A new rhodamine b-based fluorescent probe for ph detection and bioimaging under strong acidic conditions

Article abstract of DOI:10.1002/bkcs.10888

A new pH-sensitive rhodamine derivative was designed and synthesized using coupling reaction of rhodamine hydrazine with commercially available 3-bromo-4-hydroxybenzaldehyde, and characterized via NMR, HRMS, UV and fluorescent spectra. The obtained probe

Full text of DOI:10.1002/bkcs.10888

Article
DOI: 10.1002/bkcs.10888
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Q. Yang et al.
KOREAN CHEMICAL SOCIETY
A New Rhodamine B-based Fluorescent Probe for pH Detection
and Bioimaging under Strong Acidic Conditions
Quan Yang,† Jingrong Zou,†,‡ Sridhar Chirumarry,†,§ Chang Huo,† Linlin Tang,†,‡ Fang Zhang,†,‡
†,
Yi Xiang,† Hua Zuo,†,‡ Dong-Soo Shin,†,§, and Xiang Peng
*
*
†Department of Cardiology, The Fourth People’s Hospital of Sichuan Province, Chengdu 610-016,
China. *E-mail: dsshin@changwon.ac.kr; 1070715337@qq.com
‡College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
§Department of Chemistry, Changwon National University, Changwon 641-773, South Korea
Received April 4, 2016, Accepted July 7, 2016, Published online September 7, 2016
A new pH-sensitive rhodamine derivative was designed and synthesized using coupling reaction of rhoda-
mine hydrazine with commercially available 3-bromo-4-hydroxybenzaldehyde, and characterized via
NMR, HRMS, UV and fluorescent spectra. The obtained probe was marked with yellow fluorescence under
neutral condition and pink in strongly acidic media. It has high quantum yields, is not susceptible to metal
interference, and has high penetration ability for cell membrane and also further applicable in bioimaging.
Keywords: pH-sensitive probe, Rhodamine B derivative, Fluorescence, Bioimaging, Acidic condition
Introduction
It remains a challenge to develop fluorescent sensors that
respond to extreme acidity because of the weak fluores-
cence and chemical instability of fluorescein or coumarin
under strongly acidic conditions.23 Nevertheless, based on
the changes in spectroscopic phenomena, such as absorp-
tion and fluorescence due to the reversible changes in the
indicator’s spirocyclic structure induced by pH, rhodamine
B derivatives serve as excellent “off-on” fluorescent
probes.24–26 Given that the fascinating structure and unpar-
alleled photophysical properties, such as high quantum
yields, large Stokes shifts, large extinction coefficients, and
great photostability,18 some lower pH-sensitive rhodamine
derivatives were reported.27–29
Herein, we synthesized a novel pH-sensitive rhodamine
derivative using rhodamine hydrazine and commercially
available 3-bromo-4-hydroxybenzaldehyde. A conjugated
substituted styryl moiety was introduced to rhodamine core
and the obtained probe exhibits strong yellow and pink
fluorescence under neutral and acidic conditions, respec-
tively. In comparison to the reported results related to
highly acidic sensors,30 the new probe has higher quantum
yields, is less susceptible to metal interference, and has
high penetration ability for cell membrane.
Monitoring pH change is an essential technology required
in chemical, biological and industrial fields, such as chemi-
cal and biological analyses,1,2 environmental protection,1,3
and chemical process control.4 Although, the potentiometric
method using a glass electrode is most widely used for pH
determination, the glass pH electrode suffers from limita-
tions including the requirement for a reference electrode,3
frequent calibration, the susceptibility to electrical
interference,4 instability, possible damage and being too
bulky for many in vivo use.5 In recent years, the optical
pH-sensing technique based on the absorption or emission
of certain dyes has gained increasing attention because it
offers many advantages over the potentiometric method
and nuclear magnetic resonance (NMR), such as rapid
response time, real time sensing,3,6 electrical safety,1 a wide
selection of available indicator dyes,7–9 particularly high
selectivity and sensitivity. New fluorescent pH chemosen-
sors are also continually developed and commercially
available, such as small molecules derived from
aminophthalimide,4
cyanine,10
BODIPY
dyes,11,12
benzoxanthenes,13 fluorescein,14 and so on,15–17 but most
of them are associated with biotechnological applications
and thus concerns on pH sensors gained attention for the
physiological range.14,18–20 Relatively less attention has
been paid to fluorescent probes, which are promising candi-
dates for pH detection in the lower pH region (pH < 4).3 In
fact, some media such as those found in the human stom-
ach21,22 or environmental water3 are strongly acidic and
certain microorganisms, including Helicobacter pylori and
“acidophiles” favor such harsh conditions, therefore it is
necessary to develop chemosensors for indicating pH levels
of these highly acidic media.
Experimental
Chemicals and Apparatus. Deionized water was used
throughout the experiment. All the reagents were obtained
from commercial sources and used without further purifica-
tion. The metal salts used were Al(NO3)3ꢀ9H2O, Ba(NO3)2,
Ca(NO3)2ꢀ4H2O, Co(NO3)2ꢀ6H2O, CsNO3, Cu(NO3)2ꢀ
3H2O, Fe(NO3)3ꢀ9H2O, Hg(NO3)2, KNO3, LiNO3,
Mg(NO3)2ꢀ6H2O, Mn(NO3)2, NaNO3, Ni(NO3)2ꢀ6H2O and
Zn(NO3)2ꢀ6H2O. Britton–Robinson (B–R) buffer was
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prepared by mixing 40 mM acetic acid, 40 mM boric acid,
and 40 mM phosphoric acid. Dilute hydrochloric acid or
sodium hydroxide was used for tuning pH values. Melting
point (uncorrected) was determined on a micromelting
point apparatus (Shanghai Shenguang Instrument Co. Ltd.,
2-((3-Bromo-4-hydroxybenzylidene)amino)-30,60-bis
(diethylamino)spiro[isoindoline-1,90-xanthen]-3-one (3):
1
mp 225–228ꢁC. H NMR (400 MHz, CDCl3) δ 8.37 (s,
1H), 7.99 (d, J = 6.8 Hz, 1H), 7.66 (s, 1H), 7.57–7.34
(m, 3H), 7.10 (d, J = 6.8 Hz, 1H), 6.96 (d, J = 8.4 Hz,
1H), 6.51 (d, J = 8.8 Hz, 2H), 6.44 (s, 2H), 6.24 (d,
J = 7.6 Hz, 2H), 6.06 (s, 1H), 3.32 (dd, J = 13.4, 6.5 Hz,
8H), 1.16 (t, J = 6.8 Hz,12H). 13C NMR (101 MHz,
CDCl3) δ 153.5, 153.0, 151.9, 148.9, 145.1, 133.3,
131.0, 129.6, 129.0, 128.5, 128.2, 127.9, 123.7, 123.4,
115.8, 110.4, 108.0, 105.8, 97.9, 65.9, 44.3, 12.6.
IR(KBr): νmax 3437, 3128, 2968, 1685, 1610, 1519,
1371, 1280, 1217, 1109, 810, 769, 696 cm−1. HRESIMS
1
Shanghai, China). H and 13C NMR (nuclear magnetic res-
onance) spectra (at 400 MHz and 100 MHz, respectively)
were recorded in CDCl3 with tetramethylsilane as internal
reference on a Bruker Advance500 FT spectrometer. Chem-
ical shifts were reported in parts per million. IR spectra
were recorded on a FT-IR-6300 (JASCO, Tokyo, Japan)
and Mass spectra (MS) were measured by the ESI method
on an Agilent 6510 Q-TOF mass spectrometer. Aluminum
oxide (100–200 mesh) was used for flash column chroma-
tography. All reactions were monitored by TLC (thin-layer
chromatography) using 0.25 mm silica gel plates with UV
indicator (Shanghai Jiapeng Technology Co., Ltd., Shang-
hai, China). UV–vis spectra were recorded on a UV-2550
spectrometry (Hitachi, Tokyo, Japan). Fluorescent measure-
ments were recorded on an F-4500 FL spectrophotometer
(Hitachi). The pH measurements were performed on a
PHS-3C digital pH meter (Mettler Toledo, Greifensee,
Switzerland). Images of Escherichia coli cells were cap-
tured with a laser confocal microscope (Carl Zeiss LSM-
700, Oberkochen, Germany).
+
m/z: calcd for C35H35BrN4O3 : 638.1893, found:
639.1957 [M + H]+; C35H35BrN4O32+: 640.1893, found:
641.1878 [M + 2 + H]+.
Bacteria Culture and Imaging. Escherichia coli was
incubated at 37ꢁC in Luria–Bertani (LB) culture (tryptone
10 g/L, yeast extract 5 g/L, NaCl 10 g/L) in a table concen-
trator (AO HUA, ZD-85, Changzhou, China) at 180 rpm
for 5 h, and the culture was centrifuged (Heal Force TGL-
16M, Changsha, China) in 10 mL Eppendorf tubes at
10 000 rpm for 5 min. The sediment was then resuspended
in B–R buffer at pH 1.75, 2.30, and 4.83, respectively. The
pH probe dissolved in ethanol was added into each buffer,
with a final concentration at 25 μM after resuspension.
Escherichia coli cells with the probe were then incubated
in the table concentrator as mentioned above for 30 min,
washed by deionized water and smeared on slides. The
color changes of E. coli cells were observed at the wave-
length of 555 nm under laser confocal microscopy.
Synthesis of Probe L. The synthetic route of L is shown
in Scheme 1. The reaction of NH2NH2ꢀH2O (0.050 mmol,
2.0 equiv) with rhodamine-B 1 (0.025 mmol, 1.0 equiv) in
refluxed ethanol (50 mL) led to the formation of rhodamine
hydrazide 2.30 The solution of rhodamine hydrazide
2 (0.025 mmol, 1.0 equiv) and 3-bromo-4-hydroxy benzal-
dehyde (0.100 mmol, 4.0 equiv) in ethanol (20 mL) was
then refluxed for 16 h and monitored by TLC. After the
completion of the reaction, the mixture was evaporated to
dryness and 30 mL water was added to the residue. The
product was extracted by ethyl acetate (20 mL) from water
for three times and further recrystallization from ethyl ace-
tate afforded the target probe. The probe L was obtained as
a white solid in 85% yield and the structure was determined
by 1H NMR, 13C NMR, IR, and HRMS spectra
(Figures S1, S3–S5, Supporting Information).
Results and Discussion
Spectroscopic Properties and Optical Responses to pH.
Probe L showed high sensitivity towards strong acidic con-
dition, which is clarified in Figure 1. The fluorescence
intensity of probe L slightly increased with decreasing pH
value from 11.12 to 4.00, and it began to dramatically rise
under strongly acidic condition (pH < 3.06) and the maxi-
mum intensity appeared at 577 nm when the pH reached
1.75. It is assumed that probe L was present in the form of
spirolactam structure at high pH value and the ring quickly
opened to form a conjugated form under strong acidic con-
1
dition, which was confirmed by H NMR analysis.31,32 The
titration of probe L with 2.0 equiv trifluoroacetic acid
(TFA) in solution of CDCl3 showed the down-field shifts
for the signals of hydrogen atoms of the 2-bromo-4-(1-imi-
1
noethyl)phenol and xanthene moiety of rhodamine in H
NMR. These shifts due to the change of the electron cloud
density of rhodamine Schiff-base group were assumed to
be caused by proton promoted conversion of spirolactam
structure (nonfluorescent) to a conjugated ring-opened form
(fluorescent) under strong acidic condition (Figure S2).
The fluorescence intensity at 577 nm decreased sharply
when pH value increased from 1.75 to 4.00 according to
Scheme 1. The synthetic route of probe L.
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A New Rhodamine B-based Fluorescent Probe for pH Detection
KOREAN CHEMICAL SOCIETY
Figure 3. Fluorescence intensity changes of L (25 μM) at 577 nm
in the presence of different metal cations in solution (B–R buffer-
EtOH, v/v = 1:1) at pH 1.75.
Figure 1. The fluorescence emission spectrum of L (25 μM) in
solution (B–R buffer-EtOH, v/v = 1:1) with different pH. The
inset showed the fluorescence color of L with at pH 1.75 (left)
and 7.00 (right) (excitation wavelength: λex = 560 nm).
Figure 4. Absorption spectra of probe L (25 μM) in solution (B–
R buffer-EtOH, v/v = 1:1) at various pH values.
Figure 2. The fluorescence intensity at 577 nm vs. pH
λex = 560 nm.
properties of probe L caused by 15 ions at pH = 1.75
(Figure 3). All the co-existing ions did not show significant
interfering effect on the fluorescence intensity with probe
L even when the cations are in a much excess amount (10-
fold of the probe). These results suggested that probe L had
specific fluorescent response to acidic pH and was suitable
for fluorescent intracellular pH imaging.
Further, we examined the reversibility of fluorescence
vs. pH by neutralizing the solution of the probe at pH 1.75
to pH 7.01 and again acidifying it to 1.75 for circles. The
probe gave high fluorescence emission intensity upon the
pH value of the solution reached 1.75 and then quickly
decreased to a low intensity at neutral condition in all the
tested circles. As we expect, the coordination of the probe
with proton is excellently reversible. In addition to that, air,
light, and humidity did not show any effect on the pH-
the fluorescent pH titration (pH 1.75–11.12, λ ex = 560nm,
Figure 2) and this relationship can be quantified and fit well
to the Hendersone–Hasselbach-type mass action equation
pH = pKa + log[(Imax – I)/(I – Imin)].33 According to the
equation, the fluorescence intensity was linearly propor-
tional (R2 = 0.9884) to pH 1.70–4.00 (Figure S6), which
further allows us to calculate the pH value of the sample
within the range of pH from 1.75 to 4.00 based on their
fluorescent intensity. The results obtained in Figure 2 are
similar to the results obtained in fluorescence emission
spectrum (Figure 1).
To investigate the interference of metal ions towards
probe L, we measured the variation in the fluorescence
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A New Rhodamine B-based Fluorescent Probe for pH Detection
KOREAN CHEMICAL SOCIETY
We performed the pH bioimaging of E. coli by a laser
confocal microscope to investigate its ability to monitor the
intracellular pH value. Buffers with pH at 1.75, 2.30, and
4.83 were used to incubate the bacteria. The images were
captured after the probe L was added into above system
and then washed by deionized water. Red fluorescence was
clearly observed in E. coli under the tested acidic condi-
tions, and the strongest fluorescence intensity was found at
pH 1.75. The red fluorescence gradually disappeared when
the pH decreased to 4.83, as determined by scanning confo-
cal microscopy (Figure 5(A)), in accordance with the ten-
dency shown in the fluorescence spectra. In addition, the
quantitative analysis of fluorescence intensity in E. coli
cells gradually increased with the drop of pH (Figure 5(B)).
Conclusion
In summary, we have developed a novel fluorescent pH
probe L based rhodamine B by the introduction of conju-
gated substitution of styryl moiety to rhodamine core using
coupling reaction of rhodamine hydrazine with commer-
cially available 3-bromo-4-hydroxybenzaldehyde. The as-
synthesized probe exhibit high emission intensity at
577 nm for pH 1.75, which has been confirmed its excel-
lent selectivity and sensitivity to extremely acidic condi-
tions. Apart from the strong selectivity, the probe has
additional features like good reversibility, short response
time (<1 min) and no interference with the coexisted metal
ions. Moreover, this rhodamine B-based fluorescent probe
may be useful for the development of colorimetric and fluo-
rescent sensors to measure intracellular pH under extremely
acidic conditions by observing fluorescence imaging of
bacteria E. coli.
Figure 5. (A) Imaging acidity in E. coli cells with probe L. (a)–
(c) pH = 1.75; (d)–(f ) pH = 2.30; (g)–(i) pH = 4.83; (a), (d), and
(g) are the fluorescence images; (b), (e), and (h) are the bright field;
(c), (f ), and (i) are merged images (scale bar: 5 μm). (B) The quan-
titative analysis of fluorescence intensity in E. coli cells.
Acknowledgments. We would like to thank the Science
and Technology Support Program from the Science and
Technology Department of Sichuan Province China
(2015FZ0070 and 2014sz0004-5), and the National Research
Foundation of Korea (NRF-02016R1D1A1B01011019).
sensing properties of probe L during the reversibility experi-
ments (Figure S7). In further study on the response time of
the probe, we found that probe L quickly gave the maxi-
mum fluorescence intensity when the pH was low at 1.75
and maintained the intensity for 20 min. The low emission
intensity was given at pH 7.01 and lasted for 20 min
(Figure S8).
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
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