated as 3.58 × 10−9 M−2 (Fig. S25).
Competition experiments of Z to Cu2+ were measured to investigate
the effect of other co-existence metal ions on fluorescent signal. As
shown in Fig. 6, after addition of Cu2+ (50 μM) to the solution of Z in
the presence of 5 equiv. of other metal cations, the results showed
that both the fluorescence spectra and the ratio (F451/F498) of fluores-
cence intensity were all almost same as that of the solution of Z in the
presence of Cu2+. Moreover, in order to eliminate the effect of the differ-
ent anions (including ClO4−, Cl− and SO24−) attached with copper ion on
The fluorescence titration experiment of Z-2 with Cu2+ (Fig. S26)
was also measured in DMF/H2O (3/7, v/v, 0.01 M HEPES, pH = 13.0)
Fig. 4. (a) The fluorescence change of probe Z with different metal ions under 365 nm UV light; (b) Fluorescence spectra of Z (10 μM) with different metal ions in DMF/H2O (3/7, v/v, 0.01 M
HEPES, pH = 13.0); (c) Absorption spectra of Z (10 μM) with different metal ions in DMF/H2O (3/7, v/v, 0.01 M HEPES, pH = 13.0). λex = 390 nm.

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J.-X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
Fig. 5. (a) The color change of Z on addition of different amount of Cu2+ under 365 nm UV light in DMF/H2O (3/7, v/v, 0.01 M HEPES, pH = 13.0). Fluorescence spectral (b) and absorption
spectral (c) changes of Z (10 μM) added with different concentrations of Cu2+ in DMF/H2O (3/7, v/v, 0.01 M HEPES, pH = 13.0). (Error bar was represented as mean standard deviation,
n = 3).
to further investigate the sensing mechanism of Z. The fluorescence in-
tensity of Z-2 (10 μM) at 494 nm was gradually decreased with in-
creased addition of Cu2+, then it almost showed no variety when the
amount of Cu2+ was 10 μM. Although addition of Cu2+ resulted in a sig-
nificant decreased in fluorescence intensity of Z-2, there was no obvi-
ously blue-shift compared with that of probe Z with Cu2+ under the
same condition, indicating that the hydroxyl position was particularly
important for the detection of Cu2+ through the structure comparison
between Z and Z-2. So, a conclusion could be obtained that the hydroxyl
was in the ortho-position of the hydrazide (probe Z) was better for pro-
viding a binding site to Cu2+ than that of Z-2.
This result indicated the existence of strong interaction between
probe Z and Cu2+ through oxygen atom of the hydroxyl groups and ni-
trogen atom of the amide group.
1HNMR titration experiments (Fig. 9) were handled by addition of
different amount Cu2+ into the probe Z solution of DMSO-d6 (Contain-
ing 2 μL NaOD). The result showed that the peak of protons Ha was
firstly gradually diminished according to the comparison of data of inte-
gral area among these proton signals (Ha, Hk and Hj), and the signals of
Ha and Hk could not be detected until the addition of 2 equivalent of
Cu2+. This result suggested that both the amide group and the two hy-
droxyl group of probe Z were all taken part in the coordination with
Cu2+ which started at the position oxygen atom of hydroxyl group by
deprotonation of Ha, then with the other two groups (hydroxyl group
and carbonyl group).
IR spectrum analysis was carried out to investigate the binding sites
of probe Z with Cu2+ in alkaline condition, and the results were illus-
trated in Fig. 8. The typical stretching vibration of C_N, C_O, O\\H,
and N\\H of probe Z itself were located at 1541 cm−1, 1604 cm−1
,
Thus, according to the above experimental phenomena and analysis
(including fluorescence titration, job plot, FT-IR spectrum and 1HNMR
titration experiments), a probable binding mode and sensing mecha-
nism of Z with Cu2+ was proposed. As shown in Fig. 10, under alkaline
medium, Cu2+ first coordinated with the benzothiazole moiety which
resulted in the decrease in fluorescence intensity attributed to the
3059 cm−1 and 3258 cm−1, respectively. After addition of Cu2+ to the
solution of probe Z, the stretching vibration of O\\H and N\\H were dis-
appeared and turned into a broaden peak located at 3433 cm−1. Fur-
thermore, the stretching vibration of C_O and C_N were all
obviously decreased and red-shifted to 1584 cm−1 and 1456 cm−1
.
Fig. 6. Fluorescence spectra (a) and fluorescence intensity ratio (F451/F498) (b) of Z toward Cu2+ in the presence of 5 equiv. of other cations. λex = 390 nm.

J.-X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
7
Fig. 7. Job plot of Cu2+ complex formation. {[Cu2+]/([Cu2+] + [Z])} is the molar fraction of Cu2+ ion.
paramagnetic nature of Cu2+ which could cause intense chelation-
3.5. Effect of pH on the fluorescence intensity of probe Z
enhanced fluorescent quenching (CHEQ) [24,57]. When the amount of
Cu2+ added exceeded 1 equivalent, excess Cu2+ further coordinated
with the oxygen atom of the hydroxyl in the ortho-position of the hy-
drazide, which resulted in the significant inhibition of intramolecular
charge transfer (ICT) process from the oxygen atom of the hydroxyl to
the benzothiazole core. Hence, it was reasonable that the probe Z
displayed a significant blue-shift and decreased in fluorescent emission
after addition of Cu2+ in alkaline medium.
In order to further investigate the practical application, the fluores-
cence intensity at 498 nm and 451 nm of Z in the absence and presence
of Cu2+ were measured, respectively. In the pH range of 2–7 (Fig. S27a
and 27b), the probe Z itself had no fluorescence emission at 498 nm and
451 nm, respectively. However, the fluorescence emission at 498 nm
and 451 nm were gradually increased in the pH range of 8–13, respec-
tively. Upon the addition of Cu2+, comparing with that of Z itself,
Fig. 8. IR spectrum of probe Z and Z-Cu2+ in alkaline conditions.

8
J.-X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
Fig. 9. 1HNMR titration of probe Z upon addition of Cu2+ in DMSO-d6.
there was almost no difference in the pH range of 2–7, but the signifi-
cant changes were observed in fluorescence quenched at 498 nm
while the fluorescence turn-on at 451 nm in the pH range of 8–13.
Moreover, this change in fluorescence intensity before and after the ad-
dition of Cu2+ at different pH could be intuitively represented by color
changes (Fig. S27c). In the pH range of 8–10, the fluorescence of Z was
significantly quenched after addition of Cu2+ under 365 nm UV light,
accompanied with the fluorescence color changed from mint green to
blue in the pH range of 11–13. This result showed that the probe Z
could identify Cu2+ in a wide range of alkaline conditions (pH 8–13), es-
pecially in the range of pH = 11–13, and also had the potential applica-
tion in as a colorimetric fluorescent probe for the detection of Cu2+ in
Saline-alkali land.
conditions (pH N 7) (Fig. S28b). Therefore, we set H+ and OH– sepa-
rately as input 1 and input 2, the absorption intensity at 317 nm and
the fluorescence intensity of at 498 nm as outputs 1 and outputs 2
and the threshold value of outputs 1 and outputs 2 were considered
as 0.35 and 150 au, respectively. Thus, the XNOR logic gate based on out-
put 1 and INHIBIT logic gate based on output 2 were constructed, re-
spectively (Table 1).
Moreover, the combination of the above two logic gates could realize
a magnitude digital comparator at the molecular [58]. Output 1 was in
an ON (1) state only when the concentration of H+ was equal to the
concentration of OH−, ie, neutral. When the concentration of OH– was
more than the concentration of H+ in the medium, the output signal 2
was ON (1) state. Therefore, based on these logic gates, logic circuits
were constructed for identifying the neutrality or alkalinity of the
medium.
4. Applications
4.1. Logic gate construction
4.2. Real sample detection
According to the results of absorption titration of Z with different pH
(Fig. S18), the absorption intensity at 317 nm reached the maximum at
pH 7, and its value decreased gradually along with the enhancement of
whatever in acidity or alkalinity (Fig. S28a). However, the fluorescence
intensity of Z at 498 nm was increased significantly under alkaline
In order to further study the applicable function of the probe Z in
sensing Cu2+ in real water samples, the fluorescent detection of Cu2+
in ultrapure water and tap water in our campus were measured, respec-
tively. Amount of standard Cu2+ added to the samples, the data of fluo-
rescence intensity ratio (F451/F498) was obtained and the actual
Fig. 10. The proposed binding mechanism of probe Z and Cu2+ in alkaline medium.

J.-X. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 235 (2020) 118318
9
Table 1
The truth table for the operation of the Z-based digital comparator.
Input 1 (H+
)
Input 2 (OH−
)
Output 1 (A317
)
Output 2 (F498)
XNOR (equal)
INHIBIT (great than)
[H+] = [OH−
]
0
0
1
1
0
1
0
1
1
0
0
1
0
1
0
0
[H+] b [OH−
[H+] N [OH−
[H+] = [OH−
]
]
]
measured value of Cu2+ was found from the linear relationship calcu-
lated previously (Table 2). This result indicated that probe Z had good
recovery and accuracy in Cu2+ detection in real water samples, which
meant that it could perform trace analysis for the concentration of
Cu2+ in environment.
Zhen-Nan:Formal analysis, Investigation; Wu Ding-Qi: Validation;
Xiang Yuan- Yuan: Visualization; Li Jin-Long: Supervision.
Declaration of competing interest
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.
4.3. Application in test paper
Since the probe Z showed a significant fluorescent color change for
Cu2+ under strong alkaline conditions, a test strip experiment was con-
ducted to investigate whether this phenomenon could be achieved on
the test paper. Soaked the test paper in a probe solution containing dif-
ferent concentrations of Cu2+ (0–20 μM) for 2 h, then the test paper was
taken out and dried in the air. As shown in Fig. S29, under the 365 nm
UV light, as the concentration of Cu2+ increases, the color of the test
paper gradually changed from mint green to deep blue, which indicated
that Z could detect the Cu2+ by the test paper.
Acknowledgements
This work was supported by Students' Innovation and Entrepreneur-
ship Training Program of Northeast Agricultural University (No.
201910224325).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2020.118318.
5. Conclusions
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