A novel colorimetric and fluorescence turn-on pH sensor with a notably large Stokes shift for its application

Article abstract of DOI:10.1039/c8nj02860e

A novel naked-eye colorimetric and fluorescent turn-on pH sensor based on a naphthalenone scaffold was rationally designed and facilely synthesized. The probe exhibited strong alkaline pH-dependent (pK_a = 9.92) behavior, high selectivity, sensitivity, high quantum efficiency (Φ = 0.64) and rapid response to pH fluctuations (within 1 min) in the range of 9.0-14.0. In addition, probe DDTM displayed a notably large Stokes shift of 198 nm. The response mechanism of the fluorescent probes was proposed to be caused by OH^--induced structural changes from the nonfluorescent DDTM form to the highly emissive addition product (more than 150-fold fluorescence enhancement) along with ICT changes. Live cell imaging data revealed that probe DDTM could be used to selectively monitor pH changes with low cytotoxicity and cell membrane permeability. Therefore, this probe could be used as an effective intracellular pH imaging sensor under extremely alkaline conditions in the biomedical and biological fields.

Full text of DOI:10.1039/c8nj02860e

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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A novel colorimetric and fluorescent turn-on pH sensor with
notably large Stokes shift for its application
Received 00th January 20xx,
Accepted 00th January 20xx
a,b §
c, §
a, b
a, b
a
a
c
Yaoyao Ning,
Yongmin Zhang
Xiaoqing Wang, Kangjia Sheng, Lili Yang, Wei Han, Chaoni Xiao, Jianli Li,
a, b, d
a,b,*
and Shaoping Wu
DOI: 10.1039/x0xx00000x
A novel naked-eye colorimetric and fluorescent turn-on pH sensor based on naphthalenone scaffold was rationally
designed and facilely synthesized. The probe exhibited strong alkaline pH-dependent (pK =9.92) behavior, high selectivity,
www.rsc.org/
a
sensitivity, high quantum efficiency (Ф= 0.64) and rapid respond to pH fluctuations (within 1 min) in the range of 9.0 - 14.0.
In addition, probe DDTM displayed a notably large Stokes shift of 198 nm. The response mechanism of the fluorescent
-
probes was proposed to be caused by OH -induced structure changes from the nonfluorescent DDTM form to the highly
emissive addition product (more than 150-fold fluorescence enhancement) along with ICT changes. Live cell imaging data
revealed that probe DDTM could selectively monitor pH changes with low cytotoxicity and cell membrane permeability.
Therefore, this probe could be acted as an effective intracellular pH imaging sensor under extreme alkaline condition in
the biomedical and biological fields.
Raman sensor [14] and so on. Hence, it is essential for the
health professional to have simple and effective
methodologies for the rapid, selective, and sensitive detection
1
. Introduction
It is well known that the pH values are uniquely of high
importance among the few chemical parameters. Specially,
intracellular pH plays an important role in cell function and
regulation such as cell volume regulation [ 1 ], vesicle
trafficking, cellular metabolism [2], cell membrane polarity [3],
cellular signalling, cell activation, growth, proliferation and
apoptosis [4]. As an important indicator of cellular health,
abnormal pH values of intracellular could lead to cellular
functional disorders and serious diseases such as cancer [5,6],
stroke [ 7 ], Alzheimer’s disease [ 8 ], neurodegenerative
disorders [9] and cardiovascular disease [10]. Therefore, it is
extremely crucial to sense and monitor pH changes in living
cells for investigating cellular functions and further providing
insight into physiological and pathological processes.
of pH values in biological specimens. Fluorescent probes have
been found widespread application for sensing and imaging pH
values in living cells and tissues due to its high sensitivity, easy
operation, real-time and noninvasive imaging properties [15].
However, a lot of reported pH probes still have some
drawbacks such as low fluorescence quantum yields, poor
photostability and narrow Stokes shifts. For the sake of
overcoming these drawbacks, it is important to develop new
longwave fluorescent probes with large Stokes shifts, and high
turn-on fluorescence brightness.
Naphthalenone derivatives have been found to exhibit a
variety of biological activities [16,17] and have also been
surveyed for their suitability as dyes scaffold [18,19]. In
addition, aromatic heterocyclic compounds containing a five-
membered furan or thiophene unit have been extensively
studied in recent years due to their impressive optical
properties and excellent charge-transport properties [20,21].
Therefore, construction of naphthalenone derivatives
containing heterocyclic unit will be vital in synthesis of novel
organic materials with appealing photonic and electronic
functionality.
A variety of analytical techniques have been reported to
measure pH value including acid-base indicator titration [11],
potentiometric titration [12], nuclear magnetic resonance [13],
a
School of Pharmacy; Biomedicine Key Laboratory of Shaanxi Province, Northwest
University, Xi’an 710069, China
b
Joint International Laboratory of Glycobiology and Medicinal Chemistry,
Northwest University, Xi’an, Shaanxi 710069, China
c
Ministry of Education Key Laboratory of Synthetic and Natural Functional
&
Molecule Chemistry, College of Chemistry
University, Xi'an, Shaanxi 710127, China
Materials Science, Northwest As part of our ongoing program in the study of novel
fluorescent dyes [22,23], in the current work, a novel pH-
d
Sorbonne Université, Institut Parisien de Chimie Moléculaire, CNRS UMR 8232, 4
place Jussieu, 75005 Paris, France
These author contributed equally to this work.
response fluorescent probe DDTM was rationally designed and
facilely synthesized by connecting a DTC fluorophore and a
dicyanomethylidene group with double bond. In DDTM, the
§
*
Tel.: +86 029 88304569; Fax: +86 029 88304569.
Eꢀmail: wushaoping@nwu.edu.cn
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dicyanomethylidene group was used as an acceptor moiety A pH meter (Sartorius PB-10) was used to determine the pH
while the amino group acts as an electron donor to form push- value. UV-vis absorption spectra were measured at room
poll system. This probe displayed that ICT was conjugated temperature with a Shimadzu UV-2550 spectrophotometer in
organic π-systems with acceptor and donor subunits. To our 1 cm quartz. The fluorescence spectra were taken on a Hitachi
surprise, the dicyanomethylidene group of probe DDTM, one F-7000 Fluorescence spectrophotometer (Hitachi, Japan) using
of cyanide receptors, has no reaction with cyanide ion except a 5.0 nm slit width in a 1×1 cm quartz cell. The cells
-
OH [24]. Compared with other reported pH probes [25-32], fluorescence imaging was obtained by laser scanning confocal
probe DDTM exhibited strong pH-dependent behaviour, high fluorescence microscopy Olympus FV1000 (Olympus
selectivity, sensitivity and responded linearly and rapidly to Corporation, Tokyo, Japan). High resolution mass spectra
respond pH fluctuations within the range of 9.0 - 14.0. (HRMS) were recorded on a micro TOF-QII mass spectrometer.
1
13
Specially, probe DDTM displayed a notably large Stokes shift of
98 nm and high fluorescence quantum yields. The response 2000 DMX600 MHz FT NMR spectrometers using CDCl
mechanism of the fluorescent probes could be caused by the as solvent and TMS as internal standard. The chemical shifts
H NMR and C NMR spectra were obtained by Varian Gemini
1
3
/DMSO-
d
6
-
OH -induced structure changes from the nonfluorescent DDTM were represented by ppm. The quantum yield was measured
form to the highly emissive addition product along with ICT at room temperature referenced to Quinine sulphate
changes. Furthermore, live cell imaging data revealed that dehydrates in aqueous solution of 0.1 M sulfuric acid. The
probe DDTM could selectively monitor pH changes with low structures of synthesized compounds were characterized by
1
13
cytotoxicity. Therefore, this probe could be used to visualize HRMS, H NMR
,
C NMR and the related spectra are shown in
extreme alkaline within the biomedical and biological fields.
the Supplementary Information.
2
.3. Synthesis of compound 2- ((7- (dimethylamino) -4,5 –
dihydronaphtho [1,2-b] thiophen - 2-yl) methylene) malononitrile
DDTM)
A solution of DTC
(
[
33]
(0.0500 g, 0.19 mmol, 1.0 equiv.) and
malononitrile (0.0130 g, 0.19 mmol, 1.0 equiv.) in anhydrous
CH Cl (5 mL) was treated with triethylamine (0.2 mL) at room
2
2
temperature under argon. The reaction mixture was stirred at
room temperature for 3 h. The solvent was evaporated under
reduced pressure, and the residue was purified by silica gel
column chromatography. (Yield: 40.5%, R
f
= 0.5, PE: EtOAc=
1
3:1). H NMR (600 MHz, CDCl ) δ 7.58 (s, 1H), 7.45 (s, 1H), 7.36
Scheme 1. Strategies to response of pH probe.
3
(
d, J = 8.5 Hz, 1H), 6.62-6.48 (m, 2H), 3.04 (s, 6H), 2.96-2.87 (m,
1
3
2
3
H), 2.81 (dd, J = 23.5, 16.5 Hz, 2H). C NMR (151 MHz, CDCl )
2
. Materials and methods
δ 152.53, 151.97, 149.76, 139.29, 138.62, 136.61, 131.25,
1
3
3
30.63, 126.94, 118.68, 114.93, 111.54, 71.69, 40.62, 32.28,
+
2.1.Materials
0.39, 30.06, 23.90. HRMS (C H N S): calcd. for [M+H]
1
8 15 3
All chemical reagents were purchased from commercial
suppliers and used without further purification. All organic
solvents used for this work were of analytical grade also
without further purification. All reaction processes were
monitored through thin layer chromatography (TLC) which was
performed by using Merck F254 gel-60 plates. Silica gel (200
mesh) was used as the solid phase for column chromatography
by Qingdao Ocean Chemicals (China). All metal ions were
prepared from corresponding salts. All samples were prepared
at room temperature before UV-vis and fluorescence
determination. The phosphate-buffered saline (PBS) was
prepared from sodium dihydrogen phosphate and disodium
hydrogen phosphate. Probe standard solution dissolved in
DMSO and diluted to 50 mL volumetric flask. Distilled water
was prepared from a Millipore water purification system. All
glassware was cleaned with ultrapure water for three times
and dried before use.
+
06.1059; found: [M+H] 306.0987.
.4.Titration experiments of probe DDTM
2
For UV-vis and fluorescence titrations, stock solution of probe
DDTM (1.0 mM) was prepared in DMSO solution. Different
portions of the stock solution were then diluted to different
concentrations for further usage. Generally, 50 µL of probe
DDTM solution was added into 5 mL of buffer solution (10 mM
PBS buffer, 20% DMSO) in a colorimetric tube. The other ions
were dissolved in deionized water and added to the probe
solution under the same condition. The pH variations of the
solution were adjusted by adding the different volumes of
NaOH (0.1 M or 1.0 M). The resulting solution was shaken well
and incubated for
5 min at room temperature before
measurements. The spectra of these solutions were recorded
by means of fluorescence method. Fluorescence
measurements were carried out with a slit width of 5 nm (λex
80 nm) in 10 mm quartz cuvettes at room temperature and
the scan rate was 1200 nm/min.
=
3
2.2.Instrumentation and methods
2
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.5.Cytotoxicity experiments
ARTICLE
2
fluorescence emission wavelength in DMSO solvent (Fig. S1).
So DMSO was preferred to be co-solvent in the photophysical
properties experiment. In addition, the maximum emission
HeLa cells were purchased from the ATCC Cell Bank. Cell
counting kit-8 (CCK-8) was used to detect the cytotoxicity of
probe DDTM. Cells were seeded in 96-well plates at a
-
wavelength of probe DDTM toward OH was obtained in PBS
4
buffer (Fig. S2). Hence, probe DDTM could dissolve completely
in a mixture of DMSO/PBS buffer (10 mM PBS buffer, 20%
DMSO) and the corresponding solution exhibited an efficient
fluorescent turn on mode for pH value change within 1 minute.
The colour of probe DDTM in a mixture of DMSO/PBS buffer
pH 7.4 was light pink with a distinct absorption band at 430
nm. On the contrary, the colour of the mixture was changed
from light pink to yellow and the absorbance of probe DDTM
drastically increases at 430 nm when pH value increased to
concentration of 2 × 10 cells per well and cultured for 24 h in
DMEM (supplemented with 10% fetal bovine serum (FBS)) in
2
an incubator (37 ℃ , 5% CO ). After the cells were incubated
with probe DDTM at different concentrations (0, 5, 10, 15, 20
and 25 µM) for 24 h, CCK-8 (10 µL) was added to each well of
the 96-well plate for 3 h at 37 ℃ , the absorbance was measured
at 450 nm using a microplate reader. Five replicates were done
for each treatment group.
2.6.Cell imaging of probe DDTM
1
1.0 (Fig. S3). The change of colour was readily observed by
HeLa cells were placed on a 20 mm diameter glass bottomed
culture dish and allowed to adhere for 24 h for intracellular
-
imaging of OH . Before confocal scanning imaging, these cells
naked-eye.
In addition, no emission wavelength of probe DDTM was
appeared from 450 nm to 650 nm with the fluorescence
quantum efficiency of 0.01 (Quinine sulphate dehydrate in 0.1
were incubated with 10 µM of probe DDTM at 37 ℃ for 30 min
followed by washing three times with phosphate-buffered
saline (PBS, pH = 7.4). Subsequently, the cells were washed
with PBS for three times to remove excess DDTM, then in situ
treated with different pH value solution (pH 10.0 and 12.0,
respectively), cells were then visualized with a FV1000 laser
scanning confocal microscope. The excitation wavelength was
set to 405 nm and 543 nm.
2 4 S
M H SO was used as the main standard, Ф = 0.54, λex=365
nm). The low quantum efficiency was occurred by ICT process
from the amino group of naphthalenone scaffold as an
electron acceptor group to the dicyanovinyl moiety as an
electron acceptor group by its π-conjugation. To our delight,
the remarkable fluorescence enhancement was observed
more than 150-fold and the quantum efficiency increased to
0.64 due to the disruption of ICT phenomena of probe DDTM
when pH value increased to 11.0, the fluorescence colour was
changed from colourless to orange to yellow with the
maximum emission wavelength at 578 nm. The dicyanovinyl
-
group could be modulated by reacting with OH , and this
nucleophilic addition reaction could finally interrupt the π-
conjugation leading to the turn on mode of this detection.
Reagents and Conditions: (a) CH
DMF, 90℃ , reflux 5 h; (c) Na S·9H
in two steps; (d) Malononitrile, Et N, CH
3
I, K
O, Chloroacetaldehyde, K
Cl , R.T. 3 h, 41%.
2
CO
3
, DMF, 50℃ , 24 h, 54%; (b) POCl
3
,
3
.3. UV-vis absorbance and fluorescence response of probe DDTM
2
2
2
3
CO , DMF, 42%
to pH
3
2
2
Scheme 2. Synthesis of probe DDTM.
The UV-Vis and fluorescence properties of probe DDTM were
measured in a mixture of DMSO/PBS buffer system (10 mM
PBS buffer, 20% DMSO) for detecting pH changes. As shown in
Fig. 1(a), the absorbance at 430 nm was gradually increased
and had a good linear relationship with pH value in the range
3
. Results and discussion
3
.1. Synthesis of probe DDTM
The general synthetic route of probe DDTM was shown in
Scheme 2 DTC was conveniently synthesized according to our
2
from 10.5 to 13.5 (R = 0.9946) along with the pH value
increase. At the same time, the solution colour was drastically
changed from light pink to yellow accompanied with these
absorption changes. So probe DDTM could be used as naked–
eye-visible pH indicators in aqueous media due to their
sensitive colour response to solution pH change.
.
previous work. Then DTC reacted with malononitrile to obtain
the probe DDTM by Knoevenagel condensation at moderate
yield in one step. The structure of probe DDTM was
1
13
characterized and confirmed by H NMR, C NMR and HRMS
data.
3
.2. Photophysical properties of probe DDTM
Probe DDTM exhibited good solubility in common organic
solvents such as CH Cl , CHCl , THF and DMSO. On one hand,
2
2
3
probe DDTM was slightly soluble in water but DMSO could
help increase its water solubility. On the other hand, the
-
respond of probe DDTM toward OH has the maximum
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Fig. 1. (a) UV-Vis absorption spectra of probe DDTM (10 μM in whether these biomolecules could disturb the pH
0 mM PBS buffer, 20% DMSO) in accurate pH values ranging measurement efficiently in living cells, the selectivity of DDTM
from 5.0 to 14.0 at 25
. (b) Linear relationship of UV-Vis to alkaline over these species was investigated at pH 11.0, the
1
℃
absorption at 430 nm and pH values from 10.5 to 13.5. Inset: results were shown in Fig. 4(a), metal ions and reactive oxygen
the colour of probe DDTM in the different pH values under species showed very weak fluorescence intensity at 578 nm. In
contrast, the strong fluorescence signal of probe DDTM was
daylight lamp.
observed at 578 nm in alkaline solutions. Meanwhile, all
Further for fluorescent responses, as depicted in Fig. 2, probe biomolecules of fluorescence response in neutral and alkaline
DDTM itself had no fluorescence intensity at 578 nm, however, environments were evaluated to survey competition
the fluorescence intensity increased with a concomitant experiments. As shown in Fig. 4(b), addition of biomolecules
enhancement in pH changes. With the variation of pH from 8.0 has no interference on the fluorescent spectroscopic changes
to 14.0, the probe showed turn-on fluorescence. Notably, a to pH under the testing conditions. All these results indicated
2
good linearity (R = 0.9958) between the florescence intensities that probe DDTM could serve as
a highly selective
with pH in the range of 9.5- 10.7 was obtained, the regression chemosensor for pH change in extreme alkaline solution.
equation was Y=2324.5X-21250. Additionally, on the basis of
the Henderson-Hasselbalch equation (log [(Imax-I)/ (I-Imin)]=pH -
pK
by analysis of the dependency of fluorescence intensities at
78 nm (Fig. 3). These results demonstrated that probe DDTM
a a
), the pK value of probe DDTM was determined to be 9.92
5
could be used to detect pH value quantitatively in extreme
alkaline solution.
Fig. 4. (a) Emission spectra of probe DDTM in the presence of
different metal ions and biologically relevant analytes (10 μM) in
the pH 11.0 solution. (b) Fluorescence respond of probe DDTM (10
μM) in the presence of different metal ions and biologically relevant
-
-
analytes 1.0 mM in the two pH values. 1: Blank; 2: AcO ; 3: ClO ; 4:
4
2
-
-
-
-
2-
CO
3 3 3 2 2
; 5: ClO ; 6: NO ; 7: ONOO ; 8: SO ; 9: Cys; 10: GSH; 11: H O
2
+
2+
-
2+
3+
2+
(
0.5 mM); 12: Ba ; 13: Ca ; 14: CN ; 15: Cu ; 16: Fe ; 17: Hg ; 18:
+
2+
+
Fig. 2. (a) Emission spectra of probe DDTM (10 μM in 10 mM PBS K ; 19: Mg ; 20: Na ; 21: DTT; 22: H
S. (λex=380 nm, slit: 5.0 nm/5.0
2
buffer, 20% DMSO) in accurate pH values ranging from 5.0 to 14.0 nm).
at 25℃ (λ =380 nm, slit: 5.0 nm/5.0 nm). Inset: Fluorescence
ex
3
.5. Time-dependent effect and reversibility study of probe DDTM
intensity at 578 nm by pH values according to the fluorescence
titration (pH 5.0~14.0). (b) The linear relationship of fluorescence The time-dependent course analysis showed that probe DDTM
intensity at 578 nm and pH values from 9.5 to 10.7. Inset: the color could quickly respond to pH value change for less than one
of probe DDTM in the different pH values under UV 365 nm.
minute at pH 11.0. As shown in Fig. 5(a), after addition of
DDTM to PBS buffer solutions at pH 7.4 and 11.0 at room
temperature, respectively, the fluorescence intensity of the
probe instantly reached equilibrium and remained unchanged
for at least 30 min. The experimental results proved that probe
DDTM has rapid respond to pH changes in extreme alkaline
solution.
As for pH probes, the reversibility was one of the vital
requirements for practical applications. Therefore, the
reversibility of probe DDTM was explored that the pH value of
the solution was switched back and forth between pH 4.0 and
Fig. 3. Linear regression relationship between the pH value and log
1
1.0 using concentrated hydrochloric acid and aqueous
[
(Fmax-F
x
)/(F
x
-Fmin)].
sodium hydroxide. As shown in Fig. 5(b), probe DDTM
exhibited a highly reversible response to pH and the response
time in different pH values were less than 10 s. Switching
3
.4. Selectivity experiment of probe DDTM to pH
The intracellular environment is quite complex and many between the fluorescence on/off states could be repeated
biological molecules are present, such as amino acids, cysteine along with the colour change repeatedly between colourless
(Cys), homocysteine (Hcy) and glutathione (GSH), metal ions (pH 4.0) and yellow (pH 11.0). These results demonstrated that
and reactive oxygen species (ROS) and so on. In order to verify probe DDTM could show quick and reversible responses to pH
4
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changes, and also be potentially useful to real-time monitor pH angle of thiophene ring and dicyanovinyl group in the DDTM-
-
OH was 48.9°, indicating the dicyanovinyl group was twisted in
value changes in extreme alkaline solution.
-
response to OH .
-
Fig. 7. Optimized structures of DDTM and DDTM-OH at the CAM-
Fig. 5. (a) Time course of fluorescence intensity of probe DDTM (10
B3LYP/6-311+G(d, p)/PCM theoretical level.
μM in 10 mM PBS buffer, 20% DMSO) in the two pH values. (b) pH
reversibility of probe DDTM (10 μM in 10 mM PBS buffer, 20% Molecular orbital surfaces of the HOMO and LUMO for
-
DMSO) between pH 4.0 and pH 11.0 at 25 ℃ (λex=380 nm, slit: 5.0 compound DDTM and DDTM-OH were displayed in Fig. 8. For
nm/5.0 nm).
DDTM, the electron density in the HOMO was distributed over
the whole molecular while that in the LUMO was mainly
3
.6. Proposed detection mechanism and theoretical computation
concentrated on the thiophene ring and dicyanovinyl group,
-
The mechanism for the fluorescence enhancement of probe respectively. As for DDTM-OH , the electron in HOMO and
DDTM was proposed as shown in Fig. 6. In alkaline solution, LUMO orbitals was mostly spread over the moiety of DTC
.
hydroxyl ion reacted as nucleophilic reagent on the double Obviously, there was no ICT process occurred in the DDTM-
-
3
bond of dicyanovinyl group in probe DDTM to give the OH , in that a sp hybridized carbon atom existed in vinyl
addition intermediate, which could finally interrupt the π- moiety of dicyanomethylidene and dicyanomethylidene
conjugation leading to produce strong fluorescence. Then twisted. The interruption of the ICT process also resulted in the
-
hydroxyl of the addition intermediate could be occurred in increased HOMO-LUMO energy gap of DDTM-OH (6.39 eV) in
protonation process in acid condition, finally probe DDTM is comparison with that of the DDTM (4.63 eV) and is also
regenerated and the water molecule is released. To further explained for the appearance of a new absorption band at 430
-
confirm the proposed detection mechanism of probe DDTM nm in the absorption spectrum of DDTM-OH . Therefore, these
towards hydroxyl, probe DDTM was reacted with tetrabutyl DFT calculations verified that OH- was bound to the vinyl
ammonium hydroxide at room temperature for 10 min in group of probe DDTM and are in good agreement with the
MeOH solution, then the reaction solution was diluted experimental results.
DDTM
DDTM-OH
-
appropriately and tested by mass spectroscopy, an expected
signal at m/z 322.9321 corresponding to addition intermediate
-
probe DDTM + OH] was observed (Fig. S4), and these data
+
OH
-
[
were consistent with the proposed mechanism.
LUMO = ꢀ1.91 eV
LUMO = 0.07 eV
ꢁE=4.63 eV
ꢁE=6.39 eV
HOMO = ꢀ6.54 eV
HOMO = ꢀ6.32 eV
Fig. 8. HOMO-LUMO energy levels and frontier molecular orbitals of
-
probe DDTM and the adduct DDTM-OH .
Fig. 6. Proposed mechanism of probe DDTM towards pH changes.
To better understand the optical responses of probe DDTM
3
.7. Cytotoxicity and fluorescence imaging for living cells
Firstly, the cytotoxic effects of probe DDTM were studied in
order to evaluate its imaging performance in vivo. MTT, a
standard cell viability protocol, was employed to evaluate the
cytotoxicity of probe DDTM on HeLa cells by treating HeLa
cells with the probe for 48 h. The probe showed low
cytotoxicity and excellent compatibility with more than 80%
viability at concentration of 25 μM under experimental
conditions (Fig. S3).
-
upon adding with OH , density functional theory (DFT)
calculations were performed using the CAM-B3LYP/6-311+G(d,
p) level of Gaussian 09 program [34,35]. The solvent effects
were taken into account in water through the polarizable
continuum model (PCM) [36]. The optimized structures of
-
DDTM and DDTM-OH were shown in Fig. 7. The molecular
backbone of the DDTM was nearly coplanar while the dihedral
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Next, the cellular imaging assays were performed to verify 3.8. Practical application of probe DDTM in pH test strips
whether probe DDTM could be applied for determining pH
In order to further explore the practical application of probe
changes in the biological application. HeLa cells were
DDTM, pH test strips were prepared and utilized to sense
incubated with probe DDTM at pH 7.4, 10.0 and 12.0 for 5
different pH according to the excellent properties of probe
min, after that intracellular fluorescence was monitored by
DDTM. The test strip was prepared by immersing filter papers
confocal microscopy (Fig. 9). Under neutral condition, no
in probe DDTM solution (10 μM in 20% DMSO). After drying in
fluorescence was observed. On the contrary, moderate
the air, the strip displayed different colour change when the
fluorescence was observed when cells were incubated with
test strips immersed in different pH solution. As shown in Fig.
probe DDTM at pH 10.0. Furthermore, the strong blue
11, the changes of colour in different pH solution were
fluorescence was observed at pH 12.0. At the same time, the
similar results were obtained in red channel when excitation
wavelength was 543 nm. These results indicated that probe
DDTM displays sensitive fluorescence enhances in a pH-
dependent manner in living cells system.
observed in naked eyes and fluorescent light, respectively.
Therefore, the distinct colour changes promoted probe DDTM
to be conveniently used for precise pH paper preparation in
real time applications.
(a)
(b)
Fig. 11. Photographs of colorimetric responses of probe DDTM test
strips with different pH solutions. (a) Naked eye in visible light. (b)
Fluorescence colour changes under 365 nm UV lamp.
4
. Conclusions
In summary, a novel pH-sensitive colorimetric sensors based
on naphthalenone scaffold was designed and synthesized.
Probe DDTM showed a naked eye visible colour changes from
light pink to yellow, and the fluorescence colour was changed
from colourless to orange to yellow in the range pH 9.0-14.0.
In addition, the probe exhibited strong pH-dependent
behaviour, high selectivity and rapidly respond to pH
fluctuations within the range of 9.0 - 14.0. Furthermore, probe
DDTM displayed a notably large Stokes shift of 198 nm. Live
cell imaging data revealed that probe DDTM could selectively
monitor pH changes with low cytotoxicity and cell membrane
permeability. Therefore, the probe could be acted as an
effective intracellular pH imaging agent under extreme alkaline
conditions in the biomedical and biological fields.
Fig. 9. Confocal fluorescent imaging of HeLa cells incubated with
probe DDTM (10 μM) at pH 7.4 (a, b, c), 10.0 (d, e, f) and 12.0 (g, h,
i) for 5 min, respectively. Left: Excitation wavelength was 405 nm.
Right: Excitation wavelength was 543 nm. Scale bar: 40 μm.
Additionally, dynamic fluorescence imaging was also occurred
to investigate the process of DTTM diffusing into cells and the
change of fluorescence intensity. As shown in Fig. 10, the
fluorescence of DDTM was obtained at different time-points
via confocal laser scanning microscope. After addition of
DDTM in pH 12.0 alkaline solution, the fluorescence intensity
of the HeLa cells had no change and barely any fluorescence
quenching was observed until 30 min at 405 nm and 543 nm.
These cell experiments demonstrated that probe DDTM has
excellent stability and biological compatibility in living cells,
and could be used to mark the change of pH in living cells.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
We sincerely thank the Program for Changjiang Scholars and
Innovative Research Team in University (No. IRT_15R55), the
Science Foundation of Northwest University (No. 15NW17),
the International Science & Technology Cooperation Program
of Shaanxi Province (No. 2016KW-003) and Opening
Fig. 10. Dynamic fluorescence images of living HeLa cells on Foundation of Key Laboratory of Resource Biology and
incubation with 10 μM DDTM for different times 10 min (a, b, c) Biotechnology in Western China (Northwest University),
and 30 min (d, e, f) in pH 12.0 medium. Left: Excitation wavelength Ministry of Education.
was 405 nm. Right: Excitation wavelength was 543 nm. Scale bar: 40
μm.
6
| J. Name., 2012, 00, 1-3
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Notes and references
Actuat. B-Chem., 2018, 269, 322-330.
[34] T. Yanai, D. P. Tew, N. C. Handy, Chem. Phys. Lett., 2004, 393, 51-57.
[1] S. C. Burleigh, T. van de Laar, C. J. M. Stroop, W. M. J. van Grunsven, N. [35] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B., 1988, 37, 85-789.
O'Donoghue, P. M. Rudd, G. P. Davey, BMC Biotechnol., 2011, 11, 95- [36] J. Tomasi, B. Mennucci, E. Cancès, J. Mol. Struct., (THEOCHEM) 1999,
1
1.
464, 211-226.
[2] S. Humez, M. Monet, F. van Coppenolle, P. Delcour, N. Prevarskaya, Am.
J. Physiol. Cell Physiol., 2004, 287, 1733-1746.
[
[
3] A. L. Edinger, C. B. Thompson, Curr. Opin. Cell Biol., 2005, 16, 663-669.
4] A. C. Johansson, H. Appelqvist, C. Nilsson, K. Kagedal, K. Roberg, K.
Ollinger, Apoptosis., 2010, 15, 527-540.
[
5] V. Estrella, T. Chen, M. Lloyd, J. Wojtkowiak, H. H. Cornnell, A. Ibrahim-
Hashim, K. Bailey, Y. Balagurunathan, J. M. Rothberg, B. F. Sloane, J.
Johnson, R. A. Gatenby, R. J. Gillies, Cancer Res., 2013, 73, 1524-1535.
6] B. A. Webb, M. Chimenti, M. P. Jacobson, D. L. Barber, Nat. Rev. Cancer.,
[
[
[
2
011, 11, 671-677.
7] T. Myochin, K. Kiyose, K. Hanaoka, H. Kojima, T. Terai, T. Nagano, J. Am.
Chem. Soc., 2011, 133, 3401-3409.
8] T. A. Davies, R. E. Fine, R. J. Johnson, C. A. Levesque, W. H. Rathbun, K.F.
Seetoo, S.J. Smith, G. Strohmeier, L. Volicer, L. Delva, E.R. Simons,
Biochem. Biophys. Res. Commun., 1993, 194, 537-543.
[9] S. Harguindey, S. J. Reshkin, G. Orive, J. L. Arranz, E. Anitua, Curr.
Alzheimer Res., 2007, 4, 53-65.
[
10] R. D. Vaughan-Jones, K. W. Spitzer, P. Swietach, Intracellular pH
regulation in heart, J. Mol. Cell. Cardiol., 2009, 46, 318-331.
11] W. Dorota, A. Tobias, M. Colette, Anal. Chem., 2014, 86, 15-29.
12] J. Zhou, L. Zhang, Y. Tian, Anal. Chem., 2016, 88, 2113-2118.
13] S. He, R. P. Mason, S. Hunjan, V. D. Mehta, V. Arora, R. Katipally, P. V.
Kulkarni, P. P. Antich, Bioorg. Med. Chem., 1998, 6, 1631-1639.
14] L. S. Lawson, J. W. Chan, T. Huser, Nanoscale., 2014, 6, 7971-7980.
15] J. Yin, Y. Hu, J. Yoon, Chem. Soc. Rev., 2015, 44, 4619-4644.
16] C. Dax, M. Coincon, J. Sygusch, C. Blonski, Biochemistry., 2005, 44, 5430-
[
[
[
[
[
[
5
443.
[
[
17] L. W. Lawrence Woo, N. M. Howarth, A. Purohit, H. A. M. Hejaz, M. J.
Reed, B. V. L. Potter, J. Med. Chem., 1998, 41, 1068-1083.
18] G. Niu, W. Liu, H. Xiao, H. Zhang, J. Chen, Q. Dai, J. Ge, J. Wu, P. Wang,
Chem. Asian. J., 2015, 11, 498-504.
[
[
[
19] H. Chen, Y. Tang, M. Ren, W. Lin, Chem. Sci., 2016, 7, 1896-1903.
20] N. J. Greco, Y. Tor, Tetrahedron., 2007, 63, 3515-3527.
21] S. C. Rasmussen, S. J. Evenson, C. B. Mccausland, Chem. Commun.,
2
015, 51, 4528-4543.
[22] M. She, S. Wu, Z. Wang, S. Ma, Z. Yang, B. Yin, P. Liu, S. Zhang, J. Li,
Sensor. Actuat. B-Chem., 2017, 247, 129-138.
[23] X. Jin, S. Wu, M. She, Y. Jia, L. Hao, B. Yin, L. Wang, M. Obst, Y. Shen, Y.
Zhang, J. Li, Anal. Chem., 2016, 88, 11253-11260.
[24] (a) Z. Liu, X. Wang, Z. Yang, W. He, J. Org. Chem., 2011, 76, 10286-
1
2
0290; (b) X. Cheng, Y. Zhou, J. Qin, Z. Li, ACS. Appl. Mater. Interfaces.,
012, 4, 2133-2138; (c) W. Lin, S. Fang, J. Hu, H. Tsai, K. Chen, Anal.
Chem., 2014, 86, 4648-4652; (d) W. Chen, Z. Zhang, X. Li, H. Agren, J.
Su, RSC Adv., 2015, 5, 12191-12201; (e) J. Orrego-Hernandez, J. Portilla,
J. Org. Chem., 2017, 82, 13376-13385.
[
[
25] X. Li, Y. Yue, Y. Wen, C. Yin, F. Huo, Dyes Pigm., 2016, 134, 291-296.
26] Y. Ge, P. Wei, T. Wang, X. Cao, D. Zhang, F. Li, Sensor. Actuat. B-Chem.,
2
018, 254, 314-320.
[
[
[
[
[
[
[
27] J. Chao, K. Song, H. Wang, Z. Li, Y. Zhang, C. Yin, F. Huo, J. Wang, T.
Zhang, RSC Adv., 2017, 7, 964-970.
28] Y. Ge, A. Liu, J. Dong, G. Duan, X. Cao, F. Li, Sensor. Actuat. B-Chem.,
2
017, 247, 46-52.
29] G. Chen, Q. Fu, F. Yu, R. Ren, Y. Liu, Z. Cao, G. Li, X. Zhao, L. Chen, H.
Wang, J. You, Anal. Chem., 2017, 89, 8509-8516.
30] Y. Wen, W. Zhang, T. Liu, F. Huo, C. Yin, Anal. Chem., 2017, 89, 11869-
1
1874.
31] G. Niu, P. Zhang, W. Liu, M. Wang, H. Zhang, J. Wu, L. Zhang, P. Wang,
Anal. Chem., 2017, 89, 1922-1929.
32] X. Liu, Y. Su, H. Tian, L. Yang, H. Zhang, X. Song, G. W. Foley, Anal.
Chem., 2017, 89, 7038-7045.
33] Y. Ning, J. Cui, Y. Lu, X. Wang, C. Xiao, S. Wu, J. Li, Y. Zhang, Sensor.
This journal is © The Royal Society of Chemistry 20xx
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New Journal of Chemistry
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DOI: 10.1039/C8NJ02860E
A novel colorimetric and fluorescent turn-on pH sensor with
notably large Stokes shift for its application
a,b §
c, §
a, b
a, b
a
a
c
Yaoyao Ning,
Yongmin Zhang
Xiaoqing Wang, Kangjia Sheng, Lili Yang, Wei Han, Chaoni Xiao, Jianli Li,
a, b, d
a,b,*
and Shaoping Wu