In situ localization of alkaline phosphatase activity in tumor cells by an aggregation-induced emission fluorophore-based probes

Article abstract of DOI:10.1016/j.bmc.2019.115284

In situ detection of certain specific enzyme activities in cells is deeply attached to tumor diagnosis. Conventional enzyme-responsive fluorescent probes have difficulty detecting targeted enzymes in situ in cells due to the low detection accuracy caused by the spread of fluorescence probes. In order to solve this problem, we have designed and synthesized an enzyme-responsive, water-soluble fluorescent probe with AIE characteristics, which could aggregate and precipitate to produce in situ fluorescence when reacting with the targeted enzyme in cells. The AIE fluorophore (TPEQH) was utilized to design the enzyme-responsive, fluorescent probe (TPEQHA) by introducing a phosphate group on to it, which could be specifically decomposed by the targeted enzyme, namely alkaline phosphatase (ALP). In tumor cells, TPEQH was highly produced due to the interaction of phosphate on the TPEQHA and the overexpressed ALP. Water-insoluble TPEQH then precipitated and release fluorescence in situ, thereby successfully detecting the ALP. Furthermore, the expression level of ALP could be determined by the fluorescence intensity of TPEQH with higher accuracy due to the inhibition of TPEQH leak, which demonstrated a potential application of in suit ALP detection in both clinical diagnosis and scientific research of tumor.

Full text of DOI:10.1016/j.bmc.2019.115284

Journal Pre-proofs
In Situ Localization of Alkaline Phosphatase Activity in Tumor Cells by an
Aggregation-Induced Emission Fluorophore-Based Probes
Qinghua Guan, Xinmiao Lu, Yue Su, Jichen Xu, Xiaofei Liang, Peiyong Li,
Xinyuan Zhu
PII:
S0968-0896(19)31530-5
https://doi.org/10.1016/j.bmc.2019.115284
BMC 115284
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
10 September 2019
13 December 2019
18 December 2019
Please cite this article as: Q. Guan, X. Lu, Y. Su, J. Xu, X. Liang, P. Li, X. Zhu, In Situ Localization of Alkaline
Phosphatase Activity in Tumor Cells by an Aggregation-Induced Emission Fluorophore-Based Probes,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115284
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In Situ Localization of Alkaline Phosphatase Activity in
Tumor Cells by an Aggregation-Induced Emission
Fluorophore-Based Probes
Qinghua Guan,1,2, † Xinmiao Lu,1,2† Yue Su, 2 Jichen Xu,3 Xiaofei Liang,3 Peiyong Li,*,1 Xinyuan
Zhu*,2
1 Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong
University, Shanghai 200025, China
2 School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix
Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China
3 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji
Hospital, School of medicine, Shanghai Jiaotong University, No. 25/Ln 2200 Xie Tu road,
Shanghai 200032, China
† Authors who made equal contribution in this work and were considered as co-first authors.
Correspondence author: Peiyong Li, Department of Nuclear Medicine, Ruijin Hospital, School
of Medicine, Shanghai Jiao Tong University, Shanghai 200025, P. R. China. Tel: + 86-21-
34188822, fax: + 86-21-34188822, Email: peiyli@vip.sina.com
Correspondence author: Xinyuan Zhu, School of Chemistry and Chemical Engineer, Shanghai
200240, P. R. China. Tel: + 86-21-34206899, fax: + 86-21-54741297, Email: xyzhu@sjtu.edu.cn
1

ABSTRACT
In situ detection of certain specific enzyme activities in cells is deeply attached to tumor diagnosis.
Conventional enzyme-responsive fluorescent probes have difficulty detecting targeted enzymes in
situ in cells due to the low detection accuracy caused by the spread of fluorescence probes. In order
to solve this problem, we have designed and synthesized an enzyme-responsive, water-soluble
fluorescent probe with AIE characteristics, which could aggregate and precipitate to produce in
situ fluorescence when reacting with the targeted enzyme in cells. The AIE fluorophore (TPEQH)
was utilized to design the enzyme-responsive, fluorescent probe (TPEQHA) by introducing a
phosphate group on to it, which could be specifically decomposed by the targeted enzyme, namely
alkaline phosphatase (ALP). In tumor cells, TPEQH was highly produced due to the interaction of
phosphate on the TPEQHA and the overexpressed ALP. Water-insoluble TPEQH then precipitated
and release fluorescence in situ, thereby successfully detecting the ALP. Furthermore, the
expression level of ALP could be determined by the fluorescence intensity of TPEQH with higher
accuracy due to the inhibition of TPEQH leak, which demonstrated a potential application of in
suit ALP detection in both clinical diagnosis and scientific research of tumor.
KEYWORDS: AIE probe, ALP, tumor fluorescence imaging
1. INTRODUCTION
Fluorescent probes have been deemed as powerful tools for optical imaging and analysis due to its
ability to directly visualize the biological analytes at molecular level and provide delicate
information of complex biological structures and processes1-9. However, current fluorescent
probes are generally based on the aggregation-caused quenching (ACQ) fluorophores, which need
2

design as cytoplasm soluble probes in living cell imaging, such as specific imaging for a certain
enzyme.10,11 Therefore, the responsive products of ACQ probes and targeted enzyme easily diffuse
away from the original generation site, leading to the unsatisfied resolution and contrast of
images.12-14 Hence, it is imperative to develop fluorescent probes, which have capacity of
conducting high-resolution and in situ imaging and improve the imaging contrast and accuracy of
the detection results.
To address these limitations, a well-established method is to design a fluorescent probe, which
can generate insoluble fluorescent products after specific respond with target and gather in situ to
emit fluorescence. Unfortunately, ACQ fluorophores have intrinsic difficulty meeting this
requirement due to the quenched fluorescence after aggregation.15 However, with the development
of new aggregation-induced emission (AIE) fluorophores, which show exactly opposite natures to
the ACQ fluorophores, this dilemma is expected to be resolved.16,17 AIE fluorophores are a series
of fluorescence molecules showing faint or quenched fluorescence in the dilute solutions but bright
and obvious fluorescence in the state of aggregation. Obviously, this unique photophysical
phenomenon makes it an ideal precursor to design in situ fluorescent probes.
In this context, an AIE fluorescent probe to detect targeted alkaline phosphatase (ALP) in living
cells was designed based on a new AIE fluorophore TPEQH (Scheme 1). TPEQH is a typical AIE
fluorophore, which is hydrophobic and emits bright fluorescence in the state of aggregation or
solid state. Apart from that, TPEQH had long excitation wavelength and large Stokes Shift, which
contributed to high sensitivity, low background signal and high non-target contrast during
imaging.18-21 Additionally, its hydrophilicity and water solubility could be easily regulated by
modifying the hydroxyl group on the TPEQH. Meanwhile, ALP is a crucial biomarker for tumor
diagnosis in clinical because the expression level of ALP in peripheral blood is significantly
3

increased in many cancer patients. Thence, TPEQH was utilized to design an ALP-responsive,
fluorogenic probe (TPEQHA) by modifying a phosphate group onto the phenolic hydroxyl group
of TPEQH. ALP is a kind of hydrolase which are capable of hydrolyzing phosphate bond on lots
of substrates.22-28 Therefore, when phosphate bond of TPEQHA was specifically decomposed by
ALP, free TPEQH was released and precipitated in a very short time. The solid-state fluorescence
molecule could emit bright fluorescence and successfully detected and imaging ALP in living cells
with high resolution and accuracy.
Scheme 1. In-situ fluorescence imaging of tumor cells with TPEQHA a
a(A) Imaging of TPEQHA in Tumor Cell. (A) The water-soluble TPEQHA was hydrolyzed by
ALP to become a liposoluble TPEQH, which was aggregated in an aqueous solution to induce
fluorescence. (B) In tumor cells, TPEQHA was hydrolyzed by ALP on cytomembrane and
aggregated to induce fluorescence.
2. EXPERIMENTAL SECTION
4

2.1 Materials. Benzene-1,2-diamine, ethyl-2-oxopropanoate and methyl-2-bromoacetate were
bought from Tokyo Chemical Industry Co., Ltd. 4-(1,2,2-triphenylethenyl)benzaldehyde, boron
tribromide and phosphorus oxychloride were purchased from J&K Scientific Ltd. 4-(1,2,2-
Triphenylethenyl)benzaldehyde was acquired (TPE-CHO) from Zhengzhou Alfachem Co., Ltd.
Tetrahydrofuran (THF), K2CO3, ethyl acetate, N,N-dimethylformamide (DMF) and
dichloromethane (DCM) were bought from Adamas.
2.2 Synthesis of 7-Methoxy-3-methylquinoxalin-2(1H)-one (1). To obtained molecule 1,
benzene-1,2-diamine (13.8 g) and anhydrous ethanol (150 mL) were added into a beaker and the
temperature was lowered to 0 ℃. After that, ethyl pyruvate (13.92 g) dissolving in ethanol (10
mL) was added dropwise. After gradually returning to 25 ℃, the mixture kept reacting for 10 h
under magnetic stirring. During the reaction, the insoluble solid product gradually precipitated in
the solution. After the reaction, the product was then filtered and washed with anhydrous ethanol,
followed by the removal of ethanol in vacuum, 16.53 g white solid was obtained in 87% yield. 1H-
NMR and MS spectroscopy were used to identify the product, Figure S1. [LC-MS] m/z: 191.0804
[M+H]+.
2.3 Synthesis of Methyl-2-(7-methoxy-3-methyl-2-oxoquinoxalin-1(2H)-yl)acetate (2). To
synthesize compound (2), into the acetone were suspended with 3.8 g compound (1) and 3.67 g
methyl-2-bromoacetate, followed by the treatment of vigorous stirring. After that, 3.31 g K2CO3
was added, the mixture kept reacting under reflux at 62 ℃ for 12 h. After reaction, the the solid
residues were filtered and the solution was treated with rotary evaporators to remove the acetone.
The solute was then redissolved in ethyl acetate (EA, 50 mL) again and washed by deionized water
for 3 times. After washing, the organic phase was dried with anhydrous MgSO4, followed by the
filtration to remove MgSO4. The solvent was then removed in vacuum to obtain the white crude
5

product. The product was then purified by recrystallization in EA/PE, and 2.72 g product was
1
obtained in 52% yield. H-NMR and MS spectra were used to confirm the successful synthesis,
shown in Figure S2. [LC-MS] m/z: 263.1021 [M+H]+.
2.4 Synthesis of Methyl-2-(7-hydroxy-3-methyl-2-oxoquinoxalin-1(2H)-yl)acetate (3).
Eggplant bottle containing compound (2) (524 mg) was evacuated and filled with nitrogen.10 mL
DCM was added and the solution was cooled to -25 ℃. Then, BBr3/CH2Cl2 solution (1 M) was
added dropwise and the mixture gradually turned bright red. After returning to room temperature,
the mixture continued to react for 24 hours. The reaction was then quenched by adding 10 mL H2O
after the solution was cooled to 0 ℃ again. The solution was adjusted to pH 8 with NH3 solution
(5 M) and the water phase was separated in a separatory funnel. The water phase was washed with
DCM (10 mL × 3) and adjusted to pH 3 with KHSO4 solution (2 M) to induce the precipitation of
solid product. After filtration, the solid was dried in vacuum and light yellow production was
obtained after purification using silica chromatography (dichloromethane : methanol = 30:1) (213
1
mg, 43%). The product was characterized by H-NMR and MS, Figure S3. [LC-MS] m/z:
249.0867 [M+H]+.
2.5 Synthesis of Methyl(E)-2-(7-hydroxy-2-oxo-3-(4-(1,2,2-triphenylvinyl)styryl)-
quinoxalin-1(2H)-yl)acetate (4, TPEQH). Compound (3) (496 mg) were added into the flask
containing 10 mL acetic acid under magnetic stirring, followed by the addition of 4-(1,2,2-
triphenylethenyl) benzaldehyde (FTPE, 1.08 g) to obtained the suspension. After that, 100 μL
catalytic amount of concentrated H2SO4 was carefully dripped into the suspension and the mixture
kept reacting at 60 ℃ for 12 h. After reaction, the solution was cooled to room temperature and
the mixture was dissolved with 60 mL DCM. The DCM solution was then washed with deionized
water (15 mL × 3) and dried over anhydrous MgSO4, followed by filtration to remove the MgSO4.
6

The solvent wasthen removed in vacuum and solid product were obtained after pretreatment,
which was further purified by silica chromatography (EA : PE = 1 : 4) to obtain pure yellow
product (932 mg, 79% yield). The 1H-NMR and MS spectra of final product were shown in Figure
S4. [LC-MS] m/z: 591.2280 [M+H]+.
2.6 Synthesis of Methyl(E)-2-(2-oxo-7-(phosphonooxy)-3-(3-(1,2,2-triphenylvinyl)styryl)-
quinoxalin-1(2H)-yl)acetate (5, TPEQHA). TPEQH (295 mg) was suspended in dry DCM (50
mL) and treated with magnetic stirring at 0 ℃ in the nitrogen atmosphere. POCl3 (0.2 mL) and
dry pyridine (0.3 mL) were added by syringe. The mixture then slowly returned to room
temperature and reacted for 1 h. After reaction finished, ice water (50 mL) was added, and the
reaction solution was extracted with CH2Cl2/EtOH. The combined organic phase was dried with
Na2SO4 and concentrated under reduced pressure. After purified by silica gel chromatography
(CH2Cl2/EtOH, 3:1, v/v), compound HTPQA was obtained as a colorless solid (134 mg, 40%).
The NMR and MS spectra of the finical product were shown in Figure S5. [LC-MS] m/z:
688.19999 [M+NH4]+。
2.7 Spectra Characterization of the TPEQH. For the characterization of the TPEQH, the
UV-Vis and photoluminescence spectra were applied to determine the spectra of TPEQH. Firstly,
the excitation and emission wavelength of solid TPEQH were determined. Secondly, TPEOH was
dissolved in THF to investigate both UV-Vis spectrum and excitation/emission spectra in solution
(1 × 10-5 M). Furthermore, for investigating the emission spectrum in different polar solvents,
TPEQH was dissolved in acetone, THF, DMSO and MeOH (1 × 10-3 M), and measured the
fluorescence spectra.
2.8 Researches on the AIE performance of TPEQH. The AIE performance of TPEQH was
then studied in the mixture solvent of THF and H2O. Namely, the TPEQH solution (dissolved in
7

THF, 2 × 10-4 M, 100 μL) was mixed with different volume of pure THF (1900, 1700, 1300, 900,
500, 300, 100, 0 μL), followed by the addition of different volume of deionized water to adjusted
the total volume to 2 mL (1 × 10-5 M). Additionally, the AIE performance of TPEQH in pure THF
with different concentrations (1 × 10-2, 1 × 10-3, 1 × 10-4, 1 × 10-5, 1 × 10-6, 1 × 10-7 M) was also
determined. The excitation wavelength was set at 300 nm 353 nm.
2.9 Theoretical Investigations. Density functional theory (DFT) calculation was then applied
to deep understand the AIE performance of TPEQH. The calculation was conducted with the
Gaussian09 program.29 Geometry optimization of TPEQH structure was calculated at B3LYP/6-
31G* without any symmetry restriction.30-32 After that, at the same level, analytical vibration
frequencies were performed to determine the nature of the located stationary point.
2.10 The Fluorescence Response of TPEQHA to ALP. The fluorescence response of
TPEQHA to ALP was studied using TPEQHA (100 μM) dissolved in DMSO and ALP solutions
with different concentrations (0, 4, 10, 20, 40, 80, 120, 160, 200, 240, 320, 360 U/L in Tris-HCl
solution). Namely, 100 μL of TPEQHA solution was diluted with 1900 μL Tris-HCl solution. In
the TPEQHA solution (1 mL) was added into a series of ALP solution and incubated for 20 minutes
at 37℃. The fluorescence spectra after response was then determined at the excitation wavelength
of 465 nm.
2.11 In Vitro Cytotoxicity Study. HeLa and L929 cells were seeded in 96-well plates and
incubated at 37 ℃ for 12 h. Then cells were treated with TPEQH at the concentration ranging
from 0 to 20 μg/mL. After 2 d incubation, MTT solution was added into each well and incubated
with cells for another 4 h. After incubation, the medium was carefully removed, and 200 μL DMSO
was added into each well. After the addition of DMSO, plates were slightly shaken for 15 min.
8

Finally, the DMSO solution was taken out and the absorbance of solution was determined at the
wavelength of 570 nm.
2.12 Intracellular Imaging of TPEQHA. To further study the intracellular ALP response and
fluorescence imaging of TPEQHA, ALP was detected in HeLa cells and L929 cells, which were
considered as ALP positive cell and ALP negative control group respectively, using a confocal
laser scanning microscopy (CLSM) and a flow cytometry. Furthermore, in order to determine the
specificity of TPEQHA for ALP, HeLa cells treated with levamisole hydrochloride (1 × 10-3 M)
was detected as positive control group.33,34
For the flow cytometry analysis, there types of cell samples (Hela cell, HeLa cell treated with
levamisole hydrochloride and L929 cell) were seeded in 6-well plates at the density of 5 × 105 per
well. After replacing the old medium with fresh medium containing TPEQHA (10 μΜ), cells were
incubated for another 1 h. After incubation, medium was removed and cells were washed with
fresh PBS for 3 times. Finally, cells were treated with trypsin and the digested cells were measured
using a flow cytometer.
For the CLSM experiments, cells (3 × 105) were planted in the confocal dishes and cultured for
12 h in culture medium. After incubation, the original medium was removed and fresh medium
containing TPEQHA (10 μΜ) was added. After incubating for another 1 h, medium was removed
and cells were washed with fresh PBS. After washing, 1 mL PBS was added and the dishes were
applied for CLSM imaging at a 488 nm laser. Apart from that, TPEQHA was replaced with 4-
Methylumbelliferyl Phosphate (4-MUP) and observed at a 405 nm laser.
2.13 Statistical Analysis. The results are illustrated as the mean ± standard deviation (SD). The
difference was evaluated by Student’s t test, and considered statistically significant if p < 0.05 (*),
respectively.
9

3. RESULTS AND DISCUSSION
3.1 Preparation and Characterization of TPEQH and TPEQHA. The synthetic processes of
TPEQH and TPEQHA were shown in Figure 1. The TPEQH was obtained by the Aldol Reaction
of 4-(1,2,2-triphenylethenyl)benzaldehyde and methoxy quinoxalinone (MQX), while MQX was
synthesized according to the method we previously reported.48 TPEQHA was then prepared based
on TPEQH to stnthesize AIE fluorescent probe. The 1H-NMR, 13C-NMR and LC-MS were used
to prove the successful synthesis in each step (Figure S1-S6).
O
H
N
O
O
O
O
NH2
+
O
O
+
O
K2CO3
O
N
N
O
Br
EtOH
Acetone, 62℃
NH2
O
N
O
1
O
2
CHO
O
O
HO
N
N
O
O
H2SO4
HO
N
O
1M BBr/CH2Cl2
+
-60, ℃
-25 ℃
N
4
3
O
O
O
HO
O
N
O
POCl3, Prydine
CH2Cl2, 0℃
P
OH
N
5
Figure 1. Synthesis of TPEQH and TPEQHA.
After the synthesis of TPEQH, its photophysical properties including UV-vis absorption and
fluorescent spectra were investigated. As shown in Figure 2A, TPEQH presented with wide
excitation wavelength (ranged from 350 – 510 nm) and its maximum emission wavelength was
10

located at 663 nm. These results meant that TPEQH not only had excellent AIE property, but also
had a large Stokes shift. Moreover, UV-vis absorption spectrum in THF solution was shown in
Figure 2B, indicating that TPEQH exhibited strong UV-vis absorption from 350 nm to 450 nm,
which might be attributed to the π-π* transition of the TPEQH.
Finally, the solvatochromic properties of TPEQH were detected in organic solutions of different
polarities (THF < Acetone < DMSO < MeOH). The UV-Vis absorption and fluorescent emission
spectra were shown in Figure 2C and 2D. The UV-Vis absorption spectra were almost similar in
different solutions. However, with the solvent polarity increasing, a red shift was observed in the
emission spectrum, which might result from the intramolecular charge-transfer of TPEQH.49-54
Figure 2. Optical properties of TPEQH. (A) Fluorescent excitation and emission spectra of
TPEQ solid. (B) The UV-vis absorption spectrum of TPEQ in pure THF (1 × 10-5 M). (C) The
UV-Vis absorption spectrum of TPEQ (1 × 10-5 M) in different solution (THF, Acetone, DMSO,
11

MeOH). (D) The fluorescent emission spectra of TPEQ (1 × 10-3 M) in different solution (THF,
Acetone, DMSO, MeOH) with 475 nm laser.
3.2 AIE Property. To obtain the insight AIE performance of TPEQH, its fluorescence spectra
in either THF/H2O mixture or pure THF was investigated (Figure 3). The results demonstrated that
TPEQH dissolved in THF emitted dim kelly fluorescence (Figure 3A). Moreover, when the water
fraction (fw) in the mixture solvent was adjusted from 10% to 70%, the fluorescence intensities of
TPEQH kept almost constant but gradually red-shifted from 515 nm to 543 nm. Like all of other
AIE fluorophores, the weak fluorescence of TPEQH in the dilute solution was attributed to the
intramolecular free rotations of chemical bond. Additionally, with the increase of the solvent
polarity by the enhanced water content, the emission of TPEQH (with donor-acceptor structure)
was red-shifted.38 However, when the fw continuously increased to 80% and 95%, the fluorescence
intensities of TPEQH exhibited obvious enhancement due to the aggregation of TPEQH and the
restricted intramolecular free rotations, which made the pathway of radioactive-decay prior to
nonradioactive-decay for the photoexcited fluorophore to relax. To further understand the AIE
performance of TPEQH, its emissive spectra under different concentrations were studied with the
excitation wavelength of 353 nm in the THF (Figure 3B). The results indicated that, in the dilute
solution (1 × 10-7, 1 × 10-6 and 1 × 10-5 M), the fluorescence performance of TPEQH showed low
correlation to the concentrations. However, as the concentrations continued to increase (1 × 10-4,
1 × 10-3 and 1 × 10-2 M), the fluorescence intensity increased significantly, reflecting the excellent
AIE performance of TPEQH
12

Figure 3. The AIE property of TPEQHA. (A) The emission spectra of TPEQH in the mixture
of THF and deionized water. (B) The emission spectra of TPEQH with different concentrations in
pure THF.
3.3 Theoretical Investigations. Quantum chemical calculations about the TPEOH optical
behaviors when responding to the intracellular ALP were then conducted using B3LYP/6-31G*
method via Gaussian 09 program. The frontier molecular orbitals (including HOMO and LUMO)
and the optimized structures of TPEQH molecule were demonstrated in Figure 4. The results
indicated that the highest occupied molecular orbital (HOMO) was localized on the TPE unit. The
quinoxalinone structure made less contribution to the HOMO. However, the lowest unoccupied
molecular orbital (LUMO) was mainly localized on the quinoxalinone structure, but was also
influenced by the phenyl ring of the TPE. Additionally, the HOMO-1 was localized on the whole
molecule while LUMO+1 was TPE centered. The quinoxalinone structure of TPEQH had less
contribution to the LUMO+1. The localized frontier molecular orbitals of TPEQH showed that,
when encountering photoexcitation, the charge of the TPEQH molecule would redistribute.
Meanwhile, the absorbed energy of TPE could be consumed via the free rotation of chemical bonds
around the central double bond. As a result, when rotation was restricted, the fluorescence
properties of TPEQH appeared.
13

Figure 4. Optimized structures and frontier molecular orbitals for TPEQH. Calculations were
carried out by density functional theory calculations at the B3LYP/6-31G* level.
3.4 The Fluorescence Response of TPEQHA to ALP in Solutions. In order to investigate the
fluorescence response behavior of TPEQHA, the interaction between TPEQHA and different
concentrations of ALP was investigated. Without ALP response, the water soluble TPEQHA
showed dim fluorescence. When TPEQHA was cultured with ALP at different concentrations for
20 min, the phosphate group of TPEQHA was hydrolyzed and converted to TPEQH, which was
hydrophobic and self-aggregated in solution. Thus, the fluorescence in the solution was gradually
enhanced over the increase of the ALP concentration (0-180 U/L, shown in Figure 5A). Figure 5B
has shown the fluorescence intensity change with the change of ALP concentration. After further
analysis of the experimental results (Figure 5C), an approximate first-order linear correlation from
0 to 100 U/L (R2 = 0.9985) was described, thus the intracellular detection limit of ALP of (1.77
U/L, 3σ/slope rule) was deduced22. Namely, the response fluorescence of TPEQH not only
demonstrated its specificity to the ALP, but also manifested that TPEQHA was able to recognize
ALP with high efficiency.
14

Figure 5. The response of TPEQHA to ALP. (A) The fluorescence intensity changes of
TPEQHA upon the different concentrations of ALP (0-10 U/L). (B) The correlation of TPEQHA
with the different concentrations of ALP. (C) The approximate first-order linear correlation of
TPEQHA with ALP.
3.5 In Vitro Cytotoxicity and Cellular Uptake investigation. Since TPEQH was utilized for
ALP imaging in the living cells, a standard MTT assay on the L929 and HeLa cells was conducted
to estimated the potential cytotoxicity of TPEQH. HeLa cell overexpressed ALP was considered
as the model target cell, and L929 with normal ALP expression was used as the negative
control.35,36 These two cells samples were co-incubated with TPEQH (20 µM) for 48 h. As the
results demonstrated in Figure 6, the cell viability of both two cell samples remained more than
80%, indicating that TPEQH was low cytotoxicity and qualified for living cell imaging.
Figure 6. Cell viability of HeLa and L929 cells treated for 48 h with different concentrations of
TPEQH (n = 5).
15

3.6 Intracellular Imaging of TPEQH. Having demonstrated its successful ability to respond
to ALP in solution, the ALP imaging by TPEQHA was then performed in living cells (HeLa and
L929 cell). Then, the response behavior between TPEQHA and ALP in these two kinds of cell
were further analyzed using a flow cytometry and CLSM.
Figure 7. Flow cytometry analysis in Hela and L929 cells after a 1 h treatment with TPEQHA.
(A) Flow cytometry analysis of TPEQH in Hela cell. (B) Flow cytometry analysis of TPEQH in
L929 cell.
Firstly, HeLa and L929 cells were treated with TPEQHA for 1 h. Another group of HeLa cells
was pretreated with levamisole hydrochloride for 30 minutes and then incubated with fluorescent
probe for 1 hour. The results were measured by flow cytometry (Figure 7). Strong fluorescence
could be detected while in HeLa cells, while the fluorescence was weak in both L929 cells and
HeLa cells treated with levamisole hydrochloride. These results demonstrated that the phosphate
group of TPEQHA was hydrolyzed by ALP in HeLa cells and TPEQHA was converted to
hydrophobic TPEQH. TPEQH self-aggregated in the cells and emitted bright fluorescence. In two
other kinds of cells, ALP expression was either low or inhibited, which was unable to hydrolyze
phosphate. As a result, the fluorescence was weak. Namely, ALP expression could be efficiently
16

distinguished by TPEQHA in living cells, making tumor cells and normal cells be successfully
differentiated.
Figure 8. CLSM images of HeLa and L929 cells after treating with TPEQHA for 1 h. (A, B, C)
The fluorescence of TPEQH is displayed as green dots on the cytomembrance of HeLa cell by
exciting at a 488 nm laser. (D, E, F) In HeLa cell treated with levamisole, weak fluorescence of
TPEQH is observed. (G, H, I) Weak fluorescence of TPEQH is observed in L929 cell.
To obtain more intuitive fluorescence images after ALP response, the cells were observed with
CLSM. HeLa cells, L929 cells and HeLa cells treated with levamisole hydrochloride were
incubated with TPEQHA for 1 h in the confocal dishes, followed by the direct observation with
CLSM (Figure 8). The results shown in Figure 8A, B and C demonstrated that strong green
fluorescence in HeLa cells was observed. In contrast, weak fluorescence was observed in L929
cells and HeLa cells treated with levamisole hydrochloride, which were consistent with the results
of flow cytometry.
17

Figure 9. CLSM images of HeLa cell after treating with TPEQHA or 4-MUP for 1 h. The
fluorescence of TPEQH is displayed as green dots on the cytomembrane of HeLa cells by exciting
at a 488 nm laser (A, B, C). Blue fluorescence of 4-MUP diffused in HeLa cell (D, E, F). Weak
blue fluorescence of 4-MUP is observed in L929 cells (G, H, I).
Finally, to determine the in-situ imaging effect of ALP in cells with TPEQHA, a commercially
available probe 4-MUP was selected as a control, in which 4-MUP was used as an ACQ
fluorescence probe for ALP imaging in living cells. In HeLa cells treated with 4-MUP, blue
fluorescence could be detected, but diffused throughout the cell and resulted in less contrasting,
thereby gradually losing signal intensity (9D, E and F). However, when replaced with TPEQHA,
green fluorescence gathered together in HeLa cells and was presented in the form of bright
fluorescent dots, reflecting that the signal of TPEQH remained stable in situ (9A, B and C). These
results further illustrated that the solid TPEQH dye used for in-situ ALP imaging in living cells
had enhanced resolution and accuracy.
18

4. CONCLUSIONS
In conclusion, we have developed an AIE fluorescence probe (TPEQH) and exploited its
application as an enzyme-response AIE probe to detect ALP in situ in cancer cells. When dissolved
in the solvent, TPEQH showed high insolubility and splendid AIE characteristics. Its intense
fluorescence in the solid state and wide range of excitation wavelength made it particularly suitable
for in-situ imaging. In vitro, TPEQHA could image endogenous ALP with a large signal-to-
background ratio in tumor cells. Compared with the ACQ fluorescent probe 4-MUP, TPEQHA
could image ALP in situ and improve the contrast and accuracy of detection in living tumor cells.
The experiment results show that we have successfully synthesized a new fluorophore with
remarkable AIE property, and further deceloped an AIE fluorescence probe for in situ imaging
ALP to improve contrast and accuracy of detection result. We considered that the new ALP-
responsive probe had a great potential to be a powerful tool to reveal the function of ALP in various
pathological processes.
ACKNOWLEDGMENTS
This work was financially supported by the National Basic Research Program (2015CB931801)
and the National Natural Science Foundation of China (51473093, 21374062), the National Basic
Research Program (2015CB931801), Shanghai Municipal Science and Technology Commission
2019 “Science and Technology Innovation Action Plan” Funded Project (19441905900)
Author Contribution
19

Qinghua Guan and Xinmiao Lu contributed equally to this work and were considered as the co-
first author.
Conflict of Interest
The authors declare no conflicts of interest.
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Figure caption
Figure 1: Synthesis of TPEQH and TPEQHA.
Figure 2: Optical properties of TPEQH. (A) Fluorescent excitation and emission spectra of TPEQ
solid. (B) The UV-vis absorption spectrum of TPEQ in pure THF (1 × 10-5 M). (C) The UV-Vis
absorption spectrum of TPEQ (1 × 10-5 M) in different solution (THF, Acetone, DMSO, MeOH).
(D) The fluorescent emission spectra of TPEQ (1 × 10-3 M) in different solution (THF, Acetone,
DMSO, MeOH) with 475 nm laser.
Figure 3: The AIE property of TPEQHA. (A) The emission spectra of TPEQH in the mixture of
THF and deionized water. (B) The emission spectra of TPEQH with different concentrations in
pure THF.
Figure 4: Optimized structures and frontier molecular orbitals for TPEQH. Calculations were
carried out by density functional theory calculations at the B3LYP/6-31G* level.
Figure 5: The response of TPEQHA to ALP. (A) The fluorescence intensity changes of TPEQHA
upon the different concentrations of ALP (0-10 U/L). (B) The correlation of TPEQHA with the
different concentrations of ALP. (C) The approximate first-order linear correlation of TPEQHA
with ALP.
24

Figure 6: Cell viability of HeLa and L929 cells treated for 48 h with different concentrations of
TPEQH (n = 5).
Figure 7: Flow cytometry analysis in HepG2 and L929 cells after a 1 h treatment with TPEQHA.
(A) Flow cytometry analysis of TPEQH in HepG2 cell. (B) Flow cytometry analysis of TPEQH
in L929 cell.
Figure 8: CLSM images of HeLa and L929 cells after treating with TPEQHA for 1 h. (A, B, C)
The fluorescence of TPEQH is displayed as green dots on the cytomembrance of HeLa cell by
exciting at a 488 nm laser. (D, E, F) In HeLa cell treated with levamisole, weak fluorescence of
TPEQH is observed. (G, H, I) Weak fluorescence of TPEQH is observed in L929 cell.
Figure 9: CLSM images of HeLa cell after treating with TPEQHA or 4-MUP for 1 h. (A, B, C)
The fluorescence of TPEQH is displayed as green dots on the cytomembrance of HeLa cells by
exciting at a 488 nm laser. (D, E, F) Blue fluorescence of 4-MUP diffused in HeLa cell. (G, H, I)
Weak blue fluorescence of 4-MUP is observed in L929 cells.
25

Graphical Abstract
26

Supporting Information
In Situ Localization of Alkaline Phosphatase Activity in
Tumor Cells by an Aggregation-Induced Emission
Fluorophore-Based Probes
27

Y
DY20190321QHG160 308 (4.068) Cm (296:326)
1
: TOF MS ES+
191.0804
2.00e3
100
79.0205
83.0597
186.9559
192.0838
249.1094
135.1007
331.0495
350
403.1396
400
265.1017
300
0
m/z
100
150
200
250
1
1
Figure S1. The H NMR (up) and MS (down) spectra of compound (1). H NMR (400 MHz,
DMSO-d6, δ): 12.21 (s, 1H), 7.61, 7.21 (1H), 7.23, 6.74 (1H), 7.14, 6.88 (1H), 3.81 (3H), 2.4 (2H).
28

Y
DY20190321QHG164 362 (4.784) Cm (343:375)
1
: TOF MS ES+
263.1021
1.48e4
100
264.1066
285.0847
301.0623
300 350
158.9623
83.0597
186.9562
200
408.0161
400
430.8899
450 500
0
m/z
100
150
250
1
1
Figure S2. The H NMR (up) and MS (down) spectra of compound (2). H NMR (400 MHz,
CDCl3, δ): 7.76 (d, 1H), 6.92 (d, 1H), 6.48 (s, 1H), 4.99 (s, 2H), 3.87 (s, 3H), 3.77 (s, 3H), 2.56 (s,
3H).
29