A highly sensitive acidic pH fluorescent probe and its application to E. coli cells

Article abstract of DOI:10.1016/j.tet.2016.11.013

A new pH fluorescent probe 3-(N-butylcarbazol-3-dicyanovinyl) pyridine (BILE), which has a carbazole structure attached to pyridine, is synthesized for extremely acidic conditions. BILE displays excellent pH-dependent performance with a pK_aof 2.93 and responds linearly to minor pH changes in the range of 2.60–3.30, which can be used to quantitatively detect pH value based on regression equation, F = 2495.42 × pH – 6316.28. Moreover, BILE has good selectivity, large Stokes shift (122 nm), sensitivity and short response time. More importantly, BILE has been applied successfully to imaging extreme acidity in E. coli cells, indicating that the probe can afford practical application in biological systems.

Full text of DOI:10.1016/j.tet.2016.11.013

Accepted Manuscript
A highly sensitive acidic pH fluorescent probe and its application to E. coli cells
Jianbin Chao, Huijuan Wang, Kailun Song, Zhiqing Li, Yongbin Zhang, Caixia Yin,
Fangjun Huo, Juanjuan Wang, Ting Zhang
PII:
S0040-4020(16)31145-0
10.1016/j.tet.2016.11.013
TET 28227
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 22 August 2016
Revised Date: 24 October 2016
Accepted Date: 2 November 2016
Please cite this article as: Chao J, Wang H, Song K, Li Z, Zhang Y, Yin C, Huo F, Wang J, Zhang T, A
highly sensitive acidic pH fluorescent probe and its application to E. coli cells, Tetrahedron (2016), doi:
10.1016/j.tet.2016.11.013.
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ACCEPTED MANUSCRIPT
A highly sensitive acidic pH fluorescent probe and its
application to E. coli cells
Jianbin Chao*a, Huijuan Wanga,b, Kailun Songa,b, Zhiqing Lia ,b, Yongbin Zhangc ,
Caixia Yind, Fangjun Huoc, Juanjuan Wanga, Ting Zhanga.

ACCEPTED MANUSCRIPT
A highly sensitive acidic pH fluorescent probe and its
application to E. coli cells
Jianbin Chao*a, Huijuan Wanga,b, Kailun Songa,b, Zhiqing Lia ,b , Yongbin Zhangc ,
Caixia Yind, Fangjun Huoc, Juanjuan Wanga, Ting Zhanga.
aScientific Instrument Center, Shanxi University, Taiyuan 030006, P.R. China
bSchool of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China
cResearch Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
dInstitute of Molecular Science, Shanxi University, Taiyuan 030006, China
*Corresponding author.
Jianbin Chao
Scientific Instrument Center,
Shanxi University,
Taiyuan 030006,
P. R. China
Tel.: ＋86-351-721 2621
Fax: ＋86-351-721 2621.
E-mail address: chao@sxu.edu.cn (Jianbin-Chao)
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Abstract：A new pH fluorescent probe 3-(N-butylcarbazol-3-dicyanovinyl) pyridine (BILE),
which has a carbazole structure attached to pyridine, is synthesized for extremely acidic conditions.
BILE displays excellent pH-dependent performance with a pKa of 2.93 and responds linearly to
minor pH changes in the range of 2.60 -3.30, which can be used to quantitatively detect pH value
based on regression equation, F = 2495.42×pH – 6316.28. Moreover, BILE has good selectivity,
large Stokes shift (122 nm), sensitivity and short response time. More importantly, BILE has been
applied successfully to imaging extreme acidity in E. coli cells, indicating that the probe can
afford practical application in biological systems.
Keywords: pH fluorescent probe, E. coli cells, Extreme acidity, Bioimaging.
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1. Introduction
The measurement of pH is of great importance since pH is a key parameter in a wide range of
fields, such as food production, chemical process control, environmental analysis, medical
diagnosis and life sciences 1,2. Moreover, in cellular biology, pH homeostasis plays a pivotal role
5
6
9
in cell growth 3,4, proliferation , apoptosis , autophagy 7,8, ion transport , inflammation and
tumor growth 10, multidrug resistance (MDR)
and other cellular processes. As an important
11,12
indication of cellular health, minor variations of intracellular pH may cause cellular dysfunction
15
and serious diseases, such as colorectal cancer 13, breast cancer 14, cystic fibrosis
,
neurodegenerative disorders 16 and even Alzheimer's disease 17-19. Hence, sensing and monitoring
intracellular pH fluctuations is essentially important for understanding physiological and
pathological processes, and exploring cellular metabolisms.
To date, many popular techniques have been used to measure intracellular pH, including
microelectrodes, nuclear magnetic resonance (NMR), and molecular spectroscopy 20-22. Among
these techniques, fluorescence spectroscopy has attracted much more attention due to their high
selectivity and sensitivity, good spatiotemporal resolution, convenient operation and possibility of
real time monitoring 23-28
Recently, fluorescent molecular probes have shown good potential in a wide range of
applications that require sensitive election of biological analytes in living biological systems 29,30
There are different kinds of fluorescent probes with various skeleton structure, including
.
.
31
33
34
rhodamine , BODIPY 32, fluorescein , tricarbocyanine , benzo[a]phenoxazine 35,36, coumarin
37，carbazole 38,39 etc. Some special probes with pH sensitivity have been designed and synthesized
by changing functional groups to these skeletons. In general, there are two types of synthetic pH
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probes, one type for cytosol that work at a pH of 6.80–7.40 40,41, and another type for the acidic
organelles (for example, lysosomes) functioning in the pH range 4.50 to 6.00 42. Unfortunately,
little research is reported on the development of extreme-acidity pH probes (pH < 4.00). Despite
the fact that most of the living species could hardly live in highly acidic conditions, a great
number of microorganisms such as acidophiles, enteric pathogens, and Helicobacter pylori
particularly favour harsh environments 25,43. Thus, there is tremendous interests in developing
efficient pH probes for accessing the intracellular pH value under extremely acid.
Carbazole-based materials are attractive because they benefit from the advantageous
properties of the chemically stable carbazole units that can be highly emissive, electron
conducting, with good hole transporting properties and easily modified by organic synthesis. At
the same time, carbazole and its derivatives can be used as a strong electron-donating
chromophore 44,45. Hence, many studies are frequently adopted as the fluorophore to construct ICT
fluorescent probes displaying large Stokes shift 46,38. In this paper, we report a new pH-sensitive
fluorescent probe (BILE) based on the carbazole platform, which shows high sensitivity and
excellent stability for intracellular pH measurement. The pH titration indicates BILE exhibits
remarkable pH-dependent behavior in fluorescence emission and displays a notably large Stokes
shift (122 nm) under extremely acidic condition. The pH sensitive mechanism is investigated by
NMR spectroscopic techniques. Moreover, laser scanning confocal microscopy of E. coli cells
demonstrate that the probe could image acid pH values fluctuations in biological systems.
2. Experimental section
2.1. Materials and instruments
BILE was synthesized in our own laboratory. All other reagents and solvents were purchased
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from Sinopharm Chemical Reagent Beijing Co., Ltd. Twice-distilled water was used throughout
all experiments. Unless otherwise stated, all chemicals and solvents were of analytical grade and
used directly. E. coli cells were obtained from institute of cell biology in Shanxi University.
Absorption and fluorescence spectra were measured using a UV-2450 spectrophotometer
1
(Shimadzu, Japan) and a F-7000 fluorescence spectrophotometer (Hitachi, Japan). H NMR
spectra and 13C NMR (in DMSO-d6, TMS as an internal standard) were recorded on a Bruker 600
NMR spectrometer (AVANCE ׀׀׀ HD). Mass spectra were acquired with a Thermo Scientific Q
Exactive LC-MS/MS system. Fluorescent images were taken on a ZEISS LSM 880 confocal laser
scanning microscope with an objective lens (×10). pH values were recorded on a pH meter
(Mettler Toledo, Switzerland).
2.2. Synthesis and characterization of fluorescent probe
2.2.1. Synthesis of compounds N-butyl-carbazole (2) and 3-formyl-N-butylcarbazole (3)
Compounds 2 and 3 were synthesized according to a previous report 44. Experiment details
were described in the Supporting information section.
2.2.2. Synthesis of 3-(N-butylcarbazol-3-dicyanovinyl) pyridine (BILE)
3-Formyl-N-butylcarbazole (0.75 g, 3.0 mmol), 3-pyridinylacetonitrile (0.32 mL, 3.0 mmol),
and t-BuOK (0.5 g, 4.5 mmol) in dry CH3OH (20 mL) were stirred at 50 °C under an nitrogen
atmosphere. After completion of the reaction, the precipitate was collected by filtration and
washed with methyl alcohol, then dried under vacuum to afford a light yellow solid (BILE) in
54.3 % yield (0.57 g). The synthetic route of BILE was depicted in Scheme 1. 1HNMR (600 MHz,
DMSO-d6, δ/ppm) δ 9.00 (m,1H), 8.77 (s, 1H), 8.62 (d, J = 4.7 Hz, 1H), 8.29 (s, 1H), 8.20 (d, J =
8.6 Hz, 1H), 8.15 (m, 2H), 7.81 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.54 (m, 2H), 7.30
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(m, 1H), 4,46 (t, J = 7.0 Hz, 2H), 1,79 (m, 2H), 1.32 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H) (Figure S2).
13C NMR (300 MHz, DMSO-d6, δ/ppm): δ 149.76, 146.95, 146.06, 141.93, 141.15, 133.32,
131.09, 127.25, 127.09, 124.83, 124.41, 123.27, 122.72, 122.40, 120.76, 120.36, 118.83, 110.53,
110.46, 103.27, 42.79, 31.17, 20.23, 14.18 (Figure S3). HRMS calculate for C24H21N3 [M + H]+:
352.17355, found: 352.18096 (Figure S1).
CHO
DMF, POCl3
1)KOH/DMSO
2)
CH3CH2CH2CH2Br
N
N
H
N
CH2CH2CH2CH3
CH2CH2CH2CH3
2
1
3
N
NC
N
CN
CH3OH, t-BuOK
rt, 5h
N
CH2CH2CH2CH3
BILE
Scheme. 1 General routes for synthesis of BILE
2.3. General procedure for spectroscopic measurements
Both the fluorescence and absorption spectral experiments were conducted in water. The
fluorescence emission spectra were recorded at λex = 380 nm and the excitation and emission slit
widths were both set at 5 nm. A 1.0 mM BILE stock solution was prepared by dissolving in
DMSO and the solution for spectroscopic determination was obtained by diluting the stock
solution to 10.0 µM in water medium. The metal ions were provided by KCl, NaCl, CaCl2,
MgCl2·6H2O, FeCl3·6H2O, Zn(NO3)2·7H2O, CuCl2·2H2O, PbCl2, MnCl2·4H2O, HgCl2,
CoCl2·6H2O, NiCl2·6H2O, CrCl3·6H2O, AlCl3·6H2O, FeCl2·4H2O. In the pH titrations
experiments, 2.0 mL of solution was poured into a quartz optical cell of 1 cm optical path length,
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and slight pH variations of the solutions were achieved by adding the minimum volumes of HCl
(0.1, 1.0 M). The resulting solutions were shaken well at room temperature before recording its
absorption and emission spectra.
2.4. E. coli cell culture and imaging
According to the method reported in literature 47, E. coli cells were incubated at 37 ℃ in
Luria-Bertani (LB) culture medium (yeast extract 5 g/L, Trptone 10 g/L, NaCl 10 g/ L) for 17 h in
a table concentrator at 180 rpm. Then the culture was centrifuged at 5000 rpm for 5 min to collect
E. coli cells. The sediment was washed with sterile water and then resuspended with hydrochloric
acid at different pH (7.20, 4.00, 3.50, 3.00, 2.50, 2.00, 1.50), respectively. Five minutes after
resuspension, BILE was added into every tube to make the final probe concentration to be 30.0
µM. E. coli cells with BILE were incubated in a table concentrator as mentioned above for 2 h,
then smeared on slides and observed by a ZEISS LSM 880 confocal laser scanning microscope at
the wavelength of 405 nm.
3. Results and discussion
3.1. Effect of pH on UV-Vis spectra
As shown in Fig. 1, the probe BILE exhibited two obvious absorption bands at 298 nm (ε1=
45270 L·mol−1·cm−1 nm, pH = 7.20) and 391 nm (ε2 = 46540 L·mol−1·cm−1, pH = 7.20) and the
absorption peaks of BILE significantly diminished with the decrease of pH values from 7.20 to
1.10. Simultaneously, a red-shift of the absorption from 391 nm to 420 nm was observed upon
increasing the acidity of the solution and the colors of the solutions changed from yellow to
colorless. The reason why the intramolecular charge transfer (ICT) effect of BILE enhanced with
decreasing pH was that the H+ binding induced enhancement of the electron-withdrawing ability
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of pyridine.
Fig.1 Absorption properties of BILE (10.0 µM) in aqueous solutions at different pH values. Inset:
The color of the solution changed from yellow to colorless with decreasing pH.
3.2. Effect of pH on fluorescence spectra
The fluorescence pH titration of probe BILE was shown in Fig. 2. At pH 7.20, the solution of
probe BILE showed a remarkable fluorescence emission at 502 nm with a large Stokes shift of
122 nm, which were due to the extended π -conjugation. The Stokes shift had potential imaging
and sensing applications because such probes could eliminate self-quenching prevalent in
48
high-quantum-yield fluorophores . In addition, the fluorescence intensity gradually quenched at
lower pH due to the protonation of N atom in pyridine (as shown in scheme. 2). It was interesting
that the solution undergone distinct color change from green to colourless under ultraviolet light
(inset of Fig. 2). Meanwhile, a pKa value of 2.93 was obtained using a sigmoidal plot of the
fluorescence intensity at 502 nm with pH values (Fig. 3). The fluorescence intensity was linearly
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proportional (R2 = 0.9926) to pH values in the range of 2.60 - 3.30 (inset of Fig. 3) and the
regression equation was F = 2495.42×pH – 6316.28 indicating BILE could be used to
quantitatively detect pH value.
Fig. 2 Emission properties of BILE (10.0 µM) in aqueous solutions at different pH values. Inset:
The photograph of BILE solution (10.0 µM) with decreasing pH under a UV light. λex = 380 nm.
Slits: 5 nm/5 nm. Voltage = 600 V.
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Fig. 3 Sigmoidal fitting of the pH-dependent fluorescence intensity at 502 nm. Inset: The linearity
in the pH range of 2.60 -3.30.
3.3. The switching mechanism
BILE and its protonated form of HCl were prepared to study the proton-binding behavior by
1H NMR spectroscopy. Three nitrogen atoms had possibility to be protonated. As shown in Fig. 4,
when hydrochloric acid was added to BILE solution in DMSO-d6, a downfield shift was observed
for the chemical shift values of the pyridine protons (H13, H14, H15, and H16). However, the
chemical shifts of ethylhydrogen and butylcarbazole protons had almost no change, which
indicated that the protonation occurred at the pyridine N. The downfield chemical shift of these
protons was obviously due to H+ binding with N atom of pyridine, which resulted in the decrease
of electron density around these protons. The presence of acid-base equilibrium between the two
forms of BILE was given in Scheme. 2.
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Fig. 4 1H NMR spectra of BILE in DMSO-d6 before (A) and after (B) the addition of hydrochloric
acid (Portion 1H NMR spectra).
Scheme. 2. pH-dependent equilibrium of BILE
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3.4. Theoretical calculations
To provide an insight into the optical properties of BILE, time-dependent density functional
theory (TDDFT) calculations were performed with the B3LYP exchange functional employing
6-31+G* basis sets using the Gaussian 09 program. Fig. 6 showed the optimized structures and the
lowest unoccupied and the highest occupied molecular orbital (LUMO and HOMO) plots of BILE
and its protonated forms in ground state.
Protonation of BILE was a competitive process for N1, N2, N3 in an acidic solution (Fig. 5).
TDDFT calculations suggested the relative energy of N1, N2 and N3 with H+ were 0, 1.19 and 1.08
eV, respectively. The results showed that the structure of N1 with H was more stable, and the N1
atom should be protonated preferentially, which was in accordance with experiments. In addition,
the energy gap between the HOMO and LUMO of BILE+H+ was significantly smaller than that
of probe BILE (Fig. 6), which was in good agreement with the observation that BILE exhibited a
red shift at lower pH values (Fig. 1)
Fig. 5 The optimized structures of BILE.
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Fig. 6 Calculated HOMO and LUMO distributions of BILE and their protonated products.
3.5. Test for probe selectivity
On account of the complexity of the intracellular environment, we performed an additional
examination to determine whether other ions were potential interferences. It was well known that
amines could bind to many metal cations in solution 49,50
.
The selectivity of BILE to H+ over metal ions at pH 7.20 and pH 2.83 was examined to
determine whether other ions were potential interferents. Fig. 7 illustrated that the presence of
physiologically ubiquitous metal ions (K+, Na+, Ca2+ and Mg2+) over their physiological
concentrations did not give any obvious emission change on pH measurement at the two pH
values. Other metal cations, such as Fe3+, Hg2+, Al3+, Pb2+, Mn2+, Cu2+, Zn2+, Co2+, Cr3+, Ni2+ and
Fe2+ (0.2 mM) also exhibited negligible effects on the activity of probe. All these results suggested
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that BILE showed an excellent selectivity response to H+.
Fig. 7 Fluorescence responses (502 nm) of BILE (10.0 µM) in aqueous solutions at pH 2.83 and
7.20 in the presence of diverse metal ions. 1. blank; 2. K+ (150 mM); 3. Na+ (150 mM); 4. Ca2+
(10 mM); 5. Zn2+ (0.2 mM); 6. Mg2+ (0.2 mM); 7. Fe2+ (0.2 mM); 8. Al3+ (0.2 mM); 9. Pb2+ (0.2
mM); 10. Mn2+ (0.2 mM); 11. Co2+ (0.2 mM); 12. Cr3+ (0.2 mM); 13. Hg2+ (0.2 mM); 14. Fe3+
(0.2 mM); 15. Ni2+ (0.2 mM); 16. Cu2+ (0.2 mM); λex = 380 nm. Slits: 5 nm/5 nm. Voltage = 600
V.
3.6. Kinetic assays
Response time was also very important parameters of a fluorescence probe. The stability of
the probe was tested by measuring the fluorescent response during 2 h. Fig. 8 showed the time
courses of fluorescence intensity of the probe (10.0 µM) under pH 7.20, 2.93 and 1.50 at room
temperature. The fluorescence intensity was measured at λex/λem = 380/502 nm. The experiment
indicated that the probe could instantly respond to the change of H+ concentration, and the probe
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solution was stable to the medium, light and air.
Fig. 8 Changes in the fluorescence intensity at 502 nm for BILE (10.0 µM) in aqueous solutions
with times at pH 7.20, 2.93, 1.50, respectively.
3.7. Fluorescent confocal microscopy image
Encouraged by the unique optical properties of BILE, especially the sensitive response to
extreme acidity, the usefulness for biological application was investigated, focusing on living cell
pH imaging. We used E. coli cells as a model to study the fluorescence change in the periplasmic
space of E. coli cells incubated with BILE (30.0 µM) under different pH conditions (pH = 1.50,
2.00, 2.50, 3.00, 3.50, 4.00, 7.20) using confocal laser scanning microscope. The green
fluorescence (410-513 nm) was captured by 405 nm laser. As predicted, at pH 4.0 or higher pH,
the E. coli cells displayed bright green fluorescence (Fig. 9a, c), whereas the green fluorescence
faded away (Fig. 9 e, g, i, k, m) as pH decreased to 1.50. Moreover, the curve of fluorescence
intensity (I) versus pH was obtained in the pH range from 1.50 to 7.20 (Fig. 9 o), and a small pH
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fluctuation surrounding E. coli cells under the extremely acidic condition (pH < 4.0) could give
rise to a significant change of fluorescence intensity, which accorded well with the fluorescence
behavior of probe BILE in Fig. 3. These results indicated the probe was able to imaging very low
pH condition in biological system and we believed that it could work well in other real biological
systems with highly acidic conditions.
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Fig. 9 Confocal microscopy images of 30.0 µM BILE in E. coli cells clamped at pH 7.20 (a and
b), 4.00 (c and d), 3.50 (e and f), 3.00 (g and h), 2.50 (i and j) , 2.00 (k and l), 1.50 (m and n). (a, c,
e, g, i, k, m) Images in the green channel; (b) the bright-field image of panel a; (d) the bright-field
image of panel c; (f) the bright-field image of panel e; (h) the bright-field image of panel g; (j)
the bright-field image of panel i; (l) the bright-field image of panel k; (n) the bright-field image of
panel m; (o) Pseudocolor image of Igreen and corresponding pH values. the excitation wavelength
was 405 nm, and the green channel images were collected at 410–513 nm.
4. Conclusion
In summary, a new pH fluorescent probe (BILE) based on carbazole derivatives is developed
to detect pH value. With a pK a of 2.93, the fluorescence intensity of the probe BILE displays
excellent pH-dependent performance and responds linearly to minor pH changes in the range of
2.60-3.30. This probe BILE has good selectivity, large Stokes shift (122 nm), sensitivity and
extremely short response time. Also, it can resist interfering ions greatly so we can detect pH
value in complicated environment. More importantly, application of BILE to pH imaging in E.
coli cells is achieved successfully, indicating that the probe can be used to visualize intracellular
pH fluctuations in biological system. Furthermore, the detection mechanism has been verified by
1H NMR spectroscopy. All in all, BILE has good ability in detecting low pH conditions in
solutions and we believe it will be great beneficial to study chemical and biological system.
Acknowlegements
The work was supported by the National Natural Science Foundation of China (No.
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21472118), the Shanxi Province Science Foundation for Youths (Nos. 2012021009-4 and
2013011011-1), the Shanxi Province Foundation for Returnee (No. 2012-007), the Taiyuan
Technology Star Special (No. 12024703), the Program for the Top Young and Middle-aged
Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, No. 2013802), Talents
Support Program of Shanxi Province (No. 2014401) and Shanxi Province Outstanding Youth
Fund (No. 2014021002), the Scientific and Technological Innovation Programs of Higher
Education Institutions in Shanxi (20111001), and the Shanxi Province Foundation for College
Students of Science and Technology Innovation projects (No. 2013328)
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Highlights
1. A highly sensitive acidic pH fluorescent probe (BLIE) is facilely synthesized via
the ethylene bridging of 3-formyl-N-butylcarbazole and 3-pyridinylacetonitrile.
2. With a pK
a of 2.93, the fluorescence intensity of BLIE displays excellent
pH-dependent performance and responds linearly to minor pH changes in the range of
2.60-3.30.
3. BLIE has good selectivity, large Stokes shift, sensitivity and extremely short
response time.
4. BILE has been applied successfully to imaging extreme acidity in E. coli cells,
indicating that the probe can afford practical application in biological systems.