An activity-based fluorescent probe and its application for differentiating alkaline phosphatase activity in different cell lines

Article abstract of DOI:10.1039/d0cc06129h

Herein, a new fluorescent probe, AE-Phos, is reported for detecting the ALP activity with the combined advantages of aggregation-induced emission (AIE) and excited state intramolecular proton transfer (ESIPT). Further detailed fluorescence experiments demonstrated that AE-Phos exhibited excellent selectivity and sensitivity, a large Stokes shift, and a fast response towards ALP. Furthermore, AE-Phos was applied to imaging the ALP activity in different cell lines quantitatively. This journal is

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DOI: 10.1039/D0CC06129H
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An activity-based fluorescent probe and its application for
differentiating alkaline phosphatase activity in different cell lines
a‡
b‡
a‡
a
a
c,d
a
Yong He, Junli Yu, Xiangzi Hu, Shumei Huang, Lili Cai, Liu Yang, Huatang Zhang, * Yin
Received 00th January 20xx,
Accepted 00th January 20xx
a
e
c,d
Jiang, * Yongguang Jia, Hongyan Sun *
DOI: 10.1039/x0xx00000x
5
Herein, a new fluorescent probe, AE-Phos, is reported for detecting visualizing ALP activity in biological system. However, these
ALP activity with the combined advantages of aggregation-induced probes are based on the conventional fluorescent scaffolds and
emission (AIE) and excited state intramolecular proton transfer suffer from aggregation-caused quenching (ACQ) when
(
ESIPT). Further detailed fluorescence experiments demonstrated amassed in cells. In addition, the traditional “turn-on” probes
that AE-Phos exhibited excellent selectivity and sensitivity, large show obvious intrinsic fluorescence, which affects the detection
Stokes shift, and fast response towards ALP. Furthermore, AE-Phos sensitivity. Most traditional fluorophores for designing ALP
was applied to imaging ALP activity in different cell lines probes also suffer from potential self-reabsorption, photo-
quantitatively.
bleaching and background fluorescence interference because of
the short Stokes shift.
Alkaline phosphatases (ALPs) are a family of enzymes, which
regulate the phosphate metabolism and exist widely in both of
the prokaryotes and eukaryotes. Through the catalyse reaction
Fluorophores with AIE property have received increasing
attention in the field of biosensing and imaging during the past
two decades. AIE fluorophores are non-emissive when
1
6
of dephosphorylation under alkaline pH environment, ALP is
responsible for regulating a lot of momentous biological
processes. In some serious diseases such as cirrhosis, hepatitis,
dissolved in solution freely but become highly emissive in the
state of aggregation. ESIPT is a photochemical process that
occurs in the excited singlet state of a fluorophore with
2
hyperparathyroidism, Hodgkin’s lymphoma and liver cancer,
the elevated ALP activity levels in serum is a very common
7
intramolecular hydrogen bond. Because of its unique and
transient four-level photochemical process, ESIPT fluorophores
usually display a number of advantages compared with
traditional fluorophores (fluorescein, rhodamine, etc.), such as
3
phenomenon. Therefore, it is highly desirable to develop the
chemical tools that can detect ALP activity in living systems
accurately and selectively.
7a
large Stokes shift and local environment sensitiveness.
The fluorescent probes have captured the attention due to
their high sensitivity and noninvasiveness, which allow them to
be effective for in vitro and in vivo studies with minimal
perturbation.4 Several research groups including us have
reported a number of fluorescent probes for detecting and
Therefore, it will be highly desirable to design fluorophores with
both AIE and ESIPT characteristics.
In this study, we intend to design new fluorescent probes for
ALP imaging that possess both characteristics of AIE and ESIPT.
In 2009, Tong et al. reported a sequence of salicylaldehyde azine
(
SA) derivatives that exhibited AIE enhancement and ESIPT
8
characteristics. Recently, Tong and Liu et al. have designed
several fluorescent probes based on the scaffold of SA with
AIE+ESIPT mechanism. Inspired by these pioneering works, we
a. School of Chemical Engineering and Light Industry and School of Biomedical and
Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou,
Guangdong, 510006, China. E-mail: htzhang@gdut.edu.cn; junhe@gdut.edu.cn
9
b.
develop a new fluorescent probe, AE-Phos, for ALP detection
and imaging in living cells. As shown in Scheme 1, the hydroxyl
groups in AE-Phos, which is the crucial element for AIE and
ESIPT properties, is protected with the phosphate group. The
introduction of phosphate group endowed the probe with
excellent water solubility and prevent the formation of ESIPT
and AIE, leading to dramatic quenching of background
fluorescence. In the existence of ALP, the phosphate group is
Department of Ultrasonography, The Sixth Affiliated Hospital of Sun Yat-sen
University, Guangzhou, 510655, China
Department of Chemistry and Center of Super-Diamond and Advanced Films
c.
(COSDAF), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong,
China. E-mail: hongysun@cityu.edu.hk
Chengdu Research Institute, City University of Hong Kong, Chengdu, 610200,
China.
National Engineering Research Center for Tissue Restoration and Reconstruction,
South China University of Technology, Guangzhou 510006, China.
These authors contributed equally.
d.
e.
‡
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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observed for SA. This is attributed to ESIPT mechanViisewmA(rtFicilge.O1nBlin)e.
The combined AIE and ESIPT effect n lf
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quenching of fluorophore, but also improve the sensitivity of
fluorescence measurement drastically. In addition, we also
tested the fluorescence stability of AE-Phos and SA. As shown
in Fig. S2, the fluorescence intensities of AE-Phos and SA
remained unchanged over 120 min, indicating that both
Scheme 1 Synthesis route of SA, AE-OH-Phos, AE-Phos and the reaction
mechanism with ALP.
compounds possess excellent stability in 100% water.
cleaved
It is well known that the enzymatic activity of ALP is pH
sensitive. We therefore measured the effect of pH on the
reaction kinetic of AE-Phos with ALP. As shown in Fig. 1C and
S3, the fluorescence intensity of the reaction solution displayed
no obvious changes in the pH range of 4 to 7 when AE-Phos was
incubated with ALP for 20 min. This result was due to the low
enzymatic activity of ALP under acidic condition. However,
when the pH values were higher than 7, the fluorescent
intensity increased significantly with increasing pH value. It was
noted that the fluorescent intensity at pH 10 was lower than
those at pH 8 and 9. The phenomenon is not due to the lower
and the hydroxyl groups are recovered, resulting in the
formation of the fluorescent compound SA with AIE+ESIPT
characteristics. Compared with traditional ALP probes, AE-Phos
shows extremely low background fluorescence and high
fluorescent enhancement ratio due to the combined judicious
characteristics of AIE+ESIPT and the large Stokes shift. In
addition, we have successfully used the probe to differentiate
endogenous ALP activity in different cell lines by confocal
microscopy.
1
0
AE-Phos was synthesized according to the procedure
illustrated in Scheme 1 and S1. The uncaged product (SA) and reaction kinetic of ALP at pH 10. It is attributed to the
the intermediate product (AE-OH-Phos) were also synthesized deprotonation of the reaction product SA, which forms
to examine the dephosphorylation mechanism of ALP. The negatively charged ions and is released from aggregates to form
8
probe and the control compounds have been synthesized and weakly fluorescent monomers. Therefore, we chose pure Tris
characterized by 1H NMR, 13C NMR and 31P NMR in detail
buffer of pH 9.0 as the testing condition in the following
experiment (Fig. 1D).
(Supporting Information, ESI†).
Then, the reaction kinetics of AE-Phos with ALP was
investigated. The fluorescence spectra variation over time was
recorded in the presence of AE-Phos (5 μM) and a range of
different concentration of ALP (0-100 U/L) in Tris buffer. As
expected, the fluorescence intensity was enhanced in response
to increasing ALP concentration (Fig. 2A and S4). The
fluorescence intensity generally plateaued within 10 min. The
faster reaction kinetics has been observed at higher ALP
concentrations indicates higher cleavage reaction rate. We also
monitored the variation of fluorescence intensity with fixed
concentration of ALP (100 U/L) and different concentrations of
AE-Phos (0.1 to 20 µM). The fluorescent measurement data
with a given probe concentration at first 120 seconds is
displayed in Fig. 2B. These fluorescence intensities are utilized
The UV absorption and the fluorescent emission of SA, which
is the theoretical reaction product of AE-Phos and ALP, were
first measured in DMSO and water. SA dissolved well in DMSO
but it was not soluble in water. The maximum absorption
wavelength of SA was 356 nm. The fluorescence intensity of SA
enhanced when the water fraction increased. AIE effect was
observed when the water fraction was over 60% (Fig. 1A and
S1A). On the other hand, AE-Phos did not display fluorescence
emission from 0%-100% water fractions even though it
dissolved well both in water and DMSO (Fig. 1A and S1B). It is
noteworthy that a remarkable Stokes shift (180ꢀnm) was
0
to calculate the initial reaction rate (V ) at different
concentrations of probe (Fig. S5). Km and Vmax are calculated
respectively to be 7.66 μM and 0.408 μM/min based on the
Linewaver-Burk analysis. These values are comparable to those
previously reported fluorescent probes (Table S1).
Fig. 1 (A) Fluorescence intensity of SA (5 μM) and AE-Phos (5 μM) at 536 nm with
different water fractions; (B) UV-vis absorption and fluorescence emission spectra
of SA in Tris buffer; (C) Time-dependent fluorescence response of AE-Phos toward
ALP (100 U/L) in Tris buffer (50 mM) at different pHs at 37 ºC; (D) Plot of
fluorescence intensity with 536 nm AE-Phos (5 μM) after incubation with ALP (100
U/L) at 37 ºC for 30 min at different pH values. Ex:356 nm.
Fig. 2 (A) Time-dependent fluorescence intensity of AE-Phos (5 µM) at 536 nm
with various concentrations of ALP (0-100 U/L); (B) Plots of the fluorescence
intensity at 536 nm versus incubation time under different concentrations of AE-
Phos (0.5-20 µM) with 100 U/L ALP in Tris buffer (pH 9.0, 50 mM).
2
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Encouraged by the above result, we further explored
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DOI: 10.1039/D0CC06129H
Fig. 3 (A) (Fluorescence spectra of AE-Phos (5 μM) in the presence of various
concentrations of ALP (0, 1, 2, 5, 10, 20, 40, 60, 80 and 100 U/L) in Tris buffer (pH
o
9
, 50 mM) after 15 min of incubation at 37 C; (B) Fluorescence intensity changes
Fig. 4 (A) Confocal fluorescence images of different cells (MG-63, WI-38, B16F10, RAW
of AE-Phos (5 μM) at 536 nm in the presence of a series of ALP (0-100 U/L). The
insert is the linear fitting curve of fluorescence intensity at 536 nm with the
concentration of ALP from 0-10 U/L; (C) Fluorescence spectra of AE-Phos (5 μM)
toward ALP and various interfering substances in Tris buffer (pH 9, 50 mM); (D)
Fluorescence responses of AE-Phos (5 μM) at 536 nm toward ALP and various
interfering substances in Tris buffer. Esterase (200 U/L), Trypsin (200 U/L),
2
64.7, HEK 293) stained with AE-Phos (20 μM) after 2 h of incubation; (B) Confocal
fluorescence images of living MG-63 cells incubated with AE-Phos (20 μM) for 2 h, with
3 4
or without pre-incubation with 1 mM Na VO for 20 min. The columns are the average
fluorescence intensities in the cells.
2
+
2+
2+
3+
2+
2 2
Lysozyme (200 U/L), Ca /Mg /Zn /Fe /Cu (50 μM), H O (100 μM), HOCl (100
whether AE-Phos can differentiate the intracellular ALP activity
in different cell lines, such as MG-63, WI-38, B6F10, RAW264.7
and HEK293. Briefly, different cell lines were incubated with AE-
Phos (20 μM) for 2 h respectively. Fluorescence microscopy was
then used to assess the fluorescence intensity. As shown in Fig.
μM), Cys (1 mM), Hcy (1 mM), GSH (1 mM), Thr (50 μM), Pro (50 μM), ALP (100
o
U/L). All species were incubated with AE-Phos at 37 C for 30 min. Ex: 356 nm.
To examine the feasibility of using AE-Phos for quantitative
determination of ALP activity, we conducted concentration
dependent experiments and monitored dephosphorylation
reaction closely. As shown in Fig. 3A, the fluorescence intensity
increased with ALP activity in the range of 0-100 U/L after 15
min of incubation. Due to the extremely low background
fluorescence of AE-Phos in water, the enhanced fluorescence
4
A, MG-63 cells displayed the strongest fluorescence under the
same experimental condition, indicating the highest expression
level of ALP in MG-63 among these cell lines. The result is also
consistent with the reported literatures, in which the cultured
human osteosarcoma cells (MG-63) produce high level of ALP
because the transformed osteoblasts in osteosarcoma disrupt
the tight control of proliferation and progressively express the
“
turn on” ratio was about 240-fold when 100 U/L ALP was used.
This is higher than the most reported data in the literatures
Table S1). The experiment also displayed a linear correlation
1
1
cell differentiation genes such as ALP. Cells of WI-38, B16F10
and RAW 264.7 showed moderate fluorescence signal. By
contrast, no noticeable fluorescence signal was observed with
HEK 293 cells, indicating a lower level of ALP expression, whcih
(
between fluorescence signals at 536 nm and ALP activity from 0
2
to 10 U/L with the fitting equation y = 4.76x + 3.16 and R =
0
.99165 (Fig. 3B). The detection limit is estimated to be 0.012
5
a,12
agrees well with the literature.
MG-63 cells to investigate the inhibition effect of ALP inhibitor.
Specifically, 1 mM Na VO , an inhibitor of ALP, were incubated
in MG-63 cells for 20 min, followed the incubation of AE-Phos
20 μM) for another 2 h. The result showed that negligible
Furthermore, we selected
U/L based on 3σ/slope, which is better than the detection
ranges of the most of previously reported fluorescent probes.
These results together indicated that AE-Phos possess excellent
sensitivity for ALP (Table S1).
3
4
(
The specificity of AE-Phos towards ALP were also
investigated. After reacting with ALP, the fluorescence signals
of AE-Phos was greatly increased and no obvious change was
fluorescence intensity was detected with the cells served with
inhibitor, indicating the dephosphorylation of ALP activity was
inhibited by Na VO (Fig. 4B). Taken together, these results
3 4
strongly demonstrate that AE-Phos is competent to visual
imaging endogenous ALP activity in different cell lines.
2
+
2+
2+
yet observed with metal ions (Ca , Mg , Zn ), reactive oxygen
species (H , HOCl) and reactive sulfur species (Cys, Hcy, GSH)
Fig. 3C and 3D). The selectivity of AE-Phos for ALP versus some
2 2
O
(
To examine the fluorescence “turn on” mechanism of AE-
Phos, the reaction of AE-Phos towards ALP was monitored by
reversed-phase UPLC (Fig. S7A). Firstly, the retention time of
AE-Phos, AE-OH-Phos and SA was measured as the standard
references. Respectively, the retention time was recorded to be
commonly observed enzymes was examined as well. These
enzymes include esterase, trypsin and lysozyme. Similarly, no
obvious fluorescence change was obtained with 30 min of
incubation of AE-Phos and these enzymes. In addition, the
result of inhibitor test also proved that the fluorescence
increase was due to the reaction of AE-Phos and ALP (Fig. S6).
These results demonstrate that AE-Phos displays high specificity
towards ALP, indicating that the probe holds great potential for
specific detection of ALP activity in living cells.
0
.58 min, 1.32 min and 6.36 min. When AE-Phos was treated
with ALP for 5 min, three peaks belonging to AE-Phos, AE-OH-
Phos and SA were detected, which indicated AE-OH-Phos was
the intermediate product of the reaction. After extending the
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Haarhaus and P. Magnusson, Clin. Chim. Acta., 20V2iew0,A5rt0ic1le,O1n9lin8e;
d) E. E. Golub and K. B.-Battaglia, Curr. Opin. Orthop., 2007,
incubation time to 20 min, the peak belongs to SA converted to
the major product, indicating that dephosphorylation was
nearly completed. Furthermore, dynamic light scattering (DLS)
analysis revealed that the colloidal aggregates was formed with
(
1
DOI: 10.1039/D0CC06129H
8, 444; (e) K. Poelstra, W. W. Bakker, P. A. Klok, J. A. Kamps,
M. J. Hardonk and D. K. Meijer, Am. J. Pathol., 1997, 151,
163.
1
2
49.4 nm average particle diameter for the incubation mixture
3
(a) W. J. Lammers, H. R. van Buuren, G. M. Hirschfield, H. L.
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of AE-Phos and ALP (Fig. S7B). However, no colloidal aggregates
were observed with AE-Phos only in Tris buffer by DLS.
Therefore, the reaction mechanism is proposed as follows (Fig.
S7C). ALP first catalyses the dephosphorylation of AE-Phos to
produce intermediate compound AE-OH-Phos, which contains
one phosphate group and shows very weak fluorescence.
1
3
338; (b) M. Yi, F. Bodola and S. M. Lemon, Virology, 2002,
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Subsequently,
AE-OH-Phos
undergoes
the
second
4
5
dephosphorylation step and releases AE-Phos as the final
product, which forms aggregates owing to the intramolecular
hydrogen bond and increased hydrophobicity. The aggregation
state of SA exhibits strong fluorescent signal due to the
combined AIE and ESIPT mechanism.
1
2
92; (b) J. Chan, S. C. Dodani and C. J. Chang, Nat. Chem.,
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40, 220; (b) Y. Tan, L. Zhang, K. H. Man, R. Peltier, G. Chen, H.
In summary, a new fluorescent probe, AE-Phos, for ALP
activity detection in both aqueous buffer and living cells have
been constructed herein. After dephosphorylation by ALP, the
phosphate groups of AE-Phos are removed and the hydroxyl
groups are released. The free hydroxyl groups can form
intramolecular hydrogen bond with nitrogen atoms, generating
stacking of molecules and exhibiting strong fluorescence by AIE
and ESIPT effect. The reaction mechanism has been firmly
verified by different methods including fluorimeter, UPLC and
DLS. Moreover, AE-Phos exhibited good water solubility,
excellent selectivity and fast response when reacting with ALP.
Specially, after reacting with ALP, AE-Phos revealed more than
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2
40-fold turn-on ratio and 180 nm Stokes shift, testifying its
advantages over most probes in the reported literatures. Most
importantly, AE-Phos displayed the capability of differentiating
and visualizing endogenous ALP activity in different cell lines
such as human osteoblastic cells, murine melanoma cells and
macrophages.
6
7
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This work was supported by the National Natural Science
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Basic
and
Applied
Basic
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(2020A1515010986, 2020A1515011463), Sichuan Science and
Technology Program (2018JY0360), Shenzhen Basic Research
Project (JCYJ20180507181654823), the Pearl River Talent Plan
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Conflicts of interest
There are no conflicts to declare.
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