Journal of Molecular Structure 1284 (2023) 135422

Contents lists available at ScienceDirect

Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

A pyrrole-tricyanofuran-based probe for the detection of bisulfite and

viscosity in lysosomes of living cells and zebrafish
Ling Zhang a, Wei-Na Wu a,∗, Xiao-Lei Zhao a, Yun-Chang Fan a, Yuan Wang a,∗,
Zhi-Hong Xu b,c,∗
a
College of Chemistry and Chemical Engineering, Henan Key Laboratory of Coal Green Conversion, Henan Polytechnic University, Jiaozuo 454000, PR China
b
Key Laboratory of Chemo/Biosensing and Detection, College of Chemical and Materials Engineering, Xuchang University, Xuchang, 461000, PR China
c
College of Chemistry, Zhengzhou University, Zhengzhou, 450052, PR China

a r t i c l e i n f o a b s t r a c t

Article history: A novel AIE active bifunctional probe for detecting bisulfite (HSO3 − ) and viscosity synchronously was
Received 10 January 2023 developed based on pyrrole-tricyanofuran skeleton. This probe exhibited aggregation-induced emission
Revised 20 March 2023
behavior in almost pure aqueous media, and responded to HSO3 − with a “turn-off” signal and viscosity
Accepted 23 March 2023
changes with a “turn-on” signal in semi-aqueous solutions, respectively. The response mechanism of the
Available online 25 March 2023
probe associated with HSO3 − -involving C=C breakage was demonstrated via ESI-MS, 1 H NMR analysis,
Keywords: and density functional theory calculations. Furthermore, the probe has been applied to imaging endoge-
Bisulfite nous HSO3 − and viscosity changes in the lysosomes of C6 cells and zebrafish.
Fluorescent probe © 2023 Elsevier B.V. All rights reserved.
Lysosome-targeting
Viscosity changes
Zebrafish imaging

1. Introduction the specific identification of HSO3 − /SO3 2− anions by fluorescent

probe based on nucleophilic addition reactions of C=C bonds with
An abundance of evidence indicates that sulfur dioxide (SO2 ) HSO3 − /SO3 2− [9,10]. Up to now, fluorescent probes detection is
functions as a signaling molecule alongside nitric oxide, carbon widely used for separately detecting SO2 and viscosity, on account
monoxide and hydrogen sulfide [1,2]. SO2 exists in equilibrium of their favorable features, such as the high sensitivity, low bi-
with bisulfites (HSO3 − ) and sulfites (SO3 2− ) under physiological oluminescence damage, and real-time imaging [11–14]. However,
conditions, and plays a key role in numerous biological activities the bifunctional probes detecting SO2 and viscosity synchronously
[3]. However, excess SO2 would influence the concentration and are relatively rare. Particularly, some fluorophores with donor-π -
activity of lysosomal enzymes, cause irreversible damage to cells acceptor (D-π -A) system, which not only provide bridged C=C
and tissues, and even induce cardiovascular diseases and lung can- bond as the recognition site of SO2 , but also possess molecular
cer [4–6]. On the other hand, intracellular viscosity is considered as rotors for viscosity detection, have been developed as fluorescent
a key parameter of cell microenvironments by affecting the mobil- probes for both SO2 and viscosity. Nevertheless, few of them could
ity of various molecular species [7]. Generally, intracellular viscos- be used at cellular and subcellular levels, and in most cases, the
ity is inhomogeneous in subcellular organelles. Many diseases have reported probes targeted mitochondria (Table 1). In this respect, it
been speculated to be closely related to aberrant lysosomal viscos- is still urgent to design fluorescent probes with simultaneous de-
ity changes, including heart disease, aging, and lysosomal storage tection of the level of SO2 and viscosity in lysosomes.
diseases [8]. As a result, it is very crucial to monitor the changes Given the strong electron drawing ability of tricyanofuran (TCF)
of SO2 and viscosity in lysosomes for the diagnosis and treatment ring [24–26], a novel D-π -A type probe 1 (Scheme 1) was con-
of diseases caused by lysosomal SO2 and viscosity changes. structed by introducing a pyrrole ring as an electron donor for
The chemodosimeter approach is a specific and selective multiply imaging intracellular HSO3 − and viscosity. Surprisingly,
method to detect anions that have nucleophilic property, such as probe 1 exhibited aggregation-induced emission (AIE) behavior in
almost pure aqueous media. Then the red emission of probe 1
at 575 nm (excited at 500 nm) was totally quenched by HSO3 −
∗
Corresponding authors.
in semi-aqueous media, along with the obvious hypochromic ef-
E-mail addresses: [email protected] (W.-N. Wu), [email protected]
(Y. Wang), [email protected] (Z.-H. Xu).
fect of the absorption peak at 550 nm, probably due to the

https://doi.org/10.1016/j.molstruc.2023.135422
0022-2860/© 2023 Elsevier B.V. All rights reserved.
L. Zhang, W.-N. Wu, X.-L. Zhao et al. Journal of Molecular Structure 1284 (2023) 135422

Table 1
Comparison of some reported fluorescent probes for HSO3 − detection.

Ref Structure Analyte (s) Response time DL(HSO3 − ) Organelle targeting Test media

[15] HSO3 − 15 min –a Mitochondria THF/H2 O

(3/1)

[16] HSO3 − 10 min 0.1 μM No DMSO/H2 O

(1/3)

[17] HSO3 − 10 min 0.37 μM No DMSO/H2 O

(1/99)

[18] HSO3 − 36 min – Lysosomes DMSO/H2 O

(1/1)

[19] HSO3 − 10 min 0.4 μM No CH3 OH/H2 O

(1/9)

[20] HSO3 − /viscosity – 0.155 μM Mitochondria H2 O

[21] HSO3 − /viscosity 7 min 0.04 μM Mitochondria H2 O

[22] HSO3 − /viscosity 2 min 1.38 × 10−7 μM Mitochondria H2 O

[23] HSO3 − /viscosity 30 s 0.78 μM Lysosomes DMSO/H2 O

(3/7)

This work HSO3 − /viscosity 20 s 12.39 μM Lysosomes DMF/H2 O

(1/1)

a
The data is not given in the literature.

block of conjugated π system. On the contrary, the increasing vis- 2. Results and discussion
cosity of the 1 solution dramatically induced the enhancement
of red emission due to the suppression of the free rotation of 2.1. Synthesis of 1
molecular rotors. Benefiting from the terminal morpholine group
on the pyrrole moiety, the probe has been applied for tracking The probe was obtained through a condensation reac-
HSO3 − and viscosity fluctuation in lysosomes of living cells and tion between 5-formyl-2,4-dimethyl-N-(2-morpholinoethyl)−1H-
zebrafish. pyrrole-3-carboxamide [27] and 2-(3-cyano-4,5,5-trimethylfuran-

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L. Zhang, W.-N. Wu, X.-L. Zhao et al. Journal of Molecular Structure 1284 (2023) 135422

Scheme 1. Synthetic route of probe 1.

Fig. 1. (a) Fluorescence spectra of 1 (10 μM) in H2 O/DMF mixtures with increasing water fractions. (b) Fluorescence spectra of 1 (10 μM) in several solvents with distinct
polarity. λex = 500 nm.

2(5H)-ylidene)-malononitrile at room temperature [28], which has

been characterized by NMR and ESI-MS spectroscopy. The details
of methods and procedures were described in supporting informa-
tion.

2.2. AIE of 1

Considering the good solubility of probe 1 in organic solvents

(e.g., DMF) and poor solubility in H2 O solution, the fluorescence
spectra of probe 1 in various H2 O/DMF mixtures (buffered in
HEPES, 50 mM, v/v) were studied. As displayed in Fig. 1a, probe
1 was almost non-emissive in pure DMF solution (λex = 500 nm),
while the red emission of the probe was little blue-shifted and in-
creased when increasing the water level from 0 to 40% in H2 O/DMF
mixtures. Then the emission was still blue-shifted and partially
quenched in 40%−99% water contents. However, the red fluores-
cence of the probe in 99% H2 O solution was still much stronger
than that in pure DMF solution, thereby confirming the signifi-
cant AIE effect of the probe [29–31]. It’s reported that the J aggre- Fig. 2. Time resolved fluorescence spectra of 1 (10 μM) only in pure DMF and 50%
gates would produce bathochromic shifts and enhance the emis- H2 O solutions.
sion, while the H aggregates would produce hypsochromic shifts
and/or suppress the luminescence [32]. Given the literature results
[33–36], the AIE of 1 should be assigned to the combination of J- tion, and the particles became nearly irregular elliptic aggregates
type and H-type aggregates. And 1 μM probe 1 also displayed sim- (the average size more than 250 nm) in 50% H2 O solution, which
ilar AIE behavior through fluorescence spectra (Fig. S1, ESI). validated the AIE behavior of the 1 [37]. DLS analysis was per-
To investigate the aggregation behavior of probe 1, the polar- formed for probe 1 in 0% and 50% H2 O solutions as shown in Fig.
ity solvent effect was considered (Fig. 1b). The emission of probe 1 S3, ESI. The available data quality of probe 1 in 0% H2 O was so poor
was concentrated at 585 nm in DMF, DMSO, THF, EtOH, CH2 Cl2 or for distribution analysis (particle dispersion index of DLS data, PDI
CHCl3 , which blued-shifted to 580 nm in toluene or CH3 OH, and > 0.7) that it didn’t work in this DLS analysis, even though we
further to 575 nm in CH3 CN or EA, suggesting that the solvent po- tried many times. However, there is no doubt that the Z-average of
larity has a negative effect on the probe emission, further support- probe 1 in 50% H2 O was 249.6 d.nm (PDI = 0.548), which further
ing that the relative strong fluorescence of probe 1 in 99% H2 O confirmed the formation of aggregates of the 1 in 50% H2 O [38–
solution is attributed to AIE [29]. 40]. As displayed in Fig. 2 and Table S1, the probe was fitted with
Transmission electron microscope (TEM), dynamic light scatter- a single exponential decay with the lifetime of 0.48878 ns in pure
ing (DLS), and time-resolved photo-luminescence (TRPL) measure- DMF solution ( = 0.004, using rhodamine 6 G as the standard,
ments were performed to further investigate the AIE behavior of 1.  = 1.0 in ethanol [41]), whereas that increased to 0.49911 ns
As shown in Fig. S2, ESI, well-dispersed particles (with the width in 50% H2 O solution ( = 0.036). Obviously, with the addition of
about 139.46 nm) were observed for probe 1 in pure DMF solu- H2 O, the slow decay of the excited state of probe 1 remained for a

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L. Zhang, W.-N. Wu, X.-L. Zhao et al. Journal of Molecular Structure 1284 (2023) 135422

Fig. 3. The UV−Vis (a) and fluorescence (b) spectra of 10 μM probe 1 in DMF/H2 O (v/v, 1/1) solutions with 20 eq. analytes (F− , Cl− , Br− , I− , ClO− , ClO4 − , CN− , OAc− , HCO3 − ,
NO2 − , NO3 − , PPi, PO4 3− , HS− , S2 O3 2− , S2 O8 2− , SO4 2− , Ca2+ , Mg2+ , Fe3+ , H2 O2 , GSH, Cys, Hcy, and HSO3 − ) and blank. The UV−Vis (c) and fluorescence (d) spectra of 10 μM
probe 1 upon the addition of HSO3 − (0–20 eq.) in DMF/H2 O (v/v, 1/1) solutions. Insert: the absorbance at 550 nm and the fluorescence intensity at 575 nm as a function of
HSO3 − concentration with linear relationship, respectively. λex = 500 nm.

more time period. Therefore, the increased water content formed (R2 = 0.99481) against HSO3 − concentration in the range of
the aggregates of 1. 38.0 − 180.0 μM. In addition, the calculated detection limit (DL)
of probe 1 was 12.39 μM using the formula of 3δ /k [43,44]. Sim-
2.3. Spectroscopic response of 1 to HSO3 − ilar results were observed in the absorbance titration experiments
(Fig. 3c), revealing the good sensitivity of probe 1 to HSO3 − based
The spectral properties of probe 1 were analyzed in semi- on fluorescent and naked-eye detections.
aqueous media (pH = 5.0, HEPES buffer, 50 mM, containing 50% Different organelles function at different pH ranges, especially
DMF). As depicted in Fig. 3a, a significant absorption peak of the lysosomes perform in acidic environment (pH 4.5 − 5.5), thus the
probe (10 μM) was concentrated at 550 nm ascribed to its well pH effect for HSO3 − detection of probe 1 was worth being ex-
conjugated structure, which corresponded with the red solution plored [45–47]. As depicted in Fig. S5, ESI, probe 1 was effective
color. Meanwhile, excitation at 500 nm led to an emission peak in detecting HSO3 − from pH 2.0 to pH 9.0, facilitated lysosomal
of 1 at 575 nm, attributed to AIE and intramolecular charge trans- HSO3 − imaging. Besides, the response time of the probe to HSO3 −
fer (ICT) effects, along with red solution color at a 365 nm UV was 20 s, more conducive to real-time monitoring (Fig. S6, ESI).
irradiation (Fig. 3b). Adding HSO3 − (20 eq.) induced a notable This is a unique feature of probe 1 prior to some reported HSO3 −
hypochromic effect of the absorbance peak, according to the so- probes (Table 1).
lution color fading [42]. In addition, fluorescence quenching at
575 nm was observed ( = 0.075), and the solution color became 2.4. Fluorescent response of 1 to viscosity
dark under irradiation of a 365 nm UV light. Notably, the decreased
absorbance of A550 and increased fluorescence intensity of F575 To assess the viscosity response characteristics of probe 1,
are highly selective toward HSO3 − , compared with other analytes we measured its fluorescence spectra in glycerol/H2 O mixtures
(AcO− , Br− , CN− , ClO− , ClO4 − , Cl− , F− , I− , HCO3 − , NO2 − , NO3 − , PPi, with different water content (Fig. 4a). The weak emission of free
PO4 3− , S2 O3 2− , S2 O8 2− , SO4 2− , Ca2+ , Mg2+ , Fe3+ , H2 O2 , GSH, Cys, 1 (10 μM) in H2 O was observed with slit widths at 2.5/5 nm
and Hcy), except HS− (50% absorbance response of HSO3 − , couldn’t ( = 0.036). With increase in the glycerol content from 0 to
affect the fluorescence detection of HSO3 − ). In addition, the ana- 99%, the red fluorescence of 1 at 575 nm increased significantly
lytes minimally affected the intensity of probe 1 with HSO3 − (Fig. ( = 0.559), attributed to the C=C-involving restricted rotation
S4, ESI), revealing the excellent selectivity of 1 for the detection of in high viscosity media [23]. Meanwhile, a good linear trend
HSO3 − in semi-aqueous media. (R2 = 0.98306) was obtained between the F575 and viscosity range
Fluorescence titration experiments were performed to investi- of 1.0 − 956.0 cP, which indicated that the probe can detect vis-
gate the ability of quantitative detection in probe 1 (10 μM) to cosity changes via fluorescence spectra.
HSO3 − (Fig. 3d). Increasing the amount of HSO3 − led to a de- As shown in Fig. S7, ESI, most biological species induced no in-
cline in the red emission of probe 1 with a good linear trend terference in the detection of 1 toward viscosity, which was also

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L. Zhang, W.-N. Wu, X.-L. Zhao et al. Journal of Molecular Structure 1284 (2023) 135422

Fig. 4. (a) Fluorescence spectra of probe 1 (10 μM) with increased glycerol volume ratios (0%−99%). Insert: The linear relationship among the F575 and the log(η). The
η = viscosity. (b) Fluorescence spectra of probe 1 (10 μM) in various solution (DCM, CH3 CN, CH3 OH, CHCl3 , DMF, DMSO, EA, EtOH, THF, toluene, glycerol, H2 O). (c) The
corresponding fluorescence images of 1 (10 μM) with increased glycerol volume ratios (0%−99%) under a 365 nm UV lamp. λex = 500 nm.

hardly affected by pH variation (Fig. S8, ESI). Moreover, the inten- To confirm the rotation of the C=C group in the probe to
sity of probe 1 remained stable within 1 h in different viscous so- viscosity, the UV−Vis spectra of 1 were determined in different
lutions (e.g., 10%, 50% and 99% glycerol, Fig. S9, ESI). All above facts polar solvents (CH3 OH, EtOH, DMF, DMSO, CH3 CN, CHCl3 , DCM,
illustrated that probe 1 can respond to viscosity changes with high EA, H2 O, THF, toluene, and glycerol). As shown in Fig. S11, ESI,
selectivity and a wide function pH range, which represents an ex- almost no variation of absorbance peak was observed in the 1
cellent candidate for monitoring viscosity. solution with different solvent polarity, while significant fluores-
cence enhancement was observed in the fluorescence spectra of
1 + glycerol (Fig. 4b). The results indicated that the solvent po-
2.5. Sensing mechanism larity has negligible effect on the sensing mechanism of the 1 to
viscosity, and probe 1 can respond to viscosity changes due to
To demonstrate the sensing mechanism of 1 to HSO3 − , ESI-MS the C=C-involving restricted rotation in probe 1 [49,50], as shown
and 1 H NMR analysis were carried out. The spectrum of 1 + HSO3 − in Scheme 2.
displayed the appearance of a peak at m/z = 543.1963, which was
assigned to [1-SO3 H + H]+ , thereby inducting a Michael addition 2.6. Fluorescence imaging
between probe 1 and HSO3 − (Fig. 5). As shown in Fig. 6, after
the addition of HSO3 − , the Ha peak at 7.69 ppm, and Hb peak MTT assays were performed to assess the toxicity of the probe
at 6.73 ppm in the 1 H NMR spectrum of probe 1 changed to Ha ’ in C6 cells. As shown in Fig. S12, ESI, the C6 cells survival rate was
(at 4.53 ppm), and Hb ’ (at 2.90 ppm), respectively. While a new still 81.08% following incubation of the cells with probe 1 (40 μM),
Hd peak at 8.59 ppm for -OH was occurred, further indicating suggesting that probe 1 with low cytotoxicity can be used in cel-
the Michael nucleophilic addition reaction between 1 and HSO3 − . lular imaging. Treatment of 1-loaded cells with NaHSO3 (50 μM)
Moreover, the pyrrole NH peak of 1 (Hc ) at 12.25 ppm shifted resulted in fluorescence quenching, as shown in Fig. S13, ESI. Sim-
to the high field (at 10.21 ppm) in the presence of HSO3 − . The ilar results were obtained by treatment with exogenous HSO3 −
TRPL results show that the lifetime of 1 and 1+HSO3 − reached (0.5 mM glutathione (GSH) and 0.25 mM Na2 S2 O3 ). Moreover, the
0.49911 ns and 0.76604 ns respectively, suggesting that the addi- emission of 1-loaded cells in red channel increased dramatically
tion of HSO3 − affects the existence state of probe 1 (Fig. S10, ESI). upon the addition of dexamethasone (20 μM, a stimulant which
Based on previous literatures [23], a working mechanism involving can enhance the viscosity in lysosomes [7]). Therefore, probe 1 is
C=C breakage was proposed as shown in Scheme 2. a good candidate for the detection of intracellular HSO3 − and vis-
Density functional theory (DFT) calculations were performed cosity.
to further explore the sensing mechanism for 1 to HSO3 − un- Moreover, probe 1 and Lyso-Tracker Blue (a commercial lyso-
der Gaussian 09/B3LYP/6–31 G (d) [48]. The electron cloud den- some tracker) were co-incubated in C6 cells to evaluate its lyso-
sity of the probe’s HUMO was evenly distributed throughout the somal targeting ability. As displayed in Fig. S14, ESI, both red and
molecule, while the LUMO was concentrated in the pyrrole and blue channels overlapped well with an overlap coefficient of 0.83,
tricyanofuran moieties (Fig. 7). The addition of HSO3 − suspended which fully demonstrates that the probe can specifically target
the ICT effect of 1, and led to obvious fluorescence quenching. Fur- lysosomes.
thermore, the energy gap between HOMO and LUMO of 1-SO3 H Based on the encouraging results of cellular imaging, vivo imag-
(2.27 eV) was lower than that of 1 (2.78 eV), suggesting that 1- ing was performed in zebrafish. Similar to cell imaging, 1-loaded
SO3 H is more stable than probe 1. zebrafish showed significant red fluorescence, and the fluorescence

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L. Zhang, W.-N. Wu, X.-L. Zhao et al. Journal of Molecular Structure 1284 (2023) 135422

Fig. 5. ESI-MS spectral changes with the addition of HSO3 − to 1.

Fig. 6. 1
H NMR spectra of 1 with and without HSO3 − .

was quenched when treatment with either endogenous and exoge- 2.7. Application of 1 in test strips
nous HSO3 − (Fig. 8). Besides, the red fluorescence turned stronger
when the 1-loaded zebrafish was incubated with dexamethasone Test strips were used to further investigate the practical appli-
(20 μM). Therefore, probe 1 can sense SO2 and viscosity in bionic cations of probe 1. Encouraged by the red color of probe 1 solution
systems, such as zebrafish. under naked eyes, we first loaded the probe on filter papers, as

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L. Zhang, W.-N. Wu, X.-L. Zhao et al. Journal of Molecular Structure 1284 (2023) 135422

Scheme 2. The proposed reaction mechanism of 1 with HSO3 − and viscosity, respectively.

Fig. 7. Energy-minimized structures and HOMO/LUMO of 1 and 1-SO3 H by DFT calculations.

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L. Zhang, W.-N. Wu, X.-L. Zhao et al. Journal of Molecular Structure 1284 (2023) 135422

Fig. 8. Fluorescence images of probe 1 in living zebrafish: (a–f) red channel; (g–l) bright field; (m–r) the merged images of (a–f) and (g–l); (a, g, m) images of zebrafish only
for 30 min at 28 °C; (b, h, n) images of zebrafish only incubating with probe 1 (10 μM) for 30 min at 28 °C; (c, i, o) images of zebrafish incubated with probe 1 (10 μM) and
then treated with NaHSO3 (50 μM) for 30 min at 28 °C; (d, j, p) images of zebrafish pre-treated with GSH (0.5 mM) and then incubated with probe 1 (10 μM) for 30 min at
28 °C. (e, k, q) images of zebrafish pre-treated with GSH (0.5 mM) and NaS2 O3 (0.25 mM) and then incubated with probe 1 (10 μM) for 30 min at 28 °C. (f, l, r) images of
zebrafish pre-treated with dexamethasone (20 μM) and then incubated with probe 1 (10 μM) for 30 min at 28 °C. Scale bar: 200 μm.

shown in Fig. S15, ESI. When the 1-loaded test strips were dipped and Technology Department of Henan Province (No. 212102210033,
in HSO3 − solutions (from 0 to 1 mM), the color of the test strips 222300420523, and 212102210646), the Fundamental Research
faded gradually. Therefore, the probe can detect HSO3 − with naked Funds for the Universities of Henan Province (No. NSFRF230402),
eyes using test strips. and the Key Program of Xuchang University (No. 2022CXTD001).

3. Conclusion
Supplementary materials
A novel AIE active pyrrole-tricyanofuran-based bifunctional
Supplementary material associated with this article can be
probe was constructed for the synchronous detection of HSO3 −
found, in the online version, at doi:10.1016/j.molstruc.2023.135422.
and viscosity changes. Probe 1 displayed a short response time
(20 s), a wide range of working pH (2.0 − 9.0), and a detection
limit of 12.39 μM for detecting HSO3 − in semi-aqueous media. Be- References
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