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Evaluation of Cell Viability with a Single Fluorescent Probe Based on

Two Kinds of Fluorescence Signal Modes
Youbo Lai, Tengteng Zhang, Wenhui Song, Zihong Li, and Weiying Lin*
Cite This: https://doi.org/10.1021/acs.analchem.1c02911 Read Online

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ABSTRACT: Accurate evaluation of cell viability is important for

dosage tests of anticancer drugs, pathology, and numerous
Downloaded via NATL CHIAO TUNG UNIV on September 9, 2021 at 12:22:53 (UTC).

biological experiments. However, due to the serious insufficieny

of fluorescent probes, which can distinguish various cell states, the
study of cell viability is immensely limited. To resolve this issue, we
design and synthesize a new probe ACD-E to monitor cell viability
with two kinds of fluorescence signal modes, the first single
fluorescent probe that can distinguish three different cell states and
furnish accurate information in biological experiments. ACD-E can
discriminate live and dead cells in a dual-color mode by evaluating
cell mitochondrial esterase activity and can also discriminate live
and early necrosis cells by determining mitochondrial viscosity in a
“turn-on” mode in the near-infrared region. Significantly, the novel
ACD-E can also distinguish cell viability in vivo. This work establishes a robust strategy for monitoring multiple cell states using a
single fluorescent probe.

M onitoring cell viability is a necessary task for biology,1

pathology,2,3 and pharmacology,4,5 and multiple stand-
ards must be used to measure the health status of cells to
ensure that the results are repeatable and accurate. Tradition-
ally, cell viability refers to the percentage of living cells in the
total number of cells.6 Programmed cell death is usually
divided into two types: apoptosis and necrosis.7 For the
determination of cell activity, it is necessary to use a reagent to
distinguish living cells from dead cells.8 Up to now, various
techniques have been developed and applied to evaluate cell
viability.9−14 The health status of cells can be roughly
estimated by cell morphology using a light microscope. In
fact, compared with colorimetric reagents (MTT, WST),15
fluorescent probes provide a rapid, real-time, spatial resolution,
and noninvasive technique to discriminate live and dead cells.
At present, to distinguish living and dead cells, as the esterase
of dead cells is inactivated compared with that of living cells,
fluorescent probes have been widely designed with that
mechanism.16−19 Particularly, to effectively avoid the false
fluorescence signals by uneven incubation of probes and cell
autofluorescence signals caused, some dual-color probes have
been developed in succession.20−22 However, discriminating
live and dead cells is one of the key indicators of cell viability
but not the only one. Cell viability should be evaluated more
accurately to meet the needs of pharmacological and
pathological research.
The majority of cells of multicellular organisms die on their
own through the physiological process of apoptosis. However,
a small part of the cells die through necrosis, which is caused
by extreme physical, chemical, or other serious pathological
factors and the inability to maintain their own vitality.21
Notably, the ability to identify necrosis or apoptosis can
provide a new strategy for drug design and screening, as well as
tumor treatment.4,22 In recent years, various techniques have
been developed based on the mechanism of mitochondrial
depolarization in the early stage of apoptosis.23 Nevertheless,
there are few studies on the early stage of cell necrosis. It is
widely known that cell necrosis is accompanied by
inflammation, which can increase mitochondrial viscosity.24,25
The disturbance of the mitochondrial state can also provide
earlier and more sensitive indicators of slight changes in cell
health, thereby reducing the impact on the experiment.
Therefore, it is possible to observe the initial stage of cell
necrosis by evaluating the change in the viscosity of the
mitochondria.26,27 Therefore, comprehensive monitoring of
early necrosis, dead, and live cells is urgently needed.
Herein, we constructed a novel fluorescent probe ACD-E
based on the hybridization of chloro-hydroxy dihydronaph-
thalene and merocyanine dyes, which can distinguish three
kinds of cell statuses in different fluorescence signal modes.
Received: July 10, 2021
Accepted: August 18, 2021

© XXXX American Chemical Society https://doi.org/10.1021/acs.analchem.1c02911

A Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Scheme 1. Rational Design Strategy for Probe ACD-E
Hence, ACD-E can distinguish live cells from dead cells in a
dual-color mode and differentiate early necrosis cells from live
cells in a fluorescence signal “turn-on” mode. Of particular
importance is that ACD-E is capable of monitoring cell
viability in cells and zebrafish. On this account, ACD-E can
accurately evaluate multiple cell states as a unique molecular
tool.
■ EXPERIMENTAL SECTION
Materials and Instruments. The reagents or materials
used in the experiment are obtained from commercial
suppliers, and no further purification is required. Unless
otherwise specified, double-distilled water is used during the
entire analysis experiment.
Cell Culture and Cytotoxicity Assays. HeLa cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) in an
atmosphere of 5% CO2 and 95% air at 37 °C. In vitro
cytotoxicity was measured using the CCK-8 assay on HeLa
cells. Cells were seeded into a 96-well tissue culture plate in the
presence of completed DMEM supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C
and in a 5% CO2 atmosphere overnight followed by incubation
for 24 h in the presence of ACD-E and CHD-V at different
concentrations (2.5, 5, 10, 15, 20, 25, 40, 50 μM). A
commercial cell counting kit-8 (CCK-8) (TransGen Bio-
technology, China) was used to detect the cell viability, and
the assay was run following the manufacturer’s instructions.
The cell viability was determined by assuming 100% cell
viability for cells without ACD-E and CHD-V.
Synthesis of Probes ACD-E and CHD-V. The specific
synthetic route of probes is described in Figure S1. Product C
(41.6 mg, 0.2 mmol) and 60 mg of iodolium salt (0.2 mmol)
were dissolved in N-butyl alcohol/toluene (10 mL, 7:3, v/v),
and then the solution was stirred at 90 °C for 5 h in a three-
neck flask with a Dean−Stark trap. The solvent was removed in
vacuo. The brown product ACD-E was purified by column
chromatography on silica gel with a gradient elution
(CH3OH/CH2Cl2 from 1/50 to 1/20, v/v). 1H NMR (500
MHz, DMSO-d6) δ 8.12 (d, J = 13.4 Hz, 1H); 7.93 (d, J = 8.2
B https://doi.org/10.1021/acs.analchem.1c02911
pubs.acs.org/ac Article
Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H); 7.23 (t, J = 7.7 Hz, 1H);
7.11 (d, J = 9.4 Hz, 2H), 6.95 (t, J = 8.6 Hz, 2H); 5.66 (d, J =
13.4 Hz, 1H); 3.87 (q, J = 6.9 Hz, 2H); 2.93 (t, J = 6.4 Hz,
2H); 2.83 (t, J = 6.4 Hz, 2H); 2.30 (s, 3H); 1.59 (s, 6H); 1.18
(t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 183.84,
169.41, 163.92, 153.58, 145.14, 143.54, 139.55, 134.86, 132.58,
130.10, 129.01, 128.41, 123.15, 122.35, 121.53, 121.41, 120.71,
107.97, 92.32, 46.75, 37.02, 28.70, 28.23, 24.08, 21.37, 11.56.
HRMS (ESI): m/z calculated for C26H27ClNO2, 420.1733 [M
+ H]+, found: 420.1725.
Synthesis of the Probe CHD-V. CHD-V was synthesized
according to the procedure of used to produce ACD-E. 1H
NMR (500 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.01 (d, J = 13.3
Hz, 1H); 7.77 (d, J = 8.5 Hz, 1H); 7.33 (d, J = 7.2 Hz, 1H);
7.20 (t, J = 7.7 Hz, 1H); 6.95−6.85 (m, 2H); 6.73 (dd, J = 8.5,
2.3 Hz, 1H); 6.66 (d, J = 2.2 Hz, 1H); 5.60 (d, J = 13.3 Hz,
1H); 3.89−3.73 (m, 2H); 2.80 (dd, J = 19.6, 6.7 Hz, 4H); 1.57
(s, 6H); 1.16 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz,
DMSO-d6) δ 184.04, 162.66, 161.52, 145.83, 143.71, 139.42,
133.15, 129.90, 128.35, 126.87, 124.07, 122.29, 121.03, 114.58,
114.28, 107.65, 92.09, 46.50, 36.89, 30.16, 28.66, 28.63, 24.37,
11.50, 10.88. HRMS (ESI): m/z calculated for C24H25ClNO,
378.1625 [M + H]+, found: 378.1638.
■
RESULTS AND DISCUSSION
Design and Synthesis of the Probe ACD-E. In our
work, to distinguish three kinds of cell statuses, we conceived a
new fluorescent probe ACD-E based on two kinds of
fluorescence signal modes by mitochondrial esterase and
viscosity detection. As shown in Scheme 1, ACD-E was
designed with a hemicyanine moiety as the electron acceptor,
4-chloro-1,2-dihydronaphthalene as the conjugated link
structure, and an acetoxy group for esterase recognition.28,29
It could discriminate living and dead cells by monitoring the
activity of esterase with near-infrared resolution. In living cells,
the acetoxy group of ACD-E would be hydrolyzed to a strong
electron-donating hydroxyl group to transform into CHD-V,
forming a “D−π−A” molecular configuration with the hydroxyl
group. With the change of electron-donating ability, CHD-V,
as the molecular rotor, would increase the intramolecular
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry pubs.acs.org/ac Article

Figure 1. (a) Absorption spectral changes of the probe ACD-E. Inset: color change before and after treatment with esterase. (b) Fluorescence
spectral changes of the probe ACD-E. (c) Fluorescence spectral changes of the probe ACD-E with 0−0.1 U/mL esterase at 37 °C for 60 min. (d)
Linear relationship between I692 of the probe ACD-E and the concentration of esterase. All measurements were conducted in an aqueous solution
(10 mM PBS with 5% EtOH cosolvent, pH 7.4). λex = 630 nm. Slit width: 10 nm/10 nm.

Figure 2. (a) Absorption spectral changes of CHD-V in the methanol (orange line) and glycerol (blue line, 90% v/v). (b) Fluorescence spectra of
CHD-V (10 μM) change with the increase of glycerol ratio. (c) Linear relationship between log I716 and log η. (d) Fluorescence lifetime of CHD-V.

charge transfer (ICT) effect of molecules in living cells,
resulting in a red shift. However, in dead cells, ACD-E would
not be hydrolyzed to CHD-V with inactivated esterase.
Besides, CHD-V would be used to detect the changes of
mitochondrial viscosity in living cells based on the twisted
intramolecular charge transfer (TICT) progress. In early
necrosis cells, CHD-V could exhibit stronger fluorescence
than healthy cells due to the increased mitochondrial viscosity
by producing inflammation. Furthermore, it is well known that
the positively charged hemicyanine can accumulate in
mitochondria rapidly and is widely used as a target structure
of mitochondria.30,31 Therefore, the probe could target
mitochondria in a short time. The probe compound was
synthesized following the synthetic routine, as shown in Figure
S1.
Optical Properties of the Probe ACD-E Response to
Esterase and Optical Properties of the Probe CHD-V
C https://doi.org/10.1021/acs.analchem.1c02911
Response to Viscosity. First of all, the optical properties of
ACD-E responses to esterase were first evaluated in an
aqueous solution buffered (EtOH/PBS, 1:20, v/v) at
physiological pH. As shown in Figure 1a, ACD-E displayed a
main maximum absorption peak at 441 nm. After the
treatment of esterase (0.1 U/mL), the absorption at 441 nm
decreased and new peaks at 489 and 630 nm emerged
obviously. Meanwhile, the solution changed from yellow to
light brown, and the colorimetric detection of esterase could be
carried out with the naked eye (inset of Figure 1a). Besides,
the fluorescence spectrum of ACD-E exhibited a new emission
at 692 nm (Figure 1b). To test the quantitative analysis ability
of ACD-E toward esterase, we incubated ACD-E (10 μM)
under varying concentrations of esterase. As shown in Figure
1c, as the concentration of esterase increased, the fluorescence
intensity at about 692 nm increased conspicuously. In the
concentration range of 0−0.1 U/mL, the fluorescence intensity
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry pubs.acs.org/ac Article

Figure 3. (a) Fluorescence responses of ACD-E (10 μM) treated with 0.1 U/mL esterase at 37 °C for 60 min and then treated with glutathione
(GSH) (10 mM) and other various species (200 μM): 1, probe + esterase; 2, blank; 3, cellulase; 4, lysozyme; 5, pepsin; 6, α-D-glucose; 7, trypsin;
8, Cys; 9, GSH; 10, Hcy; 11, Cu2+; 12, ClO−; 13, HS−; 14, HSO3−; 15, S2O32−; 16, SCN−; 17, DNA; and 18, RNA. (b) Fluorescence responses of
CHD-V (10 μM) at λem = 716 nm in glycerol solution and other representative bioactive molecules in PBS buffer: 1, glycerol; 2, blank; 3, cellulase;
4, lysozyme; 5, pepsin; 6, α-D-glucose; 7, trypsin; 8, Cys; 9, GSH; 10, Hcy; 11, Cu2+; 12, ClO−; 13, HS−; 14, HSO3−; 15, S2O32−; 16, SCN−; 17,
DNA; and 18, RNA. (c) Effects of pH on the probe ACD-E and when reacted with 0.1 U/mL esterase. (d) Effects of pH on the probe CHD-V.

has a good linear relationship with esterase (Figure 1d). The
detection limit (3δ/k) is calculated to be 3.12 × 10−4 U/mL.
These results indicate that ACD-E can be used as a dual-color
fluorescent probe for quantitative analysis of esterase. In
addition, in the presence of esterase (1 U/mL), the
fluorescence response is significantly enhanced at pH 7.4 and
37 °C under physiological conditions (Figure 4C). Therefore,
the reaction of ACD-E with esterase can proceed well under
physiological conditions.
After that, viscosity-sensitive responses of the probe CHD-V
were evaluated in a mixture solution that contained EtOH and
glycerol with different volume ratios (10−90%). The viscosity
of the solution was increased from 0.56 cp of pure ethanol to
355 cp of 90% glycerol by adjusting the volume ratio of
glycerol to EtOH. As shown in Figure 2a, the absorption
spectra of probe CHD-V with EtOH and glycerol showed an
absorption peak at 674 nm in pure ethanol and the
fluorescence spectra of the probe CHD-V showed only a
weak fluorescence emission peak at 702 nm with excitation at
630 nm in pure ethanol. However, when the solvent was
changed to glycerol, CHD-V had an absorption peak at 676
nm and a fluorescence peak at 716 nm. When the increase of
glycerol ratio in the solvent from 10% to 90% (Figure 3b), the
fluorescence intensity was increased by about 10 times, and the
quantum yield was also significantly increased to 0.56 (Figure
S2). Besides, the fluorescence emission of the probe CHD-V
had a good linear relationship that exists (R2= 0.9950) between
log I716 and log η by the Förster−Hoffmann equation (Figure
2c). Furthermore, we measured the sensitivity of the probe by
the fluorescence lifetime in the EtOH and glycerol system and
showed that the fluorescence lifetime increased significantly
from water to glycerol (Figure 2d). This can indicate that the
nonradiation decay rate decreased with the increase of
viscosity, hence the fluorescence lifetime of CHD-V was
prolonged with the increase of viscosity, and the change of
fluorescence lifetime was positively correlated with the limited
D https://doi.org/10.1021/acs.analchem.1c02911
rotation of the CHD-V molecule. In summary, all of these
results indicated that CHD-V can be used as an excellent
fluorescent probe for detecting changes in viscosity.
Selectivity and pH Effects of Probes ACD-E and CHD-
V. To investigate probe stability, probes ACD-E and CHD-V
were tested upon addition of 10 μM and recorded for 30 min
(Figure S3). There was no obvious fluorescence fluctuation,
manifesting that the probe has good stability under the test
conditions. To demonstrate the selectivity of the probe to
esterase, the probe was incubated with various biological
substances, including common enzymes, common amino acids,
reactive oxygen species (ROS), and common anions (Figure
3a). Except for esterase, the other analytes had no obvious
fluorescence changes after incubation with the probe,
indicating that ACD-E has higher selectivity for esterase than
others. Moreover, we considered the influence of the selectivity
of the probe and the pH of the environment on the probe. As
shown in Figure 3c, the fluorescence of probe ACD-E had no
change by the pH titration. In addition, with the treatment of
esterase (1 U/mL), the fluorescence response was obviously
improved. To verify the viscosity-specific detection of CHD-V,
the fluorescence spectrum of CHD-V after coincubation with
some potential competing species was studied. As shown in
Figure 3b, compared with the other analytes, only glycerin can
induce significant fluorescence changes. Based on the above
results, it is confirmed that the fluorescence of the probe is not
affected by other biologically related substances. Then, we
designed an experiment to investigate the effect of pH on the
probe CHD-V. The pH titration showed that the fluorescence
of the probe CHD-V did not change significantly in the range
of pH 3.5−8 and that of the CHD-V probe increased slightly at
pH 7−8 (Figure 3d). All of these results indicated that the
CHD-V probe has an excellent specificity for detection of
viscosity in a complex biological environment.
Theoretical Calculation and Mechanism of CHD-V. To
further understand the influence of viscosity on CHD-V and
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
ACD-E theoretically, we first optimized CHD-V and ACD-E
with two kinds of structures and adopted density functional
theory (DFT)/time-dependent density functional theory
(TDDFT) with B3LYP/6-31G(d,p) using Gaussian 09
(Figures 4A and S4). In one kind of structure of CHD-V,
Figure 4. (A) Optimized geometries of CHD-V in the excited state.
(B) Frontier molecular orbital of CHD-V in the excited state with a
dihedral angle of 0° at around C12−C13−C14−N18. (C) Frontier
molecular orbital of CHD-V in the excited state with a dihedral angle
of 90° at around C12−C13−C14−N18. Calculations were performed
by the DFT method (polarizable continuum model, PCM model)
with a B3LYP/6-31G(d,p) basis set using Gaussian 09.
the dihedral angle of CHD-V at around C12−C13−C14−N18
is 0° in glycerol, which is a large conjugated system. Hence, the
molecular structure of the probe CHD-V is in the optimal
state, and the oscillator strength fem is 7.7732 (Figure 4B).
Furthermore, the frontier molecular orbitals (highest occupied
molecular orbital, HOMO and lowest unoccupied molecular
orbital, LUMO) of CHD-V are distributed over the whole
molecular structure, indicating that this structure state
corresponds to the activated ICT process. As shown in Figure
4C, the frontier molecular orbitals of CHD-V showed that the
dihedral angle at around C12−C13−C14−N18 is 90° in
Figure 5. (a−c) Confocal fluorescence images of the HeLa cells after incubation with 10 μM probe ACD-E at 37 °C for 30 min and 100 nM
commercial organelle trackers (MitoTracker Red) (10 μM) at 37 °C for 10 min. (a) Fluorescence images of the HeLa cells after incubation with 10
μM probe ACD-E. (b) Fluorescence images of 100 nM commercial organelle trackers (MitoTracker Red). (c) Merged image. (d) Intensity
correlation plot. (e) Intensity profile of ROI lines. Scale bar = 10 μm. Orange channel, 590−620 nm (λex = 561 nm); red channel, 690−780 nm (λex
= 630 nm).
E https://doi.org/10.1021/acs.analchem.1c02911
pubs.acs.org/ac Article
methanol. With both HOMO and LUMO centered at the 5-
chloro-7,8-dihydronaphthalen-2-ol moiety of CHD-V, the
molecule is in a twisted internal charge transfer (TICT)
state, and the oscillator strength fem is only 0.8052 (Figure
4C). The calculation results show that when glycerol is in the
conjugate plane and the ground state is excited to the excited
state, CHD-V of the probe undergoes a strong ICT process,
which leads to strong fluorescence emission. However, the
probe CHD-V molecule can rotate freely in ethanol, forming a
distorted excited state through intramolecular rotation,
resulting in weak fluorescence emission. Theoretical calcu-
lations show that the probe CHD-V can be used as a sensitive
probe for detecting changes in solvent viscosity.
Localization Experiment of the Probe ACD-E. Through
the spectrum titration experiment, we proved that probes
ACD-E and CHD-V have an excellent linear relationship with
the increase of esterase and viscosity. Therefore, to further
explore the application of probes in living systems, a
cytotoxicity experiment was constructed. As is shown in
Figure S4, probe ACD-E had low cytotoxicity to HeLa living
cells and cell viability over 90% after 24 h of culture with
different concentrations of probes. In live cells, the probe
ACD-E can be converted into a viscosity-responsive probe
CHD-V by activated esterase and indole salts can target
mitochondria. Therefore, we performed colocalization experi-
ments using commercial organelle trackers (MitoTracker Red)
to verify mitochondrial imaging selectivity of the probe CHD-
V in living HeLa cells. According to Figure 5, the Pearson’s
colocalization coefficient (Rr) was 0.9303, which clearly
indicated that mitochondrial fluorescence signals in the orange
channel with MitoTracker Red exhibited excellent correlative
fluorescence signals in the red channel with probe CHD-V.
The results indicated that the probe CHD-V was predom-
inantly located in the mitochondria.
Imaging of Early Necrosis, Live, and Dead Cells with
Two Kinds of Fluorescence Signal Modes. To verify the
ability of the ACD-E probe to distinguish live cells from dead
cells, the live cells were pretreated with 4% paraformaldehyde
for 30 min to obtain dead cells and then incubated with
probes.32 In dead cells, there was no obvious fluorescence in
the red channel and only yellow channel fluorescence was
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry pubs.acs.org/ac Article

Figure 6. Confocal fluorescence microscopy analysis for detection of viscosity in HeLa cells. (A) Images of live HeLa cells treated with 5 μM ACD-
E for 30 min. (B) Images of live HeLa cells treated with ACD-E for 30 min and incubated with 10 μM LPS for 30 min. (C) Images of HeLa cells
then pretreated with 4% paraformaldehyde for 30 min and incubated with 5 μM ACD-E for 30 min. (D) Quantification of data in (c/g), statistical
analyses were performed with a Student’s t-test. **p < 0.001. Yellow channel, 520−620 nm (λex = 470 nm); red channel, 680−780 nm (λex = 630
nm); scale bar = 20 μm.

produced (Figure 6C), indicating that ACD-E could not be

hydrolyzed to CHD-V by the inactivated esterase in dead cells.
However, weak fluorescence was observed in the near-infrared
region, when ACD-E was incubated with live cells for 30 min,
suggesting that ACD-E could be hydrolyzed to CHD-V by
activated esterase in live cells (Figure 6A). Therefore, ACD-E
can actually distinguish live and dead cells in a dual-color
mode. In addition, in early necrosis cells, the probe ACD-E
was further incubated with 10 μM lipopolysaccharide (LPS)
for 30 min, observing that the red channel had fluorescence
obviously increased by 2-fold approximately (Figure 6C),
illustrating that CHD-V can be used as a sensitive probe for
detecting changes in cell viscosity (Figure 6B) to distinguish
live and early necrosis cells in turn-on mode. Taken together,
ACD-E can be used as a cell-specific probe for identifying early
necrosis, dead, and live cells by imaging live cell esterase and
detecting viscosity. Figure 7. (A) Confocal fluorescence images of the zebrafish incubated
Imaging of ACD-E in Early Necrosis, Live, and Dead with 10 μM free probe ACD-E for 1 h. (B) Confocal fluorescence
images of the zebrafish incubated with 10 μM ACD-E for 1 h and 10
Zebrafish. We have proved that ACD-E can discriminate μM LPS for another 30 min. (C) Confocal fluorescence images of the
early necrosis, dead, and live cells. Thus, we further studied dead zebrafish incubated with 10 μM ACD-E for 1 h. Yellow channel,
imaging of ACD-E (10 μM) in living zebrafish. Zebrafish were 520−620 nm (λex = 470 nm); red channel, 680−780 nm (λex = 630
incubated with ACD-E (10 μM) for 1 h and further imaged nm).
with the fluorescence imaging for discriminating early necrosis,
live and dead zebrafish. When the probe ACD-E was incubated
for 1 h, showing weak fluorescence in the red channel (Figure by activated esterase to afford a deep red fluorescence peaked
7A). It was more intuitive to find that the probe exhibited at 692 nm. However, in dead cells, the probe has no change
higher fluorescence intensities in the red channel when the with inactivated esterase, showing yellow-color emission
zebrafish were stained with ACD-E (10 μM) for 1 h and then peaked at 574 nm. In the early necrosis cells, the fluorescence
incubated with 10 μM LPS for 30 min (Figure 7B). However, in the near-infrared region of the mitochondria was
ACD-E only displays fluorescence in the yellow channel in the significantly enhanced under the stimulation of LPS, thereby
dead zebrafish by paraformaldehyde (Figure 7C). These distinguishing the cells in early necrosis from the healthy cells
phenomena indicated that ACD-E was consistent with the in turn-on mode. Compared with the fluorescent probes that
observations of cells and could be suitable for imaging esterase can only distinguish live and dead cells, ACD-E can more
and viscosity of live cells in vivo to discriminate three kinds of accurately determine the state of cells and bring more accurate
states of zebrafish.

■
CONCLUSIONS
In summary, we have developed an unparalleled mitochondria-
targetable probe ACD-E for discriminating early necrosis,
dead, and live cells with two kinds of fluorescence signal modes
for the first time. The probe ACD-E can discriminate live and
dead cells based on a dual-color mode. In live cells, the acetate
group as the recognition site, ACD-E can convert to CHD-V
F https://doi.org/10.1021/acs.analchem.1c02911
information for biological experiments. In view of the above
characteristics, ACD-E can be further applied to zebrafish. In
summary, the characteristics of this probe provide a potential
powerful tool for clinical biological experiments.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.1c02911.
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Experimental details; additional UV−vis absorption; and
fluorescence data and imaging (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Weiying Lin − Guangxi Key Laboratory of Electrochemical
Energy Materials, Institute of Optical Materials and
Chemical Biology, School of Chemistry and Chemical
Engineering, Guangxi University, Nanning, Guangxi 530004,
P. R. China; orcid.org/0000-0001-8080-4102;
Email: weiyinglin2013@163.com; Fax: 0000-0001-8080-
4102
Authors
Youbo Lai − Guangxi Key Laboratory of Electrochemical
Energy Materials, Institute of Optical Materials and
Chemical Biology, School of Chemistry and Chemical
Engineering, Guangxi University, Nanning, Guangxi
530004, P. R. China; orcid.org/0000-0001-8821-0405
Tengteng Zhang − Guangxi Key Laboratory of
Electrochemical Energy Materials, Institute of Optical
Materials and Chemical Biology, School of Chemistry and
Chemical Engineering, Guangxi University, Nanning,
Guangxi 530004, P. R. China
Wenhui Song − Guangxi Key Laboratory of Electrochemical
Energy Materials, Institute of Optical Materials and
Chemical Biology, School of Chemistry and Chemical
Engineering, Guangxi University, Nanning, Guangxi
530004, P. R. China
Zihong Li − Guangxi Key Laboratory of Electrochemical
Energy Materials, Institute of Optical Materials and
Chemical Biology, School of Chemistry and Chemical
Engineering, Guangxi University, Nanning, Guangxi
530004, P. R. China; orcid.org/0000-0002-3086-1675
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.1c02911
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was financially supported by the National Natural
Science Foundation of China (21672083, 21877048, and
22077048), the Guangxi Natural Science Foundation
(2021GXNSFDA075003), and the startup fund of Guangxi
University (A3040051003).
■
REFERENCES
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