Ratiometric Fluorescent Probe for Lysosomal pH Measurement and Imaging in Living Cells Using Single-Wavelength Excitation

Article abstract of DOI:10.1021/acs.analchem.7b00754

A novel lysosome-targeting ratiometric fluorescent probe (CQ-Lyso) based on the chromenoquinoline chromorphore has been developed for the selective and sensitive detection of intracellular pH in living cells. In acidic media, the protonation of the quinol

Full text of DOI:10.1021/acs.analchem.7b00754

Article
A Ratiometric Fluorescent Probe for Lysosomal pH Measurement
and Imaging in Living Cells Using Single-Wavelength Excitation
Xingjiang Liu, Yuanan Su, Huihui Tian, Lei Yang, Hongyan Zhang, Xiangzhi Song, and James Walter Foley
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 May 2017
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Analytical Chemistry
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A Ratiometric Fluorescent Probe for Lysosomal pH Measurement
and Imaging in Living Cells Using Single-Wavelength Excitation
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a
a
a
a
b*
a,c*
Xingjiang Liu , Yuanan Su , Huihui Tian , Lei Yang , Hongyan Zhang , Xiangzhi Song
and
d
James W. Foley
a
College of Chemistry & Chemical Engineering, Central South University, Changsha 410083, China. Email:
song@rowland.harvard.edu.
b
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Beijing 100190, China. Email: zhanghongyan@mail.ipc.ac.cn.
c
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
d
Rowland Institute at Harvard, Harvard University, Cambridge MA 02142, USA.
ABSTRACT: A novel lysosome-targeting ratiometric fluorescent probe (CQ-Lyso) based on the chromenoquinoline
chromorphore has been developed for the selective and sensitive detection of intracellular pH in living cells. In acidic media, the
protonation of the quinoline ring of CQ-Lyso induces an enhanced intramolecular charge transfer (ICT) process, which results in
large red-shifts in both the absorption (104 nm) and emission (53 nm) spectra which forms the basis of a new ratiometric
fluorescence pH sensor. This probe efficiently stains lysosomes with high Pearson’s colocalization coefficients using
LysoTracker®Deep Red (0.97) and LysoTracker®Blue DND-22 (0.95) as references. Importantly, we show that CQ-Lyso
quantitatively measures and images lysosomal pH values in a ratiometric manner using single wavelength excitation.
Intracellular pH values play vital roles in mediating many
physiological processes such as cell metabolism, enzyme ac-
variable probe concentration, instrument sensitivity and envi-
ronmental conditions through the self-calibration of two emis-
sion bands. Among the previously reported ratiometric fluo-
1
-4
36
tivity and immunization. Lysosomes are acidic organelles
pH 4.0-5.5) in eukaryotic cells that maintain their pH value
(
rescent probes for lysosomal pH, noteworthy are the works of
Kim and colleagues who have developed two-photon rati-
+
by the inward pumping of protons (H ions) from the cytosol
across the cell membrane via proton pumps. Low pH is neces-
sary for the hydrolytic enzymes and proteins contained in ly-
29
ometric fluorescent probes, Ma’s group who have developed
a near-infrared ratiometric fluorescent probe based on the sem-
5-8
32
sosome to function at their optimum activity. For example,
icyanine skeleton in 2014, and, most recently, Lin et. al.’s
digestive enzymes in lysosomes require an acidic environment
cleverly designed probe which integrated ICT, PET and FRET
in their ratiometric fluorescent pH probe. The importance of
34
in the range of pH 4.0-5.5 to degrade ingested proteins, DNA,
9-11
polysaccharides, lipids, viruses, RNA and bacteria.
Abnor-
these contributions is that each incorporates an aspect of fluo-
rescent probe design that is generally prized by practicing life-
scientists. These include, in addition to each being ratiometric,
at least one of the following: (1) The ability to utilize a single
excitation wavelength for cell imaging because this signifi-
cantly simplifies data acquisition, minimizes background noise
associated with multiple wavelength excitations, and reduces
mal pH values in lysosomes can result in cellular dysfunction
and consequently lead to many severe lysosomal diseases such
1
2-13
as lipid storage disorders and mucolipidoses.
Because of
the critical importance of pH for lysosomes to function proper-
ly, it is essential for health professional to have simple and
effective methodologies for the rapid, selective and sensitive
determination of lysosomal pH values in biological specimens.
3
7-38
auto-fluorescence interference from cellular organelles.
(2)
Emission in the red or near infrared (NIR) spectral region be-
cause longer wavelength photons have superior tissue penetra-
tion and have minimal interference from auto-fluorescence
emitted by cellular components. For lysosome-targeting fluo-
rescent probes, a high Pearson’s coefficient is mandatory for
Due to their high sensitivity, easy operation, real-time and
noninvasive imaging properties, fluorescent probes have been
useful tools for sensing and imaging a variety of cations, ani-
39
1
4-18
ons and biomolecules in living cells and tissues.
They are
especially useful when they are able to become predominantly
localized in a single cellular component because they provide
accurate and reliable information about the micro-environment
specific staining, which is measured by colocolization experi-
40-41
ments.
To date, with the exception of Ma et. al.’s work, all
1
9-26
reported ratiometric fluorescent probes emit at wavelength
shorter than 600 nm. As a result, lysosomotropic pH fluores-
cent probes simultaneously having a high Pearson’s coefficient
in addition to the above mentioned desiderata remain in a high
demand. The scarcity of few such materials was the inspiration
for the work reported herein (shown in Scheme S1, ESI†).
within the targeted organelle.
In recent years, a number of
27
fluorescence based pH probes have been reported. However,
few of them are selective pH indicator stains for
24,27-33
lysosomes,
and fewer still specifically determine pH in
28-35
lysosomes in a ratiometric manner.
It’s known that rati-
ometric fluorescent probe can avoid data distortions caused by
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Page 2 of 9
Chromenoquinoline derivatives are mainly used as estro-
chromatography were supplied by Qingdao Ocean Chemicals
(China).
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genic agents and, with the exception of Talukdar’s and
44
Lin’s works, have seldom been subjected to photophysical
property studies. In this report, we designed and synthesized a
lysosome-targeting chromenoquinoline (CQ-Lyso) that is a
fluorescent probe for measuring the pH of lysosomes in living
cells using only single wavelength excitation (molecular struc-
ture and synthesis shown in Scheme 1). A morpholine moiety
was introduced in CQ-Lyso for lysosome-targeting purposes.
Although this probe exhibits a strong yellow florescence in
neural/basic media, when treated with an increasing gradient
of acidity, the quinoline moiety in CQ-Lyso would be ex-
Theoretical Calculations. Density functional theory (DFT)
calculations with Becke’s three parameterized Lee-Yang-Parr
(
B3LYP) exchange functional were performed for geometry
+
optimization of CQ-Lyso in the absence/presence of H using
a Gaussian 09 program.
45-46
The basis set was 6-31G (d, p).
The energy levels of the highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital (LUMO)
were obtained using time-dependent density functional theory
with the same basis sets as mentioned above.
0
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9
0
Spectral Measurements. The absorption and emission spectra
were measured in Britton-Robinson (B-R) buffer solution
pected to proportionally become protonated (shown in Scheme
31, 45
2
). Based on the previous report,
we expected that such
(
40.0 mM) with 30% CH CN (v/v). A stock solution of CQ-
3
protonation would promote an enhanced intramolecular charge
transfer (ICT) process which would concomitantly induce
significant red-shifts in both its absorption and emission spec-
tra. In this manner the pH-sensitive emission spectra of CQ-
Lyso would serve as a ratiometric fluorescent probe for lyso-
somal pH measurements.
Lyso was prepared at 1.0 mM in CH CN. The Britton-
3
Robinson buffers with pH values at 2.0, 3.0, 4.0, 4.2, 4.4, 4.6,
4
7
.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4,
.6, 7.8, 8.0 and 9.0 were prepared according to the standard
procedures. Solutions for testing the effect of possible
interfering species were prepared by dissolving Al(NO ) ,
3
3
Scheme 1. Synthetic route of CQ-Lyso.
CdCl , CoCl , CrCl , FeCl , NiCl , Pb(NO ) , ZnCl , FeCl ,
2
2
3
3
2
3 2
2
2
CuSO , BaCl , MnCl , MgCl , CaCl , NaCl, KCl, glucose,
4
2
2
2
2
GSH, Cys, Hcy, Gly, Glu, Val, Arg, Lys, Try, Thr, Asp, H O ,
2
2
NaHS, NaNO , Na PO , Na S O , NaSCN, Na SO , Na SO
4
3
3
4
2
2
3
2
3
2
-
and NaClO in twice-distilled water, respectively. ONOO was
prepared according to literature method.
47-48
For titration
experiments, a test solution of CQ-Lyso (5.0 µM) was
prepared by placing 15.0 µL of CQ-Lyso (1.0 mM) into a
mixture of B-R buffer (2.1 mL) and CH CN (0.885 mL) in a
3
quartz optical cell with 1.0 cm optical path length. For
selectivity experiments, test solutions were prepared by
placing the stock solution of CQ-Lyso into the mixture of B-R
buffer and CH CN, and then adding the appropriate amount of
(
a) 1,4-dibromobutane, acetone, K CO , 56 °C, 48 h; (b) mor-
3
2
3
the stock solution containing the corresponding analyte. NaOH
pholine, DMF, K CO , KI, 80 °C, 12 h; (c) Pd/C, H , CH OH,
2
3
2
3
(
1.0 M) or HCl (1.0 M) were used to adjust the pH of the test
6
1
5 °C, 12 h; (d) 3-bromoprop-1-yne, acetone, K CO , 56 °C,
2 3
2 h; (e) CuI, DMF, 110 °C, 4 h.
33,49
solutions.
All the resulting solutions were shaken well and
incubated at 25 °C for 15 min before absorption and
fluorescence spectral measurements.
Scheme 2. The proposed mechanism of CQ-Lyso for pH
measurements in aqueous media or lysosome.
Cell Culture and Fluorescence Imaging. The culture of
HeLa cells was conducted in DMEM (Dulbecco’s modified
Eagle’s medium) at 37 °C with 10% fetal bovine serum and
1
9
% penicillin as supplement in atmosphere of 5% CO and
5% air. When the adherent cells with stable growth in
2
50
culture flasks reached 85%-95%, cells were seeded in glass
dishes and allowed to adhere for 24 hours. Then, discard the
culture fluid and wash cells three times with PBS buffer before
EXPERIMENTAL SECTION
the
experiments.
LysoTracker®Deep
Red
and
LysoTracker®Blue DND-22 were used as the colocolization
agents. Chloroquine was used as the stimulant to induce pH
increase of lysosome.
Materials and Instruments. All commercial reagents were
used without further purification. Twice-distilled water was
used throughout all the experiments. Bruker 400 and 500
1
13
spectrometers were used to record H and C NMR spectra
with tetramethylsilane (TMS) as an internal standard for
chemical shift. A LCT Premier XE mass spectrometer (Waters)
was used to obtain high-resolution mass spectra. Absorption
and fluorescence spectra were respectively measured on a UV-
Synthesis of 1-(4-bromobutoxy)-4-nitrobenzene (1). This
5
1
compound was prepared according to a literature method. To
a solution of 4-nitrophenol (0.5760 g, 4.0 mmol) and 1,4-
dibromobutane (1.3281 g, 8.0 mmol) in acetone (6.0 mL) was
added potassium carbonate (0.8482 g, 8.0 mmol). The
resulting mixture was stirred at 56 °C for 48 h. After being
cooled to room temperature, the reaction mixture was filtered
and the filtrate was concentrated in vacuum to give an oil
residue which was purified by flash silica gel chromatography
(dichloromethane/petroleum ether as eluent, v/v = 1:2) to
afford compound 1 as a white solid (0.6012 g, 54.5% yield).
2
450 spectrophotometer and a F-280 spectrometer (Tianjin
Gangdong Sci. & Tech. Development Co. Ltd). A Leici PHS-
C meter was used for pH measurements. Fluorescence
imaging experiments were performed on an Olympus FV1000
and a Nikon ARsiMP confocal microscopes. TLC silica gel
plates and silica gel (mesh 200-300) for column
3
1
H NMR (400 MHz, CDCl ) δ 8.18 (d, J = 9.2 Hz, 2H), 6.95
3
H
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Analytical Chemistry
(
d, J = 9.3 Hz, 2H), 4.10 (t, J = 5.9 Hz, 2H), 3.51 (t, J = 6.4 Hz,
over anhydrous sodium sulfate. Following removal of the sol-
vent, the obtained crude product was further purified by flash
silica gel chromatography (ethyl acetate/petroleum ether as
1
3
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2H), 2.40-1.81 (m, 4H). C NMR (100 MHz, CDCl ) δ 163.9,
141.5, 125.9, 114.4, 67.8, 33.2, 29.2, 27.6.
3 C
eluent, v/v = 1:1) to give CQ-Lyso as a yellow solid (0.0902 g,
Synthesis of 4-(4-(4-nitrophenoxy)butyl)morpholine (2). To
a solution of compound 1 (1.0963 g, 4.0 mmol) and
morpholine (0.4482 g, 5.2 mmol) in DMF (10.0 mL) was
added potassium iodide (0.1713 g, 1.0 mmol) and potassium
carbonate (0.5531 g, 5.2 mmol). The resulting mixture was
stirred at 80 °C for 12 h. After being cooled to room
temperature, the reaction mixture was poured into 40.0 mL of
cold water. Then, the mixture was extracted with
dichloromethane (30.0 mL × 3), and the organic layers were
combined and dried over anhydrous sodium sulfate. After
removal of the solvent, compound 2 was obtained as a yellow
solid (1.1012 g, 98.2% yield). HRMS (ESI) m/z: calcd for
+
18.5% yield). HRMS (ESI) m/z: calcd for C H N O [M+1] ,
4
30 36
3
3
1
86.2757; found, 486.2756. H NMR (400 MHz, DMSO-d )
6
δH 7.89 (s, 1H), 7.81 (d, J = 9.1 Hz, 1H), 7.67 (s, 1H), 7.29 (dd,
J = 9.1, 2.8 Hz, 1H), 7.25 (d, J = 2.8 Hz, 1H), 5.24 (s, 2H),
4
2
6
.10 (t, J = 6.5 Hz, 2H), 3.62-3.49 (m, 4H), 3.20-3.16 (m, 4H),
.75 (t, J = 6.3 Hz, 2H), 2.63 (t, J = 6.5 Hz, 2H), 2.36-2.32 (m,
H), 1.91-1.88 (m, 4H), 1.83-1.77 (m, 2H), 1.65-1.58 (m, 2H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
9
0
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2
3
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5
6
7
8
9
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2
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5
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9
0
1
3
C NMR (100 MHz, CDCl ) δ 156.1, 153.7，148.4, 145.6,
3
C
1
1
2
44.4, 130.1, 129.0, 127.6, 125.1, 122.5, 121.5, 116.1, 111.0,
07.9, 106.4, 68.6, 67.9, 67.0, 58.6, 53.7, 50.1, 49.6, 27.4,
7.2, 23.1, 22.1, 21.3, 21.0.
+
1
C H N O [M+1] , 281.1501; found, 281.1512. H NMR
14
21
2
4
RESULTS AND DISCUSSION
(400 MHz, CDCl ) δ 8.21 (d, J = 9.3 Hz, 2H), 6.95 (d, J = 9.3
3 H
Hz, 2H), 4.10 (t, J = 6.3 Hz, 2H), 3.89-3.58 (m, 4H), 2.48-
2
Sensing properties of probe CQ-Lyso to pH. The optical
properties of CQ-Lyso (5.0 µM) were studied in Britton-
13
.41(m, 6H), 1.92-1.85 (m, 2H), 1.74-1.67 (m, 2H). C NMR
(100 MHz, CDCl ) δ 164.1, 141.4, 125.9, 114.4, 68.5, 67.0,
Robinson buffer with 30% CH CN as a co-solvent at different
3
C
3
5
8.5, 53.7,26.9, 22.9.
pH values. The probe featured an intense absorption band
between 350-470 nm with a maximum at 397 nm in
neutral/basic media. When the pH was made more acidic, the
397 nm band gradually disappeared and a new band in the
Synthesis of 4-(4-morpholinobutoxy)aniline (3). Compound
(0.8402 g, 3.0 mmol) was hydrogenated at slightly above the
2
atmosphere pressure in methanol (20.0 mL) at 65 °C for 12 h
with 10% Pd/C (0.1803 g) as a catalyst. Next, the reaction
mixture was filtered through celite to remove the catalyst.
Filtrate was concentrated under vacuum to yield compound 3
as a brown solid (0.6604 g, 88.0% yield). HRMS (ESI) m/z:
calcd for C H N O [M+1] , 251.1760; found, 251.1760. H
NMR (400 MHz, CDCl ) δ 6.73 (d, J = 8.8 Hz, 2H), 6.63 (d,
J = 8.8 Hz, 2H), 3.90 (t, J = 6.3 Hz, 2H), 3.73-3.71 (m, 4H),
3
range 410-580 nm (λ max = 501 nm) emerged and the color
abs
visually changed from pale yellow to red (shown in Figure S1,
ESI†). This result is the basis upon which CQ-Lyso could be
used to measure pH via spectroscopic absorptivity analysis or,
more crudely as a naked-eye pH detector. When excited at 470
nm, the probe displayed a strong yellow florescence with
+
1
1
4
23
2
2
3
H
λ max = 560 nm in neutral/basic media (Φ = 0.26, using
em
52
coumarin 102 in ethanol at 25 °C as a standard, Φ = 0.93). In
.37 (s, 2H), 2.45-2.38 (m, 6H), 1.87-1.72 (m, 2H), 1.71-1.56
1
3
contrast, when the pH of the solution was down-regulated
(m, 2H). C NMR (100 MHz, CDCl ) δ 152.1, 140.0, 116.4,
3
C
(shown in Figure 1) to more acidic values, the fluorescence
1
15.6, 68.3, 67.0, 58.7, 53.7, 27.3, 23.1.
gradually changed from yellow to red (λ max = 613 nm, Φ =
em
Synthesis of 8-hydroxy-1,2,3,5,6,7-hexahydropyrido [3,2,1-
ij]quinoline-9-carbaldehyde (4). Compound was
synthesized according to a literature procedure.
0
.12, using fluorescein in 0.1 M NaOH at 25 °C as a standard,
4
53
4
6
Φ = 0.92) under the same excitation wavelength. Thus,
increasing acidity was accompanied by red-shifts for both
absorption and emission spectra of 104 nm and 53 nm,
respectively, when the pH changed from neutral to acidic. It
was found that the ratio between the fluorescence intensities at
613 nm and 560 nm (I613 nm/I560 nm) displayed a 10-fold
enhancement (from 0.524 at pH 7.4 to 5.586 at pH 4.0,
Synthesis of 8-(prop-2-ynyloxy)-1,2,3,5,6,7-
hexahydropyrido[3,2,1-ij]quinoline-9-carbaldehyde (5). To
a solution of compound 4 (0.2173 g, 1.0 mmol) and 3-
bromoprop-1-yne (0.2380 g, 2.0 mmol) in acetone (10.0 mL)
was added potassium carbonate (0.2764 g, 2.0 mmol). The
resulting suspension was refluxed for 12 h. The reaction
mixture was filtered and the filtrate was concentrated under
vacuum to give a crude product, which was further purified
using flash silica gel chromatography (ethyl acetate/petroleum
ether as eluent, v/v = 1:3) to afford compound 5 as a yellow
solid (0.2002 g, 78.4% yield). HRMS (ESI) m/z: calcd for
C H NO [M+1] , 256.1338; found, 256.1337. H NMR (500
MHz, CDCl ) δ 10.03 (s, 1H), 7.34 (s, 1H), 4.63 (d, J = 2.4
Hz, 2H), 3.52-3.05 (m, 4H), 2.81 (t, J = 6.3 Hz, 2H), 2.73 (t, J
= 6.3 Hz, 2H), 2.55 (t, J = 2.4 Hz, 1H), 2.01-1.82 (m, 4H).
NMR (125 MHz, CDCl ) δ 187.8, 157.8, 148.8, 127.5, 117.5,
1
2
+
1
16
18
2
3
H
13
C
3
C
17.0, 112.6, 78.7, 76.1, 62.1, 50.0, 49.7, 27.3, 21.4, 21.3,
0.7.
Synthesis of CQ-Lyso. Compound 3 (0.2503 g, 1.0 mmol),
cuprous iodide (0.0573 g, 0.3 mmol) and compound 5 (0.2662
g, 1.0 mmol) were placed in 5.0 mL dry DMF. The resulting
solution was stirred at 110 °C for 4 h. After being cooled to
room temperature, the mixture was poured into 40.0 mL of
cold water and extracted with ethyl acetate (40.0 mL × 4). The
organic layers were combined, washed with water and dried
Figure 1. Fluorescence spectra of CQ-Lyso (5.0 µM) in B-R
buffers with 30% CH
wavelength was 470 nm. Inset: photographs of CQ-Lyso in B-
CN at various pH values. Excitation
3
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2
+
2+
2+
2+
R buffer with 30% CH CN at pH 7.4 (left) and pH 4.0 (right)
under 365 nm irradiation.
Fe , (15) Cu , (16) Ba , (17) Mn , (18) glucose, (19) GSH,
(20) Cys, (21) Hcy, (22) H O , (23) Gly, (24) Glu, (25) Val,
3
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2
-
(26) Arg, (27) Lys, (28) Try, (29) Thr, (30) Asp, (31) ClO ,
(32) HS , (33) NO , (34) ONOO , (35) PO , (36) S O , (37)
SCN , (38) SO , (39) SO .
Figure 2). Based on the Henderson-Hasselbalch equation:
-
-
-
3-
2-
log[(Imax-I)/(I-Imin)] = pH - pK , the pK value of CQ-Lyso was
3
4
2
3
a
a
-
2-
2-
4
9,54
calculated to be 5.0.
Notably, a good linearity (R = 0.99)
3
4
between the ratio and pH in the range of 4.0 to 6.0 was
obtained, suggesting that this probe had the potential to detect
lysosomal pH (4.0-5.5) in a ratiometric manner (shown in
Figure 2, inset). It’s noted that the single-wavelength
excitation for this ratiometric fluorescent probe simplified the
experimental measurements and improved the data accuracy in
fluorescence detection.
Selectivity studies. In order to evaluate the selectivity of CQ-
Lyso, we investigated the fluorescence spectra of CQ-Lyso
(5.0 µM) in response to relevant interfering species including
3
+
2+
2+
3+
3+
2+
2+
2+
2+
cations (Al , Cd , Co , Cr , Fe , Ni , Pb , Zn , Fe ,
2
+
2+
2+
2+
2+
Cu , Ba and Mn , 0.05 mM for each; Mg and Ca , 0.1
+
+
-
mM for each; Na and K , 1.0 mM for each), anions (HS ,
0
1
2
3
4
5
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7
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-
3-
2-
-
2-
2-
NO , PO , S O , SCN , SO , SO , 0.5 mM for each), and
3
4
2
3
3
4
small biomolecules (glucose, GSH, Cys, Hcy, Gly, Glu, Val,
Arg, Lys, Try, Thr and Asp, 1.0 mM for each) as well as
-
reactive oxygen species (H O , 1.0 mM; ONOO , 0.5 mM;
2
2
-
ClO , 0.1 mM). As shown in Figure 3, little or no interfering
effects were observed on the ratio (I613 nm/I560 nm) of CQ-Lyso
in the presence of all the above species with the exception of
ClO at pH 4.0. Under acidic condition, the strong oxidative
ability of ClO likely destroys the probe which leads to its
-
-
5
5
failure in the determination of pH (shown in Figure S10).
These results supports the probe has excellent selectivity for
pH measurement.
Figure 2. Ratio of fluorescence intensity (I613 nm/I560 nm) of CQ-
Lyso (5.0 µM) in B-R buffers with 30% CH CN at different
3
pH values. Inset: the linear relationship between I613 nm/I560 nm
and pH value (4.0 to 6.0).
Figure 4. The time course of the fluorescence intensity ratios
(
I613 nm/I560 nm) of CQ-Lyso (5.0 µM) in B-R buffer with 30%
CH CN at pH 4.0 and pH 7.4.
3
Figure 3. Fluorescence intensity ratio (I613 nm/I560 nm) of CQ-
Lyso (5.0 µM) in the presence of relevant species in B-R
Figure 5. The fluorescence reversibility of CQ-Lyso (5.0 µM)
in B-R buffer with 30% CH CN between pH 4.0 and pH 7.4.
3
buffers with 30% CH CN at pH 4.0 (a) and pH 7.4 (b). (1)
3
3
+
2+
2+
2+
3+
3+
Kinetic and reversible studies. The time-dependent
fluorescence of CQ-Lyso (5.0 µM) at pH 4.0 and 7.4 was
Blank, (2) Al , (3) Ca , (4) Cd , (5) Co , (6) Cr , (7) Fe ,
8) K , (9) Mg , (10) Na , (11) Ni , (12) Pb , (13) Zn , (14)
+
2+
+
2+
2+
2+
(
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investigated by monitoring the fluorescence intensity ratio at
613 nm and 560 nm (I613 nm/I560 nm), respectively, as a function
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of time. As shown in Figure 4, after the addition of CQ-Lyso
to B-R buffer solutions at pH 4.0 and 7.4, respectively, the
intensity ratio (I613 nm/I560 nm) of this probe rapidly reached an
equilibrium and remained unchanged for at least 10 min. In
addition, the reversibility of this probe was also studied in B-R
buffer at pH 7.4 and 4.0. As shown in Figure 5, this probe
functions well even after 6 cycles. From these results, it can be
concluded that this probe can server as a real-time, reversible
monitor for pH measurement.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
Mechanism studies
The optical performance of CQ-Lyso induced by pH changes
is consistent with the occurrence of the protonation process
depicted in Scheme 2. To support this proposed mechanism,
Figure 7. The HOMO-LUMO energy gaps and the interfacial
1
+
H NMR spectral analysis was performed on CQ-Lyso by
plots of the orbitals for CQ-Lyso and CQ-Lyso+2H .
titration studies using trifluoroacetic acid (TFA) in DMSO-
Fluorescence Imaging in Living Cells. Encouraged by the
excellent properties of CQ-Lyso in aqueous media, we
evaluated the ability of this probe to visualize intracellular pH
in living cells. First, an MTT assay was performed on CQ-
Lyso, the result of which showed that this probe was non-toxic
for living cells below 10.0 µM (shown in Figure S2, ESI†).
Next, the selectivity of CQ-Lyso for lysosomes was
determined with a colocalization experiment in living HeLa
cells using LysoTracker®Deep Red (1.0 µM) as a reference.
HeLa cells were incubated with CQ-Lyso (5.0 µM) at 37 °C
for 15 min, and followed by incubation with
LysoTracker®Deep Red (1.0 µM) at 37 °C for another 15
min. Three color channels were used for these experiments:
Red fluorescence channel (570-620 nm, pseud-colored as
yellow for clarity) and green fluorescence channel (515-550
nm) for CQ-Lyso, deep red/infrared channel (663-738 nm) for
LysoTracker®Deep Red. As shown in Figure 8, the cells
exhibited both green and red fluorescence emitted by CQ-
Lyso under a 488 nm excitation. When the cells excited at 640
nm, LysoTracker®Deep Red was observed to give deep
31
d . As illustrated in Figure 6, the addition of TFA induced
6
downfield shifts of the quinolinic protons: H2, H3, H4 and H5
in CQ-Lyso by 0.13, 0.11, 0.12 and 0.04, respectively. The
occurrence of a new peak at 9.90 was assigned to proton H6’,
suggesting the protonation of the nitrogen atom in the
chromenoquinolin moiety. Furthermore, the line shape of
proton H1 changed from a single sharp peak at 7.89 to a broad
peak at 8.10 due to the protonation process of proton H6’. In
order to better understand the photophysical properties of CQ-
+
Lyso and its protonated form CQ-Lyso+2H , the time-
dependent density functional theory (TD-DFT) calculations
were performed. As shown in Figure 7, the highest occupied+
molecular orbitals (HOMO) of CQ-Lyso and CQ-Lyso+2H
were essentially located on the entire molecular skeleton, and
the lowest unoccupied molecular orbitals (LUMO) were
mainly distributed on the quinoline moiety. The lowest energy
transition of CQ-Lyso was the same with that of CQ-
+
Lyso+2H from HOMO to LUMO, and the calculated excited
wavelengths are 403 nm and 490 nm respectively, which were
in good agreement with the experimental results (shown in
table S2). It’s found that the HOMO-LUMO energy gap of
+
CQ-Lyso+2H (2.6201 eV) was smaller than that of CQ-Lyso
(3.4410 eV), indicating the enhancement of ICT process
induced by the protonation of nitrogen atom of
4
6
1
chromenoquinoline ring. Therefore, optical studies, H NMR
analysis and theoretical calculation strongly support the
mechanism shown in Scheme 2.
Figure 8. Images of living HeLa cells co-stained with CQ-
Lyso (5.0 µM) and LysoTracker®Deep Red (1.0 µM). (a)
Bright field; (b) green fluorescence (channel 515-550 nm for
CQ-Lyso with excitation at 488 nm); (c) pseudo-yellow
fluorescence (red channel 570-620 nm for CQ-Lyso with
excitation at 488 nm, pseudo-colored as yellow color for
clarity); (d) deep red fluorescence (deep red channel 663-738
nm for LysoTracker®Deep Red with excitation at 640 nm);
(e) overlay of panels a-d; (f) the correlation of CQ-Lyso
(green channel) and LysoTracker®Deep Red (deep red
channel).
1
Figure 6. Partial H NMR spectra of CQ-Lyso before (a) and
after (b) the addition of two equivalents of trifluoroacetic acid
in DMSO-d solution.
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Figure 9 (a) Fluorescence images of CQ-Lyso (5.0 µM) in living Hela cells at various pH. Top row: bright field images; second
and third row: fluorescence images from green channel and red channel with excitation at 488 nm; fourth row: merged images of
the first, second and third row; bottom row: ratio images (Representing the ratio of fluorescence intensity between green channel
and red channel). (b) Ratios of I /Igreen at different pH.
red
Figure 10 (a) Fluorescence images of CQ-Lyso (5.0 µM) in living Hela cells with chloroquine as a stimulant (Top row: 0.0 µM,
middle row: 100.0 µM, bottom row: 200.0 µM). First column: bright field images; second column: fluorescence images from green
channel with excitation at 488 nm; third column: fluorescence images from red channel with excitation at 488 nm; fourth row:
overlayed images of the first, second and third column; fifth column: ratio images (Representing the ratio of fluorescence intensity
between green channel and red channel). (b) Average fluorescence intensity ratios (I /Igreen) between red channel and green
red
channel.
red/near infrared fluorescence signal. A Pearson’s correlation
coefficient between the green/red channel (CQ-Lyso) and
deep red/near infrared channel (LysoTracker®Deep Red)
was calculated to be 0.97, which is an unusually high value as
established. Cells were firstly incubated with CQ-Lyso (5.0
µM) in PBS buffer (pH 7.4) for 15 min at 37 °C and then
incubated in buffers with various pH values (3.0-8.0) in the
presence of 10.0 µM of nigericin for 30 min at 37 °C. As
shown in Figure 9a, when the pH changed from 3.0 to 6.0, the
green fluorescence became stronger (second row) while the
red fluorescence signal decreased (third row). The ratio of the
average fluorescence intensities from these two channels
compared to previously reported lysosome-targeted probes
24,28-35,49,54,56-60
(shown in Figures 8f, S4 and S5).
In addition,
additional co-localization experiment was performed using
LysoTracker®Blue DND-22 to confirm the above findings.
As can be seen in Figures S13 and S14, the results recorded
from this material corresponded exactly to those found for
LysoTracker®Deep Red. These results provided additional
strong support that CQ-Lyso was able to selectively stain
lysosomes in living cells with high selectivity.
To investigate the performance of CQ-Lyso for quantitative
detection of intracellular pH values, the intracellular pH
calibration profile of CQ-Lyso in living HeLa cells was
(I /Igreen) was dependent of the pH value of cells: 6.31 (pH
red
3.0), 5.45 (pH 4.0), 4.96 (pH 5.0) and 3.79 (pH 6.0), which
was consistent with the fluorescence behavior of CQ-Lyso in
aqueous media. These results support our contention that CQ-
Lyso has the potential to serve as a sensitive detector to
measure intracellular pH in a ratiometric manner.
In addition, the ability of CQ-Lyso to monitor dynamic pH
changes in living HeLa cells was evaluated. It’s known that
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the antimalarial drug chloroquine, a cell-permeable base, can
(3) Pérez-Sala, D.; Collado-Escobar, D.; Mollinedo, F. J. Biol. Chem.
1995, 270, 6235-6242.
4) Appelqvist, H.; Wäster, P.; Kågedal, K.; Öllinger, K. J. Mol. Cell
Biol. 2013, 5, 214-226.
2
6, 49
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stimulate living cells to increase lysosomal pH.
Initially,
(
we examined the effect of chloroquine (100.0 and 200.0 µM)
on the fluorescence behavior of the probe at pH 7.4 and 4.0
(shown in Figure S7) in solution. We found that in solution the
presence of chloroquine at these concentrations had little im-
pact on the fluorescence behavior of this probe. This positive
finding allows us to study the effect of chloroquine in HeLa
cells. HeLa cells were initially incubated with CQ-Lyso (5.0
µM) at 37 °C for 15 min and then incubated with varying
concentrations of chloroquine (0.0 µM, 100.0 µM and 200.0
µM, respectively) at 37 °C for 30 min. As can be seen in
Figure 10 that the chloroquine-treated cells showed an
enhanced green fluorescence (green channel) and a decreased
red fluorescence (red channel), indicating the increase of pH
in lysosomes. The intensity ratios of the green and red
(
(
5) Mindell, J. A. Annu. Rev. Physiol. 2012, 74, 69-86.
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0
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3
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fluorescence signals (I /Igreen) in the living cells were
red
calculated to be 4.38 (no chloroquine), 3.50 (100.0 µM
chloroquine) and 3.25 (200.0 µM chloroquine). Based on the
intracellular pH calibration profile of CQ-Lyso in living HeLa
cells, the pH values of chloroquine-treated cells were
determined to be 5.4 (0.0 µM), 6.5 (100.0 µM) and 6.8 (200.0
µM), respectively. These results demonstrated that CQ-Lyso
could indeed visualize the dynamic pH changes in lysosomes.
(
2
15) Chen, W.; Luo, H.; Liu, X.; Foley, J. W.; Song, X. Anal. Chem.
016, 88, 3638-3646.
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Kang, H.; Mook-Jung, I.; Kim, H. M. Chem. Sci. 2016, 4600-4606.
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X. Chem. Commun. 2014, 50, 14021-14024.
CONCLUSION
In conclusion, we have developed a chromenoquinoline-based
ratiometric fluorescent probe, CQ-Lyso, for lysosomal pH
detection and imaging having excellent selectivity, high
sensitivity and long wavelength emission. This probe can
selectively stain lysosomes with an unusually high Pearson’s
correlation coefficient (using LysoTracker®Deep Red (0.97)
and LysoTracker®Blue DND-22 (0.95) as references).
Importantly, this probe was successfully applied to
quantitatively detect lysosomal pH values in a ratiometric
manner under single wavelength excitation in living cells.
(
20) Xu, Z.; Xu, L. Chem. Commun. 2016, 52, 1094-1119.
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2313.
(
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17489.
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Lett. 2015, 17, 5532-5535.
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(
ASSOCIATED CONTENT
Chem. Int. Ed. 2016, 55, 13658-13699.
(27) Yin, J.; Hu, Y.; Yoon, J. Chem. Soc. Rev. 2015, 44, 4619-4644.
(28) Fan, J.; Lin, C.; Li, H.; Zhan, P.; Wang, J.; Cui, S.; Hu, M.;
Cheng, G.; Peng, X. Dyes Pigm. 2013, 99, 620-626.
Supporting Information
Chemical structures of compounds, additional spectral data, confocal
1
13
microscopy images, H NMR, C NMR and HRMS spectra of com-
pounds. The Supporting Information is available free of charge via
the Internet at http://pubs.acs.org.
(
1
(
29) Kim, H. J.; Heo, C. H.; Kim, H. M. J. Am. Chem. Soc. 2013, 135,
7969-17977.
30) Zhang, X.-F.; Zhang, T.; Shen, S.-L.; Miao, J.-Y.; Zhao, B.-X.
RSC Adv. 2015, 5, 49115-49121.
(31) Li, G.; Zhu, D.; Xue, L.; Jiang, H. Org. Lett. 2013, 15, 5020-
AUTHOR INFORMATION
Corresponding Author
5
(
2
(
023.
32) Wan, Q.; Chen, S.; Shi, W.; Li, L.; Ma, H. Angew. Chem. Int. Ed.
014, 53, 10916-10920.
33) Wang, Q.; Zhou, L.; Qiu, L.; Lu, D.; Wu, Y.; Zhang, X.-B.
*
Fax: +86-731-88836954; Tel: +86-731-88836954; E-mail:
song@rowland.harvard.edu
*zhanghongyan@mail.ipc.ac.cn.
Analyst 2015, 140, 5563-5569.
(34) Dong, B.; Song, X.; Wang, C.; Kong, X.; Tang, Y.; Lin, W.
Anal. Chem. 2016, 88, 4085-4091.
Notes
The authors declare no competing financial interest.
(
35) Zhang, X.-F.; Zhang, T.; Shen, S.-L.; Miao, J.-Y.; Zhao, B.-X. J.
Mater. Chem. B 2015, 3, 3260-3266.
36) Liu, X.; Yang, Q.; Chen, W.; Mo, L.; Chen, S.; Kang, J.; Song,
ACKNOWLEDGMENT
(
X. Org. Biomol. Chem. 2015, 13, 8663-8668.
(37) Pan, W.; Wang, H.; Yang, L.; Yu, Z.; Li, N.; Tang, B. Anal.
Chem. 2016, 88, 6743-6748.
This work was supported by the National Natural Science Foundation
of China (No. U1608222) and the State Key Laboratory of Fine
Chemicals (KF1606). We thank Modern Analysis and Testing Center
of Central South University for NMR spectral data.
(
6
(
38) Shi, W.; Li, X.; Ma, H. Angew. Chem. Int. Ed. 2012, 51, 6432-
435.
39) Yuan, L.; Lin, W. Y.; Zheng, K. B.; He, L. W.; Huang, W. M.
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