Two near-infrared highly sensitive cyanine fluorescent probes for pH monitoring

Article abstract of DOI:10.1016/j.cclet.2017.03.037

Two near-infrared (NIR) pH-activated heptamethine indocyanine probes with quaternary ammonium unit were designed and synthesized. The absorption and emission titrations indicate that cationic structure improves the cyanine dye's aqueous solubility and these two probes exhibit highly sensitive response to pH in acid condition. Their fluorescence intensities both gradually increase about 25-fold from pH 7.60 to 3.00 with pKa values of 4.72 and 4.45 respectively, which are suitable for studying acidic organelles in living cells. Moreover, their fluorescence intensities are linearly proportional to pH values in the range of 5.50–4.00. These results are probably attributed to the protonation of the indole nitrogen atoms, which are verified by 1H NMR spectra. Furthermore, these two probes can achieve real-time imaging of cellular pH and detection of pH in situ in living HeLa cells due to their excellent properties, including good reversibility, desirable photostability, high selectivity, low cytotoxicity and remarkable membrane permeability.

Full text of DOI:10.1016/j.cclet.2017.03.037

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Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Two near-infrared highly sensitive cyanine fluorescent probes for pH
monitoring
Jing-Ru Houa,b, Di Jina,b, Bo Chena,b, Lei-Lei Si , Yue-Hua Jin , Li-Gong Chena,b,d,
a,b
c
a,b,d
a,b,
a,b,d,
Xi-Long Yan
a
, Bo-Wei Wang *, Yang Li
*
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China
Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300350, China
Tianjin Bohai Fine Chemical Co., Ltd., Tianjin 300300, China
b
c
d
Tianjin Engineering Research Center of Functional Fine Chemicals, Tianjin 300350, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 6 January 2017
Received in revised form 20 February 2017
Accepted 26 March 2017
Available online xxx
Two near-infrared (NIR) pH-activated heptamethine indocyanine probes with quaternary ammonium
unit were designed and synthesized. The absorption and emission titrations indicate that cationic
structure improves the cyanine dye’s aqueous solubility and these two probes exhibit highly sensitive
response to pH in acid condition. Their fluorescence intensities both gradually increase about 25-fold
from pH 7.60 to 3.00 with pKa values of 4.72 and 4.45 respectively, which are suitable for studying acidic
organelles in living cells. Moreover, their fluorescence intensities are linearly proportional to pH values in
the range of 5.50–4.00. These results are probably attributed to the protonation of the indole nitrogen
atoms, which are verified by 1H NMR spectra. Furthermore, these two probes can achieve real-time
imaging of cellular pH and detection of pH in situ in living HeLa cells due to their excellent properties,
including good reversibility, desirable photostability, high selectivity, low cytotoxicity and remarkable
membrane permeability.
Keywords:
pH fluorescent probe
Near-infrared
Indocyanine
Quaternary ammonium
Biological imaging
©
2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
invasiveness, non-radioactivation, real-time imaging, sensitivity,
convenience and visualization [18–24]. Several pH probes have
As well known, pH value is a significant parameter in analytical
been reported, such as N,N-dimethylaminophenyl-2,6-dicarbox-
chemistry, biology, clinical biochemistry and environment [1–12].
Particularly, intracelluar pH plays an essential role both in cellular
behaviors and pathological conditions, such as proliferation and
apoptosis, multidrug resistance (MDR), ion transport and homeo-
stasis, enzyme activity and endocytosis [13–16]. Under normal
yethyl-1,3,5,7-tetramethylboron-dipyrromethene (DiMeN-BDP)
0
0
and its analogue [25], Dansyl acid (DA) [26], 3 ,6 -bis (diethyla-
mino)-2-(2-((5-phenyl-1,3,4-oxadiazol-2-yl) amino) ethyl) spiro
0
[isoindoline-1,9 -xanthen]-3-one [27] etc. However, absorption
maxima of these fluorescent probes were in the visible region
where tissue penetration of light was shallow, thus these probes
were less optimal for noninvasive in vivo imaging. Moreover, most
reported fluorescence pH probes are practical for near-neutral pH
range (i.e. pH 6.8–7.4) [17,28–30], while there are limited probes for
monitoring pH changes in acidic organelles (i.e. pH 4.5–6.0). These
results encourage us to develop new NIR fluorescent probes for
sensitively monitoring acid physiological pH fluctuations.
Heptamethine indocyanine dye is very famous for its NIR region
absorption and emission (650 nm–900 nm) [31]. In this region, not
only background absorption and tissue auto-fluorescence are
weak, but also the photodamage to living cells is quite limited.
Moreover, NIR emission can penetrate deeply with low light
scattering to achieve distinct image in vivo assay. In addition, the
pH-sensitive heptamethine indocyanine dyes can be divided into
+
physiological condition, extracellular H concentration is main-
tained within a very narrow limit. Deviation by 0.10–0.20 pH unit
in either direction can cause severe problems, such as cardiopul-
monary and neurologic problems (e.g., Alzheimer’s disease), a
larger variation can be fatal [17]. Therefore, it is essential to
monitor the intracellular pH precisely and conveniently. In recent
years, bioprobe has emerged and been intensively studied as a
potential tool for pH monitoring in vivo by virtue of its non-
*
Corresponding authors at: School of Chemical Engineering and Technology,
Tianjin University, Tianjin 300350, China.
E-mail addresses: bwwang@tju.edu.cn (B.-W. Wang), liyang777@tju.edu.cn
(
Y. Li).
http://dx.doi.org/10.1016/j.cclet.2017.03.037
001-8417/© 2017 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
1
Please cite this article in press as: J.-R. Hou, et al., Two near-infrared highly sensitive cyanine fluorescent probes for pH monitoring, Chin. Chem.
Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.03.037

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two types based on two different pH response mechanisms (i.e.
intramolecular charge transfer (ICT) and photoinduced electron
transfer (PET)), achieving fluorescent signal off and on [14].
Compared to the “always on” probes, “off-on” probes display high
signal-to-noise ratio. Subtle changes to the structure of these
cyanine-based probes can regulate their pKa values. However,
heptamethine indocyanine probe always suffers from poor
aqueous solubility and photostability, which hamper it from wide
application.
with chloroacetyl chloride. Lastly, compound 7 was nucleophilic
substituted by TEA and pyridine to afford 8 and 9, respectively. The
structures of target compounds and intermediates were confirmed
by H NMR, C NMR and ESI-MS analysis (Figs. S6–S18 in
Supporting information).
1
13
2.2. Spectroscopic properties and optical responses to pH
To evaluate their responses to pH, the absorption and
fluorescence spectra of the probes in ethanol-water solution were
firstly measured in the pH range of 12.00–2.00. As shown in Fig.1(a
and c), an absorption maximum at 535 nm was observed in the pH
range of 12.00–9.00. With decreasing the pH, the characteristic
peak at 535 nm was gradually reduced, whereas a new absorption
peak was evolved and progressively increased at 789 nm.
Meanwhile, the color of the solution changed from pink to pale
green, which demonstrated that both of 8 and 9 could serve as
visual pH indicators. The fluorescence spectra of probes 8 and 9
upon different pH values were presented in Fig. 1(b and d). Under
750 nm excitation, an emission band at 808 nm appeared and
underwent a concomitant monotonic increase with a decrease in
the pH of the medium from 12.00 to 2.00. Moreover, Fig. S1
illustrated the pH response of probes 8 and 9 as a function of I/Imax
vs. pH. According to the Henderson-Hasselbach-type mass action
equation [37], the pKa values were 4.72 and 4.45 for probes 8 and 9
respectively, which indicated they were very suitable for studying
acidic organelles. Probe 6 presented similar absorption and
emission spectra as probe 8 or 9, but no fluorescent signal was
observed any more when pH > 6.0 (Fig. S2 in Supporting informa-
tion). In comparison, the pH response ranges of probes 8 and 9
were extended to 12.0 from 6.0. And compared with some reported
probes [38–40], probes 8 and 9’s absorption and emission
wavelength were both in NIR region, which was more suitable
for future biological applications. Furthermore, it was worth
To the best of our knowledge, introducing cationic units into
probes’ structure may not only improve the aqueous solubility of
probes but also enhance their targeting ability to mitochondria
[32–34]. Lee et al. reported a pH-sensitive fluorescent probe with
cationic triphenylphosphonium unit as target group for diseased
mitochondria. And Mo et al. also proved the cationic group had the
ability to accumulate at the mitochondria. Based on these, we
designed two novel NIR pH-activated probes to monitor pH
fluctuation in biological system. These two probes are constructed
of non-N-alkylated indolium cyanine and quaternary ammonium
cations. The titration experiments demonstrate that these two
novel probes exhibit good aqueous solubility and sensitive
response to pH variation in the range of 5.50 to 4.00. Furthermore,
these two probes have been successfully used for fluorescence
imaging of the pH change in HeLa cells.
2. Results and discussion
2.1. Synthesis
As shown in Scheme 1, compounds 1, 2, 3 and 4 were prepared
according to our previously reported method [35,36]. A condensa-
tion reaction of 2, 3 and 4 in ethanol yielded 5. Compound 5 further
reacted with tin (II) chloride to synthesize compound 6 via a
reduction reaction. Then compound 7 was produced by acylating 6
Scheme 1. Synthesis of probe 8 and 9.
Please cite this article in press as: J.-R. Hou, et al., Two near-infrared highly sensitive cyanine fluorescent probes for pH monitoring, Chin. Chem.
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Fig. 1. (a) UV–vis spectra of probe 8 upon pH change in ethanol-water (2/8, v/v) solution. pH 2.41, 2.93, 3.37, 3.76, 4.01, 4.42, 4.60, 5.19, 5.39, 6.23, 6.53, 6.85, 7.00, 7.55, 8.79,
9
4
4
.73,11.23. Inset: visual color changes upon pH. (b) The fluorescence spectra of probe 8 upon pH change in ethanol-water (2/8, v/v) solution. pH 2.41, 2.93, 3.37, 3.76, 4.01, 4.42,
.60, 5.19, 5.39, 6.23, 6.53, 6.85, 7.00, 7.55, 8.79, 9.73, 11.23. (c) UV–vis spectra of probe 9 upon pH change in ethanol-water (2/8, v/v). pH 2.35, 2.89, 3.31, 3.61, 3.99, 4.27, 4.43,
.77, 5.13, 5.43, 6.25, 6.63, 6.86, 7.05, 7.69, 8.86, 9.70,11.11. Inset: visual color changes upon pH. (d) The fluorescence spectra of probe 9 upon pH change in ethanol-water (2/8, v/
v) solution. pH 2.35, 2.89, 3.31, 3.61, 3.99, 4.27, 4.43, 4.77, 5.13, 5.43, 6.25, 6.63, 6.86, 7.05, 7.69, 8.86, 9.70, 11.11.
mentioning that the aqueous solubility of probes 8 and 9 (ethanol/
water = 2/8, v/v) were much better than that of probe 6 (ethanol/
water = 4:6, v/v), indicating that probes 8 and 9 were more suitable
for monitoring pH in environmental and biological analysis.
In order to directly reflect the relationship between pH and
fluorescence, the relative fluorescent intensity at maximum
emission wavelength towards pH were then plotted (Fig. 2).
Remarkably, for probes 8 and 9, a sharp and almost linear
fluorescence intensity increase (about 6-fold enhancement)
appeared within the narrow range of pH 5.50–4.00. Thus, we
speculated that these two cationic probes could achieve not only
qualitative but also quantitative results in pH range of 5.50–4.00,
making these pH-sensitive probes promising fluorescence indica-
tors for monitoring slight pH fluctuation in physiological environ-
0
ment. What is more, compared with probe 6 pH-sensitive range
(pH < 4.00 (Fig. S3 in Supporting information) and other reported
probes’ pH-sensitive range (pH 6.7–7.9) [17,28], the pH-sensitive
range of probes 8 and 9 more accorded with our original concept,
particularly monitoring pH in acid organelles.
The reversibility of probes 8 and 9 was investigated [27,41]. It
was found that their response behaviors toward pH fluctuation
were reversible according to the absorption and emission titration
experiments. The absorption titration showed that the addition of
hydrochloric acid could lead to bathochromic shift of absorption
Fig. 2. The relationship between I/Imax and pH of probes 8 and 9 (a, and b). I was measured fluorescent intensity at that pH, and Imax was the maximum output of the probes.
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maxima and obvious color change. When pH was adjusted back to
.96 by sodium hydroxide solution, the absorption maxima moved
2.3. Proposed mechanism
8
back to the original state and the color of the system recovered to
pink (Fig. 3a and c). The emission titration also reflected that
fluorescent signal could be reversibly enhanced and decreased by
cyclically adding hydrochloric acid and sodium hydroxide (Fig. 3b
and d).
The responsive mechanism of probes 8 and 9 upon pH is ICT
process (probe 8 as a typical example illustrated in Scheme 2). The
pH-sensitive spectral response is possibly attributed to the
protonation and deprotonation of the indole nitrogen atoms.
The ICT process is enhanced upon the protonation of the non-
alkylated nitrogen atoms, which leads to a bathochromic shift of
absorption maxima and remarkable enhancement of the fluores-
The photostability of probes 8 and 9 was examined under
irradiation with
a 254 nm UV lamp. Their time-dependent
1
fluorescence spectra with pH = 4.01 at room temperature were
recorded over 60 min and the curves of fluorescence intensity vs.
time were depicted in Fig. 4. For probe 8, almost no reduction of the
fluorescence intensity was observed within the irradiating period,
confirming that this probe was stable to environmental factors
such as light, air and medium. However, for probe 9, the
fluorescence intensity was slightly decreased over the irradiating
time, yet the fluorescence intensity was still high. The difference
between probe 8 and probe 9 on photostability was likely
attributed to better activity of pyridinium salt than triethyl
ammonium salt. Based on these results, we could conclude that
probes 8 and 9 exhibited reversible response to pH and produced
steady signals, indicating that these two probes could serve as
switch on sensor for practical applications.
cence intensity. To confirm this, H NMR spectra of probe 8 under
+
1
different H concentrations were collected in CDCl
NMR spectra of probe 9 was shown in Fig. S4). Upon addition of
CF COOH, an obvious up-field shift was observed for the chemical
3
(Fig. 6) (The H
3
shifts of the conjugation structural prontons (H1–H11). Mean-
while, the benzene ring protons of non N -alkylated indole (H1–H3)
and the double bond protons adjoining non N -alkylated indole
(H4, H5) had larger up-field shift than the other protons (H6–H11).
The result revealed the protonation of the non-alkylated nitrogen
atoms [14], which resulted in the decrease of electron density
around the conjugation structure.
In addition, the wider pH response range of probe 8 or 9 than
probe 6 is attributed to the pKa of their alkaline sites: aniline N
atom (AN-N) > indole non-alkylated N atom (IA-N) > anilide N atom
On account of the complexity of the intracellular environment,
(
AD-N). For probe 6, proton tends to bind AN-N instead of IA-N,
ꢁ
5
the selectivity of these two probes (1.0 ꢀ10 mol/L) were studied
thus just AN-N is protonated when pH > 5.90 and there is no ICT
process as well as fluorescent signal observed. While pH < 5.90,
excess proton induces protonation of IA-N, further enhanced ICT
process and induced strong fluorescent signal. For probes 8 and 9,
proton binds to IA-N preferentially, then IA-N is protonated in pH
range from 12.00 to 2.00, namely there always exists ICT process
and the fluorescent signal in pH range from 12.00 to 2.00.
at pH = 4.01 with a series of potential competitive species,
+
+
2+
2+
2+
2+
2+
2+
including metal ions (Na , K , Mg , Zn , Cu , Ca , Mn , Co
,
2
+
and Ni ), and some biologically important species (GSH, Cys, Gly,
Trp, His, Arg, and H ). As expected (Fig. 5), no remarkable
2 2
O
changes were observed upon addition of these potential interfer-
ents, enabling them as the promising pH-sensitive probes for
practical application in complex environment.
Fig. 3. (a) Reversible changes of the absorption spectra of probe 8 between pH 8.96 and 3.02. (b) Reversible fluorescent switching of probe 8 upon pH fluctuation between pH
.96 and 3.02. (c) Reversible changes of the absorption spectra of probe 9 between pH 8.96 and 3.01. (b) Reversible fluorescent switching of probe 9 upon pH fluctuation
8
between pH 8.96 and 3.01.
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Fig. 4. (a) Time-dependent changes of the fluorescence spectra of probe 8 at pH = 4.01 over 60 min. (b) Time-dependent changes of fluorescence intensity at
lem = 815 nm of
probe 8 at pH = 4.01 over 60 min. (c) Time-dependent changes of the fluorescence spectra of probe 9 at pH = 4.01 over 60 min. (d) Time-dependent changes of fluorescence
intensity at em = 810 nm of probe 9 at pH = 4.01 over 60 min.
l
ꢁ
5
ꢁ5
Fig. 5. The fluorescence intensity at (a)
l
em = 815 nm of probe 8 (1.0 ꢀ10 mol/L) and (b)
l
em = 810 nm of probe 9 (1.0 ꢀ10 mol/L) at pH = 4.01 in ethanol-water (2/8, v/v)
ꢁ
4
+
+
2+
2+
2+
2+
2+
2+
2+
solution upon addition of different interferents (1.0 ꢀ10 mol/L).1, probe; 2, Na ; 3, K ; 4, Mg ; 5, Zn ; 6, Cu ; 7, Ca ; 8, Mn ; 9, Co ; 10, Ni ; 11, GSH; 12, Cys; 13, Gly; 14,
2 2
Trp; 15, His; 16, Arg; 17, H O .
Scheme 2. Response mechanism of probe 8 to pH.
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Fig. 6. The 1H NMR spectra of (a) probe 8 (14.4
m
mol), (b) probe 8 – TFA (2:1, mol/
mol), (c) probe 8 – TFA (1:1, mol/mol) in CDCl
3
.
2.4. Cell cytotoxicity assay
To evaluate cytotoxicity of probes 8 and 9, we performed an
MTT assay in HeLa cells with probes concentrations from
ꢁ
6
ꢁ4
1
.0 ꢀ10 mol/L to 1.0 ꢀ10 mol/L. As shown in Fig. S5, more
than 70% of cells were viable at testing concentration for both
probes 8 and 9, which clearly demonstrated that these two probes
were low-toxic to cultured cell lines under the experimental
conditions at the concentration of 1.0 ꢀ10ꢁ mol/L.
Fig. 7. Confocal imaging of probe 8 and 9 in HeLa cells at pH 4.0, 6.0, 7.0,
respectively. The lower images represent the corresponding bright field images.
5
2.5. Fluorescence imaging in living cells
4
. Experimental
Live-cell imaging studies were carried out to evaluate the
applicability of probes 8 and 9 for pH monitoring in living cells. We
measured the cell experiments using HeLa cells incubated with
probes 8 and 9 at pH 4.0, 6.0 and 7.0. As shown in Fig. 7, the cells
exhibited almost no fluorescence emission at pH 7.0, but at pH 6.0
there was weak fluorescence emission and at pH 4.0 the
fluorescence intensity obviously increased. Bright-field transmis-
sion measurements after probes 8 and 9 incubation confirmed that
the cells were viable. These data demonstrated that probes 8 and 9
had good cell-membrane permeabilities and both probes could be
used for imaging pH fluctuations under acidic condition in living
cells.
4
.1. Materials and apparatus
All the raw chemicals were purchased from commercial sources
and used without further purification.
1
13
H NMR and C NMR spectra were recorded on a Bruker Avance
00 spectrometer with TMS as an internal standard. High-
4
resolution mass spectrum was obtained using a Bruker Daltonics
microOTOF-Q II instrument. All pH measurements were carried
out with a Sartorius basic pH-meter PB-10. UV–vis spectra were
measured on an Evolution 300 UV–vis spectrophotometer.
Fluorescence spectra were recorded on
Fluorolog3 spectrophotometer.
a Jobin Yvon-Spex
3. Conclusion
4
.2. UV–vis and fluorescence spectroscopy measurement
In summary, two novel NIR cyanine fluorescent probes bearing
The stock solutions of probes 8 and 9 (1.0 ꢀ10ꢁ mol/L) were
3
quaternary ammonium unit were designed and synthesized. These
two probes were highly sensitive to pH fluctuation and especially
responded linearly and rapidly to pH in the range of 5.50 to 4.00
with pKa values of 4.72 and 4.45, respectively. Furthermore, these
two fluorescent probes could be reversibly turned off and on by
alternating pH values. These two cyanine fluorescent probes
possessed several great properties, including desirable photo-
stability, high selectivity, excellent aqueous solubility and low
cytotoxicity, which would widen their applications. The pH-
dependent fluorescence imaging experiment demonstrated that
probes 8 and 9 had excellent cell-membrane permeability and
further monitored pH fluctuations in live cells. These results
indicate that these two probes are particularly potential for
detecting pH fluctuation in biological systems.
prepared by dissolving appropriate amount of probe 8 (6.3 mg) and
probe 9 (6.0 mg) in ethanol and diluting them to the mark in the
10 mL volumetric flasks. Further dilution yielded the working
ꢁ
5
solution (1.0 ꢀ10 mol/L) by diluting 2.5 mL stock solution to
2
50 mL with ethanol-water (2:8, v/v). For measuring the optical
response of the probes towards pH, the accurate pH of the working
solution was adjusted by 0.1 mol/L NaOH and 0.1 mol/L HCl. The
absorption and emission spectra were recorded in the wavelength
range of 300 to 1000 nm. For the fluorescence measurements,
excitation and emission slits were 10 nm and 8 nm, respectively.
The excitation wavelengths for both compounds 8 and 9 were
7
50 nm.
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Acknowledgment
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Please cite this article in press as: J.-R. Hou, et al., Two near-infrared highly sensitive cyanine fluorescent probes for pH monitoring, Chin. Chem.
Lett. (2017), http://dx.doi.org/10.1016/j.cclet.2017.03.037