A novel near-infrared fluorescent probe for detecting intracellular alkaline phosphatase and imaging of living cells

Article abstract of DOI:10.1039/C8TB03230K

Development of novel near-infrared (NIR) fluorescent probes for the monitoring of active substances in living organism is desirable in biological studies. Herein, we designed a novel NIR MTR fluorophore which has a longer emission wavelength, greater Stok

Full text of DOI:10.1039/C8TB03230K

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A Novel Near-infrared Fluorescent Probe for Detecting
Intracellular Alkaline Phosphatase and Imaging of Living Cells
a
b
b
a
a
a
c
a
Longbin Xu, Xu He, Yibing Huang, Pinyi Ma,* Yanxiao Jiang, Xin Liu, Shuo Tao, Ying Sun, Daqian
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
Song, and Xinghua Wang,*
DOI: 10.1039/x0xx00000x
Development of novel near-infrared (NIR) fluorescent probes for the monitoring of active substances in living organism is
desirable in biological studies. Herein, we designed a novel NIR MTR fluorophore which has longer emission wavelength,
greater Stokes shift and higher quantum yield than the classic hemicyanine NIR fluorophore. The synthesized MTR-derived
NIR probe (MTR-P) is highly selective and sensitive to alkaline phosphatase (ALP) activity. In the presence of ALP, MTR-P
exhibited increased fluorescence signal by up to 56 folds at 723 nm, and it was determined to be 0.042 U L-1. In addition,
the mechanism of MTR-P probe was further examined by HPLC, mass spectrometry and DFT/TDDFT calculation. The NIR
probe MTR-P was successfully applied to measure the levels of alkaline phosphatase in different types of cells by
fluorescence imaging.
www.rsc.org/
Introduction
Small-molecule fluorescent probes can interact with analytes,
such as cations, anions and human enzymes; accompanied by
the changes of fluorescent spectral properties, they have
become a powerful tool that can be used to detect or examine
Figure 1 Common NIR fluorophore structures.
1,
2
Alkaline phosphatase (ALP), which can be found in mammalian
organs such as intestine, liver, bone, kidney, and placenta, is an
the analytes.
Among these probes, near-infrared (NIR)
fluorescent probe is high-demand ideal candidate for biological
tissue imaging, owing to its various attractive advantages, which
can include minimal damage to biological samples (the spectral
are obtained in NIR region) and minimal interference from
endogenous autofluorescence in living tissues.3-7 Therefore, the
15, 16
enzyme catalyzing the hydrolysis.
This enzyme is widely used as
1
7, 18
a biomarker for enzyme immunoassays and molecular biology.
Abnormal ALP levels in organisms has been found to associate with
the development and progression of various diseases such as
1
9-21
prostate and breast cancers, and bone disease.
However, it
probe not only has been extensively investigated, but also has
been widely employed in many studies, such as in the study of
remains unknown how ALP is involved in such pathological functions;
thus, tracking ALP at the cellular level is crucial for obtaining such
information. Accordingly, it is necessary to develop reliable, facile,
and sensitive ALP analysis method to understand the mechanism of
action of alkaline phosphatase in biological systems and to provide a
basis for clinical diagnosis of related diseases.
8-10
spatial sampling capability by in vivo imaging.
Recently,
numerous research have been devoted to engineer NIR
11-14
fluorophores
(Figure 1), which have a number of limitations,
including short stokes shifts, low fluorescence quantum yields,
8
poor water solubility and bad photostability. Accordingly, the
A variety of spectroscopic methods for detecting ALP levels have
fluorophores currently used for near-infrared light need to be
further studied. Moreover, it can be beneficial to study
fluorophores that show absorption and fluorescence emission
spectra in the NIR region, and that can be detected in vivo.
22
been published, and these methods can include colorimetric,
23
24
25
fluorometric, chromatographic, and electrochemical methods,
26
as well as surface-enhanced resonance Raman scattering.
Nonetheless, these methods, except the fluorescence technique,
usually require sample pretreatment and complex instrumentations,
in addition to other limitations, such as low sensitivity of
spectrophotometric methods and low repeatability of
a.College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012,
China.
b.
College of Life Sciences, Jilin University, Qianjin Street 2699, Changchun 130012,
15, 27, 28
electrochemical methods.
The use of activatable fluorescence
China.
c.
Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell
probes has a number of advantages, including high sensitivity, cost
effectiveness, simplicity, and convenientness.
Technology, School of Chemistry and Chemical Engineering, Liaocheng University,
Liaocheng 252059, China.
1
9,29-31
Therefore,
†
*
Tel: +86 431 85168399; Fax: +86 431 85168399.
E-mails: mapinyi@jlu.edu.cn (Pinyi Ma); starred.wang@gmail.com (Xinghua
small-molecule near-infrared (NIR) fluorescent probe can be
advantageously used as analytical means for the detection of alkaline
phosphatase in vivo.
Wang).
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Herein, we designed a novel NIR fluorophore MTR which has Table 1. However, both MTR and CyR have the sameVierwelAarttiicvlee Olinglihnet
longer emission wavelength, greater Stokes shift and higher stability (Figure S1). Therefore, this indDicOatI:e1s0.1t0h3a9t/Ct8hTeB03n2o3v0eKl
quantum yield than the classic hemicyanine NIR fluorophore. The NIR fluorophore MTR can have greater application potential than the
fluorophore was synthesized by replacing 2,3,3-trimethyl-3H-indole hemicyanine NIR fluorophore CyR.
in the classical hemicyanine dye with 2-methylbenzo[d]thiazole. For Table 1 Common NIR fluorophore structures.
the first time, we developed a novel water-soluble ALP-sensitive
fluorescent probe MTR-P which is used MTR as the platforms and
modified a phosphate on the hydroxyl group. The MTR-P probe is
nonfluorescent due to its phosphate group, which has both
annihilation and recognition capabilities. Upon the addition of ALP,
the emission of MTR-P at 723 nm was enhanced. Additionally, the
probe exhibits good ALP-sensing performance with a linear range of
Dye
MTR
CyR
λ
Ex
λ
Em
Stokes shift
~43 nm
Φ
680 nm
680 nm
723 nm
712 nm
0.15
0.13
~32 nm
To better understand the luminescence behavior of probe CyR
and MTR, we carried out computational studies. As shown in Figure
, the HOMO and LUMO for both CyR and MTR probes are mainly
distributed on the π-conjugated molecular framework, suggesting
that the probe itself exhibits fluorescence properties. The LUMO →
HOMO transition (S
strength (f) of 1.1945 and 1.3904, and emission wavelengths of
10.05 nm and 722.67 nm, respectively, for CyR and MTR. This
observation can be attributed to S atom in thiophene group, which
has stronger electron-donating ability. The predicted emission
spectra, obtained by TDDFT calculations, are also in good agreement
with the predicted spectra. This further confirms that the MTR has
better fluorescence character (longer emission wavelength and
higher quantum yield) than its structural analogue CyR.
-1
2
-
0
;
-8 U L (R = 0.9971) and the detection limit of as low as 0.042 U L
it also has excellent selectivity towards ALP. Moreover, MTR-P was
3
1
successfully used for detecting the exogenous and endogenous
alkaline phosphatase level in living cells.
1 0
→ S ) in both probes are allowed with oscillator
7
Results and discussion
Design and synthesis of fluorophore MTR
Recently, the use of classic hemicyanine fluorophore to construct NIR
fluorescent probes for in living cell imaging have attracted great
3
2
attention. However, these probes have disadvantages such as short
Stokes shift, low fluorescence quantum yield and interference from
scattered light. Hence, we synthesized a novel NIR fluorophore by
replacing 2,3,3-trimethyl-3H-indole in the classical hemicyanine dye
with 2-methylbenzo[d]thiazole.
The synthesized NIR fluorophore MTR (3-ethyl-2-(2-(6-hydroxy-
2
,3-dihydro-1H-xanthen-4-yl)vinyl)benzo[d]thiazol-3-ium) was then
compared with the classic hemicyanine NIR fluorophore CyR (1-ethyl-
2
3
-(2-(6-hydroxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-
H-indol-1-ium). The photophysical properties were investigated
2 3
H O/CH OH (99:1, v/v, 25 mM Tris-HCl buffer, pH=8.0), and the
fluorescence spectra were recorded at an excitation (Ex=680 nm) and
emission (Em) wavelengths between 690 nm and 850 nm (Ex/Em slit
=
7
5/5 nm). As shown in Figure 2, the emission wavelength of CyR was
12 nm, whereas that of MTR was 723 nm. With the same excitation
wavelength, a red shift to some degree of the emission peak was
observed. Additionally, MTR (~43 nm) also have larger Stokes shift
than CyR (~32 nm). Moreover, absolute quantum yield in CH
MTR (0.15) is higher than that of CyR (0.13), as demonstrated in
3
OH of
Figure 3 Optimized structures, HOMO−LUMO energy levels, wavelength, light
harvesting efficiency (%), oscillator strength (f), and molecular orbital
obtained from the emission spectra of CyR and MTR calculated by TDDFT
using the optimal structures at the first excited states.
Spectroscopic Properties of MTR-P to ALP
Because pH is a significant factor affecting enzyme reaction, the pH-
dependent response of the probe was determined in Tris-HCl buffer
(
(
Figure S2). The MTR-P probe (10 μM) exhibits good response to ALP
10 U L ) at the pH range of 2.0 to 10.0. Considering both the enzyme
-1
activity and the bioimaging, the optimum pH of 8.0 was selected for
subsequent experiments. Time, which is another factor affecting the
MTR-P probe, was also examined. After the probe was treated with
-1
ALP (10 U L ), its fluorescence emission wavelength at 723 nm was
measured within 0 to 20 min, and we observed that stable readings
of intensity could be acquired within 15 min (Figure S3). Once a
stable value was reached, the fluorescence intensity became
Figure 2 Fluorescence spectra of MTR (10 μM) and CyR (10 μM) in H
CH OH (99:1, v/v, 25 mM Tris-HCl buffer, pH = 8.0).
2
O/
3
2
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relatively stable. Therefore, the determination of fluorescence
intensity was carried out within 15 min after ALP was added.
The NIR MTR-P probe not only can detect the ALP activity, but
also can potentially be used for the screening of enzymes inhibitors.
View Article Online
DOI: 10.1039/C8TB03230K
Thus, the effect of various concentrations of Na
3 4
VO , a well-known
inhibitor of ALP, on the probe was investigated. The MTR-P probe (10
-1
μM) was added to ALP solution (10 U L ) previously incubated with
different concentrations of Na VO (0-300 μM) for 10 min. The
emission at 723 nm gradually decreases with increasing
concentration of Na VO (Figure S5). The calculated IC50 for Na VO
is 23.98 μM, which is in good agreement with the previously reported
3
4
3
4
3
4
33
value. Therefore, MTR-P can potentially be used to screen ALP
inhibitors.
Selectivity of MTR-P Probe
The selectivity of MTR-P probe to ALP was researched to
evaluate the use of MTR-P for selective binding to ALP. Fluorescent
responses of the probe to a wide range of enzymes and anions
2
+
2+
(
including ALP, ACP, Met, His, Arg, PHE, Ser, Hcy, GGT, UA, Fe , Co ,
-
-
-
-
2-
-
Figure 4 Fluorescence spectra of MTR-P (10 μM) in 25 mM Tris-HCl buffer, pH AchE, Tyr, GOD, GSH, Cys, BSA, AA, IgG, F , Cl , Br , I , SO
3
, and CN )
-
1
=
8.0, upon the addition of ALP (0, 1, 2, 3, 4, 5, 6, 7, and 8 U L ). Inset: a plot
were measured. The analytes were incubated with MTR-P for 15 min
and their fluorescent intensity changes (due to MTR-P) were
measured. Figure 5 shows the fluorescent spectra and the
corresponding bar plot. The fluorescent signal is remarkably
of fluorescence intensity versus ALP concentration.
Furthermore, the concentration-dependent response of the
probe upon stepwise addition of ALP was examined. The intensity of
fluorescence emission increases progressively by about 56-folds
after the concentration of ALP was increased up to 10 U L-1 (the
concentration of MTR-P was kept constant at 10 μM; Figure 4). The
inset of Figure 4 (Figure S4) shows that the fluorescence emission is
-1
enhanced when ALP (10 U L ) is present, and it appears that
interferences have no effect on the fluorescent signal of the probe.
Among all analytes, ADP only causes a slight enhance of
fluorescence; the change is at a much lesser extent. These results
further signify that the MTR-P probe is highly selective to ALP, and
thus has a promising potential for detecting ALP in living cells without
any interferences. Then, the competition (interference) experiments
were further conducted using the above analytes and ALP. The data
showed that the interferences by these analytes have little or no
effects on the detection of ALP, thus undoubtedly indicating that
MTR-P is highly selective to ALP.
-1
linearly proportional to the ALP concentration from 0 to 8 U L , and
-1
the detection limit was 0.042 U L (3σ/k). In addition, the regression
equation was determined as y=153.96x+63.262 with R=0.9971 and
σ=2.16. The experimental data further proves that the NIR MTR-P
probe can potentially detect ALP under the simulated physiological
conditions. The probe MTR-P exhibited preeminent sensitivity
compared to the previously reported fluorescent probes (Table S1).
-
1
-1
Figure 5 (a) Fluorescence response of MTR-P (10 μM) upon the addition of ALP (10 U L ), ACP (10 U L ), and other interferences including: 100 μM each of
2
+
2+
-
-
-
-
2-
, and CN ; 100 U L each of GGT, AchE, Tyr, and GOD; and 100 mg L-1 each of BSA
-
-1
Met, His, Arg, PHE, Ser, Hcy, UA, Fe , Co , GSH, Cys, AA, F , Cl , Br , I , SO
3
-
1
and IgG. The probe was dissolved in Tris-HCl buffer (25 mM, pH = 8.0). Inset: Fluorescence spectra of MTR-P (10 μM) in the presence of ALP (10 U L ) and other
-
1
interferences. (b) Fluorescence response of MTR-P (10 μM) upon the addition of interferences including: ACP (10 U L ); 100 μM each of Met, His, Arg, PHE,
2+
2+
-
-
-
-
2-
-
-1
-1
Ser, Hcy, UA, Fe , Co , GSH, Cys, AA, F , Cl , Br , I , SO3 , and CN ; 100 U L each of GGT, AchE, Tyr, and GOD; and 100 mg L each of BSA and IgG, in Tris-HCl
-
1
buffer (25 mM, pH = 8.0) and the presence of ALP (10 U L ).
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-
1
Figure 6 (a) Absorption and (b) fluorescence spectra of MTR (10 μM), MTR-P (10 μM) in the presence and absence of ALP (20 U L ), and MTR (10 μM) in the
-
1
presence of ALP (20 U L ) and its inhibitor Na
3
VO (100 μM). The samples were in Tris-HCl buffer (25 mM, pH = 8.0).
4
Figure 7 Theoretical molding of MTR and MTR-P calculated by TDDFT using the optimal structures of the ground and the first excited states. The structures are
illustrated with their corresponding vertical excitation energy, oscillator strength (f), recognition process, wavelength, and relevant frontier molecular orbits.
-1
Sensing Mechanism of MTR-P Toward ALP
3 4
containing ALP (20 U L ) and its inhibitor Na VO (100 μM) is similar
The photophysical properties of MTR-P were investigated in Tris-HCl to that of MTR-P. Free MTR-P probe in aqueous solution exhibited
-1
buffer solution (25 mM, pH = 8.0) with or without ALP (20 U L ). In very weak fluorescence over the emission wavelength range of 690
-1
Figure 6a, MTR-P (20 μM) in Tris-HCl buffer has a maximum to 850 nm (Figure 6b). However, with the addition of ALP (20 U L ),
-1
absorption at 588 nm. In the presence of ALP (20 U L ), the the emission spectra were enhanced to center at 723 nm after 15
absorption band MTR-P was significantly decreased to center at 588 min; this observation is the same as that observed for MTR. These
-1
nm, while the absorption band was increased to center at 658 nm. data demonstrate that the addition of ALP (20 U L ) and its inhibitor
Similarly to MTR, the color of the mixture was changed from purplish Na
3
VO
4
(100 μM) into MTR-P (20 μM) causes only slight changes in
to cyan after 15 min. The absorption spectrum of MTR-P (20 μM)
4
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fluorescence spectra, indicating that ALP is able to effectively remove the cells were cultured with 20 μM MTR-P for 24 h, cell viability
View Article Online
DOI: 10.1039/C8TB03230K
phosphate groups from MTR-P, converting it to MTR.
remained high, indicating that the probe exhibits low cytotoxicity to
the cells. Encouraged by its low cytotoxicity, MTR-P was further used
for detecting the levels of exogenous and endogenous alkaline
phosphatase in HEK 293T and BEL 7402 cells.
To further investigate the sensing mechanism of the probe,
-1
MTR-P with ALP (20 U L ) in Tris-HCl buffer was subjected to MS
analysis. The peak at m/z = 468.4279 corresponding to MTR-P was
decreased, while that at m/z = 388.4432 corresponding to MTR was
appeared (Figure S6). In addition, the HPLC analysis showed a peak
associated with MTR-P at 5.47 min, and a peak corresponding to MTR
at 7.19 min (Figure S7). After ALP was added into MTR-P, the peak at
5
7
.47 min was disappeared, and a new peak corresponding to MTR at
.33 min was appeared. These results demonstrate that the ability to
remove phosphate groups of alkaline phosphatase can transform the
MTR-P into fluorophore MTR and simultaneously causing changes in
ultraviolet and fluorescent signals (Scheme 1).
Figure 8 Fluorescence images of HEK 293T cells. From left to right: control;
-
1
cells treated with MTR-P (10 μM); and cells treated with ALP (10 U L ),
followed by MTR-P (10 μM).
Scheme 1 Proposed reaction mechanism of the fluorometric assay for ALP
activity with MTR-P.
To gain insights into the enhanced fluorescence of MTR-P in the
HEK 293T cells were used as an exogenous model because they
presence of alkaline phosphatase, the electronic and optical express low amount of ALP. As shown in Figure 8a, with the
properties, and structures of MTR-P and MTR were calculated using excitation at 635 nm, HEK 293T did not exhibit background
DFT and TDDFT methods (Figure 7). MTR has a C(1)–O(2) bond length fluorescence signal. Following the cells were incubated with 10 μM
of 127.1 pm, which is similar to the bond length of C=O double bond MTR-P for 15 min and washed thoroughly (to remove excess MTR-P)
(
~121 pm); this indicates that there is electron drawing from O(2) prior to cell imaging, the fluorescence signal inside HEK 293T cells
atom to π-conjugated system. ICT can be achieved from remains low (Figure 8b). By contrast, significant red fluorescence
benzothiazole (donor) in MTR to xanthine (receptor); as a result, a signal was observed from the cells treated with ALP (10 U L-1) for 15
push-pull electronic conjugate system was constructed. The TDDFT min, followed by MTR-P (10 μM) for 15 min (Figure 8c). The
calculation results further show that the main absorption peak of fluorescence intensities from the corresponding HEK 293T cells were
MTR is at 677.51 with f = 1.3932 (oscillator strengths), and the main shown in Figure S9. This observation indicates that the MTR-P probe
fluorescence emission peak is at 722.67 with f = 1.3904. This can be used to monitor the levels of enzymatic activity of exogenous
indicates that MTR can emit strong fluorescence signal. On the other ALP in living cells.
hand, the bond length between C(1)–O(2) in MTR-P is 139.7 p.m,
Encouraged by these results, we further used MTR-P to detect
which matches the bond length of C–O single bond (~144 p.m.). This the levels of endogenous ALP in living cells. Human hepatoma BEL
may be due to the weakening of p–π conjugation between O(2) atom 7402 cell line was chosen as a representative cell line because it has
and π-conjugated xanthylium framework. Compared to MTR, overexpression of ALP which could cause higher risk of liver disease
although contribution to the HOMO/LUMO of O(2) atom in MTR-P is 34. With the excitation at 635 nm, untreated BEL 7402 cells did not
less than that in MTR, its HOMO and LUMO energy levels is higher. exhibit background fluorescence signal (Figure 9a), whereas the cells
The results from theoretical calculations indicated that the O(2) atom treated with MTR-P (10 μM) for 15 min exhibited strong fluorescence
in MTR-P had lower electron donating ability due to phosphate, and signal (Figure 9b). To further verify whether or not the fluorescence
it can be hinder the ICT mechanism in the MTR. The TDDFT signal can be affected by ALP, and its inhibitor Na VO (50 μM), the
3
4
calculation of MTR-P showed that the main absorption and cells were treated with ALP and Na VO for 15 min prior to incubating
3
4
fluorescence emission peaks are 609.75 nm (f = 1.5589) and 677.01 with MTR-P (10 μM) for additional 15 min; and the results showed
nm (f = 1.4780), respectively. Because the emission peak is blue that the fluorescence signal was not distinctly observed (Figure 9c).
shifted to overlap with the Rayleigh scattering peak, the fluorescence On the other hand, BEL 7402 cells treated with Na VO (50 μM) for
3
4
peak for MTR-P was not observed. Altogether, the theoretical 15 min, followed by ALP (10 U L-1) for 15 min, MTR-P (10 μM) for 15
calculations are consistent with the experimental data.
min exhibited strong fluorescence signal (Figure 9d). And the
These experimental results confirm that the destruction of fluorescence intensities from the corresponding HEK 293T cells were
phosphorus and oxygen bonds in MTR-P can lead to significant shown in Figure S10. These observations suggest that the MTR-P can
increased fluorescent signals in the near-infrared region.
Fluorescence Imaging of ALP in Living Cells
efficiently and effectively detect the endogenous alkaline
phosphatase levels in living cells.
Prior to fluorescence imaging, the cytotoxicity of MTR-P to BEL
7
402 cells was examined by MTT assays. As shown in Figure S8, after
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Taken together, these results indicate that the probe MTR-P can 0.92 (5 H, t). 31P NMR (243 MHz, DMSO-d6) δ = -5.34. MS (ESI, m/z)
View Article Online
+
DOI: 10.1039/C8TB03230K
effectively and efficiently detect exogenous and endogenous ALP calcd for [C24
5
H23NO PS] : 468.10, found: 468.44.
activity in living cells.
Fluorescence Measurements
The measurement of FL and UV-vis spectra were carried out as
follows. A stock solution of MTR-P (20 μM) was prepared in Tris-HCl
buffer (25 mM, pH = 8.0, 1 mM MgCl
concentrations of ALP was carried out by dissolving ALP in Tris-HCl
buffer (25 mM, pH = 8.0, 1 mM MgCl ). Different concentrations of
2
). Preparation of various
2
amino acid, cations and other interfering substance were also
prepared using the method used for preparing ALP stock solution.
Then, the test solution was obtained by mixing 0.5 mL stock solution
of MTR-P and 0.5 mL stock solution of ALP or interfering substances.
After that, the resultant solution was shaken at 25 C for 15 min, and
its spectral properties were then measured. The fluorescence
spectra were measured with the excitation at 680 nm and emission
Figure 9 Fluorescence images of BEL 7402 cells. From left to right: control;
cells treated with MTR-P (10 μM); cells treated with Na VO
by MTR-P (10 μM); and cells treated with Na VO (50 μM), followed by ALP Theoretical Calculation and Analysis
The optimal geometrical and electronic structures of the probes
were determined by the DFT (density functional theory) and
(50 μM), followed wavelength ranges of 690 to 850 nm.
3
4
3
4
-
1
(
10 U L ) and MTR-P (10 μM).
35
B3LYP/6-31G(d) basis sets using Gaussian 16 software. The UV-vis
and FL spectra for each calculation were recorded at the time-
Experimental
Synthesis
dependent DFT (TDDFT) by optimizing S (the energy level of ground
0
Scheme 2 depicts the synthetic route to compounds, which can be state) and S (the energy level of the first excited state) structures.
1
1
13
found in the supplementary material. ( H NMR, C NMR, and MS The effect of solvent was calculated by SMD model, and water was
spectra can be found in Figures S11-S28).
used as the solvent in the calculation.
Cell Incubation and Imaging
HEK 293T and BEL 7402 cells, which was also obtained from the Life
Sciences College of Jilin University (Jilin, China), were grown in
DMEM (Dulbecco’s modified Eagle medium) supplemented with 10
%
heat-inactivated fetal bovine serum (FBS) at 37 C under 5% CO
atmosphere. After washing several times with PBS buffer (pH = 7.2-
.4), the cells were incubated with 10 μM MTR-P probe dissolved in
PBS for 15 min. As control groups, BEL 7402 cells were pretreated
2
7
with Na
3
VO
4
-
(50 μM), whereas HEK 293T cells were pretreated with
1
ALP (10 U L ). After the pretreatment for 20 min, the cells were
incubated with MTR-P (10 μM) for additional 15 min. As an additional
3 4
control group, BEL 7402 cells were also treated with Na VO (50 μM)
-1
for 20 min, followed by ALP (10 U L ) for additional 15 min. After
that, the cells were incubated with the MTR-P (10 μM) for additional
1
5 min. The cells were observed under a confocal fluorescence
microscope at an excitation wavelength of 635 nm and imaged by a
confocal fluorescence microscope with the emission wavelengths
between 680 and 750 nm.
Scheme 2. Synthetic route of the probe.
Conclusions
Synthesis of MTR-P
We successfully synthesized hemicyanine dye-mimic NIR MTR by
replacing 2,3,3-trimethyl-3H-indole with 2-methylbenzo[d]thiazole.
The synthesized NIR MTR fluorophore has longer emission
wavelength, larger stokes shifts and stronger fluorescence quantum
yields compared with hemicyanine fluorophore CyR. The MTR was
further designed (denoted as MTR-P) and applied to detect the
activity of ALP. The MTR-P probe exhibits good ALP-sensing
To a reactant of MTR (0.1 g, 0.25 mmol) in anhydrous pyridine (3 mL)
was cooled down to 0 C. After that, phosphorus oxychloride (0.1 mL)
was slowly added to the reactants under vigorous stirring. The
solution was stirred at room temperature for 6 h; after that, it was
poured into ice and then stirred for another 12 h. Subsequently, the
solution was evaporated to remove the solvent, and then purified by
silica gel column chromatography (CH
dark purple solid was obtained. Yield: 0.052 g (43.2 %). H (500 MHz,
2
Cl
2
:CH
3
OH=5:1 v/v); and a
-1
2
1
performance with the linear range of 0-8 U L (R = 0.9971) and the
-1
detection limit is 0.042 U L , which is lower than most of other ALP
probes previously reported. The luminescence and detection
3
CD OD): δ 8.53 (1H, d), 8.03 (1H, m), 7.78 (2H, d), 7.41 (1H, s), 7.33
(
1H, d), 7.10 (2H, d), 6.82 (1H, d), 5.43 – 5.29 (2H, m), 2.72 (4 H, d),
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 8
J Po lue ran sael od fo Mn aot te rai ad l jsu Cs ht emm ai rs gt ri ny sB
Journal Name
ARTICLE
2
2
2
2
0. J. A. Lorente, H. Valenzuela, J. Morote and A. GelabVeierwt,AErtuicrleoOpnelainne
mechanisms of MTR-P probe upon the addition of ALP were also
investigated by UV-vis and fluorescent spectroscopy, mass
spectrometry, theoretical calculation, and HPLC. Furthermore, the
MTR-P was successfully applied to probe the exogenous and
endogenous alkaline phosphatase activity in living cells. These
findings demonstrate the established probe has a promising
application potential in simplifying clinical diagnosis of both liver and
bone diseases.
Journal of Nuclear Medicine, 1999, 26, 625-632.
DOI: 10.1039/C8TB03230K
1. Fernandez, Nicole J. and B. A. Kidney, Veterinary Clinical
Pathology, 2007, 36, 223-233.
2. Y. Choi, N.-H. Ho and C.-H. Tung, Angewandte Chemie, 2007, 119,
721-723.
3. Y. Tan, L. Zhang, K. H. Man, R. Peltier, G. Chen, H. Zhang, L. Zhou,
F. Wang, D. Ho, S. Q. Yao, Y. Hu and H. Sun, ACS Applied Materials
&
Interfaces, 2017, 9, 6796-6803.
24. H. Takuya, S. Masaru, T. Kohei, M. Hirotaka, U. Tomonari and H.
Hiroki, Bulletin of the Chemical Society of Japan, 2006, 79, 1211-
1214.
Conflicts of interest
There are no conflicts to declare.
25. T. Murata, T. Yasukawa, H. Shiku and T. Matsue, Biosensors and
Bioelectronics, 2009, 25, 913-919.
2
2
2
6. C. Ruan, W. Wang and B. Gu, Analytical Chemistry, 2006, 78,
3379-3384.
7. H. Zhang, C. Xu, J. Liu, X. Li, L. Guo and X. Li, Chemical
Communications, 2015, 51, 7031-7034.
8. H. Zhang, R. Liu, J. Liu, L. Li, P. Wang, S. Q. Yao, Z. Xu and H. Sun,
Chemical Science, 2016, 7, 256-260.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (Nos. 21405057 and 21207047), Science and Technology 29. Y. Liu and K. S. Schanze, Analytical Chemistry, 2008, 80, 8605-
8
612.
0. T.-I. Kim, H. Kim, Y. Choi and Y. Kim, Chemical Communications,
011, 47, 9825-9827.
1. S.-J. Li, C.-Y. Li, Y.-F. Li, J. Fei, P. Wu, B. Yang, J. Ou-Yang and S.-X.
Nie, Analytical Chemistry, 2017, 89, 6854-6860.
2. S.-Y. Liu, H. Xiong, J.-Q. Yang, S.-H. Yang, Y. Li, W.-C. Yang and G.-
F. Yang, ACS Sensors, 2018, 3, 2118-2128.
Developing Foundation of Jilin Province of China (Nos.
0180201050YY and 201701011106JC), Industrial Innovation Funds
3
3
3
2
2
of Jilin Province of China (No. 2018C034-1), Project of the Education
Department of Jilin Province of China (No. 2016411).
Notes and references
33. J. Zhao, S. Wang, S. Lu, X. Bao, J. Sun and X. Yang, Analytical
Chemistry, 2018, 90, 7754-7760.
1
. X. Li, X. Gao, W. Shi and H. Ma, Chemical Reviews, 2014, 114, 590-
59.
. X. Chen, M. Sun and H. Ma, Current Organic Chemistry, 2006, 10,
77-489.
3
4. F. Zhang, X. He, P. Ma, Y. Sun, X. Wang and D. Song, Talanta,
018, 189, 411-417.
3
6
2
2
5. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb
and J. R. Cheeseman, Gaussian 16, Revision A. 03, Gaussian Inc.,
Wallingford CT, 2016.
4
3
. C.-H. Quek and K. W. Leong, Nanomaterials, 2012, 2, 92.
. C. S. Park, T. H. Ha, M. Kim, N. Raja, H.-s. Yun, M. J. Sung, O. S.
Kwon, H. Yoon and C.-S. Lee, Biosensors and Bioelectronics, 2018,
4
105, 151-158.
5
6
7
8
9
1
1
1
. F. Yu, P. Li, G. Li, G. Zhao, T. Chu and K. Han, Journal of the
American Chemical Society, 2011, 133, 11030-11033.
. F. Yu, P. Li, B. Wang and K. Han, Journal of the American Chemical
Society, 2013, 135, 7674-7680.
. Z. Lou, P. Li and K. Han, Accounts of Chemical Research, 2015, 48,
1358-1368.
. H. Chen, B. Dong, Y. Tang and W. Lin, Accounts of Chemical
Research, 2017, 50, 1410-1422.
. X. Han, F. Yu, X. Song and L. Chen, Chemical Science, 2016, 7,
5098-5107.
0. Y. Huang, F. Yu, J. Wang and L. Chen, Analytical Chemistry, 2016,
8, 4122-4129.
1. J. Deng, P. Yu, Y. Wang and L. Mao, Analytical Chemistry, 2015,
7, 3080-3086.
8
8
2. J. Wang, B. Li, W. Zhao, X. Zhang, X. Luo, M. E. Corkins, S. L. Cole,
C. Wang, Y. Xiao, X. Bi, Y. Pang, C. A. McElroy, A. J. Bird and Y.
Dong, ACS Sensors, 2016, 1, 882-887.
1
1
1
1
1
3. H. Chen, Y. Tang, M. Ren and W. Lin, Chemical Science, 2016, 7,
1896-1903.
4. P. Xu, T. Gao, M. Liu, H. Zhang and W. Zeng, Analyst, 2015, 140,
814-1816.
5. L. Zhao, S. Xie, X. Song, J. Wei, Z. Zhang and X. Li, Biosensors and
Bioelectronics, 2017, 91, 217-224.
6. X. Hou, Q. Yu, F. Zeng, J. Ye and S. Wu, Journal of Materials
Chemistry B, 2015, 3, 1042-1048.
7. F. Zheng, S. Guo, F. Zeng, J. Li and S. Wu, Analytical Chemistry,
1
2
014, 86, 9873-9879.
1
8. J. E. Coleman, Annu. Rev. Biophys. Biomol. Struct, 1992, 441-483.
9. T. Xiao, J. Sun, J. Zhao, S. Wang, G. Liu and X. Yang, ACS Applied
Materials & Interfaces, 2018, 10, 6560-6569.
1
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DOI: 10.1039/C8TB03230K
Table of contents entry
Design, Synthesis and application of fluorescent probe with novel near-Infrared fluorophore
for in vivo imaging of alkaline phosphatase.