Reversible near-infrared pH probes based on benzo[ a ]phenoxazine

Article abstract of DOI:10.1021/ac4013539

Several benzo[a]phenoxazine derivatives containing substituted N-aromatic groups are evaluated for their pH-dependent absorption and emission properties. Among the compounds exhibiting optical responses under near-neutral and subacid pH conditions, benzo[a]phenoxazine derivatives with an electron-withdrawing aromatic group attached to nitrogen of the imino group show potential application as near-infrared pH sensors. Three water-soluble pH probes based on benzo[a]phenoxazine with different pyridinium structures are designed and synthesized. Their reversible pH-dependent emissions in buffer solution containing 0.1% dimethyl sulfoxide (DMSO) locate in 625-850 nm with the fluorescent enhancement of 8.2-40.1 times, and their calculated pK_a values are 2.7, 5.8, and 7.1, respectively. A composite probe containing the three benzo[a]phenoxazines shows a linear pH-emission relationship in the range of pH 1.9-8.0. Real-time detection of intracellular pH using an in vitro assay with HeLa cells is also reported.

Full text of DOI:10.1021/ac4013539

Article
pubs.acs.org/ac
Reversible Near-Infrared pH Probes Based on Benzo[a]phenoxazine
Wu Liu,†,∥ Ru Sun,†,∥ Jian-Feng Ge,*,† Yu-Jie Xu,‡ Ying Xu,‡ Jian-Mei Lu,*,† Isaum Itoh,§
and Masataka Ihara*,§
‡
†College of Chemistry, Chemical Engineering and Material Science, School of Radiation Medicine and Protection,
Soochow University, 199 Ren’Ai Road, Suzhou 215123, People’s Republic of China
§Drug Discovery Science Research Centre, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
S
* Supporting Information
ABSTRACT: Several benzo[a]phenoxazine derivatives con-
taining substituted N-aromatic groups are evaluated for their
pH-dependent absorption and emission properties. Among the
compounds exhibiting optical responses under near-neutral
and subacid pH conditions, benzo[a]phenoxazine derivatives
with an electron-withdrawing aromatic group attached to nitro-
gen of the imino group show potential application as near-
infrared pH sensors. Three water-soluble pH probes based on
benzo[a]phenoxazine with different pyridinium structures are
designed and synthesized. Their reversible pH-dependent
emissions in buffer solution containing 0.1% dimethyl sulfoxide
(DMSO) locate in 625−850 nm with the fluorescent enhance-
ment of 8.2−40.1 times, and their calculated pKa values are 2.7, 5.8, and 7.1, respectively. A composite probe containing the three
benzo[a]phenoxazines shows a linear pH−emission relationship in the range of pH 1.9−8.0. Real-time detection of intracellular
pH using an in vitro assay with HeLa cells is also reported.
luorescent probes that exhibit red and near-infrared (NIR)
emission have received considerable attention in the past
Benzo[a]phenoxazine (Scheme 1), a deprotonated structure
of benzo[a]phenoxazinium, used to be considered an unstable
F
decade.1,2 NIR probes that emit in the range of 650−900 nm
are especially preferred for sensor design because NIR light can
penetrate tissues deeply, minimize damage to biological
samples, and decrease the influence of background autofluor-
escence in tests.34 And intracellular pH plays an important role
in organisms since it influences constituents such as cells,
enzymes, and proteins, as well as organizational units such as
muscles and nervous systems. Although lots of pH indicators
have been developed, only some of them present their full
emission spectra in the NIR region.5−8
Scheme 1. Equilibrium between Benzo[a]phenoxazine and
Benzo[a]phenoxazinium
The pH probes based on multimethylene cyanine have
received attention because of their NIR response,9−13 but the
stability of multimethylene cyanine dyes needs to be
improved.14,15 After an amino-substituted heptamethine
cyanine with improved photostability was reported,16 new
NIR probes were developed.6,7,17−19 The change of fluo-
rescence intensity of these aminocyanine-based probes with pH
in aqueous solution is usually less than 7-fold; only one
example with a change of up to 15-fold has been reported.20
Only several NIR pH sensors based on other fluorophores have
compound because of its imino group. Recent research
indicates that benzo[a]phenoxazine is a stable, nontoxic com-
pound with excellent bioactivity.27 Although alkyl-substituted
Nile blue derivatives do not show a pH-dependent optical
response in near-neutral and subacid pH conditions,28 a color
change of benzo[a]phenoxazine with an N-aromatic group has
been observed during thin-layer chromatography (TLC)
experiments. In this work, five benzo[a]phenoxazine derivatives
are selected from our bioactive dye library to evaluate their
optical response to solution pH.
been reported, for example, BF2-chelated complexes,21−23
a
styrylcyanine-based probe,24 diketo-pyrrolo-pyrrole pigments,25
and an aminoperylene compound.26 These examples have
greatly progressed the theory and application of NIR pH
probes. However, new fluorophores for use in NIR pH sensors
that are soluble in water, highly sensitive, reversible, and
biocompatible are still highly required.
Received: May 5, 2013
Accepted: July 1, 2013
Published: July 1, 2013
© 2013 American Chemical Society
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condensing 2b and iodoethanol using a similar procedure to
that described for 3a. 3b was obtained as green crystal flakes,
yield 65%, mp 232−234 °C. IR ν (KBr, cm−1): 3440, 2030,
EXPERIMENTAL SECTION
■
Materials. All reagents and solvents (analytical grade) were
purchased from TCI Development Co., Ltd. (Tokyo, Japan) or
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and
used directly. Flash chromatography was performed with silica
gel (300−400 mesh). The anion-exchange resin (717 anion-
exchange resin) was washed to the nitrate form before use.
Hydrochloric acid−citric acid buffer (pH = 0.9), disodium
hydrogen phosphate−citric acid buffer (pH = 1.6, 1.9, 2.2, 2.5,
2.7, 3.1, 3.6, 4.0, 4.5, 4.9, 5.4, 5.8, 6.3, 6.7, 7.1, 7.6, 8.0, 8.4, and
9.0), and sodium carbonate−sodium hydrogen carbonate buffer
(pH = 9.5) were used.29 The theoretical pH values of the
buffers were used for the buffer solution containing 0.1% or
1.0% dimethyl sulfoxide (DMSO). The pH values of DMSO−
H2O (v/v = 4:1) solutions were adjusted to desired values
using aqueous hydrochloric acid (1.0 M) or sodium hydroxide
(1.0 M), and their pH values were finally tested by a pH meter.
The high K+ buffer with different pH’s for live cell imaging was
prepared by the reported method.30 Compounds 1a and 1b and
2a−2c were prepared by a reported method.27,31
1
1640, 1590, 1380, 1110. H NMR (400 MHz, DMSO-d6) δ
8.74−8.73 (m, 2H, 2 × Ar-H), 8.59 (d, J = 7.8 Hz, 1H, Ar-H),
8.49 (d, J = 7.7 Hz, 1H, Ar-H), 8.20−8.11 (m, 2H, 2 × Ar-H),
7.83 (t, J = 7.3 Hz, 1H, Ar-H), 7.76 (t, J = 7.3 Hz, 1H, Ar-H),
7.60 (d, J = 9.0 Hz, 1H, Ar-H), 6.82 (d, J = 8.4 Hz, 1H, Ar-H),
6.48 (s, 1H, Ar-H), 6.34 (s, 1H, Ar-H), 5.31 (s, 1H, OH), 4.65
(br, 2H, CH2), 3.92 (br, 2H, CH2), 3.48 (q, J = 6.7 Hz, 4H, 2 ×
CH2), 1.15 (t, J = 6.3 Hz, 6H, 2 × CH3). 13C NMR (101 MHz,
DMSO-d6) δ 159.19, 151.08, 150.47, 149.51, 146.26, 139.13,
139.03, 137.40, 137.09, 131.11, 131.04, 130.85, 130.69, 130.02,
128.04, 124.89, 124.42, 123.46, 110.11, 96.71, 95.71, 63.24,
60.14, 44.45, 12.41. HRMS-ESI+: m/z = 439.2142 (calcd for
− +
[M − NO3 ] , 439.2134).
Synthesis of Probe 3c. N,N-Diethyl-5-((1-(2-hydroxy-
ethyl)pyridine-1-ium-4-yl)imino)-5H-benzo[a]phenoxazin-9-
amine Nitrate (3c). Compound 3c was synthesized by con-
densing 2c and iodoethanol using a similar procedure to that
described for 3a. 3c was obtained as green crystal flakes, yield
70%, mp 198−200 °C. IR ν (KBr, cm−1): 3410, 2030, 1630,
1580, 1380, 1130. 1H NMR (400 MHz, DMSO-d6) δ 8.72 (br,
2H, 2 × Ar-H), 8.62 (br, 1H, Ar-H), 8.41 (d, J = 7.8 Hz, 1H,
Ar-H), 7.86 (br, 1H, Ar-H), 7.78 (br, 1H, Ar-H), 7.64 (br, 1H,
Ar-H), 7.56 (d, J = 5.2 Hz, 2H, 2 × Ar-H), 6.87 (br, 1H, Ar-H),
6.56 (s, 1H, Ar-H), 6.24 (s, 1H, Ar-H), 5.25 (s, 1H, OH), 4.50
(br, 2H, CH2), 3.86 (br, 2H, CH2), 3.51 (br, 4H, 2 × CH2),
1.17 (br, 6H, 2 × CH3). 13C NMR (101 MHz, DMSO-d6) δ
165.27, 157.76, 150.87, 149.91, 146.45, 145.21, 138.32, 131.24,
131.20, 130.93, 130.24, 129.96, 125.24, 125.04, 123.44, 118.81,
110.79, 96.54, 95.73, 61.29, 60.17, 44.55, 12.42. HRMS-ESI+:
1
Apparatus. H NMR and 13C NMR spectra were recorded
on a Varian 400 MHz spectrometer, and a solvent peak was
used as an internal standard. High-resolution mass spectra were
recorded on a Finnigan MAT95 mass spectrometer in ESI+
mode. UV−vis spectra were obtained with a Shimadzu UV-3600
spectrophotometer. Fluorescence emission spectra were measured
at room temperature on a Horiba Jobin Yvon FluoroMax-4
fluorescence spectrometer; the excitation and emission slits width
are set at 5 nm. Infrared (IR) spectra were recorded on a Nicolet
5200 IR spectrometer using solid samples dispersed in KBr pellets.
Melting points were determined on an X-4 microscope electron
thermal apparatus (Taike, China). The pH values were measured
with a Lei-Ci (pH-3C) digital pH meter (Shanghai, China) using a
combined glass−calomel electrode.
− +
m/z = 439.2140 (calcd for [M − NO3 ] , 439.2134).
Preparation of the Test Solutions. Stock solutions (100
μM) of 1a and 1b were prepared with DMSO. Each test
solution (10 μM) was prepared in a volumetric flask (10 mL)
from the corresponding stock solution (1.0 mL) and DMSO−
H2O (v/v = 7:2, 9.0 mL) with different pH’s adjusted with
aqueous hydrochloric acid or sodium hydroxide to give a total
volume of 10 mL. Stock solutions (100 μM) of 3a−3c were
prepared in a volumetric flask (100 mL) with DMSO (1.0 mL)
and distilled water. Stock solutions (500 μM) of 3a and 3c were
prepared in a volumetric flask (100 mL) with DMSO (10.0 mL)
and distilled water. Each test solution (10 μM) was prepared in
a volumetric flask (10 mL) with the corresponding stock solu-
tion (1.0 mL, from 100 μM stock solution) and buffer solution
to give a total volume of 10.0 mL. The composite solution of
probes 3a−3c (27.8 μM) was prepared in a volumetric flask
(10 mL) with the composite stock solution (1.0 mL) and the
corresponding buffer solution containing 1.0% DMSO, where
the composite stock solution is a mixture of 3a (8.0 mL, from
500 μM stock solution), 3b (20 mL, from 100 μM stock
solution), and 3c (8.0 mL, from 500 μM stock solution).
Cell Culture and Imaging Methods. HeLa cells were
cultured in Roswell Park Memorial Institute culture medium
(RPMI-1640) supplemented with 10% calf serum, penicillin
(100 U·mL−1), streptomycin (100 μg·mL−1), and L-glutamine
(2.5 × 10−4 M) at 37 °C in a 5:95 CO2−air incubator. Cells
with 2 × 105 density were loaded onto a glass-bottomed
coverslip with a diameter of 35 mm and cultured for 48 h
before use. Fluorescence images of the stained HeLa cells were
obtained with a Leica SP2 laser confocal scanning microscope
equipped with a 633 nm laser head. Emission was measured for
Synthesis of Probe 3a. N,N-Diethyl-5-((1-(2-hydroxy-
ethyl)pyridine-1-ium-2-yl)imino)-5H-benzo[a]phenoxazin-9-
amine Nitrate (3a). A mixture of 2a (394.2 mg, 1.0 mmol) and
iodoethanol (0.80 mL, 10.0 mmol) in acetonitrile (50 mL) was
heated under reflux for 16 h. The solvent was removed, and the
residue was purified by column chromatography on silica gel
with gradient elution using methanol/chloroform of 1:15 to
1:5. The crude product was recrystallized from chloroform to
obtain the iodide form of 3a. A methanol solution of the iodide
salt was passed through anion-exchange resin (nitrate form).
The solvent was evaporated to obtain 3a as a dark blue power,
yield 57%, mp 236−238 °C. IR ν (KBr, cm−1): 3420, 2030,
1
1630, 1590, 1380, 1110. H NMR (400 MHz, DMSO-d6) δ
8.69 (d, J = 7.9 Hz, 1H, Ar-H), 8.65 (d, J = 6.1 Hz, 1H, Ar-H), 8.53
(d, J = 7.9 Hz, 1H, Ar-H), 8.31 (t, J = 7.6 Hz, 1H, Ar-H), 7.91 (t,
J = 7.4 Hz, 1H, Ar-H), 7.81 (t, J = 7.4 Hz, 1H, Ar-H), 7.74 (d, J =
9.1 Hz, 1H, Ar-H), 7.63 (d, J = 8.3 Hz, 1H, Ar-H), 7.53 (t, J = 6.6
Hz, 1H, Ar-H), 7.02 (d, J = 9.1 Hz, 1H, Ar-H), 6.70 (s, 1H, Ar-H),
6.66 (s, 1H, Ar-H), 5.14 (s, 1H, OH), 4.56 (br, 2H, CH2), 3.77 (br,
2H, CH2), 3.55 (q, J = 6.7 Hz, 4H, 2 × CH2), 1.18 (t, J = 6.6 Hz,
6H, 2 × CH3). 13C NMR (101 MHz, DMSO-d6) δ 161.33, 157.24,
151.77, 150.96, 146.90, 144.86, 144.27, 137.15, 131.65, 131.53,
131.34, 130.04, 129.89, 126.52, 125.58, 123.41, 119.21, 118.77,
112.32, 97.16, 95.70, 58.78, 56.93, 44.84, 12.48. HRMS-ESI+:
m/z = 439.2135 (calcd for [M − NO3−]+, 439.2134).
Synthesis of Probe 3b. N,N-Diethyl-5-((1-(2-hydroxy-
ethyl)pyridine-1-ium-3-yl)imino)-5H-benzo[a]phenoxazin-9-
amine Nitrate (3b). Compound 3b was synthesized by
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the range of 650−795 nm. HeLa cells were incubated with the
composite probe (0.8 μM, mole ratio of 3a/3b/3c = 2:1:2) in
PBS buffer (pH = 7.4, 1.03 mL) for 20 min. After removing the
nutrient fluid, the high K+ buffer (pH = 8.0, 1.0 mL) was used
to wash the cells, the basic high K+ buffer (pH = 8.0, 1.0 mL)
with nigericin (2 μg·mL−1) was added, and cells were incubated
for 10 min. The system was then changed to acidic high K+
buffer (pH = 6.0, 1.0 mL) with nigericin (2 μg·mL−1) and
incubated for a further 10 min. The system was finally changed
to acidic high K+ buffer (pH = 4.0, 1.0 mL) with nigericin
(2 μg·mL−1) and incubated for a further 10 min. Treated cells
were imaged by fluorescence microscopy. All images were
obtained using the same settings and processed with attached
software.
the optical properties of 1a (Scheme 1) with pH was first
evaluated in DMSO−H2O (v/v = 4:1) solution (Figure 1A).
The absorption maximum of 1a changes from 545 nm (pH =
9.1) to 666 nm (pH = 2.5) with an isosbestic point at 590 nm,
and a colorimetric change from pink to azure is observed. The
intensity of the emission maximum of 1a is slightly quenched at
lower pH. These results indicate that 1a is a colorimetric pH
indicator rather than a fluorescent sensor. When the electron-
donating methoxy group in 1a is replaced by an electron-
withdrawing nitro group to form 1b, both colorimetric and
fluorescent responses to pH are observed (Figure 1B). The
absorption maximum of 1b changes from 565 nm (pH = 5.9)
to 683 nm (pH = 1.4) with an isosbestic point at 605 nm, and a
colorimetric change from lilac to azure is observed. The
intensity of the emission maximum enhances by 32-fold from a
very weak band at 641 nm to a more intensive one at 720 nm
with increasing hydrion concentration.
Protonation of the π−π conjugated benzo[a]phenoxazine to
form benzo[a]phenoxazinium causes the absorption maximum
to exhibit a red shift up to 121 nm (Figure 2). The N-aromatic
substituent allowed an equilibrium between benzo[a]-
phenoxazine and benzo[a]phenoxazinium to form in near-
neutral or subacid pH conditions. The 5,9-nitrogen atoms serve
as electron donors to the benzo[a]phenoxazinium moiety of
1a + H+, and the weak emission from benzo[a]phenoxazinium
is induced by intramolecular charge transfer (ICT). The situa-
tion is changed when the electron-donating methoxy group in
1a + H+ is replaced by an electron-withdrawing nitro group in
1b + H+; specific emission is observed when ICT is weakened
by the electron-withdrawing inductive effect of the nitro group.
Thus, the introduction of electron-withdrawing groups is
required to design fluorescent pH probes. The poor solubility
of probe 1b in water will limit the in vitro application, and then
the design of water-soluble probes with electron-deficient aro-
matic groups in an attempt to produce NIR pH probes is
prompted in the next stage.
RESULTS AND DISCUSSION
■
Optical Responses of Benzo[a]phenoxazines with
N-Aromatic Group Substituents to pH. The change in
Synthesis and pH-Induced Optical Responses of N-
Pyridiniumylbenzo[a]phenoxazines. N-Pyridiniumylbenzo-
[a]phenoxazines (3a−3c) were selected to fit the above
requirements by alkylation reaction from their pyridinyl
derivatives (2a−2c, Scheme 2). Compounds 2a−2c were
heated under reflux overnight with excess iodoethanol to afford
the corresponding salts. Nitrate salts were obtained by ion
exchange using anion-exchange resin to improve their
biocompatibility. Probes 3a−3c are soluble in water, and the
imino group serves as the sensitive point to hydrions. The same
reaction with iodoethanol was performed using 1a and 1b, and
no new products were detected by TLC. This indicates the
Figure 1. Absorption and emission properties of probes 1a and 1b
(10 μM) toward different pH values in DMSO−H2O (v/v = 4:1)
solution: (A) probe 1a (λex = 590 nm); (B) probe 1b (λex = 600 nm);
insets are the absorption spectra and photographs of the samples.
Figure 2. Proposed mechanism and summary of the optical properties of probes 1a and 1b.
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Scheme 2. Synthesis of the Benzo[a]phenoxazine-Based Probes (3a−3c)
nitrogen atom in the pyridine ring is the only site reactive
toward iodoethanol in the starting material.
The absorption maximum of 3a in buffer solution containing
0.1% DMSO at 630 nm (pH = 6.3, ε = 3.3 × 104 M−1·cm−1)
shifts to 597 nm (pH = 0.9, ε = 2.9 × 104 M−1·cm−1)
(Supporting Information Figure S1A). The weak emission
maximum at 723 nm (pH = 6.3) slowly shifts to 688 nm (pH =
0.9), and the full range of emission is in 625−850 nm
(Figure 3A). The relative fluorescent intensity of 3a increases
17.3 times (Supporting Information Table S1), and the
calculated pKa value is 2.7.32 Probe 3b has an optical response
in pH range of 3.6−8.4 (Figure 3B, Supporting Information
Figure S1B). Its absorption maximum at 575 nm (pH = 8.4, ε =
2.3 × 104 M−1·cm−1) changes to 663 nm (pH = 3.6, ε = 4.0 ×
104 M−1·cm−1). As shown in Figure 3B, the relative fluorescent
intensity of 3b is enhanced by 40.1-fold (Supporting
Information Table S1) from the very weak emission at 694
nm (pH = 8.4) to remarkable emission at 697 nm (pH = 3.6).
This behavior suggests that 3b is nearly an off−on-type probe.
Its full range of emission is also located in 625−850 nm, and its
pKa is 5.8. The absorption spectra of 3c exhibit a small shift
from 593 nm (pH = 9.5, ε = 2.0 × 104 M−1·cm−1) to 599 nm
(pH = 4.5, ε = 3.4 × 104 M−1·cm−1) (Supporting Information
Figure S1C). Its weak emission at 729 nm (pH = 9.5) shifts to
694 nm (pH = 4.5) with an emission range of 625−850 nm
(Figure 3C), its relative fluorescent intensity increases by 8.2
times (Supporting Information Table S1), and its pKa is 7.1.
The pKa values of the three probes, 2.7, 5.8, and 7.1 for 3a, 3b,
and 3c, respectively, indicate that the distance-dependent
electron-withdrawing ability between the imino group and
ammonium cation in pyridinium influences the affinity between
the imino group and hydrion. Therefore, designable pKa values
could be achieved by changing the electron-withdrawing
substituent. The photochemical properties of the three probes
in water are shown in Table 1. Because the Stokes shifts of the
probes are up to 136 nm, self-absorption will be minimized in
test. The fluorescence quantum yields of the probes, which
were obtained using Oxazine 1 as a standard sample,33 are
increased by 10, 20, and 5 times for 3a, 3b, and 3c, respectively,
after protonation of the free bases.
Figure 3. Emission properties of probes 3a−3c (10 μM, λex = 600 nm)
with different pH values in buffer solution containing 0.1% DMSO:
(A) probe 3a, (B) probe 3b, (C) probe 3c; insets are the fluorescence
intensity changes with different pH at (A) 688, (B) 697, and (C) 694 nm.
Probes 3a and 3c exhibit weak emission at 723 and 729 nm
(Figure 3, parts A and C), respectively. Their emission signals
are blue-shifted under acidic conditions. In contrast, the
emission from 3b shows a red shift from 694 to 697 nm
(Figure 3B), which is similar to the emission from 1b at
different pH’s (Figure 1B). The proposed emission mechanism
is shown in Figure 4. The real structure of 3a contains
contributions from two resonance structures (i and ii).
Resonance structure ii has the characteristic of benzo[a]-
phenoxazinium, which contributes to the weak emission at
723 nm. The emission properties of 3c are similar to those of
3a. Conversely, the real structure of 3b only involves the
benzo[a]phenoxazine skeleton, so its absorption and emission
properties differ from those of 3a and 3c.
Alkyl-substituted Nile blue derivatives tend to aggregate in solu-
tion,34 resulting in new peaks in their concentration-dependent
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Table 1. Photochemical Properties of Probes 1a, 1b, and 3a−3c
a
compd
absmax (nm)
emmax (nm)
Stokes shift (nm)
extinction coefficient (M−1·cm−1
)
Φ
pKa
b
c
1a (protonated)
666
545
683
565
597
630
663
575
599
593
643
643
720
641
688
723
697
694
694
729
23
98
3.9 × 104
1.9 × 104
7.6 × 104
4.3 × 104
2.9 × 104
3.3 × 104
4.0 × 104
2.3 × 104
3.4 × 104
2.0 × 104
0.004
0.01
4.5
d
d
1a (deprotonated)
b
e
1b (protonated)
37
0.005
0.003
0.01
3.2
1b (deprotonated)
76
b
e
3a (protonated)
91
2.7
f
3a (deprotonated)
93
0.001
0.02
b
e
3b (protonated)
34
5.8
f
3b (deprotonated)
119
95
0.001
0.01
b
e
3c (protonated)
7.1
f
3c (deprotonated)
136
0.002
a
b
Oxazine 1 (Φ = 0.14, ethanol) was used as the reference for fluorescence quantum yields in measurements (ref 33). Protonated species were
c
d
tested in 0.1 M citric acid solution (pH = 1.6) containing 0.1% DMSO. pKa values were calculated by absorption intensities. Deprotonated species
were tested in DMSO. pKa values were calculated by fluorescence intensities with reported method (ref 32). Deprotonated species were tested in
e
f
0.2 M disodium hydrogen phosphate solution (pH = 9.3) containing 0.1% DMSO.
Figure 4. Proposed mechanism and summary of the optical properties of probes 3a−3c.
normalized absorption spectra. The concentration-dependent
optical properties of the N-pyridiniumylbenzo[a]phenoxazine
derivatives were examined. As shown in Supporting Informa-
tion Figures S2 and S3, the absorption maxima of probes
3a−3c in acidic or basic conditions showed a linear dependence
on concentration (2−20 μM). No obvious new peaks were
observed in normalized absorption−concentration plots
compared with those reported for alkyl-substituted benzo[a]-
phenoxazinium. When solutions containing the three probes
were changed to low or high pH, the optical properties
switched to reflect the new conditions (Figure 5, Supporting
Information Figure S4). This indicates that the stable optical
properties of these probes make their potential application in
Figure 5. Reversible fluorescence spectra of probes 3b (10 μM, λex
600 nm) in buffer solution containing 0.1% DMSO; insets are
=
real-time quantitative analysis of solution pH.
The probes 3a−3c have relatively similar structures (Scheme 2),
their emission spectra are analogous, and meanwhile the pKa
values are quite different (Figure 3). The hybrid of dyes 3a−3c
would be possible for a NIR composite probe with an extensive
pH-sensitive range. After several tries, the composite probe
with the mole ratio of 3a/3b/3c = 2:1:2 was found to fit the
requirement. As shown in Figure 6A, the composite probe with
reversible absorption spectra and photograph of the samples.
27.8 μM shows fluorescent response toward wide pH, and the
fluorescent intensities are linear agreement with the pH in the
range of pH = 1.9−8.0 with R = 99.45%. This makes the
quantitative detection of pH achievable through the composite
probe.
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The fluorescent response of the composite probe was
also evaluated by fluorescence imaging with HeLa cells
(Figure 7). HeLa cells (Figure 7A) were sequentially
incubated with the composite probe (0.8 μM) in PBS buffer
(pH = 7.4, Figure 7B), basic high K+ buffer (pH = 8.0, Figure
7C), acidic high K+ buffer (pH = 6.0, Figure 7D), and then
acidic high K+ buffer (pH = 4.0, Figure 7E). Nigericin
(2 μg·mL−1) was used to promote the acid−base balance
between HeLa cells and their conditioned medium.35 The
composite probe shows red emission (Figure 7B) since
the acidic condition inside the cancer cell in biological
conditions,36,37 NIR emission was gradually increased
(Figure 7C−E) when the concentration of the hydrion
was increasing. These in vitro experiments indicate that
the composite probe of 3a−3c can be used to sense pH
in vitro.
CONCLUSIONS
■
The absorption and emission properties of benzo[a]-
phenoxazine derivatives with an N-aromatic group substituent
induced by the equilibrium between benzo[a]phenoxazine and
benzo[a]phenoxazinium were investigated. The compounds
exhibited pH-dependent optical responses under near-neutral
and subacid conditions. Derivatives with an electron-deficient
aromatic group attached to the nitrogen in the imino group of
benzo[a]phenoxazine showed potential as pH sensors. Three
new water-soluble pH probes, 3a−3c, were designed and
synthesized. The emission maxima of 3a−3c locate at 688, 694,
and 697 nm with the full emission ranging from 625 to 850 nm;
their optical properties are reversible in aqueous solution
containing 0.1% DMSO. The pH-dependent fluorescent
intensity of 3b increased by 40.1 times in aqueous solution,
and the pKa values of 3a−3c are 2.7, 5.8 and 7.1, respectively. A
composite probe, which is mixed by 3a−3c with the mole ratio
at 3a/3b/3c = 2:1:2, has a linear pH−emission intensity
relationship in pH 1.9−8.0. The in vitro experiment with HeLa
cells indicated that the composite probe of 3a−3c can be used
for real-time pH sensing in cells. The reported method could be
useful for the design of oxazine-based pH sensors.
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectra used to characterize 3a−3c, absorption properties of
3a−3c, and other related information. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: ge_jianfeng@hotmail.com (J.-F.G); lujm@suda.edu.cn
(J.-M.L.); m-ihara@hoshi.ac.jp (M.I.).
■
Author Contributions
∥W.L. and R.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (51273136) and a project funded
by the Priority Academic Program Development of Jiangsu
Higher Education Institutions. Syntheses were supported by
the Creation and Support Program for Start-Ups from
Universities, Adaptable and Seamless Technology Transfer
Program through Target-Driven R&D, Japan Science Technol-
ogy Agency (JST).
Figure 6. Emission properties of the composite probe by 3a−3c (27.8
μM, mole ratio of 3a/3b/3c = 2:1:2). (A) Emission properties of the
composite probe (λex = 600 nm) with different pH values in buffer
solution containing 1.0% DMSO. (B) The fluorescence intensity
changes toward pH at 700 nm.
Figure 7. Fluorescence images of HeLa cells with composite probe of 3a−3c (mole ratio of 3a/3b/3c = 2:1:2): (A) bright-field image; (B)
fluorescence image of HeLa cells incubated with the composite probe (0.8 μM) in PBS buffer (pH = 7.4) for 20 min; (C) fluorescence image of
HeLa cells incubated with high K+ buffer (pH = 8.0) with nigericin (2 μg·mL−1) for 10 min; (D) fluorescence image of HeLa cells incubated with
high K+ buffer (pH = 6.0) with nigericin (2 μg·mL−1) for 10 min; (E) fluorescence image of HeLa cells incubated with high K+ buffer (pH = 4.0)
with nigericin (2 μg·mL−1) for 10 min.
7424
dx.doi.org/10.1021/ac4013539 | Anal. Chem. 2013, 85, 7419−7425

Analytical Chemistry
Article
(35) Yuan, L.; Lin, W.; Feng, Y. Org. Biomol. Chem. 2011, 9, 1723−
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