A natural hyperoside based novel light-up fluorescent probe with AIE and ESIPT characteristics for on-site and long-term imaging of β-galactosidase in living cells

Article abstract of DOI:10.1039/d0tc01981j

Fluorescence-based on-site and long-term sensing and bioimaging of biomarkers are highly desired for effective diagnosis. Aggregation-induced emission luminogens (AIEgens) with excited-state intramolecular proton transfer (ESIPT) characteristics have outstanding advantages in biological applications due to their large Stokes shift and low background signal in the aggregate state. However, most of the reported AIEgens are fabricated through rational design and complex synthesis procedures. Importantly, with rich structural skeletons and ease of access, natural products have superiority in developing promising AIEgens with ESIPT. Here, hyperoside(quercetin-3-O-β-galactoside) has been easily obtained fromHedyotis diffusa. After the incubation of hyperoside with β-galactosidase (β-Gal), the hydrolysis product, hydrophobic quercetin, is aggregatedin situ, which presents AIE and ESIPT characteristics with a large Stokes shift (170 nm). Then, a novel light-up fluorescent probe has been fabricated for the detection of β-Gal with a good linear relationship (0.03-12 U mL^?1), high sensitivity (a detection limit of 0.013 U mL^?1) and superior selectivity. Meanwhile, excellent biocompatibility, low cytotoxicity, outstanding photostability, and good intracellular retention demonstrate its superiority for on-site and long-term (about 8 h) imaging of β-Gal in SKOV-3 cells. The results demonstrate the application of hyperoside in the sensing and imaging of β-Gal in biological samples, and propose the great possibility of natural products for developing novel AIE-based fluorescence probes.

Full text of DOI:10.1039/d0tc01981j

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ARTICLE
Natural hyperoside based novel light-up fluorescent probe with
AIE and ESIPT characteristics for on-site and long-term imaging
of β-galactosidase in living cells†
Received 00th January 20xx,
Accepted 00th January 20xx
Ruiqing Long,a,‡ Cui Tang,b,‡ Zan Yang,c Qiachi Fu,a Jinju Xu,a Chaoying Tong,a Shuyun Shi,a,d,
Ying Guo,b,* and Daijie Wangd
*
DOI: 10.1039/x0xx00000x
Fluorescence-based on-site and long-term sensing and bioimaging of biomarkers are highly desired for effective diagnosis.
Aggregation-induced emission luminogens (AIEgens) with excited-state intramolecular proton transfer (ESIPT)
characteristics have outstanding advantages in biological applications for their large Stokes shift, and low background signal
in aggregated state. However, most reported AIEgens are fabricated through rational design and complex synthesis procedures.
Importantly, with rich structural skeletons and easy access, natural products have superiority in developing promising AIEgens
with ESIPT. Here, hyperoside (quercetin-3-O-β-galactoside) has been easily obtained from Hedyotis diffusa. After incubation
hyperoside with β-galactosidase (β-Gal), the hydrolysis product, hydrophobic quercetin, is in situ aggregated, which presents
AIE and ESIPT characteristics with large Stokes shift (170 nm). Then, a novel light-up fluorescent probe has been fabricated
for detection of β-Gal with a good linear relationship (0.03–12 U mL-1), high sensitivity (detection limit of 0.013 U mL-1) and
superior selectivity. Meanwhile, excellent biocompatibility, low cytotoxicity, outstanding photostability, and good
intracellular retention demonstrate the superiorities for on-site and long-term (about 8 h) imaging of β-Gal in SKOV-3 cells.
Results demonstrate the application of hyperoside in sensing and imaging of β-Gal in biological samples, and propose the
great possibility of natural products for finding of novel AIE-based fluorescence probes.
because they show strong FL in aggregate states, and can be applied
for in situ biosensing and bioimaging.10–12 Moreover, the high
intracellular retention, resistance to photo-bleaching, and robust
Introduction
Fluorescence (FL) bioimaging has become a highly powerful tool in
clinical diagnosis and biological research for its advantage of non-
invasiveness, superior sensitivity and selectivity, high temporal and
spatial resolution, and simple operation.1–4 On-site and long-term
bioimaging is in urgent need for real-time monitoring biological
targets.5 Traditional small-molecule FL probes can often diffuse away
from the detection sites to adjacent tissues or even translocate out of
cells after stimuli-response, which suffers from inaccurate in situ FL
information and difficult long-term tracking.6 In particular, some
remaining probes may still suffer from the inevitable aggregation-
caused quenching (ACQ) effect when accumulated.7–8 Therefore, it is
a tremendous need to overcome the aggregation dilemma of diffusion
resistant and ACQ effect.
biocompatibility make AIEgens more suitable for long-term
bioimaging with high fidelity.5 Heretofore, it is extremely meaningful
to make full use of the outstanding advantages of AIEgens to construct
novel FL probes, especially for those easily-obtained and low-cost.
β-Galactosidase (β-Gal) plays an important role in regulating
various physiological procedures, which is then considered as one of
the significant biomarkers in living tissues and organs.13 For example,
β-Gal is overexpressed in primary ovarian cancers.14 Thus, selective
and sensitive monitoring of β-Gal is essential for the knowledge of
pathogenesis of some diseases. Among the developed approaches
(e.g. positron emission tomography, surface-enhanced Raman
scattering,
magnetic
resonance
imaging,
colorimetry,
chemiluminescence, FL), FL methods based on synthetic β-
galactoside substrates are recognized as a powerful approach.15‒17
Apart from the traditional fluorophores (e.g. fluorescein,
Aggregation-induced emission luminogens (AIEgens),
originally reported by Tang's group,9 have attracted more attentions
a. Key Laboratory of Hunan Province for Water Environment and Agriculture
Product Safety, College of Chemistry and Chemical Engineering, Central South
University, Changsha 410083, China. E-mail: shuyshi@gmail.com
b. Department of Clinical Pharmacology, Xiangya Hospital; Hunan Key Laboratory
of Pharmacogenetics, Central South University, 410078 Changsha, China. E-mail:
guoying881212@csu.edu.cn
hydroxymethyl-N,N-diethylrhodol),18
few
AIEgens
based
fluorophores have been introduced. Tang's group designed
tetraphenylethylene-β-galactoside for light-up visualization of β-
Gal.19 Zhu and co-workers synthesized β-galactoside substituted
quinoline-malononitriles for imaging of β-Gal.5,20 Salicylaldehyde
azine-β-galactoside and hydroxyphenylquinazolinone-β-galactoside
were designed for imaging of β-Gal based on AIE and excited-state
intramolecular proton transfer (ESIPT) characteristics.21–22 Notably,
c. College of Chemistry and Chemical Engineering, Hunan Institute of Science and
Technology, Yueyang 414006, China.
d. Key Laboratory of TCM Quality Control, Shandong Analysis and Test Center,
Qilu University of Technology (Shandong Academy of Sciences), Jinan 250014,
China
‡ Ruiqing Long and Cui Tang contributed equally to this work
†Electronic Supplementary Information (ESI) available: Experimental details and Fig. AIE probes with ESIPT characteristics are found to have large Stokes
S1‒S13 as noted in the text. See DOI: 10.1039/x0xx00000x
shifts (> 150 nm) for the avoidance of self-absorption and
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Fig. 1 FL spectra of hyperoside (A) and quercetin (B) (both at 10 µg mL–1) in
different THF/H2O system; ESIPT process of quercetin calculated with TDDFT at
the level of B3LYP/6-31G* in THF solution. HOMO and LUMO molecular
orbitals are in the ground state and excited state of the enol form (C).
Scheme 1. Purification of hyperoside and the FL mechanism after reaction with β-
Gal (A); Mechanism of hyperoside as light-up FL probe for on-site and long-term
imaging of β-Gal in ovarian cancer cell (B).
(THF)/water solutions (THF as good solvent and water as poor
solvent). As shown in Fig. 1A and Fig. S2A (ESI†), hyperoside
shows only one FL emission peak at 405 nm, and FL intensities
decrease sharply with the increasing of water fractions from 0%
to 90% (v%). Quantum yields (QYs) of hyperoside in THF and
THF/H2O (20/80, v/v) are 9.36 and 2.37% (quinine sulfate as a
reference, QY at 54% in 0.05 M H2SO4). Furthermore, lifetimes
of hyperoside in THF and THF/H2O (20/80, v/v) are 1.35 and
0.69 ns. The reduction of QYs and lifetime after addition of
water indicates that hyperoside is associated with ACQ property.
In contrast, quercetin exhibits two FL emission peaks, 420 nm
for enol skeleton and 530 nm for keto skeleton (Fig. 1B). By
increasing water fractions from 0% to 80%, FL emission
intensities of keto skeleton gradually increase while those for
enol skeleton become weaker (Fig. 1B and Fig. S2B, ESI†).
Further addition of water to 90% leads to a dramatic decrease of
both FL emissions, probably resulting from the precipitation of
large-scale quercetin crystals. The QYs of keto emission of
quercetin in THF and THF/H2O (20/80, v/v) are 2.25 and 10.2%,
while the lifetimes for them are 1.13 and 2.23 ns. The
enhancement of QYs and lifetime presents the AIE property of
quercetin.
autofluorescence.8,21–22 So far, above reported FL probes are synthetic
products that commonly requires professional fabrication, time- and
cost-consuming synthetic procedures. It is thus significant to explore
new sources to easily obtain FL probes for β-Gal imaging based on
AIE and ESIPT characteristics.
Natural products contain an enormous variety of secondary
metabolites for drug leads.23‒24 In this work, we purified a flavonoid,
hyperoside (quercetin-3-O-β-galactoside), from Hedyotis diffusa
(Scheme 1A). Hydrophilic hyperoside (oil-water partition coefficient
(logP) at –1.39 by ChemBioDraw calculation) consists of a
hydrophobic aglycon, quercetin (logP at 0.35), and a β-galactoside
residue at C-ring. Hence, in the presence of β-Gal, the β-galactoside
residue is effectively and specifically cleaved, and then quercetin is
released and in situ aggregated. For hyperoside, the OH group in C-
ring is substituted by β-galactoside, resulting in the failure of ESIPT
process. We previously found that AIE and ESIPT exist when
flavonoids contain a proton acceptor (C=O) and a proton donor (OH)
in C-ring for the formation of intramolecular hydrogen bond.7
Quercetin contains AIE and ESIPT characteristics.25 Then, the FL
signal will be remarkably lighted up for on-site and long-term imaging
of β-Gal in living cells (Scheme 1). This work not only indicates that
natural products are tempting source to acquire AIEgens with
outstanding advantages, but also demonstrates a novel natural
compound for on-site and long-term imaging of β-Gal in living cells.
ESIPT mechanism for quercetin.
Dual emissions and a large Stokes shift (170 nm) are observed in
FL spectra of quercetin (Fig. 1B). Dual emissions can be the result of
several mechanisms, such as ESIPT, energy transfer, excimer
formation, as well as a charge transfer.26‒29 Excitation and UV-vis
spectra just have small overlap with emission spectrum of quercetin
in THF/H2O (20/80, v/v) (Fig. S3, ESI†), which indicate that energy
transfer is not the most match here. After increasing quercetin
concentrations in DCM and THF solutions, no new emission peak
appears, and excimer formation can be excluded (Fig. S4, ESI†). For
quercetin, charge transfer between oxygen atom and carbonyl group
results in 410 nm emission, which slightly redshifts and widens with
the addition of water. Then, emission at 530 nm might belong to
ESIPT.30 To further understand ESIPT characteristic of quercetin,
Gauss theoretical calculations were operated on time-dependent
density functional theory (TD-DFT) at the level of B3LYP/6-31G* in
THF solution (Fig. 1C). After absorbing 5.36 eV energy, the enol
Experimental section
Details of experimental section are described in Electronic
Supplementary Information (ESI†).
Results and discussion
Photophysical properties of hyperoside and quercetin.
High-resolution MS, 1H NMR and 13C NMR spectra
indicate the successful purification of hyperoside (Fig. S1,
ESI†). FL properties of hyperoside and quercetin are firstly
investigated by monitoring their FL spectra in tetrahydrofuran
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Fig. 2 TEM image of the product of hyperoside (10 µg mL−1) after incubation with
β-Gal (12 U mL–1) (A); Linear relationship between F530 and the concentration of
β-Gal (0–14 U mL–1) after incubation for 70 min at 37 °C (B).
ground state (E) of quercetin shifts to the first excited state (E*) for
HOMO→LUMO transition. At E* state, electron appears more on the
benzene group near C=O, and intramolecular hydrogen bond length
(C=O···H) is 1.92 Å, shorter than that (1.99 Å) at E state. While for
phenolic hydrogen bond (O–H), it gets a little longer from 0.98 Å (E
state) to 0.99 Å (E* state). After that, ESIPT happens, and keto
skeleton forms (K*) (Fig. S5, ESI†). Subsequently, K*→K transition
happens after releasing 2.41 eV energy. Obviously, the intramolecular
hydrogen bond mediates the tautomerization between enol and keto
skeletons in the excited state, and the E*→E and K*→K transitions
result in the dual FL emissions of quercetin, which is in good
accordance with the measured data. Moreover, the radiative decay rate
(kr) and non-radiative decay rate (knr) of quercetin in THF/H2O (20/80,
Fig. 3 Cell images of HeLa and SKOV-3 cells incubated with hyperoside (100 µg
mL‒1, 1.5 mL) for 2 h: (A1–A3) HeLa cells; (B1–B3) HeLa cells pretreated with β-
Gal (52 U mL‒1, 1.5mL) for 0.5 h; (C1–C3) SKOV-3 cells; (D1–D3) SKOV-3 cells
pretreated with D-galactose (0.2 mg mL‒1, 1.5 mL) for 0.5 h.
detection of β-Gal,19‒23 natural hyperoside has an acceptable
performance with easy-to-obtain. As a result, hyperoside can be
used as a novel and ideal light-up FL probe for sensitive, and
quantitative detection of β-Gal activity.
Stability and selectivity.
v/v) (kr = 0.0425 /ns, knr = 0.403 /ns) and THF (kr = 0.0199 /ns, knr
0.865 /ns) indicate the enhancement of radiative pathway and keto
form ratio in aggregate state.
=
The excellent stability of FL materials is crucial for long-time
detection. Therefore, the effect of pH, temperature, and UV irradiation
on the stability of hyperoside and quercetin were studied. As
presented in Fig. S10 (ESI†), both of them are stable at pH 4–8. FL
spectra of hyperoside and quercetin decrease under alkaline solution
(pH 9–12) because of the deprotonation of phenolic OH groups. Thus,
hyperoside can be applied for β-Gal detection under biological
conditions with pH at about 7.4. At the same time, FL spectra of
hyperoside and quercetin are almost unchanged within the detection
temperature (25–40 °C) (Fig. S11, ESI†), which then can be applied
for FL imaging in vivo and in vitro. Furthermore, the photostability of
hyperoside and quercetin was evaluated with time-course
fluorescence measurements under sustained UV irradiation, and the
FDA-approved near-infrared dye, indocyanine green (ICG), was
commercially achieved as control (Fig. S12, ESI†). After UV
irradiation for 100 min, the FL intensities of ICG, hyperoside, and
quercetin reduce to 10%, 82%, and 85%. Moreover, the FL half-time
for hyperoside and quercetin is 2.8 and 3.1 times longer than that of
ICG, suggesting the excellent photostability of hyperoside and
quercetin. In addition, FL intensities of hyperoside (F405) and
quercetin (F530) remain almost unchanged after storage in human
serum after 7 days (Fig. S13, ESI†). Thus, hyperoside and quercetin
present the pH and temperature independency under physiological
range, photo‐stability, and long‐term storage stability.
β-Gal activity assay by in situ generation of quercetin
nanoaggregates.
FL spectra of hyperoside after addition of β-Gal (12 U mL‒1)
at different time intervals are investigated in PBS solution (50
mM, pH 7.4) at 37 ºC (Fig. S6A, ESI†). FL emission at 405 nm
(F405) for hyperoside decreases obviously, and a new FL
emission at 530 nm (F530) concomitantly appears with a steady
increment, indicating the appearance of quercetin
nanoaggregates with average particle size at about 176 nm from
TEM and DLS experiments (Fig. 2A, Fig. S7, ESI†). F530
enhances sharply, reaches the maximum at 70 min, and keeps a
plateau over the next 50 min, which exhibits the completion of
enzymatic hydrolysis (Fig. S6B, ESI†). Accordingly, 70 min was
selected as the incubation time between hyperoside and β-Gal for
the following experiments. The maximum UV absorption band
after hyperoside reaction with β-Gal red-shifts from 356 nm to
368 nm, and the high-resolution MS spectrum affords [M–H]–
ion at m/z 301.0351 (C15H9O7, 162 Da less than that of
hyperoside) (Fig. S8, ESI†), further unambiguously confirming
the production of quercetin. It is then reasonably speculated that
quercetin is released and in situ aggregated after cleavage of β-
galactoside residue from hyperoside. Furthermore, F530 increase
with the increment of β-Gal concentration with a good linearity
in the range of 0.03–12 U mL‒1 (y = 7.01x + 6.05, R2 = 0.992)
(Fig. 2B, Fig. S9, ESI†). The limitation of detection (LOD) is
determined to be as low as 0.013 U mL‒1 (LOD = 3 σ/slope,
where σ is the standard deviation of blank measurement). By
comparison with previously reported AIE based FL probes for
The specificity of hyperoside to β-Gal was also evaluated. As
presented in Fig. S14 (ESI†), except for β-Gal, negligible changes of
FL spectra for hyperoside are observed upon treatment with 100
equiv. of common enzymes and amino acids, such as β-Glucosidase
(β-Glu), glucose oxidase (GOD), homocysteine (Hcy), glutathione
(GSH), and cysteine (Cys). Results demonstrate that hyperoside has
high selective response to β-Gal.
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Fig. 5 Time-dependent cell images of SKOV-3 cells incubated with hyperoside
(100 µg mL–1, 1.5 mL, A1–G2), and ICG (100 µg mL–1, 1.5 mL, I1–O2).
Fig. 4 Cell images for intracellular localization of β-Gal in SKOV-3 cells. Cells
were incubated with hyperoside (100 µg mL–1, 1.5 mL) for 2 h and then co-stained
with Golgi-Tracker Green (0.3 mg mL–1, 1.5 mL) for 30 min (A1–A5), Mito-
Tracker Green (0.1 mg mL–1, 1.5 mL) for 30 min (B1–B5), or Lyso-Tracker Green
(0.03 mg mL–1, 1.5 mL) for 60 min (C1–C5). Scatter plots were obtained by the
image analysis software, Image J Fiji.
It was reported that nanoparticles have slower exocytosis
rate and longer intracellular retention time than small FL
molecules.31 Here, quercetin nanoaggregates might have
difficulty to extrude out of living cells, and then realize long-
term imaging. To verify this, commercial ICG was also chosen
as control. After incubation hyperoside with SKOV-3 cells, red
FL signal gradually increases in 0–2 h, and then keeps strong
from 2 to 8 h (Fig. 5 A1–F1, A2–F2). What is more, when
prolonging incubation time to 12 h, red FL signal is slightly
attenuated within fixed imaging region (Fig. 5 G1, G2).
However, for ICG, FL intensity is barely observed when the
incubation time reaches 5 h (Fig. 5 I1–O1, I2–O2), because of
the diffusion and ACQ effect. Thus, it can be concluded that
hyperoside can be applied for long-term cell imaging.
On-site bioimaging of β-Gal in living cells.
The superior advantages of hyperoside inspire us to apply it for
bioimaging of β-Gal in living cells. SKOV-3 cells with endogenous
β-Gal overexpression and HeLa cells without β-Gal expression were
selected as the model and control cells, respectively.21–22 Firstly, the
cytotoxicities of hyperoside and quercetin were evaluated using
methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As
displayed in Fig. S15 (ESI†), viabilities of SKOV-3 and HeLa cells
are barely affected by addition of hyperoside or quercetin up to 1 mg
mL‒1 and incubation for 24 h, indicating their low cytotoxicity and
excellent biocompatibility. Subsequently, hyperoside was used to
image β-Gal in living cells (Fig. 3). After incubation with hyperoside
(100 µg mL‒1, 1.5 mL) for 2 h, Hela cells exhibit no red FL.
Significantly, enhanced red FL signal for Hela cells occurs after being
pre-treated with exogenous β-Gal (52 U mL‒1, 1.5 mL) for 0.5 h. In
contrast, SKOV-3 cells present an obvious light-up red FL. When a
well-known competitive inhibitor of β-Gal, D-galactose (1 mM, 1.5
mL), is pre-treated with SKOV-3 cells for 0.5 h, virtually no red FL
is observed. Results indicated that hyperoside can monitor not only
endogenous β-Gal but also exogenous β-Gal. Notably, just incubation
process was operated in the bioimaging process, indicating the highly
cell membrane-permeable property of hyperoside.
Obviously, the intracellular red FL does not scatter in the whole
cytoplasm (Fig. 3), indicating the on-site bioimaging of β-Gal from in
situ formation of quercetin nanoaggregates. Then the subcellular
colocalization of the FL signal was carried out in SKOV-3 cells by
co-staining hyperoside with commercially available FL probes
(Golgi-Tracker Green, Lyso-Tracker Green, and Mito-Tracker Green)
(Fig. 4). The Pearson's correlation coefficients of hyperoside with
Golgi, Mito and Lyso are found to be 0.922, 0.955 and 0.658. Thus,
results demonstrate that hyperoside can site-specially image β-Gal in
Golgi apparatus and mitochondria of living cells, which is the same
with reported synthesized AIE probe, quinoline-malononitrile-2-(2-
hydroxyphenyl) benzothiazole-β-galactoside.20
Conclusions
In summary, natural hyperoside as a novel light-up FL probe was
facilely prepared from Hedyotis diffusa. In the presence of β-Gal, β-
galactoside residue in the C-ring of hyperoside is specially cleaved,
and then quercetin with AIE and ESIPT characteristics is in situ
released. After on-site aggregation, a new FL emission at 530 nm
appears. Hyperoside exhibits a selective and sensitive light-up
response to β-Gal with a good linear relationship in broad range.
Meanwhile, excellent biocompatibility, low cytotoxicity, outstanding
photostability, large Stokes shift, self-excited aggregation emission,
and good intracellular retention demonstrate that hyperoside is very
compatible for on-site and long-term imaging of β-Gal in living cells.
This study offered a target activated strategy for fabrication of novel
AIE probes in biomedical and diagnostic applications. In addition,
results indicated that natural products are new sources to find more
FL molecules with outstanding advantages.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Long-term bioimaging of β-Gal in living cells.
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This work was financially supported by the National Natural
Science Foundation of China (31660181), and the Natural
Science Foundation of Hunan Province, China (2018JJ1043).
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