Engineering a double-rotor-based fluorescent molecule to sensitively track mitochondrial viscosity in living cells and zebrafish with high signal-to-background ratio (S/B)

Article abstract of DOI:10.1016/j.jphotochem.2020.112789

An innovative design strategy has been proposed to construct viscosity-sensitive fluorescent probes with superior sensitivity and high signal-to-background ratio (S/B) based on double-rotor molecular platform. As a proof-of-concept, a double-rotor-based f

Full text of DOI:10.1016/j.jphotochem.2020.112789

Journal Pre-proof
Engineering a double-rotor-based fluorescent molecule to sensitively
track mitochondrial viscosity in living cells and zebrafish with high
signal-to-background ratio (S/B)
Yunzhen Yang, Longwei He, Weiying Lin
PII:
S1010-6030(20)30588-8
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112789
JPC 112789
Reference:
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Journal of Photochemistry & Photobiology, A: Chemistry
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Accepted Date:
10 February 2020
12 July 2020
16 July 2020
Please cite this article as: Yang Y, He L, Lin W, Engineering a double-rotor-based fluorescent
molecule to sensitively track mitochondrial viscosity in living cells and zebrafish with high
signal-to-background ratio (S/B), Journal of Photochemistry and amp; Photobiology, A:
Chemistry (2020), doi: https://doi.org/10.1016/j.jphotochem.2020.112789
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Engineering a double-rotor-based fluorescent molecule to sensitively
track mitochondrial viscosity in living cells and zebrafish with high
signal-to-background ratio (S/B)
Yunzhen Yang, Longwei He and Weiying Lin*
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical
Engineering, School of Materials Science and Engineering, University of Jinan, Shandong 250022,
P.R. China.
E-mail: weiyinglin2013@163.com
Highlights

An innovative design strategy has been proposed to construct
viscosity-sensitive fluorescent probes (DRVPs) with superior sensitivity and
high signal-to-background ratio (S/B) based on double-rotor molecular
platform. DRVPs is more sensitive toward viscosity and performs higher S/B
ratio comparing with the traditional single-rotor-based viscosity probes
(SRVPs)


To carry out a proof-of-concept project, the probe DRVP containing two rotors,
quinolinium and carbazole moieties, was developed using a strong
intramolecular charge transfer (ICT) platform. DRVP gave rise to an obvious
fluorescence enhancement (51.8-fold) in 99% v/v glycerol solution.
The cationic probe DRVP could clearly stain the mitochondria in HepG2 cells,
and it can be successfully applied for the imaging of exogenous viscosity in
living cells and zebrafish.
1

Abstract
An innovative design strategy has been proposed to construct viscosity-sensitive
fluorescent probes with superior sensitivity and high signal-to-background ratio (S/B)
based on double-rotor molecular platform. As
a
proof-of-concept,
a
double-rotor-based fluorescent molecule (DRVP) containing two rotors, quinolinium
and carbazole moieties, was developed to track viscosity using a strong ICT platform.
The cationic probe DRVP is able to visualize the mitochondrial viscosity variations in
living cells and zebrafish.
Keywords
Fluorescent probe, High signal-to-background ratio, Viscosity, Mitochondria-targeted,
Fluorescence imaging;
2

1
. Introduction
Viscosity is an essential micro environment parameter in various
biological systems, directly affecting transmission of eukaryotic cells chemical
signals [1-5]. Changes of cell viscosity are related with some disease and
dysfunction, such as hypertension, diabetes, cancers and so on [6-10]. Detection
of intracellular viscosity remains one of the challenges in chemical biology.
Mitochondria, as an important organelle, exist in most eukaryotic cells, which
provide energy for the cells and are energy-generating compartments in cells
[
11-16]. Researches have shown that abnormal mitochondrial viscosity levels
lead to the loss of mitochondrial activation, triggering numerous diseases
including cerebral embolism, atherosclearosis, Alzheimer's disease (AD) and
diabetes [17-28]. To this end, it is vital to develop effective strategy of
monitoring the mitochondrial viscosity in biological systems for better
understanding the mechanism of physiological and pathological functions in
mitochondria.
Several analytical methods, such as chemiluminescence, capillary
electrophoresis, spectrophotometry and electrochemical, have been developed
to detect viscosity. However, the above methods fail to meet the measure of in
situ viscosity in the biological systems [29-31]. Till now, fluorescence analysis
technology based probes remains the mainstream tool for imaging of viscosity
in biological and pathology due to its high sensitivity, real-time detection, fast
response, low detection limit, and good biocompatibility [32-34]. Therefore,
fluorescent probes with excellent response characteristics, particularly high
sensitivity and S/B ratio, were proposed to be key for monitoring viscosity. It’s
well known that molecular rotor is sensitive to viscosity variations. The free
rotation of rotor moiety is one kind of non-radiation transition processes, which
would relax the excitation energy and induce the fluorescence quenching of the
molecular rotors. From this we speculated that the molecules containing
multiple rotor groups would be of avail to achieve the low emission in the
non-viscous media, and accordingly perform superior signal-to-background
ratio (S/B) in the viscous situations. Although viscosity-sensitive fluorescent
probes based on various molecular rotors have been developed, most of them
only possess single rotor and sense viscosity with low signal-to-background
ratio [35-41]. Dimer rotors have been reported, however, there is no evidence
3

that double-rotor-based fluorescent probe performs higher S/B ratio toward
viscosity, comparing with single-rotor-based viscosity probes [42]. In this work,
an innovative design strategy was proposed for constructing a fluorescent
molecular containing two rotors with high signal-to-background ratio (S/B)
toward viscosity. Notably, comparing with the traditional single-rotor-based
viscosity probes (SRVPs) consisting of single rotor and chromophore core, the
new type of double-rotor viscosity probes (DRVPs) perform the more advanced
sensitivity and S/B through alleviation of adverse interference from the
background emission signal (the emission from rotors in the non-viscous
medium). In non-viscous situation, the low fluorescence of DRVPs should be
attributed to the distortion of rotor moiety to form a non-planar platform with
the inefficient conjugation effect. However, DRVPs show strong fluorescence
in highly viscous systems because the rotation of rotor could be suppressed by
the high viscosity media.
To perform a proof-of-concept project based on this new design strategy, we
established a double-rotor-based fluorescent probe with superior S/B ratio for
monitoring viscosity in biological systems. The quinolinium moiety and
carbazole moiety were selected as two rotors, meanwhile serving as the
electron-withdrawing group and electron-donating group, respectively, which
were combined by the benzene ring building a strong intramolecular charge
transfer (ICT) platform, as well as a double-rotor-based viscosity probe
(
(
DRVP, Scheme 1B). Compared with control fluorescent viscosity probe
SRVP) based on single-rotor molecular, the probe DRVP showed a more
remarkable enhancement (51.8-fold) of the response signal induced by the
increasing viscosity. In addition, the cationic skeleton of the probe DRVP
should be an appropriate mitochondrial targeting group, which can promote the
penetration of probe into the mitochondria through the plasma membrane. The
desirable performances of turn-on response fluorescence signal were also
observed
in
the
confocal
fluorescence
images
of
living
monensin/nystatin-mediated HepG2 cells and zebrafish. Thus, the probe DRVP
is able to monitor mitochondrial viscosity variations with high sensitivity and
S/B ratio in biological systems.
4

Scheme 1. The design strategy of double-rotor-based fluorescent probes with high sensitivity and
S/B ratio toward viscosity. (A) The diagram and limitations of the traditional fluorescent viscosity
probes (SRVPs) based on the molecular rotor containing single rotor group; (B) The diagram and
advanced features of the new type of fluorescent viscosity probes (DRVPs) based on the molecular
rotor containing dual rotor groups; (C) The chemical structures of the single-rotor-based viscosity
probe (SRVP) and the double-rotors-based viscosity probe (DRVP) presented in this work.
2
. Experimental
2
.1. Synthesis of compound SRVP
Compound 1 (30 mg, 0.2 mmol), compound 3 (57 mg, 0.2 mmol), were dissolved in
ethanol (10 ml), piperidine (100 μL) and glacial acetic acid (100 μL) was added. Then,
the mixture was refluxed at 80℃ for 12 hours. After the reaction, cooling and
filtration, it was washed twice with ethanol to give a purple solid (67 mg, yield
8
0.5%), which was used in characterization and further reaction without purification.
1
H NMR (400 MHz, DMSO),  (ppm): 3.074 (s, 6H), 4.447 (s, 3H), 6.815-6.837 (d, J
4.4 Hz, 2H), 7.864-7.886 (d, J = 4.4 Hz, 2H), 7.960-8.035 (m, 2H), 8.168-8.230 (dd,
= 3.4 Hz, J = 12.4 Hz, 2H), 8.333-8.358 (m, 2H), 9.025-9.046 (d, J = 4.2 Hz, 1H),
.114-9.131 (d, J = 3.4 Hz, 1H); C NMR (100 MHz, DMSO): δ (ppm): 39.386,
9.595, 39.804, 40.012 40.219, 40.430, 40.638, 44.493, 112.368, 113.586, 114.415,
19.506, 123.527, 126.220, 126.813, 129.012, 131.251, 135.071, 139.251, 145.150,
=
J
1
2
1
3
9
3
1
1
+
+
47.241, 152.766, 153.628. HRMS (ESI) m/z calcd for C20
H
21
N
2
[M] : 289.1699.
Found 289.1696.
5

2
.2 Synthesis of the probe DRVP
Compound 3 (54.2 mg, 0.2 mmol), compound 6 (57 mg, 0.2 mmol), were dissolved in
ethanol (10 ml), piperidine (100 μL) and glacial acetic acid (100 μL) was added. Then,
the mixture was refluxed at 80℃ for 12 hours. After the reaction, the crude product
was obtained by distillation under reduced pressure. The crude product was purified
by column chromatography on silica gel using CH
2
Cl /MeOH (v/v 15:1) as eluent,
2
then the collection solution of compound DRVP was concentrated under reduced
1
pressure to afford a red solid (82 mg, yield 76.2%). H NMR (400 MHz, DMSO), 
(ppm): 4.992 (s, 3H), 7.319-7.353 (t, J = 6.8 Hz, 2H), 7.465-7.528 (q, J = 12.6 Hz,
4
H), 7.826-7.847 (d, J = 4.2 Hz, 2H), 8.066-8.104 (t, J = 3.8 Hz 1H), 8.270-8.341 (m,
H), 8.466-8.499 (m, 2H), 8.570-8.586 (d, J = 3.2 Hz, 1H), 9.106-9.127 (d, J = 4.2 Hz,
6
1
3
1
H), 9.410-9.426 (d, J = 3.2 Hz, 1H); C NMR (100 MHz, DMSO): δ (ppm): 39.391,
9.600, 39.809, 40.017, 40.226, 40.435, 40.643, 45.300, 110.257, 116.997, 119.892,
20.927, 121.094, 123.489, 126.895, 126.989, 127.299, 129.835, 131.042, 134.925,
35.525, 139.140, 139.233, 140.211, 142.233, 148.698, 152.871. HRMS (ESI) m/z
3
1
1
+
+
calcd for C30
H
23
N [M] : 411.1856. Found 411.1858.
2
2
.3. Fluorescence imaging in living cells
The monensin or nystatin (10 μM) was first added into the HepG2 cells in a glass
bottom culture dishes (Nest) for 30 min, then they were washed by PBS for three
times, after that, the probe DRVP (10 μM) was added into the HepG2 cells. The
fluorescence emission of the probe was collected at TRICT channel (570 nm-620 nm),
the excitation wavelength was 561 nm. monensin and nystatin could induce structural
changes or swelling, leading to viscosity changes in the mitochondria.
2
.4. Fluorescence imaging in zebrafish
The 10 μM monensin or nystatin was added into the zebrafish for incubated for 30
min, followed by washing away gently. Then the probe DRVP (10 μM) was put into
dishes for another 30 min. After that, the zebrafish was transferred into new glass
bottom dishes for imaging. The fluorescence emission was collected at TRICT
channel (570 nm-620 nm) upon excitation at 561 nm.
3
Results and discussion
6

3
.1. Design and synthesis of dyad DRVP and SRVP.
In order to construct a double-rotor-based fluorescent probe with high S/B
ratio, the quinolinium and carbazole moieties were selected as the two rotors,
quinolinium as well as mitochondria-targeted group, binding these two rotors
via the benzene ring to constitute the targeted double-rotor-based viscosity
probe (DRVP). To prove the superior response properties of probe DRVP
towards viscosity, we also synthesized a single-rotor-based viscosity probe
(
SRVP, Scheme 1C), which contains only one quinolinium rotor. The
compound SRVP and the target probe DRVP was successfully prepared
Scheme 2). The compounds and were prepared by the previous methods
43-44]. The control probe SRVP and target probe DRVP were simply
synthesized via the condensation reaction between the compound and
compound or , respectively. Characterization of all unknown samples was
(
3
6
[
3
1
6
obtained by NMR spectroscopy and mass spectrometry.
Scheme 2. Synthesis route of the control probe SRVP and the probe DRVP.
3
.2. The optical fluorescence properties of DRVP and SRVP towards viscosity
With the control probe SRVP and the target probe DRVP in the hands, the
optical response capability of SRVP and DRVP to the viscosity was evaluated
by emission spectra. Firstly, in order to test the influence of dimethylamino
moiety rotation on emission, we selected a rigidfluorescent quinolone dye with
a dimethylamino group (dimethylaminoquinoline-6-amine, TQA (Scheme S1))
for spectral verification. As the viscosity increases, the TQA showed a slight
fluorescence enhancement (2.09-fold) in emission spectra (Fig S1), which
should be contributed to the suppression of twisted intramolecular charge
7

transfer (TICT) effect due to the inhibiting rotation of dimethylamino group in
viscous media. Thus the quinolinium moiety should play a primary role of
rotation on compound SRVP rather than the dimethylamino group with small
weight. Then the emission spectra of SRVP and DRVP were also measured in
MeOH and glycerol. As shown in Fig. 1, both SRVP and DRVP were initially
almost non-fluorescent in methanol, but their emission significantly enhanced
with increase of the glycerol proportion in the methanol-glycerol mixture (Fig.
2
and S2). However, due to the much lower background fluorescence, probe
DRVP gave rise to a more obvious fluorescence enhancement at 640 nm
51.8-fold) than that of the reference SRVP (34.7-fold) at 680 nm in 99% v/v
(
glycerol solution. Meanwhile, 30 μM of probes DRVP and SRVP are capable
for detecting the minimum 1.29 and 1.75 cp of viscosity, respectively,
according to the linear relationship between viscosity values (η) of testing
media and response fluorescence intensity (Fig. 1C and 1D). In addition, a good
2
linear relationship between log I640 and log ƞ (R =0.9919) was obtained by the
Förster-Hoffmann equation (logI = C + xlog ƞ) [45], and the value of x (0.6154
and 0.5934), manifested that DRVP and SRVP might be used to quantitatively
monitor viscosity changes in vitro (the inset in Fig. 1C and 1D).Collectively,
those results indicated that DRVP is more sensitive toward viscosity and
performs higher S/B ratio comparing with the control probe SRVP, hence the
double-rotor-based viscosity probe (DRVP) is likely favourable in application
of sensing viscosity.
8

Fig. 1 (A) The emission spectra of SRVP (30 μM) in the MeOH (black) and MeOH-glycerol
mixture (v/v 1/99, red) with excitation at 550 nm. (B) The emission spectra of DRVP (30 μM) in
the MeOH (black) and glycerol (red) with excitation at 450 nm. The linear relationship between of
fluorescence intensity and the viscosity values (η) for SRVP (C) and DRVP (D). Inset: the linear
2
2
relationship between log I640 and log η for SRVP (R = 0.9921, K = 059348) and DRVP (R =
.9919, K = 0.6154).
0
3
.3. Optical response of DRVP to viscosity
After the initial photophysical characterisation for DRVP and SRVP towards
viscosity, then we studied the response performance of probe DRVP (30 μM)
to viscosity owing to its superior sensitivity and S/B ratio. As shown in Fig. 2A,
DRVP displayed feeble emission at 640 nm, which steadily increased with the
addition glycerol (the Φ of DRVP increased from 0.00048 to 0.01224). The
f
result of viscosity-responsive signal would be ascribed to the prohibition of
rotation in the more viscous solvents and the enhanced degree of conjugation.
What’s more, the greater enhancement (241.9-fold) of the fluorescence
intensity was achieved in methanol castor oil mixture (Fig. S3). The
fluorescence lifetime studies were also carried out in methanol and
methanol/glycerol mixture, respectively. According to the fitting curves (Fig.
S4), the fluorescence decay is double exponential. The fluorescence lifetime of
9

DRVP increased from 0.21 ns in absolute methanol to 1.51 ns in
methanol/glycerol mixture solution using the formula (τ = τ *Rel % +
*Rel %). These results indicated that the probe DRVP is a viscosity-sensitive
1
1
τ
2
2
probe. Besides, given the extremely complex intracellular environment, To
study what extent influence on emission by solvent polarity, the fluorescence
spectra of probe DRVP were tested in various solvents (DMF, DMSO,
MeOH, acetone, THF, MeCN, DCM, CHCl
with different polarity. As shown in Fig. S5, the probe exhibited weak
fluorescence intensity in most non-viscous solvents except CHCl , while it
3
, Toluene and ethylene glycol)
3
showed strong fluorescence emission in viscous solvent, glycerol. Though the
emission wavelengths were quite varied, the emission were very weak in almost
all nonviscouse solvents but glycerol. The results indicated that the emission
intensity of DRVP is mainly effected by the solvent viscosity rather than
solvent polarity.
Fig. 2 (A) The fluorescence emission spectra of DRVP (30 μM) in methanol-glycerol systems at
various ratios. λex =450 nm.
The selectivity of probe is used as an extremely significant parameter due to the
fact that cell is a complex multicomponent system [46]. Therefore, the fluorescence
spectra of DRVP were observed in the presence of competing biomolecules. The
selectivity of the probe DRVP was further assessed by co-existence with various
3
+
2+
2+
2+
2+
3+
2+
+
2+
-, NO -
,
cations (Al , Ba , Ca , Co , Cu , Fe , Mg , Na
,
Zn ), anions (NO
, CNS ), sulphur-containing (Cys, GSH, Hcy), reactive oxygen species
, HClO), and amino acids species (alanine, arginine, histidine, isoleucine,
2
3
S
2
O
3
-, SO
3
2-
-
(H
2
O
2
glutamate). As shown Fig.3A, the probe DRVP treated with these biological species
caused tiny signal fluctuations, only more viscous solvents would cause significant
fluorescence enhancement at 640 nm. These results indicated that the probe DRVP
1
0

has excellent selectivity for glycerol. What’s more, DRVP displayed almost
non-fluorescence changes at 640 nm under various pH values (Fig. 3B).With the aim
of gaining further insight into the photostability of DRVP, It was observed that the
fluorescence intensity has tiny change of at 640 nm (Fig. S6). As a result, the probe
DRVP might be stable in the biological situations and has the relative good
photostability.
Fig. 3 (A). The fluorescence intensity of DRVP (30 μM) at 640 nm in MeOH solution. 1: Blank; 2:
3
+
2+
2+
2+
2+
3+
2+
+
2+
-; 12: NO
-; 13:
Al , 3:Ba ; 4:Ca ; 5: Co ; 6: Cu ; 7: Fe ; 8: Mg ; 9: Na ; 10: Zn ; 11: NO
2
3
O -
; 14: SO
2-
; 15: CNS ; 16: Cys; 17: GSH; 18: Hcy; 19: H
-
O
; 20, HClO; 21: Ala; 22: Arg; 23:
S
2
3
3
2
2
His; 24: Ile; 25: Gln; 26: glycerol; (B) The effect of pH on the fluorescence intensity of DRVP (30
μM) at 640 nm in PBS buffer (containing 50% MeOH). The fluorescence intensity was measured
at λex = 450 nm with both excitation and emission slit widths of 5 nm, and 700 V PMT voltage.
3
.4. Mitochondrial colocalization experiment
The co-localization experiment was performed, in consideration of the
possibility that the probe DRVP has excellent membrane permeability and
specifically enrich in mitochondria. Prior to this, the cytotoxicity of probe was
evaluated by MTT assay because cytotoxicity is a momentous parameter for
fluorescent probe. The cell viability is above 75% when incubated with DRVP
(0, 5, 10, 25, or 50 μM) for 24 h, demonstrating that the influence of DRVP on
cell viability could be omitted and used for cell imaging (Fig. S7) [47]. Then,
the cell co-localization experiment of DRVP was confirmed by Mito-Tracker
Green (MTG, commercial mitochondrial dye), HepG2 cells were incubated
with DRVP (10 μM) for 30 minutes, and then cultured with 2.5 μM
Mito-Tracker Green for 5 minutes. The imaging results indicated that the
11

combined images of the Mito-Tracker Green channel and the cell channel could
overlap very well. The intensity profile variations in the green and red channels
exhibited a tendency toward synchrony. What’s more, a high correlation of 0.86
was calculated by the Pearson correlation coefficients (Fig. 4). These
experimental results indicated that DRVP with high S/B ratio could be used as
a targeting probe for specific enrichment in mitochondria.
Fig. 4 The confocal fluorescence images of HepG2 cells co-incubated with with DRVP (10
μM) and Mito-Tracker Green (2.5 μM) for 5 min. (A) Red channel image by excitation at
5
61 nm; (B) Green channel image by excitation at 488 nm; (C) Bright field image; (D)
Merged images of (A) and (B); (E) Intensity profile of the ROI indicated by arrow in (D).
Scale bar: 25 μm.
3
.5. Living cell imaging
Subsequently, we further demonstrate the ability of the probe DRVP for cell
imaging of viscosity variations. External stimuli (monensin and nystatin) are
used to induce mitochondrial disease by stimulating its viscosity increase.
When HepG2 cells were incubated with DRVP (10 μM) for 30 minutes, the
probe DRVP was observed to weak red fluorescence in the TRITC channel
(Fig. 5A). In contrast, when the cells were pre-treated with nystatin or
monensin for 30 minutes, and then stained with DRVP for 30 minutes, the red
fluorescence was significantly enhanced in the TRITC channel (Fig. 5B-C). In
addition, the cell imaging experiments using compound SRVP were also
1
2

performed as a comparison. As shown in S8, probe SRVP exhibited
.0/2.4-fold induced by monensin/nystatin stimulation in living cells. In
3
contrast, probe DRVP showed 5.9/4.0-fold and 4.3/3.5-fold increase induced
by the same drugs stimulation in living cells (Fig. 5). Overall, compared with
SRVP, probe DRVP had the advantages of high S/B ratio and high sensitivity
in living cells.
Fig. 5 Confocal fluorescence images of the probe DRVP responding to exogenous viscosity in
living cells. (A) HepG2 cells were incubated with only probe DRVP (10 μM) for 30 min. (B-C)
HepG2 cells were pretreated with (B) monensin (10 μM) or (C) nystatin (10 μM) for 30 min,
followed by incubation of probe DRVP (10 μM) for another 30 min. First line: merged images of
bright filed and TRITC channels; second line: TRITC channel (570-620 nm); Scale bar: 25 μm. (D)
Normalized intensity in TRITC channel in Fig (A2-C2).
3
.6. Zebrafish fluorescence imaging
Furthermore, we also explored the potential application of the viscosity
variations in living zebrafish. As shown in Fig 6, the zebrafish incubated with
DRVP (10 μM) showed weak red fluorescence. However, the zebrafish
successively treated with 10 μM of monensin or nystatin and probe DRVP (10
μM) showed a significant increase of fluorescence intensity. In addition, the
zebrafish imaging experiments using compound SRVP were also performed as
a comparison. As shown in Fig. S9, probe SRVP exhibited 2.9/2.3-fold
increase of fluorescence induced by monensin/nystatin stimulation in living
zebrafish, respectively. In contrast, probe DRVP showed 4.3/3.5-fold increase
induced by the same drugs stimulation in living cells and zebrafish,
1
3

respectively. The higher S/B ratio detection by probe DRVP should be
contributed to the lower background fluorescence produced by DRVP in the
relatively low viscous environment. Thus, compared with SRVP, probe DRVP
had the advantages of high S/B ratio and high sensitivity in biological systems.
What’s more, adding nystatin or monensin may changes the combining
ability of DRVP to mitochondria, leading to a higher fluorescence intensity in
living cells and zebrafish. To explain what the primary factor for leading to the
fluorescence increase, we selected quinolin fluorophore (N, N,
2
-trimethylquinolin-6-amine,
TQA
)
and
carbazole
fluorophore
(
3-aldehyde-9-ethylcarbazole, AEC) (Scheme S1), two core components being
consisted of probe DRVP, for cell and zebrafish imaging at the same operating
conditions. As expounded in Fig. S10 and S11, after stimulation of whether
nystatin or monensin, the fluorescence intensity of the cells incubated with
compound TQA or AEC hardly changed. However, after incubating with
stimulus, the fluorescence intensity of the cells treated with probe DRVP in the
TRITC channel significantly enhanced (Fig. 5). The similar change phenomena
of fluorescence induced by nystatin or monensin were also observed in
zebrafish (Fig. S12-13). Thus, the increasing viscosity of mitochondria should
the primary factor rather than increasing probe concentration for leading to the
fluorescence increase.
Fig. 6 Confocal fluorescence images of viscosity in living zebrafish. (A) Zebrafish was incubated
with probe DRVP (10 μM) for 30 min. (B-C) zebrafish were pretreated with (B) monensin (10
μM) or (C) nystatin (10 μM) for 30 min, then incubated with probe DRVP (10 μM) for another 30
min. First line: merged images of bright filed and TRITC channels; second line: TRITC channel
(570-620 nm); Scale bar: 250 μm. (D) Normalized intensity in TRITC channel in Fig (A2-C2).
1
4

4
. Conclusions
In summary, we have proposed a new design concept to construct
viscosity-sensitive fluorescent probes based on double-rotor molecular platform
for sensing viscosity with excellent sensitivity and high signal-to-background
ratio (S/B) in the complex biological system. To execute a proof-of-concept
project, the probe DRVP containing two rotors, quinolinium and carbazole
moieties, was developed using a strong ICT platform. It performs initially low
background signal in methanol solution with low viscosity, while achieves a
significant enhancement (51.8-fold) of fluorescence in viscous glycerol media.
What’s more, the cationic probe DRVP can specifically accumulate in
mitochondria, and it has been demonstrated to be able to serve as a powerful
tool for monitoring viscosity in living cells and zebrafish. We anticipate that the
design strategy proposed in this work would accelerate the development of a
diversity of fluorescent viscosity probes with superior S/B ratio and sensitivity,
providing promising prospects for the future of biological and clinical research.
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.
Author contributions
W. Lin directed the research. Y. Yang and L. He designed and performed the
experiments, analyzed the data, and wrote the paper. All the authors discussed the
results and commented on the manuscript.
Acknowledgements
This work was financially supported by NSFC (21672083, 21605059, and
2
1877048), the Taishan Scholar Foundation (TS 201511041), and the start-up
fund of the University of Jinan (309-10004 and 160100136).
1
5

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