The development of coumarin Schiff base system applied as highly selective fluorescent/colorimetric probes for Cu2+ and tumor biomarker glutathione detection

Article abstract of DOI:10.1016/j.dyepig.2019.108156

Overexpression of tumor biomarker glutathione (GSH) has been documented in numerous types of cancers, therefor GSH-activated light-up chemosensors for tumor identification require great attention. A new colorimetric/fluorescent probe (7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-4-(dimethylamino) benzohydrazide (HL) was prepared, which could be applied in discriminating Cu²⁺ and further recognizing GSH based on its Cu complex. Firstly, the probe HL toward Cu²⁺ exhibited selective fluorescence quenching and obvious color change from yellow to orange-red under visible light. Further, when GSH was introduced to the Cu²⁺-2HL system, the fluorescence recovered rapidly due to the high affinity of GSH to Cu²⁺, meanwhile the color reverted back to former yellow. Based on fluorescence titration, the detection limits were calculated as 2.40 × 10^?8 M and 1.29 × 10^?7 M for Cu²⁺ and GSH, respectively. The combination mode of HL with Cu²⁺ was investigated in detail by Job plots, ESI-MS, FT-IR, and DFT studies. Probes HL and Cu²⁺-2HL showed relatively less toxicity and were employed for biological imaging in cells and zebrafish. Remarkably, the detection for endogenous GSH by this developed sensor platform implied great potential application prospect in cancer diagnosis.

Full text of DOI:10.1016/j.dyepig.2019.108156

Journal Pre-proof
The development of coumarin Schiff base system applied as highly selective
2+
fluorescent/colorimetric probes for Cu and tumor biomarker glutathione detection
Zhi-Gang Wang, Xiao-Jing Ding, Yu-Ying Huang, Xiao-Jing Yan, Bin Ding, Qing-
Zhong Li, Cheng-Zhi Xie, Jing-Yuan Xu
PII:
S0143-7208(19)32744-5
DOI:
https://doi.org/10.1016/j.dyepig.2019.108156
DYPI 108156
Reference:
To appear in: Dyes and Pigments
Received Date: 27 November 2019
Revised Date: 18 December 2019
Accepted Date: 18 December 2019
Please cite this article as: Wang Z-G, Ding X-J, Huang Y-Y, Yan X-J, Ding B, Li Q-Z, Xie C-Z, Xu J-Y,
The development of coumarin Schiff base system applied as highly selective fluorescent/colorimetric
2+
probes for Cu and tumor biomarker glutathione detection, Dyes and Pigments (2020), doi: https://
doi.org/10.1016/j.dyepig.2019.108156.
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Author Statement
Zhi-Gang Wang: Investigation, Validation, Writing - Original Draft
Xiao-Jing Ding: Validation, Formal analysis, Resources
Yu-Ying Huang: Formal analysis, Writing - Review & Editing
Xiao-Jing Yan: Data Curation, Software
Bin Ding: Writing - Review & Editing, Funding acquisition
Qing-Zhong Li: Software
Cheng-Zhi Xie: Conceptualization, Methodology, Writing - Review & Editing
Jing-Yuan Xu: Conceptualization, Supervision, Funding acquisition

A highly selective fluorescent/colorimetric probe system for
2
+
detecting Cu and tumor biomarker glutathione.

The development of coumarin Schiff base system applied as highly
2
+
selective fluorescent/colorimetric probes for Cu and tumor
biomarker glutathione detection
a,1
a,1
a
a
b,c
Zhi-Gang Wang , Xiao-Jing Ding , Yu-Ying Huang , Xiao-Jing Yan , Bin Ding ,
d
a,b,
a,
Qing-Zhong Li , Cheng-Zhi Xie *, Jing-Yuan Xu *
a
Tianjin Key Laboratory on Technologies Enabling Development of Clinical
Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University,
Tianjin 300070, China.
b
Key Laboratory of Advanced Energy Materials Chemistry (Ministry of Education),
Nankai University, Tianjin 300071, China.
c
College of Chemistry, Tianjin Normal University, Tianjin 300387, China.
d
The Laboratory of Theoretical and Computational Chemistry, School of Chemistry
and Chemical Engineering, Yantai University, Yantai 264005, China.
1
These authors contributed equally.
Corresponding author. E-mail address: xujingyuan@tmu.edu.cn (J.-Y. Xu),
xiechengzhi@tmu.edu.cn (C.-Z. Xie)
1

Abstract:
Overexpression of tumor biomarker glutathione (GSH) has been documented in
numerous types of cancers, therefor GSH-activated light-up chemosensors for tumor
identification require great attention. A new colorimetric/fluorescent probe
(
7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-4-(dimethylamino)
benzohydrazide (HL) was prepared, which could be applied in discriminating Cu
and further recognizing GSH based on its Cu complex. Firstly, the probe HL toward
2
+
2
+
Cu exhibited selective fluorescence quenching and obvious color change from
yellow to orange-red under visible light. Further, when GSH was introduced to the
2
+
Cu -2HL system, the fluorescence recovered rapidly due to the high affinity of GSH
2
+
to Cu , meanwhile the color reverted back to former yellow. Based on fluorescence
−8
−7
titration, the detection limits were calculated as 2.40×10 M and 1.29×10 M for
2
+
2+
Cu and GSH, respectively. The combination mode of HL with Cu was investigated
2+
in detail by Job plots, ESI-MS, FT-IR, and DFT studies. Probes HL and Cu -2HL
showed relatively less toxicity and were employed for biological imaging in cells and
zebrafish. Remarkably, the detection for endogenous GSH by this developed sensor
platform implied great potential application prospect in cancer diagnosis.
2

Keywords: Fluorescent probe; “Naked-eye” detection; Glutathione monitoring; DFT
calculation; Bioimaging; Cancer diagnosis
1
. Introduction
Among the important metal ions in human body, copper plays pivotal roles in
2
+
many biological processes [1]. Abnormality of Cu in the human body may destroy
normal physiological functions and cause cancer or neurodegenerative diseases [2−4].
Elevated copper were often found in various malignancies, which is the necessary
2
+
angiogenic component in tumor growth and metastasis [5,6]. Meanwhile, Cu is also
considered to be one of environmental pollutants, for which the World Health
2
+
Organization has stipulated the value of Cu in drinking water below 2 ppm [7]. On
the other hand, thiols, such as glutathione (GSH), cysteine (Cys), and homocysteine
(Hcy), play the vital roles in maintaining biological redox homeostasis of bodies and
issues [8,9]. As the most abundant nonprotein thiol, GSH is an essential redox control
agent to resist against free radicals and toxins, and also act as biomarker for various
medical diagnoses [10]. Abnormal levels of GSH is associated with cancer,
Alzheimer’s disease, AIDS, liver damage, and other ailments [11,12]. In comparison
with normal tissues, tumors always express higher levels of GSH, which could be
used to differentiate cancer and normal cell [13,14]. Detection and imaging of tumor
biomarker GSH will greatly improve the diagnosis of cancer patients in the early
stages and contribute to precision cancer therapy [15–17]. Hence, it is very necessary
2
+
to develop efficient methods for Cu and GSH detection in biosystem, especially for
3

cancer diagnosis.
Fluorescent and colorimetric chemosensors for metal ions and cancer biomaker
have received considerable attention in recent years for their high selectivity,
sensitivity, simplicity and fast response [18–22]. Owning to the outer electronic
9
2+
arrangement (3d ) of Cu and its strong binding affinity toward thiols, copper
complexes were successfully used to detect thiols through measuring changes in
fluorescence intensity or UV-vis absorption [23–26]. The chemosensors can
2
+
specifically distinguish Cu , then generated non-emissive fluorophore-Cu(II)
complexes could further recognize thiols by displacement processes. Although this
method is simple and convenient, it is often limited by lower sensitivity, no visual
sensing and high toxicity.
It is well known that the key element of probe design is the exquisite combination
of different functional groups with appropriate properties. Coumarin dye is
extensively used as building block and chromosphere in designing fluorescent probe
owning to its well-biocompatibility, good light stability, visible emission wavelength,
high fluorescence sensitivity and readily modified backbone [27–30]. Simultaneously,
acylhydrazine Schiff base derivatives have good applications in the fields of medicine
and sensor, which can provide special coordination environment and exhibited low
toxicity as well as good water solubility [31–35]. Considering these aspects, we
designed
a
new
chemosensor
(7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-4-(dimethylamino)benzohydra
2
+
zide (HL) to detect Cu and GSH by “on-off-on” fluorescent switch strategy
4

accompanied with obvious colorimetric responses in neutral aqueous media, which
possess high sensitivity and excellent selectivity over a wide range of ions and amino
acids. Furthermore, the developed sensor platform could be successfully applied in
biological imaging and medical diagnostics with satisfied results.
Scheme 1. Syntheses of probe HL.
2
. Experimental section
2
.1. Materials and instruments
All chemicals were obtained from commercial providers and used without
purification. Zebrafish embryos and larvae were purchased from Nanjing EzeRinka
1
13
Biotechnology Co., Ltd. H-NMR and C-NMR spectra were obtained on Bruker
AVANCE III spectrometer. FT-IR spectra were measured on NICOLET IS50 FT-IR
spectrometer. UV-vis absorption spectroscopies were recorded on JASCO V-770
spectrometer. Fluorescence measurements were acquired on Hitachi F-7000
fluorescence spectrophotometer. Electrospray ionization mass spectrometries
(ESI-MS) were carried out on Finnigan LCQ electron spray mass spectrograph.
Images were performed by confocal fluorescence microscope (Olympus FV1000,
Japan). The copper contents was also determined by inductively-coupled plasma mass
5

spectrometry (ICP-MS, Optima 5300DV, PerkinElmer, USA). Steady-state
fluorescence experiment was performed on Edinburgh FS5 spectrofluorometer.
2
.2. Preparation of HL
The synthesis procedure of HL was shown in Scheme 1. First of all,
-dimethylaminobenzhydrazide [36] and 7-diethylaminocoumarine-3-aldehyde [37]
4
were synthesized followed to the reported methods. Then to a solution of
4
7
-dimethylaminobenzhydrazide (89.6 mg, 0.5 mmol) in 25 mL anhydrous methanol,
-diethylaminocoumarine-3-aldehyde (122.7 mg, 0.5 mmol) in 15 mL anhydrous
o
methanol was added slowly under stirring. The mixture was refluxed for 10 h at 80 C.
The resulting solution was cooled to room temperature, and the precipitate was
filtered, washed with methanol twice and dried under vacuum. Finally, 124 mg orange
solid
of
(7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-4-(dimethylamino)benzohydra
1
zide (HL) was obtained at 61% yield. H NMR (400 MHz, DMSO-d ): δ (ppm): 11.62
6
(s, 1H, NH), 8.47 (s, 1H, CH=N), 8.33 (s, 1H, ArCH), 7.82 (d, J = 8.9 Hz, 2H, ArCH),
7
2
1
1
1
.64 (d, J = 9.0 Hz, 1H, ArCH), 6.78 (d, J = 2.4 Hz, 1H, ArCH), 6.75 (d, J = 9.0 Hz,
H, ArCH), 6.59 (s, 1H, ArCH), 3.47 (q, J = 7.0 Hz, 4H, CH ), 3.00 (s, 6H, CH ),
2
3
1
3
.14 (t, J = 7.0 Hz, 6H, CH ) (Fig. S1). C NMR (101 MHz, DMSO-d ) δ (ppm):
3
6
60.88, 156.35, 151.13, 151.11, 148.23, 137.99, 130.69, 129.09, 119.37, 116.85,
13.01, 110.79, 109.65, 108.08, 96.38, 44.19, 40.94, 12.34 (Fig. S2). ESI-MS (m/z)
+
+
calculated [HL + H] = 407.2078, found 407.2077, [HL + Na] = 429.1897, found
6

−1
4
29.1897(Fig. S3). FT-IR (KBr, cm ): υ(hydrazide N–H): 3304.43, υ(coumarin
C=O): 1696.40, υ(hydrazine C=O): 1651.52, υ(Schiff-base C=N): 1615.94.
2
.3. Absorbance and fluorescence measurements
HL was dissolved in N,N-dimethylformamide to make a stock solution with
–
3
concentration of 10 M. All metal ion stock solutions were prepared from their
corresponding nitrate salts with methanol, meanwhile all anion stock solutions were
prepared from their corresponding sodium salts with methanol. Amino acid stock
‒
3
solutions were prepared using double distilled water. All the stock solutions (10 M)
were diluted properly for the spectrometry measurements. Job plots analysis was
performed by using UV-vis absorption spectrum, in which the proportions of HL and
2
+
2+
Cu varied meanwhile the total concentration of HL and Cu remained unchanged.
2
+
The solution of copper complex (Cu -2HL) for the detection of GSH were prepared
–
3
in situ by adding 10 M Cu(NO ) (20 µL) to 20 µM HL solution (2 mL), which
3
2
could be diluted to other concentrations.
2
.4. Toxicity and bio-imaging studies
MCF-7 (breast carcinoma cells) and HUVEC (human umbilical vein endothelial
cell) were obtained from American. The toxicity study of probe HL and its Cu
complex were determined by MTT assay. MCF-7 cells were cultured on 96-well
3
o
cell-culture plates at a density of 3 × 10 cells/well, and incubated at 37 C. 24 h later,
2
+
the cells were incubated with HL and Cu -2HL with varied concentrations for 6 h.
Then 10 µL MTT solution was added and incubated for another 4 h. Finally, the
7

supernatant was removed and 100 µL DMSO was added to dissolve the formed
®
formazan precipitate before measurements on the Infinite 200 Pro NanoQuant
Microplate Reader.
The cancerous cells MCF-7 and normal cells HUVEC were trypsinised and
o
2+
reseeded at 37 C. 12 h later, they were incubated with 10 µM Cu -2HL or HL for 30
min. GSH, N-ethylmaleimide (NEM) or Cu(NO ) with different concentration was
3
2
loaded selectively for another 30 min. After washing cells with cold PBS two times,
fluorescence images were taken.
2
+
Zebrafish embryos and larvae (3-day-old) were incubated with Cu -2HL solution
(10 µM) for 1 h, then washed with deionized water three times to remove excess
2
+
Cu -2HL. Fluorescence images of zebrafish embryos and larvae were conducted by
using confocal laser scanning microscope. For comparison, zebrafish larvae were
2+
pretreated with 50 µM NEM for 30 min to block thios, then incubated with Cu -2HL
and carried fluorescence imaging under the same condition.
3
. Results and discussion
3
.1. Colorimetric sensing studies
The absorption spectra of HL with various metal ions were measured in
CH OH/H O (v/v = 1/1) solution (PBS buffer, pH = 7.4). As shown in Fig. S4, the HL
3
2
2
+
exhibited a strong broad absorption peak at 454 nm, while upon addition of Cu , the
maximum absorption peak was red-shifted to 487 nm. The response time was below 1
minute. The absorption band with other metal ions were almost unchanged except
2
+
Ni , for which the maximum absorption slightly shifted to long-wave band (∆λ is
8

about 10 nm). In addition, all the tested metal ions didn’t cause apparent interference
2
+
on absorption spectra of the HL-Cu system (Fig. S5), which is favorite to the
2
+
colorimetric detection of Cu under complicated environment. The absorption
titration experiments have been further performed. As shown in Fig. 1a, with
2
+
increasing concentrations of Cu , the maximal peak at 454 nm gradually decreased
and a new peak increased at 487 nm concurrently with two well-defined isosbestic
2
+
points at 389 and 470 nm, revealing that only one complex formed from HL and Cu
2
+
2+
[
38]. The Cu -2HL system was prepared by adding 0.5 equiv. Cu to the solution of
2+
HL to act as a new probe for GSH. When GSH was added to the Cu -2HL system,
the maximum absorption peak at 487 nm was blue-shifted to 454 nm again (Fig. 1b).
Both of the above absorption titration displayed good linear relationship between the
2
+
2+
absorbance and Cu /GSH concentrations (within the range of 0–10 µM for Cu and
0
–25 µM for GSH) (Figs. S6, S7 and Fig. 1 insert). It was found that detection limits
−7
2+
−6
(
DL) were 1.23 × 10 M for Cu and 2.18 × 10 M for GSH. Details for DL
calculations were shown in SI, Figs. S6 and S7.
2
+
Fig. 1. Absorption titration spectra of (a) probe HL (20 µM) with increasing concentration of Cu
9

2
+
(
0–1 equiv.) and (b) probe Cu -2HL (10 µM) with increasing concentration of GSH (0–6 equiv.)
2
+
in CH OH-H O buffer solution. Inset: (a) the absorbance at 487 nm versus [Cu ]; (b) the
3
2
absorbance at 430 nm versus [GSH].
3
.2. Fluorescence sensing studies
The fluorometric analysis of probe system were determined in CH OH/H O (v/v =
3
2
1
/1) PBS buffer solution (pH = 7.4) upon excitation at 450 nm. Probe HL can emit
intense fluorescence at about 521 nm, which could be markedly quenched by the
2
+
addition of Cu due to the paramagnetic quenching effect [39] (Fig. 2a). For other
+
+
2+
2+
3+
2+
2+
3+
2+
2+
metal ions (1 equiv. Na , K , Ca , Mg , Al , Co , Ni , Fe , Zn and Cd ), most
2
+
of them did not cause obvious changes except for Ni . The fluorescence intensity for
2
+
Ni decreased by 11.9% in comparison with HL alone, while the quenching rate for
2
+
Cu reached to 89.8%. The excellent selectivity under complex condition is an
important factor for practical application of fluorescence probe. To study the
anti-interference property of probe HL, we conducted fluorescence experiments
2
+
towards Cu along with other ions. As shown in Fig. S8, most competing ions didn’t
2+
significantly interfere the quenching effect of Cu for probe HL. Notably, the
+
+
2+
2+
existence of superfluous Na , K , Mg and Ca (which are important elements in
2
+
biological activities) hardly perturbed the fluorescence of Cu -HL system.
2
+
The obtained Cu -2HL system could consequently act as a promising probe for
2
+
fluorescence “off-on” detection of GSH via Cu displacement approach. To evaluate
2
+
2+
the selectivity of fluorescence probe Cu -2HL for GSH, the response of Cu -2HL
1
0

(10 µM) to various amino acids including Arg, Asp, Cys, Glu, Gly, GSH, His, Iso,
−
−
−
−
Lys, Met, Pro, Ser, Thr, Tyr and Val, as well as various anions (F , Cl , Br , NO ,
2
−
2−
4
−
−
−
−
NO , HPO , H PO , CH COO , HS and HSO ) were measured, respectively. As
3
2
4
3
3
shown in Fig. 2b and Fig. S9, the fluorescence emission at 521 nm significantly
enhanced by about 9.2-fold with the addition of GSH, while other amino acids and
anions exerted little influence on fluorescence emission. Additionally, competition
experiments were also conducted (Figs. S10 and S11), in which other amino acids and
2
+
anions did not seriously interfere with the measurement of GSH by probe Cu -2HL,
2
+
suggesting that Cu -2HL had distinct potential to distinguish GSH in physiological
environment.
2
+
2+
Quantitative analysis of HL towards Cu , as well as Cu -2HL towards GSH,
were both studied in detail by fluorescence titration experiment (Fig. 3). With the
2
+
increase of Cu concentrations (0−1 equiv.), the fluorescence intensity of HL was
2
+
gradually reduced and reached a minimum when 0.6 equiv. of Cu was introduced,
2+
while further increase of Cu concentration did not cause significant change in the
2
+
emission intensity. For Cu -2HL system, corresponding enhancements of
fluorescence intensity at 521 nm were followed with gradual addition of GSH.
2
+
Excellent linear relationship between emission intensity and analytes (Cu and GSH)
2
+
were both observed in the low concentration region (For Cu : 0−4 µM, for GSH:
0
−10 µM; See insert of Fig. 3, Fig. S12 and Fig. S13), indicating that the chemosensor
2+
system could be used in quantitative determination of Cu and GSH. The detection
2
+
2+
limits (DL) of probe HL for Cu and Cu -2HL for GSH were calculated to be
11

−8
−7
2
.40×10 M and 1.29×10 M, respectively. Comparing with fluorescence detection
2
+
method, the content of Cu was also determined by ICP-MS, in which the relative
error was below 5% (Table S1). In addition, according to the fluorescence titration
2
+
results, the intrinsic binding constant K for Cu with HL was determined to be
5
−1
2
.63×10 M , which was calculated by the Benesi-Hildebrand equation for 2:1
complex [9]. Details for DL and K calculations were displayed in SI and Fig. S14.
2
+
Transient fluorescence titration experiment of Cu has been performed. As shown in
Fig. S15, the lifetime of Cu complex (τ = 0.29 ns) were obviously shorter than probe
HL (τ = 0.49 ns), which was accordance with the fluorescence intensity changes. The
5
-1
quenching constants K were determined as 3.77×10 M for Cu complex, according
SV
by the Stern–Volmer equation: F /F = 1+ K [Q] (Fig. S16) [40, 41]. And then,
0
SV
1
3
–
molecular quenching constants k for Cu complex was calculated to be 3.77×10 M
q
1
–1
–8
s (k = K /τ , τ = 10 s), which is much higher than the diffusion rate constant (ca.
q
SV
0
0
1
0
–1 –1
1
0 M s ), thus assures the static quenching mechanism [42].
We know that most fluorescent probes are greatly affected by pH value, so we
2
+
determined the fluorescence response of HL and Cu -2HL from pH 2 to 12 (Fig.
2
+
S17). The fluorescence intensity of HL and Cu -2HL showed the similar trend and
were stable in a broad pH range (4–12) except for a sharp downward trend from pH 3
to 4. Therefore, physiological pH value (7.4) was selected for experimental research,
which is beneficial for GSH tracking in living system.
1
2

Fig. 2. The fluorescence emission spectra of (a) probe HL (10 µM) upon addition of 1 equiv.
2
+
2+
various metal ions and (b) Cu -2HL (10 µM for HL, 5 µM for Cu ) upon addition of different
amino acids (50 µM) in CH OH-H O buffer solution (λ = 450 nm).
3
2
ex
Fig. 3. The fluorescence titration spectra of (a) probe HL (10 µM) with increasing concentration of
2
+
2+
Cu from 0 to 10 µM and (b) probe Cu -2HL system (10 µM) with increasing concentration of
2
+
GSH from 0 to 20 µM. Inset: the fluorescence intensity at 521 nm versus (a) [Cu ] and (b) [GSH]
–
7
–7
(
(
λ = 450 nm). The first points in titration experiments were set as 1 × 10 M and 2 × 10 M for
ex
a) and (b), respectively .
3
.3. “Naked-eye” detection
It should be noted that this chemosensor system has potential in “Naked-eye”
2
+
detection for Cu and GSH, which possess good practicability due to convenience
1
3

2
+
and easy-operation. As shown in Fig. 4, with addition of Cu , the color of HL
solution noticeably changed from yellow to orange-red, while no obvious response
could be observed with other metal ions. Under 365 nm UV irradiation, probe HL
2
+
exhibited green fluorescence. After adding Cu , the green fluorescence disappeared,
while no corresponding fluorescence changes occurred in the intervention of other
2
+
surveyed metal ions. With addition of GSH, the color of Cu -2HL system reverted
back to former yellow, meanwhile displayed green fluorescence under 365 nm UV
lamp (Fig. 5). Similarly, Color changes weren’t disturbed by various amino acids. It is
worth mentioning that all the response time for analytes were below 1 minute.
2
+
2+
Therefore, colorimetric probe HL and Cu -2HL can quickly detect Cu and GSH by
visual observation, respectively.
Fig. 4. The photograph of probe HL (10 µM) with visual color changes upon the addition of 1
equiv. of different metal ions in CH OH-H O buffer solution.
3
2
1
4

2
+
2+
Fig. 5. The photograph of probe Cu -2HL (10 µM for HL, 5 µM for Cu ) with visual color
changes upon the addition of 1 equiv. of GSH and different amino acids.
3
.4. Binding mode studies
2+
For determining the binding stoichiometry of HL with Cu , Job plots (Fig. S18)
was obtained by absorption spectroscopy at 487 nm. The maximum absorption was
2+
obtained at Cu molar fraction of ca. 0.33, which indicated the 2:1 combination ratio
2
+
between HL and Cu . ESI-MS experiment also proved the 2:1 binding stoichiometry
2
+
between HL and Cu (Fig. 6). The complex of HL with Cu(NO ) exhibited signal
3
2
+
peak at m/z 874.3221, corresponding to [CuL +H] (m/z: 874.3222). FT-IR
2
2
+
spectroscopy provided more evidence for the binding of HL with Cu (Figs. S19 and
–
1
S20). For HL, strong characteristic bands at 1696.40, 1651.52 and 1615.94 cm are
attributed to ν(C=O, coumarin), ν(C=O, hydrazine) and ν C=N), respectively. For
(
complex, these bands weaken and red-shifted, which certified the binding of carbonyl
and imino groups with metal ion [35].
1
5

2
+
Fig. 6. The ESI-MS spectrum of Cu -2HL.
Density functional theory (DFT) studies of HL and Cu complex were performed at
the B3LYP level using Gaussian 09 programs [43]. The LANL2DZ basis set was used
2
+
for Cu whereas 6-31+G (d,p) for the other atoms. The optimized geometries of HL
and Cu complex are presented in Fig. 7, which were both local minima in the
2
+
potential energy surfaces without constrains of symmetry. For Cu complex, Cu is
located in a distorted octahedral environment with a CuN O core involving imine
2
4
nitrogen, hydrazide carbonyl oxygen and coumarin carbonyl oxygen from two
tridentate L ligands. This coordination mode is consistent with the literature reported
before [35,44,45]. The energy band gaps between HOMO (Highest Occupied
Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) are 3.357 eV
for HL and 2.876 eV for Cu complex, respectively (Fig. S21), which is consistent with
the red-shift effect of absorption peak [35].
1
6

Fig. 7. Optimized structures of HL and its Cu complex.
3
.5. GSH sensing in biological system
To further investigate the in vivo application of probe molecule, the cytotoxicities
of HL and its Cu complex were investigated by MTT assay. MCF-7 cells were
2
+
incubated with various concentrations of HL and Cu -2HL for 6 h. As shown in Fig.
8
, cell viability was higher than 95% over the full range of staining concentrations (0–
1
00 µM) for HL and below 25 µM for Cu complex, which indicated that probe HL
2
+
(
10 µM) and Cu -2HL (10 µM) could both be used safely for luminescence
bio-imaging. It is generally believed that excessive copper ion is toxic to organisms,
2+
while the close encirclement by two chelated HL prevents the interaction of Cu with
1
7

2
+
proteins, thus results in the low cytotoxicity of Cu -2HL.
2
+
Fig. 8. Cell viability of MCF-7 cells after treatments with HL (a) and Cu -2HL (b) for 6 h.
Results are expressed as the mean ±S.D (n = 3).
Fig. 9. (a) Confocal microscope images of MCF-7 cells which were incubated with indicated
2
+
concentration of GSH followed by the treatment of probe Cu -2HL (10 µM). Top: fluorescence
images; middle: bright-field images; bottom: merged images. (b) Quantified fluorescence
intensities of cells.
2
+
As shown in Fig. 9, after MCF-7 cells were incubated with 10 µM Cu -2HL,
significant fluorescence was observed and increased in a GSH dose-dependent way
2
+
(
[GSH] = 0–20 µM). The result suggested that probe Cu -2HL was easily membrane
1
8

permeable and could detect GSH with high efficiency in live cells. Remarkably,
obvious intracellular fluorescent emission was observed in MCF-7 cells even in the
absence of exogenous GSH, which is attributed to the fact that GSH always maintains
a high level in various cells. For confirming the fluorescence is caused by GSH,
MCF-7 cells were pretreated with different concentration of NEM, which is an
irreversible chelator of GSH, to consume endogenous GSH. Then MCF-7 cells were
2
+
incubated with Cu -2HL for another 30 min and imaged under microscopy. As shown
in Fig. S22, the cellular fluorescence emission was effectively reduced by NEM in a
2
+
dose-dependent way, which verified that probe Cu -2HL could sensitively detect
2
+
endogenous GSH. In addition, probe HL could detect excessive Cu ions in MCF-7
cells through fluorescence quenching mechanism (Fig. S23).
Due to the importance of GSH level in tumor identification, we are interested in
2
+
the detection capability of Cu -2HL in differentiating endogenous GSH levels in
tumor cells and normal cells. MCF-7 and HUVEC cells were both incubated with
2
+
Cu -2HL for 30 min then imaged under the same condition. It was found that the
fluorescence intensity in MCF-7 cells was above 2-fold higher than that in HUVEC
cells, suggesting higher GSH concentration in tumor cells (Fig. 10), which is
consistent with the reports that high levels of GSH was generated in tumor cells for
resisting intrinsic oxidative stress [46,47].
1
9

Fig. 10. (a) Comparison of endogenous GSH level in MCF-7 and HUVEC cells after incubation
2
+
with fluorescent probe Cu -2HL. Left: fluorescence images; middle: bright-field images; right:
merged images. (b) Quantified fluorescence intensities of cells.
From a biological perspective, most human genes and zebrafish genes are
homologous, and studies have proved that drug response of zebrafish has a high
similarity with human being [48,49]. Due to the optical transparency, zebrafish are
commonly selected as model animal for biomolecule detection in live organisms by
2
+
fluorescence imaging. Therefore, the applicability of probe Cu -2HL to trace GSH in
2
+
zebrafish was investigated. Zebrafish larvae were incubated with 10 µM Cu -2HL,
resulting bright fluorescence signal in eyes and digestive system of zebrafish (Fig. 11
and Fig. S24). As reference experiment, when pre-treated zebrafish with 50 µM NEM,
fluorescence was obviously weaken. In addition, The zebrafish embryos treated with
2
+
1
0 µM Cu -2HL could also emit strong green fluorescence which scattered over the
whole embryos (Fig. S25). All the above results indicated the potential extensive
2
+
application of probe Cu -2HL in living systems.
2
0

2
+
Fig. 11. Fluorescence images of zebrafish larvae incubated with Cu -2HL. Bright field image
(
left), fluorescence image (middle) and overlap image (right). λ = 488 nm.
ex
4
. Conclusions
A unique chemosensor system for detecting Cu and tumor biomarker GSH has
2
+
been successfully devised, which exhibit high sensitivity, excellent selectivity and
good anti-interference over other metal ions or amino acids. The 2:1 binding mode of
2
+
Cu with HL was proven by ESI-MS, IR, Job plots analysis and DFT calculations.
The developed chemosensor system shows several significant advantages: (1)
simplicity in design and performance; (2) time-saving sensing process in which all the
detection is finished within 1 min; (3) can achieve “Naked-eye” detection; (4) can
selectively detect GSH in complicated environment including biological interferents
−
HS , Hcy and Cys. More importantly, it shows good detection ability in real sample
analysis especially for medical diagnosis. Due to its excellent characteristic, this
2
1

detection system has great potential for evaluating the tumor biomarker GSH levels in
biological and medical fields.
Acknowledgements
This work was supported by National Natural Science Foundation of China (No.
2
1
1977080), Tianjin Municipal Natural Science Foundation (No. 17JCZDJC33100,
8JCYBJC89700, 18JCYBJC91300), the Program for Innovative Research Team in
University of Tianjin (TD13-5074) and the Science &Technology Development Fund
of Tianjin Education Commission for Higher Education (2019ZD15).
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