Activatable Formation of Emissive Excimers for Highly Selective Detection of β-Galactosidase

Article abstract of DOI:10.1021/acs.analchem.9b04806

Small-molecular fluorescence sensors have become promising detection tools in many fields attributing to their high sensitivity, excellent temporal and spatial resolution, and low cytotoxicity. However, high concentration or aggregation-induced fluorescence quenching effect has usually hindered the development of traditional fluorescence dyes. Herein, a new fluorophore cyanovinylene dye BMZ with excimer emission property has been constructed. It shows an obvious enhanced and red-shift emission upon aggregation in aqueous solution, which overmatches the conventional pyrene with longer absorption and emission wavelengths. Using this unique optical property, a new fluorescence probe BMZ-Gal has been developed for trapping of β-galactosidase (β-Gal) activity with high selectivity, low limit of detection of 0.17 U, and rapid recognition, which is based on the β-Gal-induced formation of red-shift emitting excimer. β-Gal has a strong affinity for BMZ-Gal, which is verified through the Michaelis-Menten constants (K_m, 1.87 μM) and the hydrolysis efficiencies (K_cat/K_m, 1.78 × 10³ M^-1 s^-1). Furthermore, the assay system has been successfully used for detecting endogenous β-Gal in living ovarian cancer cells and can passively targeted to identify β-Gal in organelle level and determine its subcellular location with satisfactory accuracy. We anticipate that the new fluorophore cyanovinylene dye herein may inaugurate new opportunities for the development of excimer emission sensors.

Full text of DOI:10.1021/acs.analchem.9b04806

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Article
Activatable Formation of Emissive Excimers for
Highly Selective Detection of Beta-Galactosidase
Yang Li, Lulu Ning, Fang Yuan, Tian Zhang, Jianjian Zhang, Zhigang Xu, and Xiaofeng Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b04806 • Publication Date (Web): 20 Mar 2020
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Page 1 of 10
Analytical Chemistry
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Activatable Formation of Emissive Excimers for Highly Selective
Detection of Beta-Galactosidase
Yang Li,†,# Lulu Ning,‡,# Fang Yuan,† Tian zhang,§ Jianjian Zhang,†,* Zhigang Xu,§,* and Xiao-Feng
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Yang†,*
†Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Key Lab of Modern
Separation Science in Shaanxi Province, College of Chemistry and Materials Science, Northwest University, Xi’an, Shaanxi
710127, P. R. China
‡Shaanxi Provincal Key Laboratory of Papermaking Technology and Specialty Paper Development, College of Bioresources
Chemical and Materials Engineering, Shaanxi University of Science & Technology, Xi’an, Shaanxi 710021 P.R. China
§Institute for Clean Energy and Advanced Materials, School of Materials and Energy, Southwest University, Chongqing
400715, P. R. China.
*Corresponding Author: Zhang J., zhangjj@nwu.edu.cn; Xu Z. zgxu@swu.edu.cn, Yang X. xfyang@nwu.edu.cn.
ABSTRACT: Small-molecular fluorescence sensors have become promising detection tools in many fields attributing to
their high sensitivity, excellent temporal and spatial resolution, and low cytotoxicity. However, high concentration or
aggregation-induced fluorescence quenching effect has usually hindered the development of traditional fluorescence dyes.
Herein, a new fluorophore cyanovinylene dye BMZ with excimer emission property has been constructed. It shows an
obvious enhanced and red-shift emission upon aggregation in aqueous solution, which overmatches the conventional
pyrene with longer absorption and emission wavelengths. Using this unique optical property, a new fluorescence probe
BMZ-Gal has been developed for trapping of β-galactosidase (β-Gal) activity with high selectivity, low limit of detection of
0.17 U, and rapid recognition, which is based on the β-Gal-induced formation of red-shift emitting excimer. β-Gal has a
strong affinity for BMZ-Gal, which is verified through the Michaelis–Menten constants (Km, 1.87 μM) and the hydrolysis
efficiencies (Kcat/Km, 1.78 × 103 M-1s-1). Furthermore, the assay system has been successfully used for detecting endogenous
β-Gal in living ovarian cancer cells and can passively targeted to identify β-Gal in organelle level and determine its
subcellular location with satisfactory accuracy. We anticipate that the new fluorophore cyanovinylene dye herein may
inaugurate new opportunities for the development of excimer emission sensors.
Small-molecule fluorescent probes are a series of
molecules whose fluorescence behavior can be changed in
the process of molecular recognition or alteration in matrix
components.1-12 The distinctive optical properties of
fluorescent probes have spurred their potential
applications in biology, chemistry, clinical medicine, and
environmental science, which gained considerable
attention in past decades.13-14 The fluorescence-based
method possesses the particular advantages of higher
sensitivity, superior temporal and spatial resolution in cell
imaging and lower cytotoxicity. However, conventional
small-molecule fluorescent dyes, such as fluorescein,
rhodamine, cyanine, and their derivatives share a common
pitfall that only in the dilute solution (≤ 10-5 M) do they
present obvious fluorescence.15 Their fluorescence
emission is seriously quenched in highly concentrated
solutions or solid-state, due to the fact that these
fluorescent molecules tend to aggregate when combined
with the analytes or dispersed in a poor solvent, which
hinders their applications in bioimaging.
Pyrene, first discovered by Försten, is a typical excimer
that formed by the interaction between molecules in the
excited state and the same molecules in the ground state.16-
19
The fluorescence spectrum of pyrene excimer is
completely different from that of the original pyrene
molecule because the interaction between pyrene
molecules reduces the energy of the excited state and
changes the nature of the original pyrene molecule.
Compared to other fluorescent dyes, pyrene present
distinctive properties that both in dilute and high-
concentrated solutions does it display fluorescent signals.
In a dilute n-hexane solution (≤ 10-5 M), a single molecular
fluorescence band (λ ≈ 375 nm) occurs. With the increased
concentration, the fluorescence of the single molecule is
quenched and a new spectral band with approximate
Gaussian distribution appears in the longwave (λ ≈ 480
nm). The concentration-dependent unique luminescence
properties of pyrene result in the obvious redshift,
broadening, and smoothness of peak profile, which makes
pyrene excimer become a research hotspot in many fields
such as chemistry, biology, and material sciences. Several
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fluorescence dyes based on excimer property have been
2550, Japan). The XRD data was collected on Bruker D8
designed.20-22 Hadziioannou’s group21 observed a strongly
red-shifted, long lifetime emission from the cyano-
substituted oligo (para-phenylene vinylene) derivatives,
which has molecular π-stacking in the crystal lattice with
advance. Nuclear magnetic resonance (NMR) spectra were
performed using a 400 MHz INOVA-400-spectrometer
(Varian Unity, Japan). The chemical shifts (δ) were
referenced to residual DMSO (1H NMR, 2.50 ppm), CHCl3
(1H NMR, 7.26 ppm) and CH3OH (1H NMR, 3.31 ppm). The
fluorescence quantum yields measured by HAMAMATSU
C9920-03 instrument and fluorescence decay was obtained
on FLSP920 fluorescence spectrometer (Edinburgh
Instruments Ltd., U.K). High-resolution electrospray
ionization mass spectra (HRMS) were measured on a
Bruker micrOTOF-Q II mass spectrometer. Fluorescence
images of live cells were recorded by confocal laser
scanning microscope (CLSM, Zeiss 800, Germany).
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22-23
excimer emission character. Recently, Kim et. al.20,
reported a class of organic chromophores based on
excimer-forming benzothiazolyl-cyanovinylene, which
were designed for new “off-on’’ fluorescence probe for the
detection of diverse analytes. Though great effort has been
devoted to the development of excited luminescent
species, these probes have the defects such as low
photoluminescence efficiency, poor aqueous solubility,
and limited synthetic tunability,24-25 which imposes
restriction on the further extension of its applications.
In this work, a new fluorophore cyanovinylene dye
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Enzyme Kinetics. Various concentrations of BMZ-Gal
(2.5, 5, 7.5, 10, 20 μM) were incubated with β-Gal (20 U) at
o
(BMZ),
composed
of
an
electron
acceptor
37 C for 30 min in PBS buffer (20μM, pH = 4.6). After
(benzimidazole), a π-conjugated bridge (cyanovinyl), an
electron donor (N, N-Diethylaniline), and an optically
tunable benzyl group, was developed (Figure 1a). BMZ
displays a significant bathochromic-shift, long lifetime
emission upon aggregation, which was superior to the
traditional pyrene with longer absorption and emission
wavelengths.19 Furthermore, the different benzyl
substituents could regulate the spectral properties by
adjusting the degree of association between the two
molecular dyes. These unique features can be used for the
detection of different biomarkers in biological systems.
Based on this design strategy, a fluorescence sensor (BMZ-
Gal) with excimer luminescence property for β-
galactosidase (β-Gal) was constructed. The water-soluble
D-galactose attached in BMZ-Gal has two functions:
promoting the dispersion of BMZ-Gal in aqueous
solutions and acting as the β-Gal recognition group. BMZ-
Gal is non-aggregating, shows a weak background
fluorescence emission, and once encounter with β-Gal
triggers aggregate formation, resulting in the generation of
the strong, red-shift emissive excimer. The effectiveness of
the novel sensor in living cell imaging was also verified,
which validated the new fluorescent sensor design
strategy.
incubation, the fluorescence intensities were obtained for
quantification analyses (λex/λem = 470/565 nm). The initial
reaction velocity (nmol·min-1) is the slope of the linear
initial part of the progress curve. The kinetic parameters
were calculated by using Michaelis-Menten equation
shown below: V = Vmax[S] (Km + [S]), where V is initial
velocity, and [S] is substrate concentration.
Cell Culture and Cytotoxicity Test. SKOV-3 cells
were cultured at 37 oC under a humidified 5% CO2
atmosphere in Dulbecco’s modified Eagle’s medium
(DMEM), containing 10% fetal bovine serum (FSB) and 1%
penicillin/streptomycin. The cytotoxic effect of BMZ-Gal
were
assessed
by
3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2-H-tetrazolium bromide (MTT), and cells were
seeded in 96-well plates(1×104 cells/well) where BMZ-Gal
(final concentration 0, 10, 20, 50, 70, and 90 μM) was added
to. The assays were performed in six sets for each
concentration.
Cell Imaging and Confocal microscopy Imaging.
SKOV-3 cells were stained with 4',6-diamidino-2-
phenylindole (DAPI) (NucBlue Live Ready Probes Reagent,
Thermo Fisher) for the nuclei as protocols. Then the cells
were incubated with BMZ-Gal (30 μM, component of
DMSO less than 3‰) for 30 min. The cells were incubated
with D-galactose (1 mM) to suppress β-Gal levels within the
cells and incubated with hydroxyurea (HU, 20 μM) to
promote cell senescence leading to excessive secretion of
β-Gal in cells. The cells were incubated with Lyso-tracker
red (100 nM). Cell imaging excitation/emission
wavelengths were 405/490 ± 20 nm for DAPI, and 470/565
± 20 nm for BMZ-Gal. Confocal microscopy imaging
excitation/emission wavelengths were 470/565 ± 15 nm for
BMZ-Gal, and 559/595 ± 15 nm for Lyso-tracker red.
EXPERIMENTAL SECTION
Materials. β-galactosidase (β-Gal), Lysozyme, Glucose,
Cysteine (Cys) and glutathione (GSH) were purchased
from Sangon Biotech. Co., Ltd. (Shanghai, China). DL-
Dithiothreitol (DTT) was purchased from J&K (Beijing,
China). Anhydrous dichloromethane (DCM) was pre-dried
with CaH2 and sectional distilled before use. SKOV-3 cells
(Human ovarian adenocarcinoma cells) were obtained as a
gift from Faculty of Materials and Energy of Southwest
University (Chongqing, China). Unless for special needs,
all other reagents, obtained from qualified commercial
suppliers, were used without further purification.
RESULTS AND DISCUSSION
Synthesis of fluorescence dye BMZ. Figure 1a shows
the synthesis route of BMZ. 4-(diethylamino)
benzaldehyde
benzo[d]imidazol-2-yl) acetonitrile
1
was first reacted with 2-(1H-
to generate
Instrumentation. The fluorescence spectra were
2
recorded on
a
Shimadzu RF-5301 fluorescence
compound 3 via Knoevenagel condensation reaction in the
presence of piperidine. Further treatment of compound 3
with Benzyl bromide in acetonitrile (CH3CN) containing
spectrophotometer equipped with Hamamatsu (Japan)
928 PMT at the slits of 3/5 nm. Absorption spectra were
measured on a UV-visible spectrometer (Shimadzu UV-
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Analytical Chemistry
K2CO3 gave BMZ in 50% yield over two steps. The final
product BMZ was fully characterized by H NMR and
HRMS provided in the supporting information.
side of the stack, the distance between neighboring planes
is small (3.65 Å), which was a typical π-π interaction
distance.25 In the light excitation, the excimer of excited
BMZ dimer was formed, showing the luminescence
characteristics significantly different from that of single-
molecule. BMZ was further characterized by powder XRD
(PXRD) analysis (Figure S4). The characteristic peaks of
BMZ powder was coincide with that of BMZ single-crystal,
which demonstrated that BMZ powders, like BMZ single-
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To get insight into the photophysical properties of
cyanovinylene dye BMZ, fluorescence and absorption
spectra of BMZ were scanned. In dilute acetonitrile
(CH3CN) solution, BMZ shows an absorption maximum at
420 nm (ε = 3.06 × 104 M-1cm-1) and a weak fluorescence at
515 nm with low emission quantum yield (ΦF = 0.0001)
(Figure 1b, e), which is attributed to fast non-radiative
deactivation resulted from internal bond rotations of the
vinylene group.22 In contrast, it exhibits a significantly
redshifted, enhanced fluorescent signals with maximum
emission at 565, 569, and 579 nm in water solution, solid
powder, and single crystalline states, respectively. This
phenomenon is mainly attributed to the formation of
excimer via compact accumulation of cyanovinylene
molecules. BMZ can be easily dissolved by organic solvents
such as DMSO, DMF, CH2Cl2, THF, and EtOH, but it is
immiscible with water. Hence, BMZ experience gradual
aggregation in acetonitrile solutions with the increase of
the added amount of water (Figure 1d, e). The content of
the water has little effect on the fluorescence and UV
spectroscopic properties of BMZ until it reached 80%. As
the water content continues to increase to 99%, a new
significant emission band appears at 565 nm, which is
broader, and redshifted with higher 24-fold increases in
intensity compared to that in the pure CH3CN solution
(Figure S1a). On the contrary, the absorbance at 420 nm is
significantly weakened as shown in the absorption spectra,
which results in the color of the solution shifting from
yellow to light yellow (Figure S1b). These unique
fluorescence and UV spectroscopic features correspond
with the formation of suspended molecular aggregates, of
which the existence is validated by dynamic light
scattering (DLS) and transmission electron microscopy
with the estimated diameters (TEM) 92 ± 10 nm, 76 ± 20
nm respectively. (Figure 1c).
In the highly concentrated BMZ solution (500 μM)
with chloroform as a good solvent, a redshift and
attenuation of excimer emission signal at 524 nm were also
observed (Figure S2). This phenomenon revealed that
aggregation was one of the primary causes of fluorescent
change but not the only reason. In increasing
concentrations of viscous glycerol solution, the
fluorescence intensity (515 nm) of BMZ enhanced with the
emission maximum remain unchanged compared to fluid
solutions (Figure S3). Correspondingly, the fluorescence of
the solution change from non-fluorescence to strong green
was easily visible to the naked-eye under UV lamp (365
nm) irradiation (Figure S3d), which due to the
nonradiative relaxation processes were restricted.
Furthermore, the molecular structure and packing mode of
BMZ had been well defined by single-crystal X-ray
diffraction measurement (Figure S4 and S5). Molecule of
BMZ was stacking neatly in the crystal and the offset
columnar aggregation was observed with monomers
packing parallelly. The planes of mother cycles are
staggered with a displacement angle of 26.7°. Allowed by
the fact that the attached benzyl rings located the outer
crystal, are stacked in a dimeric form.
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NC
N
N
N
HN
N
Piperidine
MeOH
Br
+
H
N
K CO ,
N
MeCN
3
2
NC
NC
N
N
O
2
BMZ
1
3
b
d
c
1.2
25
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10
5
569 nm
562 nm
In MeCN
In water
Powder
Crystal
579 nm
1.0
0.8
0.6
0.4
0.2
0.0
510 nm
0
0
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Diameter (nm)
e
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fw (vol%)
fw (vol%)
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0.0
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Wavelength (nm)
Wavelength (nm)
Figure 1. (a) Synthesis route of BMZ; (b) Fluorescence spectra
of BMZ (20 μM) in 1) CH3CN solution (black line), 2) water
solution (green line), 3) solid powders (red line), and single
crystals (orange); The absolute fluorescence quantum yields
of both aggregates and solid powder were 0.9%. [BMZ (20
μM), test in 100% water solution, λex = 470 nm]. (c) DLS
profile of BMZ in water solution, Insert: representative TEM
image of BMZ. Fluorescence (d) and absorption (e) spectra of
BMZ (20 μM) in CH3CN-H2O mixtures with different water
contents (fw).
Time-dependent density-functional theory (TDDFT)26-
28 was used to predict the absorption and emission spectra
of BMZ monomer and dimer (Figure 2 and Table S1). The
main absorption band of the dimer (402.0 nm) is
bathochromically shifted relative to that of monomer
(382.0 nm), and the dimer had wider absorption spectra
(483.2 nm and 404.4 nm). Similar to the trend observed for
absorption spectra, the emission wavelength of dimer
(471.3) still longer than that of Monomer (459.2 nm). The
calculated spectra of monomer and dimer was in
agreement with the experimental spectra qualitatively.
Discrepancies of quantitative evaluations between
theoretical calculations and experiments are reasonable.
On the one hand, this is partly due to the differences
between ensemble averaged properties and dimer
properties. On the other hand, the approximation of DFT
method may contribute to the quantitative inconsistency.
Furthermore, the independent gradient model (IGM)29-30
has been employed to reveal weak interactions between
BMZ monomers and π-π stacking has been observed
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Analytical Chemistry
(Figure 2e, f). The binding energy between the two
Page 4 of 10
characterized with a short lifetime (τavg < 0.5 ns). In
contrast, BMZ in semicrystalline powder showed
biexponential decay character with a long lifetime (τavg
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7
8
monomers has been evaluated and the large binding
energy -83.5 KJ/mol supports the conclusion that BMZ
display considerable tendency to aggregation. The
conformation and binding energy both allow the formation
of excimer.
=
5.2 ns). The aggregates of BMZ suspended in water
solution also display slower decay behavior with a
biexponential feature (τavg = 4.1 ns). Based on these results,
with the improvement of stacking density, the extension of
excited-state lifetime, and the appearance of π-π
superposition in the solid-state, the origin of the new
redshift emission was completely consistent with the pre-
associated excimer emission, which was also found in the
solid-state of pyrene.19
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b
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Encouraged by the above satisfying results, we tried to
extend the potential of BMZ to biosensing applications. A
novel excimer emission BMZ-Gal was designed to detect
β-galactosidase (β-Gal), which plays an important role in
the maintenance of normal life activities by catalyzing the
hydrolysis of glycoside bonds and converting lactose into
galactose. Abnormal levels of β-Gal are involved in various
c
d
f
e
diseases such as Morquio
B
syndrome and β-
galactosialidosis.31 Hence, the detection of β-Gal is
important in the investigation and diagnosis of
interrelated diseases. Efforts have been devoted to
constructing fluorescent β-Gal probes and the molecules
are displayed in Table S2.32-42 But improvements are still
needed to decrease background fluorescence, raise signal-
to-noise ratio and shorten incubation times in order to
optimize the applications of β-Gal probes in biological
systems. To serve this purpose, BMZ-Gal was designed by
covalently connecting D-galactose residue to the benzyl
group of BMZ periphery (Scheme 1), which could be used
for the imaging of endogenous β-Gal in ovarian cancer
cells.
Figure 2. (a), (b) Initial structure of monomer and dimer; (c),
(d) Optimized structure of monomer and dimer; (e), (f) The
sideview and top view of the isosurface of IGM.
Moreover, the excimer character of BMZ was also set
up from the fluorescence lifetime in different media
(Figure S6). In pure CH3CN solution, where emission solely
took place from single-molecule dye, the excited-state
decay dynamics of BMZ was monoexponential
Strong Fluorescence
dimerization
β-Gal
12
Excimer
BMZ-Gal
Scheme 1. Schematic illustration of excimer emission responses of BMZ-Gal towards β-Gal.
As expected, the attached water-soluble D-galactose
enabled the probe BMZ-Gal to fully dissolve in solutions
composed of CH3CN and water in different proportions
(Figure S8). A single-molecule fluorescence at 520 nm was
observed with a slight decrease in intensity as water
content increases (80%-99%). However, after adding 10 U
β-Gal to the corresponding solutions, spectra at 565 nm
appeared with the phenomenal redshift, spreading and
smoothness of emission peaks. Compared to that of the
solution without β-Gal at the water content of 99%, the
excimer emission intensity at 565 nm was enhanced over
15-fold. Such obvious spectral changes were caused by the
aggregation of the insolubility compound 12, which was
released by the hydrolysis of the glycosidic bond of BMZ-
Gal by β-Gal and displayed the same characteristic of
excimer emission as BMZ did. The β-Gal-induced
hydrolysis BMZ-Gal was confirmed by High-Resolution
Mass Spectra analysis and HPLC (Figure S9), which has
been widely reported.40, 43 In the detection system of BMZ-
Gal and β-Gal, the peaks of BMZ-Gal and its hydrolysis
product were found at m/z 585.2698 and 423.2169,
respectively. All these results showed that the product 12
generated by the reaction of probe BMZ-Gal and β-Gal
aggregated in situ and gave bright excimer fluorescence.
Due to the fact that β-Gal is mainly distributed in
cytolysosome that has an acidic microenvironment (pH ≈
4.6),40 the fluorescent responses of BMZ-Gal toward β-Gal
was tested at different pH values to assess the activity of
BMZ-Gal in cytolysosome (Figure 3a, b, and S10).
Fluorescence signals are gradually enhanced at 565 nm
when pH decreased from 9.0 to 3.7. This phenomenon may
be caused by the fact that compound 12 is less soluble in
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Analytical Chemistry
acid environment than in alkaline aqueous solution. The
the microenvironment of lysosome44 and induced BMZ-
1
2
3
4
acidulous phenolic hydroxyl group can be deprotonated in
alkaline aqueous solution, which enhances the solubility of
compound 12 and inhibit its aggregation. Hence, pH 4.6 is
chosen as the experimental condition, which could mimic
Gal to be well responsive to β-Gal at the same time. At this
pH, the emission intensity of BMZ-Gal in the present of β-
Gal is 108.7, which is 11-fold higher when compared with
their initial states (9.7).
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8
b150
c
a160
β-Gal
Control
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β-Gal
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pH
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Blank and
other analytes
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pH
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d150
e
f
Concentration (μM)
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25 U
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5
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β-Gal
0
y = 11.79 + 12.13 × [β-Gal]
R2 = 0.9942
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5
10
15
20
25
β-Gal (U)
Wavelength (nm)
Time (min)
Figure 3. a) Fluorescence spectra of BMZ-Gal (20 μM) reacted with β-Gal (10 U) at different pH (3.7, 4.3, 4.6, 5.1, 5.5, 5.9,
6.3, 6.8, 7.2, 7.6, 8.0, 9.0) in phosphate buffer (20 mM). b) The β-Gal (10 U) activated Em565 of the BMZ-Gal (20 μM) as a
function of pH, λex/λem = 470/565 nm. c) Fluorescence spectra of BMZ-Gal (20 μM) reacted with various analytes (β-Gal,
lysozyme, trypsin, xanthine oxidase (XO), glucose, H2S, H2O2, Hcy, GSH, Cys, Dithiothreitol (DTT), Al3+, Fe3+, Mg2+, Ca2+,
Cu2+) in phosphate buffer (pH 4.6, 20 mM) for 30 min; (d) Time-dependent fluorescence intensity of different
concentrations of BMZ-Gal (0~20 μM) reacting with β-Gal (10 U) in PBS buffer solution (20 mM, pH 4.6) at 37 oC, λex/λem
=
470/565 nm. (e) Fluorescence spectra of BMZ-Gal (20 μM) in the presence of different concentrations of β-Gal in PBS buffer
solution (20 mM, pH 4.6) at 37 oC. (f) Em565 of BMZ-Gal as a function of the concentration of β-Gal.
The selectivity of probe BMZ-Gal targeting β-Gal has
been investigated. Interferences of various biological
species including enzymes (Lysozyme, Trypsin, and
Aldehydrase), bioactive molecules (H2O2, H2S, Hcy, GSH,
and Cys), ions (Al3+, Fe3+, Mg2+, Ca2+, and Cu2+), Glucose,
and DTT were studied systematically. The results indicate
that BMZ-Gal is free from the disturbance caused by these
substances and has a quite high selectivity for β-Gal (Figure
3c and S11). Furthermore, the sensitivity and efficiency of
β-Gal have also been examined. The time course of the
excimer emission intensity at 565 nm in the existence of β-
Gal (10 U) was established. As shown in Figure 3d (magenta
line), upon the addition of β-Gal, the fluorescence signal
was obviously increased and substantially reached a
maximum within 10 min. In addition, the hydrolysis rate
constants (Kcat) and the Michaelis–Menten constants (Km)
of β-Gal towards the probe BMZ-Gal was found to be 0.20
min-1 and 1.87 μM, respectively (Figure 3d). Hence, the
hydrolysis efficiencies (Kcat/Km) of β-Gal towards BMZ-Gal
was calculated to be 1.78 × 103 M-1s-1. A good linear
correlation between the excimer signals and the
concentration of β-Gal (0−5 U, R2 = 0.994) was obtained
with a limit of detection of ≈ 0.17 U (Figure 3e, f). In situ
formation of about 80 nm nanoaggregates was also
confirmed by using DLS and SEM techniques, and
remained stable for at least 48 hours (Figure S13). Based on
the above results, it has been verified that the probe BMZ-
Gal possesses high selectivity, sensitivity, and efficiency
toward β-Gal.
The potential utility of BMZ-Gal for imaging
endogenous β-Gal in living cells was assessed by confocal
fluorescent microscopy (Figure 4). SKOV-3 cells from
human ovarian cancer patients were selected as model
cell lines, in which the endogenous β-Gal overexpression
has been proved.26,45-47 Firstly, the cytotoxicity of BMZ-
Gal was tested by a commonly employed MTT assay. As
shown in Figure S14, according to the statistical analysis,
BMZ-Gal demonstrates good biocompatibility to
cultured cell lines with no toxicity at low concentrations,
whereas it shows mild cytotoxicity at high
concentrations(>20 μM), which may limit its application
in vivo imaging. After incubated with BMZ-Gal (30 μM)
for 30 min at 37 °C, the cells were washed and then
stained with 4',6-diamidino-2-phenylindole (DAPI) to
label nuclei in the cells with blue fluorescence, which is
in distinct contrast with green fluorescence omitting by
BMZ excimer. As shown in the top row of Figure 4b, there
is no green autofluorescence appearing in the control
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cells. In multiple panels of the second row, it was worth
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validate the possible application of BMZ-Gal, premature
senescent cells, which also experience an increased level
of β-Gal48-50, were employed. Hydroxyurea (HU),51
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noting that BMZ-Gal showed an obvious green
fluorescence in the cytoplasm region under the same
condition, which demonstrates that BMZ-Gal could
permeate into the cells and aggregate into excimers with
fluorescence characteristics. When preincubated with D-
(+)-Galactose, an effective β-Gal inhibitor, the cells
exhibited a sharply decreased fluorescence with green
color fading. Further quantification illustrated that
fluorescence intensities for the unpretreated cells was 7-
a
senescence inducer, was used to stimulate the cellular
senescence of SKOV-3. As shown in Figure 4b Row 4, the
addition of BMZ-Gal successfully images senescent
SKOV-3 with a significant increase in fluorescence
intensity, which is about 15-fold stronger than that in -
D
(+)-Galactose-pretreated cells. In contrast experiments,
no significant fluorescence signals were observed in other
types of cells (HepG2, NIH3T3, and Raw 264.7 cells)
incubating with BMZ-Gal (Figure S16). These results
indicated that BMZ-Gal has the capacity to detect β-Gal
at cell levels, which is of great significance to detect
ovarian cancer or senescent cells at early stage.
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fold higher than that for the -(+)-Galactose-pretreated
D
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cells (Figure S15), indicating that hydrolysis of BMZ-Gal
catalyzed by β-Gal is the primary cause of green
fluorescence and the inhibition of β-Gal decreases the
content of excimer’s building block BMZ. To further
Figure 4. Imaging in SKOV-3 cells. (a) Illustration of the mechanism of the formation of excimer emission from the probe BMZ-
Gal in cell lysosomes. (b) Confocal fluorescence images of endogenously produced β-Gal with probe BMZ-Gal in SKOV-3 cells.
(First row) SKOV-3 cells without any treated. (Second row) cells were incubated with BMZ-Gal (30 μM) for 30 min; (Third row)
cells pretreated with D-(+)-Galactose (1 mM) for 30 min and then incubated with BMZ-Gal (30 μM) for 30 min. (Forth row) cells
were incubated with HU (20 μM) for 12 h and then with BMZ-Gal (30 μM) for 30 min.
a
d
c
b
50 μm
50 μm
50 μm
50 μm
50k
40k
30k
20k
10k
0
e
g
LTR99
BMZ-gal
f
r=0.7136
50 μm
0
20
40
60
80
Distance (μm)
Figure 5. Confocal microscopy images of live SKOV-3 cells co-stained by LTR99 (100 nM) and BMZ-Gal (20 μM). The cells
treated with BMZ-Gal (20 μM) for 30 min at 37 °C and the medium was replaced with fresh medium containing LTR99 (100 nM)
and incubated for 30 min. Images for LTR99 (a) and BMZ-Gal (b) were then recorded using excitation wavelengths of 559 and 470
nm, and collection windows were at 580−610 nm and 550−580 nm, respectively. (c) The merged images of (a), (b) and (bright field
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Analytical Chemistry
of cells). (d) The merged images of (a) and (b). (e) The 3D perspective observation of (d). (f) Intensity profile of regions of interest
(red arrow in panel d) across SKOV-3 cells. (g) Displayed the colocalization areas of the red and green channels selected.
1
Additional information as noted in the text (Figures
2
3
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6
7
8
9
To evaluate the ability of BMZ-Gal to detect the
subcellular location of β-Gal has been determined.
S1−S28); Synthesis, characterization (1H NMR and HRMS
spectra), UV-Vis absorption, and fluorescence spectra. (PDF)
SKOV-3 cells were incubated with BMZ-Gal and LTR99
(commercially available lysosome indicator) in turn,
which was depicted in pseudo green and red colors in the
confocal microscopy images respectively (Figure 5a, b).
The green images produced by BMZ excimers almost
overlapped with the red images rendered by lysosome
indicator LTR99, with the colocalization coefficients for
cell groups higher than 0.71 (Figure 5g). This
phenomenon was consistent with previous researches
reporting that β-Gal is largely distributed in the
lysosome,40 which confirmed the location ability of BMZ-
Gal. Moreover, the fluorescence intensity profiles of
BMZ-Gal and LTR99 in the regions of interest (ROI,
given by a red line) were well-fitting (Figure 5d, f). This
observation further confirmed the fact that BMZ-Gal
entered into the cell lysosome and was activated by β-Gal
to form excimer in situ and then emitted fluorescence.
Therefore, it can be verified that the probe BMZ-Gal was
able to identify β-Gal in organelle level and determine its
subcellular location.
AUTHOR INFORMATION
Corresponding Author
*E-mail: zhangjj@nwu.edu.cn
*E-mail: zgxu@swu.edu.cn
*E-mail: xfyang@nwu.edu.cn
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ORCID
Jianjian Zhang: 0000-0002-5220-1280
Notes
#These authors contributed equally to the present work.
The authors declare no competing financial interest.
ACKNOWLEDGMENT
J.Z. thanks Northwest University for Start-Up grant and
National Natural Science Foundation of China (21904105) for
the financial support. X.Y. thanks the Natural Science
Foundation of China (Nos. 21475105, 21675123) and the Science
& Technology Department (No. 2018JM2001) of Shaanxi
Province of China.
CONCLUSIONS
REFERENCES
In summary, a novel small molecular fluorescence dye
BMZ with excimer emission property has been
constructed. The dye BMZ is composed of a principal part
(cyanovinylene) and an optically tunable benzyl group.
The structure of BMZ is determined by single crystal. BMZ
shows a weak single molecular emission in organic solvent,
while it exhibits a strong, significant bathochromic-shift
and long lifetime fluorescence in water solution due to
aggregation, which is coincide with excimer luminescence
characters. In addition, we have verified that the approach
can be easily used to develop a new kind of fluorescence
probe BMZ-Gal for trapping of β-Gal activity. BMZ-Gal is
based on β-Gal-induced formation of red-shift emitting
excimer, which can realize the detection of β-Gal activity.
More importantly, BMZ-Gal shows high selectivity, low
LOD of 0.17 U, and rapid recognition for β-Gal. The
Michaelis–Menten constants (Km) and the hydrolysis
efficiencies (Kcat/Km) of β-Gal towards the probe BMZ-Gal
are calculated to be 1.87 μM and 1.78 × 103 M-1s-1,
respectively, indicating that β-Gal has a strong affinity for
BMZ-Gal. Moreover, BMZ-Gal is successfully used for
detecting endogenous β-Gal in living ovarian cells, and
able to passively targeted to identify β-Gal in organelle
level and determine its subcellular location, which
highlights the value of the new excimer emission sensor
design.
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