A fluorescent turn-on probe for the detection of alkaline phosphatase activity in living cells

Article abstract of DOI:10.1039/c1cc13819g

Fluorescent probe 1, showing a high fluorescence turn-on signal ratio, enables the real-time imaging of endogenous alkaline phosphatase (ALP) activity in living cells, and the fast and quantitative analysis of enzyme activity at the single-cell level.

Full text of DOI:10.1039/c1cc13819g

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COMMUNICATION
A fluorescent turn-on probe for the detection of alkaline phosphatase
activity in living cellsw
a
b
Tae-Il Kim,z Hyunjin Kim,z Yongdoo Choi*b and Youngmi Kim*a
Received 27th June 2011, Accepted 21st July 2011
DOI: 10.1039/c1cc13819g
Fluorescent probe 1, showing a high fluorescence turn-on signal
ratio, enables the real-time imaging of endogenous alkaline
phosphatase (ALP) activity in living cells, and the fast and
quantitative analysis of enzyme activity at the single-cell level.
been adapted for microscopic imaging or flow cytometric
detection of ALP activity in living cells. For many of such
probes, the excretion out of cells of their water-soluble hydro-
lysis products and the high background fluorescence of the
probe result in a low signal-to-noise ratio and low sensitivity.
The commercially available, current state-of-the-art ALP probe
ELF-97 produces water-insoluble products upon enzymatic
cleavage, thereby overcoming some of the deficiencies of earlier
examples.7 The signal of the ELF-97 hydrolysis product is,
however, best visualized with a short-wavelength irradiation at
360 nm, whereas confocal laser-scanning microscopy and flow
cytometry typically use longer-wavelength Ar ion laser at
488 nm. Therefore, there is a need for new fluorescent probes
for the ALP assay that are compatible with physiologically
relevant conditions, have a high signal-to-noise ratio, and can
be excited at the 488 nm line of readily available Ar lasers. In
Alkaline phosphatases (ALPs), which catalyze the hydrolysis and
transphosphorylation of a wide variety of monophosphate
esters, consist of a group of isoenzymes that are widespread in
mammalian tissues.1 The human genome encodes four different
ALP enzyme isoforms, namely, tissue specific ALPs (intestinal,
placental, and germ cell ALP) and a tissue non-specific ALP,
which is expressed in most tissues, including liver, bone, and
kidney.2 Serum ALP activity is routinely used as a diagnostic
indicator of several human diseases, including bone diseases
(osteoblastic bone cancer, Paget’s disease, osteomalacia) and liver
diseases (cancer, hepatitis, obstructive jaundice).3 Despite the
extensive studies on ALP, its diverse physiological and patho-
logical functions have not been fully elucidated. It is only recently
that the pivotal biological roles of ALP in the maintenance of
intestinal homeostasis and protection have begun to be revealed.4
Moreover, detailed mechanisms of ALP activity regulation during
pathogenesis still remain unanswered. This is in part a consequence
of the absence of appropriate probes for the real-time tracking of
ALP activity in living systems. Therefore, the development of
sensitive and biocompatible probes with suitable intracellular
localization is highly desirable to answer unmet needs in the study
of dynamic ALP activity in various biological systems.
this communication, we report
a new iminocoumarin-
benzothiazole-based fluorophore that fully satisfies these key
requirements. We further demonstrate the application of this
probe molecule in the monitoring of ALP activity in living cells.
Scheme 1 illustrates our design rationale for ALP assays.
Fluorogenic substrates 1 and 2 are expected to display weak
emission due to fast non-radiative decay of the singlet excited
state facilitated via internal bond rotations. ALP-catalyzed
reaction triggers P–O bond cleavage in both probes 1 and 2,
and subsequent intramolecular nucleophilic attack of the
phenolic oxygen to the nitrile group results in the cyclized
iminocoumarin-benzothiazoles 3 and 4, respectively, which are
water-insoluble, and highly emissive upon photoexcitation.8
Several fluorescein- and coumarin-based substrates have been
developed and used in applications that include microplate-
based ALP assays and ALP-tagged proteins and nucleic
acids.5,6 In these the fluorescence properties change as a result
of enzymatic cleavage of a phosphate group. Although such
fluorogenic substrates have been widely used, they have rarely
a Department of Chemistry, Dankook University, 126 Jukjeon-dong,
Yongin-si, Gyeonggi-do, 448-701, Korea.
E-mail: youngmi@dankook.ac.kr; Fax: +8231-8005-3148;
Tel: +8231-8005-3156
b Molecular Imaging & Therapy Branch, National Cancer Center,
323 Ilsan-ro, Goyang-si, Gyeonggi-do 410-769, Korea.
E-mail: ydchoi@ncc.re.kr; Fax: +8231-920-2529;
Tel: +8231-920-2512
w Electronic supplementary information (ESI) available: Experimental
details and characterization and experiments using living cells. See
DOI: 10.1039/c1cc13819g
Scheme 1 Illustration of
a fluorometric assay for ALP using
z Contributed equally to this work.
iminocoumarin-benzothiazole-based probes 1 and 2.
c
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Probes 1 and 2 were synthesized in moderate yield in three
steps according to Schemes S1 and S2 (see ESIw). Both probes
1 and 2 were completely soluble in water. To demonstrate the
feasibility of the proposed detection scheme, iminocoumarin-
benzothiazole derivatives 3 and 4, which are the expected
products after reaction with ALP, were prepared according
to literature procedures.
UV-Vis absorption and emission spectra of 1 and 2 were
measured in aqueous buffer solution (10 mM Tris-HCl, pH 7.4).
As shown in Table S1 and Fig. S1 and S2 (see ESIw), probes 1
and 2 displayed a strong electronic transition at lmax,
=
abs
472 nm (e = 1.7 ꢀ 104 Mꢁ1 cmꢁ1) and 493 nm (e = 2.2 ꢀ
104 Mꢁ1 cmꢁ1), respectively. The emission spectra of 1 and 2
show maxima at 542 nm and 545 nm, respectively. The emission
quantum yields (FF) in aqueous solution are 0.002 for 1 and 0.03
for 2, which are consistent with rapid nonradiative deactivation
through free rotation about the vinylene group.9 The photo-
physical properties of iminocoumarin-benzothiazole derivatives 3
and 4 were examined in aqueous buffer solution (10 mM
Tris-HCl, pH 7.4) containing 0.5% DMSO as cosolvent (Fig. S3
and S4, see ESIw). Slight spectral changes in the absorption and
emission spectra of 3 and 4 were observed with respect to those
observed in 1 and 2, respectively: bathochromic shifts in the
absorption and hypsochromic shifts in the emission maxima.
Hydrolysis products 3 and 4 displayed higher fluorescence
quantum yields (3: FF 0.10, 4: FF 0.63) compared to that of
the precursor probes. Comparison of the quantum yields of the
probes and their hydrolysis products indicates that a higher
signal-to-noise ratio can be obtained for probe 1 than probe 2.
Consequently, our detailed studies were conducted with probe 1.
Upon incubation of probe 1 (10 mM) with ALP under
optimized assay conditions (10 mM Tris-HCl, pH 7.4, 37 1C),
the absorption spectrum showed clear bathochromic shifts
from 472 nm to 485 nm, indicating the formation of cycliza-
tion product 3 (Fig. 1a). Also, the characteristic fluorescence
signal of dye 3 at 529 nm arises and increases linearly with time
(Fig. 1b) before reaching a plateau within 30 min. A
ca. 90-fold increase in fluorescence intensity was obtained
upon incubation with ALP for 30 min. LC-MS analysis of
the assay solution confirmed that the fluorescence increase
during enzymatic reaction was due to formation of the
expected product 3 (Fig. 1c; see ESIw). The non-emissive
nature of probe 1 and the significant fluorescence turn-on of
ALP-treated probe 1 are clearly visualized, as shown in Fig. 1d.
We further investigated the ALP-catalyzed hydrolysis of
probe 1 as a function of incubation time using different
enzyme concentrations. Fig. 2 illustrates the increase in fluor-
escence intensity at 525 nm for solutions with different ALP
concentrations (0–350 nM), in which fluorescence intensity
was measured every 2 min. As expected, the fluorescence
intensity increased with increasing [ALP] within this concen-
tration range. By applying an ex situ calibration, the increase
in fluorescence intensity was shown to be quantitatively related
to the increase of [3], thus achieving real-time monitoring of
enzyme activity (see ESIw). We then determined kinetic para-
meters for the enzymatic hydrolysis reaction of probe 1 using
Lineweaver–Burk analysis, yielding values of KM = 19.2 mM
and kcat = 0.27 sꢁ1. Enzymatic efficiency of probe 1, as
Fig. 1 (a) Absorption and (b) emission spectra of probe 1 (10 mM) upon
treatment with ALP (350 nM) for different time periods (0–40 min) at
37 1C. The spectra were obtained every 5 min. (c) HPLC chromatograms
of probe 1 without treatment of ALP (top); with ALP treatment for 1 h at
37 1C (middle); 3 only (bottom). The samples were analyzed by LC-MS
with a linear gradient elution (from 0 to 50% B, A: deionized water with
1% formic acid, B: acetonitrile, flow rate 0.3 mL minꢁ1, UV: 340 nm).
The MW of the retention time at 5.5 min is 430.1, which corresponds to
[M + H]+ for probe 1 and MW of the retention time at 3.4 min is 350.1,
which corresponds to [M + H]+ for 3. [1] = [3] = 10 mM, [ALP] =
350 nM. (d) Photographs of probe 1 (10 mM) in the absence (left) and
presence (right) of ALP (350 nM) after incubation for 30 min at 37 1C
under UV light (365 nm) illumination.
that of two commercially available ALP fluorogenic sub-
strates, 4-MUP (kcat/KM = 7.7 ꢀ 103 Mꢁ1sꢁ1) and ELF-97
(kcat/KM = 3.0 ꢀ 104 Mꢁ1sꢁ1), under the same assay condi-
tions (see ESIw).
Next, the utility of probe 1 for the screening of ALP
inhibitors was investigated. The inhibition of ALP activity
was tested for two common ALP-inhibitors, p-bromotetrami-
sole (p-BT) and levamisole, at pH 7.4. ALP was pre-incubated
with different concentrations of the inhibitor for 2 min at
25 1C. Inhibitor pre-treated ALP solution was then added to
probe 1 in aqueous solution, and the enzyme reaction was
monitored by measuring the fluorescence intensity at 525 nm.
As shown in Fig. 2, the enhancement of fluorescence intensity
Fig. 2 (left) Increase in fluorescence intensity at 525 nm recorded every
2 min during a real-time turn-on assay of probe 1 (10 mM) under
varying concentrations (0–350 nM) of ALP in Tris-HCl buffer (10 mM,
pH 7.4) at 37 1C. F0 and F correspond to the fluorescence intensity of
probe 1 in the absence and the presence of ALP, respectively. (right)
Inhibition assay of ALP activity by p-BT and levamisole. Emission
intensity of probe 1 as a function of concentration of inhibitors. ALP
was incubated with an inhibitor for 2 min at 25 1C before addition of
probe 1 to final concentration of 10 mM. After 2 min incubation at
37 1C, emission intensity at 525 nm was recorded (lex 460 nm). Each
point was obtained from the average value of three measurements.
estimated by a kcat/KM = 1.4 ꢀ 104 Mꢁ1
s
ꢁ1, was between
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of probe 1 was inhibited in a dose-dependent manner. In
particular, addition of 100 mM p-BT entirely blocked the
fluorescence increase, providing confirmation that the enzy-
matic activity of ALP was required to hydrolyze probe 1. The
IC50 was calculated to be 36 mM for p-BT and 75 mM for
levamisole, which are in good agreement with the reported
literature value determined by other methods.10,11 These
results highlight the aptitude of probe 1 for both ALP activity
assays and the screening of inhibitors.
1-treated HeLa cells were 2 and 2144, respectively. On the other
hand, the cells pre-treated with levamisole and then treated with
probe 1 showed a significant shift in their histogram, resulting in
ca. 11-fold smaller mean fluorescence intensity than that of the
probe 1-treated cells (Fig. 3b). These results confirm that probe 1
is useful for fast and quantitative analysis of ALP activity at the
single-cell level, in addition to the visualization of ALP activity
using confocal fluorescence microscopy. Cell viability assays
showed that probe 1 had no cytotoxic effect on the cells at a
concentration of 10 mM after 1 h of treatment time, indicating
that probe 1 is highly biocompatible (see ESIw).
In the case of ALP-negative HT-29 cells treated with 10 mM
probe 1, no meaningful fluorescence was observed from
confocal microscopy images even after 30 min of incubation,
indicating that there is no nonspecific fluorescence increase
following intracellular uptake. In addition, FACS analysis of
the probe 1-treated HT-29 cells showed that Geo mean
fluorescence intensity is only 144 (see ESIw). Again, these
results support an increase in fluorescence in HeLa cells
exclusively due to the enzymatic activity of ALP.
We then evaluated the applicability of probe 1 for the
detection of endogenous ALP in living cells, and the quanti-
tative analysis of the ALP activity at the single cell level by
confocal fluorescence microscopy and fluorescence-activated
cell sorting (FACS) analysis. The HeLa human cervical
carcinoma cell line, which is known to express placental
ALP, and the HT-29 human colon adenocarcinoma cell line,
which is known to exhibit only trace levels of intestinal ALP,
were tested as an ALP-positive and ALP-negative control,
respectively.12 The confocal fluorescence microscopy image of
HeLa cells that were treated with 10 mM probe 1 only for
2 min clearly showed an increase in green fluorescence inside
cells, indicative of ALP activity (Fig. 3a). Meanwhile no
fluorescence was observed from cells untreated with probe 1.
When the cells pre-incubated with 10 mM levamisole were
treated with probe 1, a marked decrease in fluorescence
intensity within HeLa cells was shown. These fluorescence
images revealed that probe 1 permeated into the cells, was
activated by ALP, and accumulated in them.
In summary, we have developed a versatile ALP-inducible
fluorescent turn-on probe that is suitable for the real-time
dynamic monitoring of endogenous ALP activity in living
systems as well as for developing and characterizing ALP
inhibitors and activators. This new probe advances the mole-
cular toolkit of ALP detection, facilitating the investigation of
its biological and pathological roles in living systems.
This research was supported by a grant from the Funda-
mental R&D Program for Core Technology of Materials
funded by the Ministry of Knowledge Economy, Republic of
Korea and by a Korea Research Foundation (KRF) grant
funded by the Korea government (MEST) (No. 2010-0017220).
Probe 1 was then applied to FACS analysis of ALP activity.
As a result, probe 1-treated HeLa cells showed a distinct
histogram compared to that of the unstained control cells. Geo
mean fluorescence intensities of unstained control cells and probe
Notes and references
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Fig. 3 Application of ALP probe 1 to live cell studies. (a) Represen-
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1 for 2 min (magnification: 80ꢀ). For inhibitor-treated cells, levamisole
(10 mM) was pre-incubated for 15 min before incubation with probe 1
(10 mM). Bottom row: transmitted light images merged with the
fluorescence image above. Left: unstained control, middle: cells treated
with probe 1, right: cells treated with levamisole and probe 1.
Ex. 488 nm, Em. 505–530 nm. (b) FACS study for quantitative analysis
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c
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Chem. Commun., 2011, 47, 9825–9827 9827