A Novel Turn-on Fluorescent Probe for Highly Selective Detection of Phosphate Ion in Living Cell

Article abstract of DOI:10.1002/cjoc.201800376

A novel fluorescent probe was synthesized and studied. Fluorescent probe 1 undergoes a unique Pi induced hydrolytic reaction in DMSO- HEPES at physiological pH that produces a colorimetric change associated with a 76 nm red-shift in the UV-vis absorption maximum and up to a 210-fold enhancement in the fluorescence intensity. It has successfully been used to stain and record microscopy images of HeLa cells. Moreover, the mechanistic proposal that these spectroscopic changes are associated with the reaction of Pi and probe 1 to form 2-(2-hydroxyphenyl)benzothiazole gains support from the results of mass spectrometry studied.

Full text of DOI:10.1002/cjoc.201800376

A Novel Turn-on Fluorescent Probe for Highly Selective Detection of
Phosphate Ion in Living Cell
Jin Zhang *,a,b, Si Wangb, Changhui Liua, Guowen Hea, Tianying Penga
ABSTRACT A novel fluorescent probe was synthesized and studied. Fluorescent probe 1 undergoes a unique Pi induced hydrolytic reaction in
DMSO-HEPES at physiological pH that produces a colorimetric change associated with a 76 nm red-shift in the UV-vis absorption maximum and up to a
210-fold enhancement in the fluorescence intensity. It has successfully been used to stain and record microscopy images of HeLa cells. Moreover, the
mechanistic proposal that these spectroscopic changes are associated with the reaction of Pi and probe 1 to form 2-(2-Hydroxyphenyl)benzothiazole gains
support from the results of mass spectrometry studied.
KEYWORDS Fluorescence probe, 2-(2-hydroxyphenyl)benzothiazole, Cell imaging, Pi detection
Introduction
Results and Discussion
Over the past few decades, anions play crucial roles in a wide
To evaluate the selectivity of probe 1 for Pi over various anions,
range of chemical, biological, medical and environmental
-
-
the responses of probe 1 to NO3 , NO2 , F-, Cl-, Br-, I-, CH3COO-, Pi,
processes1-3, and numerous efforts have been made towards the
development of abiotic receptor, which are capable of selectively
recognizing and sensing these anionic species4-8. Among anions,
phosphates are not only essential for the building of the most
fundamental molecules in living organisms, but important as
components of several medicinal drugs9. Hence, the selective
detection of the different kinds of phosphates, such as inorganic
phosphate, pyrophosphate (PPi) and adenosine triphosphate (ATP)
has been a major research target. Therefore, fluorescent probe
has aroused general attention because it could offer high
selectivity and sensitivity with minimal invasiveness to the test
-
4-
SO42-, ATP, GSH, ClO4 , S2O32-, P2O7 were primarily investigated
by UV-vis spectroscopy and the fluorescence spectroscopy in
DMSO/HEPES buffered (0.02 mol/L, pH = 7.4)(V/V=9/1). As shown
in Figure 1, upon addition of 100 equiv Pi to the DMSO/HEPES
(V/V = 9:1, pH = 7.4) buffer solution, it responded with a dramatic
color change, the apparent color change from diaphanous to light
blue could be distinguished by naked eye associated with a shift
of the maximum of the major absorption band at 330 to 409 nm,
and the emission band at 465 nm undergoes a 210-fold increase
in its intensity. Interaction of probe 1 with 100 equivalents of
other anions as its sodium salt did not result in any changes in the
absorption spectrum and the fluorescent emission profile, except
for CH3COO- which caused a slight enhancement of absorbance at
409 nm and fluorescence at 465 nm. These results showed that
probe 1 can efficiently and specifically identify phosphate ions.
specimens10-12
.
PPi and ATP, both generate two molecules of inorganic phosphate
by hydrolysis under cellular conditions13-15. Although there are
many chemosensors for selectively recognition of PPi16-19 and
ATP20-23, it is still difficult to predict whether a chemosensors can
only recognize Pi because of the selectivity of Pi is hindered by the
presence of structurally similar anions in cells. Additionally, HBT is
a fluorophore with significant photostability and reasonable
fluorescent quantum yield. Moreover, HBT does have excited
state intramolecular proton transfer process, which produces a
large Stokes shift24-26. Herein, we reported a new fluorescent
probe 1, based on the 2-(2-Hydroxyphenyl)benzothiazole
fluorophore, which is not promoted by PPi, ATP, but could
triumphantly detect Pi as Pi targeted colorimetric and fluorescent
probe 1. Upon the characteristic of this, we found that the probe
1 may display almost no fluorescence, while a significant increase
of the intensity of fluorescence was observed after the addition of
Pi. The results of mass spectrometry showed that probe 1 use
ester bond as the interaction site and HBT as the fluorescent
chromophore.
Figure 1 (a)Absorption spectra of probe 1 (2.0×10-5 mol/L) in DMSO-HEPES
buffer (0.02 mol/L, pH 7.4)(V/V=9:1) with 100 equiv of NO3 , NO2 , F-, Cl-,
-
-
-
-
-
-
Br-, I-, CH3COO-, Pi, SO42 , ATP, GSH, ClO4 , S2O32 , P2O74 , (b) Absorbances of
prboe 1 (2.0×10-5 mol/L) at 330 nm after addition of 100 equiv of selected
anions. (c) Fluorescence emission spectra of probe 1 (2.0 ×10-5 mol/L) in
DMSO-HEPES buffer (0.02 mol/L, pH 7.4) (V/V = 9:1) with 100 equiv of
-
-
-
-
-
-
NO3 , NO2 , F-, Cl-, Br-, I-, CH3COO-, Pi, SO42 , ATP, GSH, ClO4 , S2O32 , P2O74 , (d)
a School of Materials and Chemical Engineering，Hunan City university, Yiyang,
Hunan 413000, People’s Republic of China
Email: tccdzdc@163.com
b Key Laboratory of Environmentally Friendly Chemistry and Applications of
Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan,
Hunan 411105, People’s Republic of China
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Fluorescence intensities of probe 1 (2.0 × 10-5 mol/L) at 465 nm after
addition of 100 equiv of selected ions.
Subsequently, to obtain a fluorescence profile for Pi, upon the
addition of different equivalents of Pi to DMSO-HEPES buffered
solution of probe 1 and recorded by using fluorescence spectra
and UV-vis spectra. As shown in Figure 2, we choose 415 nm as
the optimum excitation wavelength, probe 1 exhibited very weak
fluorescence, but with the addition of Pi that caused a significant
increase of the intensity of fluorescence at 465 nm. 210-fold
fluorescence enhancement was observed, associated with an
observable bright blue emission when the probe 1 was treated
with 100 equiv Pi. In addition, upon addition of Pi to a buffered
solution of probe 1, the absorption maximum at 330 nm
decreased while a new absorbance band at 409 nm synchronously
forms (Figure 3). Furthermore, the result in the color change from
colorless to blue could be clearly observed by the naked eye
under UV-lamp/day light at room temperature. The results also
indicated that the probe 1 is highly sensitive to Pi.
Scheme 1 Proposed response mechanism of probe 1 with Pi.
Owing to the above prospective results, we further studied the
feasibility of the probe 1 to detect Pi in living cells (Figure 4).
Fluorescent imaging was first investigated using HeLa cells that
are incubated with probe 1 (Figure 4a) and Pi (Figure 4d). No
obvious fluorescence is observed when the HeLa cells were
subsequently incubated with probe 1 for 30 min. By comparison,
a dramatic enhancement in the green emission was found when
external Pi is added to the cells (Figure 4d). These data indicate
that the probe 1 is capable of permeating into cells and reacting
with Pi to generate fluorescence.
Figure 2 (a) Fluorescence spectra of probe 1 (2.0×10−5 mol/L) after the
addition of Pi at varied concentrations in DMSO-HEPES buffer (0.02 mol/L,
pH 7.4; V/V = 9:1). λex = 415 nm. (b) Fluorescence intensity changes of
probe 1 (2.0×10−5 mol/L) at 465 nm with the amount of Pi.
Figure 4 Fluorescence imaging of the external Pi in the HeLa cells. (a)
fluorescence image of the HeLa cells treated only probe 1; (b) Bright- field
image of part a; (c) merged image of parts a and b; (d) fluorescence image
of the HeLa cells treated with probe 1 and Pi; (e) Bright- field image of part
d; (f) merged image of parts d and e. Scale bar: 10 μm.
Conclusions
In summary, we have rationally designed and synthesized a novel
probe 1 as the inorganic phosphate targeted colorimetric and
Figure 3 (a) Absorption titration spectra of probe 1(2.0×10−5 mol/L) after
the addition of Pi at varied concentrations in DMSO-HEPES buffer (0.02
mol/L, pH 7.4; V/V = 9:1). (b) Ratiometric calibration curve I409/I330 as a
function of Pi concentration.
fluorescent probe for detecting Pi. Probe
1 exhibits high
sensitivity toward Pi with 210-fold fluorescence enhancement in
the fluorescence intensity at 465 nm and a red shift in the
absorption maximum from 330 to 406 nm. Furthermore, the
probe is highly selective, which will not induce any significant
fluorescence change with other anions. In particular, it is
demonstrated that the fluorescent probe 1 displayed selective
detection of Pi, which can be easily monitored by cellular
fluorescence imaging.
Additionally, there was good linearity between the fluorescence
intensity at 465 nm and the concentration of Pi in the range of
0-2.0×10−4 mol/L, with a detection limit of 8.06 × 10-7 mol/L (SI,
Figure S5). The Job's plot of fluorescence was examined at 465nm
between probe 1 (2.0×10-5 mol/L) and Pi in DMSO-HEPES buffer
(2.0×10−2 mol/L, pH 7.4) (V/V = 9:1) (SI, Figure S6), indicating the
1:1 stoichiometric. Moreover, compound 2 showed that the
absorbance at 330 nm and emission at 465 nm in present
conditions (SI, Figure S7), and the mass spectrometry appeared
the molecular ion peak (m/z = 228.051) (SI, Figure S4). It exhibited
that the final spectral data of this sensing process by probe 1 are
exactly the same with the optical properties of compound 2,
which indicated a possible reaction of the probe 1 and Pi (Scheme
1).
Experimental
Reagents and Apparatus
Unless otherwise stated, all solvents and reagents were
commercially available and used without further purification. The
1H NMR and 13C NMR spectra (CDCl3) were performed on a
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Bruker Avance II 400 spectrometer, and using tetramethylsilane
as internal standard. The mass spectra were recorded with Bruker
Autoflex MALDI-TOF mass spectrometer. The UV-vis spectra were
measurements of emission intensity (415 nm) were performed in
the presence of Pi, and each average value of the intensities was
plotted as a concentration of Pi for determining the slope. Then
the detection limit was calculated by using detection limit 3σ /k,
where σ is the standard deviation of the emission intensity of 1
obtained
using
a
Agilent
Technologies
Carry-60
spectrophotometer. The fluorescence experiments were
conducted on a PTI QM40 Fluorescence spectrophotometer with
a 1 cm quartz cell. The pH measurements were recorded on a
Mettler-Toledo Delta 320 pH meter. Fluorescence imaging
experiments were recorded in biological samples by an Olympus
FV1000 fluorescence microscope.
and
k is the slope between the emission intensity and
concentration.
Supporting Information
The supporting information for this article is available on the
WWW under https://doi.org/10.1002/cjoc.2018xxxxx.
Synthesis of probe 1
Acknowledgement
The synthesis process was shown in Scheme 2. To a solution of
HBT (0.23 g, 1.00 mmol) and triethylamine (0.20 mL) in 10 mL of
anhydrous dichloromethane at 0℃ was dropwisely added methyl
chlorooxoacetate (0.20 mL, mixed with 10 mL of CH2Cl2) and kept
stirring at 0℃ for 30 min. Then the mixture was warmed to room
temperature and stirred overnight. The solution was diluted with
CH2Cl2 (5 mL), washed with brine (5 mL×2), and then the organic
layers was dried over anhydrous Na2SO4, filtered. The solution
was removed in vacuo to abtain a crude probe 1 as a yellow solid,
which was recrystallized in dichloromethane and petroleum ether
to obtain light yellow powder. Yield: 80%, m.p.: 115.9-116.2 ℃.
1H NMR (400 MHz, CDCl3) δ 8.19 (dd, J = 8.0, 1.6 Hz, 1H), 7.99 (d, J
= 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.56-7.52 (m, 1H), 7.52-7.44
(m, 2H), 7.44-7.38 (m, 1H), 7.33 (dd, J = 8.0, 0.8 Hz, 1H), 4.07 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 162.44, 157.59, 155.91, 153.14,
147.26, 134.92, 131.84, 130.86, 127.50, 126.51, 125.72, 125.35,
123.38, 123.34, 121.46, 54.03. TOF MS: calcd for C16H11NO4S [M +
H]+ = 314.049, found: 314.085.
We gratefully acknowledge the National Natural Science
Foundation of China (No. 21675042) and the Natural Science
Foundation of Hunan Province (No. 2017JJ2018) and for financial
support of this work.
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Entry for the Table of Contents
Page No.
A Novel Turn-on Fluorescent Probe for
Highly Selective Detection of Phosphate
Ion in Living Cell
Text for Table of Contents.
Jin Zhang *,a,b, Si Wangb, Changhui Liua,
Guowen Hea, Tianying Penga
a Department, Institution, Address 1
c Department, Institution, Address 3
E-mail:
E-mail:
b Department, Institution, Address 2
E-mail:
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