Novel ratiometric fluorescent probe for real-time detection of alkaline phosphatase and its application in living cells

Article abstract of DOI:10.1016/j.saa.2021.119953

A novel ratiometric fluorescent probe has been developed through a simple synthetic route for the detection of alkaline phosphatase(ALP) in aqueous media and for fluorescence imaging in living cells. The introduction of a spontaneous-degradation spacer in

Full text of DOI:10.1016/j.saa.2021.119953

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119953
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Novel ratiometric fluorescent probe for real-time detection of alkaline
phosphatase and its application in living cells
1
1
⇑
⇑
Xiaoqian Huang , Xiangzhu Chen , Shijun Chen, Xueyan Zhang, Lin Wang, Shicong Hou , Xiaodong Ma
College of Science, China Agricultural University, Beijing 100193, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Novel ratiometric fluorescent probe for real-time detection of alkaline phosphatase and its application in
living cells.
ꢀ
A novel ratiometric probe was
constructed to detect alkaline
phosphatase.
ꢀ
The introduction of spontaneously
degradable linker is conducive to the
construction of ratio fluorescent
probes.
ꢀ
ꢀ
The detection limit is as low as 0.16
U/L with high selectivity and
accuracy;
This probe possessing excellent
biocompatibility.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 April 2021
Received in revised form 30 April 2021
Accepted 9 May 2021
Available online 12 May 2021
A novel ratiometric fluorescent probe has been developed through a simple synthetic route for the detec-
tion of alkaline phosphatase(ALP) in aqueous media and for fluorescence imaging in living cells. The
introduction of a spontaneous-degradation spacer in the design of the fluorescent probe is beneficial
for the ratio detection method and allows the selection of a fluorophore with an amino group. Under
catalysis by ALP, the phosphate monoester bond breaks; this is followed by 1,4-elimination, decomposi-
tion of the carbamate moiety, and subsequent formation of the 4-amine-1,8-naphthalimide fluorophore.
The probe APN shows a significant fluorescence colour change from blue to green in response to ALP, and
the fluorescence intensity ratio of the probe solution (F550/F480) has a good linear relationship with the
ALP concentration in the range of 0 to 100 U L-1. Our studies have demonstrated that APN exhibits high
accuracy in recognising ALP, with a detection limit as low as 0.16 U L-1. Furthermore, the probe shows
very good biocompatibility, which is beneficial for its application in biological systems.
Keywords:
Fluorescent probe
Alkaline phosphatase
Ratiometric
Cell imaging
Ó 2021 Elsevier B.V. All rights reserved.
1
. Introduction
Alkaline phosphatase (ALP, EC 3.1.3.1) is a biological hydrolase
metabolism, signal transduction, molecular transport, and expres-
sion of genetic information [4,5]. It is widely used as a tool enzyme
in nucleic acid editing [6]. The level of ALP is an important indica-
tor in routine blood tests. Abnormal increases in alkaline phos-
phatase activity in serum are associated with many diseases,
including bone diseases (osteogenic bone cancer, Paget’s bone dis-
ease, osteomalacia), liver diseases (cancer, hepatitis, obstructive
jaundice), breast cancer, prostate cancer, and diabetes [3,7–11].
However, the level of ALP may also be reduced by metabolic disor-
ders, such as Wilson’s disease or blood system diseases, such as
aplastic anaemia and chronic myeloid leukaemia [12]. Although
ALP has been extensively studied, its various physiological and
that catalyses the dephosphorylation and transphosphorylation
of proteins, nucleic acids, and some small biomolecules [1,2]. The
enzyme plays a vital physiological role in organisms and is com-
monly found in bacteria, fungi, plants, and animals [3]. ALP is
involved in almost every type of biological process, including
⇑
Corresponding authors.
E-mail addresses: houshc@cau.edu.cn (S. Hou), dongxm@cau.edu.cn (X. Ma).
These authors contributed equally.
1
https://doi.org/10.1016/j.saa.2021.119953
386-1425/Ó 2021 Elsevier B.V. All rights reserved.
1

X. Huang, X. Chen, S. Chen et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119953
2.2. Synthesis of the probe APN
pathological functions have not been completely elucidated. In
addition, the detailed mechanism of ALP activity regulation during
pathogenesis remains unresolved. Therefore, accurate and sensi-
tive detection methods need to be established to track the activity
of ALP in living systems in real time.
To date, several classical colorimetric methods for the visual
detection of ALP have been developed based on the biological
functions of the enzyme. Substrates such as diphenyl phosphate
and p-nitrophenyl phosphate (pNPP) were used in these meth-
ods; the compounds were dephosphorylated by ALP, producing
significant colour changes, which provided a measurable rela-
tionship between the colorimetric signals and ALP activity
2.2.1. Synthesis of compound D2
3
p-Hydroxybenzaldehyde (2.5 g, 20 mmol) was dissolved in CH -
CN (100 mL) cooled to 0 °C in an ice bath. Under stirring, diethyl
chlorophosphate (4.24 g, 22 mmol) and triethylamine (0.15 mL)
were added. The reaction mixture was stirred at room temperature
overnight. After completion of the reaction, the solvent was evap-
orated to give a pale-yellow oil. The crude product was used
directly for the next step without purification. The oil was dis-
3
solved in CHCl /i-PrOH (3:1; 300 mL) and cooled to 0 °C in an ice
bath. After silica gel (10 g) was added to the reaction mixture,
sodium borohydride (1.6 g, 40 mmol) was added in portions, and
the mixture was allowed to react for 0.5 h. After filtration, the silica
[
13,14]. These methods are characterised by short reaction
times and automated procedures; they are sufficient for tradi-
tional or classic clinical trials to simply indicate advanced dis-
ease. However, to further investigate the mechanism of action
of ALP in cells and even organisms and to meet the require-
ments for higher accuracy and sensitivity at the single-cell level,
new idealised detection strategies need to be developed. Rela-
tive to traditional colorimetric methods, fluorescence analysis
methods generally have the characteristics of high sensitivity,
low sample volumes, strong anti-interference ability, and appli-
cability for in situ detection and imaging [15–16].
gel was washed with an appropriate amount of CH
phase was washed with 10% (w/w) NaHCO solution and water,
collected, and dried over anhydrous Na SO . The solvent was evap-
orated to dryness, and the residue was purified by column chro-
matography to obtain D2 as a white solid in a yield of 72%. 1
NMR (300 MHz, CDCl ): d = 7.32 (d, J = 8.7 Hz, 2H), 7.18 (d,
2 2
Cl . The organic
3
2
4
H
3
J = 7.8 Hz, 2H), 4.64 (d, J = 5.7 Hz, 2H), 4.30 – 4.11 (m, 4H), 2.45
(t, J = 5.9 Hz, 1H), 1.35 ppm (td, J = 7.1, 1.0 Hz, 6H); 13C NMR
3
(75 MHz, CDCl ): d = 149.7, 137.6, 127.9, 119.6, 64.3, 64.1,
Recently, fluorescent turn-on probes for monitoring ALP
activity have been developed [17–31]. However, the single-
wavelength response pattern limits the use of these probes.
Unlike turn-on probes, ratiometric probes provide quantitative
detection from calculations of the ratio of the emission intensi-
ties at two different wavelengths, resulting in an internal cali-
bration effect, which improves the sensitivity and accuracy of
detection [32–36]. To date, only a few ratiometric fluorescent
probes for detecting ALP have been reported [37–40]. These
have been constructed by direct attachment of a phosphate
15.7 ppm.
2.2.2. Synthesis of compound EAPN
NIN-Cl was first synthesised by following the method reported
previously [39]. NIN-Cl (1 mmol) was dissolved in dry CH
2 2
Cl
(20 mL). D2 (319 mg, 1.2 mmol) dissolved in CH Cl (5 mL) was
2
2
added dropwise to the reaction mixture at 0 °C, and the mixture
was stirred at room temperature for 5 h. After the solvent was
removed by rotary distillation, the residue was purified by column
chromatography to obtain EAPN as a yellow solid in a yield of 95%.
1
molecule to
a
hydroxy-containing fluorophore, which limits
3
H NMR (300 MHz, CDCl ): d = 8.60 – 8.48 (m, 2H), 8.39 – 8.18 (m,
the choice of fluorophore. In addition, these probes have some
limitations, such as long reaction times, requirement for organic
solvents, and background fluorescence interference, which seri-
ously affect further applications of the probes in living cells
and organisms. We expected to solve these problems by design-
ing a novel ALP ratiometric fluorescent probe.
In this study, we present a ratiometric probe for ALP (APN,
Scheme 1) and its application to quantitative analysis of ALP
activity. This probe comprises 4-amine-1,8-naphthalimide (NIN)
as the fluorophore and phosphate-derived benzyl carbamate as
the ALP hydrolytic site. We anticipated that the phosphate
monoester bond of the probe would be hydrolysed by ALP; this
would be followed by a series of spontaneous chemical degra-
dation steps, and the NIN fluorophore would be released, with
a redshift of the emission wavelength, allowing ratiometric flu-
orescence detection of ALP.
3H), 7.66 (dd, J = 8.5, 7.4 Hz, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.23 – 7.10
(m, 2H), 5.21 (s, 2H), 4.28 – 4.08 (m, 6H), 1.69 (dq, J = 15.1, 7.5 Hz,
2H), 1.47 – 1.29 (m, 8H), 0.95 ppm (t, J = 7.3 Hz, 3H); 13C NMR
3
(75 MHz, CDCl ): d = 163.8, 163.3, 153.0, 150.4, 139.0, 131.9,
130.8, 129.6, 128.5, 126.5, 125.9, 122.9, 119.7, 117.5, 117.0, 66.5,
64.3, 39.8, 29.8, 20.0, 15.7, 13.4 ppm; HRMS calcd for C28
P: [M + H ]: 555.1891; found: 555.1885.
H
31
N
2
O
8
-
+
2.2.3. Synthesis of compound APN
Under an Ar
solved in dry CH
2
atmosphere, EAPN (110 mg, 0.198 mmol) was dis-
Cl (10 mL) and the mixture was cooled to 0 °C in
2
2
an ice bath. TMS-Br (0.27 mL, 1.98 mmol) was added dropwise
with a syringe to the reaction mixture. The ice bath was removed,
and the reaction was stirred at room temperature for 48 h. After
completion of the reaction, the precipitate was collected by filtra-
tion and washed with ice-cold ethyl ether. The crude product was
then recrystallised from CH
get product as a yellow solid in a yield of 26%. H NMR (300 MHz,
-DMSO): d = 10.32 (s, 1H), 8.67 (d, J = 8.6 Hz, 1H), 8.44 (t,
3
OH and ethyl acetate to afford the tar-
1
2
. Materials and methods
D
6
J = 7.8 Hz, 2H), 8.18 (d, J = 8.3 Hz, 1H), 7.78 (dd, J = 8.5, 7.4 Hz,
1H), 7.48 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 5.22 (s, 2H),
2.1. Materials and instruments
4
.07 – 3.94 (m, 2H), 1.69 – 1.45 (m, 2H), 1.33 (dq, J = 14.4,
1
3
The reagents used in the experiments were purchased from
7.3 Hz, 2H), 0.90 ppm (t, J = 7.3 Hz, 3H); C NMR (75 MHz, DMSO):
d = 163.5, 163.0, 154.0, 151.7, 140.8, 131.7 (d, J = 2.8 Hz), 130.9,
129.9, 129.3, 128.4, 126.4, 123.9, 122.3, 120.2, 118.1, 117.1, 66.3,
commercial sources and were of analytical grade. ALP was pur-
chased from Sigma–Aldrich. UV/Vis absorption spectra were
recorded with a PerkinElmer Lambda 650S UV/Vis spectrometer.
Fluorescence spectra were recorded with a PerkinElmer LS55 fluo-
39.4, 29.8, 19.9, 13.8 ppm (s); HRMS calcd for C24
[M + H ] 499.1265; found: 499.1263.
H
23
N
2
O
8
P:
+
1
13
rescence spectrometer. H NMR and
C NMR spectra were
acquired with a Bruker Avance AVII 300 MHz spectrometer.
High-resolution mass spectra were acquired with an Agilent ESI-
Q-TOF mass spectrometer. Live-cell imaging was performed using
an Olympus IX73 inverted fluorescence microscope.
2.3. APN and NIN solution for fluorescence detection
APN and NIN were dissolved in dimethyl sulfoxide (DMSO) as a
sample stock solution (2 mM). Tris-HCl (10 mM, pH 8) buffer solu-
2

X. Huang, X. Chen, S. Chen et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119953
Scheme 1. Structure of APN and mechanism for detecting ALP.
tion was used as the solvent in spectroscopy experiments unless
otherwise stated. In the UV–vis spectrum and fluorescence spec-
3. Results and discussion
trometry experiments, 5
in 2 mL of buffer solution for measurement, and the final measured
concentration was 5 M. Because the enzyme requires a milder
and more stable environment in the organism, in order to better
detect the activity of ALP, all experiments were carried out at 37 °C.
l
L of APN and 5
l
L of NIN were dissolved
3.1. Synthesis and characterization
l
The detailed synthesis steps for APN are illustrated in Scheme 2
and described in the Materials and Methods section. Diethyl
chlorophosphate was treated with p-hydroxybenzaldehyde to
form compound D1, which was subsequently reduced with NaBH
4
to form compound D2. Previously prepared NIN-Cl was connected
to compound D2, and the product was subsequently deprotected to
give the target compound APN. The structure of the compound
APN was confirmed by H NMR, C NMR, and HRMS (see Elec-
tronic Supplementary Material (ESM) Fig. S6–8*).
2
.4. Cell culture
Live MDBK cells (Madin-Darby bovine kidney cells) and HeLa
1
13
cells (cervical cancer cells) were provided by the College of Veteri-
nary Medicine, China Agricultural University (Beijing,China). All
cells were cultured in Dulbecco’s modified Eagle’s medium
(
DMEM) containing 10% fetal bovine serum and 1% penicillin/
3.2. Fluorescence properties of the probe to ALP
streptomycin under ambient conditions of 5% CO at 37 °C.
2
The photophysical properties of the APN probe and the NIN flu-
orophore were first determined. APN and NIN exhibit absorption
maxima (kabs) at 370 nm and 430 nm, respectively, with corre-
2.5. Cytotoxicity assay
sponding emission maxima (k
fl
) at 480 nm (U = 0.19) and
The cytotoxicity of APN in cells was determined using a stan-
dard 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide
MTT) assay. The cells were seeded into a 96-well plate in the pres-
ence of 100 L of DMEM and allowed to adhere for 24 h. The cells
were then incubated with different concentrations of APN (0, 5, 10,
0, 40,80 mM, containing 0.5% DMSO) for 24 h. After the medium
was removed, 100 L of MTT (5 mg/mL) solution was added to
each well, and the cells were further cultured under the same con-
ditions for 4 h. After removing the MTT solution, 100 L of DMSO
5
50 nm ( = 0.28) (see ESM Fig. S1* and Table S1*). The higher flu-
U
orescence quantum yield of the fluorophore allows it to have
higher fluorescence brightness, and the larger Stokes shift effec-
tively prevents self-absorption of fluorescence.
The reaction of the probe with ALP to generate the fluorophore
was confirmed by HPLC, with the results corresponding to the dis-
(
l
2
l
appearance of APN (t
min) (ESM Fig. S2*); this verifies the anticipated mechanism
Scheme 1). The emission spectra of a 5 M solution of APN treated
R R
= 3.2 min) and the presence of NIN (t = 6.5-
l
(
l
was added, and the absorbance of each well was read at 570 nm.
Cell viability was calculated by assuming 100% cell viability for
the control cells without APN treatment.
with ALP in Tris-HCl buffer (10 mM, pH 8.0, 37 °C) show a gradual
increase at 550 nm with a concomitant decrease at 480 nm
(Fig. 1a). As the concentration of ALP increases, the activity of the
enzyme-catalysed reaction increases, so the time required for the
fluorescence emission intensity ratio (F550/F480) to stabilise tends
to be shorter (Fig. 1b). With 400 U L- of ALP in the solution of
APN, the F550/F480 ratio tends to be stable after approximately
12 min, and this response rate is faster than most of the reported
2
.6. Living cell imaging
1
HeLa cells were cultured in DMEM (without FBS) for 30 min
with the addition of APN (5
was first treated with the Na VO
tured for 30 min, and then further cultured for 30 min with APN
M).The residual APN was washed away and the cells were sub-
jected to imaging experiments.
l
M). Another group of HeLa cells
3
4
as a typical inhibitor of ALP, cul-
m
ALP probes. The Michaelis–Menten constant (K ) of the ALP-
catalysed reaction was determined to be 2.78 M; this low K
l
m
(
5
l
value indicates that ALP has a high affinity for the probe (ESM
Fig. S3*).
3

X. Huang, X. Chen, S. Chen et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119953
Scheme 2. Synthetic route to APN.
Fig. 1. (a) Fluorescence spectra changes of a solution of APN varied with time in the presence of 100 U L-1 of ALP. Conditions: Tris-HCl buffer (10 mM, pH 8.0, 0.25% DMSO),
7 °C, kex = 415 nm. (b) F550/F480 ratios of the fluorescence intensity of solutions of APN over time in the presence of different concentrations of ALP.
3
Fig. 2. (a) Fluorescence spectra changes of a solution of APN in the presence of different concentrations of ALP. (b) Plot of the F550/F480 ratios for APN versus the concentration
of ALP. Data were obtained 10 min after ALP addition at 37 °C in Tris-HCl buffer (10 mM, pH 8.0, 0.25% DMSO), kex = 415 nm.
Under consistent conditions (reaction at 37 °C for 10 min in
Tris-HCl buffer (10 mM, pH 8.0, 0.25% DMSO)), enzyme activity
was quantitatively detected from the F550/F480 ratio change of a
solution of APN. A plot of F550/F480 ratios against ALP concentra-
The selectivity and anti-interference of APN for a variety of
common biological enzyme species, biomolecules, and ions was
examined. Only slight fluctuations in the F550/F480 fluorescence
ratio occur upon the addition of cellulase (Cel), lysozyme (Lyso),
b-galactosidase (b-Gal), b-glucuronidase (b-Glu), bovine serum
albumin (BSA), ovalbumin (OVA), human serum albumin (HSA),
cysteine (Cys), homocysteine (Hcy), glutathione (GSH), vitamin C
-1
tions ranging from 0 to 100 U L shows a linear relationship
2
(
L
R = 0.9966), and the detection limit was calculated to be 0.16 U
-
1
(Fig. 2). Furthermore, the probe is insensitive to pH values rang-
ing from 3 to 10 (ESM Fig. S4*). These results demonstrate that the
probe can be used to sensitively detect ALP levels in biological
systems.
(Vc), hydrogen peroxide (H
MgCl , and foetal bovine serum (FBS) (Fig. 3). However, as the
ALP continues to be added to the above system in the presence
2 2 2 2
O ), hydrogen sulfide (H S), CaCl ,
2
4

X. Huang, X. Chen, S. Chen et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119953
3.3. Fluorescence imaging in living cells
Furthermore, the applicability of APN for imaging ALP activity
in live cells was investigated. First, the toxicity of the probe to
MDBK cells was investigated with an MTT assay. The probe shows
low cytotoxicity in vitro (ESM Fig. S5*); this will facilitate the use of
the probe for fluorescence imaging of ALP in living cells. HeLa cells
that can overexpress endogenous ALP[41–42] were used in cell
imaging experiments. When HeLa cells were treated with APN, a
strong signal was observed in the green channel, whereas there
was an extremely weak signal in the blue channel (Fig. 4). For com-
3 4
parison, HeLa cells were pretreated with an inhibitor (Na VO )
before treatment with the probe; in this case, dim fluorescence
was observed in the green channel, whereas very bright fluores-
cence occurred in the blue channel. These results reveal that APN
is applicable to the detection and imaging of overexpression of
endogenous ALP in HeLa cells.
Fig. 3. Selective and anti-interference experiments of the probe for various
biological enzyme species, biomolecules, and ions. Tris-HCl buffer (10 mM, pH
4
. Conclusion
8
.0, 0.25% DMSO) was used as the reaction system. Various analytes were added to
the probe solution and then allowed to react at 37 °C for 10 min. kex = 415 nm. APN
upon addition of different species (red bars), and fluorescence changes of the
mixture of APN and ALP after addition of an excess of the Interference species
In summary, a new ratiometric fluorescence assay for ALP activ-
ity was established using the novel APN probe. The use of a self-
immolative spacer increased the variety of potential fluorophores
and allowed the selection of amine-containing fluorophores. The
probe showed a marked blue-to-green change in the emitted col-
our in response to ALP. Most importantly, the probe showed a
rapid, accuracy, and selective fluorescence sensing effect for ALP
(
green bars).The concentrations of the various analytes were as follows: 1.ALP (200
-1
À1
À1
À1
À1
U L ), 2.Cel (1 U mL ), 3.Lyso (1 U mL ), 4.b-Gal (1 U mL ), 5.b-Glu (1 U mL ), 6.
BSA (100
Hcy (100
(
À1
À1
À1
l
l
g mL ), 7.OVA (100
M), 11.GSH (100
(100 M), 16.MgCl
l
l
g mL ), 8.HSA (100
M), 12.Vc (100
M), 13ÁH
(100 M), 17.FBS (100 lg mL ).
l
g mL ), 9.Cys (100
lM), 10.
l
2
O
2
(100 M), 14.H S
l
2
À1
100
l
M), 15.CaCl
2
l
2
l
-1
in Tris-HCl buffer, with a detection limit as low as 0.16 U L . This
probe possesses excellent biocompatibility and can be used to
visualise endogenous ALP in cervical cancer cells.
of ALP, the fluorescence ratio is significantly increased (Fig. 3); this
is accompanied by a distinct redshift of the fluorescence wave-
length (Fig. 2a), which corresponds to the evolution of the NIN flu-
orophore. These results indicate that APN has excellent selectivity
and interference resistance toward ALP over other competitive
analytes, which meets the need for accurate detection in actual
samples.
Funding information
This work was supported by the National Key Technology
Research and Development Program (No. 2015BAK45B01).
Fig. 4. (a) Fluorescence images of HeLa cells incubated with APN for 30 min. (b) Fluorescence images of HeLa cells pretreated with 1 mM Na
with APN for 30 min.
3 4
VO for 30 min and then treated
5

X. Huang, X. Chen, S. Chen et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119953
[
17] L. Xu, X. He, Y. Huang, et al., A novel near-infrared fluorescent probe for
detecting intracellular alkaline phosphatase and imaging of living cells, J Mater
Chem B. (2019), https://doi.org/10.1039/C8TB03230K, Ahead of Print.
CRediT authorship contribution statement
Xiaoqian Huang: Conceptualization, Methodology, Software,
Writing - original draft, Writing - review & editing, Validation.
Xiangzhu Chen: Conceptualization, Methodology, Software, Writ-
ing - original draft, Writing - review & editing, Validation. Shijun
Chen: Investigation, Formal analysis, Software. Xueyan Zhang:
Investigation, Software. Lin Wang: Software. Shicong Hou: Super-
vision, Project administration, Funding acquisition. Xiaodong Ma:
Supervision.
[18] W. Zhang, H. Yang, N. Li, et al., A sensitive fluorescent probe for alkaline
phosphatase and an activity assay based on the aggregation-induced emission
effect, RSC Adv.
C8RA01786G.
8 (27) (2018) 14995–15000, https://doi.org/10.1039/
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Declaration of Competing Interest
[
22] Y. Tan, L. Zhang, K.H. Man, et al., Reaction-Based Off-On Near-infrared
Fluorescent Probe for Imaging Alkaline Phosphatase Activity in Living Cells
and Mice, Acs Appl Mater Inter. 9 (8) (2017) 6796–6803, https://doi.org/
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
1
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[23] H.W. Liu, X.X. Hu, L. Zhu, et al., In vivo imaging of alkaline phosphatase in
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Supplementary data to this article can be found online at
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