A novel acidic pH fluorescent probe based on a benzothiazole derivative

Article abstract of DOI:10.1016/j.saa.2017.01.024

A novel acidic pH fluorescent probe 1 based on a benzothiazole derivative has been designed, synthesized and developed. The linear response range covers the acidic pH range from 3.44 to 6.46, which is valuable for pH researches in acidic environment. The evaluated pKa value of the probe 1 is 4.23. The fluorescence enhancement of the studied probe 1 with an increase in hydrogen ions concentration is based on the hindering of enhanced photo-induced electron transfer (PET) process. Moreover, the pH sensor possesses a highly selective response to H⁺ in the presence of metal ions, anions and other bioactive small molecules which would be interfere with its fluorescent pH response. Furthermore, the probe 1 responds to acidic pH with short response time that was less than 1 min. The probe 1 has been successfully applied to confocal fluorescence imaging in live HeLa cells and can selectively stain lysosomes. All of such good properties prove it can be used to monitoring pH fluctuations in acidic environment with high sensitivity, pH dependence and short response time.

Full text of DOI:10.1016/j.saa.2017.01.024

Accepted Manuscript
A novel acidic pH fluorescent probe based on a benzothiazole
derivative
Qiujuan Ma, Xian Li, Suxiang Feng, Beibei Liang, Tiqiang Zhou,
Min Xu, Zhuoyi Ma
PII:
DOI:
Reference:
S1386-1425(17)30034-3
doi: 10.1016/j.saa.2017.01.024
SAA 14877
Spectrochimica Acta Part A: Molecular and Biomolecular
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11 January 2017
11 January 2017
Please cite this article as: Qiujuan Ma, Xian Li, Suxiang Feng, Beibei Liang, Tiqiang
Zhou, Min Xu, Zhuoyi Ma , A novel acidic pH fluorescent probe based on a benzothiazole
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ACCEPTED MANUSCRIPT
A novel acidic pH fluorescent probe based on a benzothiazole derivative
Qiujuan Maa, Xian Lia, Suxiang Fenga,b,*, Beibei Lianga, Tiqiang Zhoub, Min Xua, Zhuoyi Maa
a School of Pharmacology, Henan University of Chinese Medicine, Zhengzhou 450046, PR
China
b Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment＆
Chinese Medicine Development of Henan Province, Zhengzhou 450046, PR China
*Corresponding author, E-mail: maqiujuan104@126.com; fengsx221@163.com. Tel:
+86-371-65676656; Fax: +86-371-65680028.
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Abstract
A novel acidic pH fluorescent probe 1 based on a benzothiazole derivative has been
designed, synthesized and developed. The linear response range covers the acidic pH range
from 3.44 to 6.46, which is valuable for pH researches in acidic environment. The evaluated
pKa value of the probe 1 is 4.23. The fluorescence enhancement of the studied probe 1 with
an increase in hydrogen ions concentration is based on the hindering of enhanced
photo-induced electron transfer (PET) process. Moreover, the pH sensor possesses a highly
selective response to H+ in the presence of metal ions, anions and other bioactive small
molecules which would be interfere with its fluorescent pH response. Furthermore, the probe
1 responds to acidic pH with short response time that was less than 1 min. The probe 1 has
been successfully applied to confocal fluorescence imaging in live HeLa cells and can
selectively stain lysosomes. All of such good properties prove it can be used to monitoring pH
fluctuations in acidic environment with high sensitivity, pH dependence and short response
time.
Keywords: acidic pH fluorescent probe; benzothiazole derivative; enhanced PET process; cell
imaging
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1. Introduction
The determination of pH has a great interest in different areas due to comprehensive
applications in numbers of processes, such as electrochemical sensing [1], biochemical and
medical analyses [2,3], environmental monitoring [4], isomer analysis [5], clinical analysis
[6], control and monitoring of biochemical reactions [7]. In cellular biology, pH plays pivotal
role in many biological functions such as cellular proliferation and apoptosis [8], enzymatic
activity [9,10], ion transport [11] and muscle contraction [12]. Abnormal intracellular pH
values result in cellular dysfunction and cause some serious diseases such as cancer [13] and
Alzheimer’s disease [14]. Therefore, monitoring intracellular pH changes is very critical for
the diagnosis of certain diseases and the measurement of therapeutic efficiencies.
So far, there are many approaches including electrochemistry [1], nuclear magnetic
resonance (NMR) [15], absorbance spectroscopy [16] and fluorescence spectroscopy [17] for
measuring the intracellular pH. Among these methods, fluorescence detection has attracted
much attention due to its noninvasiveness, particularly high sensitivity, convenient operation,
real-time detection and excellent specificity. As known to all, a cell can be divided into two
parts: neutral cytosol (pH: 6.8-7.4) and acidic compartments (pH: 4.5-.6.0) [18]. Consequently,
two kinds of pH fluorescent probes for the cell have been developed. One category is probes
for cytosol which work at a pH of about 6.8-7.4 [19-23]. The other one is probes for weak
acidic organelles such as lysomes and endosomes which function in the pH range of about
4.5-6.0 [24-27]. While a wide variety of fluorescent probes for the neutral pH range have been
developed, there are only few acidic fluorescent probes, especially desirable ones for studying
acidic organelles. Thus, the development of a new acidic pH fluorescent probe with high
sensitivity and selectivity, short response time and membrane permeability is still in demand.
In the past decades, benzothiazole and its derivatives have gained increasing interest as
fluorescent probes for pH [25,28-32], metal cations [33-36], anions [37-39] and small
molecules [40-43] due to their excellent photophysical properties. So far, the design of
fluorescent pH probe based on benzothiazole derivatives is mostly based on excited-state
intramolecular proton transfer (ESIPT) [25] and intramolecular charge transfer (ICT)
[28,30,32]. In order to further investigate the essential roles of acidity in cellular condition,
there is a great need to develop other strategies for designing pH fluorescent probes based on
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benzothiazole derivatives. Compared with the traditional single-atom electron donor like O, N,
Se or Te for photoinduced electron transfer (PET) process, the more electron-rich groups can
more effectively induce an “enhanced photoinduced electron transfer (PET)” process to a
fluorophore and “switch off” its fluorescence [44]. The “enhanced PET” effect which gives
lower background fluorescence and higher signal-to-noise ratio has been utilized to design the
fluorescent probes for HClO [44-46] and reactive oxygen species [47]. Therefore, the anilino
group as an electron-rich group was introduced to our target probe 1 (Scheme 1) to efficiently
quench the fluorescence of the benzothiazole fluorophore through an enhanced PET process.
The protonation of nitrogen atom of the anilino group in acidic medium results in disruption
of the enhanced PET process, and thus the restoration of the emission from the excited
fluorophore takes place.
In this article, we reported a new acidic pH fluorescent probe 1 based on enhanced PET
process. This probe 1 with pKa 4.23 can sense acid pH ranging from 3.44 to 6.46 with
excellent linear relationship, which was especially suited for quantitative determination of pH
in this acidic pH window. This probe 1 responded to acidic pH with quick response time (less
than 1 min), high selectivity and high sensitivity. Furthermore, this probe 1 has been
successfully applied to fluorescence microscopic imaging in HeLa cells and displayed low
cytotoxicity and excellent membrane permeability.
2. Experimental
2.1. Reagents and chemicals
4-(Diethylamino)salicylaldehyde and 2-aminothiophenol were purchased from Heowns
Biochemical Technology Company. 4-Chloronitrobenzene was obtained from Aladdin
Reagent Company. Anhydrous potassium carbonate, stannous chloride dihydrate and all other
reagents were of analytical reagent grade, purchased from Sinopharm Chemical Reagent
Company and used without further purification. Thin layer chromatography (TLC) was
performed on silica gel 60 F254 visualized by UV, column chromatography was conducted
over silica gel (200- 300 mesh), both of which were obtained from the Qingdao Ocean
Chemicals (Qingdao, China). Water purified by a Milli-Q system (EMD Millipore, Darmstdt,
Germany) was used for the preparation of all aqueous solutions. LysoTracker Red DND-99
was purchased from the Shanghai Qianchen Biotechnology Company.
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2.2. Apparatus
The 1H NMR and 13C NMR spectra were recorded by employing a Bruker DRX-500
spectrometer with chemical shifts expressed in parts per million and using TMS as an internal
standard. Electrospray ionization (ESI) mass spectrometry was obtained with Bruker maXis
HD mass spectrometer (Bruker, Germany). All fluorescence measurements were determined
on a Hitachi fluorescence spectrophotometer F-7000 (Tokyo, Japan). UV-vis absorption
spectra were acquired on a UV-2600 spectrophotometer using a 1 cm quartz cell (Tokyo,
Japan). Fluorescence images of living cells were obtained using an Olympus FV1200 laser
scanning confocal microscope (Japan). The pH measurements were performed on a
Mettler-Toledo Delta 320 pH meter which was calibrated with pH 4.00, pH 6.86 and pH 9.18
buffers before use. Data processing was performed on a Pentium Ⅳ computer with software
of SigmaPlot. The fluorescence intensity was measured at excitation wavelength of 360 nm
with both excitation and emission slit set at 2.5 nm.
2.3. Syntheses
The synthetic route for fluorescence probe 1 is shown in Scheme 1.
Insertion of Scheme 1
Synthesis of compound 3
The synthesis of compound 3 was accordance with the previous literature [43]. 1H NMR
(500 MHz, CDCl3), δ(ppm): 12.53 (1H, s), 7.84 (1H, d, J = 8.1 Hz), 7.79 (1H, d, J = 7.9 Hz),
7.45 (1H, d, J = 8.5 Hz), 7.41 (1H, t, J = 7.4 Hz), 7.28 (1H, t, J = 7.5 Hz), 6.27 (2H, s), 3.39
13
(4H, q, J = 7.1 Hz), 1.20 (6H, t, J = 7.1 Hz). C NMR (125 MHz, CDCl3), δ(ppm): 169.55,
159.78, 152.21, 151.30, 131.89, 129.82, 126.21, 124.14, 121.18, 120.99, 105.77, 104.13,
97.88, 44.54, 12.66. MS (ESI) m/z: 299.1212 (M+H)+.
Synthesis of compound 2
Compound 3 1.49 g (5 mmol) and anhydrous potassium carbonate 3.46 g (25 mmol) were
dissolved in 100 mL N, N-dimethylformamide (DMF). The above mixture was stirred at room
temperature for 30 min followed by the addition of 4-chloronitrobenzene 0.79 g (5 mmol),
and the reaction mixture was stirred at 80 ℃ for 9 h. The solvent was evaporated in vacuo,
and the crude product was purified by column chromatography on silica gel (petroleum ether/
ethyl acetate = 8:1, v/v) to give compound 2 as an orange solid (0.95 g, yield 45 %). 1H NMR
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(500 MHz, CDCl3), δ(ppm): 8.42 (1H, d, J = 7.9 Hz), 8.20 (2H, m), 7.98 (1H, d, J = 7.9 Hz),
7.75 (1H, d, J = 8.0 Hz), 7.40 (1H, m), 7.27 (1H, d, J = 7.4 Hz), 7.12 (2H, dd, J = 2.0 Hz, 7.3
Hz), 6.69 (1H, dd , J = 2.5 Hz, 9.1 Hz), 6.25 (1H, d, J =2.4 Hz), 3.38 (4H, q, J = 7.1 Hz), 1.18
(6H, t, J = 7.1 Hz). 13C NMR (125 MHz, CDCl3), δ(ppm): 162.67, 162.47, 153.72, 152.33,
150.87, 142.83, 135.05, 131.40, 125.99, 125.95, 124.22, 122.15, 121.10, 116.81, 113.10,
109.59, 103.04, 44.66, 12.52. MS (ESI) m/z: 420.1355 (M+H) +.
Synthesis of compound 1
Under nitrogen atmosphere, compound 2 0.46 g（1 mmol）and stannous chloride dihydrate
2.26 g（10 mmol）were dissolved in the mixture of 30 mL anhydrous ethanol and 30mL
concentrated hydrochloric acid, and the above mixture was left stirring violently at 60 ºC for 5
h. Then the anhydrous ethanol was removed, and the pH adjusted to 9.0 with 6 mol/L NaOH.
This mixture was then filtered and the filtrate was extracted by ethyl acetate (50 mL × 3).
Organic layer was collected, washed with saturated brine (50 mL × 3) and then dried by
anhydrous Na2SO4. Then the solvent was removed under reduced pressure. The crude product
was purified by column chromatography on silica gel chromatography (petroleum ether/ethyl
1
acetate = 6:1, v/v) to compound 1 as a white solid (0.30 g, yield 77 %). H NMR (500 MHz,
DMSO-d6), δ(ppm): 8.24 (1H, d, J = 9.1 Hz), 7.97 (1H, d, J = 7.7 Hz), 7.91 (1H, d, J = 8.0
Hz), 7.44 (1H, m), 7.30 (1H, m), 6.86 (2H, dd, J = 2.1 Hz, 6.7 Hz), 6.61 (2H, dd, J = 2.1 Hz,
6.6 Hz), 6.56(1H, dd, J =2.5 Hz, 9.1 Hz), 5.98 (1H, d, J = 2.5 Hz), 5.01 (2H, s), 3.27 (4H, q, J
= 7.1 Hz), 1.02 (6H, t, J = 7.1 Hz). 13C NMR (125 MHz, DMSO-d6), δ(ppm): 162.73, 158.20,
151.99, 150.33, 145.61, 144.85, 134.65, 129.98, 125.84, 123.75, 121.47, 121.36, 120.50,
114.77, 109.99, 106.48, 98.38, 43.95,12.31. MS (ESI) m/z: 390.1631 (M+H) +.
2.4. Spectrophotometric experiments
The fluorescence measurement experiments were measured in Britton–Robinson (B-R)
buffer solution. The pH of Britton–Robinson buffer solution used was between 3.00 and 8.00,
which was achieved by adding 0.04 mol/L boric acid, 0.04 mol/L phosphoricacid, 0.04 mol/L
acetic acid and 0.20 mol/L sodium hydroxide. The solutions of metal ions for fluorescence
discrimination were prepared respectively by dissolving HgCl2, CuSO4·5H2O, MnCl2·4H2O,
Pb(CH3COO)2·3H2O, ZnSO4·7H2O, CaCl2·2H2O, AgNO3, NaCl, BaSO4, AlCl3, NiCl2·6H2O,
K2CO3, MgSO4, FeCl3·6H2O, Li2CO3, CoSO4·7H2O and CrCl3·6H2O in deionized water with
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final concentrations of 5 × 10-6 mol/L for them. As well as, glutathione (GSH), cysteine (Cys),
homocysteine (Hcy), arginine (Arg), histidine (His) and alanine (Ala) solutions were prepared
respectively using twice-distilled water with final concentrations of 5 × 10-6 mol/L. All tested
samples were prepared at room temperature and rested for 5 min before UV-vis and
fluorescence determination.
2.5. Cytotoxicity assay
The cytotoxic effects of compound 1 were assessed using a tetrazolium-based colorimetric
assay (MTT assay) [48]. HeLa cells were cultured in Dulbecco̕ s Modified Eagle̕ s
medium (DMEM) containing high glucose supplemented with 10% fetal bovine serum, 100
units· mL-1 penicillin and 100 µg · mL-1 streptomycin. Approximately 1×105 cells were seeded
in a 96-well microplate to a total volume of 100 µl/ well. After 24 h of cell attachment the
culture media was removed and washed by Dulbecco̕ s phosphate buffered saline (DPBS).
The cells were then cultured in a medium with different concentrations of compound 1 for 24
h, subsequently the culture media was removed and the cells were washed with the culture
medium. Cells in a culture medium without the fluorescent probe 1 were used as the control.
Fresh medium (100 µL) containing MTT (10 µL, 5 mg · mL-1) was added to each well and
incubated for another 4 h to allow the formation of formazan dye. After removed the
supernatant carefully, 150 µL dimethyl sulphoxide (DMSO) was added to each well to
dissolve the purple products. The plate was shaken for 10 min and the absorbance values (A)
were read at a wavelength of 570 nm by microplate reader (Synergy 2, Bio Tek Instruments
Inc.). Cell viability was calculated by A/A0× 100% (A and A0 are the absorbance of
experimental group and control group, respectively).
2.6. Confocal imaging in living cells
HeLa cells were plated on 35-mm glass-bottomed dishes and cultured in DMEM containing
high glucose supplemented with 10% fetal bovine serum for 24 h. Before the experiments, the
cells were washed with DPBS, followed by treated with the medium containing 5.0 µM probe
1 and 1.0 µM LysoTracker Red DND-99 for 30 min at 37 ℃, then by washing with DPBS
three times and imaged. LysoTracker Red DND-99 (1.0 µM) was used to co-stain the cells to
lysosome. The confocal fluorescence images were observed by an Olympus FV1200 laser
scanning confocal microscope with 40× objective lens. Confocal fluorescence images were
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observed at different detection channels (probe 1 channel, λex = 405 nm; LysoTracker Red
DND-99 channel,λex = 555 nm)
3. Results and discussion
3.1. Spectroscopic analytical performance of probe 1
We studied the fluorescence spectra of probe 1 (5.0 µM) in 0.04 mol/L B-R buffer
(DMF/water=1: 99, v/v) with various pH (Figure 1). From Figure 1, one can see that probe 1
showed almost no fluorescence at pH 6.46. However, fluorescence intensity increased
dramatically when the pH value gone down from 6.46 to 3.44. It displays an approximately
19-fold increase in the emission intensities at around 432 nm within the pH range 6.46-3.44,
which is valuable for studying acidic pH.
Insertion of Figure 1
The time-dependent fluorescence responses of probe 1 (5.0 µM) were also determined at
pH 3.44, 4.96 and 6.46 (Figure S1). As shown in Figure S1, when probe 1 was added into the
solutions with different pH, the fluorescence intensity reached equilibrium in a short time
(less than 1 min). Therefore, the probe 1 can be used to detect pH of samples immediately.
The UV-vis absorption spectra of probe 1 (5.0 µM) at pH 3.44, 4.96 and 6.46 were
recorded in Figure 2. The probe 1 (5.0 µM) in 0.04 mol/L B-R buffer (DMF/water=1: 99, v/v)
with pH 6.46 exhibited weak absorption near 370 nm. At pH 3.44, the absorption band
centered 370 nm increased obviously and did not accompany the shift of the band at 370 nm.
Insertion of Figure 2
3.2. Principle of operation and the basis of quantitative assay
The ability of the probe 1 to recognize proton was investigated at an excitation wavelength
of 360 nm, with results illustrated in Figure 3. As shown in Figure 3, the linear response range
covers the acidic pH range from 3.44 to 6.46. The relationship between the pH value and the
fluorescence intensity at λem = 432 nm can be expressed quantitatively using a
Henderson-Hasselbalch type equation (Equation (1)) [49]:
Imax  I
I  Imin
log[
]  pH  pKa (1)
where I is the observed fluorescence intensity, Imax and Imin represent the maximum and
minimum of the fluorescence intensity of the probe 1 in its acid (pH 3.00) and conjugate base
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(pH 8.00), respectively.
Therefore, the linear response in Figure 3 can be expressed by the following equation (2) of
the calibration line:
Imax  I
I  Imin
log[
]  0.9912pH  4.2311 (R2=0.9853) (2)
where R is the linear correlation coefficient.
According to the equation (2), the calculated pKa value of probe 1 was 4.23.
Insertion of Figure 3
In order to determine the bonding site of proton, we got 1H NMR and 13C NMR spectra of
probe 1 + CF3COOD. The downfield shift of H(3'') and H(5'') 0.32 ppm at pH 3.44 indicated
the protonation of nitrogen atom of the primary anilino group (Figure 4). The downfield shift
of C(3'') and C(5'') 4.86 ppm at pH 3.44 also indicated the protonation of nitrogen atom of the
primary anilino group (Figure 5). The HSQC spectra of probe 1 and probe 1+CF3COOD at
pH 3.44 were shown in electronic supplementary information (ESI). To further confirm the
proton sensing mechanism, we also got the electrospray mass spectrum of probe 1 + HCOOH
in acetonitrile-water (1: 99) at pH 3.44 (Figure 6). A peak at m/z 391.2823 corresponding to
the [1 + H + H]+ was clearly observed, which indicated the formation of a 1/ H+ adduct of 1:1
stoichiometry at pH 3.44 (Figure 6). So we drew a conclusion that nitrogen atom of the
primary anilino group was protonated and the nitrogen atoms of benzothiazole ring and
diethylamino group were not protonated in acidic solution. The protonation process of probe 1
in acidic condition was shown in Scheme 2. Therefore, the primary anilino group as an
electron-rich group efficiently quenched the fluorescence of the benzothiazole fluorophore
through an enhanced PET process, giving a non-fluorescent compound. After protonation of
nitrogen atom of the primary anilino group in acidic solution, the fluorescence enhancement
of probe 1 occurred for the hindering of enhanced PET process.
Insertion of Figure 4
Insertion of Figure 5
Insertion of Figure 6
Insertion of Scheme 2
3.3. Effect of ionic strength
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For a fluorescence pH probe, ionic strength is also regarded as a limiting factor for its
application in real samples. The effect of ionic strength was examined by adjusting the buffer
solution of pH 3.44 and 6.46 to ionic strength from 0.01 mol/L to 2.0 mol/L with sodium
chloride (Figure S2). From Figure S2, it was found that with increasing the ionic strength the
fluorescence intensity remained almost constant. So it was not necessary to maintain a certain
ionic strength in later experiments.
3.4. Selectivity
In complex biological samples, a highly selective response to the target specie over other
potentially competing species is necessary. To explore if probe 1 could still work well in
complex environments, an additional examination was taken to test the selective response of
common ions, anions and amino acids which were regard as potential interference substances.
The relative fluorescence intensity of the probe 1 in the absence or presence of competitive
species was shown in Figure 7. As displayed in Figure 7 there were no metal ions, anions or
bioactive molecules cause significant changes in fluorescence intensity. These results reveal
that probe 1 exhibited high selectivity toward H+ over competitive species, which makes it
feasible for practical purposes.
Insertion of Figure 7
3.5. Cytotoxicity assays and confocal imaging in living cells
In order to explore potential applications in real biological samples. First, the
cytotoxicity of compound 1 was evaluated by the MTT assay (Figure S3). As shown in Figure
S3, about 90% cell viability could be observed after the HeLa cells were treated with
compound 1 for 24 h at concentrations between 0 µM and 16 µM, which indicated that probe
1 had almost no cytotoxicity to HeLa cells. Next, colocalization experiments were performed
in HeLa cells using probe 1 and a commercially available lysosome-specific staining probe
(LysoTracker Red DND-99) to confirm if probe 1 could be a lysosome-target probe. The
HeLa cells were incubated with probe 1 (5.0 µM) and LysoTracker Red DND-99 (1.0 µM) for
30 min at 37 ℃, then the cells were washed with DPBS three times to remove the
extracellular remaining probe 1 and imaged (Figure 8). As shown in Figure 8, the distribution
of blue fluorescence from probe 1 co-localized with red fluorescence from LysoTracker Red
DND-99, which suggest that probe 1 selectively stains lysosomes in living cells.
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Insertion of Figure 8
4. Conclusions
In conclusion, a novel acidic pH fluorescent probe 1 based on a benzothiazole derivative
has been reported. The synthesis of this probe 1 is facile and also it displays high sensitivity
and selectivity, short response time, good membrane permeability and low cytotoxicity. The
linear response range covers the acidic pH range from 3.44 to 6.46, which is applicable to
investigate acidic environment. The evaluated pKa value of the probe 1 is 4.23. The
fluorescence enhancement of the studied probe 1 with an increase in hydrogen ions
concentration is based on the hindering of enhanced PET process. Furthermore, this probe 1
was successfully applied to confocal fluorescence imaging in live HeLa cells and can
selectively stain lysosomes. All in all, this probe 1 has good ability in detecting acidic pH
conditions in solutions and it will be great beneficial to study chemical and biological
systems.
Acknowledgments
This work was supported by Henan Natural Science Foundation of Education Department
(14B150021), Program for Science & Technology Innovation Talents in Henan College of
Chinese Medicine (2013RCXCX01), the Fundamental Research Funds for the Provincial
Universities (2014KYYWF-QN04), Postgraduate Innovation Fund of Henan College of
Chinese Medicine and Program for Youth Backbone Teachers in the Universities of Henan
Province (2013GGJS-091).
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Captions of figures
Scheme 1. Synthesis of fluorescent probe 1: (a) Na2S2O5 aqueous solution, DMF, 90 ℃, 12 h,
82%; (b) 4-chloronitrobenzene, K2CO3, DMF, 80 ℃, 9 h, 45%; (c) SnCl2·2H2O, EtOH-HCl
(1:1, v/v), 60 ℃, 5 h, 77%.
Scheme 2. Proposed protonation process of probe 1 in acidic condition.
Figure 1. Fluorescence spectra of probe 1 (5.0 µM) in 0.04 mol/L Britton-Robinson buffer
(DMF/water=1: 99, v/v) with various pH: (1) 8.00, (2) 7.47; (3) 6.67, (4) 6.46, (5) 6.14, (6)
5.90, (7) 5.70; (8) 5.33, (9) 4.96, (10) 4.80, (11) 4.52, (12) 4.31, (13) 4.10, (14) 3.83, (15) 3.66,
(16) 3.44, (17) 3.20, (18) 3.00. The excitation wavelength is 360 nm. Inset: pH dependence of
the fluorescence emission intensity of probe 1.
Figure 2. UV-vis absorption spectra of probe 1 (5.0 µM) in 0.04 mol/L Britton-Robinson
buffer (DMF/water=1: 99, v/v) with pH 3.44, 4.96 and 6.46.
Figure 3. Plot of log[(Imax-I)/(I-Imin)] as a function of the pH.
Figure 4. 1H NMR spectra of probe 1 (a) and probe 1+CF3COOD at pH 3.44 (b).
Figure 5. 13C NMR spectra of probe 1 (a) and probe 1+CF3COOD at pH 3.44 (b).
Figure 6. Electrospray mass spectrum of probe 1 + HCOOH in acetonitrile-water (1: 99) at
pH 3.44.
Figure 7. Fluorescence intensity of probe 1 (5.0 µM) in the presence of various analytes in
0.04 mol/L Britton-Robinson buffer (DMF/water=1: 99, v/v) with pH 3.44 (a) and 6.46 (b).
(1) blank, (2) Li+, (3) Na+, (4) K+, (5) Mg2+, (6) Ca2+, (7) Al3+, (8) Hg2+, (9) Cu2+, (10) Pb2+,
(11) Zn2+, (12) Ag+, (13) Mn2+, (14) Ba2+, (15) Co2+, (16) Ni2+, (17) Cr3+, (18) Fe3+, (19)
-
3-
-
2-
-
2-
H2PO4 , (20) CH3COO-, (21) PO4 , (22) HCO3 , (23) Cl-, (24) SO4 , (25) NO3 , (26) CO3 ,
(27) GSH, (28) Cys, (29) Hcy, (30) Ala, (31) His, (32) Arg. The excitation wavelength is 360
nm and the emission wavelength is 432 nm. The concentration is 5.0 µM for all the analytes.
Figure 8. Fluorescence microscope images of living HeLa cells co-stained with 5.0 µM probe
1 and 1.0 µM LysoTracker Red DND-99. (a) Blue emission from probe 1; (b) red emission
from LysoTracker Red DND-99; (c) overlay of (a) and (b), areas of co-localization are
visualized in burgundy.
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Figure 4
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Figure 5
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Graphical abstract
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Highlights
1) A novel acidic pH fluorescent probe has been reported.
2) The linear response range covers the acidic pH range from 3.44 to 6.46.
3) The design of fluorescent pH probe is based on enhanced PET process.
4) The probe displays high sensitivity, high selectivity and short response time.
5) The probe has been successfully applied to fluorescence imaging in living cells.
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