A ratiometric fluorescent sensor for pH fluctuation and its application in living cells with low dark toxicity

Article abstract of DOI:10.1016/j.dyepig.2016.08.058

It is highly demanded and challenging to develop small-molecular fluorescent sensors for the ratiometric detection of pH fluctuation. A ratiometric fluorescent pH sensor was constructed by integrating a pH-sensitive fluorophore with a pH-insensitive hemicyanine group. Its linear and reversible ratiometric fluorescent response from pH 7.0 to 8.8 and 6.0 to 9.0 respectively makes this sensor suitable for the practical tracking of pH fluctuation in live cells. With this sensor, stimulated pH fluctuation has been successfully tracked in a ratiometric manner via fluorescence imaging with low dark toxicity.

Full text of DOI:10.1016/j.dyepig.2016.08.058

Accepted Manuscript
A ratiometric fluorescent sensor for pH fluctuation and its application in living cells
with low dark toxicity
Bingjie Shi, Yilin Gao, Chunxia Liu, Wei Feng, Zhanxian Li, Liuhe Wei, Mingming Yu
PII:
S0143-7208(16)30574-5
10.1016/j.dyepig.2016.08.058
DYPI 5438
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 7 July 2016
Revised Date: 16 August 2016
Accepted Date: 27 August 2016
Please cite this article as: Shi B, Gao Y, Liu C, Feng W, Li Z, Wei L, Yu M, A ratiometric fluorescent
sensor for pH fluctuation and its application in living cells with low dark toxicity, Dyes and Pigments
(2016), doi: 10.1016/j.dyepig.2016.08.058.
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ACCEPTED MANUSCRIPT
Graphical Abstract
With a ratiometric fluorescent pH sensor, stimulated pH fluctuation has been
successfully tracked in a ratiometric manner via fluorescence imaging with low dark
toxicity.

ACCEPTED MANUSCRIPT
A ratiometric fluorescent sensor for pH fluctuation and its application
in living cells with low dark toxicity
‡
a
‡b
a
b
a
a
Bingjie Shi , Yilin Gao , Chunxia Liu , Wei Feng , Zhanxian Li , Liuhe Wei , Mingming
a,
Yu *
*
Corresponding author.
a. College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou,
50001, China.
4
E-mail address: yumm@zzu.edu.cn (M. Yu).
Tel: +(86)371-67781205
Fax: +(86)371-67781205
b. Department of Chemistry, Fudan University , Shanghai, 200433, China.

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ACCEPTED MANUSCRIPT
Abstract
It is highly demanded and challenging to develop small-molecular fluorescent sensors for
the ratiometric detection of pH fluctuation. A ratiometric fluorescent pH sensor was
constructed by integrating a pH-sensitive fluorophore with a pH-insensitive hemicyanine
group. Its linear and reversible ratiometric fluorescent response from pH 7.0 to 8.8 and
6
.0 to 9.0 respectively makes this sensor suitable for the practical tracking of pH
fluctuation in live cells. With this sensor, stimulated pH fluctuation has been successfully
tracked in a ratiometric manner via fluorescence imaging with low dark toxicity.
Keywords: Ratiometric fluorescent sensor; pH fluctuation; Dark toxicity; Cell imaging.

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1
. Introduction
As a crucial physiological parameter, pH, an abbreviation for “power of hydrogen”
initially, plays a critical role in both intracellular (pH ) and extracellular (pH ) milieu,
i
e
which is influenced by diverse physiological and pathological processes. Hence
quantitatively measuring pH is useful for cellular analysis or diagnosis [1]. For example,
an acidic environment may be associated with inflammation or tumor [2]. Different from
the acidic organelles, mitochondria display a slightly basic pH value [3,4], and plays
critical roles in many physiological activities including energy production through the
respiratory chain [5,6], cell signalling via reactive oxygen species production [7],
2
+
regulation of Ca homeostasis [8–10], and the triggering of cell death [11]. The unique
function of mitochondria depends on the mitochondrial pH, and the physiological pHm
deviation is of great significance for the understanding of mitochondria physiology
although slight difference exists [12,13]. Fortunately, this minor pH deviation can be
m
concealed in the case of using turn-on fluorescent sensors since the fluorescence intensity
can be affected by local sensor concentration, microenvironment and imaging parameters,
and so on. Therefore, the development of facile and reliable methods to monitor pHi
especially the basic pH in living cells is highly demanded in understanding the
i
physiology and pathology of mitochondria.
Fluorescence imaging serves as an essential tool in the study of biological molecules,
pathways, and processes in living cells owing to its ability in providing spatial-temporal
information at microscopic, mesoscopic, and macroscopic levels [14–18]. So far, many
organic molecular fluorescent pH sensors have been reported [19–23], but most of them
are designed for acidic pH, while sensors for basic pH are rare and their design remains
challengeable.

4
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It is well known that the emission intensity may be influenced by many factors, such
as optical path length and the illumination intensity. In addition, when turn-on mode
fluorescent sensors are used in biosystem for minor pH deviation, which can be
i
concealed since the fluorescence intensity can be affected by local sensor concentration,
microenvironment and imaging parameters, etc. A ratiometric approach can eliminate the
effects of these factors and realize more effective detection via the ratio of fluorescence
intensities at two different wavelengths [24,25]. Up to now, the reported ratiometric
fluorescent sensors toward basic pH are very rare [26,27]. Therefore, it is of great interest
to design ratiometric fluorescent sensors for basic pH, especially with the delicate
detection range.
Compared with the nanoparticle-based ratiometric pH sensors, small-molecule
i
ratiometric pH sensors are more appealing due to their simple staining procedures, good
i
reproducibility, and a great number of alterative emission wavelengths for imaging
[
28,29]. Moreover, small-molecule sensors can be more readily endowed with pH
sensitivity. Given this, small-molecule sensors are among the most promising approaches
to ratiometric pH imaging and tracking [30].
i
As a very important kind of organic functional dyes, cyanine derivatives have many
merits such as much bigger conjugate plane, cationic quaternary ammonium salt, high
molar absorption coefficient, wide spectral range, and high fluorescence quantum yield.
On the other hand, the spectral wavelength of these compounds can belong to
near-infrared range, can effectively avoid the self-fluorescence of biological systems, and
improve the sensitivity and avoid interference. Therefore, cyanine-based fluorescent
chemosensors have been widely and deeply studied [31–35].
In this paper, we used a hemicyanine derivative as the fluorophore and synthesized a

5
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fluorescent molecule 1 (Scheme 1), for ratiometric pH sensing and imaging [36,37]. It
exhibits reversible pH sensing ability from pH 6.0 to 9.0 with a linear response from pH
7
.0 to 8.8. Ratiometric pH imaging via confocal microscopy has also been effectively
demonstrated.
. Experimental
.1. Methods and materials
2
2
1
13
H and C NMR spectra were recorded on a Bruker 400 NMR spectrometer. Chemical
shifts are reported in parts per million using tetramethylsilane (TMS) as the internal
standard. Mass spectra were obtained on a high resolution mass spectrometer (IonSpec4.7
Tesla FTMS-MALDI/DHB).
All of the chemicals were purchased from commercial suppliers and used without
further purification. All of the reactions were performed under an argon atmosphere using
solvents purified by standard methods. Compound 1 was synthesized according to
Scheme 1. Compound 3 was synthesized and used directly without structural
characterization.
(Insert: Scheme 1)
2
.2. Spectral characterizations
All spectral characterizations were carried out in HPLC-grade solvents at 20 °C within
a 10 mm quartz cell. UV-vis absorption spectra were measured with a TU-1901
double-beam UV-vis spectrophotometer. Fluorescence spectroscopy was determined on a
Hitachi F-4600 spectrometer.
2
.3. Cell viability assay
The dark toxicity of the material: the stock solutions were diluted with fresh medium to

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various concentrations (2, 4, 6, 8 and 10 ꢀM). The cell medium was then exchanged for
different concentrations of material medium solution. The cells were then incubated with
these solutions at 37 °C in 5% CO for 1 h before removing the material solution and
2
adding fresh medium. Subsequently, the plates were incubated at 37 °C in 5% CO for 24
2
h. The cell medium solutions were exchanged for 100 ꢀL of fresh medium, followed by
the addition of 20 ꢀL of MTT solution to each well. The culture plates were then
incubated at 37 °C in 5% CO for 4 h. The culture medium was discarded, and 100 ꢀL of
2
dimethylsulfoxide was added. The absorbance of an untreated cell population under the
same experimental conditions was used as the reference point to establish 100% cell
viability. The ratiometric confocal fluorescence images are recorded by a Nikon A1
RSiMP Confocal Microscopy with a 60 ×WI objective (NA 1.27) and 405 nm diode
laser.
2
.4. Synthesis of compound 2
,10-phenanthroquinone (320.8 mg, 1.54 mmol), terephthalaldehyde (642.5 mg, 4.79
9
mmol), and ammonium acetate (2.46 g, 31.9 mmol) were dissolved in 20 mL glacial
acetic acid. The mixture was stirred for 30 min at 100 °C and cooled to room temperature.
The crude product was obtained by filtration and being washed with acetate acid, dilute
sodium hydrogen carbonate solution and water. The final product 2 was purified by silica
gel column chromatography using acetone/petroleum (1/3 v/v) as eluent. The yield was
7
1%.
Characterization of 2: H NMR (400 MHz, DMSO-d , TMS): δ 13.73 (s, 1H), 10.10
1
6
H
(s, 1H), 8.88 (d, 2H), 8.60 (d, 2H), 8.54 (d, 2H), 8.14 (d, 2H), 7.77 (t, 2H), 7.67 (t, 2H).
1
3
C NMR (100 MHz, DMSO-d ): δ 193.03, 172.48, 148.16, 136.54, 135.89, 130.69,
6
C
1
27.75, 126.92, 126.16, 124.48, and 122.53.

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2
.5. Synthesis of compound 1
mL ethanol solution of compound
-hydroxybenzaldehyde (366 mg, 3.0 mmol) was refluxed for 3 h at 80 °C and cooled to
1
5
2
(1.035 g, 3.0 mmol) and
4
room temperature. The final product (compound 1, 637.2 mg, 31.6%) was obtained by
filtration and washed with ethanol for three times.
Characterization of 1: HRMS (EI) m/z: calcd for C H NO [M-I], 322.1443; found,
2
0
20
3
1
3
1
22.1442. H NMR (600 MHz, DMSO-d , TMS): δ 10.97(s, 1H), 8.47 (d, 1H), 8.39 (s,
6 H
13
H), 8.17 (m, 3H), 7.93 (d, 1H), 7.49 (d, 1H), 6.98 (d, 2H), 4.10 (s, 3H), 1.82 (s, 6H). C
NMR (150 MHz, DMSO-d ): δ 183.80, 167.08, 164.30, 155.91, 145.69, 143.86, 134.60,
6
C
1
3
3
31.16, 130.87, 126.53, 124.20, 117.03, 115.13, 109.76, 52.26, 34.69, and 26.06.
. Results and Discussion
.1. Spectroscopic study and pH-sensing behaviour of sensor 1
Due to the good solubility of compound 1 in water, a HEPES solution was utilized as
the medium to determine the absorption and emission spectra of 1 (HEPES solution, 1.0 ×
−
5
1
0 M) at different pH values. As shown in Fig. 1, with the increasing of pH value from
5
.0 to 9.6, the peak at 430 nm in the UV-vis spectra decreased gradually while a new band
developed at 535 nm until the maxima absorbance reached pH 9.6 (Fig. 1a). Meanwhile,
one apparent isosbestic point was observed at about 463 nm, indicating that only one
product was generated from 1 upon pH increasing. The normalized ratio of absorption at
5
25 nm to that at 430 nm, A /A increases from 0.02 to 14 (Fig. S1, Supporting
525 430
Information). Fig. 1c demonstrates that the color of HEPES solution of 1 changes from
yellow to red with pH increasing from 5.0 to 9.6, indicating that compound 1 can be as a
highly sensitive ‘naked-eye’ sensor toward pH changes. Accordingly, upon excitation at

8
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4
70 nm, the emission at 526 nm decreased and a new emission at 564 nm appeared,
which gradually increased in intensity with increased pH of the medium (Fig 1d). The
Commission Internationale de L'Eclairage (CIE) 1931 (x,y) chromaticity diagram of the
solutions with different pH are shown in Fig. 1f. With pH increasing, the fluorescence
color of the solution changed from yellow to green. Such fluorescence changes brought
from pH increase indicates that compound 1 can be employed as a ratiometric
fluorescence sensor for pH. The normalized ratio of emission at 564 nm to that at 526 nm
(
λ , 470 nm), I /I increases from 0.77 to 28.55 (Fig. S2, Supporting Information). As
ex 564 526
demonstrated in Figures 1b and 2b, in the range of pH 7.0 to 8.0, A /A and I /I of
5
25
430
564 526
1
are in good linear relationship with pH changing, implying that pH can be quantitatively
detected in a wider concentration range with both UV-vis and fluorescence spectroscopy.
The apparent pK ' was determined to be 7.87±0.14 and 7.90±0.15 via fitting the pH
a
titration profile based on the normalized A /A and I /I respectively. These results
5
25 430
564 526
led us to conclude that 1 could be an effective colorimetric and ratiometric fluorescent
sensor for pH monitoring. The high pH induced 1O formation from 1 is responsible for
the UV-vis and fluorescence spectral change (Scheme 2). The sensing mechanism was
studied by mass spectra. For pure sensor 1 in neutral solution, a characteristic peak at m/z
=
322.1442 was obtained which corresponds to the species [m-I], whilst in basic solution,
the peak at 322.1442 decreased greatly and one new peak appeared at m/z = 318.3009
corresponding to the species [m-I-4H]. Such Mass spectra data confirm that compound
1
O was formed (Scheme 2).
In addition, the enhanced I /I value of compound 1 at pH 9.00 can be recovered to
5
64 526

9
ACCEPTED MANUSCRIPT
the original I /I value at pH 6.0, and this reversible ratiometric pH sensing ability can
5
64 526
be retained for at least 5 cycles in the pH range from 6.0 to 9.0 (Fig. 2a).
(
Insert: Fig. 1)
Insert: Fig. 2)
(
3
.2. Thermal stability and photostability study of sensor 1
An ideal sensor ought to have good thermal stability and photostability, as shown in
Fig. S3, Supporting Information, with temperature increasing from 30 to 48°C, the
−
5
emission ratio I /I of compound 1 in HEPES solutions (pH = 7.4, 1.0 × 10 M)
5
64 526
decreases less than 11.6%, indicating good thermal stability. While for the photostability
experiment, an excellent result was obtained similarly, in the same condition as that of the
thermal stability experiment, the emission ratio I /I
hardly changes with time
5
64 526
extending (Fig. 2b). Such results demonstrate that compound 1 as a pH sensor has good
thermal stability and photostability.
3
.3. The anti-disturbance effect study of compound 1 for pH sensing
To further assess its utility as a pH ratiometric fluorescent sensor, its emission spectral
response to pH 7.4 in the presence of biological species including biological molecules,
metal ions, and anions (figures 2c, S4 and S5, Supporting Information) was also tested.
The emission ratio I /I exhibits a negligible change in the presence of these species.
5
64 526
All these results suggest that compound 1 might be a suitable candidate for ratiometric
intracellular pH imaging.
3
.4. Application of compound 1 in cellular imaging
The cytotoxicity of probe 1 toward HeLa cells was measured using the
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazilium bromide (MTT) assay to evaluate

1
0
ACCEPTED MANUSCRIPT
the potential application of compound 1 in live cell imagining. The cellular viability was
−
5
estimated to be greater than 90% after 1 h at the concentration below or equal 2.0 ×10
M for compound 1, suggesting the low cytotoxicity of compound 1 (Fig. 2d).
To determine a suitable concentration of compound 1 for its cellular pH imaging,
i
fluorescence imaging experiments of compound 1 of different concentrations in live
HeLa cells were carried out using confocal fluorescence microscopy with a 488 nm diode
laser. HeLa cells were first incubated with the culture medium containing compound 1
with different concentrations respectively for 20 minutes. And the confocal fluorescence
microscopy images are shown in Fig. 3. In the concentration range from 0.001 to 1.25
ꢀ
M, I564 and I526 are enhanced and the ratio of I564 to I526 hardly changes with the increase
of concentration. However, I564 and I526 decrease and the ratio of I564 to I526 is almost
unchanged with the increase of concentration in the range of 5.00 ꢀM and 10.0 ꢀM,
which might be ascribed to the quenching effect of high concentration.
(Insert: Fig. 3)
With its low cytotoxicity, compound 1 was further studied for its ratiometric pHi
imagining ability. In this study, the intracellular pH calibration was carried out in live
+
HeLa cells using a standard procedure followed by a further incubation with high K
buffers of different pH, also containing 1 mM nigericin, and based on the results from Fig.
3
, 0.63 ꢀM and 1.25 ꢀM were selected as the final concentrations to study the pHi
imagining of compound 1. As shown in Fig. S6, Supporting Information, when the
concentration of compound 1 is 0.63 ꢀM, the ratio of I564 to I526 gradually increases with
pH changing from 5.0 to 10.0, but the result is not ideal, which might be ascribed to the
insufficient amount of the dye 1 into the cell and the poor dispersion. However, when it
comes to the concentration of 1.25 ꢀM, I564 (Fig. 4, first row) in cells increases with pH

11
ACCEPTED MANUSCRIPT
value (5−10), whereas I526 (Fig. 4, second row) gradually decreases except 8. Compared
with the result of Fig. 1, we found that the results in Fig. 1 and Fig. 4 are not consistent,
which may be ascribed to the more complex situation in living cells. The ratio of I564 to
I526 increases with pH changing from 5 to 10, which indicate the ability of sensor 1 to
measure a pH-dependent signal change and the merit of ratiometric fluorescence imaging.
(Insert: Fig. 4)
4
. Conclusion
A colorimetric and ratiometric fluorescent organic molecular sensor with single
excitation wavelength was firstly synthesized for sensing both acidic and basic pH with
high sensitivity. The linear ratiometric pH response range from pH 7.0 to 8.8, the
reversible pH sensing ability, and the cell membrane permeability makes this sensor
especially suitable for the practical tracking of pH fluctuation in live cells via ratiometric
i
imaging.
Acknowledgements
We are grateful for the financial supports from National Natural Science Foundation of
China (U1504203 and J1210060), Key Laboratory of Photochemical Conversion and
Optoelectronic Materials and Zhengzhou University.
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Figure Captions
−
5
Fig. 1. (a) Absorption spectra of 1 in HEPES buffer solution (1.0 × 10 M) of
different pH values determined. (b) The linear relationship for the ratiometric response
−
5
from absorption spectra of 1 to different pH. (c) Photographs of compound 1 (1.0 × 10
M in HEPES buffer solution) at various pH conditions (5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4,
.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 from left
6
−
5
to right) in daylight. (d) Emission spectra of 1 in HEPES buffer solution (1.0 × 10 M) of
different pH values determined upon excitation at 470 nm. (e) The linear relationship for
the ratiometric response from emission spectra of 1 to different pH. (f) CIE 1931 (x,y)
chromaticity diagram of the solutions of 1 in different pH conditions derived from
fluorescence spectra (black circles); pH from left to right: 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2,
6
.4, 6.6, 6.8,7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6.
Fig. 2. (a) Emission ratio I564 /I525 of compound 1 (HEPES solution, 1.0 × 10 M) in
the same medium determined over consecutive pH cycles. (b) Emission ratio I564 /I525 of
−
5
−
5
compound 1 (HEPES solution, 1.0 × 10 M) with irradiation (λ = 470 nm) time change.
ex
−
5
(
c) Emission spectra response of compound 1 (pH 7.4 HEPES solution, 1.0 × 10 M)
upon addition of different species (10 equiv of species relative to 1), the excitation
wavelength was 470 nm. I564 and I525 represent the emission intensity at 564 nm and 525
nm. The species used were Cys, Phe, Ala, Met, Pro, Gly, Hcy, Glu, GSH, H O , Arg, Lys,
2
2
Leu, Tyr, Glc, Trp, Ser, Thr, Asp, Val, and lle. (d) Viability assay for A549 cells treated
with sensor 1 in dark.
Fig. 3. Confocal images of HeLa cells with different concentrations of compound 1 at
pH 7.4. The excitation wavelength was 488 nm and the images were collected at 564 nm
(
first row) and 526 nm (second row). Ratio images obtained from the two above channels
third row) and the corresponding contrast images (fourth row).
(

1
8
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Fig. 4. Confocal microscopy images of sensor 1 (1.25 ꢀM) in HeLa cells clamped at
pH 5 (a, g, m, s), 6 (b, h, n, t), 7 (c, i, o, u), 8 (d, j, p, v), 9 (e, k, q, w) and 10 (f, l, r, x).
The excitation wavelength was 488 nm and the images were collected at 564 nm (first
row) and 526 nm (second row). Ratio images obtained from the two above channels (third
row) and the corresponding contrast images (fourth row).
Scheme 1. Synthesis of the pH sensor 1.
Scheme 2. The proposed ratiometric pH sensing mechanism.

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−
5
Fig. 1. (a) Absorption spectra of 1 in HEPES buffer solution (1.0 × 10 M) of different
pH values determined. (b) The linear relationship for the ratiometric response from
−
5
absorption spectra of 1 to different pH. (c) Photographs of compound 1 (1.0 × 10 M in
HEPES buffer solution) at various pH conditions (5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6,
6
.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0 from left to
−
5
right) in daylight. (d) Emission spectra of 1 in HEPES buffer solution (1.0 × 10 M) of
different pH values determined upon excitation at 470 nm. (e) The linear relationship for
the ratiometric response from emission spectra of 1 to different pH. (f) CIE 1931 (x,y)
chromaticity diagram of the solutions of 1 in different pH conditions derived from
fluorescence spectra (black circles); pH from left to right: 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2,
6
.4, 6.6, 6.8,7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6.

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−
5
Fig. 2. (a) Emission ratio I564 /I525 of compound 1 (HEPES solution, 1.0 × 10 M) in the
same medium determined over consecutive pH cycles. (b) Emission ratio I564 /I525 of
−
5
compound 1 (HEPES solution, 1.0 × 10 M) with irradiation (λ = 470 nm) time change.
ex
−
5
(
c) Emission spectra response of compound 1 (pH 7.4 HEPES solution, 1.0 × 10 M)
upon addition of different species (10 equiv of species relative to 1), the excitation
wavelength was 470 nm. I564 and I525 represent the emission intensity at 564 nm and 525
nm. The species used were Cys, Phe, Ala, Met, Pro, Gly, Hcy, Glu, GSH, H O , Arg, Lys,
2
2
Leu, Tyr, Glc, Trp, Ser, Thr, Asp, Val, and lle. (d) Viability assay for A549 cells treated
with sensor 1 in dark.

2
1
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Fig. 3. Confocal images of HeLa cells with different concentrations of compound 1 at pH
.4. The excitation wavelength was 488 nm and the images were collected at 564 nm
7
(
first row) and 526 nm (second row). Ratio images obtained from the two above channels
third row) and the corresponding contrast images (fourth row).
(

2
2
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Fig. 4. Confocal microscopy images of sensor 1 (1.25 ꢀM) in HeLa cells clamped at pH 5
a, g, m, s), 6 (b, h, n, t), 7 (c, i, o, u), 8 (d, j, p, v), 9 (e, k, q, w) and 10 (f, l, r, x). The
(
excitation wavelength was 488 nm and the images were collected at 564 nm (first row)
and 526 nm (second row). Ratio images obtained from the two above channels (third row)
and the corresponding contrast images (fourth row).

2
3
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Scheme 1. Synthesis of the pH sensor 1.

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Scheme 2. The proposed ratiometric pH sensing mechanism.

ACCEPTED MANUSCRIPT
Highlights
A hemicyanine sensor was synthesized for the ratiometric detection of pH
fluctuation.
►
►
►
The linear reversible ratiometric response ranges from pH 7.0 to 8.8 and 6.0 to 9.0.
pH fluctuation was tracked via fluorescence imaging with low dark toxicity.