A Fluorescent Probe with Aggregation-Induced Emission for Detecting Alkaline Phosphatase and Cell Imaging

Article abstract of DOI:10.1002/asia.201801540

Alkaline phosphatase (ALP) is associated with many diseases, and its accurate detection is of great significance. Fluorescent compounds with aggregation-induced emission (AIE) feature show beneficial advantages for serving as fluorescent probes. Herein, a

Full text of DOI:10.1002/asia.201801540

DOI: 10.1002/asia.201801540
Full Paper
Biosensors
A Fluorescent Probe with Aggregation-Induced Emission for
Detecting Alkaline Phosphatase and Cell Imaging
Mingang Lin+, Jing Huang+, Fang Zeng,* and Shuizhu Wu*[a]
Abstract: Alkaline phosphatase (ALP) is associated with
many diseases, and its accurate detection is of great signifi-
cance. Fluorescent compounds with aggregation-induced
emission (AIE) feature show beneficial advantages for serv-
ing as fluorescent probes. Herein, an AIE-active “turn on”
probe for ALP detection was synthesized through incorpo-
rating a strong electron-withdrawing group (cyano) in the
middle and the recognition moiety phosphate group at the
end, thereby rendering a D–A–D structure with a relatively
high conjugation degree and good water solubility. It was
found that the probe TPE-CN-pho is highly sensitive to ALP
in aqueous solution. In the presence of ALP, the hydrophilic
phosphate group on the probe is rapidly removed, resulting
in a decrease in water solubility and subsequent formation
of aggregates, thereby achieving aggregation-induced emis-
sion. Moreover, the probe TPE-CN-pho has also been suc-
cessfully applied to imaging ALP in living cells.
Based on the enzyme’s ability of removing phosphate
groups from a variety of materials, various methods have been
used to study the expression levels and activities of ALP, such
as electrochemistry, chromatography, colorimetric techniques
and peptide microarrays.[5–7] Although these methods can re-
flect the expression and activity of enzymes to some extent,
they cannot be applied in living cells or organisms.[6] Fluores-
cence imaging, as an highly sensitive, fast-response and effi-
cient tool, can be used to track the state, changes, and activi-
ties of targets in cells or even some living organisms;[8–15] thus
it can be used for ALP dynamic detection. As for the fluores-
cent systems for detecting ALP, small-molecule fluorescent
probes are favored because of their good chemical modifica-
tion, biocompatibility and low cost.[16,17]
Introduction
As an important member of the phosphatase family, alkaline
phosphatase (ALP) can catalyze the hydrolysis of almost all
phosphate monoesters to form inorganic phosphates and the
corresponding alcohols, phenols, sugars and etc; and it can
also catalyze the transfer reaction of phosphate groups.[1] ALP
plays an important role in phosphorylation and dephosphory-
lation of protein metabolism in cells. Previous researches indi-
cate that the increase of endogenous ALP is closely related to
the proliferation and differentiation of osteoblasts and bone
mineralization.[2,3] In addition, cumulative clinical manifesta-
tions indicate that abnormal increase of ALP in serum is associ-
ated with many diseases, such as breast cancer, prostate
cancer, liver dysfunction, intestinal disease, bone disease, and
diabetes.[2–4] Thus examination of ALP activity/level could pro-
vide insight into how it works in a physiological environment
and how it regulates treatment under pathological conditions.
Thus, ALP has been included in the clinical routine tests.[4]
Molecules with aggregation-induced emission (AIE) feature
have beneficial advantages for serving as fluorescent
probe.[18–24] AIE fluorescent probes usually exhibit large Stokes
shifts with almost no overlap between their excitation and
emission spectra, which is conducive to fluorescence detec-
tion.[25–28] Moreover, the AIE probes have better optical stability
which is especially advantageous for long-time cell imaging
and tracking.[29] As for AIE probes, usually sufficient water solu-
bility can ensure the probe’s good solubility in aqueous solu-
tion with almost no emission; while upon the probe’s reaction
with the analyte, the resultant product has low water solubility,
thus leading to aggregation with enhanced emission.[19–29] At
present, some AIE-active probes for ALP detection have been
reported;[30–37] for most of these probes, they are based on TPE
molecule with relatively low conjugation degree, hence they
usually require short excitation wavelength (e.g. UV light) and
exhibit relatively short emission wavelength, which could
erode the detection accuracy due to autofluorescence of bio-
logical samples (which usually involves blue emission). While in
[a] M. Lin,+ J. Huang,+ Prof. F. Zeng, Prof. S. Wu
State Key Laboratory of Luminescent Materials&Devices
College of Materials Science&Engineering
South China University of Technology
Guangzhou 510640 (China)
E-mail: mcfzeng@scut.edu.cn
shzhwu@scut.edu.cn
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/asia.201801540.
This manuscript is part of a special issue on aggregation-induced emission.
A link to the Table of Contents of the special issue will appear here when
the complete issue is published.
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the case of AIE-active ALP probe, when the conjugation
degree increases, one phosphate group can hardly provide
enough water solubility to maintain the molecularly-dissolved
state of probe molecules with no emission, and it’s quite diffi-
cult to introduce multiple phosphate groups into one probe
molecule and increase the conjugation degree at the same
time. Therefore, to overcome this limitation, AIE probes for
ALP detection with longer emission wavelength and new
structure are highly sought-after.
S8). The probe TPE-CN-pho contains the ALP recognizing
group—hydrophilic phosphate group at the end, which is the
specific substrate of alkaline phosphatase. The probe shows
almost no emission in water. When the probe reacts with ALP,
the phosphate group is eliminated, which converts the phos-
phate group of the probe into hydroxyl group, and corre-
spondingly the reaction product TPE-CN-OH is generated.
Compared with TPE-CN-pho, TPE-CN-OH has poor solubility in
water, and become strongly fluorescent due to the AIE feature,
hence the detection of ALP can be achieved. To confirm the
detection mechanism, the solution of the probe TPE-CN-pho
before and after treatment with ALP for different time was in-
vestigated by HPLC (Figure 1c). Before being incubated with
ALP, the peak for TPE-CN-pho can be observed at 2.07 min,
and the peak for pure TPE-CN-OH is at 2.65 min; while after
being incubated with ALP for 30 min, the peak for TPE-CN-pho
at 2.07 min decreases and in the meantime that for TPE-CN-OH
at 2.65 min appears. After being incubated with ALP for
60 min, only the peak for TPE-CN-OH can be observed. This
result confirms the generation of TPE-CN-OH after the ALP-cat-
alyzed reaction of TPE-CN-pho. The aggregation of TPE-CN-OH
in buffer solution was confirmed by particle size analysis via
dynamic light scattering (DLS) measurements (Figure 1a and
1b). As for TPE-CN-pho, particle size of around 10 nm can be
observed (Figure 1a), while after reacting with ALP for 60 min
larger aggregates with the size of about 100 nm can be ob-
served as shown in Figure 1b, indicating the formation of ag-
gregates from TPE-CN-OH molecules. The fluorescence spectra
of pure TPE-CN-OH and the enzymatic reaction product were
displayed in Figure 1d; by comparison, it is clear that the spec-
tra are quite similar, indicating the enzymatic reaction product
is actually TPE-CN-OH. The above results confirm that, the de-
tection of ALP can be realized by TPE-CN-pho is due to the
conversion of TPE-CN-pho into TPE-CN-OH, which aggregates
in buffer solution, thereby resulting in aggregation-induced
emission with enhanced green fluorescence.
In this study, we designed a new fluorescent “turn-on”
probe for ALP detection. The scaffold of the probe is based on
the AIE-active fluorophore tetraphenylenthene (TPE); through
enhancing its conjugation degree as well as incorporating a
strong electron-withdrawing group (cyano) in the middle and
the hydrophilic recognition moiety phosphate group at the
end, this D-A-D structure has longer excitation and emission
wavelengths compared to TPE and in the meantime can main-
tain sufficient water solubility to quench fluorescence while ex-
tending conjugation length. The probe (TPE-CN-pho) shows
quite good performance for ALP detection, and has longer ex-
citation and emission wavelength compared with other TPE-
based probes for ALP. The probe has quite good water solubili-
ty, and in the absence of ALP, the probe is almost non-emis-
sive. While in the presence of ALP, phosphate group is re-
moved from the probe molecule, correspondingly the probe
turns into the enzymatic reaction product (with the same
structure as TPE-CN-OH) which has low water solubility, and
TPE-CN-OH molecules instantly form aggregates, subsequently
strong green emission can be observed. Moreover, the probe
has been successfully applied to imaging ALP in live cells.
Results and Discussion
Probe Synthesis and Detection Mechanism
The probe TPE-CN-pho was prepared via the synthetic route as
shown in Scheme S1. The chemical structure of TPE-CN-pho
was confirmed by NMR and Mass spectrometry (Figures S1–
Scheme 1. Illustration of the probe’s response toward ALP, chemical structures of the probe TPE-CN-pho and the enzymatic reaction product TPE-CN-OH.
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water content, the greater the fluorescence intensity. TPE-CN-
OH exhibits typical AIE feature. Figure 2c shows the fluores-
cence spectra of TPE-CN-pho and TPE-CN-OH in Tri-HCl buffer
pH 8.0 (containing 1% DMSO); it is clear that TPE-CN-OH ag-
gregates exhibit obvious fluorescence compared with TPE-CN-
pho.
Figure 3a shows the fluorescence spectra of the probe TPE-
CN-pho (10 mm) in Tris buffer (10 mm, pH 8.0) at different time
intervals after incubation with ALP (200 ULÀ1). The fluores-
cence intensity of the solution is very weak in the absence of
ALP. However, after incubation with ALP at 378C, the fluores-
cence intensity of the solution increases. As the incubation
time increases, the fluorescence intensity also increases. After
incubating the TPE-CN-pho solution with ALP (200 ULÀ1) for
60 min, the fluorescence intensity at 543 nm (Figure 3a) in-
creases by about 9-fold. Figure 3c shows the fluorescence
spectra of TPE-CN-pho in Tris buffer (10 mm, pH 8.0) after incu-
bation for 60 min with different concentrations of ALP (0–
200 ULÀ1). As expected, TPE-CN-pho shows little fluorescence
before addition of ALP. However, the fluorescence intensity in-
creases after incubation with ALP. When the concentration of
Figure 1. (a) Particle sizes measured by DLS for TPE-CN-pho (10 mm) without
incubation with ALP. (b) Particle sizes measured by DLS, before collecting
the DLS data, 60 minutes reaction of TPE-CN-pho and ALP at 378C in Tris
buffer (10 mm, pH 8.0) was carried out. (c) HPLC data for TPE-CN-pho and
the reaction mixture of TPE-CN-pho and ALP at different incubation time.
TPE-CN-pho without incubation with ALP (black); TPE-CN-pho
(10 mm)+200 ULÀ1 ALP after incubation for 30 min (red); TPE-CN-pho
(10 mm)+200 ULÀ1 ALP after incubation for 60 min (pink); pure TPE-CN-OH
(blue). (d) Fluorescence spectra of pure TPE-CN-OH aggregates (red line) and
enzymatically reacted product (black line) (in Tri-HCl buffer pH 8.0, contain-
ing 1% DMSO). lex =440 nm.
Fluorescence detection of ALP
First, absorption spectroscopy tests were conducted for TPE-
CN-pho and TPE-CN-OH (Figure S9). The probe TPE-CN-pho
shows absorption peak at around 375 nm, while TPE-CN-OH
(the enzymatic reaction product) displays absorption peak at
around 400 nm. Then we tested the AIE feature of the com-
pound TPE-CN-OH. Figure 2a and 2b shows the aggregation-
induced emission behavior of TPE-CN-OH in THF and water
mixture solutions. When the volume ratio of water in the
system is 80% or less, TPE-CN-OH is almost non-fluorescent
because TPE-CN-OH has a high solubility in the system and
fluorescence is quenched due to intramolecular rotation that
dissipates the excited-state energy. When the volume ratio of
water in the system is above 80%, the degree of aggregation
of TPE-CN-OH increases due to its low solubility in water, and
the solution emits strong fluorescence; and the higher the
Figure 3. (a) Fluorescence spectra of TPE-CN-pho at different time intervals
after incubation with ALP (200 ULÀ1). lex =440 nm. (b) Fluorescence intensity
at 543 nm of TPE-CN-pho at different time intervals after incubation with dif-
ferent concentration of ALP (0, 100, 200 ULÀ1). (c) Fluorescence response of
TPE-CN-pho (10 mm) upon the addition of different amounts of ALP: 0 ULÀ1
200 ULÀ1. lex =440 nm. (d) Fluorescence intensity at 543 nm upon the addi-
tion of different amounts of ALP: 0 ULÀ1–200 ULÀ1
–
.
Figure 2. (a) Fluorescence spectra of TPE-CN-OH (10 mm) in THF/water mixture at different volume ratios of water (0%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% and 99%). (b) Fluorescence intensity at 543 nm at different volume ratios of water. lex =400 nm. (c) Fluorescence spectra of pure TPE-CN-pho
(black) and TPE-CN-OH (red) (in Tri-HCl buffer pH 8.0, containing 1% DMSO) lex =440 nm.
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ALP is increased to 200 ULÀ1, the fluorescence intensity at
543 nm increases by about 9 times. The detection limit is de-
termined as 14.2 ULÀ1 (Figure S10). And the probe can function
well in the pH range of 7.4–9.5 (Figure S11). These results indi-
cate that TPE-CN-pho can effectively respond to ALP fluores-
cently.
ALP is usually overexpressed in cancer cells, so TPE-CN-pho
could be used for imaging ALP overexpressed cancer cells. In
this experiment, HeLa, L929 and HepG2 cells were incubated
with TPE-CN-pho; after washing once with PBS, the cells were
directly imaged by using a fluorescence microscope. The fluo-
rescence image of HeLa cells cultured with TPE-CN-pho shows
green fluorescence, while the control group shows almost no
fluorescence (Figure 5a). The occurrence of green fluorescence
is attributed to the excess ALP in HeLa cells which cleaves the
hydrophilic phosphate group from TPE-CN-pho thus generat-
ing TPE-CN-OH, and thereby emitting strong fluorescence
upon aggregation. To further prove that the green fluores-
cence observed in HeLa cells was caused by ALP, the cells were
incubated with the ALP inhibitor (levamisole hydrochloride)
and the probe TPE-CN-pho; and it can be observed that the
green fluorescence disappears. The results indicate that the in-
tracellular fluorescence changes in HeLa cells are indeed
caused by ALP. Moreover, we used the same method to image
HepG2 cells and obtained similar results (Figure 5b). In con-
trast, L929 cells show no significant fluorescence changes after
the addition of TPE-CN-pho because L929 does not overex-
press ALP (Figure 5c). We suppose that the probes internalized
by L929 cells do not undergo dephosphorylation due to the
lack of ALP in the cells, which is in conformity with literature
reports.[38] These results indicate that TPE-CN-pho can be used
as a fluorescent probe for detecting ALP and thereby imaging
cancer cells.
It is known that ethylene-diaminetetraacetic acid (EDTA) can
effectively inhibit the activity of ALP.[37] To verify the specific
recognition of ALP by the probe TPE-CN-pho, the inhibitor
EDTA (0.5 mm) was incubated with ALP for 20 min and then in-
cubated with the probe TPE-CN-pho for 60 min, afterwards
fluorescence spectra were measured. It can be seen from Fig-
ure 4a that after incubating with EDTA the fluorescence of the
probe is as weak as the untreated probe. While without the
addition of EDTA, TPE-CN-pho with ALP treatment displays sig-
nificant fluorescence emission. The result again confirms that
the probe can respond towards ALP.
Figure 4. (a) Fluorescence spectra of 10 mm TPE-CN-pho alone (black), and
TPE-CN-pho incubated with ALP (200 ULÀ1) in the absence of EDTA (blue)
and TPE-CN-pho incubated with ALP (200 ULÀ1) in the presence of inhibitor
EDTA (0.5 mm) (red) in Tris buffer. (b) Fluorescence response of TPE-CN-pho
(10 mm) to various species: 1: Blank, 2: BSA (1 mgmLÀ1), 3: Trypsin A
(1 mgmLÀ1), 4: ChE (200 ULÀ1), 5: GOx (1 mgmLÀ1) and 6: ALP (200 ULÀ1).
lex =440 nm.
Conclusions
A fluorescent “turn-on” probe for ALP detection was success-
fully developed with aggregation-induced emission feature
through incorporating a strong electron-withdrawing group
(cyano) in the middle and the recognition moiety phosphate
group at the end, thereby rendering a D-A-D structure with rel-
atively high conjugation degree and good water solubility and
consequently longer-wavelength emission. After reaction with
ALP, the probe TPE-CN-pho is converted to TPE-CN-OH with
low water solubility. TPE-CN-OH readily aggregates and subse-
quently emits strong green fluorescence, thereby achieving
ALP detection. Moreover, the probe TPE-CN-pho has good bio-
compatibility and has been successfully applied to cell imag-
ing. Evidently, strong green fluorescence can be observed in
HeLa cells and HepG2 cells after incubation with TPE-CN-pho
because they overexpress ALP. In contrast, no fluorescence can
be observed in L929 cells because they do not overexpress
ALP. This study may provide useful insights into devising de-
tection strategies for other enzymes.
Previous studies demonstrated that, several enzymes such
as esterase, pepsin and lysozyme won’t affect the enzymatic
reaction between ALP and the phosphate-containing sub-
strates.[17,31] Hence, in this study, the selectivity of the probe
TPE-CN-pho for ALP detection was tested through incubation
of the probe with some common biologically-relevant proteins
such as BSA (bovine serum albumin), Trypsin, ChE (cholinester-
ase), GOx (glucose oxidase) under the same conditions. As
shown in Figure 4b, TPE-CN-pho does not show fluorescence
enhancement after incubation with these species. The good
selectivity is due to the ability of ALP to specifically decom-
pose the phosphate group in TPE-CN-pho.
Imaging ALP in Live Cells
We first examined cytotoxicity of TPE-CN-pho by using 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide
Experimental Section
(MTT) assay. As shown in Figure S12, cell viabilities of HeLa
cells (human cervical cancer cells), L929 cells (mouse aneuploid
sarcoma cells) and HepG2 cells (human hepatocellular carcino-
ma cells) remain at high levels even when the TPE-CN- pho
concentration is as high as 50 mm, suggesting that TPE-CN-pho
has good biocompatibility.
Materials: Unless otherwise specified, all solvents are purified and
dried according to standard procedures. Phosphatase alkaline
(ALP) from bovine intestinal mucosa and ethylenediaminetetraace-
tic acid disodium salt (EDTA) were purchased from Sigma Aldrich.
Phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO) and
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
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(DCM), ethanol (EtOH), petroleum ether (PE) and tetrahydrofuran
(THF) were purchased from Aladdin. Other chemical reagents were
commercially available and used as received.
Instrumentation: 1H and 13C NMR spectra were recorded on a
Bruker Avance 600 MHz nuclear magnetic resonance spectrometer.
Mass spectra were recorded on a Bruker LCQ DECA XP high per-
formance liquid chromatography-mass spectrometer using the sol-
vents of the chromatographic grade methanol and deionized
water. Fluorescence spectra were recorded on a Hitachi F4600 fluo-
rescence spectrometer. The quartz cells were selected to have a
length and width of 1.0 cm. Absorption spectra and fluorescence
spectra were recorded on a Hitachi U-3010 UV/Vis spectrophotom-
eter and a Hitachi F-4600 Fluorescence Spectro-photometer. The
quartz cells were selected to have a length and width of 1.0 cm.
Fluorescence images were obtained using an Olympus IX 71 with a
DP72 color CCD. The average size distribution and particle size of
TPE-CN-pho and TPE-CN-OH solutions were determined by dynam-
ic light scattering (DLS) with particle size analyzer (Malvern Zetasiz-
er Nano-ZS90) at a fixed angle of 908 at room temperature. HPLC
analysis was determined on Agilent Technologies 1260 Infinity
High Performance Liquid Chromatography. Methanol was used as
eluents for all HPLC experiments. The flow rate was 1 mLminÀ1
.
Synthesis of TPE-CHO: 1-Bromotriphenylethylene (1005 mg,
3 mmol), (4-formylphenyl) boronic acid (675 mg, 4.5 mmol) and tet-
rakis (triphenylphosphine) palladium (73 mg, 0.09 mmol) were
placed in a reaction flask. 20 mL of a mixed solution of 1,4-dioxane
and 2m potassium carbonate solution (v/v=4:1) was added, and
the system was vacuum-filled with nitrogen three times, and
heated to 908C, reacted for 24 h. After the reaction completed, the
solvent was evaporated under reduced pressure and extracted
with dichloromethane/water, and purified by silica gel column
chromatography (petroleum ether: dichloromethane, v/v=5:1, Rf =
1
0.3), to give a white solid, 80% yield. H NMR (600 MHz, CDCl3): d=
9.90 (s, 1H), 7.61 (d, J=8.2 Hz, 2H), 7.19 (d, J=8.2 Hz, 2H), 7.13–
7.09 (m, 9H), 7.04–6.99 ppm (m, 6H).
Synthesis of TPE-CN: The TPE-CHO (115.2 mg, 0.32 mmol) and 4-
bromo-phenylacetonitrile (93.6 mg, 0.48 mmol) were put in a flask,
and a solution of NaOH (13 mg, 0.32 mmol) in anhydrous ethanol
(12 mL) was slowly added with stirring, and then reacting at room
temperature for 3 h. After completion of the reaction, the solution
was concentrated; the reaction flask was cooled in the refrigerator
until there was precipitation. The reaction solution was filtered to
obtain a precipitation solid, which was washed with ethanol and
then dried in vacuum, to give a light yellow solid, yield 75%.
1H NMR (600 MHz, CDCl3): d=7.65 (d, J=8.4 Hz, 2H), 7.57–7.52 (m,
2H), 7.51–7.47 (m, 2H), 7.39 (s, 1H), 7.15–7.09 (m, 11H), 7.07–
7.01 ppm (m, 6H).
Synthesis of TPE-CN-OH: TPE-CN (268.5 mg, 0.5 mmol) obtained
above, (4-hydroxyphenyl) boronic acid (69 mg, 0.5 mmol) and tet-
rakis (triphenylphosphine) palladium (40 mg, 0.04 mmol) were
weighed in a reaction flask. 16 mL of a mixed solution of THF and
2m potassium carbonate solution (v/v=4:1) were added, and the
system was vacuum-filled with nitrogen three times, and heated to
808C, reacted for 24 h. After the reaction completed, the solvent
was evaporated under reduced pressure and extracted with water/
dichloromethane, and then purified by silica gel column chroma-
tography (petroleum ether: dichloromethane, v/v=1:2, Rf =0.4), to
give a green solid, 80% yield. 1H NMR (600 MHz, CDCl3): d=7.66
(dd, J=8.5, 2.1 Hz, 4H), 7.58 (d, J=8.5 Hz, 2H), 7.50–7.48 (m, 2H),
7.43 (s, 1H), 7.12 (ddt, J=10.2, 6.1, 2.4 Hz, 12H), 7.06 (dd, J=6.7,
3.0 Hz, 2H), 7.05–7.02 (m, 4H), 6.93–6.91 ppm (m, 2H).
Figure 5. Bright field, fluorescence and merged image of HeLa cells (a),
HepG2 cells (b) and L929 cells (c) that had been incubated with TPE-CN-pho
(10 mm) or TPE-CN-pho (10 mm)+inhibitor (1 mm) at 378C for 60 min. Scale
bar=30 mm.
were obtained from Keygen Biotech. 1-Bromotriphenylethylene, (4-
formylphenyl) boronic acid, tetrakis(triphenyl-phosphine) palladi-
um, 4-bromophenylacetonitrile, (4-hydroxyphenyl) boronic acid,
phosphorus oxychloride, ethyl acetate (EA), dichloro-methane
Synthesis of TPE-CN-pho: TPE-CN-OH (275.6 mg, 0.5 mmol) was
weighed into a reaction flask, and dissolved in anhydrous pyridine
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(10 mL) and phosphorus oxychloride (0.466 mL, 5 mmol) was
slowly added in reaction system in ice bath and stirred for 3 h. Ice
cubes were then added to the reaction system and allowed to
react overnight. After the reaction completed, the solvent was
evaporated under reduced pressure, and then purified by silica gel
column chromatography (dichloromethane: methanol, v/v=10:1,
Rf =0.3), to give a yellow solid, 75% yield. 1H NMR (600 MHz,
DMSO): d=7.94 (s, 1H), 7.74 (dt, J=12.4, 8.6 Hz, 6H), 7.57 (d, J=
8.6 Hz, 2H), 7.19–7.10 (m, 11H), 7.05–6.98 (m, 6H), 6.88 ppm (t, J=
5.7 Hz, 2H). 13C NMR (151 MHz, CDCl3): d=155.61, 146.46, 143.36,
143.31, 143.19, 142.34, 141.42, 141.32, 140.04, 132.96, 132.67,
131.93, 131.70, 131.37, 131.32, 131.29, 128.75, 128.31, 127.91,
127.85, 127.68, 127.12, 126.90, 126.73, 126.69, 126.27, 118.16,
115.85, 110.23 ppm. 31P NMR (243 MHz, [D6]DMSO): d=À6.37 ppm.
the precipitates were re-dissolved in methanol for HPLC measure-
ment. The above four solutions were tested with Agilent Technolo-
gies 1260 Infinity High Performance Liquid Chromatography with
methanol as eluent and the flow rate was set as 1 minmLÀ1
.
Acknowledgements
This work was supported by NSFC (21788102, 21875069,
51673066 and 21574044) and the Natural Science Foundation
of Guangdong Province (2016A030312002).
Conflict of interest
General procedure for the fluorescence measurement of TPE-
CN-pho’s response to ALP: All the measurements were performed
in Tris buffer (10 mm, pH 8.0). As for the measurements involving
different concentrations of ALP, TPE-CN-pho (20 mL, 1 mm in
DMSO) was added to Tris buffer in a quartz cell (optical length:
1 cm), followed by different volumes of ALP. The concentration of
ALP in each sample was 0–200 ULÀ1. After incubation at 378C for
60 min, the fluorescence of the reaction solution was measured at
lex =440 nm (slit widths: 5 nm). A control sample that contained
no ALP was also prepared and measured under the same condi-
tions. As for the measurements involving different incubation time,
TPE-CN-pho (20 mL, 1 mm in DMSO) was added to Tris buffer in a
quartz cell (optical length: 1 cm), followed by certain volume of
ALP. After incubation at 378C for different time, the fluorescence of
the reaction solution was measured at lex =440 nm (slit widths:
5 nm). A control sample that contained no ALP was also prepared
and measured under the same conditions.
The authors declare no conflict of interest.
Keywords: aggregation-induced
emission
·
alkaline
phosphatase · biosensors · cell imaging · fluorescence ·
fluorescent probes
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Manuscript received: October 18, 2018
Revised manuscript received: November 13, 2018
Accepted manuscript online: && &&, 0000
Version of record online: && &&, 0000
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FULL PAPER
Biosensors
The joy of being a DAD: An AIE-active
“turn-on” probe, TPE-CN-pho, for alka-
line phosphatase (ALP) detection has
been developed with a donor–accept-
or–donor (D–A–D) structure, in which
phosphate acts as both the recognition
moiety and hydrophilic moiety. The
probe has been successfully applied in
detecting ALP in aqueous media as well
as imaging ALP in living cells.
Mingang Lin, Jing Huang, Fang Zeng,*
Shuizhu Wu*
&& – &&
A Fluorescent Probe with
Aggregation-Induced Emission for
Detecting Alkaline Phosphatase and
Cell Imaging
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Chem. Asian J. 2018, 00, 0 – 0
www.chemasianj.org
8
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!