A dual-functional fluorescent probe for sequential determination of Cu2+/S2? and its applications in biological systems

Article abstract of DOI:10.1016/j.saa.2020.118797

A new acylhydrazine-derived Schiff base fluorescence probe DMI based on “ON-OFF-ON” fluorescence strategy was presented in this paper. Probe DMI could detect Cu²⁺ selectively and sensitively with dramatic fluorescence quenching in CH₃OH-PBS (v/v = 3:7) mixed solution. Once the complex DMI-Cu²⁺ interacted with S^2?, 10.67-folds fluorescence increase was induced via a displacement mechanism under the same experimental conditions. The corresponding detection limits for Cu²⁺ and S^2? were calculated to be 1.52 × 10^?8 M and 1.79 × 10^?8 M, respectively. The structures of DMI and DMI-Cu²⁺ were systematically characterized by Job's plot analysis, ESI-MS, IR, X-ray diffraction and density functional theory calculations. Furthermore, fluorescence imaging in MCF-7 cells and zebrafish demonstrated the probe DMI could act as a useful tool to monitor and track intracellular Cu²⁺ and S^2?, which was encouraged by remarkable fluorescence performance and low cytotoxicity. Importantly, the complex DMI-Cu²⁺ could be applied to detect corrupt blood samples, which could estimate the time of death.

Full text of DOI:10.1016/j.saa.2020.118797

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A dual-functional fluorescent probe for sequential determination of
2
+
2−
Cu /S and its applications in biological systems
a
a
a
a
b,
a,c,
Xiao-Jing Yan , Zhi-Gang Wang , Yang Wang , Yu-Ying Huang , Hai-Bo Liu ⁎, Cheng-Zhi Xie ⁎⁎,
d
a,
Qing-Zhong Li , Jing-Yuan Xu ⁎
a
Department of Chemical Biology and Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University,
Tianjin 300070, PR China
b
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, PR China
Key Laboratory of Advanced Energy Materials Chemistry (Ministry of Education), Nankai University, Tianjin 300071, PR China
The Laboratory of Theoretical and Computational Chemistry, School of Chemistry and Chemical Engineering, Yantai University, Yantai 264005, PR China
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2020
Received in revised form 19 July 2020
Accepted 21 July 2020
Available online 06 August 2020
A new acylhydrazine-derived Schiff base fluorescence probe DMI based on “ON-OFF-ON” fluorescence strategy
2+
was presented in this paper. Probe DMI could detect Cu selectively and sensitively with dramatic fluorescence
2+
2−
3
quenching in CH OH-PBS (v/v = 3:7) mixed solution. Once the complex DMI-Cu interacted with S , 10.67-
folds fluorescence increase was induced via a displacement mechanism under the same experimental conditions.
The corresponding detection limits for Cu2+ and S were calculated to be 1.52 × 10 M and 1.79 × 10 M,
respectively. The structures of DMI and DMI-Cu2+ were systematically characterized by Job's plot analysis, ESI-
MS, IR, X-ray diffraction and density functional theory calculations. Furthermore, fluorescence imaging in MCF-
7 cells and zebrafish demonstrated the probe DMI could act as a useful tool to monitor and track intracellular
2−
−8
−8
Keywords:
Fluorescent probe
2
+
2−
Cu /S
2
+
2−
Practical application
DFT calculation
ON-OFF-ON
Cu and S , which was encouraged by remarkable fluorescence performance and low cytotoxicity. Impor-
tantly, the complex DMI-Cu could be applied to detect corrupt blood samples, which could estimate the
time of death.
2+
©
2020 Elsevier B.V. All rights reserved.
1
. Introduction
quickly hydrolyze to H
higher toxicity than sulfide itself [17–19]. Meanwhile, as a vital gaseous
signal molecule in living organisms, H S plays crucial roles in the ner-
vous system and metabolic system [20]. However, continuous exposure
to inappropriate level of H S can damage respiratory, disrupt the normal
function of human body and cause a range of sever diseases like Down's
syndrome, hypertension, diabetes, and liver cirrhosis [21–25]. Hence, it
is highly desired to develop an efficient and sensitive way for motoring
S and HS− at physiological pH, which have
2
Copper is one of the most sufficient and essential trace elements in
2
human body and takes part in a variety of fundamental biological and
physiological processes [1–3]. The concentration of copper ion in the
cell is closely related with human health [4]. Abnormal level of copper
ion may break the dynamic balance of numerous substances in the
body [5], which is connected with many neurodegenerative diseases,
including amyotrophic lateral sclerosis [6,7], Parkinson's [8],
Alzheimer's [9,10], and prion diseases [11]. At the same time, copper
ion, as a heavy metal ion, may produce potentially toxic pollutants to
the environment and exert irreversible threat to the global sustainable
2
copper ion and H
Numerous fluorescence probes have been established to detect H
and most of them are based on the following mechanisms: displace-
2
S in environmental and biological systems.
2
S
ment method based on the strong affinity of S2 to Cu
−
2+
[26,27],
development [12,13]. Similarly, hydrogen sulfide (H
gas with a smell of rotten eggs, is not only from industrial processes but
also from organic matter corruption processes [14–16]. Sulfide can
2
S), as a hazardous
azide reduction [28,29] and nucleophilic reaction [30]. Many of these
probes have drawbacks of high toxicity, long response time and rigid re-
action conditions [31–33]. However, reversible detection according to
the first strategy has attracted increasing attention [34]. On the one
hand, Cu2 has an incompletely filled d-orbital which provides an op-
portunity for quenching fluorescence [35,36]. On the other hand, the re-
+
⁎
Corresponding authors.
⁎
⁎ Correspondence to: C.-Z. Xie, Department of Chemical Biology and Tianjin Key
2−
2+
action of S
solubility (CuS: Ksp = 6.36 × 10
and Cu
forms a stable compound CuS with lower
Laboratory on Technologies Enabling Development of Clinical Therapeutics and
Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China.
E-mail addresses: hbliu@implad.ac.cn (H.-B. Liu), xiechengzhi@tmu.edu.cn (C.-Z. Xie),
xujingyuan@tmu.edu.cn (J.-Y. Xu).
−36
2+
) than common Cu -probe com-
plexes [37,38]. These two favorable factors make possibilities to turn
fluorescence off and on. Recently, several fluorescent probes have
https://doi.org/10.1016/j.saa.2020.118797
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
X.-J. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
been designed and synthesized to sequentially detect Cu2+ and S2− via
ON-OFF-ON” strategy [39,40]. The mechanism is regarded as a copper
d, ArH), 8.60 (1H, d, ArH), 8.52 (1H, s, =CH), 8.43 (1H, s, ArH), 6.58
(1H, s, ArH), 3.49–3.45 (4H, q, CH ), 1.17–1.12 (6H, t, CH ) (Fig. S1).
): δ (ppm): 167.3, 166.9, 161.8, 156.6,
“
2
3
13
sulfide precipitation strategy which can recover the fluorescence re-
sponse of free probes [41]. Nevertheless, few probes have good biocom-
patibility and water-solubility, which limit their applications in
biological research [42,43]. Thus, developing a novel fluorescent probe
with excellent performance is highly important and indispensable.
It is an efficient way to design fluorescent probes based on selective
combination of fluorescent groups and auxiliary groups [44,45]. Couma-
rin compounds and its derivatives have excellent fluorescence perfor-
mance of good optical properties and biocompatibility [46–49]. Due to
containing the C_O and C_N functional group, the acylhydrazine
Schiff base compounds can provide an appropriate binding environ-
ment to recognize target ions [50,51]. Herein, according to the above
strategies and as a continuation of our research work about “ON-OFF-
C NMR (101 MHz, DMSO‑d
6
152.3, 148.5, 146.2, 144.2, 141.6, 136.1, 133.9, 132.4, 131.3, 130.8,
128.8, 117.7, 114.4, 113.3, 110.3, 101.6, 49.4, 17.5 (Fig. S2). ESI-MS (m/
+
+
z) calculated [DMI + H] = 415.2, found 415.5, [DMI + Na]
=
−1
437.2, found 437.5 (Fig. S3). IR (KBr cm ): υ(C=O):1678.2, υ(Schiff-
base) C=N: 1617.7 (Fig. S4).
2.2. Optical measurement
The probe DMI was dissolved in DMF to obtain the stock solution
+
+
2+
3+
2+
(
1 mM). Stock solutions of various ions (Na , K , Ca , Fe , Zn
,
,
2+
3+
2+
2+
2+
2+
−
−
−
−
−
Mg , Al , Cd , Cu , Co , Ni , Br , CH
3
COO , Cl , F , H
2
PO
4
2−
−
−
−
HPO
4 3 2 3
, HSO , NO , NO ) were prepared from their corresponding ni-
ON” fluorescence detection [52,53], we developed
a novel
trate salts and sodium salts in methanol solution, respectively. The solu-
acylhydrazine-derived Schiff base receptor N′-((7-(diethylamino)-2-
oxo-2H-chromen-3-yl) methylene) isoquinoline-1-carbohydrazide
2−
tion of S
was prepared from Na
2
S. All UV–vis and fluorescence
OH-PBS (2 mL, v/v = 3:7) buffer
experiments were performed in CH
3
(
DMI) with the reaction of 7-(diethylamino)-2-oxo-2H-chromene-3-
solution at room temperature. The titration experiments were operated
carbaldehyde and isoquinoline-1-carbohydrazide. The probe DMI
2+ 2−
by gradually incremental additions of stock solutions (Cu /S ) to
could specifically detect Cu2 with dramatic fluorescence quenching
+
2+
DMI or DMI-Cu solution. Job's plot experiment is an effective way to
in CH
that the complex DMI-Cu could act as a probe to monitor H
UV–Vis and fluorescence spectra. Meanwhile, DMI and DMI-Cu
3
OH-PBS (v/v = 3:7) buffer solution. Further studies indicated
determine the coordination ratio of probe with metal ions, which was
obtained by recording absorbance with steadily concentration ratio
changes. All the measurements were carried out less than 1 min after
mixing thoroughly.
2+
2
S by
2+
could be applied in the fluorescence imaging in cells and zebrafish. In
addition, the detection of real samples could also be achieved.
2.3. X-ray crystallography
2
. Experimental section
The crystal structure of DMI was displayed in Fig. 1. X-ray data col-
2
.1. Synthesis of probe DMI
lection and structural determination were provided in the Supporting
Information. The crystallographic data for DMI were listed in Table S1.
Selected bond lengths and bond angles were listed in Table S2 and S3.
Structural information of DMI had been deposited at the Cambridge
Crystallographic Data Center (CCDC 2000671).
The synthetic procedure of probe DMI was displayed in Scheme 1.
Noline-1-carbohydrazide [54] and 7-(diethylamino)-2-oxo-2H-
chromene-3-carbaldehyde [55] were synthesized based on the previ-
ously reported route. Isoquinoline-1-carbohydrazide (374 mg,
2
mmol) was dissolved in 25 mL methanol solution, and 5 mL anhy-
drous methanol solution of 7-(diethylamino)-2-oxo-2H-chromene-3-
carbaldehyde (490 mg, 2 mmol) was added gradually with stirring.
The mixed solution was refluxed for 5 h under stirring, which was mon-
itored by TLC. When the reaction finished, the mixture was cooled to
room temperature. The precipitate was collected by filtration and
washed with methanol three times. Ultimately, an orange solid DMI
2.4. Calculation studies
The density functional theory (DFT) calculation of probe DMI and its
complex were performed on the Gaussian 09 program using Becke-3-
Lee-Yang-Parr exchange function (B3LYP). The basis set LANL2DZ and
6-31+G(d,p) had been employed to Cu2 and C, H, N, O, respectively,
and the optimized structure of DMI and its Cu complex were obtained
[56–59].
+
was obtained through drying under vacuum. Yield: 600.5 mg, 72.5%.
1
6
H NMR (400 MHz, DMSO‑d ): δ (ppm): 12.33 (1H, s, NH), 8.78 (1H,
Scheme 1. Syntheses of probe DMI.

X.-J. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
3
Fig. 1. ORTEP view of DMI molecular structure showing the crystallographic numbering scheme. Thermal ellipsoids are drawn at 30% probability level and all hydrogens are omitted for
clarity.
2
.5. Cell culture and cytotoxicity studies
(10 μM) were added into the plate and the cells were cultured for
3
0 min. For the second group, the cells were treated with 10 μM DMI
2+
MCF-7 cell line was obtained from the American Type Culture Collec-
and one equivalent of Cu
for 30 min successively. For the third
+
2−
tion (ATCC). MCF-7 cells were kept in growth medias at an atmosphere
of 5% CO . DMEM (Dulbecco's Modified Eagle Medium) was employed
to incubate cells.
group, after being incubated with DMI and Cu2 , S
(10 μM) was
2
added into the system, which were fostered for another 30 min. The
cells were washed three times with PBS and the cell imaging was re-
corded on confocal laser scanning microscope.
The cells were collected and seeded in a 96-well culture plate with a
density of 3000 cells each well. The cells were cultivated in a humidified
Zebrafish larvae were cultured three days and washed with deion-
ized water before use. Then they were incubated with DMI solution
condition containing 5% CO
2
at 37 °C for 24 h. Then, DMI and DMI-Cu2
+
2+
with different concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.5625,
(10 μM, 1 h), DMI (10 μM, 1 h) + Cu (10 μM, 1 h) as well as DMI
(10 μM, 1 h) + Cu2 (10 μM, 1 h) + S (10 μM, 1 h) at room temper-
+
2−
0
6
.78125, 0.390625 μM) were added into microplate and fostered for
h, respectively. Subsequently, the cells in each well were treated
ature, respectively. After washing zebrafish with deionized water for
three times, DMI, Cu2 and S were removed and the fluorescent im-
ages of zebrafish were observed with an Olympus FV1000 confocal
microscope.
+
2−
with MTT (10 μL, prepared in DMSO) at a concentration of 5 mg/mL.
After another 4 h, excessive MTT solution was removed. 100 μL DMSO
was added and OD values were recorded on Infinite 200-Pro Nano
Quant Microplate Reader at 570 nm. The cell viabilities were calculated
based on following formula: Cell viability (%) = (ODdrug − ODblank) /
3. Results and discussion
(
ODcontrol − ODblank) × 100%.
2
+
3.1. Selectivity of DMI to Cu
2
.6. Biological imaging
To evaluate the selectivity of DMI towards Cu2+, UV–vis absorption
MCF-7 cells were collected and reseeded in culture plates at 37 °C for
2 h. The cells were separated into three groups. For the first group, DMI
spectrum of DMI with various metal ions were investigated in CH OH-
PBS (2 mL, v/v = 3:7) buffer solution. As shown in Fig. S5, a strong
3
1
3
Fig. 2. Fluorescence emission spectra (a) and histogram (b) of probe DMI (10 μM) with addition of various metal ions (10 μM) in CH OH-PBS (2 mL, v/v = 3:7) mixed solution.

4
X.-J. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
absorption peak at about 455 nm could be observed, which was the
characteristic peak of free probe DMI. Upon addition of various metal
phenomena indicated the formation of a new complex between DMI
and Cu2
+
.
ions, Ni2 , Cu and Co resulted in a reduction of absorbance. The
+
2+
2+
Based on the fluorescence titration experiments (Fig. 3b inset), we
2
+
2+
2+
maximum peak in the presence of Cu and Co exhibited a slight red-
observed that the fluorescence intensity of DMI and Cu concentration
2
+
shift, while the absorption intensity of Cu was higher than that of
displayed an excellent linear correlation over the range of 0–10 μM. The
2+
−8
Co . Furthermore, fluorescence selectivity experiment of DMI in the
limit of detection (LOD) was calculated as 1.52 × 10 M by using the
presence of various metal ions was measured in the same system at
following equation: LOD = K × Sb1 / S (K = 3, Sb1 was standard devia-
tion of blank solution, S was the slope of linear regression equation)
[60,61]. Moreover, the association constant (K) of probe molecule DMI
4
50 nm excitation wavelength (Fig. 2). When the metal ions were
added into the DMI solution, a slight reduction in fluorescence intensity
2
+
2+
2+
was estimated to be 2.86 × 104
−1
induced by Ni /Co and negligible changes induced by other metal
towards Cu
M
according to
ions were observed, which had little influence on the detection of
Benesi-Hildebrand plot (Fig. S7) by using following equation:
2+
Cu . More importantly, the fluorescence response of DMI was
2+
Fmax−Fmin
1
K½Mꢀ
quenched distinctly in the presence of Cu , which was closely related
¼
þ 1
to the paramagnetic quenching effect of Cu2 . The fluorescence inten-
+
F−Fmin
2+
sity of probe DMI was quenched about 27.4 times by Cu . Accordingly,
the probe DMI could sense Cu2 with excellent selectivity. Fluorescence
intensity of probe DMI and its Cu complex in different proportions of
+
where [M] = Cu2+, Fmax and Fmin were the fluorescence intensity of free
2+
probe DMI and in the presence of saturated Cu , respectively. F was the
CH
3
OH and PBS was measured to select the optimal experimental con-
fluorescence intensity of Cu complex, which is associated with the con-
2+
2+
ditions. As shown in Fig. S6, the fluorescence signal of DMI-Cu were
quenched in the pure PBS, meanwhile the fluorescence emission of
DMI is not very strong. With the increasing proportion of CH
fluorescence response of probe DMI was continuously enhanced. Corre-
spondingly, when the ratio of CH OH was in the range of 0.4–1, the
centration of Cu
.
2+
3
OH, the
3.3. Binding mode studies of probe DMI towards Cu
3
To obtain a better insight into the binding mechanism of DMI with
2
+
Cu2+, Job's plot was explored from the UV–vis absorption data
quenching effect of Cu to probe DMI was not obvious. Therefore, in
2
+
order to ensure the strong fluorescence emission of probe DMI and
(Fig. S8). When the value of [DMI]/([Cu ] + [DMI]) was close to
the good quenching effect of Cu2 , CH
+
0.66, the UV–vis absorbance displayed an inflection point, which dem-
3
OH-PBS (v/v = 3:7) phosphate
2
+
buffer system was selected for the research.
onstrated the 2:1 stoichiometric binding mode of complex DMI-Cu
.
Furthermore, the mass spectra of complex DMI-Cu2 supported the
+
above result. As shown in Fig. S9, an intense peak for complex DMI-
2+
Cu was shown at m/z 890.25970, corresponding to the [Cu + 2DMI-
2
+
+
3
.2. Sensitivity of DMI to Cu
H] , calcd. m/z 890.25961. Thereafter, FT-IR data of DMI (Fig. S4) and
its complex DMI-Cu2 (Fig. S10) also confirmed binding mechanism.
+
Then, the sensitivity of probe DMI for Cu2+ were examined by the
UV–vis and fluorescence titrations experiments in CH OH-PBS (2 mL,
v/v = 3:7) mixed solvent system (10 μM). As shown in Fig. 3a, the rep-
resentative absorption peak of free probe DMI centered at 455 nm.
Upon titration of Cu (0–15 μM), the absorption peak at 455 nm was
significantly decreased with the appearance of a new absorption peaks
at around 525 nm. The formation of well-defined isosbestic points at
Compared with the FT-IR spectra of DMI in the absence and presence
of Cu2 , the C_O peak of DMI at 1678.21 cm , C_N peak at
+
−1
3
−
1
were shifted to 1605.16 cm−1 (C=O) and
1617.78 cm
−1
1573.93 cm (C=N) with the weakness of the corresponding bands,
2
+
respectively, which confirmed that the functional groups C_O and
C_N of probe DMI participated in the interaction with Cu2
+
.
2
70 nm, 335 nm, 385 nm and 492 nm demonstrated that only one com-
3.4. Density functional theoretical calculations
2+
bination mode happened between DMI and Cu . Furthermore, Fig. 3b
illustrated the change of the fluorescence response of probe DMI upon
an increasing concentration of Cu2 (0–15 μM). The fluorescence signal
of DMI was quenched gradually with addition of Cu2 . All these
In order to further clarify the electronic properties, the geometry op-
+
2+
timisations of DMI and its complex DMI-Cu were performed on the
+
Gaussian 09 program at DFT/B3LYP level. As shown in Fig. S11, the
2
+
Fig. 3. UV–vis (a) and fluorescence (b) titration spectra of probe DMI (10 μM) with addition of different Cu concentrations (0–15 μM) in CH
λ
3
OH-PBS (2 mL, v/v = 3:7) mixed solution.
2
+
ex = 450 nm. Inset: the linear relationship between the fluorescence intensity at 521 nm and the Cu concentration (Error bars, SD, n = 3).

X.-J. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
5
probe DMI possessed good planarity with an extended conjugation,
containing coumarin ring, iso-quinoline ring and hydrazine group. In
the DMI system, the dihedral angles between coumarin ring and
hydrazide group, coumarin ring and iso-quinoline ring as well as iso-
quinoline ring and hydrazide group were 0.352°, 0.162° and 0.194°, re-
spectively, which were highly close to the crystal structure of DMI. The
interaction between probe DMI and Cu2 destroyed the planarity of the
whole molecular and three dihedral angles mentioned above twisted to
+
1
2.4222°, 35.788° and 47.683°, respectively. The HOMO and LUMO or-
2
+
bital energy levels of probe DMI and its complex DMI-Cu
were
shown in Fig. 4. The HOMO-LUMO energy gap of probe DMI and Cu
complex were 3.118 eV and 2.725 eV, respectively, which were in agree-
ment with the generation of red shifted absorption band upon incre-
mentally addition of Cu2 to DMI.
+
.5. The probe DMI-Cu for detection of S2−
2+
3
Based on the strong binding affinity of Cu to S2−, we supposed
2+
Fig. 5. Fluorescence response of DMI-Cu2+ (10 μM) in the presence of different tested
analytes (10 μM) in CH OH-PBS (2 mL, v/v = 3:7) mixed solution.
3
2+
that the complex DMI-Cu could act as a probe for the detection of
2
−
2+
2−
S
. Therefore, the selectivity of DMI-Cu towards S was studied.
2
−
−
The influence of different tested analytes, including S , Br ,
CH COO , Cl , Cys, F , H PO , HPO , HSO , NO , NO and GSH
3 2 4 4 3 2 3
−
−
−
−
2−
−
−
−
were measured. As shown in Fig. 5, except a minor fluorescence en-
induced a significant change of 10.67-folds fluorescence increase.
Meanwhile, competitive experiment (Fig. S12) showed there were no
obvious changes induced by coexistent ions and biothiol, indicating
hancement upon addition of Cys and GSH, which is caused by the strong
2
+
2−
binding affinity of Cu towards sulfur-containing molecules, only S
Fig. 4. Frontier molecular orbitals of probe DMI and its complex DMI-Cu2+
.

6
X.-J. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
range (7–8) and could act as practical probes to detect Cu and S2−
2+
in living organisms.
3
3
.7. Bioimaging application
.7.1. Cytotoxicity and cell imaging
Encouraged by the high selectivity and sensitivity of DMI towards
Cu2+ as well as DMI-Cu towards S in physiological pH range, DMI
2+
2−
2+
and DMI-Cu were utilized to study potential applications on fluores-
cent bio-imaging in living MCF-7 cells [62]. Initially, the cytotoxicity of
DMI and DMI-Cu2 was evaluated by MTT method with different con-
centrations. Although the MCF-7 cells were treated by DMI and DMI-
Cu even at higher concentration (25 μM), the survival degrees were
more than 95% (Fig. 7a) and 80% (Fig. 7b), respectively, indicating DMI
and DMI-Cu2 could act as bioprobes and were suitable for intracellular
detection.
+
2+
+
Then, owing to the low cytotoxicity of DMI and DMI-Cu2+, the fluo-
rescent imaging was captured in MCF-7 cell line (Fig. 8). When the cells
were fostered with DMI (10 μM) for 30 min at room temperature, a very
strong green fluorescence signal was observed. Nevertheless, the fluo-
rescence signal was quenched upon incubation with Cu2 (10 μM) for
another 30 min, which was ascribed to the combination of DMI and
2
+
Fig. 6. Fluorescence titration spectra of probe DMI-Cu (10 μM) with addition of different
2
−
S
concentrations (0–15 μM) in CH
3
OH-PBS (2 mL, v/v = 3:7) mixed solution. λex =
50 nm. Inset: the linear relationship between the fluorescence intensity at 521 nm and
+
4
2−
the S concentration (Error bars, SD, n = 3).
2+
2−
Cu . Further treatment with S under the same experiment condition
made the fluorescence response recover. These observations indicated
that DMI was capable of penetrating cell membranes and could be
the complex DMI-Cu2+ possessed a predominant selectivity to S2−. Fur-
thermore, fluorescence titration experiment of DMI-Cu to S was in-
vestigated. As shown in Fig. 6, upon increasing S2 concentrations, the
fluorescence responses were gradually enhanced. From Fig. 6 inset,
good linearity between the fluorescence intensity and S concentra-
tion was obtained in the range of 0–14 μM. The limit of detection to
was 1.79 × 10 M (Fig. S13), which was lower than the maximum
allowable level set by WHO (15 μM). These results indicated that the
complex DMI-Cu could be employed as a probe to detect S directly
with high selectivity and sensitivity.
2+
2−
−
2+
2−
served as a probe to sense Cu and S visually in living cells.
2−
3.7.2. Zebrafish imaging
Comparing with the excellent properties of cell imaging, monitoring
Cu2+/S in living animals was more significant and meaningful. As an
advanced creature, zebrafish had the advantages of optical transpar-
ency, simple operation and genetic similarities to human beings [63].
Herein, fluorescence imaging of zebrafish was further investigated by
using confocal microscope FV1000 (Fig. 9). Incubating zebrafish with
DMI (10 μM) merely for 1 h, brightly green fluorescence signal was col-
lected in head and digestive system. Then treated zebrafish with Cu2
2−
−8
2−
S
2
+
2−
+
3
.6. Effect of pH
(
10 μM), the green fluorescence response diminished significantly.
2−
The good sensing performance of probe in suitable pH range played
After 1 h, one equivalent of S was added and the strong green fluores-
a vital role in practical applications. Therefore, the effects of pH (2–11)
cence signal was restored. These phenomena signified that DMI and
2+
2+
on fluorescence response of probe DMI and its complex DMI-Cu
were further measured in CH OH-PBS (2 mL, v/v = 3:7) mixed solution
Fig. S14). For free probe DMI, the fluorescence intensity increased and
reached to maximum at about pH 7. At the same time, the intensity of
DMI-Cu
could potentially apply in biological system to monitor
Cu and S2−, respectively.
2+
3
(
3.8. Actual sample testing
2
+
DMI-Cu decreased and reached to minimum within the same pH
Since S2 is one of the most common anions in our life, detecting S2−
−
level. These phenomena confirmed that DMI and its complex DMI-
2+
Cu possessed the best fluorescence performance at physiological pH
is of great importance. When the blood was rotten, many substances
2
+
Fig. 7. Cell viability of MCF-7 cells incubated by different concentration (0.39–25 μM) of DMI (a) and DMI-Cu (b) for 6 h at room temperature.

X.-J. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
7
Fig. 8. Confocal fluorescence imaging of living cells incubated with DMI (10 μM) for 1 h, and then incubated with Cu (10 μM) and S2− (10 μM) successively for another 1 h. Bright field
2+
image (left), fluorescence image (middle), superposition image (right). λex = 405 nm.
including hydrogen sulfide, mercaptan, sulfide, ammonia, methane, car-
excellent fluorescence properties of high selectivity and sensitivity to-
wards Cu2 /S at pH 7 in CH
+
2−
bon dioxide were produced. According to the above experiments, the
3
OH-PBS (v/v = 3:7) mixed solution. Var-
2
+
2−
probe DMI- Cu was utilized to detect S induced by blood corrup-
tion. Corrupt blood samples at different times (including 0, 2, 4, 6, 8,
1
ious experiments, including Job's plot, ESI-MS, IR spectrometry and DFT
calculations, confirmed the binding mode of DMI to Cu2 . Its application
in cell and zebrafish imaging indicated that the probe had the ability to
+
2, 24 h) were prepared and added into DMI-Cu2+ solution (10 μM).
2
+
2−
Then, the fluorescence intensity of system was measured. As shown in
Fig. S15, the fluorescence intensity increased with the time of blood cor-
ruption, indicating more hydrogen sulfide was produced. This result
demonstrated that the probe DMI-Cu2 could be applied to detect ac-
tual sulfur samples preliminarily and calculate time of death.
monitor Cu and S in living cells and biological system. The detec-
tion of S2 using DMI-Cu in corrupt blood samples could estimate
−
2+
the death time.
+
CRediT authorship contribution statement
4
. Conclusion
Xiao-Jing Yan:Conceptualization, Methodology, Investigation, Writ-
ing - original draft.Zhi-Gang Wang:Data curation, Software, Visualiza-
tion.Yang Wang:Investigation, Validation, Formal analysis.Yu-Ying
Huang:Investigation, Resources.Hai-Bo Liu:Software, Resources,
Writing - review & editing, Funding acquisition.Cheng-Zhi Xie:Method-
ology, Writing - original draft, Supervision, Funding acquisition.Qing-
In summary, a novel fluorescence probe DMI was described in this
paper, which had been successfully developed, synthesized and charac-
terized. The green emitting fluorescence of probe DMI could be
2
+
quenched by Cu , and the fluorescence response recovered with the
2
−
2+
addition of S subsequently. The probe DMI and DMI-Cu showed

8
X.-J. Yan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 243 (2020) 118797
Fig. 9. Confocal fluorescence imaging of zebrafish incubated with DMI (10 μM) for 1 h, and then incubated with Cu (10 μM) and S2− (10 μM) successively for another 1 h. Bright field
2+
image (left), fluorescence image (middle), superposition image (right). λex = 405 nm.
[
[
3] G.G.V. Kumar, M.P. Kesavan, G. Sivaraman, J. Annaraj, K. Anitha, A. Tamilselvi, S.
2+
Zhong Li:Software.Jing-Yuan Xu:Conceptualization, Supervision, Pro-
ject administration, Funding acquisition.
Athimoolam, B. Sridhar, J. Rajesh, Reversible NIR fluorescent probes for Cu ions
detection and its living cell imaging, Sensors Actuators B Chem. 255 (2018)
3235–3247, https://doi.org/10.1016/j.snb.2017.09.150.
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2
Declaration of competing interest
[5] R.A. Løvstad, A kinetic study on the distribution of Cu(II)-ions between albumin and
transferrin, Biometals 17 (2004) 111–113, https://doi.org/10.1023/B:BIOM.
0
000018362.37471.0b.
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.
[
6] Q.T. Meng, R. Zhang, H.M. Jia, X. Gao, C.P. Wang, Y. Shi, Z.Q. Zhang, A reversible fluo-
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2+
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