Construction of an alkaline phosphatase-specific two-photon probe and its imaging application in living cells and tissues

Article abstract of DOI:10.1016/j.biomaterials.2017.06.032

Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of important biological processes such as cell differentiation and bone mineralization. Monitoring the activity of ALP in serum can help diagnose a variety of diseases including bone and liver diseases. There has been growing interest in developing new chemical tools for monitoring ALP activity in living systems. Such tools will help further delineate the roles of ALP in biological and pathological processes. Previously reported fluorescent probes has a number of disadvantages that limit their application, such as poor selectivity and short-wavelength excitation. In this work, we report a new two-photon fluorescent probe (TP-Phos) to selectively detect ALP activity. The probe is composed of a two-photon fluorophore, a phosphate recognition moiety, and a self-cleavable adaptor. It offers a number of advantages over previously reported probes, such as fast reaction kinetics, high sensitivity and low cytotoxicity. Experimental results also showed that TP-Phos displayed improved selectivity over DIFMUP, a commonly utilized ALP probe. The selectivity is attributed to the utilization of an ortho-functionalised phenyl phosphate group, which increases the steric hindrance of the probe and the active site of phosphatases. Moreover, the two-photon nature of the probe confers enhanced imaging properties such as increased penetration depth and lower tissue autofluorescence. TP-Phos was successfully used to image the endogenous ALP activity of hippocampus, kidney and liver tissues from rat.

Full text of DOI:10.1016/j.biomaterials.2017.06.032

Accepted Manuscript
Construction of an alkaline phosphatase-specific two-photon probe and its imaging
application in living cells and tissues
Huatang Zhang, Peng Xiao, Yin Ting Wong, Wei Shen, Mohit Chhabra, Raoul Peltier,
Yin Jiang, Yonghe He, Jun He, Yi Tan, Yusheng Xie, Derek Ho, Yun-Wah Lam,
Jinpeng Sun, Hongyan Sun
PII:
S0142-9612(17)30432-5
10.1016/j.biomaterials.2017.06.032
JBMT 18152
DOI:
Reference:
To appear in: Biomaterials
Received Date: 12 February 2017
Revised Date: 8 June 2017
Accepted Date: 22 June 2017
Please cite this article as: Zhang H, Xiao P, Wong YT, Shen W, Chhabra M, Peltier R, Jiang Y, He Y, He
J, Tan Y, Xie Y, Ho D, Lam Y-W, Sun J, Sun H, Construction of an alkaline phosphatase-specific two-
photon probe and its imaging application in living cells and tissues, Biomaterials (2017), doi: 10.1016/
j.biomaterials.2017.06.032.
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Construction of an Alkaline Phosphatase-Specific Two-photon
Probe and Its Imaging Application in Living Cells and Tissues
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Huatang Zhang,a,b Peng Xiao,c Yin Ting Wong,d Wei Shen,b Mohit Chhabra,b Raoul
Peltier,b Yin Jiang,a Yonghe He,a Jun He,a Yi Tan,b Yusheng Xie,b Derek Ho,e Yun-Wah
Lam,b,* Jinpeng Sunc,* and Hongyan Sunb,*
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a School of Chemical Engineering and Light Industry, Guangdong University of Technology,
Guangzhou, Guang dong 510006, P. R. China.
b
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Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee
Avenue, Kowloon, Hong Kong, P. R. China.
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Department of Physiology, Shandong University School of Medicine, Jinan, Shandong
250012, P. R. China
d Department of Biomedical Sciences, City University of Hong Kong, 83 Tat Chee Avenue,
Kowloon, Hong Kong, P. R. China
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Department of Physics and Materials Science, City University of Hong Kong, 83 Tat
Chee Avenue, Kowloon, Hong Kong, P. R. China.
Corresponding Author: yunwlam@cityu.edu.hk;
sunjinpeng@sdu.edu.cn;
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hongysun@cityu.edu.hk
Abstract
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Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of
important biological processes such as cell differentiation and bone mineralization.
Monitoring the activity of ALP in serum can help diagnose a variety of diseases including
bone and liver diseases. There has been growing interest in developing new chemical
tools for monitoring ALP activity in living systems. Such tools will help further delineate the
roles of ALP in biological and pathological processes. Previously reported fluorescent
probes has a number of disadvantages that limit their application, such as poor selectivity
and short-wavelength excitation. In this work, we report a new two-photon fluorescent
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probe (TP-Phos) to selectively detect ALP activity. The probe is composed of a
two-photon fluorophore, a phosphate recognition moiety, and a self-cleavable adaptor. It
offers a number of advantages over previously reported probes, such as fast reaction
kinetics, high sensitivity and low cytotoxicity. Experimental results also showed that
TP-Phos displayed improved selectivity over DIFMUP, a commonly utilized ALP probe.
The selectivity is attributed to the utilization of an ortho-functionalised phenyl phosphate
group, which increases the steric hindrance of the probe and the active site of
phosphatases. Moreover, the two-photon nature of the probe confers enhanced imaging
properties such as increased penetration depth and lower tissue autofluorescence.
TP-Phos was successfully used to image the endogenous ALP activity of hippocampus,
kidney and liver tissues from rat.
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Keywords: Alkaline phosphatase; Selectivity; Two-photon; Ortho-functionalization; Cell
imaging.
1. Introduction
Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of
phosphate metabolism in both prokaryotes and eukaryotes. ALP can act on a variety of
biomolecules, including proteins, phosphatidates, nucleic acids and inorganic phosphates.
By catalyzing the hydrolysis and transphosphorylation of monophosphate esters at
alkaline pH, ALP is involved in regulating many important biological processes, including
cell differentiation, bone mineralization and detoxification of bacteria endotoxin [1, 2].
Studies have also shown that abnormal levels of ALP is associated with a variety of
human diseases, such as bone diseases [3, 4], liver dysfunction [5, 6], breast and
prostatic cancer [7, 8], and diabetes [9, 10]. Despite these advancements in research, the
diverse physiological and pathological functions of ALP still remain largely unknown.
Consequently there has been growing interest in developing chemical tools for accurate
and selective detection of ALP activity in living systems. Such tools will help further
delineating the roles of ALP in various biological and pathological processes. Furthermore,
developing chemical tools for sensing ALP might find useful applications in clinical
diagnosis.
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Among different methods to detect ALP activity, the fluorescent probe approach has
proven to be particularly useful, especially as a nondestructive and noninvasive way to
monitor the biological targets in real time. To date, a number of fluorescent probes have
been reported to be able to detect ALP activity. These probes are mainly based on
small-molecule fluorophores or inorganic nanomaterials [11-17]. However, the vast
majority of the currently available fluorescent probes perform at short excitation
wavelengths, usually in the UV-vis range. As a result, they have shallow tissue penetration
depths and their application in tissue imaging is limited. In addition, most of the
fluorescent probes cannot differentiate ALP from other phosphatases, such as protein
tyrosine phosphatases (PTPs), resulting in poor selectivity of the probes. For example,
commercially available ALP fluorescent probes such as MUP [18, 19], DiFMUP [20-22],
and FDP [23-25] show similar reactivity towards ALP and other phosphatases.
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O
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OH
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TP-Phos
ALP
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CO2
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TP
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Scheme 1. Chemical structure of TP-Phos probe and its proposed “turn-on” mechanism by ALP.
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To overcome the aforementioned constraints, we herein report the ALP specific
fluorescent probe with two-photon properties and its imaging application in cells and
tissues. The probe TP-Phos consists of three parts: a two-photon (TP) fluorophore, a
phosphate recognition group, and a self-cleavable linker (Scheme 1). We hypothesize the
“turn on” mechanism of the probe as follows (Scheme 1). ALP first catalyzes the
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dephosphorylation of TP-Phos to produce a phenolate intermediate. Subsequently the
intermediate undergoes 1,4-elimination reaction and releases ortho-quinone methide,
leading to the “turn on” of fluorescence [26, 27]. Compared with the traditional one-photon
fluorophore, our TP probe has a number of advantages, including increased penetration
depth, low tissue auto-fluorescence and self-absorption, high resolution, reduced photo
damage and photo bleaching [28, 29]. Furthermore, we specifically incorporated an
ortho-functionalised phenyl phosphate group in the probe design to increase the steric
hindrance between the phosphate recognition group and the active site of phosphatase.
This allows ALP to be differentiated from other phosphatases. Lastly, we found that the
probe showed excellent stability in DMSO and did not show any obvious decomposition
after being stored at -20 oC for two years (Fig S1).
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2. Material and methods
2.1. Materials and general instruments
All chemicals and solvents were purchased from commercial suppliers with the highest
grade. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS),
trypsin-EDTA and penicillin/streptomycin were purchased from Invitrogen. Calf intestinal
alkaline phosphatase was purchased from NEB. The BIOMOL Green™ Reagent for
Phosphate Detection, BML-AK111, was purchased from Enzo Life Sciences. RIPA lysis
buffer and protease inhibitor cocktail were purchased from Sigma-Aldrich. 6,
8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) was purchased from Sangon Biotech
Co. Ltd. 4-methylumbelliferyl phosphate (4-MUP) was purchased from J&K Chemical Ltd.
Other chemicals were used directly in the experiment without further purification, unless
otherwise specified. Ibidi® 8-well culture plates were purchased from ibidi GmbH. C57BL/6
mice were obtained from the Laboratory Animal Services Center of the Chinese University
of Hong Kong, and all experiments were performed in accordance with local guidelines.
Fresh tissue slices were prepared with Leica VT1000S. 1H NMR, 13C NMR, and 31P NMR
spectra were recorded on a Bruker NMR spectrometer (400 MHz or 300 MHz). Mass
spectra were obtained on a PC Sciex API 150 EX ESI-MS system using electrospray
ionization (ESI). UV absorption spectra were obtained on Shimadzu 1700 UV/Vis
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Spectrometer. Fluorescence signal was recorded with a FluoroMax-4 fluorescence
photometer. Fluorescence images were acquired using a Leica TCS SP5 Confocal
Scanning Microscope. pH value was recorded with a FiveEasy TM Fe20 pH meter.
2.2 Synthesis
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Scheme 2. Synthetic route for TP-Phos.
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2.2.1 Synthesis of diethyl (2-formylphenyl) phosphate (compound A)
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2-hydroxybenzaldehyde (2 mmol, 244 mg) and K2CO3 (10 mmol, 1.38 g) were dissolved
in THF (20 mL). Diethyl phosphorochloridate (3 mmol, 432 µL) was then added to the
solution and stirred at room temperature for 6 h. The organic solvent was evaporated.
CH2Cl2 and water were subsequently added for extraction. CH2Cl2 phase was collected
and dried with MgSO4. The crude product was purified by silica gel column
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chromatography. Compound A was obtained with a yield of 91%. H NMR (CDCl3, 400
MHz) δ 10.42 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz,
1H,), 7.31 (t, J = 8.0 Hz, 1H), 4.26 (t, J = 7.2 Hz, 4H), 1.37 (t, J = 7.2 Hz, 6H); 13C NMR
(CDCl3, 100 MHz) 188.5, 152.8, 135.7, 128.8, 127.3, 125.4, 121.1, 65.1, 16.1; 31P NMR
(CDCl3, 162 MHz) δ -6.67. ESI-MS: Calcd. for C11H16O5P [M+H]+ 259.1; found 259.4.
2.2.2 Synthesis of diethyl (2-(hydroxymethyl)phenyl) phosphate (compound B)
Compound A (1 mmol, 258 mg) was dissolved in 20 mL of mixed solvent
(EtOH/THF/H2O=100/20/1, v/v/v) and cooled in an ice bath. NaBH4 (2 mmol, 76 mg) was
added to the above solution. The reaction mixture was stirred at room temperature for 1 h.
The solvent was then removed. 1 mol/L aqueous HCl was added to the residue and
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extracted with ethyl acetate. Organic phase was evaporated and purified with silica gel
column chromatography (ethyl acetate/Hexane, from 1/5 to 2/1). Compound B was
obtained with a yield of 74%. 1H NMR (CDCl3, 400 MHz) δ 7.45 (d, J = 7.2 Hz,1H), 7.28 (t,
J = 7.2 Hz, 1H), 7.20 (t, J = 8.0 Hz, 2H), 4.65 (s, 2H,), 4.22 (m, 4H), 1.35 (t, J = 6.9 Hz, 6H);
13C NMR (CDCl3, 100 MHz) 148.3, 133.1, 131.1, 129.2, 126.0, 121.0, 65.1, 60.1, 16.1; 31P
NMR (CDCl3, 162 MHz) δ -5.06. ESI-MS: Calcd. for C11H18O5P [M+H]+ 261.1; found
261.4.
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2.2.3 Synthesis of 1-(6-hydroxynaphthalen-2-yl)ethan-1-one (compound C)
48% HBr (13.6 mL) was added to a solution containing 6-acyl-2-methoxynaphthalene
(25 mmol, 5 g) in glacial acetic acid (50 mL). The mixture was stirred at 100 oC for 12 h.
Excess acetic acid was then removed in vacuum. The residue was dissolved in ethyl
acetate and washed with dilute NaHCO3 and brine. The organic phase was dried with
MgSO4, and the crude product was purified with silica gel column chromatography
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(CH2Cl2/MeOH= 50/1). Compound C was obtained with a yield of 60%. H NMR
(DMSO-d6, 300 MHz) δ 10.21 (s, 1H), 8.53 (s, 1H), 7.97 (d, J = 9.5 Hz, 1H), 7.87 (dd, J1 =
1.7 Hz, J2 = 8.7 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.18 (m, 2H), 2.64 (s, 3H); 13C NMR
(DMSO-d6, 300 MHz) δ 197.9, 158.4, 137.6, 132.0, 131.9, 131.0, 127.1, 126.8, 124.4,
120.0, 109.3, 27.0; ESI-MS: Calcd. for C12H11O2 [M+H]+ 187.1; found 187.7.
2.2.4 Synthesis of 1-(6-(methylamino)naphthalen-2-yl)ethan-1-one (compound D)
Compound C (5 mmol, 930 mg), CH3NH2 (50% in H2O) (25 mmol, 1.86 mL), Na2S2O5
(10 mmol, 1.58 g), and H2O (10 mL) were mixed in a 15 mL stirred autoclave and heated
at 145 °C for 72 h. The product was collected by fi ltration and washed with water. The
combined filtrate was then extracted with CH2Cl2. The extract was condensed in vacuo
and subsequently purified by silica gel column chromatography (CH2Cl2/MeOH= 50/1).
Compound D was obtained with a yield of 67%. 1H NMR (CDCl3, 400 MHz) δ 8.30 (d, J =
1.4 Hz, 1H), 7.92 (dd, J1 = 1.8 Hz, J2 = 8.6 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.63 (d, J =
8.6 Hz, 1H), 6.90 (dd, J1 = 2.3 Hz, J2 = 8.8 Hz, 1H), 6.76 (d, J = 2.3 Hz, 1H), 2.97 (s, 3H),
2.67 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 197.8, 149.2, 138.1, 130.8, 130.7, 130.4,
126.0, 125.9, 124.8, 118.4, 103.1, 30.4, 26.4; ESI-MS: Calcd. for C13H14NO [M+H]+ 200.1;
found 200.6.
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2.2.5
Synthesis
of
2-((diethoxyphosphoryl)oxy)benzyl
(6-acetylnaphthalen-2-yl)(methyl)carbamate (compound E)
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Compound B (0.5 mmol, 130 mg) and pyridine (1.5 mmol, 121 µL) were dissolved in dry
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CH2Cl2 (10 mL) under nitrogen atmosphere. The solution was then cooled to 0 C in the
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ice bath. Triphosgene (0.5 mmol, 148 mg) was added slowly to the solution and stirred for
6 h. Nitrogen gas was subsequently purged into the reaction mixture for 1 h to remove
excess triphosgene. Compound D (84.87 mg, 0.55 mmol) was then added. The reaction
solution was stirred at room temperature for 24 h. After completion of reaction, CH2Cl2
was added and washed with water and brine. The organic phase was dried by MgSO4.
The crude product was purified with silica gel column chromatography (ethyl
acetate/Hexane, from 1/3 to 2/1). Compound E was obtained with a yield of 34%. 1H NMR
(CDCl3, 300 MHz) δ 8.41 (s, 1H), 8.00 (dd, J1 = 1.7 Hz, J2 = 8.6 Hz, 1H), 7.91 (d, J = 8.8 Hz,
1H), 7.80 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 1.9 Hz, 1H), 7.51 (dd, J1 = 1.9 Hz, J2 = 8.8 Hz,
1H), 7.34 (t, J = 8.5 Hz, 2H), 7.27 (td, J1 = 1.7 Hz, J2 = 7.6 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H),
5.29 (s, 2H), 4.13 (m, 4H), 3.43 (s, 3H), 2.68 (s, 3H), 1.26 (t, J = 7.1 Hz, 6H); 13C NMR
(CDCl3, 75 MHz) δ 197.9, 155.2, 148.8, 143.1, 135.8, 134.5, 130.4, 130.2, 129.9, 129.8,
129.6, 128.2, 127.5, 125.5, 125.0, 124.5, 122.5, 119.7, 64.8, 62.8, 37.7, 26.7, 16.1; 31P
NMR (CDCl3, 122 MHz) δ -4.93; ESI-MS: Calcd. for C25H29NO7P [M+H]+ 486.2; found
486.1.
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2.2.6 Synthesis of 2-(phosphonooxy)benzyl (6-acetylnaphthalen-2-yl)(methyl)carbamate
(TP-Phos)
Compound E (97 mg, 0.20 mmol) was dissolved in dry CH2Cl2 (5 mL) at room
temperature. Bromotrimethylsilane (130 ꢁL, 1.0 mmol) was then added dropwise. The
reaction mixture was stirred overnight at room temperature under nitrogen atmosphere.
MeOH (3 mL) was added when the reaction completed (monitored by TLC). After the
mixture was stirred at room temperature for 2 h, the solvent was removed under vacuum.
The crude product was purified first with silica gel column chromatography
(CH2Cl2/MeOH= 10/1) and then with PTLC (CH2Cl2/MeOH=3/1). TP-phos was obtained
with a yield of 44%. 1H NMR (DMSO-d6, 300 MHz) δ 8.65 (s, 1H), 8.10 (d, J = 8.8 Hz,1H),
7.96 (m, 3H), 7.69 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 7.29 (m, 3H), 7.09 (t, J= 8.0 Hz,1H),
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5.21 (s, 2H), 3.41 (s, 3H), 2.69 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 197.8, 154.5,
143.0, 135.3, 133.9, 130.0, 129.9, 128.9, 128.6, 128.0, 127.7, 127.6, 125.6, 124.0, 123.8,
122.1, 120.3, 62.4, 37.3, 26.7; 31P NMR (DMSO-d6, 122 MHz) δ -5.91; ESI-MS: Calcd. for
C21H21NO7P [M+H]+ 430.1; found 430.4.
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2.3. General procedure for fluorescence measurement
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TP-Phos was dissolved in DMSO to prepare 5 mM stock solution. ALP was diluted in
Tris-HCl buffer (50 mM, pH 7.4) from 10,000 U/mL stock solution, which was purchased
from NEB. Appropriate amount of TP-Phos and other analytes were added to separate
portions of the solution and mixed thoroughly. The reaction mixture was shaken uniformly
at 37 ºC before measurement was taken. The excitation wavelength was set at 365 nm.
The emission wavelength was set in the range of 400 nm to 600 nm. The slit widths of
excitation and emission wavelength were both set at 5 nm.
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2.4. Kinetic experiment
All kinetic measurements were performed in Tris-HCl buffer (50 mM, pH 7.4) at 37 °C.
The kinetic rate was measured by adding different concentrations of probe (0-20 ꢁM) to
the ALP solution (0.01 U/mL), and the fluorescence change was recorded every 20
seconds at 500 nm. The fluorescence calibration curve was used to calculate the rate.
The parameters of the kinetic enzyme reaction, Michaelis-Menten constant (Km),
maximum rate (Vmax) and catalytic rate constant (kcat) were investigated by
Lineweaver-Burk plot using software (Graphpad Prism 7.01). The results reported here
are the average values of three independent experiments.
2.5. Selectivity experiment with other phosphatases
For catalytic study in vitro, the regions encoding the catalytic domains of human PTP1B
(1-321), GLEEP (888-1180), MEG-2 (287-573), LYP (1-294), VHR (1-185), SSH2
(305-450), PPM1A (1-368), PPM1B (23-295) and PPM1G (1-546) were amplified by PCR
and subcloned into pET-15b, pNIC-CH or pT7-7 vectors. All constructs were verified by
DNA sequencing. Expression constructs were transformed into E. coli BL21 DE3 cells,
and proteins were routinely purified as described in previous papers [30-32]. Protein
purities were determined to be at least 90% by SDS-PAGE.
Phosphatase activities toward TP-Phos and 6, 8-difluoro-4-methylumbelliferyl
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phosphate (DiFMUP) were determined by inorganic phosphate assay using the BIOMOL
Green™ Reagent as described previously [33-35]. Except for metal-dependent protein
phosphatases, all reactions were conducted at 25°C in a 96-well plate in 60 ꢁL of solution
containing 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol (DTT) with an ionic
strength of 0.15 M adjusted with NaCl. For specific activity of PPM1A, PPM1B and
PPM1G, 10 mM MnCl2 was added to the reaction buffer. The reaction was stopped by
adding 120 ꢁL of BIOMOL GREEN™, and the phosphate released was determined with
an end-point reading at 620 nm. The experiment was conducted under the following
condition: TP-Phos (5 µM), DiFMUP (5 µM), ALP and other phosphatases (10 nM).
2.6. ALP assay in cell and tissue lysates
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To prepare cell lysate, HEK 293 and HeLa cells (~1 x 107) were grown for 48 h in DMEM
supplemented with 10 % FBS and 1% penicillin-streptomycin in 10 cm tissue culture plate.
After aspirating the media, the cells were washed twice with 1 mL of cold PBS buffer. RIPA
lysate buffer (500 ꢁL) with protease inhibitor cocktail was added into the plate and
incubated for 10 min on ice. Subsequently cell scraper was used to harvest the cells. The
cells were transferred to a 1.5 mL tube and then passed through a 25 gauge needle 10
times to extract cell lysates. The cell lysates were incubated on ice for another 10 min and
then clarified by centrifugation at 4 °C and 13000 x g for 10 min. Finally, the supernatant
was transferred to a clean tube on ice for subsequent fluorescence measurement.
To prepare tissue lysate, we chose heart, liver, lung and kidney of C57BL/6 mice. 10 mg
of tissue was first weighted and washed with 500 ꢁL of cold PBS buffer twice in a Dounce
homogenizer on ice. 500 ꢁL of RIPA buffer with protease inhibitor cocktail was added and
incubated for 15 min on ice. The tissue samples were then homogenized with 30-50
passes. The liquefied tissue was subsequently transferred into a 1.5 mL tube and
centrifuged at 4 °C and 13000 x g for 4 min to remove any insoluble material. Finally, the
supernatant was collected and transferred to a clean tube on ice for subsequent
fluorescent measurement.
For ALP activity test, we used the following method. Cell lysate or tissue lysate stock
solution was diluted to 0.2 mg/mL and 1 mg/ml respectively in 200 ꢁL of Tris-HCl buffer
(pH 7.4, 50 mM). TP-Phos (5 ꢁM) was then added to the solution. After 4 min of incubation
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at 37 ºC, the fluorescence intensity was measured with fluorescence photometer. The
ALP activity in cell and tissue was calculated accordingly from the standard curve.
2.7. MTT test
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HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum and appropriate amount of antibiotic (penicillin
and streptomycin). Prior to the experiments, the cells were placed at 1×105 cells per well
in a 96-well plate, followed by the addition of different concentrations of TP-Phos (0-20
µM). The cells were subsequently incubated at 37 ºC for 24 h followed by MTT assays.
2.8. Cell imaging
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HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% fetal bovine serum and appropriate amount of antibiotic (penicillin
and streptomycin). Approximately 1×105 cells were seeded in a confocal dish (20 mm)
with the medium at 37 ºC. The cells were allowed to adhere to the dish under the
atmosphere of 5% CO2 and 95% air at 37 ºC and incubated for 24 h. Subsequently, the
cells were incubated with TP-Phos (5 ꢁM) in DMEM for 20 min under the atmosphere of 5%
CO2 and 95% air at 37 ºC, and then washed with DMEM. For the control experiment, the
cells were pretreated with Na3VO4 (1 mM) for 20 min, and then incubated with TP-Phos (5
ꢁM) for 20 min at 37 ºC and washed with DMEM. Fluorescence imaging was then carried
out using Leica TCS SP5 Confocal Scanning Microscope with 40× oil and 63× water
objectives under multiphoton excitation at 720 nm.
2.9. Fresh tissue imaging
Hippocampus, kidney and liver slices were all prepared from 2-week-old rat (C57 mice).
The tissue slices were cut into thickness of 400 ꢁm slices using a vibrating-blade
microtome in artificial cerebrospinal fluid (ACSF: 124 mM NaCl, 3.0 mM KCl, 2.0 mM
CaCl2, and 1.25 mM MgSO4, 26 mM NaHCO3, 10 mM D-glucose, 1.25 mM KH2PO4). The
tissue slices were incubated with TP-Phos with final concentration of 10 ꢁM for 2 h at
37 °C. After the slices were washed and transferred to ibidi® 8-well culture plates, imaging
was performed using Leica TCS SP5 Confocal Scanning Microscope with 40× oil and 63×
water objectives under multiphoton excitation at 720 nm. For inhibition experiment,
Na3VO4 solution was first added to the slices and the mixture was incubated for 1 h.
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TP-Phos was then added to the slices and the mixture was incubated for another 2 h.
Subsequently, the slices were washed and imaged using a confocal microscope as
previously mentioned.
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3.1. Optical properties of TP-Phos towards ALP
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We first carried out absorption and emission spectra studies with the probe before and
after incubation with ALP (0.01 U/mL). TP-Phos (5 ꢁM) exhibited maximum absorption
peak at 300 nm. The peak shifted to 365 nm after incubation with ALP for 20 min in Tris
buffer (Fig. 1A). Fluorescence emission spectra of TP-Phos showed weak blue
fluorescence with maximum emission wavelength at 450 nm when excited at 365 nm (Fig.
1B). In contrast, intense green fluorescence with maximum emission wavelength at 500
nm can be observed after incubation with 0.01 U/ml ALP for 20 min. The inserted picture
in Fig. 1B, which was taken under hand UV lamp excitation before and after incubation
with ALP, also proves the “turn on” of fluorescence from weak blue to bright green. We
next determined two-photon absorption cross-section by investigating the two-photon
excited-fluorescence spectra of TP-Phos and TP with rhodamine-6G as the reference.
The product of TP-phos and ALP showed a maximal two photon cross-section of 90 GM
(GM: 10-50 cm4 photon-1) at 740 nm in Tris-HCl buffer (Fig. S2). However, the two-photon
absorption cross section value of TP-Phos in Tris-HCl was too small to be measured
accurately. Furthermore, the reaction products of TP-Phos and ALP showed similar
emission spectra under one-photon and two-photon excitations (Fig. S3).
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Fig. 1. Absorption (A) and fluorescent emission (B) spectra of TP-Phos(5 ꢁM) with or without 0.01 U/mL
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ALP in Tris-HCl buffer. Inset: photo of TP-Phos solution with or without ALP incubation under UV lamp
excitation.
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3.2. Kinetics and inhibition
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Next, we moved on to study the reaction kinetics of the probe with ALP. TP-Phos (5 ꢁM)
was incubated with 0.01 U/mL ALP in Tris Buffer, and the change of the emission spectra
over time was recorded. As expected, the fluorescence intensity at 500 nm increased
gradually for 25 min after ALP was added. It then reached equilibrium (Fig. S4). The
fluorescence intensity at 500 nm was plotted as a function of time for fixed concentration
of TP-Phos (5 µM) and various concentrations of ALP (0 to 0.1 U/mL). As expected, the
results show an increase in fluorescence intensity with increasing ALP concentration (Fig.
2A). The faster kinetics observed at higher concentrations of ALP signifies higher
cleavage reaction rate. We further monitored the fluorescence intensity as a function of
time with different concentrations of probe (0.1 to 20 µM) and fixed concentration of ALP
(0.01 U/mL). The first 100 seconds of each measurement are plotted in Fig. 2B. These
curves are used to determine the initial reaction rate of each hydrolysis reaction (Fig. S5).
By Linewaver-Burk analysis, Km and Vmax are calculated to be 8.56 ꢁM and 0.073 ꢁM/s
respectively. These parameters are close to the values of other ALP fluorescent probes
reported in the literature [21, 22, 36]. The applicability of TP-Phos as probe to determine
ALP activity was also demonstrated using a well-known inhibitor of ALP, sodium
orthovanadate (Na3VO4) [37, 38]. ALP was mixed with different concentrations of Na3VO4
at 37 oC for 10 min. TP-Phos was then added and the mixture was incubated for another
20 min (Fig. 2C). Fluorescence was measured at various concentration points of the
inhibitor. The corresponding IC50 was determined to be 7.39 ꢁM, which is in good
agreement with the value reported in the literature [39-41].
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Fig. 2. (A) Time-dependent fluorescence intensity of TP-Phos (5 ꢁM) with various concentrations of ALP
(0-0.1 U/mL). (B) Time-dependent fluorescence intensity of increasing concentrations of TP-Phos (0.1-20
ꢁM) with 0.01 U/mL ALP. (C) Inhibition study with 5 ꢁM TP-Phos in the presence of different
concentrations of Na3VO4. All reactions were performed in 50 mM Tris-HCl (pH 7.4) at 37 oC.
Fluorescence intensity in 2A-2C was measured at 500 nm with excitation at 365 nm.
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3.3. Detection limit of ALP
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Different units of ALP (0-10 U/L) were added to TP-Phos in Tris-HCl buffer. After the
reaction was incubated for 4 min, the fluorescence intensity was measured and plotted
against ALP activity. The result showed a linear relationship between fluorescence
intensity and ALP activity from 0.5 to 10 U/L with the fitting equation y = 9.16 [ALP] + 19.98
with R2=0.98 (Fig. S6). The detection limit based on 3σ/slope is estimated to be 0.30 U/L.
3.4. Selective detection of ALP
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The specificity of TP-Phos towards ALP versus other analytes was assessed using a
series of biologically relevant species that could potentially affect the probe response in
living cells, such as reactive oxygen species (ROS), reactive sulfur species (RSS), anions,
and various enzymes commonly found in cells. As shown in Fig. S7, TP-Phos displayed
excellent selectivity for ALP over these interferents. Furthermore, the selectivity of
TP-Phos for ALP versus other phosphatases was examined in detail using classical
protein tyrosine phosphatases (PTPs) (GLEPP, PTP1B, MEG-2, LYP), dual specific PTPs
(VHR, SSH2) and metal-dependent Ser/Thr phosphatases (PPM1A, PPM1B, PPM1G).
Fig. 3 shows the results obtained from TP-Phos in comparison with DiFMUP, a probe
commonly used for ALP detection. It can be seen that after 10 min of incubation, the
dephosphorylation rate of TP-Phos in the presence of ALP is more than ten folds higher
than that of the other nine phosphatases tested. DiFMUP, on the other hand, shows
comparable reactivity towards ALP and GLEPP, and a lower but still significant response
to at least two other PTPs (PTP1B and MEG-2). Similar results were obtained after 30 min
of incubation (Fig. S8). The high selectivity of our ALP probe is attributed to the
ortho-functionalised phenyl phosphate group, which increases the steric hindrance of the
probe to access the active site of phosphatases [42, 43]. Taken together, these results
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demonstrate the excellent sensitivity and selectivity of TP-Phos towards ALP, indicating
that it is a promising candidate for specific detection of ALP in live cells and tissues.
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Fig. 3. Selectivity studies of TP-Phos (A) and DiFMUP (B) with ALP and various phosphatases.
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Incubation with phosphatases was carried out at 37 C for 10 min. The dephosphoylation rate was
calculated by measuring the amount of released phosphatase over 10 min. TP-Phos (5 ꢁM), DiFMUP (5
ꢁM), ALP and various phosphatases (10 nM). All reactions were performed in 50 mM Tris-HCl (pH 7.4).
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3.5. ALP activity in cells and tissues lysate
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To investigate the capability of TP-Phos to detect ALP activity in cells and tissues, we
first prepared different cellular and tissue lysates according to the established protocol.
0.2 mg/mL cell lysate and 1 mg/mL tissue lysate were used in our study. The fluorescence
intensity of TP-Phos (5 ꢁM) was increased after the cell lysate or the tissue lysate was
added. By comparing the obtained fluorescence signal with the standard curve in Fig. S6,
the ALP activity was estimated to be 1.81± 0.49 U/L and 8.67±0.28 U/L in HEK 293 and
HeLa cells respectively (Fig. 4A). For tissue lysate, the ALP activity was measured to be
2.51±0.21, 0.80±0.07, 4.29±0.34 and 0.99±0.12 U/L in liver, lung, kidney and heart (Fig.
4B). These results confirm the capability of TP-Phos to detect ALP activity in various cell
and tissue lysates.
3.6. Two-photon imaging of ALP in cells
The potential of TP-Phos as a two-photon fluorescent probe for ALP detection was first
studied in living cells. The cytotoxicity of the probe was evaluated via the MTT assay using
HeLa cells (Fig. S9). Results show that TP-Phos at concentrations of up to 20 ꢁM remain
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Fig. 4. (A) Measurement of ALP activity in HEK293 cell and HeLa cell lysate with 5 ꢁM TP-Phos after 4
min of incubation at 37 oC. (B) Measurement of ALP activity in the tissue lysate of liver, lung, kidney and
heart with 5 ꢁM TP-Phos after 4 min of incubation at 37 oC.
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relatively non-toxic, with cell viability remaining above 85% after 24 h of incubation. At the
concentration of probe used for live cell imaging experiments (5 ꢁM), no toxicity could be
observed.
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Confocal microscopy on live HeLa cells was then performed to examine whether the
probe is able to detect endogenous ALP. As shown in Fig. 5A, cells incubated with
TP-Phos for 20 min showed strong intracellular green fluorescence when excited at 720
nm with a two-photon confocal microscope. Interestingly, the fluorescence is mainly
localized in cytoplasm. On the other hand, cells pre-incubated with 1 mM Na3VO4 inhibitor
for 20 min prior to the addition of TP-Phos did not show any fluorescence (Fig 5A).
Calculation of the pixel intensity of each image showed an approximate 8-fold reduction in
fluorescence intensity in cells pre-incubated with ALP inhibitor (Fig. 5B), which is
consistent with the results obtained under buffer condition. Further testing with different
concentrations of Na3VO4 (0 ꢁM, 250 ꢁM, 1 mM and 2.5 mM) demonstrate that the
inhibition is concentration-dependent (Fig. S11). Time-lapse fluorescence imaging of live
HeLa cells upon addition of 5 µM TP-Phos shows a gradual increase in fluorescence, and
the increase starts approximately 3 min after the addition of the probe (Fig. S12).
To establish more precise kinetics of probe activation in cells, the fluorescence
intensities in three cytosolic regions of interest were plotted over time. The results show a
linear increase of the fluorescence over time from t = 0 up to approximately 10 min. After
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this time frame, the probe signal appears to reach the maximum value (Fig. 5C and 5D).
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Together, these results demonstrate that TP-Phos is rapidly taken up by cells (within
minutes) and is able to detect ALP activity selectively and effectively in complex biological
environment.
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Fig. 5. (A) Confocal fluorescence images of live HeLa cells incubated with 5 ꢁM TP-Phos for 30 min, with
or without pre-incubation with 1 mM Na3VO4 for 20 min. (B) Fluorescence intensity of cells incubated with
or without Na3VO4 inhibitor. (C) Three regions of interest are selected in real time fluorescence
microscopy imaging experiments. Cells were treated with 5 ꢁM TP-Phos and incubated for 10 min. (D)
Real time fluorescence intensity change for the regions of interest highlighted in (C).
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Encouraged by above result, we further used TP-Phos to detect ALP activity in different
cell lines. We chose HEK 293 and HeLa cells as they are reported to have different ALP
activity [44-45]. Both cells were put under 10 min of incubation with 5 ꢁM TP-Phos and
then washed thoroughly to remove excess probes before cell imaging. As shown in Fig. 6,
HeLa cells displayed strong green fluorescence under 720 nm excitation, indicating a high
expression level of ALP. In contrast, HEK 293 did not show obvious fluorescence,
indicating a low level of ALP expression. Together with the result in lysates and in cell
imaging, we can conclude that TP-Phos is capable of practically detecting ALP level in
different cells and tissues.
The photostability of the probe was investigated with continuous irradiation at 720 nm in
tissue samples. The fluorescence intensity remained almost the same after 30 min of
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continuous irradiation at four randomly selected regions (Fig. S10). These results
demonstrated that our probe has excellent photostability under two-photon excitation.
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Fig. 6. Two-photon confocal images of HeLa cells and HEK 293 cells with magnification at 40 ×. Cells
were allowed to adhere to the dish under the atmosphere of 5% CO2 and 95% air at 37 ºC for 12 h. They
were then incubated with TP-Phos (5 ꢁM) for 10 min. Ex: 720 nm, Ex: 475-525 nm.
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3.7. Two-photon imaging of ALP in fresh tissues
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To further illustrate the advantages of our two-photon probe over previously reported
ALP fluorescent probes, we performed confocal imaging experiments to study ALP activity
in deep tissues. As reported in the literature, ALP is present in all the tissues throughout
the entire body. In particular, it is concentrated in the liver, bones and the kidney. We
performed a series of experiments to examine endogenous ALP activity using TP-Phos in
different tissues, including live hippocampus, liver and kidney from rat. First, bright-field
images of a hippocampus slice was used to reveal the Cornu Ammonis subfields CA1 and
CA3 (Fig. 7A). At 40× magnification, two-photon fluorescence images of the slice, which
was taken at a depth of 120 ꢁm, show strong fluorescence throughout the entire sample,
including CA1 and CA3. In contrast, the control tissue pre-incubated with Na3VO4 inhibitor
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prior to the addition of probe remain almost non-fluorescent (Fig. 7C). The results confirm
that the probe show selective response towards ALP.
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Subsequently, imaging of liver and kidney slices at various sample depths (30-230 µm)
was carried out using the z-scan mode of a confocal microscope. Fluorescence could be
observed throughout the entire range of depths tested in the liver slice (Fig. 7B and S13),
and up to a depth of 140 µM in the kidney slices (Fig. S14). Again, control samples
pre-treated with Na3VO4 inhibitor showed the absence of fluorescence signals. These
results show that TP-Phos is a suitable probe for selective imaging of endogenous ALP
activity in live tissues. It provides deep tissue penetration, high selectivity and low
background fluorescence.
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Fig. 7. (A) Two-photon images of endogenous ALP activities taken from a section of a fresh rat
hippocampal slice incubated with 10 ꢁM TP-Phos for 2 h. Images correspond to a depth of 120 ꢁm in the
tissue. Enlarged images correspond to CA1 and CA3 regions (40× magnification). (B) Two-photon
images of a slice of fresh rat liver incubated with 10 ꢁM TP-Phos for 2 h. Different images correspond to
different tissue depths (from 30 to 230 ꢁm). (C) Inhibition of ALP activity in fresh rat tissues of
hippocampus, kidney and liver: pictures on the left show tissues treated with 1 mM Na3VO4 for 1 h prior to
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incubation with 10 ꢁM TP-Phos for 2 h; picture on the right show tissues treated with TP-Phos only.
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Fluorescence images were taken at excitation wavelength of 720 nm and emission band of 475 to 525
nm.
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To compare the difference of resolution and penetration depth between one-photon
excitation and two-photon excitation, we used different lasers as excitation sources to test
the imaging with confocal fluorescence microscopy. The result clearly showed that
two-photon excitation displayed better resolution and deeper penetration after 2 h of
incubation of TP-Phos in rat kidney slice. For example, the imaging cannot be observed
clearly under one-photon excitation when the depth is greater than 70 ꢁm (Fig. S15),
whereas the fluorescence intensity of two-photon imaging remains quite bright when the
depth is around 140 ꢁm.
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4. Conclusion
In summary, we have developed a two-photon fluorescent probe, TP-Phos, for the
visual detection of ALP activity in biological samples in real time. TP-Phos offer a number
of advantages over previously reported probes, such as fast reaction kinetics, high
sensitivity and low cytotoxicity. In contrast with MUP, DiFMUP and FDP probes, TP-Phos
can differentiate between the activities of ALP from that of the other classes of
phosphatases. The fact that TP-Phos relies on two-photon excitement makes it
particularly attractive for tissue imaging applications. Indeed, we have shown that it is
suited for detecting endogenous ALP activity in both live cells and fresh rat tissues using
confocal microscopy. Nevertheless, our probe is not suitable for in vivo imaging because
its green emission does not permit deep tissue penetration. We hope to address this
problem by designing a two-photon probe with near-infrared emission property. We
believe that the next generation probe will be useful for in vivo investigation of biological
events involving ALP and it can help simplify clinical protocols for diagnosis of both liver
and bone diseases.
Acknowledgements
We are grateful for the financial support from the National Natural Science
Foundation of China (No. 21572190, 21471037 and 21602033), Guangdong
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Natural Science Funds for Distinguished Young Scholars (15ZK0307) and the
Research Grants Council of Hong Kong (No. 11302415 and 21300714).
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