Aqueous Zn2+ analysis: Specific recognition and instant imaging by Schiff base fluorescent probes

Article abstract of DOI:10.1016/j.molstruc.2020.129522

Two Schiff base fluorescence probes for aqueous Zn²⁺ were synthesized by introducing salicylaldehyde benzoyl hydrazine as the recognition group and C=N as the characteristic fluorescence chromophore. Spectroscopic analyses, mainly FS, illuminated the probes’ highly specific recognition towards Zn²⁺ over other metal ions, accompanied by steady optical properties at a wide pH range of 6.0 to 9.0. Moreover, cytotoxicity and fluorescence imaging in living ECV304 cells identified the probes’ instant response and membrane permeability. These results predicted the probes’ future applications for instant Zn²⁺ detection in a clinical diagnosis and dynamic analysis of Zn²⁺ in biological systems.

Full text of DOI:10.1016/j.molstruc.2020.129522

Journal Pre-proof
Aqueous Zn2 analysis: specific recognition and instant imaging by
+
Schiff base fluorescent probes
Hao He , Zhao Cheng , Lei Zheng
PII:
S0022-2860(20)31837-8
DOI:
Reference:
https://doi.org/10.1016/j.molstruc.2020.129522
MOLSTR 129522
To appear in:
Journal of Molecular Structure
Received date:
Revised date:
Accepted date:
16 August 2020
19 October 2020
26 October 2020
Please cite this article as: Hao He , Zhao Cheng , Lei Zheng , Aqueous Zn2+ analysis: specific recog-
nition and instant imaging by Schiff base fluorescent probes, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.129522
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HIGHLIGHTS
2
+


Salicylaldehyde benzoyl hydrazine was first introduced as Zn recognition group;
FS analyses proved probes’ steady optical properties at different pH and in
ions-coexisting systems;

Living ECV304 imaging identified the probes’ instant response, low toxicity and
membrane permeability.

2+
Aqueous Zn analysis: specific recognition and instant
imaging by Schiff base fluorescent probes
*
Hao He, Zhao Cheng , Lei Zheng
School of Pharmacy, Xi’an Medical University, Xi’an Shaanxi 710021, PR China
*
Corresponding author: Tel:+86 029-86177549; E-mail: chengzhao@xiyi.edu.cn
2+
Abstract: Two Schiff base fluorescence probes for aqueous Zn were synthesized by introducing
salicylaldehyde benzoyl hydrazine as the recognition group and C=N as the characteristic
fluorescence chromophore. Spectroscopic analyses, mainly FS, illuminated the probes’ highly
2+
specific recognition towards Zn over other metal ions, accompanied by steady optical properties
at a wide pH range of 6.0 to 9.0. Moreover, cytotoxicity and fluorescence imaging in living
ECV304 cells identified the probes’ instant response and membrane permeability. These results
2+
predicted the probes’ future applications for instant Zn detection in a clinical diagnosis and
2+
dynamic analysis of Zn in biological systems.
2+
Keywords: Zn probe; Schiff base; instant recognition; cytotoxicity; real-time imaging
1
Introduction
Emerging as a significant player in both neurophysiology and neuropathology, zinc is an
abundantly-existed transition metal in the body with its highest concentration occurring in the
brain. The distribution, storage, transportation and other dynamic changes of zinc [1,2], mostly
seen in abnormal accumulation or uncontrolled oxidation-reduction reactions, would disrupt the
natural balance or trigger chain reactions, and were proven to be connected with a number of
neurodegenerative diseases [3,4], such as Alzheimer’s Disease [5,6] and Parkinson’s Disease. As a
2+
consequence, fluorescent probes for Zn detection, which could provide critical information for
zinc ions’ assessments, are widely used and still in high demand.
Among the various techniques to detect metal ions [7,8], small-molecule fluorescent probes
have received much attention. It could convert the ion recognition to tangible fluorescent signals
[9] and thus exhibit lots of advantages and characteristics [10,11], such as instant response, high
selectivity, low detection limit, linear response and visual detection process.

2
+
Zn fluorescence probes were constructed mainly on APTRA (o-aminophenol-N, N,
O-triacetic acid) and DPA (diphenylamine) receptors, such as the commercial probes
APTRA-BTC [12], FluoZin-1 [13], Zinpyr-1 [14] and NG-DCF [15], etc. In this paper, a
2+
Schiff-based salicylaldehyde benzoyl hydrazine was first designed as the Zn recognition group,
2+
and two fluorescent probes for Zn were synthesized by introducing conjugated C=N chains as
the fluorescent chromophores to the Schiff receptor. Further FS analyses and ECV304 cell
imaging proved the probes’ good optical stability, instant response, low cytotoxicity, membrane
2+
permeability and specific recognition to Zn , which predicted the probes’ future applications for
2+
qualitative detection and quantitative analysis of Zn in biological systems.
2
2
Experimental
.1 Reagents and Equipments
Melting points were determined on an XT-4B micro melting point apparatus without
correction. Elemental analyses were recorded on an Elementar varioEL Ⅲ analyzer. FT-IR spectra
1
were recorded with KBr pellets on a Bruker EQUINOX-55 FT-IR spectrometer. H NMR spectra
were recorded on a Varian INOVA-400 spectrometer at 400 MHz and chemical shifts were
reported relative to the internal standard tetramethylsilane (TMS). Fluorescent spectra were
recorded on a HITACHI F-4500 fluorescence spectrophotometer. Mass Spectrometry (MS)
analyses were performed using an Agilent 1260-6460A triple quadrupole liquid
chromatograph-mass spectrometer. The cytotoxicity results were analyzed with the SoftMax Pro
Software (version 2.2.1) in a Molecular Devices SpectraMax 190 microplate reader. Cell-imaging
experiments were performed using an OLYMPUS U-LH 100HG IX73 fluorescence microscope.
All of the reagents and solvents used for synthesis were commercially available and used
without further purification unless otherwise noted. ECV304 cells were purchased from KeyGen
BioTECH (Nanjing China). Standard TSQ (HPLC, ≥98%) was purchased from Sigma-Aldrich
(US). The reaction process was monitored by thin-layer chromatography (TLC) on silica gel
GF254. The products were purified by column chromatography on a Merck silica gel (250-400
-1
mesh ASTM). A Tris-HCl buffer solution (pH 7.4) was prepared using 0.1 mol L HCl and the
-1
proper amount of a 0.1 mol L Tris stock solution (Sinopharm Chemical Reagent Company,
Shanghai China). Double distilled water was used throughout the process of solution preparing
and spectroscopic testing. Metal ion solutions were prepared from their nitrate and chloride salts,

NiCl , HgCl , Fe(NO ) •9H O, PdCl , PbCl , AlCl , Mg(NO ) •6H O, Ca(NO ) •4H O, MnCl ,
2
2
3 3
2
2
2
3
3 2
2
3 2
2
2
Cd(NO ) •2H O, SnCl2, Cu(NO ) •3H O, Cr(NO ) •9H O, NaCl, AgNO3, BaCl2 and
3
2
2
3 2
2
3 3
2
Zn(NO ) •6H O.
3
2
2
2
.2 Synthesis of P1~P2
The target probes P1 and P2 (Figure 1) were synthesized through three steps as shown in
1
Scheme 1. Structures of P1 and P2 were characterized by IR, H NMR, MS and EA.
NH N CH
HO
NH N CH
HO
O
HO
HC
OH
N
HO
HO
HC
OH
N
O
P1
P2
Figure 1 Structures of P1 and P2
OH
OH
O
NH NH2
O
CHO
NH N CH
HO
N2H4·H2O
H N
2
H N
2
H N
2
EtOH reflux
EtOH reflux
O
OH
x
u
l
f
e
r
HO
N
CHO
H
O
t
E
NH N CH
HO
HO
HC
OH
O
P1
Scheme 1 Synthetic route of the probes (shown by P1)
(1) p-Aminobenzoyl hydrazide
9
8% Hydrazine hydrate (1.0 mL, 19.8 mmol) was slowly added in a solution of
p-aminobenzoic acid methyl ester (2.00 g, 13.2 mmol) and anhydrous ethanol (20.0 mL). The
mixture was then refluxed for 4 h, cooled down, concentrated in vacuo and subsequently purified
by column chromatography (silica gel, V(EtOH) : V(dichloromethane) = 1 : 50 as eluent) to afford
white solid p-aminobenzoyl hydrazide (1.668 g, 11.0 mmol), yield 83.3%.
(2) p-Aminobenzoyl salicylaldehyde hydrazine
9
8% Salicylaldehyde (1.3 mL, 11.0 mmol) was dropwise added in a solution of
p-aminobenzoyl hydrazide (1.67 g, 11.0 mmol) and anhydrous ethanol (20.0 mL). The mixture
was then refluxed for 0.5 h, cooled down to afford the crude. After further purification in
anhydrous ethanol, yellow crystals were obtained as p-aminobenzoyl salicylaldehyde hydrazine.
(3) P1

p-Aminobenzoyl salicylaldehyde hydrazine (2.55 g, 10.0 mmol) was dissolved in anhydrous
ethanol (20.0 mL), then 2,4-dihydroxy benzaldehyde (1.38 g, 10.0 mmol) was added in and the
mixture was refluxed for 12 h, cooled down, concentrated in vacuo and subsequently purified by
column chromatography (silica gel, V (EtOAc) : V (hexane) = 1 : 3 as eluent) to afford P1.
(4) P2
p-Aminobenzoyl salicylaldehyde hydrazine (2.55 g, 10.0 mmol) was dissolved in anhydrous
ethanol (20.0 mL), then 2,3,4-trihydroxy benzaldehyde (1.54 g, 10.0 mmol) was added in, the
mixture was refluxed for 12 h, cooled down, concentrated in vacuo and subsequently purified by
column chromatography (silica gel, V (EtOAc) : V (hexane) = 1 : 3 as eluent) to afford P2.
2
.3 Spectroscopic Analysis
Stock solutions (200 μM) of P1, P2, Ni , Hg , Fe , Pd , Pb , Al , Mg , Ca , Mn ,
2+
2+
3+
2+
2+
3+
2+
2+
2+
2
+
2+
2+
3+
2+
+
2+
2+
Cd , Sn , Cu , Cr , Na , Ag , Ba and Zn were prepared in a mixed solvent (V (EtOH) : V
(
H O) = 5 : 5, Tris-HCl pH = 7.4) [16]. When used for fluorescence tests, the stock solutions were
2
usually diluted with the EtOH-H O mixture (5:5, V/V, Tris-HCl pH = 7.4) to 10 μM unless
2
otherwise noted. All the spectroscopic measurements were performed at least in triplicate and
averaged.
2
.4 Cytotoxicity
The toxicity of P1/P2 and TSQ in ECV304 cells was analyzed by Methyl Thiazolyl
Tetrazolium (MTT) assay [17,18]. ECV304 cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM) with 10% Fetal Bovine Serum (FBS). All cells in the exponential phase of
growth were used in the experiments. After digestion with 0.25% trypsin solution, cells were
4
-1
dispensed in 96-well cell culture clusters with 200 μL per well at a density of 2.5×10 cells mL ,
incubated at 37 ºC with 5% CO for 24 h. The probes P1/P2 and TSQ (100 mM in DMSO) was
2
then added to the 96-well plates to achieve a concentration gradient of 125, 250, 500, 1000, 2000
-1
and 4000 µg L , and the cells were incubated for another 24 h. Subsequently, the medium was
removed, the cells were washed with Phosphate Buffered Saline (PBS) three times and incubated
-1
with 5.0 mg mL MTT solution (20 μL MTT and 180 μL medium) at 37 ºC for 4 h. After that, the
cells were washed with PBS (1 mL per well) three times and then dissolved in DMSO (150 μL per
well). The optical density was recorded at 490 nm on a microplate spectrophotometer. All the tests
were conducted in triplicate. Data were expressed as mean ± standard deviation (SD).

2
.5 Fluorescent Imaging in Living Cells
ECV304 cells were cultured in DMEM supplemented with 10% FBS at 37 ºC in the
humidified atmosphere with 5% CO . The cells were cultured for 2 h until they plated on
2
glass-bottomed dishes. The growth medium was then removed and the cells were washed with
DMEM and incubated with 10 μM of the probes for 30 min at 37 ºC, washed three times with PBS
2
+
and imaged [19]. Then the cells were supplemented with 10 μM of Zn in the growth medium for
3
3
3
0 min at 37 ºC, washed three times with PBS and imaged.
Results and Discussion
.1 Structure Characterization
Salicylaldehyde benzoyl hydrazine, derived from the structure of Schiff base, was first
2+
designed as the Zn recognition group. By introducing conjugated C=N chains as the fluorescent
2+
chromophores to the Schiff receptor, Zn fluorescent probes P1 and P2 were synthesized.
1
Structures of P1 and P2 were characterized by IR, H NMR, MS and EA. The calculation and
1
measurement results of MS and EA were all matched, H NMR chemical shifts were in
accordance with the target structures, and characteristic absorbance peaks of υC=O and υC=N were at
-1
the reasonable ranges of ~1700 and 1600~1620 cm .
p-Aminobenzoyl salicylaldehyde hydrazine: yellow crystals, 2.30 g, yield 86.0%. IR:
-1
-1
-1
3
433.40 cm (υ- ), 1716.13 cm (υ ), 1600.14 cm (υC=N).
OH C=O
-1
P1: yellow powder, 2.6737 g, yield 71.3%, m.p.: 267-269 ºC. IR (KBr pellet, υ/cm ):
1
3
8
415.54 (υ- ), 1713.48 (υC=O), 1598.10 (υC=N). H NMR (CDCl , 400 MHz) δ: 9.69 (s, 1H, -NH-),
OH
3
.13~8.11 (m, 4H, Ar-H), 8.02 (s, 3H, Ar-H), 7.87 (d, J = 8.7 Hz, 2H, 2×=CH-), 6.68~6.62 (m, 4H,
+
Ar-H), 3.87 (s, 3H, 3×-OH). MS ((+)-ESI): m/z = 376.25 (calcd. for C H N O : 375.38, (M+H)
21
17
3
4
+
calcd.: 376.39, (M+H) found: 376.25). EA: C 67.17%, H 4.57%, N 11.21% (calcd. for
C H N O : C 67.19%, H 4.56%, N 11.19%).
2
1
17
3
4
-1
P2: yellow powder, 2.6666 g, yield 68.2%, m.p.: 273-274 ºC. IR (KBr pellet, υ/cm ):
1
3
358.00 (υ- ), 1718.39 (υC=O), 1624.19 (υC=N). H NMR (CDCl , 400 MHz) δ: 12.92 (s, 1H,
OH
3
-NH-), 8.13~8.10 (m, 3H, Ar-H), 7.45~7.42 (m, 2H, Ar-H), 7.35~7.30 (m, 3H, Ar-H), 7.30 (s, 2H,
2
×=CH-), 7.05 (d, J = 8.3 Hz, 1H, Ar-H), 6.98 (t, J = 7.6, 1.0 Hz, 1H, Ar-H), 3.95 (s, 4H, 4×-OH).
+
MS ((+)-ESI): m/z = 392.19 (calcd. for C H N O : 391.38, (M+H) calcd.: 392.38, found:
21
17
3
5
3
92.19). EA: C 64.46%, H 4.37%, N 10.77% (calcd. for C H N O : C 64.45%, H 4.38%, N
21 17 3 5

1
0.74%).
3
.2 Fluorescent Properties of P1 and P2
Fluorescent excitation and emission spectra of P1 and P2 at room temperature (1 μM in
aqueous solution, Figure 2) demonstrated a mirror symmetry [20-22], with outstanding
long-wavelength emissions and significant Stokes shifts of 103 nm (P1) and 96 nm (P2) due to
probes’ conjugated structures. In the structural design of P1 and P2, substituent position difference
in benzene rings had an obvious influence on the fluorescent properties of the probes. Due to the
electron effect and molecular rigid induced by structural differences, the maximum excitation and
emission wavelengths were located at λ /λem 433/536 nm for P1 and λ /λ 447/543 nm for P2,
ex
ex em
respectively.
Figure 2 Excitation and emission spectra of P1 and P2
.3 Metal Ion Selectivity
In the screening test for detection metal ions, P1 exhibited low fluorescence itself when
3
2
+
excited at the wavelength of 433 nm or upon adding of other ions except Zn , however, an adding
2
+
of Zn triggered almost twice enhancement in fluorescence intensity at 536 nm, as seen in Figure
a (left).
The competitive experiments were conducted by adding Zn to the solution of probe P1 in
the presence of other metal ions (P1: 10 μM, metal ions: 10 μM) excited at 433 nm. As a result,
3
2+
2+
the spectral enhancement induced by Zn was not affected by the tested background of other
metal ions (Figure 3a, right).
High selectivity is a very important parameter to evaluate the performance of a probe. The
2+
competition tests, which indicated that probe P1 was highly sensitive towards Zn , predicted the

2+
future use of P1 for Zn detection in complex environments.
And the similar phenomenon was observed at 543 nm for probe P2 when excited at the
wavelength of 447 nm (Figure 3b).
(a)
(b)
Figure 3 Fluorescence intensity changes of (a) P1 and (b) P2 upon the addition of various metal
2
+
2+
ions (left); selectivity of (a) P1 and (b) P2 to Zn in Zn -competing ions coexisting system with
mixed solvents (V (EtOH) : V (H O) = 5 : 5, Tris-HCl pH = 7.4) environment (right)
2
*
2+
2+
3+
2+
2+
3+
2+
2+
2+
2+
2+
from left to right (bar 1~17): Ni , Hg , Fe , Pd , Pb , Al , Mg , Ca , Mn , Cd , Sn ,
2
+
3+
2+
+
2+
2+
Cu , Cr , Na , Ag , Ba and Zn
2+
3
.4 Quantitative Detection of Zn
Fluorescence titration was carried out to analyze the quantitative relationship of P1/P2 and
2
+
Zn , as shown in Figure 4. Probe P1 itself displayed low fluorescence at 536 nm, while addition
2
+
of Zn immediately induced an obvious enhancement of fluorescence there and the fluorescence
2+
2+
intensity of P1-Zn system increased with the gradual addition of Zn (Figure 4a, left). When the
2+
fluorescence intensity of P1-Zn system at 536 nm was analyzed, a linear relationship between
2+
fluorescence intensity and concentration of Zn was obtained, indicating a highly quantitative
2+
2+
detection of Zn by P1 in the concentration range of 0.1~1.0 equiv. Zn towards P1 (Figure 4a,
right).

The calculation of stoichiometry was performed to further investigate the sensitivity of probe
2+
P1 and its quantitative detection towards Zn . A 1:1 stoichiometry for association between P1
2
+
and Zn was shown in Figure 5 (left), the linear response of fluorescence emission intensity at
2+
5
36 nm versus [Zn ] could be expressed by the following equation (1) [23-25]:
2+
2
P1-Zn
y＝40.179x+28.714 (R =0.9945)
(1)
Similar phenomena were observed in P2 at 543 nm (Figure 4b & Figure 5, right). As for P2,
2+
the linear fitting equation (2) of fluorescence intensity versus [Zn ] at the maximum emission
wavelength 543 nm could be expressed as:
2+
2
P2-Zn
y＝33.143x+43.13 (R =0.9994)
(2)
2+
Analyses on the association stoichiometry of probe P1/P2-Zn and calculations to obtain the
2+
respective linear relationships of P1/P2-Zn , both indicated the applications of P1/P2 for critical
2
+
quantitative detection of Zn .
(a)
(b)
2+
Figure 4 Fluorescence titration curves of (a) P1 and (b) P2 (10 μM) with Zn

2+
Figure 5 Job’s plots of P1/P2-Zn
2
+
3
.5 Influences of pH on the Quantitative Detection of Zn
Considering the complex environment in biological applications of the probes, fluorescence
stability at physiological pH [26] was examined (Figure 6). When pH value varied from 6.0 to 9.0,
2+
the fluorescence intensity of P1 and P1-Zn systems both kept steady (Figure 6, left), suggesting
2+
a relatively wide pH range from 6 to 9 appropriate for P1-Zn combination system.
The variable pH had a similar influence on the fluorescence intensity of P2, as seen in Figure
6
(right).
The high fluorescence stability of P1/P2 and P1/P2-Zn at physiological pH revealed the
probes’ applications for instant recognition and imaging in living systems.
2+
2+
Figure 6 Influence of pH on P1/P2 (10 μM) in the absence and presence of Zn (10 μM)
3
.6 MTT Assay
A standard MTT assay was performed with ECV304 cells to evaluate the potential imaging
and diagnostic applications of the two probes P1 and P2. A concentration gradient of 125, 250,
-1
5
00, 1000, 2000 and 4000 μg L for each probe were tested, the cytotoxicity of P1/P2 and TSQ in
ECV304 cells at 24 h were compiled in Table 1 and depicted in Figure 7, indicating there was no
significant cytotoxic effect of P1 and P2 on ECV304 cells when incubated for 24 h. Even when
-1
the concentration of P1/P2 reached the highest 4000 μg L in MTT assay, the cytotoxicity of

ECV304 cells remained below 20%, which indicated the relatively low cytotoxicity of P1/P2 at
common working concentrations.
Table 1 Cytotoxicity of P1/P2 and TSQ in ECV304 at 24 h
cytotoxicity (%)
-1
concentration (µg L )
P1
P2
TSQ
1
2
5
25
50
00
4.53±0.78
6.81±1.27
6.13±1.12
12.20±1.28
6.60±1.42
20.38±2.06
8.49±1.27
11.65±2.08
7.82±0.88
9.52±2.15
5.65±1.61
3.70±1.50
5.85±2.39
6.82±1.16
9.62±1.02
20.01±4.38
23.02±2.61
28.32±2.75
1
2
4
000
000
000
3
3
2
2
1
1
5
0
5
0
5
0
5
0
P1
P2
TSQ
1
25
250
500
1000
2000
4000
-
1
Concentration（μg L ）
Figure 7 The cytotoxicity of P1/P2 on ECV304 at 24 h in comparison with TSQ
2+
A commercial Zn probe TSQ [17] was also incorporated as positive control in MTT assay,
which showed similar cytotoxicity to P1/P2 as shown in Table 1 and Figure 7, indicated that
2+
P1/P2 and TSQ all showed low cytotoxicity in the MTT assay. The latest reports of Zn probes
18] with similar structures to P1/P2 were also reviewed, indicating no significant cytotoxic
effect.
.7 Recognition Mechanism
[
3

2+
As for the increase in fluorescence intensity after recognition of Zn by the probe P1/P2, the
electron effect [27] should also be considered apart from the possible structural cavity [28,29] in
recognition process (Figure 8).
In probe P1/P2, the two nitrogen atoms located at the conjugated chain act as the recognition
2+
sites to capture target Zn . The two nitrogen atoms have pairs of unbonding electrons and don’t
participate in the cyclic π bonding of benzene rings, thus there exists no obvious electron transfers
[30] inside the molecules to display strong fluorescence of the probe itself. When the appropriate
2+
structural cavity forms and captures Zn (Figure 8), the two nitrogen atoms will partly provide
2+
their unbonding electrons to fulfill the empty electron orbits of Zn through intramolecular forces
31,32], though weaker than coordination bonds, an electron transfer will then be established and
the molecular rigid reinforced, leading to better planarity of the molecules and stronger
[
2+
fluorescence intensity in the P-Zn systems.
2+
Figure 8 Recognition mechanism of probes to Zn (shown as P1)
3
.8 Bioimaging
After incubation of ECV304 with P1/P2 for 30 min (Figure 9a), 10 μM Zn was added in
and incubated for another 2 h at 37 ºC, red fluorescence was emitted from the intracellular area
2+
2
+
(
Figure 9b) in comparison with Figure 9a, demonstrating the recognition of added Zn by P1/P2
over a very short time. The overlay of fluorescence image (Figure 9b) and bright-field image
Figure 9c) revealed that the fluorescence signals were localized in perinuclear region of the
cytosol [33,34] (Figure 9d), which indicated that P1 and P2 were cell permeable and could be
(
2+
used for dynamic imaging and tracking of Zn in living cells.

Figure 9 (a) Fluorescent images of ECV304 incubated with 10 μM P1/P2 for 30 min, (b) a further
2+
incubation with 10 μM Zn for 2 h, (c) bright-field image of (b), (d) an overlay image of (b) and
(c)
4
Conclusions
Two novel Zn fluorescence probes P1 and P2 were synthesized, unlike common APTRA
2+
2+
receptor in probes for Zn , structures of P1 and P2 were based on Schiff base by introducing
2+
salicylaldehyde benzoyl hydrazine as Zn recognition group and C=N as the fluorescent
chromophore. FS analyses of P1 and P2 proved the probes’ steady optical properties at different
2+
pH environments and specific recognition to Zn in complicated ions-coexisting systems.
Moreover, MTT assay and fluorescence imaging in ECV304 cells in vitro identified the probes’
instant response and good light stability, predicting the probes’ low cytotoxicity and membrane
2+
permeability for qualitative detection and quantitative analysis of Zn in biological systems. The
2+
dynamic labelling and tracking of Zn in living cells through both instant and time-lapse imaging
would be possible with probes P1 and P2.
Acknowledgements
Project supported by Natural Science Foundation of Shaanxi Provincial Department of Education
(20JK0902).
AUTHOR STATEMENT
Hao He: Software, Validation, Data curation, Investigation, Writing-
Original draft preparation
Zhao Cheng: Conceptualization, Methodology, Writing-Review &

Editing, Funding acquisition, Supervision
Lei Zheng: Visualization, Investigation, Formal analysis
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which
may be considered as potential competing interests:
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