A fluorescent light-up probe based on AIE and ESIPT processes for β-galactosidase activity detection and visualization in living cells

Article abstract of DOI:10.1039/c5tb01938a

A novel fluorescent probe SA-βGal is reported here with light-up response to β-galactosidase. SA-βGal possesses the β-galactopyranoside group to react with β-galactosidase and releases the fluorescent salicylaldehyde azine with both aggregation induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) characteristics. The linear fluorescent response enables the in vitro quantification of β-galactosidase activity in a range of 0-0.1 U mL^-1 with a detection limit of 0.014 U mL^-1. The probe exhibits significant advantages, such as no self-quenching at high concentrations, a large Stokes shift (190 nm) and high specificity to β-galactosidase with an excellent light-up ratio of 820 fold. Moreover, thanks to its good retention in living cells, the application of SA-βGal for the imaging of cellular β-galactosidase was also achieved with high contrast.

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ARTICLE TYPE
A fluorescent lightꢀup probe based on AIE and ESIPT processes for βꢀ
alactosidase activity detection and visualization in living cells
g
Lu Peng,‡ab Meng Gao,‡c Xiaolei Cai,b Ruoyu Zhang,b Kai Li,c Guangxue Feng,b
Aijun Tong*a and Bin Liu*bc
5
Received (in XXX, XXX) Xth XXXXXXXXX 201X, Accepted Xth XXXXXXXXX 201X
First published on the web Xth XXXXXXXXX 201X
DOI: 10.1039/b000000x
A novel fluorescent probe SAꢀβGal is reported here with lightꢀup response to βꢀgalactosidase. SAꢀβGal possesses the βꢀ
galactopyranoside group to react with βꢀgalactosidase and releases the fluorescent salicylaldehyde azine with both
10 aggregation induced emission (AIE) and excitedꢀstate intramolecular proton transfer (ESIPT) characteristics. The linear
fluorescent response enables in vitro quantification of βꢀgalactosidase activity in a range of 0−0.1 U/mL with a detection
limit of 0.014 U/mL. The probe exhibits significant advantages, such as no selfꢀquenching at high concentrations, large
Stokes shift (190 nm) and high specificity to βꢀgalactosidase with an excellent lightꢀup ratio of 820 fold. Moreover, thanks
to its good retention in living cells, the application of SAꢀβGal for the imaging of cellular βꢀgalactosidase was also
15 achieved with high contrast.
25. Therefore, it is highly desirable to develop βꢀgalactosidase
1. Introduction
probes, which can accumulate in living cells without ACQ.
Contrary to many fluorescent dyes, such as fluorescein or
55 rhodamine, which show bright fluorescence in dilute solution,
but little or no fluorescence in aggregate state because of the
ACQ effect, fluorogens with aggregation induced emission
(AIEgens)26ꢀ31 are a new series of molecules emitting strong
fluorescence in their aggregate states. Heretofore, many
60 AIEgen systems based on hydrocarbons, heteroatoms or
organometallics have been constructed as fluorescent
materials for biomedical applications.32ꢀ41 Usually these
AIEgens show large Stokes shifts with almost no overlap
between their excitation and emission spectra. Besides, they
65 are normally nonꢀfluorescent in solution as a result of the nonꢀ
radiative decay of the excited states caused by intramolecular
motions, while in aggregates, this process is restricted to
produce strong fluorescence.29,35
Glycosidases are important reporter genemarkers. Among
them, βꢀgalactosidase, which is over expressed in primary
ovarian cancers, is
a molecular target for visualizing
20 peritoneal metastases from ovarian cancers1. The enzymatic
activities of βꢀgalactosidase are reporetd to be enhanced in
primary ovarian cancers compared with normal ovaries2. In
addition, abnormally accumulated βꢀgalactosidase activity has
long been reported in senescent cells, allowing βꢀ
25 galactosidase to be an important biomarker for senescence.3, 4
The sensitive detection of βꢀgalactosidase is thus of great
importance.
The fluorescent probes for various analyts have received much
interests because of the advantages such as simplicity, low
30 cost, good sensitivity, and capability of realꢀtime detection,
which enable effective inꢀvitro and inꢀvivo studies.5ꢀ16 Several
probes for βꢀgalactosidase activity assay and visualization in
living cells have been reported, but most suffer from various
disadvantages, such as poor cellular permeability,17 easy
35 diffusion away from living cells,18 small Stokes shifts (<40
Previously, it has been found that a class of salicylaldehyde
70 azines (SAs) are AIEꢀactive29, 36. Besides their AIE property,
they are also characterized with the excitedꢀstate
intramolecular proton transfer (ESIPT)37ꢀ40
.
Usually,
20
21
nm)19,
and selfꢀquenching when accumulated in cells4,
.
compounds with ESIPT characteristics are found to have high
quantum efficiency at high concentrations or in aggregated
75 state due to their large Stokes shift, which greatly reduces self
quenching of fluorescence.41 As a result, SA derivatives that
largely benefit from both AIE and ESIPT characteristics can
find their potential applications as promising fluorescent
materials.
80 As for AIEꢀactive SA derivatives, the hydroxyl groups at the
orthoꢀposition on the benzene ring of these compounds are
responsible for ESIPT and necessary for their AIE
fluorescence.36, 42 Therefore, the fluorescence of SA, which is
AIEꢀactive via ESIPT, can be quenched if the the hydroxyl
85 groups are blocked and can be subsequently activated after
they are unblocked. Recently, we have developped several
fluorescent probes based on this strategy for detection of
thiols43, lysosomal esterase44, palladium cation45, perborate45
and UV light45, 46, demonstrating the versatility of the
For example, Y. Urano and coworkers have constructed a
fluorescent probe consists of the TokyoꢀGreen scaffold, which
is a fluorescein dye, capable of monitoring βꢀgalactosidase
40 activity in living cells.18 However, the fluorescence images of
βꢀgalactosidase in cells provided by this probe is not clear,
mainly because the intracellularly fluorescent product
generated by the probe tends to diffuse across the cell
membrane, which results in a poorly defined signal. To solve
45 this problem, they replaced TokyoGreen with rhodol as the
core fluorophore because rhodol is likely to favor intracellular
accumulation in living cells and they clearly visualized βꢀ
galactosidase activity in cultured cells.19 However, rhodol
23
undergoes aggregationꢀcaused quenching (ACQ)22,
when
50 accumlated in cells, which makes the fluorescence in
aggregate state much weaker as compared to that in solution24,
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platform. In addition, the diameters of the nanoaggrates of the
AIEgens are usually in range of tens to hundreds
a
nanometers, which are suitable for cellular uptake through
endocytosis and could be well retained inside the cells.
5
Herein, we report a fluorescent probe SAꢀβGal with lightꢀup
response to βꢀgalactosidase. SAꢀβGal emits very weak
fluorescence in aqueous buffer. In the presence of βꢀ
galactosidase, the βꢀgalactopyranoside group on SAꢀβGal is
cleaved19, resulting in the formation of strong greenish yellow
10 fluorescent product (SA). The probe shows large Stokes shift,
high lightꢀup ratio and high selectivity towards βꢀ
galactosidase. More importantly, it could be well retrained in
living cells and emit strong fluorescence.
45 Scheme 2 Synthetic route to the compound SAꢀβGal.
2.3 Analytical procedures
The stock solution (1.0×10−2 M) of SAꢀβGal was prepared in
DMSO. Stock solutions of βꢀgalactosidase (100 U/mL) and other
analytes (MgCl2, CaCl2, Vitamin C, Lysozyme, Capthsin B,
50 Esterase and βꢀglucuronidase) were prepared in distilled
deionized water, respectively. In a typical detection, 10 ꢁL of SAꢀ
βGal stock solution was added into a test tube, which was diluted
to 1 mL with PBS buffer (10 mM, pH 7.4). Subsequently, a
proper amount of βꢀgalactosidase stock solution was added. The
55 fluorescence spectra were recorded before and after the addition
of βꢀgalactosidase at different time.
15 Scheme 1 The fluorescent lightꢀup probe SAꢀβGal for βꢀgalactosidase
detection.
2. Experiment
2.4 Cell imaging
C6/LacZ or HeLa cells were seeded at a density of 2 ×104/well in
the chamber (LABꢀTEK, Chambered Cover glass System) and
60 grown for 18 h at 37°C in a humidified 5% CO2 incubator. The
loading solution of SAꢀβGal was prepared by adding 5 ꢁL of its
DMSO stock solution (10 mM) into a microtube. Subsequently
995 ꢁL DMEM was added with vigorous mixing. This loading
solution of SAꢀβGal (50 ꢁM) was added to the cells and the cells
65 were incubated for 2 h at 37 oC. The fluorescence imaging of the
cells was then implemented. The images were collected upon
excitation at 405 nm, and the signals were collected using an
emission filter at 525ꢀ625 nm.
2.1 Materials and instruments
All chemicals were purchased from SigmaꢀAldrich (St. Louis,
20 USA) and used without further purification.
Absorption spectra were measured on a UV visible spectrometer
(Shimadzu UVꢀ1700). All fluorescence spectra were determined
on a fluorescence spectrometer (Perkin Elmer LSꢀ55, United
States). The determination of fluorescence quantum yield was
25 carried out on a combined steady state and fluorescence lifetime
spectrometer (Edinburgh FLS 920, United Kingdom). All NMR
spectra were recorded using a NMR spectrometer (Bruker ARX
400, Germany). The mass spectra were obtained on an ion trap
timeꢀofꢀfight mass spectrometry (Shimadzu MSꢀITꢀTOF, Japan).
30 The pH measurement was performed with a pH meter (Sartorius
PBꢀ10 basic, Germany). Fluorescence imaging was carried out on
3. Results and discussion
70 3.1 Probe Design
a
fluorescence microscope (Olympus FVꢀ1000 confocal
microscope, Japan).
As shown in Scheme 1, after the hydroxyl groups at the orthoꢀ
position on the benzene ring of salicylaldehydeazine (SA) are
blocked by βꢀgalactopyranoside, no ESIPT is possible in the
molecule (SAꢀβGal) because there are no more hydroxyl protons
75 at the orthoꢀposition. Therefore, this blocked compound (SAꢀ
βGal) should be nonꢀfluorescent. Upon the addition of βꢀ
galactosidase to remove the blocking groups, the hydroxyl and
ESIPT in the yielded SA can be recovered to regain the AIE
fluorescence.
2.2 Synthesis of compound SAꢀβGal (Scheme 2)
35 The synthetic route to SAꢀβGal is depicted in Scheme 2. The
reaction between 2ꢀhydroxybenzaldehyde 1 and 2,3,4,6ꢀtetraꢀOꢀ
acetylꢀαꢀDꢀgalactopyranosyl bromide 2 firstly formed compound
3 in 70% yield. Then compound 3 further reacted with hydrazine
hydrate and compound 4 was obtained in 90% yield. Compound
40 4 further reacted with KOH and the compound SAꢀβGal was
produced with 95% yield. The molecular structures were
confirmed to be right by the NMR spectroscopy (details can be
found in Supplementary Information).
80 3.2 The AIE characteristics of SA
The AIE characteristic of SA was investigated in water/DMSO
coꢀsolvents with different water volume fraction (fw= 0ꢀ90 vol%),
and the results are shown in Fig. 1 and Fig. S1 in the
Supplementary information. SA dissolves well in DMSO but is
85 not soluble in water. In a good solvent (fw= 0 vol%), SA disperses
2
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well and exhibits weak fluorescence. Nevertheless, in a poor
solvent (fw= 90 vol%), an intense fluorescence band at 545 nm can
be observed. The AIE effect of SA occurs when the water volume
fraction in the coꢀsolvent is more than 50%. The AIE
fluorescence intensity is also found to be enhanced by a higher
water volume fraction, which correlates well with the increased
aggregates formation in poorer solvents.
5
Fig.1 (a) Fluorescence spectra of 100 ꢁM SA in coꢀsolvent mixtures
Fig.2 (a) Fluorescence spectra of SAꢀβGal (100 ꢁM) in the presence of
various concentrations of βꢀgalactosidase (0ꢀ4.0 U/mL) in PBS buffer
solution (10 mM, pH 7.4, 37 ℃). Insets from left to right: photographs of
55 SAꢀβGal (100 ꢁM) without or with βꢀGal (4.0 U/mL) under UV light
(365 nm). (b) The corresponding fluorescence intensity (I545) change over
time and (c) concentration. (d) Calibration curve of the fluorescence
intensities (I545) versus βꢀgalactosidase concentrations.
10 containing 0~90 % PBS buffer (10 mM, pH 7.4) and 100~10% DMSO, as
well as (b) the corresponding fluorescence intensity (I545) change.
Excitation is performed at 387 nm.
3.3 Study of βꢀgalactosidase activities in solution
15 The fluorescence of SAꢀβGal in the absence and presence of βꢀ
galactosidase was first studied to prove our design principle
above (Scheme 1). As shown in Fig. S2 (Supplementary
information), SAꢀβGal (100 ꢁM) formed colloidal aggregates in
PBS buffer (10 mM, pH 7.4) containing 1% DMSO, which was
20 confirmed by dynamic light scattering (DLS) analysis, indicating
that the average particle diameter is 173 nm. The blocked
compound SAꢀβGal displays very weak fluorescence (quantum
yield Φ<0.001) in the colloid solution, suggesting successful
blocking effect of βꢀgalactopyranoside group. Upon addition of
25 βꢀgalactosidase, the fluorescence of SAꢀβGal enhanced gradually
until strong greenish yellow fluorescence (545 nm) was observed
(quantum yield Φ=0.246). The timeꢀdependent fluorescence
intensity change of SAꢀβGal in the colloid solutions was
recorded without and with the addition of βꢀgalactosidase (Fig. 2b
30 and Fig. S3), showing that the fluorescent lightꢀup effect only
occurs in the presence of βꢀgalactosidase and the reaction could
be fully completed. Thanks to the low background fluorescence
of the blocked compound SAꢀβGal, the lightꢀup ratio could be as
high as 820 fold, which is comparable to that reported by Nagano
35 (986 fold)19 and better than that reported by Tung (110 fold)21. As
shown in Fig. 2d and Fig. S4, a good linearity could be found in
the βꢀgalactosidase concentration range of 0ꢀ0.10 and 0.1ꢀ1.0
U/mL. The detection limit was calculated as 0.014 U/mL based
on the definition by IUPAC (CDL =3Sb/m) from 10 blank
40 solutions, which is similar to that reported by Urano19.
60 The selectivity of SAꢀβGal towards βꢀgalactosidase was also
studied (Fig. 3). Upon the addition of βꢀgalactosidase, the
fluorescence intensity of SAꢀβGal was greatly enhanced.
However, no change was observed with some commonly used
inorganic salts or biomolecules, such as MgCl2, CaCl2, Vitamin C,
65 Lysozyme, Capthsin B, Esterase and βꢀglucuronidase. Thus, SAꢀ
βGal shows good selectivity to βꢀgalactosidase.
Fig.3 (a) Fluorescence spectra of SAꢀβGal (100 ꢁM) upon addition of
various species: MgCl2 (1 mM), CaCl2 (1 mM), vitamin C (1 mM), and
70 enzymes (lysozyme, cathepsin B, esterase, βꢀglucuronidase and βꢀ
galactosidase, each enzyme is 4.0 U/mL) in 10 mM PBS buffer ( pH 7.4,
37 ℃). (b) The corresponding fluorescence intensity (I545).
3.4 Cellular imaging
75 The use of SAꢀβGal in its colloid solutions also has been
investigated to visualize βꢀgalactosidase activity in living cells.
The results of DLS analysis (Fig. S2, Supplementary information)
supported the formation of colloids of SAꢀβGal in aqueous
solutions. The average diameter of the particles of SAꢀβGal in
80 colloid solutions is about 173 nm, which is suitable to be uptaken
by cells through endocytosis and expected to be well retained in
cells. It is reported that βꢀgalactosidase is over expressed in rat
glial tumor C6/LacZ cells.21 Therefore, we applied probe SAꢀ
βGal for imaging βꢀgalactosidase activity in C6/LacZ cells. After
85 incubating C6/LacZ cells with the colloid solution of SAꢀβGal
for 2 h at 37 °C, strong fluorescence was observed (Fig. 4a). In
It has been reported by Urano’s group19 and others4,21 that βꢀ
galactopyranoside can be cleaved by βꢀgalactosidase to release
hydroxyl group. To verify this, the fluorescent product after
reaction between SAꢀβGal and the enzyme was isolated by
45 filtration and subsequently characterized. The results in Fig. S6
clearly suggest the formation of SA from SAꢀβGal. In addition,
the fluorescence spectrum of SAꢀβGal after reaction with βꢀ
galactosidase and that of the reference compound SA are almost
identical (see Fig. S7), supporting the hypothesis in our design
50 (Scheme 1).
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order to demonstrate the specificity of SAꢀβGal towards βꢀ
Notes and references
galactosidase activity, we undertook a control experiment (Fig.
4b). HeLa cells do not express βꢀgalactosidase,47 thus we expect
that there would be no fluorescence observed when HeLa cells
are incubated with SAꢀβGal. As can be seen in Fig. 4b, almost no
fluorescence could be observed in HeLa cells, which confirms the
specificity of probe SAꢀβGal for βꢀgalactosidase activity studies.
aDepartment of Chemistry, Beijing Key Laboratory for Analytical
Methods and Instrumentation, Key Laboratory of Bioorganic
Phosphorus Chemistry & Chemical Biology, Tsinghua University,
Beijing 100084, PR China.
45
50
55
5
Eꢀmail: tongaj@mail.tsinghua.edu.cn
bDepartment of Chemical and Biomolecular Engineering, National
University of Singapore, 4 Engineering Drive 4, Singapore 117585.
Eꢀmail: cheliub@nus.edu.sg
cInstitute of Materials Research and Engineering (A*STAR), 3
Research Link, Singapore 117602
†Electronic
Supplementary
Information
(ESI)
available:
Characterization of SAꢀβGal and supplementary Figures. This
material is available free on line. See DOI: 10.1039/b000000x/
‡ These authors contributed equally to this work
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A fluorescent probe with both AIE and ESIPT characteristics is developed for βꢀgalactosidase activity study in solution and living
cells.
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