A ratiometric fluorescent probe for quantification of alkaline phosphatase in living cells

Article abstract of DOI:10.1039/c6ra00983b

A ratiometric fluorescent probe with strong intramolecular charge transfer (ICT) character has been designed for the detection of alkaline phosphatase (ALP). In the presence of ALP, probe 1 showed a dramatic bathochromic shift from 550 to 650 nm in the fluorescence spectrum and an obvious ratiometric signal with an isoemissive point was observed. The spectral change was attributed to the hydrolysis and cleavage of a phosphate group from the probe that changed the ability of the intramolecular charge transfer. The fluorescence intensity ratio displayed a linear relationship against the concentration of ALP in the concentration range from 50 to 200 U L^-1. Other biological species, including lysozyme, trypsin, pepsin, acetyl cholinesterase, carboxylesterase, and bovine serum albumin, did not induce distinct spectral changes of the probe, indicating its selective sensing ability to ALP. Furthermore, probe 1 was also successfully applied to image endogenous ALP activity in living cells.

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FULL PAPER
A ratiometric fluorescent probe for quantification of alkaline
phosphatase in living cells
ab
*a
a
b
a
Zhenxing Lu, Jiasheng Wu, Weimin Liu, Guangyou Zhang, Pengfei Wang
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/RSC
A ratiometric fluorescent probe with strong intramolecular charge mechanism of these diseases so as to find an efficient cure method.
transfer (ICT) character has been designed for the detection of Recently, various assays for ALP detection have been developed,
alkaline phosphatase (ALP). In the presence of ALP, the probe 1 including electrochemisty, colorimetric method,
8
,9
10,11
and surface
1
2
showed a dramatic bathochromic shift from 550 to 650 nm in the enhancement Raman scattering assay. Although these provide
fluorescence spectrum and an obvious ratiometric signal with an efficient ways for ALP detection, they often need laborious
isoemissive point was observed. The spectral change was experimental procedures or expensive instruments, and thus they
attributed to the hydrolysis and cleavage of phosphate group from are not appropriate for applications in living cells. Alternatively,
the probe that changed the ability of the intramolecular charge fluorescent probes especially those based on small molecules have
transfer. The fluorescent intensity ratio displayed a linear attracted much attention due to their intrinsic advantages such as
1
3-15
relationship against the concentration of ALP in the concentration low background noise, high sensitivity and selectivity.
range from 50 to 200 U/L. Other biological species including till now, only a few fluorescent probes have been developed for ALP
lysozyme, trypsin, pepsin, acetyl cholinesterase, carboxylesterase, sensing. For example, Song and coauthors developed
However,
a
and bovine serum albumin did not induce distinct spectral changes phosphorylated tetraphenylethene derivative as a fluorescence
3
of the probe, indicating its selective sensing ability to ALP. turn-on probe based on aggregation induced emission mechanism.
Besides, the probe 1 was also successfully applied to image Kim and coauthors reported a series of fluorescent probes based on
1
6,17
endogenous ALP activity in living cells.
iminocoumarin-benzothiazole for ALP imaging in living cells.
Although these probes showed good sensing properties, their
shorter emission wavelength and single wavelength as output signal
will limit their potential applications in living cells. Therefore,
fluorescent probes with longer emission wavelength and multiple
output signals are more preferable.
Introduction
Alkaline phosphatase (ALP) is a hydrolase that is responsible for the
removal of phosphate group from many kinds of biological
substrates such as proteins, nucleic acids, carbohydrates and other
Weak ICT
Strong ICT
1
small molecules. It is widely spread in mammalian tissues but
OH
O
O
OH
2
P
mostly distributed in intestine, placenta, bone, liver and kidney.
O
OH
ALP
O
The average serum ALP range of normal adults varies from 40 U/L
3
to 150 U/L. Abnormal expression, mainly related to elevated levels
of ALP is usually in connection with some diseases, i.e.,
NC
CN
NC
CN
3
4
5
6
osteoporosis, hepatitis, prostatic cancer, and bone cancer.
Probe 1
4
Therefore, the alkaline phosphatase has been identified as an Scheme 1 Dephosphorylation of the probe 1 in the presence of ALP.
7
;
important biomarker for clinical diagnostics. Those techniques
In this communication, we have designed a dicyano-based
which could reveal the information of ALP, such as its distribution
compound with strong ICT character as a fluorescent probe for the
and activity, are very helpful for medical researchers to know the
detection of ALP (Scheme 1). The phosphate group functions as a
recognizing group that can be released from the probe in the
presence of ALP, as well as a modulator to tune the ICT ability of the
a.
Key Laboratory of Photochemical Conversion and Optoelectronic Materials,
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
phenolic hydroxyl through the hydrolysis of phosphomonoester. As
Beijing, 100190, P.R. China. Email: wujs@mail.ipc.ac.cn.
School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022,
P.R. China.
Electronic Supplementary Information (ESI) available: Additional spectra and NMR
copies. See DOI: 10.1039/x0xx00000x
expected, the probe 1 upon interaction with ALP shows a distinct
emission red shift from 550 nm to 650 nm. Therefore, the ICT
process could be regulated that can result in ratiometric signals
both in the absorption and fluorescence spectra. Ratiometric
b.
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Journal Name
fluorescent probes which employ two fluorescence intensity as was exchanged with fresh medium again. The cells were further
o
2
incubated for 24 h at 37 C under the atmosphere of 5% CO . Then
output signal are more desirable as they are free of interference
from autofluorescence, probe concentration and emission
the cell medium was exchanged with a mixture of 100 μL fresh
medium and 20 μL MTT (5 mg/ mL). After incubation for 4h under
the same condition, the cell medium was discarded and DMSO (100
μL) was added. All wells were then measured at 570 nm, and cell
viability of untreated cells was set to 100% as a reference.
1
8
intensity. The intensity ratio of the probe also shows a linear
relationship with ALP concentration through a wide concentration
range of ALP. Besides, the probe 1 was applied to endogenous
enzyme activity imaging in living cells.
ALP imaging in living cells
OH
OH
O
Na
HCl
Malonontrile
Ethyl acetate
As placental ALP is over-expressed in human uterine cervical cancer
cells, Hela cells were used as the cell experiment. Cells were
O
O
O
O
2
cultured in three confocal dishes in the culture medium overnight
OH
o
O
at 37 C under a CO (5%) atmosphere. Then three confocal dishes
2
O
p-Hydroxybenzaldehyde
were treated differently and imaged on a Nikon C1si laser scanning
confocal microscope. The images were obtained from the red
channel with an excitation at 488 nm. In the first dish, Hela cells
were imaged without treatment of the probe or sodium
orthovanadate. In the second dish, Hela cells were incubated with
the probe (10 μM) for 30 min before imaging, In the third dish, Hela
cells were imaged after incubation with ALP inhibitor sodium
orthovanadate (final concentration 1 mM) for 30 min, then with the
probe (10 μM) for another 30 min.
Diethyl chlorophosphate
NC
CN
3
NC
CN
4
O
OH
O
O
O
O
P
P
O
O
O
OH
TMSi
NC
CN
NC
CN
5
Probe 1
Scheme 2 Synthetic procedures of the probe 1.
Synthetic procedures of the probe 1
Experimental
Preparation of 2-(2-methyl-4H-chromen-4-ylidene)malononitrile
(
compound 3)
Chemicals and materials
Compound 3 was prepared in a modified way according to
literature reported. 2-Hydroxyacetophenone (0.5 g, 3.7 mmol)
and sodium (0.6 g, 26.1 mmol) were added to a 50 mL flask charged
with 20 mL anhydrous ethyl acetate. The mixture was stirred under
room temperature for about 18 h. The mixture was slowly poured
Diethyl chlorophosphate and 4-hydroxybenzaldehyde were
purchased from Energy Chemical. Iodotrimethylsilane,
19
triethylamine, calcium hydride, and 2'-hydroxyacetophenone were
purchased from J&K Chemicals. Anhydrous tetrahydrofuran was
purchased from Tianjin Heowns Biochemical Technology. into 50mL cold water then neutralized with dilute HCl solution. The
Malononitrile and 4-dimethylaminopyridine were purchased from organic portion was separated and dried with sodium sulfate,
Sino Reagent. Alkaline phosphatase from calf intestine was filtered and evaporated under reduced pressure to give the desired
product which was used directly in the next step without further
purification. The crude product was dissolved in 20mL methanol in
a 50 mL flask, and a few drops of hydrochloric acid was added. The
reaction mixture was stirred under room temperature for 4h. Then
methanol was evaporated and the residue was extracted with
dichloromethane and washed with saturated NaCl. The organic
layer was dried, filtered and purified using silica gel column
chromatography to obtain compound 2 as a yellow solid (0.3 g,
purchased from Sigma Aldrich. Other enzymes used in selectivity
experiment were purchased from Amresco LLD, Acros Organics and
BoiDee Biotechnology. Anhydrous dichloromethane was prepared
by refluxing analytical grade dichloromethane with calcium hydride.
Unless noted, all the chemicals were of analytical grade and used as
received without further purification.
Instrument
5
0%).
1
13
H NMR and C NMR spectra were recorded on a Bruker 400 MHz
The intermediate 2 (0.4 g, 2.5 mmol) and malononitrile (0.22 g,
NMR spectrometer instrument. Mass spectra were recorded on 3.0 mmol) were added to 25 mL acetic anhydride with continuous
Waters GCT Premier or Shimadzu LC-MS 2010 mass spectrometer, stirring. The mixture was heated to reflux for 14 h to complete the
respectively. Absorption spectra and fluorescence spectra were reaction, then the solvent was evaporated. Water (50 mL) was
added to the residue and the mixture was further refluxed for 30
min, followed by extraction with dichloromethane. The organic
layer was dried, filtered and purified with silica gel column
recorded on Hitachi U-3900 UV-Vis absorption spectrophotometer
and Hitachi F-4600 fluorescence spectrophotometer, respectively.
Fluorescence imaging was taken with a Nikon C1si laser scanning
confocal microscope.
chromatography to yield the intermediate 3 as an orange solid (0.15
1
g, 29%). H NMR (400 MHz, CDCl
3
, ppm): δ = 8.92 (d, J = 8.7 Hz, 1H),
7
3
.72 (t, J = 7.8 Hz, 1H), 7.46 (t, J = 7.0 Hz, 2H), 6.72 (s, 1H), 2.44 (s,
H). MS (EI): m/z = 208
Cell viability
Hela cells in a 96-well plate were firstly cultured using DMEM for 12
h. Then the cell medium was exchanged with fresh medium
containing different amount of probe (final concentration, 0, 2, 4, 6,
Synthesis of 2-(2-(4-hydroxystyryl)-4H-chromen-4-
ylidene)malononitrile (compound 4)
8, 10 μM). Hela cells were further cultured for 2 h after the medium
2
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The intermediate 4 was prepared in a modified way according to after the cleavage of phosphate group, resulting in obvious spectral
20
literature reported.
Compound 3 (0.15 g, 0.7 mmol), 4-
shift and intensity change.
hydroxybenzaldehyde (0.10 g, 0.8 mmol), together with catalytic
amount of acetic acid (0.5 mL) and piperidine (0.5 mL) were added
to a round-bottom flask with stirring anhydrous toluene (20 mL).
The mixture was refluxed for 12h then solvent was removed under
vacuum. The crude product was further purified with silica gel
1.2
(a)
1.0
ALP
0.8
0.6
column chromatography to obtain compound 4 as an orange solid
1
(
0.1 g, 44%). H NMR (400 MHz, DMSO-d , ppm): δ = 10.15 (s, 1H),
6
8
7
1
.74 (d, J = 8.4 Hz, 1H), 7.94 – 7.90 (m, 1H), 7.80 (d, J = 8.4 Hz, 1H),
.70 (d, J = 16.0 Hz, 1H), 7.65 – 7.59 (m, 3H), 7.29 (d, J = 16.0 Hz,
H), 6.96 (s, 1H), 6.86 (d, J = 8.6 Hz, 2H). MS (EI): m/z = 312.
0
0
0
.4
.2
.0
Synthesis of 4-((E)-2-(4-(dicyanomethylidene)-4H-chromen-2-
yl)ethenyl)phenyl diethyl phosphate (compound 5)
350
400
450
500
550
600
650
Wavelength (nm)
The intermediate 4 (0.20 g, 0.6 mmol) and DMAP (50 mg) were
dissolved in dry THF. Diethyl chlorophosphate (0.15g, 0.9 mmol)
and triethylamine (0.3 mL) were added under the nitrogen
atmosphere. The mixture was stirred at room temperature
overnight. The reaction mixture was condensed under reduced
pressure and purified with column chromatography to give the
400
(b)
350
300
250
ALP
1
200
50
desired 5 as a light brown solid (0.11 g, 35%). H NMR (400 MHz,
1
CDCl
3
, ppm): δ = 8.92 (d, J = 8 Hz, 1H), 7.75 (t, J = 8 Hz, 1H), 7.62 –
7
6
.56 (m, 4H), 7.46 (t, J = 8 Hz, 1H), 7.31 (d, J = 8 Hz, 2H), 6.87 (s, 1H),
100
50
0
1
3
.76 (d, J = 16 Hz, 1H), 4.29 – 4.22 (m, 4H), 1.38 (t, J = 7 Hz, 6H).
C
NMR (400 MHz, CDCl , ppm): δ = 157.42, 152.86, 152.60, 152.52,
3
500
550
600
650
700
750
800
1
1
37.81, 134.80, 131.71, 129.54, 126.03, 120.95, 118.03, 116.74,
15.66, 107.01, 64.99, 64.93, 63.43, 58.52, 18.55, 16.24, 16.18.
Wavelength (nm)
HRMS (EI): calcd m/z= 448.1188, obsd m/z = 448.1185.
Fig. 1 Time-dependent absorption (a) and emission (b) spectra of
the probe 1 (50 μM) in the presence of ALP (500 U/L) in Tirs-HCl
buffer solution (10 mM, pH = 7.4, 25 °C) recorded within 30 min.
Synthesis of 4-((E)-2-(4-(dicyanomethylidene)-4H-chromen-2-
yl)ethenyl)phenyl phosphate (the probe 1)
4
-((E)-2-(4-(dicyanomethylidene)-4H-chromen-2-yl)ethenyl)phenyl
The intermediate 4 in Tris-HCl buffer solution (10 mM, pH=7.4,
5 °C) shows a characteristic absorption band at 395 nm and an
diethyl phosphate (0.1 g, 0.2 mmol) was dissolved in anhydrous
dichloromethane. Iodotrimethylsilane (2 equiv) was added and the
mixture was stirred under room temperature for 4 h. The solvent
was removed to yield a dark solid (80 mg, 100%). H NMR (400
MHz, DMSO-d
2
emission band at 650 nm, respectively (Fig. S1). And its molar
4
-1
-1
extinction coefficient at 395 nm is 1.1 × 10 M cm . In contrast,
the probe 1 showed distinct different characteristic absorption and
emission bands peaked at 440 nm and 550 nm, respectively. The
1
6
, ppm): δ = 8.74 (d, J = 8.4 Hz, 1H), 7.93 (t, J = 8.2 Hz,
1
1
H), 7.82 – 7.73 (m, 4H), 7.62 (t, J = 8.1 Hz, 1H), 7.46 (d, J = 16.1 Hz,
H), 7.26 (d, J = 8.4 Hz, 2H), 7.03 (s, 1H), –OH (not found). C NMR
4
-
1
3
molar extinction coefficient of the probe 1 at 440 nm is 2.2 × 10 M
1 -1
cm . Emission spectrum of the probe 1 changed with different pH
(100 MHz, DMSO-d , ppm): δ = 158.58, 153.69, 153.62, 153.31,
6
conditions (Fig. S3). The emission spectra of 1 remain stable in the
pH range from 7 to 10, and it was sharply decreased in the acidic
solution with pH from 7 to 3. Compound 4 is also pH sensitive from
pH 3 to 10, as shown in Fig. S4. In this work, the physiological pH
was used for the spectral tests. In addition, fluorescence quantum
yields of 1 and 4 were calculated to be 10.5% and 7.8%, respectively,
152.44, 138.34, 135.87, 131.24, 130.13, 126.61, 125.08, 121.06,
121.01, 119.51, 119.32, 117.59, 117.52, 116.25, 107.03, 60.71.
-
HRMS (ESI): m/z = 391.0490 [M-H] .
Results and discussion
21
using rhodamine 101 as the reference compound.
Upon addition of ALP (500 U/L) to the solution of the probe 1 (50
μM), the absorption spectrum was recorded every two minutes
over 30 min (Fig. 1a). For every two minutes, the maximum
absorption decreased gradually with a concomitant increase around
The synthetic route of the probe 1 was shown in Scheme 1. The
1
13
structure of 1 has been verified by H NMR, C NMR and mass
spectra analyses. The intermediate 4 which undergoes an ICT
process between the dicyano group and phenolic hydroxyl group
was chosen as a fluorescence generator. Phosphate group was
introduced to the compound 4 through a condensation and
hydrolysis reaction to give the probe 1. The phosphate group
functions as a recognizing group which could be selectively cleaved
5
25 nm. However, its absorption around 395 nm only decreased
slightly, becoming the maximum absorption after 1 was further
incubated with ALP. After half an hour later, the reaction can reach
a platform and the absorption cannot be variable. The fluorescence
response of 1 towards ALP was recorded for half an hour under the
in the presence of alkaline phosphatase. As a weak electron same condition. As illustrated in Fig. 1b, the maximum emission
withdrawing group, the presence of phosphate group could reduce decreased gradually with the time elapsing as 1 was incubated with
the electron donating ability of the oxygen atom of phenolic ALP, and a new peak at 650 nm emerged simultaneously which
progressively became dominant after further incubation. The probe
hydroxyl group, thus different ICT intensities occurred before and
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shows a colour change from yellow-green to light orange (Fig. S5).
Journal Name
1
Obviously, bathochromic shift of the spectra and color change were
attributed to stronger an ICT process resulted from the cleavage of
phosphate group by ALP. The absorption and emission spectra of
the probe 1 after incubation with ALP for 30 min are very similar to
those of the compound 4 (Fig. S2), also indicating that 1 in the
presence of ALP can produce enzymatic product 4. To further prove
the conversion of 1 to 4 by ALP, enzymatic product has been
studied through ESI mass spectrum as follows, the probe 1 (100 μM)
was incubated with ALP in PBS buffer for 40 min, and the resulting
precipitate was analyzed (Fig. S6). It was found that the ESI peak of
enzymatic product 4 (311.0825) was clearly observed and no signals
for the probe 1 was found, indicating that the full conversion of the
probe 1 into enzymatic product 4 in the presence of ALP.
0
0
0
0
0
.8
.6
.4
.2
.0
AChE Trypsin Lysozyme Pepsin
ALP
hCEs
BSA
Fig. 3 Fluorescence changes at 650 nm of the probe 1 in the
presence of the same amount of AChE, trypsin, lysozyme, pepsin
ALP, hCEs and BSA in Tris-HCl buffer solution (10 mM, pH = 7.4, 25
(a)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
°
C).
10 U/L
8
1
2
3
4
5
0 U/L
ALP
00 U/L
00 U/L
00 U/L
00 U/L
00 U/L
Selectivity is a very important parameter for evaluating its
practical application. To verify whether this probe is selective to ALP,
fluorescence responses of 1 towards competitive biological analytes
were investigated under the same conditions (Tris-HCl buffer, 10
mM, pH=7.4) (Fig. 3). Other enzymes including pepsin, lysozyme,
trypsin, acetylcholinesterase (AChE), human carboxylesterase (hCES)
and bovine serum albumin (BSA) were added to the solution of 1,
respectively, and fluorescence responses were then recorded. The
results showed that no significant fluorescence changes were
observed, demonstrating its high selectivity toward ALP.
0
10
20
Time (min)
30
40
50
(b)
0.8
0.6
0.4
0.2
0.0
0
0
0
0
0
0
0
.7
.6
.5
.4
.3
.2
.1
40
60 80 100 120 140 160 180 200 220
ALP (U / L)
0
200
400
600
800
1000
Inhibitor concentration (µM)
Fig. 2 (a) Time-dependant fluorescence intensity ratio (F650/F550)
of the probe 1 (50 μM) in the presence of different amount of ALP
3
.5
0
mM
(10~500 U/L) in Tris-HCl buffer solution (10 mM, pH = 7.4, 25 °C). (b)
3.0
1 mM
Fluorescence intensity ratio of 1 as a function of ALP concentration
after ALP was incubated with different amount of ALP for 30 min.
2
2
.5
.0
In order to verify whether the probe 1 has the ability to quantify
ALP concentration, the fluorescence response of 1 (50 μM) in Tirs-
HCl buffer (10 mM, pH = 7.4) towards different amount of ALP (10 -
1.5
1
0
.0
.5
5
00 U/L) were recorded at room temperature. As shown in Fig. 2,
higher ALP concentration give rise to higher fluorescence ratio
F650/F550). Moreover, the fluorescence ratio was plotted versus
0.0
(
0
10
20
30
40
50
ALP concentration after the probe 1 was incubated with different
amount of ALP for 30 min, and they exhibited a good linear
relationship through a wide concentration range of 50 - 200 U/L.
The linear regression of equation is as follows: F650/F550 = 0.00325
Time (min)
Fig. 4 (a) Fluorescence intensity ratio (F650/F550) of the probe 1 in
the presence of ALP and different amount of sodium orthovanadate
after incubation for 30 min. (b) Fluorescence intensity ratio
F650/F550) change of 1 and ALP mixture in the absence and
presence of sodium orthovanadate.
×
[ALP] + 0.0297 (U/L), r = 0.99615. The detection limit calculated
from the former equation is 3.8 U/L, low enough for ALP detection
in biological samples, because in human serum the normal ALP
range is 40 - 150 U/L.
(
4
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To further verify the hydrolysis reaction of 1 in the presence of in vitro experiment. As shown in Fig. 6, Hela cells in the absence of
ALP, its inhibition effect has been studied. Sodium orthovanadate is 1 showed no fluorescence, while significant red fluorescence was
2
2
a well known inhibitor for alkaline phosphatase. Fluorescence observed after incubation with the intracellular ALP. This result
responses of 1 (50 μM) with ALP (0.5 U/mL) in the absence and indicates the existence of ALP in Hela cells. To further prove the fact
presence of different amount of sodium orthovanadate were that the fluorescence was triggered by the dephosphorylation of
recorded after the mixture were incubated for 30 min. And endogenous ALP in Hela cells, a comparison experiment was carried
fluorescence intensity ratio was plotted versus the incubation time. out in which Hela cells was incubated with ALP inhibitor (sodium
As displayed in Fig. 4, the ALP activity was inhibited in a dose orthovanadate 1 mM) for 30 min prior to the addition of 1. As
dependent manner. Particularly, in the presence of 1 mM sodium expected, only negligible fluorescence was observed, indicating that
orthovanadate, the fluorescence ratio shows no variations after 30 this probe can function well to monitor ALP activity in living cells.
min, indicating a complete inhibition of ALP activity.
Conclusion
1
00
In summary, a ratiometric fluorescent probe for the sensitive
sensing of ALP activity has been reported. In the presence of ALP,
fluorescence intensity ratio showed a ratiometric fluorescence
change, whereas other competitive biological species did not cause
distinct fluorescence changes. The probe 1 also gives a linear
relationship versus ALP concentration in a wide range from 50 to
50
2
00 U/L, making it possible to quantify the concentration in
0 -
intracellular ALP. With low cytotoxicity, the probe has been applied
in fluorescence imagining in living cells. Confocal laser scanning
imaging indicates that this probe is capable of imaging endogenous
ALP activity in Hela cells.
1
0
1
2
3
4
5
6
7
8
9
10 11
Probe concentration (µM)
Fig. 5 Cytotoxicity of the probe 1 with different concentrations in
Hela cells.
Acknowledgements
To find out whether the probe has a potential to be applied in
fluorescence imaging for ALP activity in Hela cells, MTT assay was This work was supported by Major State Basic Research
carried out to evaluate cell viability in the presence of different Development Program of China (No. 2014CB932600) and the NNSF
amount of 1. As shown in Fig. 5, the probe 1 shows very low of China (Grant Nos. 21173244).
cytotoxicity even at a concentration up to 10 μM. Thus, this probe is
biocompatible and capable for the cell culture.
Notes and references
1
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